Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation assays

OECD 471 study

The potential of LUPEROX® 520M50 to induce reverse mutations was evaluated inSalmonella typhimurium (Chevallier, 2016). The study was performed according to the international guidelines (OECD No. 471 and Commission Directive No. B.13/14) and in compliance with the principles of Good Laboratory Practice. A preliminary toxicity test was performed to define the dose-levels of the test item, diluted in ethanol, to be used for the mutagenicity experiments. The test item was then tested in two independent experiments, both with and without a metabolic activation system, the S9 mix, prepared from a liver post-mitochondrial fraction (S9 fraction) of rats induced with Aroclor 1254. Treatments were performed according to the direct plate incorporation method except for the second experiment with S9 mix, which was performed according to the pre-incubation method (60 minutes, 37°C). Five strains of bacteriaSalmonella typhimuriumwere used: TA 1535, TA 1537, TA 98, TA 100 and TA 102. 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. Since the test item was found poorly soluble in the final treatment medium but non-toxic in the preliminary test, the highest dose-level selected for the main experiments was 5000 µg/plate, according to the criteria specified in the international guidelines. 

The mean number of revertants for the vehicle and positive controls met the acceptance criteria. Also, there were five analysable dose-levels for each strain and test condition. The study was therefore considered to be valid. The selected dose-levels were 312.5, 625, 1250, 2500 and 5000 µg/plate (expressed as 2,2-di-(tert-amylperoxy) butane) for the five strains in both mutagenicity experimentswith and without S9 mix. A moderate to strong emulsion, which did not prevent any scoring, was observed in the Petri plates from the lowest selected dose-levels. No noteworthy toxicity was noted at any of the tested dose-levels, towards the five strains used, either with or without S9 mix.

LUPEROX® 520M50did not induce any biologically relevant increase in the number of revertants, in any strains or test conditions. Consequently, the results met the criteria of a negative response. 

OECD 490 study

The potential of LUPEROX® 520M50 to induce mutations at the TK (Thymidine Kinase) locus was evaluated in L5178Y TK+/-mouse lymphoma cells (Sire, 2016). The study was performed according to international guidelines (OECD guideline No. 490) and in compliance with the principles of Good Laboratory Practice. After a preliminary cytotoxicity test, the test item, diluted in ethanol, was tested in three independent 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 or control items, in the presence or absence of S9 mix (final concentration of S9 fraction 2%). During the treatment period, the cells were maintained as suspension culture in RPMI 1640 culture medium supplemented by heat inactivated horse serum at 5% (3-hour treatment) or 10% (24-hour treatment) in a 37°C, 5% CO2humidified incubator. For the 24-hour treatment, flasks were gently shaken at least once. Cytotoxicity wasmeasured 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.

Since the test item was found cytotoxic, the selection of the highest dose-level (expressed as 2,2-di-(tert-amylperoxy) butane) to be used in the main experiments was based on the level of cytotoxicity, according to the criteria specified in the international guidelines. 

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.

Experiments without S9 mix

The selected dose-levels (as 2,2-di-(tert-amylperoxy) butane)were as follows:

-7.81, 15.6, 31.3, 62.5, 125, 250 and 500 µg/mL for the 3-hour treatment in the first experiment,

- 0.391, 0.781, 1.56, 3.13, 6.25, 12.5, 18.8 and 25 µg/mL for the 3-hour treatment in the second experiment,

- 0. 313, 0.625, 1.25, 2.5, 5, 10, 20 and 40 µg/mL for the 24-hour treatment (third experiment).

 An emulsion, which did not prevent any scoring, was observed in the culture medium at dose-levels = 125 µg/mL at the end of the 3-hour treatment.

Following the 3-hour treatment in the first experiment, a 81 to 99% decrease in the Adj. RTG was observed at dose-levels = 15.6 µg/mL.

Following the 3-hour treatment in the second experiment, a 36 to 96% decrease in the Adj. RTG was observed at dose-levels = 12.5 µg/mL.

Following the 24-hour treatment (third experiment), a 65 to 87% decrease in the Adj. RTG was observed at dose-levels = 20 µg/mL.

Following the 3- and 24-hour treatments, no noteworthy increase in the mutation frequency was noted relative to the corresponding vehicle control, at any of the tested dose-levels, and no dose-related increase was observed.

These results did not meet the criteria of a positive response.

