INTERIM 1: 03/2008 United States Environmental Protection Agency Office of Pollution Prevention and Toxics VINYL CHLORIDE (CAS Reg. No. 75-01-4) INTERIM ACUTE EXPOSURE GUIDELINE LEVELS (AEGLs) ------- INTERIM 1: 3/2008 VINYL CHLORIDE (CAS Reg. No. 75-01-4) H H H Cl PROPOSED ACUTE EXPOSURE GUIDELINE LEVELS (AEGLs) ------- Vinyl chloride INTERIM 1: 3/2008 1 PREFACE 2 Under the authority of the Federal Advisory Committee Act (FACA) P. L. 92-463 of 1972, the 3 National Advisory Committee for Acute Exposure Guideline Levels for Hazardous Substances 4 (NAC/AEGL Committee) has been established to identify, review and interpret relevant toxicologic and 5 other scientific data and develop AEGLs for high priority, acutely toxic chemicals. 6 AEGLs represent threshold exposure limits for the general public and are applicable to 7 emergency exposure periods ranging from 10 minutes to 8 hours. AEGL-2 and AEGL-3 levels, and 8 AEGL-1 levels as appropriate, will be developed for each of five exposure periods (10 and 30 minutes, 1 9 hour, 4 hours, and 8 hours) and will be distinguished by varying degrees of severity of toxic effects. It is 10 believed that the recommended exposure levels are applicable to the general population including infants 11 and children, and other individuals who may be sensitive or susceptible. The three AEGLs have been 12 defined as follows: 13 AEGL-1 is the airborne concentration (expressed as ppm or mg/m3) of a substance above which it 14 is predicted that the general population, including susceptible individuals, could experience notable 15 discomfort, irritation, or certain asymptomatic, non-sensory effects. However, the effects are not disabling 16 and are transient and reversible upon cessation of exposure. 17 AEGL-2 is the airborne concentration (expressed as ppm or mg/m3) of a substance above which it 18 is predicted that the general population, including susceptible individuals, could experience irreversible or 19 other serious, long-lasting adverse health effects, or an impaired ability to escape. 20 AEGL-3 is the airborne concentration (expressed as ppm or mg/m3) of a substance above which it 21 is predicted that the general population, including susceptible individuals, could experience 22 life-threatening health effects or death. 23 Airborne concentrations below the AEGL-1 represent exposure levels that could produce mild 24 and progressively increasing odor, taste, and sensory irritation, or certain asymptomatic, non-sensory 25 effects. With increasing airborne concentrations above each AEGL level, there is a progressive increase in 26 the likelihood of occurrence and the severity of effects described for each corresponding AEGL level. 27 Although the AEGL values represent threshold levels for the general public, including sensitive 28 subpopulations, it is recognized that certain individuals, subject to unique or idiosyncratic responses, 29 could experience the effects described at concentrations below the corresponding AEGL level. in ------- Vinyl chloride INTERIM 1: 3/2008 1 TABLE OF CONTENTS 2 PREFACE iii 3 EXECUTIVE SUMMARY vii 4 1. INTRODUCTION 1 5 2. HUMAN TOXICITY DATA 2 6 2.1. Acute Lethality 2 7 2.2. Nonlethal Toxicity 3 8 2.2.1. Neurotoxicity 3 9 2.2.2. Odor 4 10 2.2.3. Irritation 5 11 2.2.4. Cardiovascular effects 6 12 2.2.5. Other Endpoints 6 13 2.3. Developmental / Reproductive Toxicity 7 14 2.4. Genotoxicity 8 15 2.5. Carcinogenicity 8 16 2.6. Summary 9 17 3. ANIMAL TOXICITY DATA 10 18 3.1. Acute Lethality 10 19 3.1.1. Rats 10 20 3.1.2. Mice 11 21 3.1.3. Guinea Pigs 12 22 3.1.4. Rabbits 12 23 3.1.5. Other Species 12 24 3.2. Nonlethal Toxicity 14 25 3.2.1. Dogs 14 26 3.2.2. Rats 14 27 3.2.3. Mice 16 28 3.2.4. Guinea Pigs 17 29 3.2.5. Rabbits 18 30 3.3. Developmental/Reproductive Toxicity 20 31 3.4. Genotoxicity 22 32 3.5. Carcinogenicity 24 33 3.6. Summary 28 34 4. SPECIAL CONSIDERATIONS 30 35 4.1. Metabolism and Disposition 30 36 4.2. Mechanism of Toxicity 32 37 IV ------- Vinyl chloride INTERIM 1: 3/2008 1 4.3. Other Relevant Information 33 2 4.3.1 PBPK-Modeling 33 3 4.3.2. Interspecies Variability 33 4 4.3.3. Intraspecies Variability 34 5 4.3.4. Concurrent Exposure Issues 35 6 5. RATIONALE AND PROPOSED AEGL-1 35 7 5.1. Human Data Relevant to AEGL-1 35 8 5.2. Animal Data Relevant to AEGL-1 35 9 5.3. Derivation of AEGL-1 36 10 6. RATIONALE AND PROPOSED AEGL-2 37 11 6.1. Human Data Relevantto AEGL-2 37 12 6.2. Animal Data Relevant to AEGL-2 37 13 6.3. Derivation of AEGL-2 38 14 7. RATIONALE AND PROPOSED AEGL-3 39 15 7.1. Human Data Relevant to AEGL-3 39 16 7.2. Animal Data Relevant to AEGL-3 39 17 7.3. Derivation of AEGL-3 40 18 8. SUMMARY OF PROPOSED AEGLs 41 19 8.1. AEGL Values and Toxicity Endpoints 41 20 8.2. Comparison with Other Standards and Criteria 42 21 8.3. Data Adequacy and Research Needs 44 22 9. REFERENCES 45 23 APPENDIX A - Derivation of AEGL values 55 24 AEGL-1 56 25 AEGL-2 57 26 AEGL-3 58 27 APPENDIX B - Time Scaling Calculations for Vinyl Chloride AEGLs 59 28 APPENDIX C - Cancer Assessment of Vinyl Chloride 67 29 APPENDIX D - Occupational epidemiological studies on carcinogenicity (focus: limited exposure 30 time) 79 31 APPENDIX E - Derivation Summary for Vinyl Chloride AEGLs 84 ------- Vinyl chloride INTERIM 1: 3/2008 1 LIST OF TABLES 2 TABLE 1: CHEMICAL AND PHYSICAL DATA 2 3 TABLE 2: SUMMARY OF ACUTE EFFECTS IN HUMANS AFTER INHALATION OF 4 VINYL CHLORIDE 7 5 TABLE 3: SUMMARY OF ACUTE LETHAL INHALATION DATA IN LABORATORY ANIMALS! 3 6 TABLE 5: QUANTITATIVE ASSESSMENT OF CARCINOGENIC POTENCY OF VC BASED ON 7 ANIMAL EXPERIMENTS 27 8 TABLE 6: METABOLIC SATURATION CONCENTRATIONS OF VC IN RATS AND 9 MONKEYS 32 10 TABLE 7: AEGL-1 VALUES FOR VINYL CHLORIDE 36 11 TABLE 8: AEGL-2 VALUES FOR VINYL CHLORIDE 39 12 TABLE 9: AEGL-3 VALUES FOR VINYL CHLORIDE 41 13 TABLE 10: SUMMARY/RELATIONSHIP OF PROPOSED AEGL VALUES 41 14 TABLE 11: EXISTENT STANDARDS AND GUIDELINES FOR VINYL CHLORIDE 43 15 LIST OF FIGURES 16 FIGURE 1: CATEGORICAL REPRESENTATION OF VINYL CHLORIDE INHALATION 17 DATA 42 18 FIGURE 2: REGRESSION ANALYSIS OF THE LOG-LOG TRANSFORMED CONCENTRATION- 19 TIME CURVE REGARDING UNCONSCIOUSNESS IN MICE AND GUINEA-PIGS 62 20 FIGURE 3: REGRESSION ANALYSIS OF THE LOG-LOG TRANSFORMED CONCENTRATION- 21 TIME CURVE REGARDING MUSCULAR INCOORDINATION IN MICE AND GUINEA- 22 PIGS 64 23 FIGURE 4: REGRESSION ANALYSIS OF THE LOG-LOG TRANSFORMED CONCENTRATION- 24 TIME CURVE REGARDING SIDE POSITION IN MICE AND GUINEA-PIGS 66 25 FIGURE 5: EXTERNAL CONCENTRATION (mg/m3) AND DOSE TO LIVER (mg/L) AS 26 CALCULATED BY PBPK-MODELING BY EPA 71 VI ------- Vinyl chloride INTERIM 1: 3/2008 1 EXECUTIVE SUMMARY 2 Vinyl chloride (VC) is a colorless, flammable gas with a slightly sweet odor. It is heavier than air 3 and accumulates at the bottom of rooms, tanks etc. Its worldwide production is approximately 27,000,000 4 tons. Most is polymerized to PVC. Combustion of VC in air produces carbon dioxide and hydrogen 5 chloride. Odor thresholds of VC were reported in the range of 10 to 25,000 ppm. Validated studies 6 providing a quantitative odor recognition and detection limit are not available. Therefore, a Level of 7 Odor Awareness (LOA) can not be derived. 8 Vinyl chloride is an anaesthetic compound. After 5 minute exposure to 16,000 ppm VC, 9 volunteers showed dizziness, lightheadedness, nausea, visual and auditory dulling (Lester et al., 1963). 10 Mild headache and some dryness of the eyes and nose were the only complaints of volunteers exposed to 11 491 ppm VC for several hours (Baretta et al., 1969). No data on developmental or reproductive toxicity of 12 VC in humans after acute exposure are available. Occurrence of chromosomal aberrations in lymphocytes 13 of humans were associated with accidental exposure to VC. After chronic occupational exposure, VC is a 14 known human carcinogen inducing liver angiosarcoma, possibly hepatocellular carcinoma and brain 15 tumors. Evidence for tumors at other locations is contradictory. Two recent epidemiological studies 16 (Mundt et al., 2000; Ward et al., 2001) did not find an increased Standard Mortality Ratio after 5 years of 17 occupational exposure to VC, whereas one other study suggested such an increase after 1 year of 18 exposure (Boffetta et al., 2003). 19 Acute exposure of experimental animals to VC results in narcotic effects (Mastromatteo et al., 20 1960), cardiac sensitization (Clark and Tinston, 1973; 1982), and hepatotoxicity (Jaeger et al., 1974). 21 Prodan et al. (1975) reported LC50 values for mice, rats, rabbits, and guinea pigs of 117,500 ppm, 150,000 22 ppm, 240,000 ppm and 240,000 ppm, respectively, after 2 hours. No investigations of reproductive or 23 developmental toxicity after single exposure are available. After repeated exposure developmental 24 toxicity in mice, rats and rabbits (e.g. delayed ossification) was only observed at maternally toxic 25 concentrations. Embryo-fetal development of rats was not affected by 2-week- exposure (6h/d) up to 26 1,100 ppm (Thornton et al., 2002). Positive results on genotoxicity after in vitro and single and repeated 27 in vivo treatment have been reported for VC. Elevated etheno-adducts were observed after single and 28 short term exposure and associated with mutational events (Swenberg et al., 2000; Barbin, 2000). Higher 29 adduct levels were seen in young animals than in adult animals after identical treatment (Fedtke et al., 30 1990; Laib et al., 1989; Ciroussel et al.,1990, Morinello et al., 2002). From a study with single exposure 31 of adult rats to 45 ppm for 6 hours, it may be concluded that no increase of relevant etheno-adducts above 32 background occurred (Watson et al., 1991). 33 Induction of liver tumors has been reported in rats after short term (5 week and 33 days, 34 respectively) exposure (Maltoni et al., 1981; 1984; Froment et al., 1994). Vinyl chloride induces lung 35 tumors in mice after single exposure to high concentrations of VC (Hehir et al., 1981). Short term 36 exposure experiments from Drew et al. (1983), Maltoni et al. (1981) and Froment et al. (1994) indicated 37 increased susceptibility of tumor formation in newborn and young animals. 38 The AEGL-1 was based on the study of Baretta et al. (1969) with 4-7 volunteers, two individuals 39 experienced mild headache during 3.5 and during 7.5 hours (3.5 hours, 0.5 hours break, 3.5 hours) of 40 exposure to 491 ppm. The time of onset of headaches is not clearly stated and was assumed to be after 3.5 41 hours. A total uncertainty factor of 3 was used. Since the AEGL-1 is based on human data no interspecies 42 extrapolation was used. The intraspecies uncertainty factor of 3 is used to account for both toxicokinetic vii ------- Vinyl chloride INTERIM 1:3/2008 1 and toxicodynamic differences among individuals. The other exposure duration-specific values were 2 derived by time scaling according to the dose-response regression equation Cn x t = k, using the default of 3 n=3 for shorter exposure periods and n=l for longer exposure periods, due to the lack of suitable 4 experimental data for deriving the value of n. The extrapolation to 10 minutes from a 3.5 hour exposure 5 is justified because exposure of human at 4,000 ppm for 5 minutes did not result in headache (Lester et 6 al., 1963). 7 The AEGL-2 was based on prenarcotic effects observed in human volunteers. After 5 minute 8 exposure to 16,000 ppm VC, 5 of 6 persons showed dizziness, lightheadedness, nausea, and visual and 9 auditory dulling. At concentrations of 12,000 ppm one of six persons showed dizziness and "swimming 10 head, reeling". No effects were observed at 4,000 ppm in this study. A single person reported slight 11 effects ("slightly heady") of questionable meaning at 8,000 ppm (this volunteer felt also slightly heady at 12 sham exposure and reported no response at 12,000 ppm) (Lester et al., 1963). 12,000 ppm was regarded 13 as the no effect for impaired ability to escape. A total uncertainty factor of 3 is used to account for 14 toxicodynamic differences among individuals. As the unmetabolized VC is responsible for the effect, no 15 relevant differences in toxicokinetics are assumed. In analogy to other anesthetics the effects are assumed 16 to be solely concentration dependent. Thus, after reaching steady state at about 2 hours of exposure, no 17 increase in effect is expected. The other exposure duration-specific values were derived by time scaling 18 according to the dose-response regression equation Cn x t = k, using an n of 2, based on data from 19 Mastromatteo et al. (1960). Mastromatteo et al. observed various time-dependent prenarcotic effects in 20 mice and guinea pigs after less than steady state exposure conditions. Time extrapolation was performed 21 from 5 to 10, 30, 60 minutes and 2 hours, where the steady state concentration was calculated. 22 The AEGL-3 was based on cardiac sensitization and the no effect level for lethality. Short term 23 exposure (5 min) of dogs to VC induced cardiac sensitization towards epinephrine (EC50: 50,000 or 24 71,000 ppm in two independent experiments) (Clark and Tinston, 1973; Clark and Tinston, 1982). Severe 25 cardiac sensitization is a life threatening effect, but at 50,000 ppm no animals died. A total uncertainty 26 factor of 3 is used to account for toxicodynamic differences among individuals. As the challenge with 27 epinephrine and the doses of epinephrine used represent a conservative scenario, no interspecies 28 uncertainty factor was used. As the unmetabolized VC is responsible for the effect, no relevant 29 differences in toxicokinetics are assumed. In analogy to other halocarbons (e.g., Halon 1211, HFC 134a) 30 which lead to cardiac sensitization the effects are assumed to be solely concentration dependent. Thus, 31 after reaching steady state at about 2 hours of exposure, no increase in effect is expected. The other 32 exposure duration-specific values were derived by time scaling according to the dose-response regression 33 equation Cn x t = k, using an n of 2, based on data from Mastromatteo et al. (1960). Mastromatteo et al. 34 observed various time-dependent prenarcotic effects (muscular incoordination, side position and 35 unconsciousness, effects which occur immediately before lethality) in mice and guinea pigs after less than 36 steady state exposure conditions. Time extrapolation was performed from 5 to 10, 30, 60 minutes and 2 37 hours, where the steady state concentration was calculated. 38 The calculated values are listed in the table below. SUMMARY TABLE OF PROPOSED AEGL VALUES FOR VINYL CHLORIDE Classificati on 10-minute 30-minute 1-hour 4-hour 8-hour Endpoint (Reference) 39 40 41 Vlll ------- Vinyl chloride INTERIM 1:3/2008 AEGL-1 (Non- disabling) AEGL-2* (Disabling) AEGL-3 (Lethal) 450 ppm 1200 mg/m3 2800 ppm 7300 mg/m3 12000 ppm* 3 1000 mg/m3 3 10 ppm 800 mg/m3 1600 ppm 4 100 mg/m3 6800 ppm* 18000 mg/m3 250 ppm 650 mg/m3 1200 ppm 3 100 mg/m3 4800 ppm* 12000 mg/m3 140 ppm 360 mg/m3 820 ppm 2100 mg/m3 3400 ppm 8800 mg/m3 70 ppm 180 mg/m3 820 ppm 2100 mg/m3 3400 ppm 8800 mg/m3 mild headaches in 2/7 humans (Baretta etal.,1969) mild dizziness in 1/6 humans (Lester etal.,1963);no effect level for impaired ability to escape cardiac sensitization (Clark and Tinston, 1982; 1973); no effect level for lethality 9 10 11 12 13 14 15 16 17 18 19 20 21 22 * The explosion limits for VC in air range from 38,000 to 293,000 ppm. The AEGL-3 values at 10 minutes, 30 minutes, and 1 hour exceed 10% of the lower explosion limit (LEL). Therefore, safety considerations against the hazard of explosion must be taken into account. # Derived AEGL-2 values do not protect for potential mutations or malignancies due to short term exposure to VC. The estimation of cancer risk was based on the study of Maltoni et al. (1981). Newborn rats were exposed from day 1 to 5 weeks of age at 6,000 or 10,000 ppm VC by inhalation (4 hr/day, 5 d/week). Liver angiosarcomas were found in 17 of 42 newborn rats exposed to 6,000 ppm and 15 of 44 newborn rats exposure to 10,000 ppm. No angiosarcomas were found in the dams exposed identically. A 6,000 ppm exposure in rats for 4 h/day, 5 d/week, for 5 weeks was found to be equivalent to a continuous human exposure of 51 ppm using a PBPK model. From this, a 1 in 10,000 risk was calculated to be at 33 (ig/m3 and 24 hour exposure was 34.7 mg/m3 (13.2 ppm). Further exposure duration calculations were done using the PBPK model for VC and are shown in the following table and Appendix C. It must be emphasized that there are substantial uncertainties in calculating cancer risk from a single exposure. IX ------- Vinyl chloride INTERIM 1:3/2008 Estimation of carcinogenic potency (10 4 risk) after single exposure Maltoni et al, 1981; from 5-weeks- study; Human equivalent dose to 6000 ppm 30-minute 1200 ppm (3100mg/m3) 1-hour 350 ppm (910mg/m3) 4-hour 81 ppm (210mg/m3) 8-hour 40 ppm (100mg/m3) 9 10 11 12 13 14 15 16 17 18 19 20 21 22 The values corresponding to 10"5 and 10"6 risk are in Appendix C. The risk for 10 minutes has not been calculated due to extreme uncertainty. The occurrence of DNA-adducts and tumorigenicity after single exposure at or below AEGL- concentrations may not be excluded. No increase of relevant etheno-adducts above background is expected at single exposure to 3.4 ppm for 8 hours. This includes extrapolation for sensitive subgroups like newborns by the use of an uncertainty factor of 10 (for details, see calculation D; Appendix C). References Barbin, A. 2000. Etheno-adduct forming chemicals: from mutagenicity testing to tumour mutation spectra. Mut. Res. 462:55-69 Baretta, E.D., R.D. Stewart, and J.E. Mutchler. 1969. Monitoring exposures to vinyl chloride vapor: breath analysis and continuous air sampling. Am. Ind. Hyg. Assoc. J. 30:537-544. Belej, M.A., D.G. Smith, and D.M. Aviado. 1974. Toxicity of aerosol propellants in the respiratory and circulatory systems. IV. Cardiotoxicity in the monkey. Toxicology 2:381-395. Boffetta, P., L. Matisane, K.A. Mundt, and L.D. Dell. 2003. Meta-analysis of studies of occupational exposure to vinyl chloride in relation to cancer mortality. Scand. J. Work Environ. Health. 29:220-229. Ciroussel, F., A. Barbin, G. Eberle, aqnd H. Bartsch. 1990. Investigations on the relationship between DNA ethenobase adduct levels in several organs of vinyl chloride-exposed rats and cancer susceptibility. Biochem. Pharm. 39:1109-1113. 23 Clark, D.G., and D.J. Tinston. 1973. Correlation of the cardiac sensitizing potential of halogenated 24 hydrocarbons with their physicochemical properties. Br. J. Pharm. 49:355-357. 25 Clark, D.G., and D.J. Tinston. 1982. Acute inhalation toxicity of some halogenated and non-halogenated 26 hydrocarbons. Hum. Toxicol. 1:239-247. ------- Vinyl chloride INTERIM 1: 3/2008 1 Drew, R.T., G.A. Boorman, J.K. Haseman, E.E. McConnell, W.M. Busey, and J.A. Moore. 1983. The 2 effect of age and exposure duration on cancer induction by a known carcinogen in rats, mice, and 3 hamsters. Toxicol. Appl. Pharmacol. 68:120-130. 4 Fedtke, N., J.A. Boucheron, V.E. Walker, and J.A. Swenberg. 1990. Vinyl chloride-induced DNA 5 adducts. II. Formation and persistence of 7-(2'oxoethyl)guanine and N2-ethenoguanine in rat tissue DNA. 6 Carcinogenesis 11:1287-1292. 7 Froment, O., S. Boivin, A. Barbin, B. Bancel, C. Trepo, and M.J. Marion. 1994. Mutagenesis of ras proto- 8 oncogens in rat liver tumors induced by vinyl chloride. Cane. Res. 54:5340-5345. 9 Hehir, R.M., B.P. McNamara, J. McLaughlin, D.A. Willigan, G. Bierbower and J.F. Hardisty. 1981. 55 5 O ' O ' J 10 Cancer induction following single and multiple exposure to a constant amount of vinyl chloride 11 monomer. Environ. Health Perspect. 41:63-72 12 Jaeger, R.J., E.S. Reynolds, RB. Conolly, M.T. Moslen, S. Szabo, and S.D. Murphy. 1974. Acute hepatic 13 injury by vinyl chloride in rats pretreated with phenobarbital. Nature 252:724-726. 14 Laib, R.J., K.P. Klein, and H.M. Bolt. 1985b. The rat liver foci bioassay: I. Age-dependence of induction 15 by vinyl chloride of ATPase-deficient foci. Carcinogenesis 6:65-68. 16 Lester, D., L.A. Greenberg, and W.R. Adams. 1963. Effects of single and repeated exposures of humans 17 and rats to vinyl chloride. Am. Ind. Hyg. Assoc. J. 24:265-275. 18 Maltoni, C., G. Lefemine, A. Ciliberti, G. Cotti, and D. Carretti. 1981. Carcinogenicity bioassays of 19 vinylchloride monomer: A model of risk assessment on an experimental basis. Environ. Health Perspect. 20 41:3-31. 21 Maltoni,C.,G. Lefemine,A. Ciliberrti,G. Cotti, and D. Carretti. 1984. Experimental Research on Vinyl 22 Chloride Carcinogenesis. Vol. II: Archives of Research on Industrial Carcinogenesis. Princeton Scientific 23 Publishers Inc., New Jersey. 24 Mastromatteo, E., A.M. Fisher, H. Christie, and H. Danziger. 1960. Acute inhalation toxicity of vinyl 25 chloride to laboratory animals. Am. Ind. Hyg. Assoc. J. 21:394-398. 26 Morinello, E.J., A.-J.L. Ham, A. Ranasinghe, J. Nakamura, P.B. Upton, and J.A. Swenberg. 2002. 27 Molecular dosimetry and repair of N(2),3-ethenoguanine in rats exposed to vinyl chloride. Cane. Res. 28 62:5189-5195. 29 Prodan, L., I. Suciu, V. Pislaru, E. Ilea, and L. Pascu. 1975. Experimental acute toxicity of vinyl chloride 30 (monochloroethene). Ann. NY Acad. Sci. 246:154-158. XI ------- Vinyl chloride INTERIM 1: 3/2008 1 Swenberg, J.A., A. Ham, H. Koc, E. Morinello, A. Ranasinghe, N. Tretyakova, P.B. Upton, and K. Wu. 2 2000. DNA adducts: effects of low exposure to ethylene oxide, vinyl chloride and butadiene. Mut. Res. 3 464:77-86. 4 Thornton, S.R, RE. Schroeder, RL. Robison, D.E. Rodwell, D.A. Penney, K.D. Nitschke, and W.K. 5 Sherman. 2002. Embryo-fetal development and reproducitve toxicology of vinyl chloride in rats. Toxicol. 6 Sci. 68:207-219. 7 Watson, W.P., D. Potter, D. Blair, and A.S. Wright. 1991. The relationship between alkylation of 8 haemoglobin and DNA in Fischer 344 rats exposed to [1,2-14C] vinyl chloride. In: Garner, R.C., Farmer, 9 P.B., Steele, G.T., Wright, A. S.: Human Carcinogen Exposure. Biomonitoring and Risk Assessment, 10 Oxford University Press, London, 421-428. xn ------- Vinyl chloride INTERIM 1: 3/2008 1 1. INTRODUCTION 2 3 Vinyl chloride (VC) is a colorless, flammable gas with a slightly sweet odor. It is heavier than air 4 and accumulates at the bottom of rooms, tanks etc. Its worldwide production is approximately 27,000,000 5 tons. Most VC is polymerized to PVC, which subsequently is used to produce packaging materials, 6 building materials, electric appliances, medical care equipment, toys, agricultural piping and tubing and 7 automobile parts. Currently the largest single use is in the building sector (WHO, 1999a). About 10,000 8 tons annually go into the production of 1,1,1-trichloroethane and other chlorinated solvents (Kielhorn et 9 al., 2000). 10 Most VC is produced either by hydrochlorination of acetylene, mainly in Eastern European 11 countries, or by thermal cracking of 1,2-dichloroethane. It is stored either under pressure at ambient 12 temperature, or refrigerated at atmospheric pressure (WHO, 1999a). Since VC does not polymerize 13 readily it is stored without additives. Combustion of VC in air produces carbon dioxide and hydrogen 14 chloride (WHO, 1999a). 15 16 Relevant chemical and physical properties are listed in Table 1. ------- Vinyl chloride INTERIM 1:3/2008 TABLE 1: CHEMICAL AND PHYSICAL DATA Parameter Molecular formula Molecular weight CAS Registry Number Physical state Color Synonyms Vapor pressure Density Melting point Boiling point Solubility in water Odor Explosion limits in air Conversion factors Value C,H,C1 62.5 g/mol 75-01-4 gaseous (at room temperature) colorless vinyl chloride monomer, monochlorethene, monochlorethylene, 1-chloroethylene, chlorethylene, chloroethene 78kPaat-20°C 165 kPa at 0 °C 333kPaat20°C 0.910g/cm3at20°C -153. 8 °C -13.4°C soluble in almost all organic solvents, slightly soluble in water slightly sweet 3.8 - 29.3 vol% in air at 20 °C 4 - 22 vol% 1 ppm = 2.59 mg/m3 at 20 °C, 101.3 kPa 1 mg/m3 =0.386 ppm Reference WHO, 1999a WHO, 1999a WHO, 1999a WHO, 1999a WHO, 1999a WHO, 1999a WHO, 1999a WHO, 1999a WHO, 1999a WHO, 1999a WHO, 1999a WHO, 1999a WHO, 1999a WHO, 1999a 10 11 12 13 14 15 16 17 2. HUMAN TOXICITY DATA 18 2.1. Acute Lethality 19 Danziger (1960) describes two deaths due to accidental exposure of workers to VC. No 20 concentration or exposure time is given, but circumstances suggest inhalation of very high concentrations. 21 Autopsy results show cyanosis, congestion of lung and kidneys and failure of blood coagulation 22 (Danziger, 1960). Citing older results from Schaumann et al., 12% VC (120,000 ppm) is given as 23 "dangerous concentrations" (Danziger, 1960; Oster et al., 1947). 24 At very high concentrations, VC causes asphyxia likely due to narcosis-induced respiratory 25 failure (NLM, 2000). ------- Vinyl chloride INTERIM 1: 3/2008 1 2.2. Nonlethal Toxicity 2 Only few data on acute human toxicity of VC after acute exposure are available. Whereas a large 3 experience on the long term effects of VC exposure at the workplace exists. Relevant data are described 4 below. 5 2.2.1. Neurotoxicity 6 Vinyl chloride has been considered as a potential anaesthetic. Narcotic limit concentration for 7 man is 7% - 10% (70,000 - 100,000 ppm) (Oster et al., 1947, Danziger, 1960, Lehmann and Flury, 1938). 8 Schauman (1934) reported somewhat higher concentrations to lead to narcosis. Exposure to unknown 9 high concentrations (e.g., during the cleaning of autoclaves) also resulted in narcotic effects (Suciu, 10 1975). 11 Acute exposure 12 Lester et al. (1963) exposed 6 volunteers - 3 men, 3 women - to 0, 0.4, 0.8, 1.2, 1.6 or 2% VC (0, 13 4,000, 8,000, 12,000, 16,000, or 20,000 ppm, nominal concentration) for 5 minutes using a plastic 14 breathing mask covering the mouth and nose. The total gas flow was 50 liters per minute. The desired 15 concentrations were obtained by metering air and VC (gas chromatography of the liquid phase indicated 16 more than 99% VC) through flow meters and passing the appropriate flows through a 2 1 mixing chamber. 17 The concentration was continuously monitored by a thermal conductivity meter (less than 5% deviation 18 from the desired concentration). All volunteers were exposed to every concentration in a randomized 19 fashion, separated by a 6-hour interval. Dizziness ("slightly heady") was experienced by 1 of 6 volunteers 20 at 8,000 ppm (the same subject reported slight dizziness at sham exposure and reported no response at 21 12,000 ppm). At 12,000 ppm 4/6 persons reported no response, one subject reported reeling, swimming 22 head and another subject was unsure of some effects. He had a somewhat dizzy feeling in the middle of 23 exposure. At 16,000 ppm 5 of 6 and at 20,000 ppm 6 of 6 persons complained of dizziness, nausea, 24 headache, and dulling of visual and auditory cues. All symptoms disappeared shortly after termination of 25 exposure; headache persisted for 30 minutes in one subject after exposure to 20,000 ppm 26 Two experimenters were exposed to 25,000 ppm (nominal concentration) for 3 minutes by 27 entering an exposure chamber which resulted in dizziness and slight disorientation as to space and size of 28 surrounding objects and a burning sensation in the feet. They immediately recovered on leaving the 29 chamber and complained only of a slight headache which persisted for 30 minutes. No further details 30 were presented (Patty et al., 1930). 