U.S. Environmental Protection Agency Hazard Characterization Document September, 2014 SCREENING-LEVEL HAZARD CHARACTERIZATION 4-Vinylcyclohexene (CASRN 100-40-3) The High Production Volume (HPV) Challenge Program1 was conceived as 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 sponsored chemicals; sponsorship entailed the identification and initial assessment of the adequacy of existing toxicity data/information, conducting new testing if adequate data did 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 Setl'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 by developing hazard characterizations (HCs). These HCs consist of an evaluation of the quality and completeness of the data set provided in the Challenge Program submissions. They are not intended to be definitive statements regarding the possibility of unreasonable risk of injury to health or the environment. The evaluation is performed according to established EPA guidance2,3 and is based primarily on hazard data provided by sponsors; however, 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. In order to determine whether any new hazard information was developed since the time of the HPV submission, a search of the following databases was made from one year prior to the date of the HPV Challenge submission to the present: (ChemID to locate available data sources including Medline/PubMed, Toxline, HSDB, IRIS, NTP, AT SDR, IARC, EXTOXNET, EPA SRS, etc.), STN/CAS online databases (Registry file for locators, ChemAbs for toxicology data, RTECS, Merck, etc.), Science Direct and ECHA4. OPPT's focus on these specific sources is based on their being of high quality, highly relevant to hazard characterization, and publicly available. OPPT does not develop HCs for those HPV chemicals which have already been assessed internationally through the HPV program of the Organization for Economic Cooperation and Development (OECD) and for which Screening Initial Data Set (SIDS) Initial Assessment Reports (SIAR) and SIDS Initial Assessment Profiles (SIAP) are available. These documents are presented in an international forum that involves review and endorsement by governmental 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. Risk Assessment Guidelines; http://cfpub.epa.gov/ncea/raf/rafguid.cfm. 4 European Chemicals Agency, http://echa.europa.eu. ------- U.S. Environmental Protection Agency Hazard Characterization Document September, 2014 authorities around the world. OPPT is an active participant in these meetings and accepts these documents as reliable screening-level hazard assessments. These hazard characterizations are technical documents intended to inform 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. 2 ------- U.S. Environmental Protection Agency Hazard Characterization Document September, 2014 Chemical Abstract Service Registry Number 100-40-3 (CASRN) Chemical Abstract Index Name Cyclohexene, 4-ethenyl- Structural Formula SMILES: C(=CCCC1C=C)C1 Summary 4-Vinylcyclohexene is a colorless liquid with high vapor pressure and moderate water solubility. It is expected to have moderate mobility in soil. Volatilization of 4- vinyl cyclohexene is high based on its Henry's Law constant. The rate of hydrolysis is expected to be negligible. The rate of atmospheric photooxidation is rapid. 4- Vinylcyclohexene is not readily biodegradable. 4-Vinylcyclohexene is expected to have moderate persistence (P2) and low bioaccumulation potential (Bl). The acute oral and dermal toxicity of 4-vinylcyclohexene is low in rats and rabbits, respectively and moderate in rats via the inhalation route. In 90-day inhalation toxicity studies in rats and mice: 4-vinylcyclohexene caused decreased body weight in males and decreased body weight gain in both sexes without concurrent changes in food consumption in rats at 6.6 mg/L; the NOAEC for systemic toxicity is 4.4 mg/L. In mice, 4-vinylcyclohexene induced mortality in both sexes at 4.4 mg/L; the NOAEC for systemic toxicity is 1.1 mg/L. In 90-day oral gavage repeated-dose toxicity studies in rats and mice with 4-vinylcyclohexene, mortality was observed in females and inflammation of the non-glandular stomach was observed in both sexes at 800 mg/kg-day; the NOAEL for systemic toxicity is 400 mg/kg-day. In the study with mice, mortality and inflammation of the stomach were observed in males at 1200 mg/kg- day (highest dose tested) and mortality in females was observed at 300 mg/kg-day; the NOAELs for systemic toxicity are 600 and 150 mg/kg-day in males and females, respectively. In a two-year NTP chronic oral gavage repeated-dose toxicity study in rats and mice with 4- vinylcyclohexene, mortality and hyperplasia of the forestomach were observed in rats (both sexes) at 200 mg/kg-day; the NOAEL for systemic toxicity is not established. In the same study, mortality and histopathological changes of the stomach was observed in mice (both sexes) at 200 mg/kg-day; the NOAEL for systemic toxicity is not