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    CAPTAN

    EXPLANATION

    First draft prepared by Dr. O. Meyer, 
    National Food Agency, Denmark

         Captan has been evaluated by the Joint Meetings on Pesticide
    Residues in 1963, 1965, 1969, 1973, 1977, 1978, 1982, and 1984
    (FAO/WHO 1964, 1965ab, 1970ab, 1974ab, 1978ab, 1979ab, 1983ab, and
    1985bc).  The data base includes data on biochemical aspects, acute
    studies in mice, rats, and rabbits, short-term studies in mice, rats,
    dogs, pigs, and chicks, long-term toxicity studies in mice and rats,
    carcinogenicity studies in mice and rats, reproduction studies in rats
    and teratogenicity studies in mice, rats, chicks, hamsters, monkeys,
    rabbits, and dogs, and several in vivo and in vitro genotoxicity
    studies.  In addition, data from observations in humans have been
    considered.  In view of concerns with respect to carcinogenicity in
    mice, the 1984 Joint Meeting desired further work or information on
    comparative study of the metabolic fate of captan in rats and mice and
    further observations in humans.  The estimate of ADI for humans is 0-
    0.1mg/kg bw established at the 1984 Joint Meeting.  Only one new study
    on teratology in rabbits was made available to the meeting.  This
    study is reviewed in the monograph addendum, and information will be
    included from a recent safety assessment of captan (Elder, 1989) and
    other relevant work published since the 1984 Joint Meeting.

    EVALUATION FOR ACCEPTABLE DAILY INTAKE

    Biological data

    Biochemical aspects

    Absorption, distribution and excretion

         When 35S-captan was administered orally to rats more than 90% of
    the radioactivity was eliminated in the urine and faeces (mainly as
    metabolites of captan) within the first 24 hours.  Between 0.01 and
    0.05% of the radioactivity remained in the body incorporated into
    protein and nucleic acids.  14C-captan was excreted in urine, faeces
    and expired air after oral administration.  Intraperitoneally treated
    rats exhibited a similar, but delayed excretion compared to orally
    dosed rats, suggesting a rapid metabolism in the gastrointestinal
    tract (Elder, 1989, Dalvi, 1989).

         Dermal application of [14C] captan showed that captan penetrates
    rat skin fairly rapidly and is excreted rapidly, primarily in the
    urine (Fisher et al., 1988).

    Biotransformation

         Captan is rapidly hydrolyzed with cleavage of the N-S bond in the
    blood and in the gut in rats.  The most typical reaction of the captan
    molecule is with thiols to form tetrahydrophthalimide and thiocarbonyl
    chloride.  The latter then reacts further with thiols and loses HCl
    yielding thiophosgene.  Thiophosgene can be detoxified by reaction
    with cysteine, glutathione, sulfite or by oxidation/or hydrolysis to
    yield CO2 (Elder, 1989).

         Degradation is likely to increase in the alkaline regions of the
    gastrointestinal tract.  Captan is also likely to be metabolized in
    the liver (Dalvi, 1989).  However, the metabolism of captan in the
    liver does not influence excretion, as the amount of radioactivity
    excreted was similar for both normal, sham-operated and partially
    hepatectomized male Sprague-Dawley rats receiving 35S-captan i.p. at
    a dosage of 6 mg/kg bw in 0.5 ml corn oil.  Captan also reacts with
    guanine to form N-7 guanine alkylation products, 7-
    (trichloromethylsulfenyl) guanine (Elder, 1989).

         In mice administered a single oral dose of 156 mg/kg of [14C-
    trichloromethyl]-labelled captan with a specific activity of 50-56
    mCi/mmol the number of trichloromethyl carbon atoms per DNA nucleotide
    ranged from 1x10-6 for testicular DNA to 8x10-5 for gastric DNA.  The
    association of radioactivity to DNA was similar in all of the tissues
    examined.  In a similar study in which both the mice and rats were
    administered higher dosages of captan with lower specific activities,
    no radioactivity was associated with tissue DNA (Elder, 1989).

         The trichloromethylthio moiety may be responsible for the
    toxicity of captan.  The trichloromethylthio moiety reacts rapidly
    with thiols.  While its interaction with soluble thiols is a
    detoxication process, the reaction with insoluble thiols is considered
    the cause of toxic action (Dalvi, 1989).

