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Genetic toxicity: in vitro

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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

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:
Xylanase, endo-1,4-
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 resultsopen allclose all
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
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
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
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
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 were2-fold (in strains TA98, TA100 and WP2 uvrA pKM101) or3-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.