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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report Date:
1998

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
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.
- Physical state: red powder
- Storage condition of test material: room temperature

Method

Target gene:
his and 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
Test concentrations with justification for top dose:
20 - 5000 µg/plate (standard plate test) and 4 - 1000 µg/plate (preincubation test)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Due to the limited solubility/insolubility of the test substance in water, DMSO was selected as the 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: 2-aminoanthracene
Remarks:
with S9 mix; all strains
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
Remarks:
without S9 mix; TA 1535 and TA 100
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
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
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
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
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 S-9 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 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:
- Toxicity: A slight decrease in the number of revertants was occasionally observed.
- Precipitation: Precipitation of the test substance was found from about 100 μg/plate onward.

Any other information on results incl. tables

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 24 37 113 114 19 20 9 10 33 40
20 23 33 101 113 20 20 9 8 27 37
100 23 28 73 107 20 21 9 9 27 35
500 22 26 104 110 19 21 8 9 23 30
2500 22 25 105 108 20 17 7 9 27 31
5000 22 24 98 106 20 19 6 7 31 27
MNNG 1145 1020
2-AA 1327 1058 219 103 291
AAC 777
NOPD 1164
ENNG 873

Results of Experiment II (Pre-incubation 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 28 36 103 112 18 18 10 11 28 35
4 20 29 101 110 14 15 11 11 28 29
20 19 28 105 121 14 15 10 11 28 32
100 18 22 103 101 11 15 9 10 31 31
500 16 24 95 101 11 12 8 10 28 31
1000 14 21 89 104 9 11 9 11 30 33
MNNG 931 1017
2-AA 959 1054 151 176 195
AAC 676
NOPD 675
ENNG 843

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)

ENNG: N-ethyl-N'-nitro-N-nitrosoguanidine (10 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:

According to the results of the present study, the test substance did not lead to an increase in the number of revertant colonies either without S-9 mix or after adding a metabolizing system in two experiments carried out independently of each other (standard plate test and preincubation assay.