31 Baretta et al. (1969) exposed 4-6 volunteers to 59, 261, 491 ppm VC (analytical concentrations) 32 for 7.5 h (including a 0.5 h lunch period; corresponding to time weighted average concentrations of 48, 33 248 or 459 ppm over a period of 7.5 h), seven persons were exposed to 491 ppm for only 3.5 hours. 34 Persons were exposed in an exposure chamber (41 feet by 6 feet wide by 7.5 feet high) with a continuous 35 positive air supply and exhaust system. Air was recirculated with a squirrel cage fan through a series of 36 inlet and outlet ducts spanning the length of the chamber. VC concentration was monitored by an infrared 37 spectrophotometer. The vapors were introduced from a pressurized storage cylinder through 6 feet of 1/8 38 inch I.D. stainless-steel tubing into a rotometer prior to entering the circulating air duct. A heating tape 39 wrapped around the stainless-steel tubing prevented condensation of the VC. Subjective and neurological 40 responses of the volunteers as well as clinical parameters were measured. The only complaints were those 41 of two subjects who reported mild headache and some dryness of their eyes and nose after exposure to the ------- Vinyl chloride INTERIM 1: 3/2008 1 highest concentration. The time of onset of headaches is not clearly stated. It is assumed that headaches 2 occurred in both experiments, after 3.5 hours and during or after 7.5 hours. 3 According to a literature review from Schottek (1969), acute human exposure to 1000 ppm for 1 4 hour leads to fatigue and vision disturbances (Lefaux, 1966). 5000 ppm for 60 minutes should lead to 5 nausea and disorientation (Oettel, 1954), with similar effects after 6000 ppm for 30 minutes (Patty et al., 6 1930). 6000 to 8000 ppm are said to lead to prenarcotic symptoms (von Oettingen, 1964). Examination of 7 the primary literature sources did not show how those figures were derived. No experimental background 8 or observation data are provided.Thus, the referred results may not be used for risk assessment. 9 Occupational exposure 10 Suciu et al. (1975) report acute effects after VC exposure from 1684 workers from two factories. 11 During periods with high air concentrations of VC between the years 1963 and 1964, acute and subacute 12 poisonings occurred: After the first breaths of exposure to "a high concentration of VC" several 13 symptoms (pleasant taste in the mouth, euphoric conditions, slow movements, giddiness, inebriety-like 14 condition) were observed. Continued exposure caused more pronounced symptoms (somnolence, 15 complete narcosis). After repeated exposures to unknown high concentrations, workers complained about 16 headaches, irritability, diminution of memory, insomnia, general asthenia, paresthesia, tingling, and loss 17 of weight. In addition to an "onset of an asthenovegetative syndrome" various other systemic and local 18 effects were observed (e.g., cardiovascular effects, hepatomegaly, digestive responses, respiratory 19 changes). Workplace concentrations in this factory were 2300 mg/m3 (about 890 ppm) in 1963 and 20 decreased in the following years. This reported VC concentration in air may have been an average 21 exposure (not specified by the authors). However, no information on peak concentrations and duration of 22 episodes with short term high concentrations of VC exposure is provided. Some of the reported activities, 23 such as cleaning autoclaves, are to be associated with very high exposures. 24 Occurrence of headache in workers chronically exposed to VC has been described by several 25 authors. However, exposure concentration and duration were not specified and always was characterized 26 as "high" (Lilis et al., 1975; Suciu et al., 1975; EPA, 1987). 27 2.2.2. Odor 28 Odor thresholds reported vary over a wide range: 10 - 25,000 ppm (26 - 65,000 mg/m3). Hori et 29 al. (1972) reported an odor threshold of 20 ppm in production workers and 10 ppm in workers from other 30 departments of polyvinyl-chloride (PVC) facilities (number of workers involved not presented). The VC- 31 odor was perceived by 50% of the "non production" workers at 200 ppm and by 50% of the "production" 32 workers at 350ppm. Odor threshold was tested by two methods. PVC was diluted with air at fixed 33 concentrations and was supplied from a glass injector to the subject's nostrils at a rate of 100 milliliters 34 over 5 to 10 seconds. This was repeated at gradually higher concentrations until the subject perceived VC. 35 The second method involved measurement of atmospheric concentrations of VC. Production workers 36 were less sensitive to VC than workers from other departments. When workders from different facilities 37 were compared even greater ranges were observed. However, inter-individual differences and 38 measurement techniques which were not strictly controlled. This odor threshold was reviewed by the 39 AIHA. The value has been rejected based on specified criteria (e.g. no calibration of panel odor 40 sensitivity, not stated whether the given limit was due to recognition or detection, number of trials not 41 stated; AIHA 1997). ------- Vinyl chloride INTERIM 1: 3/2008 1 Baretta et al. (1969) reported, that none of six subjects perceived odor entering an exposure 2 chamber at 59 ppm, while at 261 ppm all four subjects detected a very slight odor. Five of seven subjects 3 entering the exposure chamber at 491 ppm were able to detect the odor of VC, but after 5 minutes of 4 exposure the odor was no longer perceived (for study details see above). 5 Two persons who were exposed to 25,000 ppm (nominal concentration) for 3 minutes while 6 entering an experimental exposure chamber reported a "fairly pleasant odor" (Patty et al., 1930). 7 Amoore and Hautala (1983) reported an odor threshold of 3,000 ppm for VC. This value 8 represents the geometric average of three literature studies (individual studies not mentioned), studies 9 reporting extreme points and duplicate quotations were omitted. It was not stated whether this was the 10 detection or recognition threshold. 11 2.2.3. Irritation 12 13 Acute exposure 14 Irritating effects of VC are only observed after exposure to very high concentrations: lesions of 15 the eyes (wedge shaped brown discoloration of the bulbar conjunctiva, palpebral slits, conjunctiva and 16 cornea appeared dried out) were observed at autopsy in a worker who died due to inhalation of very high 17 concentrations of VC. The lesions were explained by the local effects of VC. At autopsy intensely 18 hyperemic lungs, with desquamation of the alveolar epithelium were observed (Danziger, 1960). 19 Chronic exposure 20 Tribukh et al. (1949) reported mucous irritation of the upper respiratory tract and chronic 21 bronchitis in PVC workers; however, these effects were not mentioned by Lilis et al. (1975) and 22 Marsteller et al. (1975). 23 Suciu et al. (1975) describe coughing and sneezing after exposure of workers to VC during one 24 shift; no other acute pulmonary effects or irritation are mentioned. These workers had been regularly 25 exposed to VC for an extended time period. 26 27 In chronically exposed VC workers, evidence for adverse respiratory disease is conflicting. Lung 28 function (respiratory volume and vital capacity, oxygen and carbon dioxide transfer) deteriorate over 29 time. Emphysema/chronic obstructive pulmonary disease (COPD), respiratory insufficiency, dyspnea, and 30 pulmonary fibrosis have been described (Suciu et al., 1975; Walker et al., 1976; Lloyd et al., 1984). Some 31 of these observations have been attributed to smoking as a possible confounder. ------- Vinyl chloride INTERIM 1: 3/2008 1 2.2.4. Cardiovascular effects 2 3 A slight decrease in blood pressure in VC workers has been attributed to the narcotic effects of 4 VC (Suciu et al., 1975). In older exposure experiments in human volunteers no cardiovascular parameters 5 have been measured (Lester et al., 1963). 6 Chronic exposure 1 In VC workers, Raynauds disease has been correlated to extended exposure to high VC 8 concentrations (ATSDR, 1997), with histologic alterations of small vessels (Veltman et al., 1975). Other 9 symptoms observed in VC workers are splenomegaly, hypertension, portal hypertension, generally 10 increased cardiovascular mortality, and vasospastic symptoms (ATSDR 1997; Suciu et al., 1975; Byron et 11 al., 1976). According to Kotseva, elevated occupational exposure to VC increases the incidence of arterial 12 hypertension, but there is no conclusive evidence that it is associated on its own with an increased risk of 13 coronary heart disease (Beck et al., 1973). 14 2.2.5. Other Endpoints 15 Hematology and immunology 16 Blood tests in VC victims indicated failure of blood coagulation (Danziger et al., 1960). 17 Hepatotoxicity 18 More or less pronounced hepatitis and enlargement of the liver have been reported in chronic 19 exposed workers (ECB, 2000; Marsteller et al., 1975). Others reported impaired liver function and 20 periportal liver fibrosis in workers from a PVC producing plant (no further details presented; Lange et al., 21 1974). Liver function disturbances have been reported for workers from PVC factories (Fleig and Thiess, 22 1978). Focal hepatocellular hyperplasia and focal mixed hyperplasia has been observed in VC-exposed 23 workers; some of the individuals with focal mixed hyperplasia developed liver angiosarcoma (Tamburro 24 et al., 1984). No data on liver effects after acute exposure are available. ------- Vinyl chloride INTERIM 1:3/2008 TABLE 2: SUMMARY OF ACUTE EFFECTS IN HUMANS AFTER INHALATION OF VINYL CHLORIDE Concentration (ppm) very high 25,000 ppm 20,000 ppm 16,000 ppm 12,000 ppm 8,000 ppm 4,000 ppm 3,000 ppm not specified, high 491 or 459 ppm 261 ppm 20 ppm 10 ppm Exposure Time not stated 3 min 5 min 5 min 5 min 5 min 5 min not stated not stated 3.5 h not stated not stated not stated Study type and effects irritation to the eyes dizziness, disorientation to space and size, burning sensation in feet, persisting headache 6/6 dizziness, lightheadedness, nausea, visual and auditory dulling, persisting headache in 1/6 5/6 dizziness, lightheadedness, nausea, visual and auditory dulling; no effects in one volunteer 1/6 volunteers dizzy, 1/6 "swimming head, reeling", second person was "unsure" of effects, somewhat dizzy in the middle of exposure 1/6 volunteers "slightly heady" (this volunteer felt also slightly heady at sham exposure and reported no effects at 12,000 ppm) no effects odor threshold (geometric averages of three studies, omitting extreme points and duplicate quotations) prenarcotic and narcotic effects; repeated exposure: headaches, asthenovegetative syndrome, cardiovascular effects , hepatomegaly 2/7 volunteers reported mild headache and dryness of the eyes and nose detection of the odor by 4/4 subjects odor threshold in PVC production workers odor threshold in workers from a PVC facility, not working in PVC production Reference Danziger, 1960 Patty etal., 1930 Lester etal., 1963 Lester etal., 1963 Lester etal., 1963 Lester etal., 1963 Lester etal., 1963 Amoore and Hautala, 1983 Suciuetal., 1975 Baretta et al., 1969 Baretta et al., 1969 Hori etal., 1972 Horietal., 1972 10 11 12 13 14 15 16 17 18 19 2.3. Developmental / Reproductive Toxicity 20 No data on developmental or reproductive toxicity in humans after single exposure to VC were 21 identified. 22 2.4. Genotoxicity ------- Vinyl chloride INTERIM 1: 3/2008 1 Huettner and Nikolova (1998) investigated lymphocyte chromosomal aberrations in 29 non- 2 exposed and 29 persons exposed to VC and its combustion byproducts after a train accident in 3 Schoenebeck, Germany. The authors found increased incidences of chromosomal aberrations (gaps, 4 chromatid breaks, acentric chromosomes). Blood samples were drawn 2-4 month after the accident. 5 Sixty per cent of the exposed individuals complaint of health problems ascribed to the pollutants. More 6 than 15 hours after the accident, atmospheric VC concentrations were 1-8 ppm (Huettner and Nikolova, 7 1998). Hahn et al. (1998) reported maximum VC-concentrations of 30 ppm near the center of the 8 accident. Exposure level to VC and/or other combustion products of those persons included into the 9 investigation is highly uncertain. In a follow-up study two years later in the same cohort of accidentally 10 exposed people, Becker et al. (2001) found enhanced chromosome aberrations in peripheral lymphocytes 11 as an indicator of clastogenic activity of VC, while no increased mutagenic activity (mutations in the 12 hypoxanthine-guanine-phosphoribosyl-transferase (HPRT) gene) was observed in exposed persons. 13 Chronic exposure 14 Clastogenic DNA damage has been detected by various tests in chronically VC exposed workers. 15 Chromosomal defects (inversions, translocations, rings) and/or micronuclei have been detected at 16 exposure concentrations estimated at 1 ppm (Fucic et al., 1995; short exposure spikes up to 300 ppm VC 17 were reported), and 5 ppm VC (Graj-Vrhovac et al., 1990). Also increased frequencies of sister chromatid 18 exchanges were reported (Fucic et al., 1992; Sinues et al., 1991). Awara et al. (1998) observed an 19 increased incidence of DNA damage (detection by single-cell gel electrophoresis) in workers exposed to 20 VC. The amount of DNA-damage was increasing with exposure time. Average VC concentrations were 21 highest in the production area (about 3 ppm). 22 Covalent binding to macromolecules due to VC exposure in humans has not been directly 23 assessed. However, gene mutations were found in human tumors associated with exposure to etheno- 24 adduct-forming chemicals such as VC. Specifically, in angiosarcoma of the human liver in 5 of 6 cases G- 25 >A transitions of the Ki-ras gene and A->T transitions of p53 were observed in 3 of 6 cases, which may 26 be attributed to the formation of ethenobases in DNA (Barbin, 2000). 27 2.5. Carcinogenicity 28 No data about cancer induction in humans after single exposure have been reported. From two 29 large epidemiological studies of occupational exposure of adult workers (Ward et al., 2000; Mundt et al., 30 1999), there is some evidence that risk for liver cancer or biliary tract cancer was only increased after 31 extended exposure time. However, conflicting results are also published (Weber et al., 1981) 32 demonstrating a steep increase of the Standard Mortality Rate after very limited exposure duration (for 33 details, see Appendix D). There exist no epidemiological studies which include newborn children as 34 specific risk group. 35 Chronic exposure 36 There are sufficient epidemiological data demonstrating a statistically significant elevated risk of 37 liver cancer, specifically angiosarcomas (ASL), from chronic exposure to VC (summarized in EPA, 38 2000a, b; WHO, 1999a; Boffetta et al.,2003). The possible association of brain, soft tissue, and nervous 39 system cancer with VC exposure was also reported. However, the evidence supporting a causal link 40 between brain cancer and VC exposure is limited (EPA, 2000a, b). Some other studies found an 41 association between VC exposure and cancer of the hematopoetic lymphatic systems (Simonato et al., 42 1991; Greiser et al., 1982). Lung cancer has also been associated with VC exposure, but the increased risk 43 of lung cancer observed in some cohorts may be due to exposure to PVC dust rather than VC monomer ------- Vinyl chloride INTERIM 1: 3/2008 1 (Mastrangelo et al., 2003). In angiosarcoma of the human liver, mutations were observed which may be 2 attributed to the formation of ethenobases in DNA (Barbin, 2000). 3 Quantitative risk estimates for VC based on epidemiologic studies have been derived by the 4 Netherlands (Anonymous, 1987; unit risk 1 • 10'6 per j^g/m3), the WHO (1987; 1999b; unit risk 1 • 10'6 per 5 ng/m3) and Clewell et al. (Clewell et al., 2001; unit risk 0.2 - 1.7 x lO'6). 6 2.6. Summary 7 Odor thresholds of VC were reported in the range of 10 to 25,000 ppm (Hori et al., 1972; Baretta 8 et al., 1969; AIHA, 1997; Patty et al., 1930). Amoore and Hautala (1983) reported an odor threshold of 9 3,000 ppm for VC. This value represents the geometric average of three literature studies, extreme points 10 and duplicate quotations were omitted. Validated studies detecting the recognition and the detection limit 11 are not available from literature. Vinyl chloride is an anaesthetic compound. Effects observed in acutely 12 exposed VC workers and human volunteers indicate a characteristic sequence of events from euphoria and 13 dizziness, followed by drowsiness and loss of consciousness. After five minutes exposure of volunteers, 14 health effects have been described at concentrations > 8,000 ppm, no effects were observed at 4,000 ppm 15 (Lester et al., 1963). 25,000 ppm VC for 3 minutes caused dizziness, slightly disorientation and a burning 16 sensation in feet in two volunteers (Patty et al., 1930). Mild headache and some dryness of the eyes and 17 nose were the only complaints of volunteers exposed to 491 ppm VC (the onset of headaches is not 18 specified and is assumed to have occurred after 3.5 hours of exposure) (Baretta et al., 1969). Irritation of 19 the eyes was reported in the context of an accidental exposure to lethal VC concentrations (exposure 20 concentration unknown) (Danziger et al., 1960). 21 No data on developmental or reproductive toxicity of VC in humans after acute exposure are 22 stated in the literature. 23 Occurrence of chromosomal aberrations in lymphocytes of humans accidentally exposed to VC 24 were reported by Huettner and Nikolova (1998). More than 15 hours after the accident, atmospheric VC 25 concentrations were 1-8 ppm. In a two year follow up clastogenic activity was still detectable (Becker et 26 al., 2001). 27 Vinyl chloride is a known human carcinogen inducing liver angiosarcoma and possibly brain 28 tumors. Evidence for other tumor locations including hepatocellular carcinoma is contradictory (EPA, 29 2000a, b). In angiosarcoma of the human liver mutations were observed, which may be attributed to the 30 formation of ethenobases in DNA (Barbin, 2000). Unit risk estimates based on epidemiologic studies have 31 been published (Anonymous, 1987; WHO, 1987, 1999b; Clewell et al., 2001). ------- Vinyl chloride INTERIM 1: 3/2008 1 3. ANIMAL TOXICITY DATA 2 3.1. Acute Lethality 3 Acute inhalation toxicity tests were performed in rats, mice, rabbits, and guinea pigs. However, 4 no LC50 study complying with today's standards is available. The lethality data are summarized in Table 5 3. 6 3.1.1. Rats 7 Mastromatteo et al. (1960) exposed 5 rats per group to 10, 20, 30 or 40% VC (100,000 to 400,000 8 ppm) for up to 30 minutes (purity 99.5% maximum). The animals were exposed in an inhalation chamber 9 of 56.6 liters. The VC concentration was adjusted by mixing VC and air in a flow meter outside of the 10 exposure chamber. The stream of air and VC was led to the animal chamber inlet to deliver a continuing 11 stream (flow not given, VC concentrations were not determined in the test chamber). Observations were 12 made continuously and are summarized in Table 3. No animals died after exposure to 100,000 and 13 200,000 ppm. All animals died after 15 minutes exposure to 300,000 ppm. At 300,000 ppm the lungs of 14 the animals which died revealed congestion with hemorrhagic areas, in addition congestion of the liver 15 and the kidney were observed. 16 Prodan et al. (1975) exposed rats for 2 hours in exposure chambers of the Pravdin type with 580 17 liters capacity (total of 70 rats, at least 10 animals per group, strain not given). The animals were exposed, 18 according to Krakov's method, to variable concentrations of VC. After the animals were placed in the 19 exposure chamber, the gas was introduced at the beginning at the lower part of the chamber, without any 20 ventilation. The gas was permanently stirred up by an inside pellet and was measured volumetrically with 21 a Zimmermann type spirometer. At VC-concentrations of 15, 16, 17, 20, and 21% (150,000 to 210,000 22 ppm, nominal concentration) the lethality was 23, 80, 90, 90, and 100%, respectively. The authors 23 calculated a LC50of 15% VC (about 150,000 ppm) and a LC100of 21% (about 210,000 ppm). All LC50 and 24 LC100 values from these experiments are given by the authors for 2h exposure irrespective of the time of 25 death. Findings shortly before death were general convulsions, respiratory failure, exopthalmia and 26 deflection of the head on the abdomen. Surviving animals rapidly recovered after termination of the 27 exposure. At autopsy, dead animals showed general congestion of the internal organs (lungs, liver, 28 kidney, brain and spleen); some animals (no number given) had pulmonary edema, marmorated liver and 29 kidney swelling. 30 In the context of a teratology study John et al. (1981) exposed Sprague-Dawley rats intermittently 31 with 500 or 2,500 ppm VC for 7 days. At 2,500 ppm VC 1 of 17 rats died, the exact day of death was not 32 specified by the authors (for study details see 3.3.). 33 Exposure of 18 Sherman rats (9 male; 9 female) to 100,000 VC for 8 hours resulted in deep 34 anaesthesia, with consciousness regained 5 to 10 minutes after removal to air. After two exposures one 35 female rat died and the remaining showed signs of chronic toxicity (not specified) prompting the authors 36 to lower the VC concentration to 80,000 ppm in order to minimize mortality. Despite this decrease 37 mortality was considerable especially in male rats exposed for longer than 8 days. The animals were 38 exposed in a 1100 liter steel chamber. The concentration was initially raised rapidly to the desired level by 39 admitting VC without admixture with air until the effluent from the (mixing) chamber attained the desired 40 level as noted on the thermal conductivity meter. A fan mixed the VC with the air within the (mixing) 41 chamber. Thereafter, the effluent from the 2-liter mixing vessel was admitted to the chamber, the 10 ------- Vinyl chloride INTERIM 1: 3/2008 1 throughput was 20 1/min (Lester et al., 1963). 2 Exposure of 2 Sherman rats in a 10 liter all glass exposure chamber to 150,000 ppm resulted in 3 deep anaesthesia within five minutes, one of two animals died due to respiratory failure after 42 minutes 4 (Lester et al., 1963) (study details see above). 5 3.1.2. Mice 6 Five mice were exposed to 10, 20, 30 or 40% VC (100,000 to 400,000 ppm, nominal 7 concentration) for up to 30 minutes (for study details see 3.1.1.) (Mastromatteo et al., 1960). One mouse 8 died after 25 min exposure to 200,000 ppm and all mice died after 10 min exposure to 300,000 ppm. No 9 death occurred at 100,000 ppm. At 300,000 ppm the lungs of the animals which died revealed congestion 10 of the lungs with hemorrhagic areas, in addition congestion of the liver and the kidney were observed. 11 In ventilated exposure chambers of the Pravdin type, 100,000 ppm VC was not lethal to mice 12 during 2 hours, whereas 150,000 ppm killed 46/61 mice within one hour, and all animals within 2 hours. 13 The authors calculated a LC50 of 117,500 ppm and a LC100 of 150,000 ppm for mice (for study details and 14 symptoms before death see 3.1.1.), for 2 hours. Under unstirred conditions 42,900 ppm was lethal to 70% 15 (13 of 20) of the animals within less than an hour (Prodan et al., 1975). 16 Tatrai and Ungvary (1981) exposed CFLP mice to 1,500 ppm VC for 2, 4, 8, 12 or 24 hours 17 (n=20). Animals were observed for 24 hours after exposure. In addition, 40 animals were exposed for 12 h 18 and survivors were investigated two month after the exposure. Animals were exposed in dynamic 19 exposure chambers with vertical air flow. The volume of the exposure chambers was 0.3 m3; the vertical 20 flow rate of the air was 3 m3/hour at a temperature of 20 - 23 °C and 50 - 55% relative humidity. After 24 21 hours exposure time all animals died within 24 h after exposure, 90% of the mice exposed over 12 hours 22 died. No death is reported in animals exposed for shorter periods. Exposure caused hemorrhages and 23 vasodilatation characteristic of shock in the lungs. Additionally, shock-liver developed. The authors do 24 not comment on the concentration difference between their experiments and earlier reports indicating 25 much higher total VC concentrations as lethal; however, in these studies asphyxia is given as the cause of 26 death. This effect is not conformed in other studies. 27 In a study designed to investigate long term hepatic effects of VC, Lee et al. (1977) exposed CD-I 28 mice to 1,000 ppm for 6 hr/day. Three out of seventy-two mice died between day 3 and 9; all other mice, 29 as well as replacement mice appeared healthy throughout 12 month VC exposure. Upon autopsy animals 30 had acute toxic hepatitis with diffuse coagulation type necrosis of hepatocytes, as well as tubular necrosis 31 in the renal cortex. 32 In the context of a teratology study, John et al. (1981) exposed mice to 50 or 500 ppm VC for 7 33 h/d on day 6-15 of gestation. At 500 ppm VC 5 of 29 mice died, the exact day of death was not specified 34 by the authors. 35 3.1.3. Guinea Pigs 36 Patty et al. (1930) found 15 - 25% VC (150,000 - 250,000 ppm) to be lethal to guinea pigs within 37 one hour, 40% VC (400,000 ppm) resulted in death of the animals within 10 - 20 min. Gross pathology 38 examinations of these animals revealed intense congestion and edema of the lungs and a hyperaemia of 11 ------- Vinyl chloride INTERIM 1: 3/2008 1 the kidneys and livers. The lungs were light pink in color, the cut section was uniformly light red, and 2 bled freely. The authors concluded that VC is irritating to the lungs. No eye or nasal irritation was 3 described. However, from the paper it is unclear whether sufficient mixing of the atmosphere had 4 occurred, furthermore, the number of animals per group was not mentioned. 5 Prodan et al. (1975) reported a LC50 of 238,000 ppm and a LC100 of 280,000 ppm for guinea pigs 6 exposed in a exposure chamber of the Pravdin type (the gas was permanently stirred up by an inside 7 pellet; study details are described in 3.1.1.) for 2 hours. No animals died within 2 hours at 200,000 ppm. 8 Yant (cited from Prodan et al., 1975) determined a lethal concentration of 400,000 ppm for 10 9 min for guinea pigs. 10 Exposure of guinea pigs to 10, 20, or 30% VC (100,000 - 300,000 ppm) (5 animals per group) did 11 not result in death within 30 min of exposure time, but one animal of the 300,000 ppm group died within 12 24 h following exposure. Thirty minutes exposure to 40% VC (400,000 ppm) resulted in death of one 13 animal, another animal died within 24 h following exposure whereas the other 3 animals recovered 14 (Mastromatteo et al., 1960; for study details see 3.1.1.). The liver of the animal which died at 300,000 15 ppm showed severe fatty degeneration, the liver was distended and very friable, the liver effects were less 16 pronounced at 400,000 ppm. There was marked congestion of the lungs with hemorrhages in the dead 17 animals. 