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Toxicological information

Developmental toxicity / teratogenicity

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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:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Remarks:
Conducted according to guideline in effect at the time of study conduct
Qualifier:
according to guideline
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Deviations:
no
Remarks:
Conducted according to guideline in effect at the time of study conduct
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
Remarks:
Conducted according to guideline in effect at the time of study conduct
Qualifier:
according to guideline
Guideline:
other: MAFF Japan Test Guidelines for Agricultural Chemicals 12-Nousan-8147 (2000)
Deviations:
no
Remarks:
Conducted according to guideline in effect at the time of study conduct
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Test material form:
solid
Details on test material:
Purity: 89.7% by analysis

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories International, Inc., Raleigh, North Carolina, U.S.A.
- Age at study initiation: Approximately 67 days
- Weight at study initiation: GD 0 body weights ranged from 200-225 grams at grouping
- Housing: Animals were housed in groups in solid-bottom caging with bedding and Nestlets™ as enrichment.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: At least 3 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-26ºC (68-79ºF)
- Humidity (%): 30-70%
- Air changes (per hr): Not reported
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.5% methylcellulose with 0.1% Tween® 80
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- Formulations of the test substance in the vehicle were prepared and used within the period of established stability. After an 8-day room temperature stability had been confirmed, formulations were prepared approximately weekly. The test substance was suspended in 0.5% methylcellulose with 0.1% Tween® 80. Dosing formulations were stored room temperature until used.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Not reported
- Concentration in vehicle: 0.5% methylcellulose with 0.1% Tween® 80
- Amount of vehicle (if gavage): 10 mL/kg
- The vehicle was assumed to be stable under the conditions of the study. The vehicle was stored at room temperature.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples were collected from the top, middle, and bottom of each concentration at the initial dose preparation and were analyzed to verify homogeneity and concentration (average of homogeneity samples) of the test substance in the formulations. Toward the end of the study samples were taken to verify concentration. A sample of control vehicle was analyzed together with each set of samples to verify the absence of the test substance in the vehicle. The lowest and highest concentrations of the formulations were stored at room temperature for 7 and 14 days and then re-suspended before sampling for analysis to verify the stability of the test substance in the formulations. The stability of the test substance in the vehicle was demonstrated by comparing samples stored at room temperature with the average measured values of top, middle, and bottom homogeneity samples. On days samples were taken, back-up samples were also taken and frozen for possible analysis, if required. Remaining samples were stored frozen and were subsequently discarded after satisfactory analytical results were obtained.
At the time of the analysis, the samples were diluted with an appropriate solvent and analyzed by ultra high-performance liquid chromatography (UHPLC) with ultraviolet (UV) detection.
Duration of treatment / exposure:
Beginning on gestation day (GD) 6 through GD 20
Frequency of treatment:
Daily
Duration of test:
15 days
Doses / concentrationsopen allclose all
Dose / conc.:
32 mg/kg bw/day
Dose / conc.:
125 mg/kg bw/day
Dose / conc.:
500 mg/kg bw/day
No. of animals per sex per dose:
22
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were selected based on results from a previously conducted pilot developmental toxicity study. In that study, groups of 8 rats/group were administered the test substance at 0, 50, 250, or 1000 mg/kg body weight/day. Statistically significant, treatment-related effects observed at 1000 mg/kg/day consisted of reduced body weight and food consumption parameters as compared to controls in pregnant females. Mean terminal absolute and adjusted (minus products of conception) body weight was 13% and 12% lower than the control at this level, respectively. At 1000 mg/kg/day overall mean (GD 6-21) absolute and adjusted body weight gains were 33% and 73% lower than control values, respectively, as a result of reduced body weight gains. Mean overall (GD 6-21) food consumption was 27% lower than the control. At this level mean fetal weights were also reduced as compared to the control. There were no treatment-related statistically significant adverse effects on maternal or developmental endpoints at 250 mg/kg/day or lower. There were no test substance-related deaths or gross postmortem findings in dams at any treatment level. The mean number of corpora lutea, implantation sites, resorptions and live fetuses, as well as sex ratios, were comparable across all groups. There were no test substance-related fetal external malformations or variations observed at any treatment level. Under the conditions of that study, the maternal and developmental NOAEL was 250 mg/kg/day based on the aforementioned effects on body weight and food consumption parameters in pregnant females and reduced mean fetal weights in litters at 1000 mg/kg/day.
For this study, animals were dosed orally by gavage once daily on GD 6-20. Animals were sacrificed on GD 21. The volume administered (10 mL/kg) was based on the most recent body weight.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes, at least daily during quarantine and pretest. Twice daily (AM and PM)

