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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:
2009-04-01 to 2009-05-27
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
22 January 2001
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: OECD Guidance no. 43 on Mammalian Reproductive Toxicity Testing and Assessment; 24th July 2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
416-600-4
EC Name:
-
Cas Number:
77703-56-1
Molecular formula:
C23H32N4O2
IUPAC Name:
3-butyl-1-[4-({4-[(butylcarbamoyl)amino]phenyl}methyl)phenyl]urea
Specific details on test material used for the study:
Analytical purity: > 99.4 %
Lot/batch No.: HAT-ISO 05-12-06

Test animals

Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Sándor Ferenc (formerly TETRABBIT Kft.) 2173 Kartal, Császár út 135, Hungary;
- Age at study initiation: young adult rabbits; approximately 4 month;
- Weight at study initiation: mean weight 3547 - 4227 g;
- Fasting period before study: Not stated;
- Housing: individually in AAALAC approved metal wire rabbit cages;
- Diet: PURINA Base - Lap gr. diet for rabbits; Supplier: AGRIBRANDS Europe Hungary PLC, H-5300 Karcag, Madarasi
- Water: Tap water for human consumption; via automatic water system ad libitum;
- Acclimation period: at least 5 days;


ENVIRONMENTAL CONDITIONS
- Temperature: 17.3 - 21.65 °C;
- Humidity: 32 - 98.79 % R.H.
- Air changes: not stated;
- Photoperiod: 12 hours light/12 hours dark; from 6:00 a.m. to 6:00 p.m.;


Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
The test item was administered to pregnant female rabbits daily by oral gavage on a 7 days/week basis, at similar times in the morning, from GD 6 to GD 27 (one day prior to caesarean section and necropsy examination). Prior to the insemination procedure, the animals were acclimatised for at least 5 days (see also dosing scheme). Control animals were treated concurrently with the vehicle only.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical verification was made by HPLC Analysis and detection on a DAD-Detector at 250 nm wavelength;
Details on mating procedure:
The female rabbits were artificially inseminated. The day of insemination was regarded as Day 0 of gestation (GD 0). Synchronisation of the oestrus cycle of the does was performed 48 hours prior to insemination by administration of PMSG (gonadotropin) hormone (40 IU/female, sc). The insemination procedure was performed by the breeder at LAB Research Ltd. in batches (staggered; see detailed schedule in Appendix 7). Each female was inseminated with diluted sperm containing at least 2 million spermatozoa. Simultaneously with the artificial insemination, the does’ ovulation was provoked with 1 ml buserelin-based compound (0.2 mL/animal, i.m.) (see Details of Other Materials section). The sperm originated from New Zealand White male rabbits from the same source as the females. Females were randomly assigned to dose groups on the basis of their body weight on the day of insemination.
Duration of treatment / exposure:
The test item was administered to pregnant female rabbits daily by oral gavage on a 7 days/week basis, at similar times in the morning, from GD 6 to GD 27 (one day prior to caesarean section and necropsy examination). Prior to the insemination procedure, the animals were acclimatised for at least 5 days (see also dosing scheme). Control animals were treated concurrently with the vehicle only.
Frequency of treatment:
The test item was administered to pregnant female rabbits daily by oral gavage on a 7 days/week basis, at similar times in the morning, from GD 6 to GD 27 (one day prior to caesarean section and necropsy examination).
Duration of test:
The test item was administered to pregnant female rabbits daily by oral gavage on a 7 days/week basis, at similar times in the morning, from GD 6 to GD 27
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
350 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:
24 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
The test item was administered to pregnant female rabbits daily by oral gavage on a 7 days/week basis, at similar times in the morning, from GD 6 to GD 27 (one day prior to caesarean section and necropsy examination). Prior to the insemination procedure, the animals were acclimatised for at least 5 days (see also dosing scheme). Control animals were treated concurrently with the vehicle only.

Examinations

Maternal examinations:
Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each day).
Ovaries and uterine content:
The ovaries and uterus were removed and the pregnancy status ascertained. The uterus including the cervix were weighed and examined for early and late embryonic or foetal deaths and for the number of live foetuses. Where no implantation sites were evident but corpora lutea were present, the uterus was
stretched and held in front of a light source to identify properly the implantation sites. Uteri that appeared non-gravid were further examined to confirm the non-pregnant status by Salewski staining. The corrected body weight was calculated (body weight on gestation day 28 – weight of the gravid uterus).
Fetal examinations:
The live foetuses were identified, weighed individually, and the litter mean was calculated. The crown-rump length of foetuses was measured (accuracy 1 mm) and the litter mean was calculated. All foetuses were externally and viscerally examined, and sex determined. The heads from approximately half of each litter were removed and processed for evaluation of soft tissue alterations (including eyes, brain, nasal passages and tongue), using fixation in Sanomiya mixture for Wilson-sections. After fixation the heads were examined by Wilson's free-hand razor blade method. Then all foetuses were prepared for skeletal examination. The skeletons were examined after double staining with acetic alcian blue + KOH-alizarin red S staining (cartilage and bone staining). All abnormalities (external, soft tissue and skeletal malformations, and variations) found during the foetal examinations were recorded.
Statistics:
The statistical evaluation of data was performed with the program package SPSS PC+4.0. 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 Multiple Range test was used to access the significance of inter-group differences. Getting significant result, inter-group comparisons were performed using Kruskal-Wallis and Mann-Whitney U-test.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
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:
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
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
not examined
Other effects:
no effects observed
Details on maternal toxic effects:
Maternal toxic effects: no effects observed

Details on maternal toxic effects:
There were no test-item related clinical signs noted following administration of HAT-ISO to pregnant does from GD 6 to 27, by daily oral gavage, at dose levels up to and including 1000 mg/kg bw/day HAT-ISO, at a dose volume of 2 mL/kg bw.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Basis for effect level:
other: no effects observed at highest dose tested

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
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:
not examined
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 observed

Details on embryotoxic / teratogenic effects:
No test-item related effects were noted in the number of dead/viable foetuses, or their sex distribution. The sex ratios were similar in the control and treated groups. The litter mean values of foetal weight and crown-rump length were similar in the foetuses from the treated and control does. There were no abnormalities considered toxicologically significant and/or possibly ascribed to HAT-ISO administration at external, visceral or skeletal foetal examinations.

Effect levels (fetuses)

Key result
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day
Basis for effect level:
other: no effects observed at highest dose tested

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

No mortality or test-item related clinical signs occurred during the study.

HAT-ISO administered to artificially-inseminated New Zealand White female rabbits by oral gavage at dose levels of 100, 350, and 1000 mg/kg bw/day in corn oil from GD 6 to 27, led to dose and time-dependent increases in does’ food consumption, correlated with higher body weights and/or body weight gain at 1000 mg/kg bw/day. In the absence of any maternal or developmental toxicity, these changes were not considered an adverse effect of the test item in the conditions of this study.

There were no test item related effects on any of the maternal reproductive parameters examined during the study. The number of corpora lutea and implantation sites was similar in the treated groups compared to control animals. There were no differences between the treated and control animals regarding the pre-implantation loss, early and late embryonic death, postimplantation loss, or total intrauterine mortality; the mean values and/or percentile were comparable, or lower in the HAT-ISO treated animals, with no statistically significant variations compared to the animals administered vehicle control (corn oil) only. No macroscopic findings or treatment-related changes in the gravid uterine weight were recorded in the treated pregnant does surviving to termination when compared to the controls. The placenta was normal in all the does examined.

As necropsy followed by histopathology evaluation of the females with macroscopic findings, no macroscopic or microscopic findings recorded were ascribed to HAT-ISO administration. One low dose doe administered 100 mg/kg bw/day (1/24, female 2523) aborted and was necropsied on GD27; based on the isolated incidence and in the absence of similar findings in this group, or at higher dose levels, abortion in this animal was considered spontaneous and not related to treatment.

No test-item related effects were noted in the number of dead/viable foetuses, or their sex distribution. The sex ratios were similar in the control and treated groups. The litter mean values of foetal weight and crown-rump length were similar in the foetuses from the treated and control does. There were no abnormalities considered toxicologically significant and/or possibly ascribed to HAT-ISO administration at external, visceral or skeletal foetal examinations.

In conclusion, under the conditions of this study, the no-observed-effect-level values are:

NOAEL maternal toxicity: 1000 mg/kg bw/day

NOAEL developmental toxicity: 1000 mg/kg bw/day

NOEL teratogenicity: 1000 mg/kg bw/day

Applicant's summary and conclusion

Conclusions:
HAT-ISO was tested for Developmental Toxicity / Teratology in rabbits by oral gavage during the gestation period. No mortality or test-item related clinical signs occurred during the study. The no-observed effect levels are:
NOAEL maternal toxicity: 1000 mg/kg bw/day;
NOAEL developmental toxicity: 1000 mg/kg bw/day;
NOEL teratogenicity: 1000 mg/kg bw/day.
No classification and labelling is necessary according to Regulation 1271/2008/EEC (CLP).
Executive summary:

A Prenatal Developmental Toxicity/Teratology Study was performed in the rabbit according to OECD 414 and the draft OECD guidance document 43. The objective of this study was to assess the effects of HAT-ISO on pregnant females and developing conceptuses when administered to artificially inseminated, assumed pregnant New Zealand White rabbits. Test substance was administered daily by oral gavage at dose levels of 100, 350, and 1000 mg/kg bw/day.

No mortality or test-item related clinical signs occurred during the study.

Under the conditions of this study, the no-observed-effect-level values are:

NOAEL maternal toxicity: 1000 mg/kg bw/day;

NOAEL developmental toxicity: 1000 mg/kg bw/day;

NOEL teratogenicity: 1000 mg/kg bw/day.