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:
January 31, 2005 to April 11, 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
This study followed the procedures indicated by the following internationally accepted guidelines and recommendations: "Ninth Addendum to OECD Guidelines for Testing of Chemicals", Section 4, No. 471: "Bacterial Reverse Mutation Test", adopted July 21, 1997 "Commission Directive 2000/32/EC, L1362000, Annex 4D, dated May 19, 2000

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report date:
2005

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Commission Directive 2000/32/EC, L1362000, Annex 4D, dated May 19, 2000
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Methyl 2,3-O-(1-methylethylidene)-5-O-[(4-methylphenyl)sulfonyl]-ß-D-ribofuranoside
EC Number:
609-920-2
Cas Number:
4137-56-8
Molecular formula:
C16H22O7S
IUPAC Name:
Methyl 2,3-O-(1-methylethylidene)-5-O-[(4-methylphenyl)sulfonyl]-ß-D-ribofuranoside
Details on test material:
Identity: Tosylfuranosid
Aggregate state at room temperature: Solid
Colour: White
Stability in solvent: Not indicated by the sponsor
Storage: Room temperature
Expiration Date: July 31, 2005

Method

Target gene:
Salmonella typhimurium
Strains Genotype Type of mutations indicated
TA 1537 his C 3076; rfa-; uvrB-: frame shift mutations
TA 98 his D 3052; rfa-; uvrB-;R-factor frame shift mutations
TA 1535 his G 46; rfa-; uvrB-: base-pair substitutions
TA 100 his G 46; rfa-; uvrB-;R-factor base-pair substitutions

Escherichia coli
Strains Genotype Type of mutations indicated
WP2 uvrA trp-; uvrA-: base-pair substitutions and others

Species / strain
Species / strain / cell type:
other: Salmonella typhimurium, Strains: TA 1537, TA 98, TA 1535, TA 100 and Escherichia coli, Strain WP2 uvrA
Metabolic activation:
with and without
Metabolic activation system:
Mammalian Microsomal Fraction S9 Mix - mammalian microsome enzyme activation mixture.
Test concentrations with justification for top dose:
The test item was tested at the following concentrations:
Pre-Experiment and Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II: 33; 100; 333; 1000; 2500; and 5000 µg/plate
Vehicle / solvent:
DMSO
Controls
Untreated negative controls:
yes
Remarks:
untreated
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Migrated to IUCLID6: additional positive control substances: sodium azide, NaN3, 4-nitro-o-phenylene-diamine, 4-NOPD and 2-aminoanthracene, 2-AA
Details on test system and experimental conditions:
Characterisation of the Salmonella typhimurium Strains and E. coli Strain
The histidine dependent strains are derived from S. typhimurium strain LT2 through a mutation in the histidine locus. Additionally due to the "deep rough" (rfa-minus) mutation they possess a faulty lipopolysaccharide envelope which enables substances to penetrate the cell wall more easily. A further mutation causes a reduction in the activity of an excision repair system. The latter alteration includes mutational processes in the nitrate reductase and biotin genes produced in a UV-sensitive area of the gene named "uvrB-minus". In the strains TA 98 and TA 100 the R-factor plasmid pKM 101 carries the ampicillin resistance marker.
Strain WP2 and its derivatives all carry the same defect in one of the genes for tryptophan biosynthesis. Tryptophan-independent (Trp+) mutants (revertants) can arise either by a base change at the site of the original alteration or by a base change elsewhere in the chromosome so that the original defect is suppressed. This second possibility can occur in several different ways so that the system seems capable of detecting all types of mutagen which substitute one base for another. Additionally, the uvrA derivative is deficient in the DNA repair process (excision repair damage). Such a repair-deficient strain may be more readily mutated by agents.

Regular checking of the properties of the strains regarding the membrane permeability and ampicillin resistance as well as spontaneous mutation rates is performed in RCC Cytotest Cell Research according to B. Ames et al. and D. Maron and B. Ames. In this way it was ensured that the experimental conditions set down by Ames were fulfilled.
The bacterial strains TA 1535, TA 1537, TA 98, TA 100, and WP2 uvrA were obtained from Trinova Biochem GmbH (35394 Gießen, Germany).
Evaluation criteria:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.
Statistics:
According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

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:
not determined
Vehicle controls validity:
valid
Untreated negative 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:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
This study was performed to investigate the potential of Tosylfuranosid to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA.
The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:
Pre-Experiment and Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate Experiment II: 33; 100; 333; 1000; 2500; and 5000 µg/plate
The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments.
No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation.
No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Tosylfuranosid at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher muta-tion rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.
Appropriate reference mutagens were used as positive controls and showed a distinct in-crease of induced revertant colonies.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with and without metabolic activation

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
Therefore, Tosylfuranosid is considered to be non-mutagenic in this Salmonella typhimu-rium and Escherichia coli reverse mutation assay.
Executive summary:

This study was performed to investigate the potential of Tosylfuranosid to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment and Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate Experiment II: 33; 100; 333; 1000; 2500; and 5000 µg/plate

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments.

No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Tosylfuranosid at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher muta-tion rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls and showed a distinct in-crease of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Therefore, Tosylfuranosid is considered to be non-mutagenic in this Salmonella typhimu-rium and Escherichia coli reverse mutation assay.