Toxicological evaluations

Pesticide residues in food -- 1999

Sponsored jointly by FAO and WHO
with the support of the International Programme
on Chemical Safety (IPCS)

Joint meeting of the
FAO Panel of Experts on Pesticide Residues
in Food and the Environment
and the
WHO Core Assessment Group

Rome, 20-29 September 1999

PROPYLENE THIOUREA (addendum)

First draft prepared by
Timothy C. Marrs
Department of Health, London, United Kingdom

Explanation
Evaluation for acceptable daily intake
Long-term study of toxicity and carcinogenicity
Genotoxicity
Reproductive toxicity
Special study of mechanism of action
Comments
Toxicological evaluation
References

Explanation

Propylenethiourea is a plant and animal metabolite. It is also a
degradation product of propineb and therefore forms part of the
residue to which consumers of propineb-treated produce may be exposed.
The 1977 Joint Meeting (Annex 1, reference 28) expressed concern
about the thyrotoxicity and carcinogenicity of propylenethiourea and
allocated a temporary ADI of 0-0.005 mg/kg bw to propineb; this
temporary ADI was extended by the 1980 and 1983 Meetings (Annex 1,
references 34 and 40) but was withdrawn by the 1985 Joint Meeting
(Annex 1, reference 44). An ADI of 0-0.007 mg/kg bw was allocated to
propineb by the 1993 Meeting, which also allocated a temporary ADI for
propylenethiourea of 0-0.0002 mg/kg bw on the basis of the LOAEL in a
2-year study in mice and a safety factor of 1000 (Annex 1, reference
68). The 1993 Meeting requested another long-term study of
carcinogenicity in mice, clarification of the embryotoxicity,
fetotoxicity, and teratogenicity of propylenethiourea in rodents, and
elucidation of its genotoxic potential. Studies to meet the above
requests were supplied, were evaluated at the present Meeting, and are
summarized in this monograph addendum.

Evaluation for Acceptable Daily Intake

1. Long-term study of toxicity and carcinogenicity

In a study conducted according to guidelines of the OECD, US
Environmental Protection Agency, and the European Union,
propylenethiourea (purity, 97.5-98.4%) was administered to groups of
50 male and 50 female B6C3F₁ mice in the drinking-water at
concentrations of 0, 0.25, 1.25, 6, 30, or 150 ppm for 108 weeks.
These concentrations resulted in intakes of 0, 0.040, 0.20, 0.89, 4.0,
and 18 mg/kg bw per day in males and 0, 0.051, 0.25, 1.1, 4.6, and 22
mg/kg bw per day in females. The mice were inspected twice daily,
except on weekends and public holidays, when they were inspected once
daily. Body weight and food consumption were measured weekly during

the first 13 weeks and every 4 weeks thereafter. Water consumption was
measured weekly for the first 37 weeks and then every 4 weeks.
Haematological parameters were measured at weeks 53, 79, and 104.
Animals that died during the study or were killed in extremis were
examined post mortem, and tissues other than those that were
autolysed were fixed for histological examination. At the end of the
study, body weights were determined, and the brains, livers, hearts,
spleens, both kidneys, both adrenals, and both testes were weighed;
these organs and others were then fixed, and sections were processed
for histopathology. Only the liver, oesophagus, trachea, and thyroid
(with parathyroid) were examined histologically for animals given 0.25
or 1.25 ppm propylenethiourea.

The material did not affect the animals' behaviour or mortality
rate. At 30 ppm, the mean body weights of males were lower than those
of controls up to and including week 45, and at 150 ppm, the body
weights of males were consistently lower than those of the controls
throughout the study; in females, differences were seen only at the
highest dose (150 ppm). At doses > 6 ppm, the water intake of the
animals was reduced; although the effect was ascribed to reduced
palatability, food intake was not affected by treatment. The treatment
appeared to have little effect on erythrocyte parameters, while a
slightly decreased platelet count was found in the males at the
highest dose at weeks 53 and 79. These animals also had a decreased
leukocyte count at week 53. Females at 30 ppm had an increased number
of lymphocytes and a decreased number of neutrophils at week 104, but
as this finding was not seen at the highest dose, it is of doubtful
toxicological significance. Only minor, inconsistent changes in organ
weights were seen. In males, reductions in the absolute weights of the
heart, liver, spleen, and kidney were seen at the highest dose, and
the heart weight was also reduced at the next highest dose, but these
changes probably reflect the reduction in body weight. In females,
only a small reduction in the absolute weight of the brain was seen;
although brain weight is relatively unaffected by reductions in body
weight, the reduction observed was so small that it seems unlikely to
reflect specific toxicity. The increased relative weights of the
brain, kidneys, and testes in males at the high dose almost certainly
reflect the lowered total body weight. The reduced relative weight of
the heart in males is difficult to interpret: the weight was lower in
all treated groups than in controls but was lowest at 0.25 ppm, and
there was therefore no dose-response relationship. The increased
relative weight of the kidneys observed in females at the highest dose
is probably again a reflection of the decreased body weight in that
group.

