Benzene, ethenyl-, ar-bromo derivs.

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 March to 15 July 1988

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:

1988

Report date:

1988

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

The highest dose level exceeded the limitd dose (i.e. 1600 mg/kg bw/day instead of 1000 mg/kg bw/day) and no FOBs and motor activity assessments were performed

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:

other: Crl:CD BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Portage, Michigan
- Age at study initiation: 7 weeks old
- Weight at study initiation: males: 194 to 247 g; females: 125 to 167 g
- Housing: individually in clean, wire-mesh cages suspended above cage-board
- Diet (e.g. ad libitum): Purina Certified Rodent Chow #5002 ad libitum
- Water (e.g. ad libitum): drinking water delivered by an automatic watering system ad libitum
- Acclimation period: 20 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 66 to 74 °F
- Humidity (%): 40 to 76%
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12 hrs light/12 hrs dark

IN-LIFE DATES: From: day 1 To: day 90, 91 or 92 (until the day prior to the scheduled sacrifice or the beginning of the recovery period)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Remarks:

Mazola

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: during the dosing period the appropriate amount of dibromostyrene was weighed for each group and transferred to a volumetric flask via a series of vehicle rinses. Vehicle was then added in sufficient quantity to achieve the appropriate concentration for each group. The suspensions were inverted and shaken several times and then stirred for at least 10 minutes using a magnetic plate and stir bars. After mixing the test suspensions were transferred to individual dosing bottles and frozen. The dosing suspensions were prepared weekly. One bottle per group was thawed to room temperature and mechanically stirred each day before administration to animals. Unused portion of the thawed test suspension were discarded following the completion of dosing each day.

VEHICLE: Mazola corn oil
- Concentration in vehicle: 26, 60, 140 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 initiation. These pre-initiation samples were analysed for homogeneity and stability. Samples (approx. 10 mL) were collected weekly from each dose level following test material preparation and analysed for concentration. Samples were analysed using a gas chromatography/flame ionization detection analytical method.
Analysis of pre-initiation samples showed that these dosing solutions were homogeneous and stable for 7 days when frozen. Over the course of the study, the batches of dosing solutions contained, as an average, the amount of test material specified in the protocol.

Duration of treatment / exposure:

Animals were treated daily by oral gavage. As necropsy procedures took 3 days, animals were treated for 90, 91 or 92 days (until the day prior to scheduled sacrifice or the beginning of the recovery period). Recovery animals remained untreated for ca. 30 days.

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Remarks:

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

Doses / concentrations 2

Remarks:

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

Doses / concentrations 3

Remarks:

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

Doses / concentrations 4

Remarks:

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

No. of animals per sex per dose:

20 sex/group, 5 sex/group of these assigned to the recovery period

Control animals:

yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily for mortality, moribundy, general appearance and behaviour. At the time of dosing and approx. 1 hour after dosing for clinical signs of toxicity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

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

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule: prior to study initiation and approx. after 90-day of treatment in all animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to study initiation (animals then discarded); after approx. 30 and 90 days of treatment; and after approx. 30 days of recovery
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes
- How many animals: 10 sex/group during the treatment period, all (5 sex/group) after the recovery period

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to study initiation (animals then discarded); after approx. 30 and 90 days of treatment; and after approx. 30 days of recovery
- Animals fasted: Yes
- How many animals: 10 sex/group during the treatment period, all (5 sex/group) after the recovery period

URINALYSIS: Yes
- Time schedule for collection of urine: prior to study initiation (animals then discarded); after approx. 30 and 90 days of treatment; and after approx. 30 days of recovery
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No data

NEUROBEHAVIOURAL EXAMINATION: No

OTHER:
At the time of necropsy, 2 mL of serum and 1 g samples of liver, kidney and abdominal fat were collected from all animals sacrificed at the 90-day and recovery evaluations. Serum bromide leveles were measured by electrode methodology. Tissue bromide levels were measured by neutron activation.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, all animals
HISTOPATHOLOGY: Yes, complete for control and high dose groups at both terminal and recovery sacrifices. Kidney, liver, lung, peripheral nerve, spinal cord, urinary bladder and gross lesions examined from the low and mid-dose animals.

