Registration Dossier

Administrative data

Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Qualifier:
according to
Guideline:
EPA OPPTS 870.3800 (Reproduction and Fertility Effects)
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on mating procedure:
Rats were administered the test substance for at least 70 consecutive days prior to mating.
The F0 and F1 males continued to receive the test substance throughout mating and through the day prior to euthanasia.
The F0 and F1 females continued to receive the test substance throughout mating, gestation, and lactation, and through the day prior to euthanasia.
Duration of treatment / exposure:
F0 males and females were dosed for 127–132 consecutive days, and F1 males and females were dosed for 139–148 consecutive days.
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
20 mg/kg bw/day (nominal)
Dose / conc.:
40 mg/kg bw/day (nominal)
Dose / conc.:
60 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Three groups of male and female Crl:CD(SD) rats (25/sex/group)
25 rats/sex/group received the vehicle (corn oil) on a comparable regimen.
Control animals:
yes, concurrent vehicle

Examinations

Estrous cyclicity (parental animals):
Vaginal lavages were performed daily for determination of estrous cycles beginning 21 days prior to cohabitation.

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
No test substance-related clinical observations were noted for F0 and F1 males and females in the 20 mg/kg/day group.
Test substance-related, increased incidences of red material around the nose and/or mouth were noted for F0 and F1 males and females in the 40 and 60 mg/kg/day groups and increased incidences of mucoid feces and yellow and/or brown material around the urogenital and/or anogenital areas were noted for all F0 males and females in the 60 mg/kg/day group generally throughout the treatment period; these observations were noted primarily at the time of dose administration and/or 3-4 hours following dose administration.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Lower mean body weight gains were noted in the 40 mg/kg/day group F0 males during the pre-mating period (Study Days 0–69) and in the 60 mg/kg/day group F0 males throughout the entire generation (Study Days 0–127) compared to the control group. In addition, mean body weights in the 40 and 60 mg/kg/day group F0 males were up to 6.7% and 16.3% lower, respectively, than the control group during these intervals.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Test substance-related lower mean food consumption was noted in the 40 and 60 mg/kg/day group F0 males during the first week of dosing (Study Days 0–7). For the remainder of the study, mean food consumption for F0 males in all test substance-treated groups was similar to or higher than the control group; the higher food consumption values were likely due to the high caloric content and volume of the vehicle (corn oil).
Food efficiency:
effects observed, non-treatment-related
Description (incidence and severity):
Mean food efficiency in the 40 and 60 mg/kg/day group F0 males was generally lower than the control group throughout the study. Mean F0 male mean body weights, body weight gains, and food efficiency at 20 mg/kg/day were similar to the control group. In addition, F0 female mean body weights, body weight gains, food consumption, and food efficiency during the pre-mating period, gestation, and lactation were unaffected by test substance administration.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related findings noted for F0 animals consisted of higher liver weights in the 20, 40, and 60 mg/kg/day group males and 40 and 60 mg/kg/day group females, higher adrenal gland weights and adrenal cortical hypertrophy in the 40 and 60 mg/kg/day group males and females, lower thymus weights in the 60 mg/kg/day group males, squamous cell hyperplasia of non-glandular stomach in the 20, 40, and 60 mg/kg/day group males and females, and mucosal hyperplasia of the urinary bladder in the 20, 40, and 60 mg/kg/day group males and females.
The majority of the changes (higher liver weights, mucosal hyperplasia of the urinary bladder, and squamous hyperplasia of the stomach) were attributed to the enzyme induction or irritation by the test substance. Adrenal cortical hypertrophy and increased adrenal gland weights were most likely an adaptive nonadverse change. The findings of hyperplasia of the stomach and mucosal hyperplasia of the urinary bladder were considered adverse

Details on results (P0)

There were no test substance-related effects on F0 and F1 male and female mating and fertility, male copulation, and female conception indices, estrous cycle length, pre-coital interval, or spermatogenesis parameters (motility, progressive motility, testicular and epididymal sperm numbers, sperm production rate, and sperm morphology) at any dosage level.
No test substance-related effects on parturition were noted for F0 females at 20, 40, and 60 mg/kg/day.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
parental toxicity
Sex:
male/female
Remarks on result:
not determinable
Dose descriptor:
NOAEL
Remarks:
reproductive toxicity
Effect level:
60 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance

Target system / organ toxicity (P0)

Critical effects observed:
yes
Lowest effective dose / conc.:
20 mg/kg bw/day (nominal)
Organ:
stomach
bladder
other: eye
Treatment related:
not specified
Relevant for humans:
not specified

