SCREENING-LEVEL HAZARD CHARACTERIZATION OF HIGH PRODUCTION VOLUME CHEMICALS SPONSORED CHEMICAL 2,4,6-Trimethylphenol (CAS No. 527-60-6) [9th CI Name: Phenol, 2,4,6-trimethyl-] October 2007 INTERIM Prepared by High Production Volume Chemicals Branch Risk Assessment Division Office of Pollution Prevention and Toxics Environmental Protection Agency 1200 Pennsylvania Avenue, NW Washington, DC 20460-0001 ------- SCREENING-LEVEL HAZARD CHARACTERIZATION OF HIGH PRODUCTION VOLUME CHEMICALS The High Production Volume (HPV) Challenge Program1 is a voluntary initiative aimed at developing and making publicly available screening-level health and environmental effects information on chemicals manufactured in or imported into the United States in quantities greater than one million pounds per year. In the Challenge Program, producers and importers of HPV chemicals voluntarily sponsor chemicals; sponsorship entails the identification and initial assessment of the adequacy of existing toxicity data/information, conducting new testing if adequate data do not exist, and making both new and existing data and information available to the public. Each complete data submission contains data on 18 internationally agreed to "SIDS" (Screening Information Data Set1'2) endpoints that are screening-level indicators of potential hazards (toxicity) for humans or the environment. The Environmental Protection Agency's Office of Pollution Prevention and Toxics (OPPT) is evaluating the data submitted in the HPV Challenge Program on approximately 1400 sponsored chemicals. OPPT is using a hazard- based screening process to prioritize review of the submissions. The hazard-based screening process consists of two tiers described below briefly and in more detail on the Hazard Characterization website3. Tier 1 is a computerized sorting process whereby key elements of a submitted data set are compared to established criteria to "bin" chemicals/categories for OPPT review. This is an automated process performed on the data as submitted by the sponsor. It does not include evaluation of the quality or completeness of the data. In Tier 2, a screening-level hazard characterization is developed by EPA that consists of an objective evaluation of the quality and completeness of the data set provided in the Challenge Program submissions. The evaluation is performed according to established EPA guidance2'4 and is based primarily on hazard data provided by sponsors. EPA may also include additional or updated hazard information of which EPA, sponsors or other parties have become aware. The hazard characterization may also identify data gaps that will become the basis for a subsequent data needs assessment where deemed necessary. Under the HPV Challenge Program, chemicals that have similar chemical structures, properties and biological activities may be grouped together and their data shared across the resulting category. This approach often significantly reduces the need for conducting tests for all endpoints for all category members. As part of Tier 2, evaluation of chemical category rationale and composition and data extrapolation(s) among category members is performed in accord with established EPA2 and OECD5 guidance. The screening-level hazard characterizations that emerge from Tier 2 are important contributors to OPPT's existing chemicals review process. These hazard characterizations are technical documents intended to support subsequent decisions and actions by OPPT. Accordingly, the documents are not written with the goal of informing the general public. However, they do provide a vehicle for public access to a concise assessment of the raw technical data on HPV chemicals and provide information previously not readily available to the public. The public, including sponsors, may offer comments on the hazard characterization documents. The screening-level hazard characterizations, as the name indicates, do not evaluate the potential risks of a chemical or a chemical category, but will serve as a starting point for such reviews. In 2007, EPA received data on uses of and exposures to high-volume TSCA existing chemicals, submitted