Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 Mar 2004 to 25 Mar 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
July 21, 1997
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
May 19, 2000
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
August 1998
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Appearance: Solid, powder / white
- Homogeneity: The test substance was homogeneous by visual inspection.
Specific details on test material used for the study:
TEST MATERIAL
- Name of the test substance used in the study report: DKDS - Reinkristallisat
- Batch No.: CP 203-362-04-02
- Purity: 96.8%
- Appearance: White powder
- Homogeneity: The homogeneity of the test substance was guaranteed by mixing before preparation of the test substance formulations.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature

The stability of the test substance throughout the study period was not determined analytically. However, the characterization was performed from February 18, 2004 to April 19, 2004, whereas the study was conducted from March 10, 2004 (first experiment) to March 25, 2004 (last experiment). Thus, it can be assumed that the test substance was stable throughout the study period.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The stability of the test substance at room temperature in the vehicle DMSO over a period of 4 hours and in water over a period of 96 hours was verified analytically.

Method

Target gene:
- S. typhimurium: his
- E. coli: trp
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:
- Standard plate test experiment 1: 0 (control), 20, 100, 500, 2500 and 5000 µg/plate
- Standard plate test experiment 2: 0 (control), 250, 500, 1000, 1500 and 2000 µg/plate
- Preincubation test: 0 (control), 93.75, 187.5, 375, 750 and 1500 µg/plate
Vehicle:
Due to the limited solubility 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.
Controls
Negative controls:
yes
Solvent controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
other: 2-aminoanthracene (2-AA), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 4-nitro-o-phenylendiamine (NOPD)
Details on test system and conditions:
- EXPERIMENT 1 and 2:
METHOD OF APPLICATION: in agar (plate incorporation) (SPT)

DURATION
- Exposure duration: 48-72 h at 37°C

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- decrease in the number of revertants
- clearing or diminution of the background lawn (= reduced his or trp background growth)
- reduction in the titer

- EXPERIMENT 3:
METHOD OF APPLICATION: preincubation test (PIT)

DURATION
- Preincubation period: 20 min at 37°C
- Exposure duration: 48-72 h at 37°C

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- 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:
ACCEPTANCE CRITERIA
Generally, the experiment is considered valid if the following criteria are met:
- The number of revertant colonies in the negative controls was within the normal range of the historical control data for each tester strain.
- The sterility controls revealed no indication of bacterial contamination
- The positive control articles both with and without S-9 mix induced a significant increase in the number of revertant colonies within the range of the historical control data or above.
- The titer of viable bacteria was ≥ 10E+08/mL

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 experments carried out independently of each other.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
other: TA 1535, TA 1537, TA 98 and TA 100
Remarks:
SPT
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
A bacteriotoxic effect was observed at doses ≥ 2000 µg/plate
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Key result
Species / strain:
E. coli WP2 uvr A
Remarks:
SPT
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
A bacteriotoxic effect was observed at doses ≥ 2000 µg/plate
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Species / strain:
other: TA 1535, TA 1537, TA 98 and TA 100
Remarks:
PIT
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
A bacteriotoxic effect was observed at doses ≥ 1500 µg/plate
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Species / strain:
E. coli WP2 uvr A
Remarks:
PIT
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Additional information on results:
TOXICITY
A bacteriotoxic effect (reduced his- or trp- background growth, decrease in the number of his+ or trp+ revertants, reduction in the titer) was observed in the standard plate test depending on the strain and test conditions from about 1500 µg - 2000 µg/plate onward. In the preincubation assay bacteriotoxicity was observed depending on the strain and test conditions at doses ≥ 750 µg/plate.

SOLUBILITY
No precipitation of the test substance was found.

Applicant's summary and conclusion

Conclusions:
The substance is not mutagenic in the Ames test.