UNIVERSITY OF CINCINNATI

Date: 15-Jan-2010 I, Beverly Teal Gaultney hereby submit this original work as part of the requirements for the degree of: ,

Master of Science
in

Industrial Hygiene (Environmental Health)

It is entitled:

Determination of Urinary 2-naphthol Concentration in Rubber Manufacturing Workers

Student Signature:

Beverly Teal Gaultney

This work and its defense approved by: Committee Chair:

Glenn Talaska, PhD
Glenn Talaska, PhD

Paul Succop, PhD

Paul Succop, PhD

Andrew Maier, PhD, MS

Andrew Maier, PhD, MS

2/19/2010

402

Determination of Urinary 2-Napthol Concentration in Rubber Manufacturing Workers

A thesis submitted to the Graduate School of the University of Cincinnati In partial fulfillment of the requirements for the degree of Master of Science in Industrial Hygiene (Environmental Health) in the Department of Environmental Health of the College of Medicine by

Beverly Teal Gaultney

B.S. University of Georgia December 2005

Committee Chair: Glenn Talaska, Ph.D.

ABSTRACT Polycyclic aromatic hydrocarbons (PAHs), which consist of 2 or more fused benzene rings, are formed by the incomplete combustion of fossil fuels. Although PAHs are an exposure concern in many industrial sectors, one industry of particular interest is the rubber industry. PAHs make up around 20% of the total weight of rubber products including tires. For tire manufacturing, PAH exposures arise when they are added as extender oils and they may also be present when carbon black is used to produce the master batch material (IARC, 1982). Such exposures have been of public health interest because epidemiology studies of workers exposed to PAHs have identified an increase risk of developing cancers of the lungs, urinary bladder, skin, and prostate (Bofetta et al 1997; Constantino et al 1995). Naphthalene is the simplest and most volatile member of the PAHs. The major route of elimination of naphthalene is via the formation of the hydroxylated metabolites 1- and 2- naphthol which are excreted through the urine (Onyemauwa et al 2009; Serdar et al 2004; Preuss et al 2003). The hypothesis of this thesis is that Post-shift urinary 2naphthol levels are significantly higher than pre-shift urinary 2-naphthol levels in rubber workers exposed to naphthalene.

Acknowledgements Thank you to Dr. Paul Succop, Dr. Andrew Maier, and Dr. Glenn Talaska. It was a privilege to have your assistance and advice during the completion of this thesis. I am glad that such a skilled group offered its time to helping me better this body of work. Each of you has helped me, and I appreciate all of your time and effort.

1.0

INTRODUCTION

Polycyclic aromatic hydrocarbons (PAHs), which consist of 2 or more fused benzene rings, are formed by the incomplete combustion of fossil fuels. PAHs are common environmental pollutants due to the wide range of sources that generate them, including cigarette smoke, cooked foods, forest fires, motor vehicle exhaust, air traffic, and nearby industrial plants. Workers are also exposed to PAHs present in raw materials used in the production of many products such as distillation and crystallation of coal tar, coking plants, intermediates of the pharmaceutical and chemical industries, graphite electrode plants, production and installation of fireproofing materials, and numerous other industries which have lower levels of PAH exposures (US EPA 1998 and Preuss et al 2003). Although PAHs are an exposure concern in many industrial sectors, one industry of particular interest is the rubber industry. PAHs make up around 20% of the total weight of rubber products including tires. For tire manufacturing, PAH exposures arise when they are added as extender oils and they may also be present when carbon black is used to produce the master batch material (IARC, 1982). PAHs are also formed during the incomplete combustion of organic rubber products particularly during vulcanization and curing (Talaska et al 2002, Jonsson et al 2008). Such exposures have been of public health interest because epidemiology studies of workers exposed to PAHs have identified an increase risk of developing cancers of the lungs, urinary bladder, skin, and prostate (Bofetta et al 1997; Constantino et al 1995). This increased risk of cancer among other PAH-exposed workers is consistent with the increased incidence of cancer among rubber workers, which has been well documented (Monson and Nakano 1976a, Monson and Nakano 1976b, Delzell and Monson 1981, Fine and Peters 1976, and IARC 1982).

PAH exposures are typically in the form of complex mixtures, but in some industries individual chemicals may predominate or may be able to be used as a marker for a broader PAH exposure. Naphthalene is the simplest and most volatile member of the PAHs. It consists of 2 fused benzene rings, and in most indoor and outdoor environments it is most frequently encountered as a vapor, although it may also be bound to particulate to some degree (Price et al 2008). Cigarette smoke has been found to be one of the most significant sources of naphthalene exposure in the general population (Hoffman et al 2001), and the EPA confirms that the environmental exposure to naphthalene is high compared to that of the other PAHs, with the main route of exposure being inhalation (Price et al 2008; Wilhelm et al 2008). In workplaces where PAH exposure is present, naphthalene is generally the most abundant compound present in the PAH emissions (Rappaport et al 2004; Preuss et al 2003). The amount of naphthalene exposure increases when PAH containing materials are exposed to heat, and this is of particular interest in the rubber manufacturing industry in the curing department where the rubber is heated to around 130C until the curing is complete (Fine and Peters, 1976). Rubber may continue to off gas naphthalene during the inspection process as it continues to cool. The major route of elimination of naphthalene is via the formation of the hydroxylated metabolites 1- and 2- naphthol which are excreted through the urine (Onyemauwa et al 2009; Serdar et al 2004; Preuss et al 2003).

