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Review of the U.S. Army's Health Risk Assessments for Oral Exposure to Six Chemical-Warfare Agents 6 Evaluation of the Army's Interim Reference Dose for VX THE CHEMICAL-WARFARE agent VX is an organophosphate nerve agent found at several stockpile and nonstockpile munition sites in the United States. At the request of the U.S. Army, Oak Ridge National Laboratory (ORNL) conducted a health risk assessment of VX. The assessment included a detailed analysis of VX's physical and chemical properties, environmental fate, mechanism of action, and animal and human toxicity data (see Appendix D, Health Risk Assessment of VX, ORNL 1996). On the basis of that assessment, ORNL proposed a reference dose (RfD) of 6 × 10 -7 mg/kg of body weight per day for noncancer health effects of VX exposure. Because there was no evidence that VX is carcinogenic, a slope factor was not derived. The Army's Surgeon General accepted ORNL's proposed RfD as an interim exposure value until an independent evaluation of the proposed RfD was conducted by the National Research Council (NRC). This chapter contains the NRC's independent assessment of the scientific validity of the Army's interim RfD for VX. DERIVATION OF THE ARMY'S INTERIM RFD The Army's interim RfD for VX is 6 × 10-7 mg/kg per day (0.0006 µg/kg per day). ORNL (1996) calculated that value on the basis of the lowest oral dose of VX that caused significant depression in blood-cholinesterase (ChE) activity in sheep. The lowest-observed-adverse-effect level
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Review of the U.S. Army's Health Risk Assessments for Oral Exposure to Six Chemical-Warfare Agents (LOAEL) of VX was 3 µg per day in a subchronic toxicity study in which female sheep were fed VX daily for 55 days (Rice et al. 1971). The LOAEL was weight-normalized by dividing it by 52.7 kg (the average weight of the sheep) to yield a dose of 0.06 µg/kg per day. The RfD for VX was calculated to be 6 × 10-7 mg/kg per day by dividing the adjusted LOAEL by 90, the product of the uncertainty factors and the modifying factor selected by ORNL. APPROPRIATENESS OF THE CRITICAL STUDY The critical study used by ORNL for deriving the RfD for VX was a subchronic toxicity study with yearling sheep (Rice et al. 1971). Five females per group were hand-fed pellets treated with VX at doses of 3, 9, and 15 µg for 55 days. The control group comprised 10 sheep. Whole-blood-ChE determinations were made before dosing and 16 times during the exposure period. Significant depression of ChE was observed in all dose groups by day 21, and there were no signs of toxicity. ChE concentrations stabilized at about 60% of baseline values (measurements taken before exposure) by day 31 and remained at that level for the remainder of the testing period. ORNL considered the lowest dose of 3 µg per day to be the LOAEL for the study. As described earlier, that value was weight-adjusted to 0.06 µg/kg per day. The subcommittee noted several weaknesses in the Rice et al. (1971) study, the most notable being the uncertainties about whether sheep are at least as susceptible as humans to VX. Sheep lack plasma-ChE and have lower red-blood-cell (RBC)-acetylcholinesterase (AChE) activity than humans (Ellin 1981); therefore, they might be more susceptible to VX toxicity than humans. In addition, sheep are ruminants, which means that ingested materials remain in their rumens and are later regurgitated for cud-chewing. It is unclear what effect this digestive process has on the absorption of VX. It is possible that VX is destroyed in the rumen, thereby decreasing the amount available for absorption. However, in vitro studies by Cook (1957) indicate that thiol isomers of organophosphate agents are not destroyed by rumen fluid; VX possesses a thiol group, suggesting that it would not be destroyed in the rumen. If that is the case, retention of VX in the rumen might allow increased absorption of VX through the epithelial surface of the rumen. However, there is no good evidence that shows that sheep are more sensitive than humans to VX.
