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    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.

    REFERENCES

    Bleyl, D.W. & Lewerenz, H.J. (1978) Zur teratogenen Wirkung von
    Propylenthioharnstoff bei Ratten.  Monatshefte fur Veterinarmedizin,
    33: 139-197, Jena.

    Bomhard, E. & Loeser, E. (1980) Propylenethiourea, chronic toxicity
    study on rats (two-year feeding experiment). Bayer AG Institute of
    Toxicology, Report No. 9345. Unpublished report submitted to WHO by
    Bayer AG.

    Bomhard, E. & Loeser, E. (1981) Propylenethiourea, chronic toxicity
    study with mice (feeding study over two years). Bayer AG Institute
    of Toxicology, Report No. 10102. Unpublished report submitted to WHO
    by Bayer AG.

    Herbold, B. (1980) Propylenethiourea,  Salmonella/microsome test
    for detection of point mutagenic effects. Bayer AG Institute of
    Toxicology, Report No. 9563. Unpublished report submitted to WHO by
    Bayer AG.

    Herbold, B. (1981a) Propylenethiourea,  Salmonella/microsome test
    to evaluate for point mutations employing liver homogenates from
    strain CFWI male mice. Bayer AG Institute of Toxicology, Report No.
    10116. Unpublished report submitted to WHO by Bayer AG.

    Herbold, B. (1981b) Propylenethiourea, Pol Al-test on  E. coli to
    evaluate for DNA damage. Bayer AG Institute of Toxicology, Report
    No. 10146. Unpublished report submitted to WHO by Bayer AG.

    Kimmerle, G. (1972) Comparative test of antracol, propylenethiourea,
    zineb and ethylene thiourea for thyroid action in rats. Report No
    3551, Bayer AG, Institute of Toxicology. Unpublished report
    submitted to WHO by Bayer AG.

    Klein, W. (1981) Study for the effect of PTU on DNA metabolism.
    Seibersdorf Research Centre, Institute of Biology, Project No.
    PS/2277, Bayer Study No. T 4003 443 (PTU). Unpublished report
    submitted to WHO by Bayer AG.

    Rolandi, A., De Marinis, E. & De Caterina, M. (1984) Dithiocarbamate
    pesticides: activity of propineb in the micronucleus test in mice.
     Mut. Res. 135: 193-197.

    Ruddick, J.A., Newsome, W.H. & Nash, L. (1976) Correlation of
    teratogenicity and molecular structure: Ethylene thiourea and
    related compounds.  Teratology, 13: 263-266.

    Vicari, L., de Dominicis, G., Vito, M., Placida, C., De Marinis, E.
    (1985) Teratogenic and goitrogenic activity of propineb and
    propylenethiourea in the rat.  Boll. Soc. It. Biol. 61: 271-278.

    Weber, H., Patzschke, K. & Wegner, L.A. (1978) Propylenethiourea-
    14C, biokinetic study on rats. Bayer AG Institute of
    Pharmacokinetics, Report No. 7397. Unpublished report submitted to
    WHO by Bayer AG.

    Weber, H. & Patzchke, K. (1979) Effect of long-term administration
    of propylenethiourea (PTU) on thyroid function of male and female
    rats. Bayer AG Institute of Pharmacokinetics, Report No. 8494.
    Unpublished report submitted to WHO by Bayer AG.

    Weber, H., Mager, H., Ditgens, K. & Ohs, P. (1991) PTU feeding
    study: Effect of the compound on thyroid function in male Wistar
    rats after uptake with the drinking water in the low dose range up
    to 10 ppm over a time interval up to 63 days. Bayer AG Institute of
    Toxicology. Unpublished report submitted to WHO by Bayer AG.


    See Also:
       Toxicological Abbreviations