pggn jk United States Prevention, Pesticides EPA 740-C-09-008
C, Environmental Protection and Toxic Substances October 2009
l#crn Agency ^
Endocrine Disruptor
Screening Program
Test Guidelines
OPPTS 890.1400:
Hershberger Bioassay
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NOTICE
This guideline is one of a series of test guidelines established by the Office of
Prevention, Pesticides and Toxic Substances (OPPTS), United States Environmental Protection
Agency for use in testing pesticides and chemical substances to develop data for submission to
the Agency under the Toxic Substances Control Act (TSCA) (15 U.S.C. 2601, et seq.), the
Federal Insecticide, Fungicide and Rodenticide Act (FIFRA) (7 U.S.C. 136, etseq.), and section
408 of the Federal Food, Drug and Cosmetic (FFDCA) (21 U.S.C. 346a).
The OPPTS test guidelines serve as a compendium of accepted scientific
methodologies and protocols that are intended to provide data to inform regulatory decisions
under TSCA, FIFRA, and/or FFDCA. This document provides guidance for conducting the test,
and is also used by EPA, the public, and the companies that are subject to data submission
requirements under TSCA, FIFRA and/or the FFDCA. As a guidance document, these
guidelines are not binding on either EPA or any outside parties, and the EPA may depart from
the guidelines where circumstances warrant and without prior notice. The procedures contained
in this guideline are strongly recommended for generating the data that are the subject of the
guideline, but EPA recognizes that departures may be appropriate in specific situations. You
may propose alternatives to the recommendations described in these guidelines, and the
Agency will assess them for appropriateness on a case-by-case basis.
For additional information about OPPTS harmonized test guidelines and to access the
guidelines electronically, please go to http://www.epa.gov/oppts and select "Test Methods &
Guidelines" on the left side navigation menu. You may also access the guidelines in
http://www.regulations.gov grouped by Series under Docket ID #s: EPA-HQ-OPPT-2009-0150
through EPA-HQ-OPPT-2009-0159, and EPA-HQ-OPPT-2009-0576.
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OPPTS 890.1400: Hershberger Bioassay
(a) Scope.
(1) Applicability. This guideline is intended to meet testing requirements of
the Toxic Substances Control Act (TSCA) (15 U.S.C. 2601, et seq.), the
Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) (7 U.S.C.
136, et seq.), and the Federal Food, Drug, and Cosmetic Act (FFDCA) (21
U.S.C. 346a).
(2) Background. The Endocrine Disruptor Screening Program (EDSP)
reflects a two-tiered approach to implement the statutory testing
requirements of FFDCA section 408(p) (21 U.S.C. 346a). In general, EPA
intends to use the data collected under the EDSP, along with other
information, to determine if a pesticide chemical, or other substances, may
pose a risk to human health or the environment due to disruption of the
endocrine system.
This test guideline is intended to be used in conjunction with other
guidelines in the OPPTS 890 series that make up the full screening
battery under the EDSP to identify substances that have the potential to
interact with the estrogen, androgen, or thyroid hormone (Tier 1
"screening"). The determination will be made on a weight-of-evidence
basis taking into account data from the Tier 1 assays and other
scientifically relevant information available. The fact that a substance may
interact with a hormone system, however, does not mean that when the
substance is used, it will cause adverse effects in humans or ecological
systems.
Chemicals that go through Tier 1 screening and are found to have the
potential to interact with the estrogen, androgen, or thyroid hormone
systems will proceed to the next stage of the EDSP where EPA will
determine which, if any, of the Tier 2 tests are necessary based on the
available data. Tier 2 testing is designed to identify any adverse
endocrine-related effects caused by the substance, and establish a
quantitative relationship between the dose and that endocrine effect.
(3) Source. The source material used in developing this harmonized OPPTS
guideline is Test Guideline 441 published by the Organization for
Economic Cooperation and Development (OECD) (Ref. 25).
The OECD initiated a high-priority activity in 1998 to revise existing
guidelines and to develop new guidelines for the screening and testing of
potential endocrine disrupters (Ref. 1). One element of the activity was to
develop a Test Guideline for the rat Hershberger Bioassay. After several
decades of use by the pharmaceutical industry, this assay was first
standardized by an official expert committee in 1962 as a screening tool
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for androgenic chemicals (Ref. 2). In 2001-2007, the rat Hershberger
Bioassay has undergone an extensive validation program including the
generation of a Background Review Document (Ref. 23), compilation of a
detailed methods paper (Ref. 3), development of a dissection guide (Ref.
21) and the conduct of extensive intra- and interlaboratory studies to show
the reliability and reproducibility of the bioassay. These validation studies
were conducted with a potent reference androgen (testosterone
propionate (TP)), two potent synthetic androgens (trenbolone acetate and
methyl testosterone), a potent antiandrogenic pharmaceutical (flutamide),
a potent inhibitor of the synthesis (finasteride) of the natural androgen
(dihydrotestosterone-DHT), several weakly antiandrogenic pesticides
(linuron, vinclozolin, procymidone, p,p' DDE), a potent 5a reductase
inhibitor (finasteride) and two known negative chemicals (dinitrophenol
and nonylphenol) (Refs. 4, 5, 6, 7, & 8). The OECD Test Guideline 441
was the outcome of the validation test program and is the basis of this
OPPTS Test Guideline.
Purpose. The Hershberger bioassay serves as a mechanistic in vivo screening
assay for androgen agonists, androgen antagonists and 5a-reductase inhibitors.
It is intended to be included in a battery of in vitro and in vivo tests to identify
substances with potential to interact with the endocrine system, ultimately leading
to hazard and risk assessments for human health or the environment.
Overview. The Hershberger Bioassay is a short-term in vivo screening test
using accessory tissues of the male reproductive tract. The assay originated in
the 1930's and was modified in the 1940's to include androgen-responsive
muscles in the male reproductive tract (Refs. 2 & 9-15). In the 1960s, over 700
possible androgens were evaluated using a standardized version of the protocol
(Refs. 2 & 14), and use of the assay for both androgens and antiandrogens was
considered a standard method in the 1960s (Refs. 2 & 15). The current
bioassay is based on the changes in weight of five androgen-dependent tissues
in the castrate-peripubertal male rat. It evaluates the ability of a chemical to elicit
biological activities consistent with androgen agonists, antagonists or 5 a-
reductase inhibitors. The five androgen-dependent tissues included in this Test
Guideline are the ventral prostate (VP), seminal vesicle (SV) (plus fluids and
coagulating glands), levator ani-bulbocavernosus (LABC) muscle, paired
Cowper's glands (COW) and the glans penis (GP).
In the castrate-peripubertal male rat, these five tissues all respond to androgens
with an increase in absolute weight. When these same tissues are stimulated to
increase in weight by administration of a potent reference androgen, these five
tissues all respond to antiandrogens with a decrease in absolute weight. The
primary model for the Hershberger bioassay has been the surgically castrated
peripubertal male, which was validated in Phases 1, 2 and 3 of the Hershberger
validation program.
Due to animal welfare concerns with the castration procedure, the intact
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(uncastrated) stimulated weanling male was sought as an alternative model for
the Hershberger Bioassay to avoid the castration step. The stimulated weanling
test method was validated (Ref. 24); however, in the validation studies, the
weanling version of the Hershberger Bioassay did not appear to be able to
consistently detect effects on androgen-dependent organ weights from weak
anti-androgens at the doses tested. Therefore, it was not included in this Test
Guideline.
Initial Considerations and Limitations. Androgen agonists and antagonists act
as ligands for the androgen receptor and may activate or inhibit, respectively,
gene transcription controlled by the receptor. In addition, some chemicals inhibit
the conversion of testosterone to the more potent natural androgen
dihydrotestosterone in some androgen target tissues (5a-reductase inhibitors).
Such substances have the potential to lead to adverse health hazards, including
reproductive and developmental effects. Therefore, the regulatory need exists to
rapidly assess and evaluate a chemical as a possible androgen agonist or
antagonist or 5a-reductase inhibitor. While informative, the affinity of a ligand for
an androgen receptor or transcriptional activation of reporter genes in vitro is not
the only determinant of possible hazard. Other determinants include metabolic
activation and deactivation upon entering the body, substance distribution to
target tissues, and clearance from the body. This leads to the need to screen the
possible activity of a chemical in vivo under relevant conditions and exposure. In
vivo evaluation is less critical if the chemical's characteristics regarding
Absorption - Distribution - Metabolism - Elimination (ADME) are known.
Androgen-dependent tissues respond with rapid and vigorous growth to
stimulation by androgens, particularly in castrate-peripubertal male rats. Rodent
species, particularly the rat, are also widely used in toxicity studies for hazard
characterization. Therefore, the assay version using the castrated peripubertal
rat and the five target tissues in this assay are appropriate for the in vivo
screening of androgen agonists and antagonists and 5a-reductase inhibitors.
This Guideline is based on those protocols employed in the OECD validation
study which have been shown to be reliable and reproducible in intra- and
interlaboratory studies (Refs. 4-8). Both androgen and antiandrogen procedures
are presented in this Guideline.
Although there was some variation in the dose of TP used to detect
antiandrogens in the OECD Hershberger Bioassay Validation Programe by the
different laboratories (0.2 versus 0.4 mg/kg/d sc) there was little difference
between these two protocol variations in the ability to detect weak or strong
antiandrogenic activity. However, it is clear that the dose of TP should not be too
high as to block the effects of weak androgen receptor (AR) antagonists or so
low that the androgenic tissues display little growth response even without
antiandrogen coadministration.
The growth response of the individual androgen-dependent tissues is not entirely
of androgenic origin, i.e. compounds other than androgen agonists can alter the
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weight of certain tissues. However, the growth response of several tissues
concomitantly substantiates a more androgen-specific mechanism. For example,
high doses of potent estrogens can increase the weight of the seminal vesicles;
however, the other androgen-dependent tissues in the assay do not respond in a
similar manner. Antiandrogenic chemicals can act either as androgen receptor
antagonists or 5a-reductase inhibitors. 5a-reductase inhibitors have a variable
effect, because the conversion to more potent dihydrotestosterone varies by
tissue. Antiandrogens that inhibit 5a-reductase, like finasteride, have more
pronounced effects in the ventral prostate than other tissues as compared to a
potent AR antagonist, like flutamide. This difference in tissue response can be
used to differentiate between AR mediated and 5a-reductase mediated modes
of action. In addition, the androgen receptor is evolutionarily related to that of
other steroid hormones, and some other hormones, when administered at high,
supraphysiological dosage levels, can bind and antagonize the growth-promoting
effects of TP (Ref. 13). Further, it also is plausible that enhanced steroid
metabolism and a consequent lowering of serum testosterone could reduce
androgen-dependent tissue growth. Therefore, any positive outcome in the
Hershberger Bioassay should normally be evaluated using a weight of evidence
approach, including in vitro assays, such as the AR and ER binding assays and
corresponding transcriptional activation assays, or from other in vivo assays that
examine similar androgen target tissues such as the male pubertal assay, 15-day
intact adult male assay, or 28-day or 90-day repeat dose studies.
Experience indicates that xenobiotic androgens are rarer than xenobiotic
antiandrogens. The expectation then is that the Hershberger bioassay will be
used most often for the screening of antiandrogens. However, the procedure to
test for androgens could, nevertheless, be recommended for steroidal or steroid-
like chemicals or for chemicals for which an indication of possible androgenic
effects was derived from other screening methods. Similarly, adverse effects
associated with (anti)androgenic profiles may be observed in higher tier definitive
assays, leading to the need to assess whether a substance operates by an
endocrine mode of action.
It is acknowledged that all animal based procedures should conform to local
standards of animal care; the descriptions of care and treatment set forth below
are minimal performance standards, and will be superseded by local regulations.
Further guidance of the humane treatment of animals is given by the OECD (Ref.
17).
Definitions used in this Test Guideline are given in Appendix 1.
Principle of the Test. The Hershberger Bioassay achieves its sensitivity by
using males with minimal endogenous androgen production. This is achieved
through the use of castrated males provided an adequate time after castration for
the target tissues to regress to a minimal and uniform baseline weight is allowed.
Thus, when screening for potential androgenic activity, there are low endogenous
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levels of circulating androgens, the hypothalamic-pituitary- gonad axis is
rendered unable to compensate via feedback mechanisms, the ability of the
tissues to respond is maximized, and the starting tissue weight variability is
minimized. When screening for potential anti-androgenic activity, a more
consistent tissue weight gain can be achieved when the tissues are stimulated by
a reference androgen. As a result, the Hershberger Bioassay requires only 6
animals per dose group whereas other assays with intact pubertal or adult males
suggest using 15 males per dose group.
