Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutations

The potential of 4,4-bis(tert-butyldioxy)valerate (Luperox 230M50) to induce reverse mutations was evaluated in Salmonella typhimurium (Haddouk, 2000). 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 analyzable 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 4,4-bis(tert-butyldioxy)valerate ) for the five strains in both mutagenicity experiments with and without S9 mix. A slight to marked emulsion was noted in the Petri plates when scoring the revertants generally at dose-levels >= 625 µg/plate. No noteworthy toxicity was noted at any of the tested dose-levels, towards the five strains used, either with or without S9 mix.

4,4-bis(tert-butyldioxy)valerate (Luperox 230M50) 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.

 

The potential of 4,4-bis(tert-butyldioxy)valerate (Luperox 230) to induce mutations at the TK (Thymidine Kinase) locus was evaluated in L5178Y TK+/-mouse lymphoma cells (Brient, 2016). The study was performed according to the OECD guideline No. 490 (adopted on 28 July 2015) and in compliance with the principles of Good Laboratory Practice. After a preliminary cytotoxicity test, the test item, dissolved in ethanol, was tested in two 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 was measured by assessment of Adjusted Relative Total Growth (Adj. RTG), Adjusted Relative Suspension Growth (Adj. RSG) and Cloning Efficiency following the expression time (CE2).The number of mutant clones (differentiating small and large colonies) was evaluated after expression of the mutant phenotype.

The 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. Since the test item was found not freely soluble but non-severely cytotoxic in the preliminary test, the highest dose-level selected for the main experiments was 2000 µg/mL, according to the criteria specified in the international guidelines. At the end of the 3- and 24-hour treatments, an emulsion was observed at dose-levels = 500 µg/mL in the culture medium. This emulsion did not prevent the scoring.

Experiments without S9 mix

The selected dose-levels were as follows:

.           62.5, 125, 250, 500, 1000 and 2000 µg/mL for the 3-hour treatment,

.           62.5, 125, 250, 375, 500, 1000 and 2000 µg/mL for the 24-hour treatment.

Following the 3-hour treatment, no noteworthy cytotoxicity was induced at any of the tested dose-levels, as shown by the absence of any noteworthy decrease in Adj. RTG. Following the 24-hour treatment, moderate cytotoxicity was induced at the highest tested dose-level of 2000 µg/mL, as shown by 61% decrease in Adj. RTG. Following the 3- and 24-hour treatments, increases in the mutation frequency were noted. These increases exceeded the GEF of 126 x 10-6at the highest dose-level of 2000 µg/mL. Furthermore, a dose-response relationship was demonstrated with and without S9 mix (p < 0.0001). These results met the criteria of a positive response.

Experiment with S9 mix

The selected dose-levels were 62.5, 125, 250, 500, 1000 and 2000 µg/mL.

No noteworthy cytotoxicity was induced, as shown by the absence of any noteworthy decrease in Adj. RTG. Increases in the mutation frequency were noted over the whole range of dose-levels. These increases exceeded the GEF at the dose-levels = 1000 µg/mL. Furthermore, a dose-response relationship was demonstrated with and without S9 mix (p < 0.0001). These results met the criteria of a positive response.

The dose-levels selected for the main experiments induced an emulsion in the culture medium at the end of the treatment periods, as observed at dose-levels = 500 µg/mL in each experiment. The increases in the MF (Mutation Frequency) being only observed at dose-levels showing an emulsion in the culture medium at the end of the treatment periods, they should be interpreted with caution since their biological significance is questionable. Furthermore, it is to be noted that the mouse lymphoma cells grow in suspension culture, and the presence of emulsion can interfere with the assay. Indeed, at the end of the treatment incubation of 3 hours (with or without S9 mix), the cells are washed by centrifugation and the emulsion may pellet with the cells, making the control of exposure impossible.

Overall under the experimental conditions of this study, 4,4-bis(tert-butyldioxy)valerate (Luperox 230), at concentrations higher than the limit of solubility in the culture medium, is considered to produce equivocal results regarding a potential mutagenic activity in the mouse lymphoma assay, either in the presence or absence of a rat liver metabolizing system. 

