2,6-dibromo-4-cyanophenyl heptanoate

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:

11 Jan - 30 Jan 1991

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:

1991

Report date:

1991

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EPA OPP 84-2

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), 2-aminoanthracene was used as the sole indicator of the efficacy of the S9-mix, justification for choice of vehicle and historical control data not provided

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

3,5-dibromo-4-hydroxybenzonitrile

EC Number:

216-882-7

EC Name:

3,5-dibromo-4-hydroxybenzonitrile

Cas Number:

1689-84-5

Molecular formula:

C7H3Br2NO

IUPAC Name:

3,5-dibromo-4-hydroxybenzonitrile

Method

Target gene:

His operon

Species / strain

Species / strain / cell type:

other: S. typhimurium TA 98, TA 100, TA 1535, TA 1537 and TA 1538

Metabolic activation:

with and without

Metabolic activation system:

Cofactor supplemented post-mitochondrial fraction (S9 mix) was prepared from the livers of male Sprague-Dawley rats that had been injected (i.p.) with Aroclor 1254 (200 mg per mL in corn oil) at 500 mg/kg bw (purchased from Molecular Toxicology, Inc., Annapolis, USA).
The S9 mix was prepared immediately prior to its use in any experimental procedure. One mL S9 mix solution was composed as follows: 0.70 mL water, 0.1 mL sodium phosphate buffer (1 M; pH 7.4), 0.02 mL glucose-6-phosphate (0.25 M), 0.04 mL NADP (0.1 M), 0.04 mL KCl (0.825 M) / MgCl2 (0.2 M) and 0.10 mL S9 fraction. The final concentration in culture was approximately 2 % for S9 fraction and approximately 20 % for S9 mix.

Test concentrations with justification for top dose:

Dose range-finding experiment (with and without metabolic activation): 6.67, 10.0, 33.3, 66.7, 100, 333, 667, 1000, 3330 and 5000 µg/plate
Main experiment (without metabolic activation): 10.0, 33.3, 100, 333, 1000, 3330 µg/plate
Main experiment (with metabolic activation): 3.33, 10.0, 33.3, 100, 333, 1000 µg/plate
Based on the range-finding study, in the subsequent mutagenicity assay the highest concentration of the test article was used, which gave a detectable reduction of revertants per plate and/or a thinning or disappearance of the bacterial background lawn.

Vehicle / solvent:

- Vehicle/solvent used: Dimethyl sulfoxide (DMSO)

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

sodium azide

other: ICR-191: TA 1537 2.0 µg/plate; -S9 and 2-aminoanthracene: all strains 2.5 µg/plate; +S9

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration triplicate
- Number of independent experiments : 1

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium; 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 EPA OPP 84-2 guideline

Evaluation criteria:

Acceptability
The assay is considered valid if the following criteria were met:
1. Tester strain cultures must exhibit sensitivity to crystal violet (demonstrate presence of rfa wall mutation)
2. Cultures of tester strains TA98 and TA100 must exhibit resistance to ampicillin (demonstrate presence of R-factor plasmid)
3. Tester strain cultures must exhibit a characteristic number of spontaneous revertants per plate when plated along with the vehicle under selective
conditions. The acceptable ranges for the vehicle controls are as follows: TA 98: 8 - 60, TA 100: 60 - 240, TA 1535: 4 - 45, TA 1537: 2 - 25 and TA 1538: 3 - 35
4. To demonstrate that appropriate numbers of bacteria are plated, the density of tester strain cultures must be greater than or equal to 0.5 x 10^9 bacteria per mL and/or have reached a target level of turbidity demonstrated to produce cultures with a density greater than or equal to 0.5 x 10^9 bacteria per mL.
5. The positive control substances for a respective tester strain must exhibit at least a 3-fold increase over the mean value of the vehicle control
6. A minimum of three non-toxic dose levels is required to evaluate assay data.

A test item is considered as mutagenic if:
- the assay is considered as valid
- positive response of at least a 2-fold increase (TA 98 and TA 100) or 3-fold increase (TA 1535, TA 1537 and TA 1538) in the mean revertants per plate of at least one of the tester strains over the mean revertants per plate of the appropriate vehicle control.
- the increase in the mean revertants per plate must be accompanied by a dose response to increasing concentrations of the test article

Statistics:

No statistical analysis was done.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at highest concentration level of 3330 µg/plate with S9 mix

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 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at highest concentration levels of 1000 and 3330 µg/plate with S9 mix, and at 1000 µg/plate without S9 mix

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 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at highest concentration levels of 1000 and 3330 µg/plate with S9 mix and at 1000 µg/plate without S9 mix

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 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at highest concentration levels of 1000 and 3330 µg/plate with S9 mix, and at 1000 µg/plate without S9 mix

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 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at highest concentration levels of 1000 and 3330 µg/plate with S9 mix, and at 1000 µg/plate without S9 mix

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:
The test article remained in solution at all subsequent dilutions prepared for the mutagenicity assay.

RANGE-FINDING/SCREENING STUDIES:
Ten concentrations of test article (6.67 µg/plate to 5000 µg/plate) were tested in S. typhimurium TA 100. Cytotoxicity was observed at 667 µg/plate and above in the presence of S9 and at 100 µg/plate and above in the absence of S9 as evidenced by the reduced number of revertants per plate and the thinning or disappearance of the bacterial background lawn. For details, please refer to the attachment 1.

STUDY RESULTS
All criteria for a valid experiment were met. Cytotoxicity was observed at 1000 µg/plate and above in the absence and presence of a metabolic activation system evidenced by a reduced number of revertants per plate and/or the thinning or disappearance of the bacterial background lawn. The positive and solvent controls induced the expected results, confirming the suitability and sensitivity of the assay. No positive increases in the number of histidine revertants per plate were observed with any of the tester strains either in the presence or absence of S9. For details, please refer to the attachment 2.

HISTORICAL CONTROL DATA
Historical control data were not provided.

Applicant's summary and conclusion

Conclusions:

The study was performed in compliance with GLP and according to EPA OPP 84-2 guideline, which is assumed similar to the OECD guideline 471. Main deviations to the current OECD guideline 471 (adopted 2020) are related to the use of 2-aminoanthracene as the sole indicator of the S9-mix efficacy, 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 3330 µg/plate in the S. typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 with and without metabolic activation.