α-n-butyl-α-(4-chlorophenyl)-1H-1,2,4-triazole-1-propanenitrile

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

A two-generation study in rats is available. The study is well-documented and include parameters comparable to OECD TG 416. Myclobutanil induced parental toxicity, embryotoxicity and had effects on fertility.

Link to relevant study records

Reference

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1983 to 1985

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Version / remarks:

083-4

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Remarks:

CRL:CD(SD)BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Kingston Facility, Stone Ridge, NY
- Housing: Throughout the study, the rats were housed individually (except during mating and lactation) in suspended stainless steel cages with wire mesh floors and fronts. Cages were suspended above absorbent paper liners which were changed 3 times per week.
- Diet: Purina Rodent Laboratory Chow (meal), ad libitum
- Water: Tap water, a t libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24°C
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Vehicle:

acetone

Details on exposure:

The test substance was administered in the diet which was available ad libitum. P1 parents received diet with myclobutanil from the time they were 6-8 weeks old until after their progeny were weaned. P2 animals had test substance available to them from the time they were weaned until all F2 progeny had been weaned.

Details on mating procedure:

Both the P1 and P2 animals were administered myclobutanil in the diet for a minimum of 8 weeks before mating. At the time of mating, mating pairs, consisting of one male and one female from the same treatment group, were placed together in a double sized stainless steel cage. Treatment was continued during cohabitation. Vaginal smears were made daily on all females in cohabitation. The presence of spermatozoa in a vaginal smear was considered evidence of mating and the date this occurred was designated Day 0 of gestation. The mated (presumed-pregnant) females were removed from the mating cage and placed in individual shoebox cages, containing Alpha-Dri® bedding. They were maintained in the shoebox cages throughout gestation and lactation.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The first time diets were prepared, samples from the top, middle, and bottom of each diet mix were collected. The samples were submitted for analysis of the test item to verify uniformity of mixing.

Analysis of the test item in dietary samples taken throughout the period of exposure (approximately 10 months) showed that the average concentration of active ingredient in the samples ranged from 102% to 112% of the theoretical concentration with an overall mean of 106%.

Duration of treatment / exposure:

Approximately 10 months

Frequency of treatment:

Continuous exposure via feed

Dose / conc.:

50 ppm

Remarks:

Low dose group, equivalent to approximately 4 mg/kg bw/day

Dose / conc.:

200 ppm

Remarks:

Mid dose group, equivalent to approximately 16 mg/kg bw/day

Dose / conc.:

1 000 ppm

Remarks:

High dose group, equivalent to approximately 80 mg/kg bw/day

No. of animals per sex per dose:

25

Control animals:

yes

Parental animals: Observations and examinations:

Each rat was observed daily for signs of ill health, reaction to treatment, morbidity, and mortality. Physical examinations were performed on all animals weekly until cohabitation.
The weight of each P1 rat was recorded one week prior to initiation of dosing and at weekly intervals thereafter until cohabitation.
The weight of each P2 rat was recorded at the time the animals were assigned to groups and at weekly intervals thereafter until cohabitation. Group mean body weights were calculated for each time period.
Presumed-pregnant females were weighed on nay 0, 6, 15, and 20 of gestation and lactating dams were weighed on Day 0, 4, 7, 14, and 21 of lactation. Group mean body weights were calculated for each of these time periods.

The quantity of feed consumed was determined weekly (beginning one week prior to compound administration for PI rats and at the time animals were assigned to groups for P2 rats) until cohabitation by weighing the feed cup before it was given to a rat and then reweighing the cup at the end of the week. The mean feed consumption for each group was calculated weekly and
expressed as g/animal/day.

Oestrous cyclicity (parental animals):

Time to mating (start of cohabitation to detection of sperm in a vaginal smear) was determined for males and females. The length of the gestation period (time from detection of sperm in a vaginal smear to day of delivery) was determined for each female.

Litter observations:

Each pup was observed daily for signs of ill health, reaction to treatment, morbidity, and mortality

Body weight for each pup was recorded on lactation day 0 (1), 4, 7, 14, and 21. Mean body weights were calculated as grand means for all pups in the group. All litters were adjusted to 10 pups (5 males and 5 females where possible) on day 4 of lactation. Pups to be left with the mother were randomly selected by first placing each pup in a numbered container and using a random numbers table to designate the animals (containers) to stay and those to be culled. If a litter did not have 5 pups of each sex the litter was still culled and the ratio of the sexes was made as close to 1:1 as possible. Litters with 10 pups or less were not adjusted.
Pups culled from a litter were killed and discarded without necropsy. Progeny counts and sex determination of pups were made Day 0, 4, 7, 14, and 21 of lactation. All pups were weaned when they were 22 days old.

Postmortem examinations (parental animals):

Necropsies were performed on all P1 and P2 animals found dead or killed in extremis. Following weaning of the F1b or F2b pups, all surviving P1 or P2 animals were anesthetized with ether, killed by exsanguination and necropsied. All organs, tissues, and body cavities (except the cranial vault) were examined and gross abnormalities recorded. The livers were weighed and the relative liver weight was calculated.

Histopathological examinations were performed on the following organs of both parental generations: gross lesions and liver.
For males: Epididymides, testes, prostate, seminal vesicles, coagulating glands;
For females: Ovaries, uterus, vagina, cervix.

Postmortem examinations (offspring):

Necropsies were performed on all F1 and F2 pups that were found dead after 14 days of age. Observations were recorded, but no tissues were saved from these animals.

Statistics:

Thirteen parameters were analyzed statistically:
1. Maternal weight during gestation (P1 and P2)
2. Maternal weight during lactation (P1 and P2)
3. Maternal and paternal liver weight at necropsy
4. Paternal weight (pre-mating)
5. Maternal weight (pre-mating)
6. Time to successful mating (P1 and P2 females)
7. Litter size (F1a, F1b, F2a, F2b)
8. Pup weights (F1a, F1b, F2a, F2b)
9. Neonatal survival (F1a, F1b, F2a, F2b)
10. Sex ratio of offspring
11. Time to successful mating (P1 and P2 males)
12. Incidence of stillborns (F1a, F1b, F2a, F2b)
13. Feed consumption

Feed consumption was analyzed by analysis of variance (ANOVA) and Duncan's multiple range test.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

One P0 male rat fed 200 ppm myclobutanil was killed moribund. The in-life signs observed were lethargy, ataxia, yellow staining of the ano-genital area, and black crusty material around both eyes and on the muzzle.
No other of toxicity were evident in P0 animals of either sex at dietary concentrations up to and including 1000 ppm Myclobutanil for 28 weeks.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One P0 male rat fed 200 ppm myclobutanil was killed moribund during week 23. Necropsy of this animal revealed a punctured palate due to malocclusion of its teeth. Two P0 female rats (1 at 0 ppm and 1 at 200 ppm) died during week 22, after the second mating of the P0 animals. The control (0 ppm) female died 22 days after showing positive signs of mating for the F1b pups. Necropsy of this animal revealed a 1mm cyst at one pole of the left kidney, very dark and reddened lungs, a mottled liver and 17 apparently normal fetuses in the uterus. The female at 200 ppm died during week 22, 23 days after showing positive signs of mating for the F1b pups. Necropsy revealed a pale liver and 18 apparently normal fetuses in the uterus. Neither of these females exhibited any in-life signs of toxicity. These deaths were not treatment-related.
No other deaths occurred in P0 animals of either sex at dietary concentrations up to and including 1000 ppm Myclobutanil for 28 weeks.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Mean body weights of male and female P0 rats at 50 and 200 ppm and females at 1000 ppm were comparable to those of the controls through 7 of 8 weeks of dosing prior to mating. Data for week 8 were lost due to a computer malfunction. The statistical differences between controls and
the 1000 ppm group (males) for days 7 and 14 did not persist and were judged to be unrelated to treatment.

Mean body weight of pregnant P0 females at doses of 50, 200 and 1000 ppm were comparable to those of the controls throughout the gestation and lactation periods following the first mating.
The statistically significant difference on Day 4 of lactation was judged to be a random event.

Mean body weight of P0 dams at 50, 200 and 1000 ppm was comparable to that of the controls throughout gestation. Mean body weight of P0 dams at 1000 ppm was significantly greater than controls on Day 15 of gestation, however, this difference was not attributable to treatment.
Mean body weights of the P0 dams during their lactation period were unaffected. The statistically
significant differences on Day 7 of lactation (200 ppm) and Day 21 of lactation (1000 ppm) were judged to be random events.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Feed consumption of male and female rats fed diets containing the test item at doses of 50 and 200 ppm was comparable to that of the controls through 7 of 8 weeks prior to mating. Data from Week 8 were lost due to a computer malfunction. At 1000 ppm feed consumption was significantly less than controls for weeks 1 through 4 for males and weeks 1 and 2 for females.
Feed consumption at 1000 ppm was still greater than 95% of controls and was judged to be due to lower palatability of the feed at this dose.

The mean compound intake for the P0 males during the 8 week pre-mating period ranged from 3.08 to 4.45 (mean: 3.67) mg/kg bw/day for the 50 ppm qroup, 12.1 to 17.3 (mean: 14.29) mg/kg bw/day for the 200 ppm group and 60.1 to 85.7 (mean: 70.69) mg/kg bw/day for the 1000 ppm group. For P0 females the mean compound intake ranged from 4.07 to 5.01 (mean: 4.42) mg/kg bw/day for the 50 ppm qroup, 15.7 to 19.8 (mean: 17.20) mg/kg bw/day for the 200 ppm group and 78.4 to 96.9 (85.9) mg/kg bw/day for the 1000 ppm group.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In the P0 rats the only treatment-related microscopic change was observed in the liver and consisted of hypertrophy of centrilobular hepatocytes in male and female rats fed 1000 ppm. No
treatment-related changes were observed in any of the organs or tissues examined from the 50 and 200 ppm dosage group P0 male and female rats.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

Treatment at 50 or 200 ppm myclobutanil did not affect the number of P0 females that had positive signs of mating (sperm in vaginal smear) or the number of mated females which delivered. The mean number of days needed for mating to occur and the mean length of gestation
were similar in controls and test item-treated groups. There was no suggestion of an adverse effect on fertility in male or female rats, at either dose in this mating.
In the 1000 ppm group the number of P0 females that had positive signs of mating (sperm in vaginal smear) was not reduced but the number of mated females which delivered was lower than controls. This difference was not statistically significant. The mean number of days needed for mating to occur and the mean length of gestation were similar to controls. These observations provided no evidence of an adverse effect on fertility in male or female rats in this mating.

For the second mating of the P0 animals, continued treatment at 50, 200 or 1000 ppm did not affect the number of P0 females that had positive signs of mating (sperm in vaginal smear) or the
number of mated females which delivered. The mean number of days needed for mating to occur and the mean length of gestation were similar in controls and test item-treated groups. There was no suggestion of an adverse effect on fertility in male or female rats in this mating.

Key result

Dose descriptor:

NOAEL

Remarks:

Fertility

Effect level:

70.69 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

14.29 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

histopathology: non-neoplastic

Critical effects observed:

yes

Lowest effective dose / conc.:

70.69 mg/kg bw/day (actual dose received)

