[No public or meaningful name is available]

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In a bacterial reverse mutation assay according to OECD guideline 471, the test item did not induce mutations by base pair changes or frameshifts in the genome of the strains used under the experimental conditions reported. Therefore, the test item is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli 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:

2019-04-16 to 2019-05-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

The Salmonella typhimurium histidine (his) and the Escherichia coli tryptophane (trp) reversion system measures his- → his+ and trp- → trp+ reversions, respectively. The strains are constructed to differentiate between base pair (E.coli WP2, TA 1535, TA 100) and frameshift (TA 1537, TA 98) mutations.

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- Source of S9: Phenobarbital/β-naphthoflavone induced rat liver S9
- Method of preparation of S9 mix: The S9 was prepared and stored according to the currently valid version of the SOP for rat liver S9 preparation.
- Concentration or volume of S9 mix and S9 in the final culture medium: 500 µL of S9 mix (containing 50 µL S9) in a final culture medium of 2700 µL (protein concentration of S9: 33.3 mg/mL)
- Quality controls of S9: Each batch of S9 was routinely tested for its capability to activate the known mutagens benzo[a]pyrene and 2-aminoanthracene in the Ames test.

Test concentrations with justification for top dose:

Experiment I: 3 – 5000 μg/plate
Experiment II:
- S. typhimurium TA 1537 and E.coli WP2 uvrA: 10, 33, 100, 333,1000, 2500 and 5000 μg/plate
- Remaining strains: 33, 100, 333, 1000, 2500, and 5000 μg/plate

Justification for top dose: Maximum recommended concentration for the test according to OECD guideline 471

Vehicle / solvent:

- Vehicles/solvents used: Deionized water (test item, sodium azide, methyl methane sulfonate), DMSO (4-nitro-o-phenylene-diamine, 2-aminoanthracene)

- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene (2-AA) 4-nitro-o-phenylene-diamine (4-NOPD)

Remarks:

-S9:
- Sodium azide: 10 μg/plate (TA 1535, TA 100)
- 4-NOPD: 10 μg/plate (TA 98), 50 μg/plate (TA 1537)
- Methyl methane sulfonate: 2.0 μL/plate (WP2 uvrA)
+S9:
- 2-AA: 2.5 μg/plate (S. typhimurium strains) and 10.0 μg/plate (WP2 uvrA)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration Triplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 10^8-10^9 cells/mL
- Test substance added in: Plate incorporation (experiment I), preincubation (experiment II)

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 60 minutes
- Exposure duration/duration of treatment: 48 hours at 37°C in the dark

METHODS FOR MEASUREMENT OF CYTOTOXICITY
Reduction in the number of revertants below the induction factor of 0.5

METHODS FOR MEASUREMENTS OF GENOTOXICIY
Increase in revertant colony numbers

Rationale for test conditions:

The chosen concentrations included two logarithmic decades, thus were considered to cover a wide concentration range. The top dose of 5000 µg/plate and the number of replicates were chosen according to OECD guideline 471.

Evaluation criteria:

- A test item is considered as a mutagen if a biologically relevant increase in the number of revertants of twofold or above (strains TA 98, TA 100, and WP2 uvrA) or threefold or above (strains TA 1535 and TA 1537) the spontaneous mutation rate of the corresponding solvent control is observed

- A dose dependent increase is considered biologically relevant if the threshold is reached or exceeded at more than one concentration

- An increase of revertant colonies equal or above the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment

- A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment

- Whenever the colony counts remain within the historical range of negative and solvent controls, such an increase is not considered biologically relevant

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

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, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

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, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: The test item precipitated in the overlay agar in the test tubes in experiment II at 5000 μg/plate. No precipitation of the test item was observed in the overlay agar on the incubated agar plates in both experiments.

STUDY RESULTS
- Concurrent vehicle negative and positive control data: See "Attached background material"

For all test methods and criteria for data analysis and interpretation
- Concentration-response relationship: No concentration-response relationship observed

Ames test:
- Signs of toxicity: Reduced background growth (only in experiment II for TA1537 (1000-5000 µg/plate), TA 100 (2500-5000 µg/plate) with and without metabolic activation and for WP2 uvrA with metabolic activation (5000 µg/plate)) and reduction in the number of revertants (experiment I: WP2 uvrA without metabolic activation (5000 µg/plate) ; experiment II: TA1537 with and without and WP2 uvrA without metabolic activation (5000 µg/plate))
- Individual plate counts: See "Attached background material"
- Mean number of revertant colonies per plate and standard deviation: See "Attached background material"

HISTORICAL CONTROL DATA
- Positive historical control data: See "Attached background material"
- Negative (solvent/vehicle) historical control data: See "Attached background material"

Conclusions:

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

Executive summary:

This GLP compliant study was performed according to OECD guideline 471. It was conducted to investigate the potential of the test item to induce gene mutations according to the plate incorporation test (experiment I) and the pre incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

 

Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate

Experiment II:

Strains TA 1537 and WP2 uvrA: 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate

The remaining strains: 33; 100; 333; 1000; 2500; and 5000 μg/plate

 

The test item precipitated in the overlay agar in the test tubes in experiment II at 5000 μg/plate. No precipitation of the test item was observed in the overlay agar on the incubated agar plates in both experiments. The plates incubated with the test item showed normal background growth up to 5000 μg/plate with and without S9 mix in all strains used in experiment I. In experiment II reduced background growth was observed on the plates incubated with the test item in strains TA 1537 and TA 100, both with and without S9 mix and in strain WP2 uvrA without S9. Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were observed in experiment I in strain WP2 uvrA without S9 mix and in experiment II in strains TA 1537 with and without S9 mix and WP2 uvrA without S9 mix. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

The study was performed according to OECD guideline 471. It was conducted to investigate the potential of the test item to induce gene mutations according to the plate incorporation test (experiment I) and the pre incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

 

Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate

Experiment II:

Strains TA 1537 and WP2 uvrA: 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate

The remaining strains: 33; 100; 333; 1000; 2500; and 5000 μg/plate

 

The test item precipitated in the overlay agar in the test tubes in experiment II at 5000 μg/plate. No precipitation of the test item was observed in the overlay agar on the incubated agar plates in both experiments. The plates incubated with the test item showed normal background growth up to 5000 μg/plate with and without S9 mix in all strains used in experiment I. In experiment II reduced background growth was observed on the plates incubated with the test item in strains TA 1537 and TA 100, both with and without S9 mix and in strain WP2 uvrA without S9. Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were observed in experiment I in strain WP2 uvrA without S9 mix and in experiment II in strains TA 1537 with and without S9 mix and WP2 uvrA without S9 mix. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

 

Therefore, the test item is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Justification for classification or non-classification

The available test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Thus, the test item is considered not to be classified for genotoxicity under Regulation (EC) No 1272/2008, as amended for seventeenth time in Regulation (EU) No 2021/849.