Xylanase, endo-1,4-

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:

14 January 2016 - 24 May 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Adopted 21 July 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:

Xylanase, endo-1,4-

EC Number:

232-800-2

EC Name:

Xylanase, endo-1,4-

Cas Number:

9025-57-4

Molecular formula:

Not applicable, see remarks.

IUPAC Name:

endo-1,4-beta-xylanase

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:

- Substance type: UVCB
- Physical state: liquid
- Lot/batch No.: PPQ40100
- Expiration date of the lot/batch: 03 November 2025
- Stability under test conditions: The test material and dilutions in water (10% and 100%) are stable for at least 24 hours at room temperature
- Storage condition of test material: Frozen (-20°C)

Method

Target gene:

The study describes experiments performed to assess the effect of the test material Xylanase in amino acid dependent strains of Salmonella typhimurium and Escherichia coli capable of detecting both induced frame-shift (TA1537 and TA98) and base-pair substitution mutations (TA1535, TA100 and WP2 uvrA pKM101). 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 SPF Sprague Dawley rats, manufactured at Molecular Toxicology Incorporated, USA.

Test concentrations with justification for top dose:

The following dose levels, 16, 50, 160, 500, 1600, and 5000 μg TOS/mL were tested in experiment I and 160, 300, 625, 1250, 2500, and 5000 μg TOS/mL were tested in experiment II.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Purified 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-nitrofluorene, 4-nitroquinoline 1-oxide, N-methyl-N'-nitrosoguanidine, ICR-191 mutagen and 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: 1 hr (liquid culture assay)
- Incubation time (selective incubation) : 2-3 days. The test material was incubated with the microbial cells prior to plating to avoid artifacts due to growth stimulation.

Evaluation criteria:

Individual mutation plate counts from each experiment were recorded separately and the mean and standard deviation of the plate counts for each treatment determined. Mutation plate control counts were compared with the laboratory’s historical control range.
The tests were considered to be valid as all the following criteria were met:
- The vehicle control counts fell within the laboratory’s historical control ranges for this laboratory.
- The positive control chemicals induced increases in revertant numbers of ≥2-fold (in strains TA98, TA100 and WP2 uvrA pKM101) or ≥3-fold (in strains TA1535 and TA1537) the concurrent vehicle control confirming discrimination between different strains, and an active S-9 preparation.
For valid data, the test article was considered to be mutagenic if:
- A concentration related increase in revertant numbers was ≥2-fold (in strains TA98, TA100 and WP2 uvrA pKM101) or ≥3-fold (in strains TA1535 and TA1537) the concurrent vehicle control values.
- The positive trend/effects described above were reproducible.

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

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

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

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

Positive controls validity:

valid

Test results 5

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

Positive controls validity:

valid

Additional information on results:

The test item was not toxic to the test bacteria, either in the absence or presence of S-9 mix: 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 in experiment 1.
On the basis of these results, up to 5000 µg TOS/mL was tested in experiment 2.
COMPARISON WITH HISTORICAL CONTROL DATA: The vehicle control plate counts fell within the normal historical ranges and the positive control chemicals all induced increases in revertant numbers as expected. The study thus demonstrated correct strain and assay functioning and was accepted as valid.

Applicant's summary and conclusion

Conclusions:

It was concluded, that the results of this Ames test study gave no indication of mutagenic activity of xylanase.

Executive summary:

Xylanase was assayed for mutation in four histidine-requiring strains of S. typhimurium, and one tryptophan-requiring strain of E. coli both in the absence and presence of metabolic activation by a metabolic activation system (S-9 mix), in two separate experiments. A modified ‘treat and plate’ methodology was used for all treatments in this study. All xylanase treatments in this study were performed using formulations prepared in water for irrigation (purified water).

Experiment 1 treatments used a final (nominal) concentration of 16, 50, 160, 500, 1600, and 5000µg TOS/mL Experiment 2 a narrowed concentration intervals were employed covering the range 160-5000µg TOS/mL in order to examine more closely those concentrations approaching the maximum test concentration. Following all treatments there were not clear evidence of toxicity.

Following xylanase treatment of all the test strains in the absence and presence of S-9, no clear and concentration-related increases in revertant numbers were observed that were≥2-fold (in strains TA98, TA100 and WP2 uvrA pKM101) or≥3-fold (in strains TA1535 and TA1537) the concurrent vehicle control. This study was therefore considered to have provided no evidence of any xylanase mutagenic activity in this assay system.

Based on the results obtained in this study and under the assay conditions applied, it is concluded that xylanase is not mutagenic in the Ames test.