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Toxicological information

Genetic toxicity: in vitro

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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:
2002-12-10 until 2003-01-16
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP - Guideline study
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Uvinul A Plus
- Physical state and appearance: YeIIow, pasty melt
- Analytical purity: 97.9 weight %
- Lot/batch No.: 309561121D2+1122D stripped
- Stability under test conditions and homogeneity: The stability of a comparable batch (R323/681) was verified analytically at room temperature in the vehicle DMS0 over a period of 4 hours and in water over a period of 96 hours . The homogeneity of the test substance was guaranteed by heating up to 60°C before preparation of the test substance formulations and on account of the high purity.
- Storage condition of test material: Room temperature

Method

Target gene:
For Salmonella typhimurium strains, the amino acid histidine locus is the target gene, for the E.coli WP2 uvrA strain the amino acid tryptophan locus is the target gene.
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
other: All strains have a defective excision repair system (uvrB) which results in greatly enhanced sensitivity to some mutagens and considerably reduced hydrophilic polysaccharide layer (rfa), which Ieads to an increase in permeability to lipophilic substances.
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
other: Deficient excision repair.
Metabolic activation:
with and without
Metabolic activation system:
S9- mix
Test concentrations with justification for top dose:
In the standard plate test (STP): 0; 20; 100; 500; 2500 and 5000 µg/plate
In the preincubation test (PIT): 0; 4; 20; 100; 500 and 2500 µg/plate
Vehicle / solvent:
- 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 selected as the vehicle. It has already been demonstrated that DMSO is to be suitable to be used in bacterial reverse mutation tests and historical control data exists.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2- aminoanthracene (2-AA)
Remarks:
2-aminoanthracene (2-AA) was used in all strains in the presence of S-9 mix.
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
N-ethyl-N-nitro-N-nitrosoguanidine was used in the TA 1535 and in the TA 100 strains without the presence of S-9 mix.
Positive control substance:
9-aminoacridine
Remarks:
9-aminoacridine was used in the TA 1537 strain without the presence of S-9 mix.
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
4-nitroquinoline-N-oxide was used in the E.coli strain without the presence of S-9 mix.
Positive control substance:
other: 4-nitro-o-phenylendiamine (NOPD)
Remarks:
4-nitro-o-phenylendiamine was used in the TA 98 strain without the presence of S-9 mix.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); in suspension (preincubation)

DURATION
- Preincubation period: In the plate incorporation plate test (standard plate test) there is no preincubation period (with the test substance). In the preincubation test: 20 min., before the mixture of the bacteria, test substance and medium are poured to the selective minimal agar.

SELECTION AGENT (mutation assays): Minimal agar without histidine or tryptophan.

NUMBER OF REPLICATIONS: 3

Indication for mutagenicity: An increase in the number of revertants (his+), trp(+).

DETERMINATION OGF CYTOTOXICITY:
3. Toxicity is detected by a:
- decrease in the number of revertants
- clearing or diminution of the background lawn
- reduction in the titer
All these parameters were recorded for all test groups both with and without S-9 mix in all experiments.
Evaluation criteria:
1. The test chemical is considered positive in this assay if the following criteria is 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 with or without S-9 mix.
2. A test substance is generally considered no mutagenic in this test if:
The numbers of revertants for all tester strains were within the historical negative control range under all experimental conditions in two experiments which were carried out independently of each other.

Statistics:
The mean number of revertant colonies per plate, titer, and the standard deviations were calculated for all dose groups as well as for the positive and negative (vehicle) controls in all experiments.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: A slight decrease in the number of revertants, was occasionally observed from about 2500 µg/plate onward.
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: A slight decrease in the number of revertants, indicating cytotoxicity, was occasionally observed from about 2500 µg/plate onward.
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS:
- Precipitation: Precipitation of the test substance was found from about 100 µg/plate onward. As long as precipitation did not interfere with the colony scoring, 5 mg/plate was generally selected and analyzed as the maximum dose (in the standard plate test).

Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

According to the results of the present study, the test substance Uvinul A Plus is not mutagenic in the Salmonella typhimurium/ Escherichia coli reverse mutation assay (in both tests- standard plate test and preincubation test) under the experimental conditions given.

Applicant's summary and conclusion