United States Prevention, Pesticides EPA 740-C-09-0010
&S t Ufl Environmental Protection and Toxic Substances October 2009
fircrM ^
Endocrine Disruptor
Screening Program
Test Guidelines
OPPTS 890.1600:
Uterotrophic Assay
-------
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.
-------
OPPTS 890.1600: Uterotrophic Assay
(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 guideline is Test
Guideline No. 440 published by the Organization for Economic
Cooperation and Development (OECD) (Ref. 37). 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 rodent Uterotrophic Assay. The rodent Uterotrophic Assay then
underwent an extensive validation program including the compilation of a
detailed background document (Ref. 2 & 3) and the conduct of extensive
intra- and interlaboratory studies to show the relevance and reproducibility
Page 1
-------
of the assay with a potent reference estrogen, weak estrogen receptor
agonists, a strong estrogen receptor antagonist, and a negative reference
chemical (Refs. 4, 5, 6, 7, 8, & 9). The OECD Test Guideline 440 was the
outcome of the validation test program and is the basis of this OPPTS
Test Guideline. The only differences in this OPPTS Test Guideline and
the OECD TG 440 is in the expressed preference for using the
ovariectomized rat and subcutaneous injection dosing method which
reflects the specific role of the assay in the EDSP Tier 1 Battery of
Assays.
Purpose. The Uterotrophic Assay is intended as an in vivo screening assay
providing data about a single endocrine mechanism, i.e., estrogenicity. 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 Uterotrophic Assay is a short-term screening test that originated
in the 1930's (Refs. 27 & 28) and was first standardized for screening by an
expert committee in 1962 (Refs. 32 & 35). It is based on the increase in uterine
weight or uterotrophic response (for review, see Ref. 29). It evaluates the ability
of a chemical to elicit biological activities consistent with agonists or antagonists
of natural estrogens {e.g., 17l3>-estradiol), however, its use for antagonist
detection is much less common than for agonists. The uterus responds to
estrogens in two ways. An initial response is an increase in weight due to water
imbibition. This response is followed by a weight gain due to tissue growth (Ref.
30). The uterus responses in rats and mice qualitatively are comparable.
The OECD validation program used both strong and weak estrogen agonists to
evaluate the performance of the assay to identify estrogenic compounds (Refs.
4, 5, 6, 7 & 8). Thereby the sensitivity of the test procedure for estrogen agonists
was well demonstrated besides a good intra- and interlaboratory reproducibility.
With regard to negative compounds, only one "negative" reference chemical
already reported negative by uterotrophic assay as well as in vitro receptor
binding and receptor assays was included in the validation program, but
additional test data, not related to the OECD validation program, have been
evaluated, giving further support to the specificity of the Uterotrophic Assay for
the screening of estrogen agonists (Ref. 16).
Initial Considerations and Limitations. Estrogen agonists and antagonists act
as ligands for estrogen receptors a and (3 and may activate or inhibit,
respectively, the transcriptional action of the receptors. This may have the
potential to lead to adverse health hazards, including reproductive and
developmental effects. Therefore, the need exists to rapidly assess and evaluate
a chemical as a possible estrogen agonist or antagonist. While informative, the
affinity of a ligand for an estrogen receptor or transcriptional activation of reporter
genes in vitro is only one of several determinants of possible hazard. Other
Page 2
-------
determinants can include metabolic activation and deactivation upon entering the
body, distribution to target tissues, and clearance from the body, depending at
least in part on the route of administration and the chemical being tested. This
leads to the need to screen the possible activity of a chemical in vivo under
relevant conditions, unless the chemical's characteristics regarding Absorption -
Distribution - Metabolism - Elimination (ADME) already provide appropriate
information. Uterine tissues respond with rapid and vigorous growth to
stimulation by estrogens, particularly in laboratory rodents, where the oestrous
cycle lasts approximately 4 days. Rodent species, particularly the rat, are also
widely used in toxicity studies for hazard characterization. Therefore, the rodent
uterus is an appropriate target organ for the in vivo screening of estrogen
agonists and antagonists.
This Guideline is based on those protocols employed in the OECD validation
study which have been shown to be reliable and repeatable in intra- and
interlaboratory studies (Refs. 5 & 7). Currently two methods, namely, the
ovariectomized adult female method (ovx-adult method) and the immature non-
ovariectomized method (immature method) are available. It was shown in the
OECD validation test program that both methods have comparable sensitivity
and reproducibility. However, in the EDSP battery the ovariectomized (OVX)
animal is preferred due to its increased specificity over the immature model. The
immature rat has an intact hypothalamic-pituitary-gonadal (HPG) axis may cover
a larger scope of investigation than the ovariectomized animal at the expense of
specificity because it can respond to substances that interact with the HPG axis
rather than just the estrogen receptor. The HGP axis of the rat is functional at
about 15 days of age. Prior to that, puberty cannot be accelerated with
treatments like GnRH. As the females begin to reach puberty, prior to vaginal
opening, the female will have several silent cycles that do not result in vaginal
opening or ovulation, but there are some hormonal fluctuations. If a chemical
stimulates the HGP axis directly or indirectly, precocious puberty, early ovulation
and accelerated vaginal opening result. Not only chemicals that act on the HPG
axis do this but some diets with higher metabolizable energy levels than others
will stimulate growth and accelerate vaginal opening without being estrogenic.
Such substances would not induce an uterotrophic response in OVX adult
animals as their HPG axis doesn't work.
The Uterotrophic Assay in the EDSP Tier 1 battery is intended to specifically
detect estrogenic substances and complement the rat pubertal assays in the
battery which have a functionally intact HPG axis.
The uterotrophic response is not entirely of estrogenic origin, i.e.,compounds
other than agonists or antagonists of estrogens may also provide a response.
