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Genetic toxicity in vitro

Description of key information
In conclusion, the test item Tar wood has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.
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:
07.05.2014 - 16.05.2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
Strain: TA98Genotype: trp./his mutation his D3052, type of mutation: FrameshiftMutations: cell wall: rfa / DNA-repair: uvrBMain DNA target: GCStrain: TA100Genotype: trp./his mutation hisG46, type of mutation: Base pair substitutionMutations: cell wall: rfa / DNA-repair: uvrBMain DNA target: GCStrain: TA1535Genotype: trp./his mutation his hisG46 type of mutation: Base pair substitutionMutations: cell wall: rfa / DNA-repair: uvrBMain DNA target: GCStrain: TA1537Genotype: trp./his mutation his D3052, type of mutation: FrameshiftMutations: cell wall: rfa / DNA-repair: uvrBMain DNA target: GCStrain: WP2uvrAGenotype: trp./his mutation his trpE, type of mutation: Base pair substitutionMutations: cell wall: + / DNA-repair: uvrAMain DNA target: AT
Species / strain / cell type:
S. typhimurium TA 98
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
other: Mutation in hisD3052, rfa, uvrB and pKM101
Species / strain / cell type:
S. typhimurium TA 100
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
other: Mutation in hisG46, rfa, uvrB and pKM101
Species / strain / cell type:
S. typhimurium TA 1535
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
other: Mutation in hisG46, rfa and uvrB
Species / strain / cell type:
S. typhimurium TA 1537
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
other: Mutation in hisC3076, rfa and uvrB
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
other: Mutation in trpE and uvrA
Metabolic activation:
with and without
Metabolic activation system:
post mitochondrial supernatant (S9) prepared from livers of Phenobarbital/β-naphthoflavone-induced rats
Test concentrations with justification for top dose:
5000; 1581; 500; 158; 50 and 15.8 µg/plate
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-1,2-phenylene-diamine (NPD)
Remarks:
Dose quantity/plate: 4 µg; Strain: Salmonella TA98
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Sodium azide (SAZ)
Remarks:
Dose quantity/plate: 2 µg; Strain: Salmonella TA100 and TA1535
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Dose quantity/plate: 50 µg; Strain: Salmonella TA1537
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Dose quantity/plate: 2 µL; Strain: E.coli WP2uvrA
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (2AA)
Remarks:
Dose quantity/plate: 2 µg; Strain: all of Salmonella strains
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (2AA)
Remarks:
Dose quantity/plate: 50 µg; Strain: E.coli strain
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative 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
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: other: plate incorporation method and pre-incubation method
Remarks:
Migrated from field 'Test system'.

see attached report for more information

Conclusions:
In conclusion, the test item Tar wood has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.
Executive summary:

The test item Tar wood was tested with regard to a potential mutagenic activity using the Bacterial Reverse Mutation Assay.

The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9) prepared from livers of phenobarbital/β-naphthoflavone-induced rats.

The study included a Preliminary Solubility Test, a Preliminary Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Test), and a Confirmatory Mutation Test (Pre-Incubation Test).

In the Range Finding Test as well as in the Initial Mutation Test the plate incorporation method was used.

Based on the results of the Solubility Test and the Range Finding Test the test item was dissolved in Dimethyl sulfoxide in a concentration of 100 mg/mL.

Based on the results of the preliminary Range Finding Test the following concentrations of the test item were prepared and used in the Initial and Confirmatory Mutation Tests:

5000; 1581; 500; 158; 50 and 15.8 μg/plate.

In the Initial and Confirmatory Mutation the test item concentrations, including the controls (untreated, vehicle and positive reference) were tested in triplicate.

No substantial, biological relevant increases were observed in revertant colony numbers of any of the five test strains following treatment with Tar wood at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments. Sporadic increases in revertant colony numbers compared to the vehicle control values were observed in both independently performed main experiments.

However, there was no tendency of higher mutation rates with increasing concentrations beyond the generally acknowledged border of biological relevance in the performed experiments. See details in the Section 9. RESULTS.

The highest revertant colony number increase over the spontaneous rate of the vehicle control plates was observed in the Confirmatory Mutation Test (Pre-Incubation Test) in S. typhimurium TA1537 at 158 μg/plate, without metabolic activation (-S9 Mix). The mutation rate was: 2.82*. This relatively high value was unique, remained within the laboratory’s historical control data range and remained below the genotoxicological threshold for being positive.

