U.S. Environmental Protection Agency Hazard Characterization Document December 2012 SCREENING-LEVEL HAZARD CHARACTERIZATION SPONSORED CHEMICAL Trimethyl Phosphite CASRN 121-45-9 SUPPORTING CHEMICALS Dimethyl Phosphonate CASRN 868-85-9 Methanol CASRN 67-56-1 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 Setl1'2) 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. 2 3 The evaluation is performed according to established EPA guidance ' 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. 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 December 2012 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 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 December 2012 Chemical Abstract Service Registry Number Sponsored Chemical (CASRN) 121-45-9 Supporting Chemicals 868-85-9 67-56-1 Chemical Abstract Index Name Sponsored Chemical Phosphorus acid, trimethyl ester Supporting Chemicals Phosphorus acid, dimethyl ester Methanol Structural Formula Sponsored Chemical H3C-0^ ,0-CH3 0 1 ch3 SMILES: 0(P(0C)0C)C Supporting Chemicals 0 ii A ch3 ch3 SMILES: 0=P(0C)0C CH3-OH SMILES: CO Summary TMP is a colorless liquid with high vapor pressure that reacts rapidly with water. It would be expected to have moderate mobility in soil, and volatilization would be moderate based on its Henry's Law constant; however, the rapid rate of hydrolysis suggests that these and other environmental fate pathways are not applicable for this substance. The rate of atmospheric photooxidation is slow. TMP is expected to have low persistence (PI) and low bioaccumulation potential (Bl). 3 ------- U.S. Environmental Protection Agency Hazard Characterization Document December 2012 The acute oral toxicity of TMP to rats and mice is low; acute inhalation toxicity to rats is low and the acute dermal toxicity to rabbits is moderate. In a 90-day oral (gavage) repeated-dose toxicity study in rats, TMP resulted in mortality at 160 mg/kg-day. The surviving animals at this dose showed effects on liver and male reproductive organs. The NOAEL is 80 mg/kg-day. In a 4-week oral (gavage) toxicity study in rats, effect on body weight and food consumption was seen at 164 mg/kg-day. The NOAEL is 32.8 mg/kg-day. In three separate four-week repeated-inhalation toxicity studies in rats, ocular effects were observed, including formation of irreversible cataracts at 0.53 mg/L-day in two studies and at 0.26 mg/L-day in one study. The NOAEC values in two studies are not established; the NOAEC for the third study is 0.051 mg/L-day. (In a limited study of employees of a TMP manufacturing plant showed no clinical cataracts in either exposed or non-exposed employee groups.) In a 21-dy dermal toxicity study in rabbits, TMP resulted in mortality and effects on liver at > 300 mg/kg-day; the NOAEL is not established. No reproductive toxicity studies were available for TMP, however, adverse effects on spermatogenesis and testes were observed in the 90-day oral repeated-dose toxicity study in rats. In an oral (gavage) combined reproduction/developmental toxicity screening test in rats with the supporting chemical, DMHP, effects on mating and fertility indices were seen at 270 mg/kg-day; the NOAEL for reproductive toxicity is 90 mg/kg-day. The systemic toxicity NOAEL is 90 mg/kg-day based on mortality and severe body weight effects at 270 mg/kg-day. In the same study, the NOAEL for maternal and developmental toxicity is 90 mg/kg-day, based on no effects at the highest dose at which females survived to term. In a two-generation reproductive toxicity study in rats via inhalation, the supporting chemical, methanol, showed no systemic or reproductive toxicity at 1.3 mg/L-day, highest dose tested. In the same study, the earlier time of descensus testis in pups was seen at 0.013 mg/L-day; the NOAEC for developmental toxicity is not established. In an oral prenatal developmental toxicity study in rats with TMP, decreased body weights were seen in dams at 164 mg/kg-day; the NOAEL for maternal toxicity is 49 mg/kg-day. In the same study, skeletal and soft tissue abnormalities were seen in pups at 164 mg/kg-day; the NOAEL for developmental toxicity is 49 mg/kg-day. TMP induced gene mutation in mammalian cells but not in bacteria in vitro, induced micronuclei in bone marrow in vivo, induced DNA damage in bacteria in vitro and produced point mutations, chromosomal aberrations and chromosome loss in fruit flies in vivo. TMP is irritating rabbit skin and eyes. For aquatic toxicity, all values are based on the supporting chemical DMHP. The 96-hour LC50 of supporting chemical DMHP for fish is > 15.6 mg/L. The 48-hour EC50 of supporting chemical DMHP for aquatic invertebrates is 24.8 mg/L. The 72-hour EC50 of supporting chemical DMHP for aquatic plants is >25 mg/L (growth rate). No data gaps were identified under the HPV Challenge Program. 4 ------- U.S. Environmental Protection Agency Hazard Characterization Document December 2012 The sponsor, Rhodia Inc., representing a consortium with United Phosphorus Inc. and Sabero Organic Gujarat Ltd., submitted a Test Plan and Robust Summaries to EPA for trimethyl phosphite (TMP; CAS No. 121-45-9; CA name: phosphorous acid, trimethyl ester) on December 21, 2005. EPA posted the submission on the ChemRTK HPV Challenge website on February 7, 2006 (http://www.epa.gov/HPV/pubs/summaries/viewsrch.htm). EPA comments on the original submission were posted to the website on August 11, 2008 (http://www.epa.gov/HPV/pubs/summaries/trimtphs/cl6139tc.htm). Public comments were also received and posted to the website. Justification for Supporting Chemicals Dimethyl phosphonate (DMHP; CASRN 868-85-9; CA name: phosphonic acid, dimethyl ester) is used as a supporting chemical for trimethyl phosphate. TMP hydrolyses to form DMHP and methanol. Both hydrolysis products were assessed in the OECD HPV program, and their data sets can be viewed at the following link: http://cs3-hq.oecd.org/scripts/hpv/. The first stage hydrolysis reaction occurs in minutes over a range of pH values. EPA agrees that the using DMHP and/or methanol data for addressing data gaps for human health and ecotoxicity endpoints for TMP is appropriate. 