A seminar<br />On<br />TEST FOR TERATOGENICITY<br />Submitted to JNTU, Hyderabad,<br />In the partial fulfillment for the award of degree of Master of pharmacy, Department of pharmacology.<br />PRESENTED BY<br />M.SUMANGALI<br />(10T21S0109)<br /> Under the guidance of<br /> Ms.Meenu singh; M.Pharm.<br />Associate professor<br />CMR COLLEGE OF PHARMACY<br /> Kandlakoya<br />C.M.R COLLEGE OF PHARMACY<br />(Approved by AICTE & PCI)<br />(Affiliated to JNTU)<br />Kandlakoya, Medchal.<br />2296160212090<br />CERTIFICATE<br />This is to certify that this is a bonafied record of the seminar entitle ”TEST FOR TERATOGENICITY” presented by Sumangali.M(10T21S0109), during the academic year 2010-2011 for partial fulfillment in degree of Masters of Pharmacy of Jawaharlal Nehru Technological University, Hyderabad.<br /> <br />PRINCIPAL: GUIDE:<br />Dr. K. Rajeswar Dutt, Ms.Meenu singh<br />M. Pharm., MBA, Ph.D. M. Pharm., (Ph.D.)<br /> DECLARATION<br />I hereby declare that the seminar work entitled “TESTS FOR TERATOGENICITY” submitted to Jawaharlal Nehru Technological University, Hyderabad, is a record work of seminar done by me under the guidance ofMs.Meenu singh, Associate professor of CMR College of pharmacy.<br />Sumangali.M<br />Reg No.10T21S0109.<br />TERATOGENICITY<br /> The capability of producing fetal malformation<br />INTRODUCTION<br />Developmental toxicity is any morphological or functional alteration caused by chemical or physical insult that interferes with normal growth, homeostasis, development, differentiation, and/or behavior. <br />Teratology is a specialized area of embryology. It is the study of the etiology of abnormal development (the study of birth defects).<br />Teratogens therefore are xenobiotics and other factors that cause malformations in the developing conceptus.<br />BACKGROUND<br />In 1956, thalidomide was introduced by Chemie Grunenthal, a West German pharmaceutical company, as a sedative and was marketed under the name of Contergan. By 1958, it permeated the world market, and its use expanded from simply combating insomnia. Pregnant women frequently treated their nausea of pregnancy with thalidomide. Soon thalidomide's teratogenic effects became apparent, and it was subsequently withdrawn from the market in 1961. Fortunately, thalidomide had never been approved in the United States because of concerns about the development of peripheral neuropathies. Thalidomide resurfaced in 1965 when Sheskin, a dermatologist from Israel, made a fortuitous discovery while treating his leprosy patients with thalidomide. He was prescribing thalidomide for its sedative properties, but he noticed that those with erythema nodosum leprosum had resolution or improvement of their skin lesions within 2 days of beginning treatment. This discovery has led to continued interest and research into thalidomide's immunomodulatory activities.<br /> . <br /> <br /> <br /> TERATOGEN<br />A teratogen is an agent that can produce a permanent alteration of structure or function in an organism exposed during embyronic or fetal life<br />Teratogens therefore are xenobiotics and other factors that cause malformations in the developing conceptus. Examples of teratogens may include pharmaceutical compounds, substances of abuse, hormones found in contraceptive agents, cigarette components, and heavy metals, viral agents, altered metabolic states induced by stress, and nutrient deficiencies (e.g., folic acid deficiency).<br /> Basic Characteristics of teratogen<br />A given teratogen may be organ specific.<br /> It may be species specific.<br /> It can be dose specific. <br /> <br /> <br /> TERATOLOGY<br />It is the study of abnormalities of physiological development. It is often thought of as the study of human birth defects, but it is much broader than that, taking in other non-birth developmental stages, including puberty; and other non-human life forms, including plants.<br /> Principles Of Teratology<br /> James Wilson (in 1959) proposed six principles of teratology. A simplified version of<br /> this is as follows:<br />1. Susceptibility to teratogenesis depends on the embryo’s genotype that interacts with adverse environmental factors (G × E interaction).<br />2. The developmental stage of exposure to the conceptus determines the outcome.<br />3. Teratogenic agents have specific mechanisms through which they exert there<br />pathogenic effects.<br />4. The nature of the teratogenic compound or factor determines its access to the<br />developing conceptus/tissue.<br /> 5. The four major categories of manifestations of altered development are death,malformation, growth retardation, and functional deficits. <br /> 6. The manifestations of the altered development increase with increasing dose (i.e.,no effect to lethality).