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p. 1 

Company Sanitized 

CONFIDENTIAL BUSINESS INFORMATION 

MICROBIAL COMMERCIAL ACTIVITY NOTICE (MCAN) 

(TSOVSMC2) 

CONTAINED USE OF A GENETICALLY MODIFIED 

TRICHODERMA REESEI STRAIN FOR USE IN PRODUCTION OF A 

ENZYME PREPARATION 

FILED BY 

DANISCO US, INC. 

3490 WINTON PLACE 

ROCHESTER, NY 14623 

o~u7 ~i0 ~,m~0 u0 ir0 i~NU0 iri~u0 ii2 u~i,i~uiu~~i 
Danisco US, Inc. 

MCAN TSOVSMC2 

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) Microorganism Identity Information . . . . . . . .. . . . . . . ... . . . . . . . . . . . . .. 12 

(1) Description of the recipient microorganism and the new microorganism 12 

(i) Data substantiating the taxonomy of the recipient and new microorganism to the 

strain level . . . . . . . .. . . . . . . ... . . . . . . . . . . . . .. . . . . . . ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .. . . . . . .. . . . . . . . . . . . . . . .. . 12 

(ii) Information on the morphological and physiological features of the new 

microorganism. . .. . . . . . . .. . . . . . .. . . . . . . . . . . . . . . .. . . . . . .. . . . . . . . . . . . . . . .. . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . .. . . . . . . .. . . . . . . .13 

(iii) Other specific data by which the new microorganism may be uniquely identified 

for Inventory purposes . . . . . .. . . . . . . . . . . . . . . . . . . . . . .. . . . . . . .. . . . . . . ... . . . . . . . . . . . . .. . . . . . . .. . . . . . . . .. . . . . . . .. . . . . . 13 

(2) Genetic construction of the new microorganism. . . . . . . . . . . . .. . . . . . . ... . . . . . . . . . ., . , 14 

(i) Data substantiating the taxonomy of the donor microorganisms . . . . . .. . . . 14 

(ii) Description of the traits for which the new microorganism has been selected or 

developed and other traits known to have been added or modified.. . . . . . . .. . . . . . . .. . 14 

(iii) A detailed 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 of those 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) A description 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) A description 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). .. . . . . . . . . . . . . . . . . . . . . . .. . . . . . . ... . . . . . . . . . . . . .. . . . . . . ... . . . . . . .. . . . . . . .. . . 19 
Danisco US, Inc . 

MCAN TSOVSMC2 

(f) Byproducts - A description of byproducts resulting from the manufacture, processing, 

use, and disposal of the 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 microorganism will 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 of an accidental 

release . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .31 

(vi) Procedures for disposal of any articles, waste, clothing, or other equipment in the 

activity, including procedures for inactivation of the new microorganism, containment, 

disinfection, and disposal of contaminated items. . .. . . . . . . . . . . . . . .. . . . . . . .. . . . . 32 

(2) For sites not controlled by the submitter . . . . . . . .. . . . . . . ... . . . . . . .  , 33 

(i) Identity of the site where the new microorganism will 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 of Subject 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 

microorganism that 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. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .41 
Danisco US, Inc. 

MCAN TSOVSMC2 

List of Appendices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .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. 

MCAN TSOVSMC2 

List of Appendices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .41 

Appendix 1 : Identification Kit - Trichoderma reesei . . . . . . . . . . . . . . .. . . . . . .. . . . . . .42 

Appendix 2 : Description 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. 

MCAN TSOVSMC2 

(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 belief the 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 microorganism identified in this submission. All information 

provided in this submission is complete and truthful as of the date of this submission. I am 

submitting with this submission all test data in my possession or control and a description of all 

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 of authorized official Date 

Vincent J.H. Sewalt, Ph.D. 

Director of Regulatory Affairs 

Danisco US, Inc . 

925 Page Mill Road 

Palo Alto, CA 94304 

06 '/~p A -e-' 1 P / 2 c=) c% 1/ 
p. 7 

Danisco US, Inc. 

MCAN TSOVSMC2 

(b) Executive Summary 

Danisco US, Inc. (Danisco), formerly Genencor International, Inc., has constructed a 

Trichoderma reesei strain, T. reesei 

and -

WWWWWities activ and has determined that the microorganism is 

new under Section 5 of the Toxic Substances Control Act. 

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Danisco US, Inc. 

MCAN TSOVSMC2 

Trichoderma reesei - was chosen as the starting strain for transformation, as it is a 

cellulase overproducer. Therefore, the best overall characterization of the enzyme 

preparation derived from strain 

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. 

A complete description of the production strain is provided in this submission. 

The production of the is performed under Good Industrial Large 

Scale Practice (GILSP) containment, in closed fermentation systems using pure cultures . A 

description of the 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 will be shipped from Danisco to the respective site . M 

The microorganisms 

contained in the preparation will 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 of TSCA Biotechnology Submissions for 

Microorganisms,' pp. 52-53 . 

C. General questions: 

1 . For what period of time is a claim of confidentiality being asserted? 

We request the strain information, production process and volume information be 

kept confidential for the indefinite future . Release of this information will give 

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Danisco US, Inc. 

MCAN TSOVSMC2 

competitors knowledge about key components of this proprietary strain, the 

production process, cost structure and size of the facility . This information will 

give them unfair advantage on pricing and teach them Danisco'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 of the information claimed as confidential. 

