Fatty acids C18-(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:

experimental study

Adequacy of study:

key study

Study period:

The experimental phases of the study were performed between 19 April 2010 and 21 June 2010.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

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 the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - 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:

502962-81-4

Cas Number:

502962-81-4

IUPAC Name:

502962-81-4

Constituent 2

Reference substance name:

Fatty acids, tall oil, lithium salts (LiTOFA)

IUPAC Name:

Fatty acids, tall oil, lithium salts (LiTOFA)

Details on test material:

Sponsor's identification: LiTOFA
Description : Off white solid
Batch number : SF 173
Purity : >99%
Date received : 21 January 2010
Expiry date : 21 January 2012
Storage conditions: Room temperature in the dark

The integrity of supplied data relating to the identity, purity and stability of the test material is the responsibility of the Sponsor.

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 (R0)

Properly maintained:
Yes

Periodically checked for Mycoplasma contamination:
Yes

Periodically checked for karyotype stability:
No

Periodically "cleansed" against high spontaneous background:
Yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital and beta-naphthoflavone induced rat liver, S9

Test concentrations with justification for top dose:

The maximum dose level used in the mutagenicity test was limited by test material-induced toxicity.

Vehicle and positive controls were used in parallel with the test material. Solvent (Acetone) treatment groups were used as the vehicle controls. Ethylmethanesulphonate (EMS), Sigma batch 0001423147 at 400 µg/ml and 150 µg/ml for Experiment 1 and Experiment 2 respectively, was used as the positive control in the absence of metabolic activation. Cyclophosphamide (CP) Acros batch A0164185 at 2 µg/ml was used as the positive control in the presence of metabolic activation.

Vehicle / solvent:

Vehicle used:
Vehicle (Acetone) treatment groups were used as the vehicle 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 (Acetone) treatment groups were used as the vehicle controls.

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With metabolic activation

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Vehicle (Acetone) treatment groups were used as the vehicle controls.

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

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), Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008 and the United Kingdom Environmental Mutagen Society. The technique used was a fluctuation assay using microtitre plates and trifluorothymidine as the selective agent and is based on that described by Cole and Arlett (1984). Two distinct types of mutant colonies can be recognised, i.e. large and small. Large colonies grow at a normal rate and represent events within the gene (base-pair substitutions or deletions) whilst small colonies represent large genetic changes involving chromosome 11b (indicative of clastogenic activity).

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:

RESULTS

Preliminary Toxicity Test

The dose range of the test material used in the Preliminary Toxicity Test was 4.88 to 1250 µg/ml. In all three of the exposure groups there were marked reductions in the Relative Suspension Growth (%RSG) of cells treated with the test material when compared to the concurrent vehicle controls. The toxicity curve was very steep in all three of the exposure groups. A precipitate of the test material was observed at and above 39.06 µg/ml in the 4-hour exposure groups, and at and above 156.25 µg/ml in the 24 hour exposure group. In the subsequent mutagenicity test the maximum dose was limited by test material-induced toxicity.

Mutagenicity Test

A summary of the results from the test is presented in attached Table 1.

Experiment 1

The results of the microtitre plate counts and their analysis are presented in attached Tables 2 to 7.

As was seen previously, there was evidence of marked toxicity following exposure to the test material in both the absence and presence of metabolic activation, as indicated by the %RSG and RTG values (Tables 3 and 6). The levels of toxicity observed were very similar to those of the Preliminary Toxicity Test. There was no evidence of any significant reductions in viability (%V), therefore indicating that no residual toxicity had occurred in either the absence or presence of metabolic activation. Optimum levels of toxicity were achieved in the presence of metabolic activation (Table 6). Optimum or near optimum levels of toxicity were not achieved in the absence of metabolic activation, despite using a narrow dose interval, due to the very sharp onset of test material-induced toxicity (Table 3). However, it was considered that, with no evidence of any toxicologically significant increases in mutant frequency at any of the dose levels, including a dose level that achieved the optimum level of toxicity in the presence of metabolic activation, or in the second experiment where optimum levels of toxicity were achieved in the absence of metabolic activation, the test material had been adequately tested. The excessive levels of toxicity observed at and above 60 µg/ml in the absence of metabolic activation, and at and above 100 µg/ml in the presence of metabolic activation, resulted in these dose levels not being plated for viability or TFT resistance. Acceptable levels of toxicity were seen with both positive control substances (Table 3 and Table 6).

Neither of the vehicle control mutant frequency values were outside the acceptable range of 50 to 200 x 10-6 viable cells. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional (Tables 3 and 6).

The test material did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10-6 per viable cell in either the absence or presence of metabolic activation (Tables 3 and 6). The precipitate observations varied very slightly from those of the Preliminary Toxicity Test with a precipitate of test material observed at and above 20 µg/ml in the absence of metabolic activation, and at and above 80 µg/ml in the presence of metabolic activation. However, the purpose and integrity of the study was considered unaffected.

The numbers of small and large colonies and their analysis are presented in attached Tables 4 and 7.

Experiment 2

The results of the microtitre plate counts and their analysis are presented in attached Tables 8 to 13.

As was seen previously, there was evidence of marked toxicity following exposure to the test material in both the absence and presence of metabolic activation, as indicated by the %RSG and RTG values (Tables 9 and 12). The levels of toxicity observed in the 24 hour exposure group in the absence of metabolic activation were very similar to those of the Preliminary Toxicity Test with optimum levels of toxicity being achieved (Table 9). There was also evidence of a very modest reduction in viability (%V) at the upper surviving dose level in the absence of metabolic activation, indicating some residual toxicity may have occurred at this dose level. However, the lowering of the S9 concentration to 1% in this second experiment resulted in a much steeper toxicity curve being observed when compared to 4-hour exposure groups in the presence of 2% metabolic activation in the Preliminary Toxicity Test and Experiment 1 and optimum or near optimum levels of toxicity were not achieved despite using a very narrow dose interval of 5 µg/ml (Table 12). Due to the nature of the toxicity exhibited by the test material it was considered that to achieve optimum toxicity in the presence of 1% metabolic activation would be incredibly difficult. Therefore, with no evidence of any toxicologically significant increases in mutant frequency at any of the dose levels in either the first or second experiment, including dose levels that induced optimum levels of toxicity, it was considered that the test material had been adequately tested. The excessive levels of toxicity observed at and above 70 µg/ml in the absence of metabolic activation, and at 70 µg/ml in the presence of metabolic activation, resulted in these dose levels not being plated for viability or TFT resistance. Both positive controls induced acceptable levels of toxicity (Tables 9 and 12).

The 24-hour exposure without metabolic activation demonstrated that the extended time point had no effect on the toxicity of the test material.

Neither of the vehicle control mutant frequency values were outside the acceptable range of 50 to 200 x 10-6 viable cells. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional (Tables 9 and 12).

The test material did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10-6 per viable cell in either the absence or presence of metabolic activation (Tables 9 and 12). Precipitate of test material was not observed at any of the dose levels in either the absence or presence of metabolic activation.

The numbers of small and large colonies and their analysis are presented in attached Tables 10 and 13.

Remarks on result:

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

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Please see Attached "Tables 1 to 13"

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 (migrated information):
other: 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) and Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008.

Methods. 

Two independent experiments were performed. In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test material at up to eight dose levels, in duplicate, together with vehicle (solvent) and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9). In Experiment 2, the cells were treated with the test material at up to ten dose levels using a 4 hour exposure group in the presence of metabolic activation (1% S9) and a 24 hour exposure group in the absence of metabolic activation.

The dose range of test material was selected following the results of a preliminary toxicity test and for Experiment 1 was 2.5 to 80 µg/ml in the absence of metabolic activation, and 10 to 120 µg/ml in the presence of metabolic activation. The test material dose range for Experiment 2 was 10 to 80 µg/ml in the absence of metabolic activation, and 5 to 70 µg/ml in the presence of metabolic activation.

Results. 

The maximum dose level used in the mutagenicity test was limited by test material-induced toxicity. Precipitate of test material was observed in Experiment 1, at and above 20 µg/ml in the absence of metabolic activation, and at and above 80 µg/ml in the presence of metabolic activation. The vehicle (solvent) 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.

The test material did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or the second experiment.

Conclusion. 

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