Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 13 OCT 1999 to 03 NOV 1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
with the required modification for azo-dyes (Prival modifications)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 JUL 1997
Deviations:
yes
Remarks:
1) Each batch of S9 is characterized with congo red prior to usage. 2) To evaluate the toxicity of the test item a pre-experiment was performedwith strains TA1535, TA1537, TA98, TA100 and WP2uvrA.
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
29 DEC 1992
Deviations:
yes
Remarks:
see above
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Batch: V.N0199.064
Expiry: 09.07.2001

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
uninduced hamster liver S9 mix
Test concentrations with justification for top dose:
0, 33, 100, 333, 1000, 2500, 5000 µg/plate
5000 µg/plate represents the highest concentration to be tested as indicated in the OECD guideline.
In a pre-experiment 3-5000 µg/plate were were tested. 5000 µg/plate were chosen as the top concentration since toxic effects were only observed at some of the strains at high concentrations. The pre-experiment is reported as experiment 1).
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility properties of the solvent 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:
sodium azide
Remarks:
without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylene-diamine
Remarks:
without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
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
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
congo red
Remarks:
with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation assay without and with non-induced hamster liver S9 mix for Experiment 1) and 2)

DURATION
- Preincubation period: 30° C for 30 minutes
- Exposure duration: 48 h at 37° C

NUMBER OF REPLICATIONS: 2 Experiments with 3 replicates per concentration
Rationale for test conditions:
Since the test substance is an azo-dye, the Ames test was performed with the preincubation method and uninduced hamster liver S9 mix.
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 threshold 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 and solvent controls such an increase is not considered biologically relevant.
Statistics:
Arithmetic means and standard deviation of the counted colonies were calculated.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
For details see table below
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
for details see table below
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
for details see table below
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
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 nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: results from experiment I and II

Any other information on results incl. tables

Toxic effects, evident as a reduction in the number of revertants, were observed at the following concentrations:

Strain

Experiment I [µg/plate]

Experiment II [µg/plate]

 

Without S9 mix

With S9 mix

Without S9 mix

With S9 mix

TA1535

2500-5000

/

5000

2500-5000

TA1537

5000

/

/

/

TA98

5000

/

1000-5000

/

TA100

/

/

/

/

WP2uvrA

/

/

/

/

(/ = no relevant toxic effects observed)

Applicant's summary and conclusion

Conclusions:
In a guideline study according to OECD TG 471 under GLP conditions the test item showed no mutagenic activity in both preincubation experiments with and without metabolic activation (uninduced hamster liver S9, i.e. required Prival modification for azo-dyes).
Executive summary:

In a guideline study according to OECD TG 471 under GLP conditions 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 (uninduced hamster liver S9 mix) and without metabolic activation at concentrations of 0, 33, 100, 333, 1000, 2500 and 5000 μg/plate. Due to the test items characteristic as an azo-dye the test was conducted using the Prival modification, i.e. testing the above mentioned bacterial strains in the preincubation assay without and with hamster liver S9 mix for metabolic activation.

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