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# Company Sanitized

CONFIDENTIAL BUSINESS INFORMATION MICROBIAL COMMERCIAL ACTIVITY NOTICE (MCAN) 

## (TSOVSMC2) CONTAINED USE OF AGENETICALLY MODIFIED TRICHODERMA REESEI STRAIN FORUSE IN PRODUCTION OF A ENZYME PREPARATION FILEDBY DANISCOUS, INC

. 3490WINTON PLACE ROCHESTER, NY 14623 

# o~u7 ~i0~,m~0u0ir0i~NU0iri~u0ii2u~i,i~uiu~~iDanisco US, Inc.

MCANTSOVSMC2 

### Table of Contents (a) Certification Statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5 (b) Executive Summary. .. . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . .6 (c) Confidential Business Claims .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .. . . . . . .. . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . .7 (d) Submitter Identification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .12 (1) Name. . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .12 (2) Name, address, and phone number for the principal technical contact . . . . 12 (e) MicroorganismIdentity Information. . . . . . . . . . . . . . . .. . . . . . . . . . . . . .. . 12 (1) Description of the recipient microorganismand the new microorganism 12 (i) Data substantiating the taxonomy of the recipient and new microorganismto the strain level . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . 12 (ii) Information on the morphological and physiological features ofthe new microorganism

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .13 (iii) Other specific data by which the new microorganismmay be uniquely identified for Inventory purposes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . 13 (2) Genetic construction ofthe new microorganism

. . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . ., . , 14 (i) Data substantiating the taxonomy of the donor microorganisms . . . . . . . . . . 14 (ii) Description of the traits for which the new microorganismhas been selected or developed and other traits known to have been added or modified. . . . . . . . . . . . . . . . . . . 14 (iii) Adetailed description of the genetic construction of the new microorganism, including the technique used to modify the microorganism ; including a description of the introduced genetic material, including any regulatory sequences and structural genes and the products ofthose genes, how the genetic material is expected to affect behavior of the recipient; expression, alteration, and stability of the genetic material ; methods for vector construction and introduction; and a description of the regulatory and structural genes that are components of the introduced genetic material, including genetic maps of the introduced sequences.

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .14 (3) Phenotypic and ecological characteristics . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . 16 (i) Habitat, geographical distribution, and source of the recipient microorganism

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .16 (ii) Survival and dissemination under relevant environmental conditions including a method for detecting the new or recipient microorganism(s) in the environment and sensitivity limit of detection for these techniques. . . . . . . . . . . . . . . . . . . . . . ., . . . . . . , 16 (iii) Adescription of anticipated biological interactions with and effects on target organisms and other organisms; a description of host range; a description of pathogenicity, infectivity, toxicity, virulence, or action as a vector of pathogens; and capacity for genetic transfer under laboratory and relevant environmental conditions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .17 (iv) Adescription of anticipated involvement in biogeochemical or biological cycling processes; involvement in rate limiting steps in mineral or nutrient cycling, or involvement in inorganic compounds cycling (such as possible sequestration or transformation of heavy metals) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 19Danisco US, Inc.

MCANTSOVSMC2 (f) Byproducts -Adescription of byproducts resulting fromthe manufacture, processing, use, and disposal ofthe new microorganism

. . . . . . . . . . . . . . . . . . . . . . . . . . . ,. ., . ,19 (g) Information on production volume. . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .19 (h) Use information. . .. . . . . . . .. . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . .21 (i) Worker exposure and environmental release. . .. . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . .22 (1) For sites controlled by the submitter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , 22 (i) Identity of the site where the new microorganismwill be manufactured22 (ii) Process description. . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . 22 a) Unit Operation Description. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . .. . . .. . . . . . . . . . . . . . . . . . . . . . . 24 b) Starting Materials and Feedstocks . . . . . . . . . . . . . . . . . . . . . . . . . ., . . . . . . . . . . . . . . . . . . . , . 25 c) Description of release possible points identified in process diagram . . . . . . . . . . . 25 (iii) Worker Exposure. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . 27 (iv) Environmental Release and Disposal of Subject Microorganism . . . . . . . . . . 29 (v) Emergency containment procedures to be followed in case ofan accidental release . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .31 (vi) Procedures for disposal ofany articles, waste, clothing, or other equipment in the activity, including procedures for inactivation of the new microorganism, containment, disinfection, and disposal ofcontaminated items. .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 (2) For sites not controlled by the submitter . . . . . . . . . . . . . . . .. . . . . . . . . , 33 (i) Identity ofthe site where the new microorganismwill be used. . . . . . . . . . . . . . 33 (ii) Process description. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . 33 a) Unit Operation Description. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 b) Inactivation and possible release points identified in process diagram . . . . . . . 35 (iii) Worker Exposure. . . . . .. . . . . . . . . . . . . . . .. . . . . . . . .. . . . . . . . . . . . . . . .. . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . 36 (iv) Environmental Release and Disposal ofSubject Microorganism .. . . . . . . . . 37 (j) Health and Environmental Effects Data. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .38 (1) Test data on the new microorganism- include all test data which are related to the effects on health or the environment of any manufacture, processing, distribution in commerce, use, or disposal of the new microorganism. . . . . . . . . . . . . . .. . . . . . . . . , . , 38 (2) Full report or standard literature citation must be submitted for the following types of test data: . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . .. . . . . . . . . . . . . . .. . . . . . . .. . . . . . . . . . . . . . . . . . 38 (i) Health effects data. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 38 (ii) Ecological effects data. . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .. . . . . . . . 39 (iii) Physical and chemical properties data. . . . . . . . . . . . . . . . . . . . . . .. . . 39 (iv) Environmental fate characteristics . . . . . . . . . . . . . .. . . . . . . . , 40 (v) Monitoring data and other test data related to human exposure or to environmental release of the new microorganism. . . .. . . . . . . . . . . . . . . . . . . . . 40 (3) Other data concerning the health and environmental effects of the new microorganismthat are known to or reasonably ascertainable to the submitter . 40 (i) Any data, other than test data, in the submitter's possession or contro140 (ii) Any data, including test data, which are not in the submitter's possession or control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . 40 References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .41Danisco US, Inc.

MCANTSOVSMC2 List ofAppendices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .41 Appendix 1 : Identification Kit - Trichoderma reesei . . . ., ., . . . . 42 Appendix 2: Descri tion of the Genetic Construction of Trichoderma reesei . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . .53 Appendix 3: Trichoderma reesei Safety Studies . . . . . . . . . . . . . . . .. . . . . . . . . . ., . ., . . . . . . ,gg Appendix 4

: Survival of Trichoderma reesei Strains . . . . . . . .. . . . . . . . . . . . . . . .. . . . . . . ., . . . . . . . ,. 134

### Danisco US, Inc.

MCANTSOVSMC2 List of Appendices. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .41 Appendix 1 : Identification Kit - Trichoderma reesei . . . . . . . . . . . . . . . . . . . . . . . . . . . . .42 A ppendix 2: Descri ption of the Genetic Construction of Trichoderma reesei .. . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . .. . . . . . . . . . . . . .. .. . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . .53 Appendix 3: Trichoderma reesei Safety Studies . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . gg Appendix 4: Survival of Trichoderma reesei Strains . . .. . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . .134Danisco US, Inc.

MCANTSOVSMC2 (a) Certificate Statement 

### Document Control Officer TSCA Document Processing Center (7407) Rm . L-100 Office of Pollution Prevention and Toxics U .S. Environmental Protection Agency 401 M Street, S.W

. 

### Washington, DC 20460 CERTIFICATION I certify that to the best of my knowledge and beliefthe company named in this submission intends to manufacture, import, or process for a commercial purpose, other than in small quantities solely for research and development, the microorganismidentified in this submission. All information provided in this submission is complete and truthful as ofthe date ofthis submission. I am submitting with this submission all test data in my possession or control and a description ofall other data known to or reasonably ascertainable by me as required by 40 CFR §725.160. The company identified in this notice has remitted the fee specified in 40 CFR§700.45(b) . 

### Signature ofauthorized official Date

VincentJ .H

. Sewalt, Ph.D

. Director of Regulatory Affairs Danisco US, Inc. 

### 925 Page Mill Road Palo Alto, CA94304 06 '/~p A -e-' 1 P/ 2c=)c%1/p. 7



### Danisco US, Inc. MCANTSOVSMC2 (b) Executive Summary Danisco US, Inc. (Danisco), formerly Genencor International, Inc ., has constructed a Trichoderma reesei strain, T . reesei 

### and - WWWW Wities and has determined that the microorganismis activ new under Section 5 ofthe Toxic Substances Control Act.

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Danisco US, Inc. MCANTSOVSMC2



### Trichoderma reesei - was chosen as the starting strain for transformation, as it is a cellulase overproducer. Therefore, the best overall characterization ofthe enzyme preparation derived fromstrain

The new

strain, T . reesei 3400, carries the expressed genes integrated within the genome as expression cassettes consisting only of o antibiotic resistance markers were inserted into the new microorganism

. 

### Acomplete description ofthe production strain is provided in this submission. The production ofthe is performed under Good Industrial Large Scale Practice (GILSP) containment, in closed fermentation systems using pure cultures. A description ofthe manufacturing steps including control technology is provided. 

### The preparations will be manufactured in either an existing enzyme manufacturing plant owned and operated by Danisco or on-site at individual cellulosic ethanol plants. In order to initiate on-site manufacture at cellulosic ethanol plants, viable production strain cells wil l be shipped fromDanisco to the respective site. M The microorganisms contained in the preparationwill be inactivated. The Danisco production process is similar to the process described in MCAN# previously reviewed by EPA . (c) Confidential Business Claims Danisco and DDCE are claiming certain strain construction, production process and volume information as confidential . Below are the answers to questions provided in `Points to Consider in the Preparation ofTSCA Biotechnology Submissions for Microorganisms,' pp. 52-53. C . General questions: 1 . For what period oftime is a claimofconfidentiality being asserted? We request the strain information, production process andvolume information be kept confidential for the indefinite future. Release ofthis information will give Page 7of141p. 9



### Danisco US, Inc. MCANTSOVSMC2

competitors knowledge about key components ofthis proprietary strain, the production process, cost structure and size ofthe facility. This information will give them

unfair advantage on pricing and teach themDanisco's proprietary production process. We also request that the strain construction information be kept confidential for the indefinite future. As Danisco has provided very detailed descriptions of not only the strain construction but also strain development strategies that will be employed on an ongoing basis, no finite time deadline can be identified at which CBI status will lapse. 

### 2. Briefly describe any physical or procedural restrictions within the company or institution relating to use and storage ofthe information claimed as confidential . The confidential information that relates to manufacturing operational procedures that are part ofthe company's internal operations documentation is being claimed as confidential . Procedures are written and noted for "internal" use only. There is restricted access to the plant facility limiting information exchange with outside persons . The confidential information that relates to strain construction is maintained as part ofthe intellectual property ofthe company . All Danisco and DDCE employees sign confidentiality agreements. They are informed that this type of information is confidential . Disclosure beyond employees only occurs (1) to companies that are contractually bound to preserve confidential status, and (2) to government agencies under appropriate, narrow circumstances. 3. Has the information claimed as confidential been disclosed to others outside ofthe company or institution? Process information is considered proprietary and is not disclosed to others outside the company unless under license fromDDCE. Ifvendors or others need to enter the production areas, it is encouraged to obtain a Non Disclosure Agreement to safeguard against loss of confidential information. Production strain construction information is similarly considered confidential and is not disclosed to others outside the company. 

### 4. Does the information claimed as confidential appear, or is it referred to, in any of the following: (1) Advertising or promotional materials for the microorganism or the resulting end product No. Danisco is a technology company. Our intellectual property is critical to maintain and all employees must adhere to a strict internal reviewprocess with regard to disclosure of information that is or might be valuable. 

### Page 8of141p. 10



### Danisco US, Inc. MCANTSOVSMC2

(2) Material safety data sheets or other similar material for the microorganismor the resulting end product No. Enzyme product MSDS disclosure is generally limited to exposure limits, clean-up recommendations and toxicity data. Formulation ingredients may be listed. 

### (3) Professional or trade publications No. While Danisco may publish some details of host strain characteristics, production strain construction is proprietary and not subject to publication . (4) Anyother media available to the public or to your competitors No. Equipment vendors occasionally ask ifwe can be mentioned as a user oftheir equipment - that is normally approved at corporate level . Strain development techniques and strategies are not made public to the public or competitors. (5) Patents No. While some strain construction elements claimed to be confidential are the subject of patent publications, the production strain construction details of any particular strain are proprietary and not stipulated in patent publications held by Danisco. (6) Local, State or Federal Agency public files 

### No, to the best of our ability confidential information is not disclosed in these public files. 5. Has EPA, another Federal agency, a Federal court, or a State made any confidentiality determination regarding the information claimed as confidential? No. 6 . For each type ofthe information claimed as confidential, describe the harmto the company or institution's competitive position that would result ifthis information were disclosed. There are several types ofconfidential information: (1) Production quantities; (2) Process descriptions and chemicals; (3) Unit operation descriptions &process flowdiagram ; (4) Possible organismrelease points; 

### Page 9 of 141p. 11

Danisco US, Inc. MCANTSOVSMC2



### (5) Possible worker exposure. (6) Production strain Construction, including the identity ofthe newly introduced genetic elements (1) Dissemination of planned production volumes would indicate to competitors the scale of Danisco's product introduction. Armedwith this information, knowledgeable competitors could predict the target market(s) and allow themto build focused strategies to thwart the product introduction. Our company's future is highly dependent on successful introduction ofvaluable new technologies such as this. (2) Dissemination ofa process description would advance a competitor's process and product development capabilities in general, and potentiallywould also allowthemto quickly develop a similar product. (3) Dissemination of unit operations and particularly of a process flowdiagram would also allow

a competitor to analyze the process yield and determine the cost of manufacturing. This information would give a competitor an unfair competitive advantage against Danisco. For example, a competitor armedwith Danisco's production cost information could price their product such that Daniscowould be excluded fromthe market . (4) Dissemination of possible organismrelease points tells a competitor what type ofequipment is being used to contain and process the organismand the product. This is valuable information leading to pricing and/or process determinations. (5) Dissemination of possible worker exposure as in #4 tells a competitor what type ofequipment is being used, the number of persons involved in controlling the process and the duration oftheir potential exposure. (6) Disclosure ofthe newly introduced genetic elements would teach a competitor which enzyme activities to introduce into the host organismto maximize biomass conversion efficiency; dissemination ofstrain construction techniques and strategies for developing a particular production organismtells a competitor which genes in the host organismto manipulate in order to maximize target enzyme production. In addition, such dissemination would teach competitors how to manipulate the noticed host gene sequences and inserted sequences in such ways that may overcome Danisco's competitive advantage in the use ofthe host organismas a safe and suitable enzyme production organism . 

### Making this information available to competition will allowthemto analyze Daniscos's technical capabilities, production and cost structure and give them an unfair advantage in the market place. 7. If EPAdisclosed to the public the information claimed as confidential, how difficult would it be for the competitor to enter the market for the resulting product? Competitors have already entered this emerging market. Iffully disclosed, competitors would have no trouble 'reverse-engineering' our product, process, or parts of our process, and this would allow thema competitive advantage in the specific target market for this product and in other markets. The new Page 10 of 141p. 12

Danisco US, Inc. MCANTSOVSMC2

product is avery much enhanced version ofan existing product with complementary side activities. Competitor introduction of a similar product would require specialized genetic technical expertise to construct the production microorganismand development of a specialized process to produce the intended end product. Competitors clearly have the expertise to do this type of work. Dissemination of our confidential information could very well lead to disastrous effects for Danisco in our target market.



### Page 11 of 141p. 13



### Danisco US, Inc. MCANTSOVSMC2 (d) Submitter Identification (1) Name Danisco US, Inc. Headquarters address 3490 Winton Place Rochester, NY 14623 (2) Name, address, and phone number for the principal technical contact VincentJ .H. Sewalt 

### Director of Regulatory Affairs 925 Page Mill Road Palo Alto, CA94304 (650) 846-5861 vincent .sewalt@danisco.com (e) MicroorganismIdentity Information (1) Description of the recipient microorganismand the new microorganism (i) Data substantiating the taxonomy of the recipient and new microorganismto thestrain level



### the construction of and the construction construction of construction of This recipient strain is also used inthe construction of The T . reesei recipient strain used to create the new microorganism7

: reesei is -, a derivative ofstrain RL-P37. -and 

### other derivatives of RL-P37 have a long history ofsafe use by Danisco/Genencor inthe manufacture ofcommercial cellulase preparations .

The derivation and characterization ofstrain RL-P37 has been published (Sheir-Neiss and Montenecourt, 1984), and Danisco/Genencor receivedthe strain directly fromDr. Montenecourt. Strain RL-P37 is a cellulase overproducingstrainthat was obtained through several classical mutagenesis steps fromthewild-type T

. reesei strain (QM6a) . Strain QM6a is present in several public culture collections, e.g. in theAmerican Type Culture Collection as ATCC 13631 . T

. reesei has more recently been identified as a clonal derivative or anamorph ofHypocreajecorina (Kuhls et al ., 1996; Dugan, 1998).

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Danisco US, Inc. MCANTSOVSMC2

Copies ofthese expression cassettes were i inte ated into the recipient chromosome. Th iiia gene were used as selectable marker. T . reesei 3400 is derived fromstrain RL-P37 through classical mutagenesis, gene deletions, and insertion ofthe



### The taxonomic identity ofthe donor strains is described under (e)(2)(i) . (ii) Information on the morphological and physiological features ofthe new microorganism T . reesei cultures are typically fast growing at 25-37°C. Colonies appear transparent on media such as cornmeal dextrose agar (CMD) or white to grayish-green on richer media such as potato dextrose agar (PDA) . Mycelia typically are not obvious on CMD . Conidia typically form within one week ofgrowth as compact or loose tufts in shades ofgreen or yellowor less frequently white. Yellow pigment may be secreted into the agar, especially on PDA . Acharacteristic sweet or 'coconut' odor is produced by some species. Thesefeatures also apply to the new microorganism

. The new

microorganismofthis MCAN, designated strain hereinafter called T

. reesei 3400, which is used as the production strain for manufacture of preparations, can be detected by making use ofits microbiolo ical phenotype. The strain is capable ofgrowing on plates containing 

### and the strain is (iii) Other specific data by which the new microorganismmay be uniquely identified for Inventory purposes 

### Features for unique identification for Inventory purposes are described above in (e)(1)(ii) . The new

microorganismcan be uniquely identified for Inventory purposes by using molecular biological methods such as PCRanalysis using primers specific for the inte ated gene expression cassettes plus plating the organismon source, and in the presence of

An Identification Kit guiding identification ofthe production strain is available

Page 13 of 141p. 15



### Danisco US, Inc. MCANTSOVSMC2 (2) Genetic construction ofthe new microorganism (i) Data substantiating the taxonomy of the donor microorganisms 

### The donor strain for the and the gene

used in construction ofthe new micro-organismwas T

. reesei strain RL-P37. The genes were isolated fromgenomic DNA from thewildtype T . reesei by PCRusing specific primers devised on the sequence available in Genbank database. 

### The genes were fromisolated genomic DNAfrom by PCRusing specific primers devised on the sequences available in

medium . Only the gene was used in isolated form . The_

gene was first described by The strain was not described further than at the Fungal Genetics Stock Center, Kansas Cit , USA . Also the descri tion ofthe gene in GenBank



### strain as the s M == I

ource. (ii) Description of the traits for which the new microorganismhas been selected or developed and other traits known to have been added or modified. 

### Thie new microor anis , T

. reesei 3400, was developed to overproduce 6iiiiiia involved in the hydrolysis ofpolysaccharides in biomass to simple sugars that can be fermented to ethanol . (iii) Adetailed description ofthe genetic construction of the new microorganism, including the technique used to modify the microorganism

; including a description of the introduced genetic material, including any regulatory sequences and structural genes and the products of those genes, how the genetic material is expected to affect behavior of the recipient; expression, alteration, andstability of the genetic material ; methods for vector construction and introduction;

Page 14 of 141

### Danisco US, Inc.

MCANTSOVSMC2 and a description of the regulatory and structural genes that are components of the introduced genetic material, including genetic maps of the introduced sequences. In summary, strain RL-P37 was subjected to -was selected for overproduction ofcellulase (CAS# 9012-54-8) .



### anism, we have inserted into strain

Page 15 of 141Danisco US, Inc.

MCANTSOVSMC2

All genetic elements inserted into the new strain are of Trichoderma origin with the exception of

All these modifications were performed in such a way that no bacterial vector DNAremains present in the strain. No antibiotic resistance markers were inserted into the new microorganism

. Thefinal production strain was characterized by Southern blotting analyses. 

### Adetailed description ofthe genetic construction ofthe new microorganism is attached as Appendix 2. (3) Phenotypic and ecological characteristics (i) Habitat, geographical distribution, and source of the recipient microorganism 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. (ii) Survival and dissemination under relevant environmental conditions including a method for detecting the new or recipient microorganism(s) in the environment and sensitivity limit ofdetection for these techniques Trichoderma reesei cultures are typically fast growing at 25-30° C and typically do not grow at or above 35° C

. Colonies appear transparent on Page 16 of 141p. 18

Danisco US, Inc. MCANTSOVSMC2

(iii) media such as cornmeal dextrose agar (CMD) or white on richer media such as potato dextrose agar (PDA) . Mycelia typically are not obvious on CMD . T . reesei reproduces and spreads through asexual conidia. Conidia typically form within one week ofgrowth as compact or loose tufts in shades ofgreen or yellowor less frequently white. Yellowpigment may be secreted into the agar, especially on PDA . Acharacteristic sweet or 'coconut' odor is produced by some species. These features also apply to the new microorganism

. The new

microorganismdoes not produce spores significantly during Danisco's fermentation process. Spores present in the biomass waste are not resistant (apart fromresistance to drought) and are inactivated during biomass waste treatment before disposal . It is not expected that the modifications will significantly affect the survival ofthe organism

. Survival studies conducted by Danisco indicate no difference in survival between a genetically modified endoglucanase overproducing strain derived fromRL-P37 and the host strain RL-P37 (see Appendix 1) 

### In the production strain described in the current MCANthe metabolic burden due to the overexpression of is also expected to be much higher than in the case examined in Appendix 4. If anything, the higher metabolic burden would be expected to cause this production strain to be less fit to compete in the environment . The new microorganismcan be selected for using the features described under (e)(1)(ii) and (e)(1)(iii) above,

Once candidate production strain colonies have been isolated these can be identified using molecular biological methods like PCRanalysis using primers specific for the integrated expression cassettes (see also Annex 5) . An Identification Kit guiding identification ofthe production strain is available (see Appendix 1) . Adescription ofanticipated biological interactions with and effects on target organisms and other organisms; a description of host range; a description of pathogenicity, infectivity, toxicity, virulence, or action as avector of pathogens; and capacity for genetic transfer under laboratory and relevant environmental conditions.

Page 17 of 141p. 19

Danisco US, Inc. MCANTSOVSMC2 T . reesei is a non-pathogenic fungus (see (i)(2)(i) below), and as such does not have a mammalian host nor does it have significant interactions with other organisms, other than saprophytic ones. No vector bacterial DNAused in creation ofthe new microorganism remains in the new

microorganism

. Since no vector sequences are present in the final strain, the transfer frequency ofthe integrated expression cassettes is the same as for any other chromosomal sequence. The transforming DNA consisted consisting offungal DNA :

Page 18 of 141p. 20



### Danisco US, Inc. MCANTSOVSMC2

(iv) Adescription ofanticipated involvement in biogeochemical or biological cycling processes; involvement in rate limiting steps in mineral or nutrient cycling, or involvement in inorganic compounds cycling (such as possible sequestration or transformation of heavy metals) . 

### T . reesei is a wood rot fungus. (f) Byproducts -Adescription of byproducts resulting fromthe manufacture, processing, use, and disposal ofthe new microorganism

(g) Information on production volume



### et cell mass produced per fermentor batch. (Note: Due to the poly-nucleic nature ofthe filamentous production microorganism, the cell population is difficult to quantify in terms ofcolony forming units (CFU) . Using bench scale cell density of there will be approximately The new microorganismwill be used in a batch process to manufacture the desired enzyme product. For each batch, the volume at harvest will be approximately . At a

produced in the next three years: e table below approximates the cell mass to be



### Page 19 of 141p. 21



### Danisco US, Inc. MCANTSOVSMC2

Year # of

batches DCW

(kg) per batch Total DCW

produced (kg) Wet cell

mass (kg) /batch Total wet

mass produced (kg) 

### 1 2 

# IAU

AKIIIAU 3 Production volume of new organism

The cellulosic to ethanol process in which the is used will operate at production scales that range from0.25 to 50 million gallons ofethanol per year. Ethanol production fermentor sizes will range fromabout 100m3 (-26,500 gallons; pilot facility) to about 3,800m3 (-1,000,000gallons; commercial facility) . Fermentations will operate in batch mode. The number of fermentors present at the facility will be based upon fermentation time and the upstream(e.g., pretreatment), and downstream(e.g., distillation) facilities' capacity, with the goal ofhaving sufficient fermentation capacity to permit a constant flowof material into and out of the fermentation area.

Page 20 of 141p. 22



### Danisco US, Inc. MCANTSOVSMC2

(h) Use information

Page 21 of 141p. 23



### Danisco US, Inc. MCANTSOVSMC2 (i) Worker exposure and environmental release (1) For sites controlled by the submitter (i) Identity of thesite where the new microorganismwill be manufactured The stock culture and seed vial lot preparation ofthis new microorganism will be conducted at the Danisco US manufacturing facility in Rochester, New York. The address is: Danisco US, Inc. 1700 LexingtonAvenue Rochester, NY 14606

. The sterile, sealed, cryo-protected vials will then be sent for the manufacture ofthe enzyme product to the Genencor, ADanisco Division, manufacturing facilities in Beloit, Wisconsin and Cedar Rapids, Iowa. The addresses are: Danisco US, Inc. Danisco US, Inc. 

### 2600 Kennedy Dr 1000 41st Avenue Drive, S. W . Beloit, WI 53511 ~ Cedar Rapids IA 52404

(ii) Process description Both Danisco manufacturing facilities are secure facilities that are operated 24 hours a day. People not employed by Danisco/Genencor entering the facility must check-in and be registered. The stock cultures are prepared in a controlled laboratory that is solely for that purpose; access to the lab is limited. The fermentor and processing equipment in Beloit and Cedar Rapids is located in an area ofthe plant that is separate fromoffice and laboratory space; access to the production areas is limited. The fermentor vessels used in the Danisco process for the production of

fromT . reesei 3400 are closed stainless steel tanks designed and bui lt according to American Society of Mechani cal Engineers (ASME) codes.

Page 22 of 141p. 24



### Danisco US, Inc. MCANTSOVSMC2 Piping connections such as fill line, drawline, sample port, and inoculation

port are highly secure and - Positive steampressure is maintained on the exterior side of each valve in contact with the fermentor to prevent contamination. Compressed air is filtered to remove airborne particles. All procedural steps and engineering standards described above are meant to insure that a pure culture fermentation is run. After each fermentation step is completed and the contents ofthe fermentor have been transferred, the vessel is cleaned in place (CIP) with a_ solution and the wash contents ofthe fermentor are directed to the equalization tank. Bench scale experiments have demonstrated that T . reesei is not viable after Other production equipment in a similar manner. Figure 1. Process diagramfor Danisco manufacture of using T . reesei strain 3400

Page 23 of 141Danisco US, Inc.

MCANTSOVSMC2 The major unit operation steps in the Danisco process (Figure 1) are described in detail below . 

### a) Unit Operation Description 1) Stock culture preparation - The following procedures are performed in a bio-safety cabinet by laboratory staff equipped with the typical personal protective equipment (lab coat, safety glasses, latex or nitrile gloves) . Starting with the spore suspension deposited in the Culture Collection, plates are inoculated to grow the microorganism

. After appropriate growth and spore development, the spores are harvested using 15%

glycerol, dispensed into cryogenic storage vials and stored under liquid nitrogen. Post storage quality assurance checks include 1) plating to check for the absence ofcontaminating organisms, and for spore survival and purity, 2) comparison of pre-freeze viability with the post-freeze viability to determine the survival rate and 3) testing the vials for productivity. The seed vial lots are prepared in the Culture Collection laboratory according to a Standard Operating Procedure. 2) Aseptic inoculation of production organism- The seed inoculumis prepared by adding, in a bio-safety cabinet, vials ofa stock culture spore suspension ofthe new microorganism(stored under liquid nitrogen) to flasks containing appropriate media. The flasks are then placed in an incubator. When an appropriate cell mass is achieved, the contents ofthe flask are transferred into a sterile stainless steel transfer apparatus in a bio-safety cabinet. A member ofthe dedicated laboratory staff, equipped with lab coat, latex or nitrile gloves, and safety glasses, completes the transfer ofthe flask contents to the seed fermentor via steam-sterilized hard piping. 3) Seed Fermentor - Aseed fermentation vessel, controlled at prescribed environmental conditions (temperature, pH control via ammonia addition, pressure, and airflow), is used to generate the inoculumfor the production vessel . Transfer to the production fermentor is conducted by dedicated fermentation operators and occurs via steam-sterilized hard piping. Afterthefermentation is complete, the CIP process described in (i)(1)(ii) is carried out. 4) Main Fermentor - The organisms transferred fromthe seed fermentor are allowed to grow

for (based on rate of growth and 

### Page 24 of 141

### Danisco US, Inc.

MCANTSOVSMC2 plant production schedules) . The fermentor broth is then transferred to a drop tank. 

### Criteria are in place for detecting contaminated ferm entation runs. If a run is deemed contaminated and it will not be used, is adjusted adequately to inactivate the production organism,

, and sent to the contractor for land application, anaerobic digestion or to drain and wastewater treatment. The fermentors used to grow the production organismare located in a separate area ofthe manufacturing facility and share an isolated holding tank system

. After every run, the main fermentor is washed using a CIP systemas described above in (i)(1)(ii) . Rinse material fromroutine tank cleaning is directed to the equalization tank (where the pHis brought to neutral), which flows to the municipal waste treatment facility. 5) 

### stored in a bulk storage tank prior to transfer into a tanker truck or totes for transportation to the ethanol plant. After every batch, the fermentor and drop tank units are run through a CIP wash as described above in (i)(1)(ii) . b) Starting Materials and Feedstocks The T . reesei 3400 fermentation is a contained process of a pure culture of the subject microorganism . The ingredients for the seed and main fermentors are as follows: 

### Seed flask media - Seed fermentation media - all the above plus Main fermentation media - same as the seed fermentation mediumwith

or another similar carbohydrate feedstock fed for induction of enzyme production. c) Description of possible release points (see Figure 1) 1 . Stock culture preparation - All manipulations are carried out in a biosafety cabinet to maintain sterility and to protect the laboratory technician and the environment. All disposable items (gloves, pipets, culture preparation plates, culture spreaders) are placed in a biohazard trashcan, where they are then

Page 25 of 141p. 27

Danisco US, Inc. MCANTSOVSMC2 

### autoclaved. All glassware (culture flasks, beakers) is treated with bleach before being washed. Consequently, there is no microorganismrelease expected at this stage. 2 Seed inoculumtransfer - Once the cell mass in the culture flasks is sufficient, the culture is transferred into a stainless steel transfer apparatus in a bio-safety cabinet. Empty flasks are treated with bleach and rinsed to the sink (which ends up in the equalization tank before being released to the municipal waste water treatment system .) Dedicated laboratory staff, equipped with lab coats, hard hats, gloves and safety glasses, transfer the inoculumfromthe transfer apparatus to the seed fermentor via steamsterilized hard piping. The empty transfer apparatus is caustic treated before being rinsed to the laboratory sink. Thus there are no anticipated releases ofthe new microorganismat this stage. 3 Fermentation -The fermentors used to grow the production organism are located in a separate area ofthe manufacturing facility and share an isolated holding tank system . Tanks are routinely run through a caustic wash (CIP) after every run as described in (i)(1)(ii) . Rinse material from routine tank cleaning directed to the equalization tank (where the pH is brought to neutral) that supplies the municipal waste treatment facility. a) Fermentation samples - Routine samples collected fromthe fermentor are either spot checked on the tank farmfloor (ex., pH determination) or sent to the lab (onsite) for routine analysis (ex., enzyme activity) . All samples are inactivated with either bleach or through autoclaving prior to disposal . b) Fermentor off gas - Exhaust gas passes through a droplet knockout vessel that reduces the amount oforganismreleased to the environment by impingement. The air streamflows horizontally onto a vertical plate that causes moisture condensation and droplet formation. Dropletwaste collected at the knockout vessel is discharged to the equalization tank (where the pHis brought to neutral) that supplies the municipal waste treatment system . Data from other fungal fermentations at the Beloit site have shown that no production organismis detected in the fermentor off-gas droplet waste. Therefore, there is no exposure to the production organism inside the facility fromfermentor off-gas. 4 Post fermentation processing -



### Page 26 of 141

### Danisco US, Inc.

MCANTSOVSMC2

(iii) product then goes to a storage tank. After every batch,

this unit operation is run through a CIP wash as described above in (i)(1)(ii) .

Worker Exposure 1) Stock culture preparation - Exposure to the organismis not expected during stock culture preparation due to the fact that transfers and organism handling occur in a bio-safety cabinet to maintain sterility and to protect the laboratory technician and the environment. Employee exposure to the subject microorganismis also limited at this step by the personal protective equipment worn by the laboratory technician. The stock culture preparation will require approximately I hours of lab technician time per stock culture batch and one stock culture batch will provide enough material for -



### microorganismis also limited at this step by the personal protective 2) Seed inoculumtransfer - Exposure to the organismis not expected during seed inoculumtransfer due to the fact that transfers use aseptic technique and are conducted in a bio-safety cabinet in a manner that contains the production microorganism . Employee exposure to the subject 

### equipment worn by the laboratory technician.

After transfer ofthe inoculumto the seed fermentor, the empty transfer apparatus is given a caustic treatment before being rinsed to the sump in the fermentor floor. When the apparatus is broken down for cleaning, it contains only inactivated organisms and caustic residue.

