2-oxocyclohexane-1,1,3,3-tetrapropionic acid

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

A reliable bacterial reverse mutation test is available and was performed according to OECD/EC guidelines. The Ames test turns to be negative.

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:

Study initiation: October 15, 1991 ; Experimental start date: October 25, 1991 ; Experimental termination date: November 25, 1991 and Study termination date: December 11, 1991.

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

This test system permits the detection of gene mutations induced by the test material or its metabolites in histidine-requiring strains of Salmonella typhimurium and in a tryptophan-requiring strain of Escherichia coli.

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Rat-Liver microsomal fraction S9 system.

Test concentrations with justification for top dose:

- Range in the cytotoxicity test: 20.6 - 5000 microg/plate.
- Range in the mutagenicity test without microsomal activation: 312.5 - 5000 microg/plate.
- Range in the mutagenicity test with microsomal activation: 312.5 - 5000 microg/plate.

Vehicle / solvent:

Dimethylsulfoxide (Suspension)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Dimethylsulfoxide (DMSO)

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Positive control for strain TA100 and TA1535 in the experiement without microsomal activation. (used at 5.0 microg/plate and the solvent used is bidistilled water).

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Dimethylsulfoxide (DMSO)

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Positive control for strain WP2 uvrA in the experiement without microsomal activation. (used at 2.0 microg/plate and the solvent used is DMSO).

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Dimethylsulfoxide (DMSO)

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

Positive control for strain TA98 in the experiement without microsomal activation. (used at 20.0 microg/plate and the solvent used is DMSO).

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Dimethylsulfoxide (DMSO)

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 9 (5)-aminoacridine

Remarks:

Positive control for strain TA 1537 in the experiement without microsomal activation. (used at 150.0 microg/plate and the solvent used is DMSO).

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Dimethylsulfoxide (DMSO)

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

Positive control for strain TA100, TA1535, WP2 uvrA and TA98 in the experiement with microsomal activation. (used at 2.5 microg/plate excepeted for WP2 uvrA 2-AA used at 50.0 microg/plate and the solvent used is DMSO).

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Dimethylsulfoxide (DMSO)

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

Positive control for strain TA 1535 in the experiement with microsomal activation. (used at 400.0 microg/plate and the solvent used is bidist. water).

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

Setting up of the test plates
0.1 ml of the overnight cultures were mixed with 2 ml of top agar, either 0.5 ml of 100 mM sodium phosphate buffer (experiments without activation) or 0.5 ml of the activation mixture (experiments with activation) and 0.1 ml of a solution of the test substance, the substance for the positive control or the solvent for the negative control and poured on minimal agar in Petri dishes. Each Petri dish contained about 20 ml of minimal agar(1.5% agar supplemented with 2% salts of the Vogel-Bonner Medium E and 2% glucose). The top agar was composed of 0.6% agar and 0.6% NaCl.
In the experiment with Salmonella the top agar was supplemented with 10% of 0.5 mM 1-histidine and 0.5 mM (+)biotin dissolved in water. In the experiment with E. coli it was supplemented with 10% of 0.5 mM 1-tryptophan dissolved in water.


Preparation of the bacterial cultures
Inoculates from frozen master copies were set up monthly. They were grown in liquid NB-medium overnight and then plated on NBagar.
After incubation, single colonies were taken from the plates, grown over-night in liquid NB-medium and then used for the experiment.


NUMBER OF REPLICATIONS:
- In the preliminary Toxicity/Range-Finding test: single plate
- In the mutagenicity test: Triplicate

Evaluation criteria:

Assay acceptance criteria
A test is considered acceptable if the mean colony counts of the control values of all strains are within the acceptable ranges and if the results of the positive controls meet the criteria for a positive response. In either case the final decision is based on the scientific judgement of the Study Director.

