Fatty acids, C10-20-neo, potassium 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:

31 August 2012 to 30 October 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP 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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Japanese METI/MHLW guidelines for testing of new chemical substances

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:

Naphthenic acids, potassium salts

EC Number:

266-120-2

EC Name:

Naphthenic acids, potassium salts

Cas Number:

66072-08-0

IUPAC Name:

266-120-2

Test material form:

other: liquid (unspecified)

Details on test material:

- Appearance: dark brown liquid
- Storage conditions: room temperature in the dark

Method

Target gene:

TK (thymidine kinase) +/- locus

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Cell line: L5178Y TK +/- 3.7.2c mouse lymphoma
- Cell culture: Cells were routinely cultured in RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/mL), Streptomycin (100 µg/mL), Sodium pyruvate (1 mM), Amphotericin B (2.5 µg/mL) and 10 % donor horse serum (giving R10 media) at 37 °C with 5 % CO₂ in air. The cells have a generation time of approximately 12 hours and were sub-cultured accordingly. RPMI 1640 with 20 % donor horse serum (R20) and without serum (R0) were also used during the course of the study. Master stocks of cells were tested and found to be free of mycoplasma.
- Cell Cleansing: Stocks of cells were cleansed of homozygous (TK -/-) mutants by culturing in THMG medium for 24 hours. This medium contained Thymidine (9 µg/mL), Hypoxanthine (15 µg/mL), Methotrexate (0.3 µg/mL) and Glycine (22.5 µg/mL). For the following 24 hours the cells were cultured in THG medium (i.e. THMG without Methotrexate) before being returned to R10 medium.

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital/β-naphthoflavone S9

Test concentrations with justification for top dose:

Experiment 1
In the absence of metabolic activation: 0, 10, 20, 40, 50, 60, 70, 80 and 100 µg/mL
In the presence of metabolic activation: 0, 10, 20, 40, 60, 80, 100, 120 and 160 µg/mL

Experiment 2
In the absence of metabolic activation: 0, 2.5, 5, 10, 20, 40, 60, 80 and 100 µg/mL
In the presence of metabolic activation: 0, 20, 40, 60, 80, 100, 110 and 120 µg/mL

Vehicle / solvent:

Acetone

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

PREPARATION OF TEST MATERIAL
- The test material was formulated in acetone prior to serial dilutions being prepared. The maximum dose level investigated initially was the maximum achievable dose level of 2500 µg/mL, due to a lowered dosing volume with acetone (0.5 %).
- There was no marked change in pH when the test material was dosed into media and the osmolality did not increase by more than 50 mOsm.
- The test material was formulated within two hours of it being applied to the test system.

MICROSOMAL ENZYME FRACTIONS
PB/βNF S9 was prepared from the livers of male Sprague-Dawley rats weighing approximately 250 g. These had each received, orally, three consecutive daily doses of phenobarbital/β-naphthoflavone (80/100 mg per kg per day) prior to S9 preparation on the fourth day. The S9 was stored at approximately -196 °C in a liquid nitrogen freezer.
S9-mix was prepared by mixing S9, NADP (5 mM), G-6-P (5 mM), KCl (33 mM) and MgCl₂ (8 mM) in R0.
20 % S9-mix (i.e. 2 % final concentration of S9) was added to the cultures of the Preliminary Toxicity Test and of Experiment 1. In Experiment 2, 10 % S9-mix (i.e. 1 % final concentration of S9), was added.

MUTAGENICITY TEST
EXPERIMENT 1
An exponentially growing stock culture of cells was set up. Cells were counted and processed to give 1 x 10⁶ cells/mL in 10 mL aliquots in R10 medium in sterile plastic universals. The treatments were performed in duplicate, both with and without metabolic activation at eight dose levels, vehicle and positive controls. To each universal was added 2 mL of S9-mix if required, 0.1 mL of the treatment dilutions, (0.2 mL for the positive control) and sufficient R0 medium to bring the total volume to 20 mL.
The treatment vessels were incubated at 37 °C for 4 hours with continuous shaking using an orbital shaker within an incubated hood.

EXPERIMENT 2
As in Experiment 1, an exponentially growing stock culture of cells was established. The cells were counted and processed to give 1 x 10⁶ cells/mL in 10 mL cultures in R10 medium for the 4 hour treatment with metabolic activation cultures. In the absence of metabolic activation, the exposure period was extended to 24 hours therefore 0.3 x 10⁶ cells/mL in 10 mL cultures were established in 25 cm² tissue culture flasks. The treatments were performed in duplicate, both with and without metabolic activation (S9-mix) at eight dose levels, vehicle and positive controls. To each universal was added 2 mL of S9-mix if required, 0.1 mL of the treatment dilutions, (0.2 mL for the positive control) and sufficient R0 medium to give a final volume of 20 mL (R10 is used for the 24 hour exposure group).
The treatment vessels were incubated at 37 °C with continuous shaking using an orbital shaker within an incubated hood for 24 hours in the absence of metabolic activation and 4 hours in the presence of metabolic activation.

MEASUREMENT OF SURVIVAL, VIABILITY AND MUTANT FREQUENCY
At the end of the treatment period, for each experiment, the cells were washed twice using R10 medium then resuspended in R20 medium at a cell density of 2 x 10⁵ cells/mL. The cultures were incubated at 37 °C with 5 % CO₂ in air and subcultured every 24 hours for the expression period of two days by counting and diluting to 2 x 10⁵ cells/mL.
On Day 2 of the experiment, the cells were counted, diluted to 10⁴ cells/mL and plated for mutant frequency (2000 cells/well) in selective medium containing 4 µg/mL 5-trifluorothymidine (TFT) in 96-well microtitre plates. Cells were also diluted to 10 cells/mL and plated (2 cells/well) for viability (% V) in non-selective medium.
The daily cell counts were used to obtain a Relative Suspension Growth (% RSG) value that gives an indication of post treatment toxicity during the expression period as a comparison to the vehicle control, and when combined with the Viability (% V) data a Relative Total Growth (RTG) value.

PLATE SCORING
Microtitre plates were scored using a magnifying mirror box after ten to fourteen days’ incubation at 37 °C with 5 % CO₂ in air. The number of positive wells (wells with colonies) was recorded together with the total number of scorable wells (normally 96 per plate). The numbers of small and large colonies seen in the TFT mutation plates were also recorded. Colonies are scored manually by eye using qualitative judgement. Large colonies are defined as those that cover approximately 0.25 to 0.75 of the surface of the well and are generally no more than one or two cells thick. In general, all colonies less than 25 % of the average area of the large colonies are scored as small colonies. Small colonies are normally observed to be more than two cells thick. To assist the scoring of the TFT mutant colonies, 0.025 mL of MTT solution (2.5 mg/mL in PBS) was added to each well of the mutation plates. The plates were incubated for approximately two hours.
MTT is a vital stain that is taken up by viable cells and metabolised to give a brown/black colour, thus aiding the visualisation of the mutant colonies, particularly the small colonies.

Evaluation criteria:

INTERPRETATION OF RESULTS
The normal range for mutant frequency per survivor is 50 to 170 x 10^-6 for the TK+/- locus at the testing laboratory. Vehicle control results should ideally be within this range, although minor errors in cell counting and dilution or exposure to the metabolic activation system may cause this to be slightly elevated. Experiments where the vehicle control values are markedly greater than 200 x 10^-6 mutant frequency per survivor are not normally acceptable and will be repeated.
Positive control chemicals should induce at least three to five fold increases in mutant frequency greater than the corresponding vehicle control.
For a test material to demonstrate a mutagenic response it must produce a statistically significant increase in the induced mutant frequency (IMF) over the concurrent vehicle mutant frequency value (126 x 10^-6 for the microwell method).
Therefore, any test material dose level that has a mutation frequency value that is greater than the corresponding vehicle control of 126 x 10^-6 and demonstrates a positive linear trend will be considered positive.
However, if a test material produces a modest increase in mutant frequency, which only marginally exceeds this value and is not reproducible or part of a dose-related response, then it may be considered to have no toxicological significance. Conversely, when a test material induces modest reproducible increases in the mutation frequencies that do not exceed this value then scientific judgement will be applied. If the reproducible responses are significantly dose-related and include increases in the absolute numbers of mutant colonies then they may be considered to be toxicologically significant.

Statistics:

The experimental data was analysed using a dedicated computer program, Mutant 240C by York Electronic Research, which follows the statistical guidelines recommended by the UKEMS. Small, significant increases designated by the statistical package will be reviewed, and may be disregarded at the Study Director's discretion.

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

PRELIMINARY TOXICITY TEST
The dose range of the test material used in the preliminary toxicity test was 9.77 to 2500 µg/mL.
In all three of the exposure groups(with and without metabolic activation for 4 hours; without metabolic activation for 24 hours) there was evidence of marked dose-related reductions in the Relative Suspension Growth (% RSG) of cells treated with the test material when compared to the concurrent vehicle controls.
In the 4 hour exposure groups, a cloudy precipitate was observed at and above 312.5 µg/mL in the absence and presence of metabolic activation at the dosing stage; however no precipitate was observed once the cultures had been washed. In the 24 hour exposure group, a cloudy precipitate was observed at and above 625 µg/mL at the dosing stage; however this was also not present after washing the cultures. Based on the % RSG values observed, the maximum dose levels in the subsequent Mutagenicity Test were limited by test material-induced toxicity.

MUTAGENICITY TEST
A summary of the results is presented in Table 1.

