Fatty acids, lanolin, lithium salts

Endpoint summary

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

Reference

Reference 1

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'.

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.

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.

Reference 2

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'.

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.

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.

Reference 3

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'.

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

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.

Reference 4

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'.

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

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 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.