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Toxicological information

Genetic toxicity: in vitro

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guideline
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

Test material

Constituent 1
Chemical structure
Reference substance name:
Butyl 4,4-bis(tert-butyldioxy)valerate
EC Number:
213-626-6
EC Name:
Butyl 4,4-bis(tert-butyldioxy)valerate
Cas Number:
995-33-5
Molecular formula:
C17H34O6
IUPAC Name:
butyl 4,4-bis(tert-butylperoxy)pentanoate
Test material form:
other: Colorless to yellow liquid

Method

Target gene:
Thymidine Kinase
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 medium containing L-Glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 µg/mL) and sodium
pyruvate (200 µg/mL)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes
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.

Controls
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.

Results and discussion

Test results
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.

Applicant's summary and conclusion

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.