Results: P1 (second parental generation)

General toxicity (P1)

Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related, lower mean body weights and body weight gains were noted in the 40 and 60 mg/kg/day group F1 males throughout the entire generation (PND 21–161). In the 40 and 60 mg/kg/day group F1 females, lower mean body weight gains were noted during the first 2 weeks of the pre-mating period (PND 21–28 and PND 21–35 at 40 and 60 mg/kg/day, respectively) and resulted in lower mean body weights during PND 21–35 at 40 mg/kg/day and PND 21-63 at 60 mg/kg/day; mean body weights and body weight gains in these groups were similar for the remainder of the pre-mating period.
Mean maternal body weights, body weight gains, food consumption, and food efficiency for F1 females at all dosage levels were generally similar to the control group during gestation. In the 60 mg/kg/day group F1 females, mean body weights that were up to 8.1% lower than the control group were noted during Lactation Days 1–21 and were considered test substance-related and adverse; mean body weight gains in this group were similar to the control group.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Mean food consumption (g/animal/day values) for the 60 mg/kg/day group F1 males was lower than the control group during the pre-mating period (PND 28–98).
Test substance-related, lower mean food consumption was noted in the 60 mg/kg/day group F1 females when the entire lactation period (Lactation Days 1–21) was evaluated compared to the control group; mean food efficiency in this group was generally similar to the control group. Mean body weights, body weight gains, food consumption, and food efficiency in the 20 and 40 mg/kg/day group F1 females was generally similar to the control group during lactation.
Ophthalmological findings:
effects observed, treatment-related
Description (incidence and severity):
A test substance-related increased incidence of bilateral cataracts were noted in the 40 and 60 mg/kg/day group F1 males and females during the ophthalmic examination and were considered test substance-related. These findings correlated with adverse ocular clinical and histopathological findings noted for F1 males and females at these dosage levels.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related findings noted for F1 animals consisted of lower brain weights in the 40 and 60 mg/kg/day group males and females, higher adrenal gland weights in the 40 and 60 mg/kg/day group females, higher liver weights in the 40 and 60 mg/kg/day group males and 20, 40, and 60 mg/kg/day group females, small eyes, enophthalmus, retinal dysplasia, and/or cataract formation in the 20, 40, and 60 mg/kg/day group males and 40 and 60 mg/kg/day group females, small optic nerve in the 60 mg/kg/day group males, mucosal hyperplasia of the urinary bladder in the 20, 40, and 60 mg/kg/day group males and females, and squamous hyperplasia of the non-glandular stomach in the 20, 40, and 60 mg/kg/day group males and females.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
The adverse findings included the gross and microscopic changes in the eye, small optic nerve, squamous hyperplasia of the stomach, and mucosal hyperplasia of the urinary bladder.

Details on results (P1)

There were no test substance-related effects on F0 and F1 male and female mating and fertility, male copulation, and female conception indices, estrous cycle length, pre-coital interval, or spermatogenesis parameters (motility, progressive motility, testicular and epididymal sperm numbers, sperm production rate, and sperm morphology) at any dosage level.
In the 60 mg/kg/day group, difficult parturition resulted in the moribundity of 2 F1 females (as noted previously). No other signs of dystocia were noted in F1 females at any dosage level.

Effect levels (P1)

open allclose all
Dose descriptor:
NOAEL
Based on:
test mat.
Sex:
male/female
Basis for effect level:
ophthalmological examination
histopathology: neoplastic
Remarks on result:
other: not determinable due to adverse effects observed also at the lowest dose (20 mg/kg bw/day)
Dose descriptor:
NOAEL
Effect level:
60 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance

Target system / organ toxicity (P1)

Critical effects observed:
yes
Lowest effective dose / conc.:
20 mg/kg bw/day (nominal)
Organ:
stomach
bladder
other: eye
Treatment related:
yes
Relevant for humans:
not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
In the 60 mg/kg/day group F1 pups, a test substance-related clinical observation of small stature was noted; no test substance-related clinical observations were noted in F1 pups at 20 and 40 mg/kg/day or F2 pups at any dosage level.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Lower male and female mean offspring body weight gains were noted prior to culling (PND 1-4) in F1 and F2 pups at 40 and 60 mg/kg/day and following culling (PND 4–21) in F1 pups at 40 and 60 mg/kg/day and F2 pups at 60 mg/kg/day compared to the control group and resulted in lower mean body weights in these groups generally throughout the postnatal period.
Sexual maturation:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test substance-related effects on balanopreputial separation or vaginal patency in the 20, 40, and 60 mg/kg/day group F1 weanlings.

