HEXACHLOROCYCLOPENTADIENE Ambient Water Quality Criteria Criteria and Standards Division Office of Water Planning and Standards U.S. Environmental Protection Agency Washington, D.C. ------- CRITERIA DOCUMENT HEXACHLOROCYCLOPENTADIENE CRITERIA Aquatic Life For hexachlorocyclopentadiene, the criterion to protect freshwater aquatic life, as derived using the Guidelines is 0.39 pg/1 as a 24-hour average and the concentration should not exceed 7.0 jig/1 at any time. For saltwater aquatic life, no criterion can be derived using the Guidelines, and there are insufficient data to estimate a criterion using other procedures. Human Health For the prevention of adverse effects due to the organ- oleptic properties of hexachlorocyclopentadiene in water, the criterion is 1.0 ^ug/1. ------- CRITERION DOCUMENT HEXACHLOROCYCLOPENTADIENE Hexachlorocyclopentadiene (Hex; C-56; 1,2,3,4,5,5-hexa- chlorocyclopentadiene) is a pale to greenish yellow liquid with the molecular formula, C,-Clg. Other physical properties include a molecular weight of 272.77; a solubility in water of 0.805 mg/1; a vapor pressure of 1 mm Hg at 78-79 degrees C and a density of 1.7119 (20°/4° C)(Lu, et al., 1975; Ungnade and McBee 1958). Hex was used as a chemical intermediate in the manufacture of numerous widely used chlorinated pesticides (Kirk-Othmer, 1964). Recent governmental bans on the use of chlorinated pesticides have restricted the use of Hex as a pesticide intermediate to the endosulfan(Thiodan) and decachlorobi- 2,4-cyclopentadiene-l-yl (Pentac) industries. Currently, the major use of Hex is as an intermediate in the synthesis of commerically important flame retardants (Sanders, 1978: Kirk-Othmer, 1964). Hex, though commercially important as a chemical intermediate (production levels approximate 50 million pounds per year), has no end uses of its own (Bell, et al. 1978). Environmental monitoring data for Hex are lacking except for measured levels in the vicinity of industrial sites. Hex has been identified and/or quantified in wastewater, receiving streams, rivers, fish, soil, sediment, and air surrounding pesticide plants (Spehar, et al. 1977: Swanson, 1976: Carter, 1977a). A recent incident involving the improper disposal of hex-containing industrial wastes in a Louisville, Kentucky sewer system, resulted in no apparent widespread environmental release of the chemical (Carter, 1977b). A-l ------- On the basis of the Louisville incident and laboratory and industrial observations, Hex has proven to be a potent irritant. Industrial workers have experienced irritation of the eye, irritation of the upper airway passages and headaches upon exposure to Hex vapors and burns upon contact of skin with the liquid. (Ingle, 1953; Carter, 1977b). Long-term exposure to hazardous concentrations results in systemic poisoning of laboratory animals (Treon, et al. 1955). In the laboratory, Hex has exhibited toxicity to fish and mammals (Spehar, et al. in press; Treon, et al. 1955). Hex has been reported to be nonmutagenic in laboratory tests (NCI, 1977; IBT, 1977; Litton Bionetics, 1978a,b) and there are no data available to evaluate the carcinogenicity of the compound. A-2 ------- REFERENCES Bell, M.A., et al. 1978. Review of the environmental effects of pollutants XI. Hexachlorocyclopentadiene. Unpublished report by Battelle Columbus Lab. for U.S. EPA Health Res. Lab., Cincinnati, Ohio. Carter, M.R. 1977a. Legal affidavit filed in State of Georgia, Fulton Co. dated June 14, 1977. Testimony concerning estimates of total daily discharge of hex from Velsicol Chemical Corp. Memphis plant and calculations of estimated hex concentration in Mississippi River resulting from said discharge. Carter, M.R. 1977b. The Louisville Incident. Internal report (unpublished), Serveillance and Analysis Division, Region IV, U.S. EPA, Athens, Ga. Industrial Bio-Test Laboratories, Inc. (IBT) 1977. Mutageni- city of PCL-HEX incorporated in the test medium tested against five strains of Salmonella typhimurium and as a volatilate against tester strain TA-100. Unpublished report submitted to Velsicol Chemical Corp. Ingle, L. 1953. The toxicity of chlordane vapor. Science 118: 213. Kirk-Othmer Encyclopedia of chemical technology. 2nd ed. 1964. Interscierice Publishers, New York. A-3 ------- Litton Bionetics, Inc. 1978a. Mutagenicity evaluation of hexachlorocyclopentadiene in the mouse lymphoma forward mutation assay. Unpublished report submitted to Velsicol Chemical Corp. Litton Bionetics, Inc. 1978b. Mutagenicity evaluation of hexachlorocyclopentadiene in the mouse dominant lethal assay: Final rep. Unpublished. Submitted to Velsicol Chemical Corp. Lu, PY., et al. 1975. Evaluation of environmental distribu- tion and fate of hexachlorocyclopentadiene, chlordane, hepta- chlor, and heptachlor epoxide in a laboratory model ecosystem. Jour. Agric. Food Chem. 23: 967. National Cancer Institute. 1977. Summary of data for chemical selection. Unpublished internal working paper, Chemical Selection Working Group. U.S. Dep. Health Educ. Welfare, Pub. Health Serv. Washington,D.C. Sanders, H.J. 1978. Flame retardants. Chem. Eng. News: April 24, 22. Spehar, R.L., et al. (In press). Toxicity and bioaccumulation of hexachlorocyclopentadiene, hexachloroonorbornadiene and heptachloronobornene in larval and early juvenile fathead minnows, Pimephales promelas. Bull. Environ. Contain. Toxicol. A-4 ------- Swanson, D. 1976. Discharges from Hooker Chemical Company. Internal Staff Report (unpublished), Toxic Mater. Section, Water Quality Div., Environ. Prot. Bur. Mich. Dep. Nat. Resour. Treon, J.F., et al. 1955. The toxicity of hexachlorocyclo- pentadiene. Arch. Ind. Health 11: 459. Ungnade, H.E., and E.T. McBee. 1958. The chemistry of perchlorocylopentadienes and cyclopentadienes. Chem. Rev. 58: 240. A-5 ------- AQUATIC LIFE TOXICOLOGY* FRESHWATER ORGANISMS Introduction Acute and embryo-larval data are available for the fathead minnow and hexachlorocyclopentadiene. However, no data of any type are known for invertebrate species. Acute Toxicity Henderson (1956) exposed the fathead minnow under three different conditions using two dilution waters. One had a hardness of 40 mg/1 and pH of 7.4 and the other had a hardness of 400 mg/1 and pH of 8.2. Two tests with hard water were conducted to evaluate the method used to add the chemical to the dilution water. The latter comparison was important since hexachlorocyclopentadiene is quite volatile and has an extremely low solubility in water. The chemical was added in a 0.01 percent acetone solution and a 0.001 percent suspension of an emulsion prepared in a blender. The effect of hardness, if any, was slight with unadjusted 96-hour LC50 values of 104 pg/1 in soft water and 78 pg/1 in hard water (Table 1). The test results comparing methods of addition were 78 and 59 jig/1 (Table 1) indicating little difference. Spehar, et al. (In press) determined the 96- hour LC50 value for larval fathead minnows. This result, using flow-through procedures and measured concentrations, *The reader is referred to the Guidelines for Deriving Water Quality Criteria for the Protection of Aquatic Life C43 FR 21506 (May 18, 1978) and 43 FR 29028 (July 5, 1978)3 and the Methodology Document in order to better understand the following discussion and recommendation. The following tables contain the appropriate data that were found in the literature, and at the bottom of each table are the calcu- lations for deriving various measures of toxicity as described in the Guidelines. B-l ------- is 7.0 jug/1 (Table 1). This difference between the data of Henderson (1956) and Spehar, et al. (In press) may be due to differences in test methods or in relative sensitivity of different life stages of the fathead minnow. The -Final Fish Acute Value for hexachlorocyclopentadiene is 7.0 ^ig/1 based on the geometric mean of adjusted LC50 values divided by the Guidelines sensitivity factor of 3.9. The lowest value from a flow-through test with measured concentrations is also 7.0 yug/1 (Table 1). Since there are no data for invertebrate species, the Final Fish Acute Value of 7.0 yug/1 is also the Final Acute Value. Chronic Toxicity The chronic value for the fathead minnow embryo-larval test by Spehar, et al. (In press) is 2.6 jug/1 (Table 2). This concentration is not that much lower than the 96-hour LC50 value (7.0 ^jg/1) for larval fathead minnows (Table 1). The Final Fish Acute Value is equal to 2.6 ;ug/l divided by the sensitivity factor of 6.7 or 0.39 jug/1. This also becomes the Final Chronic Value. Plant Effects No data are available on the effects of hexachlorocyclo- pentadiene on.freshwater algae or plants. Residues The bioconcentration factor for whole-body fathead minnows is 11 (Table 3) after a 30-day exposure (Spehar, et al. In press). No Residue Limited Toxicant Concentration can be determined since there is no permissible residue concentration available. B-2 ------- Miscellaneous Applegate, et al. (1957) exposed sea lamprey, rainbow trout and bluegill to concentrations of hexachlorocyclopenta- diene of 1,000 and 5,000 ;ag/l (Table 4). Death or distress was observed in one-half to one hour. The 30-day LC50 value for the fathead minnow (Spehar, et al. In press) is 6.7 jig/1 which result is only slightly lower than the 96-hour LC50 value of 7.0 ug/1 determined by the same investigators. B-3 ------- CRITERION FORMULATION Freshwater - Aquatic Life Summary of Available Data The concentrations below have been rounded to two signif- icant figures. Final Fish Acute Value = 7.0 pg/1 Final Invertebrate Acute Value = no available Final Acute Value = 7.0 /ag/1 Final Fish Chronic Value = 0.39 /ig/1 Final Invertebrate Chronic Value = not available Final Plant Value = not available Residue Limited Toxicant Concentration = not available Final Chronic Value = 0.39 ;ig/l 0.44 x Final Acute Value = 3.1 /ig/1 The maximum concentration of hexachlorocyclopentadiene is the Final Acute Value of 7.0 /ig/1 and the 24-hour average concentration is the Final Chronic Value of 0.39 jig/1. No important adverse effects on freshwater aquatic organisms have been reported to be caused by concentrations lower than the 24-hour average concentration. CRITERION: For hexachlorocylopentadiene the criterion to protect f-reshwater aquatic life as derived using the Guidelines is 0.39 jug/1 as a 24-hour average and the concen- tration should not exceed 7.0 /ag/1 at any time. B-4 ------- Table 1. Freshwater fish acute values for hexachlorocyclopentadiene Adjusted CO I ui Bloaeaay Teet Time Organ ism Method* cone.** (hrap| Fathead minnow (larva), FT M 96 Pimephales promelas Fathead minnow, S U 96 Pimephales promelas Fathead minnow, S U 96 Pimephales promelas Fathead minnow, S U 96 Pimephales promelas LC50 (uq/il 7 104 78 59 LC50 (ug/lj 7.0 56.9 42.6 32.3 deference Spehar, et al. In press Henderson, 1956 Henderson, 1956 Henderson, 1956 * S = static, FT = flow-through ** U = unmeasured, M «• measured Geometric mean of adjusted values = 27,2 iig/1 *«•'» = 7.0 ng/1 Lowest value from a flow-through test with measured concentrations = 7.0 i>g/l 27.2 ------- 03 I cn Table 2. Freshwater fish chronic values for hexachlorocyclopentadiene (Spehar, et al. In press) Chronic Limits Value Organism Test* (ug/i) Fathead minnow (larva), E-L 3.7-7.3 2.6 Pimephales promelaa * E-L = embryo-larval Geometric mean of c Lowest chronic value = 2.6 iig/1 9 £ Geometric mean of chronic values = 2.6 Mg/1 -r- =0.39 wg/1 ------- ro i Tatle 3. Freshwater residues for hexachlorocyclopentadiene (Spehar, et al. In press) .Time. Organism Eioconceutration Factor (days; Fathead minnow (juvenile), 11.0 30 Ptmephales promelas ------- Table 4. Other freshwater data for hexachlorocyclopentadiene 03 I 00 Organism Sea lamprey (larva), Petromyzon niarinus Sea lamprey (larva), Petromyzon marinus Rainbow trout (fingerling), Salmo gairdneri Rainbow trout (fIngerling). Salmo gairdneri Test Duration Ettect 24 hrs Death In 1 hr 24 hrs Distress In % hr 24 hrs Death in % hr 24 hrs Death in 1 hr Result (uq/il Fathead minnow (larva). 30 days LC50 Pimephales promelas Bluegill (flngerling), 24 hrs Death In fc hr Lepomis macrochlrus Bluegill (fingerling). 24 hrs Distress in % hr Lepomis macrochlrus 5,000 Applegate, et al. 1957 1,000 Applegate. et al. 1957 5,000 Applegate, et al. 1957 1,000 Applegate, et al. 1957 6.7 Spehar, et al. In .press 5,000 Applegate, et al. 1957 1,000 Applegate, et al. 1957 Lowest value =6.7 pg/1 ------- SALTWATER ORGANISMS Introduction No data on the effects of hexachlorocyclopentadiene on saltwater organisms are available. Criterion Formulation CRITERION: For saltwater aquatic life, no criterion for hexachlorocyclopentadiene can be derived using the Guide- lines, and there are insufficient data to estimate a criterion using other procedures. B-9 ------- REFERENCES Applegate, V.C., et al. 1957. Toxicity of 4,346 chemicals to larval lampreys and fishes. U.S. Fish and wild. Serv. Spec. Rep. Fish. No. 207. U.S. Dep. Inter. Washington, D.C. Henderson, D. 1956. Bioassay investigations for International Joint Commission. Hooker Electrochemical Co., Niagara Falls, N.y. U.S. Dep. Health Educ. Welfare, Robert A. Taft Sanitary Eng. Center, Cincinnati, Ohio. 12 p. Spehar, R.L., et al. Toxicity and bioaccumulation of hexa- chlorcyclopentadiene, hexachloronorbornadiene and heptachloro- norbornene, in larval and early juvenile fathead minnows, Pimephales promelas. Bull. Environ. Contain. Toxicol. (In press). B-10 ------- Mammalian Toxicology and Human Health Effects EXPOSURE Hexachlorocyclopentadiene, hereafter referred to as hex, is a highly reactive, highly chlorinated compound which is the key intermediate in the manufacture of many commer- cially important organochlorine pesticides and flame retar- dants for organic polymers (Kirk-Othmer, 1964). Although it has also been suggested for use as an intermediate in the manufacture of dyes, Pharmaceuticals, resins, and germi- cides, these latter uses account for only a very small per- centage of hex production. Historically, hex has been pro- duced in the United States by two companies, Hooker Chemi- cal and Plastics Corporation (Montague, Michigan) and Velsi- col Chemical Corporation (Memphis, Tennessee). In 1977, Hooker discontinued hex manufacture at the Montague plant, making Velsicol's Memphis plant the only current U.S. pro- ducer. Hex is produced at several facilities outside the U.S. Hex was formerly used in the manufacture of aldrin, endrin and dieldrin at the Shell Chemical Company plant in Denver, Colorado (Zavon, 1978). Hex has been used as a chemical intermediate in the production of numerous chlorinated pesticides, several of which have enjoyed very large usage. The list includes chlordane, aldrin, dieldrin, heptachlor, isodrin, endrin, R R mirex, Kepone, endosulfan (Thiodan ), and Pentac . With the exception of endosulfan and Pentac, both of which are C-l ------- in current use, the usage of hex-based pesticides has been banned, suspended, or severely restricted by governmental action. Although current production estimates are uncertain and highly variable, one estimate has placed annual produc- tion as high as 50 million pounds (25,000 tons) per year (Bell, et al. 1978). Recent bans or restrictions on many of the chlorinated pesticides have led to a decline in the use of hex as a chemical intermediate in the manufacture of these products; simultaneously, the use of hex in the manufacture of flame retardants has increased. Currently, a major use of hex is in the manufacture of flame retardant compounds such as chlorendic acid and chlorendic anhydride which are produced by reacting equimolar quantities of hex and maleic anhydride. These and other hex-derived chlori- nated organic compounds confer flame retardant properties to plastics, including polypropylene, polyethylene, nylon, rigid polyurethane foams, unsaturated polyesters, and other polymers including epoxy resins (Sanders, 1978). Although hex is a commercially important chemical inter- mediate with high annual production, it has essentially no end uses of its own. Consequently, hex concentrations in the environment should be negligible and limited data suggest that this indeed is the case. Small amounts of hex are occasionally present as impurities in pesticides made from it and some has undoubtedly entered the environ- ment in this way. The most likely route of entry into the environment arises from the manufacture of hex or hex-con- C-2 ------- taining products. Discharge of these industrial wastes appears to constitute the only documented sources of measur- able hex in environmental samples. Due to its infrequency in the environment and its low profile as a chemical intermediate, there have been few studies of the behavior of hex in the environment or in biological systems. By the same token, until recently, hex was not recognized as a major environmental problem nor a potential threat to humans (except for those occupa- tionally exposed). A recent incident in which scores of workers at a sewage treatment plant in Louisville, Kentucky, experienced a variety of toxic symptoms following the im- proper disposal of hex manufacturing wastes has created a great demand for information concerning the effects of hex exposure on humans. Several literature reviews on the health and environ- mental effects of hex are available. These include reviews of Equitable Environmental Health, Inc. (1976); U.S. Envi- ronmental Protection Agency (1977); National Academy of Sciences (1977); and Bell, et al. (1978). Although each of these reports is different in emphasis, they each note the unfortunate absence of epidemiologic studies of hex- exposed workers and the lack of suitable chronic exposure studies of animals (especially with respect to carcinoge- nicity). Until these types of information are available, proposed environmental criteria will necessarily be based on extrapolation of animal data to humans, a practice which C-3 ------- is invariably speculative and prone to error. Perhaps more importantly, in the absence of suitable chronic exposure studies, recommendations must be based on avoidance of rela- tively overt manifestations of toxicity (e.g., irritation) rather than less easily detectable effects (e.g., abnorma- lities in physiologic tests, increased incidence of neo- plasms, etc.) which may manifest themselves only after years of exposure. Since effects of the latter type tend to be elicited at doses lower than those causing acute toxicity, criteria based on acute responses may fail to provide ade- quate protection. Consequently, the criterion levels sug- gested in this document are presented with the understanding that they are based on decidedly inadequate chronic effects data and should be reassessed upon completion of appropriate chronic studies. Hex, Cc-Clg, is not present in the environment under natural conditions. It may be prepared in the laboratory by several alternative methods which have been described in a review article by Ungnade and McBee (1958). Commercial technical hex is a pale yellow, nonflammable liquid having a very pungent odor. It is soluble in a number of organic solvents but is relatively insoluble, about 2.0 mg/1 in water. Hex boils at 239 C (462 F) and it is sufficiently volatile at ambient temperatures to have a tendency to dis- perse into the atmosphere. This tendency to volatilize undoubtedly accounts in part for the failure to detect hex in many environmental samples. C-4 ------- Six active chlorines and two double bonds make hex a highly reactive compound which readily undergoes substi- tution and addition reactions. Its versatility is based upon its reactivity as a diene with a variety of decompo- sition. Data from the iso-octane solutions revealed no degradation after 24 hours, but a multi-peak spectrum indi- cating the presence of degradation products was obtained after 7 to 21 days' exposure. This spectrum suggested to the investigators that the compound may be susceptible to atmospheric oxidation and/or photodecomposition (National Cancer Institute, 1977). In using hex as an intermediate in the manufacture of various chlorinated pesticides (chlordane,dieldrin, hep- tachlor, etc.), it appears that although yields in all reac- tions are good, they are not quantitative. Thus, there is reason to suspect that in some cases free hex may have been present in the marketed pesticide products. An early study by Ingle (1953) provided good evidence that the reported vapor toxicity of chlordane to mice was not attributable to chlordane, but to some unreacted intermediate, chief of which was hexachlorocyclopentadiene. It is suspected that small quantities of unreacted hex may be present in other related pesticides as well. Because of the widespread use of hex as an intermediate, and the belief that hex may comprise as much as 1 percent of commercial chlordane (a pesticide with extremely great usage), laboratory studies have of olefins and polynuclear aromatic hydrocarbons in the Diels-Alder reaction. C-5 ------- Rieck's report (1977a) provides evidence of the volati- 14 lization of hex from soil. Vapors of C-hex were evolved from treated soil to the extent of 11, 13, 15, 16, 17, and 19 percent (cumulative) of the applied amounts 1, 2, 3, 5, 7, and 14 days respectively after treatment. One could, therefore, deduce that there is volatility from treated soil and that the rate decreases with time. Another distinguishing feature of hex is that it ap- pears to be strongly adsorbed to soil or soil components. Studies of hex-treated soil (Rieck, 1977a, 1977b) have demon- strated poor extractability from soil, which provides indi- rect evidence of strong adsorption. In one study (1977b), 14 soil which had been extracted was then combusted to C02* 14 Any residual but unextracted .C was then measured directly. 14 Unextracted C was found in these samples and thus was accounted for as a "bound" residue. Had it not been accounted for, it would have probably been assumed to have volatilized. Hex, unlike some of the pesticides derived from it, degrades rapidly by photolysis, giving water soluble degra- dation products. Tests on its stability towards hydrolysis at ambient temperature indicated a half-life of about 11 days at pH 3-6, which was reduced to 6 days at pH 9. In December, 1975, hex was qualitatively identified as a contaminant in the discharge of a pesticide production plant in Memphis. Later, (May, 1977) the compound was iden- tified in the air at the Hooker plant in Montague, Michigan (56 ppb), in its aqueous discharge (0.170 mg/1), and in , C-6 ------- fish tissue from the receiving stream (14-18 ppb) (Spehar, et al. 1977). Hex has also been reported to have been pre- sent in soil and bay sediments in the vicinity of a Virginia pesticide plant long after production was discontinued (Swan- son, 1976). Data on environmental concentrations of hex are minimal except for industrial discharges. Velsicol Chemical Cor- poration' s Memphis plant has been issued a National Pollu- tant Discharge System (NPDES) permit. Monitoring activities in connection with the discharge permit indicate that hexa- chlorocyclopentadiene, hexachloronorbornene, and hexachloro- bornadiene are being discharged into the City of Memphis wastewater collection system (Bennett, 1977; Marks, 1977). Sampling for the month of January, 1977 (31 consecutive days), revealed hex concentrations in wastewater ranging from 0.156 to 8.240 mg/1. U.S. Environmental Protection Agency's Water Surveillance Branch sampled Velsicol1s dis- charge February 2-3, 1977. Hex was detected at 18 mg/1. Based on the average monthly discharge by the Velsicol Chemi- cal Corporation during February, 1977 (3.16 million gallons per day), 474 pounds of hex was believed to have been dis- charged through Velsicol1s discharge outfall into the City of Memphis Wastewater Collection System and then into the Mississippi River during the period February 2-3, 1977. Calculated on the basis of the flow rate above, this dis- charge caused a concentration of hex in the Mississippi River of 0.0006 mg/1 (Carter, 1977a). C-7 ------- In a recent, well-publicized incident, an estimated 6 tons equivalent of hexachlorocyclopentadiene (hex) and octachlorocyclopentadiene (octa) dispersed in No. 4 fuel oil were dumped into the Louisville, Kentucky, municipal sewer system's Western Outfall sewer. The contaminated sludge entered the Morris Forman Wastewater Treatment Plant on March 26, 1977, causing illness among sewage treatment plant workers. Toxic effects associated with this episode forced closure of the plant with subsequent diversion of 105 million gallons per day of raw sewage into the Ohio River. There was no evidence of environmental release (out- side the immediate environs of the sewage treatment plant and contaminated sewer lines). It was, however, necessary to decontaminate the sewer.system and the treatment plant. This incident is reported in further detail in the section on "Effects - Human Studies". Several general conclusions can be made regarding the various modes of hex exposure. C-8 ------- Ingestion from Water Very little is known regarding potential hex exposures through ingestion of contaminated food or water. Hex has been detected in specific bodies of water near points of industrial discharges. Except for such source-directed sampling, it appears that there is little information on hex concentrations in surface waters. Hex is usually not detectable in water samples. Due to its low solubility and tendency to volatilize, one would not expect it to remain in flowing water. Moreover, there are no data on hex levels in drinking waterror the extent to which hex in raw (untreated) water would be passed through the water treatment process to human consumers. Hex has been identified in a few samples of fish taken from waters near the Hooker plant in Michigan (Spehar, et al. 1977). Frequently, however, hex residues have not been detected in eoTble fish deliberately exposed to hex in labora- tory experiments. According to the same investigator, the inability to recover hex in fish samples probably results from losses by vaporization during sample extraction. No reports concerning hex contamination of other foods could be located. C-9 ------- A bioconcentration factor (BCF) relates the concentration of a chemical in water to the concentration in aquatic organ- isms, but BCF's are not available for the edible.protions of all four major groups of aquatic organisms consumed in the United States. Since data indicate that the BCF for lipid-soluble compounds is proportional to percent lipids, BCF's can be adjusted to edible portions using data on percent lipids and the amounts of various species consumed by Americans, A recent survey on fish and shellfish consumption in the United States (Cordle, et al. 1978) found that the per capita consumption is 18.7 g/day. From the data on the 19 major species indentified in the survey and data on the fat content of the edible portion of. these species (Sidwell, et al. 1974), the relative consumption of the four major groups and the weighted average percent lipids for each group can be calculated: Consumption Weighted Average Group (Percent) Percent Lipids Freshwater fishes 12 4.8 Saltwater fishes 61 2.3 Saltwater molluscs 9 1.2 Saltwater decapods 18 1.2 Using the percentages for consumption and lipids for each of these groups, the weighted average percent lipids is 2.3 for consumed fish and shellfish. C-10 ------- A measured steady-state bioconcentration factor of 11 was obtained for hexachlorocyclopentadiene using fathead minnows containing about 8 percent lipids (Spehar, et al. In press). An adjustment factor of 2.3/8.0 = 0.2875 can be used to adjust the measured BCF from the 8.0 percent lipids of the fathead minnow to the 2.3 percent lipids that is the weighted average for consumed fish and shellfish. Thus, the weighted average bioconcentration factor for hexa- chlorocyclopentadiene and the edible portion of all aquatic organisms consumed by Americans is calculated to be 11 x 0.2875 = 3.2. Inhalation The heaviest and most chronic exposure to hex undoubtedly occurs among persons engaged directly in the manufacture of hex and among production workers fabricating hex-contain- ing products. Although several cohorts of hex-exposed workers C-ll ------- can be specifically identified (employees of Hooker Chemi- cals and Plastics, Michigan and Niagara Falls plants; Velsi- col Chemical Corporation, Memphis plant; Shell Chemical Company, Denver and Pernis, Netherlands, plants; an Israeli company, Makhteshim; and the Hooker plant at Genk, Bel- gium) , there have been no reports of epidemiologic studies of these workers. Inhalation of hex is the primary mode of occupational exposure. Accidental spills and illegal discharges of hex represent the primary mode of acute human exposure (e.g., the Louisville incident). Inhalation ap- pears to be the most important mode of exposure in these cases as well. Dermal According to Hooker Chemical and Plastic Corporation's Material Safety Data Sheet, hex is readily absorbed through the skin. Prolonged or repeated contract can lead to burns and manifestations of systemic toxicity not unlike those caused by inhalation. The hazards of skin contact are well recognized and industrial workers are provided with imper- vious clothing to prevent dermal contact (Hooker, 1972). Thus, dermal exposure should not be anticipated among workers familiar with hex. Persons outside the chemical industry can be exposed to hazardous contacts as a result of acci- dental spills or improper disposal of hex. C-12 ------- PHARMACOKINETICS Only two studies which address the pharmacokinetics of hex could be located (Mehendale, 1977; Kommineni, 1978). The latter study focuses upon absorption and elimination of hex while the Mehendale (1977) study is more concerned with the disposition of hex within the body and modes of elimination. The Kommineni (1978) study consisted of two parts. The first consisted of a study of rats exposed to various doses of hex by gavage while the second portion examined guinea pigs exposed to varying doses of hex via dermal appli- cation. Inferences regarding patterns of absorption, meta- bolism, and excretion are based on gross pathology findings and histopathologic findings at necropsy. In the first series, a total of 10 female rats were exposed to 0, 50, 100, 150, 200, and 300 mg/kg of hex by gavage. All animals were sacrificed 24 hours post treat- ment. The rats were necropsied and lungs, liver, spleen, kidneys, adrenals, heart, stomach, and intestines were saved for histopathology evaluation. Gross pathology of the rats exposed to 200 and 300 mg/kg revealed brown discoloration around the nostrils and anus of the rats. The urinary bladders of two of the four rats contained brown fluid. Subserosal emphysema of the nonglan- dular stomach was evident in one animal. On histopathologic examination, the lungs showed atelectasis with moderate thickening of the alveolar walls. The alveolar walls con- C-13 ------- tained moderate numbers of macrophages and neutrophils. Some bronchi contained denuded epithelium. No edema was present in the lungs. Rats receiving lower dosages showed similar, but milder, changes. The stomachs of rats receiving dosages of 200 or 300 mg/kg showed coagulative necrosis of the gastric squamous epithelium. The submucosa of the nonglandular part of the stomach showed mild neutrophilic infiltration. The supporting structures of the stomach (submucosa, submuscularis, muscularis) showed moderate edema. Epithelium of the glandular part of the stomach showed no treatment-related changes. Animals receiving lower doses showed similar changes in the stomach. Ulcers of the nonglan- dular portion of the stomach were seen in several of the animals. At all dosages, the other organs were unremarkable. The author commented that these morphological changes indicate that hex is absorbed through the squamous epithe- lium of the nonglandular part of the stomach and that the major route of elimination of hex is through the lungs. In the second part of the study, four male guinea pigs were painted on the skin (site unspecified) with hex at dosages of 0, 300, 600, and 1200 mg/kg and sacrificed 24 hours after the exposure. All animals were necropsied and the lungs, liver, pancreas, kidneys, adrenals, urinary blad- der, heart, skin, stomach, and intestines were saved for histopathologic evaluation. C-14 ------- On gross pathology, subcutaneous edema was seen extending from the inguinal area to the sternum. At the lowest dosage, the lungs were highly expanded and showed rib impressions on the parietal surface. Similar but more severe changes were seen in the animal receiving 600 mg/kg. The animal painted with 1200 mg/kg expired prior to sacrifice; the trachea was filled with frothy fluid. Histopathologic exami- nation of the lungs revealed atelectasis with thickened alveolar walls containing moderate numbers of macrophages and neutrophils. Intense congestion of all pulmonary blood vessels and occasional alveolar edema was seen in the ani- mal receiving the 1200 mg/kg dose. In the skin, moderate to marked edema disrupted the collagen bundles. Focal pockets of neutrophils were seen in the edematous dermis. Edema extended throughout the thickness of the adipose tissue layer. One animal showed partial thrombosis of medium size veins situated deep in the dermis. The skin appendages were normal. Kommineni (1978) concluded, "Hex is absorbed through the skin and probably is eliminated through the lungs. Unlike the rat stomach, the squamous epithelium of the guinea pig skin and its adnexa did not show necrotic changes. This is probably due to two factors, surface area and transit time." In the Mehendale (1977) study, radiolabeled hexachloro- 14 cyclopentadiene ( C-hex) was administered by oral intuba- tion to four male Sprague-Dawley rats in order to examine C-15 ------- absorption, metabolism, and excretion of the compound fol- 14 lowing a single oral dose. After dosing with C-hex (5 ji moles, 1 juCi per animal), the rats were maintained in metabolism cages for 7 days, during which daily urine and fecal samples were collected. After 7 days, the animals were sacrificed and the major organs were removed and radio- assayed. Urine and powdered fecal samples were radioassayed for 14 total C. An average of approximately 33 percent of the total dose was excreted in the urine after 7 days. About 87 percent of that (approximately 28.7 percent of total dose) was eliminated during the first 24 hours after the administration of the compound. Fecal excretion accounted for 10 percent of the total dose; nearly 60 percent of the 7-day fecal excretion occurred during the first day. Beyond the third day after treatment, only trace amounts of the hex-derived C were eliminated in the feces. Tis- sues retained only trace amounts of hex after 7 days. For example, the kidney retained only about 0.5 percent of the total dose and the liver less than 0.5 percent. Other organs and tissues--fat, lung, muscle, blood, etc.