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.