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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:
2014-06-06 to 2014-07-09
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

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:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes
Remarks:
WIL Research Europe B.V., Hambakenwetering 7, 5231 DD 's-Hertogenbosch, The Netherlands

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- State of aggregation: liquid
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Stability under test conditions: Stability for at least 6 hours at room temperature is confirmed over the concentration range 60-200 mg/mL
- Storage condition of test material: At room temperature in the dark

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: Females were approximately 11 weeks
- Housing: During acclimatization, females were housed in groups of maximum 5 animals/cage in Macrolon plastic cages (MIV type, height 18 cm).
- Mating: Females were placed in the males’s homecage in Macrolon plastic cages (MIII type, height 18 cm). During the weekend, mating procedures were suspended and the females were housed in groups of maximum 5 animals/cage in Macrolon plastic cages (MIV type, height 18 cm).
- Post-coitum: Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm). Sterilized sawdust as bedding material and paper as cage-enrichment/nesting material were supplied.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany)
- Water: Free access to tap-water
- Acclimation period: At least 5 days prior to pairing

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24
- Humidity (%): 40 to 70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 10 June 2014 To: 09 July 2014

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Remarks:
Polyethylene glycol 400, specific gravity 1.125 (Merck, Darmstadt, Germany).
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. Adjustment was made for the density of the test substance (1.22 g/cm^3) and the specific gravity of the vehicle (1.125). No correction was made for the purity/composition of the test substance.

VEHICLE
- Justification for use and choice of vehicle: Based on trial formulations performed at WIL Research Europe B.V.
- Amount of vehicle: 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted on a single occasion during the treatment phase (20 June 2014), according to a validated method (Project 505079; BASF Project 10Y0568/12X517). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations).
The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%.
Details on mating procedure:
Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the female was separated from the male. When sufficient mated females had been obtained for each dose group, the surplus females were removed from the study. During the weekend, mating procedures were suspended.
Duration of treatment / exposure:
From Day 6 to Day 19 post-coitum, inclusive.
Frequency of treatment:
Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 300, 1000 mg/kg
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were based on a maternal toxicity study (Project 505076; BASF Project 10R0568/12X516) in which mated rats (10 per group) were exposed by oral gavage from Day 6 to Day 19 post-coitum, inclusive, at target dose levels of 0, 300 and 1000 mg/kg bw/day. No toxicologically relevant findings were noted.

Examinations

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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily from Day 0 post-coitum onwards up to the day prior to necropsy. The time of onset, grade and duration of any observed sign was recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: Days 0, 3, 6, 9, 12, 15, 17 and 20 post-coitum.
The following weight parameters were derived:
- Corrected terminal maternal body weight (=terminal body weight - gravid uterus weight)
- Corrected body weight gain (=corrected body weight - body weight on gestation Day 6).

FOOD CONSUMPTION: Yes
Days 0-3, 3-6, 6-9, 9-12, 12-15, 15-17 and 17-20 post-coitum.

WATER CONSUMPTION: Yes
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
On Day 20 post-coitum, all animals were sacrificed using an oxygen/carbon dioxide procedure and subsequently subjected to an external, thoracic and abdominal examination, with special attention being paid to the reproductive organs. All macroscopic abnormalities were recorded, collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands).
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: half per litter
The following parameters were determined:
- The number and distribution of live and dead fetuses.
- The weight of each fetus.
- The sex of each fetus from the ano-genital distance (during necropsy) and also from gonadal inspections (during further fetal examination).
- The weight of each placenta.
Statistics:
The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-toone t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control group.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Mann Whitney test was used to compare mean litter proportions (percent of litter) of the number of viable and dead fetuses, early and late resorptions, total resorptions, pre- and postimplantation loss, and sex distribution.
- Mean litter proportions (percent per litter) of total fetal malformations and developmental variations (external, visceral and skeletal), 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 ANOVA revealed statistically significant (p < 0.05) intergroup variance, Dunn’s test was used to compare the compound-treated groups to the control group.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations might be rounded off before printing. Therefore, two groups might display the same printed means for a given parameter, yet display different test statistics values.
Indices:
- Pre-implantation loss
- Post-implantation loss
- Viable fetuses affected/litter

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No clinical signs of toxicity were noted during the observation period. For two females at 1000 mg/kg bw/day alopecia was noted on several days during the treatment period. It occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, alopecia was not considered to be related to treatment.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
There were no mortalities.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights and body weight gain of treated animals remained in the same range as controls over the study period. Likewise, corrected terminal maternal body weight and corrected body weight gain were unaffected by treatment up to 1000 mg/kg bw/day.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Description (incidence and severity):
Food consumption before or after correction for body weight for treatment up to 1000 mg/kg bw/day remained similar to the control level over the study period.
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Macroscopic observations at necropsy did not reveal any toxicologically relevant findings. Incidental findings were noted in the high dose group only. They included reddish discoloration of the thymus (one female) and alopecia (two females). These necropsy findings were not considered to be treatment related or toxicologically relevant at these low incidences.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Other effects:
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
Description (incidence and severity):
There was one dead male fetus in the control 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):
There were 0, 2, 1, and 0 non pregnant animals at 0, 100, 300 and 1000 mg/kg bw/day, respectively.
Other effects:
no effects observed
Description (incidence and severity):
There were no treatment related effects on placental weights up to 1000 mg/kg bw/day.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Basis for effect level:
other: maternal toxicity

