CTFA Microbiology Guidelines PDF

CTFA technical guidelines
CTFA Microbiology Guidelines

CTFA
Microbiology
Guidelines
CTFA
Cosmetic, Toiletry, and Fragrance Association
The Cosmetic, Toiletry, and Fragrance Association

2007
1101 17th Street, N.W., Suite 300

Washington, D.C. 20036
Phone: 202/331-1770 Fax: 202/331-1969 www.ctfa.org

CTFA
280416 Cover.indd 1 3/6/08 9:31:54 PM

CTFA
Microbiology
Guidelines
2007
EDITORS
John F. Krowka, Ph.D.
John E. Bailey, Ph.D.
PRODUCTION
Natasha Clover
PUBLISHED BY
Phone: 202/331-1770
Fax: 202/331-1969
www.ctfa.org
Copyright © 2007
No portion of the CTFA Microbiology Guidelines may be reproduced in whole or in part, in any form or by any
electronic or mechanical means, including information exchange and retrieval systems (except for the purpose of
official, nonpublic use by the United States Government), without prior written permission from The Cosmetic,
Toiletry, and Fragrance Association, Inc., 1101 17th Street, N.W., Suite 300, Washington, DC 20036-4702.
Printed in the United States of America

Table of
Contents
(Bold are new in 2007)
Acknowledgements.......................................................................................................v
Foreword ....................................................................................................................vii
Introduction .................................................................................................................1
1. Microbiological Quality Assurance for the Cosmetic Industry ...............................3
2. Microbiological Evaluation of the Plant Environment* .......................................11
3. Cleaning and Sanitization ...................................................................................29
4. Microbiology Staff Training* ...............................................................................55
5. Handling, Storage and Analysis of Raw Materials ................................................69
6. Microbiological Sampling....................................................................................73
7. Microbiological Quality for Process Water...........................................................81
8. Microbiology Laboratory Audit* .........................................................................95
9. Microbial Validation and Documentation .........................................................109
10. Maintenance and Preservation of Test Organisms ..............................................129
11. Raw Material Microbial Content.......................................................................139
12. Establishing Microbial Quality of Cosmetic Products* ......................................143
* These guidelines and methods were newly written or substantively revised for the 2005 CTFA Microbiology
Guidelines.
Table of Contents continued
(Bold are new in 2007)
13. Determination of Preservative Adequacy in Cosmetic Formulations ..................149
14. Preservation Testing of Eye Area Cosmetics .......................................................151
15. Microbiological Assessment of Product Quality After Use*................................155
16. Microbiological Risk Factor Assessment of Atypical Cosmetic Products* ...........163
17. Determination of Preservation Efficacy in Nonwoven Substrate Products .. 173
18. M-1 Determination of the Microbial Content of Cosmetic Products ................179
19. M-2 Examination for Staphylococcus aureus, Escherichia coli and Pseudomonas
aeruginosa ..........................................................................................................183
20. M-3 A Method for Preservation Testing of Water-Miscible Personal Care

Products** .........................................................................................................189
21. M-4 Method for Preservation Testing of Eye Area Cosmetics ............................197
22. M-5 Methods for Preservation Testing of Nonwoven Substrate Personal Care
Products* ..........................................................................................................207
23. M-6 A Method for Preservation Testing of Atypical Personal

Care Products* ............................................................................................ 217
24. M-7 A Rapid Method for Preservation Testing of Water-Miscible Personal

Care Products.............................................................................................. 229
25. Glossary of Microbiological Terms ....................................................................237
* These guidelines and methods were newly written or substantively revised for the 2005 CTFA Microbiology
Guidelines.
** Method M-3 underwent substantive revisions and review for the 2007 CTFA Microbiology Guidelines.
Acknowledgements
The Guidelines presented in this volume were developed by the CTFA Microbiology Committee,
with assistance from many members of the CTFA Scientific Advisory Committee. As the
development and updating effort has been a continuing one, listing all of the experts involved from
CTFA member companies would be beyond the capabilities of the current editors. Therefore,
to all who had a part, a very warm and sincere thank you. The editors also would like to thank
Michelle Duelley at CTFA and Don English at Avon for their assistance.
Foreword
In 1969, CTFA began publishing its Technical Guidelines in the CTFA Cosmetic Journal. These
guidelines were developed by the newly organized CTFA Microbiology Committee and were
concerned with microbiological issues. The benefits of having the Guidelines available in a
single volume, and presented in a standardized format, were recognized, and in 1974, the first
independent compilation of the Technical Guidelines was published.
In 1993, after several major revisions and additions to the Guidelines, CTFA responded to
requests made by the users and split the Guidelines into separate volumes so that individuals
might purchase sets relating specifically to their areas of responsibility. The Guidelines are now
published by CTFA in three volumes: Microbiology, Quality Assurance, and Safety Evaluation.
The CTFA Technical Guidelines are dynamic documents that undergo extensive development
and review prior to publication by CTFA technical committees and staff, as well as public review
by CTFA members and nonmember companies, federal government agencies, and scientific
professional societies. Comments from individuals are welcome at any time.
While CTFA has sought to ensure that these Guidelines generally satisfy applicable U.S. federal
statutory and regulatory requirements as of the date they were drafted, CTFA can assume no
responsibility for their adequacy, nor does it purport to advise as to the necessity for their use
in any particular situation. In those Guidelines that address regulatory requirements, decisions
such as when a report must be filed and what information must be included in it can be made
only by those individuals responsible for making such submissions. With regard to all of the
areas covered by CTFA Guidelines, each company must independently assume responsibility to
ensure that their conduct is consistent with all current, applicable federal, state and local laws and
regulations.
It must be emphasized to the user that these Guidelines are intended only to aid manufacturers
in developing programs that meet their individual needs. The Guidelines must not be considered
either minimum or maximum requirements of effective programs. Alternative ways to reach the
goals of the Guidelines may well exist and may be equally useful. Guidelines on any topic must, of
course, be adapted to the particular operations of the manufacturer using them.
Pamela G. Bailey John E. Bailey, Ph.D.

President & CEO Executive Vice President – Science
INTRODUCTION
Introduction
The production of quality personal care products requires a commitment from the manufacturer
to establish and maintain a total quality program. The microbiological component of such a
program is designed to ensure: (1) the product that reaches the consumer is free of microorganisms
that could affect the product quality and consumer health, and (2) during normal product use,
the quality of the product will not be affected by microbial activity.
The CTFA Microbiology Guidelines are intended to provide manufacturers with guidance regarding
establishing and maintaining a microbiological quality program within their companies. The
Guidelines are also recommended for contract packagers and suppliers of raw materials. Sections
of the Guidelines will vary as to applicability for different sectors of the industry and for individual
companies.
The Guidelines are organized into separate sections. The major provisions for an effective
microbiological quality program are outlined in the basic guideline “Microbiological Quality
Assurance for the Cosmetic Industry” (Section 1). More specific information on building and
equipment design, personnel training, cleaning, sanitization and housekeeping immediately
follows in a general section. The quality of raw materials used in cosmetic products is addressed
in guidelines that cover handling, storage, analysis and sampling. Since process water is a major
raw material in cosmetics and toiletries, a separate guideline focuses on process water systems and
quality.
Because of the increasing dependency on microbiological laboratories to provide supportive data

related to product safety and quality, guidelines are offered for evaluating laboratory practices both
in-house and in contract laboratories. “Microbial Validation and Documentation” of methods
under Section 9 offers guidance for use in the laboratory as well as the plant environment.
As an alternative to manufacturing sterile products, the consideration of rational limits to

microbiological content based on the best available information is practical and proper.
Microbiological limits for finished products as well as raw materials are covered in separate
guidelines. “Establishing Microbial Quality of Cosmetic Products” (Section 12) is the result
of the international harmonization efforts of Colipa, CTFA, and JCIA. “Microbiological Risk
Factor Assessment of Atypical Cosmetic Products” (Section 16) offers advice on conducting risk
assessment and testing of atypical and non-aqueous cosmetic formulations. An extensive glossary
defines terms used in the Guidelines.
CTFA MICROBIOLOGY GUIDELINES | INTRODUCTION | 1

These Guidelines are not intended to establish minimum industry standards for all cosmetic,
INTRODUCTION
toiletry and fragrance products. Also, the Guidelines do not cover all areas that might be addressed
under a specific category. CTFA intends to include additional topics in future updates to the
Guidelines. In the interim, cosmetic companies are encouraged to refer to other microbiology
resources. While these Guidelines can help ensure that products are microbiologically acceptable,
they cannot substitute for day-to-day familiarity with the principles of microbial control. The
Guidelines must never be taken to restrict additional activities when circumstances dictate.
Sections of these Guidelines that are new or substantively updated in 2005 or in this edition of
the CTFA Microbiology Guidelines are indicated in the Table of Contents. References including
website addresses for all sections have been updated for the 2007 edition. On November 28,
2007 the Cosmetic Toiletry and Fragrance Association changed its name to the Personal Care
Products Council. The new, broader and more contemporary name for the assocition reflects
our increasingly diverse membership. The Microbiology Guidelines will not be printed with the
new name until the next edition.
2 | INTRODUCTION | CTFA MICROBIOLOGY GUIDELINES

SECTION 1
Microbiological
Quality Assurance
for the Cosmetic
Industry
SECTION 1: QUALITY ASSURANCE

FOR THE COSMETIC INDUSTRY
INTRODUCTION
Adequate control of the microbiological quality of finished cosmetic products depends upon
the implementation of an effective microbiological quality assurance program. Although the
applicability of some aspects of such a program will vary for different types of products, processes,
and facilities, the major areas described below should be reviewed.
The reader is directed to review the “Glossary of Microbiological Terms” at the end of this
document to ensure a proper understanding of the Guidelines.
Note that these guidelines do not apply to products that have been defined as drugs or
pharmaceuticals by regulatory agencies. The Food and Drug Administration’s (FDA) Current
Good Manufacturing Procedures (CGMPs) for Finished Pharmaceuticals should be consulted
for the manufacture of drug products.1
QUALITY ASSURANCE
Quality assurance is defined as “those planned and systematic activities necessary to provide
confidence that a product satisfies given acceptance criteria.”2 The goal of an effective
microbiological quality assurance program is to assure that the finished product consistently
meets established microbiological standards.
The microbiological quality assurance program can be viewed as having several major
components:
• Personnel, including qualifications, functions, and training
• Physical environment, including plant, grounds, equipment and sanitary
procedures
• Materials, including storage, raw materials, packaging, and finished goods
• Procedures, including sampling, testing, laboratory practices, and auditing
The CTFA Quality Assurance Guidelines provides information for establishing quality assurance
programs within cosmetic manufacturing facilities, as well as establishing the control systems
designed to assure product quality and consumer safety.3
SECTION 1: QUALITY ASSURANCE FOR THE COSMETIC INDUSTRY | CTFA MICROBIOLOGY GUIDELINES | 3
PERSONNEL AND TRAINING
All personnel should have the necessary training or experience to perform their assigned functions
in manufacturing and quality control.4
Quality Assurance Microbiology Laboratory

The personnel responsible for microbiological quality control should be of adequate number and
have the necessary training and/or experience to ensure that cosmetics meet established control
limits.
A Microbiologist should have acquired by education and/or experience the expertise needed to
supervise operations and be capable of:
• Sampling raw materials, process water, bulk and finished goods
• Developing and performing test methods
• Performing sanitation inspection of plant facilities
• Performing environmental studies
• Performing documentation and record keeping
• Interpreting and reporting test results
• Developing and implementing hygiene action plans
• Participating in investigation of out of specification microbiological results
A Technician should be qualified by education and/or experience in microbiological technique.
Manufacturing/Operations
A Supervisor should be qualified by training and/or experience to properly ensure maintenance
of the microbiological integrity of the product being manufactured.
Compounders, Filling Line Operators, etc. should have an understanding of causes of
microbiological contamination, common contamination sources and their prevention.
Education Program
In order to maintain microbiological quality, it is important to instill general microbiological
awareness and to train operating employees in hygienic practices. Examples of microbiological
and hygiene training to be emphasized are listed below.
• Potential sources for product contamination by the following avenues:
− Physical contact with manufacturing equipment and formulation ingredients,
especially following poor personal hygiene
− Gross contamination from process and/or rinse water; condensation on
standing; dust and particulate matter laden with microorganisms, including
airborne spores and vegetative cells
− Unsanitary or dirty equipment
− Contaminated raw materials
4 | CTFA MICROBIOLOGY GUIDELINES | SECTION 1: QUALITY ASSURANCE FOR THE COSMETIC INDUSTRY
• Training on proper cleaning and sanitizing procedures (See “Cleaning and
Sanitization” under Section 3)
• Encouraging employees to report plant conditions that could affect product
integrity
• Personal Hygiene:
− No person with any health condition that could adversely affect products
should have direct contact with raw materials, packaging products, or
product contact surfaces
− Personnel should store personal belongings and eat, drink, or use tobacco

only in designated areas

PHYSICAL ENVIRONMENT
Plant and Grounds

Buildings and equipment should be designed for ease of cleaning and sanitization. They should
be clean and maintained in an orderly manner.
The manufacturing area should be designed to minimize the risk of contaminating raw materials,
packaging components, or products. These areas should have walls and floors that are easy to
clean and sanitize. Overhead repositories for dust, such as piping and ductwork, should be kept
to a minimum and cleaned when necessary.
Building openings, including doors, should be designed, operated, and maintained to protect
the manufacturing areas and to minimize environmental contamination. Windows should be
properly screened and each manufacturing facility should have an effective rodent and insect
control system.
Ventilation systems should include, where appropriate, changeable filters properly maintained
to restrict entry of particulate matter, insects, microorganisms, and other contaminants. Positive
air pressure should be available in areas containing easily contaminated materials. Water used for
humidifying should be of acceptable microbiological quality.
Hand-washing facilities should be provided near the production area. Signs reminding personnel
to wash hands should be prominently displayed at the washing facilities. Hand cleansers and
disposable towels should be available.
Eating and smoking should not be permitted in the manufacturing areas.
Clean containers, utensils and microbiologically acceptable water used with a disinfectant-type
cleaner should be available for general environmental cleaning. Clean containers appropriately
labeled should be provided for collecting waste and scrap materials.
Designated areas should be provided for storing raw materials and finished goods.
Machinery and Equipment
It is desirable that equipment be constructed for effective cleaning and sanitization and designed
to protect products from contamination. The CTFA Quality Assurance Guidelines “Annex 3:
Equipment, Part II-Processing” gives important pointers on construction, cleanability, and
related items.5
Possible Sources of Contamination:
• Pipes may contain crevices, pits, sharp turns, dead ends, connections,
unsanitary welded joints.
GUIDELINES
1: QUALITY ASSURANCE
• Equipment may contain pits, crevices, poorly sealed lids, leaking pump shaft
COSMETIC INDUSTRY
seals, defective sight glasses.

OF THE
• Utensils - Plastic is difficult to clean. Wood is not acceptable for most cosmetic
EVALUATION
ORGANZIATION
manufacturing applications.
• Personnel - The human body is a reservoir of large numbers of microorganisms.
Protective apparel should be worn whenever appropriate. Infected cuts or
FOR THE
SECTION
abrasions on the hands should be covered.

SAFETY
• Atmosphere - Dust is laden with airborne microorganisms.

• Other - General condensation, standing water, reused filter pads, cleaning rags
and compressed air can be sources of microbial contamination.
Recommendations:
• Pipes - Stainless steel, glass and plastic hose are the best materials; sanitary
snap joint fittings are preferred. Pipes should be graded to drain with no dead
legs.
• Equipment - Lids on compounding tanks should be tight fitting and
vented to minimize condensate formation. Drains should be at the lowest
point. Equipment should be designed to minimize backwash contamination
potential. Hard to clean equipment should be dismantled and cleaned out of
place between product changeovers.
• Utensils should be made of stainless steel and be thoroughly cleaned, rinsed,
air dried, and properly protected from contamination between uses.
• Personnel should be properly trained in personal hygiene (e.g., washing hands
after restroom use, contact with food, etc., and prior to contact with product)
and the sanitary use of equipment.
• Atmosphere - Introduction of clean air and the exclusion of particulate matter
will help.
• Other - Single-service towels should be used where possible. Compressed air
lines associated with product contact equipment should be protected with
appropriate point of use filters.
SANITARY PROCEDURES
Cleaning and sanitization procedures are essential to ensure good microbial quality in the
manufacture of cosmetics and personal care products. Written cleaning and sanitizing procedures
should be established, distributed and implemented by responsible personnel. These procedures
should be validated in order to consistently meet hygienic manufacturing requirements. Refer to
the CTFA “Cleaning and Sanitization” guideline in Section 3 for further detail.

STORAGE

Care should be taken to prevent introducing microbes when storing materials. The following are
desirable conditions of storage: dry; protected from airborne contaminants; maintained within
reasonable temperature limits (ideally above freezing and below 100°F); located within low
traffic areas; and large enough to segregate incoming materials from material already received
and approved. Materials should be stored in a manner that allows for sufficient cleaning and
inspection.
Raw material containers and storage areas should be protected from contamination by air, dust,
water, and personnel. Storage areas and raw material containers should be cleaned on schedule.
For more specific guidance for storing raw materials consult “Handling, Storage and Analysis of
Raw Materials” under Section 5.
Bulk storage of raw materials, process intermediates, and finished products should be protected
from microbial contamination. Bulk material should be properly labeled. Bulk subjected to
extended storage should be sampled and retested before use in accordance with established
procedures. A program should be established for cleaning and sanitizing bulk storage containers.
(See Section 3 “Cleaning and Sanitization”).
RAW MATERIALS
Specifications
Cosmetic manufacturers should evaluate the microbiological quality of their raw materials
and establish appropriate specifications based on the best available scientific information.
Microbiological specifications should be established for all raw materials susceptible to
contamination. (See Section 11 “Raw Material Microbial Content”). The microbiological quality
assurance program should include provisions that:
• The material is sampled immediately upon receipt from manufacturer.
• Material is held in a clean quarantine area until testing is completed.
• Rejected materials are clearly marked for prompt disposition.
• Accepted materials are so marked.
• Procedures are in place to re-sample and test susceptible raw materials stored
for prolonged periods prior to use.
Identifying Materials
All material should be clearly marked to identity.6
Sampling Plan
For sampling plans see “Microbiological Sampling” (Section 6) in the CTFA Microbiology
Guidelines and “Annex 17: Sampling” 7 in the CTFA Quality Assurance Guidelines.
PACKAGING MATERIALS AND OTHER COMPONENTS

Packaging materials (tubes, jars, bottles, caps, brushes, applicators and other components) should
be properly controlled (e.g., handling, storage, testing and proper documentation of results) to
minimize contamination and to maintain microbiological standards and specifications.
A program should be established to ensure that appropriate packing materials and product
containers conform to in-house microbiological specifications.
FINISHED GOODS
Finished goods should be sampled and tested to assure that products meet established
microbiological specifications and should not be released for distribution until the satisfactory
completion of the testing. See “Microbiological Sampling” (Section 6) and “Establishing Microbial
Quality of Cosmetic Products” (Section 12) for guidance.
LABORATORY PRACTICES
Microbiological Quality Assurance Laboratory

Several key functions of the Microbiological Quality Assurance Laboratory are:
• Analyze raw materials for microbial content and determine if microbiological
specifications are met.
• Check to ensure that plant hygiene procedures are implemented and effective
• Ensure that the microbial status of finished product meets established
specifications.
• Investigate and resolve contamination problems.
• Establish a program to routinely monitor critical control points, including
cleaning, sanitization and storage of processing and filling equipment.
• Establish appropriate documentation and record-keeping procedures for
laboratory testing.
Microbiological testing should be conducted in a laboratory specifically designed for this purpose.
Alternatively, microbiological quality assurance may be subcontracted. For additional guidance
see the “Microbiology Laboratory Audit” under Section 8.
Procedures
The following are the main suggested procedures for the microbiological laboratory:
• Sampling - It is recommended that the procedure as outlined in “Microbiological
Sampling” (Section 6) be followed.

• Testing - It is recommended that raw materials, bulk in-process, and finished
goods be tested for microbial content. See “Raw Material Microbial Content”
(Section 11) and “Establishing the Microbial Quality of Cosmetic Products”
(Section 12).
• Water - Particular attention should be given to water, as it is the most important
raw material as well as a solvent for cleaning, disinfecting and rinsing. See
“Microbiological Quality for Process Water” (Section 7).
• Preservation - The inability of microorganisms to survive in packaged
products should be verified during product development. See “Determination
of Preservative Adequacy in Cosmetic Formulations” (Section 13).
• Monitoring - Control and monitor sanitization procedures by the use of
swabs, direct contact plates, air samplers, and other means. Refer to “Cleaning
and Sanitization” (Section 3).
• Documentation/Record Keeping - Maintain accurate, detailed records
providing the history of a material. A central file should be maintained for
periodic review by a microbiologist and should be the prime record for all
testing performed. (See “Microbial Validation and Documentation” under
Section 9).
It is recommended that completed records be maintained after distribution of the batch of
manufactured product.
MONITORING
The CTFA Quality Assurance Guidelines recommends periodic self-audits.8 Periodic surveillance or
inspection of facilities, operations, practices, housekeeping and sanitation is an excellent adjunct
to a microbiological quality assurance program. Such monitoring helps to verify consistent
compliance with established procedures, confirm that the systems continue to be adequate
for provision of safe and effective products, and identify areas that may require improvement.
Appropriate measures should be taken where undesirable trends become evident or when
conditions are noted that may cast doubt on product or process integrity.
REFERENCES
1. U.S. Food and Drug Administration. 2005. 4. Bailey and Nikitakis. “Annex 1: Personnel
“Current Good Manufacturing Procedures and Training.”
(CGMPs) for Finished Pharmaceuticals.”
Title 21. Code of Federal Regulations, Part 5. Bailey and Nikitakis. “Annex 3: Equipment,
211 (21 CFR 211). http://www.fda.gov. Part II - Processing.”
2. Bailey, John E., and Nikitakis, Joanne 6. Bailey and Nikitakis. “Annex 6: Finished
M. 2007. (Ed). “Glossary of Terms Products/Lot Identification & Control.”
and Definitions”. In CTFA Quality 7. Bailey and Nikitakis. “Annex 17:
Assurance Guidelines. Washington, DC:
Sampling.”
The Cosmetic, Toiletry, and Fragrance

Association. 8. Bailey and Nikitakis. “Annex 14: Internal
Audits.”
3. Bailey and Nikitakis.
10 | CTFA MICROBIOLOGY GUIDELINES | SECTION 2: EVALUATION OF THE PLANT ENVIRONMENT

SECTION 2
Microbiological
Evaluation of
the Plant
Environment
INTRODUCTION
The cosmetic manufacturing plant environment may directly or indirectly affect the microbiological
quality of cosmetic and personal care finished products. Environmental assessment of the plant
primarily employs air and surface monitoring techniques. Evaluation of monitoring results takes
into consideration the intrinsic factors that affect the microbial environmental quality within the
facility. Changes in environmental data (i.e., trend analysis) can serve as useful indicators of the
need for investigation and possible corrective actions.
SECTION 2: EVALUATION OF THE

For facilities that manufacture products regulated as over-the-counter (OTC) drug products in
PLANT ENVIRON MENT

the United States, refer to the United States Pharmacopeia (USP),1 Parenteral Drug Association
(PDA),2 and Current Good Manufacturing Practices (cGMPs)3, 4, 5 for guidance on how to
conduct environmental monitoring for a manufacturing plant.
Since each manufacturing plant is unique, manufacturers have the responsibility of determining
what type of program is most suitable for their facilities. This guideline provides general and
specific information to aid manufacturers in designing environmental monitoring programs suited
to their own needs. Some information offered may not be directly applicable to the operations of
every facility. However, an established environmental monitoring program can provide the data,
tools, and procedures needed to maintain a well-functioning facility. An effective program can
provide information about areas of the plant that may affect the microbial quality of the finished
product.
Manufacturers may want to consider the Food and Drug Administration’s (FDA) Hazard Analysis
and Critical Control Point (HACCP), which was recently mandated for the food industry. This
program is a systematic approach for evaluating hazards and risks of various parts of a process and
places controls and systems at critical points.6
Good communication between microbiologists and facility engineers is essential in maintaining
awareness of changing environmental factors that could alter the microbiological quality of the
plant environment.
SECTION 2: EVALUATION OF THE PLANT ENVIRONMENT | CTFA MICROBIOLOGY GUIDELINES | 11

MANUFACTURING ENVIRONMENT
The quality of the manufacturing plant environment is largely influenced by five basic factors:
facilities, equipment, personnel, housekeeping, and cleaning and sanitization. Understanding
these factors is essential when developing an environmental control program. Additional guidance
on these topics is given in “Annex 2- Premises” in the CTFA Quality Assurance Guidelines7 and in
“Cleaning and Sanitization” in Section 3 of this document.
Manufacturing Facility
Design
A well-designed, well-constructed manufacturing facility can contribute to a high-quality
finished product. Proper design can minimize cross-contamination and contamination from the
surrounding environment. Contamination of the plant environment by microorganisms, dust,
and dirt can be controlled by the use of vent filters, drain traps, and tight-fitting doors and
windows. Airborne dust contamination can be minimized by the use of filtered air-handling
systems that provide adequate ventilation, temperature, and humidity controls to prevent
cross-contamination. Materials used for building interiors should be durable, easily cleaned,
and adequately maintained. Overhead utilities (pipe work) can be designed so that they do not
adversely affect the manufacturing environments. Duct work for these utility systems should be
composed of nonporous and nonflaking material.
PLANT ENVIRON MENT
General building design should include suitable barriers to separate manufacturing and packaging
areas from warehouses, offices, locker rooms, and washrooms. In particular, a good building design
provides separate areas for material receipt, storage, weighing, compounding, filling, packaging,
etc. Traffic flow of both personnel and materials (e.g., raw ingredients, packaging components,
and finished stock) can be minimized in processing and packaging areas.
When considering building design concepts, some decisions on the desired level of control may
be based on present and anticipated requirements of products and manufacturing.
Operational Influences
Both internal and external conditions are important factors that can affect the microbiological
quality of the plant environment. These diverse factors should be taken into consideration when
determining facility design as well as the frequency of microbiological monitoring.
Examples of internal influences:
• Start up of air conditioning or heating systems
• Construction
• Duct and vent cleaning
• Modifications to equipment
• Plant alterations
• Equipment maintenance
• Change in activity level
12 | CTFA MICROBIOLOGY GUIDELINES | ANNEX 2 PREMISES (FACILITY)

Examples of external influences:
• Construction
• Surrounding environment (farm lands, wet lands)
• Climate (temperature, humidity)
• Seasonal changes
Manufacturing and Filling Equipment

Equipment constructed for effective cleaning and sanitization and designed to protect products
from contamination is recommended. When designing or purchasing new manufacturing or filling
equipment, microbiology, quality, engineering, and manufacturing personnel should evaluate
this equipment for its ease of cleaning and sanitization in addition to cost and efficiency.
Some useful factors that influence sanitary maintenance of this equipment include:
• Equipment drawings and/or schematics that can provide helpful guidance for
cleaning and sanitization protocols (refer to “Cleaning and Sanitization” in
Section 3)
• Equipment manufactured from smooth, nonporous materials (e.g., 316L
stainless steel)
• Valves and gauges that are easily disassembled for cleaning

• Equipment made from materials that are compatible with products and
PLANT ENVIRON MENT

cleaning and sanitization solutions
The CTFA Quality Assurance Guidelines Annex 3, “Part I – Packaging Equipment” and “Part
II – Processing Equipment” give important direction on construction, cleanability, and related
items. 8,9
Personnel
Personnel are encouraged to practice good personal hygiene. Wearing clean uniforms and, where
appropriate, head covers, beard covers, clean gloves, or finger cots will help prevent contamination.
Adequate locker room facilities, washrooms, and eating areas should be physically separate from
the manufacturing, filling, and packaging areas of the plant.
It is recommended that employees responsible for sanitation and housekeeping be thoroughly
trained in all pertinent procedures as part of an ongoing training program. All employees involved
with manufacturing and packaging should be trained to follow cosmetic good manufacturing
practices through a regular training program.10 Training programs are most effective if documented
and conducted periodically according to a pre-planned schedule. For more information, refer to
“Cleaning and Sanitization” in Section 3.
Housekeeping
The general plant environment should be kept in a clean and orderly state. For example, cleaning
and/or sanitization of floors, walls, ceilings, vents, pipes, fixtures, and equipment exteriors should
be conducted on a regular schedule according to written operating procedures. Equipment,

hoses, tools, and other items not in use should be stored in a clean state and protected from
contamination. Cosmetic facilities should have effective programs for control of rodents and
other pests, and for proper refuse disposal. For additional guidance, refer to “Annex 2 - Premises”
in the CTFA Quality Assurance Guidelines. 7
Cleaning and Sanitization

Equipment cleaning and sanitization is carried out on an established schedule, usually between
batches of different products, according to written procedures. It is important to assure that
cleaning and sanitization is documented and validated, equipment is identified as to sanitary
status, and cleaned and/or sanitized equipment is kept dry and covered. Refer to Section 3
“Cleaning and Sanitization” for cleaning and sanitization procedures, frequency, expiration times,
and validation processes. 8
See “Annex 3-Part II - Processing Equipment”9 in the CTFA Quality Assurance Guidelines and
“Microbial Validation and Documentation” in Section 9 for additional guidance.
ENVIRONMENTAL MONITORING PROGRAM

The following are among the elements to be considered when establishing an environmental
monitoring plan.
PLANT ENVIRON MENT
Training
Personnel involved with environmental sampling should be properly trained according to a written
procedure applicable to such testing. Training materials should at least address the following:
• Methods and materials for collecting and processing samples
• Appropriate areas for monitoring
• Frequency of monitoring
• Interpretation of test results
• Determination of alert and action levels
• Proper documentation and communication of results
• Corrective action procedures
Documentation
Documentation provides an organized record of the microbiological evaluation. It is recommended
that the following information be included for proper documentation of an environmental
monitoring program:
Procedural Information
• Physical location (manufacturing area, warehouse, etc.)
• Sampling site
• Sampling method

• Collection/recovery medium used
• Incubation time and temperature
• Sampling frequency
Reported Data
• Specific site sampled
• Media quality control information
• Date and time sample was collected
• Weather conditions
• Activities occurring near the sampling site at time of sampling
• Results
• Date and time
• Signature of investigator (a microbiologist or suitably trained individual)
Additional information may be included based on in-house needs or company policies.11
Baseline Data
The objective of an environmental monitoring program is to obtain microbiological data that can
serve as indicators of change in the environment. Monitoring these indicators can help identify

the need for investigation and possible corrective actions to reduce or eliminate the contamination
PLANT ENVIRON MENT

source in the environment. Criteria are determined based on in-house needs. Prior to the setting
of microbial environmental monitoring criteria, periodic microbiological monitoring of physical
surfaces is performed to determine the baseline levels of the microbial flora within the different
areas of the manufacturing environment. There may be baseline variations in the microbial levels
depending on internal and external conditions. Operational influences are summarized in the
discussion on the “Manufacturing Environment.”
Statistical analysis of environmental historical microbiological test data may be used to set the
alert and action level criteria for deciding when to investigate a shift in the trend. It is common
practice to periodically reevaluate the alert and action levels. There are several statistical methods
for evaluating the data.12, 13
SURFACE SAMPLING
Surfaces 14,15
The microbiological quality of physical surfaces within a manufacturing environment can directly
or indirectly affect the microbial quality of finished cosmetic products. Physical surfaces coming
into direct contact with finished products may include:
• Bulk raw material storage vessels or containers
• Intermediate and finished product storage vessels
• Processing equipment
• Filling equipment

• Transfer pumps and lines
• Pumps
• Valves
• Utensils
• Ancillary equipment and other working contact surfaces
Surfaces not coming in direct contact with the product that could affect microbiological quality
may include:
• Walls
• Floors
• Ceilings
• Overhead lighting and piping
• Vertical and horizontal support beams
• Overhead walkways
• External processing and filling equipment surfaces
• External packaging materials
Monitoring Frequency
The type and frequency of microbiological monitoring of physical surfaces depend on the
PLANT ENVIRON MENT
susceptibility of the finished product to microbial contamination as well as other factors.

Additional factors to consider are the scale of manufacturing, condition, and design of the plant,
type of process, local environmental factors, and company policies. Areas in direct contact with
finished products are usually monitored more frequently than areas that are not.
In areas directly contacting a product, any increase from predetermined microbial alert and
action levels may indicate a potential microbiological problem that could affect product quality.
Increased frequency of testing, adjustments to cleaning and sanitization protocols, or changes in
other processes may be indicated if a potential microbiological problem is found to potentially
affect product quality. Supervisory personnel should routinely review the microbial test data
generated and, if required, adjust the frequency of environmental monitoring.
Methods 16,17
Sampling by means of swabs, direct contact devices, or contact plates are the most common
methods of monitoring surfaces for microbial contamination. Note that swabs and contact plates
will not recover total microbial bioburden from a surface.
Exit monitoring of rinse water can be used to evaluate interior surfaces of manufacturing and
filling equipment. However, rinse water testing may not be useful in detecting the presence of
biofilm bacteria.18 See Table 2-1 for different surface sampling methods.
Swabbing
Sterile swabs can be used to sample environmental surfaces for the presence of microbial
contamination. The sterile swab is wetted in sterile buffer, saline solution, or broth and rubbed

over a measured portion of the surface to be monitored. The swab is then either streaked across
an agar plate or placed into a sterile broth tube. The plate or tube is incubated for the appropriate
length of time.
Swabs
Swabs can be used on flat, irregular surfaces and on hard-to-reach areas. The three most common
composition materials for swabs are Dacron, cotton wool, and calcium alginate. Calcium alginate
is a fibrous material that dissolves in sodium citrate or sodium hexametaphosphate, a characteristic
that facilitates the total release of microorganisms that have been recovered on the swab from the
surface. This allows for a quantitative analysis.19 Leachables from cotton wool swabs, such as
fatty acids, may be inhibitory or detrimental to microbial growth.20 Whichever type of swab is
used, all on-going testing should be performed with the same type of swab and be processed as
soon after collection as feasible. In cases where there is a delay (e.g., swab samples need to be
shipped to a laboratory for processing and analysis), transport swabs may be used. Transport
swabs are designed to maintain the viability and numbers of microbes present at the time of
sampling until the time of processing. The swab manufacturer should be consulted for storage
and temperature conditions to determine how long after use a transport swab can maintain the
viability of microorganisms.21
Sampling

Using aseptic technique, the sample site is sampled by rubbing a premoistened swab over the
surface. Moistened swabs are essential for recovery of the highest possible numbers of bacteria,
PLANT ENVIRON MENT

molds, or yeasts. Solutions used to wet the swab may include, but are not limited to, sterile
buffer, saline, or broth.
If sanitizer or disinfectant residues are present on the surfaces being sampled, they may interfere
with the test results. All of the solutions used to wet swabs should contain a neutralizer if
disinfectant or sanitizer residues are expected.
An appropriate cleaner, such as 70% alcohol, should be used after sampling to remove swab
residue. A sterile template (e.g., 2”x 2” area) may be used to standardize the size of the surface
area sampled.
Processing Swabs
Three basic techniques are commonly used to process swabs after sampling a surface:
Direct Swab Methods
After a swab has been used to sample a test surface, it can either be streaked directly
onto an agar surface in a Petri dish or it can be added to an enrichment broth
as described below. A variety of media, both general and selective, may be used,
e.g., Trypticase Soy Agar, Pseudomonas Isolation Agar, MacConkey, Sabouraud
Dextrose, etc. If general and selective media are used, the general media should
be inoculated first. If the selective media is inoculated first, inhibitory ingredients
may be carried over to the general media and prevent growth. A neutralizer should
be included in the media if there is a concern that sanitizer/disinfectant residues
on the sampled surface may interfere with the test results.

This technique may be used for testing those surface areas on which low numbers
of microorganisms are expected and for which a quantifiable result is needed.
Streaked Petri dishes or enrichment broth are incubated for the appropriate
period and temperature.
Note: The use of selective agars when directly plating swabs can be inhibitory
to injured microorganisms. The results, either microbial growth or no growth,
may not be representative of the types of microorganisms actually present on
the surface. Unless looking for specific types of microorganisms, the use of
general microbial growth media or enrichment techniques may give more useful
information in an environmental monitoring plan.
Test results may be recorded as:
• Growth or no growth per swab
• Growth or no growth per unit area (e.g., per square inch or square
centimeter)
If low numbers are recovered, individual colonies may be counted and recorded
as the number of microorganisms per swab or unit area
Swab Enrichment Methods

This technique may be used in areas where low numbers of microorganisms are
PLANT ENVIRON MENT
expected. After sampling a test surface, aseptically transfer the used swab directly
into a test tube of enrichment broth. Include a neutralizer in the enrichment
broth if there is a concern that sanitizer/disinfectant residues on the sampled
surface may interfere with the test results. Incubate the test tube with swab for
the appropriate period and temperature.
Test results may be recorded as:
• Growth or no growth per swab, based on the presence or absence of
turbidity in a general enrichment broth
• Growth or no growth per unit area (e.g., per square inch or square
centimeter)
Standard Plate Count Methods

This technique can be used in those areas in which there could be either high
or low numbers of microorganisms. After sampling the test surface, aseptically
transfer the swab into a sterile test tube containing 10 milliliters of enrichment
broth. Enrichment broth may include neutralizer(s) for sanitizer/disinfectant
residues. Vortex the test tube to release microorganisms from the swab into
the broth. If sampling with calcium alginate swabs, sodium citrate or sodium
hexametaphosphate may be used to dissolve the swab and aid in releasing recovered
microorganisms from the swab. Remove aliquot(s) of the broth and plate onto
a general microbial growth agar medium and/or onto selective/differential agar
media. Agar media may include a neutralizer(s) for sanitizer/disinfectant residues
that may have been picked up by the swab in sampling test surfaces. If high

numbers of microorganisms are expected, further dilute the original swab dilution
sample and plate these aliquots.
To record results, count the colonies on countable plates, multiply by the dilution
factor, and record as number of microorganisms per swab or per unit area (e.g.,
per square inch or square centimeter).
Contact Sampling
Contact sampling may be performed using modified Petri dishes (i.e., RODAC™ plates), paddles,
or flexible films, which contain a solid agar culture medium whose convex surface extends above
the carrier. Selective and non-selective agar media may be used. The sterile agar surface is applied
to the test surface so that the agar makes total contact with the area being sampled. An appropriate
cleaner such as 70% alcohol is used after sampling to remove any remaining agar residue. The
sampling devices are incubated, after which the degree of microbial contamination per unit area
can be determined. Factors to be considered when choosing one of these methods are:
• Suitability for flat surfaces only
• Usefulness in remote areas under field conditions
• Commercial availability of disposable units
• Suitability for qualitative/quantitative analysis of environmental cleaning and

sanitization procedures
• Limited shelf life
PLANT ENVIRON MENT

• Cost
• Problem of confluence, with certain microorganisms, especially if agar surface
is wet
Rinse Water Method

This technique is generally used to sample either interior surfaces of equipment that cannot be
reached using a swab technique (e.g., kettles, tanks, etc.) or other hard-to-access surfaces that come
into direct contact with the finished product. The rinse water method consists of flushing the
selected surface with a suitable volume of sterile rinse water, collecting a sample of the rinse water,
and then quantitatively determining the number of microorganisms in the sample. Membrane
filtration, pour plates, spread plates, or Most Probable Number (MPN) procedures can be used
to quantify the microbial recovery. Factors to be considered include:
• Surfaces can be selectively tested using this technique
• Chance of introducing testing contamination is minimal
• Allows testing of otherwise inaccessible areas
• Can be used to monitor the efficacy of equipment-sanitizing procedures
• Rinse water monitoring may not detect biofilm bacteria that may adhere to the
interior equipment and transfer line surfaces

General Applications
Physical surfaces coming into direct contact with the product should be examined for the presence
of bacteria and fungi that are known to cause product spoilage or harm to the consumer. Indirect
surfaces such as walls and floors should also be monitored to determine background levels of
microorganisms that are intrinsic to the manufacturing environment.
In general, all equipment (processing and filling), valves, traps, and working surfaces should be
monitored on a defined and periodic basis. Transfer lines should be taken apart and tested. Viable
microbial counts should be performed to determine the levels of microorganisms present in these
areas.6
AIR SAMPLING 22-28

The selection of sites for air sampling is based primarily on the potential for adverse microbiological
effects on the finished product. Routine air monitoring may include selected environmental sites
within the facility as well as sources of compressed air used in manufacturing. Factors to take into
consideration when developing an environmental air monitoring program include room design,
airflow patterns, proximity to vents, and potential for product exposure.
Monitoring Frequency
PLANT ENVIRON MENT
The air-monitoring program establishes the frequency of routine sampling at each location based
on in-house needs, with the areas of greater microbiological concern monitored more frequently.
The schedule for air monitoring in each designated area is based upon previously determined
microbial baseline levels. Monitoring frequency is determined in part by the type of activities in
each area, such as machine operation, personnel, physical cleaning, construction, etc.
Seasonal changes and climate are also important considerations when establishing an air-sampling
program. Areas of greater microbiological concern, such as exposed product and raw materials,
are usually monitored more frequently.
Supervisory personnel, the plant microbiologist, or another suitably trained individual should
review and analyze the microbial test data generated during air sampling. These data can be
used for trend analysis and to provide a history of the plant environment, which can be used to
evaluate sampling frequency or investigate shifts in microbiological quality.
Methods 26-28
A variety of methods may be employed for environmental and compressed air sampling. Each
is designed to meet specific needs. Some sampling methods measure all particulates, including
viable and non-viable microorganisms. Others only measure viable organisms.
Consider the following factors when choosing an air-sampling method:
• Ability to determine change of air contamination over time
• Anticipated bioburden (quantity, viability, type)
• Collection medium

• Quantitative vs. qualitative measurement
• Ability to determine the number of colony-forming units per unit of time or
volume sampled
The monitoring method(s) chosen will be influenced by the requirements of the individual facility.
The requirement or need to measure only viable microorganisms versus all other particulate
matter should be determined.
Viable Methods
The most commonly used methods for measuring viable organisms, many of which are available
commercially, are listed below. Also see Table 2-2.
Settling Plate
A Petri dish containing Trypticase Soy Agar or other suitable general microbial growth agar is
directly exposed in the sampling area (i.e., placed upright in the area with the lid off ). Particles
in the air settle onto the agar surface. After a specified exposure time, the Petri dish is collected,
covered with the lid, and incubated. The number of microbial colonies is counted directly from
the plate.

Centrifugal Air Sample
PLANT ENVIRON MENT

Air is collected via centrifugation through impeller blades, and microorganisms are deposited
onto the surface of a nutrient agar medium in a strip. The growth agar strip is incubated and the
number of organisms per volume of air is calculated.
Sieve Impaction Sampler

Air is drawn into the unit through a sieve and over the surface of an agar plate. After incubation
of the plate, the number of viable microorganisms per unit of time or volume of air may be
determined.
Slit-to-Agar Sampler
Microorganisms are impinged directly onto a microbial growth agar surface in a Petri dish that
rotates beneath a slit opening. Air is drawn through this slit with a vacuum. The speed of the Petri
dish rotation and the volume of air sampled can be adjusted. After incubation of the plate, the
number of viable microorganisms per unit of time or volume of air can be calculated.
Liquid Impinger
Air is drawn through a sampler tube and particles are collected in a liquid medium. The air rises
and is removed from the system. Serial dilutions of the liquid medium are made, and duplicate
aliquots are plated into empty Petri dishes to which a sterile melted microbial growth agar is
added. The Petri dishes are allowed to solidify and are incubated. After incubation, the number
of microbial colonies is counted per Petri dish and an average is calculated for each duplicate
serial dilution.

Multi-Stage Particle Sizing Sampler
The sampler contains up to six plates, arranged vertically. A measured volume of air is drawn
through successively smaller holes in the sieve plates, resulting in acceleration of the particles at
each stage. Viable particle size distribution is calculated from the plate counts at each stage.
Membrane Filter
Air to be sampled is impinged on a gelatin membrane filter, which is then removed from the filter
holder and placed in a dish containing a general microbial growth medium. After an appropriate
incubation period, the number of microbial colonies on the membrane surface is counted. This
unit can also filter for phage and is most commonly used in clean rooms and isolators.27
Non-Viable Methods
Monitoring of non-viable airborne particulates is outside the scope of the guideline. Standards
for air based on particulate matter counts are addressed elsewhere.29, 30 A method that uses optical
particle counts is commonly employed.
Compressed Air Sampling

Compressed air that comes into direct contact with the product process or that could adversely
PLANT ENVIRON MENT
affect the manufacturing environment may be monitored. The Slit-to-Agar method has been
modified for sampling compressed air. This instrumentation, adapted for sampling compressed
gas up to pressures of 125 psi, is based on the impingement principle of particle capture. The
circular sweeping of an agar plate surface is controlled at a critical distance beneath a laser cut air
intake slit and creates a radial undulation over the area of impingement. The speed of the plate
rotation and sampling are precisely controlled so that, after a period of incubation, the growth on
the agar surface can be used to quantitatively measure microbial contamination.
EVALUATION OF RESULTS
There are no set criteria for microbiological monitoring of the environment in plants manufacturing
cosmetics. Trend analysis performed on the data from microbiological monitoring of surfaces and
air in the plant offers a useful evaluation tool. Shifts from established data patterns may indicate
changes in the environment or work practices that may have the potential to affect the microbial
quality of the finished product.
In the evaluation of environmental test results, alert and action levels should be established for
each manufacturing area. Levels set will depend on the areas monitored, historical trend data
from the area, type of monitoring, and potential effects on finished product quality.

Factors to Be Considered in Setting Alert and Action Levels
Many factors influence the microbial alert and action levels established for each production area.
These include, but may not be limited to:
• Objective of the Sampling – Whether, for example, to measure seasonal
changes in air quality to determine effectiveness of a sanitization procedure, or
to monitor changes in work practices.
• Area Sampled – Microbial criteria for air in a warehouse differ from the criteria
for air over a filling machine. Criteria for the surface of cleaned and sanitized
compounding equipment differ from those in areas that do not directly contact
product.
• Type of Product – The type of finished product manufactured, i.e., hostile
vs. microbiologically susceptible, is an important aspect. Susceptible products
likely to be more sensitive to microbial contamination by environmental
influences are expected to require more stringent controls in processing and
packaging areas.
Documentation
Documentation is important to establish the historical trend data in the plant. Trend analysis of

microbial recovery levels in the environment may be useful when:
PLANT ENVIRON MENT

• Investigating potential sources of microbial contamination in a product
• Identifying sources of microbial contamination in the plant
• Identifying potential seasonal trends
• Setting and adjusting cleaning and housekeeping schedules
• Choosing sanitizers
Once a program is in place, the results generated should be documented by keeping an organized
record system. Records of environmental monitoring should be maintained for an appropriate
length of time.
These data can be used to establish a normal range of results. Supervisory personnel, a microbiologist,
or other qualified individual should routinely review all results. Based on these results, a feedback
mechanism can be established whereby all departments involved are informed when the
environmental quality is outside of the normal range. The responsibility for documentation and
communication should be addressed by internal standard operating procedures.
Refer to “Microbial Documentation and Validation” in Section 9 and to “Annex 5 - Production
Control” in the CTFA Quality Assurance Guidelines.”31

Interpretation
Once all the data have been collected, consideration of the following information will help in its
interpretation:
• Total number of microorganisms recovered (quantitative analysis)
• Percent of positive results as compared to the total number of areas tested
(qualitative analysis); for example, this might be useful when using qualitative
swabs
• Presence or absence of objectionable organisms
Alert/Action Level Response

Regardless of the assessment method used, once a normal range of microbial recovery has been
established, the manufacturer should set alert and action levels for all areas that are routinely
monitored. If microbiological test results reach an alert level, a manufacturer can take a number
of actions that may include collecting additional samples, observing the manufacturing area,
evaluating the process, increasing the testing of finished goods, reviewing practices, etc.
Investigation
If microbiological test results reach an action level, a complete investigation is indicated, followed
by corrective actions. At a minimum, these include:

PLANT ENVIRON MENT
• Confirmation of specification results

• Microbiology laboratory interview/investigation
• Review of product logs/records
• Review of cleaning/sanitization procedures
• Review of product and area history
• Interview personnel (production and laboratory areas)
Corrective Actions
Depending on the outcome of an investigation, corrective actions may include:
• Retesting of the affected areas (depending on the extent of the problem,
manufacturing or filling may be delayed until all environmental testing is
complete.)
• Recleaning and resanitization of equipment, floors, walls, etc.
• Additional testing of the finished product to ensure its quality
• Retraining/reinforcement of cleaning and sanitization procedures
• Modification of engineering controls
• Modification of practices or processes
• Documentation of corrective action taken

CONCLUSION
A microbiological monitoring program for the plant environment is a tool to help assure the
microbiological quality of products manufactured for the cosmetic industry. An environmental
monitoring program includes the microbiological monitoring of surfaces, air, raw materials, and
finished products. The value of the program depends on adequately trained testing personnel,
documentation of test data, assessment of results, and appropriate response. Once a microbiological
profile of the plant has been established, a trend analysis can be used to develop alert and action
criteria. Changes in the trend may indicate the need for an investigation, after which a correction
action plan may be implemented. Each step of the process should be accompanied by appropriate
documentation.
Table 2-1 Surface Sampling Methods

Method Description Advantages Disadvantages
Swabs An environmental swab is a 1. Inexpensive. 1. Leachables from cotton

sampling device composed 2. Convenient. may inhibit fastidious
of a synthetic (e.g., Dacron, 3. Suitable for irregular microorganisms
calcium alginate) or cotton surfaces. 2. Microorganisms may
tip affixed to a wood or 4. Calcium alginate tips can become trapped in the
plastic stick. It is used to be dissolved in media to swab head.
sample discreet areas in release all microorganisms 3. May leave a residue or
difficult-to-reach locations collected. microbial growth agar on
or irregular surfaces.

5. Can be used for highly the surface after being
contaminated areas. sampled; the agar media
PLANT ENVIRON MENT

6. Can be utilized in residue needs to be
remote areas under field removed.
conditions.
7. Can be qualitative or
quantitative.
Contact A contact plate may be 1. Can be used in remote 1. Expensive.

Plates modified Petri dishes, areas. 2. Short shelf life under field
paddles, or flexible films 2. Samples the same size area conditions.
containing a solid microbial each time as defined by the 3. Not suitable for irregular
growth agar whose convex size of the device. surfaces..
surface extends above 3. Can be qualitative or 4. Microbial overgrowth may
the carrier. The sampling quantitative. be a problem.
device may contain any of a 5. May leave a residue or
number of various types of microbial growth agar on
microbial growth agar with the surface after being
or without a disinfectant/ sampled; the agar media
sanitizer neutralizing agent. residue needs to be
removed.
Rinse The rinse water technique 1. Can be used to test 1. Quantitative. May not
Water consists of flushing the otherwise inaccessible detect the presence of
surface to be tested with a areas such as the interior biofilm. Bacteria.
sterile rinse solution such as equipment surfaces of 2. Not suitable for many
water. manufacturing equipment. applications.
2. Larger surface areas may 3. Extensive manipulation
be sampled. may be required.
4. Sample processing may
affect test results.
Table 2-1

Table 2-2 Air Sampling Methods32
Sedimentation An open Petri dish filled 1. Easy. 1. Cannot determine the

(i.e., Settling with a microbial growth 2. Least expensive. amount of air sampled.
Plates) agar that is exposed for a 3. Constant surveillance not 2. Microbial count cannot be
specified time period (i.e., necessary. correlated with air volume.
15 to 60 minutes) that is left 4. Large numbers of areas can 3. Disposition of colonies
exposed to the air. be monitored in a short is affected by size of
amount of time. particles, temperature, and
Provides a rough estimate of 5. Any type of microbial flow/volume of air passing
airborne contaminants growth agar media can be across surface.
used. 4. Affected by air movement
6. No sampling device in area.
required. 5. If left exposed for a long
period of time, plates can
desiccate.
Centrifugal Lightweight, portable unit 1. High recovery efficiency. 1. Requires special agar strips
Impactor that measures a quantifiable 2. Portable. that are expensive and
volume of air (1 to 1,000 3. Fast and easy to operate. available only from the
liters). The sampling media 4. Excellent for areas that are manufacturer.
are agar strips. difficult to access. 2. Strips have a limited shelf
5. Measures concentration of life.
viable particles as function 3. Strips susceptible to
of time and unit volume over growth in heavily
of air. contaminated areas.

