[No public or meaningful name is available]

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

An in vitro gene mutation study in bacteria was performed according to OECD Guideline 471 (GLP, Klimisch 1).

The test item is considered to be non-mutagenic in this bacterial reverse mutation assay.

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:

October 25, 2021 to January 17, 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 21 July 1997, corrected 26 June 2020

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

1998

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

dated May 30, 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

E. coli WP2 uvr A

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9 liver microsomal fraction, obtained from Trinova Biochem GmbH, Gießen, Germany.

Test concentrations with justification for top dose:

0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 µL/plate

The test item concentrations to be applied in the main experiments were chosen according to the results of the pre-experiment. 5.0 µL/plate was selected as the maximum concentration. The concentration range covered two logarithmic decades.

Vehicle / solvent:

The test item was dissolved in A. dest. and diluted prior to treatment.

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other:

Details on test system and experimental conditions:

Preparation of Bacteria

Samples of each tester strain were grown by culturing for 12 h at 37 °C in S. typhimurium medium (Nutrient Broth) and E. coli medium (Luria Bertani), respectively, to the late exponential or early stationary phase of growth (approx. 109 cells/mL).
The S. typhimurium medium (Nutrient Broth) contains per litre of purified water:
8 g Nutrient Broth
5 g NaCl
The E. coli medium (Luria Bertani) contains per litre of purified water:
10 g tryptone
10 g NaCl
5 g yeast extract
A solution of 125 µL ampicillin (10 mg/mL) (TA98, TA100, E. coli WP2 uvrA (pKM101)) was added in order to retain the phenotypic characteristics of the strain.

Agar Plates

The Vogel-Bonner Medium E agar plates with 2% glucose used in the Ames Test were prepared by Eurofins Munich. Quality controls were performed.
Vogel-Bonner-salts contain per litre of purified water:
10 g MgSO4 x 7 H2O
100 g citric acid
175 g NaNH4HPO4 x 4 H2O
500 g K2HPO4
Sterilisation was performed for 20 min at 121 °C in an autoclave.
Vogel-Bonner Medium E agar plates contain per litre of purified water:
15 g Agar Agar
20 mL Vogel-Bonner salts
50 mL glucose-solution (40%)
Sterilisation was performed for 20 min at 121 °C in an autoclave.

The overlay agar contains per litre of purified water:
S. typhimurium:
7.0 g Agar Agar
6.0 g NaCl
10.5 mg L-histidine x HCl x H2O
12.2 mg biotin
E. coli:
7.0 g Agar Agar
6.0 g NaCl
10.2 mg tryptophan
Sterilisation was performed for 20 min at 121 °C in an autoclave.

Pre-Experiment for Toxicity
The toxicity of the test item was determined with tester strains TA98 and TA100 in a pre-experiment. Eight concentrations were tested for toxicity and induction of mutations with three plates each. The experimental conditions in this pre-experiment were the same as described below for the main experiment I (plate incorporation test).
Toxicity may be detected by a clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control.
The test item was tested in the pre-experiment at the following concentrations:
0.00316, 0.0100, 0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 µL/plate

Exposure Concentrations
The test item concentrations to be applied in the main experiments were chosen according to the results of the pre-experiment. 5.0 µL/plate was selected as the maximum concentration. The concentration range covered two logarithmic decades. Two independent experiments were performed at the following concentrations:
0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 µL/plate
As the results of the pre-experiment were in accordance with the criteria of validity (10.8), these were reported as a part of the main experiment I.

Experimental Performance
For the plate incorporation method, the following materials were mixed in a test tube and poured over the surface of a minimal agar plate:
100 µL Test solution at each dose level, solvent or negative control or reference mutagen solution (positive control),
500 µL S9 mix (for testing with metabolic activation) or S9 mix substitution buffer (for testing without metabolic activation),
100 µL Bacteria suspension (cf. Preparation of Bacteria, pre-culture of the strain),
2000 µL Overlay agar.
For the pre-incubation method 100 µL of the test item-preparation is pre-incubated with the tester strains (100 µL) and sterile buffer or the metabolic activation system (500 µL) for 60 min at 37 °C prior to adding the overlay agar (2000 µL) and pouring onto the surface of a minimal agar plate.
For each strain and dose level, including the controls, three plates were used.
After solidification the plates were inverted and incubated at 37 °C for at least 48 h in the dark.

Data Recording
The colonies were counted using a ProtoCOL counter (Meintrup DWS Laborgeräte GmbH). If precipitation of the test item precluded automatic counting the revertant colonies were counted by hand. In addition, tester strains with a low spontaneous mutation frequency like TA1535 and TA1537 were counted manually.

Evaluation of Cytotoxicity
Cytotoxicity can be detected by a clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control.

Evaluation criteria:

Evaluation of Mutagenicity
The Mutation Factor is calculated by dividing the mean value of the revertant counts by the mean values of the solvent control (the exact and not the rounded values are used for calculation).
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 (pKM101) the number of reversions is at least twice as high
- if in tester strains TA1535 and TA1537 the number of reversions is at least three times higher
as compared to the reversion rate of the solvent control.
According to the OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.
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.

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

True 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

True negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

#1 - #2: Pre-Experiment
#3 - #7: Experiment I
#8 - #12: Experiment II

Conclusions:

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used.
Therefore, the test item is considered to be non-mutagenic in this bacterial reverse mutation assay.

Executive summary:

The test item 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 TA98, TA100, TA1535, TA1537 and tester strain E. coli WP2 uvrA (pKM101).

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:

0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 µL/plate

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

No toxic effects of the test item were noted in any of the five tester strains used up to the highest dose group evaluated with and without metabolic activation in experiment I and II.

No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with the test item at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II.

All criteria of validity were met.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Based on the available data, the substance does not need to be classified and labelled according to Regulation (EC) No.1272/2008 (CLP).