Glucosidase, 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:

30-05-1990 to 16-11-1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1990

Report date:

1990

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

May 26th 1983

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Active enzyme protein of β-Glucosidase (EC no. 232-589-7, CAS no. 9001-22-3, EC name: Glucosidase, β-, Enzyme Class no. 3.2.1.21)

Molecular formula:

Not available. See remarks.

IUPAC Name:

Active enzyme protein of β-Glucosidase (EC no. 232-589-7, CAS no. 9001-22-3, EC name: Glucosidase, β-, Enzyme Class no. 3.2.1.21)

Constituent 2

Reference substance name:

Proteins as constituents of enzyme deriving from the fermentation or extraction process

Molecular formula:

Not available. See remarks.

IUPAC Name:

Proteins as constituents of enzyme deriving from the fermentation or extraction process

Constituent 3

Reference substance name:

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

Molecular formula:

Not available. See remarks.

IUPAC Name:

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

Constituent 4

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

1

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

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: No preliminary trials were carried out.
Six concentrations of the test item: 0.625, 1.25, 2.5, 5, 10 mg/mL.
The OECD 471 guideline from 1983 did not indicate a maximum dose to be used in the Ames test. 10 mg/mL was used as the maximum concentration in the laboratory.

Vehicle / solvent:

Vehicle for enzyme: Deionised 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

other: 2-Aminoanthracene, N-Methyl-N'-Nitro-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°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.

Evaluation criteria:

For Salmonella typhimurium strain TA 1535, TA 1537 and TA 98 at least a doubling of the mean control value and a dose related response was looked for. At high dose levels this may be reverted because of toxicity to the bacteria. For TA 100 a 50% reproducible increase over control value is considered as indicative of a mutagenic effect .

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 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 (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: Yes
- Negative (solvent/vehicle) historical control data: Yes

Applicant's summary and conclusion

Conclusions:

The results of the bacterial mutagenicity tests give no indication of mutagenic activity of beta-glucosidase batch DCN 0005 when tested in the presence or absence of the S-9 metabolic system. All results were confirmed by conducting two independent experiments.

Executive summary:

Beta-glucosidase batch DCN 0005 was examined for mutagenic activity using Salmonella typhimurium strains TA 1535, TA 100, TA 1537 and TA 98. A liquid culture assay was applied. Bacteria were exposed to 5 doses (0.625, 1.25, 2.5, 5, 10 mg/mL) 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 Novozym 188, either in the presence or absence of S-9 mix.

It was concluded that the results of the experiments give no indication of mutagenic activity of beta-glucosidase batch DCN 0005 in the presence or absence of metabolic activation.