EFFECT OF QUATS ON FERTILITY OF MICE 1

Effect of Quaternary Ammonium Compounds on the Fertility of Mice

by

Ansley Adcock

Shelby Bryan

A Senior Thesis Submitted in Partial Fulfillment

of the Requirements for Graduation

Central Magnet School

April 2018

Thesis Committee:

Mrs. Eve Harrison

Mrs. Laura Roland

Dr. Melanie Thomas

Dr. Brad Chesney, M.D

Mr. Chris Click

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ACKNOWLEDGEMENTS

First, we would like to thank Dr. Brad Chesney for helping us establish our ideas and

pushing us to think outside the box. Without his guidance, completing this thesis would not have

been possible. We would also like to thank our senior english teachers, Dr. Thomas and Mrs.

Roland. The feedback we have received from the both of them has helped perfect and polish our

thesis.

Next, we would like to thank our advisor, Mr. Click. Thank you for supporting us as our

advisor for the past seven years and being one of the most helpful influences on our road to

graduation. Finally, we want to thank our Biomedical Innovations teacher, Mrs. Eve Harrison,

for her guidance and support throughout this thesis journey. We are extremely grateful for her

encouragement in times of need and criticism in times that we felt we could not improve any

further.

Completing our thesis has been a journey of insight, growth, failure, and reward; we are

so thankful for these people, and those not mentioned, for being the reason we have had the

ability to complete our senior thesis.

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TABLE OF CONTENTS

Acknowledgements………………………………………………………………………………2

Abstract…………………………………………………………………………………………..

Introduction………………………………………………………………………………..…..5-8

Research Question……………………………………………………………………………...…5

Research Purpose……………………………………………………………………………...…..5

Background Information………………………………………………………………………...5-8

Hypothesis………………………………………………………………………………...……….8

Methodology………………………………………………………………………………..…8-10

Survey………………………………………………....……………………………...………....8-9

Experiment……………………………………………………………………………………….10

Results…………………………………………………………………………………………….#

Discussion………………………………………………………………………………………...#

Conclusion………………………………………………………………………………………..#

Appendices……………………………………………………………………………………….#

References………………………………………………………………………………………..#
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ABSTRACT

The purpose of this research is to observe the effects of quaternary ammonium

compounds (QUATs), such as shampoos and detergents, on the length of female mice’ Estrous

cycles as well as their ability to sustain a pregnancy. Based on prior experiments and basic

research, it was believed that the mice exposed to QUATs would experience shortened Estrous

cycles and fewer pregnancies. The differences between the control and experimental groups,

however, were expected to be slight. For one month, ten mice, five being female, were exposed

to QUATs once per day, while ten were kept separated from the chemical mix. Each morning,

the ten female mice were studied to check for pregnancy and which stage of the Estrous cycle

she was in. After the duration of the month, the data collected was analyzed, showing fewer

pregnancies and shorter Estrous cycles in the mice exposed to quaternary ammonium

compounds. While these results were expected, such a large effect was not. For further support

of these results, a larger supply of mice and a longer duration of time should be considered. A

survey was also implemented during the duration of the experiment in which women were asked

about their familiarity with QUATs. The results of this survey concluded that only a small,

insignificant amount of women had any knowledge on these chemicals. To minimize the

potential harm of quaternary ammonium compounds, restrictions and reductions on their

prevalence in household products should be considered.

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INTRODUCTION
Research Question

Does exposure to quaternary ammonium compounds (QUATs) have a negative effect on

the fertility rate of mice?

Research Purpose

The purpose of this research is to test whether quaternary ammonium compounds affect

the maternal ability to achieve and sustain pregnancy, specifically in mice. Quaternary

ammonium compounds are commonly found in shampoos, cleaners, and soaps. These chemicals

have been loosely linked to pregnancy and fertility issues. To produce further research as well as

depth to prior research, the experiment will include testing chemicals on a group of mice by

spraying them with a water and quaternary ammonium compound solution. A recent study,

published in the journal of ​Reproductive Toxicology​, exposed mice to common QUATs and

found that after an eight week period the female mice ovulated less frequently and spent less

time in heat. This study will be used as the building block of the thesis’ research and the driving

force behind its purpose. To reiterate, there is minimal solid background information on the

effects of quaternary ammonium compound exposure, the previously mentioned study being the

only, so the purpose for this research is to strengthen or discredit the relationship of certain

chemical compounds on infertility rates.