Page 27 of 141

### Danisco US, Inc.

MCANTSOVSMC2 3) Fermentation - The activities on the fermentation tank floor that involve

potential worker exposure to the subject microorganismare described below in two categories - fermentation samples and fermentor off-gas. 3a) Fermentation samples -Process technicians, equipped with uniforms, latex or nitrile gloves, and safety glasses, will occasionally take samples fromsteamlocked sample ports in the fermentor during production runs. The employee exposure to the subject microorganism is limited at this step by the personal protective equipment worn by the process technician and the small sample volumes required. Samples are carried in sealed vials to the dedicated laboratory staff for routine analysis. Exposure to the organismis also limited during this activity by the personal protective equipment that is regularly worn by the dedicated laboratory staff (lab coat, latex or nitrile gloves, and safety glasses) . All samples are inactivated with either bleach or autoclaving priorto disposal . Dueto the controls described here, handling ofthe fermentation samples represents minimal employee exposure. 3b) Fermentor off-gas - As described above in the "Description ofpossible release points identified in process diagram," the fermentor exhaust gas passes through a droplet knockout vessel before being released to the atmosphere. Droplet waste collected at the knockout vessel is discharged to a surge tank supplying the municipal waste treatment system . Asmall portion ofthe off-gas fromthe droplet knockout vessel is vented to a site in the fermentation tank area outside the operator control room

. Fromhere, the operators can routinely do a visual check on the off-gas for the express purpose ofearly detection offermentor foamouts. The best estimates on thevent volume into the fermentation tank area are ofair and small amounts of steam and fermentation broth condensate. Data from other fungal fermentations at the Beloit site have shown that no production organism is detected in the fermentor off-gas droplet waste. Therefore, exposure to the production organisminside the facility fromfermentor off-gas is not expected. 4) Post fermentation processing -

Page 28 of 141p. 30



### Danisco US, Inc. MCANTSOVSMC2

Worker Activity Protective Equipment/

Engineering Controls # of Workers Exposed Maximum Duration hrs/da Maximum

Duration da s/ r Seed safety

gloves Lab coat , , 

### Inoculum glasses, hard hat - Transfer Fermentation Lab coat and or - samples uniforms, safety Imp "&

glasses, gloves, hard 

### hat tank - Dro safety

uniforms p , aerosols glasses, gloves, hard 

### ili

hat w Summa table ofworker exp osure information

(iv) Environmental Release and Disposal ofSubject Microorganism Possible release points for and disposal ofthe new microorganismduring manufacture ofthe enzyme product fromT . reesei 3400 are discussed in Section (i)(1) (ii) c) above. Efficiency ofthe control technology at the various potential release sites is outlined in the table below .

Page 29 of 141p. 31

Danisco US, Inc. MCANTSOVSMC2 Control Technology Efficiency at Potential Sites of New MicroorganismRelease Release Number Amount of new substance released (CFU/batch) Media of Release Control Technology Efficiency To Envi- To Control ronment Technology 1) Seed cryovial ~ Water preparation - empty seed flasks 2) Seed inoculum ~ Land _ transfer - empty cryovials 3) Seed inoculum ~ Water transfer - empty seed flasks 4) Seed inoculum ~ Water - transfer - empty transfer apparatus (canisters) 5) Seed ~ Water - Fermentor 6) Main ~ Water - fermentor 7) Fermentor ~ Water - samples 8) Fermentor off Air gas - release into (Airborne atmosphere droplets) 9) Fermentor off ~ Water - gas - release into surge tank from droplet knockout vessel 10) Drop tankfor ~ Water fermentor broth Note on CFU numbers used in the above table:

Note on Fermentor off-sas:Danisco US, Inc.

MCANTSOVSMC2



### The three potential sources for release ofthe new microorganismfromthe 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 occur in the fermentor off-gas. Manufacturing Site Proximity to Drinking Water Sources The Beloit facility is located in an industrial park within the city limits of Beloit Wisconsin. Beloit has a population of 35,775 (from the 2000 census) . The closest waterway is Springbrook that flows to Turtle Creek and then the Rock River. Springbrook is approximately 0 .25 miles fromGenencor's facility. There are 10 drinking water wells within the city, each with their own aquiver. The nearest drinking water well is the 1-90 Tower, located at 2875 Cranston Road, which is approximately 0.75 miles northwest of Genencor's facility. The I-90 Tower is a single pedestal spheroid elevated tank, constructed in 1991 . The elevated tank has a storage capacity of 750,000 gallons. The Cedar Rapids facility is located at 1000 41s' Avenue Drive SW in an industrial area on the South West side of Cedar Rapids, Iowa. The primary source ofCedar Rapids' drinking water is a series ofcity wells within the Cedar River watershed. The Cedar River is located approximately 0 .8 miles from the facility. (v) Emergency containment procedures to be followed in case of an accidental release In the unlikely event ofa large spill of material containing the new microorganism, containment procedures will be followed. The action taken depends on the size ofthe spill or leak. All accidental spills and leaks greater than ten liters of fermentation broth are reported to the supervisor and personnel are evacuated fromthe area for at least thirty minutes to allow aerosolized culture to settle. Before evacuation, closing any valves that are the source of leakage stops the spill or leak. For small spills, the area is treated with hypochlorite solution then cleaned up with towels; the towels are autoclaved before disposal . Larger spills require the use of two buckets, one to mop up the spill and the other for treatment with fresh hypochlorite solution. Spills larger than those that can be handled by buckets and mops are drained and flushed to thewaste liquid collection systemfor bleach inactivation before release to the sewage treatment facility. The spill area is then treated with hypochlorite solution. The hypochlorite is left on the spill for a minimum

oftwenty minutes. The fermentor tank floor has a trench Page 31 of 141p. 33



### Danisco US, Inc. MCANTSOVSMC2 setup the feeds all run offto a sump. The sump feeds into a surge tank and is also the easiest way to access the surge tank. There is a manual isolation valve offofthe surge tank that can be closed to containvery large spills. A drumof bleach is present at the access to the sump that supplies the surge tank in case the need to inactivate a spill arises. An incident report is completed for spills greater than or equal to 10 liters. If spills are sent to the drain, the aqueous wastewill go to the respective Water pollution Control facilities ofthe City of Beloit or City ofCedar Rapids. Dischargewould be indirect to surface waters. The Water Pollution Control facilities will be notified, if the fermentation spill volume exceeds 10% of normal discharge. (vi) Procedures for disposal ofany articles, waste, clothing, or other equipment in the activity, including procedures for inactivation ofthe new microorganism, containment, disinfection, and disposal of contaminated items. 

### The procedures requested here are described above.

Page 32 of 141

### Danisco US, Inc.

MCANTSOVSMC2 Worker exposure and environmental release For sites not controlled by the submitter Identity of thesite where the new microor2anism

will be used 

### The new microorganismmay be used at various yet to be identified customer sites, including current ethanol plants that may be converted to process cellulosic biomass, new

pilot plants, and new

full scale-cellulosic ethanol plant sites. -

ii a) Process description Unit Operation Description The following is a general description of an integrated, high solids process to convert biomass to cellulosic ethanol . The process contains steps in pretreatment, saccharification, fermentation and post fermentation processing technology. Ageneral process diagramis shown below, with capital letters in parenthesis referring to the unit operations shown in that process diagram . For this documentation, the process description will focus on those steps that may contain viable T . reesei 3400

. Biomass pretreatment &saccharification 

### The biomass ofchoice can be corn cob, corn stover, sorghumstover, other agricultural (by)products and residuals, or switchgrass grown specifically as ethanol feedstock. The biomass solids are pretreated (A) with appropriate chemicals= - under conditions conducive to loosening the cell wall structure to allow access to enzymes. The pretreated solids, are then saccharified using T . reesei 3400

Main Ethanol fermentation

### Page 33 of 141

### Danisco US, Inc.

MCANTSOVSMC2

The hydrolyzate resulting fromthe pretreatment and saccharification process steps is transferred via closed pipelines or in closed totes to an anaerobic fermentor (C), where ethanol is produced utilizing ethanologen. The main fermentation is conducted under anaerobic conditions. Off-gas from the fermentor is treated by emission control technology commonly used by the bioprocessing industry (e.g., water scrubber or thermal oxidizer; D) . Downstreamprocessing 

### Further downstreamprocessing ofthe fermentation broth by

Effluent streams fromdownstreamprocessing steps have their dissolved and any remaining suspended solids concentrated and the concentrated solids disposed or beneficially reused (for example, as a fuel or via land application for agronomic purposes) . Low dissolved or suspended solids containing water from the concentration step (L) is either discharged to waste watertreatment or recycled into the process following optional physical/chemical and/or biological water polishing (M) processing steps.



### Page 34 of 141p. 36



### Danisco US, Inc. MCANTSOVSMC2

b) Inactivation and possible release points identified in process diagram The possible environmental release points for and disposal ofT . reesei 3400 and the ethanologen ofchoice are fermentor off-gas (1), solids disposal/beneficial reuse (2, 3 and 5) and treated liquid discharge (4) fromdownstreamprocessing steps. Viable cells will be used in the process under containment, which requires a combination ofengineering, mechanical, procedural, or biological controls designed and operated to restrict environmental release ofviable T . reesei 3400 from

the structure. Before leaving the containment, viable cells will be inactivatedvia exposure to heat or other appropriate means (e.g., pH or combination of heat and pH) . In addition, procedures will be in place to detect contaminated fermentation runs, which then are treated (e.g., by heat) to inactivate cells prior to discharge to waste treatment. Off-gas fromthe ethanol fermentor is treated by emission control technology commonly used in the industry (e.g., water scrubber; thermal oxidizer) that will minimize the release T . reesei 3400 to the atmosphere. Page 35 of 141p. 37

Danisco US, Inc. MCANTSOVSMC2 (iii) Worker Exposure Worker activity is focused on monitoring plant performance. The plant is controlled by a microprocessor based distributed control system . Monitoring the control system, process sampling, and process analytical work constitute the majority of routine operator activity. The estimated worker exposure to viable T . reesei 3400 is based on a 24/7 schedule with plant operators working 12 hour shifts. Worker exposure to viable T . reesei 3400 Letter in Unit Operation Unit Operation details # of Duration of Protective process Workers exposure Equipment/Eng map Exposed Controls Hrs/day Days/yr 

# 6 1 . Impervious 

# F Ir gloves, safety

k glasses / ~ Impervious

gloves, safety glasses Impervious gloves, safety glasses Impervious gloves, safety glasses Impervious gloves, safety glasses / ff IN Im ervious p gloves, safety glasses Impervious gloves, safety glasses

Page 36 of 141p. 38



### Danisco US, Inc. MCANTSOVSMC2 iv Environmental Release and Disposal of Subiect M icroorganism

Release Point Amount of new substance released CFU/batch Media of Release Control Technology Efficiency (Letters refer to process steps in To Envi- To Control diagram) ronment Technology

/ Water _ Water

, Water _ Air (Airborne droplets) Land 

# 1V C 111r6 Water



### Note on CFU numbers used above: The number for the release points "solids for treatment, disposal" and "Waste water to discharge" have been calculated by taking into consideration a 99% retention of fungal mass with the fermentation solids separated from the liquid phase in process step K . For fermentor off-gas: The amount of CFU released into the control technology, such as industry standard scrubber or thermal oxidizer, is based upon industry experience in anaerobic microbial fermentations. The efficiency ofthe control technology is based upon a conservative performance of a scrubber used in large scale fermentation (99%reduction of release of CFU) .

Page 37 of 141p. 39



### Danisco US, Inc. MCANTSOVSMC2 6) Health and Environmental Effects Data (1) Test data on the new microorganism- include all test data which are related to the effects on health or the environment of any manufacture, processing, distribution in commerce, use, or disposal of the new microorganism Danisco has not conducted tests specifically on the new microorganism, but has conducted tests on T

. reesei (the recipient microorganismspecies) . 

### T . reesei was tested for pathogenicity and toxicity and products derived from various strains ofT

. reesei were investigated for numerous toxic endpoints. These include studies initiated by Danisco/Genencor, studies obtained fromMiles Inc., and studies published in the literature (see Appendix 3) . The non-toxic and non-pathogenic status of T . reesei is well established (see (j)(2)(i) below) . The new microorganismT

. reesei 3400will be contained during use and inactivated prior to disposal . (2) Full report or standard literature citation must be submitted for the following types oftest data: 

### (i) Health effects data T . reesei is a non-pathogenic fungus as the following data will demonstrate. T . reesei is not present on the list of pathogens used by the EU (Directive Council Directive 90/679/EEC, as amended), or on culture collection lists fromGermany (Deutsche Sammlung von Microorganismen und 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. Furthermore, in 1999, the U .S. FDAaffirmed cellulase enzyme preparation derived fromT

. longibrachiatum(nowknown as T . reesei) as GRAS for use in processing food (FR 64:28658-28362; 1999 included herein by reference) . This assessment includes a determination that T . reesei is a safe host for production ofthis food-processing enzymes and that the host is nonpathogenic and non-toxigenic. Danisco/Genencor has used T . reesei strain RL-P37 and strains derived from it for years to produce commercial cellulase preparations on a large scale. Also, the parent of RL-P37, strain QM6a, has been used for the same purpose widely in industry, e.g. in Japan since the 1960's (Toyama, 1969) . T . reesei cellulase has been safely used in food, animal feed,



### Page 38 of 141p. 40



### Danisco US, Inc. MCANTSOVSMC2 pharmaceutical, wood processing and biofuel applications ever since,

demonstrating the safety of T . reesei . It is concluded that the new microorganism, T

. reesei strain 3400, is nonpathogenic and non-toxigenic. (ii) Ecological effects data 

### Asurvival study was performed which showed that an intermediate strain, M1-1 .1, does not survive better than its unmodified host RL-P37 (see Appendix 4) . (iii) Physical and chemical properties data There are no applicable physical and chemical properties data for the subject microorganism

.



### Page 39 of 141p. 41

Danisco US, Inc. MCANTSOVSMC2

(iv) Environmental fate characteristics See (j)(2)(ii) above. (v) Monitoring data and other test data related to human exposure or to environmental release ofthe new microorganism 

### No additional data are available for this particular microorganism . However, Nevalainen et al . (1994) provided a general overview of T . reesei and its safety as an industrial production organism . (3) Other data concerning the health and environmental effects of the new microorganismthat are known to or reasonably ascertainable to the submitter (i) Any data, other than test data, in the submitter's possession or control None other than that cited above. (ii) Any data, including test data, which are not in the submitter's possession or control 

### None otherthan that cited above.

Page 40 of 141

### Danisco US, Inc.

MCANTSOVSMC2 (4) References

Domsch, K

. H ., Gams, W

. andAnderson, T.-H

. 1980. Compendiumof Soil Fungi . Academic Press, London. Vol . I, p. 859 and II, p. 405. Dugan, F.M

. 1998. ANote on Trichoderma reesei and T . longibrachiatum, United States Federation for Culture Collections Newsletter. 28(2) : 1- 2. Eveleigh D

.E. 1985. Trichoderma. In: Demain, A .L. and Solomon, N

.A. (Eds.), Biology of Industrial Microorganisms. Benjamin Cunnings, London, p. 487-509.

Khuls, K, Lieckfeldt, E., Samuels, G .J ., Kovacs, W

., Meyer, 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 Hypocreajecorina, Proc. Natl . Acad. Sci . USA93: 7755- 7760. Nevalainen, H

., Suominen, P

., and Taimisto, K

. 1994. On the safety of Trichoderma reesei, J. Biotechnol . 37: 193-200 . 

### 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. Toyama, N

., 1969. Applications ofCellulases inJapan. In: Cellulases and Their Applications, R . F. Gould (Ed.), Advances in Chemistry Series 95, American Chemical Society, Washington, DC, p. 359-390. List of Appendices 1 . Identification Kit - Trichoderma reesei 3400 2 . Description ofthe Genetic Construction of Trichoderma reesei 3400 3. Trichoderma reesei Safety Studies Summaries 4 . Survival of Trichoderma reesei Strains

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## Appendix 1 : Identification Kit - Trichoderma reesei Strain 3400 1 . Introduction The methods in this document are designed to allow one to determine if an unknown fungal strain is the production strain (T . reesei 3400) . To aid in identification, one can use a combination of morphological and molecular characteristics ofthe production organism . The strain has the following key identifying characteristics,



### These identifying characteristics can be used to distinguish the T . reesei producing organismfrom

other fungi . The steps involved in characterization are described below . The first three characteristics provide a phenotypic characterization. Ifthe unknown fungal strain fails to produce the expected result with the first three characteristic, then it can be concluded that it is not the production strain 

### Ifthe unknown fun al strain does match the first three characteristic and it is desired to confirm that the strain is , then one should proceed with the molecular characterization. Ifthe unknown strain matches all ofthe above characteristics, both morphological and molecular biological, it can be concluded that it is the production strain.

