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EC number: 268-919-1 | CAS number: 68154-72-3
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
No data on genetic toxicity is available for the substance fatty acids lanolin lithium salts. Read across from dilithium sebacate and fatty acids lanolin is used to complete the in vitro gene mutation in bacteria endpoint.
Negative results were produced in two key in vitro bacterial reverse mutation assays (Ames tests) according to the OECD 471 guideline conducted with read across substances, dilithium sebacate fatty acids lanolin.
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Study period:
- Between 25 February 2010 and 26 March 2010.
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results.
- Justification for type of information:
- REPORTING FORMAT FOR THE ANALOGUE APPROACH
Further information is included under 'Attached justification' in IUCLID section 13 and 'Cross-reference'. - Reason / purpose for cross-reference:
- read-across source
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Date of inspection: 15th September 2009. Date of signature: 26th November 2009
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- Histidine operon for Salmonella
Tryptophan operon for Escherichia - Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Details on mammalian cell type (if applicable):
- Not applicable
- Additional strain / cell type characteristics:
- not applicable
- Species / strain / cell type:
- E. coli WP2 uvr A
- Details on mammalian cell type (if applicable):
- Not applicable
- Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- phenobarbitone/betanaphthoflavone
- Test concentrations with justification for top dose:
- Preliminary toxicity test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Mutation test (Experiments 1 and 2): 50, 150, 500, 1500 and 5000 µg/plate - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: The test material was insoluble in dimethyl sulphoxide, acetone, dimethyl formamide and acetonitrile at 50 mg/ml and tetrahydrofuran at 200 mg/ml in solubility checks performed in-house. The test material formed the best doseable suspension in acetone, therefore, this solvent was selected as the vehicle. Sterile distilled water was not selected as a potential vehicle following information supplied by the sponsor. - Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Acetone
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- N-ethyl-N-nitro-N-nitrosoguanidine
- Remarks:
- without S9-mix
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Acetone
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- Remarks:
- without S9-mix
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Acetone
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- Remarks:
- Without S9-mix
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Acetone
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- benzo(a)pyrene
- Remarks:
- With S9-mix
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Acetone
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-Aminoanthracene (2AA)
- Remarks:
- With S9-mix
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation)
DURATION
- Preincubation period: 10 hours
- Exposure duration: 48 hours
- Expression time (cells in growth medium): not applicable
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): 48 hours
SELECTION AGENT (mutation assays): not applicable
SPINDLE INHIBITOR (cytogenetic assays): not applicable
STAIN (for cytogenetic assays): not applicable
NUMBER OF REPLICATIONS: triplicate plating
NUMBER OF CELLS EVALUATED: not applicable
DETERMINATION OF CYTOTOXICITY
- Method: lawn deficiency and colony reduction
OTHER EXAMINATIONS:
- None - Evaluation criteria:
- Acceptance Criteria
The reverse mutation assay may be considered valid if the following criteria are met:
All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
The appropriate characteristics for each tester strain have been confirmed, eg rfa cell wall mutation and pKM101 plasmid R-factor etc.
All tester strain cultures should be in the range of 1 to 9.9 x 109 bacteria per ml.
Each mean positive control value should be at least twice the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure and the integrity of the S9-mix.
There should be a minimum of four non-toxic test material dose levels.
There should be no evidence of excessive contamination.
Evaluation Criteria
There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS (6) can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response.
A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit a definitive judgement about the test material activity. Results of this type will be reported as equivocal. - Statistics:
- Standard deviation
- 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:
- no cytotoxicity
- Remarks:
- , but tested up to the meximum recommended dose level of 5000 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:
- no cytotoxicity
- Remarks:
- , but tested up to the meximum recommended dose level of 5000 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none stated in report
- Effects of osmolality: none stated in report
- Evaporation from medium: none stated in report
- Water solubility:insoluble in water, so acetone used as solvent
- Precipitation: A particulate precipitate was noted at and above 500 µg/plate, this observation did not prevent the scoring of revertant colonies.
- Other confounding effects: none described
RANGE-FINDING/SCREENING STUDIES: The test material was non-toxic to the strains of bacteria used (TA100 and WP2uvrA-). The test material formulation and S9-mix used in this experiment were both shown to be sterile.