In 2000, the National Toxicology Program published the results of two-year inhalation cancer bioassay in F344/N rats. They concluded that under the conditions of this 2-year inhalation study, there was clear evidence of carcinogenic activity* of naphthalene in male and female F344/N rats based on increased incidences of respiratory epithelial adenoma and olfactory epithelial neuroblastoma of the nose (NTP, 2000). In addition a 1992 2-year inhalation study of male and female B6C3F1 mice found that there was some evidence of carcinogenic activity in the female was based on increased incidences pulmonary

alveolar/bronchiolar adenomas (NTP, 1992). The results of these rodent studies increased concern regarding the carcinogenic potential of naphthalene in the scientific community and led to a reclassification of naphthalene as a possible human carcinogen by the EPA and IARC (Preuss et al 2004; Preuss et al 2003; Rappaport et al 2004; EPA 2003; IARC 2002). Prior to the publication of these studies, naphthalene was not considered a significant concern for cancer in the occupational environment because it was thought to be non-carcinogenic, tolerable levels above set exposure limits were too high to be exceeded in the workplace, and sampling at workplaces revealed levels below current occupational exposure limits, which were based on non-cancer effects (Preuss et al 2003). As the potential carcinogenicity of naphthalene and its underlying mode of action have been actively debated, more emphasis has been given to sampling the compound in the workplace where it was found to be one of, if not the most, abundant chemical associated with PAH exposure. There is an increasing need to develop robust monitoring approaches for exposures to naphthalene based on the increased regulatory and public emphasis on naphthalene and its abundance in processes that involve PAHs. Urinary Naphthols have been suggested as a biomarker of general PAH inhalation exposure due to their specificity of reflecting inhalation based exposures. Urinary naphthols were not found to correlate with dietary habits, as 1HP is, but were correlated with smoking indicating an inhalation route specific marker (Yang et al 1999; Kang et al 2002; Kim et al 2001; Onyemauwa et al 2009). With bladder cancer being a prevalent illness in the rubber industry the fact that inhaled naphthalene is metabolism in the urinary bladder makes biomonitoring of interest for researchers.

In 2008 a study entitled Polycyclic Aromatic Hydrocarbon Exposure, Urinary Mutagenicity, and DNA Adducts in Rubber Manufacturing Workers DNA Adducts were seen in the urothelial cells and in the 7

peripheral blood mononuclear cells of PAH-exposed rubber workers. These adducts did not correlate to levels of 1-hydroxypyrene, a common biomarker for PAH exposure, or with measures of urinary mutagenicity. The results from this study suggest that some other DNA-reactive agent may better correlate to the observed adduct formation in these workers (Peters et al 2008). As an alternative to PAHs as reflected by 1-hydroxypyrene, naphthalene may be an inhalation exposure in these workers that would correlate better with the urinary mutagenicity and the presence of urothelial cell adducts. Since the peripheral blood mononuclear cells and urothelial cell adducts were not correlated to each other, it may suggest that there is a specific bladder carcinogen in the rubber industry which remains unidentified (Peters et al 2008). Since naphthalenes major route of elimination is through oxidized metabolites, 1- and 2-naphthol, excreted through the urine one or both of these metabolites may be measured to determine if there is a possible correlation between their concentration in urine and levels of urothelial cell DNA adducts. As a first step in investigating this possibility we evaluated whether there is an increase in mean levels of 2-naphthol from pre-shift to post-shift samples to confirm this metabolite as a valid biomarker for naphthalene exposure in rubber workers. This information may be used in future studies to examine correlations between exposure, adducts and urinary mutageniticty as described in the report by Peters et al (2008) and to compare differences in biomarker levels between differing departments within the rubber working process as a means to identify areas with greatest potential exposures.

2.0

HYPOTHESIS

Post-shift urinary 2-naphthol levels are significantly higher than pre-shift urinary 2-naphthol levels in rubber workers exposed to naphthalene.

3.0

MATERIALS AND METHODS

Materials used to perform analysis of urine samples for 2-naphthol included:

0.1M Sodium Acetate (Pure Sodium Acetate (1M solution =82.03g/l) was diluted 1:10 by adding 8.203 g and adding to 100ml water to the graduated cylinder yielding a 0.1M solution=8.203g/100ml) (Fisher Scientific, Pittsburgh, Pennsylvania) -glucuronidase/arylsulfatase (G-0876 Type h-2 from Helix pomatia, 105,000 -glucuronidase units/ml and 4,300 arlysulfatase units/ml , Sigma) 2-naphthol (CAS#135-19-3, Alfa Aesar, 98+% 2-Naphthol, stock #: A14564, lot#H4478A, Fisher Scientific, Pittsburgh, Pennsylvania ) HPLC Grade Methanol (CAS#67-56-1, Fisher Scientific, Pittsburgh, Pennsylvania) Milli-Q (ultra pure) water (double deionized water) Analytical nitrogen evaporator with nitrogen tank with nitrogen regulator turned to pressure of approximately 50psi Water bath (contained at bottom of analytical nitrogen evaporator) Refrigerator 37C Incubator (Fischer Scientific Model 630D, Fisher Scientific, Pittsburgh, Pennsylvania) Shaking platform High Performance Liquid Chromatograph (HPLC) (2695 separation module, Waters) with Fluorescence detector (730 Water Data Module fluorescence detector) connected to a PC with Empower 2 HPLC software 10

