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Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 April to 12 May 1990
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1993
Report date:
1993

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
The highest dose level exceeded the limit dose (i.e. 1600 mg/kg bw/day instead of 1000 mg/kg bw/day)
Qualifier:
according to guideline
Guideline:
other: EPA TSCA, 40 CFR Part 798.4900, May 20, 1987
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Benzene, dibromoethyl Benzene, ethenyl-, ar-bromo derivs.
EC Number:
603-094-7
Cas Number:
125904-11-2
Molecular formula:
C8 H6 Br2
IUPAC Name:
Benzene, dibromoethyl Benzene, ethenyl-, ar-bromo derivs.
Test material form:
liquid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Portage, Michigan
- Age at study initiation: approx. 14 weeks
- Weight at study initiation: 222 to 315 g
- Housing: individually in wire-mesh cages suspended above cage-board
- Diet (e.g. ad libitum): Purina Certified Rodent Chow #5002 ad libitum
- Water (e.g. ad libitum): tap water delivered by an automatic watering system ad libitum
- Acclimation period: 33 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 68 to 75 °F
- Humidity (%): 32 to 95%
These values were outside the terget ranges in a few occasions. These deviations had no impact on the outcome of the study.
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12 hrs light/12 hrs dark

IN-LIFE DATES: From: day 6 of pregnancy To: day 17 of pregnancy

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Remarks:
Mazola
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The appropriate amounts of dibromostyrene were weighed for each group and transferred to graduated cylinders via a series of vehicle rinses. Specified amounts of vehicle were then added. The preparations were inverted and shaken several times and then transferred to individual storage containers. An additional amount of vehicle was added to the storage container via a series of rinses to achieve the appropriate concentration for each group. The prepartions were stirred for at least 10 minutes using a magnetic plate and stir bars. From the storage container, a sufficient number of bottles were filled for each group. The bottles were stored frozen. The dosing suspensions were prepared weekly. One bottle per group was thawed at room temperature and mechanically stirred each day before and during administration to the animals. All test suspensions were used within one hour of thawing. Unused portions of the thawed test suspension were discarded following the completion of dosing each day.

VEHICLE: Mazola corn oil
- Concentration in vehicle: 20, 80, 160 and 320 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples were collected from the low and high dose levels prior to study intiation. These pre-samples were analysed for homogeneity and stability. Samples (approximately 10 mL) were collected weekly from each dose level following test material preparation. These samples were analysed for concentration. Samples were analysed using a gas chromatography/flame ionization detection method.
The dosing preparation were homogeneous,contained the designated amount of dibromostyrene and were stable for two weeks when stored frozen.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
From day 6 to 15 of (presumed) pregnancy
Frequency of treatment:
Daily
Duration of test:
Dams were sacrificed on day 20 of pregnancy
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
100 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
400 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
800 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1600 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
25 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: dosages were chosen on the basis of a RF study conducted at 100, 200, 400, 800 and 1600 mg/kg bw/day. A maternal response was expressed at dose levels of 800 and 1600 mg/kg bw/day (increased incidence of clinical signs and inhibition of body weight gain and food consumption). Maternal survivial was not affected. A developmental effect was equivocal at 1600 mg/kg bw/day as indicated by slightly reduced foetal body weight.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily for mortality, moribundity, general appearance and behaviour; approximately one hour after dosing for clinical signs

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily from day 0 to 20 of gestation (before dosing during the treatment period)

BODY WEIGHT: Yes
- Time schedule for examinations: days 0, 3, 6 through 16 and 20 of pregnancy

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

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #20
- Organs examined: a complete necropsy was performed, and all abnormalities were recorded. The liver of each dam was excised, trimmed, weighed and the findings recorded. Maternal tissues were retained only as deemed necessary by gross findings.
The uterus and ovaries were excised, the number of corpora lutea on each ovary was recorded; the trimmed uterus was weighed, opened and the number and location of all foetuses, early and late resorptions and the total number of implantation sites were recorded. Uteri with no macroscopic evidence of nidation were excised, opened and subsequently placed in 10% ammonium sulfite solution for detection of early implantation loss accoding to Salewsky.

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: Yes
- Number of late resorptions: Yes

OTHER: crown-rump measurements were recorded for late resorptions and the tissues were discarded
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: approx. half per litter
Statistics:
All analyses were conducted using two-tailed tests (except as noted) for a minimum significance lavel of 5% when comparing each treated group to the vehicle control group. Animals that were not gravid were not included in the claculations of group means for body weights, food consumption and other applicable parameters. The following statistical tests were performed by a digital computer (with appropriate programming):
STATISTICAL TEST PARAMETER
Chi-square test with Yates' correction factor Foetal sex ratio
Fisher's Exact test Malformations and variations
Mann-Whitney U-test Early and late resorptions, dead foetuses, postimplantation losses
One-way ANOVA with Dunnett's test Corpora lutea, total implantations, viable foetuses, foetal bw, maternal bw and bw changes,
maternal net bw chenges and gravid uterine weights, maternal food consumption and maternal
liver weight
Kruskal-Wallis test (one-tailed test) Litter proportion of intrauterine data (considering the LITTER as the experimental unit)
Historical control data:
Historical control data were included in the report. Relevent reference values were included within the text for discussion of specific findings.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
not specified
Dermal irritation (if dermal study):
not specified
Mortality:
not specified
Body weight and weight changes:
not specified
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified
Other effects:
not specified

Maternal developmental toxicity

Number of abortions:
not specified
Pre- and post-implantation loss:
not specified
Total litter losses by resorption:
not specified
Early or late resorptions:
not specified
Dead fetuses:
not specified
Changes in pregnancy duration:
not specified
Changes in number of pregnant:
not specified
Other effects:
not specified
Details on maternal toxic effects:
Maternal toxic effects:yes. Remark: including the lower dose level

