CYHEXATIN First Draft Prepared by David Clegg Health and Welfare Canada Ottawa, Ontario, Canada EXPLANATION The toxicological data available on cyhexatin (tricyclohexyltin hydroxide) were reviewed by JMPR in 1970, 1973, 1977, 1978, 1980, 1981, 1988 and 1989 (Annex I, 14, 20, 28, 30, 34, 36, 53, and 46). Cyhexatin was withdrawn from the market in a number of countries because of concerns relating to teratogenicity in the rabbit following both oral and dermal exposure. The 1989 JMPR reviewed the oral and dermal teratology studies in rabbits, but could not reconcile the available data, which comprised 2 negative oral studies, 1 negative dermal study, 1 positive oral study and 1 positive dermal study. The 1989 JMPR was informed that an additional study pertaining to rabbit teratology was in process of being completed. Consequently, the existing ADI of 0.008 mg/kg bw was maintained until the new rabbit data were available for evaluation. This study, two rat multigeneration studies, and information concerning pharmacokinetics are now available and are reviewed in the present monograph. EVALUATION FOR ACCEPTABLE INTAKE BIOLOGICAL DATA Biochemical aspects Absorption, distribution and excretion Rats Twelve female 10PS Caw rats received 3 mg cyhexatin/kg bw orally. The dose was corrected for 96% purity micronized cyhexatin used in the study. Five untreated rats served as controls. Blood samples were taken at 0.5, 1, 3, 4, 8 and 24 hours from 2 rats/time interval. Controls were bled only at 24 hours (Barrow, 1991b). Blood levels of tin peaked at 3-4 hours, declining almost to control levels by 24 hours. A group of 40 10PS Caw female rats were intubated orally with 3 mg cyhexatin technical/kg bw (dose corrected for 96% purity) in a suspension in 5 mg CMC/ml water. Five additional female rats served as controls. Treated rats (15/time interval) were bled at 0.5, 1, 3, 4, 6, 8, 12 and 24 hours post-dosing. Controls were bled at 24 hours only. Urine and faeces were collected over the 24 hour period. No mortality or clinical signs were observed. Mean blood levels (based on 2 analyses/sample) were 4.3, 6.5, 7.8, 8.8, 7.2, 5.0, 4.9, 4.9, and 3.6 µg/l at 0, 0.5, 1, 3, 4, 6, 8, 12 and 24 hrs. Mean urinary levels (5 animals) were 9.0 µg/l in controls, and 68.5 µg/l in treated rats over 24 hours. Mean faecal levels (5 animals over 24 hours) were 1.89 and 176.9 µg/g (Barrow, 1991d). In a similar study using micronized cyhexatin, mean blood levels (based on 2 analyses/ sample) were 4.17, 6.15, 8.23, 10.9, 13.8, 6.8, 5.61, 4.6 and 4.1 µg/l at 0, 0.5, 1, 3, 4, 6, 8, 12 and 24 hours. Mean values among levels were 8.5 µg/l in control, and 85 µg/l in treated rats over 24 hours. Faecal levels were 2.4 µg/g (control) and 202.8 µg/g (treated) over 24 hours (Barrow, 1991e). Fifty-five female 10PS Caw rats were injected intravenously with 0.5 mg micronized cyhexatin (96% purity)/kg bw. The cyhexatin vehicle was ethyl alcohol, and solution concentration was 1 mg/ml (i.e., injection volume was 0.5 ml/kg). Blood samples were taken from 50 animals pre-dosing, and from groups of 5 animals at 10, 15, 20, 30, 60, 120, 240, 360, 480 mins and 24 hours post-dosing. Five rats were used to obtain urinary and faecal samples, predosing and 24 hours post-dosing. Mean pretest blood levels of tin were 0.003 µg/ml. Mean blood levels of tin at the various time intervals were 0.6, 0.1, 0.2, 0.1, 0.08, 0.05, 0.038, 0.03, 0.02 and 0.01 µg/ml at 10, 15, 20, 30, 60, 120, 240, 360, 480 mins and 24 hours. Mean urine levels pretest were 4.1 µg/l, and over 24 hours post-dosing were 43 µg/l. Faecal levels, pretest, were 1.4 µg/g and over 24 hours post-dosing were 7.5 (Woehrle, 1991d). Rabbits Two groups of 8 female Hy/Cr hybrid New Zealand rabbits were administered 3.0 mg cyhexatin/kg bw. The dose was corrected for 96% purity micronized cyhexatin used in the study. Group 1 was dosed orally, and group 2, percutaneously. Blood samples were taken from 2 rabbits in each group at 0.5, 1, 3, 4, 8 and 24 hours, while urine and faeces were collected over a 24 hour period. No mortalities or clinical signs were observed (Barrow, 1991a). Analytical data (provided by the sponsor) is limited to blood levels showing evidence of absorption of tin by both routes. Distribution of tin, resulting from oral treatment of pregnant rabbits with 0 or 3 mg cyhexatin (96% purity) kg bw/day on days 6-18 inclusive of gestation were measured. Maternal blood levels (taken 2, 3, 4 and 24 hours post-dosing) were measured on days 18/19 (6 control and 6 treated rabbits). The maternal animals were sacrificed on day 19 and immediately prior to sacrifice on day 26 (10 control and 11 treated rabbits). Clinical signs were limited to one treated rabbit which showed tachypnoea immediately after dosing on day 18, clearing within 24 hours. One female aborted on day 26 of gestation. Mean body weight gain and food intake were slightly reduced in the treated group compared to the control group. On day 19, mean pup weights and placental weights were comparable between treated and control groups. At 26 days, mean pup body weight was reduced in the treated group as compared to controls (ca. 15%) but amniotic