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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:
2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
(21 Jul 1997)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
(BASF SE, Experimental Toxicology and Ecology, Ludwigshafen, Germany)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
reaction mass of: tert-alkyl(C12-C14)ammonium bis[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphthalenolato(2-)]-chromate(1-) tert-alkyl(C12-C14)ammonium bis[1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphthalenolato(2-)]-chromate(1-) tert-alkyl(C12-C14)ammonium bis[1-[[5-(1,1-dimethylpropyl)-2-hydroxy-3-nitrophenyl]azo]-2-naphthalenolato(2-)]-chromate(1-) tert-alkyl(C12-C14)ammonium [[1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphthalenolato(2-)]-[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphthalenolato(2-)]]-chromate(1-) tert-alkyl(C12-C14)ammonium [[1-[[5-(1,1-dimethylpropyl)-2-hydroxy-3-nitrophenyl]azo]-2-naphthalenolato(2-)]-[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphthalenolato(2-)]]-chromate(1-) tert-alkyl(C12-C14)ammonium ((1-(4-nitro-2-oxidophenylazo)-2-naphtholato)(1-(3-nitro-2-oxido-5-(1,1-dimethylpropyl)phenylazo)-2-naphtholato))chromate(1-)
EC Number:
938-781-3
Cas Number:
117527-94-3
Molecular formula:
C21H21N304.C16H11N304.Cr unspecified.C12-C14 chain unspec.
IUPAC Name:
reaction mass of: tert-alkyl(C12-C14)ammonium bis[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphthalenolato(2-)]-chromate(1-) tert-alkyl(C12-C14)ammonium bis[1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphthalenolato(2-)]-chromate(1-) tert-alkyl(C12-C14)ammonium bis[1-[[5-(1,1-dimethylpropyl)-2-hydroxy-3-nitrophenyl]azo]-2-naphthalenolato(2-)]-chromate(1-) tert-alkyl(C12-C14)ammonium [[1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphthalenolato(2-)]-[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphthalenolato(2-)]]-chromate(1-) tert-alkyl(C12-C14)ammonium [[1-[[5-(1,1-dimethylpropyl)-2-hydroxy-3-nitrophenyl]azo]-2-naphthalenolato(2-)]-[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphthalenolato(2-)]]-chromate(1-) tert-alkyl(C12-C14)ammonium ((1-(4-nitro-2-oxidophenylazo)-2-naphtholato)(1-(3-nitro-2-oxido-5-(1,1-dimethylpropyl)phenylazo)-2-naphtholato))chromate(1-)
Details on test material:
- Analytical purity: 100 % (HPLC at 328 nm). The IR spectrum of the test item shows the expected bands.
- Physical state: Solid / black
- Homogeneity: homogeneous by visual inspection
- The stability of the test substance under storage conditions is guaranteed until 07 Apr 2023.
- Storage conditions: Room temperature

Method

Target gene:
his+ / trp+
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital and beta-naphthoflavone induced rat liver S9 microsomal fraction
Test concentrations with justification for top dose:
0; 33; 100; 333; 1000; 2500 and 5000 μg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Due to the limited solubility of the test substance in ultrapure 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: 2-aminoanthracene (2-AA), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 4-nitro-o-phenylenediamine (NOPD), 9-aminoacridine (AAC), 4-nitroquinoline-N-oxide (4-NQO)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

Standard plate test:
- Exposure duration: 48 – 72 hours

Preincubation test:
- Preincubation period: 20 min
- Exposure duration: 48 – 72 hours


NUMBER OF REPLICATIONS: 3


DETERMINATION OF CYTOTOXICITY
- Method: decrease in the number of revertants, clearing or diminution of the background lawn, reduction in the titer


OTHER:
Titer determination: The titer was determined only in the experimental parts with S9 mix both for the negative controls (vehicle only) and for the two highest doses in all experiments.

