Amylase, β-

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 1 - Aug. 22, 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: Draft OECD guideline 487, adopted 22 July 2010

Version / remarks:

2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Active enzyme protein of beta-amylase (EC no.232-566-1, Cas no. 9000-91-3, EC name Beta-amylase, enzyme class 3.2.1.2)

Molecular formula:

n.a.

IUPAC Name:

Active enzyme protein of beta-amylase (EC no.232-566-1, Cas no. 9000-91-3, EC name Beta-amylase, enzyme class 3.2.1.2)

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.: PPY36295
- Expiration date of the lot/batch: April, 2024

Method

Species / strain

Species / strain / cell type:

lymphocytes: cultured human peripheral blood lymphocytes

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

The OECD Guideline 487 recommends a maximum test concentration for soluble non-cytotoxic substances of 5000 µg/plate (mL). Thus the test item was tested as 5000 µg total organic solids (TOS)/mL maximum dose, equivalent to 5522 µg enzyme concentrate dry matter/mL, fulfilling the recommendation.

Vehicle / solvent:

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

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: Mitomycin C (MMC) and Vinblastine (VIN) in the absence of rat liver S-9, Cyclophosphamide (CPA) in the presence of S-9.

Details on test system and experimental conditions:

METHOD OF APPLICATION: Whole blood cultures were established in HEPES-buffered RPMI medium containing 10% (v/v) heat inactivated foetal calf serum, 0.52% penicillin/streptomycin and 2% of the mitogen Phytohaemagglutinin (PHA). These cultures were incubated at approx. 37°C for 48 hours before treatment with test article. Sets of duplicate cultures were exposed to the test substance for 3 hours in the presence and absence of metabolic activation (S-9 mix) and harvested 24 hours after the beginning of treatment (3+21 hour treatment). Additionally, a continuous 24-hour treatment without S-9 mix was included with harvesting 24 hours after the beginning of treatment (24+24 hour treatment). The cultures were treated with cytochalasin-B after removal of the test substance to block cytokinesis. Six concentrations, covering an appropriate range of cytotoxicity, were selected for scoring of micronuclei by evaluating the effect of the test substance on the replication index. A minimum of 1000 cells per concentration (500 cells from each replicate culture) were scored.

DURATION
- Exposure duration: 3 and 24 hours

NUMBER OF REPLICATIONS: Sets of duplicate cultures were exposed to the test substance.

DETERMINATION OF CYTOTOXICITY
- Method: 5522 μg enzyme concentrate dry matter/mL was determined as max dose following a preliminary cytotoxicity Range-Finder Experiment. Cytotoxicity (%) was expressed as (100 – Relative replication Index (RI)).

Evaluation criteria:

A test article was considered positive if:
- the assay was valid, and
- significant increase in the frequency of MNBN cells at one or more concentrations , and
- the incidence of MNBN cells exceeded the normal range in both replicates, and
- a concentration-related increase in the proportion of MNBN cells was observed.

Statistics:

The proportion of MNBN cells for each treatment condition were compared with the proportion in negative controls by using Fisher's exact test. Probability values of p equal or less than 0.05 were accepted as significant.

Results and discussion

Test results

Key result

Species / strain:

lymphocytes: from human blood

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In the 24+24 hour treatment without S-9 mix the highest applied concentration was 4417 µg enzyme concentrate dry matter/mL due to cytotoxicity at higher concentrations. No significant cytotoxicity was seen in the 3+21 hour treatment.

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: pH of test material is neutral, 7.4
- Effects of osmolality: no marked changes in osmolality was observed (shifts of greater than 50 mOsm/kg)
- Water solubility: yes
- Precipitation: no
- Other confounding effects: none

RANGE-FINDING/SCREENING STUDIES: Preliminary cytotoxicity range-finder performed

MICRONUCLEUS EXPERIMENT: Treatment of cells with Beta amylase, batch PPY36295 in the absence and presence of S-9 resulted in frequencies of MNBN cells which were similar to and not significantly (p≤0.05) higher than those observed in concurrent vehicle controls for the majority of all concentrations analysed. Exceptions to this were noted at an intermediate concentration analysed (3313 μg enzyme concentrate dry matter/mL) following 3+21 hour –S-9 treatment and at the highest concentration (4417 μg enzyme concentrate dry matter) following 24+24 hour –S-9 treatment. However, these increases were small such that mean MNBN cell values fell within normal ranges for all concentrations. With the exception of a single replicate culture at 4417 μg enzyme concentrate dry matter/mL (24+24 hour –S-9 treatment), the MNBN cell frequency of all treated cultures (all treatments) fell within the 95th percentile of the current observed historical vehicle control (normal) ranges. The increase above normal observed in replicate ‘B’ at 4417 μg enzyme concentrate dry matter/mL was not observed in either the replicate ‘A’ culture, or, following a second score from a separate prepared slide from the ‘B’ culture (‘B2’). As such, these small statistical increases were not considered of biological importance.

Applicant's summary and conclusion

Conclusions:

It was therefore concluded that Beta amylase, batch PPY36295 did not induce biologically relevant increases in micronuclei in cultured human peripheral blood lymphocytes under the experimental conditions employed for this study.

Executive summary:

Beta amylase, batch PPY36295 was tested in an in vitro micronucleus assay using
duplicate human lymphocyte cultures prepared from the pooled blood of two female donors in a single experiment. Treatments covering a broad range of concentrations, separated by narrow intervals, were performed both in the absence and presence of metabolic activation (S-9) from Aroclor 1254-induced rats. The test article was formulated in water for irrigation (purified water) and the highest concentration tested in the Micronucleus Experiment, 5522 μg enzyme concentrate dry matter/mL (an acceptable maximum concentration for in vitro micronucleus assays according to current regulatory guidelines), was determined following a preliminary cytotoxicity Range-Finder Experiment.

Sets of duplicate cultures were exposed to the test substance for 3 hours in the presence and absence of metabolic activation (S-9 mix) and harvested 24 hours after the beginning of treatment (3+21 hour treatment). Additionally, a continuous 24-hour treatment without S-9 mix was included with harvesting 24 hours after the end of treatment (24+24 hour treatment). The cultures were treated with cytochalasin-B after removal of the test substance to block cytokinesis. Appropriatenumber of concentrations, covering an appropriate range of cytotoxicity, were selected for scoring of micronuclei. A minimum of 1000 cells per concentration were scored.

Based on a Range-Finder experiment, three or four concentrations were selected for micronucleus analysis.

Both negative and positive controls were within historical control ranges and the study was accepted as valid. 

Treatment of cells with Beta amylase, batch PPY36295 in the absence and presence of S-9 resulted in frequencies of MNBN cells which were similar to and not significantly (p≤0.05) higher than those observed in concurrent vehicle controls for the majority of all concentrations analysed. A few exceptions to this was noted in the intermediate concentration and with a single replicate culture at 24+24 hours (-S-9) at 4417 μg enzyme concentrate dry matter, but as such, these small statistical increases were not considered of biological importance.

It was therefore concluded that Beta amylase, batch PPY36295 did not induce biologically relevant increases in micronuclei in cultured human peripheral blood lymphocytes under the experimental conditions employed for this study.