Jack Berberichs Milk crate 50ml Screw cap tubes (samples were received in these) 25ml glass Scintillation vials (Fisher Scientific, Pittsburgh, Pennsylvania) Gilson Micropipettes (20ul, 5ml) (Fisher Scientific, Pittsburgh, Pennsylvania) Pipette tips (Fisher Scientific, Pittsburgh, Pennsylvania) Test tube racks (Fisher Scientific, Pittsburgh, Pennsylvania) Styrofoam holder for 25ml vials 20ml syringes (Fisher Scientific, Pittsburgh, Pennsylvania) 0.45 um filters (Fisher Scientific, Pittsburgh, Pennsylvania) 3ml syringes (Fisher Scientific, Pittsburgh, Pennsylvania) C18 Sep Pak Cartridges (Part No. WAT020515 Waters Corporation, Milford, Massachusetts) Tape (Fisher Scientific, Pittsburgh, Pennsylvania) Sharpie Markers (Fisher Scientific, Pittsburgh, Pennsylvania) Plastic tray (Fisher Scientific, Pittsburgh, Pennsylvania) Weigh boat (Fisher Scientific, Pittsburgh, Pennsylvania) Balance ( Denver Instruments model TR403, Fisher Scientific, Pittsburgh, Pennsylvania)

Sample Acquisition A total of 159 samples were received from Drs. Bo A.G. Jonsson and Roel Vermeulen. The samples were collected in 1997 from a group of rubber manufacturing workers from several Dutch facilities which manufactured rubber tires and belts, general rubber goods, and a retreading company. The urine samples collected were from exposed males with a mean age of 38.5 years. Urine samples were collected pre-shift on the Sunday prior to other sampling, and post-shift samples were collected either on Wednesday or Thursday of the same week at 4pm which was at the end of the workday (Peters et al, 2008). All samples
11

were received via a FedX shipment from Dr. Bo A.G. Jonsson in good condition, still frozen, caps secured, labels adhered and legible, with a large amount of dry-ice left in each of the two containers used to ship the samples. Dr. Glenn Talaska contacted Dr. Vermeulen to verify which samples were those of non-smokers, and these were selected as the samples used in the current study. A total of 86 individual, 43 pairs of pre and post, samples were analyzed in the study. To avoid knowledge of which samples were pre- and postshift, another student, Mr. John Jaskolka, covered the post and pre shift label indications and identification number of each sample with tape. The samples were then relabeled with a new number corresponding to the order in which they were unpacked by the student. The original numbering and pre/post shift information was recorded by the student, and the new numbering system was used throughout the experiment to ensure that the investigator, Ms. Beverly Gaultney, and advisor, Dr. Glenn Talaska, were blinded in regards to the sample status (pre or post shift as well as department).

Summary of Methods Urinary levels of 2-naphthol were determined using the solid phase extraction method of Jongeneelen et al (1987), and the HPLC analysis was carried out as in the methods used by Kim et. al, 2001. In summary, the urine was adjusted to a pH of 5.0 (+/- 0.05) by adding aliquots of 1N HCl. Next, sodium acetate and glucuronidase/arylsulfatase were added and incubated on a shaking grid for 4 hours at 37C. Following incubation SepPak cartridges were primed to remove contaminates using methanol and water; the prepared samples were then passed through the primed cartridges. Milli-Q water and methanol were once more passed through the cartridges. The sample was then eluted with methanol, and dried using a nitrogen evaporator and water bath set at 60C. Samples were resuspended with methanol and filtered into brown glass HPLC vials. The HPLC analysis was performed to detect the excitation/emission wavelengths for 2napthol at 227/355 nm (Kim et.al, 2001). The amount of 2-naphthol was estimated using a calibration curve

12

developed with standard 2-napthol solutions of varying concentrations and the determination of the peak levels seen in the HPLC analysis was assessed using the excitation/emission wavelengths of 227/355 nm.

Sample Preparation As noted above, Ms. Beverly Gaultney, and advisor, Dr. Glen Talaska, were blinded in regards to the sample status (pre or post shift as well as the department where each subject worked). Urine samples were removed from the freezer and placed in the refrigerator for 4 hours to thaw. The volume of each urine sample was recorded. The pH of each urine sample was then determined using a calibrated pH meter. Each urine sample was then adjusted to a pH of 5.0 (+/- 0.05) by adding 1N HCl one drop at a time while agitating the pH probe. The resultant pH of each sample was recorded.

Hydrolysis Five ml (per 15ml of urine) of 0.1M Sodium acetate and 8.75ul (per 15ml of urine) of glucuronidase/arylsulfatase were added to each sample. Samples with less than 15ml of urine contained proportionally adjusted amounts of Sodium acetate and -glucuronidase/arylsulfatase. The samples were then incubated with agitation at 37C for 4 hours. Once incubation was complete, the samples were refrigerated at 41F overnight.

Cartridge Priming The C18 Sep Pak Cartridges were primed to remove contaminants. Each Sep Pak Cartridge was attached to a 20mL syringe, with the plunger of each syringe removed. The syringes plus Sep Pak Cartridges were
13

held upright and 5 ml of HPLC grade methanol was added to each syringe with a pipette. The plungers were then added to the syringes and the methanol was pushed through the Sep Pak Cartridges at a rate of 2.5ml/minute. The syringe plungers were then removed and 10ml of Milli-Q water was added to each syringe using a pipette. All plungers were then replaced, and the Mill-Q water was pushed through the Sep Pak Cartridges at a rate of 2.5ml/min. This step of washing with 10ml of Milli-Q water was then repeated for a second time. Each syringe was labeled with a unique sample number.

Sample Loading After samples were incubated and cartridges were primed, the samples were then loaded onto the Sep Pak Cartridges. Syringes labeled with the corresponding number to each sample were used. The plungers were removed from each syringe and the entire volume of each sample was emptied into the syringe with the corresponding sample number. The syringe plungers were then added to the syringes and the urine sample was pushed through the Sep Pak Cartridges at the rate of 2.5ml/min. This waste was not collected and was allowed to flow down the drain the laboratorys sink. Some cartridges became clogged due to sediments in the urine and would not allow the entire sample volume to be pushed through a single Sep Pak Cartridge. These samples were then added to a second labeled syringe and Sep Pak cartridge with the corresponding sample number and the letter B. When required a 3rd, 4th, and 5th syringe and Sep Pak Cartridge were used and labeled with the sample number plus the letter C, D, or E respectively. After the entire sample was loaded onto the cartridge, the cartridge was washed to remove polar contaminants. The syringe plungers were removed with care taken to remember which plunger corresponded to each sample/syringe. This was accomplished by removing the plungers and placing them upside down (plunger facing upward, and the bottom of the handle placed on the counter top) on the counter in the same pattern that the samples were arrayed. A pipette was used to add 8ml of Milli-Q Water to each syringe and the plungers were then placed

14

in each syringe. The water was pushed through at a rate of 2.5ml/min and the waste was collected in a tray and transferred to a waste beaker.

The samples were then eluted into labeled 25 ml glass scintillation vials, by removing the syringe plunger and adding 10 ml of methanol to each syringe with Sep Pak cartridge still attached. The methanol was pushed through the Sep Pak cartridges at a rate of 2.5 ml/min. For sample numbers with multiple Sep Pak cartridges, 10 ml of methanol was pushed through the Sep Pak cartridge labeled with the letter A as described previously. The methanol and sample in the scintillation vial were then drawn up using a pipette and then place into the syringe with the Sep Pak cartridge labeled with the letter B, the methanol was then pushed through the Sep Pak cartridge at a rate of 2.5ml/min and collected in the scintillation vial. This process was repeated until all Sep Pak cartridges corresponding to the sample number were eluted into the vial. The solvent was then evaporated by placing the glass scintillation vials in a water bath at 60C and under the gentle flow of nitrogen. This process took on average 30 minutes. While the solvent was evaporating in the nitrogen flow and water bath, 0.45 um syringe filters were washed (1 for each sample) by drawing 5ml of methanol into a 3ml syringe and attaching it to each filter. The methanol was pushed through and then air was pushed through twice to dry the filters. Once the solvent was evaporated, the samples were removed from the water bath and resuspended by adding 2ml of HPLC grade methanol to each scintillation vial. The 3ml syringes were then used to draw up the resuspended sample and methanol in the vials. A 0.45 um filter was then added and the sample was pushed through into a 2ml brown glass HPLC vial labeled with the sample number. A new filter and syringe were used for each sample.

High Pressure Liquid Chromatography (HPLC) Waters 2695 separation module with 730 Water Data Module fluorescence detector)

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After all other preparation steps were complete the samples were run through the High Performance Liquid Chromatograph using the excitation/emission wavelengths of 227/355 nm (Kim et.al, 2001). The flow rate was set to 0.8ml/min and the solvent flow was set to 38% Water and 62% Methanol with the column temperature set to 35C with a range of 5C. To determine a standard curve and the peak time for 2naphthol elution a standard diluted to a concentration of 18.8 ng 2-naphthol/ul methanol was run at (5ul, 10ul, 20ul, 30ul, 40ul, 50ul,1.88ul twice, 3.76 ul twice, 5.64 ul twice, 7.52 ul twice, 9.4ul twice, and 18.8 ul twice). The 2-naphthol standard was diluted by weighing out 18.8mg of dry 2-naphthol standard on a scale, then placing the dry 2-naphthol into a 25ml glass scintillation vial and adding 10ml of methanol to yield 1.88ug/ul, another 10 ml of methanol was added to yield 18.8 ng/ul. After running the standards through the HPLC machine with the above settings, 2-naphthol was found to peak at 4.1 minutes. The standard curve was created in Microsoft Excel by taking the results from the lower end of the curve (1.88ul twice, 3.76 ul twice, 5.64 ul twice, 7.52 ul twice, 9.4ul twice, and 18.8 ul twice) and entering the amount of standard injection as the y value and the area produced at each of these levels as the x values. The function linest of Excel was used with a constant equal to the area of that sample and the stats =1. To determine the correlation coefficient between the areas and levels the stat/correlation function was used with array 1 being set as the levels and array 2 set as the areas. The results yielded a correlation coefficient of 0.9997. The slope and y intercept were determined by the Excel program with the y=to the areas and the x=to the levels to yield an equation for the standard curve line. The x value, the area produced by each sample run, was then entered manually after the sample run for each sample and the y-value was calculated by excel using this equation to yield a result of the amount of 2-naphthol in each sample. The equation produced by this process in the format y=mx+b was y=1038691*(area produced from HPLC curve)+(500684.7131).

For the sample run the following settings were used: the integration was set to inhibit integration from a start time of 0.017min to 2.3 minutes and again from 6.0 minutes to 25 minutes. The component settings
16

were set to a migration time for 2-naphthol set to 4.0 minutes with a MT Window of 1.0 minutes, and the Peak Match was set to Closet. The Y Value was set as the area, the X Value as the amount, the fit set to linear, and the weighting set to none.

4.0

RESULTS

The data revealed a possible work-related exposure to naphthalene in the rubber workers as post shift levels in 17 sample pairs were elevated when compared to the pre shift samples, while only 7 of the data sets revealed a decrease in 2- naphthol levels in the post- versus pre shift samples. Although 17 of the pairs showed an increase in the 2-napthol recovery levels in the post shift compared to the pre shift sample, this was not an adequate number of pairs to statistically determine a difference between pre and post shift samples; therefore, contrary to the hypothesis, the differences between the pre shift and post shift samples was not statistically significant. The pre and post shift differences, between the pairs were analyzed as a group, and the p-value of the differences using a 2-sided students t-test was found to be p=0.4. A sign test revealed a p value of 0.053, which was nearly statistically significant. The post-pre recovery differences were again analyzed after taking the logarithm of both measurements. The p-value for the paired t-test is slightly smaller p= 0.3, but still not significant. The post-pre differences were somewhat more normally distributed after the log transformation. The results of the statistical analysis performed by Dr. Paul Succop are found as Appendix A: Statistics Charts for Pre and Post Differences.

Table 1: contains the sample number, the pre or post shift identification, the area under the curve of the HPLC peak identified as 2-naphthol, and the amount of 2-naphthol recovered in picograms or the notation of <LOD (Limit of Detection, meaning a peak was observed but was under the limits set by the calibration curve) or ND (Non Detect, meaning no peak was seen), and a calculated amount of 2-naphthol in urine (ug/l).

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Table 1: Results of Analysis Amount Recovered per HPLC injection volume 25ul (pg) #VALUE! #VALUE! #VALUE! #VALUE! 1.741218533 15.68595846 0.226130054 2.766362529 3.606959849 0.073150991 3.177015877 #VALUE! #VALUE! 19.85982668 9.522317825 0.272028202 #VALUE! #VALUE! 2.373907083 #VALUE! 3.825519523 #VALUE! -0.098571854 0.764867674 #VALUE! #VALUE! #VALUE! #VALUE! ND ND ND ND ND <LOD ND <LOD ND ND ND ND <LOD <LOD

Sample ID 2 4 5 6 9 10 15 17 19 20 24 26 27 29 30 32 35 36 38 39 42 43 44 47 48 49 50 51

Area from HPLC Peak ND ND ND ND 2309273 16793551 735564 3374081 4247202 576666 3800623 ND ND 21128911 10391432 783238 ND ND 2966441 ND 4474218 ND 398299 1295146 ND ND ND ND

LOD/ND Value =0.28 ND ND ND ND

Decoded Sample ID (Pre or Post) 180 PRE 180 POST 179 PRE 179 POST 195 PRE 195 POST 192 PRE 192 POST 146 PRE 146 POST 232 POST 232 PRE 215 PRE 215 POST 113 POST 113 PRE 135 PRE 135 POST 230 POST 230 PRE 202 PRE 202 POST 216 PRE 216 POST 143 POST 143 PRE 126 PRE 126 POST

Change +/+/+ + + + + +/+ + +/+/-

Volume of Urine Analyzed (ml) 15 15 7.5 15 15 15 15 15 15 15 15 15 15 15 10 15 13.5 14.5 15 15 12.5 15 11 15 15 15 15 10

Calculated Amount (ug /l of urine) ND ND ND ND 0.009332931 0.084076737 0.001212057 0.014827703 0.019333305 0.000392089 0.017028805 ND ND 0.106448671 0.076178543 0.001458071 ND ND 0.012724142 ND 0.024483325 ND <LOD 0.004099691 ND ND ND ND

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52 54 56 58 59 60 61 62 63 64 65 66 69 70 74 75 79 80 83 86 87 88 90 91 93 94 97 98 99 102 107 108 111 112 117 119 120 122

5309173 ND 742203 ND ND ND ND 4008394 2952728 ND ND ND ND ND 731255 26679099 20020825 ND ND ND ND 8691446 ND ND 1006543 748752 1994284 ND 1264395 ND ND 339197 10334224 2757882 ND 7448 ND 1096201

4.629372523 #VALUE! 0.232521752 #VALUE! #VALUE! #VALUE! #VALUE! 3.377047426 2.360704891 #VALUE! #VALUE! #VALUE! #VALUE! #VALUE! 0.221981564 25.20327067 18.79301679 #VALUE! #VALUE! #VALUE! #VALUE! 7.885656153 #VALUE! #VALUE! 0.48701511 0.238826802 1.437962845 #VALUE! 0.735262146 #VALUE! #VALUE! -0.15547231 9.467240818 2.173116888 #VALUE! -0.474863689 #VALUE! 0.573333358 ND <LOD ND <LOD ND ND <LOD ND ND ND <LOD <LOD ND ND ND ND ND ND ND ND ND <LOD ND <LOD ND ND ND ND

2 PRE 2 POST 65 POST 65 PRE 62 POST 62 PRE 36 POST 36 PRE 23 PRE 23 POST 30 PRE 30 POST 6 POST 6 PRE 15 POST 15 PRE 1 POST 1 PRE 41 PRE 41 POST 4 POST 4 PRE 67 PRE 67 POST 28 POST 28 PRE 17 PRE 17 POST 58 POST 58 PRE 112 PRE 112 POST 182 POST 182 PRE 110 POST 110 PRE 90 PRE 90 POST + +/+ +/+ + +/+/+ +/+/+/+/-

