Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The genetic toxicity of subtilisin has been investigated in the Ames Assay, the In VitroChromosome Aberration Assay and theIn VitroMouse Lymphoma test. All tests have been performed according to OECD and EC guidelines, and in compliance with GLP and of high quality (Klimisch 1). No evidence for genetic toxicity was observed and thus it can be concluded that subtilisin is not mutagenic and does not induce chromosome aberration in the present test systems.


Justification for selection of genetic toxicity endpoint
No study was selected as all in vitro studies were negative.

Short description of key information:
No mutagenic activity of subtilisin could be detected in the Ames Assay, in the Mouse Lymphoma test, and no chromosomal aberrations were induced in the in vitro Mammalian Chromosome Aberration test performed with human lymphocytes - both with and without metabolic activation.

Endpoint Conclusion:No adverse effect observed (negative)

Link to relevant study records

Referenceopen allclose all

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
Qualifier:
equivalent or similar to guideline
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. QA statement)
Type of assay:
bacterial reverse mutation assay
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 / 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.
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.
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'.
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.

 

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Aug. 23 - Oct. 31, 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
1997
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
At chromosomal level.
Species / strain / cell type:
lymphocytes: human
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Highest concentration tested was 5000 µg/mL and dilutions hereof.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Sterile purified water
- Justification for choice of solvent/vehicle: Substance is water-soluble and any human exposure will be in aqueous solutions.
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
other: 4-Nitroquinoline 1-oxide
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in suspension

DURATION
- Exposure duration: 3 hours (first experiment, in absence or presence S9); 3 hours (second experiment, in presence S9); 20 hours (second experiment, in absence S9)
- Treatment plus recovery time: 20 hours

SPINDLE INHIBITOR (cytogenetic assays): Colchicine
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: Preliminary trial and two independant replicates.

NUMBER OF CELLS EVALUATED: a total of 200 cells per dose level

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
- Other: Hyperdiploid cells

Evaluation criteria:
A test article is considered as positive in this assay if:
1) the proportions of cells with structural aberrations at one or more concentration exceeds the normal range in both replicates, and
2) a statistically significant increase in the proportion of cells with structural aberrations (excluding gaps) occurs at these doses.
Statistics:
Fisher's exact test, p≤0.05 significant.
Heterogeneity between replicates evaluated by means of a binomial dispersion test, p≤0.05 significant.
Key result
Species / strain:
lymphocytes: human
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:
TEST-SPECIFIC CONFOUNDING FACTORS
Subtilisin is a proteolytic enzyme which implies, that the test substance has the potential of breaking down the metabolising system S9. In a preliminary study it was demonstrated, that the test material inactivated S9 significantly. Therefore, the main study was conducteds with heat-inactivated test substance.

RANGE-FINDING/SCREENING STUDIES: yes

COMPARISON WITH HISTORICAL CONTROL DATA:
Cells treated with Subtilisin, either in the absence of S-9, had similar numbers of abberrations to those observed in concurrent solvent controls. There were no reproducible increases in aberration frequency that were significantly higher than those observed in the negative controls.
The negative controls were withing the negative control ranges.
Normal frequencies of cells with numerical aberrations were seen under all treatment conditions.
The positive controls induced satisfactory levels of aberrations.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No significant cytotoxicity, however 30% reduction of mitotix index at the highest dose level at 20 hours treatment.
Conclusions:
Subtilisin, Batch No. PPA 6865, under the conditions of the test, did not induce chromosome aberrations in cultured human peripheral blood lymphocytes when tested to 5000 mg/mL in either the absence and presence of S-9.
Executive summary:

The clastogenic potential of Subtilisin, Batch No. PPA 6865 was evaluated by its effect on chromosomes of human peripheral blood lymphocytes according to OECD guideline 473 (July 1997). Subtilisin is a proteolytic enzyme which implies, that the test substance has the potential of breaking down the metabolising system S9. Therefore, the main study was conducted with heat-inactivated test substance. Heparinized whole blood cultures from three male donors were established, and division of the lymphocytes was stimulated by adding phytohaemagglutinin to the cultures.

Two independent experiments were performed both in the absence and presence of metabolic activation by a rat S-9 mix induced with Aroclor.

Sets of duplicate cultures were treated with the solvent (sterile purified water), test substance or positive controls (-S-9: 4-Nitroquinoline 1-oxide, +S-9: Cyclophosphamide). Treatments with subtilisin covered a broad range of doses, where the highest dose level used was 5000 ug/mL.

In the first experiment, the lymphocyte cultures were exposed to the test substance in the absence or presence of S-9 for three hours and cells were harvested 17 hours later. The second experiment included a continuous exposure for 20 hours in the absence of S-9. The test article dose levels for chromosome analysis were selected by evaluating the effect of Subtilisin on mitotic index.

Chromosome aberrations were analysed at three consecutive dose levels. Cells were arrested in metaphase by colchicine and after centrifugation and hypotonic treatment, metaphase spreads were prepared and stained with Giemsa. A total of 200 cells were scored per dose level (100 from each replicate culture) from subtilisin treatments and negative controls.

