PROPYLENETHIOUREA (PTU)
First draft prepared by
M. Watson
Pesticide Safety Directorate, Ministry of Agriculture, Fisheries and
Food
Harpenden, Hertfordshire, United Kingdom
EXPLANATION
Propylenethiourea (PTU) is a plant and animal metabolite and a
degradation product of propineb. Results of previous toxicological
evaluation of propineb (Annex I, references 28, 34, 40, and 44)
indicate that the effects of propineb on the rat thyroid may be
caused primarily by PTU. PTU is also of interest because it forms
part of the terminal residue to which consumers of produce treated
with propineb are exposed and because the levels of PTU in treated
produce generally increase during food processing as the level of
propineb decreases. A toxicological monograph has not been prepared
previously on PTU. This monograph contains summaries of relevant
studies contained in previous toxicological monographs on propineb
(Annex I, references 29, 35, and 46), as well as studies on PTU that
have become available since the last previous review of propineb.
EVALUATION FOR ACCEPTABLE DAILY INTAKE
BIOLOGICAL DATA
Biochemical aspects
Absorption, distribution and excretion
Radiolabelled 14C-PTU was orally administered to groups of 5
male Sprague-Dawley rats at dosage levels of 0.5 to 50 mg/kg bw.
Additional groups of rats were given intravenous doses of 5 or 10
mg/kg bw. Intraduodenal doses of 5 mg/kg bw were also administered
to rats with bile duct fistulas. Radioactivity in urine, faeces,
exhaled air, bile and body tissues/organs was measured for up to 10
days. Whole body autoradiograms were prepared. Total recoveries of
radioactivity in experiments ranged from 86% to 98%. More than 95%
of orally administered doses was absorbed. Absorption and excretion
of radioactivity were rapid. At 48 hours, 85-92% of orally or
intravenously administered doses was recovered in the urine, 7-14%
in the faeces, < 0.2% in exhaled air, and only 1-2% remained in the
body. The elimination half-life for the first 48 hours was about 6
hours. After 5 days, the elimination of half-life was 2-2.5 days,
which indicated a biphasic elimination pattern. Biliary excretion
was also observed, as was evidence for enterohepatic circulation.
Two hours after oral doses of 5 mg/kg bw, radioactivity was
essentially uniformly distributed in body tissues/organs, with the
exception of the thyroid, which had a 12-fold higher level of
radioactivity. Thyroid levels of radioactivity reached a peak at 8
hours, which was about double that observed at 2 hours, and
radioactivity remained considerably higher than levels in other
tissues/organs for the remainder of the 10-day study. By 48 hours,
radioactivity levels in body tissues/organs, with the exception of
the thyroid, declined about 50-fold. Somewhat higher levels of
radioactivity were observed in the kidney and liver. Selective
uptake and accumulation of radioactivity in the thyroid was apparent
throughout the 10-day study (Weber et al., 1978).
Biotransformation
No data were available to the Meeting on the metabolic fate of
PTU.
Toxicological studies
Acute toxicity
No data were available to the Meeting on the acute toxicity of
PTU.
Short-term toxicity studies (including special studies on thyroid
function)
Rats
In a 21-day comparative study of the effects of PTU and various
other thyro-suppressive agents, Wistar rats (50/sex/group) were
treated orally with 0 or 50 mg/kg bw/day of the following compounds:
pure propineb, two technical samples of propineb, PTU, ETU,
technical zineb and methyl thiouracil as a positive control. Rats
(10/sex/group) were killed after 7, 14 or 21 days treatment and
after 14 or 28 days recovery. Investigation of thyroid activity was
limited to recording of thyroid weight, no biochemical analyses or
histopathology were included in this study. PTU, ETU and methyl
thiouracil caused significant increases in absolute and relative
thyroid weight in rats of both sexes, while the three propineb
samples caused an increase in relative weight in females only.
Propineb had only a moderate effect compared to methyl thiouracil,
whereas PTU had an equivalent effect to that of methyl thiouracil in
its goitrogenic activity and was somewhat more potent than ETU. The
thyroid enlargement induced by propineb was partially reversible
during the 28-day withdrawal period (Kimmerle, 1972).
