USVI Public Records

Document text

Attachment 1 Risk Assessment Issue Paper for: Derivation of a Provisional Subchronic Inhalation RfC for Benzene (CASRN 71-43-2) INTRODUCTION To identify research reports pertinent to the derivation of a provisional subchronic RfC for benzene, EPA and ATSDR documents on benzene (U.S. EPA, 1980; 1984; 1989; 1993; 1994a,b,c; ATSDR, 1991) and the HSDB, RTECS, and TSCATS databases were reviewed; in addition, a computer search of the literature was conducted (TOXLINE, June, 1986 - February, 1992). OSHA lists an PEL TWA of 1 ppm; however, some segments of industry are exempt from the 1 ppm standard, and instead have a PEL TWA of 10 ppm (OSHA, 1989). ACGffl lists a TLV TWA of 10 ppm; however a TLV TWA of 0.1 ppm has been proposed and is awaiting verification (ACGffl, 1991). The NIOSH REL (10-hour TWA) is 0.1 ppm (NIOSH, 1991). TOXICITY IN HUMANS There is an extensive database on the toxicity of inhaled benzene. Secondary sources, as well as literature searches, were used to identify studies that defined the thresholds of toxicity. Specifically, we looked for studies that evaluated subchronic, chronic, developmental, and reproductive toxicity of inhaled benzene. When numerous studies were available, we chose those for which toxic effects were observed at low concentrations of benzene. A number of epidemiology studies were available regarding chronic toxicity of inhaled benzene; four are reported herein, covering a wide range of exposure levels and effects. Aksoy et al. (1971) examined hematological parameters in 217 apparently healthy male workers (mean age 24.7 years) exposed to 30-218 ppm benzene (96-696 mg/m3) for 3 months to 17 years, and in 100 male hospital workers and medical students (mean age 26.6 years). Peripheral blood samples were obtained for measurement of RBC, WBC, PCV, platelets, and differential counts. In 11 benzene-exposed workers known to have hematological abnormalities, bone marrow samples were obtained for determination of cellularity and myeloid and erythroid series. Twenty-four percent of the exposed workers had hematological abnormalities, including leukopenia (9.7%), thrombocytopenia (1.84%), leukopenia associated with thrombocytopenia (4.6%), pancytopenia (2.76%), acquired pseudo-Pelger-Huet anomaly (0.46%), lymphocytosis (0.46%), giant platelets (0.46%), eosinophilia (2.3%), basophilia (0.46%), and eosinophilia associated with basophilia (0.46%). Low hemoglobin levels, PCV, and MCV, indicative of mild or moderate hypochromic or normochromic anemia, were observed in 33% of the benzene- For internal use only. DRAFT - Do not cite or quote. -1- TUT O05 1307 *64562* 64562 exposed workers. In bone marrow tests, 9/11 workers had hematopoietic abnormalities, including hypercellularity (in 1 worker), hypocellularity (4), and maturation arrest (8) and vacuolization (4) in the erythroid and myeloid series. This study identifies a LOAEL of 30 ppm (96 mg/m3) for hematopoietic effects in humans. Kipen et al. (1989) examined about 18,000 peripheral blood counts from hematologic surveillance records on 459 workers employed in the rubber industry between 1940 and 1975. Mean concentrations of benzene decreased from 137 to 66 ppm between 1940 and 1948, with a mean 8-hour TWA of 75 ppm (239.6 mg/m3). WBC counts increased from 1940-1948 and were positively correlated with the decreasing benzene levels. Between 1948-1975 workers were exposed to mean 8-hour TWA concentrations of 15-20 ppm (48-64 mg/m3). WBC counts in blood samples from workers exposed from 1948-1975 were not correlated with changing benzene exposure levels. These data suggest that benzene exposure in the 75 ppm (239.6 mg/m3) range influences WBC count in exposed workers, whereas exposure to benzene at 15-20 ppm (48-64 mg/m3) does not influence WBC count. Collins et al. (1991) examined hematological parameters (peripheral blood RBC, WBC, hemoglobin, platelets, and MCV) in workers (n=200) exposed to benzene over a 10-year period. Within this 10-year period the mean length of exposure was 7.3 years. The workers were exposed to an 8-hour TWA of 0.01-1.40 ppm benzene. The mean TWA exposure was 0.045 ppm (J. Collins, 1992, personal communication). A group (n=268) of non-benzene exposed workers in the same plant were used as controls. There were statistically significant differences on demographic (age, race, sex) and personal habit (currently smokers, regular exercise) variables between the benzene-exposed workers and the control group. Multiple regression analyses were applied using the confounding factors and current exposure as independent variables. No significant correlations between cumulative exposure and hematological parameters were identified. Thus, this study identifies a free-standing NOEL of 0.045 ppm (0.14 mg/m3) for hematological effects in humans. Fishbeck et al. (1978) examined hematological parameters (RBC, WBC, hematocrit, hemoglobin, mean corpuscular volume, platelets, differential blood counts, clot retention determinations, sedimentation rate, and blood indices) in 10 employees exposed to high benzene concentrations [8-hour TWA of >25 ppm (>80 mg/m3)] for 2.5-22.9 years, with an average of 9.6 years of exposure. Concentrations of benzene in the work area were especially high in 1963, with the 8-hour TWA ranging from 37-132 ppm (118-422 mg/m3); after 1963, conditions were altered to assure that concentrations of benzene remained below 25 ppm (80 mg/m3) (the acceptable limit at that time). Examination of the 10 employees in 1963 revealed enlarged RBC's, high MCV (10/10), slightly low hemoglobin levels (9/10), and transient anemia; bone marrow was examined at this time and no abnormalities were found. After 1963, hematological values for these employees improved (in 1977, 5/10 workers had increased MCV values) and by 1978 none of the employees had developed serious health problems. The authors concluded that exposure of workers to high levels of benzene produced transient hematological effects, which did not influence the long-term overall health of the workers. For internal use only. DRAFT - Do not cite or quote. -2- TUT GO 5 13O8 TOXICITY IN ANIMALS Male C57BL/J mice (sample size not reported; initial age 8 weeks) were exposed via inhalation to vapor concentrations of 0 or 10 ppm benzene (0 or 32 mg/m3) for 6 hours/day, 5 days/week for up to 178 days (Baarson et al., 1984). After 32, 66, and 178 days of exposure, peripheral blood samples were obtained from all mice for determination of levels of RBC, lymphocytes, and neutrophils, and 5 mice/exposure level were sacrificed for measurement of erythroid progenitor cells [colony forming units - erythroid (CFU-E), burst forming units (BFU- E), nucleated red cells, and total cellularity] in bone marrow and spleen. There was a significant (p<0.05) decrease in levels of RBC (at 66 and 178 days) and lymphocytes (at all sampling times) in peripheral blood of benzene-exposed mice. CFU-E and BFU-E in bone marrow were significantly (p < 0.01) decreased at all sacrifice times and at 66 days, respectively; after 178 days of treatment, bone marrow CFU-E was 5% of controls. Splenic CFU-E (10% of controls), nucleated red cells (15%), and total nucleated cellularity (84%) were significantly (p<0.05) decreased in mice sacrificed at 178 days. This study identifies a LOAEL of 10 ppm (32 mg/m3) for depressed hematopoiesis in mice. Male CD-1 mice (11-12/exposure level/exposure duration; 8-12 weeks of age) were exposed for 6 hours/day, 5 days/week to vapor concentrations of 0 or 9.6 ppm (0 or 31 mg/m3, respectively) benzene for 10 weeks or to 0 or 302 ppm (0 or 966 mg/m3) benzene for 26 weeks (Green et al., 1981a,b). On the day of the last exposure, samples (pooled from groups of 3-4 mice) were obtained from the peripheral blood, bone marrow and the spleen to evaluate hematological and hematopoietic cells. In mice exposed to 9.6 ppm (31 mg/m3), no adverse effects were observed with respect to mortality, body weights, or cells in the peripheral blood or bone marrow. Splenic weight, total nucleated cells per spleen, and nucleated red blood cells per spleen were significantly (p < 0.05) increased in mice exposed to 9.6 ppm (31 mg/m3). Mice exposed to 302 ppm (966 mg/m3) had the following significant (p<0.05) changes: increased mortality rate; decreased numbers of lymphocytes and RBC in peripheral blood; decreased numbers of lymphocytes, granulocytes, multipotential hematopoietic stem cells, and committed granulocyte/macrophage progenitor cells in bone marrow; decreased splenic weight, and numbers of lymphocytes, multipotential hematopoietic stem cells and committed granulocyte/macrophage progenitor cells in the spleen; increased incidence of atypical cell morphology in peripheral blood, bone marrow, and spleen. This study identifies a NOAEL of 9.6 ppm (31 mg/m3) for slight hematopoietic effects in mice exposed to benzene for 10 weeks and a LOAEL of 302 ppm (966 mg/m3) for severe hematopoietic toxicity in mice exposed for 26 weeks. Sprague-Dawley rats (50/sex/group; 12 weeks of age) and CD-1 mice (150/sex/group; 9 weeks of age) were exposed to nominal vapor concentrations of 0, 1, 10, 30, or 300 ppm benzene (99.9% purity) (0, 3, 32, 96, or 959 mg/m3), 6 hours/day, 5 days/week, for 13 weeks (Ward et al., 1985). Clinical observations and body weight data were normal in both species. High-exposure level rats had leukopenia and significantly (p<0.05) decreased femoral marrow cellularity. High-exposure level mice had leukopenia, anemia, thrombocytopenia, and significant increases in MCV, MCH, glycerol lysis time, and incidence and severity of morphological changes in RBC. Relative testes weights were significantly decreased in high-dose male mice. For internal use only. DRAFT - Do not cite or quote. -3- TUT 005 13O9 High-dose mice had histological abnormalities in the thymus (atrophy), bone marrow (myeloid hyperplasia), lymph nodes (lymphoid depletion of mesenteric and mandibular lymph nodes; plasma cell infiltration into mandibular lymph node), spleen (increased incidence of extramedullary hematopoiesis; periarteriolar lymphoid sheath depletion), ovaries (bilateral ovarian cysts), and testes (bilateral atrophy/degeneration; decreases in spermatozoa in the epididymal ducts; increased numbers of abnormal sperm types); similar lesions were observed in the testes and ovaries of mice exposed to concentrations lower than 300 ppm (959 mg/m3), but the authors did not consider these effects to be biologically significant. The incidence and severity of most benzene effects were greater in male mice than in female mice. This study identifies a NOAEL of 30 ppm (96 mg/m3) and a LOAEL of 300 ppm (959 mg/m3) for these effects in rats and mice. Male C57BL/6 mice were exposed to 0 or 300 ppm benzene (0 or 960 mg/m3), 6 hours/day, 5 days/week for 9 weeks (Baarson and Snyder, 1991). Blood was withdrawn from the tail vein for differential white blood cell counts and peripheral nucleated red cell counts. Following sacrifice, femurs and the spleen were aseptically removed and placed in sterile culture dishes. Single cell suspensions were made, and the numbers of colony forming unit erythroid (CFU-E) cells and burst forming unit erythroid (BFU-E) cells were counted in control vs. exposed animals. From 1 day after beginning of exposure to benzene until the end of the treatment period, peripheral red blood cell counts were decreased. In addition, the numbers of BFU-E and CFU-E colonies were depressed to less than half the control values, in all exposed animals at days 5 and 60 of exposure. These effects represent an adverse effect on the organism, due to the potential for anemia. In this study, the combined treatment of benzene and ethanol was studied in a second group of animals, and was the thrust of the new information from this group. The exposure levels are far greater in this study than in previous work; a LOAEL of 960 mg/m3 (HEC=171 mg/m3) was established for hematotoxic effects. Similar results were seen in a study where female C57BL/6xDBA/2 Fl hybrid mice were exposed to 0 or 300 ppm benzene (0 or 960 mg/m3), 6 hours/day, 5 days/week for 6-7 weeks (Vacha et al., 1990). Indices of hematopoiesis were measured in peripheral blood (RBC and WBC count, Hb, Hct, reticulocyte, and leukocyte count), in addition to 59Fe accumulation in the erythropoietic organs (spleen and bone marrow) and in the peripheral RBC's. The distribution of developmental classes of erythroblasts was also determined. This study found that animals became anemic after 6-7 weeks of benzene exposure. The number of erythroblasts in the bone marrow was not different, however exposure to benzene shifted the population to a less mature class of cells. The number of colonies derived from BFU-E and CFU-E were decreased to 70% and 34% of controls, respectively. A LOAEL of 960 mg/m3 (HEC = 171 mg/m3) was established for hematotoxic effects. BDF1 mice were exposed to 0, 100, 300, or 900 ppm benzene (0, 320, 960, 2880 mg/m3) for up to 4 weeks (Seidel et al., 1990). The numbers of hematopoietic progenitor cells, early and late progenitors (BFU-E, CFU-E), and granuloid progenitors (CFU-C) were determined. A group was generated to establish the effect of ethanol (drinking water) on these effects. This study demonstrated that the number of CFU-E per femur was decreased in a concentration-dependent manner by benzene. This effect was evident at 300 and 900 ppm (960 For internal use only. DRAFT - Do not cite or quote. -4- iUT OO5 13.1.0 and 2880 mg/m3, respectively) concentions, however the effect of the 100 ppm (320 mg/m3) exposure group was uncertain, as the study focused on the effect of ethanol on benzene toxicity. The LOAEL/NOAEL was thus difficult to determine. Male Sprague-Dawley rats (40/group) were exposed to vapor concentrations of 0 or 100 ppm benzene (0 or 319 mg/m3), 6 hours/day/ 5 days/week, for life (American Petroleum Institute, 1983). Blood samples were obtained at 2-4 week intervals throughout the treatment period. The treatment had no adverse effects with respect to mortality rates or body weight gain. Peripheral erythrocyte and lymphocyte counts were depressed at nearly every sampling time in treated rats, but the extent of decrease was not statistically significant at p<0.05. Significantly increased incidence of splenic hyperplasia (p< 0.005) and hemosiderin pigments (p< 0.001) were observed in benzene-exposed rats. The incidences of normally rare tumors in treated rats were liver (4/40), Zymbal gland (2/40), and chronic myelogenous leukemia (1/40); the authors considered these tumors to be related to the benzene exposure. .This study identifies a LOAEL of 100 ppm (319 mg/m3) for slight hematological effects in rats. Male AKR/J (50/group) and C57BL/6J mice (40/group) were exposed to vapor concentrations of 0, 100 (319 mg/m3; AKR mice only), or 300 ppm (958 mg/m3; C57BL/J mice only) benzene, 6 hours/day, 5 days/week for life (Snyder et al., 1980). The following parameters were used to assess toxicity: clinical signs (observed daily), body weights (measured biweekly), hematology (RBC, WBC, WBC differentials, absolute neutrophil and lymphocyte; measured biweekly in 10 control and 10 treated mice from each strain), and gross and microscopic necropsy (lung, liver, spleen, kidney, and bone marrow). The treatment had no adverse effects with respect to life span, body weight, or incidence of lymphoma in AKR mice. Treated AKR mice had significant (± 2 standard errors) degrees of lymphocytopenia, neutrophilia, erythropenia, and bone marrow hypoplasia (p<0.05). Treated C57BL mice had significant (±2 standard errors) degrees of lymphocytopenia, neutrophilia, erythropenia, morphological changes in peripheral blood cells, and bone marrow and splenic hyperplasia (p<0.05). The incidence of hematopoietic neoplasms was significantly (p<0.05) increased in C57BL mice, including 6 cases of thymic lymphoma. This study identifies a LOAEL of 100 ppm (319 mg/m3) for hematopoietic effects in mice. Pregnant Swiss Webster mice (5/exposure level/progeny age group; initial age 8-12 weeks) were exposed via inhalation to nominal vapor concentrations of 0, 5, 10, or 20 ppm benzene (0, 16, 32, or 64 mg/m3) for 6 hours/day on gestation days 6-15 (Keller and Snyder, 1988). On gestation day 16 (fetuses), 2 days after birth (neonates), and 6 weeks after birth (adults), progeny (1-2 males and 1-2 females/litter) were sacrificed to determine the amounts and types of hemoglobin produced, and hemopoietic cells in the peripheral blood and hematopoietic organs. No evidence of maternal or non-hematopoietic developmental toxicity was observed in treated mice, and no adverse hematopoietic effects were observed in fetuses. The treatment had no adverse effects in any progeny with respect to peripheral blood levels of RBC, MCH, blasts, dividing granulocytes, lymphocytes, or ratio of hemoglobin A major to hemoglobin A minor. There was a concentration-related decrease in peripheral blood levels of early nucleated red cells in neonates, significant (p<0.05) at all exposure levels. High-exposure level neonates had significantly (p<0.05) increased numbers of nondividing granulocytes and decreased numbers For internal use only. DRAFT - Do not cite or quote. -5- TUT GO 5 1311 of late nucleated red cells in peripheral blood. In high-exposure level neonates, hepatic levels of blasts, dividing granulocytes, non-dividing granulocytes, and lymphocytes were significantly (p<0.05) increased and late nucleated red cells were significantly (p<0.05) decreased; hepatic levels of blasts were also significantly (p<0.05) increased