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TeratogonesisTeratogonesis- Overview

Teratogenecity refers to the capacity of a drug to cause foetal abnormalities when administered to the pregnant mother. Type of malformation depends on the drug as well as the stage of exposure to the teratogen. Teratology is the science dealing with the causes, mechanisms, and manifestations of developmental deviations of either structural or functional nature. A teratogen is an agent that can cause a defect or malformation in the development of the embryo or fetus.
Teratogens act in specific ways (e.g., cytotoxicity, mutation chromosome damage, changing enzyme activity, altering patterns of apoptosis, etc.), often in specific tissues, so they typically produce characteristic congenital anomalies.



Factors affecting the ability of a teratogen to affect a developing conceptus includenature of the agent itself, dose, the route, timing, duration of maternal exposure, the rate of placental transfer, systemic absorption, maternal and embryonic/fetal genotypes involved

Principles of Teratology
Susceptibility to teratogenesis depends on the genotype of the conceptus and the manner in which this interacts with environmental factors. Susceptibility to teratogens varies with the developmental stage at the time of exposure. Teratogenic agents act in specific ways on developing cells and tissues to initiate abnormal developmental processes. The final manifestations of abnormal development are death, malformation, growth retardation and functional disorder. The access of adverse environmental influences to developing tissues depends on the nature of the influence. Manifestations of deviant development increase in frequency and in degree as dosage increases from no effect to the 100% lethal (LD100) level.


Developmental Defects Prenatal Death (miscarriage and stillbirth) Intrauterine Growth Retardation Congenital Anomalies (Birth Defects)  Structural Defects  Macroscopic structural defects y y y Malformations Disruptions Deformations  Microscopic structural defects (dysplasias) Functional Defects     Inborn errors of metabolism Physiological disturbances Mental retardation Cellular and molecular abnormalities 4 .

insertions and rearrangements)  5 . but the majority have an unknown etiology (idiopathic conditions) or are caused by a combination of genetic and environmental factors. which are then considered risk factors that increase the liability of malformation.Causes of Malformation Some malformations have recognized genetic or teratogenic causes. Genetic Disorders Internal factors are believed to account for about 15% Single-gene defects  Chromosomal aberrations  Aneuploidy (numerical abnormalities)  Structural abnormalities (deletions.

and vitamins Infectious agents Physical agents Maternal conditions. hormones.Environmental Factors External factors (teratogens) may account for about 10%      Chemical drugs.Are presumed to account for about 75% 6 . including nutritional deficiencies and metabolic disorders Multifactorial and Idiopathic Disorders .

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growth retardation. foetal death ‡ Discloured & deformed teeth. other abnormalities ‡ Spina bifida & other neural tube defects ‡ Low IQ baby. cardiac & other abnormalities ‡ Premature closure of ductus aeteriosus. foetal loss ‡ Foetal goiter. foetal alcohol syndrome ‡ Hypoplasia of organs. Bleeding inside the skull. 8 ‡ Isotretinoin . multiple defects r lit ‡ Cleft palate. heart & CNS defects. retarded bone growth. microcephaly ‡ Various malformations ‡ Neural tube defects. eye and hand defects.H U M A N T E R A T O G E N I C D R U G S Drug ‡ Thalidomide ‡ Anticancer drugs (MTX) ‡ Tertracyclines ‡ Warfarin ‡ Phenytoin ‡ Phenobarbitone ‡ Carbamazepine ‡ Valproate sodium ‡ Alcohol ‡ ACE Inhibitors ‡ Lithium ‡ Indomethacin/Aspirin A ‡ Phocomelia. growrth retardation ‡ Hypoplastic phalanges. hydrocephalus. clept lip/palate . multiple defects. ‡ Depressed nose. growth retardation. Retarded fetal growth ‡ Craniofacial.

both isomers will later be found in the serum ² therefore. and nervous system was also seen. genitals. The (R) enantiomer is effective against morning sickness but the (S) form is teratogenic. Radial aplasia ² absence of the thumb and the adjoining bone in the lower arm. digestive tract (including lips and mouth). y Phocomelia . Similar limb malformations occurred in the lower extremities. when the limb buds of the fetus are formed. produced children with a wide range of deformities.T H A L I D O M I D E TERATOGENIC MECHANISM Thalidomide is racemic. administering only one enantiomer will not prevent the teratogenic effect. kidneys.absence of most of the arm with the hands extending flipper-like from the shoulders. y y y 9 . EFFECTS ON FETUS Mothers who had taken the drug during the first trimester. Malformations of the eyes and ears. heart. if a human is given pure (R)-thalidomide or (S)-thalidomide. The enantiomers can interconvert in vivo ² that is.

