Introduction
Cesarean Section
Fetal External Examinations
Fresh Visceral Dissection (Staple's Technique)
Wilson's Soft Tissue Sectioning Technique
Skeletal examinations
Male Fertility Evaluation

Developmental Toxicology, or Teratology, is the study of effects of compounds that cause birth defects. The field opened up after the Thalidimide controversy in the 1950s when the use of Thalidimide by pregnant women to reduce the discomfort of pregnancy resulted in the appearance of many cases of birth defects, particularly in Japan and Europe. The drug was not used in the United States not because of product testing which showed it to be dangerous, but only because the FDA had not yet finished the paperwork to approve it before the results of its use elsewhere were discovered. Due to the recognition of the dangers new drugs potentially held for pregnant women and their unborn children it was decided that new drugs should be tested for safety before use by pregnant women could be approved. In the following discussions the methods employed in testing will be detailed from the aspect of the Developmental Toxicology or necropsy technician. Test article administration to the animal model is not discussed, but is generally done in the time period from implantaion through the completion of organogenesis. The page is currently under construction and is not yet complete. Further sections will be added at a later date.

In order to determine whether a test article or substace causes malformations in offspring rats can be cesarean sectioned on the 20th or 21st day of gestation, rabbits can be cesarean sectioned on the 29th day of gestation, and mice can be cesarean sectioned on the 18th day of gestation. The animal's identity is verified following which it is euthanized and placed in a position of dorsal recumbancy. an inscision is made along the ventral midline from the area of the genital opening to the neck allowing the skin to be drawn back to reveal and subcutaneous masses. Following this the abdominal cavity is opened, the diaphragm is examined for diaphragmatic hernias, and the thoracic cavity is cut open on one or both sides of the sternum to reveal the thoracic organs.

the uterus is examined externally and then removed carefully. At this time a gravid uterine weight is taken and recorded if necessary. The uterus is laid on a flat surface and cut open on the side away from the placentae, the amniotic sacs are removed from around the fetuses and the fetuses are pulled carefully away from their implantation sites taking care not to break the umbilicus or pull the placenta free from the uterus. The uterine position and type of each implatation is noted, as well as the placement of the cervix. An implatation that shows full development and responds to the touch is noted as a live fetus, an implatation that shows full development and does not respond to the touch but does not show signs of autolysis is noted as a dead fetus, an implatation which shows fetal charachteristics but shows signs of autolysis is noted as a late resorption, and an implantation which shows no fetal charachteristics is noted as an early resorption. Following the documentation of the implantation sites the bursa is removed from each ovary and the number of corpora lutea is documented. Tere must be at least as many CLs (corpora lutea) on each ovary as there are implantations on the corresponding uterine horn due to the fact that each CL represents an ovarian follicle which has released an egg and has developed into a hormonal body, though there may be more CLs than implants. The fetuses are then cut free from the placentae and placed in individual compartments of a tray, or otherwise treated, so that each may be uniquely identified with a number corresponding to its uterine position. Following the removal of the fetuses and examination of the placentae the mother is given a gross necropsy beginning in the thoracic cavity.

The thymus is examined for size and color and the presence of hemorrhagic areas and is then gently removed allowing for examination of the trachea, esophagus, thyroid, parathyroid, and the great vessels and major vessels leaving the heart. The esophagus should descand on the left side of the trachea and there should be no obstructions or perforations in either the trachea or the esophagus. The lungs are examined for size and color and the lobes should be counted. There should be three lobes, an apical, a cardiac, and a diaphragmatic lobe, on the right side, one lobe on the left side (two, a left superior and a left inferior in rabbits), and a small intermediate lobe in the midline beneath the heart (often called the azygous lobe. This lobe is frequently absent in fetal rabbits). The heart is examined for size, color, and position. The pulmonary artery should arise from the right ventricle and the aortic arch should arise from the left ventricle. Following examination of the thoracic organs the abdominal organs are examined.

