New substances: risk assessment summary 13912
This document has been prepared to explain the regulatory decision taken under Part 6 of the Canadian Environmental Protection Act, 1999 (CEPA 1999) regarding the manufacture or import of Trichoderma reesei P345A by Iogen Corporation in a contained facility located in Ottawa.
Trichoderma reesei P345A was notified pursuant to subsection 29.11(4) of the CEPA 1999 New Substances Notification Regulations (NSNR).
The New Substances Branch of Environment Canada and the New Substances Assessment and Control Bureau of Health Canada have assessed the information submitted by Iogen Corporation and other available scientific information in order to determine whether T. reesei P345A is toxicFootnote 1 or capable of becoming toxic as described in section 64 of CEPA 1999.
Based on the hazard and exposure considerations, the joint risk assessment conducted by Environment Canada and Health Canada concluded that Trichoderma reesei P345A is not considered to be toxic to the Canadian environment or human health as described in section 64 of the CEPA 1999.
Therefore, the manufacture in or import to a contained facility of T. reesei P345A for use in the contained facility or for export only, may proceed after June 22, 2005.
This evaluation does not include an assessment of human health risk in the occupational environment nor does it include an assessment of the potential exposure and risk to humans associated with the use of the organism in or as an item that falls under the purview of the Food and Drugs Act.
NSN schedule: XVI (manufacture in or import to a contained facility of a micro-organism that is not for introduction outside a contained facility or is for export only)Footnote 2
Organism identity: Trichoderma reesei P345A
Notifier: Iogen Corporation, 310 Hunt Club Rd. East Ottawa, Ontario K1V 1C1 Canada
Date of decision: February 12, 2004
Proposed use: Commercial production, in a contained facility, of a thermophilic/alkalophilic xylanase II enzyme by genetically engineered strain Trichoderma reesei P345A.
Strain history/genetic modification
Trichoderma reesei P345A was derived from an auxotrophic mutant of the parental strain M2C38 through the introduction of the linear transformation vector pc/xHTX47a-TV. The selection cassette used in the construction of the transformation vector contains a Neurospora crassa gene functioning as a selectable marker. The expression cassette consisted of a copy of the modified version of the xylanase II (xln2) structural gene that produces a novel thermophilic/alkalophilic T. reesei xylanase II protein under the control of T. reesei regulatory sequences. Strain M2C38 (ATCC 74252) is a derivative of the T. reesei strain RUTC30 (ATCC 56765), and is a mutagenic derivative of the founder strain QM6a (ATCC 13631) which was isolated in the Solomon Islands from cotton canvas during World War II (Kuhls et al., 1996).
In addition to the information provided by the notifier, a review of in-house reference material and a comprehensive search of the scientific literature were conducted to gather information on potential harmful environmental and human health effects attributable to T. reesei.
Trichoderma species are common soil saprophytes and are metabolically versatile, aerobic, mesophilic, imperfect fungi (Nevalainen et al., 1994). The Trichoderma species are differentiated primarily by patterns of conidiophore branching and conidia morphology. They are widespread in nature, quick-growing, easy to culture and they can produce large amounts of conidia with long lifetime (Manczinger et al., 2002).
In general, large scale industrial manufacture of T. reesei enzyme preparations have a history of safe use in many industries including starch and animal feed processing, grain alcohol fermentation, malting and brewing, extraction of fruit and vegetable juices, in the pulp and paper, and textiles (Hjortkjaer et al., 1986). Based on the criteria outlined in the Organisation for Economic Co-operation and Development guidelines entitled Recombinant DNA Safety Considerations (OECD, 1986) and the European Communities Council (ECC) Directive 90/219/EEC on the contained use of genetically modified micro-organisms (ECC, 1990), Trichoderma species can be regarded as safe host organisms.
Trichoderma reesei has been shown to be non-pathogenic and non-toxic to healthy laboratory animals (Hjortkjaer et al., 1986). Trichoderma reesei is not reported to be a frank pathogen of plants or animals including humans. However, this species can act as an opportunistic pathogen to immunosuppressed animals under extreme experimental conditions (Hjortkjaer et al., 1986). Some Trichoderma species have been cited as rare and newly emerging fungal pathogens (Fleming et al., 2002). The parental strain M2C38 has been in use since 1991 and has never been associated with human infection.
While certain species of the genus Trichoderma can be used as biocontrol agents in agriculture for their ability to produce antifungal compounds against several plant pathogenic fungi, T. reesei P345A is not one of them. Some species of Trichoderma may also produce toxins under certain conditions; however, experience with T. reesei indicates that it is not likely to be toxigenic (Hjortkjaer et al., 1986). Tests conducted on commercial enzyme preparations confirm that neither antibiotics nor inhibitory substances are produced during the growth of industrial T. reesei strains (Hjortkjaer et al., 1986).
In industrial settings, allergic reactions to carbohydrases have been previously reported. Like other carbohydrases, xylanases can also be allergens. Toxicity studies on native xylanases from Aspergillus and Thermomyces administered orally to rats and mice did not result in adverse effects (Pederson and Broadmeadow, 2000). Native xylanases were not found to be mutagenic in the Salmonella typhimurium reverse mutation assay, nor did they cause chromosomal aberrations in cultured human lymphocytes (Pederson and Broadmeadow, 2000).
Previously, some allergic reactions to T. reesei M2C38 strain have been reported with plant workers but the use of appropriate personal protective equipment, such as personal ventilators, largely eliminated this problem. The notifier indicated that the notified enzyme does not contain any detectable levels of antifungal compounds or heavy metals.
Neurospora crassa, the fungal source of the selectable marker gene used in the construction of the transformation vector, is not reported to be a frank pathogen. The N. crassa gene product makes selection of T. reesei strain P345A, from a mixture of other microorganisms, easier and is unlikely to pose a risk to the environment since it has many functional equivalents in most living organisms.
Both T. reesei and N. crassa are listed as ‘Biosafety Level 1’ organisms by the American Type Culture Collection. In addition, T. reesei has been designated as a ‘Risk Group 1’ organism by the Office of Laboratory Security of the Public Health Agency of Canada.
The DNA fragments used in the construction of the transformation vector are well characterized and do not contain any large undefined fragments. According to the notifier, at least a part of the ampicillin resistance gene coding for β-lactamase has integrated into the P345A strain chromosome. However, the ampicillin resistance gene used in the construction of the transformation vector is under the control of a bacterial promoter that will not function in T. reesei. In addition, the vector DNA has been shown to be stably integrated into the chromosome without loss or rearrangement of the sequence even after several generations on non-selective media. Therefore, the potential for lateral gene transfer from this organism to humans, animals or other microbes in the environment is extremely low.
As a result of the genetic modification, the novel xylanase II has an increased thermal stability and a higher pH optimum compared to the native enzyme. Genetic modifications performed to develop T. reesei P345A do not give rise to concerns of altered virulence or pathogenicity to humans, animals, plants or altered hazards to the environment. The phenotype resulting from the modification is well characterized and is not likely to influence the normal behavior of T. reesei.
Trichoderma species, including T. reesei are common soil saprophytic fungal species found in all climate zones and are particularly prevalent in the litter of humid, mixed hardwood forests (Nevalainen et al., 1994).
Trichoderma reesei P345A is manufactured solely as an intermediate in the production of a modified xylanase enzyme in a contained facility. The notifier indicated that the manufacturing process meets the standards for the Good Large Scale Practice (GLSP) level as defined in Appendix K of the NIH Guidelines for Research Involving Recombinant DNA Molecules (NIH, 2002). The notified strain is not intended for release outside the contained facility. Consequently, the potential exposure to the general population and the environment is expected to be low.
The notifier describes procedures which will limit potential worker exposure. These include the use of protective equipment, such as National Institute for Occupational Safety and Health (NIOSH) approved respiratory masks with particulate filters, face shield, or safety goggles with side shields, rubber gloves, lab coats or overalls for workers who are chronically exposed to enzyme dusts or aerosols during procedures such as transfers of fermentation broths.
Precautions are in place and used by the notifier to ensure that exhaust and aerosols from the fermentor are decontaminated by UV irradiation to kill any organisms and volatiles and odours are removed by a cyclone and scrubber system. The fermentor is equipped with an alarm to indicate high pressure, foam-over and low-level and is diked in case of massive leakage. Trichoderma reesei P345A is not intrinsically hazardous, thus, inadvertent release from the manufacturing facility is not expected to pose significant risk to the environment and human health.
When enzyme production is complete, the spent cell mass is chemically inactivated using a quaternary ammonium compound with 99.999% effectiveness prior to disposal in a registered landfill or composting sites in accordance with provincial regulations. Laboratory studies on survival of T. reesei suggest that it can survive under cold climatic conditions (Providenti et al., 2004). Given that T. reesei P345A lacks pathogenicity and toxicity potential, the likelihood of significant harm to the environment or human health resulting from the disposal route of exposure is expected to be minimal.
ECC. 1990. Council Directive 90/219/EEC on contained use of genetically modified micro-organisms. Official Journal L 117 , 08/05/1990 P. 0001 - 0014
Fleming, R. V., Walsh, T.J. and Anaissie, E.J. 2002. Emerging and less common fungal pathogens. Infectious Clinic Diseases of North America. 16(4): 915-933.
Hjortkjaer, R.K., Bille-Hansen, V., Hazelden, K.P., McConville, M., McGregor, D.B., Cuthbert, J.A., Greenough, R.J., Chapman, E., Gardner, J.R. and Ashby, R. 1986. Safety evaluation of Celluclast®, an acid cellulase derived from Trichoderma reesei. Journal of Food and Chemical Toxicology. 24(1): 55-63.
Kuhls, K., Lieckfeld, E., Samuels, G.J., Kovacs, W., Petrini, O., Gams, W., Borner, T. and Kubicek, C.P. 1996. Molecular Evidence that the asexual industrial fungus Trichoderma reesei is a clonal derivative of the ascomycete Hypocrea jecorina. Proc. Nat. Acad. Sci. USA. 93: 7755-7760.
Manczinger, L., Antal, Z. and Kredics, L. 2002. Ecophysiology and breeding of mycoparasitic Trichoderma strains (a review). Acta Microbiologica et Immunologica Hungarica. 49(1): 1-14.
NIH. 2002. Guidelines for Research Involving Recombinant DNA Molecules -Appendix K, Department of Health and Human Services, National Institutes of Health.
Nevalainen, H., Suominen, P. and Taimisto, K. 1994. Minireview on the safety of Trichoderma reesei. Journal of Biotechnology. 37: 193-200.
Organisation for Economic Co-operation and Development (OECD). 1986. Recombinant DNA Safety Considerations: Safety Considerations for Industrial, Agricultural and Environmental Applications of Organisms Derived by Recombinant DNA Techniques. [PDF]
Pedersen, P.B. and Broadmeadow, A. 2000. Toxicological studies on Thermomyces lanuginosus xylanase expresssed by Fusarium venenatum, intended for use in food. Food Additives and Contaminants. 17(9): 739-747.
Pico Y., Fernandez, M., Rodriguez, R., Almudever, J., Manes, J., Font, G., Marin, R., Carda, C., Manzanares, P. and D. Ramon. 1999. Toxicological assessment of recombinant xylanase X22 in wine. Journal of Agricultural and Food Chemistry. 47:1597-1602.
Providenti, M.A., Mautner, I.S., Chaudhry, O., Bombardier, M., Scroggins, R., Gregorich, E. and Smith, M.L. 2004. Determining the environmental fate of a filamentous fungi, Trichoderma reesei, in laboratory-contained intact soil-core microcosms using competitive PCR and viability plating. Canadian Journal of Microbiology. 50: 623-631.
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