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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: other: chromosome aberration and aneuploidy
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03 January 2012 - 06 April 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: 1a: Compliant to GLP and testing guidelines; adequate consistence between data, comments and conclusions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD Guideline 487 (In vitro micronucleus)
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell micronucleus test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material: Tert-amyl hydroperoxide
- Physical state: colorless liquid
- Lot/batch No.: 11011D1280
- Analytical purity: 83.1%
- Water content: 12 %
- Expiry date: 01 February 2013
- Storage condition: at room temperature.

Method

Target gene:
Not applicable (not a gene mutation assay).
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: cryoprotective medium
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 mix
Test concentrations with justification for top dose:
Experiments without S9 mix
.           2.3, 4.7, 9.4, 18.8, 37.5 and 75 µg/mL in the first experiment,
.           2.5, 5, 10, 15, 20, 25, 30 and 40 µg/mL in the second experiment.

Experiments with S9 mix :
.           2.3, 4.7, 9.4, 18.8, 37.5 and 75 µg/mL for the first experiment,
.           4.7, 9.4, 18.8, 28.2, 37.5 and 75 µg/mL for the second experiment,
.           5, 10, 20, 30, 35, 40, 45 and 50 µg/mL for the third experiment.
Vehicle / solvent:
- Vehicle used: dimethylsulfoxide
- Justification for choice: using a test item concentration of 500 mg/mL in DMSO and a treatment volume of 20 µL/2 mL culture medium, the highest recommended dose-level of 5000 µg/mL was achievable.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: mitomycin C, colchicine (-S9 mix); cyclophosphamide (+S9 mix)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
see Executive summary

NUMBER OF CELLS EVALUATED: 2000/dose

DETERMINATION OF CYTOTOXICITY
- Method: population doubling
Evaluation criteria:
The biological relevance of the results should be considered first. Statistical methods are used as an aid in evaluating the test results but should not be the only determinant of a positive response. A result is considered as positive if at least a 2.5-fold increase in the number of micronucleated cells in comparison to the concurrent control is observed, with a statistically significant difference, at one or more concentrations. Concentration-related increases in the frequency of micronucleated cells and comparison to the vehicle control historical data will also be taken into account.
When inconclusive results are observed, or in case of toxic items when the highest analyzable dose-level does not exhibit about 50% toxicity, additional confirmatory experiments may be needed.

Results and discussion

Test resultsopen allclose all
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
ambiguous
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
ambiguous without metabolic activation
positive with metabolic activation

The test item induced chromosome damage, or damage to the cell division apparatus, in cultured L5178Y TK+/- mouse lymphoma cells, in the presence of a rat metabolizing system. In the absence of a rat metabolising system, its potential to induced chromosome damage, or damage to the cell division apparatus, remained equivocal.
Executive summary:

The objective of this study was to evaluate the potential of the test item to induce an increase in the frequency of micronucleated cells,in L5178Y TK+/-mouse lymphoma cells. The test was performed in accordance with OECD 487 guideline, and was GLP compliance.

 

Methods

After a preliminary toxicity test, the test item was tested in three independent experiments, with and/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

Third experiment

Without S9 mix

3 h treatment +
24 h recovery

24 h treatment +
20 h recovery

 

With S9 mix

3 h treatment +
24 h recovery

3 h treatment +
24 h recovery

3 h treatment +
24 h recovery

 

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 and quality of the cells on the slides has also been taken into account.

The test item was dissolved in dimethylsulfoxide (DMSO).

 

Results

The mean frequencies of micronucleated cells for the vehicle controls were as specified in the acceptance criteria, and positive controls showed clear unequivocal increases in the frequency of micronucleated cells. The study was therefore considered to be valid.

 

Since the test item was found severely cytotoxic in the preliminary test, the selection of the highest dose-level to be used in the main experiments was based on the level of toxicity, according to the criteria specified in the international guidelines.

 

Experiments without S9 mix

The dose-levels used for treatment were as follows:

.           2.3, 4.7, 9.4, 18.8, 37.5 and 75 µg/mL in the first experiment,

.           2.5, 5, 10, 15, 20, 25, 30 and 40 µg/mL in the second experiment.

 

Cytotoxicity

Following the 3-hour treatment, a marked to severe toxicity was observed at dose-levels = 37.5 µg/mL as shown by a 62 to 100% decrease in the PD.

Following the 24-hour treatment, a slight to marked toxicity was observed at 30 and 40 µg/mL as shown by a 32% and 72% decrease in the PD, respectively.


Micronucleus analysis

The dose-levels selected for micronucleus analysis were:

.           4.7, 9.4 and 18.8 µg/mL for the 3-hour treatment, higher dose-levels being too cytotoxic,

.           10, 20 and 30 µg/mL for the 24-hour treatment, higher dose-levels being too cytotoxic.

 

No significant increase in the frequency of micronucleated cells was noted after the 3-hour treatment.

Following the 24-hour treatment, an increase in the frequency of micronucleated cells (exceeding the threshold of 2.5-fold the vehicle control value) was observed at 30 µg/mL. This increase was statistically significant (p < 0.05). However, the corresponding micronucleated cells remained within the historical data range of the vehicle control (5 micronucleated cells in 1000 cells versus 0-5 for the historical data). Consequently, this increase is considered as equivocal.

Experiments with S9 mix

The dose-levels used for treatment were as follows:

.           2.3, 4.7, 9.4, 18.8, 37.5 and 75 µg/mL for the first experiment,

.           4.7, 9.4, 18.8, 28.2, 37.5 and 75 µg/mL for the second experiment,

.           5, 10, 20, 30, 35, 40, 45 and 50 µg/mL for the third experiment.

 

Cytotoxicity

Following the first experiment, a moderate to severe toxicity was observed at dose-levels = 37.5 µg/mL as shown by a 45 to 100% decrease in the PD.

Following the second experiment, a slight to severe toxicity was noted at dose-levels = 9.4 µg/mL as shown by a 32% to 100% decrease in the PD.

Following the third experiment, a slight to severe toxicity was observed at dose-levels = 20 µg/mL as shown by a 25% to 100% decrease in the PD.

 

Micronucleus analysis

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

.           9.4, 18.8 and 37.5 µg/mL for the first experiment, the latter inducing a 45% decrease in the PD,

.           4.7, 9.4 and 18.8 µg/mL for the second experiment, the latter inducing a 55% decrease in the PD,

.           10, 20 and 30 µg/mL for the third experiment, the latter inducing a 57% decrease in the PD.

 

In all experiments, slight increases in the frequency of micronucleated cells (exceeding the threshold of 2.5-fold the vehicle control value) were observed. These increases reached statistical significance in the first and third experiments and were dose-related. Therefore, these increases being reproducible in independent experiments, they were considered to be biologically significant.

 

Conclusion

The test item induced chromosome damage, or damage to the cell division apparatus, in cultured L5178Y TK+/-mouse lymphoma cells, in the presence of a rat metabolizing system. In the absence of a rat metabolising system, its potential to induced chromosome damage, or damage to the cell division apparatus, remained equivocal.