    Effects on enzymes and other biochemical parameters

         Captan inhibits the synthesis of DNA, RNA and protein in Ehrlich
    ascites tumour cells.  Reversal of inhibition on the synthesis was
    achieved by addition of cysteine or dithiothreitol.  In the same study
    captan was shown to inhibit glucose utilization, sulfhydryl compounds
    reversed the inhibition of DNA, RNA and protein synthesis (Elder,
    1989).

         Captan's effects on the two exonuclease activities of DNA
    polymerase 1 was determined using poly (dA-dT) and bacteriophage T7
    DNA as substrates for the enzyme activity.  One hundred M captan
    enhanced the activity of the DNA polymerase 1 holoenzyme for both
    substrates.  The enhancement by captan of the holoenzyme activity was
    related to the enzyme's 5'-3' function (Freeman-Wittig and Lewis,
    1986).

         Captan inhibits E. coli RNA polymerase activity by inhibiting its
    binding to the DNA template, probably due to its reaction with thiol
    groups on the core enzyme (Elder, 1989).

         Captan inhibited RNA synthesis by intact bovine nuclei and by
    hypotonic lysate of bovine nuclei, probably due to its sulfur moiety
    since the sulfhydryl compound dithiothreitol protected RNA polymerase
    activity from the action of captan in intact nuclei (Elder, 1989).

         Captan has been reported to inhibit microsomal cytochrome P-450
    benzphetamine N-demethylase and aniline hydroxylase.  Furthermore,
    captan is a potent inhibitor of liver and intestinal microsomal aryl
    hydrocarbon hydroxylase in rats (Dalvi, 1989).

         The carbonyl sulfide derived during the metabolism of captan may
    be a cause of liver damage through its sulfur atom which is released
    during metabolism and is bound to liver microsomal membranes (Dalvi,
    1989).

         Captan has been shown to inhibit the oxidative phosphorylation in
    rat liver mitochondria.  A correlation between mitochondrial swelling
    and loss of mitochondrial function has been indicated.  Inhibition of
    captan activated ATPase was reversed by presence of magnesium
    indicating a possible change of permeability of the mitochondrial
    membrane leading to inhibition of the Mg2+ activation of DNP-
    activated ATPase (Elder, 1989).

         Captan (10 M) inhibited the activity of the Ca2+ -transport-
    ATPase in human erythrocytes (Janik, 1986).

    Toxicological studies

    Acute toxicity studies

         The LD50 in Sherman rats dosed with captan perorally was >5000
    mg/kg bw both in adults and in weanlings (Gaines and Linder, 1986). 
    The oral LD50 for captan in rats is an order of magnitude higher than
    the LD50 for intraperitoneal (i.p.) exposure and via inhalation,
    respectively.  The i.p. LD50 and LC50 in mice are within the same
    range as for rats (Elder, 1989).

    Long-term/carcinogenicity studies

    Mice

         A study was conducted to identify and characterize early
    histologic changes in the small intestine in male CD-1 mice, 40 days
    of age (35 animals/group) dosed with captan (captan technical) by
    dietary administration at 0 and 6000 ppm for 3, 6, 9, 12, 18 or 20
    months.  In addition one group of 35 male CD-1 mice was dosed with
    6000 ppm captan in its diet for 6 months.  Ten of these animals were
    sacrificed after a recovery period of 6 months and the remaining
    animals after 12 months of recovery.  Another group of 35 male CD-1
    mice was treated in a similar way for 12 months after which 10 animals
    were sacrificed 6 months after termination of the dosing period and
    the remaining animals were sacrificed 8 months after the termination
    of dosing.

         Survival was not adversely affected by treatment.  Weight gain
    was significantly reduced in all treated mice and only slightly
    reversed during the recovery periods.

         The most characteristic pathologic findings consisted of
    necrotizing and proliferative changes in the nonglandular portion of
    the stomach (only after 3 months), dilatation of the small intestine
    and focal epithelial hyperplasia in the proximal part of the small
    intestine.  Focal epithelial hyperplasia was also found in controls,
    but the incidence was lower compared to that of treated animals, and
    the localization of these foci was more caudal than was the case for
    the captan administered mice.  Diffuse hyperplasia was found only in
    dosed animals and was not considered prerequisite for the development
    of focal hyperplasia.

         Adenomas and adenocarcinomas also developed in the small
    intestines of treated animals with the localization in the proximal 7
    cm of the small intestine, the area of localization being the same as
    for the focal hyperplasia.

     
         Removal of captan from the diet resulted in a significant
    reduction in the incidence of focal epithelial hyperplasia as compared
    to the incidence in concurrent lifetime-treated mice, and was no
    greater than that in concurrent controls.  The incidence of neoplasia,
    however, in mice in the recovery group was not significantly different
    from that of concurrent lifetime-treated mice, but increased in mice
    treated for 6 months with a recovery period of 6 months and in mice
    treated for 12 months with a recovery period of 6-8 months,
    respectively, when compared to the controls.  The latter increase was
    not found in mice treated for 6 months with a recovery period of 12
    months (Pavkov and Thomassen, 1985).

         An experiment was reported with 65 chemicals including captan
    dosed i.p. to two different strain A mice.  A slight increase in lung
    tumours was seen only in male mice in one of the employed strains. 
    However, the study revealed as poor agreement between strain A results
    from either laboratory.  In addition, there was poor agreement with 2-
    year carcinogenicity test results for the 65 chemicals used (Maronpot
    et al., 1986).

    Rats

         Groups of Wistar rats (50 males and 50 females per group) were
    administered 0, 125, 500 and 2000 ppm for 130 weeks (equal to  0, 5,
    24, and 98 mg/kg bw/day based on the actual intake levels of captan
    (analysis of diet: 0, 124, 504 and 2083 ppm).

         The body weight and food intake were reduced in the highest dose
    group throughout the study (the overall decrease was about 10% both
    for males and females) when compared to the control group.  The
    relative weight of the liver was slightly increased in males in the
    high dose group.  No other effects could be attributed to treatment
    with captan (Til, et al., 1983).

         Induction of preneoplastic glutathione S-transferase (placental
    form) positive foci by 112 compounds in partially hepatectomized
    Fischer rats dosed with diethylnitrosamine was studied.  No effect
    upon induction of foci was seen in rats dosed 3000 ppm (equivalent to
    150 mg/kg bw/day) in the diet for 6 weeks (Ito, et al., 1988).

         In two hybrid strains (C57BL/6 and C3M/AuF) of mice, 18 of each
    sex, no increased incidence of tumours was observed at 78 weeks of age
    after s.c. injections of 1000 mg/kg bw on the 28th day of life (IARC,
    1983).

    Special studies on embryo/fetotoxicity

         Four groups of mated HY/CR New Zealand White rabbits (4 to 5
    months old) were dosed with captan (91% active ingredient) in 0.5%
    carboxymethylcellulose containing 0.05% acetic acid, by oral gavage

    from day 7 to day 19 post coitum, inclusive at dosages 0 (vehicle),
    10, 40 and 160 mg/kg bw/day, respectively.  The numbers of animals in
    the groups were 18, 14, 15 and 16, respectively.

         Maternal toxicity as evidenced by reduced food intake and
    decreased body weight gain was seen in highest-dosed animals and to a
    lesser extent in the mid-dosed animals.  Fetal adverse response to
    treatment consisted of increased fetal death (post implantation loss),
    with one case of abortion following total fetal death and one case of
    total fetal death discovered at terminal necropsy.  In addition, an
    increased frequency of minor skeletal variations was recorded in
    fetuses in the high dose group.  Fetuses of dams dosed at 40 or 10
    mg/kg bw/day were not affected.  Captan did not show a potential for
    teratogenicity in the NZW rabbit in the high dosage group and did not
    cause adverse fetal effect in the absence of maternal toxicity under
    conditions of the current study (Rubin and Nyska, 1987).

    Special studies on in vitro developmental toxicity

         Captan was shown to be less effective in the presence of S-9 than
    in its absence, in an in vitro test for inhibition of
    differentiation of cultured cells derived from midbrain and limb bud
    of 34 to 36 somite rat embryos (Flint and Ortan, 1984).

         Captan was shown to inhibit the attachment of tumour cells to
    polyethylene disks coated with concanavalin, with a concentration of
    42 mg/l inhibiting attachment by 50% (Braun and Morowicz, 1983).

    Special studies on immunotoxicity

         Sprague-Dawley male rats, six to seven weeks old and six to eight
    week-old Bald/c male mice were fed a semisynthetic diet with 0.3%
    captan.  Immune effects were measured by sheep red blood cell (SRBC)
    antibody formation and by lymphocytic response in mice to stimulation
    by mitogens.  SRBC antibody production was inhibited in both rats and
    mice, the effect on the latter species being found after 42 days. 
    Captan suppressed both B and T-cell function in mice (Lafarge-
    Frayssinet and Decloitre, 1982).

         SPF-derived weanling male Wistar rats (6/dose) were fed a
    semisynthetic diet with captan at concentrations 1,000, 5,000 and
    25,000 ppm for three weeks (equivalent to 50, 250 and 1250 mg/kg
    bw/day). Captan at a dietary level of 1,000 ppm significantly
    depressed lymphocyte count and relative thymus weight, while the serum
    IgG level was significantly elevated (Vos and Krajnc, 1983).

         Rats (Wistar) were fed captan in their diets.  Pre- and postnatal
    exposure levels were 750 and 2,000 ppm (equivalent to 37.5 and 100
    mg/kg bw/day).  Significantly decreased secondary IgG response to
    tetanus toxoid in the 2,000 ppm group was the only parameter affected
    (Vos and Krajnc, 1983).

    Special studies on genotoxicity

         The genotoxicity of captan was studied in vivo in the proximal
    small intestine of male CD1 mice, aged 6-10 weeks (group sizes 5-10). 
    Orally administered captan up to 4,000 mg/kg bw/day failed to induce
    a response in the small intestine nuclear aberration assay either in
    animals pretreated with an inhibitor of glutathione biosynthesis or in
    animals not receiving this inhibitors (Chidiac and Goldberg, 1987).

         The cytogenetic effects of captan on mouse bone marrow and
    testicular cells (strain not reported) were studied.  A significant
    increase in frequency of micronuclei was seen in animals dosed orally
    with 100 mg/kg bw/day for 2 days.  The induction of chromosomal
    aberrations in bone marrow was found in animals orally dosed 400 mg/kg
    bw/day for 5 days.  The lower doses inducing mutations of primary
    spermatocyte and spermatogonia and sperm-head abnormalities were 50,
    800, and 200 mg/kg bw/day orally for 5 days, respectively (Feng and
    Lin, 1987).

         See Table 1 for a summary of genotoxicity assays with captan.

        Table 1.  Summary of genotoxicity assays with captan
                                                                                             

    I.    In vitro tests:                                 Bacteria    Yeast  Mammalian 
                                                                             cells

          1.    DNA-repair
                a)    Inhibition of growth in                 +         -      - 
                      repair-deficient bacteria
                b)    UDS                                     -         -      (+)

          2.    DNA-strand-breaks                             nt.       nt.    +

          3.    Increased recombination                       -         +      -
                a)    Gene conversion                         -         +      -
                b)    Mitotic recombination                   -         +      -
                c)    SCE                                     -         -      (+)

          4.    Point mutations                               +         (+)    (+)

          5.    Chromosomal aberrations                       -         -      (+)

          6.    Cell transformation                           -         -      

          7.    Induction of bacteriophage                             -      -

    II.   In vivo/in vitro - Salmonella/Hostmediated assay

          1.    Point mutations                               ()

    III.  In vivo - mammalian bone marrow                     

          1.    Micronucleus, bone marrow                     

          2.    Chromosomal aberrations, 
                  bone marrow                                 ()

    IV.   In-vivo germ cells                      Drosophila            Mammals

          1.    Dominant lethal mutations             nt.                 ()a

          2.    Recessive lethal mutations            +b                  -

          3.    Heritable translocation                                  c,d

          4.    Specific locus mutations              -                   
                                                                                             

         +:   Exclusively positive results
         :   Exclusively negative results
       (+):   Predominantly positive results
       ():   Predominantly negative results
       nt.:   Not tested
         -:   No test system developed
       UDS:   Unscheduled DNA-synthesis
       SCE:   Sister chromatid exchange

        a):   Mouse and rat, both i.p. and p.o.
        b):   Weak effect was not excluded
        c):   One translocation in high dose 
              group and one in the negative control group
        d):   Mouse, p.o.
    
    COMMENTS

         Biologically, captan is a very active compound.  The most common
    reaction is with thiols.  The reaction of the trichloromethylthio
    group, the biologically active moiety of captan, with insoluble thiols
    is considered to be the chemical basis of toxicity, while the
    interaction with soluble thiols is a detoxification process.

         A negative carcinogenicity study in Wistar rats further
    strengthened the previous studies in Charles River CD rats.  Captan
    has been shown to be carcinogenic in mice, but only at doses at and
    above 6000 ppm.  The induction of carcinogenicity was limited to the
    proximal part of the small intestine.  Focal proliferative changes
    preceded the induction of tumours in the intestine of mice. 
    Subcutaneous injections in Fischer 344 rats with 100 mg captan kg
    bw/day (in DMSO) for two weeks resulted in a significant increase in
    gastric mucosal thymidine kinase activity and DNA synthesis (measures
    of induction of gastric mucosal cell proliferation).  The Meeting was
    informed that an investigation of possible mechanisms of the
    neoplastic response in mice is now in progress, and would be submitted
    to WHO in 1992.

         A range of studies suggests that captan is potentially genotoxic. 
    It was positive in most in vitro genotoxicity tests, but these
    effects were reduced in the presence of the S-9 fraction from liver. 
    Although captan was negative in most in vivo genotoxicity tests,
    conflicting results have been reported.  No nuclear aberrations were
    induced in the small intestine of CD-1 male mice dosed orally with up
    to 4000 mg captan/kg bw.

         Studies in Sprague-Dawley and Wistar rats and Balb/c mice suggest
    that captan may be an immunodepressant.  Data suggest that ingested
    captan is rapidly degraded in  and excreted from the gastrointestinal
    tract.

         In 1984 the JMPR estimated the Acceptable Daily Intake for humans
    to be 0.1 mg/kg bw based upon a NOAEL in a reproduction study in rats
    of 12.5 mg/kg bw/day using a safety factor of 125.  The data available
    to the present Meeting did not alter this conclusion.

    TOXICOLOGICAL EVALUATION

    Level causing no toxicological effect

         Mouse:    800 ppm in the diet, equivalent to 120 mg/kg bw/day
         Rat:      250 ppm in the diet, equivalent to 12.5 mg/kg bw/day
                   (based on reproduction studies)
         Dog:      100 mg/kg bw/day
         Monkey:   12.5 mg/kg bw/day (based on reproduction studies)

    Estimate of acceptable daily intake for humans

         0-0.1 mg/kg bw

    Studies which will provide information valuable in 
    the continued evaluation of the compound

         -    Further observations in humans.

         -    Comparative studies on the metabolic fate of captan in rats
              and mice and its relevance for determining human safety.

         -    Further studies to elucidate the mechanism for the induction
              of gastrointestinal tract tumours in mice.

         -    Submission of studies known to be in progress.

    REFERENCES

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    See Also:
       Toxicological Abbreviations
       Captan (HSG 50, 1990)
       Captan (ICSC)
       Captan (PIM 098)
       Captan (FAO/PL:1969/M/17/1)
       Captan (WHO Pesticide Residues Series 3)
       Captan (WHO Pesticide Residues Series 4)
       Captan (Pesticide residues in food: 1977 evaluations)
       Captan (Pesticide residues in food: 1978 evaluations)
       Captan (Pesticide residues in food: 1980 evaluations)
       Captan (Pesticide residues in food: 1982 evaluations)
       Captan (Pesticide residues in food: 1984 evaluations)
       Captan (Pesticide residues in food: 1984 evaluations)
       Captan (Pesticide residues in food: 1995 evaluations Part II Toxicological & Environmental)
       Captan (IARC Summary & Evaluation, Volume 30, 1983)