Criteria for a positive response
The test substance is considered to be mutagenic in this test system if one or both of the following conditions are met:
- At least a reproducible doubling of the mean number of revertants per plate above that of the negative control at any concentration for one or more of the following strains: S. typhimurium TA 98, TA 1535, TA 1537 and E. Coli WP2 uvrA.
- A reproducible increase of the mean number of revertants per plate for any concentration above that of the negative control by at least a factor of 1.5 for strain S. typhimurium TA 100.

Generally a concentration-related effect should be demonstrable.

Statistics:

A statistical analysis was not performed. At present the use of statistical methods concerning this particular test system is not generally
recommended.

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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Toxicity test

Six concentrations of the test substance ranging from 20.6- 5000 microg/plate were tested with strains S. typhimurium TA 100 and

E.coli WP2 uvrA to determine the highest concentration to be used in the mutagenicity assay. The experiments were performed with and without microsomal activation. From the results obtained, the highest concentration suitable for the mutagenicity test was selected to be 5000 microg/plate without and with activation.

Mutagenicity test, original experiment

In the experiments performed without and with microsomal activation, treatment with the substance did not lead to an increase in the incidence of either histidine- or tryptophanprototrophic mutants in comparison with the negative control.

Mutagenicity test, confirmatory experiment

In the experiments performed without and with microsomal activation, again after treatment of the bacteria with test substance no increase in the incidence of either histidine- or tryptophan-prototrophic mutants was observed in comparison with

the negative control.

The various mutagens, promutagens, sterility checks, sensitivity and resistance tests, etc., employed to ensure the test system was

acceptable, all produced results within our established limits.

There were no known circumstances or occurrences in this study that were considered to have affected the quality or integrity of the data.

Conclusions:

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

Based on the results of these experiments and on standard evaluation criteria, it is concluded that the test substance and its metabolites did not induce gene mutations in the strains of S. typhimurium and E. coli used.

Executive summary:

The study was performed to evaluate the test compound for mutagenic activity in bacterial test systems in the absence and presence of a rat liver S9 activity.

The experiemtn was performed according to the OECD 471.

Salmonella typhimurium TA100, TA1535, TA1537, TA 98 and Escherichia coli WP2 uvrA were used.

The test item was dissolvent in Dimethysulfoxyde solvent (DMSO) at 50 mg/ml.

Concentrations applied in the mutagenicity study

The concentration range of the test item to be tested in the mutagenicity test was determined in a preliminary toxicity test.

Thus, the substance was tested for mutagenic effects without microsomal activation at five concentrations in the range of 312.5-5000 microg/plate. In the experiment carried out with microsomal activation five concentrations in the range of 312.5-5000 microg/plate were tested. In order to confirm the results, the experiments were repeated with the same concentration ranges.

Mutagenicity test, original experiment

In the original experiment performed without and with microsomal activation, none of the tested concentrations of test item led to

an increase in the incidence of either histidine- or tryptophanprototrophic mutants by comparison with the negative control. Mutagenicity test, confirmatory experiment In the confirmatory experiment performed without and with microsomal activation, again, the tested concentrations of the substamce did not lead to an increase in the incidence of either histidine- or tryptophan-prototrophic mutants by comparison with the negative control.

The test was performed with five concentrations up to 5000 microg/plate without detection of any toxic and/or mutagenic effects.

The values of the negative controls were found to be within the acceptable range and the values of the positive controls met the criteria for a positive response. These results confirm the correct performance of the test.

Conclusion

Based on the results of these experiments and on standard evaluation criteria, it is concluded that the test item and its metabolites did not induce gene mutations in the strains of S. typhimurium and E. coli used.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

The mutagenicity study has been conducted in in-vitro test systems where the Ames test turns out to be negative indicating clearly a non mutagenic potential in-vitro with or without metabolic activation.

It was judged that the test substance is not mutagenic in vitro.

Justification for selection of genetic toxicity endpoint
The study is Klimisch 1 and only this study is available.

Justification for classification or non-classification

Based on the above mentioned results the substance does not need to be classified according to CLP regulation (Regulation EC No. 1272/2008) and DSD (Directive 67/548/EEC).