PROPOSED 1: 03/ 2004 United States Environmental Protection Agency Office of Pollution Prevention and Toxics VINYL CHLORIDE ( CAS Reg. No. 75 01 4)

PROPOSED ACUTE EXPOSURE GUIDELINE LEVELS ( AEGLs)

" PUBLIC DRAFT"

Federal Register  March 2004 2 PROPOSED 1: 3/ 2004 VINYL CHLORIDE ( CAS Reg. No. 75 01 4)

PROPOSED ACUTE EXPOSURE GUIDELINE LEVELS ( AEGLs) Vinyl chloride PROPOSED 1: 3/ 2004 iii PREFACE 1 Under the authority of the Federal Advisory Committee Act ( FACA) P. L. 92 463 of 1972, the 2 National Advisory Committee for Acute Exposure Guideline Levels for Hazardous Substances 3 ( NAC/ AEGL Committee) has been established to identify, review and interpret relevant toxicologic and 4 other scientific data and develop AEGLs for high priority, acutely toxic chemicals. 5 AEGLs represent threshold exposure limits for the general public and are applicable to emergency 6 exposure periods ranging from 10 minutes to 8 hours. AEGL 2 and AEGL 3 levels, and AEGL 1 levels as 7 appropriate, will be developed for each of five exposure periods ( 10 and 30 minutes, 1 hour, 4 hours, and 8 8 hours) and will be distinguished by varying degrees of severity of toxic effects. It is believed that the 9 recommended exposure levels are applicable to the general population including infants and children, and 10 other individuals who may be sensitive or susceptible. The three AEGLs have been defined as follows: 11 AEGL 1 is the airborne concentration ( expressed as ppm or mg/ m ³ ) of a substance above which it 12 is predicted that the general population, including susceptible individuals, could experience notable 13 discomfort, irritation, or certain asymptomatic, non sensory effects. However, the effects are not disabling 14 and are transient and reversible upon cessation of exposure. 15 AEGL 2 is the airborne concentration ( expressed as ppm or mg/ m ³ ) of a substance above which it 16 is predicted that the general population, including susceptible individuals, could experience irreversible or 17 other serious, long lasting adverse health effects, or an impaired ability to escape. 18 AEGL 3 is the airborne concentration ( expressed as ppm or mg/ m ³ ) of a substance above which it 19 is predicted that the general population, including susceptible individuals, could experience life threatening 20 health effects or death. 21 Airborne concentrations below the AEGL 1 represent exposure levels that could produce mild and 22 progressively increasing odor, taste, and sensory irritation, or certain asymptomatic, non sensory effects. 23 With increasing airborne concentrations above each AEGL level, there is a progressive increase in the 24 likelihood of occurrence and the severity of effects described for each corresponding AEGL level. Although 25 the AEGL values represent threshold levels for the general public, including sensitive subpopulations, it is 26 recognized that certain individuals, subject to unique or idiosyncratic responses, could experience the 27 effects described at concentrations below the corresponding AEGL level. 28 Vinyl chloride PROPOSED 1: 3/ 2004 iv TABLE OF CONTENTS 1 PREFACE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iii 2 EXECUTIVE SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii 3 1. INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 4 2. HUMAN TOXICITY DATA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 5 2.1. Acute Lethality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 6 2.2. Nonlethal Toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 7 2.2.1. Neurotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 8 2.2.2. Odor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 9 2.2.3. Irritation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 10 2.2.4. Cardiovascular effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 11 2.2.5. Other Endpoints . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 12 2.3. Developmental / Reproductive Toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 13 2.4. Genotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 14 2.5. Carcinogenicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 15 2.6. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 16 3. ANIMAL TOXICITY DATA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 17 3.1. Acute Lethality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 18 3.1.1. Rats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 19 3.1.2. Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 20 3.1.3. Guinea Pigs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 21 3.1.4. Rabbits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 22 3.1.5. Other Species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 23 3.2. Nonlethal Toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 24 3.2.1. Dogs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 25 3.2.2. Rats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 26 3.2.3. Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 27 3.2.4. Guinea Pigs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 28 3.2.5. Rabbits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 29 3.3. Developmental/ Reproductive Toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 30 3.4. Genotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 31 3.5. Carcinogenicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 32 3.6. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 33 4. SPECIAL CONSIDERATIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 34 4.1. Metabolism and Disposition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 35 4.2. Mechanism of Toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 36 37 Vinyl chloride PROPOSED 1: 3/ 2004 v 4.3. Other Relevant Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 1 4.3.1 PBPK Modeling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 2 4.3.2. Interspecies Variability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 3 4.3.3. Intraspecies Variability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 4 4.3.4. Concurrent Exposure Issues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 5 5. RATIONALE AND PROPOSED AEGL 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 6 5.1. Human Data Relevant to AEGL 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 7 5.2. Animal Data Relevant to AEGL 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 8 5.3. Derivation of AEGL 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 9 6. RATIONALE AND PROPOSED AEGL 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 10 6.1. Human Data Relevant to AEGL 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 11 6.2. Animal Data Relevant to AEGL 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 12 6.3. Derivation of AEGL 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 13 7. RATIONALE AND PROPOSED AEGL 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 14 7.1. Human Data Relevant to AEGL 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 15 7.2. Animal Data Relevant to AEGL 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 16 7.3. Derivation of AEGL 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 17 8. SUMMARY OF PROPOSED AEGLs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 18 8.1. AEGL Values and Toxicity Endpoints . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 19 8.2. Comparison with Other Standards and Criteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42 20 8.3. Data Adequacy and Research Needs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44 21 9. REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 22 APPENDIX A  Derivation of AEGL values . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 23 AEGL 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56 24 AEGL 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57 25 AEGL 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58 26 APPENDIX B  Time Scaling Calculations for Vinyl Chloride AEGLs . . . . . . . . . . . . . . . . . . . . . . 59 27 APPENDIX C  Cancer Assessment of Vinyl Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67 28 APPENDIX D  Occupational epidemiological studies on carcinogenicity ( focus: limited exposure 29 time) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79 30 APPENDIX E  Derivation Summary for Vinyl Chloride AEGLs . . . . . . . . . . . . . . . . . . . . . . . . . . 84 31 Vinyl chloride PROPOSED 1: 3/ 2004 vi LIST OF TABLES 1 TABLE 1: CHEMICAL AND PHYSICAL DATA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 2 TABLE 2: SUMMARY OF ACUTE EFFECTS IN HUMANS AFTER INHALATION OF 3 VINYL CHLORIDE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 4 TABLE 3: SUMMARY OF ACUTE LETHAL INHALATION DATA IN LABORATORY ANIMALS13 5 TABLE 5: QUANTITATIVE ASSESSMENT OF CARCINOGENIC POTENCY OF VC BASED ON 6 ANIMAL EXPERIMENTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 7 TABLE 6: METABOLIC SATURATION CONCENTRATIONS OF VC IN RATS AND 8 MONKEYS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 9 TABLE 7: AEGL 1 VALUES FOR VINYL CHLORIDE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 10 TABLE 8: AEGL 2 VALUES FOR VINYL CHLORIDE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 11 TABLE 9: AEGL 3 VALUES FOR VINYL CHLORIDE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 12 TABLE 10: SUMMARY/ RELATIONSHIP OF PROPOSED AEGL VALUES . . . . . . . . . . . . . . . . . 41 13 TABLE 11: EXISTENT STANDARDS AND GUIDELINES FOR VINYL CHLORIDE . . . . . . . . . . 43 14 LIST OF FIGURES 15 FIGURE 1: CATEGORICAL REPRESENTATION OF VINYL CHLORIDE INHALATION 16 DATA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42 17 FIGURE 2: REGRESSION ANALYSIS OF THE LOG LOG TRANSFORMED CONCENTRATION 18 TIME CURVE REGARDING UNCONSCIOUSNESS IN MICE AND GUINEA PIGS . . . . . 62 19 FIGURE 3: REGRESSION ANALYSIS OF THE LOG LOG TRANSFORMED CONCENTRATION 20 TIME CURVE REGARDING MUSCULAR INCOORDINATION IN MICE AND GUINEA 21 PIGS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64 22 FIGURE 4: REGRESSION ANALYSIS OF THE LOG LOG TRANSFORMED CONCENTRATION 23 TIME CURVE REGARDING SIDE POSITION IN MICE AND GUINEA PIGS . . . . . . . . . 66 24 FIGURE 5: EXTERNAL CONCENTRATION ( mg/ m3) AND DOSE TO LIVER ( mg/ L) AS 25 CALCULATED BY PBPK MODELING BY EPA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71 26 Vinyl chloride PROPOSED 1: 3/ 2004 vii EXECUTIVE SUMMARY 1 Vinyl chloride ( VC) is a colorless, flammable gas with a slightly sweet odor. It is heavier than air 2 and accumulates at the bottom of rooms, tanks etc. Its worldwide production is approximately 27,000,000 3 tons. Most is polymerized to PVC. Combustion of VC in air produces carbon dioxide and hydrogen 4 chloride. Odor thresholds of VC were reported in the range of 10 to 25,000 ppm. Validated studies 5 providing a quantitative odor recognition and detection limit are not available. Therefore, a Level of Odor 6 Awareness ( LOA) can not be derived. 7 Vinyl chloride is an anaesthetic compound. After 5 minute exposure to 16,000 ppm VC, 8 volunteers showed dizziness, lightheadedness, nausea, visual and auditory dulling ( Lester et al., 1963). 9 Mild headache and some dryness of the eyes and nose were the only complaints of volunteers exposed to 10 491 ppm VC for several hours ( Baretta et al., 1969). No data on developmental or reproductive toxicity of 11 VC in humans after acute exposure are available. Occurrence of chromosomal aberrations in lymphocytes 12 of humans were associated with accidental exposure to VC. After chronic occupational exposure, VC is a 13 known human carcinogen inducing liver angiosarcoma, possibly hepatocellular carcinoma and brain 14 tumors. Evidence for tumors at other locations is contradictory. Two recent epidemiological studies ( Mundt 15 et al., 2000; Ward et al., 2001) did not find an increased Standard Mortality Ratio after 5 years of 16 occupational exposure to VC, whereas one other study suggested such an increase after 1 year of exposure 17 ( Boffetta et al., 2003). 18 Acute exposure of experimental animals to VC results in narcotic effects ( Mastromatteo et al., 19 1960), cardiac sensitization ( Clark and Tinston, 1973; 1982), and hepatotoxicity ( Jaeger et al., 1974). 