Mucorpepsin

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The genotoxic potential of mucorpepsin was tested in the Ames, chromosome aberration and mouse lymphoma assay.

- Mucorpepsin, batch PPR 3583 was examined for mutagenic activity using Salmonella typhimurium strain TA 1535, TA 100, TA 1537 and TA 98. It was concluded that the results of the experiments described in this report, gave no indication of mutagenic activity of mucorpepsin, batch PPR 3583 in the presence or absence of metabolic activation.

- Mucorpepsin was tested in an in vitro cytogenetics assay using duplicate human lymphocyte cultures from a male and female donor in 2 independent experiments. It is concluded that mucorpepsin did not induce chromosome aberrations in cultured human peripheral blood lymphocytes when tested to 5000 μg/mL (equivalent to 4490 μg enzyme concentrate dry matter/mL) in both the absence and presence of S-9.

- The study to determine the ability of mucorpepsin to induce mutations at the thymidine kinase (tk) locus in mouse lymphoma L5178YT cells using a fluctuation assay. It is concluded that rennilase, under the conditions employed in this study, has no mutagenic activity in the presence and absence of S-9.

Therefore, mucorpepsin did not show any genotoxic potential.

Link to relevant study records

Reference

Reference 1

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

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

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

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.

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.

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

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:

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

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.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22-07-1991 to 31-12-1991

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: UKEMS Test Guidelines

Qualifier:

according to guideline

Guideline:

other: EEC Annex V Test B 10.

GLP compliance:

yes

Type of assay:

other: chromosome aberrations in cultured blood lymphocytes

Species / strain / cell type:

lymphocytes: Primary cells from human blood

Details on mammalian cell type (if applicable):

From a male and female donor.

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

N/A

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Highest concentration tested was 5000 µg test item/mL and dilutions hereof. The OECD TG 473 (in effect at the time of this study) recommended a maximum test concentration for soluble non-cytotoxic substances of 5000 µg/plate (mL). Thus the test item was tested as 5000 µg as is/mL maximum dose, equivalent to 4490 µg enzyme concentrate dry matter/mL, fulfilling the recommendation.

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:

4-nitroquinoline-N-oxide

cyclophosphamide

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 hours
- Exposure duration: 3 hours
- Expression time (cells in growth medium): Cultures were incubated for an addiational 17 or 41 hours before harvesting.
- Fixation time: Approximately 1.5 hours prior to harvest, colchicine was added to arrest dividing cells in metaphase. Cultures were then cetrifuged and resuspended until the supernatants were clear (40+ min).

STAIN (for cytogenetic assays): Giemsa stain in pH 6.8 buffer.

NUMBER OF REPLICATIONS: 2 replicates.

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Cells were pelleted and resuspended in a minimal amount of fresh fixative (approximately 0.2 mL) so as to give a milky suspension. Several drops of 45% (v/v) aqueous acetic acid were added to each suspension to enhance spreading, and several drops of suspension were dropped on to clean microscope slides which had been dipped in water. After the slides had dried the cells were stained for 5 minutes in 4% (v/v) filtered Giemsa stain in pH 6.8 buffer. The slides were rinsed, dried and mounted with coverslips.

NUMBER OF CELLS EVALUATED: 200 (100 per replicate)

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
The test chemical was to be considered as clearly positive in this assay if:
1) statistically significant increases in the proportion of structurally aberrant cells (without gaps) occurred at one or more concentrations
2) the proportion of aberrant cells at such data points exceeded the normal range
3) the results were confirmed in the second independent experiment.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

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 replicate cultures, and
2. a statistically significant increase in the proportion of cells with structural aberrations (excluding gaps) occurs at these doses.

Acceptance criteria
The human lymphocyte assay was to be considered valid if the following criteria were met:
1) the binomial dispersion test demonstrated acceptable heterogeneity between replicate cultures.
2) the proportion of cells with structural aberrations (without gaps) in negative control cultures fell within the normal range.
3) at least 160 cells out of an intended 200 were analysable at each treatment level 4) the positive control chemicals induced statistically significant increases in the number of cells with structural aberrations.