<br /> <br /> CRITICAL PERIODS<br />Major fetal outcomes depend on the stage of pregnancy affected, as there are critical periods for the development of fetal processes and organs. Although embryogenesis is complex involving cell migrations, proliferation, differentiation, and organogenesis, one may divide the developmental stages in to three large categories: pre-implantation, implantation to organogenesis, and the fetal to neonatal stage. The outcomes associated with exposure during these periods vary. This is not to say there are exceptions based on the type of exposure. However, the primary outcomes are as follows:<br />STAGE OF EXPOSURE OUTCOME(S)<br />Pre-implantation Embryonic lethality<br />Implantation to time of Morphological defects<br />organogenesis <br /> <br />Fetal -> neonatal stage Functional disorders, growth retardation,<br /> Carcinogenesis<br />The sensitivity of the embryo to the induction of morphological defects is increased during the period of organogenesis. This period is essentially the time of the origination and development of the organs. The critical period graph demonstrates this point and defines the embryonic and fetal periods.<br />. <br />HISTORICAL TERATOGENS:<br />
Thalidomide<br />Thalidomide is a sedative-hypnotic drug used in Europe from 1957 to 1961.<br />It was marketed for morning sickness, nausea, and insomnia.<br />Women who had taken the drug from gestation days (GD) 35 to 50 gave birth to offspring suffering from a spectrum of different malformations, mainly Amelia (absence of limbs) or phocomelia (severe shortening of limbs).<br />Other malformations included: absence of the auricles with deafness, defects of the muscles of the eye and face, and malformations of the heart, bowel, uterus, and the gallbladder.<br />The compound was withdrawn from the market in 1961 after about 10,000 cases had occurred.<br />Once abandoned because of devastating teratogenic effects, thalidomide has reemerged as an alternative treatment in many dermatologic diseases. In 1998, thalidomide became FDA approved for the acute treatment and suppression of the cutaneous manifestations of erythema nodosum leprosum (ENL). ENL is a systemic disorder that typically occurs after several years of antileprosy treatments, usually for lepromatous leprosy. Off-label uses for thalidomide include: aphthous stomatitis, Behçet disease, pyoderma gangrenosum, chronic discoid lupus erythematosus, systemic lupus erythematosus, lichen planus, prurigo nodularis and sarcoidosis. <br />Accutane (Isotetrinoin)<br />It is used to treate acne.<br />These painful and disfiguring forms of acne do not respond to other acne treatments.<br />Accutane is very effective, but its use is associated with a number of risks including birth defects.<br />Exposure of pregnant women can lead to birth defects such as facial malformations, heart defects, and mental retardation.<br />Diethylstilbestrol (DES)<br />From 1940 to 1970, DES was used to help maintain pregnancy.<br />In utero exposure to DES has been associated with abnormal development of the uterus. It has also been associated with certain types of tumors.<br />Women who were exposed in utero often developed vaginal neoplasia, vaginal adenosis and cervical erosion.<br />Effects were not seen in offspring until they reached puberty.<br />Clear cell carcinoma of the vagina is a type of adenocarcinoma found in young women who are exposed to diethylstilbestrol in utero.<br />The reproductive organ of males can also be affected subsequent to in utero exposure. The outcomes include hypotrophic testes, poor semen volume and quality.<br /> Alcohol<br />
Fetal Alcohol Syndrome. Fetal alcohol syndrome (FAS) is a pattern of mental and physical defects that develops in some offspring when exposed to alcohol in utero. The first trimester is the most susceptible period. Some babies with alcohol-related birth defects, such as lower birth weight and body size and neurological impairments, do not have all of the classic FAS symptoms. These outcomes are often referred to as fetal alcohol effects (FAE). Currently there is not total agreement among medical scientists concerning the precise differences between
FAE and FAS. In addition to growth retardation, the most common outcomes of fetal alcohol syndrome include psychomotor dysfunction and craniofacial anomalies.
The observed growth deficiencies are associated with an inability of the baby to catch up due to a slower than normal rate of development. Other infrequent outcomes include skeletal malformations such as deformed ribs and sternum, scoliosis, malformed digits, and microcephaly.<br /> Distinctive facial anomalies have been associated with a diagnosis of fetal alcohol syndrome: small eye openings, epicanthal folds, failure of eyes to move in the same direction, short upturned nose, flat or absent groove between nose and upper lip, and thin upper lip. Visceral deformities may also be present: heart defects, genital malformations, kidney, and urinary defects. <br /> ‘‘Non Chemical’’ Teratogens<br />Teratogens are not only xenobiotics. There may be other factors having the ability to cause malformations in the developing conceptus. Restraint stress in mice (12-hour restraint during early period of organogenesis) elicits axial skeletal defects (primarily supernumerary ribs). The Rubella virus (first reported in 1941, Austria) is associated with a number of fetal outcomes depending on the stage of development that the exposure occurs. Exposure during the first and second month of pregnancy was associated with heart and eye defects. Exposure during the third month was associated with hearing defects (and mental retardation in some cases<br /> <br /> <br /> TESTING PROTOCOLS<br />Formal testing guidelines were established after thalidomide disaster.<br /> In 1966 guidelines were established by the FDA: Guidelines for Reproduction Studies for Safety Evaluation of Drugs for Human Use.<br /> Since then (1994) new streamlined testing protocols have been developed with international acceptance. This newer approach, ICH relies on the investigator to determine the model to access reproductive/developmental toxicity. <br />Under the guidelines of FDA<br />Multigenerational studies<br />Single generational studies<br />Segment I:Evaluation of Fertility and Reproductive Performance<br />Segment II: Assessment of Developmental Toxicity<br />Segment II: Postnatal Evaluation <br /> <br />Under the guidelines of ICH<br />Fertility Assessment<br />Postnatal Evaluation and Pregnancy State Susceptibility<br />Assessment of Developmental Toxicity <br />MULTIGENERATIONAL STUDY<br />Rationale for the Test:<br />The multigeneration study was designed to detect adverse effects on the integrity and performance of the male and female reproductive systems, including gonadal function, estrous cycle, mating behavior, conception, gestation, parturition,lactation, and weaning, and on the growth and development of the offspring. The adverse effects assessed in a multigeneration study also include neonatalmortality, mortality, target organs in offspring, and functional deficits in offspring,including behavior, maturation (puberty), and reproduction.<br />Principle of the Test Method:<br />The test article is administered to the parental animals (F 0 ) prior to and during<br />mating, during gestation, parturition, and lactation, and through weaning of the F 1 offspring. The test article is administered to selected F 1 offspring from weaning through adulthood, during cohabitation and mating, and through gestation, parturition,and lactation until the F 2 generation is weaned (PND 21).<br />Animal Species and Strain Selection and Rationale<br /> <br />The rat is the commonly used species for multigeneration studies. It is generally desirable to use the same species and strain as in other toxicological studies. If another mammalian species is used, then justifi cation for its use should be provided in the protocol. Within and between studies, animals should be of comparable age,weight, and parity at the start of the study. In OECD 416, the weight variation at the commencement of study should be minimal and not exceed 20% of the mean weight of each sex.<br />Procurement and Acclimation of the Test Animals<br />The test animals should be young (4 – 8 weeks of age) upon arrival. The test animals should be from the same source and strain, and the females should be nulliparous, nonpregnant, and approximately the same age and weight. The test animals should be acclimated to the laboratory conditions at least 5 days prior to study start.Each animal should be assigned a unique identification number. This is done just prior to initiation of dosing for the F 0 generation and just after weaning for the F 1 generation<br /> <br />Number of Test Animals<br />The number of animals per group should be sufficient to yield not less than 20<br />pregnancies. Routinely, 25 male and 25 female rats per group are used. Each test group should contain a similar number of mating pairs.<br /> Selection of Dosage Groups, Controls, and Route of Administration:<br />Most multigeneration studies are designed with a vehicle control and at least three dosage groups, all of which should be concurrent and housed and handled identically.Males and females should be assigned by a randomization procedure that assures that mean body weights are comparable among all test groups. The test compound and vehicle should be administered by the route that most closely<br />approximates the pattern of expected human exposure. If the route of administration differs from the expected human exposure, a justification will be required in the protocol.<br /> Selection of Dosage Levels:<br />The dosage levels should be spaced to produce a gradation of toxic effects. Unless limited by physiochemical or biological properties, the highest dose should be chosen to produce reproductive and/or systemic toxicity. For the F 0 generation, the highest dose selected should not have caused greater than 10% mortality in previous toxicity studies. The intermediate dose should only produce minimal observable toxic effects. The low dose should not produce any evidence of either systemic or reproductive toxicity (NOAEL). Two - or fourfold intervals are <br />often used for spacing of the dosage levels. For dietary studies, the dosage interval should not exceed threefold (OECD 416). Metabolism and rmacokinetics of the test article should be available from previously conducted studies to demonstrate the adequacy of the dosing regimen.A concurrent control group should always be used. If a vehicle control group is used, then the dosing volume should be equal to the highest volume used. If the test article is administered in the diet and causes reduced dietary intake, then the use of a pair - fed control group may be necessary.