4,8-Dimethyl-2,5,7,10-tetraoxaundecane

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 December 2020 to 25 January 2021

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

2016

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Test material

Test material information

Constituent 1

Reference substance name:

4,8-dimethyl-2,5,7,10-tetraoxaundecane

Cas Number:

59039-15-5

Molecular formula:

C9H20O4

IUPAC Name:

4,8-dimethyl-2,5,7,10-tetraoxaundecane

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Lambiotte & Cie
- Purity, including information on contaminants, isomers, etc.: 99.90%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 15-25°C, keep under inert atmosphere (nitrogen), in the dark
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: The stability and homogeneity of 4,8-Dimethyl-2,5,7,10-tetraoxaundecane in the vehicle were not determined as part of this study. Analysis of achieved concentration was not performed as part of this study.

Method

Target gene:

thymidine kinase locus

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
L5178Y mouse lymphoma (3.7.2c) cells (Clive and Spector, 1975), were obtained from American Type Culture Collection (ATCC), Virginia.

For cell lines:
- Absence of Mycoplasma contamination: Cell stocks are periodically checked for freedom from mycoplasma contamination.
- Periodically ‘cleansed’ of spontaneous mutants: yes; Spontaneous thymidine kinase deficient mutants, TK -/-, were eliminated from the cultures by a 24-hour incubation in the presence of methotrexate, thymidine, hypoxanthine and glycine two days prior to storage at -196 to -150°C, in heatinactivated donor horse serum (HiDHS) containing 10% DMSO

MEDIA USED
R0 RPMI 1640, buffered with 2 mg/mL sodium bicarbonate, supplemented with 2.0 mM L-glutamine and 50 g/mL gentamicin.
R10p R0, supplemented with 0.1% v/v Synperonic F68, 1.0 mM sodium pyruvate and HiDHS at 10% v/v.
R10p medium was used for cell culture unless otherwise specified.
Selective medium consisted of R10p containing 4 g/mL trifluorothymidine (TFT).

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: male Sprague-Dawley derived rats dosed with phenobarbital and 5,6-benzoflavone to stimulate mixed-function oxidases in the liver, was from Molecular Toxicology Inc and stored at -90 to -70°C.
- method of preparation of S9 mix: S9 mix contained: S9 fraction (5% v/v), glucose-6-phosphate (6.9 mM), NADP (1.4 mM) in R0. The co-factors were prepared, neutralised with 1N NaOH and filter sterilised before use.
- concentration or volume of S9 mix and S9 in the final culture medium: the final concentration of the S9 fraction was 2% v/v, if present

Test concentrations with justification for top dose:

The solubility of the test item in a vehicle compatible with this test system was assessed. 4,8-Dimethyl-2,5,7,10-tetraoxaundecane was found to be soluble at 192.25 mg/mL in water. This gave a final concentration of 1922.5 µg/mL (10 mM) when dosed at 1% v/v.
Preliminary toxicity test: 3.75, 7.51, 15.02, 30.04, 60.08, 120.16, 240.31, 480.63, 961.25 and 1922.5 µg/mL
Mutation tests: 3 hours +/- S9 mix: 120.16, 240.31, 480.63, 961.25 and 1922.5 µg/mL

Vehicle / solvent:

Water

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

methylmethanesulfonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): Duplicate cultures were prepared throughout for all cultures.
- Number of independent experiments: 1

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): Cultures contained a total of 12 x 106 cells in a final volume of 10 mL
- Test substance added in medium

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): Following the 3-hour treatment, the cells were washed once, re-suspended in R10p to nominally 2 x 10^5 cells/mL assuming no cell loss) and incubated for a further 48 hours to allow for expression of mutant phenotype.
- Selection time (if incubation with a selective agent): Mutant potential was assessed by plating 2 x 10^3 cells/well in selective medium, two plates being prepared per culture. The plates were placed in a humidified incubator at 34 to 39°C in an atmosphere of 5% CO2 in air. After the plates had been incubated for 10 to 12 days, the number of empty wells was assessed for each 96-well plate (P0).
- Method used: microwell plates for the mouse lymphoma assay.
- If a selective agent is used (e.g., 6-thioguanine or trifluorothymidine), indicate its identity, its concentration and, duration and period of cell exposure: Trifluorothymidine is used at 4 µg/ml in the selective medium for 48 hours
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: 12 x 10^6 cells in a final volume of 10ml - After 48 hours cultures with a density of more than 1 x 10^5 cells/mL were assessed for cloning efficiency (viability) and mutant potential by plating in 96-well plates.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: relative total growth (RTG)

METHODS FOR MEASUREMENTS OF GENOTOXICIY
The following criteria were applied for assessment of individual assay results using data for MF where the RTG normally exceeded 10%:
Definitions: GEF = Global Evaluation Factor. For microwell assays this is 126 x 10^-6. Providing that all acceptability criteria were fulfilled, the test item was considered to be clearly positive if, in any of the experimental conditions examined the increase in MF above the concurrent background exceeded the GEF and the increase was concentration related (i.e., there is a significant positive linear trend). The test item is then considered able to induce mutation in this test system.
Providing that all acceptability criteria are fulfilled, the test item was considered to be clearly negative if, in all experimental conditions examined there is no concentration related response or, if there is an increase in MF, it does not exceed the GEF. The test item is then considered unable to induce mutations in this test system. If the maximum concentration was based on cytotoxicity, the highest concentration aimed to achieve between 20 and 10% RTG. The consensus is that care should be taken when interpreting positive results only found between 20 and 10% RTG and a result would not be considered positive if the increase in MF occurred only at or below 10% RTG (if evaluated).
There are some circumstances under which additional information may assist in determining that a test item is not mutagenic when there is no culture showing an RTG value between 20 and 10% RTG. These situations are outlined as follows:
(1) There was no evidence of mutagenicity (e.g., no dose response, no mutant frequencies above those seen in the concurrent negative control or historical background ranges, etc.) in a series of data points within 100% to 20% RTG and there was at least one data point between 20 and 25% RTG.
(2) There was no evidence of mutagenicity (e.g., no dose response, no mutant frequencies above those seen in the concurrent negative control or historical background ranges, etc.) in a series of data points between 100% to 25% RTG and there was also a negative data point slightly below 10% RTG. In both of these situations the test item is concluded to be negative.

Statistics:

The data were analysed using Fluctuation application SAFEStat (SAS statistical applications for end users), which follows the methods described by Robinson et al. (1989). Statistics were only reported if the sum of the mean concurrent vehicle control mutant frequency and the Global Evaluation Factor was exceeded, and this was accompanied by a significant positive linear trend (p<0.05).

Results and discussion

Test results

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

No precipitate was observed by eye at the end of treatment. Exposure at concentrations from 3.75 to 1922.5 µg/mL in the absence and presence of S9 mix (3-hour exposure) resulted in RSG values from 120 to 54% and from 128 to 72% respectively.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

It was concluded that 4,8-Dimethyl-2,5,7,10-tetraoxaundecane did not demonstrate mutagenic potential in this in vitro cell mutation assay, under the experimental conditions described.

Executive summary:

4,8-Dimethyl-2,5,7,10-tetraoxaundecane was tested for mutagenic potential in an in vitro mammalian cell mutation assay. This test system is based on detection and quantitation of forward mutation in the subline 3.7.2c of mouse lymphoma L5178Y cells, from the heterozygous condition at the thymidine kinase locus (TK+/-) to the thymidine kinase
deficient genotype (TK-/-).
The study consisted of a preliminary toxicity test and two independent mutagenicity assays. The cells were exposed for 3 hours in the absence or presence of exogenous metabolic activation (S9 mix).
4,8-Dimethyl-2,5,7,10-tetraoxaundecane was found to be soluble at 192.25 mg/mL in water.
A final concentration of 1922.5 g/mL (10 mM), dosed at 1%v/v was used as the maximum concentration in the preliminary toxicity test. This is the standard limit concentration within this test system as recommended in the current OECD Guideline 490 (2016).
In the preliminary toxicity test following a 3-hour exposure to 4,8-Dimethyl-2,5,7,10-tetraoxaundecane at concentrations from 3.75 to 1922.5 µg/mL, relative suspension growth (RSG) was reduced from 120 to 54% and from 128 to 72% in the absence and presence of S9 mix respectively. The concentrations assessed for determination of mutant frequency in the main test were based upon these data, the objective being to test up the standard limit concentration within this test system as recommended in the regulatory guidelines.
In the main test following 3-hour treatment in the absence and presence of S9 mix, there were no increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the Global Evaluation Factor (GEF), within acceptable levels of toxicity. The maximum concentrations assessed for mutant frequency in both the 3-hour treatment in the absence and presence of S9 mix was 1922.5 g/mL. No significant reduction in relative total growth (RTG) was observed in either the absence and presence of S9 mix.
In all tests the concurrent vehicle and positive control were within acceptable ranges.
It was concluded that 4,8-Dimethyl-2,5,7,10-tetraoxaundecane did not demonstrate mutagenic potential in this in vitro cell mutation assay, under the experimental conditions described.