Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labeling and/or risk assessment.

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Remarks:
The study was conducted according to the guideline in effect at the time of study conduct.
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
Remarks:
The study was conducted according to the guideline in effect at the time of study conduct.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Purity: 84%

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: approximately 12 weeks
- Weight at study initiation: 228 g to 285 g
- Housing: individually housed (except during mating) in solid bottom cages (plastic maternity cages) containing ground corncob nesting material
- Diet: PMI Nutrition International, LLC Certified Rodent LabDiet® 5002, ad libitum
- Water: Reverse osmosis-purified (on-site), ad libitum
- Acclimation period: 14 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.3°C to 22.0°C)
- Humidity (%): 43.5% to 51.1%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
GAVAGE
- Solvent Used: deionized water
- Preparation frequency and procedure: A 150 mg/mL stock solution of the test substance was prepared weekly for use in preparing the test substance dosing formulations. The stock solution was stirred during preparation and overnight in the refrigerator.
The test substance formulations were prepared approximately weekly as dilutions of the stock solution as single formulations for each dosage level, divided into aliquots for daily dispensation, and stored refrigerated, stirring overnight in the refrigerator. The test substance formulations were stirred continuously throughout the preparation, sampling, and dose administration procedures.
The first dosing formulations were visually inspected by the study director’s designee and were found to be visibly homogeneous and acceptable for
administration.
- Adjusted for purity: yes

Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
Stability and resuspension homogeneity were established in a previous study. Test substance formulations were stable following 12 days of refrigerated (2-8°C) storage at concentrations of 0.01 and 100 mg/mL and were homogeneous after resuspension following 12 days of refrigerated storage (2-8°C). Therefore, stability and resuspension homogeneity analyses were not conducted in this study.

Prior to the initiation of dose administration, samples for homogeneity determination were collected from the top, middle, and bottom strata of the 1 and 100 mg/mL dosing formulations; the middle stratum of these formulations were used for concentration determination. Samples were also collected from the middle stratum of the 10 mg/mL formulation and the vehicle control for concentration analyses prior to the initiation of dose administration. Samples for concentration analyses were collected from the middle stratum of each dosing formulation (including the vehicle formulation administered to the control group) from the formulations prepared for the last week of dose administration. The analyzed dosing formulations were within the SOP range for suspensions (85% to 115%) and were homogeneous. Based on these results, the protocol-specified dosages of test article were administered to the animals. The test article was not detected in the vehicle formulation that was administered to the control group
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1
- Length of cohabitation: following positive evidence of mating
- Proof of pregnancy: the presence of a vaginal copulatory plug or the presence of sperm in a vaginal lavage was evidence of mating, that day was considered Gestation Day 0
Duration of treatment / exposure:
Gestation Days 6 to 20
Frequency of treatment:
Daily
Duration of test:
21 days
No. of animals per sex per dose:
22 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dosage levels were selected based on the results of previous studies and were provided by the Sponsor after consultation with the Study Director.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: recorded on gestation days 0 and 6-21 (daily)

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined: recorded on gestation days 0 and 6-21 (daily).

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 21
- Organs examined: thoracic, abdominal, and pelvic cavities were opened and the contents (uterus, ovaries, kidneys and liver) were examined.

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
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: all per litter
Statistics:
see Materials and Methods below

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Original Study:

One female in the 1000 mg/kg/day was found dead on gestation day 20. This female had lower mean body weight gains and/or food consumption compared to the control group during gestation days 12-18. The test substance-related liver and kidney changes (moderate coagulative necrosis in the liver and fibrin thrombi in the glomerular capillaries) noted microscopically were considered the cause of death in this animal. Four and 9 females in the 100 and 1000 mg/kg/day groups, respectively, delivered early on gestation day 21. The mortality in the 1000 mg/kg/day group and early deliveries in the 100 and 1000 mg/kg/day groups were considered test substance-related. All other females survived to the scheduled laparohysterectomy.

Test substance-related clinical findings were noted in the 1000 mg/kg/day group and consisted of yellow material on various body surfaces and salivation or evidence thereof (clear material around the mouth). No test substance-related clinical findings were observed in the 10 and 100 mg/kg/day groups.

