¦— j» United States	Prevention, Pesticides	EPA 740-C-09-012
Environmental Protection	and Toxic Substances	October 2009
%#Crri A9ency		
Endocrine Disruptor
Screening Program
Test Guidelines
OPPTS 890.1500:
Pubertal Development
and Thyroid Function in
Intact Juvenile/
Peripubertal Male Rats

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 890.1500: Pubertal Development and Thyroid Function in Intact Juvenile/
Peripubertal Male Rats
(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
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.
(b)	Purpose. The purpose of the male pubertal assay is to provide information
obtained from an in vivo mammalian system that will be useful in assessing the
potential of a chemical substance or mixture to interact with the endocrine
system. This assay is capable of detecting chemicals with antithyroid,
androgenic, or antiandrogenic [androgen receptor (AR) or steroid-enzyme-
mediated] activity or agents which alter pubertal development via changes in
gonadotropins, prolactin, or hypothalamic function.
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(c)	Endpoints.
~	Growth (daily body weight)
~	Age and body weight at preputial separation
~	Organ weights
•	Seminal vesicle plus coagulating glands (with and without fluid)
•	Ventral prostate
•	Dorsolateral prostate
•	Levator ani/bulbocavernosus muscle complex
•	Epididymides (left and right separately)
•	Testes (left and right separately)
•	Thyroid (after fixation)
•	Liver
•	Kidneys (paired)
•	Pituitary
•	Adrenals (paired)
~	Histology
•	Epididymis (one)
•	Testis (one)
•	Thyroid (colloid area and follicular cell height)
•	Kidney
~	Hormones
•	Serum testosterone, total
•	Serum thyroxine (T4), total
•	Serum thyroid stimulating hormone (TSH)
~	Clinical (serum) chemistry
•	Standard blood panel, including creatinine and blood urea nitrogen
(d)	General Conditions. Conduct the study in facilities accredited by the
Association for Assessment and Accreditation of Laboratory Animal Care
International (AAALAC) if in the U.S., or the applicable national or international
accreditation authority if outside the U.S. Care should be taken to minimize
stress from all sources including noise, other species housed nearby, or other
Rats are housed in clear plastic cages (approximately 20 x 25 x 47 cm) with
heat-treated1 laboratory-grade wood shavings other than cedar as bedding.
Corn cob bedding should not be used due to its potential to disrupt endocrine
activity2. Wire-mesh-bottomed caging should not be used due to the potential for
pup loss. Do not use polycarbonate water supply equipment.
1	to eliminate resins that induce liver enzymes
2	Markaverich BM, Alejandro MA, Markaverich D, et al. 2002. Biochem Biophys Res Commun
291 (3):692-700.
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Prior to the onset of the study, pregnant female rats are housed individually. At
weaning, pups are housed in groups of either two or three males of the same
treatment group per cage.
Animals are maintained on a balanced laboratory diet3 and deionized water4 ad
libitum, in a room with a 14:10 hour light:dark photoperiod (on at 0500 hours, off
at 1900 hours local time). Temperature, humidity, and other conditions not
specified above should be in accordance with the recommendations contained in
Guide for the Care and Use of Laboratory Animals5 or other appropriate
Report the diet that the timed pregnant females were fed by the supplier prior to
shipment to the test laboratory.
(e) Animals: Juvenile Male Rats. The Sprague-Dawley or Wistar strains of rats are
the preferred strains for this assay until a more-appropriate strain (or set of
strains) is identified and associated performance criteria developed. Results
similar to those from Sprague-Dawley rats have been produced using Wistar and
Long-Evans rats in this assay or relevant modifications of this assay, suggesting
that strain is not the major determinant of sensitivity in this assay.
Juvenile male rats are derived from individually housed pregnant females that
were bred in-house or purchased from a supplier as "timed pregnant" dams.
Dams obtained and transported from an external supplier may not be used in the
same study as dams bred in-house. All dams must be pregnant for the first time
and timed to deliver on the same day. If purchased from a supplier, all dams
should be on the same gestation day (GD) but that GD may be GD 7, 8, 9, or 10
at the time of arrival at the performing laboratory (where GD 0 = day of sperm
positive). Dams are allowed to deliver their pups naturally. Any litters with fewer
than 8 total pups/litter {i.e., including both males and females) and any litters not
delivered by GD 23 are excluded from the study. To maximize uniformity in
growth rates, the litters are standardized to 8 to 10 pups per litter between post-
natal days (PND) 3 and 5. (PND 0 is defined as the day on which the pup is first
seen, assuming that the cages are checked for new births daily, in the morning.)
All litters within a study are standardized to the same number of pups (8, 9, or
10). Reducing litter size to 8-to-10 is required when dams have more than
enough pups, but cross-fostering to raise litter size to the required number is not
acceptable. Body weights are monitored weekly and any unthrifty litters or
runted pups excluded from the study. Enough litters should be available to
assure that a sufficient number of juvenile males are available for 15 male pups
3	N.B.: Genistein-equivalent content of genistein plus daidzein (aglycone forms) of each batch must be
less than or equal to 300 ug/g, and the same batch of feed must be used for treated and control groups at
all times. ("Genistein-equivalent content" of daidzein is approximately 0.8. Owens WB, Ashby J, Odum J,
Onyon L. 2003. Environ Health Perspect 111(12):1559-1567.)
4	Deionized water is required. Tap water is not acceptable.
5	Institute of Laboratory Animal Resources. Washington, DC: National Academy Press. 1996.
