Cyclohexylidenebis[tert-amyl] peroxide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutations

LUPEROX 531 M60 was evaluated for induction of reverse mutation in Salmonella typhimurium (Sire, 2005). The study was performed according to the OECD test guideline # 471 and in compliance with the Principles of Good Laboratory Practice Regulations. A preliminary toxicity test was performed to define the dose-levels of LUPEROX 531 M60 to be used for the mutagenicity study. The test item was then 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. Both experiments were performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the preincubation method (60 minutes, 37°C). Five strains of bacteria Salmonella typhimurium: TA 1535, TA 1537, TA 98, TA 100 and TA 102 were used. Each strain was exposed to five dose-levels of the test item (three plates/dose-level). After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored. The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn. LUPEROX 531 M60 was dissolved in ethanol. All the concentrations were expressed as active item, 1,1-di(t-amylperoxy)cyclohexane, taking into account the active material content of 60.5%. The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid. Since the test item was freely soluble and non-toxic in the preliminary assay, the highest dose-level was 5000µg/plate, according to the criteria specified in the international guidelines. The selected treatment-levels were: 312.5, 625, 1250, 2500 and 5000µg/plate, for both mutagenicity experiments with and without S9 mix. A moderate to marked emulsion was observed in the Petri plates when scoring the revertants at dose-levels=625µg/plate. No noteworthy toxicity was noted towards all the strains used, with and without S9 mix. In the second experiment without S9 mix, a slight increase in the number of revertants was noted in the TA 1537 strain (up to 3.3-fold the vehicle control value). Since this slight increase was neither dose-related, nor reproducible (not observed in the first experiment performed under the same experimental conditions), and since the threshold of 3-fold the vehicle control value seems to have been reached only due to the low value of the vehicle control (near the lowest value of the vehicle control historical data range), it was considered not to be relevant. The test item did not induce any other noteworthy increase in the number of revertants, both with and without S9 mix, in any of the five strains. LUPEROX 531 M60 did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.

 

LUPEROX® 531M60 was evaluated for inductions of mutations at the TK (Thymidine Kinase) locus in L5178Y TK+/-mouse lymphoma cells (Brient, 2016). The study was performed according to the OECD test guideline # 490 and in compliance with the Principles of Good Laboratory Practice Regulations. The test item is a mixture of 61% of 1,1-di(tert-amylperoxy)cyclohexane in isododecane. The study was performed according to international guidelines OECD # 490 and in compliance with the principles of Good Laboratory Practice. After two preliminary cytotoxicity tests, the test item, dissolved in ethanol, was tested in two independent main experiments, with 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 treatments) or cultures of 50 mL at 2 x 105cells/mL (24-hour treatment) were exposed to the test item (expressed as 1,1-di(tert-amylperoxy)cyclohexane) 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, 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. With one exception which was not considered to have compromised the integrity or validity of the study, the cloning efficiencies, the mutation frequencies and the suspension growths of the vehicle controls were as specified in the acceptance criteria. For the positive control cultures, the increase in the mutation frequencies met also the acceptance criteria. In addition, the upper limit of cytotoxicity observed in the positive control cultures had an Adj. RTG greater than 10%. The study was therefore considered to be valid. The test item was found cytotoxic and poorly soluble in the preliminary tests. Cytotoxicity was observed at concentrations lower than the lowest precipitating concentration in the culture medium without S9 mix. Therefore, the selection of the highest concentration for the main experiment without S9 mix (3-hour treatment) was based on the level of cytotoxicity, according to the criteria specified in the international guidelines. Following the 24-hour without S9 mix and the 3-hour treatment with S9 mix, cytotoxicity was observed at the lowest concentration showing emulsion in the culture medium. Therefore, the selection of the highest concentration for the corresponding main experiments was based on the level of cytotoxicity and on the presence of emulsion in the culture medium, according to the criteria specified in the international guidelines.

Experiments without S9 mix

The selected concentrations were as follows:

- 1.56, 3.13, 6.25, 12.5, 25, 37.5, 50 and 100 µg/mL for the 3-hour treatment,

- 0.63, 1.25, 2.5, 5, 10, 20, 30 and 40 µg/mL for the 24-hour treatment.

