Registration Dossier

Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Link to relevant study records
Reference
Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 May 1990 to 8 February 1991
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.
Qualifier:
according to guideline
Guideline:
other: EPA TSCA, 40 CFR Part 798.4700, May 20, 1987
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Version / remarks:
1983
Deviations:
yes
Remarks:
the highest dose level exceeded the limit dose (i.e. 1600 mg/kg bw/day)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Portage, Michigan
- Age at study initiation: (P) 58 days; (F1) 21 days
- Weight at study initiation: (P) Males: 277-349 g; Females: 156-208 g; (F1) Males: 150-226 g (group mean values); Females: 126-157 g (group mean values)
- Housing: animals were housed 3 per cage by sex in suspended wire-mesh cages for a minimum of 3 days to allow for adaptation to the automatic watering system. Thereafter, animals were housed individually in wire-mesh cages suspended above cage-board. The animals were paired for mating in the home cage of the male. Following positive evidence of mating bred females were transferred to plastic maternity cages with nesting material. The dams were housed in these cages until weaning on lactation day 21 and then transferred to individual suspended wire-mesh cages till necropsy. Females for which there was no evidence of mating were placed in plastic maternity cages with nesting material upon completion of a 15-day mating period. If these animals did not deliver after 26 days, they were returned to individual suspended wire-mesh cages until necropsy. Pups form F0 generation (F1 litters) selected to become parents of the next generation were housed in suspended wire-mesh cages by sex after weaning and prior to selction (lactation day 28). Between lactation days 28 and 35, the selected F1 pups were housed 2 to 3 per cage by sex for a minimum of 3 days to allow for adaptation to the automatic watering system. Thereafter each animal was individually housed in a suspended wire-mesh cage.
- Diet (e.g. ad libitum): Purina Certified Rodent Chow #5002 ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: 33 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 67 to 78 °F
- Humidity (%): 29 to 90%
Occasional deviations from the targeted ranges were considered to have not affected the outcome of the study
- Air changes (per hr): 10-12
- Photoperiod (hrs dark / hrs light): 12 hrs light / 12 hrs dark

IN-LIFE DATES: From: 15 May 1990 (beginning of treatment of F0 generation) To: 8 February 1991 (last necropsy of F1 animals)
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 into tared glass beakers 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. The graduated cylinder was rinsed with an additional amount of vehicle, which was added to the storage container via a series 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 for one week of administration. The bottles were stored frozen. The dosing preparations were prepared weekly. One bottle per group was thawed at room temperature and mechanically stirred each day before and during administration to the animals. 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 and 320 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: 15 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- After 10 days of unsuccessful pairing replacement of first male by another male with proven fertility for additional 5 days
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged individually in plastic maternity cages with nesting material
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Before initiation of dosing, a representative series of suspensions for the low (100 mg/kg bw/day) and high (1600 mg/kg bw/day) dose levels was prepared and portions of these preparations were analysed. Duplicate aliquots (10 mL) were collected from the top, middle and bottom of thse preparations. One set of sample was analysed for homogeneity, the second set for stability after being frozen for 8 days. Samples (10 mL) were collected from each dose level (including the control) weekly for the first 4 weeks of the study and then every two weeks for the remainder of the study and 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 8 days when stored frozen.
Duration of treatment / exposure:
F0: pre-pairing period (at least 70 days), pairing period (up to 15 days), gestation and lactation periods till weaning (lactation day 21), selection of F1 generation (lactation day 28), [termination of F0 animals], pre-pairing period (at least 70 days), pairing period (up to 15 days), gestation and lactation periods till weaning (lactation day 21) [termination of F1 animals and F2 pups]
Frequency of treatment:
daily
Details on study schedule:
- F1 parental animals not mated until 9-11 weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 28 days of age.
- Age at mating of the mated animals in the study: F0: approx 19 weeks old; F1: 13 to 15 weeks old
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:
1600 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
35
Control animals:
yes, concurrent vehicle
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily for overt signs of toxicity, moribundity, mortality and signs of dystocia during the period of expected parturition. F0 parent animals were also observed for signs of toxicity at time of dosing (0-hr) and at 1, 2 and 4 hrs following dosing through study week 10. Afterward only the 1 hr post-dosing observation was mantained for the entire study