COMPARISON WITH HISTORICAL CONTROL DATA: Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and S9 mix used in both experiments was shown to be sterile. The culture density for each bacterial strain was also checked and considered acceptable. These data are not given in the report.
Results for the negative controls (spontaneous mutation rates) were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.
ADDITIONAL INFORMATION ON CYTOTOXICITY: not applicable - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results (migrated information):
negative
The test material was considered to be non-mutagenic under the conditions of this test. - Executive summary:
Introduction. Th method conforms to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF. It alsoets the requirents of the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) Number 440/2008 of 30 May 2008 and the USA, EPA (TSCA) OPPTS harmonised guidelines.
Methods. Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA-were treated with suspensions of the test material using both the Ames plate incorporation and pre-incubation methods at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate in the first experiment. The experiment was repeated on a separate day (pre-incubation method) using the same dose range as Experiment 1, fresh cultures of the bacterial strains and fresh test material formulations.
Results.The vehicle (acetone) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. A particulate precipitate was noted at and above 500 µg/plate, this observation did not prevent the scoring of revertant colonies.
No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation or exposure method.
Conclusion.The test material was considered to be non-mutagenic under the conditions of this test.
- 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:
- Between 25 February 2010 and 26 March 2010.
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results.
- Justification for type of information:
- REPORTING FORMAT FOR THE ANALOGUE APPROACH
Further information is included under 'Attached justification' in IUCLID section 13 and 'Cross-reference'. - Reason / purpose for cross-reference:
- read-across: supporting information
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Date of inspection: 15th September 2009. Date of signature: 26th November 2009
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- Histidine operon for Salmonella
Tryptophan operon for Escherichia - Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Details on mammalian cell type (if applicable):
- Not applicable
- Additional strain / cell type characteristics:
- not applicable
- Species / strain / cell type:
- E. coli WP2 uvr A
- Details on mammalian cell type (if applicable):
- Not applicable
- Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- phenobarbitone/betanaphthoflavone
- Test concentrations with justification for top dose:
- Preliminary toxicity test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Mutation test (Experiments 1 and 2): 50, 150, 500, 1500 and 5000 µg/plate - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: The test material was insoluble in dimethyl sulphoxide, acetone, dimethyl formamide and acetonitrile at 50 mg/ml and tetrahydrofuran at 200 mg/ml in solubility checks performed in-house. The test material formed the best doseable suspension in acetone, therefore, this solvent was selected as the vehicle. Sterile distilled water was not selected as a potential vehicle following information supplied by the sponsor. - Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Acetone
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- N-ethyl-N-nitro-N-nitrosoguanidine
- Remarks:
- without S9-mix
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Acetone
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- Remarks:
- without S9-mix
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Acetone
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- Remarks:
- Without S9-mix
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Acetone
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- benzo(a)pyrene
- Remarks:
- With S9-mix
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Acetone
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-Aminoanthracene (2AA)
- Remarks:
- With S9-mix
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation)
DURATION
- Preincubation period: 10 hours
- Exposure duration: 48 hours
- Expression time (cells in growth medium): not applicable
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): 48 hours
SELECTION AGENT (mutation assays): not applicable
SPINDLE INHIBITOR (cytogenetic assays): not applicable
STAIN (for cytogenetic assays): not applicable
NUMBER OF REPLICATIONS: triplicate plating
NUMBER OF CELLS EVALUATED: not applicable
DETERMINATION OF CYTOTOXICITY
- Method: lawn deficiency and colony reduction
OTHER EXAMINATIONS:
- None - Evaluation criteria:
- Acceptance Criteria
The reverse mutation assay may be considered valid if the following criteria are met:
All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
The appropriate characteristics for each tester strain have been confirmed, eg rfa cell wall mutation and pKM101 plasmid R-factor etc.
All tester strain cultures should be in the range of 1 to 9.9 x 109 bacteria per ml.
Each mean positive control value should be at least twice the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure and the integrity of the S9-mix.
There should be a minimum of four non-toxic test material dose levels.
There should be no evidence of excessive contamination.
Evaluation Criteria
There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS (6) can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response.
A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit a definitive judgement about the test material activity. Results of this type will be reported as equivocal. - Statistics:
- Standard deviation
- 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:
- no cytotoxicity
- Remarks:
- , but tested up to the meximum recommended dose level of 5000 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:
- no cytotoxicity
- Remarks:
- , but tested up to the meximum recommended dose level of 5000 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none stated in report
- Effects of osmolality: none stated in report
- Evaporation from medium: none stated in report
- Water solubility:insoluble in water, so acetone used as solvent
- Precipitation: A particulate precipitate was noted at and above 500 µg/plate, this observation did not prevent the scoring of revertant colonies.
- Other confounding effects: none described
RANGE-FINDING/SCREENING STUDIES: The test material was non-toxic to the strains of bacteria used (TA100 and WP2uvrA-). The test material formulation and S9-mix used in this experiment were both shown to be sterile.
COMPARISON WITH HISTORICAL CONTROL DATA: Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and S9 mix used in both experiments was shown to be sterile. The culture density for each bacterial strain was also checked and considered acceptable. These data are not given in the report.
Results for the negative controls (spontaneous mutation rates) were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.
ADDITIONAL INFORMATION ON CYTOTOXICITY: not applicable - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results (migrated information):
negative
The test material was considered to be non-mutagenic under the conditions of this test. - Executive summary:
Introduction. Th method conforms to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF. It alsoets the requirents of the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) Number 440/2008 of 30 May 2008 and the USA, EPA (TSCA) OPPTS harmonised guidelines.
Methods. Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA-were treated with suspensions of the test material using both the Ames plate incorporation and pre-incubation methods at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate in the first experiment. The experiment was repeated on a separate day (pre-incubation method) using the same dose range as Experiment 1, fresh cultures of the bacterial strains and fresh test material formulations.
Results.The vehicle (acetone) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. A particulate precipitate was noted at and above 500 µg/plate, this observation did not prevent the scoring of revertant colonies.
No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation or exposure method.
Conclusion.The test material was considered to be non-mutagenic under the conditions of this test.
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: GLP compliant, guideline study, available as an unpublished report.
- Justification for type of information:
- REPORTING FORMAT FOR THE ANALOGUE APPROACH
Further information is included under 'Attached justification' in IUCLID section 13 and 'Cross-reference'. - Reason / purpose for cross-reference:
- read-across: supporting information
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Deviations:
- no
- Principles of method if other than guideline:
- Each S9 batch is characterized with the mutagens Benzo-(a)-pyrene and 2-aminoanthracene, which require metabolic activation, in tester strain TA100 at concentrations of 5 μg/plate and 2.5 μg/plate, respectively Not the correct amount of 2-aminoanthracene was mentioned in the protocol. This writing error in the protocol had no effect on the results of the study.
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- - Salmonella: +Histidine
- E.Coli: Tryptophan - Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Details on mammalian cell type (if applicable):
- Not applicable.
- Additional strain / cell type characteristics:
- not applicable
- Species / strain / cell type:
- E. coli WP2 uvr A
- Details on mammalian cell type (if applicable):
- Not applicable.
- Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- Aroclor 1254 induced rat liver S9-mix
- Test concentrations with justification for top dose:
- - Preliminary Toxicity Test: 0, 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate
- Main test experiment one: 52, 164, 512, 1600 and 5000 µg/plate
- Main test experiment two: 52, 164, 512, 1600 and 5000 µg/plate - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Milli Q water for test substance and DMSO for positive controls (except sodium azelate which used saline)
- Preparation: Stock solution was treated with ultrasonic waves until the test substance had completely dissolved. Test substance concentrations were used within 2 hours after preparation. - Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates of TA1535
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Milli Q water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-Aminoanthracene: 2.5 µg/plate
- Remarks:
- With S9 mix
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates of TA1537
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Milli Q water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-Aminoanthracene: 2.5 µg/plate
- Remarks:
- With S9 mix
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates of TA98
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Milli Q water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-Aminoanthracene: 1 µg/plate
- Remarks:
- With S9 mix
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates of TA100
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Milli Q water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-Aminoanthracene: 1 µg/plate in direct plate assay and 5 µg/plate in preincubation assay
- Remarks:
- With S9 mix
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates of WP2uvrA
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Milli Q water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-Aminoanthracene: 15 µg/plate
- Remarks:
- With S9 mix
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates of TA1535
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Milli Q water
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- other: Sodium azide: 5 µg/plate
- Remarks:
- Without S9 mix
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates of TA1537
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Milli Q water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: ICR 191: 2.5 µg/plate and 2-nitrofluorene: 10 µg/plate
- Remarks:
- Without S9 mix
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates of TA98
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Milli Q water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-nitrofluorene: 10 µg/plate
- Remarks:
- Without S9 mix
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates of TA100
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Milli Q water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: Methylmethanesulfonate: 650 µg/plate
- Remarks:
- Without S9 mix
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates of WP2uvrA
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Milli Q water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 4-nitroquinoline N-oxide: 10 µg/plate
- Remarks:
- Without S9 mix
- Details on test system and experimental conditions:
- METHODS OF APPLICATION
- Experiment 1: In agar (plate incorporation)
- Experiment 2: Pre-incubation
DURATION
- Preincubation period for bacterial strains: 30 minutes
- Exposure duration: 48 hours
- Expression time (cells in growth medium): Not applicable
NUMBER OF REPLICATIONS: Triplicate plating.
DETERMINATION OF CYTOTOXICITY
- Method: The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test article precipitate to interfere with automated colony counting were counted manually. Evidence of test article precipitate on the plates and the condition of the bacterial background lawn were evaluated when considered necessary, macroscopically and/or microscopically by using a dissecting microscope.
To determine the toxicity, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were observed. - Evaluation criteria:
- Acceptability of the assay
A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
a) The vehicle control and positive control plates from each tester strain (with or without S9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at WIL Research Europe.
b) The selected dose range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
c) No more than 5% of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.
Data evaluation and statistical procedures
A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one follow up experiment.
A test substance is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is greater than three (3) times the concurrent control.
b) In case a repeat 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. - Statistics:
- No formal hypothesis testing was done. Standard deviation was determined.
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Remarks:
- Tested up to maximum recommended dose of 5000 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- 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:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Remarks:
- Tested up to maximum recommended dose of 5000 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- Results: All bacterial strains showed negative responses over the entire dose range, i.e. no significant dose-related increase in the number of revertants in two independently repeated experiments. Based on the results of this study it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Precipitate: Precipitation of the substance on the plates was not observed at the start or at the end of the incubation period in all tester strains.
Toxicity: There was no reduction in the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all tester strains in the absence and presence of S9-mix.
Mutagenicity: In the direct plate test and the pre-incubation test, no increase in the number of revertants was observed upon treatment with the test substance under all conditions tested.
Negative controls: The negative control values were within the laboratory historical control data ranges.
Positive controls: The strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly, except the response for TA1537 in the absence of S9-mix, second experiment. The purpose of the positive control is as a reference for the test system, where a positive response is required to check if the test system functions correctly. Since the value was more than 3 times greater than the concurrent solvent control values, this deviation in the mean plate count of the positive control had no effect on the results of the study. - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results (migrated information):
negative
Based on the results of this study, it is concluded that dilithium sebacate is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay. - Executive summary:
The in vitro mutagenicity of dilithium sebacate was assessed in a GLP-compliant Bacterial Reverse Mutation Test, following OECD guideline 471 (WIL 2015). S. typhimurium and E. coli strains were treated with suspensions of dilithium sebacate using both the Ames plate incorporation and pre-incubation methods at five dose levels in triplicate, both with and without the addition of a rat liver homogenate metabolising system. The vehicle and positive controls confirmed the sensitivity of the assay and the efficacy of the S9 -mix.
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: GLP compliant, guideline study, available as an unpublished report.
- Justification for type of information:
- REPORTING FORMAT FOR THE ANALOGUE APPROACH
Further information is included under 'Attached justification' in IUCLID section 13 and 'Cross-reference'. - Reason / purpose for cross-reference:
- read-across source
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Deviations:
- no
- Principles of method if other than guideline:
- Each S9 batch is characterized with the mutagens Benzo-(a)-pyrene and 2-aminoanthracene, which require metabolic activation, in tester strain TA100 at concentrations of 5 μg/plate and 2.5 μg/plate, respectively Not the correct amount of 2-aminoanthracene was mentioned in the protocol. This writing error in the protocol had no effect on the results of the study.
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- - Salmonella: +Histidine
- E.Coli: Tryptophan - Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Details on mammalian cell type (if applicable):
- Not applicable.
- Additional strain / cell type characteristics:
- not applicable
- Species / strain / cell type:
- E. coli WP2 uvr A
- Details on mammalian cell type (if applicable):
- Not applicable.
- Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- Aroclor 1254 induced rat liver S9-mix
- Test concentrations with justification for top dose:
- - Preliminary Toxicity Test: 0, 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate
- Main test experiment one: 52, 164, 512, 1600 and 5000 µg/plate
- Main test experiment two: 52, 164, 512, 1600 and 5000 µg/plate - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Milli Q water for test substance and DMSO for positive controls (except sodium azelate which used saline)
- Preparation: Stock solution was treated with ultrasonic waves until the test substance had completely dissolved. Test substance concentrations were used within 2 hours after preparation. - Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates of TA1535
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Milli Q water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-Aminoanthracene: 2.5 µg/plate
- Remarks:
- With S9 mix
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates of TA1537
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Milli Q water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-Aminoanthracene: 2.5 µg/plate
- Remarks:
- With S9 mix
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates of TA98
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Milli Q water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-Aminoanthracene: 1 µg/plate
- Remarks:
- With S9 mix
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates of TA100
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Milli Q water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-Aminoanthracene: 1 µg/plate in direct plate assay and 5 µg/plate in preincubation assay
- Remarks:
- With S9 mix
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates of WP2uvrA
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Milli Q water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-Aminoanthracene: 15 µg/plate
- Remarks:
- With S9 mix
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates of TA1535
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Milli Q water
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- other: Sodium azide: 5 µg/plate
- Remarks:
- Without S9 mix
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates of TA1537
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Milli Q water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: ICR 191: 2.5 µg/plate and 2-nitrofluorene: 10 µg/plate
- Remarks:
- Without S9 mix
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates of TA98
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Milli Q water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-nitrofluorene: 10 µg/plate
- Remarks:
- Without S9 mix
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates of TA100
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Milli Q water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: Methylmethanesulfonate: 650 µg/plate
- Remarks:
- Without S9 mix
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates of WP2uvrA
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Milli Q water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 4-nitroquinoline N-oxide: 10 µg/plate
- Remarks:
- Without S9 mix
- Details on test system and experimental conditions:
- METHODS OF APPLICATION
- Experiment 1: In agar (plate incorporation)
- Experiment 2: Pre-incubation
DURATION
- Preincubation period for bacterial strains: 30 minutes
- Exposure duration: 48 hours
- Expression time (cells in growth medium): Not applicable
NUMBER OF REPLICATIONS: Triplicate plating.
DETERMINATION OF CYTOTOXICITY
- Method: The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test article precipitate to interfere with automated colony counting were counted manually. Evidence of test article precipitate on the plates and the condition of the bacterial background lawn were evaluated when considered necessary, macroscopically and/or microscopically by using a dissecting microscope.
To determine the toxicity, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were observed. - Evaluation criteria:
- Acceptability of the assay
A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
a) The vehicle control and positive control plates from each tester strain (with or without S9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at WIL Research Europe.
b) The selected dose range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
c) No more than 5% of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.
Data evaluation and statistical procedures
A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one follow up experiment.
A test substance is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is greater than three (3) times the concurrent control.
b) In case a repeat 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. - Statistics:
- No formal hypothesis testing was done. Standard deviation was determined.
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Remarks:
- Tested up to maximum recommended dose of 5000 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- 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:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Remarks:
- Tested up to maximum recommended dose of 5000 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- Results: All bacterial strains showed negative responses over the entire dose range, i.e. no significant dose-related increase in the number of revertants in two independently repeated experiments. Based on the results of this study it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Precipitate: Precipitation of the substance on the plates was not observed at the start or at the end of the incubation period in all tester strains.
Toxicity: There was no reduction in the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all tester strains in the absence and presence of S9-mix.
Mutagenicity: In the direct plate test and the pre-incubation test, no increase in the number of revertants was observed upon treatment with the test substance under all conditions tested.
