Fatty acids, C16-18 (even numbered) saturated and C16-20 (even numbered) unsaturated, lithium salts

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

The experimental phases of the study were performed between 16 October 2001 and 14 December 2001

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no deviations from the protocol. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions. There was a deivation from GLP conditions as no analysis was conducted to determine the homogeneity, concentration or stability of the test material formulation.

Justification for type of information:

See IUCLID section 13 for read across justification

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Lithium myristate

EC Number:

243-743-8

EC Name:

Lithium myristate

Cas Number:

20336-96-3

Molecular formula:

C14H28O2.Li

IUPAC Name:

lithium myristate

Test material form:

solid

Details on test material:

- Physical state: White solid
- Purity: >99%
- Substance identity: 10634 Lithium myristate, synthetic sample
- Batch number: 10074
- Analysis code: A194/99
- Expiration date: 2013-06-30
- Storage of test material: Room temperature in the dark

Method

Target gene:

Thymidine kinase, TK ±, locus of the L5178Y mouse lymphoma cell line.

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

Type and identity of media:
RPMI 1640 (with HEPES and Glutamax-I) supplemented with heat-inactivated horse serum, sodium pyruvate and penicillin/streptomycin

Properly maintained:
Yes

Periodically checked for Mycoplasma contamination:
Yes

Periodically checked for karyotype stability:
No

Periodically "cleansed" against high spontaneous background:
No

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 liver homogenate mixed with NADPH-generating system

Test concentrations with justification for top dose:

The maximum dose level used in the mutagenicity test was limited by test material solubility and cytotoxicity.
Vehicle and positive controls were used in parallel with the test material. Solvent (DMSO) treatment groups were used as the negative controls. Methylmethanesulphonate (MMS) at 0.1 mMol/L and 3-methylcholanthrene (MCA) at 10 µg/mL were used as the positive control in the absence and presence of metabolic activation, respectively.

Vehicle / solvent:

Vehicle used: Vehicle (DMSO) treatment groups were used as the negative controls.
Justification for choice of vehicle: Formed a suspension suitable for dosing at the required concentration.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Vehicle (DMSO) treatment groups were used as the solvent control

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

Remarks:

With metabolic activation

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Vehicle (DMSO) treatment groups were used as the solvent control

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Without metabolic activation

Details on test system and experimental conditions:

This study was conducted according to a method that was designed to assess the potential mutagenicity of the test material on the thymidine kinase, TK ±, locus of the L5178Y mouse lymphoma cell line.

The use of cultured mammalian cells for mutation studies may give a measure of the intrinsic response of the mammalian genome and its maintenance process to mutagens. Such techniques have been used for many years with widely different cell types and loci. The thymidine kinase heterozygote system, TK +/- to TK -/-, was described by Clive et al., (1972) and is based upon the L5178Y mouse lymphoma cell line established by Fischer (1958). This system has been extensively validated (Clive et al., 1979; Amacher et al, 1980; Jotz and Mitchell, 1981).

The method used meets the requirements of the OECD 476 (1997). The technique used was a gene mutation assay with mouse lymphoma (L5178Y) cells which detects forward mutations at the thymidine kinase (TK) locus. If trifluorothymidine is added to the cells, this will result in non-mutant cell death but mutant cells will not make use of the trifluorothymidine and can form colonies. Two distinct types of mutant colonies can be recognised, i.e. large and small. Large colonies are produced predominantly by point mutations whilst small colonies are produced predominantly by chromosome rearrangements.

Evaluation criteria:

Please see "Any other information on materials and methods incl. tables" section.

Statistics:

Please see "Any other information on materials and methods incl. tables" section.

