Bromoacetyl bromide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 Feb - 19 Aug 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Adopted 21 July 1997, Corrected 26 June 2020

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

31 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Bromoacetyl bromide

EC Number:

209-923-5

EC Name:

Bromoacetyl bromide

Cas Number:

598-21-0

Molecular formula:

C2H2Br2O

IUPAC Name:

2-bromoacetyl bromide

Test material form:

liquid

Method

Target gene:

his operon (for S. typhimurium strains) and trp operon (for E. coli strain)

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: male Sprague Dawley rats that had been injected intraperitoneally with Aroclor 1254 500 mg/kg body weight).
- method of preparation of S9 mix:S9-mix was prepared immediately before use and kept refrigerated. S9-mix contained per 10 mL: 30 mg NADP (Randox Laboratories Ltd., Crumlin, United Kingdom) and 15.2 mg glucose-6-phosphate (Roche Diagnostics, Mannheim, Germany) in 5.5 mL Milli-Q water (Millipore Corp., Bedford, MA., USA); 2 mL 0.5 M sodium phosphate buffer pH 7.4; 1 mL
0.08 M MgCl2 solution (Merck); 1 mL 0.33 M KCl solution (Merck). The above solution was filter (0.22 μm)-sterilized. To 9.5 mL of S9-mix components 0.5 mL S9-fraction was added (5% (v/v) S9-fraction) to complete the S9-mix.
- volume of S9 mix in the final culture medium: 0.5 mL S9 mix containing 5% v/v S9 fraction
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Each S9 batch was characterized with the mutagens benzo-(a)-pyrene (Sigma) and 2-aminoanthracene, which require metabolic activation, in tester strain TA100 at concentrations of 5 μg/plate and 2.5 μg/plate, respectively.

Test concentrations with justification for top dose:

First experiment:
TA100 and WP2uvrA: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate (dose-range finding test)
TA1535, TA1537 and TA98: 17, 52, 164, 512, 1000 and 2500 μg/plate (based on cytotoxicity observed in dose-range finding test)
Second experiment:
TA1535, TA1537, TA98, TA100 and WP2uvrA: 5.4, 17, 52, 164, 512 and 1000 μg/plate (based on cytotoxicity observed in dose-range finding test)
Third experiment:
TA100: 17, 52, 100, 164, 250 and 512 μg/plate (based on cytotoxicity observed in second experiment)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dried DMSO

- Justification for choice of solvent/vehicle: Since the test item reacts violently with water, dried DMSO was selected as vehicle for the mutation experiments.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

dried DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: two with all strains with and without metabolic activation; a third independent experiment was conducted with tester strain TA100 without metabolic activation.

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 1x10E09 cells/mL
- Test substance added in agar (plate incorporation) in the first experiment; preincubation in the second and third experiments

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 30 ± 2 minutes
- Exposure duration/duration of treatment: 48 ± 4 h

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition

Rationale for test conditions:

according to test guidelines

Evaluation criteria:

In addition to the criteria stated below, any increase in the total number of revertants should be evaluated for its biological relevance including a comparison of the results with the historical control data range.

A test item is considered negative (not mutagenic) in the test if:

a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three times the concurrent control.
b) The negative response should be reproducible in at least one follow up experiment.

A test item is considered positive (mutagenic) in the test if:

a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three times the concurrent control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

Statistics:

Mean values and standard deviation were calculated.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 512 µg/plate and above

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 512 µg/plate and above (direct plate assay); at 164 µg/plate and above without S9 (pre-incubation assay)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 512 µg/plate and above

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 1600 µg/plate and above (direct plate assay); at 512 µg/plate and above (pre-incubation assay)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 512 µg/plate and above (direct plate assay); at 164 µg/plate and above without S9 (pre-incubation assay)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 6

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 1000 µg/plate and above

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Other confounding effects: the test material is known to react violently with water, resulting
in bromoacetic acid and hydrobromic acid

RANGE-FINDING/SCREENING STUDIES: see Attachment 1

Ames test:
- Signs of toxicity: Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains.
- Individual plate counts: see Attachment 2
- Mean number of revertant colonies per plate and standard deviation: see Attachment 1

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data): see Attachment 3

Applicant's summary and conclusion

Conclusions:

Based on the results of this study it is concluded that the test item is mutagenic in the Salmonella typhimurium reverse mutation assay. The test item is not mutagenic in the Escherichia coli reverse mutation assay.