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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
Non-mutagenic (OECD 471 and OECD 476) and non-clastogenic (OECD 473)
Link to relevant study records
Reference
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:
05 December 2011 to 24 January 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Date of inspection 2011-07-19 to 2011-07-21; Date of signature 2011-08-31
Type of assay:
mammalian cell gene mutation assay
Test material information:
Composition 1
Species / strain:
mouse lymphoma L5178Y cells
Details on mammalian cell lines (if applicable):
- Type and identity of media: The L5178Y TK+/- 3.7.2c mouse lymphoma cell line was obtained from the MRC Cell Mutation Unit at the University of Sussex, Brighton, UK.
- Properly maintained: yes - 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 subcultured accordingly.
- Periodically "cleansed" against high spontaneous background: yes - 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.
Metabolic activation:
with and without
Metabolic activation system:
S9 microsomal enzyme fraction
Test concentrations with justification for top dose:
Preliminary toxicity test: 0, 7.19, 14.38, 28.75, 57.5, 115, 230, 460, 920 and 1840 µg/mL

Experiment 1 (without S-9 mix): 0, 1.25, 2.5, 5, 10, 20, 40, 50 and 60 µg/mL
Experiment 1 (with S-9 mix): 0, 5, 10, 20, 40, 60, 80, 100 and 120 µg/mL

Experiment 2 (without S-9 mix): 0, 2.5, 5, 10, 20, 40, 50, 60 and 80 µg/mL
Experiment 2 (with S-9 mix): 0, 10, 20, 40, 60, 80, 100, 120 and 140 µg/mL
Vehicle:
The test material was formulated in dimethyl sulphoxide (DMSO).
Negative controls:
no
Solvent controls:
yes
Remarks:
Dimethyl sulphoxide
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Experiment 1: 400 µg/mL; Experiment 2: 150 µg/mL; without metabolic activation
Positive control substance:
cyclophosphamide
Remarks:
2 µg/mL; with metabolic activation
Details on test system and conditions:
METHOD OF APPLICATION: in medium

PRELIMINARY TOXICITY TEST
A preliminary toxicity test was performed on cell cultures at 5E+05 cells/mL using a 4 hour exposure time both with and without metabolic activation and at 1.5E+05 cells/mL using a 24 hour exposure period without S9. The dose range used in the preliminary toxicity test was 7.19 to 1840 µg/mL for all 3 of the exposure groups. Following the exposure period the cells were washed twice with R10, resuspended in R20 medium, counted and then serially diluted to 2E+05 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 2E+05 cells/mL. 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 treatment toxicity and a comparison of each treatment SG value to the concurrent vehicle control performed to give a % Relative Suspension Growth (%RSG) value.
Results from the preliminary toxicity test were used to set the test item dose levels for the mutagenicity experiments.

MUTAGENICITY TEST: EXPERIMENT 1
Cells were counted and processed to give 1E+06 cells/mL in 10 mL aliquots in R10 medium in sterile plastic universals. The treatments were performed in duplicate both with and without metabolic activation at 8 dose levels of the test item (1.25 to 60 µg/mL in the absence of metabolic activation and 5 to 120 µg/mL in the presence of metabolic activation), vehicle and positive controls. To each universal was added 2 mL of S9-mix (if required), 0.2 mL of the treatment dilutions, 0.2 mL for the positive control and sufficient R0 medium to bring the total volume to 20 mL.
The treatment vessels were incubated at 37 °C for 4 hours with continuous shaking.

MUTAGENICITY TEST: EXPERIMENT 2
Cells were counted and processed to give 1E+06 cells/mL in 10 mL duplicate cultures in R10 medium for the 4 hour treatment with metabolic activation cultures. In the absence of metabolic activation the exposure period was extended to 24 hours; therefore, 0.3E+06 cells/mL in 10 mL duplicate cultures were established in 25 cm2 tissue culture flasks. To each culture 2 mL of S9-mix was added (if required), 0.2 mL of the treatment dilutions, 0.2 mL for the positive control and sufficient R0 medium to give a final volume of 20 mL (R10 is used for the 24 hour exposure group). The dose range of the test item was 2.5 to 80 µg/mL in the absence of metabolic activation and 10 to 140 µg/mL in the presence of metabolic activation.
The treatment vessels were incubated at 37 °C with continuous shaking for 24 hours in the absence of metabolic activation and 4 hours in the presence of metabolic activation.

MICROSOMAL ENZYME FRACTION
PB/βNF S9 microsomal fraction was prepared from the livers of male Sprague-Dawley rats weighing approximately 250 g. These had each received, orally, 3 consecutive daily doses of Phenobarbitone/β-Naphthoflavone (80/100 mg/kg/day) prior to S9 preparation on day 4. Aliquots of supernatant were frozen and stored at approximately -196 °C.
The S9-mix was prepared by mixing S9, 33 mM KCl, 8 mM MgCl2, 5 mM glucose-6-phosphate and 5 mM NADP in R0.
20% S9-mix (i.e. 2% final concentration of S9) was added to the cultures of the preliminary toxicity test and of Experiment 1. In Experiment 2, 0% S9-mix (i.e. 1% final concentration of S9) was added.

DURATION
- Exposure duration: 4-hour exposure with and without metabolic activation in Experiment 1 and with metabolic activation in Experiment 2 and 24-hour exposure without metabolic activation in Experiment 2.
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): On Day 2 of the experiment, the cells were counted, diluted to 10E+04 cells/mL and plated for mutant frequency (2000 cells/well) in selective medium containing 4 µg/mL 5-trifluorothymidine (TFT) in 96-well microtitre plates. Cells were also diluted to 10 cells/mL and plated (2 cells/well) for viability (%V) in non-selective medium.