at the low-exposure level in neonates. In adults, there was a concentration-related decrease in early nucleated red cells in bone marrow, significant (p<0.05) at the high-exposure level. High-exposure level adults also had significant (p<0.05) increases in splenic levels of blasts, dividing granulocytes, and nondividing granulocytes; splenic levels of non-dividing granulocytes were also increased in low-exposure level adults. This study identifies a LOAEL of 5 ppm (16 mg/m3) for developmental hematopoietic effects in mice. Pregnant Swiss-Webster mice (5/exposure level/progeny age group; initial age 8-12 weeks) were exposed via inhalation to nominal vapor concentrations of 0, 5, 10, or 20 ppm benzene (0, 16, 32, or 64 mg/m3) for 6 hours/day on gestation days 6-15 (Keller and Snyder, 1986). On gestation day 16 (fetuses), 2 days after birth (neonates), and 6 weeks after birth (adults), progeny (1-2 males and 1-2 females/litter) were sacrificed for measurement of hematopoietic progenitor cells [colony forming units - erythroid (CFU-E), burst forming units - erythroid (BFU-E), and granulocytic colony forming cells (GC-CFU-C)] from the liver (fetuses and neonates), and bone marrow and spleen (adults). In addition, 10-week old progeny from litters in the control and mid-exposure group were exposed for 2 weeks to 10 ppm (32 mg/m3) benzene, then sacrificed for measurement of hematopoietic progenitor cells from the bone marrow and spleen. There was no evidence of maternal or non-hematopoietic developmental toxicity in benzene-exposed mice. There was a significant (p<0.05) increase in the numbers of erythroid burst forming units from livers of male and female fetuses exposed to the low- and mid-exposure level, respectively. The following significant (p<0.05) changes were observed with respect to CFU-E: in fetuses, there were increases in liver CFU-E at the low- and mid- exposure levels and decreases at the high-exposure level; in male neonates, there were increases and decreases in liver CFU-E at the mid-exposure level, and increases at the high-exposure level; in adult mice there were decreases in bone marrow CFU-E and increases in spleen CFU-E in males exposed to 10 ppm (32 mg/m3) in utero. Liver GM-CFU-C in neonates was significantly (p<0.05) decreased at the mid-exposure level (males only) and increased at the high-exposure level. Mice exposed to 10 ppm (32 mg/m3) benzene in utero and for 2 weeks as adults had significantly (p<0.05) decreased bone marrow CFU-E (males only) and splenic GM- CFU-C; mice exposed to air in utero and 10 ppm (32 mg/m3) benzene for 2 weeks as adults had no changes in bone marrow or splenic CFU-E, but had a significant (p<0.05) decrease in splenic GM-CFU-C (females only). The authors concluded that benzene treatment in utero induced hematopoietic alterations in fetuses, persisting until at least 10 weeks after birth. This study identifies a LOAEL of 5 ppm (16 mg/m3) for developmental hematopoietic effects in mice. Bred Sprague-Dawley rats (17-20/group; initial body weights 210-223 g) were exposed via inhalation to nominal vapor concentrations of 0, 10, 50, or 500 ppm benzene (0, 32, 160, and 1600 mg/m3) for 7 hours/day, on gestation days 6-15, followed by sacrifice on gestation day 20 for determination of developmental abnormalities (Kuna and Kapp, 1981). The treatment had no adverse effects on dams with respect to mortality rate, hematology, or gross necropsy. Body weight gain over gestation days 5-15 was significantly (p<0.05) decreased in mid- and high- For internal use only. DRAFT - Do not cite or quote. -6- TUT GO 3 131: exposure level dams. Fetal body weight was significantly (p<0.05) decreased at the mid- and high-exposure levels and fetal crown-rump length was decreased at the high-exposure level. The number of litters with skeletal and visceral variants was significantly (p<0.05) increased at the mid- and high-exposure levels. The skeletal and visceral abnormalities observed included execephaly, angulated ribs, dilated lateral and third ventricles of the brain, forefeet ossification out of sequence, generalized lagging ossification, and decreased numbers of caudals, and metacarpals, metatarsals, and phalanges/foot; the authors considered these abnormalities to be related to the benzene treatment. This study identifies a NOAEL of 10 ppm (32 mg/m3) and a LOAEL of 50 ppm (160 mg/m3) for maternal toxicity and developmental effects in rats. Bred Sprague-Dawley rats (26-31/group) were exposed to nominal vapor concentrations of 0, 10, or 40 ppm benzene (0, 32, or 128 mg/m3) for 6 hours/day on gestation days 6-15 (Litton Bionetics, 1978). The treatment had no adverse effects on mortality rate, body weight gain, or food consumption in dams. Pregnancy ratio, fetal weight, live litter size, and incidence of variants and malformations were similar in control and treatment groups. Benzene-exposed rats had significantly (p<0.05) decreased ratio of live fetuses/implantation site. The number of resorption sites was increased in benzene-exposed rats, but the difference was only significant (p<0.05) in the low-exposure group. This study identifies a LOAEL of 10 ppm (32 mg/m3) for developmental effects in rats. Female Sprague-Dawley rats (26/group) were exposed to vapor concentrations of 0, 1, 10, 30, or 300 ppm benzene (0, 3, 32, 96, or 958 mg/m3), 6 hours/day, 5 days/week during premating (10 weeks) and mating periods, then 6 hours/day, 7 days/week, on gestation days 1- 20, and lactation days 5-21 (Bio/dynamics, 1980). The following parameters were used to assess toxicity: clinical signs, mortality rate, body weight gain, pregnancy rates, and gestation length in dams; number alive and dead at birth, sex distribution, survival, body weights, organ weights, and gross necropsy in pups. The treatment had no adverse effects with respect to reproduction or maternal toxicity. This study identifies a NOAEL of 300 ppm (958 mg/m3) for reproductive effects and maternal and developmental toxicity in rats. Coate et al. (1984) exposed pregnant Sprague-Dawley rats (40/group) to 1, 10, 40, or 100 ppm benzene (3.2, 32, 128, or 320 mg/m3), 6 hr/day, days 6-15 of gestation. No maternal toxicity or teratogenic effects were noted. There was no deviation from controls in the number of resorptions. There was reduced fetal weight at 100 ppm (p<0.05) thus, a LOAEL of 100 ppm (320 mg/m3) can be identified along with a NOAEL of 40 ppm (128 mg/m3). Unovary and Tatrai (1985) exposed pregnant CFLP mice (15/group) and New Zealand rabbits (11-15/group) to 0, 160 or 320 ppm benzene (0, 500 or 1000 mg/m3), 24 hr/day, during days 6-15 (mice) or 7-20 (rabbits) of gestation. There were no teratogenic effects in either species. In rabbits, exposure to 320 ppm (1000 mg/m3) was associated with reduced fetal weight (p < 0.05) in the presence of reduced maternal body weight gain. The NOAEL and LOAEL for this effect was 160 ppm (500 mg/m3) and 320 ppm (1000 mg/m3), respectively. Mice exposed to concentrations >160 ppm (500 mg/m3) had reduced fetal weight, as well as skeletal deformities (maternal weight data not provided). The LOAEL for this effect was 160 ppm (500 mg/m3). For internal use only. DRAFT - Do not cite or quote. -7- TUT 005 131: DERIVATION OF PROVISIONAL SUBCHRONIC INHALATION RfC Chronic exposure of humans to benzene vapor in the work place resulted in hematological and/or hematopoietic effects at concentrations of 30-218 ppm (96-697 mg/m3) (Askoy et al., 1971; Fishbeck et al., 1978; Kipen et al., 1989). At lower concentrations (0.01-20 ppm; 0.03- 64 mg/m3), no adverse hematological effects were observed in peripheral blood of humans (Kipen et al., 1989; Collins et al., 1991). However, it is not known whether chronic exposure to low concentrations of benzene affects hematopoiesis in the bone marrow and spleen in humans. The most sensitive endpoint for long-term exposure to benzene vapor is toxicity to hematopoietic progenitor cells. The lowest LOAEL identified for this effect are at 10 ppm (32 mg/m3) in mice exposed to benzene subchronically (Baarson et al., 1984). Green et al. (1981a,b) identified NOAELs for damage to hematopoietic progenitor cells at 10 ppm (32 mg/m3). Ward et al. (1985) established a NOAEL of 30 ppm (96 mg/m3) for hematological effects, but effects on the progenitor cells were not evaluated. Lifetime studies provide evidence that mice are more sensitive to the long-term effects of benzene than are rats (Snyder et al., 1980; American Petroleum Institute, 1983). Reproductive effects (testicular lesions and ovarian cysts) were observed in mice exposed to 300 ppm (959 mg/m3) for 13 weeks, but not in mice exposed to 30 ppm (96 mg/m3) (Ward et al., 1985), or in female rats exposed to 300 ppm (959 mg/m3) for 17 weeks during premating, mating, gestation, and lactation (Bio/dynamics, 1980). The Keller and Snyder (1988) developmental toxicity study identified a LOAEL of 5 ppm (16 mg/m3), however the LOAELnEc of 16 mg/m3 is higher than the LOAELnEc of 5.7 mg/m3 from the Baarson et al. (1984) and Green et al. (1981a,b) subchronic studies. The Baarson et al. (1984) study was selected as the critical study because the exposure period was longer (25 vs. 10 weeks) at the low dose of 10 ppm (32 mg/m3) than that of the Green et al. (1981a,b) studies. a. LOAEL of 10 ppm (32 mg/m3) from the Baarson et al. (1984) studies was adjusted for intermittent exposure: • LOAELADJ = 32 mg/m3 x 6 hours/24 hours x 5 days/7 days = 5.7 mg/m3. b. Derivation of the = LOAELADJ x LA/LH where: LA = blood:air partition coefficient for benzene in male B6C3F1 mice (12.1) (Gargas et al., 1989) LH = blood: air partition coefficient for benzene in humans (8.19) (Gargas etal., 1989) For internal use only. DRAFT - Do not cite or quote. TUT OO5 1314 = 5.7 mg/m3 x 1.0 = 5.7 mg/m3; because the ratio of animal to human blood:air partition coefficients is greater than 1 (1.48), the default ratio of 1 is used (U.S. EPA, 1987). An uncertainty factor of 100 was applied to the LOAEI^Ec of 5.7 mg/m3 to yield a provisional subchronic RfC of 6 x 10"2 mg/m3. The uncertainty factor includes 3 for interspecies extrapolation using dosimetric adjustments, 10 for intraspecies variability, and 3 to extrapolate from a minimal LOAEL. Confidence in the key study (Baarson et al., 1984) is low. A small number of animals of one sex were used. Green et al. (1981a,b) identified NOAELs at the same dose level for similar endpoints. Confidence in the database is medium. A large number of studies corroborated the hematopoietic effects observed in the Baarson et. al. (1984) study. In addition, testicular lesions were reported by Ward et al. (1985); however, male reproductive performance tests and/or a multigeneration reproduction study were not identified. Reflecting the low confidence in the key study and medium confidence in the database, confidence in this provisional subchronic RfC is low. REFERENCES ACGIH (American Conference of Governmental Industrial Hygienists). 1991. Threshold Limit Values for Chemical Substances and Physical Agents and Biological Exposure Indices 1991- 1992. ACGIH, Cincinnati OH. Aksoy, M., K. Dincol, T. Akgun, S. Erdem and G. Dincol. 1971. Hematological effects of chronic benzene poisoning in 217 workers. Br. J. Industr. Med. 28: 296-302. American Petroleum Institute. 1983. Evidence for hematotoxicity and tumorigenesis in rats exposed to 100 ppm benzene. TSCA FYI Submission. OTS Fiche # OTS0000241-0. ATSDR (Agency for Toxic Substances and Disease Registry). 1991. Toxicological Profile for Benzene (Draft for Public Comment). U.S. Public Health Service. Atlanta, GA. PB/89/209464/AS. Baarson, K.A. and C.A. Snyder. 1991. Evidence for the disruption of the bone marrow microenvironment by combined exposures to inhaled benzene and ingested ethanol. Arch. Toxicol. 65: 414-20. Baarson, K.A., C.A. Snyder and R.E. Albert. 1984. Repeated exposure of C57B1 mice to inhaled benzene at 10 ppm markedly depressed erythropoietic colony formation. Toxicol. Lett. 20: 337-342. Bio/dynamics. 1980. An inhalation female fertility study with benzene in rats (Final report). For internal use only. DRAFT - Do not cite or quote. ~^~ TUT CO 5 1315 TSCA FYI Submission. OTS Fiche # OTS0000110-0. Coate, W.B., A.M. Hopberman, and R.S. Durloo. 1984. Inhalation teratology study of benzene in rats. In: Advances in Modem Environmental Toxicology. Vol. VI, Chapter 14, Princeton Scientific Publishers. Collins, J.J., P. Connor, B.R. Friedlander, P.A. Easterday, R.S. Nair and J. Braun. 1991. A study of the hematologic effects of chronic low-level exposure to benzene. J. Occup. Med. 33: 619-626. Fishbeck, W.A., J.C. Townsend and M.G. Swank. 1978. Effects of chronic occupational exposure to measured concentrations of benzene. J. Occup. Med. 20: 539-542. Gargas, M.L., R.J. Burgess, D.E. Voisard, G.H. Cason and M.E. Anderson. 1989. Partition coefficients of low-molecular-weight volatile chemicals in various liquids and tissues. Toxicol. Appl. Pharmacol. 98: 87-99. Green, J.D., C.A. Snyder, J. LoBue, B.D. Goldstein, and R.E. Albert. 198la. Acute and chronic dose/response effect of benzene inhalation on the peripheral blood, bone marrow, and spleen cells of CD-1 male mice. Toxicol. Appl. Pharmacol. 59: 204-214. Green, J.D., C.A. Snyder, J. LoBue, B.D. Goldstein, and R.E. Albert. 1981b. Acute and chronic dose/response effect of inhaled benzene on the multipotential hematopoietic stem (CFU- S) and granulocyte/macrophage progenitor (GM-CFU-C) cells in CD-1 mice. Toxicol. Appl. - Pharmacol. 59: 492-503. Keller, K.A. and C.A. Snyder. 1986. Mice exposed in utero to low concentrations of benzene exhibit enduring changes in their colony forming hematopoietic cells. Toxicol. 42: 171-181. Keller, K.A. and C.A. Snyder. 1988. Mice exposed in utero to 20 ppm benzene exhibit altered numbers of recognizable hematopoietic cells up to 7 weeks after exposure. Fund. Appl. Toxicol. 10: 224-232. Kipen, H.M., R.P. Cody and B.D. Goldstein. 1989. Use of longitudinal analysis of peripheral blood counts to validate historical reconstructions of benzene exposure. Environ. Health Perspec. 82: 199-206. Kuna, R.A. and R.W. Kapp. 1981. The embryotoxic/teratogenic potential of benzene vapor in rats. Toxicol. Appl. Pharmacol. 57: 1-7. Litton Bionetics. 1978. Teratology study in rats with benzene (Revised final report). TSCA 8E Submission. OTS Fiche # OTS0200243. NIOSH (National Institute for Occupational Safety and Health). 1991. NIOSH Pocket Guide to Chemical Hazards. U.S. Department of Health and Human Services, NIOSH, Cincinnati OH. ^ For internal use only. DRAFT - Do not cite or quote. -10- TUT 005 1,316 OSHA (Occupational Safety and Health Administration). 1989. Air Contaminants; Final Rule. 29 CFR Part 1910. Fed. Reg. 54: 2959. Seidel, H.J., R. Bader, L. Weber, and E. Barthel. 1990. The influence of ethanol on the stem cell toxicity of benzene in mice. Toxicol. Appl. Pharmacol. 105: 13-8. Snyder, C.A., B.D. Goldstein, A.R. Sellakumar, I. Bromberg, S. Laskin and R.E. Albert. 1980. The inhalation toxicity of benzene: incidence of hematopoietic neoplasms and hematoxicity in AKR/J and C57BL/6J mice. Toxicol. Appl. Pharmacol. 54: 323-331. U.S. EPA. 1980. Ambient Water Quality Criteria Document for Benzene. Prepared by the Office of Health and Environmental Assessment, Environmental Criteria and Assessment Office, Cincinnati, OH for the Office of Water Regulations and Standards, Washington, D.C. EPA- 440/50-80-18. NTISPB 81-117293. U.S. EPA. 1984. Health Effects Assessment for Benzene. Prepared by the Office of Health and Environmental Assessment, Environmental Criteria and Assessment Office, Cincinnati, OH. For the Office of Solid Waste and Emergency Response, Washington, D.C. EPA/540/1-86-037. U.S. EPA. 1987. Interim Methods for the Development of Inhalation Reference Doses. Prepared by Environmental Criteria and Assessment Office, Research Triangle Park, NC and Cincinnati, OH, Office of Health Effects and Assessment. U.S. EPA. 1989. Updated Health Effects Assessment for Benzene. Prepared by the Office of Health and Environmental Assessment, Environmental Criteria and Assessment Office, Cincinnati, OH. for the Office of Solid Waste and Emergency Response, Washington, D.C. ECAO-CIN-H037a. U.S. EPA. 1993. Chemical Assessments and Related Activities. Prepared by Office of Health and Environmental Assessment, Washington, DC. May 1993. U.S. EPA. 1994a. Integrated Risk Information System (IRIS). Online. Office of Health and Environmental Assessment, Environmental Criteria and Assessment Office, Cincinnati, OH. U.S. EPA. 1994b. Health Effects Assessment Summary Tables. Annual FY-1994. Office of Research and Development, Office of Emergency and Remedial Response, Washington, DC. NTISNo. PB94-921199. U.S. EPA. 1994c. Quarterly Status Report of RfD/RfC Work Group (as of 07/01/94). Office of Research and Development. Environmental Criteria Assessment Office, Cincinnati, OH. Unovary, G., E. Tatrai. 1985. On the embryotoxic effects of benzene and its alkyl derivatives in mice, rats, and rabbits. Arch. Toxicol. 8: 425-430. Vacha, J., V. Znojil, HJ. Seidel, and E. Barthel. 1990. Ferrokinetics and erythropoiesis in For internal use only. DRAFT - Do not cite or quote. -11- TUT GO5 1317 mice after long-term inhalation of benzene. Blut 60: 41-7. Ward, C.O., R.A. Kuna, N.K. Snyder, R.D. Alsaker, W.B. Coate and P.M. Craig. 