2-ethylhexyl salicylate

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:

May - August 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: valid guideline study performed under GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

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:

2-ethylhexyl salicylate

EC Number:

204-263-4

EC Name:

2-ethylhexyl salicylate

Cas Number:

118-60-5

Molecular formula:

C15H22O3

IUPAC Name:

p-menth-1-en-8-ol

Method

Target gene:

histidine-requiring (his-) mutants of Salmonella typhimurium

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Details on mammalian cell type (if applicable):

Strains Main mutation Additional mutations
TA 1535 His G 46 rfa uvrB -
TA 100 His G 46 rfa uvrB pKM101
TA 102 His G 428 (pAQ1) rfa - pKM101
TA 1537 His C 3076 rfa uvrB -
TA 98 His D 3052 rfa uvrB pKM101

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Arochlor 1254 induced rat liver

Test concentrations with justification for top dose:

dose-levels were 10, 100, 500, 1000, 2500 and 5000 µg/plate in pre-test
156.3, 312.5, 625, 1250, 2500 and 5000 µg/plate, for all tester strains in the first mutagenicity experiment,
312.5, 625, 1250, 2500 and 5000 µg/plate, for all tester strains in the second mutagenicity experiment.
with S9-mix:
156.3, 312.5, 625, 1250, 2500 and 5000 µg/plate, for the TA 1537 and the TA 98 strains in the first mutagenicity experiment,
15.6, 31.3, 62.5, 125, 250 and 500 µg/plate, for the TA 1535, TA 100 and TA 102 strains in the first mutagenicity experiment,
312.5, 625, 1250, 2500 and 5000 µg/plate, for the TA 1537 and TA 98 strains in the second mutagenicity experiment,
39.06, 78.13, 156.3, 312.5, 625, 1250 µg/plate, for the TA 1535 strain in the second mutagenicity experiment,
19.53, 39.06, 78.13, 156.3, 312.5, 625 µg/plate, for the TA 100 and the TA 102 strains in the second mutagenicity experiment.

Vehicle / solvent:

Dimethylsulfoxide (DMSO), Batch No.: K34181550515, Supplier: VWR, Fontenay-Sous-Bois, France.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Remarks:

without S9

Positive control substance:

sodium azide

Remarks:

1 µg/plate for TA 1537 and TA 100

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Remarks:

without S9

Positive control substance:

9-aminoacridine

Remarks:

50 µg/plate for TA 1537

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Remarks:

without S9

Positive control substance:

2-nitrofluorene

Remarks:

0.5 µg/plate for TA 98

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Remarks:

without S9

Positive control substance:

mitomycin C

Remarks:

0.5 µg/plate for TA 102

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Remarks:

with S9

Positive control substance:

other: 2-aminoanthracene

Remarks:

2 µg/plate for TA 98, TA 100, TA 1535 and TA 1537

Controls 6

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

10 µg/plate for TA 102

Details on test system and experimental conditions:

The test item was tested in a preliminary test and two mutagenicity experiments.
The preliminary test, both experiments without S9 mix and the first experiment with S9 mix were performed according to the direct plate incorporation method. The second experiment with S9 mix was performed according to the preincubation method.
The direct plate incorporation method was performed as follows: test item solution (0.05 mL), S9 mix when required or phosphate buffer pH 7.4 (0.5 mL) and bacterial suspension (0.1 mL) were mixed with 2 mL of overlay agar (containing traces of the relevant aminoacid and biotin and maintained at 45 °C). After rapid homogenization, the mixture was overlaid onto a Petri plate containing minimum medium.
The preincubation method was performed as follows: test item solution (0.05 mL), S9 mix (0.5 mL) and the bacterial suspension (0.1 mL) were incubated for 60 minutes at 37 °C, under shaking, before adding the overlay agar and pouring onto the surface of a minimum agar plate.
After 48 to 72 hours of incubation at 37 °C, revertants were scored with an automatic counter (Cardinal counter, Perceptive Instruments, Suffolk CB9 7 BN, UK).

Evaluation criteria:

In each experiment, for each strain and for each experimental point, the number of revertants per plate was scored. The individual results and the mean number of revertants, with the corresponding standard deviation and ratio (mutants obtained in the presence of the test item/mutants obtained in the presence of the vehicle), are presented in tabular form.
A reproducible 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as a positive result. Reference to historical data, or other considerations of biological relevance may also be taken into account in the evaluation of the data obtained.

Statistics:

no data

Results and discussion

Test results

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in TA 100, TA 1535 and TA 102 strains at dose-levels ≥ 500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

A moderate to strong emulsion was observed in the Petri plates when scoring the revertants mainly at dose-levels ≥ 1250 µg/plate.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Under the experimental conditions, the test item 2-ethylhexyl salicylate did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.

Executive summary:

The test item 2 -ethylhexyl salicylate (salicylate d'octyle) did not induce any noteworthy increase in the number of revertants, in any of the five tester strains and in either experiment. Thus, the substance is considerd negative (with and without metabolic activation) in the Ames-assay.