Experiment with S9 mix

The selected dose-levels (as 2,2-di-(tert-amylperoxy) butane) were as follows:

- 7.81, 15.6, 31.3, 62.5, 125, 250 and 500 µg/mL for the first experiment,

- 1.56, 3.13, 6.25, 12.5, 25, 37.5, 50 and 100 µg/mL for the second experiment.

An emulsion or precipitate, which did not prevent any scoring, was observed in the culture medium at dose-levels = 125 µg/mL.

A 36 to 99% decrease in the Adj. RTG was observed at dose-levels = 15.6 µg/mL in the first experiment and a 37 to 86% decrease in the Adj. RTG was observed at dose-levels = 25 µg/mL in the second experiment.

No noteworthy increase in the mutation frequency was noted relative to the vehicle control, at any of the tested dose-levels and no dose-response relationship was observed.

These results did not meet the criteria of a positive response.

Under the experimental conditions of this study, LUPEROX® 520M50 did not show any mutagenic activity in the mouse lymphoma assay, either in the presence or absence of a rat liver metabolizing system.

Chromosomal aberration assay

OECD 487 study

The potential of LUPEROX® 520M50 to induce an increase in the frequency of micronucleated cells was evaluated in the mouse cell line L5178Y TK+/- (Chevallier, 2016). The study design was based on the OECD guideline No. 487, adopted 26 September 2014.After a preliminary cytotoxicity test, the test item diluted 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 experiment

Second experiment

Without S9 mix

3 h treatment + 24 h recovery

3 h treatment + 24 h recovery

 

24 h treatment + 0 h recovery

24 h treatment + 0 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 culture medium during the preliminary cytotoxicity test, the selection of the highest dose-level to be used in the main experiments was based on the level of cytotoxicity and/or on the level 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, selected dose-levels ranged from 1.56 to 400 µg/mL (expressed as 2,2-di-(tert-amylperoxy) butane).An emulsion was observed in the culture medium at dose-levels superior or egal to 133 µg/mL at the end of the 3-hour treatment period in both experiments, and at the dose-level of 400 µg/mL at the end of the 24-hour treatment period in the second experiment. 

Following the 3-hour treatment in the first experiment, a marked cytotoxicity was induced at the dose-level of 200 µg/mL, as shown by a 69% decrease in the PD.

Following the 3-hour treatment in the second experiment, a slight to severe cytotoxicity was induced at dose-levels superior or egal to 25 µg/mL, as shown by a 35 to 100% decrease in the PD.

Following the 24-hour treatment in the first experiment, a slight cytotoxicity was induced at the dose-level of 50 µg/mL, as shown by a 31% decrease in the PD.

Following the 24-hour treatment in the second experiment, a moderate to severe cytotoxicity was induced at dose-levels superior or egal to50 µg/mL, as shown by a 41 to 100% decrease in the PD.

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

. 37.5, 50 and 100 µg/mL for the 3-hour treatment in the first experiment, the latter inducing no decrease in the PD and the higher dose-level being too cytotoxic,

. 33.3, 50 and 100 µg/mL for the 24-hour treatment in the first experiment, the latter inducing a 24% decrease in the PD and being the highest tested dose-level,

. 12.5, 25 and 50 µg/mL in the second experiment, the latter inducing the recommended level of cytotoxicity following the 3-hour treatment (i.e.55% decrease in the PD), or inducing a 41% decrease in the PD and higher dose-levels being too cytotoxic following the 24-hour treatment.

No noteworthy and biologically relevant increases in the frequency of micronucleated cells were notedat any of the analyzed dose-levels, in either treatment period or experiment.Despite the use of a narrower range of dose-levels in the second experiment, none of the dose-levels selected for the 24-hour treatment induced the recommended level of cytotoxicity. Considering the negative results obtained in both independent experiments performed, the overall available results were considered as suitable to allow a reliable interpretation.The overall results without S9 mix were considered to meet 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 selected were 12.5, 25, 50, 100, 200 and 400 µg/mL (expressed as 2,2-di-(tert-amylperoxy) butane).An emulsion was observed in the culture medium at dose-levels superior or egal to 200 µg/mL at the end of the treatment period.

No noteworthy cytotoxicity was induced at any of the tested dose-levels, as shown by the absence of any noteworthy decrease in the PD. 

The dose-levels selected for micronucleus analysis were 50, 100 and 200 µg/mL, the latter being the lowest dose-level showing test item emulsion in the culture medium at the end of the treatment period.

No noteworthy increases in the frequency of micronucleated cells were notedat any of the analyzed dose-levels. The results with S9 mix metthe criteria of a negative response.

Under the experimental conditions of the study, LUPEROX® 520M50 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

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 July 2016 - 13 October 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Test material information:
Composition 1
Target gene:
Histidine operon
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 mix
Test concentrations with justification for top dose:
312.5, 625, 1250, 2500 and 5000 µg/plate (expressed as 2,2-di-(tert-amylperoxy) butane)
Vehicle:
- Vehicle used: ethanol
- Justification for choice: the test item was diluted in the vehicle at a concentration of 200 mg/mL for the preliminary toxicity test and for both mutagenicity experiments.
Negative controls:
no
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: sodium azide, 9-aminoacridine, 2-nitrofluorene, mitomycin C (-S9 mix); 2-anthramine, benzo(a)pyrene (+S9 mix)
Details on test system and conditions:
METHOD OF APPLICATION: The preliminary toxicity test, both experiments without S9 mix and the first experiment with S9 mix were performed according to the direct plate incorporation method. The second experiment with S9 mix was performed according to the pre-incubation method.
DURATION
- Preincubation period: 60 minutes
- Exposure duration: 48 to 72 hours.
DETERMINATION OF CYTOTOXICITY
- Method: decrease in number of revertant colonies and/or thinning of the bacterial lawn
Evaluation criteria:
Evaluation criteriaIn all cases, biological relevance (such as reproducibility and reference to historical data) was taken into consideration when evaluating the results.The test item is considered to have shown mutagenic activity in this study if:
- 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 mean number of revertants compared with the vehicle controls is observed, in any strain, at any dose-level,
- and/or a reproducible dose-response relationship is evidenced.
The test item is considered to have shown no mutagenic activity in this study if:
- neither an increase in the mean number of revertants, reaching 2-fold (for the TA 98, TA 100 and TA 102 strains) or 3-fold (for the TA 1535 and TA 1537 strains) the vehicle controls value, is observed at any of the tested dose-levels,
- nor any evidence of a dose-response relationship is noted.
Key result
Species / strain:
other: TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to limit concentrations
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Conclusions:
LUPEROX® 520M50 did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium strains, either in the presence or absence of a rat liver metabolizing system.
Executive summary:

The potential of LUPEROX® 520M50 to induce reverse mutations was evaluated in Salmonella typhimurium. The study was performed according to the international guidelines (OECD No. 471 and Commission Directive No. B.13/14) and in compliance with the principles of Good Laboratory Practice. A preliminary toxicity test was performed to define the dose-levels of the test item, diluted in ethanol, to be used for the mutagenicity experiments. The test item was then tested in two independent experiments, both with and without a metabolic activation system, the S9 mix, prepared from a liver post-mitochondrial fraction (S9 fraction) of rats induced with Aroclor 1254. Treatments were performed according to the direct plate incorporation method except for the second experiment with S9 mix, which was performed according to the pre-incubation method (60 minutes, 37°C). Five strains of bacteria Salmonella typhimurium were used: TA 1535, TA 1537, TA 98, TA 100 and TA 102. 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. Since the test item was found poorly soluble in the final treatment medium but non-toxic in the preliminary test, the highest dose-level selected for the main experiments was 5000 µg/plate, according to the criteria specified in the international guidelines. 

The mean number of revertants for the vehicle and positive controls met the acceptance criteria. Also, there were five analysable dose-levels for each strain and test condition. The study was therefore considered to be valid. The selected dose-levels were 312.5, 625, 1250, 2500 and 5000 µg/plate (expressed as 2,2-di-(tert-amylperoxy) butane) for the five strains in both mutagenicity experimentswith and without S9 mix. A moderate to strong emulsion, which did not prevent any scoring, was observed in the Petri plates from the lowest selected dose-levels. No noteworthy toxicity was noted at any of the tested dose-levels, towards the five strains used, either with or without S9 mix.

LUPEROX® 520M50 did not induce any biologically relevant increase in the number of revertants, in any strains or test conditions. Consequently, the results met the criteria of a negative response. 

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 July 2016 - 25 October 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Test material information:
Composition 1
Target gene:
Thymidine Kinase
Species / strain:
mouse lymphoma L5178Y cells
Details on mammalian cell lines (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:
(as 2,2-di-(tert-amylperoxy) butane)
Without S9 mix : 0.313 to 500 µg/mL
With S9 mix: 1.56 to 500 µg/mL
Vehicle:
- Vehicle used: ethanol- Justification for choice: allowed solubilization of the test item at concentrations of (expressed as 2,2-di-(tert-amylperoxy) butane): - 400 mg/mL for the preliminary toxicity test, - 100 mg/mL for the first experiment, - 20 mg/mL for the second and third experiments.
Negative controls:
no
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: methylmethane sulfonate (-S9 mix); cyclophosphamide (+S9 mix)
Details on test system and conditions:
METHOD OF APPLICATION: in mediumDURATION- Exposure duration: 3 and 24 hours- Expression time (cells in growth medium): 48 hours- Selection time (if incubation with a selection agent): 11-12 daysSELECTION AGENT (mutation assays): trifluorothymidineDETERMINATION OF CYTOTOXICITY- Method: cloning efficiency; Relative Total Growth, Relative Suspension Growth.
Evaluation criteria:
IWGT recommendations were followed for the determination of a positive result, which should fulfill the following criteria:- at least at one dose-level the mutation frequency minus the mutation frequency of the vehicle control (IMF) equals or exceeds the Global Evaluation Factor (GEF) of 126 x 10-6,- a dose-response relationship is demonstrated by a statistically significant trend test. Unless an effect is considered as clearly positive, the reproducibility of a positive effect should be confirmed.Noteworthy increases in the mutation frequency observed only at high levels of cytotoxicity (Adj. RTG lower than 10%), but with no evidence of mutagenicity at dose-levels with Adj. RTG between 10 and 20%, are not considered as positive results.A test item may be considered as non-mutagenic when there is no culture showing an Adj. RTG value between 10 and 20% if:- 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:
To assess the dose-response relationship, a linear regression was performed between dose-levels and individual mutation frequencies obtained from cultures showing a mean Adj. RTG = 10%. This statistical analysis was performed using SAS Enterprise Guide software.
Key result
Species / strain:
other: mouse lymphoma L5178Y TK+/- cells mammalian cell line
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Conclusions:
LUPEROX® 520M50 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® 520M50 to induce mutations at the TK (Thymidine Kinase) locus was evaluated in L5178Y TK+/-mouse lymphoma cells. The study was performed according to international guidelines (OECD guideline No. 490 and Council Regulation) and in compliance with the principles of Good Laboratory Practice. After a preliminary cytotoxicity test, the test item, diluted in ethanol, was tested in three independent 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 105 cells/mL (3-hour treatments) or cultures of 50 mL at 2 x 105 cells/mL (24-hour treatment) were exposed to the test or control items, in the presence or absence of S9 mix (final concentration of S9 fraction 2%). During the treatment period, the cells were maintained as suspension culture in RPMI 1640 culture medium supplemented by heat inactivated horse serum at 5% (3-hour treatment) or 10% (24-hour treatment) in a 37°C, 5% CO2 humidified incubator. For the 24-hour treatment, flasks were gently shaken at least once. Cytotoxicity wasmeasured 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.

Since the test item was found cytotoxic, the selection of the highest dose-level (expressed as 2,2-di-(tert-amylperoxy) butane) to be used in the main experiments was based on the level of cytotoxicity, according to the criteria specified in the international guidelines. 

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.

 

Experiments without S9 mix

The selected dose-levels (as 2,2-di-(tert-amylperoxy) butane)were as follows:

- 7.81, 15.6, 31.3, 62.5, 125, 250 and 500 µg/mL for the 3-hour treatment in the first experiment,

- 0.391, 0.781, 1.56, 3.13, 6.25, 12.5, 18.8 and 25 µg/mL for the 3-hour treatment in the second experiment,

- 0. 313, 0.625, 1.25, 2.5, 5, 10, 20 and 40 µg/mL for the 24-hour treatment (third experiment).

 

An emulsion, which did not prevent any scoring, was observed in the culture medium at dose-levels = 125 µg/mL at the end of the 3-hour treatment.


Cytotoxicity

Following the 3-hour treatment in the first experiment, a 81 to 99% decrease in the Adj. RTG was observed at dose-levels = 15.6 µg/mL.

