Hexadecyl acetate

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:

21 June 2014 to 09 September 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was performed to a recognised guideline and used GLP - There were no deviations (unplanned changes) from the study plan.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

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:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

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:

Hexadecyl acetate

EC Number:

211-103-7

EC Name:

Hexadecyl acetate

Cas Number:

629-70-9

Molecular formula:

C18H36O2

IUPAC Name:

hexadecyl acetate

Details on test material:

- Substance appearance: White solid
- Analytical purity: 98%
- Expiry Date.: 04 June 2016
- Storage condition of test material: Room temperature in the dark

Method

Target gene:

The thymidine kinase, TK +1-, locus of the L5178Y mouse lymphoma cell line.

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media:
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 oC with 5% CO2 in air.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 was prepared in-house from the livers of male Sprague Dawley rats weighing -250g. These had each received, orally, three consecutive daily doses of phenobarbitall~-naphthoflavone(80/100 mg per kg per day) prior to S9 preparation on the fourth day.

Test concentrations with justification for top dose:

Prelim:(µg/ml) with and without S9: 5.56 to 1422.5
Experiment 1 (µg/ml) without S9: 5.63, 11.25, 22.5, 45, 67.5 & 90
Experiment 1 (µg/ml) with S9: 11.25, 22.5, 45, 67.5, 90 and 180
Experiment 2 (µg/ml) without S9: 2.81, 5.63, 11.25, 22.5, 33.75
Experiment 2 (µg/ml) with S9: 2.81, 5.63, 11.25, 22.5, 45, 67.5

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: The test item was soluble in acetone.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

400 µg/ml in Expt. 1, 150 µg/ml in Expt. 2

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

2 ug/ml in both experiments

Details on test system and experimental conditions:

METHOD OF APPLICATION: 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). 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 10^6 cells/ml in 10 ml aliquots in R10 medium in sterile plastic universals. The cells were exposed to doses of the test material, vehicle and positive control, both with and without metabolic activation. Cultures were maintained at 37 °C in a humidified atmosphere of 5 % CO2 in air.
The treatment regimes were as follows:

DURATION
- Preincubation period: Not applicable.
- Exposure duration: 4 h (Experiment 1 both with and without S9, and Experiment 2 with S9), or 24 h (without S9 Experiment 2).
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 10~14 days (plate scoring for colony formation)

SELECTION AGENT (mutation assays): 5-trifluorothymidine (TFT)
SPINDLE INHIBITOR (cytogenetic assays): Not applicable.
STAIN (for cytogenetic assays): MTT vital stain for viable cells

NUMBER OF REPLICATIONS: Duplicate

NUMBER OF CELLS EVALUATED: seeded 2000 cells/well for mutant frequency; 2 cells/well for viability.

DETERMINATION OF CYTOTOXICITY
- Method: other: Relative Suspension Growth values (RSG)

OTHER: 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. The experimental mutation frequency data were analyzed using a dedicated computer program which follows the statistical guidelines recommended by the UKEMS.

Evaluation criteria:

For a test item 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. Following discussions at an International Workshop on Genotoxicity Test Procedures in Plymouth, UK, 2002 (Moore et al 2003) it was felt that the IMF must exceed some value based on the global background MF for each method (agar or microwell). This Global Evaluation Factor (GEF) value was set following a further meeting of the International Workshop in Aberdeen, Scotland, 2003 (Moore et al 2006) at 126 x 10'6 for the microwell method. Therefore, any test item dose level that has a mutation frequency value that is greater than the
corresponding vehicle control by the GEF of 126 ^ -6 and demonstrates a positive linear trend will be considered positive. However, if a test item produces a modest increase in mutant frequency, which only marginally exceeds the GEF 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 item induces modest reproducible increases in the mutation frequencies that do not exceed the GEF 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 tobe toxicologically significant. Small significant increases designated by the UKEMS statistical package will be reviewed using the above criteria, and may be disregarded at the Study Director's discretion.

Statistics:

The experimental data was analyzed using a dedicated computer program which follows the statistical guidelines recommended by the UKEMS statistical package. Dose levels that have survival values less than 10% are excluded from any statistical analysis, as any response they give would be considered to have no biological or toxicological relevance.

