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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:
10 Feb - 19 May 1982
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1983
Report date:
1983

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
The method described complies to the prescribed method: Commission Directive 88/302/EEC. OJ No L 133. Briefly, pregnant rabbits were exposed to the test substance from gestation day (GD) 5 to 20. On GD 29, dams were sacrificed and the below mentioned examinations were performed.
GLP compliance:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
3,5-dibromo-4-hydroxybenzonitrile
EC Number:
216-882-7
EC Name:
3,5-dibromo-4-hydroxybenzonitrile
Cas Number:
1689-84-5
Molecular formula:
C7H3Br2NO
IUPAC Name:
3,5-dibromo-4-hydroxybenzonitrile

Test animals

Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River France, St. Aubin-Les-Elbeuf, France
- Age at study initiation: not reported, but sexually mature
- Weight at study initiation: approximately 3 kg
- Housing: in metal cages suspended over trays containing sawdust
- Diet: Labsure R14 pelleted diet (Christopher Hill Group (RHM), Poole, Dorset), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 14 - 23.5
- Humidity (%): not controlled
- Air changes (per hr): not reported, but forced air ventilation
- Photoperiod (hrs dark / hrs light): 10/14

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: aqueous suspensions containing 0.25% w/v gum tragacanth
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: suspensions were prepared freshly each week, and refrigerated each day after use.

VEHICLE
- Concentration in vehicle: 1.5, 3.0 and 6.0% w/v for 15, 30 and 60 mg/kg bw/day, respectively
- Amount of vehicle: 1 mL/kg bw

Positive control animals received thalidomide (150 mg/kg bw/day) orally via gelatin capsules.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Fortnightly, samples of the test suspensions were taken for each suspension concentration and analyzed for test material concentration and homogeneity of mix.
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: 1/1
- Length of cohabitation: until coitus was observed, mating was performed until a minimum of 15 pregnant females per group was obtained (for positive controls: 10 females)
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: not specified (day of coitum was defined as Day 0 post-coitum)
Duration of treatment / exposure:
Days 5 - 20 post-coitum
Frequency of treatment:
daily
Duration of test:
until Day 29 post-coitum
Doses / concentrationsopen allclose all
Dose / conc.:
15 mg/kg bw/day (nominal)
Dose / conc.:
30 mg/kg bw/day (nominal)
Dose / conc.:
60 mg/kg bw/day (nominal)
No. of animals per sex per dose:
15 (test substance groups) / 10 (positive control)
Control animals:
yes, concurrent vehicle
other: Positive control

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS/CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily

BODY WEIGHT: Yes
- Time schedule for examinations: recorded on Days 0, 5, 9, 13, 17, 21, 25 and 29 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: recorded on Days 0, 5, 9, 13, 17, 21, 25 and 29 post-coitum.

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- Organs examined: macroscopic post-mortem examination, uteri and ovaries were removed.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No data
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No data
- Number of late resorptions: No data
- Other: Number of early uterine deaths, number of late uterine deaths (early uterine deaths were defined as those implantations without, and late deaths as those with embryonal or fetal elements)
Blood sampling:
- Plasma: No
- Serum: No
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all surviving animals
- Skeletal examinations: Yes: all surviving animals
- Head examinations: Yes: all surviving animals
- Anogenital distance of all live rodent pups: not reported
Statistics:
The following statistical tests were used:
- the parametric Students 't' test for litter data, food consumption, dam gestational body weight and body weight gain.
- the modified Chi-square test for proportions and percentages for pre- and post-implantation losses and fetal abnormalities

A value of 'p' of 0.05 or less was taken as the criterion of statistical significance.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Please refer to the description provided under "Mortality".
Dermal irritation (if dermal study):
not examined
Description (incidence and severity):
not applicable
Mortality:
mortality observed, treatment-related
Description (incidence):
- 15 and 30 mg/kg bw/day: No mortalities occurred up to and including 30 mg/kg bw/day.
- 60 mg/kg bw/day: two animals died (1 was found dead on Day 13 post-coitum after a fall during dosing, 1 was found dead on Day 23 post coitum with a large volume of clear fluid in the thoracic cavity and congestion of the lung and extensive suppurative bronchopneumonia and pleurisy). The mortalities were not clearly associated with oral administration of the test material.