Castration of peripubertal male rats must be done in an appropriate manner
using approved anesthetics and aseptic technique. Analgesics should be
administered on the first few days following surgery to eliminate post-surgical
discomfort. Castration enhances the precision of the assay to detect weak
androgens and antiandrogens by eliminating compensatory endocrine feed-back
mechanisms present in the intact animal that can attenuate the effects of
administered androgens and antiandrogens and by eliminating the large inter-
individual variability in serum testosterone levels. Hence, castration reduces the
numbers of animals required to screen for these endocrine activities.
When screening for potential androgenic activity, the test substance is
administered daily by oral gavage or subcutaneous injection for a period of ten
consecutive days. Test substances are administered to a minimum of two
treatment groups of experimental animals using one dose level per group. The
animals are necropsied approximately 24 hours after the last dose. A statistically
significant increase in two or more target organ weights of the test substance
groups compared to the vehicle control group indicates that the test substance is
positive for potential androgenic activity (See section (i)). Androgens, like
trenbolone that cannot be 5 a reduced have more pronounced effects on the
LABC and GP versus TP, but all tissues should display increased growth.
When screening for potential antiandrogenic activity, the test substance is
administered daily by oral gavage or subcutaneous injection for a period of ten
consecutive days in concert with daily TP doses (0.2 or 0.4 mg/kg/d) by sc
injection. It was determined in the validation program that either 0.2 or 0.4
mg/kg/d of TP could be used as both were effective in the detection of
antiandrogens and, therefore, only one dose should be selected for use in the
assay. Graduated test substance doses are administered to a minimum of three
treatment groups of experimental animals using one dose level per group. The
animals are necropsied approximately 24 hours after the last dose. A statistically
significant decrease in two or more target organ weights of the test substance
plus TP groups compared to the TP only control group indicates that the test
substance is positive for potential antiandrogenic activity (See section (i)).
Description of the Method.
(1) Selection of Species and Strain. The rat has been routinely used in the
Hershberger Bioassay since the 1930s. Although it is biologically
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plausible that both the rat and mouse would display similar responses,
based upon 70 years of experience with the rat model, the rat is the
species of choice for the Hershberger Bioassay. In addition, since
Hershberger Bioassay data may be preliminary to a long-term
multigenerational study, this allows animals from the same species, strain
and source to be used in both studies.
This test guideline allows laboratories to select the strain of rat to be used
in the assay which should generally be that used historically by the
participating laboratory. Commonly used laboratory rat strains may be
used; however, strains that mature significantly later than 42 days of age
should not be used since castration of these males at 42 days of age
could preclude measurement of glans penis weights, which can only be
done after the prepuce is separated from the penile shaft. Thus, strains
derived from the Fisher 344 rat should not be used, except in rare cases.
The Fisher 344 rat has a different timing of sexual development compared
with other more commonly used strains such as Sprague Dawley or
Wistar strains (Ref. 16). If such a strain is to be used, the laboratory
should castrate them at a slightly older age and be able to demonstrate
the sensitivity of the strain used. The rationale for the choice of rat strain
should be clearly stated by the laboratory. Where the screening assay
may be preliminary to a repeated dose oral study, a reproductive and
developmental study, or a long-term study, preferably animals from the
same strain and source should be used in all studies.
Housing and Feeding Conditions. All procedures should conform to all
local standards of laboratory animal care. These descriptions of care and
treatment are minimum standards and will be superseded by more
stringent local regulations, when present. The temperature in the
experimental animal room should be 22°C (with an approximate range ±
3°C). The relative humidity should be a minimum of 30% and preferably
should not exceed a maximum 70%, other than during room cleaning.
The aim should be relative humidity of 50-60%. Lighting should be
artificial. The daily lighting sequence should be 12 hours light, 12 hours
dark.
Group housing is preferable to isolation because of the young age of the
animals and the fact that rats are social animals. Housing of two or three
animals per cage avoids crowding and associated stress that may
interfere with the hormonal control of the development of the sex
accessory tissue. Cages should be thoroughly cleaned to remove
possible contaminants and arranged in such a way that possible effects
due to cage placement are minimized. Cages of a proper size (~2000
square centimeters) will prevent overcrowding.
Each animal should be identified individually (e.g., ear mark or tag) using
a humane method. The method of identification should be recorded.
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Laboratory diet and drinking water should be provided ad libitum.
Laboratories executing the Hershberger Bioassay should use the
laboratory diet normally used in their chemical testing work. In the
validation studies of the Bioassay, no effects or variability were observed
that were attributable to the diet. The diet used will be recorded and a
sample of the laboratory diet should be retained for possible future
analysis.
(3) Performance Criteria for Androgen-dependent Organ Weights.
During the validation study, there was no evidence that a decrease in
body weight affected increases or decreases in the growth of the
mandatory tissue weights.
Among the different strains of rat used successfully in the validation
program, androgen-dependent organ weights are larger in the heavier rat
strains than in the lighter strains. Therefore, the Hershberger Bioassay
performance criteria do not include absolute expected organ weights for
positive and negative controls.
Because the Coefficient of Variation (CV) for a tissue has an inverse
relationship with statistical power, the Hershberger Bioassay performance
criteria are based on maximum CV values for each tissue (Table 1 )1. The
CVs are derived from the OECD validation studies. In the case of
negative outcomes, laboratories should examine the CVs from the control
group and the high dose treatment group to determine if the maximum CV
performance criteria have been exceeded.
The study should be repeated when: 1) three or more of the ten possible
individual CVs in the control and high dose treatment groups exceed the
maximums designated for agonist and antagonist studies in Table 1 and
2) at least two target tissues were marginally insignificant, i.e., p values
between 0.05 and 0.10.
1 The threshold CV for a given tissue was identified from a graph of CV values - arranged from smallest
sequentially to largest - for all means from all experiments in the validation exercise using a specific
model (agonist or antagonist). The threshold CV was read from the point at which the increments
between to the next highest CVs in the series are dramatically larger than the preceding few CV.s- the
"breakpoint". It should be noted that although this analysis identified relatively reliable "breakpoints" for
the antagonist model of the assay, CV curves for the agonist assay showed a more uniform increase
making identification of a threshold CV by this method somewhat arbitrary.
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Table 1. Maximum allowable CV.s Determined for the Mandatory Sex Accessory
Tissues for the castrate model in the OECD Validation Studies.
Tissue
Antiandrogenic effects
Androgenic effects
Seminal vesicles
40%
40%
Ventral prostate
40%
45%
LABC
20%
30%
Cowper.s glands
35%
55%
Glans penis
17%
22%
(g) Procedure.
(1) Regulatory Compliance and Laboratory Verification. Unlike the
uterotrophic assay (OPPTS Test Guideline 890.1600), a demonstration of
laboratory competence prior to the initiation of the study is not necessary
for the Hershberger assay because concurrent positive (Testosterone
Propionate and Flutamide) and negative controls are run as an integral
part of the assay.
(2) Number and Condition of Animals. Each treated and control group
should include a minimum of 6 animals. This applies to both the
androgenic and antiandrogenic protocols.
(3) Castration. There should be an initial acclimatization period of several
days after receipt of the animals to ensure that the animals are healthy
and thriving. Since animals castrated before 42 days of age or postnatal
day (pnd) 42 may not display preputial separation, animals should be
castrated on pnd 42 or thereafter, not before. The animals are castrated
under anesthesia by placing an incision in the scrotum and removing both
testes and epididymides with ligation of blood vessels and seminal ducts.
After confirming that no bleeding is occurring, the scrotum should be
closed with suture or autoclips. Animals should be treated with analgesics
for the first few days after surgery to alleviate any post-surgical discomfort.
If castrated animals are purchased from an animal supplier, the age of
animals and stage of sexual maturity should be assured by the supplier.
(4) Acclimatization After Castration. The animals should continue
acclimation to the laboratory conditions to allow for the regression in the
target tissue weights for a minimum of 7 days following castration.
Animals should be observed daily, and any animals with evidence of
disease or physical abnormalities should be removed. Thus, treatment
with initiation of dosing (on study) may commence as early as pnd 49 days
of age, but not later than pnd 60. Age at necropsy should not be greater
than pnd 70. This flexibility allows a laboratory to schedule the
experimental work efficiently.
(5) Body Weight and Group Randomization. Differences in individual body
weights are a source of variability in tissues weights both within and
among groups of animals. Increasing tissue weight variability results in an
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increased coefficient of variation (CV) and decreases the statistical power
of the assay (sometimes referred to as assay sensitivity). Therefore,
variations in body weight should be both experimentally and statistically
controlled.
Experimental control involves producing small variations in body weight
within and among the study groups. First, unusually small or large
animals should be avoided and not placed in the study cohort. At study
commencement the weight variation of animals used should not exceed ±
20% of the mean weight {e.g. 175g ± 35g). Second, animals should be
assigned to groups (both control and treatment) by randomized weight
distribution, so that mean body weight of each group is not statistically
different from any other group. The block randomization procedure used
should be recorded.
Because toxicity may decrease the body weight of treated groups relative
to the control group, the body weight on the first day of test substance
administration could be used as the statistical covariate, not the body
weight at necropsy.
Dosage. In order to establish whether a test substance can have
androgenic action in vivo, two dose groups of the test substance plus
positive and vehicle (negative) controls (See section (c)) are normally
sufficient, and this design is therefore preferred for animal welfare
reasons. If the purpose is either to obtain a dose-response curve or to
extrapolate to lower doses, at least 3 dose groups are needed. If
information beyond identification of androgenic activity (such as an
estimate of potency) is required, a different dosing regime should be
considered. To test for antiandrogens, the test substance is administered
together with a reference androgen agonist. A minimum of 3 test groups
with different doses of the test chemical and a positive and a negative
control (See subsection (g)(12)) should be used. Except for treatment
with the test substance, animals in the control group should be handled in
an identical manner to the test group subjects. If a vehicle is used in
administering the test substance, the control group should receive the
vehicle in the highest volume used with the test groups.
All dose levels should be proposed and selected taking into account any
existing toxicity and (toxico-) kinetic data available for the test substance
or related materials. The highest dose level should first take into
consideration the LD50 and/or acute toxicity information in order to avoid
death, severe suffering or distress in the animals (Ref. 17, 18, 19, & 20)
and, second, take into consideration available information on the doses
used in subchronic and chronic studies. In general, the highest dose
should not cause a reduction in the final body weight of the animals
greater than 10% of control weight. The highest dose should be either 1)
the highest dose that ensures animal survival and that is without
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significant toxicity or distress to the animals after 10 consecutive days of
administration up to a maximal dose of 1000 mg/kg/day (See subsection
(g)(7)); or 2) a dose inducing (anti)androgenic effects, whichever is lower.
As a screen, large intervals, e.g., one half log units (corresponding to a
dose progression of 3.2) or even one log units, between dosages are
acceptable. If there are no suitable data available, a range finding study
(See subsection (g)(8)) may be performed to aid the determination of the
doses to be used.
Limit Dose Level. If a test at the limit dose of 1000 mg/kg body
weight/day and a lower dose using the procedures described for this
study, fails to produce a statistically significant change in reproductive
organ weights, then additional dose levels may be considered
unnecessary. The limit dose applies except when human exposure data
indicate the need for a higher dose level to be used.
Considerations for Range Finding. If necessary, a preliminary range
finding study can be carried out with few animals to select the appropriate
dose groups. The objective in the case of the Hershberger Bioassay is to
select doses that ensure animal survival and that are without significant
toxicity or distress to the animals after ten consecutive days of chemical
administration up to a limit dose of 1000 mg/kg/d as noted in subsection
(g)(6) and (g)(7). In this respect an OECD Guidance Document (Ref. 17)
may be used defining clinical signs indicative of toxicity or distress to the
animals. If feasible within this range finding study after ten days of
administration, the mandatory target tissues may be excised and weighed
approximately 24-hours after the last dose is administered. These data
could then be used to assist the selection of the doses in the main study.
Reference Substances and Vehicle. The reference androgen agonist
should be Testosterone Propionate (TP), CAS No 57-82-5. The reference
TP dosage may be either 0.2 mg/kg-bw/d or 0.4 mg/kg-bw/d. The
reference androgen antagonist should be Flutamide (FT), CAS No 1311-
84-7. The reference FT dosage should be 3 mg/kg-bw/d, and the FT
should be coadministered with the reference TP dosage.
