[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 vehicle DMSO, was tested in a Bacterial Reverse Mutation Assay according to OECD
Guideline 471 under GLP.
Under test conditions, test article was not mutagenic 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)

Rationale for reliability incl. deficiencies:

guideline study

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:

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.

Test concentrations with justification for top dose:

50, 100, 500, 1000 and 5000 μg/plate

Vehicle / solvent:

Dimethyl Sulfoxide (DMSO)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene (2AA)

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:

methylmethanesulfonate

Remarks:

E. coli WP2 uvrA without S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Acridine, 6-chloro-9-(3-((2-chloroethyl)amino)propyl)amino-2-methoxy, dihydrochloride (ICR-191)

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:

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:

other: Daunomycin (DM)

Remarks:

S. typh. TA-98 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: 48-72 hours
- Harvest time after the end of treatment (sampling/recovery times):

METHODS FOR MEASUREMENT OF CYTOTOXICITY and GENOTOXICIY
- Method, e.g.: 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 determ
ine cytotoxicity of the test article.

Evaluation criteria:

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, ag
ain 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, but tested up to precipitating concentrations

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, but tested up to precipitating concentrations

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, but tested up to precipitating concentrations

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, but tested up to precipitating concentrations

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, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

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 vehicle DMSO, was tested in a Bacterial Reverse Mutation Assay according to OECD
Guideline 471 under GLP.
Under test conditions, test article was not mutagenic 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.