THIOPHANATE-METHYL (addendum) JMPR 1998
First draft prepared by
C. Bowes and C.E Moase
Health Evaluation Division, Pest Management Regulatory Agency
Ottawa, Ontario, Canada
Explanation
Evaluation for acceptable daily intake
Biochemical aspects
Absorption, distribution, and excretion
Biotransformation
Toxicological studies
Developmental toxicity
Comments
Toxicological evaluation
References
Explanation
Thiophanate-methyl was evaluated toxicologically by the Joint
Meeting in 1973, 1975, 1977, and 1995 (Annex 1, references 20, 24, 28,
and 74). An ADI of 0-0.08 mg/kg bw was allocated in 1973, on the basis
of a NOAEL of 8 mg/kg bw per day in a three-generation study of
reproductive toxicity in rats and a safety factor of 100. This ADI was
confirmed in 1975 and 1977. Additional data that became available were
reviewed at the 1995 Joint Meeting within the CCPR periodic review
programme, at which time an ADI of 0-0.02 mg/kg bw was established on
the basis of a NOAEL of 2 mg/kg bw per day in a study of developmental
toxicity in rabbits. New information on the metabolism of
thiophanate-methyl and the results of a second study of developmental
toxicity in rabbits have become available and were reviewed at the
present Meeting.
Evaluation for Acceptable Daily Intake
1. Biochemical aspects
(a) Absorption, distribution, and excretion
The absorption, tissue distribution, and excretion in urine and
faeces of [phenyl-U-14C]thiophanate-methyl (purity, 96%) was
investigated in groups of five Fischer 344 rats of each sex after
administration by gavage of a single dose of 14 or 170 mg/kg bw or
doses of 14 mg/kg bw per day for 15 days. Thiophanate-methyl was
rapidly absorbed from the gastrointestinal tract, reaching a maximum
serum concentration by 4 h after administration. The extent of
absorption may be dose-dependent, decreasing with increasing dose. The
highest residual tissue levels were found in the liver, thyroid, and
kidneys 96 h after dosing. Thiophanate-methyl was excreted rapidly,
with > 90% elimination via the urine and faeces within 24 h of
administration. At the low dose, the principal route of elimination
was urinary, whereas at the high dose excretion was predominantly
faecal. Repeated exposure to thiophanate-methyl also tended to shift
excretion from the urinary to the faecal route (Tanoue, 1992 a,b).
(b) Biotransformation
There was no indication of potential bioaccumulation. The dose or
duration of administration did not affect the metabolites identified
in the urine. The major urinary metabolite was
5-(2-methoxycarbonylamino) benzimidazolyl sulfate (21-42%);
5-hydroxy-1 H-benzimidazol-2-yl carbamate and
4-hydroxythiophanate-methyl each represented approximately 2% of the
radiolabel. The major faecal metabolites were
4-hydroxythiophanate-methyl (6-10%) and
5-hydroxy-1 H-benzimidazol-2-yl carbamate (~2-5%). Unchanged
thiophanate-methyl accounted for approximately 20-24% of the
administered radiolabel after repeated low doses and and 50% after the
high dose. Carbendazim was a minor metabolite (2-3%) in rats. In
plants, unchanged thiophanate-methyl and carbendazim accounted for
approximately 65 and 30% of the residue, respectively, 7-14 days after
application. A new metabolic pathway for thiophanate-methyl in rats
has been proposed and is presented in Figure 1 (Tanoue, 1992a,b).
2. Toxicological studies
(a) Developmental toxicity
Rabbits
Groups of 15 female New Zealand white rabbits received
thiophanate-methyl (purity, 96.2%) at a dose of 0, 2, 6, or 20 mg/kg
bw per day by gavage on days 6-19 of gestation. The concentrations of
thiophanate-methyl were found to be acceptable in samples of test
solutions taken during the first and last weeks of treatment. The
animals were killed on day 29 of gestation. No treatment-related
maternal deaths or clinical effects and no subsequent effects were
seen. Dose-related losses in maternal body weight were observed mainly
at the beginning of treatment (days 6-8 of gestation) with 6 mg/kg bw
per day and during days 6-14 of gestation at 20 mg/kg bw per day. The
food consumption of animals treated with 20 mg/kg bw per day was less
than that of the control group from the start of treatment, and large
reductions seen during days 6-12 of gestation were generally
associated with subsequent abortion or sacrifice in extremis. Water
consumption was unaffected. One of 12 animals treatedwith 2 mg/kg bw
per day, one of 14 animals treated with 6 mg/kg bw per day, and two of
13 animals in the control group aborted. One control animal and one
animal treated with 20 mg/kg bw per day had only one implantation,
which was resorbed early. No treatment-related trends were observed in
resorptions, preimplantation losses, or fetal or placental weights.
The numbers of viable young were slightly decreased in animals treated
with 20 mg/kg bw per day, due to abortions and total litter losses.
Gross examination of the fetuses showed an apparent
treatment-related trend in skeletal abnormalities of the ribs,
vertebrae, and pelvis in animals treated with 6 or 20 mg/kg bw per
day, that was generally close to, or slightly greater than, the upper
limit of values for historical controls. The treatment-related effects
included increased incidences of 13 pairs of ribs, incomplete or
asymmetric ossification of costal elements of the sacral vertebrae, 27
presacral vertebrae, and asymmetric pelvises associated with various
sacral vertebrae. At 20 mg/kg bw per day, the incidence of one or more
ribs thickened at the costal cartilage was significantly increased.
The NOAEL for both maternal and developmental toxicity was 2 mg/kg bw
per day on the basis of a decrease in maternal growth rate and
increased incidences of skeletal abnormalities at higher doses (Tesh
et al., 1986, Annex 1, reference 76).
The data on fetuses from the above study were reanalysed by
Christian (1997), who noted that the slight decrease in viable young
and the skeletal abnormalities were probably associated with poor
maternal health, technical problems, and the relatively few animals
available for evaluation. Although the increased incidences of
supernumerary ribs were identified by the study author as an effect at
doses of 6 and 20 mg/bw per day, reanalysis of the data showed that
these incidences were within the expected normal range and were not
statistically significant. All other variations in skeletal
ossification identified in the study also occurred at incidences that
were not statistically significant, indicating that they were
unrelated to treatment. The NOAEL for maternal toxicity was 6 mg/kg bw
per day, and the NOAEL for developmental toxicity was 20 mg/kg bw per
day. Owing to the deficiencies in the study of Tesh et al. (1986), the
Committee considered that it should be reclassified as a range-finding
study.
Groups of 20 naturally bred New Zealand white rabbits
[Hra:(NZW)SPF] were given technical-grade thiophanate-methyl (purity,
97.28%) in 1% methylcellulose by gavage at doses of 0, 5, 10, 20, or
40 mg/kg bw per day on days 6-28 of gestation, the day of mating being
considered day 0. The rabbits were sacrificed on day 29 of gestation.
The does were observed for viability, clinical signs, body weight,
food consumption, and the number of corpora lutea, and the thoracic,
abdominal, and pelvic viscera were examined. Their uteri were excised
and examined for the number and distribution of implantation sites,
live and dead fetuses, and early and late resorptions. The fetuses
were observed for sex, body weight, and gross external, visceral,
brain, and skeletal alterations. At a dose of 20 mg/kg bw per day,
there was a transient but significant reduction in maternal
body-weight gain and statistically significant reduced absolute and
relative feed consumption. At a dose of 40 mg/kg bw per day, faecal
output was reduced, in conjunction with significantly reduced maternal
body-weight gain and absolute and relative feed consumption; however,
body-weight gain and food consumption recovered after the initial week
of dosing. It is likely that the observed reduction in feed
consumption during this period contributed to the observed decreases
in body weight, body-weight gain, and faecal output. In the fetuses,
the dose of 40 mg/kg bw per day was associated with supernumerary
thoracic ribs only at the two higher of maternally toxic doses. In
addition, there appeared to be an increased incidence of resorptions
in rabbits treated with 40 mg/kg bw per day, the numbers of litters
with > 20% resorptions per litter being 2/20 in controls, 2/17 at 5
mg/kg bw per day, 2/18 at 10 mg/kg bw per day, 0/17 at 20 mg/kg bw per
day, and 5/20 at 40 mg/kg bw per day. The historical control range for
dead or resorbed conceptuses per litter was 0-18.3%.
