Pullulanase

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:

20 March 2013 - 14 June 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

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:

The ‘treat and plate’ treatment 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 (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Pullulanase

EC Number:

232-983-9

EC Name:

Pullulanase

Cas Number:

9075-68-7

Molecular formula:

not available

IUPAC Name:

Pullulanase

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.

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.

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.

IUPAC Name:

Lipids as a constituent of enzyme deriving from the fermentation or extraction process

Test material form:

liquid

Details on test material:

- Name of test material (as cited in study report): Pullulanase, PPY34283
- Substance type: UVCB
- Physical state: liquid
- Lot/batch No.: PPY34283
- Expiration date of the lot/batch: Stable at least until 24 October 2022.
- pH: 5.6
- Stability under test conditions: The undiluted test material and 10% solutions in water are stable for at least 24 hours at room temperature or 5 degrees Celcius
- Storage condition of test material: minus 18 degrees of C

Method

Target gene:

The study describes experiments performed to assess the effect of the test material Pullulanase in amino acid dependent strains of Salmonella typhimurium capable of detecting both induced frame-shift (TA1537 and TA98) and base-pair substitution mutations (TA1535 and TA100); and strain of Escherichia coli WP2uvrApKM101 that can detect substitution at AT to GC or in G-C pair. The test system is a reverse mutation of amino acid dependent bacterial strains.

Species / strain

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 induced Sprague Dawley rats , was obtained crom Cappel/MP Biomedicals, LLC, and Moltox.

Test concentrations with justification for top dose:

Six doses 156, 313, 625, 1250, 2500 and 5000 μg dry matter/mL) were tested. Highest dose 1.6 mg active enzyme protein (aep)/mL.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Deionized water
- Justification for choice of solvent/vehicle: The test substance is water-soluble and any human exposure will be in aqueous solutions.

Controls

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-Nitroflurene, Acridine mutagen ICR-191, 1-Methyl3-Nitro-N-NitrosoGuanidine, 2-Aminoanthracene

Details on test system and experimental conditions:

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

DURATION
- Exposure duration: 3 hrs (liquid culture assay)
- Incubation time (selective incubation) : 64-72 hrs
DETERMINATION OF CYTOTOXICITY
- Method: Viable cell count

Evaluation criteria:

The numbers of revertant colonies at each treatment test point were compared to the corresponding solvent control values 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 numbers of revertant colonies at each treatment test point in the main tests were compared to the corresponding solvent control values. The test substance would be considered as positive if 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.

Results and discussion

Test results

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

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: In the first experiment, the test substance was not toxic to the bacteria: no reductions in the growth of the background lawn of non-revertant bacteria were observed in any of the test series. In the second experiment, toxicity was observed in TA100, TA1535 and TA1537 at the one to three highest dose levels (1250-5000 µg/mL), especially in the absence of S9 mix. This could be seen from reductions in the growth of the background lawn and the presence of micro-colonies of non-revertant bacteria, accompanied by reductions in the viable cell counts. The difference in toxicity between the first and second experiment, most pronounced in TA100 and TA1535 without S9 mix, could indicate a technical error in one of the experiments. Therefore, a third experiment with those two test series was included. In accordance with the first experiment, no signs of toxicity were observed in the third experiment.
On the basis of these results, 5000 ug test substance/plate was chosen as the highest dose level for the main tests.
COMPARISON WITH HISTORICAL CONTROL DATA: The solvent and positive control values were compatible with the historical control values for this laboratory.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Pullulanase, batch PPY34283, was not mutagenic when tested under the conditions applied in this bacterial reverse mutation test.

Executive summary:

Pullulanase was tested in three independent main tests. The ‘treat and plate’ treatment method was used in each test to avoid the possibility that bio-available histidine in the test item might compromise the test. Six dose levels ( 156, 313, 625, 1250, 2500 and 5000 μg dry matter/mL) were tested. Pullulanase dilluted in deionized water was added to bacteria growth medium. After 3 hr treatment were bacteria washed, plated and incubated for 64 -72 hrs. Negative control plates were treated with DI water without Pululanase. The treatments were performed both with and without a metabolic activation system (S-9 mix).

The test item was considered not toxic to the test bacteria, either in the absence or presence of S-9 mix: no reductions in the growth of the background lawn of non-revertant bacteria or the number of revertant colonies were observed were observed in two independent experiments.

No increases in the number of revertant colonies were observed at any dose level of the test item in either main test.

The results obtained with the solvent and positive controls demonstrated the sensitivity of the tests and the efficacy of the S-9 mix metabolic activation system.

Based on the results obtained in this study, it is concluded that Pullulanase, batch PPY34283 did not show evidence of mutagenic activity when tested under the conditions applied in this bacterial reverse mutation test.