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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance is negative in the Ames test. Hexadecylacetate did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells or chromosome aberrations in human lymphocytes and is therefore considered to be non-mutagenic/clastogenic.

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Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07 August 2014 to 07 November 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
NA
Species / strain / cell type:
lymphocytes:
Details on mammalian cell type (if applicable):
Cells were grown in Eagle's minimal essential medium with HEPES
buffer (MEM), supplemented "in-house" with L-glutamine, penicillin/ streptomycin, amphotericin B and 15% foetal calf serum
Cytokinesis block (if used):
yes, democolcine
Metabolic activation:
with and without
Metabolic activation system:
phenobarbitone/β-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Experiment 1 (μg/ml) without S9: 0, 5.63, 11.25, 22.5, 45, 90, 180 0, 5.63, Experiment 1 (μg/ml) with S9: 11.25, 22.5, 45, 90, 180 μg/ml
Experiment 2 (μg/ml) without S9: 0, 5.63, 11.25, 22.5, 45, 90, 180, 360
Experiment 2 (μg/ml) with S9: 0, 5.63, 11.25, 22.5, 45, 90, 180, 360

Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: The test item was not sufficiently soluble in water or DMSO.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
acetone
Positive controls:
yes
Positive control substance:
not specified
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; preincubation; in suspension; as impregnation on paper disk
With Metabolic Activation: Cultures were established approximately 48 hours prior to treatment. Cultures were incubated at 37°C for 4 hours in the presence of the test material prior to washing.

Without Metabolic Activation: Cultures were established approximately 48 hours prior to treatment. In Experiment 1, cultures were incubated at 37°C for 4 hours in the presence of the test material prior to washing. In Experiment 2, the cultures were incubated in the presence of the substance at 37°c for 24 hours

DURATION
- Preincubation period: 48
- Exposure duration: 4 or 24 hours
- Expression time (cells in growth medium): 20 or 0 hours
- Selection time (if incubation with a selection agent): N/A
- Fixation time (start of exposure up to fixation or harvest of cells): Mitosis was arrested by addition of democolcine two hours prior to the required harvest time and the cells were harvested and fixed

SELECTION AGENT (mutation assays): N/A
SPINDLE INHIBITOR (cytogenetic assays): yes
STAIN (for cytogenetic assays): 5% Gurrs Giemsa
NUMBER OF REPLICATIONS: Treatments performed in duplicate.
NUMBER OF CELLS EVALUATED: A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value. Where possible the first 100 consecutive well-spread metaphases from each culture were counted.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes in comparison to controls
- Determination of endoreplication: If the chromosomes are arranged in closely apposed pairs, ie. 4 chromatids instead of 2, the cell is scored as endoreduplicated
Evaluation criteria:
A positive response was recorded for a particular treatment if the % cells with aberrations, excluding gaps, markedly exceeded that seen in the concurrent control, either with or without a clear dose relationship.
For modest increases in aberration frequency a dose response relationship is generally required and appropriate statistical tests may be applied in order to record a positive response.
Statistics:
The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.
Species / strain:
lymphocytes: primary culture
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
concentrations limited by the presence of precipitate, see text
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Preliminary Toxicity Test
The dose range for the Preliminary Toxicity Test was 0, 11.11, 22.23, 44.45, 88.91, 177.81, 355.63, 711.25, 1422.5, 2845 μg/ml.
The maximum dose was the 10 mM concentration. A precipitate of the test item was observed at and above 44.45 μg/ml in both non-S9 exposure groups and at and above 88.91 ug/ml in the with-S9 exposure group.
Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present at up to 2845 ug/mL in both of the 4(20)-hour exposure groups. In the 24-hour exposure group metaphase cells were present up to 2845 ug/mL. The selection of the
maximum dose level was based on toxicity and also on the onset of the formation of a greasy/oily precipitate, where it was considered that the maximum exposure to the test material was occurring.
Experiment 1
No inhibition of mitotic index was observed in the absence of 2% S9 or in the presence of S9. All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control items induced statistically significant increases in the frequency of cells with aberrations indicating that the sensitivity of the assay and the efficacy of the S9-mix were validated. The test item did not induce any statistically significant increases in the frequency of cells with aberrations, either in the absence or presence of metabolic activation. There was no significant increase in the incidence of polyploidy.

Experiment 2
A dose-related inhibition of mitotic index was observed in both exposure groups. In the absence of S9, 25% mitotic inhibition was observed at 180 ug/mL. In the presence of 1% S9, a maximum of 28% mitotic inhibition was achieved at 90 ug/mL. The maximum dose level selected for metaphase analysis was based on toxicity and precipitate in both exposure groups.

Results
All vehicle (acetone) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control items induced statistically significant increases in the frequency of cells with aberrations indicating that the sensitivity of the assay and the efficacy of theS9-mix were validated. The test item was slightly toxic and exposure was limited by the solubility of the test substance. The test substance did not induce any statistically significant increases in the frequency of cells with aberrations, in either of two separate experiments.
Conclusions:
The test item was considered to be non-clastogenic to human lymphocytes in vitro.
Endpoint:
in vitro gene mutation study in mammalian cells
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:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
GLP compliance:
yes
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Target gene:
TK
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
mammalian cell line
Details on mammalian cell lines (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

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 phenobarbital/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.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
acetone
Positive controls:
yes
Positive control substance:
not specified
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.

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 to be 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.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
see text
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Range-Finding
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.

Experiment 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. 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.
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.
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. 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.

Experiment 2
There was evidence of marked toxicity in both the absence and presence of metabolic activation, as indicated by the %RSG and RTG Values. 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. 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. 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 of toxicity were seen with both positive control substances.
The 24-hour exposure without metabolic activation (S9) treatment, demonstrated that the extended time point had a marked effect on the toxicity of the 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.
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. 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.
Conclusions:
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.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
other: abstract
Adequacy of study:
weight of evidence
Study period:
01 December 2014
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
abstract
Justification for type of information:
The test has been performed by a recognized GLP laboratory, but only a one page abstract is available
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
not specified in the abstract
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
His/Trp
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Species / strain:
other: all strains tested
Metabolic activation:
not specified
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Conclusions:
The substance as tested was negative in the Ames test
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

Based on the information available, the substance does not need to be classified for mutagenicity according to EC Regulation 1272/2008 (CLP).