6. Selective/differential agar
PLANT ENVIRON MENT
strips are available.
Sieve Impaction Air is drawn through a 1. Colony overlapping May be cumbersome if used
Sampler uniformly perforated surface minimal. with a vacuum.
and is distributed over an 2. Large air volumes possible.
agar surface. 3. Portable.
4. Air flow can be calibrated.
5. May be used to sample
compressed air when used
with a vacuum.
6. Choice of agar dish size and
media is flexible.
Slit to Agar Air is pulled through a slit 1. Measures concentration of 1. Vacuum source required.
Sampler over a revolving plate. viable particles as function 2. Not easily portable.
of volume of air. 3. Large numbers of sampling
2. No serial dilution or plating areas are needed, very time
required. consuming.
3. Wide application in 4. Electrical connection
surveillance of ambient air required.
contamination. 5. Best suited for clean rooms
4. Volume and speed 6. Some systems require
adjustable. 150 millimeter (mm) agar
5. Constant surveillance not plates.
necessary.
6. Remote sampling probe
can be used.
Table 2-2

Table 2-2 Air Sampling Methods32 continued
Liquid Air is drawn through a 1. Samples with high viable 1. Low sampling rate.
Impingement sampler tube and particles counts can be diluted for 2. Vacuum source required.
are collected in liquid enumeration. 3. Time consuming.
medium. Microbial counts 2. Quantitation is good for 4. Requires dilution and
are determined in the liquid. spores and vegetative cells. plating.
3. Inexpensive. 5. Breaks up bacterial
particles.
6. Device may consist
of breakable glass
components.
Sieve A specific volume of air is 1. Determines size of 1. Limited sampling duration

Multi-Stage drawn through a series of particles. does not provide entire
Particle Sizing sieve plates, resulting in 2. Particles. picture.
Sampler particle size separation. This 3. Measures concentration 2. Requires many plates.
allows plate counts at each of viable particles as a 3. Vacuum required.
stage. function of time. 4. Not well adapted for
4. No serial dilution or plating heavily contaminated
required. Comparable to areas.
the impingers. 5. Agar desiccation.
Membrane Air is drawn through a 1. Large volume of air. 1. Equipment cumbersome.

Filter gel filter disc, which is 2. 3μm pore size retains 2. Requires additional

then placed on an agar Coli-phages. manipulation of
PLANT ENVIRON MENT

surface for enumeration of 3. Gelatin overcomes membranes.
microorganisms. desiccation.
Table 2-2
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D., Amedick, G., Lutticken, R. 2001. Stress on Microbial Recovery on an Agar

IRRITATION
“Comparison of two sampling methods Surface.” Applied and Environmental

for the detection of Gram-positive Microbiology 61(4): 1232-1239.
EVALUATION
and Gram-negative bacteria in the 28. Sartorius. http://www.sartorius.com.

environment: moistened swabs versus Click on “For Your Laboratory” and then
RODAC plates.” International Journal of “Air Monitoring.”
Hygiene and Environmental Health. 203, 29. International Organization for
245-248. Standardization. 1999. “Cleanrooms and
18. U.S. Food and Drug Administration. associated controlled environments – Part
1993. Inspection References. Guide to 1: Classification of air cleanliness.” ISO
Inspections Validation of Cleaning Processes. 14644-1. Geneva, Switzerland. http://
http://www.fda.gov/ora/inspect_ref/igs/ www.iso.org.
valid.html. 30 International Organization for
19. Parenteral Drug Association, Inc. Standardization. 2000. “Cleanrooms and
Supplement TR 13 55(5): 20. associated controlled environments – Part
20. Betty A. Forbes, Daniel F. Sahm, and 2: Specifications for testing and monitoring
Alice S. Weissfeld. 1978. Bailey and Scott’s to prove continued compliance with
Diagnostic Microbiology. St. Louis, MO: ISO 14644-1.” ISO 14644-1. Geneva,
The C.V. Mosby Company. Switzerland. http://www.iso.org.
21. Wilson, D.A., M.S. Tuohy, and G.W. 31. Bailey and Nikitakis. “Annex 5 -
Propcop. 2001. “Effects of storage Production Control.”.
temperature on the recovery of bacteria 32. Hess, Kathleen. 1996. Environmental
from three swab transport system: BD Sampling for Unknowns. Boca Raton, FL:
CultureSwab and BD Culturette (BD CRC Lewis Publishers.

SECTION 3
Cleaning
and
Sanitization
INTRODUCTION
Cleaning and sanitization procedures are essential to ensure microbial quality in the manufacture
of cosmetics and personal care products. These procedures should be validated in order to
consistently meet hygienic manufacturing requirements. The design of these procedures should
take into account the product formulation and all aspects of manufacturing.
GENERAL CONSIDERATIONS
Specific internal programs for cleaning and sanitization should be established. These programs
are essential to:
• Assure the microbiological quality of the product
• Meet legal regulations where required
• Minimize the microbial load contributed by processing, filling, and storage
equipment
• Avoid the cost associated with microbial failure
• Help maintain the company commitment to quality
CLEANING AND SANITIZATION

If cosmetics and over-the-counter (OTC) drugs are manufactured with the same equipment,
refer to FDA guidelines for the manufacture of OTC drugs.1, 2, 3
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• Cleaning is the process of removing product residue and contaminants such
as dirt, dust, and grease from the surface and is the essential first step in any
cleaning and sanitization procedure.
• Sanitization is the process utilized to reduce viable microbial contaminants to
an acceptable level. All surfaces must be clean for the sanitization procedure to
be effective.
• Validation is the process of substantiating and verifying that the process does
what it purports to do.
• Documentation is the process of organizing all relevant information in
an orderly and easily understood format. This documentation is required
to validate a process and maintain an historical record of the process and
equipment usage.
SECTION 3: CLEANING AND SANITIZATION | CTFA MICROBIOLOGY GUIDELINES | 29

Guidance for the development of operating procedures is addressed in each section below. Written
protocols are required prior to attempting to validate any process. For more information, see the
“Microbial Validation and Documentation Guidelines for the Cosmetic Industry.” (Section 9).
TRAINING
Personnel should be properly trained and supervised in the cleaning and sanitization of the facility
and equipment. A document should be written to outline the training process. Ongoing training
should be conducted according to a pre-planned schedule. Performance should be monitored to
verify that the training is effective and proper procedures are followed.
Purpose
Training should be used to:
• Bring new employees to the required level of competency
• Introduce new cleaning and sanitization methods and products to all
employees
• Reinforce existing programs
Re-training should be conducted according to a predetermined schedule, with more frequent
training if needed.
Content
A training program should impart an understanding of the elements of cleaning and sanitization
and their effect on product quality. 4, 5 Training should include:
• Overall microbiological awareness and basic microbiology
• Basic concepts of microbial contamination, common contamination sources,
and their avoidance
• Consequences of microbial contamination
• Sanitary practices
• The risks associated with not following appropriate sanitary practices
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• Good housekeeping
• Personal hygiene
• Basic equipment operation and design
• Importance of cleaning and sanitization and a clear understanding of each
process
• Product type and proper procedure based on product formula ingredients
• Proper and safe use of cleaning and sanitizing agents
• Concentration, dilution, and contact time of cleaners and sanitizers
• Product and chemical residues, including cleaners and sanitizers
30 | CTFA MICROBIOLOGY GUIDELINES | SECTION 3: CLEANING AND SANITIZATION

Training should also include:
• A review of the procedure for proper cleaning and sanitization of the equipment
and areas in the employees’ care
• Effects of changes to process, formula or equipment changes on cleaning and
sanitization requirements (i.e., change control)
• Recordkeeping of cleaning and sanitization performed
• A mechanism for reporting to appropriate personnel any observations that
indicate a potential for contamination
Documentation
Training should be documented. At a minimum, this written record should include:
• Name of the trainer
• Attendees
• Date/time of training
Additional items such as training materials and any tools used to measure comprehension and
understanding may also be included.
DOCUMENTATION
Documentation is the keeping of all essential records of cleaning and sanitization. All these records
should be complete, clear, and concise. In addition to the training documentation discussed
above, manufacturing facilities should document validation and ongoing operations.
Validation
All cleaning and sanitization procedures should be validated. Validation documentation consists
of two components, a protocol and a summary document.

Protocol
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The cleaning or sanitization procedure protocol consists of:
• A written description of the objective of the validation study and acceptance
criteria
• A written explanation of the process
This documentation should include a detailed description of the product, process, and equipment
involved, as well as the protocol and test procedures to be used.

Summary
The summary document consists of:
• A report summarizing the raw data supporting conformance to acceptance
criteria, with raw data either attached or available
• A conclusion to include at least a statement of acceptance, any recommendations,
and revalidation requirements
Cosmetic Microbiology: A Practical Handbook, edited by Daniel K. Brennan provides a useful
reference for further guidance.4
Routine Documentation
Routine or ongoing documentation includes routine logs necessary to maintain a history of the
equipment usage and are an essential part of any investigation. This information can also serve
as part of a validation information package. It can be used for trend analysis, evaluating cycle
reduction, and improving efficiency.
Logs
The routine log should include the following information for each cleaning, sanitizing or
changeover activity:
• Date, start and end times of the cleaning
• Date, start and end times of the sanitization, including expiration time
• Product and batch preceding the cleaning and sanitization
• Operating procedure, SOP, or procedure number for the cleaning and
sanitization being carried out
• Any variation from the established operating procedure
• Sign off by operator
• Review, approval and sign off by verifier/reviewer
• Time, date and identity of next batch start up
• Date and description of any repairs or equipment down time

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Status
In addition to permanent logs, current cleaning and sanitization status should be clearly displayed
on equipment. Examples of status designation labels are:
• Contents and Batch or Lot Number
• Empty Needs Cleaning
• Needs Cleaning
• Clean Needs Sanitizing
• Sanitized
Information on equipment status should also note the date sanitized and the expiration time and
date.

MANUFACTURING FACILITY
The environment of the manufacturing facility strongly influences the microbial quality of the
finished product. Appropriate building design and maintenance are critical. Standard procedures
for facility cleaning and sanitization should be written and a record of their implementation
should be maintained.5, 6
Design and Maintenance

Buildings should be designed for ease of cleaning and sanitization to allow for the sanitary
manufacture of product. This design should minimize cross-contamination and contamination
from the surrounding environment. Maintenance of the building should maintain the integrity
of the sanitary design.
The building layout should be organized to accommodate a rational flow of materials, clean
operations, and adequate supporting activities in the facility. Separate areas should be maintained
for material receipt, storage, weighing, compounding, filling, packing, etc., to prevent cross-
contamination.
It is critical that there be access to all surfaces for cleaning and sanitizing. These surfaces include
equipment, walls, storage cupboards, piping, under stairs, behind tanks, etc.
The following are important points to be considered in the design of a facility:
• Exterior walls, entryways and other building openings should be designed
to prevent access to pests, vermin, birds, insects, and other potential sources
of contamination. They should be kept in good repair. Options may include
automatic closing doors, screens on windows, screened vents, sealed pipe entries,
and loading bays designed to minimize environmental contamination.
• Building systems, including heat, air conditioning, and compressed air, should
be monitored to assure that they do not contribute to contamination. Scheduled
preventative maintenance such as filter changes should be performed.
• Building leaks should be repaired immediately.
• Adequate drainage should be provided to get rid of wastewater effectively

because stagnant or standing water allows for microbial growth.
• Positive air pressure can be used to reduce the risk of contamination in areas
where product is openly exposed to the environment, as in compounding and
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filling operations.
• Equipment should be raised from the floor or otherwise constructed so that
floors can be kept clean.
• To avoid extraneous material from contaminating product, piping, wiring,
transport belts and other potential sources of contamination should not be
positioned above tanks or filling lines.
• Floor, wall and ceiling surfaces should be free from cracks, crevices and open
joints.
• Finishes should be smooth and non-porous to allow for easy cleaning and
sanitization. Peeling paint should be removed.

• There should be access to all wall areas to allow cleaning and eliminate
conditions that contribute to buildup of debris.
• Adequate storage should be provided for items not in use to minimize clutter.
• Areas where equipment is washed should be of sanitary design and properly
maintained.
• Adequate lavatories with hand-washing facilities should be provided.
• Locker areas and lunchrooms should be separate from the manufacturing
area.
Manufacturing and Production Areas

The frequency of cleaning and sanitization is determined by the types of activities conducted in
any given area. Qualified personnel should visually monitor each area routinely. Cleaning and
sanitizing schedules can be adjusted, and remedial action taken as needed. Precautions should
be taken to minimize airborne dust during all cleaning. Any spills of raw materials, product, or
packaging components should be cleaned up promptly. Recommendations are given below for
some specific areas.
Walls, ceilings, pipes, and fixtures

Clean and/or sanitize on a scheduled basis (for example, monthly, quarterly or more frequently,
if needed). Vacuum to remove loose material.
Floors
Clean floors on a scheduled basis and include the following:
• Vacuum and/or sweep frequently
• Wet-mop or machine scrub on a predetermined schedule
• Sanitize as appropriate
Cleaning equipment and supply storage

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Store cleaning equipment and supplies properly in a clean area. Maintain the supply area in
an orderly manner. Separate supplies and equipment for lavatory cleaning from other cleaning
supplies.
Warehouse Areas
Raw materials, packaging components, finished products and equipment should be stored in
warehouse areas under acceptable environmental conditions. Precautions should be taken to
prevent contamination from any source.

General guidance for the warehouse area includes the following:
• Aisles should be kept neat and clean by sweeping, damp mopping or machine
scrubbing. An appropriate, freshly prepared cleaner should be used.
• An established, monitored, and documented insect and rodent control program
should be in place.
• Stored materials and containers should be kept clean, orderly, protected and
correctly identified.
• Container exteriors should be cleaned before transferring material into
manufacturing areas.
Waste Disposal Area

Provisions should be made for regular, timely removal and disposal of waste from the proximity
of finished products, components, and manufacturing areas to minimize the risk of microbial
contamination. Suggestions for handling waste include:
• Place refuse for disposal in designated containers using plastic liners. Construct
containers so they are leakproof and rustproof, and cover whenever possible.
• Empty refuse containers a minimum of once daily and clean when necessary
prior to reuse.
• Clean spills immediately and remove debris from the manufacturing areas.
• Use disposable towels and discard immediately after single use. Avoid the use
of rags.
• Isolate waste disposal areas from manufacturing, and routinely clean and
sanitize to minimize odors.
• Dispose of product or process waste in accordance with current government
regulations.
• Handle all hazardous waste, including spills, per the facility hazardous waste
management plan.

Documentation
In general, the written procedures for the cleaning and sanitization of the floors, walls, ceilings,
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pipes, and general building environment should include the following:
• Type(s) of cleaner and/or sanitizer
• Instructions for the preparation of the proper concentration of cleaner and/or
sanitizer
• Instructions for the proper use of cleaning and sanitizing equipment
• Written schedule for the routine cleaning/sanitizing of each area including:
− Areas to be treated
− Method(s) of treatment
− Frequency of treatment

The cleaning and sanitizing procedures performed should also be documented. The record should
be signed by the individual doing the work as it is completed and routinely checked and initialed
by a qualified individual.
MANUFACTURING AND FILLING EQUIPMENT

Manufacturing and filling equipment has direct contact with product. The following are important
considerations with this equipment:7, 8
• Documentation of cleaning and sanitization of equipment
• Sanitary equipment design
• Frequency and monitoring of cleaning and sanitization
• Procedure for cleaning and sanitization
• Special equipment and procedures
• Criteria for cleaning and sanitization
Documentation of Equipment Cleaning and Sanitization
Operating Procedures
Operating procedures for each piece of equipment should be in place and include the
following:
• Equipment identification
• Equipment disassembly instructions, where necessary
• Product-specific instructions where applicable
• Type of cleaners and sanitizers to be used
• Instructions for preparation of the proper concentrations of cleaning and/or
sanitizing solutions
• Proper application technique, rinse procedure, contact times, and temperature
for cleaning and sanitizing solutions

• Proper storage, labeling and protection of equipment once it has been cleaned
and/or sanitized
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• Time limit between sanitization of equipment and use

• Safety considerations
Equipment status log

For each piece of equipment, a cleaning and sanitizing log should be prepared, maintained, and
made readily available. This log should include the product to be made, name of the equipment,
and the cleaning and sanitization date. In addition, the log should be signed and dated after
each procedure. It should be routinely checked and initialed by the operator and a qualified
individual. Equipment status tags are recommended to assure proper identification. See “Routine
Documentation” under Section 3.

Sanitary Equipment Design
It is essential that process and filling equipment have good drainage and be designed for ease of
cleaning and sanitization. In addition, the equipment should be durable enough to withstand
sanitizing chemicals and/or physical agents.
General equipment design

The following guidance on overall equipment design is intended to minimize conditions that
may lead to microbial growth in the equipment. It also offers suggestions to reduce the potential
degradation of the equipment by the effects of the sanitizers and cleaners used.
• Design process and filling equipment to minimize the retention of residual
product and/or wash water. Residual water will dilute product and/or sanitizer
which can lead to microbial growth and the development of adaptable
microorganisms.
• Minimize condensation in equipment. This can dilute product and create an
environment for microbial growth.
• Design equipment so that internal and external surfaces are accessible and
easily cleanable. All surfaces should be as free as possible of crevices that can
harbor product or microorganisms.
• Choose equipment and its surface finish that are easily cleanable and durable.
316 or 316L stainless steel with a 140 grit or better finish, a type 2B finish, or
equivalent quality, is recommended for susceptible products.
• Choose materials of construction that are not easily degraded, etched, or
reacted when in contact with the product or sanitizers.
• Routinely inspect and replace gaskets when necessary, as they are easily
contaminated.
• Use sanitary welding techniques to avoid creating crevices or rough surfaces
that are difficult to clean. Orbital welding and/or gas tungsten arc welding are
recommended.
Common sanitary design practices for specific types of equipment are listed in the following

sections.
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Tanks/Vessels
• Minimize sharp corners because they are difficult to clean.
• Avoid narrow recesses that could trap product and water.
• Design tanks with a domed head to minimize condensation.
• Choose tanks and vessels with conical or dish shaped bases if possible, with a
center drain, to allow for complete draining.
• Design vessel openings and surfaces to be cleanable.
• Design and maintain covers to fit well and close easily.
• Design vents to minimize debris.
• Eliminate unused drop leg pipes.

Transfer Pipes
• Minimize the length of pipe runs to make cleaning easier and reduce the risk
of biofilm formation.
• Slope pipe runs to be self-draining and cleanable with no dead legs/ends.
• Design piping systems to have a minimal number of T-fittings.
• Use sanitary welding techniques to avoid the creation of difficult-to-clean
crevices and rough surfaces.
• Use sanitary fittings for all connections.
• Avoid screw-threaded piping that comes in contact with the product.
Valves
Valves should be easily cleanable with no dead spaces to collect product residue or water. An
example of a sanitary valve is a diaphragm valve.
Pumps
Sanitary pumps are recommended. The design and installation should allow for complete
drainage. Pumps should be easily accessible for inspection, cleaning, and sanitization.
Filling Equipment
Fillers should be designed to be easily cleaned and sanitized. Avoid drip pans and water-lubricated
belts. If positive air is used in filling equipment, microbial air filters and air line dryers should be
monitored to prevent air line condensate from contaminating finished products.
Gaskets
Gaskets are potential sites for contamination. Gasket materials should be compatible with the
product as well as the cleaning and sanitizing solutions. Non-porous, chemically inert materials
are recommended. Care should be taken to assure that gaskets are properly installed.
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Hoses
Transfer hoses should be of a material that is compatible with product, cleaners and sanitizers
used. They should have sanitary fittings. Cleaned and sanitized hoses should be drained to dry
and capped when not in use.

Schedule
Frequency
All equipment should have a regular cleaning and sanitization schedule. The frequency should be
determined based on several factors:
• Product vulnerability to contamination
• Type of equipment used
• Difficulty in removing product from the equipment
• Whether continuous process batching is being performed
Validation
Cleaning and sanitization schedules should be validated. Ideally, cleaning and sanitization
between batches of products and/or at the end of the day’s production is preferable. Continuous
process of the same product may alter this frequency. An additional determination of an effective
time interval between equipment sanitization and start-up should also be made. This is achieved
by validating the process.
Expiration limit
A validated time or expiration limit should be set for each equipment-sanitizing procedure. This
will depend on equipment and method of sanitization. This expiration limit reflects the allowable
time a piece of sanitized equipment can stand before requiring resanitization.
Monitoring
Personnel

A microbiologist or suitably trained individual should review the entire processing system to
determine potential areas for microbial contamination.
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Areas
Areas may include processing lines, storage and mixing vessels, fillers, pumps, pipe connections,
flexible hoses, pressure relief valves, pigging systems, strainers, utensils, and other related
equipment. Most probable areas to be monitored include low-point drains, internal seams and
gaskets, internal filler nozzles, and the interior pump.

Procedures
Visually inspect dismantled equipment for residual product or water. Determine the presence of
microbial contamination by a method appropriate to the equipment. This monitoring should be
documented.
If a chemical sanitizer is used for sanitization, a neutralizing medium specific for that sanitizer
must be employed in the equipment monitoring procedure. Methods used to determine the
presence of microbial contamination may include swabbing, direct contact, or testing the final
rinse water.
If steam or hot water is used for sanitization, temperature recording devices or thermal strips
should be employed in the equipment monitoring procedure.
Methods
Sampling by means of swabs or contact plates is used to monitor surfaces. Exit monitoring of
rinse water can be used to sample interior surfaces.
Swabbing
A sterile cotton or calcium alginate swab is wetted in sterile buffer, saline solution
or broth and rubbed over a measured portion of the surface of the sanitized
equipment. The swab is then either streaked across an agar plate or placed into
a sterile broth tube. The plate or tube is incubated for the appropriate length of
time.
Examination of the plate will give an organism count, and the individual colonies
can be lifted from the plate and identified. Tubes are examined for turbidity;
this is a pass/fail test. Swabbing is very useful for irregular surfaces or curved
equipment.
Direct Contact
Contact plates contain agar, which has a convex surface. These plates are pressed
against the surface of equipment, then incubated. Examination of the plate will
give an organism count and individual colonies can be lifted from the plate and
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identified. The surface of the equipment touched by the contact plate must be
cleaned of any agar residue after sampling. Contact plates cannot be used for
irregular surfaces and are practical only for flat surfaces.
Final Rinse Test

Water of known microbiological quality and volume is rinsed through the
equipment. The water is recovered and filtered via membrane filtration technique.
The membrane is placed onto a plate and incubated. Examination of the plate
will give an organism count and individual organisms can be identified. Note
that rinse water analysis may not detect the presence of biofilm on equipment
surfaces.

Validation
Validation of equipment sanitization efficacy can be done via swabbing onto hardened agar
plates. Monitoring frequency may be based on the manufacturing history of the product or at
the discretion of the microbiologist or hygienist, e.g., it can be done after each sanitization or on
a periodic basis. If a chemical sanitizer is used, analytical testing may be used to detect residues.
General Procedures
Water
Water utilized in the cleaning and sanitizing processes may be described as:
• Make water - The water used to make up cleaners and sanitizers should have
a low microbial bioburden to avoid contaminating the cleaner and to avoid
consuming the sanitizer.
• Rinse water for cleaned equipment - Water used to rinse cleansers from
cleaned equipment should be fresh, potable water that has a microbiological
quality that meets EPA drinking water quality standards.9
• Rinse water for sanitized equipment - Water used to rinse chemical sanitizers
from sanitized equipment must have no higher microbial bioburden than the
microbial specifications of the product to be made in that equipment.4
When water is used to rinse equipment, the equipment must be drained and used within a
validated expiration time.
Equipment cleaning and sanitization

Clean and sanitize all lines; processing, storage and filling equipment; pumps; pipe connections;
flexible hoses and utensils, as well as plant facilities in the immediate processing and filling areas
as follows:
1. Remove product residue from all contact surfaces by thoroughly rinsing with
water or water/detergent solution between 120° F (49 C) and 180° F (82 C).

Temperature is dependent on product type and equipment compatibility.
Note: Non-aqueous-type product residues should be removed by appropriate
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predetermined methods.
2. Assure thorough cleaning and sanitization of pigging equipment and of the pig
itself. When not in use, pigs must be handled and stored under sanitary, dry
conditions.
Note: Pipeline pigs are devices made of non-porous materials used for recovery
of product, product separation, and cleaning of manufacturing pipelines. The
pig launcher and receiving station must be sanitary in design as this equipment
can easily harbor microbial contaminants.
3. Circulate a cleaning solution for a period of time and at a temperature capable
of effectively removing soil residue in the circuit and/or equipment. All surfaces
not accessible by this cleaning procedure should be cleaned manually and/or
by using special equipment or methods.

4. Rinse the cleaning solution thoroughly from the system with microbiologically
acceptable water, as determined by in-house standards.
5. Before use, equipment should be sanitized according to the written procedure
for the piece of equipment involved. See “Special Equipment and Methods”
below.
6. It is suggested that rinse water for sanitized equipment contain no higher
microbial content than the limits established for the formulated products.
Used process equipment should be cleaned as soon after processing as possible to facilitate removal.
Product dried and hardened on equipment surfaces can be difficult to remove thoroughly.
Cleaned/sanitized equipment should be properly stored before use to prevent recontamination. In
general, equipment should be drained dry with open ends covered to prevent recontamination.
Special Equipment and Procedures

Special cleaning and sanitizing equipment and methods may be employed for processing and
filling apparatus. The equipment and methods are generally designed to fit the individual needs
of each manufacturing facility. There are several methods for cleaning and/or sanitizing.
Manual
Manual methods involve the preparation of cleaning solution and the scrubbing
of equipment or parts using a brush, single-use cloth or pad. It is an effective but
highly time consuming method.
Soak
This method involves immersing utensils or equipment parts in containers of
detergent solution for extended periods of time. Generally, this method is used
in combination with manual cleaning.
Spray
Low or high-pressure sprays are used to remove soil. In most cases, the cleaning
action of the pressure sprays is enhanced by the use of detergents. High-pressure
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spray nozzles such as spray balls or injectors may be permanently installed in

mixing or storage tanks. High-pressure spray wand equipment is also widely used.
This type of equipment is mobile and/or portable. It is used for general surface
cleaning. Spray pressures developed should range from 200 to 1000 pounds per
square inch (psi).
Fog
Fogging is a method of generating a mist for the application of sanitizers. Large
areas of equipment surfaces can be treated by fogging in a very short time, using
small amounts of sanitizers. This method should only be used in closed systems
by properly trained personnel.

Clean In Place (CIP)
CIP is a semi- or fully automated, self-contained system for cleaning and sanitizing
equipment. Cleaning and sanitizing solutions are circulated for a specific time at
specified temperatures. No disassembly of equipment is necessary. Each system is
unique and to work well, should be properly designed, evaluated and controlled.
Factors to consider when using CIP are: detergent/sanitizer type; detergent/
sanitizer concentration; temperature; and design of equipment. Some equipment
design factors include type, number, positioning of spray balls, type of pump,
velocity rates, baffles that may shadow areas of a tank, etc.
Acceptance Criteria
Prior to validation of the cleaning and sanitization process for each piece of equipment, acceptance
criteria should be selected. Criteria should take into account the types of products processed by
the equipment. Typically, criteria include microbial bioburden that meet specific requirements
or limits of the products, the absence of pooled water, limitations on product residue, absence of
objectionable organisms.
Alert and action levels for microorganisms should be established by quality assurance based on
finished product specifications.
CLEANERS (See Table 3-1)

A cleaner can be defined as a chemical or blend of chemicals formulated to remove undesirable
soils from a contact surface. These chemicals may be solvents, acids, bases, detergents, and/or
water-based chemical blends. Industry has focused on aqueous cleaners because of concerns for
the environment and employee exposure.
Aqueous cleaners are defined as blends of water-soluble chemicals designed to remove soils into
a water-based solution with a water continuous phase during cleaning. These consist of surface
active ingredients and other cleaning chemicals that use detergency to lift soils from surfaces

by displacing the soil with the surface active materials. This occurs because the surface active
ingredients have a higher affinity for the surface than they do for the soil.7,10
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Characteristics of an Efficient Cleaner
Aqueous cleaners are typically formulated to contain several ingredients to allow for maximum
cleaning effectiveness. The ingredient requirements depend on the intended use of the cleaner.
Efficient aqueous cleaners utilize surfactants (anionic, nonionic, cationic and/or amphoteric),
dispersants, emulsifiers, wetting agents, builders, chelating agents, sequestering agents,
corrosion-inhibiting agents and stabilizers. The surfactants are used for emulsification, wetting
and penetration; builders for neutralizing hard water interferences; chelating inorganic soils
and saponification of natural oils; and additives for corrosion inhibition, anti-redisposition and
good rinseability. See Table 3-1 for information on specific chemical cleaners. For additional
information, see References 7, 8 & 10 at the end of this section.

Characteristics essential to a good cleaner include:
• Compatibility with equipment, i.e., non-corrosive
• Quick solubility
• Good wetting action
• Good penetration properties
• Good emulsification and soil-dispersion properties
• Good rinsing properties
• Economical and readily available
• Environmentally friendly and non-hazardous
Table 3-1 gives examples, descriptions and advantages/disadvantages of several different
cleaners.
Selection of a Cleaner
Although the characteristics of an efficient cleaner may be more general, the selection of a
particular cleaner for a particular cleaning task requires specific information. The most important
considerations include knowledge of the type of substrate to be cleaned and the type of soil to be
removed. The cleaner type should be matched to the surface to be cleaned (metal, glass, plastic,
etc.), the soil type (organic, inorganic, oils, heavy soils, light soils) and the desired cleaning method
(manual, soaking, CIP, power spray wand, etc.). The cleaner should also be widely available and
economical. Information on the level of cleanliness required (acceptance criteria) should also be
known. Several questions can be asked prior to the selection of a cleaning system:
• Does the cleaner have good detergency on the type of soil to be removed?
• Is the cleaner recommended for the cleaning process to be used?
• Is the cleaner free-rinsing?
• Is the cleaner hazardous or environmentally unfriendly?
• Is the cleaner economically priced at the use level and widely available?
Variables Affecting Efficiency

Besides the selection of an efficient cleaner, several other factors are extremely relevant to the
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success of a cleaning process. Beyond the cleaner itself, cleaning efficiency is influenced by cleaner
concentration, agitation, temperature, cleaning/contact time, rinse method and drying method.
These process variables must be considered, specified, and controlled to ensure a consistent and
optimized cleaning process.
Cleaner Concentration
The concentration of the cleaner should be selected through consultation with the manufacturer
followed by in-house validation. Optimizing cleaning temperature, time or agitation may reduce
the concentration of cleaner required.

Temperature
Usually the higher the cleaning process temperature, the more efficient the cleaning. However,
this depends on the soil type (melt-point issues). Temperature should be optimized for the soil
being cleaned and validated using in-house methods. Safety considerations should be included
when personnel exposure is possible.
Time
In general, the longer the cleaning process, the more thorough the cleaning. Cleaning time is
dependent on the other factors of the process, including agitation, temperature and cleaner
aggressiveness. Soaking may take hours, whereas high-pressure sprays may require from seconds
to minutes. Cleaning time should be considered in the validation of the entire cleaning process/
system.
Agitation
Depending on the product/soil to be cleaned, the range of applied mechanical fluid energy
required for effective cleaning will vary. Equipment can simply be soaked or immersed in a cleaner
solution, manually scrubbed, or cleaned with direct impingement using dynamic spray balls or
jet-spray devices. In general, increased agitation and turbulence improves cleaning efficiency.
Rinse
It is important that the rinse procedure completely removes debris detached from the equipment
during cleaning. The specified volume of rinse water should be validated for each particular rinse
program. A general recommendation is that the rinse water volume be at least three times the
volume of the cleaner solution used. There should be no cleaner residue.7
Drying

To reduce the potential for corrosion, eliminate the opportunity for microbial regrowth, and
prevent dilution of chemical sanitizers, equipment should be properly drained and dried after
rinsing. Evaporation is the simplest and least-expensive drying method. Other methods include
SECTION 3
circulated hot air, vacuum-drying, and forced-air blow drying. For these methods, the air source
may be filtered to provide high-quality air for drying.
Use of 70% alcohol as a finishing step can aid in the evaporation of water. Alcohol can be used
as a dryer/sanitizer and is especially useful for anhydrous products where it is essential that no
moisture remain on the equipment. Caution should be used when using alcohol on equipment
that could present a fire hazard.
Testing to Measure Cleaning Process for Efficacy

The development of a testing and measurement system is key to optimizing and validating the
effectiveness of a specific cleaning process. The method selected for measuring the effectiveness

of the cleaning process should provide information needed to determine that key criteria are met.
Testing of the cleaning process initially requires the development of a baseline level of cleanliness
and an effective method to measure cleanliness. In many cases, visual assessments of equipment or
simple gravimetric analysis will suffice. Alternatively, video scopes, chemical tracer measurements
(fluorescent whiteners, total organic carbon (TOC) in residual water, or conductivity) may be
used. Various methods involve the extraction of the contaminating soil from the surface followed
by quantitative chemical analysis.7 The simplest method that provides appropriately sensitive
results should be used.
After the cleaning system has been selected, it should be validated against the targeted product and
on the equipment where the production will occur. Either a quantitative or qualitative method
may be used to judge the cleaning process, and then acceptance criteria should be established.
Experimentation may occur initially on a smaller bench or pilot-plant scale; however, the cleaning
system should be validated on the actual equipment due to concerns with scale-up. Each variable
of the cleaning process (cleaner concentration, time, temperature, agitation, etc.) should be
considered to determine the optimal conditions.
SANITIZERS
Definition
A sanitizer is either a chemical or physical agent that is effective in reducing microbial contamination
on product contact surfaces. A sanitizer should achieve a 99.9% (3 log10) reduction of pathogenic
or unacceptable microorganisms and reduce other organisms to a minimal acceptable level. A
sanitizer may be considered effective if it reduces microorganisms to acceptable levels, with no
detectable objectionable microorganisms, as determined by the cleaning and sanitization protocol.
7, 8, 11, 12
Characteristics and Selection of an Efficient Sanitizer

The following are desirable characteristics of a sanitizer:

• Effective against a broad range of microorganisms
• Provides adequate microbial reduction; 99.9% effective against organisms of
SECTION 3
concern
• Effective in a relatively short contact time
• Stable and efficacious over time, both in concentrate form and at use levels
• Economical to use
• Non-toxic at use levels
• Non-corrosive at use levels
• Compatible with products and equipment
• Free from objectionable odors and residue
• Meets regulatory requirements
• Biodegradable

Chemical Sanitizers
Combined cleaner/sanitizer agents are available. However, these agents can have reduced detergent
and/or disinfectant activity compared to each agent alone.
Additionally, cleaners have a high optimal pH, whereas most chemical sanitizers are more effective
at neutral or acidic pH.
Some useful chemical sanitizing agents are chlorine, iodophores, quaternary ammonium
compounds, ethyl alcohol, phenolic compounds, formalin, phosphoric acid, hydrogen peroxide,
peracetic acid, and ozone. See Table 3-2 for information on frequently used chemical sanitizers.
Physical Sanitizers
The most common physical sanitizer is thermal energy, either in the form of steam or hot water
(180°F or 82°C minimum). A major advantage of heat is its ability to penetrate into small
cracks and crevices. Heat is also non-corrosive, cost-effective, measurable with recording devices
or thermal strips, efficient, effective against a broad range of microorganisms, and leaves no
residue.
See Table 3-3 for information on frequently used physical sanitization methods.
Factors Affecting Efficacy

Cleaning must always precede sanitization. In-house validation of each specific piece of equipment
is needed to assure sanitizer efficacy. Roughness of surface, bad welds or other defects can make
the equipment difficult to sanitize.
Care should always be taken to follow label directions and manufacturer instructions and
recommendations.
Water incorporated into sanitizers should be of acceptable microbial quality.
Operators should be properly trained. Improper use may give ineffective results, release toxic
fumes, or corrode equipment.

The following process variables should be considered, specified, and controlled to ensure consistent
sanitizer performance:
SECTION 3
• Condition of equipment surfaces
• Materials of construction
• Concentration of sanitizer
• Contact time
• Temperature
• Optimal pH range
• Mechanical energy (pressure and flow rate)

Measurement and Validation of Sanitization Effectiveness
Prior to validation of the sanitization process, the acceptance criteria should be selected for specific
equipment and products. Typical or suggested criteria include microbial bioburden that meets
specific requirements or limits and the absence of pooled water or product residue.
A suggested approach for validating the sanitization procedure effectiveness:
1. Sanitize the equipment.
2. Break down the equipment.
3. Evaluate microbial bioburden and organism type on product flow surfaces
including difficult-to-reach areas such as gaskets, valves, pumps, etc.
4. Check both microbial levels and organism types.
The validation of a sanitization procedure should not be performed immediately after cleaning
but at the longest potential time the equipment will stand before use. This gives an expiration
time for sanitization after which the equipment must be resanitized.
See the section above on “Monitoring” under MANUFACTURING AND FILLING
EQUIPMENT.
SUMMARY
The selection and effective use of a cleaning or sanitizing agent and/or method is dependent
on several factors: the manufacturing facility, the type of product processed, and the design and
layout of the equipment. All cleaning and sanitizing procedures should be properly designed and
their use documented and validated. Personnel should receive adequate instruction and training
in these areas.
With attention to these details, a cleaning and sanitizing program will positively contribute to
achieving a sanitary manufacturing facility.
SECTION 3

Table 3-1 Chemical Cleaners
Refer to manufacturer’s use directions and material safety data sheets (MSDS)
Cleaner Type pH Range Soils Removed Examples Advantages/Disadvantages
Mineral-Acid 0.2 - 5.5 Heavy scales to 1. Strong acids: 1. Good for acid-soluble soils
and Mild Acid inorganic salts Hydrochloric acid 2. Efficient for metal oxide
Cleaners Soluble metal Sulfuric acid removal
complexes Phosphoric acid
3. May be harsh on hands
2. Weak acids (dilute
solutions of organic 4. May have toxicity,
acids): environmental and
Acetic acid handling issues
Citric acid
Neutral 5.5 - 8.5 Light oils Mild, unbuilt surfactant 1. Rely on dissolution and
Cleaners Small particulate solutions (may include emulsification, rather than
water-miscible solvents aggressive chemical attack
such as alcohols or glycol 2. Lowered toxicity and
ethers) corrosivity concerns
Mild Alkaline 8.5 - 12.5 Oils Ammonium hydroxide 1. Alkalinity promotes

and Alkaline Fats Sodium carbonate a. saponification
Grease Sodium phosphate b. solubilization of alkaline-
Particulates Borax solutions soluble soils
Films c. hydrolysis
Corrosive 12.5 - 14 Heavy grease and Sodium hydroxide 1. Work best when soil
Alkaline oils Potassium hydroxide can be hydrolyzed; i.e.
Sodium silicates saponification of fatty soils
2. Harsh on hands
3. Some exposure hazards and
product toxicity hazards
4. Corrosivity
Table 3-1

SECTION 3

Table 3-2 Chemical Sanitizers
General types and uses are listed below. Refer to manufacturer’s use directions and material safety data sheets (MSDS).
Suggested
Type Description Concentration & Advantages Disadvantages
Contact Times
Chlorine Sodium 200 ppm as free 1. Excellent activity 1. Odor

hypochlorite chlorine 2. Readily available 2. Chlorine is less reactive
Calcium 30 minutes 3. Can be used alone in as pH increases
hypochlorite Somewhat cold water on clean 3. Inactivated by organics
Lithium temperature- equipment 4. Reactive with metal
hypochlorite dependent (higher 4. Rapid, sensitive test surfaces - corrosive if
Chlorine gas temperature increases available to determine misused; must carefully
Chloramines biocidal effect) concentration during regulate exposure time
Chlorocyan- Chlorine-releasing sanitization and to verify 5. Sensitive to light and
urates compounds may removal of residual after temperature
require other rinsing 6. NIOSH recommended
conditions of use, employee exposure
e.g., pH contact time, limit 0.5 ppm ceiling for
concentration 15 minutes13
Cationic Quaternary 200 ppm at time 1. Cleans (has excellent 1. Not sporicidal
surfactants ammonium recommended by detergent properties) 2. Most effective against
compounds manufacturer 2. Excellent activity microorganisms at
(normally in 3. Noncorrosive neutral or slightly
combination alkaline pH
with 4. Can be used alone in
water 3. Hard-water tolerance
nonionics) may vary
5. Deodorizes
6. Residual activity 4. Residue may be
incompatible with
7. Odorless product
8. Very stable 5. Inactivated by anionic
cleaners
6. May not be compatible
with nonionics
7. Exit monitoring requires
titration
Iodophors Iodine in 12.5 - 25 ppm 1. Cleans as formulated 1. Poor sporicidal activity

nonionic 10 minutes 2. Excellent activity 2. May stain
surfactants 3. Residual activity 3. Usually formulated
with H3PO4 4. Non-toxic at use 4. Rinsing required

concentrations
5. Stable at use
SECTION 3
concentrations
Alcohols Isopropyl 60-70% isopropyl 1. No rinsing 1. Not effective against
Ethyl alcohol for 15 minutes 2. Readily available bacterial spores
60-95% ethyl alcohol 3. Fast-drying
for 15 minutes; some 4. Used alone
applications to 30%
Table 3-2

Table 3-2 Chemical Sanitizers continued
Suggested
Contact Times
Phenols Phenyl and/or 1:200 solution 1. Cleans 1. Must be formulated

(Phenolic chlorinated 2. Excellent activity 2. Rinsing required
derivatives) phenols 3. Deodorizes 3. Used solution may be
unstable (use within 2-3
hours)
4. Worker exposure limits
5. Activity reduced by
presence of organic
matter
Pine Pine oils Per manufacturer’s 1. Cleans 1. Must be formulated

formulated use directions 2. Excellent activity 2. Odor may be
with soap or 3. Deodorizes incompatible with
surfactants certain products
4. Degreases
Formalin 37% w/v 1% (as formaldehyde) 1. Excellent activity 1. Odor

solution 30 minutes 2. Readily available 2. Highly reactive
(aqueous, as 3. Can be used alone 3. Toxicity
free formalde-
hyde) 4. Should be used cold in a
closed system
5. Skin protection required
6. NIOSH/OSHA exposure
limit to formaldehyde is
airborne concentration
ceiling of 0.1 ppm, 15
minute contact time13
Phosphoric acid H3PO4 Varies, refer to 1. Good activity 1. Used under acidic
solution manufacturer’s use 2. Stainless steel conditions to be
directions 3. Used cold effective.
4. Short contact time 2. Most used in
combination with
iodophors

Hydrogen Purchased as 1.5% of a 35% Effective vs. organics 1. Explosive at high levels
peroxide a stabilized solution for 30 2. Reactive
solution minutes 3. Minimal disinfection
SECTION 3
capacity
Chlorine Mixture of 1-10 ppm Cl02 100- 1. Strong oxidizing 1. Sensitive to light and
dioxide oxychloro 200 ppm expressed as chemical temperature
species: chlorine dioxide 2. More tolerant of organic 2. NIOSH recommended
(chlorite/ matter than chlorine employee exposure
chlorate/ 3. Less corrosive to limit to chlorine is
oxychloro stainless steel 0.5 ppm ceiling for
species, 15 minutes13
chlorine 4. Less pH sensitive
dioxide)
Table 3-2

Table 3-2 Chemical Sanitizers continued
Suggested
Contact Times
Peroxy- Peroxyacetic Refer to 1. Low residue 1. Metal ion sensitivity

hydrogen acid manufacturer’s 2. Environmentally 2. Corrosive to soft metals
peroxide Peracetic acid instructions responsible 3. Odor of concentrate
3. Broad spectrum - 4. Varied activity against
bacteria fungi
4. Generally non-corrosive 5. Corrosive and toxic
to stainless steel and only in concentrated
aluminum solutions (>40%)
5. Relative tolerance to 6. Potential of fire hazard
organic soil
6. Active at up to pH 7.5
7. Good activity against
biofilms
Acid anionics Anionic Minimum 100 ppm 1. Stable 1. pH sensitive (optimal

surfactants 2. Generally non-corrosive pH 2-3)
and acids 3. Non-staining 2. Limited and varied
4. Low odor antimicrobial activity
(poor vs. mold & yeast)
5. Not affected by hard-
water minerals 3. High foaming
6. Removes and controls
mineral films
Ozone14 Oxidizing gas 1-3 ppm 1. Powerful oxidizing gas 1. Unstable

30 minutes 2. Broad-spectrum activity 2. pH sensitive (optimal
3. Fast acting pH 6-8.5)
4. Deodorizes 3. Temperature sensitive
5. Minimal handling 4. Corrosive
5. No residual
6. Must be generated at
site
7. OSHA airborne
exposure limit 0.1 ppm
ozone13
Table 3-2
SECTION 3

Table 3-3 Physical Sanitization Methods
Suggested
Type Description Concentration & Advantages Disadvantages*
Contact Times
Steam Heat Water at 100°C 30 minutes 1. High product 1. Possible residues

Temperature must be compatibility (boiler/pipes)
reached at furthest 2. Easy availability 2. Excessive dwell time
point in system 3. Efficacious 3. High energy
4. Breaks down biofilm consumption
5. Non-selective 4. Condensation
5. High humidity
Hot Water 80° - 100°C 30 minutes 1. High product 1. Volume required

compatibility 2. High energy
(70° - 80°C) (2 hours) 2. Easy availability consumption
3. Effective over long 3. High humidity
distances of pipes 4. Condensation
4. Exit monitoring simple 5. Excessive dwell time
5. Not corrosive
6. No residue
7. Non-selective to all
microbial genera
Direct Heat Electrical heat In combination with Effective for hard-to-reach Not for general use
tape other methods equipment or piping
(specialized or limited use)
* Heat may cause equipment damage by expansion of close-fitting and/or moving parts.
Heat must be used with thermally stable materials.
Scalding water poses a potential hazard.
Table 3-3

SECTION 3

REFERENCES
9. U.S. Environmental Protection Agency.
1. U.S. Food and Drug Administration.
1998. “National Primary Drinking
2006. “Current Good Manufacturing
Water Regulations: Disinfectants and
Practice in Manufacturing, Processing,
Disinfection Byproducts Notice of Data
Packing, or Holding of Drugs; General.”
Availability.” 40 CFR Part 141. http://
In 21 CFR, Part 210. http://www.fda.
www.epa.gov/safewater/standard/v&e-
gov.
frn.pdf.
2. U.S. Food and Drug Administration. 21
10. McLaughlin, Malcolm C., and Alan S.
CFR, Part 211.
Zisman. 1998. The Aqueous Cleaning
3. U.S. Food and Drug Administration. 2000. Handbook: A Guide to Critical-Cleaning
“Good Manufacturing Practice Guide for Procedures, Techniques and Validation.
Active Pharmaceutical Ingredients.” In Rosemont, NJ: Morris-Lee Publishing
Draft ICH Consensus Guideline. http:// Group.
www.fda.gov/cder/guidance/4011dft.pdf.
11. American Society for Testing and
4. Brannan, Daniel K. (Ed.). 1997. Cosmetic Materials. 2000. “E1153-94 Standard
Microbiology: A Practical Handbook. Test Method for Efficacy of Sanitizers
Florida: CRC Press. Recommended for Inanimate Non-Food
Contact Surfaces,” In: ASTM Standards:
5. Bloomfield, S. F., and R. M. Baird. Biological Effects and Environmental Fate;
(Ed). 1996. Microbial Quality Assurance Biotechnology; Pesticides, vol. 11.05. West
in Cosmetics, Toiletries and Non-Sterile Conshohocken, PA.
Pharmaceuticals. Philadelphia: Taylor &
Francis. 12. Ecolab Inc. Food and Beverage Division.
2003. “Making the Right Choices -
6. Bailey, John E., and Nikitakis, Joanne Sanitizers”.
M. (Ed). 2007. “Annex 2 – Premises”.
In CTFA Quality Assurance Guidelines. 13. U.S. Department of Health and Human
Washington, DC: The Cosmetic, Toiletry, Services (NIOSH). 2005. NIOSH Pocket
and Fragrance Association. Guide to Chemical Hazards. http://www.
cdc.gov/niosh/npg/.
7. Block, Seymour Stanton. 2000.
Disinfection, Sterilization, and Preservation. 14. Olsen, Wayne P. “Ozone.” 1999. In:
Philadelphia: Lippincott Williams & PDA Journal of Pharmaceutical Science
Wilkins. and Technology 53: 125. Bethesda, MD:
Parenteral Drug Association.

8. Russell, A. D., W. B., Hugo, G. A., Ayliffe.
1999. Principle and Practices of Disinfection,
Preservation and Sterilization. Vermont:
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Blackwell Scientific Publications.

SECTION 4
Microbiology
Staff
Training
INTRODUCTION
The staff of the microbiology department has an essential role in maintaining product quality that
meets development specifications, marketing design, and customer expectations. The knowledge
and skills of this group are crucial. Microbial test results must be accurate and reliable so that
decisions based on the test data can be made with confidence.
Training of the microbiology laboratory staff should cover the following general areas:
• Following documented procedures
• Qualifying staff to perform the analysis
• Adhering to aseptic technique
• Checking equipment function
• Performing routine equipment maintenance
• Laboratory controls and documentation
This training provides confidence that test results are accurate and can be relied upon during the
decision-making process.
Many different types of microbiological tests may be performed in a cosmetic microbiology
laboratory. These can include content testing of microbiologically susceptible raw ingredients
and finished products, preservative challenge testing of product formulations, and the analysis of
environmental test samples such as cleaning and sanitization swabs, air, or water samples from
a cosmetic manufacturing facility. If cosmetics and over-the-counter (OTC) drugs are tested in
the same laboratory, refer to FDA guidelines for the manufacture of OTC drugs and to relevant
chapters in the United States Pharmacopeia (USP).1,2,3
There are two goals in having a training program for the employees in a cosmetic microbiology
laboratory: First, to provide an in-depth, well-rounded program in how and why a certain
microbiological test is to be conducted on a particular test sample; and second, to ensure that the
microbiological testing for a particular type of sample will be performed exactly the same way
by each employee every time a sample is received for testing. The purpose of this guideline is to
MICROBIOLOGY STAFF TRAINING
provide information regarding requirements for a microbiology staff training program.

