[Federal Register: April 24, 2008 (Volume 73, Number 80)]
[Notices]
[Page 22186-22191]
[DOCID:fr24ap08-88]

-----------------------------------------------------------------------

SECURITIES AND EXCHANGE COMMISSION

[Release No. 34-57681; File No. SR-FINRA-2008-011]

Self-Regulatory Organizations; Financial Industry Regulatory
Authority, Inc.; Notice of Filing of Proposed Rule Change To Amend the
Trade Reporting Structure and Require Submission of Non-Tape Reports To
Identify Other Members for Agency and Riskless Principal Transactions

April 17, 2008.
    Pursuant to Section 19(b)(1) of the Securities Exchange Act of 1934
(``Act'') \1\ and Rule 19b-4 thereunder,\2\ notice is hereby given that
on March 28, 2008, the Financial Industry Regulatory Authority, Inc.
(``FINRA'') (f/k/a the National Association of Securities Dealers, Inc.
(``NASD'')) filed with the Securities and Exchange Commission
(``Commission'') the proposed rule change as described in Items I, II,
and III below, which Items have been substantially prepared by the
FINRA. The Commission is publishing this notice to solicit comments on
the proposed rule change from interested persons.
---------------------------------------------------------------------------

    \1\ 15 U.S.C. 78s(b)(1).
    \2\ 17 CFR 240.19b-4.
---------------------------------------------------------------------------

I. Self-Regulatory Organization's Statement of the Terms of Substance
of the Proposed Rule Change

    FINRA is proposing to amend its trade reporting rules applicable to
over-the-counter (``OTC'') equity transactions \3\ to: (1) Replace the
current market maker-based trade reporting framework with an
``executing party'' framework; and (2) require that any member with the
trade reporting obligation under FINRA rules that is acting in a
riskless principal or agency capacity on behalf of one or more other
members submit non-tape report(s) to FINRA, as necessary, to identify
such other member(s) as a party to the trade. The text of the proposed
rule change is available at FINRA, the Commission's Public Reference
Room, and http://www.finra.org.
---------------------------------------------------------------------------

    \3\ Specifically, OTC equity transactions are: (1) Transactions
in NMS stocks, as defined in Rule 600(b) of Regulation NMS under the
Act, effected otherwise than on an exchange, which are reported
through the Alternative Display Facility (``ADF'') or a Trade
Reporting Facility (``TRF''); and (2) transactions in ``OTC Equity
Securities,'' as defined in NASD Rule 6610 (e.g., OTC Bulletin Board
and Pink Sheets securities), Direct Participation Program (``DPP'')
securities and PORTAL equity securities, which are reported through
the OTC Reporting Facility (``ORF''). The ADF, TRFs and ORF are
collectively referred to herein as the ``FINRA Facilities.''
---------------------------------------------------------------------------

II. Self-Regulatory Organization's Statement of the Purpose of, and
Statutory Basis for, the Proposed Rule Change

    In its filing with the Commission, FINRA included statements
concerning the purpose of, and basis for, the proposed rule change and
discussed any comments it received on the proposed rule change. The
text of these statements may be examined at the places specified in
Item IV below. FINRA has prepared summaries, set forth in Sections A,
B, and C below, of the most significant aspects of such statements.

A. Self-Regulatory Organization's Statement of the Purpose of, and
Statutory Basis for, the Proposed Rule Change

1. Purpose
Trade Reporting Structure
    Currently, the following structure is in place for purposes of
reporting most OTC equity transactions to FINRA: (1) In transactions
between two market makers, the sell-side reports; (2) in transactions
between a market maker and a non-market maker, the market maker
reports; (3) in transactions between two non-market makers, the sell-
side reports; and (4) in transactions between a member and either a
non-member or customer, the member reports.\4\ This reporting structure
can result in confusion, delays and double-reporting, as the parties to
a trade attempt to determine which party has the trade reporting
obligation. Today, a firm's status as a market maker may not always be
apparent to the contra-party to a trade and, increasingly, firms'
proprietary desks (other than their market making desks) are handling
and executing transactions in equity securities. In addition, members
are required to report whether any applicable exception or exemption to
Rule 611 of Regulation NMS (the Order Protection Rule) applies to a
transaction, which is information that may not be readily known to the
party with the reporting obligation if it is not the executing broker
to the transaction, e.g., whether the executing broker has routed

intermarket sweep orders in compliance with Rule 611(b)(6).
---------------------------------------------------------------------------

    \4\ See NASD Rules 4632(b) and 6130(c) relating to the NASD/
Nasdaq TRF; 4632A(b) relating to the ADF; 4632C(b) and 6130C(c)
relating to the NASD/NSX TRF; 4632E(b) and 6130E(c) relating to the
NASD/NYSE TRF; and 6130(c) and 6620(b) relating to the ORF.
    For purposes of reporting transactions in DPP securities to
FINRA, NASD Rule 6920(b) requires that in a transaction between two
members, the member representing the sell-side report and in a
transaction between a member and customer, the member report.
---------------------------------------------------------------------------

    Accordingly, FINRA is proposing to adopt a simpler, more uniform
structure for purposes of reporting OTC equity transactions to FINRA.
Specifically, FINRA is proposing to amend NASD Rules 4632(b), 4632A(b),
4632C(b), 4632E(b), 6620(b) and 6920(b) to require that for
transactions between members, the ``executing party'' report the trade
to FINRA. For transactions between a member and a non-member or
customer, the member would report the trade.\5\
---------------------------------------------------------------------------

    \5\ In addition, FINRA is proposing to amend NASD Rules 6130(c),
6130C(c) and 6130E(c) to delete the duplicative rule provisions in
subparagraphs (1) through (4) and cross-reference NASD Rules 4632(b)
and 6620(b), 4632C(b) and 4632E(b), respectively.
    FINRA also notes that the proposed executing party reporting
structure would apply to the reporting of transactions in PORTAL
equity securities to FINRA. Pursuant to NASD Rule 6732(a)(3), the
member with the obligation to report such transactions to FINRA is
determined in accordance with NASD Rule 6620(b).
---------------------------------------------------------------------------

    FINRA is proposing to define ``executing party'' as the member that
receives an order for handling or execution or is presented an order
against its quote, does not subsequently re-route the order, and
executes the transaction. In certain limited circumstances, it may not
be clear which member should be deemed the executing party for trade
reporting purposes (e.g., manually negotiated trades via the
telephone). Accordingly, FINRA is proposing to require expressly that
for transactions between two members where both members may satisfy the
definition of executing party, the member representing the sell-side
shall report the transaction to FINRA, unless the parties agree
otherwise and the member representing the sell-side contemporaneously
documents such agreement. In such instances, the sell-side will be
presumed to be the member with the trade reporting obligation unless it
can demonstrate there was an agreement to the contrary, e.g.,
contemporaneous notes of a telephone conversation or notation on the
order ticket. FINRA believes that this approach will establish an
objective standard for determining the reporting obligation in these
circumstances, while affording the parties flexibility if, for example,
the member representing the buy-side is the party that knows the
material terms and details of the trade and thus is in the better
position to report the trade.
    Under the proposed rule change, alternative trade systems
(``ATSs''), including electronic communications networks (``ECNs''),
would be the executing party and have the reporting obligation where
the transaction is executed on the ATS. If an ATS routes an order to
another member for handling and/or execution, then the other member
would be the executing party and have the reporting obligation under
the proposed rule change. If an ATS routes an order to a non-member
that is executed OTC, then the ATS would report the trade. Accordingly,
FINRA is proposing to delete subparagraphs (5) through (7) from NASD
Rules 6130(c), 6130C(c) and 6130E(c) relating to trade reporting by a
``Reporting ECN.'' \6\ Under the current rules, a Reporting ECN is
required to ensure that trades are reported in accordance with one of
three enumerated methods and must notify FINRA in writing of the method
of reporting for each of its subscribers.\7\ FINRA notes that today,
most ATSs elect to report transactions to FINRA using the first
reporting method, i.e., the ATS submits the trade report and identifies
itself as the Reporting Party. Thus, FINRA believes that the proposed
rule change would clarify the reporting requirements for ATSs and would
better align the rules with current trade reporting practices.
---------------------------------------------------------------------------

    \6\ ``Reporting ECN'' generally is defined in NASD Rules 6110,
6110C and 6110E as an electronic communications network or
alternative trading system, as those terms are defined in SEC Rule
600(b) of Regulation NMS.
    \7\ FINRA notes that the three reporting methods apply only for
purposes of reporting trades to a TRF or the ORF. There is no
comparable provision relating to reporting trades to the ADF.
---------------------------------------------------------------------------