5 ------- U.S. Environmental Protection Agency Hazard Characterization Document December 2012 1. Chemical Identity 1.1 Identification and Purity TMP is a colorless liquid with high vapor pressure that reacts rapidly with water. Where indicated in robust summaries, the purity of the tested substance is >98%. Since TMP hydrolyzes rapidly, it was protected from hydrolysis with nitrogen during the studies. 1.2 Physical-Chemical Properties The physical-chemical properties of TMP (CASRN 121-45-9) and DMHP (CASRN 868-85-9) are summarized in Table 1. The structures are provided in the Appendix. Table 1. Physical-Chemical Properties of Trimethyl Phosphite and Dimethyl Phosphonate1 Property Trimethyl Phosphite (TMP) Dimethyl Phosphonate (DMHP) CASRN 121-45-9 868-85-9 Molecular Weight 124.08 110.05 Physical State Clear, colorless, water-like liquid Colorless liquid2 Melting Point -78°C (measured) 29°C (measured)2 Boiling Point 111-112°C (measured) 170.5°C (measured)2 Vapor Pressure 24.1 mm Hg at 25°C (measured) 1.5 mm Hg at 20°C (measured) Dissociation Constant (pKa) Not applicable Not applicable Henry's Law Constant Not applicable due to hydrolysis 2.2><10"6 atm-m3/mole (estimated)3 Water Solubility Not applicable due to hydrolysis 100,000 mg/L at 20°C (measured)2 Log Kow Not applicable due to hydrolysis -1.13 (estimated)3 'The Trimethyl Phosphite Consortium. 2005. Test Plan and Robust Summary for Trimethyl Phosphite. Available online at http://www.epa.gov/oppt/chemrtk/pubs/summaries/trimtphs/cl6139tc.htm as of June 4, 2012. 2Hazardous Substance Databank (HSDB). 2012. Available online at http://toxnet.nlm.nih.gov/cgi-bin/sis/htmlgen7HSDB as of June 1,2012. 3U.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 June 4, 2012 2. General Information on Exposure 2.1 Production Volume and Use CASRN 121-45-9 had an aggregated production and/or import volume in the United States between 1 to 10 million pounds during calendar year 2005. Trimethyl phosphate is used as a solvent for aromatic halogenations and nitrations as required for the preparation of pesticides and pharmaceuticals. 6 ------- U.S. Environmental Protection Agency Hazard Characterization Document December 2012 2.2 Environmental Exposure and Fate Environmental Fate Characterization TMP is expected to have moderate mobility in soil, and volatilization is expected to be moderate; however, the rapid rate of hydrolysis indicates that volatilization, mobility in soil and biodegradation will not be important environmental fate processes. The hydrolysis half-life of TMP is reported <5 minutes at pH 6 at 0°C. Its hydrolysis product, DMHP was degraded 50% as measured by dissolved organic carbon (DOC) loss after 28 days using the modified OECD (OECD 301E) test. DMHP achieved 48% of its theoretical biochemical oxygen demand (BOD) using an activated sludge inoculum and the MITI (OECD 301C) test after a 28 day incubation period. It also undergoes hydrolysis to produce monomethyl phosphonate which slowly hydrolyzes to phosphorous acid and methanol. The rate of atmospheric photooxidation of TMP is slow. TMP is expected to have low persistence (PI) and low bioaccumulation potential (Bl). The environmental fate characteristics of TMP (CASRN 121-45-9) and DMHP (CASRN 868-85-9) are summarized in Table 2. Table 2. Environmental Fate Characteristics of Trimethyl Phosphite and DMHP1 Property Trimethyl phosphite (TMP) Dimethyl phosphonate (DMHP) CASRN 121-45-9 868-85-9 Photodegradation Half-life 1.3 days (estimated)2 1.9 days (estimated)2 Hydrolysis Half-life 21 minutes at pH 10 and 25°C; <5 minutes at pH 6 and 0°C 10 days at 25 °C and 19 days at 20°C at an unspecified pH3 Biodegradation Not applicable due to hydrolysis 50% after 28 days (not readily biodegradable); 48% after 28 days (not readily biodegradable)3 Bioaccumulation Factor Not applicable due to hydrolysis BAF = 0.9 (estimated)2 Log Koc Not applicable due to hydrolysis 3.7 (estimated)2 Fugacity (Level III Model)2'4 Air (%) Water (%) Soil (%) Sediment (%) 98.7 0.6 0.6 <0.1 3.7 42.8 53.5 <0.1 Persistence5 PI (low) PI (low) Bioaccumulation5 Bl (low) Bl (low) 7 ------- U.S. Environmental Protection Agency Hazard Characterization Document December 2012 'The Trimethyl Phosphite Consortium. 2005. Test Plan and Robust Summary for Trimethyl Phosphite. Available online at http://www.epa.gov/oppt/chemrtk/pubs/summaries/trimtphs/cl6139tc.htm as of June 1,2012. 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 June 1, 2012 3Hazardous Substance Databank (HSDB). 2012. Available online at http://toxnet.nlm.nih.gov/cgi-bin/sis/htmlgen7HSDB as of June 1,2012. 4Half-lives of 0.08 hours were used for the water, soil, and sediment compartments while a half-life of 15 hours was used for the atmosphere compartment. 'Federal Register. 1999. Category for Persistent, Bioaccumulative, and Toxic New Chemical Substances. Federal Register 64, Number 213 (November 4,1999) pp. 60194-60204. Conclusion: TMP is a colorless liquid with high vapor pressure that reacts rapidly with water. It would be expected to have moderate mobility in soil, and volatilization would be moderate based on its Henry's Law constant; however, the rapid rate of hydrolysis suggests that these and other environmental fate pathways are not applicable for this substance. The rate of atmospheric photooxidation is slow. TMP is expected to have low persistence (PI) and low bioaccumulation potential (Bl). 3. Human Health Hazard A summary of health effects data are provided in Table 3. Acute Oral Toxicity Trimethyl phosphite (CAS No. 121-45-9) (1) Sprague-Dawley rats (2/sex/dose) were administered TMP (purity not stated) via gavage at 900, 1350, 2025 or 3038 mg/kg and observed for 14 days. Mortality was 0/4, 2/4, 4/4 and 4/4 at 900, 1350, 2025 or 3038 mg/kg, respectively. LD50 = 1350 mg/kg (2) In seven studies, Wistar rats (5/sex/dose for each study) were administered TMP (purity 98.5-99%) via gavage at five doses (in each study) between 500 and 3000 mg/kg and observed for 14 days. Mortality was observed at > 1000 mg/kg. LD50 = 1500 - 2240 mg/kg (3) ICR mice (5/sex/dose) were administered TMP (purity 98.5%) via gavage at 3000, 3500, 4000, 4500 or 5000 mg/kg and observed for 14 days. Mortality was 2/10, 2/10, 3/10, 7/10 and 8/10 at 3000, 3500, 4000, 4500 or 5000 mg/kg, respectively. LD50 = 4280 mg/kg Acute Inhalation Toxicity Trimethyl phosphite (CAS No. 121-45-9) Male rats (5/concentration, strain not stated) were exposed via whole body inhalation to TMP (purity not stated) at 9000 ppm (45.7 mg/L) for 4 hours. The observation period is not stated in the robust summary. There was no mortality. LC50 > 45.7 mg/L 8 ------- U.S. Environmental Protection Agency Hazard Characterization Document December 2012 Acute Dermal Toxicity Trimethylphosphite (CAS No. 121-45-9) (1) New Zealand White rabbits (minimum of 2/sex/dose) were administered TMP (purity not stated) via the dermal route on intact and abraded sites at 266.7, 400, 600, 900, 1350, 2025, 3000 or 4556 mg/kg under occluded conditions for 24 hours. All residual test substance ewas removed following the exposure period. The animals were observed for a total of 14 days. Mortality occurred at > 400 mg/kg. LD5o = 934 mg/kg (2) Male New Zealand White rabbits (4/dose) were administered TMP (purity 98.5%) via the dermal route at 6000, 7500 or 9200 mg/kg and observed for up to 14 days. Exposure duration, abrasion and occlusion conditions were unspecified. Mortality occurred at > 7500 mg/kg. LD50 = 7500 mg/kg Repeated-Dose Toxicity Trimethyl phosphite (CAS No. 121-45-9) Oral In a subchronic study, Sprague-Dawley rats (15/sex/dose) were administered TMP (purity 98.5%, protected from hydrolysis with Nitrogen) by gavage (as 10% solution in corn oil) at 0, 40, 80 or 160 mg/kg-day for 90 consecutive days. At 160 mg/kg-day, treatment-related mortality was seen (4 males and 3 females). In two animals tremors were seen in week 11. Body weight and food consumption was significantly decreased (significance not provided); no consistent pattern was seen in food utilization. No differences were observed in hematology, clinical chemistry or urinalyses parameters. Significantly increased (significance not provided) absolute weights of adrenal, brain and kidneys (both sexes) and significantly increased (significance not provided) relative (to body) weights of heart and spleen were seen in females. At gross pathology examination, irregular thickening of the stomach (one animal) and decreased spleen size (two animals) were seen at 160 mg/kg-day. Histopathological examination at 160 mg/kg-day revealed minor fatty changes in the liver of both sexes and occasional hepatocytes containing eosinophilic material in males. Hypoplasia of gonads, seen in 11 of 11 males at 160 mg/kg-day, was characterized by reduced spermatogenesis and reduction in size and number of spermatogonia and other spermatogenic cells. However, density of sperm in the epididymis was "not greatly or uniformly reduced" at 160 mg/kg-day. No significant effects were seen at lower doses. LOAEL = 160 mg/kg-day (based on mortality and histopathological changes in liver and male reproductive tissues) NOAEL = 80 mg/kg-day (2) In a sub-acute study, Sprague-Dawley rats (5/sex/dose) were TMP (purity 98.5%, protected from hydrolysis with Nitrogen) by gavage at 0, 32.8, 164 or 328 mg/kg-day for 21 consecutive days. Treatment-related mortality was seen at 328 mg/kg-day (4 males and 4 females). At 9 ------- U.S. Environmental Protection Agency Hazard Characterization Document December 2012 328 mg/kg-day, clinical signs included decreased locomotor activity, abnormal gait and rough coat in all animals. Decreased locomotor activity was also seen in animals at 164 mg/kg-day. Body weights in females were significantly decreased (significance not provided) at >164 mg/kg-day during weeks 2 and 3. Food consumption was significantly decreased (significance not provided) in females at 164 (week 3) and 328 (weeks 2 and 3) mg/kg-day. Food utilization was also decreased at these doses. There were no effects on hematology, clinical chemistry and urinalyses parameters. Gross pathology examination showed congestion of stomach in all animals and loss of mucosal surface (2 animals) at 328 mg/kg-day. Histopathological examination was not conducted. LOAEL = 164 mg/kg-day (based on mortality and effect on body weight and food consumption) NOAEL = 32.8 mg/kg-day Inhalation (1) In a 4-week study, Sprague-Dawley rats (10/sex/concentration) were exposed via whole-body inhalation to TMP at 0, 104, 292 or 581 ppm (equivalent to 0, 0.53, 1.48 or 2.95 mg/L—effective inhaled concentrations) 6 hours/day, 5 days/week. Treatment-related deaths (not reported by sex) occurred in 2/20 rats at 292 ppm and 7/20 rats at 581 ppm. Clinical observation showed yellow perianal staining at all concentrations, labored breathing, excessive lacrimation and chromodacryorrhea at 292 and 581 ppm and reduced activity, coldness of body, general poor condition and closed eyes at 581 ppm. Lower body weight and body weight gain occurred in both sexes at 581 ppm. Increased kidney-to-body weight and kidney-to brain weight ratios were seen at 292 and 581 ppm in females; absolute kidney weights in females were increased at 292 ppm and decreased at 581 ppm. Decreased absolute and relative brain weights (to body weight) were also seen at 581 ppm. Red blood cell count was elevated in males at 581 ppm. Gross pathology revealed scattered lung abnormalities in all treated groups and increased lung discoloration and stomach abnormalities at 581 ppm. Ophthalmic examination at study termination, showed corneal opacity at 104 ppm, corneal opacity and cloudy eyes at 292 ppm and corneal opacity, cloudy eyes and film covering one or both eyes at 581 ppm. Histological examination revealed cataracts at 292 and 581 ppm (incidences not given). Based on the ocular effects seen in this study, the same laboratory conducted two follow-up studies that focused on examination of the eyes more frequently, in greater detail and with more animals/group [see summaries (2) and (3) below]. In the follow-up studies only mortality, clinical signs, body weight, lung weight, ophthalmoscopy and histological examination of lungs, eyes and gross lesions were conducted. LOAEC = 0.53 mg/L-day (based on ocular effects) NOAEC = Not established (2) Sprague-Dawley rats (20/sex in control and low-concentration groups; 36/sex in high- concentrations group) were exposed via whole-body inhalation to TMP at 0, 105 or 600 ppm (equivalent to 0, 0.53 or 3.04 mg/L) 6 hours/day, 5 days/week, for 28 days followed by 8 weeks of post-exposure observation. Deaths occurred in 31 males and 20 females at 600 ppm. No relevant non-ocular clinical signs were recorded. Mean body weights and body weight gains were lower at 600 ppm throughout weeks 1- 5. Absolute and relative lung weights were increased at 600 ppm, accompanied by a high incidence of lung abnormalities at gross observation. Histopathology associated the deaths at 600 ppm with bronchopneumonia in 10 ------- U.S. Environmental Protection Agency Hazard Characterization Document December 2012 23 of the males and 14 of the females—pulmonary granulomatous foci were seen in many of these animals (22 males and 10 females). Ophthalmoscopy showed reversible striate opacities in 7/30 animals at 105 ppm and irreversible cataracts in almost all animals at 600 ppm. Histopathology confirmed the presence of irreversible cataracts at 600 ppm. LOAEC = 0.53 mg/L-day (based on ocular effects) NOAEC = Not established (3) Sprague-Dawley rats (20/sex/group) were exposed via whole-body inhalation to TMP at measured concentrations of 0, 10, 51 or 101 ppm (equivalent to 0, 0.051, 0.26 or 0.51 mg/L, respectively), 6 h/day, 5 days/week, for 4 weeks followed by 8 weeks of post-exposure observation. No treatment-related deaths occurred and no non-ocular clinical signs were recorded. Treatment did not affect body weight, lung weight or findings at gross observation. Using ophthalmoscopy, ocular lesions (irregularities of the corneal surface) were first observed at 4 weeks at 51 ppm (3/15 males and 7/17 females) and 101 ppm (5/15 males and 7/17 females). Lenticular opacities in females were seen at 51 and 101 ppm at week 2 post-exposure and at 101 ppm at weeks 4 and 8 post-exposure. Histopathology showed mild inflammatory changes in corneas of approximately 60% of males and females sacrificed after 4 weeks of exposure to 51 or 101 ppm; these effects were not observed in animals sacrificed at 2 or 8 weeks post-exposure. The robust summary concluded that TMP is cataractogenic and a corneal irritant at 51 and 101 ppm. LOAEC = 0.26 mg/L (based on ocular effects) NOAEC = 0.051 mg/L Dermal New Zealand white rabbits (6/sex/dose) were administered undiluted TMP (purity 98.5%) dermally at 0, 300, 600 or 1200 mg/kg-day 6 hours/day for 21 days. The test substance was applied to the clipped trunks of the animals; the skin of half of the animals was abraded. After the exposure period each day, the test substance was removed by gentle washing with warm water. Mortality was 0/12 1/12, 2/12 and 11/12 at 0, 300, 600 or 1200 mg/kg-day, respectively. Vocalization, decreased locomotor activity (at 300 and 600 mg/kg-day) and loss of righting reflex was seen at 600 mg/kg-day. Treated animals showed dose-related irritation (edema and Erythema) with severity increasing in animals with abraded skin with increasing time. Body weights were significantly lower (significance not provided) at 1200 mg/kg-day. At necropsy, a dose-related increased incidence of light-colored spots on lungs was seen. Histopathology examination showed lung congestion and edema and advanced parenchymatious degeneration with chronic round cell infiltration in liver at 600 mg/kg-day. At 300 mg/kg-day, lung congestion and edema was seen at same degree as that for 600 mg/kg-day animals; effect on liver was less marked than for 600 mg/kg-day. LOAEL = 300 mg/kg-day (based on mortality and histopathology of liver and lungs) NOAEL = not established 11 ------- U.S. Environmental Protection Agency Hazard Characterization Document December 2012 Reproductive Toxicity Trimethylphosphite (CAS No. 121-45-9) No reproductive toxicity study data were submitted for TMP, but it produced adverse effects on spermatogenesis and testes histology in the 90-day repeated-dose