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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:
Aug - Sep 1990
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1991
Report date:
1991

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
May 12, 1981
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Reference substance name:
67359-57-3
EC Number:
614-052-2
Cas Number:
67359-57-3
IUPAC Name:
67359-57-3
Test material form:
liquid
Details on test material:
- Chemical name: mixture of N,N-Dimethydecan-1-amide and N,N-Dimethyloctan-1-amide
- Physical state: liquid
- Storage condition of test material: at room temperature in the dark
- Batch number: 903069
- Stable until November 17, 1990
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: 903069
- Expiration date of the lot/batch: November 17, 1990

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature, in the dark
- Stability of the test substance in the solvent/vehicle: at least 2 hours at room temperature

Test animals

Species:
rabbit
Strain:
Chinchilla
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Dr. Karl Thomae GmbH / Birkendorferstrasse 65 / D-W-7950 Biberach / Riss
- Age at pairing: between 4 and 6 months
- Weight at study initiation: 2810 - 4825 grams (day 0 post coitum)
- Housing: individually
- Diet: ad libitum; Pelleted standard Kliba 341 rabbit maintenance diet ("Kliba" / Klingentalmuehle AG / CH 4303 Kaiseraugst / Switzerland)
- Water: Tap water was available ad libitum by an automatic system.
- Acclimation period: minimum of 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 3°C
- Humidity (%): 40 - 70%
- Air changes (per hr): 10-15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours artificial fluorescent light/12 hours dark with background music played at a centrally defined low volume for at least 8 hours during the light period.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: Bi-distilled water with 0.5 % Cremophor (BASF)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The mixtures of the test article and vehicle were prepared daily before administration. The test article was weighed into a glass beaker on a tared precision balance (Mettler PE 360) and the vehicle added (w/w). The mixtures were prepared using a homogenizer. During the daily administration period,
homogeneity was maintained using a magnetic stirrer.

VEHICLE
- Concentration in vehicle: 25 mg/ml, 75 mg/ml, 250 mg/ml
- Amount of vehicle (if gavage): adjusted to a total application volumen 4ml/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Photometric analytical verification (UV/Vis)
Concentrations (25, 75, 250 mg/ml) and stability in bidestilled water with 0.5% Cremophor was verified.
Result: Recovery between 95.3 and 101.0%
Homogeneity varies from -5 to + 3%
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
until copulation was observed. The day of mating was designated as day 0 post coitum.
Duration of treatment / exposure:
- The test article was administered orally, by gavage, once daily in the morning from day 6 through to day 18 post coitum.
- Dose volume: 4 ml/kg body weight (daily adjustment of the individual volume to the actual body weight)
Frequency of treatment:
daily from day 6 through day 18 post coitum
Duration of test:
28 days (from successful mating to termination, excluding acclimatisation etc)
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:
16 mated female rabbits
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dosages were based on the results of the dose range-finding study.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations checked: mortalities, signs of reactions of treatment and/or symptoms of ill health

DETAILED CLINICAL OBSERVATIONS: no

BODY WEIGHT: Yes
- Time schedule for examinations: daily from day 0 until day 28 post coitum

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule: days 0-6, 6-11, 11-15, 15-19, 19-24 and 24-28 post coitum

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: yes
- Sacrifice on day 28 post coitum
- Post mortem examination, including gross macroscopic examination of all internal organs, with emphasis on the uterus, uterine contents, position of fetuses in the uterus and number of corpora lutea
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 (embryonic resoption): Yes
- Number of late resorptions (fetal resorption): Yes
- Number of Pre-Implantation loss: yes
- Number of Post-Implantation loss: 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:
- Univariate one-way analysis of variance was used to assess the significance of intergroup differences.
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-one t-test), based on a pooled variance estimate, was applied for the comparison between the treated groups and the control group.
- The Steel-test (many-one rank test) was applied when the data could not be assumed to follow a normal distribution.
- Fisher's Exact test for 2x2 tables was applied if the variables could be dichotomized without loss of information.
Indices:
see "Any other information on materials and methods incl. tables"
Historical control data:
Historical data of Chinchilla Rabbits (Hybrids, SPF Quality) from 1987, 1988 and 1989:
- Reproduction data of dams
- Spontaneous abnormal findings of fetuses (external, vsceral or skeletal examination)
- Skeletal examination of fetuses (stage of development) on fetus basis or on litter basis

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
- group 4 (1000 mg/kg): in 1 female slight dyspnoea and ventral recumbency a few hours prior to death on day 12 post coitum and 1 female with dyspnoea on day 9 post coitum during 5 hours

These isolated findings were considered to be incidental and not test article related effects.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
- group 2 (100 mg/kg): 3 females died during the dosing period; 1 on day 7 post coitum (2nd day of dosing) and 1 on day 15 post coitum (10th day of dosing) to intubation errors, 1 on day 12 post coitum (7th day of dosing) spontaneously without any signs of toxic effects
- group 3 (300 mg/kg): 1 female was found dead in the morning of day 28 post coitum (day of Caesarean Section, ten days after the last dosing) without any previous signs of toxic effects.
- group 4 (1000 mg/kg): 1 female died on day 12 post coitum (7th day of dosing) without any previous signs of toxic effects.

