Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000

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:
It cannot be entirely excluded that the test material used was the mono potassium salt rather than the di potassium salt.
Specific details on test material used for the study:
It cannot be entirely excluded that the test material used was the mono potassium salt rather than the di potassium salt. However, based on the very similar properties of these compounds, the toxicological data presented is considered valid and suitable to describe the toxicological property of the di potassium salt even if generated with the mono salt.

Method

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:
0; 20; 100; 500; 2500 and 5000 µg/plate (SPT)
0; 4; 20; 100; 500 and 2500 µg/plate (PIT)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: see "details in test system" for positive controls
Details on test system and experimental 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 DMS0

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 / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Test substance precipitation was found from about 100 µg/plate onward.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
A weak bacteriotoxic effect (slight decrease in the number of revertants and/or slight reduction in the titer) was observed depending on the strain and test conditions at doses ≥ 2,500 µg/plate.

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 18 18 107 137 11 13 22 32 32 38
20 16 18 97 133 9 10 24 29 34 37
100 16 16 121 129 7 11 24 30 26 30
500 13 17 120 142 7 7 23 30 25 28
2500 12 17 84 135 3 4 16 24 23 25
5000 8 7 87 104 4 3 10 16 23 31
positive control* 894 195 659 1024 683 166 830 698 696 358

- 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 17 119 117 9 13 27 31 31 38
4 15 16 112 104 11 11 23 23 28 28
20 17 15 116 110 11 9 24 23 28 31
100 12 12 95 112 7 9 27 30 25 28
500 11 14 86 106 5 10 19 25 22 29
2500 11 9 96 110 5 8 17 24 19 27
positive control* 741 115 974 677 477 94 693 628 527 225

*see above for details

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 ccli reverse mutation assay under the experimental conditions chosen here.
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 20 - 5000 µg/plate in the standard plate test and 14 -2500 µg/ml in the preincubation test. Precipitation of the test substance was found from about 100 µg/plate onward. A weak bacteriotoxic effect was observed depending on the strain and test conditions from about 2500 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.