--contained even less of the radiolabel. Such findings suggest that at least half of the administered hex was eliminated by routes other than urine and feces. The author felt that the respiratory tract is probably the major route of excretion. C-16 ------- The nature of the radioactivity excreted in the urine was examined searching for possible metabolites. It was found that about 70 percent of the radioactivity in the urine was extractable using a hexane:isopropanol (9:1) mix- ture. The organic solvent was concentrated, applied to thin-layer chromatography (TLC) plates, and developed in three solvent systems. The radioactive spots were visual- ized by auto-radiography on medical x-ray film. The results suggested the presence of at least four metabolites; how- ever, at the time of this writing they had not been identi- fied and characterized. Disposition and biliary excretion of C-hex.was stu- 14 died by injection of approximately 1 jjCi (5 >i mole) of C- hex into the femoral vein of anesthetized rats. Timed sam- ples of blood and bile were collected for 1 hour from the femoral artery and common bile duct which had been cannu- lated prior to dosing. Approximately 9 percent of the admini- stered dose was excreted in the bile in 1 hour. Because this quantity is equivalent to that excreted in the feces over 7 days, enterohepatic circulation of this compound is probable. The nature of the compound present in the bile is not yet known. At the end of the above experiments, the animals were sacrificed and the liver and kidneys were removed. Tissue homogenates from these organs were radioassayed and the distribution of the radioactivity among the various subcel- lular fractions was examined by assaying the various centri- C-17 ------- fugation fractions. Kidney cytosol accounted for 93 percent of the radioactivity in the total kidney homogenate. This behavior is consistent with rapid urinary excretion. Simi- larly, 68 percent of the radioactivity in the liver homo- genate was associated with the liver cytosol fraction, once again consistent with rapid excretion. Pre-exposure of some of the rats to hex (50 mg/kg/day) for 3 days prior to the experiment did not affect blood decay curves and biliary excretion; however, an increased concentration in the kidneys after a single challenge with 14 C-hex was observed. Whitacre (1978) reported that Velsicol has contracted 14 an independent metabolism study in rats and mice using C- hex. The metabolism of hex was determined both after single acute dosing and repeated administration over a period of about 30 days. The results of these studies have not yet been officially reported although verbal appraisal of some results has been provided to Velsicol. It appears that results of this study do not agree closely with the Mehendale study. The recent study shows hex to be eliminated from mammals (mice and rats) mainly by the fecal route and with no more than about 15 percent being eliminated in urine. Further, these studies do not indicate any significant amounts of pulmonary elimination of hex or its metabolites. Whitacre (1978) believes that the poor recoveries in feces in the Mehendale study may be the result of volatility of hex or its metabolites before removal for analysis. Losses during sample preparation undoubtedly further complicate the analysis of fecal matter. C-18 ------- EFFECTS Acute, Sub-acute, and Chronic Toxicity The classic studies of hex toxicity to mammals were conducted in the mid 1950's by Treon, et al. (1955). This series of investigations reported on both acute and subacute toxicity of hex to various species of mammals under a vari- ety of exposure regimens. Oral, dermal, and inhalation modes of exposure were included in Treon's experiments. Mammalian toxicity studies subsequent to the 1950's could not be located in the open literature, probably due to the rather low profile of hex relative to other pesticide chemi- cals. More recent, proprietary studies of the oral and dermal toxicity have now become available. In general, these findings agree remarkably well with those of Treon. It is most unfortunate that no truly long-term (i.e., = 6 months) studies of chronic effects have been conducted. Until data on the potential effects of long-term, chronic exposure (especially carcinogenicity) becomes available, any recommendations regarding environmental criteria must be regarded as tentative. 1) Acute Toxicity. a. Oral. Acute toxicity of hex was determined by Treon, et al. (1955) by administering dosages of 180, 280, 340, 420, 520, 620, 940, 1400, and 2100 mg/kg of hex in peanut oil directly into the stomachs of several groups of rabbits and rats. The data on rabbits indicate that the median lethal oral dose (LD50) administered as described above, lies in the range between 420 and 620 mg/kg of body weight. C-19 ------- Rats showed variation in minimum lethal dose depending on sex. Male rats were somewhat more sensitive in that the lethal dose was somehwat less than 280 mg/kg body weight, whereas for females the dosage causing death was greater than 280 mg/kg. The LD50 for male rats was determined to be 505 mg/kg with 95 percent confidence limits of 387-623. Also noteworthy is the fact that very few of the test ani- mals survivied longer than a week after oral administration of hex. The International Research and Development Corporation (IRDC, 1972) conducted similar studies of the acute oral toxicity of hex. Twenty-five albino rats of each sex were given hex dissolved in corn oil at dosages of 315, 500, 794, 1250, and 1984 mg/kg. Five rats of each sex were used at each dosage level. An LD50 of 530 mg/kg was determined for female rats and 630 for male rats. The combined oral LD50 for both sexes was determined to be 584 mg/kg. Note that this is the reverse of the sex differential reported by Treon, et al. (1955). Naishstein and Lisovskaya (1965) reported a LD50 of 600 mg/kg for white rats. This value is comparable to the upper part of the range (420-620 mg/kg) reported by Treon, et al. (1955). Thus, the true LD50 is probably about 600 mg/kg. b. Dermal. In this series of experiments, 93.3 percent hexachlorocyclopentadiene was applied to the intact skin of rabbits using the technique of Draize et al., described by Treon et al. (1955). It was determined that the lethal dosage lies between 430 and 630 mg/kg body weight. Such a finding is notable in that hex appears to be just as toxic via dermal application as by ingestion. C-20 (ft ------- More recently, the irritant properties of hex were exa- mined in a study conducted by the International Research and Development Corporation (IRDC, 1972). These tests were commissioned by Velsicol Chemical Corporation in accordance with the regulations of the Federal Hazardous Substances Act. IRDC (1972) reported the results of an investigation of acute dermal toxicity of hex to rabbits. Four male and four female New Zealand white rabbits were used in this test. The hair was removed from the back of each rabbit with electric clippers. Two male and two female rabbits were used at each of two dosage levels. The test compound was applied in a single administration to the back of each rabbit at a dosage of 200 or 2000 mg/kg body weight. The area of application was wrapped with a gauze bandage and occluded with Saran Wrap. Twenty-four hours later, the bandages were removed and the backs were washed with water. The rabbits were observed for mortality for a period of 14 days. All of the animals which received 2000 mg/kg dosage died withi-n 24 hours after application of the compound. At the 200 mg/kg dosage, both male rabbits died but both female rabbits survived although they both exhibited weight loss over the 14-day period. The male rabbits that died showed weight loss also. In addition, cachexia, marked dermal irritation and hypoactivity was observed. Skin at the site of application turned purple in color within a C-21 ------- few hours after hex application. Based on these results, hex was concluded to be "a highly toxic material by the dermal route of exposure" in accordance with the criteria established under the Federal Hazardous Substances Act. c. Inhalation. Treon, et al. (1955) exposed various animal species to vapors formed by bubbling a stream of air through liquid hex contained in a bubbling tower. This air was then mixed with clean air to achieve the desired concentration. The stream of air, conditioned with respect to temperature, dust content, and humidity, was then passed into a plywood exposure chamber in which the test animals were confined. A series of hex concentrations in the air in the exposure chamber were used; these varied from 0.15 to 73.6 ppm. Test species were guinea pigs, rats, mice, and rabbits. The authors reported that hex vapors were very toxic to all four species of animals. Exposure to the concentra- tion of 13.0 ppm (an intermediate level in this experiment) for 15 minutes produced fatalities in all species except guinea pigs. Of the four species, rabbits appeared to be the most susceptible. Mice, rats, and guinea pigs followed in order of decreasing susceptibility. Table 1 depicts that results of the inhalation experiments. The values tabulated correspond to the concentration in ppm which: (1) permitted all animals to survive; (2) killed 50 percent of the animals; and (3) produced 100 percent lethal con- di tions. C-22 ------- TABLE 1 Dose Response Data: Inhalation of Hex Vapors (Treon, et al. 1955) Species of Animal Guinea pigs . . . Rats . . . Mice ... Rabbits . . . Fatalities , Percent 0 50 100 0 50 100 0 40 100 0 67 100 Hex Concentration of Test 1-Hour Exposure 7.2 13.8 20. Oa 3.1 7.2 20. Oa 1.4 7.2 13.8 1.4 3.1 7.2 (in ppm) Lethal to Percent Animals Indicated 3-1/2-Hour Exposure 3.1 7.1 12.4 1.4 3.1 7.1 1.4d 3.1e 7.1 -- 6.4 7.1 7-Hour Exposure 1.5 3.2 6.7 1.5b 3.2C 6.7 -- 1.56 3.2 -- -- 7.5 ^Duration of exposure was 1.25 hours. 25 percent of group died. ^75 percent of group died. 80 percent of group died. e20 percent of group died. C-23 ------- Animals of the following species died regularly when exposed to hex vapors at the following concentrations and durations: rabbits - 1.5 ppm for 7 hours; mice -1.4 ppm for two 7-hour periods; rats - 1.0 ppm for five 7-hour periods or 3.2 ppm for two 7-hour periods; and guinea pigs - 3.2 ppm for two 7-hour periods. IRDC (1972) also reported the results of acute inhala- tion experiements in rats. The test animals were exposed to atmospheric concentrations of approximately 176.2 and 17,624 ppm of the test compound for 4 hours. Ten rats were tested at each dosage level. Due to the extremely high dosages employed, little information could be derived from the study. No justification of the choice of dosages was given. All of the animals receiving the test compound at either exposure level died within 48 hours. All rats at the 17,624 ppm dosage level died during the 4-hour exposure period. At the 176.2 ppm atmospheric concentration, one rat died during the exposure period, eight more were dead within 24 hours, and the remaining rat died on the second day of observation. Signs seen during the exposure period included eye squint, dyspnea, cyanosis, salivation, lacrimation, and nasal dis- charge. Gross necropsy showed gray coloration of the skin, severe hemorrhage of the lungs, and hydrothorax among rats exposed to 17,624 ppm. Rats exposed to 176.2 ppm revealed congestion of the lungs in all cases. C-24 ------- Based on these results, the investigators concluded that hex is highly toxic material by the inhalation route of administration. Table 2 summarizes the results of acute toxicity studies of hex. 2) Subacute Toxicity. a) Oral. To date, there has not been a satisfactory study of subacute or chronic oral toxicity of hex. One portion of the Treon, et al. (1955) study attempted to examine subacute/chronic oral toxicity but reported that dosages of 180-2000 mg/kg were fatal within such a short period of time that the investigators were unable to establish an oral dosage which could be tolerated without mortality over an extended period. Similarly, Naish- stein and Lispvskaya (1965) reported that oral administration as little as 20 mg/kg for 6 months was fatal .to 20 percent of white rats. b) Dermal. Treon, et al. (1955) examined effects of sublethal concentrations of hex applied to the skin of rabbits and monkeys. In rabbits, dosages as low as 250 mg/kg induced extreme irritation, purplish-black discolora- tion of the skin and subcutaneous edema. Although the skin lesions healed eventually, damage to the skin in the area of application persisted for many days and the damage varied in severity and extent with the amount (dosage) of the ma- terial applied. A slightly different procedure was employed in the cuta- neous exposures of the monkeys. In this case, a series of hex concentrations (0.001, 0.01, 0.1, 1.0, and 10.0 per- C-25 ------- TABLE 2 Acute Toxicity of Hexachlorocyclopentadiene By Various Modes of Exposure (Treon, et al. 1955) Oral administration' Rabbits Rats Ratsd Females Males Rats Males LD 50 420-620 mg/kg 505 mg/kg 530 mg/kg 630 mg/kg Minimum Lethal Dose 280 mg/kg Dermal application Rabbits LD 50 420-610 mg/kg Inhalation0 - (Dosage Expressed as Vapor Concentration, ppm) Guinea pig Rats Mice Rabbits 13.8 ppm 7. 2 ppm 7. 2 ppm 3.1 ppm Hex dissolved in peanut oil, administered by gavage. 