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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:
23 December 1999 - 10 March 1999
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:
1999
Report Date:
1999

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:
· name:2.5.DIMETHYL-2,5-DI-(Z-ETHYLHEXANOYLPEROXY) HEXANE

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:
S. typhimurium TA 102
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:
preliminary tes, with and withou S9-mixt: 10, 100, 500, 1000, 2500 and 5000 µg/plate.

Experiments without S9 mix:
The selected treatment-levels were:
312.5, 625, 1250, 2500 and 5000 µg/plate, for all tester strains in both mutagenicity experiments, except for the TA 98 strain in the second experiment, 625, 1250, 2500, 3750 and 5000 µg/plate, for the TA 98 strain in the second experiment.

Experiments with S9 mix:
The selected treatment-levels were:
312.5, 625, 1250, 2500 and 5000 µg/plate, for all tester strains In both mutagenicity experiments.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility
Batch No. 1743850 731 (Merck Clevenot, 77500 Chelles, France).
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: see below
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 60 min.
- Exposure duration: 48/72 hours
- Expression time (cells in growth medium): 48/72 hours

SELECTION AGENT (mutation assays): histidine

NUMBER OF REPLICATIONS: triplicate

NUMBER OF CELLS EVALUATED: revertants were scored with an automatic counter (Anek counter, model 880, OS1, 750] 5 Paris, France).

DETERMINATION OF CYTOTOXICITY: The evaluatiDn of the tDxicity was perfonned Dn the basis of the observation Df the decrease in the number of revertant cDlonies andlDr a thinning of the bacterial lawn.
Evaluation criteria:
Acceptance criteria
This study would be considered valid since the following criteria are fully met:
· the number of revertants in the vehicle controls is consistent with our historical data,
· the number of revertants in the positive controls is higher than that of the vehicle controls and is consistent with our historical data.

Evaluation criteria
A reproducible two-fold increase in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as a positive result. Reference to historical data, or other considerations of biological relevance may also be taken into account in the evaluation of the data obtained.
Statistics:
None

Results and discussion

Test resultsopen allclose all
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
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
Species / strain:
S. typhimurium TA 98
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
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
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
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 1537
Metabolic activation:
with and without
Genotoxicity:
negative
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 102
Metabolic activation:
with and without
Genotoxicity:
negative
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 substance was soluble in the vehicle (DMSO) at 50 mg/ml. With a maximum dose volume of 100 µg/plate. No precipitate was observed in the Petri plates when scoring the revertants at all dose-levels. No toxicity was noted towards the three strains used, with and without S9 mix.

Any other information on results incl. tables

In the second experiment (preincubation method), at dose-levels ≥ 625 µg/plate, a 1.8-2.4 fold and a 2-3.7 fold increase in the number of revertants were noted in the TA 98 and TA 100 strain, respectively.

Applicant's summary and conclusion

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

Under our experimental conditions, the test substance induced mutagenic activity in the bacterial reverse mutation test on TA 98 and TA 100 Salmonella typhimurium strains, with metabolic activation.
Executive summary:

A preliminary toxicity test was performed to define the dose-levels to be used for the mutagenicity study. The test substance was then tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254. Both experiments were performed according to the direct plate incorporation method except the second with S9 mix, which was performed according to the preincubation method (60 minutes, 37C).

Five strains of bacteria Salmonella typhimurium: TA 1535, TA 1537, TA 98, TA 100 and TA102 were used. Each strain was exposed to five dose-levels of the test substance (three plates/dose-level).

After 48 to 72 hours of incubation at 37C, the revertant colonies were scored.

The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn. The test substance was dissolved in dimethylsulfoxide (DMSO).

The test substance was soluble in the vehicle at 50 mg/ml. Consequently, with a maximum dose volume of 100 µg/plate, the dose-levels for the preliminary toxicity test were: 10, 100, 500, 1000, 2500 and 5000 µg/plate. Since the test substance was non-toxic in the preliminary test, the highest dose-level was 5000 µg/plate, according to the criteria specified in the international guidelines.

Experiments without S9 mix:

The selected treatment-levels were:

312.5. 625, 1250. 2500 and 5000 µg/plate for all tester strains in both mutagenicity experiments except for the TA 98 strain in the second experiment. 625, 1250.2500, 3750 and 5000 µg/plate for the TA 98 strain in the second experiment. A slight to moderate emulsion was hoted in the plates at dose levels ≥ 1250 µg/plate. No or a slight toxicity was noted depending on the tester strain and the dose-level. No mutagenic effect was noted in all the tester strains.

Experiments with S9 mix:

The selected treatment-levels were:

312.5. 625. 1250. 2500 and 5000 µg/plate for all tester strains in both mutagenicity experiments.

A slight to moderate emulsion was generally noted in the plates at dose-levels ≥1250 µg/plate. No or a slight to moderate toxicity was noted. depending on the tester strain, the dose-levels and experimental conditions.

In the second experiment (preincubation method), at dose-levels ≥ 625 µg/plate, a 1.8-2.4 fold and a 2-3.7 fold increase in the number of revertants were noted in the TA 98 and TA 100 strain, respectively.

The number of revertants of the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid.

Under our experimental conditions, the test substance induced mutagenic activity in the bacterial reverse mutation test on TA 98 and TA 100

Salmonella typhimurium strains, with metabolic activation.