The confidential information that relates to manufacturing operational procedures 

that are part of the 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 of the intellectual property of the 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 of the 

company or institution? 

Process information is considered proprietary and is not disclosed to others outside 

the company unless under license from DDCE. If vendors 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 review process with 

regard to disclosure of information that is or might be valuable. 

Page 8of141 
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Danisco US, Inc . 

MCAN TSOVSMC2 

(2) Material safety data sheets or other similar material for the microorganism or 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) Any other media available to the public or to your competitors 

No. Equipment vendors occasionally ask if we can be mentioned as a user of their 

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 of the information claimed as confidential, describe the harm to the 

company or institution's competitive position that would result if this information 

were disclosed. 

There are several types of confidential information: 

(1) Production quantities; 

(2) Process descriptions and chemicals; 

(3) Unit operation descriptions & process flow diagram; 

(4) Possible organism release points ; 

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Danisco US, Inc. 

MCAN TSOVSMC2 

(5) Possible worker exposure. 

(6) Production strain Construction, including the identity of the newly introduced 

genetic elements 

(1) Dissemination of planned production volumes would indicate to competitors the 

scale of Danisco's product introduction . Armed with this information, 

knowledgeable competitors could predict the target market(s) and allow them to 

build focused strategies to thwart the product introduction. Our company's 

future is highly dependent on successful introduction of valuable new 

technologies such as this . 

(2) Dissemination of a process description would advance a competitor's process 

and product development capabilities in general, and potentially would also 

allow them to quickly develop a similar product. 

(3) Dissemination of unit operations and particularly of a process flow diagram 

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 armed with 

Danisco's production cost information could price their product such that 

Danisco would be excluded from the market . 

(4) Dissemination of possible organism release points tells a competitor what type 

of equipment is being used to contain and process the organism and 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 

of equipment is being used, the number of persons involved in controlling the 

process and the duration of their potential exposure. 

(6) Disclosure of the newly introduced genetic elements would teach a competitor 

which enzyme activities to introduce into the host organism to maximize 

biomass conversion efficiency; dissemination of strain construction techniques 

and strategies for developing a particular production organism tells a competitor 

which genes in the host organism to 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 of the 

host organism as a safe and suitable enzyme production organism. 

Making this information available to competition will allow them to analyze 

Daniscos's technical capabilities, production and cost structure and give them 

an unfair advantage in the market place. 

7. If EPA disclosed 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. If fully disclosed, 

competitors would have no trouble 'reverse-engineering' our product, process, 

or parts of our process, and this would allow them a competitive advantage in 

the specific target market for this product and in other markets . The new 

Page 10 of 141 
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Danisco US, Inc. 

MCAN TSOVSMC2 

product is a very much enhanced version of an existing product with 

complementary side activities . Competitor introduction of a similar product 

would require specialized genetic technical expertise to construct the production 

microorganism and 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 141 
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Danisco US, Inc. 

MCAN TSOVSMC2 

(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 

Vincent J.H. Sewalt 

Director of Regulatory Affairs 

925 Page Mill Road 

Palo Alto, CA 94304 

(650) 846-5861 

vincent .sewalt@danisco.com 

(e) Microorganism Identity Information 

(1) Description of the recipient microorganism and the new microorganism 

(i) Data substantiating the taxonomy of the recipient and new 

microorganism to the strain level 

the construction of 

and the construction 

construction of 

construction of 

This recipient strain is also used in the construction of 

The T. reesei recipient strain used to create the new microorganism 7: reesei 

is -, a derivative of strain RL-P37. - and 

other derivatives of RL-P37 have a long history of safe use by 

Danisco/Genencor in the manufacture of commercial cellulase preparations . 

The derivation and characterization of strain RL-P37 has been published 

(Sheir-Neiss and Montenecourt, 1984), and Danisco/Genencor received the 

strain directly from Dr. Montenecourt. Strain RL-P37 is a cellulase overproducing strain that was obtained through several classical mutagenesis steps 

from the wild-type T. reesei strain (QM6a). Strain QM6a is present in several 

public culture collections, e.g . in the American Type Culture Collection as 

ATCC 13631 . T. reesei has more recently been identified as a clonal derivative 

or anamorph of Hypocrea jecorina (Kuhls et al., 1996; Dugan, 1998). 

Page 12 of 141 
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Danisco US, Inc. 

MCAN TSOVSMC2 

Copies of these expression cassettes were 

i 

inte ated into the recipient chromosome. Th 

iiia gene were used as selectable marker. 

T. reesei 3400 is derived from strain RL-P37 through classical mutagenesis, 

gene deletions, and insertion of the 

The taxonomic identity of the donor strains is described under (e)(2)(i) . 

(ii) Information on the morphological and physiological features of the 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 

of growth as compact or loose tufts in shades of green or yellow or less 

frequently white . Yellow pigment may be secreted into the agar, especially 

on PDA. A characteristic sweet or 'coconut' odor is produced by some 

species. These features also apply to the new microorganism . 

The new microorganism of this 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 of its microbiolo ical phenotype. The strain is 

capable of growing on plates containing 

and the strain is 

(iii) Other specific data by which the new microorganism may be uniquely 

identified for Inventory purposes 

Features for unique identification for Inventory purposes are described 

above in (e)(1)(ii) . 

The new microorganism can be uniquely identified for Inventory purposes 

by using molecular biological methods such as PCR analysis using primers 

specific for the inte ated gene expression cassettes plus plating the 

organism on source, and in the presence of 

An Identification Kit guiding identification of the 

production strain is available 

Page 13 of 141 
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Danisco US, Inc. 