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per treatment group. (If parallel male and female pubertal studies are being
conducted, males and females from the same litter can be used in their
respective studies.) Also, enough litters should be available to avoid the need for
placing littermates in the same experimental group.
The pups are weaned on PND 21. Also on day 21, male pups are marked by
litter, and then all of the male pups from all of the litters are weighed individually
to the nearest 0.1 g and ranked by body weight. A population of male pups that
is as homogeneous as possible is selected for the study by eliminating an equal
number of pups from the heavy end and the light end of the distribution, leaving
the number of animals needed for the study in the middle. In this way, one
nuisance variable (viz., body weight at weaning) is experimentally controlled.
The male pups are assigned to treatment groups such that the mean body
weights and variances for all groups are similar. Avoid placing littermates in the
same group.
After assignment to treatment groups, male offspring belonging to the same
treatment group are housed in groups of either 2 or 3 per cage, such that each
cage has the same number of animals. (In the case of housing two per cage and
a planned N of 15, it will be necessary to add an additional rat to the last cage.)
As described in more detail later in this document, the preferred procedure is to
kill all the animals on a single day to close the in-life portion of the study, but if
the number of planned necropsies is considered too large to allow careful
measurement of endpoints on one day (e.g., when multiple chemicals are being
tested simultaneously) kills may be conducted over two days rather than one,
with half of each group killed on each day. Day of kill is assigned to individuals at
the time of distribution of the pups to groups; kills over two days must not be
adopted as a matter of convenience on the day of necropsy. If kills over two
days are planned, the body weights are distributed across kill groups such that
the mean body weights and variances for all groups are similar, and, when
possible, litter mates are not in the same group.
Experimental Design. This protocol uses a randomized complete block design
(time-separated necropsy is the blocking factor) with fifteen male rats in each
treatment group. The treatment conditions are (1) vehicle-treated; and (2)
xenobiotic-treated (two dose levels). The highest dose level should be at or just
below the Maximum Tolerated Dose (MTD) level but need not exceed the limit
dose of 1 g/kg/day. A dose level will generally be considered to be at or just
below the Maximum Tolerated Dose level if it causes a statistically significant
reduction in terminal body weight gain in treated animals vs. controls, the
reduction is no greater than approximately 10% of the mean terminal body weight
for the controls, and no clinical signs of toxicity associated with the dose level are
observed throughout the study. In addition, abnormal blood chemistry values at
termination (particularly creatinine and blood urea nitrogen (BUN)) may indicate
that MTD was exceeded, even in the absence of a reduction in terminal body
weight compared to controls. Histopathology of the kidney (or any other organ
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where gross observations indicate damage) may be used as evidence that MTD
was exceeded.
The second dose level should be one half of the highest dose level being tested
unless justification is provided for testing at a different level.
If necessary, the study can be conducted in time-separated blocks rather than at
one time. In this case, each block should contain all treatment groups and be
balanced with respect to numbers of animals and body weight at weaning.
Test substance. Chemical purity and stability in vehicle must be known prior to
testing so that dose levels are correctly prepared.
The test substance is dissolved or suspended in a suitable vehicle. In choosing
a vehicle, consideration should be given to the following characteristics: effects
on the absorption, distribution, metabolism, or retention of the test substance;
effects on the chemical properties of the test substance which may alter its toxic
characteristics; and effects on the food or water consumption or the nutritional
status of the animals. Use of vehicles with potential intrinsic toxicity should be
avoided (e.g., acetone, DMSO). If corn oil is used, it must be clear and free of
sediment. It should have a bland odor, free from rancid, musty, metallic, putrid or
any other undesirable odor.
Other solvents such as water or carboxymethylcellulose may be used where
appropriate. Gentle warming may be used to assist solubilization but the solution
must not be administered warm and the solution should be checked to make sure
that precipitation did not occur upon cooling. Use of intermediate solvents (e.g.,
ethanol) to assist in solubilization is not appropriate. If the test substance is not
soluble in any of the conventional solvents, it is administered as a suspension.
Sonication may be used to assist in suspending particles. It is important that the
dosing solution or suspension be well-mixed to keep the chemical well-distributed
prior to and throughout dosing, and care must be taken to ensure that the particle
size of insoluble substances does not interfere with delivery of the full dose
through the gavage tube or needle tip.
Treatment. Each animal is weighed daily, prior to treatment, and the body
weight recorded. Clinical observations are also recorded daily. Animals which
are found dead or which must be euthanized because they are near the point of
death are removed from the cage. Endpoint measures (organ weights, hormone
levels, histology, etc.) are not taken from these animals.
Treatments are administered daily by oral gavage from PND 23 through PND 53.
This duration of treatment is unnecessary to detect androgenic chemicals, but is