1985. Subchronic inhalation toxicity of benzene in rats and mice. Amer. J. Indust. Med. 7: 457-473. For internal use only. DRAFT - Do not cite or quote. -12- TUT GO 5 1318 Benzene Subchronic RfC Principal/Supporting Studies - Inhalation Exposure Study Species/ Route Co-critical Studies: Baarson et al. C57BL/J 1984 (chronic) mice Green et al. CD-1 mice (1981a,b) (acute and chronic) Supporting Studies: Aksoy et al. Human 1971 Collins et al. Human 1991 Cone, (ppm/ mg/m3) 0/0 10/32 0/0 9.6/31 0/0 302/966 0/0 30/96 218/696 0/0 0.045/0.14 Duration Critical NOAEL LOAEL Effect (mg/m1) (mg/m9) 6 h/d Deer, ability of None 32 5 d/wk mouse marrow 1 78 d progenitor cells to form colonies 6 h/d Hematological 31 None 5 d/wk 10 wk 26 wk None 966 3 m to Hematological None 96 17yr 7 yr None 0.14 None (free- standing) NOAEUadj] HEC LOAEUadj] (mg/m*) (mg/m') 5.7 L 5.7 L 5.5 N 5.5 N 173 L 173 L 34 L 0.05 N •-G For internal use only. DRAFT - Do not cite or quote. -13- Study Special/ Cone. Rout* (ppm/ mg/m1) Ward at al. 1985 CD- 1 mica 0/0 (aubchronic) 1 /3 10/32 30/96 300/959 Rats Baarson and C57BL/6 0/0 Snydar 1991 mala mica 300/960 (subchronic) Duration Critical NOAEL Effact (mg/m1) 6h/d Hematological 96 5 d/wk effects (deer. 1 3 wk R8C, WBC, platelets, Hb, M/E ratios, and Hot.; Histological affects (91 d postexp): testicular atrophy, abn. sperm, deer. spermatozoa, etc. Deer. 96 lymphocyte count, incr. neutrophils; Histological effects: deer. femoral marrow cellularity (at 7 d exposure) 6 h/d Decreased RBC None 5 d/wk count; deer. 9 wk ability of mouse marrow progenitor cells to form colonies LOAEL NOAEUadJ] HEC (mg/m1) LOAEUadj] (mg/m*) (mg/m1) 959 17 N 17 N 171 L 171 L 959 17 N 17 N 171 L 171 L 960 171 L 171L -i '—H For internal use only. DRAFT - Do not cite or quote. -14- REFERENCES Cohen, S., D. Davis, and R. Kramkowski. 1974. Clinical manifestations of chromic acid toxicity: nasal lesions in electroplate workers. CUTIS 13: 558-568. Glaser, U., D. Hochrainer, H. Kloppel, and H. Khumen. 1985. Low level chromium (VI) inhalation effects on alveolar macrophages and immune functions in Wistar rats. Arch. Toxicol. 57: 250-256. Gomes, E.R. 1972. Incidence of chromium-induced lesions among electroplating workers in Brazil. Indust. Med. 41(12): 21-25. Johansson, A., B. Robertson, T. Curstedt, and P. Camner. 1986. Rabbit lung after inhalation of hexa-and trivalent chromium. Environ. Research 41: 110-119. Lindberg, E. and G. Hedenstierna. 1983. Chrome plating: symptoms, findings in the upper airways, and effects on lung function. Arch. Environ. Health 38(6): 367-374. Attachment 3 For internal use only. DRAFT - Do not cite or quote. -19- TUT OO5 1321 REFERENCES Cohen, S., D. Davis, and R. Kramkowski. 1974. Clinical manifestations of chromic acid toxicity: nasal lesions in electroplate workers. CDTIS 13: 558-568. Glaser, D., D. Hochrainer, H. Kloppel, and H. Khumen. 1985. Low level chromium (VI) inhalation effects on alveolar macrophages and immune functions in Wistar rats. Arch. Toxicol. 57: 250-256. Gomes, E.R. 1972. Incidence of chromium-induced lesions among electroplating workers in Brazil. Indust. Med. 41(12): 21-25. Johansson, A., B. Robertson, T. Curstedt, and P. Camner. 1986. Rabbit lung after inhalation of hexa- and trivalent chromium. Environ. Research 41: 110-119. Lindberg, E. and G. Hedenstierna. 1983. Chrome plating: symptoms, findings in the upper airways, and effects on lung function. Arch. Environ. Health 38(6): 367-374. Attachment 3 For internal use only. DRAFT - Do not cite or quote. -19- Attachment 3 (8-2S-93) Risk Assessment Issue Paper for: Derivation of a Provisional Subchronic RfC for Di(2-ethylliexyl)phthalate (CASRN 117-81-7) Pregnant Wi star rats (2S/group) were exposed to aerosol concentrations of 0, 0.01, O.OS, and 0.3 mg/L di(2-ethylhexyl) phthalate (DEHP) (0, 10, SO, and 300 mg/rn"), 6 hours/day, on gestational days 6-1S, by head/nose exposure (Merkle et al., 1988). An MMAD SO% of < 1.2 J.'m and slope factors of 7.3, 16.8, and S.8 were determined for 10, SO, and 300 mg/rrr' concentrations, respectively. These animals were acclimatized by sham exposure to air without DEHP during days 0-6 of gestation prior to the exposure period. At the end of the exposure period, 20 animals/group were subjected to cesarean section on • gestational day 20. The remaining S rats/group were allowed to deliver and rear the pups until weaning (postnatal day 21). No difference in maternal body weight gain or behavior was seen between controls and exposed groups except for a significant reduction in body weight (9 %) in the dams exposed to 300 mg/rrr' DEHP at postnatal day 21 compared to controls. This effect on body weight was not concentration-dependent. Furthermore, the food consumption was not measured. Macroscopic examination revealed no treatment-related toxic effects in exposed animals. The conception rate was 90%, 9S%, 8S%, and 80% with increasing exposure to DEHP, but there was no significant difference among the groups. The number of corpora lutea and mean uterine weights among groups was similar. Early resorptions were reportedly seen throughout all groups, but there was no concentration-response trend. In the SO mg/rrr' group, a statistically significant decrease in the number of mean live fetuses/dam (11.68 in the SO mg/rn" group vs. 1200 in controls) and live implantations/dam (94.74% vs. 92.37%) was observed. Skeletal examination showed a low incidence of anomalies, variations, and retardations in exposed animals. The incidence of fetuses with retardations (gross and skeletal)/litter was 18.83%,23.22%,26.46%, and 32.63% (primarily due to renal pelvis dilatations) with increasing concentration, but it was not significant for any exposure level. The investigators considered the renal pelvic dilatations unrelated to DEHP exposure because it is common for this strain of rat and was observed at a high incidence in historical controls (no data reported). The incidence of litters with gross retardations was 16.67%, 33.33 %, 31.2S%, and S6.2S% for the control, 10-, SO-, and 3OO-mg/m3 groups, respectively; this finding was significant at the high concentration. There was a slight concentration-related increase, although not significant, in the percentage of litters with fetuses having skeletal abnormalities; 16.67%, 21.0S%, 25%, and 37.5% for 0, 10,50, and 300 mg/rn" DEHP, respectively. The litters with skeletal retardations were not significantly different from controls. A NOAEL of 10 and a LOAEL of 50 mg/rn" DEHP was determined based on developmental effects, specifically increased incidence of litters with gross retardations. For internal use only. DRAFT - Do not cite or quote. -20- Although the effect was not significant in the 10- and 30-mg/m3 groups, the incidence was higher than the controls and showed an increasing trend. Physical development was assessed in the pups who were raised until postnatal day 21. Survival rate, viability and lactation indices, righting test (postnatal day 6), gripping reflex (day 13), pupillar reflex (day 20), hearing test (day 21), eye/ear auricle, incisivi, fur development, and body weight gain were evaluated. Exposed animals did not show alterations for any of these parameters. Therefore, DEHP did not affect the development of the offspring (postexposure and lactation period) of the exposed rats. Male Wistar rats (27/group) were exposed in a head-nose inhalation system to 0, 0.01, 0.05, or 1.0 mg/L DEHP (0, 10, 50, and 100 mg/rrr'), 6 hours/day, 5 days/week, for 4 weeks (Klimisch et al., 1991). The particle size of the aerosols had a MMAD of ~ 1.2 fLm. Another group (15/sex/group) was exposed and observed for a longer period to evaluate reversibility of effects. Some of the exposed males were mated with untreated female rats 2 and 6 weeks after the end of exposure. No clinical effects were observed in any group. A significant increase in the relative lung weight, accompanied by foam cell proliferation and thickening of alveolar septi, was observed in the two high-concentration males groups compared to the control group. Relative liver weights were increased in both sexes, but there was no corresponding histopathologic changes. Effects were reversible. No reproductive effects were observed. A NOAEL of 50 mg/rn" and a LOAEL of 100 rng/rrr' was determined for increased lung weight and changes to the alveoli septum. The study is limited because it is reported in an abstract and details are lacking. Male ICR mice (20/group) were exposed to air saturated with unspecified amounts of DEHP vapors at room temperature, 2 hours/day, 3 days/week, for 4-16 weeks (Lawrence et al., 1975). Mice (5/group) were sacrificed at the end of 4, 8, 12, and 16 weeks. The lungs and several other tissues were examined for histopathologic changes. There were no consistent lung abnormalities attributed to the DEHP exposure. The investigators reported that preliminary work in the laboratory had suggested a toxic effect on the lungs of mice exposed to phthalate vapors; however, this study failed to confirm the preliminary findings. No other details were reported regarding this study. Pregnant Fischer 344 rats (23-26/group) received 0%,0.25%,0.5% or 1% DEHP (equivalent to 0, 164, 313 or 573 mg/kg-day) in the diet during gestational days 0-20 (Price et al., 1986). Doses were based on the Wolkowski-Tyl et al. (1983a) study on Fischer 344 rats. Maternal food consumption and weight gain during the treatment period were reduced in a dose-related manner; decreased food consumption was significant in the 0.5%- and 1%- DEHP exposed groups and reduced weight gain was significant in the 1%-dosed group. There were no statistically significant DEHP-related effects observed for the percent of fertile matings, live litters, and viable litters. A significant increase in postimplantation mortality (7.8%, 8.57%, 21.4%, 19.52% with increasing doses) occurred only in the intermediate dose. Dose-related decreases in average litter size (9.47, 9.3, 8.18, and 8 per litter, respectively) and average pup weight per litter (4.91, 4.86, 4.75, and 4.52 g, respectively) occurred; the effects were significant only in the high-dose group. No major incidence of For internal use only. DRAFT - Do not cite or quote. -21- malformations in the fetuses of exposed animals were evident although significance was not reported. A NOAEL of 0.5 % DEHP (313 mg/kg-day) for reproductive effects was determined based on decreased average litter size and pup weight per litter. Tomita et al. (1982) and Yagi et al. (1980) conducted a study in which pregnant mice (3-8 females/group) were administered a single oral dose of 0.05, 0.1, and 1 mL/kg DEHP (99% purity) on gestational day 6, 7, 8, 9 or 10. Maternal and reproductive parameters were evaluated. The animals receiving 10 mL/kg DEHP on gestational day 7 or 8 had decreased body weight, although significance was not reported. Exposure on day 9 or 10 did not affect body weights. The average body weight of fetuses was significantly reduced in all dose levels and for all gestational days of exposure except for the lO-mL/kg DEHP group exposed on gestational day 9. Resorption occurred in exposed animals depending on the dose and day of exposure. However, no statistical analyses were given. High incidences of gross and skeletal abnormalities were observed for exposures on gestational day 7 or 8. Exposure to 2.5 mL/kg DEHP on day 7 caused 80% gross and 60% skeletal anomalies and exposure to 7.5 mL/kg on day 8 caused 65.5% gross and 82.8% skeletal anamolies, respectively. For day 9 or 10, gross abnormalities were 20% and 0%, respectively, with exposure to 10 mL/kg DEHP. No statistical analyses were conducted for these values. Common malformations reported were exencephaly, open eyelid, club foot and bent or no tail, and abnormal thoracic lumbar, sacral and caudal vertebrae. In a study by Tyl et al. (1988) (unpublished report by Wolkowski-Tyl et al., 1983b), pregnant CD-1 mice were exposed to 0,0.025%,0.05%, 0.1 %, and 0.15% DEHP (0,250, 500,1,000, and 1,500 ppm) in the diet during gestational days 0-17. Treatment-related clinical signs included piloerection, lethargy, and rough coat in the 0.05 %-, 0.1 %-, and 0.15 %-DEHP groups. Food consumption was significantly higher in the 0.15 % group relative to controls. A significant dose-related decrease in maternal body weight was measured on gestational days 12, 16, and 17, with significantly lower weights in the two highest dose levels compared to the controls. Maternal weight gain was also significantly lower with these dose levels. A significant dose-related increase in relative liver weight was exhibited with significance in the 0.1 %- and 0.15 %-exposed groups. The percentages of resorption, non-live and affected implantslIitter were increased, in a dose-dependent manner, with values significant for the 0.1 %- and 0.15%-exposed mice (Tyl et al., 1988; Wolkowski-Tyl et al., 1983b). Decreased fetal body weights/litter was dose-related; values were significant for the high-dose group relative to controls. A significant dose-related increase in the percentage of malformed fetuses/litter was also reported; significant for 0.05%,0.1 %, and 0.15% groups. There was an increased incidence of external malformations (e.g., exophthalmia, exencephaly), visceral malformations (e.g., malformed arteries, aorta), and skeletal defects (e.g., fused and branches ribs), however, statistical analyses were not conducted. It was concluded that DEHP produced maternal toxicity and fetotoxicity in CD-l mice exposed to 0.1 % and 0.15% diets during gestation. Teratogenicity was observed in animals at these doses, as well as with 0.05 %-exposed animals. Therefore, a NOAEL of 0.025% DEHP for teratogenicity and a NOAEL of 0.05% DEHP for maternal and reproductive toxicity were determined. The study was limited For internal use only. DRAFT - Do not cite or quote. -22- because only the liver weight was measured and histopathological examinations were not conducted in the dams. Shiota and Nishimura (1982) evaluated pregnant ICR mice given a diet containing 0.05%,0.1 %, 0.2%, 0.4%, and 1 wt-% DEHP throughout gestation. Maternal weight gain decreased and percentage of fetal resorptions increased significantly for the 0.2%-, 0.4%-, and 1%-DEHP groups compared to the controls. All implanted ova died early in the rats exposed to 0.4% and 1% DEHP. Therefore, no fetuses were available for examination. A significant decrease in fetal weight and an increase in fetal malformations were reported for the 0.2 %-DEHP exposed mice. These effects were not dose-related. A NOAEL of 0.1 % DEHP for maternal, reproductive, and teratogenic effects was determined in this study. DERIVATION OF A PROVISIONAL SUBCHRONIC RfC A NOAEL of 10 mg/rn" was determined for developmental toxicity (increased incidence of litters with gross retardations) from an inhalation rat study by Merkle et al. (1988). Therefore, this study was used to derive the provisional subchronic RfC. Calculation of the human equivalent concentration for the NOAEL is as follows: NOAE~c - NOAEL x RDDR(ER) where: the NOAELHECwas calculated for a partic1e:extrarespiratory effect and RDDR(ER) = 0.0089 x 70/0.297 [(BW)H/(BW)A' female Wistar rats] for MMAD = 1.0 J..tm and sigma g = 2.4, based on dosimetric modeling as described in u.S. EPA (1989), RDDR(ER) = 2.1. NOAELHEC= 10 mg/rn? x 2.1 = 21 mg/rrr' Subchronic RfC = NOAELHEc/(UFx MF) = 21 mg/rn" 1 (100 x 1) = 2E-l mg/rrr' An uncertainty factor of 100 reflects 3 to extrapolate from rats to humans, 10 to protect sensitive human subpopulations, and 3 for a deficient database. The resulting provisional subchronic RfC is 2E-l mg/rrr'. The study is given a medium confidence rating because the sample size was adequate and statistical significance was reported. For internal use only. DRAFT - Do not cite or quote. -23- Although this specific end point was not observed in any other studies, this finding was corroborated by oral mice reproductive studies that reported teratogenic effects, primarily skeletal malformations. The database was given low confidence because there were no human toxicity studies and limited chronic and subchronic inhalation animal studies. A low confidence in the RfC follows. REFERENCES: Lawrence, W.H., M. Malik, J.E. Turner, A.R. Singh, and J. Autian. 1975. A toxicological investigation of some acute, short-term, and chronic effects of administering di- 2-ethylhexyl phthalate (DEHP) and other phthalate esters. Environ. Res. 9(1): 1-11. Merkle, 1., H. Klimisch, and R. Jackh. 1988. Developmental toxicity in rats after inhalation exposure of di-2-ethylhexylphthalate. Toxicology Letters 42(2): 215-223. Price, C., R. Tyl, M. Marr, and B. Sadler. 1986. Reproduction and fertility evaluation of diethylhexyl phthalate in Fischer 344 rats exposed during gestation. NTP-86-309. 243 pp. Shiota, K. and H. Nishimura. 1982. Teratogenicity of di(2-ethylhexyl) phthalate and di-n- butyl phthalate in mice. Environ. Health Perspect. 45: 65-70. Tomita, I, Y. Nakamura, Y. Yagi, and K. Tutikawa. 1982. Teratogenicity/fetotoxicity of DEHP in mice. Environ. Health Perspect. 45: 71-71. Tyl, R.W., C. Price, M. Marr, and C. Kimmel. 1988. Developmental toxicity evaluation of dietary di(2-ethylhexyl) phthalate in Fischer 344 rats and CD-l mice. Fundam. Appl. Toxicol. 10(3): 395-412. Wolkowski-Tyl, R., C. Jones-Price, M.C. Marr, and C.A. Kimmel. 1983b. Teratologic evaluation of diethylhexyl phthalate in CD-l mice. Report (RTI-61): 253 pp. Yagi, Y., Y. Nakamura, I. Tomita, K. Tsuchikawa, and N. Shimoi. 1980. Teratogenic potential of di- and mono-(2-ethylhexyl)phthalate in mice. J. Environ. Pathol. Toxicol. 