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Diss Factsheets

Administrative data

Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
other information
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: HJ/T 153-2004: 416 Two-Generation Reproduction Toxicity Test
Version / remarks:
Second Edition, 2013
GLP compliance:
yes
Remarks:
The test complied with the Principles of Good Laboratory Practices (GLP) of the Certification and Accreditation Administration of the People’s Republic of China (2013 revised edition).
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
2-nitro-4-(trifluoromethyl)benzonitrile
EC Number:
212-298-1
EC Name:
2-nitro-4-(trifluoromethyl)benzonitrile
Cas Number:
778-94-9
Molecular formula:
C8H3F3N2O2
IUPAC Name:
2-nitro-4-(trifluoromethyl)benzonitrile

Test animals

Species:
rat
Strain:
Sprague-Dawley
Remarks:
SPF grade
Sex:
male/female

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
The test sample was administered every day at around the same time. After 10 weeks of continuous exposure, the F0 and F1 generations were mated, and exposure continued in the mating period. Parent generation female rats were exposed throughout pregnancy until weaning of the offspring rats. The offspring generation was only exposed to the test sample indirectly through breast milk. In the F1 generation, exposure started after weaning.
Doses / concentrationsopen allclose all
Dose / conc.:
2.5 mg/kg bw/day
Dose / conc.:
10 mg/kg bw/day
Dose / conc.:
40 mg/kg bw/day
No. of animals per sex per dose:
24
Control animals:
yes, concurrent vehicle
Positive control:
no

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In male rats exposed to 40 mg/kg bw/day, at 51 - 65 days body weight declined, and it was statistically significant (P ≤ 0.05, P ≤ 0.01). The female animals did not show any statistical significant changes in body weight when compared to controls.
Compared to the control group, in F0 generation rats exposed for 10 weeks, in female rats exposed to 40 mg/kg bw/day, at 51 and 65 days weight gain was reduced, and it was statistically significant (P ≤ 0.05, P ≤ 0.01). Although there was no statistical difference compared to F0 generation female rats at the corresponding number of weeks, in the late stages of exposure female rat weight gain slowed – consistent with the slowed weight gain in male rats in the same time period. The impact of the test sample was not eliminated.
Compared to the control group, in the F0 generation exposed for 10 weeks, weight gain was reduced in male rats exposed to 2.5 mg/kg bw/day at day 30 and it was statistically significant (P ≤ 0.01). There was no dose-response relationship, and there was no biological significance. Weight gain was reduced in male rats exposed to 40 mg/kg bw/day at days 30, 51, and 65, and it was statistically significant (P ≤ 0.05, P ≤ 0.001).
Compared to the control group, for F0 generation mother rats in all dose groups there was no statistical significance in body weight during pregnancy. Compared to the control group, for F0 generation mother rats in all dose groups there was no statistical significance in body weight during the nursing period.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Compared to the control group, F0 generation female rats exposed to 40 mg/kg bw/day had elevated food intake at day 16. In all dose groups at days 51 and 58 food intake was reduced, which was statistically significant (P ≤ 0.05, P ≤ 0.01, P ≤ 0.001). There was no dose-response relationship and no biological significance.
Compared to the control group, F0 generation male rats exposed to 40 mg/kg bw/day had elevated food intake at day 30, those exposed to 10 and 40 mg/kg bw/day had reduced food intake at day 51, and those exposed to 2.5 mg/kg bw/day had reduced food intake at days 58 and 65; which were statistically significant (P ≤ 0.01, P ≤ 0.001). There was no dose-response relationship and no biological significance.
Compared to the control group, for F0 generation mother rats in all dose groups there was no statistical significance in food intake during pregnancy.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
Compared to the control group, F0 generation female rats exposed to 40 mg/kg bw/day had reduced food utilisation rates at day 65, which was statistically significant (P ≤ 0.05). The analysis was that it was the result of the corresponding reduction in weight gain.
Compared to the control group, F0 generation male rats exposed to 2.5 and 40 mg/kg bw/day had reduced food utilisation rates at day 30; they were statistically significant (P ≤ 0.01, P ≤ 0.001). There was no dose-response relationship and no biological significance.
Compared to the control group, F0 generation male rats exposed to 40 mg/kg bw/day had reduced food utilisation rates at day 65, which was statistically significant (P ≤ 0.001). The analysis was that it was the result of the reductions in weight gain and body weight in F0 generation male rats at day 65. As food intake quantity declined, food utilisation rates were affected, but the root cause was the suppression of weight gain.
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, non-treatment-related
Description (incidence and severity):
All pathologies after histopathological examination of organs were counted and differences between the high dose group and the control group were not significant and were not statistically significant (P > 0.05). All pathologies were spontaneous or sporadic changes at this age, and they were not related to the test sample. Among the F0 generation there were 4 males and 4 females that did not conceive and did not develop. After histopathological examination, there were no findings of histopathological changes associated with the animals’ lack of conception and lack of development, thus no cause was found for the lack of conception and lack of development.
Histopathological findings: neoplastic:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
PREGNANCY DAYS
Compared to the control group, the number of pregnancy days was increased in F0 parent generation animals at 40 mg/kg bw/day, which was statistically significant (P ≤ 0.05). Combined with comprehensive analysis of other reproduction indicators, there was no biological significance.
F0 generation 4 animals were considered successfully mated, but uterine implantation count was 0.