required for the detection of pubertal delay and antithyroid effects. Test
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chemicals are administered in 2.5 to 5.06 ml vehicle/kg body weight at 0700-0900
daily using an 18 gauge gavage needle (1 to 1% inch length, 2.25 mm ball) and a
1 cc (disposable) tuberculin syringe for each treatment. Needle gauge may be
optimized to animal size but must be constructed of metal to avoid the potential
for absorption by or leaching of substances from rubber or plastic tubing. The
treatments are administered on a mg/kg body weight basis using the current
day's weight, and volume of the dose administered is recorded each day.
In the absence of other clinical signs that would normally lead to removal of an
animal from the study, failure to gain weight at the same rate as controls is
generally not a reason to remove a treated animal during the course of the study.
However, it is recognized that severe failure to grow may be a reason to
disqualify an animal even in the absence of other signs of toxicity. As general
guidance, EPA suggests that a reduction in body weight gain when compared to
controls of more than 20% in the absence of other signs of toxicity may justify
(i) Preputial Separation. Beginning on PND 30, males are examined daily for
preputial separation (PPS). The appearance of partial and complete preputial
separation, or a persistent thread of tissue between the glans and prepuce are all
recorded on the days they are observed. The day of complete preputial
separation is the endpoint used in the analysis for the age at preputial
separation. However, if any animal within any treatment group shows incomplete
separation (including persistent threads) for greater than three days, a separate
analysis is conducted using the ages at which partial separation was first
observed. Documentation of a thread even if PPS otherwise appears complete
is important. It is also critical that "initiation" of PPS be recorded. It is preferred
but not critical that PPS observations be taken after the daily dosing. Whether
collected before or after dosing, the PPS observations must be collected at
approximately the same time each day.
(j) Necropsy. Males are killed on PND 53. If necessary, one half of the males may
be killed on PND 53 and the remaining males on PND 54 as long as the animals
in each treatment group are equally dispersed between the two necropsy days.
Animals killed on PND 54 are dosed and treated on the day of kill just like the
animals killed on PND 53 with regard to time of dosing, collection of PPS
information, etc. All animals are dosed between 0700 and 0900 hours local time,
and killed beginning 2 hours following dosing. It is critical that kills are completed
by 1300 hours due to normal diurnal fluctuation in thyroid hormone levels.
On the day before the kills are to be performed, the animals are moved to a
holding room separate from the room in which the kills and/or necropsies are
performed. On the day of kill, dosing is done in the holding room. During kills,
the holding room should be undisturbed except for the removal of the next
6 Dosage volume per kg body weight must be the same for all treated animals in the experiment but the
value chosen for the study may be anywhere in the specified range.
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individual to be killed. Only the animal which is to be killed next should be
transferred from the holding room to the room in which the kill is performed, and
the time for transfer and kill should be as brief as possible.
The preferred method of kill is by decapitation without any form of anesthesia, to
minimize the potential for release of testosterone during CO2 asphyxiation or
interference by other anesthetics. Decapitation is also considered more humane
than CO2 asphyxiation. If CO2 is used, it must be given for no more than 60
seconds prior to decapitation, even if the animal has not fully succumbed in that
time. Decapitation has generally not be found to interfere with the integrity of the
thyroid, which must be maintained in order to obtain thyroid weight and histology
The order of necropsy is randomized or otherwise evenly distributed across all
groups being necropsied that day. That is, do not necropsy all animals in one
group before moving to the next group. When two or more test chemicals use
the same control group, it is particularly important to intersperse the control
animal necropsies across the entire time span in which all of the necropsies for
all the test chemicals and dose levels are conducted.
Blood from the trunk of the animal is collected immediately (e.g., by inversion
over a funnel). Collect the blood in serum separation tubes (i.e., without EDTA or
heparin). The amount of blood needed is specified by the hormone kits'
manufacturers. After collection, the blood is centrifuged at 3000 X g for 30
minutes. The serum is pipetted into siliconized microcentrifuge tubes7 and
stored at -20 °C or colder for subsequent hormone and blood chemistry
At necropsy, the testes, epididymides, ventral prostate, dorsolateral prostate,
seminal vesicle with coagulating glands and fluid, levator ani plus
bulbocavernosus muscles, thyroid (with attached portion of trachea), liver,
kidneys, pituitary, and adrenals are removed and the weights of each except the
thyroid/trachea recorded in milligrams to one decimal place with the exception of
kidney and liver, which are recorded in grams, to two decimal places. (Kidneys
and adrenals are weighed as pairs. The left and right testes, and the left and right
epididymides, are weighed individually.) Care must be taken to remove
mesenteric fat with small surgical scissors from these tissues such that the fluid
in the sex accessory glands is retained. After weighing the seminal vesicle with
coagulating glands and fluid, the fluid is removed and the seminal vesicle weight
with coagulating glands but without fluid is also recorded. Small tissues such as
the adrenals and pituitary, as well as tissues that contain fluid, should be
weighed immediately to prevent tissues from drying out prior to weighing.
Measures to prevent drying out may be necessary if such organs cannot be
weighed immediately. For example, the organs may be placed in a weigh-boat
7 If there is a greater volume of blood than will fit in one microcentrifuge tube, prepare as many separate
aliquots as appropriate before freezing. Do not freeze in large aliquots, to avoid excessive freeze/thaw
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and a moist paper towel used to cover the weigh-boat, but the paper towel must
not come into contact with the organs at any time.
The thyroid (with attached portion of the trachea) and a single testis and
epididymis from each animal are prepared for histological examination. Either
the left or the right testis/epididymis may be chosen for a study but the choice
must be applied consistently to all animals in the study and the choice must be
reported. The testis and epididymis should both be from the same side but if that
is not possible, use the other of the pair and note the reason. (The testis and
epididymis not chosen for histology are discarded after weighing.) Testis and
epididymis are placed in Bouin's fixative overnight (no longer than 24 hours)8,
after which they are rinsed and stored in 70% ethanol until embedded in paraffin.
They are then stained with hematoxylin and eosin (H&E) for subsequent
histological evaluations. The thyroid, with attached trachea, is fixed in 10%
buffered formalin for at least 24 hours. Then the thyroid (with parathyroids) is
dissected from the trachea, blotted and weighed to the nearest 0.01 mg, placed
in 70% ethanol until embedded in paraffin, stained with H&E, and histologically
evaluated. Kidney, like thyroid, is fixed in 10% buffered formalin for at least 24
hours, then placed in 70% ethanol until embedded in paraffin, stained with H&E,
and histologically evaluated.
(k) Hormonal Assays. Hormonal measurements can be conducted using
radioimmunoassay (RIA), immunoradiometric assay (IRMA), enzyme-linked
immunosorbent assay (ELISA), or time-resolved immunofluorescent procedures.
Regardless of which is used, always include multiple quality control (QC)
samples run in duplicates that are dispersed among the test samples. Any
measurement kit that is used must be shown to yield appropriate values for
control rats at the laboratory performing the pubertal assay. This includes
demonstrating that QC was performed as described by the kit manufacturer and
that the performance falls within the range defined by the manufacturer. If the kit
does not provide or specify a standard control, then the lab should use its own
historical quality control samples. The lab's criteria for evaluating the kit's
performance must be included in the study report. If the laboratory has never
had experience with the kit for making measurements specifically in the rat, it
should test the kit in one or more untreated rats outside of the pubertal assay
before relying on it for the full study.
(I) Blood Chemistry. Any standard panel of blood chemistry tests that includes
creatinine and blood urea nitrogen (BUN) may be used as long as the
measurements are calibrated for rats and the normal ranges for controls are
reported. The normal ranges for controls may be from the literature (in which
case the reference should be given), or from historical controls.
8 Fixing the testis and epididymis in 10% buffered formalin for at least 24 hours may be used in place of
fixing in Bouin's solution for less than 24 hours, but Bouin's is preferred due to the relative ease of
handling that results.
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(m) Histology. Testis, epididymis, thyroid, and one kidney are evaluated for
pathologic abnormalities and potential treatment-related effects.
Thyroid sections are subjectively evaluated for follicular cell height and colloid
area using a five point grading scale (1 = shortest/smallest; 5 = tallest/largest)9
and any abnormalities/lesions noted. A minimum of two sections of each of the
two lobes of the thyroid are evaluated. Example photomicrographs are attached.
The examples illustrate the magnitude of differences that are typically evaluated
as separate scores, but the reader will need to establish the range appropriate
for the particular study being evaluated.
Guidance for histological evaluation of the testis and epididymis is given in EPA's
Health Effects Test Guideline OPPTS 870.3800: Reproduction and Fertility
Effects10: "Besides gross lesions such as atrophy or tumors, testicular
histopathological examination should be conducted in order to identify treatment-
related effects such as retained spermatids, missing germ cell layers or types,
multinucleated giant cells, or sloughing of spermatogenic cells into the lumen ....
Examination of the intact epididymis should include the caput, corpus, and
cauda, which can be accomplished by evaluation of a longitudinal section, and
should be conducted in order to identify such lesions as sperm granulomas,
leukocytic infiltration (inflammation), aberrant cell types within the lumen, or the
absence of clear cells in the cauda epididymal epithelium."
(n) Statistical Analysis. Consideration should be given to whether there are any
data points that should be excluded from the data set, and whether any data
points that are identified as outliers by an appropriate statistical test should
actually not be excluded, based on toxicological judgment. Values due to
obvious technical errors are excluded. Justification for exclusion of each data
point must be given. Outliers must be specified in the raw data. Do not test
incidence data, e.g., from histopathology evaluation, for outliers.
All data except histology evaluation {i.e., initial body weight [PND 23], body
weight gain11, age and body weight at preputial separation, body and organ
weights at necropsy, and serum hormones) are analyzed by Analysis of Variance
