UNITED STATES ENVIRONMENTAL PROTECTION AGENCY

WASHINGTON, D.C. 20460

 

2 June 1997

OFFICE OF POLLUTION PREVENTION AND TOXICS

POINTS TO CONSIDER IN THE PREPARATION

OF TSCA BIOTECHNOLOGY SUBMISSIONS FOR MICROORGANISMS

 I. PURPOSE

 II. OVERVIEW OF THE FINAL BIOTECHNOLOGY RULE UNDER 

SECTION 5 OF TSCA

 III. ADMINISTRATIVE DETAILS FOR MICROORGANISM SUBMISSIONS

 IV. POINTS TO CONSIDER FOR MICROORGANISM SUBMISSIONS

Prepared by: EPA Office of Pollution Prevention and Toxics

 401 M. Street, S.W.

 Washington, D.C. 20460

 

For further information contact: New Chemicals Notice 

Management Branch Chemical Control Division,(202) 260-3749
2

I. PURPOSE

Living microorganisms are sufficiently different from the

traditional chemical substances reviewed under §5 of the Toxic

Substances Control Act (TSCA) to warrant development of separate

regulations and guidance. The new regulations under TSCA

applicable to biotechnology submissions were published in the

Federal Register on April 11, 1997 (62 FR 17910-17958), and

guidance for these regulations is contained in this “Points to

Consider” document. These new TSCA regulations (as well as the

proposed TSCA regulations for biotechnology), and this “Points to

Consider” document, are accessible through the EPA Office of

Pollution Prevention and Toxics’ Home Page at

http://www.epa.gov/opptintr/biotech/.

The Agency expects the submitter to provide information, as

explained in this document, for two purposes: (l) to identify and

list the microorganism on the TSCA Chemical Substances Inventory,

and (2) to assess the risks of the microorganism to human health

and the environment, given its intended use. Information

regarding new microorganisms should not be submitted on the

standard TSCA Premanufacture Notice (PMN) form for new chemicals,

as this form is not applicable to microbial products. Rather,

the format for the submission for microorganisms subject to TSCA

is outlined in the April 11 Federal Register document. The

guidance discussed within this “Points to Consider” document is

designed to elicit this information and is meant to present

points to consider rather than requirements to fulfill. 
3

II. OVERVIEW OF THE FINAL BIOTECHNOLOGY RULE UNDER SECTION 5 OF

TSCA

Section 5(a)(1) of TSCA requires any person who intends to

manufacture or import a new chemical substance to submit

information to EPA at least 90 days before manufacture or import

commences. The final rule establishes all reporting requirements

under §5 of TSCA for manufacturers and processors of

microorganisms subject to TSCA jurisdiction, that are

manufactured for commercial purposes, including research and

development (R&D) for commercial purposes. 

Manufacturers are required to report certain information to

EPA before commencing the manufacture of new microorganisms. 

“New microorganisms” are intergeneric microorganisms that are not

listed on the TSCA Inventory. Intergeneric microorganisms are

those which have been formed by the deliberate combination of

genetic material originally isolated from organisms of different

taxonomic genera. The term “intergeneric microorganism” includes

a microorganism which contains a mobile genetic element which was

first identified in a microorganism in a genus different from the

recipient microorganism. A further explanation of how

“intergeneric” should be interpreted for mobile genetic elements

is contained in Attachment 1. The rule also defines “small

quantities for research and development” for microorganisms, the

effect of which is to require §5 reporting for certain R&D

activities. 

TSCA §5 only applies to microorganisms that are

manufactured, imported, or processed for commercial purposes. 

EPA has defined manufacture or process for commercial purposes as

“manufacture or process for purposes of obtaining an immediate or

eventual commercial advantage.” Whether an activity has an

immediate or eventual commercial advantage is determined by

indicia of commercial intent. R&D activities are for commercial

purposes, and thus subject to reporting, if tests are directly

funded, in whole or in part, by a commercial entity; or, if the

R&D activities are not directly funded by a commercial entity, if

the researcher intends to obtain an immediate or eventual

commercial advantage (see 40 C.F.R. §725.205). In addition, all

post-R&D activities are considered manufacture or processing for

a commercial purpose.

An examination of the major sections of the final rule, and

the associated submissions noted under each section, is helpful

in understanding the rule and how the “Points to Consider”

compliments the new rule:
4

Subpart B of part 725 contains administrative

procedures that have been adapted with little change

from provisions in 40 CFR parts 720 and 721 of TSCA. 

Subpart C of part 725 contains requirements for

claiming confidential business information (CBI). 

These requirements were largely adapted from provisions

in part 720. 

Subpart D of part 725 establishes the reporting

program for new microorganisms manufactured or imported

for distribution into commerce and requires submission

of a Microbial Commercial Activity Notice (MCAN)90 days

prior to initiating manufacture or import of the new

microorganism. This subpart codifies the requirements

for information to be included in the MCAN at §725.155

and §725.160. Any manufacturer, importer, or processor

of a living microorganism, who is required to report

under §5 of TSCA must file a MCAN with EPA, unless the

activity is eligible for one of the specific

exemptions. 

Subpart E of part 725 establishes the exemptions

from full MCAN reporting for R&D activities. At

§725.205(b), EPA defines "commercial purposes" for R&D

activities to include all R&D directly funded in whole

or in part by a commercial entity, and all R&D

activities, regardless of funding source, for which the

researcher intends to obtain immediate or eventual

commercial advantage. Subpart E establishes, at

§725.232, a complete exemption from TSCA §5 for

certain R&D activities which are conducted in contained

structures, and are subject to regulation by another

Federal agency. Under §725.232, EPA has established a

complete exemption from EPA review, reporting and

record keeping requirements for contained research

conducted by researchers who are required to comply

with the NIH guidelines. All other manufacturers

conducting contained TSCA research and development

activities may qualify for a more limited exemption

under §725.234 and §725.235. The exemption for R&D in

contained structures specifies factors which the

technically qualified individual must consider in

selecting the appropriate containment. The

manufacturer is required to keep records to document

both compliance with the containment requirements and

compliance with the notification process for employees

involved in the R&D process. 
5

Subpart E also establishes at §725.238 and 

§725.239 the TSCA Experimental Release Application

(TERA) exemption process for R&D activities, primarily

those involving intentional environmental release. EPA

has revised requirements in §725.239 to limit the

antibiotic resistance markers that may be used in the

microorganisms eligible for the TERA exemption. For

researchers conducting small-scale field tests with the

two eligible microorganisms, Bradyrhizobium japonicum

and Rhizobium meliloti, the final rule creates an

exemption from EPA review, providing certain conditions

are met. The field testing must occur on no more than

10 terrestrial acres; the introduced genetic material

must comply with certain restrictions, and appropriate

containment measures must be selected to limit

dissemination. 

If a submitter does not meet the requirements for

the field test exemption under §725.238 and §725.239,

he or she may submit a TERA under Subpart E. The TERA

is essentially an abbreviated MCAN submission which is

submitted for an individual field test application. 

EPA's review period for these applications is reduced

to 60 days, although EPA may extend the period for good

cause. EPA must approve the test before the researcher

may proceed, even if the 60-day period expires. EPA's

approval is limited to the conditions outlined in the

TERA notice or approval. The requirements for

information that must be included in the TERA are

codified at §725.255 and §725.260.

Although a manufacturer may submit a MCAN for any

R&D activity, EPA expects that most researchers will

instead choose to submit a TERA. In addition to the

longer review period for a MCAN, EPA expects that,

because of the limited information at the R&D stage,

the Agency would likely issue a §5(e) consent order

with such an MCAN to impose conditions to address the

uncertainties. These MCAN consent orders would need to

be modified each time the manufacturer wanted to vary

the terms of the order.

Subpart F contains the requirements for exemptions

for test marketing activities. These requirements have

been adapted, with little change, from provisions in

part 720.
6

Subpart G establishes two exemptions from MCAN

reporting for certain microorganisms used in closed

systems. To qualify for the Tier I exemption, a

manufacturer must use one of the recipient organisms

listed at §725.420, and implement specific physical

containment and control technologies as noted in §

725.422. In addition, the DNA introduced into the

recipient microorganisms must be well-characterized,

limited in size, poorly mobilizable, and free of

certain sequences (see §725.421). In the Tier I

exemption, if these three requirements are met,

manufacturers only need to notify EPA that they are

manufacturing a new microorganism that qualifies for

this exemption 10 days before commencing manufacture,

and to keep certain records. A manufacturer is not

required to wait for EPA approval before commencing

manufacture.

A manufacturer, who otherwise meets the conditions

of the Tier I exemption, may modify the specified

containment restrictions, but must submit a Tier II

exemption notice under subpart G. The Tier II

exemption requires manufacturers to submit an

abbreviated notice describing the modified containment,

and provides for a 45 day period, during which EPA

would review the proposed containment. The

manufacturer may not proceed under this exemption until

EPA approves the exemption.

Subpart L establishes procedures for reporting

significant new uses of microorganisms. These

requirements have been adapted, with little change,

from provisions in part 721. Subpart M is reserved for

requirements for significant new uses for specific

microorganisms; however, none are being promulgated at

this time.

The regulatory text also amends existing

regulations regarding the collection of fees from

submitters of notices under §5 of TSCA (40 CFR part

700), to reflect the fee structure for the notices and

applications that have been developed in the final

rule. Additional amendments to parts 720, 721, and 723

consolidate TSCA §5 review of microorganisms into part

725.

Further information on the types of submissions under TSCA can

be found in § III of this document.
7

III. ADMINISTRATIVE DETAILS FOR MICROORGANISM SUBMISSIONS

III.A. Prenotice Consultations:

EPA recommends that persons planning to provide EPA with any

of the following submissions discuss their plans in advance with

a Biotechnology Program Manager in the New Chemicals Notice

Management Branch, Chemical Control Division of the Office of

Pollution Prevention and Toxics (202-260-3749): a Microbial

Commercial Activity Notice (MCAN), a TSCA Experimental Release

Application (TERA) for Research and Development activities, a

Test Marketing Exemption (TME), a Tier II exemption for a

fermentation application, a petition to add microorganisms to the

list of recipient microorganisms at 725.420, or a bona fide

submission. It is important to begin discussions with EPA staff

early in the submission planning process to identify data

requests and preliminary concerns associated with the specific

microorganism. Any meetings and relevant written communications

may be claimed confidential. Persons who are unsure as to

whether their microorganisms are subject to TSCA should consult

with EPA before preparing any submission, including a bona fide. 

In many cases submitters' questions can be addressed in

telephone conversations. However, it is often useful for

potential submitters to meet with a broader range of Agency staff

prior to submission of documents for Agency review. This is true

if the microorganism product is novel in construction or in

intended use, or if the submitter is unfamiliar with the TSCA

submission process. Although the overall format for the

submissions noted in the above paragraph should follow the

structure outlined in this document, a prenotice meeting should

result in guidance which is more specifically tailored to the

preparation of a document for the submitter's microorganism. 

Such specific guidance often results from discussion of section

IV of this document with Agency scientific, legal, and policy

staff. This may save significant time later in the review

process. 

With reference to research and development activities (R&D),

EPA recognizes that research proceeds through various stages.

There are a number of exemptions for research and development

noted in Subpart E of the 1997 Rule (62 FR 17910-17958).

For more information on prenotice consultations, call the 

New Chemicals Notice Management Branch, Chemical Control Division

at (202) 260-3749. 
8

III.B. Microorganism Bona Fide submissions

 Companies planning to manufacture, import, or process

intergeneric microorganisms for commercial purposes subject to

TSCA may submit a bona fide notice to determine their reporting

obligations under §5 of TSCA. This is because confidential,

specific identities and/or uses of microorganisms are not listed

on the public version of the TSCA Inventory. If a company’s

microorganism is listed on the confidential version of the

Inventory and is not subject to a Significant New Use Rule

(SNUR), then no §5 submission would be necessary. The review

process for a bona fide notice generally takes 30 days after

receipt of sufficient information from the submitter. For more

information on bona fide submissions, see Attachment 2.

III.C. Confidential Business Information (CBI)

EPA's confidential business information (CBI) policy is

designed to provide effective public participation by making

meaningful information available. CBI claims may be made and must

be substantiated at the time of submission for MCANs, TMEs, Tier

I certifications, and Tier II exemption requests. Upfront

substantiation is not required for TERAs. In developing

confidentiality provisions for submissions, EPA has balanced the

need to provide nonconfidential information to the public in a

reasonable period of time, to obtain the information it needs to

respond to Freedom of Information Act (FOIA) requests, and to

allow persons to assert CBI claims with the minimum burden. A

claim of confidentiality may be asserted for any information

submitted to EPA, with certain exceptions. However, submitters

are encouraged to minimize the amount of CBI in biotechnology

submissions, so that the public may participate as fully as

possible in the review process. All CBI claims must be asserted

at the time that the information is submitted. 
9

C.1. Submission of confidential business claims 

a. Indicate what information in the submission is claimed as

confidential on a cover sheet (i.e., submitter, identity,

microorganism identity, volume, process, etc.)

b. Circle the specific information which is claimed as

confidential and mark the page on which that information

appears with an appropriate designation such as "trade

secret," "TSCA CBI," or "confidential business information."

c. Attachment 3 provides the questions a company should answer

to substantiate CBI. 