MCAN TSOVSMC2 

(2) Genetic construction of the new microorganism 

(i) Data substantiating the taxonomy of the donor microorganisms 

The donor strain for the and the gene 

used in construction of the new micro-organism was T. reesei 

strain RL-P37 . The genes were isolated from genomic DNA from the wildtype T. reesei by PCR using specific primers devised on the sequence available 

in Genbank database . 

The genes 

were from isolated genomic 

DNA from by PCR using 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 

of the gene in GenBank 

strain as the s

M == I 

ource. 

(ii) Description of the traits for which the new microorganism has been 

selected or developed and other traits known to have been added or 

modified. 

T 

hi 

e new microor anis , T. reesei 3400, was developed to overproduce 

6iiiiiia involved in the hydrolysis of polysaccharides in 

biomass to simple sugars that can be fermented to ethanol . 

(iii) A detailed 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 of those 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; 

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MCAN TSOVSMC2 

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 of cellulase (CAS# 9012-54-8). 

anism, we have inserted into strain 

Page 15 of 141 
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MCAN TSOVSMC2 

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 DNA remains present in the strain. No antibiotic resistance markers 

were inserted into the new microorganism. The final production strain was 

characterized by Southern blotting analyses. 

A detailed description of the genetic construction of the 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 of detection 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 141 
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Danisco US, Inc. 

MCAN TSOVSMC2 

(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 of growth as compact or loose tufts in shades of green 

or yellow or less frequently white. Yellow pigment may be secreted into the 

agar, especially on PDA. A characteristic sweet or 'coconut' odor is 

produced by some species. These features also apply to the new 

microorganism. 

The new microorganism does not produce spores significantly during 

Danisco's fermentation process. Spores present in the biomass waste are not 

resistant (apart from resistance to drought) and are inactivated during 

biomass waste treatment before disposal . 

It is not expected that the modifications will significantly affect the survival 

of the organism. Survival studies conducted by Danisco indicate no 

difference in survival between a genetically modified endoglucanase 

overproducing strain derived from RL-P37 and the host strain RL-P37 (see 

Appendix 1) 

In the production strain described in the current MCAN the 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 microorganism can 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 PCR analysis using 

primers specific for the integrated expression cassettes (see also Annex 5). 

An Identification Kit guiding identification of the production strain is 

available (see Appendix 1) . 

A description 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. 

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Danisco US, Inc. 

MCAN TSOVSMC2 

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 DNA used in creation of the new microorganism 

remains in the new microorganism. Since no vector sequences are present in 

the final strain, the transfer frequency of the integrated expression cassettes 

is the same as for any other chromosomal sequence. The transforming DNA 

consisted consisting of fungal DNA: 

Page 18 of 141 
p. 20 

Danisco US, Inc . 

MCAN TSOVSMC2 

(iv) A description 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). 

T. reesei is a wood rot fungus. 

(f) Byproducts - A description of byproducts resulting from the manufacture, 

processing, use, and disposal of the new microorganism 

(g) Information on production volume 

et cell mass produced per fermentor batch. (Note: Due to 

the poly-nucleic nature of the filamentous production microorganism, the cell population is 

difficult to quantify in terms of colony forming units (CFU). Using bench scale 

cell density of there will be approximately 

The new microorganism will 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 141 
p. 21 

Danisco US, Inc. 

MCAN TSOVSMC2 

Year # of 

batches 

DCW 

(kg) per 

batch 

Total DCW 

produced 

(kg) 

Wet cell 

mass (kg) 

/batch 

Total wet 

mass 

produced (kg) 

1 

2 

3 I AU AKI II AU 

Production volume of new organism 

The cellulosic to ethanol process in which the is used will operate at 

production scales that range from 0 .25 to 50 million gallons of ethanol per year. 

Ethanol production fermentor sizes will range from about 100m3 (-26,500 gallons; pilot facility) to 

about 3,800m3 (-1,000,000 gallons; 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 of having sufficient fermentation capacity to permit a constant flow of material into and out of 

the fermentation area. 

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MCAN TSOVSMC2 

(h) Use information 

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MCAN TSOVSMC2 

(i) Worker exposure and environmental release 

(1) For sites controlled by the submitter 

(i) Identity of the site where the new microorganism will be manufactured 

The stock culture and seed vial lot preparation of this new microorganism 

will be conducted at the Danisco US manufacturing facility in Rochester, 

New York. The address is : 

Danisco US, Inc. 

1700 Lexington Avenue 

Rochester, NY 14606. 

The sterile, sealed, cryo-protected vials will then be sent for the manufacture 

of the enzyme product to the Genencor, A Danisco Division, manufacturing 

facilities in Beloit, Wisconsin and Cedar Rapids, Iowa. The addresses are : 

Danisco US, Inc. Danisco US, Inc. 

2600 Kennedy Dr 1000 41 st 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 of the plant that is separate from office and 

laboratory space ; access to the production areas is limited. 

The fermentor vessels used in the Danisco process for the production of 

from T. reesei 3400 are closed stainless steel tanks designed 

and built according to American Society of Mechanical Engineers (ASME) 

codes. 

Page 22 of 141 
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Danisco US, Inc . 

