EPA-650/1-73-001
September 1974
Environmental Health Effects Research Series
!'X'!'-S8W
..::!S:;:::?:!^S!iiJ:i^i*^:i
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Research reports of the Office of Research and Development, Environmental Protection
Agency, have been grouped into five series. These five broad categories were estab-
lished to facilitate further development and application of environmental technology.
Elimination of traditional grouping was consciously planned to foster technology trans-
fer and a maximum interface in related fields. The five series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
This report has been assigned to the ENVIRONMENTAL HEALTH EFFECTS RESEARCH
series. This series describes projects and studies relating to the tolerances of man for
unhealthful substances or conditions. This work is generally assessed from a medical
viewpoint, including physiological or psychological studies. In addition to toxicology
and other medical specialities, study areas include biomedical instrumentation and
health research techniques utilizing animals - but always with intended application to
human health measures.
Copies of this report are available free of-charge to Federal employees, current con-
tractors and grantees, and nonprofit organizations - as supplies permit - from the Air
Pollution Technical Information Center, Environmental Protection Agency, Research
Triangle Park, North Carolina 27711; or, for a fee, from the National Technical Infor-
mation Service, Springfield, Virginia 22151.
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EPA-650/1-73-001
TRAINING MANUAL
FOR
TERATOLOGY
by
K.D. Courtney and N. Chernoff
Pesticides and Toxic Substances Effects Laboratory
National Environmental Research Center
Research Triangle Park, North Carolina 27711
Prepared at the request of
National Center for Toxicological Research
Jefferson, Arkansas
OFFICE OF RESEARCH AND DEVELOPMENT
NATIONAL ENVIRONMENTAL RESEARCH CENTER
U.S. ENVIRONMENTAL PROTECTION AGENCY
RESEARCH TRIANGLE PARK, N.C. 27711
September 1974
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This report has been reviewed by the Office of Research and Development, EPA, and
approved for publication. Approval does not signify that the contents necessarily
reflect the views and policies of the Agency, nor does mention of trade names or
commercial products constitute endorsement or recommendation for use.
Publication No. EPA-650/1-73-001
11
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CONTENTS
Page
ABSTRACT v
INTRODUCTION 1
BASIC TECHNIQUES 2
Experimental Animals 2
Strains of Mice 2
Handling of Mice 2
Techniques of Compound Administration 2
Safety Procedures. 3
Caging 3
Coding by Ear Punch 3
Condition of Colony 3
Compound Preparation 4
Solutions 4
Suspensions 5
Color Coding of Solutions and Mice 5
Dosage Calculations 5
Calculations for Making a Solution . 6
Practice Problems 7
Fetal Fixation and Staining 7
Fetal Fixation. 7
Alizarin Staining 7
EXPERIMENT DESIGN 9
Maternal Data 9
Experiment Schedule 9
Protocol Sheet A - Before Sacrifice. 10
Protocol Sheet A - Day of Sacrifice 12
Fetal Data 13
Examination of Fetuses by Necropsy 13
Examination of Alizarin-Stained Fetuses 13
Protocol Sheet B 14
Glossary of Malformations . 14
SAMPLE EXPERIMENT: VITAMIN A 19
Experiment Design 19
111
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ABSTRACT
This report is an introduction to the basic techniques involved in tera-
tology testing. General topics covered include the handling of animals, compound
preparation and administration, dosage calculations, and coding techniques. Tera-
tology techniques include fetal fixation, alizarin staining, and the examination
of fixed and stained fetuses for soft tissue and skeletal anomalies. Suitable
protocol sheets covering data obtained before sacrifice, after sacrifice, and from
subsequent fetal examinations are included. A glossary of the most common malfor-
mations and their description is present. The outlines of a teratology experiment
with Vitamin A is included as a suggested training exercise. This experiment and
the techniques described in the manual may be used to train laboratory personnel
in the techniques and designs of teratology"experiments.
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TRAINING MANUAL
FOR
TERATOLOGY
INTRODUCTION
Teratology is the study of birth defects. It is derived from the Greek word
"teratos," which means monster, and indeed in ancient times children with birth
defects were considered monsters and evil omens. It is now known that birth defects
can occur any time there is interference with, or malfunctioning of, the normal
processes of development. The causes of birth defects are as varied as the types of
defects themselves. Genetic factors, maternal disease, too much or too little of
vital nutrients, radiation, oxygen deprivation, or the presence of foreign chemicals
may all contribute to the formation of birth defects. The defects can be external
such as shortened limbs or cleft lip, or internal such as heart or kidney malfor-
mations. Cellular defects may also occur and can make their presence known by mal-
functioning organs or mental retardation.
From day to day, human beings come in contact with large numbers of diverse chem-
icals, including food additives, drugs, and other natural and synthetic compounds pre-
sent in the environment. There is great need for the proper testing of these sub-
stances to determine their ability to cause birth defects since seemingly innocuous
drugs (e.g., thalidomide) and environmental contaminants (e.g., methylmercury) are
known to have caused serious malformations.
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BASIC TECHNIQUES
EXPERIMENTAL ANIMALS
Strains of Mice
Mice are classified according to strains. An inbred strain is homogeneous for a
number of specific genes while a random-bred strain js genetically heterogeneous. The
studies described here utilize the random-bred, all purpose, white laboratory mouse.
Handling of Mice
The description below is for right-handed people; left-handed people should re-
verse the procedure.
To handle mice properly, the worker should always he firm but gentle and never
frighten the animals. A mouse may be picked up by the tail but should not be sus-
pended in this position for too long. For transportation across short distances
(e.g., from cage to scale) it is best to place the animal on your left forearm while
maintaining your hold on the tail with your right hand.
For administration of a compound to the mouse by any route, a firm grasp is need-
ed to prevent the animal from either escaping or nipping. This is achieved by hold-
ing the mouse by the tail and placing it on a cage top or other rough surface that it
can grab. One then attempts to have the mouse extend its body by cither allowing it
to pull away from one while holding the tail or by gently pulling the mouse backwards
after it has grabbed the cage top. Once the mouse has extended its body, all the skin
at the nape of the neck should be firmly grasped with the thumb and index finger of
the left hand so that the animal's forelimbs are immobilized. Care must be taken not
to choke the mouse. The tail should then be placed between the ring finger and little
finger of your left hand. The right hand is now free to handle a syringe.
Techniques of Compound Administration
Compounds are administered by oral intubation or by subcutaneous injections. l:or
oral intubation, a feeding needle is used on the syringe. An 18-gauge needle with a
blunt end or a needle with an 18-guuge ball tip works well. These needles should he
approximately 2 inches long. The intubation needle is put into the most posterior
portion of the oral cavity, causing the mouse to swallow. When the mouse swallows,
the needle is guided down the esophagus and into the stomach. The proper volume of
vehicle, the substance in which the compound is dissolved or suspended to facilitate
administration, is expelled from the syringe, and the needle gently withdrawn.
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When the compound is to be injected subcutaneously, a hypodermic needle is used
on the syringe. A needle of 25 gauge and 1 inch length works well. The mouse is
grasped and held in the same manner described above. The needle is inserted into
the skin at the nape of the neck, which is held between the thumb and index finger.
The needle should always enter the skin at an angle of 45 degrees or less. It should
be inserted at least 0.5 inch to minimize leaking upon withdrawal. The needle should
be withdrawn at precisely the same angle to prevent tearing of the skin.
Safety Procedures
A strong point must be made concerning safety procedures. It must be remembered
that many compounds are being studied for their teratogenic potential because so little
is known about them. Thus, at all times, rubber gloves are worn when one is working
with test compounds or animals. Volatile compounds or solvents are to be used in the
hood. It may even be necessary to administer a volatile compound in a hood. All
pipetting is done by mechanical devices, not by mouth. All intubation needles are
to be rinsed with acetone after they are washed. This step is important since most
of the compounds are not soluble in water. 'Fhe washing procedure will mechanically
remove any suspensions, and a rinse with acetone will ensure that any residual will
be washed away and the needle will be dry. Because hypodermic needles will be dis-
posable, they will not require this attention.
Caging
Pregnant mice are caged at a density of no more than six and no less than two
mice per cage, with each cage numbered sequentially. The mice within the cage are
identified individually by coding 1 through 6, applied with an ear punch. The mouse
number is the cage number followed by the ear punch code number; e.g., the mouse in
cage 12 with the ear code 5 is mouse number 12-5.
Coding by Ear Punch
Individual mice within a single cage are identified by ear coding. To ear code,
one should first hold the mouse firmly in the left hand as described for compound
administration. With the ear punch in the right hand., a swift, sure movement will
produce a small hole in the ear. The placement of the hole will determine the mouse
ear code number, as depicted in the scheme in Figure 1.
Condition of Colony
It must be remembered at all times that your experiment is only as good as the
animals you use. Clean cages are necessary to minimize outbreaks of diseases or ill-
ness that can spread throughout the whole colony. Also, food and water should be
available for the mice at all times. If an animal has been without food, water, or
both, it should be immediately noted in the records. This notation is extremely im-
portant since it has been shown that mice deprived of food produce malformed Fetuses.
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MOUSE NUMBER 1
MOUSE NUMBER 2
MOUSE NUMBER 3
MOUSE NUMBER 4 MOUSE NUMBER 5 MOUSE NUMBER 6
Figure 1. Scheme for determining mouse ear code number.
COMPOUND PREPARATION
Upon arrival in the laboratory, all compounds are logged by sequential number in
a book indicating the date of arrival, source, batch number, purity, storage, and
any other pertinent information. This log number is marked on the container of the
compound.
Solutions
Either solutions or suspensions may be used for oral administration; only solu-
tions are used for subcutaneous administration. When a solution is used, all of the
compound must be dissolved, and the solution well mixed. If the compound is stable
in the solution, one solution may be made for the entire experiment. If there is any
chance of decomposition of the compound in the solution, then a new solution must be
made daily. A solution should always be stored in the dark to prevent photoclecompo-
sition. Storage in the cold is also recommended. If a solution is stored in the
refrigerator or freezer, it should be allowed to warm up to room temperature before
being administered to an animal. Extreme caution should be used if heat is applied
to these solutions since they could decompose.
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When a solution is to be injected, the pH should be within the biological range
of pH 6.0 to 8.0 in order to prevent discomfort and'stress to the mouse, as well as
lesions and fibrotic areas. Solvents that may be injected are water, saline, buffers,
and dimethylsulfoxide (DMSO). Corn oil, organic solvents, and suspending media are
not compatible with tissue fluids and, therefore, should be intubated into the stomach.
Suspensions
In order to make a suspension that will stay suspended long enough to enable the
syringe to be filled and the mice to be treated, one probably needs to grind the com-
pound to a small particulate size. The smaller the size of the particle, the longer
it stays in suspension. An ordinary mortar and pestle is adequate for most compounds.
Occasionally a motor-driven pestle is necessary. The suspending agent may be water
or saline, but usually a more viscous substance, such as corn oil, honey, sugar
solutions, or carboxymethylcellulose, is used.
Suspensions must be made daily. This can easily be achieved by weighing the
daily supply of compound in as many vials as are needed for the experiment. The
vials are then stoppered, labeled, and stored appropriately. Each morning, sufficient
suspending agent is added to a vial, and the suspension thoroughly mixed. It is
necessary to make daily suspensions in order to minimize the error in dosage created
by improper sampling of suspensions.
When suspensions ai-e used, it is recommended that the syringe be filled directly
from the vial and then the needle be placed on the syringe. In this way, it is easier
to remove air bubbles from the syringe.
Color Coding of Solutions and Mice
Each solution or suspension is labeled with colored tape. The cage containing
the mice that receive that solution or suspension is also labeled with the same col-
ored tape, thus minimizing the need for written labels as well as minimizing errors.
Dosage Calculations
The dose is always expressed in terms of kilograms (kg) of mouse per day. The
average body weight of the mice in a cage on gestational day 6 is used for the calcu-
lations. Thus, on day 6 of gestation, the mice in cage 12 are weighed. The numeri-
cal average of these weights is calculated as shown in the following example:
Mouse no. Weight, grams
12-1 28.1
12-2 22.2
12-3 24.6
12-4 23.7
12-5 27.2
12-6 25.4
151.2 = total
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151 2
g =25.2 grams « average mouse weight in cage 12
The mice in cage 12 have been assigned compound (cmpd) Z at a dose of 150 milligrams
(ing)/kg/day. To make the appropriate solution for the mice in cage 12, the following
calculations are used:
150 mg of cmpd Z = X mg of cmpd Z
1000 g of mouse = 25.2 g of mouse
This equation should be read as follows: if 150 mg of compound Z are required for
1000 g of mouse (= 1 kilogram), then how many mg of the compound (X) are required for
25.2 g of mouse. This equation is solved by cross multiplying as follows:
150 mg cmpd Z ^><^-»' X mg cmpd Z
1000 g mouse ~-><--*. 25.2~g of mouse
(1000 g mouse) (X mg cmpd Z) = (150 mg cmpd Z) (25.2 g mouse)
1000 X = 150 x 25.2
1000 X = 3780.0
y _ 5780.0
1000
/
X * 3.78 mg of compound Z
Thus each mouse in that cage receives 3.78 mg of compound Z every day.
Assuming that compound Z is soluble and stable in the vehicle, then a solution
is made as follows:
1. Each mouse receives an injection of 0.1 milliliter (ml) daily for 10
days. Thus each mouse needs 10 x 0.1 ml or 1.0 ml of solution for the
experiment.
2. Since there are six mice in the cage, the quantity of solution needed
for the experiment is six times 1.0 ml of solution or 6.0 ml.
3. As to the total volume required, allowances must be made for the amount
that is needed to fill the syringe properly. Thus, to have adequate
solution for an individual cage of mice for the whole experiment, a total
volume of 10.0 ml is more than enough.
Calculations for Making a_ Solution
The quantity of compound Z in a volume of 0.1 ml needed for each mouse for each
day is 3.78 mg. To make 10.0 ml of solution, the problem is approached in the same
manner as the following:
3.78 mg cmpd Z _ X mg cmpd Z
0.1 ml solvent ~ 10.0 ml solvent
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To solve this equation, first cross multiply as follows:
3.78 mg cmpd Z"--. x
0 . 1 ml solvent 10.0 ml solvent
(0.1 ml solvent) (X mg cmpd Z) « (3.78 mg cmpd Z) (10.0 ml solvent)
(0.1) (X) = (3.78) (10.0)
0.1. X =37.8
Y 37.8
A 0.1
X = 378 mg of compound Z
Thus, 378 mg of compound Z should be weighed out and dissolved in 10.0 ml of solvent.
Each injection will use 0.1 ml of the solution.
Practice Problems
In the following problems, determine the solution or suspension that would be
required.
1. Compound D will be administered at a dose of 84 mg/kg as a stable solu-
tion. The mice in the cage weigh: 22.4, 23.6, 25.8, 26.1, 24.7, and
23.2 g.
2. Compound E will be administered at a dose of 52 mg/kg as a suspension.
The mice in the cage weigh: 23.8, 24.3, 24.6, 25.2, and 25.9 g.
3. Compound F will be administered at a dose of 115 mg/kg as a nonstable
solution. The mice weigh: 24.2, 24.7, 25.8, 26.1, 22.0, and 23.3 g.
FETAL FIXATION AND STAINING
Fetal Fixation
Fetuses should be hardened to ensure proper necropsy. Bouin's solution is a pre-
servative and fixative which accomplishes this. It consists of a saturated solution of
picric acid mixed with formaldehyde and glacial acetic acid. The solution will harden
a mouse fetus in about 3 days. Fetuses left in this solution for more than 3 weeks
become too brittle for necropsy.
Modified Bouin's Solution:
1. Prepare picric acid: 30 g dissolved in 1900 ml H20.
2. Add 1900 ml of formaldehyde.
3. Add 190 ml of glacial acetic acid.
4. Mix well.
Alizarin Staining
Alizarin red S is used to stain the skeleton, permitting examination for skeletal
defects. Fetuses from an entire litter are placed in a jar with 70 percent propyl
or ethyl alcohol for at least 24 hours. They may be stored indefinitely in 70 per-
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cent propyl or ethyl alcohol. The amount of alcohol should be approximately 10 times
the volume of the fetuses. The fetuses are put into the alcohol to fix or preserve
the tissues.
When the fetuses are sufficiently fixed by the alcohol, usually in 1 to 3 days,
all internal organs are removed with small forceps. The heart and lungs are removed
through an opening made in the diaphragm, leaving the rib cage intact. Also, a fat
pad located on the back of the neck and between the shoulder blades is carefully re-
moved with scissors and forceps.
After the internal organs have been removed, the fetuses are placed in a 1 per-
cent potassium hydroxide (KOH) solution until the bones are visible through the skin.
This step may take from 1 to 7 days. The KOH solution is then poured off and replaced
with the following solution:
distilled H20 800 ml
II KOH solution 35 ml
glycerin 200 ml
alizarin solution 10-15 drops
Solutions are as follows:
II KOH solution - 1 g of potassium hydroxide in 100 ml of
distilled water.
501 glycerin - equal amounts of glycerin and distilled water
mix well.
alizarin solution - 500 mg alizarin red S in 100 ml of distilled
water.
Within 1 to 2 days the fetal bones should be stained red. When the desired
depth of coloring of the bones has occurred, the staining solution is poured off;
the fetuses are rinsed with water and placed in a 50 percent glycerin solution.
After 1 or more days in the glycerin solution, the fetuses can be examined for skele-
tal defects. Fetuses that are kept for long indefinite periods should be preserved
in 100 percent glycerin, to which some crystals of thymol have been added to prevent
fungal growth.
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EXPERIMENT DESIGN
MATERNAL DATA
Experiment Schedule
The schedule shown in Table 1 was designed for use with CD-I mice; with some
compounds or with other strains, variations of this design could be necessary. The
mice may be bred by a commercial supplier and delivered to the teratology laboratory
before they are 5 days pregnant, or bred at the laboratory.
The mice are mated according to the following plan. One group of mice are mated
on Friday night with detection of a vaginal plug on Saturday morning. From this
breeding, there should be 100 pregnant mice, with the first day of gestation being on
Saturday as outlined in Table 1. Then, on the following Thursday (day 6 of gesta-
tion), these mice are weighed and coded by ear punch. The compound they are to re-
ceive is prepared for the entire experiment. The compound is administered daily for
the next 10 days, starting on Friday (day 7 of gestation). On gestational day 17,
Monday, these animals are weighed and sacrificed. The fetuses are removed, weighed,
and stored in Bouin's solution until necropsied. Some litters are stored directly in
70 percent propyl alcohol for staining the skeleton-with alizarin red S. (See earlier
Table 1. SCHEDULE SHOWN FOR 4 WEEKS FOR GROUPS OF MICE A, 8, C, D, E, F
This schedule will continue as such.
Sun
Sun
Sun
treat A
treat B
Sun
treat B
treat C
treat D
Mon
Mon
weigh A
Mon
treat A
treat B
weigh C
Mon
kill B
treat C
treat D
weigh E
Tues
mate A
'rues
treat A
mate C
Tues
treat A
treat B
treat C
mate E
Tues
treat C
treat D
treat E
mate G
Wed
day 1 A
Wed
treat A
day 1 C
Wed
treat A
treat B
treat C
day 1 E
Wed
treat C
treat D
treat E
day 1 G
Thurs
Thurs
treat A
weigh B
Thurs
treat A
treat B
treat C
weigh D
Thurs
treat C
treat D
treat E
weigh F
Fri
mate B
Fri
treat A
treat B
mate D
Fri
kill A
treat B
treat C
treat D
mate F
Fri
kill C
treat D
treat E
treat F
mate H
Sat
day 1 B
Sat
treat A
treat B
day 1 D
Sat
treat B
treat C
treat D
day 1 F
Sat
treat D
treat E
treat F
day 1 H
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section entitled Fetal Fixation and Staining for preparation of Bouin's solution and
alizarin staining technique.) Other strains of mice may be sacrificed on gestational
day 18 or 19 since they have longer gestational periods. The purpose is to sacrifice
the mice 1 or 2 days before delivery to recover the fetuses.
Another group of mice are mated on Tuesday so that there will be 150 pregnant
mice with a day 1 of gestation on Wednesday. Then on the following Monday (day 6 of
gestation) these mice are weighed, and ear coded; the compound is then prepared for
this group. On Tuesday (day 7 of gestation) , the treatment begins and continues for
10 days. On Friday (day 17 of gestation), the mice are sacrificed as indicated above.
Protocol Sheet A - Before Sacrifice
Protocol sheet A, a sample of which is provided on the next page as Figure 2,
is filled out when the pregnant mice arrive in the teratology laboratory. The sheet
should be completed as follows:
The cage number is placed in the upper right-hand corner. The cages are numbered
sequentially. Assignment of the compound and the doses is done on a random basis;
however, all the mice in any single cage receive the same treatment.
Thg date of day 1 means the date (month/day) of the first day of gestation, which
is defined as the date when a vaginal plug was found in the female.
The compound and its log number are entered.
The vehicle is defined as the substance in which the compound is dissolved or
suspended to facilitate administration. There are always animals that receive only
the vehicle; they serve as controls for those receiving the compound. For these con-
trol animals, the vehicle is entered and the compound listed as NONE.
The route of administration (injection, intubation) is listed as well as the
volume administered in a single treatment.
dose is entered in terms of quantity of compound per kg of mouse per day.
Both the dates of treatment (e.g., March 12-21) and the days of gestation (i.e.,
days 7-16) will be listed.
As above, the date the mouse is sacrificed and the day of gestation are entered
by date and day sacrificed. The days of treatment and sacrifice could vary with the
compounds tested and the strain of mouse utilized.
On gestational day 6, the animals are weighed and coded. This weight is placed
in the column under the appropriate mouse number across from day 6 weight.
During the course of treatment, the dates of any maternal deaths are entered in
the appropriate boxes across from died (date) .
10
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Cage No.
Mouse strain.
Date of day 1.
Compound.
Vehicle.
Route.
Dates and days treated.
Date and day sacrificed.
Log no..
Dose.
Volume.
./kg/day
_ml/day
Mouse ear no.
Day 6 weight
Day sacrifice
weight
Liver weight
Gravid uterus
weight
Total live
litter weight
No. live fetuses
No. dead and
resorbed fetuses
Non-pregnant
Died (date)
1
2
3
-
4
5
6
Figure 2. Protocol sheet A.
1.1
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Protocol Sheet A_- Day of Sacrifice
The day the animals are sacrificed, they are weighed and this weight is entered
on the sheet under day sacrifice weight.
After sacrifice, the animals are placed on their backs on plastic-backed absor-
bant paper. The fur on the abdomen is thoroughly wetted with a dilute solution of
detergent from a squeeze bottle, preventing the fur from flying when the abdomen is
opened. With the use of a pair of dissecting scissors, the abdomen of the animal is
opened by cutting through the skin and muscle from the region of the bladder to the
thorax. This incision exposes the uterus, liver, and other maternal organs. The
maternal liver and the gravid uterus are removed, each is weighed independently, and
the weights are entered under liver weight and gravid uterus weight. The gravid
uterus is the entire uterine mass (both horns), with fetuses (live and dead) and
amniotic fluid contained within.
After the weighing, the gravid uterus is carefully opened and the fetuses re-
moved. Care should be exercised not to damage the fetuses. The live fetuses from a
single litter are counted and entered as no_. live fetuses. They are then weighed,
and this value is entered under total live litter weight.
After the weighing, the litter destined to be necropsied is placed in a single
compartment of a plastic tray that contains Bouin's solution. For large fetuses, a
transverse abdominal cut may be necessary to permit the Bouin's solution to penetrate
the internal organs fast enough to prevent rotting. The cage number and specific mouse
number should be noted, with wax pencil, on the lid of the compartment on a piece of
tape. Thus, the litter from mouse number 5 in cage 12 would have a piece of tape
reading 12-5 above its compartment. The cage number always is first, followed by the
mouse number.
A litter destined for alizarin red S staining is counted, weighed, and then
placed in a jar containing 70 percent propyl alcohol. A transverse abdominal cut may
be made in the fetuses to enhance fixation. The jar, not the cap, should be labeled
with the mouse number.
In addition to live fetuses, a gravid uterus may contain dead fetuses or resorp-
tion sites. A dead fetus generally appears as a well formed, pale fetus with no
response to stimulation and no apparent umbilical circulation. A resorption site
appears as a mascerated fetus or a small dot of black tissue. This is the result of
fetal death and subsequent autolysis (dissolving) and resorption. At the time of
examination of the uterus, the dead fetuses may vary greatly in size and form, de-
pending upon the length of time they have been autolyzing. They may appear as pale
fetal forms or very small masses termed resorption sites.
When found, dead fetuses and/or resorption sites are counted, and the number is
entered under no. dead and resorbed fetuses.
12
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Uteruses that do not appear to contain fetuses are carefully examined for the
presence of small resorption sites. If none are found after careful examination, the
animal is considered non-pregnant and should be so indicated by placing an "X" in the
appropriate box.
FETAL DATA
Examination of Fetuses by Necropsy
Fetuses stored in Bouin's solution may be necropsied when they have become firm.
Generally 3 days is sufficient for this to happen. Before T^rropsy, the fetuses are
rinsed in water to remove excess Bouin's solution since it is an eye, nose, and throat
irritant. Gloves are worn at all times during necropsy to avoid contact with both
the test chemicals and the Bouin's solution.
The fetus is initially examined for external defects. Then the top of the head
is cut off by inserting the- scalpel into the mouth above and parallel to the tongue
and slicing through the back of the skull. The palate and tongue are observed. Cor-
onal sections are made through the nares, eyes, and cerebrum, and these portions are
examined. The internal organs of the fetus are exposed by cutting, with a pair of
scissors or scalpel, through the ventral bodv wall from the region of the bladder up
through the thorax, and then across the abdomen at the level of the liver. By gently
pulling the fore and hind limbs apart, the examiner inspects the heart and lungs.
The liver and intestines should be examined and removed so that the sex of the fetus
can be determined. This step is accomplished by the identification of either the
testes or uterus. The testes are spherical bodies, one on each side of the bladder
and against the posterior body wall. The uterus is a flattened "V"-shaped tube against
the posterior body wall.,
Each kidney is then sliced open so that the internal structure can be observed.
During the examination of a fetus, the size, shape, and location of organs or
structures are carefully noted. Deviations from normal of any of these factors de-
scribe the majority of the malformations that will be observed.
Notations on specific anatomical malformations are found in the glossary at the
end of this section.
Examination of Alizarin-Stained Fetuses
Alizarin-stained fetuses should remain in 50 percent glycerin during examination.
At this stage of development, the bones of the cranium are almost completely ossified.
The bones of the arms, legs, digits, ribs, and spinal cord are ossified while the car-
pals, tarsals, and sternebrae may not be.
The skeleton should be examined for the presence or absence of bones as well as
the shape or length of the bones and degree of ossification.
13
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Protocol Sheet B
Protocol sheet B, a sample of which may be found on the next page (Figure 3), is
utilized at the time of fetal examination by necropsy or alizarin staining. It is
designed to record descriptions of abnormal fetuses. The mouse number is placed on
the upper right-hand corner. When the litter has been stained with alizarin red S,
an "X" is placed after alizarin.
If all the fetuses in a litter are found upon examination to be normal, an "X"
is placed after all normal.
In litters with both normal and abnormal fetuses, the normal fetuses are sexed
and tallied. Each abnormal fetus is arbitrarily assigned a number, and its descrip-
tion occupies a single column on the protocol sheet.
There are seven columns on the protocol sheet that will accommodate seven abnor-
mal fetuses. In a litter in which there are more than seven abnormal fetuses, two
protocol sheets are used. This fact should be indicated by placing an "X" after two
sheets used in the upper right hand corner.
The types of defects and their descriptions are found in the glossary. A single
fetus may have more than one defect, e.g., clubfoot and cleft palate. There can also
be more than one defect for each fetal structure. All defects are entered in the
column across from the appropriate structure, by the use of the acceptable abbrevia-
tions as they appear in the glossary. For defects not listed in the glossary, a
standard medical dictionary should be consulted.
All fetuses, both normal and abnormal, should be sexed, and the total number of
males and females for the litter placed in the upper left corner under no. females,
no. males. The sex of abnormal fetuses should also be entered as M or F in the line
labeled sex/uterus/testes.
Glossary of_ Malformations
The malformations listed in the glossary are arranged in groups as they appear
on protocol sheet B. Each malformation is followed by the appropriate abbreviation
to be used on the sheet. Some malformations are not listed in the glossary since they
occur infrequently. If any are observed, reference should be made to a textbook or
medical dictionary and the full name of the malformation should be entered under the
appropriate fetal column in one of the blank boxes.
Brain/Cranium
encephalocele - EN - protrusion of the brain through an opening of the skull.
The cerebrum is well formed and covered by transparent
connective tissue.
exencephaly - EX - lack of skull with disorganized outward growth of the brain.
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Mouse no.
No. females.
All normal
No. males.
Alizarin
Two sheets used
Abnormal fetus no.;
Brain/cranium
Right eye
Left eye
Jaw
Palate
Right front limb
Left front limb
Right front digits
Left front digits
Right hind limb
Left hind limb
Right hind digits
Left hind digits
Ribs
Spinal cord
Tail
Intestines
Heart
Lung
Sex/uterus/testes
Right kidney
Left kidney
-
Figure 3. Protocol sheet B.
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microcephaly - M - small head on normal sized'body.
hydrocephaly - H - marked enlargement of the ventricles of the cerebrum.
craniorachischisis - CR - exposed brain and spinal cord.
Nose
enlarged naris - EN - enlarged nasal cavities.
single naris - SN - a single naris, usually median.
Eye
microphthalmia - M - small eye.
anophthalmia - A - lack of eye.
open eye - 0 - no apparent eyelid, eye is open.
Ear
anotia - A - absence of the external ear.
microtia - M - small ear.
Jaw
micrognathia - M - small lower jaw.
agnathia - A - absence of lower jaw.
aglossia - AG - lack of tongue.
astomia - AS - lack of mouth opening.
bifid tongue - BT - forked tongue.
cleft lip - CL - may be either unilateral or bilateral cleft
of the upper lip.
Palate
cleft palate - CP - a cleft or separation of the median portion of the palate.
Limbs
clubfoot - C - a foot that has grown in a twisted manner, resulting in an abnor-
mal shape or position. It is possible to have a malposition of
the whole limb.
micromelia - M - abnormal shortness of the limb.
hemimelia - H - absence of any of the long bones resulting in a shortened limb.
phocomelia - P - absence of all the long bones of a limb, the feet being attached
directly to the body.
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Digits
polydactyly - P - presence of extra digits. Since five is the normal number in
the mouse, a polydactylous fetus would have six or more.
syndactyly - S - fusion of two or more digits.
oligodactyly - 0 - absence of one oivmore digits.
brachydactyly - B - smallness of one or more digits.
Ribs
wavy ribs - W - ribs may be any aberrant shape.
extra ribs - E - may have extra ribs on either side.
missing ribs - M - may be missing on either side.
fused ribs - F - may be fused anywhere along length of rib.
branched ribs - B - single base and branched.
Spinal Cord
spina bifida - B - nonfusion of spinal processes. Usually the covering ectoderm
is missing, and the spinal cord is evident. Vertebrae may be
missing, fused, or malshaped.
Tail
short tail - S - short tail, usually lack of vertebrae.
missing tail - A - absence of tail.
corkscrew - C - a corkscrew-shaped tail.
Intestines
umbilical hernia - UH - protrusion of the intestines into the umbilical cord.
ectopic intestines - El - extrusion of the intestines outside the body wall.
Heart
dextrocardia - D - rotation of the heart axis to the right.
enlarged heart - E - either the atrium or the ventricle may be enlarged.
Lung
enlarged lung - L - all lobes are usually enlarged.
small lung - S - all lobes are usually small. Lobes may have immature appearance,
e.g., look like a bunch of grapes.
Sex/uterus/testes
sex - M or F - the sex of every abnormal fetus should be entered.
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undescended testes - U - testes are located anterior to the bladder instead of
lateral. May be bilateral or unilateral.
agenesis of testes - AT - one or both testes may be missing.
agenesis of uterus - AU - one or both horns of the uterus may be missing.
Kidney
hydronephrosis - H - fluid-filled kidney, often grossly enlarged. This condi-
tion may be accompanied by hydroureter.
fused - F - kidneys fused, appearing as one misshapen kidney with two ureters.
agenesis - A - one or both kidneys missing.
misshapen - M - small, enlarged (normal internally), spherical, or odd-shaped
kidneys.
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SAMPLE EXPERIMENT: VITAMIN A
Vitamin A was selected as a model compound that can be used to produce many types
of anomalies. Single large doses of vitamin A are teratogenic, and the day of gesta-
tion on which it is administered determines the types of anomalies produced. The
following experiment with vitamin A has been designed to produce a large spectrum of
types of anomalies.
EXPERIMENT DESIGN
Vitamin A palmitate is administered to CD-I mice as a suspension in corn oil by
oral intubation. The initial dose is 25,000 to 40,000 Units/mouse in 0.1-ml volume.
Vitamin A is administered on. a single day of gestation during the period of organ
formation, days 8 - 13. All mice are sacrificed on day 17 of gestation.
On Friday, Saturday, Sunday, Monday, and Tuesday, mice are mated, resulting in the
following groups:
Day 1 of gestation Number of mice Group
Saturday 64 A
Sunday 64 B
Monday' 64 C
Tuesday 48 D
Wednesday 48 R
The groups are treated as follows:
Group A - 32 mice are nontreated controls. Treat 32 mice on gestational day
14; 24 with vitamin A and 8 with corn oil.
Group B - treat 32 mice on gestation day 11; 24 with vitamin A and 8 witli corn
oil. Treat the remaining 32 mice on gestational day 13; 24 with vitamin A and 8 with
corn oil.
Group C - treat 32 mice on gestational day 10; 24 with vitamin A and 8 with corn
oil. Treat remaining 32 mice on gestational day 12; 24 with vitamin A and 8 with corn
oil.
Group D - treat 48 mice on gestational day 9; 32 with vitamin A and 16 with corn
oil.
Group E - treat 48 mice on gestational day 8; 32 with vitamin A and 16 with corn
oil.
All treatments occur on either Wednesday or Friday as may be seen in the follow-
ing schedule:
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Group
Wednesday
B
B
C
C
D
D
E
E
Friday
A
A
B
B
C
C
Day of gestation
11
11
10
10
9
9
8
8
14
14
13
13
12
12
No. of nice
24
8
24
8
32
16
32
16
24
8
24
8
24
8
Treatment
vitamin A
corn oil
vitamin A
corn oil
vitamin A
corn oil
vitamin A
corn oil
vitamin A
corn oil
vitamin A
corn oil
vitamin A
corn oil
Beginning on the following Monday, the mice are sacrificed on gestational day
17 according to the following schedule. The fetuses arc stored in Bouin's solution
until necropsied.
Day they were No. of
Weekday Group treated mice
Monday A nontreated 64
controls + day 14
Tuesday B day 1.1 5 13 64
Wednesday C day 10 § 12 64
Thursday D day 9 48
Friday E day 8 48
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
EPA-650/1-73-001
2.
3. RECIPIENT'S ACCESSION-NO.
4. TITLE AND SUBTITLE
Training Manual for Teratology
5. REPORT DATE
September 1974
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
K. Diane Courtney and Neil Chernoff
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Pesticides and Toxic Substances Effects Laboratory
Research Triangle Park, N. C. 27711
10. PROGRAM ELEMENT NO.
1EA078
11. CONTRACT/GRANT NO.
12. SPONSORING AGENCY NAME AND ADDRESS
Office of Research and Development
National Environmental Research Center
Environmental Protection Agency
Research Triangle Park, N. C. 27711
13. TYPE OF REPORT AND PERIOD COVERED
Final
14. SPONSORING AGENCY CODE
15. SUPPLEMENTARY NOTES
16. ABSTRACT
The report is an introduction to the basic techniques involved in teratology
testing. General topics covered include the handling of animals, compound pre-
paration and administration, dosage calculations, and coding techniques. Teratology
techniques include fetal fixation, alizarin staining, and the examination of fixed
and stained fetuses for soft tissue and skeletal anomalies. Suitable protocol
sheets covering data obtained before sacrifice, after sacrifice, and subsequent
fetal examinations are included. A glossary of the most common malformations and
their description is present. The outlines of a teratology experiment with
Vitamin A is included as a suggested training exercise. The above experiment
and the techniques described in the manual may be used to train laboratory
personnel in the techniques and designs of teratology experiments.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
teratology
techniques
training
b.IDENTIFIERS/OPEN ENDED TERMS
c. COSATI Held/Group
18. DISTRIBUTION STATEMENT
Release Unlimited
19. SECURITY CLASS (This Report!
Unclassified
21. NO. OF PAGES
25
20. SECURITY CLASS (Thispage)
Unclassified
22. PRICE
EPA Form 2220-1 (9-73)
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