Trypsin

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 July 2007 - 28 November 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Principles of method if other than guideline:

The ‘treat and plate’ treatment method was used in each test to avoid the possibility that bio-available histidine in the test item might compromise the test.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Active enzyme protein of trypsin (CAS no. 9002-07-7, EC no. 232-650-8, EC name trypsin, enzyme class 3.4.21.4)

Molecular formula:

n.a.

IUPAC Name:

Active enzyme protein of trypsin (CAS no. 9002-07-7, EC no. 232-650-8, EC name trypsin, enzyme class 3.4.21.4)

Constituent 2

Reference substance name:

Protein as a constituent of enzyme deriving from the fermentation or extraction process

Molecular formula:

Not available

IUPAC Name:

Protein as a constituent of enzyme deriving from the fermentation or extraction process

Constituent 3

Reference substance name:

Inorganic salts as a constituent of enzyme deriving from the fermentation or extraction process

Molecular formula:

Not available. See remarks.

IUPAC Name:

Inorganic salts as a constituent of enzyme deriving from the fermentation or extraction process

Constituent 4

Reference substance name:

Carbohydrates constituent of enzyme deriving from the fermentation or extraction process

Molecular formula:

Not available. See remarks.

IUPAC Name:

Carbohydrates constituent of enzyme deriving from the fermentation or extraction process

Constituent 5

Reference substance name:

Lipids as a constituent of enzyme deriving from the fermentation or extraction process

Molecular formula:

Not available. See remarks.

IUPAC Name:

Lipids as a constituent of enzyme deriving from the fermentation or extraction process

Test material form:

liquid

Details on test material:

- Lot/batch No.: PPF 26813
- Expiration date of the lot/batch: Stable at least until 2017 (10 years).
- pH: 6.2
- Stability under test conditions: The prepared formulations were stable for at least 24 hrs when stored at 4ºC or at room temperature in the dark.
- Storage condition of test material: -18°C

Method

Target gene:

The study was performed to assess the effect of the test material SP 387/TL1 in amino acid dependent strains of Salmonella typhimurium capable of detecting both induced frame-shift (TA1537 and TA98) and base-pair substitution mutations (TA1535 and TA100); and strain of Escherichia coli WP2uvrApKM101 that can detect substitution at AT to GC or in G-C pair. The test system is a reverse mutation of amino acid dependent bacterial strains.

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

S9 from Aroclor 1254

Test concentrations with justification for top dose:

Six doses 156, 313, 625, 1250, 2500 and 5000 μg substance/mL) were tested.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DI water
- Justification for choice of solvent/vehicle: The test 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:

9-aminoacridine

2-nitrofluorene

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 109 cells/mL

DURATION
- Preincubation period: 3 hours
- Exposure duration: Same as preincubation for treat and plate
- Expression time (cells in growth medium): 64 hours

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: Viable cell count

Evaluation criteria:

A test substance is considered as 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 (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

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

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

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

Slight cytotoxicity at the highest conc. (+S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

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

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

Slight cytotoxicity at the highest conc. (+S9)

Vehicle controls validity:

not valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

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:

Weak numerical increases were observed in some of the test series. However they were all without any dose relation and coincide with increases in the viable count due to growth stimulating effect of the test substance.
Cytotoxicity was observed at the highest concentration of 5000 μg substance/mL in experiment 1 in strain TA1537 in the presence of S9; and in experiment 2 in strains TA1537 and TA1535 in the presence of S9.

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: Yes
- Precipitation: Precipitation is a concentration limiting factor, but not an issue in this study
- Definition of acceptable cells for analysis: Viability and gene type control

RANGE-FINDING/SCREENING STUDIES: No

HISTORICAL CONTROL DATA
- Positive historical control data: Yes
- Negative (solvent/vehicle) historical control data: Yes

Applicant's summary and conclusion

Conclusions:

It was concluded that the results of the experiments gave no indication of mutagenic activity of SP 387/TL1, batch PPF26813 in the presence or absence of metabolic activation, up to 5000 µg/mL under the conditions employed in this study.

Executive summary:

SP 387/TL 1 (batch PPF26813) was examined for mutagenic activity in the bacterial reverse mutation assay using Salmonella typhimurium strain TA1535, TA100, TA1537, TA98 and Escherichia coli W P2uvr A. Crude enzyme preparations, like the present batch of SP387/TL 1, contain the free amino acid histidine, most often in an amount, which exceeds the critical concentration for incorporation in the direct standard assay. To overcome this problem all Salmonella typhimurium strains were exposed to SP387/TL 1 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.

Usually the content of tryptophan in enzyme preparations is low and insignificant. Therefore the part of the study comprising Escherichia coli was conducted with the strain WP2uvrA using the direct plate incorporation assay. 6 doses of the test substance were applied with 5 mg (dry matter) per plate as the highest dose level followed by successive bi-sections between doses. 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.

A test substance was considered as positive when it had 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 was dose related and reproducible. If a dose-related numerical increased below a doubling but at least 50% higher than the solvent control was observed then the result was considered as equivocal and would need further clarification.

Cytotoxicity was observed at the highest concentration of 5000 μg substance/mL in experiment 1 in strain TA1537 in the presence of S9; and in experiment 2 in strains TA1537 and TA1535 in the presence of S9. No treatments of any of the Salmonella typhimurium and E. coli strains with SP 387/TL 1, batch PPF26813 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 SP 387/TL1, batch PPF26813 in the presence or absence of metabolic activation, when tested under the conditions employed in this study.