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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:
December 2002
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report date:
2002

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Batch number ZD48725 LOT002

Method

Target gene:
four histidine-requiring strains and one tryptophan-requiring strain
Species / strainopen allclose all
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
Obtained from the UK NCTC
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Obtained from the UK NCTC
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9)
Test concentrations with justification for top dose:
An initial range finding experiment was carried out in strain TA100 only, using final concentrations of BMS 528235-01 at 1.6, 8, 40, 200, 1000 and 5000 ug/plate, plus negative (solvent) adn positive contols. Experiment 1 treatments of the remained tester strains retained teh same dose series. Experiment 2 treatments of all the tester strains retained 5000 ug/plate as the max test dose. A narrowed dsoe range was employed for treatment of all strains (78.125 to 5000 ug/plate), in order to more closely examine the highest concentration considered most likely to provide evidence of any mutagenic activity.
Vehicle / solvent:
Sterile anhydrous analytical grade dimethyl solphoxide (DMSO)
Controls
Untreated negative controls:
yes
Remarks:
(see solvent controls)
Negative solvent / vehicle controls:
yes
Remarks:
in DMSO
Positive controls:
yes
Remarks:
Treatments with the appropriate stock positive control solution
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
other: 2-aminoanthracene
Details on test system and experimental conditions:
A preliminary range finding experiment was conducted with strain TA100 at 5 concentrations from 1.6 to 5000 ug/plate plus negative (solvent) controls. No evidence of toxicity was observed following this treatment. These data were considered acceptable for mutation assessment and were used to comprise the TA100 mutagenticity data for Experiment 1. Experiment 1 treatments fo the remaining tester strains retained the same dose series as used in teh Range-finder experiment. No evidence of toxicty was observed following any Experiment 1 treatments.
Experiment 2 treatments of all teh teser strains retained 5000 ug/plate as the max test dose. A narrowed dsoe range was employed for treatment of all strains (78.125 to 5000 ug/plate), in order to more closely examine the highest concentration considered most likely to provide evidence of any mutagenic activity. In addition all treatments in teh presence of S-9 were further modified by teh inclusion of a pre-incubation step. In this way it was hoped to increase the range of mutagenic chemicals that cold be detected using this assy system. Evidence of toxicisty was observed following the top dose treatments of some of the strains in teh presnced ofn S-9 only. Preciipitation of the test article was observed followign teh top dose treatment of S. tryphimurium strains in teh presenced of S-9 only.
Negative (solvent) and poistive control treatments were included fora ll strains in both experiments. Teh mean numbers of revertant colonies on negative control plates all fell within acceptable ranges, and were significantly evelvated by postive control treatments
Evaluation criteria:
The assay was considered valid if the following crieteria were met:
1. The mean negative control counts fell within the normal ranges
2. The positive control chemicals induced clear increases in revertant numbers confirming discrimination between different strains, and an active S-9 preparation.
3. No more than 5% of the plates were lost through contamination or some other unforseen event.

The test article was considered to be mutagenic if:
1. The assay was valid (see above)
2. Dunnett's test gave a significant response (p< 0.01) and the data set(s) showed a significant dose correlation
3. The positive responses described above were reproducible.

Results and discussion

Test resultsopen allclose all
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
up to 5000 ug/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
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 nor precipitates, but tested up to recommended limit concentrations
Remarks:
u ot 5000 ug/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
No statistically significant, dose-related and reproducible increases in revertant numbers were observed following any strain treatments in the absenced or presence of metabolic activation, and therefore this study was considered to have provided no evidence of any BMS 528235-01 mutagenic activity.
It was concluded that BMS 528235-01 did not induce mutation in four histidine-requiring strains of S. typhimurium (TA98, TA1000, TA1535 adn tA1537), adn one tryptophan-requiring strain of Escherichia coli (WP2 uvrA) when tested under the conditions of this study. These conditions included treatments at concentrations up to 5000 ug/plate in teh absence and in the prsence of a rat liver metabolic activation sysatem (S-9)

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