Cysteine conjugate $ lyase has been isolated and purified from human kidney cytosol ( Lash et al., 1990), and the human $ lyase gene has been cloned and expressed ( Perry et al., 1995). $ lyase activity has been demonstrated in the human kidney ( Green et al., 1990) and human proximal tubular cells ( Chen et al., 1990). Collectively, these human studies suggest that humans have the ability to metabolize HCBD to toxic metabolites. However, the activity of HCBD metabolizing enzymes, particularly renal $ lyase, may be many fold lower in humans than the corresponding enzymes in rat ( Lock, 1994; Lash et al., 1990; Anders and Dekant, 1998).

Werner et al. ( 1995b) demonstrated that human liver microsomes are capable of oxidizing N acetyl S( 1,1,2,3,4 pentachlorobutadienyl) L cysteine to the corresponding sulfoxide ( N AcPCBCSO In contrast to the male specific formation of N AcPCBC SO in rats described above, formation of the sulfoxide was detected in human microsomes prepared from both male and female donors. Inhibitor studies suggest that formation of the sulfoxide is catalyzed by members of the cytochrome P450 3A subfamily. Since this subfamily constitutes a major fraction of cytochrome P450 content in human liver, the formation of the sulfoxide is expected to occur in humans exposed to HCBD. Incubation of N AcPCBC SO with purified human glutathione S transferase M1 1 (: class) catalyzes the formation of ( S) N acetyl S( 4 glutathion S yl 1,2,3,4 tetrachlorobutadienyl) Lcysteine sulfoxide and ( R) N acetyl S( 4 glutathion S yl 1,2,3,4 tetrachlorobutadienyl) L cysteine sulfoxide, the same products formed in the presence of rat : class glutathione S transferase ( Rosner et al., 1998). HCBD   February 2003 6 7 6.4 Excretion HCBD and its metabolites are excreted in urine, feces, and exhaled air. In rodents, oral exposure to HCBD results in fairly rapid urinary and fecal excretion. The half life of elimination of HCBD related radioactivity bound to tissue proteins was 22 hours in both liver and kidney ( Reichert et al., 1985). Total excretion within 72 hours was found to be at least 65% of a single oral dose of up to 100 mg/ kg in rats and mice ( Reichert and Schutz, 1986; Dekant et al., 1988a). At higher doses ( 30 to 200 mg/ kg), 5 to 11% of the radiolabel was excreted in the urine ( Dekant et al., 1988a; Nash et al., 1984; Reichert and Schutz, 1986; Reichert et al., 1985; Payan et al., 1991), while a dose of 1 mg/ kg resulted in urinary excretion of approximately 18.5 to 30% of the administered radioactivity ( Payan et al., 1991; Reichert et al., 1985). Payan et al. ( 1991) attributed the fractional decrease in urinary excretion with increase in dosage to a saturation of hepatobiliary excretion or a reduction of biliary metabolite reabsorption. Exhaled unchanged labeled HCBD accounted for approximately 5% of oral doses of 1 and 50 mg/ kg when measured within 72 hours of administration, while exhaled labeled carbon dioxide accounted for 1% or 4% of the administered dose ( Reichert et al., 1985). The authors did not indicate the metabolic pathway for carbon dioxide formation.

Fecal excretion is the main pathway of elimination for HCBD and HCBD metabolites. Reichert et al. ( 1985) reported that elimination of HCBD in the feces represented 42 or 69% of the radioactivity orally administered to female Wistar rats at doses of 1 or 50 mg/ kg, respectively. The difference in recovery was attributed to an apparent absorption saturation threshold. Dekant et al. ( 1988a) found that 67 to 77% of the radioactivity in an oral 30 mg/ kg dose in corn oil was present in the feces of mice 72 hours after administration. Payan et al. ( 1991) administered oral doses of 14CHCBD in polyethylene glycol to male rats. After 72 hours, the feces and contents of the gastrointestinal tract contained 62% of a 1 mg/ kg dose and 72% of a 100 mg/ kg dose.

Enterohepatic circulation has been demonstrated in animals following oral administration of HCBD. Nash et al. ( 1984) administered 200 mg/ kg 14C HCBD to rats with and without cannulated bile ducts. Feces and urine were collected over a 5 day period. Over the course of the experiment, the non cannulated animals excreted 39% of the administered radioactivity in feces. On each of the first two days post dosing, approximately 5% and 3.5% of the administered radioactivity were found in the feces and urine, respectively. In contrast, bile excretions collected from the cannulated animals on each of the first 2 days post dosing contained 17   20% of the administered radioactivity ( with 35% total excretion by this route over two days). These findings indicate extensive reabsorption of biliary metabolites.

Payan et al. ( 1991) also compared excretion patterns in bile duct cannulated and noncannulated rats orally dosed with 1 mg/ kg 14C HCBD. Urinary excretion after 72 hours accounted for 18% of the administered radioactivity in intact animals, but just 11% of the radioactivity in the cannulated rats. In comparison, fecal excretion represented 62% and 3%, of the dose administered to non cannulated and cannulated animals, respectively. In cannulated rats, 67% of the dose was excreted into the bile. When bile excretions ( isolated from bile duct cannulated rats orally dosed with 100 mg/ kg HCBD) were directly infused into the duodenum, approximately 80% of the biliary metabolites are reabsorbed, with only 20% remaining in the feces and gastrointestinal tract. HCBD   February 2003 6 8 Several studies have reported the identity of excreted metabolites following exposure to 14CHCBD Metabolites identified in the urine of treated rats or mice include S( 1,1,2,3,4 pentachlorobutadienyl) glutathione, S( 1,1,2,3,4 pentachlorobutadienyl) L cysteine, N acetyl S ( 1,1,2,3,4 pentachlorobutadienyl) L cysteine, 1,1,2,3,4 pentachlorobutadienyl sulfenic acid, 1,1,2,3,4 pentachlorobutadiene methylthioether, 1,1,2,3,4 pentachlorobutadiene carboxymethylthioether, and 1,1,2,3 tetrachlorobutenoic acid ( Dekant et al., 1988a; Nash et al., 1984; Reichert and Schultz, 1986; Reichert et al., 1985). As noted previously, the novel metabolite N acetyl S( 1,1,2,3,4, pentachlorobutadienyl) L cysteine sulfoxide has been detected in the urine of male, but not female, rats following oral administration of HCBD ( Birner et al., 1995).

Comparatively few data are available on the identity of fecal metabolites. Dekant et al. ( 1988a) administered a single 30 mg/ kg gavage dose of 14C HCBD in corn oil to male and female NMRI mice. The feces were collected over a 72 hour period following dose administration. Approximately 80% of the fecal radioactivity was associated with HCBD. About 10% of the radiolabel was associated with the HCBD metabolite S( 1,1,2,3,4 pentachlorobutadienyl) glutathione. The remainder of the fecal radioactivity was present as polar metabolites which could not be structurally identified. HCBD   February 2003 7 1 7.0 HAZARD IDENTIFICATION 7.1 Human Effects Limited information is available on the human health effects associated with exposure to HCBD. A review of the available literature did not identify case reports describing the outcome of accidental or intentional HCBD exposure, or reports of systemic toxicity following oral or dermal HCBD exposure. A number of studies have evaluated health effects in workers occupationally exposed to HCBD via inhalation, and these studies are described below.

7.1.1 Short Term Studies No short term studies describing HCBD health effects in humans were located.

7.1.2 Long Term and Epidemiological Studies General Population No general population studies of HCBD toxicity were located.

Sensitive Populations No studies concerning HCBD toxicity in sensitive populations were located.

Occupational Exposure Studies German ( 1986) conducted two cytogenetic studies of workers employed in an HCBD production facility. The exposure levels reported by the manufacturer ranged from 1.6 to 16.9 mg/ m3. The investigators found an increased frequency of chromosomal aberrations in the peripheral lymphocytes of exposed workers. However, the frequency of aberrations was not associated with duration of employment in the HCBD manufacturing facility ( WHO, 1994), suggesting that factors other than HCBD exposure contributed to the observed effects.

Additional occupational studies have evaluated health effects in workers exposed to HCBD. However, in each case concurrent exposure of workers to other chemicals limits the usefulness of the data for evaluation of HCBD human health effects. Krasniuk et al. ( 1969), for example, evaluated health effects in 153 farm workers intermittently exposed over a period of four years to soil and grape fumigants containing HCBD. When compared to a control population of 52 unexposed workers, HCBD exposed workers exhibited increased incidence of arterial hypotension, myocardial dystrophy, chest pains, upper respiratory tract changes, liver effects, sleep disorders, hand trembling, nausea, and disordered olfactory functions ( U. S. EPA, 1991a). Interpretation of these data is confounded by concurrent exposure of these workers to polychlorobutane 80. HCBD   February 2003 7 2 Burkatskaya et al. ( 1982) reported adverse health effects in vineyard workers exposed to fumigants containing HCBD. However, the role of HCBD could not be evaluated because the workers were concurrently exposed to other agricultural chemicals ( WHO, 1994).

Driscoll et al. ( 1992) determined the concentrations of individual serum or plasma bile acids in workers exposed to chlorinated hydrocarbons, including HCBD, carbon tetrachloride, and perchloroethylene. These investigators reported increases in four serum bile acid parameters in workers exposed via inhalation to 0.005   0.02 ppm HCBD. The study found no significant relation between bile acid parameters or liver function tests and exposure. As in the studies above, the specific contribution of HCBD exposure to the observed effects could not be evaluated.

7.2 Animal Studies 7.2.1 Acute Toxicity Oral Exposure Schwetz et al. ( 1977) reported a single dose oral LD 50 value ( the dose that produces lethality in 50% of the experimental animals) of 65 mg HCBD/ kg for male weanling rats and 46 mg/ kg for female weanling rats. A single dose oral LD 50 value of 90 mg/ kg was reported for adult rats ( Kennedy and Graepel, 1991). These data suggest that age and gender may be significant variables in the acute toxicity of HCBD. Single dose LD 50 values reported for other rodents were 80 to 116 mg/ kg for mice and 90 mg/ kg for guinea pigs ( U. S. EPA, 1991a).

Three studies have evaluated the non lethal acute effects of oral HCBD exposure. Nash et al. ( 1984) administered a single oral dose of 200 mg/ kg HCBD in polyethylene glycol to six male Wistar derived rats. Treatment with HCBD increased plasma urea concentration and decreased plasma alanine aminotransferase activity. Analysis of urine revealed significantly ( p > 0.01) increased levels of glucose, protein, alkaline phosphatase, N acetyl ß D glucosaminidase ( NAG), ( glutamyl transpeptidase and alanine aminopeptidase over control levels. These are commonly used biochemical markers of kidney damage.

Jonker et al. ( 1993a) investigated the acute oral toxicity of HCBD in 12 week old male Wistar rats. The investigators administered single doses of 0, 10, 100, or 200 mg/ kg HCBD in corn oil by gavage to five rats per treatment group. Urine was collected at intervals of 0 to 6 and 6 to 24 hours. All rats were sacrificed at 24 hours. No treatment related effects were observed at the 10 mg/ kg dose. HCBD induced a variety of adverse effects at the two highest dose levels. Kidney weight, blood plasma creatinine level, urinary pH and occult blood, number of epithelial cells in the urine, urinary lactate dehydrogenase and NAG activity were significantly increased ( p< 0.05) at 100 and 200 mg/ kg. Additional effects observed in the 200 mg/ kg dose group included reduced body weight, reduced food intake, elevated plasma urea level, and increased urinary volume. Increased levels of urinary protein, glucose, and potassium, and increased activity of urinary ( glutamyltransferase and alkaline phosphatase were also observed at 200 mg/ kg. Histopathological examination of the kidneys revealed limited focal necrosis at 100 mg/ kg and extensive tubular HCBD   February 2003 7 3 necrosis at 200 mg/ kg. The study authors identified 10 mg/ kg and 100 mg/ kg as the acute " No Nephrotoxic Effect Level" and " Minimum Nephrotoxic Effect Level", respectively.

Payan et al. ( 1993) administered single oral doses of 0, 100, or 200 mg/ kg HCBD in polyethylene glycol to male Sprague Dawley rats ( 4 to 5 animals per dose). All rats were sacrificed 24 hours after exposure, and the right kidneys were subjected to microscopic examination. Nephrotoxicity was also evaluated by determination of the following biochemical urinary parameters of renal impairment: urinary concentration of $ 2 microglobulin ($ 2 m), urinary ( glutamyl transpeptidase (   GT) activity, urinary aspartate aminotransferase ( AST) activity, and urinary Nacetyl $ glucosamine ( NAG) activity At 100 mg/ kg, 4/ 5 of the rat kidneys exhibited mild or moderate lesions, compared with 0/ 4 in the controls. Significant increases in   2 m ( 2 fold) and AST ( 19 fold) were seen at 100 mg/ kg, and in all biochemical markers of renal toxicity at 200 mg/ kg. A Lowest Observed Adverse Effect Level ( LOAEL) of 100 mg/ kg was identified in this study on the basis of kidney lesions and a increased urinary   2 m and AST excretion.

Lock et al. ( 1996) administered a single oral dose of 50 mg HCBD/ kg to a calf to evaluate toxic effects on the kidney and bone marrow. The administered dose resulted in the death of the animal 5 days after treatment. Prior to death, blood urea nitrogen, plasma aspartate aminotransferase and plasma alkaline phosphatase were elevated, but no changes were observed in circulating white cells or platelets. The liver and kidneys appeared pale and swollen at necropsy. Histopathological examination revealed midzonal necrosis in the liver. Extensive areas of necrosis were evident in the kidney, and were accompanied by hyaline and granular cast formation.

Inhalation Exposure De Ceaurriz et al. ( 1988) evaluated the effects of HCBD inhalation exposure on male Swiss OF1 mice ( 6 mice/ dose). The mice were exposed to HCBD vapor at concentrations between 83 and 246 ppm ( 886 and 2,625 mg/ m3 ) for 15 minutes. Decreased respiratory rates ( reflex bradypnea) were observed at concentrations of 155 ppm ( 1,652 mg/ m3 ) or greater. An EC 50 ( concentration producing an effect in 50% of the population) of 211 ppm ( 2,250 mg/ m3) was calculated for this effect.

De Ceaurriz et al. ( 1988) investigated the effects of a 4 hour whole body exposure to HCBD at measured concentrations of 2.75, 5, 10, 25 ppm ( or 29.3, 53.4, 106.7, 266.8 mg/ m3) on male Swiss OF1 mice ( 10 animals/ dose). An HCBD related increase in the percentage of damaged renal tubules, as determined by alkaline phosphatase staining, was observed at all exposure levels. The EC 50 for this response was 7.2 ppm ( 76.8 mg/ m3).

Gehring and MacDougall ( 1971) exposed rats to 161 ppm ( 1,716 mg/ m3) HCBD for 0.88 hour or 34 ppm ( 362 mg/ m3) for 3.3 hours. All rats survived the treatment. Exposure of guinea pigs or cats under the same conditions resulted in the death of most animals. Inhalation exposure of rats to 133 to 150 ppm ( 1,418 to 1,600 mg/ m3) for 4 to 7 hours was lethal for some or all animals ( NTP, 1991). HCBD   February 2003 7 4 Dermal Exposure Gradiski et al. ( 1975) evaluated the dermal toxicity and sensitization potential of HCBD in rabbits. Dermal application of a 10% solution of HCBD ( solvent not indicated) to rabbits caused slight dermal irritation. Guinea pigs exhibited delayed allergic reactions to dermal HCBD application ( U. S. EPA, 1991a).

