Reaction mass of 2-butylheptanoic acid and 2-ethylnonanoic acid and 2-methyldecanoic acid and 2-propyloctanoic acid

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

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:

940-601-3

EC Number:

940-601-3

IUPAC Name:

940-601-3

Constituent 2

Reference substance name:

Reaction mass of n-undecanoic-acid and 2-methyl-decanoic-acid and 2-ethyl-nonanoic-acid and 2-propyl-octanoic-acid and 2-butyl-heptanoic-acid

IUPAC Name:

Reaction mass of n-undecanoic-acid and 2-methyl-decanoic-acid and 2-ethyl-nonanoic-acid and 2-propyl-octanoic-acid and 2-butyl-heptanoic-acid

Details on test material:

- Name of test material (as cited in study report): Reaction mass of n-undecanoic-acid and 2-methyl-decanoic-acid and 2-ethyl-nonanoic-acid and 2-propyl-octanoic-acid and 2-butyl-heptanoic-acid (Isocarb L 11)
- Substance type: pure active substance
- Physical state: liquid
- Expiration date of the lot/batch: 2014-10-01
- Storage condition of test material: at room temperature, protected from light

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

S9 liver microsomal fraction, derived from male Wistar rats, phenobarbital (80 mg/kg bw) / ß-naphthoflavone (100 mg/kg bw) induced for three consecutive days by oral route

Test concentrations with justification for top dose:

Pre-experiment: 0.00316, 0.0100, 0.0316, 0.100, 0.316, 1.0, 2.5, 5.0 µL/plate
Experiment I: 0.00316, 0.0100, 0.0316, 0.100, 0.316, 1.0, 2.5, 5.0 µL/plate
Experiment II: 0.00316, 0.0100, 0.0316, 0.100, 0.316, 1.0, 2.5 µL/plate (additionally 5.0 µL/plate for E. coli WP2 uvrA only)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was compatible with the survival of the bacteria and the S9 activity.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

A. dest., treated in the same way as all dose groups

Negative solvent / vehicle controls:

yes

Remarks:

DMSO, treated in the same way as all dose groups

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: S. typhimurium strains TA 100, TA 1535: sodium azide (10µg/plate); S. typhimurium strains TA 98, TA 1537: 4-nitro-o-phenylene-diamine (10 µg/plate for TA 98, 40 µg/plate for TA 1537); E.coli WP2 uvrA: methylmethanesulfonate (1 µg/plate)

Remarks:

without metabolic activation

Controls 2

Untreated negative controls:

yes

Remarks:

A. dest.

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2- aminoanthracene (2.5 µg/plate for all S. typhimurium strains, 10 µg/plate for E. coli WP2 uvrA)

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) in the pre-experiment and in experiment I, preincubation in experiment II

DURATION
- Preincubation period: 60 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3 plates

DETERMINATION OF CYTOTOXICITY
- Method: clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approx. ≤ 0.5 in relation to the solvent control

OTHER:
The properties of the S. typhimurium and E. coli strains with regard to membrane permeability, ampicillin- and tetracycline-resistance as well as normal spontaneous mutation rates are checked regularly according to Ames et al.
The colonies were conted using a ProtoCOL counter (Meintrup DWS Laborgeräte GmbH, Germany). Tester strains with a low spontaneous mutation frequency (TA 1535 and TA 1537) were counted manually.

Evaluation criteria:

Evaluation of Mutagenicity:
The Mutation Factor is calculated by dividing the mean value of the revertant counts through the mean values of the solvent control.
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs
in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:
- if in tester strains TA98, TA100 and E. coli WP2 uvrA the number of reversions is at least twice as high
- if in tester strains TA1537 and TA1537 the number of reversions is at least three times higher
as compared to the reversion rate of the solvent control
A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.

Criteria of validity:
A test is considered acceptable if for each strain:
- the bacteria demonstrate their typical responses to ampicillin (TA98, TA100)
- the mean values of the spontaneous reversion frequencies of the control plates with and without S9 mix are within the historical control data range
- corresponding background growth on both negative control and test plates is observed
- the positive controls show a distinct enhancement of revertant rates over the control plate.

Statistics:

A statistical evaluation of the results was not regarded as necessary as the biological relevance of the results is the criterion for the interpretation of results (according to guideline).

Results and discussion

Test results

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at concentrations 0.316 µl/plate and higher, depending on the particular tester strain

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation of the test item was obseved an any tester strain used in experiment I and II with and without metabolic activation.

RANGE-FINDING/SCREENING STUDIES: The toxicity of the test item was determind with tester stains TA 98 and TA 100. As the experimental conditions and the tested concentrations were the same as for experiment I, the results of the pre-experiment were included as a part of experiment I.

COMPARISON WITH HISTORICAL CONTROL DATA: The control plates with and without S9 mix were within the historical control data range.

ADDITIONAL INFORMATION ON CYTOTOXICITY: Toxic effects were noted in all tester strains evaluated:
Experiment I: tester strains TA 98, TA 100, TA 1535, TA 1537 and E. coli WP2 uvrA at concentrations of 1.0 µL/plate and higher with and without metabolic activation
Experiment II: tester strains TA 98 and E coli WP2 uvrA at concentrations of 1.0 µL/plate and higher with and without metabolic activation;
tester strains TA 100 and TA 1537 at concentrations of 0.316 µL/plate and higher with metabolic activation and at concentrations of 1.0 µL/plate and higher without metabolic activation;
tester strain TA 1535 at concentrations of 0.316 µL/plate and higher with and without metabolic acitvation.

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

The test substance was considered to be non-mutagenic in this bacterial reverse mutation assay.

Executive summary:

The test item Reaction mass of n-undecanoic acid and 2-methyl-decanoic-acid and 2-ethyl-nonanoic-acid and 2-propyl-octanoic-acid and 2-butyl-heptanoic-acid was investigated for its potential to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and tester strain E. coli WP2 uvrA. The study was designed to be compliant with OECD Guideline No. 471 and in accordance with the OECD Principles of Good Laboratory Practices.

In two independent experiments several concentrations of the test item were used. Each assay was conducted with and without metabolic activation. The concentrations, including the controls, were tested in triplicate. The following concentrations of the test item were prepared and used in the experiments:

Experiment I: 0.00316, 0.01, 0.0316, 0.1, 0.3165, 1.0, 2.5 and 5.0 µL/plate

Experiment II: 0.00316, 0.01, 0.0316, 0.1, 0.316, 1.0 and 2.5 µL/plate (additionally 5.0 µL/plate for E. coli WP2 uvrA only)

No precipitation of the test item was observed in any tester strain used in experiment I and II with and without metabolic activation.

Toxic effects of the test item were noted in all tester strains used in experiment I and II. In experiment I toxic effects of the test item were observed at concentrations of 1.0 µL/plate and higher with and without metabolic activation depending on the particular tester strain. In experiment II toxic effects of the test item were noted at concentrations of 0.316 µL/plate and higher with and without metabolic activation, depending on the particular tester strain.

No biologically relevant increases in revertant colony number of any of the five tester strains were observed following treatment with the test substance in experiment I and II.

The reference mutagens induced a distinct increase or revertant colonies indication the validity of the experiments.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test substance did not cause gene mutations by base pair changes or frameshifts in the genome of the test strains used.

Therefore, the test substance was considered to be non-mutagenic in this bacterial reverse mutation assay.