18 3.1.4. Rabbits 19 Rabbits were exposed for 2 h in exposure chambers of the Pravdin type. 200,000 ppm did not 20 result in death of 4 animals. 50% of the animals (2/4) exposed to 240,000 ppm died within the first hour 21 of exposure and all animals (4/4) exposed to 280,000 ppm (Prodan et al., 1975) (for details see 3.1.1.). 22 In the context of a teratology study, John et al. (1981) exposed rabbits intermittently to 500 or 23 2,500 ppm VC for 7 days. At 2,500 ppm VC, 1 of 7 rabbits died, the exact day of death was not specified 24 by the authors. 25 3.1.5. Other Species 26 No data on acute lethality in other species are available. 27 12 ------- Vinyl chloride INTERIM 1:3/2008 TABLE 3: SUMMARY OF ACUTE LETHAL INHALATION DATA IN LABORATORY ANIMALS Species mouse mouse mouse mouse mouse mouse mouse mouse mouse rat rat rat rat rat rat rabbit rabbit rabbit guinea pig guinea pig guinea pig guinea pig guinea pig guinea pig guinea pig guinea pig Concentration (ppm) 500 1000 1500 1500 1500 100000 117500 150000 300000 100000 150000 150000 200000 210000 300000 200000 240000 280000 100000 200000 240000 150,000 to 250,000 280000 300000 400000 400000 Exposure Time several days for 7 h/d at least 3 x 6 h 8h 12 h 24 h 2h 2h 2h 10 min 8h 2h 2h 30 min 2h 15 min 2h 2h 2h 6h 2h 2h 18 - 55 min 2h 30 min 10 - 20 min 30 min Number of animals 29 72 20 60 20 40 39 61 5 18 10 2 5 10 5 4 4 4 not stated 4 12 not stated 4 5 not stated 5 Effect LC17 •'-'Mow LC0 -LL-90 -L^-ioo LC0 LC50 -L^-ioo -L^-ioo LC0 LC50 LC50 LC0 -L^-ioo -L^-ioo LC0 LC50 -L^-ioo LC0 LC0 LC50 T r a J-'Moo -L^-ioo -LL-20 T r a J-'MOO -LL-40 Reference Johnetal., 1977; 1981 Leeetal., 1977 Tatrai and Ungvary, 1981 Tatrai und Ungvary, 1981 Tatrai und Ungvary, 1981 Prodanetal., 1975 Prodanetal., 1975 Prodanetal., 1975 Mastromatteo et al., 1960 Lester etal., 1963 Prodanetal., 1975 Lester etal., 1963 Mastromatteo et al., 1960 Prodanetal., 1975 Mastromatteo et al., 1960 Prodanetal., 1975 Prodanetal., 1975 Prodanetal., 1975 Patty et al., 1930 Prodanetal., 1975 Prodanetal., 1975 Patty et al., 1930 Prodanetal., 1975 Mastromatteo et al., 1960 Patty et al., 1930 Mastromatteo et al., 1960 29 a: number of animals per group and animals that died not stated 30 3.2. Nonlethal Toxicity 13 ------- Vinyl chloride INTERIM 1: 3/2008 1 3.2.1. Dogs 2 Oster et al. (1947) exposed 2 beagle dogs to 50% VC/50% oxygen for induction of anesthesia (no 3 time given) and subsequently with 7% VC (70,000 ppm) in oxygen for narcosis maintenance (no further 4 study details described). Narcosis induction was rapid, all animals showed salivation. Muscle relaxation 5 was incomplete with good relaxation of the abdomen, and rigidity and uncoordinated movements in legs. 6 The recovery period was prompt but accompanied by violent excitation. In four dogs anesthetized with 7 10% VC (100,000 ppm) mixed with oxygen, no effects on blood pressure were observed, but cardiac 8 irregularities (intermittent tachycardia, extraventricular systoles and vagal beats) were observed. All 9 symptoms disappeared rapidly upon change to ethyl ether, as well as after termination of narcosis. 10 Cardiac sensitizing potential of VC was tested in beagle dogs. Conscious dogs (4-7 per dose 11 group) were exposed to VC by means of a face mask for 5 minutes. Oxygen was added when high 12 concentrations were used. During the last 10 seconds of the exposure period, a bolus injection of 13 epinephrine (5|J,g/kg) was given via a cephalic vein and the ECG changes were recorded. A further 14 injection of adrenaline was also given 10 minutes after the end of exposure. Cardiac sensitization was 15 deemed to have occurred when ventricular tachycardia or ventricular fibrillation resulted from the 16 challenge injection of epinephrine. An increased number of ventricular ectopic beats was not regarded as 17 evidence of sensitization since they could often be produced by a challenge injection of epinephrine 18 during control air exposures. The EC50 for cardiac sensitization was 50,000 ppm (95 % CI: 37,000 - 19 68,000 ppm). The post exposure injection of epinephrine did not result in arrhythmias (Clark and Tinston, 20 1973). 21 A second study on cardiac sensitization to epinephrine in beagle dogs ( 6 male or female, not 22 further specified) after 5 minutes exposure to VC was published by Clark and Tinston (1982). Methods 23 were apparently identical to the study published in 1973 (Beck et al., 1973). The EC50 for cardiac 24 sensitization was 71,000 ppm (95% CI: 61,000 - 83,000 ppm). These concentrations were below the 25 concentrations which caused effects on the central nervous system in rats (EC50: 38,000 ppm after 10 26 minutes exposure). The authors did not comment on their earlier findings which indicated a lower EC50 27 for cardiac sensitization. The authors discussed, that cardiac sensitization is unlikely to occur in man in 28 the absence of any effects on the CNS and that dizziness should act as an early warning that a dangerous 29 concentration was reached. 30 3.2.2. Rats 31 In rats exposed to 100,000 ppm, increased motor activity occurred after 5 min, pronounced 32 tremor, unsteady gait and muscular incoordination occurred after 15 min, side position occurred at 20 33 min, and deep narcosis occurred after 30 min. When the VC concentration was increased, deep narcosis 34 occurred at 200,000 ppm after 15 min and at 300,000 ppm after 5 min and muscular incoordination after 2 35 or 1 min, respectively. At autopsy, lungs of the animals of the 100,000 ppm group showed a very slight 36 hyperemia even 2 weeks after exposure; at 200,000 ppm congestion of the lung in all animal and some 37 fatty infiltration in the liver of one rat were observed. Irritation (not further explained) was described to 38 occur immediately after onset of exposure to 10, 20, or 30% VC (Mastromatteo et al., 1960). 39 Lester et al. (1963) exposed Sherman rats for up to 2 hours with 50,000 - 150,000 ppm VC. The 40 total gas flow was 50 liters per minute. The desired concentrations were obtained by metering air and VC 41 (gas chromatography of the liquid phase indicated more than 99% VC) through flow meters and passing 14 ------- Vinyl chloride INTERIM 1: 3/2008 1 the appropriate flows through a 2 1 mixing chamber. The desired concentration was passed through a 10- 2 liter all-glass exposure chamber containing 2 rats. The concentration was continuously monitored by a 3 thermal conductivity meter (less than 5% deviation from the desired concentration). At 50,000 ppm VC 4 for 2 hours moderate intoxication was observed, but the righting reflex was lost; at 60,000 ppm for 2 5 hours intoxication was more intense but the righting reflex was still present (lost at 70,000 ppm). The 6 corneal reflex was lost at 100,000 ppm VC. On removal from the chamber the animals returned to the pre- 7 exposure state rapidly. Exposure to 150,000 VC resulted in deep anesthesia within 5 minutes, one of two 8 animals died after 42 minutes by respiratory failure. Autopsy revealed edema and congestion of the lungs. 9 The second rat recovered quickly after removal from the exposure chamber. 10 Exposure of 18 Sherman rats to 100,000 VC for 8 hours resulted in deep anesthesia, with con- 11 sciousness regained 5 to 10 minutes after removal to air. After two exposures one female rat died and the 12 remaining showed signs of toxicity (not specified) (Lester et al., 1963; study details presented in 3.1.1.). 13 Male Holtzman rats were exposed once to 0.5, 5 or 10% VC (5,000, 50,000, or 100,000 ppm) for 14 6 h in a dynamic inhalation chamber. Animals were killed 24 hours after the exposure (no further details 15 described). Exposure to 0.5% or 5% for a single 6 h period did not cause a substantial rise in serum 16 alanine-a-ketoglutarate transaminase (AKT) or sorbitol dehydrogenase (SDH), two cytoplasmic liver 17 enzymes whose appearance in serum correlates with liver injury. Only after exposure to 10% VC was a 18 slight increase in either parameter of hepatoxic response and centrilobular hepatocellular vacuolization 19 noted. At the lower dose levels livers were histologically normal. During exposure to 10% VC animals 20 appeared to be anesthetized (Jaeger et al., 1974). 21 Rats exposed to 30,000 ppm VC for 4 hours were slightly soporific (Viola et al., 1970). No other 22 acute toxicity data were reported, animals were exposed for total of 12 month. 23 Tatrai and Ungvary (1981) exposed CFY rats to 1,500 ppm VC for 24 hours (n=20; study details 24 are presented in 3.1.2.). Livers were investigated by histochemical methods. No morphological changes 25 were observed. 26 Fischer 344 or Sprague-Dawley rats were treated for 1 h with 50, 500, 5,000 or 50,000 ppm VC 27 (about 90 animals per group). The chambers were Rochester type, stainless steel, 1,000 liter, constructed 28 to provide laminar air flow and ensure uniform exposures to VC to test animals. The concentration of gas 29 in the inhalation chamber was monitored by a gas chromatograph. No remarkable signs of toxicity were 30 observed. Upon removal from the test atmosphere, all animals recovered to normal appearance within 24 31 hours (Hehir et al., 1981). Viola et al. (1971) also reported that exposure of rats to 50,000 ppm for one 32 hour did not result in toxicity. 33 Effects after repeated exposure 34 Pregnant rats exposed to 1,500 ppm for 7 or 9 days (day 1-9 or 8 - 14 of gestation) showed 35 increased absolute and relative maternal liver weight, without light microscopic visible changes (liver 36 weight to body weight ratio (%), exposure day 1-9 of gestation: control: 3.71; exposed: 4.25). This effect 37 was not observed in animals treated from day 14-21 of gestation. Additionally, an increased number of 38 resorbed fetuses and fetal losses were observed in animals exposed during the first 9 days of pregnancy 39 (Ungvary et al., 1978, for study details see 3.3.). 40 Intermittent exposure of rats to 500 ppm or 2,500 ppm VC during day 6-15 of pregnancy 15 ------- Vinyl chloride INTERIM 1: 3/2008 1 resulted in increased relative and absolute maternal liver weights and an increased number of absorbed 2 fetuses and fetal losses at 2,500 ppm (NOAEL 500 ppm) (absolute liver weight: control: 14.27 g; 2500 3 ppm: 15.55 g; relative liver weight: control: 34.4 mg/g bw; 2500 ppm: 37.8 mg/g bw). One dam died at 4 2,500 ppm (John et al., 1977, 1981; for details see 3.3). 5 6 After repeated inhalation exposure (4 weeks) of rats to 5,000 ppm VC (7h/day, 5 days/week) 7 vacuolized hepatocytes with swollen mitochondria were found in male and female animals (Feron et al., 8 1979). After 13 weeks inhalation exposure even at the lowest dose level (10 ppm VC) an increase of the 9 relative liver weight was seen in male rats and centrilobular hypertrophy in females (Thornton et al., 10 2002). 11 3.2.3. Mice 12 Mice exposed to 100,000 ppm VC for 30 min showed increased motor activity after 5 min, 13 twitching of extremities after 10 min and pronounced tremor, unsteady gait and muscular incoordination 14 occurred after 15 min, side position at 20 min, and deep narcosis occurred after 30 min. When the VC 15 concentration was increased deep narcosis occurred at 200,000 ppm after 15 min (side position after 5 16 min) and at 300,000 ppm after 5 min (lethal after 10 min). Mice of the 100,000 ppm group showed slight 17 hyperemia of the lungs, one of five animals showed degenerative changes in the tubular epithelium of the 18 kidney with hydropic swelling. 200,000 ppm for 30 min resulted in congestion of the lungs persisting for 19 2 weeks. Irritation (no further details) was described to occur immediately after onset of exposure to 10, 20 20, or 30% VC (Mastromatteo et al., 1960). 21 Prodan et al. (1975) exposed white mice (no strain specified) for 2 hours to 90,000 to 200,000 22 ppm VC with ventilation in a exposure chamber (for study details see 3.1.1.); no shorter exposure time 23 was reported. Salivation and lacrimation appeared shortly after onset of exposure, with narcosis reached 24 within less than one hour in the majority of the animals. Typical narcosis stages of excitement with tonic - 25 clonic convulsions and muscular contractions, tranquility and relaxation were described. Other symptoms 26 were accelerated respiration, proceeding to bradypnea, Cheyne-Stokes type of respiration and respiratory 27 failure. No differentiation of the symptoms according to the single exposure levels were made. Concen- 28 trations of 110,000 and higher were lethal. In surviving mice all symptoms were rapidly reversible. 29 Male mice exposed to 50,000 ppm VC for 1 h exhibited hyperventilation after 45 min, with 30 twitching and ataxia. Female mice became hyperactive after 40 min exposure and respiratory difficulty 31 and ataxia was observed in approximately 25% of the female mice after 55 min. At 5,000 ppm no mice 32 were visibly affected. Study details are presented in 3.2.2 (Hehir et al., 1981). 16 ------- Vinyl chloride INTERIM 1: 3/2008 1 Tatrai and Ungvary (1981) exposed CFLP-mice to 1,500 ppm VC for 2 to 24 hours. After 2 2 hours, histology demonstrated circulation stasis in the liver, with concomitant decreases in enzyme 3 activities (succinic dehydrogenase and acid phosphatase), subcellular damage, and centriobular necrosis. 4 After 24 h shock liver developed. Severity of changes increased with exposure time; after 12 hours the 5 lungs showed hemorrhages and vasodilatation as signs of circulatory disturbances. No changes were 6 observed in brain and kidney. 90% of the animals died after exposure for 12 hours, and 100% after 24 7 hours. 8 Kudo et al. (1990) exposed male ICR mice (4 or 5 per group) to 5,000 and 10,000 ppm VC for 4 9 hours on 5 or 6 successive days, respectively. Basophilic stippled erythrocytes indicating disturbances in 10 erythropoiesis appeared in peripheral blood smears on the second day indicating possible bone marrow 11 damage after a single exposure; no difference between the doses was observed, reticulocyte numbers were 12 also increased, albeit not statistically significant. The authors discuss that the increase was partly due to 13 repeated blood sampling and was not entirely due to VC-exposure. Exposure at lower concentrations, i.e. 14 30 - 40 ppm induced basophilic stippled erythrocytes after 3 days. 15 Lee et al. (1977) exposed CD-I mice with 1,000 ppm for 6 hr/day in the context of a long term 16 hepatotoxicity and carcinogenicity study. Besides 5% short term mortality within the first days due to 17 acute toxic hepatitis no sign of VC toxicity was observed in the other animals. 18 Aviado and Belej (1974) reported that exposure of mice (male, Swiss strain) to 100,000 ppm VC 19 for 6 minutes did not cause arrhythmia, whereas 200,000 ppm induced a 2nd degree block and ventricular 20 ectopics (animals were anesthetized with sodium pentobarbital). Cardiac sensitization was observed after 21 6 min exposure to 100,000 ppm VC (animals were anesthetized with sodium pentobarbital). Mice were 22 exposed through a face mask which was in contact with a 6 1 flaccid bag. The inhalation gas was balanced 23 with oxygen in order to prevent asphyxia. The number of animals per dose group was not presented. For 24 testing cardiac sensitization the animals received 6 |-lg/kg adrenaline hydrochloride intravenously. 25 3.2.4. Guinea Pigs 26 Guinea pigs exposed to 100,000 ppm VC for 30 min showed increased motor activity after 5 min, 27 unsteady gait and muscular incoordination occurred after 15 min, tremors and twitching of extremities 28 after 20 min, and side position with tremors after 30 min- one unconscious. When the VC concentration 29 was increased deep narcosis occurred at 200,000 ppm and 300,000 ppm after 30 min and at 400,000 ppm 30 after 5 min. Guinea pigs of the 100,000 ppm group showed only slightly hyperemic lungs 2 weeks after 31 exposure. At 200,000 ppm congestion of the lungs was observed. At 300,000 and 400,000 ppm survivors 32 showed marked pulmonary congestion with hemorrhagic areas and edema. In one animal of the 400,000 33 ppm group the tracheal epithelium was completely absent. In the same animals blood was unable to clot. 34 Irritation (no further details) was described to occur immediately after onset of exposure to 400,000 ppm 35 of VC, but irritation was not described at lower dose levels (Mastromatteo et al., 1960). 36 Prodan et al. (1975) exposed Guinea pigs (no strain given) to 20 - 28% VC (200,000 - 280,000 37 ppm) for 2 hours. Symptoms of progressing anesthesia as described for mice were observed in a time 38 depending manner; muscular contractions were more pronounced in guinea pigs than in mice. Lethality 39 increaed with increasing concentration, in surviving animals all symptoms were rapidly reversible. 40 Concentrations of 200,000 ppm were not lethal within 2 h (n=4). Observation of the animals did not 41 exceed 2 h. 17 ------- Vinyl chloride INTERIM 1: 3/2008 1 Exposure of guinea pigs to 5,000 or 10,000 ppm for up to 8 h did not produce any visible 2 symptoms. 25,000 ppm resulted in apparent unconsciousness and deep narcosis after 90 min and a slow, 3 shallow respiration within 6 to 8 h. No deaths were observed within 8 h lasting exposure. Similar 4 symptoms were observed at 50,000 ppm (unconsciousness within 50 min, slow, shallow respiration within 5 360 min, no death within 6 h). 100,000 ppm lead to an incomplete narcosis already 2 minutes after onset 6 of exposure, none of the animals died within the 6 h lasting exposure period (Patty et al., 1930). 7 3.2.5. Rabbits 8 Prodan et al. (1972) exposed rabbits (no strain given) to 20 - 28% VC (200,000 - 280,000 ppm) 9 for 2 hours. Symptoms of progressing anesthesia as described for mice were observed in a time dependent 10 manner, rabbits showed heavy respiration, salivation and muscular contractions. Lethality increased with 11 increasing VC concentrations, all symptoms were rapidly reversible in survivors. No death was observed 12 within 2 hours (n=4). 13 Tatrai and Ungvary (1981) exposed 20 New-Zealand-rabbits to 1,500 ppm VC for 24 hours. No 14 acute clinical effects or pathological changes of the liver were noted 24 h after exposure. 15 3.2.6 Monkeys 16 In monkeys, only myocardial depression after inhalation of 2.5-10% VC was observed. Rhesus 17 monkeys were anesthetized by i.v. injection of 30 mg/kg sodium pentobarbital. An electrocardiograph 18 was implanted for continuous monitoring. 3 monkeys received 2.5, 5, or 10% of VC. The inhalation 19 period lasted 5 minutes, alternating with room air for 10 minutes. The myocardial force was reduced by 20 2.3, 9.1 and 28.5% respectively, with a significant effect only at 10% VC. There was no effect on the 21 heart rate in compasison to controls. It is not clearly stated whether an addition challenge with epinephrine 22 was applied or not (Belej et al., 1974). 18 ------- Vinyl chloride INTERIM 1:3/2008 TABLE 4: SUMMARY OF NON-LETHAL EFFECTS IN LABORATORY ANIMALS Species dog dog dog mouse mouse mouse mouse mouse mouse mouse mouse mouse mouse rat rat rat rat rat rat rat Concentration (ppm) 50000 71000 100000 1500 5000 50000 100000 100000 100000 100000 100000 200,000 200000 500 1500 1500 30000 50000 50000 50000 Exposure Time 5 min 5 min not stated 2h Ih 40 min 6 min 6 min 15 min 30 min 2h 6 min 30 min 10x7h 24 h 9x24h 4h Ih 2h 6h Effect EC50 cardiac sensitization towards epinephrine EC50 cardiac sensitization towards epinephrine anesthesia accompanied by cardiac arrhythmia stasis of blood flow, decreasing enzyme activities in liver, subscellular liver damage, centrilobular necrosis no clinical signs of toxicity twitching, ataxia, hyperventilation, hyperactivity no induction of cardiac arrhythmia cardiac sensitization towards adrenaline pronounced tremor, unsteady gait and muscular incoordination unconsciousness, side position already after 20 min; lung hyperemia persisting for > 2 weeks intense salivation and lacrimation immediately after onset of exposure, narcosis within 1 h Induction of cardiac arrhythmia (2nd degree block, ventricular ectopics) deep narcosis, side position after 5 min, congestion of the lung for > 2 weeks no effects on liver weight (LOAEL: 2,500 ppm) (exposure: day 6-15 of pregnancy) no acute toxicity reported increased relative and absolute liver weight; increased number of absorbed fetuses and fetal losses (exposure: day 1-9 of pregnancy) slightly soporific no clinical signs of toxicity moderate intoxication (not further specified), loss of righting reflex no clinical and histological signs of Reference Clark and Tinston, 1973 Clark and Tinston, 1982 Osteretal., 1947 Tatrai and Ungvary, 1981 Hehiretal., 1981 Hehiretal., 1981 Aviado andBelei, 1974 Aviado andBelei, 1974 Mastromatteo et al., 1960 Mastromatteo et al., 1960 Prodanetal., 1975 Aviado andBelei, 1974 Mastromatteo et al., 1960 Johnetal., 1977 Tatrai and Ungvary, 1981 Ungvary etal., 1978 Viola etal., 1970 Viola etal., 1971; Hehiretal. 1981 Lester etal., 1963 Jaeger etal., 1974 19 ------- Vinyl chloride INTERIM 1:3/2008 TABLE 4: SUMMARY OF NON-LETHAL EFFECTS IN LABORATORY ANIMALS Species rat rat rat rat rat rat rat rat guinea pig guinea pig guinea pig guinea pig guinea pig guinea pig guinea pig guinea pig rabbit monkey Concentration (ppm) Exposure Time 60000 100000 100000 100000 100000 100000 200000 200000 10000 25000 25000 25000 100000 100000 200000 200000 200000 25,000-100,000 2h 15 min 30 min 2h 6h 8h 2 min 30 min 8h 5 min 90 min 6-8h 15 min 30 min 30 min 2h 2h 5 min Effect hepatic toxicity intense intoxication, righting reflex still present tremor, ataxia deep narcosis; persisting lung hyperemia for > 2 weeks deep anesthesia, loss of cornea! reflex, no visible gross pathology anesthesia, liver centrilobular vacuolization, slight increase of AKT and SDH activity in serum deep anesthesia muscular incoordination deep narcosis, fatty liver infiltration, lung congestion for > 2 weeks no visible effects ataxia, unsteadiness on feet quiet, apparent unconsciousness narcosis, slow and shallow respiration, unsteadiness unsteady gait and muscular incoordination unconsciousness, slightly hyperemic lungs persisting for 2 weeks after exposure congestion of the lung even 2 weeks after exposure deep narcosis deep narcosis myocardial depression Reference Lester etal., 1963 Mastromatteo et al., 1960 Mastromatteo et al., 1960; Jaeger etal., 1974 Lester etal., 1963 Jaeger etal., 1974 Lester etal., 1963 Mastromatteo et al., 1060 Mastromatteo et al., 1960 Patty etal., 1930 Patty etal., 1930 Patty etal., 1930 Patty etal., 1930 Mastromatteo et al., 1960 Mastromatteo et al., 1960 Mastromatteo et al., 1960 Prodanetal., 1975 Prodanetal., 1975 Belejetal., 1974 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 3.3. Developmental/Reproductive Toxicity 42 No studies concerning the effect of single VC exposure on developmental or reproductive toxicity 43 have been identified. John et al. (1977, 1981) exposed pregnant CF-1-mice to 50 or 500 ppm VC, 44 Sprague-Dawley-rats and New-Zealand-rabbits to 500 or 2,500 ppm VC during organogenesis (7 h/day, 45 days 6-15 for mice and rats and days 6 - 18 in rabbits). Exposure of bred animals was conducted in 46 stainless steel chambers of 3.7 m3 volume under dynamic conditions. The atmosphere of VC was 47 generated by diluting gaseous VC with filtered room air at a rate calculated to give the desired 20 ------- Vinyl chloride INTERIM 1: 3/2008 1 concentration. The actual atmosphere was measured with an infrared spectrophotometer (no further details 2 presented). Animals were sacrificed on day 18 (mice), 21 (rats) or 29 (rabbits) and a variety of parameters 3 determined. 4 Exposure to 500 ppm VC was maternally toxic to mice (5 of 29 bred females died), weight gain, 5 food consumption, and the absolute liver weight were decreased. Maternal toxicity was not evident in 6 mice exposed to 50 ppm. In mice exposed to 500 ppm VC, the number of live fetuses per litter and fetal 7 weight were decreased, this was probably due to the increased maternal toxicity, and fetal resorption was 8 increased. Moreover, fetal resorption was within the range of historical controls. Fetal crown rump-length 9 was significantly increased in mice exposed to 50 ppm VC, but not in mice of the 500 ppm group. 10 Delayed ossifications in skull and sternum bones and unfused sternebrae were observed at 500 ppm in 11 mice fetus. 12 Rats exposed to 500 ppm gained less weight than controls, but the body weight was not 13 significantly different from the control. At 2,500 ppm, one maternal death among 17 bred females, 14 decreased food consumption and an increase in absolute and relative liver weight were observed. No 15 significant changes were observed in rat fetuses, except for reduced fetal body weight and increased 16 crown-crump length at 500 ppm (both effects not observed at 2,500 ppm). At 2,500 ppm the incidence of 17 dilated ureter was significantly increased in comparison to the control group and the number of lumbar 18 spurs was increased at 500 ppm but not at 2,500 ppm. 19 One of seven bred female rabbits died at 2,500 ppm, rabbits exposed to 500 ppm showed a 20 decreased food consumption, but body weight was not significantly affected. The number of live fetuses 21 per litter was slightly decreased as compared to concurrent air controls among litters of rabbits exposed to 22 the lower level of 500 ppm (live fetuses/litter: 8 and 7 at 0 and 500 ppm, respectively), but no effect on 23 litter size resulted from exposure to 2,500 ppm of VC. Ossification of the sternebrae was delayed at 500 24 ppm, but not at 2,500 ppm. 25 Most of the observed effects were exaggerated when feeding 15% ethanol in the drinking water 26 indicating an additive fetotoxic effect of ethanol and VC. This difference between species should be 27 correlated to the doses which in rats and rabbits exceed the threshold of metabolic saturation, whereas in 28 mice this threshold likely has not been reached. The authors attribute the observed developmental changes 29 to maternal toxicity "exposure to VC did not cause significant embryonal or fetal toxicity and was not 30 teratogenic...". 31 CFY rats were exposed to 1,500 ppm VC for 24 h/d during the first (day 1-9)), second (day 8-14) 32 or third trimester (day 14 to 21) of gestation. The volumes of the inhalation chambers were 0.13 m3, the 33 vertical flow rate of the air 2 m3/h at a regulated temperature of 24 - 25 °C and 50 - 55% relative humidity. 