1,2,4-Oxadiazole, 3-phenyl-5-(2-thienyl)-

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

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.

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

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

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

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.

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)

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: 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

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.

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)

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

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.

Reproductive effects observed:

not specified

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).

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

60 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

High quality database.

Additional information

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).

Short description of key information:
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.

Justification for selection of Effect on fertility via oral route:
The study followed internationally accepted guideline and conducted in compliance with GLP.

Effects on developmental toxicity

Description of key information

In the absence of any evidence of developmental toxicity, a dose level of 200 mg/kg bw/day (the highest dose level tested) was considered to be the NOAEL for prenatal development when the test substance was administered orally by gavage to pregnant Crl:CD(SD) rats.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From October 11, 2010 to November 23, 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to OECD Guideline 414 and OPPTS Guideline 870.3700, in compliance with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Sprague Dawley [Crl:CD(SD)] rats

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC
- Age at study initiation: Approximately 13 week
- Weight: Ranged from 225 g to 290 g on gestation Day 0
- Housing: Rats were individually housed in clean, stainless steel wire-mesh cages suspended above cage-board. The cage-board was changed at least three times per week. The rats were paired for mating in the home cage of the male. Following positive evidence of mating, the females were returned to individual suspended wire-mesh cages; nesting material was not required as the females were euthanized prior to the date of expected parturition.
- Diet:PMI Nutrition International, LLC Certified Rodent LabDiet® #5002, provided ad libitum throughout the acclimation period and during the study
- Water: Reverse osmosis-purified (on-site) drinking water, delivered by an automatic watering system, ad libitum
- Acclimation period: 14 d

ENVIRONMENTAL CONDITIONS
- Temperature: 70.1°F to 70.7°F (21.2°C to 21.5°C)
- Humidity: 36.8% to 53.3%
- Air changes: 10 per h
- Photoperiod: 12 h light/12 h dark cycle

IN-LIFE DATES: From: October 11, 2010 To: October 28, 2010

Route of administration:

oral: gavage

Vehicle:

other: 0.5% carboxymethylcellulose in water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The vehicle suspension was prepared approximately weekly for administration to the control group (Group 1) and for preparation of the test substance formulations; aliquots were prepared for daily dispensation to the control group and were stored refrigerated (2ºC to 8ºC), protected from light. The vehicle was mixed throughout preparation, sampling, and dose administration procedures. The test substance formulations were prepared approximately weekly as single formulations for each dose level, divided into aliquots for daily dispensation, and stored refrigerated (2ºC to 8ºC), protected from light. The test substance formulations were stirred continuously throughout the preparation, sampling, and dose administration procedures.

The dosing formulations were not adjusted for purity of test substance.

VEHICLE
- Vehicle: 0.5% carboxymethylcellulose (medium viscosity), USP in water
- Concentration in vehicle: 0, 1.0, 5.0, 20 mg/mL for dose level 0, 10 , 50 and 200 mg/kg bw/day respectively
- Amount of vehicle: 10 mL/kg bw
- Lot no.: YK1264 (Source: Spectrum Chemical Manufacturing Corporation, Gardena, CA)
- Exp. date: July 07, 2012

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples for homogeneity determination were collected from the top, middle, and bottom strata of the control, 1.0, 5.0, and 20 mg/mL dosing formulations. In addition, samples for resuspension homogeneity were collected from the top, middle, and bottom strata of an aliquot taken from the 1.0, 5.0, and 20 mg/mL dosing suspensions following refrigerated storage for nine days. The aliquots were stirred at room temperature for approximately 30 minutes using a magnetic stirrer prior to sample collection. Samples for concentration analysis were collected from the middle stratum of each dosing formulation (including the control group). One set of samples from each collection was subjected to the appropriate analyses. The remaining set of samples was stored as back-up. All analyses were conducted by the WIL Research Analytical Chemistry Department using a validated high performance liquid chromatography method with ultraviolet absorbance detection.

The analyzed dosing formulations were within 85% to 115% of the target concentration. The test substance was not detected in the vehicle formulation that was administered to the control group.

Details on mating procedure:

- Impregnation procedure: Cohoused
- M/F ratio per cage: 1:1
- Proof of pregnancy: Vaginal plug / presence of sperm in a vaginal lavage referred to as Day 0 of pregnancy
- The day on which evidence of mating was identified was termed gestation Day 0.

