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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:
17 Nov - 24 Dec 1985
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions

Data source

Referenceopen allclose all

Reference Type:
other company data
Title:
Unnamed
Year:
1987
Report date:
1987
Reference Type:
study report
Title:
Unnamed
Year:
1987
Report date:
1987

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted 2018
Deviations:
yes
Remarks:
methodological limitations (no analysis of T3, T4 and TSH, and thyroid gland (organ weight and histopathology); anogenital distance of fetuses not determined, number of implantation sites not determined, no head examinations.)
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted 1981
Deviations:
no
GLP compliance:
yes
Limit test:
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:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories Ltd., UK
- Age at study initiation: not reported
- Weight at study initiation: group means ranged between 165 - 175 g and between 190 - 200 g
- Housing: individually caged in type RM2 cages with high-density polypropylene body with stainless steel grid floor and lid
- Diet: Altromin 1314 (Altromin Spezialfutterwerke GmbH, Lage, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 11 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 22
- Humidity (%): 40 - 70
- Air changes (per hr): 15

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
acidified with 0.5% acetic acid
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test material was formulated fresh daily as suspensions in 0.5% CMC containing 0.5% acetic acid.

VEHICLE
- Justification for use and choice of vehicle (if other than water): 0.5% CMC containing 0.5% acetic acid was used since the test material was unstable under alkaline conditions.
- Amount of vehicle: 5 mL/kg bw/day
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Short-term stability (4 h) and homogeneity of the test material in the vehicle was assessed via HPLC for 0.8, 2.5 and 8.0 mg/mL. The test substance was stable over 4 h in the dosage form used and homogeneity was confirmed.
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: 1/1
- Length of cohabitation: 1 night
- Further matings after two unsuccessful attempts: not specified
- Verification of same strain and source of both sexes: no
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as Day 0 of pregnancy
Duration of treatment / exposure:
from Day 6 - Day 15 post coitum
Frequency of treatment:
daily
Duration of test:
until Day 20 post coitum
Doses / concentrationsopen allclose all
Dose / conc.:
4 mg/kg bw/day
Dose / conc.:
12.5 mg/kg bw/day
Dose / conc.:
40 mg/kg bw/day
No. of animals per sex per dose:
22
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dosages for this study were based upon the results of a preliminary teratology study in which treatment at 140 mg/kg bw/day resulted in maternal lethality (4/4 animals). Treatment at 45 mg/kg bw/day resulted in reduced maternal food consumption and body weight gain, reduced fetal weight and length, and in enlarged placentas, with prominent craterfom depressions on their admetral surfaces.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS/CLINICAL SIGNS: Yes
- Time schedule: daily
- parameters observed: signs of ill-health or reaction to treatment.

BODY WEIGHT: Yes
- Time schedule for examinations: Days 0, 3, 6-15, 17 and 20 of gestation

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined:
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: No data
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Blood sampling:
- Plasma: No
- Serum: No
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: No
- Anogenital distance of all live rodent pups: No
Statistics:
Group mean values and standard deviations were calculated for maternal body weight and food consumption, fetal weight, fetal length, and placental weight (from individual means).

Maternal gross observational signs during gestation were tested using the Fisher exact test. Maternal food consumption and body weight data, uterine, fetal and placental weights, fetal length and numbers of corpora lutea and live fetuses and percent pre- and post-implantation loss (Freeman-Tukey transformed data) were tested using Students 't'-test using pooled within group error variance.
Fetal data (observations at necropsy, at free-hand sectioning and at skeletal examination) were tested using the Chi-square test, for cases where all cells were > 5, or the Fisher exact test, for cases where not all cells were > 5. Litter mean proportions for these data were tested nonparametrically, using the Mann-Whitney U-test, with correction for tied ranks, applied as a two-tail test.
Numbers of litters with one or more affected fetus were tested using the Chi-square test or the Fisher exact test.
Historical control data:
Historical control data regarding different parameters provided in attachment 6 (M-346732-01-1).

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
No differences were observed between control and treatment groups.
Dermal irritation (if dermal study):
not examined
Description (incidence and severity):
not applicable
Mortality:
no mortality observed
Description (incidence):
All dams survived the scheduled course of study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- 4 and 12.5 mg/kg bw/day: No differences regarding body weight occurred up to and including 12.5 mg/kg bw/day.
- 40 mg/kg bw/day: reduced body weight in comparison to controls both during the dosing period and after the cessation of dosing (from -12.2 to -15.5%), decreased body weight gain (p < 0.05, from Day 0 to 20 and from Day 6 to 20 corresponding to 12.6% and 14.4% respectively).