20 Prodan et al. ( 1975) reported LC50 values for mice, rats, rabbits, and guinea pigs of 117,500 ppm, 150,000 21 ppm, 240,000 ppm and 240,000 ppm, respectively, after 2 hours. No investigations of reproductive or 22 developmental toxicity after single exposure are available. After repeated exposure developmental toxicity 23 in mice, rats and rabbits ( e. g. delayed ossification) was only observed at maternally toxic concentrations. 24 Embryo fetal development of rats was not affected by 2 week exposure ( 6h/ d) up to 1,100 ppm ( Thornton 25 et al., 2002). Positive results on genotoxicity after in vitro and single and repeated in vivo treatment have 26 been reported for VC. Elevated etheno adducts were observed after single and short term exposure and 27 associated with mutational events ( Swenberg et al., 2000; Barbin, 2000). Higher adduct levels were seen in 28 young animals than in adult animals after identical treatment ( Fedtke et al., 1990; Laib et al., 1989; 29 Ciroussel et al., 1990, Morinello et al., 2002). From a study with single exposure of adult rats to 45 ppm 30 for 6 hours, it may be concluded that no increase of relevant etheno adducts above background occurred 31 ( Watson et al., 1991). 32 Induction of liver tumors has been reported in rats after short term ( 5 week and 33 days, 33 respectively) exposure ( Maltoni et al., 1981; 1984; Froment et al., 1994). Vinyl chloride induces lung 34 tumors in mice after single exposure to high concentrations of VC ( Hehir et al., 1981). Short term exposure 35 experiments from Drew et al. ( 1983), Maltoni et al. ( 1981) and Froment et al. ( 1994) indicated increased 36 susceptibility of tumor formation in newborn and young animals. 37 The AEGL 1 was based on the study of Baretta et al. ( 1969) with 4 7 volunteers, two individuals 38 experienced mild headache during 3.5 and during 7.5 hours ( 3.5 hours, 0.5 hours break, 3.5 hours) of 39 exposure to 491 ppm. The time of onset of headaches is not clearly stated and was assumed to be after 3.5 40 Vinyl chloride PROPOSED 1: 3/ 2004 viii hours. A total uncertainty factor of 3 was used. Since the AEGL 1 is based on human data no interspecies 1 extrapolation was used. The intraspecies uncertainty factor of 3 is used to account for both toxicokinetic 2 and toxicodynamic differences among individuals. The other exposure duration specific values were 3 derived by time scaling according to the dose response regression equation Cn x t = k, using the default of 4 n= 3 for shorter exposure periods and n= 1 for longer exposure periods, due to the lack of suitable 5 experimental data for deriving the value of n. The extrapolation to 10 minutes from a 3.5 hour exposure is 6 justified because exposure of human at 4,000 ppm for 5 minutes did not result in headache ( Lester et al., 7 1963). 8 The AEGL 2 was based on prenarcotic effects observed in human volunteers. After 5 minute 9 exposure to 16,000 ppm VC, 5 of 6 persons showed dizziness, lightheadedness, nausea, and visual and 10 auditory dulling. At concentrations of 12,000 ppm one of six persons showed dizziness and " swimming 11 head, reeling". No effects were observed at 4,000 ppm in this study. A single person reported slight effects 12 (" slightly heady") of questionable meaning at 8,000 ppm ( this volunteer felt also slightly heady at sham 13 exposure and reported no response at 12,000 ppm) ( Lester et al., 1963). 12,000 ppm was regarded as the 14 no effect for impaired ability to escape. A total uncertainty factor of 3 is used to account for toxicodynamic 15 differences among individuals. As the unmetabolized VC is responsible for the effect, no relevant 16 differences in toxicokinetics are assumed. In analogy to other anesthetics the effects are assumed to be 17 solely concentration dependent. Thus, after reaching steady state at about 2 hours of exposure, no increase 18 in effect is expected. The other exposure duration specific values were derived by time scaling according to 19 the dose response regression equation Cn x t = k, using an n of 2, based on data from Mastromatteo et al. 20 ( 1960). Mastromatteo et al. observed various time dependent prenarcotic effects in mice and guinea pigs 21 after less than steady state exposure conditions. Time extrapolation was performed from 5 to 10, 30, 60 22 minutes and 2 hours, where the steady state concentration was calculated. 23 The AEGL 3 was based on cardiac sensitization and the no effect level for lethality. Short term 24 exposure ( 5 min) of dogs to VC induced cardiac sensitization towards epinephrine ( EC50: 50,000 or 71,000 25 ppm in two independent experiments) ( Clark and Tinston, 1973; Clark and Tinston, 1982). Severe cardiac 26 sensitization is a life threatening effect, but at 50,000 ppm no animals died. A total uncertainty factor of 3 27 is used to account for toxicodynamic differences among individuals. As the challenge with epinephrine and 28 the doses of epinephrine used represent a conservative scenario, no interspecies uncertainty factor was 29 used. As the unmetabolized VC is responsible for the effect, no relevant differences in toxicokinetics are 30 assumed. In analogy to other halocarbons ( e. g., Halon 1211, HFC 134a) which lead to cardiac sensitization 31 the effects are assumed to be solely concentration dependent. Thus, after reaching steady state at about 2 32 hours of exposure, no increase in effect is expected. The other exposure duration specific values were 33 derived by time scaling according to the dose response regression equation Cn x t = k, using an n of 2, 34 based on data from Mastromatteo et al. ( 1960). Mastromatteo et al. observed various time dependent 35 prenarcotic effects ( muscular incoordination, side position and unconsciousness, effects which occur 36 immediately before lethality) in mice and guinea pigs after less than steady state exposure conditions. Time 37 extrapolation was performed from 5 to 10, 30, 60 minutes and 2 hours, where the steady state 38 concentration was calculated. 39 The calculated values are listed in the table below. 40 Vinyl chloride PROPOSED 1: 3/ 2004 ix SUMMARY TABLE OF PROPOSED AEGL VALUES FOR VINYL CHLORIDE 1 Classificati 2 on 3 10 minute 30 minute 1 hour 4 hour 8 hour Endpoint ( Reference)

AEGL 1 4 ( Non 5 disabling) 6 450 ppm 1200 mg/ m3 310 ppm 800 mg/ m3 250 ppm 650 mg/ m3 140 ppm 360 mg/ m3 70 ppm 180 mg/ m3 mild headaches in 2/ 7 humans ( Baretta et al., 1969)

AEGL 2# 7 ( Disabling) 8 2800 ppm 7300 mg/ m3 1600 ppm 4100 mg/ m3 1200 ppm 3100 mg/ m3 820 ppm 2100 mg/ m3 820 ppm 2100 mg/ m3 mild dizziness in 1/ 6 humans ( Lester et al., 1963); no effect level for impaired ability to escape AEGL 3 9 ( Lethal) 10 12000 ppm* 31000 mg/ m3 6800 ppm* 18000 mg/ m3 4800 ppm* 12000 mg/ m3 3400 ppm 8800 mg/ m3 3400 ppm 8800 mg/ m3 cardiac sensitization ( Clark and Tinston, 1982; 1973); no effect level for lethality * The explosion limits for VC in air range from 38,000 to 293,000 ppm. The AEGL 3 values at 11 10 minutes, 30 minutes, and 1 hour exceed 10% of the lower explosion limit ( LEL). Therefore, 12 safety considerations against the hazard of explosion must be taken into account. 13 14 # Derived AEGL 2 values do not protect for potential mutations or malignancies due to short term 15 exposure to VC. 16 The estimation of cancer risk was based on the study of Maltoni et al. ( 1981). Newborn rats were 17 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). 18 Liver angiosarcomas were found in 17 of 42 newborn rats exposed to 6,000 ppm and 15 of 44 newborn 19 rats exposure to 10,000 ppm. No angiosarcomas were found in the dams exposed identically. A 6,000 20 ppm exposure in rats for 4 h/ day, 5 d/ week, for 5 weeks was found to be equivalent to a continuous human 21 exposure of 51 ppm using a PBPK model. From this, a 1 in 10,000 risk was calculated to be at 33

g/ m3 22 and 24 hour exposure was 34.7 mg/ m3 ( 13.2 ppm). Further exposure duration calculations were done 23 using the PBPK model for VC and are shown in the following table and Appendix C. It must be 24 emphasized that there are substantial uncertainties in calculating cancer risk from a single exposure. 25 Vinyl chloride PROPOSED 1: 3/ 2004 x Estimation of carcinogenic potency ( 10 4 risk) after single exposure 1 30 minute 1 hour 4 hour 8 hour Maltoni et al., 1981; from 5 weeks 2 study; Human equivalent dose to 6000 3 ppm 4 1200 ppm ( 3100 mg/ m3) 350 ppm ( 910 mg/ m3) 81 ppm ( 210 mg/ m3) 40 ppm ( 100 mg/ m3)

The values corresponding to 10 5 and 10 6 risk are in Appendix C. The risk for 10 minutes has not been 5 calculated due to extreme uncertainty. 6 The occurrence of DNA adducts and tumorigenicity after single exposure at or below AEGL 7 concentrations may not be excluded. No increase of relevant etheno adducts above background is expected 8 at single exposure to 3.4 ppm for 8 hours. This includes extrapolation for sensitive subgroups like 9 newborns by the use of an uncertainty factor of 10 ( for details, see calculation D; Appendix C). 10 References 11 Barbin, A. 2000. Etheno adduct forming chemicals: from mutagenicity testing to tumour mutation spectra. 12 Mut. Res. 462: 55 69 13 Baretta, E. D., R. D. Stewart, and J. E. Mutchler. 1969. Monitoring exposures to vinyl chloride vapor: 14 breath analysis and continuous air sampling. Am. Ind. Hyg. Assoc. J. 30: 537 544. 15 Belej, M. A., D. G. Smith, and D. M. Aviado. 1974. Toxicity of aerosol propellants in the respiratory and 16 circulatory systems. IV. Cardiotoxicity in the monkey. Toxicology 2: 381 395. 17 Boffetta, P., L. Matisane, K. A. Mundt, and L. D. Dell. 2003. Meta analysis of studies of occupational 18 exposure to vinyl chloride in relation to cancer mortality. Scand. J. Work Environ. Health. 29: 220 229. 19 Ciroussel, F., A. Barbin, G. Eberle, aqnd H. Bartsch. 1990. Investigations on the relationship between 20 DNA ethenobase adduct levels in several organs of vinyl chloride exposed rats and cancer susceptibility. 21 Biochem. Pharm. 39: 1109 1113. 22 Clark, D. G., and D. J. Tinston. 1973. Correlation of the cardiac sensitizing potential of halogenated 23 hydrocarbons with their physicochemical properties. Br. J. Pharm. 49: 355 357. 24 Clark, D. G., and D. J. Tinston. 1982. Acute inhalation toxicity of some halogenated and non halogenated 25 hydrocarbons. Hum. Toxicol. 1: 239 247. 26 Vinyl chloride PROPOSED 1: 3/ 2004 xi Drew, R. T., G. A. Boorman, J. K. Haseman, E. E. McConnell, W. M. Busey, and J. A. Moore. 1983. The 1 effect of age and exposure duration on cancer induction by a known carcinogen in rats, mice, and hamsters. 2 Toxicol. Appl. Pharmacol. 68: 120 130. 3 Fedtke, N., J. A. Boucheron, V. E. Walker, and J. A. Swenberg. 1990. Vinyl chloride induced DNA adducts. 4 II. Formation and persistence of 7( 2' oxoethyl) guanine and N2 ethenoguanine in rat tissue DNA. 5 Carcinogenesis 11: 1287 1292. 6 Froment, O., S. Boivin, A. Barbin, B. Bancel, C. Trepo, and M. J. Marion. 1994. Mutagenesis of ras proto 7 oncogens in rat liver tumors induced by vinyl chloride. Canc. Res. 54: 5340 5345. 8 Hehir, R. M., B. P. McNamara, J. McLaughlin, D. A. Willigan, G. Bierbower and J. F. Hardisty. 1981. 9 Cancer induction following single and multiple exposure to a constant amount of vinyl chloride monomer. 10 Environ. Health Perspect. 41: 63 72 11 Jaeger, R. J., E. S. Reynolds, R. B. Conolly, M. T. Moslen, S. Szabo, and S. D. Murphy. 1974. Acute hepatic 12 injury by vinyl chloride in rats pretreated with phenobarbital. Nature 252: 724 726. 13 Laib, R. J., K. P. Klein, and H. M. Bolt. 1985b. The rat liver foci bioassay: I. Age dependence of induction 14 by vinyl chloride of ATPase deficient foci. Carcinogenesis 6: 65 68. 15 Lester, D., L. A. Greenberg, and W. R. Adams. 1963. Effects of single and repeated exposures of humans 16 and rats to vinyl chloride. Am. Ind. Hyg. Assoc. J. 24: 265 275. 17 Maltoni, C., G. Lefemine, A. Ciliberti, G. Cotti, and D. Carretti. 1981. Carcinogenicity bioassays of 18 vinylchloride monomer: A model of risk assessment on an experimental basis. Environ. Health Perspect. 19 41: 3 31. 20 Maltoni, C., G. Lefemine, A. Ciliberrti, G. Cotti, and D. Carretti. 1984. Experimental Research on Vinyl 21 Chloride Carcinogenesis. Vol. II: Archives of Research on Industrial Carcinogenesis. Princeton Scientific 22 Publishers Inc., New Jersey. 23 Mastromatteo, E., A. M. Fisher, H. Christie, and H. Danziger. 1960. Acute inhalation toxicity of vinyl 24 chloride to laboratory animals. Am. Ind. Hyg. Assoc. J. 21: 394 398. 25 Morinello, E. J., A. J. L. Ham, A. Ranasinghe, J. Nakamura, P. B. Upton, and J. A. Swenberg. 2002. 26 Molecular dosimetry and repair of N( 2), 3 ethenoguanine in rats exposed to vinyl chloride. Canc. Res. 27 62: 5189 5195. 28 Prodan, L., I. Suciu, V. Pislaru, E. Ilea, and L. Pascu. 1975. Experimental acute toxicity of vinyl chloride 29 ( monochloroethene). Ann. NY Acad. Sci. 246: 154 158. 30 Vinyl chloride PROPOSED 1: 3/ 2004 xii Swenberg, J. A., A. Ham, H. Koc, E. Morinello, A. Ranasinghe, N. Tretyakova, P. B. Upton, and K. Wu. 1 2000. DNA adducts: effects of low exposure to ethylene oxide, vinyl chloride and butadiene. Mut. Res. 2 464: 77 86. 3 Thornton, S. R., R. E. Schroeder, R. L. Robison, D. E. Rodwell, D. A. Penney, K. D. Nitschke, and W. K. 4 Sherman. 2002. Embryo fetal development and reproducitve toxicology of vinyl chloride in rats. Toxicol. 5 Sci. 68: 207 219. 6 Watson, W. P., D. Potter, D. Blair, and A. S. Wright. 1991. The relationship between alkylation of 7 haemoglobin and DNA in Fischer 344 rats exposed to [ 1,2 14C] vinyl chloride. In: Garner, R. C., Farmer, 8 P. B., Steele, G. T., Wright, A. S.: Human Carcinogen Exposure. Biomonitoring and Risk Assessment, 9 Oxford University Press, London, 421 428. 10 Vinyl chloride PROPOSED 1: 3/ 2004 1 1. INTRODUCTION 1 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 VC is polymerized to PVC, which subsequently is used to produce packaging materials, 5 building materials, electric appliances, medical care equipment, toys, agricultural piping and tubing and 6 automobile parts. Currently the largest single use is in the building sector ( WHO, 1999a). About 10,000 7 tons annually go into the production of 1,1,1 trichloroethane and other chlorinated solvents ( Kielhorn et al., 8 2000). 9 Most VC is produced either by hydrochlorination of acetylene, mainly in Eastern European 10 countries, or by thermal cracking of 1,2 dichloroethane. It is stored either under pressure at ambient 11 temperature, or refrigerated at atmospheric pressure ( WHO, 1999a). Since VC does not polymerize readily 12 it is stored without additives. Combustion of VC in air produces carbon dioxide and hydrogen chloride 13 ( WHO, 1999a). 14 15 Relevant chemical and physical properties are listed in Table 1. 16 Vinyl chloride PROPOSED 1: 3/ 2004 2 TABLE 1: CHEMICAL AND PHYSICAL DATA 1 Parameter 2 Value Reference Molecular formula 3 C2H3Cl WHO, 1999a Molecular weight 4 62.5 g/ mol WHO, 1999a CAS Registry Number 5 75 01 4 WHO, 1999a Physical state 6 gaseous ( at room temperature) WHO, 1999a Color 7 colorless WHO, 1999a Synonyms 8 vinyl chloride monomer, monochlorethene, monochlorethylene, 1 chloroethylene, chlorethylene, chloroethene WHO, 1999a Vapor pressure 9 78 kPa at  20 oC 165 kPa at 0 oC 333 kPa at 20 oC WHO, 1999a Density 10 0.910 g/ cm3 at 20 oC WHO, 1999a Melting point 11  153.8 oC WHO, 1999a Boiling point 12  13.4 oC WHO, 1999a Solubility in water 13 soluble in almost all organic solvents, slightly soluble in water WHO, 1999a Odor 14 slightly sweet WHO, 1999a Explosion limits in air 15 3.8  29.3 vol% in air at 20 oC 4  22 vol% WHO, 1999a Conversion factors 16 1 ppm = 2.59 mg/ m3 at 20 oC, 101.3 kPa 1 mg/ m3 = 0.386 ppm WHO, 1999a 2. HUMAN TOXICITY DATA 17 2.1. Acute Lethality 18 Danziger ( 1960) describes two deaths due to accidental exposure of workers to VC. No 19 concentration or exposure time is given, but circumstances suggest inhalation of very high concentrations. 20 Autopsy results show cyanosis, congestion of lung and kidneys and failure of blood coagulation ( Danziger, 21 1960). Citing older results from Schaumann et al., 12% VC ( 120,000 ppm) is given as " dangerous 22 concentrations" ( Danziger, 1960; Oster et al., 1947). 23 At very high concentrations, VC causes asphyxia likely due to narcosis induced respiratory failure 24 ( NLM, 2000). 25 Vinyl chloride PROPOSED 1: 3/ 2004 3 2.2. Nonlethal Toxicity 1 Only few data on acute human toxicity of VC after acute exposure are available. Whereas a large 2 experience on the long term effects of VC exposure at the workplace exists. Relevant data are described 3 below. 4 2.2.1. Neurotoxicity 5 Vinyl chloride has been considered as a potential anaesthetic. Narcotic limit concentration for man 6 is 7%  10% ( 70,000  100,000 ppm) ( Oster et al., 1947, Danziger, 1960, Lehmann and Flury, 1938). 7 Schauman ( 1934) reported somewhat higher concentrations to lead to narcosis. Exposure to unknown high 8 concentrations ( e. g., during the cleaning of autoclaves) also resulted in narcotic effects ( Suciu, 1975). 9 Acute exposure 10 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, 11 4,000, 8,000, 12,000, 16,000, or 20,000 ppm, nominal concentration) for 5 minutes using a plastic 12 breathing mask covering the mouth and nose. The total gas flow was 50 liters per minute. The desired 13 concentrations were obtained by metering air and VC ( gas chromatography of the liquid phase indicated 14 more than 99% VC) through flow meters and passing the appropriate flows through a 2 l mixing chamber. 15 The concentration was continuously monitored by a thermal conductivity meter ( less than 5% deviation 16 from the desired concentration). All volunteers were exposed to every concentration in a randomized 17 fashion, separated by a 6 hour interval. Dizziness (" slightly heady") was experienced by 1 of 6 volunteers 18 at 8,000 ppm ( the same subject reported slight dizziness at sham exposure and reported no response at 19 12,000 ppm). At 12,000 ppm 4/ 6 persons reported no response, one subject reported reeling, swimming 20 head and another subject was unsure of some effects. He had a somewhat dizzy feeling in the middle of 21 exposure. At 16,000 ppm 5 of 6 and at 20,000 ppm 6 of 6 persons complained of dizziness, nausea, 22 headache, and dulling of visual and auditory cues. All symptoms disappeared shortly after termination of 23 exposure; headache persisted for 30 minutes in one subject after exposure to 20,000 ppm 24 Two experimenters were exposed to 25,000 ppm ( nominal concentration) for 3 minutes by entering 25 an exposure chamber which resulted in dizziness and slight disorientation as to space and size of 26 surrounding objects and a burning sensation in the feet. They immediately recovered on leaving the 27 chamber and complained only of a slight headache which persisted for 30 minutes. No further details were 28 presented ( Patty et al., 1930). 29 Baretta et al. ( 1969) exposed 4  6 volunteers to 59, 261, 491 ppm VC ( analytical concentrations) 30 for 7.5 h ( including a 0.5 h lunch period; corresponding to time weighted average concentrations of 48, 248 31 or 459 ppm over a period of 7.5 h), seven persons were exposed to 491 ppm for only 3.5 hours. Persons 32 were exposed in an exposure chamber ( 41 feet by 6 feet wide by 7.5 feet high) with a continuous positive 33 air supply and exhaust system. Air was recirculated with a squirrel cage fan through a series of inlet and 34 outlet ducts spanning the length of the chamber. VC concentration was monitored by an infrared 35 spectrophotometer. The vapors were introduced from a pressurized storage cylinder through 6 feet of 1/ 8 36 inch I. D. stainless steel tubing into a rotometer prior to entering the circulating air duct. A heating tape 37 wrapped around the stainless steel tubing prevented condensation of the VC. Subjective and neurological 38 responses of the volunteers as well as clinical parameters were measured. The only complaints were those 39 Vinyl chloride PROPOSED 1: 3/ 2004 4 of two subjects who reported mild headache and some dryness of their eyes and nose after exposure to the 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 or 8 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 symptoms 13 ( pleasant taste in the mouth, euphoric conditions, slow movements, giddiness, inebriety like condition) were 14 observed. Continued exposure caused more pronounced symptoms ( somnolence, complete narcosis). After 15 repeated exposures to unknown high concentrations, workers complained about headaches, irritability, 16 diminution of memory, insomnia, general asthenia, paresthesia, tingling, and loss of weight. In addition to 17 an " onset of an asthenovegetative syndrome" various other systemic and local effects were observed ( e. g., 18 cardiovascular effects, hepatomegaly, digestive responses, respiratory changes). Workplace concentrations 19 in this factory were 2300 mg/ m3 ( about 890 ppm) in 1963 and decreased in the following years. This 20 reported VC concentration in air may have been an average exposure ( not specified by the authors). 21 However, no information on peak concentrations and duration of episodes with short term high 22 concentrations of VC exposure is provided. Some of the reported activities, such as cleaning autoclaves, 23 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 as 26 " 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 al. 29 ( 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 were 36 less sensitive to VC than workers from other departments. When workders from different facilities were 37 compared even greater ranges were observed. However, inter individual differences and measurement 38 techniques which were not strictly controlled. This odor threshold was reviewed by the AIHA. The value 39 has been rejected based on specified criteria ( e. g. no calibration of panel odor sensitivity, not stated whether 40 Vinyl chloride PROPOSED 1: 3/ 2004 5 the given limit was due to recognition or detection, number of trials not stated; AIHA 1997). 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 the 15 eyes ( wedge shaped brown discoloration of the bulbar conjunctiva, palpebral slits, conjunctiva and cornea 16 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 Marsteller 22 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 time. 29 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. 32 Vinyl chloride PROPOSED 1: 3/ 2004 6 2.2.4. Cardiovascular effects 1 2 A slight decrease in blood pressure in VC workers has been attributed to the narcotic effects of VC 3 ( Suciu et al., 1975). In older exposure experiments in human volunteers no cardiovascular parameters have 4 been measured ( Lester et al., 1963). 5 Chronic exposure 6 In VC workers, Raynauds disease has been correlated to extended exposure to high VC 7 concentrations ( ATSDR, 1997), with histologic alterations of small vessels ( Veltman et al., 1975). Other 8 symptoms observed in VC workers are splenomegaly, hypertension, portal hypertension, generally 9 increased cardiovascular mortality, and vasospastic symptoms ( ATSDR 1997; Suciu et al., 1975; Byron et 10 al., 1976). According to Kotseva, elevated occupational exposure to VC increases the incidence of arterial 11 hypertension, but there is no conclusive evidence that it is associated on its own with an increased risk of 12 coronary heart disease ( Beck et al., 1973). 13 2.2.5. Other Endpoints 14 Hematology and immunology 15 Blood tests in VC victims indicated failure of blood coagulation ( Danziger et al., 1960). 16 Hepatotoxicity 17 More or less pronounced hepatitis and enlargement of the liver have been reported in chronic 18 exposed workers ( ECB, 2000; Marsteller et al., 1975). Others reported impaired liver function and 19 periportal liver fibrosis in workers from a PVC producing plant ( no further details presented; Lange et al., 20 1974). Liver function disturbances have been reported for workers from PVC factories ( Fleig and Thiess, 21 1978). Focal hepatocellular hyperplasia and focal mixed hyperplasia has been observed in VC exposed 22 workers; some of the individuals with focal mixed hyperplasia developed liver angiosarcoma ( Tamburro et 23 al., 1984). No data on liver effects after acute exposure are available. 24 Vinyl chloride PROPOSED 1: 3/ 2004 7 TABLE 2: SUMMARY OF ACUTE EFFECTS IN HUMANS AFTER INHALATION OF 1 VINYL CHLORIDE 2 Concentration 3 ( ppm) 4 Exposure Time Study type and effects Reference very high 5 not stated irritation to the eyes Danziger, 1960 25,000 ppm 6 3 min dizziness, disorientation to space and size, burning sensation in feet, persisting headache Patty et al., 1930 20,000 ppm 7 5 min 6/ 6 dizziness, lightheadedness, nausea, visual and auditory dulling, persisting headache in 1/ 6 Lester et al., 1963 16,000 ppm 8 5 min 5/ 6 dizziness, lightheadedness, nausea, visual and auditory dulling; no effects in one volunteer Lester et al., 1963 12,000 ppm 9 5 min 1/ 6 volunteers dizzy, 1/ 6 " swimming head, reeling", second person was " unsure" of effects, somewhat dizzy in the middle of exposure Lester et al., 1963 8,000 ppm 10 5 min 1/ 6 volunteers " slightly heady" ( this volunteer felt also slightly heady at sham exposure and reported no effects at 12,000 ppm) Lester et al., 1963 4,000 ppm 11 5 min no effects Lester et al., 1963 3,000 ppm 12 not stated odor threshold ( geometric averages of three studies, omitting extreme points and duplicate quotations) Amoore and Hautala, 1983 not specified, 13 high 14 not stated prenarcotic and narcotic effects; repeated exposure: headaches, asthenovegetative syndrome, cardiovascular effects , hepatomegaly Suciu et al., 1975 491 or 459 ppm 15 3.5 h 2/ 7 volunteers reported mild headache and dryness of the eyes and nose Baretta et al., 1969 261 ppm 16 not stated detection of the odor by 4/ 4 subjects Baretta et al., 1969 20 ppm 17 not stated odor threshold in PVC production workers Hori et al., 1972 10 ppm 18 not stated odor threshold in workers from a PVC facility, not working in PVC production Hori et al., 1972 2.3. Developmental / Reproductive Toxicity 19 No data on developmental or reproductive toxicity in humans after single exposure to VC were 20 identified. 21 Vinyl chloride PROPOSED 1: 3/ 2004 8 2.4. Genotoxicity 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. Sixty 5 per cent of the exposed individuals complaint of health problems ascribed to the pollutants. More than 15 6 hours after the accident, atmospheric VC concentrations were 1  8 ppm ( Huettner and Nikolova, 1998). 7 Hahn et al. ( 1998) reported maximum VC concentrations of 30 ppm near the center of the accident. 8 Exposure level to VC and/ or other combustion products of those persons included into the investigation is 9 highly uncertain. In a follow up study two years later in the same cohort of accidentally exposed people, 10 Becker et al. ( 2001) found enhanced chromosome aberrations in peripheral lymphocytes as an indicator of 11 clastogenic activity of VC, while no increased mutagenic activity ( mutations in the hypoxanthine guanine 12 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 exposure 16 concentrations estimated at 1 ppm ( Fucic et al., 1995; short exposure spikes up to 300 ppm VC were 17 reported), and 5 ppm VC ( Graj Vrhovac et al., 1990). Also increased frequencies of sister chromatid 18 exchanges were reported ( Fucic et al., 1992; Sinués 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 assessed. 23 However, gene mutations were found in human tumors associated with exposure to etheno adduct forming 24 chemicals such as VC. Specifically, in angiosarcoma of the human liver in 5 of 6 cases G> A transitions of 25 the Ki ras gene and A> T transitions of p53 were observed in 3 of 6 cases, which may be attributed to the 26 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) demonstrating 32 a steep increase of the Standard Mortality Rate after very limited exposure duration ( for details, see 33 Appendix D). There exist no epidemiological studies which include newborn children as specific risk group. 34 Chronic exposure 35 There are sufficient epidemiological data demonstrating a statistically significant elevated risk of 36 liver cancer, specifically angiosarcomas ( ASL), from chronic exposure to VC ( summarized in EPA, 2000a, 37 b; WHO, 1999a; Boffetta et al., 2003). The possible association of brain, soft tissue, and nervous system 38 cancer with VC exposure was also reported. However, the evidence supporting a causal link between brain 39 cancer and VC exposure is limited ( EPA, 2000a, b). Some other studies found an association between VC 40 Vinyl chloride PROPOSED 1: 3/ 2004 9 exposure and cancer of the hematopoetic lymphatic systems ( Simonato et al., 1991; Greiser et al., 1982). 1 Lung cancer has also been associated with VC exposure, but the increased risk of lung cancer observed in 2 some cohorts may be due to exposure to PVC dust rather than VC monomer ( Mastrangelo et al., 2003). In 3 angiosarcoma of the human liver, mutations were observed which may be attributed to the formation of 4 ethenobases in DNA ( Barbin, 2000). 5 Quantitative risk estimates for VC based on epidemiologic studies have been derived by the 6 Netherlands ( Anonymous, 1987; unit risk 1  10 6 per µ g/ m3), the WHO ( 1987; 1999b; unit risk 1  10 6 per 7 µ g/ m3) and Clewell et al. ( Clewell et al., 2001; unit risk 0.2  1.7 x 10 6). 8 2.6. Summary 9 Odor thresholds of VC were reported in the range of 10 to 25,000 ppm ( Hori et al., 1972; Baretta 10 et al., 1969; AIHA, 1997; Patty et al., 1930). Amoore and Hautala ( 1983) reported an odor threshold of 11 3,000 ppm for VC. This value represents the geometric average of three literature studies, extreme points 12 and duplicate quotations were omitted. Validated studies detecting the recognition and the detection limit 13 are not available from literature. Vinyl chloride is an anaesthetic compound. Effects observed in acutely 14 exposed VC workers and human volunteers indicate a characteristic sequence of events from euphoria and 15 dizziness, followed by drowsiness and loss of consciousness. After five minutes exposure of volunteers, 16 health effects have been described at concentrations  8,000 ppm, no effects were observed at 4,000 ppm 17 ( Lester et al., 1963). 25,000 ppm VC for 3 minutes caused dizziness, slightly disorientation and a burning 18 sensation in feet in two volunteers ( Patty et al., 1930). Mild headache and some dryness of the eyes and 19 nose were the only complaints of volunteers exposed to 491 ppm VC ( the onset of headaches is not 20 specified and is assumed to have occurred after 3.5 hours of exposure) ( Baretta et al., 1969). Irritation of 21 the eyes was reported in the context of an accidental exposure to lethal VC concentrations ( exposure 22 concentration unknown) ( Danziger et al., 1960). 23 No data on developmental or reproductive toxicity of VC in humans after acute exposure are stated 24 in the literature. 25 Occurrence of chromosomal aberrations in lymphocytes of humans accidentally exposed to VC 26 were reported by Huettner and Nikolova ( 1998). More than 15 hours after the accident, atmospheric VC 27 concentrations were 1  8 ppm. In a two year follow up clastogenic activity was still detectable ( Becker et 28 al., 2001). 29 Vinyl chloride is a known human carcinogen inducing liver angiosarcoma and possibly brain 30 tumors. Evidence for other tumor locations including hepatocellular carcinoma is contradictory ( EPA, 31 2000a, b). In angiosarcoma of the human liver mutations were observed, which may be attributed to the 32 formation of ethenobases in DNA ( Barbin, 2000). Unit risk estimates based on epidemiologic studies have 33 been published ( Anonymous, 1987; WHO, 1987, 1999b; Clewell et al., 2001). 34 Vinyl chloride PROPOSED 1: 3/ 2004 10 3. ANIMAL TOXICITY DATA 1 3.1. Acute Lethality 2 Acute inhalation toxicity tests were performed in rats, mice, rabbits, and guinea pigs. However, no 3 LC50 study complying with today` s standards is available. The lethality data are summarized in Table 3. 4 3.1.1. Rats 5 Mastromatteo et al. ( 1960) exposed 5 rats per group to 10, 20, 30 or 40% VC ( 100,000 to 400,000 6 ppm) for up to 30 minutes ( purity 99.5% maximum). The animals were exposed in an inhalation chamber 7 of 56.6 liters. The VC concentration was adjusted by mixing VC and air in a flow meter outside of the 8 exposure chamber. The stream of air and VC was led to the animal chamber inlet to deliver a continuing 9 stream ( flow not given, VC concentrations were not determined in the test chamber). Observations were 10 made continuously and are summarized in Table 3. No animals died after exposure to 100,000 and 200,000 11 ppm. All animals died after 15 minutes exposure to 300,000 ppm. At 300,000 ppm the lungs of the 12 animals which died revealed congestion with hemorrhagic areas, in addition congestion of the liver and the 13 kidney were observed. 14 Prodan et al. ( 1975) exposed rats for 2 hours in exposure chambers of the Pravdin type with 580 15 liters capacity ( total of 70 rats, at least 10 animals per group, strain not given). The animals were exposed, 16 according to Krakov` s method, to variable concentrations of VC. After the animals were placed in the 17 exposure chamber, the gas was introduced at the beginning at the lower part of the chamber, without any 18 ventilation. The gas was permanently stirred up by an inside pellet and was measured volumetrically with a 19 Zimmermann type spirometer. At VC concentrations of 15, 16, 17, 20, and 21% ( 150,000 to 210,000 ppm, 20 nominal concentration) the lethality was 23, 80, 90, 90, and 100%, respectively. The authors calculated a 21 LC50 of 15% VC ( about 150,000 ppm) and a LC100 of 21% ( about 210,000 ppm). All LC50 and LC100 22 values from these experiments are given by the authors for 2h exposure irrespective of the time of death. 23 Findings shortly before death were general convulsions, respiratory failure, exopthalmia and deflection of 24 the head on the abdomen. Surviving animals rapidly recovered after termination of the exposure. At 25 autopsy, dead animals showed general congestion of the internal organs ( lungs, liver, kidney, brain and 26 spleen); some animals ( no number given) had pulmonary edema, marmorated liver and kidney swelling. 27 In the context of a teratology study John et al. ( 1981) exposed Sprague Dawley rats intermittently 28 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 29 specified by the authors ( for study details see 3.3.). 30 Exposure of 18 Sherman rats ( 9 male; 9 female) to 100,000 VC for 8 hours resulted in deep 31 anaesthesia, with consciousness regained 5 to 10 minutes after removal to air. After two exposures one 32 female rat died and the remaining showed signs of chronic toxicity ( not specified) prompting the authors to 33 lower the VC concentration to 80,000 ppm in order to minimize mortality. Despite this decrease mortality 34 was considerable especially in male rats exposed for longer than 8 days. The animals were exposed in a 35 1100 liter steel chamber. The concentration was initially raised rapidly to the desired level by admitting VC 36 without admixture with air until the effluent from the ( mixing) chamber attained the desired level as noted 37 on the thermal conductivity meter. A fan mixed the VC with the air within the ( mixing) chamber. 38 Thereafter, the effluent from the 2 liter mixing vessel was admitted to the chamber, the throughput was 20 39 Vinyl chloride PROPOSED 1: 3/ 2004 11 l/ min ( Lester et al., 1963). 1 Exposure of 2 Sherman rats in a 10 liter all glass exposure chamber to 150,000 ppm resulted in 2 deep anaesthesia within five minutes, one of two animals died due to respiratory failure after 42 minutes 3 ( Lester et al., 1963) ( study details see above). 4 3.1.2. Mice 5 Five mice were exposed to 10, 20, 30 or 40% VC ( 100,000 to 400,000 ppm, nominal 6 concentration) for up to 30 minutes ( for study details see 3.1.1.) ( Mastromatteo et al., 1960). One mouse 7 died after 25 min exposure to 200,000 ppm and all mice died after 10 min exposure to 300,000 ppm. No 8 death occurred at 100,000 ppm. At 300,000 ppm the lungs of the animals which died revealed congestion of 9 the lungs with hemorrhagic areas, in addition congestion of the liver and the kidney were observed. 10 In ventilated exposure chambers of the Pravdin type, 100,000 ppm VC was not lethal to mice 11 during 2 hours, whereas 150,000 ppm killed 46/ 61 mice within one hour, and all animals within 2 hours. 12 The authors calculated a LC50 of 117,500 ppm and a LC100 of 150,000 ppm for mice ( for study details and 13 symptoms before death see 3.1.1.), for 2 hours. Under unstirred conditions 42,900 ppm was lethal to 70% 14 ( 13 of 20) of the animals within less than an hour ( Prodan et al., 1975). 