fluid and placental weights were comparable. Brain and liver weights in pups were comparable to controls but kidney weights were depressed (ca. 15%) (Woehrle, 1991a). Peak maternal blood levels of tin (1 gram of tin being equivalent to 3.25 g of cyhexatin) were observed at about 3 hours post-dosing, but were elevated compared to controls at all time intervals on days 18/19, in the treated group. By day 26, blood tin levels were comparable in both groups. The calculated half-life of tin in maternal blood was stated to be 8.17 ± 1.59 hours. Tissue levels on day 19 were increased in maternal liver and kidney, but not in brain. Levels for all animals were generally comparable by day 26 in all organs. In the fetal tissues, at day 19, tin levels were significantly increased in amniotic fluid, placenta and whole fetuses as compared to controls. Brain levels were also elevated in day 19 fetuses as compared to brain levels in day 26 controls. Tissue levels on day 26 (liver, kidney, amniotic fluid and placenta) were comparable between control and treated groups. Brain levels remained slightly elevated (Oxam Italia, 1991; Salmona & Gagliardi, 1991). Twenty female Hy/Cr New Zealand hybrid rabbits were dosed with 3 mg cyhexatin technical/kg bw (corrected for 96% purity) as a suspension in 5 mg CMC/ml water. Groups of 4 rabbits were bled at 0.25, 0.5, 1, 4, 8, 24, 32 and 48 hours. Urine and faeces were collected at 24 and 48 hours. Mean blood levels of tin were 5.4, 5.4, 6.6, 6.2, 14.0, 11.6, 8.0, 8.0 and 5.4 µg/l at 0, 0.25, 0.5, 1, 4, 8, 24, 32 and 48 hours. Faecal levels were high at 24 hours (6.2-37.8 µg/g) decreasing to 1.8-8.4 µg/g after 48 hours. Urine levels at 24 hours were 38.9-112.7 µg/l (mean 74.75) µg/l) and at 48 hours, 58.9-108.4 µg/l (mean, 83.37 µg/l) (Barrow, 1991c). An identical study using micronized cyhexatin also resulted in mean tin levels in blood. Faecal levels were elevated at 24 hours (mean, 21-23 range and 15.2-25.3 µg/g) but had dropped to a mean of 5.7 µg/g, range 3.4-11.8 µg/g by 48 hours. Urinary levels were 62.7-98.9 µg/l (mean 76.3 µg/l) at 24 hours and 100.2-140.9 µg/l (mean 119.7 µg/l) at 48 hours (Barrow, 1991d). Twenty female hybrid Hy/Cr White New Zealand rabbits were each treated percutaneously with 3 mg cyhexatin technical (corrected for 96% purity)/kg bw in 5 mg carboxymethylcellulose/ml, 0.25 ml/kg on a 130 sq cm area on the dorsum. Blood samples were taken prior to treatment, and in groups of 4 rabbits at 0.5, 1, 4, 8, 24, 32, 48 and 56 hours post-treatment. Faecal and urine samples were collected at 24, 48 and 54 hours. Mean pre-treatment levels of tin in blood were 6.5 µg/l, and at 0.5, 1, 4, 8, 24, 32, 48 and 56 hours, 8.0, 7.0, 8.3, 10.9, 8.6, 7.9, 8.6 and 6.7 µg/l. Mean faecal tin levels were 1.5, 2.9 and 2.0 µg/g at 24, 48 and 54 hours, and mean urine levels were 65.8, 82.5 and 43.5 µg/l at 24, 48 and 54 hours (Barrow, 1991f). In a similar experiment, but using micronized cyhexatin, mean blood levels were 5.9 µg/l in pretreated rabbits, and 7.5, 7.1, 7.9, 11.1, 9.2, 7.5, 6.1 and 6.6 µg/l at 0.5, 1, 4, 8, 24, 32, 48 and 54 hours. Mean faecal levels were 5.9, 1.9 and 1.6 µg/g at 24, 48 and 54 hours, while urine levels at these time intervals were 81.3, 61.8 and (based on 2 rabbits only) 89 µg/l (Barrow, 1991g). Reports on intravenous administration of 96% purity micronized cyhexatin to Hy/Cr hybrid New Zealand white rabbits are available. In the first study, 15 female rabbits were injected with 3 mg micronized cyhexatin/kg bw, and groups of 2 female rabbits received either 1 mg or 0.5 mg micronized cyhexatin/kg bw. The cyhexatin was dissolved in absolute alcohol to give dose concentrations of 15 (3 mg/kg dose) or 5 mg cyhexatin/ml. Injection volumes were 0.2 ml/kg (3 and 1 mg cyhexatin/kg bw) or 0.1 ml/kg bw. Blood samples were taken from all high dose animals prior to testing (mean tin levels, 0.03 mg/l). At 3 mg/kg bw, the animals were divided into 3 groups of 5, blood being taken at 10, 30 and 240 mins post-dosing in group 1; 5, 60 and 360 mins in group 2, and 20, 120 and 480 mins in group 3. Mortalities or inability to obtain blood samples resulted in analyses in 3 rabbits at 10 minutes (mean tin levels 0.95 mg/l), five samples at 15 mins, (mean tin levels 0.77 mg/l), five samples at 20 mins (mean tin levels 0.68 mg/l) one sample at 30 mins, (tin level, 0.45 mg/l), two samples at 60 mins (mean tin level, 0.45 mg/l, and one sample at 120 mins (tin level 0.39 mg/l). At 240 mins, all animals receiving 3 mg micronized cyhexatin were dead. Of the 2 rabbits receiving 1 mg micronized cyhexatin/kg bw, analyses are available only at 20 mins (mean tin level, 0.503 mg/l) all animals being dead at 24 hours. No data on blood levels are available for the 2 rabbits receiving 0.5 mg micronized cyhexatin/kg bw (Woehrle, 1991b). A second report indicates 20 Hy/Cr New Zealand white rabbits received 0.5 mg micronized cyhexatin (96% purity)/kg bw intravenously, in absolute ethyl alcohol. Solution concentration was 2.5 mg/ml, and injection volume, 0.2 ml/kg bw. Blood samples were taken from 15 rabbits, pre-test (mean tin levels, 0.02 mg/l), from 5 rabbits at 5, 20, 120 and 480 