Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 Jun 2016 - 07 Jul 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
1998
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Xanthylium, 9-(2-carboxyphenyl)-3,6-bis(diethylamino)-, hydrogen bis[3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-2-hydroxy-5-nitrobenzenesulfonato(3-)]chromate(3-), compd. with 3-[(2-ethylhexyl)oxy]-1-propanamine
EC Number:
284-723-9
EC Name:
Xanthylium, 9-(2-carboxyphenyl)-3,6-bis(diethylamino)-, hydrogen bis[3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-2-hydroxy-5-nitrobenzenesulfonato(3-)]chromate(3-), compd. with 3-[(2-ethylhexyl)oxy]-1-propanamine
Cas Number:
84962-27-6
Molecular formula:
C32H20CrN10O14S2.1.5(C28H31N2O3).1.5(C11H25NO)
IUPAC Name:
Xanthylium, 9-(2-carboxyphenyl)-3,6-bis(diethylamino)-, hydrogen bis[3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-2-hydroxy-5-nitrobenzenesulfonato(3-)]chromate(3-), compd. with 3-[(2-ethylhexyl)oxy]-1-propanamine
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/ß-naphthoflavone induced rat liver S9 fraction and uninduced hamster liver S9 fraction
Test concentrations with justification for top dose:
1st Experiment
Strains: TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA
Doses: 0; 33; 100; 333; 1000; 2500 and 5000 μg/plate
Type of test: Standard plate test with and without S9 mix

2nd Experiment
Strains: TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA
Doses: 0; 10; 33; 100; 333; 1000 and 2500 μg/plate
Prival preincubation test with and without S9 mix
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Due to the insolubility of the test substance in water, DMSO was used as vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: see below
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation;

DURATION
- Exposure duration: 48 – 72 hours in the dark

NUMBER OF REPLICATIONS: 3 test plates per dose or per control

DETERMINATION OF CYTOTOXICITY
• decrease in the number of revertants (factor ≤ 0.6)
• clearing or diminution of the background lawn (= reduced his- or trp- background growth)

- POSITIVE CONTROLS
With rat liver S9 mix
• 2-aminoanthracene (2-AA)
- 2.5 μg/plate, dissolved in DMSO, strains: TA 1535, TA 100, TA 1537, TA 98
- 60 μg/plate, dissolved in DMSO, strain: Escherichia coli WP2 uvrA

With hamster liver S9 mix
• 2-aminoanthracene (2-AA)
-10 μg/plate, dissolved in DMSO, strains: TA 1535, TA 100, TA 1537, TA 98, Escherichia coli WP2 uvrA
• Congo red (CoR)
- 210 μg/plate, dissolved in DMSO, strain: TA 98

Without S9 mix
• N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)
- 5 μg/plate, dissolved in DMSO, strains: TA 1535, TA 100
• 4-nitro-o-phenylenediamine (NOPD)
- 10 μg/plate, dissolved in DMSO, strain: TA 98
• 9-aminoacridine (AAC)
- 100 μg/plate, dissolved in DMSO, strain: TA 1537
• 4-nitroquinoline-N-oxide (4-NQO)
- 5 μg/plate, dissolved in DMSO, strain: E. coli WP2 uvrA
Evaluation criteria:
The test substance was considered positive in this assay if the following criteria were met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. at least doubling (bacteria strains with high spontaneous mutation rate, like TA 98, TA 100 and E.coli WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA 1535 and TA 1537) of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.
A test substance was generally considered non-mutagenic in this test if:
• The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in at least two experiments carried out independently of each other.

Results and discussion

Test results
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Test substance precipitation was found from about 1000 μg/plate onward with and without S9 mix.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
In this study with and without S9 mix, the number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain. In addition, the positive control substances both with and without S9 mix induced a significant increase in the number of revertant colonies within or above the range of the historical positive control data or above.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
A bacteriotoxic effect (decrease in the number of his+ or trp+ revertants) was observed in the standard plate test depending on the strain and test conditions from about 333 μg/plate onward. In the prival preincubation assay bacteriotoxicity (reduced his- or trp- background growth, decrease in the number of his+ or trp+ revertants) was observed depending on the strain and test conditions from about 1000 μg/plate onward.

Any other information on results incl. tables

STANDARD PLATE TEST

Mean revertants per plate
Strain TA 1535 TA 100 TA 1537 TA 98 WP2uvrA
Dose (µg/plate) -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9
DMSO 15.7 11.0 106.3 110.0 10.0 12.0 24.7 28.0 27.0 24.0
33 11.7 15.0 127.0 112.3 8.3 10.3 22.3 30.0 19.7 31.0
100 14.3 13.0 94.0 112.0 8.3 8.7 25.3 30.0 24.7 27.3
333 12.7 10.3 95.7 103.0 6.7 9.3 23.0 24.7 17.3 22.7
1000 6.7 12.0 71.3 79.3 3.3 4.3 21.7 20.7 11.7 24.0
2500 9.3 7.7 33.7 40.7 4.3 2.0 5.0 4.7 10.0 17.7
5000 4.3 3.3 2.0 2.0 0.0 0.0 1.7 1.3 0.0 8.7
positive control 4606.0 311.0 4396.3 2296.7 922.3 217.7 980.0 1729.3 1082.3 204.0

PRIVAL PREINCUBATION TEST

Mean revertants per plate
Strain TA 1535 TA 100 TA 1537 TA 98 WP2uvrA
Dose (µg/plate) -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9
DMSO 15.7 11.3 99.0 91.7 6.0 6.7 16.3 33.3 15.3 17.7
10 12.3 11.7 103.3 104.0 6.0 9.0 16.0 29.3 16.3 19.7
33 18.3 10.7 97.3 100.3 5.3 7.0 16.7 40.3 18.0 20.7
100 14.7 9.7 101.7 102.3 8.3 7.7 16.0 36.7 20.3 17.3
333 13.7 11.7 75.7 86.3 7.3 6.3 15.0 44.0 14.0 16.7
1000 9.3 9.0 57.7 85.7 3.0 9.3 14.3 28.3 10.0 14.7
2500 0.0 8.7 0.0 62.3 0.0 4.7 4.0 8.3 10.3 7.7
positive control 1470.0 453.7 1139.3 1328.7 2748.3 333.3 1069.3 1247.3 271.7 1181.3
positive control #2 466.7

Applicant's summary and conclusion

Conclusions:
Under the experimental conditions of this study, the test substance is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.
Executive summary:

The test substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay (Ames standard plate test and Prival preincubation test). Dose ranges were 33 μg - 5000 μg/plate in the standard plate test and 10 μg - 2500 μg/plate in the preincubation test, both with and without metabolic activation. Precipitation of the test substance was found from about 1000 μg/plate onward with and without S9 mix. A bacteriotoxic effect was observed depending on the strain and test conditions from about 333 μg/plate onward A relevant increase in the number of his+ or trp+ revertants (factor ≥ 2: TA 100, TA 98 and E.coli WP2 uvrA or factor ≥ 3: TA 1535 and TA 1537) was not observed in the standard plate test or in the prival preincubation test either without S9 mix or after the addition of a metabolizing system. Thus, under the experimental conditions of this study, the test substance is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.