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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:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
From 02 MAR 2005 to 17 MAR 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD TG471) with Prival modification for azo-dyes

Data source

Reference
Title:
Unnamed
Year:
2005
Report date:
2005

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Commission Directive 2000/32/EC, L1362000, Annex 4D
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
4-[(2,5-dichlorophenyl)azo]-3-hydroxy-N-phenylnaphthalene-2-carboxamide
EC Number:
227-930-1
EC Name:
4-[(2,5-dichlorophenyl)azo]-3-hydroxy-N-phenylnaphthalene-2-carboxamide
Cas Number:
6041-94-7
IUPAC Name:
4-[(2,5-dichlorophenyl)diazenyl]-3-hydroxy-N-phenyl-2-naphthamide

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 or hamster liver S9
Test concentrations with justification for top dose:
Experiment I (Plate incorporation method): 0, 3, 10, 33, 100, 333, 1000, 2500, 5000 µg/plate
Experiment II (Pre-incubation method): 0, 33, 100, 333, 1000, 2500, 5000 µg/plate
Vehicle / solvent:
- Vehicle/ Solvent used: DMSO
- Justification for choice of solvent/ vehicle: solubility properties and its relative non-toxicity to the bacteria
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 1535 and TA 100), 4-Nitro-o-phenylene-diamine (TA 1537 and TA 98), methyl methane sulfonate (WP2 uvrA)
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
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 1535, TA 100, TA 1537 and WP2 uvrA), congo red (TA98)
Remarks:
with metabolic activation (hamster liver S9 mix)
Details on test system and experimental conditions:
METHOD OF APPLICATION:
Experiment I: plate incorporation method without and with induced rat liver S9 mix (induction with phenobarbital/beta-naphthoflavone)
Experiment II: pre-incubation method without and with non-induced hamster liver S9 mix.

DURATION:
-preincubation period (experimentII): 30 °C, for 30 min
- exposure duration: 48 h at 37 °C in the dark

NUMBER OF REPLICATIONS: 3 plates per strain and dose level including the controls
Evaluation criteria:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, WP2 uvrA) or thrice (strains TA 1535, TA 1537) the colony count of the corresponding solvent colony is observed.

A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.

An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the thresshold is regarded as an indication of mutagenic potential if reproduced in an independent second experiment. however, whenever the colony counts remain within the historical range of negative annd solvent controls such as an increase is not considered biologically relevant.
Statistics:
Arithmetic mean, standard deviation and the ratio of treated versus solvent were calculated.

Results and discussion

Test resultsopen allclose all
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:
minor toxic effects were observed in strain TA 1537 at 1000 and 5000 µg/plate with metabolic activation in experiment II (reduction in the number of revertants).
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
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:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: occurred at concentrations of 333 µg/plate and more (Experiment I), or 1000 µg/plate and more (Experiment II). Nevertheless the colonies could be counted manually.

COMPARISON WITH HISTORICAL CONTROL DATA: in experiment I the number of colonies did not reach the lower limit of the historical control data in strain WP2 uvrA in the ng. control. 0Since the deviation is rather small this effect is judged to be based upon statistical fluctuations , which has no detrimental impact on the outcome of the study.
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):
negative

The test item did not exert mutagenic activity in the reverse bacterial mutation assay (plate incorporation assay and pre-incubation 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 and TA100 as well as Escherichia coli strain 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 without and with uninduced hamster liver S9 mix for metabolic activation. This test was performed using the following concentrations: 0, 33, 100, 333, 1000, 2500, 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.