[Federal Register Volume 82, Number 84 (Wednesday, May 3, 2017)]
[Notices]
[Pages 20648-20652]
[FR Doc No: 2017-08902]

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SECURITIES AND EXCHANGE COMMISSION

[Release No. 34-80545; File No. SR-IEX-2017-03]

Self-Regulatory Organizations; Investors Exchange LLC; Notice of
Filing of Amendment No. 1 and Order Granting Accelerated Approval of a
Proposed Rule Change, as Modified by Amendment No. 1, To Amend IEX Rule
16.135 To Adopt Generic Listing Standards for Managed Fund Shares

April 27, 2017.

I. Introduction

    On January 19, 2017, Investors Exchange LLC (``IEX'' or
``Exchange'') filed with the Securities and Exchange Commission
(``Commission''), pursuant to Section 19(b)(1) of the Securities
Exchange Act of 1934 (``Act'') \1\ and Rule 19b-4 thereunder,\2\ a
proposed rule change to amend IEX Rule 16.135 to adopt generic listing
standards for Managed Fund Shares. The proposed

rule change was published for comment in the Federal Register on
February 8, 2017.\3\ On March 16, 2017, the Commission designated a
longer period within which to approve the proposed rule change,
disapprove the proposed rule change, or institute proceedings to
determine whether to approve or disapprove the proposed rule change.\4\
On March 21, 2017, IEX filed Amendment No. 1 to the proposed rule
change.\5\ The Commission has received no comments on the proposal. The
Commission is publishing this notice to solicit comments on Amendment
No. 1 from interested persons and is approving the proposed rule
change, as modified by Amendment No. 1, on an accelerated basis.
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    \1\ 15 U.S.C. 78s(b)(1).
    \2\ 17 CFR 240.19b-4.
    \3\ See Securities Exchange Act Release No. 79940 (February 2,
2017), 82 FR 9858.
    \4\ See Securities Exchange Act Release No. 80257, 82 FR 14779
(Mar. 22, 2017). (designating May 9, 2017 as the date by which the
Commission shall either approve or disapprove, or institute
proceedings to determine whether to disapprove, the proposed rule
change).
    \5\ In Amendment No. 1, the Exchange proposes to add certain
continued listing requirements for Managed Fund Shares based on
those adopted by the Nasdaq Stock Market LLC (``Nasdaq''). The
Exchange also makes technical changes to the requirements in IEX
Rule 16.135 regarding firewalls and written surveillance procedures.
Amendment No. 1 is available at: https://www.sec.gov/comments/sr-iex-2017-03/iex201703-1708027-150143.pdf.
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II. Description of the Proposed Rule Change

    The Exchange proposes to adopt generic listing criteria and
continued listing standards for Managed Fund Shares. The Exchange
represents that the proposed rule change is substantially identical to
Nasdaq Rule 5735.\6\
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    \6\ See Amendment No. 1, supra note 5, at 33.
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A. Proposed Generic Listing Criteria

    IEX proposes generic listing criteria that would permit the
Exchange to list and trade Managed Fund Shares pursuant to Rule 19b-
4(e),\7\ rather than by filing a proposed rule change under Section
19(b) of the Act.\8\ The Exchange's listing standards establish
requirements for the various types of assets that may be held in the
portfolio of a generically listed, actively managed exchange traded
fund (``Portfolio'').
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    \7\ See 17 CFR 240.19b-4(e). Rule 19b-4(e) permits self-
regulatory organizations (``SROs'') to list and trade new derivative
securities products that comply with existing SRO trading rules,
procedures, surveillance programs, and listing standards, without
submitting a proposed rule change under Section 19(b). See
Securities Exchange Act Release No. 40761 (Dec. 8, 1998), 63 FR
70952 (Dec. 22, 1998).
    \8\ The Exchange would file separate proposed rule changes
before the listing and trading of Managed Fund Shares that do not
satisfy the proposed generic listing criteria. See proposed IEX Rule
16.135(b)(1).
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1. Equity Components of the Portfolio
    Proposed IEX Rule 16.135(b)(1)(A) establishes the criteria
applicable to the equity securities included in a Portfolio. Equity
securities include the following kinds of securities: U.S. Component
Stock (defined in IEX Rule 16.105); Non-U.S. Component Stock, (defined
in IEX Rule 16.105); Exchange Traded Derivative Securities (defined in
proposed IEX Rule 16.135(c)(6)); \9\ Linked Securities (defined in IEX
Rule 16.110); and each of the equivalent security types listed on
another national securities exchange. Additionally, proposed IEX Rule
16.135(b)(1)(A) provides that no more than 25% of the equity weight of
the Portfolio can include leveraged or inverse-leveraged Exchange
Traded Derivative Securities or Linked Securities and that, to the
extent a Portfolio includes convertible securities, the equity
securities into which such securities are converted must meet the
criteria of proposed IEX Rule 16.135(b)(1)(A) after converting.
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    \9\ Proposed IEX Rule 16.135(c)(6) defines ``Exchange Traded
Derivative Securities'' as the securities described in IEX Rules
16.105(a) (Portfolio Depository Receipts); 16.105(b) (Index Fund
Shares); 16.120 (Trust Issued Receipts); 16.111(d) (Commodity-Based
Trust Shares); 16.111(e) (Currency Trust Shares); 16.111(f)
(Commodity Index Trust Shares); 16.111(g) (Commodity Futures Trust
Shares); 16.111(h) (Partnership Units); 16.111(i) (Trust Units);
16.135 (Managed Fund Shares); and 16.111(j) (Managed Trust
Securities).
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    Proposed IEX Rule 16.135(b)(1)(A)(i) requires that U.S. Component
Stocks (except as mentioned below) meet the following criteria
initially and on a continuing basis:
    (1) Component stocks (excluding Exchange Traded Derivative
Securities and Linked Securities) that in the aggregate account for at
least 90% of the equity weight of the Portfolio (excluding Exchange
Traded Derivative Securities and Linked Securities) each shall have a
minimum market value of at least $75 million;
    (2) Component stocks (excluding Exchange Traded Derivative
Securities and Linked Securities) that in the aggregate account for at
least 70% of the equity weight of the Portfolio (excluding Exchange
Traded Derivative Securities and Linked Securities) each shall have a
minimum monthly trading volume of 250,000 shares, or minimum notional
volume traded per month of $25,000,000, averaged over the previous six
months;
    (3) The most heavily weighted component stock (excluding Exchange
Traded Derivative Securities and Linked Securities) must not exceed 30%
of the equity weight of the Portfolio, and, to the extent applicable,
the five most heavily weighted component stocks (excluding Exchange
Traded Derivative Securities and Linked Securities) must not exceed 65%
of the equity weight of the Portfolio;
    (4) Where the equity portion of the Portfolio does not include Non-
U.S. Component Stocks, the equity portion of the Portfolio shall
include a minimum of 13 component stocks; provided, however, that there
would be no minimum number of component stocks if (a) one or more
series of Exchange Traded Derivative Securities or Linked Securities
constitute, at least in part, components underlying a series of Managed
Fund Shares, or (b) one or more series of Exchange Traded Derivative
Securities or Linked Securities account for 100% of the equity weight
of the Portfolio of a series of Managed Fund Shares;
    (5) Except as provided in proposed IEX Rule 16.135(b)(1)(A)(i),
equity securities in the Portfolio must be U.S. Component Stocks listed
on a national securities exchange and must be NMS Stocks as defined in
Rule 600 of Regulation NMS; and
    (6) American Depositary Receipts (``ADRs'') may be exchange traded
or non-exchange traded, but no more than 10% of the equity weight of
the Portfolio shall consist of non-exchange traded ADRs.
    Proposed IEX Rule 16.135(b)(1)(A)(ii) requires that Non-U.S.
Component Stocks must meet the following criteria initially and on a
continuing basis:
    (1) Non-U.S. Component Stocks each shall have a minimum market
value of at least $100 million;
    (2) Non-U.S. Component Stocks each shall have a minimum global
monthly trading volume of 250,000 shares, or minimum global notional
volume traded per month of $25,000,000, averaged over the last six
months;
    (3) The most heavily weighted Non-U.S. Component Stock shall not
exceed 25% of the equity weight of the Portfolio, and, to the extent
applicable, the five most heavily weighted Non-U.S. Component Stocks
shall not exceed 60% of the equity weight of the Portfolio;
    (4) Where the equity portion of the Portfolio includes Non-U.S.
Component Stocks, the equity portion of the Portfolio shall include a
minimum of 20 component stocks; provided, however, that there shall be
no minimum number of component stocks if (a) one or more series of
Exchange Traded Derivative Securities or Linked Securities constitute,
at least in part, components