C.2. Submission of a Non-confidential 'sanitized' version 

If information being submitted is claimed as CBI and is

being submitted, then the company must also provide a sanitized

copy of the submission (and any attachments). This copy is

complete except that all information claimed as confidential must

be deleted and the places marked where the deletions occurred. 

The sanitized copy will be placed in the public files. 

C.3. Generic information

Submitters who claim microorganism identity and/or use as

CBI also must provide generic information for release to the

public. By requiring generic identity and use information, EPA

would meet its obligation to provide the public with important

information related to the potential risks of new microorganisms

without revealing CBI. Generic information would also be used to

prepare Federal Register notices which announce EPA's receipt of

submissions and to develop generic Inventory listings. For more

information on preparing generic designations, see Attachment 3.

III.D. Microorganism Reporting Mechanisms Under TSCA

If a microorganism is subject to TSCA and is not considered

to be on the TSCA Inventory, the manufacturer or importer is

required under TSCA to notify EPA from 45 to 90 days before they

manufacture or import for commercial purposes a “new” chemical

substance or manufacture, import, or process a chemical substance

for a “significant new use”. Specific time frames for individual

submissions are noted in Sections III.D.1 through III.D.5 of this

document. The Final Rule under §5 of TSCA (62 FR 17910-17958),

establishes all reporting requirements for manufacturers and

processors of microorganisms subject to TSCA jurisdiction, that

are manufactured for commercial purposes, including research and

development for commercial purposes. The Rule establishes a
10

number of mechanisms for reporting to EPA, including a number of

specific exemptions. 

Certain microorganisms which are subject to TSCA do not

require reporting to EPA prior to manufacture, and are not

addressed by this document: research and development conducted

solely within a contained structure by researchers required to

comply with the NIH guidelines (§725.232), other research and

development conducted solely within a contained structure (§

725.234), and certain types of R&D field tests involving

Rhizobium meliloti and Bradyrhizobium japonicum under the TERA

exemption (§s 725.238 and 725.239).

In addition to the guidance provided for bona fide

submissions under Item III.B., this document provides guidance on

how to meet the reporting needs for the following seven types of

MCAN and 5(h)(4) Exemption submissions: the Microbial Commercial

Activity Notice (MCAN), including MCANs for significant new uses

of microorganisms (Item III.D.2.), the TSCA Experimental Release

Application (TERA) and the TERA exemption for field tests with

certain rhizobia (Item III.D.3), the test marketing exemption

(TME; Item III.D.4.), the Tier I and II exemptions for

fermentation applications (item III.D.5), and petitions to add

microorganisms to the list of recipient microorganisms at §

725.420 applicable to both Tier I and Tier II exemptions (item

III.D.5). 

This document also includes guidance on how to submit

information provided to other agencies related to the TSCA

submission (Item III.D.6) and guidance for the preparation of

5(d)2 Notices (Item III.D.7).

D.1. General Administrative Requirements, Fees, and Notification

in the Federal Register Applicable to MCANs and Exemptions 

The Administrative requirements for the seven types of MCAN

and Exemption submissions noted above can be found in Subpart B

of the Final Rule. This Subpart contains information specific to

the submission of MCANs and Exemptions, including information on

the general format of these submissions (§s 725.25 and 725.27)

and recordkeeping for these submissions (§725.65).

A fee of $2,500 is required for each MCAN or consolidated

MCAN submitted, unless the company submitting the MCAN is a small

business (see §700.45). A small business concern must remit $100

with each MCAN or consolidated MCAN. Persons are exempt from

remitting any fee for exemptions (the TERA, TME, the Tier I and

Tier II fermentation exemptions, and petitions for adding to the
11

recipient list for the Tier I and Tier II fermentation

exemptions). 

The Toxic Substances Control Act requires the Agency to

publish a notice in the Federal Register announcing the receipt

of each MCAN and Exemption submission. See item III.D.7 for

further information on preparation of this notice.

D.2. Information Requirements for MCANs

Any manufacturer, importer, or processor of a living

microorganism, who is required to report under §5 of TSCA must

file a Microbial Commercial Activity Notice (MCAN) with EPA under

§725.105, unless the activity is eligible for one of the specific

exemptions noted in item III.D. above. Further, a MCAN submission

is required under §725.105(c) for any person who intends to

manufacture, import, or process for commercial purposes a

microorganism identified as having one or more significant new

uses, and who intends to engage in a designated significant new

use of the microorganism or intends to distribute it in commerce. 

Microorganisms subject to MCAN reporting could be either for

fermentation applications or for intentional release to the

environment. A MCAN must be submitted 90 days before manufacture,

import, or processing of the microorganism for commercial

purposes.

In addition to the general administrative items and fees

noted in item III.D.1 above, certain technical information is

required for each MCAN submission as noted in §s 725.155 and

725.160. In order to provide complete information for EPA

review, the following portions of item IV of the Points to

Consider can be used as a guide for MCAN information submitted

under §s 725.155 and 725.160: 

Item 725.155(d)(1)Description of the recipient microorganism

and the new microorganism subject to TSCA: see applicable

portions of item IV.A.

Item 725.155(d)(2) Genetic construction of the new

microorganism subject to TSCA: see item IV.B.

Item 725.155(d)(3) Phenotypic and ecological

characteristics: see item IV.D., and include information as

suggested in item IV.G. if the MCAN is for a field test.

Item 725.155(e) Byproducts: see item IV.E.2.
12

Item 725.155(f) Total production volume: see item IV.E.1.

Item 725.155(g) Use information: see item IV.E.3.

Item 725.155(h) Worker exposure and environmental release:

for worker exposure, see either items IV.F.1.b. or

IV.F.2.b.; for environmental release, see either item

IV.F.1.c. or IV.F.2.b.

Item 725.160 Health Effects Information: see item IV.C.

D.3. Information Requirements for the TSCA Experimental Release

Application (TERA) Exemption for Research and Development

As noted in III.D. above, there are two types of TERA

exemptions which can be submitted instead of a MCAN. First, there

is a TERA exemption for certain types of R&D field tests

involving Rhizobium meliloti and Bradyrhizobium japonicum

(see §s 725.238 and 725.239). This exemption requires no upfront

reporting to EPA, although a certification statement and

recordkeeping are required. Since guidance on how to submit a

certification statement to EPA, and on the recordkeeping

requirements for field tests with these bacteria, is provided

under §725.238, additional guidance is not offered here. 

Second, there is a TERA exemption from MCAN reporting for

field tests with microorganisms which allows for a 60-day review

by EPA, following receipt of the information described in §

725.250. The documentation provided by the submitter for this

TERA exemption is similar in format and content to an MCAN

submission for an individual field test, but is abbreviated

relative to an MCAN submission. For example, persons who submit a

TERA would not have to include information on all commercial

manufacture, processing, transport, use, and disposal activities

that may involve the new microorganism as is the case for a MCAN. 

EPA will, however, still review any general commercial uses of

these new microorganisms through the MCAN process.

If a submitter believes sufficient information is available

which could justify adding another recipient to the list exempted

microorganisms at §725.239, please consult with the Agency. 

In addition to the general administrative items noted in

item III.D.1 above, certain technical information is required for

each TERA submission as noted in 725.238, 725.250, and 725.255. 

The following portions of item IV of the Points to Consider can
13

be used as a guide for TERA information submitted under 725.255. 

The information should be organized in accordance with 725.255.

Item 725.255(c) Microorganism identity information required

for MCANs: see items IV.A and IV.B.1

Item 725.255(d) Phenotypic and ecological characteristics as

they relate directly to the conditions of the proposed

research and development activity:

- Habitat, geographical distribution, and source of the

recipient: See item IV.D.2.a. for habitat and

geographical distribution, and item IV.A.2.b. for

source of the recipient. 

- Survival, dissemination, and detection of the subject

microorganism: See item IV.D.2. for survival and

dissemination; see item IV.G.7. for detection

methodology descriptions.

- Anticipated adverse ecological effects: See item

IV.D.1.

- Involvement in biogeochemical cycling processes: See

item IV.D.1.b.

Item 725.255(e)(1) A detailed description of the proposed

research and development activity:

Item (e)(1)(ii): Methods of application or release: see

applicable portions of IV.G.1, IV.G.3., IV.G.5, and

IV.G.8.

Item (e)(1)(iii): Characteristics of the test site: see

applicable portions of items IV.G.2. and IV.G.4.

Item 725.255(e)(2) Information on monitoring, confinement,

mitigation, and emergency termination procedures: see items

IV.G.4, IV.G.5., IV.G.6, and IV.G.7. 

D.4. Information Requirements for Test Marketing Exemptions

Test marketing activities usually involve limited sale or

distribution of a substance within a predetermined period of time

to determine its competitive value when its market is uncertain. 

In general, EPA suggests that manufacturers who intend to test

market a new microorganism file a MCAN rather than request a Test

Marketing Exemption(TME; see §725.300). However, there may be
14

situations in which this exemption is appropriate, such as for

microorganisms which were previously reviewed by EPA at the R&D

stage.

In addition to the general administrative requirements noted

in item III.D.1 above, certain technical information is required

for each TME submission as noted in 725.350 and 725.355. The

following portions of item IV of the Points to Consider can be

used as a guide for TME information submitted under 725.355, and

the information should be organized in accordance with 725.355.

Item 725.355(c) Microorganism identity information: see

applicable portions of item IV.A. for description of the

recipient and subject microorganisms, and item IV.B. for

genetic construction of the subject microorganism.

Item 725.355(d) Phenotypic and ecological characteristics: 

- Habitat, geographical distribution, and source of the

recipient: See item IV.D.2.a. for habitat and

geographical distribution, and item IV.A.2.b. for

source of the recipient. 

- Survival, dissemination, and detection of the subject

microorganism: See item IV.D.2. for survival and

dissemination; see item IV.G.7. for detection

methodology descriptions.

- Anticipated adverse ecological effects: See item

IV.D.1.

- Involvement in biogeochemical cycling processes: See

item IV.D.1.b.

Item 725.355(e)(1) Maximum quantity of microorganism, and

duration and route of exposure of persons to microorganism:

see items IV.E.1. on quantity of microorganism, either item

IV.F.1.b or IV.F.2.b. for worker exposure, and IV.E.3. for

consumer exposure.

Item 725.355(e)(2) Health and environmental effects

information: see item IV.C. for health effects information.
15

D.5. Information Requirements for Tier I and II Exemptions for

Closed Fermentation Systems

Certain microorganism applications for closed fermentation

systems can fall under a TSCA §5(h)(4) exemption from MCAN

reporting as described under §725.400. These exemptions can fall

under one of three categories: (1) a Tier I exemption for an

intergeneric microorganism based on a recipient microorganism

listed under §725.420, (2) a petition to add a recipient

microorganism to the list at §725.420, and (3) a Tier II

exemption for an intergeneric microorganism submitted under §

725.428. The Tier I exemption requires only a brief certification

and information statement to be forwarded to EPA. Since guidance

on how to submit these certification statements for Tier I

exemptions is provided under §725.424, additional guidance is not

offered here.

The process for filing a petition to add a recipient

microorganism to the list at 725.420 is addressed under item

V.B.2.b. of the Preamble of the Final Rule (and further under

Unit III.C.2 of the Response to Comments document). EPA expects

that petitions to add microorganisms to the list of recipient

microorganisms at 725.420 will be preceded by several MCANs so

that sufficient information is available to determine whether the

microorganism should be added. EPA would make this determination

after evaluating information which addresses the six criteria for

recipients noted in the Proposed Rule: (1) clear taxonomic

identification of the microorganism, (2) availability of

information to evaluate the relationship between the recipient

and closely related microorganisms which have a potential for

adverse effects, (3) a history of safe commercial use for the

recipient, (4) commercial uses which indicate that the

recipient’s potential products might be subject to TSCA, (5)

studies are available which indicate the potential for the

microorganism to cause adverse effects, and (6) studies are

available which indicate the survival characteristics of the

recipient in the environment.

The third microorganism submission under §725.428 is one in

which (1) the recipient microorganism is listed in §725.420; (2)

the introduced genetic material meets the criteria under

§725.421; and (3)adequate physical containment is proposed, even

though the containment does not meet the requirements at

§725.422. The review period for a Tier II exemption is 45 days,

beginning from the date the Agency determines that the submission

is complete; the Agency may extend the review period for good

cause. The following portions of item IV of the Points to

Consider can be used as a guide for Tier II information submitted
16

under 725.455. The information should be organized in accordance

with 725.455.