Statistics:

Statistical method used was Fisher's exact test. Probability values of p ≤0.05 were accepted as significant.

Key result

Species / strain:

lymphocytes: from human blood

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
- Effects of pH: No change
- Effects of osmolality: No change
- Evaporation from medium: Not specified
- Water solubility: Yes
- Precipitation: Precipitation is a concentration limiting factor but did not occur in this study.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: Not given
- Negative (solvent/vehicle) historical control data: Yes

Conclusions:

It was concluded that rennilase did not induce chromosome aberrations in cultured human peripheral blood lymphocytes when tested to 5000 μg/mL (equivalent to 4490 μg enzyme concentrate dry matter/mL) in both the absence and presence of S-9.

Executive summary:

Rennilase was tested in an in vitro cytogenetics assay using duplicate human lymphocyte cultures from a male and female donor in 2 independent experiments. The highest dose level used, 5000 μg/mL (equivalent to 4490 μg enzyme concentrate dry matter/mL), was considered to be an appropriate upper dose level for in vitro cytogenetics assays. Treatments covering a broad range of doses, separated by narrow intervals, were performed both in the absence and presence of metabolic activation (S-9).

In Experiment 1, treatment in the absence of S-9 was continuous for 20 hours. Treatment in the presence of S-9 was for 3 hours only followed by a 17 hour recovery period prior to harvest. The test compound dose levels for chromosome analysis were selected by evaluating the effect of rennilase on mitotic index. Chromosome aberrations were analysed at 3 consecutive dose levels. The highest concentrations chosen for analysis at this time, 3250 and 5000 μg/mL, induced approximately 69% and 0% mitotic inhibition in the absence and presence of S-9 respectively. Experiment 2 included a delayed sampling time. Treatment in the absence of S-9 was continuous for 20 or 44 hours. Treatment in the presence of S-9 was for 3 hours followed by a 17 or 41 hour recovery period. The highest concentrations chosen for analysis at 20 hours, 2560 and 5000 μg/mL, induced approximately 66% and 0% mitotic inhibition in the absence and presence of S-9 respectively. The effects of single concentrations only, 2048 μg/mL without and 5000 μg/mL with S-9 were investigated at the delayed harvest at which time 53% and 0% mitotic inhibition was induced.

Appropriate negative (solvent) control cultures were included in the test system in both experiments at both sampling times. The proportion of cells with structural aberrations in these cultures fell within historical solvent control ranges. 4-Nitroquinoline 1-oxide (NQO) and cyclophosphamide (CPA) were employed as positive control chemicals in the absence and presence of liver S-9 respectively. Cells receiving these were sampled in each experiment 20 hours after the start of treatment; both positive control compounds induced statistically significant increases in the proportion of cells with structural aberrations.

Treatment of cultures with rennilase in both the absence and presence of S-9 resulted in frequencies of cells with aberrations which were similar to and not significantly different from those observed in concurrent negative control cultures. Numbers of cells with aberrations in all rennilase-treated cultures fell within normal historical control ranges.

It was concluded that rennilase did not induce chromosome aberrations in cultured human peripheral blood lymphocytes when tested to 5000 μg/mL (equivalent to 4490 μg enzyme concentrate dry matter/mL) in both the absence and presence of S-9.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 16 1991 - December 20 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)

Deviations:

no

GLP compliance:

yes

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):

Cells were obtained from the American Type Culture Collection.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Highest concentration tested was 5000 µg/mL (equivalent to 4490 μg enzyme concentrate dry matter/mL) and 5 dilutions hereof.

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:

4-nitroquinoline-N-oxide

benzo(a)pyrene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 2 hours
- Expression time (cells in growth medium): 2 days

DETERMINATION OF CYTOTOXICITY
- Method: The plates were incubated at 37°C in a humidified incubator gassed with 5% v/v CO2 in air for 1-2 weeks. Wells containing viable clones were identified by eye using background illumination and counted.

Evaluation criteria:

Evaluation criteria
The test substance was considered to be mutagenic if:
1) the assay was valid.
2) the mutant frequency at 1 or more doses was significantly greater than that of the negative control.
3) there was a significant dose-relationship as indicated by the linear trend analysis.
4) the effects described above are reproducible.