93.3 percent hex solution in Utrasene, applied to intact skin for 24 hours. Loop's based on 1-hour vapor exposure. Based on data reported by International Research and Development Corp. (1972). Hex dissolved in corn oil. C-26 ------- cent) dissolved in Ultrasene were applied to five sites of the abdominal skin. Dosage of each of the solutions was 0.01 ml. No irritation or other changes were noted; however, when 0.05 ml of the 10 percent solution was ap- plied to the back of a monkey for three consecutive days, the skin became severely irritated and necrotic. Subsequent experiments used more concentrated solutions (20, 40, 60, and 90 percent) which were applied (dosage of 0.05 ml) on separate areas of the monkeys' backs. At all concentrations there was discoloration of the skin, ranging from very light to dark tan as the concentration increased. The discolora- tion was followed by swelling which varied from slight to severe, again depending on concentration. The highest concen- tration caused cracking, oozing, and serious discharge from the treated areas; intermediate concentrations produced hardening and swelling of the skin. Among guinea pigs, application of solutions containing 0.01, 0.10, and 1.0 percent hex caused no alterations of the skin, but more concentrated solutions (40, 60, and 90 percent) resulted in discoloration, hardening, and necrosis of the skin at the application site. Based on these tests, it appears that the threshold concentration at which hex in Ultrasene induces irritation of the intact skin lies between 10 and 20 percent for monkeys and between 1.0 and 40 percent for guinea pigs. C-27 ------- Hex was tested for eye irritancy by instilling 0.1 ml of the "test compound" (which was presumably undiluted liquid hex) into the eyes of New Zealand white rabbits (IRDC, 1972). The test material was placed into the conjunctival sac of the right eye of each rabbit; the left eye served as an untreated control. Damage to the eye was evaluated by instillation of sodium fluorescein into the eye, fol- lowed by examination of the corneal surface for evidence of ocular damage under ultraviolet light. A graded scale was used to quantify the extent and severity of damage. The eyes of the rabbits were checked for corneal lesions at periodic intervals (at 1, 24, 48, and 72 hours post-expo- sure and at 7, 14, and 21 days post-exposure). Examina- tions at 14 and 21 days were precluded by the deaths of all of the rabbits on or before the ninth day of the obser- vation period. IRDC investigators attributed the deaths to the effects of the test compound, but unfortunately did not conduct post-mortem examinations to rule out other pos- sible causes of death. Based on the severity of the ocular lesions produced in the rabbits, hex was concluded to be "an extreme irri- tant and probable corrosive substance" in the 5-minute test and "an extreme irritant and corrosive substance" in the 24-hour wash test (IRDC, 1972). These classifications are set in accordance with standards set under The Federal Haz- ardous Substances Act, specifically Part 191, Hazardous Substances Test for Eye Irritants, Food and Drug Admini- stration. C-28 ------- c) Inhalation. When mice, rats, rabbits, and guinea pigs were exposed to 0.34 ppm in air for 7 hours a day for 5 days per week, none of the mice or rats survived more than 20 such exposures (Treon, et al. 1955). Two-thirds of the rabbits had died by the end of the twenty-fifth period; however, the guinea pigs survived through 30 periods. At 0.15 ppm, some animals from all four species survived 150, 7-hour exposures over a period of 216 days. Eight percent of the mice did not survive the prolonged intermittent expo- sure. Details of these findings are discussed in the next section under the heading "chronic toxicity". 3) Chronic Toxicity. a) Oral. In the Treon, et al. (1955) study, rabbits and rats given various dosages of hex ranging from 180-2100 mg/kg tended not to survive long enough at these dosages to provide acceptable data on chronic oral toxicity. Consequently, these investigators were unable to establish an oral dosage which could be tol- erated (e.g., without mortality) over an extended period of time. Studies in the Soviet Union reported by Naishstein and Lisovskaya (1965) appear to provide the only source of information on the effects of long-term, low-dose exposure to hex. Daily administration of 1/30 of the median lethal dose (20 mg/kg) for 6 months killed only 2 animals out of 10, even though the cumulative dose received was 1.5 times the acute LD^QQ, and six times the LD50. Although some changes were noted in the weight coefficients of internal organs of the animals, the authors judged the cumulative effects of hex to be weak. No observations of neoplasms or other abnormalities were reported. C-29 ------- b) Dermal. High concentrations, 430-6130 mg/kg, ap- plied to the skin of rabbits were frequently fatal within a few hours. Six rabbits who survived for 7-21 days after application of hex were killed and autopsied. Degenerative changes were seen in the brain, liver, kidneys, and adre- nal glands of these animals in addition to chronic skin inflammation, acanthosis; hyperkeratosis; and epilation. Visceral lesions due to dermal hex application reported by Treon, et al. (1955) are described in the section on toxic symptoms and pathological effects. Naishstein and Lisovskaya (1965) also investigated the effects of multiple, low-dose dermal exposures to hex. These experiements consisted of applying 0.5-0.6 ml of a concentration of 20 ppm hex in aqueous solution to the shaved skin of rabbits daily for a period of 10 days. No differ- ences were detected between the skin of the experimental animals and that of the controls. Treon, et al. (1955) reported that dosages of less than 10 percent hex appeared to be tolerated without irritative effects in monkeys and probably also in guinea pigs. Unfor- tunately, neither investigation continued the low-dose regi- men for a sufficient period to observe chronic effects. c) Inhalation. Treon, et al. (1955) exposed guinea pigs to hex vapors at a concentration of 0.34 ppm hex for 7 hours per day, 5 days a week. All of them survived until they reached 30 periods of exposure in 6 weeks. Rats and mice exposed to this concentration survived only five per- iods of exposure; however, survival of the rabbits was intermediate; two-thirds had died before the end of the fifth week (25 exposure periods). C-30 ------- A lower concentration, 0.15 ppm hex, was tolerated by guinea pigs, rabbits, and rats throughout 150, 7-hour per- iods of exposure extending over a period of approximately 7 months. Four of five mice died within this period. Al- though guinea pigs, rabbits, and rats appeared to grow nor- mally during this period, slight degenerative changes were observed in the livers and kidneys of these animals. These changes are discussed in the following section. 4) Toxic Symptoms and Pathologic Effects. a) Oral Administration. Rats and rabbits exposed to hex in the Treon, et al. (1955) acute toxicity study exhibited diarrhea, lethargy, and retarded respiration. The odor of hex could also be detected in the feces of these animals and on their bodies, presumably from fecal contamination. Rabbits which died following exposure to moderately high doses of hex (280-2100 mg/kg hex in corn oil) showed diffuse degenera- tive changes in the epithelium of the renal tubules. As in the study of Kommineni (1978), the lungs of these animals were congested and edematous. The same types of degenera- tive changes were also noted in the rats. In addition, some of the rats showed acute necrotic gastritis. Animals which survived the oral tests and were later sacrificed exhibited residual degenerative changes of the type described above, suggesting that the pathological changes are persis- tent. The severity of the lesions diminished as the length of the post-exposure survival interval increased, however. C-31 ------- Equitable Environmental Health (1976) reported the re- sults of a 90-day subacute oral toxicity study in rats con- ducted for Hooker Chemical and Plastics Corporation by Indus- trial Bio-Test Laboratories (IBT). Equitable reported that rats exposed to hex (mixed in food) at concentrations of 0, 30, 100, and 300 ppm showed no treatment-related effects in any of the parameters measured: growth, food intake, mortality, abnormal behavior, hematology, clinical blood i studies, and urinalysis. Gross pathologic examination also failed to reveal any abnormalities which could be attributed to ingestion of hex. Similarly, organ weights and ratios and microscopic examination of tissues and organs failed to show treatment-related abnormalities. The IBT study is unusual in that hex was mixed into the animal's food rather than administered by gavage (hex in corn oil solution). Due to the volatility of hex, the actual dose present in feed at the time of ingestion is unknown. Therefore the dosages above must be regarded as maximum hex content; it is likely that the dosages ingested were well below this. Other factors, including more direct contact of the hex solution with stomach tissues in the gavage experiments, may also explain the apparent discrepancy in toxicity thresh- old between the IBT study and the gavage experiments reported earlier. Naishstein and Lisovskaya (1965) reported results of a chronic oral toxicity experiement on 90 white rats. Rats were given daily peroral doses of 0.002, 0.0002, and 0.00002 C-32 ------- (0.04, 0.004, and 0.0004 mg/1) in aqueous solution for a period of 6 months. The first dose was 30 times greater than the threshold concentration with respect to aftertaste and smell (0.0013 mg/1); the second dose corresponded to the practical limit of detection by smell, and the third dose was 10 percent of the second. No deviations were ob- served in the behavior of the rats or in their weights through- out the 6-month experimental period. Likewise, no signi- ficant changes were seen in hemoglobin, red blood cells, white blood cells, or peripheral reticulocyte counts in the experimental groups as opposed to the controls. In animals receiving the highest dose, 0.002 mg/kg, neutro- penia and a tendency toward lymphocytosis were noted. The peripheral blood of animals receiving the two lower dosages did not show any alterations relative to controls. The authors concluded that daily peroral administration of doses of 0.0002 and 0.00002 mg/kg (0.004 and 0.0004 mg/1 in aqueous solution) caused no changes in peripheral blood cells, ascor- bic acid content, conditioned reflexes, or histologic struc- ture of the organs. Based on these tests and the threshold level for organoleptic noxious effects (smell and aftertaste in water), Naishstein and Lisovskaya (1965) recommended a maximum permissible concentration of 0.001 mg/1 hex in water. b) Dermal Application. Treon, et al. (1955) showed that dermal application of very low dosages of hex (0.25 mg/kg) were extremely irritating and induced local discolora- tion and edema. The skin became hard, encrusted, and fis- sured several days after application. The extent of the local damage varied directly with the size of the dose applied. C-33 ------- At autopsy rabbits exhibited visceral lesions similar in appearance to those seen after oral administration of hex. Again, diffuse degenerative changes were seen in the brain, heart, adrenals, liver cells, and kidney tubules. Pulmonary hyperemia and edema were also noticed. Animals killed 7-21 days post-application of the compound showed evidence of the same type of degenerative changes. Monkeys dosed with various concentrations of hex in solution exhibited discoloration of the skin which increased directly as the concentration of hex applied increased. Swelling, oozing, and encrustation similar to that described above for rabbits was seen. Healing eventually took place, but scarring and hair loss in the area of application ap- peared to be permanent. Industrial Bio-Test Laboratories also reported results of a 28-day subacute dermal toxicity study using Albino rabbits (Equitable Environ. Hlth., 1976). Hex solution was allowed to contact the shaved, unoccluded skin of rab- bits for an unlimited period of time (test material not washed off). The test animals were dosed 5 days a week for a period of 4 weeks, or 20 applications in all. The concentration of hex in Group I was 0.1 (percent weight/- volume); in Group II, the concentration was 0.5. None of the animals died and no pharmacotoxic symptoms were noted; however, both hex solutions were extremely irritating to the skin and slight losses in body weight occurred in some of the rabbits receiving the higher concentration (Group C-34 ------- II). No adverse effects were noted in hematological studies, clinical blood chemistry studies, and urinalysis. No signi- ficant gross or microscopic pathology was noted except the local skin lesions. Gross skin changes were characterized by fibrosis, escharosis (scarring), and slight-to-severe desquamation. Microscopic examination revealed acanthosis and hyperkeratosis involving the epidermis. This effect was seen in a few of the animals in Group I and most of the animals in Group II. Such findings were thought to be attributable to the irritant action of hex. c) Inhalation Exposures. Rats, rabbits, guinea pigs, and mice exposed to vapors of hex showed signs of extreme irritation of the eyes and mucous membranes (Treon, et al. 1955). At very high concentrations (46.5 ppm) animals re- sponded by rubbing their noses with their forefeet, closing their eyes and retracting their heads. This behavior was accompanied by sneezing, tearing, and irregular breathing. In less than 30-60 minutes the animals were gasping for breath. Lower concentration of hex vapor (12.4 and 13.8 ppm) produced similar irritation of the mucous membranes, al- though somewhat milder in degree. The same symptoms were even seen at the low dosages (1.0 and 1.6 ppm), but the symptoms developed over a period of hours rather than min- utes. Exposure to very low concentrations (0.33 ppm and 0.15 ppm) resulted in some irritation of the eyelids and increased respiratory rate. In the case of the latter dos- C-35 ------- age (0.15 ppm), irritation was seen only in the mice, which developed mild respiratory changes (Treon, et al. 1955). Rats which survived the vapor exposure sessions lost weight and many of these animals failed to regain their initial weights as long as 6 to 8 weeks after cessation of the expo- sures. At autopsy Treon, et al. (1955) reported degenerative changes similar to those described above (oral and dermal administration experiments) in all species of animals tested. Prolonged intermittent exposure to vapor concentrations as low as 0.15 ppm hex induced slight degenerative changes in the livers and kidneys in all species of animals employed. Industrial Bio-Test Laboratories (1ST) also conducted two vapor toxicity studies: an acute test and a 28-day subacute test. Results of these studies were reported by Equitable Environmental Health (1976). In the acute vapor toxicity tests, Charles River rats were exposed for 4 hours to varying concentrations of hex in air. An acute LD50 of 3.67 ppm was reported. Complete necropsies were performed at death for those which died shortly after exposure and at the termination of the study for those which survived. Acute pneumonia was observed in the rats which died follow- ing exposure to the test material. Treated rats which sur- vived to the end of the study showed emaciation and chronic proliferative inflammatory changes in the lungs. C-36 ------- The second Industrial Bio-Test Laboratories study con- sisted of a 28-day subacute vapor inhalation study using albino rats (Equitable Environ. Hlth., 1976). Two groups of 10 rats each were exposed to hex vapor for 6 hours per day, 5 days per week for 4 weeks. A third group of 10 rats served as untreated controls. One group was exposed to vapor containing 0.529 ppm hex and the other experimental group was exposed to 1.23 ppm. Hematologic and clinical chemistry studies and urinalysis were performed on days 0 and 28. On the twenty-eighth day the animals were sacri- ficed. No mention was made of mortality in either exposure group so presumably all animals survived until the time of sacrifice. Neither hematologic nor clinical studies or urinalysis revealed any abnormalities directly attributed to the hex vapor. Increases in absolute liver weight and liver-to-brain weight ratios were seen among male rats and increased liver-to-body weight ratios were seen for both sexes of rats exposed to 1.23 ppm. Gross pathological exami- nations failed to reveal abnormalities attributable to hex, but microscopic examination of tissue revealed hepatocyto- megaly and necrotizing hepatitis. These effects were thought to be attributable to hex exposure. Synergism and/or Antagonism There does not appear to be any information available on synergistic or antagonistic effects between hex and other compounds. C-37 ------- Teratogenicity International Research and Development Corporation (Int. Res. Dev. Corp., 1978) has recently completed a pilot tera- tology study using pregnant Charles River (CD) rats. Nega- tive findings with respect to teratogenic effects were re- ported for oral hex dosages up to 100 mg/kg/day. The test protocol