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### Danisco US, Inc. MCANTSOVSMC2 2 . Materials and Methods 2.1 Strains Negative Control Parental Strain: T . reesei QM6A Pilot plant Production Strain: T . reesei - 

### 2.2 Media - Potato Dextrose Agar (PDA) is used to culture the organismfor phenotypic characterization.

Potato Dextrose Agar (PDA) 

### Available commercially, composed of, Potatoes, infusion from Dextrose Agar Per Liter 200g 20 g 15g Mix well in 50-80%final volume ofdiH20, then bring to 100%final volume. Autoclave, cool to 55 °C and pour.



### Part 1 in 500ml : 1000x salts (see below) Noble agar Adjust to pH6 Autoclave Store sterile media at 55 °C 

### Part 2 in 500 ml : CsCl Glucose KH2P04 MgS04*71-120 CaC12*21-120 Adjust pHto 4.5, filter Add to warm

Part 1 . Pour plates. 1 ml 20 g

1 .68 g 20.00 g 15.00 g 0.60 g 0 .60 g sterilize through 0

.2 micron filter.

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### 1000x Salts Per Liter FeS04*71-120 5 g 

### ZnS04*7H20 1 .4 g MnS04*41-120 1 .6g CoCl2*61-120 1 .0 g Add deionized water, mix, filter sterilize through 0.2 micron filter.

Vogel's MinimalAgar 50XVogel's Stock solution 20 ml Agar 20g Deionized water to 1 L 

### Combine and autoclave. Cool tojust above gelling temperature (-45 °C) and pour plates. 50XVogel's Stock solution 20 ml Agar 20 g Deionizedwater to 1L Combine and autoclave. Cool tojust above gelling temperature (-45 °C) and pour plates. 50XVogel's Stock Solution Prepare 3 separate stocks 

### Stock 1 : Sodiumcitrate 150g MgS04*7H20 log CaC12*21-120 5 g Dissolve in deionized water 300 ml 

### Stock 2: KH2P04 250 g Dissolve in deionized water 500 ml Stock 3: NH4N03 100g Dissolve in deionizedwater 200 ml When all 3 solutions are completely dissolved, combine and filter sterilize (0.2 micron) . Page 44 of 141p. 46



### Danisco US, Inc. MCANTSOVSMC2 Add Vogel's Trace Element solution (see below) 5 ml Vogel's Trace Element solution g/L Citric acid 50 ZnS04*7H20 50 Fe(NH4)2S04*6H20 10 CuS04*51-120 2 .5 MnS04*4H20 0 .5 

### 0.5 H3B03 

### NaMo04*2H20 0.5 Filter-sterilize using a 0.2 micron filter.

2 .3. Morphological and Physiological Characterization Determination of Colony morphology: 1 . Label separate PDAplates for each strain to be tested. 2. Using a sterile loop, streak the production strain in the center ofa plate. 3. Using a sterile loop, streak the unknown fungi in the center of a second plate. 4. Incubate at 28 °C for approximately 5 days. 1 . Label sections ofan MMAplate for each strain to be compared. 2 . Inoculate the control strains, T . reesei QM6A in the center oftwo sections. 3. Repeat with the production strain, 4. Repeat with the unknown fungus. 5. Incubate the plates at 28 °C for 5-6 days. 

### 1 . Label sections of an VMACE plate for each strain to be compared. 2 . Inoculate the control strains, and T . reesei QM6Ain the center of two sections. 3. Repeat with the production strain, 4 . Repeat with the unknown fungus. 5 . Incubate the plates at 28 °C for 5-6 days.

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### 2.4 Molecular Biology Characterization Chromosomal DNAExtraction and Purification fromMycelia: l . Grow

fungi overnight in 50 ml YEG(5 g/L yeast extract, 20 g/L glucose), 30°C, 250 rpm . 2 . Harvest mycelia by filtration through sterile Miracloth (CalBiochem) ; rinse mycelia in TE; squeeze to remove excess liquid. Freeze paste at -80°C until extraction. 3 . Grind frozen mycelia to a powder, with dry ice, in a coffee grinder. 4. Transfer ground mycelia to a phenol-resistant 50 ml centrifuge tube. Add 22 ml TE and 4 ml of20%

SDS. Vortex vigorously and let sit at roomtemperature for 10 minutes. 5. Add 10 ml each of neutralized phenol and chloroform(or 25 :24:1 phenol :chloroform

:isoamyl alcohol) . Mix by shaking and centrifuge at roomtemperature, -5000 rpm, 10 minutes. 6 . Transfer aqueous upper layer to a clean tube. Repeat extraction ifaqueous phase is cloudy or contains debris. Add 1-4 mg proteinase K ; incubate 55°C for 20 minutes. 

### 7. Extract, as above, with an equal volume ofphenol :chloroformand transfer aqueous layer to a clean centrifuge tube. 8. Add 1/10 volume of 3M sodiumacetate, pH 5.2, mix, and add 0.8 volumes isopropanol . Incubate on ice for 30minutes. 9. Centrifuge for 15 minutes at >10,000 rpm, at room temperature. Thoroughly drain tube of isopropanol and resuspend the pellet in 6 ml TE. 10 . Add - 2mg RnaseA, mix, and incubate at 37°C for 30 minutes. Then add - 2mg proteinase Kand incubate at 55°C for 10 minutes. 11 . Extract twice with phenol :chloroform . Transfer aqueous phase to a clean tube. Add 1/10 volume of 3 M

sodiumacetate, pH 5.2. Mix and add 0 .8 volumes cold isopropanol . Incubate on ice for 30 minutes. Centrifuge at >10,000rpm for 15 minutes. Drain off isopropanol thoroughly. 12. Dissolve in 0

.5 ml TE. Add 1/10 volume 3M sodiumacetate, mix, and add 1 ml cold 95% ethanol . Incubate on ice for 30 minutes. Centrifuge at >10,000 rpm for 15 minutes. Aspirate ethanol andwash pellet in 0.5 ml 70% ethanol . Aspirate ethanol and air dry pellet for 15 minutes. 13. Redissolve pellet in up to 0 .5 ml TE.

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### Polymerase Chain Reaction (PCR) procedure: This protocol is to be used with purified chromosomal DNA . Each strain, the negative control strain QM6A, and the unknown(s) should be tested for amplification

. 

### Mix the following components in a Hot Start 100tube (Molecular Bio-Products, Inc. Catalog # 6005) : Water 83 ul or 81 ul

lOX Pfu Ultra 11 Reaction Buffer (Stratagene) 10 ul IOmM dNTPs (Roche) 2 ul Forward Primer (20 uM) 1 ul Reverse Primer (20 uM) 1 ul Chromosomal DNATemplate or control plasmid 1 ul or 2 ul (depending on the

concentration) 

### Pfu Ultra Il DNAPolymerase (Stratagene) 1 ul Total Volume: 100 ul Place the tube in a programmed thermal cycler. 

### Program the automated thermal cycler according to manufacturers' instructions for the following program

: 94 °C for 2 min; 94 °C for 20 sec, 58 °C for 30 sec, 72 °C for 15 sec (28 cycles) Hold at 4 °C until ready to run gel . Electrophoresis of DNAsamples: To analyze the DNAproducts, load 5 pl ofthe PCRreaction and a Molecular Weight Marker into separate wells ofa 1 .2 % E-gel (Invitrogen Catalog No. G5000-01) . Run the gel for 30 minutes at 70 volts. Illuminate under a UV light.

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### 3. Expected Results for pilot plant strain 3.1 Colony Characteristics Production strain produces spores of white color on PDAplates. In contrast to strain QM6A, which produces green colored spores on PDAplates. See Figure 1 .

Ifan unknown fungal strain does not produce the expected result for the three henoty ic characteristics, then it can be concluded that it is not the production strain 

### These results can be confirmed by proceeding with the molecular characterization. 3.3 PCRConfirmation 

### The primer pair described above will amplify

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# Figures

Figure 1. Growth on Potato Dextrose Agar APDAplate was streaked in two parts with the T . reesei strain and the wild type strain T . reesei QM6A

. The plate was incubated at 28 °C for 5 days. The T . reesei wild type strain QM6Ais characterized by green conidia. The T . reesei strain -~displays conidiation that is white in color. Intensity of spore color is highly dependent on culture conditions .

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Figure 2

. T. reesei



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### Fi2ure 3.

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### Danisco US, Inc. MCANTSOVSMC2

Fii!ure 4

.

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## Appendix 2: Description of the Genetic Construction of Trichoderma reesei 3400 Introduction Trichoderma reesei is a mesophilic deuteromycete. It is awoodrot fungus and naturally secretes cellulase and xylanase enzymes. Several commercial cellulase andxylanase preparations are manufactured by Genencor and sold for anumber ofapplications. Cellulase is a generic name for a groupofenzymes and T . reesei produces at least seven separate cellulase enzymes each ofwhich is known to be a separate gene product. These enzymes are twocellobiohydrolases (IiTBMB 3.2.1 .91), CBHI and CBHII ; four endoglucanases (IUBMB 3.2.1 .4), EGI and EGII (previously known as EGIII in the literature) EGIII and EGV ; and a (3-glucosidase (IUBMB 3.2.1 .21), BGLI . The concerted activity ofthis group ofenzymes will degrade the cellulose polymer to release the monomer glucose . 

### Methods fortransformation of T . reesei with exogenously added plasmid DNA have been published (Penttila et al ., 1986; Gruber etal ., 1990; Smith et al ., 1991) . Stable transformants arise by integration ofplasmid DNAinto the chromosomes ofthe host. Integration can be at sites in the genome that have homology with aregion ofthe plasmid DNA or can be at non-homologous sites. anism, we have inserted into this

The host strain usedwas T . reesei strain e derivation and characterization of RL-P37 has been published previously (Sheir-Neiss and Montenecourt, 1984), and Genencor received the strain directly fromDr. Montenecourt . It is a cellulase over-producing strain that has been obtained as a result ofseveral mutagenesis steps fromthewell knownwild-type strain (QM6a) . 1)

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16) p. 65

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### REFERENCES Barnett, C.C., Berka, R.M ., Fowler, T. (1991) Biotechnology (NY) 9: 562-7 Bower, B., Kodama, K . et al . (1998) . In: Carbohydrases from Trichoderma reesei and Other Microorganisms. (The proceedings ofthe Tricel '97 meeting held in Ghent, Belgiumon 28-31 August 1997) . Editor Marc Claeyssens. Cambridge, UK, The Royal Society of Chemistry: 327-334 . 

### Brosius, J. (1989) DNA8:759. Bundock, P., Den Dulk-Ras, A ., Beijersbergen, A

., and Hooykaas, P.J.J . (1995) EMBOJ 13: 3206- 3214 Chen, C.M

., Gritzali, M

. and Stafford, D

.W . (1987) Biotechnolo 5:274-278. De Groot, M

.J .A., Bundock, P., Hooykaas, P.J.J. and Beijersbergen, A .G .M . (1998) Nature Biotechnol . 16: 839-842 Gruber, F., Visser, J., Kubicek, C .P. and de Graaf, L.H. (1990) Curr. Genet. 18:447-451 . Hajdukiewicz P., Svab, Z., Maliga, P. (1994) Plant Mol . Biol . 25: 989-994 Hood, E.E., Gelvin, S.B., Melchers, L.S., and Hoekema, A . (1993) Transgenic Res. 2: 208-218 Kado, C .I. (1991) Crit. Rev. Plant Sci . 10: 1-32 Ooi, T., Shinmyo, A

., Okada, H

., Hara, S., Ikenaka, T., Murao, S. andArai, M . (1990a) C'.urr. Genet. 18:217-222. Ooi, T., Shinmyo, A

., Okada, H

., Murao, S., Kawaguchi, T. and Arai, M . (1990b) Nucl . Acids. Res. 18:5884 . Penttila, M

., Lehtovaara, P

., Nevalainen, H

., Bhikhabhai, R

. and Knowles, J . (1986) Gene 45:253- 263. Penttila, M

., Nevalainen, H

., Ratto, M

., Salminen, E. and Knowles, J. (1987) Gene 61 :155-164. 

### Saarilahti, H

.T., Henrissat, B

. and Palva, E.T. (1990) Gene 90:9-14. Saloheimo, M

., Lehtovaara, P

., Penttila, M

., Teeri, T

.T., Stahlberg, J., Johansson, G ., Pettersson, G

., Claeyssens, M

., Tomme, P

. and Knowles, J .K.C. (1988) Gene 63:11-21 . 

### Schulein, M

. (1988) Methods in Enzymology 160:234-242. Sheir-Neiss, G

. and Montenecourt, B

.S. (1984) Appl . Microbiol . Biotechnol . 20:46-53. Page 67 of 141p. 69

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### Shoemaker, S., Schweickart, V ., Ladner, M

., Gelfand, D

., Kwok, S., Myambo, K . and Innis, M

. (1983) Biotechnology 1 :691-696. Smith, J .L., Bayliss, F.T. and Ward, M . (1991) Curr. Genet. 19:27-33. Strathmann M

., Hamilton B

.A., Mayeda C

.A ., Simon M

.I., Meyerowitz E.M

., Palazzolo M

.J. (1991) Proc Natl Acad Sci USA . 88: 1247-50 Teeri, T., Salovuori, l . and Knowles, J. (1983) Biotechnolo 

### 1 :696-699. Teeri, T .T., Lehtovaara, P

., Kauppinen, S., Salovuori, l . and Knowles, J. (1987) Gene 51 :43-52. 

### Ujiie, M ., Roy, C . and Yaguchi, M

. (1991) Appl . Environ. Microbiol . 57:1860-1862. Van Arsdell, J .N., Kwok, S., Schweickart, V.L., Ladner, M .B., Gelfand, D

.H . and Innis, M

.A. (1987) Biotechnology 5:60-64.

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: Figure 4: Figure 5: Figure 6: Figure 7: Figure 8

: Figure 9: 

### Figure 10

: Figure 11 : Figure 12: Figure 13: Figure 14: Figure 15: Figure 16: 

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## Appendix 3: Trichoderma reesei Safety Studies The pathogenicity and toxigenicity potential ofTrichoderma reesei were tested by various investigators and are summarized in this Appendix. I . STUDIES SPONSORED BY DANISCO/GENENCOR 1 . Toxicity/pathogenicity testing ofTrichoderma reesei following acute intraperitoneal challenge in rats. IIT Research Institute, No. 1182 SN3, dated September 2000. This study was conducted in accordance with the US. EPA Microbial Pesticide Test Guideline OPPTS 885.3200and the US. EPA Good Laboratory Practice Standards (40CFR.160) . In this investigation, the pathogenicity potential of Trichoderma reesei [strain EG1-EP9, derived from RLP-37 to overproduce EGl using rDNA techniques] was tested in male and female CD rats following an acute intraperitoneal injection of 5.6x 106 colony forming units (CFU) . Groups of animals of both sexes were sacrificed on Days 0, 7, 21, and 35 after injection ofthe test substance for microbial enumeration. The results were compared to those obtained fromheatkilled test substance group, naive control group and shelfcontrol group. No deaths occurred and no adverse clinical observations were noted. The major necropsy findings were enlarged spleens noted in treated females on Days 7 and 21 and in treated males on Day 21 . On Day 7, a significant decrease in relative liver weight was noted in treated males but was no longer observed on subsequent necropsy days. Astatistical difference in body weight was noted in treated males between Days 0-7 but all animals showed complete recovery thereafter. By Day 21, Trichoderma reesei was detected in the peritoneal lavage fluid and masses oftreated male rats and from the liver and peritoneal masses oftreated female rats. By Day 35, the test substance was detected only from the peritoneal masses. Complete clearance was estimated to be 67 and 43 days for male and female rats, respectively. Under the conditions ofthis investigation, Trichoderma reesei [strain EG1-EP9] was not found to be toxic or pathogenic in rats. 2 . Acute intraperitoneal toxicity/pathogenicity study in rats with Trichoderma longibrachiatum(reesei) . MABioServices No. M97BL78 .6P41 .01, datedJanuary 26, 1998. This studywas conducted in compliance with the Good Laboratory Practice Standards as published by the OECD Guidelines, the US. FDAand the US. EPA . In this investigation, Trichoderma reesei [strain A83, derived fromRLP-37 through mutation and selection] was administered as a single intraperitoneal dose of 2 .2 x 107 cfu to 15 male and 15 female Sprague Dawley rats. Five additional animals per sex were used as untreated control group . Groups ofanimals were killed on Days 4, 8, 15, 22 and 32 for microbial enumeration and necropsy observations. No deaths occurred and no adverse clinical observations were noted. No treatment related differences in body weights or body weight changes were observed at any time point, when compared to controls. No abnormal findings were noted at necropsy. By Day 15, Trichoderma Page 88 of 141p. 90

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### reesei was detected in only the spleen and liver ofboth males and females and, by Day 22, in only the spleen ofboth male and female rats. Total clearance ofthe test microbe from the spleen was achieved by Day 32. Under the conditions ofthis investigation, Trichoderma reesei [strain A83] was not found to be toxic or pathogenic in rats. 

