1,3-diisopropylbenzene

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

adopted July 21, 1997 - new OECD Guideline for this kind of test: OECD Guideline 490 (issued 2015)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

August 1998

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

dated May 30, 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Test material

Test material information

Constituent 1

Reference substance name:

1,3-diisopropylbenzene

EC Number:

202-773-1

EC Name:

1,3-diisopropylbenzene

Cas Number:

99-62-7

Molecular formula:

C12H18

IUPAC Name:

1,3-bis(propan-2-yl)benzene

Test material form:

other: colorless liquid

Method

Target gene:

thymidine kinase

Species / strain

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 µg/mL hypoxanthine
15.0 µg/mL thymidine
22.5 µg/mL glycine
0.1 µg/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:

mammalian microsomal fraction S9 homogenate Type and composition of metabolic activation system:
- method of preparation of S9 mix : 8 mM MgCl2 + 33 mM KCl + 5 mM Clucose-6-phosphate + 5 mM NADP in 100 mM sodium-phosphate buffer pH 7.4.
- concentration or volume of S9 mix and S9 in the final culture medium: 0.75 mg/mL in the cultures
- quality controls of S9: Biological activity, sterility test

Test concentrations with justification for top dose:

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

The toxicity of the test item was determined in pre-experiments up to a maximum concentration of 9. 6 mM.
The results of the pre-tests are shown in attachment. Due to high cytotoxicity of the test item only dose groups up to 0.48 mM are reported.
Pre-Experiment for Toxicity
The toxicity of the test item was determined in pre-experiments up to a maximum concentration of 9.6 mM (The correction factor of 1.042 was not applied to correct for 100 % purity of the test item. Due to this in the in pre-experiment I the maximum concentration was 9.6 mM instead of 10 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-term 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 in the main test.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle:
A solubility test was performed with different solvents and vehicles up to a concentration of 9.6 mM. It was decided to use ethanol.
- Justification for percentage of solvent in the final culture medium: 0.5 % v/v

Controls

Untreated negative controls:

yes

Remarks:

treatment medium

Negative solvent / vehicle controls:

yes

Remarks:

0.5 % v/v ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

ethylmethanesulphonate

methylmethanesulfonate

Details on test system and experimental conditions:

Experiment I with and without metabolic activation and Experiment II with metabolic activation were performed as a 4 h short-term exposure assay.
Experiment II without metabolic activation was performed as a 24 h long-term exposure assay

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): 4
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): The 1 x 10E7 cells/ 11 mL (short-term assay), 5 x 10E6 cells/ 25 mL (long-term assay)
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 4 h (short-term assay), 24 h (long-term assay)
- Harvest time after the end of treatment (sampling/recovery times): 2 d

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 2 d
- Selection time (if incubation with a selective agent): 14 d
- Fixation time (start of exposure up to fixation or harvest of cells):
- Method used: microwell plates
- If a selective agent is used (e.g., 6-thioguanine or trifluorothymidine), indicate its identity, its concentration and, duration and period of cell exposure.: 200 µL trifluorothymidine, 14 d exposure
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: 2000 cells/well

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method.: relative total growth (RTG), relative cloning efficiency

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 10E6 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 32-180 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 10E6 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 10E6 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 10E6 cells
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.

Statistics:

non-parametric Mann-Whitney test

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES (if applicable): please refer to attached tables

STUDY RESULTS
- Concurrent vehicle negative and positive control data : please refer to attached tables

Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements: please refer to attached tables

- Genotoxicity results: please refer to attached tables

Applicant's summary and conclusion

Conclusions:

The test item 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 genetic toxicity test - an in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells - was performed originally according to OECD guideline 476 (the test method used fully corresponds to the new OECD Guideline 490, which was issued in 2015), EU method B.17 and EPA OPPTS 870.5300 with GLP compliance. 

The test item was assessed for its potential to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y. 

The test item meta-Diisopropylbenzene (m-DIPB) 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 the pre-experiments.

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 without metabolic activation were performed as a 4h 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:

0.10,0.20, 0.40,0.80, 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, 0.10, 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 of 21.5 %. 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 [13,14.15]) 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 biological[y 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.

 

In conclusion, in the described mutagenicity test under the experimental conditions reported, the test item meta-DiisopropyIbenzene (m-DIPB) is considered to be non-mutagenic in the in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells.