Diethyl tartrate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames Test (OECD 471, GLP, rel. 1): non mutagenic up to 5000 µg/plate in S. typhimurium TA 97a, TA 98, TA 100, TA 102 and TA 1535.

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:

From July 14 to 30, 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study conducted according to OECD 471 Guideline without any deviation.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other 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

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected on October 13 and 14, 2014 / signed on April 08, 2015

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine gene for Salmonella typhimurium

Species / strain / cell type:

S. typhimurium, other: TA 97a, TA 98, TA 100, TA 102 and TA 1535

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9-mix, rat liver S9-mix induced by Aroclor 1254

Test concentrations with justification for top dose:

Experiment 1 (Range-finding test): 50, 150, 500, 1500 and 5000 μg/plate in TA 97a, TA 98, TA 100, TA 102 and TA 1535 strains, with and without S9 mix using plate incorporation method

Main test
Experiment 2a: 156, 313, 625, 1250, 2500 and 5000 μg/plate TA 102 and TA 1535 strains, with and without S9 mix using pre-incubation method
Experiment 2b: 156, 313, 625, 1250, 2500 and 5000 μg/plate TA 97a, TA 98, TA 100 strains, with and without S9 mix using pre-incubation method

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Demineralised water
- Justification for choice of solvent/vehicle: In a non-GLP pre-test, the solubility of the test item was determined in demineralised water, dimethyl sulfoxide (DMSO) and ethanol. The test item was soluble in a concentration of 50 g/L in demineralised water, DMSO and ethanol. Therefore, on the day of the respective experiment, a stock solution containing 50 g/L was prepared in demineralised water for all experiments.
The stock solution was used to prepare a geometric series of the concentrations to be tested. Each solution was membrane filtrated to accomplish sterility.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

other: 4-Nitro-1,2-phenylene Diamine

Remarks:

without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: 2-Amino-Anthracene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

SOURCE OF TEST SYSTEM: Salmonella typhimurium (all strains used) were obtained from TRINOVA BioChem, Germany (batch of the bacteria strains: TA97a: 4904D, TA98: 4903D, TA100: 4902D, TA102: 4872D, TA1535: 4908D) and were stored as lyophilisates in the refrigerator at 2-8 °C.

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 37 ±1 °C for 20 min
- Exposure duration: 37 ±1 °C for 48-72 h

NUMBER OF REPLICATIONS:
- Experiment 1 (Range-finding test): 3 plates/dose
- Experiment 2a and Experiment 2b: 3 plates/dose

DETERMINATION OF CYTOTOXICITY
- Method: Evaluation of the toxicity was performed on the basis of the reduction of the growth of the bacterial background lawn.

OTHERS:
- After incubation for 48–72 h, the revertants were counted and the numbers for each plate were recorded. The colonies were counted visually, the numbers were recorded.

Evaluation criteria:

A test item is considered to have mutagenic potential, if a significant, reproducible increase of revertant colonies per plate (increase factor ≥ 2) in at least one strain can be observed.
A concentration-related increase over the range tested can also be taken as a sign of mutagenic activity, as well as an increase in one or more concentrations. Also, the biological relevance must be assessed when compared to in-house controls.

Statistics:

None

Species / strain:

S. typhimurium, other: TA 97a, TA 98, TA 100, TA 102 and 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

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test item was dissolved in demineralised water. A stock solution containing 50 g/L in demineralised water was prepared.
- Precipitation: In this experiment, the test item showed no precipitates on the plates in all tested concentrations.

RANGE-FINDING/SCREENING STUDIES:
- In this experiment, the test item showed no precipitates on the plates in all tested concentrations. No signs of toxicity towards the tested strains could be observed. No visible reduction in the growth of the bacterial background lawn at any dose level. No significant increase of the number of revertant colonies in the treatments with and without metabolic activation could be observed. No concentration-related increase over the tested range was found. To verify this result, a second experiment was performed using the pre-incubation method.

