Registration Dossier
Registration Dossier
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EC number: 209-235-5 | CAS number: 562-74-3
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Ames test (2018); GLP; according to OECD 471; strains used: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E.coli WP2 uvrA (+/- S9); 33-5200 µg/plate. All S. typhimurium strains negative, E.coli positive (+/- S9).
Ames test (2019); GLP; according to OECD 471; strains used: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E.coli WP uvrA (+/- S9); 33 -5200 µg/plate. Negative results; no mutagenic potential observed.
Ames (2000); GLP; according to OECD 471; strains used: S. typhimurium strains TA1535, TA97a, TA98, TA100 and TA102 (+/- S9); 0.016 -5 mg/plate; negative results, no mutagenic potential observed.
Micronucleus test (2015); GLP; according to OECD 487; human peripheral blood lymphocytes; concentrations up to 1540 µg/ml; negative results, no clastogenic potential observed.
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- his
- Species / strain / cell type:
- S. typhimurium TA 1535
- Species / strain / cell type:
- S. typhimurium, other: 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 102
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9
- Test concentrations with justification for top dose:
- 0.016-1.6 mg/plate for TA97a
0.016-5 mg/plate for TA98 and TA102
0.016-1.6 mg/plate (without S9) and 0.0.16-5 mg/plate (with S9) for TA100 and TA1535 - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: none given - Untreated negative controls:
- not specified
- Negative solvent / vehicle controls:
- not specified
- Positive controls:
- not specified
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- not specified
- Species / strain:
- S. typhimurium, other: TA97a
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- not specified
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- not specified
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- not specified
- Species / strain:
- S. typhimurium TA 102
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- not specified
- Conclusions:
- Under the conditions of the study, the test material was found to have no mutagenic effects.
- Executive summary:
The mutagenic potential of the test substance was tested in a reverse bacteria mutation assay (Ames) according to OECD 471 in compliance with GLP in 2000. Strains used for the test were S. typhimurium TA 1535, TA 97a, TA 98, TA 100, and TA 102. Strains were treated with the test substance in dimethyl sulfoxide (DMSO) with concentrations ranging from 0.016 mg/plate to 5 mg/plate. TA 97a was treated with 0.016 -1.6 mg/plate, TA 98 and TA 102 were treated with 0.016 -5 mg/plate, and TA 100 and TA 1535 were treated with 0.016 -5 mg/plate. All strains were tested with and without metabolic activation (S9). Neither in the presence nor in the absence of S9 did any tested dose increase the mean number of revertant colonies in any strain tested.
- Endpoint:
- in vitro cytogenicity / micronucleus study
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Reason / purpose for cross-reference:
- reference to same study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian cell micronucleus test
- Species / strain / cell type:
- lymphocytes: human peripheral blood lymphocytes
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9
- Test concentrations with justification for top dose:
- up to 1540 µg/ml
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: none given - Untreated negative controls:
- not specified
- Negative solvent / vehicle controls:
- not specified
- Positive controls:
- not specified
- Details on test system and experimental conditions:
- DURATION
- Exposure duration: 4 h and 24 h - Species / strain:
- other: human peripheral blood lymphocytes
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- not specified
- Conclusions:
- Under the conditions of the study, the test substance was considered to be non-clastogenic in the in vitro micronucleus test.
- Executive summary:
The clastogenic potential of the test substance was tested in an in vitro micronucleus test according to OECD 487 in compliance with GLP in 2015. Human peripheral blood lymphocytes were treated with the test substance in dimethyl sulfoxide (DMSO) at concentrations up to 1540 µg/ml. Tests were conducted both with and without metabolic activation (S9) at the 4 h and 24 h time points. The test substance did not induce binucleated cells with micronuclei in any test concentration chosen neither in the presence nor in the absence of metabolic activation. Cytotoxic levels of the test substance were reached.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- Feb 2018 - Mar 2018
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- Jul 1997
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- May 2008
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
- Version / remarks:
- Aug 1998
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source: BASF SE, Ludwigshafen, Germany
- Batch No.of test material: 01767-1010
- Identitiy: Confirmed
- Purity: 97.3%
- Physical state, appearance: liquid, colorless, clear
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature (under light exclusion)
- Homogeneity: Was ensured by mixing before preparation of the test substance solution
- Stability under test conditions: The stability is guaranteed
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test substance was handled under light protection conditions. The test substance was weighed and topped up with the chosen vehicle to achieve the required concentration of the stock solution.
