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Genetic toxicity: in vitro

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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:
From 22 FEB 2005 to 29 MAR 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD 471) with Prival modifications for azo-dyes, GLP compliant

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report date:
2005

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
2-[(4-chloro-2-nitrophenyl)azo]-N-(2-methoxyphenyl)-3-oxobutyramide
EC Number:
236-852-7
EC Name:
2-[(4-chloro-2-nitrophenyl)azo]-N-(2-methoxyphenyl)-3-oxobutyramide
Cas Number:
13515-40-7
Molecular formula:
C17H15ClN4O5
IUPAC Name:
2-[(4-chloro-2-nitrophenyl)diazenyl]-N-(2-methoxyphenyl)-3-oxobutanamide
Test material form:
solid: nanoform, no surface treatment

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9; hamster liver S9
Test concentrations with justification for top dose:
0, 3, 10, 33, 100, 333, 1000, 2500, 5000 µg/plate (plate incorporation test)
0, 33, 100, 333, 1000, 2500, 5000 µg/plate (pre-incubation test)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility properties of the solvent
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: sodium azide (TA 100, TA1535), 4-nitro-o-phenylene-diamine (TA 1537, TA 98), methyl methane sulfonate (WP2uvrA)
Remarks:
without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (all strains)
Remarks:
with metabolic activation (rat liver S9 mix)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (TA 100, TA 1535, TA 1537, WP2uvrA), congo red (TA 98)
Remarks:
with metabolic activation (hamster liver S9 mix)
Details on test system and experimental conditions:
METHOD OF APPLICATION:
- plate incorporation assay without and with induced rat liver S9 mix (15% (v/v); induction with Phenobarbital/beta-Naphthoflavone)
- preincubation assay without and with uninduced hamster liver S9 mix (30% (v/v))

DURATION
- Preincubation period: ca. 30 minutes at 30 °C
- Exposure duration: at least 48 hours

NUMBER OF REPLICATIONS: 3 plates per strain and dose level, including controls

Evaluation criteria:
A test compound is classified as mutagenic if it has either of the following effects:
- it produces at least a 2-fold increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control at complete bacterial background lawn
- it induces a dose-related increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control in at least two to three concentrations of the test compound at complete bacterial background lawn
- an increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent secnd experiment

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
in strain TA1537 and TA100 in the pre-incubation test at 5000 µg/plate without metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: visible at 333 to 5000 µg/plate (plate incorporation assay with and without metabolic activation), at 1000 to 5000 µg/plate (pre-incubation assay without metabolic activation), at 33 to 5000 µg/plate (pre-incubation assay with metabolic activation); in the plate incorporation assay the highest test concentration of 5000 µg/plate could not be analysed in TA100 (without metabolic activation) and in TA 98, TA 100 and WP2uvrA (with metabolic activation), due to densely coloured plates


ADDITIONAL INFORMATION ON CYTOTOXICITY:
- in strain TA1537 and TA100 minor reductions in the number of revertants were observed in the pre-incubation test at 5000 µg/plate without metabolic activation
- the reduction in the number of revertants in strain TA 1535 at 333 µg/plate with S9 mix in the pre-incubation test was judged by the authors of the study to be based on biological fluctuations


Any other information on results incl. tables

In the plate incorporation assay with metabolic activation TA 98 showed a minor increase in revertant colony numbers at 2500 µg/plate. To verify the results of this experiment an independent repeat experiment was performed under indentical conditions with strain TA 98 in the presence of metabolic activation. No increase in the number of revertant colonies occurred in the repeat experiment and the effect observed in the first experiment was judged by authors of the study as biologically irrelevant.

Revertant colony counts in TA 98 (plate incorporation assay with metabolic activation)

Control group

Test item concentration (µg/plate)

First experiment (mean +/- SD)

Repeat experiment (mean +/- SD)

DMSO

 

34 +/- 9

40 +/- 13

Untrated

 

44 +/- 7

46 +/- 4

 

3

30 +/- 4

37 +/- 1

 

10

36 +/- 7

37 +/- 7

 

33

35 +/- 4

45 +/- 4

 

100

47 +/- 4

38 +/- 5

 

333

37 +/- 12

42 +/- 3

 

1000

50 +/- 2

53 +/- 5

 

2500

70 +/- 5

50 +/- 3

 

5000

Analysis not possible

49 +/- 3

Applicant's summary and conclusion

Conclusions:
Interpretation of results:
negative

The test item did not exert mutagenic activity in the reverse bacterial mutation assay (plate incorporation assay and preincubation assay according to Prival) with and without metabolic activation.
Executive summary:

Mutagenic activity of the test item was investigated in Salmonella typhimurium strains TA 1535, TA 1537, TA98, TA100 and E. coli WP2 uvrA with (induced rat liver S9 mix) and without metabolic activation at concentrations of 0, 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate using the plate incorporation assay. Due to the test items characteristic as an azo-dye the test was also conducted using the Prival modification, i.e. testing the above mentioned bacterial strains in the preincubation assay with uninduced hamster liver S9 mix for metabolic activation. This test was performed using the concentrations 0, 33, 100, 333, 1000, 2500 and 5000 µg/plate.

The test item did not reveal any mutagenic activity under the conditions tested. The appropriate reference mutagenes showed distinct positive mutagenic effects.