Duration of treatment / exposure:

Gestation Days 6-19

Frequency of treatment:

Daily

Duration of test:

20 d

Remarks:

Doses / Concentrations:
0, 10, 50, and 200 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

25 females/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on the results of a range-finding prenatal developmental study in rats (Stump, 2012, WIL-50381).
- Rationale for animal assignment: The bred females were assigned to groups using a WTDMS™ computer program which randomized the animals based on stratification of the gestation Day 0 body weights in a block design.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily, once in the morning and once in the afternoon

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: From gestation Days 0 through 20 (prior to dose administration during the treatment period). Animals were also observed for signs of toxicity approximately 1 h following dose administration

BODY WEIGHT: Yes
- Time schedule for examinations: Individual maternal body weights were recorded on gestation days 0 and 6-20 (daily)
- - Gravid uterine weight was collected and net body weight (the gestation Day 20 body weight exclusive of the weight of the uterus and contents) and net body weight change (the gestation Day 0-20 body weight change exclusive of the weight of the uterus and contents) were calculated and presented for each gravid female at the scheduled laparohysterectomy.


FOOD CONSUMPTION: Yes
- Food consumption for each animal determined: Yes
- Time schedule: on gestation Day 0 and Days 6-20 (daily)
- In addition, food efficiency (body weight gained as percent of feed consumed) was calculated


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation Day 20
- Organs examined: The thoracic, abdominal, and pelvic cavities were opened by a ventral mid-line incision, and the contents were examined.

OTHER:
Maternal tissues with gross lesions were preserved in 10% neutral-buffered formalin for possible future histopathologic examination. Representative sections of corresponding organs from a sufficient number of control animals were retained for comparison. In addition, the adrenal glands, kidneys, and liver from each female were weighed and preserved in 10% neutral-buffered formalin for possible future histopathologic examination. The carcass of each female was then discarded.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Uteri with no macroscopic evidence of implantation were opened and subsequently placed in 10% ammonium sulfide solution for detection of early implantation loss.

Fetal examinations:

Fetal examinations were performed blind to treatment group. Each viable fetus was examined externally, individually sexed, weighed, euthanized by a subcutaneous injection of sodium pentobarbital, and tagged for identification. The detailed external examination of each fetus included, but was not limited to, an examination of the eyes, palate, and external orifices, and each finding was recorded. Crown-rump measurements and degrees of autolysis were recorded for late resorptions, a gross external examination was performed (if possible), and the tissues were discarded.

Each viable fetus was subjected to a visceral examination using a modification of the Stuckhardt and Poppe fresh dissection technique to include the heart and major blood vessels. The sex of each fetus was confirmed by internal examination. Fetal kidneys were examined and graded for renal papillae development. Heads from approximately one-half of the fetuses in each litter were placed in Bouin's fixative for subsequent soft-tissue examination by the Wilson sectioning technique. The heads from the remaining one-half of the fetuses were examined by a midcoronal slice. All carcasses were eviscerated and fixed in 100% ethyl alcohol.

Following fixation in alcohol, each fetus was macerated in potassium hydroxide and stained with Alizarin Red S and Alcian Blue External, visceral, and skeletal findings were recorded as developmental variations (alterations in anatomic structure that are considered to have no significant biological effect on animal health or body conformity and/or occur at high incidence, representing slight deviations from normal) or malformations (those structural anomalies that alter general body conformity, disrupt or interfere with normal body function, or may be incompatible with life).

The fetal developmental findings were summarized by: 1) presenting the incidence of a given finding both as the number of fetuses and the number of litters available for examination in the group; and 2) considering the litter as the basic unit for comparison and calculating the number of affected fetuses in a litter on a proportional basis.

