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    IPRODIONE (addendum)

     First draft prepared by
     E. Bosshard,
     Federal Office of Public Health, Food Science Division,
     Schwerzenbach, Switzerland

    Explanation
    Evaluation for acceptable daily intake
         Toxicological studies
              Long-term toxicity and carcinogenicity
              Special studies
                   Mechanism of action
         Comments
         Toxicological evaluation
    References

    Explanation

         Iprodione, a dicarboximide fungicide, was first evaluated in
    1977, when an ADI of 0-0.3 mg/kg bw was allocated (Annex I,
    reference 28). The ADI was reduced to 0-0.2 mg/kg bw in 1992 on the
    basis of new data from a study of reproductive toxicity in rats, a
    study of teratogenicity in rabbits, and a one-year study of toxicity
    in dogs, and applying a safety factor of 100 (Annex I, reference 65).
    The results of two additional studies of long-term toxicity and
    carcinogenicity in rats and mice and studies of the mechanism of
    carcinogenesis have now become available. These results are summarized
    and discussed in this monograph addendum.

    Evaluation for acceptable daily intake

    Toxicological studies

    (a)  Long-term toxicity and carcinogenicity

         Previous studies at dietary concentrations of 0, 200, 500, or
    1250 ppm in mice and 0,125, 250, or 1000 ppm in rats revealed no
    evidence of tumorigenic activity in either species (Hastings &
    Huffmann, 1975; Hastings  et al., 1976). Two additional studies
    conducted at higher doses have become available.

    Mice

         Iprodione (purity, 95.7%) was fed in the diet at concentrations
    of 0, 160, 800, or 4000 ppm to groups of 50 male and 50 female Crl:
    DC-1 (ICR) Br mice for 99 weeks. Satellite groups of 15 animals of
    each sex received the same doses and were used for blood sampling,
    biochemical investigations, and interim sacrifice after one year of
    study. Dietary sampling conducted before the study confirmed the
    homogeneity and stability of the diet. Treatment caused no clinical
    signs of toxicity and no increase in mortality; haematological
    parameters were not affected. The group mean body-weight gain was no
    different in treated and untreated animals for the first 18 weeks, but
    after 45 weeks of treatment the body-weight gains of animals at
    4000 ppm were lower than those of the controls, by 3% in females and
    5% in males. The food consumption of females at this dose was slightly
    increased from week 19 to termination of the study. In clinical
    chemical examinations conducted during week 52 in 10 animals of each
    sex in the satellite groups, the only treatment-related changes were
    increased levels of aspartate and alanine aminotransferases in animals
    of each sex at 4000 ppm.

         At interim sacrifice, changes in organ weights were seen in
    animals at the highest dose, including increased liver weights
    (adjusted for body weight by covariance analysis) in animals of each
    sex and increased adrenal weights (absolute) which were statistically
    significant only in males. Macroscopic changes observed in satellite
    animals included liver enlargement in both males and females at
    4000 ppm and accentuated lobular markings in males at 800 and 4000 ppm
    and in females. Microscopic examination revealed various non-neo-
    plastic findings in the liver, adrenals, ovaries, and testes of
    animals at the highest dose. In the liver, there was an increased
    incidence of hepatocellular enlargement in animals of each sex, and
    females in this group also had centrilobular hepatocyte vacuolation.
    The changes in the adrenals consisted of hypertrophy of the cells of
    the zona fasciculata in females. In testes, generalized vacuolation
    and hypertrophy of the interstitial cells were observed. In a number
    of females at the highest dose, luteinization of the interstitial
    cells of the ovary was noted. No treatment-related changes in tumour
    incidence were seen at the interim sacrifice.