Negative controls: The negative control values were within the laboratory historical control data ranges.
Positive controls: The strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly, except the response for TA1537 in the absence of S9-mix, second experiment. The purpose of the positive control is as a reference for the test system, where a positive response is required to check if the test system functions correctly. Since the value was more than 3 times greater than the concurrent solvent control values, this deviation in the mean plate count of the positive control had no effect on the results of the study. - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results (migrated information):
negative
Based on the results of this study, it is concluded that dilithium sebacate is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay. - Executive summary:
The in vitro mutagenicity of dilithium sebacate was assessed in a GLP-compliant Bacterial Reverse Mutation Test, following OECD guideline 471 (WIL 2015). S. typhimurium and E. coli strains were treated with suspensions of dilithium sebacate using both the Ames plate incorporation and pre-incubation methods at five dose levels in triplicate, both with and without the addition of a rat liver homogenate metabolising system. The vehicle and positive controls confirmed the sensitivity of the assay and the efficacy of the S9 -mix.
Referenceopen allclose all
The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. A particulate precipitate was noted at and above 500 µg/plate, this observation did not prevent the scoring of revertant colonies.
No significant increases in the frequency of revertant colonies were recorded for any of the strains of bacteria, at any dose level either with or without metabolic activation or exposure method.
All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.
The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. A particulate precipitate was noted at and above 500 µg/plate, this observation did not prevent the scoring of revertant colonies.
No significant increases in the frequency of revertant colonies were recorded for any of the strains of bacteria, at any dose level either with or without metabolic activation or exposure method.
All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.
Table 1: Dose range finding test - Mutagenic response in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay
Mean number of revertant colonies/3 replicate plates (±S.D.) with one strain of Salmonella typhimurium and one Escherichia coli strain
Without S9 | With S9 | |||
Dose | TA100 | WP2uvrA | TA100 | WP2uvrA |
Positive control | 787 ± 55 | 1603 ± 80 | 1377 ± 31 | 163 ± 16 |
Solvent control | 116 ± 6 | 25 ± 6 | 112 ± 11 | 29 ± 7 |
1.7 | 107 ± 13 | 37 ± 8 | 109 ± 4 | 26 ± 3 |
5.4 | 110 ± 9 | 31 ± 3 | 106 ± 4 | 32 ± 4 |
17 | 107 ± 25 | 31 ± 10 | 126 ± 6 | 37 ± 8 |
52 | 115 ± 12 | 29 ± 2 | 117 ± 4 | 28 ± 5 |
164 | 111 ± 9 | 26 ± 7 | 111 ± 16 | 31 ± 2 |
512 | 116 ± 20 | 31 ± 9 | 120 ± 13 | 32 ± 10 |
1600 | 107 ± 4 | 27 ± 5 | 119 ± 6 | 36 ± 7 |