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Review of the U.S. Army's Health Risk Assessments for Oral Exposure to Six Chemical-Warfare Agents The Rice et al. (1971) study used a small number of animals and the control animals were older than the experimental animals, but concern about those factors was lessened by a study showing no significant differences in blood-ChE activities among various age and gender groups of sheep (Halbrook et al. 1992). The exposure duration of the Rice et al. (1971) study was subchronic (8 weeks) rather than chronic (104 weeks), but the subcommittee believes that the data from other studies (Goldman et al. 1988, see below) indicate that significant effects are unlikely with longer exposures. Other weaknesses in the Rice et al. (1971) study include lack of dose adjustments for body weight and large variation in animal weights (10-kg range). The subcommittee believes that it would be useful to review the raw data from the study to determine whether animal weight might explain an appreciable amount of the residual variance in ChE concentrations. The data could be evaluated with each animal serving as its own control, obviating potential problems with noncomparability because of the ages of test and control animals. Another weakness of the study was that the method used to measure ChE activity was not ideal (see Appendix G). The subcommittee considered other possible critical studies for the derivation of the RfD for VX. In a study by Goldman et al. (1988), VX was administered to Sprague-Dawley rats (25 males and 25 females per group) by subcutaneous (s.c.) injection at doses of 0, 0.25, 1.0, and 4.0 µg/kg for 5 days per week for up to 90 days. A dose-dependent decrease in RBC-AChE concentrations was observed in male and female rats compared with controls. Plasma ChE was significantly depressed at day 30 in both sexes administered VX at a dose of 1.0 µg/kg per day, and at days 30, 60, and 90 in both sexes at a dose of 4.0 µg/kg per day. The data from rats exposed for 30 days was reanalyzed by ORNL using analysis of variance and Dunnett's and Scheffe's comparisons. ORNL reported that RBC-AChE activity was significantly lower in both sexes in all dose groups. The subcommittee found several weaknesses in using the Goldman et al. (1988) study to identify a NOAEL or LOAEL. The study involved the s.c. route of exposure, which is not a relevant route of exposure for determining an RfD, and the study was subchronic in duration (exposures ranging from 4 to 13 weeks) rather than chronic (104 weeks). In addition, the methods used to measure ChE activity was not ideal (see Appendix G). Another study the subcommittee considered that might serve as the
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Review of the U.S. Army's Health Risk Assessments for Oral Exposure to Six Chemical-Warfare Agents critical study involved exposure of humans to VX (Sim et al. 1964). Sixteen male volunteers were given drinking-water solutions containing VX at concentrations of 1.43 µg/kg daily for 7 days. Mean depression of RBC-AChE activity was 60% of baseline values, and no signs or symptoms of toxicity occurred. The weaknesses noted in the Sim et al. (1964) study include the small number of subjects and the much shorter exposure duration than is typical in subchronic or chronic exposure studies in animals. The data on GB (McNamara et al. 1973) indicate that the effects on ChE concentrations reach a plateau more quickly for VX than for GB, in part because the half-life for aging is longer for VX than for many other organophosphates and there is some reversibility of the binding of VX to ChE before aging occurs. Although none of the three studies described above is sufficient to form an ideal basis for deriving the RfD for VX, the subcommittee believes that data from human studies should be used whenever possible. Thus, the subcommittee believes that the study of humans by Sim et al. (1964) is the most appropriate of the available studies for derivation of the RfD for VX. APPROPRIATENESS OF CRITICAL END POINT The LOAELs in the critical study used by ORNL (Rice et al. 1971) and the critical study recommended by the subcommittee (Sim et al. 1964) were based on the lowest dose of VX that caused a significant depression in RBC-ChE activity. The subcommittee notes that ChE inhibition is typically considered a biomarker of exposure to organophosphate agents rather than an adverse effect. However, it is generally agreed that inhibition of RBC and plasma ChE contributes to the overall hazard identification of ChE-inhibiting agents. The U.S. Environmental Protection Agency (EPA) has used ChE inhibition to establish RfDs for several organophosphate pesticides, such as malathion (EPA 1992) and ethion (EPA 1989). The subcommittee agrees with ORNL that ChE inhibition is a valid end point on which to base the RfD for VX but recommends that data on ChE inhibition be taken from the human study by Sim et al. (1964) rather than from the sheep study by Rice et al. (1971). The subcommittee considered other possible critical end points, notably neurotoxicity, associated with VX exposure. Organophosphate compounds like VX might act directly on nerve cell receptors or, by inhibiting neural AChE, interfere with neuromuscular transmission and produce
