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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
Study period:
Nov 2013 - Mar 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was performed according to OECD guidelines and according to GLP principles.

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Specific details on test material used for the study:
EXP1200078
Lot no. E00048-256
Exp. date: 31 December 2014
Dark brown, opaque, viscous liquid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Portage, MI, USA
- Age at study initiation: females were 13 weeks old when paired for breeding
- Weight at study initiation: 205-296 g on gestation day 0
- Housing: Upon arrival and until pairing, all rats were individually housed in clean, stainless steel wire-mesh cages. 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.
- Diet (ad libitum): basal diet used in this study, PMI Nutrition International, LLC Certified Rodent LabDiet® 5002
- Water (ad libitum): municipal water
- Acclimation period: at least 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.3 to 23.2
- Humidity (%): 39.2 to 54.7
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 03 December 2013 To: 10 January 2014

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: mineral oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test substance formulations were prepared approximately weekly as single formulations for each dosage level, divided into aliquots for daily dispensation, and stored at room temperature, protected from light. The test substance formulations were stirred continuously throughout the preparation, sampling, and dose administration procedures.

VEHICLE
- Lot/batch no.: 2BK0397 and 1CK0210
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Homogeneity and stability of the test substance formulations in the vehicle at concentrations of 0.1 and 400 mg/mL following 12 days of room temperature storage were previously established in a separate study (Coffee, 2013, WIL-537029). Therefore, homogeneity and stability assessments were not conducted in the current study.
Samples for concentration analysis were collected from the middle stratum of each dosing formulation (including the control group) prepared during the first and last weeks of the in-life phase of the study. One set of samples from each collection was subjected to the appropriate analyses. The remaining set of samples was stored at room temperature as back-up. All analyses were conducted by the WIL Research Analytical Chemistry Department using a validated high performance liquid chromatography method with fluorescence detection.
Details on mating procedure:
- Impregnation procedure: cohoused 1:1 with each animal judged to be in good health and meeting acceptable body weight requirements was placed in a suspended wire-mesh cage with a resident male from the same strain and source for breeding. Resident males were untreated, sexually mature rats. These rats were maintained under similar laboratory conditions as the females.
- M/F ratio per cage: 1/1
- Proof of pregnancy: vaginal plug or sperm in vaginal lavage referred to as day 0 of pregnancy
Duration of treatment / exposure:
Gestation days 6-19
Frequency of treatment:
Once daily
Duration of test:
Gestation day 0 through 20
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Vehicle control
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dosage levels were selected based on the results of a previous 28-day toxicity study in rats (Haas, 2013, WIL-537026). In that study, the test substance was administered orally by gavage once daily to male and female rats for 28 consecutive days at dosage levels of 100, 300, and 1000 mg/kg/day. All animals survived to the scheduled euthanasia and there were no treatment-related effects on body weights or food consumption at any dosage level tested. Test substance-related higher mean liver weights were noted in the 1000 mg/kg/day group males and females at the scheduled necropsy, but were not considered adverse. As a result, dosage levels of 100, 300, and 1000 mg/kg/day (the same dosage levels used in the previous study) were assessed in the current study.
- 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 and DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily, once in the morning and once in the afternoon, for moribundity and mortality. Individual clinical observations were recorded daily from gestation days 0 through 20. Animals were also observed for signs of toxicity approximately 1 hour following dose administration.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual maternal body weights were recorded on gestation days 0 and 6-20 (daily). Group mean body weights were calculated for each of these days. Mean body weight changes were calculated for each corresponding interval and also for gestation days 6-9, 9-12, 12-15, 15-20, and 6-20.

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
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

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. In all instances, the postmortem findings were correlated with the antemortem observations, and any abnormalities were recorded. Maternal tissues were preserved in 10% neutral-buffered formalin for possible future histopathologic examination only as indicated by the gross findings. Representative sections of corresponding organs from a sufficient number of control animals were retained for comparison.
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: placentae examined, 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:
- 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]
Half of the littr by Wilson's sectioning technique and hlaf by midcoronal slice.
Statistics:
All statistical tests were performed using WTDMS™ unless otherwise noted. Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test substance-treated group to the control group. Data obtained from nongravid animals were excluded from statistical analyses.
Maternal body weights (absolute and net), body weight changes (absolute and net), and food consumption, 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. 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. If the nonparametric 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:
Postimplantation Loss/Litter = (No. Dead Fetuses, Resorptions (Early/Late)/Group) / (No. Gravid Females/Group)
Summation Per Group (%) = (Sum of Postimplantation Loss/Litter (%)) / (No. Litters/Group)
Postimplantation Loss/Litter (%) = (No. Dead Fetuses, Resorptions (Early/Late)/Litter) / (No. Implantation Sites/Litter) x 100
Summation per Group (%) = (Sum of Viable Fetuses Affected/Litter (%)) / (No. Litters/Group)
Viable Fetuses Affected/Litter (%) = (No. Viable Fetuses Affected/Litter) / (No. Viable Fetuses/Litter) x 100

Historical control data:
These are available.

