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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06 Dec 2011 - 19 Dec 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Principles of method if other than guideline:
The Prival preincubation test is a modification of the standard Ames reverse mutation assay, in which flavin mononucleotide (FMN), liver S9 mix from uninduced hamsters and a preincubation step are used to facilitate azo reduction and the detection of the resulting mutagenic aromatic amines.
GLP compliance:
yes (incl. certificate)
Remarks:
BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Physical state: solid, red
- Storage condition of test material: room temperature
Test materials used in this dossier are all considered to fall under the definition of nano-materials according to the European Commission Recommendation 2011/696/EU as the synthesis and manufacturing of this pigment always yields particulate material with a fine particle size distribution.

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
Metabolic activation:
with and without
Metabolic activation system:
Aroclor-induced rat liver S-9 mix and uninduced hamster liver S-9 mix
Test concentrations with justification for top dose:
33 μg - 5 000 μg/plate (SPT)
33 μg - 5 000 μg/plate (PIT)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Due to the limited solubility 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
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: with rat S9 (SPT): 2-aminoanthracene (all strains) - with hamster S9 (PIT): 2-aminoanthracene (all strains), Congo red (TA98), benzidine (TA98)
Remarks:
with S9 mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: N-methyl-N'-nitro-N-nitrosoguanidine (TA1535, TA100), 4-nitro-o-phenylendiamine (TA98), 9-aminoacridine (TA1537), 4-nitroquinoline-N-oxide (E. coli WP2 uvrA)
Remarks:
without S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); Prival preincubation
The Prival preincubation test is a modification of the standard Ames reverse mutation assay, in which flavin mononucleotide (FMN), liver S9 mix from uninduced hamsters and a preincubation step are used to facilitate azo reduction and the detection of the resulting mutagenic aromatic amines

DURATION
- Preincubation period: 30 min
- Exposure duration: 48 – 72 hours

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Evaluation criteria:
The test chemical 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 results
Species / strain:
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:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Test substance precipitation was found from 333 μg/plate onward with and without S9 mix.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
A weak bacteriotoxic effect (slight decrease in the number of his+ revertants) was occasionally observed in the Ames standard plate test depending on the strain and test conditions from 2500 μg/plate onward. In the Prival preincubation assay weak bacteriotoxicity (slight decrease in the number of his+ or trp+ revertants) was observed depending on the strain and test conditions at 5000 μg/plate.

Any other information on results incl. tables

According to the results of the present study, the test substance did not lead to a biologically relevant increase in the number of revertant colonies either without S9 mix or after adding a metabolizing system in two experiments carried out independently of each other (Ames standard plate test and Prival preincubation assay). Besides, the results of the negative as well as the positive controls performed in parallel corroborated the validity of this study, since the values fulfilled the acceptance criteria of this study. 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 the range of the historical positive control data or above.

Results of Experiment I (Standard Plate Test with and without rat liver S-9 mix):

TA98 TA100 TA1535 TA1537 WP2 uvrA
concentration -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9
DMSO 20 22 79 84 11 14 6 7 47 48
33 19 23 76 82 11 15 6 7 45 51
100 19 24 79 78 13 15 6 8 44 53
333 21 21 87 81 10 15 6 6 43 49
1000 20 25 86 75 12 16 6 8 19 49
2500 17 19 74 78 11 14 5 5 46 52
5000 20 16 73 70 14 14 4 5 47 47
MNNG 675 1176
2-AA 864 790 174 123 237
AAC 363
NOPD 531
4-NQO 608

Controls:

MNNG: N-methyl-N'-nitro-N-nitrosoguanidine (5 µg/plate)

2 -AA: 2-aminoanthracene (2.5 µg/plate for TA 98, 100, 1535, 1537; 60 µg/plate for WP2 uvrA)

AAC: 9-aminoacridine (100 µg/plate)

NOPD: 4-nitro-o-phenylendiamnine (10 µg/plate)

4 -NQO: 4-nitroquinoline-N-oxide (5 µg/plate)

Results of Experiment II (Pre-incubation Test with and without hamster liver S-9 mix):

TA98 TA100 TA1535 TA1537 WP2 uvrA
concentration -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9
DMSO 28 40 120 133 16 18 8 10 51 51
33 29 36 125 1217 16 17 8 11 45 52
100 26 38 113 130 16 16 8 13 49 46
333 26 31 123 130 18 20 8 9 50 46
1000 26 34 119 117 16 17 6 10 50 47
2500 26 31 118 115 19 16 7 9 39 57
5000 21 37 105 102 14 13 7 10 49 38
MNNG 854 740
2-AA 852 1695 742 142 252
AAC 478
NOPD 708
4-NQO 235
CONGOR. 639
BENZID. 655

Controls:

MNNG: N-methyl-N'-nitro-N-nitrosoguanidine (5 µg/plate)

2 -AA: 2-aminoanthracene (10 µg/plate)

AAC: 9-aminoacridine (100 µg/plate)

NOPD: 4-nitro-o-phenylendiamnine (10 µg/plate)

4 -NQO: 4-nitroquinoline-N-oxide (5 µg/plate)

CONGOR: Congo red (0.3 µmol/plate)

benzid: benizidine (0.3 µmol/plate)

Applicant's summary and conclusion

Conclusions:
Under the experimental conditions chosen here, it is concluded that the test article is not a mutagenic substance in the bacterial reverse mutation test (Ames standard plate test and Prival preincubation 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 Salmonella typhimurium strains TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA in a reverse mutation assay (Ames standard plate test and Prival preincubation test). The Prival preincubation test is a modification of the standard Ames reverse mutation assay, in which flavin mononucleotide (FMN), liver S9 mix from uninduced hamsters and a preincubation step are used to facilitate azo reduction and the detection of the resulting mutagenic aromatic amines .This test is therefore the most appropriate method for the investigation of azo-dyes and diazo compounds such as the test compound. Both assays were performed in the presence and absence of a metabolic activation system at a concentration range of 33 – 5000 µg/plate. Precipitation of the test substance was found from a concentration of 333 μg/plate onward with and without S9 mix. A weak bacteriotoxic effect (slight decrease in the number of his+ revertants) was occasionally observed in the Ames standard plate test depending on the strain and test conditions from 2500 μg/plate onward. In the Prival preincubation assay weak bacteriotoxicity (slight decrease in the number of his+ or trp+ revertants) was observed depending on the strain and test conditions at 5000 μg/plate. According to the results of the present study, the test substance did not lead to a biologically relevant increase in the number of revertant colonies either without S9 mix or after adding a metabolizing system in two experiments carried out independently of each other (Ames standard plate test and Prival preincubation assay). Besides, the results of the negative as well as the positive controls performed in parallel corroborated the validity of this study, since the values fulfilled the acceptance criteria of this study. 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 the range of the historical positive control data or above. Thus, under the experimental conditions of this study, the test substance is not mutagenic in the Ames standard plate test and in the Prival preincubation test in the absence and the presence of metabolic activation.