Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
August 1997
Reliability:
1 (reliable without restriction)

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
EEC Methods for Determination of Toxicity, Annex to Directive 92/69/EEC, (OJ No.
L3 83A, 29.12.92), Part B, Method B.14. Other effects - Mutagenicity: Salmonella
typhimurium - Reverse Mutation Assay,
GLP compliance:
yes
Remarks:
Good Laboratory Practice in the testing of Chemicals OECD, ISBN 92-64-12367-9, Paris 1982
Type of assay:
bacterial reverse mutation assay

Test material

1
Chemical structure
Reference substance name:
4-[2-Ethoxy-5-(4-methyl-1-piperazinylsulfonyl)benzamido]-1-3-propyl-1H-pyrazole-5-carboxamide
EC Number:
639-263-7
Cas Number:
200575-15-1
Molecular formula:
C22H32N6O5S
IUPAC Name:
4-[2-Ethoxy-5-(4-methyl-1-piperazinylsulfonyl)benzamido]-1-3-propyl-1H-pyrazole-5-carboxamide
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
Off white powder with 95% purity

Method

Target gene:
four histidine requiring strains
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S9 lliver fraction - Aroclor 1254 induced rats
Test concentrations with justification for top dose:
In the preliminary toxicity test 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: 1500, 500, 150 and 50 ug/plate.
Vehicle / solvent:
dimethyl sulphoxide
Controls
Untreated negative controls:
yes
Remarks:
The chosen solvent, dimethyl sulphoxide, was used as the negative control.
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
N-ethyl-N-nitro-N-nitrosoguanidine
other: 2-Aminoanthracene
Remarks:
2-Nitrofluorene, 9-Aminoacridine and N-Ethyl-N'-nitro-N-nitrosoguanidine positive controls tested in the absence of S9 mix and diluted in Dimethyl sulphoxide solvent
Details on test system and experimental conditions:
Four concentrations of test substance were assessed for toxicity using the four tester strains. The highest concentration was 50 mg/ml of test substance in the chosen solvent, which provided a final concentration of 5000 ug/plate. Three 10-fold serial dilutions of the highest concentration were also tested. The chosen solvent, dimethyl sulphoxide, was used as the negative control. An aliquot of 0.1 ml of a 10 hour bacterial culture and 0.5 ml S-9 mix or 0.5 ml 0.1 M phosphate buffer (pH 7.4) were placed in glass bottles. An aliquot of 0.1 ml of the test solution was added, followed immediately by 2 ml of histidine deficient agar. The mixture was thoroughly shaken and overlaid onto previously prepared petri dishes containing 25 ml minimal agar. A single petri dish was used for each dose level. Plates were also prepared without the addition of bacteria in order to assess the sterility of the test substance, S9 mix and phosphate buffer. All plates were incubated at 37°C for 3 days. After this period the appearance of the background bacterial lawn was examined.
Revertant colonies were counted using a Seescan Automatic Colony Counter.
Any toxic effects of the test substance can be detected by a substantial reduction in revertant colony counts or by the absence of a complete background bacterial lawn. In the absence of any toxic effects the top concentration used in the main tests is the same as that used in the preliminary toxicity test.
If toxic effects are observed a lower concentration may be chosen for the main assays. Ideally the concentrations chosen for the mutation tests should include a minimum of three non-toxic
concentrations
Evaluation criteria:
The mean number of revertant colonies for all treatment groups is compared with those obtained for solvent control groups. The mutagenic activity of a test substance is assessed by applying the following criteria:
(a) 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 S9 mix, it is considered to show evidence of mutagenic activity in this test system. No statistical analysis is performed.
(b) 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.
(c) If the results obtained fail to satisfy the criteria for a clear "positive" or "negative" response given in paragraphs (a) and (b), the following approach is taken in order to resolve the issue of the test substance's mutagenic activity in this test system.
(i) Repeat tests may be performed using modifications of the experimental method. These modifications include (but are not restricted to), the use of a narrower dose range than that already tested; the use of different levels of liver homogenate S-9 fraction in the S9 mix.
Should an increase in revertant colony numbers be observed which satisfies paragraph (a) the test substance is considered to show evidence of mutagenic activity in
this test system. No statistical analysis is performed.

Results and discussion

Test results
Species / strain:
other: S. typhimurium TA 1535 hisG46 tia uvrB S. typhimurium TA 1537 hisC3076 tfa uvrB S. typhimurium TA98 hisD3052 tja uvrB pla1101 S. typhimurium TA100 hisG46 tfa uvrB pI(M101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
up to a concentration of 5000ug/plate
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
All strains tested negative with and without metabilic activation

Applicant's summary and conclusion

Conclusions:
When tested in dimethyl sulphoxide, UK-220,955 shows no evidence of mutagenic activity in this bacterial system.
Executive summary:

In this in vitro assessment of the mutagenic potential of UK-220,955, histidine dependent auxotrophic mutants of Salmonella typhimurium (strains TA1535, TA1537, TA98 and TA100) were exposed to the test substance, diluted in dimethyl sulphoxide 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 toxicity test with dose levels of up to 5000 yg/plate no toxicity was observed. A top dose level of 5000 yg/plate was chosen for the subsequent mutation study. Other dose levels used in the mutation assays were: 1500, 500, 150 and 50 Ag/plate. No evidence of mutagenic activity was seen at any dose level of UK-220,955 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, when tested in dimethyl sulphoxide, UK-220,955 shows no evidence of mutagenic activity in this bacterial system.