A mixture of Propanoic acid, -1-methyl, 2-hydroxy-, (C12-14)-alkyl ester, Propanoic acid, 2-hydroxy-, 2-(C12-14-alkyloxy)-1-methyl-2-oxoethyl ester, Propanoic acid, 2-hydroxy-, 2-(2-(C12-14-alkyloxy)-1-methyl-2-oxoethoxy)-1-methyl-2-oxoethyl ester, Propanoic acid, 2-hydroxy-, 2-(2-(2-(C12-14-alkyloxy)-1-methyl-2-oxoethoxy)-1-methyl-2-oxoethoxy)-1-methyl-2-oxoethyl ester, Propanoic acid, 2-hydroxy-, 2-(2-(2-(2-(C12-14-alkyloxy)-1-methyl-2-oxoethoxy)-1-methyl-2-oxoethoxy)-1-methyl-2-oxoethoxy)-1-methyl-2-oxoethyl ester, Propanoic acid, 2-hydroxy-, 2-(2-(2-(2-(2-(C12-14-alkyloxy)-1-methyl-2-oxoethoxy)-1-methyl-2-oxoethoxy)-1-methyl-2-oxoethoxy)-1-methyl-2-oxoethoxy)-1-methyl-2-oxoethyl ester, Propanoic acid, 2-hydroxy-, 2-(2-(2-(2-(2-(2-(C12-14-alkyloxy)-1-methyl-2-oxoethoxy)-1-methyl-2-oxoethoxy)-1-methyl-2-oxoethoxy)-1-methyl-2-oxoeth oxy)-1-methyl-2-oxoethoxy)-1-methyl-2-oxoethyl ester, Propanoic acid, 2-hydroxy-, 2-(2-(2-(2-(2-(2-(2-(C12-14-alkyloxy)-1-methyl-2-oxoethoxy)-1-methyl-2-oxoethoxy)-1-methyl-2-oxoethoxy)-1-methyl-2-oxoeth oxy)-1-methyl-2-oxoethoxy)-1-methyl-2-oxoethoxy)-1-methyl-2-oxoethyl ester and Propanoic acid, 2-hydroxy-, 2-(2-(2-(2-(2-(2-(2-(2-(C12-14-alkyloxy)-1-methyl-2-oxoethoxy)-1-methyl-2-oxoethoxy)-1-methyl-2-oxoethoxy)-1-methyl-2-oxoeth oxy)-1-methyl-2-oxoethoxy)-1-methyl-2-oxoethoxy)-1-methyl-2-oxoethoxy)-1-methyl-2-oxoethyl ester

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro gene mutation in bacteria: The substance was negative with and without metabolic activation in a reverse mutation assay performed using Salmonella typhimurium (strains TA1535, TA1537, TA98 and TA100) and Escherichia coli (WP2uvrA) according to OECD TG 471 and EU Method B.13/14.

Link to relevant study records

Reference

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:

2007-05-30 to 2007-06-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Gene of histidine-requiring S. typhimurium bacterial strains resulting in histidine-indpendant strains, and a gene of tryptophan-requiring E.coli bacterial strain resulting in a tryptophan-independent strain.

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: rfa: deep rough (defective lipopolysaccharide cellcoat), gal: mutation in the galatose metabolism, chl: mutation in notrate reductase, bio: defective biotin synthesis, uvrB: loss of the excision repair system (deletion of the ultraviolet-repair B gene).

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital and beta-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Dose range finding (+/- S9): 3 to 5000 µg/plate
Ist Expt (+/- S9): 3 - 1000 µg/plate
2nd Expt (+/- S9): 10 - 1000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: not specified

Untreated negative controls:

yes

Remarks:

Vehicle, DMSO, only

Negative solvent / vehicle controls:

yes

Remarks:

DMSO only (negative control)

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: See attached document: 07-006A; Positive control data

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: Not applicable
- Exposure duration: 48 h
- Expression time (cells in growth medium): 10E09 cells/mL
- Selection time (if incubation with a selection agent): Not applicable

NUMBER OF REPLICATIONS: 3

NUMBER OF CELLS EVALUATED:

DETERMINATION OF CYTOTOXICITY
- Method: reduction in bacterial background lawn, increase in size of the microcolonies and reduction of revertant colonies

Evaluation criteria:

A response is considered negative if:
- the total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 and WP2uvrA is not greater than three (3) times the concurrent control.
- the negative response should be reproducible in at least one independently repeated experiment.
A response is considered positive if
- The total number of revertants in tester strain TA100 is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is greater than three (3) times the concurrent control.
- In case a positive response will be repeated, the positive response should be reproducible in at least one indepentently repeated experiment.
The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.

Statistics:

Not stated.

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:

cytotoxicity

Remarks:

See attached document; 07-006A cytotoxicity data

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

See attached document; 07-006A cytotoxicity data

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitaion of the test material on the plates was observed at the start of the incubation period at concentrations of 333 µg/plate and upwards and at 1000 µg/plate and upwards at the end of the incubation period.