Statistics:

All statistical tests were performed using the WIL Toxicology Data Management System (WTDMS™) using two-tailed tests (except as noted otherwise) at minimum significance levels of 5% and 1%, comparing each test substance-treated group to the control group. Each mean was presented with the standard deviation (S.D.) and the number of animals (N) used to calculate the mean. Data obtained from nongravid animals were excluded from statistical analyses. Where applicable, the litter was used as the experimental unit. Mean maternal body weights (absolute and net), body weight changes (absolute and net), food consumption, and food efficiency, absolute organ weights, gravid uterine weights, numbers of corpora lutea, implantation sites, and viable fetuses, and fetal body weights (separately by sex and combined) were subjected to a parametric one-way ANOVA to determine intergroup differences between the control and test substance-treated groups. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunnett's test was used to compare the test substance-treated groups to the control group. Mean litter proportions (percent per litter) of prenatal data (viable and nonviable fetuses, early and late resorptions, total resorptions, pre- and postimplantation loss, and fetal sex distribution), total fetal malformations and
developmental variations (external, visceral, skeletal, and combined) and each particular external, visceral, and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences to determine intergroup differences between the control and test substance-treated groups. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunn’s test was used to compare the test substance-treated groups to the control group.

Indices:

Intrauterine data were summarized using two methods of calculation.
1.Group Mean Litter Basis
Postimplantation Loss/Litter = [No. Dead Fetuses, Resorptions (Early/Late)/Group] / [No. Gravid Females/Group]

2.Proportional Litter Basis
Summation Per Group (%) = [Sum of Postimplantation Loss/Litter (%)] / [No. Litters/Group]
Where
Postimplantation Loss/Litter (%) = [[(No. Dead Fetuses, Resorptions (Early/Late)/Litter)] / [(No. Implantation Sites/Litter]] x 100

The fetal developmental findings were summarized by:
1. Presenting the incidence of a given finding both as the number of fetuses and the number of litters available for examination in the group; and
2. Considering the litter as the basic unit for comparison and calculating the number of affected fetuses in a litter on a proportional basis as follows:
Summation per Group (%) = [Sum of Viable Fetuses Affected/Litter (%)] / [No. Litters/Group]
Where
Viable Fetuses Affected/Litter (%) = [[No. Viable Fetuses Affected/Litter] / [No. Viable Fetuses/Litter]] x 100

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Test substance-related clinical findings of hair loss around the rump, hindlimbs, dorsal abdominal area, and/or ventral neck, thoracic, and/or abdominal areas were noted for females in the 200 mg/kg bw/day group during gestation Days 8-20. In addition, test substance-related, statistically significantly lower mean body weight gains and/or mean body weight losses were observed in this group during gestation Days 6-9 and 15-20 and when the overall treatment period (gestation Days 6-20) was evaluated. These changes resulted in mean body weights that were statistically significantly lower than the control group during gestation Days 7-20. In addition, mean net body weight and net body weight gain for this group were statistically significantly lower than the control group. Correspondingly, mean food consumption in the 200 mg/kg bw/day group was lower than the control group throughout the treatment period and was considered to be test substance-related. Mean food efficiency in this group was similar to the control group for the overall treatment period. A test substance-related, statistically significantly lower mean adrenal gland weight was noted at 200 mg/kg bw/day. Test substance-related, statistically significantly lower mean body weight gains were noted in the 50 mg/kg bw/day group following the initiation of treatment (gestation Days 6-9) and when the overall treatment period (gestation Days 6-20) was evaluated. These changes corresponded with test substance-related, statistically significantly lower mean food consumption during gestation Days 6-9, 9-12, and 6-20. Mean food efficiency at 50 mg/kg bw/day was similar to the control group for the overall treatment period. Mean net body weight gain for this group was statistically significantly lower than the control group. Mean body weights and mean net body weight at 50 mg/kg bw/day were unaffected by test substance administration. Mean body weight and food consumption parameters at 10 mg/kg bw/day were unaffected by test substance administration. There were no test substance-related effects on mean liver, kidney, or adrenal gland weights at 10 or 50 mg/kg bw/day.

Dose descriptor:

NOAEL

Effect level:

10 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Intrauterine growth and survival were unaffected by test substance administration at dose levels of 10, 50, and 200 mg/kg bw/day. Parameters evaluated included postimplantation loss, live litter size, mean fetal body weights, and fetal sex ratios. Mean numbers of corpora lutea and implantation sites and the mean litter proportions of pre-implantation loss were similar across all groups.

