Registration Dossier

Data platform availability banner - registered substances factsheets

Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Four studies are available for assessment of genetic toxicity of 1,3(4)-bis-(t-butyl peroxyisopropyl) benzene and one is available for the meta isomer. They did not show any mutagenic activity.

 

Gene mutations assay

In a key study (Zieger et al., 1988), the potential of the substance to induce reverse mutation in Salmonella typhimurium (strains: TA 1535, TA 1537, TA 98, TA 100) was evaluated according to Haworth et al. (1975) procedure, by direct plate incorporation (the first experiment and the second without S9mix), at six dose-levels (0, 100, 333, 1000, 3333, 1000 µg/plate), for the substance named CAS 25155 -25 -3 (no further information about the substance was given). Any mutagenic activity was recorded, with and without activation, for any of the 4 test strains.

 

In a supporting study (Willems, 1976), the potential of 1,3 phenylenebis(1-methylethylidene)]bis[tert-butyl] peroxide to induce reverse mutation in Salmonella typhimurium (strains: TA 1535, TA 1537, TA 98, TA 100 and TA 1538) was evaluated according to Ames et al. (1975), by direct plate incorporation (the first experiment and the second without S9mix), at four dose-levels (0.2, 2, 20, 500 µg/plate), for the meta substance only. No mutagenic activity was recorded, with and without activation, for any of the 5 test strains.

 

The potential of 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) (Sire, 2010a). 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. 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 concentrations were 0.063, 0.125, 0.25, 0.5, 1 and 2 mM (3-hour treatment) and 0.004, 0.008, 0.016, 0.031, 0.063, 0.125 and 0.25 mM (24-hour treatment) without S9 mix and 0.063, 0.125, 0.25, 0.5, 1 and 2 mM with S9 mix. No noteworthy increase in the mutation frequency was induced at any dose-levels either with or without metabolic activation.1, 3(4)-bis(tert-butylperoxyisopropyl)benzene did not show any mutagenic activity in the mouse lymphoma assay.

 

Chromosomal aberrations assay

The potential of 1, 3 (4)-bis(tert-butylperoxyisopropyl)benzene to induce chromosome aberrations in cultured human lymphocytes was evaluated according to the international guidelines (OECD 473, Commission Directive No. B10) (Sire, 2010b). It was tested in two independent experiments, both with and without a liver metabolizing system (S9 mix), obtained from rats previously treated with Aroclor 1254. The highest dose-level for treatment in the first experiment was selected on the basis of pH, osmolality and solubility. For selection of the dose-levels for the second experiment, any toxicity indicated by the reduction of mitotic index (MI) in the first experiment was also taken into account. For each culture, heparinized whole blood was added to culture medium containing a mitogen (phytohemagglutinin) and incubated at 37°C, for 48 hours. In the first experiment, lymphocyte cultures were exposed to 0.052, 0.104, 0.208, 0.417, 0.834, 1.67, 3.34 and 6.67 mM, both with and without S9 mix, for 3 hours. Cells were harvested 20 hours after the beginning of treatment, corresponding to approximately 1.5 normal cell cycles. In the second experiment, lymphocyte cultures were exposed to 0.104, 0.208, 0.417, 0.834, 1.67, 3.34 and 6.67 mM, without S9 mix, cells were exposed continuously until harvest, and with S9 mix, cells were exposed for 3 hours and then rinsed. Cells were harvested 20 hours and 44 hours after the beginning of treatment, corresponding to approximately 1.5 normal cell cycles and 24 hours later, respectively. No significant increase in the frequency of cells with structural chromosomal aberrations was noted in either experiment and at either harvest times.1, 3(4)-bis(tert-butylperoxyisopropyl)benzene did not induce chromosome aberrations in cultured human lymphocytes.

Link to relevant study records

Referenceopen allclose all

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:
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
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
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
Target gene:
Thymidine Kinase
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.
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.
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'.
Conclusions:
Interpretation of results (migrated information):
negative

Under the 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 the experimental conditions, the test item 1,3(4)-bis(tert-butylperoxyisopropyl)benzene did not show any mutagenic activity in the mouse lymphoma assay.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
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
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
Not applicable.
Species / strain / cell type:
lymphocytes: human lymphocytes
Details on mammalian cell type (if applicable):
5 mL of RPMI 1640 medium containing 20% fetal calf serum, L-glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 µg/mL) and phytohemagglutinin (PHA: a mitogen to stimulate lymphocyte division).
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
With a treatment volume of 15 µL/5.5 mL culture medium, the treatment-levels were as follows:
. 0.052, 0.104, 0.208, 0.417, 0.834, 1.67, 3.34 and 6.67 mM for the first experiment, both with and without S9 mix,
. 0.104, 0.208, 0.417, 0.834, 1.67, 3.34 and 6.67 mM for the second experiment, both with and without S9 mix.
Vehicle / solvent:
- Vehicle used: acetone.
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:
mitomycin C
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
(acetone)
True negative controls:
no
Positive controls:
yes
Remarks:
(with S9mix)
Positive control substance:
cyclophosphamide
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium.

DURATION
In the first experiment, lymphocyte cultures were then exposed for 3 hours to the test or control items, both in the absence and presence of S9 mix, then rinsed. One and a half hours before harvest, each culture was treated with a colcemid solution (10 µg/mL) to block cells at the metaphase-stage of mitosis. Harvest time was 20 hours after the beginning of treatment, corresponding to approximately 1.5 normal cell cycles.

The second experiment was performed as follows:
. without S9 mix, cells were exposed continuously to the test or control items, until harvest,
. with S9 mix, cells were exposed to the test or control items for 3 hours and then rinsed.
One and a half hours before harvest, each culture was treated with a colcemid solution (10 µg/mL) to block cells at the metaphase-stage of mitosis. Harvest times were 20 hours and 44 hours after the beginning of treatment, corresponding to approximately 1.5 normal cell cycles and 24 hours later.

SPINDLE INHIBITOR (cytogenetic assays): colcemid
STAIN (for cytogenetic assays): Giemsa

NUMBER OF CELLS EVALUATED: 200 metaphases/dose-level, with 100 metaphases/culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
Evaluation criteria:
A reproducible and statistically significant increase in the frequency of cells with structural chromosome aberration for at least one of the dose-levelsand one of the two harvest times was considered as a positive result. Reference to historical data or other considerations of biological relevance, was also taken into account in the evaluation of the findings.
Statistics:
For each test and for each harvest time, the frequency of cells with structural chromosome aberration (excluding gaps) in treated cultures was compared to that of the vehicle control cultures. If necessary, the results were compared using the chi 2 test, in which p = 0.05 was used as the lowest level of significance.
Species / strain:
lymphocytes: human lymphocytes
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
During the solubility assay, the test item was found soluble in acetone at 826.63 mg/mL. Using a maximal treatment volume of 15 µL/5.5 mL culture medium, the highest achievable dose-level of 6.67 mM showed no precipitate in the culture medium. At this dose-level, the pH and the osmolality values were equivalent to those of the vehicle control culture.

A slight to marked precipitate (test item adhering to the wall of the tubes) was observed at the end of the treatment period, generally at dose levels >= 0.417 mM.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Experiments without S9 mix
Following the 3-hour treatment, a marked decrease in the mitotic index was noted at dose levels >= 3.34 mM (64-66% decrease).
Following the 20-hour treatment, no noteworthy decrease in the mitotic index was noted at any tested dose-levels.
Following the 44-hour treatment, a moderate decrease in the mitotic index was noted at the dose levels of 3.34 mM only (43% decrease).

Experiments with S9 mix
At the 20-hour harvest time in the first experiment, a moderate decrease in the mitotic index was observed at the dose-level of 3.34 mM only (54% decrease).
At the 20-hour harvest time in the second experiment, a slight to moderate decrease in the mitotic index was observed at dose-levels >= 0.834 mM (37-46% decrease).
At the 44-hour harvest time (second experiment), no noteworthy decrease in the mitotic index was observed at any tested dose-levels.

Remarks on result:
other: strain/cell type: human lymphocytes
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

Under the experimental conditions, the test item 1,3(4)-bis(tert-butylperoxyisopropyl)benzene (batch No. 307090713, purity: 97.6%) did not induce chromosome aberrations in cultured human lymphocytes.
Executive summary:

The potential of the test item 1,3(4)-bis(tert-butylperoxyisopropyl)benzene to induce chromosome aberrations in cultured human lymphocytes was evaluated according to the international guidelines (OECD 473, Commission Directive No. B10) and in compliance with the Principles of Good Laboratory Practice.

Methods

The test item was tested in two independent experiments, both with and without a liver metabolizing system (S9 mix), obtained from rats previously treated with Aroclor 1254.

The highest dose-level for treatment in the first experiment was selected on the basis of pH, osmolality and solubility. For selection of the dose-levels for the second experiment, any toxicity indicated by the reduction of mitotic index (MI) in the first experiment was also taken into account.

For each culture, heparinized whole blood was added to culture medium containing a mitogen (phytohemagglutinin) and incubated at 37°C, for 48 hours.

In the first experiment, lymphocyte cultures were exposed to the test or control items (with or without S9 mix) for 3 hours then rinsed. Cells were harvested 20 hours after the beginning of treatment, corresponding to approximately 1.5 normal cell cycles.

The second experiment was performed as follows:

.           without S9 mix, cells were exposed continuously to the test or control items until harvest,

.           with S9 mix, cells were exposed to the test or control items for 3 hours and then rinsed.

Cells were harvested 20 hours and 44 hours after the beginning of treatment, corresponding to approximately 1.5 normal cell cycles and 24 hours later, respectively. One and a half hours before harvest, each culture was treated with a colcemid solution (10 µg/mL) to block cells at the metaphase-stage of mitosis. After hypotonic treatment (KCl 0.075 M), the cells were fixed in a methanol/acetic acid mixture (3/1; v/v), spread on glass slides and stained with Giemsa. All the slides were coded for scoring. The test item 1,3(4)-bis(tert-butylperoxyisopropyl)benzene was dissolved in acetone.

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

.  without S9 mix, Mitomycin C: 3 µg/mL (3 hours of treatment) or 0.2 µg/mL (continuous treatment),

. with S9 mix, Cyclophosphamide: 12.5 or 25 µg/mL.

Results

During the solubility assay, the test item was found soluble in acetone at 826.63 mg/mL. Using a maximal treatment volume of 15 µL/5.5 mL culture medium, the highest achievable dose-level of 6.67 mM showed no precipitate in the culture medium. At this dose-level, the pH and the osmolality values were equivalent to those of the vehicle control culture.

With a treatment volume of 15 µL/5.5 mL culture medium, the treatment-levels were as follows:

. 0.052, 0.104, 0.208, 0.417, 0.834, 1.67, 3.34 and 6.67 mM for the first experiment, both with and without S9 mix,

. 0.104, 0.208, 0.417, 0.834, 1.67, 3.34 and 6.67 mM for the second experiment, both with and without S9 mix. The frequency of cells with structural chromosome aberrations of the vehicle and positive controls was as specified in acceptance criteria. The study was therefore considered valid.

 

A slight to marked precipitate (test item adhering to the wall of the tubes) was observed at the end of the treatment period, generally at dose-levels >= 0.417 mM.

Experiments without S9 mix

Cytotoxicity

Following the 3-hour treatment, a marked decrease in the mitotic index was noted at dose-levels >= 3.34 mM (64-66% decrease).

Following the 20-hour treatment, no noteworthy decrease in the mitotic index was noted at any tested dose-levels.

Following the 44-hour treatment, a moderate decrease in the mitotic index was noted at the dose-levels of 3.34 mM only (43% decrease).

Metaphases analysis

The dose-levels selected for the metaphases analysis were as follows:

. 0.834, 1.67 and 3.34 mM, for the 3-hour treatment, the latter inducing a 66% decrease in the mitotic index,

. 1.67, 3.34 and 6.67 mM for the 20-hour treatment,

. 6.67 mM for the 44-hour treatment. No significant increase in the frequency of cells with structural chromosomal aberrations was noted after 3-, 20- as well as 44-hour treatments.

Experiments with S9 mix

Cytotoxicity

At the 20-hour harvest time in the first experiment, a moderate decrease in the mitotic index was observed at the dose-level of 3.34 mM only (54% decrease).

At the 20-hour harvest time in the second experiment, a slight to moderate decrease in the mitotic index was observed at dose-levels >= 0.834 mM (37-46% decrease).

At the 44-hour harvest time (second experiment), no noteworthy decrease in the mitotic index was observed at any tested dose-levels.

Metaphases analysis

The dose-levels selected for the metaphases analysis were as follows:

. 1.67, 3.34 and 6.67 mM for the 20-hour harvest time in both experiments,

.  6.67 mM, for the 44-hour harvest time.

 

No significant increase in the frequency of cells with structural chromosomal aberrations was noted in either experiment and at either harvest times.

Conclusion

Under the experimental conditions, the test item 1,3(4)-bis(tert-butylperoxyisopropyl)benzene (batch No. 307090713, purity: 97.6%) did not induce chromosome aberrations in cultured human lymphocytes.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: 2d: test procedure in accordance with national standard methods with acceptable restrictions.
Qualifier:
no guideline followed
Principles of method if other than guideline:
The preincubation assay was performed as described by Haworth et al., 1983, with some differences.
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
4 kinds of S9 (prepared from rats or hamsters induced by Arochlor 1254, at two concentrations: 10% or 30 %)
Test concentrations with justification for top dose:
0, 100, 333, 1000, 3333, 10000 µg/plate
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: because it was an interlaboratory test with 300 substances involved, there were many positive controls.
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
other: Because it was an interlaboratory test with 300 substances involved, then it can be considered that there were many negative controls.
Positive controls validity:
other: Because it was an interlaboratory test with 300 substances involved, then it can be considered that there were many positive controls.
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
other: Because it was an interlaboratory test with 300 substances involved, then it can be considered that there were many negative controls.
Positive controls validity:
other: Because it was an interlaboratory test with 300 substances involved, then it can be considered that there were many positive controls.
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
other: Because it was an interlaboratory test with 300 substances involved, then it can be considered that there were many negative controls.
Positive controls validity:
other: Because it was an interlaboratory test with 300 substances involved, then it can be considered that there were many positive controls.
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
other: Because it was an interlaboratory test with 300 substances involved, then it can be considered that there were many negative controls.
Positive controls validity:
other: Because it was an interlaboratory test with 300 substances involved, then it can be considered that there were many positive controls.
Conclusions:
Interpretation of results (migrated information):
negative

Under these experimental conditions, Bis(t-nutyldioxyisopropyl)benzene did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.
Executive summary:

The potential of Bis(t-nutyldioxyisopropyl)benzene to induce reverse mutation in Salmonella typhimurium (strains: TA 1535, TA 1537, TA 98, TA 100) was evaluated according to Haworth et al. (1975) procedure: direct plate incorporation (the first experiment and the second without S9mix)

Bacterias were exposed to Bis(t-nutyldioxyisopropyl)benzen at six dose-levels (0, 100, 333, 1000, 3333, 1000 µg/plate). The revertant colonies were scored and reported.

No increase in the number of revertants was observed for all doses with and without metabolic activation (10 % or 30 %) on the 5 tested strains.

Under these experimental conditions,Bis(t-nutyldioxyisopropyl)benzene did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Study period:
1976
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
2d: Study was conducted in accordance with recognized published scientific procedure for examining the genotoxicity potential of a test compound in S. typhimurium bacteria strains. Test method utilized recognized positive controls that gave the expected positive responses, confirming the sensitivity of the method.
Qualifier:
no guideline followed
Principles of method if other than guideline:
According to Ames method: Ames, B.N., McCann, J. and Yamasaki, E. (1975). Methods for detecting carcinogens and mutagens with Salmonella/Mammalian-Microsome Mutagenicity Test. Mutation Research, 31, 347-364.
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Target gene:
- The set of Salmonella typhimurium was a gift of Dr B.N. Ames, Berkeley, California, USA, and was checked for histidine requirement, for sensitivity crystal violet, deoxychloate, and UV.
Species / strain / cell type:
S. typhimurium, other: TA 1535, TA 1537, TA1538, TA 98 and TA 100
Additional strain / cell type characteristics:
other: TA 98 and TA 10 were tested for ampicilline resistance
Metabolic activation:
with and without
Metabolic activation system:
S9: it was prepared from 2 male rats induced with 0.1 % phenobarbitone in their drinking water for 5 days. Activation propertieswere checked with  TA1538 and 4-BP. The optimum activity was produced with 100 ul/plate with 4-aminobiphenyl.
Test concentrations with justification for top dose:
Doses used in Exp. 1 were: 0.2, 2, 20 and 500 ug/plate.
Doses used in Exp. 2 were: 0.02 and  0.2 ug/plate.
Vehicle / solvent:
Solvant used: dimethylsulfoxide
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: methylmethanesulfonate (TA 1535, TA 100); N-Methyl-N-Nitro-N-Nitrosoguanidine (MNNG)(TA 1535, TA 100);  9-Aminoacridine (9-AA) (TA1537), 4-aminobiphenyl (TA100, TA98, TA1538)
Remarks:
with S9
Positive control substance:
other: methylmethanesulfonate (TA 100); N-Methyl-N-Nitro-N-Nitrosoguanidine (MNNG) (TA 1535, TA 100);  9-Aminoacridine (9-AA) (TA1537)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk


DURATION
- Preincubation period:
- Exposure duration:
- Expression time (cells in growth medium):
- Selection time (if incubation with a selection agent):
- Fixation time (start of exposure up to fixation or harvest of cells):


SELECTION AGENT (mutation assays):
SPINDLE INHIBITOR (cytogenetic assays):
STAIN (for cytogenetic assays):


NUMBER OF REPLICATIONS:


NUMBER OF CELLS EVALUATED:


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other:


OTHER EXAMINATIONS:
- Determination of polyploidy:
- Determination of endoreplication:
- Other:


OTHER:
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 98, TA1538 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
other: The positive controls were valid but for 9-AA the positive response was slight
Conclusions:
Under these experimental conditions, Perkadox 14R did not induce reverse mutation in Salmonella typhimurium (strains: TA 1535, TA 1537, TA 98, TA 100 and TA 102).
Executive summary:

The potential of the peroxide to induce reverse mutation in Salmonella typhimurium (strains: TA 1535, TA 1537, TA 98, TA 100 and TA 1538) was evaluated according to Ames et al. (1975): direct plate incorporation (the first experiment and the second without S9mix)

Bacterias were exposed to the substance at four dose-levels (0.2, 2, 20, 500 g/plate). The revertant colonies were scored and reported after substraction of spontaneous revertants. Up to 500 µg Perkadox 14 R per plate did not reveal any mutagenic activity with and without activation.

No increase in the number of revertants was observed for all doses with and without metabolic activation on the 5 tested strains.

Under these experimental conditions, the substance did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

1,3(4)-bis-(t-butyl peroxyisopropyl) benzene was not genotoxic in vitro. According to regulation (EC) No 1272 -2008, the substance is not classified.