- Dissolved in: DMSO. Test substance preparation was shaken thoroughly.
- Final dilution of a dissolved stock liquid: The further concentrations were diluted from the stock solution according to the planned doses. - Target gene:
- his and trp
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Species / strain / cell type:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix derived from rat liver induced phenobarbital and beta-naphthoflavone
- Test concentrations with justification for top dose:
- In this study, due to the purity of the test substance 5.2 mg/plate was used as top dose in all experiments.
1st experiment: 0, 33, 100, 333, 1000, 2600, and 5200 µg/plate (standard plate test with and without S9)
2nd experiment: 0, 500, 1000, 1500, 2000, 2500, 3000, and 5200 µg/plate (standard plate test with and without S9); increase of trp+ revertants was observed in the standard plate test - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test substance is insoluble in water. DMSO had been demosntrated to be suitable in bacterial reverse mutation tests and historical data are available - Untreated negative controls:
- yes
- Remarks:
- Sterility control (Soft agar, S9, buffer, vehicle, test substance)
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Vehicle control (with and without S9 mix and only vehicle)
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- 9-aminoacridine
- other: 2-aminoanthracene (2.5 µg/plate for TA 1535, TA 100, TA 1537, TA 98; 60 µg/plate for E.coli); N-methyl-N'-nitro-N-nitrosoguanidine (5 µg/plate for TA 1535, TA 100); 4-nitro-o-phenylenediamine (10 µg/plate for TA98)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation); preincubation;
- Cell density at seeding (if applicable):
DURATION
- Exposure duration: 48-72 h
SELECTION AGENT (mutation assays): not specified
NUMBER OF REPLICATIONS: 3
DETERMINATION OF CYTOTOXICITY
- Method: decrease in the number of revertants (factor ≤ 0.6), clearing or diminution of the background lawn (= reduced his- or trp- background growth)
MUTAGENICITY:
- individual plate counts, the mean number of revertant colonies per plate and the standard deviations were given for all groups - Evaluation criteria:
- The test substance was considered positive in this assay if the following criteria were met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. at least doubling (bacteria strains with high spontaneous mutation rate, like TA 98, TA 100 and E.coli WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA 1535 and TA 1537) of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.
A test substance was generally considered non-mutagenic in this test if:
• The number of revertants for all tester strains were within the range of the historical negative control data under all experimental conditions in at least two experiments carried out independently of each other. - Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with
- Genotoxicity:
- positive
- Remarks:
- Increase in the number of revertants
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at 5200 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- without
- Genotoxicity:
- positive
- Remarks:
- Increase in the number of revertants
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at and above 2500 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at 5200 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at and above 2600 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at 5200 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at and above 2600 µg/plate
- 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:
- cytotoxicity
- Remarks:
- at and above 2600 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at 5200 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at and above 2600 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- No test substance precipitation was found with and without S9 mix.
- Conclusions:
- Under the experimental conditions of this study, the test substance is mutagenic in the Escherichia coli reverse mutation assay in the absence and in the presence of metabolic activation.
- Executive summary:
The test substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay.
STRAINS: TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA
DOSE RANGE: 33 μg - 5200 μg/plate (SPT)
TEST CONDITIONS: Standard plate test (SPT) with and without metabolic activation (liver S9 mix from induced rats).
SOLUBILITY: No precipitation of the test substance was found with and without S9 mix.
TOXICITY: A bacteriotoxic effect was observed depending on the strain and test conditions from about 2500 μg/plate onward
MUTAGENICITY:
A biologically relevant increase in the number of his+ revertants (factor ≥ 2: TA 100 and TA 98 or factor ≥ 3: TA 1535 and TA 1537) was not observed in the standard plate test using tester strains TA 1535, TA 100 TA 1537 and TA 98 both with and without S9 mix.
A relevant, reproducible and partly dose dependent increase in the number of trp+ revertants exceeding a factor of 2 compared to the concurrent vehicle control was observed with E. coli WP2 uvrA with and without metabolic activation.