Duprat and Gradiski ( 1978) evaluated the acute toxicity of dermally applied HCBD in female New Zealand rabbits ( 10 animals/ dose) following an 8 hour exposure period. Undiluted HCBD was applied at doses of 0.25, 0.5, 0.75 and 1.0 mL/ kg under occluded conditions. Four hours after termination of exposure, the epidermis and subcutaneous tissue revealed edema and polymorphonuclear leukocyte infiltration at the two highest doses. Three to five days after treatment at the three highest doses, necrotic changes were noted at the site of application. A few animals from the two highest dose groups died within 24 hours from respiratory and cardiac failure. Indications of systemic toxicity included renal epithelial necrosis and fatty liver degeneration. The LD 50 for the eight hour exposure was 0.72 mL/ kg ( Duprat and Gradiski, 1978). Based on a specific gravity of 1.675 for HCBD ( U. S. EPA, 1991a), a dermal LD 50 of 1,206 mg/ kg was calculated.

Acute Ocular Toxicity Gradiski et al. ( 1975) reported that instillation of a 10% solution of HCBD ( solvent not reported) into rabbit eyes resulted in slight ocular conjunctival irritation ( U. S. EPA, 1991a).

Intraperitoneal Injection Bai et al. ( 1992) exposed male Sprague Dawley rats ( 4 animals/ dose) by intraperitoneal injection 0, 10.4, 52.2, or 104 mg/ kg day HCBD for three days. Serum bilirubin and alkaline phosphatase activity was increased ( p< 0.05) at the two higher doses, indicating disturbance in liver function. The concentration of total serum bile acids was elevated at the highest dose. No histopathological examination of the livers was conducted.

Lock and Ishmael ( 1979) administered single intraperitoneal doses of HCBD in corn oil to male albino rats ( 3 to 19 animals/ dose). The doses administered were 0, 20, 50, 100, 200, 300, 400, 500, and 1,000 mg/ kg. The effects of treatment were assessed 24 hours after administration. Three of four animals treated with 500 mg/ kg died. All rats in the 1,000 mg/ kg dose group died. Rats in the highest dose group exhibited piloerection, sedation, hunching, incoordination, loss of muscle tone and hypothermia prior to death.

Wolf et al. ( 1983) conducted studies to examine the effects of HCBD on the cytochrome P450 content and related monooxygenase activities in the kidneys of male and female rats and male mice. Adult male and female Wistar rats were administered either 200 or 400 mg/ kg HCBD in corn oil by intraperitoneal injection. Male Swiss derived mice were administered 50 mg/ kg HCBD in corn oil by intraperitoneal injection. Control animals received corn oil alone ( 5 ml/ kg). A subset of the animals were treated with 100 mg/ kg   naphthoflavone ( BNF) by intraperitoneal injection daily for 3 days before HCBD administration. Following HCBD administration, animals were fasted for 24 HCBD   February 2003 7 5 hours, and renal and hepatic microsomal fractions were obtained. Renal cytochrome P450 concentrations dropped significantly ( p < 0.01) in both male and female rats at both doses without significant losses of cytochrome b5 or NADPH cytochrome c. In rat renal microsomes, HCBD increased metabolism of aldrin and 7 ethoxycoumarin significantly ( p < 0.05), while metabolism of p nitroanisole was only slightly reduced and metabolism of lauric acid was unaffected. No effect was found on rat hepatic cytochrome P450 levels, and no reductions in rat hepatic monooxygenase activity were found, although metabolism of 7 ethoxycoumarin was significantly ( p < 0.05) increased. In BNF treated rats, HCBD decreased renal cytochrome P450 concentrations 33%, decreased metabolism of aldrin significantly ( p < 0.05), and increased metabolism of 7 ethoxycoumarin and 7 ethoxyresorufin. In naive mice, HCBD treatment led to an almost complete loss ( 15% of control remaining) of cytochrome P450 and associated monooxygenase activity ( 0 28% of control metabolism). In BNF treated mice, a similar loss of cytochrome P450 ( 17% of control remaining) resulted in similar decreases in metabolism ( 0 40% of control metabolism), except for metabolism of 7 ethoxycoumarin ( 67% of control metabolism) and 7 ethoxyresorufin ( 117% of control metabolism). These findings suggest that the cytochrome P450 subtypes which are decreased are localized in regions where HCBD damage occurs, while BNF induced subtypes are not..

Hook and colleagues ( Hook et al., 1982, 1983; Kuo and Hook, 1983) conducted a series of experiments to characterize HCBD toxicity in four different strains of rats. Single doses of 25 to 400 mg/ kg HCBD were administered by intraperitoneal injection. Following treatment, relative kidney weight increased in all dose groups. Organic ion transport was evaluated by analysis of the anion paminohippurate ( PAH) and the cation tetraethylammonium ( TEA) in renal cortical slices. Renal efflux rates of PAH and TEA were unaffected. However, accumulation of the PAH anion was decreased, while accumulation of the TEA cation was unaffected. This pattern suggests a specific impact of HCBD on the renal anion uptake system ( Hook et al., 1982; Kuo and Hook, 1983). Kidney to body weight ratios increased in all dosage groups. Blood urea nitrogen levels were elevated in all dose groups, with a more pronounced effect noted in young rats ( Kuo and Hook, 1983). Adult male rats were less susceptible to HCBD induced renal effects than were female adult rats or young male rats. The investigators attributed this pattern to age and sex related differences in the renal and hepatic enzymes responsible for activation and detoxification of HCBD.

Lock et al. ( 1984) investigated the effect of age, strain, sex, and monooxygenase inhibitors on the acute toxicity of HCBD to five different strains of mice ( 6 or more animals/ dose group). HCBD in corn oil was administered as single intraperitoneal doses ranging from 6.3 to 50 mg/ kg. Toxicity was evaluated 24 hours after treatment. Histopathological examination of the kidneys from adult Swiss derived mice of both sexes revealed extensive, dose dependent proximal tubular necrosis at doses of 12.5 mg/ kg and above. At 6.3 mg/ kg, tubular necrosis was only observed occasionally in a small number of animals. A significant increase in plasma urea occurred in adult Swiss derived mice at doses of 25 to 50 mg/ kg. No evidence for a gender difference in response was observed. Young male mice responded to lower doses of HCBD than adults, with an increase in plasma urea and a decrease in organic ion transport in renal slices evident at 12.5 mg/ kg. Prior administration of the monooxygenase inhibitor piperonyl butoxide or the monooxygenase inducers phenobarbitone or $ naphthoflavone did not modify the extent of HCBD induced renal damage. Intraperitoneal administration of the glutathione or N acetylcysteine conjugates resulted in a pattern of renal necrosis similar to that observed for HCBD. Evaluation of the comparative susceptibility of five mouse HCBD   February 2003 7 6 strains indicated that the BALB/ c strain was slightly more susceptible to HCBD toxicity than the C57BL/ 10J, C3H, DBA/ 2J, and Swiss derived strains.

Ishmael et al. ( 1984) investigated the time course of histopathology and functional impairment in adult Swiss derived mice following a single 50 mg/ kg intraperitoneal dose of HCBD in corn oil. Histopathological examination of the kidneys by light and electron microscopy revealed mitochondrial swelling within 1 hr after dosing, followed by nuclear pyknosis and increased cytoplasmic eosinophilia at 4 hr, extensive tubular necrosis after 16 hr, and active tubular regeneration within 5 days. Treated animals exhibited significantly increased plasma urea, decreased renal non protein sulfhydryl content, and increased renal water content.

7.2.2 Short Term Studies Oral exposure Kociba et al. ( 1971) conducted a study of HCBD toxicity, the results of which were published as Kociba et al. ( 1977) and Schwetz et al. ( 1977). Female Sprague Dawley rats ( 4 animals/ dose group) were fed diets containing HCBD at doses of 0, 1, 3, 10, 30, 65, or 100 mg/ kg day for 30 days. Renal toxicity in the form of increased relative kidney weight as well as renal tubular degeneration, necrosis and regeneration was observed in rats receiving doses of 30, 65 or 100 mg/ kg day. Minimal hepatocellular swelling was noted at a dose of 100 mg/ kg day. Other observed effects included decreased food consumption, reduced body weight gain, and increased hemoglobin concentration at doses of 10, 30, 65 or 100 mg/ kg day. No effects were observed in rats receiving 3 mg/ kg day. This study identified a No Observed Adverse Effect Level ( NOAEL) of 3 mg/ kg day and a LOAEL of 10 mg/ kg day.

Harleman and Seinen ( 1979) administered diets containing nominal concentrations of 0, 50, 150, or 450 ppm HCBD to weanling Wistar derived rats ( 6 animals/ sex/ dose group) for 14 days. Based on mean body weight and food consumption data in the study, the mean HCBD doses were calculated to be 0, 4.6, 14.0 and 35.3 mg/ kg day. Body weight and food conversion efficiency were decreased in a dose related manner. Food consumption was decreased at 35.3 mg/ kg day. Relative kidney weights were significantly increased at the two highest dose levels. A dose related degeneration of renal tubular epithelial cells was observed in all treated animals, particularly in the straight limbs ( pars recta) of the proximal tubules located in the outer medulla. No indications of liver toxicity were observed. The low dose of 4.6 mg/ kg day represented the LOAEL.

Stott et al. ( 1981) conducted an oral exposure study in adult male Sprague Dawley rats. Five animals per treatment group were given daily doses of HCBD ( 0, 0.2 or 20 mg/ kg day) in corn oil for three weeks. In animals exposed to 20 mg/ kg day, a decrease in body weight gain and an increase in relative kidney weight were observed. Histopathological examination of the kidneys revealed damage in the middle and inner cortical regions, with loss of cytoplasm, nuclear pyknosis, increased basophilia and mitotic activity, and increased cellular debris. No indications of toxicity were observed in animals exposed to 0.2 mg/ kg day, the NOAEL for this study. HCBD   February 2003 7 7 The National Toxicology Program ( NTP) conducted a 2 week oral exposure study in B6C3F 1 mice ( NTP, 1991; Yang et al., 1989). Groups of mice ( 5 animals/ sex/ dose group) received diets containing nominal concentrations of 0, 30, 100, 300, 1,000 or 3,000 ppm HCBD for 15 days. Target concentrations were verified under experimental conditions by gas chromatography. The estimated daily intake calculated from feed consumption and mean body weights were 0, 3, 12, and 40 mg/ kgday for the 0, 30, 100, and 300 ppm dietary concentrations, respectively, for male mice, and 0, 5, 16, and 49 mg/ kg day for female mice. All mice provided with the 1,000 and 3,000 ppm diets died within seven days. Mice receiving 100 and 300 ppm HCBD lost weight. HCBD related clinical signs observed at doses of 300 ppm or greater included lethargy, rough hair coats, hunched position, and incoordination. Marked reductions in thymus and heart weights were noted in mice consuming the 300 ppm diet. Kidney lesions attributed to HCBD exposure were observed in all treated mice of both sexes ( Yang et al., 1989). Severe necrosis of the cortex and outer medulla was observed in the two doses that caused deaths among the experimental animals. Necrosis was less severe and regeneration was prominent in the pars recta of mice receiving lower doses of HCBD. Histopathologic changes were also observed in liver, lymphoid tissues, and testis of mice in the 1,000 and 3,000 ppm dose groups, but were not clearly related to HCBD toxicity. Minimal to mild depletion of bone marrow in the femur was observed in 2 to 5 mice per dose group in animals receiving diets containing 300 ppm or higher levels of HCBD. This study identified a LOAEL of 3   5 mg/ kg day in male and female mice, respectively, based on renal tubular necrosis and cellular regeneration in animals in the lowest dose groups ( Yang et al., 1989).

Jonker et al. ( 1993b) investigated the toxicity of HCBD in 10 week old male and female Wistar rats ( 5 rats/ sex/ dose). HCBD was provided in the diet at levels of 0, 25, 100, or 400 ppm for a duration of four weeks. Based on mean body weight and food intake data in the study, these concentrations correspond to average daily doses of 0, 2.25, 8, and 28 mg/ kg day. Treatment induced signs of toxicity were observed at doses of 8 and 28 mg/ kg day in both sexes. The observed signs included decreased liver weight, tubular cytomegaly, decreased plasma creatinine, decreased body weight ( 10% in males and 15% in females), and decreased adrenal weight. Increased plasma aspartate aminotransferase activity and bilirubin were observed at the 28 mg/ kg day dose. The NOAEL and LOAEL identified from this study were 2.25 and 8 mg/ kg day, respectively.

Lock et al. ( 1996) conducted two short term experiments on the effects of HCBD in calves. Each experiment evaluated toxicity in a single animal. In the first experiment, a dose of 5 mg/ kg day was administered orally for 7 days. An increase in blood urea nitrogen was noted after the fifth dose, and levels remained high until the animal was euthanized nine days after initiation of treatment. Plasma levels of aspartate transaminase and alanine aminotransferase were elevated, but no changes were observed in hematological parameters. At necropsy, perirenal edema was observed in the kidneys and the liver was pale and swollen. Histopathological examination revealed midzonal necrosis of the liver and extensive swelling of the tubular epithelium and degenerative changes in the kidney. Casts were evident in the tubules of the medulla.

In the second short term experiment conducted by Lock et al. ( 1996), a calf was dosed with 2.5 mg HCBD/ kg day for 10 days and the blood was monitored for 20 days for urea and platelet count. The dose was subsequently increased to 5 mg/ kg day, with 8 doses administered over 12 days. A marginal increase in blood urea nitrogen was observed on day 14. Aspartate transaminase and HCBD   February 2003 7 8 alanine aminotransferase were increased on day 7, and gradually decreased to normal levels on day 15. The calf was euthanized and necropsied 18 days after the start of the dosing regimen. Histopathological examination revealed slight disruption of the midzonal architecture of the liver, while mild renal tubular degeneration was evident in the kidney. The results of the experiments conducted by Lock et al. ( 1996) indicate that HCBD is both a nephro and hepato toxicant in calves.

Nakagawa et al. ( 1998) exposed male Wistar rats ( 3 animals/ dose group) to 0, 0.008, 0.04, or 0.2% HCBD in the diet for 3 weeks. Assuming a food consumption factor of 0.09 kg/ kg/ day ( U. S. EPA, 1988), these dietary levels correspond to approximate daily doses of 0, 7.2, 36, and 180 mg/ kgday Rats ingesting the 0.04% and 0.2% diets had lower mean body weight ( decreases of 15% and 46%, respectively) at the termination of the experiment. Kidney weight was unaffected. Histopathological examination of rats in the 180 mg/ kg day group revealed indications of extensive regeneration in the straight portion ( pars recta) of the proximal tubule. Similar lesions were not evident at lower doses. These data suggest a NOAEL of 7.2 mg/ kg day based on absence of effect on weight gain or renal histopathology.

Inhalation exposure NIOSH ( 1981) reported 100% mortality in mice exposed to HCBD vapors for 5 days, 7 hours/ day, at a concentration of 50 ppm ( 533 mg/ m3), but no deaths in animals exposed to 10 ppm ( 106.6 mg/ m3).