MCAN TSOVSMC2 

Piping connections such as fill line, draw line, sample port, and inoculation 

port 

- 

are highly secure and 

Positive steam pressure 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 of the fermentor 

have been transferred, the vessel is cleaned in place (CIP) with a _ 

solution and the wash contents of the 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 diagram for Danisco manufacture of using 

T. reesei strain 3400 

Page 23 of 141 
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MCAN TSOVSMC2 

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 of contaminating 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 inoculum is 

prepared by adding, in a bio-safety cabinet, vials of a stock culture spore 

suspension of the 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 of the 

flask are transferred into a sterile stainless steel transfer apparatus in a 

bio-safety cabinet. A member of the dedicated laboratory staff, 

equipped with lab coat, latex or nitrile gloves, and safety glasses, 

completes the transfer of the flask contents to the seed fermentor via 

steam-sterilized hard piping. 

3) Seed Fermentor - A seed fermentation vessel, controlled at prescribed 

environmental conditions (temperature, pH control via ammonia 

addition, pressure, and airflow), is used to generate the inoculum for the 

production vessel . Transfer to the production fermentor is conducted by 

dedicated fermentation operators and occurs via steam-sterilized hard 

piping . 

After the fermentation is complete, the CIP process described in (i)(1)(ii) 

is carried out. 

4) Main Fermentor - The organisms transferred from the seed fermentor 

are allowed to grow for (based on rate of growth and 

Page 24 of 141 
Danisco US, Inc. 

MCAN TSOVSMC2 

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

drop tank. 

Criteria are in place for detecting contaminated fermentation 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 organism are located in a 

separate area of the manufacturing facility and share an isolated holding 

tank system . After every run, the main fermentor is washed using a CIP 

system as described above in (i)(1)(ii) . Rinse material from routine tank 

cleaning is directed to the equalization tank (where the pH is 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 medium with 

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 

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Danisco US, Inc. 

MCAN TSOVSMC2 

autoclaved. All glassware (culture flasks, beakers) is treated with bleach 

before being washed. 

Consequently, there is no microorganism release expected at this stage. 

2 Seed inoculum transfer - 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 inoculum from the transfer apparatus to 

the seed fermentor via steam sterilized hard piping . The empty transfer 

apparatus is caustic treated before being rinsed to the laboratory sink. 

Thus there are no anticipated releases of the new microorganism at this 

stage. 

3 Fermentation - The fermentors used to grow the production organism 

are located in a separate area of the 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 from the 

fermentor are either spot checked on the tank farm floor (ex., pH 

determination) or sent to the lab (on site) 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 of organism released to the 

environment by impingement. The air stream flows horizontally 

onto a vertical plate that causes moisture condensation and droplet 

formation. Droplet waste collected at the knockout vessel is 

discharged to the equalization tank (where the pH is brought to 

neutral) that supplies the municipal waste treatment system. 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, there is no exposure to the production organism 

inside the facility from fermentor off-gas. 

4 Post fermentation processing - 

Page 26 of 141 
Danisco US, Inc . 

MCAN TSOVSMC2 

(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 organism is 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 microorganism is 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 - 

microorganism is also limited at this step by the personal protective 

2) Seed inoculum transfer - Exposure to the organism is not expected 

during seed inoculum transfer 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 of the inoculum to 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. 

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

potential worker exposure to the subject microorganism are 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 

from steam locked 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 organism is 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 prior to 

disposal . Due to the controls described here, handling of the 

fermentation samples represents minimal employee exposure. 

3b) Fermentor off-gas - As described above in the "Description of possible 

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. A small portion of the off-gas from the droplet knockout vessel 

is vented to a site in the fermentation tank area outside the operator 

control room. From here, the operators can routinely do a visual check 

on the off-gas for the express purpose of early detection of fermentor 

foam outs. The best estimates on the vent volume into the fermentation 

tank area are of air 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 organism inside the facility from fermentor off-gas is 

not expected. 

4) Post fermentation processing - 

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Danisco US, Inc . 

MCAN TSOVSMC2 

Worker 

Activity 

Protective Equipment/ 

Engineering Controls 

# of Workers 

Exposed 

Maximum 

Duration 

hrs/da 

Maximum 

Duration 

da s/ r 

Seed Lab coat , gloves , safety 

Inoculum glasses, hard hat - 

Transfer 

Fermentation Lab coat and or 

- samples uniforms, safety 

glasses, 

hat 

gloves, hard Imp "& 

Dro p tank - uniforms , safety 

aerosols glasses, gloves, hard 

ili 

hat 

w 

Summa table of worker exposure information 

(iv) Environmental Release and Disposal of Subject Microorganism 

Possible release points for and disposal of the new microorganism during 

manufacture of the enzyme product from T. reesei 3400 are discussed in 

Section (i)(1) (ii) c) above. Efficiency of the control technology at the 

various potential release sites is outlined in the table below. 

Page 29 of 141 
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Danisco US, Inc. 

MCAN TSOVSMC2 

Control Technology Efficiency at Potential Sites of New Microorganism Release 

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 tank for ~ Water 

fermentor broth 

Note on CFU numbers used in the above table : 

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

MCAN TSOVSMC2 

The three potential sources for release of the new microorganism from the 

manufacturing facility are via solid, liquid or gaseous emissions . During 

normal operation, no release is expected via solid or liquid waste emissions ; 

a negligible amount of release may occur in the fermentor 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 from Genencor'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 of Cedar Rapids' drinking water is a series of city 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 of a large spill of material containing the new 

microorganism, containment procedures will be followed. The action taken 

depends on the size of the spill or leak. All accidental spills and leaks 

greater than ten liters of fermentation broth are reported to the supervisor 

and personnel are evacuated from the 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 the waste liquid collection system for 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 of twenty minutes. The fermentor tank floor has a trench 

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setup the feeds all run off to 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 off of the surge tank that can be closed to contain very large spills . A 

drum of 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 waste will go to the respective 

Water pollution Control facilities of the City of Beloit or City of Cedar 

Rapids. Discharge would 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 of any articles, waste, clothing, or other 

equipment in the activity, including procedures for inactivation of the 

new microorganism, containment, disinfection, and disposal of 

contaminated items. 

