[No public or meaningful name is available]

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:

From November 09, 2020 to March 01, 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

29 July 2016

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Method B67 of Commission Regulation (EC) No. 440/2008

Version / remarks:

26 September 2019

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:

Reaction products of hexadecyl dihydrogen phosphate, dihexadecyl hydrogen phosphate, hexadecan-1-ol, stearic acid, esters of C18 (branched and linear) fatty acids with C18 (branched and linear) alcohols, and potassium hydroxide

Molecular formula:

Not available - UVCB

IUPAC Name:

Reaction products of hexadecyl dihydrogen phosphate, dihexadecyl hydrogen phosphate, hexadecan-1-ol, stearic acid, esters of C18 (branched and linear) fatty acids with C18 (branched and linear) alcohols, and potassium hydroxide

Test material form:

other: White pastilles

Method

Target gene:

Thymidine kinase, TK +/-, locus

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

L5178Y TK+/- 3.7.2c mouse lymphoma cell line was obtained from Dr. J. Cole of the MRC Cell Mutation Unit at the University of Sussex, Brighton, UK.

Metabolic activation:

with and without

Metabolic activation system:

The S9 Microsomal Enzyme Fractions were purchased from Moltox, Lot no 4272 with the expiry date of 16 July 2022, and Lot no 4217 with the expiry date of 05 March 2022, were used in this study. The protein content was adjusted to approximately 20 mg/ml prior to use. The S9 mix was prepared by mixing S9 with 100 mM phosphate buffer containing NADP (5 mM), G6 P (5 mM), KCl (33 mM) and MgCl2 (8 mM) to give a 20% S9-mix concentration. The final concentration of S9 when dosed at a 10% volume of S9-mix was 2% for the Preliminary Toxicity Test and the Mutagenicity Test.

Test concentrations with justification for top dose:

Preliminary test: 0, 0.63, 1.25, 2.5, 5, 10, 20, 40, 80, and 160 μg/mL with and without metabolic activation (S9)
Main experiment: 0, 0.63, 1.25, 2.5, 5, 10, 20, 40, and 80 μg/mL with and without metabolic activation (S9)

The dose range of test item used in the main test was selected following the results of a preliminary toxicity test. The maximum dose levels in the Mutagenicity Test were limited by the onset of test item precipitate in both the absence and presence of metabolic activation, as recommended by the OECD 490 guideline.

Vehicle / solvent:

Acetone
Supplier: Acros
Batch number: 1911510
Purity: 99.9%
Expiry date: 29 July 2021

Controls

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

Test System

Cell Line
The L5178Y TK+/- 3.7.2c mouse lymphoma cell line was obtained from Dr. J. Cole of the MRC Cell Mutation Unit at the University of Sussex, Brighton, UK. The cells were originally obtained from Dr. D. Clive of Burroughs Wellcome (USA) in October 1978 and were frozen in liquid nitrogen at that time.

Cell Culture
The stocks of cells are stored in liquid nitrogen at approximately -196 °C. 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), 10% donor horse serum (R10), and without serum (R0), are used during the course of the study. All donor horse serum was purchased heat inactivated from the supplier. Master stocks of cells were tested and found to be free of mycoplasma.

Cell Cleansing
The TK +/- heterozygote cells grown in suspension spontaneously mutate at a low but significant rate. Before the stocks of cells were frozen they 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.

Test Item Preparation
The test item was a UVCB*, therefore, the maximum proposed dose level in the solubility test was set at 5000 μg/mL, the maximum recommended dose level and no correction for the purity of the test item was applied to the formulations. The test item was found to be immiscible in RO medium at 50 mg/mL, and DMSO and acetone at 500 and 250 mg/mL. The test item was miscible in acetone at 125 mg/ml. Acetone was therefore selected as the solvent. However, acetone is toxic to L5178Y cells at dose volumes greater than 0.5% of the total culture volume. Therefore, the test item was formulated at 125 mg/mL and dosed at 0.5% to give a maximum achievable dose level of 625 μg/mL There was no significant change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm (Scott et al., 1991).