Details on results (F1)

Lower mean F1 and F2 postnatal survival was noted in the 40 and 60 mg/kg/day groups during PND 0–1 and PND 1–4 (pre-selection) and resulted in lower postnatal survival from birth to PND 4 (pre-selection) compared to the control group. In addition, F2 postnatal survival in the 60 mg/kg/day group was lower than the control group during PND 4 (post-selection) to PND 7 and resulted in lower postnatal survival from PND 4 (post-selection) to PND 21. Postnatal survival of the F1 and F2 pups in the 20 mg/kg/day group was unaffected by parental test substance administration.
There were no test substance-related effects on the number of F1 and F2 pups born, live litter size, or percentage of males at birth at any dosage level.

Effect levels (F1)

open allclose all
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
20 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
other: bilateral cataracts
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
60 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: reproductive performance

Target system / organ toxicity (F1)

Critical effects observed:
yes
Lowest effective dose / conc.:
40 mg/kg bw/day (nominal)
System:
eye
Treatment related:
yes
Relevant for humans:
not specified

Results: F2 generation

General toxicity (F2)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
In the 60 mg/kg/day group F1 pups, a test substance-related clinical observation of small stature was noted; no test substance-related clinical observations were noted in F1 pups at 20 and 40 mg/kg/day or F2 pups at any dosage level.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Lower male and female mean offspring body weight gains were noted prior to culling (PND 1-4) in F1 and F2 pups at 40 and 60 mg/kg/day and following culling (PND 4–21) in F1 pups at 40 and 60 mg/kg/day and F2 pups at 60 mg/kg/day compared to the control group and resulted in lower mean body weights in these groups generally throughout the postnatal period.
Sexual maturation:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test substance-related effects on balanopreputial separation or vaginal patency in the 20, 40, and 60 mg/kg/day group F1 weanlings.

Details on results (F2)

Lower mean F1 and F2 postnatal survival was noted in the 40 and 60 mg/kg/day groups during PND 0–1 and PND 1–4 (pre-selection) and resulted in lower postnatal survival from birth to PND 4 (pre-selection) compared to the control group. In addition, F2 postnatal survival in the 60 mg/kg/day group was lower than the control group during PND 4 (post-selection) to PND 7 and resulted in lower postnatal survival from PND 4 (post-selection) to PND 21. Postnatal survival of the F1 and F2 pups in the 20 mg/kg/day group was unaffected by parental test substance administration.
There were no test substance-related effects on the number of F1 and F2 pups born, live litter size, or percentage of males at birth at any dosage level.

Effect levels (F2)

Dose descriptor:
NOAEL
Generation:
F2
Effect level:
20 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
other: bilateral cataracts

Target system / organ toxicity (F2)

Critical effects observed:
yes
Lowest effective dose / conc.:
40 mg/kg bw/day (nominal)
System:
eye
Treatment related:
yes
Relevant for humans:
not specified

Overall reproductive toxicity

Reproductive effects observed:
no
Lowest effective dose / conc.:
60 mg/kg bw/day (nominal)

Applicant's summary and conclusion

Conclusions:
Lower mean body weights and body weight gains were noted for F0 males and F1 males and females at 40 and 60 mg/kg/day. Histopathologic changes consisted of mucosal hyperplasia of non-glandular stomach and urinary bladder noted in F0 males and females at all dosage levels and gross and microscopic changes in the eye (small eyes, enophthalmus, retinal dysplasia, and/or cataract formation), small optic nerve, squamous hyperplasia of the stomach, and mucosal hyperplasia of the urinary bladder noted in F1 males and females in the 20, 40, and/or 60 mg/kg/day groups. Based on these results, a no-observed-adverse-effect level for F0 and F1 parental toxicity was not established in this study.
No adverse effects on F0 and F1 reproductive performance (mating, fertility, copulation and conception indices, estrous cyclicity, and spermatogenic endpoints) were noted. Based on these results, a dosage level of 60 mg/kg/day was considered to be the NOAEL for F0 and F1 reproductive toxicity.
Based on adverse reductions in F1 and F2 postnatal survival, lower offspring body weights and body weight gains, and bilateral cataracts for F1 animals at 40 and 60 mg/kg/day, the NOAEL for developmental/neonatal toxicity was considered to be 20 mg/kg/day.