Reaction mass of 1,3-diisopropylbenzene and 1,4-diisopropylbenzene

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

DIPB was negative in Ames and Chromosomal aberrations in vitro. Additionally the 1,3-DIPB isomer was considered to be non-mutagenic in the in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration 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

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Principles of method if other than guideline:

Study performed according to Guidelines for Screening Mutagenicity Testing of Chemicals (Japan) and OECD Guideline No. 473

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

other: Chinese hamster lung cells (CHL/IU)

Metabolic activation:

with and without

Metabolic activation system:

S9 mix from rat liver (induction with phenobarbital and 5,6-benzoflavone)

Test concentrations with justification for top dose:

-S9 mix (continuous treatment): 0, 0.0038, 0.0075 and 0.015 mg/mL
-S9 mix (short-term treatment): 0, 0.0019, 0.0038 and 0.0075 mg/mL
+S9 mix (short-term treatment): 0, 0.03, 0.06 and 0.12 mg/mL

Vehicle / solvent:

Acetone

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

Positive controls:

yes

Remarks:

see below

Positive control substance:

other: see below

Details on test system and experimental conditions:

Positive controls:
-S9 mix: Mitomycin C
+S9 mix: Cyclophosphamide

Continuous treatment: 24 and 48 hrs
Short-term treatment: 6 hrs
2 plates/test

Evaluation criteria:

according to OECD Guideline No. 473

Statistics:

Yes

Species / strain:

other: Chinese hamster lung cells (CHL/IU)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

See Additional information on results

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Cytotoxicity:
Short-term treatment: >= 0.015 mg/mL
Continuous treatment: >= 0.03 mg/mL

Conclusions:

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

Executive summary:

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

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)

Deviations:

no

Principles of method if other than guideline:

Pre-incubation method; Study performed according to Guidelines for Screening Toxicity Testing of Chemicals (Japan) and OECD Guidelines No. 471 and 472

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9 mix from rat liver (induction with phenobarbital and 5,6-benzoflavone)

Test concentrations with justification for top dose:

-S9 mix:
0, 0.195, 0.391, 0.781, 1.56, 3.13 and 6.25 ug/plate (TA 1537)
0, 0.781 - 50.0 ug/plate (TA 100, TA 1535, TA 98 [Test 1])
0, 0.391 - 12.5 ug/plate (TA 1535 [Test 2])
0, 0.781 - 25.0 ug/plate (TA 100, TA98 [Test 2])
0, 156 - 5000 ug/plate (E. coli WP2 uvrA)

+S9 mix:
0, 6.25 - 200 ug/plate (TA 100, TA 1535, TA 98, TA 1537)
0, 19.5 - 625 ug/plate (E. coli WP2 uvrA)

Vehicle / solvent:

DMSO

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Remarks:

see below

Positive control substance:

other: see below

Details on test system and experimental conditions:

Positive controls:
-S9 mix:
TA 100, TA 98, E. coli WP2 uvrA: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide
TA 1535: Sodium azide
TA 1537: 9-aminoacridine

+S9 mix:
All strains: 2-aminoanthracene

Evaluation criteria:

according to OECD Guidelines No. 471 and 472

Statistics:

Yes

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

see below

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

see below

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Cytotoxicity:
-S9 mix:
TA 100: >= 12.5 ug/plate
TA 1535: >= 6.25 ug/plate
TA 98: >= 12.5 ug/plate
TA 1537: >= 6.25 ug/plate
E. coli: 5000 ug/plate

+S9 mix:
TA 100, TA 1535, TA 98 and TA 1537: >= 100 ug/plate
E. coli: 625 ug/plate

Conclusions:

The TS 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 performed according to OECD Guidelines No. 471 and 472.

The test concentrations were given as follows:

-S9 mix:

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

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

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

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

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

+S9 mix:

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

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

Cytotoxic effects were seen at the following concentrations:

-S9 mix:

TA 100: >= 12.5 ug/plate

TA 1535: >= 6.25 ug/plate

TA 98: >= 12.5 ug/plate

TA 1537: >= 6.25 ug/plate

E. coli: 5000 ug/plate

+S9 mix:

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

E. coli: 625 ug/plate.

The TS 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.

Reference 3

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:

from 12 Semtember 2012 to 3 January 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

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:

mammalian 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

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: pH-value was winthin the physiological range
- Precipitation and time of the determination: 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 witohut metabolic activiation no precipitation was noted.

STUDY RESULTS
- Concurrent vehicle negative and positive control data

Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements:
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 activiation was 0.50 mM with a RTG of 21.5%. In experiment II with metabolic activation the RTG was 15.7% for the highest concentration (0.40 mM) evaluation. The highest concentration evaluated without metabolic activation was 0.20 mM with a RTG of 7.9%.

- Genotoxicity results:
In experiment I and II no biologically relevant increase of mutants was found after treatment with the test item (with and without metabolic activiation). The Global Evaluation Factor (GEF) was not exceeded by the induced mutant freqency at any concentration.
No dose-response relationship was observed.
In experiment I and II colony sizing showed no clastogenic effects induced by the the test item under the experimental conditions (with and without metabolic activiation).
EMS, MMS and B[a]P were used as positive controls and showed distinct and biologically relevant effects in mutation freqency. 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.

see attachement

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)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

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 and 472.

The test concentrations were between 0 and 200 µg/plate (limited by cytotoxic effects) in S. typhimurium and up to 5000 µg/plate in E. coli.

DIPB 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.

DIPB 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.

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

Additional results with the 1,3-DIPB isomer (pure):

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 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

In experiment I and II no biologically relevant increase of mutants was found after treatment with the test item (with and without metabolic activation). 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.

Justification for classification or non-classification

According to CLP (EC regulation 1272/2008) no classification of DIPB for germ cell mutagenicity is required. Based on the available key studies, there is no mutagenic potential in vitro.