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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Serine endopeptidase, batch PPA26797 was tested in the AMES assay.

No treatments of any of the S. typhimurium and E.coli strains with serine endopeptidase, batch PPA26797 either in the presence or absence of S-9 mix, resulted in any increases in revertant numbers that meets these criteria for a positive response. It was concluded, that there was no indication of mutagenic activity of serine endopeptidase, batch PPA26797 in the presence or absence of metabolic activation, when tested under the conditions employed in this study in concentrations up to 5000 μg/mL.
Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17-09-2007 to 08-04-2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
July 1997
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine and tryptophan locus in the genome of five strains of bacteria
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Metabolic activation system:
S-9 mix from Aroclor 1254-induced rat liver
Test concentrations with justification for top dose:
Preliminary test: No preliminary trials were carried out.
Six concentrations of the test item, 156, 313, 625, 1250, 2500, 5000 μg/mL.
Vehicle / solvent:
Vehicle for enzyme: Purified water.
Justification for choice of solvent/vehicle: Substance is water-soluble and any human exposure will be in aqueous solutions.
Solvent for the positive control: DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
N-ethyl-N-nitro-N-nitrosoguanidine
other: 2-aminoanthracene, N-Methyl-N'-Nitrosoguanidine
Details on test system and experimental conditions:
METHOD OF APPLICATION: Preincubation in suspension, followed by plating on agar plates (treat and plate method)
- Cell density at seeding (if applicable): Overnight culture of approximately 1x 10^9 cells/mL

DURATION
- Exposure duration, pre-incubation: The incubation mixtures were incubated with shaking at 37 ± 1 °C for 3 hours (treat and plate).
- Incubation time (selective incubation): about 64 hours

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: Viable cell count and observation of the bacterial background lawn. 0.1 mL aliquots of a 10^6 dilution of each bacterial suspension were poured on to minimal glucose agar plates in duplicates.
Evaluation criteria:
The test substance was considered as positive when it has induced at least a doubling in the mean number of revertant colonies per plate compared to the appertaining solvent control in one or more of the strains, in the presence or absence of S9 mix, if this response is dose related (at least 3 doses) and reproducible. If a numerical increase below a doubling is observed the result is considered as equivocal and need further clarification if the increase is dose related and reproducible and it is not accompanied by significant increases in the viable bacterial count.
Statistics:
N/A
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Test with S-9 incorporation but only at the highest dose levels.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Test with S-9 incorporation but only at the highest dose levels.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: Yes
- Precipitation: Precipitation is a concentration limiting factor, but no issues were reported in this study.
- Definition of acceptable cells for analysis: Viability and gene type control.

HISTORICAL CONTROL DATA
- Positive historical control data: Yes. In the second experiment with TA 1535 with S-9 the solvent control just exceeded the range experienced in our laboratory in the near past. However it is well within the corresponding level without S-9 and the level for the standard plate incorporation method as reported in the literature and experienced in our laboratory.
- Negative (solvent/vehicle) historical control data: Yes

Based on the viable counts of the treated culture no distinct toxicity was observed in the present study. Reduced viabilities were observed mainly in tests with S-9 incorporation and with the two base-pair substitution strain TA 100 and TA 1535 of Salmonella but only at the highest dose levels. As a consequence the number of connected revertant colonies was relatively low.

Conclusions:
No treatments of any of the S. typhimurium and E. coli strains with serine endopeptidase, batch PPA26797 either in the presence or absence of S-9 mix, resulted in any increases in revertant numbers that meets these criteria for a positive response. It was concluded, that there was no indication of mutagenic activity of serine endopeptidase, batch PPA26797 in the presence or absence of metabolic activation, when tested under the conditions employed in this study in concentrations up to 5000 μg/mL.
Executive summary:

Serine endopeptidase, batch PPA26797 was examined for mutagenic activity in the bacterial reverse mutation assay using Salmonella typhimurium strain TA 1535, TA 100, TA1537, TA98 and Escherichia coli WP2uvrApKM101. The study was conducted, using the treat and plate assay, in the presence and absence of metabolic activation - a liver preparation from male rats, pre-treated with Aroclor 1254, and

the co-factors required for mixed function oxidase activity (S9 mix). All bacterial strains were tested at concentration of the test article ranging from 156 to 5000 μg per plate. All results were confirmed by conducting two complete and independent experiments.

A test substance is considered positive in this treat and plate assay 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. If a dose related numerical increase below a doubling but at least 50% higher than the solvent control is observed then the result is considered as equivocal and need further clarification.

No treatments of any of the S. typhimurium and E.coli strains with serine endopeptidase, batch PPA26797 either in the presence or absence of S-9 mix, resulted in any increases in revertant numbers that meets these criteria for a positive response.

It was concluded, that the results of the experiments, described in this report, gave no indication of mutagenic activity of serine endopeptidase, batch PPA26797 in the presence or absence of metabolic activation, when tested under the conditions employed in this study.

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

Additional information

Justification for classification or non-classification

Not classified.