New substances risk assessment summary: 11017

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This document has been prepared to explain the regulatory decision taken under Part 6 of the Canadian Environmental Protection Act, 1999 (CEPA 1999)Footnote 1 regarding the manufacture or import of Trichoderma reesei1391A by Iogen Corporation  in a contained facility.

T. reesei 1391A was notified pursuant to subsection 29.11(4) of the CEPA 1999 New Substances Notification (NSN) Regulations.

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 1391A is toxic or capable of becoming toxic as described in section 64 of CEPA 1999.

Regulatory Decision:

Based on the hazard and exposure considerations, the joint risk assessment conducted by Environment Canada and Health Canada concluded that Trichoderma reseei 1391A is not considered to be toxic to the Canadian environment or human health as described in section 64 of the CEPA 1999.Therefore, manufacture in a contained facility or import to a contained facility of T. reesei 1391A for use in the contained facility or for export only, may proceed after September 30, 2001. 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 a contained facility or import to a contained
facility a micro-organism that is not for introduction outside a
contained facility or is for export only)
Organism Identity: Trichoderma reesei1391A
Notifier: Iogen Corporation, 8 Colonnade Rd, Ottawa, Ontario, K2E 7M6,
Canada
Date of decision: September 30, 2001
Proposed use: Production, in a contained facility, of a novel xylanase II enzyme with
enhanced thermal stability by genetically engineered Trichoderma reesei 1391A.

Strain History/Genetic Modification:

T. reesei 1391A was derived from an auxotrophic mutant of the parental strain, M2C38, by the introduction of a fragment of the transformation vector pFPX#2-TV. The parental strain M2C38 is a derivative of the public T. reesei strain RUTC30.  The selection cassette used in the construction of the transformation vector contains a Neurospora crassa gene functioning as a selectable marker. The expression cassette consists of the modified T. reesei xylanase II structural gene (xln2) under the control of T. reesei regulatory sequences.  RUTC30 was obtained from the American Type Culture Collection (ATCC), and is a derivative of the founder strain QM6a (ATCC 13631), which was isolated in the Soloman Islands from cotton canvas during World War II  (Kuhls et al., 1996). 

Hazard considerations:

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 metabolically versatile, aerobic, mesophilic, imperfect fungi (Nevalainen et al., 1994). The Trichoderma species are differentiated primarily by patterns of conidiophore branching and conidia morphology.

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, pulp and paper industry, and textile industry (Hjortkjaer et al., 1986).

T. reesei has been shown to be non-pathogenic and non-toxic to healthy laboratory animals (Hjortkjaer et al., 1986).  However, this species can act as an opportunistic pathogen to immunosuppressed animals under extreme experimental conditions (Hjortkjaer et al., 1986). T. reesei is not reported to be a frank pathogen of plants or animals including humans.

Some species of Trichoderma may produce toxins under certain conditions; however, experience with T. reeseiindicates 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). Furthermore, tests conducted on this commercial enzyme preparation produced by strain 1391A confirms adherence to recommendations regarding non-detectable amounts of the mycotoxin aflatoxin B1,.

While certain species of the genus Trichoderma can be used as biocontrol agents in agriculture since they are known antagonists to several plant pathogenic fungi, T. reesei1391A is not one of them. 

N. 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 this strain, 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 ATCC. 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.

DNA fragments used in the construction of the transformation vector are well characterized and do not contain any large undefined fragments. Sequences encoding ampicillin resistance, used for the construction of the transformation vector, have been removed from the final construct. The genetic modifications performed to develop T. reesei 1391A 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.

Toxicity studies on native xylanasesfrom 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).

Exposure considerations:

T. reesei is a common soil saprophytic fungal species. Trichoderma species are common in soil in all climate zones and are particularly prevalent in the litter of humid, mixed hardwood forests (Nevalainen et al., 1994).

T. reesei 1391A is manufactured solely as an intermediate in the production of a novel xylanase II enzyme in a contained facility, and is not intended to be released out of the contained facility. Consequently, the potential exposure of the general population and the environment is expected to be low.

According to Health Canada’s Laboratory Biosafety Guidelines (1996), the recommended safe handling, storage, and transportation procedures for Risk Group 1 are as follows:  Appropriate personal protective equipment for handling the organism is recommended, including lab coat, closed-toe shoes and gloves. No special design features beyond those suitable for a well-designed and functional laboratory with good microbiology practices are required.  Recommended storage and transportation in an appropriately labeled container designed, constructed, filled, closed, secured and maintained is also required so that under normal conditions of transport, including handling, there will be no accidental release.

The manufacturing process meets the standards for the Good Large Scale Practices (GLSP) level as defined in Appendix K of the Guidelines for Research Involving Recombinant DNA Molecules (NIH Guidelines). The notifiers describe procedures which will limit potential worker exposure. These include the use of protective equipment 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 such procedures as transfers or 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 and cyclone and scrubber system.  The fermentor is equipped with an alarm to indicate high pressure, foam-over and low-level and is diked in the case of massive leakage. Spent cell mass is chemically treated using quaternary ammonium compound with 99.999% effectiveness prior to disposal in registered landfill or composting sites in accordance with provincial regulations. Given that T. reesei 1391A is not intrinsically hazardous, the likelihood of significant harm to the environment or human health resulting from this route of exposure is expected to be minimal.

References:

Health Canada, Laboratory Centre for Disease Control.  1996. Laboratory Biosafety Guidelines, 
Second Edition.  Kennedy, M.E., ed.  84 pp.

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.

Nevalainen, H., Suominen, P., and Taimisto, K. 1994. Minireview on the safety of Trichoderma
reesei. Journal of Biotechnology.  37: 193-200.

Pedersen, P.B. and Broadmeadow, A. 2000. Toxicological studies on Thermomyces
lanuginosus xylanase expressed in Fusarium venenatum, intended for use in food. Food
Additives and Contaminants. 17(9): 739-747.

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