         At terminal sacrifice, an analysis of organ weights (for most
    organs, both adjusted and absolute weights were reported) revealed
    increased liver weights in animals of each sex at the highest dose.
    Slight increases in thyroid weights (statistically significant in
    males) and kidney weights (statistically significant in females) were
    seen, and females also had decreased uterine weights. Macroscopic
    examination revealed a higher incidence of liver masses in animals of
    each sex at 4000 ppm and in males at 800 ppm in comparison with the
    control animals, and liver enlargement was seen in male and female
    mice at 4000 ppm. Further macroscopic changes at 4000 ppm included a
    decrease in the incidence of thickened uteri in females and increased
    incidences of thickened forestomachs in animals of each sex. Kidneys
    with irregular cortical scarring and altered shape were observed at a
    higher incidence in females at 4000 ppm. The testes had a high
    incidence of masses, and there was an increased prevalence of small
    testes at 4000 ppm. Microscopic examination revealed increased
    incidences of benign and malignant liver tumours in animals of each
    sex at the highest dose; the incidences in males were 14, 12, 20, and
    52% in the controls and in animals at 160, 800, and 4000 ppm,
    respectively, and those in females were 4, 4, 4, and 42%,
    respectively. The incidence in males at the highest dose clearly
    exceeded the historical incidence, reported to be 12-21%. In females,
    the historical control incidence was reported to be 0-2%. The liver
    tumour incidences in females in the control, 160-ppm, and 800-ppm
    groups were thus slightly higher than this range, and at the highest
    dose the incidence markedly exceeded it. The slight, non-dose-related
    increases in incidences observed in the concurrent controls and in
    animals at 160 and 800 ppm were not considered to be biologically
    relevant. When all four treatment groups were considered, the trend
    was significant, but when the highest dose was excluded from the
    analysis the trend was not significant. The ovaries of females at the
    highest dose showed an increased incidence of luteoma, with incidences
    of 0, 4, 2, and 10% at 0, 160, 800, and 4000 ppm, respectively. The
    historical control range was reported to be 0-8%. When all four groups
    were considered, the trend was significant, but when the group at the
    highest dose was excluded from the analysis it was not significant. No
    increased incidences were found of other tumour types, including
    testicular tumours.

         Non-neoplastic findings at terminal sacrifice found in various
    organs in animals at 800 or 4000 ppm confirmed the observations made
    at the interim sacrifice. In the liver, an increased incidence of
    enlarged eosinophilic and fat-containing hepatocytes was observed in
    animals of each sex at the highest dose, and centrilobular hepatocyte
    enlargement was seen in females at 800 ppm and in animals of each sex
    at 4000 ppm; pigmented macrophages and centrilobular hepatocyte
    vacuolation were found in males at 4000 ppm. The testes of males at
    800 and 4000 ppm showed an increased prevalence of generalized

    vacuolation and hypertrophy of the interstitial cells. In females at
    4000 ppm, luteinization, the absence of corpora lutea, and a decreased
    incidence of endometrial hyperplasia were reported. Males at the two
    higher doses showed hyperkeratosis of the non-glandular stomach.
    Haemosiderosis in the spleen, amyloidosis, and cortical scarring in
    the kidneys were reported in female mice at the highest dose. No
    treatment-related change in the adrenals was found at termination of
    the study. The NOAEL was 160 ppm, equal to 23 mg/kg bw per day in
    males and 27 mg/kg bw per day in females, based on microscopic
    changes, particularly in liver and testes at higher doses, and 800 ppm
    equal to 115 mg/kg bw per day in males and 138 mg/kg bw per day in
    females, for tumorigenicity in the liver and ovary (Chambers  et al.,
    1993).

    Rats

         Groups of 60 male and 60 female Crl:CD(SD)BR rats were fed diets
    containing iprodione (purity, 94.5-95.7%) at concentrations of 0, 150,
    300, or 1600 ppm. Satellite groups consisting of 12 animals of each
    sex at each dose were used for blood sampling at various intervals and
    for interim sacrifice after 52 weeks of treatment. The homogeneity and
    stability of the test compound in the diet was checked by chemical
    analysis. The treatment did not result in clinical signs, no
    dose-related increase in mortality was observed, and the survival rate
    of animals at the highest dose was greater than that of the other
    groups. Ophthalmic, haematological, and biochemical investigations and
    urinalysis performed several times during and at the end of the study
    revealed no consistent treatment-related changes. The body-weight gain
    of animals of each sex at the highest dose was lower than that of
    controls during various periods of treatment, resulting in a 5% lower
    overall body weight at the end of the study in females and 10% in
    males. The food consumption of males was slightly lower throughout the
    treatment period and that of females during some weeks of the study.