Background Information

To begin, there is a significant lack of research on the effects of quaternary ammonium

compounds, specifically on women and mice. In women, infertility is a prevalent, alarming and

highly discussed topic. According to the CDC, around 6% of women suffer with complete

infertility while 12% suffer with low fertility (“Infertility”, 2016). While the 6% of completely
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infertile women often suffer from conditions such as Polycystic Ovary syndrome, Diminished

Ovarian Reserve, or Functional Hypothalamic Amenorrhea, the 12% of women with decreased

fertility could simply smoke, use alcohol excessively, gain or lose weight too quickly, or suffer

from a period of prolonged physical or emotional stress. Gynecologists and IVF doctors are

aware of these precursors to fertility loss and warn their patients, especially those trying to

conceive, to avoid them. However, the rate of infertility is still steadily increasing, rising from

5.4% (1984) to roughly 18% (2016). Doctors still struggle with finding the answer as to why

infertility occurs because there have been very few tests and experiments to find the root of

diminishing fertility. Recently, however, scientists conducted a study with results that have

linked a chemical group in common household products (such as shampoo, conditioner,

moisturizer, detergent, and hand soap) called quaternary ammonium compounds (QUATs) to a

loss in fertility in women and a spike in miscarriages and birth defects. The first study looked at

two commonly used QUATs- alkyl dimethyl benzyl ammonium chloride and didecyl dimethyl

ammonium chloride. Rats were exposed to these compounds while their fertility rates,

pregnancies, and offspring’s abnormalities were recorded. The results showed that the

chemically tested rats were less fertile and more likely to suffer from pregnancy related issues

(such as spontaneous abortions and birth defects). ​The birth defects mirrored similar defects

found in humans including Spina Bifida​ and Anencephaly​ (“​Chemicals Found in Detergents,

Shampoos Linked to Birth Defects”,​ ​2017). ​ ​Scientists predict that women in the healthcare and

food professions are at a heightened risk of the negative effects of these chemicals due to their

constant exposure to soaps and sanitizers, which is significant because it may provide an answer

to decreasing fertility.
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In another article, a chemical called TCC was tested on female lab mice. TCC, otherwise

known as triclocarban, is often found in soaps and hand sanitizers. Recently, however, the

chemical has been found in drinking water. In the study, scientists found that TCC from a

mother’s body can be transferred to the offspring and interfere with lipids, which then interferes

with the offspring’s developing organs and motor skills. The experiment tested the milk from a

mother mouse and portions of the placenta for TCC. The test was positive, as stated, and showed

that TCC exposed mice passed on chemicals to their offspring, and the offspring, and as a result,

the offspring had adverse reactions and birth defects (“Common Soap’s Chemical Exposure

During Pregnancy May Harm Babies in Womb”, 2017​)​. Lab animals were used in this

experiment because their reactions mirror that of humans, so these results are alarming because if

women are exposed to these QUATs as much as the mice were, it can be concluded that their

pregnancies and offspring would be affected in similar ways. Mice are the most common way to

test the effects of quaternary ammonium compounds because they are easily accessible and have

similar responses to humans without posing the liability that humans do. Another experiment that

used mice was conducted by researchers at Virginia Tech and Washington State University.

They fed the mice QUAT infused food daily and recorded their mating and pregnancy behaviors.

The scientists found that QUATs caused mice to have issues in their pregnancies and less

successful mating seasons as well as shortened estrous cycles. The estrous cycle in mice is a four

to five day length occurrence that has four main stages and hormone peaks (Byers, Wiles, Dunn,

Taft, 2012). If the estrous cycle is successful, a mouse will become pregnant. The scientists

found that many of the tested mice had unsuccessful estrous cycles, which indicates that they
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were most likely less fertile. The estrous cycle is similar to the menstrual cycle in women, which

makes applying the results of the experiment to humans accurate.

Another experiment related to mice estrous cycles in QUAT exposed mice found that after

eight weeks of testing, not only were female mice less fertile, male mice had also shown a

decrease in sperm concentration (Geller, 2015). A low sperm concentration, also known as

oligospermia, is the leading cause of infertility in men. This study concluded that QUAT

exposure is detrimental to the fertility of both genders of mice (Male Infertility and Sexual

Medicine Specialists, 2018).

Quaternary ammonium compounds are often seen in sanitizing products, but they can also

be found in cosmetics, which are most often used by women. Phthalates, a QUAT specifically

found in cosmetics has been linked to issues with hormone levels and fertility (“Chemicals in

cosmetics can cause infertility”, 2017). Exposure to these chemicals puts women at a much

greater risk for miscarriage and makes the baby more likely to have a birth defect.

Fertility is a pressing and frightening issue for women in modern society, and much is

still unknown about its origin and prevalence. Quaternary ammonium compounds are seemingly

everywhere- hand sanitizers, makeup, shampoos, detergents, and even drinking water. If the

effects of these chemicals are as threatening as the abundance of tests prove that they are, women

should be warned of their effects and their use in products should be as limited as possible.