4(2- 3): 533-544. For internal use only. DRAFT - Do not cite or quote. -24- Attachment 4 (12/22/93) Risk Assessment Issue Paper for: Evaluation of Subchronic Inhalation Systemic Toxicity for Vinyl Chloride (CASRN 75-01-4) METABOLISM Absorption of vinyl chloride in humans after inhalation exposure is rapid. A study was conducted using five young adult male volunteers to measure percent retention and rate of elimination of vinyl chloride by the human lung (Krajewski et al., 1980). In this study, volunteers were exposed to 7.5, 15, 30, or 60 mg/m! vinyl chloride for 6 hours via gas mask, with GC measurement of vinyl chloride concentration in inspired and expired air. Retention of vinyl chloride was calculated as the difference between inspired and expired air concentrations. It was estimated that these subjects retained 42 % of inhaled vinyl chloride in the lung, that maximum retention was reached within 15 minutes, and that the percent retention was independent of inspired vinyl chloride concentration. After cessation of exposure, the vinyl chloride concentration in expired air decreased rapidly within 30 minutes to 4% of the inhaled concentration. Again, this occurred independent of exposure concentration. This study was limited by the lack of information regarding accuracy of reproducibility of vinyl chloride assays, verification of chamber concentration measurement and if a steady state had been established, or information regarding the test subjects (body weight, physical condition, smoking status). Human volunteers (5 males) were exposed to 2.9 to 23.5 ppm vinyl chloride for 6 hours. In the first 30 minutes after cessation of exposure, the mean expired air concentrations of vinyl chloride ranged from 0.20 to 1.11 ppm (3.6-4.73% of the inhaled concentration) (Krajewski et al., 1980). The authors suggest that exhalation of unmetabolized vinyl chloride was not an important pathway of elimination at low exposure concentrations, which is consistent with studies in animals. In animal studies, the importance of the lungs as an excretion route for vinyl chloride varies according to the exposure concentration. This is a reflection of saturation of vinyl chloride metabolic processes (discussed above). In rats, the polar metabolites of vinyl chloride are excreted primarily via the urine; smaller amounts are excreted in the expired air as CO2 and in the feces (Hefner et al., 1975). When metabolic pathways are saturated, substantial amounts of unmetabolized vinyl chloride are exhaled and appear to follow first- order kinetics regardless of exposure concentrations (ATSDR, 1991). Humans exposed to 7.5 to 60 mg/rn" for 6 hours expired mean air concentrations from undetectable to 2.84 mg/rn", representing up to 3.6 to 4.73% of the inhaled concentration (Krajewski et al., 1980), suggesting that exhalation of unmetabolized vinyl chloride is not an important route of elimination. In rats, it has been demonstrated that as the dose of vinyl chloride increases, a markedly greater proportion of vinyl chloride was expired unaltered while the percentage of For internal use only. DRAFT - Do not cite or quote. -25- metabolites in the urine decreased substantially (Watanabe et al., 1976a). Rats exposed to 10 ppm for 6 hours eliminated 68% of the absorbed radioactivity in their urine and 2 % in expired air, while 6 hour exposures to 1,000 ppm resulted in 56% of the dose appearing in the urine and 12% in expired air (Watanabe et al., 1976a). The urinary excretion of radioactivity was biphasic, with the second or slow phase accounting for less than 3% of the total urinary excretion (Watanabe et al., 1976a, 1978a). Rats (number and strain unspecified) were exposed to 10,000 or 20,000 ppm radiolabeled vinyl chloride (25,561 or 51,122 mg/rrr') for 5 minutes, sacrificed, and frozen in liquid nitrogen, in order to establish the distribution pattern using whole-body autoradiography (Duprat et al., 1977). It was shown that vinyl chloride was rapidly absorbed through the lungs, and immediately accumulated in the liver. No quantitative information of percent absorption were provided in this general review. Rat studies show that the distribution of vinyl chloride is rapid and widespread, but the storage of vinyl chloride in the body is limited by its rapid metabolism and excretion (Bolt et al.,1977). Male Wistar rats were pretreated with 50 mg/kg 6-nitro-l,2,3- benzothiadiazole i.p. to block cytochrome P-450-dependent metabolism, then were exposed to 25 to 10,000 ppm radiolabeled 14C-vinyl chloride for 5 hours (Buchter et al., 1977). The highest concentration of labeled vinyl chloride was in the fat (8.3 nMol vinyl chloride/g tissue), followed by a relatively uniform, 10- to 16-fold lower distribution to the blood, liver, spleen, kidneys, and muscle (average 0.52 nMol vinyl chloride/g tissue). Without pretreatment with the metabolic inhibitor, vinyl chloride metabolites were found preferentially in the liver and kidneys, with uniform lower distribution to the spleen, muscle, and fat. Male Wistar rats (3/group) were exposed to 50 ppm vinyl chloride, with and without pretreatment with 6-nitro-l,2,3-benzothiadiazole (Bolt et al., 1976). As in the above experiment, when animals were not pretreated, distribution of the radiolabel to major organs immediately after the 5-hour exposure was highest in the kidney and liver, and was found in decreasing quantities in the spleen, muscle, fat, and brain. Distribution of vinyl chloride in the rat has been observed autoradiographically to concentrate in the liver, bile duct, digestive lumen, and kidney within 10 minutes after a 5 minute inhalation exposure to high concentrations of vinyl chloride (20000 ppm, also discussed above). No quantitative analysis was made in this study (Duprat et al., 1977). Male Sprague-Dawley rats (4/group) were exposed by inhalation to 10 or 1,000 ppm vinyl chloride for 6 hours and followed for 72 hours postexposure to determine vinyl chloride disposition (Watanabe et al., 1976a). The fate of 14C-vinylchloride was found to be concentration-dependent, with an increasing proportion eliminated by the lungs with higher exposure concentrations. After exposure to 10 or 1,000 ppm vinyl chloride, expired radiolabeled vinyl chloride was 2% and 12% of total recovered radioactivity, respectively. Radioactivity in the urine did not change significantly with inspired concentration (68% and 56% of total recovered radioactivity, respectively). After inhalation of 10 ppm, distribution For internal use only. DRAFT - Do not cite or quote. -26- was as follows: liver (14%), kidney (8%), skin (7%), lung (7%), muscle, carcass, plasma (5%), and fat (3%). After 1,000 ppm exposure, distribution was to the liver (15%), skin (12%), kidney (6%), carcass, lung (5%), muscle (4%), plasma, and fat (not detectable). Finally, there were no differences in distribution or rate of excretion between repeated- versus single-dose exposure of rats to 5,000 ppm vinyl chloride (Watanabe et al., 1978b). The distribution of vinyl chloride in tissues after single vs. repeated exposures was as follows: liver (12%, 16%), kidney (6%,7%), skin (5%, 8%), carcass (3%,4%), fat (not detected, not detected). Placental transfer of vinyl chloride occurred rapidly in rats. Female rats exposed to 0, 5,500, 18,000, 33,000 mg/rrr' vinyl chloride (0, 2,000, 7,000, or 13,000 ppm) for 2.5 hours on gestational day 18 had the highest concentration of vinyl chloride in maternal blood, followed by fetal blood, then amniotic fluid (Ungvary et al., 1978). Single oral doses of 0.05, 1, or 100 mg/kg radiolabeled vinyl chloride in corn oil (95- 96% purity) were administered to male Sprague-Dawley rats in a well-conducted study (Watanabe et al., 1976b). Concentration of 14C-vinylchloride and 14C-C02were measured in expired air, and urine and feces were collected in metabolism cages for measurement of labeled excretion products in 30 minute intervals for the first 4 hours, and in 12-hour intervals for the remainder of 72 hours. As the administered dose of vinyl chloride increased, the proportion of expired unchanged vinyl chloride increased dramatically in expired air (1.4% and 2.3% in the lower doses, 66.6% in the high dose), illustrating the dose-dependency of vinyl chloride metabolism and distribution. Distribution in major organs of the animals was measured after 72 hours. The liver contained the highest concentration of radioactive label after 72 hours post-dosing. Distribution of the radiolabel to the liver ranged from 2- to 5-fold higher in all groups compared to the concentration found in the fat, lung, muscle, plasma, or carcass of these animals. Coincident with the greater metabolism in the lower dose groups, the proportion of vinyl chloride in the tissues after 72 hours was much higher than in the 100 mg/kg dose group. The metabolism of vinyl chloride was studied in male Sprague-Dawley rats exposed for 6 hours to concentrations of 1.4 to 4,600 ppm vinyl chloride (Gehring et al., 1978). Rats were sacrificed after exposure, and the total radioactive content of the carcass was determined in order to estimate the total amount of metabolized vinyl chloride (less the exhaled labelled compounds). In this study, metabolism was determined to be a concentration-dependent process characterized by Michaelis-Menten type kinetics. It was emphasized that the toxicity of vinyl chloride exposure is related to its concentration- dependent, saturable conversion to its toxic metabolites, and is not necessarily a function of the vinyl chloride exposure concentration. This study is flawed by lack of experimental validation. Kinetic studies were performed on groups of male Sprague-Dawley rats exposed to 51 to 1,167 ppm vinyl chloride for durations of 53 to 356 minutes by a nose-only apparatus (Hefner et al., 1975). The inhalation chamber was monitored continuously using an in-line infrared analyzer, and the decline in vinyl chloride from the inhalation chamber was For internal use only. DRAFT - Do not cite or quote. -27- determined for each concentration of vinyl chloride used. The rate of decline of vinyl chloride monomer concentration from the closed system occurred via metabolism of vinyl chloride by the rats after being corrected for background loss from the system. Additional groups of rats were treated with ethanol (for inhibition of alcohol dehydrogenase activity) or SKF 525-A (for inhibition of microsomal oxidase activity). Results of this study indicate that metabolism followed first-order kinetics at concentrations less than 100 ppm, with a t'h of 86 minutes. At >220 ppm, metabolism was decreased to a t'h of 261 minutes, suggesting saturation of the metabolic pathway at below this concentration. Pretreatment with ethanol depressed the rate of metabolism by approximately 83% at less than 100 ppm but by approximately 47% at greater than 1,000 ppm, suggesting that at lower concentrations, alcohol dehydrogenase-dependent metabolism occurs. Pretreatment with SKF 525-A, however, had no effect at less than 100 ppm but depressed metabolism by 19% at greater than 1,000 ppm. This implies that at higher concentrations of vinyl chloride, metabolism may occur via pathways other than that of alcohol dehydrogenase, such as by microsomal oxidases. Saturation of metabolic pathways occurred at exposure concentrations of 250 ppm vinyl chloride in male Wi star rats and 200 ppm in Rhesus monkeys; at concentrations below this, a straight, first-order decline in radioactivity was observed (Bolt et al., 1977; Buchter et al., 1980). In male Wistar rats, the metabolic rate (VmaJ of 110 mol/hour/kg was estimated (Bolt et al., 1977). In rhesus monkeys, a Vmalt of 50 mol/hour/kg was estimated, and was believed to be a close estimate of the theoretical Vmax for humans. (Buchter et al., 1980). The metabolic rate in rats, mice, and gerbils is 5-12 times greater than that in humans. Evidence was also provided that repeated inhalation exposures of rats to the range of 50 to 15,000 ppm vinyl chloride reduces the nonprotein sulfhydryl concentration of the liver (Hefner et al., 1975). This reduction was not dependent on exposure concentration. This is consistent with a saturable mechanism for vinyl chloride metabolism followed by conjugation of the vinyl chloride metabolites with glutathione and/or cysteine (Bolt et al., 1976; Jedrychowski et al., 1984; Watanabe et aI., 1978b). Three alternative pathways for vinyl chloride metabolism have been postulated (Bolt et al., 1980; Hefner et al., 1975). At low concentrations (i.e. < 100 ppm), vinyl chloride is primarily metabolized by sequential oxidation to 2-chloroethanol, 2-chloroacetaldehyde, and 2-chloroacetic acid by the alcohol dehydrogenase pathway (inhibited by pretreatment with ethanol). Little 2-chloroacetic acid was formed, however, probably because 2-chloroacetaldehyde conjugates rapidly with ubiquitous sulfhydryl groups (such as glutathione and cysteine). When the alcohol dehydrogenase pathway becomes saturated, 2-chloroethanol can be oxidized by catalase in the presence of hydrogen peroxide (H202) to a peroxide (2-chloroethylhydroperoxide), which undergoes subsequent dehydration to form 2-chloroacetaldehyde. An alternative pathway involves oxidation by mixed-function oxidase to form a highly reactive epoxide intermediate, 2-chloroethylene oxide, which spontaneously rearranges to form 2-chloroacetaldehyde. These intermediates are detoxified mainly through conjugation with glutathione catalyzed by glutathione S-transferase. The conjugated products are excreted in urine as substituted cysteine derivatives and include thiodiglycolic acid, S- For internal use only. DRAFT - Do not cite or quote. -28- formyl-methylcysteine, and N-acetyl-S-(2-hydroxyethyl)cysteine (Bolt et al., 1980; Hefner et al., 1975). Urinary metabolites identified in rats exposed by inhalation include polar compounds at low exposure concentrations (Hefner et al., 1975; Watanabe et al., 1976a) and 2-chloroacetic acid at high exposure concentrations (Hefner et al., 1975). Studies have demonstrated the binding of metabolites of 14C-vinylchloride to liver macromolecules in vitro, and in rats exposed by inhalation (Guengerich and Watanabe, 1979; Guengerich et al., 1979, 1981; Kappus et al., 1976; Watanabe et al., 1978a, 1978b). In single-exposure experiments at concentrations ranging from 1 to 5,000 ppm 14C-vinyl chloride, the binding to macromolecules increased proportionately with increasing metabolites of vinyl chloride, and disproportionately with vinyl chloride exposure concentration (Watanabe et al., 1978a). The extent of macromolecular binding increased with repeated exposures to 5,000 ppm vinyl chloride (Watanabe et al., 1978b), and by phenobarbital pretreatment (Guengerich and Watanabe, 1979). Macromolecular binding is attributed to the reactive epoxide intermediate, 2-chloroethylene oxide, which has been shown to bind to DNA and RNA, and to its product 2-chloroacetaldehyde, which has been shown to bind to protein molecules (Guengerich and Watanabe, 1979; Guengerich et al., 1979, 1981; Kappus et al., 1976; Watanabe et al., 1978a, 1978b). These epoxide intermediates have been hypothesized to alkylate liver macromolecules, and thus be responsible for the carcinogenicity and toxicity associated with vinyl chloride (Bolt, 1986). Urinary metabolites, in particular 2-chloroethylene oxide and 2-chloroacetaldehyde, have been identified after oral exposure to 14C-Iabeledvinyl chloride (Green and Hathaway, 1975, 1977; Watanabe and Gehring, 1976; Watanabe et al., 1976b). These metabolites were not different than those produced after inhalation exposure. The Watanabe et al. (1976b) study has been discussed previously, therefore only conclusions will be mentioned. The proportions of the above metabolites in the urine were not dependent on the dose, however expiration of vinyl chloride was clearly dose-dependent, increasing dramatically with the highest dose administered (0.05 to 100 mg/kg). It was concluded that vinyl chloride metabolism is a dose-dependent, saturable process, and that metabolic saturation occurred between the doses of 1 and 100 mg/kg vinyl chloride (Watanabe et al., 1976b). Urinary metabolites identified in rats exposed by inhalation include polar compounds resulting from conjugation with sulfhydryl groups at low exposure concentrations (Hefner et al., 1975; Watanabe et al., 1976a) and 2-chloroacetic acid at high exposure concentrations (Hefner et al., 1975). The urinary metabolites identified from rats orally exposed to 14C_ vinyl chloride are consistent with the metabolic pathways postulated for inhalation exposure, in particular with the formation of 2-chloroethylene oxide and 2-chloroacetaldehyde (Watanabe et al., 1976b). Metabolic saturation appears to occur with a single gavage dose in excess of 1 and less than 100 mg/kg-day (Watanabe et al., 1976b). In rats, urinary metabolites include N-acetyl-S-(2-hydroxyethy1cysteine) and thiodiglycolic acid (Watanabe et al., 1976a). In the Watanabe study discussed above (l976b), within 72 hours of dosing, a total of 10.3%, 15.4%, and 69.1 % of the radioactivity administered was detected in expired air (as For internal use only. DRAFT - Do not cite or quote. -29- unchanged vinyl chloride or CO2) for the 0.05, 1, and 100 mg/kg dose groups, respectively. Pulmonary excretion of vinyl chloride was estimated to be a monophasic process at doses =:;; 1 mg/kg, and biphasic at the higher doses. Urinary excretion was predominant after administration of 0.05 mg/kg (68%), and was of similar magnitude after 1 mg/kg (59%), but decreased to 11% with administration of 100 mg/kg due to the disproportionately high amount of exhaled vinyl chloride at this dose level. This indicated that there was metabolic saturation above 1 mg/kg. Total recovery of radioactivity was rather low in this study (82 to 91 % if