         At interim sacrifice, analysis of organ weights (for most organs,
    absolute, adjusted, and relative weights were reported) revealed a
    non-dose-related decrease in adrenal weights in females it all doses
    in comparison with controls. Since macroscopic examination revealed
    enlarged adrenals in females at 0, 150, and 300 ppm, the reduction in
    adrenal weights is probably due to an unusually high mean control
    value. Microscopic examination revealed a dose-related increase in the
    incidence of centrilobular hepatocyte enlargement at 300 and 1600 ppm
    in animals of each sex. Increased incidences of extramedullary
    haematopoiesis and haemosiderosis were seen in the spleens of females
    at the highest dose. All male and female rats at this dose showed
    enlargement of cells of the zona glomerulosa and vacuolation in the
    zona fasciculata and reticularis of the adrenals. No neoplastic
    findings were noted at interim sacrifice.

         At terminal sacrifice, increased liver weights were seen in males
    at 300 and 1600 ppm, and the latter also had increased testicular
    weights. The macroscopic changes included masses in the testes at the
    highest dose, increased incidences of small seminal vesicles,
    irregular cortical scarring in the kidneys of males, petechiae in the
    lungs, and an increased incidence of uterine thickening. Microscopic
    examination did not confirm the hepatocellular enlargement observed at
    the interim sacrifice. A significantly increased incidence of
    interstitial-cell tumours in the testis (25%) was seen in animals at
    1600 ppm; the incidence in the other groups was 5-12%, but with no
    clear dose-response relationship. The historical control range was
    reported to be 0-10%. Statistical analysis of the results revealed a
    highly significant trend when all four treatment groups were included.

         Non-neoplastic changes seen in the testes of males at 300 and
    1600 ppm consisted of an increased incidence of interstitial-cell
    hyperplasia. The authors reported that proliferative changes of the
    interstitial cells of the testis are age-related alterations which may
    have been associated with the increased survival of males at the
    highest dose. Further changes observed were atrophy of the
    seminiferous tubules, an increased incidence of reduced or absent
    spermatozoa, atrophy of the prostate, and reduced secretion or absence
    of secretory colloid in seminal vesicles, some of these changes
    occurring at > 300 ppm. In the kidneys, there was a dose-related
    increase in the incidence of basophilic, dilated cortical tubules
    containing eosinophilic colloid at 300 and 1600 ppm. This lesion is
    reported to be present in the early stage of progressive
    glomerulonephrosis and is known as an age-related finding; the
    incidence was not dose-related. Changes in the adrenals similar to
    those observed at interim sacrifice were seen, including enlargement
    of the cells of the zona glomerulosa and vacuolation in the zona
    fasciculata and zona reticularis, in male rats at 1600 ppm and to a
    lesser degree at 300 ppm. In females at the highest dose, a higher
    incidence of focal enlargement of cells of the zona glomerulosa was
    found in some animals. The NOAEL was 150 ppm, equal to 6 mg/kg bw per
    day in males and 8 mg/kg bw per day in females, based on changes in
    liver weight and histopathological findings in the liver, kidneys,
    adrenals, testes, and accessory glands at higher doses, and 300 ppm,
    equal to 12 mg/kg bw per day, for tumorigenicity in testicular
    interstitial cells (Chambers  et al., 1992).

    (b)  Special studies

    Mechanism of action

         Androgen receptors were isolated from the ventral prostate of
    previously untreated rats and incubated with a fixed concentration of
    a high-affinity radiolabelled standard ligand (tritiated methyl-
    trienolone) in the competitive binding assay  in vitro. In this
    assay, increasing concentrations of the potential competitors
    (dihydrotestosterone, testosterone, flutamide, hydroxyflutamide,