34 VC concentration in the inhalation chamber was determined by a gas chromatograph. Section was 35 performed on the 21st day of gestation. Treatment resulted in significantly increased frequency of 36 resorptions in the group exposed during the first trimester (2 fetuses resorbed in the control group vs. 12 37 fetuses in the exposed group; fetal loss in %: 1.7 in the control group and 5.5 in the exposed group). Two 38 cases of central nervous system malformations were recorded in treated animals (not significant), no 39 increase in other malformations were detected. The absolute and relative maternal liver weight was 40 increased in animals treated in the first and second week of pregnancy without light microscopic visible 41 changes, but not in animals exposed during the third week of pregnancy (Ungvary et al., 1978). 42 A study investigating embryo-fetal/developmental toxicity and reproduction (2-generation) was 43 conducted by Thornton et al. (2002). In the developmental toxicity study, Sprague-Dawley rats were 21 ------- Vinyl chloride INTERIM 1: 3/2008 1 exposed during day 6-19 of gestation to VC-concentrations of 0, 10, 100 or 1100 ppm for 6 h/day. 2 During exposure animals were housed in stainless steel, wire mesh cages within a 6000 liter stainless steel 3 and glass exposure chamber. Placement of the animals was rotated at each exposure. No feed was 4 provided during exposure, but water was available ad libitum. The temperature was 16-28 degree Celsius; 5 the relative humidity was 29-79 %; the air flow rate was 1200 liters per minute. VC was delivered from a 6 compressed gas cylinder to a Scott Specialty Gases regulator equipped with inlet and outlet back pressure 7 gauges, gas test atmosphere was analyzed hourly with an Ambient Air analyzer equipped with a strip 8 chart recorder. Maternal body weight gains were slightly, but statistically significantly suppressed at all 9 exposure levels during GD 15-20 and 6-20. At 100 ppm the relative kidney weight and at 1100 ppm the 10 relative kidney and liver weights were statistically significantly increased in maternal animals. No further 11 adverse effects were observed in this study. 12 In the 2 generation study, (Thornton et al., 2002) exposure started 10 weeks pre-mating. Other 13 experimental details are provided above. One male rat in the 10 ppm group and one female rat in the 14 control group died. Mating indices and pregnancy rates for the FO generation were comparable between 15 control and VC exposed groups. The live birth index was significantly decreased while the number of 16 stillborn pups was significantly increased in the FO generation group exposed to 1100 ppm (the authors 17 did not regard these effects as exposure related as they were not dose dependent and in the range of the 18 historical controls). In the FO generation male rats, absolute and relative liver weights were significantly 19 increased in all exposure groups. Absolute epididymis and kidney weights were increased in 100 ppm 20 male rats of the FO. Whereas there were no changes in the liver weight of female FO rats, there were 21 histological alterations in the liver at all dose groups (hepatocytes were enlarged with increased 22 acidophilic cytoplasm within the centrilobular areas of the liver). Centrilobular hypertrophy was observed 23 in male and female rats exposed to 100 and 1100 ppm and in 2 females of the 10 ppm group. 24 One male rat in the control group of the Fl died due to unknown reasons. In the F2 litters, there 25 was a statistically significant decrease in the mean number of pups delivered in the 1100 ppm group. The 26 authors regarded this effect not as exposure related as the values were lower than respective Fl control 27 group values, but comparable to the FO control group values. In the Fl there was a statistically significant 28 increase in the absolute and relative liver weight for male rats exposed to 100 and 1100 ppm (absolute 29 liver weight also increased in female rats, but not statistically significant). Also the absolute and relative 30 spleen weight was increased in male rats of the highest dose group. Male (100 and 1100 ppm) and female 31 (all dose groups) rats showed centrilobular hypertrophy. Additionally, altered foci (acidophilic, basophilic 32 and clear cell foci) were observed in male and female rats of the Fl of the 1100 ppm group, sometimes 33 even at the 100 ppm group (foci were also observed in 2 male rats of the FO at 1100 ppm). 34 3.4. Genotoxicity 35 The mutagenic properties of VC have been tested in a variety of bacteria with the Ames test. S. 36 typhimurium TA 100 and TA 1535 yield positive results at high concentrations and long exposure times, 37 especially with metabolic activation systems added. In other test systems VC is genotoxic only after 38 metabolic activation, e.g. in forward mutation assays and gene conversion assays in yeast, cell 39 transformation assays, UDS or SCE assays in mammalian cells (summarized in WHO, 1999a). The tests 40 were performed either with 5 - 100% VC in the atmosphere or 0.025 - 50 mM VC in the culture medium. 41 In vivo assays for genotoxicity were performed with mice, rats, and hamsters. VC has also been 42 tested in Drosophila melanogaster. Increased host-mediated forward mutations were observed after oral 43 VC exposure, whereas dominant lethal assays in mice exposed by inhalation and rats as well as a mouse 44 spot test gave negative results. Micronucleus formation in mice (50,000 ppm, 4 - 6h, 1,000 ppm 2 x 4h), 22 ------- Vinyl chloride INTERIM 1: 3/2008 1 cytogenetic aberrations in rats (1,500 ppm for 1 - 12 weeks) and hamsters (25,000 ppm for 6-24 hours) 2 and loss of sex chromosomes in Drosophila melanogaster (50,000 ppm for 48 hours) indicated dose 3 related chromosomal abnormalities. Also, increased DNA damage was demonstrated by alkaline elution 4 assays in mice and SCE formation in hamsters (summarized in WHO, 1999a). Further experiments with 5 known VC metabolites indicate that genotoxic effects are likely mediated by reactive intermediates with 6 chloroethylene oxide being most effective. 7 DNA adducts of VC metabolites with miscoding properties have been directly detected after 8 incubation of bacterial or phage DNA in vitro or in E. coli cells with DNA adduct indicator systems in 9 vivo with activated VC (summarized in WHO, 1999a). Covalent binding has been frequently observed 10 after single and short term exposure. 11 12 Bolt et al. (1980) detected irreversible attachment of radioactivity [1,2-14C] VC to hepatic 13 macromolecules in the rat. After single exposure of adult rats to 250 ppm [14C] VC for 5 hours the total 14 amount metabolized per individual rat was 37 |J,mol. 23 pmol VC-metabolites/ 100 mg liver wet weight 15 were irreversibly bound to DNA. d-guanosine alkylation products amounted to 0.35 pmol. 16 Laib et al. (1989) exposed adult Wistar rats to 700 ppm [1,2-14C]VC. The animals received either 17 a single 6-h exposure, or 2 single 6-h exposures separated by a treatment free interval of 15h. The 18 following amounts of [14C]VC-derived radioactivity in liver DNA was observed: after a single exposure of 19 male rats the activity was 3.6±0.2 pmol 7-(2'-oxoethyl)guanine (OEG) /mg DNA, after 2 exposures 20 (female rats): 5.2±0.5 pmol OEG/mg DNA±SD. 21 Watson et al. (1991) exposed adult male Fisher 344 rats (nose only) for 6 hours to atmospheres 22 containing nominally 1, 10, or 45 ppm [1,2-14C] VC. The alkylation frequencies of OEG in liver DNA 23 were 0.026, 0.28 and 1.28 residues OEG per 106 nucleotides respectively. These data indicate a linear 24 relationship between exposure dose and DNA dose in rats. There was no evidence to indicate the 25 formation of the cyclic adducts l,N6-ethenoadenine (sA) or 3,N4-ethenocytosine (sC). The threshold for 26 detection of these adducts were about 1 adduct per 1 x 10s nucleotides. 27 Swenberg et al. (2000) reported dose-dependent data on etheno-adducts using a new combination 28 of immunoaffmity /GC-high resolution MS. Adult F344 rats were exposed to 0, 10, 100, 1100 ppm VC 29 for 6 hours/day, 5 days/week for 1 or 4 weeks. The mean for N2,3-ethenoguanine (sG) in a mixed liver 30 cell suspension from unexposed control rats was 90 ± 40 fmol/|J,mol guanine. Exposure to 10 ppm VC for 31 1 or 4 weeks resulted in 200 ±50 and 530 ± 11 fmol/|J,mol guanine, while exposure to 100 ppm VC 32 caused 680 ± 90 and 2280 ±180 fmol / |J,mol guanine at 1 or 4 weeks, respectively. A much lesser effect 33 was evident for the 11-fold greater exposure of 1100 ppm due to saturation of metabolic activation, with 34 1250 ± 200 and 3750 ± 550 fmol/|J,mol guanine being present in liver. 35 In addition to these studies, there exist several investigations on the differences in sensitivity of 36 young (preweanling) vs. adult animals. Laib et al. (1989) tested 11-day-old and adult Wistar rats by 37 exposure to 700 ppm [1,2-14C]VC. Adult rats received either a single 6-h exposure, or 2 single 6-h 38 exposures separated by a treatment free interval of 15h. Pups received 2 single 6h-exposures, according to 39 the same treatment schedule. The following amounts of [14C]VC-derived radioactivity in liver DNA was 40 observed after 2 exposures (female adults, male and female pups): 5.2±0.5 pmol OEG/mg DNA±SD 41 (adults), 25.5±3.0 pmol OEG/mg DNA( pups). After a single exposure of adult male rats the activity 42 (3.6±0.2 pmol OEG/mg DNA) was close to the observation after two exposures. 23 ------- Vinyl chloride INTERIM 1: 3/2008 1 After a five day exposure of F344 rats to 600 ppm (4h/d) the adduct levels in the liver were 162 ± 2 36 pmol OEG/ |J,mol guanine and 1.81 ±0.25 pmol sG / |J,mol guanine for the pups and 43 ± 7 pmol 3 OEG/ |J,mol guanine and 0.47 ± 0.14 pmol sG / |_imol guanine for the adult animals (Swenberg et al., 4 1999). 5 Ciroussel et al. (1990) compared the levels of l,N6-ethenodeoxyadenosine (sdAdo) and 3,N4- 6 ethenodeoxycytidine (sdCyd) in BD VI rats with pups (7 days old) vs. adults (13-week-old animals). 7 These rats had been exposed to 500 ppm VC for 2 weeks (7h/d, 7d/w). The molar ratios (x 10"7) in the 8 liver were 1.30, 1.31 (two analyses; sdAdo/dAdo) and 4.92, 4.67 (sdCyd/dCyd) for the newborn 9 compared to 0.19 (sdAdo/dAdo) and 0.8 (sdCyd/dCyd) for the adult animals. 10 Fedtke et al. (1990) measured the sG content in the liver of lactating Sprague-Dawley rats and 11 their 10 days old pups exposed to VC (600ppm, 5 days, 4h/d). sG concentrations found in DNA livers of 12 the dams were 470 ±140 (adults) compared with 1810 ±250 fmol/|J,mol (pups). The mean background 13 found in the control DNA was 60 ±40 fmol/|J,mol (background subtracted from sG concentration). 14 Similarly, Morinello et al. (2002) demonstrated higher sG-adduct levels in hepatocytes after exposure of 15 weanling rats to 10 ppm for 1 week (6h/d) compared to adult animals (control adult: 0.55 ±0.14 mol sG / 16 107 mol guanine; pups: 0.16 ±0.01; exposed adult: 1.4 ±0.4; pups: 4.1 ±0.8). Adducts largly persisted after 17 recovery over 5 weeks. 18 Etheno adducts may be repaired by DNA glycolases, but a) did not fully return to background 19 levels after a exposure free period of 14 days (sG: directly after exposure 1,8 pmol/|J,mol, after 14 days: 20 0,47 pmol/|J,mol; control level: 90 fmol/|J,mol), b) have a miscoding potential in vitro and in vivo 21 (Swenberg etal., 1999). 22 Gene mutations were found in animal tumors associated with exposure to etheno-adduct-forming 23 chemicals such as VC. Specifically, in rat hepatocellular carcinoma in 7 of 8 cases A->T mutations of the 24 Ha-ras gene have been found and in angiosarcoma of the rat liver in 10 of 25 cases various base pair 25 substitutions as mutations ofp53 were observed, which may be attributed to the formation of ethenobases 26 in DNA (Barbin, 2000). 27 3.5. Carcinogenicity 28 Inhalation exposure of rats to VC causes liver tumors, especially angiosarcomas and 29 hepatocellular carcinoma and neoplastic liver nodules. Furthermore, angiosarcomas of other sites are 30 reported. Additionally, tumors at other locations are found, e.g. Zymbal gland, neuroblastoma and 31 nephroblastoma in rats; lung tumors in mice; mammary gland tumors in rats, mice, and hamsters, and skin 32 tumors in rabbits and hamsters (summarized in WHO, 1999a, ATSDR 1997). Similar tumor localizations 33 are observed after oral exposure. There is evidence that liver tumors are induced in female rats at lower 34 doses than in males. There is also evidence, that animals are more susceptible to tumor induction early in 35 life (WHO, 1999a). 36 Short term exposure experiments from Drew et al. (1983) and Maltoni et al. (1981) indicate 37 increased susceptibility of newborn and young animals. Drew et al. (1983) found increased incidences of 38 tumors in rats, mice and hamsters when exposed for the first 6 month in life, but not at later exposure 39 times, e.g. exposure of rats to 100 ppm VC during month 0-6 or 6-12 resulted in a tumor incidence 40 (hemangiosarcoma of the liver) of 5.3% or 3.8%, respectively, but no tumors occurred when rats were 41 exposed during month 12 - 18 or 18 to 24. 24 ------- Vinyl chloride INTERIM 1: 3/2008 1 Maltoni et al. (1981, 1984) exposed newborn rats postnatally from day 1 to 5 weeks of age to 2 6,000 ppm or 10,000 ppm VC by inhalation (4 h/d; 5 d/w). At 6,000 ppm the number of exposed animals 3 were 42 (18 male; 24 female); at 10,000 ppm the respective number was 44 (24 male; 20 female). The 4 number of respective breeders were 6 for each exposure concentration. No direct control group was used; 5 however, in parallel experiments breeders and newborn animals without exposure were included (see 6 Experiment BT 4001, 4006). The newborn animals were simultaneously exposed to milk from exposed 7 dams (D. Soffritti, Laboratory of Prof. Maltoni, personal communication, August, 2003). The authors 8 found liver angiosarcomas in newborn SD rats in 17/42 and 15/44 animals respectively, exposed to 6,000 9 ppm or 10,000 ppm, but none in their mothers which were treated identically. No angiosarcoma were 10 found in a control group of 304 rats (parallel experiment). Additionally, hepatoma incidence was 11 increased in newborn rats (20/42 and 20/42, respectively), but no hepatoma were observed in their 12 mothers. Only 1 hepatoma were found in a control group of 304 rats (parallel experiment). Results were 13 provided after 124 weeks of observation. The internal concentration of VC may have been influenced by 14 oral uptake from milk from exposed dams. However, due to the very high inhalation exposure and due to 15 saturation of metabolism, the oral uptake by contaminated milk may have contributed only a limited 16 amount to the overall organ concentration of VC metabolites. 17 Froment et al. (1994) exposed 4 female Sprague-Dawley rats together with their pups (22 males 18 and 22 females) for 8h/d, 6d/w to 500 ppm VC from day 3 through 28 postpartum. At day 28 postpartum, 19 the young animals were weaned, and the males and females were separated and exposed for further 2 20 weeks (total exposure: 33 days). The surviving animals were all sacrificed at 19 month of age. In the 44 21 VC-exposed rats 66 hepatic lesions were identified including nodular hyperplasia, endothel cell 22 hyperplasia, peliosis, adenomas, benign cholangiomas, angiosarcoma of the liver (ASL) and 23 hepatocellular carcinoma (HCC). Liver tumors included 8 HCC, 15 ASL and 2 benign cholangioma. No 24 further details were provided. It is assumed that oral exposure via mothers'milk and inhalation exposure 25 occurred simultaneously. 26 Maltoni et al., (1981, 1984) also exposed rats 30 breeders/ exposure group to 6,000 and 10,000 27 ppm for 1 week (4h/d; 12th until 18th day of pregnancy). 32 (13 males; 19 females) and 51 (22 males; 29 28 females) offsprings were investigated after exposure to the lower or the higher concentration, respectively. 29 Angiosarcoma of the liver and hepatoma were not increased in the transplacentally exposed offsprings. 30 However, Zymbal gland carcinoma and nephroblastoma were found elevated after transplacental 31 exposure. Differences between pre- and postnatal exposure and carcinogenic outcome may possibly be 32 explained by hepatic CYP2E1 activity, which is expressed to a lower extent prenatally than postnatally, 33 both in rats (Carpenter et al., 1997) and in humans (Cresteil, 1998). 34 Hehir et al. (1981) found increased lung tumor incidences in ICR mice exposed once for 1 h to 35 VC (age of the animals not stated). Animals were exposed in an inhalation chamber to single one-hour 36 doses of VC ranging from 50 to 50,000 ppm (Rochester type inhalation chambers, 1,000 liter with laminar 37 air flow) and were then observed for the remainder of their lives. Tumor response was dose related: 38 Adenoma of the lung increased from 12/120 to 14/139, 18/139, 24/143, 45/137 respectively for exposure 39 to 0, 50, 500, 5000, 50000 ppm. For carcinoma of the lung, there was only a slight occurrence of 0/120, 40 0/139, 1/143, 3/137 (data from both sexes, combined). A slight increase in hepatic cell carcinoma 41 occurred in male mice, but without dose response (2/50; 2/64; 9/67; 6/68; 4/63). No increase in tumor 42 incidence was observed in liver and lung of rats treated in an identical fashion. Additional studies in A/J 43 mice which were exposed to 500 ppm VC for 1 h/d over 10 days or 50 ppm VC for 1 h/d over 100 days 44 revealed that for short term exposure the concentration may be the most critical factor. In both 45 experiments primarily pulmonary adenomas were observed. However, the incidence in the induction of 46 adenomas and progression to carcinoma are considered only marginal and not statistically significant in 25 ------- Vinyl chloride INTERIM 1: 3/2008 1 mice exposed to 50 ppm for 100 times (44.1% exposed; 34.5% control) whereas a significant increase of 2 pulmonary adenomas was observed in animals exposed to 500 ppm for 10 days (about 74% exposed; 3 34.4% control). 4 Suzuki (1983) also reported that short term exposure (6 h/d; 5 d/w; 4 weeks) of young CD1 mice 5 (5-6 weeks old at first exposure) to VC resulted in tumor formation. At sacrifice 12 weeks after exposure 6 pulmonary tumors were observed in the two highest dose groups (300 and 600 ppm). Forty or 41 weeks 7 after exposure pulmonary tumors were observed in all animals exposed (1 ppm to 600 ppm) but not in 8 control mice. In addition, subcutaneous and hepatic hemangiosarcoma were found. The angiosarcoma of 9 the liver occurred in one animal exposed to 600 ppm for 4 weeks as observed at necroscopy 56 weeks 10 after exposure (Suzuki, 1981). 11 After a single 12 hour exposure to 1,500 ppm of mice a hepatocellular adenoma developed. The 12 respective concentration was lethal to most of the animals (Tatrai and Ungvary, 1981). However, the 13 observed effects (asphyxiation) were not seen in other studies with similar concentrations. 14 15 In addition to angiosarcoma of the liver several studies with limited exposure duration to VC 16 confirm the occurrence of hepatocellular carcinomas and/or other preneoplastic parenchymal changes in 17 adult animals (Feron et al, 1979; Thornton et al, 2002). However, these changes were seen to a much 18 lesser extent than angiosarcoma in the adult animals or hepatocellular changes in young animals (see 19 below). 20 In accordance with these investigations in newborn rats, Laib et al. (1985a,b) reported that 21 hepatocellular ATPase-deficient foci (pre-malignant stages) were observed in rats which were exposed to 22 VC. The exposure regimen was a) Wistar -rats for 10 weeks starting on day 1 after birth (10 to 2,000 ppm; 23 5 d/w; 8 h/d) (Laib et al., 1985a), b) Wistar and Sprague-Dawley rats to 2.5 to 80 ppm VC for 3 weeks 24 (8h/d) starting on day 3 of life (Laib et al., 1985a), c) Wistar rats exposed to 2,000 ppm VC for 5,11,17,47 25 or 83 days (8h/d; 7d/w) with different ages (after birth or from an age of 7 or 21 onwards) at the start of 26 exposure (Laib et al.,1985b). Exposure to 2,000 ppm did not result in ATPase deficient foci in very young 27 (exposure period: day 1 to 5) or in adult animals (exposure period: from day 90 to 160). However, 28 relevant foci areas were demonstrated for short periods during animal growth, eg., exposure for 11 days 29 (exposure period: from day 1 to 1 l)or for 21 days (from day 7-28). The foci persisted until evaluation at 30 the age of 4 month (Laib et al., 1985b). After exposure over 10 weeks, induction of ATPase deficient foci 31 was dose dependent (nearly linear) for concentrations between 10 ppm and 500 ppm and it was shown for 32 both strains of rat, Wistar and Sprague-Dawley. This finding is in accordance with the findings that VC- 33 metabolism follows first order kinetics until saturation occurs at high exposure concentrations (Laib et al., 34 1985a). 35 Quantitative risk assessments based on animal experiments have been published by several 36 authors and are summarized in Table 5. TABLE 5: QUANTITATIVE ASSESSMENT OF CARCINOGENIC POTENCY OF VC BASED ON ANIMAL EXPERIMENTS Author ChenundBlancato, 1989 EPA, 2000 a, b Unit Risk (per M.g/m3) 6.5xlO-7-1.4xlO-6 8.8 x 10-6 37 38 39 40 41 26 ------- Clewell etal, 1995 Clewell etal, 2001 Reitz etal., 1996 6 x lO'7 - 2 x lO'6 l.lxlO'6 5.7 x 10'7 Vinyl chloride INTERIM 1: 3/2008 1 2 3 4 These risk estimates are based on the experimental data in adult animals exposed for lifetime 5 published by Maltoni et al. (1981; 1984). There are only slight differences in the human cancer risk 6 estimated by Clewell and Reitz who both used pharmacokinetic (PBPK)-models for the transfer of the 7 animal data on the human situations. These data are in good agreement with the unit risk estimates derived 8 from epidemiologic data, confirming the order of magnitude. However, these risk estimates were only 9 validated with data from adult animals and epidemiologic data from the workplace. A higher sensitivity of 10 children was not incorporated into quantification (see data from Drew et al., 1983; Maltoni et al., 1981). 11 Chen and Blancato (1989) use a modified multistage model for risk estimation on base of liver 12 tumors, considering pharmacokinetic models. Additionally, increased sensitivity in early life stages has 13 been considered. They evaluated female and male animals separately, expressed by the range of tumor 14 incidences. 15 The most recently published risk estimate by EPA (2000a, b) is based on the animal experiments 16 published by Maltoni et al. (1981, 1984). Differences in the metabolism between animals and humans 17 have been taken into consideration by use of a pharmacokinetic model. The increased sensitivity of 18 children was taken into consideration. Additionally, tumors in sites other than the liver were considered. 19 Unit risk estimates based on epidemiologic studies were regarded as uncertain due to the shortcomings of 20 the epidemiologic studies. Besides the unit risk estimate for full lifetime exposure (birth through death) of 21 8.8 x 10~6 per |o,g/m3, EPA provided an estimate of risk for early life exposure of 4.4 x 10~6 per |o,g/m3 and 22 an estimate of risk for adult only exposure of 4.4 x 10~6 per |o,g/m3. This unit risk for adults is based on the 23 PBPK-modeling from Clewell et al. (2001), with only slight modifications in some parameters. 24 3.6. Summary 25 Acute exposure of experimental animals towards VC results in narcotic effects, cardiac 26 sensitization, and hepatotoxicity. Narcotic effects are characterized by a typical sequence of events from 27 euphoria and dizziness, followed by drowsiness and loss of consciousness. Finally, animals die due to 28 respiratory failure. Prodan et al. (1975) reported LC50 values for mice, rats, rabbits, and guinea pigs of 29 117,500 ppm, 150,000 ppm, 240,000 ppm and 240,000 ppm, respectively, for 2 hours. Dead animals 30 showed congestion of the internal organs (especially lung, liver and kidney), lung edema and hemorrhagia 31 (Prodan et al., 1975; Mastromatteo et al., 1960). No lethality was seen in mice after exposure to 100,000 32 ppm for 2 hours (Prodan et al., 1975). However, Tatrai and Ungvary (1981) reported that exposure of 33 mice to 1,500 ppm for 24 h resulted in death of all animals, reduction of exposure time to 12 h resulted in 34 death of 90% of the animals. These results are not in accordance with other lethality data. 35 Short term exposure (up to 30 minutes) of experimental animals to VC-concentrations of 100,000 36 to 300,000 ppm resulted mainly in ataxia, motor activity, side position and unconsciousness, slow and 37 shallow respiration, the typical reactions observed before the onset of narcosis (Mastromatteo et al., 38 1960). Narcosis was observed in rats and mice after 30 min exposure to 200,000 ppm VC (Mastromatteo 39 et al., 1960). Short term exposure (5 min) of dogs to VC induced cardiac sensitization towards 40 epinephrine (EC50: 50,000 or 71,000 ppm in two independent experiments) (Clark and Tinston, 1973; 41 1982). These effects were also seen in mice at higher concentrations (Aviado and Belej, 1974). In 27 ------- Vinyl chloride INTERIM 1: 3/2008 1 monkeys, only myocardial depression after inhalation of 2.5-10% VC was observed. It is not clearly 2 stated whether an addition challenge with epinephrine was applied or not (Belej et al., 1974). Single 3 inhalation exposure of rats for 6 hours to 100,000 ppm VC resulted in histopathological changes of the 4 liver (vacuolization), but was not observed in lower concentrations (50,000 ppm) (Jaeger et al., 1974). 5 However, in mice Tatrai and Ungvary (1981) reported that stasis of the liver developed 2 and 4 h after the 6 beginning of inhalation. The authors observed decreasing enzyme activities in liver and subcellular liver 7 damage at exposure concentrations of 1,500 ppm VC for 2 h; after 24 h shock liver developed. Repeated 8 exposure of rats to 1,500 ppm VC for up to 9 days during pregnancy caused increased relative and 9 absolute liver weights without light microscopic visible changes (Ungvary et al., 1978). In another 10 developmental study increased absolute and relative liver weights have been observed in rats exposed 11 intermittently to 2,500 ppm from day 6-15 of pregnancy, the NOAEL was 500 ppm (John et al., 1977; 12 1981). In rats exposed to 5,000 ppm for 7 hours/day and 5 days/week after 4 weeks vacuolized liver cells 13 were observed (Feron et al., 1979). 14 No investigations of reproductive or developmental toxicity after single exposure are published. 