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Administrative data

Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From November 08, 2011 to July 25, 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to OECD Guideline 416 and EPA OPPTS Guideline 870.3800, in compliance with GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3800 (Reproduction and Fertility Effects)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
3-phenyl-5-(2-thienyl)-1,2,4-oxadiazole
EC Number:
810-533-8
Cas Number:
330459-31-9
Molecular formula:
C12H8N2OS
IUPAC Name:
3-phenyl-5-(2-thienyl)-1,2,4-oxadiazole
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): MON 102100

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC
- Age at study initiation: Approximately 6 weeks (F0)
- Body weight: 200 g - 250 g (males); 130 g - 170 g (females)
- Fasting period before study: no
- Housing: All F0 and F1 parental test animals were housed individually, except during the mating period, in clean, stainless steel wire-mesh cages suspended above cage-board
- Diet: PMI Nutrition International, LLC Certified Rodent LabDiet #5002, ad libitum
- Water: Reverse osmosis-purified (on-site) drinking water, ad libitum
- Acclimation period: 14 d

ENVIRONMENTAL CONDITIONS
- Temperature: 70.3°F to 71.4°F (21.3°C to 21.9°C)
- Humidity: 38.5% to 55.3%
- Air changes: A minimum of 10 fresh air changes per h
- Photoperiod: 12 h light/12 h dark cycle

IN-LIFE DATES: From: November 08, 2011 To: July 25, 2012

Administration / exposure

Route of administration:
oral: feed
Vehicle:
other: PMI Nutrition International, LLC Certified Rodent LabDiet® #5002
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet: Approximately weekly. Diet concentrations were adjusted as necessary based on mean body weight and food consumption data.
- Mixing appropriate amounts with: PMI Nutrition International, LLC Certified Rodent LabDiet® #5002
- Storage temperature of food: Room temperature
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 14 d
- Proof of pregnancy: vaginal plug or the presence of sperm in a vaginal lavage referred to as Day 0 of pregnancy
- When evidence of mating was not apparent after 14 d, the female was placed in a plastic maternity cage with nesting material, with no further opportunity for mating.
- After successful mating each pregnant female was caged: in an individual plastic cage with nesting material
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentration analyses were performed on samples from the first four weeks of the study and once a month thereafter. Samples were collected from the top, middle, and bottom of each formulation and analyzed for concentration and homogeneity.
The analyzed dietary formulations at concentrations ranging between 31.3 ppm to 1328.6 ppm were homogeneous. However, the pre-test diet prepared at a target of 29.1 ppm (the lowest projected admixture concentration for the Group 2 females) was determined not to be homogeneous (RSD ≥10). Diets were subsequently prepared at a concentration of 29.3 ppm and were determined to meet acceptance criteria for homogeneity. Therefore, the diets offered to the animals on this study were considered to be homogeneous.
Duration of treatment / exposure:
F0 males and females: exposed for 128-133 consecutive days
F1 males and females: exposed for 133-147 consecutive days (minimum of 70 consecutive days prior to mating and through the day of euthanasia).
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 5, 20, and 60 mg/kg bw/day
Basis:
nominal in diet
No. of animals per sex per dose:
30 rats/sex/dose (F0 and F1)
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: The highest exposure level for this study was based upon a weight-of-the-evidence evaluation of results from a 90 d rat study and a range-finding reproductive toxicity study with test substance. Excessive toxicity at 100 mg/kg bw/day was observed in the range-finding reproductive toxicity study. Based on bone and kidney effects, the United States EPA concluded that 750 ppm in the 90 d study (approximately 47 mg/kg bw/day in males and 56 mg/kg bw/day in females) represented a maximum tolerated dose for a long-term study. Considering the above, the highest exposure level selected for this study was 60 mg/kg bw/day. Other exposure levels were selected by the Sponsor to provide a graduation of responses and to ensure a no-observed-adverse-effect level in the current study.