mins, from 5 rabbits at 10, 30, 240 mins and 24 hrs and from 5 rabbits at 15, 60 and 360 mins. Mean tin levels in blood increased from 0.02 µg/l at time 0 to 0.28 µg/l after 5 minutes. After one hour, the level had declined to 0.08 µg/l and after 3 hours, to 0.07 µg/l. Urine levels (measured in 3 rabbits) indicated mean tin levels of 52.7 µg/l at 24 hrs and 67.0 µg/l at 48 hours. Pretest levels in 5 rabbits indicated mean tin levels of 31 µg/l. Faecal levels (measured in 3 rabbits) indicated mean tin levels of 12.56 µg/g at 24 hours and 14.3 µg/g at 48 hours. Pretest levels in 5 rabbits indicated a mean tin level of 5.9 µg/g (Woehrle, 1991c). In the above studies, in which a 0.5% carboxyl-methyl cellulose suspension was used, suspension content and homogeneity were within 10% of nominal values (Fraschini, 1991). Toxicological studies Reproduction study Four groups of 30 Charles River SD rats/sex were fed diets for the duration of a two-generation study (single litter in the first generation (F1a), and 2 litters in the second generation (F2a, F2b), to yield doses of 0, 0.1, 0.5, or 6.0 mg/kg bw/day. The test material was stated to be 95.6% pure, and to be stable over a 16-month period (no data). Impurities were indicated to be dicyclohexyltin oxide, 1.6-2.2%; tetracyclohexyltin, 1.7-1.9%; dicyclohexyl-xylyltin hydroxide, 0.4-0.5% and tricyclohexyl-tetrahydrofuran tin, 0.3%. The test compound was stated to be homo-genously admixed with diet, stability in diet being at least 28 days. Test diet analyses indicated that for male targeted doses, actual doses ranged from 73-114%, with mean values of 89% (0.1 mg/kg bw/day) and 97% (0.5 and 6.0 mg/kg bw/ day). Targeted doses for females ranged from 63% (1 analysis at 0.1 mg/kg bw/ day) to 126%. Mean values were 98, 94 and 103% at 0.1, 0.5 and 6.0 mg/kg bw/ day. The pairing of F0 animals was preceded by 70 days treatment and the first pairing of the F1a adults was preceded by 91 days treatment. Brother/sister matings were avoided. Pairing was on a 1 male:1 female basis, with a maximum of 3 X 7 day cohabitation using different males. Day 0 of gestation was the day of detection of sperm in vaginal smears. At day 4 post-partum, all litters were culled to 8 pups (4 male and 4 female, where possible). Culled pups and discarded weanlings were grossly examined. Gross necropsy of 10 pups/sex/dose were performed on F1a, F2a and F2b at weaning. All adults dying or surviving to scheduled necropsy from F0 and F1 generations were subject to gross necropsy. Histopathology on adults was limited to liver, kidney and reproductive organs of control and high-dose animals, and livers only of low and mid-dose animals. Gross lesions noted in F1 adults were also subject to histopathological examination. No clinical signs of toxicity were observed in either F0 or F1 adults. No compound-related mortality was apparent except for 1 F0 female which showed stomach erosion at 6 mg/kg bw/day. Body weight in F0 adults was decreased in both sexes at 6.0 mg/kg bw/day, and in females at 0.5 mg/kg bw/day. During gestation, female body weight gain was decreased during the first week (significantly at 0.5 and 6.0 mg/kg bw/day), but exceeded control values after gestation. During lactation body weight gains were generally increased during days 1-14, but decreased at 0.1 and 0.5 mg/kg bw/day during days 14-21. There was no consistent dose/effect relationship on body weight gain during the lactation period. In F1 adults body weight gain was depressed at 6 mg/kg bw/day in both sexes. During gestation and lactation, body weight gains in both breeding periods were affected only at 6 mg/kg bw/day. Food intake in F0 adults was slightly decreased in both sexes at 6.0 mg/kg bw/day, and in females at 0.5 mg/kg bw/day. In F1 adults, a slightly decreased food intake was noted in males sporadically at 6.0 mg/kg bw/day, especially in the latter half of the study. No adverse effects were seen on female food intake. Gross pathological examination of F0 and F1 adults indicated decreased abdominal fat and dark livers of both sexes at 6.0 mg/kg bw/day. Histopathology showed increased bile duct hyperplasia and periductular inflammation, as well as reduced hepatic glycogen in both sexes at 6.0 mg/kg bw/day. In the F0/F1a breeding no dose-related effects were observed on mating index, female conception index, male mating index, conception index, gestation index, survival indices (day 1, 4, 7, 14 and 21), sex ratio, duration of gestation, litter size, incidence of still births, incidence of external malformations in culled pups or evidence of internal malformations in grossly examined culled pups or sacrificed weanlings. Pup weights were, however, depressed at 6.0 mg/kg bw/day on days 7-21 of lactation. F1 weanling pathology was normal in all groups. In the F1/F2a and F1/F2b breeding, similar results were obtained. In addition, a slight decrease (probably not biologically significant) in pup survival was noted at 6.0 mg/kg bw/day on days 14 and 21 in the F1/F2b breeding. Overall, reproductive parameters appear to be unaffected at doses up to 6 mg/kg bw/day despite adult toxicity expressed as decreased body weight gain