Item 725.455(b)(1) Microorganism identity information which

indicates that its taxonomic name is listed under 725.420:

see item IV.A., and provide a brief summary of the

information as noted under IV.A.

Item 725.455(b)(2) Type of genetic modification and the

function of the introduced genetic material: see item

IV.B.2. for preparation of a final construct illustration,

and Item IV.B.3. which assists in describing the function of

the introduced genetic material. Provide a brief summary (a

one-paragraph description of construct), using these

sections of item IV.

Item 725.455(b)(3) Site of insertion: see item IV.B. 

Item 725.455(c)Production volume: see item IV.E.1.

Item 725.455(d)(1)Process and containment information: See

item IV.E. No additional guidance is offered in this

document on justifications for lower levels of containment

than that specified for the Tier I exemption. If the

containment anticipated is less than that of the Tier I

exemption, see EPA reference No. 4 in the Final Rule and

consult with the Agency for presentation of containment

information.

D.6. Submissions to Other Agencies

EPA requests that a company submit to EPA any information it

has provided to other U.S. federal agencies or foreign

governments to obtain regulatory approval of the microorganisms

in question. Information submitted as nonconfidential to other

U.S. Federal Agencies (e.g., the Food and Drug Administration)

cannot be claimed as confidential when submitted to EPA.

D.7. 5(d)2 Notice:

The Toxic Substances Control Act requires the Agency to

publish a notice announcing to the public the receipt of each

MCAN or TSCA §5(h)(4) exemption submission that requires

reporting (see item III.D. above). This is accomplished through a

5(d)2 notice published in the Federal Register. A sample 5(d)2

notice can be found at 52 FR 24527 (Wednesday, July 1, 1987). 

The submitter may consider summarizing this information in the

MCAN or exemption submission.
17

IV. POINTS TO CONSIDER FOR MICROORGANISM SUBMISSIONS

This section presents a suggested format and information to

be included, where applicable, for the seven kinds of

microorganism submissions noted in Section III.D.: The Microbial

Commercial Activity Notice (MCAN; including MCANs for significant

new uses of microorganisms), the TSCA Experimental Release

Application (TERA), the TERA exemption for field tests with

certain rhizobia, the Test Marketing Exemption (TME), the Tier I

and Tier II exemptions for fermentation applications, and

petitions to add microorganisms to the list of recipient

microorganisms for Tier I and Tier II exemptions. The information

items listed under Section IV. are intended to provide data that

would assist EPA in the review of these seven submission types.

Since not all information items listed under Section IV. are

applicable to each of the submission types, Section III.D. should

be examined prior to reviewing item IV. so that only the

necessary information under item IV. is submitted to EPA. Section

III.D. links the specific sections of the Final Rule (62 FR

17910-17958) applicable to each of the seven submission types

with the relevant portions of item IV. of the Points to Consider

document. 

If the information in a TSCA submission is inadequate for

the Agency's review process, EPA staff will contact the submitter

for clarification or further information. Prior to preparation of

a microorganism submission, it may be beneficial for the company

to consult with EPA staff (see Section III.A.). 

TERMINOLOGY

The following terms are used to describe different

microorganisms and their roles in the development of a

microorganism which may be reportable under TSCA section 5. 

Information about these microorganisms is essential to EPA's

review and will be discussed under the appropriate headings

below. 

Subject microorganism is the subject of the microbial

submission. The seven types of submissions which persons provide

for EPA review/documentation are noted in III.D. 

Recipient microorganism is the strain into which the

intergeneric DNA is introduced and which generally determines the

taxonomic designation of the subject microorganism. 

Donor microorganisms are those which contribute DNA to the

subject microorganism, or those which contribute DNA to

intermediate microorganisms used to construct the final subject

microorganism. 
18

Intermediate microorganisms do not contribute DNA, but may

be used to temporarily contain a vector used to construct the

subject microorganism, to aid in a triparental mating, etc.

Final construct is the term which describes the DNA

sequences contributed by the donor microorganisms, and where

applicable, the recipient’s DNA sequences immediately flanking

the inserted donor microorganisms’ DNA. The construct includes

such sequences as structural genes (usually genes which encode an

enzyme that is of commercial importance), vector DNA, and marker

genes (such as antibiotic resistance). Information on the final

construct should focus on the intergeneric DNA, and intrageneric

DNA that affects the expression, stability, and mobility of the

intergeneric DNA. 

TABLE OF CONTENTS

A. Recipient Organism Characterization

B. Subject Organism Characterization

C. Predicted Human Health Effects of the Subject Microorganism

D. Predicted Environmental Effects and Fate of Subject

Microorganism

E. Predicted Production Volume, Byproducts, Use, and Consumer

Exposure

F. Predicted Releases due to Manufacturing of the Subject

Microorganism, and Worker Exposure to Subject Microorganism

G. Information Applicable to Field Tests of the Subject

Microorganism
19

IV.A RECIPIENT ORGANISM CHARACTERIZATION

Section IV.A. provides guidance on information useful to the

Agency in identifying the recipient microorganism (used to

produce the subject microorganism). Many of the traits associated

with the recipient will be considered in assessing the potential

for the subject microorganism to cause adverse effects in humans

and/or the environment. Hence, taxonomic classification is a key

component in enabling an accurate assessment of the potential

concerns for the subject microorganism. Item IV.A.1. provides

general guidance for microbial classification, while item IV.A.2

suggests more specific information directly applicable to

microbial submissions under TSCA.

A.1. Taxonomy: General

The following provides some general information relative to the

use of taxonomy in the TSCA biotechnology review process:

(1) Taxonomy is a means of organizing items and showing

their relative relatedness. (Vandamme et al., 1996)

illustrate that taxonomy has three components: (I.)

classification, or the ordering of organisms in groups,

(ii.) nomenclature, or the labeling of units in those

groups and (iii.) identification, or the determination

that an unknown belongs in one of the labeled groups.

For the purposes of this document and submission of

notices under TSCA, the last meaning has the greatest

relevance.

(2) Usually, the bulk of the genetic information in any

subject microorganism is derived from the recipient

microorganism. It is therefore likely that any added

features from a donor organism, will be insufficient to

warrant a species name for the subject microorganism

different than that of the recipient parent. Thus, the

following discussions concerning reporting of the

taxonomic designation of the recipient microorganism

are usually also relevant for the subject microorganism

itself. An exception would be for products of cell

fusion, where the differentiation between donor and

recipient is not obvious and the resultant chimeric

organism may not resemble either parent. In such cases,

identification of both contributors to the genome of

the resultant organism is needed. 
20

(3) For a few taxa, the choice of identification methods,

and the process of identification may be simple. For

many others, this task is far from trivial. As there

are no universally applicable methods for identifying

microorganisms, it is up to the submitter, or its

agent, to select the most appropriate ones for

submitted organism(s). 

(4) Traditional methods based on phenotype may be the best

for some taxa. Numeric taxonomy, which uses statistical

analyses of an array of characteristics to determine

the similarities and differences between a given

isolate and closely related taxa, has been successfully

used for identifying a submitted organism to the

species level. However, some common genera do not lend

themselves to unequivocal application of these

techniques. 

(5) Modern classification schemes for bacteria rely on

nucleic acid analyses to a great extent and, for many

taxa, they are slowly supplanting phenotypic analysis

as the primary means of identification. Recent

developments in the use of genetic methods have

resulted in the successful application of some of these

to otherwise difficult-to-identify taxa. Several of

these methods were first applied to the classification

of groups of taxa, and are only recently being employed

to identify individual isolates. Since many of these

newer tools have not yet received widespread commercial

application, and because there are some taxa for which

they may not be most appropriate, EPA cannot recommend

specific genetic methods at this time. Nevertheless,

EPA believes that these methods warrant close

consideration for difficult taxa. EPA is currently

cosponsoring research in this area, and hopes to

provide more definitive guidance with future revisions

of these points-to-consider. 

A.2. Taxonomy: Specific issues.

The following provides specific observations on the kinds of

information needed to support the identification of the recipient

microorganism associated with the subject microorganism. Except

as indicated, the following applies primarily to bacteria. These

data are not expected to be provided for exempt organisms under

§5(h)4 of TSCA. Such information that is relevant to establishing

that the recipient organism belongs in the class of exempt

recipients should be retained in the submitter’s records.
21

A.2.a. §725.12 requires that a taxonomic designation for both

donor and recipient be provided. For the recipient

microorganism, this identification would include genus,

species, and strain designations, when known. If

applicable, description at a lower taxonomic rank

(subspecies, biovar, pathovar, serovar, etc.) should be

provided. The most current nomenclature should be used. 

(1) For bacteria and viruses, the name should be the most

current. 

The international bacteriological community has adopted

a naming convention based on the existence of bacterial

names on the Approved List of Bacterial Names (1980),

or on subsequent lists of validly published names as

found in current issues of the International Journal of

Systematic Bacteriology. All other names for a

bacterium are considered synonyms. 

In a similar way, the international virology community

has adopted guidance for naming viruses. While not a

formal code, guidance on naming has been provided in

the International Code of Virus Classification and

Nomenclature (1990) and the Sixth Report of the

International Committee on Taxonomy of Viruses (1995). 

(2) It is better to identify the organism to the taxonomic

level for which data support a positive name assignment

and indicate the alternative subtaxa believed to be

nearest the isolate, than to attempt to provide a name

at a lower level that is unsupported by data. 

Not all microorganisms may be identifiable to the

species level. Some isolates may be intermediate

between members of accepted taxa, but do not fit the

descriptions of a named species. In this case, it is

appropriate to provide information on the features,

phenotypic or genetic or both, which illustrate the

relationships between the isolate and the alternative

named organisms to which it appears closely related.

EPA will take this into account when describing the

subject organism for the TSCA Inventory.

(3) For fungi, the most common currently used names should

be listed and a reference to a readily available

source that will provide a cross-reference to other

synonyms, if they exist, should be provided. Where
22

applicable, names of both teleomorphic and anamorphic

forms should be given.

There is no comparable taxonomic authority for fungi,

as for bacteria and viruses. Please contact the Agency

for the most current guidance on accepted taxonomy for

fungi.

(4) For other eukaryotic organisms, commonly used names

should be provided. 

As for fungi, naming conventions have not been codified

for higher microorganisms as they have for bacteria and

viruses. Please contact the Agency for the most current

guidance on accepted taxonomy for microorganisms such

as protozoa and algae. 

(5) For any subject organism, provide taxonomic synonyms

commonly used for the recipient. 

A.2.b. Information substantiating the taxonomy of the

recipient is requested. 

A submitter is responsible for understanding the

underlying assumptions behind any identification

techniques they, or their identification supplier, use.

A description of the source from which the recipient

was originally isolated (environmental medium such as

lake water, particular plant, etc.) is helpful in

understanding the nature and ecology of the recipient.

Please provide details on the source, such as location

of lake where water sample was obtained, if known.

The submitter should be able to explain why one of

several alterntative approaches to identification was

chosen. §725.12 provides two means of supplying

substantiating information. This information can take

two forms: (1) a letter from a culture collection

establishing an organism’s identification or (2)

data/analyses used by the submitter or its agent in

establishing the identity of the submitted organism.

For the latter, this may take the form of a report from

an expert on the taxon of the submitted organism,

confirming the identification of the organism. In the

cases where either a service culture collection or an

expert provide the identification, EPA assumes that the

submitter has access to, and could provide relevant
23

data utilized by the collection or expert, if requested

by EPA. 

A.2.c. When phenotypic methods are most appropriate, analysis

methods should be described and data from these methods

should be provided, either directly, or by easily

accessible reference. 

Types of data appropriate for these methods can include

morphological, biochemical, immunological, or

physiological characteristics. Method description can

also be supplied by reference if these are readily

accessible. 

The selection of specific tests in a battery used for

numeric taxonomy for bacteria is generally dependent on

the suspected taxon of the organism. Such tests could

comprise a wide range of traits including, but not

limited to, presence of flagella, optimum temperature

for growth, oxygen requirements, fatty acid profiles,

serology, energy sources utilized, and life cycle

information such as dormant stages. Experts have

compiled these test matrices for specific purposes and,

thus, they should not be used without a complete

understanding of the function of each test within the

matrix, and its individual utility in differentiating

taxa. This information is usually available in

publications regarding the specific test batteries, or

from the supplier of the identification based on such

tests. It is not necessary to supply EPA with a

rationale for the selection of each test in a battery

unless the test system has been modified by the

submitter from one that has been published, or for

which the relevant information could not be otherwise

readily obtained by EPA. Rather, EPA suggests that the

submitter be familiar with the rationale for choice of

a specific identification matrix and statistical

method, in order to have confidence that the

identification of its organism was appropriate. In a

similar way submitters should be aware of alternative

typing methods for fungi, which often are based on

interpretations of morphological features. 

