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    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    Cyhexatin was withdrawn from the market in a number of countries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 g/l at 0, 0.5, 1, 3, 4, 6, 8, 12 and 24 hrs.  Mean urinary levels
    (5 animals) were 9.0 g/l in controls, and 68.5 g/l in treated rats
    over 24 hours.  Mean faecal levels (5 animals over 24 hours) were 1.89
    and 176.9 g/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 g/l at 0, 0.5, 1, 3,
    4, 6, 8, 12 and 24 hours.  Mean values among levels were 8.5 g/l in
    control, and 85 g/l in treated rats over 24 hours.  Faecal levels
    were 2.4 g/g (control) and 202.8 g/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 g/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 g/ml at 10, 15, 20, 30,
    60, 120, 240, 360, 480 mins and 24 hours.  Mean urine levels pretest
    were 4.1 g/l, and over 24 hours post-dosing were 43 g/l.  Faecal

    levels, pretest, were 1.4 g/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 g/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
    g/g) decreasing to 1.8-8.4 g/g after 48 hours.  Urine levels at 24
    hours were 38.9-112.7 g/l (mean 74.75) g/l) and at 48 hours,
    58.9-108.4 g/l (mean, 83.37 g/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 g/g) but had dropped to a mean of
    5.7 g/g, range 3.4-11.8 g/g by 48 hours.  Urinary levels were
    62.7-98.9 g/l (mean 76.3 g/l) at 24 hours and 100.2-140.9 g/l (mean
    119.7 g/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 g/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 g/l.  Mean faecal tin levels were 1.5,
    2.9 and 2.0 g/g at 24, 48 and 54 hours, and mean urine levels were
    65.8, 82.5 and 43.5 g/l at 24, 48 and 54 hours (Barrow, 1991f).

         In a similar experiment, but using micronized cyhexatin, mean
    blood levels were 5.9 g/l in pretreated rabbits, and 7.5, 7.1, 7.9,
    11.1, 9.2, 7.5, 6.1 and 6.6 g/l at 0.5, 1, 4, 8, 24, 32, 48 and 54
    hours.  Mean faecal levels were 5.9, 1.9 and 1.6 g/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 g/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 g/l at time 0 to
    0.28 g/l after 5 minutes.  After one hour,  the level had declined to
    0.08 g/l and after 3 hours, to 0.07 g/l.  Urine levels (measured in
    3 rabbits) indicated mean tin levels of 52.7 g/l at 24 hrs and 67.0
    g/l at 48 hours.  Pretest levels in 5 rabbits indicated mean tin
    levels of 31 g/l.  Faecal levels (measured in 3 rabbits) indicated
    mean tin levels of 12.56 g/g at 24 hours and 14.3 g/g at 48 hours. 
    Pretest levels in 5 rabbits indicated a mean tin level of 5.9 g/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)