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):
A test substance-related absence of body weight gain (0 g) was noted in the 1000 mg/kg/day group following the onset of dose administration (gestation day 6-7) and resulted in a lower mean body weight gain in this group during the gestation day 6-9 cumulative interval; differences from the control group were significant (p<0.01). Although mean body weight gains in the 1000 mg/kg/day group were similar to the control group throughout the remainder of the treatment period (gestation days 9-12, 12 15, and 15-20), the initial decrement was of sufficient magnitude to result in a significantly (p<0.01) lower mean body weight gain when the entire treatment period (gestation days 6-20) was evaluated. In addition, mean body weights in this group were up to 5.6% lower than the control group during the treatment period; the differences were significant (p<0.05) during gestation days 9-11 and 14-20. Therefore, the effect on mean body weight gain noted at the initiation of dose administration was considered to be adverse. Mean net body weight and net body weight gain in the 1000 mg/kg/day group were significantly (p<0.05 or p<0.01) lower than the control group, while mean gravid uterine weight in this group was similar to the control group.

In the 300 mg/kg/day group, sporadically lower mean body weight gains, primarily during gestation days 6-9 and 15-20, were noted during the treatment period compared to the control group; the differences were significant (p<0.05) on gestation day 17-18 only. Although these sporadic lower mean body weight gains resulted in a significantly (p<0.05) lower mean body weight gain when the entire treatment period (gestation days 6-20) was evaluated, these differences were not of sufficient magnitude to affect mean absolute body weights. Therefore, the lower mean body weight gains noted in the 300 mg/kg/day group were considered to be test substance-related but not adverse.

Mean maternal body weight gains in the 100 mg/kg/day group were unaffected by test substance administration during the treatment period; differences from the control group were generally slight and not statistically significant. Despite the absence of effects on mean body weight gain, significantly (p<0.05) lower (4.7% to 5.2%) mean body weights were noted in this group during gestation days 9-11 and 14-16 compared to the control group.

Significantly (p<0.05 or p<0.01) lower mean net body weight gains were noted in the 100 and 300 mg/kg/day groups compared to the control group, resulting in a significantly (p<0.05) lower mean net body weight at 100 mg/kg/day and a slightly lower (not statistically significant) mean net body weight at 300 mg/kg/day. In the absence of a dose response, the effects on mean net body weight and net body weight gain were not considered test substance-related. Mean gravid uterine weights in these groups were comparable to the control group.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
Lower (generally significant, p<0.05 or p<0.01) mean food consumption, evaluated as g/animal/day and g/kg/day, was noted in the 1000 mg/kg/day group during the study (gestation days 6-9, 12-15, and/or 15-20) and resulted in significantly (p<0.01) lower mean food consumption when the entire treatment period (gestation days 6-20) was evaluated. The lower mean food consumption noted during gestation days 6-9 and 6-20 corresponded to the lower mean body weight gains noted during these intervals, and therefore was considered to be test substance-related and adverse.

Mean food consumption in the 100 and 300 mg/kg/day groups were unaffected by test substance administration. In the 300 mg/kg/day group, mean food consumption was significantly (p<0.05) lower than the control group on gestation day 17-18 (evaluated as g/animal/day only), which corresponded to a lower mean body weight gain on this day.

However, the lower mean food consumption on this day was not considered to be test substance-related, due to the transient nature. Furthermore, mean food consumption in the 300 mg/kg/day group was similar to the control group during the cumulative intervals (gestation days 6-9, 9-12, 12-15, and 15-20) and when the entire treatment period (gestation days 6-20) was evaluated. In the 100 mg/kg/day group, significantly (p<0.05) lower mean food consumption (evaluated as g/animal/day only) was noted during gestation days 6-9 and subsequently when the overall treatment period (gestation
days 6-20) was evaluated. However, due to the slight nature of the differences (1 g to 2 g) and in the absence of effects on body weight gain in the group, the lower mean food consumption noted in the 100 mg/kg/day group was not considered to be test substance related. No other statistically significant differences were noted in the 100 and 300 mg/kg/day groups.