Following the 3-hour treatment in the second experiment, a 36 to 96% decrease in the Adj. RTG was observed at dose-levels = 12.5 µg/mL.

Following the 24-hour treatment (third experiment), a 65 to 87% decrease in the Adj. RTG was observed at dose-levels = 20 µg/mL.

 

Mutagenicity

Following the 3- and 24-hour treatments, no noteworthy increase in the mutation frequency was noted relative to the corresponding vehicle control, at any of the tested dose-levels, and no dose-related increase was observed.

These results did not meet the criteria of a positive response.

 

Experiment with S9 mix

The selected dose-levels (as 2,2-di-(tert-amylperoxy) butane) were as follows:

- 7.81, 15.6, 31.3, 62.5, 125, 250 and 500 µg/mL for the first experiment,

- 1.56, 3.13, 6.25, 12.5, 25, 37.5, 50 and 100 µg/mL for the second experiment.

An emulsion or precipitate, which did not prevent any scoring, was observed in the culture medium at dose-levels = 125 µg/mL.

 

Cytotoxicity

A 36 to 99% decrease in the Adj. RTG was observed at dose-levels = 15.6 µg/mL in the first experiment and a 37 to 86% decrease in the Adj. RTG was observed at dose-levels = 25 µg/mL in the second experiment.

 

Mutagenicity

No noteworthy increase in the mutation frequency was noted relative to the vehicle control, at any of the tested dose-levels and no dose-response relationship was observed.

These results did not meet the criteria of a positive response.

Under the experimental conditions of this study, LUPEROX® 520M50 did not show any mutagenic activity in the mouse lymphoma assay, either in the presence or absence of a rat liver metabolizing system.

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 July 2016 -- 30 September 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The historical data used for the validation of long treatment period without S9 mix (24 hours treatment + 0 hour recovery) were generated with non-audited data from non-GLP studies. These data were performed in compliance with CiToxLAB France’s standard operating procedures. Since CiToxLAB France is a Test Facility certified by the French National Authorities for Good Laboratory Practice, and the procedures undertaken are the same, this deviation is considered not to prejudice the overall GLP status of the study and the scientific reliability of the study conclusions. Moreover, the corresponding mean frequency of micronucleated cells in the vehicle control was 2.5‰ in the first experiment and 0.5‰ in the second experiment, therefore = 5‰ as specified in the acceptance criteria.
Reference:
Composition 0
Qualifier:
according to
Guideline:
other: OECD Guideline 487 (In vitro micronucleus)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell micronucleus test
Test material information:
Composition 1
Target gene:
Not applicable (not a gene mutation assay).
Species / strain:
mouse lymphoma L5178Y cells
Details on mammalian cell lines (if applicable):
- Type and identity of media: RPMI 1640 medium containing 10% 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: yesL5178Y TK+/- cells are an established cell line recommended by international regulations for in vitro mammalian cell gene mutation test and for in vitro micronucleus test. Indeed, they are suitable to reveal chemically induced micronuclei. The average cell cycle time is approximately 10-12 hours. L5178Y TK+/- cells were obtained from ATCC (American Type Culture Collection, Manassas, USA), by the intermediate of Biovalley (Marne-La-Vallée, France). The cells were stored in a cryoprotective medium (10% horse serum and 10% dimethylsulfoxide (DMSO)) at -80°C and each batch of frozen cells was checked for the absence of mycoplasma.
Additional strain characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 mix
Test concentrations with justification for top dose:
(expressed as 2,2-di-(tert-amylperoxy) butane)
Without S9 mix: 1.56 to 400 µg/mL
With S9 mix 12.5 to 400 µg/mL
Vehicle:
- Vehicle used: ethanol
- Justification for choice: the test item was diluted in the vehicle at concentrations of:
- 400 mg/mL for the preliminary cytotoxicity test,
- 80 mg/mL for both main cytogenetic experiments.
Negative controls:
no
Solvent 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 conditions:
METHOD OF APPLICATION: in medium
With a treatment volume of 0.5% (v/v) in culture medium, selected dose-levels ranged from 1.56 to 400 µg/mL (expressed as 2,2-di-(tert-amylperoxy) butane).An emulsion was observed in the culture medium at dose-levels superior or egal to 133 µg/mL at the end of the 3-hour treatment period in both experiments, and at the dose-level of 400 µg/mL at the end of the 24-hour treatment period in the second experiment.
DURATION
Preliminary cytotoxicity 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

First cytogenetic experiment:
Without S9 mix:
3 h treatment + 24 h recovery
24 h treatment + 0 h recovery
With S9 mix:
3 h treatment + 24 h recovery.
Second cytogenetic experiment:
Without S9 mix:
3 h treatment + 24 h recovery
24 h treatment + 0 h recovery
With S9 mix: None.