E. coli WP2 uvrA
without S9 mix: Increase at a concentration of 2600 μg/plate in the 1st Experiment and at concentrations of 2000 and 2500 μg/plate in the 2nd Experiment.
with S9 mix: Increase at a concentration of 2600 μg/plate in the 1st Experiment and at concentrations of 1000, 1500, 2000, 2500 and 3000 μg/plate in the 2nd Experiment.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- Apr 2019 - May 2019
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- 1997
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- 2008
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
- Version / remarks:
- 1998
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source and test substance No.: DRT, 17/0495-2
- Batch identification: 213199
- Expiration date of the lot/batch: The stability of the test substance under storage conditions is guaranteed until 10 Oct 2020
- Purity: 96.2%
- Physical state, appearance: liquid, colorless, clear
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature (under light exclusion)
- Homogeneity: Was ensured by mixing before preparation of the test substance solutions.
- Stability under storage conditions: 10 Oct 2020
- Stability under test conditions: The stability is guaranteed
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test substance was handled under light protection conditions. The test substance was weighed and topped up with the chosen vehicle to achieve the required concentration of the stock solution.
- Dissolved in: DMSO. Test substance preparation was shaken thoroughly.
- Final dilution of a dissolved stock liquid: The further concentrations were diluted from the stock solution according to the planned doses.
- Target gene:
- his and trp
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Species / strain / cell type:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix derived from rat liver induced phenobarbital and beta-naphthoflavone
- Test concentrations with justification for top dose:
- Test concentrations:
1st Experiment (with and without S9): 0, 33, 100, 333, 1000, 2600 and 5200 µg/plate (not considered for evaluation)
2nd Experiment (with and without S9): 0, 33, 100, 333, 1000, 2600 and 5200 µg/plate
3rd Experiment (with and without S9): 0, 10, 33, 100, 333, 1000 and 2600 µg/plate (top dose adjusted due to observed toxicity)
Due to the purity of the test substance 5.2 mg/plate was used as top dose in the standard plate tests. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: DMSO had been demonstrated to be suitable in bacterial reverse mutation tests and historical control data are available.
- Justification for percentage of solvent in the final culture medium: - Untreated negative controls:
- yes
- Remarks:
- Sterility control (Soft agar, S9 mix, buffer, vehicle and test substance without addition of tester strains)
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Vehicle control (with and without S9 mix and only vehicle)
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- 9-aminoacridine
- other: With S9 mix: 2-aminoanthracene: 2.5 µg/plate (TA 1535, TA 100, TA 1537, TA 98), 60 µg/plate (E. coli WP2 uvrA); Without S9 mix: N-methyl-N'-nitro-N-nitrosoguanidine: 5 µg/plate (TA 1535, TA 100), 4-nitro-o-phenylenediamine: 10 µg/plate (TA 98)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation); preincubation;
DURATION
- Exposure duration: 48-72 h
SELECTION AGENT (mutation assays): not specified
NUMBER OF REPLICATIONS: 3
DETERMINATION OF CYTOTOXICITY
- Method: decrease in the number of revertants (factor ≤ 0.6), clearing or diminution of the background lawn (= reduced his- or trp- background growth)
MUTAGENICITY:
- individual plate counts, the mean number of revertant colonies per plate and the standard deviations were given for all groups - Evaluation criteria:
- The test substance was considered positive in this assay if the following criteria were met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. at least doubling (bacteria strains with high spontaneous mutation rate, like TA 98, TA 100 and E.coli WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA 1535 and TA 1537) of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.
A test substance was generally considered non-mutagenic in this test if:
• The number of revertants for all tester strains were within the range of the historical negative control data under all experimental conditions in at least two experiments carried out independently of each other. - Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at and above 2600 µg/plate
- 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:
- cytotoxicity
- Remarks:
- at and above 1000 µg/plate
- 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:
- cytotoxicity
- Remarks:
- at and above 2600 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at and above 2600 µg/plate
- 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:
- cytotoxicity
- Remarks:
- at and above 1000 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- No test substance precipitation was observed with and without S9 mix.
- Conclusions:
- Under the evaluated conditions the test item was not mutagenic in the bacterial reverse mutation test with or without metabolic activation.
- Executive summary:
According to the results of the present study, the test substance did not lead to a relevant increase in the number of revertant colonies without S9 mix or after adding a metabolizing system in two valid experiments carried out independently of each other (standard plate test and preincubation assay).
The results of the negative as well as the positive controls performed in parallel corroborated the validity of this study, since the values fulfilled the acceptance criteria. The number of revertant colonies in the negative controls, with and without S9 mix, were within the range of the historical negative control data for each tester strain. In addition, the positive control substances with and without S9 mix induced a significant increase in the number of revertant colonies compatible with the range of the historical positive control data
Referenceopen allclose all
According to the results of the present study, the test substance led to a relevant and partly dose depending increase in the number of trp+ revertants with tester strain E.coli with and without metabolic activation. The increase of trp+ revertants was reproducible in two experiments carried out independently of each other. In all other tester strains, no relevant increase of revertants was observed. Based on the assessment criteria described, the test substance has to be considered positive. Besides, the results of the negative as well as the positive controls performed in parallel corroborated the validity of this study, since the values fulfilled the acceptance criteria.
In this study with and without S9 mix, the number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain.
In addition, the positive control substances with and without S9 mix induced a significant increase in the number of revertant colonies within the range of the historical positive control data.
Table 1: Results of the standard plate test WITHOUT metabolic activation - 1st experiment
Strain |
Test group |
Dose [µg/plate] |
Mean revertants per plate |
Standard deviation |
Factor |
Individual revertant colony counts |
E. coli |
DMSO |
- |
27.7 |
3.8 |
- |
32, 26, 25 |
|
Test item |
33 |
25.0 |
5.6 |
0.9 |
26, 30, 19 |
|
|
100 |
26.7 |
4.0 |
1.0 |
26, 23, 31 |
|
|
333 |
25.0 |
6.2 |
0.9 |
20, 23, 32 |
|
|
1000 |
19.3 |
6.4 |
0.7 |
12, 24, 22 |
|
|
2600 |
57.7 |
3.1 |
2.1 |
55 B, 57 B, 61 B |
|
|
5200 |
0.0 |
0.0 |
0.0 |
0 B, 0 B, 0 B |
|
4-NQO |
5 |
680.3 |
73.4 |
24.6 |
636, 765, 640 |
TA 1537 |
DMSO |
- |
8.3 |
2.3 |
- |
7, 7, 11 |
|
Test item |
33 |
8.0 |
2.0 |
1.0 |
8, 10, 6 |
|
|
100 |
7.3 |
4.9 |
0.9 |
13, 5, 4 |
|
|
333 |
7.0 |
0.0 |
0.8 |
7, 7, 7 |
|
|
1000 |
10.7 |
1.2 |
1.3 |
12, 10, 10 |
|
|
2600 |
1.7 |
1.5 |
0.2 |
0 B, 2 B, 3 B |
|
|
5200 |
0.0 |
0.0 |
0.0 |
0 B, 0 B, 0 B |
|
AAC |
100 |
886.0 |
62.8 |
106.3 |
945, 820, 893 |
TA 1535 |
DMSO |
- |
9.3 |
3.1 |
- |
12, 6, 10 |
|
Test item |
33 |
11.3 |
3.5 |
1.2 |
15, 8, 11 |
|
|
100 |
9.7 |
2.1 |
1.0 |
12, 8, 9 |
|
|
333 |
11.0 |
6.0 |
1.2 |
11, 17, 5 |
|
|
1000 |
9.7 |
6.1 |
1.0 |
3, 15, 11 |
|
|
2600 |
8.3 |
4.2 |
0.9 |
13 B, 5 B, 7 B |
|
|
5200 |
0.0 |
0.0 |
0.0 |
0 B, 0 B, 0 B |
|
MNNG |
5.0 |
3958.3 |
33.6 |
424.1 |
3942, 3997, 3936 |
TA 100 |
DMSO |
- |
105.3 |
6.8 |
- |
103, 113, 100 |
|
Test item |
33 |
98.3 |
17.5 |
0.9 |
103, 79, 113 |
|