Gage ( 1970) conducted an inhalation study in Alderley Park SPF rats. Groups of adult rats ( 4/ sex/ dose) were exposed to nominal HCBD concentrations of 53, 107, or 267 mg/ m3 for 15 days, 6 hours/ day ( duration adjusted concentrations of 13, 27, or 67 mg/ m3); 1,067 mg/ m3 for 12 days, 6 hours/ day ( 267 mg/ m3 duration adjusted); or 2,668 mg/ m3 for 2 days, 4 hours/ day ( 445 mg/ m3 duration adjusted). Petroleum ether was used as a solvent for concentrations below 1,067 mg/ m3. No indications of toxicity were observed at the lowest level of exposure, suggesting a NOAEL of 53 mg/ m3 ( 13 mg/ m3 duration adjusted). Two of the four female rats exposed to 1,067 mg/ m3 HCBD died. Pale enlarged kidneys, adrenal regeneration, and renal cortical tubular degeneration with epithelial regeneration were noted at autopsy. Surviving females at this concentration were slightly anemic. The weight gain of female rats was reduced at 107 and 267 mg/ m3. At 1,067 mg/ m3, rats of both sexes lost weight. Irritation of the eyes and nose was observed at the two highest levels of exposure. Respiratory distress occurred at concentrations of 267 mg/ m3 or greater. At the termination of the experiment, enlarged pale kidneys were evident in the 267 and 1,067 mg/ m3 treatment groups. Enlarged adrenals were observed in animals exposed to 1,067 mg/ m3. Histopathological analysis revealed degeneration in the adrenal cortex and proximal tubules of the kidneys at concentrations of 267 mg/ m3 or greater ( WHO, 1994).

7.2.3 Subchronic Studies Schwetz et al. ( 1977) fed male ( 10  17 per dose group) and female ( 20  34 per dose group) Sprague Dawley rats a diet containing 0.2, 2.0, or 20 mg/ kg day HCBD for evaluation of reproductive effects. HCBD was provided in the diet before and during mating, and throughout gestation and lactation, for a total study duration of 148 days. Adult rats from the 20 mg/ kg day dose HCBD   February 2003 7 9 level had decreased body weight gain along with decreased food consumption. At necropsy, relative kidney weights were increased in high dose males and females. Relative liver weight was increased in high dose males, and relative brain weight was increased in high dose females. The kidneys of males exposed to 2 ( 1 of 10 examined) or 20 ( 3 of 15 examined) mg/ kg day were roughened and had a mottled cortex. Histopathological examination revealed dose related increases in tubular dilation and regeneration in animals exposed to 2 or 20 mg/ kg day ( Table 7 1). These results indicate a NOAEL of 0.2 mg/ kg day for male and female rats, based on the absence of observed renal histopathology or other toxic effects at this dose.

Table 7 1. Histopathological Findings in Adult Rats Fed Diets containing Hexachlorobutadiene.

Male Rats Female Rats Administered Dose ( mg/ kg day) 0 0.2 2 20 0 0.2 2 20 Moderate Proteinaceous Casts in Dilated Renal Tubules 1/ 5 0/ 5 3/ 5 4/ 5 0/ 5 0/ 5 1/ 5 0/ 5 Moderate Focal Renal Tubular Collapse and Atrophy 1/ 5 0/ 5 3/ 5 4/ 5 0/ 5 0/ 5 4/ 5 0/ 5 Dilation and Hypertrophy of the Tubules in the Kidney Cortex 1/ 5 0/ 5 0/ 5 4/ 5 0/ 5 0/ 5 1/ 5 4/ 5 source: Schwetz et al. ( 1977)

Harleman and Seinen ( 1979) exposed groups of weanling Wistar derived rats ( 10/ sex/ dose group) to daily oral gavage doses of 0, 0.4, 1.0, 2.5, 6.3, or 15.6 mg/ kg day HCBD in a peanut oil vehicle. The study duration was 13 weeks. Reductions in body weight gain, food consumption, and food utilization efficiency were noted in the two highest dose groups. Dose related increases in relative kidney weights were noted in all treatment groups of male mice, and in females administered 6.3 or 15.6 mg/ kg day. Proximal tubular degeneration was noted in males treated with doses of 6.3 mg/ kg day and above, and in females treated with doses of 2.5 mg/ kg day and above. This effect was characterized by hyperchromatic nuclei, hypercellularity, vacuolation and focal necrosis of renal epithelial cells, and a diminished brush border. Polyuria and decreased urine osmolarity were noted in females receiving doses equal to or greater than 2.5 mg/ kg day, and in males receiving 15.6 mg/ kg day. Relative liver weights were increased in females at 15.6 mg/ kg day and in males at 6.3 and 15.6 mg/ kg day. Histological examination of the liver revealed increased cytoplasmic basophilia only in males treated with 6.3 mg/ kg day and above. Relative spleen weights were increased in males at 15.6 mg/ kg day, and in females at the two highest doses. The study authors identified NOAEL values of 1.0 mg/ kg day for females and 2.5 mg/ kg day for males.

NTP ( 1991) conducted a 13 week oral exposure study in B6C3F 1 mice. Groups of mice ( 10 animals/ sex/ dose group) received diets containing 0, 1, 3, 10, 30, or 100 ppm HCBD. Target concentrations were verified under experimental conditions by gas chromatographic analysis. Based HCBD   February 2003 7 10 on average food consumption and body weight data, these concentrations corresponded to dose levels of 0, 0.1, 0.4, 1.5, 4.9 or 16.8 mg/ kg day for males and 0, 0.2, 0.5, 1.8, 4.5 or 19.2 mg/ kg day for females. No HCBD related clinical signs or deaths were observed. Food consumption of treated and control animals was similar. Reduced body weight gain was reported in males exposed to diets containing 30 or 100 ppm HCBD ( decreases of 49% and 56%, respectively) and in females exposed to the 100 ppm diet ( 47%). Relative kidney weight was significantly decreased ( p< 0.01) in the three highest dose groups of males, and in females in the highest dose group. High dose males also exhibited decreased relative heart weight, although no histologic lesions were reported in this organ.

Histopathological changes were noted in the kidneys of treated animals. Necropsy revealed treatment related increases in renal tubular cell regeneration. This lesion was characterized as a diffuse increase in epithelial nuclei and increased basophilic staining. Female mice appeared to be more susceptible than male mice to the formation of this lesion following exposure to HCBD, with occurrence noted at dose levels of 0.2 mg/ kg day and above. Incidence of regeneration in female mice was 0% at control, 10% at 0.2 mg/ kg day, 90% at 0.6 mg/ kg day, and 100% at higher doses. Lesions were observed in male mice at dose levels of 4.9 mg/ kg day and above, with incidences of 100% in these groups ( NTP, 1991; Yang et al., 1989). In contrast to results from the 2 week study conducted by the same investigators, no evidence of necrosis was observed. Based on the histopathologic evaluation of the kidney, the authors identified a NOAEL of 1.5 mg/ kg day for male mice. Because tubular regeneration occurred in 1 of 10 females in the lowest dose group ( 0.2 mg/ kgday the study authors concluded that a NOAEL for female mice could not be identified from these data ( NTP, 1991). However, others have concluded that the effect observed at 0.2 mg/ kg day is not statistically significant, and therefore considered this dose to be the NOAEL for female mice ( WHO, 1994; U. S. EPA, 1998a). Further statistical analysis shows that, although the 0.2 mg/ kg day dose fails a Fisher's exact test against control ( p = 0.5), the data passes a Mantel Haenszel trend test ( p < 0.001), suggesting that a benchmark dose analysis would be appropriate.

Nakagawa et al. ( 1998) administered 0.1% HCBD in the diet to male Wistar rats ( 21 rats/ group) for 30 weeks in conjunction with a cancer promotion study ( discussed in Section 7.2.7). Assuming a food consumption factor of 0.09 kg/ kg/ day ( U. S. EPA, 1988), this dietary level corresponds to an average daily dose of 90 mg/ kg day. HCBD treatment resulted in decreased final body weight, and increased relative kidney weight. No significant differences were noted in serum and urine biochemical parameters. Simple hyperplasia of renal tubular structures was observed, but the incidence did not differ significantly from the control. Histopathological examination did not reveal adenomatous hyperplastic foci or renal tumors.

7.2.4 Neurotoxicity Data from distribution studies indicate that HCBD accumulates in brain tissue ( Reichert et al., 1985). This observation raises the possibility that HCBD exposure may affect neurological function. Studies designed to specifically evaluate neurotoxicological endpoints following HCBD exposure were not identified in the available literature. However, neurological effects have been observed in a number of oral and dermal exposure studies. Kociba et al. ( 1977) reported increased relative brain weights in female rats fed 20 mg/ kg day HCBD for 2 years. This increase occurred in a dose group with depressed body weights and was not accompanied by histopathological changes HCBD   February 2003 7 11 in the brain. Similarly, Schwetz et al. ( 1977) noted depressed body weight and increased relative brain weight in female rats fed 20 mg/ kg day for 148 days in a reproductive study. Concurrent changes in behavior or brain histopathology were not observed in the affected animals. An increase in relative brain weights with decreased body weights was also observed in male and female B6C3F 1 mice fed 16.8 to 19.2 mg/ kg day HCBD in their diet in a 90 day subchronic study ( NTP, 1991).

Treatment associated neurotoxic effects were observed by Harleman and Seinen ( 1979), who provided female Wistar rats ( 6 animals/ dose) with diets containing 0, 150, or 1,500 ppm HCBD ( corresponding to average daily doses of 0, 15 or 150 mg/ kg day). The duration of exposure ranged from 10 to 18 weeks. Indications of neurotoxicity observed at the 150 mg/ kg day dose included ataxia, incoordination, weakness of the hind legs, and unsteady gait. Histopathological examination revealed demyelination and fragmentation of femoral nerve fibers in high dose animals. No treatment related histopathological changes were observed in the brain.

In Russian studies cited through secondary sources ( WHO, 1994), Badaeva ( 1983) and Badaeva et al. ( 1985) observed that daily oral administration of 8.1 mg/ kg day HCBD to pregnant rats throughout gestation resulted in histopathological changes in nerve cells and myelin fibers of the spinal cord in the dams and their offspring. Increased levels of free radicals were detected in the brain and spinal cord of the offspring of treated dams ( U. S. EPA, 1991a).

Neurotoxicity has also been observed following HCBD exposure by the dermal route. Duprat and Gradiski ( 1978) observed central nervous system depression manifested as stupor in rabbits following application of 0.25 to 1.0 ml/ kg ( 418 to 1,675 mg/ kg) in an acute dermal toxicity test. Stupor was observed throughout the 8 hour exposure period, and during a 2 hour period immediately following exposure.

7.2.5 Developmental/ Reproductive Toxicity Oral Exposure Schwetz et al. ( 1977) provided male and female Sprague Dawley rats ( 30 to 51 animals/ dose group) with a diet containing HCBD at levels corresponding to doses of 0, 0.2, 2.0, or 20 mg/ kg day HCBD. The HCBD containing diet was administered for 90 days prior to mating, 15 days during mating, 22 days during gestation, and 21 days during lactation. Adults in the two higher dose groups exhibited multiple signs of toxicity, including decreased food consumption, decreased body weight gain, and renal tubular degeneration. No HCBD related effects on pregnancy rate, time to delivery, neonatal survival, neonate sex ratio, weanling histopathology, or incidence of neonatal external, visceral, or skeletal anomalies were observed. Slightly decreased pup weight ( p< 0.05) was observed in the 20 mg/ kg day treatment group at postnatal day 21. The identified NOAEL and LOAEL for developmental effect were 2 and 20 mg/ kg day, respectively.

Harleman and Seinen ( 1979) provided female rats ( 6 animals/ dose) with a diet containing HCBD at levels of 0, 150, or 1,500 ppm for 3 weeks prior to mating, 3 weeks during mating, and throughout gestation and lactation. Assuming a food consumption factor of 0.1 kg/ kg/ day ( U. S. EPA, 1988), these concentrations correspond to average daily doses of 0, 15, or 150 mg/ kg day. 1 Although the Guidelines for Developmental Toxicity Risk Assessment ( U. S. EPA, 1991b) recommended against dosimetric adjustment of developmental toxicity data, the draft Review of the Reference Dose and Reference Concentration Processes ( U. S. EPA, 2002) recommends that duration adjustment procedures to continuous exposures be used for inhalation developmental toxicity studies as for other health effects from inhalation exposure.

HCBD   February 2003 7 12 High dose females were sacrificed at week 10 of the study, and low dose females were sacrificed at week 18. Maternal toxicity occurred in both dose groups, and observed effects included reduced body weight gain, increased relative kidney weight, and histopathological changes in kidneys. Neurological effects, including ataxia, incoordination, weakness of the hind legs, and unsteady gait, were observed in the dams at the 150 mg/ kg day dose. Furthermore, at the 150 mg/ kg day dose no conceptions occurred, the ovaries showed little follicular activity, and no uterine implantation sites were observed. At 15 mg/ kg day, fertility and litter size were reduced, but the effect was not statistically significant. Pup weights in this treatment group were significantly reduced on postnatal days 0, 10, and 20. No gross abnormalities were noted. The LOAEL identified was 15 mg/ kg day.

In Russian studies cited in a secondary source ( WHO, 1994), Badaeva and colleagues ( Badaeva, 1983; Badaeva et al., 1985) conducted two studies in which pregnant female rats were orally administered 8.1 mg/ kg day HCBD throughout gestation. Offspring of HCBD treated dams had lower body weight and shorter crown rump length when compared to controls ( U. S. EPA, 1991a). Histological changes in the nerve cells and myelin fibers of the spinal cord were noted in both dams and offspring. Neurological changes reported in the offspring included ultrastructural changes in neurocytes and increased levels of free radicals in the brain and spinal cord ( U. S. EPA, 1991a).

In addition to the reproductive and developmental toxicity studies discussed above, two longer term toxicity studies have evaluated reproductive endpoints following oral exposure to HCBD. No treatment related lesions in reproductive organs were observed in rats that received lifetime exposures of up to 20 mg/ kg day HCBD ( Kociba et al., 1977). No significant changes were noted in sperm count, the incidence of abnormal sperm, estrual cyclicity, or the average estrous cycle length in mice administered 100 ppm HCBD in the diet for 13 weeks. Sperm motility in treated mice was significantly lower, though not in a dose related manner, than that observed for controls ( NTP, 1991).

Inhalation Exposure Saillenfait et al. ( 1989) exposed pregnant Sprague Dawley rats ( 24 animals/ dose) to HCBD vapor at nominal concentrations of 0, 21, 53, 107, or 160 mg/ m3 ( 0, 2, 5, 10, or 15 ppm) for 6 hours/ day from gestation days ( GD) 6   20, resulting in duration adjusted concentrations1 of 0, 5, 13, 27, or 40 mg/ m3. Concentrations were monitored by gas chromatography. Animals were sacrificed on GD 21. Decreased body weight gain was occurred in dams exposed to 53 or 160 mg/ m3. Body weight was decreased ( p< 0.01) in male ( 9.5%) and female ( 12.9%) fetuses in the 160 mg/ m3 treatment group. Fetal body weight was unaffected at lower doses. The mean number of implantation sites, total fetal losses, resorptions, number of live fetuses, pregnancy rate, and fetal sex ratio were HCBD   February 2003 7 13 comparable in the treated and control groups. No exposure related external, visceral, or skeletal anomalies were noted in any dose group. The NOAEL for maternal toxicity is at 5 mg/ m3.

