4-Quinazolinamine, N-(3-chloro-4-fluorophenyl)-6-nitro-7-[[(3S)-tetrahydro-3-furanyl]oxy]-

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 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

Test material information

Constituent 1

Reference substance name:

N-(3-chloro-4-fluorophenyl)-6-nitro-7-[(3S)-oxolan-3-yloxy]quinazolin-4-amine

EC Number:

925-395-5

Cas Number:

314771-88-5

Molecular formula:

C18 H14 Cl F N4 O4

IUPAC Name:

N-(3-chloro-4-fluorophenyl)-6-nitro-7-[(3S)-oxolan-3-yloxy]quinazolin-4-amine

Method

Species / strain

Species / strain 1

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 2

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 results

Test results 1

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

Test results 2

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

Test results 3

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

Test results 4

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".