2,6-dibromo-4-cyanophenyl octanoate

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:

05 Jul - 19 Jul 1993

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1993

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 1983

Deviations:

no

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 2020

Deviations:

yes

Remarks:

no strain with AT base pair at the primary reversion site used (e.g. TA 102 or E. coli WP2 uvrA), no justification for choice of vehicle and no historical control data provided

GLP compliance:

yes

Type of assay:

bacterial forward mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2,6-dibromo-4-cyanophenyl octanoate

EC Number:

216-885-3

EC Name:

2,6-dibromo-4-cyanophenyl octanoate

Cas Number:

1689-99-2

Molecular formula:

C15H17Br2NO2

IUPAC Name:

2,6-dibromo-4-cyanophenyl octanoate

Method

Target gene:

His operon (Salmonella strains)

Species / strain

Species / strain / cell type:

other: TA 1535, TA 1537, TA 1538, TA 98 and TA 100

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of male Fischer 344 rats treated with Aroclor 1254 i.p. at a dose of 500 mg/kg bw in corn oil; the cofactor solution was composed as follows: 0.05 M phosphate buffer (pH 7.4), 8 mM MgCI2x6H2O, 33 mM KCI, 25 mM glucose-6-phosphate (disodium salt), 4 mM NADP (disodium salt). The S9 mix was composed of 9 parts Cofactor solution and 1 part S9 fraction. The final concentration in culture was approximately 1.85% for S9 fraction and 18.5% for S9 mix.

Test concentrations with justification for top dose:

Toxicity experiment with TA 100 only:
with and without metabolic activation: 33, 100, 333, 1000, 3333, 10000 µg/plate

Mutation experiment 1 and 2:
with and without metabolic activation: 33, 100, 333, 1000, 3333, 10000 µg/plate
No toxicity to the bacteria was observed up to 10000 µg/plate, but precipitation of the test material was observed at this concentration. Therefore, based on the results of the cytotoxicity experiment, the highest dose selected for the mutation test was 10000 µg/plate.

Vehicle / solvent:

- Vehicle/solvent used: Dimethyl sulphoxide (DMSO)

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-Aminoanthracene (2-AAN): TA 1535 and TA 1537 2 µg/plate and TA 1538, TA 98 and TA 100 0.5 µg/plate; + S9

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added: in agar (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: approximately 48 h

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: decrease in the number of revertant colonies per plate and/or by a thinning or disappearance of the bacterial background lawn

Rationale for test conditions:

according to OECD guideline 471

Evaluation criteria:

Acceptability
A test was considered acceptable if for each strain:
- the bacteria demonstrated their typical responses to crystal violet, ampicillin and UV light
- at least 2 of the vehicle control plates were within the following ranges: TA 1535, 4-30; TA 1537, 1-20; TA 98, 10-60; TA 100, 60-200 and TA 1538, 5-35
- on at least 2 of the positive control plates there were x 2 the mean vehicle control mutant numbers per plate, or in the case of TA 100, x 1.5 the mean vehicle control mutant numbers per plate. If the mean colony count on the vehicle control plates was less than 10 then a value of 10 was assumed for assessment purposes. In such cases a minimum count of 20 was required on at least 2 of the positive control plates.
- no toxicity or contamination was observed in at least 4 dose levels
- in cases where a mutagenic response was observed, that no more than one dose level was discarded before the dose which gave the highest significant mean colony number

Evaluation
A significant mutagenic response was recorded if:
- for S. typhimurium strains TA 1535, TA 1537, TA 1538 and TA 98, at least a doubling of the mean concurrent vehicle control values at some concentrations of the test substance and, for S. typhimurium strain TA 100, a 1.5-fold increase over the control value were observed. If the mean colony count on the vehicle control plates was less than 10 then a value of 10 was assumed for assessment purposes. In such cases a minimum count of 20 was required before a significant mutagenic response was identified.
- a dose related response was observed, although at high dose levels this relationship could be inverted because of, e.g. (1) toxicity to the bacteria generally, (2) specific toxicity to the mutants and (3) inhibition of foreign compound metabolizing enzymes where mutagens require metabolic activation by the liver.
- a reproducible effect in independent tests was observed.

Statistics:

No statistical analysis was done.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 3333 µg/plate in the presence of S9 and tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 3333 µg/plate in the presence of S9 and tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 10000 µg/plate in the presence of S9 and tested up to precipitating concentrations

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:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 10000 µg/plate in the presence of S9 and tested up to precipitating concentrations

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
- Precipitation and time of the determination: Precipitation of the test material occurred at 10000 µg/plate in all conditions.

RANGE-FINDING/SCREENING STUDIES:
In the cytotoxicity experiment, no toxicity to the bacteria was observed up to 10000 µg/plate.

STUDY RESULTS
All criteria for a valid experiment were met. At 10000 µg/plate, cytotoxicity to the bacteria occurred in the presence of S9 mix only, in all strains, except TA1535. Cytotoxicity was also observed at 3333 µg/plate in TA1537 in both experiments and TA1538 in the second experiment only.
The test substance did not induce any significant, dose-dependent increases in the revertant numbers of any strain at any of the concentrations studied. For details, please refer to the attachment 1.
The solvent control induced the expected results and the standard positive controls induced marked increases in revertant numbers, thus, confirming the suitability and sensitivity of the assay.

HISTORICAL CONTROL DATA
Detailed historical control data were not provided.

Applicant's summary and conclusion

Conclusions:

The study was performed in compliance with GLP and according to OECD guideline 471. Main deviations to the current OECD guideline 471 (adopted 2020) are related to the use of bacterial strains with only GC base pair at the primary reversion site (instead of TA 102 or E. coli WP2 uvrA) and the lack of historical control data. However, the study is considered reliable and valid. Under the conditions of the assay, the test item was not mutagenic up to 10000 µg/plate in the S. typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 with and without metabolic activation.