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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:
1996-04-17 to 1996-08-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study

Data source

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

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
Test material form:
liquid
Details on test material:
Purity batch
> 99.0 % 7.32% active oxygenBatch 247408973
99.27 % 9407226339; 247408999
99.6 % 0410105225413
> 97 % 4289070300; 0419101226056
99.1 % 11600 0010-108
99.3 % 247413988
99.4 % 7.35 % active oxygen Batch 247409800
98.9 % 0707518256
> 99.1 % 247412384
98.5 % 0707603153
99.0 % 1703465433
99.5 % 150726; 247413309
99.3 % 000055055

Method

Target gene:
Tester strains TA 98 and TA 1537 are reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frame shift mutagens. Tester strains TA 1535 and TA 102 are reverted mutagens that cause base pair substitutions. Tester strain TA 100 is reverted by mutagens that cause both frame shift and base pair substitution mutations. Specificity of the reversion mechanism in E.coli is sensitive to base pair substitution mutations, rather than frame shift mutations.
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
NA
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 102
Details on mammalian cell type (if applicable):
NA
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Details on mammalian cell type (if applicable):
NA
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
other: WP2 pKM101
Details on mammalian cell type (if applicable):
NA
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 mix
Test concentrations with justification for top dose:
without S9 mix: 0, 100, 333, 1000, 3333 and 5000 µg
with S9 mix: 0, 33, 100, 333, 1000 and 5000 µg
Vehicle / solvent:
- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: DMSO was selected as the solvent of choice based on solubility of the test article and compatibility with the target cells. The test article was soluble in DMSO at 500 mg/mL, the maximum concentration tested.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: see below
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
For the S. typhimurium strain TA 1537, TA 1537, TA 100, TA 98 and the E.coli strain WP2 uvrA (pKM101); with metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: sterigmatocystin
Remarks:
For the S.typhimurium strain 102 and the E. coli WP2 (pKM101) strain; with metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
For the S. typhimurium strain TA 98, without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
For the S. typhimurium strain TA 1535, TA 100; without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
For the S. typhimurium strain TA 1537; without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cumene hydroperoxide
Remarks:
For the S. typhimurium strain TA 102; without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
For the E. coli WP2 (pKM101) and WP2 uvrA (pKM101) strain; without metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation)


Treatment of test system
- 0.5 mL of S9 mix or Sham mix, 100 µL tester strain and 50 µL of vehicle, test article dilution or positive control will be added to 2 mL of molten selective top agar at 45 ± 2 °C. When necessary to achieve the target concentration, aliquots of other than 50 µL of test article/ vehicle/ positive control will be plated. The mixture will be vortex mixed and overlaid onto the surface of 25 mL of minimal bottom agar. After the overlay has solidified, the plates will be inverted and incubated for approximately 48 to 72 hours at 37 ± 2 °C. Plates that are not counted immediately following the incubation period will be stored at 4 ± 2°C.

Colony Counting
- The condition of the bacterial background lawn will be evaluated for evidence of test article toxicity and precipitate. Evidence of toxicity will be scored relative to the vehicle control plate and recorded along with the revertant count for that plate.


NUMBER OF REPLICATIONS: All dose levels of test article, vehicle controls and positive controls were run in triplicate.



Evaluation criteria:
Evaluation of results:
For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and are reported. Statistical tests are not indicated.

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 with a minimum of two increasing concentrations of test article. Data sets for strains TA 1535 and TA 1537 were judged positive if the increase in mean revertants at the peak of 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.
Statistics:
For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and are reported. Statistical tests are not indicated.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
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:
valid
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:
valid
Positive controls validity:
valid
Key result
Species / strain:
other: WP2 pKM101
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
other: TA 1535 and TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
In Experiment B1, the initial assay, positive responses were observed with tester strains TA100 (2.3-fold, maximum increase, TA 102 (7.8-fold maximum increase), WP uvrA (pKM101) (5.3-fold, maximum increase) and WP2 (pKM101) (20.7-fold, maximum increase) in the presence of S9 activation. No positive responses were observed in any of the remaining tester strain/activation combinations.
In Experiment B2, the independent repeat assay, positive responses were observed with tester strains TA 100 (2.0-fold, maximum increase), WP uvrA (pKM101) (3.6-fold, maximum increase) and WP2 (pKM101) (15.4-fold, maximum increase) in the presence of S9 activation. Due to possible tester strain culture contamination, tester strain TA98 in the presence and absence of S9 activation was not evaluated but was retested in Experiment B3. Due to an unacceptable vehicle control value, tester strain TA 102 in the presence of S9 activation was not evaluated but was retested in Experiment B3. No positive responses were observed in any of the remaining tester strain/activation combinations.

In Experiment B3, no positive response was observed with tester strain TA98 in the presence and absence of S9 activation. Due to an unacceptable vehicle control value, tester strain TA 102 in the presence of S9 activation was not evaluated but was retested in Experiment B4.

In Experiment B4, a positive response was observed with tester strain TA102 (3.8-fold, maximum increase) in the presence of S9 activation.

Applicant's summary and conclusion

Conclusions:
All criteria for a valid study were met as described in the protocol. The results of the Salmonella/Escherichia coli Mutagenicity Assay indicate that under the conditions of this study, tert.-Butylperoxy- 2-ethylhexanoat did cause positive responses with tester strains TA100, TA102, WP2 uvrA (pKM101) and WP (pKM101) in the presence of Aroclor-induced rat liver S9.
Executive summary:

In a reverse gene mutation assay in bacteria, strains of Salmonella typhimurium (TA 98, TA 100, TA 1535, TA 1537 and TA 102) and at the tryptophan locus of Escherichia coli tester strains WP2 (pKM101) and WP uvrA (pKM101) were exposed to tert.-Butylperoxy- 2-ethylhexanoat in the presence and absence of S9 activation according to OECD guideline 471. The assay was performed in two phases, using the plate incorporation method. The first phase, the preliminary toxicity assay, 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 article.

DMSO was selected as the solvent of choice based on solubility of the test article and compatibility with the target cells. The test article was soluble in DMSO at 500 mg/mL, the maximum concentration tested.

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 µL plating aliquot. Based on the findings of the toxicity assay, the maximum doses plated in the mutagenicity assay was 5000 µg per plate for all tester strains in the presence and absence of S9 activation.

In the mutagenicity assay positive responses were observed. No precipitate was observed and toxicity was generally observed at ≥ 1000 to 5000 µg per plate in the presence of S9 activation. In Experiment B1, the initial assay, positive responses were observed with tester strains TA100, TA102, WP2 uvrA and WP2 (pKM101) in the presence of S9 activation. No positive responses were observed in any of the remaining tester strain/activation. In Experiment B2, the independent repeat assay, positive responses were observed with tester strains TA100, TA102, WP2 uvrA and WP2 (pKM101) in presence of S9 activation. No positive responses were observed in any of the remaining tester strain/activation.

In Experiment B3, no positive response was observed with tester strain TA98 in presence and absence of S9 activation. In Experiment B4, a positive response was observed with tester strain TA102 in the presence of S9 activation.

Under the conditions of this study, test article tert.-Butylperoxy- 2-ethylhexanoat was concluded to be positive in the Salmonella/ Escherichia coli Mutagenicity Assay.