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Toxicological information

Genetic toxicity: in vitro

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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

Constituent 1
Chemical structure
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:
2-ethylhexyl 2-hydroxybenzoate
Constituent 2
Reference substance name:
SALICYLATE D’OCTYLE
IUPAC Name:
SALICYLATE D’OCTYLE
Test material form:
other: colorless liquid

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.
Controlsopen allclose all
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
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
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
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
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
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.