For example, relatively high doses of progesterone, testosterone, or various
synthetic progestins may all lead to a stimulative response (Ref. 30). Any
response may be analyzed histologically for keratinization and cornification of the
vagina (Ref. 30). Irrespective of the possible origin of the response, a positive
outcome of an Uterotrophic Assay should normally initiate actions for further
Page 3
-------
clarification. Additional evidence of estrogenicity could come from in vitro
assays, such as the ER binding assays and transcriptional activation assays, or
from other in vivo assays such as the female pubertal assay.
Taking into account that the Uterotrophic Assay serves as an in vivo screening
assay, the validation approach taken, served both animal welfare considerations
and a tiered testing strategy. To this end, effort was directed at rigorously
validating reproducibility and sensitivity for estrogenicity - the main concern for
many chemicals-, while little effort was directed at the antiestrogenicity
component of the assay. Only one antiestrogen with strong activity was tested
since the number of substances with a clear antiestrogenic profile (not obscured
by some estrogenic activity) is very limited. Thus this Test Guideline is dedicated
to the estrogenic protocol, while the protocol describing the antagonist mode of
the assay is included in a guidance document. The reproducibility and sensitivity
of the assay for substances with purely anti-estrogenic activity will be more
clearly defined later on, after the test procedure has been in routine use for some
time and more substances with this modality of action are identified.
It is acknowledged that all animal based procedures will 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.
25).
The intended use of the Uterotrophic Assay is as a component of the EDSP Tier
1 Battery of Assays. Other uses should consider whether the data are truly
necessary prior to the start of the assay and whether another experimental option
is appropriate.
Definitions used in this Test Guideline are given in Appendix 1.
Principle of the Test. The Uterotrophic Assay relies for its sensitivity on an
animal test system in which the hypothalamic-pituitary-ovarian axis is not
functional, leading to low endogenous levels of circulating estrogen. This will
ensure a low baseline uterine weights and a maximum range of response to
administered estrogens. A young adult female after ovariectomy with adequate
time for uterine tissues to regress is preferred for use in the EDSP Tier 1. For
other purposes, an immature female after weaning and prior to puberty is an
option.
The test substance is administered daily preferably by subcutaneous injection.
Alternatively, oral gavage may be considered. Subcutaneous injection is
preferred for the assay's screening role in EDSP to provide ADME insight and
contrast with the rat pubertal assays which employ oral gavage. Graduated test
substance doses are administered to a minimum of two treatment groups (see
subsection (h)(5) for guidance) of experimental animals using one dose level per
group and an administration period of a minimum administration period of three
Page 4
-------
consecutive days for ovx-adult method and three consecutive days for the
immature method. The animals are necropsied approximately 24 hours after the
last dose. For estrogen agonists, the mean uterine weight of the treated animal
groups relative to the vehicle group is assessed for a statistically significant
increase. A statistically significant increase in the mean uterine weight of a test
group indicates a positive response in this assay.
Description of the Method.
(1) Selection of Animal Species. Commonly used laboratory rodent strains
may be used. As an example, Sprague-Dawley and Wistar strains of rats
were used during the validation. Strains with uteri known or suspected to
be less responsive should not be used. The laboratory should
demonstrate the sensitivity of the strain used as described in section (g).
The rat and mouse have been routinely used in the Uterotrophic Assay
since the 1930s. The OECD validation studies were only performed with
rats based on an understanding that both species are expected to be
equivalent and therefore one species should be enough for the world-wide
validation in order to save resources and animals. The rat is the species
of choice in most reproductive and developmental toxicity studies. Taking
into consideration that a vast historical data base exists for mice and thus
to broaden the scope of the Uterotrophic Assay Test Guideline in rodents
to the use of mice as test species, a limited follow-up validation study was
carried out in mice (Ref. 16). A bridging approach with a limited number
of test chemicals, participating laboratories and without coded sample
testing has been selected in keeping with the original intent to save
resources and animals. This bridging validation study shows for the
Uterotrophic Assay in young adult ovariectomized mice that qualitatively
and quantitatively, the data obtained in rats and mice correspond well with
each other. Where the Uterotrophic Assay result may be preliminary to a
long-term study, this allows animals from the same strain and source to be
used in both studies. The bridging approach was limited to the OVX mice
and the report doesn't provide a robust data set to validate the immature
model, thus the immature model for mice is not considered under the
scope of the current Test Guideline.
Thus, in some cases mice may be used instead of rats. A rationale should
be given for this species, based on toxicological, pharmacokinetic, and/or
other criteria. Modifications of the protocol may be necessary for mice.
For example, the food consumption of mice on a body weight basis is
higher than that of rats and therefore the phytoestrogen content in food
should be lower for mice than for rats (Refs. 9, 20, & 22).
(2) Housing and Feeding Conditions. All procedures should conform with
local standards of laboratory animal care. These descriptions of care and
treatment are minimum standards and will be superseded by local
Page 5
-------
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.
Laboratory diet and drinking water should be provided ad libitum. Young
adult animals may be housed individually or be caged in groups of up to
three animals. Due to the young age of the immature animals, social
group housing is recommended.
Very high levels of phytoestrogens in laboratory diets have been known to
increase uterine weights in rodents to a degree enough as to interfere with
the Uterotrophic Assay (Refs. 13, 14, & 15). High levels of
phytoestrogens and of metabolized energy in laboratory diets may also
result in early puberty, if immature animals are used. The presence of
phytoestrogens results primarily from the inclusion of soy and alfalfa
products in the laboratory diets. Body weight is an important variable, as
the quantity of food consumed is related to body weight. Therefore, the
actual phytoestrogen dose consumed from the same diet may vary among
species and by age (Ref. 9). For immature female rats, food consumption
on a body weight basis may be approximately double that of
ovariectomized young adult females. For young adult mice, food
consumption on a body weight basis may be approximately quadruple that
of ovariectomized young adult female rats.
Uterotrophic Assay results (Refs. 9, 17, 18, &19), however, show that
limited quantities of dietary phytoestrogens are acceptable and do not
reduce the sensitivity of the assay. As a guide, dietary levels of
phytoestrogens should not exceed 350 |jg of genistein equivalents/gram of
laboratory diet for immature female Sprague Dawley and Wistar rats
(Refs. 6 & 9). Such diets should also be appropriate when testing in
young adult ovariectomized rats because food consumption on a body
weight basis is less in young adult as compared to immature animals. If
adult ovariectomized mice or more phytoestrogen-sensitive rats are to be
used, proportional reduction in dietary phytoestrogen levels must be
considered (Ref. 20). In addition, the differences in available metabolic
energy from different diets may lead to time shifts for the onset of puberty
(Refs. 21 & 22)
Prior to the study, careful selection is required of a diet without an
elevated levels of phytoestrogens (for guidance see Refs. 6 & 9) or
metabolizable energy, that can confound the results (Refs. 15, 17, 19, 22,
& 36). Ensuring the proper performance of the test system used by the
laboratory as specified in section (g) is an important check on both of
these factors. As a safeguard consistent with GLP, representative
Page 6
-------
sampling of each batch of diet administered during the study should be
conducted for possible analysis of phytoestrogen content {e.g., in the case
of high uterine control weight relative to historic controls or an inadequate
response to the reference estrogen, 17-alpha ethynyl estradiol). Aliquots
should be analyzed as part of the study or frozen at -20°C or in such a
way as to prevent the sample from decomposing prior to analysis.
Some bedding materials may contain naturally occurring estrogenic or
antiestrogenic substances {e.g., corn cob is known to affects the cyclicity
of rats and appears to be antiestrogenic). The selected bedding material
should contain a minimum level of phytoestrogens.
(3) Preparation of Animals. Experimental animals without evidence of any
disease or physical abnormalities are randomly assigned to the control
and treatment groups. Cages should be arranged in such a way that
possible effects due to cage placement are minimized. The animals
should be identified uniquely. Preferably, immature animals should be
caged with dams or foster dams until weaning during acclimatization. The
acclimatization period prior to the start of the study should be about 5 days
for young adult animals and for the immature animals delivered with dams
or foster dams. If immature animals are obtained as weanlings without
dams a shorter duration of the acclimatization period may become
necessary as dosing should start immediately after weaning (see
subsection (h)(2)).
Verification of Laboratory Proficiency.
(1) Two different options can be used to verify laboratory proficiency:
~ Periodic verification, relying on an initial baseline positive control study
(see subsection (g)(2)). At least every 6 months and each time there is a
change that may influence the performance of the assay {e.g., a new
formulation of diet, change in personnel performing dissections, change in
animal strain or supplier, etc.), the responsiveness of the test system
(animal model) should be verified using an appropriate dose (based on the
baseline positive control study described below) of a reference estrogen:
17a-ethynyl estradiol (CAS No. 57-63-6) (EE).
~ Use of concurrent controls, by including a group administered with an
appropriate dose of reference estrogen in each assay.
If the system does not respond as expected the experimental conditions
should be examined and modified accordingly. It is recommended that
this dose of reference estrogen to be used in either approach be
approximately the ED70 to 80.
Page 7
-------
(2) Baseline Positive Control Study. Prior to the study, laboratory
proficiency should be demonstrated by testing the responsiveness of the
animal model, by establishing the dose response of a reference estrogen:
17a-ethynyl estradiol (CAS No. 57-63-6) (EE) with a minimum of four
doses. The uterine weight response will be compared to established
historical data (see Ref. 5). If this baseline positive control study does not
yield the anticipated results, the experimental conditions should be
examined and modified.
Procedure.
(1) Number and Condition of Animals. Each treated and control group
should include at least 6 animals (for both immature and ovx-adult method
protocols).
(2) Age of Immature Animals. For the Uterotrophic Assay with immature
animals the day of birth must be specified. Dosing should begin early
enough to ensure that, at the end of test substance administration, the
physiological rise of endogenous estrogens associated with puberty has
not yet taken place. On the other hand, there is evidence that very young
animals may be less sensitive. For defining the optimal age each
laboratory should take its own background data on maturation into
consideration.
As a general guide, dosing in rats may begin immediately after early
weaning on postnatal day 18 (with the day of birth being postnatal day 0).
Dosing in rats preferably should be completed on postnatal day 21 but in
any case prior to postnatal day 25, because, after this age, the
hypothalamic-pituitary-ovarian axis becomes functional and endogenous
estrogen levels may begin to rise with a concomitant increase in baseline
uterine weight means and an increase in the group standard deviations
(Refs. 2, 4, 10, 11, & 12)
(3) Procedure for Ovariectomy. For the ovariectomized female rat and
mouse (treatment and control groups), ovariectomy should occur between
6 and 8 weeks of age. For rats, a minimum of 14 days should elapse
between ovariectomy and the first day of administration in order to allow
the uterus to regress to a minimum, stable baseline. For mice, at least 7
days should elapse between ovariectomy and the first day of
administration. As small amounts of ovarian tissue are sufficient to
produce significant circulating levels of estrogens (Ref. 3), the animals
should be tested prior to use by observing epithelial cells swabbed from
the vagina on at least five consecutive days (e.g., days 10-14 after
ovariectomy for rats). If the animals indicate any evidence entering
oestrous, the animals should not be used. Further, at necropsy, the
ovarian stubs should be examined for any evidence that ovarian tissue is
present. If so, the animal should not be used in the calculations (Ref. 3).
Page 8
-------
The ovariectomy procedure begins with the animal in ventral recumbency
after the animal has been properly anesthetized. The incision opening the
dorso-lateral abdominal wall should be approximately 1 cm lengthways at
the mid point between the costal inferior border and the iliac crest, and a
few millimetres lateral to the lateral margin of the lumbar muscle. The
ovary should be removed from the abdominal cavity onto an aseptic field.
The ovary should be disconnected at the junction of the oviduct and the
uterine body. After confirming that no massive bleeding is occurring, the
abdominal wall should be closed by a suture and the skin closed by
autoclips or appropriate suture. The ligation points are shown
schematically in Figure 1. Appropriate post operative analgesia should be
used as recommended by a veterinarian experienced in rodent care.
Body Weight. In the ovx-adult method, body weight and uterine weight
are not correlated because uterine weight is affected by hormones like
estrogens but not by the growth factors that regulate body size. On the
contrary, body weight is related to uterine weight in the immature model,
while it is maturing (Ref. 34). Thus, at the commencement of the study
the weight variation of animals used, in the immature model, should be
minimal and not exceed ± 20% of the mean weight. This means that the
litter size should be standardized by the breeder, to assure that offspring
of different mother animals will be fed approximately the same. 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. Consideration should be given
to avoid assignment of littermates to the same treatment group as far as
practicable without increasing the number of litters to be used for the
investigation.
Dosage. In order to establish whether a test substance can have
estrogenic action in vivo, two dose groups and a control 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 estrogenic activity (such as an estimate of potency) is
required, a different dosing regime should be considered. 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 same amount of vehicle used with the treated groups (or highest
volume used with the test groups if different among groups).
The objective in the case of the Utertrophic Assay is to select doses that
ensure animal survival and that are without significant toxicity or distress
to the animals after three consecutive days of chemical administration up
to a maximum dose of 1000 mg/kg/d. All dose levels should be proposed
Page 9
-------
and selected taking into account any existing toxicity and (toxico-) kinetic
data available for the test compound 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 (Refs. 24, 25, & 26). The highest dose should represent the
maximum tolerated dose (MTD); a study conducted at a dose level that
induced a positive uterotrophic response would be accepted too. As a
screen, large intervals (e.g., one half log units corresponding to a dose
progression of 3.2 or even up to one log units) between dosages are
generally acceptable. If there are no suitable data available, a range
finding study may be performed to aid the determination of the doses to be
used.
Alternatively, if the estrogenic potency of an agonist can be estimated by
in vitro (or in silico) data, these may be taken into consideration for dose
selection. For example, the amount of the test chemical that would
produce uterotrophic responses equivalent to the reference agonist
(Ethynyl estradiol) is estimated by its relative in vitro potencies to ethynyl
estradiol. The highest test dose would be given by multiplying this
equivalent dose by an appropriate factor e.g., 10 or 100.
Considerations for Range Finding. If necessary, a preliminary range
finding study can be carried out with few animals. In this respect, OECD
Guidance Document No. 19 (Ref. 25) may be used defining clinical signs
indicative of toxicity or distress to the animals. If feasible within this range
finding study after three days of administration, the uteri may be excised
and weighed approximately 24-hours after the last dose. These data
could then be used to assist the main study design (select an acceptable
maximum and lower doses and recommend the number of dose groups).
Administration of Doses. As noted in section (e), the preferred method
of test compound administration for the EDSP is by subcutaneous
injection. However, animal welfare considerations as well as toxicological
aspects such as 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), the physical/chemical properties of
the test material, existing toxicological information, data on metabolism
and kinetics {e.g., need to avoid first pass metabolism, better efficiency via
a particular route), and especially the intended use of the assay {e.g.,
EDSP Tier 1 battery) should also be taken into consideration when
choosing the route of administration.
It is recommended that, wherever possible, the use of an aqueous
solution/suspension be considered first. But as most estrogen 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). However, these oils have different caloric and fat content,
Page 10
-------
thus the vehicle might affect total metabolizable energy (ME) intake,
thereby potentially altering measured endpoints such as the uterine weight
especially in the immature method (Ref. 33). Thus, prior to the study, any
vehicle to be used should be tested against controls without vehicles.
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 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.
Dosage timing will depend of the model used (refer to subsection (h)(2) for
the immature model and to subsection (h)(3) for ovx-adult model).
Immature female rats are dosed with the test substance daily for three
consecutive days. A three-day treatment is also recommended for
ovariectomized female rats but longer exposures are acceptable and may
improve the detection of weakly active substances. With ovariectomized
female mice, an application duration of 3 days should be sufficient without
a significant advantage by an extension of up to seven days for strong
estrogen agonists, however, this relation was not demonstrated for weak
estrogens in the validation study (Ref. 16) thus dosage should be
extended up to 7 consecutive days in ovx-adult mice. The dose should be
given at similar times each day. They should be adjusted as necessary to
maintain a constant dose level in terms of animal body weight (e.g., mg of
test substance per kg of body weight per day). Regarding the test volume,
its variability, on a body weight basis, should be minimized by adjusting
the concentration of the dosing solution to ensure a constant volume on a
body weight basis at all dose levels and for any route of administration.
When the test substance is administered by gavage, this should be done
in a single daily dose to the animals using a stomach tube or a suitable
intubation cannula. 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.
When the test substance is administered by subcutaneous injection, this
should be done in a single daily dose. Doses should be administered to
the dorsoscapular 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 5
Page 11
-------
ml/kg body weight, divided into 2 injection sites, except in the case of
aqueous solutions where 10 ml/kg body weight may be used.
Observations.
(1) General and 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 are to be observed for mortality, morbidity
and general clinical signs such as changes in behaviour, skin, fur, eyes,
mucous membranes, occurrence of secretions and excretions and
autonomic activity (e.g., lacrimation, piloerection, pupil size, unusual
respiratory pattern).
