Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation in bacteria (OECD 471): not mutagenic

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 Mar 2017 - 13 Apr 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
July 21, 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
Official Journal of the European Union No. L142, 31 May 2008
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: EM97160115
- Expiration date of the lot/batch: 27 January 2018
- Purity test date: 07/07/2016

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: not indicated
- Solubility and stability of the test substance in the solvent/vehicle: not indicated

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Radia MNKE was dissolved in dimethyl sulfoxide.
- Final dilution of a stock liquid: not specified

FORM AS APPLIED IN THE TEST (if different from that of starting material):Radia MNKE was dissolved in dimethyl sulfoxide.

Target gene:
histidine locus
tryptophan locus
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: Tester strains contained additional mutations: rfa: defective lipopolysaccharide cellcoat, gal : mutation in the galactose metabolism, chl : mutation in nitrate reductase, bio : defective biotin synthesis, uvrB: deletion of the ultraviolet-repair B gene)
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Rat liver microsomal enzymes (S9 homogenate)
Test concentrations with justification for top dose:
Dose range: Eight concentrations, 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate were tested in triplicate. The highest concentration of Radia MNKE used in the subsequent mutation assays was the level at which the test item inhibited bacterial growth or the level at which the test item exhibited limited solubility.
First mutagenicity test: 1.7, 5.4, 17, 52, 164 and 512 μg/plate.
Second mutagenicity test: 27 to 492 μg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: ICR-191
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation)
- Cell density at seeding: optical density of bacteria 1.0 ± 0.1 at 700 nm (109 cells/ml)

DURATION
- Preincubation period: none, direct plate incorporation
- Exposure duration: 48 ± 4 h

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies

Rationale for test conditions:
According to the designated guidelines
Evaluation criteria:
A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent vehicle control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent vehicle control.
b) The negative response should be reproducible in at least one follow-up experiment.

A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is greater than two
(2) times the concurrent vehicle control, or the total number of revertants in tester strains TA1535, TA1537, TA98 is greater than three (3) times the concurrent vehicle control.
b) In case a follow up experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up
experiment.
Key result
Species / strain:
other: TA98, TA100, TA1535, and TA1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation:
Dose-range finding test: precipitation of Radia MNKE on the plates was observed at the start of the incubation period at concentrations of 1600 and 5000 μg/plate and at 5000 μg/plate at the end of the incubation period.
First mutagenicity test: at the dose level of 512 μg/plate in the absence of S9-mix in the tester strains TA1535, TA1537, TA98 and TA100 and in the presence of 5% (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98, Radia MNKE did not precipitate on the plates.
Second mutagenicity test: Precipitation of Radia MNKE on the plates was observed at the start of the incubation period at concentrations of 2800 and 5000 μg/plate. At the end of the incubation period, the test item precipitated on the plates at the highest dose level tested in the tester strains TA1535 and TA100 in the presence of S9-mix and in strains TA1537 and WP2uvrA in the absence and presence of S9-mix. In addition, in tester strain TA98 the test item precipitated at dose levels of 154 μg/plate and upwards in the absence of S9-mix.
- Other confounding effects:In the second mutation experiment, no dose level with precipitate or toxicity was tested in the tester strain TA98 in the presence of S9-mix.
Evaluation: Although toxicity was observed at the dose levels of 164 and 512 μg/plate in the first mutation experiment, and precipitation was observed at 492 μg/plate in the tester strains TA1535, TA1537 and TA100 in the second experiment, no toxicity or precipitate was observed at 492 μg/plate in the second mutation experiment. Since clear negative responses were observed in all tester strains tested, and in the first and second mutation experiments the test item was tested up to dose levels with toxicity or precipitate, the lack of a dose level with toxicity or precipitate had no influence on the study.

RANGE-FINDING/SCREENING STUDIES:
In the dose-range finding test, the test item was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. Radia MNKE precipitated on the plates at the dose level of 5000 μg/plate. Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in tester strain TA100 at dose levels of 17 and 164 μg/plate and upwards in the absence and presence of S9-mix, respectively. In tester strain WP2uvrA, no toxicity was observed at any of the dose levels tested.


NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture:
- Indication whether binucleate or mononucleate where appropriate:

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
- Negative (solvent/vehicle) historical control data:

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: [complete, e.g. CBPI or RI in the case of the cytokinesis-block method; RICC, RPD or PI when cytokinesis block is not used]
- Other observations when applicable: [complete, e.g. confluency, apoptosis, necrosis, metaphase counting, frequency of binucleated cells]
Conclusions:
An increase in the number of revertants was not observed in the Salmonella typhimurium Reverse Mutation Assayand the Escherichia coli Reverse Mutation Assay neither with nor without S-9 mix or after the addition of a metabolizing system. Thus, under the experimental conditions chosen here, it is concluded that Radia MNKE (MNK 2-undecanone) is not a mutagenic agent and does not need to be classified for mutagenicity in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).
Executive summary:

The objective of this study was to determine the potential of Radia MNKE and/or its metabolites to induce reverse mutations at the histidine locus in several strains of Salmonella typhimurium (S. typhimurium; TA98, TA100, TA1535, and TA1537), and at the tryptophan locus of Escherichia coli (E. coli) strain WP2uvrA in the presence or absence of an exogenous mammalian metabolic activation system (S9).

In the dose-range finding test, the test item was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA.

Based on the results of the dose-range finding test, the test item was tested in the first mutation assay at a concentration range of 1.7 to 512 μg/plate in the absence of S9-mix in the tester strains TA1535, TA1537, TA98 and TA100 and in the presence of 5% (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98.

In a follow-up experiment of the assay with additional parameters, the test item was tested at a concentration range of 27 to 492 μg/plate in the tester strains TA1535, TA1537, TA98 and TA100 and at 492 to 5000 μg/plate in the tester strain WP2uvrA in the absence and presence of 10% (v/v) S9-mix.

Radia MNKE did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in the tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experiment. The negative and positive controls were valid, indicating that the test conditions were adequate and that the metabolic activation system functioned properly. In conclusion, based on the results of this study it is concluded that Radia MNKE is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The objective of this study was to determine the potential of Radia MNKE and/or its metabolites to induce reverse mutations at the histidine locus in several strains of Salmonella typhimurium (S. typhimurium; TA98, TA100, TA1535, and TA1537), and at the tryptophan locus of Escherichia coli (E. coli) strain WP2uvrA in the presence or absence of an exogenous mammalian metabolic activation system (S9).

In the dose-range finding test, the test item was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA.

Based on the results of the dose-range finding test, the test item was tested in the first mutation assay at a concentration range of 1.7 to 512 μg/plate in the absence of S9-mix in the tester strains TA1535, TA1537, TA98 and TA100 and in the presence of 5% (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98.

In a follow-up experiment of the assay with additional parameters, the test item was tested at a concentration range of 27 to 492 μg/plate in the tester strains TA1535, TA1537, TA98 and TA100 and at 492 to 5000 μg/plate in the tester strain WP2uvrA in the absence and presence of 10% (v/v) S9-mix.

Radia MNKE did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in the tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experiment. The negative and positive controls were valid, indicating that the test conditions were adequate and that the metabolic activation system functioned properly. In conclusion, based on the results of this study it is concluded that Radia MNKE is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Justification for classification or non-classification

An increase in the number of revertants was not observed in the Salmonella typhimurium Reverse Mutation Assay and the Escherichia coli Reverse Mutation Assay neither with nor without S-9 mix or after the addition of a metabolizing system. Thus, under the experimental conditions chosen here, it is concluded that Radia MNKE (MNK 2-undecanone) is not a mutagenic agent and does not need to be classified for mutagenicity in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).