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Administrative data

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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guidelineopen allclose all
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

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: pellets
Details on test material:
- Name of test material (as cited in study report): MON 102100

Test animals

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

Administration / exposure

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
Doses / concentrations
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.

Examinations

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

Results and discussion

Results: maternal animals

Maternal developmental toxicity

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.

Effect levels (maternal animals)

open allclose all
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

Results (fetuses)

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.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

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).