Potassium neodecanoate

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

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Neodecanoic acid

EC Number:

248-093-9

EC Name:

Neodecanoic acid

Cas Number:

26896-20-8

Molecular formula:

C10H20O2

IUPAC Name:

2-ethyl-2,5-dimethylhexanoic acid

Method

Target gene:

thymidine kinase

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction

Test concentrations with justification for top dose:

0, 107, 142, 190, 253, 338, 450, 600, and 800 micrograms/mL without activation
0, 26.7, 53.4, 107, 142, 190, 253, 338, 450, 600, and 800 micrograms/mL with activation and 4 hour exposure
0, 26.7, 53.4, 107, 142, 190, 253, 338, and 450 micrograms/mL with activation and 24 hour exposure

Vehicle / solvent:

Dimethylsulfoxide

Controls

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

yes

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

methylmethanesulfonate

Details on test system and experimental conditions:

Prior to use in the assay, L5178Y/TK+/- cells were cleansed to reduce the frequency of spontaneously occurring TK-/- cells. Using the procedure described by Clive and Spector (1975), L5178Y cells were cultured for 24 hours in the presence of thymidine, hypoxanthine, methotrexate and glycine to poison the TK-/- cells. L5178Y/TK+/- cells will be prepared in 50% conditioned F0P supplemented with 10% horse serum and 2 mM L-glutamine (F10P) and 50% Fischer's Media for Leukemic Cells of Mice with 0.1% Pluronics F 68 (F0P).

The preparation and addition of the test article dosing solutions was carried out under yellow lighting during the exposure period. Treatment was carried out by combining 100 µL of dosing solution of test or control article in vehicle or vehicle alone, F0P medium or S9 mix with 6 million L5178Y/TK+/- cells in a total volume of 10 mL. All pH adjustments were performed prior to adding S9 or target cells to the treatment medium. Each S9-activated 10 mL culture containws 4 mL S9 mix (final S9 concentration of 1.0%). Cultures were capped tightly and incubated with mechanical mixing at 37 ± 1 Centigrade for 4 or 24 hours.

At the end of the exposure period the cells were washed with culture medium and collected by centrifugation. The cells were resuspended in 20 mL F10P, and incubated at 37 ± 1 Centigrade for two days following treatment. Cell population adjustments to 0.3 million cells/mL were made as follows:
• 4 hour treatment – 1 and 2 days after treatment.
• 24 hour treatment – immediately after test article removal, and 2 and 3 days after treatment.

Cells from selected dose levels were cultured in triplicate with 2-4 micrograms TFT/mL at a density of 1 million cells/100mm plate in cloning medium containing 0.22% to 0.24% agar. For estimation of cloning efficiency at the time of selection of those same cultures, 200 cells/100mm plate were cultured in triplicate in cloning medium without TFT (viable cell (VC) plate). Cultures will be incubated under standard conditions (37 ± 1 Centigrade in a humidified atmosphere of 5 ± 1% carbon dioxide in air) for 10-14 days.

The total number of colonies per culture were determined for the VC plates and the total relative growth calculated. The total number of colonies per TFT plate were then determined for those cultures with 10% or greater total growth. Colonies were counted and the diameter of the TFT colonies from the positive control and vehicle control cultures will be determined over a range from 0.2 to 1.1 mm.

Evaluation criteria:

In evaluation of the data, increases in induced mutant frequency which occur only at highly toxic concentrations (i.e., less than 10% total growth) were not considered biologically relevant. All conclusions were based on scientific judgment; however, the following criteria are presented as a guide to interpretation of the data (Moore et al., 2006):
-A result was considered positive if a concentration-related increase in mutant frequency is observed in the treated cultures and one or more treatment conditions with 10% or greater total growth exhibit induced mutant frequencies of <90 mutants per million clonable cells (based on the average mutant frequency of duplicate cultures). If the average vehicle control mutant frequency is >90 mutants per million clonable cells, a doubling of mutant frequency over the vehicle will also be required (Mitchell et al., 1997).
-A result was considered negative if the treated cultures exhibit induced mutant frequencies of <90 mutants per million clonable cells (based on the average mutant frequency of duplicate cultures) and there was no concentration-related increase in mutant frequency.
Situations in which a chemical may be considered negative when there is no culture showing between 10-20% survival (Office of Food Additive Safety, 2001) include:
-There is no evidence of mutagenicity (e.g. no dose response or any induced mutant frequencies between 45 and 89 mutants per million) in a series of data points within 100% to 20% survival and there is at least one negative data point between 20% and 25% survival.
-There is no evidence of mutagenicity (e.g. no dose response or any induced mutant frequencies between 45 and 89 mutants per million) in a series of data points between 100% to 25% survival and there is also a negative data point between 10% and 1% survival. In this case it is acceptable to count the TFT colonies of cultures exhibiting <10% total growth.

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

Positive controls validity:

valid

Remarks on result:

other: strain/cell type: L5178Y/TK+/- mouse lymphoma cells

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Preliminary Toxicity Assay

A preliminary toxicity assay evaluated neodecanoic acid at concentrations of 3.4 to 1740 µg/mL using a 4-hour treatment with and without S9, and a 24 hour treatment without S9 (the maximum concentration tested approximated the 10 mM limit dose for this assay). Precipitate was observed at concentrations ≥435 µg/mL at the beginning of treatment only. The test article did not adversely impact the pH or osmolality of the cultures (454 and 446 mmol/kg for the solvent control and 218 µg/mL, the highest soluble concentration at the beginning of treatment, respectively). Relative suspension growth (RSG) was 52, 5 and 29% at concentrations of 435 µg/mL (4-hour treatment with S9), 870 µg/mL (4 hour treatment without S9), and 218 µg/mL (24-hour treatment without S9), respectively. RSG at all higher concentrations in all treatment groups was 0%.

 

Mutagenesis Assays

Cultures were treated in the mutagenicity assay at concentrations of 26.7, 53.4, 107, 142, 190, 253, 338, 450, 600 and 800 µg/mL (4-hour treatment with S9), 107, 142, 190, 253, 338, 450, 600 and 800 µg/mL (4-hour treatment without S9), and 26.7, 53.4, 107, 142, 190, 253, 338 and 450 µg/mL (24-hour treatment without S9) based on the preliminary toxicity assay results. No visible precipitate was observed during treatment. The test article did not adversely impact culture osmolality (444 and 433 mmol/kg for the solvent control and the highest treatment condition, respectively). Culture treatment concentrations of 107, 190, 338, 600 and 800 µg/mL (4-hour treatment without S9) produced RSGs of 11 to 96% and were cloned, while cultures treated at concentrations of 26.7, 53.4, 107, 190 and 253 µg/mL (24 hour treatment without S9) produced RSGs of 28 to 105% and were cloned. Cultures treated at other lower or higher concentrations were discarded prior to cloning because a sufficient number of higher concentrations was available or due to excessive toxicity. One culture treated at 800 µg/mL for 4-hours without S9 was excluded from evaluation of mutagenicity due to excessive toxicity. The remaining cloned cultures had relative total growth (RTG) of 10 to 69% (4-hour treatment without S9) and 23 to 96% (24 hour treatment without S9). Induced mutant frequency (IMF) did not increase under either treatment condition. The average mutant frequency of the negative controls with S9 exceeded the acceptance criteria (not shown) and that portion of the assay was repeated.

A re-test was conducted under identical conditions using a 4-hour treatment with S9. No visible precipitate was observed during treatment. Culture treatment concentrations of 53.4, 142, 253, 450 and 600 µg/mL resulted in 44 to 93% RSG and were cloned (cultures treated at other lower or higher concentrations were discarded prior to cloning because a sufficient number of higher concentrations was available or due to excessive toxicity). RTG of the cloned cultures ranged from 34 to 91%. No increases in IMF were observed.

The trifluorothymidine-resistant colonies for the positive and solvent control cultures were sized according to diameter over a range from approximately 0.2 to 1.1 mm. Colony sizing for the MMS and DMBA positive controls yielded the expected increase in small colonies (verifying the adequacy of the methods used to detect small colony mutants) and large colonies.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Neodecanoic acid is not mutagenic in the mouse lymphoma test.

Executive summary:

Neodecanoic acid was evaluated in vitro for potential to cause gene mutations in mammalian cells using the mouse lymphoma L5178Y assay according to OECD Test Guideline 476. Rat liver S9 fraction was used for metabolic activation. Cells were exposed to concentrations from 0 to 800 micrograms/mL for 4 hours with and without metabolic activation and from 0 to 450 micrograms/mL for 24 hours with metabolic activation. Evidence for cytotoxicity (% relative growth less than 50%) was observed at concentrations of 218 micrograms/mL and higher. Positive controls (methylmethanesulfonate and 7,12-dimethylbenzanthracene) performed as expected, increasing induced mutant frequency ~5 to 20-fold in a concentration-related manner.

These results indicate that neodecanoic acid was negative in the L5178Y/TK^+/-Mouse Lymphoma Mutagenesis Assay under the conditions, and according to the criteria, of the test protocol.