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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
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:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
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 / strainopen allclose all
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 / 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
Controlsopen allclose all
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
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
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
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
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 resultsopen allclose all
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
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.