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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1999-10-19 to 1999-10-28
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report Date:
1999

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted July 21, 1997
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:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9-mix induced with Aroclor 1254
Test concentrations with justification for top dose:
3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate (based on 75 % test substance), equivalent to 2.25, 7.5, 24.75, 75, 249.75, 750, 2497.5 and 3750 µg/plate of pure substance.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with metabolic activation for all strains
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
TA1535: without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
TA1537: Without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: daunomycine
Remarks:
TA98, without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
TA100, without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
no
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
E.coli WP2 uvr A: Without metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION:

- Dose range finding test:
Selection of an adequate range of doses was based on a dose range finding test with strain TA100 and WP2uvrA strain, both with and without S9-mix. Eight concentrations were tested in triplicate. The highest concentration of the test item used in the subsequent mutation assay was the recommened 5 mg/plate.

- Mutation assay:
At least five different doses (increasing with approximately half-log steps) of the test substance were tested in triplicate in each strain.
The test substance was tested both in the absence and presence of S9-mix in each strain.
Top agar in top agar tubes was molten and heated to 45 °C. The following solutions were successively added to 3 mL molten top agar: 0.1 mL of a fresh bacterial culture (10xe9 cells/mL) of one of the tester strains, 0.1 mL of dilution of the test substance in ethanol and either 0.5 mL S9 mix (in case of activation assays) or 0.5 mL 0.1 M phosphate buffer ( in case of non-activation assays). The ingredients were mixed on a Votex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were turned and incubated in the dark at 37 °C for 48 hours. Inadvertently, the plates of the tester strains TA1535, TA1537 (in absence of S9-mix) and TA98 were incubated for 72 hours. Since, the values of the negative and positive controls were within our historical control data range, this has no further effect on the integrity of the results. After this period revertant colonies (histidine independent for Salmonella typhimurium bacteria and tryptophan idependent for Escherichia coli) were counted.

Evaluation criteria:
No formal hypothesis testing was done.
A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in any tester strain at any concentration is not greater than two times the solvent value, with or without metabolic activation.
b) The negative response should be resproducible in at least one independently repeated experiment.

A test substance is considered positiv (mutagenic) in the test if:
- It induces a number of revertant colonies, dose related, greater than two-times the number of revertants induced by the solvent control in any of the tester strains, either with or without metabolic activation. However, any mean plate count of less than 20 is considered to be not significant.

The proceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no 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:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no 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
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at the test item concentration of 1000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with
Genotoxicity:
positive
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:
RANGE-FINDING/SCREENING STUDIES:
The test item was tested in the tester strains TA100 and WP2uvrA with concentrations of 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate (75 % substance in solvent) in the absence and presence of S9-mix.
Precipitate: The test substance precipitated in the top agar at concentrations of 1000 µg/plate and upwards. Precipitation of the test item on the plates was observed at the start of the incubation period at concentrations of 3330 and 5000 µg/plate. At the end of the incubation no precipitation on the plates was visible anymore.
Toxicity: To determine the toxicity of the test item, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were observed.
No reduction of the bacterial background lawn and no decraese in the number of revertant colonies were observed.

COMPARISON WITH HISTORICAL CONTROL DATA: The negative and strain-spefic positive control values were within the laboratory background historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

MUTATION ASSAY
Precipitate: The test item preciptated in the top agar at the concentrations of 1000 µg/plate and upwards. Precipitation of the test item on the plates was observed at the start of the incubation period at concentrations of 3330 and 5000 µg/plate. At the end of the incubation no precipitation on the plates was visible anymore.
Toxicity: In the absence of S9-mix, the bacterial background lawn was not reduced at all concnetrations tested and no decrease in the number of revertants was observed.
In the presence of S9-mix in tester strain TA1535, a moderate reduction of the bacterial background lawn was observed at the test substance concentrations of 3330 and 5000 µg/plate. In tester stratins TA98, a slight reduction of te bacterial background lawn was observed at the test substance concentration of 1000 µg/plate. an exterme reduction of the bacterial background lawn and an increase in the size of the microcolonies was observed at test substance concentrations of 3330 and 5000 µg/plate. In tester strain TA 1537, TA 100 and WP2uvrA no toxicity observed.
Mutagenicity:
In the absence of S9-mix in tester strain TA1535, the test item induced an up to 2.7-fold, dose related, increase in the number of revertant colonies compared to the solvent control. In tester strain TA1537, the test item induced an up to 6.4-fold, dose related, increase in the number of revertant colonies compared to the solvent control. In tester strain TA98, the test item induced an up to 2.2-fold, dose related, increase in the number of revertant colonies compared to the solvent control.
In the tester strains TA100 and WP2uvrA, the test item did not induce a dose-related increase in number of revertant colonies.
In the presence of S9-mix in tester strain TA1537, the test item induced an up to 6.0-fold, dose related, increase in the number of revertant colonies compared to the solvent control. In tester strain TA98, the test item induced an up to 2.4-fold, dose related, increase in the number of revertant colonies compared to the solvent control. In tester strain TA100, the test item induced an up to 5.3-fold, dose related, increase in the number of revertant colonies compared to the solvent control. In tester strain WP2uvrA, the test item induced an up to 4.6-fold, dose related, increase in the number of revertant colonies compared to the solvent control.
In the tester strains TA1535, the test item did not induce a dose-related increase in number of revertant colonies.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Based on the results of this study it is concluded that tert-butyl peroxyneodecanoate is mutagenic in the Salmonella typhiurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Executive summary:

Tert-butyl peroxyneodecanoate (75 % in solvent) was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA100 and TA98) and in Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli WP2uvrA according to OECD guideline no.471.

The test item was tested up to concentrations of 5000 µg/plate corresponding to 3750 µg/plate of pure substance in the absence and presence of S9 -mix. The test item did not precipitate on the plates at this dose-level. The test item showed toxicity in the tester strains TA98 and TA1535 in the presence of S9 -mix. In the other tester strains, no toxicity was observed.

In the absence of S9-mix in tester strain TA1535, the test item induced an up to 2.7-fold, dose related, increase in the number of revertant colonies compared to the solvent control. In tester strain TA1537, the test item induced an up to 6.4-fold, dose related, increase in the number of revertant colonies compared to the solvent control. In tester strain TA98, the test item induced an up to 2.2-fold, dose related, increase in the number of revertant colonies compared to the solvent control.

In the tester strains TA100 and WP2uvrA, the test item did not induce a dose-related increase in number of revertant colonies.

In the presence of S9-mix in tester strain TA1537, the test item induced an up to 6.0-fold, dose related, increase in the number of revertant colonies compared to the solvent control. In tester strain TA98, the test item induced an up to 2.4-fold, dose related, increase in the number of revertant colonies compared to the solvent control. In tester strain TA100, the test item induced an up to 5.3-fold, dose related, increase in the number of revertant colonies compared to the solvent control. In tester strain WP2uvrA, the test item induced an up to 4.6-fold, dose related, increase in the number of revertant colonies compared to the solvent control.

In the tester strains TA1535, the test item did not induce a dose-related increase in number of revertant colonies.

Based on the results of this study it is concluded that tert-butyl peroxyneodecanoate is mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.