Registration Dossier

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:
1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and guideline compliant

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)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes
Remarks:
Department of Toxicology of BASF Aktiengesellschaft, D-67056 Ludwigshafen/Rhein, GER
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Physical state: Darkred powder
- Storage condition of test material: Room temperature

Method

Species / strain
Species / strain:
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:
0; 21; 105; 525; 2,625 and 5,250 µg/plate (SPT)
0; 21; 105; 525; 2,625 and 5,250 µg/plate (PIT)
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO;
- Justification for choice of solvent/vehicle: Limited solubility
Controls
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
see "details in test system" for positive controls
Details on test system and conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 20 minutes (only PIT)
- Incubation duration: 48-72 h at 37°C in the dark

NUMBER OF REPLICATIONS: triplicates

DETERMINATION OF CYTOTOXICITY
- Method: decrease in revertants, background growth, reduction in the titer

POSITIVE CONTROLS With S-9 MIX
- TA 1535, TA 100, TA 1537, TA 98: 2-amninoanthracene (2-AA), 2.5 µg/plate, dissolved in DMSO
- E. coli WP2 uvrA: 2-amninoanthracene (2-AA), 60 µg/plate, dissolved in DM80

POSITIVE CONTROLS Without S-9 MIX
- TA 1535: TA 100: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 5 µg/plate, dissolved in DMSO
- TA 98: 4-nitro-o-phenylendiamine (NOPD), 10 µg/plate, dissolved in DMSO
- TA 1537: 9-aminoacridine (AAC), 100 µg/plate, dissolved in DMSO
- E. coli WP2 uvrA: 4-nitroquinoline-N-oxide (4-NQO), 5 µg/plate, dissolved in DMSO
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 nonmutagenic 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:
yes
Remarks:
A weak bacteriotoxic effect was occasionally observed from 2625 µg/plate and higher
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Test substance precipitation was found from about 525 µg/plate onward.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
A weak bacteriotoxic effect (slight decrease in the number of revertants, slight reduction in the titer) was occasionally observed depending on the strain and test conditions from about 2,625 µg/plate onward.

Any other information on results incl. tables

EXPERIMENTAL RESULT

- 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
solvent control 20 21 107 122 10 10 24 39 27 33
21 16 19 111 146 8 10 21 33 25 33
105 18 17 109 150 8 10 22 31 26 28
525 16 14 114 124 8 8 26 34 27 31
29625 13 15 98 130 6 8 23 30 25 26
5250 8 12 90 112 5 6 19 22 22 24
positive control* 679 182 787 1326 716 169 928 1097 657 180

- Preincubation Test

TA 1535 TA 100 TA 1537 TA 98 WP2uvrA
Dose (µg/plate) -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9
solvent control 17 13 101 112 9 10 20 27 36 34
21 12 17 97 102 8 8 22 25 39 27
105 13 14 106 102 8 9 22 26 40 24
525 12 9 98 114 8 7 19 20 36 25
29625 8 10 98 120 7 9 19 23 38 24
5250 10 11 79 98 7 9 19 23 32 22
positive control 1360 263 1628 910 759 104 547 748 1530 267

*see above for details

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Under the experimental conditions chosen here, it is concluded that the test article is not a mutagenic agent in a bacterial reverse mutation test.
in vitro.
Executive summary:

The test substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains in a reverse mutation assay. The tester strains (TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA) were tested in a standard plate test (SPT) and preincubation test (PIT) both with and without metabolic activation (Aroclor-induced rat liver S-9 mix). The dose range used was 21 - 5250 µg/plate in both assays. Precipitation of the test substance was found from about 525 µg/plate onward. A weak bacteriotoxic effect was occasionally observed depending on the strain and test conditions from about 2,625 µg/plate onward. 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 S-9 mix or after the addition of a metabolizing system. According to the results of the present study, the test substance is not mutagenic in the Salmonella typhimurium/Escherichia ccli reverse mutation assay under the experimental conditions chosen here.