The procedures requested here are described above. 

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Worker exposure and environmental release 

For sites not controlled by the submitter 

Identity of the site where the new microor2anism will be used 

The new microorganism may 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. A general process diagram is 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 of choice can be corn cob, corn stover, sorghum stover, 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 

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The hydrolyzate resulting from the 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). 

Downstream processing 

Further downstream processing of the fermentation broth by 

Effluent streams from downstream processing 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 water treatment or recycled into the process following 

optional physical/chemical and/or biological water polishing (M) processing steps. 

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b) Inactivation and possible release points identified in process diagram 

The possible environmental release points for and disposal of T. reesei 3400 and the 

ethanologen of choice are fermentor off-gas (1), solids disposal/beneficial reuse (2, 3 and 

5) and treated liquid discharge (4) from downstream processing steps. Viable cells will be 

used in the process under containment, which requires a combination of engineering, 

mechanical, procedural, or biological controls designed and operated to restrict 

environmental release of viable T. reesei 3400 from the structure. Before leaving the 

containment, viable cells will be inactivated via 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 from the 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. 

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MCAN TSOVSMC2 

(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 p ervious 

gloves, safety 

glasses 

Impervious 

gloves, safety 

glasses 

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MCAN TSOVSMC2 

iv Environmental Release and Disposal of Subiect Microorganism 

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 of the 

control technology is based upon a conservative performance of a scrubber used in 

large scale fermentation (99% reduction of release of CFU). 

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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 microorganism species) . 

T. reesei was tested for pathogenicity and toxicity and products derived from 

various strains of T. reesei were investigated for numerous toxic endpoints . These 

include studies initiated by Danisco/Genencor, studies obtained from Miles 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 microorganism T. reesei 3400 will be contained during 

use and inactivated prior to disposal . 

(2) Full report or standard literature citation must be submitted for the following 

types of test 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 

from Germany (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 . FDA affirmed cellulase enzyme preparation 

derived from T. longibrachiatum (now known 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 of this 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, 

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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 

A survival 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. 

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(iv) Environmental fate characteristics 

See (j)(2)(ii) above. 

(v) Monitoring data and other test data related to human exposure or to 

environmental release of the 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 

microorganism that 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 other than that cited above. 

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(4) References 

Domsch, K. H., Gams, W. and Anderson, T.-H. 1980 . Compendium of Soil Fungi. Academic 

Press, London. Vol. I, p. 859 and II, p. 405 . 

Dugan, F.M. 1998 . A Note 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 Hypocrea jecorina, Proc. Natl. Acad. Sci. USA 93 : 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 of the secreted cellulases of 

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

Biotechnol. 20: 46-53 . 

Toyama, N., 1969. Applications of Cellulases in Japan. 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 of the 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 of the production organism. The 

strain has the following key identifying characteristics, 

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

producing 

organism from other fungi. The steps involved in characterization are described below. 

The first three characteristics provide a phenotypic characterization . If the 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 

If the 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 . If the unknown strain matches all of the above characteristics, both 

morphological and molecular biological, it can be concluded that it is the 

production strain . 

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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 organism for phenotypic characterization . 

Potato Dextrose Agar (PDA) 

Available commercially, composed of, 

Potatoes, infusion from 

Dextrose 

Agar 

Per Liter 

200 g 

20 g 

15g 

Mix well in 50-80% final volume of diH20, then bring to 100% final volume. Autoclave, 

cool to 55 °C and pour. 

Part 1 in 500 ml : 

1000x salts (see below) 

Noble agar 

Adjust to pH 6 

Autoclave 

Store sterile media at 55 °C 

Part 2 in 500 ml: 

CsCl 

Glucose 

KH2P04 

MgS04*71-120 

CaC12*21-120 

Adjust pH to 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 .6 g 

CoCl2*61-120 1 .0 g 

Add deionized water, mix, filter sterilize through 0.2 micron filter . 

Vogel's Minimal Agar 

50X Vogel's Stock solution 20 ml 

Agar 20 g 

Deionized water to 1 L 

Combine and autoclave. Cool to just above gelling temperature (-45 °C) and pour plates. 

50X Vogel's Stock solution 20 ml 

Agar 20 g 

Deionized water to 1 L 

Combine and autoclave. Cool to just above gelling temperature (-45 °C) and pour plates . 

50X Vogel's Stock Solution 

Prepare 3 separate stocks 

Stock 1 : 

Sodium citrate 150 g 

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 100 g 

Dissolve in deionized water 200 ml 

When all 3 solutions are completely dissolved, combine and filter sterilize (0.2 micron). 

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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 

H3B03 0.5 

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 PDA plates for each strain to be tested . 

2 . Using a sterile loop, streak the production strain in the center of a 

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 of an MMA plate for each strain to be compared. 

2. Inoculate the control strains, T. reesei QM6A in 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. 

1 . Label sections of an VMACE plate for each strain to be compared. 

