FOLPET
Explanation
First draft prepared by Dr O. Meyer,
National Food Agency, Denmark
Folpet was evaluated for acceptable daily intake by the Joint
Meeting in 1969, and reviewed in 1973, 1982, 1984, and 1986 (Annex 1,
FAO/WHO 1970a, 1974a, 1983a, 1985b, and 1986d). A toxicological
monograph was prepared by the Joint Meeting in 1969 (Annex 1, FAO/WHO,
1970b) and monograph addenda were prepared in 1973, 1984, and 1986
(Annex 1, FAO/WHO, 1974b, 1985c, and 1987a). The data that have been
reviewed include data on biochemical aspects, acute studies in mice,
rats, and rabbits; short-term studies in rats and dogs, long-term
toxicity studies in rats, carcinogenicity studies in rats and mice,
reproduction studies in rats; teratogenicity studies in rats,
hamsters, monkeys, rabbits, and chicks and several in vivo and
in vitro genotoxicity studies. In addition, data from observations
in humans have been considered.
EVALUATION FOR ACCEPTABLE DAILY INTAKE
Biological data
Biochemical aspects
Absorption, distribution and excretion
Mice (108) and rats (36) were dosed 0, 50, and 5,000 ppm folpet
in their diet for 21 consecutive days. Following a p.o. pulse dose of
14C-folpet (14C in the trichloromethylthiol moiety) the animals were
killed either 2, 4 or 6 hours post-dose and the gastrointestinal tract
removed.
Quantitation of the amount of radioactivity present as a
percentage of the administered dose at various times showed that the
mouse removed a greater proportion of radioactivity from the
gastrointestinal tract (GI) than did the rat, and the removal of
radioactivity also occurred at a faster rate in the mouse.
Unchanged folpet was present in a lower proportion in mice when
compared to rats at each time interval in both doses. The proportion
of the radioactive dose actually in the tissues of the
gastrointestinal tract was between 1% and 3% in both species.
Gastrointestinal transit time (from stomach to caecum) was less than
2 hours and 4-6 hours for mouse and rat, respectively.
At each time interval the major portion of parent compound at the
low dose level was recovered from the stomach in both species.
However, the lower amounts found for the mouse indicate a faster rate
of stomach emptying in this species. Following the high dose the
largest proportion of unchanged folpet was located in the caecum of
the mouse and in the stomach in the rat.
The proportion of residual radioactivity (considered to be
"covalently" bound to tissues) presented as the ratio of bound
radioactivity between the high and low dose (expressed in terms of µg
equivalents) varied between 14:1 and 30:1 in the stomach, up to 115:1
and 180:1 in ileum of rat and mouse, respectively.
Folpet is rapidly degraded in the GI-tract. This occurs to a
greater extent at lower than at the higher dose levels and in the
mouse more than in the rat. The greater amounts of radioactivity
"covalently" bound after the high dose level indicates lack of
sufficient GSH for thiophosgene removal (Chasseaud and Waller, 1990).
An excretion-radioactive-balance study over 5 days with mice (36)
and rats (12) dosed as in the above study showed that following a
"pulse dose" of 14C-folpet, 14C was excreted in expired air, urine
and faeces. Urinary excretion of radioactivity during the first 24
hours was lower in the rats treated with 50 ppm (41.8%) than those
treated with 5,000 ppm (51.5%). Greater urinary excretion was
observed at the low dose level in the mouse (50 ppm 59.1% and 5,000
ppm, 44.3%).
Examination of the faecal excretion of radioactivity indicated
that gastrointestinal transit-times in the rat were slower (48 hours)
than that observed in the mouse (24 hours), and at 5,000 ppm mice
excreted 5 fold greater amount of radioactivity than did rats.
Biliary excretion of 14C-folpet was about 2% in rats and less
than 0.1% in mice (Chasseaud and Waller, 1990).
Biotransformation
Mice (108) and rats (36) were dosed 0, 50, and 5,000 ppm folpet
in their diet for 21 consecutive days. Following a p.o. pulse dose of
14C-folpet (14C in the trichloromethylthiol moiety) the animals were
killed either 2, 4 or 6 hours post-dose and the gastrointestinal tract
removed.