In dominant lethal tests in CD ( Sprague Dawley derived) rats, exposure to HCBD vapors at 10 or 50 ppm ( 107 or 533 mg/ m3) for 5 consecutive days, 7 hours/ day, did not affect fertility indices, number of corpora lutea or implantations, or the frequency of early death ( NIOSH, 1981).

For B6C3F 1 mice that were exposed to HCBD vapors at 107 or 533 mg/ m3, all animals in the high dose group died during the 5 week post treatment period ( NIOSH, 1981). The frequency of abnormal sperm morphology in the low dose group did not differ significantly from controls.

Intraperitoneal Injection Mated female Sprague Dawley rats ( 10   15 animals/ group) received 10 mg/ kg day HCBD in corn oil via intraperitoneal injection, during gestation days 1 to 15 ( Hardin et al., 1981). Maternal toxicity consisted of changes in two organ weights ( no further details provided). Decreased pre and post implantation survival was also noted. Developmental effects included decreased fetal weight or length, a 1 to 2 day delay in heart development, and dilated ureters. Gross external and internal examinations revealed no malformations ( WHO, 1994).

Harris et al. ( 1979) exposed pregnant female rats to 10 mg/ kg day HCBD from gestation days 1 to 15 via intraperitoneal injection. A 3 fold increase in soft tissue anomalies was reported in offspring of treated dams. No particular type of anomaly predominated ( U. S. EPA, 1991a).

7.2.6 Chronic Toxicity Data from a single chronic oral exposure study are available in the published literature. Kociba et al. ( 1977) provided male and female Sprague Dawley rats ( 39 to 40/ sex/ dose level; 90/ sex for controls) with diets that contained 0, 0.2, 2, or 20 mg/ kg day HCBD ( 99% pure) for 22 months ( males) or 24 months ( females). Parameters monitored included appearance and demeanor, body and organ weights, food consumption, hematologic and urine analysis, urinary porphyrins, serum clinical chemistry, and histopathology of major organs. The investigators reported significantly increased mortality in high dose males ( p< 0.05), but incidences were not given. Decreased body weight gain was noted in high dose males and females, with significant differences ( p< 0.05) from controls evident after day 27 ( females) or day 69 ( males). There were no apparent treatment related effects on food consumption. High dose animals had increased relative brain weights ( females) and relative testes weights ( males).

An important observation in the Kociba et al. ( 1977) study is the clear dose response relationship for HCBD induced renal toxicity. No discernible effects were noted at the 0.2 mg/ kgday dose. Effects noted at the intermediate dose of 2 mg/ kg day included increased urinary coproporphyrin excretion ( females only, days 427 428), and increased renal tubular epithelial hyperplasia. Lifetime ingestion of the 20 mg/ kg day dose resulted in increased urinary excretion of coproporphyrin and increased terminal weight of the kidneys in rats of both sexes ( females  days 728 729, males  days 377 378). The physiological relationship of a change in coproporphyrin HCBD   February 2003 7 14 excretion to renal toxicity is not currently clear. Microscopic examination revealed histopathological changes in the kidney, including hyperplasia and neoplasia of the renal epithelium. No incidence data was provided for the non neoplastic effects. HCBD related neoplastic changes are further discussed in Section 7.2.7. The lowest dose of 0.2 mg/ kg day was identified as the NOAEL in this study. The LOAEL was 2 mg/ kg day.

7.2.7 Carcinogenicity Oral Exposure Kociba et al. ( 1977) observed the tumorigenic potential of HCBD in male and female rats fed 0, 0.2, 2, or 20 mg/ kg day in a 2 year oral exposure study. No adverse effects attributable to HCBD were observed in the low dose group. Ingestion of the intermediate 2 mg/ kg day dose resulted in signs of renal tubular epithelial hyperplasia, but no evidence of neoplasia was observed. Ingestion of 20 mg/ kg day for 2 years resulted in development of renal tubular adenomas and adenocarcinomas. Neoplasms were observed in approximately 23% ( 9/ 39) of the males and 15% ( 6/ 40) of the females at 20 mg/ kg day, compared with 1.1% ( 1/ 90) and 0% ( 0/ 90) in control male and female rats and 0% at 0.2 or 2 mg/ kg day. Of the observed neoplasms, 7 in the high dose males and 3 in the high dose females were malignant, the rest being benign. Combined incidence of renal tubular benign and malignant tumors was significantly increased when compared to controls ( p< 0.05) for both males and females. Metastasis to the lungs was observed in two of the treated animals. An important observation in this study was that HCBD induced neoplasms occurred only at a dosage level that caused substantial renal tissue injury. Additional details of this study are provided in Section 7.2.6.

In a Russian study cited through secondary sources ( USEPA, 1991a), Chudin et al. ( 1985) conducted an HCBD oral exposure study in male Wistar rats for approximately 1 year. The doses of HCBD, administered by gavage in sunflower oil, were 0.6, 5.8 or 37 mg/ kg day ( n= 43, 43, and 41, respectively). Control rats were either untreated ( n= 90) or received only the sunflower oil vehicle ( n= 46). Some benign tumors of the kidney and liver were reported.

Nakagawa et al. ( 1998) investigated the effect of HCBD on renal carcinogenesis in male Wistar rats pre treated with N ethyl N hydroxyethylnitrosamine ( EHEN). EHEN is a known nephrocarcinogen in rats, where it selectively induces renal tubular cell tumors. The purpose of this study was to evaluate the ability of HCBD to act as a promoting stimulus following subthreshold exposure to EHEN. HCBD was administered for 30 weeks at a concentration of 0.1% by weight ( dose calculated to be1,000 mg/ kg) in the diet of rats ( 12/ treatment group) that had previously received 0.1% EHEN in the drinking water. The combined treatment with HCBD and EHEN resulted in a significantly higher renal tumor incidence than did administration of EHEN alone. Rats treated with HCBD alone did not develop renal tumors under the conditions used in this investigation. Significantly increased levels of bromodeoxyuridine ( BrdU) labeling indicated increased cell proliferation in the outer stripe and cortex of kidneys from HCBD treated rats. In a parallel experiment, immunostaining for proliferating cell nuclear antigen ( PCNA) was used to estimate nuclear DNA synthesis in defined renal tubular segments of HCBD treated rats. A significant increase in the number of PCNA positive cells was noted only in the outer stripe. These results are HCBD   February 2003 7 15 consistent with the outer stripe as a site for renal lesions induced by HCBD. Nakagawa et al. ( 1998) concluded that the ability of HCBD to induce EHEN initiated carcinogenesis appears to be associated with nephropathy and subsequent cell proliferation.

Dermal Exposure Van Duuren et al. ( 1979) evaluated the carcinogenicity of dermally applied HCBD in female Swiss mice ( 30 animals/ group). The investigators applied 6.0 mg of HCBD in acetone to shaved dorsal skin three times per week for a duration of 440 to 594 days. The treatment did not increase the incidence of papillomas or carcinoma at the site of application, or the incidence of tumors at distant sites such as the lung, stomach or kidney.

Van Duuren et al. ( 1979) also evaluated HCBD in an initiation promotion experiment. Female Swiss mice ( 30 animals/ group) received a single application of 15.0 mg HCBD in acetone on shaved dorsal skin. Fourteen days after HCBD application, dermal applications of 5 µ g of the tumor promoter 12 o tetradecanoylphorbol 13 acetate ( TPA) were administered to the test site three times per week for a total duration of 428 to 576 days. The incidence of skin papillomas in HCBDtreated animals was comparable to that in controls.

Intraperitoneal Injection Theiss et al. ( 1977) investigated the carcinogenic potential of HCBD by assessment of the pulmonary tumor response in male strain A/ St mice. Twenty animals per dose group were given intraperitoneal injections of 4 or 8 mg/ kg HCBD in tricaprylin, three times per week for a total of 13 and 12 injections, respectively. The total injected dose was 52 or 96 mg HCBD per animal. All surviving animals were killed 24 weeks after the first injection, and were examined for pulmonary surface adenomas. The tumor incidences were similar in treated and control groups. However, the use of this study for the evaluation of the carcinogenicity of HCBD is limited by the use of a mouse strain that is highly predisposed to spontaneous lung cancer, the small number of animals per dose group, the parenteral route of administration, the limited scope of histopathological evaluation ( WHO, 1994), and short study duration ( 6 months).

7.3 Other Key Data 7.3.1 Mutagenicity/ Genotoxicity The mutagenicity of HCBD has been evaluated in an array of in vivo and in vitro assays. The results of these tests are summarized below by category. No information was located regarding the genotoxic effects of HCBD in humans.

Bacterial Test Systems Test results from bacterial assays of mutagenicity are summarized in Table 7 2. Most tests in standard S. typhimurium reverse mutation assays have been negative, with or without S9 activation ( Rapson et al., 1980; Reichert et al., 1983; Stott et al., 1981; DeMeester et al., 1981; Haworth et al., HCBD   February 2003 7 16 1983; Chudin et al., 1985) except for a study by Simmon ( 1977) in which a positive response in Salmonella typhimurium was reported in the presence of metabolic activation induced by rat liver S9 fraction ( U. S. EPA, 1991a). Conflicting results in standard assays may be due to contaminants in technical and even analytical grade HCBD ( Reichert et al., 1984; Vamvakas et al., 1988). Vamvakas et al. ( 1988) observed 98% pure HCBD was a direct acting mutagen in S. typhimurium TA 100; but after HCBD was purified to 99.5%, a negative mutagenic response was obtained.

Positive results have been reported for HCBD when pre incubated with both rat liver microsomes and glutathione, but not when either was omitted ( Vamvakas et al., 1988; Roldan Arjona et al., 1991). The mutagenic response was increased by additional inclusion of rat kidney microsomes, mitochondria, or cytosol as ( glutamyl transpeptidase and dipeptidase sources, and reduced by addition of a   lyase inhibitor ( Vamvakas et al., 1988). Reichert et al. ( 1984) also reported a positive response in S. typhimurium when a " fortified" S9 mix, containing 3 fold more S9 protein than standard Ames test protocols, was utilized.

Test results for mutagenicity assays of HCBD metabolites are summarized in Table 7 3. Positive results in bacterial reverse mutation assays have been obtained for the mono glutathione and mono cysteine conjugates of HCBD ( Green and Odum, 1985; Dekant et al., 1986; Vamvakas et al., 1988) and the mercapturic acid, N acetyl S pentachlorobutadienyl L cysteine ( Reichert and Schutz, 1986; Wild et al., 1986). Other potential HCBD metabolites that gave positive results in reverse mutation assays were pentachloro 3 butenoic acid and pentachloro 3 butenoic acid chloride ( Reichert et al., 1984), in the absence of S9 activation. When rat kidney fractions were used for metabolic activation, the addition of a specific inhibitor of $ lyase ( aminooxyacetic acid) to the system reduced the mutagenic response ( Vamvakas et al., 1988), indicating that HCBD metabolites mediated mutagenesis in these assays. These results are consistent with the proposed mechanism for bioactivation of HCBD in animals ( Figure 6 1, Section 6.3).

In Vitro Mammalian Cell Test Systems Data for mutagenicity assays in mammalian test systems are summarized in the upper portion of Table 7 4. Treatment with HCBD did not increase the frequency of chromosome aberrations in Chinese hamster ovary ( CHO) cells ( Galloway et al., 1987) or cultured human lymphocytes ( German, 1988). However, Galloway et al. ( 1987) observed a significant increase in sister chromatid exchange in CHO cells treated with HCBD. Schiffman et al. ( 1984) reported unscheduled DNA synthesis ( UDS) activity and morphological transformation in Syrian hamster embryo fibroblasts with and without metabolic activation. Stott et al. ( 1981) reported negative results in a rat primary hepatocyte UDS assay.

Vamvakas et al. ( 1989) evaluated the genotoxicity of the HCBD metabolite S( 1,2,3,4,4 pentachlorobutadienyl) glutathione ( PCBG) in cultured porcine kidney LLC PK cells. Incubation of confluent monolayers with PCBG resulted in a dose dependent induction of DNA repair. Addition of either acivicin, an inhibitor of ( glutamyl transpeptidase, or aminooxyacetic acid, an inhibitor of cysteine conjugate $ lyase, prevented PCBG induced genotoxicity. These results are consistent with the hypothesis that renal metabolism plays a key role in PCBG induced genotoxicity. HCBD   February 2003 7 17 Table 7 2. Mutagenicity of HCBD in Salmonella typhimurium Test Systems.

Strain Conditions Resultsb Reference With S9 Activation Without S9 Activation TA100 TA 1535  + + Simmon ( 1977)

TA100 purity not reported nd  Rapson et al. ( 1980)

TA100 purity > 99%   Stott et al. ( 1981)

TA98 TA100 TA1530 TA1535 TA1538 purity 98%, plate incorporation   De Meester et al. ( 1981)

TA100 purity > 99%, plate incorporation   Stott et al. ( 1981)

TA98 TA100 purity not reported, suspension test   Reichert et al. ( 1983)

TA98 TA100 TA1535 TA1537 purity not reported, preincubation test   Haworth et al. ( 1983)

+ fortified S9* a,

purity > 99.5%, preincubation test + nd TA100 TA1535 TA1538 +/ rat liver S9, purity not reported, plate incorporation   Chudin et al. ( 1985)

TA100 +/ rat liver S9, preincubation test   Reichert et al. ( 1984)

+ rat liver S9*, preincubation test +

TA100 + rat liver S9*, purity 99.5%, preincubation test + c nd Wild et al. ( 1986) Table 7 2 ( continued)

Strain Conditions Resultsb Reference With S9 Activation Without S9 Activation HCBD   February 2003 7 18 TA100 purity 99.5% nd + Vamvakas et al. ( 1988)

purity 99.5%, preincubation test nd

TA100 + rat liver microsomes without additional GSH  nd Vamvakas et al. ( 1988)

+ rat liver microsomes and additional GSH,

purity 99.5%,

plate incorporation + d nd TA100 purity 98%, preincubation test  + Roldan Arjona et al. ( 1991)

Source: adapted from WHO ( 1994)

a S9* = a fortified S9 mix containing 3 times the normal protein concentration; GSH = reduced glutathione b + = $ twice the background rate or, in the case of bacterial studies, a reproducible dose related increase in the number of revertants per plate;  = negative; nd = not determined c 0.23 revertants per nmol d Addition of rat kidney microsomes further increased the number of revertants; positive results were inhibited by the $ lyase inhibitor aminooxyacetic acid HCBD   February 2003 7 19 Table 7 3. Mutagenicity of HCBD Metabolites.

