i @) 

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 Pennsylvania Avenue, N. W. 

Washington, DC 20460 

925 Page Mill Road 

Palo Alto, (alifornia 94304 

650.846.7500 

650.845.6500 fax 

www.genencor.com 

RE: Letter of Application to list Trichode171Ul reesei as exempt under subpart G of 

40 CFR Part 725- Reporting Requirements and Review Processes for 

Microorganisms 

Dear Ms. Cool: 

Genencor International, Inc. is submitting this letter of application to list Trichodenna 

reese! as exempt under subpart G of 40 CFR Part 725- ReportingRequirements and 

Review Processes for Microorganisms. As required by 40 CFR §725.67(2) any activities 

affected by the requested exemption will not present an unreasonable risk to health or the 

environment. Information to support this request is given in the enclosed attachment. 

We urge 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 

not hesitate to contact me. 

Sincerely, 

Gu.:u d' W.~ Alice J. Caddow 

Vice President of Regulatory 

And Environmental Affairs 

acaddow@genencor.com 

650·846·7557 

Enc. 
Rebecca S. Cool. 

March 17, 2005 

Page 2 

RE: Letter of Application to list Trichoderma reesei as exempt under subpartG of 

40 CFR Part 725- Reporting Requirements and Review Processes for 

Microorganisms 

The information to document this request is described in the following paragraphs: 

(i) The effects of the new microorganism on health 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 probably plays a natural role in the turnover of organic matter 

due to its ability to produce cellulases and xylanases. There are no reports 

of its having a negative impact on the environment. 

There is no evidence in the literature that T. reesei is a human pathogen. 

When studied in mice, guinea pigs and rabbits, it was also found to be . 

non-pathogenic, only causing disease in irnmuuo~suppressed animals 

given a large spore inoculum. This finding was considered similar to 

those in studies on other well-known fungi used for enzyme production, 

such as Rhizomucor miehei (previously Mucor miehei) and Aspergillus 

spp. (Hjortkjaer et al., 1986). 

T. reesei is not present on the list of pathogens used by the EU (Directive 

Couucil Directive 90/679/EEC, as amended), or on 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). It is listed as being suitable for the construction of Genetically 

Modified Microorganisms (GMMs) of Risk Group 1 in Germany and the 

Netherlands. 

Genencor has conducted several toxicity tests on T. reesei and enzymes 

produced from it that were summarized in MCANs J-04-1, J-04-05, J-05-

01, included herein by reference. These tests confirm that T. reesei is non- . 

pathogenic and non-toxicogenic. 

Trichoderma reesei is not known to be a plant pathogen nor is it known 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 for 
Rebecca S. Cool 

March 17, 2005 

Page 3 

production of this food-processing enzyme and that the host is nonpathogenic and non-toxigenic. T. reesei is also listed as a source of food 

processing enzymes in the Australia New Zealand Food Standards Code 

(http://www.foodstandards.gov.auJ srcfiles/Standard 1 3 3 Processing 

Aids v751.pdf), in the list of Existing Food Additives in Japan and 

approved as a source of food 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 in these countries 

determined that T. reesei is a safe source for food and feed enzymes. 

(ii) The magnitude of exposure to human beings and the environment to the new 

microorganism. 

The only potential exposure to human beings 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 that the organism is not in the product. Genencor has 

included exposure estimates in the MCANs filed with the Agency that 

indicated minimal exposure to workers. 

The potential release of the organism to the environment was also 

discussed and a table indicating potential release points was included. The 

three potential sources for release of the 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 of release may occur in the fermentor offgas. 

As the Genencor information only relates to one company, alternatively, 

the exposure assessments utilized by the Agency in 1996 to list the 

original organisms as exempt in 40 CRF §725. 420 are also applicable 

here. For example, see http://www.epa.govlbiotech .rule/fra/fra006.htrn, 

IV. Exposure Assessment (included herein by reference). The Agency's 

conclusion from these assessments that the worst case estimates do not 

suggest high levels of exposure to either workers or the public resulting 

from normal fermentation operations is valid for the fermentation use of T. 

reesei. 

(iii) The benefits of the new microorganism for various uses and the availability of 

substitutes for such uses. 

T. reesei has been used for over forty years as a source of cellulases and 

xylanases. Several enzyme companies have used this organism to produce 
Rebecca S. Cool 

March 17; 2005 

Page 4 

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 of its ability to 

express high levels of protein to 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 herein by reference). 