15 Tátrai and Ungváry ( 1981) exposed CFLP mice to 1,500 ppm VC for 2, 4, 8, 12 or 24 hours 16 ( n= 20). Animals were observed for 24 hours after exposure. In addition, 40 animals were exposed for 12 h 17 and survivors were investigated two month after the exposure. Animals were exposed in dynamic exposure 18 chambers with vertical air flow. The volume of the exposure chambers was 0.3 m3; the vertical flow rate of 19 the air was 3 m3/ hour at a temperature of 20  23 ° C and 50  55% relative humidity. After 24 hours 20 exposure time all animals died within 24 h after exposure, 90% of the mice exposed over 12 hours died. No 21 death is reported in animals exposed for shorter periods. Exposure caused hemorrhages and vasodilatation 22 characteristic of shock in the lungs. Additionally, shock liver developed. The authors do not comment on 23 the concentration difference between their experiments and earlier reports indicating much higher total VC 24 concentrations as lethal; however, in these studies asphyxia is given as the cause of death. This effect is not 25 conformed in other studies. 26 In a study designed to investigate long term hepatic effects of VC, Lee et al. ( 1977) exposed CD 1 27 mice to 1,000 ppm for 6 hr/ day. Three out of seventy two mice died between day 3 and 9; all other mice, as 28 well as replacement mice appeared healthy throughout 12 month VC exposure. Upon autopsy animals had 29 acute toxic hepatitis with diffuse coagulation type necrosis of hepatocytes, as well as tubular necrosis in the 30 renal cortex. 31 In the context of a teratology study, John et al. ( 1981) exposed mice to 50 or 500 ppm VC for 7 h/ d 32 on day 6  15 of gestation. At 500 ppm VC 5 of 29 mice died, the exact day of death was not specified by 33 the authors. 34 3.1.3. Guinea Pigs 35 Patty et al. ( 1930) found 15  25% VC ( 150,000  250,000 ppm) to be lethal to guinea pigs within 36 Vinyl chloride PROPOSED 1: 3/ 2004 12 one hour, 40% VC ( 400,000 ppm) resulted in death of the animals within 10  20 min. Gross pathology 1 examinations of these animals revealed intense congestion and edema of the lungs and a hyperaemia of the 2 kidneys and livers. The lungs were light pink in color, the cut section was uniformly light red, and bled 3 freely. The authors concluded that VC is irritating to the lungs. No eye or nasal irritation was described. 4 However, from the paper it is unclear whether sufficient mixing of the atmosphere had occurred, 5 furthermore, the number of animals per group was not mentioned. 6 Prodan et al. ( 1975) reported a LC50 of 238,000 ppm and a LC100 of 280,000 ppm for guinea pigs 7 exposed in a exposure chamber of the Pravdin type ( the gas was permanently stirred up by an inside pellet; 8 study details are described in 3.1.1.) for 2 hours. No animals died within 2 hours at 200,000 ppm. 9 Yant ( cited from Prodan et al., 1975) determined a lethal concentration of 400,000 ppm for 10 min 10 for guinea pigs. 11 Exposure of guinea pigs to 10, 20, or 30% VC ( 100,000  300,000 ppm) ( 5 animals per group) did 12 not result in death within 30 min of exposure time, but one animal of the 300,000 ppm group died within 24 13 h following exposure. Thirty minutes exposure to 40% VC ( 400,000 ppm) resulted in death of one animal, 14 another animal died within 24 h following exposure whereas the other 3 animals recovered ( Mastromatteo 15 et al., 1960; for study details see 3.1.1.). The liver of the animal which died at 300,000 ppm showed severe 16 fatty degeneration, the liver was distended and very friable, the liver effects were less pronounced at 17 400,000 ppm. There was marked congestion of the lungs with hemorrhages in the dead 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 of 21 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 28 Vinyl chloride PROPOSED 1: 3/ 2004 13 TABLE 3: SUMMARY OF ACUTE LETHAL INHALATION DATA IN LABORATORY ANIMALS 1 Species 2 Concentration ( ppm) Exposure Time Number of animals Effect Reference mouse 3 500 several days for 7 h/ d 29 LC17 John et al., 1977; 1981 mouse 4 1000 at least 3 x 6 h 72 LClow Lee et al., 1977 mouse 5 1500 8 h 20 LC0 Tátrai and Ungváry, 1981 mouse 6 1500 12 h 60 LC90 Tátrai und Ungváry, 1981 mouse 7 1500 24 h 20 LC100 Tátrai und Ungváry, 1981 mouse 8 100000 2 h 40 LC0 Prodan et al., 1975 mouse 9 117500 2 h 39 LC50 Prodan et al., 1975 mouse 10 150000 2 h 61 LC100 Prodan et al., 1975 mouse 11 300000 10 min 5 LC100 Mastromatteo et al., 1960 rat 12 100000 8 h 18 LC0 Lester et al., 1963 rat 13 150000 2 h 10 LC50 Prodan et al., 1975 rat 14 150000 2 h 2 LC50 Lester et al., 1963 rat 15 200000 30 min 5 LC0 Mastromatteo et al., 1960 rat 16 210000 2 h 10 LC100 Prodan et al., 1975 rat 17 300000 15 min 5 LC100 Mastromatteo et al., 1960 rabbit 18 200000 2 h 4 LC0 Prodan et al., 1975 rabbit 19 240000 2 h 4 LC50 Prodan et al., 1975 rabbit 20 280000 2 h 4 LC100 Prodan et al., 1975 guinea pig 21 100000 6 h not stated LC0 Patty et al., 1930 guinea pig 22 200000 2 h 4 LC0 Prodan et al., 1975 guinea pig 23 240000 2 h 12 LC50 Prodan et al., 1975 guinea pig 24 150,000 to 250,000 18  55 min not stated LC100 a Patty et al., 1930 guinea pig 25 280000 2 h 4 LC100 Prodan et al., 1975 guinea pig 26 300000 30 min 5 LC20 Mastromatteo et al., 1960 guinea pig 27 400000 10  20 min not stated LC100 a Patty et al., 1930 guinea pig 28 400000 30 min 5 LC40 Mastromatteo et al., 1960 a: number of animals per group and animals that died not stated 29 Vinyl chloride PROPOSED 1: 3/ 2004 14 3.2. Nonlethal Toxicity 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 10% 7 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 group) 11 were exposed to VC by means of a face mask for 5 minutes. Oxygen was added when high concentrations 12 were used. During the last 10 seconds of the exposure period, a bolus injection of epinephrine ( 5 µ g/ kg) was 13 given via a cephalic vein and the ECG changes were recorded. A further injection of adrenaline was also 14 given 10 minutes after the end of exposure. Cardiac sensitization was deemed to have occurred when 15 ventricular tachycardia or ventricular fibrillation resulted from the challenge injection of epinephrine. An 16 increased number of ventricular ectopic beats was not regarded as evidence of sensitization since they could 17 often be produced by a challenge injection of epinephrine during control air exposures. The EC50 for 18 cardiac sensitization was 50,000 ppm ( 95 % CI: 37,000   68,000 ppm). The post exposure injection of 19 epinephrine did not result in arrhythmias ( Clark and Tinston, 1973). 20 A second study on cardiac sensitization to epinephrine in beagle dogs ( 6 male or female, not 21 further specified) after 5 minutes exposure to VC was published by Clark and Tinston ( 1982). Methods 22 were apparently identical to the study published in 1973 ( Beck et al., 1973). The EC50 for cardiac 23 sensitization was 71,000 ppm ( 95% CI: 61,000   83,000 ppm). These concentrations were below the 24 concentrations which caused effects on the central nervous system in rats ( EC50: 38,000 ppm after 10 25 minutes exposure). The authors did not comment on their earlier findings which indicated a lower EC50 for 26 cardiac sensitization. The authors discussed, that cardiac sensitization is unlikely to occur in man in the 27 absence of any effects on the CNS and that dizziness should act as an early warning that a dangerous 28 concentration was reached. 29 3.2.2. Rats 30 In rats exposed to 100,000 ppm, increased motor activity occurred after 5 min, pronounced tremor, 31 unsteady gait and muscular incoordination occurred after 15 min, side position occurred at 20 min, and 32 deep narcosis occurred after 30 min. When the VC concentration was increased, deep narcosis occurred at 33 200,000 ppm after 15 min and at 300,000 ppm after 5 min and muscular incoordination after 2 or 1 min, 34 respectively. At autopsy, lungs of the animals of the 100,000 ppm group showed a very slight hyperemia 35 even 2 weeks after exposure; at 200,000 ppm congestion of the lung in all animal and some fatty infiltration 36 in the liver of one rat were observed. Irritation ( not further explained) was described to occur immediately 37 after onset of exposure to 10, 20, or 30% VC ( Mastromatteo et al., 1960). 38 Lester et al. ( 1963) exposed Sherman rats for up to 2 hours with 50,000  150,000 ppm VC. The 39 Vinyl chloride PROPOSED 1: 3/ 2004 15 total gas flow was 50 liters per minute. The desired concentrations were obtained by metering air and VC 1 ( gas chromatography of the liquid phase indicated more than 99% VC) through flow meters and passing the 2 appropriate flows through a 2 l mixing chamber. The desired concentration was passed through a 10 liter 3 all glass exposure chamber containing 2 rats. The concentration was continuously monitored by a thermal 4 conductivity meter ( less than 5% deviation from the desired concentration). At 50,000 ppm VC for 2 hours 5 moderate intoxication was observed, but the righting reflex was lost; at 60,000 ppm for 2 hours 6 intoxication was more intense but the righting reflex was still present ( lost at 70,000 ppm). The corneal 7 reflex was lost at 100,000 ppm VC. On removal from the chamber the animals returned to the pre exposure 8 state rapidly. Exposure to 150,000 VC resulted in deep anesthesia within 5 minutes, one of two animals 9 died after 42 minutes by respiratory failure. Autopsy revealed edema and congestion of the lungs. The 10 second rat recovered quickly after removal from the exposure chamber. 11 Exposure of 18 Sherman rats to 100,000 VC for 8 hours resulted in deep anesthesia, with con 12 sciousness regained 5 to 10 minutes after removal to air. After two exposures one female rat died and the 13 remaining showed signs of toxicity ( not specified) ( Lester et al., 1963; study details presented in 3.1.1.). 14 Male Holtzman rats were exposed once to 0.5, 5 or 10% VC ( 5,000, 50,000, or 100,000 ppm) for 15 6 h in a dynamic inhalation chamber. Animals were killed 24 hours after the exposure ( no further details 16 described). Exposure to 0.5% or 5% for a single 6 h period did not cause a substantial rise in serum 17 alanine   ketoglutarate transaminase ( AKT) or sorbitol dehydrogenase ( SDH), two cytoplasmic liver 18 enzymes whose appearance in serum correlates with liver injury. Only after exposure to 10% VC was a 19 slight increase in either parameter of hepatoxic response and centrilobular hepatocellular vacuolization 20 noted. At the lower dose levels livers were histologically normal. During exposure to 10% VC animals 21 appeared to be anesthetized ( Jaeger et al., 1974). 22 Rats exposed to 30,000 ppm VC for 4 hours were slightly soporific ( Viola et al., 1970). No other 23 acute toxicity data were reported, animals were exposed for total of 12 month. 24 Tátrai and Ungváry ( 1981) exposed CFY rats to 1,500 ppm VC for 24 hours ( n= 20; study details 25 are presented in 3.1.2.). Livers were investigated by histochemical methods. No morphological changes 26 were observed. 27 Fischer 344 or Sprague Dawley rats were treated for 1 h with 50, 500, 5,000 or 50,000 ppm VC 28 ( about 90 animals per group). The chambers were Rochester type, stainless steel, 1,000 liter, constructed to 29 provide laminar air flow and ensure uniform exposures to VC to test animals. The concentration of gas in 30 the inhalation chamber was monitored by a gas chromatograph. No remarkable signs of toxicity were 31 observed. Upon removal from the test atmosphere, all animals recovered to normal appearance within 24 32 hours ( Hehir et al., 1981). Viola et al. ( 1971) also reported that exposure of rats to 50,000 ppm for one 33 hour did not result in toxicity. 34 Effects after repeated exposure 35 Pregnant rats exposed to 1,500 ppm for 7 or 9 days ( day 1 9 or 8  14 of gestation) showed 36 increased absolute and relative maternal liver weight, without light microscopic visible changes ( liver 37 weight to body weight ratio (%), exposure day 1 9 of gestation: control: 3.71; exposed: 4.25). This effect 38 was not observed in animals treated from day 14 21 of gestation. Additionally, an increased number of 39 Vinyl chloride PROPOSED 1: 3/ 2004 16 resorbed fetuses and fetal losses were observed in animals exposed during the first 9 days of pregnancy 1 ( Ungváry et al., 1978, for study details see 3.3.). 2 Intermittent exposure of rats to 500 ppm or 2,500 ppm VC during day 6  15 of pregnancy resulted 3 in increased relative and absolute maternal liver weights and an increased number of absorbed fetuses and 4 fetal losses at 2,500 ppm ( NOAEL 500 ppm) ( absolute liver weight: control: 14.27 g; 2500 ppm: 15.55 g; 5 relative liver weight: control: 34.4 mg/ g bw; 2500 ppm: 37.8 mg/ g bw). One dam died at 2,500 ppm ( John 6 et al., 1977, 1981; for details see 3.3). 7 8 After repeated inhalation exposure ( 4 weeks) of rats to 5,000 ppm VC ( 7h/ day, 5 days/ week) 9 vacuolized hepatocytes with swollen mitochondria were found in male and female animals ( Feron et al., 10 1979). After 13 weeks inhalation exposure even at the lowest dose level ( 10 ppm VC) an increase of the 11 relative liver weight was seen in male rats and centrilobular hypertrophy in females ( Thornton et al., 2002). 12 3.2.3. Mice 13 Mice exposed to 100,000 ppm VC for 30 min showed increased motor activity after 5 min, 14 twitching of extremities after 10 min and pronounced tremor, unsteady gait and muscular incoordination 15 occurred after 15 min, side position at 20 min, and deep narcosis occurred after 30 min. When the VC 16 concentration was increased deep narcosis occurred at 200,000 ppm after 15 min ( side position after 5 min) 17 and at 300,000 ppm after 5 min ( lethal after 10 min). Mice of the 100,000 ppm group showed slight 18 hyperemia of the lungs, one of five animals showed degenerative changes in the tubular epithelium of the 19 kidney with hydropic swelling. 200,000 ppm for 30 min resulted in congestion of the lungs persisting for 2 20 weeks. Irritation ( no further details) was described to occur immediately after onset of exposure to 10, 20, 21 or 30% VC ( Mastromatteo et al., 1960). 22 Prodan et al. ( 1975) exposed white mice ( no strain specified) for 2 hours to 90,000 to 200,000 ppm 23 VC with ventilation in a exposure chamber ( for study details see 3.1.1.); no shorter exposure time was 24 reported. Salivation and lacrimation appeared shortly after onset of exposure, with narcosis reached within 25 less than one hour in the majority of the animals. Typical narcosis stages of excitement with tonic clonic 26 convulsions and muscular contractions, tranquility and relaxation were described. Other symptoms were 27 accelerated respiration, proceeding to bradypnea, Cheyne Stokes type of respiration and respiratory failure. 28 No differentiation of the symptoms according to the single exposure levels were made. Concentrations of 29 110,000 and higher were lethal. In surviving mice all symptoms were rapidly reversible. 30 Male mice exposed to 50,000 ppm VC for 1 h exhibited hyperventilation after 45 min, with 31 twitching and ataxia. Female mice became hyperactive after 40 min exposure and respiratory difficulty and 32 ataxia was observed in approximately 25% of the female mice after 55 min. At 5,000 ppm no mice were 33 visibly affected. Study details are presented in 3.2.2 ( Hehir et al., 1981). 34 Vinyl chloride PROPOSED 1: 3/ 2004 17 Tátrai and Ungváry ( 1981) exposed CFLP mice to 1,500 ppm VC for 2 to 24 hours. After 2 hours, 1 histology demonstrated circulation stasis in the liver, with concomitant decreases in enzyme activities 2 ( succinic dehydrogenase and acid phosphatase), subcellular damage, and centriobular necrosis. After 24 h 3 shock liver developed. Severity of changes increased with exposure time; after 12 hours the lungs showed 4 hemorrhages and vasodilatation as signs of circulatory disturbances. No changes were observed in brain 5 and kidney. 90% of the animals died after exposure for 12 hours, and 100% after 24 hours. 6 Kudo et al. ( 1990) exposed male ICR mice ( 4 or 5 per group) to 5,000 and 10,000 ppm VC for 4 7 hours on 5 or 6 successive days, respectively. Basophilic stippled erythrocytes indicating disturbances in 8 erythropoiesis appeared in peripheral blood smears on the second day indicating possible bone marrow 9 damage after a single exposure; no difference between the doses was observed, reticulocyte numbers were 10 also increased, albeit not statistically significant. The authors discuss that the increase was partly due to 11 repeated blood sampling and was not entirely due to VC exposure. Exposure at lower concentrations, i. e. 12 30  40 ppm induced basophilic stippled erythrocytes after 3 days. 13 Lee et al. ( 1977) exposed CD 1 mice with 1,000 ppm for 6 hr/ day in the context of a long term 14 hepatotoxicity and carcinogenicity study. Besides 5% short term mortality within the first days due to acute 15 toxic hepatitis no sign of VC toxicity was observed in the other animals. 16 Aviado and Belej ( 1974) reported that exposure of mice ( male, Swiss strain) to 100,000 ppm VC 17 for 6 minutes did not cause arrhythmia, whereas 200,000 ppm induced a 2nd degree block and ventricular 18 ectopics ( animals were anesthetized with sodium pentobarbital). Cardiac sensitization was observed after 6 19 min exposure to 100,000 ppm VC ( animals were anesthetized with sodium pentobarbital). Mice were 20 exposed through a face mask which was in contact with a 6 l flaccid bag. The inhalation gas was balanced 21 with oxygen in order to prevent asphyxia. The number of animals per dose group was not presented. For 22 testing cardiac sensitization the animals received 6 µ g/ kg adrenaline hydrochloride intravenously. 23 3.2.4. Guinea Pigs 24 Guinea pigs exposed to 100,000 ppm VC for 30 min showed increased motor activity after 5 min, 25 unsteady gait and muscular incoordination occurred after 15 min, tremors and twitching of extremities after 26 20 min, and side position with tremors after 30 min one unconscious. When the VC concentration was 27 increased deep narcosis occurred at 200,000 ppm and 300,000 ppm after 30 min and at 400,000 ppm after 28 5 min. Guinea pigs of the 100,000 ppm group showed only slightly hyperemic lungs 2 weeks after 29 exposure. At 200,000 ppm congestion of the lungs was observed. At 300,000 and 400,000 ppm survivors 30 showed marked pulmonary congestion with hemorrhagic areas and edema. In one animal of the 400,000 31 ppm group the tracheal epithelium was completely absent. In the same animals blood was unable to clot. 32 Irritation ( no further details) was described to occur immediately after onset of exposure to 400,000 ppm of 33 VC, but irritation was not described at lower dose levels ( Mastromatteo et al., 1960). 34 Prodan et al. ( 1975) exposed Guinea pigs ( no strain given) to 20  28% VC ( 200,000  280,000 35 ppm) for 2 hours. Symptoms of progressing anesthesia as described for mice were observed in a time 36 depending manner; muscular contractions were more pronounced in guinea pigs than in mice. Lethality 37 increaed with increasing concentration, in surviving animals all symptoms were rapidly reversible. 38 Concentrations of 200,000 ppm were not lethal within 2 h ( n= 4). Observation of the animals did not exceed 39 Vinyl chloride PROPOSED 1: 3/ 2004 18 2 h. 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 symptoms 4 were observed at 50,000 ppm ( unconsciousness within 50 min, slow, shallow respiration within 360 min, 5 no death within 6 h). 100,000 ppm lead to an incomplete narcosis already 2 minutes after onset of 6 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 Tátrai and Ungváry ( 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 was 18 implanted for continuous monitoring. 3 monkeys received 2.5, 5, or 10% of VC. The inhalation period 19 lasted 5 minutes, alternating with room air for 10 minutes. The myocardial force was reduced by 2.3, 9.1 20 and 28.5% respectively, with a significant effect only at 10% VC. There was no effect on the heart rate in 21 compasison to controls. It is not clearly stated whether an addition challenge with epinephrine was applied 22 or not ( Belej et al., 1974). 23 Vinyl chloride PROPOSED 1: 3/ 2004 19 TABLE 4: SUMMARY OF NON LETHAL EFFECTS IN LABORATORY ANIMALS 1 Species 2 Concentration ( ppm) Exposure Time Effect Reference dog 3 50000 5 min EC50 cardiac sensitization towards epinephrine Clark and Tinston, 1973 dog 4 71000 5 min EC50 cardiac sensitization towards epinephrine Clark and Tinston, 1982 dog 5 100000 not stated anesthesia accompanied by cardiac arrhythmia Oster et al., 1947 mouse 6 1500 2 h stasis of blood flow, decreasing enzyme activities in liver, subscellular liver damage, centrilobular necrosis Tátrai and Ungváry, 1981 mouse 7 5000 1 h no clinical signs of toxicity Hehir et al., 1981 mouse 8 50000 40 min twitching, ataxia, hyperventilation, hyperactivity Hehir et al., 1981 mouse 9 100000 6 min no induction of cardiac arrhythmia Aviado and Belej, 1974 mouse 10 100000 6 min cardiac sensitization towards adrenaline Aviado and Belej, 1974 mouse 11 100000 15 min pronounced tremor, unsteady gait and muscular incoordination Mastromatteo et al., 1960 mouse 12 100000 30 min unconsciousness, side position already after 20 min; lung hyperemia persisting for > 2 weeks Mastromatteo et al., 1960 mouse 13 100000 2 h intense salivation and lacrimation immediately after onset of exposure, narcosis within 1 h Prodan et al., 1975 mouse 14 200,000 6 min Induction of cardiac arrhythmia ( 2nd degree block, ventricular ectopics) Aviado and Belej, 1974 mouse 15 200000 30 min deep narcosis, side position after 5 min, congestion of the lung for > 2 weeks Mastromatteo et al., 1960 rat 16 500 10 x 7 h no effects on liver weight ( LOAEL: 2,500 ppm) ( exposure: day 6 15 of pregnancy) John et al., 1977 rat 17 1500 24 h no acute toxicity reported Tátrai and Ungváry, 1981 rat 18 1500 9 x 24 h increased relative and absolute liver weight; increased number of absorbed fetuses and fetal losses ( exposure: day 1 9 of pregnancy) Ungváry et al., 1978 rat 19 30000 4 h slightly soporific Viola et al., 1970 rat 20 50000 1 h no clinical signs of toxicity Viola et al., 1971; Hehir et al. 1981 rat 21 50000 2 h moderate intoxication ( not further Lester et al., 1963 Vinyl chloride PROPOSED 1: 3/ 2004 TABLE 4: SUMMARY OF NON LETHAL EFFECTS IN LABORATORY ANIMALS Species Concentration ( ppm) Exposure Time Effect Reference 20 specified), loss of righting reflex rat 22 50000 6 h no clinical and histological signs of hepatic toxicity Jaeger et al., 1974 rat 23 60000 2 h intense intoxication, righting reflex still present Lester et al., 1963 rat 24 100000 15 min tremor, ataxia Mastromatteo et al., 1960 rat 25 100000 30 min deep narcosis; persisting lung hyperemia for > 2 weeks Mastromatteo et al., 1960; Jaeger et al., 1974 rat 26 100000 2 h deep anesthesia, loss of corneal reflex, no visible gross pathology Lester et al., 1963 rat 27 100000 6 h anesthesia, liver centrilobular vacuolization, slight increase of AKT and SDH activity in serum Jaeger et al., 1974 rat 28 100000 8 h deep anesthesia Lester et al., 1963 rat 29 200000 2 min muscular incoordination Mastromatteo et al., 1060 rat 30 200000 30 min deep narcosis, fatty liver infiltration, lung congestion for > 2 weeks Mastromatteo et al., 1960 guinea pig 31 10000 8 h no visible effects Patty et al., 1930 guinea pig 32 25000 5 min ataxia, unsteadiness on feet Patty et al., 1930 guinea pig 33 25000 90 min quiet, apparent unconsciousness Patty et al., 1930 guinea pig 34 25000 6  8 h narcosis, slow and shallow respiration, unsteadiness Patty et al., 1930 guinea pig 35 100000 15 min unsteady gait and muscular incoordination Mastromatteo et al., 1960 guinea pig 36 100000 30 min unconsciousness, slightly hyperemic lungs persisting for 2 weeks after exposure Mastromatteo et al., 1960 guinea pig 37 200000 30 min congestion of the lung even 2 weeks after exposure Mastromatteo et al., 1960 guinea pig 38 200000 2 h deep narcosis Prodan et al., 1975 rabbit 39 200000 2 h deep narcosis Prodan et al., 1975 monkey 40 25,000 100,000 5 min myocardial depression Belej et al., 1974 3.3. Developmental/ Reproductive Toxicity 41 No studies concerning the effect of single VC exposure on developmental or reproductive toxicity 42 have been identified. John et al. ( 1977, 1981) exposed pregnant CF 1 mice to 50 or 500 ppm VC, Sprague 43 Vinyl chloride PROPOSED 1: 3/ 2004 21 Dawley rats and New Zealand rabbits to 500 or 2,500 ppm VC during organogenesis ( 7 h/ day, days 6  15 1 for mice and rats and days 6  18 in rabbits). Exposure of bred animals was conducted in stainless steel 2 chambers of 3.7 m3 volume under dynamic conditions. The atmosphere of VC was generated by diluting 3 gaseous VC with filtered room air at a rate calculated to give the desired concentration. The actual 4 atmosphere was measured with an infrared spectrophotometer ( no further details presented). Animals were 5 sacrificed on day 18 ( mice), 21 ( rats) or 29 ( rabbits) and a variety of parameters determined. 