Benzene, ethenyl-, ar-bromo derivs.

Toxicity to reproduction

Administrative data

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1993

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: EPA TSCA, 40 CFR Part 798.4700, May 20, 1987

Deviations:

no

Test guideline 2

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

Test material

Test material information

Constituent 1

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

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)

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 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

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
100 mg/kg bw/day
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
400 mg/kg bw/day
Basis:
actual ingested

Doses / concentrations 3

Remarks:

Doses / Concentrations:
1600 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

35

Control animals:

yes, concurrent vehicle

Examinations

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

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

Details on results (P0)

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).

Effect levels (P0)

1

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)

2

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)

3

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: parental systemic toxicity

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

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.

Effect levels (F1)

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

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

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.