A.2.d. If the results of phenotypic analyses are submitted in

support of the taxonomic designation, the test system

used should be referenced in such a way that the

individual tests used can be clearly determined.
24

Phenotypic information is usually comprised of either

“binary” or numeric data. Binary data are results of

tests where the organism either exhibits the trait or

it does not; in numeric taxonomy, certain statistical

treatments can also take into account equivocal or

absent data of this type. Numeric data are results of

tests where a graduated response is expected. “Grows

at 37EC” is binary; ”minimum inhibitory concentration

(MIC) of ampicillin is ___μg/ml.” is numeric. Different

laboratories often use different sets of tests for

identifying similar organisms. Occasionally, different

test methods will receive identical descriptive names

due to these interlaboratory differences. To avoid this

problem and aid in describing individual tests used in

phenotypic analyses, there exists an international

system for encoding these data in a uniform shorthand

way. The RKC format (Rogosa et al., 1986) would encode

the above binary data example as “017015" and the

numeric example as “535081". The individual codes for

this system can be found in searchable format at the

following sites on the World Wide Web:

http://bioinfo.ernet.in/cgi-bin/asearch/rkc/rkc_wais_read

http://fragrans/riken/go/jp/htbin/RKCCODE.pl

http://sun/im.ac.cn/cgi-bin/RKC.pl 

A.2.e. In a similar way, when genetic methods are used,

analysis methods should be described and data from

these methods should be provided, either directly, or

by easily accessible reference. 

An alternative approach to traditional phenotypic

analysis consists of analysis of relatedness among

microbial taxa by comparing their nucleic acids rather

than, or in addition to, comparing phenotypic

information. Many different types of nucleic acid

analysis have been utilized in research settings. For

the purpose of classifying groups of bacteria, DNA:DNA

homology has long been established as a standard.

However, this has not been considered an appropriate

method for routine bacterial species identification.

Newer methods have moved away from homology methods

based on annealing of DNA strands from different

organisms, to sequence comparisons and restriction

analyses. Some of these are just beginning to become

available as commercial methods as well. The most

promising techniques for bacteria are variations on the

theme of ribosomal RNA (rRNA) analysis. Variability in

certain conserved regions of rRNA sequences has become
25

well established as a means to reveal genus level

differences among isolates. Recent work utilizing

other portions of the rRNA or related DNA sequences has

evolved into a variety of tools for separation of

bacteria at species or lower taxonomic levels of

organization. EPA cannot recommend any one method as

appropriate for all species. EPA will continue to

monitor the progress of development of these newer

identification tools and may issue additional guidance

when these methods become established as generally

available and reliable tools.

A.2.f. Genetic data should also be submitted in a manner that

allows comparison with similar data. Sequences should

be provided where applicable. Relevant references

should also be submitted. If the sequence is publicly

available from the data bank, accession numbers may be

provided in lieu of the sequence data.

Genetic information may take a variety of forms. See

“Illustrations of Construct Information” below for

details of graphical representations of these data.

Sequence data are commonly presented as linear strings

of base, or amino acid units, usually arrayed as a

matrix. Sequences submitted to generally available

sequence data banks will be given specific accession

numbers by the curators of those data banks.

REFERENCES.

A. Vandamme, P. Pot, M. Gillis, P. De Vos, K. Kersters and J.

Swings. 1996. Polyphasic taxonomy, a consensus approach to

bacterial systematics. Microbiol. Rev. 60:407-438]

Franki, R., C.M. Fauquet, D. L. Knudson, and F. Brown. 1990.

Classification and nomenclature of viruses. Arch. Virology Suppl. 

2:1-445.

Murphy, F.A., C.M. Fauquet, D.H.L. Bishop, S.A. Ghabrial, A.W.

Jarvis, G. Marinelli, M. A. Mayo, and M. D. Summers (eds.). 1995.

Arch Virology Suppl. 10 Springer Verlag ,N. Y. 586 pp.).

Rogosa, M., M.I. Krichevsky, R. Colwell. 1986. Coding

Microbiological Data for Computers. Springer Verlag, N.Y. 299 pp.
26

IV.B. SUBJECT ORGANISM CHARACTERIZATION

Item IV.B. is intended to provide information to the Agency

that is useful in identifying the taxonomies of the subject and

donor organisms (IV.B.1.), the final construct (item IV.B.2), the

genetic manipulations done to derive the final construct (item

IV.B.3.), and properties of the subject organism (item IV.B.4.). 

This information is useful to the Agency for listing of the

organism on the TSCA Inventory and for assessment of the

potential risks of the organism.

Examples of illustrations and text which describe the final

construct and the genetic manipulations done to arrive at the

final construct are provided in Attachment 4 of this document,

and referenced in the text of item IV.B. These illustrations and

their accompanying detailed text descriptions are intended to aid

in the identification of major sources of DNA used to construct

and/or introduce the intergeneric DNA sequence(s) contained in

subject microorganisms. 

Major sources of DNA may include the recipient microorganism

and donor microorganism(s). Although the Agency will focus its

risk assessment on the intergeneric DNA, all introduced DNA

sequences (both intra- and intergeneric sequences which have been

placed in the subject microorganism by using genetic

manipulations) need to be identified. This allows the Agency to

fully compare the subject microorganism to the recipient in order

to assess any altered risk posed by the subject microorganism. 

Identification of all introduced DNA allows the Agency, for

example, to determine how the intrageneric introduced DNA will

affect the expression, mobility, and transfer potential of the

intergeneric DNA.

In order to conduct the most efficient review of the genetic

manipulations done, full reference citations noted in association

with the construction schemes and final construct illustrations

should be provided. It is also helpful if the full text of

references cited is provided for documents such as internal

company documents and other which do not appear in peer-reviewed

journals, foreign journals, hard-to-access theses, and patents,

all of which may be difficult for the Agency to obtain. 

 

B.1. Taxonomies of the Subject and Donor Microorganisms

B.1.a. If genetic manipulation is so extensive that a subject

microorganism might be more appropriately assigned to a

different taxon than the recipient parent, the

submitter should provide equivalent support for the

designation that applies to the final construct as that
27

provided for the designation of the recipient parent. 

Such support should apply to the microorganism intended

to be used as the final production strain.

B.1.b. A generic name for the subject microorganism is needed

if the name of the subject microorganism is to be

considered as Confidential Business Information (CBI).

Any trade name under which the subject microorganism

might be marketed should also be supplied.

B.1.c. Taxonomic characterization of donor microorganisms

which contribute intergeneric DNA to the subject

microorganism, or provide intrageneric DNA that may

affect the expression or stability/transfer of the

intergeneric DNA is needed. Characterization could

include identification of a genus, species, and strain

designation for each donor microorganism. Generally,

support information for these taxonomic designations

need not be provided in detail as suggested for the

recipient microorganism under item IV.A. Taxonomic

information on the microorganisms from which the

nucleic acid sequences were first isolated may also be

helpful, if their taxonomies differ from that of the

donor microorganisms used to construct the subject

microorganism. 

B.2. Final Construct

Provide an illustration of the final construct which is in

the subject microorganism identified in item IV.B.1., and again

note the taxonomy of the subject microorganism. Provide a

detailed legend to support the final construct illustration.

Often, the final construct can be classified into one of three

different general types: (1) an intergeneric plasmid which is

extrachromosomal, (2) an intergeneric plasmid which is integrated

into the chromosome, or (3) an intergeneric insert, possibly with

associated vector material, which is integrated into the

recipient’s genome. Combinations of these general types of

constructs, as well as other construct types, are possible. Two

examples of final construct diagrams accompanied by respective

legends, are illustrated in Attachment 4 (Figure 1 refers to the

pPCB plasmid and illustrates the first general construct type;

Figure 2 refers to the dct/omega/nifA insertion and illustrates

the third general construct type).

The legend which accompanies the final construct illustration

should focus on the intergeneric DNA, and intrageneric sequences

which could affect expression or genetic transfer of the
28

intergeneric DNA. For example, for general construct types (1)

and (2) above, the introduced DNA should be described in terms of

the original host source, followed by size and function of the

DNA. Introduced intrageneric structural genes, promoters,

leaders, repressors, antibiotic markers, transposon or transposon

fragments, other gene fragments, and cloning sites (which affect

the expression, stability, or mobility of the intergeneric DNA)

should be identified in the introduced DNA. The level of detail

for the legend which accompanies the final construct is

illustrated by the legends for Figures 1 and 2 of Attachment 4.

B.3. Construction of the Subject Microorganism

In this section, the methods and sources used to produce the

final construct in the subject microorganism should be described. 

The description should consist of a flow diagram and its

associated text in which the flow diagram is discussed. The flow

diagram and associated text should describe the names, functions,

and sources of donor, recipient, and vector DNA which have been

manipulated to produce the subject microorganism. Source

microorganisms for the introduced DNA are those microorganisms

from which the DNA sequences were first identified in the

literature. Again, the flow diagram should focus on the original

sources for the intergeneric DNA, and other sequences associated

with its expression and stability/transfer, including the items

listed below. Figure 3 of Attachment 4 and its associated text

provide an example of the level of detail helpful to the EPA

evaluation of the cloning techniques involved in production of

the subject microorganism. 

3.a. A brief summary of the construction strategy should be

presented. The summary should indicate why the genetic

manipulations were done and their effect(s) relative to the

recipient microorganism. The strategy, as indicated by the

text associated with the construction of plasmid pPCB in

Attachment 4, should provide an adequate level of detail. 

This summary should allow the Agency to follow the

manipulations done, beginning with the original sources of

the introduced DNA, to arrive at the final construct.

3.b. Final recipient microorganism characterization:

(1.) Prior modifications (deletions, additions)

(2.) Presence of plasmids and their ability to promote

mobility/transfer, or affect the expression, of the

introduced DNA

(3.) Location of endogenous gene(s) homologous to the

introduced DNA that could promote mobility/transfer of the

introduced DNA
29

(4.) Characterization of the insertion site for the

introduced DNA

3.c. Reference to any prior submission to EPA (or other Federal

Agencies) which is directly related to construction of the

subject microorganism (provide submission number if

available).

3.d. A key which contains full names for abbreviations used in

the diagram.

3.e. As many circular plasmid/vector maps of intermediate

constructs as necessary to clearly show genetic

manipulations and gene modification. A linear portion of a

plasmid representing only the changes is adequate for

plasmids which have been illustrated in their entirety

earlier in the diagram. These intermediate construct

illustrations should be sufficient to trace and verify the

origins of intergeneric DNA shown in the final construct

illustration. The level of detail of the flow diagram

should be similar to Figure 3 of Attachment 4. 

3.f. Sizes of important gene fragments retained and lost,

sequences altered, and addition and/or deletion of

restriction sites.

3.g. Restriction enzymes used, including whether resulting cuts

are full or partial.

3.h. Detailed map of the final cloning vector if the vector, or

portions of that vector, are to be retained in the subject

microorganism.

3.i. Methods for isolating and identifying the DNA used to modify

the recipient microorganism.

3.j. Preparation or modification of DNA including procedures such

as deletion, insertion, directed mutagenesis, and

rearrangement.

3.k. Procedures for selection of intermediate hosts including

methods and results for determining the success of

insertion, deletion, and/or rearrangement.

3.l. Characterization of vectors so that their function (cloning,

expression, or shuttle) is noted.
30

3.m. Transfer or integration techniques such as transformation,

filter matings, triparental matings, and recombination

events critical to the construction of the subject

microorganism.

3.n. Catalogue references for commercial systems used such as

special recipient strains, plasmids, cosmids, etc.

3.o. Literature references to original sources of important

sequences used in both intermediate and final constructs. 

B.4. Properties of the Subject Microorganism

In this section, please provide a brief description of the

characteristics of the subject microorganism. Such

characteristics may be available for the recipient microorganism,

but applicable to the subject microorganism. Such information

could include the items below. 

4.a. Methods and results used to verify the final construct,

including verification of the location of the intergeneric DNA

and its copy number.

4.b. Nucleotide and protein sequences of major structural

gene(s), if appropriate.

4.c. Description of gene regulation and expression in the subject

microorganism and the characteristics of the product encoded for

the intended use.

4.d. Possibility of unexpected gene expression or suppression in

the subject microorganism as a result of the genetic

modification, both during use and subsequent to release to the

environment.

4.e. Indication of whether the major gene product is

extracellular or intracellular.

4.f. Methods and results for determining stability of the

introduced DNA.

4.g. Likelihood of genetic transfer of the introduced DNA by

transformation, conjugation, transduction, and/or transfection

during use and subsequent to release to the environment.

4.h. Growth characteristics in laboratory and environmental media

such as generation time, growth temperature (optimum and range),
31

pH (optimum and range), oxygen requirements (optimum and range),

preferred energy and carbon sources, etc.

4.i. Factors limiting growth, survival, or reproduction (such as

auxotrophy, asporogenicity, debilitation from continuous

culturing, etc.).

4.j. Subject microorganism antibiotic profile.