underlying a series of Managed Fund Shares, or (b) one or more series
of Exchange Traded Derivative Securities or Linked Securities account
for 100% of the equity weight of the Portfolio of a series of Managed
Fund Shares; and
    (5) Each Non-U.S. Component Stock shall be listed and traded on an
exchange that has last-sale reporting.
2. Fixed Income Components of the Portfolio
    Proposed IEX Rule 16.135(b)(1)(B) establishes criteria for fixed
income securities that are included in a Portfolio. Fixed income
securities are debt securities that are notes, bonds, debentures, or
evidence of indebtedness that include, but are not limited to, U.S.
Department of Treasury securities (``Treasury Securities''),
government-sponsored entity securities (``GSE Securities''), municipal
securities, trust preferred securities, supranational debt and debt of
a foreign country or a subdivision thereof, investment grade and high
yield corporate debt, bank loans, mortgage and asset backed securities,
and commercial paper.\10\ To the extent that a Portfolio includes
convertible securities, the fixed income securities into which such
securities are converted shall meet the criteria of proposed IEX Rule
16.135(b)(1)(B) after converting.\11\
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    \10\ See proposed IEX Rule 16.135(b)(1)(B).
    \11\ See id.
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    Under proposed IEX Rule 16.135(b)(1)(B), fixed income securities
that are part of a Portfolio must satisfy the following criteria
initially and on a continuing basis:
    (1) Components that in the aggregate account for at least 75% of
the fixed income weight of the Portfolio must each have a minimum
original principal amount outstanding of $100 million or more;
    (2) No component fixed-income security (excluding Treasury
Securities and GSE Securities) shall represent more than 30% of the
fixed income weight of the Portfolio, and the five most heavily
weighted fixed income securities in the Portfolio (excluding Treasury
Securities and GSE Securities) shall not in the aggregate account for
more than 65% of the fixed income weight of the Portfolio;
    (3) A Portfolio that includes fixed income securities (excluding
exempted securities) shall include a minimum of 13 non-affiliated
issuers; provided, however, that there shall be no minimum number of
non-affiliated issuers required for fixed income securities if at least
70% of the weight of the Portfolio consists of equity securities as
described in IEX Rule 16.135(b)(1)(A);
    (4) Component securities that in aggregate account for at least 90%
of the fixed income weight of the Portfolio must be: (a) From issuers
that are required to file reports pursuant to Sections 13 and 15(d) of
the Act; (b) from issuers each of which has a worldwide market value of
its outstanding common equity held by non-affiliates of $700 million or
more; (c) from issuers each of which has outstanding securities that
are notes, bonds, debentures, or evidence of indebtedness having a
total remaining principal amount of at least $1 billion; (d) exempted
securities as defined in Section 3(a)(12) of the Act; or (e) from
issuers that are a government of a foreign country or a political
subdivision of a foreign country; and
    (5) Non-agency, non-GSE, and privately issued mortgage-related and
other asset-backed securities shall not account, in the aggregate, for
more than 20% of the weight of the fixed income portion of the
Portfolio.
3. Cash and Cash Equivalent Portfolio Components
    Proposed IEX Rule 16.135(b)(1)(C) provides that a Portfolio may
include cash and cash equivalents. Cash equivalents are defined as
short-term instruments with maturities of less than three months.\12\
The Exchange defines short-term instruments to include the following:
(1) U.S. Government securities, including bills, notes, and bonds
differing as to maturity and rates of interest, which are either issued
or guaranteed by the U.S. Treasury or by U.S. Government agencies or
instrumentalities; (2) certificates of deposit issued against funds
deposited in a bank or savings and loan association; (3) bankers'
acceptances, which are short-term credit instruments used to finance
commercial transactions; (4) repurchase agreements and reverse
repurchase agreements; (5) bank time deposits, which are monies kept on
deposit with banks or savings and loan associations for a stated period
of time at a fixed rate of interest; (6) commercial paper, which are
short-term unsecured promissory notes; and (7) money market funds.\13\
The Exchange does not propose to limit to the amount of cash or cash
equivalents that may be held in a Portfolio.\14\
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    \12\ See proposed IEX Rule 16.135(b)(1)(C).
    \13\ See proposed IEX Rule 16.135(b)(1)(C)(ii).
    \14\ See proposed IEX Rule 16.135(b)(1)(C)(i).
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4. Derivatives in the Portfolio
    Proposed IEX Rule 16.135(b)(1)(D) establishes criteria for the
portion of a Portfolio that consists of listed derivatives, such as
futures, options, and swaps overlying commodities, currencies,
financial instruments (e.g., stocks, fixed income securities, interest
rates, and volatility), or a basket or index of any of the foregoing.
The Exchange does not propose to limit the percentage of a Portfolio
that may be composed of such holdings, provided that, in the aggregate,
at least 90% of the weight of holdings in listed derivatives
(calculated using the aggregate gross notional value) must, on both an
initial and continuing basis, consist of futures, options, and swaps
for which the Exchange may obtain information via the Intermarket
Surveillance Group from other members or affiliates or for which the
principal market is a market with which the Exchange has a
comprehensive surveillance sharing agreement (``CSSA'').\15\
Additionally, the aggregate gross notional value of listed derivatives
based on any five or fewer underlying reference assets shall not exceed
65% of the weight of the Portfolio (including gross notional
exposures), and the aggregate gross notional value of listed
derivatives based on any single underlying reference asset shall not
exceed 30% of the weight of the Portfolio (including gross notional
exposures).\16\
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    \15\ See proposed IEX Rule 16.135(b)(1)(D)(i).
    \16\ See proposed IEX Rule 16.135(b)(1)(D)(ii).
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    Proposed IEX Rule 16.135(b)(1)(E) establishes a limit on over-the-
counter (``OTC'') derivatives. Specifically, no more than 20% of the
weight of the Portfolio may be invested in OTC derivatives.\17\ For
purposes of calculating this limitation, a Portfolio's investment in
OTC derivatives will be calculated as the aggregate gross notional
value of the OTC derivatives.\18\
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    \17\ OTC derivatives include: forwards, options, and swaps
overlying commodities, currencies, financial instruments (e.g.,
stocks, fixed income securities, interest rates, and volatility), or
a basket or index of any of the foregoing. See proposed IEX Rule
16.135(b)(1)(E).
    \18\ See id.
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    Finally, proposed IEX Rule 16.135(b)(1)(F) provides that, to the
extent that listed or OTC derivatives are used to gain exposure to
individual equities and/or fixed income securities, or to indexes of
equities and/or fixed income securities, the aggregate gross notional
value of such exposure shall meet the criteria set forth in IEX Rules
16.135(b)(1)(A) and 16.135(b)(1)(B), respectively.

B. Other Proposed Changes to IEX Rule 16.135

    With respect to proposals to list and trade shares of actively
managed funds that do not satisfy the proposed generic listing
criteria, proposed IEX Rule 16.135(b)(1) provides that statements or
representations in those 19b-4s regarding the following constitute
continued listing standards: (1) The description of the portfolio; (2)
limitations on portfolio holdings or reference assets; (3)
dissemination and availability of the reference asset or intraday
indicative values; or (4) the applicability of IEX rules and
surveillance procedures.
    The Exchange also proposes to expand to definition of ``Disclosed
Portfolio'' to require that the Web site for each series of Managed
Fund Shares must disclose the following information, to the extent
applicable: ticker symbol, CUSIP or other identifier, a description of
the holding, identity of the asset upon which the derivative is based,
the strike price for any options, the quantity of each security or
other asset held as measured by select metrics, maturity date, coupon
rate, effective date, market value, and percentage weight of the
holding in the portfolio.\19\
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    \19\ See proposed IEX Rule 16.135(c)(2).
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    Additionally, the Exchange proposes to amend the continued listing
requirements in Rule 16.135(d)(2)(A) by changing the requirement that
an Intraday Indicative Value (``IIV'') for Managed Fund Shares be
widely disseminated by one or more major market data vendors at least
every 15 seconds during the time when the Managed Fund Shares trade on
the Exchange to a requirement that an IIV be widely disseminated by one
or more major market data vendors at least every 15 seconds during the
Regular Market Session, as defined in IEX Rule 1.160(gg).
    The Exchange proposes to require that every issue of Managed Fund
Shares have a stated investment objective and that it be adhered to
under normal market conditions.\20\
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    \20\ See proposed IEX Rule 16.135(d)(1)(C). ``Normal market
conditions'' includes, but is not limited to, the absence of trading
halts in the applicable financial markets generally; operational
issues (e.g., systems failure) causing dissemination of inaccurate
market information; or force majeure type events such as a natural
or man-made disaster, act of God, armed conflict, act of terrorism,
riot or labor disruption, or any similar intervening circumstance.
See proposed IEX Rule 16.135(c)(5).
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    Further, the Exchange also seeks to amend Rule 16.135(d)(2)(C) to
provide that IEX will consider suspension of trading and will initiate
delisting proceedings under the IEX Rule Series 14.500 with respect to
a series of Managed Fund Shares (rather than only considering removing
a series from listing) under the following new or revised
circumstances:
    1. If, following the initial twelve-month period after commencement
of trading on IEX of a series of Managed Fund Shares, there are fewer
than 50 beneficial holders of the series of Managed Fund Shares.
    2. If an interruption to the dissemination of the value of the IIV
persists past the trading day in which it occurred or is no longer
calculated or available.
    3. If the Disclosed Portfolio is not made available to all market
participants at the same time.
    4. If the series is not in compliance with any statements or
representations included in the applicable rule proposal under Section
19(b) of the Act regarding: (a) The description of the portfolio or
reference assets; (b) limitations on portfolio holdings or reference
assets; (c) dissemination and availability of the reference asset or
intraday indicative values; or (d) the applicability of IEX rules and
surveillance procedures.
    5. If any of the requirements of IEX Rule 16.135 are not
continuously maintained.
    Further, the Exchange proposes to amend Rule 16.135(g) to provide
that, if an investment adviser to the investment company issuing
Managed Fund Shares is affiliated with a broker-dealer, the investment
adviser must erect and maintain a ``fire wall'' between the investment
adviser and the broker-dealer with respect to access to information
concerning the composition and/or changes to such investment company
portfolio.

III. Discussion and Commission's Findings

    After careful review, the Commission finds that the Exchange's
proposal to amend IEX Rule 16.135 to, among other things, adopt generic
listing criteria and continued listing requirements, is consistent with
the Act and the rules and regulations thereunder applicable to a
national securities exchange.\21\ In particular, the Commission finds
that the proposed rule change is consistent with Section 6(b)(5) of the
Act,\22\ which requires, among other things, that the Exchange's rules
be designed to prevent fraudulent and manipulative acts and practices,
to promote just and equitable principles of trade, to remove
impediments to and perfect the mechanism of a free and open market and
a national market system, and, in general, to protect investors and the
public interest.
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    \21\ In approving this proposed rule change, the Commission has
considered the proposed rule's impact on efficiency, competition,
and capital formation. See 15 U.S.C. 78c(f).
    \22\ 15 U.S.C. 78f(b)(5).
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    IEX's proposal is substantively identical with respect to Managed
Fund Shares to proposals recently approved by the Commission (``Prior
Orders'').\23\ Accordingly, for the reasons discussed in Prior Orders,
the Commission finds that the proposed rule change, as modified by
Amendment No. 1, is consistent with Section 6(b)(5) of the Act \24\ and
the rules and regulations thereunder applicable to a national
securities exchange.
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    \23\ See Securities Exchange Act Release Nos. 78918 (Sep. 23,
2016), 81 FR 67033 (Sep. 29, 2016) (SR-NASDAQ-2016-104); 78396 (Jul.
22, 2016), 81 FR 49698 (Jul. 28, 2016) (SR-BATS-2015-100); and 78397
(Jul. 22, 2016), 81 FR 49320 (Jul. 27, 2016) (SR-NYSEArca-2015-110)
(orders approving generic listing standards for Managed Fund
Shares). See also Securities Exchange Act Release Nos. 80189 (Mar.
9, 2017), 82 FR 13889 (Mar. 15, 2017) (SR-NYSEArca-2017-01); 80169
(Mar. 7, 2017), 82 FR 13536 (Mar. 13, 2017) (SR-BatsBZX-2016-80);
and 79784 (Jan. 12, 2017), 82 FR 6664 (Jan. 19, 2017) (SR-NASDAQ-
2016-135) (orders approving certain continued listing standards).
    \24\ 15 U.S.C. 78f(b)(5).
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    In support of its proposal, the Exchange represents the following:
    (1) Managed Fund Shares listed and traded on IEX will conform to
the initial and continued listing criteria under Rule 16.135;
    (2) The Exchange's surveillance procedures are adequate to continue
to properly monitor the trading of the Managed Fund Shares in all
trading sessions and to deter and detect violations of Exchange rules;
\25\
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    \25\ Specifically, the Exchange intends to utilize its existing
surveillance procedures applicable to derivative products, which
will include Managed Fund Shares, to monitor trading in the Managed
Fund Shares.
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    (3) Prior to the commencement of trading of a particular series of
Managed Fund Shares, the Exchange will inform its members in an
information circular (``Circular'') of the special characteristics and
risks associated with trading the Managed Fund Shares, including
procedures for purchases and redemptions of Managed Fund Shares,
suitability requirements under Rules 3.150 and 3.170, the risks
involved in trading the Managed Fund Shares during the Pre-Market and
Post-Market Sessions when an updated IIV will not be calculated or
publicly disseminated, information regarding the IIV and the Disclosed
Portfolio, prospectus delivery requirements, and other trading
information; \26\
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    \26\ In addition, the Circular will disclose that the Managed
Fund Shares are subject to various fees and expenses, as described
in the applicable registration statement, and will discuss any
exemptive, no-action, and interpretive relief granted by the
Commission from any rules under the Act. Further, the Circular will
disclose that the net asset value for the Managed Fund Shares will
be calculated after 4 p.m., ET, each trading day.

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    (4) The issuer of a series of Managed Fund Shares will be required
to comply with Rule 10A-3 under the Act for the initial and continued
listing of Managed Fund Shares, as provided under the IEX Rule Series
14.400;
    (5) The Exchange, on a periodic basis and no less than annually,
will review issues of Managed Fund Shares generically listed pursuant
to Rule 16.135 and will provide a report to the Regulatory Oversight
Committee of the Exchange's Board of Directors regarding the Exchange's
findings;
    (6) The Exchange will provide the Commission staff with a report
each calendar quarter that includes the following information for
issues of Managed Fund Shares listed during such calendar quarter under
Rule 16.135(b)(1): (a) Trading symbol and date of listing on the
Exchange; (b) the number of active authorized participants and a
description of any failure of an issue of Managed Fund Shares or of an
authorized participant to deliver shares, cash, or cash and financial
instruments in connection with creation or redemption orders; and (c) a
description of any failure of an issue of Managed Fund Shares to comply
with Rule 16.135;
    (7) Prior to listing pursuant to proposed Rule 16.135(b)(1), an
issuer would be required to represent to the Exchange that it will
advise the Exchange of any failure by a series of Managed Fund Shares
to comply with the continued listing requirements;
    (8) Pursuant to its obligations under Section 19(g)(1) of the Act,
the Exchange will monitor for compliance with the continued listing
requirements; and
    (9) If a managed fund is not in compliance with the applicable
listing requirements, the Exchange will commence delisting procedures
under IEX Rule Series 14.500.

This approval order is based on all of the Exchange's representations,
including those set forth above and in Amendment No. 1. For the
foregoing reasons, the Commission finds that the proposed rule change,
as modified by Amendment No. 1, is consistent with Section 6(b)(5) of
the Act \27\ and the rules and regulations thereunder applicable to a
national securities exchange.
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    \27\ 15 U.S.C. 78f(b)(5).
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IV. Accelerated Approval of Amendment No. 1

    As noted above, in Amendment No. 1, the Exchange proposed to adopt
certain continued listing requirements for Managed Fund Shares. The
Commission believes that the changes to the Managed Fund Shares listing
standard proposed in Amendment No. 1: (1) Clarify how the Exchange will
interpret and administer its listing requirements; (2) make Managed
Fund Shares listed on the Exchange less susceptible to manipulation by
adding the firewall provision discussed above; and (3) enhance
consistency between the Exchange's Managed Fund Shares listing criteria
and the requirements for Managed Fund Shares recently adopted by other
national securities exchanges. Accordingly, the Commission finds good
cause, pursuant to Section 19(b)(2) of the Act, to approve the proposed
rule change, as modified by Amendment No. 1, on an accelerated basis.

V. Solicitation of Comments on Amendment No. 1

    Interested persons are invited to submit written data, views, and
arguments concerning whether Amendment No. 1 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 email to<EMAIL_ADDRESS>Please include
File Number SR-IEX-2017-03 on the subject line.

Paper Comments

     Send paper comments in triplicate to Brent J. Fields,
Secretary, Securities and Exchange Commission, 100 F Street NE.,
Washington, DC 20549-1090.

All submissions should refer to File Number SR-IEX-2017-03. This file
number should be included on the subject line if email 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 Web site viewing and
printing in the Commission's Public Reference Room, 100 F Street NE.,
Washington, DC 20549, on official business days between the hours of
10:00 a.m. and 3:00 p.m. Copies of the filing also will be available
for inspection and copying at the principal office of the Exchange. 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-IEX-2017-03 and should be
submitted on or before May 24, 2017.

VI. Conclusion

    It is therefore ordered, pursuant to Section 19(b)(2) of the
Act,\28\ that the proposed rule change (SR-IEX-2017-03), as modified by
Amendment No. 1, be, and it hereby is, approved on an accelerated
basis.\29\
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    \28\ 15 U.S.C. 78s(b)(2).
    \29\ 17 CFR 200.30-3(a)(12).

    For the Commission, by the Division of Trading and Markets,
pursuant to delegated authority.
Eduardo A. Aleman,
Assistant Secretary.
[FR Doc. 2017-08902 Filed 5-2-17; 8:45 am]
 BILLING CODE 8011-01-P


[Federal Register Volume 88, Number 52 (Friday, March 17, 2023)]
[Rules and Regulations]
[Page 16378]
[FR Doc No: 2023-05479]

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DEPARTMENT OF HOMELAND SECURITY

Coast Guard

33 CFR Part 165

[Docket No. USCG-2023-0181]

Safety Zone; Fireworks Displays Within the Fifth Coast Guard
District

AGENCY: Coast Guard, DHS.

ACTION: Notification of enforcement of regulation.

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

SUMMARY: The Coast Guard will enforce a safety zone for a fireworks
display at The Wharf DC on April 1, 2023, to provide for the safety of
life on navigable waterways during this event. Our regulation for
Fireworks Displays within the Fifth Coast Guard District identifies the
safety zone for this event in Washington, DC. During the enforcement
period, vessels may not enter, remain in, or transit through the safety
zone unless authorized to do so by the COTP or his representative, and
vessels in the vicinity must comply with directions from the Patrol
Commander or any Official Patrol displaying a Coast Guard ensign.

DATES: The regulation in 33 CFR 165.506 will be enforced for the
location identified in line no. 1 of table 2 to 33 CFR 165.506(h)(2)
from 7:30 p.m. until 9:30 p.m. on April 1, 2023.

FOR FURTHER INFORMATION CONTACT: If you have questions about this
notification of enforcement, call or email MST2 Courtney Perry, Sector
Maryland-NCR, Waterways Management Division, U.S. Coast Guard:
telephone<PHONE_NUMBER>, email [email protected].

SUPPLEMENTARY INFORMATION: The Coast Guard will enforce the safety zone
regulation for a fireworks display at The Wharf DC from 7:30 p.m. to
9:30 p.m. on April 1, 2023. This action is being taken to provide for
the safety of life on navigable waterways during this event. Our
regulation for Fireworks Displays within the Fifth Coast Guard
District, Sec.  165.506, specifies the location of the safety zone for
the fireworks show, which encompasses portions of the Washington
Channel in the Upper Potomac River. During the enforcement period, as
reflected in Sec.  165.506(d), if you are the operator of a vessel in
the vicinity of the safety zone, you may not enter, remain in, or
transit through the safety zone unless authorized to do so by the COTP
or his representative, and you must comply with directions from the
Patrol Commander or any Official Patrol displaying a Coast Guard
ensign.
    In addition to this notification of enforcement in the Federal
Register, the Coast Guard plans to provide notification of this
enforcement period via the Local Notice to Mariners and marine
information broadcasts.

    Dated: March 13, 2023.
David E. O'Connell,
Captain, U.S. Coast Guard, Captain of the Port, Sector Maryland-
National Capital Region.
[FR Doc. 2023-05479 Filed 3-16-23; 8:45 am]
BILLING CODE 9110-04-P


Qualifier, value, source (may use more than once)	Describe any further
information about the hybridization in a structured manner.	When
additional information is available that would be useful to base queries
on

	Non-specific

Table B.6  Measurements

(MIAME distinguishes between three levels of data processing: image (raw
data), image analysis and quantitation, gene expression data matrix
(normalized and summarized data).

MIAME	Description	When applicable	Notes	Values

B.6.1 Raw data	Each hybridization has at least one image.

	Scanner image file	The image file including header	Always

TIFF,

JPEG

Scanning protocol

	Steps taken for scanning array and generating an image

	Always

Description of the scanning instruments and the parameter settings.

B.6.2 Image analysis and quantitation	Each image has a corresponding
image quantitation table, where a row represents an array design element
and a column represents different quantitation types

	Mean or median pixel intensity.

Image analysis output	The complete image analysis output for each image.
Always.

Spreadsheet or tab-delimited file

Image analysis protocol

	Documentation of the set of steps taken to quantify the image

	Always.

	 	Image analysis software, the algorithm and all the parameters used

B.6.3 Normalized and summarized data

	Several quantitation tables are combined using data processing metrics
to obtain the ‘final’ gene expression measurement table (gene
expression data matrix) associated with the experiment.

	Data processing protocol

	Documentation of the set of steps taken to process the data. 	When
normalization has been performed

Normalization strategy and the algorithm used to allow comparison of all
data.

Final gene expression table (s)

	Derived measurement value summarizing related elements and replicates,
providing the type of reliability indicator used.	When a value used for
a reliability indicator has been generated	Should be consistent with
quality control description and replicate description	Replicates of the
elements on the same or different arrays or hybridizations, as well as
different elements related to the same entity (e.g., gene).

Reliability indicator for each data point (e.g., standard deviation)

Qualifier, value, source (may use more than once)	Describe any further
information about the measurements in a structured manner	When
additional information is available that would be useful to base queries
on

Appendix C:  Genomics Data Evaluation Record (gDER) Template

Genomics

DATA EVALUATION RECORD

STUDY TYPE:

PC CODE:	DP BARCODE:

	SUBMISSION NO.:

TEST MATERIAL (PURITY):

SYNONYMS:

CITATION:

SPONSOR:

EXECUTIVE SUMMARY:

COMPLIANCE:

I. MATERIALS AND METHODS

A. MATERIALS:

1. Test Material:

	Description:

Lot/Batch #:

Purity:

	CAS # of TGAI:

	[Structure]

2. Control Materials:

	Negative control (if not vehicle) :

Final Volume:

Route:

	Vehicle:

3. Test animals:

	Species:

	Strain:

	Age/weight at study initiation:

	Source:

	No. animals used per dose per duration:

	Properly Maintained?

4. Compound administration:

a. Test material

		Dose levels:

		Route:

		Method:

b. Vehicle control:

c. Positive control:

B. TEST PERFORMANCE

1. Treatment and Sampling Times:

	Duration of dosing:

	Frequency of dosing:

	Total number of doses:

	Timing and frequency of sampling:

	Time elapsed between dosing and sampling:

2. Tissues and Cells Examined:

3. Details of tissue harvest:

4. Detail of tissue storage:

5. Sample Preparation:

	a	RNA Isolation, Labelling, Amplification:

Histology:

Immunochemistry:

Western blot analysis:

Array analysis:

Characteristics of the arrays:

Methods:

Data Analysis:

Evaluation Criteria/Statistical Analysis

II. REPORTED RESULTS

A. IMMUNOCHEMISTRY:

B. WESTERN BLOT ANALYSIS:

C. MICROARRAY ANALYSIS:

III. DISCUSSION and CONCLUSIONS

A. INVESTIGATORS’ CONCLUSIONS:

B. REVIEWER COMMENTS:  	.

C. STUDY DEFICIENCIES:

REFERENCES

ATTACHMENTS and TABLES

Appendix D:  Genomics Data Evaluation Record (gDER) for Alachlor
(Sample)

Genomics

DATA EVALUATION RECORD

STUDY TYPE: Mode of Action In vivo Genomic Analysis in Rat Olfactory
Mucosa

PC CODE:	DP BARCODE:

	SUBMISSION NO.:

TEST MATERIAL (PURITY): Alachlor (Purity not listed)

SYNONYMS:

CITATION:	Genter, M.B., Burman, D.M., Vijayakumar, S., Ebert, C.L.,
Aronow, B.J.  (2002). Genomic analysis of alachlor-induced oncogenesis
in rat olfactory mucosa. Physiol. Genomics 12:35-45.

SPONSOR:

EXECUTIVE SUMMARY:

-catenin (gene/product associated with the wnt signaling pathway),
immunochemistry was also performed on sections prepared for  histology
using an anti-hensin antibody or a commercial antibody; intestinal
sections served as the positive control for antibody staining.  CYP2A3
levels were assessed by Western blot analysis.  For the array analysis,
total RNA was reverse transcribed followed by second-strand cDNA
synthesis.  The resulting cRNA was biotinylated and hybridized to the
Affymetrix GeneChip Rat U34A.

Based on an independent review of qualitative data only (presented as
graphs or photographic copies of tissue sections), it was concluded that
alachlor induces olfactory nasal carcinomas through a nongenotoxic mode
of action (i.e., oxidative stress).  Support for this conclusion comes
from data showing upregulation (2-fold increase over untreated
control) of genes correlated with the following steps in the
carcinogenic process: oxidative stress and damage to DNA  (heme
oxygenase, glutathione and metalothionein, GADD 45,
apurinic/apyramidinic endonuclease);progression  of adenomas to
malignant adenocarcinomas (activation of the wnt signaling pathway), and
transformation to adenocarcinomas (activation of nuclear -catenin
genes, also associated with the wnt signaling pathway).

This study is classified as acceptable (non-guideline) but does not
satisfy current regulatory data  requirements for pesticides.  Although
guidelines do not exist for genomic data, the results presented in this
published article provided critical information that enhances the
understanding of the nongenotoxic mode of action for olfactory mucosal
tumors induced by alachlor.

COMPLIANCE: Not applicable; the publication, however, comes from a
reputable, peer-reviewed  scientific journal.

I. MATERIALS AND METHODS

A. MATERIALS:

1. Test Material:	 Alachlor

	Description:	Not provided

	Lot/Batch #:	Not reported

	Purity:	        % a.i. Not reported

	CAS # of TGAI:

	[Structure]

2. Control Materials:

	Negative control

 	(if not vehicle) :	Final Volume: NA	Route: NA

	Vehicle:		Harlan powder diet

3. Test animals:

	Species:	Rat

	Strain:	Long-Evans

	Age/weight at study initiation:	Not specified

	Source:	Harlan, Indianapolis, IN

	No. animals used per dose per duration	1-2   males;   0  females

	Properly Maintained?	Not specified

4. Compound administration:

a. Test material

		Dose levels: 126 mg/kg/day 	Route Oral – Feeding

	Preliminary: Not performed, referred to a citation (Genter et al.,
(2000).

	Main Study: Dietary administration of 0 or 126 mg/kg/day (tumorigenic
dose as per EPA 1985)

b. Vehicle control:

		Untreated Harlan powdered feed

c. Positive control:

		See Immunochemistry

B. TEST PERFORMANCE

1. Treatment and Sampling Times:  Male rats were fed dietary
preparations of 0 or 126 mg/kg/day for 1 day to 126 mg/kg.

	Sampling (after last dose): 1 and 4 days, 3, 4 and 5 month.  Tumors
were harvested from rats treated for 3, 4, 5, 11 or 18 months.

2. Tissues and Cells Examined:  Ethmoid turbinates and/or oflactory
mucosal tumors

3. Details of tissue harvest: Rats were sacrificed by a pentobarbitol
overdose and decapitated.  Ethmoid turbinates were rapidly removed and
frozen in liquid N2 until use.  RNA samples of olfactory mucosal tumors
derived from two different rats were also harvested.

4. Detail of tissue storage: Stored in liquid N2 until use.

5. Sample Preparation:

	a	RNA Isolation:  Selected frozen tissues were homogenized and total
RNA was precipitated with ethanol/sodium acetate, resuspended in
DEPC-treated water and screened for RNA quality using an Agilent
Bioanalyzer.  Acceptable samples had cut-off ratios of 1.8 for the
28S:18S ribosomal subunits.  Duplicate samples were prepared for 2
rats/group at each sampling time (3, 4, 5 months) and one sample was
used for single rats at days 1 and 4.

-catenin (associated with the 	activation of the wnt signaling
pathway)was localized with antibody from BD-	Transduction Laboratories,
Lexington, KY and visualized using an HRP-	conjugated anti-mouse
secondary antibody and TSA amplification as described.

d	Western blot analysis:  Gene expression changes in the olfactory
specific 	cytochrome P-450 enzyme (CYP2A3),  were assessed by Western
blot analysis of 	5 g of olfactory mucosal microsomal protein per
lane.  Visualization was 	achieved with HRP-conjugated secondary
antibody, enhanced chemiluminescence 	and exposure to X-ray film.

e	Array analysis:  Characteristics of the arrays: The total number of
probe sets 	(genes or expressed sequence tags, ESTs) interrogated was
not reported.  	However, the study authors provided the following
information: ESTs represented 	on the U34A GeneChip were derived from
the Rat Unigene Build No. 34 	assembly.  All clones represented on the
chip were ESTs on gene lists of interest 	and were subjected to
re-annotation by use of Unigene and execution of the 	National Center
for Biotechnology Information (BLASTN) searches 	(  HYPERLINK
"(http://www.ncbi.nlm.gov/BLAST)"  http://www.ncbi.nlm.gov/BLAST)
against non-redundant nucleotide databases 	during February-April, 2002.
 Gene category information was based on all 	publically available gene
ontology information from the Gene and Ontology 	Consortium (  HYPERLINK
"(http://www.geneontology.org)"  http://www.geneontology.org)  as
harvested from SWISS-PROT, 	GeneCards, Compugen. LocusLinks, and
GeneBank as well as exhaustive 	Medline literature searches.

Methods:  Total DNA was reverse transcribed with an oligo-dT primer and
second-strand cDNA was synthesized.  The resulting T7 RNA
polymerase-mediated cRNA was biotin-labeled and hybridized on the
Affymetrix GeneChip Rat U343A using the recommended protocol provided by
Affymetrix

Data Analysis:

The study authors provided the following information.  “MicroArray
Suite 5.0 software was used to scan and quantitate the GeneChip data
using a default scan setting; intensity data were scaled to target
intensity of 1,500, and results were analyzed using the MicroArray Suite
5.0 and GenSpring 4.1.5 software.  Data values used for filtering and
clustering were “signal”, signal confidence”, “absolute call”
(absent or present), and “change” (increased, decreased, unchanged)
as implemented in MicroArray Suite 5.0.  Data were normalized as
follows: the 50th percentile of all measurements was used as the
positive control for each array.  Each measurement for each gene was
divided by this synthetic positive control, assuming that this was at
least 10.  The bottom 10th percentile signal level was used as a test
for correct background subtraction.  The measurement of each gene in
each sample was divided by the corresponding value in untreated samples,
assuming that the value was at least 0.01.  Genes regulated across the
experimental study were identified by data filtering for those over-or
under- expressed in at least two samples whose signal strength was
greater than 500 in two samples, and were also called “present” in
at least two samples.  An additional approach combined those with genes
that could predict length of alachlor exposure or histological responses
using Kruskal-Wallis ANOVA at p<0.001 and a Benjamini-Hochberg multiple
testing correction as implemented in GeneSpring.  K-means analysis were
similarly executed in  GeneSpring to organize genes into clusters based
on similar expression across the treatment time course.”

Evaluation Criteria/Statistical Analysis

Initial Filter Criteria: The study authors indicated that 4,777 probe
set elements (a pool of genes that fulfill a series of initial filter
criteria)  were called “present” by the Affymetrix algorithm on at
least one of 26 chips, 998 probe set elements were overexpressed by 1.8X
or more in at least 2 samples, whereas 584 were underexpressed 0.5 X in
at least 2 samples.  Additionally, significant gene regulation was
detected using a Welch t-test with a cutoff of p<0.001 (without
correction for false discovery rate error).  Using this approach,
alachlor-exposed samples could be distinguished from untreated controls
based on differential expression of 644 genes.

Cluster analysis: 1392 probe sets elements were provided for cluster
analysis by combining the under and over expressed genes along with the
alachlor-regulated genes and then restricting these to only genes that
met the ‘present’ criteria.  Using the K-means algorithm, 16 set
were determined to serve as excellent representations of the prominent
patterns in the data set.  Clusters with “highly chaotic” patterns
were eliminated from further analysis.  Accordingly, 1,265 genes whose
variance was well represented by 16 K-means sets were found.  These
K-means sets were grouped into the following behavior patterns:

Sets that were upregulated acutely

Sets that were upregulated only in alachlor-induced tumors

Sets that were downregulated following alachlor treatment

Sets that were downregulated in alachlor-induced tumors

Sets that were persistently upregulated across the treatment intervals.

II. REPORTED RESULTS

A.  IMMUNOCHEMISTRY:  The study authors stated that ebnerin was highly
expressed after 4 months of treatment with 126 mg/kg/day alachlor in
nasal respiratory mucosa.  Tissue sections of olfactory mucosal tumors
induced by alachlor and control nasal mucosa were provided to support
the study authors’ claim that this gene product was detected in
vehicle control nasal respiratory tissue but was absent in the control
olfactory mucosa.  Increased gene expression of this protein was also
noted in the progression of alachlor-induced tumors.  In the olfactory
tumors, ebnerin was displayed on the surface and in the ductal lumens of
the tumor. In addition, nuclear localization of -catenin was also
confirmed using immunochemical staining.  It was stated that
alachlor-induced polyps and early adenomas did not exhibit nuclear
localization of -catenin but more advanced adenocarcinomas displayed
abundant cytoplasmic and nuclear -catenin; a tissue section of
olfactory mucosal tumors induced by alachlor was presented to support
this claim.

B.  WESTERN BLOT ANALYSIS:  K-mean analysis indicated that after
exposure of the rats to alachlor for 2 or 4 days or 1 month, 137 genes
were downregulated; included among these genes was the olfactory
predominant cytochrome P-450 enzyme, CYP2A3 and CYP2F1, an olfactory
marker protein.  Western blot analysis, depicted in graphs and
accompanied by histological alterations,  also indicated that these
genes/products returned to background levels in the presence of foci of
respiratory metaplasia (3- and 4-  month samples), in the presence of
more pronounced epithelial atypia and small neoplasms present in 25%
of the animals (5-month samples).  CYP2A3 and CYP2F1 were downregulated
in the presence of numerous tumors, some of which were invasive.  These
results were supported by the composite graphs of the 16 K-means sets
presented in the publication.

3-fold increase in the normalized intensity value of 1.0) with acute
(1 day to 1 month) exposure to alachlor were identified.  These include
genes associated with the control of extracellular matrix such as matrix
metalloproteinase-9 (MMP-9, upregulated 9-fold),  carboxypeptidase Z
(upregulated 7-fold) and tissue inhibitor of metalloproteinase-1
(upregulated 3-fold); immune system functions; cell  proliferation/ cell
cycle regulation, including apoptosis-related genes; calcium
homeostasis/signaling; olfactory-related; nervous system-related;
oncogene-related; transporters; and structural machinery.  These genes,
subgrouped according to the key functional categories listed above are
presented in Table 1 of the article (see Attachment 1).  Other genes
mentioned by the study authors as being upregulated 2-fold following
acute exposure included multiple genes which encode proteins associated
with oxidative stress; these included heme oxygenase, glutathione
synthase and metallothionein (MT)-1 and MT-2.  Additionally, the GADD 45
gene (associated with mutagenesis possibly caused by oxidative damage to
DNA) was listed as one of the most highly regulated genes by alachlor.

	An additional, 417 genes and ESTs were identified, based on a 2-fold
upregulated expression, in alachlor-induced tumors as compared to the
untreated mucosa.  These genes included several immune response genes
(i.e., neutrophil defensin, mast cell proteases, squamous cell carcinoma
antigens and major histocompatible complex antigens and genes associated
with cell proliferation (e.g., nucleolin, the major nucleolar protein in
exponentially-growing eukaryotic cells).  Another set of highly
expressed gene were axin2 and frizzled.  The study authors claim that
the increased expression of these genes is suggestive of activation of
the wnt signaling pathway.  This pattern is consistent with the results
of immunochemical staining confirming nuclear localization of
-catenin late in the carcinogenesis process.  Primary normalized
data, gene lists and K-means groups can be obtained from   HYPERLINK
"http://genet.chmcc.org"  http://genet.chmcc.org  in the U34A folder
listed under Genter et al., 2002.

III. DISCUSSION and CONCLUSIONS

A.  INVESTIGATORS’ CONCLUSIONS:  Based on these analyses, the study
authors concluded that “initiation and progression of alachlor-induced
olfactory mucosal tumors is associated with alterations in extracellular
matrix components, induction of oxidative stress, upregulation of
ebnerin, and final transformation to a malignant state by wnt pathway
activation.”

2-fold increase) of genes consistent with a mutagenic response
possibly as a result of oxidative damage to DNA (GADD 45,
apurinic/apyramidinic endonuclease).  While the exact role of GADD
(growth arrest and DNA-damage inducible) gene products is not known,
this gene group is upregulated in response to stress to allow cells time
to repair macromolecular damage or to lead cells into apoptosis so that
a genetic defect is not propagated.  Types of environmental stress that
induce GADD genes include UV irradiation, alkylating agents and glucose
starvation (Takahashi et al., 2001; Jackman et al., 1994).  Stokes et
al. (2002) also demonstrated that GADD 45 gene induction occurs in
response to reactive oxygen species (ROS) and quinones and is abolished
in the presence of the antioxidant, ascorbic acid.  It is of note that
quinones, which are operationally non-genotoxic (Clayson et al., 1994),
are highly redox active molecules which can redox cycle with their
semiquinone radicals, leading to formation of ROS, including superoxide,
hydrogen peroxide, and ultimately the hydroxyl radical.  Production of
ROS can cause severe oxidative stress within cells through the formation
of oxidized cellular macromolecules, including lipids, proteins and DNA
(Bolton et al., 2000).  Supporting the hypothesis of oxidative stress,
Genter et al., also observed upregulation of other genes associated with
oxidative stress,[ i.e., heme oxygenase (Otterbein et al., 2000),
glutathione synthase and metallothionein (Andrews 2000)].

Progression from histologically altered olfactory mucosa to the
development of adenomas

	The study authors stated that this step was accompanied by expression
of genes 	indicating inhibition of apoptosis [Bid3(AI102299)] and
enhancement of cell 	proliferation (zyxin).  However, no data were
provided to support this claim.  	Nevertheless, it is of note that
Sarafian and Bredesen (1994) state that ROS can serve as  	common
mediators of apoptosis.

Progression to a malignant adenocarcinoma phenotype

This phase was indicated by induction of genes (i.e., axin2 and
frizzled) related to activation of the wnt signaling pathway, which are
generally upregulated late in the carcinogenesis process.

Transformation to adenocarcinomas

In the late stages of tumor progression, the activation of nuclear
ß-catenin genes, which is critical for tumor formation in other organs
and is associated with mutations in the wnt pathway.

20% depletion of the olfactory mucosa antioxidant, GHS followed by a
significantly (p<0.001) increased expression of genes associated with
increased GHS production after 2 and 4 days of treatment.  A return to
control values was seen by 10 days of treatment.  A pattern somewhat
similar to GSH  was observed for ascorbate in the olfactory tissue of
126-mg/kg/day male rats (i.e., initially, a significant decrease 1 day
post-treatment, followed by  significant increases 2 and 4 days after
dosing).  In contrast to the GSH data, there was a reduction in
ascorbate at 10 days.  We noted, however, that the response with either
antioxidant was not dose related.  From these results, the investigators
concluded that,  “Despite the fact that GHS levels recovered, acute
antioxidant perturbations may have been sufficient to trigger other
steps in the carcinogenic process.  Therefore, acute depletion of GSH
and ascorbate may trigger more sustained events involved in both the
initiation and promotion of the carcinogenic process.”

DNA damage tissue damage) with a 2-fold increase in DNA-SSB and a
3-fold increase in LDH leakage.  Although olfactory nasal tissue was not
examined in this series of assays, the ability of alachlor to generate
ROS with subsequent DNA damage and tissue damage both  in vivo and in
vitro has been established.  Finally, Bagchi et al. cite the work of
Akubue and Stohs (1991) showing that the oral administration of 800
mg/kg alachlor to rats caused the increased urinary excretion of the
“oxidative lipid metabolites, malondialdehyde, formaldehyde,
acetaldehyde and acetone”.

DNA damage tissue damage  cell proliferationolfactory nasal
tumors) in rats is plausible and coherent.  An additional factor
favoring this MOA is the evidence of weak and sporadic mutagenic
effects, generally seen only at concentration near or at cytotoxic
concentrations.

C. STUDY DEFICIENCIES:

	The independent review of the data presented in this publication was
limited to the analysis of qualitative results presented in graphs or
photographs copies of tissue sections.  Attempts to access the link for
raw data provided in the article failed.  Additionally, there were no
data to support the study authors’ claim of upregulation of genes
associated with apoptosis or cell proliferation.  These data would
complete the sequence of key events in the carcinogenic process for
alachlor. Access to the primary microarray data through a functioning,
public website would have been preferable.

	Based on an independent review of qualitative genomic data (presented
as graphs or photographic copies of tissue sections) in conjunction with
the conventional data, it was concluded that alachlor induces olfactory
nasal carcinomas through a nongenotoxic mode of action (i.e.,
cytotoxicity manifested through oxidative stress).  Partial support for
this conclusion comes from data showing upregulation (2-fold increase
over untreated control) of genes correlated with the following steps in
the carcinogenic process: oxidative stress and damage to DNA progression
of adenomas to malignant adenocarcinomas, and transformation to
adenocarcinomas.  Although guidelines do not yet exist for genomic data,
the results presented in this DER provided critical information that
enhanced the understanding of the nongenotoxic mode of action for
olfactory mucosal tumors induced by alachlor in the rat.

REFERENCES

Andrews, G.K., .2000. Regulation of metallathionein gene expression by
oxidative stress and metal ions.  Biochem. Pharm. 59: 95-104.

Bagchi, D., Bagchi, M., Hassoun, E.A., Stohs, S.J. 1995..  In vitro and
in vivo generation of ROS, DNA damage and lactate dehydrogenase leakage
by selected pesticides.  Toxico. 104: 129-140.

Bolton, J.L., Trush, M.A., Penning, T.M., Dryhurst, G. Monks, TJ. 2000.
Role of quinones in toxicology.  Chem. Res. Toxicol. 13:135-160.

Burman, D.M., Shertzer, H.G., Senft, A.P., Dalton, T., Genter, M.B.
2003. Antioxidant perturbations in the olfactory mucosa of
alachlor-treated rats.  Biochem Pharm 66:1707-1715.

Clayson, D.B., Mehta, R., Iverson, F. 1994. Oxidative DNA damage - The
effect of certain genotoxic and operationally non-genotoxic carcinogens.
 Mutat. Res.317: 25-42.

