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    Pesticide residues in food -- 1999



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



    Toxicological evaluations




    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    references  34 and  40) but was withdrawn by the 1985 Joint Meeting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 g/plate    99.5          Negative  S9     Herbold (1995)
                        TA1535, TA1537, 
                        TA100, TA98

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

    Forward mutation    Chinese hamster          312.5-5000 g/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