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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:
29 Feb - 28 Mar 1996
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Comparable to guideline study with acceptable restrictions. The test substance was only administered during organogenesis and not through the entire period of gestation to the day before caesarean section. Body weights were not determined in 3-day intervals during treatment period.
Justification for type of information:
refer to category justification provided in IUCLID section 13

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report Date:
1997

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
test substance was only administered during organogenesis and not through the entire period of gestation to the day before caesarean section; body weights were not determined in 3-day intervals during treatment period
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
other: Sprague Dawley, CD
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Wiga GmbH, Sulzfeld, Germany
- Age at study initiation: 8 weeks
- Weight at study initiation: 203 g (mean)
- Housing: individually in Makrolon Type M3 cages (EBECO, Castrop-Rauxel, Germany) with standard softwood bedding (ARWI-Center, Essen, Germany)
- Diet: pelleted Atromin Maintenance Diet 1324 (ALTROMIN GmbH, Lage, Germany), ad libitum
- Water: community tap water (Düsseldorf, Germany), ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23
- Humidity (%): 40-66
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: the test substance was prepared daily before administration by dissolving appropriate amounts in distilled water.

VEHICLE
- Concentration in vehicle: 1, 3 and 10% (v/v), i.e. 10, 30 and 100 mg/mL
- Amount of vehicle: 10 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The formulations of test substance were analysed once by High Performance liquid chromatography (HPLC). The determined analytical concentrations of 0.94, 2.88 and 9.23% verified the nominal concentrations of 1, 3 and 10% of the test substance in solution.
Details on mating procedure:
- Impregnation procedure: purchased timed pregnant
Duration of treatment / exposure:
(P) Females: Day 6-15 of gestation (organogenesis period)
Frequency of treatment:
once daily in the morning
Duration of test:
20 days
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
24 P females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on the results of previous toxicological examinations

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: animals were checked at least twice daily (working days) for clinical signs and mortality.

BODY WEIGHT: Yes
- Time schedule for examinations: body weights were determined on Day 0, 6, 16 and 20

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: all maternal organs with emphasis on uterus and its content
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: position of fetuses in the uterus, distribution of implantations, live or dead fetuses
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: No
- Other: sex, weight incl. placenta
Statistics:
Mean values and standard deviations were calculated. Statistical analyses at significance levels of 5% and 1% were performed on the following parameters and using the following statistical tests:
- Steel test: implantation sites, embryonic and fetal resorptions, live and dead fetuses
- Fishers exact test (Bonferroni-Holm-corrected): pre- and post-implantation loss, total embryonic deaths, total and malformed fetuses, skeletal parameters, sex of fetuses
- Dunnett-test based on pooled variance: weights of live fetuses, placenta and uteri, body weight of dams
Indices:
Percentage of implantation sites: (no. of implantations / no. of corpora lutea) * 100
Percentage of pre-implantation loss: [(no. of corpora lutea - no. of implantations) / no. of corpora lutea] * 100
Percentage of post-implantation loss: [(no. of implantations - no. of live foetuses) / no. of implantations] * 100
Percentage of embryonic resorptions: (no. of embryonic resorptions / no. of implantations) * 100
Percentage of fetal resorptions: (no. of fetal resorptions / no. of implantations) * 100
Percentage of total fetuses: (no. of total fetuses / no. of implantations) * 100
Percentage of malformed fetuses: (no. of malformed fetuses / no. of total fetuses) * 100
Percentage of male fetuses: (no. of male fetuses / total no. of live fetuses) * 100
Percentage of female fetuses: (no. of female fetuses / total no. of live fetuses) * 100

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
The animal of the 100 mg/kg bw/d test group found dead on Day 11 showed impeded respiration, lethargy, and a decrease in body weight on Day 10. One animals of the high dose group (1000 mg/kg bw/d) showed impeded respiration on Day 15 and 16 and lethargy on Day 16. However, these findings were not related to treatment.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No deaths occurred in dams receiving the vehicle or the test substance at doses of 300 mg/kg bw/d. Due to an application error, test substance-unrelated mortalities were found on Day 11 and 13 each in one animal of the 100 and 1000 mg/kg bw/d test group.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Maternal body weights were not affected by treatment. The mean body weights of the high dose group showed a slight increase on Day 6 compared to the controls. Mean corrected body weight gain (corrected for uterus weight) of the treated groups was comparable to the control group.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
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:
no effects observed
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
At scheduled necropsy, no macroscopic changes were observed between treated and control animals. Incidental findings in two animals either comprised white nodules in the left inguinal region or haematoma in the region of larynx.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

