Bromotrifluoromethane

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1977

Report date:

1977

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Only three strains used and three concentrations of the test material tested. No pre-incubation method used and only one positive control used (for strain 1538 with metabolic activation system).

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Bromotrifluoromethane

EC Number:

200-887-6

EC Name:

Bromotrifluoromethane

Cas Number:

75-63-8

Molecular formula:

CBrF3

IUPAC Name:

bromotrifluoromethane

Test material form:

gas under pressure: liquefied gas

Details on test material:

- Name of test material (as cited in study report): PYROFORANE 1301
- Lot/batch No.: 531942Y

Method

Target gene:

his

Species / strain

Species / strain / cell type:

other: S. thyphimurium TA 1535, TA 1537 and TA 1538

Metabolic activation:

with and without

Metabolic activation system:

liver S9-mix prepared from Aroclor 1254-induced Anglia Laboratory rats

Test concentrations with justification for top dose:

10, 30 and 50% (v/v) in air

Vehicle / solvent:

air

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

Remarks:

only for strain 1538 with metabolic activation system

Details on test system and experimental conditions:

The strains were obtained from Professor B. N. Ames, Biochemistry Dept., University of California, Berkeley, California, U.S.A. Sub-cultures were prepared in nutrient broth (Difco) and incubated at 37°C for 18 hours. This overnight culture provides approximately 2E+9 organisms/mL.

Procedure
Two sets of bottles were prepared for each tester strain and 0.1 mL of one of the standard bacterial suspensions was added to each of the bottles. Into each of one of the duplicate sets of bottles was placed 0.5 mL of the S-9 liver microsome mix (Appendix 1). In the other set the S-9 mix was replaced by 0.5 mL volumes of sterile physiological saline.

2.8 mL volumes of histidine deficient agar, melted and cooled to 45°C were added to each of the bottles, thoroughly mixed and then over layed onto previously prepared plates containing 15 mL of minimal agar. Duplicate sets of plates were used for each concentration of test material.

Pyroforane 1301 was tested as a percentage mixture in compressed air. Three dose-levels were used, 50%, 30% and 10% gas mixture respectively.

To obtain the correct gas mixture two Rotameters were used to measure the gas flow of the test materials and compressed air. The gas was passed to a 100 mL pyrex mixing vessel via silicone rubber tubing and allowed to equilibrate inside the mixing vessel for 2 minutes. After equilibration the gas mixture was passed from the mixing vessel to standard Mc Intosh and Fildes jars which contained the petri dishes seeded with tester strain and, where applicable, the liver microsomal extract. The test gas mixture was applied until the jar was full of the test gas mixture.

The gas flow rates used to provide the required gas mixtures are as indicated below:
- 50% of gas mixture for a flow rate (in cc/min) of 150/150 (Air/Test gas)
- 30% of gas mixture for a flow rate (in cc/min) of 150/72.5 (Air/Test gas)
- 10% of gas mixture for a flow rate (in cc/min) of 135/15 (Air/Test gas)
- Air for a flow rate (in cc/min) of 150/0 (Air/Test gas)

When the gas jars were filled with the test gas mixture, the jars were sealed and placed inside a 37°C incubator for 48 hours. The number of revertant bacterial colonies arising on each plate was counted.
This procedure was repeated for each of the bacterial tester strains on the test gas of Pyroforane 1301.

A further set of plates was prepared without the tester strains to ensure that extraneous contamination had not occurred during the preparation of the materials and test plates.

Finally, serial ten-fold dilutions of the standardised suspensions were prepared and 0.3 mL aliquots of some dilutions were mixed with 2.7 mL volumes of agar and overlayed onto minimal agar plates. The 1E-1 dilutions was mixed with histidine deficient agar to check the spontaneous mutation rate, and the 1E-6 to 1E-8 dilutions were mixed with the histidine rich agar to confirm the viability of the prepared suspensions.

Evaluation criteria:

After incubation for 48 hours at 37°C the numbers of revertant (histidine independent) colonies were counted and compared with negative controls. The numbers of colonies developing on the first set of plates indicated the degree of reverse mutation caused by the test material itself and the numbers developing on the second set of plates (with rat liver microsomal fraction) indicated the degree of reverse mutation due to metabolites of the test compound.

Results and discussion

Test results

Species / strain:

other: S. thyphimurium TA 1535, TA 1537 and TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Low colony counts at 1E-1 dilution when cells were plated onto histidine deficient agar indicate that the strain is maintaining its low spontaneous mutation rate. High colony counts at the 1E-6 to 1E-7 dilutions indicate that the viability of the strain is good. The positive control provoked increases in the number of revertant colonies.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Bromotrifluoromethane did not show a mutagenic response under the conditions of the test.

Executive summary:

Mutagenic activity of bromotrifluoromethane was evaluated by using 3 histididine dependent strains of Salmonella typhimurium (TA 1535, TA 1537 and TA 1538) .  Tests were carried out in presence and in absence of Rat liver derived S-9 mix activation system. Cells were incubated with  10, 30 and 50% (v/v in air)  HFC-32 for 48 hours at 37°C. 2 -amino positive controls were used in the presence (2 -aminoanthracene) and in the absence (nitrosoguanidine, acridine, daunorubicine and aminoanthracene) of S-9 mix. No significant cytotoxicity was evidenced and no significant increases in the number of revertant colonies were observed in any experiment, whatever the strain and dose with and without metabolic activation.