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly until evidence of copulation or the end of pairing period. Female body weights were recorded on gestation days 0, 7, 14 and 20, and on lactation days 0, 4, 7, 14 and 21.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, weekly, with the exception of the pairing period. Females food consumption measured on the same days of bw recording during gestation and lactation.

Oestrous cyclicity (parental animals):
Not performed (not required according to relevant guidelines of that time)
Sperm parameters (parental animals):
An evaluation of gross morphology and an estimate of sperm motility were performed for F0 and F1 males that did not sire a litter. Sperm evaluations were also performed for a single male each in the 100 and 400 mg/kg bw/day group which sired litters.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were weighed, examined externally, killed and preserved for possible future examination (approx. one half per litter placed in Bouin's fixative, the remaining half eviscerated, fixed in in 95% isopropyl alcohol, macerated in potassium hydroxide and stained with Alzarin Red S).

PARAMETERS EXAMINED
The following parameters were examined in F1 / F2 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals were necropsied following weaning of the F1 or F2 pups
- Maternal animals: All surviving animals were necropsied following weaning of the F1 or F2 pups

GROSS NECROPSY
- Gross necropsy consisted of complete necropsy including examination of external surface, all orifices, the cranial cavity, the external and cut surfaces of the brain and spinal cord, and the thoracic, abdominal and pelvic cavities including viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
Microscopic examination was performed for adult animals of the control and high dose groups on the following tissues/organs: all gross lesions, epididymides, kideney, liver, ovaries, pituitary, prostate, seminal vesicles, testes, uterus and vagina. Kidneys (F0 and F1 generations) and livers (F1 generation) were also examined from all adult animals of the low and mid-dose groups.
The ovaries or testes, kidneys and livers from all F0 and F1 parent animals were weighed at the time of necropsy.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 28 and 21 days of age, respectively.
- These animals were subjected to postmortem examinations (macroscopic examination)

