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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:
Echa Approval Provided. Study period May 2015- JUne 2015
Reliability:
1 (reliable without restriction)

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid at 15-25 ºC
Details on test material:
Batch number 1140045
Name of test substance : Robac As100
Appearance: Yellow liquid
Purity: 98%

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
Species and strain: Hannover Wistar rats (CRLHan)
Source: Charles River Laboratories, Research Models and
Services, Germany GmbH, Sandhofer Weg 7, D-
97633, from SPF colony
Justification of strain: The rat is regarded as a suitable rodent species for
reproduction studies and the test guideline states it is
the preferred rodent species. The Hannover Wistar rat
was selected due to experience with this strain in
teratology studies.

Animal health: Only healthy animals were used for the test, as certified
by the staff Veterinarian

Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 18.2-22.8°C (target: 22 ± 3 °C)
Relative humidity: 34-66 % (target: 30-70%)
Ventilation: 15-20 air exchanges/hour

Administration / exposure

Route of administration:
oral: gavage
Details on exposure:
A constant volume of 4 mL/kg bw was administered to all dose groups, including the
controls. The individual volume of the treatment was based on the most recent
individual body weight of the animals.
The control or test item dose formulations were administered to mated, sperm positive
assumed pregnant female rats daily by oral gavage on a 7 days/week basis,
approximately at similar times, from Gestation Day (GD) 6 to GD19, according to the
following dosing scheme:
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability of the AS100 in the vehicle (peanut oil) was assessed in the conditions
employed on the study during the validation study (according to CiToxLAB study
code 14/497-901AN). According to the results, the test item is stable in vehicle in
concentration range of 5 mg/mL - 100 mg/mL for 14 days when stored refrigerated (2-
8ºC).
Analysis of test item formulations for concentration and homogeneity was performed
in the Test Site (Fumoprep Ltd.) using a quantitative IR spectroscopic method (Test
Site study code: FPBSTUDY-121-VAL1, Ref. 4). Duplicate samples (top, middle and
bottom samples) were taken from the test item formulations twice during the study
(during the first and last weeks of treatment). Similarly, one sample (duplicate) was
taken on each occasion from the Group 1 (control) solution for concentration
measurements.
Details on mating procedure:
The oestrus cycle of female animals was examined shortly before start of pairing.
After acclimation, the females were paired according to their oestrus cycle with males
in the morning for approximately 2 hours (1 male: 1 females) until at least 24 sperm
positive females/group are attained. After the daily mating period, a vaginal smear
was prepared and stained with 1% aqueous methylene blue solution. The smear was
examined with a light microscope; the presence of a vaginal plug or sperm in the
vaginal smear was considered as evidence of copulation (GD0). Sperm positive
females were separated and caged individually
Duration of treatment / exposure:
The dams (one control and 3-treated
groups) were treated daily by oral (gavage) administration, from gestation day GD6 up
to and including GD19 (sperm positive day = 0 day of pregnancy, GD0). Caesarean
sections, necropsy of dams and examination of uterine contents were performed on
GD20. The control animals were treated with vehicle only ( peanut oil)
Frequency of treatment:
Daily treatment GD 6 to GD19
Duration of test:
Dosing scheme
Acclimatisation period 5 days- no dosing
Gestation days 5 to 19
Caesarean section and necroscopy Gestation day 20

Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
23mg/kg/bw/day
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
70mg/kg/day
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
210 mg/kg/day
Basis:
analytical conc.
No. of animals per sex per dose:
26-27 females per dose and control group
The number of confirmed pregnant, evaluated dams in the dose groups treated was 26
in the control group and 25 at treated groups.
Control animals:
yes, concurrent vehicle
Details on study design:
accord tp OECD 414