DETAILED CLINICAL OBSERVATIONS: Yes, on GD 4; twice daily on GD 6-20 (during weighing and at least 2 hours post-dosing); clinical signs observed at other times were recorded by exception.

BODY WEIGHT: Yes
- Time schedule for examinations: Daily on GD 4 and GD 6-21

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, GD 4, 6, 8, 10, 12, 14, 16, 18, 20, and 21
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 21
- Organs examined: Whole body
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: For each female with visible implantation sites, the types of implantations (live and dead fetuses, early and late resorptions) and their relative positions in the uterus were recorded. The body weight of each live fetus was recorded.
Fetal examinations:
- External examinations: Yes [all per litter]
- Soft tissue examinations: Yes [all per litter]
- Skeletal examinations: Yes [all per litter]
- Head examinations: Yes [half per litter]
Statistics:
See any other information on materials and methods section

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
There were treatment-related, adverse effects observed on body weight gain parameters at 500 mg/kg/day. These effects were most pronounced during the first two days of dosing (GD 6-8) when animals lost an average of 1.8 grams compared to an average gain of 9.8 grams in the control group. Mean maternal body weights were also statistically reduced (4% lower than control) on gestation day 8 at this level. Some recovery was evident from gestation days 8-12 for these animals, however the overall (GD 6-21) absolute mean body weight gains were 11% (statistically significant) lower than control group means. These overall reductions were attributed primarily to the lower body weight gains observed during the first few days of dosing. Mean adjusted (minus products of conception) body weight gains, were 17% lower than the control group mean at 500 mg/kg/day.
Treatment-related, potentially adverse reductions in mean body weight gain were observed at 125 mg/kg/day. Mean body weight gains were generally lower than control group means during the study. However, these reductions were not statistically significantly different (p ≤ 0.05) from control and the magnitude of change (% difference from control) for overall absolute body weight gains was minimal (6%) at this treatment level. When the overall body weight gains were calculated excluding the products of conception, mean body weight gains were 14% lower (not statistically significant) than the control group mean. As previously mentioned, there were no adverse, treatment-related effects on body weight parameters in a previously conducted pilot developmental toxicity study up to 250 mg/kg/day. However, comparing the similar magnitude of effects observed at 500 mg/kg/day and 125 mg/kg/day, these effects at 125 mg/kg/day were considered marginal, but potentially adverse.
There were no adverse treatment-related effects on mean maternal body weight parameters at any level tested.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Treatment-related, adverse effects on food consumption were observed at 500 mg/kg/day. As stated above for body weight parameters, the effects on food consumption were most pronounced during the first few days of dosing when mean food consumption was up to 31% lower than the control group mean. Mean food consumption at this level was also statistically reduced (compared to control) on gestation days (GD) 8-10 (12% lower than control), GD 14-16 (8% lower than control, and GD 18-20 (7% lower than control). Overall (GD 6-21) mean food consumption was 9% lower (statistically significant) than the control group as a result.
Treatment-related, but non-adverse effects on food consumption were observed at 125 mg/kg/day. Mean food consumption was up to 11% lower than the control group at this level from GD 6-10. In addition, there was a 6% reduction in mean food consumption as compared to the control group mean on GD 18-20. However, overall mean food consumption was not significantly reduced at this level. Therefore, the treatment-related effects noted at 125 mg/kg/day were not considered adverse.
Gross pathological findings:
no effects observed

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
32 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake

Results (fetuses)

Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Mean fetal weights were 4.6% lower than the control group (statistically significant) at 500 mg/kg/day. This finding was related to the maternal weight and nutritional effects reported above; however, the mean fetal weight observed at this level (5.67 grams) was within the facility’s historical control ranges (mean 5.85 grams; mean ranges 5.56 to 6.01 grams; 17 studies; 2010 to present). Therefore, this reduction in mean fetal weight at 500 mg/kg/day, while test substance-related, was not considered adverse as it was minimal in nature. Mean fetal weights were comparable to control group mean at ≤125 mg/kg/day.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
> 500 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: highest dose tested

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

Historical Control Data for Mean Fetal Weights:

 Study Route  Study Start Mean fetal weight (grams) 
 1 gavage 3/8/2015 5.94
 2 gavage 11/9/2014 5.98 
 3 gavage 10/26/2014 5.97 
 4 gavage 10/12/2014 5.76 
 5 gavage  8/31/2014 6.01 
 6 gavage 8/11/2013 5.71 
 7 gavage 7/14/2013 5.90 
 8 gavage 6/9/2014 5.91 
 9 gavage 6/9/2013 5.89 
 10 gavage 5/5/2013 5.72 
 11 gavage 9/30/2012 5.85 
 12 gavage 5/1/2011 6.01
 13 gavage 1/17/2010 5.72 
 14 gavage 5/4/2014 5.84 
 15 gavage 4/7/2014 5.56 
 16 gavage 10/20/2013 5.89 
 17 gavage 3/11/2013 5.78 
Mean 5.85 
SD 0.13
Min 5.56
 Max 6.01

Applicant's summary and conclusion

Conclusions:
NOAEL (Maternal): 32 mg/kg/day
NOAEL (Developmental): >500 mg/kg/day
Executive summary:

The purpose of this study was to evaluate the potential maternal and developmental toxicity of the test substance in pregnant rats. The test substance, was suspended in 0.5% methylcellulose and a small percentage of Tween® 80 was used to aid dispersion. The formulations were administered by gavage to timed-mated animals daily beginning on gestation day (GD) 6 through GD 20. The day of mating was defined as gestation day 0.

Dose levels were selected based on results from a previously conducted pilot developmental toxicity study. Based on data from that study, the dose levels for the current study were selected to be 0, 32, 125, and 500 mg/kg/day. The dose volume was 10 mL/kg for all groups. Samples of the dosing formulations were collected and analyzed near the beginning of dose administration to verify homogeneity and concentration and then towards the end of the dosing period to verify concentration. The results of these analyses confirmed that the formulations were at the targeted concentrations, uniformly mixed, and stable under the experimental conditions used during the study.

During the in-life portion of the study, body weights, food consumption, and clinical observations before and after dosing were collected at regular intervals. All dams were euthanized on GD 21. Gross necropsy included an examination and description of uterine contents, including counts of corpora lutea, implantation sites, resorptions, and live and dead foetuses. All live foetuses were examined externally and euthanized. Following euthanasia, fresh visceral and head examinations were performed on selected foetuses. The foetal carcasses were then processed and examined for skeletal alterations.

Under the conditions of this study, there was adverse maternal toxicity at 500 mg/kg/day consisting of reduced mean body weight and food consumption parameters in females. Treatment-related effects were noted at 125 and consisted of effects on body weight and food consumption parameters. The changes in body weight parameters at this level were considered potentially adverse.

There was no adverse developmental toxicity observed at any level tested. Mean fetal weight was reduced 4.6% at 500 mg/kg/day compared to the concurrent control mean, but the reduction was not considered to be adverse because it was minimal in magnitude and was within the range of the test facility historical control group means. There was no early mortality or adverse treatment-related effects on clinical observations, maternal gross observations, mean number of implantation sites, resorptions, live foetuses, foetal sex ratio, foetal weight, or foetal alterations. Therefore, the no-observed-adverse effect level (NOAEL) for maternal toxicity was considered to be 32 mg/kg/day. The NOAEL for developmental toxicity was considered to be greater than 500 mg/kg/day.