Kasai, H.1997. Analysis of a form of oxidative DNA damage,
8-hydroxy-2'-deoxyguanosine, as a marker of cellular oxidative stress
during carcinogenesis. Mutat. Res. 387:147-163.

Jackman, J., Alamo  I.Jr.,, Forance, A.J. Jr. 1994. Genotoxic stress
confers preferential and coordinate messenger RNA stability on the five
gadd genes.  Cancer Res. 54:5656-5662.

Otterbein, L.E., Augustine, M.K.C. 2000.  Heme oxygenase: colors of
defense against cellular stress.  Am. J. Physiol. Lung Cell. Mol.
Physiol. 279: 1029-1037.

Sarafian, T.A. and Bredesen, D.E. 1994. Is apoptosis mediated by ROS?
Free Rad. Res. 21:1-8.

Stokes, A.H., Freeman, W.M., Mitchell, S.G., Burnette, T.A., Hellman,
G.M., Vrana, K.E.  2002. Induction of GADD 45 and GADD153 in
Neuroblastoma Cells by Dopamine-Induced Toxicity.  Neuro.Toxicol.
23:675-684.

Takahashi, S., Saito, S., Ohtani, N., Sakai, T. 2001.  Involvement of
the Oct-1 regulatory Element of the gadd45 Promoter in the
p53-independent Response to Ultraviloet Irradiation.  Cancer Res.
61:1187-1195.

Appendix E: MIAME Glossary

For the most recent version of the MIAME glossary, please see:
HYPERLINK "http://www.mged.org/Workgroups/MIAME/miame_glossary.html"
http://www.mged.org/Workgroups/MIAME/miame_glossary.html

Age:  The time period elapsed since an identifiable point in the life
cycle of an organism. (If a developmental stage is specified, the
identifiable point would be the beginning of that stage. Otherwise the
identifiable point must be specified such as planting) [MGED Ontology
Definition]

Amount of nucleic acid labeled:  The amount of nucleic acid labeled

Amplification method:  The method used to amplify the nucleic acid
extracted

Array design:  The layout or conceptual description of array that can be
implemented as one or more physical arrays. The array design
specification consists of the description of the common features of the
array as the whole, and the description of each array design elements
(e.g., each spot). MIAME distinguishes between three levels of array
design elements: feature (the location on the array), reporter (the
nucleotide sequence present in a particular location on the array), and
composite sequence (a set of reporters used collectively to measure an
expression of a particular gene)

Array design name:  Given name for the array design, that helps to
identify a design between others (e.g., EMBL yeast 12K ver1.1)

Array dimensions:  The physical dimension of the array support (e.g. of
slide)

Array related information:  Description of the array as the whole

Attachment:  How the element (reporter) sequences are physically
attached to the array (e.g. covalent, ionic)

Author, laboratory, and contact:  Person(s) and organization (s) names
and details (address, phone, FAX, email, URL)

Biomaterial manipulation:  Information on the treatment applied to the
biomaterial

Bio-source properties:  Information on the source of the sample

Cell line:  The identifier for the immortalized cell line if one was
used to derive the BioMaterial [MGED Ontology Definition]

Cell type:  Cell type used in the experiment if non mixed. If mixed the
targeted cell type should be used [MGED Ontology Definition]

Clone information:  For each reporter, the identity of the clone along
with information on the clone provider, the date obtained, and
availability

Common reference:  A hybridization to which all the other hybridizations
have been compared

Composite sequence information:  The set of reporters contained in the
composite sequence. The nucleotide sequence information for each
composite element: number of oligonucleotides, oligonucleotide sequences
(if given), and the reference sequence accession number (from relevant
databases)

Composite sequence related information:  Information on the set of
reporters used collectively to measure an expression of a particular
gene

Compound:  A drug, solvent, chemical, etc., that can be measured [MGED
Ontology Definition]

Contact details for sample:  The resource (e.g., company, hospital,
geographical location) used to obtain or purchase the BioMaterial and
the type of specimen [MGED Ontology Definition]

Control elements position:  The position of the control features on the
array

Control elements related information:  Array elements that have an
expected value and/or are used for normalization

Control type:  The type of control used for the normalization and their
qualifier

Data processing protocol:  Documentation of the set of steps taken to
process the data, including: the normalization strategy and the
algorithm used to allow comparison of all data

Developmental stage:  The developmental stage of the organism's life
cycle during which the BioMaterial was extracted [MGED Ontology
Definition]

Disease state:  The name of the pathology diagnosed in the organism from
which the BioMaterial was derived. The disease state is normal if no
disease has been diagnosed [MGED Ontology Definition]

Element dimensions:  The physical dimensions of each features

Experiment description:  Free text description of the experiment and
link to an electronic publication in a peer-reviewed journal

Experiment design:  Experiment is a set of one or more hybridizations
that are in some way related (e.g., related to the same publication
MIAME distinguishes between: the experiment design (the design, purpose
common to all hybridizations performed in the experiment), the sample
used (sample characteristics, the extract preparation and the labeling),
the hybridization (procedures and parameters) and the data (measurements
and specifications)

Experiment type (s):  A controlled vocabulary that classify an
experiment

Experimental design:  Design and purpose common to all hybridizations
performed in the experiment

Experimental factor (s):  Parameter (s) or condition (s) tested in the
experiment

Extraction method:  The protocol used to extract nucleic acids from the
sample

Features related information:  Information on the location of the
reporters on the array

Final gene expression table (s):  Derived measurement value summarizing
related elements and replicates, providing the type of reliability
indicator used

Gene name:  The gene represented at each composite sequence: name and
links to appropriate databases (e.g. SWISS-PTOR or organism specific
database)

Genetic variation:  The genetic modification introduced into the
organism from which the BioMaterial was derived. Examples of genetic
variation include specification of a transgene or the gene knocked-out
[MGED Ontology Definition]

Growth conditions:  A description of the isolated environment used to
grow organisms or parts of the organism [MGED Ontology Definition]

Hybridization protocol:  Documentation of the set of steps taken in the
hybridization, including: solution (e.g. concentration of solutes);
blocking agent and concentration used; wash procedure; quantity of
labelled target used; time; concentration; volume, temperature, and
description of the hybridization instruments

Hybridization extract preparation:  Information on the extract
preparation for each extract prepared from the sample

Hybridizations:  Procedures and parameters for each hybridization

Image analysis and quantitation:  Each image has a corresponding image
quantitation table, where a row represents an array design element and a
column to a different quantitation types (e.g. mean or median pixel
intensity)

Image analysis output:  The complete image analysis output for each
image

Image analysis protocol:  Documentation of the set of steps taken to
quantify the image including: the image analysis software, the algorithm
and all the parameters used

In vitro treatment:  The manipulation of the cell culture condition for
the purposes of generating one of the variables under study and the
documentation of the set of steps taken in the treatment

In vivo treatment:  The manipulation of the organism for the purposes of
generating one of the variables under study and the documentation of the
set of steps taken in the treatment

Individual genetic characteristics:  The genotype of the individual
organism from which the BioMaterial was derived [MGED Ontology
Definition]

Individual number:  Identifier or number of the individual organism from
which the BioMaterial was derived. For patients, the identifier must be
approved by Institutional Review Boards (IRB, review and monitor
biomedical research involving human subjects) or appropriate body [MGED
Ontology Definition]

Label incorporation method:  The label incorporation method used

Label used:  The name of the label used

Measurements:  MIAME distinguishes between three levels of data
processing: image (raw data), image analysis and quantitation, gene
expression data matrix (normalized and summarized data)

Normalized and summarized data:  Several quantitation tables are
combined using data processing metrics to obtain the ‘final’ gene
expression measurement table (gene expression data matrix) associated
with the experiment

Nucleic acid type:  The type of nucleic acid extracted (e.g. total RNA,
mRNA)

Number of elements on the array:  The number of features on the array

Number of hybridizations:  Number of hybridizations performed in the
experiment

Organism:  The genus and species (and subspecies) of the organism from
which the BioMaterial is derived [MGED Ontology Definition]

Organism part:  The part or tissue of the organism's anatomy from which
the BioMaterial was derived MGED Ontology Definition]

Platform type:  The technology type used to place the biological
sequence on the array

Production protocol:  A description of how the array was manufactured

Provider:  The primary contact (manufacturer) for the information on the
array design

Qualifier, value, source (may use more than once):  Describe any further
information about the array in a structured manner

Quality control steps:  Measures taken to ensure or measure quality:
replicates (number and description), dye swap (for two channel
platforms) or others (unspecific binding, low complexity regions, polyA
tails)

Raw data:  Each hybridization has at least one image

Relationship between samples and arrays:  Relationship between the
labelled extract (related to which sample which extract) and arrays
(design, batch and serial number) in the experiment

Reporter and location:  The arrangement and the system used to specify
the location of each features on the array (e.g. grid, row, column,
zone)

Reporter approximate length:  The approximate length of the reporter’s
sequence

Reporter generation protocol:  A description of how the reporters were
generated

Reporter related information:  Information on the nucleotide sequence
present in a particular location on the array

Reporter sequence information:  The nucleotide sequence information for
reporter: sequence accession number (from DDBJ/EMBL/GenBank), the
sequence itself (if known) or a reference sequences (e.g. for
oligonucleotides) and PCR primers pair information (if relevant)

Reporter type:  Physical nature of the reporter (e.g. PCR product,
synthesized oligonucleotide)

Sample:  The biological material from which the nucleic acids have been
extracted for subsequent labelling and hybridization. MIAME
distinguishes between: source of the sample (bio-source), its treatment,
the extract preparation, and its labeling

Sample labeling:  Information on the labeling preparation for each
labelled extract

Scanner image file:  The TIFF file including header

Scanning protocol:  Documentation of the set of steps taken for scanning
the array and generating an image including: description of the scanning
instruments and the parameter settings

Separation technique:  Technique to separate tissues or cells from a
heterogenous sample (e.g. trimming, microdissection, FACS)

Sex:  Term applied to any organism able to undergo sexual reproduction
in order to differentiate the individuals or type involved. Sexual
reproduction is defined as the ability to exchange genetic material with
the potential of recombinant progeny [MGED Ontology Definition]

Single or double stranded:  Whether the reporter sequences are single or
double stranded

Spike type and qualifier:  The type of spike used (e.g. oligonucleotide,
plasmid DNA, transcript) and its qualifier (e.g. concentration, expected
ratio, labeling methods)

Spiking control:  External controls added to the hybridization extract
(s)

Spiking control feature:  Position of the feature (s) on the array
expected to hybridize to the spiking control

Strain or line:  Animals or plants that have a single ancestral breeding
pair or parent as a result of brother x sister or parent x offspring
matings [MGED Ontology Definition]

Surface and coating specification: Type of surface and name for the type
of coating used

Targeted cell type:  The targeted cell type is the cell of primary
interest. The BioMaterial may be derived from a mixed population of
cells although only one cell type is of interest [MGED Ontology
Definition]

Treatment type:  The type of manipulation applied to the BioMaterial for
the purposes of generating one of the variables under study [MGED
Ontology Definition]

Appendix F:  Additional Glossary from Genomics White Paper

Allele:  An alternative form of a gene or any other segment of a
chromosome

Bioinformatics:  The analysis of biological information using computers
and statistical techniques; the science of developing and utilizing
computer databases and algorithms to accelerate and enhance biological
research.