IV.C. POTENTIAL HUMAN HEALTH EFFECTS OF THE SUBJECT 

MICROORGANISM

Potential effects on fermentation workers, those who apply

the microorganisms in the field, and/or consumers are assessed by

the Agency for some of the categories of submissions reviewed

under TSCA. In order to examine these potential effects, it is

helpful to provide a review of the ability of the subject

microorganism (or the recipient) to cause diseases, or be

associated with disease, in humans. This section identifies

information which is helpful in assessing both the potential for

the subject microorganism to cause pathogenicity (IV.C.1) and

toxicity (IV.C.2.) in humans.

C.1. Pathogenicity of Subject Microorganism

1.a. Nature and degree of pathogenicity, virulence, or

infectivity, in humans.

1.b. Results of pathological tests on effects of the subject 

microorganism in mammalian species, if relevant and 

available.

1.c. Ability to colonize humans (e.g., the skin, the gut, etc.).

1.d. Ability to grow at human body temperature, 37 C. o

1.e. Susceptibility to control measures such as antibiotics or

disinfectants, substrate requirements, or physical and

chemical control methods. 

C.2. Toxicity and Immunological Effects of Subject

Microorganism or Its Products

a. Nature and degree of toxicity to humans.
32

b. Results of toxicological tests on effects of the subject

microorganism or its products in mammalian species, such as

allergenicity, or immunological responses after exposure via

ingestion, inhalation, or dermal contact.

IV.D. PREDICTED ENVIRONMENTAL EFFECTS AND FATE OF SUBJECT

MICROORGANISM

Potential effects on organisms other than humans outside the

fermentation facility, and/or in the vicinity where the

microorganism or its products are applied for commercial purposes

need to be assessed by the Agency for some of the categories of

submissions under TSCA. The potential for exposure to organisms

in these areas may also need to be assessed. In order to examine

these potential effects, it is helpful to provide a review of the

ability of the subject microorganism (or the recipient) to cause

diseases, or be associated with disease, in organisms other than

humans. A review of the known ability for the subject (or

recipient) microorganism to survive and spread in the environment

is also helpful. This section identifies information which is

helpful in assessing both the potential for the subject organism

to cause adverse ecological effects (item IV.D.1.) and its

ability to survive and spread in the environment (item IV.D.2.)

D.1. Ecological Effects

1.a. Nature and degree of pathogenicity, virulence, or

infectivity to mammals, fish, insects and other

invertebrates, and plants; including host range.

1.b. Toxicity of microbially-produced toxins to mammals, fish,

insects and other invertebrates, and plants.

1.c. Involvement in or effects on biogeochemical processes (e.g.,

effects on nutrient cycling, particularly C,N,P, and S;

effects on primary production (CO2 fixation); utilization of

complex carbon substrates, such as cellulose and lignin

degradation; effects on nitrogen fixation; effects on

nitrification).

1.d. Known or predicted effects on other organisms including

microorganisms in the environment, including effects on

competitors, prey, hosts, symbionts, predators, parasites,

pathogens; effects on community structure or species

diversity.
33

1.e. Identification and description of target organisms, e.g.

taxonomy, agricultural uses, and the anticipated mechanism

of interaction between microorganism and target organism.

1.f. Existence of nontarget organisms or alternate hosts (e.g., 

nitrogen fixing bacterial inoculants often have an intended

legume host, but may be able to infect other leguminous

plants).

1.g. Known or expected substrate range of degradative gene

protein products including both contaminant compounds and

environmental substrates (e.g., lignin).

1.h. Known or expected metabolic pathways of xenobiotic

contaminant(s) present.

1.i. Nature and degree of toxicity of metabolites (dead-end or

intermediate metabolites produced by metabolism of a

xenobiotic contaminant) to mammals, fish, insects and other

invertebrates, and plants. Toxicity should be compared with

the toxicity of the parent contaminant compound.

1.j. Resident antibiotic production levels.

D.2. Survival and Fate

2.a. Natural habitats and geographical distribution of the

recipient microorganism.

2.b. Laboratory studies comparing survival of the subject

microorganism and the unmodified parental recipient strain

in soil or water samples taken from the release site.

2.c. Survival/persistence in other environmental media aside from

that found at the release site (e.g. other types of soil,

water, and/or air). 

2.d. Known and predicted environmental conditions that may affect

survival, multiplication, dissemination. 

2.e. Method of detection and detection limits of microorganism in

soil and/or water (particularly in the intended

environmental media or release site). 

2.f. Prevalence of gene exchange in natural populations.
34

IV.E. PREDICTED PRODUCTION VOLUME, BYPRODUCTS, USE, AND CONSUMER

EXPOSURE

This section provides guidance on submission of information

on production volume of microorganisms, the types of byproducts

from microbial production which may pose hazards, the ultimate

commercial use of the subject microorganism or its products, and

likely consumer exposures to the subject microorganism or its

products.

E.1. Information on production volume

1.a. For batch processes, the batch volume, the maximum

cells/batch, and the maximum CFU/batch that are likely

to be produced in the first year of production; and the

maximum cells/batch and the maximum CFU/batch that are

likely to be produced during any 12-month period during

the first three years of production. Provide also the

maximum cell density of the fermentation broth in

CFU/ml, the number of hours needed to produce each

batch, and the number of batches per year.

1.b. For continuous processes, the maximum cells/day and

CFU/day that are likely to be produced during any 12-

month period during the first three years of

production, the # hrs/day and # days/yr, and the

maximum cell density of the fermentation broth in

CFU/ml.

E.2. Information on Byproducts

In addition to information on the amounts of viable

cells produced per year, it may also be helpful to know

about the concentration of proteins, DNA, or other materials

produced as byproducts of the manufacturing process, if such

material may pose hazards to humans or the environment. 

Please indicate amounts of byproducts which may pose hazards

to man or the environment as was done for viable cells under

item E.1.

E.3. Use Information and Consumer Exposure

3.a. Describe the intended use(s) of the microorganism for the

particular processes for which it is intended (such as waste

degradation) or products it is intended to produce (such as

enzymes for detergent use).

3.b. Estimate the percent of the production volume for each use.
35

3.c. Estimate the concentration of the microorganism in the

formulations for each use identified.

3.d. Generic use if the use is claimed as CBI.

3.e. Based on the intended use(s), identify products in which the

subject microorganism or its product will be present.

3.f. Presence of subject microorganism in consumer products

(estimate amount if possible).

IV.F. PREDICTED RELEASES DUE TO MANUFACTURING OF THE SUBJECT

MICROORGANISM, AND WORKER AND CONSUMER EXPOSURES TO THE

SUBJECT MICROORGANISM

This section provides guidance on information useful to the

Agency in developing a quantitative assessment of the potential

for releases and exposures of the microorganism. This assessment

is used to develop EPA’s risk assessment for the microorganism 

Most of the information listed below is requested of any

submitter of a pre-manufacture notice (PMN) for a new chemical

substance under the traditional chemical program. This

information has also been requested of previous submitters of

biotechnology PMNs under EPA’s 1986 policy and was found to be

helpful in developing a quantitative exposure and release

assessment. As per the standard PMN guidance for chemical PMNs,

the information requested is organized according to whether the

manufacturing site is owned by (a) the company which is

submitting information on the subject microorganism or (b)

someone other than the company submitting information on the

subject microorganism.

F.1. Industrial Sites Controlled by the Submitter

1.a. Operation description

1.a.1. Identity - identity of the site at which the operation

will occur to include the name, site address and city,

county, state and zip code.

1.a.2. An indication of whether the operation is best

described as manufacturing, processing, or use

1.a.3. Process description
36

- A diagram of the major unit operation steps such as

provided in the attached example (Attachment 5).

- As part of the diagram, an indication of the identity, the

approximate weight (by kg/day or kg/batch) and entry point

of all starting materials or feedstocks, and of all

products, recycle streams, wastes and any chemicals used for

inactivation or cleaning.

- Identification of the points of release of the subject

microorganism, including small or intermittent releases, to

the environment outside the fermentation facility.

1.b. Occupational Exposure

1.b.1. Description of activities in which workers may be

exposed to the recombinant microorganisms

1.b.2. A description of any protective equipment and

engineering controls used to protect workers during

these activities

1.b.3. Estimates of the maximum number of workers involved in

each activity

1.b.4. Estimates of the maximum duration of the activity in

hours per day and days/year

1.b.5. An example of a summary table for items 1b.1.-1.b.4. 

is provided below.

Worker Activity Protective Equipment/ # of Maximum Maximum

Engineering Controls Workers Duration Duration

Exposed (hrs/day) (days/yr)
37

1.b.6. Results of any personnel or area monitoring conducted

during the production process.

1.c. Environmental Release and Disposal

For each release point identified in the process description:

1.c.1. An estimate of the amount of the subject microorganism

released (a) directly to the environment and (b) into

control technology, in CFU/day or CFU/batch

Note: a control technology is any combination of

engineering, mechanical, procedural, or biological method

designed and operated to restrict environmental release of

viable microorganisms from a structure. For air waste

streams such as the off gas from the fermentor, examples

include: HEPA filters, mist eliminators, caustic scrubbers,

and ozone treatment. For liquid and solid waste streams

such as the biomass from separation processes, examples

include: steam sterilization, treatment with caustic or

acidic solutions, or other antimicrobial chemicals. 

1.c.2. Indication of the media (air, water, or land) to which

the new substance will be released from that release

point

1.c.3. For each release point released into control

technology:

- A description of the control technology that will be used

to limit the release of the subject microorganisms into the

environment

- An estimate of the efficiency of the control technology

- An estimate of the amount of subject microorganisms

released to the environment after control technology in

CFU/day, and CFU/unit volume of liquid or gas

- An example of a summary table for the items listed 

above is provided below.
38

Release Amount of new substance Media of Control Efficiency

Number released (CFU/day or CFU/bt) Release Technology

To To Control

Environment Technology

 

1.c.4. The basis for the estimates of release and control

technology efficiency.

1.c.5. For air modeling purposes, information on the

configuration of off-gassing vents if applicable, e.g.

height above ground level would be helpful.

1.c.6. For environmental fate and transport assessment,

information on the location of the waste disposal site

for solid wastes; and for aqueous wastes, determination

of whether releases are direct to surface waters or

indirect to surface waters via POTW, name of receiving

stream, and NPDES numbers (for manufacturing site

and/or POTW), where applicable.

 

1.c.7 For the purpose of modeling potential exposures to the

general population resulting from releases outside the

plant, an indication of the proximity of the site to

population areas, ground water aquifers, surface water

and drinking water sources would be helpful. 

F.2. Industrial Sites Controlled by Others

2.a. Operation Description

2.a.1. A diagram of the major unit operation steps, including

interim storage and transport containers. On the

diagram, indicate where the major unit operations are

by labeling with a letter, etc and provide a brief

description of worker activities associated with each

one of these labeled operations.
39

2.a.2. An estimate of the number of sites at which the

operation will occur.

2.a.3. As part of the diagram, an indication of the identity,

the approximate weight (by kg/day or kg/batch) and

entry point of all starting materials or feedstocks,

and of all products, recycle streams, wastes. Include

cleaning chemicals.

2.a.4. Identification of the points of release, including

small or intermittent releases, to the environment of

the new microorganism.

2.b. Worker Exposure/Environmental Release

2.b.1. Completion of items 2.b.1 to 2.b.4. for each worker

activity described.

2.b.2. An estimate of the number of workers exposed for all

sites combined.

2.b.3. An estimate of the typical duration of exposure per

worker in (a) hours per day and (b) days/year

2.b.4. A description of any protective equipment and

engineering controls used to protect the workers.

2.b.5. Completion of items 2.b.6 and 2.b.7 for each release

point identified in the diagram.

2.b.6. An estimate of the amount of subject microorganisms

released (a) directly into the environment or (b) into

control technology

2.b.7. A description of the media of release i.e., stack air,

fugitive air, surface water, on-site or off-site land

or incineration, POTW or other (specify) and control

technology, if any, that will be used to limit the

release of the subject microorganisms into the

environment.

2.b.8. An example of a summary table for items 2.b.1 to 2.b.7

is provided below.
40

Letter # of Duration Protective Release Amount of Control

of Workers of Equipment/Eng Number New Technology

Act- Exposed Exposure Controls Substance

ivity Released

 

 

2.b.8. The basis for the estimates of release and control

technology efficiency

IV.G. INFORMATION APPLICABLE TO FIELD TESTS OF THE SUBJECT

MICROORGANISM

G.1. Objectives

1.a. List the objectives of the field trial and describe the

rationale which requires the environmental release of

the subject microorganism.

1.b. Describe the possible benefits and risks of the

proposed field test.

1.c. Approximate start date and the duration of the field

test.

G.2. Nature of the site

2.a. Location and size of the test area

2.b. Describe why this site was selected (relying on items such

as those listed in 2.c. - 2.g. below).