PTU of about 99% purity was administered in the diet of rats
(Wistar: 40/sex/dose) for 24 months at concentrations of 0, 1, 10,
100, or 1000 ppm. The homogeneity, stability and accuracy of
admixture of the test material into the test diet was not checked
during the study. At 3, 7, and 14 days, and at 1, 3, 6, 12, and 24
months, 5 rats/sex/group were subjected to thyroid function tests.
Twenty-four hours following oral administration of 2 µCi of 125I-NaI
tracer, the rats were sacrificed. Accumulation of iodine by the
thyroid was determined by measuring radioactivity in excised and
weighed thyroid tissue. PBI was also assayed by measuring
radioactivity in plasma protein precipitated by trichloroacetic
acid.
PTU inhibited thyroid function in a dose-related manner. Both
sexes were affected to about the same degree. Initial effects were
observed at all dose levels at 3 days, as evidenced by dose-related
decreases in iodine uptake by the thyroid and by decreased PBI
levels. By 7 days, compensatory reactions were observed in the 1000
ppm and 100 ppm groups. In the 1000 ppm group, thyroid weights
increased approximately 6-fold over control weights, reaching peak
levels at 6 months in females and at 12 months in males. Iodine
uptake by the thyroid, expressed as the amount of radioactive iodine
per mg of tissue, decreased to about 10% of the control levels at
these times. PBI, after an initial decrease to approximately 20% of
the control levels, later rose to control levels by 1-3 months. Mean
body weights in this group were significantly decreased from 7 days
to the end of the study. In the 100 ppm group, thyroid weights
increased 2-3-fold over control weights, reaching peak levels at 6
months. Iodine concentration in the thyroid, however, remained
similar to control levels throughout the study. PBI levels that were
initially decreased, later increased to 2-3 times control levels at
1 month. At 10 ppm, initially reduced iodine uptake and PBI levels
recovered to control levels at 7 days and remained similar to
control levels for the remainder of the study. However, this is
considered to be a normal physiological response, without lasting
adverse effect, and the NOAEL in this study was thus 10 ppm,
equivalent to 0.5 mg/kg bw/day (Weber & Patzschke, 1979).
The effect of PTU on thyroid function was investigated in a 63-
day study in Wistar rats. Groups of 36 males received PTU (> 97%
purity) in the drinking water at concentrations of 0, 0.1, 0.3, 1 or
10 ppm. Stability and accuracy of admixture in the drinking water
was checked during the study and found to be adequate. On days 7, 21
and 63, thyroid function was investigated in groups of 12 rats by
determination of thyroid weight, measurement of 131I incorporation
into the thyroid and by radioimmunoassay determination of T3, T4
and TSH in the serum. Food and water intake, as well as weight gain,
were not affected by treatment. Thyroid weights were unaffected by
treatment at any of the examinations. Although minor intergroup
differences in results of other investigations attained a level of
statistical significance when compared with controls, there were no
consistent, dose-related effects. Histopathological examination of
thyroids revealed no treatment-related effects. The NOAEL in this
study was 10 ppm, the highest dose tested, equal to 1 mg/kg bw/day
(Weber et al., 1991).
Long-term toxicity/carcinogenicity studies
Mice
PTU of 99.3% purity was administered in the diet to groups of
CF1/W74 mice (60/sex/dose) at levels of 0, 1, 10, or 100 ppm for 24
months. An additional group of mice received 1000 ppm in the diet,
which was regularly alternated on a weekly basis with control diet.
The stability of the admixture of the test material into the test
diet was reported checked and found to be adequate before the start
of the study. General examinations did not indicate any notable
differences between test and control animals. Food consumption of
1000 ppm mice was increased compared to control mice. Mean body
weights for 1000 ppm male mice were consistently lower than those of
control male mice from week 16 to termination of the study. No other
meaningful body-weight differences were observed for any of the
groups. Mortality was equivalent in all test and control groups.
Haematological examinations did not reveal any meaningful
differences between control and test group male or female mice.
Plasma ALP activity at 1000 ppm was increased at 12 and 24 months.
ALP levels in other dose groups were generally similar to control
levels. Cholesterol levels were increased at 1000 ppm at 12 and 24
months.
Gross necropsies on mice sacrificed at 12 months revealed
clearly enlarged thyroids in 1000 ppm male mice. For mice dying or
sacrificed during the study, increased incidences of enlarged or
swollen livers, sometimes with nodules, were observed in 10, 100,
and 1000 ppm groups. For mice sacrificed at 24 months, dose-related
increased incidences of enlarged or swollen livers and/or nodular
alterations were observed in all treated male and female groups.
These livers also tended to be partly hardened and brittle. Thyroid
weights were significantly increased in 1000 ppm male mice, but not
in female mice at 12 months. Liver weights of 1000 ppm male mice
were significantly increased at 12 months and at 24 months, and of
female mice at 24 months. Liver weights of 100 ppm male and female
mice were also significantly increased at 24 months. Kidney weights
from female mice were significantly increased at 24 months at 1000
ppm, at 100 ppm, and possibly also at 10 ppm.
Histopathological examination revealed increased incidences of
thyroid hypercellularity in the high-dose male mice at the interim
12-month sacrifice and at the terminal sacrifice. Clearly increased
incidences of hepatocellular tumours were observed in treated male
and female groups. For male mice, percentages of animals with
adenomas 0, 11, 10, 7, and 26%, and of animals with carcinomas 0,
13, 29, 31, and 21% for the control, 1, 10, 100, and 1000 ppm
groups, respectively. For female mice, comparable percentages of
adenomas were 0, 10, 13, 32, and 18%, and of carcinomas 2, 2, 5, 26,
and 38%, respectively. Historical control data were supplied, but
the Meeting concluded that the figures were not helpful in
considering the results of this study. Hepatic tumour incidence was
variable from one study to another and the slides had been read by
different pathologists from a number of different institutions. In
the thyroid, only 2 adenomas (both in the one ppm male group) and 2
adenocarcinomas (one in the 100 ppm female group and one in the 1000
ppm female group) were observed. The Meeting concluded that it was
not possible to establish a NOAEL in this study since liver tumour
incidence in all treated groups was higher than in controls.
However, there was evidence of a dose-response relationship and the
lowest dose level (equal to 0.2 mg/kg bw/day) was considered to be a
marginal effect level (Bomhard & Loeser, 1981).
Rats
PTU of approximately 99% purity was fed to Wistar TNO/W74 rats
for 24 months at dosage levels of 0, 1, 10, 100, or 1000 ppm. The
homogeneity, stability and accuracy of admixture of the test
material into the test diet was not checked during the study. The
control group consisted of 100 rats/sex/group and each of the test
groups of 50 rats/sex/group. Additional satellite groups of 25
rats/sex/group were used for clinical laboratory studies. From the
third week onward, animals in the 1000 ppm group gained little or no
body weight and were cachectic. Food consumption was substantially
below that of control animals. Most rats had a ruffled hair coat
and/or heavy loss of hair. The skin of some rats was covered with a
brownish scale. Mortalities commenced at 3 months. For male rats,
31/50 died by 6 months and 34/50 by 12 months. For female rats,
38/50 died by 6 months and 40/50 by 12 months. At 65 weeks the last
survivors in this group, 12 male and 3 female rats, were sacrificed.
Haematological examinations on male and female rats in this dose
group revealed a marked and widespread decrease in haematopoiesis,
and differential white blood cell counts revealed a relative
increase in mature polymorphonuclear neutrophils and a relative
decrease in lymphocytes. Clinical chemistry revealed highly
increased plasma ALP activities and urea, creatinine and cholesterol
levels were increased. Gross necropsies revealed enlarged thyroids
in some animals.
Histopathological examination of organs/tissues revealed
nodular and generalized hyperplasia of the thyroid, calculi and
vacuolation of tubular cells in kidneys, aplasia and decreased
haematopoiesis in marrow (particularly in males), vacuolation of
hepatocytes in liver (particularly in males), and atrophy and
reduced spermatogenesis in testes. Nine male rats (of 42 examined)
had thyroid adenomas. Two female rats (of 25 examined) also had
thyroid adenomas and one additional female rat had a thyroid
cystadenoma. Daily observations of animals receiving lower dietary
levels indicated no differences from the control animals. Food
consumption was also similar in these test and control groups. Body
weights of 100 ppm male rats were consistently lower than those of
control male rats from week 20 to termination of the study. Body
weights of all other test groups were similar to those of the
respective control groups. Slightly increased mortality in the 100
ppm male group was observed at 18 and 24 months. Mortalities were
equivalent in all other test and control groups. The number of male
rats surviving to termination of the study were 85/100, 41/50,
44/50, and 38/50 for the control, 1, 10, and 100 ppm groups,
respectively. The respective numbers for female rats were 83/100,
40/50, 42/50, and 43/50.
Haematological examinations and clinical chemistry, including
determination of PBI in plasma, did not reveal any indication of
reaction to treatment at 1, 10 or 100 ppm. Gross necropsies revealed
enlarged thyroids in numerous rats treated with 100 ppm. Thyroid
weights from rats treated with 100 ppm were significantly increased.
Histopathological examination of the thyroids revealed nodular
hyperplasia in 3 males treated with 1 ppm and 5 males and 2 females
treated with 100 ppm (in addition to 6 males and 4 females treated
with 1000 ppm). Historical control data were not presented, but the
report states that "this change may be expected in normal control
rats". The presence of large follicles and colloid cysts in the
thyroids of rats treated with 100 ppm was probably treatment-
related. Inter-group differences in incidence of other findings in
the thyroid were probably not treatment-related: vacuolated cells
lining the follicles, often seen in active thyroids, were present in
2 control males and 4 treated with 100 ppm, and in no control
females, compared with 1 at 10 ppm and 3 at 100 ppm. Small follicles
with little colloid were seen in 1% of control females, 22% at
1 ppm, 46% at 10 ppm, and 2% at 100 ppm. In males the corresponding
incidences were 7%, 18%, 20% and 16%. In summary, at 1000 ppm, a
clearly excessive dosage level, thyroid tumours were definitely
related to the test material. At 100 ppm non-neoplastic thyroid
changes and reduced body-weight gain were seen. The NOAEL in this
study was 10 ppm, equal to 0.56 mg/kg bw/day (Bomhard & Loeser,
1980).
Reproduction studies
No data were available to the Meeting regarding multigeneration
reproduction studies with PTU.
Special studies on embryo/fetotoxicity
Rats
A teratology study of propineb and PTU is described in the
published literature in which groups of 15 Sprague-Dawley rats
received PTU by gavage on days 6 to 16 of pregnancy at levels of 0,
11, 23, 45 or 90 mg/kg bw/day. PTU showed teratogenic effects at
doses which did not show any maternal toxicity (45 and 90 mg/kg
bw/day). However, slightly reduced body-weight gain and increased
thyroid weights were reported at 45 and 90 mg/kg bw/day and the
Meeting considered these findings to be evidence of slight maternal
toxicity. Results were not reported in sufficient detail to fully
evaluate this study (Vicari et al., 1985).
Earlier studies showed that PTU had teratogenic potential in
rats following one or several doses (> 20 mg/kg bw/day) on days 12
or 13 of gestation (Ruddick et al., 1976; Bleyel & Lewerenz,
1978).
No data were available to the Meeting regarding special studies
with PTU on embryo/fetotoxicity in species other than rats.
Special studies on genotoxicity
The results of genotoxicity studies are summarized in Table 1.
Observations in humans
No information available.
Table 1. Results of genotoxicity assays on PTU
Test system Test Object Concentration Purity Results Reference
Ames test S. typhimurium up to 12.5 mg/plate 99.5% Negative Herbold, 1980
TA98, TA100, TA1535,
TA1537
Ames test S. typhimurium up to 12.5 mg/plate 99.5% Negative Herbold, 1981a
TA98, TA100, TA1535,
TA1537
Pol A1 E. coli p3478, W3110 up to 1 mg/plate 99.5% Negative Herbold, 1981b
Micronucleus test Mouse ? ? Negative Rolandi et al., 1984
DNA metabolism and CF1 mice 100 mg/kg bw 99.5% Negative Klein, 1981
binding
COMMENTS
Following oral administration to rats, PTU was rapidly absorbed
and eliminated in the urine and faeces. Less than 0.2% of the
administered dose was detected in the exhaled air and after 10 days
only 1-2% remained in the body. Biliary excretion was also
observed, as was evidence for enterohepatic recirculation.