    Finally, FINRA is proposing to make certain technical conforming
changes, including to (1) delete NASD Rules 4632(b)(5), 4632C(b)(5),
4632E(b)(5), 6620(b)(5) and 6920(b)(3) relating to reporting by a
Reporting ECN; (2) delete the definitions of, and references to,
``Reporting ECN,'' ``Reporting Market Maker'' and ``Reporting Order
Entry Firm'' in NASD Rules 6110, 6110C and 6110E, which terms would be
obsolete as a result of the proposed rule change; and (3) amend NASD
Rules 6130(d)(5), 6130C(d)(5) and 6130E(d)(5) to replace the terms
``Market Maker side'' and ``Order Entry side'' with ``MMID or Reporting
Party side'' and ``OEID or non-Reporting Party side,'' respectively.
    FINRA believes that the proposed rule change would result in more
accurate and timely trade reporting and make the trade reporting
process less cumbersome for members. The proposed rule change would
ensure that the member with the trade reporting obligation is the party
that knows the material terms and details of the transaction, including
any exceptions or exemptions to the Order Protection Rule that may
apply to the trade. Furthermore, many members have entered into
agreements to permit the executing party to report on behalf of the
member with the reporting obligation under FINRA's current rules. Thus,
FINRA believes that, to a large extent, the proposed rule change would
be consistent with current trade reporting practices.
Submission of Non-Tape Reports To Identify Other Members for Agency and
Riskless Principal Transactions
    As a general matter, FINRA trade reporting rules require that a
member that is a party to an OTC trade be identified in trade reports
submitted to FINRA. Each trade report submitted for public
dissemination purposes (or ``tape report'') generally only allows for
the identification of two parties. Thus, where a FINRA member executes
a trade in a riskless principal \8\ or agency capacity on behalf of
another member, or matches, as agent, the orders of two or more
members, the tape report will not identify all members involved in the
trade. In such circumstances, additional ``non-tape reports,'' i.e.,
reports that are not submitted to the tape for public dissemination,\9\
would need to be submitted to identify all members involved in the
trade.
---------------------------------------------------------------------------

    \8\ For purposes of FINRA trade reporting rules applicable to
equity securities, a ``riskless principal'' transaction is a
transaction in which a member, after having received an order to buy
(sell) a security, purchases (sells) the security as principal and
satisfies the original order by selling (buying) as principal at the
same price.
    \9\ Non-tape reports can be (1) ``non-tape, non-clearing,''
meaning that the report is submitted to FINRA solely for regulatory
purposes, or (2) ``clearing-only,'' meaning that the report is
submitted to FINRA for clearing, i.e., for submission by FINRA to
the National Securities Clearing Corporation (and perhaps also
regulatory purposes).
---------------------------------------------------------------------------

    Today, some members submit non-tape reports to FINRA identifying
the other members involved in the trade, while other members do not.
FINRA trade reporting rules generally are not specific in this regard
because, for the most part, they reflect the traditional two-party
trade model where a broker-dealer acts as principal or as agent for a
non-broker-dealer customer. Industry business models have evolved to
include more trades where one broker-dealer acts as agent or riskless
principal for another broker-dealer and order management systems and
ATSs can simultaneously match one or more broker-dealer orders on one
or both sides of a trade.
    To address these changes, FINRA is proposing to adopt NASD Rules
4632(d)(4), 4632A(e)(1)(D), 4632C(d)(4), 4632E(d)(4), 6620(d)(4) and
6920(d)(5) to require that any member with the

obligation to report the trade under FINRA rules that is acting in a
riskless principal or agency capacity on behalf of one or more other
members submit to FINRA one or more non-tape report(s) identifying such
other member(s) as a party to the transaction, if such other member(s)
is not identified on the initial trade report or a report submitted to
FINRA to reflect the offsetting leg of a riskless principal
transaction. In addition, FINRA is proposing to amend NASD Rule
6732(a)(3), which currently cross-references the trade reporting
structure in NASD Rule 6620(b), to also cross-reference NASD Rule
6620(d), thereby making the proposed reporting requirement applicable
to PORTAL equity security transactions. A member that matches, as
agent, the orders of multiple members on one or both sides of the trade
would be required to submit multiple non-tape reports, as necessary, to
identify all members on whose behalf the member was acting.
    For example, where Member A, as agent or riskless principal on
behalf of Member B, executes an OTC trade with Member C, and Member A
has the obligation to report the trade to FINRA, Member A also would be
required to submit a non-tape report to FINRA to indicate that it was
acting on behalf of Member B. By way of further example, where Member A
matches, as agent, the orders of Member B and Member C and submits to
FINRA a tape report between Member A and Member C, Member A also would
be required to submit a non-tape report to identify Member B as a party
to the trade. In this example, if Member A were to report the trade to
the tape as an agency cross (such that neither Member B nor Member C is
identified on the tape report), then Member A would be required to
submit two non-tape reports to identify Members B and C. In these
examples, Member A can satisfy its reporting obligation under the
proposed rule change by submitting a clearing-only report, if necessary
to clear the offsetting leg(s) of the transaction through a FINRA
Facility. However, if the parties do not need to clear the offsetting
leg(s) of the transaction through a FINRA Facility, then Member A would
be required to submit a non-tape, non-clearing report(s). Additionally,
if Member A is required to submit a non-tape report to comply with
applicable riskless principal reporting requirements under FINRA rules
\10\ and such report identifies Member B, then Member A would have no
separate reporting obligation under the proposed rule change.
---------------------------------------------------------------------------

    \10\ If an OTC riskless principal transaction is not reported to
FINRA in a single tape report properly marked as riskless principal,
then two separate reports must be submitted: (1) A tape report to
reflect the initial leg of the transaction and (2) a non-tape report
to reflect the offsetting, ``riskless'' leg of the transaction, with
the correct capacity of riskless principal. See NASD Rules
4632(d)(3)(B), 4632A(e)(1)(C)(ii), 4632C(d)(3)(B), 4632E(d)(3)(B)
and 6620(d)(3)(B).
---------------------------------------------------------------------------

    The proposed reporting requirement would only apply to the member
that has the responsibility under FINRA rules to report the trade to
FINRA (i.e., the ``executing party'' in a trade between two members, as
discussed above). For example, where Member A, as agent on behalf of
Member B, and Member C execute an OTC trade, and Member C has the
obligation to report the trade to FINRA, Member A would not be required
under the proposed rule change to submit a non-tape report to indicate
that it was acting on behalf of Member B.
    However, the proposed rule change expressly would not negate or
modify the requirements for reporting riskless principal transactions
under FINRA rules. Thus, drawing on the example in the paragraph above,
if Member A is acting as riskless principal (as opposed to agent) on
behalf of Member B, Member A currently is required to submit a non-tape
report to reflect the offsetting leg of the transaction under FINRA
riskless principal rules, if the tape report does not properly reflect
Member A's capacity as riskless principal.\11\ This requirement would
not change under the proposed rule change. Additionally, the proposed
rule change would not change the reporting requirements applicable to
riskless principal transactions with a customer.
---------------------------------------------------------------------------

    \11\ If Member A's capacity is properly marked as riskless
principal on the tape report, Member A would not be required to
submit a non-tape report to FINRA.
---------------------------------------------------------------------------

    FINRA notes that the proposed reporting requirement would not apply
to transactions that are executed on and reported through an exchange.
Today, where the initial leg of a riskless principal or agency
transaction is executed on an exchange, members are not required to
report either leg of the transaction to FINRA. The initial leg of the
transaction is reported through the exchange (and therefore must not be
reported to FINRA), and members have the option of submitting a non-
tape (typically, a clearing-only) report to FINRA for the offsetting
leg of the transaction. Pursuant to the proposed rule change, members
would continue to have the option of submitting a non-tape report for
riskless principal and agency transactions where the initial leg is
executed on an exchange; however, there would continue to be no
obligation to submit a non-tape report for such trades. Thus, for
example, where Member A, as agent or riskless principal on behalf of
Member B, executes a trade on an exchange, the trade will be reported
to the tape by the exchange and, under the proposed rule change, Member
A would not be required to submit a non-tape report to FINRA to
indicate that it was acting on behalf of Member B. However, Member A
would be permitted to submit a clearing-only report to clear the
offsetting leg of the transaction between Member A and Member B through
a FINRA Facility.\12\
---------------------------------------------------------------------------

    \12\ See FINRA Regulatory Notice 07-38 (August 2007).
---------------------------------------------------------------------------

    To clarify the scope and application of the proposed reporting
requirement, FINRA is proposing to include several examples in the
proposed rule text. FINRA notes that these examples are not intended to
represent all possible trade reporting scenarios under the proposed
rule change. Additionally, consistent with the definition of ``riskless
principal'' in other FINRA rules applicable to OTC equity trade
reporting, FINRA is proposing to amend the definition of ``riskless
principal transaction'' in NASD Rule 6910 to clarify that a member may
act in a riskless principal capacity on behalf of another broker-dealer
as well as a customer.\13\
---------------------------------------------------------------------------

    \13\ FINRA also is proposing a technical change to insert
paragraph headings for ease of reference in NASD Rules 4632(d),
4632A(e)(1), 4632C(d), 4632E(d), 6620(d) and 6920(d).
---------------------------------------------------------------------------

    Finally, FINRA notes that because members would be submitting non-
tape reports, the 90-second reporting requirement under FINRA trade
reporting rules would not apply. Thus, members generally would have
until the end of the day on trade date to submit the requisite non-tape
reports.\14\
---------------------------------------------------------------------------

    \14\ In certain circumstances, however, members must submit non-
tape reports contemporaneously with trade execution, e.g., to
qualify for the exemption from the requirements of IM-2110-2
(Trading Ahead of Customer Limit Order) for riskless principal
transactions.
---------------------------------------------------------------------------

    FINRA believes that the proposed rule change would enhance FINRA
staff's ability to create a complete and accurate audit trail and
assist in the automated surveillance of various customer protection and
market integrity rules.
    Many members today submit clearing-only reports to FINRA in
instances where the proposed reporting requirement would apply, e.g.,
if a member needs to clear the offsetting leg of an agency transaction
through a FINRA Facility or if a member elects under FINRA rules to
report an OTC riskless principal trade in related tape and non-tape
reports. Thus, for some

members, the proposed rule change may not require any changes to
current reporting practices and systems. For other members, however,
the proposed rule change would require systems changes, e.g., if a
member does not need to clear the offsetting leg of an agency
transaction through a FINRA Facility. Additionally, where a member
reports a riskless principal transaction to FINRA in a single properly
marked tape report, a non-tape report would be required under the
proposed rule change if the member is acting on behalf of another
member.
    FINRA will announce the operative date of the proposed rule change
on its website. In recognition of the technological changes that the
proposed rule change will require, the operative date will be (1) at
least 90 days following Commission approval for transactions executed
on ATSs, including electronic communications networks; and (2) at least
180 days following Commission approval with respect to all other
transactions. FINRA believes that a shorter implementation period is
appropriate for ATSs because, as noted above, most ATSs currently are
the reporting party for transactions executed on the ATS and some
voluntarily submit non-tape reports to reflect all FINRA members that
are parties to a trade.
2. Statutory Basis
    FINRA believes that the proposed rule change is consistent with the
provisions of Section 15A(b)(6) of the Act,\15\ which requires, among
other things, that FINRA rules be designed to prevent fraudulent and
manipulative acts and practices, to promote just and equitable
principles of trade, and, in general, to protect investors and the
public interest. FINRA believes that the proposed rule change to amend
the trade reporting structure will result in more accurate and timely
trade reporting and thus enhance market transparency. Additionally,
FINRA believes that the proposed rule change to require the submission
of non-tape reports to identify other members for agency and riskless
principal transactions will promote a more complete and accurate audit
trail.
---------------------------------------------------------------------------

    \15\ 15 U.S.C. 78o-3(b)(6).
---------------------------------------------------------------------------

B. Self-Regulatory Organization's Statement on Burden on Competition

    FINRA does not believe that the proposed rule change will result in
any burden on competition that is not necessary or appropriate in
furtherance of the purposes of the Act.

C. Self-Regulatory Organization's Statement on Comments on the Proposed
Rule Change Received From Members, Participants or Others

    In September 2007, FINRA published Regulatory Notice 07-46
(``Notice'') soliciting comment on a proposal to adopt a simpler and
more uniform trade reporting structure. Nine comment letters were
received in response to the Notice.\16\
---------------------------------------------------------------------------

    \16\ See Letters from Liquidnet, Inc., to Office of the
Corporate Secretary, FINRA, dated October 26, 2007 (``Liquidnet'');
Archipelago Trading Services, Inc., to Barbara Z. Sweeney, Office of
the Corporate Secretary, FINRA, dated November 6, 2007
(``ArcaEdge''); Financial Information Forum, to Barbara Z. Sweeney,
Office of the Corporate Secretary, FINRA, dated November 8, 2007
(``FIF''); Pipeline Trading Systems LLC, to Barbara Z. Sweeney,
Office of the Corporate Secretary, FINRA, dated November 12, 2007
(``Pipeline''); Automated Trading Desk, LLC, to Barbara Z. Sweeney,
Office of the Corporate Secretary, FINRA, dated November 12, 2007
(``ATD''); TD AMERITRADE, Inc., to Barbara Z. Sweeney, Office of the
Corporate Secretary, FINRA, dated November 15, 2007 (``TD
AMERITRADE''); UBS Securities LLC, to Barbara Z. Sweeney, Office of
the Corporate Secretary, FINRA, dated November 15, 2007 (``UBS'');
The Securities Industry and Financial Markets Association, Barbara
Z. Sweeney, Office of the Corporate Secretary, FINRA, dated November
16, 2007 (``SIFMA''); and BNY ConvergEx Execution Solutions LLC,
Charles Schwab & Co., Inc., National Financial Services LLC and
Pershing LLC, to Barbara Z. Sweeney, Office of the Corporate
Secretary, FINRA, dated November 30, 2007 (``BNY'').
---------------------------------------------------------------------------

    All of the commenters support the adoption of a new trade reporting
structure, asserting that the current structure can be confusing and
create delays and reporting errors. Seven of the nine commenters
support the proposed executing party reporting structure, asserting
that this structure is the most logical and efficient approach.\17\
These commenters assert that the executing party knows the material
terms and details of the transaction, as well as any Order Protection
Rule exceptions or exemptions that apply to the trade,\18\ and thus is
in the best position to report in a timely manner \19\ and to correct
reporting errors.\20\ In addition, several commenters note that
industry practice is for executing parties to trade report; most
executing parties already have established systems to trade report and
many firms have entered give-up agreements to replicate the executing
party reporting structure.\21\
---------------------------------------------------------------------------

    \17\ FIF, Pipeline, ATD, TD AMERITRADE, UBS, SIFMA and BNY.
    \18\ FIF, ATD, UBS and SIFMA.
    \19\ Pipeline and UBS.
    \20\ ATD.
    \21\ ATD, TD AMERITRADE and BNY.
---------------------------------------------------------------------------

    One commenter states that it is unclear whether the advantages of
Qualified Service Representative (QSR) agreements would remain under
the proposed executing party reporting structure and strongly urges
that any changes continue to keep the QSR process intact.\22\ FINRA
notes that a QSR agreement is a National Securities Clearing
Corporation agreement and, for FINRA purposes, merely establishes that
one party can send a trade to clearing on behalf of the other party. A
give up agreement still is required for a member to report trade
information to a FINRA Facility on behalf of another member, even if
the parties have a QSR agreement in effect.\23\ This proposed rule
change would not change the QSR process or member obligations with
respect to give up agreements.
---------------------------------------------------------------------------

    \22\ TD AMERITRADE.
    \23\ See NASD Member Alert: Notice to All TRF, ADF and Other
NASD Facility Participants Regarding AGU and QSR Relationships
(January 25, 2007).
---------------------------------------------------------------------------

    In the Notice, FINRA specifically requested comment on how
``executing party'' should be defined. The commenters generally suggest
that the ``executing party'' should be defined as the party that
receives the order electronically for execution, does not subsequently
re-route the order, and agrees to execute the trade, or in other words,
the broker that is the ``final recipient'' and determines the
price.\24\ One commenter states that in the electronic marketplace, the
identity of the order entry broker generally will be readily apparent
based on which party is initiating or seeking an execution, and the
executing party's identity will be equally apparent based on which
party is receiving the order for execution.\25\ This commenter provides
the following example: A displays a limit order to sell 100 shares at
$10. B routes an order to buy 100 shares against A's displayed order.
In this example, it is clear that A is the executing broker and B is
the order entry broker; B initiated and sought out an execution against
A's displayed limit order.\26\ As discussed above, FINRA is proposing
to define ``executing party'' substantially as proposed by these
commenters.
---------------------------------------------------------------------------

    \24\ FIF, ATD, UBS, SIFMA and BNY.
    \25\ ATD.
    \26\ ATD.
---------------------------------------------------------------------------

    In instances of telephone orders, three commenters believe that the
same approach should be followed (i.e., the executing party is the
``answering'' or ``receiving'' or ``responding'' broker), unless the
parties agree to the contrary.\27\ One commenter believes that in the
case of telephone trades, the sell-side member should be the reporting
party,\28\ while another commenter

asserts that the current trade reporting structure should apply in such
instances.\29\ Additionally, one commenter asserts that the executing
party may not be clear when a member requests a quote from another
member, receives a quote and then agrees to trade at the quoted price,
and suggests that the member responding to the request for a quote
(i.e., the price-making firm) should be deemed the executing party.\30\
As discussed above, FINRA is proposing to require that where it may be
difficult to determine which member satisfies the definition of
``executing party,'' such as telephone and other manually negotiated
trades, the member representing the sell-side report, unless the
parties agree otherwise. Several commenters note that in today's
market, the number of telephone negotiated trades is relatively small
compared to the number of trades involving the routing of electronic
orders, and thus the instances where it would not be clear which member
is the executing party should be limited.\31\ In the words of one
commenter, ``[a]ll but a tiny fraction of orders in the current
marketplace are routed electronically'' and as such, ``in the vast
majority of transactions, there is no doubt about which entity is the
Executing Broker.'' \32\
---------------------------------------------------------------------------

    \27\ ATD, SIFMA and BNY.
    \28\ FIF.
    \29\ UBS.
    \30\ BNY.
    \31\ UBS, SIFMA and BNY.
    \32\ BNY.
---------------------------------------------------------------------------

    Two commenters support a sell-side reporting structure, whereby the
member representing the sell-side would report a trade between
members.\33\ One commenter asserts that in all cases, it would be clear
which party is selling and which party is buying, but the distinction
between the executing party and introducing broker could be unclear in
certain cases.\34\ FINRA disagrees and believes that where Member A, an
introducing broker, routes an order for handling and/or execution to
Member B, and Member B does not re-route the order and executes the
trade, it is clear that Member B is the executing party. This commenter
also asserts that in a trade between two brokers, the selling broker
should be the reporting party, but the brokers should have full
flexibility to override this default rule and designate the buyer as
the reporting party.\35\ FINRA believes that the determination of which
member has the trade reporting obligation should not be subject to
agreement between the parties, except in limited circumstances as
discussed above, as that approach would result in confusion and
possible under or double reporting. FINRA notes, however, that members
can enter into give up agreements under FINRA rules, whereby one member
can trade report on behalf of the other member, while the member with
the reporting obligation under FINRA rules remains responsible for
trades submitted on its behalf.
---------------------------------------------------------------------------

    \33\ Liquidnet and ArcaEdge.
    \34\ Liquidnet.
    \35\ Liquidnet.
---------------------------------------------------------------------------

    The second commenter supports sell-side reporting in light of the
problems with the current market maker-based reporting structure,
noting that these problems are compounded in the context of ATS trades,
where non-subscribers may not recognize that the reporting
responsibility lies with the ATS.\36\ As discussed above, under the
proposed executing party structure, it would be clear that an ATS has
the reporting responsibility where the trade is executed on the ATS.
---------------------------------------------------------------------------