NUMBER OF CORPORA LUTEA
There was no statistically significant effect on the number of implantations and number of corpora lutea.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
general toxicity
Effect level:
10 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
organ weights and organ / body weight ratios
Dose descriptor:
NOAEL
Remarks:
reproductive toxicity
Effect level:
40 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other:

Target system / organ toxicity (P0)

Critical effects observed:
yes
Lowest effective dose / conc.:
40 mg/kg bw/day (actual dose received)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:
no effects observed
Mortality:
mortality observed, non-treatment-related
Description (incidence):
At 38 weeks of exposure, one animal died unexpectedly after the offspring rats were weaned; the cause of death was not discovered.
Apart from the above-described animal, no anomalies were seen in other animals during the test.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In male rats exposed to 40 mg/kg bw/day, at 37 - 65 days body weight declined, and it was statistically significant (P ≤ 0.05, P ≤ 0.01, P ≤ 0.001).
In male rats exposed to 10 mg/kg bw/day at day 58 weight gain was reduced and in those exposed to 40 mg/kg bw/day at days 2, 16, 23, 51, and 58 weight gain was reduced, which were statistically significant (P ≤ 0.05, P ≤ 0.01, P ≤ 0.001).
No effect on body weight and body weight gain was noted in females.
Compared to the control group, for F1 generation mother rats in all dose groups there was no statistical significance in body weight during pregnancy.
Compared to the control group, for F1 generation mother rats in all dose groups there was no statistical significance in body weight during the nursing period.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Compared to the control group, F1 generation female rats exposed to 2.5 mg/kg bw/day had reduced food intake at days 9, 51, and 58, those exposed to 10 mg/kg bw/day had reduced food intake at days 9, 16, 44, 51, and 58, which were statistically significant (P ≤ 0.05, P ≤ 0.01, P ≤ 0.001). There was no dose-response relationship and no biological significance.
Compared to the control group, F1 generation male rats in all dose groups had reduced food intake at day 23 of exposure, in those exposed to 40 mg/kg bw/day food intake was reduced at day 30, which were statistically significant (P ≤ 0.05, P ≤ 0.01, P ≤ 0.001).
Compared to the control group, for F1 generation mother rats in all dose groups there was no statistical significance in food intake during pregnancy.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
Compared to the control group, in F1 generation female rats exposed for 10 weeks, there was no statistical significance in food utilisation rates for all dose groups.
Compared to the control group, F1 generation male rats exposed to 40 mg/kg bw/day had reduced food utilisation rates at days 2 and 51, which were statistically significant (P ≤ 0.01, P ≤ 0.001). The cause was the corresponding reduction in weight gain and it was biologically significant.
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):
Compared to the control group, in F1 parent generation female rats exposed to 40 mg/kg bw/day, liver organ coefficient, left kidney organ weight and organ-brain ratio relative weight were elevated, and left kidney and right kidney organ-body ratio relative weights were elevated, which were statistically significant (P ≤ 0.05, P ≤ 0.01, P ≤ 0.001). They were biologically significant and toxicologically significant.
Compared to the control group, in the F1 parent generation female rat exposure groups, adrenals and ovaries on both sides, individual organ weights, and organ coefficients were elevated or reduced to varying degrees, which were statistically significant (P ≤ 0.05, P ≤ 0.01, P ≤ 0.001). There was no consistent trend, no dose-response relationship, and no biological significance.
Compared to the control group, in F1 parent generation female rats exposed to 10 mg/kg bw/day, uterine organ coefficients were elevated and brain weight was reduced and these were statistically significant (P ≤ 0.05). There was no dose-response relationship and no biological significance.
Compared to the control group, in F1 parent generation male rats exposed to 40 mg/kg bw/day, body weight at necropsy was reduced, which was statistically significant (P ≤ 0.001). Combined with reduced body weight at 10 weeks of exposure, they possessed a consistent trend, biological significance, and toxicological significance.
Compared to the control group, in F1 parent generation male rats exposed to 10 and 40 mg/kg bw/day, liver weight and organ coefficients were elevated, and the increase was larger than 10%. They were statistically significant (P ≤ 0.05, P ≤ 0.001), biologically significant, and toxicologically significant.
Compared to the control group, in F1 parent generation male rats exposed to 40 mg/kg bw/day, left kidney and right kidney organ-body ratio relative weights were elevated while, in those exposed to 10 mg/kg bw/day, left kidney and right kidney organ-brain ratio relative weights were elevated, and they were statistically significant (P ≤ 0.05, P ≤ 0.001). Although there was no dose-response relationship, when combined with comprehensive analysis of the trend towards elevation in kidney weight for F0 and F1 parent generation animals, it was biologically significant.
Compared to the control group, in F1 parent generation male rats exposed to 40 mg/kg bw/day, right adrenal and left and right epididymal weights and organ-brain ratio relative weights were reduced and they were statistically significant (P ≤ 0.05, P ≤ 0.001). There was no dose-response relationship and no biological significance.
Compared to the control group, in F1 parent generation male rats exposed to 40 mg/kg bw/day, right testis to brain and body ratio relative weights were elevated. They were statistically significant (P ≤ 0.01, P ≤ 0.001) and the cause was reduced body weight at necropsy. There was no biological significance.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Small right testis occurred in 1/24 animals in the high dose group, with corresponding manifestation of mild multifocal deformation of the seminiferous tubules and severe diffuse atrophy of the seminiferous tubules under microscopy. Small right epididymis was noted in 1/24 animals in the high dose group, with corresponding manifestation of mild multifocal fragmentation of the seminiferous epithelial cells and serious diffuse lack of sperm in the epididymal tube under microscopy. Enlarged left testis was noted in 1/24 animals in the control group, with corresponding manifestation of mild diffuse expansion of the seminiferous tubules under microscopy. Missing kidney occurred in 1/24 animals in the control group, no slides were prepared. Enlarged uterus was noted in 1/24 animals in the high dose group, with corresponding manifestation of uterine expansion under microscopy. No anomalies were seen in any other animal organs.
All differences in necropsy findings of organs with anomalous changes were not significant, and they were not statistically significant (P > 0.05). All macroscopic changes were spontaneous or sporadic changes at this age, and they were not related to the test sample.
Dead female animals: There were no significant differences among organs that had anomalous changes in the necropsy findings and none were statistically significant (P > 0.05).
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
After histopathological examination of all organs, the differences were not significant, and none were statistically significant (P > 0.05). All pathologies found were spontaneous or sporadic changes at this age, and they were not related to the test sample. Among F1 generation there were 12 males and 12 females that did not conceive and did not develop. After histopathological examination, there were no findings of histopathological changes in animals that did not conceive and did not develop, thus the cause of the lack of conception and lack of development was not discovered.
Differences after calculation of primordial follicle quantitative and qualitative results in F1 generation female animals that did not conceive and did not develop
were not significant, and they were not statistically significant (P > 0.05). The results indicated that there was no follicular depletion.
Dead female animals: Differences after histopathological examination of all organs were not significant and none were statistically significant (P > 0.05).
Histopathological findings: neoplastic:
no effects observed

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
REPRODUCTIVE INDICATORS
7 animals were considered successfully mated, but uterine implantation count was 0. Four other animals were considered unsuccessfully mated.

NUMBER OF CORPORA LUTEA
Compared to the control group, the number of corpora lutea was elevated in F1 parent generation animals exposed to 2.5 mg/kg bw/day, which was statistically significant (P ≤ 0.01). There was no dose-response relationship and no biological significance.

PRIMORDIAL FOLLICLES: The normal range for primordial follicle count in the control group was 12.289 - 28.791. In this dose group, primordial follicle count ≥ 29 was an increase in follicle count and primordial follicle count ≤ 12 was a decrease in follicle count. The normal range for primordial follicle count in the high dose group was 10.400 - 25.180. In this dose group, primordial follicle count ≥ 26 was an increase in follicle count and primordial follicle count ≤ 10 was a decrease in follicle count. Differences after calculating primordial follicle qualitative results in F1 generation female animals were not statistically significant (P > 0.05).

Effect levels (P1)

open allclose all
Dose descriptor:
NOAEL
Remarks:
general toxicity
Effect level:
2.5 mg/kg bw/day
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain
organ weights and organ / body weight ratios
Dose descriptor:
NOAEL
Remarks:
general toxicity
Effect level:
10 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
organ weights and organ / body weight ratios
Dose descriptor:
NOAEL
Remarks:
reproductive toxicity
Effect level:
40 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other:

Target system / organ toxicity (P1)

open allclose all
Critical effects observed:
yes
Lowest effective dose / conc.:
10 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Critical effects observed:
yes
Lowest effective dose / conc.:
40 mg/kg bw/day (actual dose received)
System:
urinary
Organ:
kidney
Treatment related:
not specified
Dose response relationship:
not specified
Relevant for humans:
not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
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:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Compared to the control group, F1 generation offspring rats exposed to 2.5, 10, and 40 mg/kg bw/day had elevated birth survival rates; which was statistically significant (P ≤ 0.05). Owing to the fact that all birth survival rates were 100% in the exposure groups, it was not biologically significant.
Compared to the control group, and with the exception of birth survival rates of F1 generation offspring rats in all dose groups, mating success rates, conception rates, live birth rates, birth survival rates, survival rates, and nursing survival rates for all of the other parental generation and offspring generation animals were not statistically significant.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
40 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other:

Target system / organ toxicity (F1)

Critical effects observed:
no

Results: F2 generation

General toxicity (F2)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
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:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed

Developmental neurotoxicity (F2)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:
not examined

Effect levels (F2)

Dose descriptor:
NOAEL
Generation:
F2
Effect level:
40 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other:

Target system / organ toxicity (F2)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
Under the conditions of this test, there were no findings that 2-nitro-4-(trifluoromethyl)benzonitrile had toxicity to Sprague Dawley rat reproduction and development.
Based on the results of the present test, the no observed adverse effect level (NOAEL) in parent generation female rats was 10 mg/kg bw/day, the no observed adverse effect level (NOAEL) in parent generation male rats was 2.5 mg/kg bw/day, and the no observed adverse effect level (NOAEL) in offspring generation female and male rats was 40 mg/kg bw/day.