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Genencor International, Inc. OJ

March 17, 2005 Rebecca S, Cool, Branch Chief New Chemicals Notice Management Branch 7405M USEP A Headquarters Ariel Rios Building 1200 PennsylvaniaAvenue, N. W. Washington, DC 20460 925 Page Mill Road Palo Alto,(alifornia 94304 650.846.7500 650.845.6500fax www.genencor.com



### RE: Letter ofApplication tolist 

### Trichode171Ul reesei as exempt under subpart G of 40 

### CFR Part725- ReportingRequirements and Review Processes for Microorganisms
Dear Ms. Cool: Genencor International, Inc. is submitting this letter ofapplication to list 

### Trichodenna reese! as exemptunder subpart G of40 CFRPart 725- ReportingRequirements and Review Processes for Microorganisms. As required by40 CFR §725.67(2) any activities affected bythe requested exemption willnot present an unreasonable risk to health orthe environment. Information to support this request is given inthe enclosed attachment.
Weurge the EPA to act on this request, as it will be beneficial to both industry and the Agency, saving time from reviewing unnecessary MCANs on this microorganism. We look forward to your attention to this request. Should you have any questions, please do nothesitate to contact me. Sincerely, 

# Gu.:u d'**W.~** Alice 

### J. Caddow Vice President ofRegulatory And Environmental Affairs acaddow@genencor.com 650·846·7557
Enc.Rebecca S. Cool. March 17, 2005 Page 2



### RE: Letter ofApplication to list Trichoderma reesei 

### as exemptunder subpartGof 40 CFRPart725- ReportingRequirements and Review Processes for Microorganisms

The information to document this request is described in the following paragraphs: (i) The effects ofthe new microorganism onhealth and the environment. Trichoderma reesei is a filamentous fuugus first isolated from canvas in the Solomon Islands during World War II. It is found in soils in the tropics and probablyplays a natural role in the turnover oforganic matter due to its abilityto produce cellulases and xylanases. There are noreports ofits having a negative impact onthe environment. There is no evidence inthe literature that T. reesei is a human pathogen. Whenstudied in mice, guinea pigs and rabbits, itwas also found tobe

. non-pathogenic, only causing disease in**irnmuuo~suppressed** animals given a large spore inoculum. This finding was considered similarto those in studies on other well-known fungi used for enzyme production, such as Rhizomucormiehei (previouslyMucormiehei) andAspergillus spp. (Hjortkjaer et al., 1986).
T. reesei is notpresent on the list ofpathogens usedbythe EU (Directive Couucil Directive 90/679/EEC, as amended), oron culture collection lists from Germany (Deutsche Sammluug von Microorganismen uud Zellkulturen, DMSZ), the Netherlands (Centraal Bureau Schimmelculturen, CBS), or USA (American Type Culture Collection, ATCC). Itis listed as being suitable for the construction ofGenetically Modified Microorganisms (GMMs) ofRisk Group 1 inGermany and the Netherlands. Genencor has conducted several toxicity tests on T. reesei and enzymes produced from itthat were summarized in MCANs J-04-1, J-04-05, J-05- 01, includedhereinbyreference. These tests confirm that T. reesei is non- . pathogenic and non-toxicogenic. Trichoderma reesei is notknown to be a plant pathogen nor is itknown to have-significw'1t economic impact-as a food spoilage organism. Furthermore, in 1999, the U.S. FDA affirmed cellulase enzyme preparation derived from T. longibrachiatum (now kn()wn as T. reesei) as GRAS for use in processing food (PR 64:28658-28362; 1999). This assessment includes a determination that T. reesei is a safe host forRebecca S. Cool March 17, 2005 Page 3
production ofthis food-processing enzyme and that the host is nonpathogenic and non-toxigenic. 

