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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:
24 May 2011 - 12 September 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The results from the present study are used to support the evaluation of the test substance dextransucrase IUBMB 2.4.15 by read-across from the in vitro gene mutation study in bacteria performed on cellulase IUBMB 3.2.1.4

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Report date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Active enzyme protein of Cellulase (EC no. 232-734-4, CAS no. 9012-54-8, EC name: Cellulase, Enzyme Class no. 3.2.1.4)
Molecular formula:
Not applicable, see remarks.
IUPAC Name:
Active enzyme protein of Cellulase (EC no. 232-734-4, CAS no. 9012-54-8, EC name: Cellulase, Enzyme Class no. 3.2.1.4)
Constituent 2
Reference substance name:
Protein as a constituent of enzyme deriving from the fermentation or extraction process
Molecular formula:
Not available. See remarks.
IUPAC Name:
Protein as a constituent of enzyme deriving from the fermentation or extraction process
Constituent 3
Reference substance name:
Carbohydrates constituent of enzyme deriving from the fermentation or extraction process.
Molecular formula:
Not available. See remarks.
IUPAC Name:
Carbohydrates constituent of enzyme deriving from the fermentation or extraction process.
Constituent 4
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
Constituent 5
Reference substance name:
Lipids as a constituent of enzyme deriving from the fermentation or extraction process
Molecular formula:
Not available. See remarks.
IUPAC Name:
Lipids as a constituent of enzyme deriving from the fermentation or extraction process
Test material form:
other: Fermentation liquid
Details on test material:
Substance type: UVCB
Physical state: fermentation liquid (brown)
Stability under test conditions: the undiluted test material and dilutions in water (50 and 25%) for at least 5 hours at room temperature. The undilutedtest material is stable at least 7 days at 4 degrees Celsius.
Storage conditions of test material: approximately minus 20 degrees Celsius in the dark

Method

Target gene:
Histidine or tryptophan locus in the genome of five strains of bacteria
Test concentrations with justification for top dose:
Preliminary test: Concentrations tested were 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500, 5000 ug total protein/plate
Experiment 1: Five concentrations of the test item (50, 150, 500, 5000 ug total protein/plate, based on total protein content of 130.11 mg/ml)
Experiment 2: Five concentrations of the test item (50, 150, 500, 5000 ug total protein/plate, based on total protein content of 130.11 mg/ml)
Vehicle / solvent:
sterile distilled water
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
sterile distilled water
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
Details on test system and experimental conditions:
The test item was fully soluble in the Sponsor selected vehicle (sterile distilled water) at 50 mg total protein/ml in solubility checks performed in-house. Prior to the start of each experiment, the test item was removed from storage and placed at approximately 4C to thaw.
The test item was accurately measured out and approximate half-log dilutions prepared in sterile distilled water by autovortexing on the day of each experiment. All formulations were used within four hours of preparation and were assumed to be stable.
Evaluation criteria:
Any, one, or all of the following can be used to determine the overall result:
1) A dose-related increase in mutant frequency over the dose range tested. (De Serres and Shelby (1979))
2) A reproducible increase at one or more concentrations.
3) Biological relevance against in-house historical control ranges.
4) Statistical analysis of data as determined by UKEMS. (Mahon et al (1989))
5) Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response).

Results and discussion

Test results
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
Additional information on results:
The test item, Optimash BG (Trichoderma reesei), was considered to be non-mutagenic under the conditions of this test.

Applicant's summary and conclusion

Conclusions:
Cellulase is not mutagenic in the Ames assay in both the presence and absence of metabolic activation
Executive summary:

The objective of this assay was to assess the potential of Cellulase to induce point mutations (frame-shift and base-pair) in four strains ofSalmonella typhimurium TA 98, TA 100, TA 1535 and TA 1537 andE. coliWP2 uvrA. The test material was tested both in the presence and absence of a metabolic activation system (Aroclor 1254-induced rat liver; S9- mix). A pre-experiment test was performed first for dose selection. Subsequently, one independent main test was performed with all 5 strains in both the presence and absence of S9-mix. Triplicate plates were used at each test point. All dose levels were expressed in terms of total protein (TP). 

The test item was fully soluble in the vehicle (sterile distilled water) at 50 mg TP/ml. In the preliminary assay, 10 dose levels ranging from 0.15 to 5000 µg TP/plate were used. µNo precipitation and/or cytotoxicity were noted up to the highest tested concentration of 5000 µg TP/plate. In experiment 1, five dose levels of the test item ranging from 50 to 5000 µg TP/plate (equivalent to 61 to 6100 µg TOS/plate) were assayed in triplicate using the direct plate incorporation method. The highest dose level tested (5000 µg TP/plate) is the maximum required by the OECD guideline. In experiment 2, five dose levels of the test item ranging from 50 to 5000 µg TP/plate were assayed in triplicate using the pre-incubation method. Sterile distilled water served as vehicle control. Appropriate positive controls were selected for assays with and without S9-mix. The study was conducted in accordance with OECD guideline No. 471 (1997) and complied with all standard GLP.

Cellulase was not toxic to the test bacteria up to and including the highest dose level (5000 µg TP/plate) in both the absence and presence of S9-mix. No biologically significant increases in the number of revertant colonies were observed at any dose level tested in both presence and absence of S9-mix in either vehicle control or test item cultures. Statistically significant increases in the mean number of revertant colonies were noted in all assays with positive control chemicals.

 

Under the conditions of this assay, cellulase shall be classified as “Non-Mutagenic” up to the highest concentration of 5000 µg/plate.