(ANOVA). If the study was conducted in blocks, then the analysis is a two-way
ANOVA with Block and Treatment as main effects. Age and body weight at PPS,
and all organ weights are also to be analyzed by Analysis of Covariance
(ANCOVA) using the body weight at PND 21 as the covariate12. When
9	see Capen CC, Martin SL. 1989. The effects of xenobiotics on the structure and function of thyroid
follicular and C-cells. Toxicol Pathol 17(2):266-93.
10	USEPA, Office of Prevention, Pesticides, and Toxic Substances. August 1998. Health Effects Test
Guidelines 870.3800: Reproduction and fertility effects. EPA 712-C-98-208.
11	Use the body weights on the last day all the animals were weighed. Specifically, if kills were performed
over two days, do not use the day when only the last half of the animals were available.
12	The covariate is body weight on the day of weaning rather than on the day of kill because ANCOVA
assumes that the covariate that is being adjusted for is not affected by the treatment, whereas in this
assay endocrine-active substances may affect the overall body weight gain and thus body weight at kill.
Using body weight at kill as covariate could mask a potentially endocrine effect on an organ. The Agency
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statistically significant effects are observed (p < 0.05), treatment means are
examined further using appropriate pairwise comparison tests to compare the
control with each dose group13. Where there is heterogeneity of variance, data
are to be transformed appropriately prior to ANOVA/ANCOVA, or analyzed using
an appropriate nonparametric test. Non-parametric analysis should be the
method of last resort since it does not allow analysis of covariation. In addition to
ANOVA and ANCOVA, examine the unadjusted and adjusted values for linear
trend with dose level.
In cases where preputial separation has not occurred prior to necropsy, use the
last day of observation +1 as the age at PPS when determining the mean for
each group. For example, if the animal was killed on PND 53 without PPS, use
PND 54 as the value for that animal when determining the mean for the
treatment group.
When possible, appropriate statistical analysis should be applied to the histology
(o) Data Summary. The Agency requests that all raw data and data summaries be
provided in electronic format (spreadsheet or comma-separated values), along
with all formatting information that is necessary to read the data. The Agency
intends to provide a suggested template with the posting of this guideline on the
Agency's Web site.14 Provide the following figure and tables for each test
chemical along with the respective control. Be sure to use the units shown in
the example tables. Provide values to one decimal place for the organs
reported in milligrams, and to two decimal places for those organs that are
required to be reported in grams.
Prepare an executive summary describing the number and strain of rats used in
the study, the dose levels and chemicals tested, and the effects, with levels of
statistical significance for all endpoints except histology. Include a summary of
the histological findings.
Electronic and hard copies of spreadsheets containing the raw data from all
animals for each endpoint are to be submitted to the EPA. Also provide the full
report of the histological findings with photomicrographs of significant
understands that using body weight at kill as covariate might identify which organs are more sensitive (or
less sensitive) than body weight gain to potentially endocrine effects, but has chosen to maximize the
potential of identifying organ-specific effects rather than relying on bodyweight as an indicator of potential
endocrine activity.
13	Comparison of the means of dose groups to each other is not required.
14	Available on-line at: http://www.epa.gov/oppts (select "Test Methods & Guidelines" on the left side
navigation menu). You may also access the guidelines in htto://www.reaulations.gov under Docket ID #:
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Figure 1. Graph the mean body weight +/- Standard Deviation (SD) for
each day during dosing for each treatment group, including vehicle
control. (If animals were necropsied over 2 days, do not include the body
weight from the last day of necropsy since only half of the animals are
available.) Place an arrow at the mean age of the controls at PPS. All
three groups (control, dose level 1, and dose level 2) are plotted on the
same graph but are distinguished from each other by point and line styles.
Table 1. Preputial Separation; General Growth. Report the mean,
standard deviation, coefficient of variation, number of animals (N), and p-
value for the following endpoints, for each test-chemical dose group and
control, both unadjusted (U) and adjusted (A) for body weight on PND 21:
~	Age at PPS.
~	Body weight at the age of PPS.
~	Initial body weight (PND 23).
~	Body weight on the last day all the animals were weighed {i.e., if kills
were performed over two days, do not use the day when only the last
half of the animals were available).
~	Final body weight as percent of control (leave control column blank).
~	Body weight gain from first dose to the (first) day of necropsy.
Mark endpoints that show an effect (by ANOVA/ANCOVA or a non-
parametric test, as appropriate) with an asterisk in the "Effect" column.
List the transformation (if any) used to eliminate heterogeneity of variance,
or the non-parametric test used, in the "Transform or nonparam" column.
Name the pairwise test used to compare the means of dosed groups to
the mean of controls in the "Pairwise test" column.
Mark means that are significantly different from control means (p < 0.05)
by shading the cell (rather than by the traditional asterisk).
Show the proportion of animals in which PPS had not occurred by the time
of necropsy {e.g., X/15), and explain that age-at-necropsy-plus-one {e.g.,
54) was used for those animals when calculating the mean.
Page 11