 

Chromosomal aberrations

The potential of 4,4-bis(tert-butyldioxy)valerate (Luperox 230M50) to induce chromosome aberrations was evaluated in cultured human lymphocytes (Haddouk, 2002). The test item was tested in two independent experiments, both with and without a liver metabolising system (S9 mix), obtained from rats previously treated with Aroclor 1254. No preliminary cytotoxicity test was performed. 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, toxicity indicated by the reduction of mitotic index (MI) in the first experiment, if any, was also taken into account. For each culture, heparinised whole blood was added to culture medium containing a mitogen (phytohaemagglutinin) 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. As this first experiment was negative, a second experiment was performed as follows:

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

. 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 hour 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. 4,4-bis(tert-butyldioxy)valerate (Luperox 230M50) was dissolved in ethanol. All the dose-levels were expressed as active item, taking into account the active material content of 50.1%. 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: 25 or 50 µg/mL.

In the culture medium, the dose-level of 1148 µg/mL (obtained with treatment volume of 15 µL of the test item) showed a slight emulsion. At this dose-level, the pH and the osmolality values were equivalent to those of the vehicle control culture. Both with and without S9 mix, with a treatment volume of 15 µL/5.5 mL culture medium, the treatment-levels were as follows: 8.97, 17.93, 35.87, 71.73, 143.5, 286.9, 573.9 and 1148 µg/mL, for the first experiment and 35.87, 71.73, 143.5, 286.9, 573.9 and 1148 µg/mL, for the second experiment. A slight emulsion was observed at the end of the treatment period, at dose-levels=573.9 µg/mL. Cytotoxicity: Except for a moderate decrease in the mitotic index noted at 1148 µg/mL, following the 44 hours treatment without S9 mix, no noteworthy toxicity was noted. The dose-levels selected for metaphase analysis, both with and without S9 mix, were as follows: 286.9, 573.9 and 1148 µg/mL, for the 20-hour harvest time and 1148 µg/mL, for the 44-hour harvest time. No noteworthy increase in the frequency of cells with structural chromosomal aberrations which could be considered as relevant was induced, at both harvest times, both with and without S9 mix. The frequencies of cells with structural chromosome aberrations of the vehicle and positive controls were as specified in acceptance criteria. The study was therefore considered valid. 4,4-bis(tert-butyldioxy)valerate (Luperox 230M50) 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
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 January 2016 -- 02 March 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 sodium
pyruvate (200 µg/mL)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 mix
Test concentrations with justification for top dose:
Preliminary test: 4, 40, 200, 400, 1000 and 2000 µg/mL.
Main experiments:
Without S9 mix :
- 62.5, 125, 250, 500, 1000 and 2000 µg/mL for the 3-hour treatment,
- 62.5, 125, 250, 375, 500, 1000 and 2000 µg/mL for the 24 hour treatment.
With S9 mix
The selected dose-levels were 62.5, 125, 250, 500, 1000 and 2000 µg/mL.
Vehicle / solvent:
- Vehicle used: ethanol, batch No. V4D543054E.
- Justification for choice: according to solubility data, the test item was dissolved in the vehicle at a concentration of 400 mg/mL for the preliminary toxicity test and for both mutagenicity experiments in order to reach the highest recommanded dose-level of 2000 µg/mL.

Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
methylmethanesulfonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in culture medium

DURATION
- Exposure duration: 3 or 24 hours, depênding on the experiment and experimental conditions.
- 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 x 10-6.
- A dose response relationship is demonstrated by a statistically significant trend test.

Unless clearly positive, the reproducibility should be confirmed.

