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Toxicity of Selected Contaminants 63 Using this dose for a 70-kg human drinking 2 liters of water daily and an uncertainty factor of 1,000, and assuming that 205'o of exposure is pro- vided by water, one can calculate the chronic SNARL as: 150 mg/kg X 70 kg X 0 2 = 1 ns m~/liter- 1,000 X 2 liters The uncertainty factor of 1,000 was used because the issue of carcinogenic- ity has not yet been resolved and also because the Johnson (1971) study does not provide enough information for a complete judgment of its ade- quacy. Picloram is relatively nontoxic in rats fed concentrations as high as 3,000 ppm in the diet through three generations. It is not teratogenic in the rat or mutagenic in two out of four bacterial tester strains with or without micro- somal activation. The results of carcinogenicity testing in rats and mice are equivocal. At levels of 14,875 ppm, benign hepatomas were observed in female rats. Pi- cloram-related tumors were not reported in male rats or mice of both sexes. ROTENONE [l]benzopyrano[3,4-bifuro [2,3-h][llbenzopyran-6(6H) one, 1,2,12,12a-tetrahydr~8,9~imethoxy-2-(1-methylethenylY, [2R12CY, 6aa,2aaF CAS No. 83-794 OCH3 CH3O ~r~ CH2 H , Car H CH3 In the form of ground derris root, rotenone has been used as a nonpersis- tent insecticide to control pests on plants and animals and as a fish poison to manage or to eliminate undesirable species in reservoirs, lakes, and streams (Fukami et al., 1967~. First isolated in 1895, world consumption is

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64 DRINKING WATER AND HEALTH now estimated to be 10,000 to 20,000 kg/year (Haley, 1978~. When ex- posed to light and air, rotenone undergoes rapid decomposition. Colorless solutions in organic solvents become successively yellow, orange, and, fi- nally, deep red as the result of oxidation, and they may deposit crystals containing dehydrorotenone and rotenone (Windholz, 1976~. METAB O LI S M A literature review by Haley (1978) indicates that rotenone functions as an inhibitor of the mitochondrial oxidative phosphorylation-electron trans- port system, but the chemical also was found to be a potent in vitro antago- nist of slow-reacting substance of anaphylaxis (Ashack et al., 1980~. Studies of the metabolism of rotenone have not been extensive. The committee found no literature on the in ViVQ kinetics of rotenone absorp- tion or excretion. In vitro studies with microsomal preparations and in vivo studies with mice and houseflies indicated that there are hydroxylation me- tabolites of rotenone designated as rotenolone I; rotenolone II; dehydroro- tenone I; dehydroxyrotenone; 8'-hydroxyrotenolone; 6',7'-dehydro- 6',7'-dihydrorotenone; and other uncharacterized polar compounds (Fukami et al., 1967; Haley, 19781. HEALTH ASPECTS Observations in Humans Toxicity in humans has not been well studied. Large inhaled or ingested doses may cause numbness of oral mucous membranes, nausea and vomit- ing, muscle tremors, convulsions, tachypnea, and respiratory paralysis at lethal doses. The lethal oral dose has been estimated at 0.3 to 0.5 g/kg (Gosselin et al., 1976~. Chronic poisoning may result in fatty changes in the liver and kidney. Direct contact may cause irritation of the skin or con- juncti~ra. No human fatalities have been reported (Haley, 1978; Windholz, 1976~. Observations in Other Species Acute Effects In rats, the intravenous, intraperitoneal, and oral LDSo values for rotenone in ethanol or acetone solution were reported to be 0.20, 1.60, and 60 mg/kg, respectively (Santi and Toth, 1965~. Depending on the route of administration, toxic signs appeared within 2 to 20 minutes. Death from respiratory failure occurred within minutes after intravenous injection and within 1 day after intraperitoneal or oral administration.