|
100 |
92.0 |
7.0 |
0.9 |
92, 99, 85 |
|
|
333 |
105.3 |
15.5 |
1.0 |
118, 110, 88 |
|
|
1000 |
114.0 |
4.4 |
1.1 |
112, 119, 111 |
|
|
2600 |
54.7 |
24.4 |
0.5 |
82 B, 47 B, 35 B |
|
|
5200 |
0.0 |
0.0 |
0.0 |
0 B, 0 B, 0 B |
|
MNNG |
5.0 |
2847.3 |
228.1 |
27.0 |
2646, 3095, 2801 |
TA 98 |
DMSO |
- |
16.7 |
1.5 |
- |
17, 18, 15 |
|
Test item |
33 |
17.7 |
5.7 |
1.1 |
16, 13, 24 |
|
|
100 |
16.0 |
1.0 |
1.0 |
17, 16, 15 |
|
|
333 |
17.7 |
1.5 |
1.1 |
19, 16, 18 |
|
|
1000 |
22.0 |
1.0 |
1.3 |
23, 22, 21 |
|
|
2600 |
9.7 |
1.5 |
0.6 |
10 B, 8 B, 11 B |
|
|
5200 |
0.0 |
0.0 |
0.0 |
0 B, 0 B, 0 B |
|
NOPD |
10 |
996.7 |
122.7 |
59.8 |
855, 1068, 1067 |
Table 2: Results of the standard plate test WITH metabolic activation - 1st experiment
Strain |
Test group |
Dose [µg/plate] |
Mean revertants per plate |
Standard deviation |
Factor |
Individual revertant colony counts |
E. coli |
DMSO |
- |
20.3 |
1.2 |
- |
19, 21, 21 |
|
Test item |
33 |
21.0 |
10.4 |
1.0 |
33, 15, 15 |
|
|
100 |
20.7 |
3.2 |
1.0 |
23, 22, 17 |
|
|
333 |
28.7 |
10.7 |
1.4 |
23, 22, 41 |
|
|
1000 |
32.0 |
10.5 |
1.6 |
42, 33, 21 |
|
|
2600 |
53.3 |
9.1 |
2.6 |
63, 52, 45 |
|
|
5200 |
0.0 |
0.0 |
0.0 |
0 B, 0 B, 0 B |
|
2-AA |
60 |
78.3 |
5.8 |
3.9 |
85, 75, 75 |
TA 1537 |
DMSO |
- |
6.0 |
1.0 |
- |
7, 5, 6 |
|
Test item |
33 |
6.3 |
2.3 |
1.1 |
5, 5, 9 |
|
|
100 |
7.3 |
0.6 |
1.2 |
7, 7, 8 |
|
|
333 |
8.0 |
2.0 |
1.3 |
10, 8, 6 |
|
|
1000 |
10.3 |
2.5 |
1.7 |
10, 13, 8 |
|
|
2600 |
9.3 |
2.1 |
1.6 |
10 B, 7 B, 11 B |
|
|
5200 |
0.0 |
0.0 |
0.0 |
0 B, 0 B, 0 B |
|
2-AA |
2.5 |
81.7 |
14.0 |
13.6 |
93, 66, 86 |
TA 1535 |
DMSO |
- |
10.7 |
5.0 |
- |
6, 10, 16 |
|
Test item |
33 |
8.0 |
1.7 |
0.8 |
7, 10, 7 |
|
|
100 |
8.7 |
1.5 |
0.8 |
7, 10, 9 |
|
|
333 |
7.7 |
2.9 |
0.7 |
11, 6, 6 |
|
|
1000 |
10.0 |
3.6 |
0.9 |
6, 11, 13 |
|
|
2600 |
11.7 |
4.0 |
1.1 |
16, 11, 8 |
|
|
5200 |
0.0 |
0.0 |
0.0 |
0 B, 0 B, 0 B |
|
2-AA |
2.5 |
240.7 |
4.9 |
22.6 |
235, 244, 243 |
TA 100 |
DMSO |
- |
99.3 |
6.1 |
- |
94, 98, 106 |
|
Test item |
33 |
90.7 |
14.2 |
0.9 |
82, 107, 83 |
|
|
100 |
81.3 |
6.4 |
0.8 |
85, 85, 74 |
|
|
333 |
98.3 |
8.3 |
1.0 |
89, 101, 105 |
|
|
1000 |
119.7 |
15.3 |
1.2 |
108, 114, 137 |
|
|
2600 |
114.0 |
12.5 |
1.1 |
115, 126, 101 |
|
|
5200 |
0.0 |
0.0 |
0.0 |
0 B, 0 B, 0 B |
|
2-AA |
2.5 |
1853.0 |
168.0 |
18.7 |
2044, 1728, 1787 |
TA 98 |
DMSO |
- |
26.0 |
3.6 |
- |
25, 23, 30 |
|
Test item |
33 |
25.7 |
4.0 |
1.0 |
30, 22, 25 |
|
|
100 |
22.3 |
5.5 |
0.9 |
17, 28, 22 |
|
|
333 |
26.3 |
3.5 |
1.0 |
26, 23, 30 |
|
|
1000 |
35.7 |
6.8 |
1.4 |
28, 41, 38 |
|
|
2600 |
32.0 |
9.6 |
1.2 |
28, 25, 43 |
|
|
5200 |
0.0 |
0.0 |
0.0 |
0 B, 0 B, 0 B |
|
2-AA |
2.5 |
931.0 |
62.0 |
35.8 |
1000, 913, 880 |
Table 3: Results of the standard plate test WITHOUT metabolic activation - 2nd experiment
Strain |
Test group |
Dose [µg/plate] |
Mean revertants per plate |
Standard deviation |
Factor |
Individual revertant colony counts |
E. coli |
DMSO |
- |
25.0 |
6.2 |
- |
18, 27, 30 |
|