Statistics:
All analyses were conducted using two-tailed tests (except as noted) for a minimum significance level of 5% when comparing each treated group to the vehicle control group. The following statistical tests were performed by a digital computer (with appropriate programming):
STATISTICAL TEST PARAMETER
Chi-square test with Yates' correction factor Pup sex ratio, parental mating and fertility indices, numbers of stillborn and dead pups,
pups viability index
One-way ANOVA with Dunnett's test Parental weekly body weights and weight changes, gestation and lactation body weights and
body weight changes, parental food consumption, mean gestation length, pup body weights,
absolute and relative organ weights, live litter size
Kolmogorov-Smirnov test Histopathological findings
Reproductive indices:
Male fertility index = No. of males siring at least one litter/total No. of males used for mating x 100
Female fertility index = No. of females with confirmed pregnancy/total No. of females used for mating x 100
Male (female) mating index = No. of males (females) with evidence of mating/total No. of males (females) used for mating x 100
Offspring viability indices:
Live litter size = total viable pups day 0/No. of litters with viable pups day 0
Viability index before culling = pups viable on day (N) before culling/pups viable on day 0 x 100
Viability index after culling = pups viable on day (N)/pups viable on day 4 after culling x 100
Livebirth index = No. of pups born alive/total No. of pups born x 100
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
at and above 400 mg/kg bw/day
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
at and above 400 mg/kg bw/day
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
at and above 400 mg/kg bw/day
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
kidney
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
a potential adverse effect on male fertility in the F1 generation at 1600 mg/kg bw/day
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
A total of 3 males and 14 females treated at 1600 mg/kg bw/day died or were euthanised moribund in the F0 generation. Two females treated at 1600 mg/kg bw/day died in the F1 generation. Treatment-related clinical signs occurred in animals treated at 400 and 1600 mg/kg bw/day in both generations. These included salivation, clear staining around the mouth and light yellow staining in the urogenital area, limited to the F0 generation for the 400 mg/kg bw/day animals. At 1600 mg/kg bw/day hypoactivity, impaired equilibrium and lethargy during the first week of dosing were also noted in the F0 generation.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
During the 1st week of treatment a reduced mean body weight gain and reduced food consumption values were recorded in the 400 mg/kg bw/day F0 males and a mean body weight loss and reduced food consumption values occurred in the F0 1600 mg/kg bw/day males. No further adverse effects were noted on food consumption but statistically reduced mean body weight gains for F0 males at 1600 mg/kg bw/day were recorded periodically and all mean weekly body weights remained statistically reduced through necropsy. Statistically significant reduced mean body weight gains and reduced food consumption values occurred in the 1600 mg/kg bw/day F1 males during the initial 3-4 weeks of dosing and statistically reduced mean body weight were recorded throughout the treatment period. In the F1 1600 mg/kg bw/day females, mean body weight gain was inhibited during the last week of gestation, resulting in reduced mean body weights on gestation day 20 and throughout lactation. Food consumption in these females was statistically significantly reduced during lactation. A statistically significantly reduced mean body weight gain was also recorded in F1 males treated at 400 mg/kg bw/day during the first week of dosing.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Reproductive parameters were not affected in the F0 generation. A potential adverse effect on male fertility was observed in the F1 generation at 1600 mg/kg bw/day. Fertility index was 65.7% (vs 80.0% in the controls), 12/35 (34%) males did not sire a litter, 31/35 (89%) males had lower testes weights and 10/12 (83%) males which did not sire a litter had low testes weights. in addition, fertility and/or testes weight data for these F1 males differed markedly from F1 males of the other groups and from F0 1600 mg/kg bw/day males.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Increased absolute and relative liver weights (400 and 1600 mg/kg bw/day F0 and F1 animals); increased mean absolute and relative kidney weights (F0 males treated at 400 mg/kg bw/day and F0 animals at 1600 mg/kg bw/day); increased absolute and relative kidney weights (F1 males treated at 400 and 1600 mg/kg bw/day); decreased absolute testes weight in F1 males at 1600 mg/kg bw/day. No effects on liver and kidney weight in F0 animals treated at 100 mg/kg bw/day; mean liver and kidney weights (absolute and relative) in F1 males, but not females, treated at 100 mg/kg bw/day were also increased.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Kidney changes were noted in F0 and F1 animals treated at 1600 mg/kg bw/day. These were characterised by tubular dilatation (13/32 males and 5/21 females in F0; 15/35 males and 13/33 females in F1), followed by papillar necrosis (7/32 males and 1/21 females in F0, 7/35 males and 1/33 females in F1).

Dose descriptor:
NOAEL
Effect level:
400 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: reproductive toxicity
Remarks on result:
other: Generation: both generations (migrated information)
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: neonatal toxicity
Remarks on result:
other: Generation: both generations (migrated information)
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: parental systemic toxicity
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
at and above 400 mg/kg bw/day
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
at and above 400 mg/kg bw/day
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
at and above 400 mg/kg bw/day
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
VIABILITY (OFFSPRING)
Mean live F1 litter size was slightly decreased (non-significant) at 400 and 1600 mg/kg bw/day; mean live F2 litter size was slightly decreased (non-significant) at 1600 mg/kg bw/day. The number of F1 dead pups on lactation day 0 was statistically increased in the 400 and 1600 mg/kg bw/day, resulting in a decreased viability index on lactation days 1 and 4 (before culling) at 1600 mg/kg bw/day. Viability indices were significantly decreased in F2 at 400 mg/kg bw/day (lactation days 7, 14 and 21) and at 1600 mg/kg bw/day (lactation days 1 and 4, before culling).