<br /> Treatment Timing and Test Duration:<br />The F 0 animals are dosed daily beginning at 5 – 9 weeks of age. Daily dosing of them selected F 1 generation offspring commences at weaning. For both generations (F 0 and F 1 ) dosing should be continued for at least 10 weeks prior to mating. Daily dosing of the F 0 and F 1 animals should continue until termination.<br />Mating Procedures, Detection of Mating, and Housing of Mated Females:<br />A mating ratio of 1:1 from the same dosage group allows identification of both<br />parents of a litter. The mating pair is cohoused until evidence of copulation is<br />observed or either three estrous periods or 2 weeks have elapsed. Animals should be separated as soon as evidence of copulation is observed .Once they are pregnant, females should be housed individually to allow for monitoring of feed and, if necessary, water consumption. Feed and water should be provided ad libitum . Pregnant animals should be provided with nesting materials near the end of gestation.<br /> <br /> <br />F 0 GENERATION<br />F 0 Maternal Observations<br /> Maternal Body Weight, Feed Consumption, Behavior, and Clinical Signs Clinical signs and mortalities should be recorded at least once daily throughout the test period. Body weight values should be recorded on the fi rst day of dosing and at least weekly, but during gestation and lactation body weights are routinely collected at protocol - specifi ed intervals (e.g., GD 0, 7, 14, and 21, and LD 0, 4, 7, 14, and 21). Food consumption should be measured at least once weekly and at the same interval as the body weight collection through LD 14. Water consumption should be measured weekly if the test article is administered in the drinking water. Estrous cycle length and pattern should be determined from vaginal smears during a minimum of 3 weeks prior to mating and throughout cohabitation.<br /> F 0 Parturition Dams should be housed in litter boxes with bedding material no later than DG 20 (2 days prior to expected parturition, earlier if required by protocol).The dams are observed periodically for signs of parturition onset. Individual pup observations should not be made until delivery is completed. Dead pups should be removed from the nesting box to preclude cannibalization by the dam. <br />Assessment of the F 0 Dam at Necropsy<br /> Unscheduled Deaths Any mated females that appear moribund should be euthanized appropriately. Any mated females that are found dead should be necropsied to investigate the cause of death. Any evidence of pregnancy and of embryonic development should be evaluated. A vaginal smear should be taken and the stage of estrus determined. Scheduled Deaths All F 0 females Should be terminated when they are no longer needed for assessment of reproductive effects. Dams are routinely necropsied on LD 21. A vaginal smear should be taken and the stage of estrus determined. The dam is weighed and euthanized, her ovaries and uterus are examined, and she is subjected to a complete necropsy. <br />Inspection of F 0 Maternal Viscera<br /> <br />Unscheduled Deaths If necropsy occurs prior to parturition, then the gravid uterus should be removed and weighed. The uterus should be examined to determine if the animal was pregnant, and the relative numbers of live and dead conceptuses and their relative positions in the uterus should be recorded. If the uterus shows no signs of implantation, it should be treated with a reagent, such as 10%ammoniumsulfide, to reveal any early resorptions. The number of corpora lutea should be recorded for each ovary, as described earlier. The remaining maternal abdominal and thoracic organs should be inspected grossly for evidence of toxicity. The following organs should be weighed from all F 0 females: uterus with oviducts, ovaries, brain, pituitary, liver, kidneys, adrenal glands, spleen, and any known target organs.The vagina, uterus with oviducts, cervix, ovaries, pituitary, and adrenal glands should be removed, fi xed, and stored in appropriate medium for histopathological examination. If gross organ lesions are noted, the affected organs should be preserved for possible histopathological examination.<br /> Scheduled Deaths If necropsy occurs as scheduled, then the uterus should be removed and weighed. The number of corpora lutea should be recorded for each<br />ovary as described earlier. The remaining maternal abdominal and thoracic organs should be inspected grossly for evidence of toxicity. The organs for organ weight collection and possible histopathological examination are listed below. If gross organ lesions are noted, the affected organs should be preserved for possible histopathological examination.