(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 Uterus Weight. Twenty-four hours
after the last treatment, the rats will be humanely killed. Ideally, the
necropsy order will be randomized across groups to avoid progression
directly up or down dose groups that could subtly affect the data. The
assay objective is to measure both the wet and blotted uterus weights.
The wet weight includes the uterus and the luminal fluid contents. The
blotted weight is measured after the luminal contents of the uterus have
been expressed and removed.
Before dissection the vagina will be examined for opening status in
immature animals. The dissection procedure begins by opening the
abdominal wall starting at the pubic symphysis. Then, uterine horn and
ovaries, if present, are detached from the dorsal abdominal wall. The
urinary bladder and ureters are removed from the ventral and lateral side
of uterus and vagina. Fibrous adhesion between the rectum and the
vagina is detached until the junction of vaginal orifice and perineal skin
can be identified. The uterus and vagina are detached from the body by
incising the vaginal wall just above the junction between perineal skin as
shown in Figure 2. The uterus should be detached from the body wall by
gently cutting the uterine mesentery at the point of its attachment along
the full length of the dorsolateral aspect of each uterine horn. Once
removed from the body, uterine handling should be sufficiently rapid to
avoid desiccation of the tissues. Loss of weight due to desiccation
becomes more important with small tissues such as the uterus (Ref. 23).
If ovaries are present, the ovaries are removed at the oviduct avoiding
Page 12
-------
loss of luminal fluid from the uterine horn. If the animal has been
ovariectomized, the stubs should be examined for the presence of any
ovarian tissue. Excess fat and connective tissue should be trimmed away.
The vagina is removed from the uterus just below the cervix so that the
cervix remains with the uterine body as shown in Figure 2.
Each uterus should be transferred to a uniquely marked and weighed
container {e.g., a petri-dish or plastic weight boat) with continuing care to
avoid desiccation before weighing {e.g., filter paper slightly dampened with
saline may be placed in the container). The uterus with luminal fluid will
be weighed to the nearest 0.1 mg (wet uterine weight).
Each uterus will then be individually processed to remove the luminal fluid.
Both uterine horns will be pierced or cut longitudinally. The uterus will be
placed on lightly moistened filter paper {e.g., Whatman No. 3) and gently
pressed with a second piece of lightly moistened filter paper to completely
remove the luminal fluid. The uterus without the luminal contents will be
weighed to the nearest 0.1 mg (blotted uterine weight).
The uterus weight at termination can be used to assure that the
appropriate age in the immature intact rat was not exceeded, however, the
historical data of the rat strain used by the laboratory are decisive in this
respect (see paragraph 56 for interpretation of the results).
(4) Optional Investigations. After weighing, the uterus may be fixed in 10%
neutral buffered formalin to be examined histopathologically after
Haematoxylin & Eosin (HE)-staining. The vagina may be investigated
accordingly (see section (d)). In addition, morphometric measurement of
endometrial epithelium may be done for quantitative comparison.
Data and Reporting.
(1) Data. Study data should include:
~ The number of animals at the start of the assay;
~ The number and identity of animals found dead during the assay or
killed for humane reasons and the date and time of any death or
humane kill;
~ The number and identity of animals showing signs of toxicity, and a
description of the signs of toxicity observed, including time of onset,
duration, and severity of any toxic effects; and
~ The number and identity of animals showing any lesions and a
description of the type of lesions.
Individual animal data should be recorded for the body weights, the wet
uterine weight, and the blotted uterine weight. One-tailed statistical
analyses for agonists should be used to determine whether the
administration of a test substance resulted in a statistically significant (p <
Page 13
-------
0.05) increase in the uterine weight. Appropriate statistical analyses
should be carried out to test for treatment related changes in blotted and
wet uterine weight. For example, the data may be evaluated by an
analysis of covariance (ANCOVA) approach with body weight at necropsy
as the co-variable. A variance-stabilizing logarithmic transformation may
be carried out on the uterine data prior to the data analysis. Dunnett and
Hsu's test are appropriate for making pair wise comparisons of each
dosed group to vehicle controls and to calculate the confidence intervals.
Studentised residual plots can be used to detect possible outliers and to
assess homogeneity of variances. These procedures were applied in the
OECD validation program using the PROC GLM in the Statistical Analysis
System (SAS Institute, Cary, NC), version 8 (6)(7).
(2) Final Report. A final report shall include:
~ Testing Facility:
• Responsible personnel and their study responsibilities.
• Data from the Baseline Positive Control Test and periodic positive
control data (see section (g)).
~ Test Substance:
• Characterization of test substances.
• Physical nature and where relevant physicochemical properties.
• Method and frequency of preparation of dilutions.
• Any data generated on stability.
• Any analyses of dosing solutions.
~ Vehicle:
• Characterization of test vehicle (nature, supplier and lot).
• Justification of choice of vehicle (if other than water).
~ Test Animals:
• Species and strain and justification for their choice.
• Supplier and specific supplier facility.
• Age on supply with birth date.
• If immature animals, whether or not supplied with dam or foster
dam and date of weaning.
• Details of animal acclimatization procedure.
• Number of animals per cage.
• Detail and method of individual animal and group identification.
~ Assay Conditions:
• Details of randomization process {i.e., method used).
• Rationale for dose selection.
• Details of test substance formulation, its achieved concentrations,
stability and homogeneity.
• Details of test substance administration and rationale for the
choice of exposure route.
Page 14
-------
• Diet (name, type, supplier, content, and, if known, phytoestrogen
levels).
• Water source (e.g., tap water or filtered water) and supply (by
tubing from a large container, in bottles, etc.).
• Bedding (name, type, supplier, content).
• Record of caging conditions, lighting interval, room temperature
and humidity, room cleaning.
• Detailed description of necropsy and uterine weighing
procedures.
• Description of statistical procedures.
