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 µM 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 µM) 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.
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