D,L-DIETHYL TARTRATE

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Ames Test (OECD 471, GLP, K, rel. 1): non mutagenic up to limit concentration in S. typhimurium TA 1535, TA 1537, TA 98, TA 100 & E. coli WP2 (uvr A-) (pKM101)

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 June to 27 July 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted according to OECD Guideline 471 without deviation.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

2 November 2016

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature

Target gene:

Histidine gene for Salmonella typhimurium and tryptophan gene for Escherichia coli

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

10 % (v/v) S9 mix: S9 fraction prepared from liver homogenates of Sprague Dawley rat.

Test concentrations with justification for top dose:

Experiment 1: 50, 150, 500, 1500 and 5000 µg/plate in S. typhimurium TA 98, TA 100, TA 1535 & TA 1537 and E. coli WP2 (uvr A-) (pKM101), with and without metabolic activation using plate incorporation method
Experiment 2: 50, 150, 500, 1500 and 5000 µg/plate in S. typhimurium TA 98, TA 100, TA 1535 & TA 1537 and E. coli WP2 (uvr A-) (pKM101), with metabolic activation (pre-incubation method) and without metabolic activation (plate incorporation method).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Ethanol
- The item was soluble in ethanol. A stock solution on the day for each assay was prepared at 100 mg/mL in ethanol. All formulations were used within two hours and were assumed to be stable for the period. The highest dose tested was 5000 µg/plate.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: cis-Platinum (II) Diammine Dichloride

Remarks:

Without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

9,10-dimethylbenzanthracene

other: 2-Anthramine

Remarks:

With metabolic activation

Details on test system and experimental conditions:

SOURCE OF TEST SYSTEM
Strains of S. typhimurium and E. coli were obtained from MOLTOX™.

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

DURATION
- Preincubation period: Test tubes, which contained mixtures of bacteria, S9 mix and test dilution, were incubated at 37 °C for 30 minutes with shaking before the addition of the agar overlay.
- Exposure duration: Plates were incubated at 37 °C for 48-72 h

NUMBER OF REPLICATIONS: Triplicate plates per dose level.

DETERMINATION OF CYTOTOXICITY
- Method: The cytotoxicity is based upon the survival: in case bacteriostatic activity is detected, the highest concentration to be retained is that exhibiting a bacteriostatic activity of 75 % or less.

OTHERS:
SOURCE OF TEST SYSTEM
Strains of S. typhimurium and E. coli were obtained from MOLTOX™.

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

DURATION
- Preincubation period: Test tubes, which contained mixtures of bacteria, S9 mix and test dilution, were incubated at 37 °C for 30 minutes with shaking before the addition of the agar overlay.
- Exposure duration: Plates were incubated at 37 °C for 48-72 h

NUMBER OF REPLICATIONS: Triplicate plates per dose level.

DETERMINATION OF CYTOTOXICITY
- Method: The cytotoxicity is based upon the survival: in case bacteriostatic activity is detected, the highest concentration to be retained is that exhibiting a bacteriostatic activity of 75 % or less.

OTHERS:
- Sterility tests: Test item and the corresponding dilutions were added to 2 mL of top agar maintained at 45 °C, and poured after homogenization on the bottom agar (20 mL) onto a Petri plate (90 mm in diameter) (n = 3). Plates were incubated for 48 - 72 hours at 37 °C and then examined. There should be no bacterial growth on any plate. S9-mix sterility was checked using the same protocol.
- After a 48-72 hour incubation period at 37 °C, revertant colonies were counted in each plate.
The following ratio was calculated:
R = Number of revertant colonies in the presence of the test item / Number of revertant colonies in the absence of the test item

Rationale for test conditions:

Experiment 1 - Maximum concentration was 5000 μg/plate (the maximum recommended dose level).
Experiment 2 - Maximum concentration was 5000 μg/plate (the maximum recommended dose level).
Up to five test item dose levels per bacterial strain were selected in the second mutation test in order to achieve both a minimum of four non-toxic dose levels and the toxic limit of the test item.

Evaluation criteria:

The result of the test is considered as negative if the revertant number is below three fold the number of spontaneous reversions, for TA 1535 and TA 1537 strains, and below two fold the number of spontaneous reversions for TA 98, TA 100 and Escherichia coli WP2(uvrA-) (pKM 101) strains without and with metabolic activation.
The result of the test is considered positive if a dependent relationship concentration is obtained in one, or several of the 5 strains, without and/or with metabolic activation, a mutagenic effect being taken into account for a given dilution of test item if the number of revertant colonies is at least two fold that of spontaneous revertant colonies for TA 98, TA 100 and Escherichia coli WP2(uvrA-) (pKM 101), and three fold for TA 1535 and TA 1537.
All results must be confirmed in an independent experiment.

