Hexyl salicylate

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:

February 28th to May 30th 2000.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

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:

Hexyl salicylate

EC Number:

228-408-6

EC Name:

Hexyl salicylate

Cas Number:

6259-76-3

Molecular formula:

C13H18O3

IUPAC Name:

hexyl salicylate

Constituent 2

Reference substance name:

0006259-76-3

Cas Number:

0006259-76-3

IUPAC Name:

0006259-76-3

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): Hexyl Salicylate
- Physical state: Colourless to pale yellowish liquid
- Lot/batch No.: 50554361
- Storage condition of test material: Cool and dry

Method

Target gene:

Histidine loci

Species / strain

Species / strain 1

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Not applicable as bacterial cells

Additional strain / cell type characteristics:

other: Strains TA 1535 and TA 100: base pair substitution mutation hisG46. Strain TA 1537: histidine frameshift mutation his3076. Strain TA 98: histidine frameshift mutation his3052.

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 102

Details on mammalian cell type (if applicable):

Not applicable as bacterial cells

Additional strain / cell type characteristics:

other: Strain TA 102 contains the unrevertable deletion hisG8476 in the histidine operonof the bacterial chromosome but offers the revertible mutation hisG428 on the plasmid pAQ1.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix from rat liver homogenates

Test concentrations with justification for top dose:

Presence of S9 mix: 5 - 5000 µg per plate
Absence of S9 mix: 15 - 5000 µg per plate

Vehicle / solvent:

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

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

Dissolved in DMSO

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Dissolved in distilled water

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Dissolved in distilled water

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

Dissolved in DMSO

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Dissolved in distilled water

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Expression time (cells in growth medium): 48 - 72 hours

NUMBER OF REPLICATIONS:
Three parallel plates were prepared for each experimental point.

DETERMINATION OF CYTOTOXICITY
- Method: other: Number of revertant colonies per plate

OTHER:
To confirm reversion properties and specificity of the bacterial strains as well as the activity of the liver homogenates, positive mutagenesis controls were run simultaneously. The genotypes of the tester strains (histidine requirement, spontaneous reversion frequency, sensitivity to UV light, crystal violet and ampicillin) were checked in each experiment. The Vogel-Bonner minimal plates, the test compound and the S9 mix were checked for sterility.

Evaluation criteria:

When there was a question about the nature of colonies scored as revertants and when positive mutagenic results were obtained, the genotype of revertant colonies was spot-checked by picking and streaking histidine free plates.

Statistics:

No information provided

Results and discussion

Test results

Test results 1

Key result

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:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Observed at 1500 and 5000µg/plate

COMPARISON WITH HISTORICAL CONTROL DATA:
The number of spontaneous revertants observed using each of the 5 strains was similar to those previously established.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the presence of S9 mix, hexyl salicylate was bacteriotoxic towards the strain TA 1537 at 500ug/plate, towards the strains TA 100 and TA 102 at 1500ug/plate and towards the strains TA 98 and TA 1535 at 5000ug/plate. In the absence of S9 mix, hexyl salicylate was bacteriotoxic towards the strain TA 1537 at 500ug/plate and towards the strains TA 98, TA 100 and TA 102 at 5000 ug/plate.

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions of this study hexyl salicylate was not considered to be mutagenic to Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 in the presence and absence of metabolic activation.

Executive summary:

Hexyl salicylate was investigated in an Ames test using Salmonella typhimurium strains TA 1535, TA 153, TA 100, TA 98 and TA 102. The study was conducted using the standard plate incorporation method, both in the presence and absence of metabolic activation (S9 fraction). The test substance was tested at concentrations from 5-5000 µg/plate in the presence of metabolic activation and 15-5000 µg per plate in the absence of metabolic activation. Appropriate positive controls were also used to confirm the sensitivity of the assay. Hexyl salicylate did not induce a significant increase in the mutation frequency of the tester strains in the presence or absence of metabolic activation.