The lobes of the liver are examined for size, shape, texture, and color. There should be a left lateral lobe, a median lobe, a right lateral lobe, a caudate lobe, and a papillary process with two sections, one on each side of the stomach. The median lobe is divided into two parts by a fissure with some laboratories naming one portion the median and the other portion the quadrate. Further some laboratories name the caudate lobe as the right inferior lobe and the papillary process as the caudate lobe. In rabbita and mice a gallbladder will be found on the underside of the median lobe. Rats have no gallbladder. The stomach is opened along the greater curvature and the contents and mucosal surface examined. Any reddened areas, erosions, or ulcerations are noted as are thickenings or thinnings of the lining. The intestines are examined for amount and color and any reddening, necrotic areas, or fistulas are noted. The adreanals are examined for size, shape, position, and color. The ureters are examined for distention and the presence of red fluid, granular white material, or calculi. The kidneys are examined externally for size, shape, and color as well as the presence of clear fluid-filled cysts, cortical cysts, pitting, or granular appearance and then sectioned with a sharp scalpel or razor blade to examine the pelvis for distention or the presence of calculi or white granular material. The left kidney is sectioned with one longitudinal slice just off center and the right kidney is sectioned with one transverse slice directly through the papilla. The capsule, cortex, medulla, renal papilla, and renal pelvis should all be present and the pelvis should not be distended with fluid. The urinary bladder is examined for reddening, thickening, and the presence of calculi or white granular material.

Any abnormalities or lesions should be photographed and saved in 10% neutral buffered formalin for possible histopathological examination with the exception of calculi, which should bre saved in a seperate dry container. Following the gross necropsy examination the carcass is discarded in an appropriate manner.

Each fetus is examined externally for malformations or developmental variations beginning with the head. The head is examined for size and shape. Two eye bulges should be present, as well as two nares, five facial papillae, and two pinnae. The mouth should be examined for size and shape and opened gently with a dull probe to examine the palate for any cleft or fleshy protrusions and the tongue for presence and size. The head should also be examined for any closure defects. The ventral aspect is examined for any closure defects and the limbs are examined for presence, size, and shape as well as medial rotation or flexion of the elbows, paws, or shoulders. The digits are counted. There should be five digits (a pollux and four digits) on each forepaw and five digits on each hindpaw (except rabbits which will have only four digits on each hindpaw). The digits should be examined for presence, length, webbing, or fusion. The fetus should be turned over and examined for any dorsal closure defects and the size and shape of the tail should be examined. Short, thread-like, bent, coiled, or absent tails should be noted. The tail should be gently lifted and the anal opening examined. At this time the mouse or rat fetuses can be externally sexed by examination of the distance between the anal opening and the genital tubercle (females show a short distance, males show a longer distance). Rabbit fetuses cannot be sexed externally. Fetuses should also be weighed at this time.

The following terms may be used to describe fetal findings:

Acephaly- absence of the head
Microcephaly- small head
Anencephaly- a closure defect of the head revealing the brain, which does not protrude
Exencephaly- a closure defect of the head revealing the brain, which does protrude
Cranioschisis- a closure defect at the back of the head revealing the brainstem

Microphthalmia- depressed or small eye bulges (eyes)
Macrophthalmia- large eye bulges (eyes)
Anophthalmia- absent eyes
Circumcorneal hemorrhage- hemmorrhagic iris
Coloboma- an opening in the cornea

Microstomia- small oral opening
Macrostomia- large oral opening
Astomia- absent oral opening

Microtia- small pinna
Macrotia- large pinna
Anotia- absent pinna

Microglossia- small tongue
Macroglossia- large or protruding tongue
Aglossia- absent tongue

Micrognathia- short jaw (noted as mandibular for the lower jaw)
Macrognathia- large jaw
Agnathia- absent jaw

Ectrodactyly- short digits
Brachydactyly- long digits
Syndactyly- webbing between the digits
Adactyly- absent digits

Micromelia- short limbs
Macromelia- large limbs
Amelia- absent limbs
Carpal or Tarsal felxure- fore or hindpaws flexed (respectively)
Rigid Flexure- flexure of the shoulder, elbow, or knee (with location noted)

Thoracoschisis- closure defect of the ventral thorax
Gastroschisis- closure defect of the ventral abdomen
Thoracogastroschisis- closure defect of the ventral thorax and abdomen
Omphalocele- umbilical hernia
Ectocardia- the heart lies outside of the thoracic cavity with the skin sealed around it
Rachischisis- closure defect of the spinal column
Craniorachischisis- closure defect of the back of the skull and the spinal column
Spina Bifida- closure defect of the spinal column revealing the spinal chord
Meningocele- meninges protrude from skull or spinal column.

The preceding list is not to be considered complete but is only a guide to MARTA terms. The current practice appears to be away from using the MARTA terminology, many of which terms are diagnostic, and toward using the descriptive terms instead.

