Endocrine Disruptor Screening Program; Proposed Statement of Policy

Federal RegisterDec 28, 1998

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SUMMARY: In this notice, EPA is providing additional details and an

opportunity for public comment on its Endocrine Disruptor Screening

Program (EDSP). The Agency first set forth the basic components of the

EDSP in the August 11, 1998, Federal Register. The EDSP is required by

the Federal Food, Drug, and Cosmetics Act (FFDCA), as amended by the

Food Quality Protection Act (FQPA). In developing the EDSP, EPA

considered recommendations of the Endocrine Disruptor Screening and

Testing Advisory Committee (EDSTAC), a panel chartered pursuant to the

Federal Advisory Committee Act. EDSTAC recommended expansion of the

screening program beyond the statutory minimum to include not only

pesticides but commercial chemicals regulated under the Toxic

Substances Control Act (TSCA), certain natural products, non-pesticide

food additives, and cosmetics. EDSTAC also recommended that EPA screen

for effects on the androgen and thyroid systems and for effects on fish

and wildlife. This notice describes the major elements of EPA's EDSP,

as well as its implementation. EPA is seeking public comment on the

EDSP in this notice.

DATES: Written comments on this proposed policy must be received by

EPA on or before February 26, 1999.

The joint meeting of the EPA Science Advisory Board (SAB) and

Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) Scientific

Advisory Panel (SAP) to review EPA's proposal for the EDSP will be held

March 30 through April 1, 1999. A document announcing the meeting sites

and times will be published in the Federal Register.

ADDRESSES: Each comment must bear the docket control number OPPTS-

42208. All comments should be sent in triplicate to: OPPT Document

Control Officer (7407), Office of Pollution Prevention and Toxics,

Environmental Protection Agency, 401 M St., SW., Room G-099, East

Tower, Washington, DC 20460.

Comments and data may also be submitted electronically to: oppt.

[email protected]. Follow the instructions under Unit IX. of this notice. No

Confidential Business Information (CBI) should be submitted through e-

mail.

All comments which contain information claimed as CBI must be

clearly marked as such. Three sanitized copies of any comments

containing information claimed as CBI must also be submitted and will

be placed in the public record for this rulemaking. Persons submitting

information on any portion of which they believe is entitled to

treatment as CBI by EPA must assert a business confidentiality claim in

accordance with 40 CFR 2.203(b) for each such portion. This claim must

be made at the time that the information is submitted to EPA. If a

submitter does not assert a confidentiality claim at the time of

submission, EPA will consider this as a waiver of any confidentiality

claim and the information may be made available to the public by EPA

without further notice to the submitter.

FOR FURTHER INFORMATION CONTACT: For general information or copies of

the EDSTAC Final Report: TSCA Hotline, Environmental Assistance

Division (7408), Office of Pollution Prevention and Toxics,

Environmental Protection Agency, 401 M St., SW., Washington, DC 20460;

telephone (202) 554-1404, TDD (202) 554-0551; e-mail address: TSCA-

H[email protected]. For technical information, please contact Anthony

Maciorowski, Office of Pesticide Programs, telephone: (202) 260-3048,

e-mail address: [email protected] or Gary Timm, Chemical

Control Division, Office of Pollution Prevention and Toxics, telephone:

(202) 260-1859, e-mail address: [email protected].

SUPPLEMENTARY INFORMATION

Table of Contents

I. General Information

A. Does this notice apply to me?

B. How can I get additional information or copies of this notice or

other support documents?

1. Electronically. You may obtain electronic copies of this notice

and various support documents from the EPA Home Page at http://

www.epa.gov/. On the EPA Home Page select ``Laws

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and Regulations'' and then look up the entry for this notice under

``Federal Register--Environmental Documents.'' You can also go directly

to the ``Federal Register'' listings at http://www.epa.gov/fedrgstr/.

The complete EDSTAC Final Report is available on the worldwide web

at: www.epa.gov/opptintr/opptendo/whatsnew.htm. Paper copies of the

EDSTAC Final Report can be obtained upon request from the TSCA Hotline

at the address listed under ``FOR FURTHER INFORMATION CONTACT'' section

of this notice.

2. In person or by phone. If you have any questions or need

additional information about this action, please contact the technical

person identified under ``FOR FURTHER INFORMATION CONTACT.'' A public

version of this record, including printed, paper versions which does

not include any information claimed as CBI, is available for inspection

in the TSCA Nonconfidential Information Center, Rm. NE-B607, 401 M St.,

SW., Washington, DC, 12 noon to 4 p.m., Monday through Friday,

excluding legal holidays. The telephone number of the TSCA Docket is

(202) 260-7099.

II. Background

A. Concern Regarding Endocrine Disruptors

The endocrine system consists of glands and hormones which are

found in all mammals, birds, fish, and invertebrates. Hormones are

biochemical substances produced in glands and released into the blood

stream to act on an organ in another part of the body. Over 50 hormones

have been identified in humans and other vertebrates. Hormones control

or regulate many biological processes and are often produced in

exceptionally low amounts within the body. Examples of such processes

include blood sugar control (insulin); differentiation, growth, and

function of reproductive organs (testosterone (T) and estradiol); and

body growth and energy production (growth hormone and thyroid hormone).

Much like a lock and key, many hormones act by binding to receptors

that are produced within cells. The hormone-receptor complex switches

on or switches off specific biological processes in cells, tissues, and

organs.

Scientific evidence has been accumulating that humans, domestic

animals, and fish and wildlife species have exhibited adverse health

consequences from exposure to environmental chemicals that interact

with the endocrine system. To date, such problems have been detected in

domestic or wildlife species with relatively high exposure to

organochlorine compounds (e.g., 1,1,1-trichloro-2,2-bis(p-chlorophenyl)

ethane (DDT) and its metabolite dichorodiphenyldichloroethylene (DDE),

polychlorinated biphenyls (PCBs), and dioxins) or to some naturally

occurring plant estrogens. But effects from exposure to low levels of

endocrine disruptors has been observed as well (e.g., parts per

trillion levels of tributyl tin have caused masculinization of female

marine molluscs such as the dog whelk and ivory shell). Adverse effects

have been reported for humans exposed to relatively high concentrations

of certain contaminants. However, whether such effects are occurring in

the human population at-large at concentrations present in the ambient

environment, drinking water, and food remains unclear. Several

conflicting reports have been published concerning declines in the

quality and quantity of sperm production in humans over the last 4

decades, and there are reported increases in certain cancers (e.g.,

breast, prostate, testicular). Such effects may have an endocrine-

related basis, which has led to speculation about the possibility that

these endocrine effects may have environmental causes. However,

considerable scientific uncertainty remains regarding the actual causes

of such effects. Nevertheless, there is little doubt that small

disturbances in endocrine function, particularly during certain highly

sensitive stages of the life cycle (e.g., development, pregnancy,

lactation) can lead to profound and lasting effects (Kavlock et al.,

1996. EPA, 1997).

Taken collectively, the body of scientific research on human

epidemiology, laboratory animals, and fish and wildlife provides a

plausible scientific hypothesis that environmental contaminants can

disrupt the endocrine system leading to adverse-health consequences. A

critical issue is whether ambient environmental levels are sufficiently

high to exert adverse effects on the general population. Various types

of scientific studies (epidemiology, mammalian toxicology, and

ecological toxicology) are necessary to resolve many of the scientific

questions and uncertainty surrounding the endocrine disruptor issue.

Many such studies are currently underway by government agencies,

industry, and academia.

B. The Food Quality Protection Act, Safe Drinking Water Act, and Other

Environmental Legislation

In 1996, Congress amended the FFDCA with the FQPA. FFDCA section

408(p) requires EPA to develop a program ``to determine whether certain

substances may have an effect in humans that is similar to an effect

produced by a naturally occurring estrogen, or such other endocrine

effects as [EPA] may designate'' (FFDCA section 408(p) (21 U.S.C.

346a(p))).

When carrying out the program, EPA ``shall provide for the testing

of all pesticide chemicals'' and ``may provide for the testing of any

other substance that may have an effect that is cumulative to an effect

of a pesticide chemical if the Administrator determines that a

substantial population may be exposed to such a substance'' (21 U.S.C.

346a(p)(3)).

In addition, Congress amended the Safe Drinking Water Act (SDWA)

and gave EPA authority to provide for the testing, under the FQPA

Screening Program, ``of any other substance that may be found in

sources of drinking water if the Administrator determines that a

substantial population may be exposed to such substance'' (SDWA

Amendments of 1996, section 136 (42 U.S.C. 300j-17)).

This notice describes the major elements of the program EPA has

developed to comply with the requirements of FFDCA section 408 (p) as

amended by FQPA. EPA initially set forth the Program in an August 11,

1998, Federal Register notice (63 FR 42852) (FRL-6021-3). The screening

program described in this notice is ambitious. EPA is considering

87,000 substances as potential candidates for testing. EPA believes

that the FFDCA and SDWA provide authority to require the testing of

many of these substances. EPA will use other testing authorities under

the FIFRA and TSCA to require the testing of those chemical substances

that the FFDCA and SDWA do not cover. EPA also plans to work with other

Federal agencies and departments to ensure that substances not covered

under any of EPA's authorities are tested.

As described in detail in this unit, the EDSP is divided into

several stages, including a priority-setting stage, a stage involving

screening tests (Tier 1 screening), and a stage involving confirmatory

testing (Tier 2 testing). EPA believes that the results from the entire

battery of tests required in the Tier 1 screening and Tier 2 testing

stages (or their equivalents) are necessary to make the statutory

determination of whether a particular

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substance ``may have an effect in humans that is similar to an effect

produced by a naturally occurring [hormone]''(21 U.S.C. 346a(p)). In

other words, a positive result in the Tier 1 screening assays would not

be adequate to make the determination ``whether a substance may have an

effect in humans that is similar to an effect produced by a naturally

occurring [hormone].'' Id. Conversely, a negative result in all Tier 1

screening tests will be adequate to determine that a particular

substance is not likely to have an effect on the estrogen, androgen,

and thyroid hormone systems (EAT) and, therefore, is not a priority for

testing in Tier 2. The confirmatory tests in the Tier 2 testing stage

are necessary to determine whether a substance may have an effect

similar to that of a naturally occurring hormone.

C. The EDSTAC

Recognizing the expertise available outside the Agency on endocrine

disruptor issues, as well as the evolving nature of the science

surrounding endocrine disruption, EPA chartered an advisory committee

under the Federal Advisory Committee Act to advise it on developing a

program to comply with FFDCA section 408(p) requirements. The Advisory

Committee, known as the EDSTAC, was comprised of members representing

the commercial chemical and pesticides industries, Federal and State

agencies, worker protection and labor organizations, environmental and

public health groups, and research scientists. EPA charged the EDSTAC

with providing advice and recommendations to the Agency regarding a

strategy for testing chemical substances to determine whether they may

have an effect in humans similar to an effect produced by naturally

occurring hormones. Specifically, EPA charged EDSTAC with developing

the following:

Methods for chemical selection and priorities for screening.

1. A set of available, validated screening tests for early

application.

2. Ways to identify new and existing screening tests and mechanisms

for their validation.

3. Processes and criteria for deciding when additional tests beyond

screening would be needed and how to validate such tests.

4. Processes for communicating to the public about the EDSTAC's

agreements, recommendations, and information developed during priority

setting, screening, and testing.

In response to this charge, EDSTAC reached consensus on a set of

recommendations for the Agency. These recommendations are contained in

the EDSTAC Final Report (EDSTAC, 1998). Considering EDSTAC's diverse

membership--including individuals from industry, labor, environmental

justice groups, public health and environmental groups, academia, and

Federal and State agencies--EPA found its consensus compelling. More

importantly, EPA found the advice contained in the EDSTAC Final Report

scientifically rigorous. As such, EPA relied heavily on EDSTAC's advice

and recommendations in developing its EDSP. EPA has not further

developed recommendations in areas where EDSTAC recommended further

stakeholder involvement. However, in other areas, EPA has added

additional refinements which are highlighted under ``Issues for

Comment'' in Unit VII. of this notice.

D. Key Terms and Definitions

For the purposes of this notice, EPA will use the following

definitions.

Chemical or chemical substance as used in this notice includes

naturally occurring and synthetic chemicals and elements.

Commercial chemical is defined as chemical substances subject to

the provisions of TSCA (15 U.S.C. 2602 et seq.).

Exempted chemicals are pesticide chemicals that have been given an

exemption under FFDCA section 408(p) or commercial chemicals that the

Agency determines to exempt from the requirements of screening and are

therefore not subject to the EDSP.

Functional equivalency--an assay, test, or endpoint may be defined

as being ``functionally equivalent'' to another assay, test, or

endpoint when it provides equivalent information for each endpoint

being studied. For purposes of the EDSP, assays, tests, and endpoints

must be standardized and validated prior to use. The standardization

and validation process will provide data and information that will

allow EPA to develop guidance on the use of functionally equivalent

assays, tests, and endpoints prior to the implementation of the

screening program.

Hazard assessment is defined to include identification of the

chemical substances and mixtures that have endocrine-disruption effects

(which is often referred to as hazard identification) and establishment

of the relationship between dose and effect (which is often referred to

as dose-response assessment).

Mixtures refers to combinations of two or more chemical substances,

including those found in the environment. This definition is the

ordinary definition applied by chemists and differs from the legal

definition under TSCA section 3. The TSCA definition of mixture

excludes natural products and chemical reaction products that may be a

combination of two or more chemical substances.

Pesticide chemical means any substance that is a pesticide within

the meaning of FIFRA, including all active and inert ingredients of

such pesticide and all impurities.

Polymer is defined as a chemical substance consisting of one or

more types of monomer units and comprising a simple weight majority of

molecules containing at least three monomer units which are covalently

bound to at least one other monomer unit or other reactant and which

consists of less than a simple weight majority of molecules of the same

molecular weight. Such molecules must be distributed over a range of

molecular weights wherein differences in the molecular weight are

primarily attributable to differences in the number of monomer units.

Priority setting is defined as the collection, evaluation, and

analysis of relevant information, including the results of HTPS, to

determine the general order in which chemical substances or mixtures

will be subjected to screening and testing.

Screening is defined as the application of short-term assays to

determine whether a chemical substance or mixture may interact with the

endocrine system. As these are preliminary assays, a positive result

during screening does not mean that a chemical substance may have an

effect in humans, fish, or wildlife that is similar to the effect

produced by naturally occurring hormones.

Sorting is the separation of chemicals into groups prior to

priority setting for the purpose of distinguishing chemicals needing

Tier 1 screening from those needing Tier 2 testing, hazard assessment,

and those for which endocrine screening, testing, or hazard assessment

is not warranted at this time.

Testing is defined as a customized combination of long-term assays

and endpoints designed to determine whether a chemical substance or

mixture may cause effects in humans, fish, or wildlife that are similar

to effects caused by naturally occurring hormones and to identify,

characterize, and quantify these effects. Tests are designed to confirm

and further define the results obtained in Tier 1 screens.

Weight-of-evidence refers to the process by which trained

professionals judge the strengths and weaknesses of a collection of

information to render an

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overall conclusion that may not be evident from consideration of the

individual data.

III. Overview of the Screening Program

A. Scope

Based on the body of available scientific information, EDSTAC

recommended that EPA's EDSP address both human and ecological (fish and

wildlife) effects; examine effects to EAT-related processes; and

include chemical substances and representative mixtures. EPA fully

agrees with the EDSTAC that this is the appropriate scope for the

initial EDSP.

For the reasons stated in this unit, EPA is proposing that the EDSP

include the following:

1. Human and ecological (fish and wildlife) effects. Adverse

effects on wildlife and fish can serve as an early warning of potential

health risks for humans. There is strong evidence for endocrine

disruption observed in natural wildlife and fish populations. Moreover,

wildlife and fish are inherently valuable components of ecosystems, and

they act as sentinels for the relative health of the environment that

they share with humans.

2. Effects on EAT-related processes. Initially, the EDSP will focus

on EAT effects. These three hormone systems are presently among the

most studied of the approximately 50 known vertebrate hormones. In

vitro and in vivo test systems to examine EAT effects exist, and are

currently the most amenable for regulatory testing. Further, inclusion

of EAT effects will cover aspects of reproduction, development, and

growth.