established. A two- generation oral gavage reproductive toxicity study in mice with 4-vinylcyclohexene showed a decrease in body weight and increased liver weights; decreased pup weights, decreased sperm concentration and histopathological changes in the ovaries at 500 mg/kg-day; the NOAEL for systemic, reproductive and developmental toxicity is 250 mg/kg-day. In the 90-day repeated- dose toxicity studies previously mentioned, female mice treated via inhalation showed ovarian atrophy at 4.4 mg/L, and female mice treated orally showed a decrease in the number of primary follicles and mature graafian follicles in the ovaries at 1200 mg/kg-day. In the two- 3 ------- U.S. Environmental Protection Agency Hazard Characterization Document September, 2014 year NTP oral repeated-dose toxicity studies previously mentioned, female mice treated with 200 mg/kg-day of 4-vinylcyclohexene showed tubular cell, hyperplasia granulosa cell and papillary hyperplasia of the ovaries. 4-Vinylcyclohexene induced gene mutations in mouse lymphoma cells but did not induce gene mutations in bacteria cells in vitro. In a sister chromatid exchange and chromosome aberrations study, 4-vinylcyclohexene did not induce chromosomal aberrations in mammalian cells in vitro. In micronucleus assays in mice and rats, 4-vinylcyclohexene did not induce micronuclei in vivo. 4-Vinylcyclohexene is irritating to mice skin, and rabbit skin and eyes. 4-Vinylcyclohexene increased the incidence of tumors in rats and mice. For 4-vinylcyclohexene the 96-h LC50 for fish is 4.60 mg/L and the 48-h EC50 for aquatic invertebrates is 1.87 mg/L. There were no adequate data submitted to evaluate toxicity to aquatic plants. The 21-d EC50, NOEC and LOEC were 0.915, 0.227 and 0.513 mg/L, respectively, for chronic toxicity to aquatic invertebrates. The toxicity to aquatic plants endpoint was identified as a data gap under the HPV Challenge Program. 4 ------- U.S. Environmental Protection Agency Hazard Characterization Document September, 2014 The sponsor, 4-Vinylcyclohexene Group, submitted a Test Plan and Robust Summaries to EPA for 4-vinylcyclohexene (CASRN 100-40-3; CA index name: cyclohexene, 4-ethenyl-) on November 14, 2006. EPA posted the submission on the ChemRTK HPV Challenge website on December 7, 2006 (http://www.epa.gov/hpv/pubs/summaries/4vinvlhx/cl6390tc.htm). EPA comments on the original submission were posted to the website on April 30, 2009. Public comments were also received and posted to the website. The sponsor submitted a response to EPA's comments on July 20, 2009, which was posted to the ChemRTK website on December 7, 2009. 1. Chemical Identity 1.1 Identification and Purity 4-Vinylcyclohexene is an intermediate chemical used to produce styrene, flame retardants, fragrances, solvents, polyoleftn products, and specialty chemicals such as vinylcyclohexene diepoxide. Where specified the purity of 4-vinylcyclohexene is >98%. 1.2 Physical-Chemical Properties The physical-chemical properties of 4-vinylcyclohexene are summarized in Table 1. 4-vinylcyclohexene is a colorless liquid with high vapor pressure and moderate water solubility. Table 1. Physical-Chemical Properties of 4-Vinylcyclohexene1 Property Value CASRN 100-40-3 Molecular Weight 108.18 Physical State Colorless liquid Melting Point -108.9°C (measured) Boiling Point 128.9°C (measured) Vapor Pressure 15.7 mm Hg at 25°C (measured) Dissociation Constant (pKa) Not applicable Henry's Law Constant 4.5x 10"2 atm-m3/mole (estimated)2 Water Solubility 50 mg/L at 25°C (measured) Log Kow 3.93 (measured) 'Expcricn Health Sciences. Inc. 2009. Test Plan and Robust Summary for 4-Vinylcyclohexene. Available online at http://www.epa.gov/chemrtk/pubs/summaries/4vinvlhx/cl6390tc.htm as of May 22, 2012. 2U.S. EPA. 2012. Estimation Programs Interface Suite™ for Microsoft® Windows, v4.10. U.S. Environmental Protection Agency, Washington, DC, USA. Available online at http://www.epa.gov/opptintr/exposure/pubs/episuitedl.htm as of May 22, 2012. 5 ------- U.S. Environmental Protection Agency Hazard Characterization Document September, 2014 2. General Information on Exposure 2.1 Production Volume and Use Pattern 4-Vinylcyclohexene (CASRN 100-40-3) had an aggregated production and/or import volume in the United States between 1-10 million pounds during calendar year 2005. Non-confidential information in the IUR indicated that the industrial processing and uses of the chemical include petrochemical manufacturing as fuels. Non-confidential commercial and consumer uses of this chemical include transportation products. 