<br />Histopathology for F 0 Females<br /> The vagina, uterus with oviducts, cervix, ovaries, pituitary and adrenal glands, and any target organs and gross lesions should be fi xed and stored in a suitable medium for histopathological examination. Complete histological evaluation is performed on the listed organs from ten randomly selected control and high dose F 0 females. Organs demonstrating treatment - related changes should be examined from the remainder of the high dose and control animals, and for all F 0 females in the low and mid - dose groups. Additionally, reproductive organs of the low and mid - dose females suspected of reduced fertility (those that failed to mate, conceive, or deliver healthy offspring, or for which estrous cyclicity was affected) should be subjected to histopathological evaluation as described previously (Segment <br />I). Histopathological examination should detect qualitative depletion of the primordial follicle population; however, reproductive study guidelines require a quantitative evaluation of primordial follicles of the F 0 females <br />.F 0 Paternal Observations<br /> Paternal Body Weight, Feed Consumption, Behavior, and Clinical Signs Clinical signs and mortalities should be recorded at least once daily. Body weight values should be recorded on fi rst day of dosing and at least weekly thereafter. Food consumption should be measured at least once weekly at the same interval as the body weight collection. Water consumption should be measured weekly if the test article is administered in the drinking water.<br />Assessment of the F 0 Males at Necropsy <br />Unscheduled Deaths Any males that are found dead or appear moribund are subjected to a complete necropsy, and their visceral contents are examined to investigate the cause of death.<br />Scheduled Deaths Males are routinely euthanized and necropsied after the<br />outcome of mating is known. In the event of an equivocal mating result, males can be mated with untreated females to ascertain their fertility or infertility. The males treated as part of the study may also be used for evaluation of toxicity to the male reproductive system if dosing is continued beyond mating and sacrifi ce is delayed.<br />The male is weighed, euthanized, and subjected to a complete necropsy.<br />Inspection of F 0 Male Viscera <br />The testes, epididymides (paired and individually), seminal vesicles (with coagulating glands and their fl uids), prostate, brain, pituitary, liver, kidneys, adrenal glands, spleen, and any known target organs are removed and weighed. For all F 0 males, sperm from one testis and one epididymis should be <br />collected for enumeration of homogenization - resistant sperm and caudal epididymal sperm reserves, respectively. In addition, sperm from one cauda epididymis (or vas deferens) should be collected for evaluation of sperm motility and morphology. <br />The remaining abdominal and thoracic organs should be inspected grossly for evidence of toxicity. If gross organ lesions are noted, the affected organs should be preserved for possible histopathological examination.<br />Histopathology for F 0 Males <br />One testis (preserved in Bouin ’ s fi xative or a comparable preservative), one epididymis, seminal vesicles, prostate, coagulating, pituitary, and adrenal glands, and any target organs and gross lesions should be fi xed and stored in a suitable medium for histopathological examination. Complete histological evaluation is performed on the listed organs from ten randomly selected control and high dose F 0 males. Organs demonstrating treatment - related changes should be examined from the remainder of the high dose and control animals, and from all F 0 males in the low and mid - dose groups. Additionally, reproductive organs of the low and mid - dose males suspected of reduced fertility (those that failed to mate or impregnate or had abnormal sperm number, motility, or morphology) should be subjected to histopathological evaluation as described earlier (Segment I).<br />F 0 Sperm Evaluation<br /> The sperm quality parameters are sperm number, spermmotility, and sperm morphology. Sperm evaluation will be performed on all control and high dose group F 0 males unless treatment - related effects have been observed; in that case, the lower dosage groups should be evaluated. The sperm evaluation methodologies were described earlier (Section 12.3.10 ). A morphological evaluation of epididymal (or vas deferens) sperm sample should be performed. If sperm evaluation parameters have already been evaluated as part of a systemic toxicity study of at least 90 days ’ duration, the evaluations need not be repeated in this study.<br /> <br />F 1 GENERATION<br />F 1 Litter Evaluations<br /> The general appearance of the entire F 1 litter and aternal and pup nesting behavior is observed as described earlier for Segment III.<br />F 1 Pup Evaluations <br />The litter is evaluated as soon as possible after delivery to<br />establish the number and sex of pups, stillbirths, live births, and the presence of gross anomalies. Pups that are found dead should be examined for possible defects and cause of death. Pups that have been cannibalized to the extent that their gender cannot be determined are recorded as “ sex unable to be determined due to degree of cannibalization ”).