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Review of the U.S. Army's Health Risk Assessments for Oral Exposure to Six Chemical-Warfare Agents delayed-onset subjunctional muscle damage. VX at concentrations of 10 picomolar has been shown to depress gabanergic transmission in the central nervous system (Rocha et al. 1998) and this could have profound implications for behavioral effects in laboratory animals and humans. Some organophosphate compounds cause a neurotoxic effect (organophosphate-induced delayed neuropathy, or OPIDN) that is not associated with ChE inhibition. However, OPIDN has not been observed in humans exposed to acutely toxic concentrations of VX (Munro et al. 1994). The subcommittee notes that additional human data, which were not included in ORNL's assessment, are available. Data summaries from human experimentation conducted in the 1950s and 1960s were evaluated by the NRC in a series of reports titled Possible Long-Term Health Effects of Short-Term Exposure to Chemical Agents (NRC 1982, 1984, 1985). The reports include an evaluation of health records of volunteer soldiers who were exposed intravenously or intramuscularly to chemical-warfare agents, as well as a follow-up morbidity study conducted by the NRC in 1985. The NRC found no long-term health effects from short-term exposure to any specific chemical-warfare agent, but there was some evidence of an increase in malignant neoplasms among men exposed to anti-ChE agents. Although these studies are not directly applicable to deriving RfDs, they add to the completeness of the data base on VX. Provided that appropriate assays were used, the subcommittee finds no reason at this time to alter the practice of using RBC-ChE or plasma-ChE inhibition as the critical toxicity end point and agrees with ORNL that such inhibition is the best available critical noncancer end point on which to base the calculation of the RfD for VX. APPROPRIATENESS OF UNCERTAINTY FACTORS Because the subcommittee recommends the use of the human study by Sim et al. (1964) as the basis for deriving the RfD for VX, it assigned values to the uncertainty factors and the modifying factor with respect to that study below. EXTRAPOLATION FROM ANIMAL TO HUMAN Because the Sim et al. (1964) study involved exposure to humans, the subcommittee assigned a factor of 1 to the uncertainty factor for extrapolation of data from animals to humans (UFA)
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Review of the U.S. Army's Health Risk Assessments for Oral Exposure to Six Chemical-Warfare Agents PROTECTING SUSCEPTIBLE SUBPOPULATIONS The subcommittee believes that a factor of 10 should be assigned to the uncertainty factor for protecting susceptible subpopulations (UFH) because some individuals have a genetic polymorphism causing their serum-ChE activity to be abnormally low (Evans et al. 1952; Harris and Whittaker 1962). For homozygous individuals, the activity can be as low as 8–21% of the normal mean (Bonderman and Bonderman 1971). Genetic polymorphisms are also recognized for butyrylcholinesterase and paraoxonase, enzymes that might function in the sequestration and metabolism of organophosphate nerve agents (Loewenstein-Lichtenstein et al. 1995; Maekawa et al. 1997; Furlong et al. 1998). Individuals with those polymorphisms might be unusually susceptible to organophosphate anti-ChE compounds (Morgan 1989). EXTRAPOLATION FROM LOAEL TO NOAEL The subcommittee assigned a factor of 10 to the uncertainty factor for extrapolation from a LOAEL to a NOAEL (UFL) because the 60% inhibition of ChE observed with the LOAEL was near levels where physical signs of clinical toxicity might occur. The subcommittee notes that ChE inhibition is a biomarker of exposure rather than a toxic effect. Although it could be argued that a dose of VX that significantly induces ChE inhibition in the absence of toxic effects is indicative of a NOAEL rather than a LOAEL, the subcommittee believes that given the 60% ChE inhibition observed at the LOAEL, a factor of 10 is a prudent choice for UFL. EXTRAPOLATION FROM SUBCHRONIC TO CHRONIC EXPOSURES The subcommittee notes that in the derivation of RfDs for other organophosphate compounds, EPA (1989, 1992) used NOAELs for ChE inhibition that were based on subchronic data without adjustment for chronic exposures, because ChE inhibition is unlikely to change over time. Hence, a factor of 1 was used for the uncertainty factor for extrapolation from subchronic to chronic exposures (UFS). For example, studies with VX indicate that maximal ChE inhibition occurs after 30–60 days of exposure and then levels off and sometimes shows signs of recovery
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Review of the U.S. Army's Health Risk Assessments for Oral Exposure to Six Chemical-Warfare Agents (Goldman et al. 1988). The subcommittee believes, however, that because the exposure duration of the critical study (7 days) is too short to be considered a subchronic exposure study, a factor of 10 should be assigned to UFS to account for the greater uncertainty involved with extrapolating such data to chronic exposures. DATA-BASE ADEQUACY The subcommittee notes that with the exception of the Rice et al. (1971) study, subchronic or chronic toxicity studies of oral exposures to VX are lacking. Because it is unclear whether sheep (ruminants) are a relevant animal model for estimating the toxicity of VX to humans, the subcommittee believes that a factor of 3 should be assigned to the uncertainty factor for data-base adequacy (UFD) to account for the absence of long-term oral studies of VX in