2.c. Describe the history of site use. Describe any prior

agricultural uses of the site, if applicable. Include items

such as cropping history, tillage management systems,

fertilizer and pesticide applications, and other factors,

conditions, or practices which might influence

characteristics of the site. If the site is not an

agricultural site, describe other prior uses of the site
41

that would influence survival, distribution, and effects of

the subject microorganism such as wastes present in surface

or subsurface soils, surface or underground installations,

etc.

2.d. Describe physical characteristics of the site related to

surface and ground water such as distance to surface or

ground water including public and private drinking water

sources. For groundwater include temperature, flow velocity,

dissolved oxygen, dissolved organic carbon, suspended

solids, direction of flow, volume, depth, width of aquifer,

and pH.

2.e. Describe physical/chemical characteristics of the soil for

agricultural and other applications such as its pH, organic

carbon content, texture (silt loam, clay loam, sand, etc)

and cation exchange capacity. Include, if available,

information on the soil series and associated soil

classification employing current soil taxonomic schemes (see

USDA references at the end of Section IV.G.).

2.f. Evaluate the possibility of dissemination to adjacent

ecosystems and other characteristics of the site that would

influence containment or dispersal e.g. relation to flood

plain, slope, average wind speed and direction, annual

rainfall.

2.g. Identification and description of nontarget human and

nonhuman populations of concern that may be exposed, e.g.

distance to nearest dwellings and population density around

site.

G.3. Field test design

3.a. Rationale for field test design, description of proposed

statistical analyses, and explanation of how the

statistical analysis will answer the field test objectives.

3.b. Describe the control treatments being used for comparison

purposes.

3.c. Submit a diagram of the plot layout and describe the

procedure for randomization of the test plots.

3.d. In a table: summarize the types of samples that will be

collected, where the samples will be taken, how frequently

each type of sample will be taken, and how it will be stored

prior to analysis.

3.e. Describe the proposed management of the site. If the subject

microorganism is intended for an agricultural application,

describe the planting and spacing of the test crop, width
42

and placement of border rows, and, pesticide applications,

crop rotation, crop harvesting schedule, etc. 

G.4. On-site containment practices

4.a. Describe the procedures that will be followed for packaging

and transporting the subject microorganism to the site.

4.b. Describe the procedures that will be followed for the

packaging and transport of samples from the field site to

the laboratory for processing, including the labeling of

such containers

4.c. Describe procedures for cleaning or disinfecting of

planting, sampling, earthmoving and excavation equipment.

4.d. Describe procedures for the disposal of field test samples

and materials that contain the subject microorganism

strain(s).

4.e. Describe physical containment features such as the disposal

of crop plants or dikes to contain water runoff. 

4.f. Biological containment features of the field test (e.g. the

use of trap plants) and the subject microorganism(s). 

4.g. Describe access and security measures to be observed during

the field test. 

4.h. Describe containment procedures and training of personnel

allowed on site. 

4.i. Describe frequency and type of observations (e.g. ambient

conditions or adverse effects) that will be made on site,

submit sample observation forms. 

G.5. Application methods

5.a. Describe the application methods and precautions that will

be followed to control dissemination during the initial

release. 

5.b. Describe any personal protective measures that will be

followed to reduce human exposure. 

5.c. Describe waste materials handling and disposal procedures. 

5.d. Describe method, amount, frequency, and duration of

application of microorganisms. 
43

5.e. Describe methods of cultivation after application if any. 

5.f. Describe number of workers involved in application or

subsequent activities, duration and routes of exposure. 

5.g. Describe worker safety procedures during application and

cultivation. 

G.6. Termination and mitigation procedures

6.a. Procedure to be followed after the field test is completed

such as: level of the subject microorganism population at

which no containment measures are necessary, use of plots

after the field test is terminated. 

6.b. Define the type of unexpected effects and the quantitative

level (if possible) which would necessitate the emergency

termination of the field test. 

6.c. Describe the emergency termination procedures to be followed

if adverse environmental effects are observed during the

course of the field test. 

6.d. Describe how spills or leaks will be handled. 

G.7. Monitoring endpoints & procedures for isolating subject

microorganism

7.a. Relate the monitoring endpoints that will be evaluated to

samples that are collected, such as population trends in

soil or rhizosphere samples, or based on aerial

dissemination during application as indicated by gravity

plate samples. 

7.b. Describe comparisons between the subject microorganism

strain and the unmodified parent that will be monitored in

the field. 

7.c. Describe the techniques used to isolate the subject

microorganism from test samples and the rationale for this

procedure. Include information on positive or negative

controls used with sampling technique, if applicable.

7.d. Describe the selectivity or specificity of the monitoring

technique based on experimental observations under

conditions similar to the field test site.
44

7.e. Describe the sensitivity and reliability of the isolation

and identification procedure based on experimental data.

7.f. Include experimental data on the efficiency of recovery for

each of the sampling techniques.

G.8. Sampling procedure

8.a. For each objective or monitoring endpoint identify the

following: how, where and when samples will be taken.

8.b. Describe how the samples will be labeled so that they can

later be traced back to their source.

8.c. Include the standard procedures for preserving, processing,

and analyzing samples.

8.d. Describe methods of measurement, equipment used, the

precision and accuracy of the method of analysis.

8.e. Describe methods for the statistical analysis of field data.

G.9. Record keeping & reporting test results

9.a. Describe the frequency of reports on the field experiment

and the proposed format of the reports.

9.b. Outline the contents of the progress reports including a

summary statement, statistical analysis procedures and

presentation of the raw data.

9.c. Describe procedures for filing raw data and information on

procedures followed for the analysis of the samples.

9.d. Describe compliance with Good Laboratory Practices.

REFERENCES FOR IV.G.

Soil Survey Division Staff. 1993. Soil survey manual. USDA

Handbook number 18. USDA-SCS. U.S. Government Printing Office,

Washington, D.C.

Soil Survey Staff. 1975. Soil taxonomy: A basic system of soil

classification for making and interpreting soil surveys. USDA-SCA

Agricultural Handbook 436. U.S. Government Printing Office,

Washington, D.C.
45

ATTACHMENT 1

DEFINITION OF NEW MICROORGANISM

MOBILE GENETIC ELEMENTS POLICY

Excluded from the definition of “new” microorganism are

naturally occurring microorganisms and genetically modified

microorganisms other than intergenerics. EPA also specifically

excluded from the definition of “new” microorganisms, those

microorganisms that have resulted from addition of intergeneric

material consisting of well-characterized non-coding regulatory

regions such as operators, promoters, origins of replication,

terminators, and ribosome-binding regions.

In the course of implementing the Agency’s regulatory

policies for microbial products of biotechnology, it became

apparent that a policy was needed to address certain genetic

elements which can be transferred between microorganisms of

different genera. These are termed mobile genetic elements

(MGEs) and include plasmids, transposons, and viruses. 

For MGEs, the major consideration was the source of the

original isolation of the MGE. The genus of the original source

microorganism for the MGE was identified as that genus which was

first noted (in the literature, etc.) as bearing the MGE. 

Microorganisms were considered “new” and thus subject to

reporting requirements, if the MGE was originally isolated from a

microorganism in a genus different from the recipient genus. 

Microorganisms were considered intrageneric, and hence not

subject to reporting requirements, if the MGE was originally

isolated from a microorganism in the same genus as the recipient. 

This policy, which distinguishes which microorganisms with

deliberately introduced MGEs are subject to reporting under TSCA,

has been retained in the 1997 Rule (62 FR 17910-17958).

Microorganisms subject to TSCA with such intergeneric MGEs should

be reported using one of the seven submission types noted in item

III.D., or are subject to recordkeeping and other requirements of

TSCA if exempted from reporting. 

EPA has adopted this interpretation for reasons of

regulatory clarity and uncertainty about the possibility of the 

subject microorganism exhibiting new traits. For example, some

MGEs may contain genetic material that normally is not expressed

in one microorganism but, when inserted into another

microorganism, may be expressed and result in a new trait. 
46

ATTACHMENT 2

GUIDANCE FOR BONA FIDE NOTICES

 OF INTENT TO 

MANUFACTURE/IMPORT INTERGENERIC MICROORGANISMS

INTRODUCTION

A "new" microorganism is one that is not included on the TSCA

Inventory. In the final rule (62 FR 17910-17958) the EPA defines a

"new" microorganism as one which is constructed to contain genetic

material originally isolated from a different genus (intergeneric). 

This includes microorganisms which are constructed to contain

material from a mobile genetic element (MGE) which originally was

isolated from a host of a genus different from that of the

recipient microorganism.

 

To determine reporting obligations for unreported "new"

microorganisms, manufacturers/importers usually consult the public

version of the TSCA Inventory. There, microorganisms which are on

the Inventory are listed. However, when any portion of a

microorganism's identity or use has been claimed as TSCA

confidential business information (CBI), that information does not

appear on the public version of the Inventory. Generic identity or

use information is listed instead to identify the microorganism. 

In fact, several of the new microorganisms which have been added to

the Inventory since 1986 will have generic listings on the public

version of the Inventory. This is because portions of the specific

identity information, e.g., taxonomic designation, genotypic traits

or phenotypic traits, are claimed as CBI. When generic information

is listed on the public Inventory, manufacturers/importers may

request that the Agency search the confidential version of the

Inventory.

To assist manufacturers/importers in determining their

reporting obligations, they may file a submission establishing a

bona fide intent to manufacture, process or import a new

microorganism and request that EPA determine whether their

particular, unreported microorganism is listed on the confidential

version of the Inventory. The Agency will determine whether a

microorganism or a specific use which is identified in a bona fide

notice is the same as, or is equivalent to, a

microorganism/specific use that is listed on the confidential TSCA

Inventory.
47

The Agency will process bona fide notices and conduct a search

of the Master Inventory File only if the submitter demonstrates a

bona fide intent to manufacture or import the microorganism for a

TSCA purpose, and if sufficient information is provided to indicate

the specific identity and use of the microorganism. The Agency

will make every effort to complete its review and notify the

submitter of the Inventory status of an unreported microorganism

within 30 days.

Information provided in a bona fide notice may be claimed as

CBI. 

INFORMATION REQUIREMENTS

The information which should be included in a bona fide notice

is described in the Final Rule. These requirements were developed

specifically for microorganisms. However, due to the complex

nature of microorganisms, EPA may request additional information to

document the unique identity of a microorganism described in a bona

fide notice. In those cases, a notice will be considered

incomplete until the requested information is provided. 

The following information should be included:

1. A signed statement certifying that the submitter intends to

manufacture, process or import the microorganism for commercial

purposes.

Certification statements must be made by the person (company)

who intends to manufacture, process or import an intergeneric

microorganism. Consultants or law firms retained by the company

may not sign the certification statement; cover letters may be

written by such legal representatives.

The submitter must be incorporated, licensed, or doing

business in the United States. If the microorganism is to be

imported, the authorized agent or importing company must submit the

notice. Bona fide notices submitted by foreign companies with no

identifiable domestic subsidiary or agent will not be accepted or

processed. 

2. Taxonomic designation and supplemental information for the

subject microorganism, the recipient (host) microorganism and the

source(s) organism (donor(s) of introduced genetic material).
48

Information on taxonomic designations for the recipient and

donor(s) organisms should include the following:

- Taxonomic designation (including strain designations, if

possible) for recipient and donor organisms, including

synonyms for designations;

- Information substantiating taxonomic designations, especially

that of the recipient microorganism, which may include a

letter from a culture collection which verifies the taxonomic

designation, literature references, or the results of tests

conducted to determine phenotypic characteristics for purposes

of taxonomic classification; and

- For commercially available plasmids, identify the source from

which it was originally isolated, provide a genetic map that

shows both coding and regulatory sequences, and provide the

name of the commercial supplier.

Information that provides the specific identity of the 

microorganism(s), and the development methods used should include

the following:

- Analytical data which describe and document the genotypic and

phenotypic traits of the new microorganism, e.g., genetic

map(s) of the vectors used to introduce genetic material into

the recipient microorganism (see Attachment 4). A vector map

should clearly indicate DNA sequences, their sources, and the

functions they encode. The cellular location (chromosomal or

extrachromosomal) and number of copies of introduced genetic

material maintained in the subject microorganism should also

be noted.

- An indication of those traits known to be added, modified, or 

deleted;

- A description of genotypic and phenotypic characteristics of

the source microorganisms that are known/determined by the

submitter; and

- A description of methods used to construct the expression

and/or selection vector(s), to introduce the vectors into the

recipient microorganism (e.g., cell fusion, injection of DNA,

transformation), and any mutagenesis, selection, chemical

treatment, and/or processing (e.g., covalent attachment of new

microorganism to other organisms, chemicals, or physical

structures) used to manipulate the subject microorganism. 
49

3. Indication of whether a related microorganism has been reviewed

by the Agency.

A submitter may think that the microorganism is related to one

previously reviewed by the Agency as a part of Premanufacture (PMN)

or Microbial Commercial Application Notifications (MCAN). In such

cases, the bona fide notice should include the following

information:

- The PMN or MCAN submission number; and 

- A description of ways in which the bona fide microorganism is

similar to and/or differs from the microorganism described in

a previous PMN/MCAN submission, e. g.,

similarities/differences in source strains and vectors used

and in steps and methods used in the development of the bona

fide microorganism.

