Registration Dossier

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:
13.02.2008 - 22.02.2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD ) and in compliance with GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Report Date:
2008

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): N-Methyl-4-nitroaniline
- Physical state: yellow to orange crystalline substance or powder
- Analytical purity: 99.3%
- Purity test date: 06 February 2008 date of CoA
- Lot/batch No.: 304269
- Expiration date of the lot/batch: 30 November 2008
- Storage condition of test material: At room temperature in the dark (ambient humidity)

Method

Species / strain
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
100, 300, 1000, 3000 and 5000 µg/plate
Vehicle:
- Vehicle(s)/solvent(s) used:DMSO
Controls
Negative controls:
yes
Remarks:
Dimethylsulfoxide (DMSO)
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
mitomycin C
other: 2-Aminoanthracene
Details on test system and conditions:
METHOD OF APPLICATION: soft agar

DURATION

- Exposure duration: 2 days (TA 102: 3 days)


NUMBER OF REPLICATIONS: 3


Evaluation criteria:
A reproducible, concentration-dependent increase in the number of revertants of at least one
tester strain over the vehicle control value and/or outside the historical control range is
indicative of genotoxic activity. Since the results were unequivocal, no detailed statistical
evaluation was performed.

Assay acceptance criteria
All tester strains of S. typhimurium exhibited a characteristic number of spontaneous
revertants per plate. The historical control ranges are based on ca 100 experiments conducted
in our laboratory (not filed in the raw data). In the absence of a distinct difference between
non-activation and activation, the mean control values were combined and given as ranges:
TA 1535: 5-22
TA 1537: 3-39
TA 98: 16-68
TA 100: 49-164
TA 102: 249-531

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity:
yes
Remarks:
≥3000 μg/plate
Vehicle controls valid:
not specified
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

SOLUBILITY AND TOXICITY

N-Methyl-4-nitroaniline precipitated and was bacteriotoxic primarily at concentrations ≥3000 μg/plate.

MUTAGENICITY

The OD600 of the individual overnight bacterial cultures varied between 2.04 and 2.84 (raw

data). These values correspond to a bacterial titer of ca 100 millions/0.1 mL.

N-Methyl-4-nitroaniline induced a concentration-dependent increase in the number of mean

revertants in S. typhimurium TA 98 starting at 300 μg/plate with and without S9 mix. The

increase was 4-5-fold at 1000 μg/plate. In addition, there was a 4-fold increase in the number

of revertants in strain TA 1537 at 1000 μg/plate in absence of S9 mix. No mutagenic

response was seen in the strains TA 1535, TA 100 and TA 102. In view of the clear

mutagenic response in TA 98 and TA 1537, no repeat experiment was performed. However,

the Ames test is known to be oversensitive for aromatic nitro compounds and therefore,

further investigations are needed to evaluate the biological relevance of this in vitro result.

The validity of this study is given since the vehicle control plates showed spontaneous

revertants in different tester strains of S. typhimurium at frequencies similar to those

described in the literature and within the historical control range experienced in our

laboratory. The diagnostic mutagens NaN3, 9-AA, 2-NF, MMC and 2-AA showed the

expected strain specific responses in the absence and presence of a metabolic activation

system.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
positive

N-Methyl-4-nitroaniline, an intermediate in the synthesis of BIBF 1120 ES, induced a
concentration-dependent increase in the number of revertants in S. typhimurium strain TA 98
starting at 300 μg/plate with and without metabolic activation. There was also a clear increase
at 1000 μg/plate in strain TA 1537 without metabolic activation. Therefore, it was concluded
that N-Methyl-4-nitroaniline induces frameshift mutations in the Ames test when tested up to
insoluble and bacteriotoxic concentrations and has to be classified as "Ames positive" under
the conditions of this study. Nevertheless, in view of the oversensitivity of the Ames test for
aromatic nitro compounds, the biological relevance still needs to be determined in an in vivo
experiment