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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
January to November 21, 1994
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study conducted under Good Laboratory Practices, met validity criteria, only protocol deviation was a change in the archive location

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
EPA OPP 84-2
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Bromine
EC Number:
231-778-1
EC Name:
Bromine
Cas Number:
7726-95-6
Molecular formula:
Br2
IUPAC Name:
dibromine
Details on test material:
- Name of test material (as cited in study report): Bromine
- Physical state: red liquid
- Analytical purity: 99.99%
- Stability under test conditions: tested in an independent 72 hour stability test of aqueous solutions
- Storage condition of test material: room temperature
- Other:

Method

Target gene:
Thymidine kinase locus of L5178Y TK+/- mouse lymphoma cells
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: F10P (Fischer's Medium for Leukemia Cells of Mice with 10% heat inactivated horse serum, 0.1% Pluronics and 0.22% sodium pyruvate)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
other: obtained from Dr. D. Clive, Burroughs Wellcome Company
Metabolic activation:
with and without
Metabolic activation system:
Aroclor-induced rat liver homogenate (S-9 fraction) and cofactor pool
Test concentrations with justification for top dose:
Range finding test: with and without activation: 100, 50, 10, 5.0, 1.0, 0.5, 0.1, 0.05, 0.01, and 0.005 µL/mL
Definitive test:
Assay B1: 0.004 to 0.05 µL/mL without activation, 0.025 to 0.5 µL/mL with activation
without activation: 0.004, 0.006, 0.008, and 0.01 µL/mL were used for cloning based on growth after 2 days expression period
with activation: 0.025, 0.05, 0.075, and 0.1 µL/mL were used for cloning based on growth after 2 day expression period
Assay B3: without activation 0.001 to 0.3 µL/mL with activation 0.005 to 0.2 µL/mL
without activation: 0.002, 0.004, 0.006, 0.008, and 0.01 µL/mL were cloned
with activation: 0.01, 0.25, 0.05, 0.075, and 0.1 µL/mL were cloned
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water used to make lower dilutions of test article; acetone used to dissolve positive control substance DMBA, and DMSO was used to dissolve the positive control substance Hycanthone
- Justification for choice of solvent/vehicle: Bromine oxidants were shown to be most stable in sterile, deionized distilled water. Solvents for the positive control substances are standard for those materials.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
sterile, deionized distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
Used at 2.5 and 5.0 µg/mL in the activated system

Migrated to IUCLID6: DMBA
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
sterile, deionized distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: hycanthone methanesulfonate
Remarks:
Used at 5.0 and 10 µg/mL in the non-activated system
Details on test system and experimental conditions:
METHOD OF APPLICATION: Test article was applied directly to cultures for concentrations of 1.0 µL/mL, and as aqueous solutions for lower concentrations

DURATION

- Exposure duration: 4 hours and then exposure stopped by centrifugation, pouring off the supernatent and resuspension.
- Expression time (cells in growth medium): 2 days (Growth was checked at 20 and 44 hours, and cultures were selected for cloning based on suspension growth)
- Cloning time: 10 to 12 days after plating

NUMBER OF REPLICATIONS: three plates per culture, three assays (one assay was invalidated as solvent and positive controls did not meet criteria)

NUMBER OF CELLS EVALUATED:

DETERMINATION OF CYTOTOXICITY
- Method: relative suspension growth, observation of cultures


OTHER:
Evaluation criteria:
Valid assay:
Solvent Control cultures: average cloning efficiency must be 50% or higher, average of the mutant frequency must be less than 100 per 10^6 via ble cells
Positive Control cultures: treated cultures must have mutation frequencies at least 3 times the average of their solvent controls, Solvent controls mu st have an average cloning efficiency of 505 or greater, colony size distribution analysis must indicate sufficient recovery of small colonies
Evaluation of test results:
Criteria for a negative result: all cultures exhibiting total growth of 10% and greater have mutant frequencies that are less than twice that of the me an mutant frequency of the corresponding solvent control cultures and the response is not dose dependent.
Criteria for a positive response: at least one culture has a mutant frequency that is two times or more greater than the average mutant frequency o f the corresponding solvent control cultures and the response is dose-dependent.
Criteria for an equivocal response: Does not fulfill the criteria for negative or positive response, and/or the Study Director does nt consider the re sponse to be either positive or negative.

Results and discussion

Test results
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Other confounding effects: The results of the first mutation assay indicated that bromine had an extremely narrow toxic response curve as indicated by the values for relative suspension growth. Doses for subsequent assays were selected within an extremely tight concentration gradient.

RANGE-FINDING/SCREENING STUDIES: Immediately after addition of test article to the test cultures, it was observed that the cultures treated with 100, 50 and 10 uL/mL bromine turned greenish blue in color, and after a few minutes, turned yellow. After one hour of incubation on the roller drum, clumps of material were observed in the cultures. This was probably precipitated cellular debris and horse serum from oxidation by the test article. At about 20 hours post exposure, cultures treated with 0.5 uL/mL and higher were dead.

Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Under the conditions of the test, bromine produced a positive response in cultures treated in the absence and presence of exogenous metabolic activation.
Executive summary:

Bromine was tested for it's potential to induce mutations at the thymidine kinase locus of L5178Y TK+/- mouse lymphoma cells both in the presence and absence of exogenous metabolic activation (Aroclor 1254 -induced male Sprague Dawley rat liver). Doses for the Mutation Assays were chosen from results in a range finding test that produced varying degrees of reduction in cell growth. Under the conditions of this test, bromine produced a positive response in cultures treated in the absence and presence of exogenous metabolic activation.