Distribution in body tissues was essentially uniform, with the
exception of the thyroid which had a 12-fold higher level than other
tissues.
The results of toxicity studies clearly indicate that PTU has a
goitrogenic effect in rats. In a 21-day comparative study of the
effects of propineb, PTU, ETU, zineb and methyl thiouracil on
thyroid weight, PTU had an equivalent effect to that of methyl
thiouracil, and was somewhat more potent than ETU. The thyroid
enlargement was partially reversible during a 28-day withdrawal
period and the results suggested that the effects of propineb on the
thyroid in rats may be caused primarily by the metabolite, PTU. In
a 63-day study in male rats, in which PTU was administered in the
drinking water at levels of 0, 0.1, 0.3, 1 or 10 ppm, no consistent
effects on thyroid function were seen at doses up to 10 ppm, the
highest dose tested, which was equal to 1 mg/kg bw/day. In a 24-
month study on thyroid function, using dietary levels of 0, 1, 10,
100 or 1000 ppm, the NOAEL (based on increased thyroid weight at
higher doses) was 10 ppm in the diet, equivalent to 0.5 mg/kg
bw/day.
In long-term studies, treatment-related alterations in tumour
incidence were seen in rats and mice. In a 2-year study in mice,
using dietary levels of 0, 1, 10, 100 and 1000 ppm, it was
considered not possible to establish a NOAEL since liver tumour
incidence in all treated groups was higher than in controls.
However, there was evidence of a dose-response relationship and the
lowest dose level (equal to 0.2 mg/kg bw/day) was considered to be a
marginal effect level. Thyroid tumour incidence was not affected by
treatment of mice with PTU. In a 2-year study in rats, also using
dietary levels of 0, 1, 10, 100 or 1000 ppm, the NOAEL was 10 ppm in
the diet, equal to 0.56 mg/kg bw/day. Treatment-related thyroid
tumours were seen at 1000 ppm. This dietary level was accompanied by
increased mortality, while non-neoplastic thyroid changes and
reduced body-weight gain were seen at 100 ppm.
A published article indicated that PTU showed teratogenic
effects in rats at 45 and 90 mg/kg bw/day, doses which showed slight
maternal toxicity. Results were not, however, reported in
sufficient detail to fully evaluate this study. No information was
available to the Meeting regarding special studies with PTU on
embryo/fetotoxicity in species other than rats.
PTU is not mutagenic in bacteria and does not cause damage to
mouse DNA in vivo. The Meeting could not reach any conclusion
regarding the genotoxicity of PTU because of the limited database.
A temporary ADI was allocated to PTU, which was based on the
marginal effect level in the 2-year study in mice (1 ppm in the
diet, equal to 0.2 mg/kg bw/day). The Meeting felt reassured that,
in view of the metabolic conversion of propineb to PTU, the long-
term study in mice with PTU identified the same target organ as the
long-term study in mice with propineb. However, in view of the
overall inadequacy of the toxicological data for PTU, the Meeting
concluded that a 1000-fold safety factor was necessary.
TOXICOLOGICAL EVALUATION
Level causing no toxicological effect
Mouse: 1 ppm in the diet, equal to 0.2 mg/kg bw/day
(marginal effect level in 2-year study)
Rat: 10 ppm in the diet, equivalent to 0.5 mg/kg bw/day
(2-year thyroid function study)
10 ppm in the diet, equal to 0.56 mg/kg bw/day
(2-year study)
Estimate of temporary acceptable daily intake for humans
0-0.0002 mg/kg bw
Studies without which the determination of a full ADI is impractical
Results to be submitted to WHO by 1998:
1. Long-term carcinogenicity study in mice, identifying a
NOAEL.
2. Clarification of the genotoxic potential of PTU.
3. Clarification of embryo/fetotoxicity and teratogenicity
potential of PTU in rodents.
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