Transcript What can EDSP screening tell us that the FIFRA guidelines

James T. Stevens, PhD, DABT, ATS, ERT
Wake Forest University Health Sciences
1



To briefly review some of the history of crop
protection chemical industry’s response to EDSTAC
proposal to endocrine screening and testing, and
the history of the FIFRA guidelines.
To review the FIFRA guideline mammalian
toxicology tests in regard to their ability to detect an
endocrine - active chemicals (EACs).
To give my candid assessment in regard to value –
added by the EDSP Tier 1 screens to the crop
protection registration process.
2
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NACA (CLA’s former name) position in 19971: …“ the
FIFRA testing procedures have been demonstrated to
identify endocrine modulators of sufficient potency to
represent a concern to human health.”

ECPA position in 19982: …“these screening tests do
not add value to the current regulatory battery… the
regulatory safety paradigm has a low likelihood of
missing potential endocrine-active chemicals…”
1Stevens
J, Tobia A, Lamb J, Tellone C, and O’neil F.(1997). FIFRA Subdivision F Testing Guidelines: Are These Tests
Adequate to Detect Potential Hormonal Activity For Crop Protection Chemicals. J Toxicol Environ Health, 50: 415-31.
J, Gfeller W, Machemer L, and Leist KH. (1998). Adequacy of Required Regulatory Hazard Testing for the
Detection of Potential Hormonal Activity of Crop Protection Chemicals. J Toxicol Environ Health, Part B, 1: 59-79.
2Stevens
The Federal insecticide, Fungicide and Rodenticide Act (FIFRA) was passed
by Congress in 1947.
 The EPA was granted authority to administer FIFRA in 1970.
 FIFRA defines the requirement of the registration of a pesticide under the
Code of the Federal Regulations (40 CFR part 150-189).
 Some of Part 158 testing requirements:
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Subdivision D - Product Chemistry

Subdivision E - Hazard Evaluation: Wildlife & Aquatic Organisms
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Subdivision F - Hazard Evaluation: Humans & Domestic Animals

Subdivision G – Product Performance

Other subdivisions cover experiment use permits, nontarget plants and insects,
reentry, biorational pesticides, environmental fate, residue chemistry, spray drift,
and application exposure monitoring
The Food Quality Protection Act (FQPA) of 1996 amended FIFRA to include,
among other things, the development of assays to detected substances that
may cause estrogenic effects.
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The EDSTAC and the EPA EDSP have set the FIFRA OPPTS two
generation reproduction study in the rat as the definitive EDSP
mammalian Tier II test.

But how about, the other FIFRA subdivision F studies that may
detect EACs?
 Acute testing (oral, dermal, inhalation, irritation, sensitization) are
designed to determine single exposure hazard estimates and used for
product labeling purposes.
 Subchronic studies [90-day neurotoxicity (neuroendocrine); 90-day
Feeding studies-rodent and non-rodent (organ toxicity); 21-day Dermal
/90-day Dermal (organ toxicity); and 90-day Inhalation (organ toxicity) by
design can and have yielded information on EACs.
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The Design of a FIFRA Subchronic Study Affords the
Potential to Identify EACs.
Typical Experimental Design
Group
number of animals/sex/group
Control
DE11
DE22
½ MTD
MTD
20
20
20
20
20
Animals weighed weekly, observation, clinical signs, and mortality
recorded daily
Clinical Pathology (Hematology and Clinical chemistry), Urinalysis,
and eye examinations made as specified in the protocol
Necropsy, gross pathology, organ harvest and preservation, organ
weights, tissue fixation, slide preparation, histopathology performed
as specified in the protocol
1100x
the anticipated crop residue; 2100x anticipated worker exposure

At minimum, the test substance (minimum of 3 doses +
control) is administered daily from implantation to the day 20
(prior to the expected day of parturition) to 20 pregnant
females/test group. Litters are harvested by caesarian.

Pregnancy status, gravid uterus (with cervix) weighed and
uterine contents should be examined for embryonic or fetal
deaths and the number of viable fetuses.

For each fetus, the gender, body weight, and malformations as
well as skeletal and soft tissue anomalies are determined.
Additional reproduction / developmental
and genotoxicity tests that are on the
FIFRA books
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OPPTS 870.3550 (OECD 421) Reproduction/developmental
toxicity screening test.
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OPPTS 870.3650 (OECD 422) Combined repeated dose toxicity
study with the reproduction/developmental toxicity screening
test.
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The OECD 422 study was designated as an acceptable screen
by the EPA to use of evaluating the potential reproductive
and/or developmental toxicity for over 100 inerts used in
pesticide formulations (Joint Inerts Task Force/Exponet).

OPPTS 870.5450 Rodent dominant lethal assay (Optional
Genotoxicity Tests)
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The combined chronic toxicity/carcinogenicity study was
designed to determine the potential chronic toxicity
and/or tumorigenicity of a chemical administered over the
lifetime of a rat.
Endpoints included in this study are designed to measure
the potential to produce neurological, physiological,
endocrinologic, biochemical, hematological, and
pathological change.
Tumors of the of the endocrine tissues , i.e., testes, ovary,
uterus, and mammary gland may yield the key to
detecting weak hormonally mediated effects.
Endpoints in FIFRA Subdivision F Tests Methods
for the Detection of Hormonally Mediated
Responses in Adult Animals
Endpoint
Subchronic
Develop
870.3550
OECD 421
Reproduction-fertility
2-gen
Repro
Yes
Yes
Fecundity (litter size)
Yes
Yes
Yes
Gestation length
Yes
Yes
Yes
Abortion / resorption
Yes
Yes
Yes
Premature delivery
Yes
Yes
Yes
Difficult labor
Yes
Yes
Time to mating
Yes
Yes
Mating and sexual behavioral
Yes
Yes
Chronic/
cancer
Estrous cyclicity
Optional
Optional
Yes
Yes
Optional
Ovulation
Optional
Optional
Yes
Yes
Yes
Spermatogenesis
Optional
Yes
Yes
Yes
Sperm count and Production
Optional
Optional
Optional
Optional
Reproductive life span
Yes
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Endpoints in FIFRA Subdivision F Tests Methods
for the Detection of Hormonally Mediated
Responses in Adult Animals (cont’d)
Endpoint
Gonadal development (size.
morphology and weight)
Sex accessory organ function
(secretory chemicals)
Subchronic
Develop
870.3550
OECD 421
Yes
2-gen
repro
F1, Yes
Optional
Chronic/
cancer
Yes
Optional
Secondary sexual characteristics
(muscle mass, etc)
Yes
F1, Yes
Yes
Gross path of reproductive organs
Yes
F1, Yes
Yes
Histopath of reproductive tissues
Yes
F1, Yes
Yes
Hormone levels
Major differences between sexes
Tumor (incidence and latency) in
reproductive/endocrine organs
Optional
Optional
Optional
Optional
Yes
Yes
Yes
Yes
Yes
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Endpoints in FIFRA Subdivision F Tests Methods
for the Detection of Hormonally Mediated
Responses in Offspring
Endpoint
Sexual differentiation
Subchronic
Develop
Yes
Yes
870.3550
OECD 421
Offspring sex ratio
2-gen
repro
Yes
Yes
Yes
Yes
Gonadal development (size, weight)
Yes
Accessory sex organ development
Yes
Accessory sex organ function
Yes
Yes
Secondary sexual characteristics
Yes
Yes
Sexual developmental (vo, ps, testes
descent, nipple develop.)
Yes
Anomalies and malformations of the
genital tract
Yes
Yes
Yes
Gross path of reproductive organs
Yes
Yes
Yes
Histopath of reproductive tissues
Optional
Yes
Yes
Viability of the conceptus (losses)
Yes
Yes
Yes
Growth of the conceptus (weight)
Yes
Yes
Yes
Optional
Yes
Yes
Yes
Yes
Growth of the offspring (weight)
Major differences between sexes
Chronic/
cancer
Yes
Yes 12
EAC
FIFRA test
Endpoints
Reference
Diethylstilbesterol
Subchronic
↑uterine weight in female rats
Burin et al., 1993
DDT
Subchronic
↑uterine weight in female rats
Burin et al., 1993
Estrone
Developmental
50 % in pregnancies
IARC, 1979
Ethinyl estradiol
Developmental
50 % in pregnancies
Blye, 1970
Cyproterone acetate
Developmental
live embryos
Eibs et al., 1984
Tamoxifen
Developmental
↑Embryonic loss, and fetal
resorption, litter size
Furr et al, 1976
DDT
2-gen repro
↑length of gestation,lactation
Smith, 1991
Chlordecone
2-gen repro
Persistent estrus
Gray et al., 1989
Methoxychlor
2-gen repro
Early vo, fertility
Uphouse et al.,
1984
Procymidone
2-gen repro
Hypospadias
Ravenzwaay,1992
Vinclozolin
2-gen repro
Feminization of male pups
Ravenzwaay,1992
Estradiol -17B
Chronic/cancer
↑cervical, mammary, and
uterine tumors
IARC, 1979
A Nonylphenol 3-Generatiion Study with 17-β
Estradiol as a Positive Control (Tyl et al., 2006)
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Twenty-five rats/sex/group were administered concentrations
of 0, 20, 200, 650 and 2000 ppm of nonylphenol in Purina 5002
diet and 0 and 650 ppm of nonylphenol in NIH-07 diet. 17-β
Estradiol was used as a positive control at 2.5 ppm in Purina
5002 diet over 3 generations.
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No effects were seen on any reproductive parameter with
nonylphenol over three generations. Ovarian weights were
reduced at 2000 ppm and kidney toxicity at 650 and 2000 ppm
in males.
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An 800-fold lower dose of 17-β Estradiol produced renal,
reproductive, and developmental (lactational and peripubertal
effects) toxicity in all three generations.
Did the FIFRA Mammalian Toxicology Test Identify Possible EACs
in the Process of Registration and Re-registration of Crop
Protection Chemicals?
No.1
Develop.
Tox.2
2-gen.
repro.
Combined
Tox. / Carc.3
Fungicides
34
7
4
6
Insecticides
48
4
5
1
Herbicides
88
4
3
14
170
15
12
21
Totals
1 The
majority of crop protection chemicals registered for use in the US were reviewed (Breckenridge
and Stevens (2007) Chapter 16, Principles and Methods of Toxicology. 5th Ed. AW Hayes, Ed. Pp.727839.
2 Some
of the developmental effects may be associated with maternal toxicity.
3 Identified
by an increased incidence of ovarian, mammary, testicular or thyroid tumors; however,
some of these may be due to overdosing.
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What Value Does the EDSP Tier 1 Screening
Battery Add to the Crop Protection Crop Registration
and Registration Process?

To the registrant who already conducted the FIFRA
definitive guideline tests in order to register a
product, the EDSP Tier 1 screens brings little to no
value to the process.

Yet 58 crop protection chemicals, each of which
have already been using the Tier II FIFRA 2generation reproduction study as well as the other
FIFRA tests previously considered, are scheduled to
be evaluated in the EDSP Tier 1 screening battery.