Positive controls:

With S9 mix: 2-aminoanthracene (2-AA), 2.5 μg/plate, dissolved in DMSO / TA 1535, TA 100, TA 1537, TA 98; 60 μg/plate, dissolved in DMSO / Escherichia coli WP2 uvrA
Without S9 mix: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 5 μg/plate, dissolved in DMSO / TA 1535, TA 100; 4-nitro-o-phenylenediamine (NOPD), 10 μg/plate, dissolved in DMSO / TA 98; 9-aminoacridine (AAC), 100 μg/plate, dissolved in DMSO / TA 1537; 4-nitroquinoline-N-oxide (4-NQO) (SIGMA, N-8141), 5 μg/plate, dissolved in DMSO / E. coli WP2 uvrA
Evaluation criteria:
Acceptance criteria:
Generally, the experiment is considered valid if the following criteria are met:
• The number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain.
• The sterility controls revealed no indication of bacterial contamination.
• The positive control substances both with and without S9 mix induced a distinct increase in the number of revertant colonies within the range of the historical positive control data or above.
• Fresh bacterial culture containing approximately 10^9 cells per mL were used. For approval the titer of viable bacteria was ≥ 10^8 colonies per mL.

Assessment criteria:
The test substance is considered positive in this assay if the following criteria are met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.

A test substance is 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 resultsopen allclose all
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
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
Increase of revertants at concentrations of 33, 333, 1000, 2500 and 5000 μg/plate (without S9) or at all concentrations (with S9)
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
Increase of revertants at concentrations of 333, 1000, 2500 and 5000 μg/plate
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
at all concentrations
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 of the test substance was found from 333 μg/plate onward with and without S9 mix.

COMPARISON WITH HISTORICAL CONTROL DATA: yes

ADDITIONAL INFORMATION ON CYTOTOXICITY: No bacteriotoxic effect was observed under all test conditions.

Any other information on results incl. tables

Tests without S9 -mix

TA 1535: No relevant increase in the number of his+ revertants.

TA 100: Increase in the number of revertants at 333, 1000, 2500 and 5000 μg/plate (factors 2.2, 3.1, 4.9 and 5.8, respectively).

TA 1537: Increase in the number of revertants at 33, 100, 333, 1000, 2500 and 5000 μg/plate (factors 3.5, 2.9, 4.8, 7.3, 13.4 and

20.8, respectively).

TA 98: Increase in the number of revertants at 33, 100, 333, 1000, 2500 and 5000 μg/plate (factors 12.1, 14.6, 19.5, 26.2, 30.6

and 38.3, respectively).

E. coli WP2 uvrA: No relevant increase in the number of trp+ revertants.

Tests with S9 -mix

TA 1535: No relevant increase in the number of his+ revertants.

TA 100: Increase in the number of revertants at 333, 1000, 2500 and 5000 μg/plate (factors 2.4, 4.0, 5.5 and 5.6, respectively).

TA 1537: Increase in the number of revertants at 33, 100, 333, 1000, 2500 and 5000 μg/plate (factors 5.0, 6.0, 8.9, 11.8, 14.7 and

19.1, respectively).

TA 98: Increase in the number of revertants at 33, 100, 333, 1000, 2500 and 5000 μg/plate (factors 11.8, 13.9. 20.7, 28.8, 33.4

and 35.9, respectively).

E. coli WP2 uvrA: No relevant increase in the number of trp+ revertants.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
positive

The substance is mutagenic in the Ames test.
Executive summary:

The Ames test (OECD 471, GLP) was performed with Salmonella typhimuriam strains TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA with doses between 33 μg - 5000 μg/plate in the standard plate assay with and without metabolic activation (liver S9 mix from induced rats). Precipitation of the test substance was found from 333 μg/plate onward with and without S9 mix. No bacteriotoxic effect was observed under all test conditions. A biologically 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 either with or without S9 mix using tester strains TA 1535 and E.coli WP2uvrA.

A distinct and dose dependent increase in the number of his+ revertants exceeding a factor of 2 compared to the concurrent vehicle control was observed with TA 100 and TA 98 with and without metabolic activation. Using tester strain TA 1537, a distinct and dose depending increase of revertants exceeding a factor of 3 compared to the concurrent vehicle control was observed in the standard plate test with and without metabolic activation.