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

Developmental toxicity / teratogenicity

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1984

Report date:

1984

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

410-400-0

EC Name:

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

Cas Number:

88671-89-0

Molecular formula:

C15H17ClN4

IUPAC Name:

2-(4-chlorophenyl)-2-[(1H-1,2,4-triazol-1-yl)methyl]hexanenitrile

Test material form:

liquid: viscous

Remarks:

brown oil

Test animals

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%

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

31.3 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

93.8 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

312.6 mg/kg bw/day

Doses / concentrations 4

Dose / conc.:

468.9 mg/kg bw/day

No. of animals per sex per dose:

25 females

Control animals:

yes, concurrent vehicle

Examinations

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

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

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.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

93.8 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

clinical signs

Maternal abnormalities

Abnormalities:

no effects observed

Results (fetuses)

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.

Effect levels (fetuses)

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

Fetal abnormalities

Abnormalities:

no effects observed

Overall developmental toxicity

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

Any other information on results incl. tables

Results are provided in the tables below.

Applicant's summary and conclusion

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.