STAIN: To assist the scoring of the TFT mutant colonies 0.025 mL of MTT solution (2.5 mg/mL in PBS) was added to each well of the mutation plates. The plates were incubated for approximately two hours.

SELECTION AGENT (mutation assays): 5 trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: Each treatment was performed in duplicate (A + B), both with and without metabolic activation (S9-mix).

MEAUREMENT OF SURVIVAL, VIABILITY AND MUTANT FREQUENCY
At the end of the treatment period, for each experiment, the cells were washed twice using R10 medium then resuspended in R20 medium at a cell density of 2E+05 cells/mL. The cultures were incubated at 37 °C with 5% CO2 in air and subcultured every 24 hours for the expression period of 2 days by counting and diluting to 2E+05 cells/mL. On Day 2 of the experiment, the cells were counted, diluted to 10E+04 cells/mL and plated for mutant frequency (2000 cells/well) in selective medium containing 4 µg/mL 5-trifluorothymidine (TFT) in 96-well microtitre plates. Cells were also diluted to 10 cells/mL and plated (2 cells/well) for viability (%V) in non-selective medium.
The daily cell counts were used to obtain a %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 %V data a Relative Total Growth (RTG) value.

PLATE SCORING
Microtitre plates were scored after 10 to 14 days incubation at 37 °C with 5% CO2 in air. To assist the scoring of the TFT mutant colonies 0.025 mL of MTT solution (2.5 mg/mL in PBS) was added to each well of the mutation plates. The plates were incubated for approximately two hours.

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Evaluation criteria:
The normal range for mutant frequency per survivor is 50-200 x 10E-06 for the TK+/- locus in L5178Y cells. Vehicle control results should ideally be within this range.
Positive control chemicals should induce at least 3 to 5 fold increases in mutant frequency greater than the corresponding vehicle control.
For a test material 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.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Additional information on results:
PRELIMINARY TOXICITY TEST
In all 3 of the exposure groups there was evidence of marked dose-related reductions in the %RSG of cells treated with the test item when compared to the concurrent vehicle controls. The steep nature of the toxicity curve was taken to indicate that achieving optimum toxicity would be difficult. A greasy/oily precipitate was observed at and above 230 µg/mL at the end of the exposure period in all 3 of the exposure groups. In addition, an increase in intensity was associated with an increase in dose concentration. Based on %RSG values observed, the maximum dose levels in the subsequent mutagenicity test were limited by test item induced toxicity.

MUTAGENICITIY TEST - EXPERIMENTS 1 AND 2:
Based on the %RSG and RTG values observed, it was considered that optimum levels of toxicity had been achieved in the presence of metabolic activation and very near optimum levels of toxicity in the absence of metabolic activation. Acceptable levels of toxicity were seen with both positive control substances.
The 24 hour exposure in Experiment 2 without metabolic activation demonstrated that the extended time point had no marked effect on the toxicity of the test item.
Neither of the vehicle control mutant frequency values were outside the acceptable range. Both of the positive controls produced marked increases in the mutant frequency per viable cell.
The test item did not induce any statistically significant or dose related increases in the mutant frequency x10E-06 per viable cell in either the absence or presence of metabolic activation.

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitate of test item was not observed at any of the dose levels in either Experiment.
Conclusions:
Interpretation of results (migrated information):
negative with and without metabolic activation

The test item, 9-decenoic acid, methyl ester (9DAME) 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 conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

In vitro gene mutation study in bacteria:

In a reliable GLP OECD guideline 471 study (Harlan Laboratories Ltd, 2012), 9-decenoic acid, methyl ester (9-DAME) was tested in a bacterial reverse mutation assay in Salmonella typhimurium (strains TA98, TA100, TA1535 and TA1537) and Escherichia coli WP2 uvrA with and without metabolic activation (S9). The concentrations tested were between 5 and 5,000μg/plate in the first experiment and between 0.15 and 5000 µg/plate in Experiment 2. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation or exposure method. The test item, 9-decenoic acid, methyl ester (9-DAME) was therefore considered to be non-mutagenic.

 

In vitro chromosome aberration study in mammalian cells:

In a reliable GLP OECD guideline 473 study (Harlan Laboratories Ltd, 2012), 9-decenoic acid, methyl ester (9-DAME) was tested for its ability to induce structural chromosomal aberrations in cultured mammalian cells (human lymphocytes) in the presence and absence of metabolic activation. The test item did not induce any statistically significant increases in the frequency of cells with aberrations, in either of two separate experiments, using a dose range that included a dose level that induced approximately 50% mitotic inhibition. 9-decenoic acid, methyl ester was therefore considered to be non-clastogenic to human lymphocytes in vitro.

 

In vitro gene mutation study in mammalian cells:

In a reliable GLP OECD guideline 476 study (Harlan Laboratories Ltd, 2012), 9-decenoic acid, methyl ester (9-DAME) was tested for its ability to induce mutations in mouse lymphoma L5178Y cells in the presence and absence of metabolic activation. The test item did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or second experiment. 9-decenoic acid, methyl ester (9-DAME) was therefore considered to be non mutagenic to L5178Y cells under the conditions of the test.

 


Justification for selection of genetic toxicity endpoint
The study has been conducted according to OECD Guideline 476 and to GLP and is adequately reported. As such the study as been assigned a reliability 1.

Justification for classification or non-classification

The results of an in vitro gene mutation study in bacteria, an in vitro chromosome aberration study in mammalian cells and an in vitro gene mutation study in mammalian cells were all negative. Therefore, it is concluded that 9-decenoic acid, methyl ester (9-DAME) is not genotoxic and does not warrant classification for mutagenicity according to CLP Regulation EC 1272/2008.