in accordance with the requirements of the Inventory Update Reporting (IUR) rule. For the chemicals in the HPV Challenge Program, EPA will review the IUR data to evaluate exposure potential. The resulting exposure information will then be combined with the screening-level hazard characterizations to develop screening-level risk characterizations4'6. The screening-level risk characterizations will inform EPA on the need for further work on individual chemicals or categories. Efforts are currently underway to consider how best to utilize these screening-level risk characterizations as part of a risk- based decision-making process on HPV chemicals which applies the results of the successful U.S. High Production Volume Challenge Program and the IUR to support judgments concerning the need, if any, for further action. 1 U.S. EPA. High Production Volume (HPV) Challenge Program; http://www.epa.gov/chemrtk/index.htm. 2 U.S. EPA. HPV Challenge Program - Information Sources; http://www.epa.gov/chemrtk/pubs/general/guidocs.htm. 3 U.S. EPA. HPV Chemicals Hazard Characterization website (http://www.epa.gov/hpvis/abouthc.html). 4 U.S. EPA. Risk Assessment Guidelines; http://cfpub.epa.gov/ncea/raf/rafguid.cfm. 5 OECD. Guidance on the Development and Use of Chemical Categories; http://www.oecd.org/dataoecd/60/47/1947509.pdf. 6 U.S. EPA. Risk Characterization Program; http://www.epa.gov/osa/spc/2riskchr.htm. 2 ------- SCREENING-LEVEL HAZARD CHARACTERIZATION 2,4,6-Trimethylphenol (CAS No. 527-60-6) Introduction The sponsor, General Electric Company, submitted a Test Plan and Robust Summaries to EPA for 2,4,6- trimethylphenol (CAS No 527-60-6; 9th CI name: Phenol, 2,4,6-trimethyl-) on December 30, 2002. EPA posted the submission on the ChemRTK HPV Challenge website on January 30, 2003 (http://www.epa.gov/chemrtk/pubs/summaries/246trime/cl4218tc.htm'). EPA comments on the original submission were posted to the website on June 16, 2003. Public comments were also received and posted to the website. The sponsor submitted updated/revised documents on August 5, 2003 and December 29, 2005, which were posted to the ChemRTK website on September 5, 2003 and March 16, 2006, respectively. This screening-level hazard characterization is based primarily on the review of the test plan and robust summaries of studies submitted by the sponsor(s) under the HPV Challenge Program. In preparing the hazard characterization, EPA considered its own comments and public comments on the original submission as well as the sponsor's responses to comments and revisions made to the submission. A summary table of SIDS endpoint data with the structure(s) of the sponsored chemical(s) is included in the appendix. The screening-level hazard characterization for environmental and human health toxicity is based largely on SIDS endpoints and is described according to established EPA or OECD effect level definitions and hazard assessment practices. Sum m an-Conclusion The lou k i»f 2.4.<>-iiiniclh\ Iphenol indicates that lis potential to bioacciininlatc is c\pectcd to he low. 2.4.(<- l riincl11> Iphenol is not readiK biodegradable, indicating dial il has ihc potential to persist in ilie en\ iroiiniciit. The e\ ahiatioii of a\ ailable to\icit> dala lor fish. aquatic 11in eriebrales and aquatic plants indicates that the potential acute ha/aid of 2.4.<>-iiiniclh\ Iphenol to aquatic organisms is moderate \cnte oral and dermal to\icit\ of 2.4.<>-iiiniclh\ Iphenol is low In a combined repeated- dose reproducee de\elopniental to\icit> screening test in rats, the follow ing effects were noted at the highest dose tested, increased serum potassium in I'u males, decreased serum albumin and cholesterol mi I'n males, increased lh\ nms-io-bod\ weight ratios and increased th\ niiis-io-brain weight ratios in I'I females, increased hematocrit and decreased tail pinch response in I'u females, increased glucose in I I adult females, decreased bod> weight in I I females at post-natal da> 22 2.4.<>-Tiinicth\ Iphenol was not mutagenic in viini in bacterial cells and results from in viini mouse l\ niphonia assa\ were et|in\ ocal 2.4.