- Rationale for animal assignment: At the conclusion of the acclimation period, all available F0 animals were weighed and examined in detail for physical abnormalities. At the discretion of the Study Director, each animal judged to be in good health and meeting acceptable body weight requirements was selected for use in the computerized randomization procedure based on body weight stratification randomized in a block design.

- The offspring of the F0 and F1 generations (F1 and F2 litters, respectively) in the present study were potentially exposed to the test substance in utero, as well as via the milk while nursing and via direct consumption of the diet during the latter portion of the lactation period. The F1 pups selected for mating (30 sex/group) were directly administered the test diet following weaning (beginning on Postnatal Day (PND) 21).

-Selection for the F1 generation: A computerized randomization procedure was used to select a minimum of one male and one female per litter, when available. An additional male and/or female were selected from a litter, if necessary, to obtain 30 males and 30 females for each group.

Examinations

Parental animals: Observations and examinations:
All animals were observed twice daily for appearance and behavior; detailed physical examinations were conducted weekly. Body weights and food consumption were recorded weekly, beginning one week prior to the start of test diet exposure; food consumption was not recorded during the mating period. Following evidence of mating for females, body weights and food consumption were recorded on gestation Days 0, 7, 14, and 20 and lactation Days 1, 4, 7, 11, 14, and 21.
Oestrous cyclicity (parental animals):
Vaginal lavages were performed daily for determination of estrous cycles beginning 21 d prior to pairing.
Sperm parameters (parental animals):
Spermatogenic endpoints (sperm motility [including progressive motility], morphology, and numbers) were recorded for all F0 and F1 males.
Litter observations:
All F0 and F1 females were allowed to deliver and rear their pups until weaning on lactation Day 21. Clinical observations and body weights for F1 and F2 pups were recorded on PND 1, 4, 7, 14, and 21; pups were sexed on PND 0, 4, 14, and 21. Developmental landmarks (balanopreputial separation and vaginal patency) were evaluated for the selected F1 rats. Nonselected F1 pups and all surviving F2 pups were necropsied on PND 21.
Postmortem examinations (parental animals):
Each surviving F0 and F1 parental animal were subjected to a complete detailed gross necropsy following the completion of weaning of the F1 and F2 pups, respectively.

Macroscopic examination (F0 and F1 parental animal): Adrenals (2), Aorta, Bone with marrow, Femur, Sternum, Brain, Cerebrum level one, Cerebrum level two, Cerebellum with medulla/pons, Coagulating gland, Eyes with optic nerve (2), Gastrointestinal tract, Esophagus, Stomach, Duodenum Jejunum, Ileum, Cecum, Colon, Rectum, Heart, Kidneys (2), Liver (sections of two lobes), Lungs (including bronchi,, fixed by inflation with fixative), Lymph node, Axillary, Mandibular, Mesenteric, Ovaries and oviducts (2), Pancreas, Peripheral nerve (sciatic), Pituitary gland, Prostate gland, Salivary gland [mandibular (2), Seminal vesicles (2), Skeletal muscle (rectus femoris), Skin with mammary glandc, Spinal cord (cervical), Spleen Testes with epididymides (1), and vas deferens, Thymus gland, Thyroids [with parathyroids, if present (2)], Trachea, Urinary bladder, Ureters, Uterus with cervix and vagina, All gross lesions (all groups).

The organs weighed from F0 and F1 adults were: adrenals, brain, epididymides (total and cauda), kidneys, liver, ovaries, pituitary gland, prostate gland, seminal vesicles with coagulating glands (with accessory fluids), spleen, testes, thyroid gland, thymus gland, uterus with oviducts and cervix
Designated tissues from all F0 and F1 parental animals in the control and high-exposure groups were examined microscopically. In addition, the kidneys were examined for all F0 and F1 males and females in the 5 and 20 mg/kg bw/day groups and the liver, femur, and adrenal cortex were examined for F0 and F1 males in the 5 and 20 mg/kg bw/day groups. A peer review of the vacuolar changes in the adrenal cortex of F0 and F1 males was also performed without knowledge of the animals’ dose group assignments.