total dose), and may be partly explained by the lack of special precautions for measuring volatile compounds in the urine and feces. Administration of single oral doses of 14C-vinyl chloride in com oil (0.05, 0.25, 1, 20, 100, and 450 mg/kg) gave similar large increases in the exhalation of unchanged vinyl chloride at doses > 20 mg/kg in other oral studies, suggesting again metabolic saturation at this approximate dose (Green and Hathaway, 1975; Watanabe and Gehring, 1976). In these studies, there was also a compensatory decrease in urinary and fecal excretion of radioactive label. At concentrations lower than 1 mg/kg, urinary excretion of polar metabolites predominated. Urinary excretion was judged to be biphasic in nature, having half-lives of approximately 4.5 hours for doses of 0.05 to 100 mg/kg-day. Green and Hathaway (1975) also performed longer-term oral studies that showed that although exhalation of unchanged vinyl chloride was complete within hours, excretion of metabolites in the urine continued for days. These metabolites have been identified as thiodiglycolic acid and N-acetyl-S-(2-hydroxyethylcysteine) (Watanabe et aI., 1976b). HUMAN TOXICITY Vihko et al. (1984) attempted to assess the early hepatotoxicity of vinyl chloride- exposed workers by measuring liver enzymatic activities, and quantitating serum primary bile acids by radioimmunoassay. A total of 76 workers were exposed to vinyl chloride air concentrations varying up to 2.6 mg/m! for a mean duration of 3 years. All subjects were fasted overnight and refrained from alcohol consumption for at least 1 week prior to blood analysis. A statistically significant increase in serum chenodeoxycholic acid was noted; however, all other parameters examined were normal in comparison to the healthy reference population. This study is limited because the exposure concentrations were not well characterized, and the methodology for estimating the vinyl chloride air concentration was not reported. The investigators suggest that determination of serum bile acid concentrations might be useful indicators of early hepatotoxicity. Several epidemiology and case studies have associated chronic occupational exposure with impaired liver function and/or biochemical or histological evidence of liver damage, notably subcapsular, portal and perisinusoidal fibrosis, hyperplasia of hepatocytes and sinusoidal cells and portal hypertension (Buchancova et al., 1985; Doss et al., 1984; Gedigk et al., 1975; Lilis et al., 1975; Marsteller et al., 1975; Popper and Thomas, 1975; Tamburro et al., 1984). Focal hepatocellular hyperplasia and focal mixed (hepatocytes and sinusoidal cells) hyperplasia are early histological alterations indicative of vinyl chloride exposure For internal use only. DRAFT - Do not cite or quote. -30- (Popper and Thomas, 1975), and are the principal anatomic lesion in vinyl chloride- associated liver disease (Berk et al., 1976). Doss et al. (1984) reported coproporphyinuria in 46 males occupationally exposed to vinyl chloride for 18 months to 21 years. Gedigk et al. (1975) correlated liver damage manifested as parenchymal damage, fibrosis and proliferation of the sinusoidal cells with duration of exposure to vinyl chloride in 51 patients. The severity of degenerative lesions increased with increasing duration of exposure, and appeared to be reversible upon exposure cessation. Another study reported the progressive nature of the liver changes which resulted in "chronic hepatitis" (Lilis et al., 1975). Thresholds for hepatotoxicity cannot be identified because data regarding exposure concentrations and duration were not available. The symptoms and signs of liver disease associated with occupational exposure to vinyl chloride include pain or discomfort in the right upper quadrant of the abdomen, hepatomegaly, splenomegaly, thrombocytopenia, in addition to fibrosis, cirrhosis and portal hypertension; however, these observations are not pathognomonic for vinyl chloride-induced liver disease (Lilis et al., 1975; Marsteller et al., 1975; Popper and Thomas, 1975). Fibrosis frequently occurs in the elderly and patients with diabetes mellitus (Popper and Thomas, 1975). An occupational study attempted to correlate the effects of vinyl chloride on the liver function of exposed workers (77 total), as measured by the plasma clearance of the 99mTc-N- (2,4-dimethylacetanilido)iminodiacetate (HEPIDA) complex (Studniarek et al., 1989). The duration of exposure varied from 3 to 17 years. Personal air samplers were used to determine the mean vinyl chloride concentrations in 1982 at various regions of the plant. Polymerization operators (n= 13) had the highest mean exposure to vinyl chloride, 30 mg/rrr', with a mean duration of employment of 10 years. Autoclave cleaners (n=9), and auxillary personnel (n= 12) in polymerization rooms were exposed to mean concentrations of 9 mg/rn" for a mean duration of 8 and 12 years, respectively, while technical supervisors (n=6) had the lowest mean vinyl chloride exposure of 6 mg/rrr' for a mean duration of 13 years. The investigators found a significant correlation between degree of exposure to vinyl chloride and the frequency of low clearance values; however, no concentration-response relationship was detected among the groups with respect to plasma clearance of 99mTc-HEPIDA. This study is of limited value since personal air sampling was conducted for only one year. The yearly geometric means of vinyl chloride atmospheric concentrations in various departments of the plant were provided, but these concentrations dramatically fluctuated between 0.1 to 600 rng/rrr' from 1974 to 1982. ANIMAL TOXICITY SUBCHRONIC EXPOSURE Hong et al. (1981) exposed groups of CD rats (4-16/sex/group) and CD-l mice (8- 28/sex/group) by inhalation to 0, 50, 250, or 1000 ppm vinyl chloride 6 hour/day, 5 day/week for 1, 3, 6, or 10 months (rats only). Animals were sacrificed 12 months after termination of exposure. Clinical signs, biweekly body weight and weekly food consumption measurements, hematological (RBC, reticulocyte, platelet, and WBC counts, hemoglobin, For internal use only. DRAFT - Do not cite or quote. -31- hematocrit, methemoglobin, and Heinz bodies) and clinical chemistry [SGPT and BUN (both species), prothrombin time, SGOT, alkaline phosphatase, bilirubin, creatinine, LDH, immunoglobulin IgA, 19B-A, IgB-B, and IgM, total protein, albumin, globulin, and collagen contents of liver and lungs (rats only)] parameters, gross examination of major tissues and organs, and histopathological examination of mammary gland, lung, liver, spleen, kidney and tissues with gross pathological changes were used to assess toxicity. During the recovery period for rats, significant (p<0.05, Fisher exact test performed by EPA) increases in mortality were observed in rats exposed to 1000 ppm for 6 months and all groups of rats exposed to vinyl chloride for 10 months. Increased incidence of neoplastic nodules (mid- concentration group exposed for 6 or 10 months), hemangiosarcomas in the liver (mid- concentration group exposed for 10 months), fibroadenoma (low-concentration group exposed for 6 or 10 months), and bronchioloalveolar tumor and hemangiosarcomas in the lungs (mid- and high-concentration groups) were observed. During the recovery phase for mice, significant (p< 0.05, Fisher exact test performed by EPA) dose- and duration-related increases in mortality were observed. Increased mortality was observed in mice exposed to 1000 ppm for 1 month, in the 250 and 1000 ppm groups exposed for 3 months, and all vinyl chloride exposed groups exposed for 6 months. Rough coat hair, lethargy, and the appearance of external tumor masses were observed in animals dying early. Increased incidence of bronchioloalveolar tumors (mid- and high-concentration groups exposed for 1 month; all vinyl chloride exposed groups after 3 and 6 months of exposure), hemangiosarcoma in the liver (mid- and high-concentration groups after 3 and 6 months of exposure), and adenocarcinoma/carcinoma in mammary gland (low-, mid- and high- concentration females after 3 months of exposure and mid-and high-concentration females after 6 months of exposure) were observed. Bi et al. (1985) reported a concentration-related elevation in relative liver weight and testicular degeneration in Wistar rats (8-30/sex/concentration) exposed to 0, 10, 100, or 3,000 ppm vinyl chloride (99.99% pure), 6 hours/day, 6 days/week (duration adjusted to 0, 5.5, 55, 1,643 mg/rn", respectively) for up to 12 months. The dynamic exposure chambers were controlled for temperature and relative humidity. Gross and histopathological examinations were performed on the testes, lungs, liver, heart, kidneys, spleen and brain. After 6 months, rats exposed to 10 ppm (duration adjusted to 5.5 mg/rrr') had significantly elevated relative liver, spleen and heart weights (p<0.01). The magnitude of these changes were 14%, 8%, and 12% above controls, respectively. The results of histopathology were not reported for any organ except the testes (including the lung). Kidney-to-body weight ratios were significantly increased after 3 months exposure to 3,000 ppm (duration adjusted to 1,643 mg/rn"). The incidence of damage to the testicular seminiferous tubules in rats (n=74) exposed to 0, 10, 100 and 3,000 ppm groups were 18.9, 29.7,36.5 and 56%, respectively. The incidence was statistically elevated at 100 and 3,000 ppm (duration adjusted to 55 and 1,643 mg/m", respectively) (p<0.05 and p<O.OOI, respectively) compared to controls and appeared to be concentration-related. This damage consisted of cellular alterations, degeneration and necrosis. The testes-to-body weight ratio was also significantly elevated at 100 ppm (duration adjusted to 55 mg/rrr'); measurement of this organ weight was limited to 6 months exposure. Thus, 10 ppm (duration adjusted to 5.5 mg/rrr'; For internal use only. DRAFT - Do not cite or quote. -32- HEC=5.5 mg/rrr') is considered a LOAEL for liver, kidney and heart organ weight changes, and for biologically significant testicular degeneration. Several species of animals were exposed to either 0, 50, 100, 200 or 500 ppm vinyl chloride via inhalation for up to 6 months (Torkelson et al., 1961). Hematologic determinations, urinalysis, organ weight measurement and histopathology examination were conducted. Rats (24/sex/group), guinea pigs (12/sex/group), rabbits (3/sex/group) and dogs (l/sex/group) exposed to 50 ppm (127.8 mg/rn'), 7 hours/day for 130 days in 189 days (duration adjusted to 26.6 rng/nr') did not exhibit toxicity as judged by appearance, mortality, growth, hematology, liver weight and pathology. At concentrations of 100 ppm administered 138-144 times in 204 days (duration adjusted to 53.25 mg/rrr'), a statistically significant increase in the liver weight of female rats was noted (approximately 12% above controls). Repeated exposure (138-144 times in 204 days) for 6 months to 200 ppm (duration adjusted to 106.5 mg/m") resulted in histological changes (characterized as granular degeneration and necrosis with some vacuolization and cellular infiltration) in the centrilobular area of the liver of rabbits, but not in rats, guinea pigs or dogs. The liver weight of rats (both sexes) were also significantly increased at this level (7-13% above controls). Histopathological lesions of the liver (centrilobular granular degeneration) and increased organ weight occurred in rats exposed to 500 ppm (duration adjusted to 266 mg/rrr'), although biochemical parameters of liver status were within normal limits at all exposure concentrations. Slightly elevated relative liver weights appeared to be the most sensitive indicator of hepatotoxicity and were observed in both sexes of rats (n=5) at 100 and 200 ppm, exposed for 2-4 hours/day· (duration adjusted to 15 to 30 and 30 to 60 mg/rn", respectively). The small sample size made these findings statistically insignificant in the 100 ppm males, however female rats had significantly higher relative liver weight. The highest concentration without any detectable effect on any species was 50 ppm (duration adjusted to 26.2 mg/rn"), therefore this dose is designated as a NOAEL for liver effects. Another subchronic inhalation study exposed male Wistar rats (7-l0/group) under dynamic conditions to nominal concentrations of 50, 500, and 20,000 ppm vinyl chloride (99.99% pure) or to air only, 5 hours/day, 5 days/week (duration adjusted to 127.8, 190, 7,607 mg/rrr', respectively) for 10 months (Wisniewska-Knypl et al., 1980). Examinations were limited to the liver. Concentrations of 50 ppm (duration adjusted to 127.8 mg/rrr') for 10 months resulted in hepatocellular changes characterized by proliferation of smooth endoplasmic reticulum. Rats exposed to 500 ppm (duration adjusted to 190 mg/rrr') for 3 months exhibited hypertrophy of the smooth endoplasmic reticulum, distension of canals of rough-surfaced membranes, swelling of mitochondria and an increased number of lipid droplets in cytoplasm; these changes were more intensive in the 20,000 ppm (duration adjusted to 7,607 mg/rn") exposure group. Pathological alterations in liver did not occur earlier than after 6-month exposure to 500 and 20,000 ppm (duration adjusted to 190 and 7,607 mg/rn", respectively), and not before lO-month exposure to 50 ppm (duration adjusted to 127.8 mg/rn'). This study identifies a LOAEL of 50 ppm (duration adjusted to 127.8 mg/rrr') for liver effects. For internal use only. DRAFT - Do not cite or quote. -33- Lee et al. (1977) observed no adverse effects on clinical chemistry parameters of rats or mice exposed to less than 250 ppm (duration adjusted to 114 mg/rn'), and failed to identify a NOAEL for hepatotoxicity. Albino CD-1 mice (36/sex/group) and CD rats (36/sex/group) were exposed to concentrations of 0, 50, 250, or 1,000 ppm vinyl chloride 6 hours/day, 5 days/week (duration adjusted to 22.6, 114, 456 mg/rrr', respectively) for up to 12 months. Parameters of toxicity evaluated included general appearance, food consumption, body weight, hematology, clinical chemistry, macrophage counts of pulmonary washings, cytogenic examination of bone marrow cultures, senographic radiography of the long bones of the limbs, gross necropsy on all tissues including the respiratory tract, selected organ weights and histopathologic examination of a comprehensive set of organs and tissues. Several mitotic figures, indicating increased rate of cell division, were observed in the livers of mice exposed to 50 or 1,000 ppm (duration adjusted to 22.67 or 456 mg/rrr', respectively) at 8 to 9 months, and increased rate of DNA synthesis was observed at 50 ppm (duration adjusted to 22.67 mg/rrr') in male mice. Since the liver is a known target organ for the toxicity of vinyl chloride, 50 ppm (duration adjusted to 22.67 mg/rrr') is considered an (LOAEL) effect level in this study. No persistent changes were noted in mice of either sex compared to controls with respect to hematology, clinical blood chemistry, cytogenic analysis of bone marrow cultures, x-ray examination of extremities, and serum alpha-fetoprotein. No significant changes were noted in rats for the same parameters, and in addition for: macrophage count, collagen contents in liver and lung, serum ALA synthetase, and urinary ALA level. Abnormalities observed in the mice included body weight loss at 1,000 ppm (duration adjusted to 456 mg/m") after 8 months, and elevated pulmonary macrophage count in mice from all exposure groups that had bronchioloalveolar adenoma; however, because of its association with lung tumors, this in not considered a noncarcinogenic toxic effect. Only a few mice exposed to 50 ppm (duration adjusted to 22.67 mg/rrr') survived for 12 months, no mice survived after 9 months in the 250 or 1,000 ppm (duration adjusted to 114 and 456 mg/rrr', respectively) exposure groups. Due to the high mortality among mice, 50 ppm (duration adjusted to 22.67 mg/m3) is considered a FEL. Histopathology of 5 unscheduled deaths of mice exposed to 1,000 ppm (duration adjusted to 456 mg/rn') for 3-9 days revealed acute toxic hepatitis, and marked tubular necrosis. After the 7th month, the general health of mice exposed to vinyl chloride deteriorated. Rats were more resistant to the toxic effects of vinyl chloride. Rats exposed to 1,000 ppm (duration adjusted to 456 mg/rrr') had reduced body weights compared with controls, but no other non-neoplastic effects. Exposure to 1,000 ppm (duration adjusted to 456 mg/rrr') also resulted in the death or sacrifice of 21136 rats during 8-12 months; of those exposed to 250 ppm (duration adjusted to 114 mg/rrr'), 14/35 died or were terminated. CHRONIC EXPOSURE Male Wi star rats (7-34/sex/group) (2-months old) were exposed to vinyl chloride in dynamic inhalation chambers at concentrations of 50, 500 and 20,000 ppm for 5 hours/day, 5 days/week (duration adjusted to 19, 190, 7,607 mg/rn", respectively) for 10 months (Sokal et al., 1980). Control animals received ambient air only. The animals were examined for hematological indices, and urinalysis, and histopathology was conducted on all major organs, For internal use only. DRAFT - Do not cite or quote. -34- -, including the lungs. Treatment-related histological changes developed in the liver and testes; however, the liver appeared to be the more sensitive target organ. After 10 months, the rats exposed to 500 and 20,000 ppm (duration adjusted to 190 and 7,607 mg/rn", respectively) exhibited a significant increase in scanty histological changes in the liver, characterized as increased polymorphism of hepatocytes and proliferation of reticulo-endothelial cells lining the sinusoids. In addition, damage to the spermatogenic epithelium was significant (p < 0.05) following exposure to 500 ppm (duration adjusted to 190 mg/rn') comp.ared to controls, but these effects were not clearly concentration-related. At 50 ppm (duration adjusted to 19 mg/rn'), relative spleen and heart weights were significantly elevated (9% and 4% above control, respectively). In addition, body weight gain was significantly decreased at concentrations >50 ppm. At 500 ppm and 20,000 ppm (duration adjusted to 190 and 7,607 mg/rrr', respectively) relative spleen, kidney and liver weights were significantly elevated; relative testis and heart weights were also significantly increased at 20,000 ppm (duration adjusted to 7,607 mg/rn'). Increased relative spleen and kidney weights were not accompanied by any histopathological changes and the indices of kidney function disorders were negative. The increased relative heart weight was not concentration-dependent. No statistically differences were observed for urinalysis, hematological or biochemical indices. The incidence of histopathological changes in the liver and testes did not correlate with the exposure level. No adverse effects on the lung were reported. The authors postulate that the enzymatic pathways become saturated at 20,000 ppm (duration adjusted to 7,607 mg/rrr'), resulting in reduced vinyl chloride metabolism, and thus less active metabolite formation. A LOAEL of 50 ppm (duration adjusted to 19 mg/rn') has been identified for decreased body weight gain, and increased relative weights of the spleen and heart. A NOAEL of 50 ppm (duration adjusted to 19 mg/rrr') has been identified for hepatocellular changes. Maltoni et al. (1980, 1981) exposed Wistar rats intermittently to 1 to 30,000 ppm 4 hours/day, 5 days/week (duration adjusted to 0.3 to 9,129 mg/rrr') for 52 weeks, and mice and hamsters to 50 to 30,000 ppm (duration adjusted to 127.8 to 9,129 mg/rn") for 30 weeks followed by an observation period. A statistically significant increase in tumor incidence, including liver angiosarcoma was observed in all three species at 50 ppm (duration adjusted to 15.2 mg/rrr'). This study primarily investigated the development of tumors. However, the incidence of preneoplastic lesions including hepatomas, neoplastic liver nodules, nodular hyperplasia of the liver, and diffuse hyperplasia of the liver were presented. Diffuse hyperplasia was the most significant observation which was evident in most exposure groups but did not appear to be concentration-related. The incidence for diffuse hyperplasia among controls, 1, 50, 250, 500, 2,500, 6,000 and 10,000 ppm (duration adjusted to 0.3, 15.2, 76, 152,760, 1,826, 3,043 mg/rrr', respectively) exposure groups were 2.3%,4.2%, 10%, 16.7%,3.3%, 6.7%, 6.7%, and not reported, respectively. Statistical significance and non- neoplastic results were not reported for the other organs examined, including the lung. Thus, 50 ppm (duration adjusted to 15.2 mg/rrr') was designated as a LOAEL for diffuse hyperplasia. This endpoint was not recommended for calculation of the RfC, however, due to the pre-neoplastic nature of the lesion. For internal use only. JJRAFT - Do not cite or quote. -35- The liver appears to be the critical target organ for animals orally exposed to vinyl chloride (Feron et al., 1981; Til et aI., 1983)." Feron et al. (1981) administered Wistar rats (60-80/sex/group) dietary concentrations of 0, 1.7,5.0, or 14.1 mg vinyl chloride/kg-day (99.97% pure) for a lifetime. A statistically significant increased incidence of several histopathologic lesions (foci of cellular alterations) were observed in the livers of rats exposed to 1.7, 5.0 and 14.1 rug/kg-day. Rats exposed to 1.7 rug/kg-day exhibited a series of changes in the hepatic parenchyma, characterized by an increased incidence of cellular alteration, liver polymorphism, cysts, neoplastic nodules and a few hepatocellular carcinomas. The liver lesions were most pronounced in the 14.1 mg/kg-day group. Liver- to-body weight ratios were higher in the 14.1 rug/kg-day dose group compared to controls. This study observed an increased incidence of neoplastic nodules of the liver and/or hepatocellular carcinoma in male rats administered 1.7 mg/kg-day, and in females exposed to 5.0 mg/kg-day. In a lifetime dietary study, Wistar rats (100/sex/dose) were administered doses of 0, 0.014,0.13 or 1.3 mg vinyl chloride/kg-day for 149 weeks (Til et al., 1983). Relative organ weights were not evaluated. An increased incidence of basophilic foci were observed in both sexes at 1.3 mg/kg-day and only in females in the two lower dosage groups. Since the basophilic foci lacked a dose-related increase, and histopathological alterations at 0.13 mg/kg-day, basophilic foci in the liver of rats of one sex may be considered a nonadverse, although a compound-related effect. Rats exposed to 1.3 rug/kg-day also had a significantly increased incidence of liver cell polymorphism, hepatic cysts, neoplastic nodules, and hepatocellular carcinoma. DEVELOPMENTAL TOXICITY Insufficient data exist to evaluate the teratogenicity of vinyl chloride in humans. Several epidemiology studies have investigated the effects of vinyl chloride exposure on the incidence of fetal loss and birth defects (Edmonds et al., 1978; Hatch et al., 1981; Infante et al., 1976; Theriault et al., 1983; Waxweiler et al., 1977), however no solid association has been found. Using a questionnaire, Infante et al. (1976) and Waxweiler et al. (1977) associated increased fetal loss and birth defects with paternal exposure to vinyl chloride. They studied the outcome of pregnancies of wives of 95 vinyl chloride polymerization workers, a control group of 158 unexposed rubber workers and polyvinyl chloride fabricators exposed to "very low" levels of vinyl chloride monomer. Data were obtained for the exposed cohort regarding pregnancies that occurred before and during employment in a vinyl chloride-contaminated atmosphere. Subsequent to the husband's exposure, the most significant observation was that "age adjusted" fetal loss occurred in 8.8% of the pregnancies of wives of controls and in 15.8% of the pregnancies of wives of exposed workers. The most significant difference occurred in wives of men under age 30, where fetal loss was 5.3% (7/131) for controls and 20% (14170) for exposed workers. The exposure data were not quantified (Infante et al. 1976). A published evaluation by Hatch et al. (1981) severely criticized the conduct and statistical analysis of the Infante et al. (1976) study. Women with For internal use only. DRAFT - Do not cite or quote. -36- multiple spontaneous abortions were included in the exposed group of this study. Since one spontaneous abortion is' associated with a 66% increase in the risk of subsequent abortions, use of Chi-square for statistical analysis is incorrect because a woman's first pregnancy and her subsequent pregnancies can not be considered independent. Hatch et al. (1981) concluded that the study showed no association of paternal occupational exposure to vinyl chloride with increased fetal loss and that the study lacked statistical power to do so. It appears that the data of Infante et al. (1976) are suggestive of an effect, but additional studies are warranted. This association was contradicted by other case-control studies (Edmonds et al., 1978; Theriault et al., 1983) which found no association between parental residence in a region with a vinyl chloride plant and the incidence of birth defects in the exposed community. Edmonds et al. (1978) compared the incidence rates of CNS defects in a West Virginia county in which a polyvinyl chloride polymerization plant was located with those for other regions in the United States with no exposure to vinyl chloride. The incidence rates of the index county exceeded those of control areas by a factor of 1.5 to 2. By comparing data from parents of deformed infants with randomly chosen matched controls living in the index county (46 matched pairs), no correlation was noted for parental occupation, for proximity to the polyvinyl chloride plant, or for patterns of wind direction and air pollution. Furthermore, one major and several smaller chemical plants were located in the area. Vinyl chloride does not appear to be teratogenic in animals. Inhalation experiments in animals have not associated vinyl chloride with developmental toxicity at concentrations below those associated with maternal toxicity. John et al. (1977) examined the effects of inhaled vinyl chloride on the fetuses of mice, rats, and rabbits. Pregnant CFl mice (30- 40/group) were exposed to 0, 50 or 500 ppm vinyl chloride on gestational days 6 to 15. Sprague-Dawley rats (20-35/group) and New Zealand white rabbits (15-20/group) were administered 0, 500 or 2,500 ppm vinyl chloride, 7 hours/day on gestational days 6 through 15 for rats and 6 to 18 for rabbits. Parameters of maternal and developmental toxicity were evaluated; both the fetuses and litter were evaluated. Mice were more sensitive to the toxic effects of vinyl chloride than either rats or rabbits. In mice, concentrations of 500 ppm induced maternal effects which included increased mortality, reduced body weight, and reduced absolute, but not relative liver weight. Fetotoxicity, also occurred in mice at 500 ppm, and was manifested as significantly increased fetal resorption, decreased fetal body weight, reduced litter size, and retarded cranial and sternebral ossification; however, there was no evidence of a teratogenic effect in mice at either concentration. Maternal effects restricted to reduced body weight gain were noted in rats exposed to 500 ppm but not to 2,500 ppm. Maternal effects in rats at 2,500 ppm were death of one rat, elevated absolute and relative liver weights, and reduced food consumption. A significant reduction in fetal body weight and an increase in the incidence of lumbar spurs were observed among rats exposed to 500 ppm but not 2,500 ppm and are not considered signs of vinyl chloride- induced fetotoxicity. At 2,500 ppm, an increased incidence of dilated ureters was observed which may represent a chemical-induced effect. No signs of maternal or developmental toxicity were observed in rabbits at either dose. This study identifies a NOAEL of 50 ppm for maternal and fetotoxicity in mice and a NOAEL of 2,500 ppm for rabbits. For internal use only. DRAFT - Do not cite or quote. -37- Ungvary et al. (1978) exposed groups of pregnant CFY rats continuously to 1,500 ppm (4,060 mg/m") on gestational days 1 to 9, 8 to 14 or 14 to 21 and demonstrated that vinyl chloride is not teratogenic and has no embryotoxic effects when administered during the second or last third of pregnancy. During the first third of pregnancy, maternal toxicity was manifested by increased relative liver weight; increased fetal mortality and embryo toxic effects were evident. Slightly reduced body weight gain was noted in dams exposed on days 14 to 21. Vinyl chloride does not appear to produce germinial mutations as manifested by a dominant lethal effect in male rats. In an dominant lethal study, Short et al. (1977) exposed male CD rats to 0, 50, 250, or 1,000 ppm vinyl chloride 6 hours/day, 5 days/week. Eleven weeks later the exposed males were mated with untreated females, and there was no evidence of either preimplantation or postimplantation loss in pregnant females. However, reduced fertility was observed in male rats exposed to 250 and 1,000 ppm vinyl chloride. CONCLUSION The occupational study (Vihko et al., 1984) was a subchronic study and lacks experimental details related to measurement of the dosage concentration and other biochemical and clinical indices. The subchronic study by Bi et al. (1985) was conducted in one species (rats) in the appropriate numbers of animals, and measured several endpoints. There are several corroborative inhalation studies which observed effects on the liver and testes (Lee et al., 1977; Maltoni et al., 1980, 1981; Sokal et al., 1980; Torkelson et al., 1961; Wisniewska-Knypl et al., 1980) in rodents and rabbits following subchronic inhalation exposure. Numerous human occupational studies observed hepatotoxicity as the endpoint. Two developmental inhalation studies were located which did not observe teratogenicity in mice, rats or rabbits (John et al., 1977; Ungvary et al., 1978), and one reproductive study examining the dominant lethal effect of vinyl chloride was located (Short et al., 1977). The Hong et al. (1981) study showed that significantly more mice exposed by inhalation to 50 ppm vinyl chloride 6 hour/day, 5 day/week for 6 months (equivalent oral dose of 19 mg/kg-day) died 6-12 months after exposure termination as compared to the control group. No significant increases in the incidence of neoplastic tumors were observed in this group of mice (Fisher exact test performed by EPA). Non-neoplastic effects were not reported, however, it is not known if the tissues were examined for neoplastic endpoints only. No deaths were reported during exposure. Similar effects were observed in rats. However, mice may be more sensitive than rats to the lethal effects of vinyl chloride. The Hong et al. (1981) study suggests that the data are not adequate for the derivation of a subchronic value for vinyl chloride because subchronic exposure to 300 mg/kg may result in increased mortality several months after exposure termination. Furthermore, data from chronic exposure studies should not be used to derive a subchronic value. For internal use only. DRAFT - Do not cite or quote. -38- The health effects data for vinyl chloride were reviewed and determined to be inadequate for the derivation of a subchronic inhalation RfC. The status for this chemical is currently not verifiable. For internal use only. DRAFT - Do not cite or quote. -39- Summary Table of the Toxicity Data for Vinyl Chloride Study Species Effect UF/MF RfD (mg/kg/day) Reported dose or concentration Dose (mg/kg/day) Subchronic oral toxicity Feron et al., 1975 rat Chronic oral toxicity Til et al., 1983 rat decreased lifespan and liver damage at 1.3 mg/kg/day Feron et al., 1981 rat decreased lifespan and liver damage at ~1.7 mg/kg/day Knight and Gibbons, 1987 rat decreased lifespan and increased collagen in the skin at 30 mg/kg/day Subchronic inhalation toxicity studies Bi et al., 1985 testicular damage rat Hong et al., 1981 increased mortality; increased incidence of lung tlJllOrs rat Lee et al., 1977, 1978; increased mortality rat o (mg/kg, 6 d/wk) 30 100 300 0 (ppm) 10 100 L 3000 0 (ppm) 50 F 250 1000 0 (ppm) 50 250 F 1000 o 26 86 257 0 0.014 0.13 1.3 0 1.7 5.0 14.1 214' 0 3 30 300 Ob 3 30 L 888 oc 10 51 F 204 For internal use only. DRAFT - Do not cite or quote. -40- Study Species Effect Reported dose or Dose UF/MF RfD concentration (mg/kg/day) (mg/kg/day) Lee et al., 1977, 1978; mouse increased mortality 0 (ppm) Od 50 F 20 F 250 98 1000 392 Inhalation developmental toxicity John et al., 1981 rat developmental toxicity o (ppm) O' 500 L 194 L 2500 968 John et al., 1981 mouse developmental toxicity and maternal o (ppm) 0' toxicity 50 31 500 L 312 L John et al., 1981 rabbit developmental toxicity o (ppm) og 500 70 2500 351 • gavage dose of 300 mg/kg 5 day/week b mg/kg/day doses were calculated by multiplying the duration expanded concentration (mg/m3) by the inhalation rats of 0.2136 m3/day (using allometric equation, u.S. EPA, 1987) and dividing by a TYA body weight of 0.200 kg. This mg/kg/day dose was multiplied by the ratio of the inhalation absorption rate of 0.42 (Krajewski et al., 1980) and oral absorption rate of 0.83 (Feron et al., 1981). C mg/kg/day doses calculated same as in b, inhalation rate of 0.5116 m3/day and body weight of 0.580 kg d mg/kg/day doses calculated same as in b, inhalation rate of 0.0537 m3/day and body weight of 0.032 kg • mg/kg/day doses calculated same as in b, inhalation rate of 0.2565 m3/day and initial body weight of 0.250 kg , mg/kg/day doses calculated same as in b, inhalation rate of 0.0414 m3/day and initial body weight of 0.025 kg g mg/kg/day doses calculated same as in b, inhalation rate of 1.303 m3/day and initial body weight of 3.5 kg For internal use only. DRAFT - Do not cite or quote. -41- REFERENCES ATSDR. 1991. Agency for Toxic Substances and Disease Registry. Draft for Public Comment. Revised Toxicological Profile Report on Vinyl Chloride. Prepared by Clement International Corp., October, 1991. Berk, P.D., J.F. Martin, R.S. Young, J. Creech, I.J. Selikoff, H. Falk, P. Watanabe, H. Popper, and L. Thomas. 1976. Vinyl chloride-associated liver disease. Ann. Intern. Med. 84(6): 717-31. Bi, W.F., Y.S. Wang, M. Y. Huang, and D.S. Meng. 1985. Effect of vinyl chloride on testis in rats. Ecotoxicol. Environ. Safety 10(3): 281-9. Bolt, H.M. 1986. Metabolic activation of vinyl chloride, formation of nucleic acid adducts and relevance to carcinogenesis. IARC Sci. Publ. 70:261-268. Bolt, H.M., J.G. Filser, R.J. Laib, and H. Ottenwaelder. 1980. Binding kinetics of vinyl chloride and vinyl bromide at very low doses. Quantitative aspects of risk assessment in chemical carcinogenesis. Arch. Toxicol. Suppl. 3: 129-42. Bolt, H.M., H. Kappus, A. Buchter, and W. Bolt. 1976. Disposition of [1,2-14C] vinyl chloride in the rat. Arch. Toxicol. 35: 153-62. Bolt, H.M., R.J. Laib, H. Kappus, and A. Buchter. 1977. Pharmacokinetics of vinyl chloride in the rat. Toxicology 7: 179-88. Buchancova, J., I. Reznak, V. Horak, P. Altmann, L. Svehlova, E. Suchova, and E. Sramkova. 