2.2 Environmental Exposure and Fate The environmental fate properties are provided in Table 2. Table 2. Environmental Fate Properties of 4-Vinylcyclohexene1 Property Value CASRN 100-40-3 Photodegradation Half-life 1.4 hours (estimated reaction with hydroxyl radicals)2 1.3 hours (estimated reaction with ozone)2 Hydrolysis Half-life Stable Biodegradation 0% after 28 days (not readily biodegradable) Bioaccumulation Factor BCF = 83-211 (measured in carp at 0.1 mg/L); BCF = 100-208 (measured in carp at 0.01 mg/L); BAF = 138.4 (estimated)2 Log Koc 2.6 (estimated)2 Fugacity (Level III Model)2 Air (%) Water (%) Soil (%) Sediment (%) 1.2 74 23.5 1.3 Persistence3 P2 (moderate) Bi oaccumul ati on3 Bl (low) 'Expcricn Health Sciences. Inc. 2009. Test Plan and Robust Summary for 4-Vinylcyclohexene. Available online at http://www.epa.gov/chemrtk/pubs/summaries/4vinvlhx/cl6390tc.htm as of May 22, 2012. 2U.S. EPA. 2012. Estimation Programs Interface Suite™ for Microsoft® Windows, v4.10. U.S. Environmental Protection Agency, Washington, DC, USA. Available online at http://www.epa.gov/opptintr/exposure/pubs/episuitedl.htm as of March 16, 2012. 3Federal Register. 1999. Category for Persistent, Bioaccumulative, and Toxic New Chemical Substances. Federal Register 64, Number 213 (November 4, 1999) pp. 60194-60204. 6 ------- U.S. Environmental Protection Agency Hazard Characterization Document September, 2014 4-Vinylcyclohexene is expected to have moderate mobility in soil. 4-Vinylcyclohexene achieved 0% of its theoretical biochemical oxygen demand (BOD) over the course of a 28-day incubation period using an activated sludge inoculum during the modified MITI (OECD 301C) test and is considered not readily biodegradable. This substance is usually produced with an inhibitor to prevent oxidation and polymerization; therefore, it is not expected to be highly persistent in the environment. Volatilization of 4-vinylcyclohexene is high based on the Henry's Law constant. The rate of hydrolysis is expected to be negligible. The rate of atmospheric photooxidation is rapid. 4-Vinylcyclohexene is expected to have moderate persistence (P2) and low bioaccumulation potential (Bl). Conclusion: 4-Vinylcyclohexene is a colorless liquid with high vapor pressure and moderate water solubility. It is expected to have moderate mobility in soil. Volatilization of 4- vinylcyclohexene is high based on its Henry's Law constant. The rate of hydrolysis is expected to be negligible. The rate of atmospheric photooxidation is rapid. 4-Vinylcyclohexene is not readily biodegradable. 4-Vinylcyclohexene is expected to have moderate persistence (P2) and low bioaccumulation potential (Bl). 3. Human Health Hazard A summary of the human health toxicity data submitted for SIDS endpoints is provided in Table 3. Acute Oral Toxicity Carworth-Wistar rats (five/sex/dose) were administered 4-vinylcyclohexene (purity unknown) via oral gavage at 1660 or 3320 mg/kg and observed for 14 days following dosing. Mortality was observed at 3320 mg/kg. OTS0522363 LDso = 2560 mg/kg Acute Dermal Toxicity Male New Zealand White rabbits (four/dose) were administered 4-vinylcyclohexene (purity unknown) via the dermal route at 5, 10 or 20 mL/kg (4150, 8299 or 16,598 mg/kg) under occluded conditions for 24 hours and observed for 14 days following dosing. Mortality was observed at doses > 4150 mg/kg. OTS0522363 LD50 = 16,598 mg/kg Acute Inhalation Toxicity (1) Albino rats (six males or females/dose) were exposed to 4-vinylcyclohexene (purity unknown) as a vapor at nominal concentrations of 4000, 8000 or 16,000 ppm (17.7, 35.4 or 70.8 mg/L; ~ 3202, 6404 or 12,808 mg/kg) for 4 hours and observed for 14 days. Mortality was observed in 4/6 rats at 6404 mg/kg. OTS0522363 LCso = 17.7 - 35.4 mg/L 7 ------- U.S. Environmental Protection Agency Hazard Characterization Document September, 2014 Repeated-Dose Toxicity (1) Sprague-Dawley Crl:CD BR rats (10/sex/dose) were exposed whole-body to 4- vinylcyclohexene (99.8% purity) via inhalation as a vapor at nominal concentrations of 0, 250, 1000 or 1500 ppm (0, 1.1, 4.4 or 6.6 mg/L) for 6 hours/day, 5 days/week for 13 weeks (90-days). Mean measured concentrations were identical to nominal concentrations. At 6.6 mg/L, effects included lethargy in both sexes, statistically significant lower body weights in males and lower body weight gains in both sexes. At > 4.4 mg/L, liver weights were increased in both sexes and kidney weights were increased in males. In the absence of histopathological effects, changes in liver and kidney weights were not considered adverse effects. Increased accumulation of hyaline droplets was observed in the kidneys of male rats at all treatment doses5. No effects were observed on food consumption, hematology, clinical chemistry or urinalysis. Additional details were obtained from TSCATS (OTS0573005). LOAEC = 6.6 mg/L (based on decreased body weights in males and decreased body weight gains in both sexes without concurrent changes in food consumption) NOAEC = 4.4 mg/L (2) B6C3F1 mice (10/sex/dose) were exposed whole-body to 4-vinylcyclohexene (99.8 % purity) via inhalation as a vapor at nominal concentrations of 0, 50, 250 or 1000 ppm (0, 0.22, 1.1 or 4.4 mg/L) for 6 hours/day, 5 days/week for 13 weeks (90-days). Mean measured concentrations were 0, 53, 250 and 1000 ppm. Exposure to 4.4 mg/L resulted in the death of all males and 