The proportion of cells with structural aberrations in all cultures of the solvent controls (purified water) was within the limits of the historical ranges. The positive controls induced statistically significant increases in the proportion of cells with structural aberrations, thus demonstrating the sensitivity of the test procedure and the metabolic activity of the S-9 mix employed.

Cells treated with subtilisin, either in the absence and presence of S-9, had similar numbers of aberrations to those observed in concurrent solvent controls.

Subtilisin, batch No. PPA 6865,did not induce chromosome aberrations in cultured human blood lymphocytes when tested up to a concentration of 5000 ug/mL in either the absence or presence of S-9.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Dec. 12 1990 - Feb. 12, 1991
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
1984
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay
Target gene:
HPRT (6-thioguanine resistance)
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: Fischer's Medium 10 (10% horse serum)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Highest concentration tested was 5000 µg/mL and dilutions hereof.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Distilled water
- Justification for choice of solvent/vehicle: Substance is water-soluble and any human exposure will be in aqueous solutions.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 4-nitroquinoline-1-oxide, benzo(a)pyrene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; growth in suspension; selection phase is performed in microtitre plates

DURATION
- Exposure duration: 2 hours
- Expression time (cells in growth medium): 7days
- Selection time (if incubation with a selection agent): At the end of the expression time, the culteres were counted and diluted appropriately and placed into microtitre wells. Incubation performed until scorable


SELECTION AGENT : 6-TG

NUMBER OF REPLICATIONS: Preliminary trial and two independant replicates.

DETERMINATION OF CYTOTOXICITY
- Method: Cell density by counting viable cells, expressed as relative survival

Evaluation criteria:
A test article was considered positive if:
- The assay was valid, and
- Significant induced mutation (i.e the lower 95 percentile of a treated culture exceeded the upper 95 percentile of a control culture) occurred at consecutive doses in at least one experiment, and
- Dose-related increases in mutation could be confirmed by regression analysis in both experiments.
Statistics:
The mutation frequency was evaluated statistically by using logaritthmic tranformation of the variances of the number of clones observed on viability and mutation plates as described by E.E. Furth et al., Anal Biochem 110: 1-8, 1981
Key result
Species / strain:
mouse lymphoma L5178Y cells
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:
TEST-SPECIFIC CONFOUNDING FACTORS
Subtilisin is a proteolytic enzyme which implies, that the test substance has the potential of breaking down the metabolising system S9. In a preliminary study it was demonstrated, that the test material inactivated S9 significantly. Therefore, the study was conducted with heat-inactivated test substance in the presence of S-9.
- Water solubility: yes



RANGE-FINDING/SCREENING STUDIES: Preliminary range finder performed

COMPARISON WITH HISTORICAL CONTROL DATA:
Cells treated with Subtilisin, either in the absence and presence of S-9, had similar mutation frequencies as those observed in concurrent solvent controls. The negative controls were within the historical negative control ranges.



Conclusions:
Subtilisin, Batch No. PPA 3366, under the conditions of the test, had no mutagenic activity in cultured mouse lymphoma cells when tested to a concentration of 5000 ug/mL in either the absence or presence of S-9.
Executive summary:

Subtilisin batch PPA 3366 was assayed for its ability to induce mutation at the HPRTlocus (6-thioguanine resistance) in mouse lymphoma cells using a fluctuation protocol. The study consisted of two independent experiments, each conducted in the absence and presence of metabolic activation (S-9 mix). A preliminary experiment established that Subtilisin (a protease) inactivated the enzymes of the S-9 mix. The positive control substance benzo(a)pyrene did not give a positive mutagenic response in the presence of Subtilisin and S-9. Therefore Subtilisin was inactivated for all treatments in the presence of S-9. Following a wide range of treatments, separated by half-log intervals and reaching 5000 ug/mL, cells survived all doses of Subtilisin showing 91% and 128% relative survival in the absence and presence of S-9 respectively, at the top dose. This dose together with the next four lower doses, was plated for viability and 6-thioguanine resistance 7 days after treatment. In the second experiment a narrower dose range was used to maximise the chance of detecting any dose related effects. The top dose plated in this experiment was again 5000 ug/mL in the absence and presence of S-9, which yielded 86% and 99% survival, respectively. Negative (solvent) and positive control treatments were included in each experiment in the absence and presence of S-9. Mutation frequencies in negative control cultures fell within normal ranges, and statistically significant increases in mutation were induced by the positive control chemicals 4-nitroquinoline 1-oxide (without S-9) and benzo(a)pyrene (with S-9). Therefore the study was accepted as valid. No Subtilisin treatment, either in the absence or presence of S-9, resulted in a statistically significant increase in mutation frequency. Therefore when tested up to 5000 ug/mL, Subtilisin failed to induce mutation at the HPRT locus of L5178Y mouse lymphoma cells, both in the absence and presence of S-9 (active enzyme tested in the absence of S-9 and inactivated enzyme in the presence of S-9). It was concluded that Subtilisin, under the conditions employed in this study, had no mutagenic activity in this test system.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Justification for classification or non-classification

Due to the lack of genetic toxicity subtilisin is not classified.