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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

1,1-dimethylpropyl 1-methoxycyclohexyl peroxide is negative in the Bacterial Reverse Mutation Assay.

1,1-dimethylpropyl 1-methoxycyclohexyl peroxide (Luperox XPS-TP) was tested in the Bacterial Reverse Mutation Assay using Salmonella typhimurium tester strains TA98, TAl00, TA1535 and TA1537 and Escherichia coli tester strain WP2 uvrA in the presence and absence of Aroclor-induced rat liver S9 (Wagner & Van Dyke, 2006). The assay was performed in two phases, using the plate incorporation and preincubation methods. The first phase, the preliminary toxicity assay, was used to establish the dose-range for the mutagenicity assay. The second phase, the mutagenicity assay (initial and confirmatory or independent repeat assays), was used to evaluate the mutagenic potential of the test article. The preincubation method was used in the confirmatory mutagenicity assay only. Ethanol (EtOH) was selected as the solvent of choice based on solubility of the test article and compatibility with the target cells. The test article formed a soluble and clear solution in ethanol (EtOH) at approximately 500 mg/mL, the highest concentration tested. In the preliminary toxicity assay, the dose levels tested were 6.7, 10, 33, 67, 100, 333, 667, 1000, 3333 and 5000 µg per plate. No precipitate was observed. Toxicity was observed with Salmonella tester strains TA98, TAl00 and TA1537 without S9 activation and with TAl00 and TA1535 with S9 beginning at 667, 3333 or at 5000 µg per plate. Based on the findings of the preliminary toxicity assay, the maximum dose tested in the mutagenicity assay was 5000 µg per plate. In the Initial Mutagenicity Assay via Plate Incorporation, no positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. The dose levels tested were 15, 50, 150, 500, 1500 and 5000 µg per plate. No precipitate was observed. No appreciable toxicity was observed but a reduction in revertant count was observed at 5000 µg per plate with tester strain TA 1535 in the presence of S9 activation. In a Confirmatory Mutagenicity Assay via Preincubation, no positive mutagenic responses were observed with any of the tester strains in the presence of S9 activation and with tester strain WP2 uvrA in the absence of S9 activation. The dose levels tested were 15, 50, 150, 500, 1500 and 5000 µg per plate. No precipitate was observed. Toxicity was observed beginning at 50, 150 or 500 µg per plate with the Salmonella tester strains. Due to excessive toxicity with Salmonella in the absence of S9 activation, these conditions were not evaluated but were retested in a new experiment. In the Retest of the Confirmatory Mutagenicity Assay via Preincubation, no positive mutagenic responses were observed with any of the Salmonella tester strains in the absence of S9 activation. The dose levels tested were 0.50, 1.5, 5.0, 15, 50 and 150 µg per plate. No precipitate was observed. Toxicity was observed beginning at 50 or at 150 µg per plate. Under the conditions of this study, Luperox XPS-TP was concluded to be negative in the Bacterial Reverse Mutation Assay with an Independent Repeat Assay.

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine operon (Salmonella typhimurium) and tryptophan locus (Escherichia coli)
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Aroclor-induced rat liver S9.
Test concentrations with justification for top dose:
0, 15, 50, 150, 500, 1500 and 5000 µg per plate
Vehicle / solvent:
Ethanol
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
methylmethanesulfonate
other: 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: plate incorporation and preincubation methods

DURATION
- Preincubation period: 60 min

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: bacterial background lawn
Evaluation criteria:
For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and are reported.
For the test article to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test article.
Data sets for tester strains TA1535 and TA1537 were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 3.0-times the meanvehicle control value. Data sets for tester strains TA98, TA100 and WP2 uvrA were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 2.0-times the mean vehicle control value.
An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose-responsive increase that does not achieve the respective threshold cited above or a non-dose responsive increase that is equal to or greater than the respective threshold cited.
A response will be evaluated as negative, if it is neither positive nor equivocal.
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
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 examined
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
ln the preliminary toxicity assay, the maximum dose tested was 5000 µg per plate; this dose was achieved using a concentration of 100 mg/mL and a 50 µL plating aliquot. The dose levels tested were 6.7, 10, 33, 67, 100, 333, 667, 1000, 3333 and 5000 µg per plate. No precipitate was observed. Toxicity was observed with Salmonella tester strains TA 98, TA 100 and TA 1537 without S9 activation and with TA 100 and TA1535 with S9 beginning at 667, 3333 or at 5000 µg per plate. Based on the findings of the preliminary toxicity assay, the maximum dose tested in the mutagenicity assay was 5000 µg per plate. This dose was achieved using a concentration of 200 mg/mL and a 25 µL plating aliquot. The plating aliquot was changed in preparation for the confirmatory mutagenicity assay, which required a 25 µL plating aliquot when using ethanol (EtOH) during a 60 minute preincubation.

MAIN STUDY
ln Experiment B 1 (Initial Mutagenicity Assay via Plate Incorporation), no positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. The dose levels tested were 15, 50, 150, 500, 1500 and 5000 µg per plate. No precipitate was observed. No appreciable toxicity was observed but a reduction in revertant count was observed at 5000 µg per plate with tester strain TA1535 in the presence of S9 activation.

ln Experiment B2 (Confirmatory Mutagenicity Assay via Preincubation), no positive mutagenic responses were observed with any of the tester strains in the presence of S9 activation and with tester strain WP2 uvrA in the absence of S9 activation. The dose levels tested were 15, 50, 150, 500, 1500 and 5000 µg per plate. No precipitate was observed. Toxicity was observed beginning at 50, 150 or 500 µg per plate with the Salmonella tester strains. Due to excessive toxicity with Salmonella in the absence of S9 activation, these conditions were not evaluated but were retested in Experiment B3.

In Experiment B3 (Retest of the Confirmatory Mutagenicity Assay via Preincubation), no positive mutagenic responses were observed with any of the Salmonella tester strains in the absence of S9 activation. The dose levels tested were 0.50, 1.5, 5.0, 15, 50 and 150 µg per plate. No precipitate was observed. Toxicity was observed beginning at 50 or at 150 µg per plate.
Conclusions:
Luperox XPS-TP was concluded to be negative in the Bacterial Reverse Mutation Assay
Executive summary:

Luperox XPS-TP was tested in the Bacterial Reverse Mutation Assay using Salmonella typhimurium tester strains TA98, TAl00, TA1535 and TA1537 and Escherichia coli tester strain WP2 uvrA in the presence and absence of Aroclor-induced rat liver S9. The assay was performed in two phases, using the plate incorporation and preincubation methods. The first phase, the preliminary toxicity assay, was used to establish the dose-range for the mutagenicity assay. The second phase, the mutagenicity assay (initial and confirmatory or independent repeat assays), was used to evaluate the mutagenic potential of the test article. The preincubation method was used in the confirmatory mutagenicity assay only. Ethanol (EtOH) was selected as the solvent of choice based on solubility of the test article and compatibility with the target cells. The test article formed a soluble and clear solution in ethanol (EtOH) at approximately 500 mg/mL, the highest concentration tested. In the preliminary toxicity assay, the dose levels tested were 6.7, 10, 33, 67, 100, 333, 667, 1000, 3333 and 5000 µg per plate. No precipitate was observed. Toxicity was observed with Salmonella tester strains TA98, TAl00 and TA1537 without S9 activation and with TAl00 and TA1535 with S9 beginning at 667, 3333 or at 5000 µg per plate. Based on the findings of the preliminary toxicity assay, the maximum dose tested in the mutagenicity assay was 5000 µg per plate. In the Initial Mutagenicity Assay via Plate Incorporation, no positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. The dose levels tested were 15, 50, 150, 500, 1500 and 5000 µg per plate. No precipitate was observed. No appreciable toxicity was observed but a reduction in revertant count was observed at 5000 µg per plate with tester strain TA 1535 in the presence of S9 activation. In a Confirmatory Mutagenicity Assay via Preincubation, no positive mutagenic responses were observed with any of the tester strains in the presence of S9 activation and with tester strain WP2 uvrA in the absence of S9 activation. The dose levels tested were 15, 50, 150, 500, 1500 and 5000 µg per plate. No precipitate was observed. Toxicity was observed beginning at 50, 150 or 500 µg per plate with the Salmonella tester strains. Due to excessive toxicity with Salmonella in the absence of S9 activation, these conditions were not evaluated but were retested in a new experiment. In the Retest of the Confirmatory Mutagenicity Assay via Preincubation, no positive mutagenic responses were observed with any of the Salmonella tester strains in the absence of S9 activation. The dose levels tested were 0.50, 1.5, 5.0, 15, 50 and 150 µg per plate. No precipitate was observed. Toxicity was observed beginning at 50 or at 150 µg per plate. Under the conditions of this study, Luperox XPS-TP was concluded to be negative in the Bacterial Reverse Mutation Assay with an Independent Repeat Assay.

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 possible according to CLP/GHS criteria.