oral study in rats described above. These data alone are inadequate to satisfy the reproductive toxicity endpoint. Reproductive toxicity data for the hydrolysis products of TMP, DMHP and methanol, are used to address the reproductive toxicity endpoint for TMP. Dimethylphosphonate (CAS No. 868-85-9, supporting chemical) In a combined reproduction/developmental toxicity screening study, Wistar rats (12/sex/dose) were administered DMHP by gavage at 0, 30, 90 or 270 mg/kg-day from 2 weeks before mating to the end of gestation and up to 4 - 5 days of lactation. Males were sacrificed after at least 28 days of treatment. Females and pups were sacrificed on days 4-5 postpartum. At 270 mg/kg-day, animals of both sexes exhibited clear clinical signs of systemic toxicity (poor general state, apathy, high stepping gait, squatting position, bloody muzzle, piloerection, emaciation, tremor and/or desiccation of skin) and severe body weight loss. Two males of this group were found dead with discolored liver and/or lungs, and all females of this group had to be sacrificed in moribund condition during mating or gestation. At 90 mg/kg-day, soft feces and/or diarrhea were noted in both sexes more frequently than in the control group. No treatment- related effects were noted at 30 mg/kg-day. At 270 mg/kg-day, relative testis weight was increased and absolute epididymidis weight was decreased; however, no pathological changes were found at macroscopic and microscopic examinations of these organs. No treatment-related histological changes were reported for ovaries. At 270 mg/kg-day, the number of females with corpora lutea and implantation sites and the frequency and severity score of "large corpora lutea" and of granular luteal cells were decreased and mating and fertility indices were reduced. At 30 and 90 mg/kg-day, no effects were found on reproductive endpoints (mating parameters, fertility index, gestation indices, gestation length, prenatal loss, number of implantation sites, macroscopically visible corpora lutea, live birth index, sex ratio, pup birth weight, litter size, pup weight development, viability and lactation of F1 rats). Sex ratio, mortality and weights of F1 pups were not affected by treatment up to and including 90 mg/kg-day, while evaluation was not possible at higher doses as there were no surviving pups. No externally malformed pups were observed. Summarized from "Dimethyl Phosphonate", OECD SIDS, "868859.pdf' at http://cs3-hq.oecd.org/scripts/hpv/. LOAEL (systemic toxicity) = 270 mg/kg-day (based on mortality and severe body weight loss) NOAEL (systemic toxicity) = 90 mg/kg-day LOAEL (reproductive toxicity) = 270 mg/kg-day (based on reduced insemination and fertility indices) NOAEL (reproductive toxicity) = 90 mg/kg-day 12 ------- U.S. Environmental Protection Agency Hazard Characterization Document December 2012 Methanol (CAS No. 67-56-1, supporting chemical) In a two-generation reproductive toxicity study, Sprague-Dawley rats (30/sex/group for F0 animals) were exposed 20 hours/day via whole-body inhalation to methanol at concentrations of 0 (untreated control), 0.013, 0.13 or 1.3 mg/L. For F0 animals, the premating exposure period was 60 days. Exposure durations were as follows: F0 males, age 8 weeks through mating; F0 females, age 8 weeks through end of lactation; F1 males, from birth through end of mating; F1 females, from birth through weaning of F2 pups; and F2 males and females, from birth to day 21. Mating periods were up to 21 days. No treatment-related alterations of reproductive endpoints, including sexual cycle, mating time, fertility and pregnancy rate, were seen in F0 or F1 animals. The developmental parameter descensus testis occurred 0.5 tol day earlier than control in F1 and F2 pups at 1.3 mg/L and occurred 0.5 days earlier than control in F2 pups at 0.13 mg/L. At 1.3 mg/L, absolute and relative (to body) brain weights were decreased in F1 and F2 pups, but this was not accompanied by histological changes. Summarized from "Methanol", OECD SIDS, "38958562.zip" at http://cs3-hq.oecd.org/scripts/hpv/. NOAEC (F0 parental toxicity) = 1.3 mg/L-day (highest concentration tested) NOAEC (reproductive toxicity) = 1.3 mg/L-day (highest concentration tested) LOAEC (developmental toxicity) = 0.013 mg/L-day (based on earlier time of descensus testis) NOAEC (developmental toxicity) = Not established Trimethylphosphite (CAS No. 121-45-9) In the 90-day gavage study of trimethyl phosphite in Sprague-Dawley rats described previously, hypoplasia of gonads, seen in 11 of 12 males at 160 mg/kg-day, was characterized by reduced spermatogenesis and reduction in size and number of spermatogonia and other spermatogenic cells. However, density of sperm in the epididymis was "not greatly or uniformly reduced" at 160 mg/kg-day. No adverse effects on male reproductive organs were seen at the other doses tested (40 and 80 mg/kg-day). Trimethyl phosphite reduced spermatogenesis and produced adverse histological changes in the testes in this repeated-dose oral study. Developmental Toxicity Trimethyl phosphite (CAS No. 121-45-9) In a prenatal developmental toxicity study, pregnant Sprague-Dawley rats (25/dose) were administered TMP by gavage in corn oil at 0, 16, 49 or 164 mg/kg-day from day 6 through day 15 of gestation and were sacrificed on gestation day 21. No treatment-related maternal clinical signs or mortalities occurred. At 164 mg/kg-day, maternal body weight was reduced during days 6 - 10 of gestation and at day 20; dam body weight minus gravid uterus weight was also reduced at this dose. Developmental effects at 164 mg/kg-day included abnormalities