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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
The substance is considered to be not genotoxic.
Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10.10.2013 - 25.04.2014
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
Target gene:
Histidine (Salmonella strains) or Tryptophan (E. coli strain)
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
- Source: Dr. Bruce Ames, Department of Biochemistry, University of California (Berkeley, CA).
- Strains TA98 and TA100: contain pKM101 plasmid to further increase sensitivity to some mutagens. Suggested mechanism: modification of an existing bacterial DNA repair polymerase complex involved with the mismatch-repair process.
- Strains TA98 and TA1537: reverted from histidine dependence to histidine independence by frameshift mutagens.
- Strains TA100, TA1535: reverted from auxotrophy to prototrophy by base substitution mutagens.
Additional strain / cell type characteristics:
other: Additional mutations: uvrB and rfa
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
- Source: The National Collection of Industrial Bacteria, Torrey Research Station, Scotland (United Kingdom).
- Reverted from auxotrophy to prototrophy by base substitution mutagens.
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Initial mutagenicity assay: 5, 16, 50, 160, 500, 1600 and 5000µg/plate.
Confirmatory mutegenicity assay: 78, 156, 313, 625, 1250 and 2500µg/plate.
Vehicle / solvent:
DMSO
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
Dimethylsulfoxide (DMSO) purity ≥ 99.9%
Positive controls:
yes
Remarks:
for TA98 strain without S9 activation
Positive control substance:
2-nitrofluorene
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
Dimethylsulfoxide (DMSO) purity ≥ 99.9%
Positive controls:
yes
Remarks:
for TA1537 strain without S9 activation
Positive control substance:
other: ICR-191
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
Dimethylsulfoxide (DMSO) purity ≥ 99.9%
Positive controls:
yes
Remarks:
for TA100, TA1535 strains without S9 activation
Positive control substance:
sodium azide
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
Dimethylsulfoxide (DMSO) purity ≥ 99.9%
Positive controls:
yes
Remarks:
for WP2uvrA strain without S9 activation
Positive control substance:
4-nitroquinoline-N-oxide
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
Dimethylsulfoxide (DMSO) purity ≥ 99.9%
Positive controls:
yes
Remarks:
for TA98 strain with S9 activation
Positive control substance:
benzo(a)pyrene
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
Dimethylsulfoxide (DMSO) purity ≥ 99.9%
Positive controls:
yes
Remarks:
for TA100, TA1535, TA1537 and WP2uvrA strains with S9 activation
Positive control substance:
other: 2-aminoanthracene
Details on test system and experimental conditions:
DURATION
- Preincubation period: overnight
- Exposure duration: 52+-4hours
- Expression time (cells in growth medium): not defined

TEST SYSTEM
- Culture Density: target OD650 (1:4)=0.4 to 0.6; ≥ 1x10E9 cells/mL.

SELECTION AGENT (mutation assays): not defined
NUMBER OF REPLICATIONS: at least 2
NUMBER OF CELLS EVALUATED: not defined
DETERMINATION OF CYTOTOXICITY: relative total growth
Evaluation criteria:
A. Criteria of test validity: (1) Histidine/Tryptophan Auxotrophy: Salmonella tester strains must have ability to grow on minimal bottom agar plates supplemented with histidine/biotin. Tester strain WP2uvrA must be able to grow on bottom agar plates supplemented with Tryptophan/biotin. (2) rfa Cell Wall Mutation: Tester strains must have sensitivity to crystal violet, while tester strain WP2uvrA must exhibit resistance to crystal violet. (3) pKM101 Plasmid: Tester strains TA98 and TA100 must demonstrate resistance to ampicillin, while tester strains TA1535, TA1537 and WP2uvrA must be sensitive. (4) uvrA and uvrB Mutation: All tester strains must demonstrate their sensitivity to ultraviolet light. B. Characteristic Number of Spontaneous Revertants: average revertants/plate of the vehicle control cultures must be within the acceptance limits based upon historical data and published reports. (TA98: 8 - 60; TA100: 60 - 240; TA1535: 4 - 45; TA1537: 2 - 25; WP2uvrA: 5 - 40) C. Positive Controls: must induce a >=3-fold increase in revertants/plate compared to the concurrent vehicle controls. D. Criteria for a Positive Response: dose-dependent increase in revertant frequency is >= 2.0-fold vehicle control values for tester strains TA98, TA100, and WP2uvrA, or >=3.0-fold for tester strains TA1535 and TA1537. E. Criteria for a Negative Response: no dose-dependent increases
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at concentration 2500 µg/plate
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
at any concentration tested
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
- Cytotoxicity: in the absence of S9 all Salmonella tester strains ≥1600 µg/plate, for WP2uvrA at 5000 µg/plate in the absence of S9. In the presence of S9 very thin background lawn was observed at 5000 µg/plate in all tester strains. No precipitate was observed at any tested concentration.
- Genotoxicity: no increases in the mean number of revertant colonies at any tested concentration in the presence or absence of S9
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'. Remarks: Main mutagenicity assay
Conclusions:
Interpretation of results: negative

