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EC number: 213-147-2 | CAS number: 927-07-1
Genetic toxicity: in vitro
Administrative data
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 1995-10-17 to 1995-12-12
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 1 996
- Report date:
- 1996
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- May 1983
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- 1992
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
Test material
- Reference substance name:
- tert-butyl peroxypivalate
- EC Number:
- 213-147-2
- EC Name:
- tert-butyl peroxypivalate
- Cas Number:
- 927-07-1
- Molecular formula:
- C9H18O3
- IUPAC Name:
- tert-butyl 2,2-dimethylpropaneperoxoate
- Reference substance name:
- 2,2,4,6,6-pentamethylheptane
- EC Number:
- 236-757-0
- EC Name:
- 2,2,4,6,6-pentamethylheptane
- Cas Number:
- 13475-82-6
- Molecular formula:
- C12H26
- IUPAC Name:
- isododecane
- Test material form:
- liquid
Constituent 1
additive 1
Method
Species / strainopen allclose all
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
- Species / strain / cell type:
- E. coli WP2 uvr A pKM 101
- Species / strain / cell type:
- E. coli, other: WP2 (pKM101)
- Metabolic activation:
- with and without
- Metabolic activation system:
- Aroclor 1254-induced rat liver S9
- Test concentrations with justification for top dose:
- - 100, 333, 450, 1000, 5000 µg/plate (for all strains without S9-mix)
- 10, 33, 100, 333, 450, 1000 µg/plate (for strains TA98, TA100, TA1535, T1537)
- 33, 100, 333, 450, 1000, 5000 µg/plate (for strains WP2 uvrA(pKM101) and WP2) - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: acetone
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 for strains TA98, TA100, TA1535, TA1537
- Remarks:
- with S9-mix
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: sterigmatocystin for WP2 (pKM101) and TA102
- Remarks:
- with S9-mix
- 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
- 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
- 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
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cumene hydroperoxide
- Remarks:
- without S9-mix
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- methylmethanesulfonate
- Remarks:
- without S9-mix
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation) Test item dilutions were prepared immediately before use. One-half (0.5) mililiter of S9 or Sham mix, 100 µL of tester strain and 50 µL of the vehicle or test item were added to 2.0 mL of molten selective top agar at 45 +/-2°C. After vortexing, the mixture was overlaid onto the surface of 25 mL of minimal bottom agar. When plating the positive controls, the test article aliquot was replaced by a 50 or 100 µL aliquot of appropriate positve control. After the overlay had solidified, the plates were inverted and incubated for approximately 48 to 72 hours at 37 +/-2°C. Plates that were not counted immediately following the incubation period were stored at 4+/-2°C until colony counting could be conducted.
DURATION
- Exposure duration: 48-72 hours
SELECTION AGENT (mutation assays): Histidine
NUMBER OF REPLICATIONS: 1 in triplicates
DETERMINATION OF CYTOTOXICITY
- Method: cell count
OTHER EXAMINATIONS:
- Determination of polyploidy: no
- Determination of endoreplication: no - Evaluation criteria:
- For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and reported.
For the test item to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of a least one tester strain with a minimum of two increasing concentrations of the test item. Data sets for strains TA 1535 and TA 1537 were judged positive if the increase in mean revertants at the dose response is equal to or greater than three times the mean vehicle control value. Data sets for strains TA 98, TA 100, TA 102, WP2 uvrA (pKM101) and WP2 (pKM101) were judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than two times the mean vehicle control value.
Results and discussion
Test resultsopen allclose all
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 102
- Metabolic activation:
- with
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- E. coli, other: WP2 uvrA (pKM101) and WP2 (pKM101)
- Metabolic activation:
- with
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- 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 applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 102
- Metabolic activation:
- without
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- E. coli, other: WP2 (pKM 101)
- Metabolic activation:
- without
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- without
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- 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 applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- 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 applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- RANGE-FINDING/SCREENING STUDIES: In the preliminary toxicity assay, the maximum dose tested was 5000 µg per plate; this dose was achieved using a stock concentration of 100 mg/mL and a 50 µg plating aliquot. Precipitate was observed at >= 3333 µg per plate and toxicity was generally observed from 667 to 5000 µg per plate. Based on the findings of the toxicity assay, the maximum doses plated in the mutagenicity assay were 1000 µg per plate for Salmonella in the presence of S9 activation and 5000 µg per plate for the remaining tester strain/activation conditions.
Applicant's summary and conclusion
- Conclusions:
- Tert-butyl peroxypivalate did cause positive responses with tester strains TA 98, TA 100, TA 1537, TA 102, WP2 uvra (pKM101) and WP2 (pKM101) in the presence of acroclor-induced rat liver S9 and with tester strains TA 100, TA 1537, TA 102 and WP2 (pKM101) in the absence of S9.
- Executive summary:
Tert-butyl peroxypivalate was tested in the bacterial reverse mutation assay using S. typhimurium tester strains TA 98, TA 100, TA 1535, TA 1537 and TA 102 and E.coli tester strains WP2 uvrA (pKM101) and WP2 (pKM101) in the presence and absence of Aroclor-induced rat liver S9 according to OECD guideline no.471 and EU method B.13/14. The assay was performed in two phases, using the plate incorporation method. The first phase, the dose range-finding study, was used to establish the dose range for the mutagenicity assay. The second phase, the mutagenicity assay, was used to evaluate the mutagenic potential of the test item.
Ethanol was selected as solvent of choice based on solubility of the test item and compatility with the target cells.
In the preliminary toxicity assay, the maximum dose tested was 5000 µg per plate; this dose was achieved using a stock concentration of 100 mg/mL and a 50 µg plating aliquot. Precipitate was observed at >= 3333 µg per plate and toxicity was generally observed from 667 to 5000 µg per plate. Based on the findings of the toxicity assay, the maximum doses plated in the mutagenicity assay were 1000 µg per plate for Salmonella in the presence of S9 activation and 5000 µg per plate for the remaining tester strain/activation conditions.
In the mutagenicity assay positiv responses were observed. Precipitate was generally observed at >= 3333 µg per plate and toxicity was generally observed at >= 1000 µg per plate in the presence of S9 activation.
Tert-butyl peroxypivalate did cause positive responses with tester strains TA 98, TA 100, TA 1537, TA 102, WP2 uvra (pKM101) and WP2 (pKM101) in the presence of acroclor-induced rat liver S9 and with tester strains TA 100, TA 1537, TA 102 and WP2 (pKM101) in the absence of S9.
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