MAIN TEST:
Experiments 2a and 2 b
In both experiments, the test item showed no precipitates on the plates in all tested concentrations. No signs of toxicity towards the tested strains could be observed. No visible reduction in the growth of the bacterial background lawn at any dose level. No significant increase of the number of revertant colonies in the treatments with and without metabolic activation was observed in both experiments. No concentration-related increase over the tested range was found.

COMPARISON WITH HISTORICAL CONTROL DATA:
- Results were compared with historical control data.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Confirmation of the Criteria and Validity

The treatments for the sterility control and the determination of the titre did not show any inconsistencies. The determined values for the spontaneous revertants of the solvent controls were within the normal range of the test laboratory.

All positive controls (diagnostic mutagens) showed mutagenic effects with and without metabolic activation.

 

Acceptability of Study

Nearly all spontaneous revertants (one outlayer) and all positive control values were within the range of the historical data. Difference of revertants lying outside the range is marginal. Therefore, the study is considered valid.

Conclusions:

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

Under the test conditions, the test material is not mutagenic with and without metabolic activation in S. typhimurium (TA 97a, TA 98, TA 100, TA 102 and TA 1535 strains) according to the criteria of the Annex VI of the Regulation (EC) No 1272/2008 (CLP).

Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, strains of Salmonella typhimurium (TA 97a, TA 98, TA 100, TA 102 and TA 1535) were exposed to the test item diluted in water both in the presence and in the absence of a metabolic activation system (S9-mix, rat liver S9-mix induced by Aroclor 1254) for 48 -72 h. Experiment 1 (Range-finding test) was performed at 50, 150, 500, 1500 and 5000 μg/plate in TA 97a, TA 98, TA 100, TA 102 and TA 1535 strains, with and without S9 mix using plate incorporation method. In the main test, Experiment 2a was performed at 156, 313, 625, 1250, 2500 and 5000 μg/plate in TA 102 and TA 1535 strains, with and without S9 mix using pre-incubation method and Experiment 2b was performed at 156, 313, 625, 1250, 2500 and 5000 μg/plate in TA 97a, TA 98, TA 100 strains, with and without S9 mix using pre-incubation method. Negative, vehicle and positive control groups were also included in mutagenicity tests. 

 

The sterility control and the determination of the titre did not show any inconsistencies. The determined values for the spontaneous revertants of the negative controls (vehicle) were in the normal range. All positive controls showed mutagenic effects with and without metabolic activation. Therefore, the sensitivity of the assay and the efficacy of the S9-mix were validated.

 

The test item showed no precipitates on the plates in all tested concentrations. No signs of toxicity towards the tested strains could be observed, no visible reduction in the growth of the bacterial background lawn at any dose level. No significant increase of the number of revertant colonies in the treatments with and without metabolic activation was observed in any of the experiments. No concentration-related increase over the tested range was found.

 

Under the test condition, the test material is not considered mutagenic with and without metabolic activation to S. typhimurium (TA 97a, TA 98, TA 100, TA 102 and TA 1535 strains) according to the criteria of the Annex VI of the Regulation (EC) No. 1272/2008 (CLP).

This study is considered as acceptable and satisfies the requirement for reverse gene mutation endpoint.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

A Bacterial Reverse mutation Assay (Ames test) was performed according to OECD test guideline No 471 with the substance. No significant or biologically relevant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose, either in the presence or absence of metabolic activation.The substance does not induce gene mutations in bacteria under the test conditions whereas all positive control chemicals (with and without metabolic activation) induced significant increase of colonies. The substance is therefore considered as non-mutagenic according to the Ames test.

Justification for selection of genetic toxicity endpoint
Only one GLP compliant and of very high quality Ames study available (Klimish score = 1).

Justification for classification or non-classification

Harmonized classification:

The substance has no harmonized classification for human health according to the Regulation (EC) No. 1272/2008 including ATP7.

Self classification:

Based on the available data, no additional classification is proposed regarding genetic toxicity according to the Annex VI of the Regulation (EC) No. 1272/2008 (CLP).