The numbers of fetuses (litters) available for morphological evaluation were 390(25), 401(25), 413(25), and 346(23) in the control, 10, 50, and 200 mg/kg/day groups, respectively. Malformations were observed in 3(3), 0(0), 2(2), and 1(1) fetuses (litters) in the same respective groups, and were considered to be spontaneous in origin. When the total malformations and developmental variations were evaluated on a proportional basis, no statistically significant differences from the control group were noted. Fetal malformations and developmental variations, when observed in the test substance-treated groups, occurred infrequently or at a frequency similar to that in the control group, did not occur in a dose-related manner, and/or were within the WIL historical control data ranges. Based on these data, no fetal malformations or developmental variations were attributed to the test substance.

Developmental toxicity was not observed at dose levels of 10, 50, and 200 mg/kg bw/day when administered orally to maternal animals during gestation Days 6-19.

Abnormalities:

not specified

Developmental effects observed:

not specified

Conclusions:

Based on clinical findings of hair loss and lower mean adrenal gland weights at 200 mg/kg bw/day and mean body weight losses or lower body weight gains with corresponding lower food consumption at 50 and 200 mg/kg bw/day, the lowest-observed-adverse-effect level (LOAEL) was considered to be 50 mg/kg bw/day. A dose level of 10 mg/kg bw/day was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity. In the absence of any evidence of developmental toxicity, a dose level of 200 mg/kg bw/day (the highest dose level tested) was considered to be the NOAEL for prenatal development when the test substance was administered orally by gavage to pregnant Crl:CD(SD) rats (Stump, 2012).

Executive summary:

A study was conducted to assess the developmental toxicity potential of the test substance according to OECD Guideline 414 and OPPTS Guideline 870.3700, in compliance with GLP. The test substance, in the vehicle (0.5% carboxymethylcellulose in water), was administered at the dose levels of 10, 50 and 200 mg/kg bw/day orally by gavage to three groups of 25 bred female Crl: CD(SD) rats once daily from GD 6 through 19. All animals were observed twice daily for mortality and moribundity, and individual clinical observations were recorded from GD 0 through 20. Animals were also observed for signs of toxicity approximately 1 h following dose administration. Body weights and food consumption were recorded on GD 0 and 6-20 (daily). On GD 20, a laparohysterectomy was performed on each female, and the liver, kidneys and adrenal glands were weighed. These tissues and any tissues with gross lesions were preserved for possible future histopathologic examination. The uteri, placentae and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations and corpora lutea were recorded. Gravid uterine weights were recorded, and net body weights and net body weight changes were calculated. The fetuses were weighed, sexed and examined for external, visceral, skeletal malformations and developmental variations. All females survived to the scheduled necropsy on GD 20. Several females in the 200 mg/kg bw/day group were noted with test substance-related hair loss around the rump, hindlimbs, dorsal abdominal area, and/or ventral neck, thoracic, and/or abdominal areas during GD 8 through 20. No other test substance-related clinical findings were noted at any dose level. Test substance-related lower mean body weight gain and/or mean body weight losses and reduced food consumption were observed in a statistically significant and dose-dependent manner in the 50 and 200 mg/kg bw/day groups as early as GD 6-9 and when the overall treatment period (GD 6-20) was evaluated. The effects on mean body weight gain resulted in lower mean body weights in the 200 mg/kg bw/day group but were not of sufficient magnitude to affect mean body weight at 50 mg/kg bw/day. Furthermore, mean net body weight gains at 50 and 200 mg/kg bw/day and mean net body weight at 200 mg/kg bw/day were statistically significantly lower than the control group. Food efficiency at 50 and 200 mg/kg bw/day was similar to the control group for the overall treatment period. There were no test substance-related effects on mean body weights, body weight gains, net body weight gain, food consumption, or food efficiency in the 10 mg/kg bw/day group, net body weight at 10 and 50 mg/kg bw/day, or mean gravid uterine weights at any dose level. A lower mean adrenal gland weight was noted for females in the 200 mg/kg bw/day group relative to the control group. There were no organ weight effects noted at 10 or 50 mg/kg bw/day. No test substance-related macroscopic findings were noted at any dose level. There were no test substance-related effects on intrauterine growth and survival and fetal morphology at dose levels of 10, 50 and 200 mg/kg bw/day. Based on clinical findings of hair loss and lower mean adrenal gland weights at 200 mg/kg bw/day and mean body weight losses or lower body weight gains with corresponding lower food consumption at 50 and 200 mg/kg bw/day, the LOAEL was considered to be 50 mg/kg bw/day. A dose level of 10 mg/kg bw/day was considered to be the NOAEL for maternal toxicity. In the absence of any evidence of developmental toxicity, a dose level of 200 mg/kg bw/day (the highest dose level tested) was considered to be the NOAEL for prenatal development when the test substance was administered orally by gavage to pregnant Crl: CD(SD) rats (Stump DG, 2012).

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

200 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

High quality database.

Additional information

A study was conducted to assess the developmental toxicity potential of the test substance according to OECD Guideline 414 and OPPTS Guideline 870.3700, in compliance with GLP. The test substance, in the vehicle (0.5% carboxymethylcellulose in water), was administered at the dose levels of 10, 50 and 200 mg/kg bw/day orally by gavage to three groups of 25 bred female Crl: CD(SD) rats once daily from GD 6 through 19. All animals were observed twice daily for mortality and moribundity, and individual clinical observations were recorded from GD 0 through 20. Animals were also observed for signs of toxicity approximately 1 h following dose administration. Body weights and food consumption were recorded on GD 0 and 6-20 (daily). On GD 20, a laparohysterectomy was performed on each female, and the liver, kidneys and adrenal glands were weighed. These tissues and any tissues with gross lesions were preserved for possible future histopathologic examination. The uteri, placentae and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations and corpora lutea were recorded. Gravid uterine weights were recorded, and net body weights and net body weight changes were calculated. The fetuses were weighed, sexed and examined for external, visceral, skeletal malformations and developmental variations. All females survived to the scheduled necropsy on GD 20. Several females in the 200 mg/kg bw/day group were noted with test substance-related hair loss around the rump, hindlimbs, dorsal abdominal area, and/or ventral neck, thoracic, and/or abdominal areas during GD 8 through 20. No other test substance-related clinical findings were noted at any dose level. Test substance-related lower mean body weight gain and/or mean body weight losses and reduced food consumption were observed in a statistically significant and dose-dependent manner in the 50 and 200 mg/kg bw/day groups as early as GD 6-9 and when the overall treatment period (GD 6-20) was evaluated. The effects on mean body weight gain resulted in lower mean body weights in the 200 mg/kg bw/day group but were not of sufficient magnitude to affect mean body weight at 50 mg/kg bw/day. Furthermore, mean net body weight gains at 50 and 200 mg/kg bw/day and mean net body weight at 200 mg/kg bw/day were statistically significantly lower than the control group. Food efficiency at 50 and 200 mg/kg bw/day was similar to the control group for the overall treatment period. There were no test substance-related effects on mean body weights, body weight gains, net body weight gain, food consumption, or food efficiency in the 10 mg/kg bw/day group, net body weight at 10 and 50 mg/kg bw/day, or mean gravid uterine weights at any dose level. A lower mean adrenal gland weight was noted for females in the 200 mg/kg bw/day group relative to the control group. There were no organ weight effects noted at 10 or 50 mg/kg bw/day. No test substance-related macroscopic findings were noted at any dose level. There were no test substance-related effects on intrauterine growth and survival and fetal morphology at dose levels of 10, 50 and 200 mg/kg bw/day. Based on clinical findings of hair loss and lower mean adrenal gland weights at 200 mg/kg bw/day and mean body weight losses or lower body weight gains with corresponding lower food consumption at 50 and 200 mg/kg bw/day, the LOAEL was considered to be 50 mg/kg bw/day. A dose level of 10 mg/kg bw/day was considered to be the NOAEL for maternal toxicity. In the absence of any evidence of developmental toxicity, a dose level of 200 mg/kg bw/day (the highest dose level tested) was considered to be the NOAEL for prenatal development when the test substance was administered orally by gavage to pregnant Crl: CD(SD) rats (Stump DG, 2012).

Justification for selection of Effect on developmental toxicity: via oral route:
The study followed internationally accepted guideline and conducted in compliance with GLP.

Justification for classification or non-classification

Based on absence of adverse effects in a two generation reproductive toxicity study and a developmental toxicity study, no classification under CLP criteria (EC 1272/2008) or Directive 67/548/EEC was warranted.