Amylase, gluco-

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 December 2008 - 30 January 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Principles of method if other than guideline:

Crude enzyme preparations, like the present batch of glucoamylase 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 glucoamylase in liquid culture (“treat and plate assay”).

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Active enzyme protein of Amylase, gluco- (EC no. 232-877-2, CAS no. 9032-08-0, EC name: glucan 1,4-alpha-glucosidase, Enzyme Class no. 3.2.1.3)

IUPAC Name:

Active enzyme protein of Amylase, gluco- (EC no. 232-877-2, CAS no. 9032-08-0, EC name: glucan 1,4-alpha-glucosidase, Enzyme Class no. 3.2.1.3)

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

Test material form:

liquid

Details on test material:

Substance type: UVCB
- Physical state: liquid
- Lot/batch No.: trg08050/52
- Expiration date of the lot/batch: At least stable until January 2011
- Stability under test conditions: The test material and dilutions in water (25% and 50%) are stable for
at least 5 hours at room temperature, 7 days at 4 degrees Celcius and 90 days at minus 20 degrees
of Celcius
- Storage condition of test material: minus 20 degrees of Celcius

Method

Target gene:

The study describes experiments performed to assess the effect of glucoamylase in amino acid dependent strains of Salmonella typhimurium capable of detecting both induced frame-shift (TA1537 and TA98) and base-pair substitution mutations (TA1535, TA100, and TA102). The test system is a reverse mutation of amino acid dependent bacterial strains.

Species / strain

Species / strain / cell type:

bacteria, other: Salmonella typhimurium TA1537, TA98, TA1535, TA100, TA102

Metabolic activation:

with and without

Metabolic activation system:

S9 from Aroclor 1254 induced SPF Wistar rats obtained from Taconic Europe A/S, Ejby, DK-5623 Lille Skensved, Denmark.

Test concentrations with justification for top dose:

50, 160, 500, 1600 and 5000 µg test substance (total protein) per plate, with and without the metabolic activation.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: sterile saline (0.9% NaCl, Fresenius Kabi Norge A/S)
- Justification for choice of solvent/vehicle: substance is water-soluble and any human exposure will be in aqueous solutions.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: with S-9 mix: cumene hydroperoxide, sodium azide, 2-nitrofluorene, 9-aminoacridine; without S-9 mix: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium before plating, i.e a liquid culture assay (treat and plate assay).

DURATION
- Exposure duration: 3 hours
- Incubation time (selective incubation): 3 days

DETERMINATION OF CYTOTOXICITY
- Method: Viable cell count

Evaluation criteria:

The numbers of revertant colonies at each treatment test point were compared to the corresponding negative control values using the Analysis of Variance test. When this test showed statistically significant differences in the data, Dunnett’s test was used to determine the statistical significance of increases and decreases in the numbers of revertant colonies for each set of triplicate plates.
The tests were considered to be valid as all the following criteria were met:
- negative and positive control data were consistent with the historical control data for this laboratory
- the positive control data showed marked increases over the concurrent negative control values
- the evaluation of the data was not restricted by loss of plates (e.g. through contamination).
The test item would have been considered to have shown evidence of mutagenic activity in this study if all of the following criteria had been met:
- increases in the numbers of revertant colonies were observed at one or more test points
- the mean number of revertant colonies at the test point showing the largest increase was more than twice the corresponding negative control value
- there was a credible scientific explanation for the observed dose-response relationship that involved a mutagenic effect of the test item
- the increases were reproducible between replicate plates and were observed in both main tests (when treatment conditions were the same)
- the increases were statistically significant
- the increases were not directly related to increased growth of the non-revertant bacteria.

Statistics:

The statistical analyses were performed with SAS® procedures (version 8.2) described in SAS/STAT® User’s Guide, SAS OnlineDoc®, 1999, SAS Institute Inc., Cary, North Carolina 27513, USA.

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 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

Test results 4

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 5

Species / strain:

S. typhimurium TA 102

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:

RANGE-FINDING/SCREENING STUDIES: The number of revertant colonies on the control plates were generally lower than the historical control range for this laboratory, but the data were considered to be adequate to assess the toxicity of the test item. The test item was not toxic to the test bacteria: no marked reductions in the number of revertant colonies or growth of the background lawn of non-revertant bacteria were observed, compared to the negative control plates.
On the basis of these results, 5000 μg/plate was chosen as the highest dose level for the main tests.

COMPARISON WITH HISTORICAL CONTROL DATA: The negative and positive control values were compatible with the historical control values for this laboratory. One of the positive control values in TA 102 without S-9 mix in the second test was slightly lower than the historical control range and one plate at this test point had microcolonies. One of the negative control values for TA 1537 without S-9 mix in the second test was higher than the historical control range. These values are considered to be acceptable. The large increases in the number of revertant colonies produced by the positive control treatments demonstrated the sensitivity of the tests and the efficacy of the S-9 mix metabolic activation system.

Applicant's summary and conclusion

Conclusions:

Based on the results obtained in this study, it is concluded that the test substance was not mutagenic in the Ames test.

Executive summary:

Glucoamylase was examined for mutagenic activity in the bacterial reverse mutation assay using Salmonella typhimurium strain TA1535, TA100, TA1537, TA98 and TA102.

Crude enzyme preparations, like the present batch contain the free amino acid histidine and tryptophan, 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 5 doses of the test substance in a phosphate buffered nutrient broth for 3 hours with 5 mg (total protein) per 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 rats, pre-treated with Aroclor 1254, and the co-factors required for mixed function oxidase activity (S9 mix).

Two identical and independent experiments were conducted.

The treatment of the Salmonella strains with the test substance, in the presence or absence of S9 mix, did not result in any increases in revertant numbers. The test substance was found not mutagenic.