1H-Indole-6-carboxylic acid, 2,3-dihydro-2-oxo-, methyl ester

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Jan 29 - Feb 01, 2002

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Guideline:

other: instruction manual for the Ames II (Xenometrix, Boulder/USA)

Principles of method if other than guideline:

The chemical intermediate BIBF 1120/CDBC 0167 XX, a chemical intermediate of the BIBF 1120 ES - synthesis was investigated in a modified bacterial mutagenicity test as described by Ames et al. (1975). This high throughput microtiter-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) as a traditional Ames I assay 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:

methyl 2-oxo-2,3-dihydro-1H-indole-6-carboxylate

EC Number:

604-263-8

Cas Number:

14192-26-8

Molecular formula:

C10 H9 N O3

IUPAC Name:

methyl 2-oxo-2,3-dihydro-1H-indole-6-carboxylate

Test material form:

solid: particulate/powder

Method

Species / strain

Species / strain / cell type:

S. typhimurium, other: TA 7001 - 7006 and TA 98

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

1 to 5000 µg/ml (7 concentration levels: 1, 4, 20, 100, 500, 2500, 5000µg/ml)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

other: 2-aminoanthracene

Details on test system and experimental conditions:

Test Design
The Ames II test was conducted in presence and absence of a metabolic activation system (S9mix: microsomalliver enzymes from rats, Aroclor 1254-induced and co-factors). Following base-pair and frameshift-specific tester strains were used: S. typhimurium TA Mix (TA7001, 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).

Techniques
The assay was performed according to the instruction manual for the Ames II (Xenometrix,
Boulder/USA). 0.01 ml ofthe vehicle, test article or positive control were incubated with
0.24 ml bacterial ovemight 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.

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
Our historical control range: 0-7/48 wells
A concentration-dependent increase of revertant wells (mean of triplicate) over the vehicle control is indicative of genotoxic activity.

Evaluation criteria:

The pH indicator bromocresol purple tums the colour ofthe 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 spontaneaus
revertant wells ofthe vehicle control. Each test point contains 48 wells of a 384-well plate. In
each 48-well section, the wells were scored for the number ofrevertant wells (yellow) and the
mean value of the triplicates was calculated.

Results and discussion

Test results

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

BIBF 1120 Indol precipitated at concentrations of >= 2500 μg/ml following the incubation
for 90 min at 37 °C. Bacteriotoxicity, as seen by a reduced background lawn, was
observed at concentrations of >= 2500 μg/ml only in the absence of S9 mix.

Applicant's summary and conclusion

Conclusions:

Based on the described results it is concluded, that BIBF 1120 Indol when tested up to maximum recommended 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 compounds are, therefore, classified as "Ames II negative".

Executive summary:

BIBF 1120 Indol was tested whether it induces mutations inS. typhimurium TA Mix (TA 7001, TA 7002, TA 7003, TA 7004, TA 7005, TA 7006 sensitive to base-pair substitution) and TA 98 (susceptible to frameshift mutagens) bacterial tester strains. BIBF 1120/CDBC 0167 XX was dissolved in dimethylsulfoxide (DMSO) and added to bacterial suspensionsbothin presence and absence of a metabolic activation system (S9 mix: rat liver preparation and co-factors) to give final concentrations ranging from 1 to 5000 flg/ml. The bacteria were transferred to 384-well plates after 90 minutes incubation at 37°C and further incubated for 48 hours at 37°C when revertant growth was quantified colorimetrically.

 

BIBF 1120 Indol precipitated at concentrations of >/= 2500 μg/ml following the incubation for 90 min at 37 °C. Bacteriotoxicity, as seen by a reduced background lawn, was observed at concentrations of >/= 2500 μg/ml only in the absence of S9 mix.

 

BIBF 1120 Indol did not consistently increase the number of positive (revertant)

wells in the different tester strains compared to the vehicle control neither in presence nor

absence of S9mix. The number ofrevertants from the vehicle and positive controls remained

within the range of our historical controls showing the validity of the study.