Inhibitory effect of the test item was obtained in the Confirmatory Mutation Test following the pre-incubation procedure. The inhibitory effect of the test item was indicated by lower revertant colony numbers than the revertant colony numbers of the vehicle controls (that were often below the corresponding historical control data ranges) and affected background lawn development: absent, reduced or slightly reduced background lawn.

The revertant colony numbers of vehicle control (Dimethyl sulfoxide, DMSO) plates with and without S9 Mix demonstrated the characteristic mean number of spontaneous revertants and were in line with the corresponding historical control data ranges.

The spontaneous revertant colony numbers of the DMSO vehicle control plates were slightly lower (the actual average number: 16) than the characteristic mean numbers agreed with the actual historical control data range (historical control range: 17-49) in the Initial Mutation Test in Salmonella typhimurium TA98 in the presence of metabolic activation (+S9 Mix). The lower counts were evaluated as acceptable without any influence on the final conclusion of the study.

The reference mutagen treatments (positive controls) showed the expected, biological relevant increases in induced revertant colonies in all experimental phases, in all tester strains.

The reported data of this mutagenicity assay show (see Appendix I to IV) that under the experimental conditions applied, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

* Mutation rate (MR): The mutation rate is the quotient of the mean revertant of test item treatment and the mean revertant of the vehicle control. In the case of S. typhimurium TA1537 a biologically relevant increase (positive result) is when the number of reversions is at least three times higher than the reversion rate of the vehicle control (Mutation rate ≥ 3.00).

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

Additional information

Additional information from genetic toxicity in vitro:

The test item Tar wood was tested with regard to a potential mutagenic activity using the Bacterial Reverse Mutation Assay.

The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9) prepared from livers of phenobarbital/β-naphthoflavone-induced rats.

The study included a Preliminary Solubility Test, a Preliminary Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Test), and a Confirmatory Mutation Test (Pre-Incubation Test).

In the Range Finding Test as well as in the Initial Mutation Test the plate incorporation method was used.

Based on the results of the Solubility Test and the Range Finding Test the test item was dissolved in Dimethyl sulfoxide in a concentration of 100 mg/mL.

Based on the results of the preliminary Range Finding Test the following concentrations of the test item were prepared and used in the Initial and Confirmatory Mutation Tests:

5000; 1581; 500; 158; 50 and 15.8 μg/plate.

In the Initial and Confirmatory Mutation the test item concentrations, including the controls (untreated, vehicle and positive reference) were tested in triplicate.

No substantial, biological relevant increases were observed in revertant colony numbers of any of the five test strains following treatment with Tar wood at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments. Sporadic increases in revertant colony numbers compared to the vehicle control values were observed in both independently performed main experiments.

However, there was no tendency of higher mutation rates with increasing concentrations beyond the generally acknowledged border of biological relevance in the performed experiments. See details in the Section 9. RESULTS.

The highest revertant colony number increase over the spontaneous rate of the vehicle control plates was observed in the Confirmatory Mutation Test (Pre-Incubation Test) in S. typhimurium TA1537 at 158 μg/plate, without metabolic activation (-S9 Mix). The mutation rate was: 2.82*. This relatively high value was unique, remained within the laboratory’s historical control data range and remained below the genotoxicological threshold for being positive.

Inhibitory effect of the test item was obtained in the Confirmatory Mutation Test following the pre-incubation procedure. The inhibitory effect of the test item was indicated by lower revertant colony numbers than the revertant colony numbers of the vehicle controls (that were often below the corresponding historical control data ranges) and affected background lawn development: absent, reduced or slightly reduced background lawn.

The revertant colony numbers of vehicle control (Dimethyl sulfoxide, DMSO) plates with and without S9 Mix demonstrated the characteristic mean number of spontaneous revertants and were in line with the corresponding historical control data ranges.

The spontaneous revertant colony numbers of the DMSO vehicle control plates were slightly lower (the actual average number: 16) than the characteristic mean numbers agreed with the actual historical control data range (historical control range: 17-49) in the Initial Mutation Test in Salmonella typhimurium TA98 in the presence of metabolic activation (+S9 Mix). The lower counts were evaluated as acceptable without any influence on the final conclusion of the study.

The reference mutagen treatments (positive controls) showed the expected, biological relevant increases in induced revertant colonies in all experimental phases, in all tester strains.

.

Justification for classification or non-classification

The reported data of this mutagenicity assay show (see Appendix I to IV in the report) that under the experimental conditions applied, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

In conclusion, the test item Tar wood has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.