EPA recognizes that there is a great deal of ongoing research

related to other hormones and test systems. As more scientific

information becomes available, EPA will consider expanding the scope of

the EDSP to other hormones. For now, however, the EAT effects and test

systems represent a scientifically reasonable focus for the Agency's

EDSP.

3. Evaluate endocrine disrupting properties of chemical substances

and common mixtures. The universe of chemicals and mixtures to be

prioritized for endocrine-disruptor screening and testing numbers more

than 87,000 and includes commercial chemicals, active pesticide

ingredients, ingredients in cosmetics, nutritional supplements, and

food additives. Commercial chemicals are being included because

chemicals like PCBs and other non-pesticidal chemicals have been

implicated as endocrine disruptors. Nutritional supplements are known

to contain certain naturally occuring phytoestrogens. In addition, EPA

plans to screen representative examples of six different types of

mixtures (i.e., combinations of two or more chemicals). The inclusion

of the representative mixtures was viewed to be a pragmatic, achievable

first look at a highly complex problem. Testing mixtures will determine

whether mixtures cause different endocrine effects from those of the

individual component chemicals. While pharmaceuticals will not be

tested per se since they are already tested and highly regulated for

human or animal use, they may be tested as pollutants if found to be

present in the environment.

B. Program Elements

EPA will use a tiered approach for determining whether a substance

may have an effect in humans that is similar to an effect produced by

naturally occurring EAT. The core elements of the tiered approach

include: Sorting, priority setting, Tier 1 screening, and Tier 2

testing. The purpose of Tier 1 is to identify substances that have the

potential to interact with the endocrine system. The purpose of Tier 2

is to determine whether the substance causes adverse effects, identify

the adverse effects caused by the substance, and establish a

quantitative relationship between the dose and the adverse effect. At

this stage of the science, only after completion of Tier 2 tests will

EPA be able to determine whether a particular substance may have an

effect in humans that is similar to an effect produced by a naturally

occurring EAT, that is, that the substance is an endocrine disruptor.

Therefore, both Tier 1 and Tier 2 are essential elements of the

screening program mandated by the FQPA. Moreover, this tiered approach

is the most effective strategy for using available resources to detect

endocrine-disrupting chemicals and quantify their effects. The core

elements of the program are introduced in this overview section and

presented in greater detail in subsequent sections.

Some of the major implementation steps and estimated completion

dates are:

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

Implementation steps Estimated completion dates

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

EDSTAC Final Report and Recommendations Completed

Development of EPA's EDSP Completed

Public comment on EPA's EDSP February 22, 1999

SAB/SAP Peer Review Processes April 1, 1999

HTPS Demonstration February 1999

HTPS June 2000

EDPSD June 2000

Priority Setting for Tier 1 Phase 1 November 2000

Tier 1 Standardization and Validation 2001

September

Tier 1, Phase 1 TSCA Test Rule Notice of December 2001

Proposed Rulemaking (NPRM) and FQPA

Orders

Tier 1, Phase 1 TSCA Final Test Rule June 2003

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

IV. Sorting and Priority Setting

A. The Universe of Chemicals Included in the EDSP

As stated earlier, EPA is concerned about the endocrine disrupting

potential of more than 87,000 chemical substances, including pesticide

chemicals, commercial chemicals, ingredients in cosmetics, food

additives, nutritional supplements, and certain mixtures. Testing of

all of these chemicals cannot be supported at the same time because,

even if EPA and industry had the resources to do so, there are not

enough laboratories or other facilities capable of conducting the

testing. Consequently, EPA has included a priority-setting phase as

part of its EDSP. During the priority-setting phase, EPA will use

existing information, and in some cases, preliminary test results, to

prioritize chemicals for testing. While EPA believes that the FFDCA and

SDWA provide authority to require the testing of many of these

substances, EPA also will use other testing authorities under FIFRA and

TSCA to require the testing of those chemical substances that the FFDCA

and SDWA do not cover. EPA also plans to work with other Federal

agencies and departments to ensure that these substances also are

tested. EPA will use appropriate authority to obtain testing of the

chemical.

B. Sorting

Chemicals under consideration for EAT screening will undergo

sorting based on existing, scientifically relevant information. The

sort would identify chemicals for HTPS as well as place chemicals into

categories 1-4.

1. Category 1--Hold--Chemicals with sufficient, scientifically

relevant information to determine that they are not likely to interact

with the EAT. If

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EPA is able to determine, based on scientifically relevant information,

that a specific chemical is not likely to interact with the EAT, it

will place that chemical in a hold category. Chemicals in this hold

category will have the lowest priority for further analysis and may not

undergo further analysis unless new and compelling information suggests

that the chemical may interact with the endocrine system. Although EPA

will place chemicals in the hold category during the initial sorting

phase of the screening program, it may add chemicals to this category

if, during a later phase of the EDSP (Tier 1 screening, or Tier 2

testing), the Agency determines that a particular chemical is not

likely to interact with the endocrine system.

Currently, EPA believes it is appropriate to assign two groups of

chemicals to the hold category:

i. Polymers.

ii. Exempted chemicals.

These substances would not be subject to HTPS or to priority

setting for screening at this time (See Fig. 1).

[GRAPHIC] [TIFF OMITTED] TN28DE98.002

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i. Polymers. EPA anticipates placing most polymers with a number

average molecular weight (NAMW) greater than 1,000 daltons in the hold

category. These polymers are not likely to cross biological membranes

and therefore are not likely to be biologically available to cause

endocrine-mediated effects. EPA will not place polymers that are

pesticide chemicals, and therefore must be tested under the FFDCA, in

this category. In addition, EPA will not place monomer and oligomer

components of polymers in this hold category. Instead, it will

prioritize them for Tier 1 screening or Tier 2 testing.

ii. Exempted chemicals. Exempted chemicals are pesticides given an

exemption under FFDCA 408(p) and other chemicals that the Agency

determines to exempt from the requirements of screening. These

substances would not be included in the HTPS and would be placed in the

hold category (see Unit. VI.L. of this notice).

2. Category 2--Priority Setting/Tier 1 Screening--Chemicals for

which there is insufficient, scientifically relevant information to

determine whether or not they are likely to interact with the EAT. If

EPA is not able to determine, based on scientifically relevant

information, whether or not a chemical is likely to interact with the

EAT, it will place that chemical into a category of chemicals needing

Tier 1 screening. Category 2 chemicals are those for which there is

insufficient scientifically relevant information to be placed on hold

(Category 1), or assigned to Tier 2 testing (Category 3) or hazard

assessment (Category 4). Category 2 chemicals will be subjected to

formal priority setting, and Tier 1 screening, and as appropriate (i.e.

positive results in Tier 1 screening), Tier 2 testing.

3. Category 3--Tier 2 Testing--Chemicals with sufficient,

scientifically relevant information comparable to that provided by the

Tier 1 screening. Recognizing the need for flexibility, EPA has

included the possibility of bypassing Tier 1 screening. For example, if

sufficient, scientifically relevant information already exists

regarding a specific chemical, EPA may move that chemical directly into

Tier 2 testing. In addition, EPA may allow a chemical to bypass Tier 1

if the chemical's producer or registrant chooses to conduct Tier 2

testing without performing Tier 1 screening.

4. Category 4--Hazard Assessment--Chemicals with sufficient,

scientifically relevant information to bypass Tier 1 screening and Tier

2 testing. For certain chemicals, there already may be sufficient,

scientifically relevant information regarding their interaction with

EAT--information comparable to that derived from Tier 1 screening and

Tier 2 testing--to move them directly into hazard assessment. These

chemicals, thus, will bypass both Tier 1 screening and Tier 2 testing.

EPA anticipates that this will be a relatively small number of

chemicals.

C. Information Required for Sorting and Priority Setting

Relevant scientific information is essential to sort and prioritize

chemicals for endocrine-disruptor testing. EPA plans to use three main

categories of information to set priorities: Exposure-related

information, effects-related information, and statutory criteria. EPA

is in the process of developing a relational data base to manage the

information that it will use to set priorities. A relational data base

is one that can link with other data bases thus allowing EPA to access

and manipulate data from other existing data bases.

1. Exposure-related information and criteria. EPA proposes to use

several types of existing exposure-related information and criteria for

initial sorting and priority setting. These include at least four

exposure information categories and one fate and transport information

category. The four exposure-related information categories are:

Biological sampling data for humans and other biota; environmental

monitoring data, and information on occupational , consumer product,

and food-related exposures; data on environmental releases; and data on

production volume and use. Note that the data categories are listed

from most robust (actual presence in biological tissue confirming that

exposure has occurred) to least robust (amounts produced which may or

may not result in exposure).

This unit describes the nature of the information included in each

exposure-related information category, the strengths and limitations of

the type of information in each category, and a set of guiding

principles that EPA will generally apply to complete the task of

setting priorities for endocrine-disruptor screening and testing.

i. Biological sampling data. Biological sampling refers to the

monitoring of tissues from live or dead organisms for chemicals to

document actual human or animal exposure. Biological sampling

information falls into two subcategories: Human biomonitoring and

monitoring of other biota. Human biomonitoring includes human tissues

and media (e.g., blood, breast milk, adipose tissue, and urine).

Monitoring of other biota encompasses a wide range of species

(invertebrates, vertebrates such as fish and other wildlife) and sample

matrices (e.g., carcass, liver, kidney, egg, feathers, etc.) for

exposure to environmental contaminants. EPA will be guided by the

following principles when using biological sampling data for sorting

and priority setting.

a. Greater weight is generally given to data sets that provide

relevant information on large populations, disproportionately exposed

subpopulations, or particularly susceptible subpopulations.

b. Greater weight is generally given to non-detect data when it is

associated with low analytical detection limits for organisms that are

likely to be exposed.

ii. Environmental, occupational, consumer product, and food-related

data. Environmental, occupational, consumer product, and food-related

data include: Monitoring data for chemical contaminants found in a

variety of environmental media to which humans and animals are exposed,

such as water (surface, ground, and drinking), air, soil, sediment, and

food; and use information for chemicals, when it is available. EPA will

be guided by the following principles when using environmental,

occupational, consumer product, and food-related data for initial

sorting and priority.

a. Greater weight is generally given to validly measured data than

to estimates.

b. Greater weight is generally given to data that demonstrate that

a chemical is more likely to be internalized by an organism from its

environment.

c. Greater weight is generally given to data sets that provide

relevant information on large populations, disproportionately exposed

subpopulations, or particularly susceptible subpopulations.

d. Greater weight is generally given to non-detect data when it is

associated with low analytical detection limits for organisms that are

likely to be exposed.

In the absence of monitoring data, estimates from the National

Occupational Environment Survey, Permissible Exposure Limits (PELs) and

similar estimates will be used to infer potential exposure levels.

These estimates are much less robust than monitoring data but will be

used unless actual monitoring data are submitted.

iii. Environmental releases. Environmental release information

includes data on chemicals released to the environment to which humans

and environmental species may be exposed, such as permitted industrial

discharges to air or water and accidental release or spill data. EPA

may use data from its Toxic Release Inventory (TRI) and the Agency for

Toxic Substances Disease Registry's (ATSDR's) Hazardous

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Substance Emergency Surveillance System. EPA will be guided by the

following principles when using environmental release data for sorting

and priority setting.

a. Greater weight is generally given to validly measured data than

to estimates.

b. Greater weight is generally given to data demonstrating that an

environmental release will more likely lead to organism exposure.

(e.g., EPA will give greater weight to TRI releases to air and water

than TRI releases to permitted landfills, etc.).

c. Greater weight is generally given during priority setting to

data sets that provide relevant information on large populations,

disproportionately exposed subpopulations, or particularly susceptible

subpopulations.

iv. Production volume data. Production volume data are generally

available for existing chemicals, but not for polymers, inorganics, or

chemicals under 10,000 pounds of annual production. (These latter

substances have been exempted from EPA's quadrennial TSCA Inventory

Update Rule (40 CFR part 710, subpart B)). For new chemicals, the only

production volume information available is estimates and it is not

relevant for environmental contaminants. EPA will be guided by the

following principles when using production volume data for sorting and

priority setting.

a. Production volume provides only a very rough indication of

potential human and environmental exposure.

b. Production data generally should be combined with other data

(e.g., use and physical properties data) in an effort to minimize some

of the inherent weaknesses of using production data as a surrogate for

exposure.

c. Production information generally should not be used to compare

existing industrial chemicals, pesticides and new chemicals because

production volume ranges are too divergent. For example, production

volumes for high-volume industrial chemicals are several orders of

magnitude higher than those for either new chemicals or pesticides.

v. Fate and transport data and models. The fate and transport

information category includes chemical and/or physical properties that

may be used to predict or estimate the medium or media where a chemical

is likely to be found and whether or not a chemical is likely to remain

in the environment over time.

Environmental fate and transport information is available from

various reference sources, including data bases, textbooks, and

monographs. Numerous sources of data and models are listed in Appendix

G of the EDSTAC Final Report (EDSTAC, 1998). The sheer volume of

environmental fate and transport data makes it necessary to identify

those data useful for sorting and prioritization purposes. EPA will

focus attention on three subcategories of environmental fate and

transport information including: Persistence, mobility, and

bioaccumulation.

EPA will consider the following characteristics of fate and

transport data: Hydrolysis half-life persistence; biodegradation

persistence; photooxidation persistence; volatility (Henry's Law)

mobility; adsorption coefficient (Koc ) mobility; and

octanol: water partition coefficient (Kow/LogP) mobility and

bioaccumulation. EPA may use a multimedia fate and partitioning model

to combine this information in a meaningful manner. EPA will be guided

by the following principles when using fate and transport data and

models for initial sorting and priority.

a. Air, water, and soil environmental compartments generally should

be considered when using fate and transport data to help set priorities

for screening.

b. Greater weight generally should be given to fate and transport

characteristics based on laboratory or field tests than on estimates.

2. Effects-related information and criteria. EPA generally plans to

rely on HTPS data, toxicological laboratory studies, epidemiological

studies, and predictive structure activity models to assist the Agency

in setting priorities for screening.

i. Toxicological and epidemiological studies. Toxicological

laboratory studies include information related to the laboratory study

of toxic effects of commercial chemicals, pesticides, contaminants, or

mixtures on living organisms or cell systems including humans,

wildlife, or laboratory animals. Epidemiological and field studies

range from hypothesis-generating descriptive studies, such as case

reports and ecological field analyses, to prospective cohort studies

and rigorously controlled hypothesis-testing clinical trials.

Empirical toxicological and epidemiological data are reported in

numerous peer-reviewed scientific journals. Published studies are

conducted and described in varying degrees of methodological rigor and

data are reported in widely varying detail. To rely on this

information, EPA would be required to review it and determine its

applicability and adherence to generally acceptable investigatory

practices. The search and review of this primary literature would be

too resource intensive to be part of the prioritization process.

Instead EPA will rely on data bases containing studies addressing the

endpoints of interest. In response to EPA's proposed Priority List,

public commenters can submit studies that EPA will review. If the

submitted studies indicate that the priority should be changed or they

meet the requirements of portions of Tier 1, EPA will change the

priority or screening requirements for that chemical, as appropriate.

EPA will be guided by the following principles when evaluating

toxicological and epidemiological data:

a. Negative epidemiological studies generally will not override

positive toxicological studies. Positive epidemiological studies

generally will override negative toxicological studies for priority-

setting purposes.

b. EPA generally will give greater weight to in vivo studies with

relevant endpoints than to in vitro studies.

ii. Predictive structure-activity models. Predictive biological

activity or effects models attempt to identify the correlation between

chemical structure and biological activity, including those that can be

identified through in vitro and in vivo screens. Models can be useful

when biological data are unavailable. While EPA believes this approach

will be of limited success early in the screening program, it believes

that the refinement of models as more screening results become

available may increase their utility as a predictive tool for priority

setting and may actually replace some of the more mechanistic Tier 1

assays.

3. Statutory criteria. The FFDCA, as amended, requires that EPA

provide for the testing of all ``pesticide chemicals.'' Under the

FFDCA, ``pesticide chemical'' includes ``any substance that is a

pesticide within the meaning of FIFRA, including all active and inert

ingredients'' (21 U.S.C. 321(q)(1)). It also includes impurities. The

statute does not restrict testing to pesticides used on foods. As part

of priority setting, EPA will ensure that all substances that must be

tested pursuant to the FFDCA--i.e., pesticide chemicals--are tested in

a timely manner.

