Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Kaiser-Friedrich-Straße 7, D-55116 Mainz
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: 33470-153-FK

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature.
- Stability under test conditions: The stability of the test substance under storage conditions throughout the study period was guaranteed by the sponsor.
- Solubility and stability of the test substance in the solvent/vehicle: DMSO was used as vehicle due to the limited solubility in water. The stability of the test substance in the vehicle DMSO was not determined analytically. However, the suitability of DMSO as vehicle had been demonstrated in bacterial reverse mutation tests and historical control data are available.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The substance was dissolved in dimethylsulfoxide (DMSO). To achieve a solution of the test substance in the vehicle, the test substance preparation was shaken thoroughly.

FORM AS APPLIED IN THE TEST (if different from that of starting material): solution in DMSO

Method

Target gene:
his gene, trp gene
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 1254-induced rat liver S9 mix
Test concentrations with justification for top dose:
20, 100, 500, 2500 and 5000 μg/plate (SPT)
312.5, 625, 1250, 2500 and 5000 μg/plate (PIT)
In agreement with the recommendations of current guidelines 5 mg/plate or 5 μL/plate are generally selected as maximum test dose at least in the 1st Experiment.
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Due to the limited solubility of the test substance in water, DMSO was used as vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available.
Controls
Negative controls:
other: not applicable
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
other: 2-aminoanthracene (2-AA) with S9 mix for strains: TA 1535, TA 100, TA 1537, TA 98, WP2. N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) without S9 mix for strains: TA 1535, TA 100. 4-nitro-o-phenylendiamine (NOPD) without S9 mix for strain T98.
Details on test system and conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 20 min (PIT)
- Exposure duration: 48 - 72 (SPT and PIT)

DETERMINATION OF CYTOTOXICITY
- Method: Toxicity detected by a decrease in the number of revertants, clearing or diminution of the background lawn (= reduced his- or trp- background growth) and/or reduction in the titer is recorded for all test groups both with and without S9 mix in all experiments
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 S9 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 at least two experiments carried out independently of each other.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
weak bacteriotoxic effect from 2500 ug/plate onwards, precipitation at 500 ug/plate and above
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
weak bacteriotoxic effect from 2500 ug/plate onwards, precipitation at 500 ug/plate and above
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
weak bacteriotoxic effect from 2500 ug/plate onwards, precipitation at 500 ug/plate and above
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
weak bacteriotoxic effect from 2500 ug/plate onwards, precipitation at 500 ug/plate and above
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Species / strain:
E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
weak bacteriotoxic effect from 2500 ug/plate onwards, precipitation at 500 ug/plate and above
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Test substance precipitation was found from about 500 μg/plate onward with and without S9 mix.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: the positive control substances both with and without S9 mix induced a significant increase in the number of revertant colonies within the range of the historical positive control data or above.
- Negative (solvent/vehicle) historical control data: In this study with and without S9 mix, the number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain.

Any other information on results incl. tables

Table 1: Summary of Results

Experiments Metabolic activation Strains Result
Standard plate test Tests without S9 mix TA 1535 No relevant increase in the number of his+ or trp+ revertants.
TA 100
TA 1537
TA 98
E. coli WP2 uvrA
Tests with S9 mix TA 1535 No relevant increase in the number of his+ or trp+ revertants.
TA 100
TA 1537
TA 98
E. coli WP2 uvrA
Preincubation test Tests without S9 mix TA 1535 No relevant increase in the number of his+ or trp+ revertants.
TA 100
TA 1537
TA 98
E. coli WP2 uvrA
Tests with S9 mix TA 1535 No relevant increase in the number of his+ or trp+ revertants.
TA 100
TA 1537
TA 98
E. coli WP2 uvrA

Applicant's summary and conclusion

Conclusions:
The test material is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in absence and presence of metabolic activation.
Executive summary:

The test material was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay.

STRAINS: TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA

DOSE RANGE: 20 μg - 5000 μg/plate (SPT) and 312.5 μg - 5000 μg/plate (PIT)

TEST CONDITIONS: Standard plate test (SPT) and preincubation test (PIT) both with and without metabolic activation (Aroclor-induced rat liver S9 mix).

SOLUBILITY: Precipitation of the test substance was found from about 500 μg/plate onward with and without S9 mix.

TOXICITY: A weak bacteriotoxic effect was occasionally observed depending on the strain and test conditions from about 2500 μg/plate onward.

MUTAGENICITY: 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 S9 mix or after the addition of a metabolizing system.

CONCLUSION: According to the results of the present study, the test material is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay under the experimental conditions chosen here.