Metabolitea Conditions Resultc Reference With S9 Activation Without S9 Activation PCBG no activation nd  Green & Odum ( 1985)

+ rat kidney S9 + nd Green & Odum ( 1985)

+/ rat kidney fractions + d + d Vamvakas et al. ( 1988)

TCBG +/ rat kidney fractions   Vamvakas et al. ( 1988)

TCBC +/ rat kidney fractions   Vamvakas et al. ( 1988)

PCBC +/ rat kidney S9 + e + e Green & Odum ( 1985)

no activation nd + f Dekant et al. ( 1986)

N AcPCBC  rat liver S9 nd  Wild et al. ( 1986)

+ rat liver S9 + nd + rat liver S9 + nd Reichert and Schutz ( 1986)

PCCMT + rat liver S9* b  nd Wild et al. ( 1986)

PCMT + rat liver S9  nd Wild et al. ( 1986)

PCBA + rat liver S9 + + Reichert et al. ( 1984)

PCBAC + rat liver S9 + + Reichert et al. ( 1984)

Source: modified from WHO ( 1994)

aAbbreviations: PCBG S( 1,1,2,3,4 pentachlorobutadienyl) glutathione TCBG 1,4 bis( 1,2,3,4 tetrachlorobutadienyl) glutathione TCBC 1,4 bis ( 1,2,3,4 tetrachlorobutadienyl) L cysteine PCBC S( 1,1,2,3,4 pentachlorobutadienyl) L cysteine N AcPCBC N acetyl S( 1,1,2,3,4 pentachlorobutadienyl) L cysteine PCCMT 1,1,2,3,4 pentachlorobutadiene carboxymethylthioether PCMT 1,1,2,3,4 pentachlorobutadiene methylthioether PCBA 2,2,3,4,4 pentachloro 3 butenoic acid PCBAC 2,2,3,4,4 pentachloro 3 butenoic acid chloride b S9* = a fortified S9 mix containing 3 times the normal protein concentration; GSH = reduced glutathione c + = twice background rate;  = negative; nd = not detectable d Mutagenic potency enhanced by rat kidney microsomes or mitochondria and less so by cytosol; positive results e Mutagenic potency enhanced by rat kidney microsomes or mitochondria and less so by cytosol; positive results f Mutagenic potency enhanced by the   lyase inhibitor aminooxyacetic acid HCBD   February 2003 7 20 Table 7 4. Genotoxicity of HCBD in Eukaryotic Assay Systems.

Species/ Strain/

Cell Type Compound End Point Comments Resultsa Reference In Vitro Assays Chinese hamster ovary cells HCBD Chromosome aberrations +/ rat liver S9  Galloway et al. ( 1987)

Human lymphocytes HCBD Chromosome aberrations +/ rat liver S9  German ( 1988)

Chinese hamster ovary cells HCBD Sister chromatid exchange +/ rat liver S9 + Galloway et al. ( 1987)

Syrian hamster embryo fibroblast HCBD Unscheduled DNA synthesis +/ activation + Schiffmann et al. ( 1984)

Porcine kidney cells PCBG Unscheduled DNA synthesis Addition of acivicin or aminooxyacetic acid gave negative results + Vamvakas et al. ( 1989)

Syrian hamster embryo fibroblasts HCBD Morphological transformation +/ activation + Schiffmann et al. ( 1984)

Rat primary hepatocyte HCBD Unscheduled DNA synthesis  Stott et al. ( 1981)

In Vivo Assays Mouse; bone marrow cells HCBD Chromosome aberrations Inhalation, 4h + German ( 1988)

Mouse; bone marrow cells HCBD Chromosome aberrations Oral gavage + German ( 1988)

Rat; bone marrow cells HCBD Chromosomal aberration Dietary doses of up to 20 mg/ kg day for 148 days  Schwetz et al. ( 1977) Table 7 4 ( continued)

Species/ Strain/

Cell Type Compound End Point Comments Resultsa Reference HCBD   February 2003 7 21 Rat; bone marrow cells HCBD Chromosomal aberrations 10 or 50 ppm, 7 hrs/ day,

1 or 5 days  NIOSH ( 1981)

Rat HCBD Dominant lethality 10 or 50 ppm, 7 hrs/ day,

5 days  NIOSH ( 1981)

Rat kidney cells HCBD DNA alkylation 20 mg/ kg by gavage + Stott et al. ( 1981)

Rat kidney cells HCBD DNA repair 20 mg/ kg by gavage + Stott et al. ( 1981)

Mouse HCBD DNA binding Single dose, 30 mg/ kg + b Schrenk and Dekant ( 1989)

Drosophila melanogaster HCBD Gene mutation (

sexlinked recessive lethal)  NIOSH ( 1981)

Drosophila melanogaster HCBD Sex linked lethals Feeding or injection  Woodruff et al. ( 1985)

Source: modified from ATSDR ( 1994) and WHO ( 1994).

a + = $ twice the background rate or, in the case of bacterial studies, a reproducible dose related increase in the number of revertants per plate;  = negative;

nd = not determined;

b Binding was predominately to mitochondrial DNA HCBD   February 2003 7 22 In Vivo Test Systems Results of in vivo HCBD genotoxicity tests are summarized in the lower portion of Table 7 4. Both negative ( NIOSH, 1981; Schwetz et al., 1977) and positive ( German, 1988) results have been reported for chromosome aberration assays conducted in HCBD treated mice or rats. Negative findings have been reported in a dominant lethal assay in rats ( NIOSH, 1981), and in the Drosophila melanogaster sex linked recessive lethal mutation assay with exposure via either injection or feeding ( NIOSH, 1981; Woodruff et al., 1985). However, Stott et al. ( 1981) reported a small ( 1.25 to 1.54 fold) increase in UDS activity and DNA alkylation ( 0.78 alkylation per 106 nucleotides) in kidney cells from rats fed 20 mg/ kg day HCBD in the diet for 3 weeks, suggesting that HCBD exhibited a minor degree of renal genotoxicity.

Schrenk and Dekant ( 1989) evaluated the covalent binding of HCBD metabolites to renal and hepatic DNA in NMRI mice. A low level of covalent binding ( covalent binding index ( CBI) = 27) was observed with nuclear DNA ( nDNA) isolated from the kidney, while covalent binding was undetectable in nDNA isolated from liver. Significantly higher levels of covalent binding were observed with mitochondrial DNA ( mtDNA), with CBIs of 513 and 7,506 determined for liver and kidney, respectively. Analysis of covalent binding to renal mtDNA identified three 14C labeled compounds that appeared to be DNA bases altered by HCBD metabolites.

7.3.2 Immunotoxicity The immunological effects of HCBD have not been systematically evaluated in humans, and there are currently no case reports that describe immunological abnormalities occurring in humans exposed to HCBD.

Animal data on the immunological effects of HCBD are limited. In a 2 week oral exposure study conducted by NTP ( 1991), depletion ( atrophy) and necrosis of lymphoid tissues of the spleen, thymus, and lymph nodes was observed in B6C3F 1 mice administered lethal concentrations of 1,000 and 3,000 ppm of HCBD. However, the investigators noted that these lesions may have been secondary to chemical induced stress. Similar lesions were not observed in mice administered 19.2 mg/ kg day in a subsequent 13 week study conducted by NTP ( 1991).

In a 13 week gavage study, relative spleen weights were significantly increased in male rats orally administered HCBD at 15.6 mg/ kg day and in females at 6.3 mg/ kg day and above ( Harleman and Seinen, 1979). Treatment related lesions in lymphoid organs ( thymus, lymph nodes, spleen) have not been reported in terminal necropsy of mice or rats in other HCBD subchronic and chronic oral exposure studies at doses up to 100 mg/ kg day ( Harleman and Seinen, 1979; Kociba et al. 1977; Schwetz et al., 1977). Immune function screening batteries in HCBD treated animals has not been evaluated.

Delayed hypersensitivity reaction was exhibited in guinea pigs to dermal HCBD application ( Gradiski et al., 1975). HCBD   February 2003 7 23 7.3.3 Hormonal Disruption No studies were identified that associate HCBD exposure with endocrine disruption.

7.3.4 Physiological or Mechanistic Studies Many studies have been undertaken to investigate the mechanisms underlying the effects of HCBD and, in particular, its toxicity in the proximal renal tubule. The mode of action for HCBD as determined from these studies and implications for cancer assessment are discussed in Section 7.4.3 The proximal tubule specific toxicity of HCBD is likely determined by two factors: 1) the distribution of enzymes required for its bioactivation, and 2) the ability of this region to concentrate precursors of the ultimate toxic species ( Dekant et al., 1990). The enzyme cysteine conjugate $ lyase is believed to catalyze the conversion of HCBD cysteine conjugates to a highly reactive thioketene metabolite ( Figure 6 1, Section 6.3). Multiple investigators have addressed the localization of $ lyase and its relationship to nephrotoxicity. MacFarlane et al. ( 1989) demonstrated by immunocytochemical technique that the region of highest cytosolic $ lyase activity in untreated rats coincides with the site of HCBD induced necrosis in the pars recta region of the proximal tubule. However, Jones et al. ( 1988) and Kim et al. ( 1997) detected $ lyase in the entire proximal tubule. Trevisan et al. ( 1998) detected histopathological changes and increased levels of $ lyase activity in the urine following treatment of rats with S 3 and S 1 S 2 specific nephrotoxicants, which were cited as evidence for distribution of the enzyme along the entire length of the proximal tubule. These data suggest that additional factors may contribute to selective damage in the pars recta.

The ability of the proximal tubule to concentrate HCBD metabolites has been investigated as a factor in renal toxicity. Nash et al. ( 1984) administered a single dose of radiolabeled HCBD and observed that radioactivity was concentrated in the renal cortex shortly after dosing. Renal cells that concentrated the radiolabeled compounds were subsequently observed to undergo necrosis. In mammals, ( glutamyltranspeptidase, the enzyme that together with dipeptidase catalyzes the conversion of the glutathione conjugate to the cysteine conjugate, is concentrated in the brush border membrane of the proximate tubular cells. The distribution of this enzyme may also contribute to an increase in the concentration of cysteine conjugates in the proximal renal tubules.

The probenecid sensitive organic anion transporter that is present on the basolateral side of proximal tubule cells appears to play a role in the accumulation of HCBD metabolites ( Dekant 1996). Probenecid is a competitive inhibitor of organic anion transport, and is reported to be without effect on energy metabolism, transport carrier synthesis, or uptake of other substances actively transported by the kidney ( Lock and Ishmael, 1985; Dekant, 1996). Haloalkene derived mercapturates have the highest affinity for the organic anion transporter, but glutathione and cysteine S conjugates with lipophilic substituents on sulfur are also substrates for transport ( Dekant, 1996).

The effect of probenecid on development of HCBD induced renal toxicity has been investigated in in vivo and in vitro studies. Lock and Ishmael ( 1985) administered a single intraperitoneal dose of 16 or 64 : mol/ kg 14C radiolabeled N acetyl pentachlorobutadienyl L cysteine to female Alpk/ AP rats and observed acute renal necrosis within four hours. Prior administration of HCBD   February 2003 7 24 up to 500 : mol/ kg probenecid reduced renal cortical concentrations of radioactivity and provided protection against nephrotoxicity in a dose dependent manner as assessed by plasma urea concentration and renal histopathology. Pretreatment with probenecid also reduced or prevented the toxic effects of intraperitoneally injected HCBD and its glutathione and cysteine conjugates. Thus, the selective toxicity to the pars recta in rats is thought to result in part from transport of HCBD metabolites into cells of this region via a probenecid sensitive transport system.

Bach et al. ( 1986) confirmed antagonism of probenecid in HCBD metabolite induced toxicity in freshly isolated rat proximal tubule fragments. Incorporation of 3H proline into acid precipitable protein was utilized as an indicator of synthetic capacity of the tubular fragment. Addition of 2 mM N acetyl pentachlorobutadienyl L cysteine to the incubation medium reduced 3H proline incorporation to 34% of the control value. The inclusion of 400 : M probenecid in the incubation medium almost completely restored ( to 95%) 3H proline incorporation.

Multiple studies suggest that renal cortical mitochondria are a primary subcellular target for HCBD toxicity. Jones et al. ( 1986) investigated the toxic effects of pentachlorobutadienylglutathione ( PCBG) in isolated rat renal epithelial cells. Exposure to PCBG decreased cell viability and reduced the concentration of intracellular thiols. Other PCBG related effects included depletion of Ca2+ from the mitochondrial compartment, an elevation of cytosolic Ca2+ concentration, inhibition of respiration, and decreased levels of ATP. Prevention of PCBG bioactivation by inhibition of ( glutamyl transpeptidase or $ lyase provided complete protection against cytotoxicity. The authors hypothesized that PCBG induced renal cell injury results from selective effects on mitochondrial function, including inhibition of respiration, depression of ATP synthesis, and release of mitochondrial calcium ( II) ions.

Wallin et al. ( 1987) studied S pentachlorobutadienyl L cysteine ( PCBC) toxicity in mitochondria isolated from the rat kidney cortex. Respiring mitochondria exposed to PCBC showed a dose dependent loss of ability to retain calcium. This effect was associated with a collapse of mitochondrial membrane potential. A slow nonenzymatic depletion of mitochondrial glutathione was also observed. Preincubation with aminooxyacetic acid, an inhibitor of $ lyase, effectively counteracted the loss of glutathione, suggesting that an interaction of the reactive thioketene with the mitochondrial inner membrane was responsible for the observed effects.

Schnellmann et al. ( 1987) investigated the mechanism of PCBC induced toxicity in renal proximal tubules isolated from New Zealand rabbits. Suspensions of isolated tubules were exposed to concentrations of 20 to 500 : M PCBC in the presence or absence of chemicals which targeted the activity of ion channel ATPases ( nystatin, ouabain), Cytochrome c ( ascorbate, tetramethylphenylenediamine), cytochrome oxidase ( carbonyl cyanide ptrifluoromethoxyphenylhydrazone and/ or ATP synthase ( atractyloside or oligomycin). Fifteen minutes of exposure to 200 : M PCBC caused an increase in basal and ouabain insensitive respiration but not nystatin treated respiration. However, sixty minutes of PCBC exposure inhibited basal, nystatin stimulated and ouabain insensitive respiration, and resulted in a 79% decrease in glutathione concentration. In addition, at 60 minutes, an 11% decrease in lactate dehydrogenase retention was observed, suggesting that cell viability was decreased over time as a result of treatment. The changes in respiration observed at 60 minutes appeared to result from gross HCBD   February 2003 7 25 mitochondrial damage characterized by inhibition of state 3 ( aerobic) respiration, depression of cytochrome c/ cytochrome oxidase activity, and inhibition of electron transport. The results of these studies suggest that alterations in mitochondrial function are an early event in PCBC mediated toxicity.

A similar pattern of events was observed by Groves et al. ( 1991). These workers investigated the relationship between uptake and covalent binding of the HCBD metabolite pentachlorobutadienyl L cysteine ( PCBC) in rabbit renal proximal tubules, renal membrane vesicles, and isolated renal cortical mitochondria. Their findings confirmed the PCBC induced pattern of mitochondrial dysfunction previously observed by Schnellmann et al. ( 1987) in rabbit proximal tubule suspensions. Furthermore, Groves et al. ( 1991) demonstrated the rapid accumulation of 35SPCBC in renal proximal tubule cells and its metabolism to a reactive intermediate that bound to tubular protein. An estimated 70 to 90% of the intracellular radioactivity was bound to protein. Mitochondria isolated from renal proximal tubules also metabolized 35S PCBC to a reactive intermediate that bound to mitochondrial protein, consistent with the mitochondrion being a critical subcellular target for HCBD induced toxicity. Addition of the $ lyase inhibitor aminooxyacetic acid ( AOAA) reduced covalent binding to tubular proteins, and blocked the toxic effects of PCBC on isolated mitochondria. However, AOAA decreased but did not prevent the toxic effects PCBC on respiration and cellular ATP levels induced by PCBC exposure.

