Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study conducted under Good Laboratory Practices. Only deviation was relocation of study archives.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1994
Report Date:
1994

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EPA OPP 84-2
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Bromine
- Stability under test conditions: Determined in separate study that stability of bromine oxidants for 72 hours was best in sterile, deionized distilled water.
- Storage condition of test material: room temperature

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, North Carolina, USA
- Age at study initiation: 42 days old
- Weight at study initiation: males 28 to 36 grams females 21 to 26 grams
- Assigned to test groups randomly: yes, computer-generated random matrix
- Fasting period before study:
- Housing: three to five per cage in cages with hardwood chip bedding
- Diet (e.g. ad libitum): ad libitum Purina certified rodent chow
- Water (e.g. ad libitum): ad libitum tap water
- Acclimation period: 7 days prior to range find and micronucleus test

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 25 degrees C
- Humidity (%): 49 to 65%
- Photoperiod (hrs dark / hrs light): 12 hours/12 hours

IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: stability work showed bromine oxidants most stable in sterile, distilled deionized water
- Concentration of test material in vehicle: 6.2 mg/ml, 3.1 mg/ml, 1/24 mg/ml nominal; 4.56 mg/ml, 1.92 mg/ml and 0.819 mg/ml measured. Concentrations measured by outside lab one day after preparation
- Amount of vehicle: 10 ml/kg
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: prepared shortly before dosing by dilution of appropriate amount of test article liquid with water

Duration of treatment / exposure:
Single intraperitoneal injection
Frequency of treatment:
Once
Post exposure period:
Animals sacrificed at 24, 48, or 72 hours after injection of test article. Animals sacrificed at 24 hours after treatment with positive control.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0.004 mL/kg
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
0.01 mL/kg
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
0.02 mL/kg
Basis:
nominal conc.
No. of animals per sex per dose:
5 mice per sex per group
Control animals:
yes, concurrent vehicle
Positive control(s):
triethylenemelamine
- Justification for choice of positive control(s): Causes micronuclei
- Route of administration: Intraperitoneal
- Doses / concentrations: 1.0 mg/kg (0.1 mg/mL x 10 mL/kg)

Examinations

Tissues and cell types examined:
Femoral bone marrow cells; Polychromatic erythrocytes (PCE) and normochromatic erythrocytes (NCE) in approximately 1000 erythrocytes determined. Also, number of micronucleated PCE (MPCE) per 1000 PCE determined.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Deaths occurred in the range finder at doses of 0.05 mL/kg and above. All dose levels caused weight loss in the range finder on day 1, except 0.01 mL/kg, but that level did cause weight loss in males, but not females on day 2 post exposure. No weight loss differences from control were seen on day 3 post exposure in the 0.01 mL/kg group

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Animals sacrificed at 24, 48, and 72 hours post treatment using CO2 asphyxiation. Femurs were recovered from the animals and bone marrow cavity flushed with 1.0 ml of fetal bovine serum. After cells from all animals were collected, tubes were centrifuged at 800 ppm for 5 minutes. Supernatent was removed leaving about 0.1 ml above the cell pellet. Cells were resuspended by flicking the tube.

DETAILS OF SLIDE PREPARATION: A small drop of suspension was placed just below the frosted end of a precleaned slide and spread along the length of the slide. Slides were air dried adn then fixed in methanol for 15 minutes before air drying again. Slides were stained with Wright Giemsa stain for 3 minutes, rinsed in distilled water, allowed to air dry, and mounted in permount using # 1 cover glasses.

METHOD OF ANALYSIS: Slides were scored "blind". first the total number of polychromatic erythrocytes (PCE) and normochromatic erythrocytes (NCE) in approximately 1000 erythrocytes were determined. In addition, the number of micronucleated PCE (MPCE) per 1000 PCE were determined.

Evaluation criteria:
Criteria for valid test: 1. In vehicle control, average number of MPCE per 1000 PCE should not exceed 5 2. In positive control, increase in average number of MPCE per 1000 PCE over the average number of MPCE for the vehicle control should be statistically significant. 3. At least 3 animals from each sex must be alive at the time of sacrifice for each dose level

Criteria for a Positive Response: 1. Test article shows a positive dose-response trend and a statistically significant increase over that of the concurrnet vehicle control in the number of MPCE at one or more dose levels. In the event that the test article caused a statistically significant increase in the number of MPCE (less than 0.05%) in the concurrent vehicle control, the data from that dose may be compared to historical vehicle control data. 2. In the event there is no poritive dose-response trend, at least two consecutive test doses show a statistically significant increase in the number of MPCE.

Criteria for Negative Response: If no indication pf a positive dose-response trend and none of the test doses had a statistically significant increase in number of MPCE when compared to the vehicle control.


Statistics:
Data were analyzed separately for male and female animals. Unless otherwise indicated, the frequency of micronucleated PCE in each dose group was compared to that in the respective vehicle control using a two-tailed Student's t-test. Results were considered significant if the p value is = or < 0.05.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
slight weight loss, clinical signs that reversed 2 hours after dosing
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 0.001 mL/kg to 10 mL/kg
- Clinical signs of toxicity in test animals: death, prostration, piloerection, convulsions and irregular breathing in high dose group before death. No deaths or clinical symptoms were observed in the 0.001 or 0.01 mL/kg or vehicle control groups. Surviving animals showed weight loss. On day 3 after treatment, only the female 0.05 and 0.1 mL/kg groups still showed weight loss.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): No evidence of significant increase in proportion of MPCE per 1000 PCE at any harvest time compared to concurrent vehicle control groups (Vehicle mean 0.4, low dose mean 0.6, mid dose mean 0.6, high dose mean 0.2, positive control mean 82.0
- Ratio of PCE/NCE (for Micronucleus assay): Vehicle mean 1.52; low dose mean 1.76, mid dose mean 1.54, high dose mean 1.38, positive control mean 0.37
- Appropriateness of dose levels and route: Oral route would have been inappropriate due to caustic nature of test article. Dose levels did not cause mortality, but did cause slight effects on weight, and clinical signs
- Statistical evaluation: P values for MPCE did not show statistical significant increase for treated groups

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Under the conditions of this micronucleus assay in mice, bromine did not satisfy the criteria for a positive test.
Executive summary:

Bromine was tested for its potential to induce micronucleated polychromatic erythrocytes in bone marrow cells of male and female CD-1 mice. The test article was dissolved in sterile distilled water and administered as a single intraperitoneal dose at the levels of 0.004, 0.01, and 0.02 mL/kg (5 male/5 female mice per dose group). The micronucleated polychromatic erythrocyte (MPCE) frequency was determined at 24, 48, and 72 hours after dose administration. There was no statistically significant increase in the number of MPCE in the test article treated groups at any harvest time compared to the concurrent vehicle control groups. The results of the assay indicated that under the conditions of the test, and according to the criteria set for evaluation, bromine was negative in the Micronucleus Assay.