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SECTION 4: MICROBIOLOGY STAFF TRAINING | CTFA MICROBIOLOGY GUIDELINES | 55

ESTABLISHING A PROGRAM
The establishment of a training program should include, but not be limited to, the understanding
of microbiological concepts, review of Standard Operating Procedures (SOPs), and review of
test methods or procedures. It is important that the individual have full understanding of the
principles of aseptic technique. Internal or external training classes can be provided as part of
the training program for an employee. It is recommended that hands-on training be included
to demonstrate proficiency in using laboratory equipment and conducting microbiological test
methods. It is recommended that a knowledgeable, qualified individual possessing appropriate
academic and work experience should train new employees to the laboratory.
Training Frequency
All new laboratory employees should receive training prior to beginning work in the laboratory. In
addition, it is recommended that all current staff employees receive periodic re-training at intervals
appropriate to keep them current and proficient in performing the various procedures for which
they are responsible. It is the responsibility of management to ensure that each staff member is
updated or trained according to the company’s policy or Standard Operating Procedures.
Documentation
For each employee in a cosmetic microbiology laboratory, a training record or log should be
established. The documentation should include, but not be limited to, training and dates when
proficiency has been demonstrated for each particular test method, technique, and policy or
procedure used by that individual during a workday. It is important that no laboratory staff
member be allowed to perform any laboratory task until documentation is established indicating
sufficient training was received and proficiency was demonstrated.
The trainer should either initial or sign and date the training record or log to verify that the training
was received and completed for that task. Each training record or log should be periodically
reviewed and initialed or signed and dated by the supervisor of the testing laboratory. Records
should be kept for an appropriate length of time.
It is also important that proper documentation exists verifying that the trainer has the necessary
experience and knowledge to conduct a particular microbial test method or use a particular piece
of laboratory equipment.
Topics
The training topics will often depend upon the laboratory equipment utilized, testing methods
performed, laboratory function, and individual job responsibilities. The tables in the sections
that follow suggest topics and elements that should be included in a microbiology staff training
program.
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56 | CTFA MICROBIOLOGY GUIDELINES | SECTION 4: MICROBIOLOGY STAFF TRAINING

LABORATORY ORIENTATION
A general orientation should be given to any new individual as an introduction for entering
the microbiology laboratory. The topics covered during orientation should remain general in
scope, give an overview of Standard Operating Procedures (SOPs), and cover guidelines within
the laboratory as an introduction. The topics listed in Table 4-1 may be included in a general
orientation. Other topics may be added at the discretion of the person developing the training.
More specific topics are discussed in detail in sections that follow.
MICROBIOLOGY LABORATORY
Equipment
Equipment availability and usage will vary depending on the testing performed in each laboratory.
Most laboratories will contain many of the instruments listed below. Employees should be trained
in the safe and effective use of each piece of equipment needed to fulfill their job function. The
list below is not exhaustive; however, it does contain many of the basic pieces of equipment
requiring calibration. Each laboratory will need a customized list depending on their particular
testing requirements. Common microbiology laboratory equipment includes:
• Balances
• Sterilizers/Autoclaves
• pH Meters
• Water Baths
• Incubators
• Refrigerators
• Low temperature freezers
• Automatic pipetting/dispensing devices (e.g. pipettors, micropipettors,
dispensing pumps, etc.)
• Laminar flow hoods/biological safety cabinets
• Microscopes
• Stereoscopes
• Laboratory water system
• Bunsen burners
• Colony counters
• Sample mixing devices (e.g., vortexes, Waring® Blenders, etc.)
• Laboratory shakers
• Centrifuges
• Laboratory ovens
• Air samplers
• Stopwatches
• Spectrophotometers
SECTION 4:
• Lyophilizers

• Automated microbial identification systems
• Automated microbial counting devices
• Water activity instruments
• Dishwashers
• Automated data collection systems
Calibration of Laboratory Equipment

Every testing laboratory has pieces of equipment that require periodic calibration to verify that
they are maintained and operated in accordance with the manufacturer’s specifications. Training
in verification of the operational status of the equipment, including its calibration, is important.
Besides learning how to use a piece of laboratory equipment, an employee should be trained in
how to recognize when an instrument is not operating correctly.
The list below is not exhaustive; however, it contains the basic equipment that needs periodic
calibration and is found in most microbiology laboratories. Additional information on calibration
of microbiological equipment is given in Table 8-1 in “Microbiology Lab Audit” in Section 8.
• Balances (e.g., weight checks)
• pH Meters (e.g., daily)
• Micropipettors
• Thermometers (e.g., test and standard)
• Temperature recorders
• Water activity instruments
• Sterilizers/Autoclaves
• Timers
• Temperature recorders
• Chamber pressure gauges
• Heat distribution and penetration of chamber and chamber loads
• Stopwatches
• Laminar/Biological safety cabinets
• Air samplers
• Automated microbial identification systems
• Automated microbial counting devices
LABORATORY TECHNIQUES
Common Techniques
Table 4-2 contains common key elements to be included in a training program for an individual
responsible for conducting tests in a microbiology laboratory. The list contains key microbiological
SECTION 4:

techniques that may be employed in the laboratory; however, it is not inclusive of all the different
types of techniques that might be used in every laboratory. Additional techniques performed in
your laboratory should be added to your training program. Specific tests are discussed in detail
in sections that follow.
Microbial Content Testing

Microbial content testing is performed on raw ingredients, packaging components, and finished
goods that are susceptible to microbial contamination. It is important that an individual performing
these types of tests be trained and have demonstrated proficiency in using the techniques listed
in Table 4-3.
Preservative Effectiveness Testing

With the exceptions of the preparation of microbial challenge inocula and inoculated test
samples, many techniques used for conducting preservative effectiveness tests are common
to the routine analysis of test samples for microbial content. In addition to these laboratory
manipulations, training should include the calculation of percentage or logarithmic reduction
and the interpretation of acceptance criteria.
ENVIRONMENTAL MONITORING
General
To effectively monitor the quality of the cosmetic manufacturing plant environment, laboratory
employees with the responsibility for conducting environmental monitoring should be trained
in all methods currently in use. Environmental testing comprises three major categories: surface
sampling, air sampling, and water analysis. Refer to “Microbiological Evaluation of the Plant
Environment” (Section 2) in these guidelines for information on conducting environmental
monitoring in a manufacturing plant.
Training should be based on written procedures which include:
• Methods and materials
• Suggested sites to monitor
• Frequency of testing
• Interpretation of results to include specification levels, where applicable
• Determination of alert and action levels, documentation
• Communication of results
• Corrective action procedures
SECTION 4:

Environmental Monitoring Test Methods
Surfaces
For monitoring the microbial content of surfaces in a manufacturing plant, a laboratory employee
should be trained in how to use one or more of the following surface sampling test methods:
• Swab
• Contact plate (e.g., RODAC plates)
• Flexible films or contact slides
• Final rinse water
Air
For monitoring the microbial content of air in different locations of a manufacturing plant, a
laboratory employee should be trained in how to use one or more of the following air sampling
methods:
• Settling plate (sedimentation plate)
• Centrifugal air sampler
• Sieve impaction sampler
• Slit-to-agar sampler
• Liquid impinger
• Multi-stage particle sizing sampler
• Membrane filter
• Compressed air
Water
For determining the microbial content of water samples in a manufacturing plant, a laboratory
employee should be trained on how to perform the activities listed in one or more of the areas in
Table 4-4.
IDENTIFICATION OF MICROBIAL ISOLATES

Microorganisms are often isolated in environmental, microbial content or preservative challenge
test samples. At times, there may be a need to identify microbial isolate to either the genus or
species level. Results may determine whether a sample passes or fails and may provide information
on the source of the contamination. It is expected that the individual conducting the testing
has the necessary educational background and proper training to correctly identify a microbial
isolate to the genus or species. It is strongly recommended that this individual demonstrate
proficiency in performing microbial identifications. Table 4-5 contains key microbiological tests
that may be employed to identify microbial isolates. The list may not be all inclusive. Additional
microbial identification tests performed on isolates in different laboratories should be added to
the laboratory training program.
SECTION 4:

Proper cleaning and sanitization of the manufacturing equipment and facility are vital to ensure
microbial quality in the manufacture of cosmetic and personal care products. Refer to “Cleaning
and Sanitization” (Section 3) for detailed information for a cosmetic manufacturing facility.
Depending upon the structure of the company, the role of the microbiologist and laboratory staff
may include the following:
• Advising on hygienic equipment design
• Cleaning and sanitization procedures and validation
• Performing equipment monitoring to analyze for microbial bioburden
• Auditing cleaning and sanitization procedures
• Interpreting test results
• Advising on action steps
The microbiology department is, by function, an integral part of the cleaning and sanitization
program. It is recommended that training include the following:
• Aseptic sampling
• Testing methods such as:
− Swabbing
− Direct contact
− Final rinse water
• Validation protocol
• Ongoing environmental monitoring procedures
• Documentation
− Documentation of validation and qualification of cleaning and sanitization
procedures
− Logs for equipment cleaning and sanitization history
• Basic understanding of:
− Cleaning
♦ Chemicals
♦ Physical methods
− Sanitizers
♦ Physical methods
♦ Chemical (including pH range, soil effects, concentration, and contact
time)
SECTION 4:

• Basic Understanding of sanitary equipment design and equipment function
− Process water system
− Processing equipment
♦ Mixers/kettles
♦ Transfer pumps
♦ Transfer pipes and hoses
♦ Valves and gaskets
♦ Storage tanks and vessels
♦ Ancillary and associated equipment (including scoops, pitchers, funnels)
♦ Packaging equipment
CONCLUSION
It must be realized that the topics listed above and the suggested elements for a training program
for a microbiology laboratory cannot be all-inclusive. These elements are only for guidance on the
components of a microbiology staff training program. If a microbial technique, procedure, or a
piece of laboratory equipment is not listed here and is being performed or used in a microbiology
laboratory, then it should be included in the training record or log for each employee whose job
duties include using the equipment or performing the procedure.
Proficiency testing, as a means of demonstrating competence, is an integral part of a training
program.
SECTION 4:

Table 4-1 General Orientation to the Microbiology Laboratory
Topics Areas to be Covered
Organizational Structure • Vice-president

• Director
• Lab manager
• Microbiologists
• Technicians
• Contract temporary staff
• Customers
Introduction to Laboratory • Director

Personnel and Customers • Laboratory manager
• Microbiologists
• Technicians
• Contract temporary staff
• Relevant customer staff
Types of Microbiological • Environmental monitoring

Testing Conducted • Microbial content testing
• Preservative challenge testing
• Selective media
• Culture identification
• Other tests
Laboratory Rules and Safety4 • Laboratory safety manual

• Occupational safety training (29 CFR)
Introduction to Current Cosmetic Good • Documentation of methods and test results

Manufacturing Practices (GMPs)5
• Record keeping rules
• Out-of-Specification investigations and documentation
• Labeling
• Dating
• Signatures
• Expiration dates
• Lot numbers
• Other items as appropriate
Good Laboratory Housekeeping • General organization and cleanliness

• Cleaning schedule
• Cleaning checklist
• Waste handling and disposal
• Others where applicable

SECTION 4:
Table 4-1

Table 4-2 Key Microbiological Techniques
Technique Detailed LIst
General • Aseptic technique

• Use of controls (e.g., positive and negative)
• Check expiration dates of media, reagents, etc.
Media and Broth Preparation • Medium/broth selection for application

Diluent Preparation • Ingredient/component weighing
• Water selection
• Equipment selection
• Sterilization
• Sterility/growth promotion controls
• Shelf life
• Documentation
Organism Identification • Isolation

• Gram stain
• Spore stain
• Lacto phenol cotton blue – mold
• Automated methods (as applicable)
• Catalase, oxidase, coagulase testing
Maintenance of Microbial • Lyophilized or frozen culture reconstitution

Culture Stocks • Rotation and generation criteria, including identification
criteria
• Preparation of slants
• Documentation for traceability of stocks
Sample Preparation and Testing • Inocula preparation and enumeration

• Sample weighing
• Pour plates
• Streaking
• Broth enrichment
• Incubation temperatures and times
• Colony counting
Waste Disposal • Autoclaving/sterilization

• Labeling
• Chemical and biological hazardous waste handling
SECTION 4:
Table 4-2

Table 4-3 Microbial Content Testing
Technique Detailed LIst
How to Sample • Liquid or granular/solid raw ingredients

• Packaging components (e.g., applicators, sponges, etc.)
• Bulk finished products
• Finished products
Sample Preparation • Water-miscible/dispersible raw ingredients and finished

products
• Water immiscible raw ingredients and finished products
• Packaging components
Enumeration • Bacterial and yeast/mold plate count procedures

— Membrane filtration method
— Pour plate method
— Automated methods
Detection • Enrichment testing
Microbiological Acceptance Criteria • Release test specifications/reject procedures

— Raw ingredients
— Packaging components
— Finished goods
Verification of Test Methods • Demonstrate that enumeration and detection methods

are capable of recovering microorganisms from test
samples
Table 4-3
Table 4-4 Water Monitoring Activities

Area of Training Detailed LIst
Sample Collection • Key sites in applicable water system:

— Non-circulating hot/cold (with or without chlorine)
— Circulating hot or cold
— Other
• Importance of timing – holding of sample
Use of Chlorine Inactivators • Where Required
Test Methods: • Pour plate method

Total Count • Membrane filtration method
• Paddle Testers (e.g., Membrane Dip Samplers, Agar Dip
Slides)
Coliform Screening • Membrane filtration

• Most Probable Number (MPN)
• Presence/Absence Enrichment
• Differential/Selective Agar
Pseudomonas detection • Pour Plate Method

SECTION 4:
• Membrane Filtration Method

Table 4-4

Table 4-5 Microbial Identification Tests
Identification of Bacteria Test
Staining • Gram stain (e.g., Gram-negative and Gram-positive)

• Potassium Hydroxide (KOH) Test - for use on inconclusive
Gram stain results for a bacterial isolate to separate them
into Gram-negative and Gram-positive bacteria groups.
• Morphology (e.g., bacillus or cocci)
• Spore Stain
Biochemical Tests for Gram-negative Bacilli • Cytochrome Oxidase Test -to separate Gram-negative
bacilli into fermentor and non-fermentor groups.
• Oxidation/Fermentation Test -Glucose for Gram-negative
bacilli
Biochemical Tests for Gram-positive Cocci • Catalase Test - to separate Gram-positive cocci into
Catalase Positive and Negative groups.
• Coagulase Test - to separate Catalase Positive Gram-
positive cocci into Coagulase Positive and Negative
groups.
• Hemolytic Reactions - to identify the various members of
Catalase-negative Gram-positive cocci species.
Specific Biochemical Reactions • Assimilation/Utilization of Specific Chemicals
• Fatty Acid Cell Wall Analysis
Genotypic Methods • DNA Analysis System
Identification of Yeast Test
Staining • Morphology
Morphology • Germ Tube Test
Use of Specific Biochemical Reactions • Assimilation of Specific Chemicals
Identification of Mold Test
Examination • Morphology/Slide Preparation
Table 4-5
SECTION 4:

ADDITIONAL INFORMATION
Akers, M. 1993. “cGMP Education and Instruction: A Corporate Approach to Employee Training
Worldwide.” Pharmaceutical Technology. 17: 51-60.
Beauchemin, K., D. Gallup, and M. Gillis. 2001. “Read and Understand vs. a Competency-
Based Approach to Designing, Evaluation, and Validating SOP Training.” PDA Journal of
Pharmaceutical Science and Technology, 55 (1): 10-15.
Deluca, P.P. 1983. “Microcontamination Control: A Summary of an Approach to Training.”
PDA Journal of Pharmaceutical Science and Technology, 37(6): 218-224.
Gallup, D., K. Beauchemin, and M. Gillis. 1999. “A Comprehensive Approach to Compliance
Training in a Pharmaceutical Manufacturing Facility.” PDA Journal of Pharmaceutical Science and
Technology, 53(4): 163-167.
Gallup, D., K. Beauchemin, and M. Gillis. 1999. “Competency-Based Training Program Design.”
PDA Journal of Pharmaceutical Science and Technology, 53(5): 240-246.
Levechck, J.W. 1991. “Training for GMPs.” Journal of Parenteral Science and Technology, 45(6):
270-275.
Parenteral Drug Association, Inc. 2001. “Technical Report No. 35, A Proposed Training Model
for the Microbiological Function in the Pharmaceutical Industry.” PDA Journal of Pharmaceutical
Science and Technology, 55(6).
REFERENCES
4. U.S. Department of Health and Human
1. U.S. Food and Drug Administration.
Services, Centers for Disease Control and
2006. “Current Good Manufacturing
Prevention and National Institutes of
Practice in Manufacturing, Processing,
Health. 2007. Biosafety in Microbiological
Packing, or Holding of Drugs General.”
and Biomedical Laboratories (BMBL): 5th
21 CFR, Part 210.
Edition.” Washington, DC. http://www.
2. U.S. Food and Drug Administration. cdc.gov.
July 2000. “Good Manufacturing
5. Bailey, John E., and Nikitakis, Joanne
Practice Guide for Active Pharmaceutical
M. (Ed). 2007. CTFA Quality Assurance
Ingredients.” Draft ICH Consensus
Guidelines. Washington, DC: The
Guideline. http://www.fda.gov/cder/
Cosmetic, Toiletry, and Fragrance
guidance/4011dft.pdf.
Association.
3. United States Pharmacopeia. 2007. United
States Pharmacopeia and the National
Formulary. USP30 - NF25. Rockville,
MD. http://www.usp.org.
SECTION 4:

SECTION 4:

SECTION 5: HANDLING, STORAGE &
ANALYSIS OF RAW MATERIAL
SECTION 5
Handling,
Storage and
Analysis of
Raw Material
INTRODUCTION
Raw materials used by the cosmetic industry are not expected to be sterile as received. Some
commodities, especially those of natural origin, may contain large microbial populations.
The incorporation of such raw materials into product formulations is undesirable because the
organisms introduced could:
• Contaminate equipment and environment
• Present a health hazard
• Produce undesirable changes in products
• Reduce preservative effectiveness
CATEGORIES
A program to control organisms in raw materials should consider the physical and chemical
nature of the raw materials as well as the subsequent processing involved in the manufacture of
quality products. In general, raw materials may be categorized as:
• Hostile - A raw material that will not support and may inhibit the growth of
microorganisms.
• Inert - A raw material that may act as a carrier of microorganisms but ordinarily
will not promote microbial proliferation.
• Supportive - A raw material that serves as a nutritional substrate and supports
microbial growth.
• Preserved - A raw material to which antimicrobial substances have been added
to inhibit microbial growth.
SECTION 5: HANDLING, STORAGE & ANALYSIS OF RAW MATERIALS | CTFA MICROBIOLOGY GUIDELINES | 69
STABILITY
Raw materials have various degrees of stability throughout their shelf life, which may be affected by
the presence of microorganisms. To monitor changes in microbial content, raw materials should
be examined upon receipt and on a regular basis by acceptable microbiological procedures.
EXPIRATION
Expiration dates should be established as determined by history and in-house experience. An
appreciable change from the normal microbial profile indicates a possible problem, which should
be investigated.
RECEIPT
Raw materials received should be properly labeled, placed on quarantine status, and held until
released by Quality Assurance. For further guidance, refer to “Annex 17 - Sampling” in the CTFA
Quality Assurance Guidelines.1
STORAGE
Raw materials should be stored under conditions that minimize the possibility for microbial
contamination. Among the various factors to consider are:
• Control of environmental factors such as temperature, humidity, ventilation
and light
• Proper housekeeping practices
• Rodent, small animal and insect-control programs
• Quarantine systems
• Special storage conditions where indicated
Procedures for the control of raw materials should be adequately outlined for the department
responsible and reviewed and approved by qualified personnel. Once established, the procedures
should be reviewed on a periodic basis.
TRANSFER
Transfer systems for raw materials include sanitized containers, transfer lines, pumps, and related
equipment. These systems should be evaluated on an individual basis depending on the specific
raw material involved. The raw material categories listed above will aid in this evaluation. For
example, a supportive raw material will require greater consideration (i.e., stringent, sanitary
handling) and more monitoring than a hostile one.
70 | CTFA MICROBIOLOGY GUIDELINES | SECTION 5: HANDLING, STORAGE & ANALYSIS OF RAW MATERIALS
SAMPLING
Appropriate control procedures are required for sampling raw materials.
• Personnel - Personnel responsible for sampling raw materials should be trained
in aseptic sampling techniques, preferably by a qualified microbiologist.
Individuals with contagious illnesses or open lesions should not touch or
otherwise contact materials being sampled.2
• Containers - All sampling containers should be sterile and of suitable size.
• Utensils - All sampling utensils should be sterile and suited to the particular raw
material. Long-handled dippers, syringes, sampling tubes, “thieves,” spatulas,
spoons, and pipettes are all examples of sampling utensils. Some of these are
commercially available as presterilized items.
• Techniques - To ensure that samples are representative of the lot or batch, a
logical sampling plan should be developed.1
When samples are obtained for microbiological analysis the following procedures should be
observed.
• Sanitize sample sites externally.
• Obtain subsurface samples of dry raw materials.
• Mix liquids for homogeneity.
• Where possible, take representative samples from the top, middle and bottom
of bulk tanks.
• Properly seal containers.
TESTING
Microbiological testing of raw materials can be accomplished according to “M-1 Determination
of the Microbial Content of Cosmetic Products” (Section 18) or other appropriate method. The
nature of the raw material will determine the method used. This method or any departure from
it must be validated through appropriate testing.
REFERENCES
1. Bailey, John E., and Nikitakis, Joanne 2. Bailey and Nikitakis. “Annex 1 - Personnel
M. (Ed). 2007. “Annex 17 – Sampling”. and Training.”
In CTFA Quality Assurance Guidelines.
Washington, DC: The Cosmetic, Toiletry,
and Fragrance Association.
SECTION 5: HANDLING, STORAGE & ANALYSIS OF RAW MATERIALS | CTFA MICROBIOLOGY GUIDELINES | 71
72 | CTFA MICROBIOLOGY GUIDELINES | SECTION 5: HANDLING, STORAGE & ANALYSIS OF RAW MATERIALS
SECTION 6
Microbiological
Sampling
MICROBIOLOGICAL SAMPLING
SECTION 6:
INTRODUCTION
Appropriate microbiological techniques are needed for sampling raw materials, bulk in-process,
packaging components, and finished goods to ensure cosmetic product quality. Although each
area has its own specific needs, there are basic similarities that are vital to all. From the time raw
materials arrive until the finished product emerges, product history and proper identification are
essential. In general, aseptic techniques should be followed for valid evaluations of samples. The
frequency, sampling and screening methods may vary, but the need for monitoring by qualified
personnel is of utmost importance.
CATEGORIES OF SUSCEPTIBILITY
All raw materials, packaging components, bulk in-process, and finished goods differ in
susceptibility to microbial growth. In order to assess the risk of growth occurring in a material, it
is helpful to establish categories of susceptibility. These categories of susceptibility influence the
extent of sampling and testing required for each material.
Category 1 (High Susceptibility)

High-susceptibility materials include:
• Eye products (aqueous and semi-aqueous)
• Emulsions
• Geriatric and pediatric preparations
• Cream lip preparations (water-based emulsions)
• Water-based products
• Raw materials of natural origin
SECTION 6: MICROBIOLOGICAL SAMPLING | CTFA MICROBIOLOGY GUIDELINES | 73

Category 2 (Medium Susceptibility)
Medium-susceptibility materials include:
• Pressed powders (compact powders, blushing powders)
• Stick preparations, make-up sticks
• Loose powders (face)
• Bath powders (dusting talc)
• Some aerosol products
• Eye powders, pressed and loose, and stick preparations
Category 3 (Low Susceptibility)

SECTION 6:
Low-susceptibility materials include:

• Alcoholic preparations (≥ 20%)
• Deodorants and anti-perspirants
• Bath salts
• Many aerosol products
• Raw materials with antimicrobial activity
Category 4: (Nonsusceptible)
A nonsusceptible material is one that by nature of its components, exclusive of preservatives, will
not support the survival of vegetative organisms.
NOTE: The susceptibility of packaging components and other raw materials should be evaluated
based on their composition and the nature of the product with which they are used.
The above categories are based on the following:
• History - Necessity and frequency of testing a material are based on past
microbiological profiles. Determining the microbial content of a designated
number of batches over a period of time helps to establish the susceptibility
category.
• Susceptibility Tests - Materials may be challenged with microorganisms and
tested for susceptibility.
SAMPLING DEVICES
The following devices for sampling, available from scientific supply houses, may be used:
• Sterile Thief can be used for liquid and/or powder. Glass is not
recommended.
• Sterile Scoops are used for powders. Glass is not recommended.
• Sterile Cups can be used for liquid and/or powder. Avoid contact of hands
with products.
74 | CTFA MICROBIOLOGY GUIDELINES | SECTION 6: MICROBIOLOGICAL SAMPLING

PERSONNEL
The individual(s) responsible for sampling should be trained in aseptic sampling technique by
a microbiologist or other qualified person and should be familiar with visual characteristics of
containers and/or materials to be sampled.
RAW MATERIALS
Designated personnel should be notified of the receipt of each shipment of raw materials. The
shipment should be inspected for physical damage as indicated by leakage of liquids or powders,
rusty or dented containers, and broken or torn containers that expose the contents to outside
contamination. Tank car shipments may be inspected through the top for gross contamination.
SECTION 6:
Any container damaged in such a manner that the contents could be contaminated should be
rejected and the supplier notified.1 Each container should be properly labeled.2
Sampling Technique
Aseptic technique should be followed at all times by trained personnel. Sampling should
be performed with sterile equipment, which can be of stainless steel, plastic, or any other
microbiologically acceptable material. Devices for sampling include ladles, cups, spatulas, scoops,
and spoons. In general, glass devices should be avoided because of the danger of breakage. Each
container should be sampled with a separate sterile device.
Techniques for Specific Containers

Bags
1. Place the bag in a flat position.
2. Clean and sanitize the area to be opened.
3. Aseptically make an opening in the bag.
4. Aseptically remove the sample, transfer to a sterile container, and cap the
container.
5. Close the opening of the bag carefully and seal it.
6. Label, initial and date the sample container.
7. Identify each bag sampled.2
Tank Car and Storage Tanks

Tank cars and storage tanks present unique problems in sampling. As it is imperative to obtain
a representative sample, it is necessary to sample top and bottom. For example, a “thief,” such
as a sterile plastic bottle (of unreactive material such as polypropylene) held in a modified water
sampler holder, may be used to transfer the sample to a sterile, properly labeled container.

Drums Containing Liquids
1. Clean and sanitize the cover.
2. If the entire lid is removable, a ladle-type device may be employed. When there
is only a small opening, a dipper-like device is preferred.
3. Transfer the sample to a sterile, properly labeled container, and cap the
container.
4. Identify each container sampled (label, initial and date).2
Drums Containing Dry Materials

1. Clean and sanitize the cover.

2. Sample from the container using a sterile sampling device.
SECTION 6:
3. Transfer the sample to a sterile, properly labeled container and cap the
container.
4. Identify each container sampled (label, initial and date).2
Sample Properties
The intrinsic properties and microbiological history (internal monographs developed from previous
microbiological assessments) of a raw material are of prime importance in ascertaining sampling
frequency. The type and homogeneity of the material will also play a role in this determination.
A microbiologically nonhomogeneous material generally requires a greater number of samples.
Raw material lots, depending on the type of material, amount ordered, and/or the supplier, are
received in various forms: boxcars, truckloads, bags, boxes and drums. A determination of the
number of samples per lot to be taken (whether the lot is in the form of a single boxcar or in the
form of many bags) should be made. Typical sampling plans can be found in the CTFA Quality
Assurance Guidelines.2
In most cases, 30-100 grams of sample are aseptically taken from each container or area of the
container chosen by one of the above methods. It is also feasible and practical to test composite
samples of the same lot number of certain raw materials, which are by previous analysis and/
or nature considered microbiologically homogeneous. If composites of a lot are shown to be
unacceptable by in-house standards, then all previously sampled containers should be reassayed.1
More extensive sampling and testing may also be necessary.
Stability
Raw materials should be investigated to determine susceptibility to microbial proliferation,
including the effect of storage conditions. Retest intervals should be scheduled to determine the
continued adherence to microbial content specifications.
Stored Unopened Containers

“Low susceptibility” raw materials should be sampled as necessary. “High susceptibility” raw
materials should be sampled on a defined periodic basis or prior to use. If no history is available,
the raw materials should be retested just prior to use in manufacturing.
76 | CTFA QUALITY ASSURANCE GUIDELINES | ANNEX 6 FINISHED PRODUCTS/LOT ID AND CONTROL

Partially Used Containers
Partially used and resealed raw materials, especially of “high susceptibility,” which have been
stored for a defined, preestablished period of time, should be retested just prior to use in
manufacturing.
PACKAGING COMPONENTS
Components should be inspected before shipment by the supplier. The burden of correcting
problems and minimizing defects should be the responsibility of the supplier; however, it is
still the cosmetic manufacturer’s responsibility to have an acceptable component to give the
consumer.
SECTION 6:
Sampling
Upon receipt of a shipment of components, a trained quality-assurance sampler should check for
proper identification.
The quality-assurance sampler randomly samples the shipment. The shipment is then sent to
a designated area until it has been released. A visual examination should be done for obvious
defects, such as mold, dust, dirt, insects, or other extraneous materials. If there is any evidence of
these, a microbiological examination should be done. As a rule, most components are considered
microbiologically safe and not routinely tested except for applicators, brushes and puffs and, in
predetermined cases, those that are in direct contact with “highly susceptible” products. Only
surface areas that come in direct contact with the product are tested.
Sampling Techniques
1. Clean and sanitize area of carton or containers to be opened.
2. Aseptically remove a sufficient number of pieces to ensure that a representative
sample is submitted.
3. Place samples in a suitable bag or container and properly seal to prevent
contamination.
4. Place identification label on the outside of the sample containing the following
information:
− Name of item
− Supplier
− Date received
− Date submitted to microbiology department
− Lot number
− All other pertinent information needed for the identification of the
sample.
5. Properly identify, initial and date each carton or container sampled.

Storage
When the sampling of components meets all inspecting criteria, the shipment is then accepted.
The warehouse is notified so that the balance of the shipment can be stored in the proper place.
Components should be kept elevated from the ground in a relatively clean dust-free area with a
good rotation system.
BULK IN PROCESS
Bulk products should be sampled and tested to ensure acceptability of the product before filling,
as a secondary check on sanitary manufacturing practices, to build a product profile, and as an
economic control to save on labor and component cost.
SECTION 6:
If at any time after manufacture an adjustment is made to the batch, samples should be resubmitted
for microbiological testing. This applies to both hot and cold mixes.
Types of Mixes
1. Cold Mix - No heat is applied at any time during manufacture. Sample in
accordance with the procedures stated previously.
2. Hot Mix - Sample after cooling.
3. Aerosols - Sample the concentrate in the same manner as for hot and cold
mixes.
In the above three types of mixes, approximately the same sample size should be obtained.
NOTE: If composites of a lot of bulk mixed products are shown to be unacceptable by in-house
standards, then all samples should be retested individually and if necessary all previously sampled
containers should be reassayed. More extensive sampling and testing may also be necessary. Tanks
should be sampled from the top and bottom before mixing or stirring the contents. Special
attention should be given to the interface of the possible moisture layer on the top of the material.
Low-susceptibility materials (Category 3) should be sampled on a defined periodic basis or prior
to use.
Sampling of Bulk Product

Because bulk products are usually stored in tanks, drums, carboys and cartons, a representative
sample should be taken regardless of the container size and shape.
A representative sample may be 50-100 grams of a well-mixed product collected in a sterile
container.
• These samples should be sufficient in size to perform all necessary tests,
including confirmatory tests.

• Drums and all sub-units of a manufactured batch of high- and medium-
susceptibility materials may be sampled according to typical plans as outlined
in CTFA Quality Assurance Guidelines, “Annex 17 - Sampling.”2
• Stored bulk material should be resampled and retested on a defined periodic
basis or prior to use.
FINISHED GOODS
Sampling Intervals and Quantities
Samples for testing should be taken at the beginning, middle and end of each shift. If more than
SECTION 6:
one shift fills a batch, samples from each shift should be submitted. In determining the number of
samples taken, consideration should be given to multiple filling lines, container size and extended
downtime and product susceptibility. It is suggested that for possible future reference at least two
unopened samples per batch and/or lot be retained. Retention time should be comparable to that
normally required for other quality control purposes.2
Composites
Composite samples of products from each sampling time may be made; i.e., equal portions of
samples at the beginning, at the middle, and at the end of the run would be combined to provide
three composite samples.
Frequency of Testing
Samples should be tested as soon as possible after manufacture. In general, the frequency of
testing is determined by the nature of the preparation, efficacy of any antimicrobial agent present,
manufacturing process, and experience gained as a result of previous microbiological evaluation.
In practice, it is recommended that, with few exceptions, all susceptible finished products be tested
with the same frequency. This will permit the detection of microbiological problems resulting
from formula changes, errors in compounding or failure of good manufacturing practices.
Microbial Limits
The microbial content for products should follow “Establishing Microbial Quality of Cosmetic
Products” (Section 12), in-house specifications, or other appropriate criteria.
Product Release
Finished products should not be released for consumer use until all microbiological testing has
been completed and products are approved for release.

REFERENCES
1. Bailey, John E., and Nikitakis, Joanne

M. (Ed). 2007. “Annex 8 - Discrepant
Materials Control.” In CTFA Quality
Assurance Guidelines. Washington, DC:
The Cosmetic, Toiletry, and Fragrance
Association.
2. Bailey and Nikitakis. “Annex 17 –
Sampling.”
SECTION 6:

SECTION 7
Microbiological
Quality for
Process Water
INTRODUCTION
This overview of process water system design, treatment and sanitization methods also includes
concepts about process water validation. It is intended for use by microbiologists and other
technical personnel involved with the installation, qualification, and maintenance of process
water systems for cosmetic manufacture.
Process water is purified water that has been obtained by distillation, ion-exchange treatment,
reverse osmosis, or other suitable means to remove chemical and physical impurities. The
QUALITY FOR PROCESS WATER

SECTION 7: MICROBIOLOGICAL
microbiological content of process water should be defined and controlled because:
• It is a major raw material in cosmetics and toiletries.
• It can be a major source of contamination for the entire manufacturing
system.
• The presence of specific microorganisms can pose a potential health hazard.
• Microbial contaminants in process water can produce physical changes in odor,
color and clarity in product formulations. These effects may be present even
after the organisms are destroyed.
• A high microbial load introduced by process water may reduce preservative
activity or cause preservative failure in the final product.
The microbiological quality of cosmetic process water varies and can be influenced by conditions
of manufacture such as pH, temperature, equipment, and the presence of chemicals. It should be
noted that seasonal variation in feed water may also alter process water quality.
In general, it is suggested that process water contain no higher microbial load than the limits
established for the finished product. At a minimum, the microbiological quality of process water
should meet EPA drinking water quality standards.1, 2
A manufacturer should be familiar with the microbiological profile of the water purification
and distribution system. Alert and action levels should be established for the microbiological
acceptability of process water.3, 4, 5
SECTION 7: MICROBIOLOGICAL QUALITY FOR PROCESS WATER | CTFA MICROBIOLOGY GUIDELINES | 81

DESIGN AND MAINTENANCE
Water supply, storage and distribution systems can be major sources of microbial contamination.
Bacterial growth and subsequent biofilm formation can occur as a result of poor design, inadequate
maintenance, and improper cleaning and sanitization procedures.6
Note: Bacterial species of the genus Pseudomonas tend to be the most problematic in contaminating
process water systems.
Common sources of microbiological problems are deionization beds, water softeners, carbon
or sand filters, storage tanks, water meters, valves, lines, dead ends, and return lines. The water
quality should be monitored downstream from these points. The system should be cleaned and
sanitized routinely.
The product manufacturer is responsible for making sure the system is designed for ease of
maintenance and sanitization. It is extremely important that the microbiologist be involved in
the design, installation, and modification of new or existing systems.
The entire system should be designed for proper drainage. Elbows, tees and bends should be
kept to a minimum. U-bends should be avoided unless they can be inverted so as not to form a
pocket where water can stagnate. Unused valves or branch lines should be removed, since these
could result in “dead legs” where microbial proliferation could occur. Areas that restrict or reduce
the flow of water, such as water meters and filter housings, are frequently sites of microbiological
contamination.
Fittings such as unions and valves should be of the sanitary type for ease of cleaning and accessible
to facilitate their removal when necessary. Long runs of welded pipe can be incorporated into a
water system if hygienic design, materials and construction have been used.
Consideration should be given to the materials of construction. Not all materials are compatible
with certain sanitizing agents. As examples, hypochlorite reacts with silver solder; glutaraldehyde
and quaternaries react with rubber. In addition, some plumbing materials are capable of supporting
microbial growth, especially certain plastic tubing, packing and jointing compounds.7 It has been
determined that Teflon® is better than unplasticized polyvinyl chloride (PVC), which is better
than high-density polyethylene (HDPE), which is better than plasticized PVC for inhibiting
the development of mold and bacterial biofilms on plumbing material.8 To minimize biofilm
formation and for ease of sanitization, 316 stainless steel should be used whenever possible.
Service and maintenance of system components should be assessed. When deionizing systems
are used, regeneration and general maintenance should be the responsibility of a competent
individual who is familiar with the equipment. The equipment vendor could be consulted for
advice. In-line filters, ultraviolet lamps, and other equipment requiring special attention should
be serviced according to the vendor’s specifications to maintain them at peak operating efficiency.
Microbial growth can build up on a filter and be a source of contamination, as a result of bacterial
grow-through, long before water flow rate is affected.9
When process water is held in a storage tank, steps should be taken to control the growth of
microorganisms. This can be accomplished by circulating the water through an ultraviolet light
source. Another effective method is the use of heat (65-80°C or 150-176°F) to control microbial
levels in storage tanks.2 Other methods are listed below under “Sanitization of Process Equipment
and Lines.”
82 | CTFA MICROBIOLOGY GUIDELINES | SECTION 7: MICROBIOLOGICAL QUALITY FOR PROCESS WATER

It is the responsibility of the manufacturer, with the cooperation of the microbiologist and the
engineering department, to establish and document methods and procedures for maintaining the
microbial purity in water storage tanks and water distribution systems.
If the system is also designed to remove minerals, it should be examined at regular intervals to
insure maximum efficiency in removal of inorganic materials.
Further information on design and maintenance of process water systems can be found in
References 10-13.
VALIDATION AND MONITORING

The water system is a critical part of any cosmetic manufacturing facility. Appropriate validation
and operation of the system is necessary to maintain product quality. Refer to “Microbial
Validation and Documentation” (Section 9) for additional information.
Validation is the process that shows the system can consistently produce water of the quality
required. It is an overall program which yields documented evidence that the system does what it
purports to do.5 For water systems, this may take the form of one of three conceptual approaches:
prospective, concurrent, or retrospective validation.
Prospective validation involves the execution of an experimental plan, the validation protocol,

before the process is implemented. This approach typically consists of three elements. First, data
are developed to support standard operating procedures (installation qualification or “IQ”).
Next, procedures show how water of the specified quality is consistently produced (operational
qualification or “OQ”). Finally, data are developed to demonstrate that seasonal variations in feed
water do not adversely affect process water quality.2
Concurrent validation is accomplished during the actual implementation of the water treatment
process. Concurrent validation shows a system to be in a state of control through the use of
validated methods to evaluate representative samples taken at strategic sites in the water system.
It involves ongoing monitoring of water microbial quality over an extended time.
Retrospective validation, the third approach, entails the collection and documentation of key
historic data to prove the system performs as specified.
Once a system is validated and its typical microbiological profile is established, a control plan
can be implemented to maintain the performance of the water treatment system.14 If ongoing
monitoring of water quality reveals microbial counts beyond specified quality levels, appropriate
remedial action should be taken.
Monitoring usually includes the identification of alert and action levels.14 Alert levels are those
microbial levels that, when exceeded, signal a potential drift from usual operating conditions, but
not sufficient to compromise the quality of water as an ingredient. In contrast, action levels signal
an excessive drift that requires remedial action.
The control plan should also include a change control system that evaluates the effects of
subsequent changes to the water system and provides for the implementation of appropriate
action or revalidation, if necessary, to maintain water system quality.

WATER TREATMENT METHODS (See Table 7-1)
Depending on the chemical and microbial content of incoming water, many different methods
or combinations of methods may be used to produce microbiologically acceptable process water.
Selection of the appropriate methods and the point of application should be based on a thorough
knowledge of the composition of the raw water and the applicability of each process for correction
of each problem. It should be noted that bacterial tolerance or resistance to chemical agents
can occur. Furthermore, biofilm formation in the process water system can reduce the efficacy
of water treatment methods. Refer to “Cleaning and Sanitization” (Section 3) for additional
information.
The water treatment method may be selected based on factors other than microbiological quality,
as for example, the removal of chemical impurities by distillation.
Chlorination
Chlorine and chlorine-donating compounds are widely used because they are relatively
inexpensive, easily monitored, and effective at low concentrations (2-10 ppm).15 The biocidal
effects of gaseous chlorine and chlorine compounds are inactivated in the presence of organic or
inorganic residuals, and at pH levels above 8.5. Chlorine compounds provide residual activity for
transport and storage conditions, but residual chlorine may have to be removed from the water
prior to its use in manufacturing.
Chlorine is a respiratory irritant and is corrosive at high concentrations. The National Institute
for Occupational Safety and Health (NIOSH) occupational health guidelines recommend an
exposure limit for employees of a 0.5 ppm chlorine ceiling for 15 minutes.16
Ozonation
Ozone has a strong oxidizing effect which is rapidly lost over time as it decomposes to oxygen.
Validation system control should take this into account.
A dissolved ozone level of less than 0.5 mg/l effectively kills bacteria and viruses.17 It is less
affected by temperature and pH changes than is chlorine. In addition, ozone provides residual
activity for transport and storage conditions.
Dissolved ozone in water must be removed prior to use of the water for manufacturing purposes.
Ultraviolet lights or granular-activated carbon are commonly used for this. Routine maintenance
of these systems will be required
Ozone cannot be used successfully in water containing more than 0.5 ppm of soluble manganese
because manganese is converted to an insoluble form.
The NIOSH/OSHA occupational health guidelines stipulate that the permissible exposure limit
for employees is 0.1 ppm (0.2 mg/m3) ozone.16

Filtration
Submicron filtration, reverse osmosis, and ultrafiltration methods are mechanical means of
removing microorganisms from water. These methods introduce no chemical residues and do
not pose a safety hazard. Filtration methods can be highly effective. However, if not properly
maintained, a microbial build-up can occur on filter membranes that can lead to downstream
contamination.
Submicron Filtration
Submicron filters (pore size 0.22-0.45 micron) are nominally rated to remove all microorganisms
and are most effective at points of use. These filters should be tested for integrity to assure absence
of defects. Extensive pretreatment of water may be necessary to reduce the replacement frequency
of submicron filters.
Reverse Osmosis
Reverse osmosis removes greater than 99% of microbial contamination.18 In an appropriate
position in the process water lines, it can replace deionization beds that are often a source of
microorganisms. Initial reverse osmosis costs are higher than chemical treatment but lower than
distillation. Reverse osmosis membranes are somewhat fragile; the high operating pressures render
them susceptible to rupture. Care should be taken to control the pH of incoming water so as not
to destroy the membrane.

Ultrafiltration
Ultrafiltration, like reverse osmosis, removes most microorganisms.19 Unlike reverse osmosis, the
ultrafiltration membrane may be regenerated, and operating pressures are much lower. Costs
are lower than are those for reverse osmosis methods, but are still considerably higher than for
chemical methods.
Recirculation
Recirculation of water is a process that controls microbial levels because constant motion
eliminates stagnation and markedly diminishes microbial proliferation on surfaces. However, it
should not be considered alone as a method for treatment of contaminated water. Recirculation
should be used in conjunction with other procedures such as ultraviolet irradiation or heat. Flow
rates of 1-2 m/sec are recommended for water systems to minimize microbial adhesion.20
Distillation
Distillation effectively removes microorganisms without introducing residual chemicals. This
method requires a substantial initial capital investment with continued need for large amounts
of energy. The distillation process is more efficient for products that are formulated at high
temperatures. Distillation results in considerable loss of water yields, and the high temperatures
that are generated pose a potential safety hazard.

Heat Treatment
Heat treatment (80°C or 175°F), like distillation, represents a large initial capital investment with
continued needs for large amounts of energy. There is some loss of water through evaporation,
and contact time should be carefully controlled so as to assure biocidal effects. As with distillation,
the high temperatures generated could also represent a potential safety problem.
Ultraviolet Irradiation
Ultraviolet irradiation (UV) in the range of 250-260 nm16 destroys most vegetative microorganisms
if the absorbed dose is sufficient. The absorbed dose depends on depth and turbidity of water,
flow rate, lamp intensity, and temperature. UV irradiation becomes less effective as the bioburden
increases. UV irradiation does not introduce chemical residues. It can be used at various points in
the system as well as at the point of water use.
To maintain maximum effectiveness of the UV light source, the UV cell housing must be cleaned
regularly. Lamp intensity must be monitored to ensure sufficient energy output. Personnel must
be shielded from irradiation to prevent eye damage. Manufacturer’s recommendations should be
followed when designing a maintenance schedule.
SANITIZATION OF PROCESS EQUIPMENT AND LINES (See Table 7-2)

These methods are guidelines and should be used according to the particular need and
compatibility with the process water system. For final selection, the efficacy of the concentration
and contact time should be experimentally verified. Any sanitization method must be validated
for the intended process. Suggested concentrations reflect percent active ingredient. For certain
methods, rinsing may be required to assure that there is no residual sanitizer remaining in
equipment or lines.
Chemical Methods - Most Commonly Used Sanitizers
Chlorine
Chlorine and chlorine compounds are readily available, inexpensive and easily monitored. Target
concentration of 200-500 ppm of available chlorine for 10 minutes contact time has generally
been found effective over a pH range of 6-8.5.
Chlorine is rapidly inactivated by trace organic residuals and at pH levels above 8.5. With
prolonged contact, stainless steel and other metals are attacked by chlorine or chlorine compounds.
Deionization resins can be oxidized in the presence of free chlorine. At full strength, chlorine and
chlorine compounds are respiratory irritants and appropriate protective measures for personnel
must be provided.16
Ozone
Although the main application of ozone is in water treatment, it may be used as a sanitizer at 10
to 50 ppm for a contact time of approximately ten minutes.21 Its activity is not as pH-dependent
as is chlorine, and operating and maintenance costs are low.

It is not suitable where water contains more than 0.5 ppm of manganese. Residual ozone can be
removed by granular activated carbon (GAC) filters and UV lights. GAC filters, if used, may be
a source of microbial growth and should be monitored.
Ozone requires electric energy and cooling water, and the large initial capital costs may make
it impractical if used only occasionally as a sanitizer. For personnel protection, NIOSH/OSHA
guidelines stipulate a permissible exposure limit of 0.1 ppm ozone.16
Peroxygen Compounds
Peroxide and peroxyacetic acid are two strong oxidizers employed as process water equipment
sanitizers. A 1.5% solution of hydrogen peroxide for a one-hour contact time provides excellent
sanitization of process water equipment. Hydrogen peroxide has recently gained wide acceptance
since the innocuous end products, oxygen and water, can be readily disposed of via the sewer
without adversely affecting the environment. The presence of organic matter can decrease the
antimicrobial activity of hydrogen peroxide.
Peracetic or peroxyacetic acid is an effective biocide with no toxic residuals. Peroxyacetic acid
solutions show broad spectrum effectiveness at low concentrations, even in the presence of
organic matter. At room temperature, solutions of 0.05% to 3.0% peroxyacetic acid demonstrate
excellent sporicidal activity at 15 minutes to 15 seconds contact, respectively.15 Peracetic acid is
commercially available as a 15% aqueous solution and poses no environmental hazards since it
decomposes into acetic acid and water.

When compared to hydrogen peroxide, peracetic acid vapor has a potential fire and explosion
hazard when heated (56°C or 133°F). At high temperatures, peracetic acid decomposes and is
corrosive and toxic. Concentrated solutions of peroxygen compounds should be treated with
caution as they are irritating to skin, mucous membranes, and eyes. Adequate protection should
be employed during handling.
Less Commonly Used Sanitizers

Iodine or Iodophors
At a concentration of 25 ppm with a contact time of 10 minutes, elemental iodine and most
iodophors destroy microorganisms.15 Iodine, like chlorine, is easily monitored, but unlike
chlorine, it is less susceptible to inactivation by organic residuals. Elemental iodine is effective
over a wide pH range, but some iodophors should be used under the controlled conditions of pH
concentration and contact time as specified by the supplier.
Elemental iodine is a respiratory irritant and corrosive at high concentrations. For personnel
protection, NIOSH/OSHA guidelines stipulate a permissible exposure ceiling of 0.1 ppm iodine
vapor.16 Iodophors are generally easier to handle and much less toxic and irritating. Iodophors
can also be used as residual sanitizers at slightly higher concentrations.
Formalin (37% Formaldehyde)

Formalin at a concentration of 1-2% formaldehyde for a contact tome of 2-3 hours is an effective
sanitizing agent. Deionization resin beds can be sanitized simultaneously provided the particular
resin is compatible with formaldehyde.

Since formaldehyde is a respiratory irritant and skin sensitizer, personnel must be adequately
protected during its use. The current NIOSH recommendation is 0.016 ppm averaged over an
8-hour day with a 0.1 ppm 15-minute ceiling. OSHA stipulates an exposure limit of 0.75 ppm
formaldehyde.16 Questions about formaldehyde toxicity have caused many manufacturers to
discontinue use of this chemical.
Quaternary Compounds
Quaternary surfactants are active over a wide pH range at concentrations of 200-300 ppm. They
are less sensitive to organic residuals than are halogenated compounds. Quaternary compounds
may be mixed with cationic or nonionic detergents for cleaning action. They are incompatible
with anionics. Their compatibility with deionization resins varies and should be ascertained for
particular resins. At very high concentrations, quaternaries are toxic.
Detergent-Sanitizers
The detergent-sanitizer combinations provided by various manufacturers require careful adherence
to the instructions for use. If sanitizing effects are satisfactory, a significant saving of time is
possible for combining washing and sanitizing in one operation. Toxicity and safety hazards vary
and must be considered individually.
Glutaraldehyde
Glutaraldehyde can be used as a sanitizer at diluted concentrations of 0.1% to 0.25% for a five-
minute contact time.22 Glutaraldehyde is effective against a broad spectrum of microorganisms
over a wide temperature range on a variety of surfaces. It is most effective, although not very
stable, in an alkaline solution.15 It is compatible with common materials of construction that
can tolerate exposure to water. It is an effective sanitizer even in extremely hard water. Closed
systems are recommended. Glutaraldehyde is inactivated by ammonia, primary amines, bisulfites
and proteins. It is a respiratory irritant and personal protective equipment is required when
handling.
Physical Methods
Hot Water
Sanitizing with hot water requires temperatures of 80°C or 175°F for at least 30 minutes contact
time. Lower temperatures may be used, but longer times would be required. Although this
method leaves no residual material, energy requirements are high. Care should be taken to be sure
that high temperatures are attained throughout the process water system. Also, scalding water
temperatures create a potential personnel hazard.
Steam
Steam, like hot water, has the advantage of not requiring rinsing or cleaning to remove residual
material. However, a large energy output is needed and high temperatures should be present
throughout the system to destroy microorganisms in remote or less accessible areas. Filtration
may be required to remove foreign materials from the steam source prior to use. If the addition
of boiler treatment chemicals is warranted, potential residual effects should be assessed.

SAMPLING AND TESTING METHODS
To assure that water treatment methods and sanitization procedures are effective, routine sampling
and testing for microbiological quality should be conducted. A sampling program should be
established which takes into consideration the purification system, water consumption rate, size
of the system, validation data, and any other factors that can affect water quality.
Sample Collection Method

Prior to obtaining a sample, flush the valve for a sufficient period of time to remove any stagnant
water (minimum of two to three minutes).23 Where warranted, rinse or sanitize the surface of the
valve with 70% alcohol (or other suitable sanitizer), flush the valve to remove residual sanitizer,
and obtain a sample. Use sterile wide-mouth glass or plastic containers having a suitable volume
capacity.
Sampling must be thorough and representative of the system and done under strict aseptic
conditions. Some specific critical sampling areas are: points of use, storage tanks, before and
after deionization beds, before and after UV lights, water meters, filters, and return water. In
general, sampling areas should also include any area in which the flow of water is reduced and
microorganisms might proliferate.
Testing Procedure

Samples should be tested within one hour after collection if not refrigerated, or within 24 hours
if refrigerated. The microbiological testing of process water can be accomplished by different
methods that can be selected on an individual basis. Plate count, most probable number, and
membrane filtration are methods commonly employed in most laboratories. The method
employed when establishing a microbiological history of a water system should be validated to
assure the accuracy of results due to technique or media. “Standard Methods for the Examination
of Water and Wastewater” is recommended for guidance in establishing test procedures.23
Acceptability Criteria
Alert and action levels should be established for purified water, taking into consideration such
factors as processing conditions, susceptibility of the product to contamination, and intended
use of the product. It is the responsibility of the manufacturer to assure that microbial limits for
process water are appropriate and achievable. Microbial limits that have been defined should be
documented.4, 5, 14

Table 7-1 Microbiological Control Methods - Water Treatment Methods
Method Advantages Restrictions/Disadvantages Toxicity/Safety Hazards
Chlorination 1. Low level residual required 1. Rapidly inactivated by trace 1. Respiratory irritant and
(as provided (target 2-10 ppm) organic residuals corrosive liquid at full
by aqueous 2. Sensitive test available to 2. Chlorine is less reactive as strength.
OCI, CI2, HOCI monitor concentration pH increases 2. Reaction with trace
solutions and 3. Relatively inexpensive and 3. Chlorine is more reactive as organic residuals can form
CIO2 ) readily available temperature increases trihalomethanes which are
4. Relatively short contact 4. Most frequently designed human carcinogens.
time to precede deionization, 3. NIOSH recommended
5. pH range 6.0-8.5 consequently water will be employee exposure limit
susceptible to contamination 0.5 ppm ceiling for 15 min.
6. Not affected by hard water
7. Residual activity useful 5. May require method to
for transport/storage remove residual (e.g., carbon
conditions filters).
6. Chlorine can be corrosive to
gaskets and stainless steel
processing equipment.
Ozonation 1. Not as pH-dependent as 1. Not applicable to waters OSHA airborne exposure limit
chlorine. possessing high (>0.5 ppm) 0.1 ppm
2. Low operating and Mn++ concentration.
maintenance costs. 2. Residual organic materials
3. Half-life of ozone is 20 may be biodegraded
minutes-decomposes which could lead to biofilm
rapidly to oxygen. formation.