15 John et al. (1977, 1981) investigated developmental effects after repeated exposure in mice, rats and 16 rabbits. Developmental toxicity (e.g. delayed ossification) was only observed at maternal toxic 17 concentrations. Ungvary et al. (1978) reported that in maternal rats which were exposed to 1,500 ppm VC 18 for 24 h/d during the first (day 1-9) or second (day 8-14) trimester of gestation maternal liver toxicity 19 occurred. Frequency of resorptions was significantly increased in the group exposed during the first 20 trimester. A recently published developmental toxicity study in rats (exposure on day 6-19 of gestation 21 towards 10, 100 or 1100 ppm VC, 6 h/d) indicated that up to 1100 ppm embryo-fetal development was 22 not affected by VC exposure. The only toxic effects observed were an increased relative organ to body 23 weight ratio for the kidney and liver at 1100 ppm and for the kidney at 100 ppm in dams (Thornton et al., 24 2002). In a 2-generation study in rats no adverse effects on embryo-fetal development or reproductive 25 capability were observed over 2 generations in concentrations up to 1100 ppm (F0: exposure: 10 weeks 26 premating, 3-weeks mating, gestation, lactation; F^ identical exposure pattern; F2: until postnatal day 21). 27 The primary target organ of VC, the liver, was affected as evidenced by an increase in liver weight and/or 28 histopathologically identified cellular alterations, such as centrilobular hypertrophy and induction of 29 altered hepatocellular foci at 100 and 1,000 ppm, with increased incidence in the Fl generation (Thornton 30 et al., 2002). 31 Positive results on genotoxicity after in vitro and single and repeated in vivo treatment (e.g. 32 induction of micronuclei, 4 - 6 h, 50,000 ppm; chromosomal aberrations, 6 - 24 h, 25,000 ppm) have been 33 reported for VC (WHO, 1999a). Elevated DNA-adducts were seen after single 5 hour exposure of adult 34 rats to 250 ppm (Bolt ,1976). Watson et al. (1991) exposed adult male Fisher 344 rats for 6 hours to 35 atmospheres containing 1, 10, 45 ppm VC. The alkylation frequencies of 7-(2'-oxoethyl)guanine (OEG) in 36 liver DNA were 0.026, 0.28 and 1.28 residues OEG per 106 nucleotides respectively. With these air 37 concentrations, there was no evidence to indicate the formation of the cyclic adducts l,N6-ethenoadenine 38 (sA) or 3,N4-ethenocytosine (sC). The threshold for detection of these adducts were about 1 adduct per 1 39 x 10s nucleotides. Adult rats repeatedly (5 days) exposed to 10 ppm VC for 6 hours/day showed slightly 40 elevated etheno-adducts for N2,3-ethenoguanine (sG) compared to control (200 ± 50 vs. 90 ± 40 finol/ 41 |J,mol guanine) (Swenberg et al., 2000). Higher adduct levels were seen in young animals than in adult 42 animals after identical treatment (Fedtke et al., 1990; Laib et al., 1989; Ciroussel et al. (1990). OEG are 43 not likely to cause mutations, however, the cyclic adducts sA, sC, sG have miscoding potential; 44 respective mutations (e.g., G->A transitions, A->T transitions) were observed in VC-induced tumors 45 (Barbin, 2000). Despite relevant repair, no full reduction to background was observed for these adducts 46 two weeks after a 5 day exposure (4 hours/day) to 600 ppm (Swenberg et al., 2000). 28 ------- Vinyl chloride INTERIM 1: 3/2008 1 Induction of liver tumors has been reported in rats after subacute (5 week and 33 days, 2 respectively) exposure (Maltoni et al., 1981; 1984; Froment et al. , 1994). The liver is the primary 3 localization of tumors after chronic exposure (for review see EPA, 2000a, b).Vinyl chloride induces lung 4 tumors in mice after single one hour exposure to 5,000 ppm or 50,000 ppm (Hehir et al., 1981). After a 5 single 12 hour exposure to 1,500 ppm of mice a hepatocellular adenoma developed. The respective 6 concentration was lethal to most of the animals (Tatrai and Ungvary, 1981). Suzuki (1983) reported that 7 short term exposure (6 h/d; 5 d/w; 4 weeks) of young CD 1-mice (5-6 weeks old at first exposure) to VC 8 resulted in lung tumor formation. Additionally, subcutaneous and hepatic hemangiosarcoma were found 9 in this study. Short term exposure experiments from Drew et al. (1983), Maltoni et al. (1981) and Froment 10 et al. (1994) also indicated increased susceptibility of newborn and young animals towards tumor 11 formation. Hepatoma (Maltoni et al., 1981) or hepatocellular carcinoma (Froment et al., 1994) developed 12 to a greater extent in young than in adult animals. Laib et al. reported that hepatocellular ATPase-deficient 13 foci (pre-malignant stages) were observed in rats which were exposed to VC. Relevant foci areas were 14 demonstrated after short periods of exposure during animal growth, eg., exposure to 2,000 ppm for 11 15 days (exposure period: from day 1 to 11) or for 21 days (from day 7-28). The foci persisted until 16 histological examination at the age of 4 month (Laib et al., 1985b). 29 ------- Vinyl chloride INTERIM 1: 3/2008 1 4. SPECIAL CONSIDERATIONS 2 4.1. Metabolism and Disposition 3 Krajewski et al. (1980) estimated the retention of VC after inhalation through a gasmask in 5 male 4 human volunteers by measuring the difference between inhaled and exhaled concentrations. Exposure to 5 concentrations between 3 and 24 ppm VC for 6 hours revealed an average retention of 42%, independent 6 from VC concentration. Thirty minutes after the beginning initially higher retention values (maximum 7 46% on average) dropped down and stayed on a relative constant level. Interindividual retention rates 8 varied from 20.2% to 79% at 12 ppm. Immediately after cessation of inhalation the VC concentration in 9 the expired air dropped rapidly. After 30 minutes less than 5% of the initial chamber concentration could 10 be measured. Buchter et al. (1978) determined a retention rate of 26 - 28% at 2.5 ppm VC in two 11 individuals 3-5 min after the start of inhalation. Given the variability of VC retention found by 12 Krajewski these values may be attributed to interindividual differences. WHO (1999a) reports an average 13 of 30 - 40% absorption after inhalation, without citing the relevant studies. 14 An absorption of inspired VC of about 40% was calculated for rats (calculation based on the 15 decline of 14C-VC in a closed system) (Bolt et al., 1976). In Rhesus monkeys VC is also efficiently 16 absorbed after inhalation as can be deduced from data on the metabolic elimination (no further 17 quantification) (Buchter et al., 1980). 18 Whole body (excluding the head) exposure of rhesus monkeys to radioactive VC indicated that 19 only very little VC was absorbed through the skin (about 0.031% and 0.023% at 800 and 7,000 ppm, 20 respectively after 2 - 2.5 h) (ATSDR, 1997). No further data on dermal absorption are available. 21 The percentage of the dose remaining in the carcass after oral application after 72 h was 10, 11, 22 and 2% for the 0.05, 1 and 100 mg/kg doses. The data suggest that almost complete elimination of VC 23 occurred (Watanabe et al., 1976b). Seventy two hours after exposure to 10 and 1,000 ppm radioactive VC 24 14 and 15%, respectively, of the recovered 14C-activity remained in the carcass of rats, VC per se was not 25 found in tissues. Radioactivity was detected in the liver, skin, plasma, muscle, lung fat and kidney, 26 representing non volatile metabolites of VC (Watanabe et al., 1976a) or incorporation into Q-pool (Laib 27 etal., 1989). 28 Data on serum concentrations of VC are scarce. Ungvary et al. (1978) exposed pregnant rats to 29 2,000 - 12,000 ppm VC; they determined blood concentrations ranging from 19 |J.g/ml at 2,000 ppm to 30 48.4 |-lg/ml at 12,000 ppm VC indicating no direct proportionality between air VC concentration and 31 blood concentration. Feron et al. (1975) reported a peak blood concentration of 1.9 |-lg/ml 10 min after 32 gavage of 300 mg/kg VC; this value is much smaller than expected compared to blood concentrations 33 after inhalation which might be due to the effective presystemic hepatic clearance of VC after oral uptake. 34 Similar to other inhalation anaesthetics, maximal blood concentration of VC after inhalation 35 exposure depends on the partial pressure of VC in the air. Blood respectively brain concentration, which 36 directly correlates with the depth of narcosis (see below) and - presumably - with cardiac sensitization 37 level, can be controlled by changing the concentration of VC in the air, i.e. by changing the partial 38 pressure of VC in the air. If equilibrium is reached between the partial pressure of VC in the air and in the 39 blood (steady state), no further increase of VC concentration in the blood is possible, even if the exposure 40 time is prolonged (Forth et al., 1987). The time necessary to set up steady state mainly depends on the 41 blood/air partition coefficient of a substance. The blood/air partition coefficient of VC in humans is 1.2 42 (Csanady and Filser, 2001), similar to that of the inhalation anaesthetic isoflurane (1.4; Forth et al., 1987). 43 For this substance the equilibrium is reached after about 2 hours, derived by graphical extrapolation of the 30 ------- Vinyl chloride INTERIM 1: 3/2008 1 data on isoflurane (Goodman and Oilman, 1975). For VC, in much lower concentrations an elimination 2 half-time of VC of 20.5 minutes has been derived (Buchter, 1979; Bolt et al, 1981). From that, for low 3 concentrations a steady state concentration for VC in blood of about 5 x 20.5 = 102.5 minutes can be 4 calculated by standard estimation rules. Thus, in high or low concentrations a relevant increase of internal 5 concentrations of VC is not to be expected after more than 2 hours of exposure. However, for shorter 6 periods of exposure a relevant influence of time on the built-up of VC on internal concentrations has to be 7 taken into account. 8 VC is oxidized by cytochrome P450 2E1 to the highly reactive epoxide 2-chloroethylene oxide 9 which can directly interact with DNA and proteins or spontaneously rearrange to 2-chloroacetaldehyde 10 which might bind to proteins and DNA. 2-Chloroethylene oxide can also be transformed to glycol 11 aldehyde by epoxide hydrolase or react with glutathione leading to the formation N-acetyl-S-(2- 12 hydroxyethyl)-cysteine. Chloroacetaldehyde is oxidized by aldehyde dehydrogenase to 2-chloroacetic 13 acid which reacts with glutathione leading to the formation of thiodiglycolic acid (which leads to the 14 liberation of carbon dioxide). Comparison of in-vitro metabolism with rat liver microsomes and in-vivo 15 experiments in rats show that virtually all the metabolic activation of VC in vivo occurs in the liver 16 (WHO, 1999a). After low doses VC is metabolically eliminated and non volatile metabolites excreted 17 mainly in the urine. At doses that saturate the metabolism, the major route of excretion is exhalation of 18 unchanged VC. Excretion of metabolites via feces is only a minor route, independent of applied dose 19 (WHO, 1999a). 20 Buchter et al. (1980) exposed rhesus monkeys with 100 - 800 ppm VC and measured the time- 21 dependent disappearance of VC from the atmosphere. The maximum metabolic rate was determined at 45 22 |J,mol/kg-hr; this turnover is obtained at 400 ppm VC; no attempt was made to identify the metabolites 23 formed. From the decrease in atmospheric VC concentration metabolic clearance rates were calculated in 24 liter air/hour/kg body weight. Clearance rates for monkeys, rabbit and humans are 2.0 - 3.55 1/hr-kg, for 25 gerbils and rats 11.0 to 12.5, and 25.6 1/hr-kg for mice, indicating major species differences, which are in 26 accordance with allometric scaling. 27 After oral ingestion of 0.05, 1.0 or 100 mg/kg body weight, male rats metabolize VC to the 28 epoxide which is further metabolized (e.g. to thiodiglycolic acid: about 25% of the 14C containing urinary 29 metabolites). Of the total dose, 9, 13.3 and 2.5% are excreted as CO2 or 1.4, 2.1 or 66.6% VC, 30 respectively at the low, mid and high dose (Watanabe et al., 1976b). At 100 mg/kg bw pulmonary 31 elimination showed a biphasic clearance with an initial half life of 15 min and a terminal half life of 41 32 min. After 0.05 and 1 mg/kg VC only monophasic pulmonary clearance could be observed with half life 33 values of 53 - 58 min (Watanabe et al., 1976b). Initial urinary excretion of metabolites followed first 34 order-kinetics with half life values of 4.5 - 4.6 hours, followed by a slow terminal phase (Watanabe et al., 35 1976b). Thus, the equilibrium concentration for metabolites will not be reached within 8 hours or less. 36 The ratio of the metabolites excreted in the urine does not vary in dependence on dose. 37 Vinyl chloride metabolism is saturated at concentrations exceeding 380 ppm in Rhesus monkeys 38 (Buchter et al., 1980) (see table 6). In humans, 24 ppm appears to be below the threshold of saturation 39 (Krajewski et al., 1980) since no difference in pulmonary retention could be observed between 3, 6, 12 40 and 24 ppm VC. When exposing rats in a closed system with 50 - 1,000 ppm VC metabolic clearance was 41 slowed at concentrations above 220 ppm as evidenced by longer half lives (Hefner et al., 1975). Bolt et al. 42 (1977) exposed rats in a similar system and found metabolic saturation to occur at 250 ppm (see table 6). 43 These data are in accordance with the data from Watanabe et al. (1976a): after inhalation of 1,000 ppm in 44 rats metabolism was saturated, whereas at 100 ppm VC saturation was not evident (no intermediate 45 concentration was tested). 31 ------- Vinyl chloride INTERIM 1: 3/2008 1 Saturation of the metabolism has also been observed after oral application: at high doses (100 2 mg/kg) metabolism was saturated as is evident from the increase in VC expiration from 2.1% at 1 mg/kg 3 to 66,6% at 100 mg/kg (Watanabe et al, 1976b). TABLE 6: METABOLIC SATURATION CONCENTRATIONS OF VC IN RATS AND MONKEYS Rhesus Monkey Rat about 380 ppm (Buchter et al., 1980) 250 ppm (Bolt etal., 1977) 7 VC metabolites are assumed to destroy cytochrome P450 enzymes responsible for its epoxidation 8 (Du et al., 1982; Pessayre et al., 1979). On the other hand activity of glutathione-S-transferase and 9 glutathione reductase is elevated after VC exposure of rats (glutathione content is reduced) representing 10 an early hepatocellular adaption to VC exposure (Du et al., 1982). 11 4.2. Mechanism of Toxicity 12 Acute neurotoxicity by inhalation of high VC concentrations is likely dependent upon VC 13 concentrations and independent of VC metabolism. This assumption is supported by comparison of 14 narcotic concentrations which are similar for the four species guinea pig, mouse, rabbit and rat (Prodan et 15 al., 1975; Mastromatteo et al., 1960). Vinyl chloride has been investigated as a possible human anesthetic 16 (Oster et al., 1947; Peoples and Leake, 1933) but was abandoned because of its induction of cardiac 17 arrhythmia. 18 Acute toxicity/lethality is mainly accompanied by congestion of all internal organs, pulmonary 19 edema, liver and kidney changes (up to necrosis) (Prodan et al., 1975). The mechanism of action is not 20 evident, toxic effects are possible mediated by reactive metabolites. 21 VC genotoxicity and carcinogenicity has been attributed to formation of reactive metabolites, 22 especially 2-chloroethylene oxide and 2-chloroacetaldehyde (see WHO, 1999a). 2-Chloroethylene oxide 23 interacts directly with DNA and produces alkylation products (Fedtke et al., 1990). This alkylation results 24 in a highly efficient base-pair substitution that leads to neoplastic transformation (ATSDR, 1997). VC- 25 DNA ethenobases are shown to lead to miscoding and are found in VC-induced tumors in animals and 26 humans (Barbin, 2000). Despite relevant repair, no full reduction to background was observed for these 27 adducts two weeks after a 5 day exposure (4 hours/day) to 600 ppm (Swenberg et al., 1999). For vinyl 28 fluoride, when all of the data for rats and mice on sG and hemangiosarcomas were compared by 29 regression analysis, a high correlation was seen (^=0.88) (Swenberg et al.,1999). However, in case of VC 30 there is a close correlation in the occurrence of sA, sC, eG and there are indications that also sA might be 31 related to tumor formation (Barbin, 1999; Barbin, 2000). In adults, nonparenchymal cells have a higher 32 rate of proliferation than hepatocytes. Thus, this cell population is more likely to convert promutagenic 33 DNA adducts into mutations (Swenberg et al., 1999). During rapid growth of the liver this relationship 34 may be changed: Young animals demonstrate a high rate of etheno-adducts in the liver and a high rate of 35 preneoplastic foci of the liver. These foci persisted over several month even after short durations of 36 exposure (Laib et al., 1989). In young animals a high rate of hepatoma and hepatocellular carcinoma have 37 been found after short term exposure to VC (Maltoni et al., 1981; 1984; Froment et al., 1994). 38 In humans occupationally exposed to VC ,,vinyl chloride disease" (characterized by Raynaud's 39 phenomena and scleroderma) is a common finding after prolonged exposure. No similar observations 40 have been made in experimental animals after single exposition experiments. These effects are probably 32 ------- Vinyl chloride INTERIM 1: 3/2008 1 due to immunological abnormalities (caused by interaction of reactive VC metabolites with proteins) as 2 has been proposed by Grainger et al. (1980) and Ward et al. (1976), however, no definitive mechanism 3 has been elucidated to date. 4 4.3. Other Relevant Information 5 4.3.1 PBPK-Modeling 6 Physiology-based pharmacokinetic (PBPK) models have been proposed to predict VC metabolism 7 and cancer risk (Reitz et al., 1996; Clewell et al., 1995 and Clewell et al., 2001). PBPK models have been 8 developed to account for physiological differences among species relevant to VC uptake, distribution, 9 metabolism and excretion and should allow a better comparison across species. 10 Current models use four compartments (liver, fat, slowly perfused tissues, richly perfused tissues) 11 and partition coefficients determined in vitro. Metabolism is modeled by one (Reitz et al., 1996) or two 12 (Clewell et al., 1995) saturable pathways. The model of Clewell et al. (1995, 2001) uses one high affinity, 13 low capacity pathway likely pertaining to cytochrome P450 2E1, and one low affinity, high capacity 14 pathway tentatively assigned to cytochromes P450 2C11/6 and 1A1/2). Since VC readily reacts with 15 glutathione (GSH) and is known to deplete hepatic GSH stores, description of the GSH kinetics was also 16 included. 17 4.3.2. Inter species Variability 18 A comparison of the metabolic activity across species indicates mice to be the metabolically most 19 active species with a first order metabolic clearance rate for VC of 25.6 1/h per kg bw at VC 20 concentrations below metabolic saturation (Buchter et al., 1980). Clearance of rats, rhesus monkey, 21 rabbits and men is lower (11.0, 3.55, 2.74 and 2.02 1/h per kg bw, respectively). Because the metabolism 22 of VC is perfusion-limited (Filser and Bolt, 1979), comparison of clearance rates on body weight basis is 23 not satisfying. If clearance is compared on a body surface area basis these mammalian species exhibit 24 similar clearance rates (WHO, 1999a). 25 Comparison of lethal concentrations (lethality occurring in the context of narcosis) in mice, rats, 26 rabbits and guinea pigs point to certain interspecies variations with the guinea pig and rabbit being less 27 sensitive than mice and rats. Comparing the most sensitive species (mouse) with the at least sensitive 28 species (rabbit and guinea pig) point to a factor of 2. 33 ------- Vinyl chloride INTERIM 1: 3/2008 1 LC50 mouse; exposure time 2 h: 117,500 ppm (Prodan et al, 1975) 2 LC50 rat; exposure time 2 h: 150,000 ppm (Prodan et al., 1975; Lester et al., 1963) 3 LC50 rabbit; exposure time 2 h: 240,000 ppm (Prodan et al., 1975) 4 LC50 guinea pig; exposure time 2 h: 240,000 ppm (Prodan et al., 1975) 5 Concerning non lethal, pre-narcotic effects marginal interspecies differences are observed 6 indicating that rats and mice are a little bit more sensitive than guinea pigs: e.g. thirty minutes exposure of 7 guinea pigs, rats and mice to 100,000 ppm VC resulted in the same symptoms: unconsciousness (in all 8 rats and mice but only in one of five guinea pigs) and a lung hyperaemia persisting for more than 2 weeks, 9 rats and mice fell aside after 20 min exposure and guinea pigs showed side position after 30 min exposure 10 (Mastromatteo et al., 1960). No comparable data on humans are available. Concerning hepatic effects 11 mice seem to be more sensitive than rats and rabbits: Exposure of mice to 1,500 ppm VC for 2 h caused 12 severe liver effects, resulting in shock liver and death of the mice. But no hepatic and lethal effects were 13 observed in rats and rabbits treated identically for 24 h (Tatrai and Ungvary, 1981). The reasons for these 14 interspecies differences are not known. Data on acute hepatic effects of VC in humans are not available. 15 Concerning the similar clearance rates of VC on a body surface area there does not seem to be a 16 large toxicokinetic difference between various species. Due to these findings we suggest to use a reduced 17 interspecies factor of 3, accounting for toxicodynamic differences, in cases where the toxicity of VC is 18 mediated by VC metabolites. 19 With respect to lethality and VC induced (pre-) narcotic symptoms there seem to be only minimal 20 interspecies differences. Use of a reduced extrapolation factor of 3 is recommended in this context. 21 4.3.3. Intraspecies Variability 22 Cytochrome P450 isoenzyme 2E1 is the key enzyme converting VC to 2-chloroethylene oxide. 23 CYP2E1 activity in human liver microsomes may vary up to 12-fold between individuals (substrate: p- 24 nitrophenol; Seaton et al., 1994). These data indicate a potential interindividual variability in VC 25 metabolism. 26 Investigation of VC retention in the lung of human volunteers revealed large interindividual 27 differences in VC retention (minimum 20.2% of the exposure concentration; maximum 79% of the 28 exposure concentration; Krajewski et al., 1980). 29 Interindividual differences in the response of human subjects to varying concentrations of VC 30 were observed by Lester et al. (1963): 8,000 ppm VC did not cause any response in 5 individuals, but one 31 person felt ,,slightly heady". Other subjects complained about adverse health effects at concentrations of > 32 12,000 ppm, indicating that there are only small interindividual differences in the response to neurotoxic 33 effects of VC. 34 Relevant interindividual differences were not described in animal experiments. 35 Due to these observations a factor of 3 is used for the characterization of intraspecies variabilities 36 in the context with neurotoxic effects or cardiac sensitization. A factor of 10 is used to describe 37 intraspecies differences which are mediated by metabolites of VC. 38 4.3.4. Concurrent Exposure Issues 34 ------- Vinyl chloride INTERIM 1: 3/2008 1 Concurrent administration of ethanol and VC leads to an increase of liver angiosarcoma in rats in 2 comparison to animals exposed only to VC. This effect may be due to the interaction of ethanol (a known 3 CYP2E1 inducer) with VC metabolism (WHO, 1999a). 4 Induction of certain enzymes of the mixed-function oxidase system by pretreatment with 5 phenobarbital or the mixture of poly chlorinated biphenyls enhanced acute hepatotoxicity in rats as 6 measured by increased activity of hepatic enzymes and /or focal hepatic necrosis. On the other hand, 7 inhibitors of the mixed-function oxidase system like SKF-525A have an opposite effect (WHO, 1999a). 8 5. RATIONALE AND PROPOSED AEGL-1 9 5.1. Human Data Relevant to AEGL-1 10 Detection of 261 ppm VC by entering the exposure chamber was reported by Baretta et al. (1969). 11 The authors also described that 5 of 7 persons detected the odor of VC entering a chamber with 491 ppm 12 VC, but after 5 minutes of exposure detection was not any longer possible. 13 Amoore and Hautala (1983) reported an odor threshold of 3,000 ppm for VC. This value 14 represents the geometric average of three studies, extreme points and duplicate quotations were omitted. It 15 was not stated whether it is the detection or recognition threshold. 16 A "fairly pleasant odor" was reported by two persons exposed to 25,000 ppm for 3 minutes. At 17 these concentrations dizziness and slight disorientation occurred (Patty et al., 1930). 18 Hori et al. (1972) reported an odor threshold for VC of 10 - 20 ppm (20 ppm in production 19 workers and 10 ppm in workers from other sites). This value was reviewed by the AIHA and the value has 20 been rejected because of several shortcomings of the experimental procedure (e.g. no calibration of panel 21 odor sensitivity, not stated whether the given limit was due to recognition or detection, number of trials 22 not stated). 23 Irritating effects of VC are only observed at very high concentrations: accidental exposure to 24 lethal concentrations was accompanied by lesions of the eyes (Danziger, 1960). 25 Baretta et al. (1969) exposed 4-6 volunteers to 59, 261, 491 ppm VC (analytical concentrations) 26 for 7.5 h (including a 0.5 h lunch period; corresponding to time weighted average concentrations of 48, 27 248 or 459 ppm over a period of 7.5 h), seven persons were exposed to 491 ppm for only 3.5 hours. The 28 only complaints were those of two subjects who reported mild headache and some dryness of their eyes 29 and nose during exposure to the highest concentration (the time of onset of headaches is not specified and 30 is assumed to have occurred after 3.5 hours of exposure). 31 5.2. Animal Data Relevant to AEGL-1 32 Lacrimation occurred shortly after onset of exposure in animals exposed to VC (exposure of mice, 33 rats, guinea pigs, and rabbits to concentrations between 42,900 ppm to 280,000 ppm, no differentiated 34 evaluation according to lacrimation). Lethal effects have been observed in mice and rats even in the 35 lowest exposure concentrations (42,900 ppm without ventilation in mice and 150,000 ppm with 36 ventilation in rats) (Prodan et al., 1975). Mastromatteo et al. (1960) described that irritation (no further 37 details) was occurring immediately after onset of exposure to 100,000, 200,000 or 300,000 ppm VC in 38 rats and mice; in guinea pigs irritation was not described in concentrations below 400,000 ppm VC. 35 ------- Vinyl chloride INTERIM 1:3/2008 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 However, 100,000 ppm VC already resulted in unconsciousness of the animals. No other data on irritation of VC in animals are available from literature. 