An emulsion, remaining in the culture medium, was observed at concentrations = 30 µg/mL at the end of the 24 -hour treatment, without preventing any scoring.

No emulsion was observed in the culture medium at the end of the 3-hour treatment.

Following the 3-hour treatment, a moderate to marked cytotoxicity was induced at concentrations = 25 µg/mL, as shown by a 73 to 84% decrease in Adj. RTG.

Following the 24-hour treatment, a severe cytotoxicity was induced at concentrations = 30 µg/mL, as shown by a 100% decrease in Adj. RTG.

Following the 3-hour treatment, no noteworthy increase in the mutation frequency was induced at any of the tested concentrations, as shown by the absence of any noteworthy increase in the IMF, and no dose response relationship was evidenced (p > 0.05). These results met thus the criteria of a negative response.

Following the 24-hour treatment, increases in the mutation frequency were observed at concentrations up to 30 µg/mL. These increases remained below the GEF to the concentration of 20 µg/mL, which showed Adj. RTG > 10%.

It is to be noted that the increase observed at the concentration of 30 µg/mL was above the GEF (i.e.IMF of 131), but this increase was observed at a too cytotoxic concentration (i.e.Adj < 10%). Consequently, this increase was considered as not biologically relevant.

Furthermore, the statistical analysis performed did not demonstrate a linear trend between the mutation frequencies and the tested concentrations (p > 0.05). These results met thus the criteria of a negative response.

Experiments with S9 mix

The selected concentrations were 3.13, 6.25, 12.5, 25, 50, 100, 200 and 400 µg/mL.

An emulsion, remaining in the culture medium, was observed at concentrations = 200 µg/mL, without preventing any scoring.

A slight to moderate cytotoxicity was induced at concentrations of 100 and 200 µg/mL, as shown by a 43 to 75% decrease in Adj. RTG.

No noteworthy increase in the mutation frequency was induced at any of the tested concentrations, as shown by the absence of any noteworthy increase in the IMF, and no dose-response relationship was evidenced (p > 0.05). These results met thus the criteria of a negative response.

Under the experimental conditions of this study, LUPEROX® 531M60 did not show any mutagenic activity in the mouse lymphoma assay, either in the presence or absence of a rat liver metabolizing system.

 

Chromosomal aberrations

LUPEROX® 531M60 was evaluated for the induction of an increase in the frequency of micronucleated cells in the mouse cell line L5178Y TK+/- (Brient, 2016). The study was performed according to the OECD test guideline # 487 and in compliance with the Principles of Good Laboratory Practice Regulations. The test item is a mixture of 61% of 1,1-di(tert-amylperoxy)cyclohexane in isododecane and all the concentrations were expressed as active item, 1,1-di(t-amylperoxy)cyclohexane. After a preliminary cytotoxicity test, the test item LUPEROX®531M60, prepared as a solution in ethanol, was tested in two independent experiments, with or without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254, as 3 h treatment + 24 h recovery (tests I and II +/- S9), 24 h treatment + 0 h recovery (test II –S9). Each treatment was coupled to an assessment of cytotoxicity at the same dose-levels. Cytotoxicity was evaluated by determining the PD (Population Doubling) of cells. Then, after the final cell counting, the cells were washed and fixed. Then, cells from at least three dose-levels of the test item treated cultures were dropped onto clean glass slides. The slides were air-dried before being stained in 5% Giemsa. Slides from vehicle and positive controls cultures were also prepared as described above. All slides were coded before analysis, so that the analyst was unaware of the treatment details of the slide under evaluation (”blind” scoring). For each main experiment (with or without S9 mix), micronuclei were analyzed for three dose-levels of the test item, for the vehicle and the positive controls, in 1000 mononucleated cells per culture (total of 2000 mononucleated cells per dose). Number of cells with micronuclei and number of micronuclei per cell were recorded separately for each treated and control culture. Since the test item was found cytotoxic and poorly soluble in the preliminary test, the selection of the highest dose-level to be used in the main experiments was based on the level of cytotoxicity and/or the presence of emulsion, according to the criteria specified in the international guidelines. The mean population doubling and the mean frequencies of micronucleated cells for the vehicle controls were as specified in the acceptance criteria. Also, positive control cultures showed clear statistically significant increases in the frequency of micronucleated cells. The study was therefore considered to be valid.