1985. Scintigraphic pictures of the liver in workers after a long-term exposure to vinyl chloride. Pracov. Lek. 37(6): 190-94. Buchter, A., H.M. Bolt, H. Kappus, and W. Bolt. 1977. [The distribution of 1,2-14C-vinyl chloride in rat tissue.] Int. Arch. Occup. Environ. Health 39: 27-32. (German, translation) Buchter, A., J.G. Filser, H. Peter, and H.M. Bolt. 1980. Pharmacokinetics of vinyl chloride in the rhesus monkey. Toxicol. Lett. 6: 33-6. Doss, M., C.E. Lange, and G. Veltman. 1984. Vinyl chloride-induced hepatic coproporphyrinuria with transition to chronic hepatic porphyria. Klin. Wochenschr. 62(4): 175-8. Duprat, P., J.P. Fabry, D. Gradiski, and J.L. Magadur. 1977. Metabolic approach to industrial poisoning: blood kinetics and distribution of 14C-vinylchloride monomer (V.C.M.). Acta Pharmacol. Toxicol. Supp. (Kbh) 41: 143-3. For internal use only. DRAFT - Do not cite or quote. -42- Edmonds, L.D., C.E. Anderson, J.W. Flynt Jr., and L.M. James. 1978. Congenital central nervous system malformations and vinyl chloride monomer exposure: A community study. Teratology 17(2): 137-42. Feron, V.J., A.J. Speek, M.I. Willems, D. van Battum and A.P. de Groot. 1975. Observations on the oral administration and toxicity of vinyl chloride in rats. Food Cosmet. Toxicoi. 13: 633-638. Feron, V.J., C.F.M. Hendriksen, A.J. Speek, H.P. Til, B.J. Spit. 1981. Lifespan oral toxicity study of vinyl-chloride in rats. Food Cosmet. Toxicol, 19(3): 317-33. Gedigk, P., R. Muller, and H. Bechtelsheimer. 1975. Morphology of liver damage among polyvinyl chloride production workers. A report on 51 cases. Ann. N. Y. Acad. Sci. 246: 279-85. Gehring, P.J., P.G. Watanabe, and C.N. Park. 1978. Resolution of dose-response toxicity data for chemicals requiring metabolic activation: Example-vinyl chloride. Toxicol. Appl. Pharmacol. 44: 581-91. Green, T., and D.E. Hathway. 1975. The biological fate in rats of vinyl chloride in relation to its oncogenicity. Chern. Biol. Interact. 11: 545-62. Green, T., and D.E. Hathway. 1977. The chemistry and biologenesis of the S-containing metabolites of vinyl chloride. Chern. BioI. Interact. 17: 137-50. Guengerich, F.P., W.M. Crawford Jr., and P.G. Watanabe. 1979. Activation of vinyl chloride to covalently bound metabolites: Roles of 2-chloroethylene oxide and 2- chloroacetaldehyde. Biochemistry 18: 5177-82. Guengerich, F.P., P.S. Mason, W.T. Stott, T.R. Fox, and P.G. Watanabe. 1981. Roles of 2-haloethylene oxide and 2-haloacetaldehydes derived from vinyl bromide and vinyl chloride in irreversible binding to protein and DNA. Cancer Res. 41: 4391-8. Guengerich, F.P., and P.G. Watanabe. 1979. Metabolism of e4C)- and e6CI)-labeled vinyl chloride in vivo and in vitro. Biochem. Pharrnacol. 28: 589-96. Hatch, M., J. Kline, and Z. Stein. 1981. Power considerations in studies of reproductive effects of vinyl chloride and some structural analogs. Environ. Health Perspect. 41: 195- 201. Hefner, R.E., Jr., P.G. Watanabe, and P.J. Gehring. 1975. Preliminary studies of the fate of inhaled vinyl chloride monomer in rats. Ann. N. Y. Acad. Sci. 246: 135-48. Hong, C.B., J.M. Winston, L.P. Thornburg, C.C. Lee and J.S. Woods. 1981. Follow-up study on the carcinogenicity of vinyl chloride and vinylidene chloride in rats and mice: For internal use only. DRAFT - Do not cite or quote. -43- tumor incidence and mortality subsequent to exposure. J. Toxicol. Environ. Health. 7: 909- 924. Infante, P.F., J.K. Wagoner, and R.J. Waxweiler. 1976. Carcinogenic, mutagenic, and teratogenic risks associated with vinyl chloride. Mut. Res. 41: 131-42. Jedrychowski, R.A., J.A. Sokal, and J. Chmielnicka. 1984. Influence of exposure mode on vinyl chloride action. Arch. Toxicol. 55: 195-198. John, J.A., F.A. Smith, B.K.J. Leong, and B.A. Schwetz. 1977. The effects of maternally inhaled vinyl chloride on embryonal and fetal development in mice, rats, and rabbits. Toxicol. Appl. Pharmacol. 39: 497-513. John, J.A., F.A. Smith and B.A. Schwetz. 1981. Vinyl chloride: inhalation teratology study in mice, rats, and rabbits. Environ. Health Perspect. 41: 171-177. Kappus, H., H.M. Bolt, A. Buchter, and W. Bolt. 1976. Liver microsomal uptake of [14C] vinyl chloride and transformation to protein alkylating metabolites in vitro. Toxicol. Appl. Pharmacol. 37: 461-71. Knight, K.R. and R. Gibbons. 1987. Increased collagen synthesis and cross-link formation in the skin of rats exposed to vinyl chloride monomer. Clin. Sci. 72: 673-678. Krajewski J., M. Dobecki, and J. Gromiec. 1980. Retention of vinyl chloride in the human lung. Br. J. Ind. Med. 37(4): 373-74. Lee, C.C., J.C. Bhandari, J.M. Winston, W.B. House, and P.J. Peters. 1977. Inhalation toxicity of vinyl chloride and vinylidene chloride. Environ. Health Perspectives. 21: 25-32. Lee, C.C., J.C. Bhandari, J.M. Winston, W.B. House, R.L. Dixon and J.S. Woods. 1978. Carcinogenicity of vinyl chloride and vinylidene chloride. J. Toxicol. Environ. Health 4: 15-30. Lilis, R., H. Anderson, W.J. Nichloson, S. Daum, A.S. Fischbein, and I.J. Selikoff. 1975. Prevalance of disease among vinyl chloride and polyvinyl chloride workers. Ann. NY. Acad. Sci. 246: 22-41. Maltoni, C., G. Lefemine, A. Ciliberti, G. Cotti and D. Carretti. 1980. Vinyl Chloride carcinogenicity bioassays (BT project) as an experimental model for risk identification and assessment in environmental and occupational carcinogenesis. Epidemiol. Anim. Epidemiol. Hum.: Cas Chlorure Vinyle Monomer (Reun Club Carcerog Chim) 20th meeting date 1979, pp. 11-112 PubI Essent, Paris, France. For internal use only. DRAFT - Do not cite or quote. -44- Maltoni, C., G. Lefemine, A. Ciliberti, G. Cotti and D. Carretti. 1981. Carcinogencity bioassays of vinyl chloride monomer: A model or risk assessment on an experimental basis. Environ. Health Perspect. 41: 3-29. (Basis of cancer potency factor) Marsteller, H.J., W.K. Lelbach, R. Muller, and P. Gedigk. 1975. Unusual spenomegalic liver disease as evidenced by peritoneoscopy and guided liver biopsy among polyvinyl chloride production workers. Ann. N.Y. Academ. Sci. 95-134. Popper, H. and L.B. Thomas. 1975. Alterations of liver and spleen among workers exposed to vinyl chloride. Ann. NY Acad. Sci. 246: 172-194. Short, R.D., J.L. Minor, J.M. Winston, and C. Lee. 1977. Dominant lethal study in male rats after repeated exposures to vinyl chloride or vinylidene chloride. J. Toxicol. Environ. Health. 3: 965-968. Sokal, J.A., B. Baranski, J. Majka, R. Rolecki, J. Stetkiewicz, L. Ivanovachemishanska, T. Vergieva, G. Antonov, E. Mirkova, Kolakowski, S. 1980. Experimental studies on the chronic toxic effects of vinyl chloride in rats. J. Hyg. Epidem. Microbiol. Immunol. 24: 285-94. Studniarek, M., K. Durski, J. Liniecki, D. Brykalski, A. Poznanska, and M. Gluszcz. 1989. Effects of vinyl chloride on liver function of exposed workers, evaluated by measurements of plasma clearance of the 99mTc-N-2,4-dimethylacetanilido-iminodiacetate complex. J. Appl. Toxicol. 9(4): 213-8. Tamburro, C.H., L. Makk, and H. Popper. 1984. Early hepatic histologic alterations among chemical (vinyl monomer) workers. Hepatology 4(3): 413-8. Theriault, G., H. Iturra, and S. Gingras. 1983. Evaluation of the association between birth- defects and exposure to ambient vinyl-chloride. Teratology 27(3): 359-70. Til, H.P., H.R. Immel, and V.I. Feron. 1983. Lifespan oral carcinogenicity study of vinyl chloride in rats. Final report. Civo Institutes. TNO. Report No. V 93.285/291099. Torkelson, T.R., F. Oyen, and V.K. Rowe. 1961. The toxicity of vinyl chloride as determined by repeated exposure of laboratory animals. Am. Ind. Hyg. Assoc. J. 22: 354- 61. U.S. EPA. 1987. Health Advisory for Vinyl Chloride. Prepared by Office of Drinking Water, Washington D.C. Ungvary, G., A. Hudak, E. Tatrai, M. Lorincz. 1978. Effects of vinyl chloride exposure alone and in combination with trypan blue applied systematically during all thirds of pregnancy on the fetuses of CFY rats. Toxicology 11(1): 45-54. For internal use only. DRAFT - Do not cite or quote. -45- Vihko, R., P. Vihko, O. Maentausta, A. Pakarinen, O. Janne, and E. Yrjanheikki. 1984. Assessment of Early Hepatotoxicity. Biological Monitoring and Surveillance of Workers Exposed to Chemicals, Aitio, A., V. Riihimaki, and J. Vainio, Editors; Hemisphere Publishing Co., Washington D.C., pp. 309-13. Watanabe, P.G. and P.J. Gehring. 1976. Dose-dependent fate of vinyl chloride and its possible relationship to oncogenicity in rats. Environ. Health Perspect. 17: 145-52. Watanabe, P.G., G.R. McGowan, E.O. Madrid, and P.J. Gehring. 1976a. Fate of 14C- vinyl chloride following inhalation exposure in rats. Toxicol. Appl. Pharmacol. 37(1): 49- 59. Watanabe, P.G., G.R. McGowan, and P.J. Gehring. 1976b. Fate of [14C]vinyl chloride after single oral administration in rats. Toxicol. Appl. Pharmacol. 36: 339-52. Watanabe, P.G., LA. Zempel, D.G. Pegg, and P.L Gehring. 1978a. Hepatic macromolecular binding following exposure to vinyl chloride. Toxicol. Appl. Pharmacol. 44: 571-79. Watanabe, P.G., J.A. Zempel, and P.J. Gehring. 1978b. Comparison of the fate of vinyl chloride following single and repeated exposure in rats. Toxicol. Appl. Pharmacol. 44: 391- 99. Waxweiler, R.J., H. Falk, A. McMichael, J.S. Mallov, and A.S. Grivas. 1977. A cross- sectional epidemiologic survey of vinyl chloride workers. NTIS PB274193. Wisniewska-Knypl, J.M., J. Kliinczak, and J. Kolakowski. 1980. Monooxygenase activity and ultrastructural changes of liver in the course of chronic exposure of rats to vinyl chloride. Int. Arch. Occup. Environ. Health. 46(3): 241-9. For internal use only. DRAFT - Do not cite or quote. -46- Attachment 5 (11/9/93) Risk Assessment Issue Paper for: Evaluation of Subchronic Oral Systemic Toxicity for Vinyl Chloride (CASRN 75-01-4) SUBCHRONIC ORAL TOXICITY Groups of 15 weanling male and female Wistar rats were administered 0, 30, 100, or 300 mg/kg vinyl chloride (purity not reported) in soybean oil by gavage 6 day/week for 13 weeks (Feron et al., 1975). The duration expanded doses are 0, 26, 86, and 257 mg/kg-day. Weekly body weight measurements, hematological parameters (blood clotting time, hemoglobin, packed cell volume, and erythrocyte and leukocyte counts), clinical chemistry parameters (glucose, SGOT, SGPT, BUN, total protein, albumin), organ weights, histopathology of major organs and tissues (control and high-dose groups only), and electron microscopy of the liver (2/sex/group) were used to assess toxicity. In addition, urinalysis (specific gravity and GOT in the urine) was conducted in 10 rats/sex in the control and high dose group: Urinary pH, glucose, protein, occult blood, ketones and microscopic constituents were measured from pooled urine samples from 10 rats/sex in each group. Slight but significant (p<0.05) decreases in blood glucose, total leukocytes (mid- and high- dose females), SGOT, SGPT, and urinary GOT (high-dose males) levels were observed. Increases (p< 0.05) in relative liver and adrenal (males only) weights were observed in the high-dose group. Foci of hyperbasophilic hepatocytes were observed in 2/30 in each of the 100 and 300 mg/kg groups, this slight increase was not statistically significant. This study identifies a NOAEL of 300 mg/kg. CHRONIC ORAL TOXICITY Groups of 110 female and 110 male Wistar rats were fed diets containing 0, 0.014, or 0.13 mg/kg-day vinyl chloride and groups of 60 female and 60 male Wistar rats were fed 1.3 rug/kg-day vinyl chloride for over 3 years (Til et al., 1983). The diets were prepared by incorporating polyvinyl chloride powder with varying proportions of vinyl chloride monomer-containing powder (4600 ppm) or vinyl chloride-free powder. The concentration of polyvinyl chloride in the diet was 1%. The rats had access to the diet for 4 hour/day. Til et al. (1983) calculated mg/kg-day doses from the rate of evaporation of vinyl chloride. monomer from the diet, food intake, and body weight data. After 9 and 18 months, 5 female and 5 male rats in each group were killed for measurement of glutathione levels in the liver. Daily observations of clinical signs, monthly body weight and bimonthly food consumption measurements, hematological measurements (thrombocyte count and prothrombin time) in 10 rats/sex/group after 9 and 18 months of exposure, gross examination of major tissues and organs, and histopathological examination of the liver were used to assess toxicity. Increased mortality was observed in the high dose group during the last 6 and 9 months of the study. No compound-related effects on general appearance, food intake, body weight, hematological parameters, or glutathione levels were observed. The only For internal use only. DRAFT - Do not cite or quote. -47- compound-related alteration observed during the gross examination was a significant (p< 0.05) increase in the incidence of liver cysts in the high dose group. An increase in the incidence of foci of basophilic cellular alterations in the liver were observed in all groups of vinyl chloride-exposed female rats, the incidence was not dose-related. Other compound- related liver alterations observed in the high-dose group include cell polymorphism, eosinophilic cellular alterations, clear cell foci of alterations, and cysts. A statistically significant increase in the incidence of neoplastic tumors in the liver and mammary glands were observed in the high-dose group. Groups of 80 (control and high dose groups) or 60 male and 80 or 60 female Wistar rats were fed diets containing 0, 1.7, 5.0, or 14.1 mg/kg-day vinyl chloride for 135 (males) or 144 (females) weeks (Feron et al., 1981). The diets were prepared by combining polyvinyl chloride powder (10% in diet) and varying proportions of vinyl chloride monomer. Actual levels of vinyl chloride monomer in the diet were measured. The diet was available for 4 hours each day; another control group that had ad libitum access to a diet containing 10% polyvinyl chloride was also used. A group of 80 male and 80 female rats received 300 mg/kg vinyl chloride in corn oil by gavage 5 day/week for 2 years (expanded dose of 214 mg/kg-day). The following measurements were used to assess toxicity: monthly body weights and bimonthly food consumption measurements; hematological (hemoglobin, hematocrit, thrombocyte, RBC, and WBC counts, BUN, blood glucose) and clinical chemistry (alkaline phosphatase, SGOT, SGPT, total protein, alpha-fetoprotein, and albumin) parameters (blood samples taken from 10 rats/sex/group in week 13, 26, 52, 78, and 94); urinalysis (urine samples collected same as blood samples); and gross and histopathological examinations (major organs and tissues - 20 rat/sex in control, 14.1 and 214 mg/kg-day groups at termination; liver, Zymbal glands, lungs, kidneys, spleen, pituitary, thyroid, and adrenals - all rats killed at termination; and liver, kidneys and Zymbal glands - 10 rats/sex in control, 14.1 and 214 mg/kg-day groups killed after 26 or 52 weeks). The data for animals in the 214 mg/kg-day group were not analyzed statistically because no corresponding control group was included in the study. Rats in the 214 mg/kg-day gavage group died or were killed in a moribund condition by week 84. Lethargy and severe lesions in the liver, lungs and other organs, were observed in this group. In the 5.0 and 14.1 mg/kg-day groups, lethargy, emaciation, and general poor condition were observed after 18 months of exposure. A significant (p<0.05) increase in mortality was observed in the female rats in the 5.0 and 14.1 mg/kg-day groups after 80 weeks and in the 1.7 mg/kg-day group after 143 weeks; in the male rats increased mortality was observed in the 14.1 mg/kg-day group after 80 weeks of exposure and in the 5.0 mg/kg-day group after 134 weeks of exposure. A decrease in body weight gain was observed in the 214 mg/kg-day group compared with the ad libitum control group. No differences in the body weight gain of the other groups were observed. Decreased (p< 0.05) prothrombin times and increased alpha-fetoprotein levels were observed in the 14.1 and 214 mg/kg-day groups after 26 (prothrombin time only) and 52 weeks of exposure. Significant (p < 0.05) increases in the incidence of clear cell foci, neoplastic nodules (females only), and cystic proliferation of the bile ducts (females only) were observed in rats exposed to 14.1 mg/kg-day for 26 or 52 weeks. In animals killed at termination, dying early, or killed in extremis, statistically significant increases in the incidence of clear-cell, basophilic, and eosinophilic foci of cellular alterations, neoplastic For internal use only. DRAFT - Do not cite or quote. -48- nodules, cysts, and liver cell polymorphism (all treated groups), and hepatocellular carcinomas, extensive necrosis, cysts, and focal hematopoiesis (14.1 mg/kg-day) were observed in the liver. The severity and incidence of the lesions were generally higher in the females than the males. Marked hematopoietic activity was observed in the spleen in rats exposed to the 2 highest concentrations. Significant increases of neoplastic tumors were also observed in the lung, abdomen, and pitui~ gland. Male and female Wistar rats (total of 10-20 rats/group) received daily gavage doses of 0, 3, 30, or 300 mg/kg-day of vinyl chloride in com oil for 95-125 weeks (Knight and Gibbons, 1987). Increased mortality was observed in the 300 mg/kg-day group after 60 days. Mortality in the low- and mid-groups was 1/15 and 5/16, respectively. The incidence of liver tumors, primarily hepatic angiosarcomas, were 1116, 11115, and 10/10 in the low-, mid-, and high-dose groups, respectively; the incidence in the control group was not reported. The chemical composition of the skin from 9 control group rats and 8 rats exposed to 30 mg/kg-day was