No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. The test item was formulated within two hours of it being applied to the test system; it is assumed that the formulation was stable for this duration. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Test Procedure

Preliminary Toxicity Test
A preliminary toxicity test was performed on cell cultures at 1x10E7 cells/mL, using a 4 hour exposure period both with and without metabolic activation (S9). Due to the precipitate observed in the solubility check, the maximum dose concentration was limited to 160 μg/mL for both exposure groups and the dose range used in the preliminary toxicity test was 0, 0.63, 1.25, 2.5, 5, 10, 20, 40, 80, and 160 μg/mL for both of the exposure groups. Following the exposure periods the cells were washed twice with R10, resuspended in R20 medium, counted and then serially diluted to 2x10E5 cells/mL. The cultures were incubated at 37 °C with 5% CO2 in air and sub-cultured after 24 hours by counting and diluting to 2x10E5 cells/mL in R20 medium. After a further 24 hours the cultures were counted and then discarded. The cell counts were then used to calculate Suspension Growth (SG) values. The SG values were then adjusted to account for immediate post exposure toxicity, and a comparison of each exposure SG value to the concurrent solvent control performed to give a percentage Relative Suspension Growth (%RSG) value.

Results from the preliminary toxicity test were used to set the test item dose levels for the mutagenicity experiments. Maximum dose levels were selected using the following criteria:
i) For non-toxic test items the upper test item concentrations will be 10 mM, 2 mg/mL or 2 μL/mL whichever is the lowest. When the test item is a substance of unknown or variable composition (UVCB*) the upper dose level may need to be higher and the maximum concentration will be 5 mg/mL.
ii) Precipitating dose levels will not be tested beyond the onset of precipitation regardless of the presence of toxicity beyond this point.
iii) In the absence of precipitate and if toxicity occurs, the highest concentration should lower the Relative Total Growth (RTG) to approximately 10 to 20 %. This optimum upper level of toxicity was confirmed by an IWGT meeting in New Orleans, USA (Moore et al., 2002).

Mutagenicity Test
Several days before starting the experiment, an exponentially growing stock culture of cells was set up so as to provide an excess of cells on the morning of the experiment. The cells were counted and processed to give 1 x 107 cells/mL in 10 mL aliquots in R10 medium in sterile plastic universals for the 4-hour exposure groups in both the absence and presence of metabolic activation. The exposures were performed in duplicate (A + B), both with and without metabolic activation (2% S9 final concentration) at eight dose levels of the test item (0, 0.63, 1.25, 2.5, 5, 10, 20, 40, and 80 μg/mL in both the absence and presence of metabolic activation), solvent and positive controls. To each universal was added 2 mL of S9 mix if required, 0.1 mL of the exposure dilutions, (0.2 mL or 0.15 mL for the positive controls), and sufficient R0 medium to bring the total volume to 20 mL. The exposure vessels were incubated at 37 °C for 4 hours with continuous shaking using an orbital shaker within an incubated hood.

Evaluation criteria:

Dose selection for the mutagenicity experiments was made using data from the preliminary toxicity test in an attempt to obtain the desired levels of toxicity. This optimum toxicity is approximately 20 % survival, but no less than 10% survival (90% toxicity). Relative Total Growth values are the primary factor used to designate the level of toxicity achieved by the test item for any individual dose level. However, under certain circumstances, %RSG values may also be taken into account when designating the level of toxicity achieved. Dose levels that have RTG survival values less than 10% are excluded from the mutagenicity data analysis, as any response they give would be considered to have no biological or toxicological relevance.

An approach for defining positive and negative responses is recommended to assure that the increased MF is biologically relevant. In place of statistical analysis generally used for other tests, it relies on the use of a predefined induced mutant frequency (i.e. increase in MF above the concurrent control), designated the Global Evaluation Factor (GEF) of 126 x 10-6, i.e. the mutant frequency of the concurrent solvent control plus 126, which is based on the analysis of the distribution of the solvent control MF data from participating laboratories.

Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if, in any of the experimental conditions examined, the increase in MF above the concurrent background exceeds the GEF and the increase is concentration related. The test chemical is then considered able to induce mutation in this test system.

Providing that all acceptability criteria are fulfilled, a test chemical is 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 chemical is then considered unable to induce mutations in this test system.