5.3. Derivation of AEGL-1 Vinyl chloride is a compound with poor odor warning properties. Reports on odor threshold vary over a wide range (10 to 25,000 ppm). There is no qualified study determining the detection or recognition threshold. According to the report of Baretta et al. (1969) people seem to get used to the odor of VC. In humans and animals irritation is only reported in the context of exposure to very high concentrations which are lethal or cause unconsciousness. So, derivation of AEGL-1 values on base of the odor detection or irritation is not possible. Occurrence of headache has been reported by Baretta et al. (1969) in two subjects after acute exposure (the time of onset of headaches is not specified and is assumed to have occurred after 3.5 hours of exposure) . These findings are supported by data from occupationally exposed persons who developed headache after VC exposure (Lilis et al., 1975; Suciu et al., 1975). The endpoint "mild headache" in the study from Baretta et al. (1969) can be regarded as a no effect level for notable discomfort (491 ppm for 3.5 h). An intraspecies factor of 3 is employed: it is assumed that the effects are due to VC itself and not due to a metabolite, so only small interindividual differences are expected. The relationship between concentration and duration of exposure as related to lethality was examined by Ten Berge et al. (1986) for approximately 20 irritant or systemically-acting vapors and gases. The authors subjected the individual animal data sets to Probit analysis with exposure duration and exposure concentration as independent variables. An exponential function (Cn x t = k), where the value of n ranged from 0.8 to 3.5 for different chemicals was found to be an accurate quantitative descriptor for the chemicals evaluated. Approximately 90 percent of the values of n range between n=l and n=3. Consequently, these values were selected as the reasonable lower and upper bounds of n to use when data are not available to derive a value of n. A value of n=l is used when extrapolating from shorter to longer time periods because the extrapolated values are conservative and therefore, reasonable in the absence of any data to the contrary. Conversely, a value of n=3 is used when extrapolating from longer to shorter time periods because the extrapolated values are conservative and therefore reasonable in the absence of any data to the contrary. The default values for "n" are used, as the mechanism for the induction of headache is not well understood. The extrapolation to 10 minutes from a 3.5 hour exposure is justified because exposure of human at 4,000 ppm for 5 minutes did not result in headache (Lester et al., 1963). TABLE 7: AEGL-1 VALUES FOR VINYL CHLORIDE AEGL Level AEGL-1 10-minute 450 ppm 1200 mg/m3 30-minute 3 10 ppm 800 mg/m3 1-hour 250 ppm 650 mg/m3 4-hour 140 ppm 360 mg/m3 8-hour 70 ppm 180 mg/m3 6. RATIONALE AND PROPOSED AEGL-2 6.1. Human Data Relevant to AEGL-2 Lester et al. (1963) reported that 5 min exposure to 8,000 ppm caused dizziness in 1/6 subjects (the same subject reported slight dizziness at sham exposure and no effect at 12,000 ppm). No complaints were made by any volunteer at 4,000 ppm. At 12,000 ppm one subject reported clear signs of discomfort (reeling, swimming head) and another subject another was unsure of some effect; he had a "somewhat 36 ------- Vinyl chloride INTERIM 1: 3/2008 1 dizzy" feeling in the middle of exposure. At 16,000 ppm (5/6) and 20,000 ppm (6/6) persons complained 2 about dizziness, nausea, headache, dulling of visual and auditory cues. All symptoms disappeared shortly 3 after termination; headache persisted for 30 minutes in one subject after exposure to 20,000 ppm. 4 Three minutes exposure to 25,000 ppm resulted in dizziness and slight disorientation as to space 5 and size of surrounding objects and a burning sensation in the feet in two persons. They immediately 6 recovered on leaving the chamber and complained only of a slight headache which persisted for 30 7 minutes (Patty et al, 1930). 8 Baretta et al. (1969) exposed 4-6 volunteers to 59, 261, 491 ppm VC (analytical concentrations) 9 for 7.5 h (including a 0.5 h lunch period; corresponding to time weighted average concentrations of 48, 10 248 or 459 ppm over a period of 7.5 h), seven persons were exposed to 491 ppm for only 3.5 hours. The 11 only complaints were those of two subjects who reported mild headache and some dryness of their eyes 12 and nose during exposure to the highest concentration (the time of onset of headaches is not specified and 13 is assumed to have occurred after 3.5 hours of exposure) . 14 6.2. Animal Data Relevant to AEGL-2 15 Animal toxicity after short term exposure is characterized by cardiac sensitization, (pre-) narcotic 16 and hepatic effects. Short term exposure (5 min) of dogs to VC induced cardiac sensitization towards 17 epinephrine (EC50: 50,000 or 71,000 ppm in two independent experiments) (Clark and Tinston, 1973; 18 1982). This observation is confirmed in higher concentrations by additional experimental data). 19 Hehir et al. (1981) reported that single exposure of mice to 50,000 ppm VC caused twitching, 20 ataxia, hyperventilation and hyperactivity, beginning 40 min after start of exposure. Consistent with these 21 data Mastromatteo et al. (1960) reported that 100,000 ppm VC induced pronounced tremor, unsteady gait 22 and muscular incoordination in mice 15 min after onset of exposure. Exposure of mice to 1,500 ppm VC 23 for 2 h resulted in stasis of blood flow, decreasing enzyme activities in the liver, subcellular liver damage, 24 and shock liver after 24 h of exposure (Tatrai and Ungvary, 1981). 25 Viola et al. (1970) reported that rats exposed to 30,000 ppm for 4 h/d were slightly soporific (no 26 further details). At higher concentrations (50,000 ppm for 2 h) moderate intoxication and loss of righting 27 reflex and intense intoxication at 60,000 ppm for 2 h (but righting reflex still present) have been reported 28 by Lester et al. (1963). Intoxication was not further characterized. Higher VC concentrations (100,000 29 ppm) resulted in a loss of the corneal reflex (exposure for 2 h) (Lester et al., 1963). Already 15 min after 30 onset of exposure to 100,000 ppm tremor and ataxia were observed by Mastromatteo et al. (1960).Guinea 31 pigs exposed to 25,000 ppm for 5 min showed motor ataxia, unsteadiness on feet, after 90 min the animals 32 were unconscious (NOAEL 10,000 ppm) (Patty et al., 1930). Mastromatteo et al. (1960) reported the 33 unsteady gait and muscular incoordination in guinea pigs exposed for 15 min to 100,000 ppm. 34 Single inhalation exposure of rats for 6 hours to 100,000 ppm VC resulted in histopathological 35 changes of the liver (vacuolization), but was not observed in lower concentrations (50,000 ppm) (Jaeger et 36 al., 1974). However, in mice Tatrai and Ungvary (1981) reported that stasis of the liver developed 2 and 4 37 h after the beginning of inhalation. The authors observed decreasing enzyme activities in liver and 38 subcellular liver damage at exposure concentrations of 1,500 ppm VC for 2 h; after 24 h shock liver 39 developed. Repeated exposure of rats to 1,500 ppm VC for up to 9 days during pregnancy caused 40 increased relative and absolute liver weights without light microscopic visible changes. Also, no 37 ------- Vinyl chloride INTERIM 1: 3/2008 1 histopathological effects were observed in rabbits treated identically (Ungvary et al, 1978). In another 2 developmental study increased absolute and relative liver weights have been observed in rats exposed 3 intermittently to 2,500 ppm from day 6-15 of pregnancy, the NOAEL was 500 ppm (John et al., 1977; 4 1981). 5 6 6.3. Derivation of AEGL-2 7 Short term exposure (5 min) of dogs to VC induced cardiac sensitization towards epinephrine 8 (EC50: 50,000 or 71,000 ppm in two independent experiments) (Clark and Tinston, 1973; 1982). A 9 NOAEL for this effect can be reasonably estimated by using a factor of 3 on EC50 (50,000 ppm) resulting 10 in a concentration of about 17,000 ppm. This concentration already leads to CNS-effects in humans after 11 5 minutes exposure (Lester et al., 1963). Thus, the endpoint of cardiac sensitization would not be the 12 critical effect for AEGL-2 derivation. However, the AEGL-2 derived below is supported by the data on 13 cardiac sensitization. 14 Liver toxicity is a major endpoint after long term exposure to VC and may possibly be linked to 15 tumor development in young animals (see section 4.2. for further discussion). The NOAEL for 16 irreversible effects to the liver after single exposure is 50,000 ppm (6h, rat data). The effects seen in lower 17 concentrations (liver weight changes) may not be regarded as key studies for AEGL-2 derivation. 18 Narcotic effects seem to predominate in rats, mice and guinea pigs acutely exposed to high 19 concentrations of VC. These effects are relevant AEGL-2 endpoints as they impair the possibility to 20 escape. Although guinea pigs seem to be less sensitive than rats and mice concerning lethality (see 7.2) 21 they are more sensitive than rats and mice with regard to early signs of narcotic effects: exposure of 22 guinea pigs for 5 min to 25,000 ppm resulted in early signs of narcotic effects (motor ataxia, unsteadiness 23 on feet), after 90 minutes animals were unconscious (NOAEL 10,000 ppm) (Patty et al., 1930). Rats 24 exposed to 30,000 ppm VC for 4 h were only slightly soporific (Viola et al., 1970), and single exposure of 25 mice to 50,000 ppm VC caused twitching, ataxia, hyperventilation and hyperactivity, beginning 40 min 26 after start of exposure (Hehir et al., 1981) . 27 The observations in animals are in good accordance with the effects observed in humans: 28 dizziness, reeling, swimming head, nausea etc., which can be regarded as early signs of narcosis, have 29 been reported in humans exposed to VC in concentrations > 12,000 ppm for 5 min. No effects were 30 reported at 4,000 ppm (Lester et al., 1963). The effects observed at 12,000 ppm (dizziness, reeling, 31 swimming head) were only seen in 1 or 2 of 6 persons (one person was unsure of an effect) and do not yet 32 impair the capability to escape, whereas, the effects observed at concentrations > 16,000 ppm (dizziness, 33 nausea, headache, dulling of visual and auditory cues) might possibly impair escape. Therefore, 12,000 34 ppm is interpreted as the no effect level for impaired ability to escape and is used to derived the AEGL-2 35 values. 36 By analogy to other anaesthetics the effects are assumed to be solely concentration dependent. 37 Thus, after reaching steady state at about 2 hours of exposure, no increase in effect is expected. The other 38 exposure duration-specific values were derived by time scaling according to the dose-response regression 39 equation Cn x t = k, using a factor of n=2, based on data from Mastromatteo et al. (1960). Mastromatteo et 40 al. observed various time-dependent prenarcotic effects in mice and guinea pigs after less than steady state 41 exposure conditions (For details see Appendix B). With this, time extrapolation was performed from 5 to 42 10, 30, 60 minutes and 2 hours, where the steady state concentration was calculated. However, the 38 ------- Vinyl chloride INTERIM 1:3/2008 resulting AEGL-2 values may not provide a sufficient margin safety to avoid mutational events or malignancies after short-term exposure to VC. The calculations of exposure concentrations scaled to AEGL-2 time points are shown in Appendix A. The data are listed in the table below. TABLE 8: AEGL-2 VALUES FOR VINYL CHLORIDE AEGL Level AEGL-2 10-minute 2,800 ppm (7300 mg/m3) 30-minute 1,600 ppm (4 100 mg/m3) 1-hour 1,200 ppm (3 100 mg/m3) 4-hour 820 ppm (2 100 mg/m3) 8-hour 820 ppm (2 100 mg/m3) 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 7. RATIONALE AND PROPOSED AEGL-3 7.1. Human Data Relevant to AEGL-3 Only two cases of accidental death due to VC exposure are described in literature. Exposure concentrations and exposure time are unknown, but circumstances suggest inhalation of very high concentrations. At autopsy cyanosis, congestion of lung and kidneys and blood coagulation failure were observed (Danziger, 1960). 7.2. Animal Data Relevant to AEGL-3 LC50 values reported for mice, rats, rabbits and guinea pigs indicate similar sensitivity of mice and rats and of rabbits and guinea pigs. According to the data presented by Prodan et al. (1975) the following LC50 values were obtained: mice 117,500 ppm rats 150,000 ppm rabbits 240,000 ppm guinea pigs 240,000 ppm The findings in rats are supported by the data of Lester et al. (1963) who described that exposure of 2 rats to 150,000 ppm for 2 hours resulted in the death of one rat whereas the other rat recovered on removal to air. The following LC00 values have been reported for these species. mice 100,000 ppm (2 h, Prodan et al., 1975) rats 100,000 ppm (8 h, Lester et al., 1963) 200,000 ppm (0,5 h, Mastromatteo et al., rabbits 200,000 ppm (2 h, Prodan et al., 1975) guinea pigs 100,000 ppm (6 h, Patty et al., 1930) 200,000 ppm (2 h, Prodan et al., 1975) 1960) In addition, relevant data on cardiac sensitization exist: Short term exposure (5 min) of dogs to VC induced cardiac sensitization towards epinephrine (EC50: 50,000 or 71,000 ppm in two independent 39 ------- Vinyl chloride INTERIM 1:3/2008 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 experiments) (Clark and Tinston, 1973; 1982). These effects were also seen in mice at higher concentrations (Aviado and Belej, 1974). In monkeys, only myocardial depression after inhalation of 2.5- 10% VC was observed. It is not clearly stated whether an addition challenge with epinephrine was applied or not (Belej etal., 1974). 7.3. Derivation of AEGL-3 Lethality data provide AEGL-3 values that are marginally higher than those derived based on cardiac sensitization. Thus, animal data (Clark and Tinston, 1973; 1982) on cardiac sensitization after exposure for 5 minutes were used to derive AEGL-3. Severe cardiac sensitization is a life threatening effect, but at 50,000 ppm no animal died in the reported study and is used to derive AEGL-3 values. A total uncertainty factor of 3 is used to account for toxicodynamic differences among individuals. As the challenge with epinephrine and the doses of epinephrine used represent a conservative scenario, no interspecies uncertainty factor was used. As the unmetabolized VC is responsible for the effect, no relevant differences in toxicokinetics are assumed. In analogy to other halocarbons (e.g., Halon 1211, HFC 134a) which lead to cardiac sensitization the effects are assumed to be solely concentration dependent. Thus, after reaching steady state at about 2 hours of exposure, no increase in effect is expected. The other exposure duration-specific values were derived by time scaling according to the dose-response regression equation Cn x t = k, using an n of 2, based on data from Mastromatteo et al. (1960). Mastromatteo et al. observed various time-dependent prenarcotic effects (muscular incoordination, side position and unconsciousness, effects which occur immediately before lethality) in mice and guinea pigs after less than steady state exposure conditions. With this, time extrapolation was performed from 5 to 10, 30, 60 minutes and 2 hours, where the steady state concentration was calculated. The values are listed in the table below. TABLE 9: AEGL-3 VALUES FOR VINYL CHLORIDE AEGL Level AEGL-3 10-minute 12,000 ppm (31,000mg/m3) 30-minute 6,800 ppm (18,000 mg/m3) 1-hour 4,800 ppm (12,000 mg/m3) 4-hour 3,400 ppm (8,800 mg/m3) 8-hour 3,400 ppm (8,800 mg/m3) 26 27 28 29 30 31 32 33 34 35 36 8. SUMMARY OF PROPOSED AEGLs 8.1. AEGL Values and Toxicity Endpoints The derived AEGL values for various levels of effects and durations of exposure are summarized in Table 10. AEGL-1 values have been derived based on mild headaches observed in volunteers (Baretta et al., 1969); odor threshold was not determined in a validated manner and seems to vary over a wide range. AEGL-2 values are based on CNS-effects, which may impair escape capacity (Lester et al.,1963). Data on cardiac sensitization (Clark and Tinston, 1982; 1973) are supported by lethality concentrations (LC00) in slightly higher concentrations (Prodan et al.,1975) and are used for AEGL-3 derivation. TABLE 10: SUMMARY/RELATIONSHIP OF PROPOSED AEGL VALUES Classification 10-minute 30-minute 1-hour 4-hour 8-hour 40 ------- Vinyl chloride INTERIM 1:3/2008 AEGL-1 (Non-disabling) AEGL-2 (Disabling) AEGL-3 (Lethal) 450 ppm 1200 mg/m3 2,800 ppm 7,300 mg/m3 12,000 ppm (31,000mg/m3) 3 10 ppm 800 mg/m3 1,600 ppm 4, 100 mg/m3 6,800 ppm (18,000 mg/m3) 250 ppm 650 mg/m3 1,200 ppm 3, 100 mg/m3 4,800 ppm (12,000 mg/m3) 140 ppm 360 mg/m3 820 ppm 2, 100 mg/m3 3,400 ppm (8,800 mg/m3) 70 ppm 180 mg/m3 820 ppm 2,100 mg/m3 3,400 ppm (8,800 mg/m3) 7 Inhalation data are summarized in Figure 1 below. The data were classified into severity 8 categories chosen to fit into definitions of the AEGL level health effects. The category severity definitions 9 are "No effect"; "Disabling"; "Lethal" and "AEGL". 41 ------- Vinyl chloride INTERIM 1:3/2008 1000000 100000 10000 Q. Q_ 1000 100 Chemical Toxicity - TSD All Data Vinyl chloride Human - No Effect Human - Discomfort Human - Disabling O Animal -No Effect O Animal - Discomfort Animal - Disabling Animal - Partially Lethal Animal - Lethal 60 120 180 240 Minutes 300 360 420 480 1 FIGURE 1: CATEGORICAL REPRESENTATION OF VINYL CHLORIDE INHALATION 2 DATA (data where the exposure time exceeded 500 min are not included) 3 8.2. Comparison with Other Standards and Criteria 4 Other standards and guidance levels for workplace and community exposures are listed in Table 5 11. 42 ------- Vinyl chloride INTERIM 1:3/2008 TABLE 11: EXISTENT STANDARDS AND GUIDELINES FOR VINYL CHLORIDE Guideline AEGL-1 AEGL-2 AEGL-3 PEL-TWA (OSHA) a STEL (OSHA) b TLV-TWA (ACGIH) c TEEL-0 (CSP) d TEEL-1 (CSP) e TEEL-2 (CSP) f TEEL-3 (CSP) B TRK (Germany) h Einsatztoleranzwerte (Greim, Germany) ' Storfallbeurtei-lungswert (VCI) ] Exposure Duration 10-minute 3 10 ppm 2,800 ppm 12,000 ppm 5 ppm [for 15 min] 30-minute 3 10 ppm 1,600 ppm 6,800 ppm 1-hour 250 ppm 1,200 ppm 4,800 ppm 1 ppm 5 ppm 5 ppm 75 ppm 1,000 ppm 4-hour 140 ppm 820 ppm 3,400 ppm 100 ppm 8-hour 70 ppm 820 ppm 3,400 ppm 1 ppm 5 ppm 2 (3) ppm 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 a OSHA PEL-TWA (Occupational Health and Safety Administration, Permissible Exposure Limits - Time Weighted Average) (OSHA, 2002) is the time-weighted average concentration for a normal 8-hour workday and a 40-hour work week, to which nearly all workers may be repeatedly exposed, day after day, without adverse effect. 22 23 24 25 26 " OSHA PEL-STEL (Permissible Exposure Limits - Short Term Exposure Limit) (OSHA. 2002) is defined as a 15 minute TWA exposure which should not be exceeded at any time during the workday even if the 8-hour TWA is within the PEL-TWA. Exposures above the PEL-TWA up to the STEL should not be longer than 15 minutes and should not occur more than 4 times per day. There should be at least 60 minutes between successive exposures in this range. 27 c ACGIH TLV-TWA (American Conference of Governmental Industrial Hygienists, Threshold Limit Value - 28 Time Weighted Average) (ACGIH, 1998). The time-weighted average concentration for a normal 8-hour 29 workday and a 40-hour work week, to which nearly all workers may be repeatedly exposed, day after day, 30 without adverse effect. The value was based on a calculation of the carcinogenic potency of vinyl chloride 31 by Gehring and coworkers. The TLV-Committee concluded that a TLV-TWA of 5 ppm should not result in 32 a detectable increase in the incidence of occupational cancers, specifically angiosarcoma of the liver. 43 ------- Vinyl chloride INTERIM 1: 3/2008 d TEEL-OflJ.S. department of Energy's Chemical safety Program, Temporary Emergency Exposure Limit) 2 (CSP, 2002). The threshold concentration below which most people will expenence no appreciable nsk of 3 health effects. 4 e TEEL-1 (U.S. department of Energy's Chemical safety Program, Temporary Emergency Exposure Limit) 5 (CSP, 2002). The maximum concentration in air below which it is Believed nearly all individuals could be 6 exposed without experiencing other than mild transient adverse health effects or perceiving a clearly defined 7 objectionable odor. 8 f TEEL-2 (U.S. department of.Energy's Chemical safety Program. Temporary Emergency Exposure Limit) 9 (CSP, 2002). The maximum concentration in air below which it is believed nearly all individuals could be 10 exposed without experiencing or developing irreversible or other serious health effects or symptoms that 11 could impair their abilities to take protective action. g TEEL-3 (U.S. department of Energy's Chemical safety Program, Temporary Emergency Exposure Limit) 13 (CSP, 2002). The maximum concentration in air below which it is believed nearly all individuals could be 14 exposed without experiencing or developing life-threatening health effects. h f RK (Technische Richtkonzentrationen [Technical Guidance Concentration], Deutsche 16 Forschungsgemeinschaft [German Research Association], Germany) (DFG, 2001). TRK is defined as 17 the air concentration of a substance which can be achieved with the current technical standards. TRK-values 18 are given for those substances for which no maximum workplace concentration can be established. 19 Compliance of the TRK should minimize the risk of health effects, but health effects cannot be excluded 20 even at this concentration. (A value of 3 ppm is given for existing plants and the production of VC and 21 PVC, in all other cases 2 ppm should not be exceeded.) 1 Einsatztoleranzwert JAction Tolerance Levels] (Vereinigung zur Forderung des deutschen Brandschutzes 23 e.V. [Federation for the Advancement of German Fire Prevention]) (Greim, 1995/1996) constitutes a 24 concentration to which unprotected firemen and the general population can be exposed to for up to 4 hours 25 without any health risks. The value is based on the observation that no acute toxic effects or irritating effects 26 have been observed during exposure to 500 ppm for 4 hours. J Storfallbeurteilungswert [Emergency Assessment Value] (VCL Verband der Chemischen Industrie, 28 Deutschland [Association of the Chemical Industry in Germany]) (VCI, 1990).These values have been 29 set for an exposure time of up to 1 h. Considering that VC leads to anaesthesia in concentrations of 7%, to 30 pre-narcotic syndroms at 0.5%, and to respiratory arrest the Emergency Assessment Value has been set at 31 1,000 ppm. 32 8.3. Data Adequacy and Research Needs 33 As VC has only poor warning properties there is only a very limited data base to derive AEGL-1. 34 Additional studies with volunteers may not be performed due to ethical reasons. AEGL-2 values are based 35 on animal experiments regarding CNS-effects. The respective concentration range is well established but 36 excludes potential mutagenic or carcinogenic effects after short term exposure, which might occur in 37 lower concentrations. However, quantitative estimates of the respective risk are highly uncertain. For 38 derivation of AEGL-3 values, the dogs studies on cardiac sensitization are in good accordance with 39 lethality data in slightly higher concentrations. 40 9. REFERENCES 44 ------- Vinyl chloride INTERIM 1: 3/2008 1 ACGIH (American Conference of Governmental Industrial Hygienists). 1998. Documentation of the 2 Threshold Limit Values and Biological Exposure Indices. Cincinnati, OH. 3 AIHA (American Industrial Hygiene Association). 1997. 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J. 1:936-938. 11 Watanabe, P.O., G.R. McGowan, E.G. Madrid, and P.J. Gehring. 1976a. Fate of [14C] vinyl chloride 12 following inhalation exposure in rats. Toxicol. Appl. Pharmacol. 37:49-59. 13 Watanabe, P.G., G.R. McGowan, and P.J. Gehring. 1976b. Fate of [14C] vinyl chloride after single oral 14 administration in rats. Toxicol. Appl. Pharmacol. 36:339-352. 15 Watson, W.P., D. Potter, D. Blair, and A.S. Wright. 1991. The relationship between alkylation of 16 haemoglobin and DNA in Fischer 344 rats exposed to [1,2-14C] vinyl chloride. In: Garner, R.C., Farmer, 17 P.B., Steele, G.T., Wright, A. S.: Human Carcinogen Exposure. Biomonitoring and Risk Assessment, 18 Oxford University Press, London, 421-428. 19 Weber, H, W. Reinl, and E. Greiser. 1981. German investigations on morbidity and mortality of workers 20 exposed to vinyl chloride. Environ. Health Perspect. 41:95-99. 21 WHO, World Health Organization. 1987. Air Quality Guidelines for Europe. WHO Regional 22 Publications. European Series, No. 23. Kopenhagen, 1987. 23 WHO, World Health Organization. 1999a. Environmental Health Criteria 215, Vinyl Chloride. IPCS, 24 International Programme on Chemical Safety. World Health Organization, Geneva, 1999. 25 WHO, World Health Organization. 1999b. Update and Revision of WHO Air Quality Guidelines for 26 Europe, http://www.who.dk/envhlth/airqual.htm (accessed: 29/10/1999). 54 ------- Vinyl chloride INTERIM 1: 3/2008 APPENDIX A - Derivation of AEGL values 55 ------- Vinyl chloride INTERIM 1:3/2008 AEGL-1 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 Key study: Toxicity endpoint: Uncertainty/ modifying factors: Time Scaling: Calculations: 10-minute AEGL-1 30-minute AEGL-1 1-hour AEGL-1 4-hour AEGL-1 8-hour AEGL-1 Barettaetal. (1969) Mild headache in 2 subjects during exposure to highest concentration (i.e. 491 ppm for 3.5 h) Total uncertainty factor of 3 for intraspecies variability C3 x t = k for extrapolation to 1-hour and 30-minute (10-minute = 30-minute value); C1 x t = k for extrapolation to 4- and 8-hour k = (491 ppm)3 x 210 min = 2.49 x 10E+10 ppm3 min k = (491 ppm)1 x 210 min = 103110 ppm min C3 x 10 min = 2.49 x 10E+10 ppm3 min C = 1355 ppm 10-min AEGL-1 = 1355 ppm/3 =450 ppm (= 1170 mg/m3) C3 x 30 min = 2.49 x 10E+10 ppm3 min C = 939.25 ppm 30-min AEGL-1 =939 ppm/3 = 310 ppm (=810 mg/m3) C3 x 60 min = 2.49 x 10E+10 ppm3 min C = 745.48 ppm 1-h AEGL-1 = 745 ppm/3 =250 ppm (= 640 mg/m3) C x 240 min = 103110 ppm min C = 429.63 ppm 4-h AEGL-1 =430 ppm/3 = 140 ppm (= 370 mg/m3) C x 480 min = 103110 ppm min C = 214.81 ppm 8-h AEGL-1 = 214 ppm/3 = 70 ppm (= 190 mg/m3) 56 ------- Vinyl chloride INTERIM 1:3/2008 AEGL-2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 Key study: Toxicity endpoint: Lester etal. (1963) Uncertainty/ modifying factors: Time Scaling: Calculations: 10-minute AEGL-2 30-minute AEGL-2 1-hour AEGL-2 2-hour steady state 4-hour AEGL-2 8-hour AEGL-2 Prenarcotic effects were observed in human volunteers. After 5 minute exposure to 16,000 ppm VC 5 of 6 persons showed dizziness, lightheadedness, nausea, visual and auditory dulling. At concentrations of 12,000 ppm one of six persons showed "swimming head, reeling". Another individual was unsure of some effect and was somewhat dizzy. A single person reported slight effects ("slightly heady") of questionable meaning at 8,000 ppm (this volunteer felt also slightly heady at sham exposure and reported no response at 12,000 ppm). No effects were observed at 4,000 ppm. (Lester et al., 1963). 