employed in the pilot teratology study involved administration of various dosages of hex to 30 female Charles River (CD) rats approximately 12 weeks of age. Females were mated with male rats of the same strain. After mating, the females were assigned to six groups, one control and five treatment groups of five rats each. Hex was dissolved in corn oil and administered by gavage from day 6 through day 15 of gestation. Dosage levels of 3, 10, 30, 100, and 300 mg/kg/day were administered to the test groups and the control group was given the vehicle (corn oil) on a comparable regimen of 10 ml/kg/day. During gestation, the females were observed for clini- cal signs of toxicity, mortality, and body weight gains. They were then sacrificed on gestation day 20 and the ute- rine contents examined for viable and nonviable fetuses, early and late resorptions, and total implantations. There were no differences in the four treatment groups given 100 mg/kg/day or less when compared to the control group in terms of number of viable or nonviable fetuses, resorptions, implantations, or corpora lutea. Rats receiving doses of 3 or 10 mg/kg/day showed no treatment-related changes in C-38 ------- appearance or behavior. Rats receiving 30 mg/kg/day or higher showed staining of the anogenital area and reduced body weight gains. The females in the 100 mg/kg/day group had body weight losses during the first 3 days of treatment and reduced weight gains for the remainder of the study. Survival was 100 percent for all rats given 100 mg/kg/day or less. All rats in the 300 mg/kg/day group were dead by gestation day 10. Various reproductive parameters examined in the pilot teratology study are shown in Table 3. Mutagenicity Hex has. been tested for mutagenicity and reported non- mutagenic in both short-term _in vitro mutagenic assays (Na- tional Cancer Institute, 1977; Industrial Bio-Test Labora- tories, 1977; Litton Bionetics, 1978a) and in a mouse domi- nant lethal study (Litton Bionetics, 1978b). The National Cancer Institute (1977) reported that preli- minary results indicated that hex was non-mutagenic in Esche- richia coli K12 (mutation site not specified) in the pre- sence of a mammalian metabolic activation system containing mouse liver microsomes. Negative results were also reported by Industrial Bio- Test Laboratories (1977) using a test protocol almost iden- tical to the Ames Mutagenic Assay (Ames, et al. 1975). The tests used four strains of Salmonella typhimurium with and without metabolic activation. Hex was dissolved in acetone and added to the microbial assay plates in dosages C-39 ------- TABLE 3 Pilot Teratology Study in Rats: Cesarean Section Data For Individual Females (Int. Res. Dev. Corp., 1978b) Dosage Level Dam Number Control: 73662 77334 77336 77428 77428 Total Mean 3 mg/ kg/day: 73642 77342 77343 77426 77428 JPotal plean 10 mg/kg/day: 4K304 W309 77346 77427 77436 Total Mean 30 mg/kg/day: 77310 77313 77350 77438 77450 Total Mean 100 mg/kg/day: 73673 77302 77314 77415 K7439 "otal Mean 2flO mg/kg/day; Viable Fetuses 13 14 12 11 15 65 13.0 16 17 16 12 15 76 15.2 17 13 11 12 15 68 13.6 14 13 6 11 12 56 11.2 16 14 15 11 12 68 13.6 Nonviable Fetuses 0 0 0 0 0 0 0.0 0 0 0 0 0 0 0.0 0 0 0 0 0 0 0.0 0 0 0 0 0 0 0.0 0 0 0 0 0 0 0.0 Late Resorptions 0 0 0 0 0 0 0.0 0 0 0 0 0 0 0.0 0 0 0 0 0 0 0.0 0 0 0 0 0 0 0.0 0 0 0 0 0 0 0.0 Early Resorptions 1 1 1 1 0 4 0.8 0 0 0 1 0 1 0.2 0 0 3 0 0 3 0.6 0 1 0 0 0 1 0.2 0 0 2 0 0 2 0.4 Post Implantation Loss 1 1 1 1 0 4 0.8 0 0 0 1 0 1 0.2 0 0 3 0 0 3 0.6 0 1 0 0 0 1 0.2 0 0 2 0 0 2 0.4 Implan- tations 14 15 13 12 15 69 13.8 16 17 16 13 15 77 15.4 17 13 14 12 15 71 14.2 14 14 6 11 12 57 11.4 16 14 17 11 12 70 14.0 Corpora Lutes 14 15 13 22 16 80 16.0 16 17 16 18 15 82 16.4 18 13 14 13 15 73 14.6 14 16 7 14 14 65 13.0 16 14 17 11 12 70 14.0 r58 Died, 77324 Died, 77333 Died, 77417 Died, 77445 Died, gestation day 9 - gravid gestation day 10 - gravid ,gestation day 10 - gravid gestation day 10 - gravid gestation day 10 - gravid C-40 ------- from 10-5000 pg/10 pi. Concentrations greater than 10 jug/10 pi produced a bacteriocidal effect in three of the four strains tested; a possible lethal effect occurred at 2500 pg/10 pi or greater in the fourth strain. A repressive effect was noted in three of the four strains at concentra- tions below 10 pg/10 pi. Volitilate (volatile vapors) of hex was also tested on one strain using the vapor from hex concentrations of up to 2500 pg/10 pi and exposure times of up to 2 hours. Results from two successive assays in the absence of rat liver enzymes (hex concentrations 10, 25, 50, 75, and 100 pg/10 pi) were negative in all four tester strains. Two assays using the same dosages in the presence of rat liver microsomes were reported non-mutagenic; similarly, negative results were obtained for the hex effu- sate as well. The investigators expressed concern over the repressive effect of hex on the test bacteria, stating "It appears that hex is probably non-mutagnic and that some toxic effect prevailed with respect to the tester strains required for this assay. Analysis of variance and multiple comparison of the data confirms this observation". Litton Bionetics (1978a) conducted a mouse lymphoma cell assay in order to evaluate the capability of hex in inducing specific locus forward mutation. The indicator cells used in the assay were Fischer mouse lymphoma cells derived from cell line L5178Y. These cells are heterozygous for a specific autosomal mutation at the TK locus and are bromodeoxyuridine (BUdR) sensitive. Scoring for mutation C-41 ------- is based on selecting cells which have undergone forward mutation from a TK+/- to a TK-/- genotype by cloning them in soft agar with BUdR. Cells were maintained in Fischer's medium for leukemic mouse cells with 10 percent horse serum and sodium pyruvate. The dosages used in the test were predetermined by exposing the cells to a wide range of hex concentrations and measuring the reduction of growth poten- tial following a 4-hour exposure at each dose. The max- imum dose selected was that which produced a 50 percent reduction in growth. The actual hex dosages employed were: .00040 >jl/ml; .00048 >il/ml; .00056 jJl/ml; .00064 jul/ml; and .00125 jjl/ml in the activated series (mouse liver microsomes were added to the growth medium). A nonactivated series using somewhat lower dosages was included also. Both negative and positive controls were used; the negative control for both series was the solvent dimethyl- sulfoxide (DMSO), whereas ethylmethane-sulfonate (EMS) and dimethylnitrosamine (EMN) were used as positive controls in the nonactivated and activated systems, respectively. Hex was added to the cells in the growth medium for 4 hours. The cells were then washed and allowed to express in the growth medium for 3 days. After the expression period, results were evaluated by counting the TK-/- mutants after cloning the cells in a selection medium (soft agar with BUdR). C-42 ------- Hex dissolved in DMSO was evaluated over the concentra- tion range of 0.0000025 jjl/ml to 0.00125 jjl/ml. Consider- able toxicity occurred at concentrations greater than this and the extent varied according to the presence of the mouse liver activation system as shown in Table 4. No cells treated with hex (at the concentrations shown) survived in the non- activated system. Hexachlorocyclopentadiene did not induce forward muta- tion in L5178Y cells. The data presented in Table 4 show the concentrations of the test compound employed, the number of mutant clones obtained, surviving populations after the expression period, and calculated mutation frequencies. No dose-related trends in either absolute number of mutants or mutant frequencies were observed, and at no level did any of the test parameters increase significantly over the spontaneous level. Consequently, hex was considered to be nonmutagenic under the conditions of this assay. The mutagenic properties of hex were also evaluated in a dominant lethal study of mice (Litton Bionetics, 1978b). The dominant lethal assay provides a means of determining whether a compound is capable of inducing damage in the germ cells of treated male mice. Dominant lethality is manifested in various forms of fetal wastage, both pre-and post-implantation. Positive dominant lethal assays indicate that a compound is able to reach the developing germ cells. Chromosome aberrations including breaks, rearrangements, and deletions as well as ploidy changes and nondisjunction C-43 ------- n i A. B. C. NOTE: TEST TABLE 4 Summary of Mouse Lymphoma (L5^_gx) Results (Litton Bionetics, 1978a) Name or code designation of the test compound: Hexachloropentadiene Solvent: DMSO Test date: 12/18/77 Concentrations are given in microliters (UL) or micrograms (UG) or nanoliters (NL) per milliliter. Relative Relative Suspension Total Total Cloning Percent S-9 Daily Counts Growth (% Mutant Viable Efficiency Relative Source Tissue (Cells/ml x 10ES) of control) Clones Clones (% of control) Growth* NOHACTIVATION Solvent control Negative control EMS .5UL/ML ACTIVATION Solvent control Negative control DMN .5UL/ML Test compound 0.00002 UL/ML 0.00004 UL/ML 0.00008 UL/ML 0.00016 UL/ML 0.00032 UL/ML mouse mouse mouse mouse mouse mouse mouse mouse liver liver liver liver liver liver liver liver 1 16.8 13.2 9.0 15.2 14.2 7.2 16.8 13.0 12.4 13.6 18.2 2 10.2 12.0 9.2 9.6 13.0 7.6 9.0 12.4 9.8 13.8 9.0 3 13.8 15.0 11.8" 13.2 10.6 8.2 10.6 9.6 16.2 7.4 10.0 100.0 100.5 41.3 100.0 101.6 23.3 83.2 80.3 102.2 72.1 85.0 48.0 48.0 597.0 55.0 39.0 322.0 99.0 50.0 55.0 45.0 38.0 257.0 234.0 89.0 281.0 293.0 55.0 288.0 269.0 194.0 359.0 309.0 100.0 91.1 34.6 100.0 104.3 19.6 102.5 95.7 69.0 127.8 110.0 100.0 91.5 14.3 100.0 105.9 4.6 85.3 76.9 70.6 92.1 93.5 Mutant Frequency** (X 10E-6) 18.7 20.5 670.8 19.6 13.3 585.5 34.4 18.6 28.4 12.5 12.3 (Relative suspension growth X relative cloning efficiency: / 100 (Mutant clones / viable clones) X 10E-6 ------- are believed to produce positive results on this test. Since substances capable of producing gross chromosomal lesions are probably capable of producing more subtle bal- anced lesions or specific locus mutations, the test also provides suggestive evidence of nonlethal mutations transmis- sible to future generations as well. Litton Bionetics reported negative results, that is, there was no evidence of significant dominant -lethal acti- vity by hex in mice. The test protocol called for the assign- ment of ten random bred male mice to one of five groups. Three test groups received hex dosages of 1.0 mg/kg, 0.3 nig/kg, and 0.1 mg/kg, respectively. These dosages were determined by deriving an LD50 level (1.0 mg/kg) and taking one-third and one-tenth of that dose. A fourth group re- ceived only the solvent and the fifth group served as a positive control. Hex was mixed in the feed of the three experimental groups and the solvent control group for five consecutive days. The positive control group received a known mutagen, triethylenemelamine (TEM) in a single intra- peritoneal injection. Two days following treatment, each male was caged with two unexposed virgin females. At the end of seven days, these females were removed and replaced by two unexposed virgin females. This mating cycle was continued for seven weeks. Each pair of female mice was killed two weeks after mating and necropsied. Their uterine contents were examined for dead and living fetuses, resorp- tion sites, and total implantations. All test parameters C-45 ------- (fertility index, average implantations per pregnancy, aver- age resorptions (dead implants) per pregnancy, proportion of females with one or more dead implantations, proportion of females with two or more dead implantations, and the ratio of dead implantations to total implantations) were within normal limits based on historical and concurrent control levels for this test. Thus, there was no evidence of dominant lethal activity in any of the hex treated groups. The positive control group, however, did show the expected dominant lethal activity. Carcinogenici ty Various types of evidence may be used in evaluating the possible carcinogenic activity of a substance. In order of preference, these include: (1) human data; (2) animal data; (3) short-term (in vitro) tests; (4) metabolic pat- tern; and (5) structure-activity relationships. This section summarizes what is known about each of the above. No epidemiologic studies or case reports examining the relationships between exposure to hex and cancer incidences could be found in the literature. As indicated previously, Hooker Chemicals and Plastics Corporation reports that an in-house study of the mortality patterns of hex-exposed workers is now underway; however, the study is far from being completed (Zavon, 1978, personal communication). Other in-house studies of workers employed in the manufac- ture of pesticides (including hex) are reportedly being conducted by Velsicol Chemical Corporation. We were unable to obtain any further information on the current status or findings of these studies. C-46 ------- The National Cancer Institute concluded that toxicologic studies of hex in animals have not been adequate for evalua- tion of carcinogenicity (National Cancer Inst., 1977). Chronic toxicity studies as reported in section 4 Toxic Symptoms and Pathologic Effects, were based on too few ani- mals in some cases and/or the duration of the experiments was too short for appropriate evaluation of chronic effects, including carcinogencity. Only one short term in vitro test of hex for carcino- genic activity could be identified. Litton Bionetics (1977) reported the results of a test to determine whether hex could induce malignant transforma- tion in BALB/3T3 cells in vitro. The cells and methodology of the test were those of Dr. T. Kakunaga, described else- where (Int. Jour. Cancer 12:463, 1973). The basic rationale of the test and its validity as an indicator of carcinogenic activity was described by the investigators as follows: The endpoint of carcinogenic activity is deter- mined by the presence of fibroblastic-like colo- nies which are altered morphologically in com- parison to the cells observed in normal cultures. These (transformed) cells grow in criss-cross, randomly oriented fashion with overlapping at the periphery of the colony. The colony exhibits dense piling up of cells. On staining, the foci are deeply stained and the cells are basophilic in character and variable in size. These changes are not observed in normal cultures, which stain uniformly. Cell cultures with very little or no spontaneous trans- formation are maintained for use in these tests. The data generated at each dose level of the test material are ana- lyzed using the t statistic. A significant set of data C-47 ------- for any dose level may be sufficient to indicate a positive , response. Because this assay is still nonroutine, and defi- nitive criteria for evaluation have yet to be developed, scientific judgement and expert consultation are needed for appropriate interpretation of results. The BALB/3T3 cells used in the test were grown in Eagle's minimal essential medium (EMEM) supplemented by 10 percent fetal calf serum. Cultures were passaged weekly in 60 mm culture dishes. Approximately 10,000 cells were seeded into 50 ml sterile tissue culture flasks and incubated in EMEM to permit attachment. After the cells were attached, the control and test compounds were added to the plates. Dosages of 0.00001 pi/ml; 0.00002 jul/ml; 0.000039 jul/ml; 0.000078 jul/ml; and 0.000156 pi/ml of hex were employed. The maximal dosage, 0.000156 /il/ml, was determined by se- lecting from preliminary cytotoxicity tests the maximum dosage which permitted survival of at least 80 percent of the cells. 