RANGE-FINDING/SCREENING STUDIES:
The test material was tested up to 5000 µg/plate, +/- S9 in the strains TA100 and WP2uvrA. In tester strain TA100, toxicity was observed at dose levels of 100 µg/plate, with and without S9. With WP2uvrA, no toxicity was observed at any dose levels tested.

COMPARISON WITH HISTORICAL CONTROL DATA:
All control values were within laboratory historical control data ranges confirming that the test conditions were adequate and that the metabolic activation system functioned properly.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

See attached document: 07-006A; Mutagenicity Response Tables

Conclusions:

Interpretation of results (migrated information):
negative

The test material is not mutagenic with or without metabolic activiation in bacteria under the conditions of the study.

Executive summary:

The test material dissolved in dimethyl sulfoxide (DMSO) was evaluated in the Salmonella typhimurium (strains TA1535, TA1537, TA98 and TA100) reverse mutation assay and the Escherichia coli (WP2uvrA) reverse mutation assay (with independent repeat) in the absence and presence of phenobarbital and beta-naphthoflavone induced rat liver S9 following OECD 471 and EC B13/14 Guidelines.

In a dose range-finding study the test material was tested up to 5000 µg/plate, +/- S9 in the strains TA100 and WP2uvrA. In tester strain TA100, toxicity was observed at dose levels of 100 µg/plate, with and without S9. With WP2uvrA, no toxicity was observed at any dose levels tested.

The test material precipitated at dose levels of 1000 µg/plate and above.

In the first experiment the test material was tested at a concentration range of 3 to 1000 µg/plate with and without 5% (v/v) S9 in strains TA1535, TA1537 and TA98. Toxicity was only observed in the tester strain TA1535 with and without S9 and in TA1537 without S9.

In the repeat assay with additional parameters the test material was tested in the concentration range 10 to 1000 µg/plate in the absence and presence of 10% (v/v) S9 -mix in strains TA1535, TA1537, TA98 and WP2uvrA and at 3 to 666 µg/plate (+/- S9) in tester strain TA100. Toxicity was observed in tester strains TA1535 without S9, and TA1537 and TA100 with and without S9.

The test material did not induce a signficant dose-related increase in the number of revertant (His+) colonies in each of the four S typhimurium tester strains and in the number of revertant (Trp+) colonies in the E.coli tester strain both with and without S9.

All control values were within laboratory historical control data ranges confirming that the test conditions were adequate and that the metabolic activation system functioned properly.

Based on the results of this study it is concluded that the test material is not mutagenic in vitro in bacteria with or without metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

In vitro gene mutation in bacteria:

The substance, dissolved in dimethyl sulfoxide (DMSO) was evaluated in the Salmonella typhimurium (strains TA1535, TA1537, TA98 and TA100) reverse mutation assay and the Escherichia coli (WP2uvrA) reverse mutation assay (with independent repeat) in the absence and presence of phenobarbital and beta-naphthoflavone induced rat liver S9 following OECD TG 471 and EU Method B13/14.

In a dose range-finding study the substance was tested up to 5000 µg/plate, +/- S9 in the strains TA100 and WP2uvrA. In tester strain TA100, toxicity was observed at dose levels of 100 µg/plate, with and without S9. With WP2uvrA, no toxicity was observed at any dose levels tested.

The substance precipitated at dose levels of 1000 µg/plate and above.

In the first experiment the substance was tested at a concentration range of 3 to 1000 µg/plate with and without 5% (v/v) S9 in strains TA1535, TA1537 and TA98. Toxicity was only observed in the tester strain TA1535 with and without S9 and in TA1537 without S9.

In the repeat assay with additional parameters the substance was tested in the concentration range 10 to 1000 µg/plate in the absence and presence of 10% (v/v) S9 -mix in strains TA1535, TA1537, TA98 and WP2uvrA and at 3 to 666 µg/plate (+/- S9) in tester strain TA100. Toxicity was observed in tester strains TA1535 without S9, and TA1537 and TA100 with and without S9.

The substance did not induce a signficant dose-related increase in the number of revertant (His+) colonies in each of the four S typhimurium tester strains and in the number of revertant (Trp+) colonies in the E.coli tester strain both with and without S9.

All control values were within laboratory historical control data ranges confirming that the test conditions were adequate and that the metabolic activation system functioned properly.

Based on the results of this study it is concluded that the substance is not mutagenic in vitro in bacteria with or without metabolic activation.

Justification for selection of genetic toxicity endpoint
Only study available at Annex VII. It is a GLP guideline study.

Justification for classification or non-classification

On the basis of the available data (i.e. an in vitro gene mutation assay in bacteria) the substance does not require classification. The classification will be revisited when Annex VIII data are available.