Without S9 mix and a treatment volume of 0.5 % (v/v) in culture medium, the dose-levels used for treatment were 9.38, 18.8, 37.5, 75, 150 and 300 µg/mL in the 3-hour treatment of the first experiment, 4.69, 9.38, 18.8, 28.2, 37.5, 56.3, 75 and 200 µg/mL in the 24-hour treatment of the first experiment, and 0.35, 0.69, 1.39, 2.78, 5.55, 11.1, 33.3 and 100 µg/mL in the 3-hour treatment of the second experiment.

An emulsion was observed at dose-levels superior or equal to 75 µg/mL at the end of the 3-hour treatments, and at 200 µg/mL at the end of the 24-hour treatment. Following the 3-hour treatment of the first experiment, an unexpected marked to severe cytotoxicity was observed for all the tested dose-levels, as shown by a 75 to 100% decrease in the PD. Due to this excessive cytotoxicity, none of the tested dose-levels could be selected for micronucleus analysis. Therefore, a second experiment was implemented in the same experimental conditions, but using a lower range of dose-levels. Following the 3-hour treatment of the second experiment, a moderate cytotoxicity was observed at 33.3 µg/mL, as shown by a 58% decrease in the PD. Following the 24-hour treatment of the first experiment, a slight to severe cytotoxicity was observed at dose-levels = 18.8 µg/mL, as shown by a 35 to 100% decrease in the PD. Due to the excessive cytotoxicity observed following the 3-hour treatment of the first experiment, none of the tested dose-levels was selected for micronucleus analysis. The dose-levels selected for micronucleus analysis were 5.55, 11.1 and 33.3 µg/mL for the 3-hour treatment (second experiment), the latter inducing the recommended level of cytotoxicity (i.e.58% decrease in the PD) and 4.69, 9.38 and 18.8 µg/mL for the 24-hour treatment, the latter inducing a 34% decrease in the PD and higher dose-levels being too cytotoxic.

Increases in the frequency of micronucleated cells were noted at 11.1 and 33.3 µg/mL after the 3-hour treatment (second experiment). A dose-response relationship was noted. However, none of these increases were statistically significant when compared to the corresponding vehicle control, and the frequencies of micronucleated cells for each replicate culture remained within the vehicle historical range. Consequently, these results are considered to meet the criteria of a negative response. No increase in the frequency of micronucleated cells were noted after the 24-hour treatment. Furthermore, no dose-response relationship was observed, and the frequencies of micronucleated cells for each replicate culture remained within the vehicle historical data. These results met the criteria of a negative response.

With S9 mix and a treatment volume of 0.5% (v/v) in culture medium, the dose-levels used for treatment were 9.38, 18.8, 37.5, 75, 150 and 300 µg/mL. An emulsion was observed at the end of the treatment period, at dose-levels superior or equal to 150 µg/mL. A slight cytotoxicity was induced at the highest tested dose-level of 300 µg/mL, as shown by a 28% decrease in the PD. The dose-levels selected for micronucleus analysis were 75, 150 and 300 µg/mL, the latter inducing a 28% decrease in the PD and showing emulsion in the culture medium at the end of the treatment period which did not prevent any scoring. Increases in the frequency of micronucleated cells were noted at 75 and 300 µg/mL. However, these increases were neither statistically significant nor dose-related, and none of the analyzed dose-levels showed frequency of micronucleated cells of each replicate culture above the vehicle control historical range. These results met the criteria of a negative response.

Under the experimental conditions of the study, LUPEROX®531M60, did not induce any chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, using L5178Y TK+/-mouse lymphoma cells, either in the presence or absence of a rat liver metabolizing system.

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21st July 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine operon

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254

Test concentrations with justification for top dose:

312.5, 625, 1250, 2500 and 5000 µg/plate

Vehicle / solvent:

Ethanol

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

other: 2-Anthramine

Details on test system and experimental conditions:

METHOD OF APPLICATION: direct plate incorporation method except for the second test with S9 mix, which was performed according to the preincubation method (60 minutes, 37°C)

NUMBER OF REPLICATIONS: 3

Evaluation criteria:

A reproducible 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as a positive result. Reference to historical data, or other considerations of biological relevance may also be taken into account in the evaluation of the data obtained.

Key result

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

A moderate to marked emulsion was observed in the Petri plates when scoring the revertants at dose-levels . 625 ƒÊg/plate.
No noteworthy toxicity was noted towards all the strains used, with and without S9 mix.
In the second experiment without S9 mix, a slight increase in the number of revertants was noted in the TA 1537 strain (up to 3.3-fold the vehicle control value).
Since this slight increase was neither dose-related, nor reproducible (not observed in the first experiment performed under the same experimental conditions), and since the threshold of 3-fold the vehicle control value seems to have been reached only due to the low value of the vehicle control (near the lowest value of the vehicle control historical data range), it was considered not to be relevant.

Conclusions:

1,1-DI(t-AMYLPEROXY)CYCLOHEXANE did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.

Executive summary:

LUPEROX 531 M60 was evaluated for induction of reverse mutation in Salmonella typhimurium. The study was performed according to the international guidelines (OECD 471, Commission Directive No. B13/14) and in compliance with the Principles of Good Laboratory Practice Regulations. A preliminary toxicity test was performed to define the dose-levels of LUPEROX 531 M60 to be used for the mutagenicity study. The test item was then 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. Both experiments were performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the preincubation method (60 minutes, 37°C). Five strains of bacteria Salmonella typhimurium: TA 1535, TA 1537, TA 98, TA 100 and TA 102 were used. Each strain was exposed to five dose-levels of the test item (three plates/dose-level). After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored. The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn. LUPEROX 531 M60 was dissolved in ethanol. All the concentrations and dose-levels were expressed as active item, 1,1-DI(t-AMYLPEROXY)CYCLOHEXANE, taking into account the active material content of 60.5%. The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid. Since the test item was freely soluble and non-toxic in the preliminary assay, the highest dose-level was 5000µg/plate, according to the criteria specified in the international guidelines. The selected treatment-levels were: 312.5, 625, 1250, 2500 and 5000µg/plate, for both mutagenicity experiments with and without S9 mix. A moderate to marked emulsion was observed in the Petri plates when scoring the revertants at dose-levels=625µg/plate. No noteworthy toxicity was noted towards all the strains used, with and without S9 mix. In the second experiment without S9 mix, a slight increase in the number of revertants was noted in the TA 1537 strain (up to 3.3-fold the vehicle control value). Since this slight increase was neither dose-related, nor reproducible (not observed in the first experiment performed under the same experimental conditions), and since the threshold of 3-fold the vehicle control value seems to have been reached only due to the low value of the vehicle control (near the lowest value of the vehicle control historical data range), it was considered not to be relevant. The test item did not induce any other noteworthy increase in the number of revertants, both with and without S9 mix, in any of the five strains. LUPEROX 531 M60 did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 March 2016 - 23 September 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

26 September 2014

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

Not applicable (not a gene mutation assay).

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640 medium containing 10% (v/v) heat inactivated horse serum, 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

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 mix

Test concentrations with justification for top dose:

Since the test item was found cytotoxic and poorly soluble in the preliminary test, the selection of the highest dose-level to be used in the main experiments was based on the level of cytotoxicity and/or the presence of emulsion, according to the criteria specified in the international guidelines.
With a treatment volume of 0.5% (v/v) in culture medium, the dose-levels used for treatment were as follows:
- Experiments without S9 mix: 9.38, 18.8, 37.5, 75, 150 and 300 µg/mL for the 3-hour treatment in the first experiment, 0.35, 0.69, 1.39, 2.78, 5.55, 11.1, 33.3 and 100 µg/mL for the 3-hour treatment in the second experiment and 4.69, 9.38, 18.8, 28.2, 37.5, 56.3, 75 and 200 µg/mL for the 24-hour treatment.
- Experiment with S9 mix: 9.38, 18.8, 37.5, 75, 150 and 300 µg/mL.