humans or a relevant animal model. MODIFYING FACTOR FOR ADDITIONAL UNCERTAINTY The subcommittee considers the uncertainties of the data on VX to be represented adequately by the values assigned to the uncertainty factors above and believes a modifying factor (MF) of 1 is appropriate. SUMMARY Table 6-1 presents the uncertainty factors recommended by the subcommittee on the basis of the Sim et al. (1964) study. Using those uncertainty factors, and a LOAEL of 0.00143 mg/kg per day, the RfD for VX is 5 × 10-7 mg/kg per day (0.00143 mg/kg per day ÷ 3,000). WEIGHT AND STRENGTH OF EVIDENCE On the basis of the Sim et al. (1964) study, the subcommittee believes that the weight of evidence for an RfD of 5 × 10-7 mg/kg per day is moderately good. It is likely that the LOAEL (0.00143 mg/kg per day) for VX used to calculate the RfD might not be an accurate LOAEL for ChE
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Review of the U.S. Army's Health Risk Assessments for Oral Exposure to Six Chemical-Warfare Agents TABLE 6-1 Uncertainty Factors Used to Calculate the RfD for VX on the Basis of the Sim et al. (1964) Study Uncertainty Factor Description NRC UFA For animal-to-human extrapolation 1 UFH To protect susceptible subpopulations 10 UFL For LOAEL-to-NOAEL extrapolation 10 UFS For subchronic-to-chronic extrapolation 10 UFD For data-base adequacy 3 MF Modifying factor for additional uncertainty 1 TOTAL UF 3,000 Abbreviations: LOAEL, lowest-observed-adverse-effect level; MF, modifying factor; NOAEL, no-observed-adverse-effect level; NRC, National Research Council; RfD, reference dose; UF, uncertainty factor inhibition in humans, because 60% ChE inhibition was observed at that dose and lower doses were not tested. The subcommittee believes that because ChE inhibition is a biomarker of exposure rather than a toxic effect, use of this end point overestimates the oral toxicity of VX. CONCLUSIONS The subcommittee disagrees with ORNL that the Rice et al. (1971) study should be used as the basis for calculating an RfD for VX. The subcommittee found several weaknesses and uncertainties that undermined its confidence in this study. The subcommittee believes that data from human studies should be used to derive the RfD whenever possible; thus, the subcommittee recommends that the human study by Sim et al. (1964) be used instead. Although the Sim et al. (1964) study also had a number of weaknesses, the subcommittee prefers to use it rather than data from a questionable animal model. On the basis of the Sim et al. (1964) study, the RfD for VX is 5 × 10-7 mg/kg per day, which is similar to the Army's interim RfD of 6 × 10-7 mg/kg per day. DATA GAPS AND RESEARCH RECOMMENDATIONS The major gap in the available information on VX is the lack of an oral subchronic or chronic toxicity study that demonstrates a clear dose-
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Review of the U.S. Army's Health Risk Assessments for Oral Exposure to Six Chemical-Warfare Agents response relationship between VX exposure and ChE inhibition. The absence of that type of data could be addressed by conducting a subchronic oral toxicity study that assesses anti-ChE activity in RBCs and plasma in one or preferably two species. At least one dose between 0 and 0.00143 mg/kg per day should be used. If further research reveals that significant toxic effects can be induced by any of the nerve agents evaluated (i.e., GA, GB, GD, or VX) at doses below those that cause significant ChE inhibition, new studies should be conducted to reassess the safety of the recommended RfD for VX. REFERENCES Bonderman, R.P., and D.P. Bonderman. 1971. A typical and inhibited human serum pseudocholinesterase. A titrimetric method for differentiation. Arch. Environ. Health 22:578–581. Cook, J.W. 1957. In vitro destruction of some organophosphate pesticides by bovine rumen fluid. Agric. Food. Chem. 5:859–863. Ellin, R.I. 1981. Anomalies in Theories and Therapy of Intoxication by Potent Organophosphorus Anticholinesterase Compounds. Special Publication USABML-SP-81-003. DTIC AD-A101364. U.S. Army Medical Research and Development Command, Biomedical Laboratory, Aberdeen Proving Ground, Edgewood, Md. EPA (U.S. Environmental Protection Agency). 1989. Ethion Reference Dose for Chronic Oral Exposure. Integrated Risk Information System (IRIS). Online file. http://www.epa.gov/iris/ (Accessed July 22, 1998). EPA (U.S. Environmental Protection Agency). 1992. Malathion Reference Dose for Chronic Oral Exposure. Integrated Risk Information System (IRIS). Online file. http://www.epa.gov/iris/ (Accessed July 22, 1998). Evans, F.T., P.W.S. Gray, H. Lehmann, and E. Silk. 1952. Sensitivity to succinylcholine in relation to serum cholinesterase. Lancet i:1129–1230. Furlong, C.D., W.F. Li, L.G. Costa, R.J. Richter, D.M. Shih, and A.J. Lusis. 1998. Genetically determined susceptibility to organophosphorus insecticides and nerve agents: Developing a mouse model for the human PON1 polymorphism. Neurotoxicology 19:645–650. Goldman, M., B.W. Wilson, T.G. Kawakami, L.S. Rosenblatt, M.R. Culbertson, J.P. Schreider, J.F. Remsen, and M. Shifrine. 1988. Toxicity Studies on Agent VX. Final Report. DTIC AD-A201397. Prepared by the Laboratory for Energy-Related Health Research, University of California, Davis, Calif., for the U.S. Army Medical Research and Development Command, Fort Detrick, Frederick, Md. Halbrook, R.S., L.R. Shugart, A.P. Watson, N.B. Munro, and R.D. Linnabary.