At 2 hours after dosing, the disulfonic acid was the major
metabolite in the rat duodenum with both the thiazolidine and the
glutathione conjugate of thiophosgene being present. At 4 hours the
disulfonic acid predominated and the thiazolidine metabolite was not
seen. In the mouse, the same metabolites were seen, but the
thiazolidine metabolite becomes more prominent in the later timed
samples indicating that the mouse relied more than did the rat on GSH
conjugation for the detoxification of the "active metabolite" of
folpet (Chasseaud and Waller, 1990).
An excretion-radioactive-balance study over 5 days with mice (36)
and rats (12) dosed as in the above study, showed quantitative
differences in the urinary excretion of metabolites. The sulphonic
acid metabolite predominated in rat while the thiazolidine metabolite
predominated in the mouse after the high folpet dose. This indicated
a possibly greater utilization of, and requirement for, GSH in the
mouse (Chasseaud and Waller, 1990).
Effects on enzymes and other biochemical parameters
Mice (72) and rats (24) were dosed 0, 50, and 5,000 ppm folpet in
their diet for 21 consecutive days. Mean concentration of glutathione
(GSH) in liver was similar to the controls in rats dosed with 50 and
5,000 ppm folpet whereas a small decline was observed in mice dosed
5,000 ppm.
In both rats and mice, GSH concentrations were significantly
increased after 50 ppm and 5,000 ppm in both duodenum and jejunum. A
similar effect observed in ileum was more pronounced in mice. These
results suggest an initial depletion of GSH followed by increasing
de novo synthesis of GSH resulting in a "rebound" elevation in
tissue GSH concentrations in the GI-tract.
Tissue weights of stomach, duodenum and jejunum were increased in
both species. Mean cytosolic protein concentrations (total mg/tissue)
were also increased in duodenum and jejunum in both species with the
increase being greater in rats. Total cytosolic protein in the mouse
liver declined to 86% of control value.
Glutathione-S-transferase levels (with 1-chloro-2,4-
dinitrobenzene used as substrate) increased significantly in tissue
from duodenum, jejunum and ileum in both species, in liver in rats and
in stomach in mice after treatment with 5,000 ppm. This led to a
greater capacity to enzymatically conjugate thiophosgene with GSH.
A marked reduction in the concentrations of lipid peroxides (the
concentration of malondialdehyde was used as indicator of the overall
lipid peroxidation state of the mucosal cells) was noted in the
duodenum of both species receiving 5,000 ppm folpet in their diets.
The reduction in the concentration of lipid peroxides was only
statistically significant in the stomach of mice fed 5,000 ppm folpet.
No alteration was found in the level of intracellular conjugated
dienes in either species when compared to those of the animals in the
control groups.
The non-selenium dependent glutathione peroxidase activity (i.e.
that due to the peroxidase activity of the glutathione S-transferase)
was increased in duodenum, jejunum and ileum in both species after
receiving 5,000 ppm folpet in their diet, and in the stomach of rats
in either dose level (Chasseaud and Waller, 1990).
In mice (240) and rats (48) dosed as in the above study mean
microsomal protein (total mg/tissue) was significantly increased in
rat ileum, jejunum and duodenum. Although increases were observed in
the mouse ileum, duodenum and jejunum, they were not statistically
significant. Hepatic total microsomal protein declined significantly
in rat but remained unchanged in the mouse. Cytochrome P450 was
reduced in the liver in both species but the reduction was
statistically significant only in mice receiving 5,000 ppm folpet in
their diet.
Both aniline hydroxylase and 7-ethyloxycoumarin o-deethylase were
reduced in hepatic microsomes in both species following exposure to
either dose level, the reduction of aniline hydroxylase being
statistically significant in both species dosed at 5,000 ppm
(Chasseaud and Waller, 1990).
In an experiment with mice (90) and rats (54) treated as in the
above study, a statistically significant decrease in pH was observed
in duodenum and jejunum of mice dosed with 5,000 ppm folpet.
Incorporation of 3H-thymidine into the mucosal DNA in mice and
rats was reduced in most tissues of both species. In neither species
was there any evidence of a dose related increase in DNA synthesis
(Chasseaud and Waller, 1990).
Mice (150 CD1 males, 30-35 g) and rats (75 Sprague Dawley males,
230-300 g) received one single dose of 0, 7, 7, 72, and 668 mg
folpet/kg bw by oral gavage. At various times thereafter animals were
killed and GSH concentrations measured in their livers and in
different regions of their gastrointestinal tracts.
Depletion of hepatic and gastrointestinal concentrations of GSH
was observed in both rats and mice, the latter species showing the
most pronounced effect. The depletion was evident after half an hour
in duodenum, jejunum and ileum, with a clear dose-dependent response
in mice. By 2 hours after dosing, the effects on GSH depletion in
duodenum of rat and mouse were similar, and after 6 hours the GSH
levels were elevated compared to the controls. The degree of GSH
rebound in duodenum and jejunum was higher in the mouse than that
observed in the rat (Chasseaud and Waller, 1990).
Studies in both young (33 days old) and adult (82 days old)
Fischer 344 rats with dermal application of [14C] folpet has shown
that folpet penetrates rat skin, and that dose-absorption was similar
(Hall et al., 1988).
Male Sprague-Dawley rats, 200-250 g were dosed with folpet either
by i.p. injection up to 100 mg/kg (suspended in 0.5 ml corn oil) or
orally in doses up to 1,000 mg/kg bw (in corn oil). Fifty mg folpet
dosed i.p. for up to 24 hours caused a significant decrease in
benzphetamine N-demethylase and cytochrome P-450, while SGOT activity
significantly increased. Oral doses of folpet up to ten times that of
the i.p. doses did not cause any similar adverse effect (Ashley,
et al., 1982).
In vitro incubation of folpet with rat liver microsomes with
and without NADPH showed that folpet may not require metabolism to
exert inhibitory effect on the microsomal enzymes. The inhibition of
hepatic microsomal cytochrome P-450 by folpet in vitro could be
prevented by prior addition of reduced glutathione in the incubation
media (Ashley, et al., 1982).
Folpet (5µM) inhibited the activity of the Ca2+-transport-ATP-
ase in human erythrocytes in vitro (Janik, 1986).
Toxicological studies
Acute toxicity studies
The LD50 in Sherman rats dosed with folpet perorally was >
5,000 mg/kg bw for adult males and females and for female weanlings
(Gaines and Linder, 1986).
Short-term studies
Dogs
Groups of five male and five female beagle dogs were dosed with
folpet (technical grade) by oral capsule administration at dosages of
0, (empty capsules), 325, 650, and 1,300 mg/kg bw/day for 52 weeks.
No mortalities occurred during the treatment period. Decreased
body weight was detected in the high and intermediate dose group in
both sexes during the dosing period. A concomitant decrease in food
intake was found in both males and females in the high dose group.
The clinical signs observed in all treated groups were vomiting,
diarrhoea and salivation, the latter effect only observed in the first
8 weeks in the low dose group. The effect was most pronounced in the
high and intermediate groups. Dogs of the high and intermediate dose
groups were poorer in condition as compared to their controls.
Haematological parameters were affected in all treated females
after the first third of the study. These effects included a decrease
in packed cell volume, haemoglobin and erythrocyte counts. Clinical
chemistry changes (e.g. reduced total protein, cholesterol, glucose
and urea) were observed during the treatment period relating to poor
physical condition of the dogs. Chloride level was increased in the
males, mainly in the high dosage group, and calcium levels were
decreased in the high dosage group, the latter effect also observed in
the high and intermediate dosed females but only up to 25 weeks of
treatment.
Urine volume was lowered in the high dosed females after 13 weeks
of treatment. Urine acidity of the male and female groups was
increased, this effect persisted in males in high and intermediate
groups throughout the study. Tubular testicular degeneration
associated with no spermatozoa in the epididymides was found in two
male dog in the high dosage group, one of these having moderate
atrophy of the prostatic glands. Absolute testes weights were
decreased in the high dosage male group. Changes in relative organ
weights were recorded in adrenals (all males and intermediate dosed
females), brain and kidney (high dosed males and intermediate dosed
females), liver (high dosed females and intermediate dosed males) and
thyroid (high dose female and intermediate male group). The no
observed adverse effect level is 325 mg folpet/kg bw per day (Waner
and Nyska, 1988).
Long-term/carcinogenicity studies
Groups of 20 male and 20 female rats (F344) were fed a diet
containing folpet (Technical grade, purity, 91.1%) at concentrations
of 0, 250, 1,500, and 5,000 ppm for two years. Average concentrations
measured on a weekly basis were calculated at 0, 190.4, 1287.7, and
4532.3 ppm, respectively.
No effect of the test compound on longevity was found. In the
high dosage group, mean body weight and food intake were depressed in
both males and females (up to 10% in males and 6% in females). Water
consumption was depressed for high dose animals with females effected
to a greater extent than males.
Reductions in alkaline phosphatase and alanine aminotransferase
were apparent in treated groups throughout the study. Sporadic
decreases in aspartate aminotransferase, creatinine phosphokinase and
gamma-glutamyl transferase were found. Blood cholesterol was
significantly lowered in highest dosed animals, in both sexes,
throughout the dosing period. Total plasma protein was reduced in the
male high dosage group during the first year of treatment. Phosphate
was elevated in the male high dosage group at most examinations. Urea
was raised in the highest dosage female group up to 18 months. Most
male treated groups excreted a more concentrated urine of smaller
volume at the 3 and 6 month assay.
Treatment was associated only with non-neoplastic lesions
consisting of diffuse hyperkeratosis of the oesophageal and gastric
squamous epithelium. The no-observed-effect level was 190 ppm
(equivalent to 9.5 mg/kg bw/day (Crown, et al., 1989).
Reproduction studies
Four groups of CD rats, 25 males and 25 females (F0) were fed a
diet containing folpet (technical grade, purity 91%) in concentrations
0, 250, 1,500, and 5,000 ppm, respectively.
Body weight gain and food consumption for the top dosage group
was reduced in parental animals in F0 and F1 -generations. A minor
decrease in body weight was observed in F1 and F2 offspring.
The principal histopathological effect was hyperkeratosis of the
nonglandular gastric mucosa in high and intermediate dosage group in
both F0 and F1 -generations, oesophageal hyperkeratosis in the high
and intermediate dosage groups of the F1 generation and increased
incidence of basophilic renal tubules in the high dosage males of the
F0 generation. Folpet is concluded not to be a specific reproductive
toxin in the current test system (Rubin and Nyska, 1986).
Special studies on embryo/fetotoxicity
In an in vitro test, the inhibition of attachment by tumour
cells to polyethylene disks coated with concanavalin was found with
folpet. The dose of 4.8 mg/l inhibited attachment by 50% (Braun and
Howicz, 1983).
Special studies on genotoxicity
The effects on chromosomal structure of exposure to folpet
(technical grade, purity 90.1%) were studied in cultured human
lymphocytes. The concentrations of folpet tested in the cytogenetic
assay was 1, 2 and 3 µg/ml for 24 hours with and without S-9 mix and
solvent (DMSO) and cyclophosphamide and chlorambucil were used as
negative and positive controls, respectively. In addition, a second
cytogenetic test using 3 and 5 µg/ml folpet for 2 hours with and
without S-9 mix was performed.
There was some evidence of weak clastogenic potential of folpet
following 24 hours of exposure at 3 µg/ml. However, in view of the
questionable biological significance of gap-type aberrations, the lack
of response in activated cultures following 24 hours of exposure, and
lack of response in all cultures following 2 hours of exposure, no
clear, biologically significant evidence of clastogenic potential was
found (Bootman, et al., 1987).
Folpet (technical grade, purity, 90.1%) was examined for
mutagenic potential by measuring its ability to induce mutation in
Chinese hamster cells (V 79) at the hypoxanthine-guanine-
phosphoribosyl transferase (HGPRT) locus. The concentrations of
folpet were 0.125, 0.25, 0.5, 1, and 2 µg/ml in presence of S-9 mix
for three hours. DMSO served as negative control, and ethylmethane-
sulfonate and 7,12 dimethylbenzanthracene as positive controls.
Folpet induced no significant increases in mutation frequency at the
HGPRT gene locus with or without S-9 mix (Bootman, et al., 1986).