8/10 females. Lethargy was observed at 4.4 mg/L. The only compound-related histopathological finding was ovarian atrophy, which was observed at 4.4 mg/L. No effects were observed on body weight, food consumption, hematology or clinical chemistry. Additional details were obtained from TSCATS (OTS0573005). LOAEC = 4.4 mg/L (based on mortality in both sexes) NOAEC = 1.1 mg/L (3) Fischer 344 rats (10/sex/dose) were administered 4-vinylcyclohexene (> 99% pure) in corn oil via oral gavage at 0, 50, 100, 200, 400 or 800 mg/kg-day for 5 days/week for 13 weeks (90- days). One male died at 400 mg/kg-day and one female died at 800 mg/kg-day however, the mortalities were not dose-dependent. Decreased body weight was observed in males at 400 mg/kg-day and in females at 800 mg/kg-day (significance not specified). Hyaline droplet degeneration of the proximal convoluted tubule of the kidney was observed in males at 800 mg/kg-day5. Inflammation of the submucosa of the non-glandular stomach (significance not specified) was observed in one male and three females at 800 mg/kg-day. No other treatment- related histological abnormalities were observed. LOAEL = 800 mg/kg-day (based on mortality in females and inflammation of the nongrandular stomach in both sexes) NOAEL = 400 mg/kg-day (4) B6C3F1 mice (10/sex/dose) were administered 4-vinylcyclohexene (purity unknown) in corn oil via oral gavage at 0, 75, 150, 300, 600 or 1200 mg/kg-day for 5 days/week for 13 weeks (90- days). A high level of mortality (9/10 males and 4/10 females) was observed at 1200 mg/kg-day, 5 In the absence of corroborating evidence of kidney toxicity, EPA does not consider the presence of hyaline droplets in the kidney to be an adverse effect for the purposes of determining a NOAEL or LOAEL. 8 ------- U.S. Environmental Protection Agency Hazard Characterization Document September, 2014 with lower mortality (2/10) in females at 300 mg/kg-day. Other deaths during the study consisted of 1-2/10 females from 150-600 mg/kg-day which were stated in the Robust Summary as being due to dosing errors. Body weight was reduced in females (~ 5%) at 600 mg/kg-day and in the sole surviving male (~ 7%) at 1200 mg/kg-day. Mild acute inflammation of the stomach was detected microscopically in both sexes at 1200 mg/kg-day. The number of primary follicles and mature Graafian follicles was reduced in the ovaries of females at 1200 mg/kg-day. No other treatment-related histopathological abnormalities were observed. LOAEL (males) = 1200 mg/kg-day (based on mortality and inflammation of the stomach) NOAEL (males) = 600 mg/kg-day LOAEL (females) = 300 mg/kg-day (based on mortality) NOAEL (females) = 150 mg/kg-day (5) In an NTP study, B6C3F1 mice (50/sex/dose) were administered 4-vinylcyclohexene (> 98% pure) in corn oil via oral gavage at 0, 200 or 400 mg/kg-day for 5 days/week for 103 weeks (2- years). Increased mortality was observed in both sexes, 43/50 males and 33/50 females at 400 mg/kg-day. Mortality at the low dose was comparable to the control. A decrease in body weight was also observed in females (12%) at 400 mg/kg-day by study end and a 13% decrease in male body weight at the same dose. A significant treatment-related increase in ulcers (15%), mild inflammation (14 and 35%) and epithelial hyperplasia (14 and 15%) of the forestomach were observed in males at > 200 mg/kg-day. A significant increase in inflammation (22%) was observed in females at 400 mg/kg-day. Significant treatment-related tubular cell (21 and 28%) and granulosa cell (10%) hyperplasia of the ovary was observed in female mice at > 200 mg/kg- day. A significant increase in congestion of the lung was observed at 400 mg/kg-day in both sexes (72% males and 40% females). A significant increase in atrophy of the splenic red pulp (22%) was observed in males at 400 mg/kg-day. Significant increases in histopathological abnormalities of the adrenal gland, including alteration of the adrenal cortex (49 and 29%) and congestion of the adrenal gland (17%) were observed in females at > 200 mg/kg-day. A 14% increase in hepatic centrilobular congestion was observed in males at 400 mg/kg-day. Neoplastic lesions were observed in the ovary, lung, hematopoietic system and adrenal gland. In the ovary, mixed benign tumors, granulosa cell tumors and carcinomas increased in a dose-related manner in female mice. Alveolar/bronchial adenomas and malignant lymphomas were observed in male mice at 400 mg/kg-day. Capsular adenomas were observed in the adrenal gland of female mice at 400 mg/kg-day. LOAEL = 200 mg/kg-day (based on histopathological changes of the stomach in both sexes, histopathology of the ovary and adrenal gland in females, adenomas and malignant lymphomas in males and mortality at the higher dose) NOAEL = Not established (6) In an NTP study, Fischer 344 rats (50/sex/dose) were administered 4-vinylcyclohexene (purity unknown) in corn oil via gavage at 0, 200 or 400 mg/kg-day for 5 days/week for 103 weeks (2-years). Mortality was observed in the control and all doses in males: control (17/50); 200 (37/50); and 400 (45/50); females: control (10/50); 200 (22/50); and 400 (36/50) atP<0.001 for all groups except 200 mg/kg-day in which P=0.022. Mortality statistical significance was noted in both sexes at 400 mg/kg-day. Mean body weight of treated animals was comparable to the controls except decreased body weights (5-14%) in males at 400 mg/kg-day at study week 9 ------- U.S. Environmental Protection Agency Hazard Characterization Document September, 2014 72. The incidence of epithelial hyperplasia of the forestomach was increased at > 200 mg/kg- day. No other non tumor microscopic lesions were reported. LOAEL = 200 mg/kg-day (based on mortality and epithelial hyperplasia of the forestomach in both sexes) NOAEL = Not established Reproductive Toxicity (1) In a two-generation reproductive toxicity study, CD-I (ICR) BR outbred Swiss albino mice (25/sex/dose) were administered 4-vinylcyclohexene (purity unknown) in corn oil via gavage. The doses for the first generation (F0) were 0, 100, 250 or 500 mg/kg-day, but only the highest dose and control were continued for a second generation (Fl). Reproductive/developmental parameters were measured for each generation and included mating success, number of litters/pair, duration of gestation, number of live and dead pups/litter, pup weight, sex ratio and pup survival and growth to post-natal day 21. Parental endpoints included food and water consumption, body weight, organ weights, histopathology of ovaries and sperm parameters (motility, concentration and morphology). No treatment-related mortality was observed. At 500 mg/kg-day, an 8% decrease in body weights was observed in F0 females and Fl males and females with no concurrent changes in food and water intake. A statistical significant increase in liver weights was observed in Fl animals at 500 mg/kg-day. Statistical significant reductions in pup weights were observed in F0 offsprings at 500 mg/kg-day during weeks 77 and 117. An increase in sperm motility and a decrease in sperm concentration were observed at 500 mg/kg- day in Fl males. Examination of Fl ovaries revealed reductions in the number of primordial oocytes/follicles by 33%, growing follicles by 55% and antral follicles by 33% in females receiving 500 mg/kg-day. LOAEL (systemic toxicity) = 500 mg/kg-day (based on reduced body weights and elevated liver weights) NOAEL (systemic toxicity) = 250 mg/kg-day LOAEL (reproductive toxicity) = 500 mg/kg-day (based on decreased sperm concentration and histopathological changes in the ovaries) NOAEL (reproductive toxicity) = 250 mg/kg-day LOAEL (developmental toxicity) = 500 mg/kg-day (based on reduced pup weights) NOAEL (developmental toxicity) = 250 mg/kg-day (2) In the 90-day inhalation repeated-dose toxicity study in mice described previously, ovarian atrophy was observed at 4.4 mg/L. (3) In the 90-day oral repeated-dose toxicity study in mice described previously, the number of primary follicles and mature Graafian follicles was reduced in the ovaries of females at 1200 mg/kg-day. (4) In the 2-year NTP oral repeated-dose toxicity study in mice described previously, tubular cell hyperplasia, granulosa cell hyperplasia and papillary hyperplasia of the ovary were observed at > 200 mg/kg-day. 10 ------- U.S. Environmental Protection Agency Hazard Characterization Document September, 2014 Developmental Toxicity In the two-generation reproductive toxicity study in mice previously described, a decrease in pup weight was observed at 500 mg/kg-day. Genetic Toxicity — Gene Mutation In vitro (1) In a National Toxicology Program (NTP) study, Salmonella typhimurium strains TA97, TA98, TA100, TA104 and TA1535 were exposed to 4-vinylcyclohexene (purity unknown) in dimethyl sulfoxide (DMSO) at concentrations up to 10,000 |ig/plate with and without metabolic activation. Positive and negative controls were used and yielded the expected results. Cytotoxicity was observed at 333 |ig/plate with and without activation. No increase in mutation frequency was observed. NTP study #609542 4-Vinylcyclohexene was not mutagenic in this assay. (2) In a NTP study, Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 were exposed to 4-vinylcyclohexene (purity unknown) in DMSO at concentrations up to 1000 |ig/plate with and without metabolic activation. Positive and negative controls were used and yielded the expected results. Cytotoxicity was observed at 1000 |ig/plate with activation. No increase in mutation frequency was observed. NTP study #777152 4-Vinylcyclohexene was not mutagenic in this assay. (3) In a NTP study, mouse lymphoma L5178Y TK+/- cells were exposed to 4-vinylcyclohexene (purity unknown) in ethanol at concentrations ranging from 20 to 150 |ig/mL with and without metabolic activation. Positive and negative controls were used and yielded the expected results. Elevated mutation frequencies were observed in cells exposed to 4-vinylcyclohexene with activation. NTP study #971117 4-Vinylcyclohexene was mutagenic in this assay. Genetic Toxicity — Chromosomal Aberrations In vitro (1) In a sister chromatid exchange assay conducted by the NTP, Chinese hamster ovary (CHO) cells were exposed to 4-vinylcyclohexene (purity unknown) in DMSO at 5, 16.7, 50 and 166.7 |ig/mL with and without metabolic activation. Positive and negative controls were used and yielded the expected results. Exposure to 4-vinylcyclohexene did not produce an increase in sister chromatid exchange in CHO cells with or without activation. NTP study #169960 4-Vinylcyclohexene did not induce sister chromatid exchange in this assay. (2) In a chromosomal aberration test conducted by the NTP, CHO cells were exposed to 4- vinylcyclohexene (purity unknown) in DMSO at 25, 49.9, 99.8 or 149.5 |ag/m L without metabolic activation or 12.5, 25, 49.9 or 99.8 |ig/mL with activation. Positive and negative controls were used and yielded the expected results. Exposure to 4-vinylcyclohexene did not 11 ------- U.S. Environmental Protection Agency Hazard Characterization Document September, 2014 produce an increase in chromosomal aberrations in CHO cells with or without activation. Additional details were obtained from NTP study #169960. 4-Vinylcyclohexene did not induce chromosomal aberrations in this assay. In vivo (1) In a bone marrow micronucleus assay, B6C3Fl/CrlBR mice (5/sex/dose) were exposed to 4- vinylcyclohexene (purity unknown) via inhalation at nominal concentrations of 0, 50, 250 or 1000 ppm for 6 hours/day for 5 days/week for 13 weeks. Mean measured concentrations were 0, 53, 250 and 1000 ppm. Mortality was observed at 1000 ppm for all male mice and half of the female mice. Positive controls were used and responded appropriately. Exposure to 4-vinylcyclohexene did not produce an increase in the frequency of micronucleated polychromatic erythrocytes. 4-Vinylcyclohexene did not induce micronuclei in this assay. (2) In a bone marrow micronucleus assay, Sprague-Dawley Crl:CD BR rats (5/sex/dose) were exposed to 4-vinylcyclohexene (purity unknown) via inhalation at nominal concentrations of 0, 250, 1000 or 1500 ppm for 6 hours/day, 5 days/week for 13 weeks. Mean measured concentrations were identical to nominal concentrations. No mortality was observed. Positive controls were used and responded appropriately. Exposure to 4-vinylcyclohexene did not produce an increase in the frequency of micronucleated polychromatic erythrocytes. 4-Vinylcyclohexene did not induce micronuclei in this assay. Additional Information Eye Irritation Rabbits (5/sex unspecified) were administered 0.5 mL of undiluted 4-vinylcyclohexene (purity unknown) in one eye; the other eye served as a control. Eye irritation, consisting of minimal corneal irritation, was scored as 2 on a scale from 1 to 10. Additional details were obtained from TSCATS (OTS0522363). 4-Vinylcyclohexene was slightly irritating to rabbit eyes in this study. Skin Irritation (1) Albino rabbits (5/sex unspecified) were administered 0.01 mL of undiluted 4- vinylcyclohexene (purity unknown) via the dermal route under open conditions for 24 hours. Skin irritation, ranging from moderate capillary injection to marked erythema, was scored as 4 on a scale from 1 to 10. Additional details were obtained from TSCATS (OTS0522363). 4-Vinylcyclohexene was moderately irritating to rabbit skin in this assay. (2) In a 54-week carcinogenicity study described below, male Swiss mice exposed to 4- vinylcyclohexene via the dermal route displayed extensive skin damage. 4-Vinylcyclohexene was strongly irritating to mouse skin in this study. 12 ------- U.S. Environmental Protection Agency Hazard Characterization Document September, 2014 (3) In the acute dermal toxicity study described previously, New Zealand White rabbits administered 4-vinylcyclohexene via the dermal route exhibited reddened scaly or scabby skin. 4-Vinylcyclohexene induced a crying response immediately following application, which was interpreted as a reaction to pain. 4-Vinylcyclohexene was irritating to rabbit skin in this study. Carcinogenicity (1) In the 103-week oral repeated-dose NTP study in rats described previously, neoplastic lesions were observed in the skin, urinary bladder, pituitary, preputial gland and clitoral gland. In the skin, squamous cell papillomas and squamous carcinomas increased in a dose-dependent manner. Neoplasms in the urinary bladder included a transitional cell papilloma and a transitional cell carcinoma. Increases in the incidence of adenomas and carcinomas were elevated in the anterior pituitary gland of females at 200 mg/kg-day. In the clitoral gland, increased incidence of adenoma or squamous cell carcinoma was observed in females at 200 mg/kg-day. 4-Vinylcyclohexene increased the incidence of tumors in rats in this study. (2) In the 103-week oral repeated-dose NTP study in mice described previously, neoplastic lesions were observed in the