4. Description of Research and Development (R&D) conducted with

the microorganism described in the bona fide notice, and a

demonstration of the submitter's ability to produce or obtain the

intergeneric microorganism from a foreign manufacturer. 

Statements about R&D conducted with the microorganism

described in the bona fide notice should contain any of the

following information which applies: 

- A statement that testing, e.g., genetic, biochemical, etc., of

the microorganism has been conducted;

- A statement that the microorganism has been produced on

laboratory scale;

- Description of research on possible uses for the

microorganism; and

- A statement regarding whether the microorganism has already

been manufactured outside the United States, and, if so, by

whom, the quantity (volume, cell number, or colony forming

units / year), and for how long.

5. A specific description of the major intended application or

use of the microorganism.

 

If a microorganism will be imported and the foreign supplier

considers the identity of the microorganism proprietary, then the
50

foreign supplier should submit identity information directly to the

Agency. This constitutes a joint submission of a bona fide notice. 

However, the U. S. importer still must submit the bona fide notice,

indicating that information concerning the identity of the

microorganism is proprietary, and that the foreign supplier will

provide this information to the Agency. It is the responsibility

of the importer to ensure that this information is submitted to the

Agency. 

In response to joint submissions, the Agency informs only the

submitter of a bona fide notice of the TSCA Inventory status of the

microorganism. Proprietary information is treated as confidential 

business information. The Agency will not disclose proprietary

information submitted by a foreign supplier to the submitter of the

bona fide notice.
51

ATTACHMENT 3

CONFIDENTIAL BUSINESS INFORMATION (CBI) CLAIMS

SUBSTANTIATION QUESTIONS &

GUIDANCE FOR PREPARING GENERIC INFORMATION

I. CBI SUBSTANTIATION QUESTIONS

EPA's general procedures for processing and reviewing

confidentiality claims are published at 40 CFR Part 2. The basic

points that should be covered in CBI substantiation are set out at

40 CFR §2.204(e)(4)(I) through (ix). To ensure that substantiation

responses are appropriate for submissions involving microorganisms,

EPA has developed a set of questions based on the points in 40 CFR

Part 2. These questions are designed to reduce the burden of

substantiation by focussing the inquiry on points relevant to a

biotechnology product. Upfront substantiation is required for CBI

claims in MCANs, TMEs, Tier I certifications, and Tier II exemption

requests.

A. Prior to commencement of manufacture or import. 

1. MCAN, TME, Tier I, Tier II Submissions. Any person who submits

a MCAN, TME, Tier I exemption request, or Tier II exemption request

should limit confidentiality claims to that information which is

confidential and proprietary to the business. If any information

in the submission is claimed as confidential business information,

the submitter must substantiate each claim by submitting written

answers to the questions in sections C., D., and E. at the time the

information is submitted. If the submitter does not provide

written substantiation, the submission will be considered

incomplete and the review period will not begin.

2. TERA Submission. Any person who submits a TERA should limit

confidentiality claims to that information which is confidential

and proprietary to the business. If any information in such a

submission is claimed as confidential business information, the

submitter must substantiate each claim by preparing written answers

to the questions in sections C., D., and E. and submitting them to

the Agency upon EPA's request. 

B. After commencement of manufacture or import. 

If a submitter claimed portions of the microorganism identity

confidential in the MCAN and does not wish to have the specific

identity listed on the public version of the Inventory, the claim
52

must be reasserted and substantiated at the time the Notice of

Commencement is submitted. Otherwise, EPA will list the specific

microorganism identity on the public Inventory. The submitter must

substantiate the claim for confidentiality of the microorganism

identity by answering all of the questions in sections C., D., and

E. below. In addition, the following questions must be answered: 

1. What harmful effects to the company or institution's

competitive position, if any, would result if EPA publishes the

identity of the microorganism on the public Inventory? How could a

competitor use such information given the fact that the identity of

the microorganism otherwise would appear on the Inventory with no

link between the microorganism and the company or industry? What

is the causal relationship between the disclosure and the harmful

effects? How substantial would the harmful effects of disclosure

be?

2. Has the identity of the microorganism been kept confidential to

the extent that competitors do not know it is being manufactured or

imported for general commercial use by anyone?

C. General questions 

The following questions must be answered in detail for each

confidentiality claim: 

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

asserted? If the claim is to extend until a certain event or point

in time, indicate that event or time period. Explain why the

information should remain confidential until such point.

2. Briefly describe any physical or procedural restrictions within

the company or institution relating to the use and storage of the

information claimed as confidential. What other steps, if any,

apply to use or further disclosure of the information?

3. Has the information claimed as confidential been disclosed to

individuals outside of the company or institution? Will it be

disclosed to such persons in the future? If so, what restrictions,

if any, apply to use or further disclosure of the information?

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.
53

(2) Material safety data sheets or other similar materials for

the microorganism or the resulting end product.

(3) Professional or trade publications.

(4) Any other media available to the public or to your 

competitors.

(5) Patents.

(6) Local, State, or Federal agency public files. 

If the answer is yes to any of these questions, indicate where

the information appears and explain why it should nonetheless be

treated as confidential.

5. Has EPA, another Federal agency, a Federal court, or a State

made any confidentiality determination regarding the information

claimed as confidential? If so, provide copies of such

determinations.

6. For each type of information claimed confidential, describe

the harm to the company or institution's competitive position that

would result if this information were disclosed. Why would this

harm be substantial? How could a competitor use such information? 

What is the causal connection between the disclosure and harm? 

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? Consider such constraints as

capital and marketing cost, specialized technical expertise, or

unusual processes.

D. Microorganism identity and production method. 

If confidentiality claims are asserted for the specific

identity of the microorganism or information on how the

microorganism is produced, the following questions must be

answered:

1. Has the microorganism or method of production been patented in

the U.S. or elsewhere? If so, why is confidentiality necessary?

2. Does the microorganism leave the site of production or testing

in a form which is accessible to the public or to competitors? 

What is the cost to a competitor, in time and money, to develop

appropriate use conditions? What factors facilitate or impede

product analysis?
54

3. For each additional type of information claimed as

confidential, explain what harm would result from disclosure of

each type of information if the identity of the microorganism were

to remain confidential.

E. Health and safety studies of microorganisms. 

If confidentiality claims are asserted for information in a

health and safety study of a microorganism, the following questions

must be answered: 

1. Would the disclosure of the information claimed confidential

reveal: (I) Confidential process information, or (ii) information

unrelated to the effects of the microorganism on human health and

the environment? Describe the causal connection between the

disclosure and harm.

2. Does the company or institution assert that disclosure of the

microorganism identity is not necessary to interpret any health and

safety studies which have been submitted? If so, explain how a

less specific identity would be sufficient to interpret the

studies.

II. GENERIC INFORMATION FOR CBI CLAIMS

The generic information must reveal the identity and use of

the microorganism to the maximum extent possible without revealing

proprietary information. Submitters are encouraged to review the

guidelines for preparing generic descriptions listed below and

consult with EPA regarding appropriate generic information prior to

submitting a notice. A description for a microorganism must be

specific enough to allow clear interpretation of any accompanying

health and safety data. When the location of the release site is

claimed as CBI, a generic description for use must include

information regarding the type of environment into which the

microorganism will be released. 

A. MICROORGANISM IDENTITY

Subject microorganisms cannot be specifically identified by a

taxonomic designation alone. The microorganism identity used to

describe such microorganisms on the Inventory will be based, in

part, on the supplemental information provided which defines the

specific identity of the subject microorganism. The description

used to identify microorganisms on the TSCA Inventory may include

the following information:
55

1. Taxonomic designation of genetically modified microorganism: 

genus, species, strain.

2. Description of the key phenotypic traits of the microorganism,

including a description of anticipated behavior of

microorganism. 

3. Description of the key genotypic traits and modifications of

the microorganism, including the following. Discussion of

genotypic traits should focus on the intergeneric DNA in the

final construct, and the intrageneric DNA that affects the

expression, stability, and mobility of the intergeneric DNA:

b. Introduced genes: genes introduced into, and retained in, 

the recipient microorganism. 

c. Vector construct: introduced genetic material used to

transfer intergeneric DNA into recipient microorganism. 

d. Cellular location of introduced genes: stability of

introduced genes and location of introduced genetic

material in recipient (on chromosomes or on extrachromosomal material). 

e. Number of genes introduced: number of copies of

introduced genes present in recipient microorganism.

f. Method of construction: how the genes were assembled and

introduced into the recipient microorganism. 

 

Generic names for microorganism identity should be created by

masking only the portion of the information above which the

submitter considers to be proprietary. Generic microorganism

identity should be specific enough to allow clear interpretation of

any accompanying health and safety data.

Taxonomic Designation. Submitters are encouraged always to

reveal the taxonomic designation of the microorganism, since that

information alone is not a sufficient generic description for a

subject microorganism, unless a strain designation has been

developed to describe that microorganism and its new traits. If

the submitter claims the taxonomic designation as CBI, it can be

masked by substituting a description of the microorganism that is

as specific as possible without revealing CBI. 

Description of the Microorganism. In most cases, changes in

taxonomic designation of the subject microorganism have not been

necessary scientifically as a result of such modifications. As
56

indicated above, some description of the microorganism, beyond the

taxonomic designation, will be required for the specific and

generic names. Given that a new microorganism is the result of the

introduction of genetic material, EPA has determined that the

generic description of the microorganism identity must, at a

minimum, include a statement regarding the function and stability

of the genetic construct. This includes an indication of whether

the introduced genes are present on the chromosome or remain extrachromosomal. Additional descriptive information should only be

masked or not included in the generic description insofar as the

submitter is claiming this as CBI. 

B. MICROORGANISM USE

A generic description of use should describe the use or

proposed use as precisely as possible without revealing proprietary

information. However, the description should indicate to the

maximum extent possible how the use may result in human exposure to

the microorganism or its release to the environment. 

Alternatively, the generic use description may provide an

indication of potential exposure or can describe the degree of

containment of the microorganism. 

When a subject microorganism is intended for use in the

environment, a generic description for use must include information

regarding the type of environment into which the microorganism will

be released (e.g., description of field test site). If the exact

location of the proposed environmental release is claimed as

confidential, the submitter should provide a description of the

site that will allow for an ecological analysis. While the exact

information necessary for such an analysis will vary, such

information could include: elevation, climate, slope and aspect,

proximity to open water, groundwater level, vegetation type, and

proximity to habitations and roads. 

C. Identity in health and safety studies. 

TSCA section 14(b) states that EPA is not prohibited from

disclosing health and safety studies of substances for which TSCA

§5 notification is required, unless disclosure reveals confidential

information on process or mixture. EPA has determined that the

regulations developed to address chemical identity in health and

safety studies can also be applied to microorganism identity. In

this regard, if any health and safety information has been

submitted for the microorganism in question, the specific

microorganism identity will be held confidential only if disclosure

would reveal confidential process or mixture information or if the

specific microorganism identity is not necessary to interpret any

of the information. Under this approach, companies that claim
57

specific microorganism identity confidential in their submissions

and wish to argue that knowledge of the specific identity is not

necessary to interpret their health and safety information are

encouraged to choose generic names which are sufficiently specific

to allow interpretation of such information. Sufficiently specific

generic names will tend to support arguments that disclosure of the

specific microorganism identity is not necessary to understand the

health and safety information.
58

ATTACHMENT 4

SUMMARY ILLUSTRATIONS FOR CONSTRUCT ANALYSIS

Guidance on how to prepare construct analysis is provided

below so that only key information needed to fully characterize the

genetic manipulations is provided. Illustrations provided include a

text discussion and accompanying flow diagram and two illustrations

for final construct. Flow diagram and final constructs are central

to the description of the genetic manipulations. 

The diagrams can be made using appropriate software, or

legibly hand-drawn if such software is not available. For sequence

information, such as analysis of open reading frames (ORFs),

computerized references such as a GenBank sequence accession

number, or electronic files of sequences, could be supplied.

This Attachment provides guidance under Item 1. on preparation

of a final construct illustration and description. See Figures 1

and 2 and its associated legends for more information on final

construct illustration and text preparation. Item 2 provides

guidance on preparation of a flow diagram and its associated

descriptive text. See Figure 3 and its associated text for

examples of information useful in the review of the construction

strategy for the subject microorganism.

1. Final Constructs (Figures 1 and 2)

Figure 1 provides an example of the plasmid construct pPCB

where the entire plasmid is retained in the recipient to produce

the subject microorganism. Chromosomal final construct

illustrations (such as Fig. 2) would contain the same level of

detail and include information on the sequences flanking the site

of the chromosomal insertion. Figure 2 provides a dct/omega/nifA

cassette integration into a recipient’s megaplasmid by homologous

recombination in which the vector sequence was not inserted.

