Amylase, β-

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The genetic toxicity potential of beta-amylase, batch PPY36295 was tested in the AMES test and the in vitro micronucleus test.

It was concluded that beta-amylase, batch PPY36295 showed no evidence of mutagenic activity in this bacterial system nor did it show any evidence of causing an increase in the induction of micronuclei in cultured human lymphocytes, in either of the in vitro test systems described in this section.

The conclusion is further confirmed by read-across to mouse lymphoma test performed with enzymes closely related to beta-amylase i.e two other amylases. In both tests no evidence of genetic toxicity was observed supporting the conclusion, that beta-amylase is not genotoxic.

Link to relevant study records

Reference

Reference 1

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

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

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

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.

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.

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.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09-04-2014 to 11-07-2014

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:

The OECD Guideline 471 recommends a maximum test concentration for soluble non-cytotoxic substances of 5000 µg/plate (mL). 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.

Preliminary test: No preliminary trials were carried out.
Six concentrations of the test item (173, 345, 690, 1380, 2761 and 5522 μg enzyme concentrate dry matter/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:

2-nitrofluorene

other: Acridine mutagen (ICR-191), 1-Methyl-3-Nitro-N-NitroGuanidine, 2-Aminoanthracene

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): about 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. The plates were inverted and incubated at 37 ± 2°C for about 72 hours and scored.

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:

Not performed.

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:

cytotoxicity

Remarks:

in the absence of S9

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

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

In experiment 2 with TA1537 treated at 1380 μg enzyme concentrate dry matter/mL in the absence of S9 mix, an increase only just exceeding 50% of the solvent control was seen. However, no obvious dose relationship was present and the increase was not reproduced in experiment 1. Therefore this increase is considered to be of no biological importance.

The test substance was slightly toxic to some of the bacterial strains at the one to three highest dose levels, most pronounced with TA100 in the absence of S9 mix, as seen by reductions in the viable cell counts, accompanied by minor reductions in the revertant colony counts.

Conclusions:

Based on the results obtained in this study, it was concluded that beta-amylase, batch PPY36295 did not show any evidence of mutagenic activity when tested under the conditions applied in this bacterial reverse mutation test, in concentrations up to 5522 μg enzyme concentrate dry matter/mL in the presence and absence of S9.

Executive summary:

Beta-amylase, batch PPY36295 was examined for mutagenic activity in the bacterial reverse mutation assay using Salmonella typhimurium strain TA1535, TA100, TA1537, TA98 and Escherichia coli WP2uvrApKM101. Crude enzyme preparations, like the present test substance contain the free amino acids histidine and tryptophan, most often in an amount, which exceeds the critical concentration for incorporation in the direct standard assay. To overcome this problem all strains were exposed to the test substance 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 5.5 mg enzyme concentrate dry matter/L as the highest concentration. After incubation the test substance was removed by centrifugation prior to plating.

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). All results were confirmed by conducting two complete and independent experiments. The test substance contains an abundance of various nutrients, and composes a rich growth medium to the test bacteria. These circumstances are reflected in the present study.

The test substance was slightly toxic to some of the bacterial strains at the one to three highest dose levels, as seen by reductions in the viable cell counts accompanied by minor reductions in the revertant colony counts. No treatments of any of the S. typhimurium and E. coli strains with the test substance resulted in any increases in the number of revertant colonies that meet the criteria for a positive or equivocal response.

Based on the results obtained in this study, it was concluded that beta-amylase, batch PPY36295 did not show any evidence of mutagenic activity when tested under the conditions applied in this bacterial reverse mutation test, in concentrations up to 5522 μg enzyme concentrate dry matter/mL in the presence and absence of S9.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Not classified.