CLINICAL SIGNS (OFFSPRING)
The general physical condition of the F1 and F2 pups was affected at 400 mg/kg bw/day (body cool to the touch for F1 offspring, body cool to the touch, pups small in size, dehydration, laboured respiration and hair loss on the dorsal head for F2 offspring) and 1600 mg/kg bw/day (body cool to the touch and pups small in size for F1 offspring and body cool to the touch for the F2 offspring).

BODY WEIGHT (OFFSPRING)
Mean F1 pup body weights were statistically reduced beginnning day 28 post partum at 400 mg/kg bw/day and day 1 post partum at 1600 mg/kg bw/day. These reduction continued through day 42 post partum. Mean F2 pup body weights were statistically decreased at 400 mg/kg bw/day on lactation day 21 and at 1600 mg/kg bw/day from lactation day 1 through 21.

GROSS PATHOLOGY (OFFSPRING)
Necropsy findings for F1 and F2 surplus pups were not suggestive of any correlation with parental treatment.
Dose descriptor:
LOAEL
Generation:
F1
Effect level:
< 100 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: parental systemic toxicity
Reproductive effects observed:
not specified
Conclusions:
Based on results of this 2-generation study, the NOAEL for reproductive toxicity was 400 mg/kg bw/day based on possible adverse effect on F1 male fertility observed at 1600 mg/kg bw/day. The NOAEL for neonatal toxicity was 100 mg/kg bw/day based on decreases in viability indices and pup body weights recorded at 400 and 1600 mg/kg bw/day. The NOAEL for parental systemic toxicity was 100 mg/kg bw/day in the F0 generation, and less than 100 mg/kg bw/day in the F1 generation.
Executive summary:

In a two-generation study, groups of 35 male and female Sprague-Dawley rats were treated daily, by oral gavage, at dosages of 100, 400 and 1600 mg/kg bw/day. A concurrent vehicle control group received Mazola corn oil at 5 mL/kg bw. The F0 parent animals were treated for at least 70 days prior to mating. Treatment continued throughout all subsequent phases of the study until one day prior to the scheduled necropsy for each animal. All females were allowed to deliver and rear their pups to weaning (lactation day 21, culling performed on lactation day 4). One litter was produced in each generation. Offspring from the F0 animals (F1 litters) were selected to constitute the F1 generation. The study design for the F1 generation was identical to the F0 generation; 35 animals per sex were selected for each dose group. Beginning day 22 post partum, the selcted F1 parent animals were treated for at least 70 days prior to the pairing and throughout all subsequent phases of the study until one day prior to the scheduled necropsy for each animal.

Decreased male fertility in the F1 generation at 1600 mg/kg bw/day was considered to be a potential effect of dibromostyrene administration. Although no morphological changes were observed in the reproductive tract of these males, the mean absolute testes weight was signficantly reduced. Other reproductive parameters (gestation length and parturition) were unaffected by treatment in the F0 and F1 generations at any dose level.

Survival was affected at 1600 mg/kg bw/day; 17 F0 animals (3 males and 14 females) and 2 F1 females died or were euthanised moribund. Nine of these F0 females died due to an apparent enhanced toxicity of the test substance due to water deprivation on one day. Treatment-related clincial signs (primarily salivation, clear staining around the mouth and/or yellow staining in the urogenital area) occurred in the F0 and F1 animals at 400 and 1600 mg/kg bw/day. Body weight gain and/or food cinsumption were inhibited in the 400 mg/kg bw/day males (F0 and F1 during the 1st week of dosing), 1600 mg/kg bw/day males (F0 and F1 primarily during the initial one to four weeks of dosing) and 1600 mg/kg bw/day females (F1 at the end of the gestation and during the lactation period).