(1) Figure 1 (plasmid final construct) and its legend provide

an example of the level of detail necessary for a plasmid final

construct illustration. The plasmid map should have a scale so

that approximate location and size of DNA segments on the plasmid

can be located on the map. First, the molecule itself has a number

of details relevant to the EPA review. The coding genes of primary

commercial interest are the bph genes, driven by the kanamycin

promoter (Kp). The bph sequence also contains 3 open reading

frames (ORFs). The third one, ORF2, has been determined recently
59

to encode four enzymes. The technique used to identify ORFs should

be noted, along with any possible functions for these putative

genes. The marker gene (Tc) and direction of transcription are

also identified. Vector material originally from plasmid pRK2 with

a description of which genes are present that serve plasmid

maintenance and transfer functions is noted. Also, key restriction

enzymes are noted on the molecule (5 EcoRI sites on the bph gene

insert). The PCB degradative genetic unit (bphA to bphD) is within

a 12.4 kb A. eutrophus EcoRI restriction fragment. It has a 5'

flanking sequence of about 1.4 kb containing an open reading frame

(ORF0) but the 3' flanking sequence (<500 bp) can be considered as

part of bphD gene since this sequence is essential for the

expression of bphD.

 Second, brackets and labels surround the molecule which

along with the Figure 1 legend, identify the original sequence

sources used to construct the plasmid. Since the entire plasmid

pPCB remains in the recipient, every gene segment on the plasmid

should be identified by its original host, size, and function. 

These segments refer to the EcoRI fragment containing the bph

genes, the two kanamycin fragments (one contains the promoter), 

the vector sequence carrying the tetracycline antibiotic marker,

and the MCS sites. In this case, intergeneric DNA comes from two

sources: The bph genes from A. eutrophus and the vector

sequence/antibiotic marker from K. aerogenes. The source of

promoter, although it is intergeneric, is excluded since it is a

noncoding regulatory gene and is well characterized. For plasmids

and transposons, the original host is defined as that organism from

which the DNA was isolated first, according to the literature. For

example, many broad host range plasmids can be maintained in any

number of bacteria, but the original host is the one from which

that plasmid was first obtained according to the literature,

submitter records, etc. The legend contains additional information

including the full name of the gene identified in Figure 2, as well

as their size and function. Since the entire plasmid pPCB is

retained in the new microorganism, a similar detailed plasmid map

of vector, plasmid pCL1, is also required in the submission.

(2) Figure 2 (gene cassette final construct without plasmid vector

sequence) and its legend is an example of another type of final

construct illustration. Chromosomal insertion would contain the

same level of detail and include information on sequences flanking

the site of the chromosomal insertion. In this figure, the dct

genes are from an intrageneric source (another species of

Rhizobium) and nifA, from Rhizobium meliloti, (same as the

recipient). The omega marker makes this new microorganism

intergeneric. The omega marker lacks the direction of
60

transcription in this example, although this information would be

useful.

The diagrams and descriptions in the two legends are not a

fixed format but a guide to the submitter.

2. Construction of Subject Microorganism -- Text discussion and

Flow diagram (Figure 3)

The original source of the final plasmid example in construct

analysis (flow diagram in Figure 3 and final construct in Figure 1)

is a Section 5 submission to OPPT of a microorganism containing a

recombinant plasmid pPCB for degradation of polychlorinated

biphenyls. The two diagram illustrations can be compared and

contrasted to the information provided in the open literature

citation by Lajoie, Layton and Sayler, 1994 (Appl. & Environm.

Microbiol., V60(8):2826-2833). 

In the flow diagram, sufficient circular plasmid maps have

been presented to clearly show the genetic manipulations through

the intermediate plasmids and vectors. Particularly important is

the size of intermediate plasmids/vectors and final plasmid

(pPCB)/cloning vector (pCL1). Since the map of vector pRK248 and

the restriction enzyme used to cut the vector for the construction

of pRK2501 are not available, the submitter should verify that the

EcoRI/SalI kanamycin marker fragment from pRK2501 used in the

construction of pRK293 contains only E. coli and RK2 sequences. The

lines on pRK290 and pRK2501 mark the location of restriction enzyme

fragment replacement to obtain the next plasmid, pRK293. Note the

size of the XhoI/HindIII fragment removed from E. coli Tn903

kanamycin resistance gene. In this case, having the nucleotide

sequence of the kanamycin resistance transposon Tn903 is very

helpful to verify the size of the kanamycin fragments deleted and

retained. The restriction enzymes for the 40 bp multiple

restriction site were identified. The partial EcoRI fragment

containing the A. eutrophus PCB genes cloned in plasmid pUC19 is

about 22 kb and that on plasmid pCL1 is about 12.4 kb. The map of

cloning vector, plasmid pCL1, is presented in the flow diagram but

the final plasmid pPCB is not since it is identical to the plasmid

final construct in Figure 2.
61

 

ATTACHMENT 4

SUMMARY ILLUSTRATIONS FOR CONSTRUCT ANALYSIS

Figure 1: Final plasmid construct illustration
62

Legend for Figure 1

Kp (360 bp) -- Region from Tn903 which includes kanamycin

promoter, one 18 bp repeat sequence, and 29 bp of

kanamycin structural gene (original host E. coli).

Bph genes -- A EcoRI Alcaligenes eutrophus DNA fragment,

approximately 12.4 kb) -- responsible for

degradation of polychlorinated biphenyls. The are 5

EcoRI restriction sites on the fragment, 3 of them

at internal positions. The first EcoRI site starts

at about 1.4 kb from bphA1 gene and the last EcoRI

site ends at <500 bp beyond bphD gene. The 3'

flanking sequence is essential for bphD gene

function. Approx. size: bphA (bphA1 1376 bp, bphA2

566 bp, bphA3 318 bp, and bphA4 1225 bp); bphB

830 bp; bphC 897 bp; bphD 861 bp; encodes

biphenyl dioxygenase; biphenyl-2,3-dihydrodiol-2,3-

dehydrogenase; biphenyl-2,3-diol-1,2-dioxygenase;

and 2-hydroxy-6-oxo-6-phenylhexa-2,3-dienoate

hydrolase respectively.

ORFs -- approx. Size: ORF0 738 bp, may involve in the

regulation of the bph operon; ORF1 416 bp, function

unknown; ORF2 3.5 kb, this region was characterized

in 1994 reference to contain closely spaced cistrons

(bphKHJI) encoding a glutathione S-transferase

(GST), a 2-hydroxypenta-2,4-dienoate hydrolase, an

acetaldehyde dehydrogenase (acylating) and a 4-

hydroxy-2-oxovalerate aldolase, respectively.

Km (549 bp) -- region from Tn903 which includes one 18 bp repeat,

and 265 bp of kanamycin structural gene. Region

also includes 34 bp of a synthetic MCS. 

Tc (approx. 2000 bp) -- functional tetracycline resistance gene.

pRK290 genes (approx. 20000 bp) -- plasmid RK2

sequence including Tc gene and a number of genes r

for plasmid maintenance genes including the

vegetative (OriV) and transfer (OriT) genes (pRK2

original host: K. Aerogenes).
63

ATTACHMENT 4

SUMMARY ILLUSTRATIONS FOR CONSTRUCT ANALYSIS

Figure 2: Final linear construct illustration
64

Legend for Figure 2

dctABD-- a 5.645 kb BglII/PstI Rhizobium leguminosarum

chromosomal DNA. dctABD, 4.8 kb, involves in

the C4 carboxylate transport important for

nitrogen fixation. DctBD are regulatory genes.

Bj nifD promoter-- 565 bp including approx. 200 bp E. coil pBR322

tetracycline resistance sequence upstream of

promoter.

Rm nifH leader-- 69 bp synthetic.

Rm nifA-- 1.6 kb coding sequence for the gene which

activates the production of nitrogenase enzyme

which is required for symbiotic nitrogen

fixation.

Omega fragment-- aadA gene, 2kb, specifying

streptomycin/spectinomycin resistances, derived

from antibiotic plasmid R100 of Shigella

flexneri. The gene is flanked by transcription

termination sequences from bacteriophage T4,

synthetic translation terminators and

polylinkers.

T1T2 terminator-- a 1.1 kb tandem repeat of two 500 bp DNA

fragment from E. coli rrnB gene which encode 5S

ribosomal RNA. The 1.1 kb includes a 100 bp

pBR322.
65

ATTACHMENT 4

SUMMARY ILLUSTRATIONS FOR CONSTRUCT ANALYSIS

Figure 3: Flow diagram illustration
66
67
68

Construction of Pseudomonas putida IPL5 (pPCB)

Text-Discussion for Fig. 3

The PCB-degradative genes (bphABCD) were cloned from

Alcaligenes eutrophus strain ENV307. Large fragments (20 to 30 kb)

from Sau3A partial digests of chromosomal DNA from ENV307 were

ligated into the BamHI site of the cosmid cloning vector pHC79

(Bethesda Research Laboratories, Gaithersburg, Md.). The cosmids

were then packaged into bacteriophage lambda with an in vitro

packaging system. The resulting lambda phage were used to

transduce E. coli MC1061, and ampicillin positive transductant

clones were selected. Clones containing PCB-degradative genes were

selected on the basis of their ability to convert 2,3-

dihydroxybiphenyl (DHB) to a yellow meta-cleavage product. The

partial EcoRI restriction enzyme fragment containing the PCB

degradative genes were subcloned into pUC19 which was cut with

EcoRI to give pUC19PCB. The partial EcoRI fragment containing the

subcloned PCB genes was about 22 kb. 

The plasmid backbone of pPCB plasmid is plasmid pCL1 which is

derived from cloning vector plasmid pRK290, a broad host plasmid. 

Plasmid pRK290 is derived from plasmid RK2, a 50 kb natural plasmid

in Klebsiella aerogenes. Plasmid RK2 is identical to plasmid RP1

and RP4 carried in Pseudomonas aeruginosa. During the development

of pRK290, the genes for plasmid self-transmission were removed so

that the plasmid can only be transferred to a new host by

triparental mating using a helper plasmid. The kanamycin

resistance gene from Tn903 was inserted into plasmid pRK290 to

create plasmid pRK293. This was achieved by replacing the small

EcoRI/SalI fragment on plasmid pRK290 with EcoRI/SalI kanamycin

fragment from plasmid pRK2501, another RK2-derived plasmid. 

Plasmid pRK2501 was obtained by cloning a 1.4 kb HaeII fragment,

containing the Tn903 kanamycin gene from plasmid pMK20, into

plasmid pRK248. Transposon Tn903 carrying the kanamycin resistance

gene was originally isolated from plasmid R6-5 onto plasmid pSC101

to obtained plasmid pSC105). Subsequently, the kanamycin

resistance gene was carried on pLM2, then on pCR1 , and finally on

pMK20. E. coli R6-5 is a spontaneous mutant of E. coli R6. The

complete sequence of Tn903 has been published. The 1.4 kb HaeII

fragment contains all of the kanamycin resistance gene and promoter

and 2 of the 4 18-bp inverted repeats. A 520 bp XhoI/HindIII

fragment was eliminated from the central region of the kanamycin

resistance gene on plasmid pRK293 and a 40-bp XhoI-HindIII fragment

from the multiple cloning site (MCS) of plasmid pGEM-7f(-) (Promega

Corp.) was inserted into the XhoI/HindIII site on plasmid pRK293 to

create the cloning vector (plasmid pCL1). The insertion of the

MCS leaves 360 bp Km fragment including one 18 bp repeat, the

kanamycin promoter, and 29 bp of coding and 549 bp Km fragment
69

including another 18 bp repeat and 265 bp of coding sequence. 

Plasmid pCL1 contains two EcoRI sites, one of which is in the MCS

downstream from the kanamycin promoter and is the site for the

insertion of the PCB degradative genes. Thus, the bph genes are

controlled by the kanamycin promoter. The other EcoRI site is on

the plasmid pRK290 vector near the oriV.

Plasmid pPCB was obtained by inserting partially EcoRIdigested pUC19 containing the PCB genes into partially EcoRI

digested plasmid pCL1. The size of Alcaligenes eutrophus DNA on

pCL1 is approximate 12.4 kb. The cloned PCB genes from strain

ENV307 have not been sequenced, but restriction mapping analysis

indicates that these genes are extremely similar if not identical

to the PCB genes from Pseudomonas LB400. The entire bph locus from

strain LB400 has been sequenced. 

Plasmid pPCB in E. coli DH5" was transferred to Pseudomonas

putida IPL5 by triparental mating using E. coli containing the

helper plasmid (plasmid pRK2013). The positive clones were

identified by tetracycline resistance and by turning yellow when

they were sprayed with DHB. Plasmid pPCB is maintained in the

recipient as an extrachromosomal plasmid. The new strain is

designated as Pseudomonas putida IPL5(pPCB).
70

ATTACHMENT 5

Diagram for Fermentation Facility Releases