Details on maternal toxic effects:
100 mg/kg bw/day: limited to a decreased body weight gain (not statistically significant) during the first 3 days of treatment and dose-related reduction of food consumption
400 mg/kg bw/day: clinical signs (soft stool, hair loss and yellow or brown matting on several body surfaces); decreased body weight gain during the first 6 days of treatment, accompanied by reduced food consumption
800 mg/kg bw/day: clinical signs (soft stools, decreaed defecation, yellow faeces, bright yellow urine, hair loss and yellow or brown matting on several body surfaces; decreased body weight gain during the first 6 days of treatment, accompanied by reduced food consumption
1600 mg/kg bw/day: marked. Mortalities (5 of 25 animals + one animal sacrificed moribund), clinical signs (lethargy, high carriage, impaired equilibrium, soft stools, mucoid faeces, diarrhea, decreased defecation and urination, orange or yellow faeces, bright yellow or red urine, hair loss on several body surfaces, tan, brown and yellow matting on several body surfaces, red material around the nose, mouth and eyes, and tan staining around the nose and mouth); decreased body weight gain throughout the treatment period; decreased food consumption during the first 6 days of dosing.

Effect levels (maternal animals)

open allclose all
Key result
Dose descriptor:
LOEL
Effect level:
400 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Maternal abnormalities

Key result
Abnormalities:
not specified

Results (fetuses)

Fetal body weight changes:
not specified
Reduction in number of live offspring:
not specified
Changes in sex ratio:
not specified
Changes in litter size and weights:
not specified
Changes in postnatal survival:
not specified
External malformations:
not specified
Skeletal malformations:
not specified
Visceral malformations:
not specified
Other effects:
not specified
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
400 mg/kg bw/day: increased incidence of a skeletal specific variation (7th cervical rib)
800 mg/kg bw/day: increased incidence of skeletal variations (sternebrae #5 and/or #6 unossified and 7th cervical rib). These variations were within HC ranges but likely dose related because similar to those observed at the highest dose level. 1 foetus with hydrocephaly was also noted (within HC range)
1600 mg/kg bw/day: decreased mean foetal body weight and increased post-implantation loss (with corresponding decrease in the number of mean viable foetuses). Statistically significant increased number of litters with external malformations, mainly cranio-facial defects (i.e. microphthalmia and/or anopthalmia, total of 37 foetuses from 9 litters, exceeding HC values; mandibular agnathia and aglossia in 3 foetuses, one of which had also microstomia and malpositioned pinnae, from 3 litters, exceeding HC values, cleft palate in one foetus exceeding HC value on a litter basis). Soft tissue malformations were also observed (single foetuses with folded retina and common truncus arteriosus, and two foetuses from the same litter with hydrocephaly - all exceeding HC values). Increased incidence of skeletal variations (sternebrae #5 and/or #6 unossified and 7th cervical rib).

Effect levels (fetuses)

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
Key result
Dose descriptor:
LOAEL
Effect level:
400 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
skeletal malformations

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
400 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to maternal toxicity:
not specified
Dose response relationship:
yes
Relevant for humans:
not specified

Applicant's summary and conclusion

Conclusions:
Treatment of pregnant rats with dibromostyrene during the organogensis period at dosages of 100, 400, 800 and 1600 mg/kg bw/day caused both maternal and developmental toxic effcts. Maternal toxicity was slight at the lowest dose level of 100 mg/kg bw/day however, based on the slight decreased body weight gain during the first 3 days of dosing and a dose-related decrease in food consumption during the first 6 days of dosing, this dose level was consedered to be a LOAEL. Maternal toxicity increased with increasing dosages as evidenced by the number of clinical signs and effects on body weight gains and food consumption. Maternal toxicity was marked at the highest dose level of 1600 mg/kg bw/day, where 5 dams died and another one was sacrificed muribund. Related to the degree of maternal toxicity, effects on foetuses consisted mainly in increases of of specific skeletal variations at 400 mg/kg bw/day and above, and teratogenic effects, primarily cranio-facial defects, at the highest dose level of 1600 mg/kg bw/day. The NOAEL for developmental toxicity was 100 mg/kg bw/day.
Executive summary:

In a teratogenicity study in rats, groups of 25 presumed pregnant female rats were traeted daily, from day 6 to 15 of gestation, at dosages of 100, 400, 800 or 1600 mg/kg bw/day. A concurrent vehicle control group received corn oil alone, at a volume of 5 mL/kg bw.

Maternal toxicity was noted at all dose level, expressed by reduced body weight gains and reduced food consumption primarily during the first 3 -6 days of treatment. Clinical signs, increasing in severity and number of occurences, were noted in animals dosed at 400 mg/kg bw/day and above. The highest dose level of 1600 mg/kg bw/day was severely toxic to the dams, causing the death of 5 of them and and the sacrifice moribund of another one. Similarly, developmental toxicity was marked at 1600 mg/kg bw/day, by a decrease in mean foetal weights and an increase in post-implantation loss. in addition, deveopmental toxicity was also noted at dosages of 400 mg/kg bw/day and higher, expressed by external malformations (1600 mg/kg bw/day dose level) and specific skeletal variations (400 mg/kg bw/day and higher). Although body weight gain of females dosed at 100 mg/kg bw/day was only slightly reduced during the first 3 days of dosing, and food consumption was reduced during the first 6 days of dosing, this dose level was set as the LOAEL for maternal toxicity. 100 mg/kg bw/day was NOAEL for deveopmental toxicity.