NUMBER OF CELLS EVALUATED: 2000 mononucleated cells per 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.
When the criteria of a positive response were only partially met, results were evaluated on a case by case basis, taking into account other parameters such as reproducibility between experiments. When the highest analyzable dose-level did not exhibit about 55% cytotoxicity (in case of cytotoxic items), or if results remained inconclusive, additional confirmatory experiments were performed.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to precipitating concentrations
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Conclusions:
LUPEROX® 520M50 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 LUPEROX® 520M50 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, adopted 26 September 2014. After a preliminary cytotoxicity test, the test item diluted 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 experiment

Second experiment

Without S9 mix

3 h treatment + 24 h recovery

3 h treatment + 24 h recovery

 

24 h treatment + 0 h recovery

24 h treatment + 0 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 culture medium during the preliminary cytotoxicity test, the selection of the highest dose-level to be used in the main experiments was based on the level of cytotoxicity and/or on the level 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, selected dose-levels ranged from 1.56 to 400 µg/mL (expressed as 2,2-di-(tert-amylperoxy) butane). An emulsion was observed in the culture medium at dose-levels superior or egal to 133 µg/mL at the end of the 3-hour treatment period in both experiments, and at the dose-level of 400 µg/mL at the end of the 24-hour treatment period in the second experiment.  

Following the 3-hour treatment in the first experiment, a marked cytotoxicity was induced at the dose-level of 200 µg/mL, as shown by a 69% decrease in the PD.

Following the 3-hour treatment in the second experiment, a slight to severe cytotoxicity was induced at dose-levels superior or egal to 25 µg/mL, as shown by a 35 to 100% decrease in the PD.

Following the 24-hour treatment in the first experiment, a slight cytotoxicity was induced at the dose-level of 50 µg/mL, as shown by a 31% decrease in the PD.

Following the 24-hour treatment in the second experiment, a moderate to severe cytotoxicity was induced at dose-levels superior or egal to 50 µg/mL, as shown by a 41 to 100% decrease in the PD.

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

. 37.5, 50 and 100 µg/mL for the 3-hour treatment in the first experiment, the latter inducing no decrease in the PD and the higher dose-level being too cytotoxic,

. 33.3, 50 and 100 µg/mL for the 24-hour treatment in the first experiment, the latter inducing a 24% decrease in the PD and being the highest tested dose-level,

. 12.5, 25 and 50 µg/mL in the second experiment, the latter inducing the recommended level of cytotoxicity following the 3-hour treatment (i.e. 55% decrease in the PD), or inducing a 41% decrease in the PD and higher dose-levels being too cytotoxic following the 24-hour treatment.

No noteworthy and biologically relevant increases in the frequency of micronucleated cells were notedat any of the analyzed dose-levels, in either treatment period or experiment. Despite the use of a narrower range of dose-levels in the second experiment, none of the dose-levels selected for the 24-hour treatment induced the recommended level of cytotoxicity. Considering the negative results obtained in both independent experiments performed, the overall available results were considered as suitable to allow a reliable interpretation. The overall results without S9 mix were considered to meet 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 selected were 12.5, 25, 50, 100, 200 and 400 µg/mL (expressed as 2,2-di-(tert-amylperoxy) butane). An emulsion was observed in the culture medium at dose-levels superior or egal to 200 µg/mL at the end of the treatment period.

No noteworthy cytotoxicity was induced at any of the tested dose-levels, as shown by the absence of any noteworthy decrease in the PD.  

The dose-levels selected for micronucleus analysis were 50, 100 and 200 µg/mL, the latter being the lowest dose-level showing test item emulsion in the culture medium at the end of the treatment period.

No noteworthy increases in the frequency of micronucleated cells were notedat any of the analyzed dose-levels. The results with S9 mix metthe criteria of a negative response.

Under the experimental conditions of the study, LUPEROX® 520M50 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.

 

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

No classification is warranted according to CLP and GHS criteria.