2. Inoculate the control strains, and T. reesei QM6A in 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 DNA Extraction and Purification from Mycelia: 

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 of 20% SDS. Vortex vigorously and let sit at room temperature 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 room temperature, 

-5000 rpm, 10 minutes. 

6. Transfer aqueous upper layer to a clean tube . Repeat extraction if aqueous 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 of phenol : chloroform and transfer aqueous layer to 

a clean centrifuge tube . 

8 . Add 1/10 volume of 3M sodium acetate, pH 5 .2, mix, and add 0 .8 volumes isopropanol. 

Incubate on ice for 30 minutes. 

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 

K and 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 sodium acetate, pH 5 .2 . Mix and add 0.8 volumes cold isopropanol. 

Incubate on ice for 30 minutes . Centrifuge at >10,000 rpm for 15 minutes. Drain off 

isopropanol thoroughly. 

12 . Dissolve in 0.5 ml TE . Add 1/10 volume 3M sodium acetate, mix, and add 1 ml cold 95% 

ethanol. Incubate on ice for 30 minutes. Centrifuge at >10,000 rpm for 15 minutes. 

Aspirate ethanol and wash 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 100 tube (Molecular Bio-Products, Inc. Catalog # 

6005) : 

Water 83 ul or 81 ul 

l OX 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 DNA Template or control plasmid 1 ul or 2 ul (depending on the 

concentration) 

Pfu Ultra Il DNA Polymerase (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 DNA samples: 

To analyze the DNA products, load 5 pl of the PCR reaction and a Molecular Weight Marker 

into separate wells of a 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 PDA plates . In 

contrast to strain QM6A, which produces green colored spores on PDA plates . See Figure 1 . 

If an 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 PCR Confirmation 

The primer pair described above will amplify 

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Figures 

Figure 1. Growth on Potato Dextrose Agar 

A PDA plate 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 

QM6A is 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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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 a wood rot fungus and naturally 

secretes cellulase and xylanase enzymes. Several commercial cellulase and xylanase preparations are 

manufactured by Genencor and sold for a number of applications . Cellulase is a generic name for a 

group of enzymes and T. reesei produces at least seven separate cellulase enzymes each of which is 

known to be a separate gene product. These enzymes are two cellobiohydrolases (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 of this group of enzymes will degrade the cellulose polymer to release the monomer glucose. 

Methods for transformation of T. reesei with exogenously added plasmid DNA have been 

published (Penttila et al., 1986; Gruber et al., 1990 ; Smith et al., 1991). Stable transformants arise by 

integration of plasmid DNA into the chromosomes of the host. Integration can be at sites in the 

genome that have homology with a region of the plasmid DNA or can be at non-homologous sites. 

anism, we have inserted into this 

The host strain used was 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 from Dr. 

Montenecourt. It is a cellulase over-producing strain that has been obtained as a result of several 

mutagenesis steps from the well known wild-type strain (QM6a). 

1) 

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2) 

3) 

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4) 

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5) 

6) 

7) 

8) 

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9) 

10) 

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12) 

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13) 

14) 

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16) 

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18) 

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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 of the Tricel '97 meeting held in Ghent, Belgium on 28-31 August 

1997). Editor Marc Claeyssens. Cambridge, UK, The Royal Society of Chemistry: 327-334. 

Brosius, J. (1989) DNA 8 :759. 

Bundock, P ., Den Dulk-Ras, A., Beijersbergen, A., and Hooykaas, P.J .J . (1995) EMBO J  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. and Arai, 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 . 

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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 1 : 

Figure 2 : 

Figure 3: 

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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Figure 1 - 

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Figure 7- 

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Figure 8 - 

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Figure 10 - 

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Figure 11- 

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Appendix 3 : Trichoderma reesei Safety Studies 

The pathogenicity and toxigenicity potential of Trichoderma reesei were tested by various 

investigators and are summarized in this Appendix. 

I. STUDIES SPONSORED BY DANISCO/GENENCOR 

1 . Toxicity/pathogenicity testing of Trichoderma 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 .3200 and 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 5.6 x 106 colony forming units (CFU). Groups 

of animals of both sexes were sacrificed on Days 0, 7, 21, and 35 after injection of the test 

substance for microbial enumeration. The results were compared to those obtained from heatkilled test substance group, naive control group and shelf control 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. A statistical 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 of treated male rats and from the liver and peritoneal masses of treated 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 of this 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) . MA BioServices No. M97BL78.6P41 .01, dated January 26, 

1998. 

This study was conducted in compliance with the Good Laboratory Practice Standards as 

published by the OECD Guidelines, the US . FDA and the US . EPA. 

In this investigation, Trichoderma reesei [strain A83, derived from RLP-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 of animals 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 

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reesei was detected in only the spleen and liver of both males and females and, by Day 22, in 

only the spleen of both male and female rats. Total clearance of the test microbe from the 

spleen was achieved by Day 32. 

Under the conditions of this 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, 

dated June 13, 1990. 

Trichoderma reesei was administered as a single intravenous injection to groups of rabbits . 

Other groups of rabbits that received either cell free medium, uninoculated culture medium or 

uninoculated phosphate buffer saline served as controls . All animals were observed for overt 

signs of toxicity 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 of viable spores in liver and spleen 14 days following the 

intravenous injection. 

Under the conditions of this 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. MTD 0160, 

dated June 14, 1990 . 

Trichoderma reesei was administered as a single intravenous injection to groups of mice. 

Other groups of mice, which received either cell free medium, uninoculated culture medium or 

uninoculated phosphate buffer saline served as controls . All animals were observed for overt 

signs of toxicity 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 of viable spores in liver and spleen 14 days following the 

intravenous injection. 

Under the conditions of this investigation, Trichoderma reesei was not found to be toxic or 

pathogenic in mice. 

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III. STUDIES PUBLISHED IN THE 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 of a large dose of cortisone 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 inoculum given to immuno-suppressed 

animals - the microorganism could be pathogenic. However, this finding is not unexpected 

due to the nature of the immunological condition of the animals prior to test substance 

administration. 

Nevalainen et al. (1994) provided an overview of Trichoderma reesei and its safety as a 

production organism for 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 of celluclast, an acid cellulase derived from Trichoderma 

reesei. Food Chem Tox, 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 dated January 26, 

1998. 

Miles Inc., Study No. MTD 0159: Pathogenicity study in rabbits with Trichoderma reesei . Final 

report dated June 13, 1990 . 

Miles Inc., Study No. MTD 0160 : Pathogenicity study in mice with Trichoderma reesei . Final 

report dated June 14, 1990 . 

Modeweg-Hansen L. : Acute oral toxicity of cellulase SP122 to rats, mice, and dogs. Novo 

Reports, 1978 . 

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Nevalainen H et al. : On the safety of Trichoderma reesei, J Biotechnology, Vol . 37, 193-200, 

1994. 

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Trichoderma reesei Safety Studies Summaries from 

Laboratory Reports 

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STUDY TITLE 

IUXICITYrPATII(KF;NICITYTFSTI!JGOr Tra~hrxtermareesei 

1 "'ClI .1 .nWING ACUTE INTRAPERITONEAL CHALLENGE IN RATS 

PERFORMED ACCORDING 

I.1,S EPA Microhial Pesticide Test Guideline: OPPTS 885 .3200 

AI! TNnR 

William M . Mega, B .S . 

4 TUDY COMPILE ION 

Fo-Ttember .1000 (Final Rqwti) 

PERFORMING LABORATORY 

I IT Research Institute 

Life Sciences Operation 

10 West 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 NUMBER OF PAGES IN THE REPORT). 

p. 94 

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'1'OXICITYIPATHl1GEN!('_tTY TESTING OF Tricbmdermrn reesei 

FOLC,OWING ACUTE INTRAPERITONEAL CHALLENGE IN RATS 

GLP C.'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 raw data 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 : 

William A9, 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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TOJiICITYJPATHt1GENICITY TESTING OF Trichodernwf rernei 

FOLLOWING ACUTf'. 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 

William M. 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 raw data and a copy of the final 

rept.xt will be archived in the IITRI Archives . 

Respectfully suhmitted, 

L ZZ, ' 2 00 

William M. 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 AND MEl'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 fcw Dosing . . . . . . . . . . . . . . . . . . . . . . . 

H . Sensitivity of Detection Method tiir Test Substance . . . . . . . . . . . . . . . . . . . . . . . 

I Test Substance Titer, Contaminant, "fest Medium Growth 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 Medium Growth and Hornogeneity . . . . . . . 

1 Test Substance Titer, llomogenettv and Contaminant . . . . . . . . , . . . . . 

:, Test Medium (irowth . . . . . . . . . . . . . . . . . . . . . . . . . 

. 

. . . . . . 

li Sensitivity of Ucteetion of Test 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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, 

0 

© 

0 

. 

a 

. 

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 of Intraperitontal Administration of Trieheufermcr ree9ei 

on Body Weight Gains of Rats . . . . . . . . . . . . . . . . . . . . . . . . . , . 34 

Table 5 . Effect of lntraperitoneal Administration of TriclaWernra reesei on 

Clinical Observ'ations of Rats . . . . . . . . . . . . . . . . . _i ' 

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 Trichnderxm rersei on 

Relative (}rgan"''ri ssue Weights of Female Rats . . . . . . . . . . . . . . . . . 

Table 9- Recovery of TrichMdrrma reeset Following Intraperitoneul 

Administration from Selected Tissues of Male Rats . . . . . . . . . . . . . . . . . _'+= 

Table ICl Recovery of Trichnderrrru reesei Following Intraperitnncal 

Administration from Selected Tissues of Female Rats ±4 

VI11 FIGURES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 

Figurc I . Clearance of Mass-Associated Tric~hWerrsq reescl ftom Malt 

Rats Following Intraperitoneal Challenge . . . . . . . . . . . . . . . . . . 3? 

Figure 2. C .'Icaraix:e of Mass-Associated Trichai'erma rresei from Female 

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 - Ccrtificaw of Analysis to 

Appendix C - Protocol aiKi I'rolocol Amendment . - . . . . . . . 't [ 

IIT RE.tiF.ARCH INSTCI'11TE 

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. 

. 

0 

,, 

0 

1 . . 