Vehicle / solvent:

- Vehicle used: ethanol
- Justification for choice: based on available solubility data, the test item was found to be not soluble in water for injections and dimethylsulfoxide (DMSO), but a solution was obtained in ethanol and the highest recommended dose-level could be reached using the highest practicable volume with ethanol.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: mitomycin C, colchicine (-S9 mix); cyclophosphamide (+S9 mix)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
Preliminary cytoxicity test:
Without S9 mix:3 h treatment + 24 h recovery
24 h treatment + 0 h recovery
With S9 mix:
3 h treatment + 24 h recovery
Main cytogenetic test
- first experimentWithout S9 mix:
3 h treatment + 24 h recovery
24 h treatment + 0 h recovery
With S9 mix:3 h treatment + 24 h recovery
Main cytogenetic test
- second experiment
Without S9 mix:3 h treatment + 24 h recovery
With S9 mix:None.

NUMBER OF CELLS EVALUATED: 2000 mononucleated cells/dose

DETERMINATION OF CYTOTOXICITY
- Method: population doubling

Evaluation criteria:

The biological relevance of the results was always taken into account when evaluating results. Evaluation of a positive response: a test item is considered to have clastogenic and/or aneugenic potential, if all the following criteria were met: - a dose-related increase in the frequency of micronucleated cells was demonstrated by a statistically significant trend test,- for at least one dose-level, the frequency of micronucleated cells of each replicate culture was above the corresponding vehicle historical range,- a statistically significant difference in comparison to the corresponding vehicle control was obtained at one or more dose-levels.Evaluation of a negative response: a test item is considered clearly negative if none of the criteria for a positive response was met.

Key result

Species / strain:

other: mouse lymphoma L5178Y TK+/- 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

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Effects of osmolality: none
- Evaporation from medium: none
- Precipitation: >= 75 µg/mL
- Definition of acceptable cells for analysis:
Analysis was performed under a microscope (1000 x magnification), on the basis of the recommendations of Miller et al. (1995) (e), according to the following criteria:
. micronuclei should be clearly surrounded by a nuclear membrane,
. the micronucleus area should be less than one-third of the area of the main nucleus,
. non-refractility of the micronuclei,
. micronuclei should not be linked to the main nucleus via nucleoplasmic bridges,
. micronuclei should be located within the cytoplasma of the cell,
. only mononucleated cells with a number of micronuclei = 5 should be scored to exclude apoptosis and nuclear fragmentation.
- Other confounding effects: none

RANGE-FINDING/SCREENING STUDIES:
Based on available solubility data, the test item was prepared as a solution in ethanol at a concentration of 400 mg/mL. Therefore, using this stock solution and a treatment volume of 0.5% (v/v) in culture medium, the highest recommended dose-level of 2000 µg/mL was achievable. Thus, the dose-levels selected for the treatment of the preliminary test were 4, 40, 200, 400, 1000 and 2000 µg/mL.

At the highest dose-level of 2000 µg/mL, the pH of the culture medium was approximately 7.4, as for the vehicle control. Furthermore, none of the tested dose-levels induced a significant increase in the osmolality when compared to the vehicle control (up to 399 mOsm/kg H2O vs. 394 mOsm/kg H2O for the vehicle control). Therefore, none of the tested dose-levels was considered to produce extreme culture conditions.

An emulsion was observed in the culture medium at the end of the treatment periods, at dose-levels >= 200 µg/mL.

Following the 3-hour treatment without S9 mix, a slight cytotoxicity was observed at dose-levels >= 1000 µg/mL, as shown by a 30 to 36% decrease in the PD.
Following the 24-hour treatment without S9 mix, a moderate to severe cytotoxicity was observed at dose-levels >= 40 µg/mL, as shown by a 52 to 100% decrease in the PD.
Following the 3-hour treatment with S9 mix, no noteworthy cytotoxicity was noted, as shown by the absence of any noteworthy decrease in the Population Doubling (PD).

