Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

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:
Sept. 18 - Nov. 10, 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
Principles of method if other than guideline:
Crude enzyme preparations, like the present batch of Subtilisin contain the free amino acid histidine and tryptophan, most often in an amount, which exceeds the critical concentration for incorporation in the direct standard assay.
To overcome this problem, all strains were exposed to Subtilisin in liquid culture (“treat and plate assay”).
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
liquid
Details on test material:
- Substance type: UVCB
- Physical state: Liquid
- Lot/batch No.: PPA 28009
- Purity test date: 2008-04-30
- Expiration date of the lot/batch: 2018-04-30, i.e. at least 10 years from production, or as long as enzyme activity is preserved
- Stability under test conditions: Solutions in water are stable for at least 24 hours at room temperature or 4 degrees Celcius
- Storage condition of test material: 4 degrees of C

Method

Target gene:
The study describes experiments performed to assess the effect of subtilisin in amino acid dependent strains of Salmonella typhimurium and Escherichia coli capable of detecting both induced frame-shift (TA1537 and TA98) and base-pair substitution mutations (TA1535, TA100, and WP2uvrA). The test system is a reverse mutation of amino acid dependent bacterial strains.
Species / strain
Species / strain / cell type:
bacteria, other: Salmonella typhimurium TA1537, TA98, TA1535, TA100, Escherichia coli WP2uvrA
Metabolic activation:
with and without
Metabolic activation system:
S9 from Aroclor 1254 induced Spraque Dawley rats obtained from MP Biomedicals, LLC. 29525 Fountain Parkway Solon, Ohio 44139.
Test concentrations with justification for top dose:
5000 µg test substance (dry matter) per ml and 2-fold dilutions, with and without the metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: sterile deionised water
- Justification for choice of solvent/vehicle: substance is water-soluble and any human exposure will be in aqueous solutions.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: N-Methyl-N'-nitro-N-nitrosoguanidine, 2-aminoanthracene, 2-nitrofluorene, 9-aminoacridine
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium before plating, i.e a liquid culture assay (treat and plate assay).

DURATION
- Exposure duration: 3 hours
- Incubation time (selective incubation) : 64 hours

DETERMINATION OF CYTOTOXICITY
- Method: Viable cell count

Evaluation criteria:
A test substance is regarded as positive when it has induced at least a doubling in the mean number of revertants per plate compared to the appropriate solvent control in one or more of the strains, in the presence or absence of S9, if this response is dose related and reproducible.
Statistics:
No statistics performed.

Results and discussion

Test results
Key result
Species / strain:
bacteria, other: as specified above
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The test material is a fluid enzyme preparation. It contains an abundance of various nutrients, and composes a rich growth medium to the test bacteria. This means, that comparison of viable counts between exposed cultures and control culture in a “treat and plate” assay reflects growth stimulation/inhibition as well as cell killing. It is our experience, that in a treat and plate assay, where bacteria are exposed to different doses of such a test substance in separate liquid cultures for a certain time, the spontaneous revertant levels fluctuate more than in the direct "plate incorporation assay."
No significant toxicity was evident in the majority of the tests with and without metabolic activation. Reduced viabilities are evident at the three highest doses in tests with TA100 and TA1535 without the presence of S-9, mainly in the first experiment. These observations have no significant influence on the overall evaluation of the results.
No treatments of any of the Salmonella and E.coli strains with subtilisin resulted in any increases in revertant numbers that meets these criteria for a positive or equivocal response.
The results of the controls in the study were within the historical ranges.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
No indication of the presence of mutagenic components in the test material with and without metabolic activation.
Executive summary:

Subtilisin (Batch Number: PPA 28009) was examined for mutagenic activity in the bacterial reverse mutation assay using Salmonella typhimurium strain TA1535, TA100, TA1537, TA98 and Escherichia coli WP2uvrA.

Crude enzyme preparations, like the present batch contain the free amino acid histidine and tryptophan, which exceeds the critical concentration for incorporation in the direct standard assay. To overcome this problem all strains were exposed to PPA 28009 in liquid culture (“treat and plate assay”).

Bacteria were exposed to 6 doses of the test substance in a phosphate buffered nutrient broth for 3 hours with 5 mg (dry matter) per mL as highest concentration. After incubation the test substance was removed by centrifugation prior to plating.

The study was conducted with and without the metabolic activation system S9 - a liver preparation from male rats, pre-treated with Aroclor 1254, and the co-factors required for mixed function oxidase activity (S9 mix).

Two identical and independent experiments were conducted.

The treatment of the Salmonella and E.coli strains with Batch PPA 28009, in the presence or absence of S9 mix, did not result in any increases in revertant numbers. Batch number PPA 28009 was found not mutagenic.