    \36\ ArcaEdge.
---------------------------------------------------------------------------

    The commenters opposing the sell-side reporting structure assert
that this approach would be less efficient and could increase the rate
of unreported or inaccurately reported trades.\37\ These commenters
further assert that a sell-side broker that is not also the executing
party will not have access to necessary information, such as exceptions
and exemptions under the Order Protection Rule, may not be able to
easily obtain this information and will not be able to independently
verify this information.\38\ Additionally, another commenter asserts
that while an originating broker would be the seller if its sale were
executed by the first broker to whom it routed its orders, frequently
re-routed orders could make it difficult to determine which party has
the reporting responsibility under a sell-side structure.\39\
Furthermore, the commenters assert that a sell-side reporting structure
would be costly because it would require members that currently do not
trade report to implement trade reporting systems.\40\ FINRA agrees
with these commenters, and as discussed above, is proposing to adopt
the executing party trade reporting structure.
---------------------------------------------------------------------------

    \37\ FIF, Pipeline and BNY.
    \38\ FIF, SIFMA and BNY.
    \39\ Pipeline.
    \40\ TD AMERITRADE and BNY.
---------------------------------------------------------------------------

III. Date of Effectiveness of the Proposed Rule Change and Timing for
Commission Action

    Within 35 days of the date of publication of this notice in the
Federal Register or within such longer period (i) as the Commission may
designate up to 90 days of such date if it finds such longer period to
be appropriate and publishes its reasons for so finding or (ii) as to
which FINRA consents, the Commission will:
    (A) By order approve such proposed rule change, or
    (B) Institute proceedings to determine whether the proposed rule
change should be disapproved.

IV. Solicitation of Comments

    Interested persons are invited to submit written data, views, and
arguments concerning the foregoing, including whether the proposed rule
change is consistent with the Act. Comments may be submitted by any of
the following methods:

Electronic Comments

     Use the Commission's Internet comment form (http://
www.sec.gov/rules/sro.shtml); or
     Send an e-mail to<EMAIL_ADDRESS>Please include
File Number SR-FINRA-2008-011 on the subject line.

Paper Comments

     Send paper comments in triplicate to Nancy M. Morris,
Secretary, Securities and Exchange Commission, 100 F Street, NE.,
Washington, DC 20549-1090.

All submissions should refer to File Number SR-FINRA-2008-011. This
file number should be included on the subject line if e-mail is used.
To help the Commission process and review your comments more
efficiently, please use only one method. The Commission will post all
comments on the Commission's Internet Web site (http://www.sec.gov/
rules/sro.shtml). Copies of the submission, all subsequent amendments,
all written statements with respect to the proposed rule change that
are filed with the Commission, and all written communications relating
to the proposed rule change between the Commission and any person,
other than those that may be withheld from the public in accordance
with the provisions of 5 U.S.C. 552, will be available for inspection
and copying in the Commission's Public Reference Room, 100 F Street,
NE., Washington, DC 20549, on official business days between the hours
of 10 a.m. and 3 p.m. Copies of the filing also will be available for
inspection and copying at the principal office of FINRA. All comments
received will be posted without change; the Commission does not edit
personal identifying information from submissions. You should submit
only information that you wish to make

available publicly. All submissions should refer to File Number SR-
FINRA-2008-011 and should be submitted on or before May 15, 2008.

    For the Commission, by the Division of Trading and Markets,
pursuant to delegated authority.\41\
---------------------------------------------------------------------------

    \41\ 17 CFR 200.30-3(a)(12).
---------------------------------------------------------------------------

Nancy M. Morris,
Secretary.
[FR Doc. E8-8872 Filed 4-23-08; 8:45 am]

BILLING CODE 8010-01-P


1 UNITED STATES ENVIRONMENTAL PROTECTION AGENCY WASHINGTON, D. C. 20460 JAN 30 2004 OFFICE OF PREVENTION, PESTICIDES AND TOXIC SUBSTANCES MEMORANDUM SUBJECT: Review of Product Characterization and Safety Data for Phosphomannose Isomerase, an Inert Marker Gene from Syngenta Seeds, Inc.

TO: Mike Mendelsohn Regulatory Action Leader Microbial Pesticides Branch, Biopesticides and Pollution Prevention Division ( 7511C)

FROM: John L. Kough, Ph. D., Biologist Microbial Pesticides Branch, Biopesticides and Pollution Prevention Division ( 7511C)

ACTION REQUESTED: To review the data submitted for a determination of dietary safety for phosphomannose isomerase ( PMI) an inert ingredient ( or marker gene) in plant incorporated protectant. s.

CONCLUSION: The data presented shows a lack of acute oral toxicity at a high dose of purified PMI ( 3080mg/ kg BW). The PMI protein was also shown to be rapidly degraded by simulated gastric fluid or simulated intestinal fluid and to lose significant enzymatic activity when incubated at 65

C. PMI is not derived from a source known to have induced allergy and has no significant amino acid sequence homology to known toxins or allergens. These data suggest that the is a reasonable certainity of no harm resulting from the cumulative exposure to PMI.

DATA REVIEW RECORD Active Ingredient: Cry1Ab protein and PMI protein marker gene in Event 3243M Product Name: Event 3243M derived Cry1Ab Corn Company Name: Syngenta Seeds, Inc., Research Triangle Park, NC 2 Company Number: 67979 Petition No: 3E6748 Chemical Number: 706505 Decision Number: 330482 DP Number: 292501 MRID No: 459344 02; 459344 05 thru 09 BACKGROUND: The phosphomannose isomerase ( PMI) is a new marker gene employing unusual carbohydrate metabolism to allow for selection of transformants in cell culture. Use of this marker addresses some of the complaints received from the public about the possible adverse effects of using antibiotic resistance genes as selection markers.

DISCUSSION: The data relating the PMI protein expressed in corn to the bacterially expressed protein had one curious difference. The bacterial protein was expressed with an additional 17 amino acids ( a 14 AA T7 tag plus 3 AA from a polylinker region) compared to that stated as being in the plant. While the N termianl tag would not be expected to alter the toxicity of PMI, it is unclear why there was no detectable difference between these two forms in SDS PAGE.

RECOMMENDATION: The company should identify the 92 known and hypothetical proteins that are stated in MRID 459344 05 as sharing significant homology with PMI. This should include the known source of these proteins as well as any activities or functions that are associated with them. It would also be helpful for the company to explain the sensitivity of the SDS PAGE/ western blot assay to detect the 17 AA difference between the bacterial and plant expressed proteins.

SUMMARY OF DATA SUBMITTED:

459344 02 This is a summary of the data provided elsewhere on phosphomannose isomerase ( PMI) enzyme and the genetic material required for production of this protein in transgenic corn plants derived from transformation event 3243M. PMI is a ubiquitous, essential enzyme from a nonallergenic source and has conceivably always been in the human diet in low quantities. The results of the equivalence studies indicate similar molecular weight by SDS PAGE between the corn silk expressed PMI and that produced in an E. coli expression vector. This is somewhat unusual in that the E. coli form of PMI contained an additional 17 amino acids which should be distinguishable by western blot analysis. The enzymatic activity is also similar based on the detectable level of PMI by ELISA. These data suggest that the two sources produce PMI protein that is similar enough that the bacterial PMI source can be used for further biochemical and toxicological tests to establish dietary safety. CLASSIFICATION: Acceptable.

459344 05 The PMI amino acid sequence showed no significant homology to known protein toxins. CLASSIFICATION: Supplemental. Can be upgraded to acceptable with identification of the 92 known and hypothetical proteins identified as having significant amino acid sequence homology to PMI along with the source of those proteins. If the PMI homologues have other activities or 3 functions these should also be listed when identifying the 92 proteins.

459344 06 The PMI protein showed no significant amino acid homology with known or putative allergenic proteins using either an 8 amino acid sequence stepwise comparison or and an 80 amino acid fragment comparison. CLASSIFICATION: Acceptable.

459344 07 The oral LD 50 of PMI 0198 protein for males, females, and combined was greater than 3080 mg/ kg. CLASSIFICATION: ACCEPTABLE  TOXICITY CATEGORY III 459344 08 Full length PMI 0198 protein was degraded to undetectable levels in Coomassie blue stained SDS PAGE gels after incubation in simulated gastric and intestinal fluids. CLASSIFICATION: Acceptable.

459344 09 The phosphomannose isomerase ( PMI) enzyme derived from E. coli was prepared at 0.44 mg/ mL in standard buffer ( 50 mM Tris HCl, pH 7.0) and incubated at ambient temperature ( 25

C), 37, 55, 65 and 95

C for 30 minutes. PMI enzymatic activity was monitored by measuring NADPH production using a spectrophotometer. Results indicate that incubation at ambient temperature ( 25

C) and at 37 and 55

C for 30 minutes had little effect on PMI. However, incubation at 65 and 95

C for 30 minutes inactivated PMI. CLASSIFICATION: Acceptable. 4 DATA EVALUATION RECORD ________________________________________________________________________________ Reviewer: Anthony Q. Armstrong, M. S., Oak Ridge National Labs EPA Reviewer: John L. Kough, Ph. D., Biologist ________________________________________________________________________________ STUDY TYPE: Product Identity [ Selectable Marker Phosphomannose Isomerase ( PMI) Characterization and Safety] MRID NO: 459344 02 DP BARCODE: D292501/ 3E6748 TEST MATERIAL: Cry1Ab and PMI ( Corn Event 3243M) PROJECT NO: NA SPONSOR: Syngenta Seeds, Inc., 3054 Cornwallis Road, RTP, North Carolina 27709 2257 TESTING FACILITY: Syngenta Seeds, Inc., 3054 Cornwallis Road, RTP, North Carolina 27709 2257 TITLE OF REPORT: Characterization and Safety of Phosphomannose Isomerase ( PMI), a Selectable Marker Expressed in Event 3243M Derived Maize ( Corn) Plants AUTHOR: Demetra Vlachos and Robert Joseph STUDY COMPLETED: April 28, 2003 GOOD LABORATORY PRACTICE: Non GLP compliant CONCLUSION: This is a summary of the data provided elsewhere on phosphomannose isomerase ( PMI) enzyme and the genetic material required for production of this protein in transgenic corn plants derived from transformation event 3243M. PMI is a ubiquitous, essential enzyme from a non allergenic source and has conceivably always been in the human diet in low quantities. The results of the equivalence studies indicate similar molecular weight by SDS PAGE between the corn silk expressed PMI and that produced in an E. coli expression vector. This is somewhat unusual in that the E. coli form of PMI contained an additional 17 amino acids which should be distinguishable by western blot analysis. The enzymatic activity is also similar based on the detectable level of PMI by ELISA. These data suggest that the two sources produce PMI protein that is similar enough that the bacterial PMI source can be used for further biochemical and toxicological tests to establish dietary safety. CLASSIFICATION: Acceptable.

This submission by Syngenta summarizes data and information relevant to the characterization and safety of the phosphomannose isomerase ( PMI) enzyme and the genetic material required for production of this protein in transgenic corn plants derived from transformation event 3243M. PMI is considered an inert ingredient with respect to pesticidal activity and is present in plants as a selectable marker. The company stated that it is not feasible to extract sufficient PMI protein directly from Event 3243 M plants to conduct safety studies. Therefore, PMI was produced in a 5 recombinant E. coli over expression system for testing purposes and the bacterially produced substance was compared to the plant version.

The results of the studies showing the equivalence of the bacterial and plant produced protein are found in appendix A in this submission and discussed below.

Comparison of PMI protein produced in event 3243 corn and recombinant E. coli ( appendix A) Proteins purified from corn silks and E. coli expression systems were compared by apparent size, immunoreactivity and enzymatic activity. The corn silks were collected from event 3243M derived 10 week old corn ( genotype H8431 x( NP2499 x NP2500) 3243M) and shipped to Syngenta Seeds. The silks were powdered with a mortar and pestle in liquid nitrogen, lyophilized and labeled 3243MPMI silk. Similar corn silk powders were prepared from two samples of isogenic but transgenic negative corn. The bacterial protein was labeled PMI 0198 and consisted of a purified preparation isolated from E. coli strain BLRDE3 transformed with the pET 3a vector containing the manA ( pmi) gene. The PMI protein expressed is similar to the corn protein except for an additional N terminal 13 amino acids in the T7 Tag   and 3 amino acids from the poly linker sequence.

The bacterial PMI was purified by differential ammonium sulfate precipitation followed by hydrophobic interaction chromatography ( phenyl sepharose or toyopearl ether packed columns) then ion exchange chromatography. The appropriate collected fractions were pooled prior to dialysis against a 50mM NH 4 HCO 3 pH 8.0 buffer then subjected to lyophilization. The protein content of the lyophilized product was found to be approximately 93% PMI (

45,000 molecular weight, enzymatically active) and was given the designation PMI 0198. The lyophilized prep was stored at  20

C.

One tenth gram of the dry silk tissue preparations was suspended in 3 ml. of extraction buffer ( 500mM CAPS, 1M NaCl, 20 mM EDTA, 1mM AEBS, 2mM DTT, 1 µ M leupeptin, pH 10.0) and further extracted with a polytron homogenizer. This extract was centrifuged at 10,000 x g for 15 minutes. The supernatant was used for further PMI comparison testing by ELISA, western blot and enzyme activity. Protein concentration of these silk extracts were determined by the BCA method after iodoacetamide treatement. The concentration of PMI enzyme in the preps was determined by ELISA using protein A purified rabbit polyclonal IgG and an immunoaffinity purified goat antibody preparation specific for PMI.

The PMI containing samples from corn silk were separated by SDS PAGE followed by electroblotting. The sample amounts from the silk extracts loaded into the wells were matched for apparent PMI concentration ( 5ng) with those of the bacterial preps employing the results of the ELISA test. A similar amount of non transgenic corn silk extract ( 194 µ g) was loaded as a control in an adjacent well. After blotting, the membrane was probed with immunoaffinity purified goat antibody generated with PMI from an E. coli over expressing the manA gene. The blot was visualized by reaction with a rabbit anti goat HRP conjugate and developed with a chemiluminescent substrate.

PMI enzymatic activity was measured by an SOP not included that measured the production of NADPH from the conversion of mannose 6 phosphate to 6 gluconolactone and NADPH in the 6 presence of phosphoglucose isomerase and glucose 6 phospahte dehydrogenase. This NADPH product was measured by absorbance change at 340nm ( molar extinction coefficient 6.2). A unit of PMI activity is defined as 1 µ mole of NADPH reduced per minute.

The results of the western blot indicate that the PMI enzyme purified from both corn silk tissue and E. coli are of similar molecular weight and immunoreactivity ( see attached blot). The enzymatic activity results indicate similar activity levels per unit weight of PMI enzyme concentration ( 109.78 + 3.55units/ mg PMI in silk extract and 93.21 + 18.23 units / mg PMI in E. coli extract).

Source and Function of PMI Protein in Event 3243M Plants The PMI protein produced in Event 3243M derived plants is encoded by the native pmi gene ( also referred to elsewhere as the " manA" gene) from E. coli ( strain K 12). The pmi gene ( GenBank Accession NO. M15380; NCBI, 2003) encodes a protein ( Genbank Accession No. AAA24109.1) of 391 amino acids and ca. 45,000 molecular weight. PMI catalyzes the reversible interconverison of mannose 6 phosphate and fructose 6 phosphate. The PMI reaction is specific for mannose 6 phosphate and fructose 6 phosphate with a Keq near 1.0.

Selection of PMI Expressing Plant Cells by Mannose Plant cells that produce PMI are able to convert mannose to a readily metabolized compound, fructose 6 phosphate thus improving the energy status of cells and avoiding the accumulation of derivatized mannose. PMI has been shown to be an effective selectable marker via multiple transformation methods in several plants including maize, wheat, barley, sugar beets, tomato, rice and cassava. The use of PMI as a selectable marker and mannose as the sole carbon source and selective agent offers an efficient alternative selection system for a number of plant species to the more traditional markers that confer resistance to antibiotics or herbicides. However, use of this selection marker is limited to those species that do not already express a native PMI enzyme.

Lack of Unintended Effects of PMI Expression in Plants Expression of the pmi gene in transformed plants does not appear to adversely affect plant morphology, growth or agronomic characteristics in data referred to for corn and sugar beets. Studies indicate that the presence of PMI activity in transformed plants does not affect the concentration of simple carbohydrates.

PMI Enzymes in Other Organisms PMI enzymes of varying degrees of amino acid homology occur widely among prokaryotes and eukaryotes. Although PMI has not been detected in some plants, PMI enzymes have been found in such diverse plant species as tobacco, pine, walnut, lily bulbs, Brassica and Arabidopsis as well as soybeans and other legumes. Putative genes for PMI have been found in rice and guar. PMI enzymes have also been purified and characterized from many other organisms including bacteria, yeast, rats, pigs and humans. There are three classes of PMI enzyme with the subject of this review being type I. This type of PMI is also found in Salmonella typhimurium, Saccharomyces cerevisiae, Aspergillus nidulans, Candida albicans and humans. The amino acid sequence homology assessment results given in table 2 ( Attached) are unclear since the company states the different PMI types are determined by homology yet these types do not seem to relate to the groups shown or the sequence overlap. 7 Mammalian Toxicology Assessment of PMI Protein 1. An extensive bioinformatics search was performed to determine whether the amino acid sequence of the PMI protein shows homology with proteins known to be toxins. PMI showed no significant homology with any proteins identified as or known to be toxins. ( MRID 459344 05)

2. Because it was not feasible to extract sufficient PMI protein from Event 3243M derived corn plants for toxicology studies, PMI protein was produced in recombinant E. coli by over expressing the same pmi ( E. coli manA) gene that was introduced into Event 3243M corn. Equivalence studies between the corn derived PMI and E. coli PMI ( appendix A) were conducted to establish the similarity of the protein from the two sources and are reviewed above.