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
There were no toxicologically relevant findings on fetal body weight. At 1000 mg/kg bw/day, mean fetal body weights for females were significantly lower than controls (3.3 gram versus 3.5 gram). Also for male fetuses in the high dose group there was a trend towards slightly lower body weights as compared to controls (3.5 gram versus 3.7 gram). This difference was at least in part caused by the lower litter size in controls as compared to the high dose group (group mean of 10.7 versus 12.0 fetuses/litter). As for fetal body weights the difference from controls was only slight and group mean litter weights per sex remained within the available historical data range (i.e. minimum values of 3.1 and 3.3 gram for females and males, respectively), no toxicologically relevance was attached to the fetal body weight findings.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Description (incidence and severity):
There were no treatment-related effects on the fetal sex ratio in any group.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
The number of litters with viable fetuses available on Day 20 post-coitum were 22, 20, 21 and 22 for the groups at 0, 100, 300 and 1000 mg/kg bw/day, respectively.
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
There were no external malformations or variations seen for any live fetus in any group. For the dead fetus in the control group, generalized subcutaneous edema was noted. This finding was not test substance related.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment related effects on skeletal morphology following treatment up to 1000 mg/kg bw/day. Sternoschisis, costal cartilage anomaly, malaligned sternebra(e) #3-#5 (severe) and anomaly of the vertebral centra was observed in the highest dose group in one fetus each. Different fetuses from different litters were affected and the incidence of 0.3% per litter for each of these skeletal malformations remained within the available historical control data range (Max: 0.8%, 0.5%, 0.5% and 0.5%, respectively). As such they were not considered treatment-related. Remaining skeletal malformations observed in fetuses of test substance treated groups were bent limb bone(s), vertebral anomaly with or without associated rib anomaly or malpositioned metacarpal(s) and/or metatarsal(s). Neither of these findings was considered to be treatment related as they occurred infrequently, were observed at lower frequencies as compared to the concurrent control group, or occurred without dose relationship. Skeletal variations seen in treated groups were not considered to be treatment related as they occurred infrequently and/or in the absence of a dose-related incidence trend, were observed either at same frequencies as in the concurrent control group or at frequencies that were within the range of available historical control data, or were noted for two control fetuses only.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment related effects on visceral morphology following treatment up to 1000 mg/kg bw/day. One fetus at 300 mg/kg bw/day had situs inversus, a visceral malformation. As this was an isolated observation and in the absence of a dose-related incidence, it was considered as chance finding and thus not to be related to treatment. The slightly higher incidence of dark red discoloration of the adrenal glands (either general discoloration or dark red foci) recorded for Groups 2 and 3 was not considered to be toxicologically relevant as the incidence in the highest dose Group 4 was the same as for controls. Remaining visceral variations seen in treated groups were not considered to be treatment related as they did not follow a dose-related trend.
Other effects:
no effects observed

Effect levels (fetuses)

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

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
Based on the results in this prenatal developmental toxicity study the maternal and developmental No Observed Adverse Effect Level (NOAEL) for the test article was established as being at least 1000 mg/kg bw/day.
Executive summary:

In a GLP-compliant prenatal developmental toxicity study following OECD guideline 414, groups of mated Wistar Han (10 per group) rats were dosed by oral gavage from Day 6 to Day 19 post-coitum, inclusive, at target dose levels of 0, 300 and 1000 mg/kg bw/day. The rats of the control group received the vehicle, polyethylene glycol 400, alone. Females were checked daily for the presence of clinical signs. Body weight and food consumption of females were determined at periodic intervals. Formulations prepared on one day during treatment were analyzed for accuracy and homogeneity, and formulations prepared after completion of the in-life phase were analyzed for stability. On Day 20 post-coitum, all animals were subjected to an examination post-mortem and external, thoracic and abdominal macroscopic findings were recorded. A laparohysterectomy was performed on each surviving female of the groups. The uteri, placentae and ovaries were examined, placenta weights were determined, and the numbers of fetuses, early and late resorptions, total implantations and corpora lutea were recorded. (Gravid) uterine weights were recorded, and corrected body weights (changes) were calculated. The fetuses were weighed, sexed, and examined for external malformations and developmental variations. All live fetuses were euthanized. One half of the fetuses were decapitated and the heads were fixed in Bouin’s fixative. All fetuses were dissected and examined for visceral anomalies and subsequently fixed in 96% aqueous ethanol and stained with Alizarin Red S for skeletal examinations.

Accuracy, homogeneity and stability of formulations were demonstrated by analyses. No maternal toxicity was observed in the 100, 300 and 1000 mg/kg bw/day groups. No developmental toxicity was noted in the 100, 300 and 1000 mg/kg bw/day groups. Based on the results in this prenatal developmental toxicity study the maternal and developmental No Observed Adverse Effect Level (NOAEL) for the test substance was established as being at least 1000 mg/kg bw/day.