Biomarker: A molecular indicator of a specific biological property; a
biochemical feature or facet that can be used to measure the progress of
disease or the effects of treatment.

Complementary DNA (cDNA):  DNA made from a messenger RNA (mRNA)
template. The single-stranded form of cDNA is often used as a probe in
physical mapping.

Biotechnology: Set of biological techniques developed through basic
research and now applied to research and product development. In
particular, biotechnology refers to the use by industry of recombinant
DNA, cell fusion, and new bioprocessing techniques.

Computational Toxicology (Comp Tox): Word used first in EPA’s Interim
Policy on Genomics - “Computational Toxicology is defined as the
application of models from computational and mathematical biology and
computational chemistry for prediction and understanding mechanisms” -
Computational Toxicology Framework Document, ORD, April 2003.

DER:  Data Evaluation Record

Deoxyribonucleic acid (DNA):  Nucleic acid that constitute the genetic
material of all cellular organisms and DNA viruses.  The genetic
information is used in the synthesis of ribonucleic acids (RNAs) from
DNA templates (transcription), and in the synthesis of proteins from
messenger RNA (mRNA) templates (translation).

DNA Microarray:. Microarray is a tool used to sift through and analyze
the information contained within a genome. A microarray consists of
different deoxyribonucleic acid (DNA) probes that are chemically
attached to a substrate, which can be a microchip, a glass slide or a
microsphere-sized bead.

Expressed sequence tag:  A unique stretch of DNA within a coding region
of a gene that is useful for identifying full-length genes and serves as
a landmark for mapping.

FACS:  Fluorescence Activated Cell Sorter

Gene:  The fundamental physical and functional unit of heredity. A gene
is an ordered sequence of nucleotides located in a particular position
on a particular chromosome that encodes a specific functional product
(i.e., a protein or RNA molecule).

Gene chip technology:  Development of cDNA microarrays from a large
number of genes. Used to monitor and measure changes in gene expression
for each gene represented on the chip.

Gene expression:  Process by which a gene's coded information is
converted into the structures present and operating in the cell.
Expressed genes include those that are transcribed into mRNA and then
translated into protein and those that are transcribed into RNA but not
translated into protein (e.g., transfer and ribosomal RNAs).

Genetics:  Study of inheritance patterns of specific traits.

Genetic testing:   Analyzing an individual's genetic material to
determine predisposition to a particular health condition or to confirm
a diagnosis of genetic disease.

Genomics:  Comprehensive study of whole sets of genes, gene products and
their interaction.

Genome:  All the genetic material in the chromosomes of a particular
organism; its size is generally given as its total number of base pairs.

Genotype:  The genetic composition of an organism or a group of
organisms; a group or class of organisms having the same genetic
constitution.

Hazard Assessment:  The process of determining whether exposure to an
agent can cause an increase in the incidence of a particular adverse
health effect (e.g., cancer, birth defect) and whether the adverse
health effect is likely to occur in humans.

Hazard Characterization:  A description of the potential adverse health
effects attributable to a specific environmental agent, the mechanisms
by which agents exert their toxic effects, and the associated dose,
route, duration, and timing of exposure.

Hazard identification:  The process of determining whether it is
scientifically correct to infer that toxic effects observed in one
setting will occur in other settings (e.g., whether substances found to
be carcinogenic or teratogenic in experimental animals are likely to
have the same results in humans).

In Vitro: A biological study is one which is performed in isolation from
a living organism (in contrast to In Vivo studies).

In Vivo:  A biological study is one which is performed within a living
biological organism (as opposed to an In Vitro study).

Knockout:  Inactivation of specific genes. Knockouts are often created
in laboratory organisms such as yeast or mice so that scientists can
study the knockout or null organism as a model for a particular disease.

MAGE:  MicroArray and Gene Expression; the group aims to provide a
standard for the representation of microarray expression data that would
facilitate the exchange of microarray information between different data
systems.

MAGE-OM: Microarray Gene Expression:  Object Model

MGED:  The Microarray Gene Expression Data (MGED) Society is an
international organization of biologists, computer scientists, and data
analysts that aims to facilitate the sharing of microarray data
generated by functional genomics and proteomics experiments.

Mapping:  Charting the location of genes on chromosomes.

Mass spectrometry:  A method used to determine the masses of atoms or
molecules in which an electrical charge is placed on the molecule and
the resulting ions are separated by their mass to charge ratio.

Metabolome:  Entire complement of all the small molecular weight
metabolites inside a cell suspension (or other sample) of interest
(Aberystwyth, University of Wales Web site-
http://dbk.ch.umist.ac.uk/metabol.htm). This profile is a product of the
genome of the organism, the expression of that genome, and the operation
of the metabolism is a particular part of the organism, in a particular
environment.

Metabolomics:  Involves the systematic estimation of metabolomes from a
range of organisms, followed by statistical analyses and other
investigations of that large quantity of data.

Metabonomics:  Study of the endogenous composition of biofluids and
tissues of an organism in order to probe the metabolic state in
homeostasis, and when under interventional stress.  Hector Keun
(Biological Chemistry and Biological Sciences, Imperial College,
London);  Metabolic Profiling: Application to Toxicology and Risk
Reduction.  International Conference, May 14-15, 2003, NIEHS, Research
Triangle Park, North Carolina.

MIAME: Minimum Information About a Microarray Experiment that is needed
to enable the interpretation of the results of the experiment
unambiguously and potentially to reproduce the experiment

Microarray:  A tool used to sift through and analyze the information
contained within a genome. A microarray consists of different nucleic
acid probes that are chemically attached to a substrate, which can be a
microchip, a glass slide or a microsphere-sized bead.

Mode of Action:  Key events and processes, starting with the interaction
of an agent with a cell, through functional and anatomical changes
observed on the progression to toxicity

MOPS-EDTA:  [MOPS] 3-(N-Morpholino) propanesulfonic acid], [EDTA]
ethylenediaminetetraacetic acid

Northern blot:  A technique used to separate and identify RNA.

Nucleotide:  A subunit of DNA or RNA. To form a DNA or RNA molecule,
thousands of nucleotides are joined in a long chain.

“Omics”:   Term including genomics, proteomics, metabonomics (some
differentiate this term from metabolomics), transcriptomics, and
associated bioinformatics (Environmental Health Perspectives, 110: 2002,
1047-1050; Meeting Report: Use of Genomics in Toxicology and
Epidemiology: Findings and recommendations of a workshop). Carol J.
Henry and Vanessa Vu, first and last authors, respectively.

Omics Technologies: A quote often cited describes this phrase“…are
based on comprehensive biochemical and molecular characterizations of an
organism, tissue or cell type” Sumner et. al. 2003.

Phenotype:  The observable physical or biochemical traits of an
organism, as determined by genetics and the environment; the expression
of a given trait based on phenotype; an individual or group of organisms
with a particular phenotype.

PMT:  Photomultiplier tube; used in the capture of raw data

Polymorphism:  The quality or character of genes occurring in several
different forms.

Proteome:  All of the proteins produced by a given species, just as the
genome is the totality of the genetic information possessed by that
species.

Proteomics:  Study of the function of all expressed proteins (Nature,
422: 2003, 193-197).

Quality policy statement:   Describing the specific objectives and
commitment of the laboratory and its management to quality and data
integrity.  An ethics statement may be included at this point.

RNA:  Nucleic acid found in all living cells that plays a role in the
transfer of information from DNA to the protein-forming system of the
cell. The base sequence of an RNA is specified by the base sequence of a
section of the DNA (a Gene) which is used as the template for RNA
synthesis (transcription). (Dorland’s Medical Dictionary)

Risk Assessment (in the context of human health):  The evaluation of
scientific information on the hazardous properties of environmental
agents (hazard characterization), the dose-response relationship
(dose-response assessment), and the extent of human exposure to those
agents (exposure assessment). The product of the risk assessment is a
statement regarding the probability that populations or individuals so
exposed will be harmed and to what degree (risk characterization).

Signal transduction pathway:  The course by which a signal from outside
a cell is converted to a functional change within the cell.

Single nucleotide polymorphism (SNP): A change in which a single base in
the DNA differs from the usual base at that position.

Standard operating procedures (SOPs): listing all routine laboratory
operations documented and signed by management which are available to
clients upon request and readily accessible to staff.  Also known as
laboratory operating procedures and protocols.

Susceptibility: Increased likelihood of an adverse effect, often
discussed in terms of relationship to a factor that can be used to
describe a human subpopulation (e.g. life stage, demographic feature, or
genetic characteristic).

Susceptible Subgroups:  May refer to life stages, for example, children
or the elderly, or to other segments of the population, for example,
asthmatics or the immune-compromised, but are likely to be somewhat
chemical-specific and may not be consistently defined in all cases.

Systems Biology:  A holistic approach to the study of biology with the
objective of simultaneously monitor all biological processes operating
as an integrated system.  Sumner et. al., 2003.

Systems Toxicology:  “…involves the study of perturbation of
organisms by chemicals and stressors, monitoring changes in molecular
expression and conventional toxicological parameters, and iteratively
integrating biological response data to describe the functioning
organism”.

Throughput:  Output or production, as of a computer program, over a
period of time.

Toxicity:  Deleterious or adverse biological effects elicited by a
chemical, physical, or biological agent.

Toxicology:  The study of harmful interactions between chemical,
physical, or biological agents and biological systems.

Toxicogenomics:  The collection, interpretation, and storage of
information about gene and protein activity in order to identify toxic
substances in the environment, and to help treat people at the greatest
risk of diseases caused by environmental pollutants or toxicants.  Study
of the roles that genes play in the biological responses to
environmental toxicants and stressors (Environmental Health Perspective
Toxicogenomics (NIEHS).

Transgenic:  Having genetic material (DNA) from another species. This
term can be applied to an organism that has genes from another organism.