These Isolated cases of spontaneous deaths In any dose groups were considered to be incidental because no relation to the number of administrations or to the dosages was evident.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- group 4 (1000 mg/kg): slightly reduced mean body weight gain during the dosing period (in particular between days 6 and 11 post coitum (1st and 6th day of dosing))
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- group 4 (1000 mg/kg): reduction in food consumption (-21.2% compared to vehicle control) during the dosing period (days 6 to 19 post coitum), statistically significant between days 11-15 post coitum
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:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
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
Description (incidence and severity):
- the total post-implantation loss in two females of group 3 (300 mg/kg) was considered to be incidental, because no female with total post-implantation loss was evident in group 4 (1000 mg/kg)
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 specified
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 specified
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Remarks:
general toxicity
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
- groups 1 (vehicle control) and 3 (300 mg/kg): 2 runts (body weight <19.0 g)
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, non-treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): - groups 1 (vehicle control) and 3 (300 mg/kg): 2 runts (body weight <19.0 g)
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
no dead fetuses
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
not examined
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
1/158 fetuses in 1/16 litters in group 1 (vehicle control);
2/145 fetuses in 2/14 litters in group 2 (100 mg/kg);
3/120 fetuses in 3/12 litters in group 3 (300 mg/kg);
1/147 fetus in 1/15 litters in group 4 (1000 mg/kg).

The findings noted were: thoracic vertebral bodies and/or arches (hemicentric, missing or fused), sternebrae abnormally ossified and/or fused, rib(s) bifurcated or fused and caudal vertebrae hemicentric or bipartite.

The types and the incidences of the abnormal findings noted did not indicate test article specific effects.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
- group 2 (100 mg/kg): 1/145 fetuses with dilated aorta and missing arch of aorta
- group 3 (300 mg/kg): 1/120 fetuses with hemidiaphragm and 1 fetus with oval foramen in the diaphragm
- group 4 (1000 mg/kg): 1/147 fetuses with hydronephrosis (both kidneys)

These isolated abnormal findings noted did not indicate an association with administration of the test article.
Other effects:
no effects observed

Effect levels (fetuses)

open allclose all
Dose descriptor:
NOAEL
Remarks:
fetotoxicity
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
The fetal parameters were not affected up to and including the highest dose level of 1000 mg/kg body weight/day.
Dose descriptor:
NOAEL
Remarks:
teratogenicity
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
The fetal parameters were not affected up to and including the highest dose level of 1000 mg/kg body weight/day.

Fetal abnormalities

Abnormalities:
no effects observed

Overall developmental toxicity

Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
The test substance did not reveal any teratogenic potential up to and including the highest dose level of 1000 mg/kg body weight/day.
The no-observed adverse effect level for the maternal organism was considered to be 300 mg/kg and for the fetal organism 1000 mg/kg body weight/day.
Executive summary:

The purpose of this study was to assess the effects of the test substance on embryonic and fetal development in pregnant Chinchilla rabbits.

Each group consisted of 16 mated female rabbits. The test substance was administered orally by gavage once daily from day 6 through to day 18 post coitum, at dose levels of:

Group 1:       0 mg/kg body weight/day (vehicle control)

Group 2:       100 mg/kg body weight/day

Group 3:       300 mg/kg body weight/day

Group 4:       1000 mg/kg body weight/day

The dosages were based on the results of a dose range-finding study. A standard dose volume of 4 ml/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (bi-distilled water with 0.5 % Cremophor).

Females were sacrificed on day 28 post coitum and the fetuses were removed by Caesarean section. The examination of the dams and fetuses was performed in accordance with international recommendations.

The following results were obtained:

MATERNAL DATA

GENERAL TOLERABILITY

- There were no deaths, clinical signs or necropsy findings in the females of any dose group which were considered to be related to administration of the test substance.

- Test article-related reduced food consumption (statistically significant between days 11-15 post coitum) was evident during the dosing period at 1000 mg/kg. During this period, the body weight gain was slightly reduced (without statistical significance).

No effects on food consumption and body weight development were noted at 100 mg/kg or at 300 mg/kg.

REPRODUCTION PARAMETERS

- None of the differences between the vehicle control group and any dose group noted were considered to be an effect of administration of the test substance. The differences evident were considered to be incidental because of missing dose-relation.

FETAL DATA

- During external and fresh visceral examination as well as at examination of fetal heads by Wilson technique and skeletal examination of fetuses, no abnormal findings were evident which indicated test article-related effects.

The isolated common abnormal findings (with the lowest incidence at 1000 mg/kg) were within the normal range of spontaneously occurring findings in this rabbit strain and were considered to be incidental.

- The other fetal parameters recorded - sex ratios, body weights and stage of skeletal development - resulted in similar values in all groups.