Glucanase, endo-1,3(4)-β-

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:

07-03-2005 04-08-2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

July 1997

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Active enzyme protein of Beta-glucanase, endo-1,3(4) (CAS no, EC no., EC name endo-1,3(4)-beta-glucanase, enzyme class 3.2.1.6)

Molecular formula:

Not applicable

IUPAC Name:

Active enzyme protein of Beta-glucanase, endo-1,3(4) (CAS no, EC no., EC name endo-1,3(4)-beta-glucanase, enzyme class 3.2.1.6)

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.: PPB24534
- Expiration date of the lot/batch: 21 January 2015
- Storage condition of test material: Frozen in the dark

Method

Target gene:

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

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

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

Controls

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: 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): 48-72 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. In case of a dose related and reproducible numerical increase, which is below a doubling but at least 50% higher than the solvent control, the result is considered as equivocal and needs 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

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

+S9, a dose-related increase in revertant colony count just exceeding a doubling at the highest dose level was observed, but was not reproducible.

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
- Negative (solvent/vehicle) historical control data: Yes

Any other information on results incl. tables

Some changes in viability were seen for TA98 and TA1537, however, no dose-response effect was observed.

Applicant's summary and conclusion

Conclusions:

The results of the bacterial mutagenicity tests described in this report gave no indication of the presence of mutagenic components in this preparation of beta-glucanase, batch PPB24534, when tested under the conditions employed in this study, in concentrations up to 5000 μg/mL in the presence and absence of S9.

Executive summary:

Beta-glucanase batch PPB24534 was examined for mutagenic activity in the bacterial reverse mutation assay using Salmonella typhimurium strain TA 1535, TA 100, TA 1537, T A98 and Escherichia coli WP2uvrA. Crude enzyme preparations, like the present beta-glucanase, contain the free amino acid L-histidine, most often in an amount, which exceeds the critical concentration for incorporation in the direct standard assay. To overcome this problem all S. typhimurium strains were exposed to beta-glucanase 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)/mL as highest concentration. After incubation the test substance was removed by centrifugation prior to plating. Two identical and independent experiments were conducted.

Usually the content of tryptophan in enzyme preparations is low and insignificant. Therefore the part of the study comprising Escherichia coli was initially 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. 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). The positive control substances induced significant responses in the appropriate strains in similar conditions as the test article. In general beta-glucanase was not toxic to any of the test strains applied.

No treatments of any of the S. typhimurium strains with beta-glucanase resulted in any increases in revertant numbers that meets these criteria for a positive or equivocal response.

In the first experiment with the E.coli WP2uvrA strain with S9 incorporated, a dose-related increase in revertant colony count just exceeding a doubling at the highest dose level was observed. In the similar second and a third repeated test series, this increase was not reproduced. The increase in the first experiment was therefore considered spurious and of no biological significance. It may have been caused by formation of additional spontaneous revertants due to the low content of tryptophan in the test substance.

As no treatments of any of the bacterial strains with beta-glucanase in the treat and plate assay resulted in any dose related and reproducible increase in revertant numbers compared to the solvent control, the criteria for a positive or equivocal response was not met in this study.

The results of the bacterial mutagenicity tests described in this report gave no indication of the presence of mutagenic components in this preparation of beta-glucanase, batch PPB24534, when tested under the conditions employed in this study, in concentrations up to 5000 μg/mL in the presence and absence of S9.