Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
17 September 2009 to
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: compliant to GLP and testing guideline; adequate coherence between data, comments and conclusions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): 1,3(4)-bis(tert-butylperoxyisopropyl)benzene
- Physical state: slightly yellow pieces
- Molecular weight: 338 g.mol-1
- Peroxide content: 97.6%
- Purity test date: 28 July 2009
- Lot/batch No.: 307090713
- Expiration date of the lot/batch: July 2010
- Storage conditions of test material: at room temperature (not exceeding 30°C) and protected from light

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
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Experiments without S9 mix:
. 0.063, 0.125, 0.25, 0.5, 1 and 2 mM for the first experiment (3-hour treatment),
. 0.004, 0.008, 0.016, 0.031, 0.063, 0.125 and 0.25 mM for the second experiment (24-hour treatment).

Experiments with S9 mix:
. 0.063, 0.125, 0.25, 0.5, 1 and 2 mM for both mutagenicity experiments.
Vehicle / solvent:
- Vehicle used: acetone.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
(acetone)
True negative controls:
no
Positive controls:
yes
Remarks:
(without S9 mix)
Positive control substance:
methylmethanesulfonate
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
(acetone)
True negative controls:
no
Positive controls:
yes
Remarks:
(with S9 mix)
Positive control substance:
cyclophosphamide
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium.

DURATION
- Exposure duration: 3 hours (with and without S9 mix) and 24 hours (without S9 mix only)
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 11-12 days

SELECTION AGENT (mutation assays): Trifluorothimidine

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 equals or exceeds the global evaluation factor (126 x 10-6 for the microtiter method),
. and a dose-related trend is demonstrated by a statistically significant trend test.

Unless 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 (RTG lower than 10%), but with no evidence of mutagenicity at dose-levels with RTG between 10 and 20%, is not considered as positive result.
A test item is determined to be non-mutagenic when there is no culture showing an Adj. RTG value between 10-20% if:
. there is at least one negative data point between 20 and 25% Adj. RTG and no evidence on mutagenicity in a series of data points between 100 to 20% Adj. RTG,
. there is no evidence of mutagenicity in a series of data points between 100 to 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:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: moderate precipitate at the final dose-level of 5 mM (corresponding to 1690 µg/mL) ; pH of 7.4 and osmolality of 320 mOsm/kg H2O

RANGE-FINDING/SCREENING STUDIES:
To assess the cytotoxicity of the test item, at least six dose-levels (one culture/dose-level) were tested both with and without metabolic activation.
A treatment of 3 hours (with and without S9 mix) and 24 hours (without S9 mix) was performed using a final concentration and conditions as described below for the mutagenicity experiment. At the end of treatment, cells were washed and then cell concentrations were adjusted to 2 x 10EXP5 cells/mL and cultured for 2 days as for the mutagenicity experiment. Two days after the end of the treatment, cultures were adjusted in order to seed an average of 1.6 cells per well in the 96 well microtiter plates.
Approximately one week after incubation at 37°C in a humidified atmosphere of 5% CO2/95% air, the clones were counted on the plates.

In the preliminary test, since the test item was non-severely toxic following the 3-hour treatment, but poorly soluble in the final treatment medium,the highest dose-level was based on the level of precipitate, according to the criteria specified in the international guidelines.
Since the test item was toxic following the 24-hour treatment, 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).

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Experiments without S9 mix
Using a treatment volume of 50 µL/20 mL, the selected dose-levels were as follows:
. 0.063, 0.125, 0.25, 0.5, 1 and 2 mM for the first experiment (3-hour treatment),
. 0.004, 0.008, 0.016, 0.031, 0.063, 0.125 and 0.25 mM for the second experiment (24 hour treatment).

Following the 3-hour treatment, no noteworthy toxicity was observed at any of the tested dose levels.
Following the 24-hour treatment, a 83-100% decrease in the Adj. RTG was induced at dose levels ≥ 0.063 mM.


Experiments with S9 mix
Using a treatment volume of 50 µL/20 mL, the selected dose-levels were as follows: 0.063, 0.125, 0.25, 0.5, 1 and 2 mM for both mutagenicity experiments.

In the first experiment, a 28-50% decrease in the Adj. RTG was noted at dose-levels ≥ 0.25 mM.
In the second experiment, no noteworthy decrease in the Adj. RTG was induced.