Web-based Glossary Sources

1- National Center for Toxicogenomics (NCT, NIEHS) Glossary
<http://www.niehs.nih.gov/nct/glossary.htm>

2- Human Genome Project Information Web Glossary
<http://www.ornl.gov/sci/techresources/Human_Genome/glossary/>

3- Cambridge Healthtech Institute
<http://www.genomicglossaries.com/CONTENT/omes.asp>

4- The Physical and Theoretical Chemistry Laboratory, Oxford University
Chemical and Other Safety Information <http://ptcl.chem.ox.ac.uk/MSDS/>

5- NIH Glossary
<http://www.accessexcellence.org/AE/AEPC/NIH/gene27.html>

6- Integrated Risk Information Systems (IRIS, EPA) Glossary
<http://www.epa.gov/iris/gloss8.htm>

Appendix G:  Content and Instructional Goals for the Three Levels of
EPA Genomics Technical Training:

Level I:   Introductory Modules – Molecular Biology Concepts

Modules 1-8

Goal:    Provide the basic information necessary for understanding the
more intricate assessments of cellular functions at the molecular level.
 Introduce gene arrays and discuss how genomics data may affect risk
assessments in the future – this module will tie into EPA’s current
Genomics Policy.  Issues relating to how to communicate genomics
information to risk managers and the public will be addressed.

Target Audience:   Non-scientists and/or technical staff without
training in biological sciences, such as:

	Managers from Office of Research and Development, Regional and Program
Offices

Regional Risk Managers (e.g., Remedial Project Managers, On Scene
Coordinators)

Attorneys

Staff from Regional Office Programs (e.g., Air, Water, Waste,
Pesticides, Community Involvement, Tribal Program)

Staff from States and Tribes

Components:  	Cell structure and function

DNA structure and replication

RNA – Types, functions, transcription (gene expression)

Proteins – General features, formation (translation)

	Gene Arrays – General principles and types

Risk Assessment Concepts – Cancer and non-cancer risk, how genomics
data may affect risk assessments in the future

Regulatory Framework and Risk communication (different regulatory
applications)

Level II: Intermediate Level Modules – Techniques in Molecular Biology

Modules 9-12

Goal:  Provide a general understanding of all of the various
applications that may be currently considered by programs throughout EPA
and is intended to support human health and ecological risk assessors.
Specific modules for individual program applications are considered
separately (see Level II Modules – Specific Applications for Molecular
Tools)

Target Audience:  Scientists and/or those likely to use genomics data
generated by risk assessors are the audience. Modules are intended for
staff who need a more in-depth understanding of how genomics data is
generated, but do not necessarily need to generate that data to support
decision-making.  Modules for specific applications will be developed
(e.g., microbial source tracking, homeland security, field inspectors).
Examples include:

	Laboratory Staff

Regional Laboratories

Office of Research and Development

Enforcement/Compliance Staff (e.g., Water programs, TMDLs, FIFRA)

	Risk Assessors - Human Health and Ecological

Regional Offices

Office of Research and Development

Program Offices

Components:	Background on molecular techniques, such as:

		Microarrays

		DNA amplification using PCR and RT-PCR

		Isolation kits

		Restriction enzymes

		Electrophoresis

		DNA fingerprinting

		Protein Analysis

	Laboratory exercises using various molecular techniques (see above)

	Techniques for specific applications, such as:

		Microbial source tracking

		Homeland security

		Field inspection

	Molecular Biology Approaches in Quantitative Risk Assessment

Level II:  Intermediate Level Modules – Specific Applications for
Molecular Tools

Module 13: Homeland Security

Module 14: Microbial and/or Bacterial Source Tracking

Module 15: Molecular Techniques to Assess Exposure in Environmental
Media

Module 16: Molecular Techniques for Genetically Modified Crop Plant
Inspectors

Goal:  Reinforce information and techniques learned in the Level II
Modules – Techniques in Molecular Biology, and to provide more
in-depth knowledge and skills in the performance of molecular
techniques.  Each of these modules is focused on a separate and specific
application of the molecular tools (introduced in modules 8-11) to
support different programs and needs of the Agency and its staff.  Each
module is intended to provide technical training to staff to increase
the breadth of scientific understanding that will assist in improving
job competencies with respect to science in their particular program
area.

Target Audience:  Same as Level II Modules – Techniques in Molecular
Biology.

Components:   Technical training in particular program areas, focusing
on research and tools currently under development by or through ORD.
For example, Module 13: Microbial and/or Bacterial Source Tracking will
use a newly developed Guide on Tools for Microbial Source Tracking
(Jorge Santodomingo, in preparation), which compares a number of
molecular (RT-PCR, DNA finger printing) and non-molecular (antibiotic
resistance) techniques for identifying pathogenic bacteria from in
water.  This information may be supplemented by laboratory exercises.

Level III: Advanced Modules

Module 17: Data Analysis (1) – Statistical Analysis

Module 18: Data Analysis (2) – Bioinformatics Approaches,
Computational Toxicology

Module 19: Use of Molecular Biology in Mode-Of-Action Determinations

Module 20: Using Genomics Data in Chemical Hazard/Risk Assessment

Overall Goal:  Provide advanced-level knowledge on specific technical
needs that scientists performing research or developing hazard/risk
assessments associated with chemical registrations and other regulatory
activities may face.  Due to the novel and continually evolving nature
of the genomics field, the advanced training modules will be flexible to
account for these potential dynamic changes.  As new technologies and
applications appear, additional or existing training modules will be
developed, enhanced and/or revised.  Modules
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Modules 17 & 18 (Data Analysis 1 & 2)

Goal:  Provide information to research scientists and program office
risk assessors on computational toxicology, bioinformatics and
statistics.  The modules will focus on how to identify and interpret
patterns within the large volumes of genomics data and assess data
significance and accuracy, offering insight into the critical
evaluation, including pros, cons and limitations of possible approaches.

Components – Module 17:

Statistical approaches to microarray data analysis including, but not
limited to:

 	Bayesian statistics

	Correlation

	Clustering

	Principle component analysis

Components – Module 18:

Computational toxicology and bioinformatic approaches and tools used to
analyze genomics data.

Models and molecular biological applications used to predict effects and
understand the cascade of events leading to an effect and how
statistical analyses fits together with other information to form a
bigger picture.

Bioinformatics tools (algorithms and statistics) that will be used to
discriminate unique signature and families of signatures indicative of
stressors and groups of stressors.

Data access (i.e. data mining) and management of data

Module 19:  Use of Molecular Biology in Mode-Of-Action Determinations

Goal:  Introduce the approaches for and limitations of data
interpretation.  This module will provide a link between molecular
biology methods and information and the risk assessment process.

Components:

            The module will present the general concept that an
understanding of the key events associated with the production of
adverse health outcomes at the molecular level could enhance our ability
to predict these outcomes in a qualitative and quantitative sense. In
addition, variability and other uncertainties (e.g., adaptive responses
and homeostatic compensation) surrounding the analysis and
interpretation of microarray data for making quantitative conclusions
about effect/response levels will be discussed.  The concept of
mode-of-action (MOA including key events) will also be introduced.  The
different classes of MOA will be discussed:  these will include
genotoxicity, mutagenicity, receptor-mediated, cell killing regenerative
cell proliferation, and mitogenic responses.  Each of these will be
discussed in terms of the current understanding at the molecular level.
For example, what is cell signaling and how do changes affect cell
function; what is apoptosis and how is it induced; what controls the
cell cycle and how can it be abrogated; what is the mechanism for the
induction of mutations and chromosome changes and the role of DNA repair
and replication?  These molecular underpinnings will allow for examples
of key event pathways to be discussed and how chemicals might
potentially impact the various pathways.

Module 20: Using Genomics Data in Chemical Hazard/Risk Assessment

Goal:  Provide guidance on the incorporation of genomics (microarray)
data in a weight-of-evidence approach for hazard assessment.  Present
principles and pitfalls using simple case studies.  Case studies will be
flexible to meet the needs of the programs and offices, for example,
case studies may focus on homeland security and microbial source
tracking applications.

Components:

Case studies such as:

Examples where microarray data quality is high

Examples that demonstrate data concerns which could lead to erroneous
conclusions

Case Studies should be developed to support the need of the programs and
Regional offices, e.g., homeland security, microbial source tracking,
ambient water quality monitoring, etc. to support the use of microarray
data or for other molecular-biology-based or “omics” approaches.
Examples include, but are not limited to:

Demonstration of purported evidence that a particular chemical belongs
to a particular class of hepatotoxins

Demonstration of purported evidence that chemical has characteristics of
a certain class of hormonally active substances

  These Case Studies should include the following elements:

Purpose

Overall (microarray or other “omics” approach) study design

Purported mode of action and details of how data support proposal,
including purported rationale for utility of microarray data; arguments
to support conclusions

Conventional mechanistic support:  histopathology, clinical chemistry,
metabolic profile, time course to appearance of critical elements,
dose-response information, special studies, etc.

Microarray data:  summary gene expression profile data presentation and
necessary supporting raw data, proposed up and down regulated and
constituent gene identification, rationale for platform and chip design,
demonstration of reproducibility, analysis of variability,  positive and
negative controls, dose response/temporal elements analysis, statistical
analysis; RNA stability

Correlation and comparison:  between conventional and microarray data to
support argument; phenotypic anchoring

Other Evidence:  Structure-Activity Relationship, etc.

Any perceived data gaps

Potential relevance to humans

Weight-Of-Evidence Conclusion

Genter, MB, Burman, DM, Dingeldein, MW, Clough, I , Bolon, B (2000).
Characterization of cell proliferation and immunochemical markers of
alachlor-induced olfactory mucosal tumors in Long-Evans rats.  Toxicol
Pathol 28:770-781.

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External Review Draft of the Interim Guidance for Microarray-Based
Assays

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External Review Draft of the Interim Guidance for Microarray-Based
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External Review Draft of the Interim Guidance for Microarray-Based
Assays

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External Review Draft of the Interim Guidance for Microarray-Based
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External Review Draft of the Interim Guidance for Microarray-Based
Assays

Performance Approach to

Quality Assessment

David Lattier, ORD

Susan Lundquist, OEI

Data Submission

Greg Miller, OPEI

Doug Wolf, ORD

Data Analysis

David Dix, ORD

Brenda Groskinsky, Region 7 RSL

Data Management

Susan Hester, ORD

Joseph Retzer, OEI

Training

Bobbye Smith, Region 9 RSL

Julian Preston, ORD

Microbial Source Tracking

Jorge Santo Domingo, ORD

Ron Landy Region 3 RSL

William H. Benson

Office of Research and Development

Kathryn Gallagher

Office of the

Science Advisor

J. Thomas McClintock

Office of Pollution Prevention and Toxics

Kerry Dearfield

Office of the Science Advisor

(2004 - June 2005)