None of the criteria for a positive result are met.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
ambiguous
Remarks:
positive at concentrations higher than the limit of solubility in the culture medium
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
>=500 µg/ml, emulsion observed in the culture medium at the end of treatment
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
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. Since the test item was found not freely soluble but non-severely cytotoxic in the preliminary test, the highest dose-level selected for the main experiments was 2000 µg/mL, according to the criteria specified in the international guidelines. At the end of the 3- and 24-hour treatments, an emulsion was observed at dose-levels = 500 µg/mL in the culture medium. This emulsion did not prevent the scoring.  
Experiments without S9 mix
The selected dose-levels were as follows:
.            62.5, 125, 250, 500, 1000 and 2000 µg/mL for the 3-hour treatment,
.            62.5, 125, 250, 375, 500, 1000 and 2000 µg/mL for the 24-hour treatment.
Following the 3-hour treatment, no noteworthy cytotoxicity was induced at any of the tested dose-levels, as shown by the absence of any noteworthy decrease in Adj. RTG. Following the 24-hour treatment, moderate cytotoxicity was induced at the highest tested dose-level of 2000 µg/mL, as shown by 61% decrease in Adj. RTG. Following the 3- and 24-hour treatments, increases in the mutation frequency were noted. These increases exceeded the GEF of 126 x 10-6 at the highest dose-level of 2000 µg/mL. Furthermore, a dose-response relationship was demonstrated with and without S9 mix (p < 0.0001). These results met the criteria of a positive response.
Experiment with S9 mix
The selected dose-levels were 62.5, 125, 250, 500, 1000 and 2000 µg/mL.
No noteworthy cytotoxicity was induced, as shown by the absence of any noteworthy decrease in Adj. RTG. Increases in the mutation frequency were noted over the whole range of dose-levels. These increases exceeded the GEF at the dose-levels = 1000 µg/mL. Furthermore, a dose-response relationship was demonstrated with and without S9 mix (p < 0.0001). These results met the criteria of a positive response.
The dose-levels selected for the main experiments induced an emulsion in the culture medium at the end of the treatment periods, as observed at dose-levels = 500 µg/mL in each experiment. The increases in the MF (Mutation Frequency) being only observed at dose-levels showing an emulsion in the culture medium at the end of the treatment periods, they should be interpreted with caution since their biological significance is questionable. Furthermore, it is to be noted that the mouse lymphoma cells grow in suspension culture, and the presence of emulsion can interfere with the assay. Indeed, at the end of the treatment incubation of 3 hours (with or without S9 mix), the cells are washed by centrifugation and the emulsion may pellet with the cells, making the control of exposure impossible.
Conclusions:
The dose-levels selected for the main experiments induced an emulsion in the culture medium at the end of the treatment periods, as observed at dose-levels = 500 µg/mL in each experiment.
The increases in the MF (Mutation Frequency) being only observed at dose-levels showing an emulsion in the culture medium at the end of the treatment periods, they should be interpreted with caution since their biological significance is questionable.
Furthermore, it is to be noted that the mouse lymphoma cells grow in suspension culture, and the presence of emulsion can interfere with the assay. Indeed, at the end of the treatment incubation of 3 hours (with or without S9 mix), the cells are washed by centrifugation and the emulsion may pellet with the cells, making the control of exposure impossible.
Overall under the experimental conditions of this study, the test item, at concentrations higher than the limit of solubility in the culture medium, is considered to produce equivocal results regarding a potential 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 230 to induce mutations at the TK (Thymidine Kinase) locus was evaluated in L5178Y TK+/- mouse lymphoma cells.  

The study was performed according to the OECD guideline No. 490 (adopted on 28 July 2015) and in compliance with the principles of Good Laboratory Practice. After a preliminary cytotoxicity test, the test item, dissolved in ethanol, was tested in two 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 was measured by assessment of Adjusted Relative Total Growth (Adj. RTG), Adjusted Relative Suspension Growth (Adj. RSG) and Cloning Efficiency following the expression time (CE2). The number of mutant clones (differentiating small and large colonies) was evaluated after expression of the mutant phenotype.

The 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. Since the test item was found not freely soluble but non-severely cytotoxic in the preliminary test, the highest dose-level selected for the main experiments was 2000 µg/mL, according to the criteria specified in the international guidelines. At the end of the 3- and 24-hour treatments, an emulsion was observed at dose-levels = 500 µg/mL in the culture medium. This emulsion did not prevent the scoring.  

Experiments without S9 mix

The selected dose-levels were as follows:

.            62.5, 125, 250, 500, 1000 and 2000 µg/mL for the 3-hour treatment,

.            62.5, 125, 250, 375, 500, 1000 and 2000 µg/mL for the 24-hour treatment.

Following the 3-hour treatment, no noteworthy cytotoxicity was induced at any of the tested dose-levels, as shown by the absence of any noteworthy decrease in Adj. RTG. Following the 24-hour treatment, moderate cytotoxicity was induced at the highest tested dose-level of 2000 µg/mL, as shown by 61% decrease in Adj. RTG. Following the 3- and 24-hour treatments, increases in the mutation frequency were noted. These increases exceeded the GEF of 126 x 10-6 at the highest dose-level of 2000 µg/mL. Furthermore, a dose-response relationship was demonstrated with and without S9 mix (p < 0.0001). These results met the criteria of a positive response.

Experiment with S9 mix

The selected dose-levels were 62.5, 125, 250, 500, 1000 and 2000 µg/mL.

No noteworthy cytotoxicity was induced, as shown by the absence of any noteworthy decrease in Adj. RTG. Increases in the mutation frequency were noted over the whole range of dose-levels. These increases exceeded the GEF at the dose-levels = 1000 µg/mL. Furthermore, a dose-response relationship was demonstrated with and without S9 mix (p < 0.0001). These results met the criteria of a positive response.

