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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
"Ninth Addendum to OECD Guidelines for Testing of Chemicals", adopted July 21, 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: ISO-10993-1; 2009 (E) / ISO-10993-3; 2003 (E) / ISO-10993-12, 2007 (E) / EN 7405 (2008) / DIN EN ISO (2009-6)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Reaction mass of 2-{[(2-{3-[2-({[2-(methacryloyloxy)ethoxy]carbonyl}amino)propan-2-yl]phenyl}propan-2-yl)carbamoyl]oxy}propyl methacrylate and 1,3-phenylenebis(propane-2,2-diylcarbamoyloxyethane-2,1-diyl) bis(2-methylacrylate) and 1,3-phenylenebis(propane-2,2-diylcarbamoyloxypropane-2,1-diyl) bis(2-methylacrylate)
EC Number:
943-136-4
Molecular formula:
C26H36N2O8, C27H38N2O8, C28H40N2O8
IUPAC Name:
Reaction mass of 2-{[(2-{3-[2-({[2-(methacryloyloxy)ethoxy]carbonyl}amino)propan-2-yl]phenyl}propan-2-yl)carbamoyl]oxy}propyl methacrylate and 1,3-phenylenebis(propane-2,2-diylcarbamoyloxyethane-2,1-diyl) bis(2-methylacrylate) and 1,3-phenylenebis(propane-2,2-diylcarbamoyloxypropane-2,1-diyl) bis(2-methylacrylate)

Method

Target gene:
The Salmonella typhimurium histidine (his) and the Escherichia coli tryptophan (trp) reversion system measures his- -> his+ and trp- -> trp+ reversions, respectively. The Salmonella typhimurium and Escherichia coli strains are constructed to differentiate between base pair (TA 1535, TA 100, and WP2 uvrA) and frameshift (TA 1537, TA 98) mutations.
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
The histidine dependent strains are derived from Salmonella 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 this way it was ensured that the experimental conditions set down by Ames were fulfilled.
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/ß-naphthoflavone induced rat liver S9 mix
Test concentrations with justification for top dose:
Pre-Experiment/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 test item precipitated in the overlay agar in the test tubes from 1000 to 5000 µg/plate.
Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 2500 to 5000 µg/plate. The undissolved particles had no influence on the data recording.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria.
Controls
Untreated negative controls:
other: Concurrent untreated and solvent controls were performed.
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine (4-NOPD); 2-aminoanthracene (2-AA)
Details on test system and experimental conditions:
METHOD OF APPLICATION: Experiment I was performed as a plate incorporation assay. Since a negative result was obtained in this experiment, experiment II was performed as a pre-incubation assay.

DURATION
- Preincubation period: 60 minutes
- Exposure duration: 48 hours

SELECTION AGENT (mutation assays): histidine

NUMBER OF REPLICATIONS: For each strain and dose level including the controls, three plates were used. 2 experiments were performed.

DETERMINATION OF CYTOTOXICITY
- Method: Toxicity of the test item results in a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.

OTHER: According to the direct plate incorporation or the pre-incubation method the bacteria are exposed to the test item with and without metabolic activation and plated on selective medium. After a suitable period of incubation, revertant colonies are counted.
To establish a dose response effect at least 6 dose levels with adequately spaced intervals were tested. The maximum dose level was 5000 µg/plate.
To validate the test, reference mutagens were tested in parallel to the test item.
Evaluation criteria:
Acceptability of the Assay
The Salmonella typhimurium and Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
- regular background growth in the negative and solvent control
- the spontaneous reversion rates in the negative and solvent control are in the range of historical data
- the positive control substances should produce a significant increase in mutant colony frequencies

Evaluation of Results
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:
Not performed; according to the OECD Guideline 471, a statistical analysis of the data is not mandatory.

Results and discussion

Test results
Key result
Species / strain:
other: 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:
no cytotoxicity
Remarks:
tested up to 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
other: see vehicle control
Positive controls validity:
valid
Additional information on results:
The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in both experiments.
No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), 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 the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.
Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies.

Applicant's summary and conclusion

Conclusions:
The test material was considered to be non-mutagenic under the conditions of this test; in both experiments no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.
Executive summary:

This GLP study according to OECD Guideline 471 and EU Method B.13/14 was performed to investigate the potential of the test item 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/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 (below the indication factor of 0.5), 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 the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). Appropriate reference mutagens were used as positive controls and showed a distinct increase 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 and therefore, is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

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