Acceptance criteria
The assay was considered valid if the following criteria were met:
1) the mutant frequencies in the negative (solvent) control cultures fell within the normal range.
2) at least 1 concentration of each of the positive control chemicals induced a clear increase in mutant frequency.

Statistics:

Statistical significance of mutant frequencies (total wells with clones) was carried out according to the UKEMS guidelines. Thus, the control log mutant frequency (LMF) was compared with the LMF from each treatment dose, and secondly the data was checked for a linear trend in mutant frequency with treatment dose. These tests required the calculation of the heterogeneity factor to obtain a modified estimate of variance.

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

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not specified
- Effects of osmolality: There was no significant increase in osmolality (>50 mOsm/kg) in cultures treated with rennilase at the top dose of 4490 μg enzyme concentrate dry matter/mL.
- Evaporation from medium: Not specified
- Water solubility: The enzyme is water soluble
- Precipitation: Could be a confounding factor, but not an issue in this study.

RANGE-FINDING/SCREENING STUDIES: 14.2 - 4490 μg/mL

HISTORICAL CONTROL DATA
Sufficient data using identical test conditions were not available to enable historical mean values to be quoted. However, the mutant frequencies obtained in this study for the negative control cultures were in line with those obtained for recent assays performed in this laboratory. Furthermore, the positive control chemicals induced a clear increase in mutant frequency at all doses tested.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: The percentage relative survival (%RS) in each test culture was therefore determined by comparing plating efficiencies in test and control cultures thus:
%RS = [PE (test)/PE (control)] x 100

In the presence of S-9 no statistically significant increases were obtained in Experiment 1 or 2 following rennilase treatment. Although there were no significant increases in mutant frequency at any dose level in Experiment 1, a significant linear trend was indicated in this experiment. However, in Experiment 2, rennilase was tested over the same dose range and no linear trend was obtained. Therefore the dose-relationship indicated in Experiment 1 was not reproducible and is attributed to a chance effect of no biological significance.

Conclusions:

It was concluded that rennilase, under the conditions employed in this study, had no mutagenic activity in the presence and absence of S-9.

Executive summary:

Rennilase was assayed for its ability to induce mutation at the tk locus (5-trifluorothymidine resistance) in mouse lymphoma cells using a fluctuation protocol. The study consisted of a preliminary experiment followed by 2 independent experiments, each conducted in the absence and presence of metabolic activation by an Aroclor 1254 induced rat liver post-mitochondrial fraction (S-9). The preliminary experiment established that rennilase did not inactivate the enzymes of the S-9 mix and it could be tested as supplied.

Following a wide range of treatments, separated by 2-fold intervals and reaching 5000 μg/mL (equivalent to 4490 μg enzyme concentrate dry matter/mL), cells survived all doses of rennilase in Experiment 1 yielding 98.6% relative survival in the absence and 101 .4% relative survival in the presence of S-9. Accordingly, the top 5 doses were plated for viability and 5- trifluorothymidine resistance 2 days after treatment. In the second experiment, the same dose range was selected, excluding the bottom dose. The top dose plated in this experiment was again 5000 μg/mL in the absence and presence of S-9, which yielded 58.1% and 77.4% relative survival, respectively. All 5 doses were plated for the determination of mutant frequency.

Negative (solvent) and positive control treatments were included in each experiment in the absence and presence of S-9. Mutant 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.

In the absence of S-9, no significant increases in mutant frequency were observed following treatment with rennilase at any dose level in either experiment. Similarly, in the presence of S-9 no statistically significant increases were obtained in Experiment 1 or 2 following rennilase treatment. Although there were no significant increases in mutant frequency at any dose level in Experiment 1, a significant linear trend was indicated in this experiment. However, in Experiment 2, rennilase was tested over the same dose range and no linear trend was obtained. Therefore the dose-relationship indicated in Experiment 1 was not reproducible and is attributed to a chance effect of no biological significance.

It was concluded that rennilase, under the conditions employed in this study, had no mutagenic activity in the presence and absence of S-9.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Not classified.