Additional studies have investigated interactions between the reactive intermediate generated by metabolism of PCBC and cellular macromolecules. Lock and Schnellmann ( 1990) examined the ability of reactive thiols formed by the action of $ lyase on cysteine conjugates of several haloalkenes, including HCBD, to inhibit renal enzymes. The activities of glutathione reductase ( a cytosolic enzyme) and lipoyl dehydrogenase ( a mitochondrial enzyme) were assayed for this purpose. Administration of 200 mg/ kg HCBD to male rats by intraperitoneal injection resulted in inhibition of both enzymes. The authors suggested that such inhibition is a general outcome of PCBC exposure, and is likely to occur with a diverse range of renal enzymes.

Schrenk and Dekant ( 1989) investigated covalent binding of 14C labeled HCBD metabolites to mouse DNA after a single oral dose of 30 mg/ kg 14C HCBD. HCBD metabolites bound extensively to mitochondrial DNA. In contrast, little binding to nuclear DNA was observed. The study authors suggested that proximity to high $ lyase concentration in the mitochondrial membrane and the absence of associated histones make mitochondrial DNA a more vulnerable target for reactive HCBD metabolites.

As noted above ( Section 6.3), the cysteine derivative of HCBD is a substrate for cysteine conjugate $ lyase. The activity of $ lyase leads to formation of an enethiol intermediate which is rapidly converted to thioketene, a potent acylating agent ( Dekant et al., 1990). In rats, the enethiol intermediate may be detoxified by methylation to form pentachlorobutadienyl methylthioether. Morel et al. ( 1999) investigated the role of S adenosyl methionine ( SAM) dependent thiol methylation in prevention of HCBD induced nephrotoxicity in male Swiss OF1 mice. The mice were treated with a single intraperitoneal dose of periodate oxidized adenosine ( ADOX) prior to administration of a single intraperitoneal dose of 80 or 100 mg HCBD/ kg. Pretreatment with ADOX increased the level of SAM in the liver and kidney approximately four fold, but did not modify the HCBD   February 2003 7 26 nephrotoxicity of HCBD as determined by histopathological evaluation of renal proximal tubules. This result was interpreted by the study authors as evidence that SAM dependent thiol methylation does not play a role in detoxification of HCBD derived enethiol in mice.

Chemically induced " 2: globulin nephropathy represents a potential alternative mechanism for HCBD toxicity in rats. Since " 2: globulin synthesis is androgen dependent in the liver, this form of nephropathy occurs exclusively in male rats, and is characterized by the accumulation of hyaline droplets in proximal tubule cells. Binding of the chemical to " 2: globulin is a prerequisite for development of nephrotoxicity. Because HCBD induced renal toxicity occurs in both male and female rats, it is evident that " 2: globulin nephropathy is not required for nephrotoxicity. However, there is limited evidence to suggest that the " 2: globulin mechanism may contribute to HCBDinduced nephrotoxicity observed in male rats. Birner et al. ( 1995) observed that unmetabolized HCBD was excreted in the urine of male, but not female, Wistar rats following exposure to a single gavage dose of 14C HCBD in corn oil. The study authors also noted more pronounced necrotic changes in the proximal tubules of male rats when examined 48 hours after treatment. Slight liver damage was observed only in male rats. In a subsequent experiment in the same laboratory, Pähler et al. ( 1997) orally administered 200 mg/ kg 14C HCBD in corn oil to Sprague Dawley ( SD) and NCI Black Reiter rats ( NBR), an " 2: globulin deficient strain.. 14C HCBD was present only in the urine of male SD rats, but not NBR rats. The study authors determined that the excreted HCBD detected in the urine of male SD rats was associated with its binding to " 2: globulin. Histopathological examination 48 hours after treatment revealed the formulation of hyaline droplets indicative of " 2: globulin accumulation in renal epithelial cells of male SD rats, but not in male NBR rats. In addition, microscopic examination confirmed the occurrence of more extensive nephropathy in male than in female animals as previously observed in Wistar rats.

Saito et al. ( 1996) established that dose dependent levels of kidney type " 2: globulin (" G K) in the urine are a reliable predictor of " 2: globulin accumulation in the kidney. These investigators subsequently administered 100 mg/ kg day HCBD to adult male Sprague Dawley rats for five consecutive days. No increase in urinary " G K was detected following exposure, suggesting that HCBD treatment did not induce a marked accumulation of " 2: globulin. However, histopathological examination revealed some epithelial cells showing hyaline droplet related degeneration. The size of the hyaline droplets formed following HCBD treatment were generally smaller than those observed after treatment with the well characterized " 2: globulin nephropathy inducing agent dlimonene

No significant increase in " 2: globulin was observed in kidney cytosol prepared from Fischer 344/ N rats treated with a single oral dose of 200 mg HCBD/ kg when assayed by Western blot and capillary electrophoresis ( Pähler et al., 1999).

" 2: globulin accumulation is unique to male rats, and when in excess is associated with nephropathy, cytotoxicity, cellular proliferation, and consequent renal tumors. HCBD does not cause an accumulation of " 2: globulin in the male rats ( Saito et al., 1996; Pähler et al., 1999). Moreover, HCBD clearly causes kidney damage in both male and female rats and in mice ( Kociba et al., 1977; NTP, 1991). With respect to kidney tumors, Kociba ( 1977) shows that kidney tumors are found in both HCBD treated male and female rats. Based upon current EPA guidelines ( U. S. EPA, 1991e), HCBD   February 2003 7 27 " 2: globulin accumulation does not appear to be a mode of action of HCBD induced kidney toxicity in rats or relevant to human risk.

7.3.5 Structure Activity Relationship HCBD appears to share a common target tissue with several structurally related haloalkenes, including perfluoropropene, trichloroethene, tetrachloroethene, and trichlorotrifluoropropene. All of these chemicals demonstrate dose dependent toxicity in the proximal tubule. Trichloroethene, tetrachloroethene, tetrafluoroethylene, and HCBD have all been found to induce neoplasia of the proximal tubule in rats. The common basis for toxicity may be bioactivation of these compounds by a multistep pathway which is initiated by conjugation with glutathione, resulting in the formation of a glutathione S conjugate. Metabolism to the corresponding cysteine S conjugates, and subsequent degradation by renal cysteine conjugate $ lyase, yields reactive electrophiles that are believed to be ultimately responsible for renal toxicity. These electrophiles alkylate mitochondrial macromolecules, resulting in cellular energy deficit, loss of membrane potential, and disruption of calcium homeostasis.

When haloalkenes are considered as a group, the extent of conjugation is much higher with liver microsomes than with liver cytosol, in contrast to results observed with most other substrates. This effect is attributed to the preferential distribution of the highly lipophilic haloalkenes into lipid membranes, thus providing high substrate concentrations for membrane bound glutathione Stransferase ( Dekant et al., 1990). In vitro studies suggest that rates of haloalkene conjugation correlate well with the chemical reactivity of the individual compounds ( Dekant et al., 1990). For example, substitution with chlorine results in stabilization of a B bond. Chloroalkenes are thus reported to be more resistant to metabolism by glutathione conjugation than are fluoroalkenes.

Investigations have compared the toxicity of structurally related haloalkene conjugates. Lock and Schnellmann ( 1990) investigated the effect of HCBD and other haloalkene cysteine conjugates on renal glutathione reductase and lipoyl dehydrogenase activity, and concluded that inhibition of these enzymes by the reactive thiols formed by $ lyase cleavage of haloalkene cysteine conjugates represented a general mechanism of toxicity.

Green and Odum ( 1985) investigated the nephrotoxicity and mutagenicity of the cysteine conjugates of halogenated alkenes in rat kidney slices. Compounds investigated included the chloroalkenes HCBD, trichloroethylene and perchloroethylene, and the fluoroalkenes hexafluoropropene ( HFP) and tetrafluoroethylene ( TFE). All of these conjugates had a marked effect on the uptake of both the organic anion p aminohippuric acid ( PAH) and the cation tetraethylammonium bromide ( TEA) into rat kidney slices. This observation was considered to be consistent with the known nephrotoxicity of HCBD, TFE and HFP in vivo. Each of the conjugates was metabolized by rat kidney slices and by semi purified rat kidney $ lyase to pyruvate, ammonia, and an unidentified reactive metabolite. Although all of the conjugates were activated by $ lyase and had a similar effect on ion transport, their mutagenicity differed. The conjugates of HCBD, trichloroethylene and perchloroethylene were mutagenic in the Ames bacterial mutation assay when activated by rat kidney S9 fraction. In contrast, the conjugates of TFE and HFP were not mutagenic either in the presence or absence of rat kidney S9 fraction. HCBD   February 2003 7 28 Birner et al. ( 1997) compared the nephrotoxicity of cysteine S conjugates derived from trichloroethene, tetrachloroethene, and HCBD. Male and female rats received identical intravenous doses of S( 1,2 dichlorovinyl) L cysteine ( 1,2 DCVC), S( 2,2 dichlorovinyl) L cysteine ( 2,2 DCVC), S( 1,2,2 trichlorovinyl) L cysteine ( TCVC), or S( 1,2,3,4,4 pentachlorobutadienyl) Lcysteine ( PCBC). Assessment of the relative nephrotoxic potency of the conjugates by histopathological examination and excretion of ( glutamyltranspepidases in urine indicated a decrease in the order TCVC > 1,2 DCVC > PCBC $ 2,2 DCVC.

7.4 Hazard Characterization 7.4.1 Synthesis and Evaluation of Major Noncancer Effects There are no unconfounded reports of human health effects following HCBD exposure by any route. Oral exposure studies of HCBD toxicity in animals are summarized in Table 7 5. A distinctive feature of HCBD toxicity in animals is its selective effect on the kidney, regardless of the route of administration. Toxicity within the kidney is also selective, with damage restricted to the proximal tubule. In rats, damage is further localized to the pars recta region of the proximal tubule.

Subchronic and chronic studies in rodents present a clear picture of dose related renal damage. Progressive events over time include changes in kidney weight, renal tubular degeneration, necrosis and regeneration, hyperplasia, focal adenomatous proliferation, and tumor formation. Tumor formation occurs exclusively in the kidney, and only at doses that cause extensive cytotoxicity.

Evidence from metabolic enzyme inhibitor studies, cannulation experiments, and analysis of urinary metabolites indicates that the nephrotoxicity of HCBD is dependent on a multistep bioactivation mechanism involving both liver and kidney enzymes. The initial step in HCBD metabolism is the glutathione S transferase mediated biosynthesis of a glutathione conjugate ( PCBG) in the liver. After elimination into the bile, PCBG undergoes subsequent metabolism to a cysteine conjugate ( PCBC) in the bile, gut or kidneys. PCBC may be acetylated by renal N acetyltransferases to form a N acetyl cysteine conjugates ( N AcPCBC). Both PCBC and N AcPCBC are concentrated in renal cells via an active transport system ( Dekant, 1990). N AcPCBC can be excreted in the urine or de acetylated to regenerate PCBC. PCBC is a substrate for $ lyase dependent activation to a highly reactive thioketene in the kidney. Covalent binding of this reactive species to cellular macromolecules is believed to initiate the damage that ultimately results in renal cell toxicity.

Potential molecular targets for binding of the reactive thioketene include enzymes, membrane proteins, glutathione, phospholipids, and mitochondrial DNA. Localized damage to the proximal tubule is believed to reflect high $ lyase concentration in this region. Evidence from studies using the selective inhibitor probenecid suggests that accumulation of the cysteine and N acetyl cysteine conjugates via anion transport systems localized in this segment of the proximal tubule may account for this selective pattern of toxicity.

In vitro studies suggest that cortical mitochondria are the critical subcellular target for toxicity of the bioactivated sulfur conjugates of HCBD. Susceptibility of mitochondria to HCBD toxicity is linked to high concentrations of $ lyase associated with mitochondrial membranes. The HCBD   February 2003 7 29 Table 7 5. Summary of Principal HCBD Toxicity Studies.

Species/ Strain Number/

Sex/ Dose Route Frequency/

Duration Doses ( mg/ kg day) NOAEL ( mg/ kg day) LOAEL ( mg/ kg day) Effect( s) Reference ACUTE EXPOSURE Rat 6 M G Single Dose 24 hours 200  200 Increased plasma urea;

increased proteins and metabolites in urine Nash et al.

( 1984)

Rat / Wistar 5 M G Single Dose 24 hours 0, 10, 100,

200 10 100 Limited focal necrosis of the kidneys and other signs of renal damage in urine and blood Jonker et al.

( 1993a)

Rat / Sprague

Dawley 4 5 M G Single Dose 24 hours 0, 100, 200  100 Histological kidney lesions and impaired kidney function Payan et al.

( 1993)

SHORT TERM EXPOSURE Rat / Sprague

Dawley 4 F F Daily 30 days 0, 1, 3, 10, 30,

65, 100 3 10 ( reduced body wt.

gain; increased hemoglobin concentration) Increased relative kidney weight; renal tubular degeneration, necrosis and regeneration; increased hemoglobin concentration reduced body weight gain Kociba et al.

( 1971)

Rat / Wistarderived 6 M 6 F F Daily 14 days 0, 4.6, 14,

35.3  4.6 Degeneration of renal tubular epithelial cells,

localized to pars recta Harleman and Seinen ( 1979)

Rat / Sprague

Dawley 5 M G Daily 3 weeks 0, 0.2, 20 0.2 20 Histopathological indications of renal cortical damage; decreased body weight gain; increased relative kidney weight Stott et al.

( 1981) Table 7 5 ( continued)

Species/ Strain Number/

Sex/ Dose Route Frequency/

Duration Doses ( mg/ kg day) NOAEL ( mg/ kg day) LOAEL ( mg/ kg day) Effect( s) Reference HCBD   February 2003 7 30 Mouse /

B6C3F 1 5 M 5 F F Daily 15 days M: 0, 3, 12, 40 F: 0, 5, 16, 49  3 5 Renal necrosis and cellular regeneration NTP ( 1991)

Yang et al.

( 1989)

Rat / Wistar 5 M 5 F F Daily 4 weeks 0, 2.25, 8, 28 2.25 8 Renal tubular cytomegaly;

decreased plasma creatine;

decreased body weight,

decreased liver and adrenal weight Jonker et al.

( 1993b)

Rat / Wistar 12 M F Daily 3 weeks 0, 7.2, 36, 180 7.2 36 ( body weight)

180 ( kidney lesions) Lower mean body weight ( 15% decrease); extensive regeneration at 180 but not 36 mg/ kg day Nakagawa et al. ( 1998)

SUBCHRONIC EXPOSURE Rat / Wistarderived 10 M 10 F G Daily 13 weeks 0, 0.4, 1.0,

2.5, 6.3, 15.6 2.5 ( M)

1.0 ( F) 6.3 ( M)

2.5 ( F) Proximal tubular degeneration Harleman and Seinen ( 1979)

Mouse /

B6C3F 1 10 M 10 F F Daily 13 weeks M: 0, 0.1, 0.4,

1.5, 4.9, 16.8 F: 0, 0.2, 0.5,

1.8, 4.5, 19.2 1.5 ( M)

0.2? ( F) 4.9 ( M)

0.5 ( F) Renal tubular regeneration NTP ( 1991) Table 7 5 ( continued)

Species/ Strain Number/

Sex/ Dose Route Frequency/

Duration Doses ( mg/ kg day) NOAEL ( mg/ kg day) LOAEL ( mg/ kg day) Effect( s) Reference HCBD   February 2003 7 31 Rat / Sprague

Dawley Gross Pathology:

10 12 M,

20 24 F ( HCBD) 17 M, 34 F ( control) Histopatho logy:

5 M 5 F F Daily 0.2, 2.0, 20 0.2 2 Renal tubular hyperplasia Schwetz et al. ( 1977)

Rat / Wistar 21 M F Daily 30 weeks 90  90 Decreased final weight and increased relative kidney weight Nakagawa et al. ( 1998)

CHRONIC EXPOSURE Rat / Sprague

Dawley 39M, 40F F Daily 22 months ( M)

24 months ( F) 0, 0.2, 2, 20 0.2 2 Increased urinary coproporphyrin excretion ( females); increased kidney weight; renal tubular epithelial hyperplasia Kociba et al.

( 1977) Table 7 5 ( continued)

Species/ Strain Number/

Sex/ Dose Route Frequency/

Duration Doses ( mg/ kg day) NOAEL ( mg/ kg day) LOAEL ( mg/ kg day) Effect( s) Reference HCBD   February 2003 7 32 DEVELOPMENTAL AND REPRODUCTIVE STUDIES Rat / Sprague

Dawley 10 12 M,

20 24 F ( HCBD)

17M, 34 F ( control) F Daily 148 days ( 90 days prior to mating through postnatal day 21) 0, 0.2, 2, 20 2 20 Slightly decreased neonatal weight Schwetz et al. ( 1977)

Rat 6 F F Daily 3 weeks prior to mating; 3 weeks during mating; throughout gestation and lactation 0, 15, 150  15 Maternal toxicity ( renal,

neurological). Reduced pup weights on days 0, 10, and 20 Harleman and Seinen ( 1979)

Rat F NS NS 8.1  8.1 Reduced body weight;

shorter crown rump length;

ultrastructural chances in neurocytes; increased levels of free radicals in brain and spinal cord Badaeva ( 1983)

Abbreviations: Sex: M = male Route: G = gavage NS = Not specified F = female F = feed study HCBD   February 2003 7 33 reactive metabolite formed by $ lyase cleavage of sulfur conjugates is thought to interact with components of the inner mitochondrial membrane. Disruption of respiration and uncoupling of oxidative phosphorylation leads to a marked reduction of ATP levels in susceptible kidney cells, and ultimately necrosis.

The mechanism described above is believed to contribute to the renal damage observed in both male and female rats. However, additional mechanisms may contribute to nephrotoxicity of HCBD in male rats. Current evidence suggests that at least two discrete male specific pathways may participate in the more pronounced necrotic changes observed in the renal tubules of male rats in some studies. Formation of hyaline droplets indicative of " 2: globulin accumulation has been observed in the kidney of HCBD treated male rats. The significance of this finding for HCBDinduced nephrotoxicity remains to be determined. A second potential mechanism for male specific toxicity involves the cytochrome P450 3A mediated formation of an N acetylated cysteine conjugate sulfoxide.

Other noncancer effects associated with HCBD exposure in animals include developmental effects and neurotoxicity. Reproductive effects were observed only at maternal toxic dose. In one study, female Wistar rats were administered a diet containing 0, 15 or 150 mg/ kg day) HCBD for 3 weeks prior to mating, 3 weeks during mating and throughout gestation and lactation. Maternal toxicity was evident in treated groups. No conceptions occurred for the high dose group, the ovaries showed little follicular activity, and no uterine implantation sites were observed. At 15 mg/ kg day, pups exhibited lower birth weights and reduced growth compared to controls ( Harleman and Seinen, 1979). In a Russian study cited through secondary sources, pregnant rats administered 8.1 mg/ kg day of HCBD during gestation gave birth to pups with lower body weights and shorter crown rump lengths ( Badaeva, 1983).

Harleman and Seinen ( 1979) observed ataxia, incoordination, weakness of the hind legs, and unsteady gait in conjunction with demyelination and fragmentation of femoral nerve fibers in female rats consuming dietary dose of 150 mg/ kg day HCBD for 10 to 18 weeks. No neurotoxic effects were reported for rats consuming 15 mg/ kg day. In a Russian study cited through secondary sources, daily oral administration of 8.1 mg/ kg day HCBD to pregnant rats throughout gestation resulted in histopathological changes in nerve cells and myelin fibers of the spinal cord in treated dams and their offspring ( Badaeva et al., 1985).

The mode of neurotoxic action has not been studied. Toxicokinetic studies in animals following oral administration demonstrated that HCBD and its metabolites distributed to the brain and adipose tissues in addition to the kidney and the liver ( Reichert, 1983; Reichert et al., 1985; Dekant et al., 1988a). Thus, reported toxicity at targets other than the kidney may be related to the distribution of HCBD and/ or its metabolites to these targets and subsequent covalent binding of the reactive metabolites to cellular macromolecules.

An important issue in the evaluation of the hazard posed by HCBD concerns the applicability of mechanistic data obtained in rodent studies to humans. Limited data from in vitro studies with human renal cytosol and cultured human proximal tubule cells suggest that humans have the ability HCBD   February 2003 7 34 to form HCBD glutathione conjugates and to metabolize HCBD cysteine conjugates to a toxic metabolite.

7.4.2 Synthesis and Evaluation of Carcinogenic Effects No studies of the potential for HCBD carcinogenicity in humans have been reported. In animals, one lifetime exposure carcinogenicity study has been performed. Kociba et al. ( 1977) observed increased incidence of renal tumor formation in male and female rats following lifetime exposure to HCBD in the diet. Neoplastic changes occurred only at the highest dose, which exceeded the maximum tolerated dose ( MTD). There was increased mortality, significant weight loss ( greater than 10%), and severe renal toxicity. This pattern suggests that tumor formation may be secondary to HCBD induced cytotoxicity. This conclusion is supported by the study of Nakagawa et al. ( 1998), who found increased cell proliferation and increased DNA synthesis in the outer stripe and cortex of kidneys from HCBD treated rats.

However, available data must be considered as too limited to support a conclusion with high confidence. The widely spaced doses ( a 10 fold spacing between the highest and next lower dose) in the Kociba et al. ( 1977) study, for example, did not provide the opportunity to confirm that pronounced cytotoxicity is a prerequisite for tumorigenesis. Additional limitations in the database include the absence of cell proliferation studies and limited in vivo data for mutagenesis. These limitations prevent the use of cell kinetic multistage ( CKM) models in the analysis of cancer risk ( Bogen, 1989).

Results from mutagenicity studies with HCBD are mixed. In the presence of appropriate metabolic activation conditions, HCBD and its metabolites are mutagenic in some, but not all, studies. Thus, a genotoxic mode of action must be considered. The observation that high doses of HCBD metabolites can bind to DNA weakly in vivo in mice ( Schrenk and Dekant, 1989) strengthens this conclusion somewhat. There is, however, still some question as to whether genotoxicity is the primary mode of action at the lower concentrations at which nephrotoxic effects are first observed. The weight of evidence suggests that both genotoxic and nongenotoxic effects contribute, possibly at different dose levels, and that the current database is insufficient to disentangle the relative contributions of these effects.

7.4.3 Mode of Action and Implications in Cancer Assessment Both sustained cytotoxic damage and irreversible DNA binding have been proposed as events in HCBD renal carcinogenesis in rodents ( Stott et al., 1981) An. A lifetime oral study in rats showed kidney tumors at a very high dose that exceeded the MTD, suggesting that HCBD induced cytotoxicity may lead to tumor formation ( see Section 7.4.2). Studies in rats and mice indicate that kidney is the target organ. Progressive toxicological changes are observed in kidney over time: decreased and increased kidney weight, increased excretion of coporphyrin ( kidney dysfunction), renal tubular degeneration, necrosis and regeneration, hyperplasia, focal adenomatous proliferation, and finally tumor formation. HCBD   February 2003 7 35 On the other hand, in the presence of metabolic activation, HCBD and its reactive metabolites are mutagenic in some ( Simmon, 1977; Reichert et al., 1984; Reichert and Schutz, 1986; Wild et al., 1986), but not all, studies ( See Section 7.3.1). Those studies done either on kidney cells ( in vivo) or with kidney microsomes ( in vitro) appear to represent most of the mutagenic response to HCBD. Thus, a mutagenic mode of action cannot be ruled out ( Dekant et al., 1990; Lock, 1994).

The hypothesis that both cytotoxicity and mutagenic mode of action may be operating is consistent with the findings that the adverse effects of HCBD are dependent on a multistep pathway of bioactivation. The ultimate step in this pathway is a $ lyase mediated degradation of a HCBD metabolite that generates a highly reactive thioketene in proximal tubule cells. Covalent binding of this thioketene to DNA, proteins and other macromolecules is considered to be the mechanism responsible for the observed cytotoxic and mutagenic effects of HCBD and its metabolites. Restriction of these effects to the proximal tubule most likely reflects both uptake processes that concentrate the cysteine conjugate substrate in epithelial cells, and localization of ( glutamyltranspeptidase and $ lyase activity to this region of the kidney.

In vitro studies ( Schnellman et al., 1987; Groves et al., 1991; Jones et al., 1986; Wallin et al., 1987) indicate that mitochondria in renal tubular epithelial cells are the major target for HCBD metabolite induced toxicity. The reactive metabolite formed by $ lyase cleavage of cysteine conjugate is thought to interact with components of the mitochondrial inner membrane. The initial effect is an uncoupling of oxidative phosphorylation and prevention of generation of ATP. The decrease in renal tubular ATP secondary to mitochondrial dysfunction in turn limits ATP dependent active transport in the tubules, inhibiting reabsorption processes ( Jaffe et al., 1983). Later effect involves gross mitochondrial damage characterized by inhibition of cytochrome c cytochrome oxidase activity, and inhibited electron transport. This sequence of events in the renal proximal tubules ultimately leads to cell death. Other studies indicate that the reactive species generated by $ lyase mediated degradation of HCBD metabolites interact directly with mitochondrial DNA ( mtDNA) from mouse kidney ( Schrenk and Dekant, 1989). Renal mtDNA may be the preferential target due to the high concentration of $ lyase in the mitochondrial membrane, the lack of protective histones associated with mitochondrial DNA ( Borst & Grivell, 1978), and an inadequate repair function ( Mansouri et al., 1997). Mutations in the mtDNA can lead to a respiratory chain deficiency and cell dysfunction when the percentage of the mutants reach a certain level ( Schapira, 1999).

Three important aspects of mitochondrial oxidative phosphorylation involved in the pathogenesis of mitochondrial dysfunction are: generation of cellular energy in the form of ATP; generation of reactive oxygen species ( ROS); and regulation of apoptosis or programmed cell death ( Wallace, 1999). The process of oxidative phosphorylation produces significant amounts of ROS which are toxic byproducts of respiration. Chronic exposure to ROS can result in oxidative damage to mitochondrial and cellular proteins, and mutations in the mtDNA. Because mtDNA codes for 22 transfer RNAs ( tRNA) and 2 ribosomal RNAs ( rRNA) for synthesis of important mitochrondrial proteins involved in the oxidative phosphorylation, functional mtDNA is critical to the normal function of a cell. Mutations in mtDNA may lead to overexposure to ROS and decreased energy production. Apoptosis is initiated when the mitochondrial permeability transition pore ( mtPTP) in the inner membrane opens and cell death promoting factors such as the caspases are released ( Wallace, 1999). Opening of the mtPTP and the accompanying cell death can be initiated by the HCBD   February 2003 7 36 mitochondrion's excessive uptake of Ca2+, increased exposure to ROS, or decline in energetic capacity. Therefore, a marked reduction in mitochondrial energy production and a chronic increase in oxidative stress could activate the mtPTP and initiate apoptosis.

Numerous mtDNA mutations have been associated with human mitochondrial disease. Mitochondrial disease is a disruption of the proper function of the mitochondria, resulting in a variety of clinical manifestation. This disruption can include an inhibition of the electron transport chain, a disruption of oxidative phosphorylation and an increase in the production of reactive oxygen species. MtDNA mutations could contribute to neoplastic transformation by changing cellular energy capacities, mitochondrial oxidative stress, and/ or modulating apoptosis ( Wallace, 1999). Thus, it may be postulated that mutations of renal mtDNA induced by HCBD may result in reduction in energy production, increase in oxidative stress, and initiation of apotosis, leading to tumor formation.

Mitochondrial dysfunction may also result from interaction of highly reactive HCBD metabolites with components of the mitochondrial inner membrane, such as enzymes related to cell function. Subsequent energy depletion may trigger the renal cytotoxicity that is the putative mechanism for HCBD mediated carcinogenesis. Thus, HCBD induced cytotoxicity and tumorigensis may be ultimately the consequence of mitochondrial dysfunction resulting from exposure.

Recent evidence suggests that HCBD induced " 2: globulin accumulation contributes to renal injury in male rats. However, renal tubular necrosis and renal tubular tumors were observed in both male and female rats following HCBD exposure ( Kociba et al., 1977), and renal necrosis and regeneration were also observed in male and female mice ( NTP, 1991). Therefore, " 2: globulin accumulation cannot be the sole mechanism for HCBD induced carcinogenesis.

7.4.4 Weight of Evidence Evaluation for Carcinogenicity No human carcinogenicity data are available for HCBD.

A single lifetime study of HCBD carcinogenicity in rats ( Kociba et al., 1977) is available for evaluation. This study revealed statistically significant increases in the incidence of tumors in male and female rats following oral HCBD exposure. Although human carcinogenicity data are unavailable, evidence exists that the metabolic enzymes responsible for conversion of HCBD to the reactive and toxic thioketene occur in humans, albeit at levels lower than that in the rat ( see Section 6.3). In accordance with EPA's 1986 Guidelines for Carcinogen Risk Assessment ( U. S. EPA, 1986), HCBD is best classified as Group C, possible human carcinogens, based on limited evidence of carcinogenicity in one animal study, and no data in humans. Based on the proposed guidelines for Carcinogen Risk Assessment ( U. S. EPA, 19991996a), HCBD is classified as " likely to be carcinogenic to humans by the oral route of exposure, but whose carcinogenic potential by the inhalation and dermal routes of exposure cannot be determined because there are inadequate data to perform an assessment". This descriptor is considered appropriate when there are no or inadequate data in humans, but the combined experimental evidence demonstrates the production or anticipated production of tumors in animals by modes of action that are relevant or assumed to be relevant to humans, tempered by the lack of adequate inhalation or dermal studies. Mechanistic studies performed in vitro and in vivo suggest that either genotoxic or non genotoxic modes of action may HCBD   February 2003 7 37 underlie or contribute to the carcinogenic potential of HCBD. However, the strongly non linear doseresponse in the Kociba et al. ( 1977) study cannot be ignored. At present, the mode of action information still lacks identification of the sequence of key events and a quantitative description of the doses at which those key events begin to occur. In such cases, EPA's proposed cancer guideline revisions ( U. S. EPA, 1999) support consideration of both linear and nonlinear extrapolation to lower doses. In the absence of adequate data to exclude a linear mechanism( s) of tumor formation, the quantitative cancer risk assessment of HCBD should conservatively be conducted using the linear default model. Therefore, both linear and non linear approaches are presented.

7.4.5 Sensitive Populations Sensitive populations are those which experience more adverse effects at comparable levels of exposure, or which experience adverse effects at lower exposure levels, than the general population. The enhanced response of these sensitive subpopulations may result from intrinsic or extrinsic factors. Factors that may be important include, but are not limited to: impaired function of detoxification, excretory, or compensatory processes that protect against or reduce toxicity; differences in physiological protective mechanisms; genetic differences in metabolism; developmental stage; health status; gender; or age of the individual.

Human populations that exhibit greater sensitivity to HCBD have not been identified. However, it has been generally observed that existing nephropathy or age related kidney degeneration can increase the risk of renal injury or exacerbate nephrotoxicity in humans ( WHO, 1991). Evidence that existing nephropathy increases sensitivity to HCBD toxicity has been obtained in a study conducted in male Wistar rats ( Kirby and Bach, 1995). Nephrosis was induced by pretreatment with adriamycin ( ADR), and rats were subsequently exposed to HCBD. Damage to the proximal tubule was more severe and renal cortical repair capacity was decreased in ADR treated rats when compared to rats exposed to HCBD without prior ADR exposure. These results suggest that individuals with existing kidney damage or the elderly may be potentially sensitive populations for HCBD exposure.

Studies in animals showed that the young rats and mice experience acute effects at significantly lower doses than do adults ( Hook et al., 1983; Lock et al., 1984), suggesting that infants may represent a potentially sensitive subpopulation for acute HCBD exposure, perhaps as a result of immature organ systems. Additionally, female rodents were apparently more sensitive than males to acute HCBD exposure ( Kociba, 1977). The mechanism underlying this sensitivity is not currently clear. 2 Note that the literature has used the terms BMD and BMDL in a confusing way ( Crump, 1984,1995). The EPA benchmark dose software ( BMDS version 1.3.1) and EPA technical guidance on this subject ( U. S. EPA, 2000a), use the term " BMD" to refer to the central or maximum likelihood estimate ( MLE) of the dose that is expected to yield the BMR. " BMC" ( benchmark concentration) is a term that is sometimes used, as opposed to " BMD," to distinguish between inhalation and oral benchmarks. " BMDL" or " BMCL" refer to the lower end of a one sided confidence interval for that central estimate. " BMD" will be used to refer to the entire process. The POD for low dose extrapolation on for setting the RfD/ RfC will be the BMDL or BMCL. To simplify further discussion in this document, we will use BMD or BMDL generically to mean oral or inhalation values, unless stated otherwise.

HCBD   February 2003 8 1 8.0 DOSE RESPONSE ASSESSMENT 8.1 Dose Response for Noncancer Effects 8.1.1 RfD Determination The reference dose ( RfD) for a chemical is " an estimate ( with uncertainty spanning approximately an order of magnitude) of a daily exposure to the human population ( including sensitive subgroups) that is likely to be without appreciable risk of deleterious effects over a lifetime" ( U. S. EPA, 1993). Data on the non cancer effects of HCBD from chronic and subchronic studies were used to estimate a RfD value using the benchmark dose ( BMD2) approach ( U. S. EPA, 1995).

Choice of Principal Study and Critical Effect There are no reliable dose response data for humans exposed to HCBD. The NTP ( 1991) subchronic study on mice was chosen as the principal study, with the Kociba et al. ( 1977) and Schwetz et al. ( 1977) studies on rats as supporting studies, due to their exposure durations and sensitivity of endpoints observed. Hyperplasia and regeneration of the renal tubular cell epithelial cells was selected as the critical effect, based upon its observation at the lowest doses. The RfD for HCBD is derived from a BMDL of 0.1 mg/ kg day for renal tubular epithelial cell hyperplasia/ regeneration from the NTP study.

In the Kociba et al. ( 1977) lifetime oral exposure study of rats to HCBD, a NOAEL of 0.2 mg/ kg day and a LOAEL of 2 mg/ kg day were identified, based on an increase in renal tubular epithelial cell hyperplasia/ regeneration. In the Schwetz et al. ( 1977) 148 day oral exposure study of rats to HCBD, a NOAEL of 0.2 mg/ kg day and a LOAEL of 2 mg/ kg day were identified, based on an increase in the severity of renal tubular dilation, collapse, and atrophy. In the 13 week feeding study by NTP ( 1991), the study authors identified a NOAEL of 1.5 mg/ kg day for male mice, and did not identify a NOAEL for female mice because renal tubular regeneration occurred in 1 of 10 females in the lowest dose group ( 0.2 mg/ kg day). However, others ( U. S. EPA, 1998a; WHO, 1994) have concluded that the effect observed at 0.2 mg/ kg day is not statistically significant, and therefore considered this dose to be the NOAEL.

In order to avoid the uncertainty in the LOAEL/ NOAEL classification of the 0.2 mg/ kg day dose in the NTP study, the most sensitive response found, a benchmark dose ( BMD) analysis was 3 A copy of BMDS can be obtained from the Internet at http:// www. epa. gov/ ncea/ bmds. htm.

4 A benchmark response ( BMR) of 10% was used for all BMD analyses.

HCBD   February 2003 8 2 conducted on the NTP female mouse renal tubular regeneration response ( Table 8 1). Using BMDS3, version 1.3.1, the data was fit to all available dichotomous models ( Table 8 2). The data was best fit by a Weibull model ( p = 1.00, AIC = 17), resulting in a BMD4 of 0.2 mg/ kg day and a BMDL of 0.1 mg/ kg day ( Figure 8 1). This dose was therefore taken as the point of departure for further calculations.

Table 8 1. Incidence of Renal Tubular Regenerative Response in Mice Treated with HCBD for 13 Weeks.

Test Organism Administered Dose in Feed ( ppm) Approximate Daily Dose ( mg/ kg day) Renal Tubule Regeneration Incidence Male Mice 0 0 0/ 10 ( 0%)

1 0.1 0/ 10 ( 0%)

3 0.4 0/ 10 ( 0%)

10 1.5 0/ 9 ( 0%)

30 4.9 10/ 10 ( 100%)

100 16.8 10/ 10 ( 100%)

Female Mice 0 0 0/ 10 ( 0%)

1 0.2 1/ 10 ( 10%)

3 0.5 9/ 10 ( 90%)

10 1.8 10/ 10 ( 100%)

30 4.5 10/ 10 ( 100%)*

100 19.2 10/ 10 ( 100%)*

* Not used in BMD analysis. source: NTP ( 1991). 5 Akaike's Information Criterion ( AIC) =  2L + 2p, where L is the log likelihood at the maximum likelihood estimates for the parameters, and p is the number of model degrees of freedom. This can be used to compare models with different numbers of parameters using a similar fitting method ( for example, least squares or a binomial maximum likelihood). Although such methods are not exact, they can provide useful guidance in model selection.

HCBD   February 2003 8 3 Table 8 2. Benchmark Dose Estimates from NTP ( 1991) Female Mouse Renal Tubular Regeneration Response.

Model BMD BMDL Chisquare p value AIC5 Weibull 0.200 0.099 1.00 17 Gamma 0.200 0.111 1.00 17 Log Probit 0.200 0.126 1.00 17 Log Logistic 0.200 0.123 1.00 17 Probit 0.203 0.112 0.99 17 Logistic 0.209 0.119 0.97 17 Multistage ( 2) 0.123 0.056 0.68 17 Quantal Quadratic 0.123 0.094 0.68 17 Quantal Linear 0.042 0.026 0.14 22 HCBD   February 2003 8 4 Figure 8 1. Benchmark Dose Estimate Using Weibull Model. 6 A single generation reproductive/ developmental study misses important windows of vulnerability, lactational exposure, and latent effects that only become evident as the F1 generation reaches maturity. That is the rationale for the uncertainty factor.

HCBD   February 2003 8 5 different areas of uncertainty. A composite uncertainty factor ( UF) of 300 was used in the derivation of the RfD. The composite UF included a factor of 10 to account for extrapolation from animals to humans; a factor of 10 for protection of sensitive subpopulations; and a factor of 3 for database deficiencies ( lack of a 2 generation reproductive study6, and developmental toxicity studies in only one species). Although some studies suggest that humans may have lower rates of formation of putatively toxic metabolites than rodents, others suggest that the rate is the same ( Section 6.3).

Calculation of RfD Using the BMDL of 0.1 mg/ kg day from the NTP ( 1991) study, the RfD is derived as follows:

RfD = ( 0.1 mg/ kg day) = 3 × 10 4 mg/ kg day 300 where:

0.1 mg/ kg day = BMDL, based on the histopathological effects in kidneys of mice exposed to HCBD in the diet for up to 24 months ( NTP, 1991).

300 = uncertainty factor. This is based on a factor of 10 to account for extrapolation from animals to humans; a factor of 10 for protection of potentially sensitive human subpopulations; and a factor of 3 for database deficiencies ( lack of a two generation reproductive study).

8.1.2. RfC Determination RfC for HCBD is not derived. No subchronic or chronic inhalation exposure studies are available for the determination of RfC.

8.2 Dose Response for Cancer Effects 8.2.1 Choice of Study As noted previously, only one lifetime oral carcinogenicity study of HCBD was located ( Kociba et al., 1977). In this study, Sprague Dawley rats ( 40 animals/ sex/ dose group and 90 animals/ sex in the control group) were dosed with 0, 0.2, 2 or 20 mg/ kg day HCBD via the diet for 22 months ( males) or 24 months ( females).

Neoplastic changes were found only at the highest dose, which exceeded the maximum tolerated dose. There was a significant increase in mortality in males, a greater than 10% decrease in body weights for both sexes, and other severe renal toxicity effects were observed. The incidence HCBD   February 2003 8 6 of renal tubular neoplasms was increased only in the high dose group of both males and females, as shown in Table 8 3.

Table 8 3. Incidence of Renal Tubular Neoplasms in Rats Treated with HCBD for 2 Years.

Test Organism Administered Dose ( mg/ kg day) Human Equivalent Dosea ( mg/ kg day) Renal Tubular Neoplasm Incidence Male rats 0 0 1/ 90 ( 1.1%)

0.2 0.062 0/ 40 ( 0%)

2.0 0.62 0/ 40 ( 0%)

20 5.8 9/ 39 ( 23%)

Female rats 0 0 0/ 90 ( 0%)

0.2 0.054 0/ 40 ( 0%)

2.0 0.55 0/ 40 ( 0%)

20 5.3 6/ 40 ( 15%)

a Human Equivalent Dose = Animal dose ( Animal body weight/ Human body weight) 1/ 4 source: Kociba et al. ( 1977)

Increased renal tubular hyperplasia and renal tubular adenomas and adenocarcinomas ( some of which metastasized to the lungs), were found in rats exposed to 20 mg/ kg day of HCBD for up to 2 years. Lesser degrees of toxicity, including an increase in renal tubular hyperplasia, were found in rats ingesting 2 mg/ kg day for up to 2 years. A composite dose related change in the rodent kidney leading to tumor formation is shown in Table 8 4. This pattern is consistent with the hypothesis that renal tumor formation may require, and be secondary to, renal cytotoxicity induced by exposure to HCBD. The mode of action established for HCBD implies that, following exposures less than those which produce overt renal damage, no significant excess carcinogenic risk can be attributed to HCBD.

8.2.2 Dose Response Characterization The Kociba et al. ( 1977) and NTP ( 1991) studies were used to quantify the cancer risk from ingested HCBD, as discussed below.

1986 Guidance: Linearized Multistage Model The current IRIS file contains a carcinogenicity assessment of HCBD based on EPA's 1986 Guidelines for Carcinogen Risk Assessment ( U. S. EPA, 1986). The dose response data for male rats were fitted to the linearized multistage model. To estimate human equivalent dose from an animal HCBD   February 2003 8 7 Table 8 4. Dose Related Changes in the Rodent Kidney after Oral Exposure to HCBD, Chronic Study  Rat ( Kociba et al., 1977).

Dose ( mg/ kg day) 0.2 2 20 terminal kidney weight increase ( abs. & rel.) _ _ +

hyperplasia  multi focal _ ? +

hyperplasia adenomatous _ + (& only) +

tumors _ _ +

study, the doses administered to animals were adjusted by a scaling factor of ( body weight) 2/ 3. The resulting cancer slope factor is 7.8 × 10 2 ( mg/ kg day) 1. This slope factor corresponds to a drinking water unit risk of 2.2 × 10 6 per : g/ L ( U. S. EPA, 1997a), and the drinking water concentration that corresponds to a lifetime excess cancer risk of 1 × 10 6 is 0.5 : g/ L.

1996, 1999 Proposed Guidance The draft Ambient Water Quality Criteria for hexachlorobutadiene ( U. S. EPA, 1998a) utilized the methodology discussed in EPA's 1996 Proposed Guidelines for Carcinogen Risk Assessment ( U. S. EPA, 1996a) to evaluate the carcinogenicity of HCBD. This has been updated in the 1999 draft Guidelines for Carcinogen Risk Assessment ( U. S. EPA, 1999). Under these guidelines, two approaches can be used for dose response extrapolation for quantification of cancer risk, depending on what is known about the mode of action for carcinogenicity and the shape of the dose response curve. A linear approach is used for a chemical when available evidence indicates the chemical has direct DNA mutagenic activity or is DNA reactive, or when the evidence supports another mode of action that is anticipated to be linear. An inference of linearity may also be supported if existing human exposure is high and near doses associated with key events in the carcinogenesis process. The linear approach is used as a matter of policy if there is an absence of sufficient mode of action information on tumorigenesis. The nonlinear approach may be used when the tumor mode of action supports nonlinearity ( e. g., some cytotoxic and hormonal agents) and the chemical does not demonstrate mutagenic effects consistent with linearity. The nonlinear approach is also selected when a mode of action supporting nonlinearity has been demonstrated, and the chemical has some indication of mutagenic activity, but is judged not to play a significant role in tumor causation. As a matter of science policy, nonlinear probability functions are not fitted to tumor response data to extrapolate quantitative low dose risk estimates because different models can lead 7 This modeling was carried out using the Global 86 multistage model software.

HCBD   February 2003 8 8 to a wide range of results, and there is currently no basis to choose among them. In these cases, the RfD is generally used for protection of cancer effects.

Because both linear ( mutagenic) and nonlinear ( toxicity associated) mode of action for carcinogenicity of HCBD may be operating in vivo, both of these approaches were evaluated in the draft Ambient Water Quality Criteria for hexachlorobutadiene ( U. S. EPA, 1998a) for characterizing the carcinogenic hazard of HCBD, as discussed below. DNA adduct formation in mouse kidney was weak, even at an oral dose of 30 mg/ kg. However, hypertrophy and regeneration of renal tubule epithelial cells precede neoplastic effects in both dose ( 0.2 vs. 20 mg/ kg day) and time ( subchronic vs. chronic responses). This suggests that nonlinear effects may underlie or contribute to the carcinogenic potential of HCBD, although a role for genotoxic mechanisms cannot confidently be eliminated. Based on these considerations, the linear method for quantitative cancer risk assessments of HCBD would be the default approach.

Linear Approach Because there are limited data which suggest that HCBD might be genotoxic and mutagenic ( see Section 7.3.1), this approach is considered in the dose response extrapolation for HCBD.

Under the proposed guidelines, the cancer risk from a chemical is assessed in two steps. The first step involves curve fitting of the cancer dose response data within the observable range to derive a point of departure ( Pdp) ( U. S. EPA, 1999). The dose at the point of departure is expressed as the human equivalent dose. The dose that causes a 10% increase in extra risk is referred to as the ED 10. The point of departure is defined as the 95% lower confidence limit on the ED 10, and is referred to as the LED 10. The second step in the process is linear extrapolation of the dose response curve from the LED 10 to the origin, and determination of the slope of that line.