3. The susceptibility of PMI to proteolytic degradation was evaluated in simulated mammalian gastric fluid ( SGF) containing pepsin and also in simulated mammalian intestinal fluid ( SIF) containing pancreatin. The E. coli derived PMI was rapidly degraded in SGF such that no intact PMI was detected upon immediate analysis of the reaction mixture by both protein presence and enzymatic activity. Likewise, the E. coli derived PMI was also rapidly degraded in SIF with no intact PMI detected after 2 min. of incubation at 37

C. These data indicate that PMI will be readily digested as conventional dietary protein under typical mammalian gastric conditions. Furthermore, in the unlikely event that PMI protein survives the gastric environment, it will rapidly be degraded in the intestinal environment. ( MRID 459344 08)

4. An acute mouse oral toxicity study was conducted using the E. coli derived PMI. PMI was administered to 7 male and 6 female young mice via gavage at a dose of 5050 mg/ kg body weight equivalent to ca. 3080 mg pure PMI/ kg body weight. No test substance related mortalities or clinical signs of toxicity occurred during the study. Gross necropsy of the remaining mice at study termination revealed no observable abnormalities. The NOEL was ca. 3080 mg PMI protein/ kg body weight. The estimated LD 50 was greater than 3080 mg PMI protein/ kg body weight, the single dose tested. ( MRID 459344 07)

Potential for Exposure to PMI via Food or Feed 1. Current Exposure to PMI Although Syngenta Seeds is unaware of documented dietary exposure to PMI, it is conceivable that small amounts of PMI proteins from various sources have always been present in the food and feed supply due to ubiquitous occurrence of PMI enzymes in nature, including in food plants and animals. Human PMI, which has significant structural and functional similarity to the PMI in Event 3243Mderived corn, exists in several body tissues and is an essential enzyme. Furthermore, PMI is present in naturally occurring E. coli strains and other gram negative bacteria that colonize the human intestine and it is expected that these PMI proteins are highly homologous or identical to PMI present in Event 3243M derived corn.

2. Potential for Dietary Exposure to PMI via Event 3243M Derived Corn PMI is present in the grain of Event 3243M derived plants at a concentration of ca. 2 ppm on a dry weight basis. Although trace quantities of PMI were detected in 15 day old corn silage prepared from whole, chopped Event 3243M derived corn plants, PMI was not detectable in 29 day old silage. Cooking and most forms of food processing would be expected to degrade and/ or inactivate 8 PMI in corn products and by products.

Evidence that PMI is Unlikely to Become a Food Allergen Although the probability that any specific novel protein will become a food allergen is small, the potential allergenicity of the PMI protein was evaluated using a weight of evidence approach. First, food products derived from Event 3243M corn are expected to contain little or no PMI protein. As previously noted, the level of PMI in corn grain is not expected to exceed 2 ppm. Second, PMI protein is not derived from a know source of allergens. The pmi gene was cloned from E. coli which has no history of allergenicity. Third, PMI does not have amino acid sequence homology to known allergens. Extensive bioinformatics searches were conducted to determine whether the amino acid sequence of the PMI protein shows homology with proteins known or suspected to be allergens. Comparisons among protein databases containing allergens or putative allergens was compared by contiguous PMI peptides of 80 amino acids as well as comparisons using shorter sequences of 8 amino acids. Results from the searches indicate no similarity of the PMI protein to any known or putative allergens for which amino acid sequences were available. ( MRID 459344 06)

Fourth, PMI protein is not highly stable like many food allergens. Incubation of PMI at 65

C for 30 minutes essentially inactivated the enzyme. Additionally, the PMI protein is unlikely to be stabilized by disulfide bonds since there are only two cysteine residues in the protein. Stabilized bonds appear to contribute to the allergenicity of some food proteins. Fifth, PMI protein is readily degraded in simulated mammalian gastric and intestinal fluids ( see Mammalian Toxicity section). Sixth, PMI protein is not likely to be post translationally glycosylated because its expression in corn is not targeted to a cellular glycosylated pathway. PMI expressed in E. coli is not glycosylated and mass spectrometric analysis of human PMI indicates no post translational modifications.

In summary, PMI is a ubiquitous, essential enzyme from a non allergenic source and has conceivably always been in the human diet in low quantities. The non allergenic characteristics of the PMI protein, combined with the minimal potential for additional dietary exposure via Event 3243M corn indicate that the potential for PMI to become a food allergen is extremely low.

Reviewer's Conclusion The results of the equivalence study are the only new and essential information provided in this submission. The other parts are a reiteration of information provided elsewhere or derivative information used by the company to estimate dietary exposure of PMI expressed in corn. The results of the equivalence studies indicate similar molecular weight by SDS PAGE between the corn silk expressed PMI and that produced in an E. coli expression vector. This is somewhat unusual in that the E. coli form of PMI contained an additional 17 amino acids which should be distinguishable by western blot analysis. The enzymatic activity is also similar based on the detectable level of PMI by ELISA. These data suggest that the two sources produce PMI protein that is similar enough that the bacterial PMI source can be used for further biochemical and toxicological tests to establish dietary safety. 9 DATA EVALUATION RECORD ________________________________________________________________________________ Primary reviewer: H. Tim Borges, Ph. D., MT ( ASCP), DABT, Oak Ridge National Lab EPA Reviewer: John L. Kough, Ph. D., Biologist ________________________________________________________________________________ STUDY TYPE: Amino acid sequence comparison to known protein toxins MRID NO: 459344 05 DP BARCODE: D292501/ 3E6748 TEST MATERIAL: Event 3243M Corn EUP PROJECT REPORT NO: SSB 016 03 TESTING FACILITY: Syngenta Seeds, Inc., Product Registration Group, P. O. Box 12257, 3054 Cornwallis Road, Research Triangle Park, NC 27709 2257 TITLE OF REPORT: Phosphomannose isomerase protein: assessment of amino acid homology with known toxins AUTHOR: J. Zawodny STUDY COMPLETED: April 23, 2002 CONCLUSION: The PMI amino acid sequence showed no significant homology to known protein toxins. CLASSIFICATION: Supplemental. Can be upgraded to acceptable with identification of the 92 known and hypothetical proteins identified as having significant amino acid sequence homology to PMI along with the source of those proteins. The other activities of PMI homologues should also be listed. GOOD LABORATORY PRACTICE: Non GLP study ________________________________________________________________________________

A. STUDY PURPOSE: The study was done to determine whether E. coli derived phosphomannose isomerase protein ( PMI) has any significant amino acid homology with known toxic protein sequences.

B. MATERIALS AND METHODS: The 391 amino acid sequence of PMI was compared using the v. 2.2.6 BLASTP program ( Altschul et al., 1997) to known amino acid protein sequences contained in National Center for Biotechnology Information ( NCBI) GenBank Database ( 2003 posting). This program develops an " Expect" ( E) ratio where comparisons between highly homologous proteins yield ratios approaching zero indicating the probability of similarities are not occurring by chance. To determine if any of the " hits" identified with E ratios < 10 were had random similarities or if they were homologous, the amino acid sequence of PMI was shuffled into five additional versions each containing the same 391 amino acids. These shuffled sequences represented the background control incidence of random hits for the amino acid composition.

C. RESULTS: The shuffled PMI sequences returned E ratios ranging from 0.42 to 8.0. To be considered potentially homologous, the E ratio for the intact PMI sequence would need to be < 0.42. The BLASTP program identified 92 protein sequences in the GenBank database as potentially homologus with PMI. Of these, 77 proteins were PMI homologs with E ratios ranging from 0.0 to 0.42; one protein was a Neisseria menigitidis RNA polymerase   factor not 10 known to be toxic, and 14 hypothetical protein sequences. Therefore, the PMI amino acid sequence showed no significant homology to known protein toxins. 11 DATA EVALUATION RECORD ________________________________________________________________________________ Primary reviewer: H. Tim Borges, Ph. D., MT ( ASCP), DABT, Oak Ridge National Lab EPA Reviewer: John L. Kough, Ph. D., Biologist ________________________________________________________________________________ STUDY TYPE: Amino acid sequence comparison to known allergens MRID NO: 459344 06 DP BARCODE: D292501/ 3E6748 TEST MATERIAL: Event 3243M Corn EUP PROJECT REPORT NO: SSB 014 03 TESTING FACILITY: Syngenta Seeds, Inc., Product Registration Group, P. O. Box 12257, 3054 Cornwallis Road, Research Triangle Park, NC 27709 2257 TITLE OF REPORT: Phosphomannose isomerase protein: assessment of amino acid homology with known allergens AUTHOR: J. Zawodny STUDY COMPLETED: April 23, 2002 CONCLUSION: The PMI protein showed no significant amino acid homology with known or putative allergenic proteins using either an 8 amino acid sequence stepwise comparison or and an 80 amino acid fragment comparison. CLASSIFICATION: Acceptable GOOD LABORATORY PRACTICE: Non GLP study ________________________________________________________________________________

D. STUDY PURPOSE: The study was done to determine whether E. coli derived phosphomannose isomerase protein ( PMI) has any significant amino acid homology with known allergenic sequences.

B. MATERIALS AND METHODS: The 391 amino acid sequence of PMI was compared to the Syngenta Biotechnology, Inc. ( SBI) Allergen Database. The SBI Allergen Database contains the amino acid sequences of known or putative allergens and was initially compiled from the following sources: the publically available GenPept, PIR protein database ( 2001 version); the SWISS PROT database ( 2001 version); the List of Allergens database ( International Union of Immunological Societies, 2001 version); and the Food Allergy Research and Resource Program ( 2001 version). In addition, the database has been updated with putative allergens identified in the scientific literature. The SBI Allergen Database contains 1114 entries and was last updated in February 2003.

Two types of comparisons of the PMI protein sequence were done. For the first comparison, the similarity of sequential PMI protein 80 amino acid peptide blocks were compared to the allergens using the FASTA search algorithm. Each successive 80 amino acid block was offset from the previous block by one amino acid. Any sequence with > 35% homology to an allergen sequence was considered positive.

For the second comparison, a computer program developed by Syngenta compared 8 or more 12 contiguous amino acids of the PMI protein to every possible 8 contiguous amino acids of allergens in the SBI Allergen Database. The purpose of this study was to screen for short, local regions of amino acid identity that could indicate IgE binding epitopes.

