[No public or meaningful name is available]

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Test article in the TCH2O vehicle, was tested in a Bacterial Reverse Mutation Assay according to OECD Guideline 471. The test article at 50 to 5000 μg/plate, with or without S9, did not cause a significant increase or a dose-dependent increase of the number of revertants of any bacterial tester strain, indicating that the test article is negative for mutagenicity in the 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:

From 20 Dec 2016 to 15 Feb 2017

Reliability:

1 (reliable without restriction)

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

Species / strain / cell type:

S. typhimurium TA 97a

Species / strain / cell type:

S. typhimurium TA 98

Species / strain / cell type:

S. typhimurium TA 100

Species / strain / cell type:

S. typhimurium TA 1535

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: An activation buffer containing 10% S9 obtained from the livers of Aroclor 1254-treated adult Sprague Dawley® rats was prepared according to the manufacturer’s (Moltox) instructions.
- method of preparation of S9 mix
- concentration or volume of S9 mix and S9 in the final culture medium: For the activation portion of the test, 0.5 ml of S9 mixture was added last.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability)

Test concentrations with justification for top dose:

Five concentrations (50, 100, 500, 1000 and 5000 μg/plate)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Tissue culture water (TCH2O)
- Justification for choice of solvent/vehicle: Prior to the cytotoxicity screen, solubility of the test article was checked at 50 mg/ml in TCH2O, DMSO, 95% ethanol, and acetone. The test article was freely soluble in TCH2O, but was not soluble in the other vehicles tested. The Study Director, in consultation with the Sponsor, chose TCH2O as the vehicle for the study.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other:

Remarks:

All bacterial strains with exogenous metabolic activation (S9)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other:

Remarks:

S. typh. TA-97a without S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other:

Remarks:

S. typh. TA-98 without S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

S. typh. TA-100 and TA-1535, without S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

E. coli WP2 uvrA without S9

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): triplicate
- Number of independent experiments: 1

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable):
- Test substance added in agar (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable:
- Exposure duration/duration of treatment: The plates were incubated at 37±2°C for 48 to 72 hours.
- Harvest time after the end of treatment (sampling/recovery times): 72 hours
Revertant Colony Count:
Counting of the revertants per plate was performed using an AlphaImager™ 2200 (Alpha Innotech
Corporation, San Leandro, CA) fluorescence imager. Proper function of the imager was verified against a standard template (e.g. high (1000), medium (100) and low (10) counts) prior to each daily use. The number of revertants was recorded, along with observations of cytotoxicity. Routine examination (under a light microscope) of the bacterial background lawn was used to determine cytotoxicity of the test article. The plates were also examined visually for test article precipitate.

Evaluation criteria:

Plates were scored based on the number of revertant colony-forming units present per plate. The
number of revertants of each test article plate were averaged and plotted versus concentration of the test article. The mean number of revertants of each dose was divided by the mean for the vehicle control value to obtain a ratio to vehicle. In evaluating the data, cytotoxicity of the test article as well as quality checks of the assay were taken into account.
In general, a 2-fold increase with or without metabolic activation is considered a positive response. Doserelated increases approaching a 2-fold increase are deemed equivocal.
A negative result is determined by the absence of a dose-related increase in all five tester strains, again taking into account cytotoxicity of the test article as well as the quality checks of the assay.
Positive results from the bacterial reverse mutation test indicate that the material induces point mutations by base substitutions or frame shifts in the genome of either Salmonella typhimurium and/or Escherichia coli. Negative results indicate that under the test conditions, the test material is not mutagenic in the tested strains.

Key result

Species / strain:

S. typhimurium TA 97a

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle 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

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

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

Positive controls validity:

valid

Key result

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

Positive controls validity:

valid

Additional information on results:

Sterility Test:
Solutions and reagents used in the assay were tested for sterility in the main assay. Samples were
added to minimal glucose agar plates and incubated at 37 ± 2°C for 48 to 72 hours. No contaminating microorganisms were detected in any of the reagents used in the assay. The sterility test passed the quality check.
Vehicle Controls:
All vehicle controls passed the quality check.
Positive Controls:
All positive controls passed the quality check.
Main Assay:
No diminution or clearing of the bacterial background lawn was observed, indicating no or minimal cytotoxicity of the test article under test conditions. There was no significant increase or dose-dependent increase of the number of revertants in any tester strain treated with the test article in the presence or absence of S9. All positive and negative control values were within acceptable ranges, and all criteria for a valid study were met.

Conclusions:

Under test conditions, test article was not mutagenic in the Bacterial Reverse Mutation Assay.

Executive summary:

Test article in the TCH2O vehicle, was tested in a Bacterial Reverse Mutation Assay according to OECD Guideline 471. The test article at 50 to 5000 μg/plate, with or without S9, did not cause a significant increase or a dose-dependent increase of the number of revertants of any bacterial tester strain, indicating that the test article is negative for mutagenicity in the Bacterial Reverse Mutation Assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Ames test, OECD 471, negative.
Therefore in accordance with Regulation (EC) No. 1272/2008 Table 3.5.1, the test substance should not be classified as germ cell mutagens.