Mucorpepsin

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:

1991-10-31 to 1992-01-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Active enzyme protein of mucorpepsin (CAS no.148465-73-0, EC no 642-981-3, enzyme class 3.4.23.23)

Molecular formula:

Not applicable

IUPAC Name:

Active enzyme protein of mucorpepsin (CAS no.148465-73-0, EC no 642-981-3, enzyme class 3.4.23.23)

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:

solid: particulate/powder

Details on test material:

- Lot/batch No.: PPR 3583
- Expiry data of the lot/batch: 2002-12-15

Method

Target gene:

Histidine and tryptophan locus in the genome of four strains of bacteria

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

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: Preliminary and 1. test: 10, 3.3, 1.0, 0.33, 0.10 and 0.03 mg enzyme concentrate dry matter/mL incubation mixture.
Six concentrations of the test item (100, 50, 25, 12.5, 6.3 and 3.1 mg enzyme concentrate dry matter/mL)

Vehicle / solvent:

Vehicle: DMSO
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

Remarks:

purified water

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

other: 2-Aminoanthracene, N-Methyl-N'-NitroNi trosoguanidine, 2- Nitrofluorene

Details on test system and experimental conditions:

METHOD OF APPLICATION: Preincubation in suspension, followed by plating on agar plates (treat and plate method).

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

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: 0.1 mL aliquots of a 10^-6 dilution of each bacterial suspension were poured on to nutrient agar plates. All platings with exposed bacteria were in triplicates and the negative control in 5 plates.

Evaluation criteria:

The test substance was considered as positive when it had 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. In case of a dose related and reproducible numerical increase, which was below a doubling but at least 50% higher than the solvent control, the result was considered as equivocal and would need further clarification.

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

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

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 can be a concentration limiting factor, but not in this study.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: No
- Negative (solvent/vehicle) historical control data: No

Applicant's summary and conclusion

Conclusions:

It was concluded that the results of the experiments described in this report, gave no indication of mutagenic activity of rennilase (Batch No. PPR 3583) in the presence or absence of metabolic activation.

Executive summary:

Rennilase, batch PPR 3583 was examined for mutagenic activity using Salmonella typhimurium strain TA 1535, TA 100, TA 1537 and TA 98. A liquid culture assay was applied. Bacteria were exposed to 6 doses of the test substance in a phosphate buffered nutrient broth for 3 hours. After incubation, the test substance was removed by centrifugation prior to plating. The number of revertants to prototrophy and viable cells were estimated. The test was conducted 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 (S-9 mix).

The sensitivity of the individual bacterial strains was confirmed by significant increases in number of revertant colonies induced in similar conditions by diagnostic mutagens. All results were confirmed by conducting two independent experiments. No dose-related and reproducible increases in revertants to prototrophy were obtained with any of the bacterial strains exposed to rennilase either in the presence or absence of S9-mix.

It was concluded that the results of the experiments described in this report, gave no indication of mutagenic activity of rennilase, batch PPR 3583 in the presence or absence of metabolic activation.