of long bones, increases in dilated ventricles and undescended testes and teratogenic effects including exencephaly, spina bifida and scoliosis and cleft palate. No developmental effects were noted in mid- and low-dose groups. LOAEL (maternal toxicity) = 164 mg/kg-day (based on reduced body weight) NOAEL (maternal toxicity) = 49 mg/kg-day LOAEL (developmental toxicity) = 164 mg/kg-day (based on skeletal and soft tissue abnormalities) NOAEL (developmental toxicity) = 49 mg/kg-day 13 ------- U.S. Environmental Protection Agency Hazard Characterization Document December 2012 Dimethylphosphonate (CAS No. 868-85-9, supporting chemical) In a combined reproduction/developmental toxicity screening study described above, Wistar rats (12/sex/dose) were administered DMHP by gavage at 0, 30, 90 or 270 mg/kg-day from 2 weeks before mating to the end of gestation and up to 4 - 5 days of lactation. Males were sacrificed after at least 28 days of treatment. Females and pups were sacrificed on days 4-5 postpartum. At 270 mg/kg-day, the number of females with corpora lutea and implantation sites and the frequency and severity score of "large corpora lutea" and of granular luteal cells were decreased and mating and fertility indices were reduced. At 30 and 90 mg/kg-day, no effects were found on reproductive endpoints (mating parameters, fertility index, gestation indices, gestation length, prenatal loss, number of implantation sites, macroscopically visible corpora lutea, live birth index, sex ratio, pup birth weight, litter size, pup weight development, viability and lactation of F1 rats). Sex ratio, mortality and weights of F1 pups were not affected by treatment up to and including 90 mg/kg-day, while evaluation was not possible at higher doses as there were no surviving pups. No externally malformed pups were observed. Summarized from "Dimethyl Phosphonate", OECD SIDS, "868859.pdf' at http://cs3-hq.oecd.ore/scripts/hpv/. NOAEL (maternal/developmental toxicity) = 90 mg/kg-day (based on no effects at highest dose in which females survived to term) Genetic Toxicity — Gene Mutation In vitro Trimethylphosphite (CAS No. 121-45-9) (1) Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538 were exposed to TMP at 0.5, 3, 15, 30 or 50 |iL/plate with and without metabolic activation. Positive and negative controls were tested concurrently and responded appropriately. TMP was cytotoxic at 50 |iL/plate without activation and 30 |iL/plate with activation. Trimethyl phosphite was not mutagenic in this assay. (2) Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538 and Sacromyces cerevisiae D4 were exposed to trimethyl phosphite at 0.001, 0.01, 0.1, 1 or 5 |iL/plate with and without metabolic activation. Positive and negative controls were tested concurrently and responded appropriately. No cytotoxicity was seen at any concentration tested. Trimethyl phosphite was not mutagenic in this assay. (3) In three studies, mouse lymphoma cells (L5178Y/TK+/-) were exposed to trimethyl phosphite at various concentrations (0.18 - 2.7 |iL/mL without metabolic activation and 0.24 - 3.2 |iL/mL with activation in the first study; 0.13-1.8 |iL/mL without metabolic activation and 0.24 - 3.2 |iL/mL with activation in the second study; and 0.18 - 2.7 |iL/mL without metabolic activation and 0.24 - 3.2 |iL/mL with activation in the third study). Positive and negative controls were tested concurrently and responded appropriately. Trimethyl phosphite was cytotoxic at 1 |iL/mL in the first study and 10 |iL/mL in the other two studies. In all three studies, trimethyl phosphite induced increases in mutation with and without metabolic activation. Trimethyl phosphite was mutagenic in these assays. 14 ------- U.S. Environmental Protection Agency Hazard Characterization Document December 2012 In vivo Trimethylphosphite (CAS No. 121-45-9) In three studies, fruit flies (Drosophila melanogaster) were exposed to TMP (purity 99.51%) at multiple concentrations (0.07 mL as an aerosol in 25 mL flasks for 1 minute or 30 seconds, respectively, in the first and second studies; 0.3 mL in a 50-mL volume for an unspecified interval in the third study). Positive and negative controls were tested concurrently and responded appropriately. In the first study (1-minute exposure), no effects occurred for the point-mutation endpoints (induction of sex-linked lethals and white-ivory somatic reversions) and the chromosome aberrations and loss endpoints (induction of dominant lethal mutations, Y chromosome loss and bithorax test of Lewis) that were examined. Because levels of sterility and mortality were unacceptable after 1 minute of exposure in the first study, the exposure period was reduced to 30 seconds in the second study. In this study, TMP was positive in a test for point mutations (induction of sex-linked lethals) and positive for two tests for chromosome aberrations and loss (induction of dominant lethal mutations and the bithorax test of Lewis). In the third study, TMP was positive for the same endpoints as in the second study and, in addition, induced Y chromosome loss. Trimethyl phosphite induced point mutations, chromosomal aberrations and chromosome loss in these assays. Genetic Toxicity — Chromosomal Aberrations In vivo Dimethylphosphonate (CAS No. 868-85-9, supporting chemical) (1) In micronucleus assay, B6C3F1 mice were administered DMHP via intraperitoneal injection at 0, 250 and 500 mg/kg-day once daily for three consecutive days. Twenty four hours after last treatment, the polychromatic erythrocytes from bone marrow were analyzed for micronuclei. The number of micronucleated polychromatic erythrocytes (PCEs) per 1000 PCEs score was elevated in the first trial at 500 mg/kg-day. In the second trial, this result could not be reproduced. The results were inconclusive. Dimethyl phosphonate induced micronuclei in this assay. (2) In another micronucleus assay NMRI mice were administered a single dose of DMHP at 200 mg/kg. The incidences of micro-nucleated polychromatic erythrocytes (PCEs) per 1000 PCEs scored were measured 16, 24 and 48 hours after i.p. injection of DMP. There was a statistically non-significant doubling of micro-nucleated PCEs after 48 hours (negative controls 1.3 ± 1.1, 16h0.8 ± 1.1, 24h 1.8 ± 1.5, 48 h 2.7 ± 3.1). Although statistically significant, the values for the positive control group (cyclophosphamide) were unusually low (7.3 ± 5.5 as compared to the laboratory's historical positive control range of 10.2 - 25.1). It is therefore not certain, whether this test was sufficiently sensitive. The study authors concluded the results of this assay as negative. Dimethyl phosphonate did not induce micronuclei in this assay. 15 ------- U.S. Environmental Protection Agency Hazard Characterization Document December 2012 Genetic Toxicity - Other In vitro Trimethylphosphite (CAS No. 121-45-9) In DNA damage and repair studies, Escherichia coli WP2/WP100 and Salmonella typhimurium TA1978/TA1538 strains were exposed to TMP at 0.3 - 50 |iL/plate in two studies and at 10 |iL/plate in a third study; all studies were conducted with and without metabolic activation. Positive and negative controls were tested concurrently and responded appropriately. Cytotoxicity occurred at 50 |iL /plate. Preferential killing of repair-deficient strains was observed with and without activation in the two studies that tested up to 50 |iL/plate, whereas no preferential killing was observed in the third study (maximum concentration of 10 |iL/plate). Trimethyl phosphite induced DNA damage in these assays. Additional Information Skin Irritation Trimethyl phosphite (CAS No. 121-45-9) (1) Six New Zealand albino rabbits were administered undiluted TMP (0.5 g 98.5% purity), moistened with a minimal amount of water, via the dermal route on intact and abraded sites under occluded conditions for 24 hours and observed at 24 and 72 hours following dosing. The subtotals for mean scores for erythema and edema were 2.6 for abraded and 2.5 for unabraded skin. The primary dermal irritation index (PDII) was 1.3. Trimethyl phosphite was irritating to rabbit skin in this study. (2) Six New Zealand albino rabbits were administered undiluted TMP (0.5 g 98.5% purity), moistened with a minimal amount of water and protected from hydrolysis by N2, via the dermal route to intact and abraded sites under occluded conditions for 24 hours and observed at 24 and 72 hours following dosing. No erythema, edema or eschar formation was observed. The PDII was 0. Trimethyl phosphite was not irritating to rabbit skin in this study. Eye Irritation Trimethyl phosphite (CAS No. 121-45-9) (1) Six New Zealand albino rabbits were administered undiluted TMP (0.1 mL, purity not indicated) by instillation into the conjunctival sac of one eye of each rabbit. Ocular reactions were graded after 1, 24, 48 and 72 hours and 7 days. Irritation was observed in the iris at 1 hour and the conjunctiva at 1 and 24 hours. No reaction was noted in the cornea during the study or the iris after 1 hour and the conjunctiva after 24 hours. Trimethyl phosphite was irritating to rabbit eyes in this study. 16 ------- U.S. Environmental Protection Agency Hazard Characterization Document December 2012 (2) Six New Zealand albino rabbits were administered undiluted TMP (0.1 mL of 98.5% purity, protected from hydrolysis by N2) by instillation into the conjunctival sac of one eye of each rabbit. Ocular reactions in cornea, iris and conjunctiva were graded after 1, 24, 48 and 72 hours and 4 and 7 days. Trimethyl phosphite was not irritating to rabbit eyes in this study. Conclusion: The acute oral toxicity of TMP to rats and mice is low; acute inhalation toxicity to rats is low and the acute dermal toxicity to rabbits is moderate. In a 90-day oral (gavage) repeated-dose toxicity study in rats, TMP resulted in mortality at 160 mg/kg-day. The surviving animals at this dose showed effects on liver and male reproductive organs. The NOAEL is 80 mg/kg-day. In a 4-week oral (gavage) toxicity study in rats, effect on body weight and food consumption was seen at 164 mg/kg-day. The NOAEL is 32.8 mg/kg-day. In three separate four-week repeated-inhalation toxicity studies in rats, ocular effects were observed, including formation of irreversible cataracts at 0.53 mg/L-day in two studies and at 0.26 mg/L-day in one study. The NOAEC values in two studies are not established; the NOAEC for the third study is 0.051 mg/L-day. (In a limited study of employees of a TMP manufacturing plant showed no clinical cataracts in either exposed or non-exposed employee groups.) In a 21-dy dermal toxicity study in rabbits, TMP resulted in mortality and effects on liver at > 300 mg/kg-day; the NOAEL is not established. No reproductive toxicity studies were available for TMP, however, adverse effects on spermatogenesis and testes were observed in the 90-day oral repeated-dose toxicity study in rats. In an oral 9gavage) combined reproduction/developmental toxicity screening test in rats with the supporting chemical, DMHP, effects on mating and fertility indices were seen at 270 mg/kg-day; the NOAEL for reproductive toxicity is 90 mg/kg-day. The systemic toxicity NOAEL is 90 mg/kg-day based on mortality and severe body weight effects at 270 mg/kg-day. In the same study, the NOAEL for maternal and developmental toxicity is 90 mg/kg-day, based on no effects at the highest dose at which females survived to term. In a two-generation reproductive toxicity study in rats via inhalation, the supporting chemical, methanol, showed no systemic or reproductive toxicity at 1.3 mg/L-day, highest dose tested. In the same study, the earlier time of descensus testis in pups was seen at 0.013 mg/L-day; the NOAEC for developmental toxicity is not established. In an oral prenatal developmental toxicity study in rats with TMP, decreased body weights were seen in dams at 164 mg/kg-day; the NOAEL for maternal toxicity is 49 mg/kg-day. In the same study, skeletal and soft tissue abnormalities were seen in pups at 164 mg/kg-day; the NOAEL for developmental toxicity is 49 mg/kg-day. TMP induced gene mutation in mammalian cells but not in bacteria in vitro, induced micronuclei in bone marrow in vivo, induced DNA damage in bacteria in vitro and produced point mutations, chromosomal aberrations and chromosome loss in fruit flies in vivo. TMP is irritating rabbit skin and eyes. 17 ------- U.S. Environmental Protection Agency Hazard Characterization Document December 2012 Table 3. Summary of the Screening Information Data Set under the U.S. HPV Challenge Program - Human Health Data Endpoint SPONSORED CHEMICAL TMP (121-45-9) Supporting Chemical DMPH (868-85-9) Supporting Chemical Methanol (67-56-1) Acute Toxicity Oral LD5o (mg/kg) 1350 - 4200 — — Acute Toxicity Inhalation LC50 (mg/L) >45.7 Acute Toxicity Dermal LD50 (mg/kg) 934 - 7500 — — Repeated-Dose Toxicity NOAEL/LOAEL Oral (mg/kg-day) (rat, 90 d) NOAEL = 80 LOAEL = 160 (rat, 4 wk) NOAEL = 32.8 LOAEL 164 Repeated-Dose Toxicity NOAEC/LOAEC Inhalation (mg/L-day) (rat, 4 wk) NOAEC = not established LOAEC = 0.53 Repeated-Dose Toxicity NOAEL/LOAEL Dermal (mg/kg-day) (rabbit, 21 d) NOAEL = not established LOAEL = 300 Reproductive Toxicity (oral) N O AEL/N O AE C/LO AEL/LO AE C (mg/kg-day/mg/L/day) Systemic Toxicity No data NOAEL = 90 LOAEL = 270 NOAEL = 90 LOAEL = 270 — Reproductive Toxicity NOAEL = 90 LOAEL = 270 (RA) In the 90-day repeated-dose oral toxicity study, adverse effects on spermiogenesis and testes histopathology NOAEL = 90 LOAEL = 270 18 ------- U.S. Environmental Protection Agency Hazard Characterization Document December 2012 were seen. Reproductive Toxicity (Inhalation) NOAEC/LOAEC (mg/L/day) Systemic Toxicity Reproductive Toxicity No data NOAEC = 1.3 (highest conc. tested) NOAEC = 1.3 (highest conc. tested) (RA) NOAEC = 1.3 (highest conc. tested) NOAEC = 1.3 (highest conc. tested) Developmental Toxicity NOAEL/LOAEL Oral (mg/kg-day) Maternal Toxicity NOAEL = 49 LOAEL = 164 Developmental Toxicity NOAEL = 49 LOAEL = 164 Genetic Toxicity - Gene Mutation In vitro Negative (bacteria) Positive (mammalian cells) Genetic Toxicity - Chromosome Aberrations In vivo (Drosophila) Positive Genetic Toxicity - Other DNA Damage and Repair In vitro Positive Additional Information Skin Irritation Eye Irritation Irritating Irritating Bold-Indicates experimental data; RA = Read Across; - Data not needed/used for this assessment 19 ------- U.S. Environmental Protection Agency Hazard Characterization Document December 2012 4. Environmental Effects - Aquatic Toxicity A summary of ecotoxicity data for SIDS and other endpoints is provided in Table 4. The table also indicates where data for the supporting chemical are read-across (RA) to the sponsored substances. Acute Toxicity to Fish Trimethylphosphite (CAS No. 121-45-9) No data. Dimethylphosphonate (CASRN 868-85-9, supporting chemical) (1) Fathead minnows (Pimephalespromelas) were exposed to DMHP at unspecified concentrations under static conditions for 96 hours. 96-h LC50 = 225 mg/L (2) Zebrafish (Brachydanio rerio) were exposed to DMHP at 100 mg/L (nominal concentration) under static conditions for 96 hours. The mean measured concentration was 15.6 mg/L over 24 - 96 hours. No mortalities were observed. 96-h LC50 > 15.6 mg/L Acute Toxicity to Aquatic Invertebrates Trimethyl phosphite (CAS No. 121-45-9) No data. Dimethyl phosphonate (CASRN 868-85-9, supporting chemical) Daphnia magna were exposed to DMHP at nominal concentrations of 0, 6.25, 12.5, 25, 50 or 100 mg/L under static conditions for 48 hours. Immobilization was seen at > 25 mg/L. 48-h EC50 = 24.8 mg/L Toxicity to Aquatic Plants Trimethyl phosphite (CAS No. 121-45-9) No data. Dimethyl phosphonate (CASRN 868-85-9, supporting chemical) Green algae (Desmodesmus subspicatus) were exposed to DMHP at 0 or 100 mg/L (nominal concentration) under static conditions for 72 hours. The measured concentration of monomethyl phosphonate was -25 mg/L. Growth rate inhibition relative to controls did not occur. 72-h EC50 (growth rate) > 25 mg/L 20 ------- U.S. Environmental Protection Agency Hazard Characterization Document December 2012 Conclusion: For aquatic toxicity, all values are based on the supporting chemical DMHP. The 96-hour LC50 of supporting chemical DMHP for fish is > 15.6 mg/L. The 48-hour EC50 of supporting chemical DMHP for aquatic invertebrates is 24.8 mg/L. The 72-hour EC50 of supporting chemical DMHP for aquatic plants is >25 mg/L (growth rate). Table 4. Screening Information Data Set as Submitted under the U.S. HPV Challenge Program—Aquatic Toxicity Data Endpoints SPONSORED CHEMICAL Trimethyl phosphite (121-45-9) SUPPORTING CHEMICAL Dimethyl phosphonate (868-85-9) Fish 96-h LC50 (mg/L) No Data >15.6 (RA) > 15.6 Aquatic Invertebrates 48-h EC50 (mg/L) No Data 24.8 (RA) 24.8 Aquatic Plants 72-h EC50 (mg/L) (growth) No Data >25 (RA) >25 Bold-Indicates experimental data; RA = Read Across 21 ------- |