12,000 ppm was regarded as a concentration below AEGL-2 level and taken as NOAEL. Total uncertainty factor of 3 for intraspecies variability C2 x t = k for extrapolation 2-hour, 1-hour, 30-minute, and 10-minute, flatlining from 4h to 8 h (based on 2 hours steady state concentration) k = (12,000 ppm)2 x 5 min = 7.2 x 10E+8 ppm2 min C2 x 10 min = 7.2 x 10E+8 ppm2 min C = 8485.28 ppm 10-min AEGL-2 = 8485 ppm/3 = 2800 ppm (= 7300 mg/m3) C2 x 30 min = 7.2 x 10E+8 ppm2 min C = 4898.98 ppm 30-min AEGL-2 = 4899 ppm/3 = 1600 ppm (= 4100 mg/m3) C2 x 60 min = 7.2 x 10E+8 ppm2 min C = 3464.11 ppm 1-h AEGL-2 = 3464 ppm/3 = 1200 ppm (= 3100 mg/m3) C2 x 120 min = 7.2 x 10E+8 ppm2 min C = 2449.49 ppm 2-h steady state= 2450/3 ppm/3 = 820 ppm (=2100 mg/m3) = 2-hour steady state/3 = 820 ppm (=2100 mg/m3) = 4-hour AEGL-2 =820 ppm (= 2100 mg/m3) 57 ------- Vinyl chloride INTERIM 1:3/2008 1 2 3 4 5 6 7 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 Key study: Toxicity endpoint: Uncertainty/ modifying factors: Time Scaling: Calculations: 10-minute AEGL-3 30-minute AEGL-3 1-hour AEGL-3 2-hour steady state 4-hour AEGL-3 8-hour AEGL-3 AEGL-3 Clark and Tinston, 1973; 1982 Short term exposure (5 min) of dogs to VC induced cardiac sensitization towards epinephrine (EC50: 50,000 or 71,000 ppm in two independent experiments) (Clark and Tinston, 1973; 1982). These effects were also seen in mice at higher concentrations (Aviado and Belej, 1974). 50,000 ppm was used as NOAEL for life threatening effects Combined uncertainty factor of 3 1 for interspecies variability 3 for intraspecies variability C2 x t = k for extrapolation to 2-hour, 1-hour, and 30-minute and 10-minutes; flatlining from 4h to 8 h (based on 2 hours steady state concentration) k = (50,000 ppm)2 x 5 min = 1,25 10E+10 ppm2 min C2 x 10 min = 1,25 10E+10 ppm2 min C = 35,355.34 ppm 30-min AEGL-2 = 35,355 ppm/3 = 12,000 ppm (=31,000 mg/m3) C2 x 30 min = 1,25 10E+10 ppm2 min C = 20,412.41 ppm 30-min AEGL-2 = 20,412 ppm/3 = 6,800 ppm (= 18,000 mg/m3) C2 x 60 min = 1,25 10E+10 ppm2 min C= 14433.76 ppm 1-h AEGL-2 = 14434 ppm/10 = 4,800 ppm (= 12,000 mg/m3) C2 x 120 min = 1,25 10E+10 ppm2 min C= 10,206.21 ppm 2-h steady state = 10,206 ppm/3 = 3,400 ppm (= 8,800 mg/m3) = 2-h steady state/3 =3,400 ppm (= 8,800 mg/m3) = 4-h AEGL-3 =3,400 ppm (= 8,800 mg/m3) 58 ------- Vinyl chloride INTERIM 1: 3/2008 APPENDIX B - Time Scaling Calculations for Vinyl Chloride AEGLs 59 ------- Vinyl chloride INTERIM 1: 3/2008 1 Time Scaling for Vinyl Chloride AEGLs 2 The relationship between dose and exposure time to produce a toxic effect for any given chemical 3 is a function of the physical and chemical properties of the substance and the unique toxicologic and 4 pharmacologic properties of the individual substance. Historically, the relationship according to Haber 5 (1924), commonly called Haber's rule (i.e., C x t = k, where C = exposure concentration, t = exposure 6 duration, and k = a constant) has been used to relate exposure concentration and duration to a toxic effect 7 (Rinehart and Hatch, 1964). This concept states that exposure concentration and exposure duration may 8 be reciprocally adjusted to maintain a cumulative exposure constant (k) and that this cumulative exposure 9 constant will always reflect a specific quantitative and qualitative response. This inverse relationship of 10 concentration and time may be valid when the toxic response to a chemical is equally dependent upon the 11 concentration and the exposure duration. However, an assessment by ten Berge et al. (1986) of LC50 data 12 for certain chemicals revealed chemical-specific relationships between exposure concentration and 13 exposure duration that were often exponential. This relationship can be expressed by the equation Cn x t = 14 k, where n represents a chemical-specific and even a toxic endpoint-specific exponent. The relationship 15 described by this equation is basically the form of a linear regression analysis of the log-log 16 transformation of a plot of C vs. t (NRC, 2001). 17 Acute CNS-toxicity and lethality of VC are dominated by its narcotic effects characterized by a 18 typical sequence of effects (increased motor activity, tremor, muscular incoordination, side position, 19 unconsciousness, resulting in deep narcosis). The occurrence and time sequence of these effects in rats, 20 mice and guinea pigs has been described by Mastromatteo et al. (1960). These experimental data are used 21 for the derivation of values of n by linear regression analysis of the log-log transformed plot of C vs. t. 22 Three data sets of toxic effects in mice and rats or guinea pigs described by Mastromatteo et al. 23 (1960) were analyzed. As the time-concentration relationships for mice and rats were identical the 24 following evaluation concentrates on the data obtained in mice and guinea pigs. Regression analysis has 25 been performed for the endpoints unconsciousness, muscular incoordination, and side position. The time- 26 concentration relation ships are described below. 27 Time dependency is only true as long as no steady state is reached. Similar to other inhalation 28 anesthetics, maximal blood concentration of VC after inhalation exposure depends on the partial pressure 29 of VC in the air. Blood respectively brain concentration, which directly correlates with the depth of 30 narcosis (see below) and - presumably - with cardiac sensitization level, can be controlled by changing the 31 concentration of VC in the air, i.e. by changing the partial pressure of VC in the air. If equilibrium is 32 reached between the partial pressure of VC in the air and in the blood (steady state), no further increase of 33 VC concentration in the blood is possible, even if the exposure time is prolonged (Forth et al., 1987). The 34 time necessary to set up steady state mainly depends on the blood/air partition coefficient of a substance. 35 The blood/air partition coefficient of VC in humans is 1.2 (Csanady and Filser, 2001), similar to that of 36 the inhalation anesthetic isoflurane (1.4; Forth et al., 1987). For this substance the equilibrium is reached 37 after about 2 hours, derived by graphical extrapolation of the data on isoflurane (Goodman and Oilman, 38 1975). For VC, in much lower concentrations an elimination half-time of VC of 20.5 minutes has been 39 derived (Buchter, 1979; Bolt et al., 1981). From that, for low concentrations a steady state concentration 40 for VC in blood of about 5 x 20.5 = 102.5 minutes can be calculated by standard estimation rules. Thus, in 41 high or low concentrations a relevant increase of internal concentrations of VC is not to be expected after 42 more than 2 hours of exposure. However, for shorter periods of exposure a relevant influence of time on 43 the built-up of VC on internal concentrations has to be taken into account: 60 ------- Vinyl chloride Unconsciousness: INTERIM 1:3/2008 The time after which unconsciousness was observed in mice after exposure to 100,000, 200,000 or 300,000 ppm VC was 25 min, 10 min, and 5 min, respectively: Time min 5 10 25 Concentration ppm 300000 200000 100000 Log time 0.699 1 1.398 Log Concentration 5.477 5.301 5 The time after which unconsciousness was observed in guinea pigs after exposure to 100,000, 200, 000, 300,000, and 400,000 ppm VC was 30 min, 10 min, 5 min and 5 min, respectively: Time min 5 5 10 30 Concentration ppm 400000 300000 200000 100000 Log time 0.699 0.699 1 1.477 Log Concentration 5.602 5.477 5.301 5 10 11 12 13 14 15 Regression analysis of the data is shown in figure 2: 61 ------- Vinyl chloride INTERIM 1:3/2008 5.7 5.6 5.5 | 5.4 .g +j £ 5.3 I o 0 5.2 5.1 • Unconsciousness-gpg A Unconsciousness-mouse \ ^ y = -0.6957X + 6.01 9 R2 = 0.9586 y = -0.6865X + 5.968 R2 = 0.9951 ^ 4.9 0.0 0.2 0.4 0.6 0.8 1.0 log time [min] 1.2 1.4 1.6 1 FIGURE 2: REGRESSION ANALYSIS OF THE LOG-LOG TRANSFORMED 2 CONCENTRATION-TIME CURVE REGARDING UNCONSCIOUSNESS IN MICE AND 3 GUINEA-PIGS (DATA FROM MASTROMATTEO ET AL., 1960) 4 The slope of the regression line was -0.6865 and -0.6957 in mice and guinea pigs, respectively, 5 corresponding to a value of 1.46 and 1.44 for n. 62 ------- Vinyl chloride Muscular incoordination: INTERIM 1:3/2008 The time after which muscular incoordination was observed in mice after exposure to 100,000, 200,000 or 300,000 ppm VC was 15 min, 2 min, and 1 min, respectively: Time min 1 2 15 Concentration ppm 300000 200000 100000 Log time 0 0.301 1.176 Log Concentration 5.477 5.301 5 The time after which muscular incoordination was observed in guinea pigs after exposure to 100,000, 200,000, 300,000, or 400,000 ppm VC was 15 min, 5 min, 2 min, and few seconds, respectively: Time min few seconds* 2 5 15 Concentration ppm 400000 300000 200000 100000 Log time ~ 0.301 0.699 1.176 Log Concentration 5.602 5.477 5.301 5 10 11 12 13 14 15 16 *: this value was not regarded in regression analysis Regression analysis of the data is shown in figure 3: 63 ------- Vinyl chloride INTERIM 1:3/2008 5.7 5.6 • Muscular incoordination-gpg A Muscular incoordination-mouse 5.5 - O •4= 5.3 - c 01 O O 0 5.2 O) O 5.1 - 5 - 4.9 y =-0.5481 x +5.6569 R2 = 0.9905 y =-0.3919x +5.4 R2 = 0.9845 0.2 0.4 0.6 0.8 log time [min] 1.2 1.4 1 FIGURE 3: REGRESSION ANALYSIS OF THE LOG-LOG TRANSFORMED 2 CONCENTRATION-TIME CURVE REGARDING MUSCULAR INCOORDINATION IN MICE 3 AND GUINEA-PIGS (DATA FROM MASTROMATTEO ET AL., 1960) 4 The slope of the regression line was -0.3919 and -0.5481 in mice and guinea pigs, respectively, 5 corresponding to avalue of 2.6 and 1.8 forn. 64 ------- Vinyl chloride Side position: INTERIM 1:3/2008 The time after which side position was observed in mice after exposure to 100,000, 200,000 or 300,000 ppm VC was 20 min, 5 min, and 2 min, respectively: Time min 2 5 20 Concentration ppm 300000 200000 100000 Log time 0.301 0.699 1.301 Log Concentration 5.477 5.301 5 The time after which side position was observed in guinea pigs after exposure to 100,000, 200,000, or 300,000 ppm VC was 30 min, 10 min, 2-5 min (set to 3.5), respectively: Time min 35 10 30 Concentration ppm 300000 200000 100000 Log time 0.544 1 1.477 Log Concentration 5.477 5.301 5 10 11 12 13 14 Regression analysis of the data is shown in figure 4: 65 ------- Vinyl chloride INTERIM 1:3/2008 5.6 5.5 5.4 Q. Q. ?5-3 I • Side position-gpg A Side position-mouse 5.1 y =-0.5123x +5.7753 R2 = 0.9814 y = -0.479X + 5.6268 R2 = 0.9989 4.9 0.2 0.4 0.6 0.8 log time [min] 1.2 1.4 1.6 1 FIGURE 4: REGRESSION ANALYSIS OF THE LOG-LOG TRANSFORMED 2 CONCENTRATION-TIME CURVE REGARDING SIDE POSITION IN MICE AND GUINEA- 3 PIGS (DATA FROM MASTROMATTEO ET AL., 1960) 4 The slope of the regression line was -0.479 and -0.5123 in mice and guinea pigs, respectively, 5 corresponding to a value of 2.1 and 2.0 for n. 6 Regarding the three different endpoints and the data obtained for mice and guinea pigs values for 7 n were in the range of 1.44 to 2.6 (1.44; 1.46; 1.8; 2.0; 2.1; 2.6; arithmetic mean: 1.9+/-0.4). Based on 8 these data it is justified to use a value of n=2 for the time extrapolation for AEGL-2 (CNS-effects) and 9 AEGL-3 (cardiac sensitization) values up to two hours. Concentrations for these "less-than-steady-state" 10 durations (i.e. 10, 30, 60 and 120 minutes) should be calculated according to 11 C2*t = const. 66 ------- Vinyl chloride INTERIM 1: 3/2008 APPENDIX C - Cancer Assessment of Vinyl Chloride 67 ------- Vinyl chloride INTERIM 1: 3/2008 1 Cancer Assessment of Vinyl Chloride 2 3 The most recently published risk estimate from the US EPA seems to be the best unit risk estimate 4 currently available (US EPA 2000 a, b). The values are 8.8 x 10"6 ((ig/m3)"1 for continuous lifetime 5 exposure, including childhood, and 4.4 x 10"6 ((ig/m3)"1 for continuous exposure as an adult. These risk 6 values indicate that exposure during childhood results in a similar tumor incidence as exposure as an 7 adult. The EPA unit risk calculation was derived by using the PBPK model of Clewell et al. (1995, 8 2002). These risk values are based on model-derived estimates of internal dose of the active metabolite in 9 animals and the continuous external exposure in humans that would result in these same internal dose of 10 the active metabolite. 11 Several calculations for cancer risk are presented below. These are: 12 Calculation A: based on the unit risk for continuous lifetime exposure from EPA (2002 a, b), 13 transformed to a single 24 hour exposure estimate by the default procedure recommended 14 in the SOP on AEGL development (that is, linear transformation, correction by a factor of 15 6 to account for the relevance of sensitive stages in development). Exposures of less than 16 24 hours are derived using the PBPK model of Clewell et al. (1995, 2002). 17 Calculation B: based on the unit risk for childhood exposure only (possibly the first 10 years of age) as 18 estimated by US EPA (2002 a, b), transformed to a single 24 hour exposure estimate by 19 the default procedure recommended in the SOP on AEGL development (that is, linear 20 transformation, correction by a factor of 6 to account for the relevance of sensitive stages 21 in development). Exposures of less than 24 hours are derived using the PBPK model of 22 Clewell etal. (1995,2002). 23 Calculation C: based on the cancer incidence as evident from a five-weeks animal study from Maltoni et 24 al. (1981), assuming that 5 weeks of exposure of animal is equivalent to about 150 weeks 25 exposure of humans, with linear transformation to a single 24 hour exposure without 26 further correction for potential sensitive stages of tumor development. Exposures of less 27 than 24 hours are derived using the PBPK model of Clewell et al. (1995, 2002). 28 Calculation D: based on the NOAEL for DNA adducts after single in vivo exposure of adult animals and 29 the application of an uncertainty factor for intraspecies variability. 30 Calculation C is judged to be the best basis for estimation of the risk for carcinogenic effects after single 31 exposure and is included into the main part of the TSD. However, substantial uncertainties on risk 32 quantification exist. 33 Calculation A: based on the unit risk for continuous lifetime exposure from EPA (2002 a, b), 34 transformed to a single 24 hour exposure estimate by the default procedure recommended 35 in the SOP on AEGL development (that is, linear transformation, correction by a factor of 36 6 to account for the relevance of sensitive stages in development). Exposures of less than 37 24 hours are derived using the PBPK model of Clewell et al. (1995, 2002). 38 AEGL SOP Calculation 68 ------- Vinyl chloride INTERIM 1: 3/2008 1 The US EPA's unit risk estimate for continuous lifetime exposure (inclusive of childhood) is 8.8 2 x ID'6 (ng/m3)-1. This unit risk was derived using the PBPK model of Clewell et al (1995, 2002) which 3 relates liver tumor incidence in animals with the lifetime average daily dose of the vinyl chloride 4 metabolite in the liver believed responsible for the tumor response (that is, the internal dose of the 5 metabolite). The model then uses human parameters to transform that internal dose to an external 6 exposure concentration for humans. 7 Unit risk for continuous lifetime exposure: 8.8 x 10"6 per (ig/m3 8 Exposure at a risk of 1 in 10,000: 11.36 (ig/m3 9 To convert a 70 year exposure to a 24 hour exposure, the exposure is multiplied by the number of days in 10 70 years. Under this strict c x t assumption, these exposures are considered equipotent. 11 11.36 (ig/m3x 25,600 =291 mg/m3 12 To account for uncertainty regarding the variability in the stage of the cancer process at which VC or its 13 metabolites may act, a multistage factor of 6 is applied (NRC, 2001). 14 291 mg/m3 x 1/6 = 48.5 mg/m3 (18.4 ppm) 15 Based on this transformation, a 24 hour VC exposure at this concentration would result in a 10"4 risk. For 16 10"5 and 10"6 risk, the 10"4 value is reduced by 10- and 100-fold, respectively. This estimate is based on the 17 assumption of a strict c x t relationship. 18 PBPK model calculations for an exposure less than 24 hours 19 As mentioned above, the basis of US EPA's risk estimate is the internal dose, the lifetime average 20 daily dose(LADD) of VC metabolite in the liver. For numerous reasons this metric may be quite different 21 after a single exposure of less than 24 hours. Rather than make any assumption about the extent to which 22 c x t may or may not be operative, the PBPK model was used to estimate directly the internal dose to the 23 liver under different external exposure regimes. These data are shown in the table and figure below. 24 From above, the external exposure corresponding to a 10"4 risk with a 24 hour exposure is 48.5 25 mg/m3. Values for less than 24 hour exposure are determined by interpolation using Table 1. The internal 26 dose metric (mg/L Liver) corresponding to a 10"4 risk with a 24 hour exposure is 51.4 mg/L (48.5 mg/m3 27 divided by 100 mg/m3 times 106 mg/L. The external exposure necessary to give 51.4 mg/L Liver after an 28 8 hour exposure is 147 mg/m3 (51.4 mg/L divided by 35.0 mg/L times 100 mg/m3). A corresponding 29 calculation was made for each exposure duration (0.5 hours, 1 hr, 4 hrs, and 8 hrs) and each risk level (10" 30 4, 10-5, and 10-6). 31 Dose to the liver (mg/L) of active metabolite at 24 hours after exposure to VC 32 mg/m3 0.5 hr 1 hr 4 hr 8 hr 24 hr/70 yrs 33 1 0.022 0.044 0.176 0.352 1.07 34 10 0.220 0.441 1.76 3.52 10.7 35 100 2.19 4.38 17.5 35.0 106 36 200 4.36 8.72 34.8 69.4 211 37 300 6.50 13.0 51.8 103 313 38 400 8.61 17.2 68.4 136 413 69 ------- Vinyl chloride INTERIM 1:3/2008 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 500 600 700 800 900 1000 2000 3000 4000 5000 6000 7000 8000 9000 10000 10.7 12.7 14.6 16.5 18.2 19.9 30.4 35.7 39.7 43.3 46.6 49.7 52.3 54.7 57.0 21.3 25.2 29.1 32.7 36.1 39.3 57.7 65.8 71.9 77.2 82.1 86.7 91.1 95.3 99.3 84.5 100 115 129 142 153 211 231 243 254 264 273 279 284 289 169 199 229 256 282 304 412 442 461 476 490 502 513 523 533 510 604 692 775 850 917 1220 1300 1350 1390 1420 1460 1490 1520 1540 16 Figure 5 shows the PBPK modeling results graphically (with a cut-off for the external concentration at 17 2000 mg/m3). 500 0 500 1000 1500 External concentration (mg/m3) 2000 70 ------- Vinyl chloride INTERIM 1:3/2008 1 FIGURE 5: EXTERNAL CONCENTRATION (mg/m3) AND DOSE TO LIVER (mg/L) AS 2 CALCULATED BY PBPK-MODELING BY EPA (Personal Communication, Gary Foureman, US 3 EPA, NCEA-RTP, June 2003) 4 5 6 7 8 9 10 If the exposure is limited to a fraction of a 24-hour period, the exposure corresponding to the various risk levels are presented in the table below. Exposure Duration 8 hours 4 hours 1 hour 30 minutes 10 4 risk 147 mg/m3 (55. 9 ppm) 298mg/m3(113ppm) 1780 mg/m3 (676 ppm) 7870 mg/m3 (2990 ppm) 10 5 risk 14.6 mg/m3 (5.55 ppm) 29.2 mg/m3 (11.1 ppm) 117 mg/m3 (44.5 ppm) 236 mg/m3 (89.7 ppm) 10 6 risk 1.46 mg/m3 (0.555 ppm) 2.92 mg/m3 (1.11 ppm) 1 1.6 mg/m3 (4.45 ppm) 23. 3 mg/m3 (8. 97 ppm) 71 ------- Vinyl chloride INTERIM 1:3/2008 1 2 3 4 5 6 7 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 Calculation B: based on the unit risk for childhood (possibly first 10 years of age) as estimated by EPA (2000 a,b), transformed to a single exposure estimate by the default procedure, recommended in the SOP on AEGL development (i.e. linear transformation, correction by a factor of 6 to account for the relevance of sensitive stages in development). Exposures of less than 24 hours derived using the PBPK model of Clewell et al. (1995, 2002). The unit risk calculation of EPA is based on the occurrence of angiosarcoma in newborn rats (5 weeks exposure) which were observed with similar incidences as in adult female rats (52 weeks exposure beginning at 13 weeks of age; see Table Cl). Thus, the unit risk for adults (long term study) was directly calculated and was assumed to be roughly identical for childhood (first 10 years of exposure). unit risk for continuous childhood exposure: 4.4 x 10"6 per |o,g/m3 (first 10 years) dose at risk 1 : 10,000: 22.73 To convert a 10 year exposure (= 10 x 365.7 = 3657) to a 24 hours exposure, the dose is multiplied by the number of days in 10 years: 22.73 |^g/m3 x 3657 = 83.1 mg/m3 To account for uncertainty regarding the variability in the stage of the cancer process at which VC or its metabolites may act, a multistage factor of 6 is applied (NRC, 2001): 83.1 mg/m3 x 1/6 = 13.85 mg/m3 Therefore, based upon the potential carcinogenicity of VC during early life, a 24 h exposure corresponding to a 10"4 risk would be 13.85 mg/m3 (5.26 ppm). For 10"5 and 10"6 risk levels, the 10"4 values are reduced by 10-fold and 100-fold, respectively. If the exposure is limited to a fraction of a 24-hour period, the exposure corresponding to the various risk levels are presented in the table below. These values were calculated using the PBPK model for vinyl chloride as described above for calculation A. Exposure Duration 8 hours 4 hours 1 hour 30 minutes 10 4 risk 42.1 mg/m3 (16.0 ppm) 84.5 mg/m3 (32.1 ppm) 342 mg/m3 (130 ppm) 709 mg/m3 (269 ppm) 10 5 risk 4.21 mg/m3 (1.60 ppm) 8. 41 mg/m3 (3 .20 ppm) 33.6 mg/m3 (12. 8 ppm) 67.5 mg/m3 (25.7 ppm) 10 6 risk 0.421 mg/m3 (0.160 ppm) 0.840 mg/m3 (0.329 ppm) 3. 36 mg/m3 (1.28 ppm) 6.72 mg/m3 (2.55 ppm) 29 Calculation C: based on the cancer incidence as evident from a five-weeks animal study from Maltoni et 30 al. (1981), assuming that 5 weeks of exposure of animal is equivalent to about 150 weeks 31 exposure of humans, with linear transformation to a single 24 hour exposure without 72 ------- Vinyl chloride INTERIM 1:3/2008 4 5 6 7 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 further correction for potential sensitive stages of tumor development. Exposures of less than 24 hours are derived using the PBPK model of Clewell et al. (1995, 2002). The study seems to be relevant, as • investigations were performed with newborn rats which represent a sensitive subgroup for the endpoint carcinogenesis exposure was over a short period of time endpoints (incidence of liver angiosarcoma) are relevant for humans. Data are shown in table C1: TABLE Cl Administered 4 hours/day, 5 INCIDENCE OF TUMORS IN THE STUDIES FROM MALTONI ET AL., 1981, (EXPERIMENTS BT 14 AND BT 1), CITED FROM EPA, 2000a concentration (ppm) Angiosarcoma Hepatoma days/week for 5 weeks starting at day 1 (BT 14) 6000 10000 4 hours/day, 5 20/42 (48%), all* 17/42 (40.5%) ,LAS* 18/44 (41%), all* 15/44 (34. 1%), LAS* 20/42 (47,6 %) 20/44 (45,4 %) days/week for 52 weeks starting at age 13 weeks (BT 1) 6000 10000 22/42 (52%), all* 13/42(31%), LAS* 13/46 (28 %), all* 7/46 (15%), LAS* 1/27 (3,7%) 1/24 (4,2%) * Angiosarcoma, all sites include extra-liver angiosarcoma, including angioma; LAS: liver angiosarcoma (only those were taken for further risk quantifications) Derivation on the Inhalation Unit Risk Exposure concentration: liver angiosarcoma 6,000 ppm 40.5 % 6,000 ppm corresponds to a human equivalent concentration of 51 ppm (132 mg/m3), based on the PBPK model published by Clewell et al. (1995). Corresponding data are shown in table C2 (note that rats exposure is intermittent (4hours/day; 5 days/week) compared to HEC (human equivalent exposure) which is given for continuous exposure (24 hours/day)). Note further that saturation in rats leads to only minor increases of metabolite concentrations, when exposure exceeds 250 ppm (intermittent exposure). The derivation of the Inhalation Unit Risk is based on the assumption that the tumor response is a linear function of the concentration of the active metabolite in the liver (HEC). See Table C2. 30 132 mg/m3 = 40.5%; 73 ------- 8 9 10 11 12 13 14 15 16 17 18 19 20 Vinyl chloride INTERIM 1: 3/2008 1 => 3.3 mg/m3 = 1%; 2 => 33 [ig/m3 = 0.01%= 1:10,000 3 dose at risk (1:10,000): 33.0 [ig/m3 4 conversion from 5 weeks to 24 h exposure: 5 Newborn rats grow about 30 times faster than newborn humans (NRC, 1993), which is similar to the ratio 6 of lifetime 75 years (human): 2.5 years (rat) = 30. 5 x 7 x 30 =1050 7 33,0 [ig/m3 x 1050 days = 34.7 mg/m3 (14 ppm) An additional factor to adjust for uncertainties in assessing potential cancer risks under short term exposures is not applied, as exposure was short-term in the underlying study. Therefore, based upon the potential carcinogenicity of VC during early life, a 24 h exposure corresponding to a 10"4 risk would be 34.7 mg/m3 (13.2 ppm). For 10"5 and 10"6 risk levels, the 10"4 values are reduced by 10-fold and 100-fold, respectively. If the exposure is limited to a fraction of a 24-hour period, the exposure corresponding to the various risk levels are presented in the table below. These values were calculated using the PBPK model for vinyl chloride as described above for calculation A. Exposure Duration 8 hours 4 hours 1 hour 30 minutes 10 4 risk 106 mg/m3 (40. 3 ppm) 213 mg/m3 (80. 9 ppm) 922 mg/m3 (350 ppm) 3 110 mg/m3 (1180 ppm) 10 5 risk 10.5 mg/m3 (3.99 ppm) 2 1.0 mg/m3 (7.98 ppm) 84.4 mg/m3 (32.1 ppm) 170 mg/m3 (64.6 ppm) 10 6 risk 1.05 mg/m3 (0.399 ppm) 2. 10 mg/m3 (0.798 ppm) 8. 40 mg/m3 (3. 19 ppm) 16.8 mg/m3 (6.38 ppm) 21 22 23 24 25 26 27 28 29 30 31 A similar result is obtained if the tumor data from Froment et al. (1994) are used. Froment et al. exposed the newborn animals to only 500 ppm. Hence, fewer extrapolations were needed compared to the Maltoni et al. data. (Data and calculation not shown). For both calculations, relevant uncertainty on the influence of the oral uptake of mothers'milk has to be stated. Because of metabolic saturation at high level inhalation exposure, this influence may have been limited. However, no estimate of the quantitative consequences of this multi pathway exposure may be given. TABLE C2: CONVERSION OF ADMINISTERED VC DOSE TO A HUMAN EQUIVALENT CONCENTRATION (data from EPA, 2000a, b) Admin, cone. (ppm)a 0 1 Metabolite (mg/L liver)b 0 0.59 HEC (ppm)c 0 0.2 74 ------- Vinyl chloride INTERIM 1:3/2008 5 10 25 50 100 150 200 250 500 2500 6000 2.96 5.9 14.61 31.27 55.95 76.67 90 103.45 116.94 134.37 143.72 1 2 4.6 10.1 19 26 31 35 40 48 51 a Animals exposed 4 hours/day, 5 days/week for 52 weeks. b Dose metric (lifetime average delivered dose in female rats) calculated from PBPK modeling of the administered animal concentration. c Continuous human exposure concentration over a lifetime required to produce an equivalent mg metabolite/L of liver. 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 Calculation D: based on the NOAEL for DNA adducts after single in vivo exposure of adult animals and the application of an uncertainty factor for intraspecies variability. DNA-adducts seem to be relevant and quantitatively linked to carcinogenic potency of VC: ethenobases were shown to possess miscoding properties (Barbin, 2000) and are slowly repaired (Morinello et al., 2002a) ethenobases generate mainly base pair substitution mutations (Barbin, 2000) • ethenobases assumed to be initiating lesions in carcinogenesis (Barbin, 2000) • high correlation between DNA-adducts formation (sG) and incidence of haemangiosarcoma in mice after exposure to vinyl fluoride (Swenberg et al., 1999) Elevated DNA-adducts were seen after single 5 hour exposure of adult rats to 250 ppm VC (Bolt et al., 1980). Watson et al. (1991) exposed adult male Fisher 344 rats for 6 hours to atmospheres containing 1, 10, or 45 ppm VC. The alkylation frequencies of 7-(2'-oxoethyl)guanine (OEG) in liver DNA were 0.026, 0.28 and 1.28 residues OEG per 106 nucleotides respectively. With these air concentrations, there was no evidence to indicate the formation of the cyclic adducts l,N6-ethenoadenine (sA) or 3,N4-etheno- cytosine (sC). The threshold for detection of these adducts were about 1 adduct per 1 x 10s nucleotides. Swenberg et al. (1999) reported a factor 1/10 - 1/100 to calculate the amount of N2,3-ethenoguanine (sG) in relation to OEG. Thus, sG would be lower than 0.1 - 0.01 per 106 nucleotides at 45 ppm. This would equal the reported background of sG (Swenberg et al., 1999). It may be concluded that single exposure to 45 ppm VC (6 hours) would not lead to an increase of relevant cyclic adducts (sA, sC, sG) in adult rats. 75 ------- Vinyl chloride INTERIM 1:3/2008 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 With higher DNA-adduct levels (at higher single exposure, or in young rats, or after repeated short term exposure) there apparently is a relevant correlation to mutations, foci or carcinogenicity: Adult rats repeatedly (5 days) exposed to 10 ppm VC for 6 hours/day showed slightly elevated etheno-adducts (sG) compared to control (Swenberg et al, 2000). Higher adduct levels were seen in young animals than in adult animals after identical treatment (Fedtke et al., 1990; Laib et al., 1989; Ciroussel et al.,1990, Morinello et al., 2002a). Respective mutations (e.g., G->A transitions, A->T transitions) were observed in VC-induced tumors (Barbin, 2000). Despite relevant repair, no full reduction to background was observed for these adducts two weeks after a 5 day exposure (4 hours/day) to 600 ppm (Swenberg et al., 1999).DNA-adducts formation (sG) in whole liver DNA or hepatocytes increased linearly from 5 days to 8 weeks after exposure of rats to 500 ppm or 10 ppm VC (Morinello et al.,2002a). Table C3 presents the data for relevant DNA-adducts after short term exposure to VC for different concentrations and exposure durations and gives an indication about the reversibility. TABLE C3: DNA-ADDUCTS AFTER SINGLE AND SHORT TERM VC EXPOSURE VC-inhalation (ppm) 7-(2'-oxoethyl)guanine (OEG) adducts/ nucleotides]1 l,N6-ethenoadenine (sA)1 3,N4-ethenocytosine (sC)1 N2,3-ethenoguanine (sG)* 0 1 0.026/106 10 0.28/106 45 1.28/106 <1/108 <1/108 -1/108 for comparison2: sG- Background (rat) sG, 5 days eG, 20 days sG, 4h/d, 5d, immed. after exposure sG, 4h/d, 5d, 14 days after exposure sG- Background (human) 0.9/107 6/108-7/107 2/107 5.3/107 100 6.8/107 2.3/106 600 3.8/106 4.7/107 * estimated (eG) by the authors of the TSD from ratio ~ 1/100 OEG/eG in other VC experiments 1 data from Watson et al.,1991;2 data from Swenberg et al.,1999 76 ------- Vinyl chloride INTERIM 1:3/2008 TABLE C4: ADDUCTS RATIO NEONATE: ADULT FOR VINYL CHLORIDE Swenberg et al, 1999 (OEG) 600 ppm 5d, 4h/d, rat 162/43-3.8 Swenberg et al., 1999 (sG) 600 ppm 5d, 4h/d, rat 1.81/0.47-3.9 Ciroussel et al., 1990 (sdAdo/dAdo) 500 ppm 2 weeks, 7h/d, rat 1.3/0.19-6.8 Ciroussel et al., 1990 (sdCyd/dCyd) 500 ppm 2 weeks, 7h/d, rat 4.92/0.8-6.15 2 3 4 5 6 7 9 10 11 12 13 14 15 16 17 18 calculation of an practical threshold ("NAEL") for short term exposure: Intraspecies: Because of the high sensitivity of young animals an intraspecies factor of 10 is regarded as necessary. This is supported by comparisons between effects at different ages based on tumors, foci or DNA-adducts. For DNA-adducts a comparison is shown in table C4. Interspecies: There is no apparent higher sensitivity of men compared to rats, which is supported by the comparison of unit risks derived from animal data respectively human data (Clewell et al., 2001). This leads to an uncertainty factor for interspecies differences of 1 (EPA, 2000a). Exponent for time extrapolation: Steady state is not reached within 8 hours as evidenced by the longer halftime of metabolites. Thus, default time extrapolation should be performed based on the observed NOAEL at 6 hours exposure. This leads to an estimated close to background level as quantified by the calculations below: 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 Key study: Toxicity endpoint: Uncertainty/ modifying factors: Time Scaling: 30-minute: 1-hour: Watson et al.,1991; Swenberg et al., 1999; Barbin, 2000 DNA-adducts; background adduct levels at single 45 ppm exposure of rats is taken as practical "NAEL" (6 hours) Combined uncertainty factor of 10 1 for interspecies variability 10 for intraspecies variability C3 x t = k for extrapolation to 4-hour, 1-hour, and 30-minute; k = (45 ppm)3x 360 min = 3,2 x 10E+7 ppm3 min C1 x t = k for extrapolation to 8-hours; k = 45 ppmx 360 min = 16,200 ppm1 min C3 x 30 min = 3,2 x 10E+7 ppm3 min C = 103 ppm 30-min NAEL = 103 ppm/10 = 10 ppm (= 26 mg/m3) C3 x 60 min =3,2 x 10E+7 ppm3 min C = 81.8 ppm 1-h NAEL = 81.8 ppm/10 = 8.2 ppm (= 21 mg/m3) 77 ------- Vinyl chloride 4-hour: 8-hour: INTERIM 1:3/2008 C3 x 240 min =3,2 x 10E+7 ppm3 min C = 51.5 ppm 4-h NAEL = 51.5 ppm/10 = 5.1 ppm (= 13 mg/m3) C x 480 min = 16200 ppm min C = 33.75 ppm 8-h NAEL = 34 ppm/10 = 3.4 ppm (= 8.8 mg/m3) Concluding remark: Table C5 provides an overview of the calculations on carcinogenic potency after single exposure as derived above compared to the AEGL- values derived based on nonmalignant effects. TABLE C5: COMPARISON OF AEGL VALUES (VC) BASED ON NONMALIGNANT EFFECTS AND DIFFERENT ESTIMATIONS OF CARCINOGENIC RISK AFTER SINGLE EXPOSURE fppml AEGL-l(Baretta et al., UF:3; n=3,l) AEGL-2 (Lester et al.,UF:3; n=2 to 2h; 2h=4h=8h) AEGL-3 (Clark & Tinston; UF:3; n=2 to 2h; 2h=4h=8h) 10-minute 450 2800 12000 30-minute 310 1600 6800 1-hour 250 1200 4800 4-hour 140 820 3400 8-hour 70 820 3400 estimation of carcinogenic potency (10~4 risk): CALCULATION A (unit risk) default SOP; linear transformation ifetime unit risk x 6 CALCULATION B (unit risk) linear transformation, early life=10 years, x 6 CALCULATION C (Maltoni et al., 1981, risk-direct from 5w-study); Human equivalent dose to 6000 ppm; growth rate rat/hum: 30 CALCULATION D (Watson et al., (DNA)), UF:3; n=3: 30,60, 120,480 min; n=l: 8h; 10min=30min. 2990 269 1180 10 676 130 350 8.2 113 32.1 80.9 5.1 55.9 16 40.3 3.4 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 Calculation C is judged to be the best basis for estimation of the risk for carcinogenic effects after single exposure and is included into the main part of the TSD. However, substantial uncertainties on risk quantification persists. 78 ------- Vinyl chloride INTERIM 1: 3/2008 1 APPENDIX D - Occupational epidemiological studies on carcinogenicity (focus: limited exposure 2 time) 79 ------- Vinyl chloride INTERIM 1: 3/2008 1 Two large studies of workers employed in the VCM/PVC industry prior to 1974 were completed. 2 Both studies were retrospective cohort mortality studies. The first study was done in Europe and included 3 study populations in Italy, Norway, Sweden and United Kingdom. The second study included plants in 4 the United States and Canada. Each study has been updated multiple times and has been the subject of 5 numerous papers. Only the results from the most recent updates are discussed here. The focus is to review 6 the liver cancer incidence in workers exposed to VCM for relatively short time periods or where the 7 cumulative dose (ppm-years) was known to have been low. Both studies have more deaths than expected 8 from ASLs among workers with high and/or long exposure to VCM (Ward et al., (2000) and Mundt et al., 9 (1999)). A third study from Weber et al. (1981) with epidemiologic data from Germany shows 10 conflicting results to the above cited large studies. 11 European Study 12 The European study includes approximately 12,700 men with at least one year of employment in 13 the VCM/PVC industry from 1955 to 1974 (Ward et al., 2000). Three of the 19 plants had incomplete 14 records and thus the starting date for these three plants ranged from 1961 to 1974. The vital status follow- 15 up was complete through 1997. Age- and calendar period-specific mortality rates for males from Italy, 16 Norway, Sweden and United Kingdom were used to calculate the Standardized Mortality Ratios (SMR) 17 and Confidence Intervals (CI). Typical exposure scenarios were estimated by industrial hygienists based 18 on job exposure matrices. These job exposure matrices were based primarily on job title and were 19 reviewed by two other industrial hygienists with several years of experience in the VC industry. 20 Information provided in the job exposure matrix was used to develop a ranked level of exposure index. 21 Quantitative estimates of exposure were obtained for 82% of the cohort. 22 The total number of person-years at risk by the cohort is 324,701. The work force was classified 23 by duration of employment, <3, 3-6, 7-11, 12-18 and 19+ ppm-years. The SMR (CI) for liver cancer for 24 workers with less than 3 years experience was 62 (2-345), below the expected value (Table Dl). For 25 workers exposed to VCM/PVC for a longer time period, the incidence of liver cancer was higher than 26 expected. In general, the incidence of liver cancer increased with years of employment in the VCM/PVC 27 industry. 28 In addition, Ward et al., (2000), examined cumulative exposure for the cohort (Table D2). Again, 29 the work force was subdivided into 0-734, 735-2379, 2380-5188, 5189-7531 and 7532+ ppm-years. The 30 SMR (CI) was 107 (54-192) based on 11 observed liver cancers and 10.26 expected. Assuming workers 31 are employed in the industry for up to 30 years, to be included in this first category, the highest average 32 concentration the worker would have been exposed to was -25 ppm. Workers with shorter work histories 33 may have been exposed to much higher concentrations. Under this scenario there was no increase in the 34 incidence of liver cancer. As previously noted, the incidence of liver cancer increased with cumulative 35 exposure with an SMR(CI) of 1140 (571-2050) for those workers with a cumulative exposure of 7532+ 36 ppm-years. However, of the 11 liver cancers observed in the 0-734 ppm-year cumulative exposure 37 group, fours were angiosarcomas. These four angiosarcomas occurred in individuals with 287- 38 734 ppm-years cumulative exposure (Ward et al., 2001). There were no angiosarcomas reported 39 in workers with less than 287 ppm-years cumulative exposure. 80 ------- Vinyl chloride INTERIM 1:3/2008 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 North American Study The North American study consists of approximately 10,100 men employed for at least one year in the VCM/PVC industry from 1942-1974 (Mundt et al, 1999). This group was followed through December 31, 1995. Thus, most workers have been followed for at least twenty one years. Since the VCM/PVC industry was located in 16 states and one Province of Canada, mortality rates for 16 states were used to calculate SMR's. For the Province of Canada, mortality rate data from the state of Michigan was used since it was geographically the closest to the plant. As of December 31, 1995, 30% of the study group were deceased Although the authors of previous studies had attempted to categorize individuals by exposures, no consistent criteria had been used and thus no attempt was made to estimate exposure levels in this study. The age at first exposure, duration of exposure and year of first exposure appeared to be related to cancer of the liver and biliary tract (data not shown). Of these, duration of exposure had the greatest significance and appeared to be independent of age at first exposure and year of first exposure (Table D3). Mundt categorized the cohort into groups working 1-4, 5-9, 10-19 or 20+ years in the VCM/PVC industry. Nearly half of the cohort worked for less than 5 years in the VCM/PVC industry with fewer workers in each of the subsequent groups. This data shows that working in the VCM/PVC industry for 1-4 years resulted in a slightly lower liver cancer rate than expected. Working in this industry for longer periods of time resulted in higher death rates than expected for liver and biliary tract cancer. Mundt et al. (2000) also examined the incidence of angiosarcomas based on duration of exposure. Three individuals working in the VCM/PVC industry for 1-4 years have ASLs. No further information on exposure or job classification was provided. Both of these studies have shown that working in the VCM/PVC industry for <3 years or to a low, but still relevant, estimated concentration of VCM resulted in liver cancer rates very close to expected values. A low incidence of ASLs was reported by both Ward et al. (2000) and Mundt et al. (2000) but based on the Ward study appeared to be related to higher ppm-years exposure. TABLE Dl: LIVER CANCER INCIDENCE FOR ALL EUROPEAN COUNTRIES BY DURATION OF EMPLOYMENTA Duration of Incidence Employment (years) <3 3-6 7-11 12-18 19+ Total Number of Individuals'5 10961 8999 6919 4610 2006 12700 Number of person years 91970 79747 65789 55149 32050 324706 (Observed/ Expected) 1/1.61 3/1.44 7/1.35 5/1.42 13/1.46 29/7.29 SMR (95%CI)C 62 (2-345) 208 (43-609) 517(208-1060) 352 (114-821) 893 (475-1530) 398 (267-572) a From Tables T1.7 and D7 of Ward et al., (2000). b Number of individuals cited for various employment intervals add up to greater than 12,700 since individuals can meet more than one criteria as defined by the author. c SMR = Observed/Expected *100. CI = Confidence Intervals. 40 TABLE D2: LIVER CANCER INCIDENCE FOR ALL EUROPEAN COUNTRIES BY 41 CUMULATIVE EXPOSUREA 81 ------- Vinyl chloride INTERIM 1:3/2008 Cumulative Exposure (ppm-years) Unknown 0-734 735-2379 2380-5188 5189-7531 7532+ Total Number of Individuals'5 2243 9552 2772 1463 515 215 12700 Number of person years 52300 188204 43174 26480 9274 5274 324706 Incidence (Observed/ Expected) 2/3.19 11/10.26 9/3.32 10/2.62 10/1.77 11/0.96 53/22.11 SMR (95%CI)C 63 (8-227) 107(54-192) 271(124-515) 382(183-703) 566(271-1040) 1140(571-2050 240(1800-3140) 1 2 3 4 5 6 7 9 10 11 12 13 a From Tables 12 and D7 of Ward et al., (2000). b Number of individuals cited for various employment intervals add up to greater than 12,700 since individuals can meet more than one criteria c SMR = Observed/Expected * 100. CI = Confidence Intervals. TABLE D3: LIVER AND BILIARY TRACT CANCER INCIDENCE FOR THE UNITED STATES BY DURATION OF EMPLOYMENTA Duration of Employment (years) 1-4 5-9 10-19 20+ Total Number of Individuals 4774 2383 1992 960 10109 Number of person years 136200 71806 69015 39524 Incidence (Observed/ Expected) 7/8.43 10/4.65 39/5.74 24/3.49 SMR (95%CI)b 83 (33-171) 215 (103-396) 679 (483-929) 688 (440-1023) 14 15 16 17 18 19 20 21 22 23 24 a From Tables 21 and 23 of Mundt et al., (1999). b SMR = Observed/Expected * 100. CI = Confidence Intervals. 25 Study from Weber et al.. 1981 26 Three German cohorts were investigated: Group 1 (VCM/PVC production; 7021 persons; 73734 27 person years, Group 2, (reference group, 4910 persons; 76029 person years), Group 3 (PVC processing, 28 4007 persons; 52 896 person years). West German reference mortality rates were used for comparison. 29 Malignant tumors of the liver occurred in 12 cases (VCM/PVC production; SMR=1523) or 4 cases in the 30 reference group (SMR=401) or 3 cases in PVC processing (SMR=434). No confidence intervals were 31 provided. No exposure concentration is known. The subclassification according to duration of 32 employment demonstrates increased mortality already after little more than 1 year of exposure (Table 33 D4). Results from this study together with the results from the studies cited above are included in a meta- 82 ------- Vinyl chloride INTERIM 1:3/2008 9 10 11 12 analysis from Boffetta et al. (2003) and illustrated by graphical presentation (see figure 1; Boffetta et al., 2003) showing the conflicting information about minimum exposure duration for adult workers to have a increased tumor risk. TABLE D4: LIVER CANCER IN VCM/PVC-PRODUCTION AND DURATION OF EXPOSURE3 Duration of Employment (months) <12 13-60 61-120 >121 Total Cases 0 2 3 7 12 SMR - 874 1525 2528 - beyond 95th confidence interval beyond 99th confidence interval beyond 99th confidence interval 1 From Table 3, Weber et al., 1981. 83 ------- Vinyl chloride INTERIM 1: 3/2008 APPENDIX E - Derivation Summary for Vinyl Chloride AEGLs 84 ------- Vinyl chloride ACUTE EXPOSURE GUIDELINES FOR VINYL CHLORIDE (CAS Reg. NO. 75-01-4) INTERIM 1:3/2008 10 minutes 450 ppm 30 minutes 310 ppm 1 hour 250 ppm 4 hours 140 ppm 8 hours 70 ppm AEGL-1 VALUES Reference: Baretta, E.D., R.D. Stewart, J.E. Mutchler, 1969. Monitoring exposures to vinyl chloride vapor: breath analysis and continuous air sampling. American Industrial Hygiene Association Journal, 30, 537-544. Test Species/Strain/Sex/Number: human volunteers, male, 4-7 individuals Exposure Route/Concentrations/Durations: inhalation; 3.5 hours; 459 - 491 ppm, 3.5 - 7.5 hours Effects: mild headache, some dryness of eyes and nose in 2/7 subjects Endpoint/Concentration/Rationale: Endpoints relevant for the derivation of AEGL-1 values for VC have are: a) headache, b) odor recognition or detection, c) irritation. Occurrence of mild headache has been reported by Baretta et al. (1969) in two subjects after acute exposure, an endpoint which can be regarded as NOAEL for AEGL-1. No qualified studies on odor recognition or detection are reported for VC. Irritation in humans or animals is only reported in the context of exposure to very high concentrations which are lethal or cause unconsciousness. The mechanism by which headaches developed are not clearly understood. The derived AEGL-1 does not necessarily exclude mutagenic or tumorigenic effects by VC at similar or lower concentrations. Uncertainty Factors/Rationale: The intraspecies uncertainty factor of 3 is used to compensate for both, toxicokinetic and toxicodynamic differences between individuals. For headaches, no or only very slight effects would be expected for the general public after inclusion of an intraspecies factor of 3 on the "mild" effects observed in volunteers. Modifying Factor: not applicable Animal to Human Dosimetric Adjustment: not applicable Time Scaling: The duration-specific values were derived by time scaling according to the dose-response regression equation Cn x t = k, using the default of n=3 for shorter exposure periods and n=l for longer exposure periods, due to the lack of suitable experimental data for deriving the concentration exponent. The extrapolation to 10 minutes from a 3.5 hour exposure is justified because exposure of human at 4,000 ppm for 5 minutes did not result in headache (Lester et al., 1963). Data Adequacy: The study of Baretta et al. (1969) has been regarded as qualified for the derivation of AEGL-1 values and the endpoint is supported by several findings from occupational studies (Lilis et al., 1975; Suciu et al., 1975; EPA, 1987). Confirmation of the observed effects in other studies with controlled exposure would be helpful, but may not be performed due to ethical reasons. 85 ------- Vinyl chloride ACUTE EXPOSURE GUIDELINES FOR VINYL CHLORIDE (CAS Reg. NO. 75-01-4) INTERIM 1:3/2008 10 minutes 2,800 ppm 30 minutes 1,600 ppm 1 hour 1,200 ppm 4 hours 820 ppm 8 hours 820 ppm AEGL-2 VALUES Reference: Lester, D., L.A. Greenberg, W.R. Adams, 1963. Effects of single and repeated exposures of humans and rats to vinyl chloride. American Industrial Hygiene Association Journal, 24, 265-275; Clark, D.G., D.J. Tinston, 1973. Correlation of the cardiac sensitizing potential of halogenated hydrocarbons with their physicochemical properties. Br. J. Pharm., 49, 355-357. Mastromatteo, E., A.M. Fisher, H. Christie, H. Danziger, 1960. Acute inhalation toxicity of vinyl chloride to laboratory animals. Am. Ind. Hyg. Assoc. J., 21, 394-398. Test Species/Strain/Sex/Number: human male (n=3) and female (n=3) volunteers, 6 persons Exposure Route/Concentrations/Durations: Inhalation, single exposure, 0, 4,000, 8,000, 12,000, 16,000, 20,000 ppm for 5 minutes Effects: After 5 minute exposure to 16,000 ppm VC 5 of 6 persons showed dizziness, lightheadedness, nausea, visual and auditory dulling. At concentrations of 12,000 ppm one of six persons reported "swimming head, reeling", another was unsure of an effect and felt somewhat dizzy. A single person reported slight effects ("slightly heady") of questionable meaning at 8,000 ppm (this volunteer felt also slightly heady at sham exposure and reported no response at 12,000 ppm). No effects were observed at 4,000 ppm. 12,000 ppm was regarded as a concentration below AEGL-2 level and taken as NOAEL. Derived AEGL-2 levels are supported by the an assumed NOAEL for cardiac sensitization of 17,000 ppm in dogs after epinephrine challenge (5 minutes exposure; Clark and Tinston, 1993), leading to similar values. However, the resulting AEGL-2 values may not provide a sufficient margin of safety to avoid mutational events or malignancies after short-term exposure to VC. Endpoint/Concentration/Rationale: Severe dizziness may influence capability to escape and thus is relevant as endpoint for AEGL-2. At 12,000 ppm no such effects were seen.Derived AEGL-2 levels are supported by the an assumed NOAEL for cardiac sensitization of 17,000 ppm in dogs after epinephrine challenge (5 minutes exposure; Clark and Tinston, 1993), leading to similar values. Uncertainty Factors/Rationale: A total uncertainty factor of 3 is used to compensate for both, toxicokinetic and toxicodynamic variability, with small interindividual differences in case of CNS- effects. As the unmetabolized VC is responsible for the effects no relevant differences in kinetics are assumed. Total uncertainty factor: 3 Interspecies: 1 Intraspecies: 3 Modifying Factor: Not applicable Animal to Human Dosimetric Adjustment: Not applicable 86 ------- Vinyl chloride INTERIM 1:3/2008 9 10 11 Time Scaling: In analogy to other anaesthetics the effects are assumed to be solely concentration dependent. Thus, after reaching steady state (about 2 hours), at 4 and 8 hours no increase of effect-size by duration is expected. The other exposure duration-specific values were derived by time scaling according to the dose-response regression equation Cn x t = k, using a factor of n=2, based on data from Mastromatteo et al. (1960). Mastromatteo et al. observed various time-dependent prenarcotic effects in mice and guinea pigs after less than steady state exposure conditions. With this, time extrapolation was performed from 5 to 10, 30, 60 minutes and 2 hours, where the steady state concentration was calculated. Data Adequacy: The overall quality of the key study (Lester et al., 1963) is medium. There is an observed dose-/response relationship supporting the quantitative figures. Subjective reporting of effects leads to limited preciseness. 87 ------- Vinyl chloride ACUTE EXPOSURE GUIDELINES FOR VINYL CHLORIDE (CAS Reg. NO. 75-01-4) INTERIM 1:3/2008 10 minutes 12,000 ppm 30 minutes 6,800 ppm 1 hour 4,800 ppm 4 hours 3,400 ppm 8 hours 3,400 ppm AEGL-3 VALUES References: Clark, D.G., D.J. Tinston, 1973. Correlation of the cardiac sensitizing potential of halogenated hydrocarbons with their physicochemical properties. British Journal of Pharmacology, 49, 355-357. Clark, D.G., D.J. Tinston, 1982. Acute inhalation toxicity of some halogenated and non- halogenated hydrocarbons. Human Toxicology, 1, 239-247., Aviado, D.M., M.A. Belej, 1974. Toxicity of aerosol propellants in the respiratory and circulatory systems. I. Cardiac arrhythmia in the mouse. Toxicology, 2, 31-42.; Belej, M.A., D.G. Smith, D.M. Aviado, 1974. Toxicity of aerosol propellants in the respiratory and circulatory systems. IV. Cardiotoxicity in the monkey. Toxicology, 2, 381-395; Prodan, L., I. Suciu, V. Pislaru, E. Ilea, L. Pascu, 1975. Experimental acute toxicity of vinyl chloride (monochloroethene). Ann. NY Acad. Sci., 246, 154-158. Mastromatteo, E., A.M. Fisher, H. Christie, H. Danziger, 1960. Acute inhalation toxicity of vinyl chloride to laboratory animals. Am. Ind. Hyg. Assoc. J., 21, 394-398. Test Species/Strain/Sex/Number: dog, beagle, sex not reported, 4-7 dogs/dose level (Clark and Tinston, 1973) Exposure Route/Concentrations/Durations: inhalation /"several doses" / 5 minutes (Clark and Tinston, 1973) Effects: Short term exposure (5 min) of dogs to VC induced cardiac sensitization towards epinephrine (EC50: 50,000 or 71,000 ppm in two independent experiments; Clark and Tinston, 1973; 1982). The lower reported EC50 (50,000 ppm) was taken as NOAEL for life threatening effects. These effects were also seen in mice at higher concentrations (Aviado and Belej, 1974). In monkeys, only myocardial depression after inhalation of 2.5-10% VC was observed. It is not clearly stated whether an addition challenge with epinephrine was applied or not (Belej et al., 1974). Severe cardiac sensitization is a life threatening effect, but at 50,000 ppm no animal died in the reported study, providing a NOAEL for AEGL-3 derivation. Endpoint/Concentration/Rationale: Considering possible sensitive subpopulations and increased excitement in case of emergency reaction epinephrine induced cardiac reactions may occur and may be enhanced by high exposure concentrations to VC. The respective effects are well known for certain unsubstituted and halogenated hydrocarbons. The test method using beagle dogs is well established. Supported by lethality data in slightly higher concentrations (Prodan et al., 1975). Uncertainty Factors/Rationale: A total uncertainty factor of 3 is used to compensate for both, toxicokinetic and toxicodynamic differences between individuals and interspecies differences. As the challenge with epinephrine and the doses of epinephrine used represent a conservative scenario an interspecies factor of 1 was employed. As the unmetabolized VC is responsible for the effects no relevant differences in kinetics are assumed. Total uncertainty factor: 3 Interspecies: 1 Intraspecies: 3 88 ------- Vinyl chloride INTERIM 1:3/2008 Modifying Factor: Not applicable Animal to Human Dosimetric Adjustment: Insufficient data 3 4 5 6 7 8 9 10 11 12 13 Time Scaling: In analogy to other halocarbons (e.g., Halon 1211, HFC 134a) which lead to cardiac sensitization the effects are assumed to be solely concentration dependent. Thus, after reaching steady state (about 2 hours), at 4 and 8 hours no increase of effect-size by duration is expected. The other exposure duration-specific values were derived by time scaling according to the dose-response regression equation Cn x t = k, using a factor of n=2, based on data from Mastromatteo et al. (1960). Mastromatteo et al. observed various time-dependent prenarcotic effects (muscular incoordination, side position and unconsciousness, effects which occur immediately before lethality) in mice and guinea pigs after less than steady state exposure conditions. With this, time extrapolation was performed from 5 to 10, 30, 60 minutes and 2 hours, where the steady state concentration was calculated. Data Adequacy: Due to some discrepancies between the two studies from Clark and Tinston (1973, 1982) the data quality is judged to be medium with adequate data from human experience lacking. 89 ------- |