5000 | 108 ± 15 * | 27 ± 9 * | 120 ± 16 * | 35 ± 4 * |
* No precipitate and normal bacterial background lawn
Table 2: Experiment 1 - Mutagenic responnse in Salmonella typhimurium reverse mutation assay - Direct plate assay
Mean number of revertant colonies/3 replicate plates (±S.D.) with different strains of Salmonella typhimurium
Without S9 | With S9 | |||||
Dose | TA 1535 | TA 1537 | TA 98 | TA 1535 | TA 1537 | TA 98 |
Positive control | 795 ± 41 | 477 ± 31 | 848 ± 64 | 275 ± 17 | 442 ± 33 | 1106 ± 55 |
Solvent control | 17 ± 2 | 10 ± 8 | 20 ± 4 | 11 ± 9 | 12 ± 3 | 27 ± 4 |
52 | 16 ± 6 | 3 ± 2 | 21 ± 2 | 11 ± 4 | 13 ± 3 | 33 ± 3 |
164 | 20 ± 7 | 4 ± 1 | 22 ± 3 | 14 ± 8 | 11 ± 4 | 29 ± 7 |
512 | 17 ± 6 | 5 ± 2 | 22 ± 8 | 13 ± 4 | 12 ± 5 | 26 ± 4 |
1600 | 19 ± 1 | 9 ± 3 | 20 ± 2 | 17 ± 9 | 11 ± 1 | 20 ± 3 |
5000 | 13 ± 2 * | 5 ± 2 * | 27 ± 6 | 14 ± 2 * | 9 ± 2 * | 25 ± 8 * |
* No precipitate and normal bacterial background lawn
Table 3: Experiment 2 - Mutagenic response in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay - Pre-incubation assay
Mean number of revertant colonies/3 replicate plates (±S.D.) with different strains of Salmonella typhimurium and one Escherichia coli strain
Without S9 | With S9 | |||||||||
Dose | TA 1535 | TA 1537 | TA 98 | TA 100 | WP2uvra | TA 1535 | TA 1537 | TA 98 | TA 100 | WP2uvra |
Positive control | 772 ± 66 | 52 ± 15 | 988 ± 309 | 623 ± 70 | 206 ± 38 | 142 ± 6 | 170 ± 27 | 425 ± 57 | 1814 ± 48 | 312 ± 24 |
Solvent control | 13 ± 3 | 6 ± 2 | 25 ± 18 | 112 ± 11 | 28 ± 5 | 10 ± 4 | 5 ± 2 | 19 ± 7 | 102 ± 10 | 35 ± 4 |
52 | 11 ± 3 | 6 ± 2 | 20 ± 4 | 113 ± 18 | 25 ± 8 | 12 ± 5 | 7 ± 4 | 26 ± 6 | 117 ± 5 | 29 ± 7 |
164 | 15 ± 1 | 8 ± 5 | 17 ± 5 | 106 ± 10 | 21 ± 2 | 8 ± 2 | 7 ± 3 | 30 ± 7 | 111 ± 13 | 27 ± 7 |
512 | 14 ± 4 | 6 ± 1 | 11 ± 0 | 116 ± 7 | 25 ± 8 | 8 ± 2 | 7 ± 1 | 25 ± 7 | 103 ± 016 | 26 ± 6 |
1600 | 11 ± 6 | 3 ± 2 | 16 ± 6 | 104 ± 10 | 20 ± 6 | 14 ± 6 | 9 ± 6 | 25 ± 9 | 101 ± 11 | 27 ± 8 |
5000 | 12 ± 2 * | 6 ± 2 * | 15 ± 4 * | 100 ± 10 * | 21 ± 2 * | 10 ± 3 * | 7 ± 3 * | 33 ± 10 * | 115 ± 4 * | 32 ± 8 * |
* No precipitate and normal bacterial background lawn
Table 1: Dose range finding test - Mutagenic response in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay
Mean number of revertant colonies/3 replicate plates (±S.D.) with one strain of Salmonella typhimurium and one Escherichia coli strain
Without S9 | With S9 | |||
Dose | TA100 | WP2uvrA | TA100 | WP2uvrA |
Positive control | 787 ± 55 | 1603 ± 80 | 1377 ± 31 | 163 ± 16 |
Solvent control | 116 ± 6 | 25 ± 6 | 112 ± 11 | 29 ± 7 |
1.7 | 107 ± 13 | 37 ± 8 | 109 ± 4 | 26 ± 3 |
5.4 | 110 ± 9 | 31 ± 3 | 106 ± 4 | 32 ± 4 |
17 | 107 ± 25 | 31 ± 10 | 126 ± 6 | 37 ± 8 |
52 | 115 ± 12 | 29 ± 2 | 117 ± 4 | 28 ± 5 |
164 | 111 ± 9 | 26 ± 7 | 111 ± 16 | 31 ± 2 |
512 | 116 ± 20 | 31 ± 9 | 120 ± 13 | 32 ± 10 |
1600 | 107 ± 4 | 27 ± 5 | 119 ± 6 | 36 ± 7 |
5000 | 108 ± 15 * | 27 ± 9 * | 120 ± 16 * | 35 ± 4 * |
* No precipitate and normal bacterial background lawn
Table 2: Experiment 1 - Mutagenic responnse in Salmonella typhimurium reverse mutation assay - Direct plate assay
Mean number of revertant colonies/3 replicate plates (±S.D.) with different strains of Salmonella typhimurium
Without S9 | With S9 | |||||
Dose | TA 1535 | TA 1537 | TA 98 | TA 1535 | TA 1537 | TA 98 |
Positive control | 795 ± 41 | 477 ± 31 | 848 ± 64 | 275 ± 17 | 442 ± 33 | 1106 ± 55 |
Solvent control | 17 ± 2 | 10 ± 8 | 20 ± 4 | 11 ± 9 | 12 ± 3 | 27 ± 4 |
52 | 16 ± 6 | 3 ± 2 | 21 ± 2 | 11 ± 4 | 13 ± 3 | 33 ± 3 |
164 | 20 ± 7 | 4 ± 1 | 22 ± 3 | 14 ± 8 | 11 ± 4 | 29 ± 7 |
512 | 17 ± 6 | 5 ± 2 | 22 ± 8 | 13 ± 4 | 12 ± 5 | 26 ± 4 |
1600 | 19 ± 1 | 9 ± 3 | 20 ± 2 | 17 ± 9 | 11 ± 1 | 20 ± 3 |
5000 | 13 ± 2 * | 5 ± 2 * | 27 ± 6 | 14 ± 2 * | 9 ± 2 * | 25 ± 8 * |
* No precipitate and normal bacterial background lawn
Table 3: Experiment 2 - Mutagenic response in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay - Pre-incubation assay
Mean number of revertant colonies/3 replicate plates (±S.D.) with different strains of Salmonella typhimurium and one Escherichia coli strain
Without S9 | With S9 | |||||||||
Dose | TA 1535 | TA 1537 | TA 98 | TA 100 | WP2uvra | TA 1535 | TA 1537 | TA 98 | TA 100 | WP2uvra |
Positive control | 772 ± 66 | 52 ± 15 | 988 ± 309 | 623 ± 70 | 206 ± 38 | 142 ± 6 | 170 ± 27 | 425 ± 57 | 1814 ± 48 | 312 ± 24 |
Solvent control | 13 ± 3 | 6 ± 2 | 25 ± 18 | 112 ± 11 | 28 ± 5 | 10 ± 4 | 5 ± 2 | 19 ± 7 | 102 ± 10 | 35 ± 4 |
52 | 11 ± 3 | 6 ± 2 | 20 ± 4 | 113 ± 18 | 25 ± 8 | 12 ± 5 | 7 ± 4 | 26 ± 6 | 117 ± 5 | 29 ± 7 |
164 | 15 ± 1 | 8 ± 5 | 17 ± 5 | 106 ± 10 | 21 ± 2 | 8 ± 2 | 7 ± 3 | 30 ± 7 | 111 ± 13 | 27 ± 7 |
512 | 14 ± 4 | 6 ± 1 | 11 ± 0 | 116 ± 7 | 25 ± 8 | 8 ± 2 | 7 ± 1 | 25 ± 7 | 103 ± 016 | 26 ± 6 |
1600 | 11 ± 6 | 3 ± 2 | 16 ± 6 | 104 ± 10 | 20 ± 6 | 14 ± 6 | 9 ± 6 | 25 ± 9 | 101 ± 11 | 27 ± 8 |
5000 | 12 ± 2 * | 6 ± 2 * | 15 ± 4 * | 100 ± 10 * | 21 ± 2 * | 10 ± 3 * | 7 ± 3 * | 33 ± 10 * | 115 ± 4 * | 32 ± 8 * |
* No precipitate and normal bacterial background lawn
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
The in vitro bacterial reverse mutation tests (Ames tests) with read across substances, dilithium sebacate and fatty acids lanolin were conducted according to OECD guideline 471 (2015, 2010). No increase in revertant colonies was observed, either in the presence or absence of S9 metabolic activation system, and both substances were considered to be non-mutagenic under the conditions of the OECD studies.
Therefore, no classification is considered relevant for this endpoint for substance fatty acids lanolin lithium salts.
Both studies are GLP-compliant, guideline studies, and have been assigned a Klimisch score of 1.
Justification for classification or non-classification
Negative results were produced in an in vitro reverse mutation test in bacterial cells with two read across substances, dilithium sebacate and fatty acids lanolin.
Therefore, no classification is considered relevant for this endpoint for substance fatty acids lanolin lithium salts.
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