### II . STUDIES OBTAINED BY DANISCO/GENENCOR l . Pathogenicity study in rabbits with Trichoderma reesei . Miles Inc. Report No. MTD0159, datedJune 13, 1990

. 

### Trichoderma reesei was administered as a single intravenous injection to groups ofrabbits. Other groups ofrabbits that received either cell free medium, uninoculated culture mediumor uninoculated phosphate buffer saline served as controls. All animals were observed for overt signs oftoxicity and sacrificed on Day 14 for microbial enumeration and necropsy observations. No deaths occurred and no adverse clinical observations were noted. Therewere no overt signs of infections or toxicosis and no abnormal findings were found at necropsy. Tissue cultures confirmed the presence ofviable spores in liver and spleen 14 days following the intravenous injection. 

### Under the conditions ofthis investigation, Trichoderma reesei was not found to be toxic or pathogenic in rabbits. 2 . Pathogenicity study in mice with Trichoderma reesei . Miles Inc. Report No. MTD0160, datedJune 14, 1990. Trichoderma reesei was administered as a single intravenous injection to groups of mice. Other groups ofmice, which received either cell free medium, uninoculated culture mediumor uninoculated phosphate buffer saline served as controls. All animals were observed for overt signs oftoxicity and sacrificed on Day 14 for microbial enumeration and necropsy observations. No deaths occurred and no adverse clinical observations were noted. There were no overt signs of infections or toxicosis and no abnormal findings were found at necropsy. Tissue cultures confirmed the presence ofviable spores in liver and spleen 14 days following the intravenous injection. Under the conditions ofthis investigation, Trichoderma reesei was not found to be toxic or pathogenic in mice.

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### Danisco US, Inc. MCANTSOVSMC2 III . STUDIES PUBLISHED INTHE LITERATURE Published data indicated that Trichoderma reesei is non-toxigenic and non-pathogenic and toxins were not found in the culture broth, the tox batch or the commercial enzyme preparation (Hjortkjaer et al ., 1986) . The potential pathogenicity of Trichoderma reesei in immunosuppressed animals was investigated in mice, guinea pigs and rabbits by the subcutaneous administration ofa large dose ofcortisone acetate (5 mg) 2 days before the administration of 107 or 105 viable or 107 killed spore suspensions. All animals were observed for 40 days then sacrificed (Modeweg-Hansen, 1979 as cited in Hjortkjaer et al ., 1986) . The authors reported that spores were microscopically and microbiologically identified in different organs 40 days after administration of viable Trichoderma reesei by intravenous injection. No effects were seen after intraperitoneal administration of 105 viable spores. These investigations suggested that under special conditions - a large inoculumgiven to immuno-suppressed animals - the microorganism could be pathogenic. However, this finding is not unexpected due to the nature ofthe immunological condition ofthe animals prior to test substance administration. 

### Nevalainen et al . (1994) provided an overviewof Trichoderma reesei and its safety as a production organismfor cellulase enzymes. CONCLUSION In conclusion, there is a body of information to assert the lack of pathogenicity and toxicity of Trichoderma reesei (formerly longibrachiatum) . 

### REFERENCES: Hjortkjaer RK et al . : Safety evaluation ofcelluclast, an acid cellulase derived fromTrichoderma reesei . Food ChemTox, Vol . 24, pp 55-63, 1986. IIT Research Institute, Study No. 1182 SN3: Toxicity/pathogenicity testing of Trichoderma reesei following acute intraperitoneal challenge in rats. Final report dated September 2000. MA BioServices, Study No. M97BL78 .6P41 .01 : Acute intraperitoneal toxicity pathogenicity study in rats with Trichoderma reesei (formely longibrachiatum) . Final report datedJanuary 26, 1998. Miles Inc., Study No. MTD0159: Pathogenicity study in rabbits with Trichoderma reesei . Final report dated June 13, 1990. Miles Inc., Study No. MTD0160: Pathogenicity study in mice with Trichoderma reesei . Final report datedJune 14, 1990. Modeweg-Hansen L. : Acuteoral toxicity ofcellulase SP122 to rats, mice, and dogs. Novo Reports, 1978. 

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### Nevalainen Het al . : On the safety ofTrichoderma reesei, J Biotechnology, Vol . 37, 193-200, 1994.

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# Trichoderma reesei Safety Studies Summaries from

Laboratory Reports

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S TU DYTITLE IUXICITYrPATII(KF;NICITYTFSTI!JGOr Tra~hrxtermareesei 1"'ClI .1 .nWINGACUTE INTRAPERITONEAL CHALLENGE IN RATS

PERFORMEDACCORDING I.1,S EPA Microhial Pesticide Test Guideline: OPPTS 885.3200 AI! TNnR WilliamM

. Mega, B.S. 4 TUDY COMPILE ION Fo-Ttember .1000 (Final Rqwti) PERFORMING LABORATORY IIT Research Institute Life Sciences Operation 10West 35th Strect Chicago, Illinois 60616-3ty9 LABORATORY PROJECT ID IITRl Project No.: 1182 SN3 Sponsor Stody Nn : 1

.95 SPL}7VSOR GerwncurInlcrnational, Inc 925 Page Mill Road Palu Alto. CA 94304-1011

since 1936 (100 IS THE TOTAL NUMBEROF PAGES IN THE REPORT) . p. 94



# I

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'1'OXICITYIPATHl1GEN!('_tTY TESTING OF Tricbmdermrn reesei FOLC,OWING ACUTE INTRAPERITONEAL CHALLENGE IN RATS

GLPC.'OMPI .IANCE STATEMENT

. T'hi5 study was conducted in accordance with U,S. F .PA (iood lshoratorn Practice Standards (40('FR l60) . The characterization and stability of the bulk test substance are the responsibility of the Sponsor . The rawdata have been reviewed by the Study Director, who certifies that tht information contained in this report represents an appropriate and accurate conclusion within the context of the study design and evaluation criteria.

SUBMITTED BY; Study Director :

WilliamA9, Mega. B S Rcscarch Biologist Life Sciences Operation Spouaor . Beth A. Carn

;obt' Sponsors Representative Date

IIT RFtiEARCH INSTITC"fE 11 S'SN.~

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TOJiICITYJPATHt1GENICITYTESTING OF Trichodernwf rernei FOLLOWINGACUTf' . INTRAPERITONEAL CHALLENGE IN ItA7'S .J

.

. FOREWORD I'his report describes a study conducted bti " the l.ifc Sciences Operation, IIT Research Institute, for C)enenatr International, Inc., during the period April d, 2401) to June 6, 2000 . 7he study was performed under lIllil Project No. 1182. The Sponsor Representative for the study way Ms Beth A c'oncoby WilliamM

. Mega, B 5 ,scn~ed as Studyllircectnr . J . Brooks Harder . D,V.M .. Staff Veterinarian,was resportsible for animal care l3attholnmew C Wacek, M .S., Technical F

:ditior, helped in the preparation of the final report Ronald A. Boyne, B.S., was nesprntsible for Quality Assurance. Fxperimental data are recorded m a hinder labeled 1182 SN3. All rawdata and a copy ofthe final rept.xt will be archived in the IITRI Archives. Respectfully suhmitted,

L 

# Z Z 

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. Mefia. B.S. ' " Date Study Director Research Biologist Lite Sciences Operation Approved by:

Robert l . Sher,vcwd, Ph.U . Date Assistant Vice President and Manager Microbiology & Immunology Division l .ifc Sciences Operation

IIT RESEARCH INSTITUTE ; 3 J 182"

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TABLE OF CONTENTS c_iLP CO .ti1PI .lANCE STATEh1F'1J'r . [,'ORE-WARD . . . . . . . . . . . , 4 SUMMARY 1 . iNTROUIICTION . . . . . . . . . . . . . [1. MATF?RIALS ANDMEl'HO[ri . A Test Substance Receipt and Handling . . . . . . . . . . . . . , . . . . . . . . . . . . . . . D. Animals and Animal Cart: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Animal Quarantine and Randomization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D . Preliminary Challenge Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E Experimental Design . . . . . . . . . . . . . . . . . . . . . . . . . , ., . . . . . . . . . . . . . F, Test Substance 4iabilitv Dctermination . . . . . . . . . . . . . , . . . . . . . . . . . . . G. Ptrparation of Test Substancc fcwDosing . . . . . . . . . . . . . . . . . . . . . . . H. Sensitivity ofDetection Method tiir Test Substance . . . . . . . . . . . . . . . . . . . . . . . I Test Substance Titer, Contaminant, "fest MediumGrowth and Homogeneity. . . . . J . Body Weights . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . K. Clinical Observations . . . . . . . . . . . . -- . . . . . . . . . . . . . . . L. Nccropsy . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . M . Microbial Enumeratum. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . N. Statistics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I . Microbial Enumeration . . . . . . . . . . . . . . . . . . . . . .- . . . . . . . . . 2. Body Weights , . . . . . . . . . . . ., . . . . . . . . . . . . . . . . . . . . 3. Organ Weights . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . 0 Arrhives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S 8 4 9 9 9 la 10 ip tl 11 11 It 12 12 12 I? 13 lit . RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 A Test Substance Titer, Contaminant, Test MediumGrowth and Hornogeneity.. . . . . . 1 Test Substance Titer, llomogenettv and Contaminant . . . . . . . . , . . . . . :, Test Medium(irowth . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . li Sensitivity of Ucteetion ofTest Substancc . , . . . . . . . . . . . . . . . . . . . t' Test Substance Mw Administration . . . . . . . . . . . . . . . , . . . . . [) Body Weights and Body Weight Gains . . . . . . . . . . . . . . . . , . . . . . . . . 1? Clinical Observation, ; . . . . . F. (iroas Nr-.ropsp (hccr+-axiom . . . . . . . . . G Organ Weights H Enumeration . . . . _ . . . . . . . . . 13 13 13 I :i IS 15 15 15 Ib IV. DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . ., Ifi 'Y' . CONCLUSIONS . . . . . . . . . . . . . . I'

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. TABLE OF CC)IYTf' .NTS (coat.l Rav

Vl . QLIALITY ASSURANCE STATEMF :N"r . . . . . . . . . . - . . _ , , . . . . . . IS VII TAf3LGS . . . . . . . . , . . . . . . . . . . . . . . . . . . . 19 Table 1 . Animal Identification Numbers . . . . . . . . . . . . . , . . . . . . . . . . . 24 Table 2. Sensitivity of Doection of 7'richcWrrma reesei . . . . . . . . . . . . . . 21 Table a . Effect of lntraperitoneal Administration of Trichxferma reesei

on Rody Weights of Rats . . . . . . . . . . . . . . . , . . . . . . . . . . . . . 2 t Table a Effoct ofIntraperitontal Administration of Trieheufermcr ree9ei

on Body Weight Gains of Rats . . . . . . . . . . . . . . . . . . . . . . . . . , . 34 Table 5. Effect of lntraperitoneal Administration of TriclaWernra reesei on _i

Clinical Observ'ations of Rats . . . . . . . . . . . . . . . . . ' Table 6. Effect of Intraperitoneal Administration of Trirhrxiermrr reesej on

Gross Nccropsy Observations in Rats . . . . . . . . . . ., . . . . . . . . ., . 26 Table ?. Effect of Intraperitoneal Administration of Trichrkierrna reesei on

Relative Organ/Tissue Weights of Male Rats . . . . . . . . . . . . . . . . . . . . 48 Table 8. Effect of IntrVeritoneal Administration of Trichnderxmrersei on

Relative (}rgan"''rissue W eights ofFemale Rats . . . . . . . . . . . . . . . . . Table 9- Recovery of TrichMdrrma reeset Following Intraperitoneul _'+=

Administration fromSelected Tissues of Male Rats . . . . . . . . . . . . . . . . . Table ICl Recovery of Trichnderrrru reesei Following Intraperitnncal

Administration fromSelected Tissues of Female Rats ±4 VI11 FIGURES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 Figurc I . Clearance of Mass-Associated Tric~hWerrsq reescl ftomMalt

Rats Following Intraperitoneal Challenge . . . . . . . . . . . . . . . . . . 3? Figure 2. C.'Icaraix:e ofMass-Associated Trichai'erma rresei fromFemale

R91S Following lntraperitoneal Challenge . . . . . . . . . . . . . . . . . . . 

### IX. APPENDICES . . . . . . . . . ., . . . . .- . . . . . , . . . . . . . . . . . . . . . 39 Appendix A- Individual Animal Data . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . 40 t3cKiy W'cights . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . _ . 41 Body Weights Gains . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . -i3 Clinical Observations . . . . . . 45 Necropsy f)h5ervations . . . . . . . . . . . . . , . . . . . . . . . . . . . . . 47 C1rg<tn \Vctghts aad T issuc:C?rgan Enumeration . . . . . . . . . . . . . . . . . 51 Appoiulix I3 - CcrtificawofAnalysis to Appendix C - Protocol aiKi I'rolocol Amendment . - . . . . . . . 't [

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# 1 .. TOXICITYIPATHOGENI( .'I7Y TFSTING OF Trfckarlrrrrra reetrf FOLLOWING ACUTE INTRAPERITONEAL CHALLENGE IN RA1'S SUMMARY Triih{ulcrin.r reesei . a microbe, was tested for toxicity and patfiogenicity in male and female C[1" rats hy obsrnation of the effects following intraperitotteal administration once on Day 0 of approximately 5_0x 106colony forming units (cfu)of viable technical grade (TG) test substance at a volume of 1 nil per rat. 'The dose by the intrsperitoneal routewas consistent with U.S: EPAt)PPTS Guideline, 885_3200, in that it was the highest deliverable dose ofneat test substance. In addition, another group at"male and female animals were similariy challenged on Day 0 with 5 0x I Or' crlis ofhcat-k died technical grade (KTG) test substance. Anaive control (NC) group and a shelf control (SC) group (housed with the TG gtoupj for each sex wvrc also included. Following dosing on I)ay . 0, designated male and female TO, KTGand NCrats in scheduled groups weft sacrificed at four time points (0, ", 21 and 35days) for inicmbial enumeratiort- 5C tats wwc sacrificed on Day 35. Obsened or measured parameters consisted of clinical and necropsy observations, body weights, body weight gain--,, organ weights and microbial enumeration. The results indicated no evidence of toxicity or pathogenicity following intraperitoneal administration of Trichodernttr rer5ei to male and female CDt` rats. ?`o deaths occurred and no adverseclinical observations were noted Astatistically significant reduction in body weight gain in male TG- and KTG-dosedtats hetween Days 0 and 7appeared to he treatment related. However, the effect was rxu ofan,, toxicological significance since no similar effects were noted in femalcs durinq the same petiod and all male treatment groups showed complete recovery. The major necropsy findings were enlarged spleens, cysts in the liver and masses and accumulations in the heritonegl cavity of TC .i- and KTG-doscd rats. Statistically significant increases m relative spleen weightx, compared to the naive controlwere ohsetvcd in femaleTG-dosed rats on Days 7and 21 and in male TG-dosed rats on Day 21 . Also TG-dosed male rats showed statistically significant dcxrea5kNl rclativc liver weight on Day " Test suhstance wasdetected m both male and female TGdosed rats front the peritoncal Image fluid, spleen, liver, mesenteric lymph nodes and ceacumand from111c lidncvs ofT(i~dosed male rats on Day0 . Iletoctittrt oftcst substance fromthese Organs on Day 0 was tlir result of surface ctnitamination due to the dose route and not because of internal incorporation of the test cubctancc. By Day ' 1, test substance was oniy detected in the peritrtncal lii~~awc fluid and massesofmale fG-dosed rats andfromthe liverand pet ik7ncal mjsses ofTG-dosed fernale rats. On Uat 35. tcNr substance wasdetected only fromthe peritonea] masses of 1-6-closed malc and trrnale rats. ( ompletc clearance of test substance from TG-dosod Mt .r was estimated to IIT RESEARCH INSTITl1TE 11x2sN3

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r occurw ithin approrimately 6' days for male rats and 43days for female rats. Notest suhtitance w3s detected in \X', KTC~ and SC anirnah. Within the study design parameters measured, Triclux{ernra reesei . was not found to he tnxic v4 pathogenic at thc dosc administcrod by iiytraperitoneal injection to the male and female CD' rats employ,ed in the study .