6 Exposure to 500 ppm VC was maternally toxic to mice ( 5 of 29 bred females died), weight gain, 7 food consumption, and the absolute liver weight were decreased. Maternal toxicity was not evident in mice 8 exposed to 50 ppm. In mice exposed to 500 ppm VC, the number of live fetuses per litter and fetal weight 9 were decreased, this was probably due to the increased maternal toxicity, and fetal resorption was 10 increased. Moreover, fetal resorption was within the range of historical controls. Fetal crown rump length 11 was significantly increased in mice exposed to 50 ppm VC, but not in mice of the 500 ppm group. Delayed 12 ossifications in skull and sternum bones and unfused sternebrae were observed at 500 ppm in mice fetus. 13 Rats exposed to 500 ppm gained less weight than controls, but the body weight was not 14 significantly different from the control. At 2,500 ppm, one maternal death among 17 bred females, 15 decreased food consumption and an increase in absolute and relative liver weight were observed. No 16 significant changes were observed in rat fetuses, except for reduced fetal body weight and increased crown 17 crump length at 500 ppm ( both effects not observed at 2,500 ppm). At 2,500 ppm the incidence of dilated 18 ureter was significantly increased in comparison to the control group and the number of lumbar spurs was 19 increased at 500 ppm but not at 2,500 ppm. 20 One of seven bred female rabbits died at 2,500 ppm, rabbits exposed to 500 ppm showed a 21 decreased food consumption, but body weight was not significantly affected. The number of live fetuses per 22 litter was slightly decreased as compared to concurrent air controls among litters of rabbits exposed to the 23 lower level of 500 ppm ( live fetuses/ litter: 8 and 7 at 0 and 500 ppm, respectively), but no effect on litter 24 size resulted from exposure to 2,500 ppm of VC. Ossification of the sternebrae was delayed at 500 ppm, 25 but not at 2,500 ppm. 26 Most of the observed effects were exaggerated when feeding 15% ethanol in the drinking water 27 indicating an additive fetotoxic effect of ethanol and VC. This difference between species should be 28 correlated to the doses which in rats and rabbits exceed the threshold of metabolic saturation, whereas in 29 mice this threshold likely has not been reached. The authors attribute the observed developmental changes 30 to maternal toxicity " exposure to VC did not cause significant embryonal or fetal toxicity and was not 31 teratogenic...". 32 CFY rats were exposed to 1,500 ppm VC for 24 h/ d during the first ( day 1 9)), second ( day 8 14) 33 or third trimester ( day 14 to 21) of gestation. The volumes of the inhalation chambers were 0.13 m3, the 34 vertical flow rate of the air 2 m3/ h at a regulated temperature of 24  25 ° C and 50  55% relative humidity. 35 VC concentration in the inhalation chamber was determined by a gas chromatograph. Section was 36 performed on the 21st day of gestation. Treatment resulted in significantly increased frequency of 37 resorptions in the group exposed during the first trimester ( 2 fetuses resorbed in the control group vs. 12 38 fetuses in the exposed group; fetal loss in %: 1.7 in the control group and 5.5 in the exposed group). Two 39 cases of central nervous system malformations were recorded in treated animals ( not significant), no 40 increase in other malformations were detected. The absolute and relative maternal liver weight was 41 Vinyl chloride PROPOSED 1: 3/ 2004 22 increased in animals treated in the first and second week of pregnancy without light microscopic visible 1 changes, but not in animals exposed during the third week of pregnancy ( Ungváry et al., 1978). 2 A study investigating embryo fetal/ developmental toxicity and reproduction ( 2 generation) was 3 conducted by Thornton et al. ( 2002). In the developmental toxicity study, Sprague Dawley rats were 4 exposed during day 6 19 of gestation to VC concentrations of 0, 10, 100 or 1100 ppm for 6 h/ day. During 5 exposure animals were housed in stainless steel, wire mesh cages within a 6000 liter stainless steel and 6 glass exposure chamber. Placement of the animals was rotated at each exposure. No feed was provided 7 during exposure, but water was available ad libitum. The temperature was 16 28 degree Celsius; the 8 relative humidity was 29 79 %; the air flow rate was 1200 liters per minute. VC was delivered from a 9 compressed gas cylinder to a Scott Specialty Gases regulator equipped with inlet and outlet back pressure 10 gauges, gas test atmosphere was analyzed hourly with an Ambient Air analyzer equipped with a strip chart 11 recorder. Maternal body weight gains were slightly, but statistically significantly suppressed at all exposure 12 levels during GD 15 20 and 6 20. At 100 ppm the relative kidney weight and at 1100 ppm the relative 13 kidney and liver weights were statistically significantly increased in maternal animals. No further adverse 14 effects were observed in this study. 15 In the 2 generation study, ( Thornton et al., 2002) exposure started 10 weeks pre mating. Other 16 experimental details are provided above. One male rat in the 10 ppm group and one female rat in the control 17 group died. Mating indices and pregnancy rates for the F0 generation were comparable between control and 18 VC exposed groups. The live birth index was significantly decreased while the number of stillborn pups 19 was significantly increased in the F0 generation group exposed to 1100 ppm ( the authors did not regard 20 these effects as exposure related as they were not dose dependent and in the range of the historical controls). 21 In the F0 generation male rats, absolute and relative liver weights were significantly increased in all 22 exposure groups. Absolute epididymis and kidney weights were increased in 100 ppm male rats of the F0. 23 Whereas there were no changes in the liver weight of female F0 rats, there were histological alterations in 24 the liver at all dose groups ( hepatocytes were enlarged with increased acidophilic cytoplasm within the 25 centrilobular areas of the liver). Centrilobular hypertrophy was observed in male and female rats exposed to 26 100 and 1100 ppm and in 2 females of the 10 ppm group. 27 One male rat in the control group of the F1 died due to unknown reasons. In the F2 litters, there 28 was a statistically significant decrease in the mean number of pups delivered in the 1100 ppm group. The 29 authors regarded this effect not as exposure related as the values were lower than respective F1 control 30 group values, but comparable to the F0 control group values. In the F1 there was a statistically significant 31 increase in the absolute and relative liver weight for male rats exposed to 100 and 1100 ppm ( absolute liver 32 weight also increased in female rats, but not statistically significant). Also the absolute and relative spleen 33 weight was increased in male rats of the highest dose group. Male ( 100 and 1100 ppm) and female ( all dose 34 groups) rats showed centrilobular hypertrophy. Additionally, altered foci ( acidophilic, basophilic and clear 35 cell foci) were observed in male and female rats of the F1 of the 1100 ppm group, sometimes even at the 36 100 ppm group ( foci were also observed in 2 male rats of the F0 at 1100 ppm). 37 3.4. Genotoxicity 38 The mutagenic properties of VC have been tested in a variety of bacteria with the Ames test. S. 39 typhimurium TA 100 and TA 1535 yield positive results at high concentrations and long exposure times, 40 especially with metabolic activation systems added. In other test systems VC is genotoxic only after 41 metabolic activation, e. g. in forward mutation assays and gene conversion assays in yeast, cell 42 Vinyl chloride PROPOSED 1: 3/ 2004 23 transformation assays, UDS or SCE assays in mammalian cells ( summarized in WHO, 1999a). The tests 1 were performed either with 5  100% VC in the atmosphere or 0.025  50 mM VC in the culture medium. 2 In vivo assays for genotoxicity were performed with mice, rats, and hamsters. VC has also been 3 tested in Drosophila melanogaster. Increased host mediated forward mutations were observed after oral VC 4 exposure, whereas dominant lethal assays in mice exposed by inhalation and rats as well as a mouse spot 5 test gave negative results. Micronucleus formation in mice ( 50,000 ppm, 4  6h, 1,000 ppm 2 x 4h), 6 cytogenetic aberrations in rats ( 1,500 ppm for 1  12 weeks) and hamsters ( 25,000 ppm for 6  24 hours) 7 and loss of sex chromosomes in Drosophila melanogaster ( 50,000 ppm for 48 hours) indicated dose related 8 chromosomal abnormalities. Also, increased DNA damage was demonstrated by alkaline elution assays in 9 mice and SCE formation in hamsters ( summarized in WHO, 1999a). Further experiments with known VC 10 metabolites indicate that genotoxic effects are likely mediated by reactive intermediates with chloroethylene 11 oxide being most effective. 12 DNA adducts of VC metabolites with miscoding properties have been directly detected after 13 incubation of bacterial or phage DNA in vitro or in E. coli cells with DNA adduct indicator systems in vivo 14 with activated VC ( summarized in WHO, 1999a). Covalent binding has been frequently observed after 15 single and short term exposure. 16 17 Bolt et al. ( 1980) detected irreversible attachment of radioactivity [ 1,2 14C] VC to hepatic 18 macromolecules in the rat. After single exposure of adult rats to 250 ppm [ 14C] VC for 5 hours the total 19 amount metabolized per individual rat was 37 µ mol. 23 pmol VC metabolites/ 100 mg liver wet weight 20 were irreversibly bound to DNA. d guanosine alkylation products amounted to 0.35 pmol. 21 Laib et al. ( 1989) exposed adult Wistar rats to 700 ppm [ 1,2 14C] VC. The animals received either 22 a single 6 h exposure, or 2 single 6 h exposures separated by a treatment free interval of 15h. The 23 following amounts of [ 14C] VC derived radioactivity in liver DNA was observed: after a single exposure of 24 male rats the activity was 3.6 ± 0.2 pmol 7( 2' oxoethyl) guanine ( OEG) / mg DNA, after 2 exposures ( female 25 rats): 5.2 ± 0.5 pmol OEG/ mg DNA ± SD. 26 Watson et al. ( 1991) exposed adult male Fisher 344 rats ( nose only) for 6 hours to atmospheres 27 containing nominally 1, 10, or 45 ppm [ 1,2 14C] VC. The alkylation frequencies of OEG in liver DNA were 28 0.026, 0.28 and 1.28 residues OEG per 106 nucleotides respectively. These data indicate a linear 29 relationship between exposure dose and DNA dose in rats. There was no evidence to indicate the formation 30 of the cyclic adducts 1, N6 ethenoadenine (  A) or 3, N4 ethenocytosine (  C). The threshold for detection of 31 these adducts were about 1 adduct per 1 x 108 nucleotides. 32 Swenberg et al. ( 2000) reported dose dependent data on etheno adducts using a new combination of 33 immunoaffinity / GC high resolution MS. Adult F344 rats were exposed to 0, 10, 100, 1100 ppm VC for 6 34 hours/ day, 5 days/ week for 1 or 4 weeks. The mean for N2,3 ethenoguanine (  G) in a mixed liver cell 35 suspension from unexposed control rats was 90 ± 40 fmol/ µ mol guanine. Exposure to 10 ppm VC for 1 or 36 4 weeks resulted in 200 ± 50 and 530 ± 11 fmol/ µ mol guanine, while exposure to 100 ppm VC caused 680 37 ± 90 and 2280 ± 180 fmol / µ mol guanine at 1 or 4 weeks, respectively. A much lesser effect was evident 38 for the 11 fold greater exposure of 1100 ppm due to saturation of metabolic activation, with 1250 ± 200 39 and 3750 ± 550 fmol/ µ mol guanine being present in liver. 