and liver histopathological changes. Post-natal pup development is also affected at 6.0 mg/kg bw/day, as expressed by reduced pup weight gain, although weanling histo-pathology is apparently normal. There was no evidence of induced abnormal pup development at any dose level. The NOAEL for this study appears to be 0.1 mg/kg bw/day, with some indication of decreased female body weight gain in F0 adults at 0.5 mg/kg bw/day. It should be noted that data provided in the report of this study was limited to mean values (with standard deviations). No detailed individual data were available except those on histopathological examination (Breslin et al, 1987). A second multigeneration study has been reported. In this study two generations were used with one litter in the first generation, and 2 litters in the second generation, the second of these litters was terminated prior to parturition and examined following caesarian section. Prior to commencing the multigeneration study, a palatability study was performed in three groups of 5 rats/sex/ dose. These groups were fed 0, 125 ppm technical cyhexatin (96% purity) or 125 ppm micronized cyhexatin (96% purity). It is stated that no deaths or clinical signs of toxicity occurred. Body weight gain was reduced for both cyhexatin groups during week 1 in males, and weeks 1 and 2 in females. Food consumption was reduced markedly in the first week (both sexes, both cyhexatin groups) and slightly in subsequent weeks. Food intake per kg bw was greater than control intakes in males in weeks 2-4 inclusive, but was depressed throughout the study in females. Water intake was reduced throughout the study in all cyhexatin groups. The severity of effects on the limited parameters measured was generally comparable for both cyhexatin technical and cyhexatin micronized (Barrow, 1991h) Groups of 25 OFA 5D (10PS Caw) rats/sex/dose level were fed diets containing 0, 10, 30 or 100 ppm of cyhexatin. Purity and physical characteristics (other than a statement that the cyhexatin was micronized) are not available. Analyses of the diets (all dose levels, weeks 1, 2, 4, 6-15 and 31-45 and at 10 ppm every week to week 45, except weeks 3, 5, and 28) indicate mean percentage of nominal concentrations to be 105.4 (range 87.0-138.2)% at 10 ppm, 103.9 (range 90.9-119.9)% at 30 ppm and 106.0 (89.9-138)% at 100 ppm (Masini, 1990 reported on analysis only). Samples were taken for analyses at the time of diet preparation, except the first week, when samples were analysed after 8 days post-diet preparation to assess stability. The initial pre-pairing treatment period of the F0 parents was 10 weeks. F1a parents were 13-16 weeks of age at pairing. Pairing was on a 1 male:1 female basis, with the same male throughout the 3 week period of pairing. Day 0 of gestation was considered to be the day on which a positive vaginal smear was observed. All litters were culled to 8 (4/sex where possible) on day 4 post-partum. Offspring were sacrificed at weaning (intended to be 21 days post-partum) unless required as parent animals for a subsequent generation. Prior to sacrifice, 2 pups/sex/litter were examined for pupillary reflex and auditory response. Sibling matings were avoided in the F1 parents. Pairing of F1 parents was, in both pairings, as for F0 pairings. In both pairings, the same male and female were paired. The interval between weaning of the F2a and pairing for the F2b litters was 2 weeks. Male body weight, recorded weekly from the time of selection until necropsy in both generations, was reduced at 100 ppm in the F0 generation. Weight gain was markedly reduced during the first week of treatment, and continued to be slightly reduced thereafter. In the F1a males, at selection, the body weight showed a dose-related decrease in all groups (statistically significant at 30 and 100 ppm). Weight gain during the study was very slightly reduced at 100 ppm, but was comparable to controls at 10 and 30 ppm. Female body weight (recorded weekly during pre-mating and mating periods, and on day 0, 7, 14 and 20 of gestation and days 1, 4, 7, 14 and 21 of lactation) was reduced in all F0 treated groups in a dose-related pattern, achieving statistical significance at 30 and 100 ppm by the end of week 1, and at 10 ppm by the end of week 2 of treatment. During gestation, body weight gain was reduced at 100 ppm on days 0-7, and 14-21. During lactation, the body weight gain at 100 ppm was reduced during days 4-7 and 7-14. In the F1 parents, body weight was reduced at the time of selection at 30 and 100 ppm (statistically significant). Body weight gain was increased at 100 ppm during the pre-mating period but was reduced during gestation, and days 7-14 of lactation. In the second mating, the 30 ppm and the 100 ppm group had reduced body weight compared to controls at the start of gestation and showed reduced body weight gain during gestation. Male food consumption was reduced at 100 ppm in the F0 generation (statistically significant in weeks 1, and 6-9 inclusive). In the F1 parents, food intake was significantly reduced every week at 100 ppm and in the first week at 30 ppm. Female food intake in F0 parents was depressed