No tretment-related internal findings were observed in the F0 and F1 parent animals that survived to the scheduled necropsies. Increased liver weights were apparent in the F0 and F1 generations at 400 and 1600 mg/kg bw/day. Increased weights and treatment-related microscopic lesions were observed in the kidney at 1600 mg/kg bw/day (F0 and F1); the microscopic lesions consited of tubular dilatation, nephrosis and/or papillary necrosis. Increased kidney weights, but no microscopic lesions, were also noted in males at 400 mg/kg bw/day (F0 and F1).

In the F1 and F2 neonates, adverese effects were apparent at 400 and 1600 mg/kg bw/day. Live litter sizes were slightly decreased (non-significant) in the 400 mg/kg bw/day F1 pups and the 1600 mg/kg bw/day F1 and F2 pups. Pup viability was reduced at 400 mg/kg bw/day (F2) and 1600 mg/kg bw/day (F1 and F2). Changes in clinical condition of the pups (primarily body cool to the touch) were observed in both generations at 400 and 1600 mg/kg bw/day. Neonatal body weights were reduced at 400 mg/kg bw/day beginning days 28 (F1) or 21 (F2) post partum and at 1600 mg/kg bw/day beginning day 1 post partum (F1 and F2).

At 100 mg/kg bw/day, kidney and liver weights were increased in the F1 males. No other adverse effects were apparent on the F0, F1 or F2 animals.

The NOAEL for reproductive toxicity was 400 mg/kg bw/day. The NOAEL for neonatal toxicity was 100 mg/kg bw/day. The NOAEL for parental systemic toxicity was 100 mg/kg bw/day for the F0 generation, and less than 100 mg/kg bw/day for the F1 generation.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The quality of the database is high.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

The key study for this endpoint, Nemec MD (1993), was performed in accordance with the current internationally accepted guideline OECD 416 and in compliance with GLP. The study was assigned a reliability score of 1 and considered adequate for assesment. The NOAEL from the study was selected for the assessment of reproductive toxicity as a protective level against all reported effects.

Short description of key information:

Based on results of the 2-generation study (Nemec MD (1993)), the NOAEL for reproductive toxicity was 400 mg/kg bw/day based on possible adverse effect on F1 male fertility observed at 1600 mg/kg bw/day. The NOAEL for neonatal toxicity was 100 mg/kg bw/day based on decreases in viability indices and pup body weights recorded at 400 and 1600 mg/kg bw/day. The NOAEL for parental systemic toxicity was 100 mg/kg bw/day in the F0 generation, and less than 100 mg/kg bw/day in the F1 generation.

Justification for selection of Effect on fertility via oral route:

A single good quality two-generation reproductive toxicity study was available for the assessment of effects on fertility.

Effects on developmental toxicity

Description of key information

Treatment of pregnant rats with dibromostyrene during the organogenesis period at dosages of 100, 400, 800 and 1600 mg/kg bw/day caused both maternal and developmental toxic effects. 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 considered 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 moribund. Related to the degree of maternal toxicity, effects on foetuses consisted mainly in increases 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.

Link to relevant study records
Reference
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.
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
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
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
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.
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.
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
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.
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
Key result
Abnormalities:
not specified
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).
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
Abnormalities:
not specified
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
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.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
High. Two good quality studies were available using two standard species.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

The Lamb IC (1993) study in rats was selected as the key study over the study in rabbits (by the same author) as it provided the lowest NOAEL of the two studies and therefore conferred a higher level of protection. The rat study was selected as the key study was conducted to the guideline OECD 414 and in compliance with GLP. As such, the study was assigned a reliability score of 1 and considered adequate for assessment.

The supporting study, in rabbits, by Lamb IC (1993) was conducted in compliance with GLP and according to guideline OECD 414 and was assigned a reliability score of 1.

Justification for selection of Effect on developmental toxicity: via oral route:

The study with rats was selected as the key study as the NOAEL derived in the study was lower than that in the rabbit study, and was therefore selected on a worst-case basis.

Justification for classification or non-classification

According to the criteria in directive 67/548/EEC and regulation (EC) No 1272/2008, the substance does not meet the criteria for classification for reproductive or developmental toxicity.

Additional information