Fetal fresh visceral dissections follow much the same path as the maternal gross necropsy (see CESAREAN SECTION) with the following changes. The head is either removed and saved in Bouins solution for later Wilson's slicing or it is sliced at the level of the frontal/parietal suture and gently held open to examine the brain for distention of the lateral and third ventricles or fluid buildup between the brain and the cranial vault (internal or external hydrocephaly). The examination of the heart and vessels includes a fetal hert dissection. There should be three vessels rising from the aortic arch. From right to left they should be the innominate (which branches into the right subclavian and the right carotid), the left carotid (which occasionally arises from the innominate), and the left subclavian. The Ductus arteriosis should be apparent and patent between the pulmonary trunk and the aortic arch. The heart is gently held with microforceps and the right ventricle is opened near the apex. The microdissection scissors are onserted into the opening and a cut is made on the ventral aspect which goes up into the pulmonary trunk, past the semilunar valves. The muscular flap is held open to allow for examination of the interventricular septum for septal defects, the papillary muscles, the chordae tendonae, the tricuspid (atrio-ventricular) valve, and the semilunar valves. The flap is put back in place and the left ventricle is cut at the apex. The microdissection scissors are inserted and a cut is made with the scissors rotated slightly to the left up through the semilunar valves and into the aorta. This cut will cross and sever the pulmonary artery just above the semilunar valves. The left ventricle is held open to allow for examination of the septum, the papillary muscles, the chordae tendonae, the mitral or bicuspid (artio-ventricular) valve, and the semilunar valves. A small red spot should be apparent in one of the cusps of the semilunar valves where the carotid artery arises. The testes of male fetuses and the uteri of female fetuses are examined to internally verify the fetal sex.

Fetuses used for the Wilson's soft tissue sectioning technique are first fixed in Bouin's solution, which is a mixture of saturated picric acid, formaldehyde, and glacial acetic acid. The purpose of the fixative is to fix the tissues, harden the soft tissues, and soften the bones in order to preserve the specimens and make it possible to slice them cleanly with a razor blade into sections of approximately 1mm (or less) thickness. One drawback in this fixed tissue technique is that the original coloration of the tissues is lost and all tissues appear as a shade of yellow with the exception of blood which appears brown and the liver which appears olive green. The fetuses remain in the fixative for a period of two weeks minimum and are then rinsed in alcohol prior to slicing. Since the formaldehyde and acetic acid fumes from the fixAtive are irritating and present a carcinogenic danger it is recommended that the rinsing and slicing be done under a fume hood or in a well ventilated area. As Bouins solution stains tissues those working with these specimens should also wear latex gloves, preferably two pair.

Fetuses are sliced with a sharp razor blade held in a Lipshaw or Pathco handle. The blades should be changed frequently in order to insure even slices with a smooth face and care should be taken to slice with as few strokes as possible. Eccesive downward pressure with the blade, or squeezing of the fetus in the grip, will cause the internal organs to be pushed out resulting in uneven slices and slices which fall apart into pieces.

The identity of the fetus is first verified after which it is given an external examination. The forelimbs are removed with either the razor blade or a sharp pair of scissors, and the head is sliced with between four and eight slices, the minimum being a slice at the beginning of the nasal passage just behind the nares, a slice at a point just before the eye bulges to examine the nasal passages (turbinates), the palate, and the nasal septum, a slice through the eye bulges to allow the examination of the lenses and upon removal of the lenses the retinas, and a slice behind the eye bulges at the level of the frontal/parietal suture in order to examine the lateral and third ventricles of the brain. If only these four slices are taken it is recommended that the brain be carefully removed from the last section to allow an examination of the cerebellum and meninges. If eight slices are taken, then the final slice should allow examination of the fourth ventricle of the brain. Following these slices the bottom of the head is removed from the fetus by slicing through the neck and the structures of the inner ear are examined.

The remainder of the fetus is sliced as thinly and evenly as possible and the sections laid on the wax block with the cranial side upward. The trachea, esophagus, and blood vessels are followed through the slices and the spinal chord and organs of the neck are examined. The right carotid, the right subclavian, left carotid, and left subclavian should be apparent and the right arotid and subclavian should be seen to converge to form the innominate. Care should be taken to have a cut transect the top of the aortic arch. From the top of the aortic arch through the apex of the heart the technician should attempt to slice thinly enough to produce ten slices in which the following structures should be apparent: the aortic arch, the ductus arteriosus, the pulmonary artery, the artia, the semilunar valves, the tricuspid valve, the mitral valve, the ventricles, and the interventricular septum.

Following the slices through the heart there are two possible techniques. The first technique continues slicing thinly through the abdomen reveaing the liver lobes, stomach, and intestines until the level where the slices transect the kidney through the pelvis following which the remaining viscera are gently pulled back to internally verify the sex of the fetus. The second technique stops thoracic slicing at a point just before the diaphragm is reached and gently removes the remainder of the lungs in order to examine the diaphragm fro diaphragmatic hernias. Following this a single abdominal slice is made from the level of the umbilicus on the ventral aspect to a point just abouve the kidneys and adrenals on the dorsal aspect. The sections of liver lobe in the upper section are gently pulled apart and examined, following which the remaining portions of liver and the intestines are pulled out of the lower section to reveal the kidneys and sex organs. The kidneys are sliced through the pelvis and the feal sex is internally verified.

The effect of the Wilson's soft tissue sectioning technique is much like taking a CAT scan by hand. All the internal organs are examined as they are revealed in the succeeding slices. The slices, once made, are examined under a dissecting microscope, any abnormalities or developmental variations are documented, and the sections can be saved if desired either in seperate scintillation vials or tissue cassettes.

Following the fetal gross examination fetuses are eviscerated. Rabbit fetuses are skinned. Fetuses of all species are skinned if double staining is to be performed. The eviscerated and skinned fetuses are placed in 95% alcohol for a period of approximately two weeks. When the fetuses have been fixed they are removed from the alcohol and mascerated in potassium hydroxide, then placed in a solution of alizarin red stain. Following staining the fetuses are cleared in a dilute solution of glycerine prior to reading.

The ossified structures expected to be present in a specimen include:
Skull;
right and left nasals, right and left frontals, right and left parietals, right and left premaxillae, right and left maxillae, right and left squamosals, right and left zygomatics (called the jugals in some other laboratories), supraoccipital, right and left exoccipitals, basioccipital, hyoid, right and left alae (in rabbits only- not ossified in rats, called the hyoid arches in some other laboratories), pterygoid process (of the basisphenoid), right and left mandibles.
Sternum;
manubrium, sternebrae (commonly four in number), and xyphiod. (In some laboratories these are simply numbered as sternebrae 1-6)
Vertebral column;
cervical vertebrae (7), thoracic vertebrae (12-13 in rabbits, 13-14 in rats), lumbar vertebrae (6-7 in rabbits, 5-6 in rats), sacral vertebrae (4 in adults but frequently only 3 are noted to lie within the limits of the ilia in fetuses), caudal vertebrae (variable in number. Few are present in rat fetuses but more are present in rabbit fetuses)
At some laboratories it is expected that the common number of combined thoracic and lumbar vertebrae is 19 while at other laboratories this is noted by expecting the common number of presacral vertebrae to be 26.
Each vertebra is expected to be made of three parts, one vertebral centrum on the ventral aspect and two vertebral arches on the dorsal aspect.
Pectoral bones;
right and left clavicles and right and left scapulae.
Forelimb bones (listed for one forelimb, noted as right or left);
humerus, radius, ulna, carpals (usually not ossified in rats), metacarpals (5 per forepaw), phalanges (1,1,1,1,1 in rats; 2,3,3,3,3 in rabbits)
Pelvic bones;
right and left ilia, right and left ischia, right and left pubes.
Hindlimb bones (listed fore hindlimb, noted as right or left);
femur, patella, tibia, fibula (in a rabbit the tibia and fibula are fused into one bone noted in some other laboratories as the tibiofibula), tarsals (usually not ossified in rats; usually two present in rabbits, the talus and the calcaneous), metatarsals (5 per hindpaw in rats, 4 per hindpaw in rabbits), phalanges (1,1,1,1,1 in rats; 3,3,3,3 in rabbits)

Ossified structures which show retarded development or incomplete ossification, or which show malformations, are noted. Common findings are seventh cervical ribs, bifid vertebral centra, wavy ribs, fused or forked ribs, fused or absent vertebrae, bent limb bones, bent scapulae, malpositioned pelvic bones, accessory skull bones found in the suture lines, etc.