Wissenschaftlich und logisch? Nein!
1This
is my personal opinion which does reflect my more than a decade of
service on EDSP advisory subcommittees/committees, but does not reflect the
opinion of Wake Forest University or my former employers!
Release in 1st Qtr of 2010
Pre-lease sales on Informahealthcare, Amazon, Target, Flipkart,
Keenzo and Chaos.
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Table of Contents

J. Charles Eldridge, James T. Stevens (WFUHS) – Preface

James C. Lamb, Karin L. Hantz (Exponent, Alexandria, VA) - Chapter 1: Impact of
Endocrine Disruptor Policies and Regulations

J. Charles Eldridge (WFUHS), Susan C. Laws (NHEERL, ORD, US EPA) - Chapter
2: The US EPA'S Tier 1 Screening Battery for Endocrine Disruptor Compounds

Derek V. Henley, Kenneth S. Korach (LRDT, NIEHS) - Chapter 3: Modulation of
Estrogen Receptor Signaling by Endocrine-Disrupting Chemicals

Terry R. Brown (Johns Hopkins University) - Chapter 4: Androgen Receptor
Binding and Transactivation Assays Identify Environmental Chemicals as
Endocrine Disruptors

James Devillers (Centre de Traitement de l'Information Scientifique, France)
Chapter 5: Structure-Activity Modeling of Endocrine Disruptors

Stephen Safe, Gayathri Chadalapaka, Indira Jutooru (Texas A&M University) Chapter 6: Aryl Hydrocarbon Receptor Ligands: Toxic, Biochemical and
Therapeutic Effects
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Table of Contents
(continued)

Tammy E. Stoker, Leah M. Zorrilla (NHEERL, ORD, US EPA) - Chapter 7: The
Effects of Endocrine Disrupting Chemicals on Pubertal Development in the Rat:
Use of the EDSP Pubertal Assays as a Screen

Tomoya Yamada (Sumitomo Chemical Co., Japan) - Chapter 8: Male Reproductive
Endpoints and the Rodent Hershberger Assay

Jerome C. Goldman, Ralph L. Cooper (NHEERL, ORD, US EPA) – Chapter 9: The
Impact of Centrally-Acting Pesticidal/Environmental Toxicants on the
Neuroendocrine Regulation of Reproductive Function in the Female Rodent:
Relevance to Human Reproductive Risk Assessment?

E. Keith Inskeep (West Virginia University) – Chapter 10: Time Dependent
Embryotoxicity of the Endogenous Luteolysin, Prostaglandin F2alpha, in
Ruminants
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Michael C. Henson, Martina Piasek, P. Jorge Chedrese (Purdue University
Calumet), V. Daniel Castracane (Texas Tech University)- Chapter 11: Metal Toxicity
in Mammalian Reproduction

Gerald A. LeBlanc (North Carolina State University) – Chapter 12: Overview of
Endocrine Disruptor Ecotoxicity in Wildlife
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Table of Contents
(continued)

Leslie W. Touart (OPPTS, OSCP, US EPA), Mary Ann Ottinger (University of
Maryland) – Chapter 13: Avian Endocrine Toxicology

Stephen J. Jordan, Christy M. Foran, Erin R. Bennett, Erin M. Snyder, William H.
Benson. (Gulf Ecology Division, ORD, US EPA) – Chapter 14: EndocrineDisrupting Compounds in Aquatic Ecosystems

Gerald T. Ankley, Geraldine Cripe, Sigmund J. Degitz, Mary L. Haasch, Kathleen
M. Jensen, Rodney D. Johnson, Allen W. Olmstead, Joseph E. Tietge (MidContinent Ecology Division, US EPA) - Chapter 15: Overview of US EPA Tests with
Aquatic Vertebrates for Detecting and Assessing Endocrine Disrupting
Chemicals

Werner Kloas (Humboldt University, Germany) and Ilka Lutz (Leibniz Institute,
Germany) - Chapter 16: The Hypothalamic-Pituitary-Gonadal Axis in Anuran
Amphibians

Benjamin D. Stanford, Mark J. Benotti, Shane A. Snyder (Southern Nevada Water
Authority) – Chapter 17: Impact of Endocrine Disruptors to the Water Industry
20