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Toxicity of Selected Contaminants 65 These workers suggested that both the lethal and pharmacological effects of rotenone are due to inhibited mitochondrial metabolism. Haley (1978) reported that the acute oral toxicity of rotenone is more variable and less toxic. This investigator reported acute toxicity doses as 0.6 g/kg in rats, 1.5 g/kg in guinea pigs, and 3 g/kg in rabbits. In this study, dogs were not affected at 2 g/kg, but oral ingestion near the feeding time produced eme- SiS. Short-term administration of rotenone in sunflower oil, injected intra- peritoneally in doses of 0.1 mg/kg/day for 5 days into female rats, pro- duced a marked elevation in serum growth hormone concentration and a decrease in serum prolactin (Gosalvez et al., 1979~. These alterations and transient elevations in concentrations of estrogens, progesterone, and cor- ticosterone suggested to the authors that rotenone was stimulating the hy- pophysis and that the physiopathogeny of rotenone-induced mammary tu- mors is indirect and hormonal. Subchronic Effects A 28-day repeated dose and 6-month (26-week) study in beagle dogs has been completed by Ellis et al. (1980~. In this study, animals were dosed daily with the rotenone preweighed into gelatin capsules on a weekly basis for each animal. The capsules were stored at freezer temperature until administered to the dogs. The chemical was ana- lyzed at greater than 99~o purity; the 6-month study was conducted with only one lot of chemical. In the 28-day range-finding studies, one male and one female each received one of five levels of rotenone: 0.08, 0.4, 2, 10, or 50 mg/kg/day. The animals were observed daily, and their feed consump- tion and body weights were detennined weekly. At 10 and 50 mg/kg the primary toxic effect was emesis, which was more pronounced and pro- longed in females; at the high dose, excessive salivation was noted in the female. Despite the emesis, all the dogs generally gained weight, but the number of animals was too small and duration of exposure too short to draw definite conclusions about weight gain. In the 6-month study, groups consisting of six male and six female bea- gles received rotenone at dose levels of 0, 0.4, 2, or 10 mg/kg/day. The doses were administered approximately 1 hour before daily feeding. Ani- mals were observed daily, body weight was determined weekly, and both feed and water consumption were estimated 6 days/week. Hematological and clinical chemistry and urinalysis were performed twice during a pre- treatment period and after 2, 4, 6, 13, 17, 21, and 26 weeks during the dosing period. Animals were necropsied at the end of the 26-week expo- sure, major organs were weighed, and approximately 35 tissues from each animal were retained and examined histologically. The principal toxic sign was emesis, which occurred primarily during

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66 DRINKING WATER AND HEALTH the first week at the high-dose level (10 mg/kg/day); males appeared to be more affected than the females in this phase of the study. The dogs receiv- ing the high dose had a higher incidence of diarrhea and soft stools throughout the study. The body weight of high-dose males and females decreased during the first 2 months, but remained stable thereafter. At 2 mg/kg/day, body weights of females were consistently lower than those of controls after 2 months. Effects on feed consumption generally paralleled body weight responses. At high doses, hematology and blood biochemistry appeared to be adversely affected in high dose males and females. In com- parison to controls, hemoglobin and hematocrit values were significantly reduced. Erythrocyte counts were also generally lower than in controls, but seldom was the difference statistically significant. Other consistent effects were decreases in glucose, total lipids, and cholestrol. Urinalysis failed to reveal consistent effects that could be attributed to the test chemical. At necropsy, average absolute major organ weights were generally similar in all groups. Relative to body weight, the weights of the kidney (males), heart (females), and brain (males and females) were significantly higher in the high-dose group than in controls, but these differences were attributed to decreased body weight in the high-dose group. At necropsy, the most frequent lesions observed were hemorrhagic patches in one or more sec- tions of the small intestine. This lesion was more prevalent in the high-dose dogs but was also observed in controls; hence, it could not be definitely associated with exposure to the test chemical. The only microscopically observed lesion that might have been related to the rotenone treatment was mild hemorrhagic enteritis of the small intestine in one low- and one high- dose female; however, the incidence was not dose-dependent and was much too low to be of significance. Other findings in all dose groups were not associated with exposure to the compound. Chronic Effects The committee found no recent studies on chronic ef- fects of rotenone. Studies conducted during the 1930's and 1940's were reviewed by Haley (1978), but the quality of the studies and their results are questionable. In general, these early studies indicated that chronic in- gestion of cube, derris root, or rotenone by rats led to suppressed growth, and 75 ppm caused liver injury. Rats maintained on high-fat diets contain- ing derris root or rotenone for 140 days had nonnal growth curves and no histological evidence of organ damage (Haley, 1978~. In a 2-year study, rats and dogs were given cube powder, the commercial natural product (Hansen et al., 1965), which contained 5.85ro rotenone by analysis. The powder was added to the diet of rats at levels of 0, 50, 100, 250, 500, or 1,000 ppm and to the diet of dogs at levels of 0, 50, 150, or 400 ppm. Each experimental group consisted of 25 male and 25 female rats

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Toxicity of Selected Contaminants 67 and two male and two female beagle dogs. Unfortunately, the diets were not analyzed to verify rotenone content, the stability of the compound in the diets was not verified, and the frequency of the diet preparation was not stated. Otherwise, the basic experimental design was not unreasonable for a chronic study conducted at that time. The number of animals, espe- cially rats, was too small, based on present standards for a carcinogenic evaluation, which was a major purpose of the study. Results of this study appeared to be reasonably straightforward. The test powder at all levels, except 50 ppm, inhibited the growth of the rats. Histological lesions in rats were attributable to the test material, and the incidence of gross mammary tumors, nephritis, and pituitary lesions at higher dose levels were lower than in the controls. No clinical or hematological effects were noted in the dogs. Mutagenicity No unscheduled DNA synthesis (UDS) was observed in human fibroblast cultures (VA4) in the presence and absence of a rat S9 liver enzyme activation system when rotenone was tested at 1, 10, and 1,000 mM concentrations (Ahmed et al., 1977~. Negative results were also obtained in a rat UDS hepatocyte assay (Protest and Hill, 1980~. The col- ony-fo~ming ability of continuously cultivated bovine cells (T4) derived from normal ovarian tissue was reduced to 50~o in the presence of 3.5 X 10-7 M concentration of rotenone (Malcolm et al., 19731. Alkali-labile single strand DNA breakage was observed when mouse L1210 leukemia cells were exposed to 10-7 M rotenone. Hilton and Walker (1977) sug- gested that the DNA strand breaks might be an indirect effect of ro- tenone's effect on adenosine triphosphate (ATP) depletion, which could result in increased nucJeolytic activity. Tomkins et al. (1980, abstract) ob- served no sister chromatic exchanges (SCE) in Chinese hamster ovary cells in the presence or absence of a rat liver S9 metabolic activation system. The maximum dose level used was the dose that reduced the proportion of dividing cells to Who. Rotenone added to Chinese hamster cells in vitro increased the mitotic index, and mitotic cells contained monopolar spindles with chromosomes grouped around centriole pairs near the cell center (Haley, 1978~. The compound was also found to arrest mitosis in cultured mammalian cells by inhibition of the spindle microtubule assembly (Barham and Brinkley, 1976; De Brabander et al., 1976; Meisner and Sorensen, 1966~. A 4.6 X 10-7 M (0.18 ~g/ml) concentration of rotenone inhibited the rate of rat neurotubulin polymerization in vitro by 505to (De Brabander et al., 1976~. The effective inhibition of the in vi~o fonnation of microtubules from tu- bulin has been suggested by Marshall and Himes (1978) to be the mecha- nism by which rotenone inhibits mitosis.

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68 DRINKING WATER AND HEALTH In summary, the data regarding the mutagenicity of rotenone are incon- clusive. Carcinogenicity The potential carcinogenicity of rotenone was evalu- ated by Innes et al. (1969) in two strains of mice by oral intubation of the test materials in a O.5~O suspension in a gelatin vehicle. A dose of 1 mg/kg was administered daily to 7-day-old mice until they were 4 weeks old. The weanlings were then fed diets containing rotenone concentrations of 3 mg/kg for approximately 18 months. Histological examination of major organs and of grossly observed lesions supported the conclusion that oral administration of rotenone did not cause a significant increase in tumors. In the early 1970's, a brief communication by Gosalvez and Merchan (1973) indicated that mammary tumors were produced in albino rats (strain unidentified) given rotenone in sunflower oil by daily intraperito- neal injection of 1.7 mg/kg for 42 days. The rotenone was reported as having less than 1 To impurities, which consisted mainly of other rotenoids. Mammary tumors developed in the dosed animals within 6 to 11 months; no tumors of this type were present in controls after 19 months. The tu- mors were diagnosed as adenomas with interstitial fibrosis and "areas with adenocarcinomatous transformation." They were encapsulated, and some were successfully transplanted to normal rats. In a later study, rotenone administered to female Wistar rats in the diet at concentrations of 5, 10, or 20 ppm for 8 to 12 months resulted in a 40~o incidence of mammary tu- mors. However, higher concentrations of rotenone (50 or 100 ppm) did not increase mammary tumors in treated rats above the To incidence ob- served in controls (Haley, 1978~. In other publications, the same group of investigators described the morphology of rotenone-induced breast tumors (Merchan et al., 1978) and reported a possible mechanism for the induc- tion of the tumors (Gosalvez et al., 1979~. Clarification of these findings was attempted by the U.S. Environmen- tal Protection Agency through contractual laboratory testing and evalua- tion of the carcinogenic potential of rotenone in Syrian golden hamsters and Wistar and Sprague-Dawley rats (Leber and Persing, 1979; Leber and Thake, 19791. These studies were conducted with 98~o pure rotenone con- taining traces of other rotenoids. In the hamster study, histological exami- nations were conducted on 30 tissue/organ samples from approximately 30 animals of each sex and experimental group that had received rotenone at doses of 0, 125, 250, 500, or 1.000 ppm in the diet for as long as 18 months. There was no evidence that rotenone was carcinogenic to the hamsters. In the Sprague-Dawley rat study, rotenone in corn oil was administered daily by intraperitoneal injection to 25 animals of each sex at doses of 0,