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Hrial Report NN1-\ tittidti No_ hlr}TR1 .7R.i.;l'-ilJ)I ACUTE INTRAPERITONEAL TOXIC'ITY/PATIIOGENICITY STUDY IN RATS

I; Lst Article I nchrtdcrma lonvi.hrichiatum rcesci

Data RNuiremen Part 158.740 of' Eitle 40 of the Code of Federal Regulation-, (40 CFR 158

.740) Ciutdcluni 5uW- t\ ision r4 1 S2A-13 Author Visa S ci . Sha

:lton, Ph.f). Study

. (.-pmplet ed On J :uuarti '_6

. 1998 t'erformine [,;ihoratot} :41A Rtotiervicec. Inc. (MA) 46±11 Medical Center Drive RockvilIc, MD '20850 I,,ahc,ratqry Prqjcct Identifi cation h9l17Fi1 .78.6P41 .01 fit,c,nenr tittuly

_Nuoilx .-r '_411 Srx~tiuor Gencncor Intcrnutic,nnl . Inc. 92i Page Mill Road falo Alln, CA 9a?()4-It)1? I'agc ) of 95 

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Danisco US, Inc. MCANTSOVSMC2 [ :trial Report MA Studv \!o. N19?131.?$.irP4I .Of EXECUTIVE SUMMARY I rictnaderma IonLibracht atunt (rc-~~t , whrn administered inttaperiton~:a1LF -it ? 2x lc}' ltrlom Forming itnits (( .'hl I) per aniunal . prtxluccd no aclverk ef~feet, due it, toxicity Infectivity in nlntuat all o1 tlte selected tissues was drtectetl on rest Day -i . l otal clearance was demonstrated in all (if' the selected tissues by study termination. Since the inlcctivitN detected resulted in no apparent sign, (if disease in term, of gross pathology or abnormal syrnptontatoloiy and shows a f>attcrn of clearance, Tzi chtxlenna lonetbrachiattumtreeset was cottsidcr4d ncmpathos;enic and nontoxic in Spragtac-C),twlcy rats under the conditions of this stud}' . 1. StIM'1+IARY .+1 single rntraperitoneal dove of 2?r 107 C'htJ per animal of Frtch+xic~ ]c~nkilxacl»atunt Lrccst" i1 was administered to 15 male :utd 1 _5 fetnale Spragtrc-Dawlcy rats. Five additional animals per sex %vcrc used as an untreated control group. All animal ., were oh3erv~ed for clinical signs of toxicity, moribundity and mortality, twice within the first 6 hcturs after dosing I:apprxrximatcly I and 3 hours) and once daily thereafter. .A detailed "hands on" examination was perfbrtued at weekly in.tervais Body w~rights were recorded prior to dosing, weekly. and at each sacrifice Animals were sacrificed lbr combined sterile necropsy and microbial analysis on Vest Days 4. R, 15, ? 2 and 12. The gross necropsy procedure included external examination and a gross examination of specified tissues. For test rnicrobcs enumeration, hlocxl, ncsenteric lymph nodes, spleen, kidney, liver, lungs and brain tissues were collected aseptically . All animals survived to scheduled sacrifice, and no unusual clinical signs were observed. No treutrnent-related differences in body weights or body weight changes were ol>served in the treated att'tntals at any time point, when compared to controls_ During the course of the study, there was no apparent treatment~ rclatcd toxicity or evidence of pr,thogenicity . No abnornaal fndin~, attributed to iesi article treatment were noted in any of the animals at nccropKy A growth preparution ot the test tmcroUc, l ;rl~IUl~ ionl ;ihracitiatutn rccac't , ucts uscd to generate frozen stocks for tt<sr in preliminary experiments and For dosing in the main study. Based on test ailiclcltissuc homogcrultc mixing experiments conducted in rilro, the test nucr«he was recovered with fair rflicirncy (St7-RO% oh,ervetL'expo.~ .ted) from all sclcctea tissues and blood (hereafter also referred to as a "tissue") . ]it the main study, the test microhe was detected in the spleen . kidney and liv-rr of treated rnale rats and in the lyntph, blood, spleen, kidney. liver and lungs of treated female rats on Day ~1 On Day R, the test microbe was detected in lymph, splccn, liver and lungs of treated tnales and spleen and liver of treated frtnales. By I)ay 15. the test microbe was detected in only the splecn and liver ctt h<tth treated ntale and fentale rats and. by My 22, in only the ipleen (if ixrth trt:atcd males and I-cntalcs. 'l ota6 clearance of the test microbe fromthe p1cen wa, achieved by Day i_' . 1 i,inp the definition of infectivity as the invasion by and.'oi multiplication of' un organism within thrc : hom tissue, it %ra, concludcd that animals intectc.~cl with l rirhodertna Iongihrachianirn frcr,,et) demonstrated itt) detectable infrxtivitv by the end of thc test laerlcKi utxlcr the conditions (if this studv . Page 8 

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1-ut.tl I{elx>rt M~1 titttdy No. N147Ji1-7t;Gl'41 .Ut Since clearance ot intectaon wati iletnun .,U-ateci . and no signs uf tr+t article-rclatcd toxicity Or hathogcnicitr weri observed at the end of the test periad, { rich~ odenna lon~ihrac

hiatutn (reL.kl H't.lb Considered to be non-toxic and non-pathogenic in thts text >ystent. 

## 11. INTRODUCTION The purlwat of this study was to provide infitrnt:dton oil the toxicit> . infeY;tivity and hathugenicity ofa test nucrobe in rats using a single high dose exposure by intrttperitnne ;jl injection anti an :uieyuatc post-exposure observation petii>d. III, MATERIALS AND METHODS .4. Test Article 'Crichoderma lotlisihra<:hiatum reesci . a white cloudy spore suspension - not viscous, was received frozen oil July 22. 1997 and assigned A4 :1 chemical number 97BL78. It was stored frozen at s--60'C, Fhe Sponsor was responsible for the determination and documentation of the idcntification. composition and Ix)tency of the test article. The concentration of test microbe in the bulk test material was given by the 5ponroras 7x1()' CF'U+mL. Mcthod,of production of this test article are documented and retained by the Shx-onsor. A certilicatc of analysis on the test article is included in Appendix (.'- B . Microbiology 1. 141edia and Incubation Procedures 'fhc plating mediumspecified by the Sponsor wa., Potato Lk :Xtrose Agar (NC)A) (Remel . Richmond . VA), This medium Has used for plating all tissue-., and blood . All plates tiwtlx

: main study were incubated at 30±Z°(' for at least 4 days before reporting a negative rcsult . Otherwise, plates were incubated until colonies developed to an appropriate size for counting, giving the muximutu number (It' Cl .l! :platc, usually between ' and , day, . Serial dilutic,n.v were made ax necccsary to bracket plates with 30-100 CI :lYs for counting. Thrcrugluwt this report- unless noted otherwise . all plate ~;ounts and concentrations oh the test article and tissue ,ample5 are bascd oil enumeration of the tc,t article active ingredient Frichoderm ;4 loneibrachiatum

(r,ce~.uij which is referred to as -test microbe. .

Pa~~ .y 

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Report No. : M'I'D0159 Date : June 13, 1990

Safety Evaluation of Trichoderma reesez :

Intravenous Pathogenicity and Toxigenicity Study in the Rabbit

Authors M .C. Porter, V. Jasty, D.E . Reese*,

and R.E . Hartnagel Jr .

Performing _Lr+boratorv Toxicology Department Miles Inc. P.O. Box 40 Elkhart, IN 46515 *Microbiology Research Biotechnology Products Division

Miles Inc . Elkhart, IN 46515

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AasTxAcT Safety Evaluation of Trichoderma raesei : Intravenous Pathogenicity and Toxigenicity Study in the Rabbit Lntroductl=: The Biotechnology Group of Miles Inc . has an interest in Trichoderma reesei that has the capability to produce T .R. Cellulase, a carbohydrase enzyme with potential for food use . This study was conducted to determine the potential of this organism to induce a pathogenic or toxigenic response in rabbits and to provide data directed toward assuring product safety . Study Groups of 4 male and 4 female New Zealand white rabbits received either a viable or heat-killed spore suspension of T . rsesei, while groups of 3 males and 3 females received either the cell-free supernatant fluid in which the test organismwas grown or the uninoculated culture medium . Spore suspensions, supernatant fluid, and uninoculated culture medium were administered intravenously as a single dose of 1 .0 ml . Following administration of the test materials, animals were observed daily and body weights were recorded on days 0, 7, and 14 . Twenty of the 28 rabbits were sacrificed on day 14. Two males and 2 females in the viable and heat-killed spore groups were allowed to remain on study until sacrificed on day 28. Additional body weights were recorded for these animals on days 21 and 28 . At termination of the study on days 14 and 28, each animal was killed with euthanasia solution . Immediately following death, a blood sample was obtained from all 16 rabbits that received the viable or heat-killed spore suspensions and from the first male and first female in each of the other 2 groups . Samples were obtained via open chest cardiac puncture, using aseptic technique, and were cultured for T . roesei in 50 ml of potato dextrose broth. Additionally, 3 gross pathologic examination was performed on each dnimal . Specimens of .liver and spleen from rabbits from which blood was obtained were excised and cultured for the test organism . All blood and tissue cultures were then subcultured onto potato dextrose agar on days -2- NTUO15'3

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1, 4, and ?, and resulting growth identified by standard methods . Result ~ " Intravenous administration of viable and heatkilled spore suspensions of T. reesei, as well as the supernatant fluid in which T. rsesei had been grown to the highest obtainable concentration, elicited no overt indications of toxicosis . Tissue cultures confirmed the presence of viable spores in liver and spleen 14 days 7following i .v . administration of a high dose (=1o spores) of viable T . reesei . . Findings at 28 days postdosing, compared to those at 14 days, suggested gradual elimination of fungal spores from tissues . Additionally, the absence of tissue reaction, as determined by microscopic examination, indicated that T . reesei was nonpathogenic under the conditions of this study . C,qDS:lusions : The presence of fungal spores in tissues following parenteral administration is to be expected . Work with T. reesei in another laboratory showed the presence of fungal spores in liver, kidney, and spleen of mice until day 57 postdosing (Hjortkjaer et a1 ., 1986) . No attempt should be made to extrapolate these findings to other routes of exposure . Ingestion (the anticipated route for po3aikle accidental exposure of humans) of the test organism certainly would not be expected to pose any health threat . The final product, : .R. cellula5e, caused no adverse effects when administered orally to rats at an extremely high dose of 5 qrarns per kilogram of body weight (Porter et al ., 1989) .

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TABLE OF CONTENTS Paclg

INTRODUCTION . . . . . . . . . . . . . . . . . . . . . MATERIALS AND METHODS Culturing the Test Organism . . . " . - . " " " ~ " Pathogenicity/Toxigenicity . . . . . . . - . . - ~ " Culturing of Blood and Tissue Samples . . . . . . . Histopathologic Evaluation . . . . . . . " . . ~ " RESULTS AND DISCUSSION . . . . . . . " . " - " " " " TABLE I " Body Weights . . . . . - . " " " . " . . . . 11 REFERENCES . . . . . . . . . . . . . . . . " " . " . . 12 QUALITY ASSURANCE STATEMENTS . . . . . . . - - . . . . 13 APPENDICES A Culturing of Tissues and Blood for Trichoderna reesei . . . . . . . . . . . " " " 15 B Microscopic Tissue Examination . - . . . . - ~ . " 21

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INTRODUCTION The Biotechnology Group of Miles Inc . has an interest in .Trichoderma reesei that has the capability to produce T .R. Cellulase, a carbohydrase enzyme with potential for food use . This studyl was conducted in compliance with FDA Good Laboratory Practice Regulations (21 CFR, Part 58) to determine the potential of this organism to induce a pathogenic or toxiqenic response in rabbits and to provide data directed toward assuring product safety. MATERIALS AND METHODS Culturitl gthe Test Organism . A culture of T . reesei was qrowCl at 27°C in wheat bran medium to the highest obtainable concentration . The culture was centrifuged and filtered through a 0.2 A filter to obtain a cell-free supernatant fluid . A viable spore suspension was prepared by isolating spores from potato dextrose agar and resuspendiny them in phosphate-buffered saline (0.9% NaCl + 0.01 M Na2HPO4 to pH 7 .4) to a concentration of approximately 2 x

t Thr stuWi.as ccrduete0 in the lttles 10xiC0iagY Department rrau 3/4/D9 to 4/6/89 fcf the 3jnt0chnolopy Group ofcordirq to texicology PfOTaCOI a923 . All data relatiny to fungal cultues nnri r .ultirin9 of btaod and tfgt.ua saaples can be fcuid in Bintechnaicgp notebook RB16720, pages LO~fJ . Ail nther data relatirvg to this study Clnct Oirg notebooks 1163 and 1169 ; wet tissues, si idez, !nd btccks ; and a copy of The fitni rU can be taird +n the Toiticology Drptrtaent Archi we .

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107 spores/ml . A heat-killed spore suspension was prepared in the same way, except spores were autoclaved 15 minutes at 121°c . The final preparations administered parenterally to rabbits included the following : 1 . A cell-free median consisting of the supernatant fluid (wheat bran medium) in which T . reesei had been grown to the highest obtainable concentration 2 . A viable spore suspension containing approximately 2 x 107 spores/ml in phosphate-buffered saline 3 . Uninoculated sterile culture medium (wheat bran medium) 4 . A heat-killed spore suspension containing approximately 2 x 107 spores/ml in phosphate-buffered saline spore counts were made and each test and control article was administered within hours of its preparation . A imore detailed description of culturing procedures can be found in Appendix A. Pathogen 3,..r

;,j,tv/ToxiaPn.igitv . Following a 5-week acclimation period, 14 male and 14 female 3- to 4-month-old (body weights of 2 .86 to 3 .89 kg) New Zealand white (specific pathogen free) rabbits`, judged to be in good physical condition, were randomly3 assigned to 2 groups of 3 males and 3 females and 2 groups of 4 males and 4 females . The

Xwtsu(s were abtarned frdn Mcdnt~ Valley RalbitrY Inc' . LU

.K?rmt(le, OH (this is a ~1eviation trnx the Protocol that doe-, lot ON act the qua(Ity or inteqrlrY of the study) ard were rtu"ntainsed fn rrn¢ Miles Corporate Anvnil Facility, wesich is registered boy the U6t10 . and fut(y acOred,ted by thT Aocr :can Assoc-800n for Accreditrt(on of taboratory Animl Care (AAAA[) 3 Tozicoluyy Ccpartnmt Method (TOM) Ms. 25; srracrfioa randoni :ation by body wrGht

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rabbits were individually identified4 and caged, and received a single intravenous (via the marginal ear vein) dose of 1 .0 ml of either the viable or heat-killed spore suspension, the cell-free supernatant fluid, or uninoculated culture medium . Water was available ad Iibituu and approximately 130 grams of a commercial rabbit food5 was offered to each rabbit daily . Following administration of the test materials, rabbits were observed daily for 14 or 28 days and body weights were recorded for survivors on days 0, 7, 14, 21, and 28 . Twenty of the 28 rabbits were sacrificed on day 14 . Two males and 2 females in the viable and heat-killed spore groups were allowed to remain on study until sacrificed on day 28 . At termination of the study on days 14 and 28, survivors were killed with euthanasia solution . Immediately following death, a 3 ml sample of blood was obtained from all rabbits that received the viable or heat-killed spore suspension and from the first male and first female in each of the other 2 groups . Blood samples were obtained by open chest cardiac puncture, using aseptic technique, to be cultured for

. reesei . Additionally, a gross pathologic examination was performed on each rabbit, using aseptic technique . Specimens of liver and spleen from rabbits from which blood was obtained were excised and cultured for the test organism .

q ;ra. M" ' : oatat ~ .' lag S put~,,a C~ .rtjYjod Rabait Chor 2SS'?. Rzlston PurimCclperh', St_ Lcuis, f4J

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specimens of kidney, lung, and brain from the same animals were excised and retained in formalin . cultur i na of Blood and TiSSUG Samples . Blood, liver, and spleen samples were cultured for T . reesei in 50 ml of potato dextrose broth plus antibiotic supplement at 27°C on a 250 rpm rotary shaker for 7 days . Each flask was subcultureel onto potato dextrose agar plus antibiotic supplement at 1, 4, and 7 days and maintained at 270C. organisms were identified by standard methods . Precise details can be found in Appendix A.

'sto 'atho ovic Evaluation . Following thoroi2gh nocropsy of each animal, tissue specimens from liver and spleen were obtained from all rabbits that received the viable or heat-killed spore suspensions and from the first male and female in each of the other z groups to be fixed by immersion in modified Millonig's phosphate-buffered lot formalin (Carson, et al ., 1973) . The specimens were processed for paraffin embedding, sectioned between 4 and 6 4m, stained with hematoxylin and eosin, and examined with a light microscope . Additional sections of liver and spleen from all rabbits that received viable or hoat-killed spores as well as from 1 rabbit that received only culture medium (negative control) were stained by Crocott's method for fungi (Grocott, 1y55) . A detailed description of histopatholoqic evaluation is presented in Appendix B.

