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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:
1996-01-19 - 1996-02-16
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Only two replicates per group were used, method for measuring toxicity was not specified, no historical control data provided, and the procedure (preincubation or plate incorporation) used for the preliminary reverse mutation test was not provided. However, all other parts of OECD guideline 471 were followed.

Data source

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

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
Only two replicates per group were used, method for measuring toxicity was not specified, no historical control data provided, and procedure (preincubation or plate incorporation) used for the preliminary reverse mutation test was not provided.
GLP compliance:
yes
Remarks:
Japanese Good Laboratory Practice
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Jeffcat DMP
Specific details on test material used for the study:
- Name of test material (as cited in study report): N,N'-dimethylpiperazine (DMP)
- Molecular weight (if other than submission substance): 114.19
- Physical state: colorless liquid
- Analytical purity: 99.0 % or more
- Impurities (identity and concentrations): water, 0.1 %
- Lot/batch No.: U3-1-1294
- Stability under test conditions: stable at room temperature; stability in solvent: stable in distilled water
- Storage condition of test material: at room temperature, shield under from light

Method

Target gene:
Histidine locus (Salmonella strains) and tryptophan locus (E. coli strain)
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: TA100 and TA1535 are base pair substitution detecting strains and TA98 and TA1537 are frameshift detecting strains
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
other: base pair substitution detecting strain
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital and 5,6-benzoflavone induced rat liver S9
Test concentrations with justification for top dose:
313, 625, 1250, 2500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: distilled water
- Justification for choice of solvent/vehicle: the test substance was soluble in water at concentrations of 100 g/L or more. From these results, distilled water was used as the solvent for the test substance.
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
distilled water
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide-used for strains TA100 and WP2 uvrA at 0.01 ug/plate and TA98 at 0.1 ug/plate
Remarks:
without activation
Untreated negative controls:
yes
Remarks:
distilled water
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without activation used for strain TA1535 at 0.5 ug/plate
Untreated negative controls:
yes
Remarks:
distilled water
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without activation used for strain TA1537 at 80 ug/ plate
Untreated negative controls:
yes
Remarks:
distilled water
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene-used for strain TA1535 at 2 ug/plate and WP2 uvrA at 10 ug/plate
Remarks:
with activation
Untreated negative controls:
yes
Remarks:
distilled water
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
with activation used for strains TA100, TA98 and TA1537 at 5 ug/plate
Details on test system and experimental conditions:
METHOD OF APPLICATION: pre-incubation


DURATION
- Preincubation period: 30 minutes
- Exposure duration: 48 hours
- Selection time (if incubation with a selection agent): 48 hours (simultaneous with exposure)


SELECTION AGENT (mutation assays): L-histidine (Salmonella strains), L-tryptophan (E.coli strain)


NUMBER OF REPLICATIONS: 2


DETERMINATION OF CYTOTOXICITY
- Method: cytotoxicity was determined but no information was given on how it was determined.


Evaluation criteria:
The number of revertant colonies at each dose was compared with that of the solvent control group for each bacterial strain with and without metabolic activation. The test substance was considered as positive when significant dose-related and more than two-fold increases in the number of revertant colonies were observed with sufficient reproducibility.
Statistics:
No statistical analysis was performed for evaluation of the test results.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: A preliminary reverse mutation test was performed in each of the strains listed above with and without metabolic activation to determine the suitable dosage of the test substance to be applied in the reverse mutation test. The maximum dose level for the preliminary test was set at 5000 µg/plate and six dose levels (10, 50, 100, 500, 1000 and 5000 µg/plate) were employed.

Neither toxic effect nor more than two-fold increases in the number of revertant colonies were observed in any test substance treated groups with and without metabolic activation, while the dose related increase in the number of revertant colonies was observed in TA100 with and without metabolic activation. Considering these results, the maximum dose level for the reverse mutation test was set at 5000 µg/plate, and five different amounts of the test substance were prepared by serial dilution with a factor of 2 from the maximum dose.




Remarks on result:
other: strain/cell type: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Any other information on results incl. tables

Neither toxic nor more than two-fold increase in the number of revertant colonies were observed in any test substance treated groups with and without metabolic activation, while the dose related increase in the number of revertant colonies was observed in TA100 with and without metabolic activation. On the other hand, all of the positive controls produced more than two-fold increases in the number of the revertant colonies in comparison with that of the solvent control with all bacterial strains with and without metabolic activation. These results indicated that the test had been properly carried out.

Applicant's summary and conclusion

Conclusions:
The test substance was evaluated for mutagenic potential in the reverse mutation test in the presence and absence of metabolic activation using S. typhimurium strains TA100, TA98, TA1535, TA1537 and E. coli strain WP2 uvrA. Based on the results of the assay, the test substance was considered to be negative under the test conditions employed up to a dose level of 5000 µg/plate. According to the criteria laid down in the CLP Regulation, the substance is considered not classified as mutagenic.