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)