It is recommended that, wherever possible, the use of an aqueous
solution/suspension be considered first. However, since many androgen
ligands or their metabolic precursors tend to be hydrophobic, the most
common approach is to use a solution/suspension in oil {e.g. corn, peanut,
sesame or olive oil). Test substances can be dissolved in a minimal
amount of 95% ethanol or other appropriate solvents and diluted to final
working concentrations in the test vehicle. The toxic characteristics of the
solvent must be known, and should be tested in a separate solvent-only
control group. If the test substance is considered stable, gentle heating
and vigorous mechanical action can be used to assist in dissolving the test
substance. The stability of the test substance in the vehicle should be
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determined. If the test substance is stable for the duration of the study,
then one starting aliquot of the test substance may be prepared, and the
specified dosage dilutions prepared daily using care to avoid
contamination and spoilage of the samples.
Administration of Doses. TP should be administered by subcutaneous
injection, and FT by oral gavage.
The test substance is administered by oral gavage or subcutaneous
injection. Animal welfare considerations and the physical/chemical
properties of the test substance need to be taken into account when
choosing the route of administration. In addition, toxicological aspects like
the relevance to the human route of exposure to the chemical {e.g. oral
gavage to model ingestion, subcutaneous injection to model inhalation or
dermal adsorption) and existing toxicological information and data on
metabolism and kinetics {e.g. need to avoid first pass metabolism, better
efficiency via a particular route) should be taken into account before
extensive, long-term testing is initiated if positive results are obtained by
injection.
The animals should be dosed in the same manner and time sequence for
ten consecutive days at approximately 24 hour intervals. The dosage
level should be adjusted daily based on the concurrent daily measures of
body weight. The volume of dose and time that it is administered should
be recorded on each day of exposure. Care must be taken in order not to
exceed the maximum dose described in subsection (g)(6) to allow a
meaningful interpretation of the data. Reduction of body weight, clinical
signs, and other findings should be thoroughly assessed in this respect.
For oral gavage, a stomach tube or a suitable intubation cannula should
be used. The maximum volume of liquid that can be administered at one
time depends on the size of the test animal. Local animal care guidelines
should be followed, but the volume should not exceed 5 ml/kg body
weight, except in the case of aqueous solutions where 10 ml/kg body
weight may be used. For subcutaneous injections, doses should be
administered to the dorsoscapular and or lumbar regions via sterile needle
{e.g., 23- or 25-gauge) and a tuberculin syringe. Shaving the injection site
is optional. Any losses, leakage at the injection site or incomplete dosing
should be recorded. The total volume injected per rat per day should not
exceed 0.5 ml/kg body weight.
Specific Procedures for Androgen Agonists. For the test for androgen
agonists, the vehicle is the negative control, and the TP-treated group is
the positive control. Biological activity consistent with androgen agonists
is tested by administering a test substance to treatment groups at the
selected doses for 10 consecutive days. The weights of the five sex
accessory tissues from the test substance groups are compared to the
vehicle group for statistically significant increases in weight.
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(12) Specific Procedures for Androgen Antagonists and 5a-reductase
Inhibitors. For the test for androgen antagonists and 5a-reductase
inhibitors, the TP-treated group is the negative control, and the group
coadministered reference doses of TP and FT is the positive control.
Biological activity consistent with androgen antagonists and 5a-reductase
inhibitors is tested by administering a reference dose of TP and
administering the test substance for 10 consecutive days. The weights of
the five sex accessory tissues from the TP plus test substance groups are
compared to the reference TP-only group for statistically significant
decreases in weights.
Observations.
(1) Clinical Observations. General clinical observations should be made at
least once a day and more frequently when signs of toxicity are observed.
Observations should be carried out preferably at the same time(s) each
day and considering the period of anticipated peak effects after dosing.
All animals should be observed for mortality, morbidity and general
clinical signs such as changes in behavior, skin, fur, eyes, mucous
membranes, occurrence of secretions and excretions and autonomic
activity {e.g., lacrimation, piloerection, pupil size, unusual respiratory
pattern).
Any animal found dead should be removed and disposed of without further
data analysis. Any mortality of animals prior to necropsy should be
included in the study record together with any apparent reasons for
mortality. Any moribund animals should be humanely terminated. Any
moribund and subsequently euthanized animals should be included in the
study record with apparent reasons for morbidity.
(2) Body Weight and Food Consumption. All animals should be weighed
daily to the nearest 0.1 g, starting just prior to initiation of treatment i.e.,
when the animals are allocated into groups. As an optional measurement,
the amount of food consumed during the treatment period may be
measured per cage by weighing the feeders. The food consumption
results should be expressed in grams per rat per day.
(3) Dissection and Measurement of Tissue and Organ Weights.
Approximately 24 hours after the last administration of the test substance,
the rats should be euthanized and exsanguinated according to the normal
procedures of the conducting laboratory, and necropsy carried out. The
method of humane killing should be recorded in the laboratory report.
Ideally, the necropsy order should be randomized across groups to avoid
progression directly up or down dose groups that could affect the data.
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Any finding at necropsy, i.e., pathological changes/visible lesions should
be noted and reported.
The five androgen-dependent tissues (VP, SV, LABC, COW, GP) are
mandatory measurements. These tissues should be excised, carefully
trimmed of excess adhering tissue and fat, and their fresh (unfixed)
weights determined. Each tissue should be handled with particular care to
avoid the loss of fluids and to avoid desiccation, which may introduce
significant errors and variability by decreasing the recorded weights.
Several of the tissues may be very small or difficult to dissect, and this will
introduce variability. Therefore, it is important that persons carrying out
the dissection of the sex accessory tissues are familiar with standard
dissection procedures for these tissues. A standard operating procedure
(SOP) manual for dissection is available from the OECD (Ref. 23).
Careful training according to the SOP guide will minimize a potential
source of variation in the study. Ideally the same prosector should be
responsible for the dissection of a given tissue to eliminate inter-individual
differences in tissue processing. If this is not possible, the necropsy
should be designed such that each prosector dissects a given tissue from
all treatment groups as opposed to one individual dissecting all tissues
from a control group, while someone else is responsible for the treated
groups. Each sex accessory tissues should be weighed without blotting to
the nearest 0.1 mg, and the weights recorded for each animal.
Several of the tissues may be very small or difficult to dissect, and this will
introduce variability. Previous work has indicated a range of coefficient of
variations (CVs) that appears to differ based upon the proficiency of the
laboratory. In a few cases, large differences in the absolute weights of the
tissues such as the VP and COWS have been observed within a particular
laboratory.
Liver, paired kidney, and paired adrenal weights are optional
measurements. Again, tissues should be trimmed free of any adhering
fascia and fat. The liver should be weighed and recorded to the nearest
0.1 g, and the paired kidneys and paired adrenals should be weighed and
recorded to the nearest 0.1 mg. The liver, kidney and adrenals are not
only influenced by androgens; they also provide useful indices of systemic
toxicity.
Measurement of serum luteinizing hormone (LH), follicular stimulating
hormone (FSH) and testosterone (T) is optional. Serum T levels are
useful to determine if the test substance induces liver metabolism of
testosterone, lowering serum levels. Without the T data, such an effect
might appear to be via an antiandrogenic mechanism. LH levels provides
information about the ability of an antiandrogen to not only reduce organ
weights, but also to affect hypothalamic-pituitary function, which in long
term studies can induce testis tumors. FSH is an important hormone for
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spermatogenesis. Serum T4 and T3 also are optional measures that
would provide useful supplemental information about the ability to disrupt
thyroid hormone homeostasis. If hormone measurements are to be made,
the rats should be anesthetized prior to necropsy and blood taken by
cardiac puncture, and the method of anesthesia should be chosen with
care so that it does not affect hormone measurement. The method of
serum preparation, the source of radioimmunoassay or other
measurement kits, the analytical procedures, and the results should be
recorded. LH levels should be reported as ng per ml of serum, and T
should also be reported as ng per ml of serum.
The dissection of the tissues is described as follows with a detailed
dissection guide with photographs published as supplementary materials
as part of the validation program (Ref. 21). A dissection video is also
available from the Korea Food and Drug Administration web page (Ref.
22).
~ With the ventral surface of the animal upwards, determine if the prepuce
of the penis has separated from the glans penis. If so, then retract the
prepuce and remove the glans penis, weigh (nearest 0.1 mg), and record
the weight.
~ Open the abdominal skin and wall, exposing the viscera. If the optional
organs are weighed, remove and weigh liver to nearest 0.1 g, remove the
stomach and intestines, remove and weigh the paired kidneys and paired
adrenals to the nearest 0.1 mg. This dissection exposes the bladder and
begins the dissection of the mandatory male accessory tissues.
~ To dissect the VP, separate bladder from the ventral muscle layer by
cutting connective tissue along the midline. Displace the bladder
anteriorly towards the seminal vesicles (SV), revealing the left and right
lobes of the ventral prostate (covered by a layer of fat). Carefully tease the
fat from the right and left lobes of the VP. Gently displace the VP right
lobe from the urethra and dissect the lobe from the urethra. While still
holding the VP right lobe, gently displace the VP left lobe from the urethra
and then dissect; weigh to nearest 0.1 mg and record the weight.
~ To dissect the SVCG, displace the bladder caudally, exposing the vas
deferens and right and left lobes of the seminal vesicles plus coagulating
glands (SVCG). Prevent leakage of fluid by clamping a hemostat at the
base of the SVCGs, where the vas deferens joins the urethra. Carefully
dissect the SVCGs, with the hemostat in place trim fat and adnexa
away, place in a tared weigh-boat, remove the hemostat, and weigh to
the nearest 0.1 mg and record the weight.
~ To dissect the levator ani plus bulbocavernosus muscles (LABC), the
muscles and the base of the penis are exposed. The LA muscles wrap
around the colon, while the anterior LA and BC muscles are attached
to the penile bulbs. The skin and adnexa from the perianal region
extending from the base of the penis to the anterior end of the anus are
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removed. The BC muscles are gradually dissected from the penile bulb
and tissues. The colon is cut in two and, the full LABC can be
dissected and removed. The LABC should be trimmed of fat and
adnexa, weighed to the nearest 0.1 mg, and record the weight.
~ After the LABC has been removed, the round Cowper's or
bulbourethral glands (COW) are visible at the base of, and slightly
dorsal to, the penile bulbs. Careful dissection is required to avoid nicking
the thin capsule in order to prevent fluid leakage. Weigh the paired COW
to the nearest 0.1 mg, and record the weight.
~ In addition, if fluid is lost from any gland during the necropsy and
dissection, this should be recorded.
If the evaluation of each chemical requires necropsy of more animals than
is reasonable for a single day, the study start may be staggered on two
consecutive days, resulting in the staggering of the necropsy and the
related work over two days. If staggered in this manner, one-half of the
animals per treatment group should be used per day.
Carcasses should be disposed of in an appropriate manner following
necropsy.
Reporting.
(1) Data. Data should be reported individually {i.e., body weight, accessory
sex tissue weights, optional measurements and other responses and
observations) and for each group of animals (means and standard
deviations of all measurement taken). The data should be summarized in
tabular form. The data should show the number of animals at the start of
the test, the number of animals found dead during the test or found
showing signs of toxicity, a description of the signs of toxicity observed,
including time of onset, duration and severity. Data are requested to be
submitted in a machine-readable (electronic) form.
(2) Final Report. A final report shall include:
~ Testing facility:
• Name of facility, location.
• Study director and other personnel and their study responsibilities.
• Dates the study began and ended, i.e., first day of test substance
administration and last day of necropsy, respectively.
~ Test substance:
• Source, lot/batch number, identity, purity, full address of the supplier
and characterization of the test substance(s).
• Physical nature and, where relevant, physicochemical properties.
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• Storage conditions and the method and frequency of dilution
preparation.
• Any data generated on stability.
• Any analyses of dosing solutions/suspensions.
~ Vehicle:
• Characterization of the vehicle (identity, supplier and lot #).
• Justification of the vehicle choice (if other than water).
~ Test animals and animal husbandry procedures:
• Species/strain used and rationale for choice.
• Source or supplier of animals, including full address.
• Number and age of animals supplied.
• Housing conditions (temperature, lighting, and so on).
• Diet (name, type, supplier, lot number, content and if known,
phytoestrogens levels).
• Bedding (name, type, supplier, content).
• Caging conditions and number of animals per cage.
~ Assay Conditions:
• Age at castration and duration of acclimatization after castration.
• Individual weights of animals at the start of the study (to nearest 0.1 g).
• Randomization process and a record of the assignment to vehicle,
reference, test.
• Substance groups, and cages.
• Mean and standard deviation of the body weights for each group for
each weigh day throughout the study.
• Rationale for dose selection.
• Route of administration of test substance and rationale for the choice
of exposure route.
• If an assay for antiandrogenicity, the TP treatment (dose and volume).
• Test substance treatment (dose and volume).
• Time of dosing.
• Necropsy procedures, including means of exsanguinations and any
anesthesia.
• If serum analyses are performed, details of the method should be
supplied. For example, if RIA is used, the RIA procedure.