3-Methylcholanthrene at 5 pg/ml was used as a positive control and the test compound solvent was used as a negative control. Ten replicates per dose level were prepared and chemical exposure was maintained for 48 hours. Plates were then washed free of the compound and replenished with fresh growth medium. The plates were then incubated for an additional 3-4 weeks with twice weekly medium changes. Cell integrity was monitored by daily observations. Cells were separated from the medium, washed with saline, and stained. They were examined for stained foci; all poten- tial foci were examined microscopically. Results were pre- sented as the number of foci per set of replicate plates at each dosage level. C-48 ------- The test material was quite toxic to cells as indicated in the preliminary range-finding tests. No significant carcinogenic activity for hex was reported under the condi- tions of this test. A low level of spontaneous transforma- tion was observed on all of the plates. Only the 3-methyl- cholanthrene treated plates showed a significantly higher number of transformed foci than the negative control. It should be noted that in this and other cell culture tests, extremely low dosages of hex were used. Because hex is relatively toxic to cells in culture and test proto- cols normally require a high survival rate, the applicabi- lity of test results to environmental conditions is unclear. Taken together, however, the mutagenicity and carcinogeni- city tests conducted by Litton (1977, 1978a) suggest that outright toxicity, rather than chronic effects, is perhaps the critical effect of hex, even at very low dosages. Ex- tremely poor survival has also been problematic in several subchronic tests of hex in mammalian species. A very recent study involving chronic dietary exposure of rats to hexachlorobutadiene also provides some insight into the relationship between direct toxic effects and chro- nic effects (i.e., carcinogenesis) in this related compound (Kociba, et al. 1977). Male and female Sprague-Dawley rats were maintained on diets supplying 20, 2.0, 0.2, or 0 mg/kg/day of hexa- chlorobutadiene (HCBD) for up to 2 years. Rats ingesting 0.2 mg/kg/day had no discernible ill effects that could C-49 ------- be attributed to this dose level. Ingestion of the inter- mediate dose level of 2.0 mg/kg/day caused some degree of toxicity, affecting primarily the kidney in which increased renal tubular epithelial hyperplasia was noted. Urinary excretion of coproporphyrin was also increased at this dose (level. Ingestion of the highest dose level of 20 mg/kg/day (caused a greater degree of toxicity. Effects included de- creased body weight gain and length of survival, increased urinary excretion of coproporphyrin, increased weights of kidneys, and renal tubular adenomas and adenocarcinomas, some of which metastasized to the lung. In this study irre- versible toxicological effects, such as the development of neoplasms, occurred only at a dose level which caused significant tissue injury and other manifestations of toxi- city. No neoplasms resulted with dose levels which caused no injury or only mild, reversible injury. Little information is available on the metabolism of hex. Although at least four metabolites were found in the Mehendale (1977) study, at the time of this writing they had not been identified. Thus, the metabolic pathway is uncertain. As far as structure/activity relationships are concerned, the National Cancer Institute (1977) speculated that as a cyclopentene vinyl halide, hex potentially may be meta- bolized to an electrophile. In addition, hex is related to the pesticides dieldrin, heptachlor, and chlordane which have been found to induce liver tumors in mice following oral administration (National Cancer Inst., 1977). C-50 ------- Hex has recently been selected for testing in the Na- tional Cancer Institute's test program (National Cancer Inst., 1977). The reasons given for its selection include: (1) its high potential for exposure (as an industrial inter- mediate used in the manufacture of pesticides, flame retar- dants and dyes, Pharmaceuticals, resins, and germicides); (2) its suspect chemical structure; and (3) the relative lack of information on the effects of chronic exposure to this compound. Extremely limited data are available concerning the effects of hex exposure on humans. That which is known about acute human toxicity is based largely upon isolated spills or other accidental incidents involving pesticide workers, laboratory technicians, or others having occupa- tional contact with hex. A recent incident in which approxi- mately 200 sewage treatment plant workers were exposed to acutely toxic levels of hex from the illegal disposal of large quantities of the compound has done much to elucidate the potential health effects of acute human exposures. Due to the accidental and episodic nature of these incidents and the lag time in setting up environmental monitoring equipment in response to the incidents, it has not been possible to measure environmental concentrations of hex at the exact time workers report symptoms (post-exposure sampling results are sometimes available). Thus, while there is information regarding the range and variety of toxic responses, the exact dose which elicited a given re- sponse remains unknown. It is obvious that reliable dose- response estimates require accurate measurement of both dose and response parameters. C-51 ------- There is virtually no information regarding the health effects of chronic exposure to hex. Despite the fact that there are a number of potential cohorts for retrospective epidemiologic studies (see section 2), studies of chronically exposed workers have not been reported to date. Hooker Chemicals and Plastics Corporation, a manufacturer of hex, reports that they are presently conducting a mortality study of hex-exposed workers, but the study is in its initial stages and very likely would not be completed until 1980 (Zavon, 1978, personal communication). Likewise, virtually nothing is known regarding the poten- tial effects resulting from chronic exposure to environ- mental sources of hex. Potential modes of environmental exposure (e.g., through ingestion of contaminated air or water) are uncertain at this time. According to Hooker's material safety data sheet for hexachlorocyclopentadiene (Hooker Industrial Chemicals Divi- sion, 1972), the compound is very irritating to the eyes and mucous membranes causing lacrimation, sneezing, and salivation. Repeated contact with the skin can cause blis- tering burns, and inhalation can cause pulmonary edema. Hex is readily absorbed through the skin. Ingestion can cause nausea, vomiting, diarrhea, lethargy, and retarded respiration. Recommendations for safe use include: (1) good general ventiliation plus local exhaust at points of potential fume emission; (2) respiratory protection of the organic vapor-acid gas canister type and full-face self- C-52 ------- contained breathing apparatus for emergencies; (3) elbow length neoprene gloves; (4) eye protection including chemi- cal safety glasses, plus face shield where appropriate; (5) protective clothing including full length clothing fas- tened at neck and wrist, rubber safety shoes or boots, rubber or other impervious clothing or aprons as needed for splash protection. According to Treon et al. (1955), a very faint odor of hex was detectable in air by some individuals at concen- trations as low as 0.15 ppm which was the lowest concentra- tion employed in their experiments. At approximately twice that concentration (0.33 ppm), a very pronounced, pungent odor was present. Treon, et al. (1955) observed that headaches developed among laboratory workers following incidental exposure to hex vapor from the respiratory chambers used for their vapor inhalation experiments. The exact concentration of hex escaping into the laboratory from the opening of the respi- ratory chamber is unknown; however, the chamber was not opened until the contaminated air had been exhausted and the chamber flushed for some time with clean air. Thus, the ambient concentration producing headhaches among the laboratory workers was well below the dosages employed in the animal experiments. Because no mention is made of any other irritative symptoms (e.g., lacrimation, etc.), it seems reasonable to speculate that the concentration of hex present was somewhere in the range between 0.15 ppm- 1.0 ppm, above the detection threshold but below the level producing acute symptoms of irritation. C-53 ------- Irritant effects are elicited at a vapor concentration greater than that shown to produce chronic toxicity in ani- mals. Thus, Treon et al. (1955) concluded that the irri- tant effects of hex vapors are not sufficiently pronounced to serve as a warning that a hazardous level of hex vapor is present and/or that hazardous exposure is taking place. According to Naishstein and Lisovskaya (1965), hex may be detected by taste and smell at very low concentrations in water. They placed the threshold level for altering the organoleptic qualities of water at 0.0014-0.0010 mg/1. 4) Epidemiologic Studies. To date, the only well documented incident of the acute toxicity of hex to humans occurred at the Morris Forman Wastewater Treatment Plant (MFWTP) in Louisville, Kentucky. The problem apparently began about the middle of March, 1977, when an unknown chemi- cal, later identified as a mixture of hex and octachlorocyclo- pentene (C-58), began entering the Morris Forman sewage treatment facility. An exact date of initial appearance at the plant, and hence, the initial date of worker exposure is unknown. However, unusual odors became evident around March 17, 1977. The odor gradually intensified over the next 2 weeks. From March 25-28, an odoriferous, sticky material entered the plant and gummed the barscreens and grit collection systems in the primary treatment area. Attempts to dislodge the material with steam produced a blue gas which permeated the grit removal and sludge handling areas. Workers exposed C-54 ------- to this vapor complained of severe irritation of the eyes, nose, throat, and lungs (Carter, 1977b). Approximately 20 workers sought medical treatment for tracheobronchial irritation. These workers were treated in the local emer- gency room; none was hospitalized (Singal, 1978). A sample of the material from the Screen and Grit Build- ing was sent to the U.S. EPA Laboratory in Athens, Georgia, for analysis. The primary contaminants in the samples were identified as hexachlorocyclopentadiene (hex) and octachloro- cyclopentene (octa). (Octa is a waste by-product in the manufacture of hex whose toxicity is presently unknown). Table 5 shows the results of the analysis. Due to the ap- parent potential toxicity of hex (and the unknown toxic potential of octa), the sewage treatment plant was evacuated and closed on March 29, 1977. Thereafter, until the partial reopening in June, 1977, 105 million gallons per day of domestic and industrial wastes were diverted directly to the Ohio River. Estimates of the extent of contamination indicate that about 60 million gallons (25,000 tons) of hex-contaminated material were present at the Morris Forman plant. Of this, approximately 6 tons of hex and octa were thought to be present in the contaminated waste. U.S. EPA's analysis revealed hex concentrations up to 1000 ppm in the sewage water at the time of the plant closure. The route of chemi- cal contamination was traced to one large sewer line which passed through several heavily populated areas. Wastewater C-55 ------- TABLE 5 Analysis of a Sludge Sample Obtained in the Screen and Grit Building on April 2, 1977, Morris Forman Wastewater Treatment Plant, Louisville, Kentucky (Singal, 1978) COMPOUND' Concentration - % by weight Octachlorocyclopentene Hexachlorocyclopentadiene Hexachlorobenzene Pentachlorobenzene Octachloronaphthalene Heptachloronaphthalene Hexachloronaphthalene Mirex 9 4 0. 0. 0. 0. 0.007 (estimated) (estimated) (not quanti- tated) (estimated) Analysis was conducted by the U.S. Food and Drug Administration, Division of Chemical Technology, Chemical Industry Practices Branch. 2 The sample was analyzed using gas chromatography interphased with mass spectroscopy for positive identification of each compound. C-56 ------- in this sewer showed hex and octa in concentrations ranging up to 100 ppm. Samples from the sewer showed air concen- trations ranging up to 0.4 mg/1 for hex and up to 0.03 mg/1 of octa. Thus, it was decided to investigate the health of not only the workers at the sewage treatment plant but also residents of the area surrounding the sewer line (Morse, et al. 1978). A cooperative investigation involving Region IV U.S. EPA (Surveillance and Analysis Division), Center for Di- sease Control (CDC), National Institute for Occupational Safety and Health (NIOSH), Jefferson County (Kentucky) Health Department, and the Kentucky State Health Department was initiated. Information on both aspects of the investigation (i.e., community residents on one hand and exposed workers on the other) is thus far unpublished but preliminary drafts of reports were made available by Dale Morse, M.D., who headed the initial epidemiologic studies conducted by the Center for Disease Control (Morse, et al. 1978) and by Mitchell Singal of the Hazard Evaluation and Technical Assistance Branch of NIOSH who reported on the follow-up investigations of workers during cleanup operations at the sewage treatment facility (Singal, 1978, personal communication). Findings from these drafts are reported below; however, they should be regarded as preliminary. C-57 ------- a) Plant Employee Health Effects Evaluation. i) Ini- tial studies. The Center for Disease Control investigation began by identifying all sewage treatment employees who worked at the plant for 2 or more days during the period from March 14-29, 1977. Health effects evaluations, includ- ing mailed questionnaires, physical examination, and blood and urine testing, were conducted appropriately to exposed individuals who agreed to participate. The questionnaire covered demographic information, a detailed work-area his- tory, symptoms and history of chemical poisoning, personal habits, and other sources of chemical exposure. Routine tests were performed on blood and urine speciments. Addi- tional samples were sent to NIOSH laboratories for potential toxic chemical analysis. Of 193 plant employees who had worked during the latter half of March, questionnaire data was obtained from 145. Seventy-five percent of the questionnaire respondents indi- cated that they detected an unusual odor at the plant some- time during March. A few individuals reported detecting unusual odors as early as March 1, 1977; the percentage reporting the odor by March 14 was noticeably increased. From March 15 onward, the percentage of workers who reported noticing the odor steadily increased until the plant was closed on March 29. A comparison between the time of odor detection and the onset of eye irritation, the most common symptom, showed that irritation developed on the same day in 45 percent of individuals, within 1-5 days in 28 percent, and after 5 days in 21 percent. Only 6 percent of employees reported onset of symptoms prior to noticing an unusual odor at the plant. C-58 ------- Eye irritation, headache, and throat irritation were the most common symptoms, with 59 percent, 45 percent, and 27 percent of employees reporting these symptoms, respec- tively. Data for these and other symptoms is reported in Table 6. Of 41 workers physically examined, five had signs of eye irritation (tearing and/or redness) and five had signs of skin irritation. Forty-two persons were interviewed and provided blood and urine samples. This included 24 of 29 (83 percent) of the workers who had been previously evaluated by local physicians, 17 of 164 other plant employees (a 10 percent random sample) as well as one non-employee accidentally exposed to the contamined sludge. Abnormalities were found in laboratory analysis of some of the workers (e.g., LDH elevations in 27 percent and proteinuria in 15 percent of those examined). These results are suggestive of either a transitory abnormality or a problem with the laboratory analysis. No LDH or urin- alysis abnormalities were corroborated on repeat tests run 3 weeks later by another laboratory. Also, no abnormalities were reported among individuals seen at the local hospital or by the plant physician. Analysis of data according to employee work areas revealed that symptoms occurred in workers of all job categories and in all work areas. Data for attack rates in employees by main work area is reported in Table 7. Only small differ- ences in case rates appeared by work area although the highest C-59 ------- TABLE 6 n i o\ o Symptoms of 145 Plant Employees, Louisville, Kentucky, March, 1977 (Morse, et al. 1978) Percent Symptom With Symptom With Symptom Eye irritation Headache Throat irritation Nausea Skin irritation Cough Chest pain Difficult breathing Nervousness Abdominal cramps Decreased appetite Decreased memory