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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:
1995-10-17 to 1995-12-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study; EU and OECD guidelines were followed

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1996
Report Date:
1996

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
May 1983
Deviations:
no
Qualifier:
according to
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
Name:
Unnamed
Type:
Constituent
Test material form:
other: clear liquid

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

Migrated to IUCLID6: for TA98
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 TA 100, TA 1535
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 TA 1537
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

Migrated to IUCLID6: for TA 102
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
Species / strain:
S. typhimurium, other: TA 98, TA 100, TA 1537
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
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 examined
Positive controls validity:
valid
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 examined
Positive controls validity:
valid
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 examined
Positive controls validity:
valid
Species / strain:
S. typhimurium, other: TA 100, TA 1537, TA 102
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
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 examined
Positive controls validity:
valid
Species / strain:
other: S. typhimurium: TA 98, TA1535; E. coli: WP2 uvrA (pKM101)
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
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.
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):
positive

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.