C. RESULTS: No significant similarities between any of the sequential PMI 80 amino acid peptides with peptides in the SBI Allergen Database were found using the FASTA algorithm. In addition, no identical alignments of 8 or more contiguous amino acids between PMI protein and known or putative allergens in the SBI Allergen Database were found. 13 DATA EVALUATION RECORD ________________________________________________________________________________ Primary Reviewer: Susan Chang, M. S., Oak Ridge National Lab Secondary Reviewer: John L. Kough, Ph. D., Biologist ________________________________________________________________________________ STUDY TYPE: Acute Oral Toxicity  Mice ( OPPTS 870.1100) MRID NO: 459344 07 DP BARCODE NO: D292501 CASE NO: Not reported SUBMISSION NO: Not reported TEST MATERIAL: PMI 0198 Protein containing ~ 60% phosphomannose isomerase PROJECT NO: 4708 98 SPONSOR: Novartis Seeds, Inc.  Field Crops  NAFTA TESTING FACILITY: Stillmeadow, Inc., Sugar Land, TX TITLE OF REPORT: Acute Oral Toxicity Study in Mice AUTHOR: Janice O. Kuhn, Ph. D., D. A. B. T. STUDY COMPLETED: August 11, 1999 GOOD LABORATORY PRACTICE: GLP Compliant CONCLUSION: The oral LD 50 of PMI 0198 protein for males, females, and combined was greater than 3080 mg/ kg. CLASSIFICATION: ACCEPTABLE  TOXICITY CATEGORY III ________________________________________________________________________________

I. STUDY DESIGN:

1. Test material: PMI 0198 Protein containing ~ 60% phosphomannose isomerase 2. Test animals: Thirteen male and eleven female HSD: ICR mice were received from Harlan Sprague Dawley, Inc., Indianapolis, IN, were assigned, and weighed 22.2 28.2 g ( males) and 18.6 24.7 g ( females) on the day of dosing. The young adult animals, approximately 4 weeks old, were housed individually in suspended, wire top, polycarbonate cages. The animals were fed PMI Feeds Inc. Formulab N. 5008, ad libitum, except for approximately 16 hours before dosing. Municipal water was available ad libitum. The environmental conditions of the animal room were as follows: temperature, 22

± 3

C; humidity, 30 80%; air changes, 10 12 per hour; and photoperiod, 12 hour light/ dark cycle.

3. Methods: Mice were ear tagged: males ( Test group  Nos. 11 M to 16 M and 28 M; Control group  Nos. 1 M to 5 M and 27 M) and females ( Test group  Nos. 21 F to 26 F; Control group  Nos. 6 F to 10 F). The mice were quarantined for 5 days and fasted 16 hours prior to dosing. The test material ( 5050 mg/ kg body weight) was dosed as a 20% w/ v suspension in a 0.5% w/ v aqueous CMC by gavage at a volume of 25.25 mL/ kg to seven test males and five test females ( Table 1). The control group ( six males and five females) was dosed with 25.25 mL/ kg of 0.5% CMC. Body weights were recorded prior to dosing, on day 14 or at death. The test animals were observed for clinical signs of toxicity at least three times post dosing and at least daily for 14 days. All decedent or euthanized animals were necropsied. 14 II. RESULTS:

1. Mortality: Mortality is given in Table 1. Two test males ( Nos. 11 M and 16 M) died on the day of dosing and one control male ( No. 3 M) was dead on day 1. One replacement animal was available for each group. All other rats survived the study.

TABLE 1. Doses, mortality/ animals treated Dose ( mg/ kg) Males Females Combined 0 1/ 6 0/ 5 1/ 11 5050 2/ 7 0/ 5 2/ 12 Data taken from Table 1, p. 10 11, MRID 45934407.

2. Body weights: All surviving animals gained weight during the study.

3. Clinical observations: No clinical signs of toxicity were noted from any surviving animal.

4. Gross necropsy: The decedents had perforated esophaguses due to gavage error. No gross abnormalities were noted from any survivor.

III. DISCUSSION: The mouse oral LD 50 for males, females, and combined was greater than 5050 mg/ kg of dosing solution or 3080 mg/ kg of PMI protein. This places PMI 0198 Protein in TOXICITY CATEGORY III. The packet classification is ACCEPTABLE.

Appendix A. Characterization of the Test Substance PMI 0198: Similar information is to be found in MRID 459344 02.

The test substance protein ( sample PMI 0198) consists of a purified preparation of phosphomannose isomerase ( PMI) isolated from E. coli strain BLRDE3 transformed with the pET 3a vector containing the manA ( pmi) gene. The PMI protein expressed is similar to that found in corn except for an additional 16 amino acids at the N terminus ( 13 amino acids in the T7 Tag   and 3 amino acids from the poly linker sequence).

I. MATERIALS AND METHODS Purification: The bacterial PMI was purified by differential ammonium sulfate precipitation followed by hydrophobic interaction chromatography ( Phenyl Sepharose ® or Toyopearl Ether ® 650M packed columns) then ion exchange chromatography on a Sepharose Q ® column. The appropriate collected fractions were pooled prior to dialysis against a 50mM NH 4 HCO 3 pH 8.0 buffer then subjected to lyophilization. The protein content of the lyophilized product was found to be approximately 93% PMI (

45,000 molecular weight, enzymatically active) and was given the 15 designation PMI 0198. The lyophilized prep was stored at  20

C. An aliquot of PMI 0198 was dissolved in 50mM Tris HCl, pH 7.0 to a final concentration of 10 mg/ ml.

Protein quantification: The PMI 0198 prep was subjected to BCA   procedure for determining total protein against an ovalbumin standard.

PMI quantification: The actual amount of PMI enzyme present was determined by ELISA ( SOP 2.43, not submitted) using immunoaffinity purified polyclonal rabbit antibodies and protein G purified goat antibodies specific for PMI.

Molecular weight and densitometric determination: SDS PAGE ( 14% polyacrylamide) with Novex molecular weight markers followed by Coomassie blue staining was used to determine the molecular weight and purity of the PMI 0198 prep. There was a disagreement between the protein determinations by BCA and ELISA assays for the percent amount of PMI present. The company claims this indicates that ovalbumin is an inappropriate standard to use in BCA total protein assays for the PMI prep. Therefore, an additional test of purity of the PMI 0198 was undertaken as a densitometric scan of the Coomassie blue SDS PAGE gel ( SOP 2.6, not submitted).

Immunoreactivity: Aliquots of the PMI 0198 ranging from 5 to 25 ng of PMI per lane prep were electroblotted after SDS PAGE ( SOPs 2.4 & 2.3, not submitted). The membranes were probed with goat polyclonal antiserum specific for PMI. Rabbit anti goat IgG horseradish peroxidase ( HRP) conjugate was used to detect the bound goat anitserum. The bound antibodies were visualized by HRP reaction on the chromogenic substrate diaminobenzidine.

Enzymatic Activity: The verify the integrity of the PMI enzyme, activity assays were performed using production of NADPH from the reaction of PMI on mannose coupled to phosphoglucose isomerase ( PGI) and glucose 6 phosphate dehydrogenase ( G6PDH) as seen below:

PMI PGI G6PDH Mannose 6 P  fructose 6 P  glucose 6 P + NADP  6 gluconolactone + NADPH NADPH production is monitored spectrophotometrically at 340nm ( SOP 2.44, not submitted). The micromolar extinction coefficient of NADPH is 6.2 with a unit of enzyme activity being defined as 1 µ mole of NADP being reduced per minute.

Lipopolysaccharide measurement: Lipopolysaccharide ( LPS) or gram negative bacterial endotoxin was determined since it is a common contaminant in preparations purified from E. coli expression systems. The E TOXATE   kit was employed to quantify the amount of LPS present in PMI 0198 following the manufacturer's 16 instruction ( SOP 2.35, not submitted).

N terminal amino acid sequencing: A sample of PMI 0198 was analyzed for the N terminal amino acid sequence to confirm the expressed sequence was the same as that encoded in the manA gene. The PMI 0198 sample was subjected to SDS PAGE, electroblotted athe stained with amido black stain. The appropriate band was cut out and subjected to Edman degradation, phenylhydantoin coupling followed by fractionation of the amino acid on HPLC ( SOP 2.34 not submitted)

Solubility determination: The solubility of PMI 0198 was measured by SOP 2.36 ( not submitted).