Hypothesis

If mice are exposed to quaternary ammonium compounds (QUATs) for a four week

period then they will experience slightly shortened estrous cycles as well as fewer pregnancies as

compared to the mice that act without an independent variable (QUATs).

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METHODOLOGY

To test our hypothesis, we ran two tests. One test was a survey while the other was an

experimental study.

Survey

Our mentor, Dr. Brad Chesney, an OB GYN at Murfreesboro Medical Clinic,

administered the survey to his patients. The only requirements of the participants in our survey

was that they were all 18 years or older and female. Each participant was given the choice to take

part in our survey. The survey was written, and participants completed it prior to their

appointment with Dr. Chesney. The survey was random, meaning there were no names linked to

the answers. This survey can be accessed in appendix A.

We will be asking these questions to observe the knowledge that women in the age of

fertility have on these potentially harmful chemicals. The survey will be active for six weeks to

ensure that we will have enough participants. After the six weeks, we will collect all responses

from Dr. Chesney and transfer the results digitally. We will be running the survey and

experimental test with mice simultaneously, which we chose to do so that one of the reasons

behind our experimental test (QUATs are harmful, yet rarely discussed) would be supported and

represented by our survey.

Experiment

To begin, 20 feeder mice were purchased from Animal City Pet Center. The feeder mice

were chosen for th

is experiment because of their small size and ability to be easily observed. Ten mice were female

and the other ten were male. The mice were separated into two cages with five males and five
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females in each cage. One cage was the control group, kept separate from the independent

variable, while the other mice were sprayed with QUATs. We placed a dot of food coloring on

each mouse to differentiate the mice for individual data analysis purposes. We used the colors

brown, yellow, blue, purple, and pink on the experimental group. Black, orange, green, red, and

no color will be used to mark the control group. The QUAT exposed mice were sprayed with a

diluted alkyl dimethyl benzyl ammonium chloride mix once a day through a spray bottle. Food

and water were administered ad libitum. The mice were kept in a climate controlled facility set to

the recommended standards with food and water administered ad libitum. We observed the

female’s cycle lengths two weeks before we start the experiment and for a week prior to the

experiment’s conclusion to determine the average initial and average final estrous cycle lengths.

Once the experiment began, the females’ vulva was observed on a daily basis to ensure proper

data was taken to determine the stage of pregnancy/ stage of estrous cycle that the mice were in.

The estrous cycle is divided into four stages (​proestrus, estrus, metestrus, and diestrus)​ and it

repeats every four-five days unless it is interrupted by pregnancy. The experiment was run for 31

days to give time for ovulation, fertilization, and pregnancy. We did not directly study the

success of the pregnancies (i.e no miscarriages, spontaneous abortions, birth defects, etc.), but

rather the fertility (appearance of pregnancies, length of estrous cycle) seen in the mice. If the

mice experienced shorter estrous cycles and/or lower pregnancy rates, which indicates lower

fertility, and an adverse effect of QUATs on mice. If the mice experienced no change to their

estrous cycles, and thus fertility, it is assumed that QUATs had no effect on them. At the end of

the 31 days, we compared the data from both the experimental and control groups to see if there

was any significance. Then, we transferred and represented our data digitally.
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RESULTS

We surveyed 122 women ranging in age from 19-68. Out of the 122, 54.9% are currently

pregnant, and 22.1% have been pregnant within the last ten years. Though there are many

potentially dangerous hygiene products, only 69.7% had been warned against using certain

products. And even more alarming, only 1.6% of the 122 women surveyed had any knowledge

on QUATs.
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Each morning for the duration of the experiment, the ten female mice were observed. The

stage of the estrous cycle in which they were in was recorded. If they had died, we marked that

day, and the rest after that, with ‘DIED’. The graph is pictured below.
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DISCUSSION

Does the exposure to quaternary ammonium compounds adversely affect the fertility and

pregnancy rates in mice? This was the driving question of our research. From the only previous

experiment related to our topic, scientists concluded that by mixing QUATs into the daily food

regimen of mice, they were less likely to successfully reproduce. From this data, we anticipated

similar results. Though we did not plan to have our mice digest the QUATs directly, we

hypothesized that any exposure to QUATs would cause reduced fertility and pregnancy rates as

it did in the previous experiment. After our 31 day experiment, including daily exposure of

QUATs on the mice, our hypothesis was confirmed. We found that there were significantly

fewer pregnancies in the mice exposed to QUATs than those that were in the control group. The

mice exposed to the independent variable also had a shorter and more irregular estrous cycle,

which is linked to low fertility.

Analysis of Data

We tested five total female mice with the QUAT mixture, and out of those five, only one

had been/was pregnant at the end of the thirty one day experiment. As of our control group, all

five female mice were impregnated at some point of our test. We also noticed that the female’s

estrous cycles in the experimental group became extremely short and irregular by the termination

of the test. While these female mice should have been going through four to six day long cycles,

spending most of those days in the metestrus stage, they were instead experiencing two to three

day long cycles that were almost impossible to determine the start and end of due to their

irregularities. At the start of our experiment, the five female mice exposed to the QUATs had an

average cycle length of 5.4 days, and by the end of their exposure to QUATs, their cycle length
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was, on average, 2.6 days. The control group started with an average cycle length of 5.3 days and

were all pregnant by the termination of our experiment, so we were unable to note any changes

in their cycle length. However, their reproductive success was 100% compared to the

experimental group’s 20%. We did have one mice die in the control group on day 11 to what we

believed was a pregnancy complication. It is important to note that we did not lose any males and

had an equal amount (five) in either cage during the entire duration of our test.

To reiterate, we surveyed 122 women ranging from ages 19-68. The majority of

participants had no knowledge of QUATs nor the harmful side effects of these chemicals. The

few that did have prior knowledge of QUATs had majored in biochemistry. The data we

accumulated through the surveys is significant as it proves the limited amount of research done

to prove QUAT exposure lowers fertility rates is inhibiting doctors from warning patients against

these chemicals.

Limitations

There we multiple limitations to our experiment. Due to limited finances and space, we

were unable to obtain the amount of mice that we had hoped. It would have made our experiment

more accurate if we could have tested double or triple the amount of mice we had, especially

females. We also had issues with the general health of our mice prior to the initiation of our

experiment. Finding the adequate caging, bedding, food, and water sources for our mice was trial

and error. The initial cedar shavings we used as bedding were insufficient and after research we

concluded that the shavings were linked to possible liver disease. We also originally used water

bowls, but quickly noticed the mice could not reach the water so we had to implement a hanging

water bottle. Before making these minor changes, we lost five mice, which we had to replace
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before the start of our experiment. Time was also a limitation for us. Our results were still

significant, but if we had a longer amount of time to test on the mice we would not only have had

more confidence in our results, but also the ability to answer our hypothesis and any further

questions in more depth.

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CONCLUSION

This experiment has shown that there is a strong effect of quaternary ammonium

compounds on the fertility rate of mice. The mice exposed to QUATs were five times less likely

to become pregnant than those unexposed and had estrous cycles shortened, on average, by 49%

as compared to their initial lengths. Though these chemicals are proven to be harmful to the

fertility and reproductive capabilities of mice, they did not cause an increase in mortality. The

reproductive nature is similar between mice and humans. To prevent any harm on fertility in

women, precautions should be implemented on the usage of QUATs in everyday household

products and possible alternatives should be sought. The survey responses indicated an alarming

lack of knowledge on QUATs from women in the reproductive age. To combat this issue,

doctors should be trained and briefed on the threats and usages of these compounds so they are

able to better explain QUATS better to their patients and possibly warn against them.

If this experiment was redone, there would need to be a greater number of mice tested, as

well as a longer period of time to acquire more significant and reliable results. I would also be

very interesting to test different types of QUATs, since this experiment only tested the effects of

alkyl dimethyl benzyl ammonium chloride. This experiment would also be better suited in a

more sophisticated setting equipped with professional tools; however, the funds were not

available for that.

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APPENDIX A
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References

Byers S.L, Wiles, M., Dunn, S., and Taft, R. (2012). Mouse Estrous Cycle Identification Tools

and Identification. Retrieved from

http://citeseerx.ist.psu.edu/viewdoc/citations;jsessionid=2EAA4CB3AFA034A545039AC11

387A980?doi=10.1.1.270.970

Center for Disease Control. (2016).​ National Infertility Rate​ [Data file]. Retrieved from

https://www.cdc.gov/nchs/fastats/infertility.htm

Chemicals found in detergents, shampoos linked to birth defects. (2017, June 17). ​IANS​.

Retrieved from

http://link.galegroup.com/apps/doc/A495734651/GPS?u=tel_k_cmsmb&sid=GPS&xid=e

13f1fae

Common soap chemical exposure during pregnancy may harm babies in womb. (2017, August

5). ​Indo Asian News Service. ​Retrieved from

http://link.galegroup.com/apps/doc/A500333509/GPS?u=tel_k_cmsmb&sid=GPS&xid=993

a45b0

Geller, S. (2017, July 31). Disinfectant Mix in Cleaning Products Linked to Birth Defects in Lab

Animals. Retrieved From

https://www.ewg.org/enviroblog/2017/07/disinfectant-mix-cleaning-products-linked-birth-d

efects-lab-animals#.WjbULrpFzug

Low Sperm Count Treatment | Increase Sperm Count | San Diego, California. (n.d.). Retrieved

March 18, 2018, from

https://www.malefertility.md/male-infertility/sperm-disorders/low-sperm-count-treatment
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