Statistics:

All analyses were conducted using two-tailed tests for minimum significance levels of 1% and 5% comparing the treatment groups to the vehicle control group by sex. All statistical tests were performed by a Digital Equipment Corporation computer with appropriate programming. Analysis of body weight, body weight changes, food consumption, clinical laboratory parameters and absolute and relative organ weight values were analysed by a one-way analysis of variance, followed by Dunnett's test.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

3 animals of the high dose group died early in the study

Mortality:

mortality observed, treatment-related

Description (incidence):

3 animals of the high dose group died early in the study

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

only in males of the high dose group

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

increased in animals of the high dose group and in males treated at 700 mg/kg bw/day

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

high dose animals, greater at the 30-day evaluation than at the 90-day

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

dose-related decreases in glucose levels at dosages of 300 mg/kg bw/day and greater

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

increased urine volume in males treated at 300 and 700 mg/kg bw/day and in animals treated at 1600 mg/kg bw/day

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

liver (abs and rel) in females at 700 and 1600 mg/kg bw/day; liver (rel) in females at 300 mg/kg bw/day; relative kidney, liver and tests weights increased in males at 1600 mg/kg bw/day related to lower bw

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

minimal to mild centrilobular hypertrophy in animals at 700 and 1600 mg/kg bw/day; areas of nephrosis in kidenys of animals at 1600 mg/kg bw/day

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
2 males and 1 female treated at 1600 mg/kg bw/day died on day 2 and 3, respectively, after showing severe clinical signs. Clinical signs thereafter were limited to salivation, considered related to the gavage administration. No signs of systemic toxicity were noted.

BODY WEIGHT AND WEIGHT GAIN
Mean body weight was significantly decreased in males treated at 1600 mg/kg bw/day during the first week of treatment and sporadically throughout the remaining treatment period. No effect in females treated at the same dose level.

FOOD CONSUMPTION
Mean food consumption values were consistently increased in animals treated at 1600 mg/kg bw/day and in males at 700 mg/kg bw/day throughout the treatment period. Food consumption in recovery animals previously treated at 1600 mg/kg bw/day remained slightly higher than controls, without attaining statistical significance.

HAEMATOLOGY
Slightly lower haemoglobin and slightly higher haematocrit and mean cell volume averages in animals at 1600 mg/kg bw/day were suggestive that a compensatory increase in red blood cell production may have occurred, although red blood cell counts remained comparable to the controls. Differences were greater at the 30-day evaluation than at the 90-day evaluation, thus indicating that the changes were transitory. In addition, examination of the peripheral blood smears and histopathologic evaluation of bone marrow gave no indication of red blood cell abnormalities. All haematologic parameters were similar to the controls at the end of the recovery period.

CLINICAL CHEMISTRY
Dose-related decreases in glucose levels were observed in both sexes at dosages of 300, 700 and 1600 mg/kg bw/day at the 30-day and 90-day evaluations. At the recovery evaluation, all values were similar to the control values.

URINALYSIS
Urine volume was increased in the 300 and 700 mg/kg bw/day males and in the 1600 mg/kg bw/day animals at the 30-day evaluation. Mean urine volumes remained increased in the 700 mg/kg bw/day males and in animals treated at 1600 mg/kg bw/day at the 90-day evaluation.The biological significance of these increases is equivocal, because no other treatment-related effect was apparent on urinalysis parameters. Urine volumes and urinalysis were comparable between treated and control groups at the recovery evaluation with the exception of an increased mean urine volume in the 700 mg/kg bw/day males.

