1H-Purine-2,6-dione, 8-bromo-7-(2-butyn-1-yl)-3,7-dihydro-3-methyl-

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:

07 - 09 Sept 2004

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The Test is well documented, but not according an OECD guidline and is Non GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

The assay was performed according to the instruction manual for the Ames II (Xenometrix, Boulder/USA). Vehicle, test substance or positive
control in a volume of 0.01 mL were incubated with 0.24 mL bacterial overnight culture (ca 10e7/mL) in exposure medium in 24-well plates
for 90 min at 37°C and 250 rpm. With metabolic activation 0.2 mL strain mixture and 0.04 mL S9-mix (30%) were used.
After 90 min the exposed cultures were diluted with pH indicator medium lacking histidine and aliquoted into 48 wells of a 384-well plate
(3 replicates) using an 8-channel pipettor.The plates were incubated for 48 h at 37°C.
To confirm the sensitivity of the tester strains and the metabolic capacity of the S9 fractions, the diagnostic mutagens 2-nitrofluorene (2-NF),
4-nitroquinoline-N-oxide (4-NQO) and 2-aminoanthracene (2-AA) were used, respectively.

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

8-bromo-7-(but-2-yn-1-yl)-3-methyl-2,3,6,7-tetrahydro-1H-purine-2,6-dione

EC Number:

613-975-8

Cas Number:

666816-98-4

Molecular formula:

C10-H9-Br-N4-O2

IUPAC Name:

8-bromo-7-(but-2-yn-1-yl)-3-methyl-2,3,6,7-tetrahydro-1H-purine-2,6-dione

Test material form:

solid: particulate/powder

Method

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 98

Species / strain 2

Species / strain / cell type:

S. typhimurium, other: TA 7001, TA 7002, TA 7003, TA 7004, TA 7005 and TA 7006

Metabolic activation:

with and without

Metabolic activation system:

0.2 mL strain mixture and 0.04 mL S9-mix (30%)

Test concentrations with justification for top dose:

1 to 2500 μg/mL

Vehicle / solvent:

DMSO

Controls

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

other: 2-aminoanthracene

Details on test system and experimental conditions:

The test system was suitably labelled to clearly identify the bacterial strain, test article concentration, positive and vehicle controls, absence or presence of S-9 mix.

Rationale for test conditions:

The experiment is regarded valid, if the vehicle control showed the normal spontaneous
revertant frequency and the diagnostic mutagens caused the expected increase in the
mutation rate. The individual test chemicals were classified according to the following
criteria:
Negative: ≤8/48 wells Equivocal: 9-12/48 wells Positive: ≥13/48 wells
Historical control range: 0-7/48 wells in ca 300 experiments (1999-up to date)
A concentration-dependent increase of revertant wells (mean of triplicate) over the vehicle
control is indicative for a genotoxic activity of the test substance.

Evaluation criteria:

The pH indicator bromocresol purple turns the colour of the cultures from blue to yellow
as the pH drops due to the accumulation of catabolites from the metabolic activity of
revertant cells. The number of positive wells (yellow) out of a total of 48 wells is an
indication of the frequency of reversion per replicate per dose and was compared to the
number of spontaneous revertant wells of the solvent control. Each test point contains
48 wells of a 384-well plate. In each 48-well section, the wells were scored for the number
of revertant wells (yellow) and the mean value of the triplicates was calculated.

Statistics:

The bacterial reverse mutation assay (Ames test) is used extensively as a routine test for
mutagenicity for more than 25 years. In the meantime a high throughput screening version
in microtiter plates is available for identifying chemical mutagens (Ames II). Based on the
high concordance (80%) with the traditional Salmonella microsome assay, the Ames II
procedure seems an effective screen for identifying bacterial mutagens.

Results and discussion

Test results

Species / strain:

S. typhimurium, other: TA 7001, TA 7002, TA 7003, TA 7004, TA 7005, TA 7006 and TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

Based on the described results it is concluded, that CD 164, when tested up to
insoluble concentrations, caused neither base-pair substitutions nor frameshift
mutations in bacteria. No evidence of genotoxic activity was observed in a series of
S. typhimurium tester strains (TA Mix and TA 98) in the absence and presence of
metabolic activation. The test article is, therefore, classified as "Ames II negative".

Executive summary:

As part of the worker's safety program, CD 164, a chemical intermediate of the BI 1356 BS synthesis, was investigated in the Ames II test under non GLP-conditions. The Ames II assay is a high throughput version showing a high correlation with the traditional Salmonella assay. Concurrently with positive and negative (vehicle) controls, the test article was tested over a concentration range from 1 to 2500 μg/mL medium with and without microsomal rat liver enzymes (Aroclor 1254-induced). The bacterial tester strains S. typhimurium TA Mix (TA 7001, TA 7002, TA 7003, TA 7004, TA 7005 and TA 7006, sensitive to base-pair substitution) and TA 98 (susceptible to frameshift mutagens) are histidine-auxotrophic and were exposed with the test substance for 90 minutes. Using the liquid fluctuation technique revertant growth was quantified colorimetrically in 384-well plates after 48 hours at 37°C.