Examinations

Maternal examinations:
Maternal Data:
- Number of animals at test start, no. of animals surviving, no. of pregnant animals,
no. of animals with total intrauterine mortality
- Clinical signs (by gestation day)
- Mortality (by gestation day), if any
- Body weight and body weight gain: mean ± S.D.
- Corrected body weight on GD20 (body weight-gravid uterine weight) and
corrected body weight gain: mean ± S.D.
- Net body weight change (Body weight on GD20 minus body weight on GD6
minus gravid uterine weight): mean ± S.D.
- Food consumption: mean ± S.D.
- Gross pathology findings
- Gravid uterine weight
- Absolute and relative to body weight liver and spleen weights
Ovaries and uterine content:
Caesarean Section and Necropsy Data:
- Number of corpora lutea: mean ± S.D.
- Number of implantations: mean ± S.D.
- Number and percent of live foetuses: mean ± S.D.
- Number and percent of intrauterine mortality: mean ± S.D.
Classified according to time of death: Pre-implantation loss, Post-implantation
mortality, Early and late embryonic, as well as foetal death
- Pre-implantation loss: %, group mean
UNumber of corpora lutea-Number of implantationsU x100
Number of corpora lutea
- Post-implantation loss: %, group mean
UNumber of implantations-Number of live foetusesU x100
Number of implantations
Fetal examinations:
Sex distribution: %, group mean
UNumber of male (female) foetusesU x100
Number of foetuses
- Foetal body weight (accuracy 0.01 g): mean ± S.D.
- External abnormalities/litter: %, group mean
UNumber of foetuses with abnormalityU x100
Number of foetuses
- Visceral abnormalities/litter: %, group mean
UNumber of foetuses with abnormalityU x100
Number of foetuses
- Skeletal abnormalities/litter: %, group mean
UNumber of foetuses with abnormalityU x100
Number of foetuses
Statistics:
Appropriate statistical method (Bartlett,
ANOVA and Duncan, Kruskal-Wallis and Mann-Whitney U tests, Chi2) using the
litter as the unit for data analysis. The homogeneity of variance between groups was
checked by Bartlett`s homogeneity of variance test. Where no significant
heterogeneity was detected a one-way analysis of variance (ANOVA) was made. If
the obtained result was significant Duncan’s Multiple Range test was used to assess
the significance of inter-group differences. Significant results with inter-group
comparisons were further compared using Kruskal-Wallis, and Mann-Whitney Utests.
Indices:
Pre-implantation loss: %, group mean
UNumber of corpora lutea-Number of implantationsU x100
Number of corpora lutea
- Post-implantation loss: %, group mean
Number of implantations-Number of live foetuses/ Number of implantations x 100
Historical control data:
Hisrorical control data was used to provide clear indications where toxicologically significant effects had occurred

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Clinical signs: Upon immediate administration of the test material to all treated groups was noted as a dose response effect. Clinical signs included piloerection, clonic convulsion, rooting in bedding and increased salivation.
Body Weight: At 23mg/kg/bw the dams had comparable body weights to control group. There was a marked initial body weight loss noted in mid and high dose groups with partial recovery over the timecourse. The corrected body weight ( adjusted for gravid uterine weight) for both mid and high dose was lower than the control attaining statistical signifcant result.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
23 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
LOAEL
Effect level:
70 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
none

Effect levels (fetuses)

open allclose all
Dose descriptor:
NOAEL
Effect level:
210 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: teratogenicity
Dose descriptor:
NOAEL
Effect level:
210 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: embryotoxicity
Dose descriptor:
NOAEL
Effect level:
210 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: fetotoxicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Mortality : There was no unscheduled mortality in the study.

Clinical signs: Upon immediate administration of the test material to all treated groups was noted as a dose response effect. Clinical signs included piloerection, clonic convulsion, rooting in bedding and increased salivation.

Body Weight: At 23mg/kg/bw the dams had comparable body weights to control group. There was a marked initial body weight loss noted in mid and high dose groups with partial recovery over the timecourse. The corrected body weight ( adjusted for gravid uterine weight) for both mid and high dose was lower than the control attaining statistical signifcant result.

The weight of liver and spleen were increased in mid and high dose groups but no statistically significant changes were seen in liver or spleen weight in low dose group.

Foetal Body weights: Mean foetal body weights were unaffected by maternal treament with test substance at any dose level. The incidence of body weight retarded foetuses was simliar in control and test item treated groups. The weight of foetuses at 23, 70 and 210 mg/kg bw/day did not differ significantly from the control mean, when evaluated by both litter mean and group mean.

Although the overall mean foetal body weights, and the mean weight of foetuses evaluated by litter mean, were statistically significantly higher at the low and mid dose levels (by 5.5 and 6.1%, respectively; all values were in the normal historical range and there was no clear dose response relationship. Therefore these statistical differences were considered not related to treatment.

Teratogenic/Embryotoxic effects: No foetal external abnormalities to the test item at all dose levels up to and including 210mg/kg/day.

No foetal visceral abnormalities are ascribed to the test item administration.

The mean number of corpora lutea and the mean number of implantation sites were comparable with the controls in all treated groups. The pre-implantation loss was at control level in all treated groups. The total intrauterine mortality was comparable with the control. The early and the late embryonic loss did not differ significantly from the control in the test item treated groups. There was no statistically significant difference in foetal death compared to the control.

Applicant's summary and conclusion

Conclusions:
At both mid and high dose groups maternal toxicity was observed in bodyweight loss and increased liver and spleen weights, however, there was no evidence of adverse treatment related effects seen at external, visceral or skeletal foetal examination at all dose groups.
Executive summary:

This developmental toxicity study was performed to assess the effects of the test item AS100 on the embryonic and foetal development (including the organogenesis period) of Hannover Wistar rats in their first pregnancy.

At both mid and high dose groups maternal toxicity was observed in bodyweight loss and increased liver and spleen weights, however, there was no evidence of adverse treatment effects seen at external, visceral or skeletal foetal examination at all dose groups.