Reaction mass of hexadecyl dihydrogen phosphate and cetyl alcohol (mono- C16 PSE and C16-OH)

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 May 26, 2017 to June 27, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

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:

adopted 29 July 2016,

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 2008

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: MITI/MHW guidelines for testing of new chemical substances.

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vitro mammalian cell gene mutation tests using the thymidine kinase gene (migrated information)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of hexadecyl dihydrogen phosphate and cetyl alcohol

Molecular formula:

C16H35O4P1 (mono- C16 PSE) C16H34O1 (cetyl alcohol)

IUPAC Name:

Reaction mass of hexadecyl dihydrogen phosphate and cetyl alcohol

Test material form:

solid

Method

Target gene:

Thymidine kinase gene

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Group 1 (4-h without S9): 1.22, 2.44, 4.88, 9.75, 19.5 and 39 μg/mL
Group 2 (4-h with S9 (2%)): 0.61, 1.22, 2.44, 4.88, 9.75 and 19.5 μg/mL
Group 3 (24-h without S9): 1.22, 2.44, 4.88, 9.75, 19.5 and 39 μg/mL

The dose range of test substance used in the main test was selected following the results of a preliminary toxicity test. A precipitate of the test substance was observed at and above 39 μg/mL in all three exposure groups. The dose levels plated for viability and expression of mutant colonies were as follows:

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

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 approximately 37°C with 5% CO2 in air. The cells have a generation time of approximately 12 h and were subcultured 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. Master stocks of cells were tested and found to be free of mycoplasma.

Microsomal enzyme fraction:
The S9-mix was prepared prior to the dosing of the test cultures and contained the S9 fraction (20% (v/v)), MgCl2 (8mM), KCl (33mM), sodium orthophosphate buffer pH 7.4 (100mM), glucose-6-phosphate (5mM) and NADP (5mM). The final concentration of S9, when dosed at a 10% volume of S9-mix into culture media, was 2%.

Test substance preparation:
Following solubility checks performed in-house, the test substance was accurately weighed and formulated in DMSO prior to serial dilutions being prepared. The test substance is a UVCB with a purity value of 100%; therefore the maximum recommended dose level was initially set at 5000 μg/mL. However the maximum achievable dose level was 2500 μg/mL due to formulation problems at 5000 μg/mL. There was no marked change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm.

Evaluation criteria:

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 Mutation Frequency (MF) above the concurrent background exceeds the Global Evaluation Factor (GEF) and the increase is concentration related (e.g., using a trend test). 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, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Remarks:

DMSO

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

Under the study conditions, the test substance was determined to be non-mutagenic in mouse lymphoma assay, with and without metabolic activation.

Executive summary:

An in vitro study was conducted to determine the genotoxic potential of the test substance, 'mono- C16 PSE and C16-OH', using the thymidine kinase gene according to OECD Guideline 490, in compliance with GLP. This study was performed to investigate the potential of the test substance to induce mutations at the thymidine kinase locus (TK1) on chromosome 11 and/or structural chromosomal aberrations in mouse lymphoma L5178Y TK+/- cells. One main Mutagenicity Test was performed. In this main test, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test substance at 8 dose levels (ranging from 0.61 to 78 μg/mL) in duplicate, together with vehicle (DMSO), and positive controls using 4 h exposure groups both in the absence and presence of metabolic activation (2% S9), and a 24 h exposure group in the absence of metabolic activation. The dose range of test substance used in the main test was selected following the results of a preliminary toxicity test. The dose levels plated for viability and expression of mutant colonies in the preliminary test ranged from 2.44 to 625 μg/mL in the three exposure groups (i.e., 4 h with and without S9 and 24 h without S9). A precipitate of the test substance was observed at and above 39 μg/mL in all three exposure groups. Therefore, the maximum dose level used for the main test was limited by precipitate.The vehicle control cultures had mutant frequency values that were acceptable for the L5178Y cell line at the TK +/- locus. The positive control substances induced marked increases in the mutant frequency, sufficient to indicate the satisfactory performance of the test and of the activity of the metabolizing system. There was evidence of moderate toxicity following exposure to the test substance in the 4 h and 24 h exposure in the absence of metabolic, as indicated by the % RSG (relative suspension growth) and RTG (relative total growth) values. There was no evidence of reductions in viability (%V) in either of the three exposure groups, therefore indicating that residual toxicity had not occurred. The dose levels of 0.61 and 78 μg/mL in both the 4 and 24 h exposure –S9 were not plated out for 5-TFT resistance and viability due to excessive toxicity. The dose levels of 39 and 78 μg/mL in the 4 h +S9 exposure again were not plated out for 5-TFT resistance and viability due to excessive toxicity. The vehicle control cultures had mutant frequency values that were acceptable for the L5178Y cell line at the TK +/- locus. The positive control substances induced marked increases in the mutant frequency, sufficient to indicate the satisfactory performance of the test and of the activity of the metabolizing system. The test substance did not induce any toxicologically significant increases in the mutant frequency x 10-6 per viable cell in either of the three exposure groups. The GEF (Global Evaluation Factor) value of the test substance dose levels were not exceeded in any of the three exposure groups, including dose levels beyond the acceptable level of toxicity in the 24 h exposure. Under the study conditions, the test substance was considered not to induce mutations in the mouse lymphoma thymidine kinase locus assay, with and without metabolic activation in the absence and presence of metabolic activation (Envigo, 2017).