A L C O H O L y y Alcohol is a teratogen in that exposure to the fetus during pregnancy can result in physical malformations of the face and head. Exposure to excessive amounts of alcohol can even cause embryonic death. Fetal alcohol syndrome (F S) represents a preventable pattern of clinical abnormalities that develop during embryogenesis due to exposure to alcohol during pregnancy. growth deficiency and mental retardation. MECHANISMS OF ACTIONS INCLUDE altered neural crest cell migration/increased neural crest cell death or general cell death by superoxide radial lysis of cells Inhibition of growth factors regulating cell proliferation and survival altered developmental regulation of gene expression. 10 .

and continued long enough. In Britain. dose and duration of exposure of a substance could be important in determining its teratogenic effect. In a three dose-level test. the recommended minimum numbers are 20 pregnant female rodents and 10 pregnant female non-rodents. three dose-levels are usually used in a teratogenic screen. it is recommended that at least 20 pregnant female rodents and 8 pregnant female non-rodents be used per group. route. The number of animals used should be large enough to satisfy statistical requirements. In Britain. Commonly used species are the rat or mouse. It has now been well established that the time. 11 . and the rabbit. Drugs should be given to test animals by the same route as they are to be administered clinically. In the United States. It is commonly recommended that drugs should be examined for teratogenic activity in two species of animals. the low dose should produce a clinically relevant effect and the third dose should be intermediate between the other two.TER TOGENIC TESTING Teratogenic testing has only come into being since the thalidomide tragedy of 1961. the high dose should be toxic but not lethal to the mother. Drug treatment should be started early enough. to cover the period of organ formation in the species used for the test.

the dose range is selected so that the highest dose causes some signs of maternal toxicity. and the outcomes compared to control untreated animals. Typically. COMMONLY USED SPECIES OF NIM LS: mouse. rat. Safety testing regulations generally require testing on two species. one of which must be a non-rodent. The usual sample size is 20 pregnant females per dose. monkey and also chick embryo. and at least one intermediate dose. but occasionally dermal or via inhalation) is given to pregnant animals during the period of embryonic organogenesis.Predicting Human Teratogens: problems with animal-based animaltesting and evaluation methods Animal-based studies of developmental toxicology provide the initial guidelines on whether a drug or chemical may present a teratogenic risk during pregnancy. 12 . hamster. the lowest causes no discernable effect in the mother or foetus. rabbit. a range of doses administered via the most appropriate route (usually oral. Most teratogenic studies are noted in lab animals under manipulated and well controlled experimental conditions and the mechanism of toxicity on animals may shed some light on possible toxic effects in relation to the human embryo or foetus.

Gestation period is short (3 wks in rats/mouse) Multiple births are the rule (hence 1 treated female will give large amount of data). 13 . This can be overcome by use of rhesus monkey and other primate as their placental structure & function & the embryological development is similar to humans. 2) 3) 4) Disadvantages1) Rodents have different structure (and possibly function) of their placenta as compared to humans. so that drugs are exposed to maternal tissues & subjected to maternal metabolism before entering fetus. of animals is simple because of smaller size & low cost.Contd« Contd« Advantages1) Like humans these mammals have placenta. Housing & maintenance of a large no.

the teratogenic agents must be given during organogenesis.Experiments in Rodents: Isolate the virgin females and determine estrous cycle by vaginal smear. 14 . Gross examination will reveal malformation& microscopic examination will reveal other defects. dead. All implanation sited are examined and every productof conception is to be accounted for as resorbed. malformed. Arrange matings at time of ovulation. live or fully developed normal foetus. The ofring must be obtained by caeserean operation in order to get reliable data. After mating the females are isolated again and treated as desired with a teratogen and sacrificed just befor term. As the timing of teratogenic treatments is extremely critical it is essential to know when conception occurs.