analyzed. Significant increases in moisture content and collagen content were observed in the vinyl chloride treated rats. A significant increase in the number of collagen cross-links was also observed in the treatment group, indicating an increase in collagen synthesis. Thus, exposure to 30 mg/kg-day vinyl chloride for 2 years resulted in a thickening of the skin due to increased collagen. Scleroderma-like syndrome has been observed in individuals exposed to vinyl chloride. Characteristics of this syndrome include thickening and increased rigidity of the skin, poor circulation in the extremities, and hypersensitivity to the cold. SUBCHRONIC INHALATION TOXICITY Hong et al. (1981) exposed groups of CD rats (4-16/sex/group) and CD-l mice (8- 28/sex/group) by inhalation to 0, 50, 250, or 1000 ppm vinyl chloride 6 hour/day, 5 day/week for 1, 3, 6, or 10 months (rats only). Animals were sacrificed 12 months after termination of exposure. Clinical signs, biweekly body weight and weekly food consumption measurements, hematological (RBC, reticulocyte, platelet, and WBC counts, hemoglobin, hematocrit, methemoglobin, and Heinz bodies) and clinical chemistry [SGPT and BUN (both species), prothrombin time, SGOT, alkaline phosphatase, bilirubin, creatinine, LDH, immunoglobulin IgA, 19B-A, IgB-B, and IgM, total protein, albumin, globulin, and collagen contents of liver and lungs (rats only)] parameters, gross examination of major tissues and organs, and histopathological examination of mammary gland, lung, liver, spleen, kidney and tissues with gross pathological changes were used to assess toxicity. During the recovery period for rats, significant (p<0.05, Fisher exact test performed by EPA) increases in mortality were observed in rats exposed to 1000 ppm for 6 months and all groups of rats exposed to vinyl chloride for 10 months. Increased incidence of neoplastic nodules (mid- concentration group exposed for 6 or 10 months), hemangiosarcomas in the liver (mid- concentration group exposed for 10 months), fibroadenoma (low-concentration group exposed for 6 or 10 months), and bronchioloalveolar tumor and hemangiosarcomas in the lungs (mid- and high-concentration groups) were observed. During the recovery phase for mice, significant (p< 0.05, Fisher exact test performed by EPA) dose- and duration-related increases in mortality were observed. Increased mortality was observed in mice exposed to For internal use only. DRAFT - Do not cite or quote.. -49- 1000 ppm for 1 month, in the 250 and 1000 ppm groups exposed for 3 months, and all vinyl chloride exposed groups exposed for 6 months. Rough coat hair, lethargy, arid the appearance of external tumor masses were observed in animals dying early. Increased incidence of bronchioloalveolar tumors (mid- and high-concentration groups exposed for 1 month; all vinyl chloride exposed groups after 3 and 6 months of exposure), hemangiosarcoma in the liver (mid- and high-concentration groups after 3 and 6 months of exposure), and adenocarcinoma/carcinoma in mammary gland (low-, mid- and high- concentration females after 3 months of exposure and mid-and high-concentration females after 6 months of exposure) were observed. Groups of male Wistar rats were exposed by inhalation to nominal concentrations of 0, 10, 100, or 3000 ppm (0, 26, 256, 7669 mg/rn") vinyl chloride 6 hour/day, 6 day/week for 12 months (Bi et al., 1985). Equivalent oral doses using a TWA body weight of 0.200 kg, an inhalation rate of 0.2136 m3/day (calculated using the allometric equation in u.s. EPA, 1987), inhalation absorption rate of 0.42 (Krajewski et al., 1980) and oral absorption rate of 0.83 (Feron et al., 1981). The equivalent oral doses are 0, 3, 30, 888 mg/kg-day, respectively. Following 3, 6, and 9 months of exposure, 8, 30, 6, and 10 rats per dose group, respectively, were killed. The remaining animals were killed 6 months after exposure termination. Histopathological examination of the testes, lungs, liver, heart, kidneys, spleen, and brain were performed. During the study, the body weights of rats in the 100 and 3000 ppm groups were greater than 20% lower (p < 0.05) than in the control group. Transient increases in relative kidney, spleen, liver, and heart weights were observed; in general, the changes were not dose- or duration-related. After 6 months of exposure, a significant (p< 0.05) decrease in relative testes weight was observed in the 100 and 3000 ppm groups; this effect was not observed after 12 months of exposure. In the 100 and 3000 ppm groups, significant increases in the incidence of damage of the testicular seminiferous tubules were observed. Significant (p<0.05) increases in the incidence of tumors (predominantly in the liver and lung) were observed in the two highest-concentration groups. A significant increase in hepatic angiosarcomas was observed in the 3000 ppm group. Groups of 36 male and 36 female CD rats and CD-l mice were exposed by inhalation to 0, 50, 250, 1000 ppm (0, 128, 639, 2556 mg/m") vinyl chloride 6 hour/day, 5 day/week for 12 months (Lee et al., 1977, 1978). Equivalent oral doses can be estimated using TWA body weights of 0.580 kg (rat) and 0.032 kg (mouse), inhalation rates of 0.5116 (rat) and 0.0537 m3/day (mouse) (calculated using the allometric equation in U.S. EPA, 1987), inhalation absorption rate of 0.42 (Krajewski et al., 1980) and oral absorption rate of 0.83 (Feron et al., 1981). The equivalent oral doses for rats are 0, 10,51, and 204 mg/kg-day, respectively, and 0, 20, 98, 392 mg/kg-day, respectively, for mice. Four animals/species/sex/group were killed after 1, 2, 3, and 9 months of exposure. Clinical signs, biweekly body weight and weekly food consumption measurements, hematological (RBC, reticulocyte, platelet, and WBC counts, hemoglobin, hematocrit, methemoglobin, and Heinz bodies) and clinical chemistry [SGPT and BUN (both species), prothrombin time, SGOT, alkaline phosphatase, bilirubin, creatinine, LDH, immunoglobulin IgA, 19B-A, IgB- B, and IgM, total protein, albumin, globulin, and collagen contents of liver and lungs (rats only)] parameters, and gross and histopathological examination of major tissues and organs For internal use only. DRAFT - Do not cite or quote. -50- were used to assess toxicity. In rats, increased mortality was observed in the vinyl chloride exposed rats; 0, 2, 14, and 21 animals died or were killed in a moribund state between months 8 and 12 in the 0, 50, 250, and 1000 ppm groups, respectively. Rough hair coats, loss of muscle tone, lethargy, and weight loss were observed in the animals prior to death. A slight decrease in body weight gain was observed in the high-concentration group (statistical significance not reported). No persistent alterations in hematological or clinical chemistry parameters were observed. Hepatic and/or pulmonary hemangiosarcomas were observed in the rats exposed to 250 and 1000 ppm vinyl chloride. In mice, increased mortality was observed in the vinyl chloride treated animals after 6 months of exposure. The number of animals dying early or killed due to morbidity in the 0, 50, 250, and 1000 ppm groups are 2, 20, 38, 34, respectively. All mice in the 1000 ppm and females in the 250 ppm groups were killed at the end of the ninth month. During the first 8 months of exposure, no differences in body weight gain were observed; in the ninth month, decreases in body weight were observed in the high concentration group. No persistent changes in hematological or clinical chemistry parameters were observed. A number of tumors including bronchiolo-alveolar adenomas, hemangiosarcoma in the liver, and mammary tumors were observed in the vinyl chloride exposed rats. The statistical significance of the tumor incidence were not reported. INHALATION DEVELOPMENTAL TOXICITY John et al. (1981) exposed groups of pregnant Sprague-Dawley rats (17-33/group) and New Zealand white rabbits (7-18/group) by inhalation to 0, 500, or 2500 ppm (0, 1278, 6391 mg/rrr') vinyl chloride for 7 hour/day on gestational days 6-15 or 6-18, respectively. Groups of 29-37 CF-l mice were exposed 7 hour/day on days 6-15 of gestation to 0, 50 or 500 ppm (0, 128, 1278 rng/rn') vinyl chloride. The pregnant rats, mice, and rabbits were sacrificed on day 21, 18, or 29 of gestation, respectively. Equivalent oral doses can be estimated using initial body weights of 0.250, 0.025, and 3.5 kg for rats, mice, and rabbits, respectively, inhalation rates of 0.2565 (rat), 0.0414 (mouse), and 1.302 (rabbit) m3/day (calculated using the allometric equations in u.S. EPA, 1987), inhalation absorption rate of 0.42 (Krajewski et al., 1980) and oral absorption rate of 0.83 (Feron et al., 1981). The equivalent oral doses are 0, 194, 968 mg/kg-day; 0, 31, 312 mg/kg-day; and 0, 70, 351 mg/kg-day for rats, mice, and rabbits, respectively. Decreased body weight gain was observed in the rat dams exposed to 500 ppm, this effect was not observed in the high- concentration group. At 2500 ppm, decreased food intake and liver weight were observed; 1117 rats died. Decreased mean fetal body weight and increased crown-rump length was observed in the 500 ppm group but not the 2500 ppm group. Lumbar spurs in the vertebrae (500 ppm) and dilated ureter (2500 ppm) were observed. Increased incidence of maternal deaths and decreases in body weight gain, food consumption and absolute liver weight were observed in mice exposed to 500 ppm. The incidence of resorption was significantly increased in the high-concentration group, but, the incidence was within the range for historical controls. A decrease in litter size was also observed in the high-concentration group. A statistically significant (p < 0.05) increase in fetal crown-rump length (50 ppm group) and decrease in mean fetal body weight (500 ppm group) were observed. Skeletal anomalies (delayed ossification in skull and sternebrae and unfused sternebrae) were observed For internal use only. DRAFI - Do not cite or quote. -51- in the offspring of mice exposed to 500 ppm vinyl chloride. In the high-concentration group, 117rabbit does died; decreased food intake was observed in the low-concentration group. A significant increase in the incidence of resorption was observed in the low-concentration group. Delayed sternebrae ossification was observed in the offspring of rabbits exposed to 500 ppm vinyl chloride. CONCLUSION One subchronic oral toxicity study was located (Feron et al., 1975). In this study, a small but significant increase in relative liver weight (approximately 10%) and slight decreases in SGPT, SGOT, and urinary GOT levels were observed in rats receiving gavage doses of 300 mg/kg (257 mg/kg-day) for 13 weeks. The 300 mg/kg dose is considered a NOAEL. The Hong et al. (1981) study showed that significantly more mice exposed by inhalation to 50 ppm vinyl chloride 6 hour/day, 5 day/week for 6 months (equivalent oral dose of 19 mg/kg-day) died 6-12 months after exposure termination as compared to the control group. No significant increases in the incidence of neoplastic tumors were observed in this group of mice (Fisher exact test performed by EPA). Non-neoplastic effects were not reported, however, it is not known if the tissues were examined for neoplastic endpoints only. No deaths were reported during exposure. Similar effects were observed in rats. However, mice may be more sensitive than rats to the lethal effects of vinyl chloride. The Hong et al. (1981) data suggest that the Feron et al. (1975) study is not adequate for the derivation of a subchronic RID for vinyl chloride because subchronic exposure to 300 mg/kg may result in increased mortality several months after exposure termination. Furthermore, data from chronic exposure studies should not be used to derive a subchronic RID. Decreased lifespan and liver damage following chronic oral exposure to vinyl chloride (Til et al., 1983; Feron et al., 1981) have been observed at dose levels greater than 2 orders of magnitude lower than the NOAEL from the Feron et al. (1975) subchronic study. References Bi, W., Y. Wang, M. Huang and D. Meng. 1985. Effect of vinyl chloride on testis in rats. Ecotoxicol. Environ. Safety 10: 281-289. Feron, V.J., A.J. Speek, M.1. Willems, D. van Battum and A.P. de Groot. 1975. Observations on the oral administration and toxicity of vinyl chloride in rats. Food Cosmet. Toxieo!. 13: 633-638. Feron, V.J., C.F.M. Hendriksen, A.J. Speek, H.P. Til and B.J. Spit. 1981. Lifespan oral toxieity study of vinyl chloride in rats. Food Cosmet. Toxieol. 19: 317-333. For internal use only. DRAFT - Do not cite or quote. -52- Hong, C.B., J.M. Winston, L.P. Thornburg, C.C. Lee and J.S. Woods. 1981. Follow-up study on the carcinogenicity of vinyl chloride and vinylidene chloride in rats and mice: tumor incidence and mortality subsequent to exposure. J. Toxicol. Environ. Health. 7: 909- 924. John, J.A., F.A. Smith and B.A. Schwetz. 1981. Vinyl chloride: inhalation teratology study in mice, rats, and rabbits. Environ. Health Perspect. 41: 171-177. Knight, K.R and R Gibbons. 1987. Increased collagen synthesis and cross-link formation in the skin of rats exposed to vinyl chloride monomer. Clin. Sci. 72: 673-678. Krajewski, J., M. Dobecki and J. Gromiec. 1980. Retention of vinyl chloride in the human lung. Br. J. Industr. Med. 37: 373-374. Lee, C.C., J.C. Bhandari, J.M Winston, et al. 1977. Inhalation toxicity of vinyl chloride and vinylidene chloride. Environ. Health Perspect. 21: 25-32. Lee, C.C., J.C. Bhandari, J.M. Winston, W.B. House, RL. Dixon and J.S. Woods. 1978. Carcinogenicity of vinyl chloride and vinylidene chloride. J. Toxicol. Environ. Health 4: 15-30. Til, H.P., H.R. Immel and V.J. Feron. 1983. Lifespan oral carcinogenicity study of vinyl chloride in rats. Conducted by CIVO Institutes TNO. Submitted by Verband Kunststofferzeugende Industrie e.V. to U.S. EPA, Office of Toxic Substances. Fiche No. FYI-AX-1084-0353. U.S. EPA. 1980. Ambient Water Quality Criteria Document for Vinyl Chloride. Prepared by the Office of Health and Environmental Assessment, Environmental Criteria and Assessment Office, Cincinnati, OH for the Office of Water Regulations and Standards, Washington, DC EPA-440/5-80-078. NTIS PB81-117889. U.S. EPA. 1984. Health Effects Assessment for Vinyl Chloride. Prepared by the Office of Health and Environmental Assessment, Environmental Criteria and Assessment Office, Cincinnati, OH for the Office of Solid Waste and Emergency Response, Washington, D.C. EPA 540/1-86-036. U.S. EPA. 1985a. Health and Environmental Effects Profile for Chloroethene. Prepared by the Office of Health and Environmental Assessment, Environmental Criteria and Assessment Office, Cincinnati, OH, for the Office of Solid Waste and Emergency Response. Washington, D.C. ECAO-CIN-PI55. U.S. EPA. 1985b. Drinking Water Criteria Document for Vinyl Chloride. Prepared by the Office of Health and Environmental Assessment, Environmental Criteria and Assessment Office, Cincinnati, OH for the Office of Emergency and Remedial Response, Washington D.C. PB86-1l8320. For internal use only. DRAFT - Do not cite or quote. -53- " U.S. EPA. 1987. Health Advisory for Vinyl Chloride. Prepared by Office of Drinking Water, Washington D.C. For internal use only. DRAFT - Do not cite or quote. -54- Summary Table of the Toxicity Data for Vinyl Chloride Study Effect UF/HF RfD (mg/kg/day) Species Reported dose or concentration Dose (mg/kg/day) Subchronic oral toxicity Feron et al., 1975 rat Chronic oral toxicity Til et al., 1983 rat decreased lifespan and liver damage at 1.3 mg/kg/day Feron et al., 1981 rat decreased lifespan and liver damage at ~1.7 mg/kg/day Knight and Gibbons, 1987 rat decreased lifespan and increased collagen in the skin at 30 mg/kg/day Subchronic inhalation toxicity studies Bi et al., 1985 testicular damage rat Hong et al., 1981 increased mortality; increased incidence of lung tunors rat Lee et al., 1977, 1978; increased mortality rat o (mg/kg, 6 d/wk) 30 100 300 0 (ppm) 10 100 L 3000 0 (ppm) 50 F 250 1000 0 (ppm) 50 250 F 1000 o 26 86 257 0 0.014 0.13 1.3 0 1.7 5.0 14.1 214· 0 3 30 300 Ob 3 30 L 888 oc 10 51 F 204 For internal use only. DRAFT - Do not cite or quote. -55- Study Species Effect Reported dose or Dose UF/MF RfD concentration (mg/kg/day) (mg/kg/day) Lee et al., 1977, 1978; mouse increased mortality D (ppm) Od 50 F 20 F 250 98 1000 392 Inhalation developmental toxicity John et al., 1981 rat developmental toxicity o (ppn) O· 500 L 194 L 2500 968 John et al., 1981 mouse developmental toxicity and maternal o (ppm) 0' toxicity 50 31 500 L 312 L John et al., 1981 rabbit developmental toxicity o (ppm) 00 500 70 2500 351 • gavage dose of 300 mg/kg 5 day/week b mg/kg/day doses were calculated by multiplying the duration expanded concentration (mg/m3) by the inhalation rats of 0.2136 m3/day (using allometric equation, u.S. EPA, 1987) and dividing by a TWA body weight of 0.200 kg. This mg/kg/day dose was multiplied by the ratio of the inhalation absorption rate of 0.42 (Krajewski et al., 1980) and oral absorption rate of 0.83 (Feron et al., 1981). C mg/kg/day doses calculated same as in b, inhalation rate of 0.5116 m3/day and body weight of 0.580 kg d mg/kg/day doses calculated same as in b, inhalation rate of 0.0537 m3/day and body weight of 0.032 kg • mg/kg/day doses calculated same as in b, inhalation rate of 0.2565 m3/day and initial body weight of 0.250 kg , mg/kg/day doses calculated same as in b, inhalation rate of 0.0414 m3/day and initial body weight of 0.025 kg o mg/kg/day doses calculated same as in b, inhalation rate of 1.303 m3/day and initial body weight of 3.5 kg For internal use only. DRAFT - Do not cite or quote. -56- •