### T. reesei is also listed as a source offood processing enzymes inthe Australia New Zealand Food Standards Code (http://www.foodstandards.gov.auJ srcfiles/Standard 1 3 3 Processing Aids v751.pdf), in the list ofExisting Food Additives in Japan and approved as a source offood additive enzymes in France. Cellulases and xylanases from 

### T. reesei are also approved for use in animal feed in the European Union, the U. S. and Canada. Authorities inthese countries determined that 

### T. reesei is a safe source for food and feed enzymes.
(ii) The magnitude ofexposure to humanbeings and theenvironment to the new microorganism. The onlypotential exposure to humanbeings is to workers in fermentation facilities handling the organism. This is because manufacturers generally inactivate the production organism after separating it from the enzyme product and assure thatthe organismis not in the product. Genencorhas included exposure estimates in the MCANs filed with the Agency that indicated minimal exposure to workers. The potential release ofthe organismto the environment was also discussed and a table indicating potential releasepoints was included. The three potential sources for release ofthe new microorganism from the manufacturing facility are via solid, liquid or gaseous emissions. During normal operation, no release is expected via solid or liquid waste emissions; a negligible amount ofrelease may occurinthe fermentor offgas. As the Genencor information onlyrelates to onecompany, alternatively, the exposure assessments utilized bythe Agency in 1996 to list the original organisms as exempt in40 CRF §725. 420 are also applicable here. Forexample, seehttp://www.epa.govlbiotech .rule/fra/fra006.htrn, IV. Exposure Assessment (includedherein by reference). The Agency's conclusion from these assessments thatthe worst case estimates do not suggesthigh levels ofexposure to either workers orthe public resulting from normal fermentation operations is valid for the fermentation use of 

### T. reesei. (iii) Thebenefits ofthe new microorganism for various uses and the availability of substitutes for such uses. 

### T. reesei has beenused for over forty years as a source ofcellulases and xylanases. Several enzyme companies have used this organism to produceRebecca S. Cool March 

## 17; 2005 Page4

its native cellulases and xylanases from traditionally derived and

.recombinant organisms (Harkki,et al, 1991; Barnett et at., 1991; KubicekPranz et aI., 1991; Ward et aI., 1991; Nevalainen et at, 1991; Uusitalo et aI., 1993; Fowler, 1993). Others have taken advantage ofits ability to express high levels ofproteinto produce heterologous gene products (Harkki et aI., 1989; Saloheimo and Niku-Paavaola, 1991; Uusitalo et al., 1991; Nyyssonen et aI., 1992; Joutsjoki et al., 1993; MCANs J-04-1, J-04- OS, J-05-01, included hereinbyreference). Recently, in 2000 and 2001 respectively, Genencor International and Novozymes both received contracts from the U. S. Department of Energy's National Renewable EnergyLab (NREL) to developT. resin as an economic source ofcellulases for use inthe biofuel industry. The concept was toreduce the cost ofthe enzymes used in biomass processing to be able to make that step inthe production offuel ethanol economical. Genencor, Novozymes and the NREL team were selected byR&D Magazine for its list ofthe Top 100 Technologically Significant Products for 2004. The organizations were cited for theirprogresstoward an economical process for convertingbiomass to ethanol. Now that this has been done, it will be difficult to easily substitute other enzymes into this process. Substitutes for some ofthe commercial applications for cellulases from T. reesei are cellulases isolated from othermicroorganisms such as Aspergillus niger, Trichodenna viride, Bacillus subtilis andHumicola insolens. (iv) Thereasonably' ascertainable economic consequences ofgranting or denying the exemption, including the effects onthe national economy, small business, and technological innovation

.

.Ifthe exemption is granted, companies will be able to file Tier 1 orTier 2 exemptions rather than MCANs when using this organism as a host. This will save time and moneyfor the companies involved and the EPA. In addition, products will be able to enter commerce faster. Innovation will be encouraged becauseT. reesei will be a recognized safe host for producing TSCAregulated chemicals.
40 CFR§725.67(3)(iii) Information regarding t.he following criteria should be addressed in an application to list a recipient microorganism under §725.420. (A) Identification and classification ofthe microorganism using available genotypic and phenotypic information;, Rebecca S. Cool March 17, 2005 PageS

Trichoderma spp. comprise a group ofmetabolically versatile aerobic mesophilic imperfect fungi, common in soil ofall climate zones (Domsch etal., 1980) and are particularly prevalent inthe litter ofhumid, mixed hardwood forests (Eveleigh, 1985). Trichoderma is a ubiquitous soil fungus and produces white, yellowish or green colonies when cultured. It is also common on wood. Trichoderma reesei is an asexually reproducing filamentous fungus. It was isolated.from cotton canvas inthe Solomon Islands during World War 

