Hexanoic acid, 2-ethyl-, C12-15-alkyl esters

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:

18 May 1992- 02 June 1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Hexanoic acid, 2-ethyl-, C12-15-alkyl esters

EC Number:

291-443-0

EC Name:

Hexanoic acid, 2-ethyl-, C12-15-alkyl esters

Cas Number:

90411-66-8

IUPAC Name:

Hexanoic acid, 2-ethyl-, C12-15-alkyl esters

Test material form:

liquid

Method

Target gene:

histodine locus

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

other: sensibility to crystalviolet, histidine necessity, sensibility to ultra-violet radiations, resistance to Atrpicitlin

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

other: sensibility to crystalviolet, histidine necessity, sensibility to ultra-violet radiations, resistance to Atrpicitlin

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 1535

Additional strain / cell type characteristics:

other: sensibility to crystalviolet, histidine necessity, sensibility to ultra-violet radiations

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 1537

Additional strain / cell type characteristics:

other: sensibility to crystalviolet, histidine necessity, sensibility to ultra-violet radiations

Species / strain 5

Species / strain / cell type:

S. typhimurium TA 1538

Additional strain / cell type characteristics:

other: sensibility to crystalviolet, histidine necessity, sensibility to ultra-violet radiations

Metabolic activation:

with and without

Metabolic activation system:

mammalian liver post-mitochondrial fraction (S-9)

Test concentrations with justification for top dose:

Dosage
The following concentrations expressed in µg/plate were used;
10.000
1.000
100
10
1
o (negative control)

Vehicle / solvent:

The test material COSMACOL EOI, was dissolved in Dimethylsulphoxide (DMSO) at a concentration of 100 mg/ml.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-aminoanthracene

Details on test system and experimental conditions:

TEST PROCEDURE

Plate test without metabolic activation

0.1 ml of the appropriate dilution of the test solution and 0.1 ml of a bacterial suspension containing approximately 108 -109 cfu/ml were added in this sequence to 2 ml of molten top agar.
The ingredients were thoroughly mixed and immediately poured into plates containing minimal medium.
At the same time, negative control and positive controls using the chosen concentration of the reference mutagens were performed.
The plates were incubated at 37°C for 48 72 hours. After incubation the revertant colonies obtained in the plates were counted. Triplicate plates were used for the test material and two for the positive controls.

Plate test with metabolic activation

0.1 ml of the appropriate dilution of the test solution, 0.1 ml of bacterial suspensions and 0.5 ml of the metabolic activation system were added in this sequence to 2 ml of molten top agar.
The ingredients were thoroughly mixed and immediately poured into plates containing minimal medium.
At the same time, negative control and positive controls using the chosen concentration of the reference mutagens were performed.
The plates were incubated at 37°C for 48 72 hours. After incubation the revertant colonies obtained in the plates were counted.
Triplicate plates were used for the test material and two for the positive controls.

Evaluation criteria:

ACCEPTANCE CRITERIA
At the end of the assay, a sterility check on S9Mix must show less two viable colonies per 0.5 ml.
At the end of the test, a sterility check of the test material must show less than two viable colonies per plate.
The positive controls must produce at least a threefold increase in the number of revertant colonies with regard to the mean value for the respective negative control.
The mean number of spontaneous revertant colonies in the negative controls must correspond to the following values:
STRAIN ACCEPTABLE RANGE
TA 1535 20 ± 15
TA 1537 20 ± 15
TA 1538 15 ± 10
TA 98 40 ± 25
TA 100 150 ± 90

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST MATERIAL MUTAGENICITY (Table n 1 and n 2 - see background attached info)
The test material induced the development of as many revertant colonies as those induced by the negative control, both in the absence and in presence of the rat liver metabolizing system, at the maximum dose tested (10 mg/plate).

CONTROLS
Preliminary tests
The genetic characteristics of the bacterial strains were found to be unaltered.

Test material toxicity
The test material did not induce toxic effects at a concentration oOf 10 mg/plate both in the absence and in presence of the metabolic activation system.

Negative control
The number of revert~~t colonies on the negative control plates fell within the normal range.

Positive control
All the positive controls induced a significant increase in the number of revertant colonies.

Sterility test
The sterility test performed on the test material and on S9Mix did not show any bacterial contamination.

Applicant's summary and conclusion

Conclusions:

The test material at maximum concentration 10 mg/petri dish produced an increase in number of revertant colonies similar to the negative control so the test material is unable to induce mutation under there conditions.

Executive summary:

A test was performed to verify the mutagenic potential of the test material COSMACOL EOI on Salmonella typhimurium.

The test was carried out on 5 strains of Salmonella typhimurium (TA1535, TA1537, TA1538, TA98 , TA100).

The mutagenic activity of the material was assessed by comparing the number of revertant colonies induced with the positive control.

This activity was tested in the presence and absence of a metabolizing system and done directly in a petri dish. The test material was tested at a concentration of 100000 µg/ml, 10000 µg/ml , 1000 µg/ml, 100 µg/ml, 10 µg/ml corresponding to the dose 10000 µg/petri dish, 1000 µg/petri dish, 100 µg/petri dish, 10 µg/petri dish and 1 µg/petri dish.

The results of the study can be summarized as follows:

The test material at maximum concentration 10 mg/petri dish produced an increase in number of revertant colonies similar to the negative control so the test material is unable to induce mutation under there conditions.