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: see attached document

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: see attached document
- Negative (solvent/vehicle) historical control data: see attached document

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: Population Doubling

Conclusions:

LUPEROX® 531M60, did not induce any chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, using L5178Y TK+/- mouse lymphoma cells, either in the presence or absence of a rat liver metabolizing system.

Executive summary:

The objective of this study was to evaluate the potential of the test item, LUPEROX^® 531M60, to induce an increase in the frequency of micronucleated cells in the mouse cell line L5178Y TK^+/-. The study design was based on the OECD guideline No. 487. After a preliminary cytotoxicity test, the test item LUPEROX^® 531M60, prepared as a solution in ethanol, was tested in two independent experiments, with or without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254, as follows:

 

	First main experiment	Second main experiment
Without S9 mix	3 h treatment + 24 h recovery 24 h treatment + 0 h recovery	3 h treatment + 24 h recovery
With S9 mix	3 h treatment + 24 h recovery	None

 

Each treatment was coupled to an assessment of cytotoxicity at the same dose-levels. Cytotoxicity was evaluated by determining the PD (Population Doubling) of cells. Then, after the final cell counting, the cells were washed and fixed. Then, cells from at least three dose-levels of the test item treated cultures were dropped onto clean glass slides. The slides were air-dried before being stained in 5% Giemsa. Slides from vehicle and positive controls cultures were also prepared as described above. All slides were coded before analysis, so that the analyst was unaware of the treatment details of the slide under evaluation ("blind" scoring). For each main experiment (with or without S9 mix), micronuclei were analyzed for three dose-levels of the test item, for the vehicle and the positive controls, in 1000 mononucleated cells per culture (total of 2000 mononucleated cells per dose). Number of cells with micronuclei and number of micronuclei per cell were recorded separately for each treated and control culture.

Since the test item was found cytotoxic and poorly soluble in the preliminary test, the selection of the highest dose-level to be used in the main experiments was based on the level of cytotoxicity and/or the presence of emulsion, according to the criteria specified in the international guidelines. The mean population doubling and the mean frequencies of micronucleated cells for the vehicle controls were as specified in the acceptance criteria. Also, positive control cultures showed clear statistically significant increases in the frequency of micronucleated cells. The study was therefore considered to be valid.

Experiments without S9 mix

With a treatment volume of 0.5 % (v/v) in culture medium, the dose-levels used for treatment were as follows:

.            9.38, 18.8, 37.5, 75, 150 and 300 µg/mL in the 3-hour treatment of the first experiment,

.            4.69, 9.38, 18.8, 28.2, 37.5, 56.3, 75 and 200 µg/mL in the 24-hour treatment of the first experiment,

.            0.35, 0.69, 1.39, 2.78, 5.55, 11.1, 33.3 and 100 µg/mL in the 3-hour treatment of the second experiment.

An emulsion was observed at dose-levels superior or equal to 75 µg/mL at the end of the 3-hour treatments, and at 200 µg/mL at the end of the 24-hour treatment. Following the 3-hour treatment of the first experiment, an unexpected marked to severe cytotoxicity was observed for all the tested dose-levels, as shown by a 75 to 100% decrease in the PD. Due to this excessive cytotoxicity, none of the tested dose-levels could be selected for micronucleus analysis. Therefore, a second experiment was implemented in the same experimental conditions, but using a lower range of dose-levels. Following the 3-hour treatment of the second experiment, a moderate cytotoxicity was observed at 33.3 µg/mL, as shown by a 58% decrease in the PD. Following the 24-hour treatment of the first experiment, a slight to severe cytotoxicity was observed at dose-levels = 18.8 µg/mL, as shown by a 35 to 100% decrease in the PD. Due to the excessive cytotoxicity observed following the 3-hour treatment of the first experiment, none of the tested dose-levels was selected for micronucleus analysis. 