### II. Kuhls et al (1996) reported it as unique because it is known only from this single isolate (QM6a). However since that time, Samuels (one ofthe authors in Kuhls) has isolated itfrom otherparts ofthe tropics (Lieckfeldt et al, 2000). According to Samuels (personal communication) "theHypocreajecorina part of T. reesei is a common,. pantropical species. But, we do not often see T. reesei as a result ofdirect isolations from substrata." Strains of T. reesei used incommerce can all be traced to QM6a (see Nevalainen, etal., 1994). T. reesei is notknoWll to undergorecombination through meiosis. Classically T. reesei and other deutoromycetes have been described by their morphological characteristics. According to Kuhls, et aI., (ibid):
T. reesei was initially distinguished from Trichoderma viride

.Pers.:Fr. and Trichoderma longibrachiatum Rifai both onthe basis ofits unusuallyhigh production ofcelluolytic enzymes and on its morphological characteristics (Simmons, 1977). Considering only morphology, Bissett (1984) synonymized 

### T. reeseiunder T. IOYfgibrachiatum. However, restriction fragment length polymorphism methods (Morawetz, et al., 1992; Meyer, et al., 1992) and combinedmorphometric and isozyme analyses (Samuels, et aI., 1994; Leuchtmann, et aI., 1996) show that 

### T. reesei and T. longibrachiatum are taxonomically separable. Moreover, considerable morphological and isozyme differences between.temperate and tropical collections ofHypocrea schweinitzii ledto the recognitionthat the tropical collectionswere actuallyHypocreajecorina Berk. & Broome. Itwas also shown that T. reesei was more closely related to Hypocreajecorina than eitherfungus is to H. schweinitzii, T. longibrachiatum, or the other species of sect. Longibrachiatum (Samuels, et al., 1994).
Kuhls et al. (ibid) studied T. reesei and its putative teleomorphic ancestor, Hypocreajecorina along with other species ofthe Trichoderma sect. Longibrachiatum by PCR-fingerprinting and sequence analyses ofthe nuclear ribosomal DNAregion containing the internal transcribed spacers (ITS-l and ITS-2) and the 5.8S rRNA gene. The differences in theRebecca S. Cool March 17, 2005 Page 6
corresponding ITS sequences allowed a grouping ofanamorphic species·

. of Trichoderma sect. Longibrachiatum into T. longibrachiatum, T. pseudokoningii, andT. reesei. The sexual species H. schweintizii andH. jecorina were also clearly separated from each other. H.jecorina and T. reesei exhibited identical ITS sequences. PCR-fingerprinting patterns supported the differentiation based on ITS sequences, the grouping ofthe strains, and the assigrunent ofthese strains to individual species. They concludedthat T. reesei is an asexual, clonal line derived from a population ofthe tropical ascomycete H. jecorina. Dr.·Samuels (personal communication) now believes "that given the TrichodermaIHypocreapair, that all new Hypocrea species shouldbe described as Trichoderma with reference onlyto a Hypocrea teleomorph that does nothave its own name." Information in the literature that supports the safety and history of T. reesei also applies to H. jecorina. (B) Information to evaluate the relationship ofthe microorganism to any other closely related microorganisms which have a potential for adverse effects on .health orthe environment; Trichoderma species are used inbiological control (papavizas, 1985; Chet, 1987), inthe production ofenzymes (Kubicek, et aI., 1990; PenttiHi, et al., 1991) and antibiotics (Ghisalberti and Sivasithamparam, 1991). Nonehave. been identified as causing harm to humans or the environment. T. reesei is not closely related to species that cause harm to humanhealth or the environment. (C) A history of safe commercial usefor the microorganism;
T. reesei strain QM6a and derivatives ofit have beenused widely in industry, e.g., inJapan since the 1960's (Toyama, 1969). Several enzyme companies haveused this organism to produce its native cellulases.and xylanases from traditionally derived and recombinant organisms (Harkki,et aI, 1991; Barnett et aI., 1991; Kubicek-Pranz et al., 1991; Ward et al., 1991; Nevalainen et aI., 1991; Uusitalo et a!., 1993; Fowler, 1993). Others have taken advantage ofits ability to expresshigh levels ofprotein to produceheterologous gene products (Harkki et aI., 1989; Saloheimo and Niku-Paavaola, 1991; Uusitalo et aI., 1991; Nyyssiinen et al., 1992; Joutsjoki et a!., 1993; MCANs J-04-1,). The safety of T. reesei for industrial enzyme production was reviewed by Nevalainen, et a!. (1994). They indicated that T. reesei has beenused to produce enzymes used in the food industry includingbaking, malting, brewing and grain alcohol production, in the animal feed industry to improve the digestibility offeed,.as digestive aids and in wood processing,Rebecca S. Cool March 17, 2005 Page 7

the textile indnstry and-in the saccharification oflignocellulosic biomass for biofuel production. (D) Commercial uses indicating thatthe microorganism products mightbe subject to TSCA; Cellulases and xylanases from T. reesei have been used in wood processing, biomass conversion, detergent, textile and biofuel applications. T. reesei has also beenthe subject ofthree MCANs fliedby Genencor International in 2004-2005. (E) Studies which indicate the potential for the microorganism to cause adverse effects to health orthe environment; The safety of T. reesdfor industrial enzyme production was reviewed by Nevalainen, etal. (1994). They discussed the organisms potential pathogenicity and allergenicity, occurrence ofantibiotics and rnycotoxins and safety in food use as follows: There is no evidence in the literature that T. reesei would be a pathogen for man. Inthepathogenicity study ofT. reesei made

. with mice, gninea"pigs and rabbits (Hjortkjaer et al., 1986), the

data obtained also confirmed that T. reesei can be regarded as nonpathogenic. Only under extreme conditions, involving a large spore inocululm given to immunocsuppressed animals, could the microorganism act as a pathogen. This finding was considered similar to those in studies on other well-known fungi used for enzymeproduction, such as Rhizomucormiehei (previouslyMucor miehei) andAspergillus spp. (Hjortkjaeret al., 1986). Several conidia ofmolds are known to cause allergic diseases (Kabi PharmaciaDiagnostics AB, 1992). There is, however, no evidence inthe literature that specifies T. reesei as a source of molds known to cause allergic diseases. The antifungal compound trichoderrnin has been isolated and a volatile unidentified component detected from a T. reesei mutant strain P-12 (Watts et aI., 1988) and a peptaibol has been isolated from T. longibrachiautm (Briickner et al., 1990). However, in the study performed by Hjortkjaer et al. (1986) ofaT. reesei strain used in manufacture ofa commercial cellulose preparation, the tests on a cellulasepreparation in accordance with the recommendations ofthe Joint FAOIWHO Expert Committee on Food Additives 1981 (JECFA), gave no indication thatinhibitoryRebecca S. Cool March 17,2005 Page 8

substances were producedby T. reesei during its growtb. The industrial enzymepreparations are confirmed bythe enzyme producers notto have antibiotic activity according to the specifications recommended byJECFA (Joint FAOIWHO Expert Committee on FoodAdditives, 1990).

It shouldbe noted that JECF A removed their general specification for mycotoxin testing (aflatoxin B, ochratoxin A, sterigmatocystin, T -2 toxin and zearalenone) ofall fungal enzyme preparations and has indicated that onlyrelevant mycotoxinsreported inthe species of close relatives shouldbe tested (JECFA, 57thmeeting, Rome 5-14 June, 2001; http://www,who.intlipcs/foodljecfalsurtnnaries/en/summary_57.pdf). However, inbetween 1981 and 2001, hundreds oflots ofenzyme products from T. reesei musthave beentested andfound to be negativefor T-2, the only relevant mycotoxin for this species (see below).'·

.. . Blumenthal (2003) also indicated thatWatts et al. (1988) had reported the isolation oftrichoderminfrom T. reesei and indicated that this may need further confirmation. She indicated that van Dijck (personal communication) is ofthe opinion thattrichodermin is only producedby T. harzianum. She went on to say that T-2 toxin has beenreportedfrom Trichoderma and should continue to betestedin commercial food use enzyme preparations from Trichoderma species. (F) Studies which indicate the survival characteristics ofthe microorganism in the environment.


### T. reesei and H.jecorina have only been isolated inthe tropics; 

### T. reesei has been reported from several locations and H.jecorina is pantropical. Neither form is reported from the United States. Genencor conducted a survival study ofa traditionally derived strain of T. reesei and a recombinant one (see Appendix 4, MCANJ-04-1). In sterile and non-sterile soil and water, both strains werefound to persist in

. numbers or decline, indicating t.1}at t.he Strai.11S are not able to proliferate, but presumablyturn into a dormant state.Rebecca S. Cool March 17, 2005 Page 9

References Bissett, J. (1984) Can. J. Bot. 62,924-931. Chet, I. (1987) inInnovativeApproaches to PlantDisease Control, ed. Chet, I. (Wiley, New York), pp. 137-160. Doi, N. & Doi, Y. (1979) Bull. Nat!. Sci. Mus. Tokyo Ser. B 5, 117-123. Domsch, K. H., Gams, W. and Anderson, T.-R.1980. Compendium ofSoil Fnngi. Academic Press, London. Vo!. I, p. 859 and II, p. 405. Esser, K. & Lemke, p, A. (1995) The Mycota (Springer, Berlin). Eveleigh D.E. 1985. Trichoderma. In: Demain, A.L. and Solomon, N.A. (Eds.), Biology ofIndustrial Microorganisms. Benjamin Cunnings, London, pp. 487-509. Gams, W. (1995) Can. 

### 1. Bot. 73, Supp!. 1, S747-S753. Ghisalberti, E. L. & Sivasithamparam, K. (1991) SoilBioI. Biochem. 23,1011-1020. Kubicek, C. P., Eveleigh, D. E., Esterbauer, R., Steiner, W. & Kubicek-Franz, E. M., eds. (1990) Trichoderma reesei Cellulases: Biochemistry, Genetics, Physiology andApplications, (R. Soc. Chern. Press, Cambridge, U.K.). Leuchtmann, A., Petrini, O. & Samuels, G. J. (1996)Mycologia 88,384-394. Lieckfeldt, E., C. Kullnig, G.J. Samuels, and C.P. Kubicek. 2000. Sexually competent, sucrose- and nitrate-assinrilating strains ofHypocreajecorina(Trichoderma reesei) from South American soils. Mycologia 92: 374-380. Meyer, W., Morawetz, R., Bo"mer, T. & Kubicek, C. P. (1992) Curro Genet. 21,27-30. 'Ko"a'we<z D r!.ruho

.. ]:1 Messno" R &**K"b;~ok** C P ( 1002) IV.l.l l.., ..L'\.._, ........1 U\"-.l, J.: "' ..I. .1 \"-.1, • ..L U .1""""''';,

. ..L • \..L

.......... Curro Genet. 21,31-36. Nevalainen, R., Suominen, P., and Taimisto, K. 1994. Onthe safety of Trichoderma reesei, J. Biotechno!. 37: 193-200., RebecCli S. Cool March 17, 2005 Page 10
Papavizas, G, C. (1985)Annu. Rev. Phytopathol. 23,23-54. Penttila··, M., Teeri, T. T., Nevalainen, H. & Knowles, 

### J. K. C. (1991) inApp/iedMolecular Genetics of Fungi, eds. Peberdy, 

### J. F., Caten, C. E., Ogden, J. E. & Bennett, J. W. (Cambridge Univ. Press, Cambridge, U.K.), pp. 85-101. Samuels, G. J., Petrini, O. & Manguin, S. (1994) Mycologia 86, 421-435. Sheir-Neiss, G. and Montenecourt, B.S. 1984. Characterization ofthe secreted cellulases of Trichoderma reesei wild type and mutants during controlled fermentations. Appl. Microbiol. Biotechnol. 20: 46-53.

. Simmons, E. G. (1977) in SecondInternational Mycological Congress, eds. Bigelow, H. E. & Simmons, E. G. (Hamilton I. Newell, Amherst, MA), Vol. 2, p; 618. Toyama, N., 1969. Applications ofCellulases inJapan. In: CellulasesandTheir Applications, R. F. Gould (Ed.), Advances in Chemistry Series 95, American Chemical Society, Washington, DC, p. 359-390.