4. Residual activity useful 3. Dissolved ozone must be
for transport/storage removed from prior water
conditions. use (e.g., UV or granulated
carbon).
Filtration 1. Submicron filters at 1. Development of defects can None
1. Submicron point of use are most permit microbial passage.
effective in removing all 2. Replacement of filters should
microorganisms. be regularly performed and
2. Integrity tests (Bubble carefully monitored.
Point) can be performed. 3. Water quality most
often requires extensive
pretreatment to minimize
replacement frequency.
2. Reverse 1. Removes >99% 1. Initial capital outlay and None

Osmosis microorganisms. maintenance is higher than
2. No chemical interference. chemical treatment.
3. Can replace deionization 2. Loss in water yield
beds which often (approximately 50%).
represent a potential site 3. Regeneration of membrane
for microbial proliferation. is limited.
4. Water quality may require
pretreatment to control pH.
3. Ultra 1. Removes most 1. Initial capital outlay and None

Filtration microorganisms. maintenance is higher than
2. No chemical interference. chemical treatment.
3. Can be used at point of 2. Loss in water yield
use (approximately 20%).
Table 7-1

Table 7-1 Microbiological Control Methods - Water Treatment Methods continued
Distillation No chemical interference. 1. Large initial capital Scalding water poses

investment and ongoing potential hazard.
requirement for large
amount of hear energy.
2. Considerable loss of water in
water yields.
Heat No chemical interference. 1. Large initial capital Scalding water poses

investment. potential hazard.
2. Ongoing requirement for
large amount of heat energy.
3. Some loss of water in yields.
4. Should have sufficient
contact time and
temperature to assure
biocidal activity.
Ultraviolet 1. No chemical interference. 1. The absorbed dose depends UV light can cause eye
Irradiation 2. Relatively easy installation on depth and turbidity damage. Eye protection is
and low maintenance. of water, flow rate, lamp required when lamps are
3. Thin films units more intensity, and temperature. replaced or inspected.
efficient than older 2. UV lamps need to be cleaned
designs. at the end of their rated

4. Most vegetative organisms hours, as the UV output
destroyed by irradiation. decays with time to an
ineffective level.
5. Can be used at point of
use. 3. High bioburden will reduce
effectiveness.
Table 7-1

Table 7-2 Sanitization Methods for Process Water Equipment and Lines
Active chlorine 1. Rapid, sensitive test 1. Rapidly inactivated by trace 1. Respiratory irritant and
compounds available to determine organic residuals. corrosive liquid at full
(includes liquid concentration during 2. Chlorine is less reactive as strength.
CI2, HCIO3, sanitization and to verify pH increases. 2. Reaction with trace
CIO2, organic removal of residual after 3. Efficacy is somewhat organic residuals can form
and nonorganic rinsing. temperature-dependent trihalomethanes which are
chloramines) 2. Material is commonly (higher temperature human carcinogens.
available and relatively increases biocidal effect). 3. NIOSH exposure limit of 0.5
Target inexpensive. 4. Will attack 316 stainless ppm ceiling for 15 min.
concentration steel and other metals with
200-500 ppm prolonged contact; carefully
available CI2; regulate exposure time.
10-minute 5. Capable of oxidizing resins
contact time; at low concentrations of free
pH range of chlorine.
6.0-8.5.
6. Chlorine-releasing
compounds may require
other conditions of use;
e.g., pH contact time,
concentration.
Ozone 1. Not as pH-dependent as 1. Not suitable for water NIOSH/OSHA exposure limit is
Target chlorine. supplies containing greater airborne concentration ceiling
than 0.5 ppm manganese. of 0.1 ppm, 15 minute contact
concentration 2. Low operating and

10-50 ppm maintenance costs. 2. Activated carbon filters time.

for about 10 3. Half-life of ozone is 20 or ultra-violet light are
minutes to minutes, decomposes necessary to remove residual
sanitize. rapidly to oxygen. ozone.
3. Electric energy is required;
cooling water may be
needed after treatment.
4. Initial capital outlay makes it
more practical as continuous
process water treatment
system than as occasional
sanitizing agent.
Peroxygen 1. Innocuous by-products. 1. Organic matter may 1. Strong oxidizers are

compounds decrease efficiency. respiratory and skin
Target 2. Unstable. irritants.
concentrations 3. Gas formation in pumps 2. Corrosive and toxic only
1.5% H2O2 for could cause pumps to seize. in concentrated solutions
60 minutes. (>40%).
3. Potential of fire hazard.
Table 7-2

Table 7-2 Sanitization Methods for Process Water Equipment and Lines continued
Iodine 1. Rapid, sensitive test is 1. Exposure of resins is not 1. Elemental iodine is

(elemental available to determine recommended. respiratory irritant and
iodine or the concentration during 2. Iodophors may require other corrosive liquid at full
iodophors) sanitization and to verify conditions of use-contact strength.
removal of residual after time, concentration, etc. 2. Iodophors represent very
rinsing. 3. Iodophors require acid pH. low toxicity.
Target
concentration 2. Less sensitive than 4. Unstable in the presence of 3. Reaction with trace
of 25 ppm chlorine to inactivation by hard water, heat, and organic organic residuals can form
residual organics. soil. trihalomethanes which are
10-minute
contact time. 3. Material is relatively human carcinogens.
inexpensive and generally 4. NIOSH/OSHA permissible
available. exposure ceiling 0.1 ppm.
4. Elemental iodine is
effective over a wide pH
and temperature range.
5. Can have a residual effect.
Formalin 1. Generally resins will not be 1. Should verify compatibility 1. Respiratory and skin irritant
(37% attacked, and beds can be with resin prior to use. sensitizer.
formaldehyde) sanitized at the same time 2. Large amounts of water 2. NIOSH/OSHA exposure limit
as piping holding systems. are necessary to flush is airborne concentration
Target
formaldehyde residues from ceiling of 0.1 ppm, 15
concentration
the system. minute contact time.
of 1-2%

formalin;
2-3 hours
contact time.
Detergent- By combining washdown As described by vendor. As described by vendor.
Sanitizers and sanitization steps,
significant time savings are
possible.
Glutaraldehyde 1. Effective broad-spectrum 1. Inactivated by ammonia, Respiratory and contact

Target activity over wide pH and primary amines, bisulfites irritant; avoid inhalation of
concentration temperature range. and proteins. vapors. Personal protective
of 0.1% to 2. Compatible with common 2. Surfaces must be thoroughly equipment required.
0.25%; 5- construction materials; cleaned prior to treatment.
minute contact effective on wide variety
time of surfaces; sanitizes even
in very hard water.
Hot water 1. May be readily available. Energy supplies may be Scalding water poses
Target 80°C 2. No chemical interference. expended to produce potential work hazard.
(175°F); contact temperatures required.
time 30
minutes.
Steam 1. No chemical interference. 1. Large energy supplies may Scalding steam poses
Flowing steam be expended to produce potential work hazard.
contact time 30 temperatures required
minutes. throughout the entire
system.
2. Significant temperature
drops at remote points may
limit efficacy.
3. Filtration of steam may be
required to remove foreign
material.
Table 7-2
REFERENCES 11. Cross, J. 1993. Designing Water Systems
that Comply With GMP. Manufacturing
1. U.S. Environmental Protection Agency. Chemist. 64: 31-33.
1998. “National Primary Drinking Water
Regulations: Disinfectants and Disinfec- 12. Brown, J., N. Jayawardena, and Y. Zelma-
tion.” 40 CFR Parts 141 and 142. http:// novich. 1991. “Water Systems for Phar-
www.epa.gov/safewater/standard/v&e- maceutical Facilities.” Pharmaceutical En-
frn.pdf. gineering. 11: 15-23.
2. U.S. Food & Drug Administration. 1993. 13. Lorch, W., (Ed) 1987. Handbook of Wa-
“FDA Guide to Inspections of High Pu- ter Purification. New York: John Wiley &
rity Water Systems.” http://www.fda.gov. Sons.
3. United States Pharmacopeia. 2007. 14. Pharmaceutical Manufacturing Associa-
<1231>. “Water for Pharmaceutical Pur- tion Deionized Water Committee. 1985.
poses.” United States Pharmacopeia and “Validation and Control System Concepts
the National Formulary. USP30 - NF25. for Water Treatment Systems.” Pharma-
Rockville, MD. 687-706. ceutical Technology, 8, 52-56.
4. Anon. 1984. “Use of Alert and Action 15. Block, S.S. 2000. Disinfection, Steriliza-
Levels in Pharmaceutical Manufactur- tion and Preservation. Philadelphia: Lea &
ing.” Pharm. Manuf. 1: 24-26. Febiger.
5. Pharmaceutical Manufacturing Associa- 16. U.S. Department of Health and Human
tion’s Deionized Water Committee. 1984. Services (NIOSH). 2005. NIOSH Pocket
“Protection of Water Treatment Systems, Guide to Chemical Hazards. http://www.

Part III: Validation and Control.” Phar- cdc.gov/niosh/npg/.
maceutical Technology. 8: 54-68.
17. “Ozone, the Process Water Sterilant.”
6. Duddridge, J. J. 1988. “Biofilm Growth 1984. Pharmaceutical Manufacturing. 16-
in Water for Cosmetics.” Manufacturing 23.
Chemist. 59: 42-44.
18. Cross, J. R. 1987. “Contemporary Tech-
7. Burman, N. P. and J. Colvourne. 1977. niques for the Production of Ultrapure
“Techniques for the Assessment of Growth Water in the Pharmaceutical Industry.”
of Microorganisms on Plumbing Materi- Drug-Dev-Ind-Pharm. 13: 9-11.
als Used in Contact with Potable Water
Supplies.” Journal of Applied Bacteriology 19. Marcus, D. L. and R. Pastrick. 1988. “Ul-
43: 137-144. trafiltration - Its Role in Today’s Water
Purification Systems.” Ultrapure Water,
8. Van der Kooij, D. and H.R. Veenendall. 40-45.
“Assessment of the Biofilm Formation:
Potential of Synthetic Material in Con- 20. Cross, J. 1995. “Water Purification.”
tact with Drinking Water During Distri- Chemical Engineering, 983: 15-16.
bution,” paper presented at the American 21. Mittelman, Marc W. 1986. “Biological
Water Works Association, 1993 Water Fouling of Purified-Water Systems: Part
Quality Technology Conference. III, Treatment.” Microcontamination 4,
9. Meltzer, T. H. et. al. 1979. ”Adsorptive 30-40.
Retention of Pseudomonas dimunuta by 22. Union Carbide Corporation. Technical
Membrane Filters.” J. Parenteral Drug As- literature provided by supplier.
sociation 33: 40-51.
23. American Public Health Association
10. Avalone, H. L. 1988. “Microbiologi- 1995. Standard Methods for the Examina-
cal Control of Topicals.” Pharmaceutical tion of Water and Wastewater 20th Edition.
Technology. 12: 55-62. Washington, DC.

SECTION 8
Microbiology
Laboratory
Audit
INTRODUCTION
Within the cosmetic industry, quality assurance, development, and contract microbiology
testing laboratories provide supportive microbiological data related to product safety and quality.
Microbiological aspects of formula development, from product conception to finished goods,
need to be addressed to determine if microbiological standards are met. Examples include the
development and evaluation of preservative systems and the examination of the microbial content
of raw materials, intermediates, finished goods, and the manufacturing environment.
Laboratory practices should be evaluated at regular intervals to ensure that the generation of data
is reproducible, accurate, and reliable. An audit serves to review existing practices, systems, and
equipment to ensure that they perform as expected. Microbiology lab audits should be conducted
by individuals familiar with the functions and processes that typically occur in a microbiology
laboratory. This document provides guidance for conducting an audit of both in-house and
contract cosmetic microbiology testing laboratories. If cosmetics and over-the-counter (OTC)
drugs are tested in the same laboratory, refer to Food and Drug Administration (FDA) guidelines
for the manufacture of OTC drugs, and to relevant chapters in the USP.1,2,3
Based on the information presented in this document, an example of a Cosmetic Microbiology
MICROBIOLOGY LABORATORY AUDIT

Laboratory Audit Checklist can be found in Table 8-1.
SECTION 8:
PERSONNEL
Supervisory personnel have the responsibility of ensuring that operating systems are consistent
with existing cosmetic regulations and current cosmetic industry good manufacturing practices
(GMPs).4
Laboratory supervisors should be familiar with applicable regulations and current industry
practices. Initial and continuing training programs are a valuable means of ensuring that employees
are qualified for their roles and responsibilities and informed about all laboratory procedures.
SECTION 8: MICROBIOLOGY LABORATORY AUDIT | CTFA MICROBIOLOGY GUIDELINES | 95

The following documentation may be considered in recording an employee’s qualifications and
is most useful when maintained on file for reference:
• Job descriptions for all laboratory positions, which may describe the
qualifications, primary and secondary responsibilities, and job functions.
• Organizational charts showing each staff position and each incumbent
identified by position, name, and reporting relationship.
• Skills checklist or applicable training documentation for each employee
detailing the microbiological techniques and procedures that an individual has
been trained and certified to perform on a routine basis (see Table 8-2).
• Records of initial and continuing training for each employee.
LABORATORY FACILITIES
Adequate laboratory facilities should be provided to minimize errors in test results due to
contamination, inaccuracy in data interpretation, equipment failure, or sampling mistakes. It is
recommended that rooms used for microbiological testing be of suitable size, construction, and
location to facilitate proper operation. In general, the attributes of an adequate microbiology
laboratory facility include, but are not limited, to the following:
• The laboratory contains separate areas for microbiological analysis, support
functions (e.g., media preparation, sample preparation, sample login, clerical
work), and storage of personal belongings.
• The area for microbiological analysis is used exclusively for testing aspects such
as sample handling, performing procedures, transfer of cultures, counting of
colonies, etc.
• All surfaces in the laboratory are nonporous, cleanable, and sanitizable.
• The facility design minimizes exposure of laboratory areas to air currents.
• Laboratory access is restricted to minimize foot traffic by non-laboratory
personnel.
• Microbiological quality of the laboratory environment is controlled by
appropriate mechanical and physical means such as use of positive-pressure

room air, germicidal lamps, high-efficiency particulate air filters, and/or
laminar flow stations.
SECTION 8:\
• Workspaces such as laboratory benches and laminar flow hoods are sanitized
at the beginning and end of the workday, as well as between individual
procedures.
• Adequate ventilation and lighting is provided, especially over work areas.
• Electrical service is appropriate for the equipment used in the laboratory and
associated areas to avoid power outages and equipment failure.
• Adequate sink areas with hot and cold water service are provided.
• Sufficient counter and shelf space are available so that all procedures can
be performed while preventing overcrowding, safety hazards, or any cross
contamination.
96 | CTFA MICROBIOLOGY GUIDELINES | SECTION 8: MICROBIOLOGY LABORATORY AUDIT

• Clean uniforms or lab coats are provided.
• Microbiologically contaminated materials are decontaminated prior to
disposal.
• Floors are kept clean by regular mopping and sanitized by using a disinfectant
detergent.
• Adequate containers that are appropriately labeled are provided for
microbiological waste, non-hazardous waste, and general trash disposal.
General trash is removed from the laboratory each working day.
SAFETY
The microbiology laboratory should have a written safety policy that addresses laboratory
personnel, equipment, and processes. This policy may reference a laboratory safety manual.5,6
LABORATORY EQUIPMENT
It is recommended that standard operating procedures as well as supporting documentation
(e.g., equipment manuals, calibration data) be readily available to personnel for each piece of
equipment.
Equipment is to be maintained in accordance with the manufacturer’s guidelines and routinely
calibrated, with clearly visible calibration stickers containing calibration date, calibrator’s name,
and next scheduled calibration date. Personnel should be adequately trained on each piece of
equipment necessary to their function prior to their routine use of that equipment. A summary
of calibration recommendations can be found in Table 8-3. The following specific attributes for
some common laboratory equipment are recommended as a minimum guideline:
Incubators

Incubators should maintain a uniform and constant temperature within predetermined limits;
±2.0ºC is recommended for most laboratory applications. An accurate thermometer with a
bulb continuously immersed in liquid (water, mineral oil, or propylene glycol) is maintained
SECTION 8:
at appropriate location(s) (e.g., hot and cold spots detected through temperature mapping) in
the incubator. Daily temperature readings are recorded on laboratory workdays. Daily humidity
readings are recorded for humidity-controlled incubators. Temperature recording devices or
maximum and minimum registering thermometers within the incubator are recommended to
record temperature variations. Provision is made for humidity control (e.g., placing a beaker of
water in the incubator). The temperature setting is appropriate for the types of organisms being
incubated.

Hot-Air Sterilizing Ovens
Hot-air sterilizing ovens maintain a uniform and constant temperature of up to 200ºC, within
predetermined limits; ±5.0ºC is suggested for most laboratory applications. Ovens are equipped
with thermometers capable of accurate measurement between 160º to 200ºC. Ovens are of
sufficient size to prevent crowding of the interior.
Autoclaves
Laboratory autoclaves are capable of maintaining a uniform and constant temperature of
121º -123ºC and reach target temperature within 30 minutes. In order to allow sufficient
heat distribution and penetration for sterilization, autoclaves are properly sized and loaded to
prevent crowding of items. Autoclaves are equipped with accurate thermometers or temperature
recorders, pressure gauges, and properly adjusted safety valves. If a time-temperature recorder is
not available, an alternative temperature monitoring device or bioindicator is used. Autoclave
tape is not an indicator of sterility. A maintenance program is in place, which includes an annual
certification of temperature and pressure gauges, timers, and temperature recording devices. In
addition, individual run records (e.g., time-temperature recording) are maintained. Records are
routinely reviewed for deviation. An autoclave log is maintained detailing run conditions and
allowing traceability of autoclave run documentation with specific loads. Temperature controllers,
temperature recording devices, pressure gauges, and timers must be certified for accuracy to insure
proper operation.
Colony Counters
A standard colony counter or comparable instrument may be used. Automated colony counters
may be used where accuracy and reliability have been validated. Automated colony counter
accuracy is checked each day of use and a log-in book is maintained.
pH Meters
DISCREPANT MATERIALS CONTROL
pH meters are capable of accurately measuring the hydrogen ion concentration to 0.1 pH
units. The pH meter should be calibrated each day of use by using at least two certified pH
buffer solutions that bracket the pH range of the sample. Buffers should not be used past their
expiration dates. Probes for taking the pH of a sample should be appropriate for the material
ANNEX 8
being analyzed.
Balances
Balances used for routine weighing (e.g., media, samples, etc.) are accurate for the utilized task.
Balances and weights are calibrated according to a regular preventative maintenance schedule
with weight (mass) standards traceable to NIST Reference standards. Weight checks should be
performed with calibrated weights.

Labware
Disposable or reusable labware is inert to the materials with which it may be used. For glassware
items, borosilicate glass is recommended because of its ability to withstand high temperatures.
The following are recommendations for selected examples of commonly used labware:
• Pipettes - sterile glass or disposable plastic pipettes may be used. The accuracy
of the pipettes is ± 5%. If used, micropipettors should be calibrated at least
annually.
• Dilution bottles and tubes - bottles and tubes are single use or are made of
autoclavable material. Screw caps are equipped with inert liners. All reusable
items are thoroughly cleaned and rinsed using a protocol that assures no
detectable detergent residue.
• Media preparation utensils -clean borosilicate glass, stainless steel, or other
suitable inert labware are recommended.
• Petri dishes - sterile borosilicate glass or disposable plastic Petri dishes are
recommended.
Microscopes
A monocular or binocular microscope suitable for the intended purpose is recommended. The
microscope should be capable of a minimum total magnification (combined ocular and objective
lenses) of 1000x. A dissecting scope may be used for lower magnification. An annual maintenance
check is recommended by which lenses, alignment, and mechanism aspects are checked.
Refrigerators/Freezers
Commercially available refrigerators are suitable unless there is a need for an explosion-proof
model. Refrigerator temperature should be maintained at 5±3ºC and routinely checked. No food
should be stored in laboratory refrigerators or freezers. Standard freezers have autodefrost. Ultra-
freezers (e.g., -80ºC) are maintained 5ºC within designed temperature.

Water Baths
SECTION 8:
Water baths have thermostatic controls to deliver temperatures from ambient to 100ºC ±2.0ºC. An
accurate, calibrated thermometer should be placed in the water bath to monitor the temperature.
Temperature readings are recorded daily when in use and also after equilibrium has been reached
following any adjustment of the temperature controls. Biocide may be added to the water to
prevent/control microbial growth.
Laminar Flow Hoods

Laminar flow hood operation is certified at least annually to verify adequacy of airflow, condition
of pre-filters, HEPA filter integrity, and particle size exclusion limit. An appropriate sanitizer
is recommended for use to disinfect interior hood surfaces before and after use. UV lights (if
installed) are maintained and operated according to manufacturer’s recommendations and
replaced or verified annually.

Biological Safety Cabinets
A biological safety cabinet is used when a material is suspected of being highly contaminated,
when manipulating high concentrations of microorganisms considered to be a biohazard, or
when there is a potential for aerosolization of microorganisms.5 Operation is certified at least
annually, or after 5,000 hours of use, to verify the cabinet performance for airflow, condition of
prefilters, HEPA filter efficiency, and particle size exclusion limit. UV lights (if installed) should
be maintained and operated according to manufacturer’s recommendations, and replaced or
verified annually.
Water Treatment Systems

The laboratory has an appropriate system for producing the desired grade of water (e.g., deionized,
distilled) for use in microbial growth media and other applications. The water treatment unit is
maintained and operated consistent with manufacturer’s recommendations. Water is periodically
monitored to ensure it meets chemical and microbiological quality. If a UV light is part of the
water system, it should be maintained and operated according to the manufacturer’s directions.
Note: If cosmetics and over-the-counter (OTC) drugs are tested in the same laboratory, refer to
FDA reference materials and the USP Guidelines for Water.1, 2, 3
Lyophilizers
Vacuum and temperature records are maintained for each lyophilization operation. Temperature,
vacuum, and timing systems should be calibrated every six months.
Thermometers
Thermometers are of appropriate range for the application. Measurement accuracy is initially
established and rechecked at least annually by using a NIST-certified thermometer.
Spectrophotometers
Calibration is performed against standard solutions every six months or as recommended by the
SECTION 8:
manufacturer.
Centrifuges
Centrifuges are calibrated for temperature and rotor speed annually.
Water Activity Devices

Devices for the measurement of water activity should be calibrated each day of use against known
standards.

Media Dispensers
The dispensing volume should be verified on a regularly scheduled basis.
For more detailed information on calibration of microbiological equipment, see “Annex 16 -
Calibration Systems” in the CTFA Quality Assurance Guidelines.4
DOCUMENTATION
The purpose of documentation is to provide the written record of a laboratory operation. A
history of operation is maintained through the retention of documents (e.g., logbooks, worksheets,
calibration records, etc.). Establishment of a document retention program is highly recommended
since it defines the policy of the company, retention time, form (microfilm, etc.), place of storage,
etc.
Any documentation should describe the function of the laboratory operation with properly
prepared and regularly updated procedures. Scheduled reviews of laboratory procedures should
be performed to ensure that procedures are as described. Discrepancies should be corrected and
procedures amended when appropriate. Laboratory personnel should document any investigations
and procedure changes.
For additional information, refer to “Microbial Validation and Documentation” (Section 9),
“Determination of the Microbial Content of Cosmetic Products” (Section 18), “Establishing
Microbial Quality of Cosmetic Products” (Section 12) and “Raw Material Microbial Content”
(Section 11), in this document.
The following quality control checks are recommended as part of laboratory procedures:
Methods Validation
All microbiological methods should be validated and documented to ensure the accuracy of the
test response.

Media
Media performance should be documented via positive and negative controls on each prepared
SECTION 8:
batch of media.
Equipment Checks (see Table 8-3)

Calibration, performance, and maintenance logs should be maintained for each piece of laboratory
equipment. All entries should be dated and initialed.
Personnel
Documentation of microbiology laboratory personnel training should be maintained as well as
the documentation described in Section 4 of this guideline. A current signature list conforming
to signatures issued in the laboratory and retraining documentation should also be maintained.

Forms
Forms used to document test results should be periodically reviewed and updated to reflect
current procedures.
Procedures
Written standard operating procedures and test methods should be reviewed and updated
on a regular basis to document changes. All procedures should include an effective date and
supersedes date to identify the current version. All changes should be communicated to laboratory
personnel.
Investigations and Discrepancies

Laboratories should have in place an out-of-specification investigation procedure that includes
the steps outlining the process, detailing the findings, and reporting the results. Any discrepancies
in the microbiological test results and/or procedures should be investigated, documented, and
evaluated by the appropriate personnel.
SECTION 8:

Table 8-1 Cosmetic Microbiology Laboratory Audit Checklist (Example)
This list gives examples of points to consider when performing an audit or preparing to receive an audit. It is not
intended to be all-inclusive or to infer that all points are applicable to all laboratories.
General
1. Is there an organizational chart showing each incumbent and reporting relationship?
2. Is the signature list up-to-date?
3. Are there written job descriptions for each laboratory position?
4. Are individual qualifications for laboratory personnel on file and updated regularly?
5. Is there a training and development program for laboratory staff members?
6. Is there formal training documentation?
Laboratory Facilities
1. Is access to the microbiology laboratory controlled?
2. Are laboratory facilities clean and orderly?
3. Is the laboratory free of dust, drafts, and temperature extremes?
4. Does the laboratory have adequate workspace, ventilation, and light?
5. Are there adequate facilities for cold storage, microbial media, and storage of samples?
6. Is the staff regularly reviewing sanitization and cleaning records?
Laboratory Safety
1. Are laboratory coats worn only in laboratory areas?
2. Are proper shoes and clothing worn in laboratory areas?
3. Is a safety committee and/or advisor established and functional?
4. Are all laboratory staff members provided with, or do they have access to, the laboratory safety manual?
5. Are the members of the janitorial staff provided appropriate training for the microbiology area?
6. Does the laboratory provide:
a. Designated containers for broken glass, sharp objects, etc.?
b. First aid kits that are easily accessible and well maintained?
c. Conveniently located and operational eyewash and deluge shower stations?
d. Up-to-date and readily accessible fire extinguishers and blankets?
e. Clearly marked emergency exits?
f. Emergency telephone numbers that are widely distributed and conveniently located?

7. Has mouth pipetting been eliminated?
8. Is microbiologically contaminated waste decontaminated before disposal?
Laboratory Equipment
1. Review records for equipment calibration and preventive maintenance.
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2. Review records for calibration of standards and equipment daily calibration check logs
(evaluate what corrective actions were taken when calibration failed).
3. Review cleaning log records (e.g., incubators, water baths, etc.).
4. Review sterilization cycle log records for autoclaves.
5. Ensure that autoclave controls are present (e.g., biological indicator test results).
6. Identify and tag each piece of laboratory equipment along with its calibration, preventive maintenance, and
operational status.
7. Review temperature monitoring log records for incubators, refrigerators, and controlled areas.
8. Review laminar flow hood HEPA filter certification records.
9. Make sure that up-to-date equipment operating instructions are available.
10. Ensure that appropriate laboratory/instrumentation is available for use in accordance with required
methodology.
11. Keep track of service contracts for equipment maintenance for each type of equipment.

Table 8-1 Cosmetic Microbiology Laboratory Audit Checklist (Example) continued
Environmental Monitoring
1. Is there a monitoring program for equipment cleaning (e.g., swab tests)?
2. Are records for environmental monitoring being reviewed?
3. Is there a periodic evaluation of trended environmental monitoring data?
4. Are environmental isolates from swabs and air samples characterized (e.g., gram stain) and/or identified?
Microbial Media, Buffers, and Reagents

1. Are expiration dates for microbial media, buffers, and reagents assigned and updated?
2. Are microbial media growth promotion test results being reviewed?
3. After sterilization, is the pH recorded for each prepared lot/batch of microbial growth media?
4. Are microbial growth media, buffers, and reagents traceable to preparation records?
5. Are quality control and quarantine practices for purchased and laboratory prepared microbial growth media
being reviewed?
6. Are records for receipt, storage, preparation, sterilization, and storage of microbial growth media, buffers, and
reagents being reviewed?

1. At the time of testing, are negative controls performed to verify the sterility of media, buffers, and materials
used as well as aseptic technique?
2. Whenever appropriate, are positive test controls (e.g., media) performed?
3. Are inoculum counts verified at the time of test (e.g., positive controls and validations)?
4. Is work performed in a laminar flow hood or biological safety cabinet?
5. Is test data analyzed for aberrant/out-of-specification (OOS) results?
6. Are test methods followed as written?
7. Are isolated microorganisms characterized as needed?
8. Has microbial growth media used in testing been released from quarantine?
9. Have the microbiological test methods for determining microbial content been validated?
10. Have the microbiological test method data been properly documented?
11. Is microbiological testing performed on susceptible raw ingredients?
12. Is in-process bulk batch testing performed?
13. Are sampling requirements for finished products being followed?
Culture Maintenance
1. Are ATCC microbial cultures verified upon receipt (prior to use)?
SECTION 8:
2. Are record keeping and traceability of microorganisms used in testing in place?

3. Is the seed lot technique/control of number of passages (e.g., not more than five passages from original) being
evaluated?
Water
1. Is there a source of distilled or deionized water?
2. Is water monitored routinely for chemical and microbiological quality?
3. Are review data generated to include evaluation of corrective action plans when test results are aberrant?
4. Are sanitization and preventive maintenance log records for laboratory water system reviewed?
5. Are procedures for taking microbiological test samples of water reviewed?
6. Are representative water isolates identified?
Table 8-1
Sample Handling
1. Are there adequate written procedures for receipt, storage, and handling of test samples?
2. Are there established sample turnaround times/targets?
3. Are sample submission forms stamped with the sample’s date and time of receipt?
4. Are samples given an unambiguous sample number when logged?
5. Does a permanent record exist for sample log-in data?
6. Are appropriate chain-of-custody procedures documented and followed when required?
7. Are there established operating procedures available for the disposal of samples?
Quality Assurance/Quality Control (QA/QC) System

1. Is the quality assurance manual readily available to all staff members?
2. Is the quality assurance manual updated regularly?
3. Does the quality assurance officer operate independently of analyses?
4. Does the laboratory have periodic system audits?
5. Does the laboratory evaluate its performance through proficiency testing?
6. Do proficiency testing programs have feedback and corrective action programs, procedures, and protocols in
place?
7. Does the laboratory provide in-house training on quality?
8. Are QA policies, protocols, and procedures documented for the following:
a. Methods?
b. Sample collection and handling?
c. Quality control for each type of test performed?
d. Procurement and inventory control?
e. Operation and calibration of laboratory equipment?
f. Preventive maintenance?
g. Records management?
9. Is data retrievable and identifiable for each test result?
10. Are applicable computer software programs documented and back-up copies secured?
11. Are any analyses subcontracted?

12. Are subcontracted laboratories evaluated for QA? Are they audited, when and how often?
Records Management
1. Is a system in place that provides for retrievability and traceability of sample source, methodology of analyses,
SECTION 8:
results, person performing analysis, and date?
2. Are records and reports adequately secured and retained for the required length of time to ensure their
integrity?
3. Are all laboratory notebooks, when completed, filed in a secure, controlled archive area from which they can
be easily retrieved?
4. Are all laboratory equipment/instrument maintenance logs uniquely identified and stored for easy retrieval?
5. If the laboratory operates a computerized data/information management system (LIMS), are there backups to
ensure integrity and availability of data/information in the event of a system/power failure?
Table 8-1
Test Reports
1. Do the laboratory’s reports accurately and clearly present test results and all other relevant information?
2. Does each test report include the following:
a. Identification of laboratory issuing the report
b. Identification of client, if applicable
c. Sample identification and description (e.g., sample name and lot number)
d. Dates/times of sample collection or receipt. Receipt and types of testing performed
e. Identification of microbiological test methods used in the analysis
f. Description of sampling procedure, where relevant
g. Any deviations, additions, or exclusions from a test method
h. Disclosure of any subcontractor used
i. Results and any failures identified
j. Identity of person accepting responsibility for the testing
k. Laboratory reports in an understandable format
l. Corrections or additions made to test reports after they were issued
m. A policy/protocol for handling inquiries and complaints about test reports and results.
n. A policy/protocol in place outlining the checking and authorization for data release to clients.
Table 8-1
Table 8-2 Microbiology Laboratory Skills Checklist (Example)

This Microbiology Skills Checklist provides examples of training areas for the skills needed and the basic duties
and responsibilities assigned to staff in the microbiology laboratory. The need for proficiency in specific tasks is
dependent on an employee’s work level and area of responsibility. The skills trainer/assessor should be proficient in
all areas being reviewed. This list may be modified as needed based on equipment and function of the lab.
Techniques and Analyses Techniques and Analyses Equipment Use and Operation
❏ Aseptic technique ❏ Microscope

❏ Pipetting – traditional and micropipettor ❏ Water bath
❏ Streak for Isolation ❏ Autoclave
SECTION 8:
❏ Gram stain/KOH string test ❏ Colony counter

❏ Catalase test ❏ Spectrophotometer
❏ Oxidase test ❏ Water activity meter
❏ Microbial identification – traditional and rapid ❏ Lyophilizer
❏ Water testing and sample collection ❏ Anaerobic chamber and jar
❏ Individual test method proficiency
Microorganisms
Media ❏ Inoculum preparation
❏ Preparation and storage ❏ Organism maintenance techniques
❏ Media quality control
❏ Media remelt in autoclave/microwave
❏ Use of differential media
Table 8-2

Table 8-3 Microbiology Lab - Equipment Calibration and Performance
Daily/Continuously
Temperature controlled devices including incubators, Verify temperature control

waterbaths, refrigerators, freezers, centrifuges
Humidified chambers Verify humidity control
Autoclave Sterility indication – each day of use
pH meters Calibration – each day of use
Water activity device Calibration – each day of use
Weekly
Treated water Chemical and microbial testing
Six months
Balances Calibration
Spectrophotometers Calibration
Dispensers Calibrate dispensing volume
Lyophilizer Vacuum, temperature, timing calibration
Annually
Autoclave Temperature calibration
Centrifuge Calibration (temperature, rotor speed)
Laminar flow/biohazard hood Certify airflow and operation
Thermometers Accuracy vs. NIST thermometer
Pipettors Dispensing accuracy and precision calibration

Stomachers Efficacy
Sonicators Efficacy
Microscope Inspection – alignment, lenses, mechanical aspects
SECTION 8:
UV lamps Certification or replacement
Table 8-3

REFERENCES
1. U.S. Food and Drug Administration. 5. Fleming, D.O., and D. L. Hunt, (Ed).
2006. “Current Good Manufacturing 2000. Biological Safety: Principles and
Practice in Manufacturing, Processing, Practices. Washington, DC: ASM Press.
Packing, or Holding of Drugs, General.”
21 CFR, Part 210. 6. U.S. Department of Health and Human
Services, Centers for Disease Control
2. U.S. Food and Drug Administration. and Prevention and National Institutes
July 2000. “Good Manufacturing Prac- of Health. 2007. “Biosafety in Micro-
tice Guide for Active Pharmaceutical In- biological and Biomedical Laboratories
gredients.” Draft ICH Consensus Guide- (BMBL)”. http://www.cdc.gov.
line. http://www.fda.gov/cder/guidance/
4011dft.pdf. 7. Davis, R.S. 1989. “NIST Measurement
Services: Mass Calibrations.” National
3. United States Pharmacopeia. 2007. Unit- Institute Standardized Technology, Spec.
ed States Pharmacopeia and the National Publ., 250–31. http://ts.nist.gov/Mea-
Formulary. USP30 - NF25. Rockville, surementServices/calibrations/upload/
MD. SP250-31.pdf.
4. Bailey, John E., and Nikitakis, Joanne 8. Wise, J.A. 1988. “NIST Measurement
M. (Ed). 2007. CTFA Quality Assurance Services: Liquid-in-Glass Thermometer
Guidelines. Washington, DC: The Cos- Calibration Service.” National Institute
metic, Toiletry, and Fragrance Associa- Standardized Technology, Spec. Publ., 250-
tion. 23. http://ts.nist.gov/MeasurementSer-
vices/calibrations/upload/SP250-23.pdf.
SECTION 8:

SECTION 9
Microbial
Validation
and
Documentation
INTRODUCTION
Validation assures that the methods and procedures used by the cosmetic microbiology laboratory
are accurate and reproducible. Documentation provides the record of the validation.
In order for results to be meaningful, methods and procedures must be validated. Otherwise, the
conclusions drawn could be erroneous. For example, failure to properly neutralize the preservative
system during testing can result in false negative results where lack of organism recovery may be
due to inhibition of the organism.
A variety of techniques for validating and documenting methods and procedures is available. In
addition to the test methods and procedures used in the laboratory, the microbiological aspects
of process water systems and of cleaning and sanitizing procedures can also be validated by the
microbiologist. The results of a validation should be documented in an organized record keeping
system.
Once these methods and procedures are validated and documented, there is a high degree of
confidence that they can consistently produce accurate and reliable results. This completes the
full circle of quality assurance and is necessary to assure product quality.
Personnel responsible for any aspect of the validation must be adequately trained by education
and/or experience.
Validation is a process designed to establish documented evidence that a method or procedure
does what it purports to do. A validation or revalidation should be performed:
• When a new method or procedure is developed.
• If there is a significant change in procedure, supplier, product formulation or
equipment.
VALIDATION AND DOCUMENTATION
• As part of a recommended revalidation schedule.

SECTION 9: MICROBIAL
SECTION 9: MICROBIAL VALIDATION AND DOCUMENTATION | CTFA MICROBIOLOGY GUIDELINES | 109

Validation Format
The validation format should contain the following elements:
Scope
The scope identifies the area being validated, describes the purpose of the validation, and tells
what it encompasses. This should include the types of validation that will be performed. Three
types of validation include:
Prospective
Prospective validation establishes documented evidence that a process does what
it purports to do based on a preplanned validation protocol. All information and
results are gathered before implementation.
Concurrent
Concurrent validation establishes documented evidence that a process does what
it purports to do based on information generated during actual implementation
of the process. The test methods, procedures (such as cleaning and sanitizing),
systems (such as deionized water system) and equipment are being used while
data are gathered to support the validation.
Retrospective
Retrospective validation consists of presenting documented evidence that, based
on a review and analysis of historical data and information, a process does what
it was meant to do and that, all things being equal, can be expected to continue
performing properly.
Concurrent and retrospective validation can be performed simultaneously.
Description
A detailed step by step description of the procedure or method is provided.
Requirements
The requirements and acceptance criteria for the areas to be validated are described. For
example:
• Equipment cleaning and sanitization requirements include specific sites,
number of swabs before and after, and defined performance criteria.
• Laboratory autoclave requirements include number of heat penetration studies,
defined acceptable results, and negative controls acceptability criteria.
• Test methods criteria include the number of replicate platings and the allowable
variance.
Protocol
A written protocol for each method or procedure to be validated should be included.
110 | CTFA MICROBIOLOGY GUIDELINES | SECTION 9: MICROBIAL VALIDATION AND DOCUMENTATION

Documentation
A validation protocol should include appropriate documentation of all methods, procedures and
results.
Conclusion
Each validation protocol should include a conclusion that indicates the results of the validation.
If the results are satisfactory, a statement may be made that the validation is satisfactory and the
method or procedure can produce accurate and reliable results. If the results are unsatisfactory, the
next steps or modification required to complete a satisfactory validation should be indicated.
A final written report is prepared once the validation has been completed. Any revalidation
information can be added as it is generated.
Documentation
Once a procedure or method is validated, all results generated during its use should be documented
by keeping an organized record system. They can be organized by product or type of test and can
include:
• Material identification/description
• Identification/code number
• Vendor/in-house lot number
• Reference to the validated procedure used
• Acceptance criteria
• Tested by
• Date tested
• Reviewed and approved by
All results should be routinely reviewed by supervisory personnel. Records should be kept for
an appropriate length of time. Refer to “Annex 5 – Production Control” in the CTFA Quality
Assurance Guidelines.1
Documented results can be maintained as part of a product or raw material profile in the
development of an historical data base. Validation procedure results should be included as part of
this data base. Results compiled in an historical data base prior to validation may be useful as part
of a retrospective validation. The data base can also be used as a guide in interpreting test results
and establishing test guidelines and requirements.
LABORATORY AUTOCLAVE
An autoclave is an instrument that uses moist heat supplied by steam under pressure to sterilize
materials. The contents, whether liquid or solid, are exposed to saturated steam at the required
temperature and period of time. Pressure serves as a mechanism for obtaining higher temperatures
than otherwise could be obtained.

Validation
Preliminary Considerations
The accurate measurement of both time and temperature are necessary to confirm the sterilization
of the autoclave chamber contents. Before an autoclave cycle can be validated, temperature
controllers, temperature recording devices, pressure gauges, and timers of the autoclave must be
certified for accuracy to insure proper operation.
The autoclave load configuration and contents are important elements. The autoclave cycle will
vary based on volume of containers, number of containers, container type, media type, etc.
Heat Measuring Devices

Biological indicators and/or chemical and/or mechanical heat measuring devices can be used
to validate an autoclave cycle. The use of biological indicators (BIs) for certain media cycles
may be problematic if minimal sterilization cycles are required. BIs are available with different
populations. Select the most appropriate population.
Mechanical Devices
These include calibrated autoclave thermometers or thermocouples.
Biological Indicators (BI)
Due to their high resistance to moist heat, Bacillus (now called Geobacillus)
stearothermophilus spores on a paper strip or as a spore suspension in a glass
ampule are the most commonly used biological indicator for monitoring autoclave
performance.
At the end of the autoclave cycle, the Bacillus stearothermophilus biological
indicators are removed and incubated per manufacturer’s directions. These are
usually incubated at 56.0-60.0°C for 7 days to account for the possibility of
slower growth following exposure to sublethal heat. Use an appropriate media
if spore strips are used. If BI are used, they should be certified against the label
claim.
Negative and positive controls should be incubated along with the autoclaved
biological indicator samples. An unautoclaved biological indicator should be
incubated as a positive control. If spore strips are used, a negative media control
should be also included.
Chemical/Physical Indicators
Chemical and physical indicators are used as a secondary check to monitor a
validated cycle. They are not intended to be used as primary indicators to validate
a cycle. Autoclave tape indicates it has been exposed to an autoclave cycle when,
for example, black stripes or the word “autoclaved” appear on the tape. Ampules
which contain a material that melts and changes color when exposed to the proper
temperature may also be used to monitor an autoclave cycle.

Heat Distribution and Penetration
Heat penetration and/or distribution studies should be performed on empty (or minimum) and
maximum load configurations of the autoclave chamber by using temperature measuring devices
and biological/chemical indicators. The biological/chemical indicators and/or the heat measuring
devices should be evenly distributed throughout the autoclave chamber for the monitoring of
representative areas. At minimum, the monitoring devices should be placed at the four corners
and center of the autoclave. The actual number may vary based on the size of the chamber and
the load pattern.
Heat Distribution
The purpose of a heat distribution study of an autoclave is to determine the
uniformity of temperature in the load. Heat distribution studies should be
performed on load configurations of the autoclave chamber by using temperature
measuring devices. It is suggested that a minimum of three heat distribution
studies be done on the load configuration being validated.
A mean chamber temperature is taken from all the distributed temperature
readings. Chamber temperature uniformity can be considered acceptable if
individual temperature readings deviate less than ±1.0°C from the mean chamber
temperature. A mean temperature deviation greater than ±2.5°C versus the set
temperature may indicate equipment malfunction. The chamber uniformity
range should be based on the manufacturer’s recommendation for the autoclave
capability.
Heat Penetration
The purpose of a heat penetration study is to assure that all the containers within
a loading pattern will consistently be exposed to a sufficient amount of heat for
sterilization. A minimum of three heat penetration studies is suggested.
Heat penetration studies may be performed by placing chemical/biological
indicators in containers of media distributed in a load pattern throughout the
autoclave. Biological and/or chemical indicators should be placed throughout the
chamber including areas considered to be the most difficult to sterilize.
Documentation
The following information should be recorded for each validation run:
• Date of validation
• Autoclave identification or number
• Run number
• Sterilization time
• Sterilization temperature
• Load contents (media, broth or agar)
• Number of containers, configuration

• Indicators used and the lot number
• Placement of indicators, temperature measuring devices

• Results of indicators, measured temperatures
• Control results.
A cycle is considered to be validated when all the chemical and/or biological indicators show
appropriate reactions or no growth after the process and all heat measuring devices read within
acceptable parameters.2
Routine Monitoring
Each autoclave cycle should be monitored for the following:
• Temperature and time - Confirm temperature and length of cycle on recording
charts or cycle printouts.
• Pressure - Observe pressure on gauge and confirm if recorded on cycle
printouts.
• Biological indicators - Monitor using BIs at least quarterly where applicable
for monitoring a validated cycle.
• Chemical/Physical indicators - Temperature-sensitive indicators, such as
autoclave tape, should be used with each load.
Preventive maintenance should be performed on a routine basis by the manufacturer or other
qualified personnel. Records of maintenance should be retained.
MEDIA
General
Microbiological culture media contain growth-promoting substances such as available sources of
carbon, nitrogen and inorganic salts. The quality of the growth characteristics of microorganisms
in culture media depends upon the care taken in the preparation of the medium. To insure
satisfactory microbial culture media for use in the microbiology laboratory, a validation and
quality control program should be established for freshly prepared or received media as well as to
establish the shelf life of the media.
This applies whether the culture media are prepared from commercially dehydrated products or
purchased from a supplier in prepared form. This program is needed to ensure that the media can
consistently perform as expected over its shelf life. This program should include the identification
and control of those factors which affect the performance of the media. Some of these factors
include:
• Preparation of media
• Temperature
• pH
• Storage conditions
• Shelf life

Validation and Quality Control Program
Elements of a media validation and quality control program include:
Inventory Control and Storage of Dehydrated Media
• Write the date received and the date opened on the containers of dehydrated
media.
• Follow the manufacturers’ expiration dates and storage conditions. Most
dehydrated microbial culture media can be stored in a cool, dry place, preferably
at a temperature below 30°C. Manufacturers recommend storing certain types
of dehydrated microbial culture media at a refrigerated temperature (2-8°C).
• Rotate laboratory stock of culture media so that the oldest container is used
first. This practice will allow a turnover in the dehydrated media containers.
It will help keep media stock fresh and within the manufacturer’s expiration
dates. Media with outdated expiration dates should be discarded.
• Protect laboratory culture media that is in dehydrated form from absorbing
additional moisture from the environment during storage. A high moisture
content could possibly cause the degradation of various ingredients of the
media.
• Supplements and additives, where used, should be stored per manufacturer’s
recommendations. For example, store under refrigeration if the material is
sensitive to heat degradation, or protect from light until use if the material is
light sensitive.
Preparation of Dehydrated Media

Preparation
Follow manufacturer’s directions for the preparation of dehydrated media. Factors
in preparing dehydrated media which may affect its performance include:
• Water for reconstitution - Use purified water, such as distilled, deionized or
reverse osmosis, to reconstitute dehydrated culture media. At a minimum,
the water should meet the Environmental Protection Agency guidelines
for drinking water standards.3 Microbiological and chemical evaluation of
purified water is recommended. The quality of purified water may be checked
for specific chemical parameters, for example using current USP. or in-house
requirements, on a regular basis.4,5
• Agar temperature - Monitor temperature when pouring agar plates for streak
plates. The correct temperature should be employed since an incorrect agar
temperature can result in the alteration of the final water content of the medium
by excessive evaporation and medium shrinkage or excessive condensate.
• Additive temperature - Temperature of additives or supplements which are
required to be added after sterilization are important. They should be added at
the correct temperature to avoid the chemical degradation or destruction of the

additive or supplement.
• pH - The pH of the microbial culture media is critical. There should be a

laboratory procedure that lists the acceptable pH ranges for a liquid or agar

medium. In general, the pH reading should be taken after autoclaving and
subsequent cooling to room temperature and meet established requirements.
Media Records
Establish a batch record for each lot of culture medium that is prepared in the
laboratory. The following information should be included on the batch sheet:
• Medium name
• Date of preparation
• Manufacturer’s lot or batch number
• Quantity prepared
• Signature of preparer
• Method of preparation
• pH after autoclaving
• pH adjustment, if required
• Sterilization time and temperature
• Volume dispensed
• Number of units dispensed
• General comments (e.g., appearance of the medium during preparation)
• Performance test results and disposition (acceptable/unacceptable).
Where available and/or appropriate obtain a certificate of analysis from the
supplier of prepared media.
Labeling and Storage of Prepared Media

Labeling
Label all microbial culture media, whether commercially prepared or prepared in
the laboratory. Include the following information:
• Date of preparation or date received of commercially prepared media
• Type of media
• Shelf life or an expiration date, either on the label or on a separate list.
Storage
Establish a shelf life, an expiration date for the culture media. In general, the
shelf life of a particular medium is dependent upon how well that medium will
continue to support the growth of test microorganisms. The shelf life can be
determined by growth promotion studies over time and storage conditions. This
can be accomplished by comparing the growth promotion abilities of the stored
versus freshly prepared microbial culture media. Factors which affect the shelf life
of prepared media include:
• Form - The formulation and packaging of the medium will decide its basic
susceptibility to deterioration during storage. In most cases, the shelf life of an

agar medium in a Petri dish will be shorter than the same dehydrated agar in a
sealed container or bottle.

• Storage temperature - The optimum storage temperature for the majority
of prepared microbial culture media is about 2-8°C. Most liquid media will
keep for many months at 2-8°C, but have a tendency to form deposits. This
is especially true for culture media at double strength. If stored at room
temperature, shelf life may be shortened.
• Light exposure - Dye-containing media may fade if exposed to light.
• Evaporation - A volume check on liquid media should be made on older stocks
which may change due to evaporation.
• Dehydration and contamination - Prepared solid media can be stored for
many months in an airtight container. The storage of agar Petri dishes presents
two main problems: dehydration and contamination. The length of storage
time that agar Petri dishes can be kept for use will depend upon the ability to
avoid microbial contamination and the loss of moisture. To prevent moisture
loss, agar Petri dishes can be wrapped in plastic bags for storage at 2-8°C.
Performance Testing of Microbial Culture Media

Performance testing is conducted to confirm that a given media yields expected results when
inoculated with applicable microorganisms.
Usually on the same batch preparation sheet or on a separate coordinated sheet, the following
performance testing details are listed:
• Medium
• Batch/preparation date
• Date tested
• Sterility evaluation
• Specific microorganisms
• Growth promoting, differential, and inhibitory ability
• Pass/Fail
• Operator’s signature and date
A sterility check should be performed for each lot of prepared microbial culture medium. The
tester should incubate a sufficient number of units at the temperature and time for which the
medium is going to be used in the laboratory testing. The sterility test for each lot of prepared
medium will document that it was sterilized properly. This test will ensure that any microbial
contamination detected during a test procedure using this lot of prepared medium was not caused
by a lot of improperly sterilized media.
Each lot of dehydrated microbial culture medium or commercial or laboratory lot should be tested,
as appropriate, for its ability to support, differentiate, or inhibit the growth of microorganisms.
For general selection media, the choice of which microorganisms used to validate the ability of that
medium to support growth is up to the individual laboratory. It is preferable to use representative
strains of different types of organisms. However, selective or differential media should utilize
microorganisms that will demonstrate the characteristics of that medium. A negative control
organism may be included to determine that there are no false positive reactions.

If all control parameters of the preparation process are validated and monitored, testing may be
done less frequently, but on a regularly scheduled basis.
Two methods used by industry to validate the growth promotion ability of a microbial culture
medium are described below.
• One method involves inoculating a culture medium by streaking solid media
or pipetting into liquid media with a dilution yielding 10-100 colony forming
units (CFU) prepared from a 24-hour culture of a known microbiological
strain.
• The second method consists of inoculating by streaking or pipetting and
spreading a solid culture medium surface with a 10-3 dilution of a 24-hour
culture of a known microorganism(s).
The choice of method is up to the individual laboratory. There are many factors to consider
including the type of test and the sensitivity and/or detection limits of the test procedures for
which the media will be used. The known microbiological strains used may be American Type
Culture Collection (ATCC) strains of a particular microorganism. In either case, the inoculated
media should be incubated at the same temperature and time for which it will be used in laboratory
testing. After incubation, the inoculated liquid media is examined for the presence of turbidity
and general solid media for the presence of growth. Selective and enrichment agars are examined
for typical color and colony morphology of the strain used to inoculate them.
If the lot is within the correct pH range, passes the sterility test, and passes the growth promotion
test, it can be used for testing in the microbiology laboratory. If the medium’s pH is out of
range, if the medium does not pass the sterility test, or if it fails to support the growth of the test
microorganism(s), the medium fails the criteria for use in the laboratory.
There may be times when performance testing is conducted concurrently with the use of the
media for laboratory testing. If a lot of media fails the performance test and was used in the
laboratory, all laboratory testing should be repeated with acceptable media.
Results of performance testing should be documented and reviewed for accuracy.
TEST METHODS
General Considerations
Validation of a microbiological method is the process by which it is established through laboratory
studies that the performance characteristics of the method meet the requirements for the intended
application. Protocols should be designed to generate reliable, accurate and reproducible results.
Microbiological methods are designed to detect and/or identify microorganisms when present.
These methods include conventional plate count/streak plate procedures, as well as rapid or
automated methods. These methods are intended to perform within a wide range of variables,
some of which are:
• Different types of product

• Different types of media
• Incubation conditions
• Organisms
• Detection limits
Elements of a Validation Protocol
A protocol should be designed to incorporate all variables inherent to the method. Each step of
the protocol or procedure should be documented and include (when applicable):
• Reference to protocol or test procedure number
• Incubation conditions (time or temperatures)
• Neutralizing agents for preservatives or other growth-inhibiting agents
• Confirmation of preservative neutralization
• Identity of personnel performing each step
• Type of media
• Media lot numbers
• Test organisms - known profile (biochemical, morphological, etc.)
• Test organism inoculum level
• Dilutions tested
• Acceptance criteria which includes test and control sample. For example, one
acceptance criterion is recovery of artificially introduced microorganisms into
a test and control sample. Data is analyzed comparing test and control sample
results.
• Test personnel’s signature upon test completion
• Reviewer’s signature
• Test organisms used should be maintained, as recommended, by the supplier
of the organism.
• Subculturing should exhibit unique, demonstrable characteristics of the
organisms to ensure a pure and known culture.
• Organism storage, transfers and subcultures should be documented.
Modifications to the protocol should be documented.
The accuracy of a test method can be validated by performing repeated tests on the same sample.
Its reproducibility is usually confirmed by designing a collaborative test whereby a number of
different laboratories perform the same procedure on material from the same batch.
A file should be maintained for all data generated by the test procedures.
In addition to the above elements and general considerations, the following items apply to specific
test procedures.

• Microbial content testing may be performed on raw materials, components,
bulk product, in-process material and finished goods.
• Microbial limit guidelines, sample size and frequency of testing should be
established.
• The established testing guidelines should have appropriate documentation.

Refer to “Determination of the Microbial Content of Cosmetic Products” (Section 18)
“Establishing the Microbial Quality of Cosmetic Products,” (Section 12) and “Microbiological
Limit Guidelines for Raw Materials” (Section 11).
Preservative Efficacy Testing

Method
The method of preservative challenge testing adopted for use should predict preservative efficacy
under routine good manufacturing practice and normal consumer use conditions. The preservative
system is not intended to compensate for poor GMPs.
Test Procedure
The type of test procedure used should be determined. For example:
• Semi-quantitative - Swab or Streak plate Method
• Quantitative - Total Plate Count
Protocol
The elements of the protocol should include:
• Length of the test
• Test organism(s)
• Media
• Sampling frequency
• Confirmation of the neutralization of the preservative
• Test parameters (incubation time, temperature, etc.)
• Sample size
• Acceptance criteria.
Refer to “M-3 The Determination of Preservation Adequacy of Water-Miscible Cosmetic and
Toiletry Formulations” (Section 20), “M-4 Method for the Preservation Testing of Eye Area
Cosmetics” (Section 21), and “M-6 A Method for the Preservation Testing of Atypical Personal
Care Products.” (Section 23).
Environmental
Ambient air, compressed air, and equipment surfaces in the manufacturing plant are monitored
for the presence of microorganisms using apparatus such as settling plates, air samplers, swabs,
and contact plates. Equipment should be evaluated by reviewing manufacturer’s technical
information.
Surface monitoring procedures can be validated in the laboratory by applying known types and
levels of organisms to sample surfaces simulating production surface materials. Recovery using

the various surface monitoring techniques can then be compared to initial types and levels of
organisms to determine the acceptability of the procedure.
• Maintain equipment maintained per manufacturer’s specifications and or
recommendations.
• Validate that the media used supports organism growth.
• Ensure that media neutralizes any sanitizer present.
• Establish action and alert levels based on historical data.
Refer to “Microbiological Evaluation of the Plant Environment” (Section 2).
ORGANISM IDENTIFICATION
Identification Systems
Microorganisms may be identified using classical methods. Identification via commercial
identification kits and/or automated systems is common practice in cosmetic microbiology
laboratories. Systems used for microbial identification should be validated to ensure they
consistently and accurately identify microorganisms tested.
A validated system should show equivalence with a known method. Introduction of an alternate
identification system requires a validation vs. the current system to ensure performance is
comparable or better than the current system(s). Some of the factors to include:
• Side-by-side comparison testing of the standard routine cultures used in the
laboratory and/or ATCC cultures.
• Side-by-side comparison testing of unknown organisms such as those isolated
from the plant product.
Standard cultures of microorganisms, such as ATCC cultures, should be used. A performance
check should be done on each new lot of kits received. It is important to follow the directions
supplied with the systems. The manufacturer of the identification systems may recommend a
series of QA microorganisms to test reliability and accuracy. QA test organisms should be chosen
to give a positive response to each of the tests. Organisms used for the QC test should include
the following:
• Positive Control - Inoculate with organisms the manufacturer claims are
identifiable by the kit.
• Negative Control/Identification System Control - If applicable, inoculate with
sterile saline or appropriate diluent. Do not inoculate with any organisms.
Follow manufacturer’s recommendations.
A system can be considered valid if the known organisms are properly and consistently identified
and the negative controls/identification system controls, as applicable, do not demonstrate any
reactions.
Records should be kept specifying the following:

• System tested
• Manufacturer
• Lot number

• Date tested
• Organisms tested
• Results
• Acceptance criteria - action taken if system or performance check not valid
• Initials or signature of test performer
• Initials or signature of reviewer, as appropriate
Refer to “M-2 Examination for Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa”
(Section 19).
MANUFACTURING, EQUIPMENT CLEANING AND

SANITIZING PROCEDURES
Introduction
Validation of cleaning and sanitization methods is a procedure to establish performance
characteristics of a cleaning and sanitizing process and to document the process will consistently
yield equipment that meets microbiological, physical and analytical chemistry requirements. The
microbiological validation process basically consists of:
• Determining requirements
• Writing a protocol
• Following the written protocol
• Testing and documenting the results
Results should consistently demonstrate that the process is in control and all steps should be
carefully documented. The protocol should be approved by the appropriate personnel including
the Microbiology Department. Performing the tests and evaluating the results should be the
responsibility of the Microbiology Department. This activity should be coordinated with analytical
chemistry, manufacturing, engineering and key personnel in other departments.
Validation Protocol
Components of the validation protocol include:
1. Determine specification or outcome desired
• What results are required to indicate that the procedure will result in
equipment that meets requirements?
2. Outline cleaning and sanitizing procedure to be validated
• Identify specific equipment and testing locations.
• What pumps, valves, filters, tanks, lines will be looked at and where?
• List cleaning methods and agents, concentration, contact time,

temperature.
• Equipment must be clean before it can be sanitized.

• List method(s) of sanitization.
• Be sure that the sanitizer has itself been evaluated.
• Map the cleaning and sanitization process; for example:
− Identify Tank #XXX and associated pipes, pumps & lines.
− Rinse with hot DI (deionized) water for XXX minutes to remove product
residue. Disassemble pumps/equipment when necessary to remove
product residue.
− Drain rinse water.
− Fill tank with ?% solution XX.
− Recirculate/mix for XXX minutes.
− Drain wash solution.
− Rinse to remove wash solution.
− Sanitize (follow directions for use of sanitizing agent).
• Establish a time limit for use of prepared sanitizing solution.
• Establish frequency of sanitization.
• Establish a time limit for resanitization.
3. Determine what steps should be evaluated, document test procedure (be specific)
and determine what data is required.
4. Determine test methods used to provide the data and acceptance criteria required
in item 3 (above) to confirm the efficacy of the cleaning and sanitizing procedures.
Several types of methods include:
• Visual examination
• Visual inspection to detect product residue
• Examination for “odor”
• Inspection to detect the odor of residual perfume or sanitizer
• Swabs
• Swabs to detect microorganisms
• RODAC plate
• Contact plates to detect microorganisms
• Rinse water
• Tests on rinse water to detect microorganisms and/or chemical residue
5. Compare results of tests performed before and after the cleaning and sanitizing
procedures to confirm the efficacy of the process.
6. Validation testing should be performed a minimum of three times.
Once the validation for a piece of equipment is completed, it need only be repeated if there is a
change in process, equipment or any parameter that may effect the outcome of the process, such
as product type. To remain aware of any changes, a Process Audit should be routinely done and
communication with key personnel maintained. There should be a routine review, for example,
annually, to insure and document that there have been no changes.

After a process is validated, routine testing, such as equipment monitoring, can be used to monitor
the process.
Refer to “Annex 3, Part 1 - Packaging Equipment” and “Annex 3, Part II - Processing Equipment,”
CTFA Quality Assurance Guidelines.1
PROCESS WATER SYSTEM
Validation
The purpose of validating a process water system is to generate documented evidence to provide a
high degree of confidence that the process water system will consistently produce water that meets
established guidelines. This is especially important with a water system since microbiological test
results may not be available before the water is used in manufacturing.
The following elements should be included:
System description
Describe the system and give an explanation of the purpose of each element. These should
include:
Type of system
• Deionizing (DI) - carbon beds, deionizing beds (separate, mixed or both)
• Reverse Osmosis
• Distillation Unit
Flow rate of the system
Description of water flow system
• Type of piping
• Number of outlets
• Points of use
• Recirculation route if applicable.
Description of the storage tanks, if applicable
Description and location of any filters present
• Micron size
• Prefilter
• Final filter
Description and location of any treatment element
• UV light with rated throughput
• Chlorination with concentration
• Heat with temperature requirements
• Ozonization with concentration.
Description and location of sampling and use points.
A schematic of the system should also be included.

System Maintenance
Describe the system maintenance. Include the following elements:
• Frequency of regeneration, backflush of carbon beds and/or changes of the DI
bed and/or filters
• Frequency of cleaning and sanitizing the system
• Method of cleaning and sanitizing, such as heat treatment or use of cleaning or
sanitizing solutions
• Description of cleaning and/or sanitizing solution(s) used
The Validation Protocol

Type of validation
Type of validation performed may be:
• Prospective
• Concurrent
• Retrospective
• Combination
Conditions
The conditions under which a revalidation will be performed should be
listed, whether periodically, after a significant system change, or under other
circumstances. Suggested revalidation requirements include:
• Perform a complete revalidation if there is a significant change such as in
equipment, procedure, etc.
• Perform a reduced or modified revalidation at a preset interval, for example,
annually, to ensure that the system is still performing as expected.
A periodic revalidation may also be accomplished by a formal review of the
data and system to document that there are no significant changes, and the
system continues to perform as intended.
Time period covered
Samples should be tested more frequently, a minimum of two or three times
per week during this period. The time period covered should include seasonal
changes so all variables are part of the validation.
Test methods
Include a detailed step-by-step description of the methods used or a reference
to established test methods. For example, tests commonly performed are:
• Chemical, as per USP4 or internal requirements.
• Microbiological, including total plate count and any other tests needed to
support the criteria as per established internal or compendial methods.

Test criteria
Include the criteria used and any action limits that are established. Also specify
what actions are to be taken.

Results
The validation protocol should include copies of all test results during the
validation test period.
Discussion of Results
A discussion of the test results should include any investigations or actions taken if results do not
meet expected criteria.
Conclusion
A determination based on an analysis of the results of whether or not the system can consistently
produce water which meets requirements if the key parameters are followed.
Documentation
Once established as part of the validation, critical system parameters, such as frequency of
regeneration, frequency of cleaning/sanitizing, names and concentrations of cleaners/sanitizers
used, and filter changes, should also be checked on a routine basis and documented to ensure
the proper operation of the system. This information is usually recorded as part of the system
maintenance record. This record should include:
• Dates the system was cleaned and sanitized
• Names and concentrations or conditions of the cleaners/sanitizers used
• Maintenance of UV light if applicable
• Dates the system was regenerated, backflushed and/or filters or resin beds were
changed
• Dates and explanation of any repairs to the system.
These parameters should be reviewed as part of an investigation if results do not conform to the
guidelines.
After the water system has been validated, samples should be tested on a routine basis to monitor
its performance. These results should be recorded and trends noted. This information should
include:
• Sampling points
• Reference to the validated test procedure/method
• Acceptance guidelines
• Any investigation results and/or actions taken if results do not conform to
guidelines
• Tested by and date tested
• Reviewed and approved by

A record tracing the use of the water (batch numbers, product) should also be maintained. This
is usually maintained as part of the manufacturing record.
Refer to “Microbiological Quality for Process Water” (Section 7).

LABORATORY EQUIPMENT AND INSTRUMENTATION
Diverse laboratory equipment and instrumentation is utilized in the microbiology laboratory
to assist in preparing and performing microbiological analyses. All instrumentation should
be regularly monitored to ensure its proper performance and reliability. A master logbook of
equipment requiring monitoring and the observed results should be maintained. In addition, a
file should be kept for each piece of equipment including:
• Name of instrument
• Model number
• Serial number
• Purchase date
• Manufacturer and/or distributor
• Maintenance and operational manuals
• Service representative information
A list of suggested equipment and instrumentation to be monitored includes but is not limited
to:
• Laboratory autoclaves (as above)
• Laminar flow hoods/biological safety cabinets
− Air flow
− Integrity of HEPA filter
− Particle size
• Balances
− Standard weights
• Centrifuges
− Rotor speed (rpm)
− Timer
• Incubators/refrigerators
− Temperature
• Water baths
− Temperature
• Microscopes
− Optical alignment
− Calibration for scale
− Cleaning and maintenance
• Automatic colony counters
− Standard template
• pH meters
− Standard pH buffers
− Standard solution
• Water activity devices
− Standardized salt solution
• Thermometers
− ASTM, NIST
Information pertaining to proper control steps for carrying out performance checks and preventive
maintenance should be found in the operator’s manual. To maintain equipment accuracy and
reliability, these tests and maintenance should be performed at regularly scheduled intervals. The
documentation for such testing should include the following:
• Frequency of performance (monitoring schedule)
• Criteria for acceptance/rejection
• Performance against standards
• Results
• Noted deficiencies
• Corrective action if necessary
• Documentation of corrective action and “fit for purpose” performance should
be reviewed and signed
• Initials or signature of test performer and reviewer.
During the validation of standard operation, all results should be recorded accurately including
deviations and corrective action initiated, if necessary. Records should be reviewed periodically
by supervisory personnel.
All equipment should receive regular preventative maintenance. Specialized checks, such as
autoclave servicing, centrifuge calibration and laminar flow hood maintenance should be
performed by trained personnel or an authorized representative of the equipment manufacturer.
Refer to “Annex 16 - Calibration Systems,” CTFA Quality Assurance Guidelines.1
REFERENCES
1. Bailey, John E., and Nikitakis, Joanne 4. United States Pharmacopeia. 2007.
M. (Ed). 2007. In CTFA Quality <1231>. “Water for Pharmaceutical
Assurance Guidelines. Washington, DC: Purposes.” United States Pharmacopeia
The Cosmetic, Toiletry, and Fragrance and the National Formulary. USP30 -
Association. NF25. Rockville, MD. 687-706.
2. Parenteral Drug Association. 1978. 5. U.S. Food & Drug Administration, 1993.
Validation of Steam Sterilization Cycles, “FDA Guide to Inspections of High Purity
Technical Monograph No. 1. Water Systems.” http://www.fda.gov.
3. EPA 40 CFR Part 141.1998. National
Primary Drinking Water Regulations:

Disinfectants and Disinfection Byproducts

Notice of Data Availability. http://www.
epa.gov/safewater/standard/v&e-frn.pdf.

PRESERVATION OF TEST ORGANISMS
SECTION 10: MAINTENANCE AND
SECTION 10
Maintenance
and Preservation
of Test
Organisms
INTRODUCTION
Cosmetic microbiology laboratories require maintenance and preservation of stock cultures for
research, educational, and industrial testing purposes. The conservation of viable, uncontaminated
cultures without variation or mutation of their original characteristics is essential for preservation
of product isolates, challenge testing of antimicrobial agents, efficacy and stability testing, quality
control evaluations, etc. Various short- and long-term methods are available for preservation of
microorganisms. Culture maintenance implies viability and purity, whereas preservation involves
retention of phenotypic and genotypic characteristics over a period of time.
All preservation methods have advantages and disadvantages that must be considered in view of
the needs of the user. Laboratories that stock few cultures that are frequently used may select an
inexpensive, short-term method such as subculturing to agar slants, immersing in mineral oil,
ordinary freezing or various drying techniques. Others who have large culture collections may
primarily use long-term methods such as freeze-drying or ultra-freezing. Species of microorganisms,
ease of manipulation, and financial restraints are additional selection criteria.
SHORT-TERM METHODS
Subculturing
Maintaining organisms on artificial media with periodic transfer is the most common and
simplest method of preserving a culture line. It has the advantage of not requiring any advanced
skills or equipment. However, it is also the method that has the greatest propensity for causing
strain variation. Therefore, the microbiologist should carefully consider which organisms are to
be maintained in this manner.
Media used for subculturing should be nutritionally minimal. By slowing metabolism,
accumulation of toxic metabolites is kept to a minimum, and the possibility of culture death or
mutation is reduced. Experience is perhaps the best determinant of how frequently subculturing
is done. Kept under refrigeration, most cultures will survive a month’s storage. However, some
fastidious strains may require shorter intervals between subculturing, while others may be more
tolerant of prolonged storage. In general, length of storage should be the maximum time that
ensures a viable, unaltered culture.
SECTION 10: MAINTENANCE/PRESERVATION OF TEST ORGANISMS | CTFA MICROBIOLOGY GUIDELINES | 129

Frequent checks of unique characteristics (e.g., pigment, colonial morphology, biochemical

traits) should be performed in addition to periodic extensive identification procedures, since this
method may create variations in strains of organisms.
Overlay Techniques
As an extension of common short-term subculture methods, an overlay of mineral oil or liquid
paraffin is often used. This has the effect of reducing availability of air to the organism, thereby
slowing the metabolic rate. After growth of the culture in an appropriate agar, as a slant or a stab,
or in broth, add sterile oil to a depth of 1-2 cm above the highest point of growth. The cultures
may then be stored at room temperature or refrigerated. A common source of contamination as
a result of this method is nonsterile oil. To prevent this, the oil should be heated to 170ºC for
1-2 hours.
Recovery of the organism is accomplished by removing a small amount of the growth with an
inoculating needle or loop and transferring to an appropriate growth medium.
This technique, used extensively over the years, has been very successful in fungal preservation.
Its chief disadvantage is that it is a messy procedure and therefore a poor method when frequent
retrieval is required.
LONG-TERM METHODS
Drying
The preservation of organisms by drying or desiccation is accomplished by the removal of
water and prevention of rehydration. There are a wide variety of drying methods that have been
extensively used.
Sand and Soil

Sand and soil have been found acceptable for preserving sporulating fungi. Sand or garden loam
having a water content of approximately 20% is put into glass bottles and filled to between half
and two-thirds capacity. The sand/soil is then sterilized by autoclaving at least twice at 121ºC for
20 minutes, allowing the contents to cool between runs.
The inoculum or spore suspension is prepared by adding 5 mL of sterile water to the culture
and gently scraping the colony to release the spores. If the isolate does not sporulate, then a
suspension of mycelia can be used.
The suspension is then dispersed in 1.0-mL amounts into the sterile soil/sand bottles.
The inoculated bottles are allowed to stand at room temperature for a period of 3-14 days
depending on the growth of the organism. This allows the fungi to utilize the moisture and for
growth to slow down for storage.
The soil/sand culture bottles are stored with loose caps in the refrigerator (4-7 ºC). The fungi can
be revived by sprinkling a few grains of soil onto a suitable medium.
130 | CTFA MICROBIOLOGY GUIDELINES | SECTION 10: MAINTENANCE/PRESERVATION OF TEST ORGANISMS

Studies have shown isolates of Fusarium and other genera to survive by this method 10-20 years.
No data are available on the preservation of bacteria and yeasts by the soil/sand method.
Silica Gel
The silica gel method has been found to be a successful drying method for fungi and yeasts as
well as bacteria.
In this method, bottles one-quarter filled with silica gel (6-22 mesh) are sterilized in a hot-air
oven at 180 ºC for 2-3 hours. The bottles are then cooled by placing the bottles in trays of water
and freezing in a deep freeze (-17 ºC to -24 ºC).
The organism suspension is prepared in sterile 5% nonfat skimmed milk. The suspension is
cooled and added to the silica gel bottles in an ice bath. Only three-fourths of the silica gel
crystals are wetted to avoid oversaturation. The bottles are left in the ice bath for 20 minutes until
the ice is melted. The bottles are kept at 25 ºC until the crystals readily separate when shaken,
between 1 and 2 weeks. After this time, the viability is checked by sprinkling a few crystals onto
medium for growth. If growth is good, the bottle caps are tightened and the bottles are stored
over indicator silica gel in an airtight container at 4ºC. The indicator gel must be replenished
from time to time.
Note: It is very important to keep all apparatus very cold during inoculation to minimize the
effect of the heat generated when the gels are hydrated.
Porcelain Penicylinders and Silica Gel Crystals

Porcelain penicylinders are inoculated with the microorganism suspension, dried on silica gel
crystals and stored in the refrigerator at 0-4ºC.
Test tubes, one-third filled with silica gel crystals (6-16 mesh) topped with a layer of glass wool,
are sterilized in a hot-air oven for 2 hours. Rubber stoppers are sterilized by autoclaving at 121ºC
for 30 minutes. Two sterile porcelain penicylinders are placed in a 48-hour microorganism
suspension and allowed to absorb for 15 minutes, then aseptically removed and placed in a
sterilized silica gel tube. The stopper is applied and the tube stored at 0-4ºC. Organisms are
revived by culturing a penicylinder into a tube of appropriate nutrient medium and incubating
for growth.
Inoculated cylinders can be stored for 6 months or longer depending on the organism.
Paper Strips or Discs

Yeasts have been successfully stored on paper. The paper replica method, developed by Basel et
al. (1977)1, has been found to maintain several hundred strains of yeasts for 3-6 years. A mature
yeast colony is suspended in a drop of sterile evaporated milk and mixed thoroughly. Sterile filter
paper discs (Watman #2 filter paper) cut into 1 cm2 sections are immersed in the suspension for a
short time. They are then transferred to sterile aluminum foil packets. Three edges of the foil are
left open to allow the discs to dry. The packets with discs are placed in a desiccator and allowed
to dry for 2-3 weeks at 4ºC. Once dry, all edges of the packets are sealed and packets are then
stored in a dry area at 4ºC.

For revival, the disc is streaked across a plate containing appropriate medium for recovery. The
disc can remain in one corner of the plate. The plate is incubated at the appropriate temperature
for growth.
Predried Plugs
A number of types of bacteria that are difficult to preserve by freeze-drying have been successfully
preserved on predried plugs. Predried plugs can be made of cotton-wool cellulose, starch,
peptone or dextran. Suspensions of organisms are dropped onto the plug. Preserving agents, such
as mixtures of 5% peptone and 5% glucose or 5% peptone and 5% sorbitol, can be added to
improve recovery at various storage temperatures. The plug is then dried in a desiccator and stored
in ampules under vacuum either at 4ºC or at room temperature. Organisms can be recovered by
rinsing the plugs with 0.5 mL broth and plating out a few drops onto appropriate solid media.
Descriptions of several modifications in technique and apparatus have been documented.
Microorganisms such as several species of Salmonella, Escherichia coli, Staphylococcus aureus and
Vibrio sp. have been successfully recovered after 3 to 6 years of storage on predried cellulose plugs.
In contrast, other species showed very poor recovery. Consequently, this method may be limited
to preservation of particular groups of microorganisms.
Gelatin Discs
Many species of bacteria have been successfully preserved by the gelatin disc method. Bacterial
growth is suspended in melted nutrient gelatin. Drops of the suspension are delivered to a Petri
dish with the aid of a ropping pipette delivering 0.02 mL. The Petri dish is covered and placed in
a deep freeze at -20ºC to -40ºC until the drops are frozen, indicated by a change from transparent
to opaque. The Petri dishes are transferred to the freeze-dryer containing desiccant trays with
phosphorous pentoxide. The freeze-dryer is turned on and cultures dried overnight. After drying,
the gelatin discs are transferred to sterile vials containing silica gel and cotton-wool and stored
at 5ºC.
To revive the organisms, the gelatin disc is placed in 1.0 mL of nutrient broth and allowed to melt
by warming in a 37ºC water bath. Once melted, a loopful of broth is transferred to a suitable
solid medium for growth.
Advantages and Disadvantages of Drying Techniques

Viability of cultures preserved by drying techniques can extend to years. Capital equipment costs
are low and the methods are not labor-intensive. Many of the methods are suitable for storing
large numbers and frequently used cultures, since aliquots of dried material can be removed from
containers without great risk of contamination.
On the other hand, these methods cannot be universally applied. Most are limited to use with
particular groups of organisms. Since there is not enough data available on the reliability of these
methods, users are advised to gain experience first, giving careful consideration to the criteria
outlined in the introduction.

Freezing
Freezing of microorganisms as a method of culture preservation is successful because water is
made unavailable to the cell. Final storage temperature and cooling and rewarming rates are
critical variables in the operation, as is selection of a suitable cryoprotective agent. In general, the
lower the storage temperature, the greater the rate and longevity of survival.
Low-temperature freezing (-70ºC to -140ºC) is better than temperatures of 0ºC to -20ºC, which
do not preserve well because the cells are exposed to high salt concentrations during the freezing
period.
Frozen Suspension
A cell suspension of the microorganism is prepared, routinely as an overnight broth culture, utilizing
a minimal medium, so as to lower the metabolic rate of the organism. After centrifugation, the
pellet is resuspended in fresh sterile broth, to which has been added an appropriate cryoprotective
agent. The most frequently used are glycerol (10-15% vol/vol) and DMSO (5-10% vol/vol). If
agar slants are used as the source of cells, the growth is washed from the slant with sterile broth
containing the cryoprotective agent. The selection of cryoprotective agent and its concentration
may be determined in advance by examining for any toxic effects on the organism. This may vary
depending on the species of microorganism.
Aliquots of the resuspended cells (0.5-5.0 mL) are placed in sterile vials, allowing sufficient space
for expansion of the liquid during freezing. Vials are placed in a bed of crushed dry ice until
frozen solid, then removed and transferred to an ultra-low-temperature freezer at -70ºC to -
90ºC. Duplicate vials of each strain should be prepared to have one set of cultures remain frozen
at all times as a permanent collection. To recover the organisms, the vial is removed from the
freezer, opened, and a small amount of material scraped from the surface with a sterile stick or
needle, inoculated into appropriate media and incubated at specified growth conditions. The vial
should not be allowed to thaw or re-warm. This can be minimized by keeping it in a bed of dry
ice when working with it.
Freezing cell suspensions at low temperatures is a reliable, efficient means of long-term preservation.
It is not labor-intensive, and large numbers of cultures can be maintained.
The high initial equipment costs and the possibility of associated mechanical freezer malfunctions
or power failures may be of concern. A contingency plan should be developed in case of such
emergencies.
Glass Bead Storage

An alternate ultra-low-temperature storage method is to coat the microbial suspension onto glass
beads. This eliminates the problem of repeated freezing and warming of frozen suspensions when
subcultures are made, because each bead may be removed without any warming of the remaining
beads. The individual bead is then used as the inoculum.
Glass beads, approximately 2 mm in diameter, are washed with detergent and rinsed with dilute
HCl to neutralize the pH, followed by a distilled-water rinse. After drying, the beads are placed in
screw-cap glass vials, the quantity per vial dependent on intended use and the vial size. The vials
with the beads are sterilized by autoclaving at 121ºC for 15 minutes. The organism is grown on

the surface of an appropriate nonselective agar plate as an overnight culture, then inspected for
any contaminants. Approximately 1 mL of an appropriate sterile broth with cryoprotective agent
(see Frozen Suspension) is added to the agar plate and mixed with the growth to form a thick
suspension. This suspension is then added to the vial containing the beads. After the beads are
wetted thoroughly, excess suspension is removed from the bottom of the vial. The vials are then
placed in an ultra-low-temperature freezer maintained at -70ºC to -90ºC.
To recover the organism, a bead is aseptically removed from the vial and placed on the surface of a
plate of solid medium and rubbed over the surface to release organisms. Alternately, the bead may
be placed in a tube of sterile broth medium. The plate or tube is then incubated at appropriate
growth parameters.
This method has the same advantages and disadvantages as frozen suspension techniques and
is easier for frequently used organisms. However, it is slightly more time-consuming due to the
extra steps of preparation and coating of the glass beads.
Ultra-Freezing (-140ºC to -196ºC)

Ultra-freezing, using liquid nitrogen, is a method that offers long-term storage of organisms,
preservation of their original characteristics, and relative simplicity in processing. Storage in
liquid nitrogen provides a stable culture collection of identical sub-units that are easily accessible
when needed or preserved indefinitely providing the storage temperature is maintained below
-130ºC.
Organisms can be stored using nitrogen in either the liquid phase at -196ºC or in the vapor
phase at -140ºC and below. Most organisms used in routine laboratory work do not require
programmable cooling rate equipment with its high capital costs. A liquid nitrogen storage
container is sufficient for maintaining a typical organism collection. It is recommended to include
a cryoprotectant such as glycerol or dimethyl sulfoxide (DMSO) in the culture suspension to
enhance freeze/thaw survival.
Generally, bacterial broth cultures should be harvested in the mid- to late-log phase, agar fungus
cultures when spores are mature. Cultures should be centrifuged, supernatant decanted, the pellet
reconstituted with broth plus cryoprotectant, and the desired aliquot distributed aseptically to
ampules. The ampules are then capped and placed on aluminum canes for storage.
To revive a frozen culture after withdrawal from liquid nitrogen, place the ampule in a container
and then rapidly place the container into a 35ºC water bath. When the culture has thawed and
warmed to 35ºC, transfer it aseptically to an appropriate growth medium. Cultures that will be
used for challenge testing should be held a minimum of two hours in a nutritive medium to allow
resuscitation before exposure to a hostile environment.
The use of liquid nitrogen requires some precaution. Ampules that are stored in the liquid phase
can present the risk of explosion if liquid nitrogen penetrates an imperfect seal and expands
rapidly when warmed. Polypropylene screw-cap ampules reduce the hazard that glass ampules
pose, as well as eliminating several steps of preparation. Safety glasses or a plastic face shield and
protective gloves should be worn when depositing or withdrawing cultures from liquid nitrogen.
Failure to maintain a minimal level of liquid nitrogen can result in the loss of an entire culture
collection. To prevent this, liquid nitrogen levels must be monitored regularly.

Freeze-Drying (Lyophilization)
Freeze-drying, or lyophilization, is widely used to successfully preserve a broad range of bacteria,
fungi, and viruses. In each case, the process for each particular microorganism should be optimized
to provide for the greatest recovery of the original population. Several factors in the process
should be considered in reaching this goal:
• Growth phase of the culture
• Temperature of growth
• Composition of the growth and suspending medium
• Freezing temperature
• Rate and duration of the freeze-drying process
• Final moisture content
• Rehydration processes
Freeze-drying is a process where water is removed by sublimation. The organisms are first
suspended in a suitable medium, usually a cryoprotectant. They are then placed in glass ampules
and a vacuum is applied to remove the water as it sublimes. After drying, the ampule is flame-
sealed under vacuum or an inert gas.
Culture Preparation and Cryoprotectants

The culture should be grown in a medium particularly suited for maintaining preservative
resistance characteristics while not decreasing viability. Use of preservative or product as a
supplement to ordinary nutrient media can be a successful means of obtaining this goal. Cells are
generally grown to late logarithmic phase. If grown on agar slants or plates, the cells are washed
off with aid of a sterile policeman or pasteur pipette and resuspended in a minimal amount of
broth media. If grown in liquid suspension, they are harvested by centrifugation and the pellet
is resuspended in a small amount of broth. To each milliliter of broth-suspended culture, one
milliliter of sterile 12% sucrose can be added, mixed, and then placed into the ampules. Other
suspending media include a mixture of inositol (5 gm) and horse serum (100 mL) or a mixture of
nutrient broth (2.5 gm), inositol (5 gm), and distilled water (100 mL). Skim milk has also been
used with success.
General Procedure
The microbial suspensions are pipetted into glass ampules, cotton plugged, frozen, and a vacuum
(0.05 to 0.2 torr) applied. The ampule can remain in a brine-ice bath or dry ice/methanol bath
while under the vacuum to control the rate of sublimation. Once dry, the ampules are flame-
sealed under vacuum. The vacuum of the sealed ampules may be checked by use of a high-
frequency spark tester. Other variations include manifold batch preparation, centrifugal drying,
or shelf drying. Vials used may be single or double vials.

Recovery
Rehydration of the dried cultures should be done with a medium of the same composition as
the suspension broth. The vials are scored, wiped clean with ethanol, broken into, and sterile
medium introduced to rehydrate the culture. The rehydrated culture is then transferred to a
broth or agar medium.
Once growth occurs, a check to ensure all the type characteristics including product/preservative
resistance should be done. If the checks prove stable and viable, the lyophilization and storage
conditions can be considered validated for the culture under those conditions of freeze-drying
and storage.
Advantages and Disadvantages

In general, stability of a microbial population’s characteristics is not adversely affected by freeze-
drying. However, some selection, particularly loss of plasmids, can occur in some bacterial species.
Also, a significant drop in viability can occur in sensitive species.
A major advantage of freeze-drying is that once the culture is lyophilized, it is stable over periods
up to 50 years without the need for special storage conditions. A major disadvantage is the high
initial capital cost for equipment.
SPECIAL CONSIDERATIONS
Microorganisms are used by the cosmetic industry to obtain a variety of important data.
Consequently, the microbiologist must have some assurance that the organisms used will retain
their original genotype. This is especially true of organisms isolated from contaminated product
or organisms known to have resistance to certain materials. In many instances, conventional
methods for maintaining such organisms may not be adequate. Spontaneous reversion may occur
if mutants are removed from their original environment to artificial media or subjected to physical
stress. Occasionally, the organism may not survive even minimal storage on artificial media. The
microbiologist should give special attention to these organisms and store them in such a manner
that their original attributes are not lost and viability is maintained. This may mean periodic
subculturing into material from which the original isolate was obtained or devising an artificial
medium that will ensure a culture that has retained the desired trait.
These organisms should also have well-documented profiles. In addition to periodic screening,
certain key traits unique to the isolate should be monitored frequently to provide an added
measure of assurance of strain stability. Any deviation from the expected biological profile may
indicate that the organism is no longer suited for use.

CONCLUDING REMARKS
The aforementioned methods are an overview of those techniques currently acknowledged
as satisfactory for maintaining and storing microorganisms. There are perhaps several other
methods less commonly employed that may be equally reliable; their absence here does not imply
otherwise. Whether published or internally developed methods are used, it is the responsibility
of the microbiologist to carefully monitor the variables in the method, periodically ascertain the
purity and integrity of each strain of microorganism, and maintain any applicable documentation
relating to a culture collection. Adherence to sound microbiological practices will ensure that data
obtained from procedures using microorganisms are accurate, reproducible, and meaningful.
Brown, Michael R.W. and Peter Gilbert. 1995. Microbiological Quality Assurance A Guide Towards
Relevance and Reproducibility of Inocula. Boca Raton, FL: CRC Press.
Downes, Frances Pouch and Keith Ito, (Eds). 2001. Compendium of Methods for the Microbiological
Examination of Foods. Washington, DC: American Public Health Association.
Gerhardt, Philipp, R.G.E. Murray, Willis A. Wood, and Noel R. Krieg, (Eds). 1994. Methods for
General and Molecular Bacteriology. Washington, DC: American Society of Microbiology.
Gherna, R.L. 1989. Practical Handbook of Microbiology. Edited by W.M. O’Leary. Boca Raton,
FL: CRC Press. 249-250.
Hill, L.R. 1981. Essays in Applied Microbiology. Edited by J.R. Norris and M.H. Richmond.
Chichester: John Wiley & Sons.
Kirsop, B.E. and A. Doyle. 1991. Maintenance of Microorganisms and Cultured Cells: A Manual
of Laboratory Methods. Burlington, MA: Academic Press.
Lapage, S.P., K. F. Redway, and R. Rudge. 1978. CRC Handbook of Microbiology. Edited by A.I.
Laskin and H.A. Lechevalier. Florida: Chemical Rubber Press. 743-758.
Simione, F.P. and E.M. Brown, (Eds). 1991. American Type Culture Collection Preservation
Methods: Freezing and Freeze-drying. Manassas, VA: ATCC. http://www.atcc.org.
REFERENCES
1. Basel, J., R. Contopoulou, R. Mortimer and S. Fogel. 1977. UK Federation for Culture
Collections Newsletter. 4: 7.


SECTION 11
Raw Materials
Microbial
Content
RAW MATERIALS MICROBIAL CONTENT

SECTION 11:
INTRODUCTION
In order to minimize the chance of contaminated finished product, it is necessary to control the
microbial content of cosmetic raw materials along with other physical and chemical attributes.
Cosmetic manufacturers should evaluate the microbiological quality of their raw materials and
establish appropriate specifications based on the best available scientific information.
Water and water supplies are addressed in the CTFA “Microbiological Quality for Process Water”
(Section 7). Water systems should be properly validated and controlled. Quality specifications for
water should be set, including alert and action levels.
When establishing acceptable levels for raw material microbial content, the following criteria
should be considered:
• Chemical composition
• Physical nature
• Origin and availability
• Lot uniformity
• Intended use of the product
• Concentration of raw material used in the product
• Manufacturing process
• Raw material history
• Storage conditions
• Water activity
Many synthetic raw materials currently used by the industry contain low microbial counts,
due to extremes in pH, low water content, or inherent antimicrobial properties. Others may be
supplied as aqueous dispersions or solutions, and may be susceptible to microbial proliferation.
Therefore, it is important to evaluate susceptible synthetic materials upon receipt to ensure that
they have not been contaminated during the manufacturing process, packaging, transportation
and storage.
SECTION 11: RAW MATERIALS MICROBIAL CONTENT | CTFA MICROBIOLOGY GUIDELINES | 139
Naturally occurring raw materials are likely to contain a high level of microorganisms that may
pose a contamination risk to the finished product if not reduced or eliminated during processing.
The microbial content may vary depending upon the type and source of the raw material. It may
be necessary to treat such materials to reduce microbial levels before use or to purchase already
treated materials.
The criteria set by the manufacturer for the microbial content of a raw material should take into
consideration the release criteria established for each finished product. For example, the absence
of Salmonella is significant if a raw material is used in an oral product. A raw material microbial
content specification is usually not greater than that for the finished product, especially when it
is used at greater than 1% in the formulation. A raw material with a microbial count greater than
that set for the finished product may be acceptable if its use does not compromise the safety and
stability of the formulation and its concentration in the finished product is low.
SECTION 11:
SPECIFIC CRITERIA
This guideline recognizes the importance of using raw materials of the highest quality in the
manufacture of cosmetics. Special conditions may allow or necessitate acceptance criteria that
vary from those recommended below. It is recommended that the minimum test portion be 1 g
or 1 mL of sample.
The following are recommended guidelines:
• All Synthetic and Natural Raw Materials not more than 102 CFU per g
or mL
Note: Interpretation of results
The inherent variability of a plate count should be taken into account, thus the interpretation
may be as follows:
• 10² - may be interpreted as 5 x 10²
In addition to these recommended numerical guidelines, no raw material should have a microbial
content recognized as either harmful to the user or able to compromise integrity of the finished
product as recovered by standard plate count, specific pathogen test, or an equivalent automated
procedure.
GENERAL RECOMMENDATIONS
As cosmetics and toiletries need not be manufactured from sterile raw materials, it is important
that raw materials are obtained from qualified suppliers and handled, stored, and used under
conditions designed to deter microbial proliferation or subsequent contamination. The CTFA
Quality Assurance Guidelines are a useful guide for the storage and handling of raw materials1 as
well as microbiological sampling techniques.
140 | CTFA MICROBIOLOGY GUIDELINES | SECTION 11: RAW MATERIAL MICROBIAL CONTENT
Sampling techniques are located in “Microbiological Sampling” (Section 6) and in “Annex 17:
Sampling” in the CTFA Quality Assurance Guidelines2.
Validated microbiological analytical methods should permit the detection of microorganisms and
ensure the inactivation of the preservative (See Section 18: “M-1 Determination of the Microbial
Content of Cosmetic Products” and the Annual Book of ASTM Standards3). The presence of
objectionable organisms can be determined by identification of isolates using procedures such
as described in “M-2 Examination for Staphylococcus aureus, Escherichia coli and Pseudomonas
aeruginosa” (Section 19).

If raw materials are found to have a microbial content greater than specified, an investigation to
identify and eliminate the source of the contamination can assist in implementing preventative
measures 4.
SECTION 11:
It is recommended4 that a qualified microbiologist or independent microbiology laboratory be
engaged to:
• Design procedures for the examination of specific raw materials
• Examine the manufacturer’s raw materials for microbial content on a continuing
basis
• Interpret assay data on a routine basis
• Periodically review and update procedures, when applicable
REFERENCES
1. Bailey, John E., and Nikitakis, Joanne M. 3. ASTM E 1054-91. 1999. “Standard Prac-
2007. (Ed). “Annex 4 – Raw Materials tices for Evaluating Inactivators of Anti-
and Packaging Materials”. In CTFA Qual- microbial Agens Used in Disinfectant,
ity Assurance Guidelines. Washington, DC: Sanitizer, Antiseptic, or Preserved Prod-
The Cosmetic, Toiletry, and Fragrance ucts.” Annual Book of ASTM Standards
Association. 11.05.
2. Bailey and Nikitakis. “Annex 17 – Sam- 4. Bailey and Nikitakis. “Annex 10 – Sub-
pling.” contractor Quality Assurance.”
SECTION 11: RAW MATERIALS MICROBIAL CONTENT | CTFA MICROBIOLOGY GUIDELINES | 141
SECTION 11:
142 | CTFA MICROBIOLOGY GUIDELINES | SECTION 11: RAW MATERIAL MICROBIAL CONTENT
SECTION 12
Establishing
Microbial Quality
of Cosmetic
Products
INTRODUCTION
Control over the microbial content of cosmetics is consistent with the control of other aspects of
quality, and is necessary for ensuring consumer safety and product stability.
Because cosmetics are applied to bacteria-populated skin, microorganisms need to be controlled
in, but not necessarily eliminated from, cosmetics. Therefore, it is appropriate to assign rational
limits to microbial content based on the best available information. Criteria such as the product’s
SECTION 12: ESTABLISHING MICROBIAL

intended use, route of administration, and target population should be taken into account
QUALITY OF COSMETIC PRODUCTS

when establishing microbial guidelines for safety and quality. The microbial quality guidelines
presented here are intended to assist manufacturers in judging the microbiological quality of their
products.
It should be recognized that the application of microbial limits alone will not guarantee product
quality, and that a microbial quality management process must be implemented to ensure that
manufacturers produce products that conform to specifications. This quality management process
encompasses correct product development to ensure consumer safety, supplier quality management,
and adherence to Good Manufacturing Practices (GMPs)1 to prevent microbiological problems
from occurring. Essential to the implementation of effective microbial quality management is a
microbial awareness education and training program for all levels of employees.
Product Development
Product Preservation
Cosmetic and toiletry formulations that can support microorganisms or are susceptible to microbial
contamination should contain preservatives to retard microbial growth. The manufacturer has
the responsibility of producing an effectively preserved product. For products conducive to
microbial growth that can result in contamination, preservation efficacy should be determined
by appropriate tests during the development phase.
The function of preservation is to protect consumers and prevent product spoilage during
normal and reasonably foreseeable product use. Preservatives should not be used in lieu of good
SECTION 12: ESTABLISHING MICROBIAL QUALITY OF PRODUCTS | CTFA MICROBIOLOGY GUIDELINES | 143
production hygiene. Understanding the causes of microbial growth and eliminating them during
production will lead to control and prevention of microbial contamination.
It must be emphasized that preservation systems cannot be chosen satisfactorily on theoretical
grounds and they require in situ determination of their efficacy by microbiological challenge tests
or other appropriate test systems during product development.
Development of Suitable Formulations

Whenever possible, the formulator should be encouraged to develop formulations that are
incapable of supporting microbial growth, hence reducing the need for the addition of a
preservative. However, if a preservative is shown to be necessary, it should be selected at an early
stage in product development and considered as an integral part of the formulation.
Water is essential for microbial growth. A preservative system should have solubility and partition
characteristics such that it is available at effective concentration in the aqueous phase of a multi-
phase system.
A preservative system should be effective against a broad spectrum of microorganisms and safe
at the concentration used. Combinations of preservatives can sometimes be more effective than
individual compounds. Storage temperature, light exposure, and prolonged storage stability are
important. The concentration of the preservative system in a product formulation should be at

levels necessary to allow for antimicrobial activity sufficient to ensure adequate preservation. The
preservative system should be compatible with other product constituents and effective at the pH
of the formulation. These determinations can all be obtained from the testing of formulations.
Raw Materials
Cosmetics and toiletries produced for use by the general public are not required to be manufactured
from sterile raw materials or under aseptic conditions. Therefore, microorganisms found in the
general environment, in raw materials, and in formulation components may be introduced into
the product during manufacture. It is important that raw materials and components be handled
and stored under conditions designed to deter microbial contamination and proliferation.
Because raw materials can contribute a significant level of microbial contamination to the
finished product, it is important to monitor and control them. The monitoring of raw materials
should be appropriate to their susceptibility to microbial contamination, as determined by a risk
assessment. If practical, manufacturers should use raw material suppliers whose products yield
the lowest population of microbial contamination.
Water is one of the major raw materials used in the formulation of cosmetic and toiletry products
and one that can be populated by large numbers of microorganisms. This may present a distinct
hazard to the microbiological stability of the finished products. Therefore, steps must be taken
to ensure that water used as an ingredient or for processing is regularly monitored and, where
necessary, appropriately treated.
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Good Manufacturing Practices
Good manufacturing practices (GMPs) are necessary to avoid accidental human or environmental
microbial contamination during product manufacture. The manufacturing equipment should be
designed for ease of cleaning and sanitizing, as well as for processing capability. It is recommended
that, wherever possible, the physical plant of the manufacturing establishment be designed and
constructed to facilitate the conduct of manufacturing operations (making, packaging, storage,
and quality control) in accordance with current GMPs.
To comply with GMP, manufacturers of cosmetics and toiletries must define and follow specific
cleaning, sanitization, and control procedures. This process should include procedures to control
microorganisms in susceptible raw materials, bulk and finished products, as well as on personnel,
equipment, and premises.
Adequate records should be maintained for all aspects of microbiological testing during the
development and manufacture of each product and for all control procedures used at the
manufacturing facility. In particular, documentation during the manufacture of products is an
essential component of GMP.
Control and Assessment of Bulk and Finished Products

Susceptible formulations should be tested for microbial content on a routine basis, with

product data periodically reviewed for trends. The testing program should take into account
microbiologically susceptible raw materials, processing steps, and storage conditions.
The manufacturer should take into account the specific nature of the product when establishing
microbiological criteria for safety. The manufacturer is responsible for assuring that:
• Any microorganisms present are incapable of growing in the product.
• The species and quantity of microbes do not present a hazard to the consumer
when using the product as directed.
• Any microorganisms present do not compromise the stability of the product.
• The packaging (including cap or closure) does not promote microbial
contamination throughout the anticipated life of the product.
A sampling plan developed for formulations that are susceptible to microbial contamination
during the manufacturing process should be appropriate to the formulation and take into
consideration the manufacturing process history.
It is further recommended that a qualified microbiologist or independent microbiology laboratory
be engaged to develop validated microbiological procedures to analyze specific products,
periodically examine manufacturing procedures, and interpret assay data.
SPECIFIC CRITERIA
Acceptance criteria for the various classes of cosmetic products should be established when
necessary. For microbiologically susceptible products, conformance to the criteria is determined
by using a suitable technique, such as a plate count procedure. A risk assessment of products that
are not considered microbiologically susceptible may either support a decision not to test some
products or indicate reduced testing levels for others.
An enumeration method for a particular product should be qualified for the type of product
being tested. The method must permit the detection at a minimum, or the growth of any relevant
microorganisms present. The test method used should adequately inactivate microbial growth
inhibitors present in the product. It is recommended that the minimum test portion be 1 g or 1
ml of sample.
No product should have a microbial content recognized as either harmful to the user or able to
compromise product integrity, as recovered by standard plate count, test for specified organism,
or an equivalent alternative procedure. Specified organisms can be determined and identified
from colonies recovered on suitable microbial growth media. Alternative procedures can also be
used to detect the presence of specified organisms.
The conditions under which cosmetics are manufactured, marketed, and used by the consumer
vary throughout the world. Alternative microbial limits, such as those set by compendia,
governmental bodies, or other recognized authorities, may be applicable in some cases. The
application of specific criteria under this guideline should be decided by each national agency,
whether government or association. Where applicable, additional criteria for microbial content
may be listed in an annex that is specific to the country, region, or other area.
Some suggested criteria for microbial content are listed below:
• Baby products - not more than 102 CFU per g or ml
• Eye area products - not more than 102 CFU per g or ml
• All other products - not more than 103 CFU per g or ml
Note: Interpretation of results: The inherent variability of a plate count should be taken into
account, thus the criteria recommended should be interpreted as follows:
10² - maximum limit of acceptance is 5 x 10².
10³ - maximum limit of acceptance is 5 x 10³.
Any microbial content exceeding acceptance criteria should be investigated to identify and
eliminate the source of the contamination. Preventive measures should then be implemented.
A variety of different methods and procedures that may be used to make sure that cosmetic
products are in conformance with the recommended limits are available2-7.
REFERENCES
1. Bailey, John E., and Nikitakis, Joanne by SCCP during 10th Plenary Meeting.
M. (Ed). 2007. CTFA Quality Assurance December 19, 2006. http://ec.europa.
Guidelines. Washington, DC: The Cos- eu/health/ph_risk/committees/04_sccp/
metic, Toiletry, and Fragrance Associa- docs/sccp_s_04.pdf.
tion.
5. Department for Business, Enterprise &
2. The European Cosmetic Toiletry and Regulatory Reform. April, 2005. “Guid-
Perfumery Association. 1997. “Colipa ance on the Implementation of the Cos-
Guidelines on Microbial Quality Man- metic Products (Safety) Regulations
agement (MQM).” Brussels. http://www. 2004.” London. http://www.dti.gov.uk/
colipa.com. files/file25422.pdf.
3. Krowka, John F. and Bailey, John E. (Ed). 6. Japan Cosmetic Industry Association
2007. CTFA Microbiology Guidelines. (JCIA), 1997. “Microbial Test Methods
Washington, DC: The Cosmetic, Toi- for Cosmetics.” Tokyor. http://www.jcia.
letry, and Fragrance Association, 2007. org.
http://www.ctfa.org.
7. U. S. Food and Drug Administration,
4. “The SCCP’s Notes of Guidance for the FDA/Industry Activities Staff Booklet,
Testing of Cosmetic Ingredients and Their 1992. Cosmetics Handbook. http://www.
Safety Evaluation”, 6th Revision. Adopted fda.gov.