Remarks on result:
other: strain/cell type: L5178Y
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Under our experimental conditions, the test item 1,3(4)-bis(tert-butylperoxyisopropyl)benzene did not show any mutagenic activity in the mouse lymphoma assay.
Executive summary:

The potential of the test item 1,3(4)-bis(tert-butylperoxyisopropyl)benzene to induce mutations at the TK (Thymidine Kinase) locus in L5178Y mouse lymphoma cells was evaluated according to the international guidelines (OECD 476, Commission Directive No. B17) and in compliance with the Principles of Good Laboratory Practice.

Methods

After a preliminary toxicity test, 1,3(4)-bis(tert-butylperoxyisopropyl)benzene was tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254.

Approximately 0.5 x 106 (3-hour treatment) or 0.15 x 106 (24-hour treatment) cells/mL in 20 mL culture medium with 5% horse serum were exposed to the test or control items, in the presence or absence of S9 mix (final concentration of S9 fraction 2%), at 37°C. For the 24-hour treatment, the incubation at 37°C was performed with a gentle shaking. Cytotoxicity was measured by assessment of adjusted relative total growth (Adj. RTG) and relative suspension growth (Adj. RSG) as well as cloning efficiency following the expression time (CE2). The number of mutant clones (differentiating small and large colonies) were checked after the expression of the mutant phenotype. The test item was dissolved in acetone.

The dose-levels for the positive controls were as follows:

. without S9 mix: methylmethane sulfonate (MMS), used at a final concentration of 25 µg/mL, (3-hour treatment) or 5 µg/mL (24-hour treatment),

. with S9 mix: Cyclophosphamide (CPA), used at a final concentration of 3 µg/mL.

Results

In the culture medium, the final dose-level of 5 mM (corresponding to 1690 µg/mL) showed a moderate precipitate. At this dose-level, the pH and the osmolality values were equivalent to those of the vehicle control culture. In the preliminary test, since the test item was non-severely toxic following the 3-hour treatment, but poorly soluble in the final treatment medium, the highest dose-level was based on the level of precipitate, according to the criteria specified in the international guidelines. Since the test item was toxic following the 24-hour treatment, 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). The cloning efficiencies CE2and the mutation frequencies of the vehicle and positive controls were mainly as specified in acceptance criteria. The study was therefore considered valid.

Experiments without S9 mix

Using a treatment volume of 50 µL/20 mL, the selected dose-levels were as follows:

. 0.063, 0.125, 0.25, 0.5, 1 and 2 mM for the first experiment (3-hour treatment),

. 0.004, 0.008, 0.016, 0.031, 0.063, 0.125 and 0.25 mM for the second experiment (24-hour treatment).

At the end of the 3-hour treatment, a slight precipitate was noted in the culture medium at 2 mM.

At the end of the 24-hour treatment, a slight precipitate was noted in the culture medium at dose-levels ≥ 0.125 mM.

Cytotoxicity

Following the 3-hour treatment, no noteworthy toxicity was observed at any of the tested dose-levels.

Following the 24-hour treatment, a 83-100% decrease in the Adj. RTG was induced at dose-levels ≥ 0.063 mM.

Mutagenicity

No noteworthy increase in the mutation frequency was induced at any dose-levels either following the 3- or the 24-hour treatment.

Experiments with S9 mix

Using a treatment volume of 50 µL/20 mL, the selected dose-levels were as follows: 0.063, 0.125, 0.25, 0.5, 1 and 2 mM for both mutagenicity experiments.

In the first experiment, a slight precipitate was observed in the culture medium at 2 mM at the end of the 3-hour treatment.

In the second experiment, a slight to moderate precipitate was observed in the culture medium at dose-levels ≥ 0.125 mM at the end of the 3-hour treatment.

Cytotoxicity

In the first experiment, a 28-50% decrease in the Adj. RTG was noted at dose-levels ≥ 0.25 mM.

In the second experiment, no noteworthy decrease in the Adj. RTG was induced.

Mutagenicity

No noteworthy increase in the mutation frequency was induced at any dose-levels in either experiment.

Conclusion

Under our experimental conditions, the test item 1,3(4)-bis(tert-butylperoxyisopropyl)benzene did not show any mutagenic activity in the mouse lymphoma assay.