<>-Tiiniclh\ Iphenol induced structural chromosomal aberrations in ( I l() cells mi the presence of metabolic acli\ ation ()\ enill. the weight of e\ idence suggest that 2.4.<>- trinielhs Iphenol has potential to cause mutagenic effects. specificalK in the presence of metabolic acli\ ation I lie potential health ha/ard of 2.4.<>-innielh\ Iphenol is modenite based on repeated-dose and reprodiicliN e de\ elopniental to\icit> \\ ailable data suggests that 2.4.(>-tiinielh\ Iphenol has the potential to be genoioMc \o data gaps were identified imder the I ll'Y ( hallenge I'rogiani 1. Physical-Chemical Properties and Environmental Fate A summary of physical-chemical properties and environmental fate data submitted is provided in Table 1. For the purpose of the screening-level hazard characterization, the review and summary of these data was limited to the octanol-water partition coefficient and biodegradation endpoints as indictors of bioaccumulation and persistence, respectively. 3 ------- Octanol-Water Partition Coefficient Log Kow: 2.73 Biodegradation In an aerobic biodegradation study using activated sludge as inoculum (0.05 ml/L) and 1.68 mg/L of 2,4,6-trimethylphenol, 11.3% 2,4,6-trimethylphenol had degraded after 28 days. 2,4,6-Trimethylphenol is not readily biodegradable. Conclusion: The log Kow of 2,4,6-trimethylphenol indicates that its potential to bioaccumulate is expected to be low. 2,4,6-Trimethylphenol is not readily biodegradable, indicating that it has the potential to persist in the environment. 2. Environmental Effects - Aquatic Toxicity Acute Toxicity to Fish Rainbow trout (Oncorhynchus mykiss) were exposed to 2,4,6-trimethylphenol at nominal concentrations of 0, 0.8, 1.7, 3.8, 8.3, 18.2 or 40 mg/L under static conditions for 96 hours. Analysis of the 0.8, 8.3 and 40 mg/L treatment solutions showed the measured concentrations were within 20% of the nominal concentrations. All fish at 18.2 and 40 mg/L died within 3 hours of exposure. 96-h LCS0 = 9.7 mg/L Acute Toxicity to Aquatic Invertebrates (1) Daphnia magna were exposed to 2,4,6-trimethylphenol at measured concentrations of 0, 0.8, 1.7, 3.8, 8.3, 18.2 or 40 mg/L under static conditions for 24 hours. A subset of test solutions were measured at the start and end of the experiment. 24-h ECS0 = 3.5 mg/L (2) Daphnia magna were exposed exposed to 2,4,6-trimethylphenol at measured concentrations of 0, 0.1, 0.35, 1.0, 3.5, 100 or 350 mg/L under static conditions for 24 hours. Acetone was used as a solvent dispersant. Test solution concentrations were not measured. 24-h ECS0 = 28.95 mg/L (3) In response to the original submission that included 24-hour daphnid test data, but not the normally required 48- hour daphnid test data, EPA requested that the submitter support the 24-hour data with structure-activity estimates or measured data for an analogous chemical. In the revised submission, neither was provided. Therefore, EPA estimated a 48-hour LC50 for Daphnia magna using the EPA's ECOSAR Program (http://www.epa. gov/oppt/newchems/tools/2 lecosar.html in order to confirm the robustness of the 24-hour data. The modeled results for the 48-hour test were in good agreement with the 24-hour results for the test conducted without a solvent and with analytical confirmation of the water concentrations. Therefore, the 24-hour data from that test were accepted for the purpose of the HPV Challenge Program. 48-h EC50 = 2.48 mg/L (estimated) Toxicity to Aquatic Plants Pseudokirchneriella subcapitata were exposed to 2,4,6-trimethylphenol at concentrations of of 0, 0.82, 2.05, 5.12, 12.8, 32.0 or 80.0 mg/L under static conditions for 96 hours. 72-h EC50 (biomass) = 2.54 mg/L 72-h EC50 (growth) = 5.59 mg/L 4 ------- Conclusion: The evaluation of available toxicity data for fish, aquatic invertebrates and aquatic plants indicates that the potential acute hazard of 2,4,6-trimethylphenol to aquatic organisms is moderate. 3. Human Health Effects Acute Oral Toxicity Albino rats (3/sex) were administered 2,4,6-trimethylphenol as a single oral dose of 2000 mg/kg-bw. No mortality was observed. Piloerection was the major clinical sign, which resolved by day 4 of the study. LDS0 > 2000 mg/kg-bw Acute Dermal Toxicity New Zealand White rabbits were administered 2,4,6-trimethylphenol as a single, occlusive dermal application of 2000 mg/kg-bw for 24 hours. After removal of the test substance 24 hours later, necrosis was observed at the site of application in 6 out of 10 animals. The necrotic areas remained visible for the duration of the study. Slight to moderate erythema and edema was observed in the remaining four animals. No mortality or overt signs of systemic toxicity were evident during the study. LDS0 > 2000 mg/kg-bw Repeated-Dose Toxicity In a combined repeated-dose/reproductive/developmental toxicity screening test, Sprague-Dawley rats (10/sex/dose) were administered 2,4,6-trimethylphenol in corn oil via gavage at doses of 0, 10, 100 or 200 mg/kg-bw/day for 2 weeks prior to mating and during a 2-week mating period through 3 weeks of gestation and lactation (F0 parents). A 2-week recovery group of five additional males and females per group (at 0 and 200 mg/kg-bw/day) was also included. Five additional females (not mated) were dosed at 0 and 200 mg/kg-bw/day for 28 days and subsequently sacrificed (28-day females). Selected F1 offspring (10/sex/treatment) were dosed from weaning through scheduled sacrifice approximately 7 weeks post-weaning (F1 adults). The study summary concluded that except for signs of a taste aversion response (dose-related incidences of rooting); no systemic toxicity was noted. The study reported a NOAEL of > 200 mg/kg-bw/day. However, the data tables presented in the robust summary show trends and noted statistically significant differences in the following parameters when compared with controls: F0Males: increased serum potassium levels at 10 (p < 0.01), 100 (p < 0.001) and 200 (p < 0.05) mg/kg-bw/day; decreased serum albumin levels at 100 (p < 0.01) and 200 (p < 0.05) mg/kg-bw/day; and decreased total protein levels at 100 (p < 0.01) and 200 (p < 0.01) mg/kg-bw/day. F0 Females: increased hematocrit at 200 mg/kg-bw/day (p < 0.05); decreased tail pinch response at 200 mg/kg- bw/day (p < 0.01). F1 Males: increased liver (relative-to-brain weight) at 200 mg/kg-bw/day (p < 0.05) and increased incidence of renal nephropathy (4/10) at 200 mg/kg-bw/day. F1 Females: increased glucose level at 200 mg/kg-bw/day (p < 0.05). Recovery Males: increased adrenal weight (absolute and relative-to-body weight) at 200 mg/kg-bw/day (p < 0.05). 28-Day females: decreased hind-limb grip strength at 200 mg/kg-bw/day (p < 0.05). LOAEL = 10 mg/kg-bw/day (based on statistically significant changes in serum potassium) NOAEL = Not established Reproductive Toxicity In the combined repeated-dose/reproductive/developmental toxicity screening test described previously, the following trends or statistically significant differences in the following parameters were seen when compared with controls: F0 Females: fluid-filled uterus in 1 of 10 animals receiving 100 mg/kg-bw/day and 'retained fetus in uterus' was reported in 1 of 10 females receiving 200 mg/kg-bw/day while no such effect was observed in control animals. Gestational length appeared to be reduced in animals receiving 200 mg/kg/day when compared to controls. Post- implantation loss/litter was reported approximately twice as many times as controls in females receiving 200 mg/kg- bw/day. 5 ------- Recovery females (FO): fluid-filled uterus was reported in 1 of 5 females dosed at 200 mg/kg-bw/day. F1 Females: fluid-filled uterus was reported in all treatment groups. There was a trend showing an increase in adjusted age at vaginal opening compared to control animals (unable to determine significance). 28-Day females: fluid-filled uterus was reported in 2 of 5 animals receiving 200 mg/kg-bw/day. LOAEL (systemic and reproductive toxicity) = 10 mg/kg/day (based on effect on fluid-filled uterus in F1 females) NOAEL (systemic and reproductive toxicity) = Not established Developmental Toxicity In the combined repeated-dose/reproductive/developmental toxicity screening test described previously, the following trends or statistically significant differences in the following parameters when compared with controls: F0 Females: see Reproductive Toxicity section. F1 Offspring (male and female pups): reduced pup growth (pup weight/litter) from postnatal day 7 to 21 at 100 and 200 mg/kg-bw/day. F1 Females (postnatal day 21): increased thymus-to-body weight ratio at 100 and 200 mg/kg-bw/day (p < 0.001); increase in thymus-to-brain weight ratio at 200 mg/kg-bw/day (p < 0.01); and a trend of increasing adjusted age at vaginal opening compared to control animals (unable to determine significance). LOAEL (maternal) = 10 mg/kg-bw/day (based on fluid-filled uterus inFl females; see reproductive toxicity section) NOAEL (maternal) = Not established LOAEL (developmental toxicity) = 100 mg/kg-bw/day (based on effects on thymus) NOAEL (developmental toxicity) = 10 mg/kg-bw/day Genetic Toxicity - Gene Mutation In vitro (1) In an Ames assay, Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 were exposed to 2,4,6- trimethylphenol at concentrations of 0, 0.03, 0.3, 3 and 30 (imol/plate; with and without metabolic activation. The number of revertants/plate at all concentrations in all tester strains was less than the number reported in the negative control group, with and without metabolic activation. Positive control response was not indicated in the robust summary. 2,4,6-Trimethylphenol was not mutagenic in this assay. (2) Mouse lymphoma L5178Y cells were exposed to 2,4,6-trimethylphenol at test concentrations of 0, 10, 30, 40, 50 or 100 (ig/mL with metabolic activation and 0, 50, 75, 100, 125 or 150 (ig/mL without metabolic activation. Mutations were equivocal in the absence of metabolic activation at 4- and 24-hour exposures and negative in the presence of metabolic activation. 2,4,6-Trimethylphenol gave equivocal results in this assay. Genetic Toxicity - Chromosomal Aberrations In vitro Chinese hamster ovary (CHO) cells were exposed for 4 hours to 2,4,6-trimethylphenol at concentrations of 0, 25, 50, 100, 150, 200, or 300 (ig/mL without metabolic activation and at concentrations of 0, 12.5, 25, 50, 100, 150 or 200 |ig/ with metabolic activation. In addition, cells were exposed for 20 hours to concentrations of 0, 25, 50, 100, 150, 200 300 or 400 (ig/mL without metabolic activation for the 20 hours. Mitomycin C and cyclophosphamide were used as the positive controls and DMSO was used as the solvent control. Cytotoxic concentrations were greater than 136 and 408 ng/mL with and without metabolic activation, respectively. 2,4,6-trimethylphenol did not induce structural and numerical chromosome aberrations without metabolic activation system. In the presence of metabolic activation, 2,4,6-trimethylphenol was negative for numerical chromosomal aberrations but induced statistically significant (p < 0.01 at 100 ng/mL and p < 0.05 at 200 ng/mL) structural chromosomal aberrations. 2,4,6-Trimethylphenol induced chromosomal aberrations in this assay. 6 ------- In vivo Male and female mice were administered 2,4,6-trimethylphenol at concentrations of 0, 500, 1000, 1200, 1400 and 1600 mg/kg-bw via intraperitoneal injection. The vehicle was corn oil and the positive control was cyclyphosphamide. The number of micronucleated polychromatic erythrocytes (MPCEs) per 1000 PCEs in treated groups was not statistically increased relative to the respective vehicle controls in either male or female mice, regardless of dose level or bone marrow collection time. Positive and negative (vehicle) control results indicated appropriate responses. 2,4,6-Trimethylphenol did not increase MPCEs in this assay. Conclusion: Acute oral and dermal toxicity of 2,4,6-trimethylphenol is low. In a combined repeated- dose/reproductive/developmental toxicity screening test in rats, the following effects were noted at the highest dose tested: increased serum potassium in F0 males, decreased serum albumin and cholesterol in F0 males, increased thymus-to-body weight ratios and increased thymus-to-brain weight ratios in F1 females, increased hematocrit and decreased tail pinch response in F0 females, increased glucose in F1 adult females, decreased body weight in F1 females at post natal day 22. 2,4,6-trimethylphenol was not mutagenic in vitro in bacterial cells and results from in vitro mouse lymphoma assay were equivocal. 2,4,6-trimethylphenol induced structural chromosomal aberrations in CHO cells in the presence of metabolic activation. Overall, the weight of evidence suggest that 2,4,6- trimethylphenol has potential to cause mutagenic effects, specifically in the presence of metabolic activation. The potential health hazard of 2,4,6-trimethylphenol is moderate based on repeated-dose and reproductive/developmental toxicity. Available data suggests that 2,4,6-trimethylphenol has the potential to be genotoxic. 4. Hazard Characterization The log Kow of 2,4,6-trimethylphenol indicates that its potential to bioaccumulate is expected to be low. 2,4,6-Trimethylphenol is not readily biodegradable, indicating that it has the potential to persist in the environment. The evaluation of available toxicity data for fish, aquatic invertebrates and aquatic plants indicates that the potential acute hazard of 2,4,6-trimethylphenol to aquatic organisms is moderate. Acute oral and dermal toxicity of 2,4,6-trimethylphenol is low. In a combined repeated- dose/reproductive/developmental toxicity screening test in rats, the following effects were noted at the highest dose tested: increased serum potassium in F0 males, decreased serum albumin and cholesterol in F0 males, increased thymus-to-body weight ratios and increased thymus-to-brain weight ratios in F1 females, increased hematocrit and decreased tail pinch response in F0 females, increased glucose in F1 adult females, decreased body weight in F1 females at post-natal day 22. 2,4,6-Trimethylphenol was not mutagenic in vitro in bacterial cells and results from in vitro mouse lymphoma assay were equivocal. 2,4,6-Trimethylphenol induced structural chromosomal aberrations in CHO cells in the presence of metabolic activation. Overall, the weight of evidence suggest that 2,4,6- trimethylphenol has potential to cause mutagenic effects, specifically in the presence of metabolic activation. The potential health hazard of 2,4,6-trimethylphenol is moderate based on repeated-dose and reproductive/developmental toxicity. Available data suggests that 2,4,6-trimethylphenol has the potential to be genotoxic. 5. Data Gaps No data gaps were identified under the HPV Challenge Program. 7 ------- APPENDIX Summary Tabic of the Screening Information Data Set as Submitted under the U.S. HPV Challenge Program Endpoints SPONSORED CHEMICAL 2,4,6-T rimethylphenol (527-60-6) Structure OH H,C^ Js. /CH, \ ll ch3 Summary of Physical-Chemical Properties and Environmental Fate Data Melting Point (°C) 73 Boiling Point (°C) 220 Vapor Pressure (hPa at 25°C) 0.07 Log K„w 2.73 Water Solubility (mg/L at 25°C) 1.01x103 Direct Photodegradation — Indirect (OH ) Photodegradation Half-life (t1/2) 8.2 h (estimated) Stability in Water (Hydrolysis) (ti/2) Not susceptible under environmental conditions Fugacity (Level III Model) Air (%) Water (%) Soil (%) Sediment (%) 0.773 31.7 67.2 0.336 Biodegradation at 28 days (%) 11.3 Not readily biodegradable Summary of Environmental Effects - Aquatic Toxicity Data Fish 96-h LCS0 (mg/L) 9.7 Aquatic Invertebrates 48-h ECS0 (mg/L) 3.5 (24-h) 28.95 (24-h) 2.48 (estimated) Aquatic Plants 72-h ECS0 (mg/L) (growth) (biomass) 5.59 2.54 8 ------- Summary Tabic of the Screening Information Data Set as Submitted underthe U.S. HPV Challenge Program Endpoints SPONSORED CHEMICAL 2,4,6-T rimethylphenol (527-60-6) Summary of Human Health Data Acute Oral Toxicity LDS0 (mg/kg-bw) >2000 Acute Dermal Toxicity LDS0 (mg/kg-bw) >2000 Repeated-Dose Toxicity NOAEL/LOAEL Oral (mg/kg-bw/day) NOAEL = Not established (54-d) LOAEL = 10 (54-d) Reproductive Toxicity NOAEL/LOAEL (mg/kg-bw/day) NOAEL = Not established LOAEL = 10 Developmental Toxicity NOAEL/LOAEL (mg/kg-bw/day) Maternal Toxicity Developmental Toxicity NOAEL = Not established LOAEL = 10 NOAEL = 10 LOAEL = 100 Genetic Toxicity - Gene Mutation In vitro Equivocal Genetic Toxicity - Chromosomal Aberrations In vitro Positive Genetic Toxicity - Chromosomal Aberrations In vivo Negative — indicates endpoint was not addressed for this chemical. 9 ------- |