II. RESULTS AND DISCUSSION The apparent molecular weight of the major band, assumed to be PMI, appearing on SDS PAGE and western blot, in both the initial and repeat run, was approximately 45,000. The protein quantification of PMI by ELISA correction of BCA value yielded an initial finding of 61% of the PMI 0198 sample as PMI protein. Re analysis after the acute oral study gave a value of 68% PMI. Analysis of the PMI percentage by densitometery from a Coomassie blue stained SDS PAGE gel yielded a PMI value of 95% initially and 93% in the repeat analysis after the oral study. The enzyme activity test gave an initial value of 7.34+ 0.17 units/ mg PMI protein and 6.26+ 0.81 units/ mg PMI protein after the study. The LPS value for the PMI 0198 sample was found to be 0.3ng/ mg. The company states that the known LD 50 values for LPS in rats by the intraperitoneal or intravenous route of exposure are 107 mg/ kg or 4.33 mg/ kg, respectively. The N terminal amino acid analysis gave the following sequence:

ASMTGGQQMGRGPIMQKL This sequence would match the expected AA sequence starting with the second amino acid in the T7 tag. It is important to note that this only includes the first 4 amino acids of the PMI protein as claimed to be expressed in plant tissue: MQKL. The solubility test gave an estimate of solubility of the PMI 0198 sample at above 150 mg/ ml. All these tests indicate that the PMI 0198 sample was stable for the duration of the acute oral study. 17 DATA EVALUATION RECORD ________________________________________________________________________________ Primary Reviewer: H. Tim Borges, Ph. D., MT( ASCP), DABT, Oak Ridge National Lab EPA Reviewer: John L. Kough, Ph. D., Biologist ________________________________________________________________________________ STUDY TYPE: In vitro simulated digestion MRID NO: 459344 08 DP BARCODE: D292501/ 3E6748 TEST MATERIAL: Event 3243M Corn EUP REPORT NUMBER: NSB 002 99 TESTING FACILITY: Novartis Seeds, Inc., Product Registration Group, 3054 Cornwallis Road, Research Triangle Park, NC 27709 2257 TITLE OF REPORT: In vitro digestibility of PMI protein under simulated mammalian gastric and intestinal conditions AUTHOR: Privalle, L. STUDY COMPLETED: June 24, 1999 CONCLUSION: Full length PMI 0198 protein was degraded to undetectable levels in Coomassie blue stained SDS PAGE gels after incubation in simulated gastric and intestinal fluids. CLASSIFICATION: Acceptable GOOD LABORATORY PRACTICES: Full compliance with 40 CFR 160, signed June 24, 1999 ________________________________________________________________________________

D. STUDY PURPOSE: The study was done to determine whether E. coli derived phosphomannose isomerase protein ( PMI) underwent proteolytic degradation in simulated mammalian gastric and intestinal fluid environments. The equivalence studies for E. coli expressed and plant expressed PMI are found in MRID 459344 02.

B. MATERIALS:

PMI Source: The test material was prepared as sample PMI 0198 by extraction and differential ammonium sulfate concentration followed by hydrophobic interaction chromatography and ion exchange chromatography from the inducible over expression pET 3a ® vector in E. coli strain BLRDE3. Dialysis into 50 mM ammonium carbonate was done following lyophilization. The sample was ~ 60% PMI protein by weight and accounted for ~ 95% of the protein in the sample.

SGF: Simulated gastric fluid ( SGF, pH 1.0 1.2) containing 2 mg/ mL sodium chloride, 14  L 6 N hydrochloric acid/ mL, and 3.2 mg/ mL ( 3200 3400 units/ mg) pepsin was prepared fresh for each assay. Simulated gastric fluid was also prepared as above without the addition of pepsin for use as a negative digestion control. To monitor digestion, SGF was prepared with 10%, 1%, 0.1%, and 0.01% the amount of pepsin as contained in the standard SGF.

SIF: Simulated intestinal fluid ( SIF, pH 7.5) containing 6.8 mg potassium phosphate, 10 mg/ mL pancreatin, and tap water was prepared daily and checked for proteolytic activity using azoalbumin as the substrate. SIF was also prepared with 10%, 1%, and 0.1% the pancreatin to 18 monitor digestion.

SDS PAGE: A 10  L aliquot from each reaction mixture was loaded on to 14% polyacrylamide gels and electrophoresed. The gels were stained with Coomassie blue for protein detection.

C. METHOD:

Digestion Studies: The digestion reactions were initiated by the addition of 20  L of 10 mg/ mL PMI 0198 to 80  L of each of the following prepared solutions: full strength concentration, 10%, 1%, or 0.1% enzyme concentrations in the SGF or SIF mixture. An aliquot was immediately removed and the reaction quenched with an equal volume Laemmli buffer followed by heating at 75

C for 10 minutes. The remainder of the reaction mixture was incubated for two minutes before quenching. To follow the time course of gastric digestion, 80  L of 10 mg/ mL PMI 0198 was added to 320  L of 0.01% SGF. Aliquots, 10  L, were removed immediately after addition, and after 2, 5, 10, 20, 30, and 60 minutes of incubation at 37

C.

Enzymatic Inactivation Studies: These studies were done to monitor loss of PMI activity in SGF. The studies were done by adding 20  L of 10 mg/ mL PMI 0198 to 80  L SGF containing 0.01% pepsin, SGF without pepsin, or 50 mM Tris HCl pH 7.0 buffer. Ten  L aliquots of each reaction mixture were removed 0, 2, 5, 10, 20, 30, and 60 minutes after substrate addition and immediately placed into PMI assay mixture and stored on ice until time of assay. ( The control " buffer alone" reaction aliquots were removed at 0 and 60 minutes only.) The enzymatic reactions were initiated by addition of mannose 6 phosphate and the change in absorbance monitored at 340 nm.

D. RESULTS:

SGF: PMI 0198 was rapidly degraded at the standard pepsin concentration and at pepsin concentrations of 10% and 1%. At a pepsin concentration of 0.1%, full length PMI 0198 was degraded to smaller fragments within two minutes. ( figure 1, attached) With a pepsin concentration of 0.01% full length PMI 0198 was degraded within 10 minutes. ( figure 3, attached) SIF: Full length PMI 0198 was degraded to smaller fragments within two minutes at the standard pancreatin concentration. At lower pancreatin concentrations degradation was not completed within the two minute incubation period. ( figure 2, attached) Enzymatic Inactivation: PMI 0198 enzymatic activity was relatively stable at 37

C in SGF without pepsin and in buffer. However, no enzymatic activity was detected after 10 minutes of incubation in the presence of 0.01% pepsin. ( figure 4, attached)

Reviewer's comments The in vitro SGF and SIF degradation of the PMI is apparent here by the absence of significant Coomassie blue stained bands after introduction of the substrate. Using lower amounts of the digestive enzymes in the buffer solution, a time course of the degradation can be visualized showing an accumulation of smaller molecular weight bands over time as the inital PMI substrate band becomes fainter until all the bands disappear ( figure 3 for SGF attached). The PMI protein 19 does not seem to be as readily degraded by SIF ( figure 2 attached).

An approximately 40,000 MW band was faint but apparent in the lanes with the full strength pepsin in SGF. The company stated the band was pepsin. Pepsin has a range of apparent molecular weights depending on the source it was isolated from.

The full strength and 10% intestinal fluid lanes had a heavily staining band at approximately 50,000 molecular weight as well as fainter bands at approximately 30,000 and 25,000 molecular weight. These bands could also represent the presence of numerous digestive enzymes present in pancreatin. Possibilities include pancreatic lipase ( 49,000 MW), trypsin ( 24,000 MW) and carboxypeptidase B ( 34,700 MW). 20 DATA EVALUATION RECORD ________________________________________________________________________________ Primary Reviewer: Anthony Q. Armstrong, M. S., Oak Ridge National Labs EPA Reviewer: John L. Kough, Ph. D., Biologist ________________________________________________________________________________ STUDY TYPE: Product Identity [ Temperature Stability of PMI] MRID NO: 459344 09 DP BARCODE: D292501/ 3E6748 TEST MATERIAL: Cry1Ab and PMI ( Corn Event 3243M) PROJECT NO: SSB 013 03 SPONSOR: Syngenta Seeds, Inc. 3054 Cornwallis Road, RTP, North Carolina 27709 2257 TESTING FACILITY: Syngenta Seeds, Inc. 3054 Cornwallis Road, RTP, North Carolina 27709 2257 TITLE OF REPORT: Effects of Temperature on Stability of Phosphomannose Isomerase ( PMI) AUTHOR: Kim Hill STUDY COMPLETED: April 25, 2003 GOOD LABORATORY PRACTICE: Not 40 CFR 160 GLP compliant CONCLUSION: The phosphomannose isomerase ( PMI) enzyme derived from E. coli was prepared at 0.44 mg/ mL in standard buffer ( 50 mM Tris HCl, pH 7.0) and incubated at ambient temperature ( 25

C), 37, 55, 65 and 95

C for 30 minutes. PMI enzymatic activity was monitored by measuring NADPH production using a spectrophotometer. Results indicate that incubation at ambient temperature ( 25

C) and at 37 and 55

C for 30 minutes had little effect on PMI. However, incubation at 65 and 95

C for 30 minutes inactivated PMI. CLASSIFICATION: Acceptable.

I. TEST MATERIAL: Purified PMI was produced from over expression of the pET 3A ® vector ( containing the manA gene) in E. coli strain BLR ( DE3). This test substance was designated PMI 0198 by Syngenta.

II. METHODS: For temperature stability analysis, 0.44 mg/ mL solution of PM 0198 was prepared in standard buffer ( 50 mM Tris HCl, pH 7.0) and incubated at ambient temperature ( 25

C), 37, 55, 65 and 95

C for 30 minutes. PMI enzymatic activity was measured by NADPH production3 using a UV 1601 Shimadzu spectrophotometer set at UV absorbance of 340 nm. Samples were assayed in triplicate and results normalized to 25

C.

III. RESULTS: Table 1 contains results from the temperature inactivation studies. Results indicate that incubation at ambient temperature ( 25

C) and at 37 and 55

C for 30 minutes had no effect on PMI. However, incubation at 65 and 95

C for 30 minutes inactivated PMI. 21 IV. REVIEWER'S CONCLUSION: The PMI enzyme activity is demonstrated by this study to be significantly reduced with 30 minute incubation at temperatures above 55

.

TABLE 1. Effect of Temperature on PMI Enzymatic Activity Temperature (

C) Specific Activity ( U/ mg PMI) Std. Dev. ( U/ mg PMI) % Activity ( normalized to 25

C)

25 11.74 2.59 100.00 37 11.10 3.01 94.55 55 10.24 0.92 87.22 65 0.27 0.09 2.30 95 0.10 0.07 0.85