    iprodione, and seven iprodione metabolites) are added, leading to
    displacement of the radiolabelled ligand from the ligand-receptor
    complex. Free labelled ligand is then separated from the receptor-
    bound labeled ligand, which is quantified by scintillation counting.
    This allows calculation of the concentration of test substance that
    causes 50% displacement of the labelled ligand. The relative binding
    affinity (percentage of competitor in relation to standard
    concentrations at 50% displacement on the standard curve) is then
    calculated for each substance, making it possible to rank all of the
    substances tested. Flutamide was used as the reference compound
    because it and its metabolite hydroxyflutamide have known
    anti-androgenic activity, with relative binding affinities to the
    androgen receptor of 0.01% for flutamide and 0.16% for hydroxy-
    flutamide. As testosterone and dihydrotestosterone have relative
    binding affinities to prostatic tissue of 35 and 100%, respectively,
    flutamide and hydroxyflutamide are much less potent. Iprodione and
    most of its metabolites had relative binding affinities of < 0.001%,
    only one metabolite having a value of about 0.006%. The study
    therefore provided no strong evidence for competitive binding or
    inhibition of the androgen receptor by iprodione (Fail  et al.,
    1994).

         Another study was performed to investigate the potential
    inhibitory effects of iprodione and its metabolites on
    steroidogenesis, using a cultured porcine Leydig-cell model to detect
    a potential inhibitory effect on testosterone secretion. The
    testosterone concentrations were determined in a radioimmunoassay.
    Iprodione and two of its metabolites inhibited gonadotropin-stimulated
    testosterone secretion after an incubation time of three days; the
    other iprodione metabolites tested had no detectable effects.
    Inhibition by iprodione was also observed after exposure for only 3 h.
    These results suggest a competitive interaction with the biosynthetic
    and/or transport pathway of steroid hormones. Ketoconazole, a known
    inhibitor of steroidogenesis, had similar effects. The inhibitory
    effect of iprodione was completely reversible after its withdrawal
    from the culture medium. The absence of cytotoxicity and the recovery
    of steroidogenesis strongly suggest interference with biochemical
    steps involved in testosterone secretion. The precise location of the
    biochemical lesions is being investigated (Benahmed, 1995).

         Sex hormones were also measured  in vivo in male rats after
    treatment with iprodione. In a range-finding study, groups of six or
    seven rats were treated twice daily at 12-h intervals by gavage with
    total daily doses of 0, 120, 300, or 600 mg/kg bw iprodione or
    150 mg/kg bw per day flutamide for 15 days. An additional group was
    given single oral doses of 300 mg/kg bw iprodione per day. Luteinizing
    hormone, follicle-stimulating hormone, testosterone, and estradiol
    were determined in a blood sample taken at necropsy. No clinical signs
    were noted in treated animals. A decrease in body-weight gain and
    reduced food consumption were observed with 300 or 600 mg/kg bw
    iprodione or 150 mg/kg bw flutamide. Absolute and relative increases

    in liver weight were found in animals receiving flutamide and in those
    given 600 mg/kg bw iprodione. Flutamide treatment also caused
    reductions in absolute testicular weight and pronounced reductions in
    the weights of the epididymides, all accessory sex organs, the
    prostate, and the seminal vesicles. Treatment with 600 mg/kg bw
    iprodione resulted in less pronounced weight reductions in the same
    organs. Peripheral plasma hormones were also affected by treatment:
    Flutamide increased the levels of luteinizing hormone, follicle-
    stimulating hormone, testosterone, and estradiol markedly, whereas
    iprodione caused a less pronounced increase in luteinizing hormone
    concentration at 600 mg/kg bw and in follicle-stimulating hormone
    concentration at 300 and 600 mg/kg bw per day.

