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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:
2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report date:
2008

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
GLP compliance:
yes (incl. QA statement)
Remarks:
BASF Aktiengesellschaft, Experimental Toxicology and Ecology
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
2,9-bis(p-methoxybenzyl)anthra[2,1,9-def:6,5,10-d'e'f']diisoquinoline-1,3,8,10(2H,9H)-tetrone
EC Number:
280-472-4
EC Name:
2,9-bis(p-methoxybenzyl)anthra[2,1,9-def:6,5,10-d'e'f']diisoquinoline-1,3,8,10(2H,9H)-tetrone
Cas Number:
83524-75-8
Molecular formula:
C40H26N2O6
IUPAC Name:
7,18-bis[(4-methoxyphenyl)methyl]-7,18-diazaheptacyclo[14.6.2.2²,⁵.0³,¹².0⁴,⁹.0¹³,²³.0²⁰,²⁴]hexacosa-1(22),2,4,9,11,13(23),14,16(24),20,25-decaene-6,8,17,19-tetrone
Details on test material:
- Physical state: Solid, black
- Storage conditions: 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+ or 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:
rat liver S9-mix (pretreated with phenobarbital iand β-naphthoflavone)
Test concentrations with justification for top dose:
20, 100, 500, 2500 and 5000 μg/plate (standard plate and preincubation test)
Vehicle / solvent:
DMSO
- 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
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
With S9-mix, all strains
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: N-methyl-N'-nitro-N-nitrosoguanidine
Remarks:
Without S9-mix, TA 1535, TA 100
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylenediamine
Remarks:
Without S9-mix, TA 98
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Without S9-mix, TA 1537
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
Without S9-mix, E. coli WP2 uvrA
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
Plate incorporation method:
- Exposure duration: ca. 48-72 hours at 37°C
Preincubation method:
- Preincubation period: 20 minutes at 37°C
- Exposure duration: ca. 48-72 hours at 37°C

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: decrease in the number of revertants, clearing or diminution of the background lawn (= reduced his- or trp- background growth), reduction in the titer
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 experiment 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
Remarks:
a weak bacteriotoxic effect was occasionally observed depending on the strain and test conditions from about 2500 μg/plate onward
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
An increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test either without S9 mix or after the addition of a metabolizing system.

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Test substance precipitation was found from 20 μg/plate onward with and without S9 mix.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
A weak bacteriotoxic effect was occasionally observed depending on the strain and test conditions from about 2500 μg/plate onward. Test substance precipitation was observed from 20 μg/plate onward with and without S9 mix.

Any other information on results incl. tables

Results of the Standard Plate Test:

TA 1535 TA 100 TA 1537 TA 98 WP2uvrA
Dose (µg/plate) -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9
0 15 14 104 111 8 9 26 37 44 38
20 14 14 94 106 7 8 23 34 35 37
100 13 14 107 100 7 8 20 31 33 43
500 11 14 98 105 8 9 20 35 34 45
2500 13 14 94 101 6 6 22 31 34 44
5000 13 12 91 101 5 5 17 19 36 40
2-AA 119 984 167 940 205
MNNG 658 754
AAC 410
NOPD 340
4-NQO 987

Results of the Preincubation Test:

TA 1535 TA 100 TA 1537 TA 98 WP2uvrA
Dose (µg/plate) -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9
0 15 19 96 114 6 7 24 30 46 47
20 15 21 98 101 12 8 22 30 32 40
100 12 23 95 105 9 11 23 26 48 29
500 13 18 104 98 9 8 23 28 38 38
2500 14 18 92 103 8 6 23 19 35 33
5000 9 14 78 81 6 5 16 19 27 35
2-AA 132 952 111 821 190
MNNG 704 1133
AAC 466
NOPD 1035
4-NQO 1367

Controls:

2-AA: 2-aminoanthracene (2.5 µg/plate for all TA strains, 60 µg/plate for WP2uvrA)

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

AAC: 9-aminoacridine (100 µg/plate)

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

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

Applicant's summary and conclusion

Conclusions:
According to the results of the present study, the test substance is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation
assay under the experimental conditions chosen here.
Executive summary:

In a GLP compliant Ames Test based on the OECD testing guideline 471, the test substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of the tester strains TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA. The substance was tested in a standard plate test and in a preincubation test at dose levels ranging from 20 μg - 5 000 μg/plate. Both tests were performed with and without metabolic activation (induced rat liver S9 mix). An increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test either without S9 mix or after the addition of a metabolizing system. Therefore, the test substance is considered to be not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay.