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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 28 SEP 2004 to 12 OCT 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD 471) with Prival modifications for azo-dyes

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-chlorophenyl)-3-oxobutyramide
EC Number:
229-355-1
EC Name:
2-[(4-chloro-2-nitrophenyl)azo]-N-(2-chlorophenyl)-3-oxobutyramide
Cas Number:
6486-23-3
Molecular formula:
C16H12Cl2N4O4
IUPAC Name:
2-[(4-chloro-2-nitrophenyl)diazenyl]-N-(2-chlorophenyl)-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 with induced rat liver S9 mix (15% (v/v); induction with Phenobarbital/beta-Naphthoflavone
- preincubation assay 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 in the plate incorporation test without metabolic activation at 5000 µg/plate
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 2500 to 5000 µg/plate (plate incorporation assay without metabolic activation; pre-incubation assay with and without metabolic activation) and at 1000-5000 µg/plate in the plate incorporation assay with metabolic activation

COMPARISON WITH HISTORICAL CONTROL DATA:
- minor deviations from the historical control range were seen in strain WP2 uvrA which were judged as fluctuation, not relevant for the test evaluation

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- in strain TA1537 in the plate incorporation test without metabolic activation at 5000 µg/plate toxic effects became evident as a reduction in the number of revertants


Any other information on results incl. tables

The test item showed no mutagenic activity in both experiments (plate incorporation assay, preincubation assay) each with and without metabolic activation.

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.

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