Positive control:
1 animal was extremely excitable, difficult to dose and was killed.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- 15 mg/kg bw/day: mean body weight gain was slightly reduced during the dosing period compared to vehicle control, but this was not statistically significant. Body weight was slightly but not significantly reduced compared to the vehicle control.
- 30 mg/kg bw/day: mean body weight gain was statistically significantly reduced during the dosing period compared to vehicle control. There was a recovery after dosing but body weight was still reduced compared to controls at sacrifice.
- 60 mg/kg bw/day: mean body weight gain was statistically significantly reduced during the dosing period compared to vehicle control. There was a recovery after dosing but body weight was still different compared to controls at sacrifice. Body weight was reduced compared to vehicle controls from Day 13 post coitum onwards.

Summarized results can be found in Attachment 1.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- 15 mg/kg bw/day: slight but not statistically significant reduction in mean food consumption throughout the dosing period, compared to the vehicle control
- 30 mg/kg bw/day: statistically significant reduction in mean food consumption during Days 9 to 13 (p < 0.05) and Days 13 to 17 (p < 0.01) post coitum with post-dosing recovery, compared to the vehicle control
- 60 mg/kg bw/day: statistically significant reduction in mean food consumption throughout the dosing period with post-dosing recovery, compared to the vehicle control

Summarized results can be found in Attachment 2.
Food efficiency:
not examined
Description (incidence and severity):
not applicable
Water consumption and compound intake (if drinking water study):
not examined
Description (incidence and severity):
not applicable
Ophthalmological findings:
not examined
Description (incidence and severity):
not applicable
Haematological findings:
not examined
Description (incidence and severity):
not applicable
Clinical biochemistry findings:
not examined
Description (incidence and severity):
not applicable
Endocrine findings:
not examined
Description (incidence and severity):
not applicable
Urinalysis findings:
not examined
Description (incidence and severity):
not applicable
Behaviour (functional findings):
not examined
Description (incidence and severity):
not applicable
Immunological findings:
not examined
Description (incidence and severity):
not applicable
Organ weight findings including organ / body weight ratios:
not specified
Description (incidence and severity):
not applicable
Gross pathological findings:
not specified
Description (incidence and severity):
not applicable
Neuropathological findings:
not examined
Description (incidence and severity):
not applicable
Histopathological findings: non-neoplastic:
not examined
Description (incidence and severity):
not applicable
Histopathological findings: neoplastic:
not examined
Description (incidence and severity):
not applicable
Other effects:
not examined
Description (incidence and severity):
not applicable

Maternal developmental toxicity

Number of abortions:
not specified
Pre- and post-implantation loss:
effects observed, treatment-related
Description (incidence and severity):
- 15 mg/kg bw/day: no differences regarding pre- and post-implantation loss
- 30 mg/kg bw/day: non-significant increases in post-implantation loss occurred
- 60 mg/kg bw/day: statistically significant increase in post-implantation loss (41% vs 12% in vehicle controls, as increase in embryo toxicity as early uterine deaths)

No significant inter-group differences were found in regard to pre-implantation losses. The effect on post-implantation loss was primarily due to an increase in early uterine deaths.
Total litter losses by resorption:
not specified
Description (incidence and severity):
not appropriate
Early or late resorptions:
not specified
Description (incidence and severity):
not appropriate
Dead fetuses:
not specified
Description (incidence and severity):
No information on dead fetuses available. However, it is stated in the report that the mean number of viable young litter was comparable among the groups.
Changes in pregnancy duration:
not specified
Description (incidence and severity):
not applicable
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
No differences between control and treatment groups.
Description (incidence and severity):
Results for the positive control
Group mean food consumption for thalidomide treated animals remained fairly constant throughout the dosing period and showed the characteristic reduction in the post-dosing period.
The positive control group was not included in the main statistical comparisons of group litter data.
The mean number of implantations in the thalidomide group was comparable to those of the vehicle control and test substance groups.
The mean number of viable young per litter in the thalidomide group was lower than any other group.
The thalidomide group did show an increase in post-implantation loss and the number of fetuses
exhibiting major malformations of a type associated with treatment of this kind and indicating the sensitivity of the strain to a known teratogen.

Effect levels (maternal animals)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
maternal general toxicity
Effect level:
15 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: No adverse effects observed at this dose level.
Key result
Dose descriptor:
LOAEL
Remarks:
maternal general toxicity
Effect level:
30 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Key result
Dose descriptor:
NOAEL
Remarks:
maternal developmental toxicity
Effect level:
30 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: No adverse effects in regard to maternal developmental toxicity observed up to this dose level.
Key result
Dose descriptor:
LOAEL
Remarks:
maternal developmental toxicity
Effect level:
60 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
pre and post implantation loss

Maternal abnormalities

Key result
Abnormalities:
effects observed, treatment-related
Localisation:
uterus
Description (incidence and severity):
increased post-implantation loss at 60 mg/kg bw/day

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
- 15 and 30 mg/kg bw/day: No differences regarding fetal body weights occurred up to and including 30 mg/kg bw/day.
- 60 mg/kg bw/day: statistically significant reduction in fetal body weight compared to vehicle controls (-18.1%)

Summarized results can be found in Attachment 3 in the attached background material.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
No statistically significant differences between control and treatment groups.
Changes in sex ratio:
not specified
Description (incidence and severity):
not applicable
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
No differences between control and treatment groups.
Anogenital distance of all rodent fetuses:
not examined
Description (incidence and severity):
not applicable
Changes in postnatal survival:
not examined
Description (incidence and severity):
not applicable
External malformations:
effects observed, treatment-related
Description (incidence and severity):
- 15 and 30 mg/kg bw/day: No differences regarding external malformations occurred up to and including 30 mg/kg bw/day.
- 60 mg/kg bw/day: abnormalities of the eye (anophthalmia, 13/106 animals in 5/13 litters, and microphthalmia, in 20/106 animals in 6/13 litters)

Summarized results can be found in Attachment 4 and 5 in the attached background material.
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
- 15 mg/kg bw/day: increased incidence of supernumerary rib variants (75.9% vs 63.7% in vehicle controls, not statistically significant)
- 30 mg/kg bw/day: increased incidence of supernumerary rib variants (79.5% vs 63.7% in vehicle controls, not statistically significant)
- 60 mg/kg bw/day: severe ossification defects of the skull, spine and ribs, increased incidence of supernumerary rib variants, 96.4% vs 63.7% in vehicle controls

Summarized results can be found in Attachment 4 and 5 in the attached background material.
Visceral malformations:
effects observed, treatment-related
Description (incidence and severity):
- 15 mg/kg bw/day: agenesis of the gallbladder (1/140 animals in 1/16 litters)
- 30mg/kg bw/day: agenesis of the gallbladder (1/134 animals in 1/16 litters)
- 60 mg/kg bw/day: agenesis of the gallbladder (in 10/106 animals in 4/13 litters), kidney agenesis (2/106 animals in 1/13 litters), abnormalities of the brain (hydrocephalus, in 28/106 animals in 9/13 litters)

Summarized results can be found in Attachment 4 and 5 in the attached background material.
Description (incidence and severity):
The thalidomide group did show an increase in the number of fetuses exhibiting major malformations of a type associated with treatment of this kind and indicating the sensitivity of the strain to a known teratogen.

Effect levels (fetuses)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Effect level:
30 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse findings at this dose level observed.
Key result
Dose descriptor:
LOAEL
Remarks:
developmental toxicity
Effect level:
60 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
external malformations
skeletal malformations
visceral malformations
other: increased incidences of malformations (anophthalmia and microphthalmia, hydrocephalus and ossification effects) and supernumerary ribs

Fetal abnormalities

Key result
Abnormalities:
effects observed, treatment-related
Localisation:
external: eye
skeletal: supernumerary rib
visceral/soft tissue: urinary
visceral/soft tissue: eye
Description (incidence and severity):
Reduced fetal weight, increased incidences of malformations (anophthalmia and microphthalmia, hydrocephalus and ossification effects) and supernumerary ribs observed at 60 mg/kg bw/day.

Overall developmental toxicity

Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
60 mg/kg bw/day
Treatment related:
yes
Relation to maternal toxicity:
developmental effects occurring together with maternal toxicity effects, but not as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
not specified

Applicant's summary and conclusion

Conclusions:
The current study was not performed under GLP conditions and conformed to no existing guideline. However, similarities to OECD guideline 414 (adopted 1981 and 2018) exist with some restrictions, especially methodological limitations such as missing examination of anogenital distance of fetuses, and blood sampling for analysis of thyroid hormones T4, T3 and TSH. Nevertheless, the study can serve as key study. Based on the obtained results, a maternal systemic NOAEL of 15 mg/kg bw/day and a developmental NOAEL of 30 mg/kg bw/day was derived in this study.