• Source of RIA kits, kit expiration dates, procedure for scintillation
counting, and standardization should be reported.
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~ Results:
• Daily observations for each animal during dosing, including:
- Body weights (to the nearest 0.1 g);
- Clinical signs (if any);
- Any measurement or notes of food consumption.
• Necropsy observations for each animal, including:
- Date of necropsy.
- Animal treatment group.
- Animal ID.
- Prosector.
- Time of day necropsy and dissection are performed
- Animal age.
- Final body weight at necropsy, noting any statistically significant
increase or decrease.
- Order of animal exsanguination and dissection at necropsy.
- Weights of five mandatory androgen dependent tissues:
+ Ventral prostate (to the nearest 0.1 mg);
+ Seminal vesicles plus coagulating glands, including fluid (paired,
to nearest 0.1 mg);
+ Levator ani plus bulbocavernosus muscle complex (to nearest
0.1 mg);
+ Cowper's glands (fresh weight - paired, to nearest 0.1 mg);
+ Glans penis in the adult castrate version (fresh weight to
nearest 0.1 mg).
- Weights of optional tissues, if performed:
+ Liver (to nearest 0.1 g)
+ Kidney (paired, to nearest 0.1 mg)
+ Adrenal (paired, to nearest 0.1 mg)
- General remarks and comments.
• Analyses of serum hormones, if performed:
- Serum LH (optional - ng per ml of serum), and
- Serum T (optional - ng per ml of serum).
• General remarks and comments.
~ Data summarization. Data should be summarized in tabular form
containing the sample size for each group, the mean of the value, and the
standard error of the mean or the standard deviation. Tables should
include necropsy body weights, body weight changes from the beginning
of dosing until necropsy, mandatory accessory sex tissues weights, and
any optional organ weights.
~ Discussion/Analysis of the Results. Necropsy body and organ weights
should be statistically analyzed for characteristics such as homogeneity of
variance with appropriate data transformations as needed. Treatment
groups should be compared to a control group using techniques such as
ANOVA followed by pairwise comparisons {e.g. Dunnett's one tailed test)
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and the criterion for statistical difference, for example, p < 0.05. Those
groups attaining statistical significance should be identified. However,
"relative organ" weights should be avoided due to the invalid statistical
assumptions underlying this data manipulation.
For androgen agonism, the control should be the vehicle-only test group.
The mode of action characteristics of a test substance can lead to different
relative responses amongst the tissues, for example trenbolone, which
cannot be 5 alpha-reduced, has more pronounced effects on the LABC
and GP than does TP. A statistically significant increase (p< 0.05) in any
two or more of the five required androgen-dependent tissue weights (VP,
LABC, GP, CG and SVCG) should be considered a positive androgen
agonist result, and all the target tissues should display some degree of
increased growth. Combined evaluation of all ASO tissue responses
could be achieved using appropriate multivariate data analysis. This could
improve the analysis, especially in cases where only a single tissue gives
a statistically significant response.
For androgen antagonism, the control should be the reference androgen
(testosterone propionate only) test group. The mode of action
characteristics of a test substance can lead to different relative responses
amongst the tissues, for example 5 alpha a-reductase inhibitors, like
finasteride, have more pronounced effects on the ventral prostate than
other tissues as compared to a potent AR antagonists, like flutamide. A
statistically significant reduction (p < 0.05) in any two or more of the five
required androgen-dependent tissue weights (VP, LABC, GP, CG and
SVCG) relative to TP treatment alone should be considered a positive
androgen antagonist result and all the target tissues should display some
degree of reduced growth. Combined evaluation of all ASO tissue
responses could be achieved using appropriate multivariate data analysis.
This could improve the analysis, especially in cases where only a single
tissue gives a statistically significant response. Data should be
summarized in tabular form containing the mean, standard error of the
mean (standard deviation would also be acceptable) and sample size for
each group. Individual data tables should also be included. The individual
values, mean, SE (SD) and CV values for the control data should be
examined to determine if they meet acceptable criteria for consistency
with expected historical values. CVs that exceed CV values listed in Table
1 (See subsection (f)(3)) for each organ weight should determine if there
are errors in data recording or entry or if the laboratory has not yet
mastered accurate dissection of the androgen-dependent tissues and
further training/practice is warranted. Generally, CVs (the standard
deviation divided by the mean organ weight) are reproducible from lab to
lab and study to study.
Data presented should include at least; ventral prostate, seminal vesicle,
levator ani plus bulbocavernosus, Cowper's glands, glans penis, liver, and
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body weights and body weight change from the beginning of dosing until
necropsy. Data also may be presented after covariance adjustment for
body weight, but this should not replace presentation of the unadjusted
data. In addition, if preputial separation (PPS) does not occur in any of
the groups, the incidence of PPS should be recorded and statistically
compared to the control group using Fisher Exact test.
Data should be summarized in tabular form containing the mean, standard
error of the mean (standard deviation would also be acceptable) and
sample size for each group. Individual data tables should also be included.
The individual values, mean, SE (SD) and CV values for the control data
should be examined to determine if they meet acceptable criteria for
consistency with expected historical values. CVs that exceed CV values
listed in Table 1 (See subsection (f)(3)) for each organ weight should
determine if there are errors in data recording or entry or if the laboratory
has not yet mastered accurate dissection of the androgen-dependent
tissues and further training/practice is warranted. Generally, CVs (the
standard deviation divided by the mean organ weight) are reproducible
from lab to lab and study to study. Data presented should include at least;
ventral prostate, seminal vesicle, levator ani plus bulbocavernosus,
Cowper's glands, glans penis, liver, and body weights and body weight
change from the beginning of dosing until necropsy. Data also may be
presented after covariance adjustment for body weight, but this should not
replace presentation of the unadjusted data. In addition, if preputial
separation (PPS) does not occur in any of the groups, the incidence of
PPS should be recorded and statistically compared to the control group
using Fisher Exact test.
When verifying the computer data entries with the original data sheets for
accuracy, organ weight values that are not biologically plausible or vary by
more than three standard deviations from that treatment group means
should be carefully scrutinized and may need to be discarded, likely being
recording errors.
Comparison of study results with OECD CV values (in Table 1) is often an
important step in interpretation as to the validity of the study results.
Historical data for vehicle control groups should be maintained in the
laboratory. Historical data for responses to positive reference substances,
such as TP and FT, should also be maintained in the laboratory.