SECTION 13
Determination
of Preservative
Adequacy
in Cosmetic
Formulations
INTRODUCTION
The design of preservation tests and the subsequent interpretation of results is a complex process.
The technical personnel responsible for preservation testing should, therefore, be professionally
educated and experienced in conducting test procedures and evaluating the data generated. It
is important to remember that microorganisms are ubiquitous and capable of adaptation and
selection. No method can guarantee adequate microbial control under all conditions. In addition,
the importance of adhering to good manufacturing procedures in the production of cosmetics
and toiletries cannot be overstated.
The following factors should be considered when designing a preservation test or recommending
a preservative system for a new formulation:
• The nature of the raw materials in the formulation
• Information on preservation of similar formulations
• Manufacturing procedures
• Types of packaging used to contain product
• Information on the product's intended use, including area of application,
frequency of use, shelf-life, etc.
• Anticipated storage and/or shipping conditions
SECTION 13: DETERMINATION OF

PRESERVATIVE ADEQUACY
Developmental Formulations
Formulations that differ considerably from each other should be tested during the developmental
stage. Where the only variable in experimental design is the preservative system, it is essential that
each formulation be prepared from microbiologically acceptable raw materials. An unpreserved
formulation should be included in the test procedure as a control to determine the need for
preservation.
In order to evaluate the stability of preservative systems under consideration, developmental
formulations should also be tested after storage at temperatures simulating warehouse, shipping,
and shelf-life conditions.
SECTION 13: DETERMINATION OF PRESERVATIVE ADEQUACY | CTFA MICROBIOLOGY GUIDELINES | 149

Pilot Batches
Preservation tests should be performed on individual pilot batches to confirm the effectiveness of
the preservative system. Tests may be run on either bulk material prior to filling or filled samples.
Where possible, these tests should be accompanied by analytical verification of the preservative(s)
concentration.
Final Package
Preservation tests should be conducted on the product in the final package to ensure package
compatibility with the preservative system. A decrease in preservative effectiveness over a period
of time can result when the preservative system is altered by the final package, reacts chemically
with it, or is absorbed into the packaging material.
RECOMMENDATIONS
Since many cosmetic and toiletry products are used on a regular basis, an effective preservative
system should ensure the reduction of microorganisms to a low and steadily decreasing level, even
after severe microbial insult. The following minimal criteria are recommended:
Bacteria
There should be at least a 99.9% reduction of vegetative bacteria within 7 days following each
challenge and no increase for the duration of the test period.
Yeasts and Molds

There should be at least a 90% reduction of yeasts and molds within 7 days following each
challenge and no increase for the duration of the test period.
CONDITIONS
The tests should be carried out for a minimum of 28 days. At least one rechallenge is recommended.
PRESERVATIVE ADEQUACY
If a product does not meet these criteria, additional evaluations should be considered. It is
the responsibility of the manufacturer to select an appropriate methodology and appropriate
criteria.
Methods for determining the adequacy of preservation of cosmetic and toiletry formulations
are described in the Methods section. In addition, AOAC INTERNATIONAL official method
998.101 offers a validated method that may be referenced.
REFERENCE
1. AOAC INTERNATIONAL. 2000. “Efficacy of Preservation of Non-Eye Area Water-
Miscible Cosmetic and Toiletry Formulations,” Official Method 998.10. In: Official Methods of
Analysis of AOAC INTERNATIONAL. Gaithersburg, MD.
150 | CTFA MICROBIOLOGY GUIDELINES | SECTION 13: DETERMINATION OF PRESERVATIVE ADEQUACY

SECTION 14
Preservation
Testing of
Eye Area
Cosmetics
INTRODUCTION
It is recognized that cosmetic products may be environments in which microorganisms can
adapt and proliferate unless proper precautions are taken during formulation and manufacture.
The intended use of eye area cosmetics makes it imperative that these products be prepared
with preservative systems that remain effective.1 The alleged incidence of corneal ulceration due
to the periocular use of bacteria-laden cosmetics2 has led the Food and Drug Administration
(FDA) to specifically address the adequate preservation of these eye area cosmetics3 and the
CTFA to recommend the same microbial limits as those indicated for baby products. (Section 12:
“Establishing the Microbial Quality of Cosmetic Products”). Eye area products are normally free
of microbial contamination when purchased. However, some products may contain organisms
representative of human skin flora after use by the consumer.4 Microorganisms may be introduced
into the product from the environment or by the consumer, who may, for example, add tap water
to a product to make it less viscous.
In evaluating the adequacy of preservation of eye area cosmetics, it is important to point out that
there is no substitute for judgment by knowledgeable microbiologists. It must also be recognized
that the addition of preservatives to cosmetics is an adjunct to, but not a substitute for, good
manufacturing practices.
Developmental Formulations
Formulations that differ in at least one ingredient, e.g., binder or surfactant, should be tested
during the developmental stage using appropriate test microorganisms. If several preservative
systems are to be evaluated, each test formulation should be prepared concurrently from the same
microbiologically acceptable raw materials.
SECTION 14: PRESERVATION TESTING
OF EYE AREA COSMETICS
SECTION 14: PRESERVATION TESTING OF EYE AREA COSMETICS | CTFA MICROBIOLOGY GUIDELINES | 151
Pilot Batches
It may be desirable to perform a preservation test on individual pilot batches of product to verify
the effectiveness of the preservative system. If feasible, these tests should be accompanied by
analytical determinations of preservative presence and concentration. Tests may be performed on
bulk material or on the filled samples.
Stability
Product should be evaluated for preservative stability in commercial packaging by testing after
storage that simulates warehouse, shipping and shelf-life conditions.
RECOMMENDATIONS
Since eye area cosmetics are usually applied daily, an effective preservation system will help ensure
a low level of microorganisms even after severe microbial insult acquired during product use or
misuse. There are many references recommending preservative efficacy in sterile ophthalmics4,
6, 7, 8
and several in aqueous eye cosmetics.5, 7 Given the daily use of eye area cosmetics, it is
recommended that multiple challenges be made to fully ensure adequacy of preservation. 9 The
following are recommended as minimal criteria for preservative performance.
Aqueous Liquid and Semi-Liquid Eye Cosmetics

Vegetative Bacteria
There should be greater than 99.9% reduction of vegetative bacteria by aerobic plate count
or quantitative spread plate methods within 7 days following each challenge and continued
reduction to a less-than-detectable level by the end of the test period.
Yeast and Molds

There should be greater than 90% reduction of yeasts and molds by aerobic plate count or
quantitative spread plate methods within 7 days following each challenge and continued reduction
for the duration of the test period.
Spore-Forming Bacteria
There should be bacteriostatic activity against spore-forming bacteria throughout the entire test
period.
Non-Aqueous Eye Products

Vegetative Bacteria
There should be a 99.9% or greater reduction of vegetative bacteria by aerobic plate count or
quantitative spread plate methods within 7 days following each challenge and continued reduction
to a less-than-detectable level by the end of the test period.
152 | CTFA MICROBIOLOGY GUIDELINES | SECTION 14: PRESERVATION TESTING OF EYE AREA COSMETICS
Yeasts and Molds
There should be at least a 90% reduction of yeasts and molds by aerobic plate count or quantitative
spread plate methods within 7 days following each challenge and the level should remain at or
below that level for the duration of the test.
Spore-Forming Bacteria
There should be bacteriostatic activity against spore-forming bacteria throughout the entire test
period.
These minimal criteria are suggested to aid manufacturers in evaluating the adequacy of
preservation of eye area cosmetics. Ultimately, it is the responsibility of the manufacturer to select
appropriate criteria that will ensure product integrity.
REFERENCES
1. Wilson, L.A., J. W. Kuehne, S. W. Hall, 5. Tenenbaum, S. 1967. “Pseudomonads
and D. G. Ahearn. 1971. “Microbial in Cosmetics.” Journal of the Society of
Contamination in Ocular Cosmetics.” Cosmetic Chemistry. 18: 797-807.
American Journal of Ophthalmology.
71(6):1298-1302. 6. Bean, H.S. 1972. “Preservatives for
Pharmaceuticals.” Journal of the Society of
2. Wilson, L.A. and D. G. Ahearn. 1977. Cosmetic Chemistry. 23: 703-720.
“Pseudomonas-Induced Corneal
Ulcers Associated with Contaminated 7. Cosmetics, Toiletry & Perfumers
Eye Mascaras.” American Journal of Association, 1983. Appendix III - CTPA
Ophthalmology. 84:112-119. Recommended Microbiological Limits and
Guidelines to Microbiological Quality
3. Madden, J.M. and G. J. Jackson. 1981. Control. London.
“Cosmetic Preservation and Microbes:
Viewpoint of the Food and Drug 8. United States Pharmacopeia. 2007. United
Administration.” Cosmetics & Toiletries States Pharmacopeia and the National
96:75-77. Formulary. USP30 - NF25. Rockville,
MD.
4. Wilson, L.A., A. I. Julian, and D. G.
Ahearn. 1975. “The Survival and Growth 9. “A Study of the Use of Re-challenge in
of Microorganisms in Mascara During Preservation Testing of Cosmetics.” 1981.
Use.” American Journal of Ophthalmology. CTFA Cosmet. Journal. 13:19-22.
79(4):596-601.
SECTION 14: PRESERVATION TESTING OF EYE AREA COSMETICS | CTFA MICROBIOLOGY GUIDELINES | 153
154 | CTFA MICROBIOLOGY GUIDELINES | SECTION 14: PRESERVATION TESTING OF EYE AREA COSMETICS
PRODUCT QUALITY AFTER USE
SECTION 15: ASSESSMENT OF
SECTION 15
Microbiological
Assessment of
Product Quality
after Use
INTRODUCTION
Every cosmetic manufacturer has a responsibility to establish the microbiological safety of its
finished products. Doing this is a two-step process. The first is to assure consumers that each
cosmetic product is free from the numbers and types of objectionable microorganisms that could
affect product quality and/or the health of the consumer. (In addition to the information provided
in this publication, users should also refer to the CTFA Quality Assurance Guidelines1.)
Secondly, cosmetic manufacturers should ensure that each cosmetic product is not affected by the
introduction of microorganisms during normal or reasonably anticipated use by the consumer.
To prevent this, manufacturers may add preservatives to cosmetic product formulations. For
most cosmetic products (e.g., water miscible), microbial challenge testing is performed to verify
that the preservative system of a formulation can prevent the growth of microorganisms. For
additional guidance on testing preservation efficacy, refer to “M-3 A Method for Preservation
Testing of Water-Miscible Personal Care Products” (Section 20), “M-4 Method for Preservation
Testing of Eye Area Cosmetics” (Section 21), and “M-6 A Method for Preservation Testing of
Atypical Personal Care Products” (Section 23).
In addition, the analyses of used test samples for microbial content may provide added assurance
in the adequacy of the preservative system during use. Used samples may be collected for microbial
analyses as part of another study being conducted, such as a clinical or sensory study.
Furthermore, there are atypical cosmetic products (e.g., anhydrous gels, waxed-based sticks,
loose or pressed powders, etc.) for which the traditional preservative challenge test may not
yield the appropriate information regarding either the microbial integrity or susceptibility to
contamination of the product by the consumer. For these types of products, analysis of samples
after an in-use study should be considered.
ESTABLISHING A PROGRAM
When developing an adequately preserved product, microbiologists should consider the nature
of the product, directions for its application, the microbial quality of the raw materials in the
product, and the manufacturing process. While the microbiologist can exercise control over these
factors, a cosmetic company cannot control how a consumer will use a finished product.
SECTION 15: ASSESSMENT OF PRODUCT QUALITY AFTER USE | CTFA MICROBIOLOGY GUIDELINES | 155
To establish a program for evaluating the microbiological integrity or susceptibility to

contamination of a cosmetic product during consumer use, it is necessary to generate information
relating to the way the product will or should be used. This information can be obtained from the
product manager or through questionnaires, consumer letters, or consumer market tests.
To determine how to structure an in-use study for a cosmetic product, the following information
should be obtained: application, handling, length-of-use, and storage. This information will aid
the investigator in selecting the appropriate test panel members, defining usage instructions, and
setting a timetable. Study design should reflect actual product usage as closely as possible.*
PANEL SELECTION
Selection of panel test members should be based on consumer habits and practices. The following
elements may be considered in structuring the panel:
• Typical consumer age, sex, geographical distribution, product usage patterns,
etc.
• Panel size should be a function of the degree of statistical sensitivity desired,
with larger panels yielding increased sensitivity. An exact size-versus-sensitivity
determination may be made by consulting an appropriate sampling table.
After the panel structure has been determined, the study director will forward a request for
participation to potential panelists. It is recommended that this request include at least 20%
more individuals than are required to participate as test panel members to allow for attrition and
the initial inability of some people to participate. The request for participation should include
the test panel’s start and termination dates and a concise, comprehensive outline of what the test
panelists would be expected to do during the study.
PRODUCT EVALUATION
Before being submitted for evaluation, any formulation should be reviewed and approved for
application and the microbial content of unused test samples tested to ensure the product is
microbiologically safe under the prescribed conditions of use. In addition, the formulation being
tested should have been evaluated for safety and cleared for usage by the appropriate product
safety department. Product identification should be recorded, including any pertinent history,
product age, and lot or batch number. To determine actual usage by a test panel member, it
is recommended that each test sample be weighed prior to distribution as well as after it is
returned.
*Note: If test samples of a cosmetic formulation are to be used in a clinical study for the establishment
of proposed product claims or to determine actual product safety, the study director is responsible
for incorporating applicable aspects of Good Clinical Practice (see http://www. fda.gov/oc/gcp/
default.htm) where appropriate.
156 | CTFA MICROBIOLOGY GUIDELINES | SECTION 15: ASSESSMENT OF PRODUCT QUALITY AFTER USE
When the test samples are distributed, a comprehensive set of use instructions should be prepared
and given to each test panel member. The instructions for using a test sample should include the
following:
• Approximate quantity of material to apply
• Method of application
• Frequency of use
• Handling of the product between uses
• Instructions for returning the product (intermittent evaluation or after final
use)
• Any other instructions pertinent to the product under review
• Name of a person to contact if any questions should arise
EVALUATION OF USED PRODUCT

When used test samples are returned to the test site, each test panelist should complete a
questionnaire or hand in a diary. In designing a questionnaire, the following points should be
considered:
• How often was the product used?
• When was the product used?
• Where was the product stored between uses?
• Were there any problems associated with the product’s use?
The questionnaire should be designed to generate information that might be helpful in pinpointing
the reasons for an aberrant test result. It may provide the investigator or microbiologist with
information regarding the product’s ability to withstand either inappropriate or normal consumer
usage. A sample questionnaire is presented in Table 15-1.
If a test panelist uses a diary, the following information should be recorded: time at which the
product was applied each day, amount of product used at each application, and the location of
the product between applications.
When evaluating returned test samples, microbiological content testing should be conducted
before any other testing is performed. This is to ensure that recovered microbial contaminants
from a test sample were introduced during consumer usage and not from subsequent handling.
The microbiological evaluation of the used product should be conducted within a reasonable
time after the last use of the product by test panelists (i.e., seven days or less). All or a portion of
the returned samples will be tested for microbiological content, the number chosen based upon
some type of statistical sampling plan (e.g., the square root plus one). If aberrant microbiological
test results are obtained, the microbiologist has the option of analyzing additional returned test
samples to confirm the result.
MICROBIAL CONTENT OF PRODUCT

The method used for determining the microbial content of used product samples depends on the
nature of the product. Several methods may be used. While the aerobic plate count method is
considered a quantitative method, many of the others are considered semi-quantitative. Whenever
possible, quantitative recovery is preferable to semi-quantitative recovery. Semi-quantitative
results should be reported as an estimate of the microbial content of the unit.
Where feasible, an aliquot of the used test sample may be aseptically removed from each
container and analyzed for microbial content using an aerobic plate count method (e.g., “M-
1 Determination of the Microbial Content of Cosmetic Products” (Section 18) and “M-2
Examination for Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa” (Section
19) or in-house method). For water immiscible products (e.g., oils or emulsions), a suitable
solubilizing agent may be incorporated into the test diluent or broth to make the sample aliquot
miscible with water in order to recover microorganisms present in the test sample.
For products where microorganisms would only be recovered from the product surface (e.g., sticks,
pressed powders, hot pour products in compacts), only the surface of the product sample should
be tested. For these “atypical products,” the following methods of recovery may be considered.
• A sterile moistened applicator may be used to sample the product surface, and
then streaked onto a Petri dish containing solid culture media.
• The product may be sampled by a direct contact method using a contact plate
(a modified Petri dish containing a solid culture medium whose convex surface
extends above the carrier), paddles, or flexible film containing solid culture
media.
Alternative test methods to those described above may also be used. Appropriate preservative
neutralizers should be incorporated into product diluents, liquid or solid media. Whatever
method is chosen, it should be verified for the recovery of microorganisms. The same method
should be applied to the control sample.
MICROBIAL CONTENT OF APPLICATORS/UNIQUE

PACKAGING ELEMENTS
Product applicators or unique packaging elements (natural or synthetic) that come into direct
contact with the product may be evaluated for estimated microbial content, as these items are the
vectors of microbial contamination into the product. The following semi-quantitative methods
may be used to determine estimated microbial content:
• Aseptically transfer the applicator to a container of sterile diluent or liquid
culture medium. After vigorously shaking or vortexing for a set period of time,
perform anaerobic plate count on an aliquot of the diluent or liquid culture
medium.
• The applicator may be sampled using a direct contact method (see “Microbial
Content of Product” above).
Alternative test methods to those described above may be used. Appropriate preservative
neutralizers, as required, should be incorporated into diluents, liquid or solid media. Whatever
method is chosen, it should be verified for recovery of microorganisms.
IDENTIFICATION OF ISOLATES
It is recommended that recovered microorganisms from test samples be identified. If multiple
types of microbial colonies are obtained, representative microbial colonies may be selected for
identification.
INTERPRETATION OF RESULTS
For convenience, it is recommended that all test results be summarized. The interpretation of
microbiological in-use test results is largely a matter of in-house specifications. The extent to
which microorganism recoveries can be considered significant must be viewed in light of what the
ultimate effect would be on the consumer, the type of product (e.g., typical or atypical product
formulation), and the manner in which the product would typically be used by the consumer
(e.g., eye versus lip).
Recovery of low levels of microorganisms may be of some significance, including both water-
in-oil and oil-in-water formulations, especially in water-based products where the potential for
proliferation may exist. Thus, the acceptance criteria for samples of water-based products returned
from in-use studies normally reflect the specification for end product release. For these products,
further investigation into recovery of low levels of microorganisms may be warranted.
However, for products with a water activity of less than 0.6, the potential for proliferation does
not exist. For these types of products, recovery of normal skin flora may be expected. Therefore,
the acceptance criteria for atypical products may differ significantly from those of water-based
products. Higher microbial counts may be acceptable in atypical products used in areas of the
body that contain higher microbial populations and where there is less potential risk to the
consumer.
To ensure that the number of organisms recovered does not increase over time, initial samples
or additional samples may be retested to confirm stasis or reduction in recoverable levels of
microorganisms. If levels of microorganisms recovered do increase, then the formulation and/or
package design should be reviewed. Aberrant results may warrant further investigation including
performance of non-microbiological testing.
In-use studies of test samples cannot give a complete picture of how well a product will withstand
consumer handling and use. However, an in-use study for a proposed product may provide a
margin of added assurance to the manufacturer, as well as alert them to potential problems that
could occur in the field. Regardless of the nature of the test data generated, consumer in-use
studies can provide meaningful information as to how a product may behave during repeated
microbiological insult during consumer usage.
Table 15-1: Sample Consumer Evaluation Questionnaire
(Information needed by microbiologist)
Product: __________________________________________________________________________________
Panelist ID: ________________________________________________________________________________
1. How often did you apply the product?

______________________________________________________________________________________
______________________________________________________________________________________
______________________________________________________________________________________
2. When did you apply the product (e.g., after bath/shower, after housework, before retiring, etc.)?
______________________________________________________________________________________
______________________________________________________________________________________
______________________________________________________________________________________
3. Did you use the product on areas other than the area specified in the instructions? If so, where?
______________________________________________________________________________________
______________________________________________________________________________________
______________________________________________________________________________________
4. Where was the product kept when not in use?

______________________________________________________________________________________
______________________________________________________________________________________
______________________________________________________________________________________
5. When was the product last used?

______________________________________________________________________________________
______________________________________________________________________________________
______________________________________________________________________________________
6. List any comments you may have.

______________________________________________________________________________________
______________________________________________________________________________________
______________________________________________________________________________________
Table 15-1
Bailey, John E., and Nikitakis, Joanne M. (Ed). 2007. CTFA Quality Assurance Guidelines.
Washington, DC: The Cosmetic, Toiletry, and Fragrance Association.
Brannan, D.K., J.C. Dille, and D.J. Kaufman. 1987. “Correlation of In-Vitro Challenge Testing
with Consumer Use Testing for Cosmetic Products.” Applied and Environmental Microbiology,
58(3):1827-1832.
Brannan, D.K., and J.C. Dille. 1990. “Type of Closure Prevents Microbial Contamination of
Cosmetics During Consumer Use.” Applied and Environmental Microbiology, 56(5):1476-1479.
CTFA Microbiology Committee. December 1985. “CTFA Survey: Correlation of the In-Vitro
Preservative Challenge Test with Consumer Use Testing,” Presented by R. Spielmaker at the
Society for Cosmetic Chemists Scientific Conference.
Farrington, J.K., et al. 1994. “Ability of Laboratory Methods to Predict In-Use Efficacy
of Antimicrobial Preservatives in an Experimental Cosmetic.” Applied and Environmental
Microbiology, 60(12): 4553-4558.
U.S. Food and Drug Administration. Guidances, Information Sheets, and Important Notices on
Good Clinical Practice in FDA-Regulated Clinical Trials. http://www.fda.gov/oc/gcp/guidance.
html.
Larson, E.L., et al. 2003. “Microbial flora of hands of homemakers.” American Journal Infect.
Control, 31(2): 72-79.
Lindstrom, S.M. 1986. “Consumer Use Testing: Assurance of Microbial Product Safety.” Cosmetics
and Toiletries, 101: 73-74.
Lindstrom, S.M., and J.D. Hawthorne. 1986. “Validating the Microbiological Integrity of
Cosmetic Products through Consumer-Use Testing.” J. Soc. Cosmet. Chem., 37: 481-428.
Orth, D.C., R.F. Barlow, and C.A. Gregory. 1992. “The Required D-Value: Evaluating Product
Preservation in Relation to Packaging and Consumer Use/Abuse.” Cosmetics and Toiletries,
107(12): 39-43.
Passaro, D.J., et al. 1997. “Postoperative Serratia marcescens Wound Infections Traced to an
Out-of-Hospital Source.” J. Infect. Diseases, 175: 992-995.
Ryan, G.M., D.J. Floumoy, and P. Schlagertre. 1994. “Microbiological Flora and Nail Polish: A
Brief Report.” J. Okla. State Med. Assoc., 87: 504-505.
Singer, S. 1987. “The Use of Preservative Neutralizers in Diluents and Plating Media.” Cosmetics
and Toiletries, 112(12): 55-60.
Trick, W.E., et al. 2002. “Impact of Ring Wearing on Hand Contamination and Comparison of
Hand Hygiene Agents in a Hospital.” Clinical and Infectious Diseases, 36:1383-1390.
Wilson, L.A., A.I. Julian, and D.G. Ahearn. 1975. “The Survival and Growth of Microorganisms
in Mascara During Use.” American Journal of Ophthalmology, 79(4): 596-601.
Yablonski, J.I., and S.E. Mancuso. 2002. “Preservation of Atypical Cosmetic Systems.” Cosmetics
and Toiletries, 117(4): 31-40.
United States Pharmacopeia. 2007. United States Pharmacopeia and the National Formulary.
USP30 - NF25. Rockville, MD: http://www.usp.org.
REFERENCES
1. Bailey, John E., and Nikitakis, Joanne M. (Ed). 2007. CTFA Quality Assurance Guidelines.
Washington, DC: The Cosmetic, Toiletry, and Fragrance Association.
SECTION 16
Microbiological Risk
Factor Assessment
of Atypical Cosmetic
Products
SECTION 16: RISK FACTOR ASSESSMENT

OF ATYPICAL COSMETIC PRODUCTS
INTRODUCTION
Every cosmetic manufacturer has a dual responsibility relative to the microbiological quality of
its products. The first is to ensure that the product, as purchased, is free from the numbers and
types of microorganisms that could affect product quality and consumer health. The second is to
ensure that microorganisms introduced during normal product use will not adversely affect the
quality or safety of the product.*
During product development, the microbiologist may use several tools to evaluate the ability
of a product to prevent the growth of microorganisms introduced during product use. The
challenge test, which involves introducing a known quantity of microorganisms into a formula
and monitoring the rate of kill over time, is frequently used. A second method of evaluating
product quality during consumer use is by evaluating the product after a use test that simulates
“real life” situations.** Finally, the microbiologist may perform a microbiological risk assessment
of the product. The risk assessment process is based on a number of factors generally accepted
as important in evaluating the spoilage potential of a product. It is intended to guide the
microbiologist and formulator in determining what level of testing is necessary to assure the
quality of the product during manufacturing and consumer use.
This guideline serves as an aid to the cosmetic microbiologist in assessing the microbiological
quality of formulations for which the normally recommended method of challenge testing,
developed for water-based formulations may not yield appropriate information. These include
anhydrous formulations, formulations with low-water content, or those products where water is
the internal phase.
* For examples, see “M-3 The Determination of Preservation Adequacy of Water-Miscible

Cosmetic and Toiletry Formulations” (Section 20), “M-4 Method for the Preservation Testing
of Eye Area Cosmetics” (Section 21), and “M-6 A Method for Preservation Testing of Atypical
Personal Care Products” (Section 23). For guidance on the methods, see “The Determination of
Preservation Adequacy of Cosmetic and Toiletry Formulations” (Section 13) and “Preservation
Testing of Eye Area Cosmetics” (Section 14).
**For guidance, see “Microbiological Assessment of Product Quality after Use” (Section 15).
SECTION 16: RISK FACTOR ASSESSMENT OF ATYPICAL PRODUCTS | CTFA MICROBIOLOGY GUIDELINES | 163
This guideline also serves as a tool to aid the microbiologist in recommending ways of
reducing product susceptibility to microbial growth. Certain cosmetic products, depending on
their composition and presentation (packaging), may have negligible potential for microbial
proliferation during use. Microbial contamination of a cosmetic product during use is a function
of the physico-chemical characteristics of the product and the way in which it is packaged (i.e.,
its presentation).
PRODUCT TYPES
Common examples of atypical products are listed below. In each example, water is not readily
available to provide an environment that supports the growth of microorganisms. Water in the
product may be surrounded by oil or silicone as the external phase, with the water being present as
small droplets and influenced by other water-soluble formula ingredients. Also, the water activity
may be too low to support growth. In some cases, the product might be totally anhydrous.1
Common examples of atypical products:
• Wax-based products
• Products with high oil/low water content
• Siloxane and siloxane derivative-based products
• Lip balms
• Pomades
• Ointments
• Powders
• Cream-to-powder makeup
In addition to products with low-water activity, products with the physico-chemical characteristics
below do not allow the proliferation of harmful microorganisms:
• Products with an alcohol content equal to or greater than 20% (vol/vol)2
• Products with a pH of less than 3 or greater than 103,4,5
PRODUCT SUSCEPTIBILITY
Atypical products may contain raw ingredients that do not support the growth of microbial
contaminants and, therefore, may prevent microorganisms from proliferating when the product
is subjected to normal consumer use. In these types of products, organisms may survive, but
cannot reproduce. This may be due to low water activity or low water activity in combination
with pH and/or antagonistic formula ingredients that are water soluble. Water droplet size may
also be critical in the water phase. If the water activity reading is low in a product formulation,
or if the formula is anhydrous, studies have shown that microorganisms will not proliferate. In
fact, this is the basis for the use of water activity as an assessment tool in determining the risk for
microbiological proliferation for food products, like cereals.6, 7
In some atypical products, microbial survival may occur on the outside of the product without
ever permeating and spreading through the product. This observation is also due to the low “free
water” content.
164 | CTFA MICROBIOLOGY GUIDELINES | SECTION 16: RISK FACTOR ASSESSMENT OF ATYPICAL PRODUCTS
Because most atypical products will not support microbial proliferation, the method of product
delivery may be the vector for transferring microbial contamination from the product back to
the consumer.
RISK FACTOR ASSESSMENT

A number of factors need to be evaluated when performing a microbial risk assessment to
determine what type of testing or preservative system may be needed for a particular formulation.

Listed below are several factors to be considered in determining a product’s potential risk of
microbial contamination during consumer use.
Water Activity
The metabolism and reproduction of microorganisms demand the presence of water in an
available form. The most useful measurement of water availability in a product formulation is
water activity (aw). Water activity is defined as the ratio of the water vapor pressure of the product
to that of pure water at the same temperature:6
aw = p/po = (n2/(n1 + n2))
where,
p is the vapor pressure of the solution,
po is the vapor pressure of pure water,
n1 is the number of moles of solute, and
n2 is the number of moles of water. \
When a solution becomes more concentrated, vapor pressure decreases and the water activity falls
from a maximum of 1.00 (aw) for pure water. As the water activity level falls below the optimum
value for each microorganism, the length of the lag phase in the microbial growth cycle will
increase toward infinity unless rehydration occurs.
Listed below are examples of the minimum water activity levels required for the growth of selected
microorganisms.3,6,8
Approximate Water Activity (aw) Required for the Growth of Selected Microorganisms 1,10
• Most bacteria 0.94 – 1.00
• Enterbacteraciae >0.93
• Pseudomonas species >0.96
• Staphylococcus aureus >0.86
• Most spoilage yeast >0.70
• Most spoilage mold >0.60
The water activity values listed on the previous page should be considered as reference points
since microbial growth may occur at lower values depending on differences in temperature or
nutrient content of the product formulation. Even though water activity values are important
in assisting in the risk analysis for microbial contamination, water activity should not be used as
the sole indicator in determining whether product testing is necessary for a particular product
formulation.
Formula Review
Every formula contains raw materials that have an impact on the susceptibility of the formula to
microbiological contamination. Raw materials can be classified as susceptible, hostile, or neutral
to microbial growth, and their concentration will affect the susceptibility of the formulation to
microbial contamination.
It is recommended that a microbiologist review formulas to determine their potential susceptibility
to microbial contamination. If a formulation tends to absorb moisture, samples of these types
of atypical products should be microbiologically evaluated (including aw) after exposure to high
humidity conditions (i.e., 50–75% for about three weeks or until equilibrium is demonstrated).
Anhydrous products may contain binders or other hygroscopic materials that can absorb moisture.
CALIBRATION SYSTEMS
In addition, consideration of water on the surface of products may occur under high humidity
ANNEX 16
conditions. Consumers may introduce water during normal use or misuse.

The physical product form will affect whether microbial contaminants will be introduced at the
product surface or mixed throughout the product during consumer use.
Factors to be considered are:
• Raw material susceptibility
• Raw material microbial load
• Percent concentration of raw material
• Presence of preservative inactivators
• Presence of preservative potentiators
• Presence of fragrance and other ingredients that may act as preservatives
• Binder level in powders (the higher the binder percentage, the more hydrophobic
the product will be)
• Product's physical form (solid or liquid; melting point)
Site of Application
Risk assessment needs may vary since the site of a product’s application is an important risk factor
in determining the level and type of microbiological testing required for a cosmetic product.
For example, an eye area product poses a much greater risk of microbiological contamination
to the consumer than a product applied to other areas of the body. Lip products, under normal
conditions, generally come into contact with higher numbers of microorganisms that are part of
the normal microflora present in the consumer’s lip area, but do not pose a health risk.
Some points to consider when determining the risk in relation to the site of a product’s application
include:
• Is the site on the body to which the formulation is to be applied an area where
microbial levels are high (lip) or low (eye)?
• Is the product applied under moist (higher risk) or dry (lower risk)
conditions?
• What is the mode of application (brush, sponge, or finger)?
• How frequently is the product used?
Applicators and the Mode of Application
The mode of application plays a large role in determining the risk factor of a product. Even
though the product may not support growth, the method of delivery may be a vector of microbial
bacterial contamination.
Applicators could provide an environment that might be suitable for microbial proliferation.
For example, porous sponge applicators may be a concern due to their ability to absorb moisture
and retain sebum and dirt from the skin. With the presence of water, sebum, and dirt from the
skin, there maybe enough water and nutrients present in a used applicator to allow for microbial

proliferation. In those applicators that are to be used in conjunction with a wet/dry product, the

incorporation of an antimicrobial agent may be considered to prevent microbial proliferation.
The preservatives system of a product must not be expected to inhibit microbial growth in or on
a product applicator.
Some typical questions need to be asked before microbiological testing is conducted on
applicators:
• Are applicators such as puffs, brushes, or pads used with the products?
• Could these applicators act as a breeding ground for microorganisms or a
vector for microbial contamination of the product and/or consumer?
• Do the product and component come in direct contact with the consumer
(lips, eyes, fingers)?
• Do these applicators contain an antimicrobial agent?
• Is the applicator stored in direct contact with the product?
• Are there directions given on how to store or clean applicators between uses?
When evaluating and determining the risk factors for microbiological contamination in product
applicators, the following additional factors are to be considered:
• Type of applicator
• Type of material used
• Treated or not treated with an antimicrobial agent
• The efficacy of a treated applicator should be tested via a zone of inhibition test
or other appropriate method.9, 10
• Wet/dry application of product
Packaging
The type of packaging used for a finished product is a critical risk factor in determining the
overall potential for microbial contamination during consumer usage. The use of packaging can
provide additional protection by restricting direct access to the product.
The following factors are among those that should be taken into consideration when assessing
product risk in regards to packaging:
• Is packaging for single or multiple use?
• What is the size of the package?
• What is the mode of dispensing?
• What is the predicted use-up rate?
• Does the package type allow for direct consumer contact?
• Is the package pressurized?
Confirmation of the microbial integrity of the applicator and finished product can be determined
by conducting an in-use study.
Manufacturing Process
Certain aspects of the manufacturing process (e.g., high temperature) may affect the microbiological
contamination susceptibility for a cosmetic product. It is useful for both the microbiologist and
cosmetic chemist to review the manufacturing process to determine the potential risk of microbial
contamination to the formulation.
Factors to be considered:
• Are there processing factors that could affect the efficacy of the preservative
system?
• What is the temperature of the manufacturing process?
• What is the microbial content of the raw materials?
• Are hostile raw materials used to make the product?
• What is the order of the addition of raw materials?
PRODUCT TESTING
General
After evaluating the above factors, the microbiologist can determine what level and type of
microbiological testing is necessary. A risk assessment may indicate that a challenge test is not
needed for some atypical products. If it is determined that microbiological testing is necessary,
the following information can be used to assist in selecting the most appropriate test method:
Challenge Tests
General Considerations
The recommended preservative challenge test methods that are used for determining the
preservative adequacy of aqueous-based products may not be suitable for evaluating certain
atypical product formulations. When testing and assessing preservative challenge test data for
atypical products, the following factors are important points to consider:
• A test in which an aqueous-based inoculum is introduced into an anhydrous
sample may change the physical dynamics of the product and, therefore, may
not predict its microbial stability.
• Most preservatives are water soluble. In emulsions, preservatives are used
in the water phase because contaminating microorganisms require water to
proliferate.
• For an emulsion in which the external phase is water immiscible and an aqueous
challenge inoculum is used, the water-soluble preservatives will be unable to
penetrate the water-immiscible phase. In these cases, the preservatives will not
be available to either inhibit proliferation or have acidal activity against each of
the challenge microorganisms.
Possible Test Method Modifications

If preservative challenge testing is performed on an atypical product formulation, aqueous-based

challenge protocols may need to be modified to take into account a number of factors. For
specific test methods see M-3 and M-4 of the CTFA Microbiology Guidelines (Sections 20 and
21). A number of possible modifications to these methods are discussed on the next page.
Reduction in Inoculum Concentration
For some atypical products, (e.g., anhydrous products), a reduction in the
challenge inoculum size to 103 to 104 Colony-Forming Units (CFU) per gram
or milliliter may be used instead of the inoculum concentration of 105 to 106
CFU per gram or milliliter that is recommended in aqueous-based challenge test
methods. By reducing the inoculum size, it is easier to measure stasis or quantify
an increase in the microbial count.
Reduction in Inoculum Volume
Reduction of the ratio of microbial inoculum suspension to the volume of
product may also be considered. The recommended ratio of inoculum suspension
for aqueous-based products is no more than 1.0% for a challenge sample. For
atypical product formulations, the ratio of inoculum suspension to product may
need to be reduced to 0.1% to minimize changes in the physical dynamics of the
product.
Surface Inoculation and Sampling
For solid atypical products, such as anhydrous sticks and powders, inoculation
and sampling of the product surface instead of the whole product more closely
simulates potential consumer contamination. This modification also maintains
the physical product integrity. In these types of products, the microorganisms are
not able to penetrate into the interior and will always be found on the outermost
layer of the product after consumer usage.
Note: If performing challenge testing on a solid anhydrous stick or powder
product, inoculate a sufficient number of samples to obtain a unique sample for
each sampling time point.
Inoculum Delivery Systems
For liquid, anhydrous, atypical product formulations, one may consider using
an oil-soluble carrier system, such as light mineral oil or other suitable oil carrier,
to deliver and disperse the microbial challenge inoculum into liquid atypical
product formulations to form a homogeneous mixture.
If using this technique, the absence of inhibitory or toxic properties of the oil-
soluble carrier system should be verified for each of the challenge organisms.11
Sampling Diluents
The recovery procedure for determining the microbial counts from inoculated
challenge samples of an atypical product may need to be modified from those
that are commonly used in aqueous-based challenge test methods. For example,
water-in-oil emulsions and anhydrous products are not readily miscible with
water. It has been demonstrated that 1.0-gram aliquots of an anhydrous product
solubilizer in a 1.0-gram aliquot of sorbitan monostearate (Tween 80) and this
mixture were dispersed further by increasing the volume with an aqueous diluent
to make a 1:10 dilution.12
Challenge Test Acceptance Criteria

It is the responsibility of the manufacturer to set the challenge test criteria to the product type
and form. In the performance of challenge testing of atypical products, the pass/fail criteria may
need to be modified in comparison to the preservative challenge test criterion that is commonly
used for aqueous-based products. For example, the challenge acceptance criteria for anhydrous
atypical products may be stasis for certain types of challenge microorganisms, because these
organisms do not need a source of water to survive.
If criteria other than the aqueous-based challenge criteria are used to show adequate preservation
for an atypical product formulation, a risk assessment needs to be conducted by the microbiologist
to justify the use of these alternate preservative challenge test criteria.
In-Use Studies
General
In addition to or in place of a product challenge test, an in-use study may provide sufficient data
to conduct a risk assessment on some products. An in-use study may be used to evaluate the
microbiological integrity of a product during consumer use. The study design should reflect actual
product use as closely as possible. For further information, refer to “Microbiological Assessment
of Product Quality after Use” in Section 15.
Testing
When samples are returned from an in-use study, an aerobic plate count must be conducted
before any other tests are performed to ensure that any microbial contaminants recovered were
introduced by the panelists and did not arise from subsequent handling in the laboratory. Useful
microbial content information may be obtained by a similar evaluation of the components (such
as applicators) that come into direct contact with the product during use.
Microbiological analysis of these samples can be conducted by using either standard in-house
methods or the CTFA methods, “M-1 Determination of the Microbial Content of Cosmetic
Products” and “M-2 Examination for Staphylococcus aureus, Escherichia coli and Pseudomonas
aeruginosa” (Sections 18 and 19). It is recommended that microbiological evaluation take place
within seven days after the last consumer use.
Criteria
The pass/fail criteria for in-use return samples normally reflect the microbiological test specifications
that are used for quality control end product release. The pass/fail criteria for atypical products or
for water-based products may vary significantly depending on the product type and area of use.
For example, it may be acceptable that products that have been used in the lip area may contain
a higher microbial level than products used in the eye when evaluated after use. To ensure that
the number of microorganisms recovered after usage does not increase over time, these types of
atypical products should be tested at a prescribed time interval. If microbial counts do increase
over time, the formulation and/or package design should be reviewed to determine what steps

could be taken to prevent them from increasing.
Brannan, D.K., J.C. Dille, and D.J. Kaufman. 1987. “Correlation of In-Vitro Challenge Testing
with Consumer Use Testing for Cosmetic Products.” Applied and Environmental Microbiology,
58(3):1827-1832.
CTFA Microbiology Committee. December 1985. “CTFA Survey: Correlation of the In-Vitro
Preservative Challenge Test with Consumer Use Testing,” Presented by R. Spielmaker at SCC
Scientific Conference.
Farrington, J.K., et al. 1994. “Ability of Laboratory Methods to Predict In-Use Efficacy
of Antimicrobial Preservatives in an Experimental Cosmetic.” Applied and Environmental
Microbiology, 60(12):4553-4558.
Kabara, J.J., and D. S. Orth. 1996. Preservative-Free and Self-Preserving Cosmetics and Drugs.
New York: Marcel Dekker, 45-73.
Lindstrom, S.M. 1986. “Consumer Use Testing: Assurance of Microbial Product Safety.” Cosmetics
and Toiletries, 101:73-74.
Lindstrom, S.M., and J.D. Hawthorne. 1986. “Validating the Microbiological Integrity of
Cosmetic Products through Consumer-Use Testing.” J. Soc. Cosmet. Chem. 37: 481-428.
Orth, D.S. 1993. Handbook of Cosmetic Microbiology. New York: Marcel Dekker, 151.
Orth, D.S. and S.R. Milstein. 1989. “Rational development of preservative systems for cosmetic
products.” Cosmetics and Toiletries, 104(10): 91-103.
Orth, D.S., R.F. Barlow, and C.A. Gregory. 1992. “The Required D-Value: Evaluating Product
Preservation in Relation to Packaging and Consumer Use/Abuse.” Cosmetics and Toiletries,
107(12): 39-43.
Wilson, L.A., A.I. Julian, and D.G. Ahearn. 1975. “The Survival and Growth of Microorganisms
in Mascara During Use.” American Journal of Ophthalmology. 79(4): 596-601.
Yablonski, J. I. 2002. “Preservation of Atypical Skin Care and Related Cosmetic Product Systems.”
Cosmetics and Toiletries, 117.
REFERENCES
1. Yablonski, J.I. and S.E. Mancuso. 2002. 7. Doyle, Michael P., Larry R. Beuchat, and
“Preservation of Atypical Cosmetic Prod- Thomas J. Montville, (Ed). 1997. Food
uct Systems.” Cosmetics and Toiletries, Microbiology Fundamentals and Frontiers.
117(4):31-40. Washington, DC: ASM Press, ISBN 1-
55581-117-5.
2. Block, S.S. 1997. “Alcohol.” In: Disinfec-
tion, Sterilization, and Preservation, editors 8. Jay, J.M., (Ed). 2000. Modern Food Mi-
Ali, Y., et al. 2001: 229-253, especially p. crobiology, Sixth Edition. Gaithersburg,
234. MD: Aspen Publishers, 38-44, especially
p. 42.
3. Brannan, D.K., 1997. Cosmetic Microbi-
ology. New York: CRC Press, 47-50. 9. Hartman, P.A. 1968. Miniaturized Mi-
crobiological Methods. Orlando, FL: Aca-
4. Kabara, J.J. 1984. “Food grade chemicals demic Press.
in a systems approach to cosmetic pres-
ervation.” In: Cosmetic and Drug Preser- 10. Curry, J. 1985. “Water Activity and Pres-
vation: Principles and Practice. New York: ervation.” Cosmetic and Toiletries, 100:
Marcel Dekker, 339-356. 53-54.
5. Kabara, J.J. and D.S. Orth. 1996. Preser- 11. ASTM E1054-02 - Standard Test Meth-
vative-Free and Self-Preserving Cosmetics ods for Evaluation of Inactivators of Anti-
and Drugs. New York: Marcel Dekker, microbial Agents. http://www.astm.org.
245-246. 12. McConville, J.F., C.H. Anger, and D.W.
6. Silliker, J.H. et al., editors. 1980. “Inter- Anderson. 1974. “Method for Performing
national Commission on Microbiological Aerobic Plate Counts of Anhydrous Cos-
Specifications for Food.” Microbial Ecol- metics Utilizing Tween 60 and Arlacel 80
ogy of Food. Orlando, FL: Academic Press, as Dispersing Agents.” Applied Microbiol-
76-91. ogy, 27, No.1: 5-7.
SECTION 17
Determination
of Preservation
Efficacy in
Nonwoven
Substrate Personal
Care Products
INTRODUCTION
Nonwoven substrate personal care products, commonly called wipes, constitute a wide and
expanding variety of items that differ significantly from other types of personal care products in

their composition, intended use, and physical characteristics.1 The nonwoven matrix or substrate
PRESERVATION EFFICACY
is composed of fibers or filaments that are bonded together mechanically, thermally, or chemically
and is used for the delivery of cosmetics or other product systems.
In view of the differences between wipes and other types of personal care products, the standard
preservation efficacy tests for aqueous-based (See “M-3 A Method for Preservation Testing
of Water-Miscible Personal Care Products” in Section 20) or atypical (“M-6 A Method for
Preservation Testing of Atypical Personal Care Products” in Section 23) personal care products
are not suitable for testing these product forms. The two major test method differences have to do
with the procedure for inoculum introduction and the procedure for the recovery of introduced
microorganisms. It is recommended that, when developing preservation efficacy methods and
testing protocols, the cosmetic microbiologist be aware of the factors listed below under “General
Considerations” and how they may affect the reliability of the test method under development.
This document is intended to be used in conjunction with “M-5 Methods for Preservation
Testing of Nonwoven Substrate Personal Care Products” in Section 22).
Training
Those who develop methods and test protocols for wipes are expected to have training and
experience in conducting and verifying the procedures (See “Microbial Validation and
Documentation” in Section 9) in evaluating the data, and interpreting the results obtained.
Standard laboratory safety procedures for microbiological testing should be followed.
SECTION 17: DETERMINATION OF PRESERVATION EFFICACY | CTFA MICROBIOLOGY GUIDELINES | 173

Components
A nonwoven personal care product is composed of the following components:
• Substrate - nonwoven carrier including coatings or finishes applied to that
carrier
• Add-ons - personal care formulation applied to a substrate; liquids and lotions
are the most common
• Packaging - final container for delivering the finished product
Any change to the composition or nature of any of these components may affect the overall
preservation efficacy of the final product and may require retesting.
Substrate
The nature and composition of substrates can have a decided effect upon preservative-substrate
interaction as well as subsequent preservative system performance. A substrate2,3 is a nonwoven
web of long and short fibers held together by some means of bonding other than weaving.
Fibers can be natural or synthetic. The substrate functions as the carrier for a variety of product-
specific add-ons. Generally, substrates are composed of any one or a combination of various fiber
types including cellulose or wood pulp, rayon or viscose, polyester and polypropylene polymer
extrusions or bicomponent materials. Bonding technologies include mechanical entanglement,
chemical or adhesive binding, thermal melting and hydrogen bonding.

Binders can significantly affect the preservative system.4,5 For example, anionic binders,
commonly used in some substrates, have the potential to inactivate or seriously disrupt most
cationic preservative systems. Alternatively, binders may contain preservatives that may result in
a more robust product.
Other substrate issues that can affect preservation efficacy may include the fiber type and
composition, the web forming process, the web bonding process, the proportion of pulp to
binder, the composition and ionic nature of the chemical binding agent, and the presence and
nature of substrate finishes or coatings. Depending on the nature and degree of reactivity of fiber
surfaces, preservatives may become chemically or physically bound, reducing their antimicrobial
activity. This may also be the case with certain finishes, coatings and other substrate surface
treatments that can react with preservatives.
Add-Ons
An add-on is the formulation applied to a nonwoven substrate. The add-on can be of varied
composition and may be in the form of a liquid, lotion, emulsion, powder, cream, ointment,
oil or other material. Although preservation efficacy demonstrated for the add-on may provide
useful information, it may not be predictive of the preservation efficacy of the final nonwoven
substrate product. Substrate and packaging may also influence preservation efficacy.
Packaging
The packaging size and type, e.g. tubs, canisters, soft packages, single pack, etc., should be taken
into consideration in developing the most appropriate protocol for the preservation efficacy
174 | CTFA MICROBIOLOGY GUIDELINES | SECTION 17: DETERMINATION OF PRESERVATION EFFICACY

testing of the final product. How the product changes over time may be dependent on the type
of package chosen, e.g. evaporation of the add-on through the package or adsorption of the add-
on to the package can occur, potentially affecting preservative stability.
Intended Use and Delivery of Product

Intended use and delivery of the product may influence the test procedure. The type of packaging,
e.g. open tub or pack, may influence the number of inoculations in the test method. The length
of the test should be representative of packaging design, with consideration given to how long
the consumer may use the product after it has been opened; e.g., a single use package versus one
containing multiple substrates. The selection of challenge organisms ideally reflects the final use
of the product. For example, challenge organisms for baby wipes (coliforms) may differ from
those for an eye area product (Pseudomonads - See Section 14).
End use and delivery of the product may determine acceptance criteria. For example, there may
be different acceptance criteria for single pack versus multi pack products.
Preservative Stability
It is recommended that preservative stability be evaluated in the finished product packaging
because of possible interactions of preservative, add-on, substrate, and package. The stability

of the preservative system in the add-on does not necessarily reflect its stability in the finished
product. It is recommended that accelerated aging studies on finished wipe products be confirmed
with real time studies. Additional information on stability testing of personal care products is
available from COLIPA6 and from the IFSCC7.
PRODUCT TESTING
Preliminary Considerations
It is recommended that the microbial content of the substrate, the add-on, and the finished
product be determined prior to starting the preservation efficacy test. Due to the unique nature
of non-woven products, molds are organisms of particular concern due to their ability to degrade
cellulose fibers and the exposure of the large surface area of the wipe to the environment during
manufacture and use. A high microbial load may reduce preservative activity or cause preservative
failure in the final product.
A sedimentation study8 to determine fluid migration through a vertical stack of wipes may be
useful information for some test protocols, for instance if the inoculum is delivered by filter
carrier. This data is useful in determining the distribution, or degree of sedimentation, of the
add-on within packages of saturated wipe products packaged in stacks.
A period of time for equilibration of the add-on and the substrate before testing is recommended.
This allows time for total saturation of the add-on and distribution of the preservative.
It is recommended that all aspects of product testing, such as organism recovery, neutralization,
inoculation, etc, be verified for method suitability. (See Section 9: “Microbial Validation and
Documentation”).

Organisms
Organism strains recognized by United States Pharmacopeia (USP)9 and/or used in the industry
for preservation efficacy testing are recommended. Additional reference strains and/or organisms
appropriate to the product, including spores, may be used where deemed necessary.
Pure or mixed culture inocula may be used. However, if different microorganisms are pooled,
antagonism among microbes may occur or it may be difficult to differentiate between types of
survivors.
Some products may not require a full preservation efficacy test. For example, dry wipes, as
defined by water activity measurement, may require limited or alternative testing. (See Section
16: “Microbiological Risk Factor Assessment of Atypical Cosmetic Products”).
Inoculation Procedures
The choice of inoculation site, inoculum volume, and distribution of the inoculum onto the
product should be determined with the anticipated consumer use and packaging of the product
in mind. The procedure used should be representative of product use.
There are several aspects to consider when choosing an inoculation procedure:
Packaging
Although testing in the final product packaging is preferred, there are situations where it is
impractical or impossible to do so. In these cases, testing outside of the final package is an
acceptable alternative. If the same product is delivered in different packaging, i.e. tubs, canisters,
soft packages, etc., it is recommended that each package type be tested.
Inoculum Volume
It is recommended that a consistent inoculum volume be chosen to achieve a set organism level.
This volume is dependent on the method of inoculation (Section 17). Keeping the inoculum
volume to a minimum will avoid dilution of the add-on.
Inoculation Site / Distribution

The nonwoven substrate can be inoculated using a variety of methods. It is important to verify
that the inoculum site, distribution, and inoculum recovery is appropriate to the final packaging
and use.
Reinoculation
Reinoculation of the nonwovens during the preservation efficacy test is determined based on
how the product is used by the consumer and whether the nonwovens are tested in or out of the
final package. If a reinoculation is performed, ensure that there are adequate numbers of wipes to
complete assays for the required test period.

Recovery Procedures
Neutralization Verification
It is recommended that neutralization studies be conducted with all challenge organisms used in
the test. See ASTM 1054 for details10.
Recovery Verification
The nonwoven substrate material is likely to entrap some microorganisms, resulting in a less
than complete recovery of the inoculum. The level of recovery may change from product to
product, depending on the combination of substrate and add-on. In most cases, mechanical or
other action (Section 17) is necessary to release microorganisms from the substrate. Addition
of a surfactant and/or multiple extractions from the same sample may be necessary to optimize
recovery. It is recommended that the interpretation of results take into account the established
recovery efficiency which is based on a time zero count.
RECOMMENDATIONS
It is recommended that a risk assessment be used to establish acceptance criteria for a specific

product (Section 16). The risk assessment should reflect the final product and its intended use
and may take into consideration many factors including, but not limited to, the following:
• Test method and microorganisms
• Nature of the add-on
• Nature of the substrate
• Degree of saturation
• Degree of microbial recovery from the substrate
• Design and size of the packaging
• Intended use and target consumer
• Performance and history of similar products
Whatever the product, an effective preservative system for a personal care wipe should prevent
proliferation of introduced microorganisms, even after repeated microbial insult.

REFERENCES
1. Lochhead, Robert Y. 2006. “Emerging 7. International Federation of Societies of
Technologies for Cosmetic and Personal Cosmetic Chemists. 1992. “Fundamen-
Care Wipes.” Cosmetics and Toiletries 121: tals of Stability Testing.” IFSCC Mono-
47-52. graph Number 2. Cranford, NJ: Micelle
Press.
2. International Nonwovens & Disposables
Association (INDA) website. http://www. 8. Cremieux, A., S. Cupferman, and C. Lens.
inda.org. 2005. “Method for the Evaluation of the
Efficacy on Antimicrobial Preservatives in
3. European Disposables and Nonwovens Cosmetic Wet Wipes.” International Jour-
Association (EDNA) website. http:// nal of Cosmetic Science 27: 223-236.
www.edana.org.
9. United States Pharmacopeia. 2007. <51>
4. Sutton, S. 1996. “Neutralizer Evaluations “Antimicrobial Effectiveness Testing.”
as Control Experiments for Antimicrobial United States Pharmacopeia and the Na-
Efficacy Tests.” In: Handbook of Disinfec- tional Formulary. USP30 – NF25. Rock-
tants and Antiseptics. Edited by J. M. As- ville, MD. 79-81.
cenzi. 43-62. Marcel Dekker, Inc.
10. ASTM International. 2003. “ASTM 1054
5. McCarthy, Terrence J. 1984. “Formulat- E 1054 - Standard Practices for Evaluat-
ed Factors Affecting Activity of Preserva- ing Inactivators of Antimicrobial Agents
tives.” In: Cosmetic and Drug Preservation Used in Disinfectant, Sanitizer, Antisep-
Principles and Practice. Edited by Jon J. tic, or Preserved Products.” In: Annual
Kabara. 359-388. Marcel Dekker, Inc. Book of ASTM Standards 11.05. West
6. The European Cosmetic, Toiletry, and Conshohocken, PA.

Perfumery Association (COLIPA) and
CTFA. 2004. Guidelines on Stability Test-
ing of Cosmetic Products. http://www.co-
lipa.com.

