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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:
17 March 2003 - 09 June 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study is performed according to OECD guidelines and GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
1,1,3,3-Tetramethylbutyl peroxy-2-ethylhexanoate
CAS# 22288-43-3

Method

Target gene:
Histidine operon
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: see below
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
other: see below
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
Results of the dose rangefinding study were used to select doses tested in the mutagenicity assay. Doses tested with all tester strains in the presence and absence of S9 mix were 100, 333, 1000, 3330 and 5000 μg per plate.

Based on the toxicity observed in the presence of S9 mix in the first trial, the doses tested in the confirmatory assay were selected. Doses tested in the confirmatory assay were 10.0, 33.3, 100, 333, 500, 750, 1000, 2000 and 5000 μg per plate in the presence of S9 mix and 100, 333, 1000, 3330 and 5000 μg per plate in the absence of S9 mix.

Vehicle / solvent:
Ethanol
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: see below
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: not applicable
- Exposure duration: 51 ± 4 hours

SELECTION AGENT (mutation assays): histidine

NUMBER OF REPLICATIONS: triplicate

NUMBER OF CELLS EVALUATED: Revertant colonies were counted by automated colony counter or by hand

DETERMINATION OF CYTOTOXICITY
Condition of the bacterial background lawn was evaluated for evidence of cytotoxicity and test article precipitate. Evidence of cytotoxicity was scored relative to the vehicle control and recorded along with the revertant counts for that dose.
Evaluation criteria:
Assay Evaluation Criteria
Once the criteria for a valid assay had been met, responses observed in the assay were evaluated as follows:

Tester Strains TA98, TA100, and WP2uvrA. For a test article to be considered positive, it had to produce at least a 2-fold increase in the mean revertants per plate of at least one of these tester strains over the mean revertants per plate of the appropriate vehicle control. This increase in the mean number of revertants per plate had to be accompanied by a dose response to increasing concentrations of the test article.

Tester Strains TA1535 and TA1537. For a test article to be considered positive, it had to produce at least a 3-fold increase in the mean revertants per plate of at least one of these tester strains over the mean revertants per plate of the appropriate vehicle control. This increase in the mean number of revertants per plate had to be accompanied by a dose response to increasing concentrations of the test article.
Statistics:
None

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
at 5000 µg/plate precipitation
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
at 5000 µg/plate precipitation
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
+ S9 mix: from 750 µg/plate, -S9-mix: 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
+ S9 mix: from 750 µg/plate, -S9-mix: 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
+ S9 mix: from 750 µg/plate, -S9-mix: 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
In the initial mutagenicity assay, Trial 24908-B1, no positive increases in the mean number of revertants per plate were observed with any of the tester strains in either the presence or absence of S9 mix. In this trial, a 2.2-fold increase was observed with tester strain WP2uvrA in the presence of S9 mix. However, this increase was not clearly dose-responsive, and therefore did not meet the criteria for a positive evaluation. In order to clarify this increase, the test article was re-tested with tester strain WP2uvrA in the presence of S9 mix in Trial 24908-D1. All data generated in Trial 24908-B1 were acceptable with the exception of tester strains TA98 and TA1537 in the presence of S9 mix, where only two non-toxic doses were observed. For this reason, the test article was re-tested with these tester strains in Trial 24908-D1.

Based on the toxicity observed in the presence of S9 mix in Trial 24908-B1 (which was not observed in the dose rangefinding study), the doses tested in the confirmatory assay were selected. Doses tested in the confirmatory assay were 10.0, 33.3, 100, 333, 500, 750, 1000, 2000 and 5000 μg per plate in the presence of S9 mix and 100, 333, 1000, 3330 and 5000 μg per plate in the absence of S9 mix.

In the confirmatory mutagenicity assay, Trial 24908-C1, all data were acceptable and a 3.0-fold positive increase was observed in the mean number of revertants per plate with tester strain WP2uvrA in the presence of S9 mix. No positive increases were observed with any other tester strains in the presence or absence of S9 mix. In this trial, a 1.8-fold increase was observed with tester strain TA100 in the presence of S9 mix. However, this increase did not meet the 2-fold criteria to be considered a positive response. In order to clarify this increase, the test article was re-tested with tester strain TA100 in the presence of S9 mix in Trial 24908-D1.

In the repeat mutagenicity assay, Trial 24908-D1, all data were acceptable positive increases were observed in the mean number of revertants per plate with tester strains TA100 (3.0-fold) and WP2uvrA (2.3-fold)in the presence of S9 mix. In addition, increases in the mean number of revertants per plate were observed with tester strain TA1537 in the presence of S9 mix, however, these increases were not clearly dose-related. In addition, all observed values were within the acceptable vehicle control range for this strain. The observed increases appear to be the result of a lower than routinely observed TA1537 mean vehicle control value and were not
considered to be biologically relevant. No positive increases were observed with tester strain TA98 in the presence of S9 mix.

All criteria for a valid study were met.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
positive with metabolic activation TA100 and WP2uvrA

The results of the Salmonella-Escherichia coli/Mammalian-Microsome Reverse Mutation Assay with a Confirmatory Assay indicate that under the conditions of this study, the test article, 1,1,3,3-Tetramethylbutyl peroxy-2-ethylhexanoate, did cause positive increases in the mean number of revertants per plate with tester strains TA100 and WP2uvrA in the presence of S9 mix. No positive increases were observed with any other tester strain either in the presence or absence of microsomal enzymes prepared from Aroclor™-induced rat liver (S9).
Executive summary:

The objective of this study was to evaluate the test article, 1,1,3,3-Tetramethylbutyl peroxy-2 - ethylhexanoate, for its ability to induce reverse mutations either in the presence or absence of mammalian microsomal enzymes at 1) the histidine locus in the genome of several strains of

Salmonella typhimurium and at 2) the tryptophan locus of Escherichia coli tester strain WP2uvrA.

The doses tested in the mutagenicity assay were selected based on the results of a dose rangefinding study using tester strains TA100 and WP2uvrA and ten doses of test article ranging from 6.67 to 5000 μg per plate, one plate per dose, both in the presence and absence of S9 mix.

The tester strains used in the mutagenicity assay were Salmonella typhimurium tester strains TA98, TA100, TA1535, and TA1537 and Escherichia coli tester strain WP2uvrA. The assay was conducted in both the presence and absence of S9 mix along with concurrent vehicle and positive controls using three plates per dose. The doses tested in the mutagenicity assay with all tester strains in both the presence and absence of S9 mix were 100, 333, 1000, 3330 and 5000 μg per plate. An independent confirmatory experiment was conducted with all tester strains at doses of 10.0, 33.3, 100, 333, 500, 750, 1000, 2000 and 5000 μg per plate in the presence of S9 mix and at doses of 100, 333, 1000, 3330 and 5000 μg per plate in the absence of S9 mix. The results of the Salmonella-Escherichia coli/Mammalian-Microsome Reverse Mutation Assay with a Confirmatory Assay indicate that under the conditions of this study, the test article, 1,1,3,3-Tetramethylbutyl peroxy-2-ethylhexanoate, did cause positive increases in the mean number of revertants per plate with tester strains TA100 and WP2uvrA in the presence of S9 mix. No positive increases were observed with any other tester strain either in the presence or absence of microsomal enzymes prepared from Aroclor™-induced rat liver (S9).