Summarized results can be found in Attachment 1.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- 4 and 12.5 mg/kg bw/day: No differences regarding food consumption occurred up to and including 12.5 mg/kg bw/day.
- 40 mg/kg bw/day: markedly reduced food consumption during the first days of dosing (Days 7 to 9 post coitum, -27.5%), moderately reduced from Day 10 to Day 13 (-8.2%), increased consumption from Day 14-16 and 17-20 (+14.5 and +10.8%, respectively).

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:
effects observed, non-treatment-related
Description (incidence and severity):
- 4 mg/kg bw/day: No differences regarding gross pathology occured at 4 mg/kg bw/day.
- 12.5 mg/kg bw/day: 1 placenta was markedly enlarged and appeared mottled, 1 additional placenta was likewise somewhat enlarged, but of normal appearance.
- 40 mg/kg bw/day: 1 placenta was grossly enlarged and showed irregular depressions on its admetral surface.

Due to the absence of histopathological findings, no clear correlation to treatment is present.
Neuropathological findings:
not examined
Description (incidence and severity):
not applicable
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No differences were observed regarding placenta histopathology between control and treatment groups. Briefly, all placentae examined from control and high dosage groups, including one placenta descibed at necropsy with ‘irregular depressions on admetral surface’ were histologically normal.
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:
no effects observed
Description (incidence and severity):
No differences were observed between control and treatment groups.
Pre- and post-implantation loss:
effects observed, treatment-related
Description (incidence and severity):
- 4 mg/kg bw/day: No differences regarding pre- and post-implantation loss occurred at 4 mg/kg bw/day.
- 12.5 mg/kg bw/day: elevated post-implantation loss, compared to controls
- 40 mg/kg bw/day: elevated post-implantation loss, compared to controls

Pre-implantation loss was elevated at 12.5 mg/kg bw/day, but not among animals in the high dosage group. It was therefore considered incidental.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
No differences were observed between control and treatment groups.
Early or late resorptions:
no effects observed
Description (incidence and severity):
No differences were observed between control and treatment groups.
Dead fetuses:
no effects observed
Description (incidence and severity):
No differences were observed between control and treatment groups.
Changes in pregnancy duration:
not examined
Description (incidence and severity):
not applicable
Changes in number of pregnant:
not specified
Description (incidence and severity):
not applicable
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Maternal body weight nett of the gravid uterus was depressed among animals dosed at 40 mg/kg/day, indicating that the maternal compartment of the maternal-fetal complex was affected at this dosage.

Effect levels (maternal animals)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
maternal general toxicity
Effect level:
12.5 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:
40 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:
4 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 developmental toxicity
Effect level:
12.5 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 was observed at 12.5 mg/kg bw/day. Maternal body weight nett of the gravid uterus was depressed among animals dosed at 40 mg/kg bw/day, indicating that the maternal compartment of the maternal-fetal complex was affected at this dosage.

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
- 4 and 12.5 mg/kg bw/day: No differences regarding fetal body weight occurred up to and including 12.5 mg/kg bw/day.
- 40 mg/kg bw/day: fetal weight and fetal length were significantly reduced compared to controls.

Summarized results can be found in Attachment 3.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
No differences observed between control and treatment groups.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
No differences observed between control and treatment groups.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
No differences observed 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):
- 4 and 12.5 mg/kg bw/day: No differences regarding external malformations occurred up to and including 12.5 mg/kg bw/day.
- 40 mg/kg bw/day: anophthalmia (6 fetuses in 3 litters), microphthalmia (2 fetuses in 2 litters)

Anophthalmia also occurred in a single low dosage fetus, associated with hydrocephalus and right aortic arch. In contradistinction to anophthalmic and microphthalmic fetuses in the high dosage groups, this low dosage fetus was dramatically runted. This fetus was found at the distal (ovarian) end of the uterus, an implantation site often associated with reduced fetal size. Thus, it would be difficult to attribute the malformations observed in this fetus solely to the action of the test material.

If data from fetuses allocated to free-hand sectioning and those allocated to skeletal evaluation are pooled, the overall incidence of anophthalmia/microphthalmia in the high dosage group is 19/319, or 6%.

Summarized results can be found in Attachment 4.
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
- 12.5 mg/kg bw/day: adverse effect on development of the spinal and thoracic bones
- 40 mg/kg bw/day: reduced ocular orbits (11 fetuses), adverse effect on development of the spinal and thoracic bones, higher incidences of incidence of unossified, bipartite or/and incompletely fused thoracic vertebral centra, increased incidence of lumbar ribs, higher incidences of unossified and/or irregular sternebrae, ossification of the metatarsal bones was inferior, all compared to controls. Moreover, 2 fetuses had scoliosis caused by unequal numbers of right and left vertebral arches (associated with fused ribs).

Ambiguous findings:
Increased incidence of large anterior fontanelles compared to controls, possibly indicating retarded cranial ossification, was found in the low and intermediate dosage groups. In the high dosage group, small fontanelles occurred more frequently. Reduced ossification of the parietal and/or interparietal bones was more often found in the 4 and 12.5 mg/kg bw/day groups compared to controls, but fetuses of the 40 mg/kg bw/day group did not show any such signs (only in the case of the frontal bone was there a significant increase in reduced ossification).

Summarized results can be found in Attachment 5.
Visceral malformations:
effects observed, treatment-related
Description (incidence and severity):
- 40 mg/kg bw/day: diaphragmatic hernias (2 fetuses in 2 litters), 1 fetus showed narrowing of the aortic arch (accompanied by esophageal atresia and absence of the trachea), anophthalmia (6 fetuses in 3 litters), microphthalmia (2 fetuses in 2 litters), increased Incidence of short renal papillae.

A case of pronounced narrowing of the aortic arch occurred among fetuses treated at 12.5 mg/kg bw/day as well; the possibility of treatment-related malformations at this dosage can thus not be excluded.

Not related to treatment:
- 4 mg/kg bw/day: 1 fetus with a right aortic arch (observed in historical control data and therefore probably not related to treatment), the fetus also had anophthalmia and was severely runted (1.56 g) with hydrocephalus. This fetus was implanted at the distal (ovarian) terminus of the uterus, a fact which may have contributed to the occurrence of runting and of other malformations.
- 12.5 mg/kg bw/day: umbilical hernia (observed in historical control data and therefore probably not related to treatment)

Summarized results can be found in Attachment 3 and 4.
Details on embryotoxic / teratogenic effects:
A number of fetal anomalies often indicative of generalised developmental retardation, consistent with reduced fetal weight and length were observed. These include shortened renal papillae and unossified vertebral centra, sternebrae, metacarpals and metatarsals. The occurrence of lumbar ribs among high and intermediate dosage fetuses, while not a major malformation, does indicate a deleterious effect of the test compound upon morphogenesis.
While none of the following observations achieved statistical significance, their overall pattern of incidence might be considered as suggestive for treatment relation: diaphragmatic hernia, narrowing of the aortic arch and fusion of ribs associated with scoliosis (the last two were in one case associated with esophageal atresia and absence of trachea).

Effect levels (fetuses)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Effect level:
4 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed at this dose level.
Key result
Dose descriptor:
LOAEL
Remarks:
developmental toxicity
Effect level:
12.5 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
skeletal malformations

Fetal abnormalities

Key result
Abnormalities:
effects observed, treatment-related
Localisation:
skeletal: rib
Description (incidence and severity):
increases in the incidence of 14th thoracic ribs (further details are summarised in Attachment S)

Overall developmental toxicity

Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
12.5 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 performed under GLP conditions and in accordance with OECD TG 414 (adopted 1981). The guideline was updated 2018, so that the study has some deviations to the current guideline. Nevertheless, the study is reliable and valid.
Based on the results it may be concluded that the test substance was found to be more toxic to fetuses than to pregnant female rats. Maternal animals were only affected at the highest dose of 40 mg/kg bw/day in regard to systemic maternal toxicity, evident by reduced body weight, feed consumption and body weight nett of the gravid uterus. Therefore, the NOAEL for maternal (systemic) toxicity was determined to be 12.5 mg/kg bw/day. Based on increased post-implantation loss and fetuses with malformations including increases in the incidence of 14th thoracic ribs found at 12.5 and 40.0 mg/kg bw/day the fetal (developmental) NOAEL was 4 mg/kg bw/day in CD rats.