<br /> Live pups should be counted, sexed, and weighed individually at birth, and on LD 4, 7, 14, and 21. If a pup cannot be found during the daily litter count, the pups are sorted by gender in order to determine the sex of the missing pup(s) by process of elimination. The missing pup is recorded as “ missing, presumed cannibalized. ” If the pups have been tattooed or otherwise identifi ed, the actual identifi cation number of the missing pup(s) should be recorded.<br /> The pup survival indices (e.g., Live Birth Index, Survival Index, and Sex Ratio) should be calculated as described previously<br />Standardization of F 1 Litter Sizes<br /> Animals should be allowed to litter normally and rear their offspring to weaning. Standardization of litter sizes at LD 4 is optional.<br />Weaning<br /> Weaning is usually performed on LD 21. At weaning, one male and one female offspring<br />per litter are selected for rearing to adulthood and mating to assess reproductive competence.<br />Test Article Administration <br />The test article is administered to selected F 1 offspring from weaning through adulthood, during cohabitation and mating, and<br />through gestation, parturition, and lactation until the F 2 generation is weaned.<br />F 1 Postweaning Developmental Landmarks<br /> Sexual maturation landmarks are required in multigeneration studies where the selected offspring are raised to adulthood. The OECD 416 guideline recommends that additional developmental parameters be evaluated for supplementary information. Other functional investigations are also recommended before and/or after weaning, such as motor activity, sensory function, and refl ex ontogeny. These observations should not be performed on the pups selected for mating. Described<br />F 1 Mating Procedure<br /> A mating ratio of 1 : 1 from the same dosage group but from different litters allows identifi cation of both parents of a litter. The mating pair is cohoused as described above. Pregnant animals should be provided with nesting materials near the end of gestation. In instances of poor reproductive performance in the controls or in the event of a treatment - related alteration in the litter size, the adults of that generation may be remated to produce an F 1b litter. The dams should not be remated until approximately 1 – 2 weeks following weaning of the F 1a litter.<br />F 1 Maternal Observations<br />Maternal Body Weight, Feed Consumption, Behavior, and Clinical Signs Maternal body weight, feed consumption, behavior, and clinical signs should be recorded at the same intervals as the F 0 maternal observations. Estrous cycle length and pattern should be determined from vaginal smears during a minimum of 3 weeks prior to mating and throughout cohabitation. Observations that have proved of value in other toxicity studies, as well as duration of pregnancy, parturition, and nursing behaviors, are recorded.<br />F 1 Parturition <br />Dams should be housed in litter boxes with bedding material no<br />later than DG 20 .The dams are observed periodically throughout the day for signs of parturition onset. As soon as the fi rst pup is found, parturition is considered initiated and should be observed. <br />Assessment of F 1 Weanlings <br />Not Selected for Continuation At the time of termination or death during the study, when the litter size permits, at least three pups per sex per litter should be examined macroscopically for structural abnormalities or gross lesions, paying special attention to the reproductive organs. Dead pups or pups that are terminated in a moribund condition should be examined for possible defects and cause of <br />death. The following organs should be weighed from one randomly selected pup per sex per litter: brain, spleen, and thymus. The following organs should be fixed and stored for histopathological examination: gross lesions and any target organs.<br />Assessment of the Parental F 1 Female at Necropsy <br />Same as assessment of the parental f 0 female at necropsy, as described above.F 1 Male Observations are also same.<br /> F 2 GENERATION<br />F 2 Litter and Pup Evaluations<br /> The general appearance of the entire F 2 litter and maternal and pup nesting behavior should be observed as described earlier for the F 1 pups. The litter is evaluated as soon as possible after delivery to establish the number and sex of pups, stillbirths, live births, and the presence of gross anomalies as described earlier for the F 1 pups. Live F 2 pups should be counted, sexed, and weighed individually at birth, and on LD 4, 7, 14, and 21 as described for F 1 pups. Pup survival indices should be calculated and reported as described earlier <br />Assessment of F 2 Pups at Weaning<br /> At weaning or death during the study, when the litter size permits, at least three pups per sex per litter should be examined macroscopically for any structural abnormalities or pathological changes, paying special attention to the reproductive organs. Dead pups or pups that are terminated in a moribund condition should be examined for possible defects and/or cause of death. The following organs should be weighed from one randomly selected pup per sex per litter: brain, spleen, and thymus. The following organs should be fixed and stored for histopathological examination: any gross lesions and targetorgans.