D. Use of a HTPS to Assist Priority Setting

For the majority of chemicals, EPA does not believe that any

endocrine-disruptor effects data exists. This lack of data makes it

difficult to set priorities for screening and testing. To help solve

this problem, EPA plans to conduct two of the Tier 1 screening tests

(see Units V.A. and VI.B. and C. of this notice) on approximately

15,000 chemicals in a high-speed, automated fashion. Since these assays

are being run before the

[[Page 71549]]

Tier 1 screening is conducted, EPA refers to this testing as HTPS. HTPS

test results will provide information on the interaction of chemicals

with the estrogen and androgen receptor. The automated, low-cost nature

of HTPS allows EPA to test a large number of chemicals in a short

period of time. HTPS will provide EPA with preliminary information

relating to one of several possible mechanisms by which a chemical may

affect the endocrine system. Thus, EPA will use HTPS to assist in

setting priorities for further screening; the Agency will not use HTPS

alone to decide whether a chemical should or should not move to the

next phase in the EDSP.

E. Setting Priorities for Tier 1 Screening

EPA plans to use existing, available information, HTPS data, and

the EDPSD to establish Tier 1 screening priorities. EPA anticipates,

however, that the quantity and quality of exposure and effects

information will be uneven for the majority of chemicals. Thus, to

ensure the integrity of the priority-setting process and avoid an

``apples'' to ``oranges'' comparison, EPA plans to adopt a

``compartment-based approach'' to priority setting. The term

``compartment'' refers to the particular information category or

criterion or combinations of information or criteria that defines a set

of chemicals, just as a group of parameters defines a set of numbers in

mathematics. All members of the set must possess the properties

required for membership in the compartment and thus will have these

elements in common as the basis for comparison. Operationally, EPA will

establish a limited number of compartments and sort chemicals into

those compartments based on the criteria defining each compartment. EPA

will then prioritize chemicals within each of the compartments

according to criteria related to those for membership in the

compartment. Finally, EPA will recombine the highest priority chemicals

in each compartment to form the group of chemicals going into phase 1

of the screening program.

EPA has not identified all of the specific compartments. Examples

of compartments, however, may include HPVCs, chemicals in consumer

products, chemicals found in biological tissue, pesticide-active

ingredients, formulation ingredients in pesticides, and chemicals found

in sources of drinking water. A chemical could fall into more than one

compartment. To help develop the list of priority-setting compartments,

EPA plans to convene a priority-setting workshop for multi-

stakeholders. The document announcing the priority-setting workshop is

published elsewhere in this issue of the Federal Register.

Pesticides present a special difficulty in priority setting because

data on both inert formulation ingredients and active ingredients need

to be available at the time of a pesticide's evaluation. This will

present some logistical difficulties in prioritizing the screening of

pesticide formulations since pesticides with the same active ingredient

may contain significantly different formulation inert ingredients.

Although EPA has not identified all priority-setting compartments,

it has decided on some compartments. EPA plans to have a ``mixtures''

compartment, a ``naturally occurring non-steroidal estrogen''

compartment; and a ``nominations'' compartment. Each of these

compartments is described in detail in this unit.

1. Nominations. The priority-setting process generally will give

high priority to chemicals with widespread exposure at the national

level. However, there are chemicals that result in disproportionately

high exposure to identifiable groups, communities, or ecosystems. For

these, EPA plans to establish process by which affected citizens can

nominate chemicals with regional or local exposure to receive priority

for Tier 1 screening (see Unit VI.E. of this notice).

2. Mixtures. Mixtures, defined as a combination of two or more

chemicals, will need special attention during the initial stages of

sorting and prioritization because they present unique challenges for

testing and hazard assessment. Consequently, EDSTAC recommended that

EPA determine the technical feasibility and, where feasible, screen and

test representative samples of mixtures from six distinct types of

mixtures, including: Contaminants in human breast milk; phytoestrogens

in soy-based infant formula; mixtures of chemicals commonly found at

hazardous waste sites; pesticide/fertilizers mixtures; disinfection

byproducts; and gasoline.

EPA will investigate the technical feasibility for screening and

testing mixtures as recommended by EDSTAC. This will include an

evaluation of whether it is possible to identify a reasonable number of

representative samples of mixtures from each of the recommended six

types of mixtures, as well as the ability to send the representative

samples of mixtures through HTPS, Tier 1 screening, and Tier 2 testing

depending on their physical properties, and validation and

standardization of the results.

3. Naturally occurring non-steroidal estrogens (NONEs). Another

special class of chemicals of interest to EPA are naturally occurring

NONEs. These are natural products derived from plants (phytoestrogens)

and fungi (mycotoxins). These chemicals occur widely in foods and have

the potential to act in an additive, synergistic, or antagonist fashion

with other hormonally active chemicals. EPA will work with the Food and

Drug Administration (FDA) and the National Toxicology Program to obtain

testing of the seven specific NONEs that were identified by EDSTAC.

F. Bypassing Tier 1 Screening

Recognizing the need for flexibility in applying the screening and

testing requirements, EPA plans to permit chemicals to bypass Tier 1

screening under certain circumstances. If sufficient, scientifically

relevant information exists regarding a specific chemical, EPA may move

that chemical directly into Tier 2 testing. In addition, EPA may allow

a chemical to bypass Tier 1 screening if the chemical's producer or

registrant chooses to conduct Tier 2 testing without performing Tier 1

screening. Each of these two scenarios has different implications for

the information requirements associated with completing Tier 2 testing.

1. Chemicals that have previously been subjected to 2-generation

reproductive toxicity tests. This scenario includes chemicals that have

previously been subjected to mammalian and wildlife developmental

toxicology and/or reproductive testing, but where the tests did not

include endocrine sensitive endpoints included in the most recent

Office of Prevention, Pesticides, and Toxic Substances (OPPTS) or

Organization for Economic Cooperation and Development (OECD) test

guidelines (See Tables 2, 3, and 4 in Unit V.B. of this notice). Food-

use pesticides fall into this category, as do a small number of certain

other pesticides and industrial chemicals. Chemicals and non-food-use

pesticides that meet this criterion also will likely be candidates for

alternative approaches to Tier 2 testing.

Chemicals that have data from tests that meet the requirements of

the new mammalian guidelines, but not the new wildlife tests, would be

subjected to the wildlife testing requirements unless scientifically

sound reasons are provided to limit testing.

2. Chemicals for which there is limited prior toxicology testing.

The second bypass scenario includes chemicals whose manufacturer or

[[Page 71550]]

registrant has decided to voluntarily complete Tier 2 testing without

having completed the full Tier 1 screening battery or any prior 2-

generation reproductive toxicity testing. Chemicals that bypass Tier 1

screening under this scenario must be evaluated using the entire Tier 2

battery (i.e., the mammalian and non-mammalian multi-generation tests

with all the recommended test species and endpoints) unless

scientifically sound reasons are provided to limit testing.

EPA will generally follow the guidance set forth in this unit when

setting Tier 2 testing priorities for chemicals that bypass Tier 1

screening:

i. If a chemical is deemed to be high priority for Tier 1 screening

and the manufacturer or registrant of the chemical decides to

voluntarily bypass Tier 1, it should also be high priority for Tier 2

testing. Voluntary action on the part of registrants/manufacturers

should expedite testing.

ii. To the extent practicable, pesticides should be tested on the

schedule EPA has established for tolerance reassessments, pesticide re-

registration and registration renewal under the FFDCA and FIFRA, unless

HTPS or other data indicate that the pesticide should be tested in a

shorter timeframe. EPA does not intend to delay tolerance

reassessments, re-registration or registration renewal actions to await

implementation the EDSP.

G. Mixtures

For purposes of the EDSP, EPA defines ``mixture'' as a combination

of two or more chemicals. EPA will consider most commercial chemicals

(class 1 and class 2 substances under TSCA) to be chemicals even though

they may contain other substances in them as impurities or exist as

complex reaction products. In some cases a commercial product is in

reality a complex mixture of unidentified composition in which no

single substance predominates. These complex products have Chemical

Abstract Service (CAS) numbers and will be regarded as chemicals from a

legal and policy perspective but may need to be treated as mixtures

from a scientific perspective in the EDSP. This determination will be

made case by case.

EPA recognizes that the science of evaluating mixtures remains

complex and unclear, but believes that it should begin to confront the

issues raised by them. EPA will sponsor some screening of mixtures

after the demonstration of the HTPS and validation of the Tier 1

screening battery on single chemicals.

Initially, EPA plans to include a few mixtures in the HTPS. EDSTAC

has recommended that one or more representative samples from each of

the following high priority mixtures would be tested:

1. Contaminants in human breast milk.

2. Phytoestrogens in infant soy formula.

3. Mixtures of chemicals found at hazardous waste sites.

4. Pesticide and fertilizer mixtures.

5. Disinfection byproducts.

6. Gasoline.

EPA also plans to evaluate some mixtures in the Tier 1 screen. If

results of Tier 1 are positive for a mixture, the Agency will face a

choice of testing the mixture in Tier 2 or determining what substances,

or combination of substances, are responsible for the activity. The

Agency likely will choose this latter course of action and test the

individual active chemical or active fraction in Tier 2.

H. Categories of Chemicals

In its first TSCA proposed test rule (45 FR 48524, July 18, 1980),

EPA outlined three approaches for testing chemicals belonging to a

chemical category:

1. Test members of a category as individual chemicals.

2. Select test substances to represent the structural and chemical

variation of the category as a whole.

3. Subdivide the category into subgroups and choose a

representative from each as a surrogate for the entire subgroup.

For the HTPS, EPA plans to screen all members of a category that

are produced in quantities over 10,000 pounds. The Agency will make a

case-by-case decision regarding whether all of these chemicals will be

required to go through Tier 1. However, it is likely that the HPVCs

would be screened in Tier 1 regardless of the strategy used. As

Quantitative Structure Activity Relationship (QSAR) modeling becomes

more reliable, the two sampling approaches (approaches 2 and 3 as

described in this unit) may become more viable alternatives.

V. Screening Program

EPA recognizes that a huge number of chemicals could be evaluated

under the EDSP. EPA is adopting EDSTAC's recommendation of a two-tiered

system to make the evaluation process more efficient. In Tier 1, a

screening battery of assays will identify those chemical substances and

mixtures capable of interacting with EAT. Tier 1 covers only screening

tests and these alone are not sufficient to determine whether a

chemical substance may have an effect in humans that is similar to an

effect produced by naturally occurring hormones. The purpose of Tier 2

tests is to determine whether a chemical substance or mixture may cause

endocrine-mediated effects for EAT, determine the consequences to the

organism of the activities observed in Tier 1, and establish the

relationship between the doses of the endocrine-active substance

administered in the test and the effects observed.

A. Tier 1 Screening

Chemical substances or mixtures can alter endocrine function by

affecting the availability of a hormone to the target tissue, and/or

affecting the cellular response to the hormone. Mechanisms regulating

hormone availability to a responsive cell are complex and include

hormone synthesis, serum binding, metabolism, cellular uptake (e.g.,

thyroid), and neuroendocrine control of the overall function of an

endocrine axis. Mechanisms regulating cellular response to hormones are

likewise complex and are tissue specific. Because the role of receptors

is often crucial to cellular responsiveness, specific nuclear receptor

binding assays are included. In addition, tissue responses that are

particularly sensitive and specific to a hormone are included as

endpoints for Tier 1 screens. In order for the Tier 1 screening battery

to discriminate between substances likely to affect the endocrine

system and those not likely to affect it, the screening battery should

meet the following criteria:

1. Detect all known modes of action for the endocrine endpoints of

concern. All chemicals known to affect the action of EAT should be

detected.

2. Maximize sensitivity to minimize false negatives while

permitting a level of as yet undetermined, but acceptable, false

positives. The screening battery should not miss potential EAT active

materials.

3. Include a sufficient range of taxonomic groups among the test

organisms. There are known differences in endogenous ligands,

receptors, and response elements among taxa that may affect endocrine

activity of chemical substances or mixtures. The screening battery

should include assays from representative vertebrate classes to reduce

the likelihood that important pathways for metabolic activation or

detoxification of parent chemical substances or mixtures are not

overlooked.

4. Incorporate sufficient diversity among the endpoints and assays

to reach conclusions based on ``weight-of-evidence'' considerations.

Decisions based on the screening battery results

[[Page 71551]]

will require weighing the data from several assays.

EPA's Tier 1 screening battery meets these criteria. The proposed

Tier 1 screening battery and alternative assays for possible inclusion

are:

Proposed Tier 1 Screening Battery

In Vitro

1. Estrogen Receptor (ER) Binding/Transcriptional Activation Assay.

2. Androgen Receptor (AR) Binding/Transcriptional Activation

Assay. 1

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

1The ER and AR transcription activitation assays are in the

HTPS. Those chemicals which go through the HTPS program, if it is

technically feasible and validated, would not be required to

separately undergo the first two in vitro assays at the bench.

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

3. Steroidogenesis Assay with Minced Testis.

In Vivo

1. Rodent 3-Day Uterotrophic Assay (Subcutaneous (sc)).

2. Rodent 20-Day Pubertal Female Assay with Thyroid.

3. Rodent 5-7-Day Hershberger Assay.

4. Frog Metamorphosis Assay.

5. Fish Gonadal Recrudescence Assay.

Alternative Assays for Possible Inclusion in Tier 1

In Vitro

1. Placental Aromatase Assay.

In Vivo

1. Modified Rodent 3-Day Uterotrophic Assay (Intraperitoneal).

2. Rodent 14-Day Intact Adult Male Assay With Thyroid.

3. Rodent 20-Day Thyroid/Pubertal Male Assay.

EPA plans to include the alternative assays in the standardization

and validation program. Combinations of the alternative assays, if

validated and found to be functionally equivalent, could potentially

replace three of the component assays in the recommended Tier 1

screening battery (in vitro steroidogenesis assay with testis, 20-day

pubertal female assay, and 5-7-day Hershberger assay), thereby possibly

reducing the overall time, cost, and complexity while maintaining

equivalent performance of the overall Tier 1 screening battery.

1. In vitro assays. EPA has identified two categories of in vitro

assays that may be used in Tier 1 screening to assess the binding of

test substances to receptors, i.e., cell-free assays for receptor

binding and transfected cells designed to detect transcriptional

activation. The specific assays chosen, whether done ``at the bench''

or as a HTPS should have the following characteristics:

a. Evaluate binding to estrogen and androgen nuclear receptors.

b. Evaluate binding to the receptor in the presence and absence of

metabolic capability (e.g., one or more of the P450 isozymes, e.g.,

cyp1A1, cyp3A4).

c. Distinguish between agonists and antagonists in functional

assays.

d. Yield dose responses for relative potency of chemical substances

or mixtures exhibiting endocrine activity.

In vitro evaluations can provide both false positive and false

negative results. In vitro false positives (i.e., active in vitro but

not in vivo) arise when a chemical is not absorbed or distributed to

the target tissue, is rapidly metabolically inactivated and/or

excreted, and/or when some other form of toxicity predominates in vivo.

False negatives are considered to be of greater concern if in vitro

tests were used to the exclusion of in vivo methods. In vitro

evaluations can result in false negatives due to their inability, or

diminished capacity, to metabolically activate toxicants. As a result,

EPA's proposed screening battery includes in vivo methods in

conjunction with in vitro techniques. Nevertheless, some in vitro

assays may offer distinct advantages over in vivo assays when

investigating the activity of specific metabolites.

The estrogen and androgen receptor binding assays provide an

indication of the potential of a substance to disrupt ER or AR function

in vivo. In the receptor binding assays the test chemical competes for

binding at the receptor with the natural ligand or other strongly

binding substance. EPA strongly prefers stably transfected

transcriptional-activation assays over receptor binding assays. In

addition to binding, there is a consequence to the binding with the

transcriptional-activation assay, i.e., transcription (synthesis of

messenger Ribonucleic Acid (mRNA)) of a reporter gene and translation

of the mRNA to an identifiable detectable protein such as firefly

luciferase or beta-galactosidase. This assay can distinguish between

agonists and antagonists and can be run with and without metabolic

activation.

The third in vitro assay in the screening battery is the

steroidogenesis assay. This assay utilizes minced testes and detects

the ability of substances to interfere with the endocrine system by

inhibiting the activity of P450 enzymes in the steroid pathway.