Increased saliva 86 65 39 31 29 28 28 23 21 17 13 6 6 59 45 27 21 20 19 19 16 14 12 9 4 4 ------- TABLE 7 Attack Rates in Employees, by Louisville, Kentucky, March, 1977 Main Work Area, (Morse, et al. 1978) o i a\ Main Work Area Primary treatment Throughout plant Vacuum filtration Secondary aeration chamber Administration and laboratory Final effluent pump station Low pressure oxidation I ncineration Number of Employees 19 71 19 14 30 10 13 17 Number Reporting Symptoms 17 54 15 12 22 5 10 10 Percentage of Employees Re- porting Symptoms 89 76 79 86 73 50 77 50 Percentage of Cases of Those Reporting Symptoms 59 48 47 42 41 40 30 20 Totals 193 145 75 44 ------- attack rates occurred in workers in the primary treatment area where the level of hex was presumably highest. Attack rates were significantly higher by X test for individuals who had been exposed to the screen and grit chamber (p = .0001) and to the primary settling area (p = .02) than for workers not exposed to these areas. The initial investigation demonstrated that 64 of 145 (44 percent) of current employees questioned at the waste- water treatment plant had experienced headache and mucous membrane, skin, and respiratory tract irritation after expo- sure to airborne hex. Highest attack rates occurred among workers in the primary treatment area where exposure was highest and ventilation poorest. In most cases symptoms were transient, but in some workers, they persisted for several days. This episode clearly demonstrates the volati- lity of hex and its potential for having a toxic effect on humans. Results of the follow-up investigation of the sewage treatment plant workers and the community survey are reported below. ii) Follow-up Survey. After the initial health evalua- tion survey was completed (April 3, 1977), NIOSH assumed the responsibility for follow-up of the sewage treatment workers exposed during the March, 1977, episode. NIOSH was also responsible for medical monitoring of those involved in the cleanup operations prior to reopening the Morris Forman plant. NIOSH1s activities consisted of the following: (1) administering follow-up questionnaires to all plant C-62 ------- employees to determine how persistent symptoms had been after the initial chemical exposure in March; (2) review of the medical records of the 90 employees who had seen the plant physician from late March through May 10, 1977; (3) collection of repeat biologic samples on the 23 employees who had shown some abnormality on the testing done by the CDC physicians (March 31-April 2, 1977, tests); (4) biolo- gical monitoring of EPA and NIOSH industrial hygienists and environmental technicians exposed to the chemicals in the sewer system during cleanup; and (5) medical monitoring of Morris Forman plant employees who were actively involved in the plant cleanup. Results of each of these aspects of the investigation are reported below. Usable responses were obtained from 182 individuals on the follow-up questionnaire. The frequency of symptoms among those who completed the questionnaire is shown in Table 8. In decreasing order of frequency, these symptoms included eye irritation, headache, fatigue, chest discomfort, sore throat, cough, nausea, and skin rash. These symptoms were surprisingly persistent. Except for eye irritation and sore throat, 25-45 percent of those who exhibited symp- toms during the last 2 weeks of March, 1977, still had them 6 weeks later. Although symptoms occurred in workers in all areas of the plant, maintenance department personnel consistently reported the highest number of symptoms. C-63 ------- TABLE 8 Symptoms Reported on Follow-up Questionnaire,* Morris Forman Wastewater Treatment Plant, Louisville, Kentucky (Singal, 1978) Symptom % with symptoms Persistence of Symptoms** (% of those c symptoms) Headache Eye Irritation Sore Throat Cough o OA Chest Discomfort Skin Irritation Nausea Fatigue in last 2 weeks of March** 55% 62% 30% 24% 34% 21% 22% 34% Gone within 1 day 19% 36% 15% 14% 11% 18% 18% 8% Gone within 1 Week 30% 23% 49% 27% 20% 18% 23% 16% Gone within 2 Weeks 18% 16% 13% 16% 21% 10% 18% 24% Still at time 32% 15% 18% 36% 39% 46% 25% 45% present of survey *Distributed and Collected last 2 weeks of May 1977 Excludes employees actively involved in cleanup, since their symptoms could relate to exposure during cleanup instead of to exposure prior to the plant shutdown. **% Persistences do not quite add to 100% due to some employee confusion about the need to fill in questionnaire completely. ------- A review of medical records of the 90 workers examined by the plant physician (mid-March to May 10, .1977) revealed symptom reports similar to those reported on the NIOSH and CDC questionnaires. Fatigue, headache, and mucous membrane irritation were the predominant complaints; respiratory and skin problems were also reported. Seven of the 90 workers reported transient memory loss ranging from a few minutes to a few days. These are believed to represent a transient state of confusion, rather than true amnesia (Singal, 1978, personal communication). Although several workers reported neurologic symptoms, the plant physician found no one with any objective neurologic signs. Seven persons had rash on exposed areas of face and arms. Respiratory tract symp- toms, cough, and chest discomfort were commonly reported. Twenty-eight persons, including those with respiratory symp- toms, received chest x-rays. Essentially all of the x-rays were normal. Sixteen persons received blood gas determina- tions, none of which showed an elevated pCO^ or a pC^ below 70 mmHg. Pulmonary function tests were done on 22 indivi- duals but no significant pattern of abnormalities was seen. Cholinesterase levels on 27 workers were negative. Several workers had elevated liver function tests; these were mainly minor elevations of lactic dehydrogenase (LDH) and alkaline phosphatase which are difficult to interpret. More specific liver function tests such as serum glutamic oxalacetic trana- minase (SCOT) and serum glutamic pyruvic transaminase (SGPT) were elevated in three persons. Six elevations of bilrubin, C-65 ------- two elevations of serum creatinine, and six mild proteinurias were detected. Unfortunately/ the specimens were analyzed by at least three different laboratories and comparison/inter- pretation of these results is uncertain. Attempts to develop a technique to isolate and identify concentrations of hex in specimens of blood or urine at the time of the investiga- tion were unsuccessful (Morse, et al. 1978). Biological monitoring of NIOSH and U.S. EPA personnel who were actively involved in the cleanup effort showed no significant abnormalities. Repeat laboratory tests were done on 20 of the 23 sewage treatment plant workers who had abnormalities on the blood and/or urine tests at the time of plant shutdown. Three of these people continued to have persistent abnormalities in liver function tests on one or more occasions but there were no persistent urinary abnormalities. Exposure levels of the cleanup crew were monitored by taking samples of breathing zone concentrations (inside masks) of hex and octa. These values are reported in Tables 9 and 10. Biological monitoring of the cleanup crew was also car- ried out by NIOSH. Due to continuous turnover of crew mem- bers, it was not possible to obtain pre-exposure baseline studies on more than 54 percent of the workers. Symptoms reported by crew members were similar to those reported on the NIOSH and CDC questionnaire surveys of the plant employees in March. Headache and eye irritation were the C-66 ------- TABLE 9 ms of Hexachlorocyclopentadiene (HCCPD) and i the Grit Loading and Screen and Grit Buildings ater Treatment Plant, Louisville, Kentucky, 1977 igal, 1978) Sample Volume Airborne ling 57 - 59 - 28 - 44 - 36 - 43 - D5 - 08 - 43 - D6 - 97 - 55 - 45 - 46 - 45 - 35 - 30 - 30 - 36 - 35 - 30 - 17 - 50 - 33 - 49 - 18 - L7 - 46 - 58 - Period 1322 1533 1919 2222 1907 2220 0905 1120 1519 0909 1120 1515 1803 1805 2253 0119 0440 0740 0120 0441 0741 0857 1112 1359 0851 1113 1405 1715 2234 liters 6 12 16 13 10 4 10 12 14 14 8 12 7 HCCPD 1.5 3 7 0.7 0.5 2.3 8 ' 6 1 5 7 0.8 1.4 10 2 Concentration - ppb OCCP 2.4 1.2 0.9 1.1 0.8 3.7 2 4 1.0 1.1 1.9 1.2 2.2 None Established Loading Building and PBZ-SGB denotes personal breathing zone sampled by volume. suit in the breathing zone of the worker. ------- TABLE 10 Personal Breathing Zone Concentrations of Hexachlorocyclopentadiene (HCCPD) and Octachlorocyclopentene (OCCP) Measured Inside the Protective Suits Worn by Persons Involved with the High Pressure Water Washdown of the Screen and Grit Building, Morris Forman Wastewater Treatment Plant, Louisville, Kentucky, 1977 (Singal, 1978) o 1 o> 00 Sample Date 4-22 4-22 4-22 4-23 4-23 4-23 4-25 4-25 4-25 Environmental Sample No. CR-058 CR-059 CR-060 CR-078 CR-079 CR-080 CR-081 CR-082 CR-083 Criteria Sampling Period 0945 - 1225 - 0946 - 1225 - 0947 - 1226 - 0850 - 1248 - 0851 - 1253 - 1045 - 1252 - 1245 - 1438 - 1308 - 1438 - 1246 - 1117 1534 1122 1540 1120 1540 1145 1600 1145 1601 1145 1604 1401 1534 1405 1537 1402 Sample Volume liters 12 15 13 18 9 8 32 23 29 Airborne Concentrations - ppb HCCPD 0.8 0.6 0.7 0.5 1.0 1.0 0.3 0.4 0.3 10 OCCP 4 0.9 1.1 0.8 1.8 1.4 0.4 0.6 0.5 None Established "Parts of contaminant per billion parts of contaminated air sampled by volume. ------- predominant symptoms; sore throat, fatigue, nausea, dizzi- ness, chest discomfort, cough, and skin irritation were also reported. Physical examinations on cleanup crew members were unremarkable except for conjunctival irritation in workers wearing half-face respirators. Of 97 crew members tested, 18 (19 percent) showed some elevation on one or more of the five occasions testing was done. These elevations were generally small (see Table 11), but once they appeared, they tended to persist over several weeks (Table 12). A small number of abnormalities appeared on renal function tests but generally these were small and non-reproducible on serial testing. Likewise, abnormalities in complete blood counts were also minor and non-reproducible. It should be noted that the laboratory results on cleanup workers are difficult to interpret due to lack- of adequate controls. Essentially all of the plant employees, including many of the cleanup workers, had been exposed in March prior to the plant shutdown. As indicated, there were no environ- mental samples taken at the time of the acute exposure episode. Although exposure levels of the cleanup workers were well below the current occupational standard for hex (0.01 ppm) , one cannot rule out the possibility that abnormalities among the cleanup crew are reflective of earlier, unspecified exposures. Interpreting the significance of variations in liver function tests of the magnitude seen in this group of workers is difficult. First, many of the abnormalities C-69 ------- TABLE 11 Abnormalities in Lab Tests on Cleanup Workers, Morris Forman Wastewater Treatment Plant, Louisville, Kentucky (Singal, 1978) Lab Test Ranges of values Number of Persons Normal Results in Range Range 1 SCOT - (serum glutamate- oxalacetic acid transaminase) 2 Serum alkaline phosphatase 3 Serum total Bilirubin 4 Serum LDSH (lactate dehydrogenase) 5 Serum creatinine 40-49 50-59 60-69 70-79 80-89 90-99 100-109 110-119 120-129 1.0-1.9 230-239 1.3-1.9 5 1 4 0 1 1 3 1 1 7-40 mp/ml 30-100 mp/ml 0.15-1.0 mg% 100-225 mjj/ml 0.5-1.3 mg/dl C-70 ------- TABLE 12 Liver Function Abnormalities in Cleanup Workers, Morris Forman Wastewater Treatment Plant, Louisville, Kentucky (Singal, 1978) Patient No. 4/8 4/12 4/20 Date of Visit 5/5 Did 5/19 Hours spent Lab Abnormality in cleanup result in removal from cleanup? 1 2 3 4 ? 5 H 6 7 8 9 10 11 12 13 14 15 16 17 18 SCOT 461,, Bili 0.9 SCOT 48 SCOT 35 Bili ., 1.6 Alk phos 117 SCOT 47 SCOT 66 LDH4 239 Bili 1.4 SCOT SCOT Alk phos Alk phos LDH SCOT 31 43 105 88 232 59 SCOT 51 SCOT SCOT SCOT SCOT 44 Alk phos 96 Alk phos LDH SCOT SCOT 63 31 52 103 159 42 54 SCOT Alk phos SCOT Alk phos SCOT SCOT Alk phos Alk phos SCOT SCOT 45 100 42 113 60 39 120 101 87 46 SCOT SCOT SCOT Alk phos SCOT SCOT SCOT 43 63. 39 129 93 47 48 40 56 115 150 11 100 5 80 110 40 80 60 80 15 32 108 40 140 Yes Yes Yes Yes No Yes No No Yes Yes No No Yes Yes No Yes No Yes 1 SCOT = Serum glutamate-oxaloacetate transterase in mJJ/ml - Normal range = 7-40 mJU/ml 2 Bili = Total serum bilirubin in mg% - Normal range = 0.15-1.0 mg% 3 Alk phos - Serum Alkaline phosphatase in mJJ/ml - Normal range = 30-100 mJU/ml 4 LDH = Serum Lactate dehydrogenase in mjU/ml - Normal range = 100-224 mJJ/ml ------- seen are relatively nonspecific, that is such changes may be caused by a variety of conditions and thus are not neces- sarily attributable to exposure. Second, there is little consensus concerning what constitutes the normal range in some of these tests. Despite these problems in analysis, Dr. Singal expressed the opinion that these data suggest that exposure to the mixture of chemicals contaminating the sewage treatment plant may be associated with some mild liver injury (Singal, 1978). iii) Community Survey. CDC workers administered a ques- tionnaire to a systematically selected sample of residents in a 48-block area surrounding the contaminated sewer line (Morse, et al. 1978). One home per block was surveyed by administering a questionnaire to the head of each household. In all, 212 occupants of the 48-block area were surveyed. Questions were asked concerning basic demographic data, history of unusual odors, and any symptoms noted by household members within the past 2 weeks. Results of the community survey were essentially negative. Eight of the 212 persons (3.8 percent) reported noticing an unusual odor at some time during the preceding 2 weeks. While some of the respondents reported symptoms compatible with hex exposure (headache, 4.7 percent; burning or watering eyes, 4.7 percent), no symptom occurred at greater than background rates. Symptoms not associated with hex were reported just as frequently as those possibly related to exposure. Furthermore, there was no association between symptom rates and distance from the sewer line. Subsequent air sampling failed to show a significant ambient concentra- tion of hex in the sewer line area. C-72 ------- CRITERION FORMULATION Existing Guidelines and Standards The Occupational Safety and Health Administration (OSHA) has not set a standard for occupational exposure to hex. On the other hand, the American Conference of Governmental Industrial Hygienists (ACGIH) has adopted both a threshold limit value (TLV) and a Short Term Exposure Limit (STEL) for hexachlorocyclopentadiene. The current occupational TLV for hex is set at 0.01 ppm (0.11 mg/m ), which, according to ACGIH "represents a time-weighted average concentration for a normal 8-hour workday or 40-hour workweek to which nearly all workers may be repeatedly exposed, day after day, without adverse effect" (Am. Conf. Govt. Ind. Hyg., 1977). The Short Term Exposure Limit (STEL) for hex is set at 0.03 ppm (0.33 mg/m ). This level represents the maximal concentration to which workers can be exposed for a period up to 15 minutes without suffering from irrita- tion; chronic or irreversible tissue damage; or narcosis of sufficient degree to increase accident proneness, impair self-rescue, or materially reduce work efficiency. The STEL should be considered a maximum allowable concentration or absolute ceiling not to be exceeded at any time in the 15 minutes. Up to four excursions up to the STEL are permit- ted per day provided that at least 60 minutes between excur- sions up to the STEL (Am. Conf. Govt. Ind. Hyg., 1977). C-73 ------- In selecting the TLV and STEL values for hex, the ACGIH emphasizes that these particular levels were selected on the basis of preventing irritant effects rather than chronic toxicity. The U.S.S.R. has recommended a tenfold lower limit (0.001 ppm) for occupational exposures. No nonoccupational exposure limits have been established or recommended except for one Soviet study which proposed a maximum concentration of 0.001 mg/1 in water to prevent "organoleptic effects" (i.e., adverse effects on the taste and odor of water). There is a serious lack of data to support nonoccupational exposure limits or environmental criteria for hex. Specifically lacking are: (1) epidemio- logic studies of individuals having known and quantifiable hex exposures; (2) long-term animal