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
Intrauterine growth and survival were unaffected by test substance administration at dosage levels of 100, 300, and 1000 mg/kg/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. Differences from the control group were slight and not statistically significant.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
Differences from the control group were slight and not statistically significant.
Early or late resorptions:
no effects observed
Description (incidence and severity):
Differences from the control group were slight and not statistically significant.
Dead fetuses:
no effects observed
Description (incidence and severity):
None found in controls or any dose group.
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
Description (incidence and severity):
With the exception of 1 and 2 females in the control and 300 mg/kg/day groups, respectively, all females were determined to be gravid.
Other effects:
no effects observed
Details on maternal toxic effects:
Maternal toxic effects:yes. Remark: lower mean body weight gain

Details on maternal toxic effects:
No mortality nor adverse clinical signs were observed: increased incidences of clear and red material around the mouth were noted for females in the 1000 mg/kg/day group, starting at gestation day 12 and continued sporadically through gestation day 19. These findings did not persist to the daily examinations the following day and therefore were not considered adverse.
Body weight: A statistically significant lower mean body weight gain in the 1000 mg/kg/day group was noted during gestation days 6-9.This resulted in a significant lower mean body weight gain for the entire treatment period, although mean body weight gains in the 1000 mg/kg/day group were similar the the control group from gestation days 9 onwards. Mean body weights in this group were statistically significantly decreased during the treatment period: 5.6% lower compared to controls. In addition, some effects on mean body weight and/or body weight gain were observed in the 100 and 300 mg/kg/day dose groups. However, no dose response was observed, and the actual mean body weights were only approx 4% lower compared to controls. Therefore, these effects are not considered substance related.
A significantly lower mean food consumption was observed in the 1000 mg/kg/day dose goup.
At necropsy no test substance related findings were observed in any dose group.

Effect levels (maternal animals)

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food efficiency
Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Maternal abnormalities

Key result
Abnormalities:
no effects observed
Description (incidence and severity):
No abnormalities / malformations detected in any soft tissue, skeletal exam, external or any other parameter.

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): 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
Other effects:
no effects observed
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
No significant changes in the laparohysterectomy data were observed. Malformations were observed in all treatment (and control) groups, and were considered spontaneous in origin. No statistically significant difference from the control group was noted, no dose related manner was observed, and/or effects were within the historical control data ranges.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks:
Test material given to maternal animals.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
changes in sex ratio
fetal/pup body weight changes
changes in litter size and weights
changes in postnatal survival
external malformations
skeletal malformations
visceral malformations
other:
Remarks on result:
not determinable due to absence of adverse toxic effects

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Treatment related:
no

Any other information on results incl. tables

The analyzed dosing formulations were within the range 85% to 115%. The test substance was not detected in the analyzed vehicle formulation (Group 1).

Results of the analyses of dosing formulations are summarized below.

Results of Concentration Analyses

 

Mean Concentration, mg/mL (% of Target)

Date of Preparation

Group 2

(20 mg/mL)

Group 3

(60 mg/mL)

Group 4

(200 mg/mL)

19 December 2013

20.2 (101)

60.6 (101)

207 (104)

02 January 2014

20.7 (104)

61.7 (103)

207 (103)

 

Applicant's summary and conclusion

Conclusions:
In an OECD414 test gudeline study, female rats orally dosed with EXP1200078 during gestation days 6 through 19, did show adverse effects at the highest dose tested and no effects were observed on the fetuses. Therefore, the NOAEL for maternal toxicity was determined to be 300 mg/kg bw/day and for developmental toxicity the NOAEL is >=1000 mg/kg bw/day.
Executive summary:

EXP1200078 was administered by daily oral gavage to female Sprague Dawley rats during gestation days 6 through 19 at dose levels of 0, 100, 300 or 1000 mg/kg bw/day according to the OECD 414 guideline. On gestation day 20 a laparohysterectomy was performed on each female. All females in the 0, 100, 300 and 1000 mg/kg/day groups survived to the scheduled necropsy. A lower mean body weight gain was noted in the 1000 mg/kg/day group during gestation days 6 -9 resulting in a lower mean body weight gain and body weight mean over the entire treatment period. Mean food consumption in this group was lower than the control group throughout the treatment period. Mean gravid uterine weight in the 1000 mg/kg/day group was similar to the control group. No test substance related macroscopic findings were observed at any dose level. Therefore, the NOAEL for maternal toxicity is determined to be 300 mg/kg/day.

Intrauterine growth, survival and fetal morphology were unaffected by maternal test substance administration at all dose levels. Therefore, the NOAEL for embryo/fetal development was considered to be >=1000 mg/kg bw/day based on the absence of adverse effects.