The genetic activity profile of 65 pesticides was tested and
folpet was active in inducing point/gene mutations and primary DNA
damage in both pro- and eukaryotes. In addition folpet was shown to
cause chromosomal effects in Chinese hamster ovary cells and mouse
bone marrow and cardiac blood. Folpet was not found to induce
unscheduled DNA synthesis in human lung fibroblasts W1-38 or dominant
lethality in mouse (Garrett, et al., 1986).
In a computer-assisted analysis of structure-genotoxicity
relationship, a correlation was found between some structural
fragments, one of these being C1-C-S-N-C=0 present in folpet and
Salmonella typhimurium histidine reversion assay, the thymidine kinase
gene mutation assay using mouse lymphoma L51784 cells, prokaryotic DNA
repair assays using respectively, pairs of Escherichia coli polA+ and
polA+ and Bacillus subtilis rec+ and rec+ strains and mitotic
recombination in strain D3 of the yeast Saccharomyces cerevisiae
(Klopman, et al., 1985).
In a review of the sex-linked recessive lethal test for
mutagenesis in Drosophila melanogaster, folpet is listed as mutagenic
(Lee, et al., 1983).
COMMENTS
The results of a recently-submitted short-term study in beagle
dogs confirmed earlier findings in this species. However, a higher
NOAEL was observed in the present study. The present study involved
52 weeks of dosing, while in the earlier study the dogs were
administered folpet for only 13 weeks. Tolerance to the effects of
folpet could partly explain the differences in toxicity observed.
The results of a recently submitted long-term study in rats
support earlier findings observed in previous long-term studies in
this species. The NOAEL in the present study was 190 ppm folpet,
equivalent to 10 mg/kg bw/day. Effects were observed at the next
highest dose of 1290 ppm.
Folpet did not cause reproductive effects in a two-generation 2-
litter/generation rat study. The NOAEL in this study was 250 ppm
equal to 13.7 mg/kg bw/day.
A range of genotoxicity studies suggest that folpet is
potentially genotoxic. The presence of the S-9 fraction from liver
reduced the in vitro effects of folpet. Folpet was negative in the
dominant lethal test in mice.
The importance of glutathione (GSH) in the metabolic degradation
of folpet was demonstrated. It is apparently a more important pathway
in the mouse than the rat. This difference in metabolism coupled with
the relatively higher exposure in the mouse than in the rat may
explain the different susceptibility to tumour induction by folpet in
the two species. Data suggest that the upper small intestine is the
site of tumour formation provided that sufficient levels of the active
metabolite(s) of folpet are available to exceed the biological defence
provided by GSH and its associated GSH-S-transferase.
In 1986 the JMPR allocated a temporary ADI of 0-0.01 mg/kg bw
based upon a NOAEL in a short-term toxicity study in dogs of 10 mg/kg
bw/day and using a safety factor of 1000.
The recently-submitted data does not influence the present
estimate of the acceptable daily intake for humans. Since a NOAEL in
the mouse has not been established and since the mechanism of tumour
induction in this species has not been defined, the ADI remains
temporary. However, the sponsors are to be commended for their
efforts to elucidate the toxicological mechanisms associated with
folpet.
TOXICOLOGICAL EVALUATION
Level causing no toxicological effect
Rat: 800 ppm equivalent to 40 mg/kg bw/day
Dog: 10 mg/kg bw/day
Estimate of temporary acceptable daily intake for humans
0-0.01 mg/kg bw
Studies without which the determination of a full ADI is impracticable
To be submitted to WHO by 1992:
- Results of further investigation of the relevance of the
metabolic data in animals for humans.
- Further studies to elucidate the mechanism for the induction
of gastrointestinal tract tumours in mice.
- Studies designed to establish a NOAEL in mice.
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assessment of the clastogenic activity of Folpan tech in cultured
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Makhteshim Chemical Works Ltd., Beer-Sheva, Israel.
See Also:
Toxicological Abbreviations
Folpet (HSG 72, 1992)
Folpet (ICSC)
Folpet (FAO/PL:1969/M/17/1)
Folpet (WHO Pesticide Residues Series 3)
Folpet (WHO Pesticide Residues Series 4)
Folpet (Pesticide residues in food: 1984 evaluations)
Folpet (Pesticide residues in food: 1986 evaluations Part II Toxicology)
Folpet (Pesticide residues in food: 1995 evaluations Part II Toxicological & Environmental)