Inhibition of mammalian-steroid synthesis can potentially result in a

broad spectrum of adverse effects in vivo, including abnormal serum

hormone levels, pregnancy loss, delayed parturition, demasculinization

of male offspring, lack of normal male and female mating behavior,

altered estrous or menstrual cyclicity, and altered reproductive organ

sizes and weights. Interference with other enzymes involved in the

synthesis of specific hormones will be detected in the in vivo assays.

2. In vivo assays. The value of each individual assay cannot be

considered in isolation from the other assays in the screening battery,

as they have been combined in a manner such that limitations of one

assay are complemented by strengths of another. In vivo assays

complement in vitro assays in several important ways. In vivo methods

in Tier 1 can help reduce false negatives related to absorption,

distribution, metabolism, and excretion of a chemical substance in the

absence of knowledge of its pharmacokinetics. In vivo assays typically

cover a broader range of mechanisms of action than in vitro assays. It

would be impractical to try to include an in vitro assay for every

mechanism of action and in some cases it would be impossible as the

mechanism would be expressed only in whole animal systems. It is clear

that a combination of in vivo and in vitro assays is necessary in order

to detect EAT alterations that act via the ER, AR, thyroid receptor

(TR), inhibition of steroid hormone synthesis, and/or alterations of

the hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituitary-

thyroid (HPT) axes. The screening battery, once validated, should

detect all chemicals with the potential to disrupt the EAT systems,

including xeno(anti)estrogens (that act via the ER or inhibition of

aromatase by oral or parenteral administration), xeno(anti)androgens

(via AR or hormone synthesis), altered HPG axis, and antithyroid action

(via synthesis, metabolism and transport, and the TR). However, results

of even the most specific in vivo assays can be affected by endocrine

mechanisms other than those directly related to ER, AR, and TR action.

The lack of specificity of in vivo assays is a limitation if the goal

is to only identify ER, AR, and TR alterations. In contrast, this lack

of specificity could be considered an advantage if a broader, more

apical screening strategy is desired.

i. Uterotrophic assay. An increase in uterine weight is generally

considered to be one of the best indicators of estrogenicity when

measured in the ovariectomized (ovx) or immature female rat or mouse

after 1-3 days of treatment. EPA is planning to require as part of the

program a 3-day uterotrophic assay using the ovx adult female rat (the

duration can be extended if so desired) with 10 animals per group. EPA

will require sc treatment because most of the

[[Page 71552]]

historical data are collected in this manner and there are relatively

few data concerning the effects of other routes of administration at

this time. EPA is also planning to use this assay to detect

antiestrogens. When run to detect antiestrogens, a control and

xenobiotic-treated group are co-administered with estradiol. The

uterotrophic assay is an in vivo check on the ER binding and ER

reporter gene assays.

ii. 20-Day pubertal female with thyroid. The 20-day pubertal female

assay is the most comprehensive assay in the screening battery. It can

detect thyroid effects, aromatase inhibitors, estrogens, antiestrogens,

and agents which interfere with one of the hormone feedback loops that

controls maturation and reproduction, the HPG axis. Next to in utero

development, the pubertal stage is the most sensitive and vulnerable

life stage.

Exposure of weanling female rats to environmental estrogens can

result in alterations of pubertal development (Ramirez and Sawyer

1964). Exposure to a weakly estrogenic pesticide after weaning and

through puberty induces pseudoprecocious puberty (accelerated vaginal

opening without an effect on the onset of estrous cyclicity) after only

a few days of exposure (Gray et al. 1989). Pubertal alterations are

also observed in girls exposed to estrogen-containing creams or drugs,

which induce pseudoprecocious puberty and alterations of bone

development (Hannon et al. 1987).

In the pubertal female assay, oral dosing is initiated in weanling

rats at 21 days of age (10 per group, selected for uniform body weights

at weaning to reduce variance). The animals are dosed daily, 7 days a

week, and examined daily for vaginal opening (one could also check for

age at first estrus and onset of estrous cyclicity). Dosing continues

until vaginal opening is attained in all females (typically 2 weeks

after weaning, unless delayed). The advantage over the uterotrophic

assay is that one test detects both agonists and antagonists, it

detects xenoestrogens like methoxychlor that are almost inactive via sc

injection, it detects aromatase inhibitors, altered HPG function, and

unusual chemicals like betasitosterol. In addition, at necropsy one

should weigh the ovary (increased in size with aromatase inhibitors,

but reduced with betasitosterol), save the thyroid for histopathology,

take serum for T4, and measure thyroid-stimulating hormone (TSH). In

addition to estrogens, the age at vaginal opening and uterine growth

can be affected by alteration of several other endocrine mechanisms,

including alterations of the HPG axis (Shaban and Terranova 1986; and

Gonzalez et al. 1983). In rats, this event can also be induced by

androgens (Salamon 1938; and EGF (Nelson et al. 1991). In the last 20

years there have been over 200 publications which demonstrate the broad

utility of this assay to identify altered estrogen synthesis, ER

action, growth hormone, prolactin, follicle-stimulating hormone (FSH)

or luteinizing hormone (LH) secretion, or central nervous system (CNS)

lesions.

iii. Rodent 5-7 day Hershberger assay. This assay is designed to

detect androgenic and antiandrogenic effects. In this in vivo assay,

sex accessory gland weights (ventral prostate and seminal vesicle

separately) are measured in castrated, T-treated adult male rats after

4-7 days of treatment by gavage with the test compound. The advantage

of this assay is that it is fairly simple, short term, and relatively

specific for direct androgenic/antiandrogenic effects compared to other

in vivo procedures. To detect both agonists and antagonists the assay

requires two-dosing regimes:

a. Castrated male rat + Xenobiotic (to detect agonist)

b. Castrated male rat + T + Xenobiotic (to detect antagonist)

Although the androgens, T, and dihydrotestosterone (DHT), play a

predominant role in the growth and maintenance of the size of these

accessory gland structures, several other hormones and growth factors

can influence sex organ weights including the thyroid and growth

hormones, prolactin, and epidermal growth factor (EGF). Exposure to

estrogenic pesticides can also reduce sex accessory gland size;

however, it is unclear to what degree these reductions result from

direct versus indirect action of the chemical. Other useful endpoints

that help reveal the mechanism of action include serum hormone levels

of T, DHT, LH, AR distribution, TRPM2/C3 gene activation, ornithine

decarboxylase (ODC), and 5-alpha-reductase activity in the prostate.

The prostate and seminal vesicles should be weighed separately

because these organs differ with respect to the androgen that controls

their growth and differentiation. The prostate is dependent upon

enzymatic reduction of T to DHT, whereas the seminal vesicle is less

dependent upon this conversion. Hence, effects on 5-alpha-reductase can

be distinguished from AR-mediated mechanisms by determining whether the

prostate is preferentially affected. Growth of the levator ani muscle

is T dependent, having little capacity to convert T to the more potent

androgen DHT. Weight of this muscle is useful in identifying anabolic

androgens and antiandrogens, and for this reason has been used

extensively in the pharmaceutical industry. In order to detect

androgenic rather than antiandrogen action one would simply delete the

hormone administration from the protocol.

iv. Frog metamorphosis assay. This assay is in the screening

battery to detect thyroid (increase in tail resorption rate) and

antithyroid (decrease in tail resorption rate) effects. It also

broadens the taxonomic representation of the screening battery. This

assay employs intact larval (tadpole) stages of the African clawed frog

(Xenopus laevis) exposed over a 14-day time period, 50-64 days of age,

to observe the rate of tail resorption (Fort and Stover 1997). Tail

resorption can be easily quantified with computer-aided video image

processing (Fort and Stover 1997). The molecular mechanisms involved in

tail resorption are well characterized (Brown et al. 1995; Hayes 1997a)

and this assay is, therefore, considered to be a simple and specific

assay for thyroid action. Because evidence also suggests that thyroid

action on tail resorption is regulated by corticoids, estrogens, and

prolactin (Hayes 1997b), this assay will address distinctive modulating

pathways and, in tandem with the 20-day mammalian pubertal assay, a

comprehensive screen for thyroid hormone activity is achieved.

v. Fish gonadal recrudescence assay. This assay is in the Tier 1

screening battery because as a group, fish are the most distant from

mammals within the vertebrates, and it provides an additional safeguard

that endocrine disruptors will not pass through the screen undetected.

Intact mature fish maintained under simulated ``winter'' conditions

(short-day length, cool temperatures) exhibit regressed secondary sex

characteristics and gonad maturation.

In this assay, intact fish of both sexes (fathead minnow,

Pimephales promelas, or other appropriate species) are simultaneously

subjected to an increasing photoperiod/temperature regime and test

substance to determine potential effects on maturation from the

regressed position (recrudescence). The primary endpoints examined in

the assay include morphological development of secondary sexual

characteristics, ovary and testis development (weight increases),

gonadosomatic index (ratio of gonadal weight to body weight), final

gamete maturation (ovulation, spermiation), and induction of

vitellogenin. This assay is sensitive to HPG axis effects in

[[Page 71553]]

addition to androgen- and estrogen-related activity.

Having diverse taxa in Tier 1 may give some information on the

homology of the endocrine system across species and likelihood of

consistent response across taxa and among organisms of the same species

and when one must be concerned about variability.

3. Alternative assays for possible inclusion. These assays are

being developed and validated (see Unit VI.F. of this notice) and may

be acceptable cost effective substitutes for some of the assays in the

primary Tier 1 screening battery of recommended by EDSTAC.

i. Placental aromatase assay. Aromatase converts T to estradiol. If

an assay using a male is substituted for the 20-day pubertal female

assay it will be necessary to add this assay to the screening battery

since aromatase is present at very low levels in the testis. It is

present at higher levels in the ovary, uterus, and placenta. Human

placental aromatase is commercially available and could be used in

vitro to assess the effects of toxicants on this enzyme.

ii. Modified rodent 3-day uterotrophic assay (Intraperitoneal). The

intraperitoneal (ip) injection method may enhance the sensitivity of

the uterotrophic assay and is capable of detecting the estrogenic

potential of methoxychlor, which has been cited as an example of a

compound not detectable by the sc route. This is an in vivo assay

(O'Conner et al. 1996) for estrogenic activity in ovx female rats. It

can detect certain antiestrogens with mixed activity, i.e., some

agonistic activity (e.g., tamoxifen).

The rats are injected intraperitoneally with the test agent daily

for 3 days. The females are necropsied either 6 hours or 24 hours after

the final treatment, depending on the protocol employed by the

laboratory. Vaginal cytology is evaluated by vaginal lavage to

determine whether the epithelium has become cornified, indicative of

estrus. Presence of fluid in the uterine lumen is noted and recorded,

and the number of animals that have fluid in the uterus is reported.

Fluid imbibition (uptake) is indicative of estrogenic potential. The

uterus is excised and weighed. It is then preserved in an appropriate

fixative for subsequent histological evaluation, if needed. Subsequent

histological evaluation will be triggered by an equivocal uterine

weight or uterine fluid response (i.e., an increase that is not

statistically significant). This evaluation will consist of a

characterization of the appearance of the uterine epithelium, a

measurement of uterine epithelial cell height, and epithelial mitotic

index or proliferating cell nuclear antigen (PCNA)

immunohistochemistry. Uterine cell height and cell proliferation are

sensitive indicators of estrogenic potential.

iii. 14-Day intact adult male assay. This in vivo assay is intended

to detect effects on male reproductive organs that are sensitive to

antiandrogens and agents that inhibit T synthesis or inhibit 5-alpha-

reductase (Cook et al. 1997). The proponents of this assay believe that

the duration of the assay is sufficient to detect effects on thyroid

gland activity. The rats are anatomically intact and mature; therefore,

they have an intact HPG axis, allowing an assessment of the higher

order neuroendocrine control of male reproductive function and the

thyroid. This assay coupled with the aromatase assay could potentially

replace the Hershberger and the pubertal female assays in the

recommended screening battery. Empirical assessment of this assay has

shown it to be sensitive to agents that are directly antiandrogenic,

inhibit 5-alpha-reductase, inhibit T synthesis, or affect thyroid

function. The sensitivity of this assay, as defined as the ability to

detect a hazard, may be comparable to other assays that have been

recommended.

Young adult male rats (70-90 days of age) are used in this assay.

They are dosed daily with the test agent for 14 days. The recommended

route of administration is ip, which may, in some cases, maximize the

sensitivity of the assay. They are necropsied 24 hours after the final

dose. Immediately after sacrifice, one cauda epididymis is weighed and

processed for evaluation of sperm motility and concentration. The

following organs are weighed: Testes, epididymides, seminal vesicles,

and prostate. The following are fixed and evaluated histologically: One

testis and epididymis and the thyroid. The following hormones are

measured in blood plasma: T4, TSH, LH, T, DHT, and estradiol.

iv. Rodent 20-day thyroid/pubertal male assay. This assay (in

conjunction with the aromatase assay) is another candidate to replace

the pubertal female and Hershberger assays in the screening battery.

The thyroid/pubertal male assay detects androgens and antiandrogens in

vivo in a single stage-apical test. ``Puberty'' is measured in male

rats by determining age at preputial separation (PPS). Preputial

separation and sex accessory gland weights are sensitive endpoints.

However, a delay in PPS is not pathognomonic for antiandrogens.

Pubertal alterations result from chemicals that disrupt hypothalamic-

pituitary function (Huhtaniemi et al. 1986), and, for this reason,

additional in vivo and in vitro tests are needed to identify the

mechanism of action responsible for the pubertal alterations. For

example, alterations of prolactin, growth hormone, gonadotrophin (LH

and FSH) secretion, or hypothalamic lesions alter the rate of pubertal

maturation in weanling rats. Sex accessory gland weights in intact-

adult male rats also can be affected directly or indirectly by toxicant

exposure. The HPG axis in an intact animal is able to compensate for

the action of antiandrogens by increasing hormone production, which

counteracts the effect of the antiandrogen on the tract (Raynoud et al.

1984; Edgren 1994; Hershberger 1953).

Delays in male puberty result from exposure to both estrogenic and

antiandrogenic chemicals including methoxychlor (Gray et al. 1989),

vinclozolin (Anderson et al. 1995b and dichlorodiphenyldichloroethylene

(p,p' DDE) (Kelce et al. 1995). Exposing weanling male rats to the

antiandrogenic pesticides p,p' DDE or vinclozolin delays pubertal

development in weanling male rats as indicated by delayed PPS and

increased body weight (because they are older and larger) at puberty.

In contrast to the delays associated with exposure to estrogenic

substances, antiandrogens do not inhibit food consumption or retard

growth (Anderson et al. 1995). Antiandrogens cause a delay in PPS and

affect a number of endocrine and morphological parameters including

reduced seminal vesicle, ventral prostate, and epididymal weights. It

is apparent that PPS is more sensitive than are organ weights in this

assays. In addition, responses of the HPG are variable. In studies of

vinclozolin, increases in serum LH were a sensitive response to this

antiandrogen, whereas serum LH is not increased in males exposed to

p,p' DDE during puberty (Kelce et al. 1997). Furthermore, a systematic

review of the literature indicates that the sex accessory glands of the

immature intact-male rat are consistently more affected than in the

adult intact-male rat.

Animals are dosed by gavage beginning 1 week before puberty (which

occurs at about 40 days of age) and PPS is measured. Androgens will

accelerate and antiandrogens and estrogens will delay PPS. The assay

takes about 3 weeks and allows for comprehensive assessment of the

entire endocrine system in one study. The animals (10 per group,

selected for uniform body weights to reduce variance) are dosed daily,

7 days a week, and examined daily for PPS. Dosing continues until 53

[[Page 71554]]

days of age; the males are then necropsied. The body, heart (thyroid),

adrenal, testis, seminal vesicle plus coagulating glands (with fluid),

ventral prostate, and levator ani plus bulbocavernosus muscles (as a

unit) are weighed. The thyroid is retained for histopathology and serum

is taken for T4, T3, and TSH. Testosterone, LH, prolactin, and DHT

analyses are optional. These endpoints take several weeks to evaluate

and are affected not only by estrogens but by environmental

antiandrogens, drugs that affect the hypothalamic-pituitary axis

(Hostetter and Piacsek 1977; Ramaley and Phares 1983), and by prenatal

exposure to 2,3,78-tetrachlorodibenzo-p-dioxin (TCDD) (Gray et al.

1995a; Bjerke and Peterson 1994) or dioxin-like PCBs (Gray et al.

1995b). In contrast to these other mechanisms, only peripubertal

estrogen administration accelerates this process in the female and

delays it in the male. Preputial separation in the male rodent is easy

to measure and this is not a terminal measure (Korenbrot et al. 1977).

Age and weight at puberty, reproductive organ weights, and serum

hormone levels can also be measured.

As indicated in this unit, the determination of the age at

``puberty'' in the male rat uses endpoints that already have gained

acceptance in the toxicology community. Preputial separation in the

male is a required endpoint in the new EPA 2-generation reproductive

toxicity test guideline. In this regard, this assay would be easy to

implement because these endpoints have been standardized and validated

and PPS data are currently being collected under Good Laboratory

Practice (GLP) conditions in most toxicology laboratories. In addition,

PPS data are reported in many recently published developmental

reproduction studies (i.e., see studies from R.E. Peterson's, J.

Ashby's, R. Chapin's, and L.E. Gray's laboratories on dioxins, PCBs,

antiandrogens, and xenoestrogens).

4. Selection of doses in screening assays. All in vitro screening

assays (including the steroidogenesis assay) will involve multiple-dose

levels, whether performed by HTPS or bench level methods, so a dose-

response curve and assessment of relative potencies can be developed.

EDSTAC recommended that in vivo screening assays be conducted at a

single-dose level to save testing resources. In comments on the draft

EDSTAC Report the SAB/SAP raised concern that relying on a single-dose

level might give false negative results. EPA believes this question can

be resolved in the standardization and validation program. EPA will

require one-, two-, or three-dose levels for in vivo screens depending

upon the results of the standardization and validation program.

Information to assist in selecting the doses in the in vivo screens

includes:

i. Prior information, such as that available during the priority-

setting phase.

ii. Results from the HTPS (or its equivalent bench-level assays).

iii. Results from range-finding studies, utilized for T1S dose

selection.

Results from the HTPS (or its equivalent) will provide potency

information (i.e., EC 50) relative to a positive control such as 17-

beta estradiol (E2), diethylstilbestrol (DES), or T for those chemical

substances or mixtures which bind to the estrogen or androgen

receptors. Information on the in vitro effective doses of E2, DES, or

T, can be used to set the dose level(s), based on the validation

process, for the in vivo Tier 1 screening assays for these chemical

substances or mixtures.

It may be more cost effective to conduct the shortest of the in

vivo screening assays at several doses without the intermediate step of

a range finding study since repeating the study at different doses in

the event that inappropriate doses are used would be relatively

inexpensive. A range-finding study can be performed at multiple dose

levels (at least five) with a few animals per dose level and a limited

number of relevant endpoints. In general, range-finding studies should

meet the following guidelines:

i. Use of the same species strain, sex(es), and age in the assay

for which it is being performed (principal study).

ii. Use of the same route of administration, vehicle, and duration

of dosing as in the principal study.

iii. Use of multiple dose levels; the number of dose levels will

depend on the availability and extent of prior information.

iv. Use of multiple animals per dose level which may be fewer than

the number used per group in the assay.

v. Use of relevant endpoints, which may be more limited than those

in the main assay; for example, the range-finding study for the

uterotrophic assay may employ only body weights and uterine wet weight,

while the full screening assay may also evaluate uterine gland height,

serum hormone levels, and/or vaginal cornification, etc.

vi. Use of comparable animals, e.g., ovarectomized females for the

uterotrophic range-finding study or castrated males for the Hershberger

range-finding assay. However, there may be circumstances under which

exceptions occur, e.g., use of intact males in the range-finding study

for the Hershberger assay to define doses producing systemic toxicity

and any effects on the reproductive system as a first pass

approximation.

vii. Use of more than one range-finding study if the initial

version does not identify the dose level(s) to be used in the specific

Tier 1 screening assay if necessary by extrapolation or interpolation.

The doses to be selected for the in vivo assays should not result

in excessive systemic toxicity, but should result in effects useful for

detection of potential EAT disruption. However, no-dose level higher

than one gram/kilogram body weight/day (i.e., a ``limit'' dose) should

be utilized. The rationale for selection of dose levels for each range-

finding study, all of the results for such studies, and the logic

employed to select the dose level(s) for the principal study should be

included in the submission of study results for evaluation by the

Agency as to the appropriateness of the study design, conduct, and

conclusions.

B. Tier 2 Testing

The purpose of Tier 2 testing is to characterize the likelihood,

nature, and dose-response relationship of the endocrine disruption of

EAT in humans, fish, and wildlife. To fulfill this purpose, the tests

are longer-term studies designed to encompass critical life stages and

processes, a broad range of doses, and administration of the chemical

substance by a relevant route of exposure, to identify a more

comprehensive profile of biological consequences of chemical exposure

and relate such results to the dose or exposure which caused them. Dose

selection, specifically the use of environmentally relevent low doses

for endocrine disruptor testing, has not been conclusively resolved.

The EPA will continue its collaborations with other Federal agencies,

industry, and environmental and public health organizations regarding

low-dose research projects to resolve outstanding scientific questions.

Effects associated with endocrine disruption may be latent and not

manifested until later in life or may not appear until the reproductive

period is reached. Unless a rationale exists to limit the test to 1

generation, tests for endocrine disruption will usually encompass 2

generations including effects on fertility and mating, embryonic

development, sensitive neonatal growth and development, and

transformation from the juvenile life stage to sexual maturity.

The outcome of Tier 2 is designed to be conclusive in relation to

the outcome

[[Page 71555]]

of Tier 1 and any other prior information. Thus, a negative outcome in

Tier 2 will supersede a positive outcome in Tier 1. Furthermore, each

full test in Tier 2 has been designed to include those endpoints that

will allow a definitive conclusion as to whether or not the tested

chemical substance or mixture is or is not an endocrine disruptor for

EAT in that species/taxa. Conducting all five tests in the Tier 2

testing battery would provide a more comprehensive profile of the

effects a chemical substance or mixture could induce via EAT disruption

mode(s)/mechanism(s) of action than would be the case if only a subset

of tests or less comprehensive tests were performed. Considerations for

determining whether the full battery of comprehensive tests should be

implemented include an understanding of mechanisms of action,

environmental fate and transport, persistence, potential for

bioaccumulation, and potential exposure. EPA plans to require that all

tests be performed in Tier 2 with all endpoints, unless compelling

information is presented to show why testing should be limited.

Despite the design of Tier 2 to be as definitive as possible, there

will always be situations in which ambiguous results are obtained. In

some of these cases a weight of evidence approach using Tier 1 and Tier

2 data together may resolve the ambiguity. In others, it may be

necessary to conduct additional special studies or to repeat a test to

resolve the data interpretation issues.

1. Tier 2 tests. EPA is proposing that the Tier 2 test battery

include the following tests: 2-Generation Mammalian Reproductive

Toxicity Study, Avian Reproduction, Fish Reproduction, Amphibian

Reproduction and Developmental Toxicity, and Invertebrate Reproduction.

Except for the amphibian reproduction and developmental toxicity

study, these tests are routinely performed for pesticides with

widespread outdoor exposures that are expected to affect reproduction.

Modifications to each may be necessary to enhance the ability to detect

endocrine-related effects. The amphibian test, though not standardized,

is important because of the extensive fundamental knowledge base on

amphibian development and the realization that amphibians may serve as

key indicators of the health of the environment.

There is utility in considering the results of the entire battery

when assessing human risk. For instance, if the results from different

taxa produce similar results, one can feel more confident that the

results are generally applicable to humans. If the results are widely

divergent, either qualitatively or quantitatively, it indicates greater

biological variability and perhaps additional caution in conducting a

hazard assessment.

i. Mammalian reproductive toxicity. The 2-generation reproductive

toxicity study in rats (40 CFR 799.9380; OPPTS Guideline 870.3800; OECD

Guideline No. 416, 1983; FIFRA, Subdivision F, Guidelines 83-4) is

designed to evaluate comprehensively the effects of a chemical on

gonadal function, estrous cycles, mating behavior, fertilization,

implantation, pregnancy, parturition, lactation, weaning, and the

offspring's ability to achieve adulthood and successfully reproduce,

through 2 generations, one litter per generation. While administration

is usually oral (dosed feed, dosed water, or gavage), other routes are

acceptable if justified (e.g., inhalation). In addition, the study also

provides information about neonatal survival, growth, development, and

preliminary data on possible teratogenesis.

In the existing 2-generation reproductive toxicity test, a minimum

of three-treatment levels and a concurrent control group are required.

At least 20 males and sufficient females to produce 20 pregnant females

must be used in each group as prescribed in this current guideline. The

highest dose must induce toxicity (or meet the limit dose requirement)

but not exceed 10% mortality. In this study, potential hormonal effects

can be detected through behavioral changes, ability to become pregnant,

duration of gestation, signs of difficult or prolonged parturition,

apparent sex ratio (as ascertained by anogenital distances) of the

offspring, feminization or masculinization of offspring, number of

pups, stillbirths, gross pathology and histopathology of the vagina,

uterus, ovaries, testis, epididymis, seminal vesicles, prostate, and

any other identified target organs.

Table 2 provides a summary of the endpoints evaluated within the

framework of the experimental design of the updated 2-generation

reproductive toxicity test (and some recommended additional endpoints

for validation and inclusion to cover EAT concerns). These endpoints

are comprehensive and cover every phase of reproduction and

development. Tests that measure only a single dimension or component of

hormonal activity, (e.g., in vitro or short-term assays) provide

supplementary and/or mechanistic information cannot provide the breadth

of information that is critical for risk assessment.

Additionally, in this study type, hormonally induced effects such

as abortion, resorption, or premature delivery as well as abnormalities

and anomalies such as masculinization of the female offspring or

feminization of male offspring, can be detected. Substances such as the

phytoestrogen, coumesterol, and the antiandrogen cyproterone acetate,

which possess the potential to alter normal sexual differentiation,

were similarly detected in this study test system (i.e., 1982

Guideline).

Table 2 contains two types of lists: First, those endpoints

required in current EPA harmonized 1998 test guidelines; second,

additional endpoints recommended by EDSTAC for validation and inclusion

in both the recommended 2-generation test, as well as the alternative

mammalian tests discussed in Unit V.B.3. of this notice. These

additional endpoints will detect EAT effects.

The default assumption is that all of these endpoints would be

evaluated unless the conditions which are set forth in the guidelines

for determining the selection of endpoints are met.

Table 2.--Mammalian Tier 2 Test Endpoints

Current Guideline Endpoints Sensitive to Estrogens/Antiestrogens

sexual differentiation

gonad development (size, morphology, weight) accessory

sex organ (ASO) development

ASO weight fluid; histology

sexual development and maturation: Acquisition of vaginal patency

(VP), PPS

fertility

fecundity

time to mating

mating and sexual behavior

ovulation

estrous cyclicity

gestation length

abortion

premature delivery

dystocia

spermatogenesis

epididymal sperm numbers and morphology; testicular spermatid head

counts; daily sperm production (DSP); efficiency of DSP

gross and histopathology of reproductive tissues

anomalies of the genital tract

viability of the conceptus in utero (prenatal demise)

survival and growth of offspring

maternal lactational behaviors (e.g., nursing, pup retrieval, etc.)

Current Guideline Endpoints Sensitive to Androgens/Antiandrogens

altered apparent sex ratio (based on AGD)

malformations of the urogenital system

altered sexual behavior

changes in testis and ASO weights

effects on sperm numbers, morphology, etc.

retained nipples in male offspring

[[Page 71556]]

altered AGD (now triggered from PPS/VP)

reproductive development; PPS/VP (puberty)

male fertility

agenesis of prostate

changes in androgen-dependent tissues in pups and adults (not

limited to sex accessory glands)

Recommended Additional Estrogen/Androgen Endpoints for Validation

and Inclusion

ASO function (secretory products)

sexual development and maturation (nipple development and retention)

androgen and estrogen levels

LH and FSH levels

testis descent

Current Guideline Endpoints Sensitive to Thyroid Hormone

Agonists/Antagonists (general)

growth, body weight

food consumption, food efficiency

developmental abnormalities

perinatal mortality

testis size and DSP

VP; PPS

Recommended Additional Thyroid Endpoints for Validation and

Inclusion

neurobehavioral deficits (see developmental landmarks in this unit)

TSH, T4, thyroid weight and histology (e.g., goiter)

developmental landmarks:

prewean includes pinna detachment, surface righting reflex, eye

opening, acquisition of auditory startle, negative geotaxis, mid-air

righting reflex, motor activity on PND 13, 21, etc.

postwean includes motor activity PND 21 and postpuberty ages (sex

difference); learning and memory PND 60--active avoidance/water maze

brain weight (absolute), whole and cerebellum

brain histology

ii. Avian reproduction test. While birds are not included as

subjects in the Tier 1 screening battery, it is important to evaluate

the effects of exposure of birds to chemical substances or mixtures

with endocrine activity.

EPA is planning to modify its Avian Reproduction Test guideline

(OPPTS Guidelines 850.2300) for use in the endocrine disruptor testing

program. The modification include: The additional endpoints presented

in this unit to make the test more sensitive to chemical substances or

mixtures with endocrine activity. Table 3 provides a summary of the

endpoints evaluated within the framework of the Avian Reproduction Test

(and recommended additional endpoints for validation and inclusion to

cover EAT concerns). Two important extensions of this guideline include

modification and standardization of the husbandry and dosing of the

offspring from EPA's Avian Reproduction Test guidelines (OPPTS

Guidelines 850.2300) to create a 2-generation avian reproduction test

and evaluation of an additional exposure pathway (i.e., direct topical

exposure, which is common in the wild, by dipping eggs). The extensions

to the guideline are outlined in Appendix Q in the EDSTAC Final Report

(EDSTAC, 1998).

In the current Avian Reproduction Test guidelines, two species are

commonly used, mallards and northern bobwhite. Exposure of adults

begins prior to the onset of maturation and egg laying and continues

through the egg-laying period; their offspring are exposed, in early

development, by material deposited into the egg yolk by the females.

These offspring can be used efficiently to test for the effects of

chemical substances or mixtures on avian development. There are several

endpoints currently required (see OPPTS Guidelines 850.2300(c)(2)) that

are particularly relevant to disruption of endocrine activity,

including: Eggs laid, cracked eggs, eggshell thickness, viable embryos,

and chicks surviving to 14 days. EPA is extending the guidelines to

require: Additional measurements of circulating steroid titers, thyroid

hormones, major organ (including brain) weights, gland weights, bone

development, leg and wing bone lengths, and ratios of organ weights to

bone measurements; skeletal x-rays; histopathology; functional tests;

and assessment of reproductive capability of offspring (Baxter et al.

1969; Bellabarba et al. 1988; Dahlgren and Linder 1971; Emlen 1963;

Cruickhank and Sim 1986; Fleming et al. 1985a; Fleming et al. 1985b;

Fox 1976; Fox et al. 1978; Freeman and Vince 1974; Hoffman and Eastin

1981; Hoffman and Albers 1984; Hoffman 1990; Hoffman et al. 1993;

Hoffman et al. 1996; Jefferies and Parslow 1976; Kubiak et al. 1989;

Maguire and Williams 1987; Martin 1990; Martin and Solomon 1991;

McArthur et al. 1983; McNabb 1988; Moccia et al. 1986; Rattner et al.

1982; Rattner et al. 1987; Summer et al. 1996; Tori and Mayer 1981).

Table 3.--Avian Reproduction Test Endpoints

Current Guideline Endpoints Sensitive to Estrogens/Antiestrogens,

Androgens/Antiandrogens, and/or HPG Axis

egg production

eggs cracked

viable embryos (fertility)

eggshell thickness

fertilization success

live 18-day embryos

hatchability

14-day-old survivors

Recommended Additional Endpoints for Validation and Inclusion

sex ratio

major organ (including brain) weights

gland weights

histopathology

plasma steroid concentrations

neurobehavioral test (e.g., nest attentiveness)

Current Guideline Endpoints Sensitive to Thyroid Hormone Agonists/

Antagonists

body weight of adults

food consumption of adults

body weight of 14-day-old survivors

developmental abnormalities

Recommended Additional Endpoints for Validation and Inclusion

plasma T3/T4

thyroid histology

bone development (skeletal x-ray)

ratio of organ weights to bone measurements

neurobehavioral test (cliff test)

cold stress test

iii. Fish reproduction test. Fish are the most diverse of all

vertebrates. Reproductive strategies extend from oviparity, to

ovoviviparity, to true viviparity. The consequences of an endocrine

disruptor may be quite different across the many families of fishes. As

a first step though, EPA plans to require use of fathead minnows, or in

special cases, sheepshead minnows in the Fish Life Cycle Test. The Fish

Life Cycle Test consists of continuous exposure from fertilization

through development, maturation, and reproduction, and early

development of offspring with a test duration of up to 300 days. EPA

also anticipates use of the fathead minnow in the Tier 1 fish gonadal

recrudescence assay, and as such, the relevance of any activity

detected in the screening assay would be evaluated. If exposure to a

particular chemical substance or mixture is predominantly estuarine or

marine, EPA may require use of the estuarine sheepshead minnow

(Cyprinodon variegatus) in the test. However, EPA will permit

flexibility to species selection with appropriate justification as to

species choice by the test sponsor.

The Fish Life Cycle Test (OPPTS 850.1500) follows procedures

outlined in (Benoit 1981) for the fathead minnow and (Hansen et al.

1978) for the sheepshead minnow. In general, the test begins with 200

embryos distributed among eight incubation cups in each treatment

group. When hatching is completed, the number of larvae are reduced to

25 individuals, if available, which are released to each of four

replicate larval growth chambers. Four weeks following their release

into the larval growth chambers, the number of juvenile fish are

reduced again and 25 individuals, if available, distributed to each of

two replicate adult test chambers. When fish reach sexual

[[Page 71557]]

maturity, fish are separated into spawning groups (pairs or one male/

two females) with a minimum of eight breeding females. Remaining adults

will be maintained in the tank but will be segregated from the spawning

groups. Adults will be allowed to reproduce, at will, until the 300th

day of exposure. Alternatively, the test may be continued past 300 days

until 1 week passes in which no eggs from any group have been laid. The

embryos and fish are exposed to a geometric series of at least five

test concentrations, a negative (dilution water) control, and, if

necessary, a solvent control.

Assessment of effects on offspring of the parental group (first

filial or F1 generation) will be made by collecting two groups of 50

embryos from each experimental group and incubating those embryos. When

embryos hatch, the number of larvae hatched from each group will be

impartially reduced to 25, if available, and released into the larval

growth chambers. After 4 weeks of exposure, lengths, and weights of

surviving individuals will be recorded.

Observations are made of the effects of the test substance on

embryo hatching success, larvae-juvenile-adult survival, growth of

parental and F1 generation, and reproduction of the adults. Table 4

provides a summary of the endpoints evaluated within the framework of

the Fish Life Cycle Test (and recommended additional endpoints for

validation and inclusion to cover EAT concerns).

Table 4.--Fish Reproduction Test Endpoints

Current Guideline Endpoints Sensitive to Estrogens/Antiestrogens,

Androgens/Antiandrogens, and/or HPG Axis

viability of embryos

time to hatch

spawning frequency

egg production

fertilization success

Recommended Additional Endpoints for Validation and Inclusion

sexual differentiation (tubercle formation, gonadal histology)

sex ratio

gonadosomatic index

gamete maturation (production, final oocyte maturation, sperm

motility test, etc.)

vitellogenin

plasma steroid concentrations

in vitro gonadal steroidogenesis

Current Guideline Endpoints Sensitive to Thyroid Hormone Agonists/

Antagonists

growth, length, and body weight

developmental abnormalities

Recommended Additional Endpoints for Validation and Inclusion

plasma T3/T4

thyroid histopathology

bone development (skeletal x-ray)

ration of organ weights to bone measurements

neurobehavioral test (cliff test)

cold stress test

iv. Invertebrate reproduction test. Although invertebrates do not

generate EAT, EPA plans, through use of this test, to examine in more

depth invertebrate hormones that are functionally equivalent to EAT.

The species of choice would be mysids or daphnia.

Although neither the daphnia nor the mysid chronic test was

designed to examine endocrine-specific endpoints, both species are

crustaceans and therefore share common physiology. Ecdysone is a

steroid hormone that regulates growth and molting in arthropods, and

exhibits some functional and structural similarities to estrogen. The

central role of ecdysone makes it an attractive candidate for examining

endocrine effects in invertebrates; however, other possibilities also

exist. Morphogenetic and reproductive development of arthropods is

controlled in part by juvenile hormone (JH). Methyl farnesoate is a JH

like compound that may play a role in reproduction and development

(Borstet et al. 1987; Laufer et al. 1987a,b).

Invertebrate hormones are beyond the immediate scope of the EDSTAC

which has focused on the vertebrate EAT. Nevertheless, invertebrate

hormones that are functionally equivalent to EAT need to be examined in

more depth. More importantly, chemicals that affect these vertebrate

hormones may also affect invertebrate hormones resulting in altered

reproduction, development, and growth.

Chemicals with estrogenic properties are reported to have altered

normal function of ecdysone systems (Mortimer 1993, 1994, 1995a, 1995b;

Chu et al. 1997). Satyanarayana et al. 1994 showed stimulation of

vitellogenin in insect prepupae and pupae by methoprene, a JH mimic

with retinoid properties. Whether vitellogenin production is controlled

through either an estrogen receptor or an alternative mechanism is not

crucial for obtaining test results that show alteration occurs.

Therefore, the mysid shrimp chronic life cycle test (OPPTS

850.1350) may be adapted to determine whether chemicals that affect

hormonal activity in vertebrates also affect arthropods. Once adapted

to include reproductive and developmental endpoints relevant to the

EDSP, the test could be a useful component in screening and testing.

The other common invertebrate bioassay, one using the water flea,

daphnia, is used internationally (OECD Guideline No. 202). It

incorporates life cycle assessment and reproductive and developmental

endpoints, albeit applied quite differently in this group of animals.

Reproduction is usually parthenogenic in the laboratory in these

animals, limiting the applicability to endpoints identified in this

report. The particular aspect of this system is that the daphnia is

sensitive to estrogenic compounds (Baldwin et al. 1995; Baldwin et al.

1997; Shurin and Dodson 1997), and possesses receptors for T, making

the system sensitive to another vertebrate hormone. Again, this

bioassay would have to be adapted for the endpoints and processes of

interest in the EDSP as a protocol for including invertebrate species

in the endpoints addressed by the EDSP screening and testing batteries.

Other invertebrates, such as molluscs, crayfishes, and echinoderms, do

have EAT, but again relevant standardized tests for evaluating the

consequences of interfering with these systems are not currently

available. It is simply not known whether one (mysid) or two (mysid and

daphnia) Tier 2 tests will provide sufficiently valid information for

other invertebrate groups not tested. This is a source of uncertainty,

potentially leading to Type II errors of unknown magnitude. These

issues will be addressed during the development and validation of this

assay.

v. Amphibian development and reproduction. A definitive amphibian

test, which exposes larvae through metamorphosis and reproduction, is

important to evaluate the consequences of endocrine disruption in

poikilothermic oviparous vertebrate distinct from fishes. A rich

literature on metamorphosis, growth, and reproduction exists for frogs.

No established method has been identified which is suitably

comprehensive to serve as a Tier 2 test at this time but a promising

method is under development by EPA.

2. Alternative test procedures--i. Alternative Mammalian

Reproduction Test (AMRT). One alternative to the 2-generation test

procedure in Unit V.B.1.i. of this notice is the AMRT. The objectives

of this test are to describe the consequences of in utero and/or

lactational exposure on reproduction and development from compounds

that displayed EAT activity in the Tier 1 screens. If validated, this

test may be used, under certain defined circumstances, instead of the

recommended 2-generation reproductive toxicity test (TSCA guidelines,

1997) in Tier 2 tests. In this regard, the test will be conducted with

[[Page 71558]]

at least three treatment groups plus a control and include endpoints

sensitive to chemicals that alter development via EAT activities. As

with the 2-generation mammalian reproductive toxicity study, the

default assumption is that all of the endpoints would be evaluated in

the AMRT, unless the conditions set forth in the guidelines for

determining the selection of endpoints are met.

The AMRT involves exposure of maternal rats (designated F0

generation) from gestational day 6 (time of implantation), through

parturition (birth), and through the lactation period until weaning of

offspring (designated F1 generation) on post-natal day 21. F1 offspring

(both sexes) are retained after weaning with no exposures for 10 weeks

and then mated within groups. F1 males are necropsied after the mating.

F1 females and their litters (designated the F2 generation) are

retained until the F2 generation is weaned. F0 females (and a subset of

F1 weanlings) are necropsied with organ weights and possible

histopathology. F1 animals are evaluated for reproductive development

(VP, PPS), estrous cyclicity, and, at necropsy, for organ weights,

possible histopathology, andrological assessments, and T3/T4 (with TSH

triggered). F2 weanlings are counted, sexed, weighed, examined

externally, and discarded.

The AMRT differs from the ``standard'' 2-generation study design in

that it:

a. Does not include exposures prior to mating, during mating, or

during the early pre-implantation stage of pregnancy in the dams.

b. Does not include exposures to parental males.

c. Does not include direct exposure to the postweanling offspring;

potential exposure is limited to in utero transplacental and/or

lactational routes.

The AMRT differs from the 1-generation test (see Unit V.B.2.ii. of

this notice) in that its study design provides for:

a. Exposure to the F0 dam only from gestational day 6 through

weaning of the F1 offspring on post-natal day 21.

b. No exposure to parental males.

c. Mating of the F1 animals (who have not been directly exposed) to

produce F2 offspring.

d. Following the F2 offspring to weaning (post-natal day 21).

ii. 1-Generation reproduction toxicity test. A second alternative

to the standard 2-generation reproductive toxicity test is a 1-

generation reproductive toxicity test, which has been used in rats and

mice. The 1-generation reproductive toxicity test has been used as a

range-finding study prior to performance of a guideline 2-generation

(or more) study for the last 10 years under EPA (TSCA/FIFRA) GLPs; the

design is similar to that used by Sharpe et al. 1996. This is a

shortened, scaled-down version of the new draft OPPTS and Final TSCA

guidelines for reproductive toxicity testing. As with the 2-generation

mammalian reproductive toxicity study, the default assumption is that

all of the endpoints would be evaluated in the 1-generation test,

unless the conditions set forth in the guidelines for determining the

selection of endpoints are met.

The 1-generation test is a less comprehensive evaluation of

functional reproductive development than the AMRT (since it does not

follow F1 animals through production of F2 offspring), but it has the

advantage of assessing post-natal development and adult reproductive

capacity after in utero lactational and post-lactational exposure. In

the presence of continued exposure, the post-natal component of the

test is extended to evaluate acquisition of VP, PPS, estrous cyclicity,

and andrological assessments in the F1 offspring. Inappropriate

retention of Mullerian duct derivations (e.g., oviducts) in males and

of Wolffian duct derivatives (e.g., seminal vesicles, epididymides) in

females can be identified in all three proposed tests (with or without

satellite F0 females and examination of term fetuses).

The 1-generation test involves a short prebreed-exposure period for

male and female rats of the initial parental generation (designated

F0), and exposure continues through mating, gestation, and lactation of

F1 litters. F0 males are necropsied after F1 deliveries; F0 females are

necropsied after F1 weaning. Postweanling F1 animals are directly

exposed for a 10-week postwean period and are then necropsied. F1

animals are evaluated for reproductive development (VP, PPS), estrous

cyclicity and at necropsy for organ weights, possible histopathology,

andrological assessments, and T3/T4 (TSH triggered). F0 animals will

undergo the same necropsy assessments.

The 1-generation test differs from the ``standard'' 2-generation

study design in that it:

a. Is shorter (basic design calls for 2 weeks but it can be

extended) than the standard 2-generation study (10 weeks to encompass

one full spermatogenic cycle in rats), though it does include a

prebreed-exposure period.

b. Does not evaluate effects of in utero and/or lactational

exposure (and beyond) on generation of F2 offspring though it does

include direct exposure of F1 offspring after weaning, including

exposure through puberty and sexual maturation. F1 male and female

reproductive organs (weight/histology), estrous cyclicity, and

andrological endpoints are assessed at scheduled necropsy on post-natal

day 90 2.

The 1-generation test differs from the AMRT in that its study

design provides for:

a. Exposure to both male and female F0 parental animals prior to

mating, during mating, and during gestation and lactation of F1

offspring (F0 males are necropsied after F1 deliveries, F0 females are

necropsied after F1 weaning).

b. Direct exposure of postweanling F1 offspring after lactation

until termination.

c. No mating of F1 animals to produce F2 offspring.

C. Route of Administration

As part of the test guideline, EPA will provide guidance on a route

of administration for each screen and test. Tier 1 screening assays may

employ dosing routes that maximize the likelihood of detecting

endocrine activity such as ip. Conversely, Tier 2 tests will employ

routes of administration based upon the most ecologically relevant

exposure pathway to provide data relevant for risk assessment.

The route of administration for the uterotrophic assay is sc

injection while the route for the modified uterotrophic assay and 14-

day intact adult male assay with thyroid is an ip injection. The route

for all other mammalian in vivo assays is gavage (orogastric

intubation). The parenteral (non-oral) routes avoid the first-pass

metabolic effect of the liver and will permit detection of potential

endocrine disruptors that are active as parent compounds and which

undergo significant first-pass metabolism. Hepatic xenobiotic

metabolism does occur eventually after parenteral administration

(substantially with ip), so the potential effects of metabolites will

be evaluated as well by these routes. Compounds are occasionally

metabolized by the gut microflora; this type of metabolism has been

shown to be important for some plant-derived estrogens. The oral route

of exposure will allow for this type of metabolism.

VI. Implementation

This section of the Federal Register notice discusses the

implementation steps for the EDSP and many of the issues EPA must deal

with in its implementation.

[[Page 71559]]

A. Overview of Implementation Steps and Timeline

There are many elements associated with the development and

implementation of the EDSP. A timeline that shows the key elements and

their relationship to each other is provided in Figure 2.

They include:

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

Implementation steps Estimated completion dates

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

EDSTAC Final Report and Recommendations Completed

Development of EPA's EDSP Completed

Public comment on EPA's EDSP February 26, 1999

SAB/SAP Peer Review Processes April 1, 1999

HTPS Demonstration February 1999

HTPS June 2000

EDPSD June 2000

Priority Setting for Tier 1 Phase 1 November 2000

Tier 1 Standardization and Validation 2001

September

Tier 1, Phase 1 TSCA Test Rule Notice of December 2001

Proposed Rulemaking (NPRM) and FQPA

Orders

Tier 1, Phase 1 TSCA Final Test Rule June 2003

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

BILLING CODE 6560-50-F

[[Page 71560]]

[GRAPHIC] [TIFF OMITTED] TN28DE98.001

BILLING CODE 6560-50-C

[[Page 71561]]

As noted, the recommendations of EDSTAC form the basis for EPA's

endocrine-disruptor screening and testing strategy. Today, EPA is

soliciting comments on its strategy for screening and testing

substances for their potential to disrupt the EAT. These comments and

the Agency's proposal will be reviewed by a joint meeting of the EPA

SAB and FIFRA SAP in March 1999. Notice of the meeting site and

specific times will be published in the Federal Register.

EPA plans to begin running chemicals through the HTPS in August

1999.

The Agency will submit a report to Congress and plans to issue a

notice in the Federal Register in the year 2000 adopting final policies

for the screening program based on comments of the SAP/SAB and the

comments received in response to this notice. The year 2000 notice will

also propose the Priority List of chemicals and mixtures for Tier 1

screening. The proposed screening Priority List will be based on

information in the EDPSD including the results of the HTPS. EPA may

also issue a procedural rule that describes the procedures related to

implementation of the EDSP.

EPA plans to publish the results of the standardization and

validation effort for the screening battery along with guidelines for

the screening assays that flow from this effort in the Federal Register

in 2001. The standardization and validation of Tier 2 tests will be

undertaken approximately in parallel with that of the screening

battery. However, the test validation program is anticipated to take

longer than the screening validation program because the Tier 2 tests

take much longer to run than the Tier 1 screening assays.

In late 2001, EPA plans to issue testing orders to the first group

of pesticides and other chemical substances that are subject to the

authority provided to EPA under the FFDCA and SDWA. In parallel to

these activities, EPA may propose a TSCA test rule to require screening

of chemicals that may not be covered by the FFDCA/SDWA. EPA could

propose the TSCA test rule in 2001 and promulgate it in mid 2003. The

screening program will operate in phases so as to not overwhelm

resources. The number of phases and length of time between phases will

depend on available resources and the number of chemicals proposed for

screening in each phase. EPA plans to review its initial prioritization

of chemicals and issue a separate proposed rule for each screening

phase. This would allow the results from the first phase of screening

to improve the priority setting for the second phase of screening.

Tier 2 testing of chemicals that are part of the first phase of

Tier 1 screening would begin after review of screening data indicated

that testing was warranted. Standardization and validation of Tier 2

tests will take from 2 to 5 years. EPA plans to require tests as soon

as they are available and not wait for the full battery to initiate

Tier 2 testing. Orders under FFDCA, FIFRA, or SDWA would be issued on

individual chemicals as their review is completed. TSCA rules would be

issued for a group of chemicals, probably on an annual basis.

B. HTPS Demonstration

EPA has initiated a demonstration program to validate use of HTPS

technology to screen chemical substances for EAT disrupting properties.

The demonstration program is projected to be completed in February

1999. If EPA successfully validates HTPS through the demonstration

program, it could begin running chemical substances through HTPS in

August of 1999.

C. HTPS Priority-Setting Project

After completion of the HTPS demonstration and validation project,

EPA plans to conduct the HTPS on approximately 15,000 chemicals

(commercial chemicals produced in amounts greater or equal to 10,000

pounds per year and all pesticides) to supplement existing information.

EPA will fund the actual screening of these compounds and is soliciting

industry cooperation in supplying samples of pesticides and

commercially produced chemicals. One major issue in HTPS is how to deal

with the need for analytical characterization of so many chemicals. The

cost of chemical analysis is more than an order of magnitude greater

than the cost of the HTPS battery.

Option One is to require full analysis on each chemical prior to

HTPS. This is the usual requirement for toxicological testing.

Option Two is to perform chemical analysis after HTPS on those

substances that test positive.

Option Three is to rely on the chemical identity and composition

claims of the chemical supplier.

EPA favors Option Two as a cost effective alternative to full

analysis of every chemical. Nevertheless, every sample submitted to EPA

should be accompanied by some information regarding its analytical

characterization. It should at a minimum state whether the material is

a technical grade, analytical grade, etc., to what extent it has been

characterized, and note the concentration or percentage of the sample

comprised by the test substance.

EPA plans to subject chemicals to HTPS that will bypass Tier 1

screening as well as those that need screening. The rationale for

conducting HTPS on these chemicals is:

1. Data generated from the HTPS assays will be valuable for

receptor-binding mechanisms even though such data by itself cannot be

used to determine whether or not a chemical may be an endocrine

disruptor.

2. As an ancillary benefit, the data can be used to improve and

validate QSAR models.

3. For food-use pesticides that will probably undergo

reregistration and tolerance reassessments prior to the availability of

validated Tier 2 tests, HTPS data can be used along with other relevant

testing information to help determine if and when they should undergo

any additional endocrine-disruptor testing.

D. Priority-Setting Data Base (EDPSD) Development

As described in Unit IV.C. of this notice, EPA plans to use

existing exposure, effects and statutory-related data and information

to sort and prioritize chemicals for endocrine-disruptor screening and

testing. To maximize its resources, EPA will rely upon data excerpted

in electronic format instead of primary literature. Recognizing the

numerous data bases of potential utility to initial sorting priority

setting (see Appendix H of the EDSTAC Final Report), EPA plans to

assemble the relevant and useful data sources into a single-relational

data base. Development of this data base was initiated by the EDSTAC

but not completed due to time and resource constraints of the EDSTAC

process. EPA has resumed efforts to complete development of the

prototype EDPSD initiated by EDSTAC. EPA is publishing elsewhere in

this issue of the Federal Register a document announcing a priority-

setting workshop for multi-stakeholders and the use of the EDPSD during

the comment period.

The purpose of the workshop is to provide stakeholders an

opportunity for input into the design and implementation of the

priority-setting system. The focus of the workshop is to discuss the

basic structure and functioning of the priority-setting system.

Specifically, the workshop will address the definition of compartments,

principles and approaches for developing rankings within compartments,

and for assigning overall

[[Page 71562]]

weighting factors to the various compartments and categories.

E. Process for Public Nominations for Chemical Screening

Chemical nominations from the public were considered to be an

important part of the nominations process by EDSTAC because they

provide a mechanism to identify and screen chemicals which may result

in high exposures in local communities but which do not receive

national attention. EPA proposes to establish a nomination process. The

nominations process could be a formal petition process or an informal

one such as a letter submitted to the Agency. EPA belives that any

nomination should be signed and should include the following

information:

Statement that it is nominating a chemical for screening in the

EDSP, identification of the chemical.

Statement of the reasons for its nomination.

Although EPA does not believe it can legally protect the identity

of nominators, employees in the chemical industry are protected by law

against reprisals from employers for reporting a chemical under TSCA

(15 U.S.C. 2622) and any threats or reprisal of any kind should be

reported to the U.S. Secretary of Labor with a copy of the threat or

reprisal report to the EPA Administrator.

F. Standardization and Validation of Assays, Screening Battery, and

Tests

Validation is the scientific process by which the reliability and

relevance of an assay method are evaluated for the purpose of

supporting a specific use (ICCVAM, 1997). Relevance refers to the

ability of the assay to measure the biological effect of interest.

Measures of relevance can include sensitivity (the ability to detect

positive effects), specificity (the ability to give negative results

for chemicals that do not cause the effect of interest), statistically

derived correlation coefficients, and determination of the mechanism of

the assay response with the toxic effects of interest. Reliability is

an objective measure of a method's intra- and inter-laboratory

reproducibility. The process of validation includes standardization,

that is, definition of conditions under which the assay is run

(species, strain, culture medium, dosing regimen, etc.).

Standardization is critical to ensure reliability, that is, valid,

consistent results between laboratories.

FFDCA as amended by the FQPA requires EPA to ``develop a screening

program, using appropriate validated test systems and other

scientifically relevant information, to determine whether certain

substances may have an effect in humans that is similar to an effect

produced by a naturally occurring estrogen, or such other endocrine

effect as the Administrator shall designate.''

EPA convened a meeting of the Domestic Validation Task Force (Task

Force) comprised of experts and representatives of major stakeholders

on August 6, 1998, and is scheduled to meet on a bimonthly basis during

1999. The Task Force is made up of members from Federal agencies,

industry, and public interest groups. The purpose of the Task Force is

to implement the validation program for the screens and tests. In March

1998 and November 1998, the OECD Endocrine Disruptor Testing and

Assessment Workgroup met to initiate an international validation

program for endocrine-disruptor screening and testing. The

international validation program is important in developing an

internationally harmonized approach to endocrine-disruptor screening

and testing. An internationally harmonized approach saves money by

reducing duplicative testing. EPA anticipates that some, but by no

means all, of the assays it is proposing will be included in the

international validation program. The majority of the screening assays

and the screening battery itself will have to be validated in the

domestic validation program.

Standard protocols for most of the screening assays and tests are

now being developed. Most of these should be ready for Task Force

review and approval in 1999. EPA is inviting laboratories to

participate in the validation program. Laboratories that are interested

in the participating in any aspect of the validation program should

contact Anthony Maciorowski (see the ``FOR FURTHER INFORMATION

CONTACT'' section of this notice). Participating laboratories will

receive a standard protocol for each assay they want to conduct and

appropriate control and test chemicals from the EPA or its agent. EPA

is planning to begin the laboratory phase in the spring of 1999. Some

assays which need further development will not begin validation until

late 1999 or the year 2000.

G. Implementation Mechanisms

As stated previously, EPA believes that the FFDCA and SDWA provide

authority to require the testing of many of the approximately 87,000

chemical substance that it wishes to test. As appropriate, EPA also

will use other testing authorities, such as those under FIFRA and TSCA.

Likewise, to the extent that EPA is concerned about the endocrine

disrupting potential of other chemical substances, it will work with

other Federal agencies and departments to ensure that these substances

also are tested. EPA will determine under which authority it will

require testing of specific chemicals on a case-by-case basis. A brief

description of EPA's major testing authorities and guidance on their

application to the EDSP are set forth in this unit.

1. FFDCA testing authority. Under the FFDCA, as amended by FQPA,

EPA has authority to order registrants, manufactures, or importers to

test certain chemical substances, including pesticide chemicals and any

other substance that may have an effect that is cumulative to an effect

of a pesticide chemical if EPA determines that a substantial population

may be exposed to such substances.

Under the FFDCA, ``pesticide chemical'' includes ``any substance

that is a pesticide within the meaning of FIFRA, including all active

and inert ingredients.'' It also includes impurities (see 40 CFR

177.81). The testing requirement is not restricted to pesticides used

on foods.

EPA is still working out how to determine whether a substance ``may

have an effect that is cumulative to the effect of a pesticide

chemical.'' However, at a minimum, EPA believes that if the mechanism

of action of a pesticide chemical and a nonpesticide chemical is the

same, their effects are additive and therefore may be cumulative.

Likewise, when the metabolic detoxification or clearance process of a

pesticide chemical and a nonpesticide chemical are the same, exposure

to the nonpesticide chemical may slow the clearance of the pesticide,

and therefore, increase the pesticide chemical's toxicity. This is an

example of a cumulative effect even when the two chemicals do not

operate by the same mechanism of toxicity or cause the same toxic

effect. The same argument would also apply to enzyme poisons or

noncompetitive inhibitors of pesticide metabolism that slow or

completely block the metabolic pathway of a pesticide. EPA is

interested in receiving comment on these and other examples or on

methods to determine whether a substance may have an effect that is

cumulative to the effect of a pesticide chemical.

The phrase ``substantial population'' is used in FFDCA section

408(p)(3)(B) and in SDWA section 1457 but is not defined in either of

these statutes. Based upon EPA's experience under TSCA, it is necessary

for the Agency to define this term. Under TSCA section 4(a)(1)(B) EPA

defined ``substantial human

[[Page 71563]]

exposure'' in terms of numbers of persons exposed based on a sliding

scale that reflected that more direct exposures would require smaller

numbers of persons exposed in order to be substantial than less direct

exposures would (58 FR 28736, May 14, 1993). EPA is offering no

definition of ``substantial population'' for SDWA and FIFRA purposes at

this time but seeks public comment on an appropriate definition.

2. SDWA testing authority. Congress amended SDWA to give EPA

authority to provide for the testing, under the FFDCA Screening

Program, ``of any other substance that may be found in sources of

drinking water if the Administrator determines that a substantial

population may be exposed to such substance'' (42 U.S.C. 300j-17).

Drinking water contaminants may include, but may not be limited to,

pesticide active and inert ingredients and their degradates, commercial

chemicals and their degradation products, substances formerly

manufactured and used as pesticides or commercial chemicals (orphan

chemicals), or natural substances.

3. FIFRA testing authority. FIFRA section 3(c)(2)(B) provides EPA

authority to require pesticide registrants to submit to EPA additional

data regarding a pesticide if EPA determines that the additional data

are required to maintain in effect an existing pesticide registration.

Under this provision, EPA could require submission of endocrine effects

data for registered pesticides and for chemicals that may have an

effect that is cumulative to that of a pesticide. FIFRA sections

3(c)(2)(A), 3(c)(5), 3(c)(7), and 3(d) also give EPA authority to

require testing.

4. TSCA testing authority. TSCA section 4 provides EPA with

authority to require testing of certain chemical substances, not

including pesticides or food additives among other things, if the

Agency finds that the chemical substance or mixture:

i. May present an unreasonable risk of injury to health or the

environment.

ii. There are insufficient data and experience from which the

Agency can determine the effects of such substance or mixture on health

or the environment.

iii. Testing with respect to such substance or mixture with respect

to such effects is necessary to develop such data.

Alternatively, EPA can require testing if the Agency finds that:

i. A chemical substance or mixture is or will be produced in

substantial quantities and:

a. It enters or may reasonably be anticipated to enter the

environment in substantial quantities, or

b. There is or may be significant or substantial human exposure to

such substance or mixture.

ii. There are insufficient data and experience which from which the

Agency can determine the effects of such substance or mixture on health

or the environment.

iii. Testing with respect to such substance or mixture with respect

to such effects is necessary to develop such data.

EPA achieves TSCA testing through rulemaking and enforceable

consent agreements (ECAs). For more information on EPA's TSCA testing

authority see 40 CFR part 790.

Some chemicals might be subject to more than one testing authority.

Inert pesticide ingredients will frequently have TSCA uses in addition

to their use as inert ingredients in pesticide formulations and could

be screened or tested under TSCA or FFDCA/FIFRA authorities. TSCA

chemicals found in drinking water sources could also be screened or

tested under SDWA or TSCA. Compared with order authority under FIFRA,

FFDCA, or SDWA, a test rule is a slow and labor intensive mechanism.

Therefore, the Agency believes that when a choice is possible it is in

the public interest to require screening and testing under its FIFRA,

FFDCA, or SDWA authorities, rather than under TSCA, when it has that

option.

H. Data Compensation Issues

The FFDCA, as amended, requires EPA ``to the extent practicable,''

to ``minimize duplicative testing of the same substance for the same

endocrine effect, [and] develop, as appropriate, procedures for fair

and equitable sharing of test costs.''

To meet these requirements, EPA is planning to adopt procedures

similar, but not identical, to both TSCA's and FIFRA's data

compensation procedures. If EPA knows that there is more than one

registrant, manufacturer, and/or importer of a specific chemical, it

will order each to test the chemical. As part of the order, it will

include a list of all of the parties who receive equivalent orders and

require the parties to work together to minimize duplicative testing

and share testing costs. The parties may notify EPA of other parties

not listed who also manufacture or import the chemical. Alternatively,

or in addition, EPA will publish the order in the Federal Register and

require parties not listed to self identify. If the parties are unable

to work out testing and data compensation responsibilities, they will

be required to submit to binding arbitration. If a party fails to

comply with an arbitrator's decision, it will be subject to the

penalties described in FFDCA section 408(p)(5)(C).

If, after completion of the testing, another party seeks to use the

resulting data in support of a pesticide registration, it will be

required to comply with FIFRA sections 3(c)(1)(F) or 3(c)(2)(B) which

require compensation for data. Likewise, TSCA requires parties to

compensate test sponsors if they manufacture or import a substance

covered by a test rule within 5 years of the submission of the last

required study. Chemicals being tested pursuant to a rulemaking under

TSCA will follow the TSCA procedures for reimbursement under 40 CFR

part 791.

I. Data Submission and Collection

EPA is proposing to post an electronic form for the capture of data

from screening and testing so that these data can be easily uploaded

into the Endocrine Knowledge Base (EKB) being developed by the FDA's

National Center for Toxicological Research. The EKB will be the

repository of all data from the EDSP as well as other sources of

endocrine effects testing and research. The data base will thus serve

research and regulatory purposes. As the data base is further

developed, EPA will provide guidance on how to submit data

electronically to be compatible with the EKB.

J. Data Release and CBI

FFDCA section 408(p)(5)(B) requires that EPA, to the extent

practicable, develop, as necessary, procedures for handling CBI

submitted as part of the EDSP. EPA anticipates that much of the

information that registrants and manufacturers submit under the

auspices of its EDSP will be health and safety information that

generally does not warrant CBI protection. Nevertheless, EPA is

interested in receiving comments from potential data submitters

concerning whether they think any of the information will deserve CBI

protection. If data submitters believe that certain information will be

deserving of protection, the Agency is interested in receiving comments

on the specific types of information that might need protection and on

procedures that the Agency could develop to verify the validity of CBI

claims and to ensure protection of valid CBI. EPA also is interested in

receiving comments on whether current procedures under FIFRA and TSCA

would be adequate and, if so, how they should be applied.

[[Page 71564]]

EPA is considering adopting FIFRA CBI procedures for data submitted on

pesticide active ingredients and TSCA CBI procedures for all other

substances. If necessary, EPA will develop additional procedures to

ensure that any valid confidential business information is protected

from disclosure.

