Tar, wood

Endpoint summary

Key value for chemical safety assessment

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

Reference 1

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

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:

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).

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

yes

Type of assay:

comet assay

Specific details on test material used for the study:

Name: Scansmoke R909
Batch No: 03/2004

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

3.7.2C cells
ATCC CRL-9518
lot 1661603

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

Test 1
-S9: 0, 250, 400, 550, 650, 750 and 850 µg/mL
+S9: 0, 500, 750, 1000, 1200, 1300 and 1400 µg/mL

Test 2
-S9: 0, 250, 350, 450, 550, 600 and 650 µg/mL
+S9: 0, 500, 750, 1000, 1200, 1300 and 1400 µg/mL

Vehicle / solvent:

- Vehicle used: RPMI5 medium

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

cyclophosphamide

Details on test system and experimental conditions:

DURATION
Test 1: Exposure during 3 h with and without metabolic activation
Test 2: Exposure during 3 h with and 24 h without metabolic activation

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

dose associated at 450 to 650 µg/mL after 3 h treatment

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Positive controls validity:

valid

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

dose associated at 1000 to 1400 µg/mL after 24 h treatment

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Positive controls validity:

valid

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Specific details on test material used for the study:

Name: Scansmoke R909
Batch No: 03/2004

Species:

mouse

Strain:

NMRI

Details on species / strain selection:

Strain: CRL: NMRI BR mice

Sex:

not specified

Route of administration:

oral: unspecified

Vehicle:

distilled water

Dose / conc.:

500 mg/kg diet

Dose / conc.:

1 000 mg/kg diet

Dose / conc.:

2 000 mg/kg diet

Control animals:

yes

Positive control(s):

Cyclophosphamide (ip. administration)

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Executive summary:

No increases in the frequency of micronucleated polychromated erythrocytes (MCPE) in male and female mice at either 24 or 48 hours after treatment compared to the vehicle control.

Considerable differences in the ratio of polychromatic and normochromatic erythrocytes were not found after the treatment.

Biologically significant depression of PCE:NCE ratio was not observed in the study. The positive control showed significantly increased MCPE numbers compared to the control.

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.