         In the main study, replicate groups of nine male rats were
    treated daily with doses of 0 or 600 mg/kg bw iprodione by gavage for
    30 days. A pair-fed group was also included. A positive control group
    was treated daily with 150 mg/kg bw flutamide. Five rats fed iprodione
    died during the experiment. Weight loss was observed during the first
    seven days of the study, and reduced body-weight gain was seen
    thereafter in all treated groups, corresponding to reduced food
    consumption. Changes in absolute and relative organ weights, similar
    to those observed in the 15-day pilot study, consisted of increased
    liver weights in rats treated with iprodione and flutamide and marked
    increases in adrenal weights, especially in those receiving iprodione.
    Flutamide-treated animals showed pronounced weight reductions in the
    epididymides, all accessory sex organs, prostate, and seminal
    vesicles; those treated with iprodione had similar but less pronounced
    reductions in these organs. The histopathological findings in animals
    treated with flutamide consisted of changes in the testes
    (degeneration of the seminiferous tubules, interstitial-cell
    hyperplasia), epididymides (presence of atypical luminal cells and
    hypospermia), seminal vesicles, and prostate (glandular atrophy); they
    also had liver-cell hypertrophy. In rats given iprodione, the
    histopathological lesions included an increased incidence of glandular
    atrophy of the seminal vesicles and prostate gland over that in the
    control group. The incidence was similar to that in the pair-fed
    group, but the severity of the atrophy in the seminal vesicles was
    more pronounced. Iprodione-treated rats had higher incidences of
    cytoplasmic vacuolization within the cortex of the adrenal glands and
    of centrilobular hepatocellular hypertrophy than those treated with
    flutamide. There were marked increases in the mean concentrations of
    luteinizing hormone, follicle-stimulating hormone, testosterone, and
    estradiol in flutamide-treated rats during and at the end of the
    study, whereas in iprodione-treated animals only the estradiol
    concentrations were increased. Subtle changes in the pattern of
    secretion of testosterone and luteinizing hormone were noted, e.g.
    prolongation of decreased basal concentrations of testosterone and
    increased pulse frequency in most concentration ranges of luteinizing
    hormone (Fail  et al., 1994).

    Comments

         In a study of carcinogenicity in mice, iprodione was administered
    over 99 weeks at dietary concentrations at 0, 160, 800, or 4000 ppm.
    At 800 ppm, non-neoplastic lesions were seen that included
    hepatocellular enlargement and hypertrophy of interstitial cells in
    the testis. At 4000 ppm, reduced body-weight gain, increased liver
    weights and increased levels of alanine and aspartate transaminases
    were observed. An increased incidence of liver tumours in animals of
    each sex and an increased incidence of luteomas of the ovaries were
    observed at 4000 ppm. The NOAEL was 160 ppm, equal to 23 mg/kg bw per
    day.

         In a 104-week study of carcinogenicity in rats, the dietary
    concentrations were 0, 150, 300, or 1600 ppm of iprodione. At 300 ppm,
    increased liver weights, changes in the male reproductive system
    including an increased incidence of interstitial-cell hyperplasia in
    the testis, and hypertrophic changes in the adrenals of male rats were
    observed. At 1600 ppm, reduced body-weight gain and an increased
    incidence of interstitial-cell tumours of the testis were noted. The
    NOAEL was 150 ppm, equal to 6 mg/kg bw per day.

         A number of studies have been conducted  in vitro and  in vivo
    to investigate the possible mechanism of tumorigenicity. Two studies
     in vitro to investigate the competitive binding capacity of
    iprodione to rat androgen receptors and possible inhibition of
    gonadotrophin-stimulated testosterone secretion in porcine Leydig
    cells indicated that iprodione may act by both mechanisms. The results
    of endocrine studies in rats  in vivo also provide some evidence that
    iprodione may interfere with androgen biosynthesis.

         An ADI of 0-0.06 mg/kg bw was established on the basis of an
    NOAEL of 6 mg/kg bw per day in the most recent two-year study of
    carcinogenicity in rats and a safety factor of 100.

    Toxicological evaluation

     Levels that cause no toxic effect

    Mouse:    160 ppm, equal to 23 mg/kg bw per day (99-week study of
              toxicity and carcinogenicity)

    Rat:      300 ppm in the diet, equal to 21 mg/kg bw per day
              (two-generation study of reproductive toxicity) 150 ppm
              equal to 6 mg/kg bw per day (104-week study of toxicity and
              carcinogenicity)

    Rabbit:   20 mg/kg bw per day (maternal toxicity in study of
              developmental toxicity)

    Dog:      400 ppm, equal to 18 mg/kg bw per day (one-year study of
              toxicity)