The test item was negative in the Bacterial Reverse Mutation Assay in the presence and absence of S9 metabolic activation under the conditions of this protocol.
Executive summary:

In the current study the ability of the test substance to induce reverse mutations in four strains of Salmonella typhimurium, TA98, TA100, TA1535, TA1537, and one Escherichia coli strain WP2uvrA, in the presence or absence of an exogenous mammalian activation system (S9) containing microsomal enzymes. The study was performed according to OECD 471 guideline and under GLP without significant deviations.

The test substance was evaluated in an initial and an confirmatory Ames assay (main test) using plate incorporation method. All validity criteria were met. The number of revertant colonies produced by the positive and vehicle controls were within the acceptable historical range.

In the initial mutagenicity assay all tester strains were evaluated at dose levels of 5, 16, 50, 1600 and 5000 µg/plate with and without S9. No precipitation in presence or absence of S9 fraction was observed.

Cytotoxicity was observed in all Salmonella typhimurium strains starting at a concentration of 1600 µg/plate in the absence of S9. In the Escheria coli strain cytotoxicity was observed at 5000 µg/plate in the absence of S9. In the presence of S9, cytotoxicity was observed in all strains at a concentration of 5000 µg/plate. No genotoxicity was observed in any of the strains at any concentration tested.

Based on the results of the initial data, an independent confirmatory mutagenicity assay (main test) was conducted in all tester strains at dose levels of 78.0, 156, 313, 625, 1250, and 2500 µg/plate with and without S9. No precipitate was observed at any tested concentration in the presence or absence of S9.

Cytotoxicity was observed at 2500 µg/plate in all Salmonella typhimurium strains in the presence and absence of S9. In the Escheria coli strain cytotoxicity was not observed in the presence of S9, not in the absence of S9. No genotoxicity was observed in any of the strains at any concentration tested.

In conclusion, the results indicate that the test item is not genotoxic towards bacteria cells under the current conditions.

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

Additional information

Additional information from genetic toxicity in vitro:

For this endpoint, one study is available assessing the ability of the substance to induce gene mutations in bacteria.

In the study four Salmonella typhimurium strains (TA98, TA100, TA1535 and TA1537) and 1 Escheria coli strain (WP2uvrA), in the presence and absence of an exogenous activation system (S9).

In an initial mutagenicity test the tested doses were 5, 16, 50, 160, 500, 1600 and 5000 µg/plate. Cytotoxicity was observed in all Salmonella typhimurium strains starting at a concentration of 1600 µg/plate in the absence of S9. In the Escheria coli strain cytotoxicity was observed at 5000 µg/plate in the absence of S9. In the presence of S9, cytotoxicity was observed in all strains at a concentration of 5000 µg/plate. No genotoxicity was observed in any of the strains at any concentration tested.

In the confirmatory mutagenicity assay the dose levels were 78, 156, 313, 625, 1250 and 2500 µg/plate. Cytotoxicity was observed at 2500 µg/plate in all Salmonella typhimurium strains in the presence and absence of S9. In the Escheria coli strain cytotoxicity was not observed in the presence of S9, not in the absence of S9. No genotoxicity was observed in any of the strains at any concentration tested.

All validity criteria were met and therefore, the results indicate that the test item is not genotoxic towards bacteria cells under the current conditions.


Justification for selection of genetic toxicity endpoint
The study is well documented and according to GLP and internationally accepted guidelines.

Justification for classification or non-classification

According to section 3.5.2.3. of Annex I of the CLP Regulation N° 1272/2008 the test results of in vitro genotoxic test shall be considered for the classification of the test item for mutagenicity. As both the initial and the confirmatory mutagenicity test are negative, the test substance needs not to be classified as mutagenic based on the available information.