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two permanent murine cell lines are used to assess teratogenic potential . ‡ the inhibition of D3 cell growth. then released from LIF and allowed to form embryo bodies that differentiate into cardiomyocytes.In Vitro Alternatives to Animal-Based Teratology AnimalThe Embryonic Stem Cell Test (EST) In the EST. The D3s are maintained in an undifferentiated stage in the presence of leukaemia inhibiting factor (LIF). and 3T3 fibroblast cells represent adult tissue. Embryonic stem (ES) cells known as D3 cells represent embryonic tissue. The cultured D3 and 3T3 cells are then exposed to a range of concentrations of the potentially embryotoxic substance. 17 . ‡ the inhibition of 3T3 cell growth. after 10 days of culture three endpoints are assayed: ‡ the inhibition of D3 cell differentiation.

Three values are derived: (i) the concentration of substance at which there is 50% inhibition of D3 differentiation (ID50). --weak embryotoxic.Contd«. 18 . --not embryotoxic. Contd«. (iii) the concentration at which there is 50% inhibition of 3T3 growth (IC50 3T3. And --strong embryotoxic. ‡ These values are then channelled through a number of equations in the ¶Improved Prediction Model· the results of which allow the classification of the substance under examination into one of three classes. (ii) the concentration at which there is 50% inhibition of D3 growth (IC50 D3).

differentiation. essentially reproducing cartilage histogenesis. cell-to-cell communication and interactions with the extracellular matrix. In practice. Used for the screening and identification of strong embryotoxic chemicals only. when cultured in small volumes at high density. though subsequent experiments produced more variable results. or strongly embryotoxic. form foci of differentiating chondrocytes within a background of non-differentiated cells Cultured embryonic rat midbrain cells forming foci of neurons were also examined.THe Micromass (MM) Test The MM test is based upon rat embryonic limb mesenchyme cells which. weakly. a fundamental step in skeletal morphogenesis. the end-point used in the MM test is a 50% inhibition of differentiation (ID50) as assessed by the staining of cartilage cells with an alcian blue dye a prediction model similar to that for the EST was then used to classify test substances as non-. The foci of differentiating chondrocytes exhibit cell proliferation. movement. 19 .

Metabolic activation systems may also be included. y y y y y y 20 . and ICNOEC for TMS (the maximum concentration that has no observable effect on the total morphological score (TMS). such as S9 or microsomal fractions from liver.Whole Embryo Culture (WEC) y Teratogen screening systems using whole mouse . embryonic growth and differentiation are assayed in addition to dysmorphogenesis. or co-culture with hepatocytes In terms of appropriate end-points and to correctly classify the embryotoxic potentials of test chemicals. adverse effects on yolk sac development. during which time the test substance is added. Two prediction models were developed (PM1 and PM2) PM1 involves two end-points. leaving the visceral yolk sac and ectoplacental cone intact. rat and rabbit embryos cultured for short periods during the phase from fertilization to the end of organogenesis have been described. The conceptus is then cultured in an appropriate high-serum medium for 24-48 hours. parietal yolk sac and Reichert·s membrane. These methods involve the dissection of embryos at the head-fold or early somite stage away from maternal tissue. IC50 for malformation (the concentration of test substance at which 50% of embryos are malformed).

its the Intraamniotic injection eliminates the problem of continuous exposure of the embryo because the test substance is readily distributed to the extraembryonic compartments. Therefore. The presence of the compound and its stability in the culture medium must be verified. One general problem with CHEST has been the inability to distinguish general toxicity from specific developmental effects.Other test includeincludeThe chick embryotoxicity screening test (CHEST).48). and its cellular distribution. In vivo target concentrations are dependent on maternal absorption of the compound. and death as well as dose-response and stage-response relationships and malformation spectra are easily determined. metabolism. and excretion. the tissues and cells in culture. Growth retardation. toxicokinetic and metabolism studies are of crucial importance for the design and interpretation of developmental toxicity studies with both in vitro and in vivo methods (47. along with an assessment of its transport to. 21 . malformation. The production of a direct effect on the developing organism depends on the concentration/ time relationship of the chemical and/or its active metabolite(s) in the target cells. and its placental transfer and distribution in the embryo. and uptake by. Toxicokinetic studies are also important in vitro. its metabolic activation.

Controlled studies in women fail to demonstrate a risk to the foetus . and the possibility of foetal harm appears remote. EXAMPLES      folic acid. EXAMPLES: ‡ ‡ ‡ ‡ ‡ Acetaminophen Aspartame Famotidine Prednisone Insulin 22 . vitamin B6. depending on their safety of use during pregnancy. simethicone terbutaline azithromycin Category B. or animalreproduction studies have shown an adverse effect (other than a decrease in fertility) that was not confirmed in controlled studies in women.The US FDA has a categorization of drugs. Category A.Either animal-reproduction studies have not demonstrated a foetal risk but there are no controlled studies in pregnant women.

EXAMPLES: ‡ ‡ ‡ ‡ ‡ ‡ Prochlorperzaine.g. if the drug is needed in a life-threatening situation or for a serious disease for which safer drugs cannot be used or are ineffective).Contd« Contd« Category C. but the benefits from use in pregnant women may be acceptable despite the risk (e. 23 ..There is positive evidence of human foetal risk. EXAMPLES: ‡ ‡ ‡ ‡ Lithium Phenytoin Alcohol Chemotherapy drugs to treat cancer.Either studies in animals have revealed adverse effects on the foetus (teratogenic or embryocidal or other) and there are no controlled studies in women. Sudafed Fluconazole Ciprofloxacin Diclofenac Rifampicin Category D. or studies in women and animals are not available. Drugs should be given only if the potential benefit justifies the potential risk to the fetus.

The drug is contraindicated in women who are or may become pregnant. or there is evidence of foetal risk based on human experience or both. and the risk of the use of the drug in pregnant women clearly outweighs any possible benefit.Contd« Contd« Category X. EXAMPLES: ‡ ‡ ‡ ‡ Accutane Diethylstilbestrol Thalidomide Tegison or Soriatane 24 .Studies in animals or human beings have demonstrated foetal abnormalities.

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but by comparing effective drug concentrations with human therapeutic concentrations they can be differentiated from true-positives. A similar comparison is not possible after in vivo testing because for most compounds there are major pharmacokinetic differences between humans and experimental animals. This problem can be overcome in the in vitro system by adding the metabolite directly at the desired concentration either with or without the parent compound. Adverse embryonic outcomes (malformations or embryotoxicity) are directly related to the serum concentration of the compound being tested and can be compared to the serum concentration in the human. y y y y 26 . whole embryo culture offers distinct advantages over in vivo teratogenicity testing. This restricts the range of malformations that can be induced and may render the testing system unsuitable for compounds that are likely to exert their major toxicological effect late in gestation. In this respect in vivo testing is severely deficient.y Whole embryo culture appears to be an excellent method to screen chemicals for teratogenic hazard. It is likely that these species differences are due to metabolic differences between species and it is possible that if the proximate teratogen/s of thalidomide were identified they would be teratogenic in rat embryo culture. The embryo culture testing system would also be expected to produce many false-positives. Compared to in vivo testing it is cheap and rapid and does not involve experimentation on live adult animals. There is only one major disadvantage to in vitro testing and that is the limited period of embryogenesis. Whole embryo culture remains a very powerful technique that should continue to contribute to the determination of the safety of drugs and other chemicals during pregnancy. Over 2000 chemicals have been reported to be teratogenic in experimental animals exposed in vivo In comparison only about 20 chemicals are known to cause birth defects in the human. In vivo testing is also limited by the possibility that metabolites that occur in the human do not occur in the test animal. This large number of in vivo false-positive cannot easily be distinguished from true-positives. Thalidomide remains an important index chemical because it is not teratogenic in rats or mice but is teratogenic in the rabbit and human.

IS THERE SPECIFICITY OF ACTION IN EXPERIMENTAL TERATOGENESIS? y Time-specificity has been well established in numerous experiments in which a teratogenic agent has been shown to cause different malformations when applied at different times in development. Such action can be localized. each agent acts by interfering with a particular metabolic process in a specific way in the differentiating and growing embryo. Recent experiments have indicated that. depending on the distribution within the embryo of the process concerned. many teratogenic agents seem to produce distinctive patterns of anomalies which differ qualitatively and quantitatively from those caused by other agents.These time-specific effects are related to definite stages or events in embryonic development which might be regarded as periods of special susceptibility. generalized or selectively distributed. irrespective of time. Excessive doses tend to obscure time-specificity by causing teratogenic effects at times other than during periods of special susceptibility. y y 27 . The association of a particular group of malformations with a particular agent may be termed agent-specificity.

size. On the other hand. those agents well accepted as human teratogens have been shown to be teratogenic in one or more laboratory species. developmental patterns. A number of factors relate to the inability to predict accurately and the impreciseness in extrapolating from one species to another. and variability in diet. no single species has clearly distinguished itself as being more advantageous in the detection of human teratogens over any other. primates offered a higher level of predicability than others. the mouse and rat produced the greatest number of concordant defects. it is seen that the predictive value of animal teratogenicity tests in extrapolating results in terms of human safety is imperfect. It seems likely that variations in metabolic pathways are a major cause of species differences 28 .Yet. Among the species used for testing. but they also were responsible for the most noncorcordant responses as well. it is concluded that safety decisions should be based on all reproductive and developmental toxicity data in light of the agent's known pharmacokinetic. to generally subteratogenic exposure levels. intercurrent disease processes. Regarding concordance of target malformations. placental transfer. the rat and mouse most successfully model the human reaction. with the exception of the coumarin anticoagulant drugs. Since no other species is clearly more predictive of the human response. etc. metabolic and toxicologic parameter The extrapolation of animal data to the human is the foundation of safety evaluation of chemicals and drugs prior to human exposure. and include genetic heterogeneity (affecting absorption. However.Among species less commonly used for testing. or to the lack of an appropriate means of identifying human teratogens. metabolism and excretion of a given chemical). it remains to be determined if the unresponsiveness of humans is due to lessened sensitivity. but the rabbit is less likely than other species to give a false positive finding.Species Sensitivities and Prediction of Tetratogenic Potential Many chemicals shown to be teratogenic in laboratory animals are not known to be teratogenic in humans.

6).While the laboratory rat has been the most frequently used rodent species. The rat and rabbit are less prone to stressinduced teratogenicity. the susceptibility of this species to putative teratogens has been variable. Mice have also been used frequently in teratology studies despite the marked variability of responses observed with different strains. and be economically housed and easily handled. thalidomide (5. the maternal-placental-embryonic relationship as characterized in mammals is essential. in a manner similar to that in human beings. In addition. the ideal animal model should be able to be easily bred. including transplacental crossing. the patterns of embryonic and fetal structural and metabolic development should parallel those in man. 29 . metabolic rates as well as the pathways of xenobiotic metabolism should be comparable to those of man.9). and y certain teratogens such as cortisone (4. produce large litters. Finally. trimethadione (10). Rabbits have been used routinely as the nonrodent species required by most regulatory agencies. Parent compounds and their respective intermediates should undergo distribution. Recently it has been suggested that hamsters & guinea pig may serve as an appropriate rodent species for teratogenicity testing. Also. have a short gestation. and lithium carbonate (11) have elicited a poor teratogenic response.

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and toxicologic data.The relevancy of the route of exposure and existence of a dose-response relationship are important for all species.y Assessment of the safety of drugs and other chemicals regarding teratogenic potential must take into account the following points. 31 . disposition. not just data on malformations. The greater the number of species with positive results. the greater the likelihood of an adverse effect in humans. including pharmacologic. Data from any species must be used in the context of the total data base for the agent. All reproductive and developmental data should be used to predict safety.