15 15 15 15 15 11 15 15 15 15 15 15 15 4 15 15 15 0.85 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 5 15 15 15 12.5

0.024813437 ND 0.001246317 ND ND ND ND 0.018100974 0.012653378 ND ND ND ND ND 0.001189821 0.135089531 0.10073057 ND ND ND ND 0.042267117 ND ND 0.002610401 0.001280112 0.007707481 ND 0.003941005 ND ND <LOD 0.050744411 0.03476987 ND <LOD ND 0.003669333

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123 125 126 128 129 132 133 134 139 140 141 143 145 147 148 152 153 156 158 159

161735 ND 2741090 ND ND 4317974 ND ND 1254370 ND ND 436851 ND ND 4043984 ND ND ND 6252048 2090257

-0.326323866 #VALUE! 2.156950388 #VALUE! #VALUE! 3.675095598 #VALUE! #VALUE! 0.725610577 #VALUE! #VALUE! -0.061455913 #VALUE! #VALUE! 3.411311702 #VALUE! #VALUE! #VALUE! 5.537125513 1.53036086

<LOD ND ND ND ND ND ND ND <LOD ND ND ND ND ND

128 PRE 128 POST 14 POST 14 PRE 84 PRE 84 POST 103 PRE 103 POST 144 POST 144 PRE 154 POST 154 PRE 95 POST 95 PRE 86 POST 86 PRE 161 PRE 161 POST 92 POST 92 PRE + +/+ +/+/+ +/+ + +/-

15 15 15 15 10.5 11 15 15 12.5 5 15 10 15 15 12.5 2.5 15 15 7.5 15

<LOD ND 0.011561254 ND ND 0.026754696 ND ND 0.004643908 ND ND <LOD ND ND 0.021832395 ND ND ND 0.058915015 0.008202734

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The change in individual urinary 2-naphthol levels is shown in Figure 1: Urinary 2-napthol levels (ug/l) paired by pre and post shift samples, which plots each of the paired data sets that had either a change in pre and post shift recoveries of urinary 2-naphthol. This presentation shows the trend (as reflected in the near significant sign test discussed above) of increased 2-naphthol levels in the post-shift samples.

Figure 1: Urinary 2-napthol levels (ug/l) paired for each individual by pre and post shift samples

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Figure 2 displays the mean values of the recovered amounts of pre and post shift samples above the limit of detection. The values under the limit of detection and non detects were not included in this figure. The 25th quartile was found to be 0.008 ug/l for pre shift samples and 0.004 ug/l for post shift samples. The 75th quartile value for pre-shift samples was calculated as 0.02 ug/l and for post-shift samples the number increased to a value of 0.05 ug/l.

Figure 2: Mean values of Recovered Pre and Post Shift Naphthol (ug/l)

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Figure 3 displays the mean values of the urinary 2-naphthol levels in ug/l in pre and post shift samples with the values under the Limit of Detection Value included in the calculated values as equal to one half the limit of detection (LOD=0.28pg).

Figure 3: Calculated Mean values of Recovered Pre and Post Shift Naphthol (ug/l) including LOD values in calculations.

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5.0

DISCUSSION AND CONCLUSIONS

The data revealed a possible work-related exposure to naphthalene in the rubber workers as post shift levels in 17 sample pairs were elevated when compared to the pre shift samples, while only 7 of the data sets revealed a decrease in 2- naphthol levels in the post- versus pre shift samples. Using a one-sided significance sign test of post results greater than pre results p=0.053. This would indicate that the results are extremely close to a statistically significant level of p=0.05. The difference in the two values is approximately the difference between the odds of the data occurring to chance being 18.9 to 1 versus 20 to 1 odds at the p=0.05 level. These results support the workday exposure to naphthalene hypothesized to be expected among the rubber workers.

The post shift samples ranged from non-detected and <limit of detection (=0.28pg recovered) to a maximum recovered value of 0.11 ug/l urine and for pre shift samples the samples ranged from ND/LOD to a maximum value of 0.14 ug/l urine with the next highest value equal to 0.03 ug/l for the pre shift samples. It is possible that the outlier in the pre-shift samples which is higher than the post shift samples may actually be a mislabeled post-shift sample as the post shift sample for the same individual decreased to an amount of 0.0012 ug/l which would be more in line with the values collected as pre-shift samples. Another possibility is that this individual may have smoked or been exposed to second hand smoke prior to the pre- shift sample, but the individual was reported as a nonsmoker. With this data pair removed from the data set, the post-shift average of the paired samples from the remaining rubber workers is roughly twice that of the pre-shift averages. This adjusted data may be viewed below in Figure 4: Mean Urinary Naphthol Levels with Outlying Pair Excluded. This figure does not include values at, or under, the limit of detection.

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Figure 4: Mean Urinary Naphthol Levels with Outlying Pair Excluded.

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Figure 5 also excluded the outlying sample pair, and included the limit of detection values and those values under the limit of detection were set equal to the LOD to calculate the mean urinary naphthol levels. The figure displays an increase in the post shift recovery amounts of 2-naphthol when compared to the pre shift recovery amounts.

Figure 5: Mean Urinary Naphthol Levels with Outlying Pair Excluded and Limit of Detection Values Included in the Calculations.