Recently, in 2000 and 2001 respectively, Genencor International and 

Novozymes both received contracts from the U. S. Department of 

Energy's National Renewable Energy Lab (NREL) to developT. resin as 

an economic source of cellulases for use in the biofuel industry. The 

concept was to reduce the cost of the enzymes used in biomass processing 

to be able to make that step in the production of fuel ethanol economical. 

Genencor, Novozymes and the NREL team were selected by R&D 

Magazine for its list of the Top 100 Technologically Significant Products 

for 2004. The organizations were cited for their progress toward an 

economical process for converting biomass to ethanol. Now that this has 

been done, it will be difficult to easily substitute other enzymes into this 

process. 

Substitutes for some of the commercial applications for cellulases from T. 

reesei are cellulases isolated from other microorganisms such as 

Aspergillus niger, Trichodenna viride, Bacillus subtilis and Humicola 

insolens. 

(iv) The reasonably' ascertainable economic consequences of granting or denying 

the exemption, including the effects on the national economy, small business, 

and technological innovation . 

. If the exemption is granted, companies will be able to file Tier 1 or Tier 2 

exemptions rather than MCANs when using this organism as a host. This 

will save time and money for 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 TSCA regulated 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 of the microorganism using available genotypic 

and phenotypic information; 
, Rebecca S. Cool 

March 17, 2005 

PageS 

Trichoderma spp. comprise a group of metabolically versatile aerobic 

mesophilic imperfect fungi, common in soil of all climate zones (Domsch 

et al., 1980) and are particularly prevalent in the litter of humid, 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 in the 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 

of the authors in Kuhls) has isolated it from other parts of the tropics 

(Lieckfeldt et al, 2000). According to Samuels (personal 

communication) "the Hypocrea jecorina part of T. reesei is a common, . 

pantropical species. But, we do not often see T. reesei as a result of direct 

isolations from substrata." Strains of T. reesei used in commerce can all 

be traced to QM6a (see Nevalainen, etal., 1994). T. reesei is notknoWll 

to undergo recombination 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 on the basis 

of its unusually high production of celluolytic enzymes and on its 

morphological characteristics (Simmons, 1977). Considering only 

morphology, Bissett (1984) synonymized T. reesei under T. 

IOYfgibrachiatum. However, restriction fragment length 

polymorphism methods (Morawetz, et al., 1992; Meyer, et al., 

1992) and combined morphometric 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 of Hypocrea 

schweinitzii led to the recognition that the tropical collections were 

actually Hypocreajecorina Berk. & Broome. It was also shown 

that T. reesei was more closely related to Hypocrea jecorina than 

either fungus 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, 

Hypocrea jecorina along with other species of the Trichoderma sect. 

Longibrachiatum by PCR-fingerprinting and sequence analyses of the 

nuclear ribosomal DNA region containing the internal transcribed spacers 

(ITS-l and ITS-2) and the 5.8S rRNA gene. The differences in the 
Rebecca S. Cool 

March 17, 2005 

Page 6 

corresponding ITS sequences allowed a grouping of anamorphic species· 

. of Trichoderma sect. Longibrachiatum into T. longibrachiatum, T. 

pseudokoningii, andT. reesei. The sexual species H. schweintizii and H. 

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 of the 

strains, and the assigrunent of these strains to individual species. They 

concluded that T. reesei is an asexual, clonal line derived from a 

population of the tropical ascomycete H. jecorina. Dr.· Samuels (personal 

communication) now believes "that given the TrichodermaIHypocrea pair, 

that all new Hypocrea species should be described as Trichoderma with 

reference only to a Hypocrea teleomorph that does not have 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 of the microorganism to any other 

closely related microorganisms which have a potential for adverse effects on 

.health or the environment; 

Trichoderma species are used in biological control (papavizas, 1985; 

Chet, 1987), in the production of enzymes (Kubicek, et aI., 1990; 

PenttiHi, et al., 1991) and antibiotics (Ghisalberti and Sivasithamparam, 

1991). None have. been identified as causing harm to humans or the 

environment. T. reesei is not closely related to species that cause harm to 

human health or the environment. 

(C) A history of safe commercial use for the microorganism; 

T. reesei strain QM6a and derivatives of it have been used widely in 

industry, e.g., in Japan since the 1960's (Toyama, 1969). Several enzyme 

companies have used 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 of its ability to express high levels ofprotein 

to produce heterologous 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 been used to 

produce enzymes used in the food industry including baking, malting, 

brewing and grain alcohol production, in the animal feed industry to 

improve the digestibility of feed,. as digestive aids and in wood processing, 
Rebecca S. Cool 

March 17, 2005 

Page 7 

the textile indnstry and -in the saccharification of lignocellulosic biomass 

for biofuel production. 

