Cellulase

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The present test substance, cellulase IUB 3.2.1.4, has been investigated in two in vitro test systems, the Ames test and the in vitro chromosome aberration test and in a cultured human peripheral blood lymphocyte micronucleus assay. All tests have been performed according to current OECD guidelines, and in compliance with GLP. No evidence for genetic toxicity was observed. These results are supported by read-across from three in vitro gene mutation studies in L5178Y mouse lymphoma cells performed on three different amylases belonging to the same subclass of glycosidases (IUBMB class 3.2.1.) as cellulase.

Link to relevant study records

Reference

Reference 1

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

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

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) .
The highest dose is equivalent to 6.37 mg enzyme concentrate dry matter/mL.

Vehicle / solvent:

sterile distilled water

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).

Species / strain:

E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

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.

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 of Salmonella 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. No 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. The highest dose is equivalent to 6.37 mg enzyme concentrate dry matter/mL. 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 which is equivalent to 6.37 mg enzyme concentrate dry matter/mL.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 May 2011 - 03 August 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Vehicle / solvent:

Rat liver homogenate metabolising system (S9)
Mitomycin C (MMC) (Sigma, Batch No. 098K1681)
Cyclophosphamide (CP) (Acros, Batch No. A0164185)

Untreated negative controls:

yes

Positive controls:

yes

Details on test system and experimental conditions:

Test item was accurately measured and dissolved in minimal essential medium (MEM) and serial dilutions prepared. Prior to the start of each experiment, the test item was removed from storage and placed at approximately 4C to thaw. There was no significant change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm. Ten concentrations of Cellulase were used in the preliminary assay for the selection of dose levels in the main studies. Three doses were then selected for the two main assays with the highest dose level clearly inducing a toxic effect (50% reduction in mitotic index). In the absence of cytotoxicity, the highest dose selected would be 5000 µg Total Protein/ml (equivalent to 6100 µg TOS/ml) as recommended by the OECD guideline.

Duplicate cultures of human lymphocytes, treated with the test material, were evaluated for chromosome aberrations at 3 dose levels, together with negative control (minimal essential medium) and positive controls (Mitomycin C for assays in the absence of S9-mix and cyclophosphamide for assays with S9- mix).  The treatment conditions used for the study were: In Experiment 1, 4-hour exposure to the test item without S9-mix followed by 20-hour expression period and 4-hour exposure to the test item with S9-mix followed by 20-hour expression period. In Experiment 2, 24-hour continuous exposure to the test item without S9-mix and 4-hour exposure to the test item with S9-mix followed by 20-hour expression period. Mitosis was arrested by addition of Colcemid (0.1 µg/ml) 2 hours before the required harvest time.

Evaluation criteria:

Where possible the first 100 consecutive well-spread metaphases from each culture were counted, where there was approximately 30 to 50% of cells with aberrations, slide evaluation was terminated at 50 cells. If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommeded in the 1983 UKEMS guidelines for mutagenicity testing.

Statistics:

The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.

Key result

Species / strain:

lymphocytes: Human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Additional information on results:

The test item induced evidence of toxicity in the 24-hour continuous exposure group at and above 2500 ug total protein/ml. There was also a very modest reduction in the mitotic index of the 4(20)-hour exposure group in the absence of S9 at and above 2500 ug total protein/ml.
Experiment 1: The test item did not induce any statistically significant increases in the frequency of cells with aberrations either in the absence or presence of metabolic activation.
Experiment 2: The test item did not induce any statistically significant increases in the frequency of cells with chromosome aberrations either in the absence of presence of metabolic activation.

Conclusion: Non-clastogenic to human lymphocytes in vitro.

Conclusions:

Cellulase is not clastogenic in the in vitro cytogenetic test using cultured human lymphocytes in both the presence and absence of metabolic activation.

Executive summary:

The objective of this assay was to investigate the potential of Cellulase (lot No. E21003SU3421Z1; 130.11 mg total protein/ml; 15.86% TOS) to induce numerical and/or structural changes in the chromosome of mammalian systems (i.e., human peripheral lymphocytes) in both the presence and absence of metabolic activation (Aroclor 1254-induced rat liver; S9-mix). This assay was conducted in accordance with OECD guideline No. 473 (1977) and complied with all standard GLP. Ten concentrations of Cellulase were used in the preliminary assay for the selection of dose levels in the main studies. Three doses were then selected for the two main assays with the highest dose level clearly inducing a toxic effect (50% reduction in mitotic index). In the absence of cytotoxicity, the highest dose selected would be 5000 µg Total Protein/ml (equivalent to 6100 µg TOS/ml) as recommended by the OECD guideline. The highest dose is equivalent to 6.37 mg enzyme concentrate dry matter/mL.

 

Duplicate cultures of human lymphocytes, treated with the test material, were evaluated for chromosome aberrations at 3 dose levels, together with negative control (minimal essential medium) and positive controls (Mitomycin C for assays in the absence of S9-mix and cyclophosphamide for assays with S9- mix).  The treatment conditions used for the study were: In Experiment 1, 4-hour exposure to the test item without S9-mix followed by 20-hour expression period and 4-hour exposure to the test item with S9-mix followed by 20-hour expression period. In Experiment 2, 24-hour continuous exposure to the test item without S9-mix and 4-hour exposure to the test item with S9-mix followed by 20-hour expression period. Mitosis was arrested by addition of Colcemid (0.1 µg/ml) 2 hours before the required harvest time.