ovary, lung, hematopoietic system and adrenal gland. In the ovary, mixed benign tumors, granulosa cell tumors and carcinomas increased in a dose-related manner in female mice. Alveolar/bronchial adenomas and malignant lymphomas were observed in male mice at 400 mg/kg-day. Capsular adenomas were observed in the adrenal gland of female mice at 400 mg/kg-day. The NTP concluded that the interpretation of the findings for males (tumors in the lungs and hematopoietic system) was confounded by the poor health and low survival of the animals, which may have resulted in artifactual temporal and statistical associations between treatment and tumor incidence in animals dying of unrelated/undefined causes. In females, 4- vinylcyclohexene increased the incidence of several types of uncommon ovarian tumors in a manner that was independent of survival. It was unclear whether the incidence of adrenal gland tumors was a direct effect of exposure or secondary to altered ovarian function. 4-Vinylcyclohexene increased the incidence of tumors in mice in this study. (3) Male Swiss mice (30) were administered 4-vinylcyclohexene in 50% benzene via the dermal route at 45 mg/day for 3 days/week from 8 weeks of age until their death (~ 54 weeks). Tumors (benign and malignant) were observed in 20% of the mice receiving 4-vinylcyclohexene and in 5% of untreated mice. Only one malignant tumor (a squamous cell cancer) was observed in the test population and in the negative controls. 4-Vinylcyclohexene increased the incidence of tumors equivocally in this study. Conclusion: The acute oral and dermal toxicity of 4-vinylcyclohexene is low in rats and rabbits, respectively and moderate in rats via the inhalation route. In 90-day inhalation toxicity studies in rats and mice, 4-vinylcyclohexene decreased body weight in males and decreased body weight gain in both sexes without concurrent changes in food consumption in rats at 6.6 mg/L; the NOAEC for systemic toxicity is 4.4 mg/L. In mice, 4-vinylcyclohexene induced mortality in 13 ------- U.S. Environmental Protection Agency Hazard Characterization Document September, 2014 both sexes at 4.4 mg/L; the NOAEC for systemic toxicity is 1.1 mg/L. In 90-day oral gavage repeated-dose toxicity studies in rats and mice with 4-vinylcyclohexene, mortality was observed in females and inflammation of the non-glandular stomach was observed in both sexes at 800 mg/kg-day; the NOAEL for systemic toxicity is 400 mg/kg-day. In the study with mice, mortality and inflammation of the stomach were observed in males at 1200 mg/kg-day (highest dose tested) and mortality in females was observed at 300 mg/kg-day; the NOAELs for systemic toxicity are 600 and 150 mg/kg-day in males and females, respectively. In a two-year NTP chronic oral gavage repeated-dose toxicity study in rats and mice with 4-vinylcyclohexene, mortality and hyperplasia of the forestomach were observed in rats (both sexes) at 200 mg/kg- day; the NOAEL for systemic toxicity is not established. In the same study, mortality and histopathological changes of the stomach was observed in mice (both sexes) at 200 mg/kg-day; the NOAEL for systemic toxicity is not established. A two-generation oral gavage reproductive toxicity study in mice with 4-vinylcyclohexene showed a decrease in body weight and increased liver weights; decreased pup weights, decreased sperm concentration and histopathological changes in the ovaries at 500 mg/kg-day; the NOAEL for systemic, reproductive and developmental toxicity is 250 mg/kg-day. In the 90-day repeated-dose toxicity studies previously mentioned, female mice treated via inhalation showed ovarian atrophy at 4.4 mg/L, and female mice treated orally showed a decrease in the number of primary follicles and mature graafian follicles in the ovaries at 1200 mg/kg-day. In the two-year NTP oral repeated-dose toxicity studies previously mentioned, female mice treated with 200 mg/kg-day of 4- vinylcyclohexene showed tubular cell, hyperplasia granulosa cell and papillary hyperplasia of the ovaries. 4-Vinylcyclohexene induced gene mutations in mouse lymphoma cells but did not induce gene mutations in bacteria cells in vitro. In a sister chromatid exchange and chromosome aberrations study, 4-vinylcyclohexene did not induce chromosomal aberrations in mammalian cells in vitro. In micronucleus assays in mice and rats, 4-vinylcyclohexene did not induce micronuclei in vivo. 4-Vinylcyclohexene is irritating to mice skin, and rabbit skin and eyes. 4- Vinylcyclohexene increased the incidence of tumors in rats and mice. 14 ------- U.S. Environmental Protection Agency Hazard Characterization Document September, 2014 Table 3. Summary Table of the Screening Information Data Set as Submitted under the U.S. HPV Challenge Program - Human Health Data Endpoints 4-Vinylcyclohexene 000-40-3) Acute Oral Toxicity 2560 LDso (mg/kg) Acute Dermal Toxicity 16,598 LD50 (mg/kg) Acute Inhalation Toxicity 17.7-35.4 LC50 (mg/L) Repeated-Dose Toxicity (rat; 