<br />Analysis of Maternal and Litter/Pup Data<br />Values from control and test groups of animals should be compared statistically Historical control data should be used to enhance interpretation<br />of study results<br /> SINGLE-GENERATION STUDIES<br /> Single-generation studies are short-term studies conducted in three segments:<br /> Segment I:<br /> Evaluation of Fertility and Reproductive Performance.<br /> Male rodents are treated for 70 days (to expose for one spermatogenic cycle), and nonpregnant females for 14 days (to exposure for several estrous cycles). Treatment is continued in the females during mating, pregnancy, and lactation. Fifty percent of the females are killed and the fetuses are examined for presence of malformations. The other 50% are allowed to give birth. After weaning, these offspring are killed and necropsied.<br />Segment II: <br />Assessment of Developmental Toxicity. <br />This involves the treatment of pregnant females only during the period covering implantation through organogenesis (typically from gestational days 6 to 15 in mice with 18-day gestational periods). One<br />day prior to birth, the animals are killed and fetuses examined for viability, body weight, and presence of malformation.<br />Segment III:<br /> Postnatal Evaluation.<br /> Pregnant animals are treated from the last trimester of pregnancy until weaning. Evaluated are parturition process, late fetal development, neonatal survival, and growth as well as presence of any malformations.<br /> <br /> UNDER THE GUIDELINES OF ICH<br />International Conference of Harmonization (ICH) of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH)—US FDA, 1994<br />New streamlined testing protocols with international acceptance have been developed. Below is a description of these guidelines as it relates to similarity to a particular segment-type study<br />Fertility Assessment.<br /> This study duration is typically shorter than segment I studies. Males are exposed for four weeks before mating and females two weeks before mating. Male reproductive organs are carefully evaluated: organ weights, histological analysis and sperm count, and mobility evaluation. For the females, fertility, litter size, and viability of conceptus are evaluated.<br /> Postnatal Evaluation and Pregnancy State Susceptibility.<br /> This study protocol is similar to the segment III study. Maternal toxicity is evaluated by comparing the degree of toxicity of the nonpregnant female to that of the pregnant female. Postnatal viability and growth are also evaluated. Offspring are also evaluated to assess functional development (i.e., presence of behavioral and reproductive deficits).<br /> Assessment of Developmental Toxicity. <br />This is almost identical to the segment II study protocol. Pregnant animals are exposed from implantation through organogenesis. The parameters measured in the segment II study are similar. However, the study is usually conducted using at least two species. More specifically, at least one rodent and one nonrodent species.<br /> ALTERNATIVE TEST METHODS<br />A number of alternative test methods have been developed to reduce the number of whole animals used in studies and/or to obtain more rapid information concerning the potential of a compound to be a reproductive/developmental toxicant. Validation of many of the methods has been problematic, since they do not address the contribution of maternal factors or multiorgan contributions to outcomes. Some of these alternative methods include the use of cell or embryo culture. For example, the micromass culture involves the use of limb bud cells from rat embryos grown in micromass culture for five days. The processes of differentiation and cell proliferation are assessed. In the Chernoff/Kavlock Assay, pregnant rodents are exposed during organogenesis and allowed to deliver. Postnatal growth, viability, and gross morphology of litters are recorded (detailed skeletal evaluations are not performed). Other alternative tests involve the use of nontraditional test species such as Xenopus embryos (FETAX) and Hydra. Xenopus embryos are exposed for 96 hours and then evaluated for morphological defects, viability, and growth. The cells of Hydra aggregate to form artificial embryos. The dose response in these “embryos” is compared to that of the adult Hydra.<br /> CONCLUSION<br />Understanding the mechanisms of the induction of birth defects is key to determine<br />how to prevent these effects. Further, increasing the accuracy of experimental animal<br />extrapolation will aid in the interpretation of experimental data in order to more<br />accurately determine the risk of a given compound to elicit birth defects in humans<br />Please, give only good fertilizer… <br />REFERENCES<br />