significantly (dose-related) every week at 30 and 100 ppm, and during weeks 4-7 at 10 ppm. During gestation, food intake was also significantly reduced at all dose levels, and a dose-related decreased intake was seen during lactation, achieving statistical significance at 30 and 100 ppm. In the F1 females, throughout the premating, gestation and lactation periods, food intake was reduced at 100 ppm. In the pre-mating period food intake was reduced during the first 2 weeks (statistically significant) and during the lactation period. The reductions in food intake were dose-related. In the second mating, food intake was reduced at 30 and 100 ppm (dose-related, statistically significant) during gestation. In the F0 - F1a reproduction phase, incidence of mating and pregnancy were comparable in all groups. The number of females with live pups was reduced at 100 ppm as were gestation, viability and weaning indices. Incidence of total litter loss during lactation was 0, 2, 3 and 4 at 0, 10, 30 and 100 ppm. Survival to weaning was reduced in 30 and 100 ppm groups (dose-related), and pup weights at weaning also showed a dose-related reduction at 30 and 100 ppm (resulting in up to 13 days delay in the weaning of some litters). Litter size and implantations/ dam were reduced at 100 ppm. Reduced litter size noted at 10 ppm was considered to be of questionable relationship to exposure to cyhexatin because of the absence of such an effect at 30 ppm. Pup eye opening was delayed by about 2 days in the 100 ppm group. In the F1 - F2a breeding, insemination rates, successful pregnancies and incidence of litters with live pups were comparable in all groups. The pre-coital interval between pairing and mating was increased at 100 ppm but was still within 1 oestrus cycle. Duration of gestation and total litter losses during lactation were also comparable in all groups. At 100 ppm, litter size and implants/dam were reduced, incidence of stillbirths was slightly increased and pup survival to weaning was decreased. Pup weights at birth were comparable, but during lactation pup weight gain was markedly reduced at 100 ppm, and slightly reduced at 30 ppm. Sex ratios were normal in all groups. Eye opening and incisor eruption were delayed at 100 ppm. Pupillary reflex was absent in 7 pups from 4 litters at 21 days post-partum at 100 ppm. In the F1 - F2b breeding, numbers of corpora lutea were reduced at 100 ppm, but pre-implantation losses were comparable in all groups. Neither were post-implantation losses different between groups. No dead fetuses were observed in any group. Fetal sex ratio was unaffected as was fetal weight. The only malformation (cleft palate and thoracic blood vessel effects) occurred at 30 ppm. The absence of similar malformations at other dose levels, especially at 100 ppm, mitigates against a relationship between this malformation and exposure to the compound. Uterine dilation and torsion at 100 ppm was increased compared to contemporary controls but the incidence was within historical control values. Skeletal variants showed an increased incidence of sternal ossification defects at 30 and 100 ppm but a decrease in the incidence of asymmetric sternebrae. Overall, there was no evidence of induction of developmental abnormalities (Barrow, 1990). A study utilizing 22-25 pregnant females/group was performed to determine the result of reduced food intake. The control group received food ad libitum with consequent intake of 26, 28, 31, 36, 57 or 73 g/day on gestation days 6-11, 11-16, 16-20 and lactation days 0-7, 7-14, and 14-21. Group 2 was terminated by caesarian section, food intake being limited to 20 g/day from day 6-20 of gestation. Group 3 was similarly limited during gestation and during lactation received 22, 30 and 33 g/day on lactation days 0-7, 7-14 and 14-21. Mean female body weight was significantly reduced from gestation day 11 until termination. Pup weights were also reduced at caesarian section (4.4 ± 0.4 in historical controls versus 3.3 ± 0.4 in Group 2) and throughout lactation. Litter size was unaffected, and survival to weaning was comparable to Group 1. It seems probable, therefore, that observed effects were, in many cases, related to reduced food intake. However, the reduced survival of pups at 30 and 100 ppm in the F0-F1a offspring, although not observed in subsequent pairings, support an NOAEL of 10 ppm. Special studies on embryotoxicity and teratology Rabbit Seven groups of 14 to 17 artificially inseminated pregnant female NZW rabbits were dosed orally, daily, on days 6-19 inclusive of gestation. Day 0 was considered to be the day of insemination. Dosage volume was 1 ml/kg bw. Group one received 0.5% w/v methyl-cellulose mucilage, which was used as the sus-pending agent for the remaining groups. Groups 2, 3 and 4 received 97% purity cyhexatin, manufactured in Kentucky, USA, at dose levels of 0.75, 1.5 or 3 mg/kg bw/dose, and groups 5, 6 and 7 received similar doses, but of 98% purity cyhexatin manufactured in The Netherlands. Two additional groups of pregnant artificially inseminated NZW rabbits received 99.7% purity cyhexatin. Treatment duration was 6-19 days of gestation: administered