glucan 1,4-alpha-maltohydrolaseIUBMB 3.2.1.133

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Glucan 1,4-alpha-maltohydrolase was tested for genotoxicity using the Ames assay.

The results of the bacterial mutagenicity tests gave no indication of the presence of mutagenic components in amylase, batch PPY32896, when tested under the conditions applied in this study, in the presence and absence of S-9.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 July 2012 - 8 November 2012

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

Principles of method if other than guideline:

The ‘treat and plate’ method was used in each test to avoid the possibility that bio-available histidine in the test item might compromise the test.

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, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

S9 from Aroclor 1254

Test concentrations with justification for top dose:

Six doses 156, 313, 625, 1250, 2500 and 5000 μg TOS/mL were tested. Highest dose enzyme activity.

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

Positive controls:

yes

Positive control substance:

2-nitrofluorene

other: Acridine mutagen (ICR-191), 1-Methyl-3-Nitro-N-NitrosoGuanidine, 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: 0.1 mL aliquots of a 10^6 dilution of each bacterial suspension were poured on to minimal glucose agar plates (added the required amino acids in excess) in duplicates.

Evaluation criteria:

A test substance is considered positive when it has induced at least a doubling in the mean number of revertant colonies per plate compared to the appropriate 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, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

Some cytoxocity was seen in the absence of S9 for TA100 in the highest concentration and TA1537 in the highest 3 concentrations.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Some cytotoxicity was seen in the absence of S9 for TA100 in the highest concentration and TA1537 in the highest 3 concentrations.
Growth stimulation (feeding effect) was present in a few test series especially in with the E. coli strain with addition of S9 as demonstrated by increases in the viable count of exposed cultures compared to the solvent control. As a consequence, weak increases in the number of spontaneous revertant colonies were present which obviously is without biological significance.

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

Conclusions:

The results of the bacterial mutagenicity tests gave no indication of the presence of mutagenic components in amylase, batch PPY32896, when tested under the conditions applied in this study, in the presence and absence of S-9.

Executive summary:

Amylase, batch PPY32896 was examined for mutagenic activity in the bacterial reverse mutation assay using Salmonella typhimurium strains TA1535, TA100, TA1537, TA98 and Escherichia coli WP2uvrApKM101. Crude enzyme preparations, like the present batch of amylase contain the free amino acid 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 amylase 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 5000 μg TOS/mL as 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.

Amylase contains an abundance of various nutrients, and composes a rich growth medium to the test bacteria. These circumstances are reflected in the present study. No toxicity of the test substance to the bacteria was observed.

Growth stimulation (feeding effect) was present in a few test series especially in with the E. coli strain with addition of S9 as demonstrated by increases in the viable count of exposed cultures compared to the solvent control. As a consequence weak increases in the number of spontaneous revertant colonies were present which obviously is without biological significance.

The results of the bacterial mutagenicity tests gave no indication of the presence of mutagenic components in amylase, batch PPY32896, when tested under the conditions applied in this study, in the presence and absence of S-9.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Not classified.