EXPERIMENT 1
There was evidence of marked dose-related toxicity following exposure to the test material in both the absence and presence of metabolic activation, as indicated by the RTG and % RSG values. There was evidence of a marked reduction in viability (% V) in the absence of metabolic activation therefore indicating that residual toxicity had occurred at this dose level (70 µg/mL), however this dose level was discarded due to excessive toxicity. No reductions in viability (% V) were observed in the presence of metabolic activation, therefore indicating that residual toxicity had not occurred. Based on the % RSG and RTG values observed, it was considered that near to optimum levels of toxicity had been achieved in both the absence and presence of metabolic activation. The toxicity observed varied from the preliminary toxicity test and the test material induced toxicity proved that it would be hard to achieve optimum toxicity. The excessive toxicity observed at and above 80 µg/mL in the absence of metabolic activation resulted in these dose levels not being plated for viability or 5-TFT resistance. A dose level (70 µg/mL) that exceeded the upper acceptable limit of toxicity as indicated by the % RSG was plated out as there was sufficient cell culture available. This dose level was later excluded from statistical analysis as the RTG value also fell below the acceptable level of toxicity and this also displayed that the mutant frequency did not increase with high levels of toxicity. Acceptable levels of toxicity were seen with both positive control substances.

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.

The test material did not induce any statistically significant or dose related (linear-trend) increase in the mutant frequency x 10^-6 per viable cell in the absence or presence of metabolic activation. No precipitate of the test material was observed at any dose level.

EXPERIMENT 2
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 RTG and % RSG values. There was no evidence of any reductions in viability (% V) in either the absence or presence of metabolic activation, therefore indicating that residual toxicity had not occurred. Based on the % RSG and RTG values observed, it was considered that optimum levels of toxicity had been achieved in the absence of metabolic activation. Based on the % RSG and RTG a plateau of toxicity can be observed in the presence of metabolic activation therefore optimum levels of toxicity could not be achieved in this instance. It was considered that the lowering of the S9 concentration in Experiment 2 and a shift in toxic response may be responsible for this effect. With adequate toxicity being observed in the presence of S9 in Experiment 1 it was considered that the test material had been adequately tested and there was no impact on the integrity of the study. Acceptable levels of toxicity were seen with both positive control substances.

The 24 hour exposure without metabolic activation demonstrated that the extended time point had a marked 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.

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. No precipitate of the test material was observed at any dose level in either in the absence or presence of metabolic activation.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1 Summary of Results

Experiment 1

Treatment (µg/mL)	4 Hour Exposure without S9			Treatment (µg/mL)	4 Hour Exposure with S9
	% RSG	RTG	MF		% RSG	RTG	MF
0 10 20 40 50 60 70† 80* 100*	100 105 102 83 65 36 4 3 4	1.00 1.12 1.13 0.99 0.67 0.42 0.01	119.69 88.42 85.64 111.70 113.55 117.40 94.07	0 10 20 40 60 80 100 120* 160*	100 91 93 81 71 58 24 1 1	1.00 0.95 1.02 0.98 0.82 0.63 0.30	121.07 122.98 99.69 110.76 108.83 116.07 83.34
Linear trend - Not significant				Linear trend - Not significant
EMS 400	83	0.52	1482.19	CP 2	56	0.37	1301.32

Experiment 2

Treatment (µg/mL)	24 Hour Exposure without S9			Treatment (µg/mL)	4 Hour Exposure with S9
	% RSG	RTG	MF		% RSG	RTG	MF
0 2.5 5 10 20 40 60 80* 100*	100 86 81 57 44 31 14 8 1	1.00 0.96 0.86 0.73 0.63 0.51 0.32	143.11 131.37 146.45 128.14 148.23 156.34 135.57	0 20* 40 60 80 100 110 120	100 84 96 79 68 48 53 49	1.00   0.89 0.82 0.62 0.50 0.53 0.53	133.35   138.76 129.11 144.22 106.16 155.71 146.25
Linear trend - Not significant				Linear trend - Not significant
EMS 150	60	0.53	964.97	CP 2	51	0.31	1194.01

% RSG = Relative Suspension growth

RTG = Relative Total Growth

MF = 5-TFT resistant mutants/10⁶ viable cells 2 days after treatment

†Treatment excluded from test statistics due to toxicity

*Not plated for viability or 5-TFT resistance

EMS = Ethylmethanesulphonate

CP = Cyclophosphamide

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Under the conditions of this study, the test material is considered to be non-mutagenic in the L5178Y mouse lymphoma assay.

Executive summary:

The potential mutagenicity of the test material on the thymidine kinase (TK +/-) locus of the L5178Y mouse lymphoma cell line was investigated in accordance with the standardised guidelines OECD 476, EU Method B.17, EPA OPPTS 870.5300 and the Japanese METI/MHLW guidelines for testing of new chemical substances.

Two independent experiments were performed; the maximum dose levels were limited by test material-induced toxicity. In Experiment 1, cells were treated with the test material at eight dose levels (10 to 100 µg/mL in the absence of metabolic activation, 10 to 160 µg/mL in the presence of metabolic activation), in duplicate, together with 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 eight 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 was 2.5 to 100 µg/mL in the absence of metabolic activation and 20 to 120 µg/mL in the presence of metabolic activation.

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. The vehicle and positive controls were acceptable.

Under the conditions of this study, the test material is considered to be non-mutagenic in the L5178Y mouse lymphoma assay.