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Genetic toxicity in vitro

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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
Reliability:
1 (reliable without restriction)
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Target gene:
thymidine kinase
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
Mouse Lymphoma L5178Y cells have been used successfully in in vitro experiments for many years. These cells are characterised by their high proliferation rate (10-12 h doubling time of the BSL BIOSERVICE stock cultures) and their cloning efficiency, usually more than 50%. The cells obtain a near diploid karyotype (40 ± 2 chromosomes). They are heterozygous at the Thymidine Kinase (TK) locus in order to detect mutation events at the TK-locus. To prevent high backgrounds arising from spontaneous mutation, cells lacking TK can be eliminated by culturing cells in RPMI 1640 supplemented with: 9.0 ug/mL hypoxanthine
15.0 ug/mL thymidine
22.5 ug/mL glycine
0.1 ug/mL methotrexate
The cells are resuspended in medium without methotrexate but thymidine, hypoxanthine and glycine for 1-3 days. Large stock cultures of the cleansed L5178Y cell line are stored over liquid nitrogen (vapour phase) in the cell bank of BSL BIOSERVICE. This allows the repeated use of the same cell batch in experiments. Each cell batch is routinely checked for mycoplasma infection.

Thawed stock cultures are maintained in plastic culture flasks in RPMI 1640 complete medium and subcultured three times per week.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
mmammalian microsomal fraction S9 homogenate
Test concentrations with justification for top dose:
The toxicity of the test item was determined in pre-experiments up to a maximum concentration of 9. 6 mM.
Reason:
For the solubility experiment and for pre-experiment I the correction factor of 1.042 was not applied to correct for 100% purity of the test item. Due to this in the solubility experiment and in pre-experiment I the maximum concentration was 9.6 mM instead of 10 mM. However, both experiments still provided the information required and results were used for evaluation of solubility and cytotoxicity of the test item.
Vehicle / solvent:
ethanol (0.5% v/v)
Untreated negative controls:
yes
Remarks:
treatment medium
Negative solvent / vehicle controls:
yes
Remarks:
0.5% v/v ethanol
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
ethylmethanesulphonate
methylmethanesulfonate
Details on test system and experimental conditions:
Pre-Experiment for Toxicity
The toxicity of the test item was determined in pre-experiments up to a maximum concentration of9.6 mM. For experiment I six concentrations [0.19, 0.48, 2.4, 4.8, 7.2 and 9.6 mM] were tested with and without metabolic activation. Due to high cytotoxicity of the test item only dose groups up to 2.4 mM are reported. For the 24 h long~terrn exposure assay (experiment II, without metabolic activation) eight concentrations [0.0048, 0.0096, 0.019, 0.048, 0.096, 0.48, 0.96, 1.92 mM] were tested. Due to high cytotoxicity of the test item only dose groups up to 0.48 mM are reported. The experimental conditions in these pre-experiments were the same as described below under "experimental performance".

Exposure Concentrations
The selection of the concentrations used in the main experiments was based on data from the pre-experiment. In experiment I 3.00 mM (with metabolic activation) and 0.50 mM (without metabolic activation) were selected as the highest concentrations. In experiment II 0.40 mM (with metabolic activation) and 0.20 mM (without metabolic activation) were selected as the highest concentrations. Experiment I with and without metabolic activation and experiment II with metabolic activation were performed as 4 h short-term exposure assay. Experiment II without metabolic activation was performed
as a 24 h long-term exposure assay. The test item was investigated at the following concentrations:
Experiment I with metabolic activation:
0.1 0, 0.20, 0.40, 0.80, 1.20, 1.60, 2.00, 2.40, 2.80 and 3.00 mM
Experiment I without metabolic activation:
0.05, 0.08, 0.10, 0.15, 0.20, 0.30, 0.40 and 0.50 mM
Experiment II with metabolic activation:
0.01, 0.02, 0.05, 0.10, 0.20, 0.30, 0.35 and 0.40 mM
Experiment II without metabolic activation:
0.002, 0.005, 0.01, 0.02, 0.05, 0.10, 0.15 and 0.20 mM
According to OECD Guidelines at least 8 concentrations of the test item were set up in the experiments with and without metabolic activation.

Cell Culture Media
For preparation of the different media horse serum (HS) was heat-inactivated for 30 min. at 56 degrees C prior usage.

Complete Culture Medium (RPMI 1640 medium) supplemented with:
10 % Horse serum (HS)
1 00 U/1 00 ug/mL penicillin/streptomycin
1 mM sodium pyruvate
2 mM L-glutamine
25 mM HEPES
2.5 ug/mL amphotericin B

Treatment Medium (RPMI 1640 medium) supplemented with:
5% HS (in case of short-term exposure)
7.5 % HS (in case of long-term exposure)
100 U/100 J.lg/mL penicillin/streptomycin
1 mM sodium pyruvate
2 mM L-glutamine
25 mM HEPES
2.5 ug/mL amphotericin B

Selective Medium (RPMI 1640 medium) supplemented with:
20% HS
100 U/100 ug/mL penicillin/streptomycin
1 mM sodium pyruvate
2 mM L-glutamate
25 mM HEPES
2.5 1-tg/mL amphotericin
5 ug/mL TFT


Evaluation criteria:
Acceptability of the Assay
A mutation assay is considered acceptable if it meets the criteria mentioned in current international guidelines and the current recommendations of the IWGT (11, 12, 13, 14, 15):
- At least three out of four 96-well plates from the TFT resistance-testing portion of the experiment are scorable.
- The cloning efficiency of the negative and/or solvent controls is in the range 65%-120%.
- The spontaneous mutant frequency in the negative and/or solvent controls is in the range 50-170 mutants per 1 06 cells
- The cell number of the negative/solvent controls should undergo 8-32 fold increase during a 2 day growth period (short-term treatment) or 32-1 80 fold increase during a 3 day growth period (long-term treatment).
- The clastogenic positive controls (MMS and B[a]P) have to produce an induced mutant frequency (total mutant frequency minus concurrent negative control mutant frequency) of at least 300 mutants per 106 cells with at least 40% of the colonies being small colonies or with an induced small
colony mutant frequency of at least 150 mutants per 106 cells The RTG must be greater than 10%.

Evaluation of Results
The test item is considered mutagenic if following criteria are met (13, 14, 15):
The induced mutant frequency meets or exceeds the Global Evaluation factor (GEF) of 126 mutants per 106 celts
A dose-dependent increase in mutant frequency is detected.
Besides, combined with. a positive effect in the mutant frequency, an increased occurrence of small colonies (~40% of total colonies) is an indication for potential clastogenic effects and/or chromosomal aberrations.

According to the OECD guideline, the biological relevance is considered first for the interpretation of results. Statistical methods might be used as an aid in evaluation the test result. A test item is considered to be negative if the induced mutant frequency is below the GEF and the trend test is negative.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
above 2.4 mM
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: strain/cell type:
Remarks:
Migrated from field 'Test system'.
Conclusions:
Eastman ™ meta-Diisopropylbenzene is considered to be non-mutagenic in the in vitro mammalian cell gene mutation assay (thymidine kinase locus)
in mouse lymphoma L5178Y cells.
Executive summary:

The test item Eastman ™ meta-Diisopropylbenzene was assessed for its potential to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y. The selection of the concentrations used in the main experiments was based on data from a pre-experiments. The test item was investigated at the following concentrations: Experiment I with metabolic activation: 0.10, 0.20, 0.40, 0.80, 1.20, 1.60, 2.00, 2.40, 2.80 and 3.00 mM and without metabolic activation: 0.05, 0.08, 0.10, 0.15, 0.20, 0.30, 0.40 and 0.50 mM Experiment II with metabolic activation: 0.01, 0.02, 0.05, 0.1 0, 0.20, 0.30, 0.35 and 0.40 mM and without metabolic activation: 0.002, 0.005, 0.01, 0.02, 0.05, 0.10, 0.15 and 0.20 mM Precipitation of the test item was noted in the pre-experiment I with and without metabolic activation at a concentration of 2.4 mM and higher. In the experiments I and II with and without metabolic activation and in the pre-experiment II without metabolic activation no precipitation was noted. Growth inhibition was observed in experiment I and II with and without metabolic activation. In experiment I with metabolic activation the relative total growth (RTG) was 12.3% for the highest concentration (3.00 mM) evaluated. The highest concentration evaluated without metabolic activation was 0.50 mM with a RTG of21.5%. In experiment II with metabolic activation the relative total growth (RTG) was 15.7% for the highest concentration (0.40 mM) evaluated. The highest concentration evaluated without metabolic activation was 0.20 mM with a RTG of 7.9%. In experiment I and II no biologically relevant increase of mutants was found after treatment with the test item (with and without metabolic activation). The Global Evaluation Factor (GEF; defined as the mean of the negative/vehicle mutant frequency plus one standard deviation; data gathered from ten laboratories) was not exceeded by the induced mutant frequency at any concentration. No dose-response relationship was observed. Additionally, in experiment I and II colony sizing showed no clastogenic effects induced by the test item under the experimental conditions (with and without metabolic activation). EMS, MMS and B[a]P were used as positive controls and showed distinct and biologically relevant effects in mutation frequency. Additionally, MMS and B[a]P significantly increased the number of small colonies, thus proving the efficiency of the test system to indicate potential clastogenic effects.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

The mixed isomers of m-DIPB and p-DIPB were evaluated in a bacterial reverse mutation (Ames) assay using Salmonella typhimurium TA 100, TA 1535, TA 98, TA 1537 and Escherichia coli WP2 uvrA, with or without an exogenous metabolic activation system. Data indicate that the mixed isomers of DIPB were not mutagenic, either in the presence or absence of metabolic activation.

 

The mixed isomers of m-DIPB and p-DIPB also were evaluated in a chromosomal aberration assay in Chinese hamster lung cells (CHL/IU), with or without an exogenous metabolic activation system. Data indicate that the mixed isomers of DIPB did not produce either chromosome aberrations or polyploidy.

m-DIPB also was evaluated in a mammalian cell (mouse lymphoma) mutagenesis assay, with or without an exogenous metabolic activation system. Data indicate that m-DIPB was not mutagenic, either in the presence or absence of metabolic activation.

Justification for classification or non-classification

Based upon negative findings from exposure of m-DIPB (in combination with p-DIPB) in a bacterial reverse mutation (Ames) assay and a mammalian cell chromosome aberration assay and negative findings from exposure of m-DIPB in a mammalian cell mutagenesis assay, it was determined that m-DIPB does not satisfy the criteria for classification as an ocular irritant according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008 or UN Globally Harmonized System of Classification and Labelling of Chemicals (GHS).