Microscopic evaluations (F0 and F1 parental animals): Adrenal glands, Bone with marrow, Femur, Sternum, Brain, Cervix, Coagulating gland, Epididymis (right): caput,, corpus, and cauda, Kidneys, Liver Ovaries, Oviducts, Pituitary gland, Prostate gland, Seminal vesicles, Spleen, Testis (right), Thymus gland, Urinary bladder, Ureters, Uterus, Vagina Vas deferens, All gross (internal) lesions (all groups).
Postmortem examinations (offspring):
Nonselected F1 pups and all surviving F2 pups were necropsied on PND 21; selected organs from one pup/sex/litter were weighed. Each surviving F0 and F1 parental animal received a complete detailed gross necropsy following the completion of weaning of the F1 and F2 pups, respectively; selected organs were weighed.

The organs weighed from F1 and F2 weanlings were: brain, spleen, and thymus.
Statistics:
Analyses were conducted using two-tailed tests at minimum significance levels of 1% and 5%, comparing each treated group to the control group by sex. Each mean was presented with the standard deviation (S.D.) and the number of animals (N) used to calculate the mean. Parental mating, fertility, copulation, and conception indices were analyzed using the Chi-square test with Yates’ correction factor. Mean parental (weekly, gestation, and lactation) and offspring body weights and body weight changes, parental food consumption, and food efficiency data, estrous cycle lengths,
pre-coital intervals, gestation lengths, former implantation sites, live litter sizes, unaccounted-for sites, numbers of pups born, balanopreputial separation data (day of attainment and body weight), vaginal patency data (day of attainment and body weight), absolute and relative organ weights, sperm production rates, epididymal and testicular sperm numbers, and ovarian primordial follicle counts were subjected to a parametric one-way ANOVA to determine intergroup differences. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunnett's test was used to compare the treated groups to the control group. Mean litter proportions (percent per litter) of postnatal pup survival and pup sexes at birth (percentage of males per litter), percentages of motile and progressively motile sperm, and percentages of sperm with normal morphology were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunn’s test was used to compare the treated groups to the control group. Histopathological findings in the treated groups were compared to the control group using a two-tailed Fisher’s Exact test.
Reproductive indices:
Mating, fertility, copulation, and conception indices
Offspring viability indices:
Mean live litter size, Postnatal survival between birth and PND 0 or PND 4 (pre-selection) (% per litter), Postnatal survival for all other intervals (% per litter)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
No remarkable clinical observations were noted at any exposure level tested in the F0 and F1 generations.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At an exposure level of 60 mg/kg/day, test substance-related reductions in mean body weight, body weight gain, food consumption, and food efficiency were noted for F0 and F1 males throughout each generation.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At an exposure level of 60 mg/kg/day, test substance-related reductions in mean body weight, body weight gain, food consumption, and food efficiency were noted for F0 and F1 males throughout each generation.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
No test substance-related effects were observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS): F0 and F1 parental survival was unaffected by test diet administration at all exposure levels. No remarkable clinical observations were noted at any exposure level tested in the F0 and F1 generations.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS): At an exposure level of 60 mg/kg bw/day, test substance-related reductions in mean body weight, body weight gain, food consumption, and food efficiency were noted for F0 and F1 males throughout each generation. Parental body weight and food consumption parameters were unaffected by test substance exposure at exposure levels of 5 and 20 mg/kg bw/day for F0 and F1 males and females and 60 mg/kg bw/day for F1 females. Slightly lower (not statistically significant) mean maternal body weight gains were noted for the F0 females in the 60 mg/kg bw/day group throughout gestation (gestation Days 0-20). As a result of these changes, mean body weight in this group was statistically significantly lower (4.8% to 5.7%) than the control group on gestation Days 14 and 20. In addition, slightly lower (generally significant at p<0.01) mean food consumption was noted in the F0 females in the 60 mg/kg bw/day group compared to the control group throughout gestation when evaluated on a g/animal/day basis. However, because of the transient nature of these findings, the lower gestation body weights and food consumption in the 60 mg/kg bw/day F0 females were not considered to be adverse.

REPRODUCTIVE FUNCTION: No test substance-related effects were observed on F0 and F1 reproductive performance (estrous cycles, mating, fertility, copulation or conception indices, the mean number of days between pairing and coitus and the mean length of gestation), parturition, or the mean numbers of implantation sites and unaccounted-for sites.
Spermatogenic endpoints (mean testicular and epididymal sperm numbers, sperm production rate, motility, progressive motility, and the percentage of morphologically normal sperm) were also unaffected by test diet exposure.

HISTOPATHOLOGY (PARENTAL ANIMALS): Test substance-related microscopic findings were present in the femur, kidney, liver, and adrenal gland. Minimal hyperostosis of the metaphyseal region of the femur was observed in the 60 mg/kg bw/day group F0 and F1 males. A minimal amount of foreign material (brown to gray, granular, small aggregates) was present in the tubular lumina of the kidney cortex of F0 and F1 males and females in the 20 and 60 mg/kg bw/day groups and was not considered to be an adverse excretory product of the test substance. Similarly, a small amount of a granular brown pigment was observed in renal tubular epithelial cells from two F0 males in the 60 mg/kg bw/day group. The composition of the foreign material and brown pigment were undetermined, but these findings were not considered to be adverse effects because there was no microscopic evidence of renal toxicity. Minimal to mild hepatocellular cytoplasmic alteration, consisting of increased fine eosinophilic granularity in the cytoplasm of centrilobular hepatocytes, occasionally extending into midzonal and periportal regions, was observed in the livers of the 20 and 60 mg/kg bw/day group F0 and F1 males and correlated with higher liver weights relative to final body weight in these groups.While considered to be treatment-related, the higher liver weights and corresponding histologic correlate were considered a nonadverse effect of test substance administration. In the adrenal cortex, an increase in the incidence of minimal to mild vacuolation of the zona fasciculata was observed in the 20 and 60 mg/kg bw/day group F0 and F1 males.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
20 mg/kg bw/day
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Based on lower mean body weight gains, body weights, food consumption, and/or food efficiency for F0 and F1 males at 60 mg/kg bw/day.
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
60 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Due to the absence of toxicity noted for the F0 and F1 females throughout the study.
Dose descriptor:
NOAEL
Remarks:
reproductive toxicity
Effect level:
60 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on the absence of effects on F0 and F1 reproductive performance (mating, fertility, copulation and conception indices, estrous cyclicity, and spermatogenic endpoints) and on the F1 and F2 litters and offspring.
Dose descriptor:
NOAEL
Remarks:
neonatal toxicity
Effect level:
60 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on the absence of effects on the F1 and F2 litters and offspring.
Remarks on result:
other: Generation: F1 and F2 litters and offspring (migrated information)

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed

Details on results (F1)

No test substance-related effects were observed on the mean number of F1 or F2 pups born, the pup sex ratio, pup survival, offspring body weights, or the general physical condition of the pups during the pre-weaning period. No test substance-related macroscopic findings were observed in F1 and F2 pups that were found dead, euthanized in extremis, or at the scheduled necropsy. There were no test substance-related effects on F1 or F2 pup organ weights on PND 21.
A test substance-related higher incidence in adrenal vacuolation of the zona fasciculate was noted in the 20 and 60 mg/kg bw/day group males. This test substance-related effect was not considered adverse in this study because the vacuolation was noted at a severity level of minimal to mild which was considered not unusual for normal rats and not indicative of abnormal adrenal histology.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The NOAEL for F0 and F1 male systemic toxicity was considered 20 mg/kg bw/day based on lower mean body weight gains, body weights, food consumption, and/or food efficiency for F0 and F1 males at 60 mg/kg bw/day. Due to the absence of toxicity noted for the F0 and F1 females throughout the study, the highest exposure concentration of 60 mg/kg bw/day, was considered to be the NOAEL for F0 and F1 female systemic toxicity. Based on the absence of effects on F0 and F1 reproductive performance (mating, fertility, copulation and conception indices, estrous cyclicity, and spermatogenic endpoints) and on the F1 and F2 litters and offspring, an exposure level of 60 mg/kg bw/day was considered to be the NOAEL for F0 and F1 reproductive toxicity, as well as F1 and F2 neonatal toxicity of the test substance when administered continuously in the diet to Crl:CD(SD) rats.
Executive summary:

A two-generation study was conducted to determine the potential adverse effects of the test substance on reproduction according to OECD Guideline 416 and EPA OPPTS Guideline 870.3800, in compliance with GLP. Four groups of male and female Crl: CD®(SD) rats (30/sex/group) were offered either basal diet or the test substance, continuously in the diet for at least 70 consecutive days prior to mating. The test substance doses were adjusted weekly to provide target concentrations of 0, 5, 20, and 60 mg/kg bw/day for the F0 and F1 generations. The test diet was administered to the offspring selected to become the F1 generation following weaning (beginning on PND 21). The F0 and F1 males continued to receive the test substance throughout mating and continuing through the day of euthanasia. The F0 and F1 females continued to receive the test substance throughout mating, gestation, lactation and through the day of euthanasia. Offspring (30/sex/group) from the pairing of the F0 animals were selected on PND 21 to constitute the F1 generation. F0 males and females were exposed for 128-133 consecutive days, and F1 males and females were exposed for 133-147 consecutive days. F0 and F1 parental survival was unaffected by test diet administration at all exposure levels. No remarkable clinical observations were noted at any exposure level tested in the F0 and F1 generations. At 60 mg/kg bw/day, test substance-related reductions in mean body weight, body weight gain, food consumption and food efficiency were noted for F0 and F1 males throughout each generation. No test substance-related effects were observed on F0 and F1 reproductive performance (estrous cycles, mating, fertility, copulation or conception indices, the mean number of days between pairing and coitus and the mean length of gestation), parturition or the mean numbers of implantation sites and unaccounted-for sites. Spermatogenic endpoints (mean testicular and epididymal sperm numbers, sperm production rate, motility, progressive motility and the percentage of morphologically normal sperm) were also unaffected by test diet exposure. There were no test substance-related macroscopic findings in F0 and F1 males and females at 5, 20 and 60 mg/kg bw/day. In addition, there were no test substance-related microscopic findings in the male and female reproductive tissues for either generation. A test substance-related higher incidence in adrenal vacuolation of thezona fasciculatawas noted in the 20 and 60 mg/kg bw/day group males. This effect was not considered adverse in this study because the vacuolation was noted at a severity level of minimal to mild, which was considered not unusual for normal rats and not indicative of abnormal adrenal histology. No test substance-related effects were observed on the mean number of F1 or F2 pups born, the pup sex ratio, pup survival, offspring body weights or the general physical condition of the pups during the pre-weaning period. No test substance-related macroscopic findings were observed in F1 and F2 pups that were found dead, euthanized in extremis or at the scheduled necropsy. There were no test substance-related effects on F1 or F2 pup organ weights on PND 21. Therefore the NOAEL for F0 and F1 male systemic toxicity was considered to be 20 mg/kg bw/day based on lower mean body weight gains, body weights, food consumption and/or food efficiency for F0 and F1 males at 60 mg/kg bw/day. Due to the absence of toxicity noted for the F0 and F1 females throughout the study, the highest exposure concentration of 60 mg/kg bw/day was considered to be the NOAEL for F0 and F1 female systemic toxicity. Based on the absence of effects on F0 and F1 reproductive performance (mating, fertility, copulation and conception indices, estrous cyclicity and spermatogenic endpoints) and on the F1 and F2 litters and offspring, an exposure level of 60 mg/kg bw/day was considered to be the NOAEL for F0 and F1 reproductive toxicity, as well as F1 and F2 neonatal toxicity of the test substance when administered continuously in the diet to Crl: CD(SD) rats (Stump, 2014).