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

presumably yes

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Only some animals that died showed the clinical signs ante mortem.
Male, 50 ppm: Lethargy, laboured breathing, brown-stained muzzle, black crusty material around both eyes and scant faecal droppings
Male, 200 ppm: Black crusty material on the muzzle and around both eyes; scant faecal droppings.
Female, 50 ppm: Ataxia, lethargy, gasping, brown-stained muzzle and emaciation.
Female, 50 ppm: Yellow-staining of anogenital area, dehydration, emaciation, brown-stained muzzle.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Two male rats (1 at 200 ppm and 1 at 50 ppm) and 4 female rats (1 at 1000 ppm, 1 at 200 ppm, 2 at 50 ppm) died. The deaths which occurred were distributed across all dose groups and were considered random events unrelated to treatment. There was no evidence of a dose-response.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weights of P1 females at 50, 200 and 1000 ppm were comparable to those of the controls throughout the 8 week dosing period prior to mating. Mean body weights of P2 males at 50 and 200 ppm were also comparable to those of the controls for the same 8 week dosing period. Mean body weight of male P1 rats fed myclobutanil at 1000 ppm was significantly decreased throughout the 8 weeks of dosing prior to mating. This lower mean weight was present at initiation of the dosing and was the result of lower body weights during the first 3 weeks of postnatal growth. The observed weight differences were ascribed to treatment.
Mean body weights of P1 dams at 50, 200 and 1000 ppm were comparable to controls throughout gestation and lactation.
Mean body weights of P1 dams at 50, 200, and 1000 ppm of myclobutanil were not different than controls throughout gestation and lactation. The statistically significant difference at 50 ppm on Day 6 of gestation was not attributable to treatment.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Mean body weights of P1 dams at 50, 200 and 1000 ppm were comparable to controls throughout gestation and lactation. Mean body weights of P1 dams at 50, 200 and 1000 ppm were not different than controls throughout gestation and lactation. The statistically significant difference at 50 ppm on Day 6 of gestation was not attributable to treatment.

Feed consumption of male and female rats fed diets containing the test item at doses of 50 and 200 ppm was comparable to that of the controls throughout the 8 week exposure period prior to mating. The statistically significant difference in feed consumption at 50 ppm in Week 5 was incidental and not related to treatment. At 1000 ppm statistical differences were noted. The difference in feed consumed between controls and the high dose was slight (approximately 1 g/day)t but is consistent and may reflect a modest degree of poor palatability.

The mean compound intake for the P1 males during the approximately 8 week pre-mating period ranged from 2.95 to 4.75 (mean: 3.64) mg/kg bw/day for the 50 ppm qroup, 12.2 to 19.7 (mean: 15.13) mg/kg bw/day for the 200 ppm qroup, and 61.2 to 100.5 (mean: 76.43) mg/kg bw/day for the 1000 ppm group. For P1 females the mean compound intake ranged from 3.66 to 4.86 (mean: 4.17) mg/kg bw/day for the 50 ppm group, 14.8 to 20.6 (mean: 17.5) mg/kg bw/day for the 200 ppm qroup, and 77.2 to 101.0 (88.04) mg/kg bw/day for the 1000 ppm group.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

When liver weights of P1 animals were statistically analyzed using ANCOVA (main effects of treatment and sex plus interaction with body weight as a covariate) a significant dose-effect was observed. Individual comparisons of treated groups to controls revealed that male rats fed 200 and 1000 ppm and female rats fed myclobutanil at 1000 ppm had significantly increased liver weights. Liver weights at 50 ppm were comparable to controls. Similar results were obtained when relative liver weights were analyzed by ANCOVA and Duncan's multiple range test. Absolute weights were not significantly different by this analysis.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Male rats in all treatment groups and controls had livers with prominent lobular architecture (not observed in females). Kidneys with slight to moderate dilation of the renal pelvis were observed in both sexes at 0, 50 and 200 ppm, but only males at 1000 ppm had this anatomical variation. The
frequency of these findings was not dose-related and the gross observations at necropsy were judged to be spontaneous occurrences. Other gross lesions of the non-reproductive organs of both sexes and the reproductive organs of the females were distributed as low incidences or they occurred sporadically in both treated and control groups and were not ascribed to treatment.
At 50 and 200 ppm gross findings in the reproductive organs of the males were distributed as single incidences which were not treatment-related. At 1000 ppm, 8 of 25 males had small and flaccid testes. These were ascribed to treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

No treatment-related microscopic changes were observed in males or females fed 50 ppm of myclobutanil. Centrilobular hepatocellular hypertrophy occurred in male rats at 200 and 1000 ppm and in females at 1000 ppm. An increased incidence of rats with multifocal or diffuse testicular atrophy was observed in male rats fed 1000 ppm of myclobutanil. Other associated and probably secondary lesions to the testicular atrophy in the high-dosage group male rats were decreased amounts of spermatozoa and the presence of necrotic spermatocytes and spermatids in the epididymal tubules and prostatic atrophy. Testicular atrophy, prostatic atrophy, and epididymal changes comparable to that seen in the high-dosage group male rats were seen in a few control, 50 and 200 ppm dosage group P1 male rats, but the incidence in the low- and mid-dosage groups was not considered to be different from the controls.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

Treatment with myclobutanil at 50, 200 or 1000 ppm did not affect the number of P1 females that had positive signs of mating (sperm in vaginal smear) or the number of mated females which delivered. The mean number of days needed for mating to occur and the mean length of gestation
were similar in controls and treated groups. There was no suggestion of an adverse effect on fertility in male or female rats in this mating.

For the second mating of the P1 animals, continued treatment with myclobutanil at 50 or 200 ppm did not affect the number of P1 females that had positive signs of mating (sperm in vaginal smear) or the number of mated females which delivered. The mean number of days needed for mating to occur and the mean length of gestation were similar in controls and treated groups. There was no suggestion of an adverse effect on fertility in male or female rats in this mating.

In the 1000 ppm group the number of P1 females that had positive signs of mating (sperm in vaginal smear) was slightly reduced and the number of mated females which delivered was also lower than controls. The mean number of days needed for mating to occur was higher than controls due to 2 mating pairs which took 16 and 20 days to mate. Only one other mating pair
required longer (8 days) than one estrus cycle (5 days) to mate. The mean length of gestation was similar to controls. These observations provided no clear evidence of an adverse effect on fertility in male or female rats in this mating.