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The levels of urinary 2-naphthol recovered in the post shift samples were below those seen in nonsmokers in other industries with naphthalene exposure. The highest value recovered in this analysis for the post-shift samples of 0.11ug/l was lower than the mean value obtained in studies from the following industries: converter infeed, coal-tar distillation, coking plant, production of fire-proof materials, production of graphite electrodes, shipyard workers, and steel workers. The results of these studies may be seen below in Table 2: Comparison of urinary 2-naphthol levels recovered in non-smoking populations in various exposed industries. In the production of fire-proof materials study, the production of graphite electrodes, and the steel workers the values did include a range that encompasses lower values such as the ones produced in this analysis (Preuss et al,2005; Onyemauwa et al, 2009; Kim et al 2001). This may indicate that the department of an industry, processes involved in, and type of exposure could greatly affect naphthalene exposure and thus the urinary 2-naphthol levels.

Table 2: Comparison of post-shift urinary 2-naphthol levels recovered in non-smoking populations in various exposed industries Number of Mean Value of 2-naphthol Range (ug/l) Industry Participants (ug/l) Source
Converter Infeed (Preuss et al 2005) Coal-tar Distillation (Preuss et al 2005) Coking Plant (Preuss et al 2005) Production of Fireproof Materials (Preuss et al 2005) Production of Graphite Electrodes (Preuss et al 2005) Shipyard Workers (Kim et al 2001) Steel Workers (Onyemauwa et al 2009) Current Analysis 6 7 18 28 70.2 74.1 13.4 14.0 1.0-190.4 5.6-334.2 3.0-36.2 <LOD-127.0

34

13.9

<LOD-212.8

65 57

2.46 7.12

0.20-20.37 NA

43

0.03

<LOD-0.11

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In summary, urinary naphthols are a promising biomarker for exposure to PAHs; however, many questions remain regarding their use and further research is needed to determine if they are an adequate marker of PAH exposure especially in an occupational setting.

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Boeffetta P, Jourenkova N, Gustavsson P (1997) Cancer risk from occupational and environmental exposure to polycyclic aromatic hydrocarbons. Cancer Causes Control 8: 444-472 Constantino JP, Redmond CK, Bearden A (1995) Occupationally related cancer risk among coke oven workers: 30 years of follow-up. Journal of Occupational and Environmental Medicine 37: 597-604 Dezell E, Monson RR (1982) Mortality among rubber workers: V. Processing workers. Journal of Occupational Medicine 24(7): 539-545 Fine, L and Peters, J (1976) Respiratory morbidity in rubber workers I. Prevalence of respiratory symptoms and disease in curing workers. Archives of Environmental Health January/February: 5-9 IARC (International Agency for Research on Cancer)(1982); IARC Monographs on the Evaluation of Carcinogenic Risks to Humans: Some Traditional Herbal Medicines, Some Mycotoxins, Naphthalene and Styrene. Vol 82, IARC, Lyon IARC (International Agency for Research on Cancer)(2002); IARC Monographs on the Evaluation of Carcinogenic Risks to Humans: Some Traditional Herbal Medicines, Some Mycotoxins, Naphthalene and Styrene. Vol 82, IARC, Lyon Jonsson L S, Broberg K, Axmon A, Bergendorf U, Littorin M, Jonsson B A G (2008). Levels of 1hydroxypyrene, symptoms and immunologic markers in vulcanization workers in the southern Sweden rubber industries. International Archives of Occupational and Environmental Health 82: 131-137. Kim H, Cho S-H, Kang J-W, Kim Y-D, Nan H-M, Lee C-H, Lee H, Kawamoto T (2001) Urinary 1hydroxypyrene and 2-naphthol concentrations in male Koreans. International Archives Occupational Environmental Health 74: 59-62 Monson RR, Fine LJ (1978) Cancer mortality and morbidity among rubber workers. JNCI 61(4):1047-1053 Monson RR, Nakano KK (1976) Mortality among rubber workers. I. White male union employees in Akron, Ohio. American Journal of Epidemiology 103(3):284-296 National Institute of Occupational Safety and Health: Special NIOSH hazard review: Rubber products manufacturing industry. DHHS (NIOSH) Publication No. 93-106. US Department of Health and Human Services, Public Health Service, National Institutes of Health, Research Triangle Park (NC), 2003.

