1H-Imidazole, 5,5'-[1,1'-biphenyl]-4,4'-diylbis[2-(2S)-2-pyrrolidinyl-, hydrochloride (1:4)

Genetic toxicity: in vitro

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:

January 31st, 2007 to February 20th, 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:

2007

Report date:

2007

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-[(2S)-pyrrolidin-2-yl]-5-(4'-{2-[(2S)-pyrrolidin-2-yl]-1H-imidazol-5-yl}-[1,1'-biphenyl]-4-yl)-1H-imidazole tetrahydrochloride

EC Number:

807-342-7

Cas Number:

1009119-83-8

Molecular formula:

C26H32Cl4N6

IUPAC Name:

2-[(2S)-pyrrolidin-2-yl]-5-(4'-{2-[(2S)-pyrrolidin-2-yl]-1H-imidazol-5-yl}-[1,1'-biphenyl]-4-yl)-1H-imidazole tetrahydrochloride

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Powder stored at room temperature, in the dark, under nitrogen.

Method

Target gene:

five histidine-requiring strains

Species / strain

Species / strain / cell type:

other: Salmonella typhimurium: TA1535, TA1537, TA98, TA100, Escherichia coli: WP2 uvrA (pKM101)

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: 5, 15, 50, 150, 500, 1500, 5000 ug/plate
Second test: 1.5, 5, 15, 50, 150, 500 ug/plate

Vehicle / solvent:

Vehichle: water
Solvent: DMSO (A.C.S. spectrophotometric grade)

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

water

Controls 2

Positive controls:

yes

Remarks:

In the absence of S9 mix

Positive control substance:

sodium azide

Remarks:

2 µg/plate for strains TA100 and TA1535

Controls 3

Positive controls:

yes

Remarks:

In the absence of S9 mix

Positive control substance:

9-aminoacridine

Remarks:

50 µg/plate for strain TA1537

Controls 4

Positive controls:

yes

Remarks:

In the absence of S9 mix

Positive control substance:

2-nitrofluorene

Remarks:

2 µg/plate for strain TA98

Controls 5

Positive controls:

yes

Remarks:

In the absence of S9 mix

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

2 µg/plate for strain WP2 uvrA (pKM101)

Controls 6

Positive controls:

yes

Remarks:

In the presence of S9 mix

Positive control substance:

other: 2-Aminoanthracene

Remarks:

5 µg/plate for strains TA100 and TA1535 10 µg/plate for strain WP2 uvrA (pKM101)

Controls 7

Positive controls:

yes

Remarks:

In the presence of S9 mix

Positive control substance:

benzo(a)pyrene

Remarks:

5 µg/plate for strains TA98 and TA1537

Details on test system and experimental conditions:

MUTATION TEST PROCEDURE
First test
Aliquots of 0.1 mL of the test substance solutions (seven concentrations up to 5000 µg/plate),
positive control or negative control were placed in glass vessels. The negative control was the
chosen vehicle, water. S9 mix (0.5 mL) or 0.1 M pH 7.4 phosphate buffer (0.5 mL) was added,
followed by 0.1 mL of a 10-hour bacterial culture and 2 mL of agar containing histidine (0.05 mM),
biotin (0.05 mM) and tryptophan (0.05 mM). The mixture was thoroughly shaken and overlaid
onto previously prepared Petri dishes containing 25 mL minimal agar. Each Petri dish was
individually labelled with a unique code, identifying the contents of the dish. Three Petri dishes
were used for each treatment. Plates were also prepared without the addition of bacteria in order to
assess the sterility of the test substance, S9 mix and sodium phosphate buffer. All plates were
incubated at approximately 37°C for ca 72 hours. After this period, the appearance of the
background bacterial lawn was examined and revertant colonies counted using an automated colony
counter (Perceptive Instruments Sorcerer). Some plates in the first were scored manually because
of the presence of precipitate.
Any toxic effects of the test substance would be detected by a substantial reduction in mean
revertant colony counts or by a sparse or absent background bacterial lawn. In the absence of any
toxic effects, the maximum concentration selected for use in the second test would be the same as
that used in the first. If toxic effects were observed, a lower concentration might be chosen,
ensuring that signs of bacterial inhibition were present at this maximum concentration. Ideally a
minimum of four non-toxic concentrations should be obtained. If precipitate were observed on the
plates at the end of the incubation period, at least four non-precipitating concentrations should be
obtained, unless otherwise justified by the Study Director.
Second test
As a clear negative response was obtained in the first test, a variation to the test procedure was used
for the second test. The variation used was the pre-incubation assay in which the tubes, which
contained mixtures of bacteria, buffer or S9 mix and test dilution, were incubated at 37°C for 30
minutes with shaking before the addition of the agar overlay. Because of toxicity observed in the
first test, the maximum concentration chosen for the Salmonella strains was 500 µg/plate, and the
maximum concentration chosen for the E. coli strain was 1500 µg/plate, and only six
concentrations were used

Evaluation criteria:

If exposure to a test substance produces a reproducible increase in revertant colony numbers of at
least twice (three times in the case of strains TA1535 and TA1537) the concurrent vehicle controls,
with some evidence of a positive dose-response relationship, it is considered to exhibit mutagenic
activity in this test system. No statistical analysis is performed.

Statistics:

If exposure to a test substance does not produce a reproducible increase in revertant colony
numbers, 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, even
after additional testing, the test data may be subjected to analysis to determine the statistical
significance of any increases in revertant colony numbers. The statistical procedures used are those
described by Mahon et al (1989) and are usually Dunnett’s test followed, if appropriate, by trend
analysis. Biological importance should always be considered along with statistical significance. In
general, treatment-associated increases in revertant colony numbers below two or three times the
vehicle controls (as described above) are not considered biologically important. It should be noted
that it is acceptable to conclude an equivocal response if no clear results can be obtained.

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:

cytotoxicity

Remarks:

500 ug upwards

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The absence of colonies on sterility check plates confirmed the absence of microbial contamination
of the S9 mix, buffer and test substance formulation.
The total colony counts on nutrient agar plates confirmed the viability and high
cell density of the cultures of the individual organisms.
The mean revertant colony counts for the vehicle controls were within or close to the 99%
confidence limits of the current historical control range of the laboratory. Appropriate
positive control chemicals (with S9 mix where required) induced substantial increases in revertant
colony numbers with all strains in all reported tests, confirming sensitivity of the cultures and
activity of the S9 mix.

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

It is concluded that BMS-749445-02 showed no evidence of mutagenic activity in this bacterial
system under the test conditions employed.