Glucanase, endo-1,3(4)-β-

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The present test substance, beta-glucanase, has been investigated in two in vitro test systems, the Ames test and the in vitro chromosome aberration test. No evidence for genetic toxicitywas observed.  The results are supported by read-across from in vitro gene mutation studies in L5178Y mouse lymphoma cells performed with alpha-amylase.

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:

07-03-2005 04-08-2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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

Target gene:

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

Species / strain / cell type:

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

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

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

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

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

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

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.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Jan 20 - Aug 15, 2005

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)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

At chromosomal level.

Species / strain / cell type:

lymphocytes: human

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Highest concentration tested was 5000 µg/mL and dilutions hereof.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Sterile purified water
- Justification for choice of solvent/vehicle: Substance is water-soluble and any human exposure will be in aqueous solutions.

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

other: 4-Nitroquinoline 1-oxide

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in suspension

DURATION
- Exposure duration: 3 hours (first experiment, in absence and presence of S9); 3 hours (second experiment, in presence S9); 20 hours (second experiment, in absence S9)
- Treatment plus recovery time: 20 hours

SPINDLE INHIBITOR (cytogenetic assays): Colchicine
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: Preliminary trial and two independant replicates.

NUMBER OF CELLS EVALUATED: a total of 200 cells per dose level

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
- Other: Hyperdiploid cells

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

Statistics:

Fisher's exact test, p≤0.05 significant.
Heterogeneity between replicates evaluated by means of a binomial dispersion test, p≤0.05 significant.

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

COMPARISON WITH HISTORICAL CONTROL DATA:
Treatment of cultures with beta-glucanase, batch PPB24534 in the absence and the presence of metabolic activation (S-9) (Experiments 1 and 2) resulted in frequencies of cells with structural aberrations, which were similar to those observed in concurrent vehicle control cultures for all concentrations analysed.
Moreover, the aberrant cell frequency of all test item treated cultures fell within current historical negative control (normal) ranges.

Conclusions:

Beta-glucanase, batch PPB24534, under the conditions of the test, did not induce chromosome aberrations in cultured human peripheral blood lymphocytes when tested to 5000 ug/mL in either the absence and presence of S-9.

Executive summary:

Beta-glucanase, batch PPB24534 was tested in an in vitro cytogenetics assay using duplicate human lymphocyte cultures prepared from the pooled blood of three male donors in two independent experiments. Treatments covering a broad range of doses, separated by narrow intervals, were performed both in the absence and presence of metabolic activation (S-9). The test article was formulated in water for injection (purified water) and the highest dose level used was 5000 μg/mL.

Appropriate control cultures were included in the test system in both experiments under each treatment condition. The proportion of cells with structural aberrations in these cultures fell within historical solvent control ranges.

In Experiment 1, treatment in the absence and presence of S-9 was for 3 hours followed by a 17-hour recovery period prior to harvest (3+17). The S-9 used was prepared from a rat liver post-mitochondrial fraction (S-9) from Aroclor 1254 induced animals. The test article dose levels for chromosome analysis were selected by evaluating the effect of the test item on mitotic index. Chromosome aberrations were analysed at three dose levels. The highest concentration chosen for analysis, 5000 μg/mL, induced approximately 38% and 23% mitotic inhibition (reduction in mitotic index) in the absence and presence of S-9 respectively.

In Experiment 2, 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 (3+17). Chromosome aberrations were analysed at three dose levels and the highest concentration chosen for analysis, 5000μg/mL, induced approximately 11% and 10% mitotic inhibition in the absence and presence of S-9 respectively.

It was concluded that Beta-glucanase, batch PPB24534 did not induce chromosome aberrations in cultured human peripheral blood lymphocytes when tested up to 5000mg/mL in both the absence and presence of a rat liver metabolic activation system (S-9).

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 June 1989 - 10 October 1989

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:

HGPRT (6-thioguanine resistance)

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: Fishers medium (10% horse serum)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

The highest concentration tested was 5000 µg/ml (weighed out as received).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Distilled water
- Justification for choice of solvent/vehicle: Substance is water-soluble and any human exposure is in aqueous solutions.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 4-nitroquinoline-1-oxide (without S-9) and benzo(a)pyrene (with S-9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium, growth suspension. Selection phase was performed in microtitre plates.

DURATION
- Exposure duration: 2 hours
- Expression time (cells in growth medium): With exception of experiment 1 treatments in the absence of S-9 (where cultures were maintained for eight days) cultures were maintained for 7 days.
- Fixation time (start of exposure up to fixation or harvest of cells): At least 7 days after treatment.

SELECTION AGENT (mutation assays): 6-TG

NUMBER OF REPLICATIONS: duplicate

DETERMINATION OF CYTOTOXICITY
- Method: Cell density by counting viable cells, expressed as percentage relative survival (RS%)

Evaluation criteria:

A test article was considered positive if:
- The assay was valid, and
- Significant induced mutation (i.e. the lower 95 percentile of a treated culture exceeded the upper 95 percentile of a control culture) occurred at consecutive doses in at least one experiment, and
- Dose-related increases in mutation could be confirmed by regression analysis in both experiments.

Statistics:

The mutation frequency was evaluated statistically by using logarithmic transformation of the variances of the number of clones observed on viability and mutation plates as described by E.E. Furth et al., Anal Biochem 110: 1-8, 1981

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

Conclusions:

Alpha-amylase, batch PPY2693, under the conditions of the test, had no mutagenic activity in cultured mouse lymphoma cells when tested to a concentration of up to 5000 µg/mL in either the absence or presence of S-9.

Executive summary:

Alpha-amylase, batch PPY2693, was assayed for its ability to induce mutation at the HGPRT locus (6-thioguanine resistance) in mouse lymphoma cells using a fluctuation protocol. The study consisted of two independent experiments, each conducted in the absence and presence of metabolic activation (S-9 mix).

Following a wide range of treatments, separated by half-log intervals and reaching 5000 µg/mL, cultures surviving the top dose of 5000 µg/mL in the absence and in the presence of S-9showed 55% and 53% survival respectively. These, together with the next 3 lower doses, were plated for viability and 6-thioguanine resistance eight (treatments in the absence of S-9) or seven (treatments in the presence of S-9) days after treatment. In the second experiment a narrower dose range was used to maximise the chance of detecting any dose related effects. The top dose plated in this experiment was again 5000 µg/mL in the absence and presence of S-9, which resulted in 50% and 117% survival respectively.

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

The treatment did not result in any statistically significant increases in mutation frequency, neither in the absence or presence of S-9.

Therefore, when tested to a concentration of 5000 µg/mL in the absence and presence of S-9, the present alpha-amylase, batch PPY2693 failed to induce mutation at the HGPRT locus of L5178Y mouse lymphoma cells in two independent experiments.

It was concluded that alpha-amylase had no mutagenic activity in this test system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Not classified.