TOXICITYIPATHOGENI( .'I7Y TFSTING OF Trfckarlrrrrra reetrf 

FOLLOWING ACUTE INTRAPERITONEAL CHALLENGE IN RA1'S 

SUMMARY 

T rii h{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_0 x 106 colony forming units (cfu) of viable technical grade (TG) test substance at 

a volume of 1 nil per rat . 'The dose by the intrsperitoneal route was consistent with U.S : EPA t)PPTS 

Guideline, 885_3200, in that it was the highest deliverable dose of neat test substance . In addition, 

another group at "male and female animals were similariy challenged on Day 0 with 5 0 x I Or' crlis 

of hcat-k died technical grade (KTG) test substance. A naive 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, KTG and NC rats in scheduled groups 

weft sacrificed at four time points (0, ", 21 and 35 days) for in icmbial 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 

adverse clinical observations were noted A statistically significant reduction in body weight gain 

in male TG- and KTG-dosed tats hetween Days 0 and 7 appeared to he treatment related. However, 

the effect was rxu of an,, 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 

wei ghtx, compared to the naive control were ohsetvcd in female TG-dosed rats on Days 7 and 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 was detected m both male and female TGdosed rats front the peritoncal Image fluid, spleen, liver, mesenteric lymph nodes and ceacum and 

from 111c lidncvs of T(i~dosed male rats on Day 0. Iletoctittrt of tcst substance from these 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 masses of male fG-dosed rats and from the liver and pet ik7ncal mjsses of TG-dosed 

fernale rats. On Uat 35 . tcNr substance was detected only from the peritonea] masses of 1-6-closed 

malc and trrnale rats . ( ompletc clearance of test substance from TG-dosod Mt .r was estimated to 

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rt 

r 

occur w ithin approrimately 6' days for male rats and 43 days for female rats . No test 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 . 

11'r RESEARCH INSTITUTE 

' 1182SN3 

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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 lon vi.hrichiatum rcesci 

Data RNuiremen 

Part 158.740 of' Eitle 40 of the Code of Federal Regulation-, 

(40 CFR 158.740) 

Ciutdc luni 

5uW- t\ ision r4 1 S2A-13 

Author 

Visa S ci . Sha:lton, Ph.f). 

Study. (.-pmplet ed On 

J :uuarti '_6. 1998 

t'erformin e [,;ihoratot} 

:41A Rtotiervicec . Inc . (MA) 

46±11 Medical Center Drive 

RockvilIc, MD '20850 

I,,ahc,ratqry Prqjcct Identifi cation 

h9l17Fi1 .78 .6P4 1 .01 

fit,c,nenr tittuly _Nuoilx.-r 

'_ 411 

Srx~tiuor 

Gencncor Intcrnutic,nnl . Inc . 

92i Page Mill Road 

f alo Alln, CA 9a?()4-It)1? 

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[ :trial Report 

MA Studv \!o . N19?131.?$.irP4I .Of 

EXECUTIVE SUMMARY 

I rictnaderma IonLibracht atunt (rc-~~t , whrn administered inttaperiton~:a1LF -it ? 2x lc}' l trlom 

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, Tzichtxlenna lonetbrachiattum treeset 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 from the 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 Iongihra chianirn frcr,,et) demonstrated itt) detectable infrxtivitv 

by the end of thc test laerlcKi utxlcr the conditions (if this studv . 

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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~ihrachiatutn 

(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 of a 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 medium specified 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 tiw tlx: 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 

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

Pa~~.y (P MA BioSERVicesPage 102 of 141 
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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 

Pages 1 to 78 

-1- MTD0159 

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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 organism was 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 

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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 study l 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 stuW i.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 

Arch i we . 

fiT D0 159 
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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 randomly 3 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 (AAA A[) 

3 Tozicoluyy Ccpartnmt Method (TOM) Ms . 25 ; srracrfioa randoni :ation by body wrGht 

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rabbits were individually identified 4 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 food 5 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 Purim Cclperh', St_ Lcuis, f4J 

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specimens of kidney, lung, and brain from the same animals 

were excised and retained in formalin . 

culturina 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 m an 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 study i 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 

mice 2 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 identified 4 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. ; Foac ~d ;totn 

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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 . Food 5 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. st . lcvi ., ml 

-7 - r~~rmt 6 u 
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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 Gain C 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 

d weight gain thr"xiph d&y 28 for animals rct sec-if iced at day '4 

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Table I I 

Individjal Body weight s (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 

d 

Ytight gain through day :9 for anirtald -At "cretieed at day 14 

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fadte )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) 

b Cetl-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 rdfm toa+troU 

"wt-t'. s~per~pnt flvid 

-ia- t+rTDoi6o 

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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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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 raw data 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 

Date 1 

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 of the 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 from the 15 cm top 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 20 minutes at 120°C. 

The natural water used was obtained from a canal and was of pH7. 

The following 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 medium and grown for 48 hours at 250 rpm and 30°C. Production medium was 

inoculated at 10% with this inoculum culture, and grown for 5 days at 250 rpm and 30°C. 

Cells were collected by centrifugation, washed 3 times with 0.85% NaCl and suspended in 

0.85% NaCl . Aliquots of these suspensions were added to the environmental samples. 

b. A soil sample taken randomly was divided into 1-gram portions . To each soil sample, 0 .2 ml 

of the 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 106 to 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°C for variable time 

periods . Cell concentrations were then determined as described below (d). 

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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 gram for 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 . 

From these figures a number of observations 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 equilibrium with 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 of the 

sterilization . 

5 . In non-sterile water the endogenous microflora shows a fluctuating pattern, perhaps 

indicating a succession of various species of microflora. Certainly the water sample is viable, 

indicating the validity of the 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 

4 - --m- + P37 

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 

-~- + E3C 16 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 

M 7 

6 

5 

v 4 

0 3 J 2 

1 

~ Control 

++ P37 

-~- + E3C 16 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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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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