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National Toxicology Program 1992: Toxicology and carcinogenesis studies of naphthalene (CAS No.:9120-3) in B6C3F1 Mice (inhalation studies). NIH Publication No. 92-3141. US Department of Health and Human Services, Public Health Service, National Institutes of Health, Research Triangle Park (NC), 1992. National Toxicology Program 2000: Toxicology and carcinogenesis studies of naphthalene (CAS No.:9120-3) in F344/N rats (inhalation studies). Technical report Series No. 91-20-3. NIH Publication No. 014434. US Department of Health and Human Services, Public Health Service, National Institutes of Health, Research Triangle Park (NC), 2000. Onyemauwa F, Rappaport S M, Sobus J R, Gajdosova D, Wu R, Waidyantha (2009) Using liquid chromatography-tandem mass spectrometry to quantify monohydroxylated metabolites of polycyclic aromatic hydrocarbons in urine. Journal of Chromatography B 887: 1117-1125 Peters S, Talaska G, Jonsson Bo A G, Kromhout H, Vermeulen R (2008) Polycyclic Aromatic Hydrocarbon exposure, urinary mutagenicity, and DNA adducts in rubber manufacturing workers. Cancer Epidemiology Biomarkers and Prevention 17 (6): 1452-1459 Preuss R, Angerer J, Drexler H (2003) Naphthalene-an environmental and occupational toxicant. International Archives of Occupational Environmental Health 76: 556-576 Preuss R, Kock H M, Wilhelm M, Pischetsrieder M, Angerer J (2004) Pilot study on the naphthalene exposure of German adults and children by means of urinary 1- and 2-napthol levels. Internation Journal of Hygiene and Environmental Health 207: 441-445 Preuss R, Drexler H, Bottcher M, Wilhelm M, Bruning T, Angerer J (2005) Current external and internal exposure to naphthalene of workers occupationally exposed to polycyclic aromatic hydrocarbons in different industries. International Archives of Occupational Environmental Health 78: 355-362 Price P, Jayjock M (2008) Available data on naphthalene exposures: Strengths and limitations. Regulatory Toxicology and Pharmacology 51: S15-S21 Rappaport S M, Waidyanatha S, Serdar B (2004) Naphthalene and its biomarkers as measures of occupational exposure to polycyclic aromatic hydrocarbons. Journal of Environmental Monitoring 6: 413-416 Serdar B, Egeghy P P, Gibson R, Rappaport S M (2004) Dose-dependent production of urinary naphthols among workers exposed to jet fuel (JP-8). American Journal of Industrial Medicine 46: 234-244 Talaska G, Maier A, Henn S et al. (2002) Carcinogen biomonitoring in human exposures and laboratory research: validation and application to human occupational exposures. Toxicology Letters 134: 39-49 U.S. EPA (U.S. Environmental Protection Agency)IRIS Toxiocological Review and Summary Documents of Naphthalene (1998). US Environmental Protection Agency, Washington D.C. U.S. EPA (U.S. Environmental Protection Agency), electronic resource 2003: http://www.epa.gov/ttn/atw/hlthef/naphthal.html#ref7. 30

Wilhelm M, Hardt J, Schulz C, Angerer J (2008) New reference value and the background exposure for the PAH metabolites 1-hydroxypyrene and 1- and 2-naphthol in urine of the general population in Germany: Basis for validation of human biomonitoring data in environmental medicine. International Journal of Hygiene and Environmental Health 211: 447-453

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Appendix A: Statistics Charts for Pre and Post Differences.

t test and sign test for post - pre differences 311 13:51 Tuesday, July 14, 2009 The UNIVARIATE Procedure Variable: diff_recovered Moments N Mean Std Deviation Skewness Uncorrected SS Coeff Variation 43 0.93630298 6.58091931 -0.2925947 1856.65348 702.862159 Sum Weights Sum Observations Variance Kurtosis Corrected SS Std Error Mean 43 40.261028 43.308499 7.11031036 1818.95696 1.00358079

Basic Statistical Measures Location Mean Median Mode 0.936303 0.000000 0.000000 Variability Std Deviation Variance Range Interquartile Range 6.58092 43.30850 44.50310 2.09391

Tests for Location: Mu0=0 Test Student's t Sign Signed Rank -Statistict M S 0.932962 4.5 43.5 -----p Value-----Pr > |t| Pr >= |M| Pr >= |S| 0.3562 0.1078 0.2497

Quantiles (Definition 5) Quantile 100% Max 99% 95% 90% 75% Q3 50% Median 25% Q1 10% 5% 1% 0% Min Estimate 19.57983 19.57983 13.94474 7.29412 2.09391 0.00000 0.00000 -3.32696 -4.34937 -24.92327 -24.92327

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t test and sign test for post - pre differences 312 13:51 Tuesday, July 14, 2009 The UNIVARIATE Procedure Variable: diff_recovered Extreme Observations ------Lowest-----Value -24.92327 -7.60566 -4.34937 -3.54552 -3.32696 Obs 6 3 2 39 31 ------Highest----Value 7.29412 9.24232 13.94474 18.51302 19.57983 Obs 36 25 38 1 40

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t test and sign test for ln(post) - ln(pre) differences 441 13:51 Tuesday, July 14, 2009 The UNIVARIATE Procedure Variable: diff_ln_recovered Moments N Mean Std Deviation Skewness Uncorrected SS Coeff Variation 43 0.30336206 1.92397563 -0.2275838 159.42788 634.217622 Sum Weights Sum Observations Variance Kurtosis Corrected SS Std Error Mean 43 13.0445685 3.70168222 0.30006635 155.470653 0.29340353

Basic Statistical Measures Location Mean Median Mode 0.303362 0.000000 0.000000 Variability Std Deviation Variance Range Interquartile Range 1.92398 3.70168 8.76160 1.47168

Tests for Location: Mu0=0 Test Student's t Sign Signed Rank -Statistict M S 1.033941 4.5 27.5 -----p Value-----Pr > |t| Pr >= |M| Pr >= |S| 0.3071 0.1078 0.4706

Quantiles (Definition 5) Quantile 100% Max 99% 95% 90% 75% Q3 50% Median 25% Q1 10% 5% 1% 0% Min Estimate 4.26166 4.26166 3.52660 2.50006 1.47168 0.00000 0.00000 -2.55583 -2.80539 -4.49994 -4.49994

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t test and sign test for ln(post) - ln(pre) differences 442 13:51 Tuesday, July 14, 2009 The UNIVARIATE Procedure Variable: diff_ln_recovered Extreme Observations ------Lowest----Value -4.49994 -3.33801 -2.80539 -2.61466 -2.55583 Obs 6 3 2 39 31 -----Highest----Value 2.50006 2.57454 3.52660 4.20645 4.26166 Obs 18 17 25 1 40

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