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

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

It was concluded that did not induce biologically relevant increases in mutation at the TK +/- locus of L5178Y mouse lymphoma cells that exceeded the Global Evaluation Factor (GEF) when tested for 4 h in the absence and presence of a rat liver metabolic activation system (S-9). Solvent, vehicle and positive control treatments were included in each mutation experiment in the absence and presence of S9 metabolism. The solvent controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive controls produced marked increases in the mutant frequency per viable cell achieving the acceptability criterion.

Remarks on result:

other: no mutagenic potential

Any other information on results incl. tables

Result

Preliminary Cytotoxicity Test

The dose range of the test item used in the preliminary toxicity test was 0.63 to 160 μg/mL. There was evidence of very modest dose-related reductions in the Relative Suspension Growth (%RSG) of cells treated with the test item in both of the exposure groups. Precipitate of the test item was observed at and above 40 μg/mL in both the absence and presence of metabolic activation at the end of the exposure period. Therefore, following the recommendations of the OECD 490 guideline, the maximum dose levels in the subsequent mutagenicity test were limited by the onset of test item precipitate in both the absence and presence of metabolic activation.

Mutagenicity Test

There was no evidence of any toxicity, in either the absence or presence of metabolic activation, following exposure to the test item, as indicated by the %RSG and RTG values. There was also no evidence of any marked reductions in cloning efficiency (%V) in either the absence or presence of metabolic activation, therefore indicating that residual toxicity had not occurred. Test item precipitate was observed at 80 μg/mL in both the absence and presence of metabolic activation. Acceptable levels of toxicity were seen with both positive control substances. The solvent controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive controls produced marked increases in the mutant frequency per viable cell achieving the acceptability criterion recommended by the OECD guideline, indicating that the test system was operating satisfactorily, and that the metabolic activation system was functional. The test item did not induce any increases in the mutant frequency at any of the dose levels in the main test that exceeded the Global Evaluation Factor (GEF), using a dose range that included the lowest precipitating dose level in both the absence and presence of metabolic activation, and at least four analysable dose levels in each exposure group, as recommended by the OECD 490 guideline.

Conclusion

The test item did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the Global Evaluation Factor (GEF), consequently it is considered to be non-mutagenic in this assay in which all acceptability criteria were met.

Applicant's summary and conclusion

Conclusions:

Under the study conditions, the test substance was considered to be non-mutagenic to L5178Y mouse lymphoma cells at the TK +/- locus, in the absence and presence of metabolic activation.

Executive summary:

A study was conducted to determine the mutagenic potential of the test substance on thymidine kinase, TK +/-, locus of L5178Y mouse lymphoma cells, according to the OECD Test Guideline 490, in compliance with GLP.  L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) cells were counted and processed to give 1 x 10E7 cells/mL in 10 mL aliquots in test medium in sterile plastic universals for the 4-hour exposure groups in both the absence and presence of metabolic activation. The exposures were performed in duplicate, both with and without metabolic activation (2% S9 final concentration) at eight dose levels (0, 0.63, 1.25, 2.5, 5, 10, 20, 40, and 80 μg/mL) in duplicate, both the absence and presence of metabolic activation (2% S9), solvent and positive controls for 4 hour exposure groups both in the absence and presence of metabolic activation (2% S9). The exposure vessels were incubated at 37 °C for 4 hours with continuous shaking using an orbital shaker within an incubated hood. There was no evidence of any toxicity, in either the absence or presence of metabolic activation, following exposure to the test substance, as indicated by the %RSG and RTG values. There was also no evidence of any marked reductions in cloning efficiency in either the absence or presence of metabolic activation, therefore indicating that residual toxicity had not occurred. Test substance precipitate was observed at 80 μg/mL in both the absence and presence of metabolic activation. Acceptable levels of toxicity were seen with both positive control substances. The solvent controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive controls produced marked increases in the mutant frequency per viable cell achieving the acceptability criterion recommended by the OECD guideline, indicating that the test system was operating satisfactorily, and that the metabolic activation system was functional. The test substance did not induce any increases in the mutant frequency at any of the dose levels in the main test that exceeded the Global Evaluation Factor (GEF), using a dose range that included the lowest precipitating dose level in both the absence and presence of metabolic activation, and at least four analysable dose levels in each exposure group, as recommended by the OECD 490 guideline. Under the study conditions, the test substance was considered to be non-mutagenic to L5178Y mouse lymphoma cells at the TK +/- locus, in the absence and presence of metabolic activation (Willey, 2022).