 

No visible precipitation of the test material in the culture medium was observed. Mitotic inhibition of 38% and 23% was achieved at 5000 µg Total Protein/ml in the presence and absence of S9-mix. Consequently, in all treatment conditions, the dose range of cellulase used was 156.25 to 5000 µg Total Protein/ml. No biologically or statistically significant increases in cells with chromosomal aberrations were noted either in the presence or absence of metabolic activation. No statistically significant increase in the numbers of polyploidy cells was noted at any dose level either in the presence of absence of S9-mix. All of the negative control cultures had frequencies of cells with chromosomal aberrations within the expected range of historical control data. The positive control chemicals induced statistically significant increases in the frequency of cells with aberrations.

 

Cellulase shall be classified as non-clastogenic in this in vitro cytogenetic test using cultured human lymphocytes cells both in the presence and absence of metabolic activation.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

other information

Justification for type of information:

According to the ECHA Guidance Chapter R 7a: Endpoint specific guidance (version 2, Nov 2012), the following studies on genetic toxicity are required: In vitro gene mutation study in bacteria and one of the following, in vitro cytogenicity study in mammalian cells or an in vitro micronucleus study. In case these studies are both negative, an in vitro gene mutation study in mammalian cells is requested in addition. The present test substance, cellulase, has been investigated in two in vitro test systems, the Ames test and the in vitro chromosome aberration test. The tests have been performed according to current OECD guidelines, and in compliance with GLP. No evidence for genetic toxicitywas observed. The results are supported by read-across from three in vitro gene mutation studies in L5178Y mouse lymphoma cells performed on three different amylases belonging to the same subclass of glycosidases (IUBMB class 3.2.1.) as cellulase. The safety of the production strain is fully documented to belong to a safe strain lineage (Pariza and Johnson, 2001; Enzymes REACH Consortium, 2009) and the enzyme test material was well characterized. All enzyme classes are hydrophilic and readily biodegradable and in general, non-protease enzymes exhibit the same toxicological properties and although they are potential respiratory sensitizers, they are considered to be of low toxicity, confirmed by toxicity studies performed and published by the industry (summarized in Basketter et al. 2012a and 2012b). The physico-chemical properties of enzymes including logPow are very similar. They are further proteins built up of amino acids and the type, order and number of the amino acids in the polymer differs between enzymes, determining the 3-dimensional structure, the activity and specificity of the individual enzyme type. Industrial production strains typically have a history of safe use for many years in the production of technical and also often food grade enzymes. Because all enzymes are built up of the same amino acids the physical and chemical characteristics will be very similar for different enzymes, and hence read-across from other enzymes (e.g. amylase) should be fully applicable. The overall conclusion is that cellulase is not mutagenic and does not induce genotoxicity in the present test systems.
References - Pariza, M. W., and Johnson, E. A. (2001). Evaluating the Safety of Microbial Enzyme Preparations Used in Food Processing: Update for a New Century. Regulatory Toxicology and Pharmacology, 33: 173-186. - Enzymes REACH Consortium: Safety evaluation of technical enzyme products with regards to the REACH legislation. Document from Manufacturers, Importers and/or Only Representatives of one or more enzymes, who are subject to the registration requirements pursuant to REACH, 2009. http://www.enzymes-reach.org/documents.html - D. Basketter; N. Berg; F. Kruszewski; K. Sarlo; B. Concoby. The Toxicology and Immunology of Detergent Enzymes. 2012a. J. Immunotox 9(3): 320-6. - Basketter D., Berg N., Broekhuizen C., Fieldsend M., Kirkwood S., Kluin C., Mathieu S. and Rodriguez C. Enzymes in Cleaning Products: An Overview of Toxicological Properties and Risk Assessment/Management. 2012b. Reg. Toxicol. Pharmacol, 64/1: 117-123

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

The genetic toxicity of cellulase has been investigated in two test systems, the Ames test and the in vitro chromosome aberration test. All tests have been performed according to current OECD guidelines, and in compliance with GLP. No evidence for genetic toxicity was observed. The results are supported by read-across from three in vitro gene mutation studies in mouse lymphoma cells performed on three different amylases belonging to the same group of glycosidases as cellulase. The safety of the production strain is fully documented to belong to a safe strain lineage (Pariza and Johnson, 2001) and the enzyme test material was well characterized. Because enzymes are built up of the same amino acids, the physical and chemical characteristics will be very similar for different enzymes, and hence read-across from other non-proteolytic enzymes (e.g. amylase) should be fully applicable.

In conclusion, cellulase is not mutagenic and does not induce genotoxicity in any of the presented test systems.

Reference:

Pariza, M. W., and Johnson, E. A. (2001). Evaluating the Safety of Microbial Enzyme Preparations Used in Food Processing: Update for a New Century. Regulatory Toxicology and Pharmacology, 33: 173-186.

Justification for selection of genetic toxicity endpoint:
No genetic toxicity of cellulase could be detected in the in vitro gene mutation study in bacteria and the in vitro cytogenicity study in mammalian cells. The production strains of cellulase further meet the criteria for safe strain production micro-organisms. The conclusion that cellulase is non-genotoxic is supported by the read-across from in vitro gene mutation studies in mouse lymphoma cells of three other enzymes belonging to the IUBMB subclass 3.2.1 (glycosidases), where cellulase also belongs.

Justification for classification or non-classification

Due to the lack of genetic toxicity cellulase is not classified.