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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:
15 November 1994 - 29 November 1994
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report Date:
1997

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Remarks:
Statement of Compliance
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
liquid

Method

Target gene:
His-gene: Amino acid histidine, and Trp-gene: amino acid tryptophan
Species / strain
Species / strain / cell type:
other: S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 , TA 100 and E. coli WP2 uvrA
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
other: S. typhimurium TA1535, TA1537, TA1538: rfa, uvrB; S. typhimurium TA98, TA100: rfa, uvrB, pKM101; E. coli WP2: uvrA
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9 induced by Aroclor 1254
Test concentrations with justification for top dose:
5000, 2500, 1250, 625, and 312.5 ug/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: the test substance was virtually insoluble in water.
Controls
Untreated negative controls:
yes
Remarks:
untreated
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: -S9: S. typhimurium TA 1535, TA 100, E. coli WP2 uvrA: N-Ethyl-N'-nitro-N-nitrosoguanidine; S. typhimurium TA 1537: 9-Aminoacridine; S. typhimurium TA 1538, TA 98: 2-Nitrofluorene. +S9: all strains: 2-Aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Selection time (if incubation with a selection agent): 3 days

SELECTION AGENT (mutation assays): agar containing Histidine or Tryptophan

NUMBER OF REPLICATIONS: 3 plates per concentration

NUMBER OF CELLS EVALUATED: 10*9 viable cells

DETERMINATION OF CYTOTOXICITY
- Method: determination of a concentration related reduction in the mean number of revertants per plate and/or the reduction of the microcolony background lawns

OTHER EXAMINATIONS: Preliminary toxicity/Dose range finding test at 5, 50, 500, and 5000 ug/plate.
Evaluation criteria:
If treatment with a test substance produces an increase in revertant colony numbers of at least twice the concurrent solvent controls, with some evidence of a positive dose-relationship, in two separate experiments, with any bacterial strain either in the presence or absence of S-9 mix, it is considered to show evidence of mutagenic activity in this test system. No statistical analysis is performed.
If treatment with a test substance does not produce reproducible increases of at least 1.5 times the concurrent solvent controls, at any dose level with any bacterial strain, it is considered to show no evidence of mutagenic activity in this test system. No statistical analysis is performed.
If the results obtained fail to satisfy the criteria for a clear "positive" or "negative" response, the following approach is taken in order to resolve the issue of the substance's mutagenic activity in this test system.
- Repeat tests may be performed using modifications of the experimental method.
- If no clear "positive" response can be obtained the test data may be subjected to analysis to determine the statistical significance of any observed increases in revertant colony numbers.
Statistics:
Mean and Standard Deviation

Results and discussion

Test results
Key result
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 , TA 100 and E. coli WP2 uvrA
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:
RANGE-FINDING/SCREENING STUDIES: OTNE was not toxic towards the tester strains in the preliminary toxicity test. Therefore 5000 ug/plate was chosen as the top dose level in the mutation tests.
Remarks on result:
other: all strains/cell types tested

Applicant's summary and conclusion

Conclusions:
No substantial increases in revertant colony numbers of any of the tester strains were observed following treatment with OTNE at any dose level, in the presence or absence of S-9 mix, in either mutation test. The concurrent positive control compounds demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations. It is concluded that OTNE tested in DMSO shows no evidence of mutagenic activity in this bacterial system.
Executive summary:

In this in vitro assessment of the mutagenic potential of OTNE, histidine dependent auxotrophic mutants of S. typhimurium (strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100) and a tryptophan dependent mutant of E. Coli (WP2uvrA) were exposed to the test substance, diluted in DMSO which was also used as a negative control. Two independent mutation tests were performed, in the presence and absence of liver preparations from Aroclor 1254-induced rats. In the preliminary dose range finding study with dose levels of up to 5000 ug/plate, no toxicity was observed. A top dose level of 5000 ug/plate was chosen for the subsequent mutation study. Other dose levels used in the mutation assays were: 2500, 1250, 625, and 312.5 ug/plate. No evidence of mutagenic activity was seen at any dose level of OTNE in either mutation test. The concurrent positive control compounds demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations. It is concluded that OTNE tested in DMSO was not mutagenic in this bacterial system.