K. Reporting Requirements Under TSCA 8(e) and FIFRA 6(a)(2)

The following provides EPA's guidance on the reporting obligations

under the TSCA section 8(e) and FIFRA section 6(a)(2) with respect to

results from certain priority-setting studies and in vitro screening

assays that industry or others may conduct voluntarily or as part of

EPA's EDSP. TSCA section 8(e) requires that ``[a]ny person who

manufactures, processes, or distributes in commerce a chemical

substance or mixture and who obtains information which reasonably

supports the conclusion that such substance or mixture presents a

substantial risk of injury to health or the environment shall

immediately inform [EPA] of such information'' (15 U.S.C. 2607(e)).

Likewise, FIFRA section 6(a)(2) requires registrants that, after

registration of a pesticide, have additional factual information

regarding unreasonable adverse effects on the environment of the

pesticide to submit the information to EPA ( 7 U.S.C. 136d(a)(2)).

EPA will likely adopt as part of its EDSP both in vitro and in vivo

assays that assess selected hormonal endpoints. Based on the current

state of the science, EPA considers the results of endocrine disruptor

in vitro screening assays to be indicators of potential endocrine

activity. Whether performed at the bench or in a high throughput mode,

results from in vitro assays may suggest some mechanisms of endocrine

activity (e.g., hormone receptor binding, binding plus transcription,

cell proliferation, steroidogenesis, etc.). Thus, the results of these

in vitro assays are arguably within the scope of TSCA section 8(e) and

FIFRA section 6(a)(2). At this time, however, EPA can not conclude that

the results of these in vitro assays translate into an understanding of

particular health or environmental hazards and risks in vivo.

Therefore, based on the current state of the knowledge, EPA will not,

at this time, require submission of TSCA section 8(e) or FIFRA section

6(a)(2) reports containing only the results of these in vitro assays.

Registrants, manufactures, or importers are, nevertheless, encouraged

to submit the data voluntarily. If these test results are included with

other information reportable under TSCA section 8(e) or FIFRA section

6(a)(2), then they must be reported.

L. Exemptions

There are several circumstances in which exemptions from screening

or testing requirements are appropriate. The FFDCA section 408(p)

provides for exemptions from its requirements if EPA determines that a

substance is anticipated not to produce any effect in humans similar to

an effect produced by a naturally occurring estrogen. Although EPA has

not determined when or under what circumstances it will grant

exemptions from FFDCA 408(p) requirements, examples of the types of

chemicals that might warrant such exemptions include class 4 pesticide

formulation inerts--those inert ingredients in pesticide formulations

judged by EPA to be virtually non-toxic (for example cookie crumbs)--

and strong mineral acids and strong mineral bases, which would likely

interact with tissue at the portal of entry giving rise to localized

lesions rather than systemic effects. The strong reactivity of these

substances would cause interaction with membranes and other biological

chemicals before the chemical reached the endocrine receptors.

EPA is considering establishing a petition process as a means of

establishing exemptions from screening. The details of this process

could be set forth in the procedural rule EPA is considering issuing

for the EDSP. EPA is asking for comments on criteria that might form

the basis for granting exemptions.

Exemptions under FFDCA 408(p) are not the same as exemptions under

FFDCA section 408(c). Please note also that the term exemption as used

under FFDCA section 408(p) is different from, and should not be

confused with, the use of this term under TSCA section 4(c). An

exemption under FFDCA section 408(p) means that testing requirements do

not apply. However, under TSCA section 4(c) an exemption is a mechanism

for avoiding duplicative testing. Under TSCA section 4(c) an exemption

can be granted when data are being or have been generated by a

responsible party and, therefore, other responsible parties can

reimburse the test sponsor for a portion of the cost. A similar cost

sharing provision exists for data compensation among registrants under

FIFRA (see Unit VI.H. of this notice). Unless otherwise indicated, the

term exemption used in this notice will be used in the sense in which

it is used under FFDCA section 408(p), that is, a waiver of all testing

obligations.

M. Use of Significant New Use Rules (SNURs) Under TSCA

During the EDSTAC deliberations, concern was expressed that under

certain circumstances less than the full Tier 2 testing would be

permitted on chemicals based on their limited use and exposure profile.

For instance, a pesticide registered for contained use only may result

in human exposure but negligible or no environmental exposure.

Therefore, performing the 2-generation mammalian reproductive effects

test may be all that is needed to assess the hazards of this substance.

Granting permission to limit Tier 2 testing does not present a problem

for pesticides because pesticide registration limits the uses of the

pesticide to those contained in the registration application. If a

pesticide registrant wants to expand the uses and therefore potentially

the exposure to a pesticide, the registrant must apply to register the

expanded uses. The same is not true for chemicals under TSCA, since

TSCA is not a registration statute. Once a commercial chemical is on

the market it can ordinarily be used freely for any purpose resulting

in exposures that were not occurring at the time testing requirements

were promulgated. A potential solution to this dilemma lies in EPA's

authority under TSCA section 5(a)(2) to issue SNURs.

A SNUR defines certain uses of a chemical as new uses. Before a

manufacturer or processor can use a chemical for one of the defined new

uses, the manufacturer or processor must notify EPA of such intention

at least 90 days before commencement of the new use. A SNUR thus

subjects an existing chemical that triggers a new use to the same

review that a new chemical receives. Submission and review of the new

use can be tied to the performance of testing and submission of test

data to EPA if there is a test rule that covers that chemical.

EPA is considering the development of a SNUR based on a

manufacturer's showing of limited use and exposure as a condition for

granting a waiver for limited Tier 2 testing for TSCA chemicals (i.e.,

permission to perform fewer than the five tests in Tier 2 based upon

exposure considerations). If the manufacturer's claims for limited use

and exposure are refuted in the significant new use rulemaking process

by someone who is already using the chemical in such a manner, the SNUR

will not be valid and the manufacturer will be required to perform the

full battery of Tier 2 tests required in the test rule issued for that

chemical under the EDSP.

[[Page 71565]]

N. Relationship Between the EDSP and Related Actions Under TSCA

Several other testing actions under TSCA may affect chemicals in

the EDSP. Actions planned or underway include the Hazardous Air

Pollutants (HAPs) test rule (61 FR 33178, June 26, 1996) (FRL-4869-1)

as amended, the Children's' Health test rule, the Agency for Toxic

Substances and Disease Registry (ATSDR) test rule, the High Production

Volume (HPV) testing initiative and the Screening Information Data Set

(SIDS) Program on HPV chemicals. None of the EDSP Tier 1 screening

assays is being considered for by these actions. The SIDS and HPV

testing programs do not meet either the screening or testing

requirements of the EDSP. The only likely overlap in testing

requirements is the 2-generation mammalian test, which is proposed in

the HAPs rule and being considered in the Children's Health test rule

and ATSDR test rule. The reproductive effects testing for these

programs will meet the Tier 2 mammalian reproductive effects testing

requirement for the EDSP if the 1998 or later guideline for a 2-

generation mammalian reproductive effects study is used. The results

from some of these testing programs likely will be available before

final testing decisions are made under the EDSP. It is possible that if

the results of the 2-generation test (with endocrine-sensitive

endpoints including thyroid) generated under one of these other testing

programs is negative that only the fish gonadal recrudescence assay

would need to be performed to satisfy the testing requirements of the

EDSP. The correlation of various test results in the validation study

will provide more information on which to make this judgment. If the

mammalian 2-generation test were positive, the other Tier 2 tests would

have to be run depending upon the exposure profile of the chemical in

question.

O. Analysis of Data in the EDSP

EPA discussed use of HTPS data for priority setting for Tier 1

screening and as part of the weight of evidence consideration to

determine when a chemical should be tested in Tier 2. These data may

also used in conjunction with other data to help determine if adverse

effects observed in Tier 2 are due to endocrine disruption or from

another cause. The Tier 1 data will also serve a dual purpose. They

will be used to make the determination of which chemicals receive Tier

2 testing and will also be used to help interpret positive results

observed in Tier 2 testing.

More detailed guidance regarding the assessment of hazards due to

endocrine disruption must await both the results of the standardization

and validation program and ongoing research. EPA intends to review the

need for revising its standard evaluation procedures for interpreting

studies and its human health and ecological risk assessment guidelines

as relevant data from these programs become available.

VII. Issues for Comment

1. The FFDCA, as amended, requires EPA to screen pesticides for

estrogenic effects that may affect human health. EPA has decided that

it is scientifically appropriate to focus on EAT effects, not just

estrogenic effects. Is this an appropriate scope for the EDSP?

2. Are there classes of chemicals besides the ones identified in

Unit VI.L. of this notice that should be exempted (excluded) from the

EDSP? What criteria and what burden of proof should be applied to

claims of persons seeking to exempt chemicals from screening? What type

of process should EPA establish?

3. As discussed in Unit IV.E. of this notice, EPA is proposing a

compartment-based (or set-based) approach to priority setting as a way

of accommodating the real world situation of uneven data. Under the

compartment-based approach, EPA will group the chemicals into sets

based on the existence of factual information in a given area. Thus,

priority ranking can be made fairly among chemicals, i.e., chemicals

will compete for priority with other chemicals on the basis of

comparable data and will not be assigned lower priority for lack of

information. Are these principles and the compartment-based approach to

priority setting reasonable? Are there alternatives to the compartment-

based approach which EPA should consider?

4. As recommended by EDSTAC, EPA is proposing that polymers with an

average number molecular weight greater than 1,000 daltons be excluded

from priority setting and screening unless they are pesticide chemicals

or unless their monomers, oligomers, or leachable components are shown

to have endocrine-disrupting potential in Tier 1 screening. Is this

approach scientifically sound?

5. EPA is developing a relational data base to assist in setting

priorities for screening. The relational data base is intended to

import existing data and information and allow its synthesis, as well

as the estimation of certain parameters through modeling. EPA and

EDSTAC consider the relational data base to have great value in helping

to identify the specific compartments under the compartment-based

priority-setting approach. The data base will also be helpful in

selecting chemicals for the first and subsequent rounds of screening.

The data fields currently in the data base are defined in Chapter 4 of

the EDSTAC Final Report. What additional data fields or types of data

should EPA include as it further develops the relational data base?

6. EPA is soliciting industry's cooperation in supplying chemicals

for the HTPS. Is this an appropriate role for industry and is industry

willing to do so?

7. EPA plans to screen and, if appropriate, test representative

mixtures to which large or identifiable segments of the population are

exposed. The high-priority mixture categories include: Chemicals in

breast milk, phytoestrogens in soy-based infant formulas, mixtures

commonly found at Superfund sites, common pesticide/fertilizer mixtures

found in ground and surface water, disinfection byproducts, and

gasoline. EPA plans to screen and test one representative mixture from

each category.

a. Can standardized representative mixtures be developed? If so,

how should the chemical combinations, ratios, and doses be selected for

mixtures?

b. Is the proposal a reasonable way to address the practicality of

screening and testing mixtures?

c. Are the six categories of mixtures the most appropriate to

address first?

d. Are there other mixture categories that should be included in

addition to, or instead of those identified (e.g., Should fish tissue

contaminants be one of the first mixtures)?

e. If a mixture is positive in Tier 1, should the whole mixture be

tested in Tier 2 or should EPA attempt to identify the active

component(s) and test it (them) in Tier 2?

8. EPA has identified a screening battery consisting of in vitro

and in vivo assays to address EAT effects. Will the battery, once

validated, be capable of detecting such effects in a consistent and

reliable manner?

9. EPA is planning to require that the Tier 1 screening in vivo

assays be conducted at one dose, with appropriate use of range finding

studies and other information (i.e., HTPS results) to inform dose

selection. The single-dose approach was adopted to save testing

resources. The SAB/SAP in a preliminary consultation raised concern

about relying on only one dose level and suggested that EPA require a

minimum of two doses and preferably three to ensure that tests did not

result in false negatives. Does the potential risk of

[[Page 71566]]

false negatives outweigh the cost savings of running the Tier 1

screening in vivo assays with only one dose?

10. EDSTAC could not identify existing practical vertebrate

endocrine disruptor screening assays that incorporated exposure in

utero or in ovo. Do such screening assays exist?

11. Is adequate coverage of the thyroid provided in the recommended

Tier 1 screening battery? Does the Tier 1 screening battery provide

adequate coverage of non-receptor mediated pathways?

12. EPA is proposing a Tier 2 testing battery to delineate dose-

response relationships of chemicals that yield positive results in the

screening battery. Do the tests provide sufficient rigor to identify

adverse effects and establish dose response for disruption of the EAT?

13. Will the Tier 2 tests be adequate to detect all known EAT

endpoints in chemicals that bypass Tier 1 screening?

14. Tier 2 tests will identify the adverse effects due to endocrine

disruption as well as reproductive and developmental effects caused by

non-endocrine mechanisms of toxicity. Thus, it may not be possible to

determine that a substance is an endocrine disruptor if it bypasses

tier 1 screening. Is it important to be able to identify substances as

endocrine disruptors from the standpoint of conducting a hazard

assessment?

15. If the results of the 2-generation test (with endocrine-

sensitive endpoints including thyroid) generated under one of these

other testing programs is negative what additional screening or testing

should be required to demonstrate that the chemical is not an endocrine

disruptor?

16. FFDCA gives EPA authority to test pesticides and substances

``that are cumulative to the effect of a pesticide.'' EPA is interested

in receiving comment on how the term ``cumulative to the effect of a

pesticide'' should be applied in defining additional substances which

can be tested under FFDCA.

17. How should EPA define substantial population as used in FFDCA

section 408(p) and SDWA section 1457?

8. Is EPA's proposal to adopt FIFRA cost sharing provisions for

data received under FIFRA and TSCA cost sharing provisions for all

other substances feasible and practical?

19. Is EPA's proposal to adopt FIFRA CBI procedures for active

pesticide ingredients and TSCA CBI procedures for all other substances

feasible and practical? TSCA makes health and safety data freely

available. The chemical portion of chemical substances comprising

formulated products is confidential under both statutes.

20. Should EPA permit chemicals to receive less than the full Tier

2 testing battery under certain circumstances? Should EPA issue a SNUR

for TSCA chemicals that are subject to limited Tier 2 testing?

21. Should EPA issue a procedural rule codifying many of the

procedures discussed in Unit VII. of this notice?

VIII. References

The Agency's actions are supported by the references listed in this

unit and cited in this notice. These references are available in the

public record for this notice under docket control number OPPTS-42208

in the TSCA Docket, see the ``ADDRESSES''section in this notice.

1. Anderson, S., S. Pearce, P. Fail, B. McTaggert, R. Tyl, and L.E.

Gray (1995) ``Validation of the alternative reproductive test protocol

(ART) to assess toxicity of methoxychlor in rats.'' The Toxicologist,

15, pp. 164.

2. Anderson, S., S. Pearce, P. Fail, B. McTaggert, R. Tyl, and L.

Gray (1995b) ``Testicular and adrenal response in adult Long-Evans

Hooded rats after antiandrogenic vinclozolin exposure.'' Journal of

Andrology, 16, pp. 43.

3. Baxter, W.L., R.L. Linder, and R.B. Dahlgren, (1969) ``Dieldrin

Effects in Two Generations of Penned Hen Pheasants.'' Journal of

Wildlife Management, 33(1), pp. 96-102.

4. Bellabarba, D., S. Belisle, N. Gallo-Payet, and J.G. Lehoux

(1988) ``Mechanism of Action of Thyroid Hormones During Chick

Embryogenesis.'' American Zoologist, 28, pp. 389-399.

5. Bjerke, D., and R. Peterson (1994) ``Reproductive toxicity of

2,3,7,8 tetrachlorodibenzo-p-dioxin in male rats: Different effects of

in utero versus lactational exposure.'' Toxicology and Applied

Pharmacology, 127, pp. 241-249.

6. Brown, D.D., Z. Wang, A. Kanamori, B. Eliceiri, J.D. Furlow, and

R. Schwartzman (1995) ``Amphibian metamorphosis: a complex program of

gene expression changes controlled by the thyroid hormone.'' Recent

Progress in Hormone Research, 50, pp. 309-315.

7. Cruickhank, J.J., and J.S. Sim (1986) ``Morphometric and

Radiographic Characteristics Of Tibia Bone of Broiler Chickens with

Twisted Leg Disorders,'' Avian Diseases, 30(4), pp. 699-708.

8. Dahlgren, R.B., and R.L. Lind

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