1,6-dibromohexane

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

Study period:

From January to June 1989

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD guideline

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1989

Report date:

1989

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Dibromo-1,6 Hexane

IUPAC Name:

Dibromo-1,6 Hexane

Details on test material:

- Name of test material (as cited in study report): Dibromo-1,6 Hexane
- Substance type: Colorless liquid
- Physical state: Liquid
- Analytical purity: 98.4%
- Purity test date: 98.4% - January 1989
- Lot/batch No.: TVV 01-07
- Storage condition of test material: In the dark and at room temperature

Method

Target gene:

Salmonella typhimurium (TA 1535, TA 1537, TA 1538, TA 98 and TA 100) contain a mutation in the operon histidine, making them auxotrophic for this amino acid.
+ rfa mutation: alters the barrier lypopolysaccharidic of the bacterial cell wall and thus confer greater membrane permeability.
+ a deletion in uvr B gene: supress the repair system by excision, so the damage caused by a mutagenic agent are not repaired by the bacteria and
can be expressed toward the phenotype.
+ the addition of a R factor of resistance to ampicillin carried by plasmid pKM 101 in strains TA 98 and TA 100, increases the sensitivity of detection of certain mutagens.

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9 mix

Test concentrations with justification for top dose:

* Test 1:
10, 50, 75, 100 and 500 µg/plate,
* Test 2
100, 250, 500 , 750 and 1000 µg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Test item dissolved in dimethylsulfoxyde (DMSO)
- Justification for choice of solvent/vehicle: standard solvent

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

yes

Positive controls:

yes

Remarks:

* Without metabolic activation: Sodium azide (TA 1535 and TA 100), 9-amino-acridine (TA 1537), 2-nitrofluorene (TA 1538 and TA 98) - With metabolic activation: 2-anthramine (TA 1535, TA 1537, TA 1538, TA 98 and TA 100). All dissolved in DMSO.

Positive control substance:

other: * Without metabolic activation: Sodium azide (TA 1535 and TA 100), 9-amino-acridine (TA 1537), 2-nitrofluorene (TA 1538 and TA 98) - With metabolic activation: 2-anthramine (TA 1535, TA 1537, TA 1538, TA 98 and TA 100). All dissolved in DMSO.

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Plate incorporation test.
The solution of the test item, for which the volume varies from 25 to 100 µl/plate, 0.5 ml of S9 mix for metabolic system and 0.1 ml of the strain are added to 2 ml of soft agar containing traces histidine and biotin and kept at 45 ° C. After brief homogenizing, the mixture is spread on a Petri dish containing a minimal medium.

DURATION
- Exposure duration: 48 hours at 37°C

BACTERIOSTATIC ACTIVITY:
To determine the maximum concentration of the test item that does not affect the growth of bacteria,.
- Replicates: 1 plate/concentration
- Concentrations tested: 10, 100, 1000, 2500, 5000 and 10000 mg/l per plate
- Strains: TA 98 and TA 100
- Methods: The bacteriostatic activity was evaluated by a clarification of the cell layer due to an inhibition of bacterial growth, and possibly a decrease in the number of colonies.

MUTAGENICITY TEST:
- Two independent experiments performed
- Replicates: 3 plates/concentration
- Concentrations:
* Test 1: 10, 50, 75, 100 and 500 µg/plate with and without S9 mix
* Test 2: 100, 250, 500, 750 and 1000 µg/plate with and without S9 mix
- Strains: TA 1535, TA 1537, TA 1538, TA 98 and TA 100
- controls:
* negative control: to check the spontaneous reversion (strains alone)
* solvent control
The strain sensitivity and the metabolism efficiency of the S9 mix are controlled in the presence of the mutagenic products (positive control).

NUMBER OF CELLS EVALUATED:
automatic counter - Artek Counter, 880 model, OSI , 75015 Paris, France

Evaluation criteria:

The product is considered mutagenic if it induced a doubling of revertants compared to the number of spontaneous mutants and / or solvent on one of the strains, if a statistically significant dose-response relationship exists and if the effect is reproduced in an independent test.

Statistics:

linear regression analysis for concentration response

Results and discussion

Test results

Test results 1

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

but not in positive strain

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'. Remarks: S. typhimurium TA 1537, TA 1538, TA 98 and TA 100

Applicant's summary and conclusion

Conclusions:

Under these experimental conditions, the test item dibromo-1, 6 hexane was genotoxic for the TA 1535 strain (substitution mutation base pairs) with and without metabolic activation.

Executive summary:

The potential ability of the test substance dibromo-l,6-hexane, to induce point mutations in bacteria was investigated by the Ames test in the five strains of Salmonella typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100, without and with a metabolic activation system prepared from a liver microsomal fraction (S9 mix) of rats pretreated with Aroclor 1254.

The test was performed according to the direct plate incorporation method.

The number of revertants induced by the positive control were higher than the spontaneous one, which demonstrated the sensitivity of the test strains and the efficacy of the metabolic activation system.

The test substance dibromo- l,6 -hexane was tested on two separate assays, respectively at the concentrations of 10, 50, 75, 100 and 500 µg/plate, then 100, 250, 500, 750 and 1000 µg/plate. The concentratrions were determiend by a preliminary bacteriotoxicity test. Higher concentrations caused considerable bacterial toxicity. Towards the TA 1537, TA 1538, TA 98 and TA 100 strains, no sinificant increase in the revertant number was observed up to toxic concentration levels. But towards the TA 1535 strain without or with S-9 mix, this test substance induced a significant does dependent and reproducible increase in the revertant number.

In conclusion, under the described experimental conditions, dibromo-1,6 -hexane genotoxic towards the TA 1535 strain (base-pair substitution mutation).