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: None
- Effects of osmolality: None

RANGE-FINDING/SCREENING STUDIES:
The dose range of the test item used in the preliminary toxicity test was 5.56 to 1422.5 µg/mL . There was evidence of marked reductions in the Relative Suspension Growth (%RSG) of cells treated with the test item when compared to the concurrent vehicle controls in all three of the exposure groups. The onset of test item-induced toxicity was more apparent in the absence of metabolic activation, with maximum exposure occurring at 88.91 µg/mL in both the 4-hour and 24-hour exposure. In the presence of metabolic activation the test item induced toxicity was less prominent, but it was considered that slight toxicity occurs around 88.91 µg/mL. A greasy oily precipitate of the test item was observed at and above 88.91 µg/mL in the 4-hour exposure group in the absence of metabolic activation and at and above 177.81 µg/mL in the presence of metabolic activation. In the 24-hour exposure group a greasy oily precipitate can be observed at and above 355.63 µg/mL. Based on the %RSG values observed and the onset of greasy oily precipitate the maximum dose level in the subsequent mutagenicity experiment was limited by test item-induced toxicity for the 24-hour exposure group and precipitate for both of the 4-hour exposure groups.

Remarks on result:

other: strain/cell type: mouse lymphoma L5178Y cells

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Experience 1

There was evidence of slight toxicity (around 45 µg/mL) following exposure to the test item in both the absence and presence of metabolic activation, as indicated by the RTG values (Tables3 and 6). There was also evidence of slight reductions in viability (%V) in both the absence and presence of metabolic activation, therefore indicating that residual toxicity had occurred.

Acceptable levels of toxicity were seen with both positive control substances (Tables 3 and 6).

The vehicle controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. 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 (Tables 3 and 6).

The test item did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency 10^(-6) per viable cell, at any of the dose levels (Tables 3 and 6). It should be noted that all mutant frequency values were within the acceptable range for a vehicle control culture and the GEF value was not exceeded. With no evidence of any toxicologically significant increases in mutant frequency in either the absence or presence of metabolic activation the test item was considered to have been adequately tested. Precipitate of the test item was observed at and above 67.5 µg/mL in the absence of metabolic activation and at 180 µg/mL in the presence of metabolic activation. It was therefore concluded that the test item had been adequately tested. The number of small and larges colonies and their analysis are presented in Table 4 and 7.

Experience 2

There was evidence of marked toxicity in both the absence and presence of metabolic activation, as indicated by the %RSG and RTG Values (Tables 9 and 12). There was evidence of a reduction in viability (%V) in both the absence and presence of metabolic activation, therefore indicating that residual toxicity had occurred (Tables 9 and 12). Based on the RTG and %RSG values observed, optimum levels of toxicity were considered to have been achieved in the absence of metabolic activation, whilst adequate levels of toxicity were achieved in the presence of metabolic activation (Tables 9 and 12). A dose level in the absence of metabolic activation (33.75 µg/mL), whilst having a %RSG below the acceptable range was plated out for expression of mutant frequency, however, the data was later discarded due to excessive toxicity. Acceptable levels oftoxicity were seen with both positive control substances (Tables 9 and 12).

The 24-hour exposure without metabolic activation (S9) treatment, demonstrated that the extended time point had a marked effect on the toxicity ofthe test item.

The vehicle (solvent) controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. 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 (Tables 9 and 12).

The test item did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10^(-6) per viable cell, at any dose level, in either the absence or presence of metabolic activation (Tables 9 and 12). The GEF value was not exceeded at any dose including one with excessive toxicity. It was therefore considered that the test item had been adequately tested. A cloudy precipitate of test item was observed at and above 67.5 µg/mL in the presence of metabolic activation. The number of small and larges colony and their analysis are presented in Table 10 and 13.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The test item did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and is therefore considered to be non-mutagenic under the conditions of the test.

Executive summary:

Introduction The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method was designed to be compatible with the OECD Guidelines for Testing of Chemicals No.476 "In Vitro Mammalian Cell Gene Mutation Tests", Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, the US EPA OPPTS 870.5300 Guideline, and be in alignment with the Japanese MITI/MHW guidelines for testing of new chemical substances. Method Two independent experiments were performed. In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at eight dose levels, in duplicate, together with vehicle (acetone) 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 item 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 of test item used in the main test was selected following the results of a preliminary toxicity test. The maximum dose level used in the main test was limited by test item induced toxicity. Results The maximum dose levels used in the Mutagenicity Test were limited by test item-induced toxicity and the onset of precipitate. Overall a precipitate of the test item was observed at and above 67.5 µg/mL. The vehicle controls (acetone) had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/— locus. The positive control treatment induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolizing system. The test item did not induce any toxicologically significant increases in the mutant frequency at any of the dose levels, either with or without metabolic activation, in either the first or the second experiment. Conclusion The test item did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells.