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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:
1991
Report Date:
1991

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)
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:
- Substance type: pigment, black powder
- Physical state: solid
- Analytical purity: > 99 %
- Storage condition of test material: 4 - 6°C

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 1254 induced rat liver S-9 mix
Test concentrations with justification for top dose:
20, 100, 500, 2500, 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Controlsopen allclose all
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: TA100 and TA1535
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylendiamine
Remarks:
without S9 mix: TA98
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: TA1537
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
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
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
Plate incorporation method:
- Exposure duration: ca. 48 hours at 37°C
Preincubation method:
- Preincubation period: 30 minutes at 30°C
- Exposure duration: 48 hours at 37°C

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

DETERMINATION OF CYTOTOXICITY
reduced his- or trp- background growth
Evaluation criteria:
In general, a substance to be characterized as positive in the bacterial tests has to fulfill the following requirements:
- doubling of the spontaneous mutation rate ( control)
- dose-response relationship
- reproducibility of the results

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:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: from about 2500 µg/plate onward

Any other information on results incl. tables

Standard Plate Test Result:

TA 1535 TA 100 TA 1537 TA 98 WP2uvrA
Dose (µg/plate) -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9
0 10 12 88 102 10 10 27 35 36 47
20 10 13 92 115 11 12 22 35 26 39
100 9 13 74 98 9 10 31 33 29 38
500 15 8 67 94 9 10 29 40 33 37
2500 14 12 90 102 9 9 26 38 29 34
5000 12 16 77 92 9 12 26 41 36 34
2-AA 320 908 121 2350 173
MNNG 1336 1327
AAC 463
NPD 1817
ENNG 199

Preincubation Test Result:

TA 1535 TA 100 TA 1537 TA 98 WP2uvrA
Dose (µg/plate) -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9
0 17 18 116 113 11 11 29 35 22 24
20 17 15 123 120 9 10 24 39 25 28
100 16 18 97 119 9 10 27 42 26 27
500 20 18 79 107 10 12 28 40 23 30
2500 14 17 85 89 10 10 21 40 25 30
5000 16 15 69 81 12 10 22 37 22 24
2-AA 151 661 109 1137 127
MNNG 879 897
AAC 230
NPD 1067
ENNG 399

Controls:

2-AA: 2-aminoanthracene (10 µg/plate for TA strains, 60 µg/plate for WP2uvrA)

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

AAC: 9-aminoacridine (100 µg/plate)

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

ENNG: N-ethyl-N'-nitro-N-nitrosoguanidin (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 Ames test and in the Escherichia coli reverse mutation assay under the experimental conditions chosen here.