Acetamide, 2-chloro-N-methyl-N-(4-nitrophenyl)-

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:

30 January - 28 February 2002

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study was performed for internal use and hence not in GLP-compliance. No guideline was followed. However, the test is very well described and is considered reliable.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

The test was performed according to the instruction manual for the test (Xenometrix, Boulder/USA).

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

BIBF 1120 / CDBE 0055 XX

IUPAC Name:

BIBF 1120 / CDBE 0055 XX

Test material form:

not specified

Details on test material:

Batch no.: PKLHUC00799
Storage: stored at rom temperature in the dark (ambient humidity)
Manufacturer: Boehringer Ingelheim Pharma KG, Biberach, Germany

Method

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

seven concentration levels between 1 - 5000 microgram/L: 1, 4, 20, 100, 500, 2500 and 5000µg/ml

Vehicle / solvent:

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

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

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:

0.01 mL of the vehicle, test article or positive control were incubated with 0.24mL bacterial overnight culture (ca 10exp.7/mL)/exposure medium in 24-well plates for 90 min at 37°C and 250rpm.
With metabolic activation 0.2mL strain mixture and 0.04mL 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 sensivity of the tester strains and the metabolic capacity of the S9 mix, the diagnostic mutagend 2-NF, 4-NQO and 2-AA were used, respectively.

The traditional Ames I test was performed by the preincubation technique. 0.1mL of the vehicle, test article or positive control solution, 0.5mL phosphate buffer or S9-mix and 0.1mL of a bacterial culture were preincubated and shaken for 20 min at 37°C. Then 2mL soft agar were added to the mixture and after vortexing overlaid onto minimal medium plate in duplicate.

Evaluation criteria:

pH indicator bromocresol was used to colour the number of replicate wells (yellow). In each 48-well section the wells were scored for the number of revertant wells and the mean value of the triplicates was calculated.

In the Ames I test the number of revertant colonies were counted using an automatic colony counter. For all replicates, the mean number of revertants per test concentration was calculated.

Results and discussion

Test results

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

> = 500 µg/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The vehicle controls showed numbers of positive wells similar to those described in literature.

The positive controls induced the expected increase in the number of positive wells (-S9). Following metabolci activation, 2-AA showed a marked mutagenic response.

The test substancecaused a clear increase of positive wells (up to 48/48 wells) in all tester strains in the presence and absence of S9 at the low concentration range (1-500 microgram/mL). Higher concentrations could not be evaluated due to toxicity. The clear mutagenicity was confirmed (up to 42-fold) in the formal Ames test using the strains TA 98 in the presence and absence of S9.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
positive

Based on the described results it is concluded that the test substance when tested up to insoluble and toxic concentrations showed a clear mutagenic response in a series of S. typhimurium tester strains: TA Mix (base-pair substitution) and TA 98 (frameshift) in the presence and absence of metabolic activation. The positive results were quantitatively confirmed in the formal Ames test using the strain TA 98.
The test article is therefore classified as "Ames positive" and miht be grouped in Category 3, R 68 "can cause irreversible damage" e.g. genetic changes. Due to the empirical correlation between mutations in bacteria and genotoxic effects leading to mutation and cancer in mammals, this substance should be handled with extreme care.