Registration Dossier

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:
August 13th, 2007 to September 9th, 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted according to OECD method and in accordance with GLP. Study material is well characterized.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
Off white crystalline solid stored at room temperature over silica gel in the dark.

Method

Target gene:
five histidine-requiring strains
Species / strain
Species / strain / cell type:
other: Salmonella typhimurium: TA1535, TA1537, TA98, TA100, Escherichia coli: WP2uvrA
Metabolic activation:
with and without
Metabolic activation system:
S-9 mix
Test concentrations with justification for top dose:
For genotoxicity experiment concentrations (with & without metabolic activation) used:
First test: 50, 150, 500, 1500, 5000 ug/plate
Second test: 50, 150, 500, 1500, 5000 ug/plate
Vehicle / solvent:
Vehicle: Dimethyl sulphoxide
Controlsopen allclose all
Positive controls:
yes
Remarks:
Plates wtihout S-9 mix
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
3 ug/plate for TA100, 5 ug/plate for TA1535 and 2 ug/plate for WP2uvrA
Positive controls:
yes
Remarks:
Plates wtihout S-9 mix
Positive control substance:
9-aminoacridine
Remarks:
80 ug/plate for TA1537
Positive controls:
yes
Remarks:
Plates wtihout S-9 mix
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
0.2 ug/plate for TA98
Positive controls:
yes
Remarks:
Plates with S-9 mix
Positive control substance:
other: 2-Aminoanthracene
Remarks:
2 AA at 1 ug/plate for TA 100, 2AA at 2 ug/plate for TA1535 and T1537, BP at 5 ug/plate for TA98, and 2AA at 10ug,plate for WP2uvrA
Positive controls:
yes
Remarks:
Plates with S-9 mix
Positive control substance:
benzo(a)pyrene
Remarks:
2 AA at 1 ug/plate for TA 100, 2AA at 2 ug/plate for TA1535 and T1537, BP at 5 ug/plate for TA98, and 2AA at 10ug,plate for WP2uvr
Details on test system and experimental conditions:
MUTATION TEST PROCEDURE
First test
Measured aliquots (0.1 ml) of one of the bacterial cultures were dispensed into sets of test tubes followed by 2.0 ml of molten, trace histidine or tryptophan supplemented, top agar, 0.1 ml of the test material formulation, vehicle or positive control and either 0.5 ml of S9-mix or phosphate buffer.
The contents of each test tube were mixed and equally distributed onto the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This
procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test material both with and without S9-mix.
All of the plates were incubated at 37°C for approximately 48 hours and the frequency of revertant colonies assessed using a Domino colony
counter. Manual counts were performed at 5000 µg/plate because of excessive test material precipitation.

Second test
The second experiment was performed using methodology as described for Experiment 1, using fresh bacterial cultures, test material and control solutions. The test material dose range was the same as Experiment 1 (50 to 5000 µg/plate).
Evaluation criteria:
Evaluation criteria:
The test material will generally be considered mutagenic to bacteria if the following criteria are achieved. If the following criteria are not achieved then the test material will be considered non­ mutagenic to bacteria.
i) In strain TA98, TAl OO or WP2uvrA-, a two-fold increase in the mean number of revertants per plate compared to the mean value of the
concurrent vehicle control.
ii) In strain TA1535 or TA1537, a three-fold increase in the mean number of revertants per plate compared to the mean value of the concurrent
vehicle control.
iii) Increases in revertant numbers for all strains must be related to increases in test material concentration.
iv) A positive response m one tester strain either with or without exogenous metabolic activation is sufficient to designate the test material
as a bacterial mutagen.
Statistics:
The individual plate counts, the mean number of revertant colonies and the standard deviations, for the test material, positive and vehicle controls,
both with and without metabolic activation were found.

Results and discussion

Test results
Species / strain:
other: all strains/cell types tested
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:
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The S9-mix used in both experiments was shown to be sterile.
The individual plate counts, the mean number of revertant colonies and the standard deviations, for the test material, positive and vehicle controls,
both with and without metabolic activation, are presented.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

BMS-801509-01 was considered to be non-mutagenic under the conditions of this test.