studies (e.g., 2-year chronic feeding studies) suitable for evaluating chronic effects, especially carcinogenicity; (3) data on current levels of human exposure from various media; and (4) suit- able methods for interpreting the significance of in vitro assays and their applicability to actual environmental condi- tions. Without these essential data it is not possible to use the model proposed by U.S. EPA's Carcinogen Assessment Group (CAG) to derive recommended exposure criteria for humans. In fact, the CAG states that "there is insufficient evidence to categorize this compound as a carcinogen or non-carcinogen." Consequently, other toxic endpoints must form the basis for recommended exposure criteria until a more adequate information base on hex is developed. C-74 ------- Special Groups at Risk As indicated earlier, it is presently unknown whether ingestion or inhalation of hex (through ingestion of hex- contaminated food, water, or air) constitute significant sources of exposure among the general population. Although it is not likely this is the case, present data on the envi- ronmental occurrence of hex are so sketchy that this possi- bility cannot be ruled out. Occupational exposures appear to constitute the only documented source of human exposure to hex. Oral contact does not appear to be a likely mode of occupational exposure. However, dermal and inhalation exposures are recognized hazards for the following groups: (1) workers engaged di- rectly in hex manufacture; (2) those engaged in the formula- tion and use of other, related pesticides where hex may be present as an impurity; (3) flame retardant workers; (4) those having "quasi-occupational" exposures such as sewage treatment workers, industrial hygienists, etc. Basis and Derivation of Criterion Notwithstanding the obvious data deficiencies, some tentative recommendations can be made in consideration of the levels of hex which produce chronic toxicity in labora- tory experiments. As indicated earlier, there are no epidemiologic studies nor suitable chronic toxicity studies in mammals from which threshold levels for chronic effects could be derived. Very little is known regarding potential hex exposures through C-75 ------- ingestion of contaminated food or water. In the environment hex has been detected only in specific bodies of water near points of industrial discharges. There are no data on hex levels in drinking or untreated water. Based on the available and cited literature, there is insufficient evidence to categorize this compound as a carci- nogen or non-carcinogen. There has not been a satisfactory study of the effects of chronic oral exposure to hex. A single study of chronic oral toxicity reported by Naishstein and Lisovskaya (1965). The test consisted of only one spe- cies (rats) and the duration of exposure was only six months. No neoplasms were reported, however the duration of the study would not have been sufficient for a proper evaluation of carcinogenicity. Hex has been tested for mutagenicity and reported non- mutagenic in both short-term in vitro mutagenic assays (NCI, 1977, IBT, 1977 and Litton Bionetics, 1978a) and in a mouse dominant lethal study (Litton Bionetics, 1978b). No epidemi- ologic studies or case reports examining the relationship between exposure to hex and cancer incidences could be found in the literature. Therefore, there is virtually no informa- tion regarding the carcinogenic potential of chronic exposure to hex. In selecting hex for future chronic toxicity testing, National Cancer Institute (1977) recognized these data voids. f Although one study (Treon, et al. 1955) reported on the effects of chronic low-dose inhalation of hex, its appli- cability in deriving water quality guidelines is unclear. C-76 ------- Furthermore, with the exception of very limited data on hex in water near points of discharge, there appears to be no information on hex levels in water bodies. What is needed is a method for converting the results of respiratory exposure experiments into equivalent dosages from water. Stokinger and Woodward (1958) describe a model by which the threshold limit values (TLV's) for industrial substances in air may be used in establishing drinking water standards. The model assumes that, for any given inhaled dose, an equiva- lent ingested dose from ingested water can be derived using reasonable estimates of daily air and water intakes and corresponding respiratory and gastrointestinal absorption rates. In the absence of suitable chronic ingestion studies of hex, the Stokinger and Woodward (1958) model will be used to estimate suitable limits for hex in water based on the established threshold limit value expressed as milli- grams per cubic meter of air. The threshold limit of 0.11 mg/m (0.01 ppm) hex repre- sents what is believed to be a maximal concentration to which a worker may be exposed for 8 hours per day, 5 days per week over his working lifetime without hazard to health or well-being (Amer. Conf. Gov1t. Ind. Hyg. , 1977). To the TLV, Stokinger and Woodward apply terms expressing respi- ratory volume during an 8-hour period (assumed to be 10 m ) and a respiratory absorption coefficient appropriate to the substance under consideration. As in the case of hex where absorption rates are unknown, 100 percent absorp- C-77 ------- tion is assumed. In addition, the 5-day-per-week occupa- tional exposure is often converted to a 7-day-per-week equiva- lent in keeping with the more continuous pattern of exposure to drinking water. According to the model, the amount of hex that may be taken into the bloodstream and presumed to be noninjurious and which, hence, may be taken in water each day is: 0.11 mg/m3 X 10 m3 X 1.0 X 5/7 week = 0.7857 mg/day (TLV) Respiratory Respiratory Proportion Maximum Intake Absorption of week Noninjurious Term Coefficient Exposed Intake To calculate the equivalent amount of hex in ambient water, the model assumes a maximal daily intake of 2 liters of water per day, the consumption of 18.7 grams of fish/shell- fish per day, a bioconcentration factor of 3.2 for fish and 100 percent absorption. (X) x (2 + 3.2(0.0187)) x 1.0 = 0.7857 Upper Oral Gastrointestinal Maximum Intake Intake Absorption Noninjurious Limit — Term Coefficient Intake Solving for X, the value derived is 0.38 mg/1 or 380 jug/1. According to Stokinger and Woodward (1958), "This derived value represents an approximate limiting concentration for a healthy adult population; it is only a first approximation in the development of a tentative drinking water criterion.... several adjustments in this value may be necessary...Other factors, such as taste, odor and color may outweigh health considerations because acceptable limits for these may be below the estimated health limit." C-78 ------- It should also be noted that the basis for the above recommended limit, the TLV for hex, is set on the basis of avoidance of irritation, rather than chronic effects (Am. Conf. Govt. Ind. Hyg., 1977). Should chronic effects data become available, both TLV's and recommendations based on them will warrant reconsideration. A single study of chronic oral toxicity in white rats reported no adverse effects (specifically changes in peri- pheral blood cells, ascorbic acid content of the adrenals, conditioned reflexes of the animals, or histological struc- ture of the organs) following daily oral administration of doses up to 4 jag/1 of hex in aqueous solution (Naishstein and Lisovskaya, 1965). Animals receiving the highest dosage, 40 jag/1, showed neutropenia and lympho-cytosis which the investigators thought possibly attributable to mobilization of the protective forces of the organism in response to this dose. Such findings imply adverse effects at levels as low as 10 percent of the tentative drinking water standard based on the Stokinger and Woodward (1958) model. Naishstein and Lisovskaya (1965) found that hex in concen- trations of 1.4 to 1.6 pg/1 in water is capable of altering the smell and taste of water. Based on these organoleptic effects, these investigators proposed a maximum permissible concentration of 1 /ag/1. Stokinger and Woodward (1975) themselves noted that oftentimes "other factors, including taste, odor and color may outweigh health considerations because acceptable limits for these may be well below the estimated health limit." C-79 ------- Because chronic effects in a mammalian species (rats) have been documented at water concentrations of hex as low as 40 jag/1, it is obvious that an acceptable water quality criterion should be well below this level. Thus, a reason- able safety factor of 10 to 100 applied to 40 ;ig/l would place an appropriate criterion recommendation in the range of 4.0 - 0.4 jag/1 in water. The level recommended by Naish- stein and Lisovskaya (1965) based on smell and aftertaste falls well within this range. No adverse effects on humans or mammals have been reported to be caused by hex concentrations lower than approximately 1.0 jug/1. Therefore, based on avoidance of alteration in smell and aftertaste in water, a criterion of 1.0 jig/1 of hex in water is tentatively suggested. This level should be adequate for protection of public health. It is to be stressed that this criterion is based on inadequate chronic effects data and should be re-evaluated upon completion of chronic oral toxicity studies. C-80 ------- REFERENCES American Conference of Governmental Industrial Hygienists. 1977, TLV's threshold limit values for chemical substances and physical agents in the workroom environment with intended changes for 1977. Cincinnati, Ohio. Ames, B.N., et al. 1975. Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutageni- city test. Mutat. Res. 31: 347. Bell, M.A., et al. 1978. Review of the environmental effects of pollutants XI. Hexachlorocyclopentadiene. Unpub- lished report by Battelle Columbus Lab. for U.S. EPA Health Effects Res. Lab. Cincinnati, Ohio. Bennett, T.B. 1977. Legal affidavit filed in State of Georgia, Clarke Co., June 15, .1977, concerning sampling of discharge at Velsicol Chemical Corp. Memphis plant. Bevenue, A., and A.Y. Yeo. 1969. Gas chromatographic characteristics of chlordane. II. Observed compositional changes of the pesticide in aqueous and non-aqueous environments. Jour. Chromatog. 42: 45. C-81 ------- Carter, M.R. 1977a. Legal affidavit filed in State of Georgia, Fulton Co. dated June 14, 1977. Testimony concerning estimates of total daily discharge of hex from Velsicol Chemical Corp. Memphis plant and calculations of estimated hex concentration in Mississippi River resulting from said discharge. Carter, M.R. 1977b. The Louisville incident. Internal report (unpublished), Surveillance and Analysis Division, Region IV, U.S. EPA, Athens, Ga. Cordle, F., et al. 1978. Human exposure to polychlorinated biphenyls and polybrominated biphenyls. Environ. Health Perspect. 24: 157. Equitable Environmental Health, Inc. 1976. Literature review of the health and ecological effects of exposure to C-56 (Hexachlorocyclopentadiene). Unpublished rep. prepared for Hooker Chemical and Plastics Corp. Harris, C.R. 1972. Behavior of Dieldrin in soil: Laboratory studies on the factors influencing biological activity. Jour. Econ. Entomol. 65: 8. Hooker Industrial Chemicals Division. 1972. Material safety data sheet: Hexachlorocyclopentadiene. Unpublished internal memo dated April, 1972. C-82 ------- Industrial Bio-Test Laboratories, Inc. 1977. Mutagenicity of PCL-HEX incorporated in the test medium tested against five strains of Salmonella typhimurium and as a volatilate against tester strain TA-100. Unpublished report submitted to Velsicol Chemical Corp. Ingle, L. . 1953. The toxicity of chlordane vapor. Science 118: 213. International Research and Development Corp. 1972. Acute toxicity studies in rates and rabbits. Unpublished report prepared for Velsicol Chemical Corp. International Research and Development Corp. 1978. Pilot teratology study in fats. Unpublished report submitted to Velsicol Chemical Corp. Kakunaga, T. 1973. A quantitative system for assay of malignant transformation by chemical carcinogens using a clone derived from BALB/3T3. Int. Jour. Cancer 12: 463. Kirk-Othmer Encyclopedia of chemical technology. 2nd ed. 1964. Interscience Publishers, New York. Kociba, R.J., et al. 1977. Results of a two year chronic toxicity study with hexachlorobuadience in rats. Toxicol. Appl. .Pharmacol. 41: 204. C-83 ------- Konunineni, C. 1978. Internal memo dated February 14, 1978, titled, Pathology report on rats exposed to hexachlorocylo- pentadiene. U.S. Dep. of Health Ed. Welfare, Pub. Health Serv. Center for Dis. Control, Natl. Inst. for Occup. Safety and Health. Litton Bionetics, Inc. 1977. Evaluation of hexachlorocyclo- pentadiene ir\ vitro malignant transformation in BALB/3T3 cells: Final rep. Unpublished report submitted to Velsicol Chemical Corp. Litton Bionetics, Inc. 1978a. Mutagenicity evaluation of hexachlorocyclopentadiene in the mouse lymphoma forward mutation assay. Unpublished rep. submitted to Velsicol Chemical Corp. Litton Bionetics, Inc. 1978b. Mutagenicity evaluation of hexachloropentadiene in the mouse dominant lethal assay: Final report. Unpublished rep. submitted to Velsicol Chemical Corp. Lu, P.Y., et al. 1975. Evaluation of environmental distri- bution and fate of hexachlorocyclopentadiene, chlordane, heptachlor, and heptachlor epoxide in a laboratory model ecosystem. Jour. Agric. Food Chem. 23: 967. C-84 ------- Marks, D.R. 1977. Letter to Donald I. Mount, Director, Environmental Research Lab, U.S. EPA, Duluth, Minnesota, dated February 3, 1977. Description and data concerning monitoring of discharge from Velsicol Chemical Corp. Memphis plant to City of Memphis wastewater collection system during January, 1977. Mehendale, H.M. 1977. The chemical reactivity - absorption, retention, metabolism, and elimination of hexachlorocylo- pentadiene. Environ. Health, Perspect. 21: 275. Metcalf, R.L. 1971. Model ecosystem for the evaluation of pesticide biodegrading and ecological magnification. Environ. Sci. Technol. 5: 708. Morse, D.L., et al. 1978. Hexachlorocyclopentadiene contami- nation of a municipal sewage treatment plant, Louisville, Kentucky. Pub. Health Serv., Center for Disease Control, Atlanta, Georgia, EPI-77-49-2. Naishstein, S.Y., and E.V. Lisovskaya. 1965. Maximum permissible concentration of hexachlorocylopentadiene in water bodies. Gigiena i Sanitariya (Translation) Hyg. Sanit. 30: 177. C-85 ------- National Academy of Sciences. 1977. Kepone/Mirex/Hexachloro- cylopentadiene: An environmental assessment (final draft). Environ. Stud. Board/ Comm. on Nat. Resour. Nat. Res. Counc. National Cancer Institute. 1977. Summary of data for chemical selection. 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A rapid assessment of the toxicity of three chlorinated cyclodiene insecticide intermediates to fathead minnows. Off. Res. Dev. Environ. Res. Lab., Duluth, Minn. U.S. Environ. Prot. Agency. Spehar, R.L., et al. (In press). Toxicity and bioaccumulation of hexachloropentadiene, hexachloronorbornadiene and hepta- chloronorbornene in larval and early juvenile fathead minnows, Pimephales promelas. Bull. Environ. Contain. Toxicol. Stokinger, H.E., and R.L. Woodward. 1958. Toxicologic methods for establishing drinking water standards. Jour. Am. Water Works Assoc, April, 1958: 515. Swanson, D. 1976. Discharges from Hooker Chemical Company. Internal Staff Report (unpublished), Toxic Mater. Section, Water Quality Div., Environ. Prot. Bur. Mich. Dep. Nat. Resour. Treon, J.F., et al. 1955. The toxicity of hexachlorocylo- pentadiene. Arch. Ind. Health 11: 459. C-87 ------- Ungnade, H.E., and E.T. McBee. 1958. The chemistry of perchlorocylopentadienes and cyclopentadienes. Chem. Rev. 58: 240. U.S. EPA. 1977. Early warning report on hexachlorocylopenta- diene and its derivatives. Internal Staff Report (unpublished), Off. Toxic Subst.,U.S. Environ. Prot. Agency, Washington, D.C. U.S. EPA. 1978. Derivation of freshwater criterion for hexachlorocyclopentadiene. Unpublished memo, Water Quality Lab., Duluth, Minn. Whitacre, D.M. 1978. Letter to R. A. Swing, Battelle Columbus Laboratories, dated August 9, 1978. Comments on document titled, Review of environmental effects of pollutants XI. Hexachlorocylopentadiene. Zavon, M.R. 1978. Letter to G. A. Lutz, Battelle Columbus Lab. March 13. /D'XJ.cn- ;1 -••„.. b/-- • * -, • • -J— -'-Lo' •'0 fu .rii Dci^om Surest C-88 ------- |