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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:
January 14-16, 2003
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report date:
2003

Materials and methods

Test guideline
Guideline:
other: AMES II
Deviations:
not specified
Principles of method if other than guideline:
The chemical intermediate BIBW 2992/CDBA 0573 BS was investigated in a modified bacterial mutagenicity test as described by Ames et al. (1975). This high throughput microtitre-based version, called Ames II, is based on the same genetic principle (base-pair substitution and frameshift mutations in the his operon of S. typhimurium) combined with the fluctuation method (Gee et al., 1998).
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
(3-Chloro-4-fluoro-phenyl)-{6-nitro-7-[(S)-(tetrahydro-furan-3-yl)oxy]-quinazolin-4-yl}-amine
EC Number:
925-395-5
Cas Number:
314771-88-5
Molecular formula:
C18 H14 Cl F N4 O4
IUPAC Name:
(3-Chloro-4-fluoro-phenyl)-{6-nitro-7-[(S)-(tetrahydro-furan-3-yl)oxy]-quinazolin-4-yl}-amine

Method

Species / strainopen allclose all
Species / strain / cell type:
other: S. typhimurium TA Mix (TA 7001, TA 7002, TA 7003, TA 7004, TA 7005, TA 7006)
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 98
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
none
Metabolic activation:
with and without
Metabolic activation system:
hepatic microsomal enzymes
Test concentrations with justification for top dose:
the test article was tested over a concentration range from 1 to
5000 µg/ml medium with and without microsomal rat liver enzymes.
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 Ames II test was conducted with and without addition of microsomal liver enzymes from
rats (Aroclor 1254-induced). Following base-pair and frameshift-specific tester strains were
used: S. typhimurium TA Mix (TA 7001, TA 7002, TA 7003, TA 7004, TA 7005 and TA
7006) and TA 98, respectively. The Salmonella strains were described by Gee et al. (1998).

The assay was performed according to the instruction manual for the Ames II (Xenometrix,
Boulder/USA). 0.01 ml of the vehicle, test article or positive control were incubated with 0.24
ml bacterial overnight culture (ca 107/ml)/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 a 8-channel pipettor. The
plates were incubated for 48 hrs at 37°C.
To confirm the sensitivity of the tester strains and the metabolic capacity of the S9 fractions,
known diagnostic mutagens were used (2-nitrofluorene/2-NF, 4-nitroquinoline-N-oxide/4-
NQO and 2-aminoanthracene/2-AA, respectively).
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.
Evaluation criteria:
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 170 experiments (1999-2002)
A concentration-dependent increase of revertant wells (mean of triplicate) over the vehicle
control is indicative for a genotoxic activity of the test article.
Statistics:
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.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 98
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with
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
Species / strain:
other: S. typhimurium TA Mix (TA 7001, TA 7002, TA 7003, TA 7004, TA 7005 and TA 7006)
Metabolic activation:
with
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
Species / strain:
other: S. typhimurium TA Mix (TA 7001, TA 7002, TA 7003, TA 7004, TA 7005 and TA 7006)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
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 BIBW 2992/CDBA 0573 BS, when
tested up to bacteriotoxic concentrations, caused neither base-pair substitution 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 compound is, therefore, classified as "Ames II negative".