Statistics:

No data

Key result

Species / strain:

S. typhimurium, other: TA 98, TA 100, TA 1535 & 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

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

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
- Precipitation: None

RANGE-FINDING/SCREENING STUDIES:
Bacteriostatic activity control: Results shown that neither original solution nor dilutions on TA 100 have bacteriostatic effect and no precipitate formation observed during the test. The test item was tested in triplicate against each tester strain at these doses (50, 150, 500, 1500 and 5000 µg/plate).

MUTAGENICITY:
- No precipitate formation and no toxicity were observed during the main test.
- There was no evidence of any increase in the number of revertant colonies in the presence of the test item stock solution and dilutions (50, 150, 500, 1500 and 5000 µg/plate) without and with metabolic activation in Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and in Escherichia coli WP2(uvrA-) (pKM 101). Results were confirmed in an independent experiment.

HISTORICAL CONTROL DATA
- Results were compared with historical data.
- There is no significant difference between the number of spontaneous reversions, the number of reversions obtained in the positive controls (without and with metabolic activation), and the mean of corresponding experimental "historical" values obtained in the laboratory.

OTHERS
Sterility controls:
Test items: The absence of any bacterial growth in the presence of the various concentrations of the test item was observed.
S9-mix: The absence of any bacterial growth in the presence of S9-mix was observed.

Cf Tables of results in attached background material

Conclusions:

Under the test condition, test material is not mutagenic with and without metabolic activation in S. typhimurium (strains TA1535, TA1537, TA98 and TA100) and E. coli WP2(uvrA-) (pKM 101).

Executive summary:

In a reverse gene mutation assay in bacteria, performed according to the OECD Guideline 471 and in compliance with GLP, Salmonella typhimurium, strains TA 1535, TA 1537, TA 98 and TA 100, and Escherichia coli, strain WP2(uvrA-) (pKM 101), were exposed to the test item diluted in ethanol at the following concentrations:

 

Experiment 1: 50, 150, 500, 1500 and 5000 µg/plate in S. typhimurium TA 98, TA 100, TA 1535 & TA 1537 and E. coli WP2 (uvr A-) (pKM101), with and without metabolic activation using plate incorporation method

Experiment 2: 50, 150, 500, 1500 and 5000 µg/plate in S. typhimurium TA 98, TA 100, TA 1535 & TA 1537 and E. coli WP2 (uvr A-) (pKM101), with metabolic activation (pre-incubation method) and without metabolic activation (plate incorporation method).

 

Metabolic activation system used in this test S9 mix (10% v/v S9 fraction): S9 fraction, prepared from liver homogenates of Sprague Dawley rat. Negative, vehicle and positive control groups were also included in mutagenicity tests.

 

Positive controls induced a significant increase in the number of revertant colonies compared to negative controls. There is no significant difference between the number of spontaneous reversions, the number of reversions obtained in the positive controls (without and with metabolic activation), and the mean of corresponding experimental "historical" values obtained in the laboratory.

 

No precipitate formation and no toxicity were observed during the main test. There was no evidence of any increase in the number of revertant colonies in the presence of the test item stock solution and dilutions (50, 150, 500, 1500 and 5000 µg/plate) without and with metabolic activation in S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and in E. coli WP2(uvrA-) (pKM 101). Results were confirmed in an independent experiment.

 

Under the test condition, test material is not mutagenic with and without metabolic activation in S. typhimurium (strains TA1535, TA1537, TA98 and TA100) and E. coli WP2(uvrA-) (pKM 101).

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

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Table 7.6/1: Summary of genotoxicity tests

Test n°	Test / Guideline Reliability	Focus	Strains tested	Metabolic activation	Test concentration	Statement
1   LEMI, 2017	Ames Test (OECD 471) K, rel. 1	Gene mutation	TA 1535, TA 1537, TA 98, TA 100 E. coli WP2 (uvr A-) (pKM101)	-S9 +S9	Up to limit concentration	-S9 : non mutagenic +S9 : non mutagenic

Gene mutation Assays (Tests n° 1):

A Bacterial Reverse mutation Assay (Ames test) was performed according to OECD guideline No. 471 with the substance (Test n°1, see Table 7.6/1). No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains under the test condition, with any dose of the substance, either in the presence or absence of metabolic activation. The substance does not induce gene mutations in bacteria 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 classification or non-classification

Harmonized classification:

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

Self-classification:

Based on the available data, no additional classification is proposed regarding germ cell mutagenicity according to the Regulation (EC) No. 1272/2008 (CLP) and to the Globally Harmonised System of classification and labelling of chemicals (GHS).