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Toxicity of Selected Contaminants 69 1.7, or 3.0 mg/kg bw for 42 days. Fifteen animals of each sex were used as vehicle controls. The animals were observed for an additional 18 months after treatment until death or sacrifice. In the Wistar rat study, rotenone in corn oil was administered by gavage to 25 animals of each sex at doses of 0, 1.7, or 3.0 mg/kg bw for 42 days. Fifteen animals of each sex were used as vehicle controls. The Wistar rats were observed for an additional 12 months after treatment until death or sacrifice. Histological examination was performed on more than 30 tissue/organ sites sampled from animals from each of these two studies. There was no evidence that intraperitoneal or oral administration of rotenone in corn oil to Sprague-Dawley or Wistar rats induced mammary neoplasia or statistically significant neoplasias at any other organ site. Evidence of the possible carcinogenicity of rotenone is limited to positive findings in one sex of one strain of rats and to one tumor type and site (i.e., adenoma of the mammary glands). The weight of evidence is further lim- ited by the lack of findings of carcinogenicity in three comparable studies using the Syrian golden hamster and the Wistar and Sprague-Dawley rats and by limited evidence in the literature indicating that rotenone is not mutagenic. The conflicting evidence suggests that there were uncontrolled variables in the testing of rotenone, e.g., the purity of the test material, the sensitivity of the strains used, and the possible effects of the sunflower oil. These types of questions and the differences in test results must be resolved before the potential carcinogenicity of rotenone can be assessed. Teratogenicity Khera et al. (1982) tested technical grade rotenone (87% rotenone and 13~o other cube extractives) for teratogenicity in preg- nant female Wistar rats. The compound was administered by gavage in corn oil at doses of 2.5, 5.0, and 10 mg/kg bw. The 10 mg/kg dose killed 12 of the 20 rats after two to nine exposures. At this dose there was also a significant decrease in the number of live fetuses per dam and an increase in the proportion of resorptions. The incidence of skeletal malformations was not different from those of controls at all doses. There was, however, an increased frequency (p c 0.05) of skeletal aberrations such as an extra rib, delayed ossification of sternebrae, and missing sternebrae in the 5 mg/kg dose group. The 2.5 mg/kg dose produced no observed maternal toxicity or any adverse effect on fetal development. These authors con- cluded that, in light of the high dosages used, the effects noted may be of little or no significance to the rat and that additional studies should be conducted in a nonrodent species. It is not possible to make a definitive assessment of the potential terato- genicity of rotenone based on this one negative study in rats.

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70 DRINKING WATER AND HEALTH CONCLUSIONS AND RECOMMENDATIONS Suggested No-Adverse-Response Level (SNARL) Chronic Exposure Until the unconfirmed report of mammary tumors in female rats can be resolved, results of the 6-month study by Ellis et al. (1980) can be used to calculate a chronic SNARL. Using the lowest appar- ent no-effect level in dogs (2 mg/kg) and an uncertainty factor of 1,000 (because this is less than a lifetime study), and assuming that a 70-kg hu- man consumes 2 liters of water daily and that 20% of intake is provided by the water, one may calculate the SNARL as: 2 mg/kg X 70 kg X 0.2 14 /li 2 liters X 1,000 Because results of acute toxicity studies have been variable, additional studies with the purified chemical, with several animal species, and with several routes of administration appear warranted. Additional teratologi- cal evaluation should also be considered, assuming that humans may be exposed to the chemical through drinking water. In addition, pharmaco- kinetic metabolism studies are needed, since rates of absorption and routes and rates of excretion would undoubtedly assist in the evaluation of toxic responses. Since rotenone enters surface waters through direct appli- cation in fishery management, definitive studies should be conducted to determine the fate (rates and routes of degradation) of the chemical in wa- ter under various environmental conditions. Since rotenone is quite sensi- tive to degradation following exposure to air and sunlight, it is necessary to determine the extent of these reactions and the ultimate products to which humans would be exposed. Of greatest concern is the uncertainty of the chemical nature of the ro- tenone being tested. The compound's potential carcinogenicity must then be resolved. Carcinogenesis bioassays have produced equivocal results that cannot be resolved on the basis of current information. More recent studies have been negative. TETRACHLOROETHYLENE ethene, tetrachIor~ CAS No. 127-18~ Cl2C = CC12 Tetrachloroethylene was evaluated in the first and third volumes of Drink- ing Water and Health (National Research Council, 1977, pp. 769-770;