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

non-mutagenic

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

The highest concentration of lithium myristate tested in the absence of S9 mix was 281 µg/mL and 500 µg/mL in the presence of S9 mix. The highest concentration of lithium myristate tested for mutagenicity was 158 µg/mL and 500 µg/mL in the absence and presence of S9-mix respectively. In the absence, as well as the presence, of S9-mix, lithium myristate was cytotoxic. This results in a decrease in initial cell yield above of concentration of 50 µg/mL, in the absence of S9-mix, and above 158 µg/mL, in the presence of S9-mix. The relative suspension growth and relative total growth were decreased above concentrations of 87 µg/mL and 119 µg/mL, in the absence and presence of S9 respectively. The toxicity curves were very sharp so it was difficult to find a concentration that resulted in relative total growth between 10 and 20%.
In the absence of S9-mix in the first assay an equivocal response in the mutant frequency was observed at the highest concentration of 158 µg/mL lithium myristate. The mutant frequency was increased by 89 mutants per 1,000,000 clonable cells compared to the negative control, while the relative total growth was 1.1%. In the second assay, duplicate cultures were used and at concentrations of 144 µg/mL the mutant frequency increased with 201 mutants per 1,000,000 clonable cells but the relative total growth was 0.7%. In the duplicate culture treated with 144 µg/mL, the mutant frequency was not increased and the relative total growth was 28%. At 117 µg/mL, the relative total growth was 12% and 15% in the duplicate cultures and there was no increase in mutant frequencies. No other cultures produced positive or equivocal responses.
In the presence of S9-mix in the first assay, an equivocal response in the mutant frequency was observed at the highest concentration of 281 µg/mL lithium myristate. The mutant frequency was increased by 70 mutants per 1,000,000 clonable cells compared to the negative control, while the relative total growth was 1.0%. In the second assay, there was no increase in the mutant frequency at any dose level. The relative total growth at the highest concentration of 252 µg/mL was 3% and 9% for duplicate cultures, while, at the next higher concentrations of 227 µg/mL, the relative total growth was 29% and 47%.
The equivocal or positive responses in mutant frequency appears only in cultures with more than 95% cytotoxicity. Therefore, these were considered to be an artefact and not indicative of mutagenicity.

- Controls: Methyl methanesulphonate (MMS) and 3-methylcholanthrene (MCA) were used as positive control substances in the absence and in the presence of S9-mix, respectively. Treatment with the positive controls yielded the expected significant increase in mutant frequency compared to the negative controls.

Remarks on result:

other: strain/cell type: Thymidine kinase, TK ±, locus of the L5178Y mouse lymphoma cell line.

Any other information on results incl. tables

Please see Attached "Full study report"

Due to the nature and quantity of tables it was not possible to insert them in this section.

Applicant's summary and conclusion

Conclusions:

Interpretation of results: Non-mutagenic
The test material did not induce any toxicologically significant increases in the mutant frequency at the TK ± locus in L5178Y cells and is therefore considered to be non mutagenic under the conditions of the test.

Executive summary:

- Introduction.: The study was conducted according to a method that was designed to assess the potential mutagenicity of the test material on the thymidine kinase, TK ±, locus of the L5178Y mouse lymphoma cell line. The method used meets the requirements of the OECD (476).

- Methods: Two independent experiments were performed.  L5178Y TK ± 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test material at up to 281 µg/mL, in the absence of S9-mix, and 500 µg/mL, in the presence of S9-mix, in duplicate, together with vehicle (solvent) and positive controls. The dose range of test material was selected following the results of a preliminary toxicity test and, for Experiment 1, was 8.9 to 281 µg/mL in the absence of metabolic activation and presence of metabolic activation. The test material dose range for Experiment 2 was 49 to 160 µg/mL in the absence of metabolic activation and presence of metabolic activation.

- Results: The maximum dose level used in the mutagenicity test was 281 µg/mL in the absence of S9-mix and 500 µg/mL in the presence of S9-mix. The controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK ± locus. The positive control materials induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system. Some equivocal responses were found at the higher test concentrations but this was only found in cultures with more than 95% cytotoxicity. Therefore, these were considered to be an artefact and not indicative of mutagenicity. 

- Conclusion: The test material was considered to be non-mutagenic to L5178Y cells under the conditions of the test.