oral dose in both groups was 3.0 mg/kg bw/dose. Group size was limited to 8 or 9 animals because of the maternally toxic responses observed. Solvent systems differed between the two additional groups, one being 0.5% w/v methyl-cellulose mucilage and the second being 1% Cremophor EL. The test materials were analysed for purity, the major impurities being Cy4Sn and Cy2Sn), present at less than 1% in the technical samples. Purified cyhexatin contained 0.3% of Cy2SnO. Particle size of the three samples was measured, with the following results: Particle size (microns) Surface area % 901 % 502 % 103 (m2/gm) Kentucky 315 161 8 0.34 Netherlands 140 38 9 0.59 High purity 80 27 7 0.60 1 90% of particles are less than this size 2 Median particle size 3 10% of particles are less than this size. Homogeneity of test solutions was measured at 0.75 and 3.0 mg/ml in methyl cellulose mucilage (Kentucky technical material) and at 3.0 mg/ml in Cremophor (pure material). No data were available for the Netherlands sample. Homogeneity was acceptable. Mean administered dose was within 10% of nominal values. Administered solutions for all groups were measured in the first and last week of the study. In the first week of the study, all concentrations were within 10% of nominal except in groups 5 and 6 (Netherlands technical, dose levels 0.75 and 1.5 mg/ml, which indicated 84.5 and 82.6% of nominal, respectively. Only one sample (Group 3, Kentucky technical at 1.5 mg/ml, with 86% of nominal concentration) was more than 10% below nominal in the analyses in the final week of the study. Maternal mortality (or animals killed in extremis) was highest with the pure cyhexatin suspended in methyl cellulose mucilage (2/9), although no maternal deaths occurred in the Cremophor EL group. The Kentucky technical caused 1/15 deaths at 3.0 mg/kg bw/day, and 1/15 possibly compound related deaths at 1.5 mg/kg bw/day. The Netherland technical caused 4/18 maternal deaths at 3.0 mg/kg bw/day. Abortions occurred with the pure cyhexatin in both methyl-cellulose (2/9) and Cremophor EL (3/7) media. The technical materials resulted in 1/14 and 1/14 at 1.5 and 3.0 mg/kg bw/day using the Kentucky technical and in 1/16 and 2/17 at 0.75 and 3.0 mg/kg bw/day using Netherlands material. Maternal body weight gain was markedly reduced during treatment in both groups receiving the pure cyhexatin. The maternal body weight gain was initially reduced in animals receiving the Kentucky technical material at 0.75 and 1.5 mg/kg bw/day (not dose-related) but after day 20, exceeded controls and was comparable to these by day 24. At 3.0 mg/kg bw/day, although weight gain was similar to controls in gestation days 10-16 it was markedly reduced during the remainder of the treatment period. After treatment withdrawal, body weight gain slightly exceeded control values. The Netherlands technical material caused a dose-related decrease in body weight gain at all dose levels during the treatment period. After termination of dosing, body weight gain was comparable to control values, except in the 3.0 mg/kg bw/day group where body weight gain exceeded that of controls. Food intake was markedly reduced, especially in the Cremophor EL group in rabbits receiving the pure cyhexatin. Following termination of dosing, both groups showed increased food intake, compared to control values. The Kentucky technical material caused a decrease in food intake at 3.0 mg/kg bw/day but not at lower doses and the Netherlands technical material caused a slight decrease at 1.5, and a more marked decrease at 3.0 mg/kg bw/day, with compensatory increases in food intake during the post-dosing period. No compound attributable findings were observed during gross necropsy of dams in any group. No abortions occurred in the control group. A possible dose-related incidence of abortion was noted at 3.0 mg/kg bw/day (2/11 rabbits) with the Netherlands technical material, and a high incidence of abortion was observed in both groups receiving purified cyhexatin. All these abortions followed marked maternal weight loss. Corpora lutea of pregnancy counts were unaffected in any treated groups. Pre-implantation loss was increased slightly at 1.5 mg/kg bw/day with Kentucky technical material when compared to concurrent controls, but not when compared to historical control data. A dose-related increased pre-implantation loss occurred at all dose levels with Netherlands technical cyhexatin (% loss, 12.9, 25.5, 27.3, 28.7% at 0, 0.75, 1.5 and 3.0 mg/kg bw/day respectively). All values are, however, within the range of historical control incidence (6.8-29.2%). With purified cyhexatin pre-implantation losses were increased with both solvents (39% and 36% for Methocel and Cremophor solvents respectively). However, the apparent increases in both solvent groups were due to extremely high levels in one individual in each group, which exerted a disproportionate effect on the mean because of the small group sizes. Post implantation losses are increased with Netherlands technical material at 3.0 mg/kg bw/day (19.5%) exceeding loss rates in historical control data (range 2.8 - 18.8%). No post-implantation loss rate changes were