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

15.13 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

histopathology: non-neoplastic

Key result

Dose descriptor:

NOAEL

Remarks:

fertility

Effect level:

15.13 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

76.43 mg/kg bw/day (actual dose received)

System:

male reproductive system

Organ:

testes

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

presumably yes

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

76.43 mg/kg bw/day (actual dose received)

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

presumably yes

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

F1a animals: The percentage of survival to Day 4 was not significantly different from controls at
50 and 200 ppm. At 1000 ppm the number of dead pups (12) was significantly higher than in controls (3). Survival to Day 4 was reduced as a consequence, but there was no increased mortality between Day 0 and 4 among pups born alive. The increase in dead pups was judged to be treatment-related.
F1b animals: At 200 and 1000 ppm the number of pups born dead (9 and 16, respectively) was elevated over controls (0). The increase in dead pups at 1000 ppm was judged to be treatment-related, the increase at 200 ppm was not as no other parameters were affected at this dosage, and a similar increase was seen in the F1a generation which was not significant. Survival to Day 4 was not different among groups for this mating due to deaths in the controls between birth and Day 4.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

F1a animals: Body weights for F1a pups in treated groups were not significantly different from controls at birth and rats exposed to 50 or 200 ppm had body weights which were comparable to control throughout the lactation period. At 1000 ppm, female pup weight was significantly less than controls on Day 4 of lactation. By Day 7 of lactation, pups of both sexes in the 1000 ppm group had lower body weights. The reduced weight gain was persistent through Day 21 of lactation and the difference in weight between pups treated with myclobutanil at 1000 ppm and controls was increased.
F1b animals: Body weights for F1b groups were not significantly different from controls at birth for either sex or combined sexes. By Day 4 of lactation, the pups in the 1000 ppm group had lower body weights. The reduced weight gain was persistent through Day 21 of lactation and the difference in weight between pups treated with myclobutanil at 1000 ppm and controls again increased. Rats exposed to 50 or 200 ppm had body weights which were comparable to controls throughout the lactation period.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Other effects:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

16 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

body weight and weight gain

Critical effects observed:

no

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

F2a animals: At 1000 ppm, the number of pups born dead (13) was higher than in controls (6). This response and the increase in dead pups were judged to be treatment-related. Survival to Day 4 was unaffected.
F2b animals: At 1000 ppm, the number of dead pups (12) was higher than in controls (5). The increase in dead pups was not statistically significant, but was judged to be treatment-related. Survival to Day 4 was significantly reduced at 1000 ppm only because of the pups born dead, but there was no increased mortality between Day 0 and 4 among pups born alive.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

F2a animals: Body weights for F2a pups were not significantly less than controls at birth for either sex or combined sexes. The statistically significant increases in Day 0 weights in treated groups were not attributed to treatment because the weights were within the normal range and a similar outcome was not observed in other matings. At 1000 ppm, reduced weight gain was readily apparent at Day 7 and persisted through Day 21 of lactation. The difference between pups treated with myclobutanil at 1000 ppm and controls increased as the pups matured. Rats exposed to 50 or 200 ppm had body weights which were comparable to controls throughout the lactation period.
F2b animals: Body weights for F2b pups were not significantly different from controls at birth for either sex or combined sexes. By Day 4 of lactation, the pups in the 1000 ppm group had lower body weights. The reduced weight gain was clearly present by Day 7 and persisted through Day 21 of lactation. The difference between pups treated with myclobutanil at 1000 ppm and controls again increased as the pups matured. Rats exposed to 50 or 200 ppm had body weights which were comparable to controls throughout the lactation period.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Other effects:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

16 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

body weight and weight gain

Critical effects observed:

no

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

80 mg/kg bw/day (actual dose received)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

presumably yes

Results are provided in the tables below.

Conclusions:

In conclusion, mycobutanil, at a dietary concentration of 1000 ppm (80 mg/kg bw/day), produced reproductive effects as evidenced by reduced numbers of females delivering litters and increased incidences of still-born pups; and general toxic effects, including testicular atrophy in P1 males and decreased weight gain of offspring during lactation. General, non-adverse systemic effects, but not reproductive effects, occurred at 200 ppm (16 mg/kg bw/day) as evidenced by increased liver weight in the P0 and P1 males, and hepatocellular hypertrophy in P1 males. These effects were detected at a dose level five times lower than the dose level at which reproductive effects were observed. The no-observed effect level (NOEL) for reproductive effects with exposure to myclobutanil was 200 ppm (16 mg/kg bw/day). The NOAEL for systemic toxicity was 200 ppm (16 mg/kg bw/day).

Executive summary:

Myclobutanil was administered in the diet to rats at concentrations of 50, 200, and 1,000 ppm (approximately 4, 16 and 80 mg/kg/day active ingredient) through two generations with two matings per generation. 

Parental toxicity: The data indicated that the NOEL in this study was 4 mg/kg bw/day and that 16 mg/kg bw/day was the minimum effect level based on slightly increased liver weight and histopathologically diagnosed liver hypertrophy.  However, these findings are not considered adverse.  At 80 mg/kg bw/day, the effect on the liver was more pronounced, body weight gain of male parents was reduced in the P1 generation and reproductive performance was affected.

At 80 mg/kg bw/day, there were adverse effects on mating performance.  In the P0/F1a mating, only 20/25 females delivered litters.  A similar response (20/25) was observed in the P1/F2a mating with a concomitant reduction in litter size.  In the P1/F2b mating, only 17/25 females delivered and there was a slight prolongation of mating time.  These reduced frequencies of deliveries were ascribed to treatment because there were no less than 22/25 females delivering litters in any other control or treated group in the first or second generation matings.

Testicular atrophy was observed at 80 mg/kg bw/day only in the P1 males while adverse reproductive effects occurred in the F1 and F2 generations.  Furthermore, other more pronounced effects were seen in P1 animals than in P0 animals.  For example, histologic changes of the liver were seen in P1 males, but not in P0 males at 16 mg/kg bw/day and reduced weight gain was seen in P1, but not P0, 80 mg/kg bw/day males.  The more pronounced responses in the P1 animals were attributed to the higher dosages the P1 animals received (in mg/kg bw/day) as a result of exposure to test diets during the nursing and early post-weaning periods.

Offspring:  The number of still-born pups was increased at 80 mg/kg bw/day in all matings and the body weight gain of pups born to dams exposed to 80 mg/kg bw/day was reduced in all matings.  The increase in dead-pups was treatment-related.  The decreased weight gains were attributed to general toxicity at 80 mg/kg bw/day.

These data supported a conclusion that, at 80 mg/kg bw/day, there were adverse reproductive effects, but only in the presence of a generalised toxic response and at 5 times the dose at which hepatic effects were first observed in the liver (16 mg/kg bw/day).

In conclusion, myclobutanil, at a dietary concentration of 80 mg/kg bw/day, produced reproductive effects as evidenced by reduced numbers of females delivering litters and increased incidences of still-born pups; and general toxic effects, including testicular atrophy in P1 males and decreased weight gain of offspring during lactation.  General, non-adverse systemic effects, but not reproductive effects, occurred at 16 mg/kg bw/day as evidenced by increased liver weight in the P0 and P1 males, and hepatocellular hypertrophy in P1 males.  These effects were detected at a dose level 5 times lower than the dose level at which reproductive effects were observed.  The no-observed effect level (NOEL) for reproductive effects with exposure to myclobutanil was 16 mg/kg bw/day.  The NOAEL for systemic toxicity was 16 mg/kg bw/day.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

16 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

The key study is reliable (Klimisch 1).

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Myclobutanil was administered in the diet to rats at concentrations of 50, 200, and 1,000 ppm (approximately 4, 16 and 80 mg/kg/day active ingredient) through two generations with two matings per generation. 

Parental toxicity: The data indicated that the NOEL in this study was 4 mg/kg bw/day and that 16 mg/kg bw/day was the minimum effect level based on slightly increased liver weight and histopathologically diagnosed liver hypertrophy.  However, these findings are not considered adverse.  At 80 mg/kg bw/day, the effect on the liver was more pronounced, body weight gain of male parents was reduced in the P1 generation and reproductive performance was affected.

At 80 mg/kg bw/day, there were adverse effects on mating performance.  In the P0/F1a mating, only 20/25 females delivered litters.  A similar response (20/25) was observed in the P1/F2a mating with a concomitant reduction in litter size.  In the P1/F2b mating, only 17/25 females delivered and there was a slight prolongation of mating time.  These reduced frequencies of deliveries were ascribed to treatment because there were no less than 22/25 females delivering litters in any other control or treated group in the first or second generation matings.

Testicular atrophy was observed at 80 mg/kg bw/day only in the P1 males while adverse reproductive effects occurred in the F1 and F2 generations.  Furthermore, other more pronounced effects were seen in P1 animals than in P0 animals.  For example, histologic changes of the liver were seen in P1 males, but not in P0 males at 16 mg/kg bw/day and reduced weight gain was seen in P1, but not P0, 80 mg/kg bw/day males.  The more pronounced responses in the P1 animals were attributed to the higher dosages the P1 animals received (in mg/kg bw/day) as a result of exposure to test diets during the nursing and early post-weaning periods.

Offspring:  The number of still-born pups was increased at 80 mg/kg bw/day in all matings and the body weight gain of pups born to dams exposed to 80 mg/kg bw/day was reduced in all matings.  The increase in dead-pups was treatment-related.  The decreased weight gains were attributed to general toxicity at 80 mg/kg bw/day.

These data supported a conclusion that, at 80 mg/kg bw/day, there were adverse reproductive effects, but only in the presence of a generalised toxic response and at 5 times the dose at which hepatic effects were first observed in the liver (16 mg/kg bw/day).

In conclusion, myclobutanil, at a dietary concentration of 80 mg/kg bw/day, produced reproductive effects as evidenced by reduced numbers of females delivering litters and increased incidences of still-born pups; and general toxic effects, including testicular atrophy in P1 males and decreased weight gain of offspring during lactation.  General, non-adverse systemic effects, but not reproductive effects, occurred at 16 mg/kg bw/day as evidenced by increased liver weight in the P0 and P1 males, and hepatocellular hypertrophy in P1 males.  These effects were detected at a dose level 5 times lower than the dose level at which reproductive effects were observed.  The no-observed effect level (NOEL) for reproductive effects with exposure to myclobutanil was 16 mg/kg bw/day.  The NOAEL for systemic toxicity was 16 mg/kg bw/day.

Effects on developmental toxicity

Description of key information

A developmental toxicity study in rats and a two-generation study in rats are available. Both studies are well-documented and include parameters comparable to guideline 414 and 416, respectively. Myclobutanil was considered to be not teratogen but showed embryotoxicity.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 May 1983 to 24 June 1983

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

no

Principles of method if other than guideline:

A teratology study was conducted with myclobutanil. The test material was
administered orally in corn oil to 25 presumed-pregnant rats in each of 5 groups. Doses of 0.0 (vehicle control), 31.3, 93.8, 312.6, and 468.9 mg/kg/day were administered on Day 6 through 15 of gestation. Dose volume was 10 mL/kg and the vehicle control group received only corn oil. Rats were observed daily for signs of ill health or reaction to treatment. Maternal body weights were recorded on Day 0, 6, 10, 13, 16, 18, and 20 of gestation. Dams were killed on Day 20 of gestation and the number of corpora lutea, resorption sites, and live or dead fetuses were recorded. Live fetuses from each 1 itter were weighed, sexed, and examined for external malformations and variations then prepared for either soft tissue examinations (1/3) or skeleton examinations (2/3).

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD(SD)BR

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratory (Kingston facility, Stone Ridge, NY.)
- Age at study initiation: Rats were approximately 85 days old when mated
- Weight at study initiation: Between 219 and 261 grams
- Housing: Rats were housed individually, except during cohabitation, in suspended wire mesh cages. Absorbent paper liners were placed beneath the cages and changed 3 times per week, except during mating when they were changed daily.
- Diet: Pelletized Purina Laboratory Rodent Chow, ad libitum
- Water: Tap water, ad libitum
- Acclimation period: 20 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 to 26°C
- Humidity (%): 44 to 72%

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

A dose volume of 10.0 mL/kg was used and the volume administered was adjusted based on the most recent body weight of the dam.

The entire sample of the test item was heated at 55°C until melted, then divided into aliquots, each containing enough test material for one day of dosinq, and each aliquot was labeled with the day it was to be used. Each day an aliquot was heated at 55°C, melted and the appropriate amount of the test material was weighed into a volumetric flask. This was mixed with preheated corn oil (55°C) on a magnetic stirring plate. The prepared solutions were cooled to room temperature, volume was adjusted to 100 mL with corn oil, and stored at room temperature until used. All prepared solutions were stirred continuously on a magnetic stirring plate during dosing.
The dose concentrations were 3.13, 9.38. 31.3 and 46.9 mg/mL.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical verification of dosing was performed on day 1, day 8 and on the final day of dosing.

Details on mating procedure:

Female rats were cohabited with males (1:1) for no longer than 5 days. Vaginal smears were taken daily and examined for the presence of sperm. Sperm-positive females were presumed to be pregnant, and the day on which sperm was found was considered Day 0 of gestation.

Duration of treatment / exposure:

10 days (Gestation Day 6 - 15)

Frequency of treatment:

Daily, as close to the same time each day as was practical

Duration of test:

Rats were sacrificed on gestational day 20

Dose / conc.:

31.3 mg/kg bw/day

Dose / conc.:

93.8 mg/kg bw/day

Dose / conc.:

312.6 mg/kg bw/day

Dose / conc.:

468.9 mg/kg bw/day

No. of animals per sex per dose:

25 females

Control animals:

yes, concurrent vehicle

Maternal examinations:

Maternal tissues with gross lesions were saved in 10% neutral buffered formalin and the remainder of the carcasses were discarded. These tissues were not processed for histopathological examination.

Ovaries and uterine content:

On Day 20 of gestation, each female was killed, the uterus and ovaries were exposed, and the number and uterine position of viable and non-viable foetuses, early resorptions and late resorptions were recorded. Corpora lutea on each ovary were counted.

Fetal examinations:

Each live foetus was weighed, examined for external malformations and variations, externally sexed and placed in appropriate fixative. Two-thirds of the foetuses from each litter were eviscerated, fixed and stained with Alizarin Red for examination of skeletal variations and malformations. The remaining foetuses/litter were placed in Bouin’s fixative, sectioned and examined for visceral variations and malformations

Statistics:

All data were visually examined for evidence of treatment-related trends. Parameters which were suggestive of an effect were statistically analyzed. Normally distributed data were analyzed by weighted analyses of variance. Proportional data were analyzed by the Jonkheere test for dose-related trends and the beta-binomial model or Fisher's exact test for group comparisons. Statistical analysis of some categories of variations was inappropriate due to the small number of observations, but each category was included as a component in the statistical analysis of all variations combined. A similar approach was used for malformations.

Indices:

Fertility Index = No. pregnant/No. presumed-pregnant;
Implantation Efficiency = No. Implantation Sites/No. Corpora Lutea;
Viability Index = No. Viable Fetuses/No. Implantation Sites

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Only the 312.6 and 468.9 mg/kg bw/day groups showed signs of ill health or reaction to treatment. At 312.6 mg/kg bw/day, rough hair coat, desquamation, and salivation were observed in 4, 1 and 3 rats, respectively. At 468.9 mg/kg bw/day, these same signs were observed in 8, 4 and 4 rats, respectively. In addition, red exudate from the mouth, red exudate from the vagina, scant faeces, and soft faeces were observed in 10, 1, 3 and 1 rats, respectively, at 468.9 mg/kg bw/day. The three incidences of urine stained urogenital area in the control and 312.6 mg/kg bw/day groups were not considered treatment related, but the 17 occurrences of the same sign at 468.9 mg/kg bw/day were judged to be signs of unkempt appearance related to exposure to myclobutanil.
Alopecia occurred in all groups and is a normal physiological response in rats progressing through gestation. No other signs occurred.

Mortality:

no mortality observed

Description (incidence):

All rats survived until scheduled necropsy.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Maternal body weights during gestation were statistically analyzed using adjusted mean weights. The adjustment was for maternal weight on Day 6, the final pre-treatment weight. All groups were compared to controls. There was no effect on maternal body weight at 31.3, 93.8 or 312.6 mg/kg bw/day. The significantly greater weight of dams at 31.3 mg/kg bw/day on Day 13 of gestation was considered an anomalous result. At 468.9 mg/kg bw/day, a statistically significantly lower maternal weight was observed on Day 10 of gestation, but not thereafter. Graphic representation of the maternal weights and weight gains suggested that the 468.9 mg/kg bw/day group incurred a slight weight decrement, following the initiation of dosing, from which the dams recovered.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Observations on pregnant does at necropsy indicated single occurrences of anatomical abnormalities in all groups, none of which were treatment-related. Reddened lungs, hydrocele on the ovary, hydrometra, and dilated kidney each occurred in a single animal in the treated groups and one control rat had reddened lungs. None of these findings were deemed to be related to treatment with the test item.

Key result

Dose descriptor:

NOAEL

Effect level:

93.8 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

clinical signs

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Body weights of male and female foetuses in the 31.3, 93.8, 312.6 and 468.9 mg/kg bw/day groups were not affected by exposure to myclobutanil.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

Sex ratio of fetuses was not affected by exposure to myclobutanil at any dose.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

The number of viable foetuses per litter (litter size) was significantly less than controls at 31.3, 93.8, 312.6 and 468.9 mg/kg bw/day myclobutanil. This effect on litter size was due primarily to the high number of foetuses per litter in the control group and the statistically significant differences in litter size were not attributed to exposure to myclobutanil. Three factors were considered. First, the number of corpora lutea and implantations in the control group was high, thus creating an increased probability that the litters would be large. Secondly, the viability of the implants was also good in the controls, thus allowing more embryos to mature.
Thirdly, the type of statistical analysis used for evaluating litter size was different than that used for other parameters reflecting gestational success.
If the frequencies of ovulation and implantation in controls had been separately compared to the frequencies in treated litters, artifactual differences would have been observed. Herein lies the importance of using statistical analyses based on proportions (i.e. Implantation Efficiency). Even when the number of corpora lutea was high (controls), the proportion of those ova which implanted was the same across groups and no statistical differences were observed. This outcome is the correct one for the analysis of ovulation and implantation.
Litter size is not proportional data, thus the number of viable foetuses in a litter is without a frame of reference (i.e. how many could there have been). The significant differences between control litters and litters exposed to myclobutanil were an artifactual result in this case. Control litters had a mean of 15.3 foetuses per litter, which is above the historical control mean of 12 to 13 rats (Shepard, 1980), and above the mean from historical control data in this laboratory. The 31.3, 93.8, 312.6 and 468.9 mg/kg bw/day groups had litters containing 13.5, 13.3, 13.2 and 13.1 viable foetuses, respectively. All of these outcomes were well within the range of historical controls. A conclusion that myclobutanil had no effect on litter size was warranted, but a conclusion that myclobutanil had no effect on foetal survival was not warranted.
There were no significant differences between fetal weights in control litters and litters from the 31.3, 93.8, 312.6, or 468.9 mg/kg groups.

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were 5 types of external malformations observed in this study, agnathia, craniorachischisis, microphthalmia, misplaced pinna, and omphalocele. There were no malformations observed in controls in the 312.6 mg/kg bw/day group. At 31.3 mg/kg bw/day, a single incidence of microphthalmia occurred; agnathia, misplaced pinna, and omphalocele occurred as single occurrences at 93.8 mg/kg bw/day. A single foetus at 468.9 mg/kg bw/day had craniorachischisis. These malformations were not related to each other, showed no dose-related trend, and were not treatment-related. Statistical analysis of single categories of external malformations was deemed inappropriate due to the small number of malformations observed, but these malformations were included as the external malformation component of the statistical analysis of all malformations combined.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Skeletal malformations observed in the study were of 2 types. The anomalies included an atlo-occipital anomaly (atlas present as a left dorsal arch only, with the occipital bone malformed on the left) at 31.3 mg/kg bw/day and an anomaly of a vertebral centra (thoracic vertebrae 9 (T9) and 10 (T10) were bipartite with the left side of T9 fused to the right side of T10) at 468.9 mg/kg bw/day. These malformations were not related, showed no dose-related trend, and were not treatment-related. Statistical analysis of single categories of skeletal malformations was deemed inappropriate due to the small number of malformations observed, but these malformations were included as the skeletal malformation component of the statistical analysis of all malformations combined.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Soft Tissue Malformations:
There were 7 types of soft tissue malformations observed, hydrocephaly, open eyelids, anophthalmia, interventricular septal defect, retroesophageal aortic arch, transposition of the great vessels and single atria with single atrioventricular valve. All of these malformations except hydrocephaly were confined to 2 foetuses in the 93.8 mg/kg bw/day group and were not treatment-related.
Two hydrocephalic foetuses appeared in the 468.9 mg/kg bw/day group and were judged to be random occurrences. Statistical analysis of single categories of soft tissue malformations was deemed inappropriate due to the small number of malformations observed, but these malformations were included as the soft tissue malformation component of the statistical analysis of all malformations combined.

All Malformations Combined:
When all malformations were considered together a marginally significant dose-related trend was noted. The control incidence was zero and required the use of Fisher’s exact test for analysis of the data. While the analysis indicated a significant increase in malformations (foetuses only) at the 468.9 mg/kg bw/day dose, it was not regarded as toxicologically significant.

Key result

Dose descriptor:

NOAEL

Effect level:

31.3 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

Abnormalities:

no effects observed

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

93.8 mg/kg bw/day

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

yes

Results are provided in the tables below.

Conclusions:

A prenatal rat study was performed in rats. The overall no observed effect level (NOEL) was 31.3 mg/kg bw/day related to decreased viability at 93.8 mg/kg bw/day and above. Maternal toxicity with myclobutanil occurred at 312.6 mg/kg bw/day, and above, any fetotoxicity occurred only at maternal toxic levels.

Executive summary:

A teratology study was conducted with myclobutanil. The test material was administered orally in corn oil to 25 presumed-pregnant rats in each of 5 groups. Doses of 0.0 (vehicle control), 31.3, 93.8, 312.6, and 468.9 mg/kg bw/day were administered on Day 6 through 15 of gestation. Dose volume was 10 mL/kg bw and the vehicle control group received only corn oil. Rats were observed daily for signs of ill health or reaction to treatment. Maternal body weights were recorded on Day 0, 6, 10, 13, 16, 18, and 20 of gestation. Dams were killed on Day 20 of gestation and the number of corpora lutea, resorption sites, and live or dead fetuses were recorded. Live fetuses from each 1 itter were weighed, sexed, and examined for external malformations and variations then prepared for either soft tissue examinations (1/3) or skeleton examinations (2/3).

Myclobutanil caused no deaths at any dose. Clinical signs of toxicity were not observed at 31.3 or 93.8 mg/kg bw/day. At 312.6 and 468.9 mg/kg bw/day compound-related signs included rough hair coat, desquamation, and salivation. At 468.9 mg/kg bw/day additional signs of reduced fecal output, red exudate from the mouth, and red exudate from the vagina were recorded. Necropsy observations on females at all doses were not remarkable. Maternal body weight during gestation was significantly depressed at 468.9 mg/kg bw/day only on Day 10 of gestation and returned to control levels by Day 13. Corpora lutea per litter and implantation sites per litter were high in the control group, but Implantation Efficiency (No. Implantation Sites/No. Corpora Lutea) was within normal limits for all groups. Fetal body weight, and fetal sex ratio were unaffected by treatment with myclobutanil. Viability Index (No. Viable Fetuses/No. Implantation Sites) was reduced at 93.8, 312.6, and 468.9 mg/kg bw/day with a concommitant increase in resorptions per litter and litters with more than 2 resorptions. A significant reduction in live fetuses per litter (litter size) at all doses was ascribed to the unusually large control litters. Incidences of 7th cervical and 14th rudimentary ribs (skeletal variations) were increased at 312.6 and 468.9 mg/kg bw/day and a significant increase in developmental variations was also observed at these doses. Those incidences indicate foetotoxicity. Myclobutanil was not teratogenic at any dose.

The overall no observed effect level (NOEL) was 31.3 mg/kg bw/day when administered to rats on Day 6 through 15 of gestation in a corn oil vehicle related to decreased viability at 93.8 mg/kg bw/day and above. Maternal toxicity with myclobutanil occurred at 312.6 mg/kg bw/day, and above, which indicated that fetotoxicity occurred only when maternal toxicity was also produced.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

31.3 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The key study is reliable (Klimisch 1).

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

A teratology study was conducted with myclobutanil. The test material was administered orally in corn oil to 25 presumed-pregnant rats in each of 5 groups. Doses of 0.0 (vehicle control), 31.3, 93.8, 312.6, and 468.9 mg/kg bw/day were administered on Day 6 through 15 of gestation. Dose volume was 10 mL/kg bw and the vehicle control group received only corn oil. Rats were observed daily for signs of ill health or reaction to treatment. Maternal body weights were recorded on Day 0, 6, 10, 13, 16, 18, and 20 of gestation. Dams were killed on Day 20 of gestation and the number of corpora lutea, resorption sites, and live or dead fetuses were recorded. Live fetuses from each 1 itter were weighed, sexed, and examined for external malformations and variations then prepared for either soft tissue examinations (1/3) or skeleton examinations (2/3).

Myclobutanil caused no deaths at any dose. Clinical signs of toxicity were not observed at 31.3 or 93.8 mg/kg bw/day. At 312.6 and 468.9 mg/kg bw/day compound-related signs included rough hair coat, desquamation, and salivation. At 468.9 mg/kg bw/day additional signs of reduced fecal output, red exudate from the mouth, and red exudate from the vagina were recorded. Necropsy observations on females at all doses were not remarkable. Maternal body weight during gestation was significantly depressed at 468.9 mg/kg bw/day only on Day 10 of gestation and returned to control levels by Day 13. Corpora lutea per litter and implantation sites per litter were high in the control group, but Implantation Efficiency (No. Implantation Sites/No. Corpora Lutea) was within normal limits for all groups. Fetal body weight, and fetal sex ratio were unaffected by treatment with myclobutanil. Viability Index (No. Viable Fetuses/No. Implantation Sites) was reduced at 93.8, 312.6, and 468.9 mg/kg bw/day with a concommitant increase in resorptions per litter and litters with more than 2 resorptions. A significant reduction in live fetuses per litter (litter size) at all doses was ascribed to the unusually large control litters. Incidences of 7th cervical and 14th rudimentary ribs (skeletal variations) were increased at 312.6 and 468.9 mg/kg bw/day and a significant increase in developmental variations was also observed at these doses. Those incidences indicate foetotoxicity. Myclobutanil was not teratogenic at any dose.

The overall no observed effect level (NOEL) was 31.3 mg/kg bw/day when administered to rats on Day 6 through 15 of gestation in a corn oil vehicle related to decreased viability at 93.8 mg/kg bw/day and above. Maternal toxicity with myclobutanil occurred at 312.6 mg/kg bw/day, and above, which indicated that fetotoxicity occurred only when maternal toxicity was also produced.

Furthermore, a two generation study is available with myclobutanil.

Myclobutanil was administered in the diet to rats at concentrations of 50, 200, and 1,000 ppm (approximately 4, 16 and 80 mg/kg/day active ingredient) through two generations with two matings per generation. 

Parental toxicity: The data indicated that the NOEL in this study was 4 mg/kg bw/day and that 16 mg/kg bw/day was the minimum effect level based on slightly increased liver weight and histopathologically diagnosed liver hypertrophy.  However, these findings are not considered adverse.  At 80 mg/kg bw/day, the effect on the liver was more pronounced, body weight gain of male parents was reduced in the P1 generation and reproductive performance was affected.

At 80 mg/kg bw/day, there were adverse effects on mating performance.  In the P0/F1a mating, only 20/25 females delivered litters.  A similar response (20/25) was observed in the P1/F2a mating with a concomitant reduction in litter size.  In the P1/F2b mating, only 17/25 females delivered and there was a slight prolongation of mating time.  These reduced frequencies of deliveries were ascribed to treatment because there were no less than 22/25 females delivering litters in any other control or treated group in the first or second generation matings.

Testicular atrophy was observed at 80 mg/kg bw/day only in the P1 males while adverse reproductive effects occurred in the F1 and F2 generations.  Furthermore, other more pronounced effects were seen in P1 animals than in P0 animals.  For example, histologic changes of the liver were seen in P1 males, but not in P0 males at 16 mg/kg bw/day and reduced weight gain was seen in P1, but not P0, 80 mg/kg bw/day males.  The more pronounced responses in the P1 animals were attributed to the higher dosages the P1 animals received (in mg/kg bw/day) as a result of exposure to test diets during the nursing and early post-weaning periods.

Offspring:  The number of still-born pups was increased at 80 mg/kg bw/day in all matings and the body weight gain of pups born to dams exposed to 80 mg/kg bw/day was reduced in all matings.  The increase in dead-pups was treatment-related.  The decreased weight gains were attributed to general toxicity at 80 mg/kg bw/day.

These data supported a conclusion that, at 80 mg/kg bw/day, there were adverse reproductive effects, but only in the presence of a generalised toxic response and at 5 times the dose at which hepatic effects were first observed in the liver (16 mg/kg bw/day).

In conclusion, myclobutanil, at a dietary concentration of 80 mg/kg bw/day, produced reproductive effects as evidenced by reduced numbers of females delivering litters and increased incidences of still-born pups; and general toxic effects, including testicular atrophy in P1 males and decreased weight gain of offspring during lactation.  General, non-adverse systemic effects, but not reproductive effects, occurred at 16 mg/kg bw/day as evidenced by increased liver weight in the P0 and P1 males, and hepatocellular hypertrophy in P1 males.  These effects were detected at a dose level 5 times lower than the dose level at which reproductive effects were observed.  The no-observed effect level (NOEL) for reproductive effects with exposure to myclobutanil was 16 mg/kg bw/day.  The NOAEL for systemic toxicity was 16 mg/kg bw/day.

Justification for classification or non-classification

Based on the available data and a harmonised classification for Myclobutanil a classification for reproductive toxicity (Cat. 2, H361d) according to the Classification, Labelling and Packaging (CLP) Regulation ((EC) No 1272/2008) is warranted.