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Review of the U.S. Army's Health Risk Assessments for Oral Exposure to Six Chemical-Warfare Agents 1992. Characterizing biological variability in livestock blood cholinesterase activity for biomonitoring organophosphate nerve agent exposure. J. Am. Vet. Med. Assoc. 201:714–725. Harris, H., and M. Whittaker. 1962. The serum cholinesterase variants. A study of twenty-two families selected via the ''intermediate" phenotype. Ann. Hum. Genet. 26:59–72. Lowenstein-Lichtenstein, Y., M. Schwarz, D. Glick, B. Norgaard-Pederson, H. Zakut, and H. Soreq. 1995. Genetic predisposition to adverse consequences of anti-cholinesterases in "atypical" BCHE carriers. Nat. Med. 1:1082–1085. Maekawa, M., K. Sudo, D.C. Dey, J. Ishikawa, M. Izumi, K. Kotani, and T. Kanno. 1997. Genetic mutations of butyrylcholine esterase identified from phenotypic abnormalities in Japan. Clin. Chem. 43:924–929. McNamara, B.P., F. Leitnaker, and F.J. Vocci. 1973. Proposed Limits for Human Exposure to VX Vapor in Nonmilitary Operations. EASP 1100-1 (R-1). AD-770434-19. U.S. Army, Medical Research Laboratory, Edgewood Arsenal, Aberdeen Proving Ground, Md. Morgan, D.P. 1989. Recognition and Management of Pesticide Poisonings, 4th Ed. EPA-540/9-88-001. Health Effects Division, Office of Pesticide Programs, U.S. Environmental Protection Agency, Washington, D.C. Munro, N.B., K.R. Ambrose, and A.P. Watson. 1994. Toxicity of the organophosphate chemical warfare agents GA, GB, and VX: Implications for public protection. Environ. Health Perspect. 102:18–38. NRC (National Research Council). 1982. Possible Long-Term Health Effects of Short-Term Exposure to Chemical Agents, Vol. 1. Anticholinesterases and Anticholinergics. Washington, D.C.: National Academy Press. NRC (National Research Council). 1984. Possible Long-Term Health Effects of Short-Term Exposure to Chemical Agents, Vol. 2. Cholinesterase Reactivators, Psychochemicals, and Irritants and Vesicants. Washington, D.C.: National Academy Press. NRC (National Research Council). 1985. Possible Long-Term Health Effects of Short-Term Exposure to Chemical Agents, Vol. 3. Final Report. Current Health Status of Test Subjects. Washington, D.C.: National Academy Press. ORNL (Oak Ridge National Laboratory). 1996. Health Risk Assessment for the Nerve Agent VX. Draft Report. Interagency Agreement No. 1769-1769-A1. Prepared by Oak Ridge National Laboratory, Life Sciences Division, Oak Ridge, Tenn., for the U.S. Department of the Army, Army Environmental Center, Aberdeen Proving Ground, Edgewood, Md. Rice, G.B., T.W. Lambert, B. Haas, and V. Wallace. 1971. Effect of Chronic Ingestion of VX on Ovine Blood Cholinesterase. Technical Report DTC 71–512. Desert Test Center, Dugway Proving Ground, Dugway, Utah. Rocha, E.S., S.R. Chebabo, M.D. Santos, Y. Aracava, and E.X. Albuquerque. 1998. An analysis of low level doses of cholinesterase inhibitors in cultured
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Review of the U.S. Army's Health Risk Assessments for Oral Exposure to Six Chemical-Warfare Agents neurons and hippocampal slices of rats. Drug Chem. Toxicol. 21(Suppl. 1):191–200. Sim, V.M., C. McClure Jr., F.J. Vocci, L. Feinsilver, and W.A. Groff. 1964. Tolerance of Man to VX-Contaminated Water. Technical Report CRDLR 3231 . U.S. Army Chemical Research and Development Laboratories, Edgewood Arsenal, Aberdeen, Md.
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