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:
not examined
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined
Changes in number of pregnant:
not examined
Other effects:
not examined
Details on maternal toxic effects:
Maternal toxic effects: no effects

MORTALITY:
No deaths occurred in dams receiving the vehicle or the test substance at doses of 300 mg/kg bw/d. Due to an application error, test substance-unrelated mortalities were found on Day 11 and 13 each in one animal of the 100 and 1000 mg/kg bw/d test group.

CLINICAL SIGNS:
No substance-related symptoms were observed at any dose level during the study. The animal of the 100 mg/kg bw/d test group found dead on Day 11 showed impeded respiration, lethargy, and a decrease in body weight on Day 10. One animals of the high dose group (1000 mg/kg bw/d) showed impeded respiration on Day 15 and 16 and lethargy on Day 16. However, these findings were not related to treatment.

BODY WEIGHTS:
Maternal body weights were not affected by treatment. The mean body weights of the high dose group showed a slight increase on Day 6 compared to the controls. Mean corrected body weight gain (corrected for uterus weight) of the treated groups was comparable to the control group.

REPRODUCTION DATA:
No substance-related effects on reproduction data were noted compared to controls.

NECROPSY:
At scheduled necropsy, no macroscopic changes were observed between treated and control animals. Incidental findings in two animals of the 300 mg/kg bw/day and 1000 mg/kg bw/day, respectively, either comprised white nodules in the left inguinal region or haematoma in the region of larynx.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Remarks:
maternal toxicity
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: no treatment-related effects observed
Remarks on result:
not determinable due to absence of adverse toxic effects

Maternal abnormalities

Key result
Abnormalities:
no effects observed

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
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
The weight of placenta and uteri including content showed no significant differences between treated and control animals.
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects: no effects

EMBRYOTOXICITY/FETOTOXICITY:
No substance-related differences in pre-and post-implantation loss, mean figures of resorptions, embryonic deaths and total fetuses were observed in treated animals compared to controls.

BODY WEIGHTS:
The weight of live fetuses exhibited not significant differences on a litter or individual basis.

PLACENTA AND UTERUS WEIGHT:
The weight of placenta and uteri including content showed no significant differences between treated and control animals.

SEX RATIOS:
The fetal sex ratio was comparable in all test groups.

EXTERNAL OBSERVATIONS:
No abnormal findings were observed that were considered to be related to treatment. Incidental findings comprised the occurrence of only one placenta for two fetuses in the control group and one fetus with hydrops in the high dose group (1000 mg/kg bw/d).

VISCERAL OBSERVATIONS:
The main figures of visceral variations (hydronephrosis, dilated and waved ureters) were similar compared to controls and variations were not considered to be related to treatment. All other findings except for one situs inversus total in the high dose group and enlarged parenchymous organs in one fetus in the controls were considered to be incidental.

SKELETAL EXAMINATION:
No substance-related abnormal findings were noted at skeletal examination of the fetuses. Spontaneous findings comprised absent or malformed cervical vertebrae arches in one mid dose fetus and luxation and malposition of the mandible in one high dose fetus when compared to the control group. Retarded ossification was found in all treatment groups and was similar to the level of skeletal ossification in the control group. The isolated statistically significant difference in the figure “14 ribs short/rudimentary bilateral” in the high dose group (100 mg/kg bw/d) was considered to be incidental and not related to treatment, since no dose-response relationship was apparent.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no treatment-related effects observed
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

Applicant's summary and conclusion

Conclusions:
A prenatal developmental toxicity study performed according to OECD 414 and in compliance with GLP with the test material and did not reveal any treatment-related effect. Thus, a NOAEL (maternal/developmental) of ≥1000 mg/kg bw/day was derived.