Treament had little effect on histopathological appearance.
Although a higher prevalence of pituitary hyperplasia was seen in
females at 30 ppm, the prevalence at the highest dose was close to
that in controls, the incidences being 4/50 in controls, 0/50 at 0.25
ppm, 0/50 at 1.25 ppm, 4/50 at 6 ppm, 11/50 at 30 ppm, and 5/50 at 150
ppm. There was no treatment-related neoplastic or non-neoplastic
effect. In particular, there were no treatment-related effects on the
thyroid or pituitary glands or on the liver. Tumours that were

observed but at incidences not related to treatment included
broncheoloalveolar adenomas and carcinomas of the lung, hepatocellular
adenomas, carcinomas, haemangiomas, and haemangiosarcomas of the
liver. Thyroid follicular-cell adenomas and carcinomas and pituitary
adenomas were seen, but there was no evidence of a dose-response
relationship. The NOAEL was 6 ppm, equal to 0.89 mg/kg bw per day,
because of effects on body-weight gain in males at the next highest
dose. Propylenethiourea was not carcinogenic (Schladt & Jekat, 1998).

2. Genotoxicity

The results of the submitted studies on the genotoxicity of
propylenethiourea are summarized in Table 1.

3. Developmental toxicity

In a study carried out according to guideline 83-3 of the US
Environmental Protection Agency, propylenethiourea (purity 99.5-99.9%)
was administered by gavage in deionized water to groups of 30
inseminated Sprague-Dawley dams at doses of 0, 1, 7, or 51.4 mg/kg bw
per day on days 6-19 of gestation. The dams were observed twice daily;
body weight was estimated on days 0, 2, 4, and 6-20 of gestation, and
food consumption was recorded on days 2, 4, 6-19, and 20. The dams
were killed on day 20 of gestation after removal of blood from 10
gravid dams at each dose for determination of triiodothyronine,
thyroxine, and thyroid-stimulating hormone. At termination, the dams
were examined externally, and a gross necropsy was carried out. The
liver, kidneys, and thyroid were excised and weighed; the ovaries were
excised, the corpora lutea counted, and the pregnancy status of the
animal was determined. The uterus was opened, resorptions and
implantations were counted, and the fetuses that were removed were
identified, sexed, weighed, and examined externally. About half the
fetuses from each litter were fixed in 70% ethanol, eviscerated,
processed, and evaluated for general skeletal development. Gross
visceral examination was carried out on the remainder of the fetuses,
which were placed in Bouin's solution and transferred to 70% ethanol
before cranial examination.

No treatment-related clinical signs were seen during the study.
Decreased body-weight gain was seen in dams at 51.4 mg/kg bw per day
on days 0-20, while food consumption was decreased on days 7-8, 17-18,
and 19-20. Dams at the two highest doses showed effects on the
pituitary and thyroid system; at 7 mg/kg bw per day, the levels of
thyroid-stimulating hormone were increased and those of thyroxine
decreased, while at the highest dose the level of thyroid-stimulating
hormone was increased and those of both triiodothyronine and thyroxine
were decreased. At the highest dose, the absolute and relative weights
of the thyroid were increased, and the absolute but not the relative
weights of the kidney and liver were decreased. No effects were found
on the gestation index, preimplantation or postimplantation loss or
reabsorptions that could be attributed to treatment, and there were no
significant effects on litter size or the number of fetuses or
implantation sites. Slightly decreased litter weight was seen at 51.4

Table 1. Results of tests for the genotoxicity of propylenethiourea

End-point Test system Concentration Purity (%) Results Reference

Reverse mutation S. typhimurium 16-5000 µg/plate 99.5 Negative ± S9 Herbold (1995)
TA1535, TA1537,
TA100, TA98

Chromosomal Chinese hamster 500-5000 µg/ml 99.5 Negative ± S9 Herbold (1996a)
aberration V79 cells

Forward mutation Chinese hamster 312.5-5000 µg/ml 99.5-98.9 Negative ± S9 Herbold (1996b)
V79 cells, hprt locus

mg/kg bw per day. The malformations in the fetuses including hydrops,
meningocoele, haematoma, domed head, exencephaly, protruding tongue,
cleft palate, micrognathia, anasarca, imperforate anus, and absence of
musculature near the umbilicus and abnormalities of the appendages,
such as malrotation and adactyly. The visceral malformations observed
included enlarged thyroid glands, hydroureter, dilated ventricles in
the brain, hydrocephalus, and malformations of the brain. The skeletal
effects included malformations of the cranial bones, lordosis, and
abnormal radii; some fetuses had multiple malformations. Increased
incidences of malformations in the fetuses at lower doses were
confined to the skeleton: e.g. incompletely ossified parietals,
interparietals, and occipitals, enlarged sagittal sutures and
fontanels, and incompletely ossified thoracic and lumbar centra,
sacral arches and sternebrae at 7 mg/kg bw. While the authors
concluded that the incidence of skeletal variations was elevated at
7 mg/kg bw per day, they reported that the increases seen at the
lowest dose were within the range seen in historical controls and that
although the fetal incidences were elevated the litter incidences were
generally not (Astroff, 1997). This conclusion ignores the increased
incidences of incompletely ossified parietal and interparietal bones
in fetuses at 1 mg/kg bw, which were statistically significant by
comparison with the controls. No NOAEL was identified for fetal
toxicity.

In a study conducted according to guidelines of the OECD, US
Environmental Protection Agency, and the European Union, 30
inseminated Sprague-Dawley rats were given propylenethiourea (purity,
99.7-99.8%) by gavage in deionized water at nominal doses of 0, 0.3,
or 1.2 mg/kg bw per day (the doses analysed were 0, 0.32, and 1.2
mg/kg bw per day) on days 6-19 of gestation. The animals were examined
daily, and weights and food consumption were recorded on days 2, 4,
6-19, and 20 of gestation. The dams were killed on day 20 of gestation
with carbon dioxide, examined grossly externally and internally, and
the livers, thyroids, kidneys, and spleens were excised and weighed;
the ovaries were excised, corpora lutea counted, and pregnancy
determined. The uterus was removed and weighed and then opened, at
which time any resorptions were characterized. Fetuses were removed,
implants noted, and placentas were trimmed and weighed. The fetuses
were identified, sexed, weighed, and examined externally. About half
the fetuses from each litter were fixed and processed for examination
of general skeletal development, and the remainder were processed for
cranial examination after gross visceral examination. No
treatment-related clinical signs were observed in the dams, and no
significant effects were seen on maternal weight, although the food
consumption of those at the highest dose was decreased on one occasion
(days 14-15). The authors did not consider this effect to be
biologically significant. At necropsy, no effects were seen on organ
(including uterine) weights or on total body weight. The compound had
no significant effect on fertility, mating, or gestation indexes. No
significant differences were seen between groups in the number of
corpora lutea or implantation sites or resorption or pre- and
post-implantation loss. No significant differences were seen in litter
sizes, the number or proportion of live fetuses in litters, fetal or

placental weights, or external or visceral fetal malformations. At the
high dose, the incidences of incompletely ossified frontal and
interparietal bones and enlarged anterior fontanels were increased,
and, although the incidences were within that of historical controls,
the authors considered that they might be dose-related. There was no
sex-related effect. At 0.3 mg/kg bw per day, the only finding of this
type was enlargement of the posterior fontanel, with no dose-response
relationship (the incidence was lower at the high dose), and the
author considered that the effect was not related to treatment. The
NOAEL for maternal effects was 1.2 mg/kg bw per day, and that for
fetal effects (skeletal variations) was 0.3 mg/kg bw per day (Young &
Astroff , 1999).

4. Special study of mechanism of action

The toxicity of propylenethiourea, ethylene thiourea,
N,N'-tetramethyl thiourea, and propyl thiouracil was studied
in vitro in a partially purified fraction of pig thyroid with a
10 000 × g supernatant from a homogenate of rat liver.
Propylenethiourea, ethylene thiourea, and N,N'-tetramethyl thiourea
did not inhibit thyroid peroxidase-catalysed oxidation of guaiacol, a
measure of peroxidase activity, while propyl thiouracil did. Like the
other three compounds, propylenethiourea temporarily suppressed
thyroid peroxidase-catalysed iodine formation in a dose-dependent
fashion, although propyl thiouracil was the least effective compound
in this respect. All four compounds also suppressed non-enzymatic and
thyroid peroxidase-catalysed iodination of tyrosine. Propylenethiourea
appeared to be only a weak inhibitor of iodothyronine deiodinase, with
1/500th of the potency of propylthiouracil. The author concluded that
propylenethiourea (and ethylene thiourea) were unlikely to interfere
with the formation of triiodothyronine from thyroxine in vivo and
that depression of thyroid hormone synthesis and consequent
stimulation of the hypothalamic-pituitary-thyroid axis caused the
thyroid lesions (Freyberger, 1996).

Comments

Propylenethiourea was administered to mice in the drinking-water
for 108 weeks. The NOAEL was 0.89 mg/kg bw per day, because of effects
on body-weight gain of males at the next higher dose.
Propylenethiourea was not carcinogenic in this study.

In a study of developmental toxicity in rats in which
propylenethiourea was administered by gavage in deionized water to
groups of inseminated dams, a NOAEL was not identified for fetal
toxicity at the lowest dose tested (1 mg/kg bw per day). In a
supplemental study of developmental toxicity with a very similar
protocol, the NOAEL for maternal effects was 1.2 mg/kg bw per day and
that for fetal effects (skeletal variations) was 0.3 mg/kg bw per day.

Propylenethiourea did not induce reverse mutation in
Salmonella typhimurium and did not induce chromosomal aberration or
forward mutation at the Hrpt locus in Chinese hamster V79 cells. The
Meeting concluded that propylenethiourea is unlikely to have genotoxic
potential.

In a study in vitro of the mechanisms of the toxicity to the
thyroid of propylenethiourea, ethylenethiourea, tetramethylthiourea,
and propylthiouracil in a partially purified fraction of pig thyroid
and a 10 000 × g supernatant from rat liver homogenate,
propylenethiourea appeared to be only a weak inhibitor of
iodothyronine deiodinase. The Meeting concluded that propylenethiourea
is unlikely to interfere with the formation of triiodothyronine from
thyroxine in vivo and that the thyroid lesions seen were due to
depression of thyroid hormone synthesis and consequent stimulation of
the hypothalamic-pituitary-thyroid axis.

An ADI of 0-0.0003 mg/kg bw was allocated on the basis of the
NOAEL of 0.3 mg/kg bw per day in the study of developmental toxicity
in rats, and a 1000-fold safety factor. The 1000-fold safety factor
was considered necessary since a multigeneration study of reproductive
toxicity was not available. In fact, the Meeting noted that the NOAEL
in a multigeneration study of reproductive toxicity of propineb, which
generates propylenethiourea as a main metabolite, was about one-tenth
of the NOAEL for developmental toxicity, and there was no evidence
that a similar difference does not exist for propylenethiourea itself.

An acute reference dose of 0.003 mg/kg bw was established, on the
basis of the NOAEL in the study of developmental toxicity in rats and
a 100-fold safety factor.

Toxicological evaluation

Levels that cause no toxic effect

Mouse: 6 ppm in drinking-water, equal to 0.89 mg/kg bw per day
(2-year study)

Rat: 10 ppm in the diet, equivalent to 0.56 mg/kg bw per day
(2-year study; evaluated by the 1993 JMPR)

1.2 mg/kg bw per day (maternal effects in a study of
developmental toxicity)

0.3 mg/kg bw per day (fetal effects in a study of
developmental toxicity)

Estimate of acceptable daily intake

0-0.0003 mg/kg bw

Estimate of acute reference dose

0.003 mg/kg bw

Studies that would provide further information useful for continued
evaluation of the compound

Studies of reproductive toxicity

References

Astroff, A.B. (1997) A developmental toxicity study with propylene
thiourea in the SpragueDawley rat. Unpublished report No. 108018
from Bayer Corporation Agriculture Division, Stillwell, Kansas,
USA. Submitted to WHO by Bayer AG, Monheim, Germany.

Freyberger, A. (1996) Propineb in vitro characterization of the
goitrogenic properties of its metabolite 1,2-propylenethiourea.
Unpublished study 24639 from Bayer AG, WuppertalElberfeld,
Germany. Submitted to WHO by Bayer AG, Monheim, Germany.

Herbold, B (1995) Propylenethiourea (PTU) Salmonella/microsome test
plate incorporation and preincubation method. Unpublished study
No. 23853, dated 21 March 1995 from Bayer AG,
Wuppertal-Elberfeld, Germany. Submitted to WHO by Bayer AG,
Monheim, Germany.

Herbold, B. (1996a) Propylenethiourea (PTU) in vitro mammalian
chromosome aberration test with Chinese hamster V79 cells.
Unpublished study No. 24604, dated 8 January 1996 from Bayer AG,
Wuppertal-Elberfeld, Germany. Submitted to WHO by Bayer AG,
Monheim, Germany.

Herbold, B. (1996b) Propylenethiourea mutagenicity study for the
detection of induced forward mutations in the V79-HPRT assay in
vitro. Unpublished report No. 25287, dated 24 July 1996 from
Bayer AG, Wuppertal-Elberfeld, Germany. Submitted to WHO by Bayer
AG, Monheim, Germany.

Schladt, L. & Jekat, F.W. (1998) PTU (propylene thiourea) oncogenicity
study in B6C3F1 mice. Administration in drinking water over 2
years. Unpublished report No. 27696 from Bayer AG. Submitted to
WHO by Bayer AG, Monheim, Germany.

Young, A.D. & Astroff, A.B. (1999) A supplimental developmental
toxicity study with propylene thiourea (PTU) in the
Sprague-Dawley rat. Unpublished report No 108018-1 from Bayer
Corporation Agriculture Division, Stillwell, Kansas, USA.
Submitted to WHO by Bayer AG, Monheim, Germany.

    See Also:
       Toxicological Abbreviations