Diisopropylbenzene hydroperoxide

Genetic toxicity: in vitro

Administrative data

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:

04 June 2012 - 02 October 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Compliant to GLP and testing guidelines; adequate consistence between data, comments and conclusions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Diisopropylbenzene hydroperoxide

EC Number:

247-988-1

EC Name:

Diisopropylbenzene hydroperoxide

Cas Number:

26762-93-6

Molecular formula:

C12H18O2

IUPAC Name:

reaction mass of 1-(3-isopropylphenyl)-1-methylethyl hydroperoxide and 1-(4-isopropylphenyl)-1-methylethyl hydroperoxide

Test material form:

other: colorless to yellowish liquid

Method

Target gene:

Thymidine Kinase

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640 medium containing L-Glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 µg/mL) and sodium
pyruvate (200 µg/mL)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 mix

Test concentrations with justification for top dose:

Experiments without S9 mix
Using a treatment volume of 1% in culture medium, the selected dose-levels were:
- 1.6, 3.1, 6.3, 12.5, 25 and 50 µg/mL for the first experiment (3-hour treatment),
- 0.6, 1.3, 2.5, 5, 10, 15 and 20 µg/mL for the second experiment (24-hour treatment).

Experiments without S9 mix
Using a treatment volume of 1% in culture medium, the selected dose-levels were:
- 1.6, 3.1, 6.3, 12.5, 25 and 50 µg/mL for the first experiment (3-hour treatment),
- 0.6, 1.3, 2.5, 5, 10, 15 and 20 µg/mL for the second experiment (24-hour treatment).

Vehicle / solvent:

Vehicle used: dimethylsulfoxide
Justification for choice: test item was soluble in the vehicle at:
- 860 mg/mL for the preliminary toxicity test,
- 20 mg/mL for the first experiment and for the second experiment with S9 mix,
- 15 mg/mL for the second experiment without S9 mix and the third experiment.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: methylmethanesulfonate (-S9 mix); cyclophosphamide (+S9 mix)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 and 24 hours
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 11-12 days

SELECTION AGENT (mutation assays): trifluorothymidine

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth.

Evaluation criteria:

IWGT recommendations were followed for the determination of a positive result, which should fulfill the following criteria:
- at least at one dose-level the mutation frequency minus the mutation frequency of the vehicle control (IMF) equals or exceeds the Global Evaluation Factor (GEF) of 126 x 10-6,
- a dose-related trend is demonstrated by a statistically significant trend test.

Unless an effect is considered as clearly positive, the reproducibility of a positive effect should be confirmed.

Noteworthy increases in the mutation frequency observed only at high levels of cytotoxicity (Adj. RTG lower than 10%), but with no evidence of mutagenicity at dose-levels with Adj. RTG between 10 and 20%, are not considered as positive results.

A test item may be considered as non-mutagenic when there is no culture showing an Adj. RTG value between 10 and 20% if (Moore et al., 2002):
- there is at least one negative data point between 20 and 25% Adj. RTG and no evidence of mutagenicity in a series of data points between 100 and 20% Adj. RTG,
- there is no evidence of mutagenicity in a series of data points between 100 and 25% and there is also a negative data point between 10 and 1% Adj. RTG.

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:

not applicable

Positive controls validity:

valid

Remarks on result:

other: strain/cell type: mouse lymphoma L5178Y cells

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The test item was not mutagenic to mammalian cells in the presence or absence of metabolic activation.

Executive summary:

The potential of the test item (diisopropylbenzene monohydroperoxide in diisopropylbenzen) was evaluated in a Mouse Lymphoma Assay. The study was performed according to international guidelines (OECD guideline No. 476 and Council Regulation (EC) No. 440/2008 of 30 May 2008, Annex, Part B.17) and in compliance with the principles of Good Laboratory Practice.

 

Methods

After a preliminary toxicity test, Diisopropylbenzene monohydroperoxide, was tested in three independent experiments, with and/or without a metabolic activation system (S9 mix) prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254.

Cultures of 20 mL at 5 x 10⁵cells/mL (3-hour treatment) or cultures of 50 mL at 2 x 10⁵cells/mL (24-hour treatment) were exposed to the test or control items, in the presence or absence of S9 mix (final concentration of S9 fraction 2%). During the treatment period, the cells were maintained as suspension culture in RPMI 1640 culture medium supplemented by heat inactivated horse serum at 5% (3-hour treatment) or 10% (24-hour treatment) in a 37°C, 5% CO₂humidified incubator. For the 24-hour treatment, flasks were gently shaken at least once.

 

Cytotoxicity was measured by assessment of Adjusted Relative Total Growth (Adj. RTG), Adjusted Relative Suspension Growth (Adj. RSG) and Cloning Efficiency following the expression time (CE₂).

The number of mutant clones (differentiating small and large colonies) was evaluated after expression of the mutant phenotype.

 

The test item was dissolved in dimethylsulfoxide (DMSO).

 

Results

With one exception which is not considered to have a biological impact on the validity of the study, the Cloning Efficiencies (CE₂), the Suspension Growths (SG) and the mutation frequencies of the vehicle controls were as specified in the acceptance criteria. Moreover, the induced mutation frequencies obtained for the positive controls met the acceptance criteria specified in the study plan. The study was therefore considered as valid.

 

Since the test item was found severely cytotoxic in the preliminary test, the choice of the highest dose-level for the main test was based on the level of toxicity, according to the criteria specified in the international guidelines (decrease in Adj. RTG).

 

Experiments without S9 mix

Using a treatment volume of 1% in culture medium, the selected dose-levels were:

- 1.6, 3.1, 6.3, 12.5, 25 and 50 µg/mL for the first experiment (3-hour treatment),

- 0.6, 1.3, 2.5, 5, 10, 15 and 20 µg/mL for the second experiment (24-hour treatment).

 

Cytotoxicity

Following the 3-hour treatment, a marked toxicity was induced at 50 µg/mL, as shown by a 66% decrease in Adj. RTG.

Following the 24-hour treatment, a moderate to severe toxicity was induced at dose-levels = 10 µg/mL, as shown by a 43 to 97% decrease in Adj. RTG.

Mutagenicity

Following the 3- or 24-hour treatments, no relevant or dose-related increases in the mutation frequency were noted in comparison to the vehicle control. The results did not meet the criteria for a positive response.

 

Experiments with S9 mix

Using a treatment volume of 1% in culture medium, the selected dose-levels were as follows:

- 6.3, 12.5, 25, 50, 100 and 200 µg/mL for the first experiment,

- 12.5, 25, 50, 100, 150 and 200 µg/mL for the second experiment,

- 25, 50, 75, 100, 116.7, 133.3 and 150 µg/mL for the third experiment.

 

Cytotoxicity

Following the first experiment, a slight to severe toxicity was induced at dose-levels = 50 µg/mL, as shown by a 31 to 100% decrease in Adj. RTG.

Following the second experiment, a marked to severe toxicity was induced at dose-levels = 100 µg/mL, as shown by a 66 to 97% decrease in Adj. RTG. Following the third experiment, a moderate to severe toxicity was induced at dose-levels = 75 µg/mL, as shown by a 48 to 98% decrease in Adj. RTG.

 

Mutagenicity

No noteworthy increases in the mutation frequency, which could be considered as biologically relevant, were induced in any of the experiments.

 

Conclusion

The test item did not show any mutagenic activity in the mouse lymphoma assay, in the presence or in the absence of a rat metabolizing system.