40 Vinyl chloride PROPOSED 1: 3/ 2004 24 In addition to these studies, there exist several investigations on the differences in sensitivity of 1 young ( preweanling) vs. adult animals. Laib et al. ( 1989) tested 11 day old and adult Wistar rats by 2 exposure to 700 ppm [ 1,2 14C] VC. Adult rats received either a single 6 h exposure, or 2 single 6 h 3 exposures separated by a treatment free interval of 15h. Pups received 2 single 6h exposures, according to 4 the same treatment schedule. The following amounts of [ 14C] VC derived radioactivity in liver DNA was 5 observed after 2 exposures ( female adults, male and female pups): 5.2 ± 0.5 pmol OEG/ mg DNA ± SD 6 ( adults), 25.5 ± 3.0 pmol OEG/ mg DNA( pups). After a single exposure of adult male rats the activity 7 ( 3.6 ± 0.2 pmol OEG/ mg DNA) was close to the observation after two exposures. 8 After a five day exposure of F344 rats to 600 ppm ( 4h/ d) the adduct levels in the liver were 162 ± 9 36 pmol OEG/ µ mol guanine and 1.81 ± 0.25 pmol  G / µ mol guanine for the pups and 43 ± 7 pmol OEG/ 10 µ mol guanine and 0.47 ± 0.14 pmol  G / µ mol guanine for the adult animals ( Swenberg et al., 1999). 11 Ciroussel et al. ( 1990) compared the levels of 1, N6 ethenodeoxyadenosine (  dAdo) and 3, N4 12 ethenodeoxycytidine (  dCyd) in BD VI rats with pups ( 7 days old) vs. adults ( 13 week old animals). These 13 rats had been exposed to 500 ppm VC for 2 weeks ( 7h/ d, 7d/ w). The molar ratios ( x 10 7) in the liver were 14 1.30, 1.31 ( two analyses;  dAdo/ dAdo) and 4.92, 4.67 (  dCyd/ dCyd) for the newborn compared to 0.19 15 (  dAdo/ dAdo) and 0.8 (  dCyd/ dCyd) for the adult animals. 16 Fedtke et al. ( 1990) measured the  G content in the liver of lactating Sprague Dawley rats and their 17 10 days old pups exposed to VC ( 600ppm, 5 days, 4h/ d).  G concentrations found in DNA livers of the 18 dams were 470 ± 140 ( adults) compared with 1810 ± 250 fmol/ µ mol ( pups). The mean background found in 19 the control DNA was 60 ± 40 fmol/ µ mol ( background subtracted from  G concentration). Similarly, 20 Morinello et al. ( 2002) demonstrated higher  G adduct levels in hepatocytes after exposure of weanling rats 21 to 10 ppm for 1 week ( 6h/ d) compared to adult animals ( control adult: 0.55 ± 0.14 mol  G / 107 mol 22 guanine; pups: 0.16 ± 0.01; exposed adult: 1.4 ± 0.4; pups: 4.1 ± 0.8). Adducts largly persisted after 23 recovery over 5 weeks. 24 Etheno adducts may be repaired by DNA glycolases, but a) did not fully return to background 25 levels after a exposure free period of 14 days (  G: directly after exposure 1,8 pmol/ µ mol, after 14 days: 26 0,47 pmol/ µ mol; control level: 90 fmol/ µ mol), b) have a miscoding potential in vitro and in vivo ( Swenberg 27 et al., 1999). 28 Gene mutations were found in animal tumors associated with exposure to etheno adduct forming 29 chemicals such as VC. Specifically, in rat hepatocellular carcinoma in 7 of 8 cases A> T mutations of the 30 Ha ras gene have been found and in angiosarcoma of the rat liver in 10 of 25 cases various base pair 31 substitutions as mutations of p53 were observed, which may be attributed to the formation of ethenobases 32 in DNA ( Barbin, 2000). 33 3.5. Carcinogenicity 34 Inhalation exposure of rats to VC causes liver tumors, especially angiosarcomas and hepatocellular 35 carcinoma and neoplastic liver nodules. Furthermore, angiosarcomas of other sites are reported. 36 Additionally, tumors at other locations are found, e. g. Zymbal gland, neuroblastoma and nephroblastoma in 37 rats; lung tumors in mice; mammary gland tumors in rats, mice, and hamsters, and skin tumors in rabbits 38 and hamsters ( summarized in WHO, 1999a, ATSDR 1997). Similar tumor localizations are observed after 39 Vinyl chloride PROPOSED 1: 3/ 2004 25 oral exposure. There is evidence that liver tumors are induced in female rats at lower doses than in males. 1 There is also evidence, that animals are more susceptible to tumor induction early in life ( WHO, 1999a). 2 Short term exposure experiments from Drew et al. ( 1983) and Maltoni et al. ( 1981) indicate 3 increased susceptibility of newborn and young animals. Drew et al. ( 1983) found increased incidences of 4 tumors in rats, mice and hamsters when exposed for the first 6 month in life, but not at later exposure 5 times, e. g. exposure of rats to 100 ppm VC during month 0 6 or 6 12 resulted in a tumor incidence 6 ( hemangiosarcoma of the liver) of 5.3% or 3.8%, respectively, but no tumors occurred when rats were 7 exposed during month 12  18 or 18 to 24. 8 Maltoni et al. ( 1981, 1984) exposed newborn rats postnatally from day 1 to 5 weeks of age to 9 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 10 were 42 ( 18 male; 24 female); at 10,000 ppm the respective number was 44 ( 24 male; 20 female). The 11 number of respective breeders were 6 for each exposure concentration. No direct control group was used; 12 however, in parallel experiments breeders and newborn animals without exposure were included ( see 13 Experiment BT 4001, 4006). The newborn animals were simultaneously exposed to milk from exposed 14 dams ( D. Soffritti, Laboratory of Prof. Maltoni, personal communication, August, 2003). The authors 15 found liver angiosarcomas in newborn SD rats in 17/ 42 and 15/ 44 animals respectively, exposed to 6,000 16 ppm or 10,000 ppm, but none in their mothers which were treated identically. No angiosarcoma were found 17 in a control group of 304 rats ( parallel experiment). Additionally, hepatoma incidence was increased in 18 newborn rats ( 20/ 42 and 20/ 42, respectively), but no hepatoma were observed in their mothers. Only 1 19 hepatoma were found in a control group of 304 rats ( parallel experiment). Results were provided after 124 20 weeks of observation. The internal concentration of VC may have been influenced by oral uptake from milk 21 from exposed dams. However, due to the very high inhalation exposure and due to saturation of 22 metabolism, the oral uptake by contaminated milk may have contributed only a limited amount to the 23 overall organ concentration of VC metabolites. 24 Froment et al. ( 1994) exposed 4 female Sprague Dawley rats together with their pups ( 22 males 25 and 22 females) for 8h/ d, 6d/ w to 500 ppm VC from day 3 through 28 postpartum. At day 28 postpartum, 26 the young animals were weaned, and the males and females were separated and exposed for further 2 weeks 27 ( total exposure: 33 days). The surviving animals were all sacrificed at 19 month of age. In the 44 VC 28 exposed rats 66 hepatic lesions were identified including nodular hyperplasia, endothel cell hyperplasia, 29 peliosis, adenomas, benign cholangiomas, angiosarcoma of the liver ( ASL) and hepatocellular carcinoma 30 ( HCC). Liver tumors included 8 HCC, 15 ASL and 2 benign cholangioma. No further details were 31 provided. It is assumed that oral exposure via mothers ´ milk and inhalation exposure occurred 32 simultaneously. 33 Maltoni et al., ( 1981, 1984) also exposed rats 30 breeders/ exposure group to 6,000 and 10,000 34 ppm for 1 week ( 4h/ d; 12th until 18th day of pregnancy). 32 ( 13 males; 19 females) and 51 ( 22 males; 29 35 females) offsprings were investigated after exposure to the lower or the higher concentration, respectively. 36 Angiosarcoma of the liver and hepatoma were not increased in the transplacentally exposed offsprings. 37 However, Zymbal gland carcinoma and nephroblastoma were found elevated after transplacental exposure. 38 Differences between pre and postnatal exposure and carcinogenic outcome may possibly be explained by 39 hepatic CYP2E1 activity, which is expressed to a lower extent prenatally than postnatally, both in rats 40 ( Carpenter et al., 1997) and in humans ( Cresteil, 1998). 41 Vinyl chloride PROPOSED 1: 3/ 2004 26 Hehir et al. ( 1981) found increased lung tumor incidences in ICR mice exposed once for 1 h to VC 1 ( age of the animals not stated). Animals were exposed in an inhalation chamber to single one hour doses of 2 VC ranging from 50 to 50,000 ppm ( Rochester type inhalation chambers, 1,000 liter with laminar air flow) 3 and were then observed for the remainder of their lives. Tumor response was dose related: Adenoma of the 4 lung increased from 12/ 120 to 14/ 139, 18/ 139, 24/ 143, 45/ 137 respectively for exposure to 0, 50, 500, 5 5000, 50000 ppm. For carcinoma of the lung, there was only a slight occurrence of 0/ 120, 0/ 139, 1/ 143, 6 3/ 137 ( data from both sexes, combined). A slight increase in hepatic cell carcinoma occurred in male mice, 7 but without dose response ( 2/ 50; 2/ 64; 9/ 67; 6/ 68; 4/ 63). No increase in tumor incidence was observed in 8 liver and lung of rats treated in an identical fashion. Additional studies in A/ J mice which were exposed to 9 500 ppm VC for 1 h/ d over 10 days or 50 ppm VC for 1 h/ d over 100 days revealed that for short term 10 exposure the concentration may be the most critical factor. In both experiments primarily pulmonary 11 adenomas were observed. However, the incidence in the induction of adenomas and progression to 12 carcinoma are considered only marginal and not statistically significant in mice exposed to 50 ppm for 100 13 times ( 44.1% exposed; 34.5% control) whereas a significant increase of pulmonary adenomas was observed 14 in animals exposed to 500 ppm for 10 days ( about 74% exposed; 34.4% control). 15 Suzuki ( 1983) also reported that short term exposure ( 6 h/ d; 5 d/ w; 4 weeks) of young CD1 mice 16 ( 5  6 weeks old at first exposure) to VC resulted in tumor formation. At sacrifice 12 weeks after exposure 17 pulmonary tumors were observed in the two highest dose groups ( 300 and 600 ppm). Forty or 41 weeks 18 after exposure pulmonary tumors were observed in all animals exposed ( 1 ppm to 600 ppm) but not in 19 control mice. In addition, subcutaneous and hepatic hemangiosarcoma were found. The angiosarcoma of 20 the liver occurred in one animal exposed to 600 ppm for 4 weeks as observed at necroscopy 56 weeks after 21 exposure ( Suzuki, 1981). 22 After a single 12 hour exposure to 1,500 ppm of mice a hepatocellular adenoma developed. The 23 respective concentration was lethal to most of the animals ( Tátrai and Ungváry, 1981). However, the 24 observed effects ( asphyxiation) were not seen in other studies with similar concentrations. 25 26 In addition to angiosarcoma of the liver several studies with limited exposure duration to VC 27 confirm the occurrence of hepatocellular carcinomas and/ or other preneoplastic parenchymal changes in 28 adult animals ( Feron et al., 1979; Thornton et al., 2002). However, these changes were seen to a much 29 lesser extent than angiosarcoma in the adult animals or hepatocellular changes in young animals ( see 30 below). 31 In accordance with these investigations in newborn rats, Laib et al. ( 1985a, b) reported that 32 hepatocellular ATPase deficient foci ( pre malignant stages) were observed in rats which were exposed to 33 VC. The exposure regimen was a) Wistar  rats for 10 weeks starting on day 1 after birth ( 10 to 2,000 ppm; 34 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 35 ( 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 36 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 37 exposure ( Laib et al., 1985b). Exposure to 2,000 ppm did not result in ATPase deficient foci in very young 38 ( exposure period: day 1 to 5) or in adult animals ( exposure period: from day 90 to 160). However, relevant 39 foci areas were demonstrated for short periods during animal growth, eg., exposure for 11 days ( exposure 40 period: from day 1 to 11) or for 21 days ( from day 7 28). The foci persisted until evaluation at the age of 4 41 month ( Laib et al., 1985b). After exposure over 10 weeks, induction of ATPase deficient foci was dose 42 dependent ( nearly linear) for concentrations between 10 ppm and 500 ppm and it was shown for both 43 strains of rat, Wistar and Sprague Dawley. This finding is in accordance with the findings that VC 44 Vinyl chloride PROPOSED 1: 3/ 2004 27 metabolism follows first order kinetics until saturation occurs at high exposure concentrations ( Laib et al., 1 1985a). 2 Quantitative risk assessments based on animal experiments have been published by several authors 3 and are summarized in Table 5. 4 TABLE 5: QUANTITATIVE ASSESSMENT OF CARCINOGENIC POTENCY OF VC BASED ON 5 ANIMAL EXPERIMENTS 6 Author 7 Unit Risk ( per µ g/ m3)