The dose-levels selected for the main experiments induced an emulsion in the culture medium at the end of the treatment periods, as observed at dose-levels = 500 µg/mL in each experiment. The increases in the MF (Mutation Frequency) being only observed at dose-levels showing an emulsion in the culture medium at the end of the treatment periods, they should be interpreted with caution since their biological significance is questionable. Furthermore, it is to be noted that the mouse lymphoma cells grow in suspension culture, and the presence of emulsion can interfere with the assay. Indeed, at the end of the treatment incubation of 3 hours (with or without S9 mix), the cells are washed by centrifugation and the emulsion may pellet with the cells, making the control of exposure impossible. Overall under the experimental conditions of this study, the test item, at concentrations higher than the limit of solubility in the culture medium, is considered to produce equivocal results regarding a potential mutagenic activity in the mouse lymphoma assay, either in the presence or absence of a rat liver metabolizing system.  

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)
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:
rat liver S9 mix
Test concentrations with justification for top dose:
312.5, 625, 1250, 2500 and 5000 µg/plate (expressed as butyl 4,4-bis(tert-butyldioxy)valerate)
Vehicle / solvent:
- Vehicle used: ethanol
- Justification for choice: the test item was diluted in the vehicle at a concentration of 100 mg/mL for the preliminary toxicity test and for both mutagenicity experiments.
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: 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:
A reproducible two-fold increase 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 applicable
Positive controls validity:
valid
Additional information on results:
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 substance was freely soluble and non-toxic, the highest dose-level was 5000 µg/plate, according to the criteria specified in the international guidelines.
A slight to marked emulsion was noted in the Petri plates when scoring the revertants generally at dose-levels >= 625 µg/plate.
No noteworthy toxicity was induced towards all the strains used, both with and without S9 mix.
The test substance did not induce any noteworthy increase in the number of revertants, both with and without S9 mix, in any of the five strains.
Conclusions:
LUPEROX® 230M50 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® 230M50 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 4,4-bis(tert-butyldioxy)valerate ) for the five strains in both mutagenicity experiments with and without S9 mix. A slight to marked emulsion was noted in the Petri plates when scoring the revertants generally at dose-levels >= 625 µg/plate. No noteworthy toxicity was noted at any of the tested dose-levels, towards the five strains used, either with or without S9 mix.

LUPEROX® 230M50 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 cytogenicity / chromosome aberration study in mammalian cells
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 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
lymphocytes:
Remarks:
human
Details on mammalian cell type (if applicable):
Human lymphocytes were prepared from whole blood samples obtained from two healthy donors and collected into heparinised sterile tubes.
Cytokinesis block (if used):
Colcemid (10 µg/mL) to block cells at the metaphase-stage of mitosis
Metabolic activation:
with and without
Metabolic activation system:
Rat S9 mix. Liver S9 homogenate was prepared from rats that have been induced with Arochlor 1254.
Test concentrations with justification for top dose:
8.97, 17.93, 35.87, 71.73, 143.5, 286.9, 573.9 and 1148 µg/mL, for the first experiment, +/- S9 mix
35.87, 71.73, 143.5, 286.9, 573.9 and 1148 µg/mL, for the second experiment, +/- S9 mix
(expressed as butyl 4,4-bis(tert-butyldioxy)valerate)
Vehicle / solvent:
- Vehicle: ethanol
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium;

DURATION
- Preincubation period: 48H
- Exposure duration:
- 3h (+/-S9) for the 1st experiment.
- Until harvest: 20-44h (-S9), 3h (+S9) for the 2nd experiment
- Fixation time (start of exposure up to fixation or harvest of cells): 20h (1st experiment - 2nd experiment with S9) ; 44h in the second experiment without S9.


SPINDLE INHIBITOR: 10 µg/mL of colcemid solution (1.5h prior harvest)

STAIN: Giemsa (time)

NUMBER OF REPLICATIONS: 2 experiments with duplicates for each experiment

NUMBER OF CELLS EVALUATED: 100 metaphases per culture (200 per dose-level). Only 50 metaphases/culture were analyzed when at
least 10% cells with structural chromosome aberration were observed.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index;