A transient mean body weight loss was observed in the 1000 mg/kg/day following administration of the first dose (gestation days 6-7), resulting in a lower mean body weight gain during gestation days 6-9. Correspondingly lower mean food consumption was also observed in the 1000 mg/kg/day group. The lower mean body weight gain early in gestation was considered test substance-related. Mean body weight gain was also lower in this group during gestation days 18-21 and when the entire treatment period (gestation days 6-21) was evaluated. The lower mean body weight gain late in gestation in this group as well as a lower mean gravid uterine weight and lower mean body weight on gestation day 21 (8.4% lower than the control group) was attributed to the test substance-related lower mean fetal body weights observed at 1000 mg/kg/day, supported by the lack of effect on mean net body weight and net body weight gain. Mean body weights, body weight gains, net body weights, net body weight gains, gravid uterine weights, and food consumption in the 10 and 100 mg/kg/day groups were similar to the control group, with the following exception. A lower mean gravid uterine weight was noted in the 100 mg/kg/day group and was attributed to the lower mean fetal weights observed in this group.

An edematous pancreas was noted in 2 females that delivered early in the 1000 mg/kg/day group at necropsy; the relationship of this finding to the test substance is uncertain. Other macroscopic findings occurred in single females and/or are not uncommon in females that deliver. Higher mean liver weights were noted in the 100 and 1000 mg/kg/day group females, and higher mean kidney weight was observed in the 1000 mg/kg/day group. There were no microscopic correlates to the higher mean kidney weight. Focal necrosis of the liver was noted in some females in the 100 and 1000 mg/kg/day groups in a dose-related manner. In addition, test substance-related hepatocellular hypertrophy was noted at 1000 mg/kg/day; hypertrophy was morphologically consistent with a PPARα agonist.

Follow-Up Study:

There was a dose-related increase in the number of dams found delivered in their cages on the morning of GD 21. There were 0 and 3 dams found delivered at 0 and 1000 mg/kg/day, respectively. Mean foetal weight, mean net maternal weight, and mean maternal food consumption was lower than controls at 1000 mg/kg/day. There was a test substance-related increased in the incidence of wet fur at 1000 mg/kg/day. Mean maternal kidney and liver weights were increased, and microscopic changes in the liver at 1000 mg/kg included increased hepatocellular hypertrophy and focal coagulative necrosis.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
10 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Mean fetal weights were 8.8% and 28.1% lower in the 100 and 1000 mg/kg/day groups, respectively. No test substance-related effects on mean fetal weight were noted in the 10 mg/kg/day group. Intrauterine survival was not affected by test substance administration at any dosage level.

There were no test substance-related fetal malformations. A higher mean litter proportion of 14th rudimentary ribs was observed in the 1000 mg/kg/day group, resulting in a higher mean litter proportion of total skeletal variations and total developmental variations. Although considered test substance-related, the increase in the number of fetuses with this finding was not considered adverse because it has been suggested that 14th rudimentary ribs are resorbed during postnatal development. No test substance-related developmental variations were observed in the 10 and 100 mg/kg/day groups.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
10 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: The no-observed-adverse-effect level (NOAEL) for developmental toxicity was considered to be 10 mg/kg/day based on early deliveries and lower mean fetal weights at 100 and 1000 mg/kg/day.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
This study and the conclusions which are drawn from it fulfill the quality criteria (validity, reliability, repeatability).
NOAEL maternal toxicity = 10 mg/kg/bw
NOAEL developmental toxicity = 10 mg/kg/bw
Executive summary:

The objective of the study was to determine the potential of the test substance to induce developmental toxicity after maternal exposure during the critical period of organogenesis, to characterize maternal toxicity at the exposure levels tested, and to determine a no-observed-adverse-effect level (NOAEL) for maternal and developmental toxicity. to 3 groups of 22 bred female Crl:CD(SD) rats once daily from gestation day 6 through 20. Dosage levels were 10, 100, and 1000 mg/kg/day. A concurrent control group of 22 bred females received the vehicle (deionized water) on a comparable regimen.The no-observed-adverse-effect level (NOAEL) for maternal and developmental toxicity was considered to be 10 mg/kg/day based on mortality and lower mean body weight gains and food consumption at 1000 mg/kg/day and early deliveries, microscopic findings in the liver (focal necrosis), and lower mean fetal weights at 100 and 1000 mg/kg/day. At 1000 mg/kg/day, there were additional test substance-related effects that were not considered adverse and consisted of higher kidney and liver weights and hepatocellular hypertrophy. To verify the findings of an apparent dose-related increase in dams that delivered litters just prior to scheduled euthanasia and caesarean section on gestation day 21, a study was conducted using the same experimental design, but was limited to a control group and a group dosed at 1000 mg/kg/day. Under the conditions of the follow-up study, test substance-related findings were observed that were consistent with and confirmed the findings of the original study. Findings included reductions in maternal body weight and food consumption, increased early deliveries, reduced foetal weight, increased maternal liver and kidney weight, microscopic hepatocellular hypertrophy and focal necrosis in the liver. These findings were consistent with those of the original study with the test material at this same dose level.