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Genetic toxicity in vitro

Description of key information

Gene mutation in bacteria


Read across to Mixture of 1,3-diisopropylbenzene and 1,4-diisopropylbenzene:


- Ames test, OECD 471, S. typhimurium TA 100, TA 1535, TA 98, TA 1537 and E. coli WP2 uvrA, +/- S9, negative


 


Cytogenicity


Read across to Mixture of 1,3-diisopropylbenzene and 1,4-diisopropylbenzene:


- Chromosome aberration test, OECD 473, Chinese hamster lung cells (CHL/IU), +/-S9, negative


 


Gene mutation in mammalian cells


- Mouse lymphoma Tk assay, OECD 476 (new OECD 490), GLP, L5178Y, +/-S9, negative

Link to relevant study records

Referenceopen allclose all

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
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
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Version / remarks:
August 1998
Deviations:
no
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
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 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
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
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
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.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
In this justification, the read-across (bridging) concept is applied. Please refer to a full version of Read-across statement attached in the section 13 "Assessment reports".

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The underlying hypothesis for the read-across is that the target and the source substance have similar toxicological properties (including the same target organs) due to their structural similarity, resemblance to their chemical reactivity, and therefore a similar mode of action.
The source substance ‘Mixture of 1,3-diisopropylbenzene and 1,4-diisopropylbenzene' is a mixture of structural isomers, 1,3- diisopropylbenzene(target substance) and 1,4-diisopropylbenzene. The physico-chemical properties are highly equivalent based on the high structural similarity. As a conclusion, it is scientifically justified to address the endpoint Genetic toxicity with data on this mixture of isomers.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source substance: Mixture of 1,3-diisopropylbenzene and 1,4-diisopropylbenzene (Diisopropylbenzene), CAS no. 25321-09-9
structural formula: C12H18
Smiles: CC(C)C1=CC=CC=C1C(C)C
Molecular weight: 162 g/mol
CAS 25321-09-9
EC No 246-835-6
purity: not specified

target substance: 1.3-diisopropylbenzene (or m-DIPB)
structural formula: C12H18
Smiles: CC(C)c1cccc(c1)C(C)C
Molecular weight: 162 g/mol
CAS 99-62-7
EC No 202-773-1
purity: not specified

No additional information is available on purity of the source and the target substances. Both substances are normally of high purity, containing only minor amounts of impurities that do not influence the read-across validity.

3. ANALOGUE APPROACH JUSTIFICATION
Please refer to the full version of the read-across statement attached in the section 13 "Assessment reports".

4. DATA MATRIX
Please refer to the full version of the read-across statement attached in the section 13 "Assessment reports".
Reason / purpose for cross-reference:
read-across source
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES (if applicable):
Diisopropylbenzene was tested with a ratio of about 3 in the range of 50.0 to 5000 μg/plate. As a result, in the S9 mix-free test, antimicrobial activity was observed at 5000 μg/plate for WP2 uvrA and at all doses for the other test bacteria. In the S9 mix addition test, antimicrobial properties were observed for WP2 uvrA at 500 μg/plate or more and for the other test bacteria at more than 150 μg/plate.
Therefore, the highest dose in this study was 5000 μg/plate for WP2 uvrA and 50.0 μg/plate for other test bacteria for S9 mix-free test. In the S9 mix addition test, WP2 uvrA was tested up to 625 μg/plate, and for the other test bacteria, 200 μg/plate was used.

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

Ames test:
- Signs of toxicity: Please refer to attached tables
- Individual plate counts: Please refer to attached tables
- Mean number of revertant colonies per plate and standard deviation: Please refer to attached tables
Conclusions:
The test substance - Mixture of 1,3-diisopropylbenzene and 1,4-diisopropylbenzene- was not mutagenic in Salmonella typhimurium TA 100, TA 1535, TA 98, TA 1537 and Escherichia coli WP2 uvrA, with or without an exogenous metabolic activation system.
Executive summary:

 


S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 as well as E. coli WP2 uvr A were tested in a pre-incubation assay with and without metabolic activation (S9) performed according to OECD Guidelines No 471.