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
Evaluation criteria:
A cell having one or more of the above-mentioned structural chromosome aberration was recorded as a single cell with structural chromosome aberration. Therefore the total frequency of cells with structural chromosome aberration was not necessarily equivalent to the total number of aberrations
Reproducible and statistically increase in the number of cells with structural chromosome aberration for at least one dose level and one of the two harvest times is considered as positive.
Reference to historical data and consideration to biological relevance may also be taken into account.
Statistics:
Frequency of cells with structural chromosome aberrations compared to that of the vehicle control. Test of “Chi-2’ is used when necessary with p=0.05 as the lowest significant level.
Key result
Species / strain:
lymphocytes: human
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 examined
Positive controls validity:
valid
Additional information on results:
The test item was freely soluble in the vehicle (ethanol). In the culture medium, the highest dose-level of 1148 µg/mL (obtained with treatment volume of 15 µL of the test item) showed a slight emulsion. At this dose-level, the pH was about 7.4 (as for the vehicle control) and the osmolality equal to 306 mOsm/kg H2O
(325 for the vehicle control). A slight emulsion was observed at the end of the treatment period, at dose-levels = 573.9 µg/mL.

- Experiments without S9 mix:
Cytotoxicity:
Except for a moderate decrease in the mitotic index noted at 1148 µg/mL following the 44 hours treatment (58% decrease), no noteworthy toxicity was noted.

Chromosomal aberration analysis:
The dose-levels selected for metaphase analysis were as follows:
. 286.9, 573.9 and 1148 µg/mL, for the 3-hour and the 20-hour treatments,
. 1148 µg/mL, 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 hours treatments.

- Experiments with S9 mix:
Cytotoxicity:
No noteworthy toxicity was induced, in both experiments and at both harvest times.

Chromosomal aberration analysis:
The dose-levels selected for metaphase analysis were as follows:
. 286.9, 573.9 and 1148 µg/mL, for the 20-hour harvest time in both experiments,
. 1148 µg/mL, for the 44-hour harvest time.
A slight significant increase in the frequency of cells with structural chromosomal aberrations was noted at 1148 µg/mL (3.5%, p < 0.05). However, this slight increase was not considered as relevant since it was not reproducible neither between donors nor between experiments.
No significant increase in the frequency of cells with structural chromosomal aberrations was noted at the 44-hour harvest time.

The frequencies of cells with structural chromosome aberrations of the vehicle and positive controls were as specified in acceptance criteria. The study was therefore considered valid.
Conclusions:
LUPEROX 230M50 did not induce chromosome aberrations in cultured human lymphocytes
Executive summary:

The potential of LUPEROX 230M50 to induce chromosome aberrations was evaluated in cultured human lymphocytes. The test item was tested in two independent experiments, both with and without a liver metabolising system (S9 mix), obtained from rats previously treated with Aroclor 1254. No preliminary cytotoxicity test was performed. 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, toxicity indicated by the reduction of mitotic index (MI) in the first experiment, if any, was also taken into account. For each culture, heparinised whole blood was added to culture medium containing a mitogen (phytohaemagglutinin) 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. As this first experiment was negative, a second experiment was performed as follows:

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

. 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 hour 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. LUPEROX 230M50 was dissolved in ethanol. All the dose-levels were expressed as active item, taking into account the active material content of 50.1%. 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: 25 or 50 µg/mL.

In the culture medium, the dose-level of 1148 µg/mL (obtained with treatment volume of 15 µL of the test item) showed a slight emulsion. At this dose-level, the pH and the osmolality values were equivalent to those of the vehicle control culture. Both with and without S9 mix, with a treatment volume of 15 µL/5.5 mL culture medium, the treatment-levels were as follows: 8.97, 17.93, 35.87, 71.73, 143.5, 286.9, 573.9 and 1148 µg/mL, for the first experiment and 35.87, 71.73, 143.5, 286.9, 573.9 and 1148 µg/mL, for the second experiment. A slight emulsion was observed at the end of the treatment period, at dose-levels=573.9 µg/mL. Cytotoxicity: Except for a moderate decrease in the mitotic index noted at 1148 µg/mL, following the 44 hours treatment without S9 mix, no noteworthy toxicity was noted. The dose-levels selected for metaphase analysis, both with and without S9 mix, were as follows: 286.9, 573.9 and 1148 µg/mL, for the 20-hour harvest time and 1148 µg/mL, for the 44-hour harvest time. No noteworthy increase in the frequency of cells with structural chromosomal aberrations which could be considered as relevant was induced, at both harvest times, both with and without S9 mix. The frequencies of cells with structural chromosome aberrations of the vehicle and positive controls were as specified in acceptance criteria. The study was therefore considered valid. LUPEROX 230M50 did not induce chromosome aberrations in cultured human lymphocytes.

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 for germ cell mutagenicity according to CLP/GHS criteria.