Results:
• For individual animals:
- All daily individual body weights (from allocation into groups
through necropsy) (to the nearest 0.1 g).
- Age of each animal (in days counting day of birth as day 0)
when administration of test compound begins.
- Date and time of each dose administration.
- Calculated volume and dosage administered and observations
of any dosage losses during or after administration.
- Daily record of status of animal, including relevant symptoms
and observations.
- Suspected cause of death (if found during study in moribund
state or dead).
- Date and time of humane killing with time interval to last
dosing.
- Wet uterine weight (to the nearest 0.1 mg) and any
observations of luminal fluid losses during dissection and
preparation for weighing.
- Blotted uterine weight (to the nearest 0.1 mg).
• For each group of animals:
- Mean daily body weights (to the nearest 0.1 g) and standard
deviations (from allocation into groups through necropsy).
- Mean wet uterine weights and mean blotted uterine weights
(to the nearest 0.1 mg) and standard deviations.
- If measured, daily food consumption (calculated as grams of
food consumed per animal).
- The results of statistical analyses comparing both the wet and
blotted uterine weights of treated groups relative to the same
measures in the vehicle control groups.
- The results of statistical analysis comparing the total body
weight and the body weight gain of treated groups relative to
the same measures in the vehicle control groups.
Page 15
-------
Table 1. Summary of the Important Guidance Facts of
the Test Guideline.
Rat
Mice
Animals
Strain
Commonly used laboratory rodent strain
Number of animals
A minimum of 6 animals per dose group
Number of groups
A minimum of 2 test groups (see paragraph 33 for guidance) and a negative
control group
For guidance on positive control groups see paragraphs 26 and 27
Housing and Feeding Conditions
T° in animal room
22°C ± 3°C
Relative humidity
50-60% and not below 30% or above 70%
Daily lighting sequence
12 hours light, 12 hours dark
Diet and drinking water
Ad libitum
Housing
Individually or in groups of up to three animals (social group housing is
recommended for immature animals)
Diet and bedding
Low level of phytoestrogens recommended in diet and bedding
Protocol
Method
Ovariectomized adult female method
(the US EDSP preferred one).
Immature non-ovariectomized method.
Ovariectomized adult female method
Age of dosing for
immature animals
PND 18 at the earliest. Dosing should
be completed prior to PND 25
Not relevant under the scope of the
current TG.
Age of ovariectomy
Between 6 and 8 weeks of age.
Age of dosing for
ovariectomized animals
A minimum of 14 days should elapse
between ovariectomy and the 1st day
of administration.
A minimum of 7 days should elapse
between ovariectomy and the 1s day
of administration.
Body weight
Body weight variation should be minimal and not exceed ± 20% of the mean
weight.
Dosing
Route of administration
Subcutaneous injection(preferred) or oral gavage.
Frequency of
administration
Single daily dose
Volume amount for
gavage and injection
< 5ml/kg body weight (or up to 10 ml/kg body weight in case of aqueous
solutions) (in 2 injection sites for subcutaneous route)
Duration of
administration
Minimum of 3 consecutive days for the
OVX model
3 consecutive days for immature
model
7 consecutive days for the OVX
model
Time of necropsy
Approximately 24 hours after the last dose
Results
Positive response
Statistically significant increase of the mean uterus weight (wet and or blotted)
Reference estrogen
17a-ethynyl estradiol
(k) Guidance for the Interpretation and Acceptance of the Results. In general, a
test for estrogenicity should be considered positive if there is a statistically
significant increase in uterine weight (p< 0.05) at least at the high dose level as
compared to the solvent control group. A positive result is further supported by
the demonstration of a biologically plausible relationship between the dose and
the magnitude of the response, bearing in mind that overlapping estrogenic and
Page 16
-------
antiestrogenic activities of the test chemical may affect the shape of the dose-
response curve.
Care must be taken in order not to exceed the maximum tolerated dose to allow
a meaningful interpretation of the data. Reduction of body weight, clinical signs,
and other findings should be thoroughly assessed in this respect.
An important consideration for the acceptance of the data from the Uterotrophic
Assay is the uterine weights of the vehicle control group. High control values
may compromise the responsiveness of the assay and the ability to detect very
weak estrogen agonists. Literature reviews and the data generated during the
validation of the Uterotrophic Assay suggest that instances of high control means
do occur spontaneously, particularly in immature animals (Refs. 2, 3, 6, & 9). As
the uterine weight of immature rats depends on many variables like strain or
body weight, no definitive upper limit for the uterine weight can be given. As a
guide, if blotted uterine weights in immature control rats are comprised between
40 and 45 mg, results should be considered as suspicious and uterine weights
above 45 mg may lead to rerun the test. However, this needs to be considered
on a case by case basis (Refs. 3, 6, & 8). When testing in adult rats incomplete
ovariectomy will leave ovarian tissue that can produce endogenous estrogen and
retard the regression of the uterine weight.
Blotted vehicle control uterine weights less than 0.09% of body weight for
immature female rats and less than 0.04% for ovariectomized young adult
females appear to yield acceptable results. If the control uterine weights are
greater than these numbers, various factors should be scrutinized including the
age of the animals, proper ovariectomy, dietary phytoestrogens, and so on, and a
negative assay result (no indication for estrogenic activity) should be used with
caution.
Historical data for vehicle control groups should be maintained in the laboratory.
Historical data for responses to positive reference estrogens, such as 17a-
ethynyl estradiol, should also be maintained in the laboratory. Laboratories may
also test the response to known weak estrogen agonists. All these data can be
compared to available data (Refs. 2, 3, 4, 5, 6, 7, & 8) to ensure that the
laboratory's methods yield sufficient sensitivity.
The blotted uterine weights showed less variability in the course of the OECD
validation study than the wet uterine weights (Refs. 6 & 7). However, a
significant response in either measure would indicate that the test substance is
positive for estrogenic activity.
