Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
August 2002 - October 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
1993
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Name of test material (as cited in study report): 4,4'-BIS[[1-[[(2,4-DIMETHYLPHENYL)AMINO]CARBONYL]-2-OXOPROPYL]AZO][1,1'-BIPHENYL]-2,2'-DISULPHONIC ACID
- Physical state: Powder
- Purity: ~100%.
- Storage condition of test material: In refrigerator (1-10°C), in the dark.
- pH (1% in water, indicative range): 6.2 - 6.4

Method

Target gene:
- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene
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-mix induced by Aroclor 1254
Test concentrations with justification for top dose:
Experiment 1
Preliminary test (without and with S9) TA100: 1.6, 8, 40, 200, 1000, and 5000 µg/plate
Main study: TA1535, TA1537, TA98 and Wp2uvrA:
Without and with S9-mix: 1.6, 8, 40, 200, 1000, and 5000 µg/plate
Experiment 2:
TA100 and TA1537 without S9-mix and TA98 and TA1537 with S9-mix: 8.192, 20.48, 51.2, 128, 320, 800, and 2000 µg/plate
TA198, TA1535 and WP2 uvrA without S9-mix and TA100, TA1535 and WP2 uvrA with S9-mix: 20.48, 51.2, 128, 320, 800, 2000, and 5000 µg/plate

The top dose for experiment 1 was based on toxicity observed in the range-finder experiment. For experiment 2, TA198, TA1535 and WP2 uvrA without S9-mix and TA100, TA1535 and WP2 uvrA with S9-mix retained 5000 µg/plate as the maximum test dose. For treatment of strains TA100 and TA1537 without S9-mix and TA98 and TA1537 with S9-mix, which had resulted in the most pronounced toxicity in experiment 1, the maximum dose was reduced to 2000 µg/plate.
Vehicle / solvent:
- solvent used: DMSO
- Justification for choice of solvent: A homogeneous suspension could be in DMSO and DMSO is accepted and approved by authorities and international guidelines
- All test item concentrations were corrected and expressed in terms of the acid form, using a correction factor of 1.05.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-nitrofluorene
Remarks:
without S9 5 µg/plate in DMSO for TA98
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without S9 2 µg/plate in water for TA100 and TA1535
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without S9 50 µg/plate in DMSO for TA1537
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without S9 2 µg/plate in DMSO for WP2uvrA
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
with S9 10 µg/plate in DMSO for TA98
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with S9 in DMSO 5 µg/plate for TA100, TA1535, and TA1537 and 10 µg/plate for WP2 uvrA
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 72 hour

Experiment 2 included a pre-incubation step. Quantities of test item or control solution, bacteria and S9 were mixed and incubated for 1 hour at 37±1°C before the addition of molten agar.

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted.

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn and the reduction of the revertant colonies.

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.
Rationale for test conditions:
Based on the negative results of experiment 1, treatments in the presence of S9 in experiment 2 included a pre-incubation step of 1 hour to increase the range of mutagenic chemicals that could be detected in the assay.
Evaluation criteria:
Acceptance criteria
The assay was considered valid if the following criteria were met:
- The mean negative control counts fell within the laboratories historical normal ranges
- The positive control chemicals induced clear increases in revertant numbers confirming discrimination between different strains, and an active S-9 preparation
- No more than 5% of the plates were lost through contamination or some other unforeseen event.

Evaluation criteria
The test item was considered to be mutagenic if:
- The assay was valid
- Dunnett's test gave a significant response (p≤0.01) and the data set(s) showed a significant dose correlation
- The positive responses described above were reproducible.
Statistics:
The m-statistic was calculated to check that the data were Poisson-distributed, and Dunnett's test (signicifance level 1%) was used to compare the counts of each dose with the control. The presence or otherwise of a dose response was checked by linear regression analysis.

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
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
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:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: precipitation was observed at dose levels of 2000 µg/plate and above with and without S9

RANGE-FINDING/SCREENING STUDIES:
- In tester strain TA100, toxicity was observed at dose levels of 5000 μg/plate in the presence of S9 and 1000 μg/plate and above in the absence of S9-mix.

COMPARISON WITH HISTORICAL CONTROL DATA:
- The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
TA1535: without S9: 5000 µg/plate and above and with S9: no cytotoxicity observed
TA1537: without S9: 800 µg/plate and above and with S9: 800 µg/plate and above
TA98: without S9: 2000 µg/plate and above and with S9: 2000 µg/plate and above
TA100: without S9: 800 µg/plate and above and with S9: 5000 µg/plate and above
WP2uvrA: without S9: no cytotoxicity observed and with S9: 5000 µg/plate and above

Applicant's summary and conclusion

Conclusions:
In an AMES test, performed according to OECD guideline and GLP principles, 4,4'-bis[[1-[[(2,4-dimethylphenyl)amino]carbonyl]-2-oxopropyl]azo][1,1'-biphenyl]-2,2'-disulphonic acid was found not to be mutagenic with or without metabolic activation.
Executive summary:

An AMES test was performed with 4,4’-bis[[1-[[(2,4-dimethylphenyl)amino]carbonyl]-2-oxopropyl]azo][1,1’-biphenyl]-2,2’-disulphonic acid according to to OECD guidelines and according to GLP principles. The study was performed at test substance concentrations up to the lower limits of toxicity and/or precipitation. The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. All bacterial strains showed negative responses up to and including 5000 ug/plate, i.e. no significant dose-related increase in the number of revertants with or without metabolic activation was seen.

Based on the results of this study it is concluded that 4,4'-bis[[1-[[(2,4-dimethylphenyl)amino]carbonyl]-2-oxopropyl]azo][1,1'-biphenyl]-2,2'-disulphonic acid is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay with or without metabolic activation.