90-d) NOAEC/LOAEC LOAEC = 6.6 Inhalation (mg/L) NOAEC = 4.4 (mouse; 90-d) LOAEC = 4.4 NOAEC = 1.1 Repeated-Dose Toxicity (rat; 90-d) NOAEL/LOAEL LOAEL = 800 Oral (mg/kg-day) NOAEL = 400 (mouse; 90-d) LOAEL = 1200 (males); highest dose tested NOAEL = 600 (males) LOAEL = 300 (females) NOAEL = 150 (females) (mouse; 2-y) LOAEL = 200 NOAEL = Not Established (rat; 2-y) LOAEL = 200 NOAEL = Not Established Reproductive Toxicity NOAEL/LOAEL Oral (mg/kg-day) LOAEL = 500 Reproductive Toxicity NOAEL = 250 Developmental Toxicity NOAEL/LOAEL (mouse) Oral (mg/kg-day) LOAEL = 500 Maternal/Developmental Toxicity NOAEL = 250 15 ------- U.S. Environmental Protection Agency Hazard Characterization Document September, 2014 Table 3. Summary Table of the Screening Information Data Set as Submitted under the U.S. HPV Challenge Program - Human Health Data Endpoints 4-Vinylcyclohexene 000-40-3) Genetic Toxicity - Gene Mutation In vitro Bacteria Mammalian Cells Negative Positive Genetic Toxicity - Chromosomal Aberrations In vitro Negative Genetic Toxicity - Chromosomal Aberrations In vivo Negative (rat and mouse) Genetic Toxicity - Other In vitro SCE Negative Additional Information Eye Irritation Skin Irritation Carcinogenicity Irritating Irritating Positive [Rat (Fischer)] Positive [Mouse (B6C3F1)] Equivocal [Mouse (Male Swiss)] 4. Hazard to the Environment A summary of aquatic toxicity data submitted for SIDS endpoints is provided in Table 4. Acute Toxicity to Fish Medaka (Oryzias latipes; 1 vessel and 10 organisms per test concentration) were exposed to 4- vinylcyclohexene (95.9 % purity) at 0 (control, solvent control), 1.0, 1.8, 3.3, 6.0, 11.0 or 20.0 mg/L under semi-static conditions for 96 hours. Dimethyl formamide and HCO-60 (polyoxyethylene hydrogenated caster oil 60) were used as solvents at 100 mg/L. Geometric mean measured concentrations were 0 (control, solvent control), 0.62, 1.07, 1.90, 3.08, 5.98 and 11.5 mg/L. The temperature ranged from 23.5 - 23.7 °C, pH ranged from 7.0 - 7.5, and dissolved oxygen concentrations ranged from 6.6 - 8.6 mg/L. Toxicity was based on nominal concentrations, http://www, safe. nite. go. i p/en gli sh/db.html 96-h LCso = 4.60 mg/L 16 ------- U.S. Environmental Protection Agency Hazard Characterization Document September, 2014 Acute Toxicity to Aquatic Invertebrates Water fleas {Daphnia magna; 5 per vessel with 4 vessels per test concentration) were exposed to 4-vinylcyclohexene (95.9 % purity) at 0 (control, solvent control), 0.25, 0.53, 1.1, 2.4 or 5.0 mg/L under semi-static conditions for 48 hours. Dimethyl formamide and HCO-60 (polyoxyethylene hydrogenated caster oil 60) were used as solvents at 25 mg/L. Geometric mean measured concentrations were 0 (control, solvent control), 0.15, 0.31, 0.60, 1.41 and 2.88 mg/L. The temperature ranged from 19.9 - 20.0 °C, pH ranged from 8.1- 8.3, and dissolved oxygen concentrations ranged from 8.4 - 8.8 mg/L. Toxicity was based on measured concentrations, http://www.safe.nite.go.ip/english/db.html 48-h ECso = 1.87 mg/L Toxicity to Aquatic Plants No adequate data were available. Chronic Toxicity to Aquatic Invertebrates Water fleas (Daphnia magna; 1 organism per vessel with 10 vessels per test concentration) were exposed to 4-vinylcyclohexene (95.9 % purity) at 0 (control, solvent control), 0.060, 0.160, 0.440, 1.20 or 3.20 mg/L under semi-static conditions (daily renewal) for 21 days. Dimethyl formamide and HCO-60 (polyoxyethylene hydrogenated caster oil 60) were used as solvents at 51.2 mg/L. Geometric mean measured concentrations were 0 (control, solvent control), 0.025, 0.067, 0.227, 0.513 and 1.45 mg/L. The temperature ranged was 20.0 ± 1°C, pH ranged from 6.7 - 8.5, dissolved oxygen concentrations ranged from 7.6 - 8.8 mg/L and water hardness ranged from 225 - 255 mg/L as CaCC>3. Toxicity was based on nominal concentrations. http://www.safe.nite.go.ip/english/db.html 21-d ECso = 0.915 mg/L (reproduction) 21-d NOEC = 0.227 mg/L (cumulative number of juveniles produced per adult) 21-d LOEC = 0.513 mg/L (cumulative number of juveniles produced per adult) Conclusion: For 4-vinylcyclohexene the 96-h LC50 for fish is 4.60 mg/L and the 48-h EC50 for aquatic invertebrates is 1.87 mg/L. There were no adequate data submitted to evaluate toxicity to aquatic plants. The 21-d EC50, NOEC and LOEC were 0.915, 0.227 and 0.513 mg/L, respectively, for chronic toxicity to aquatic invertebrates. 17 ------- U.S. Environmental Protection Agency Hazard Characterization Document September, 2014 Table 4. Summary Table of the Screening Information Data Set as Submitted under the U.S. HPV Challenge Program - Aquatic Toxicity Data Endpoint 4-Vinylcyclohexene (CASRN 100-40-3) Fish 96-h LCso (mg/L) 4.60 Aquatic Invertebrates 48-h ECso (mg/L) 1.87 Aquatic Plants 72-h ECso (mg/L) No Adequate Data Chronic Toxicity to Aquatic Invertebrates 21-day ECso (mg/L) 21-day NOEC (mg/L) 21-day LOEC (mg/L) 0.915 0.227 0.513 Bold = experimental data (i.e. derived from testing) 18 ------- |