The uterotrophic response is not entirely of estrogenic origin, however, a positive
result of the Uterotrophic Assay should generally be interpreted as evidence for
estrogenic potential in vivo, and should normally initiate actions for further
clarification (see paragraph 9 and the "OECD Conceptual Framework for the
Testing and Assessment of Endocrine Disrupting Chemicals", Annex 2).
Page 17
-------
Figure 1. Schematic Diagram Showing the Surgical Removal of the Ovaries.
Ovary
Oviduct
Cut here
Uterus
Incision
Mesometrium, vasculature
and fat pad not shown
The procedure begins by opening dorso-lateral
abdominal wall at the mid point between the costal
inferior border and the iliac crest, and a few
millimetres lateral to the lateral margin of the lumbar
muscle. Within the abdominal cavity, the ovaries
should be located. On an aseptic field, the ovaries
are then physically removed from the abdominal
cavity, a ligature placed between the ovary and
uterus to control bleeding, and the ovary detached
by incision above the ligature at the junction of the
oviduct and each uterine horn. After confirming that
no significant bleeding persists, the abdominal wall
should be closed by suture, and the skin closed,
e.g., by autoclips or suture. The animals should be
allowed to recover and the uterus weight to regress
for a minimum of 14 days before use.
Figure 2. The Removal and Preparation of the Uterine Tissues for Weight
Measurement.
The procedure begins by opening the abdominal wall
at the pubic symphysis. Then, each ovary, if present
and uterine horn is detached from the dorsal
abdominal wall. Urinary bladder and ureters are
removed from the ventral and lateral side of uterus
and vagina. Fibrous adhesion between the rectum
and the vagina are detached until the junction of
vaginal orifice and perineal skin can be identified.
The uterus and vagina are detached from the body
by incising the vaginal wall just above the junction
between perineal skin as shown in the figure. The
uterus should be detached from the body wall by
gently cutting the uterine mesentery at the point of its
attachment along the full length of the dorsolateral
aspect of each uterine horn. After removal from the
body, the excess fat and connective tissue is
trimmed away. If ovaries are present, the ovaries
are removed at the oviduct avoiding loss of luminal fluid from the uterine horn. If the
animal has been ovarectomized, the stubs should be examined for the presence of any
ovarian tissue. The vagina is removed from the uterus just below the cervix so that the
cervix remains with the uterine body as shown in the figure. The uterus can then be
weighed.
UTERINE
WEIGHT
Page 18
-------
(I) References.
1. OECD (1998). Report of the First Meeting of the OECD Endocrine Disrupter
Testing and Assessment (EDTA) Task Force, 10th-11th March 1998,
ENV/MC/CHEM/RA(98)5.
2. OECD (2003). Detailed Background Review of the Uterotrophic Assay: Summary
of the Available Literature in Support of the Project of the OECD Task Force on
Endocrine Disrupters Testing and Assessment (EDTA) to Standardise and
Validate the Uterotrophic Assay. OECD Environmental Health and Safety
Publication Series on Testing and Assessment No. 38. ENV/JM/MONO(2003)1.
3. Owens JW, Ashby J (2002). Critical Review and Evaluation of the Uterotrophic
Assay for the Identification of Possible Estrogen Agonists and Antagonists: In
Support of the Validation of the OECD Uterotrophic Protocols for the Laboratory
Rodent. Crit. Rev. Toxicol. 32:445-520.
4. OECD (2001). Final Report of the Phase 1 of the Validation Study of the
Uterotrophic Assay. [ENV/JM/TG/EDTA (2001 )1/REV1 ].
5. Kanno, J, Onyon L, Haseman J, Fenner-Crisp P, Ashby J, Owens W (2001). The
OECD program to validate the rat uterotrophic assay to screen compounds for in
vivo estrogenic responses: Phase 1. Environ Health Perspect. 109:785-94.
6. OECD (2003). OECD Draft Report of the Validation of the Rat Uterotrophic Assay.
Phase 2. Testing of Potent and Weak Oestrogen Agonists by Multiple
Laboratories. [ENV/JM/TG/EDTA (2003)1],
7. Kanno J, Onyon L, Peddada S, Ashby J, Jacob E, Owens W (2003). The OECD
program to validate the rat uterotrophic assay: Phase Two - Dose Response
Studies. Environ. Health Persp. 111:1530-1549
8.) Kanno J, Onyon L, Peddada S, Ashby J, Jacob E, Owens W (2003). The OECD
program to validate the rat uterotrophic assay: Phase Two - Coded Single Dose
Studies. Environ. Health Persp. 111:1550-1558.
9. Owens W, Ashby J, Odum J, Onyon L (2003). The OECD program to validate the
rat uterotrophic assay: Phase Two - Dietary phytoestrogen analyses. Environ.
Health Persp. 111:1559-1567.
10. Ogasawara Y, Okamoto S, Kitamura Y, Matsumoto K (1983). Proliferative pattern
of uterine cells from birth to adulthood in intact, neonatally castrated, and/or
adrenalectomized mice assayed by incorporation of [l125]iododeoxyuridine.
Endocrinology 113:582-587.
11. Branham WS, Sheehan DM, Zehr DR, Ridlon E, Nelson CJ (1985). The postnatal
ontogeny of rat uterine glands and age-related effects of 17(3-estradiol.
Endocrinology 117:2229-2237.
12. Schlumpf M, Berger L, Cotton B, Conscience-Egli M, Durrer S, Fleischmann I,
HallerV, Maerkel K, Lichtensteiger W(2001). Estrogen active UV screens. SOFW-
J. 127:10-15.
Page 19
-------
13. Zarrow MX, Lazo-Wasem EA, Shoger RL (1953). Estrogenic activity in a
commercial animal ration. Science 118:650-651.
14. Drane HM, Patterson DSP, Roberts BA , Saba N (1975). The chance discovery of
oestrogenic activity in laboratory rat cake. Fd. Cosmet. Toxicol. 13:425-427.
15. Boettger-Tong H, Murphy L, Chiappetta C, Kirkland JL, Goodwin B, Adlercreutz H,
Stancel GM, Makela S (1998). A case of a laboratory animal feed with high
estrogenic activity and its impact on in vivo responses to exogenously
administered estrogens. Environ. Health Perspec. 106:369-373.
16. OECD (2006). Validation of the Uterotrophic Assay in mice by bridging data to rats
17. Degen GH, Janning P, Diel P, Bolt HM (2002). Estrogenic isoflavones in rodent
diets. Toxicol. Lett. 128:145-157.
18. Wade MG, Lee A, McMahon A, Cooke G, Curran I (2003). The influence of dietary
isoflavone on the uterotrophic response in juvenile rats. Food Chem. Toxicol.
41:1517-1525.
19. Yamasaki K, Sawaki M, Noda S, Wada T, Hara T, Takatsuki M (2002). Immature
uterotrophic assay of estrogenic compounds in rats given different phytoestrogen
content diets and the ovarian changes in the immature rat uterotrophic of
estrogenic compounds with ICI 182,780 or antide. Arch. Toxicol. 76:613-620.
20. Thigpen JE, Haseman JK, Saunders HE, Setchell KDR, Grant MF, Forsythe D
(2003). Dietary phytoestrogens accelerate the time of vaginal opening in immature
CD-1 mice. Comp. Med. 53:477-485.
21. Ashby J, Tinwell H, Odum J, Kimber I, Brooks AN, Pate I, Boyle CC (2000). Diet
and the aetiology of temporal advances in human and rodent sexual development.
J. Appl. Toxicol. 20:343-347.
22. Thigpen JE, Lockear J, Haseman J, Saunders HE, Caviness G, Grant MF,
Forsythe DB (2002). Dietary factors affecting uterine weights of immature CD-1
mice used in uterotrophic assays. Cancer Detect. Prev. 26:381-393.
23. Thigpen JE, Li L-A, Richter CB, Lebetkin EH, Jameson CW (1987). The mouse
assay for the detection of estrogenic activity in rodent diets: I. A standardized
method for conducting the mouse assay. Lab. Anim. Sci. 37:596-601.
24. OECD (2001). Acute oral toxicity - up-and-down procedure. OECD Guideline for
the testing of chemicals, No. 425.
25. 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.
26. 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.
27. Bulbring, E., and Burn, J.H. (1935). The estimation of oestrin and of male hormone
in oily solution. J. Physiol. 85: 320 - 333.
Page 20
-------
28. Dorfman, R.I., Gallagher, T.F. and Koch, F.C (1936). The nature of the estrogenic
substance in human male urine and bull testis. Endocrinology 19: 33-41.
29. Reel, J.R., Lamb IV, J.C. and Neal, B.H. (1996). Survey and assessment of
mammalian estrogen biological assays for hazard characterization. Fundam. Appl.
Toxicol. 34: 288 - 305.
30. Jones, R.C. and Edgren, R.A. (1973). The effects of various steroid on the vaginal
histology in the rat. Fertil. Steril. 24: 284 - 291.
31. OECD (1982). Organization for Economic Co-operation and Development -
Principles of Good Laboratory Practice, ISBN 92-64-12367-9, Paris.
32. R.I. Dorfman (1962). Methods in Hormone Research, Vol. II, Part IV: Standard
Methods Adopted by Official Organization. New York, Academic Press.
33. J. E. Thigpen et al. (2004). Selecting the appropriate rodent diet for endocrine
disruptor research and testing studies. ILAR J 45(4): 401-416.
34. L.E. Gray and J. Ostby (1998). Effects of pesticides and toxic substances on
behavioral and morphological reproductive development: endocrine versus non-
endocrine mechanism. Toxicol Ind Health. 14 (1-2): 159-184.
35. Booth AN, Bickoff EM and Kohler GO (1960). Estrogen-like activity in vegetable
oils and mill by-products. Science 131:1807-1808.
36. Kato H, Iwata T, Katsu Y, Watanabe H, Ohta Y, Iguchi T (2004). Evaluation of
estrogenic activity in diets for experimental animals using in vitro assay. J. Agric
Food Chem. 52, 1410-1414.
37. OECD Test No. 440: Uterotrophic Bioassay in Rodents: A Short Term Test for
(Anti)Androgenic Properties.
Page 21
-------
Appendix 1
Definitions
Antiestroqenicitv is the capability of a chemical to suppress the action of estradiol 17(3.
in a mammalian organism.
Date of birth is postnatal day 0.
Dosage is a general term comprising of dose, its frequency and the duration of dosing.
Dose is the amount of test substance administered. For the Uterotrophic Assay, 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).
Maximum Tolerable Dose (MTD) is the highest amount of a substance that, when
introduced into the body does not kill test animals (denoted by DL0) (IUPAC, 1993).
Estroqenicitv is the capability of a chemical to act like estradiol 1713. in a mammalian
organism.
Postnatal day X is the Xth day of life after the day of birth.
Sensitivity is the proportion of all positive/active substances that are correctly classified
by the test. It is a measure of accuracy for a test method that produces categorical
results, and is an important consideration in assessing the relevance of a test method.
Specificity is the proportion of all negative/inactive substances that are correctly
classified by the test. It is a measure of accuracy for a test method that produces
categorical results and is an important consideration in assessing the relevance of a test
method.
Uterotrophic is a term used to describe a positive influence on the growth of uterine
tissues.
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.
VMG mamm is the Validation Management Group on Mammalian Testing and
Assessment.
A -1
------- |