Test item |
500 |
33.7 |
8.1 |
1.3 |
43, 29, 29 |
|
|
1000 |
34.0 |
4.4 |
1.4 |
29, 37, 36 |
|
|
1500 |
48.0 |
2.0 |
1.9 |
46, 50, 48 |
|
|
2000 |
53.7 |
14.2 |
2.1 |
70, 45, 46 |
|
|
2500 |
68.3 |
1.5 |
2.7 |
68 B, 67 B, 70 B |
|
|
3000 |
0.0 |
0.0 |
0.0 |
0 B, 0 B, 0 B |
|
|
5200 |
0.0 |
0.0 |
0.0 |
0 B, 0 B, 0 B |
|
4-NQO |
5 |
791.0 |
53.2 |
31.6 |
815, 828, 730 |
Table 4: Results of the standard plate test WITH metabolic activation - 2nd experiment
Strain |
Test group |
Dose [µg/plate] |
Mean revertants per plate |
Standard deviation |
Factor |
Individual revertant colony counts |
E. coli |
DMSO |
- |
22.3 |
1.5 |
- |
22, 24, 21 |
|
Test item |
500 |
28.3 |
9.2 |
1.3 |
39, 23, 23 |
|
|
1000 |
47.3 |
8.4 |
2.1 |
43, 57, 42 |
|
|
1500 |
57.0 |
9.5 |
2.6 |
46, 62, 63 |
|
|
2000 |
53.0 |
6.2 |
2.4 |
58, 46, 55 |
|
|
2500 |
56.7 |
8.5 |
2.5 |
48, 57, 65 |
|
|
3000 |
64.7 |
9.3 |
2.9 |
69, 54, 71 |
|
|
5200 |
0.0 |
0.0 |
0.0 |
0 B, 0 B, 0 B |
|
2-AA |
60 |
79.0 |
4.4 |
3.5 |
84, 76, 77 |
According to the results of the present study, the test substance did not lead to a relevant increase in the number of revertant colonies with or without S9 mix in two valid experiments.
The results of the negative as well as the positive controls performed in parallel corroborated the validity of this study, since the values fulfilled the acceptance criteria. The number of revertant colonies in the negative controls, with and without S9 mix induced a significant increase in the number of revertant colonies compatible with the range of the historical positive control data.
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Summary of available data:
The test substance was tested in an Ames test according to OECD 471 in compliance with GLP in 2018 using Salmonella typhimurium (TA 1535, TA 100, TA 1537, TA 98) and Escherichia coli (E. coli WP2 uvrA) strains. Doses were chosen between 33 µg/plate and 5200 µg/plate in a standard plate test. Strains were treated in the test with and without metabolic activation (liver S9 mix from phenobarbital and beta-naphthoflavone induced rats). No precipitation of the test substance was found with and without S9 mix. A bacteriotoxic effect was observed depending on the strain and test conditions from about 2500 μg/plate onward.
A biologically relevant increase in the number of his+ revertants (factor ≥ 2: TA 100 and TA 98 or factor ≥ 3: TA 1535 and TA 1537) was not observed in the standard plate test using tester strains TA 1535, TA 100 TA 1537 and TA 98 both with and without S9 mix.
A relevant, reproducible and partly dose dependent increase in the number of trp+ revertants exceeding a factor of 2 compared to the concurrent vehicle control was observed with E. coli WP2 uvrA with and without metabolic activation.
E. coli WP2 uvrA
- without S9 mix: Increase at a concentration of 2600 μg/plate in the 1st Experiment (factor 2.1) and at concentrations of 2000 and 2500 μg/plate in the 2nd Experiment (factors 2.1 and 2.7, respectively).
- with S9 mix: Increase at a concentration of 2600 μg/plate in the 1st Experiment (factor 2.6) and at concentrations of 1000, 1500, 2000, 2500 and 3000 μg/plate in the 2nd Experiment (factory 2.1, 2.6, 2.4, 2.5, and 2.9, respectively).
The mutagenic potential was tested in an Ames test according to OECD 471 in accordance with GLP in 2019. Doses ranging from 33 - 5200 µg/plate were tested for mutagenic potential with and without metabolic activation (S9 mix) in the strains Salmonella typhimurium (TA 1535, TA 100, TA 1537, TA 98) and Escherichia coli (E.coli WP2 uvrA). No increase of the mean number of revertant colonies was observed neither with or without metabolic activation (S9 mix).
The mutagenic potential of the test substance was tested in a reverse bacteria mutation assay (Ames) according to OECD 471 in compliance with GLP in 2000. Strains used for the test were S. typhimurium TA 1535, TA 97a, TA 98, TA 100, and TA 102. Strains were treated with the test substance in dimethyl sulfoxide (DMSO) with concentrations ranging from 0.016 mg/plate to 5 mg/plate. TA 97a was treated with 0.016 -1.6 mg/plate, TA 98 and TA 102 were treated with 0.016 -5 mg/plate, and TA 100 and TA 1535 were treated with 0.016 -5 mg/plate. All strains were tested with and without metabolic activation (S9). Neither in the presence nor in the absence of S9 did any tested dose increase the mean number of revertant colonies in any strain tested.
The clastogenic potential of the test substance was tested in an in vitro micronucleus test according to OECD 487 in compliance with GLP in 2015. Human peripheral blood lymphocytes were treated with the test substance in dimethyl sulfoxide (DMSO) at concentrations up to 1540 µg/ml. Tests were conducted both with and without metabolic activation (S9) at the 4 h and 24 h time points. The test substance did not induce binucleated cells with micronuclei in any test concentration chosen neither in the presence nor in the absence of metabolic activation. Cytotoxic levels of the test substance were reached.
The genotoxic potential of tea tree oil was investigated by the in vitro chromosome aberration assay using human lymphocytes derived from donors. Subjects were non-smoking, non-alcoholic, not under drug therapy and without recent history of exposure to mutagens. Human lymphocytes were treated with three concentrations ( 95, 182, and 365 µg/m) in triplicate. None of the concentrations caused a significant increase in the observed frequencies of micronuclei when compared to the negative control. The positive control mytomicin C induced a significant response. Cytotoxicity was observed at 365 µg/ml.
The genotoxic potential of tea tree oil was investigated by the in vitro chromosome aberration assay using human lymphocytes derived from donors. Subjects were non-smoking, non-alcoholic, not under drug therapy and without recent history of exposure to mutagens. Human lymphocytes were treated with three concnetrations ( 95, 182, and 365 µg/m) in duplicate. No significant differences regarding the frequencies of chromosome aberrations were observed among the concentrations tested when compared to the negative control. The positive control (colchicine) induced significant chromosome aberrations. Cytotoxicity was observed at 365 µg/ml.
Discussion of available data:
The substance p-menth-1 -en-4 -ol is a naturally occuring substance. Chemical synthesis can be achieved via different methods, however relevant amounts of impurities are to be expected for both chemical synthesis and extraction of natural compound. Potential effects of impurities must therefore always be taken into consideration. Data from two different products which were synthesized via different methods are available. While an Ames test with one test material did identify a mutagenicity concern, the second test material showed no concern for mutagenicity in the Ames test. Both tests are equally reliable as OECD testing guideline 471 studies conducted under GLP, therefore it seems likely that the divergence in test results stems from a difference in impurities present in the test materials. Additionally, the mutagenicity concern was detected in only one bacterial strain with a definite but low biological relevance at rather high doses. These findings can be additional indications of an impurity being the causative agent of the overall test result. Due to the biological complexity of the test materials, a final conclusion on the mutagenic potential of p-menth-1 -en-4 -ol cannot be drawn based on the data available and the test material was assessed as "inconclusive" with regard to its mutagenic potential.
Justification for classification or non-classification
Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. Although five negative studies (two Ames, two in vitro micronucleus assays, and one in vitro chromosome aberration test) are available, one Ames test using the E. coli WP2 uvrA strain was tested positive concerning genotoxicity. It may be assumed that the outcome of this test is due to relevant impurities with genotoxic potential, however, a final assessment on the mutagenic potential of the test substance cannot be conducted at this point.
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