Laboratories may also periodically test the response to known weak
androgen agonists and antagonists and maintain these data. These data
can be compared to available data OECD data to ensure that the
laboratory's methods yield sufficient statistical precision and power.
References.
OECD (1998). Report of the First Meeting of the OECD Endocrine Disrupter
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Testing and Assessment (EDTA) Task Force, 10th-11 th March 1998,
ENV/MC/CHEM/RA(98)5.
2. Dorfman Rl (1962). Standard methods adopted by official organization. Academic
Press, NY.
3. Gray LE Jr, Furr J and Ostby JS (2005). Hershberger assay to investigate the
effects of endocrine disrupting compounds with androgenic and antiandrogenic
activity in castrate-immature male rats. In: Current Protocols in Toxicology 16.9.1-
16.9.15. J Wiley and Sons Inc.
4. OECD (2002). Final OECD report of the initial work towards the validation of the
rat Hershberger assay. Phase 1. Androgenic response to testosterone propionate
and anti-androgenic effects of flutamide. ENV/JM/TG/EDTA(2002)1/REV2/ADD1.
5. OECD (2005). Report of the OECD Validation of the Rat Hershberger Bioassay:
Phase 2: Testing of Androgen Agonists, Androgen Antagonists and a 5a-
Reductase Inhibitor in Dose Response Studies by Multiple Laboratories.
E N V/J M/T G/E DT A(2003)4/ ADD1.
6. OECD (2007). Report of the Validation of the Rat Hershberger Assay: Phase 3:
Coded Testing of Androgen Agonists, Androgen Antagonists and Negative
Reference Chemicals by Multiple Laboratories. Surgical Castrate Model Protocol.
Environmental Health and Safety Monograph Series on Testing and Assessment
No73. ENV/JM/MONO(2007)20.
7. Owens, W, Zeiger E, Walker M, Ashby J, Onyon L, Gray, Jr, LE (2006). The OECD
programme to validate the rat Hershberger bioassay to screen compounds for in
vivo androgen and antiandrogen responses. Phase 1: Use of a potent agonist and
a potent antagonist to test the standardized protocol. Env. Health Persp. 114:1265-
1269.
8. Owens W, Gray LE, Zeiger E, Walker M, Yamasaki K, Ashby J, Jacob E (2007).
The OECD program to validate the rat Hershberger bioassay to screen
compounds for in vivo androgen and antiandrogen responses: phase 2 dose-
response studies. Environ Health Perspect. 115(5):671 -8.
9. Korenchevsky V (1932). The assay of testicular hormone preparations. Biochem J
26:413-422.
10. Korenchevsky V, Dennison M, Schalit R (1932). The response of castrated male
rats to the injection of the testicular hormone. Biochem J 26:1306-1314.
11. Eisenberg E, Gordan GS (1950). The levator ani muscle of the rat as an index of
myotrophic activity of steroidal hormones. J Pharmacol Exp Therap 99:38-44.
12. Eisenberg E, Gordan GS, Elliott HW (1949). Testosterone and tissue respiration of
the castrate male rat with a possible test for mytrophic activity. Endocrinology
45:113-119. (13)
13. Hershberger L, Shipley E, Meyer R (1953). Myotrophic activity of 19-nortestosterone
and other steroids determined by modified levator ani muscle method. Proc Soc
Exp Biol Med 83:175-180.
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14. Hilgar AG, Vollmer EP (1964). Endocrine bioassay data: Androgenic and
myogenic. Washington DC: United States Public Health Service.
15. Dorfman Rl (1969). Androgens and anabolic agents. In: Methods in Hormone
Research, volume IIA. (Dorfman Rl, ed.) New York:Academic Press, 151-220.
16. Massaro EJ (2002). Handbook of Neurotoxicology, volume I. New York: Humana
Press, p 38.
17. OECD (2000). Guidance document on the recognition, assessment and use of
clinical signs as humane endpoints for experimental animals used in safety
evaluation. Environmental Health and Safety Monograph Series on Testing and
Assessment, No. 19. ENV/JM/MONO(2000)7.
18. OECD (1982). Organization for Economic Co-operation and Development -
Principles of Good Laboratory Practice, ISBN 92-64-12367-9, Paris, France.
19. OECD (2001). Acute oral toxicity - up-and-down procedure. OECD Guideline for
the testing of chemicals, No. 425.
20. OECD (2001). Guidance document on acute oral toxicity. Environmental Health
and Safety Monograph Series on Testing and Assessment, No. 24.
ENV/JM/MONO(2001)4.
21. Supplemental materials for Owens et al. (2006). The OECD programme to validate
the rat Hershberger bioassay to screen compounds for in vivo androgen and
antiandrogen responses. Phase 1: Use of a potent agonist and a potent antagonist
to test the standardized protocol. Env. Health Persp. 114:1265-1269. See section
II, The dissection guidance provided to the laboratories. Available at
http://www.ehponline.org/docs/2006/8751/suppl.pdf.
22. Korea Food and Drug Administration. Visual reference guide on Hershberger
assay procedure, including a dissection video.
http://rndmoa.kfda.go.kr/endocrine/reference/education fr.html.
23 OECD (2008). Background Review Document on the Rodent Hershberger
Bioassay. Environmental Health and Safety Monograph Series on Testing and
Assessment, No. 90. ENV/JM/MONO(2008)17.
24. OECD (2008). Draft Validation report of the Intact, Stimulated, Weanling Male Rat
Version of the Hershberger Bioassay.
25. OECD Test 441: The Hershberger Bioassay in Rats: A Short Term Test for
(Anti)Androgenic Properties.
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Appendix 1
DEFINITIONS
Androgenic is a term used to describe a positive influence on the growth of androgen-
dependent tissues.
Antiandrogenic is the capability of a chemical to suppress the action of TP in a
mammalian organism.
Date of birth is postnatal day 0.
Dose is the amount of test substance administered. For the Hershberger Bioassay, the
dose is expressed as weight of test substance per unit body weight of test animal per
day {e.g. mg/kg body weight/day).
Dosage is a general term comprising of dose, its frequency and the duration of dosing.
Moribund is a term used to describe an animal in a dying state, i.e., near the point of
death.
Postnatal day X is the Xth day of life after the day of birth.
Sensitivity is the capability of a test method to correctly identify chemicals having the
property that is being tested for.
Specificity is the capability of a test method to correctly identify chemicals not having
the property that is being tested for.
Validation is a scientific process designed to characterize the operational requirements
and limitations of a test method and to demonstrate its reliability and relevance for a
particular purpose.
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