considered compound-related in any other test group. Litter size was reduced at all dose levels with the Netherlands technical material, with a consequent increase in litter and placental weights. A similar pattern was noted with the purified cyhexatin. Incidences of fetal skeletal aberrations which showed a dose-effect relationship included 13/13 ribs, or short 13th rib with Kentucky technical at 3.0, the incidences being within historical control ranges, increased incidence of 12/13 ribs and increased incidence of thickened ribs with Netherlands technical material at 3.0 mg/kg bw/day, the incidence in both cases exceeding historical control incidences. These effects are considered as variants. Skeletal variants were also observed using purified cyhexatin, but the numbers of pups (34 in 4 litters with Methocel and 33 in 4 litters with Cremophor) are insufficient for valid evaluation. Soft tissue examinations indicated an increased incidence of unilateral or bilateral folded retinas at all dose levels with both technical compounds, exceeding historical control ranges. This was also noted with the Cremophor solvent group with the purified cyhexatin. Incidence of slightly increased dilation of lateral 3rd ventricle of the brain was observed in 1/22 and 2/34 heads examined in the 1.5 and 3.0 mg/kg bw/day groups receiving Kentucky technical, 1/36 heads examined in the 1.5 mg/kg bw/day group receiving Netherlands technical material, and in 1/12 heads examined in the Methocel solvent group receiving purified cyhexatin. The historical control range based on 740 heads examined in 20 studies was exceeded only at 3.0 mg/kg bw/day using the Kentucky technical (6.5% v 5.6%) and by the purified cyhexatin in methocel solvent (12.5% v 5.6%) (Bailey et al., 1990). COMMENTS In rats, blood levels of tin following administrations of cyhexatin peaked in 3-4 hours and then declined almost to control values in 24 hours. An oral study in rats using technical and micronized cyhexatin resulted in peak blood levels of tin at 3 hours with technical material and 4 hours with micronized material. Levels with micronized material were higher than those with technical material. Dermal exposure of rabbits resulted in similar blood levels of tin with both technical and micronized material. In pregnant rabbits administered 3.0 mg technical cyhexatin kg bw/day on days 6-18 of gestation, peak maternal blood tin levels were achieved about 3 hours after dosing. Tin half-life in maternal blood was 8.17 ± 1.59 hours. Tin levels in amniotic fluid, placentae, and pups were significantly increased on day 19. Tin levels in pup brains were also elevated. By day 26, tin levels in treated animals were comparable to those in control animals, except in brain where levels were slightly elevated. On day 19 mean pup weights were comparable to controls, but were reduced by day 26. No fetal malformations were reported in this study. Following both oral and dermal dosing with cyhexatin in rats and rabbits, the tin was eliminated in both urine and faeces. The major route of elimination was via the urine. A rabbit teratology study using two different technical samples, one from the USA and the other from The Netherlands (the Netherlands material had a smaller particle size), and one pure sample of cyhexatin indicated differences in the severity of cyhexatin maternal toxicity, which appeared to be related to the product particle size, a smaller particle size resulting in increased toxicity. When the two technical samples were compared (high mortality with the pure material prevented valid interpretation of comparative data), pre- and post-implantation losses, fetotoxicity, and reduction in litter size followed a pattern similar to maternal toxicity. A high incidence of folded retinas (exceeding the control range) was noted with both technical samples at the lowest dose tested (0.75 mg/kg bw/day); the significance of this finding was uncertain. An increase in the occurrence of dilation of the third and/or lateral ventricle of the brain was noted with the US technical material and with the pure material at 3.0 mg/kg bw/day. There was no evidence of hydrocephaly at 0.75 mg/kg bw/day with technical cyhexatin. Two studies in rats were available. The first study utilized doses of 0, 0.1, 0.5 or 6 mg technical cyhexatin/kg bw/day in a two-generation study with 1 or 2 litters/generation. The NOAEL in this study was 0.1 mg/kg bw/day, with decreased weight gain occurring in females at 0.5 mg/kg bw/day. Reproductive parameters were unaffected in this study, except for reduced post-natal pup weight gain at 6 mg/kg bw/day. There was no evidence of induced abnormal development of pups in utero. The second study, utilizing dietary concentrations of 0, 10, 30 or 100 ppm, which incorporated a teratology component, indicated a NOEL of 10 ppm, equivalent to 0.5 mg/kg bw/day. Decreased body-weight gain in pups during lactation and reduced pup survival in F0-F1a offspring were observed at 30 ppm. There was no evidence of compound-induced developmental abnormalities. The ADI was estimated on the basis of the multigeneration study in rats (NOAEL 0.1 mg/kg bw/day), applying a 