     Estimate of acceptable daily intake for humans

         0-0.06 mg/kg bw

     Information that would be useful for tcontinued evaluation of the
     compound

         Observations in humans

        Toxicological criteria for setting guidance values for dietary and non-dietary exposure to iprodione
                                                                                                                           

    Exposure                       Route, study type, species                Result, remarks
                                                                                                                           

    Short-term (1-7 days)          Dermal, irritation, rabbit                No irritation
                                   Eye, irritation, rabbit                   Eye irritation
                                   Inhalation 4-h, lethality, rat            LC50 > 3.29 mg/litre
                                   Oral, lethality, rat                      LD50 > 2000 mg/kg bw
                                   Dermal, lethality, rabbit                 LD50 > 2000 mg/kg bw
    Medium-term (1-26 weeks)       Repeated dietary, four weeks,             NOAEL = 115 mg/kg bw per day;
                                   mouse                                     gross liver changes
                                   Repeated dietary, three months,           NOAEL = 21 mg/kg bw per day;
                                   two-generation study of reproductive      microscopic adrenal hypertrophy
                                   toxicity, rat                             and reduced parental body weight
                                   Repeated dietary, developmental           NOAEL = 20 mg/kg bw per day for
                                   toxicity rabbit                           maternal toxicity; 60 mg/kg bw per
                                                                             day for embryotoxicity. No teratogenicity
    Long-term (> one year)         Repeated dietary, carcinogenicity,        NOAEL = 6 mg/kg bw per day for
                                   rat                                       increased liver weight; interstitial-cell
                                                                             hyperplasia in testis, adrenal hypertrophy;
                                                                             interstitial-cell tumours at highest dose
                                                                                                                           

        References

    Benahmed, M. (1995) Update on the effects of iprodione and its
         metabolites on testosterone secretion in cultured Leydig cells.
         Unpublished report prepared by the Instiut National de la Santé
         et de la Recherche Médicale, Lyon, France. Submitted to WHO by
         Rhône-Poulenc, Lyon, France.

    Chambers, P.R., Crook, D., Gibson, W.A., Gopinath, C. & Ames, S.A.
         (1992) Potential tumorigenic and toxic effects in prolonged
         dietary administration to rats. Unpublished report prepared by
         Rhône-Poulenc, Lyon, France. Submitted to WHO by Rhône-Poulenc,
         Lyon, France.

    Chambers, P.R., Crook, D., Gibson, W.A., Read, R.M. & Gopinath, C.
         (1993) Potential tumorigenic effects in prolonged dietary
         administration to mice. Unpublished report prepared by
         Rhône-Poulenc, Lyon, France. Submitted to WHO by Rhône-Poulenc,
         Lyon, France.

    Fail, P.A., Anderson, StA. & Pearce, S.W. (1994) Toxicity testing of a
         fungicide, iprodione, in adult male CD Sprague Dawley rats. Part
         I: Chemistry, binding and dose-range finding in adult male CD
         Sprague Dawley rats exposed to oral iprodione. Part II: 30-Day
         endocrine toxicology screen in adult male CD Sprague Dawley rats
         exposed to oral iprodione. Unpublished report prepared by
         Research Triangle Institute, Research Triangle Park, North
         Carolina, USA. Submitted to WHO by Rhône-Poulenc, Lyon, France.

    Hastings, S.E. & Huffman, K.W. (1975) Chronic toxicologic and
         carcinogenic study with RP 26019 in mice. Unpublished report from
         Rhodia Inc., Hess and Clark Division, No. SEH 75: 223. Submitted
         to WHO by Rhône-Poulenc, Lyon, France.

    Hastings, S.E., Winbigler, J.C. & Kiggins. E.M. (1976) Chronic
         toxicologic and carcinogenic study with RP 26019 in rats.
         Unpublished report from Rhodia, Inc, Hess and Clark Division,
         No. 76:57. Submitted to WHO by Rhône-Poulenc, Lyon, France.
    


    See Also:
       Toxicological Abbreviations
       Iprodione (Pesticide residues in food: 1977 evaluations)
       Iprodione (Pesticide residues in food: 1980 evaluations)
       Iprodione (Pesticide residues in food: 1992 evaluations Part II Toxicology)