SECTION 18
M-1
Determination
of the Microbial
Content of
Cosmetic Products
1. Scope
1.1 This is an acceptable plate count procedure for determining the microbial content of
cosmetic products.
2. Applicable Documents
2.1 “M-2 Examination for Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa”
(Section 19).
3. Suggested Materials
3.1 Media for the enumeration of bacteria or fungi
3.1.1 Media for the enumeration of bacteria or fungi*
Letheen Agar (BBL, Difco)
Microbial Content Agar (Difco)
DETERMINATION OF MICROBIAL CONTENT

Nutrient Agar (BBL, Difco, Oxoid)
Standards Methods Agar with Lecithin and Polysorbate 80 (BBL)
SECTION 18: M-1

Trypticase Soy Agar (BBL)
Trypticase Soy Agar with Lecithin and Polysorbate 80 (BBL)
Tryptic Soy Agar (Difco)
Tryptone Soya Agar (Oxoid) or equivalent
*It must be demonstrated that the test method adequately inactivates the microbial growth
inhibitors present in the product. It is recommended that a neutralizer be present in the diluent
or agar or both. 1
SECTION 18: M-1 DETERMINATION OF MICROBIAL CONTENT | CTFA MICROBIOLOGY GUIDELINES | 179
3.1.2 Media for the enumeration of fungi*
Mycophil Agar with low pH (BBL)
Potato Dextrose Agar (BBL, Difco, Oxoid)
Sabouraud Dextrose Agar (BBL, Difco, Oxoid) or equivalent
3.1.3 Media used as diluents.*
Acto Tryptone (1%) (Difco).
Letheen Broth (BBL, Difco).
Nutrient Broth (BBL, Difco, Oxoid).
Trypticase Azolectin Tween Broth Base (BBL).
D/E Neutralizing Media (Difco) or equivalent.
3.1.3.1 Prepare dilution bottles containing 80 mL of diluent for water-
immiscible products and 90 mL for water-miscible products.
3.1.3.2 Sterile wide-mouth dilution bottles containing 10 mL of Polysorbate
80.
3.2 Equipment
3.2.1 Autoclave
3.2.2 Sterile Petri dishes, 15 × 100 mm
3.2.3 20 mL sterile syringes, 10 mL pipettes, 1.0 mL pipettes, spatulas and other
sampling devices
3.2.4 Water bath capable of maintaining a temperature range of 45°-50°C
3.2.5 Microbiological incubators at 20°-25°C and 30°-34°C
3.2.6 Colony counter
3.2.7 Compound light microscope with 1000X oil immersion lens
SPECIFICATIONS
3.2.8 Stereo microscope

ANNEX 18
4. Procedure for Aerobic Plate Count

4.1 Use sterile materials, equipment and aseptic techniques.
4.2 For water-miscible products, transfer by means of a syringe, pipette or spatula 10mL or
g of the well-mixed product to a dilution bottle containing 90 mL of diluent (this is a
1:10 dilution).
*It must be demonstrated that the test method adequately inactivates the microbial growth
inhibitors present in the product. It is recommended that a neutralizer be present in the diluent
or agar or both. 1
180 | CTFA MICROBIOLOGY GUIDELINES | SECTION 18: M-1 DETERMINATION OF MICROBIAL CONTENT
4.3 For water-immiscible products, transfer by means of a syringe, pipette or spatula 10 mL
or g of the well-mixed product to a dilution bottle containing 10 mL of Polysorbate 80.
Disperse the product within the Polysorbate 80 with a spatula. Volume to 100 mL with
diluent (this is a 1:10 dilution).
4.4 The precise volume or weight of sample and diluent may be varied. A dilution ratio of
1:10 with a minimum sample size of 10 mL or g is recommended.
4.5 When the same agar is used for bacterial and fungal assays, dispense 1 mL of the dilution
into each of three Petri dishes and 0.1 mL into three additional Petri dishes (to give
triplicate plates of 1:10 and 1:100 dilutions). Add 15 to 10 mL melted agar medium
kept at 44°-48°C and rotate plates sufficiently to disperse the product. Allow the agar to
solidify and invert plates. Incubate one plate of each dilution as follows:
a) At 30°-35°C for a minimum of 48 hours for the bacterial assay
b) At 20°-25°C for a minimum of 5 days for the fungal assay
c) In a refrigerator to prevent growth. Or: Dispense 1 mL of the dilution into two
Petri dishes and 0.1 mL into two additional Petri dishes (to give duplicate plates
of 1:10 and 1:100 dilutions). Add melted agar medium (as above) and incubate
one plate of each dilution as follows:
(1) At 30°-35°C for a minimum of 48 hours followed by a minimum of 48 hours
at 20°-25°C.
(2) In a refrigerator to prevent growth.
4.6 When separate agars are used for bacterial and fungal assays, dispense 1 mL of the
dilution into each of four Petri dishes and 0.1 mL into four additional Petri dishes (to
give quadruplicate plates of 1:10 and 1:100 dilutions). Add 15-20 mL of agar medium
for bacterial assay kept at 44°-48°C to two plates of each dilution. Add 15-20 mL of
agar medium for fungal assay kept at 44°-48°C to two plates of each dilution. Rotate
all plates sufficiently to disperse the product. Allow the agar to solidify and invert the
plates. Incubate one plate of each dilution as follows:

a) Bacterial assay medium at 30°-35°C for a minimum of 48 hours
b) Fungal assay medium at 20°-25°C for a minimum of 5 days
SECTION 18: M-1

c) Remaining bacterial and fungal medium plates in a refrigerator to prevent growth
4.7 Include a laboratory control using apparatus, dilution blank (without product), media
and appropriate incubation. Concurrent contamination on test and control plates
invalidates the test. Find and eliminate the source of contamination. Repeat both control
and product tests.
4.8 Count the colonies. If there is difficulty in distinguishing colonies from material,
compare to the refrigerated plates, or examine the colonies under a stereo microscope.
(With experience, the refrigerated plates can be eliminated from the procedure.)
4.9 The number of colony forming units (CFU) per mL or g is the colony count multiplied
by the appropriate dilution factor (10 or 100).
SECTION 18: M-1 DETERMINATION OF MICROBIAL CONTENT | CTFA MICROBIOLOGY GUIDELINES | 181
4.10 Neutralization of preservatives should be validated for each product tested. This may be
accomplished by streaking a 10–4 dilution of an appropriate organism onto the surface
of test plates at the end of the incubation period. Failure of the inoculum to grow
invalidates the test. Repeat the test using greater dilution of the test material.
4.11 Morphologically suspect colonies can be further identified by the methods described
in “M-2 Examination for Staphylococcus aureus, Escherichia coli and Pseudomonas
aeruginosa.”1
REFERENCES
1. 1999. “ASTM E 1054-91 – Standard Practices for Evaluating Inactivators of Antimicrobial
Agents Used in Disinfectant, Sanitizer, Antiseptic, or Preserved Products.” Annual Book of
ASTM Standards 11.05.
SECTION 18: M-1
182 | CTFA MICROBIOLOGY GUIDELINES | SECTION 18: M-1 DETERMINATION OF MICROBIAL CONTENT
SECTION 19
M-2
Examination for
Staphylococcus
aureus, Escherichia
coli and
Pseudomonas
aeruginosa
1. Scope
1.1 This is an acceptable procedure for determining whether or not bacteria isolated from
cosmetics are Staphylococcus aureus, Escherichia coli or Pseudomonas aeruginosa.
2.1 “M-1 Determination of the Microbial Content of Cosmetic Products” (Section 18).
2.2 “Microbial Limit Tests” <61>. The United States Pharmacopeia, 30th edition (2007)1
3. Suggested Materials
3.1 Media
3.1.1 Eosin-Methylene Blue Agar plates (EMB)
3.1.2 EC Broth
3.1.3 Pseudomonas Isolation Agar
3.1.4 Motility Agar
3.1.5 Trypticase Soy Broth/TSB (BBL), Tryptic Soy Broth (Difco), Tryptone Soya
Broth (Oxoid) or equivalent
3.1.6 Pseudomonas F Agar
EXAMINATION FOR S.a., E.c., AND P.a.
3.1.7 Pseudomonas P Agar

SECTION 19: M-2
3.1.8 MacConkey Agar

3.1.9 Vogel-Johnson Agar Plates (V-J)
SECTION 19: M-2 EXAMINATION FOR S.a, E.c. AND P.a. | CTFA MICROBIOLOGY GUIDELINES | 183
3.2 Reagents
3.2.1 Cytochrome Oxidase Test - Use filter paper impregnated with 1% tetraethyl
phenylenediaminedihydrochloride
3.2.2 Materials for Coagulase Test - Mammalian plasma, preferably rabbit or horse,
with or without suitable additives. (see USP 301)
3.3 Equipment
3.3.1 Water baths at 37°C, 42°C and 45.5°C
3.3.2 Gram-Stain materials
3.3.3 Ultraviolet light
3.3.4 Fermentation tubes
3.3.5 Compound light microscope with 1000X, oil immersion lens
3.3.6 Microbiological incubator: 35°-37°C
4. Procedure
4.1 The following procedures should be performed only with isolated colonies.
4.2 Gram Stain
4.2.1 If colonies are observed with 24 hours of testing, Gram Stains of representative
colonies should be performed. Colonies appearing after 24 hours should be
streaked onto plates of the same medium, incubated 18-24 hours and Gram
Stained.
4.3 Test for Staphylococcus aureus
4.3.1 Gram-positive cocci appearing in clusters should be streaked onto V-J Agar
Plates and incubated at 35° ± 2°C for 24 hours.
After 24 hours, transfer characteristic growth (see Table 19-1) from the
surface of V-J medium to individual tubes each containing 0.5 mL of a
mammalian plasma. Simultaneously assay coagulase positive and negative
cultures. Incubate in a water bath at 37°C, examining the tubes at 3 hours
and at subsequent intervals up to 24 hours. If no coagulation in any degree
is observed, the colonies are not S. aureus. If the reactions of the controls are
not correct, the assay results are invalid.
If the test is positive and a quantitative level is desired, count the type colonies
on the primary isolation medium corresponding to the positive colony
selected from that medium and planted on V-J agar. Express as CFU of S.
aureus colonies per mL or g of product.

4.4 Test for Pseudomonas aeruginosa
SECTION 19: M-2
4.4.1 Gram-negative rods should be tested for cytochrome oxidase activity. If oxidase
positive, streak colonies onto Pseudomonas Isolation Agar, Pseudomonas
184 | CTFA MICROBIOLOGY GUIDELINES | SECTION 19: M-2 EXAMINATION FOR S.a, E.c. AND P.a.
Agar F for detection of fluorescein, and Pseudomonas Agar P for detection
of pyocyanin. Incubate at 35° ± 2°C for not less than 3 days. Examine the
streaked surfaces under ultraviolet light to determine whether colonies having
the characteristics of pseudomonads are present (see Table 19-1).
Perform either a microscopic motility test or stab motility agar with growth
from selective Pseudomonas agar and observe for motility after incubating
the stab culture for 24 hours.
Transfer oxidase and motility positive colonies to TSB and incubate at
42°C for 24-48 hours. Growth at 42°C indicates P. aeruginosa. Lack of
characteristic pigmentation of colonies and failure to grow at 42°C indicates
other pseudomonads. If the test is positive, count the type colonies on the
primary isolation medium corresponding to the P. aeruginosa colony selected
from that medium and planted on selective Pseudomonas Isolation Agar.
Express as CFU of P. aeruginosa colonies per mL or g of product.
4.5 Test for Escherichia coli
4.5.1 Oxidase-negative, Gram-negative rods should be tested to determine whether
or not they are E. coli. Streak growth onto EMB and MacConkey Agar Plates
and incubate at 35° ± 2°C for 24 hours.
Transfer characteristic growth (see Tables 19-1 and 19-2) from MacConkey
and/or EMB agars to EC Broth containing fermentation tubes. Incubate at
45.5°C in a water bath for 24-48 hours. Production of gas is characteristic of
E. coli.
If the test is positive, count the type colonies on the primary isolation medium
corresponding to the positive colony selected from that medium and planted
on MacConkey and EMB agars. Express as the number of E. coli colonies per
mL or g of product.
4.6 Positive Controls
4.6.1 With each test for S. aureus, E. coli or P. aeruginosa a test should be conducted
simultaneously with known cultures as positive controls.
4.6.1.1 Staphylococcus aureus
Check each negative V-J plate after incubation by streaking with
10-4 dilution of S. aureus from a 24-hour broth culture. Failure to
grow invalidates the test.
4.6.1.2 Pseudomonas aeruginosa
Check each negative Pseudomonas Isolation Aagar plate,
Pseudomonas F Plate, Pseudomonas P Plate and TSB tube, after
incubation with a streak or inoculum of a 10-4 dilution of P.
aeruginosa from a 24-hour broth culture. A positive check requires

that P. aeruginosa grow in a specific medium and produce pigment
SECTION 19: M-2
as specified in Tables 19-1 and 19-2. Failure to do so invalidates

that portion of the examination.
4.6.1.3 Escherichia coli
Check each negative MacConkey and EMB plate and each negative
EC broth tube after incubation with a streak of inoculum of a 10-4
dilution of E. coli from a 24-hour broth culture. Failure of E. coli
to grow in a specific medium as specified in Tables 19-1 and 19-2
invalidates that portion of the examination.
4.7 Alternative Methods
4.7.1 The identification of S. aureus, P. aeruginosa or E. coli may be confirmed
by further suitable cultural and biochemical tests or by the use of rapid
identification kits.
Table 19-1: Identification of Staphylococcus aureus, Pseudomonas aeruginosa,

Escherichia coli: Presumptive Tests
PRESUMPTIVE TEST
S. aureus E. coli P. aeruginosa
Vogel-Johnson Agar Black, surrounded ND ND

by yellow zone
MacConkey Agar ND Brick red may have ND

surrounding zone of
precipitated bile
Pseudomonas Isolation Agar ND ND Generally greenish
Pseudomonas Agar F ND ND Colorless to yellow

In Normal Light
Pseudomonas Agar F ND ND Yellowish

In Ultraviolet Light
Pseudomonas Agar P ND ND Greenish

In Normal Light
Pseudomonas Agar P ND ND Blue

In Ultraviolet Light
Gram Stain Positive clusters Negative rods Negative rods

of cocci (cocco-bacilli) (slender)
ND = Not done
Table 19-1
SECTION 19: M-2
Table 19-2: Identification of Staphylococcus aureus, Pseudomonas aeruginosa,
Escherichia coli: Completed Test
COMPLETED TEST
S. aureus E. coli P. aeruginosa
Coagulase Test Positive ND ND
Cytochrome Oxidase Test ND Negative Positive
E.M.B. Agar ND Metallic sheen under Purple

reflected light and a
blue-black appearance
under transmitted light
Fermentation E.C. Broth at 45.5°C ND Positive ND
Growth at 42°C, TSB ND Growth Growth
ND = Not done
Table 19-2
REFERENCES
1. United States Pharmacopeia. 2007. <61> “Microbial Limit Tests.” United States Pharmacopeia
and the National Formulary. USP30 - NF25. Rockville, MD. 83-88.

SECTION 19: M-2
SECTION 19: M-2
WATER-MISCIBLE PERSONAL CARE
SECTION 20 M-3: TESTING
SECTION 20
M-3
A Method for
Preservation
Testing of Water-
Miscible Personal
Care Products
1. Scope
1.1 This is an acceptable procedure for determining the preservative efficacy of water-
miscible cosmetic and toiletry formulations1-4.
1.2 Aseptic techniques and sterile materials must be employed.
2.1 “Determination of Preservative Adequacy in Cosmetic Formulations” (Section 13).
3. Materials
3.1 Selection of Challenge Microorganisms
The microbial strains listed in Table 20-1 may be considered for use in developing
preservation data of Personal Care products.
Either pure or mixed microbial culture suspensions may be used to challenge test
formulations. Inocula consisting of only pure microbial cultures will yield specific data
on each test microorganism employed in the challenge study. When conducting mixed
culture challenge studies, it is recommended that closely related types of microorganisms
such as Gram-positive bacteria, Gram-negative bacteria, and yeasts and molds be pooled
separately.
3.2 Maintenance of Challenge Microorganisms
Refer to the ATCC culture maintenance recommendations, available on their website5,
and to other sources6-8.
Storage of other organisms relevant to the product in the original product or
incorporation of product into maintenance medium is often the only way to retain
its unique characteristics. This method is especially appropriate where the isolate is
subsequently inoculated into a similar material.
SECTION 20: M-3 TESTING WATER-MISCIBLE PERSONAL CARE | CTFA MICROBIOLOGY GUIDELINES | 189
3.3 Test Media

3.3.1 Inocula Suspending Fluids
Suspending fluids are used to prepare the bacterial and fungal suspensions for
inoculating the test product. The following may be used:
• Phosphate Buffer (pH 7.0)
• 0.85% Sodium Chloride Solution (Normal Saline)
• Sodium Chloride Peptone Solution (1% peptone in normal saline)
Other suitable fluids may be used. The addition of 0.05% – 0.1% polysorbate
80 or other surfactant to the suspending fluid is recommended to aid in
dispersion of mold spores.
3.3.2 Microbial Plate Count Diluents
Plating diluents serve to disperse the sample and dilute it to levels that permit
recovery of surviving microorganisms from an inoculated product formulation.
The choice of diluent depends on its ability to meet the requirements of preservative
neutralization (Section 4.1). The following are examples of diluents that may be
used:
• Buffered Sodium Chloride Peptone Solution
• Dey/Engley (D-E) Neutralizing Broth
• Eugon Broth
• Letheen Broth
• Modified Letheen Broth
• Phosphate Buffer, pH 7
• Soybean Casein Digest Medium (Tryptic Soy Broth)
• Trypticase Azolectin™ Tween™ (TAT) Broth
• Saline-Tween-Lecithin Diluent
Addition of neutralizers may be necessary to demonstrate adequate preservative
neutralization. If neutralizing systems other than those listed above are used,
absence of neutralizer toxicity should be verified.
3.3.3 Recovery Agars
Many factors affect organism viability. Therefore, it is important for the agar to
provide optimum nutritional support for the recovery of the challenge organisms.
The following have been found suitable for preservation studies:
• Eugon Agar
• Letheen Agar
• Microbial Content Agar
190 | CTFA MICROBIOLOGY GUIDELINES | SECTION 20 M-3 TESTING WATER-MISCIBLE PERSONAL CARE
• Modified Letheen Agar
• Plate Count Agar
• Soybean Casein Digest Agar (Tryptic soy agar)
• Microbial Content Agar with Tween
neutralization. Other suitable agars and neutralizing agents may be used.
If the above agars do not support the growth of fungi, one of the following agars
may be considered:
• Malt Agar
• Malt Extract Agar
• Mycological Agar
• Potato Dextrose Agar
• Sabouraud Dextrose Agar
Other suitable agars and neutralizing agents may be used.
4. Preliminary Tests
4.1 Preservative Neutralization
Carryover of antimicrobial activity from the product formulation into the plate count
diluent and recovery growth agar may occur. This may inhibit the growth of surviving
challenge test microorganisms, resulting in a false negative microbial count. To avoid a
false negative result, neutralization of the antimicrobial properties of the formulation
must take place in the plate count diluent and/or the recovery growth agar.
Antimicrobial neutralization may normally be accomplished by use of chemical
neutralizing agents, physical dilution, or a combination of both.
Verification of neutralization is generally performed by inoculating the product dilution
with a low level of challenge microorganisms and performing the enumeration method
Side-by-side dilutions with and without a product formulation are made. Enumeration
of the microorganisms from these dilutions is performed. Neutralization is verified if
microbial recoveries are similar. If one or more challenge microorganisms cannot be
recovered, the use of a higher dilution and/or the investigation of additional chemical
neutralizers may be considered9,10.
4.2 Microbial Content Test
It is recommended that that a microbial content test11, 13 be performed on the test sample
prior to performing the preservative efficacy test. Verification of neutralization of the
antimicrobial properties of the test sample should be demonstrated (See Section 4.1
above and References 9 and 10) for the microbial content test.
5. Inoculation Procedures
5.1 Preparation of Inocula
Freshly prepared cultures should be used for inoculating test samples.
In general, culture conditions in Table 20-2 should be considered when preparing the
inocula. Refer to the ATCC website5 for optimal growth media and conditions for
specific microorganisms. Inclusion of cellulose degrading molds may necessitate longer
incubation periods and require a paper source for growth.
5.1.1 Preparation of Initial Bacteria and Yeast Suspensions
Broth cultures or cultures grown on solid agar media are acceptable for use. For
reference strains such as the ATCC strains, no more than five transfers from
the stock culture are recommended12. Broth cultures should be centrifuged
and then re-suspended in the chosen suspending fluid. (See 3.3.1) Microbial
growth on a solid medium is transferred to the chosen suspending fluid.
5.1.2 Preparation of Initial Mold Suspensions
The mold inoculum is prepared by washing the sporulating agar culture with
the chosen suspending fluid (See 3.3.1) and filtering the spore suspension
through sterile gauze or glass wool. Sterile glass beads can be used as an aid in
the dispersion of spores in the suspending fluid.
5.1.3 Preparation of Bacterial Spore Suspensions
If spore-forming bacteria are to be included in the test, the inocula may
be prepared as indicated in the AOAC Sporicidal Test3. Some strains are
commercially available as prepared spore suspensions.
5.1.4 Preparation of Challenge Inocula
5.1.4.1 Inoculum levels
The recommended inoculation levels for challenge testing are:
• 1×106 Colony-Forming Units (CFU) of bacteria pergram of product
• 1×105 CFU of yeast per gram of product
• 1×105 CFU of mold spores per gram of product
The inoculum level for the challenge microorganisms should be verified
by standard microbiological techniques such as pour plate methods.
5.2 Product Challenge
5.2.1 Pure and mixed-culture challenge
Either pure or mixed cultures may be used to challenge test formulations. Pure
culture challenge, although more time-consuming, will yield specific data on
each microorganism employed in the study. Mixed-culture challenge, on the
other hand, can be used to obtain rapid pass-or-fail decisions on preservative
adequacy and reduce the workload. However, antagonism among organisms
may occur. It is recommended that broadly related types of microorganisms
such as Gram-positive bacteria, Gram-negative bacteria, or molds be pooled
separately when conducting mixed-culture challenge.
All products should be thoroughly mixed manually or mechanically after
inoculation to distribute the challenge microorganisms uniformly. It is
recommended that the volume of the inoculum be < 1% of the sample
weight and should not alter the character of the product being challenged.
Challenged formulations should then be stored at ambient temperature for
the duration of the test.
5.3 Sampling the Challenged Product
5.3.1 Sampling interval
Challenged formulations should be sampled for viable microorganisms at
selected time intervals after inoculations. The frequency of sampling should
follow a set pattern to facilitate future comparison of test results between
different product formulations or samples, for example, weekly up to 28 days
after inoculation.
5.3.2 Sampling and plating methods
The inoculated product should be thoroughly mixed just prior to sampling to
ensure that the sample is representative. In some cases, the inoculum can thrive
in “pockets” of growth in the formulation while other areas are relatively free
of microorganisms. Many aerobic microorganisms grow especially well at the
formulation-air interface. Often it is very difficult to break up the “pockets”
of growth, and special procedures are needed. The following mixing methods
have been used to overcome this problem:
• Vigorous mixing with a stirring rod
• Capping and shaking vigorously by hand
• Mixing in a vortex mixer
• Mixing with a magnetic stirrer
• Mixing with a propeller stirrer
• Mixing with a non-aerating stirrer
• Mixing in a micro blender
• Mixing in a stomacher
• Gentle mixing in a tissue grinder
Sample size will in part determine the minimum detectable level. A sample
sizeofatleastonegramoronemilliliterofproductforthequantitativepourplatemethodis
recommended. Aseptic techniques must be employed.
5.3.2.1 Quantitative pour plate method

Serial dilutions are prepared from the aliquot recovered from the
challenged sample unit. Each serial dilution is thoroughly mixed,
and an aliquot is transferred to a Petri dish. Melted agar maintained
at 44-48°C is added to the Petri dish, and the dish is rotated to
uniformly disperse the product dilution. The agar plates are
allowed to solidify, then inverted and incubated under conditions
appropriate for the test microorganisms (see Table 20-2).
After incubation, the number of microbial colonies is counted, and
the resulting figure is multiplied by the appropriate dilution factor
to obtain the number of microorganisms per sample unit.
5.3.2.2 Quantitative spread plate method
The quantitative spread plate method is performed in a manner
similar to the pour plate method; however, an aliquot of each
dilution is transferred directly onto the surface of solidified
microbial growth agar. The sample aliquot is then evenly spread
over the agar surface. The agar plates are allowed to dry, then
inverted and incubated under conditions appropriate for the test
microorganisms (see Table 20-2).
to obtain the number of microorganisms per sample unit.
6. Other Considerations
6.1 Length of Test Procedure
It is recommended that preservation tests be carried out for a minimum of 28 days. In
some cases, numbers of challenge microorganisms may be reduced below detectable
levels during the early stages of the test only to adapt to the preservative system and later
proliferate. A final judgment of preservative adequacy should not be made until all the
data are obtained.
6.2 Rechallenge
Consideration may be given to rechallenge. A rechallenge is useful for determining if a
formulation is marginally preserved and identifying which types of microorganisms may
be potential problems for that particular formulation.
6.3 Storage Stability
sIt is important that challenge tests also be conducted on samples aged in the final
container in order to determine the stability of the preservative system.
Table 20-1: Suggested Challenge Microorganisms
Type Microorganism (ATCC5 Number) Recommendation

Gram-Positive Cocci Staphylococcus aureus (6538)*
Staphylococcus epidermidis (12228) Select at least one
Fermentative Klebsiella pneumoniae (10031) Select at least one

Gram-Negative Bacilli Enterobacter cloacae (13047))
Escherichia coli (8739)*
Enterobacter gergoviae (33028)
Non-Fermentative Pseudomonas aeruginosa (9027)* Select at least one

Gram-Negative Bacilli Burkholderia cepacia (25416)
Pseudomonas fluorescens (13525)
Pseudomonas putida (31483)
Yeasts Candida albicans (10231)* Recommended
Molds Aspergillus niger (16404)* Select at least one

Penicillium species
Spore-Forming Bacilli Bacillus subtilis (6051) Optional
Other Other organisms relevant to product Optional
*Staphylococcus aureus (6538), Escherichia coli (8739), Pseudomonas aeruginosa (9027), Candida albicans (10231)
and Aspergillus niger (16404) are specified in the United States Pharmacopeia (USP) Antimicrobial Effectiveness
Testing method4.
Table 20-1
Table 20-2: Culture Conditions for Preparation of Inocula
Cultures Media** Temperature Time
Bacteria Soybean Casein Digest (Tryptic Soy) Broth/Agar 30-37°C 18-48 hours
Nutrient Broth/Agar
Eugon Agar/Broth
Yeasts Sabouraud Dextrose Agar 25-35°C 24-48 hours

Soybean Casein Digest (Tryptic Soy) Broth/Agar
Mycophil (Mycological) Broth/Agar
Molds Sabouraud Dextrose Agar 20-30°C 7-28 days

Potato Dextrose Agar
Mycophil (Mycological) Agar
Malt Extract Agar
** Available in dehydrated forms from commercial sources.
Table 20-2
REFERENCES
1. AOAC INTERNATIONAL. 2000. “Of- 7. Kirsop, B.E. and A. Doyle, (Ed). 1991.
ficial Method 998.10 - Efficacy of Pres- Maintenance of Microorganisms and Cul-
ervation of Non-Eye Area Water-Miscible tured Cells, Second Edition. New York,
Cosmetic and Toiletry Formulations.” In: NY: Academic Press.
Official Methods of Analysis of AOAC IN-
TERNATIONAL. Gaithersburg, MD. 8. Simione, F. P. 1998. “Cryopreservation
Manual.” Nalgene Nunc International,
2. American Society for Testing and Materi- http://www.nalgenelabware.com/techda-
als. 2001. “ASTM E 640-78 - Standard ta/technical/manual.asp
Test Method for Preservatives in Water-
Containing Cosmetics.” In: American So- 9. United States Pharmacopeia. 2007.
ciety for Testing and Materials. West Con- <1227> “Validation of Microbial Recov-
shohocken, PA. ery from Pharmacopeal Articles.” United
States Pharmacopeia and the National For-
3. AOAC INTERNATIONAL. 2000. “Of- mulary. USP30 – NF25. Rockville, MD,
ficial Method 966.04 - Sporicidal Activ- 684-686.
ity of Disinfectants.” In: Official Methods
of Analysis of AOAC INTERNATIONAL. 10. American Society for Testing and Materi-
Gaithersburg, MD. als. 1999. ASTM E 1054-91, “Standard
Practices for Evaluating Inactivators of
4. United States Pharmacopeia. 2007. <51> Antimicrobial Agents Used in Disinfec-
“Antimicrobial Effectiveness Testing.” tant, Sanitizer, Antiseptic, or Preserved
United States Pharmacopeia and the Na- Products.” In: Annual Book of ASTM
tional Formulary. USP30 – NF25. Rock- Standards, 11.05. West Conshohocken,
ville, MD. 79-81. PA. ASTM. http://www.astm.org.
5. The American Type Culture Collec- 11. Krowka, John F., and Bailey, John E.
tion (ATCC) website: http://www.atcc. 2007. “M-1 Determination of the Micro-
org recommends appropriate media for bial Content of Cosmetic Products.” In:
the microbial strains it provides and lists CTFA Microbiology Guidelines. Washing-
formulations for these media on its web- ton, DC: The Cosmetic Toiletry and Fra-
site: http://www.atcc.org/common/cata- grance Association.
log/media/mediaIndex.cfm. The media
formulations listed are not ready-to-use 12. Reichgott, M. 2003. “Reference Strains:
products for sale by the ATCC but in How Many Passages Are Too Many?”
some cases other commercial suppliers are ATCC Connection, 23, No. 2, http://www.
listed. atcc.org/common/documents/pdf/tb06.
pdf
6. Brown, M.R.W. and P. Gilbert, (Ed).
1995. Microbiological Quality Assurance: 13. United States Pharmacopeia. 2007. <61>
A Guide Towards Relevance and Reproduc- “Microbial Limit Tests.” United States
ibility of Inocula. Boca Raton, FL: CRC Pharmacopeia and the National Formulary.
Press. USP30 - NF25. Rockville, MD. 83-88.
SECTION 21
M-4
Method for
Preservation
Testing of Eye
TESTING OF EYE AREA COSMETICS

SECTION 21: M-4 PRESERVATION
Area Cosmetics
1. Scope
1.1 This is an acceptable procedure for determining the preservative efficacy of eye area
cosmetic products.1, 2
2.1 “Preservation Testing of Eye Area Cosmetics” (Section 14).
3. Materials
The following types of microorganisms should be given consideration in developing
preservation data:
Additional microorganisms should also be included in the test procedure if preservation
problems have been encountered with such microorganisms.
See also Section 10 and Reference 3.
Table 21-2 shows conditions recommended for culture maintenance:
Alternatively, cultures may be freeze-dried, frozen or grown on a slant and overlaid with
sterile mineral oil. Although initially more time-consuming, these methods eliminate
the necessity of frequent transfers and help ensure better culture stability. The viability
of cultures must be checked regularly.
In-house isolates may present unique maintenance problems. Storage in the original
product or incorporation of product in the maintenance medium is often the only way
to retain viability and continued resistance. This method is especially appropriate where
the isolate is subsequently inoculated into a similar product.
SECTION 21: M-4 PRESERVATION TESTING OF EYE AREA COSMETICS | CTFA MICROBIOLOGY GUIDELINES | 197
3.3 Test Media
3.3.1 Plating diluents
better recovery of the microbial population of a challenged formulation.
Ideally, the diluent should contain both neutralizing agents and a biologically
inert surface-active agent.
The following diluents have been found suitable for preservation studies:
• Williamson Buffer Suspending Fluid (modified)4
• Letheen Broth*
• Thioglycolate Broth*
• TAT Broth*
The addition of lecithin and an appropriate polysorbate to commercially
available dehydrated broth formulations is also acceptable.
3.3.2 Recovery media
It is important that the recovery medium provide adequate nutritional
support for the growth of damaged cells. It is recommended that neutralizing
agents be incorporated into the agar to counteract preservative carry-over
from the diluent to the recovery medium. Letheen agar is an example of
a commercially available medium containing neutralizers. It can be used
in the recovery of bacteria, yeasts and molds. Thioglycolate agar should be
considered for inactivation of mercury and other heavy metals, while D-E
Medium (DIFCO) is useful when the preservative system is unknown or
when several different types of preservatives are present. In most cases, the
addition of lecithin and an appropriate polysorbate to any nutritionally
adequate growth medium for bacteria, yeasts or molds is sufficient to achieve
preservative neutralization.
3.3.3 Evaluating preservative neutralization5, 6
The presence of active preservatives carried over from the challenged
formulation into the plating diluent and recovery medium may inhibit viable
organisms and result in false-negative readings. Neutralizing agents should
be incorporated into the plating diluent and/or recovery medium in order
to inactivate preservatives and permit accurate enumeration of the microbial
content. Methods to evaluate neutralizer effectiveness are as follows:
If growth is not obtained on the dilution plates after incubation, inoculate the
surface of the 10-1 and 10-2 plates with approximately 100 CFU of a mixed
culture of Gram-positive bacteria. Perform the same procedure with a mixed
culture of Gram-negative bacteria, a mixed culture of yeasts and a mixed
culture of molds. If growth is not apparent on any one of the streaks after
incubation, neutralization of the preservative system is inadequate and an
198 | CTFA MICROBIOLOGY GUIDELINES | SECTION 21: M-4 PRESERVATION TESTING OF EYE AREA COSMETICS
appropriate neutralizer must be found. Where neutralizers are not available
or effective, physical dilution or membrane filtration to recover surviving
microorganisms from the sample may be necessary.
4. Procedure
4.1 Aseptic technique should be practiced, and all test materials and media should be
sterile.
4.2 Aqueous Liquid and Semi-liquid Eye Products

4.2.1 Inoculum preparation
Fresh cultures should be used for challenging preservative systems. Either
broth cultures or cultures grown on solid media are recommended. After two
or three consecutive daily transfers, bacterial and yeast cultures may be used
to challenge the product. If log-phase cells are desired, an incubation period
of 18-24 hours is usually adequate. Broth cultures may be used directly or
centrifuged and resuspended in phosphate buffer (pH 7.0) or 0.85% saline.
Growth on a solid medium is transferred to phosphate buffer prior to use.
The mold inoculum is prepared by washing the 7- to 14-day-old agar slants
with phosphate buffer or 0.85% saline and filtering the spore suspension
through gauze. Low concentrations of polysorbates (e.g., 0.05% polysorbate
80) can be added to the saline to aid in spore dispersal. In addition, harvesting
spore suspensions with glass beads in the saline aids in their dispersal.
In-house isolates should be inoculated into the test formulation directly from
contaminated product. If this is not feasible, in-house isolates may be cultured
in broth or on solid media as described above.
Table 21-3 shows conditions that should be considered when preparing the
inoculum:
If spore-forming bacteria are to be employed, inocula can be prepared as
indicated in the AOAC sporicidal test.7
4.2.2 Sample Preparation
The amount of product required to perform a preservation study should be a
minimum of 20 grams for each test microorganism or pool of microorganisms.
Sufficient product is needed to sample at each evaluation time and to have
some material remaining in the event that additional platings are required.
When rechallenges8 are used, it will be necessary to increase the amount of
product to be tested.
During the early developmental stage, a formulation may be challenged
in glass containers. Subsequent preservation tests should be conducted on
product in the final package to ensure its compatibility with the preservative
system. It is recommended that all formulations be examined for microbial
content prior to initiating preservation studies.
4.2.3 Product challenge
4.2.3.1 Pure and mixed-culture challenge
Either pure or mixed cultures may be used to challenge test
formulations. Pure-culture challenge, although more time
consuming, will yield specific data on each microorganism
employed in the study. Mixed-culture challenge, on the other hand,
can be used to obtain rapid pass-or-fail decisions on preservative
adequacy and reduce the workload. However, antagonism among
organisms may occur. It is recommended that closely related types
of microorganisms such as Gram-positive bacteria, Gram-negative

bacteria, or yeasts and molds be pooled separately when conducting
mixed culture challenge.
Challenge levels ranging from 1 × 104 to 1 × 108 CFU per gram of
product have been reported in the literature for preservative system
evaluation.9, 10, 11 A reasonable challenge should be larger than the
total challenge expected during consumer use. On this basis, the
following levels are recommended:
• 1×106 CFU of bacteria per gram of product
• 1×105 CFU of yeast per gram of product
• 1×105 CFU of mold spores per gram of product
After inoculation, all products should be thoroughly mixed
manually or mechanically to uniformly distribute the challenge
microorganisms. The volume of the inoculum should not alter the
character of the product being challenged. Challenged formulations
should then be incubated at controlled room temperature under
appropriate conditions of humidity for the duration of the test.
4.2.4 Sampling the challenged product
4.2.4.1 Sampling intervals
Challenged formulations should be sampled for viable
microorganisms at selected time intervals after inoculations. The
frequency of sampling should follow a set pattern to facilitate future
comparison of results. Sampling is recommended immediately
after inoculation (0 hour) and at 1-3, 7, 14, 21 and 28 days. When
rechallenged, sampling should be once a week thereafter for at least
3 weeks.
4.2.4.2 Sampling methods
The inoculated product should be thoroughly mixed just prior to
sampling to ensure that the sample is representative. In some cases,
the inoculum can thrive in “pockets” of growth in the formulation
while other areas are relatively free of microorganisms. Many
aerobic microorganisms grow especially well at the formulation-
air interface. Often it is very difficult to break up the “pockets” of
growth, and special procedures are needed. The following mixing
methods have been used to overcome this problem:

Sample size will in part determine the minimum detectable level.
A sample size of one-gram or one-milliliter of product for the
quantitative pour plate method is recommended.
4.2.4.3 Plating (enumeration) methods
Quantitative pour plate method
Serial tenfold dilutions are prepared from the one-gram or one-
milliliter aliquot withdrawn from the challenged formulation.
Each dilution is thoroughly mixed, and one milliliter from each
dilution is transferred by pipette to a Petri dish. Melted agar
maintained at 43-46°C is added to the Petri dish, and the dish
is rotated to uniformly disperse the product. Solidified plates for
bacteria and yeast are incubated at 30-37°C for 48-72 hours. Mold
plates are incubated at 20-25°C for 5-7 days. After incubation, the
colonies are counted, and the resulting figure is multiplied by the
dilution factor to obtain the number of microorganisms per gram
or milliliter of product.
Quantitative spread plate method
similar to the pour plate method; however, 0.1-0.2 ml of each
dilution is pipetted directly onto the surface of solidified agar. The
sample is then evenly spread over the surface of the agar with a glass
“hockey stick.” The plates are allowed to dry and then incubated,
and colonies are counted as described above.
4.3 Non-Aqueous Eye Cosmetics
4.3.1 Inoculum preparation
Aqueous inoculum - See 4.2.1.
Emulsified aqueous inoculum - This is an aqueous inoculum emulsified with
not more than 1% of dispersing agent such as polysorbate, sorbitan oleate or
glycerol.
Oil inoculum - Challenge cultures may be resuspended in light mineral oil.
4.3.2 Inoculation methods

4.3.2.1 Mixing
See 4.2.4.2 above. A glass rod, tongue depressor, or mechanical
mixer may be necessary to uniformly disperse test microorganisms.
Inoculated product that has collected on the mixing device or on
the container’s inner surfaces or edges must be worked back into
the sample to prevent excessive loss of product.
4.3.2.2 Surface inoculation
Swabbing - A swab is dipped into an inoculum of known
concentration and swabbed across the entire product surface.
Spreading - A known volume of inoculum is pipetted onto the
surface of the product and uniformly spread using a glass rod or
other instrument.
Dipping - The product in its container is dipped into an inoculum
of known concentration for a predetermined length of time.
Spraying - The product is sprayed with a suspension of inoculum
using an atomizer. Appropriate safety precautions should be
taken.
4.3.3 Product challenge
4.3.3.1 Oils and water-in-oil emulsions
Procedure
Prepare enough of the formulation to permit adequate sampling
at each test interval. At least 20 mL or 20 grams of the product
should be challenged with each test microorganism or mixture of
test microorganisms. Use containers that can be sealed to prevent
excessive evaporation and are large enough to allow for adequate
mixing. The containers should not react with the product.
Inoculum
The inoculum volume should be 0.1% to 1.0% of the sample
volume to keep the sample as water-free as possible. The inoculum
may be an aqueous or oil suspension added as a liquid or spray.
4.3.3.2 Loose powders
Procedure
At least 20 grams of product should be challenged with each test
organism or mixture of test microorganisms. This sample size is
usually large enough to permit numerous samplings. Standardized
containers that can be loosely capped and are large enough to allow
for adequate mixing should be used. The containers should not
react with the product.

Inoculum
A fine spray or a liquid inoculum (volume 1% to 5% of the test
sample) should be added to the product and thoroughly mixed.
4.3.3.3 Pressed powders
Pressed powders treated as loose powders
Procedure
Pressed powders may be removed from containers, ground
(e.g., mortar and pestle) into fine particles, and processed as
described above. A minimum sample size of 20 grams should
be prepared for each challenge microorganism or pool of
microorganisms.
Inoculum
See 4.3.3.2.
Pressed powders in pans or cakes
Procedure
For pressed powders inoculated on the surface, a suitable
number of pans or cakes should be prepared for adequate
sampling for each sampling interval. It is suggested that each
pan or cake contain one gram of sample.
Inoculum
These samples are surface inoculated using any of the methods
under “Surface Inoculation” above.
4.3.3.4 Wax-based products
Bulk samples
Procedure
For bulk samples, a minimum size of 20 grams should be
prepared for each challenge microorganism or pool of
microorganisms. Briefly warming the bulk product to no
more than 45°C may aid in the dispersion of the challenge
inoculum.
Inoculum
An oil or emulsified aqueous inoculum is suggested. It may
be applied as a liquid or a spray and mixed as in “Mixing”
above.
Pan, cake or stick samples
Procedure
For pans, cakes or sticks that are surface inoculated, see
4.3.3.3.
Pressed powders in pans or cakes

Inoculum
See 4.3.2.2.
4.3.4 Sampling the challenged product
See 4.2.4.
Type Microorganism Recommendation

In-house Isolates As appropriate one or more
Gram-Positive Cocci Staphylococcus aureus at least one

Staphylococcus epidermidis
Fermentative Gram-Negative Rod Klebsiella pneumoniae at least two

Enterobacter cloacae
Escherichia coli
Proteus species
Enterobacter gergoviae
Non-Fermentative Pseudomonas aeruginosa at least one in addition to

Gram-Negative Rod Burkholderia cepacia P. aeruginosa
Pseudomonas fluorescens
Pseudomonas putida
Flavobacterium species
Acinetobacter species
Yeasts Candida albicans at least one

Candida parapsilosis
Molds Aspergillus niger at least one

Penicillium luteum
Spore-Forming Bacteria Bacillus subtilis optional
Table 21-1
Table 21-2: Suggested Culture Conditions
Cultures Maintenance Media* Storage Conditions Transfer Frequency
Bacteria Nutrient Agar Refrigeration 4-8°C Weekly, Biweekly, or

Tryptic (Trypticase) Soy Agar Monthly
Eugon Agar
Yeasts Tryptic (Trypticase) Soy Agar Refrigeration 4-8°C Biweekly or Monthly


Molds Sabouraud Dextrose Agar Refrigeration 4-8°C Biweekly or Monthly
* Available in dehydrated forms from Becton Dickinson Microbiology Systems, BBL Division (Cockeysville, MD 21030), DIFCO
(Detroit, MI 28401) and other commercial sources
Table 21-2
Table 21-3: Suggested Inoculum Conditions
Cultures Media* Temperature Time

Bacteria Tryptic (Trypticase) Soy Broth/Agar 30-37°C 18-48 hours
Nutrient Broth/Agar
Eugon Agar/Broth
Yeasts Tryptic (Trypticase) Soy Broth/Agar 30-37°C 24-48 hours


* Available in dehydrated forms from Becton Dickinson Microbiology Systems, BBL Division (Cockeysville, MD 21030), DIFCO
(Detroit, MI 28401) and other commercial sources.
Table 21-3
Bean, H.S. 1972. “Preservatives for Pharmaceuticals.” Journal of the Society of Cosmetic Chemistry 23:703-720.
1972.
Tenenbaum, S. 1967. “Pseudomonads in Cosmetics.” Journal of the Society of Cosmetic Chemistry 18:797-807.
Wilson, L.A., Kuehne, J.W., Hall, S.W. and Ahearn, D.G. 1971. “Microbial Contamination in Ocular Cosmetics,”
American Journal of Ophthalmology, 71(6):1298-1302.
REFERENCES
1. American Society for Testing and Mate- 6. Wilson, L.A., Julian, A.J. and Ahearn,
rials. 2001. “Standard Test Method for D.G. April 1975. “The Survival and
Preservatives in Water-Containing Cos- Growth of Microorganisms in Mascara
metics.” ASTM E 640-78. West Con- During Use”, American Journal of Oph-
shohocken, PA. thalmology 79(4): 596-601.
2. AOAC INTERNATIONAL. 2000. “Ef- 8. AOAC INTERNATIONAL. 2000. “Spo-
ficacy of Preservation of Non-Eye Area ricidal Activity of Disinfectants.” Official
Water-Miscible Cosmetic and Toiletry Method 966.04, Official Methods of Anal-
Formulations” Official Method 998.10, ysis of AOAC INTERNATIONAL, 17th

Official Methods of Analysis of AOAC IN- Ed. Gaithersburg, MD.
TERNATIONAL, 17th Ed. Gaithersburg,
MD. 9. Preservation Subcommittee of The Cos-
metic, Toiletry, and Fragrance Association.
3. Brown, M.R.W. and P. Gilbert, (Ed). 1981. “A Study of the Use of Rechallenge
1995. Microbiological Quality Assurance: in Preservation Testing of Cosmetics”.
A Guide Towards Relevance and Reproduc- CTFA Cosmet. Journal 13:19-22.
ibility of Inocula. CRC Press.
10. Wilson, L.A. and Ahearn, D.G. 1977.
4. P. Williamson and A. Klingman. 1965. “A “Pseudomonas-induced Corneal Ulcers
New Method for the Quantitative Inves- Associated with Contaminated Eye Mas-
tigation of Cutaneous Bacteria.” Journal caras” American Journal of Ophthalmology
of Invest Dermatology 45(6): 498-503. 84: 112-119.
5. “Standard Practices for Evaluating Inac- 11. Madden, J.M. and Jackson, G.J. 1981.
tivators of Antimicrobial Agents Used in “Cosmetic Preservation and Microbes:
Disinfectant, Sanitizer, Antiseptic, or Pre- Viewpoint of the Food and Drug Admin-
served Products. ASTM E 1054-91. istration”. Cosmetics and Toiletries 96:75-
77.
SECTION 22
M-5
Methods for
Preservation
Testing of Nonwoven
Substrate Personal
Care Products
1. Scope
1.1 These methods cover a variety of procedures currently used within the cosmetics
industry to evaluate preservative efficacy of different types of nonwoven substrate, wipe,
or towelette products.
NONWOVEN
EVALUATION
These methods apply to nonwoven substrate personal care products that contain an
SECTION 22:
aqueous-based add-on solution. For nonwoven personal care products containing non-
IRRITATION
aqueous add-on materials or concentrates, it is important that critical consideration
SUBSTRATE
be given to the typical use of the finished product and risk assessment and testing be
OFM-5
completed as detailed in “Microbiological Risk Factor Assessment of Atypical Cosmetic
PRIMARY
POTENTIAL
Products” (Section 16).
TESTING
PC PRODUCTS
It is recommended that the method chosen reflect consideration of the manufacturing
DERMAL
OF
process, the type of packaging used, and the end use of the product.
2.1 “Determination of Preservative Adequacy in Nonwoven Substrate Personal Care
Products” (Section 13).
3. Materials
preservation data of nonwoven substrate, wipe, or towelette products.
Refer to the American Type Culture Collection (ATCC) culture maintenance
recommendations, available on the ATCC Web site,1 and other sources.2,3,4
SECTION 22: M-5 NONWOVEN SUBSTRATE PC PRODUCTS | CTFA MICROBIOLOGY GUIDELINES | 207
3.3 Test Media
• Sodium Chloride Peptone Solution (1% Peptone in Normal Saline)
Other suitable fluids may be used. To aid in dispersion of mold spores, adding
0.05% – 0.1% polysorbate 80 or other surfactant to the suspending fluid is
recommended.
The choice of diluent depends on its ability to meet the requirements of
preservative neutralization (Under “Preliminary Tests” later in this section).
The following are examples of diluents that may be used:
PC PRODUCTS
DERMAL
• Buffered Sodium Chloride Peptone Broth

OF
POTENTIAL
TESTING

PRIMARY
SUBSTRATE
• Eugon Broth
OFM-5
IRRITATION
SECTION 22:
• Letheen Broth
EVALUATION
NONWOVEN

• Soybean Casein Digest Medium (Tryptic Soy Broth)
• Trypticase Azolectin™ Tween™ (TAT) Broth
• Saline-Tween-Lecithin Diluent
Other suitable diluents may be used.
The addition of neutralizers may be necessary to demonstrate adequate
preservative neutralization (See 4.2).
Many factors affect organism viability. Therefore, it is important for the agar
to provide optimum nutritional support for the recovery of the challenge
organisms. The following have been found suitable for preservation studies:
• Eugon Agar
• Letheen Agar
208 | CTFA MICROBIOLOGY GUIDELINES | SECTION 22: M-5 NONWOVEN SUBSTRATE PC PRODUCTS
• Soybean Casein Digest Agar (Tryptic Soy Agar)
• Microbial Content Agar with Tween
The following media are specifically recommended for the recovery of yeasts
and molds during preservation studies:
• Malt Agar
Other suitable agars may be used. The addition of neutralizers may be
necessary to demonstrate adequate preservative neutralization.
NONWOVEN SUBSTRATE PC PRODUCTS

SECTION 22: M-5 TESTING OF

4.1 Initial Count
It is recommended that all formulations be examined for microbial content prior to
initiation of preservation studies.
challenge test microorganisms resulting in a false negative microbial count. To avoid a
Antimicrobial neutralization may normally be accomplished by the use of chemical
(See Section 6.2). Side-by-side dilutions with and without a product formulation are made.
Enumeration of the microorganisms from these dilutions is performed. Neutralization
is verified if microbial recoveries are similar. If one or more challenge microorganisms
cannot be recovered, the use of a higher dilution and/or the investigation of additional
chemical neutralizers may be considered.
Refer to ASTM E1054-025 or USP6 for additional detail.
In some cases, low recovery of organisms may be due to poor recovery efficacy rather
than failure to neutralize the preservative system.
4.3 Recovery Efficiency
Recovery of microorganisms from the nonwoven substrate is a separate issue from
antimicrobial neutralization. The substrate may entrap the microorganisms resulting
in incomplete recovery of the microbial population by the use of conventional dilution
and plating techniques. Therefore, additional techniques may be used to verify the
consistent recovery of microorganisms from the substrate material (Section 6.1.3).
Freshly prepared cultures should be used for inoculating test samples. In general, culture
conditions in Table 22-2 should be considered when preparing the inocula. Refer to the
ATCC Web site3 for optimal growth media and conditions for specific microorganisms.
Inclusion of cellulose degrading molds may necessitate longer incubation periods and
require a paper source for growth.
Either broth cultures or cultures grown on solid agar media are acceptable
for use. For reference strains such as the ATCC strains, no more than five
transfers from the stock culture are recommended.7 Broth cultures should be
centrifuged and then re-suspended in the chosen suspending fluid (See 3.3.1).
Microbial growth on a solid medium is transferred to the chosen suspending
fluid.
the chosen suspending fluid (See 3.3.1) and filtering the spore suspension
through sterile gauze or glass wool. Sterile glass beads can be used as an aid in
the dispersion of spores in the suspending fluid.
be prepared as indicated in the AOAC Sporicidal Test.8 Some strains are
5.1.4.1 Inoculum Levels
• 1x106 Colony-Forming Units (CFU) of bacteria per sampling
unit of product
• 1x105 CFU of yeast per sampling unit of product
• 1x105 CFU of mold spores per sampling unit of product
The inoculum level for the challenge microorganisms should be
verified by standard microbiological techniques such as pour plate
methods.
5.1.4.2 Culture Suspensions
Either pure or mixed microbial culture suspensions may be used
to challenge test formulations. Inocula consisting of only pure
microbial cultures will yield specific data on each test microorganism
employed in the challenge study. When conducting mixed culture
challenge studies, it is recommended that closely related types of
microorganisms such as Gram-positive bacteria, Gram-negative
bacteria, and yeasts and molds be pooled separately. These
suspensions may be used directly for inoculation or dried onto
filter carriers as described below.
5.1.4.3 Dried Inoculum on Filter Carriers
Dried inoculum carriers are prepared by filtering the culture
suspensions onto 13 mm 0.45 micron membrane filters (such
as cellulose ester membranes) to achieve inoculum levels
recommended after drying. The filters are placed in a covered

