3-(chloromethyl)heptane

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:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

3-(chloromethyl)heptane

EC Number:

204-594-4

EC Name:

3-(chloromethyl)heptane

Cas Number:

123-04-6

Molecular formula:

C8H17Cl

IUPAC Name:

3-(chloromethyl)heptane

Details on test material:

- Name of test substance: 2-Ethylhexylchloride
- Batch identification: B023 000STD77L0
- CAS No.: 123-04-6
- Purity/composition: 99.4 corr. area-%
- Homogeneity: The homogeneity of the test substance was guaranteed by mixing before preparation of the test substance solutions.
- Storage stability: The stability of the test substance under storage conditions throughout the study period is guaranteed until 13 Jun 2012 as indicated by the sponsor, and the sponsor holds this responsibility.
- Date of production/supply: 13 Jun 2011
- Physical state, appearance: Liquid, colorless, clear
- Storage conditions: Room temperature

Method

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/ß-naphthoflavone-induced rat liver S9 mix

Test concentrations with justification for top dose:

0.33, 1, 3.3, 10, 33, 100, 333, 1000, 2650, and 5300 μg/plate (SPT, TA Strains)
33, 100, 333, 1000, 2650, and 5300 μg/plate (SPT, E.coli)
0.33, 1, 3.3, 10, 33, and 100 μg/plate (PIT, TA Strains)
33, 100, 333, 1000, 2650, and 5300 μg/plate (PIT, E.coli)

Vehicle / solvent:

- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: Due to the limited solubility of the test substance in water, DMSO was used as vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available.

Controls

Untreated negative controls:

yes

Remarks:

sterility control

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Remarks:

W/ S-9: 2-AA (2.5 µg/plate for TA 1535, TA 100, TA 1537, TA 98; 60 µg/plate for E. coli WP2 uvrA); w/o S-9: MNNG (5 µg/plate for TA 1535, TA 100); NOPD (10 µg/plate for TA 98); AAC (100 µg/plate for TA 1537); 4-NQO (5 µg/plate for E. coli WP2 uvrA)

Details on test system and experimental conditions:

TEST CONDITIONS: Standard plate test (SPT) and preincubation test (PIT) both with and without metabolic activation (liver S9 mix from induced rats).

NUMBER OF REPLICATIONS: 3 individual experiments (2x SPT b/c bacteriotoxicity was observed in first SPT; 1x PIT b/c no mutagenicity was observed in SPT) with 3 test plates per dose or per control

DETERMINATION OF BACTERIOTOXICITY: Toxicity was detected by a decrease in the number of revertants, the clearing or diminution of the background lawn (= reduced his- or trp- background growth), and a reduction in the titer.

OTHER EXAMINATIONS: solubility (precipitation was recorded)

Evaluation criteria:

Generally, the experiment is considered valid if the following criteria are met:
- The number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain.
- The sterility controls revealed no indication of bacterial contamination.
- The positive control substances both with and without S9 mix induced a distinct increase in the number of revertant colonies within the range of the historical positive control data or above.
- The titer of viable bacteria was ≥ 10E+08/mL.

The test substance is considered positive in this assay if the following criteria are met:
A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.
A test substance is generally considered non-mutagenic in this test if:
The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in at least two experiments carried out independently of each other.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535, TA 1537, 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:

E. coli WP2 uvr A

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

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS:
- Precipitation: No test substance precipitation was found onward with and without S9 mix.

ADDITIONAL INFORMATION ON BACTERIOTOXICITY:
A strong bacteriotoxic effect (reduced his- background growth, decrease in the number of his+ or trp+ revertants, reduction in the titer) was observed in the standard plate test depending on the strain and test conditions from about 10 μg/plate onward. In the preincubation assay bacteriotoxicity (reduced his- background growth, decrease in the number of his+ or trp+ revertants, reduction in the titer) was observed depending on the strain and test conditions from about 10 μg/plate onward.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Under the experimental conditions chosen here, it is concluded that 2-Ethylhexylchloride is not a mutagenic test substance in the bacterial reverse mutation test in the absence and the presence of metabolic activation.

Executive summary:

The study was performed according to OECD guideline 471 in compliance with GLP.

The test substance 2-Ethylhexylchloride was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay. Test strains were S. typhimurium strains TA 1535, TA 100, TA 1537, and TA 98, as well as E. coli strain WP2 uvrA. A standard plate test (SPT) and preincubation test (PIT) both with and without metabolic activation (liver S9 mix from induced rats) was performed. The substance was tested in the dose ranges 0.33 μg - 5300 μg/plate (SPT, TA Strains), 33 μg - 5300 μg/plate (SPT, E.coli), 0.33 μg - 100 μg/plate (PIT, TA Strains), and 33 μg - 5 300 μg/plate (PIT, E.coli).

No Precipitation of the test substance was found with and without S9 mix. A strong bacteriotoxic effect was observed depending on the strain and test conditions from about 10 μg/plate onward. According to the results of the present study, the test substance did not lead to a biologically relevant increase in the number of revertant colonies either without S9 mix or after adding a metabolizing system in three experiments carried out independently of each other (standard plate test and preincubation assay). Besides, the results of the negative as well as the positive controls performed in parallel corroborated the validity of this study, since the values fulfilled the acceptance criteria of this study.

Conclusion: Thus, under the experimental conditions of this study, the test substance 2-Ethylhexylchloride is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.