The dose-levels selected for micronucleus analysis were as follows:

.            5.55, 11.1 and 33.3 µg/mL for the 3-hour treatment (second experiment), the latter inducing the recommended level of cytotoxicity (i.e. 58% decrease in the PD),

.            4.69, 9.38 and 18.8 µg/mL for the 24-hour treatment, the latter inducing a 34% decrease in the PD and higher dose-levels being too cytotoxic.

Increases in the frequency of micronucleated cells were noted at 11.1 and 33.3 µg/mL after the 3-hour treatment (second experiment). A dose-response relationship was noted. However, none of these increases were statistically significant when compared to the corresponding vehicle control, and the frequencies of micronucleated cells for each replicate culture remained within the vehicle historical range. Consequently, these results are considered to meet the criteria of a negative response. No increase in the frequency of micronucleated cells were noted after the 24-hour treatment. Furthermore, no dose-response relationship was observed, and the frequencies of micronucleated cells for each replicate culture remained within the vehicle historical data. These results met the criteria of a negative response.

Experiment with S9 mix

With a treatment volume of 0.5% (v/v) in culture medium, the dose-levels used for treatment were 9.38, 18.8, 37.5, 75, 150 and 300 µg/mL. An emulsion was observed at the end of the treatment period, at dose-levels superior or equal to 150 µg/mL. A slight cytotoxicity was induced at the highest tested dose-level of 300 µg/mL, as shown by a 28% decrease in the PD. The dose-levels selected for micronucleus analysis were 75, 150 and 300 µg/mL, the latter inducing a 28% decrease in the PD and showing emulsion in the culture medium at the end of the treatment period which did not prevent any scoring. Increases in the frequency of micronucleated cells were noted at 75 and 300 µg/mL. However, these increases were neither statistically significant nor dose-related, and none of the analyzed dose-levels showed frequency of micronucleated cells of each replicate culture above the vehicle control historical range. These results met the criteria of a negative response.

Under the experimental conditions of the study, LUPEROX^® 531M60, did not induce any chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, using L5178Y TK^+/-mouse lymphoma cells, either in the presence or absence of a rat liver metabolizing system.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 March 2016 - 10 May 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

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 sodiumpyruvate (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
- 1.56, 3.13, 6.25, 12.5, 25, 37.5, 50 and 100 µg/mL for the 3-hour treatment,
- 0.63, 1.25, 2.5, 5, 10, 20, 30 and 40 µg/mL for the 24-hour treatment.
Experiments with S9 mix
- 3.13, 6.25, 12.5, 25, 50, 100, 200 and 400 µg/mL

Vehicle / solvent:

- Vehicle used: ethanol
- Justification for choice: the test item was found not soluble in water for injections or DMSO, but was found soluble in ethanol (see solubility data obtained at CiToxLAB France).

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: microwell method
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, relative suspension growth.

Evaluation criteria:

Positive result defined as:
- 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 E-6.
- A dose-related trend is demonstrated by a statistically significant trend test.Unless clearly positive, the reproducibility should be confirmed.Negative results defined as:none of the criteria for a positive response is met.
A test item may be considered as non-mutagenic when there is no culture showing an Adj. RTG value between 10 and 20% if (g):
- 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.

Statistics:

A trend test was performed to assess the linear trend between the mutation frequency and the concentrations tested. This statistical analysis was a linear regression performed using SAS Enterprise Guide software.Only individual mutation frequencies obtained from cultures showing an Adj. RTG = 10% were used for this analysis.

Key result

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

Conclusions:

LUPEROX® 531M60 did not show any mutagenic activity in the mouse lymphoma assay, either in the presence or absence of a rat liver metabolizing system.

Executive summary:

The potential of LUPEROX® 531M60 to induce mutations was evaluated at the TK (Thymidine Kinase) locus in L5178Y TK^+/-mouse lymphoma cells. The test item is a mixture of 61% of 1,1-di(tert-amylperoxy)cyclohexane in isododecane. The study was performed according to international guidelines (OECD No. 490 and Council Regulation No. 440/2008) and in compliance with the principles of Good Laboratory Practice. After two preliminary cytotoxicity tests, the test item, dissolved in ethanol, was tested in two independent main experiments, with 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 treatments) or cultures of 50 mL at 2 x 10⁵cells/mL (24-hour treatment) were exposed to the test item (expressed as 1,1-di(tert-amylperoxy)cyclohexane) 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, 5% CO₂ humidified 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.

With one exception which was not considered to have compromised the integrity or validity of the study, the cloning efficiencies, the mutation frequencies and the suspension growths of the vehicle controls were as specified in the acceptance criteria. For the positive control cultures, the increase in the mutation frequencies met also the acceptance criteria.In addition, the upper limit of cytotoxicity observed in the positive control cultures had an Adj. RTG greater than 10%. The study was therefore considered to be valid. The test item was found cytotoxic and poorly soluble in the preliminary tests. Cytotoxicity was observed at concentrations lower than the lowest precipitating concentration in the culture medium without S9 mix. Therefore, the selection of the highest concentration for the main experiment without S9 mix (3-hour treatment) was based on the level of cytotoxicity, according to the criteria specified in the international guidelines.  

Following the 24-hour without S9 mix and the 3-hour treatment with S9 mix, cytotoxicity was observed at the lowest concentration showing emulsion in the culture medium. Therefore, the selection of the highest concentration for the corresponding main experiments was based on the level of cytotoxicity and on the presence of emulsion in the culture medium, according to the criteria specified in the international guidelines.

Experiments without S9 mix

The selected concentrations were as follows:

- 1.56, 3.13, 6.25, 12.5, 25, 37.5, 50 and 100 µg/mL for the 3-hour treatment,

- 0.63, 1.25, 2.5, 5, 10, 20, 30 and 40 µg/mL for the 24-hour treatment.

An emulsion, remaining in the culture medium, was observed at concentrations = 30 µg/mL at the end of the 24 -hour treatment, without preventing any scoring.

No emulsion was observed in the culture medium at the end of the 3-hour treatment.

Following the 3-hour treatment, a moderate to marked cytotoxicity was induced at concentrations = 25 µg/mL, as shown by a 73 to 84% decrease in Adj. RTG.

Following the 24-hour treatment, a severe cytotoxicity was induced at concentrations = 30 µg/mL, as shown by a 100% decrease in Adj. RTG.

Following the 3-hour treatment, no noteworthy increase in the mutation frequency was induced at any of the tested concentrations, as shown by the absence of any noteworthy increase in the IMF, and no dose response relationship was evidenced (p > 0.05). These results met thus the criteria of a negative response.

Following the 24-hour treatment, increases in the mutation frequency were observed at concentrations up to 30 µg/mL. These increases remained below the GEF to the concentration of 20 µg/mL, which showed Adj. RTG > 10%.

It is to be noted that the increase observed at the concentration of 30 µg/mL was above the GEF (i.e. IMF of 131), but this increase was observed at a too cytotoxic concentration (i.e. Adj < 10%). Consequently, this increase was considered as not biologically relevant.

Furthermore, the statistical analysis performed did not demonstrate a linear trend between the mutation frequencies and the tested concentrations (p > 0.05). These results met thus the criteria of a negative response.

Experiments with S9 mix

The selected concentrations were 3.13, 6.25, 12.5, 25, 50, 100, 200 and 400 µg/mL.

An emulsion, remaining in the culture medium, was observed at concentrations = 200 µg/mL, without preventing any scoring.

A slight to moderate cytotoxicity was induced at concentrations of 100 and 200 µg/mL, as shown by a 43 to 75% decrease in Adj. RTG.

No noteworthy increase in the mutation frequency was induced at any of the tested concentrations, as shown by the absence of any noteworthy increase in the IMF, and no dose-response relationship was evidenced (p > 0.05). These results met thus the criteria of a negative response.

Under the experimental conditions of this study, LUPEROX® 531M60 did not show any mutagenic activity in the mouse lymphoma assay, either in the presence or absence of a rat liver metabolizing system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

No classification is warranted according to the available data and CLPcriteria.