Thiophanate-methyl was not selectively toxic to embryo or fetal
viability, growth, or morphology and was not teratogenic. The NOAEL
for maternal toxicity was 10 mg/kg bw per day and that for
developmental effects was 20 mg/kg bw per day (York, 1997a,b).
Comparison of the studies of Tesh et al. (1986) and York (1997b)
revealed a consistent profile of maternal toxicity, comprising a
dose-related, statistically significant, transient reduction in food
consumption and body weight and/or body-weight gain, accompanied by
reduced faecal output during the two-week period after the beginning
of treatment. The transient, significant loss in maternal body-weight
at 20 mg/kg bw per day in the first study was not seen at the same
dose in the second study, although a transient reduction in
body-weight gain in conjunction with reduced food consumption was
noted. In the second study, a dose of 40 mg/kg bw per day was required
to produce a significant loss in maternal body weight, which was
transient, and subsequently a significant reduction in maternal
body-weight gain. In both studies, food consumption, maternal
body-weight gain, and faecal output steadily recovered towards the end
of the study, even though the dosing period was longer in the second
study. Neither study provided maternal body weights corrected for
gravid uterine weight. The maternal effects described above occurred
at lower doses in the first study (Tesh et al., 1986), probably
because of compromised maternal health. Treatment with doses up to 20
mg/kg bw per day (Tesh et al., 1986) or 40 mg/kg bw per day (York,
1997b) did not significantly affect fetal body weights or litter size.
Neither study showed treatment-related malformations.
The study of Tesh et al. (1986) showed increased incidences of
resorptions or abortions at 20 mg/kg bw per day that were associated
with poor maternal health, which was not treatment-related, or were
within the historical control ranges. All does killed in extremis
(two, one, one, and one at 0, 2, 6, and 20 mg/kg bw per day,
respectively) had severe infections in their lungs and/or abdominal
cavities. Of the six abortions (0/1, 1/1, 1/1, and 2/3 at 0, 2, 6, and
20 mg/kg bw per day, respectively) that occurred in surviving does,
four were in does that had clear signs of infection. Nine out of 60
does either died or aborted due to severe infections in this study. In
addition, a common finding at necropsy was the presence of
precipitates or milkiness in the amniotic fluid of does that
delivered, an indication of uterine infection. Owing to the high
incidence of maternal disease in this study, the health status of the
does that delivered litters was questionable, and the increased
incidence of resorptions or abortions is unlikely to be
treatment-related. As a result of the high incidence of deaths and
abortions in the first study, only 12, 10, 12, and 9 litters were
available for analysis at doses of 0, 2, 6, and 20 mg/kg bw per day,
respectively. Removal of does with litters containing only 1 or 2
implants to reduce bias in any statistical analysis resulted in only
11, 9, 10, and 7 litters available for analysis at doses of 0, 2, 6,
and 20 mg/kg bw per day, respectively. Even without the exclusion of
does with small litters, the number of litters available for analysis
was not sufficient for evaluation under current standards.
The only treatment-related fetal effect identified by York
(1997b) was an increase in the incidence of supernumerary ribs, only
at maternally toxic doses. This variation was also noted at 80 mg/kg
bw per day, but not at 40 mg/kg bw per day, in the dose range-finding
study of York (1997a); both doses were maternally toxic. In the study
of Tesh et al. (1986), extra ribs occurred in 44/88, 48/78, 68/95, and
36/51 fetuses from 10/12, 10/10, 11/12, and 8/9 litters at 0, 2, 6,
and 20 mg/kg bw per day, respectively, with incidences of 12/13 and
13/13 rib pairs combined. Although the incidence was significantly
increased at 6 and 20 mg/kg bw per day, it was not significant on a
litter basis and was within the historical control range. When
calculated as the mean number of rib pairs per fetus per litter, as
was done in the study of York (1997b), the means were 12.58, 12.58,
12.64, and 12.63 at 0, 2, 6, and 20 mg/kg bw per day, respectively. In
the study of York (1997b), extra ribs occurred in 85/168, 76/141,
87/164, 78/115, and 142/160 fetuses from 17/19, 16/17, 18/18, 16/16,
and 19/19 litters at 0, 5, 10, 20, and 40 mg/kg bw per day,
respectively, with incidences of 12/13 and 13/13 rib pairs combined.
Expressed as the mean number of rib pair per fetus per litter, 12.45,
12.44, 12.45, 12.58, and 12.85 mean rib pairs occurred at doses of 0,
5, 10, 20, and 40 mg/kg bw per day, respectively, which was
significant at 40 mg/kg bw per day. The historical control values for
rib pairs were: litter range, 12.34-12.67; fetal mean, 12.47. The
incidence of extra ribs was considered to be treatment-related at 40
mg/kg bw per day. The two studies are consistent for this effect.
The two studies could not be compared directly with respect to
the increased numbers of presacral vertebrae, asymmetrical pelvises,
and incomplete or asymmetric ossification of the costal elements of
the sacral vertebrae, because of differences in the methods of
examination and tabulation and in terminology. However, an increased
incidence of 27 presacral vertebrae, asymmetrical pelvises, and
incomplete or asymmetric ossification of the costal elements of the
sacral vertebrae, which were believed to be interrelated findings, was
noted in the study of Tesh et al., (1986) but not in the study of York
(1997b). These findings probably represent either delays or
variablility in the ossification of the ilia, the relative position of
the iliac crest, and the degree of ossification of the sacral
vertebrae or processing artefacts such as the degree of staining
and/or realignment of the pelvis. The degree of ossification may have
been reduced by poor maternal health in the first study. No difference
in the degree of ossification at the sites evaluated was noted by
York.
Comments
Thiophanate-methyl was rapidly absorbed in rats after oral
administration. The extent of absorption may be dose-dependent,
decreasing with increasing dose; a study of biliary excretion would be
useful to confirm this hypothesis. The highest residual levels
occurred in the liver, thyroid, and kidneys. The elimination of
thiophanate-methyl was rapid, with more than 90% in the urine and
faeces within 24 h of administration. There was a shift towards faecal
elimination between the low and high doses and after repeated doses.
There was no indication of potential bioaccumulation. The major
urinary metabolite was 5-hydroxycarbendazim sulfate (21-42%); 5- and
4-hydroxythiophanate-methyl each represented approximately 2% of the
radiolabel. The major faecal metabolites were
4-hydroxythiophanate-methyl (6-10%) and 5-hydroxycarbendazim (2-5%);
carbendazim (2-3%) was also found. Unchanged thiophanate-methyl
accounted for approximately 20-24% and 50% of the administered
radiolabel after repeated low and high doses, respectively. In plants,
unchanged thiophanate-methyl and carbendazim accounted for
approximately 60 and 30% of the residue respectively, 7-14 days after
treatment.
WHO has classified thiophanate-methyl as unlikely to present an
acute hazard in normal use (WHO, 1996).
In a study of developmental toxicity in rabbits,
thiophanate-methyl was administered by gavage at 0, 5, 10, 20, or 40
mg/kg bw per day. The NOAEL for maternal toxicity was 10 mg/kg bw per
day, as doses at and above 20 mg/kg bw per day caused transient but
significant reductions in maternal body-weight gain and feed
consumption. Additionally, faecal output was reduced at 40 mg/kg bw
per day. The NOAEL for developmental toxicity was 20 mg/kg bw per day,
as the incidence of supernumerary thoracic ribs was increased at 40
mg/kg bw per day, a variation that occurred only at the higher of two
maternally toxic doses. Thiophanate-methyl did not affect embryonic or
fetal viability, growth, or morphology and was not teratogenic.
In the study of developmental toxicity in rabbits reviewed by the
1995 Joint Meeting, the NOAEL for maternal and developmental toxicity
was 2 mg/kg bw per day, on the basis of reduced maternal body-weight
gain and treatment-related increased incidences of supernumerary ribs,
ribs thickened at the costal cartilage, incomplete or asymmetric
ossification of costal elements of sacral vertebrae, 27 presacral
vertebrae, and asymmetric pelvises at 6 and 20 mg/kg bw per day.
Because of reporting deficiencies, compromised maternal health, and an
unacceptably small number of litters available for analysis, the
present Meeting concluded that the status of this study should be
reduced to that of a range-finding study. The new study submitted to
the present Meeting was considered to be more appropriate for the
identification of the NOAEL for developmental toxicity in rabbits, as
modern methods were used, the reporting was adequate, and the
population of rabbits was larger and healthier.
A two-generation study of reproductive toxicity in rats was
evaluated by the 1995 Joint Meeting, which considered 10 mg/kg bw per
day to be the LOAEL for parental toxicity; however, after re-examing
this study, the present Meeting concluded that 10 mg/kg bw per day
should be considered the NOAEL. The effects in the liver observed in
all treated groups were considered to be non-adverse at this dose, and
the slight changes in the thyroid occurred in the absence of a
measurable effect on thyroid hormones.
An ADI of 0-0.08 mg/kg bw per day was established on the basis of
the NOAEL of 8 mg/kg bw per day in a three-generation study of
reproductive toxicity in rats and in a one-year study in dogs, both of
which were evaluated at earlier meetings, and a safety factor of 100.
The Meeting concluded that an acute RfD was not required because
thiophanate-methyl is of low acute toxicity when administered orally
or dermally and is only slightly toxic when administered by
inhalation. The Meeting concluded that the acute intake of residues is
unlikely to present a risk to consumers.
Although the toxicities of thiophanate-methyl and carbendazim are
qualitatively different, carbendazim is an important metabolite in
plants but a minor metabolite in animals. The risk assessment for
residues in plants and plant products should therefore include
consideration of both thiophanate-methyl and carbendazim. The ADI for
the latter, established by the 1995 JMPR, is 0-0.03 mg/kg bw.
Toxicological evaluation
Levels that cause no toxic effect
Mouse: 150 ppm, equal to 29 mg/kg bw per day (18-month
study of toxicity and carcinogenicity)
1000 mg/kg bw per day (maternal toxicity and
teratogenicity in study of reproductive toxicity)
500 mg/kg bw per day (study of developmental
toxicity)
Rat: 200 ppm, equal to 9 mg/kg bw per day (two-year
study of toxicity and carcinogenicity)
160 ppm, equivalent to 8 mg/kg bw per day (study
of reproductive toxicity)
1000 mg/kg bw per day (study of developmental
toxicity)
300 mg/kg bw per day (maternal toxicity in a study
of developmental toxicity)
Rabbit: 10 mg/kg bw per day (maternal toxicity in a study
of developmental toxicity)
20 mg/kg bw per day (teratogenicity and
fetotoxicity in a study of developmental toxicity)
Dog: 10 mg/kg bw per day (studies of toxicity of up to
two years)
Estimate of acceptable daily intake for humans
0-0.08 mg/kg bw
Estimate of acute reference dose
Not allocated (unnecessary)
Studies that would provide information useful for continued
evaluation of the compound
1. Study of excretion in the bile
2. Further observations on humans
List of relevant end-points for setting guidance values for dietary and non-dietary exposure
Absorption, distribution, excretion and metabolism in mammals
Rate and extent of absorption Rapid (~70% by urinary excretion, 96 h)
Dermal absorption No data
Distribution Thyroid, liver, kidney
Potential for accumulation No indication of bioaccumulation
Rate and extent of excretion Rapid/complete, > 90% within 24 h
Metabolism in animals Predominantly metabolized (71-88%)
Toxicologically significant compounds Urine (rats): 5-hydroxy-carbendazim sulfate,
(animals, plants and environment) 5-hydroxy-carbendazim and
4-hydroxy-thiophanate-methyl
Faeces (rats): 4-hydroxy-thiophanate-methyl
and 5-hydroxy-carbendazim
Plants: unchanged thiophanate-methyl and carbendazim
Acute toxicity
Rat: LD50 oral 7000 mg/kg bw
Rabbit: LD50 dermal > 10 000 mg/kg bw
Rat: LC50 inhalation 1.8 mg/L air
Skin irritation Not irritating
Eye irritation Mildly irritating
Skin sensitization Sensitizing (maximization test)
Not sensitizing (Buehler test)
Short-term toxicity
Target/critical effect Thyroid, liver: hypertrophy of thyroid epithelium,
increased thyroid and liver weights (rats, dogs);
increased liver weight (mice)
Lowest relevant oral NOAEL/NOEL Dog: 10 mg/kg bw per day (capsule, overall NOAEL)
Lowest relevant dermal NOAEL/NOEL No data
Lowest relevant inhalation NOAEL/NOEL No data
Genotoxicity Not mutagenic, weak aneugenic potential
Long-term toxicity and carcinogenicity
Target/critical effect Thyroid hyperplasia, increased thyroid-stimulating
hormone, decreased thyroxine (rats, dogs); thyroid
adenoma (rats);
hepatocellular adenoma (mice)
Lowest relevant NOAEL/NOEL Dog: 8 mg/kg bw per day (1 year)
Carcinogenicity Hepatocellular adenoma (mice); thyroid adenoma (rats)
at high doses
Reproductive toxicity
Reproduction target/critical effect Increased ovary/testis weights (no histological
findings)
Lowest relevant reproductive NOAEL/NOEL Rat: 8 mg/kg bw per day (decreased litter size,
reduced pup body-weight gain in F1, F2, F3)
Developmental target/critical effect Supernumerary ribs (rabbit); not teratogenic in rat
or rabbit
Lowest relevant developmental NOAEL/NOEL Rabbit: 20 mg/kg bw per day
Neurotoxicity/Delayed neurotoxicity No data. No indication of neurotoxic potential in
other studies
Other toxicological studies
Mechanstic studies Inhibition of thyroid microsomal peroxidase involved
in thyroid hormone synthesis
Medical data No adverse effects on health of personnel involved
in manufacturing process
Summary Value Study Safety factor
ADI 0-0.08 mg/kg bw 3-generation, rat; 100
1 year, dog
Acute reference dose Not allocated
(unnecessary)
References
Christian, M.S. (1997) Critical review of two developmental
(embryo-fetal toxicity/teratogenicity) toxicity studies of thiophanate
methyl in rabbits. Unpublished report from Argus International, Inc.,
Horsham, Pennsylvania, USA. Submitted to WHO by Nippon Soda Co., Ltd.,
Tokyo, Japan.
Tanoue, T. (1992a) Thiophanate-methyl: Metabolism in rats. Unpublished
report Nisso EC-338 from Environmental Toxicology Laboratory, Nippon
Soda. Submitted to WHO by Nippon Soda, Tokyo, Japan.
Tanoue, T. (1992b) Thiophanate-methyl: Metabolism in rats.
Supplemental report to Nisso EC-338. Unpublished report Nisso EC-338
from Environmental Toxicology Laboratory, Nippon Soda. Submitted to
WHO by Nippon Soda, Tokyo, Japan.
Tesh, J.M., Ross, F.W., Wightman, T.J., Wilby, O.K. & Tesh, S.A.
(1986) Thiophanate methyl: Teratology study in the rabbit. Unpublished
report No. 86/NISO10/111 from Life Science Research, Eye, Suffolk,
United Kingdom. Submitted to WHO by Nippon Soda, Tokyo, Japan.
WHO (1996) The WHO Recommended Classification of Pesticides by
Hazard and Guidelines to Classification 1996-1997 (WHO/PCS/96.3),
International Programme on Chemical Safety, Geneva.
York, R.G. (1997a) Oral (stomach tube and dietary) dosage-range
developmental toxicity study of thiophanate-methyl in rabbits.
Unpublished report No. 914-002P from Argus Research Laboratories,
Inc., Horsham, Pennsylvania, USA. Submitted to WHO by Nippon Soda Co.,
Ltd. Tokyo, Japan.
York, R.G. (1997b) Oral (stomach tube) developmental toxicity study of
thiophanate-methyl in rabbits. Unpublished report No. 914-002 from
Argus Research Laboratories, Inc., Horsham, Pennsylvania, USA.
Submitted to WHO by Nippon Soda Co., Ltd., Tokyo, Japan.