Fatty acids, C16-18 and C18-unsatd., reaction products with diethylenetriamine, di-Me sulfate-quaternized

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2002-08-30 to 2003-01-30

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Read-across from a guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 21, 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

June 08, 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

oleic-acid based IQAC, DMS quaternised

IUPAC Name:

oleic-acid based IQAC, DMS quaternised

Constituent 2

Reference substance name:

Imidazolium compounds, 2-C17-unsatd.-alkyl-1-(2-C18-unsatd. amidoethyl)-4,5-dihydro-N-methyl, Me sulfates

IUPAC Name:

Imidazolium compounds, 2-C17-unsatd.-alkyl-1-(2-C18-unsatd. amidoethyl)-4,5-dihydro-N-methyl, Me sulfates

Method

Target gene:

This in vitro experiment assesses the potential of the test item to induce gene mutations by means of a Thymidine Kinase assay using the mouse lymphoma cell line L5178Y. The Thymidine Kinase (TK) system detects base pair mutations, frameshift mutations, small deletions as well as large, non lethal deletions and rearrangements of the relevant chromosomes.
Cells deficient in the heterozygous TK-Iocus due to the forward mutation TK+/- to TK-/- are resistant to the cytotoxic effects of pyrimidine analogues such as trifluorothymidine (TFT).

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640 medium
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

S9 fractions obtained from livers of male Wistar rats which were induced with Phenobarbital (80 mg/kg b.w.) and ß-Naphthoflavone (100 mg/kg b.w.). Final protein concentration of 0.75 mg/mI in the cultures.

Test concentrations with justification for top dose:

The selection of the concentrations was based on data from a pre-experiment.

Experiment I
- with metabolic activation: 2.50, 5.00, 7.50, 10.0, 12.5, 15.0, 20.0, 40.0 and 60.0 µg/ml
- without metabolic activation: 1.00, 3.00, 5.00, 10.0, 12.0, 16.0, 18.0, 20.0, 25.0 and 30.0 µg/ml
Experiment II
- with metabolic activation: 10.0, 15.0, 20.0, 25.0, 30.0, 35.0, 40.0, 45.0, 50.0 and 55.0 µg/ml
- without metabolic activation: 0.50, 1.00, 2.00, 4.00, 6.00, 8.00, 10.0, 12.0 and 16.0 µg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was compatible with the survival of the cells and the S9 activity.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Migrated to IUCLID6: 80 and 400 µg/ml without metabolic activation

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

Migrated to IUCLID6: 1.50 µg/ml with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: experiment I short time incubation 4 hours and experiment II long-term incubation 24 hours
- Expression time (cells in growth medium): 72 hours in experiment I and 48 h in experiment II
- Selection time (if incubation with a selection agent): 11 to 14 days
- Fixation time (start of exposure up to fixation or harvest of cells): 14 to 17 days

SELECTION AGENT (mutation assays): trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: two

NUMBER OF CELLS EVALUATED: 300000/plate seeded

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth, cloning efficiency

Evaluation criteria:

There are several criteria for determining a positive result:
- clear and dose-related increase in the mutant frequency,
- biologically relevant response (at least 2-fold increase of mutant frequencies related to the comparable negative control values and higher than the historical range of negative controls) for at least one of the dose groups.
- combined with a positive effect in the mutant frequency, an increased occurrence of small colonies (slow growth colonies) indicated by a low large/small colonies ratio (< 4), is an indication for potential clastogenic effects and/or chromosomal aberrations.

According to the OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

A test item is considered to be negative if there is no biologically relevant increase in the induction of mutant cells above concurrent control levels, at
any dose level.

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at concentrations >/= 16 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The selection of the concentrations was based on data from the pre-experiment. Precipitation of the test item in cell culture medium was observed at concentrations of 50.0 µg/ml and higher. The pH-value detected at a test item concentration of 200 µg/ml was 7.2.

TOXICITY:

Growth inhibition was observed in experiment I and II with and without metabolic activation.

In experiment I with metabolic activation the relative total growth (RTG) was 6.85 % for the highest concentration (60.0 µg/ml) evaluated. The highest biologically relevant dose group evaluated with metabolic activation was 40.0 µg/ml with a RTG of 16.19 %. The highest concentration investigated without metabolic activation was 30.0 µg/ml with a RTG of 7.62 %. The highest biologically relevant concentration evaluated without metabolic activation was 20.0 µg/ml with a RTG of 10.19 %.

In experiment II with metabolic activation the relative total growth (RTG) was 13.04 % for the highest concentration (55.0 µg/ml) evaluated. The highest biologically relevant concentration evaluated without metabolic activation was 16.0 g/ml with a RTG of 17.14 %.

MUTAGENICITY:
In experiment I with metabolic activation all mutant values found were within the historical control data of the test facility BSL BIOSERVICE (about 20-150 mutants per 10exp6 cells). No dose effect relationship could be observed. The mutation frequencies found in the groups treated with the test item did not show a biologically relevant increase as compared to the controls.

Mutation frequencies with the negative controls were 66.39 - 100.61 mutants/10exp6 cells. The values of the solvent controls were found to be 64.18 - 90.07 mutants/10exp6 cells and 46.56 - 133.57 mutants/10exp6 cells with the test item, respectively. The highest mutation factor (compared to the solvent control values) of 1.73 was found at a concentration of 60.0 µg/ml with a RTG of 6.85 %. The mutation factor found at this dose group was considered as not biologically relevant due to the high toxicity found. The highest biologically relevant concentration evaluated was 40.0 µg/ml and showed a mutation factor of 1.07.

In experiment I without metabolic activation all mutant values found were within the historical control data of the test facility BSL BIOSERVICE (about 20-150 mutants per 10exp6 cells) except the value (172.70 mutants/10exp6 cells) found at a dose of 25.0 µg/ml. No dose dependency could be observed. Due to the high cytotoxicity of 8.93 % RTG at 25.0 µg/ml the increased mutation factor (above the historical control data) was considered as not biologically relevant. Moreover, if the spontaneous mutant frequency (negative control) is > 100 mutants/10exp6 cell, a doubling of the spontaneous frequency should be required for a definitive positive response. The mutation factor found at a dose of 25.0 µg/ml was 1.45, thus below the threshold value of 2.

Mutation frequencies with the negative controls were 100.99 - 136.72 mutants/10exp6 cells. The values of the solvent controls were found to be 102.49 - 135.07 mutant/10exp6 cells and 65.22 - 172.70 mutants/10exp 6 cells with the test item, respectively. The highest mutation factor (compared to the solvent control values) of 1.45 was found at a concentration of 25.0 µg/ml with a RTG of 8.93 %. The highest biologically relevant dose group
evaluated (20.0 µg/ml) showed a mutation factor of 1.02.

In experiment II with metabolic activation all mutant values found were within the historical control data of the test facility BSL BIOSERVICE (about 20-150 mutants per 10exp6 cells). No dose effect relationship could be observed. The mutation frequencies found in the groups treated with the test item did not show a biologically relevant increase as compared to the controls.

Mutation frequencies with the negative controls were 64.80 - 68.94 mutants/10exp6 cells. The values of the solvent controls were found to be 62.71 - 103.15 mutants/10exp6 cells and 40.30 - 88.61 mutants/10exp6 cells with the test item, respectively. The highest mutation factor (compared to the solvent control values) of 1.07 was found at a concentration of 15.0 µg/ml with a RTG of 91.69 %.

In experiment II without metabolic activation all mutant values found were within the historical control data of the test facility BSL BIOSERVICE (about 20-150 mutants per 10exp6 cells). No dose effect relationship could be observed. The mutation factor of 2, which was found at a dose of 0.50 µg/ml, was considered as not relevant due to the mutant frequency of 136.84 mutants/10exp6 cells, which was within the historical control data.

Mutation frequencies with the negative controls were 76.86 - 111.59 mutants/10exp6 cells. The values of the solvent controls were found to be 64.67 - 72.05 mutants/l0exp6 cells and 49.86 - 136.84 mutants/l0exp6 cells with the test item, respectively. The highest mutation factor (compared to the solvent control values) of 2.00 was found at a concentration of 0.50 µg/ml with a RTG of 83.16 %.

EMS (80 µg/ml and 400 µg/ml) and B[a]P (1.50 µg/ml) were used as positive controls and showed distinct and biologically relevant effects in mutation frequency.

RELATIONSHIP TO LARGE AND SMALL COLONIES:

Colony sizing was performed for the highest concentrations of the test item and for the negative and positive controls. A mutation frequency above 2 in combination with an increased occurrence of small colonies (defined by slow growth and/or morphological alteration of the cell clone), indicated by a low large/small colonies ratio (< 4), is an indication for potential clastogenic effects and/or chromosomal aberrations.

In experiment I, with metabolic activation the quotients of large/small colonies of the negative controls were found to be 14.00 and 73.00. For the
solvent controls the quotients noticed were 7.43 and 10.33. The quotient of the highest dose groups were found to be 17.00 (15.0 µg/ml), 14.50
(20.0 µg/ml), 15.00 (40.0 µg/ml) and 10.38 (60.0 µg/ml). No indication of potential clastogenic effects and/or chromosomal aberrations was found in all dose groups evaluated.

In experiment I without metabolic activation the quotients of large/small colonies of the negative controls were found to be 12.60 and 26.00. For the
solvent controls the quotients noticed were 5.91 and 10.43. The quotients of the highest dose groups were found to be 14.50 (18.0 µg/ml), 7.89
(20.0 µg/ml), 4.89 (25.0 µg/mI) and 7.45 (30.0 µg/ml). No indication of potential clastogenic effects and/or chromosomal aberrations was found in
all dose groups evaluated.

In experiment II with metabolic activation the quotients of large/small colonies of the negative controls were found to be 7.38 and 11.60. For the
solvent controls the quotients noticed were 7.67 and 9.40. The quotients of the highest dose groups were found to be 18.00 (40.0 µg/ml), 6.43
(45.0 µg/ml), 17.00 (50.0 µg/ml) and 10.60 (55.0 µg/ml). No indication of potential clastogenic effects and/or chromosomal aberrations was found in all dose groups evaluated.

In experiment II without metabolic activation the quotients of large/small colonies of the negative controls were found to be 7.33 and 8.89. For the
solvent controls the quotients noticed were 4.44 and 6.70. The quotients of the highest dose groups were found to be 9.67 (8.00 µg/ml), 7.63 (10.0 µg/ml), 12.25 (12.0 µg/ml) and 5.25 (16.0 µg/ml). No indication of potential clastogenic effects and/or chromosomal aberrations was found in
all dose groups evaluated.

CONCLUSION

In conclusion, in the described mutagenicity test under the experimental conditions reported, the test item is considered to be
non-mutagenic in the mouse lymphoma thymidine kinase locus using the cell line L5178Y.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

In conclusion, in the described mutagenicity test under the experimental conditions reported, the test item oleic-acid based IQAC, DMS quaternised is considered to be non-mutagenic in the mouse lymphoma thymidine kinase locus using the cell line L5178Y.

Executive summary:

In a mammalian gene mutation assay, L5178 Y (mouse lympoma thymidine kinase locus) cells cultured in vitro were exposed to the oleic-acid based IQAC; DMS quaternised (98 % a.i.) at the following concentrations:

Experiment I

- with metabolic activation: 2.50, 5.00, 7.50, 10.0, 12.5, 15.0, 20.0, 40.0 and 60.0 µg/ml

- without metabolic activation: 1.00, 3.00, 5.00, 10.0, 12.0, 16.0, 18.0, 20.0, 25.0 and 30.0 µg/ml

Experiment II

- with metabolic activation: 10.0, 15.0, 20.0, 25.0, 30.0, 35.0, 40.0, 45.0, 50.0 and 55.0 µg/ml

- without metabolic activation: 0.50, 1.00, 2.00, 4.00, 6.00, 8.00, 10.0, 12.0 and 16.0 µg/ml

The oleic-acid based IQAC, DMS quaternised was tested up to cytotoxic concentrations (>/= 16.0 µg/ml without metaboic activation and >/= 40.0 µg/ ml with metabolic activation). The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of increased number of induced mutant colonies over background.