ORGAN WEIGHTS
Absolute and relative liver weights were increased in the 700 and 1600 mg/kg bw/day females at the 90-day evaluation. Relative liver weight was also increased in the 300 mg/kg bw/day females. Relative kidneys, liver and testes weights were significantly increased at the 90-day evaluation in males treated at 1600 mg/kg bw/day but these changes were related to the decreased final body weight of these animals. Apparent dose-related significant decreases in mean absolute heart weight in the treated group males were judged to be due to a high mean absolute heart weight value for the control males. At the recovery sacrifice, mean absolute liver weight remained increased in the 1600 mg/kg bw/day females, with mean relative weight increased 16% above the control value.

HISTOPATHOLOGY: NON-NEOPLASTIC
Test material-related microscopic changes were observed in the liver, kidneys and possibly urinary bladder of animals treated at 1600 mg/kg bw/day and in the liver of those treated at 700 mg/kg bw/day. The liver effect consisted of minimal to mild centrilobular hepatocyte hypertrophy, indicative of increased detoxyfication requirement. The kidney effect consisted of areas of nephrosis, a noninflammatory lesion characterised by tubular dilatation and degeneration and regeneration of the epithelium of cortical tubules. in addition, an increased incidence of minimal hyperplasia of the urinary bladder epithelium possibly test article-related was observed at 1600 mg/kg bw/day. These changes were not observed at the end of the recovery period.

OTHER FINDINGS
Serum bromide and tissue bromide concentrations were increased in all treated groups at the end of the treatment period. These increases were generally dose-related. At the end of the recovery period, serum bromide concentrations were comparable to the control group and tissue bromine levels were very slightly elevated in the previously treated groups.

Effect levels

Dose descriptor:

NOEL

Effect level:

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

Sex:

male/female

Basis for effect level:

other: overall effects

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The dosage of 130 mg/kg bw/day of dibromostyrene was found to be the No Observable Effect Level (NOEL) for systemic toxicity when administered orally by gavage to male and female rats for 90 consecutive days.

Executive summary:

In an oral gavage 90-day study, groups of 20 male and female rats received daily dosages of 130, 300, 700 or 1600 mg/kg bw/day. The control group received the vehicle, corn oil, at a volume of 5 mL/kg bw. Five animals/sex in each group remained on study following the dosing period for a 30 -day recovery period. The animals were observed for signs of toxicity and effects on body weight, food consumption, clinical pathology parameters, ophthalmology and organ weights. Complete necropsy examinations were performed on all animals. A complete list of organs and tissues was examined microscopically in animals of the control and high dose groups. The kidney, liver, peripheral nerve spinal cord, urinary bladder and gross lesions were examined microscopically from the low and mid dose animals.

Dibromostyrene induced decreased male body weight and body weight gain at 1600 mg/kg bw/day and increased food consumption in both sexes at 1600 mg/kg bw/day and in males at 700 mg/kg bw/day. Transitory haematologic changes, possibly indicative of compensatory increased red blood cell production were apparent at the 30 -day evaluation in both sexes at 1600 mg/kg bw/day and, to a much lesser extent in the 300 and 700 mg/kg bw/day groups; some evidence of these haematologic changes persisted to the 90 -day evaluation at the highest dose level. Treatment-related hypoglicemia was present in the 300, 700 and 1600 mg/kg bw/day groups. Serum bromide and tissue bromine levels were increased in all groups, generally in a dose-related pattern, at the 90 -day sacrifice. Test material-related liver weight increases were observed in the 700 and 1600 mg/kg bw/day females at the 90 -day sacrifice. Microscopic liver changes indicative of increased detoxification requirement were present in the 700 and 1600 mg/kg bw/day groups animals. Kidney and possibly urinary bladder changes were also observed in the 1600 mg/kg bw/day group.

Essentially all evidence of toxicity subsided by the end of the recovery period. No biologically significant differences between the control and treated groups in body weight, body weight gain, food consumption, haematology or serum chemistry values or microscopic pathology findings were apparent for recovery animals. Increased liver weights in females previously treated at 1600 mg/kg bw/day persisted to the end of recovery, however, the magnitude of the increase was less than at the 90 -day sacrifice and no microscopic finding accompanied this increase.

The dosage of 130 mg/kg bw/day was found to be the No Observable Effect Level (NOEL) for systemic toxicity.