Table 1. Preputial Separation; General Growth.
Chemical Name
Transform or
Pairwise test
Vehicle Control
(Dose Level 1)
(Dose Level 2)
Age at PPS


Body weight at PPS


Initial body weight
(PND 23, g)


Final body weight


Final body weight
(% of control)



Body weight gain
(final minus initial
body weight) (g)


Proportion unseparated (#/N)

*Means different from controls at p<0.05 are marked by a shaded cell.
Page 12

(3) Table 2. Organ Weights at Necropsy. Report the mean, standard deviation, coefficient of variation,
number of animals (N), and p-value for liver, kidneys, pituitary, adrenals, seminal vesicle plus coagulating
glands (with and without fluid), ventral prostate, dorsolateral prostate, levator ani/bulbocavernosus muscle
complex, epididymides (left and right separately), testes (left and right separately), and thyroid weights, for
each treatment group, both unadjusted (U) and adjusted (A) for body weight on PND 21. Also report the
mean, standard deviation, and p-value of the organ-weight-to-body-weight ratio for liver, kidney, adrenals,
and pituitary only. For all other organ weights, do not use relative organ to body weight ratios, and do not
adjust for body weight at necropsy.
Table 2. Organ Weights at
Chemical Name
Vehicle Control
(Dose Level 1)
(Dose Level 2)
Organ weights
Transform or
Pairwise test












Page 13

vesicle +
gland, with
fluid (mg)


vesicle +
gland, without
fluid (mg)


prostate (mg)


prostate (mg)




(left, mg)


(right, mg)


(left, mg)


(right, mg)




*Means different from controls at p<0.05 are marked by a shaded cell.
U=Unadjusted, A=Adjusted, R=Organ-to-body-weight ratio
Page 14

(4) Table 3. T4, TSH, and Testosterone Levels. Report the mean, standard deviation, coefficient of variation,
number of animals, and p-value for the T4, TSH, and testosterone levels, for each treatment group, including
vehicle control.
Table 3. T4, TSH, and Tes
osterone Levels.
Chemical Name
Vehicle Control
(Dose Level 1)
(Dose Level 2)

Transform or
Pairwise test
Serum T4, total

Serum TSH,

total (ng/ml)

"Means different from controls at p<0.05 are marked by a shaded cell.
Page 15

(5) Table 4. Blood Chemistry. Similarly to Table 3, report the mean, standard deviation, coefficient of
variation, number of animals, and p-value for each of the parameters measured. Also report the normal
range for each parameter, and indicate whether these normal values are from the literature (provide
reference) or from historical controls.
Table 4. Blood Chemistry.
Chemical Name
Vehicle Control
(Dose Level 1)
(Dose Level 2)

Transform or
Pairwise test
Normal range+



*Means different from controls at p<0.05 are marked by a shaded cell.
Page 16

(p) Performance Criteria. The following performance criteria have been
established for the vehicle-control animals. See the Data Interpretation
Procedure for use of the performance criteria. Units for the endpoints are shown
in the table. Coefficients of variation (CVs) are in percent. The "mean", "2 SDs",
"CV", and "1.5 CV" columns describe the mean, two standard deviations,
coefficient of variation, and 1.5 times the coefficient of variation for that endpoint
in historical controls. Mean values and CVs for the vehicle control group must
fall in the acceptable range of each to be considered fully acceptable.
Table 5. Performance Criteria for Controls (Sprague-Dawley and Wistar Strains).
2 SDs
Acceptable Range
1.5 CV
Top of
Ventral prostate (grams)

0.151 to 0.295
0.160 to 0.332
LABC (grams)


0.447 to 0.855
Epididymis (grams)

0.350 to 0.598
0.364 to 0.528
Seminal vesicle (grams)

0.342 to 0.810
0.295 to 0.719
Testis (grams)

1.091 to 1.591

T4 (ijg/dl)

3.314 to 7.642
4.056 to 7.376
Thyroid weight (milligrams)


14 to 26
TSH (ng/ml)10


4.212 to 24.112
Age at PPS (postnatal day, where day of birth = PND 0)

40.176 to 46.072
39.781 to 46.513
Weight at PPS (grams)

177.292 to 240.992
188.277 to 256.169
Testosterone (ng/ml)

0 to 4.658
0.260 to 3.960
Final body weight (grams)

250.24 to 333.396
259.235 to 332.059
Adrenals (milligrams)

40.829 to 68.365
15 TSH values were derived using the kit from Amersham.
Page 17

2 SDs
Acceptable Range
1.5 CV
Top of

31.842 to 61.114

Kidneys (grams)

1.966 to 3.066
2.242 to 3.050
Liver (grams)

11.196 to 16.944
9.990 to 15.350
Pituitary (milligrams)

6.117 to 9.985
7.810 to 12.898
Weaning weight (grams)

47.180 to 69.296
45.472 to 59.812
aBottom of the acceptable range for coefficient of variation is zero.
(q) Data Interpretation Procedure. The male pubertal assay is intended to be one
of a suite of in vitro and in vivo assays for determining the potential of a
substance to interact with the endocrine system (Tier 1 assays). Therefore, it is
important to emphasize that the data interpretation of a specific chemical will be
a combination of the results from a number of these Tier-1 screening assays
taken as a whole and not merely the sum of results of assays interpreted in
isolation. That said, there are certain guidelines that can be given for interpreting
data from a male pubertal assay.
First, the dose levels tested should be examined to see if a Maximum Tolerated
Dose was used. (The highest dose level need not exceed a limit dose of 1
g/kg/day, even if MTD has not been reached.) Body weight loss (compared to
controls at termination) that does not exceed approximately 10% is an indication
that MTD was approached but not exceeded. Adverse clinical observations or
histopathology of the kidney and/or other organs, and/or significant deviations of
blood chemistry values of treated animals vs. controls may be indications that
MTD was exceeded.
Negative results for interaction with the endocrine system in the pubertal assay
will generally require demonstration that the highest dose level tested was at or
near the MTD. Positive results in the assay generally require no such proof, but
will generally require demonstration that interference due to decrease in body
weight gain compared to controls perse was not a factor in generating the
results. Studies that suggest interaction with the endocrine system only at a
dose level causing more than approximately 6% decrease in body weight gain at
termination compared to controls may require additional studies and/or a weight-
of-evidence approach using other information in order to be interpretable.
The endpoint values for the control group should be compared to the
performance criteria. Comparison should be made on the basis of the measured
values, not adjusted values. Any endpoints which do not meet the performance
Page 18

criteria in controls will generally be given little weight for the test chemicals if they
are negative but may provide useful information if they are positive.
Information that is missing due to inability to meet a performance criterion is not
the same as a negative result. The more endpoints that are missing, the less
likely the study will be regarded as adequate. No firm rules can be given for the
minimum number of endpoints that must be available for evaluation since some
of the endpoints are somewhat redundant {e.g., androgen-dependent tissue
weights) while others are not (thyroid-related endpoints). In general, however,
missing one or two performance criteria will not be regarded as fatal to the study.
More emphasis will be placed on meeting performance criteria for the coefficients
of variation than for the endpoint control means. Laboratories may submit
historical data for their own colonies to substantiate claims that tissue weights or
other endpoints in the study being evaluated are in line with historical values of
controls in that laboratory.
Once the data set has been compared to the performance criteria, it is evaluated
to see if there is evidence of interaction of the test chemical with the endocrine
Due to the covariance of certain organ weights with body weight, care should be
taken in interpreting pituitary, liver, and kidney weight changes. Only if a change
in the organ weight relative to body weight is significant for these particular
organs (i.e., not all the organs) should the weights adjusted for covariance with
body weight at weaning for these particular organs be interpreted as relevant.
Do not evaluate endpoints other than pituitary, liver, and kidney weights on their
values relative to terminal body weight, nor use analysis of covariance with
terminal body weight be used for interpretation of any but pituitary, liver, and
kidney weights. Since endocrine-active agents themselves may have an effect
on body weight, it is most appropriate to adjust for covariance with body weight at
weaning, before chemical treatment began.
The judgment of the histopathologist as to whether an effect on testis,
epididymis, and/or thyroid is associated with exposure to the test chemical is
important to consider when evaluating the organ weights and hormone levels
measured in this assay. Severity and incidence of effect(s), and dose-response
relationship may also be important information to consider.
The male pubertal data provide general profiles of changes in the assay
endpoints for various modes of action such as androgen agonism, androgen
antagonism, alteration of steroidogenesis, thyroid toxicity, and interference with
HPG function. These profiles can be used to establish a "weight of evidence" for
general mechanisms of interaction of a test chemical with the endocrine system.
For example, an antiandrogen such as vinclozolin delays puberty, impairs
reproductive tract development (e.g., decreased VP, SV, LABC, epididymis
Page 19

weight) and increases testosterone at higher doses, so a test chemical with
similar responses would likely be suspected of having an antiandrogenic
interaction. A similar profile would be expected if the compound inhibits
testosterone synthesis. One way to discern a compound that inhibits
steroidogenesis from one that is antiandrogenic is to evaluate serum
testosterone (a required endpoint) as this endpoint will obviously be decreased.
The pubertal male assay includes redundant androgen-dependent endpoints and
in general, all would be expected to respond similarly. However, it is possible
that a chemical may not alter all the endpoints measured, or the effect may not
be dose dependent, or it may occur only at the high dose. In these kinds of
cases, data from the other proposed assays in the Tier 1 battery would provide
added insight into a potential effect.
Another example of how a chemical would produce a particular profile would be
the way in which the thyroid homeostasis is disrupted. For example, the
polybrominated diphenyl ether mixture, DE-71, disrupts thyroid hormones by
inducing the clearance of thyroxin by hepatic enzyme induction. The ensuing
profile of effects includes a decrease in T4, decrease in T3 (although not required
in the male pubertal) and a subsequent increase in TSH. In many cases, thyroid
weight and thyroid histology appear less sensitive than the changes in the
hormone concentration because the hormonal changes can occur more rapidly
than any frank change in histology.
Other chemicals may target the HPG axis and there are certain profiles that may
indicate altered brain-pituitary function. For example, a chemical may delay
puberty, lead reduced androgen dependent tissues, yet be negative in an
androgen receptor binding assay, negative for alterations in steroidogenesis and
the Hershberger.
It should be noted that consistency with a known "profile" is not a requirement for
determining that a test chemical interacts with the endocrine system, nor is
consistency among supposedly redundant endpoints.
In addition to the redundancy of the endpoints in the protocol to detect
reproductive and thyroid effects, the availability of a minimum of two dose levels
provides an opportunity to examine the relationship between dose and response.
If an isolated endpoint is positive at the lower dose and no effect is seen at the
higher dose, then the effect and the overall conclusions may need to be
questioned. However, since the assay provides information from only two dose
levels, the dose-response information from the male pubertal assay is sparse
and informs the weight of evidence for interaction with the endocrine system but
generally does not control it.
Page 20

Table 6. Potential Changes Indicative of Different Modes of Action that May Be
Observed in the Male Pubertal Protocol.
Steroidogenesis inhibitor
or HPG suppression
|Age of Puberty
t Age of Puberty
i t4
jVentral prostate, seminal vesicles,
LABC, epididymis
jVentral prostate, seminal vesicles,
LABC, epididymis
jTestosterone or no effect
fThyroid wt.

^Follicular cell height
iColloid area

fLiverwt (for
compounds which
induce hepatic
clearance of thyroxine)
or no effect
(r) Explanation of Thyroid Slides. The slides are coded as follows:
~	Positions 1 and 2: follicular cell height score, 1 to 5 with 5 as greatest height
~	Positions 3 and 4: colloid area score, 1 to 5 with 5 as greatest area
"F5C1" represents a follicular score of 5 and colloid score of 1.
F1 C5 is considered "normal".
These slides were taken at 20X and excluded the edge of the tissue.
The slides have been chosen to illustrate both follicular cell height and colloid area
changes in the minimum number of slides. While all of these examples show scores for
follicular cell height and colloid area that happen to add up to "6", this is not expected to
be the case for most slides.
Page 21

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