Petri dish and dried at 37°C for 20 to 30 minutes. The number of
viable microorganisms on dried carriers must be equivalent to the

recommended levels. If necessary, the volume of filtered culture
suspension may be increased to take into account mortality due to
desiccation.
5.2 Sample Preparation
In addition to the quantity required for the test, it is recommended that extra sample units
be prepared in the event they are needed. An unpreserved control should be included
if possible. If product rechallenge is desired, sufficient sample units must be prepared
prior to the start of the test. The sample reporting unit used depends on the inoculation
and sampling method chosen. If applicable, determine and record the weight and/or the
average area of the nonwoven substrate product sample, e.g., 1 g or 1 cm2 (See Section
7).
If possible, preservative challenge testing should be conducted on product in the final
package to ensure compatibility with the preservative system and to represent the
marketed product. Where the product does not lend itself to testing in the container/
package, other approaches may be employed, as detailed below in Section 5.2.4.
5.2.1 Tubs
Aseptically open packages and inoculate the product as received according to
the inoculation procedure (Section 5.3). Reseal the packages and follow the
sampling procedure under Section 6.1.
5.2.2 Soft Packages
Aseptically open packages, and inoculate the product as received according to
the inoculation procedure (Section 5.3). Some inoculation techniques may
allow for the aseptic introduction of the inocula directly into the package.
Reseal the packages and follow the sampling procedure (Under “Sampling
the Challenged Product” in Section 6.1 below).
5.2.3 Canisters
Aseptically open canister, remove the roll, and inoculate the top sheets of
the product according to an appropriate inoculation procedure (Section 5.3).
Reinsert the roll into the canister. Seal the canisters and follow the sampling
procedure (Section 6.1).
5.2.4 Transferred Samples
Aseptically open packages and transfer an appropriate number of nonwoven
substrate units to sterile, resealable containers for inoculation. Follow the
inoculation procedure (Section 5.3). Seal the containers and follow the
sampling procedure (Section 6.1).
5.3 Methods for Inoculation
Inoculation of nonwoven substrates can be accomplished in a variety of ways. Methods
for inoculating product are described below. In each case, verification of microorganism
recovery (described below) is an important component of the method verification.
5.3.1 A specific volume of an inoculum suspension is delivered by pipette using a

point delivery over the sample unit in a predetermined pattern. (For example,
place 0.1 ml in five different areas of the substrate such as the four corners
and the center.) After inoculation, the package is sealed. This inoculation
method can be used to inoculate one or a series of multiple sample units in
one package.
5.3.2 A specific volume of an inoculum suspension is delivered by multi-channel
pipette using a point delivery over the sample unit in a predetermined pattern.
After inoculation, the package is sealed. This inoculation method can be used
to inoculate one or a series of multiple sample units in one package.
5.3.3 A specific volume of an inoculum suspension is aseptically introduced onto
the substrate in a straight line down the center of the substrate sample. The
inoculum must be applied to the substrate so that uniform cross sections may
be cut off of the substrate(s) for sampling. After inoculation, the package is
sealed. This inoculation method can be used to inoculate one or a series of
multiple sample units in one package.
5.3.4 By means of a syringe, a specific volume of an inoculum suspension is aseptically
introduced into the package containing a sample unit. After inoculation, the
package is sealed, and the inoculum is well mixed by massaging the package.
This inoculation method is quantitative for single unit soft packages and
qualitative for multiple unit soft packages. This method is not suitable for
tubs or canisters.
5.3.5 In a Class 2 (or greater) biological safety cabinet, the inoculum is sprayed
evenly over the entire surface of a pre-determined area of the substrate, (e.g.,
a 9 cm2 sample in a 10 cm2 Petri dish), by using an airbrush or other suitable
spraying device. The substrate sample is sprayed for an appropriate time to
deliver the target inoculum. The quantity of inoculum delivered must be
calculated for the specific spray device. The inoculated sample should be
sealed in a plastic bag or other suitable container to prevent drying.
5.3.6 Dried inocula on membrane filter carriers are placed between two substrate
layers in the package. Placement of the filter may be determined by conducting
a sedimentation study. After inoculation, the package is resealed.
5.4 Storage of Inoculated Samples
Challenged formulations can be stored at controlled or ambient temperature
underconditions of humidity considered appropriate for the final product packaging
for the duration of the test.
6. Recovery Procedures
6.1.1 Sampling Intervals


different product formulations or samples, for example, weekly up to 28 days
after inoculation.
6.1.2 Sampling Sites
The method of sampling chosen will depend upon several factors including
the method of inoculation. Below are several sampling methods that may be
used.
6.1.2.1 For most inoculation methods, the top nonwoven substrate in the
stack or the outer most nonwoven substrate in the roll may be
sampled from a product package.
6.1.2.2 For a product challenge method where multiple nonwoven
substrates in a product package are inoculated, the inoculated
substrate units per package should be sampled at the appropriate
time.
6.1.2.3 For a product challenge method where an inoculated nonwoven
substrate is aseptically transferred to a secondary package (See
“Transferred Samples” in Section 5.3), one product package per
sampling interval may be sampled.
6.1.2.4 For a product challenge method where the inoculum is evenly
distributed across the nonwoven substrate, a uniform cross section
(e.g., 1 g) of the substrate may be sampled at each sampling
interval.
6.1.2.5 For a product challenge method using dried inocula, a membrane
filter carrier is sampled at each interval. Additionally, one or two
substrates above and one or two below the filter may be sampled
separately at each sampling interval to evaluate migration of
organisms through the sample.
6.1.3 Recovery Methods
Aseptically remove the inoculated product or membrane filter carrier from
the container(s) and thoroughly mix with the preservative neutralizing
diluent. Care must be taken to sample the areas of the product that have been
inoculated.
Organisms may be recovered from the sample using the following processing
techniques:
• Mixing with diluent and glass beads by means of a mechanical wrist
shaker or reciprocal shaker for a predetermined period.
• Mixing with diluent in a vortex mixer is recommended when sample
sizes are small, e.g., 1 g or less.
• Mixing in a Stomacher™ with a diluent, e.g., 1 to 2 minutes at medium
speed.
Other methods that may be employed include manual shaking or the use
of an orbital mixer or a blender. The addition of glass beads may improve
recovery of microorganisms, although they are not recommended for use in
plastic bags or with a blender.
6.2 Enumeration Methods
6.2.1 Quantitative Pour Plate Method
Serial dilutions are prepared from the aliquot recovered from the challenged
sample unit. Each serial dilution is thoroughly mixed and an aliquot is
transferred to a Petri dish. Melted agar maintained at 44-48°C is added to the
Petri dish, and the dish is rotated to uniformly disperse the product dilution.
The agar plates are allowed to solidify, then inverted and incubated under
conditions appropriate for the test microorganisms (see Table 22-2).
After incubation, the number of microbial colonies is counted and the
resulting figure is multiplied by the appropriate dilution factor to obtain the
number of microorganisms per sample unit.
6.2.2 Quantitative Spread Plate Method
The quantitative spread plate method is performed in a manner similar to
the pour plate method; however, an aliquot of each dilution is transferred
directly onto the surface of solidified microbial growth agar. The sample
aliquot is then evenly spread over the agar surface. The agar plates are allowed
to dry, then inverted and incubated under conditions appropriate for the test
After incubation, the number of microbial colonies is counted, and the
resulting figure is multiplied by the appropriate dilution factor to obtain the
number of microorganisms per sample unit.
7. Reporting
Calculate and report the percent reduction of inoculum counts per substrate, per gram of
product, or per unit area for each organism or organism pool.9
Type Microorganism (ATCC Numbers) Recommendation

Gram-Positive Cocci Staphylococcus aureus (6538)* Select at least one
Staphylococcus epidermidis (12228)

Fermentative Gram- Klebsiella pneumoniae (10031) Select at least one
Negative Bacilli Enterobacter cloacae (13047)
Non-Fermentative Gram- Pseudomonas aeruginosa (9027)* Select at least one

Negative Bacilli Burkholderia cepacia (25416)
Yeasts Candida albicans (10231)* Select at least one

Candida parapsilosis (22019)

Chaetomium globosum (6205)**
Trichoderma reesei (13631)**
Cladosporium oxysporum (76499)**
Penicillium species
Other In-house isolates Optional
*Staphylococcus aureus (6538), Escherichia coli (8739), Pseudomonas aeruginosa (9027), Candida albicans (10231), and
Aspergillus niger (16404) are specified in the United States Pharmacopeia (USP) Antimicrobial Effectiveness Testing
Method.5 10
**Inclusion of cellulose degrading molds may necessitate longer incubation periods and require a paper source for
growth.6
Table22-1

Bacteria Soybean Casein Digest (Tryptic Soy) 30-37°C 18-48 hours
Broth/Agar
Nutrient Broth/Agar
Eugon Broth/Agar

Soybean Casein Digest (Tryptic Soy) Broth/Agar

Malt Extract Agar
* Available in dehydrated forms from commercial sources.
Table 22-2
REFERENCES
1. The American Type Culture Collection 6. United States Pharmacopeia. 2007.
(ATCC; website: www.atcc.org ) recom- <1227> “Validation of Microbial Recov-

mends appropriate media for the microbi- ery from Pharmacopeal Articles.” United
al strains it provides and lists formulations States Pharmacopeia and the National For-
for these media on its website (http:// mulary. USP30 – NF25. Rockville, MD.
www.atcc.org/common/catalog/media/ 684-686.
mediaIndex.cfm) . The media formula-
tions listed are not ready-to-use products 7. Reichgott, M. Winter 2003. “Refer-
for sale by the ATCC but in some cases ence Strains: How Many Passages Are Too
other commercial suppliers are listed. Many?” ATCC Connection. Vol 23, No.
2, available at http://www.atcc.org/com-
2. Brown, M.R.W. and P. Gilbert, (Ed). mon/documents/pdf/tb06.pdf.
1995. Microbiological Quality Assurance:
A Guide Towards Relevance and Reproduc- 8. AOAC INTERNATIONAL. 2000. Of-
ibility of Inocula. Boca Raton, FL: CRC ficial Method 966.04, “Sporicidal Activ-
Press. ity of Disinfectants.” Official Methods of
Analysis of AOAC INTERNATIONAL.
3. Kirsop, B.E. and A. Doyle, (Ed). 1991. Gaithersburg, MD. www.aoac.org .
Maintenance of Microorganisms and Cul-
tured Cells. Orlando, FL: Academic 9. AOAC INTERNATIONAL. 2000. Of-
Press. ficial Method 998.10, “Efficacy of Pres-
ervation of Non-Eye Area Water Miscible
4. Simione, F. P. 1998. Cryopreservation Cosmetic and Toiletry Formulations.”
Manual. Rochester, NY: Nalgene Nunc Official Methods of Analysis of AOAC IN-
International. http://www.nalgenelabwa- TERNATIONAL. Gaithersburg, MD.
re.com/techdata/technical/manual.asp. www.aoac.org.
5. ASTM E 1054. 2003. “Standard Practices 10. United States Pharmacopeia. 2007. <51>
for Evaluating Inactivators of Antimicro- “Antimicrobial Effectiveness Testing.”
bial Agents Used in Disinfectant, Sani- United States Pharmacopeia and the Na-
tizer, Antiseptic, or Preserved Products.” tional Formulary. USP30 – NF25. Rock-
Annual Book of ASTM Standards, 11.05. ville, MD. 79-81.
Washington, DC: ASTM. www.astm.org.
SECTION 23
M-6
A Method for
Preservation Testing
of Atypical Personal
Care Products
1. Scope
1.1 This general method reflects a variety of approaches currently used within the cosmetics
industry and serves as an acceptable procedure for determining the preservative efficacy
of atypical personal care products, such as oils, powders, or other formulations that have
low water content and/or are not miscible with water. The recommended preservative
challenge test methods used for determining the preservative adequacy of aqueous-based
products (See References 1 and 2, and Section 20) may not be suitable for evaluating
certain atypical product formulations. When testing and assessing preservative challenge
test data for atypical products, the following factors are important points to consider
(See “Microbiological Risk Factor Assessment of Atypical Cosmetic Products” in Section
16).
• A test in which an aqueous-based inoculum is introduced into an hydrous product
may change the physical dynamics of the product and, therefore may not predict
its microbial stability.
• Most preservatives are water soluble. In emulsions, preservatives are used in the
water phase because contaminating microorganisms require water to proliferate.
• For an emulsion in which the external phase is water immiscible (emulsions in
which water is not the external or continuous phase) and an aqueous challenge
PERSONAL CARE PRODUCTS

SECTION 23: M-6 ATYPICAL
inoculum is used, the water-soluble preservatives may not be able to migrate into
the aqueous phase. In these cases, the preservatives may not be available to inhibit
proliferation or have cidal activity against each of the challenge microorganisms.
2.1 “Microbiological Risk Factor Assessment of Atypical Cosmetic Products” (Section 16).
2.2 “Determination of Preservative Adequacy in Cosmetic Formulations” (See Section 13).
SECTION 23: M-6 ATYPICAL PERSONAL CARE PRODUCTS | CTFA MICROBIOLOGY GUIDELINES | 217
3. Materials
3.1 Selection of Challenge Microorganisms.
preservation data of Personal Care products.
culture challenge studies, it is recommended that separate pools of closely related types
of microorganisms such as Gram-positive bacteria, Gram-negative bacteria, and yeasts
and molds be maintained.
Refer to the ATCC culture maintenance recommendations, available on their website,
and to other sources.3,5,6,7
Organisms appropriate to the product under test may be best stored in the original
product matrix to retain their unique characteristics. Periodic testing may be employed
to verify retention of these characteristics.
3.3 Test Media
• Sodium Chloride Peptone Solution (1% peptone in normal saline)

The choice of diluent depends on its ability to meet the requirements of
preservative neutralization (See Section 4.1 “Preservative Neutralization”).
The following are examples of diluents that may be used:
• Eugon Broth
• Letheen Broth
218 | CTFA MICROBIOLOGY GUIDELINES | SECTION 23: M-6 ATYPICAL PERSONAL CARE PRODUCTS
• Soybean-Casein Digest Medium (Tryptic Soy Broth)
• Tryptone-Azolectin-Tween® (TAT) Broth
neutralization. Other suitable diluents may be used.
• Eugon Agar
• Letheen Agar
• Soybean-Casein Digest Agar Medium (Tryptic Soy Agar)
The addition of neutralizers may be necessary to demonstrate adequate
preservative neutralization. Other suitable agars may be used.
If the above agars do not support the growth of fungi, one of the
following agars may be considered:
• Malt Agar

Other suitable agars may be used.
with a low level of challenge microorganisms and performing the enumeration method7.
of the microorganisms from these dilutions is performed as described7. Neutralization
is verified if microbial recoveries are similar. If one or more challenge microorganisms
cannot be recovered, the use of a higher dilution and/or the investigation of additional
chemical neutralizers may be considered.
It is recommended that that a microbial content test (Section 18) be performed on the
test sample prior to performing the preservative efficacy test. Verification of neutralization
of the antimicrobial properties of the test sample should be demonstrated (See Section
4.1 and Reference 7) at the same time as the microbial content test.
5. Inocula Preparation
inocula.
for use. For reference strains such as the ATCC3 strains, no more than five
transfers from the stock culture are recommended8.
5.1.1.1 Aqueous Inoculum
Broth cultures should be centrifuged and then re-suspended in the
chosen suspending fluid. (See Section 20) Microbial growth on a
solid medium is transferred to the chosen suspending fluid.
5.1.1.2 Emulsified Aqueous Inoculum
An aqueous emulsified inoculum may be prepared by adding not

more than 1% of dispersing agent such as polysorbate, sorbitan
oleate or glycerol to the aqueous inoculum.
5.1.1.3 Oil Inoculum
Challenge cultures may be resuspended in light mineral oil.
Note: If using this technique, the absence of inhibitory or toxic
properties of the dispersing agent or oil soluble carrier system
should be verified for each of the challenge organisms.
5.1.2.1 Aqueous Mold Inoculum
The mold inoculum is prepared by washing the sporulating agar
culture with the chosen suspending fluid and filtering the spore
suspension through sterile gauze or glass wool. Sterile glass beads
can be used as an aid in the dispersion of spores in the suspending
fluid.
5.1.2.2 Emulsified Aqueous Mold Inoculum
An aqueous emulsified inoculum may be prepared by adding not
more than 1% of dispersing agent such as polysorbate, sorbitan
oleate or glycerol to the aqueous inoculum.
5.1.2.3 Oil Inoculum
Challenge cultures may be resuspended in light mineral oil.
5.1.4 Inoculum Levels
Inoculum challenge levels ranging from 1 × 104 to 1 × 108 CFU per gram
or ml of product have been reported in the literature for preservative system
evaluation.10-12
For some atypical products, (e.g., anhydrous products), a reduction in the
challenge inoculum size to 103 to 104 Colony-Forming Units (CFU) per
gram or milliliter may be used instead of the inoculum concentration of 105
to 106 CFU per gram or milliliter that is recommended in the aqueous based
challenge test methods.
Note: By reducing the inoculum size, it is easier to measure stasis or quantify

an increase in the microbial count.
6. Inoculation Procedures for Test Samples

Depending on the product form, the following carriers for inocula may be considered. The
volume of the inoculum should not alter the character of the product being tested.
• Aqueous inoculum (See Section 20)
• Emulsified aqueous inoculum
• Oil inoculum
6.1 Inoculum Dispersed into Product
6.1.1 Oils, water-in-oil emulsions, water in silicone and semisolid products (<20%
water)
Procedure
Prepare enough of the formulation to permit adequate sampling at each test
interval. At least 20 mL or 20 grams of the product should be challenged with
each test microorganism or mixture of test microorganisms.
Inoculum
The inoculum volume should be 0.1% to 1.0% of the sample volume in
order to keep the sample as water-free as possible. The inoculum may be an
aqueous or oil suspension.
6.1.2 Loose Powders
Procedure
At least 20 grams of product should be challenged with each test organism or
mixture of test microorganisms. This sample size is usually large enough to
permit numerous samplings.
Inoculum
The inoculum (volume 0.1% to 1% of the test sample) should be added to
the product and thoroughly mixed.
6.1.3 Pressed Powders
Procedure
Pressed powders can be inoculated on the surface or removed from containers,
ground (e.g., mortar and pestle) into fine particles, and processed.
A minimum sample size of 20 grams should be prepared for each challenge
microorganism or pool of microorganisms.
For wet dry pressed powders, up to 5% water may first be added to the
product, prior to inoculation.
6.1.4 Mixing
A glass rod, tongue depressor, or mechanical mixer may be necessary to
uniformly disperse test microorganisms. Inoculated product that has collected
on the mixing device or on the container’s inner surfaces or edges must be
worked back into the sample to prevent excessive loss of product (See Section
20).
6.2 Surface inoculation
Swabbing
A swab is dipped into an inoculum of known concentration and swabbed across the
entire product surface.
Spreading
A known volume of inoculum is pipetted onto the surface of the product and uniformly
spread using a glass rod or other instrument.
Dipping
The product in its container is dipped into an inoculum of known concentration for a
predetermined length of time.
Spraying
The product is sprayed with a suspension of inoculum using an atomizer. Appropriate
safety precautions should be taken.
6.3 Wax based solid products
For solid atypical products, such as anhydrous sticks or pans, inoculation and sampling
of the product surface instead of the whole product more closely simulates potential
consumer contamination. This modification also maintains the physical product
integrity. In these types of products, the microorganisms are not able to penetrate
into the interior and will always be found on the outer-most layer of the product after
consumer usage. Although this type of product is not usually susceptible to microbial
contamination, surface inoculation may be used.
Note: If performing challenge testing on a solid anhydrous stick or powder product,
inoculate a sufficient number of samples to obtain a unique sample for each sampling
time-point.
6.4 Storage of Inoculated Samples
Inoculated samples should be stored under ambient conditions

different product formulations. Sampling intervals should be based on
microbial contamination risk as demonstrated by product water activities and
other microbiological related attributes.
• Water in oil and/or silicone emulsions
Three sampling points, such as 7, 14, 28 day
• Semisolid products (<20% water)
Three sampling points, such as 7, 14, 28 day
• Oil or silicone based products (anhydrous)
Three sampling points, such as 2,7,14 days.
• Loose, wet dry and pressed powders
Three sampling points, such as 2,7,14 days
• Wax based and other solid products
Three sampling points, such as 2,7,14 days.
ensure that the sample is representative. For water-immiscible products (e.g.
oils and emulsions), a suitable solubilizing agent may be incorporated into the
test diluent or broth to make the sample aliquot miscible with water in order
to recover microorganisms present in the test sample. For solid products,
the surface may be sampled by removing the top layer. For products where
microorganisms would only be recovered from the product surface (e.g.,
sticks, pressed powders, hot pour products in compacts), only the surface
of the product sample should be tested. For these “atypical products”, the
following methods of recovery may be considered.
• A sterile moistened applicator may be used to sample the product surface,
and then streaked onto a Petri dish containing solid culture medium.
• The product may be sampled by a direct contact method using a contact
plate (a modified Petri dish containing a solid culture medium whose
convex surface extends above the carrier), paddles, or flexible film
containing solid culture media.
In some cases, the inoculum can thrive in “pockets” of growth in the
formulation while other areas are relatively free of microorganisms. Many
aerobic microorganisms grow especially well at the formulation-air interface.
Often it is very difficult to break up the “pockets” of growth, and special
procedures are needed. The following mixing methods have been used to
overcome this problem:
• Hand mixing with a stirring rod

size of at least one gram or one milliliter of product for the quantitative pour
plate method is recommended. Aseptic techniques must be employed.
to obtain the number of CFU per gram or milliliter of sample.
similar to the pour plate method; however, an aliquot of each
to obtain the number of CFU per gram or milliliter of sample.

Type Microorganism (ATCC Number3) Recommendation

Fermentative Gram-Negative Bacilli Klebsiella pneumoniae (10031) Select at least one

Enterobacter cloacae (13047))


Penicillium species
Other organisms relevant Optional

to the product
*Staphylococcus aureus (6538), Escherichia coli (8739), Pseudomonas aeruginosa (9027), Candida albicans (10231) and Aspergillus
niger (16404) are specified in the United States Pharmacopeia (USP) Antimicrobial Effectiveness Testing method4.
Table23-1

Bacteria Soybean-Casein Digest Medium/Soybean- 30-37°C 18-48 hours
Casein Digest Agar Medium (Tryptic Soy Broth/Agar )
Nutrient Broth/Agar
Eugon Broth/Agar

Soybean-Casein Digest Medium/Soybean-

Malt Extract Agar
* Available in dehydrated forms from commercial sources.
Table 22-2
Table 23-3: Incubation Conditions for Recovery of Microorganisms
Cultures Media Temperature Time
Bacteria 30-37°C 24-72 hours
Yeasts For recovery agars, see Section 3.3.3 25-35°C 48-72 hours
Molds 20-30°C 3-7 days
Table 23-3
Bean, H.S. 1972. “Preservatives for Pharmaceuticals.” J. of Soc. Cosmet. Chem. 23:703-720.
Tenenbaum, S. 1967. “Pseudomonads in Cosmetics.” J. of Soc. Cosmet. Chem. 18:797-807.
Wilson, L.A., J.W. Kuehne, S.W. Hall, and D.G. Ahearn. 1971. “Microbial Contamination in
Ocular Cosmetics,” American Journal of Ophthalmology. 71(6):1298-1302.
Yablonski, J.I. and S.E. Mancuso. 2002. “Preservation of Atypical Cosmetic Systems.” Cosmetics
& Toiletries 2: 41.
REFERENCES
1. ASTM International. 2007. ASTM E 4. United States Pharmacopeia. 2007. <51>
640-78,” Standard Test Method for Pre- “Antimicrobial Effectiveness Testing.”
servatives in Water-Containing Cosmet- United States Pharmacopeia and the Na-
ics.” In: Annual Book of ASTM Standards. tional Formulary. USP30 – NF25. Rock-
West Conshohocken, PA. ville, MD. 79-81.
2. AOAC International. 2000. “Official 5. Brown, M.R.W. and P. Gilbert (Ed).
Method 998.10 - Efficacy of Preservation 1995. Microbiological Quality Assurance:

of Non-Eye Area Water-Miscible Cosmet- A Guide Towards Relevance and Reproduc-

ic and Toiletry Formulations.” In: Official ibility of Inocula. Boca Raton, FL: CRC
Methods of Analysis of AOAC International. Press.
Gaithersburg, MD. 6. Kirsop, B.E. and A. Doyle (Ed). 1991.
3. The American Type Culture Collec- Maintenance of Microorganisms and Cul-
tion (ATCC) website: http://www.atcc. tured Cells. Second edition. New York,
org recommends appropriate media for NY: Academic Press.
the microbial strains it provides and lists 7. ASTM International. 1999. ASTM E
formulations for these media on its web- 1054-91, “Standard Practices for Evaluat-
site: http://www.atcc.org/common/cata- ing Inactivators of Antimicrobial Agents
log/media/mediaIndex.cfm. The media Used in Disinfectant, Sanitizer, Antisep-
formulations listed are not ready-to-use tic, or Preserved Products.” In: Annual
products for sale by the ATCC but in Book of ASTM Standards, 11.05. West
some cases other commercial suppliers are Conshohocken, PA.
listed.
8. Reichgott, M. 2003. “Reference Strains: 11. Madden, J. M. and G.J. Jackson. 1981.
How Many Passages Are Too Many?” In: “Cosmetic Preservation and Microbes:
ATCC Connection, 23, No. 2, http://www. Viewpoint of the Food and Drug Admin-
atcc.org/common/do cuments/pdf/tb06. istration.” Cosmetics & Toiletries 96:75-
pdf 77.
9. AOAC International. 2000. Official 12. Wilson, L.A., A.J. Julian and D.G.
Method 966.04 “Sporicidal Activity of Ahearn. 1975. “The Survival and Growth
Disinfectants.” In: Official Methods of of Microorganisms in Mascara During
Analysis of AOAC International. Gaithers- Use.” Am J. Ophthal 79(4): 596-601.
burg, MD.
10. Wilson, L.A. and D.G. Ahearn. 1977.
“Pseudomonas-Induced Corneal Ulcers
Associated with Contaminated Eye Mas-
caras.” Am J. Ophthal. 84:112-119.
SECTION 24
M-7
A Rapid Method for
Preservation Testing
of Water-Miscible
Personal Care
Products
Scope
1. Introduction
1.1 This procedure allows the rapid determination of preservative performance in water-
miscible personal care products. This procedure is intended to be used as a screening test
during product development to quickly differentiate between preservative systems that
may be capable of providing adequate preservation and those which have insufficient
anti-microbial activity to protect the product. It is not intended to provide the definitive
information on the adequacy of preservation of the final formulation. This information
is obtained through standard tests, such as those described in Methods M-3 (Section
20), M-5 (Section 22), and M-6 (Section 23).
This procedure may also be used to rapidly qualify products to which minor formulation
changes have been made. However, to assure that the product is adequately preserved,
more stringent criteria for the elimination of microorganisms over the course of the test
than those used in conventional tests should be adopted.
2.1 “Determination of Preservative Adequacy in Cosmetic Formulations” (Section 13).
3. Materials
SECTION 24: M-7 WATER-MISCIBLE
preservation data on Personal Care products.


Refer to the ATCC culture maintenance recommendations, available on their website1,
and to other sources3-5.
SECTION 24: M-7 WATER-MISCIBLE PERSONAL CARE PRODUCTS | CTFA MICROBIOLOGY GUIDELINES | 229
Storage of other organisms appropriate to the product under test in the original product
or incorporation of product into maintenance medium is often the only way to retain
its unique characteristics. This method is especially appropriate where the isolate is
subsequently inoculated into a similar material.
3.3 Test Media
• 0.85% Sodium Chloride Solution
• Sodium Chloride Peptone Solution (1% peptone in 0.85% saline)
Plating diluents serve to disperse the sample and dilute it to levels that
permit recovery of surviving microorganisms from an inoculated product
formulation. The choice of diluent depends on its ability to meet the
requirements of preservative neutralization (Section 4.1). The following are
examples of diluents that may be used:
• Eugon Broth
• Letheen Broth
• Soybean-Casein Digest Medium (Tryptic Soy Broth)
• Tryptone-Azolectin-Tween® (TAT) Broth
neutralization6. Other suitable diluents may be used.
• Eugon Agar
• Letheen Agar
230 | CTFA MICROBIOLOGY GUIDELINES | SECTION 24: M-7 WATER-MISCIBLE PERSONAL CARE PRODUCTS
• Microbial Content Test Agar
• Soybean-Casein Digest Agar Medium (Tryptic Soy Agar)
neutralization6. Other suitable agars may be used.
If the above agars do not support the growth of fungi, one of the following
agars may be considered:
• Malt Agar
Other suitable agars may be used.
must take place in the plate count diluent and/or the recovery growth agar6.
of the microorganisms from these dilutions is performed. Neutralization is verified if
microbial recoveries are similar. If one or more challenge microorganisms cannot be
recovered, the use of a higher dilution and/or the investigation of additional chemical
neutralizers may be considered6.
It is recommended that that a microbial content test (See Section 18) be performed
on the test sample prior to performing the preservative efficacy test. Verification of
neutralization of the antimicrobial properties of the test sample should be demonstrated

(See Section 4.1 and Reference 6) at the same time as the microbial content test.
inocula. Refer to the ATCC website1 for optimal growth media and conditions for
specific microorganisms.
for use. For reference strains such as the ATCC strains, no more than five
transfers from the stock culture are recommended7. Broth cultures should be
centrifuged and then re-suspended in the chosen suspending fluid. Microbial
growth on a solid medium is transferred to the chosen suspending fluid.
the chosen suspending fluid (See Section 3.3.1 above) and filtering the spore
suspension through sterile gauze or glass wool. Sterile glass beads can be used
as an aid in the dispersion of spores in the suspending fluid.
• 1x105 to 1×106 Colony-Forming Units (CFU) of bacteria per
gram of product
• 1-5×105 CFU of yeast per gram of product
• 1-5×105 CFU of mold spores per gram unit of product
The inoculum level for the challenge microorganisms should be
verified by standard microbiological techniques such as pour plate
methods. It is recommended that the volume of the inoculum be <
1% of the sample weight/volume and should not alter the character
of the product being challenged.
5.2 Product Challenge
culture challenge studies, it is recommended that closely related types of microorganisms
such as Gram-positive bacteria, Gram-negative bacteria, and yeasts and molds be pooled
separately.
All products should be thoroughly mixed manually or mechanically after inoculation to
distribute the challenge microorganisms uniformly. The volume of the inoculum should
not alter the character of the product being challenged. Challenged formulations should
then be stored at ambient temperature for the duration of the test.
To obtain rapid results, it is suggested that challenged formulations be sampled
for viable microorganisms at 1, 2 or 3 and 7 days following inoculation.
ensure that the sample is representative. In some cases, the inoculum can thrive
in “pockets” of growth in the formulation while other areas are relatively free
of microorganisms. Many aerobic microorganisms grow especially well at the
formulation-air interface. Often it is very difficult to break up the “pockets”
of growth, and special procedures are needed. The following mixing methods
have been used to overcome this problem:
size of at least one gram or one milliliter of product for the quantitative pour
plate method is recommended.

to obtain the number of microorganisms per gram.
similar to the pour plate method. However, an aliquot of each
to obtain the number of microorganisms per gram.
Type Microorganism* (ATCC1 Number) Recommendation

Fermentative Gram-Negative Bacilli Klebsiella pneumoniae (10031) Select at least one

Enterobacter cloacae (13047))


Penicillium species
Other organisms relevant Optional

to the product
*Staphylococcus aureus (6538), Escherichia coli (8739), Pseudomonas aeruginosa (9027), Candida albicans (10231) and Aspergillus
niger (16404) are specified in the United States Pharmacopeia (USP) Antimicrobial Effectiveness Testing method2.
Table 24-1
Cultures Media** Temperature Time

Bacteria Soybean-Casein Digest Medium/Soybean- 30-37°C 18-48 hours
Nutrient Broth/Agar
Eugon Broth/Agar

Soybean-Casein Digest Medium/Soybean-

Malt Extract Agar
** Available in dehydrated forms from commercial sources.

Table 24-2
Table 24-3: Incubation Conditions for Recovery of Microorganisms
Cultures Media Temperature Time
Bacteria 30-37°C 24-72 hours
Yeasts For recovery agars, see Section 3.3.3 25-35°C 48-72 hours
Molds 20-30°C 3-7 days
Table 24-3
REFERENCES
1. ATCC recommends many different me- 4. Kirsop, B.E., and A. Doyle, (Ed). 1991.
dia in order to provide optimal condi- Maintenance of Microorganisms and Cul-
tions for growing its microbial cultures. tured Cells, 2nd edition. New York, NY:
The formulations for these media are part Academic Press.
of their catalog database, which can be
5. Simione, F. P. 1998. “Cryopreservation
searched for any medium recommended
Manual.” Nalgene Nunc International,
in an ATCC strain description. Formula-
http://www.nalgenelabware.com/techda-
tions for recommended cell culture media
ta/technical/manual.asp
are not included in the database, but the
on-line catalog description for each cell 6. ASTM International. 2007. ASTM E
line has details about the appropriate me- 1054-91, “Standard Practices for Evaluat-
dium. Media formulations found via the ing Inactivators of Antimicrobial Agents
internet search are not ready-to-use prod- Used in Disinfectant, Sanitizer, Antiseptic,
ucts for sale by the ATCC. The catalog is or Preserved Products.” In: Annual Book
no longer published in hard copy. Visit of ASTM Standards. West Conshohocken,
http://www.atcc.org; the search page for PA.
media formulations in the on-line catalog
is http://www.atcc.org/common/catalog/ 7. Reichgott, Michael. 2003. “Reference
media/mediaIndex.cfm . Strains: How Many Passages Are Too
Many?” ATTCC Connection 23, No. 2,
2. United States Pharmacopeia. 2007. <51> http://www.atcc.org/common/docu-
“Antimicrobial Effectiveness Testing.” ments/pdf/tb06.pdf
United States Pharmacopeia and the Na-
tional Formulary. USP30 – NF25. Rock- 8. AOAC International. 2000. Official
ville, MD. 79-81. Method 966.04, “Sporicidal Activity of
Disinfectants.” In: Official Methods of
3. Brown, M.R.W., and P. Gilbert, (Ed). Analysis of AOAC International. Gaithers-
1995. Microbiological Quality Assurance: burg, MD.
A Guide Towards Relevance and Reproduc-
ibility of Inocula. Boca Raton, FL: CRC
Press.
Glossary of
GLOSSARY
Microbiological
Terms
This glossary provides definitions for Agar

microbiological and associated terms used a gelatinous colloidal extract from algae con-
throughout the cosmetic industry. Its purpose sisting of agarose and agaropectin that is
is to assist individuals in understanding used in microbial growth media to make it a
the terms used in the CTFA Microbiology semi-solid at room temperature.
Guidelines.
Air Sampling
a technique by which the quantity of viable
microorganisms or particulate matter present
A in a volume of air is determined or isolated.
Action Level
level or range that, when exceeded, indicates
Alert Level
that a process has deviated from its normal op-
a level or range that, when exceeded, warns
erating condition, and that requires corrective
that a process may have deviated from its nor-
action to be taken to bring the process back
mal operating condition.
into its normal operating condition.
Ambient Air
Adaptation
environmental or room air.
a change or changes in an organism or pop-
ulation of organisms, through which the
Anaerobic
organism(s) become more suited to the pre-
able to grow in the absence of oxygen.
vailing environmental conditions.
Anhydrous
Add-on
without water.
in a non-woven substrate based product, the
formulation added to the substrate, e.g., a lo-
Antimicrobial
tion, solution, emulsion, oil, or other mate-
a chemical agent, either produced by a micro-
rial.
organism or by synthetic means, that is ca-
pable of killing or suppressing the growth of
Aerobic
microorganisms.
requiring oxygen for growth.
Antimicrobial Preservative Efficacy Test
See Preservation Test.
GLOSSARY | CTFA MICROBIOLOGY GUIDELINES | 237

Antiseptic Biofilm
a substance for use on living tissue that either a complex structure consisting of diverse mi-
GLOSSARY
destroys or inhibits the growth of microorgan- crocolonies of various microorganisms embed-

isms. ded in a matrix of extracellular organic poly-
mers adhering to moist surfaces.
Aqueous
containing water. Biological Indicator
a characterized preparation of a specific micro-
Aseptic organism resistant to a particular sterilization
free of microorganisms that are capable of process.
causing infection or contamination.
Biostatic Activity
Aseptic Technique the ability of a chemical agent or a physical
precautionary measures taken in microbiologi- condition that inhibits the growth of microor-
cal work to prevent the contamination by ex- ganisms.
traneous microorganisms.
Bulk in Process
Atypical Product a product in a partial state of completion or
a product in which water is not readily avail- finished goods before filling.
able to provide an environment that supports
growth of microorganisms, a product in which
the water activity is too low to support growth, Bulk Product
or a product having other physico-chemical product that has completed the processing
characteristics that do not allow growth of mi- steps up to final packaging but has not been
croorganisms. placed in the final package.
B C
Bactericide CFU
a chemical or physical agent that destroys vi- See Colony-forming Unit
able bacteria.
Calibration
Bacteriostat the set of operations and conditions that es-
an agent that inhibits the growth of bacteria. tablishes the relationship between values pro-
duced by a measuring instrument or system,
or values obtained from a material measure,
Bacterium (pl. bacteria) and the corresponding values from a known
a single-celled, prokaryotic microorganism reference standard.
that multiplies by cell division.
Challenge Organisms
Biochemical Characteristics microorganisms used in preservative challenge
biochemical reactions that are indicative for a tests.
particular microbial species.
Challenge Test
Biocide See Preservation Test.
a chemical or physical agent that destroys all
viable microorganisms.
238 | CTFA MICROBIOLOGY GUIDELINES | GLOSSARY

Chlorination Contact Time
the use of chlorine or chlorine-donating com- the time during which a microorganism or mi-
GLOSSARY
pounds to effect sanitization or to control microbial growth medium is in the presence of a
crobial levels. test surface or chemical agent.
Cleaner Contamination
a chemical or blend of chemicals formulated the presence of undesirable organisms.
to remove undesirable soils from a surface;
may be a solvent, acid, base, detergent, and/or Culture, Fresh
water-based mixture. a population of a single species of a microor-
ganism that has been recently cultivated either
Cleaning in a liquid medium or on an agar medium.
the process of separating and eliminating gen-
erally visible dirt from a surface, accomplished Culture Maintenance
using, in variable proportions, chemical ac- a process that keeps a microorganism alive,
tion, mechanical action, temperature and du- uncontaminated, and without variation or
ration of application. mutation, so that it is as close as possible to
the original isolate.
Cleaning Agent
an agent designed to remove visible and Culture Medium
non-visible foreign matter from surfaces. a nutrient-containing liquid (broth) or solid
(agar) that supports the growth of microor-
Coliform Organisms ganisms.
gram-negative, nonspore-forming bacteria of
intestinal origin that ferment lactose with gas Culture, Mixed
formation. the presence of more than one species of mi-
croorganism in a culture medium.
Colony
a macroscopically visible growth of microor- Culture Slant
ganisms on a solid culture medium. an inclined agar medium in a test tube used
for growth of microorganisms.
Colony-forming Unit (CFU)
an organism or cluster of organisms that causes Culture Stability
a visible colony. maintenance of biochemical or other desirable
characteristics of bacteria or fungi over time
Compressed Air and through repeated subcultures.
air under pressure greater than that of the at-
mosphere.
D
Concurrent Validation
the generation of current test data that will be Dead End
used to document that a process or procedure See Dead Leg.
does what it is intended to do.
Dead Leg
Contact Plate any area within a piping system that allows
See RODAC Plate. material to accumulate and then stagnate.

Deionization
a water-treatment process that removes ions Filtration
GLOSSARY
from water by passing it through cationic and the removal of particulates from a fluid by an
anionic resin beds. appropriately sized filter.
Diluent Finished Goods

a medium or vehicle used to reduce a material any manufactured cosmetic product or com-
to a less concentrated form. ponent that is suitable for use, whether or not
it is packaged or labeled.
Disinfection
the destruction of disease-causing or objec- Finished Product
tionable microorganisms, with the exception a manufactured cosmetic product that has
of spores, on inanimate surfaces by chemical undergone all stages of production, including
or physical means. packaging in its final container as placed on the
market and made available to the consumer.
Distillation
a process that consists of driving gas or vapor Formalin
from a liquid or solid by heating and then con- a clear 37% aqueous solution of formalde-
densing to liquid. hyde.
Documentation Fungicide
records containing all relevant information a chemical or physical agent that kills fungi.
organized in an orderly and easily understood
format, usually applied to a process, method, Fungistat
or equipment usage. an agent that inhibits the growth of fungi.
Fungus
E a saprophytic, symbiotic, or parasitic, hetero-
trophic, eukaryotic microorganism, i.e., a yeast
Enteric Organisms or mold
microorganisms associated with the intestinal
tract.
G
Eucaryotic
a type of cell that has a well-defined nuclear Genotype
membrane. genetic material contained in the entire com-
plement of alleles (chromosomes).
F Genus (pl. genera)

a specific biological classification of very closely
Fecal Contamination related species of organisms, ranking between
adulterated by feces, often inferred from the a family and a species.
presence of coliform bacteria, but may be pres-
ent whether or not coliforms are present. Glutaraldehyde
a saturated dialdehyde that is chemically re-
Filamentous Fungus lated to formaldehyde.
an organism that exhibits mycelial or thread-
like morphology.

Gram-negative I
bacteria that retain a red color after the Gram
GLOSSARY
stain procedure. Indigenous
occurring naturally within a particular envi-
Gram-positive ronment.
bacteria that retain a violet color after the
Gram stain procedure. Innocuous
microorganisms commonly considered harm-
Gram Stain less.
differential staining procedure used for bacte-
rial classification. Inoculation
the introduction of microorganisms into a
Growth product formulation or onto a substrate.
an increase in the number of microorganisms.
Inoculum
Growth Phase material containing living microorganisms
See Log Phase. used for inoculation.
Installation Qualification (IQ)

H
a description of the physical characteristics,
Halogenated Compounds drawings, specifications, operating manuals,
chemical compounds containing either chlo- calibration of instrumentation, and verifica-
rine, bromine, iodine, or fluorine. tion of the proper installation of utilities for a
piece of equipment.
Heat Distribution
measurement of temperature uniformity with- Iodophore
in an autoclave chamber with empty and load- a chemical complex of iodine and a surface
ed chamber configurations. active agent that functions as a disinfectant
through slow release of iodine.
Heat Penetration
measurement of temperature uniformity with- Isolate (verb)
in items for sterilization of a loaded autoclave to separate a mixed population of microorgan-
chamber configuration. isms to obtain pure cultures.
Homogeneous Isolate (Noun)

uniform throughout, in structure or composi- a microorganism cultured from an ingredient,
tion. finished product, or the environment
Hostile Environment
any material or condition unfavorable for sur- L
vival or growth of microorganisms.
Log Phase
a pattern of microbial growth in which there
is an exponential increase in the number of vi-
able cells.

Lyophilized Motility
freeze-dried. the movement of a bacterial cell in any me-
GLOSSARY
dium.
M
N
Maintenance
support and verification operations, either pe- Natural Raw Materials
riodic or unplanned, intended to keep facility substances of plant, animal, or mineral origin
and equipment in proper working condition. that may be minimally processed before use.
Membrane Filter Negative Control

a pliable filter or filter unit containing pores of a sample in a test series without the test agent
a known size used to separate out microorgan- used as a standard of comparison in judging
isms from a fluid. experimental effects.
Membrane Filtration Neutralizing Agent

removal of particulates (including microorgan- a chemical substance added to a medium to
isms, depending on the filter=s rating) using a inactivate an antimicrobial agent.
membrane.
Microbes O
microorganisms, including bacteria, yeasts and
molds. Objectionable Organism
an organism that can be harmful to the user
Microbial Content based upon the nature of the product, its in-
the number of viable microorganisms present tended use and its potential hazard, or is able
in a specific volume or quantity of material. to compromise the physical integrity or ap-
pearance of the product.
Microbial Proliferation Rate
reproduction rate of microorganisms. Operational Qualification (OQ)
Microbiological Limits a list of critical components, operating ranges,

maximum microbial content and specific mi- as defined by the specification, and actual per-
crobial type restrictions established for raw formance for a piece of equipment.
materials or finished products.
Ozone
Microbiological Specification a highly reactive allotropic triatomic form of
a statement of microbial content limits. oxygen used in disinfection and deodoriza-
tion.
Mold
filamentous fungus. Ozonation
the addition of ozone to a water system to re-
Most Probable Number duce microbial levels.
a counting technique that utilizes a multiple
Adilution@ to extinction approach in micro-
bial growth medium and a mathematical for-
mula to estimate the number of microorgan-
isms present in a sample.

P Preservative
a chemical agent that kills microorganisms or
GLOSSARY
Package Compatibility prevents microbial growth
the absence of a detrimental interaction be-
tween a product and its package. Preservative Efficacy Testing
See Preservation Test.
Pathogen
a disease-causing organism. Preservative Failure
deterioration in the effectiveness of the pre-
Peroxygen Compounds servative system in a product that allows for
chemical compounds that contain the bivalent microbial growth or survival.
group O-O and are used as sanitizing agents.
Preservative System
Pipeline Pig the agent(s) incorporated into a product to re-
a device made of non-porous materials that is duce or prevent microbial growth.
used to remove and clean product from manu-
facturing pipelines; it fits the internal radius Procaryotic
of a pipe, is inserted, launched and moved a type of cell without a nuclear membrane.
through the pipe length pushed by air, prod-
uct or water. Process Water
treated water used as a raw material in the
Plate Count manufacture of a product.
the number of viable colony-forming units
(CFU) per plate; a method of determining Process Water System
how many CFU per measure (usually per gram a manufacturing system used to make process
or ml) of the sample being evaluated. water.
Positive Control Proliferation

a sample in a test series with a test agent that microbial growth.
has a known observable effect for use as a stan-
dard of comparison. Prospective Validation
the establishment of documented evidence that
Potable Water a process does what it purports to do, based on
water that is suitable for drinking. a preplanned validation protocol.
Pour Plate Purified Water

a plate count method in which a test material process water obtained by distillation, ion-ex-
is introduced and dispersed uniformly after change treatment, reverse osmosis, or other
the addition of molten agar medium to a Petri suitable means, the quality of which may be
dish. checked for specific chemical parameters, for
example using current USP or in-house re-
Preservation Test quirements.
a method in which a material is inoculated
with selected microorganisms to determine
the antimicrobial effectiveness of a preserved
or an unpreserved formulation.

Q Sampling Devices
tools and equipment used to aseptically sample
GLOSSARY
Quality Assurance materials, surfaces, or an environment.

those planned and systematic activities neces-
sary to provide confidence that a product satis- Sampling Personnel
fies given acceptance criteria individuals trained in proper sampling tech-
niques to prevent extraneous microbial con-
Quaternary Compound tamination.
a quaternary ammonium compound in which
the ammonium hydrogen atoms have been re- Sanitary
placed with organic radicals, generally used as hygienic.
a surface active agent and/or a biocide.
Sanitary Manufacturing Practice
R guidelines to maintain clean and sanitary con-

ditions within the manufacturing environ-
Raw Material ment.
any ingredient used in the manufacture of a
product. Sanitization
the process utilized to reduce viable microor-
Resistance ganisms on a surface to an acceptable level;
the ability of microorganisms to maintain vi- surfaces must be clean for the sanitization pro-
ability in adverse environmental conditions. cedure to be effective.
Retrospective Validation Sanitizer

the use of historical test data to document that a chemical agent used for sanitization of clean
a process does what it is intended to do. surfaces.
RODAC Plate Selective medium

Replicate Organism Detection and Counting a medium that allows the growth of certain
(RODAC); an agar plate used for determining types of microorganisms in preference to oth-
surface contaminants by direct contact. ers.
Semi-quantitative
ess than quantitative measurement, often re-
S ferring to a measurement on an arbitrary scale,
e.g., 0 to ++++.
Sample
one or more representative portions or items Shelf Life
selected from a set to obtain information about a period of time during which a raw material
that set. or finished product may be stored and remain
suitable for use.
Sampling
operations relating to the taking and prepara- Species
tion of samples. one kind of microorganism; a subdivision of
a genus.

Specification Suitable Medium
clear and accurate description of the essential a medium used for optimal growth of mi-
GLOSSARY
technical requirements for items, materials, or croorganisms, or one that will suppress the
services; in the microbiology laboratory, often growth of certain organisms, while allowing
refers to a statement of microbial limits. the growth of Atarget@ organisms.
Spore Surface Monitoring

a highly resistant form of a microorganism, periodic determination of the microbial con-
e.g., mold or bacilli. tent of a surface.
Spread Plate Susceptible

a plate count method in which a small volume subject to microbial contamination.
of test material is dispersed, by means of a ster-
ile spreader, over the entire surface of a solid Swab
agar medium in a Petri dish. a wad of absorbent material usually wound
around or attached to the end of a small stick
Stabile Product or applicator and used for removing material
a finished product that maintains acceptable or microorganisms from a surface area. See
characteristics over a specified time and under also Transport Swab.
defined environmental conditions.
Swabbing
Standard Operating Procedures the process of wiping a surface with a moist,
sterile applicator, in order to collect viable mi-
written procedures detailing how to operate croorganisms.
equipment or execute a process.
Sterile T
free from viable microorganisms and spores.
Taxonomy
Sterilization the classification of organisms, based on mu-
the use of either physical or chemical agents to tual similarities.
destroy all viable microorganisms and spores
from a material or equipment. Total Plate Count
See Plate Count.
Streak Plate
a method in which inoculum is spread across Transport Swab
the surface of a prepared solid agar medium in a swab designed to maintain, under specified
a Petri dish, to produce isolated colonies. conditions of time and temperature, the vi-
ability and numbers of microorganisms recov-
Subculture ered from a sampling procedure.
preparation of a fresh culture from an existing
culture. Trend Analysis
review and analysis of routine data for patterns
Submicron Filtration and variations.
a filtration process that uses a filter with a pore
size smaller than 1 micron.

U Verification
evidence that establishes or confirms that the
GLOSSARY
Ultrafiltration specified application of a process or test meth-

a water-treatment process in which a selective od consistently and accurately does or mea-
permeable membrane filter is used to separate sures what it is intended to do or measure; evi-
dissolved molecules based on size. dence that establishes or confirms the accuracy
of a procedure or criterion.
Ultraviolet
referring to electromagnetic radiation with Viable
wavelengths shorter than visible light and lon- capable of living.
ger than X-rays, generally between 200 and
400 nanometers.
W
Ultraviolet lamp
a light that produces ultraviolet radiation for Waste
disinfection. any material or product that its holder intends
for disposal.
V Water Activity
the ratio of the vapor pressure in a product to
Validation that of pure water; this ratio is used to evaluate
substantiation and verification that a specific the susceptibility of a water-based product to
process or test method consistently does what microbial contamination.
it is intended to do.
Wipes
Validation Protocol products consisting of a nonwoven matrix or
an approved written plan that details the substrate that is composed of fibers or fila-
means by which validation will be achieved ments that are bonded together mechanically,
and defines the acceptance criteria for a pro- thermally, or chemically and used for the de-
cess or test method. livery of cosmetics or other product systems
Vectors
carriers of microorganisms. Y
Vegetative Bacteria Yeast
viable bacterial cells not in a resting state. a single-cell fungus that reproduces primarily
by budding.

CTFA technical guidelines
CTFA Microbiology Guidelines

CTFA
Microbiology
Guidelines
CTFA

2007

Phone: 202/331-1770 Fax: 202/331-1969 www.ctfa.org

CTFA
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CTFA technical guidelines

CTFA Microbiology Guidelines

The Cosmetic, Toiletry, and Fragrance Association

1101 17th Street, N.W., Suite 300

Phone: 202/331-1770 Fax: 202/331-1969 www.ctfa.org

280416 Cover.indd 1 3/6/08 9:31:54 PM

Printed in the United States of America

1. Microbiological Quality Assurance for the Cosmetic Industry ...............................3

2. Microbiological Evaluation of the Plant Environment* .......................................11

3. Cleaning and Sanitization ...................................................................................29

4. Microbiology Staff Training* ...............................................................................55

5. Handling, Storage and Analysis of Raw Materials ................................................69

7. Microbiological Quality for Process Water...........................................................81

8. Microbiology Laboratory Audit* .........................................................................95

9. Microbial Validation and Documentation .........................................................109

10. Maintenance and Preservation of Test Organisms ..............................................129

11. Raw Material Microbial Content.......................................................................139

12. Establishing Microbial Quality of Cosmetic Products* ......................................143

13. Determination of Preservative Adequacy in Cosmetic Formulations ..................149

14. Preservation Testing of Eye Area Cosmetics .......................................................151

15. Microbiological Assessment of Product Quality After Use*................................155

16. Microbiological Risk Factor Assessment of Atypical Cosmetic Products* ...........163

17. Determination of Preservation Efficacy in Nonwoven Substrate Products .. 173

18. M-1 Determination of the Microbial Content of Cosmetic Products ................179

20. M-3 A Method for Preservation Testing of Water-Miscible Personal Care

23. M-6 A Method for Preservation Testing of Atypical Personal

24. M-7 A Rapid Method for Preservation Testing of Water-Miscible Personal

25. Glossary of Microbiological Terms ....................................................................237

Pamela G. Bailey John E. Bailey, Ph.D.

Because of the increasing dependency on microbiological laboratories to provide supportive data

As an alternative to manufacturing sterile products, the consideration of rational limits to

CTFA MICROBIOLOGY GUIDELINES | INTRODUCTION | 1

2 | INTRODUCTION | CTFA MICROBIOLOGY GUIDELINES

SECTION 1: QUALITY ASSURANCE

Quality Assurance Microbiology Laboratory

SECTION 1: QUALITY ASSURANCE

FOR THE COSMETIC INDUSTRY

Plant and Grounds

seals, defective sight glasses.

abrasions on the hands should be covered.

• Atmosphere - Dust is laden with airborne microorganisms.

SECTION 1: QUALITY ASSURANCE

FOR THE COSMETIC INDUSTRY

PACKAGING MATERIALS AND OTHER COMPONENTS

Microbiological Quality Assurance Laboratory

SECTION 1: QUALITY ASSURANCE

The Cosmetic, Toiletry, and Fragrance

10 | CTFA MICROBIOLOGY GUIDELINES | SECTION 2: EVALUATION OF THE PLANT ENVIRONMENT

SECTION 2: EVALUATION OF THE

PLANT ENVIRON MENT

SECTION 2: EVALUATION OF THE PLANT ENVIRONMENT | CTFA MICROBIOLOGY GUIDELINES | 11

12 | CTFA MICROBIOLOGY GUIDELINES | ANNEX 2 PREMISES (FACILITY)

Manufacturing and Filling Equipment

SECTION 2: EVALUATION OF THE

PLANT ENVIRON MENT

SECTION 2: EVALUATION OF THE PLANT ENVIRONMENT | CTFA MICROBIOLOGY GUIDELINES | 13

Cleaning and Sanitization

ENVIRONMENTAL MONITORING PROGRAM

14 | CTFA MICROBIOLOGY GUIDELINES | SECTION 2: EVALUATION OF THE PLANT ENVIRONMENT

SECTION 2: EVALUATION OF THE

PLANT ENVIRON MENT

SECTION 2: EVALUATION OF THE PLANT ENVIRONMENT | CTFA MICROBIOLOGY GUIDELINES | 15

susceptibility of the finished product to microbial contamination as well as other factors.