(D) Commercial uses indicating that the microorganism products might be 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 been the subject of three MCANs flied by 

Genencor International in 2004-2005. 

(E) Studies which indicate the potential for the microorganism to cause adverse 

effects to health or the environment; 

The safety of T. reesdfor industrial enzyme production was reviewed by 

Nevalainen, et al. (1994). They discussed the organisms potential 

pathogenicity and allergenicity, occurrence of antibiotics 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. In the pathogenicity study of T. 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 

enzyme production, such as Rhizomucor miehei (previously Mucor 

miehei) and Aspergillus spp. (Hjortkjaeret al., 1986). 

Several conidia of molds are known to cause allergic diseases 

(Kabi Pharmacia Diagnostics AB, 1992). There is, however, no 

evidence in the 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) of aT. reesei strain 

used in manufacture of a commercial cellulose preparation, the 

tests on a cellulase preparation in accordance with the 

recommendations of the Joint FAOIWHO Expert Committee on 

Food Additives 1981 (JECFA), gave no indication that inhibitory 
Rebecca S. Cool 

March 17,2005 

Page 8 

substances were produced by T. reesei during its growtb. The 

industrial enzyme preparations are confirmed by the enzyme 

producers not to have antibiotic activity according to the 

specifications recommended by JECFA (Joint FAOIWHO Expert 

Committee on Food Additives, 1990). 

It should be noted that JECF A removed their general specification 

for mycotoxin testing (aflatoxin B, ochratoxin A, sterigmatocystin, 

T -2 toxin and zearalenone) of all fungal enzyme preparations and 

has indicated that only relevant mycotoxins reported in the species of 

close relatives should be tested (JECFA, 57th meeting, Rome 5-14 

June, 2001; 

http://www,who.intlipcs/foodljecfalsurtnnaries/en/summary_57.pdf). 

However, in between 1981 and 2001, hundreds of lots of enzyme 

products from T. reesei must have been tested and found to be 

negative for T-2, the only relevant mycotoxin for this species (see 

below).'· .. . 

Blumenthal (2003) also indicated that Watts et al. (1988) had 

reported the isolation of trichodermin from T. reesei and indicated 

that this may need further confirmation. She indicated that van 

Dijck (personal communication) is of the opinion that trichodermin 

is only produced by T. harzianum. She went on to say that T-2 

toxin has beenreported from Trichoderma and should continue to 

be tested in commercial food use enzyme preparations from 

Trichoderma species. 

(F) Studies which indicate the survival characteristics of the microorganism in the 

environment. 

T. reesei and H. jecorina have only been isolated in the 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 of a traditionally derived strain of T. 

reesei and a recombinant one (see Appendix 4, MCAN J-04-1). In sterile 

and non-sterile soil and water, both strains were found to persist in . 

numbers or decline, indicating t.1}at t.he Strai.11S are not able to proliferate, 

but presumably turn 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) in Innovative Approaches to Plant Disease 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 of Soil 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 

of Industrial Microorganisms. Benjamin Cunnings, London, pp. 487-509. 

Gams, W. (1995) Can. 1. Bot. 73, Supp!. 1, S747-S753. 

Ghisalberti, E. L. & Sivasithamparam, K. (1991) Soil BioI. 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 and Applications, (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 of Hypocreajecorina(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 IV.l.l l.., D ..L'\.._, r!.ruho ....... .1 U\"-.l, .. ]:1 J.: "' ..I. Messno" .1 \"-.1, R • & ..L K"b;~ok U .1""""''';, C . ..L P • (\..L 100.......... 2) 

Curro Genet. 21,31-36. 

Nevalainen, R., Suominen, P., and Taimisto, K. 1994. On the safety of Trichoderma 

reesei, J. Biotechno!. 37: 193-200. 
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March 17, 2005 

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Papavizas, G, C. (1985) Annu. Rev. Phytopathol. 23,23-54. 

Penttila··, M., Teeri, T. T., Nevalainen, H. & Knowles, J. K. C. 

(1991) in App/ied Molecular 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 of the secreted cellulases of 

Trichoderma reesei wild type and mutants during controlled fermentations. Appl. 

Microbiol. Biotechnol. 20: 46-53. . 

Simmons, E. G. (1977) in Second International Mycological 

Congress, eds. Bigelow, H. E. & Simmons, E. G. (Hamilton I. 

Newell, Amherst, MA), Vol. 2, p; 618. 

Toyama, N., 1969. Applications of Cellulases in Japan. In: Cellulasesand Their 

Applications, R. F. Gould (Ed.), Advances in Chemistry Series 95, American Chemical 

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