The test concentrations of Mixture of 1,3-diisopropylbenzene and 1,4-diisopropylbenzene were given as follows:


-S9 mix:


0, 0.195, 0.391, 0.781, 1.56, 3.13 and 6.25 µg/plate (TA 1537)


0, 0.781 - 50.0 µg/plate (TA 100, TA 1535, TA 98 [Test 1])


0, 0.391 - 12.5 µg/plate (TA 1535 [Test 2])


0, 0.781 - 25.0 µg/plate (TA 100, TA98 [Test 2])


0, 156 - 5000 µg/plate (E. coli WP2 uvrA)


+S9 mix:


0, 6.25 - 200 µg/plate (TA 100, TA 1535, TA 98, TA 1537)


0, 19.5 - 625 µg/plate (E. coli WP2 uvrA).


 


The test substance Mixture of 1,3-diisopropylbenzene and 1,4-diisopropylbenzene was not mutagenic in Salmonella typhimurium TA 100, TA 1535, TA 98, TA 1537 and Escherichia coli WP2 uvrA, with or without an exogenous metabolic activation system.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
In this justification, the read-across (bridging) concept is applied. Please refer to a full version of Read-across statement attached in the section 13 "Assessment reports".

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The underlying hypothesis for the read-across is that the target and the source substance have similar toxicological properties (including the same target organs) due to their structural similarity, resemblance to their chemical reactivity, and therefore a similar mode of action.
The source substance ‘Mixture of 1,3-diisopropylbenzene and 1,4-diisopropylbenzene' is a mixture of structural isomers, 1,3- diisopropylbenzene(target substance) and 1,4-diisopropylbenzene. The physico-chemical properties are highly equivalent based on the high structural similarity. As a conclusion, it is scientifically justified to address the endpoint Genetic toxicity with data on this mixture of isomers.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source substance: Mixture of 1,3-diisopropylbenzene and 1,4-diisopropylbenzene (Diisopropylbenzene), CAS no. 25321-09-9
structural formula: C12H18
Smiles: CC(C)C1=CC=CC=C1C(C)C
Molecular weight: 162 g/mol
CAS 25321-09-9
EC No 246-835-6
purity: not specified

target substance: 1.3-diisopropylbenzene (or m-DIPB)
structural formula: C12H18
Smiles: CC(C)c1cccc(c1)C(C)C
Molecular weight: 162 g/mol
CAS 99-62-7
EC No 202-773-1
purity: not specified

No additional information is available on purity of the source and the target substances. Both substances are normally of high purity, containing only minor amounts of impurities that do not influence the read-across validity.

3. ANALOGUE APPROACH JUSTIFICATION
Please refer to the full version of the read-across statement attached in the section 13 "Assessment reports".

4. DATA MATRIX
Please refer to the full version of the read-across statement attached in the section 13 "Assessment reports".
Reason / purpose for cross-reference:
read-across source
Species / strain:
Chinese hamster lung (CHL/IU)
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):
In order to determine the treatment concentration of the test substance used in the chromosomal abnormality test, the effect of the test substance on cell proliferation was examined. The inhibitory effect of the test substance on CHL/IU cells compared to the solvent control was measured using a monocellater TM, Olympus Optical Industry Co., Ltd. As a result, the concentration of 50 % inhibition of cell proliferation in continuous treatment was 0.03 mg/mL. In addition, both in the presence and absence of S9 mix in the short-term treatment were 0.06 mg/mL. The maximum treatment concentration in each series was twice the 50 % cell growth inhibitory concentration, and the other treatment concentrations were set at the ratio of 2.

STUDY RESULTS
- Concurrent vehicle negative and positive control data

Chromosome aberration test (CA) in mammalian cells:
- Results from cytotoxicity measurements:
Short-term treatment -S9: >= 0.015 mg/mL
Short-term treatment +S9: no cytotoxicity up to 0.015 mg/mL
Continuous treatment -S9: >= 0.03 mg/mL
- Genotoxicity results (for both cell lines and lymphocytes)
o Number of cells scored for each culture and concentration, number of cells with chromosomal aberrations and type given separately for each treated and control culture, including and excluding gaps: Please refer to attached Tables
Conclusions:
The test substance - Mixture of 1,3-diisopropylbenzene and 1,4-diisopropylbenzene - caused no chromosomal aberrations or polyploidy in Chinese hamster lung cells (CHL/IU), with or without an exogenous metabolic activation system.
Executive summary:

The test substance - Mixture of 1,3-diisopropylbenzene and 1,4-diisopropylbenzene - was tested in an in vitro mammalian chromosome aberration test with Chinese hamster lung cells (CHL/IU) according to OECD Guideline 473.


The test concentrations were as follows:


- S9 mix (continuous treatment; 24 or 48 hrs): 0, 0.0038, 0.0075 and 0.015 mg/mL


- S9 mix (short-term treatment; 6 hrs): 0, 0.0019, 0.0038 and 0.0075 mg/mL


+ S9 mix (short-term treatment; 6 hrs): 0, 0.03, 0.06 and 0.12 mg/mL.


 


The test substance caused no chromosomal aberrations or polyploidy in Chinese hamster lung cells (CHL/IU), with or without an exogenous metabolic activation system.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Genetic toxicity in vitro - mouse lymphoma (mammalian cell gene mutation assay)


The test item 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 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) 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.


 


Genetic toxicity in vitro - mammalian chromosome aberration test


Read across to Mixture of 1,3-diisopropylbenzene and 1,4-diisopropylbenzene:


The test substance was tested in an in vitro mammalian chromosome aberration test with Chinese hamster lung cells (CHL/IU) according to OECD Guideline No. 473.


The test concentrations were as follows:


-S9 mix (continuous treatment; 24 or 48 hrs): 0, 0.0038, 0.0075 and 0.015 mg/mL


-S9 mix (short-term treatment; 6 hrs): 0, 0.0019, 0.0038 and 0.0075 mg/mL


+S9 mix (short-term treatment; 6 hrs): 0, 0.03, 0.06 and 0.12 mg/mL.


The test substance caused no chromosomal aberrations or polyploidy in Chinese hamster lung cells (CHL/IU), with or without an exogenous metabolic activation system.


 


Genetic toxicity in bacterial reverse mutation assay


Read across to Mixture of 1,3-diisopropylbenzene and 1,4-diisopropylbenzene:


S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 as well as E. coli WP2 uvr A were tested in a pre-incubation assay performed according to OECD Guidelines No. 471.


The test concentrations were given as follows:


-S9 mix:


0, 0.195, 0.391, 0.781, 1.56, 3.13 and 6.25 µg/plate (TA 1537)


0, 0.781 - 50.0 µg/plate (TA 100, TA 1535, TA 98 [Test 1])


0, 0.391 - 12.5 µg/plate (TA 1535 [Test 2])


0, 0.781 - 25.0 µg/plate (TA 100, TA98 [Test 2])


0, 156 - 5000 µg/plate (E. coli WP2 uvrA)


+S9 mix:


0, 6.25 - 200 µg/plate (TA 100, TA 1535, TA 98, TA 1537)


0, 19.5 - 625 µg/plate (E. coli WP2 uvrA).


 


Cytotoxic effects were seen at the following concentrations:


-S9 mix:


TA 100: >= 12.5 µg/plate


TA 1535: >= 6.25 µg/plate


TA 98: >= 12.5 µg/plate


TA 1537: >= 6.25 µg/plate


E. coli: 5000 µg/plate


+S9 mix:


TA 100, TA 1535, TA 98 and TA 1537: >= 100 µg/plate


E. coli: 625 µg/plate.


 


The test substance was not mutagenic in Salmonella typhimurium TA 100, TA 1535, TA 98, TA 1537 and Escherichia coli WP2 uvrA, with or without an exogenous metabolic activation system.

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 according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No 1272/2008.