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RESULTS AND DISCUSSION Intravenous administration of viable and heat-killed spare suspensions of T . reesei,, as well as the supernatant fluid in which 2' . reesei had been grown to the highest obtainable concentration, elicited no overt indications of toxicosis and no adverse effect on body weight gains (Table 1) . Animals appeared healthy at 14- and 28-day terminations and no gross abnormalities were observed at necropsy . ?' . reesai was not recovered from blood samples, but was cultured from spleen and liver specimens from 3 of 4 animals in the viable spore group that were terminated on day 14 . The test organism was again isolated from 3 of 4 liver samples (viable spore group) on day 28, but was not present in any of 4 spleen samples from viable spore-treated rabbits terminated after 28 days . This probably reflects a gradual elimination of the fungal spores from tissues . Inability to isolate 7' . reesei from t :ssues 1 day after culturing also suggests that the spore count was quite low in the tissues . Blood and tissue cultures from animals in the other 3 groups were negative for the test organism . Detailed culture findings for rabbits in the viable spore group are presented in Appendix A . Histopathologic examination (Appendix B) corroborated culture results sugge.°.tinq spore counts to be very low in the tissues examined . Neither spores nor hyphae were seen in liver and spleen samples stained by Grocott's method for fung :: . In 3 similar study :n mice (Porter et a1 .~ 1990),

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spores were seen in tissue from all 40 mice that received either viable or heat-killed spores . Additionally, the absence of tissue reaction indicated that T . reesei was nonpathogenic under the conditions of this study . The presence of fungal spores in tissues following parenteral administration is to be expected . Work with T . rQesei in another laboratory showed the presence of viable fungal spores in liver, kidney, and spleen of mice until day 57 postdosing (Hjortkjaer et al ., 1996) . No attempt should be made to extrapolate these findings to other routes of exposure . Ingestion (the anticipated route for possible accidental exposure of humans) of the test organism certainly would not be expected to pose any health threat . The I'inal product, T .R. Cellulase, caused no adverse effects when administered orally to rats at an extrolnely high dose of 5 grarss per kilogram or body weight (Porter et al ., 1989) .

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lable 1 gpdy b7eights of Au:Ofts man tntraveroua PstheganicitY/1009enlclty Sr-.iJy with trichc+ientg [q"t!

RM~bt t usrst+pr SeA tort Flaterfal Day 0 Day 7 Day 14 Day 21 Day 29 Gain

3395 X Cell-lrae 2~86 2.92 2.94 - " 0 .08 3400 M s~+parnatant 3 .34 3.30 S.Z9 - -0 3404 p FtuLdi 3,55 3.37 3.57 - ' 0 .02 3407 F 3,79 3.69 S.82 - ' 0 " 3410 F S .52 3.51 S.52 - 0.00 3416 F 3 .48 3.49 5.48 - 0.00 3393 tl Ytobte 5po6e 3.37 3.34 3.44 : : 0.07 5395 x suspe+sion 3.28 3.27 3.30 0.02 3399 1 3.47 3.50 3.56 3 .69 3.68 0.21 :

, 3.01 2.99 3.05 3 .14 3.14 0.13 3<.09 r 3.56 3.51 3.58 - - 0.02 5412 r 3.bD 3.E6 3.91 - - 0.02 5414 F 3.55 3 .51 3.57 3 .68 3.69 0.14e S415 ~ 3.55 3 .53 3-54 3 .77 3.76 0.21e 3394 w Meat-killed 3 .37 3.36 3 .40 - - 0 .03 3397 w spore SUNNI- irnc 3 .45 3.37 3.462 - - " 0 .03 3403 p 3 .60 3.61 3.66 3.74 3.70 Me

p 3.12 3.19 3.21 3.36 3 .30 0.13 3413 F 3.8 3.30 3,39 - ° 0.10 3417 c 3.ai 3.67 3.93 - 0.04C 3419 ~ 1 .85 3.72 S.'J2 3.89 3.54 -0.01e 3420 1 S9 3.53 5 56 3.78 3.69 0.10 3396 M u"lnoca Culture S .39 3 .40 5.40 - : 0.01 3401 M V4Aiuw 5 .30 3 .27 5.31 - 0.01 3405 M S .34 3 .30 3 .35 - 0.01 3408 F S .51 3 .46 3 .S2 - - 0.01 3411 F 3.81 3 .75 3 .76 " - -0 .05 3418 F 3.27 3.29 3.31 " - 0 .04

aliimat bran nadiuo following Centrifugation end filtering of 1 . re ,+ uhice had been grown to the hlghast obtainable carr.antratlcWe b5iypemicn <;f =7 +i 107 viaple sFnrnslnl in Fi+csphete-bu1faro0 satine c.uspercicn ct " 2 . 107 he," k,',le+l qWrsitnl in pho:phate-OW t¢red sal,ne d umnewlatro .refi~=~~ for _ell~frre xi .l~rnatanT fluid e Deternincd arsr a .'0-dnY xriod

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REFERENCES Carson, F .L ., J .H. Martin, and J .A. Lynn : Am . J . C2in . P3thol . 59 :365-373, 1973 . Grocott, R .A. : Am . J . Clin . PathoI . 25:975-979, 1955. Hjortkjaer, R.K., V. Bille-Hansen, K .P. Hazelden, M . McConville, D.E . McGregor, J .A. Cuthbert, R.J . Greenough, E . Chapman, J .R. Gardner, and R . Ashby : Fd . Chvm . Toxic . 24(No .l) :55-63, 1986. Porter, M

.C ., R.E . Craigo, and R.E . Hartnagel Jr . : Miles Toxicoloqy Department Report No. MTD0091, February 21, 1989 . Porter, M

.C., V. Jasty, D.E . Reese, and R.E . Hartnagel Jr . : Miles 'roxicoloqy Department Report No. MTD0160, June 13, 1990.

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In compliance with Good Laboratory Practice Regulations, this final report has been audited by the Quality Assurance Unit . This report accurately describes the methods and standard procedures used in the Toxicology Department in the conduct of the study and the results presented accurately reflect the raw data as recorded in study notebook Nos . 1163 and 1169 .

Date of Dates of Reports to Iny,gq i on Management and Study Director 3/8/89 (protocol audit) 3/9189 3/9/89 4/6/89 4/6/89 6/4/90 (report audit) 6/4/90 This study was conducted in compliance with FDA Good Laboratory Practice Regulations .

James J . Bare Date Manager, Regulatory Compliance

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This study was conducted in compliance with the Good Labora - tory Practice Regulations . The report accurately describes the methods and procedures used in the study and the reported results accurately reflect the raw data of the study .

David E. ReQse Date Microbiologist

_ t' yt!~j ~ . b " ~~~G1u V. Jasty, I3VSc, PhD Date Veterinary Pathologist

, ' Michael C.-Porter Date Study Director

Ralph E . Hartna~el~4Y' . Director of Toxicology .'.L

c~~ Il Date

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Report No. : MTD0160 Date : June 13, 1990

Safety Evaluation of Trichoderma reesei : Intravenous Pathogenicity and Toxigenicity Study in the Mouse

Authors M .C. Porter, V. Jasty, D.E . Reese*, and R.E . Hartnagel Jr .

Performing Laboratory Toxicology Department Miles Inc . P.O . BOX 40 Elkhart, IN 46515 *Microbiology Research Biotechnology Products Division

Miles Inc . Elkhart, IN 46515

Pa es 1 to 114

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AssTRAM Safety Evaluation of Trichoderma reesei : Intravenous Pathogenicity and Toxigenicity Study in the Mouse Introduction : The Biotechnology Group of Miles Inc . has an interest in Trichoderma reesei that has the capability to produce T .R. cellulase, a carbohydrase enzyme with potential for food use . This study was conducted to determine the potential of this organism to induce a pathogenic or toxigenic response in mice and to provide data directed toward assuring product safety . Study Desian : Groups of 10 male and 10 female Charles River Crl :CFmBR mice received either a viable spore suspension of T. reesei, the cell-free supernatant fluid in which the test organism was grown, the uninoculated culture medium, or a heat-killed spore suspension . Spore suspensions, supernatant fluid, and uninoculated culture medium were administered intravenously as a single dose of 0.1 ml . Following administration of the test materials, animals were observed daily and body weights were recorded on days 0, 7, and 14 . Sixty of the so mice were sacrificed on flay 14. Five males and 5 females in the viable and heat-killed spore groups were 28. allowed to remain on study until sacrificed on day Additional body weights were recorded for these animals on days 21 and 28. At termination of the study on days 14 and 28, animals were killed with carbon dioxide . Immediately following death, a blood sample was obtained from all mice that received the viable or heat-killed spore suspensions and from the first male and first female in each of the other 2 groups . samples were obtained via open chest cardiac puncture, using aseptic technique, and were cultured for T . rpesei in 50 ml of potato dextrose broth . Additionally, a gross pathologic examination was performed on each animal . Specimens of liver and spleen from mice from which blond was obtained were excised and cultured for the test organism . All blood and tissue cultures were then subcultured onto potato dextrose agar on days 1, 4, and :, and resulting growth identified by standard methods .

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Results : Intravenous administration of viable and heatkilled spore suspensions of T . reesei, as well as the supernatant fluid in which T. reeses had been grown to the highest obtainable concentration, elicited no overt indications of toxicosis . Tissue cultures and histopathologic examinations confirmed the presence of viable spores in liver and spleen 14 and 28 days fol lowing i .v . administration of a high dose (=2 x 10 spores) of viable T . reesei . Findings at 28 days postdosing, compared to those at 14 days, suggested gradual elimination of fungal spores from tissues . Additionally, the absence of tissue reaction around the spores, as determined by microscopic examination, indicated that T . reese3 was nonpathogenic under the conditions of this study . Conclusions: The presence of fungal spores in tissues following parenteral administration is to be expected . work with T. reesei in another laboratory showed the presence of fungal spores in liver, kidney, and spleen of mice until day 5'7 postdosing (Njortkjaer et a1 ., 198s) . No attempt should be made to extrapolate these findings to other routes of exposure . Ingestion (the anticipated route for possible accidental exposure of humans) of the test organisa certainly would not be expected to pose any health threat . The final product, T .R. Cellulase, caused no adverse effects when adninistered orally to rats at an extremely high dose of 5 grams per kilogram of body weight (Porter et a1 ., 1989) .

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TABLE OF CONTENTS a e INTRODUCTION . . . . . . . . . . . . . . . . . . . . . 5 MATERIALS AND METHODS Culturing the Test organism . . . . . . . . . . . . Pathogenicity/Toxiganicity

. . . . . . . . . . . . . s Culturing of Blood and Tissue Samples . . . . . . . 8 Histopathologic Evaluation. . . . . . . . . . . . . g RESULTS AND DISCUSSION . . . . . . . . . . . . . . . , g REFERENCES . . . . . . . . . . . . . . . . . . . . . . 15 QUALITY ASSURANCE STATEMENTS . . . . . . . . . . . . . 16 TABLES I Male Body Weights - H-KSS/VSS . . . . . . . . . . 11 II Female Body Weights - H-KSS/VSS . . . . . . . . . 12 III Male Body Weights - SCM/CFS

. . . . . . . . . . . 13 IV Female Body Weights - SCM/CFS

. . . . . . . . . . 14 APPENDICES A Culturing of Tissues and Blood for Trichoderma reosei . . . . . . . . . . . . . , is B Microscopic Tissue Examination . . . . . . . . . . 24

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INTRODUCTION The Biotechnology Group of Miles Inc . has an interest in Trichoderma reasei that has the capability to produce T .R. Cellulase, a carbohydrase enzyme with potential for food use . This studyi was conducted in compliance with FDA Good Laboratory Practice Regulations (21 CFR, Part 58) to determine the potential of this organism to induce a pathogenic or toxigenic response in mice and to provide data directed toward assuring product safety . MATERIALS AND METHODS Culturing the Test Organism . A culture of T . reesei was grown at 27°C in wheat bran medium to the highest obtainable concentration . The culture was centrifuged and filtered thtough a 0 .2 u filter to obtain a cell-free supernatant fluid . A viable spore suspension was prepared by isolating spores from potato dextrose agar and resuspenfling theta in phosphate-buffered saline (0.9% NaCl + 0 .01 M Na 2HPOd to pH 7 .4) to a concentration of

1 this study was corcf.etra +n the Miles iCaicatogy .1epartrent from 319/-89 to 416/89 Or the Bio-trhmlcqy Lrcx,p acrarAin3 to Toxicology Protocol 8914. All tfita relating to fwNal cultures mj oAttlring of hinrd xrf tl,sw s,ltples can be found in Biotechrtology r4t2bCCi B$167'TB, pagcs 47 79. Al l tthwraata rclatinq to this study tincltding rv;teboot.s 1163 ard 1169; wct tissues, aid-t, ar.7 ."itrxk ; ; anc a :ope or the firut ropartl can be fa.rd in tFle 14KiCOlogy DeRrtment Arrhive . -5- MTD4160

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2 7 4 approximately 2 x l07 spores/ml . A heat-killed spore suspension was prepared in the same way, except spores were autoclaved 15 minutes at 121°C . The final preparations administered parenterally to mice included the following: 1 . A cell-free medium consisting of the supernatant fluid (wheat bran medium) in which T. reesei had been grown to the highest obtainable concentration 2 . A viable spore suspension containing approximately 2 x 107 spores/ml in phosphate-buffered saline 3 . Uninoculated sterile culture medium (wheat bran medium) S . A heat-killed spore suspension containing approximately 2 x 107 spores/ml in phosphate-buffered saline Spore counts were made and each test and control article was administered within hours of its preparation. A more detailed description of culturing procedures can be found in Appendix A . Pathogenjwity/Toxigenicitv . Following a i week acclimation period, 40 of 58 male and 40 of 52 female 42- day-old (body weights of 20.3 to 34 .9 g) Charles River Cr1 :CFMBR mice2 were selected on the basis of physical condition and randomly 3 assigned to 4 groups of 10 males and 10 females each- The mice were individually identified4 and caged, and

Antnatc uere obtained from Charles Blver Brpaditry Laboratories, Rvrte.q+, $1 .1 and 60re maintained

'.n iha Miiea Ccrparate Aniiaul facility, rOSCA ia registered by the U5CA " rutty accredited t;V the At.Prlran A£Fx iatsSn tor Accredita!ian Cf Laboratory AniriLll Care (AA4lA(1 toxico(r,qy Crnarrnrnt Mrthorl 1[GM) No. 25 ; i .,ing n;m) :roO chips "DR Uc+ . b

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received a single intravenous (via the caudal vein) dose of o .l ml of either the viable or heat-killed spore suspension, the cell-free supernatant fluid, or uninoculated culture medium

. Food5 and water were available ad 2ibitum . Following administration of the test materials, mice were observed daily for 14 or 28 days and body weights were recorded for survivors on days 0, 1, 14, 21, and 28 . Sixty of the 80 mice were sacrificed on day 14 . Five males and 5 females in the viable and heat-killed spore groups were allowed to remain on study until sacrificed on day 28. At termination of the study on days 14 and 28, survivors were killed with carbon dioxide . Immediately following death, as much blood as possible was obtained from all mice that received the viable or heat-killed spore suspension and from the first male and first female in each of the other 2 groups . Blood samples were obtained by open chest cardiac puncture_, using aseptic technique, to be cultured for T . reesei . Additionally, a gross pathologic examination was performed on each mouse, using aseptic technique . Specimens of liver and spleen from mice from which blood was obtained were excised and cultured for the test organism . Specimens of kidney, lunq, and brain from the same animals were excised and retained in formalin .

Pjrrno Ccrtrf ;,nn amti .n fn- a' :Ol?, D.~I,^,cn purira cinpa^Y

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Culturing of Blood and Tissue samples . Blood, liver, and spleen samples were cultured for T . reesei in 50 ml of potato dextrose broth plus antibiotic supplement at 27°C on a 250 tured at 1, rpm rotary shaker for 7 days. Each flask was subculonto potato dextrose agar plus antibiotic supplement 4, and 7 days and maintained at 27°C. Organisms were identified by standard methods . found in Appendix A . Histopatholoc.iic Fvaluation . Precise details can be Following thorough necropsy of Qach animal, tissue specimens from liver and spleen were obtained from all mice that received the viable or heat-killed spore suspensions and from the first male and female in each of the other 2 groups to be fixed by immersion in modified Millonig's phosphate-buffered 10% formalin (Carson, et al ., 1973) . The specimens were processed for paraffin embedding, sectioned between 4 5 ILM, stained with hematoxylin and eosin, and examined a light microscope . Additional sections of liver and spleen from that received viable or heat-killed spores as well mouse that received only culture medium (negative and with all mice as from 1 control) were stained by Grocott's nethod for fungi (Grocott, 1955) . A detailed description of histopathologic evaluation is presented in Appendix B.

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RESULTS AND DISCUSSION Intravenous administration of viable and heat-killed spore suspensions of T. reesei, as well as the supernatant fluid in which T . reesei had been grown to the highest obtainable concentration, elicited no overt indications of toxicosis . All mice wade normal body weight gains (Tables 1 - IV), although gains for animals that received heatkilled spores were generally slightly greater than those for mice dosed with viable spores . Animals appeared healthy at 14- and 28-day terminations and no gross abnormalities were observed at necropsy . T . reesei was not recovered from blood samples, but was cultured from spleen and liver specimens obtained from all animals dosed with viable spores . Blood and tissue cultures fron animals in the other ] groups were negative for the findings for mice in the viable were sugqestive of lower tissue terminated on day 28 compared to test organism . culture spore group (Appendix A) spore counts for animals those on day 14 . This is based on the fact that all 1 .4-day tissue specimens were positive for T. reesei 1 day after culturing, while 5 of 20 tissue cultures from the 28-day termination were negative for T . reesei on day 1 . 'rhis probably reflects a gradual elimination of fungal spores from tissues . ttistopathologic examination (Appendix ©) corroborated culture

. results :s liver and spleen samples stained by Grocr,tt's method for fungi showed spores in all animals that received viable or heat-killed spores, but spores appeared

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to be less numerous in tissues from animals terminated at 28 days. Additionally, the absence of tissue reaction around the spores indicated that T . reesei was nonpathogenic under the conditions of this study. The presence of fungal spores in tissues following parenteral administration is to be expected . Work with T. reesei in another laboratory showed the presence of viable fungal spores in liver, kidney, and spleen of mice until day 57 postdosing (Hjortkjaer et al ., 1986) . No attempt should be made to extrapolate these findings to other routes of exposure . Ingestion (the anticipated route for possible accidental exposure of humans) of the test organism certainly would not be expected to pose any health threat . The final product, T .R. Cellulase, caused no adverse effects when administered orally to rats at an extremely hiqh dose of 5 grams per kilogram of body weight (Porter et a! ., 1989 ; .

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Table 1 Indlvfd,rl BloOy weights (Gram) fQr Da %l# Mice Rwee4vfnR H-C54 or VSS

Animal No. Grow Osy 0 Day 7 Day 14 GainC Day 21 Day 28 Gatnd

2 R-Kes 29_0 31 .2 33.1 4.1 - - - 3 ll-¢TS 30.6 30.9 33.1 2.5 - - -

w-lcs4 28.1 29.0 30.0 1 .9 - - 5 r-¢85 27.1 28.2 29.4 2.3 - - - 23 a-¢s5 30.0 31 .3 52.9 2.9 - " - 24 W-KSS 25.7 26.0 28.1 2.4 30.4 32.3 6.6 34 M-RSS Z8.1 27.9 29.9 1 .8 32.< SG.4 6 .1 37 N-r(S4 27.8 29.0 31 .5 3.7 33.1 36.5 6 .7 36 p .KyS 2S.9 27.2 26 .7 2.8 30.1 32.3 6.4 39 R-tSS 27.2 29.5 31 .7 4 .5 33.5 36.0 8A

kean: 28.0 29.0 30.8 2.8~ 31 .9 33.9 7.Od Mss 23.< 29.6 30.9 2.5 - - " 9 v33 27.2 28.4 29.5 2.3 - - 13 v9S 27.8 28.7 30.3 2.5 - - " 17 v5S 27.9 29.4 31 .4 3.S - ' ' 19 as5 27.8 29.3 29.8 2.0 - " ' 20 vss 27.2 28.3 29.5 2.3 30.1 31 .2 <.0 21 v5s Z9.4 30.5 32.4 3.0 32.6 34 .1 <.7 22 v55 Z6 .7 28.2 30.4 3.9 39.T 7119 i,2 30 V8s 26.7 30.3 32.1 . 3.7 33.1 34.6 5.9 33 vss 29 .6 31 .4 33.0 3.4 32.5 34.1 4.5 Moan: 28.1 29.4 31 .0 2.9~ 31_8 33.2 4.9d a+teat~E,llpd spare suspension of t . Rm Viable tparo- swpmsion of T . Qeesei C Height gain tfira+Oh day 14 for all antnals dweight gain thr"xiph d&y 28 for animals rct sec-if iced at day '4

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Table I I Individjal Body weights (gram) tot Faw~lt Mice Receiving R-KSSa or V SS

.4olarl NO, Grow Day 0 Day 7 Day 14 Gain` Day 21 NY 25 Gain d

u H-KSS 21 .3 22.7 24.0 2.7 " - - 45 R-KSS 23.8 23.6 26.4 2.6 54 w-xS5 22.6 23.5 26.9 4.3 " ' 55 11-[55 22.4 23.2 26.7 2.3 - - - 63 u-KSS 21 .2 22.3 24.2 3.0 - - - 6S R"KSS 24.5 26.2 26 .9 2.6 . 26 .3 28.7 4.4 69 R~KSS 20.7 22.3 23.6 2.9 24 .0 25.2 43 72 N" KSS 21 .2 22.7 22.8 1 .6 23 .2 25.0 3.6 78 N" iCS4 19.0 20.3 21 .1 2.7 22.2 24 .7 5 .7 80 N-K51 22.6 23 .9 26.S 3.9 2S.9 29.1 6,5

Newt 21 .9 23 .1 24 .8 2.9t 24 .3 26.5 SID d 49 li55 22.3 22 .2 24.S 2.2 - 50 v55 21 .2 21 .S 23.0 1 .8 - " - $3 v38 22.9 23 .2 24.S 1 .6 - - - 62 V5S 22.2 22 .4 24.9 2.7 - - - 64 vs9 22.4 21 .6 23.6 1 .2 - - - 66 vs3 23.2 22.4 25.0 1 .8 25 .4 2S.6 2 .4 67 YSS 22.8 23.0 26.0 3.2 25 .0 26.9 4 .1 66 Y; ; 24.8 26.0 27.1 2.3 28.2 30.3 5 .5 71 Yse 24.5 24 .5 27.8 3.3 26.8 26.2 1 .7 75 v55 23.0 22.2 25.0 2.0 25.5 26.6 3.6

mean, 22.9 22,9 75.1 2.2c 2 .6.2 27.1 3.5d

aN<et-killod spare slspansion of j Ret941 Ytabie "re ciapansiorn of j__ ~PCtWj c Yeight 9ain through day 14 for alt wasiS dYtight gain through day :9 for anirtald -At "cretieed at day 14

-12- MTi}0160fadte )11 IrmSividust Bo* Unights (gram) for male Nice keceivinp SG)t or US

Anuenl Yo

. GroW Day 0 Day 7 gay 1< Gain

1 9Ch 29.4 31 .2 32.7 3 .3 10 sdl 27.0 28.8 30.4 3 .4 11 SOf 31 .1 30.3 $3.9 2 .8 1S SCJI 28 .6 29.4 30.0 1 .4 16 SL14 27.2 28 .5 30.0 2.0 25 51CGI 27.5 27.7 25 .9 1 .4 28 SON 29 .6 30

.1 31 .1 1 .5 29 SCM 31 .2 34.0 36 .3 5.1 35 13CIt 29 .10 29

.9 30.7 1 .2 36 SCU 31 .1 32.2 33 .0 1 .9

Mean: 29.2 30 .2 31 .7 Z.5 crs 28 .s 29,9 31 .2 2.3 7 crs 29 .1 29,5 29 .2 0 .1 12 US 27.6 28 .3 29.8 2.2 14 US 27.9 90.2 31 .4 3.5 18 cr6 28.9 28 .9 30.0 1 .1 26 ciS 30.2 30.0 31 .9 1 .7 27 CfS 29.9 32.0 33.< 3.5 31 us 30.7 32.6 33.2 2.5 32 Ct5 32.9 34.9 36 .3 3.4 40 CFS 29.1 31 .1 31 .6 2.5

-can ; 29.5 34.7 31 .8 2.3

A Steri(e a.(ture eeditsv (controt) bCetl-free supernatant ftuld

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fame IV lrdNrf"t Ody Yriots TIM , fgr swl. mtw

woelvin0 fd or US "lot ~e. o-v a.r 0 Ow 7 Ow

u Gain <1 ~OI MIA 21 .9 27.7 2.6 42 Opl 22 .6 2t.0 2t .0 2.Z a OQl 2Lf 22 .0 21 .2 2.9 ii SIX 2S .& I2

.9 24.8 1 .2 47 ~al 21 .8 52 .9 tc.a 2 .6 b 00~ 21 .9 l2 .7 a.i 2 .s S~ f01 21 .3 23. " 26.1 2 .0 SO ax 22.9 N .9 27. " < .1 76 m 12.71 21.4 24.6 1 .9 n sa M .2 us 31 .1 s .9 "Mm z1 .0 24.2 21.4 2 .8 51 CF: 1110.411 21.9 23.11 3 .9 52 Us 23.5 24 .< 26.7 3.4 s7 U

: t1s 21.s 21.9 s.a s9 CFI 21 .a tl .41 2t.6 60 CFt 22.~ 22 .3 25.9 3.3 61 cFS 2e.a 21 .1 22.7 2.r 70 tit 23.9 D1 .3 29 .0 5.2 73 at S3 21 .5 a6 .6 3.3 74 Cis 23.9 2a0 2r .2 3.2 79 as n.6 27.7 3t.o 3 .2 : 22.2 23.5 2s.b 3.2

. *tSsrit* aAture rdfmtoa+troU "wt-t' . s~per~pnt flvid

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asrsRSSCSs Carson, F.L., J .H. Martin, and J .A. Lynn: An. J . C1in . Pat?io2. 59:365-373, 1973. Grooott, R

.A. : Ara. J . C11 :a . Pathol . 25:975-979, 1955. Hjortkjasr, R

.R., V . Bill*-Hanson, K .P. Haseldsn, K

. lfaConvilla, D

.B. DloGraqor, J .h. Cuthbart, R .J . Greenouqh, E. Chapaan, J .A. Gardnar, and R . 1lshby: Yd. C?:om . Rbadc. 24 (No.l) :55-63, 1986. Porter, M

.C., R .B. Craiqo, and R .E . Hartnaqal Jr . : llila: TOYiaoloqy Depart :aent Report No. lrTD0091, Pabruary 21, 1989.

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### MCANTSOVSMC2

in compliance with Good Laboratory Practice Regulations, this final report has been audited by the Quality Assurance unit . This report accurately describes the methods and standard procedures used in the Toxicology Department in the conduct of the study and tAe results presented accurately reflect the rawdata as recorded in study notebook No* . 1163 and 1169 .

Date of Dates of Reports to fnocection rrawaaa,n.*Yti and Study Director 3/8/89 (protocol audit) 3/9/89 3/9/89 4/6/89 6/6/89 9/2d-2S/90 (report audit) 6/6/90 This study was conducted in compliance with FDA Good Laboratory Practice Regulations.

wiA ll - &A4--' J J . 8ari Manaqer, Etegulatory Compliance

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### //_e .04,rJ~ /9?c This study was conducted in compliance with the Good Laboratory Practice Regulations. The report accurately describes the methods and procedures used in the study and the reported results accurately reflect the rev data of the study.

Data

14 '/ . i. ~1r-tv

Dr . V. Jast~

. r Date Veterinary Pathologist

W1 041, Nichael C

. Porter Study Director 

# 1 Date

jvi!'0 //, /940 oat*

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## Appendix 4: Survival of Trichoderma reesei Strains Introduction: To determine the ability of RL-P37 descendant strains to survive in the environment, broth samples of standard fermentations were taken and added to non-sterile and sterile soil, and non-sterile water samples. Survival as a function ofthe incubation time was then measured by counting the total colony forming units (CFU) of indigenous flora plus added cells. As a control the indigenous flora alone was monitored. Materials: The soil used was obtained fromthe 15 cmtop layer of a site without grass. The soil was dried in air and sieved (2 mm) . To obtain sterile soil samples these were sterilized for 20minutes at 120°C . The natural water used was obtained froma canal andwas of pH7. Thefollowing strains were tested (see Annex 1 for a pedigree) :

- Classical host strain RL-P37 (= P37)

- Genetically Modified intermediate strain M1-1 .1, see Appendix 2) . Procedure: a. Cultures were grown on PDA plates. Colonies were used to inoculate fermentation plant

inoculum mediumand grown for 48 hours at 250 rpmand 30°C . Production mediumwas inoculated at 10%with this inoculumculture, and grown for 5 days at 250 rpmand 30°C . Cells were collected by centrifugation, washed 3 times with 0 .85%NaCl and suspended in 0.85%NaCl . Aliquots ofthese suspensions were added to the environmental samples. b. Asoil sample taken randomly was divided into 1-gramportions. To each soil sample, 0.2 ml

ofthe cell suspension was added, and the sample was incubated at 25°C or 4°C for variable time periods. To monitor only the indigenous microflora, a volume of 0 .2 ml 0.85%NaCl was added to the soil sample. The amount of inoculated cells was 106to 107 colony forming units (CFU)/g. To determine the concentration of the microorganisms in the mixtures after a certain incubation time, 1 ml of sterile 0.85%NaCl solution was added and the suspensions were shaken for one hour at 30°C . Cell concentrations were then determined as described below (d) . c. Similar to the procedure for the soil samples, 0 .2 ml of 100-fold dilutions of the cell suspensions were added to 1 ml water samples. The amount of inoculated cells was the same as for the soil samples above (b) . Samples were incubated at 25°C or 4°Cfor variable time periods. Cell concentrations were then determined as described below(d) .

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. MCANTSOVSMC2 d. Cell concentrations were determined by plating dilutions, made in 0.85% NaCl, on TSA

plates. To improve the analysis plating was also done on plates containing antibiotics preventing bacterial growth. The plates were incubated at 30°C for 48 hours before counting. With this method the detection limit is about 100 CFU per gramfor soil samples and 10 CFU/ml for water samples. Results: The results obtained were plotted in the graphs presented in figures 1 to 6 . The numbers in these graphs represent total colony forming units, not specific for a particular strain. Fromthese figures a number ofobservations can be made: l . In non-sterile soil the endogenous microflora remained at the original level, indicating the validity of the experiment . The soil is in equilibriumwith the microflora grown up to its limits. This applies to both total microflora (experiments plated on agar without antibiotics) as well as fungal microflora (experiments plated on agar containing antibiotics) . 2 . In non-sterile soil the level of M1-1 .1 and RL-P37 remain at the inoculated level, indicating that M1-1 .1 does not have any growth advantage over RL-P37, and that the strains are not able to proliferate, but presumably turn into a dormant state. 3. In sterile soil both the level of M1-1 .1 and RL-P37 decline slightly over the testing period. A

possible explanation might be a change in soil structure and/or composition as a result ofthe sterilization. 5. In non-sterile water the endogenous microflora shows a fluctuating pattern, perhaps

indicating a succession ofvarious species ofmicroflora. Certainly the water sample is viable, indicating the validity ofthe experiment. 6. In non-sterile water the levels of both M1-1 .1 and RL-P37 decline slightly over the testing

period, without any advantage of M1-1 .1 over RL-P37. 

### CONCLUSION

: Genetically modified strain MI-1 .1 exhibits an identical survival behavior as its non-genetically modified parent strain RL-P37, and does not have any growth advantage over strain RL-P37.

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## Figure 1 

# Soil, Non-Sterile, 4°C I 8 07 

# -~o 5 U 4 0 3 J 2 

# 1 .

0 20 40 60 80 100 120

Time (days) 

# Soil, Non-Sterile, 25°C 8 -~ Control ++ P37 

# ~+ E3C16 M1

. ~ -~-Control - --m- + P37 4 

# 0 3 LA + E3C16 M1 J 2 1 ~ 7 6 5 

# 0 20 40 60 80 100 120

Time (days)

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## Figure 2



# 8 IM 7 6 5 v 4 0 3 ~ 2 

# 1

8 07 

# 6 

# Soil, Non-Sterile, 4°C, + Antibiotic

. * 6 4 

# Control

-+- ~- + P37 -~+E3C16M1 0 20 40 60 80 100 120

Time (days) 

# Soil, Non-Sterile, 25°C, +Antibiotic

- -- ~-~ Control , -~- + P37 

# -~- + E3C16 M

1 5 v 4 

# 0 3 J 2 1 . 

# 0 20 40 60 80 100 120

Time (days)

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## Figure 3 

# Soil, Sterile, 4°C 8 ~ 7 6 U- 5 I v 4 

# , 0 3 I J 2 1 ~Control -~ + P37



# - .& +E3C16M1 

# J 0 20 40 60 80 100 120

Time (days) 

# Soil, Sterile, 25°C

0 3 J 2 1 8 ~ 7 6 5 4 

# J .A ; 

# --*- + P37 

# ~ + E3C16 M1 0 20 40 60 80 100 120

Time (days)

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## Figure 4 

# Soil, Sterile, 4°C, + Antibiotic 8 M7 6 5 v 4 0 3 J 2 

# 1 ~Control ++ P37 -~- + E3C16 M

1 0 20 40 60 80 100 120

Time (days) 

# Soil, Sterile, 25°C, + Antibiotic 8 a~ 7 6 - 5 v 4 0 3 

# J 2 lose 1 _L ---- * Control

. ~ + P37 !I ~+E3C16M1 ~I

. 0 20 40 60 80 100 120

Time (days)

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MCANTSOVSMC2 

## Figure 5 

# Water, Non-Sterile, 4°C

_ 8 7 6 5 v 4 

# 0 3 ~J 2 1 0 20 40 60 80 100 120

Time (days) 

# Water, Non-Sterile, 25°C

_ 8 7 6 5 v 4 

# 1 + -+- Control ~ + P37 " -~ +E3C16 M1

- 0 3 2 0 20 40 60 80 100 120

Time (days)

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## Figure 6



# _ 8 7 

# 6 5 v 4 0 3 J 2

_ 8 7 6 5 v 4 0 3 

# J 2 1 

# Water, Non-Sterile, 4°C, + Antibiotic Control -~- + P37

-"-+ E3C16 M1 0 20 40 60 80 100 120

Time (days) 

# Water, Non-Sterile, 25°C, + Antibiotic

_- -~ Control ~ + P37 ~ + E3C16 M1 

# J 0 20 40 60 80 100 120

Time (days)

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