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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2000-01-21 to 2000-02-10
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study; OECD and EU guidelines were followed

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted on 21 st July 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
31st July 1992
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9 mix (Aroclor 1254-induced enzymatic systems contained in rat liver microsomal fraction)
Test concentrations with justification for top dose:
312.5, 625, 1250, 2500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without S9 mix

Migrated to IUCLID6: for strains TA 1535, TA100
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without S9 mix

Migrated to IUCLID6: for strain TA1537
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
without S9 mix

Migrated to IUCLID6: for strain TA98
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without S9 mix

Migrated to IUCLID6: for strain TA 102
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-anthramine for all strains
Remarks:
with S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION:

Preliminary toxicity test:
To assess the toxicity of the test item to the bacteria, six dose-levels (one plate/dose-level) were tested in the TA 98, TA 100 and TA 102 strains, with and without S9 mix.
The evaluation of the toxicity was performed on the basis of the observation of the decrease in number of revertant colonies and/or a thinning of the bacterial lawn.

Main test:
In two experiments, five dose-levels of the test item (three plates/dose-level) were tested on each strain, with and without S9 mix.
In each experiment, the following controls were included using triplicate plates:
- vehicle controls: each bacterial tester strain treated with the vehicle,
- positive controls: each bacterial tester strain treated with appropriate reference mutagens.

The experiments were performed according to :
- direct plate incorporation method (preliminary toxicity test, both experiments without S9 mix, first experiment with S9 mix): test items solution (0.1 mL), S9 mix (0.5 mL) when required and bacterial suspension (0.1 mL) were mixed with 2 mL of overlay agar (containing traces of the relvant aminoacid and biotin and maintained at 45 °C). After rapid homogenization, the mixture was overlaid onto a Petri plate containing minimum medium.
- preincubation method (second experiment with S9 mix): test substance solution (0.1 mL), S9 mix (0.5 mL) and bacterial suspension (0.1 mL) were incubated for 60 minutes at 37 °C before adding the overlay agar and pouring onto the surface of a minimum agar plate.
After 48 to 72 hours of inhibition at 37 °C, revertants were scored with an automatic counter.
Evaluation criteria:
Treatment of results:
In each experiment, for each strain and for each experimental point, the number of revertants per plate was scored. The individual results and the mean number of revertants, with the corresponding standard deviation and ratio (mutants obtained in the presence of the test substance/mutants obtained in the presence of the vehicle).

Acceptance criteria:
This study is considered valid if the following criteria are fully met:
- the number of revertants in the vehicle controls is consistent with our historical data
- the number of revertants in the positive controls is higher than that of the vehicle controls and is consistent with the historical data.

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 other considerations of biological relevance may also be taken into account in the evaluation of the data obtained.

Results and discussion

Test resultsopen allclose all
Species / strain:
other: TA 98, TA 1535, TA 100
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
other: TA 1537, TA102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
other: TA 98, TA 1535, TA 100, TA 1537, TA102
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Preliminary toxicity test:
The test substance was freely soluble in vehicle (distelled water) at 50 mg/mL.
Consequently, with a maximum dose volume of 100 µL/plate, the dose-levels were: 10, 100, 500, 1000, 2500 and 5000 µg/plate.
A slight emulsion was observed in the Petri plates when scoring the revertants at the highest dose-level.
No toxicity was noted towards the three strains used, with and without S9 mix.

Mutagenicity experiments:
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.
The selected treatment-levels were 312.5, 625, 1250, 2500 and 5000 µg/plate, for both mutagenicity experiments with and without S9 mix.
A slight or moderate emulsion was observed in the Petri plates when scoring the revertants, at the highest dose-level, in both mutagenicity experiments with and without S9 mix.

Experiments without S9 mix:
Except for a slight or moderate toxicity, noted in the first experiment in the TA 1535, TA 98 and TA 100 strains at dose-levels >= 1250 µg/plate, no noteworthy toxicity was observed in both experiments.
No noteworthy increase in the number of revertants was observed in both experiment, in any of the five strains.

Experiments with S9 mix:
No noteworthy toxicity was noted all strains used, in the both experiment.
No noteworthy increase in the number of revertants was observed in both experiment, in any of the five tester strains.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The test item cyclohexylidenebis[tert-butyl]peroxide does not show mutagenic acitivity in bacterial reverse mutation test with Salmonella typhimurium.
Executive summary:

Cyclohexylidenebis[tert-butyl]peroxide was tested in the Ames test with Salmonella typhimurium TA 98, TA 100, TA 102, TA 1535 and TA 1537 according to EU method B14 and OECD guideline No.471.

A preliminary toxicity was performed to define the dose-levels of cyclohexylidenebis[tert-butyl]peroxide to be used for mutagenicity study. The test item was then tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254.

Both eyperiments were preformed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the preincubation method (60 minutes, 37 °C).

Five strains of bacteria Salmonella typhimurium TA 98, TA 100, TA 102, TA 1535 and TA 1537 were used. Each strain was exposed to five dose-levels of the test substance (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. The test item was dissolved in distelled water.

Since the test item was freely soluble and non-toxic, the highest dose-level was 5000 µg/plate.

The selected treatment-levels were 312.5, 625, 1250, 2500 and 5000 µg/plate, for both mutagenicity experiments with and without S9 mix.

Except for slight to moderate toxicity noted in the first experiment without S9 mix in the TA 98, TA 1535 and TA 100 strains, no noteworthy toxicity was observed. The test item did not induce any noteworthy increase in the number of revertants, both with and without S9 mix, in any of five strains.

The number of revertants for the vehicle and positive controls was as specified in th acceptance criteria. The study was therefore considered valid.

The test item cyclohexylidenebis[tert-butyl]peroxide does not show mutagenic acitivity in bacterial reverse mutation test with Salmonella typhimurium.