100-fold safety factor. Additional data on the particle size of micronized material was received during the Meeting but there was not sufficient time to interpret and relate these data to all the relevant studies. The Meeting recommended that cyhexatin be reviewed again in 1994 when the results of ongoing work and the studies listed below should be available. TOXICOLOGICAL EVALUATION Level causing no toxicological effect Mouse: 3 mg/kg bw/day Rat: 0.1 mg/kg bw/day (multigeneration study) Rabbit: < 0.75 mg/kg bw/day (teratology) Dog: 0.75 mg/kg bw/day Estimate of acceptable daily intake for humans 0-0.001 mg/kg bw Studies which will provide information valuable in the continued evaluation of the compound 1. Observations in humans 2. Clarification of the influence of particle size on the toxicokinetics and toxicity of cyhexatin 3. Determination of the effect of restricted food intake on reproduction parameters, preferably by a limited paired feeding study on rats during gestation and lactation. 4. Information on the particle size of cyhexatin residues on food. REFERENCES Oxam Italia (1991). Analytical determinations by Oxam Italia resulting from the in-life study reported by Woehrle, F., and submitted to WHO by Oxam Italia. Bailey, G.P., Wilby, D.K., Tesh, S.A. & Brawn, P.M. (1990). Tricyclolmexyltin hydroxide: Teratology study in the rabbit. Unpublished report of Life Science Research, submitted to WHO by Aloctom North America, Inc. Barrow, P.C. (1990). Draft report of a two-generation oral (dietary administration) reproduction study in the rat. Unpublished draft report of Hazleton, France, submitted to WHO by Oxam Italia. Barrow, P.C. (1991a). Cyhexatin - Preliminary proof of absorption investigation in the rabbit by oral and percutaneous routes. Unpublished report by Hazleton, France, submitted to WHO by Oxam Italia. Barrow, P.C. (1991b). Cyhexatin - Preliminary proof of absorption investigation in the rat by the oral route. Unpublished report by Hazleton, France, submitted to WHO by Oxam, Italia. Barrow, P.C. (1991c). Cyhexatin technical - Pharmacokinetic investigation in the rabbit by the oral route. Unpublished report by Hazleton, France, submitted to WHO by Oxam Italia. Barrow, P.C. (1991d). Cyhexatin technical. Pharmacokinetic investigation in the rat by the oral route. Unpublished report by Hazleton, France, submitted to WHO by Oxam Italia. Barrow, P.C. (1991e). Cyhexatin micronized Pharmacokinetic investigation in the rabbit by the oral route. Unpublished report by Hazleton, France, submitted to WHO by Oxam Italia. Barrow, P.C. (1991f). Cyhexatin technical. Pharmacokinetic investigation in the rabbit by the percutaneous route. Unpublished report of Hazleton, France, submitted to WHO by Oxam Italia. Barrow, P.C. (1991g). Cyhexatin micronized. Pharmacokinetic investigation in the rabbit by the percutaneous route. Unpublished report by Hazleton, France, submitted to WHO by Oxam Italia. Barrow, P.C. (1991h). Cyhexatin. Palatability study in Sprague-Dawley rats. Unpublished report by Hazleton, France, submitted to WHO by Oxam Italia. WHO. Breslin, W.J., Berdasco, N.M., Keyes, D.G. & Koceba, R.J. (1987) Cyhexatin: two-generation dietary reproduction study in Sprague-Dawley rats. Unpublished report of the Dow Chemical Co., submitted to WHO by Dow Chemical. Fraschini, C. (1991). Liquid Chromatographic analysis of cyhexatin (tricyclohexyltin hydroxide) in aqueous suspension. Unpublished report of Sepcam S.p.A., submitted to WHO by Oxam Italia. Masini, M. (1990). Analysis of dietary samples to check the content of lacgetobexyltin hydroxide. Unpublished report by Instituto de Ricerche E. Collaudi, submitted to WHO by Oxam Italia. Salmona, M. & Gagliardi, L. (1991). Appraisal of the analytical determinations anonymously reported, obtained from the in-life portion of the study reported by Woehrle, submitted by Oxam Italia. Woehrle, F. (1991a). Cyhexatin technical: Blood and tissue level determination in the pregnant rabbit and its litter after oral administrations during organogenesis. Unpublished report by Hazleton, France, submitted to WHO by Oxam Italia. Woehrle, F. (1991b). Cyhexatin micronized-Pharmacokinetic investigation in the rabbit by the intravenous route. Unpublished draft report of Hazleton, France, submitted to WHO by Oxam Italia. Woehrle, F. (1991c). Cyhexatin micronized - Pharmacokinetic investigation in the rabbit by the intravenous route. Unpublished draft report of Hazleton, France, submitted to WHO by Oxam Italia to WHO. Woehrle, F. (1991d). Cyhexatin micronized - Pharmacokinetic investigation in the rat by the intravenous route. Unpublished draft report by Hazleton France, submitted to WHO by Oxam Italia.
See Also: Toxicological Abbreviations Cyhexatin (WHO Pesticide Residues Series 4) Cyhexatin (WHO Pesticide Residues Series 5) Cyhexatin (Pesticide residues in food: 1978 evaluations) Cyhexatin (Pesticide residues in food: 1980 evaluations) Cyhexatin (Pesticide residues in food: 1981 evaluations) Cyhexatin (Pesticide residues in food: 1983 evaluations) Cyhexatin (Pesticide residues in food: 1989 evaluations Part II Toxicology) Cyhexatin (JMPR Evaluations 2005 Part II Toxicological)