Teratogenecity Edited

8/8/2019 Teratogenecity Edited

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Istst M.Pharm (Q.A)

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TeratogonesisTeratogonesis-- OverviewOverview

y 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.

y Teratology is the science dealing with the causes, mechanisms, and

manifestations of developmental deviations of either structural or

functional nature.

y A teratogen is an agent that can cause a defect or malformation inthe 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 include-

nature 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

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Principles of TeratologyPrinciples of Teratology

Susceptibility to teratogenesis depends on the genotype of the

conceptus and the manner in which this interacts with environmentalfactors.

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 indegree as dosage increases from no effect to the 100% lethal (LD100)

level.

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Causes of MalformationCauses of Malformation

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Some malformations have recognized genetic or teratogenic

causes, 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.

Genetic Disorders

Internal factors are believed to account for about 15%

Single-gene defects

Chromosomal aberrationsAneuploidy (numerical abnormalities)

Structural abnormalities (deletions, insertions and rearrangements)



Environmental FactorsEnvironmental Factors

External factors (teratogens) may account for about 10%

Chemical drugs, hormones, and vitamins

Infectious agents

Physical agents

Maternal conditions, including nutritional deficiencies and metabolic

disorders

Multifactorial and Idiopathic Disorders - Are presumed to accountfor about 75%

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Drug

Thalidomide

Anticancer drugs (MTX)

Tertracyclines

Warfarin

Phenytoin

Phenobarbitone

Carbamazepine

Valproate sodium

Alcohol

ACE Inhibitors

Lithium

Indomethacin/Aspirin

Isotretinoin

A r lit

Phocomelia, multiple defects

Cleft palate, hydrocephalus, multiple defects, foetal death

Discloured & deformed teeth, retarded bone growth.

Depressed nose, eye and hand defects, growrth retardation

Hypoplastic phalanges, clept lip/palate , microcephaly

Various malformations

Neural tube defects, other abnormalities

Spina bifida & other neural tube defects

Low IQ baby, growth retardation, foetal alcohol syndrome

Hypoplasia of organs, growth retardation, foetal loss

Foetal goiter, cardiac & other abnormalities

Premature closure of ductus aeteriosus, Bleeding inside the skull,Retarded fetal growth

Craniofacial, heart & CNS defects.8

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TERATOGENIC MECHANISM

Thalidomide is racemic. The (R) enantiomer is effective against morning sickness but the

(S) form is teratogenic.

The enantiomers can interconvert in vivo ² that is, if a human is given pure (R)-thalidomide

or (S)-thalidomide, both isomers will later be found in the serum ² therefore,administering only one enantiomer will not prevent the teratogenic effect.

EFFECTS ON FETUS

Mothers who had taken the drug during the first trimester, when the limb buds of the

fetus are formed, produced children with a wide range of deformities.

y Phocomelia - absence of most of the arm with the hands extending flipper-like from the

shoulders.

y Radial aplasia ² absence of the thumb and the adjoining bone in the lower arm.

y Similar limb malformations occurred in the lower extremities.

y Malformations of the eyes and ears, heart, genitals, kidneys, digestive tract (including lips

and mouth), and nervous system was also seen.

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AA

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y Alcohol is a teratogen in that exposure to the fetus during pregnancy

can result in physical malformations of the face and head, growth

deficiency and mental retardation.Exposure to excessive amounts of

alcohol can even cause embryonic death.y Fetal alcohol syndrome (F S) represents a preventable pattern of

clinical abnormalities that develop during embryogenesis due to

exposure to alcohol during pregnancy.

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MECHANISMS OF ACTIONS INCLUDE

altered neural crest cell migration/increasedneural crest cell death or general cell death bysuperoxide radial lysis of cells

Inhibition of growth factors regulating cell

proliferation and survival

altered developmental regulation of geneexpression.



TER TOGENICTESTINGTER TOGENICTESTING

Teratogenic testing has only come into being since the thalidomide tragedy of 1961. It has

now been well established that the time, route, dose and duration of exposure of a

substance could be important in determining its teratogenic effect.It is commonly recommended that drugs should be examined for teratogenic activity in two

species of animals. Commonly used species are the rat or mouse, and the rabbit.

In Britain, three dose-levels are usually used in a teratogenic screen. In

a three dose-level test, the high dose should be toxic but not lethal to the

mother, the low dose should produce a clinically relevant effect and the third

dose should be intermediate between the other two. Drugs should be given to test animals

by the same route as they are to be administered clinically.

Drug treatment should be started early enough, and continued long enough,

to cover the period of organ formation in the species used for the test.

The number of animals used should be large enough to satisfy statistical

requirements. In Britain, it is recommended that at least 20 pregnant female

rodents and 8 pregnant female non-rodents be used per group. In the United

States, the recommended minimum numbers are 20 pregnant female rodents

and 10 pregnant female non-rodents.

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Predicting Human Teratogens: problems with animalPredicting Human Teratogens: problems with animal--basedbased

testing and evaluation methodstesting and evaluation methods

Animal-based studies of developmental toxicology provide the initial guidelineson whether a drug or chemical may present a teratogenic risk during

pregnancy.

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.

Typically, a range of doses administered via the most appropriate route (usuallyoral, but occasionally dermal or via inhalation) is given to pregnant animals

during the period of embryonic organogenesis, and the outcomes compared to

control untreated animals.

COMMONLY USED SPECIES OF NIM LS: mouse, rat, rabbit, hamster,monkey and also chick embryo.

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.

the dose range is selected so that the highest dose causes some signs of

maternal toxicity, the lowest causes no discernable effect in the mother or

foetus, and at least one intermediate dose. 12



ContdContd««

Advantages-

1) Like humans these mammals have placenta, so that drugs are exposed to maternal

tissues & subjected to maternal metabolism before entering fetus.

2) Gestation period is short (3 wks in rats/mouse)

3) Multiple births are the rule (hence 1 treated female will give large amount of data).

4) Housing & maintenance of a large no. of animals is simple because of smaller size & low

cost.

Disadvantages-

1) Rodents have different structure (and possibly function) of their placenta as comparedto humans.

This can be overcome by use of rhesus monkey and other primate as their placental

structure & function & the embryological development is similar to humans.

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Isolate the virgin females and determine estrous

cycle by vaginal smear.

Arrange matings at time of ovulation.As thetiming of teratogenic treatments is extremelycritical it is essential to know when conceptionoccurs.

After mating the females are isolated again and treated asdesired with a teratogen and sacrificed just befor term. Theofring must be obtained by caeserean operation in order toget reliable data.

All implanation sited are examined and every productof

conception is to be accounted for as resorbed, dead, malformed,live or fully developed normal foetus.

Experiments in Rodents:

Gross examination will reveal malformation& microscopic examination

will reveal other defects. the teratogenic agents must be given during

organogenesis.



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In VitroIn Vitro Alternatives toAnimalAlternatives toAnimal--Based TeratologyBased Teratology

The Embryonic Stem Cell Test (EST)

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In the EST, two permanent murine cell lines are used to assess

teratogenic potential .

Embryonic stem (ES) cells known as D3 cells representembryonic tissue, and 3T3 fibroblast cells represent adulttissue.

The D3s are maintained in an undifferentiated stage in thepresence of leukaemia inhibiting factor (LIF), then releasedfrom LIF and allowed to form embryo bodies thatdifferentiate into cardiomyocytes.

The cultured D3 and 3T3 cells are then exposed to a rangeof concentrations of the potentially embryotoxic substance,

after 10 days of culture three endpoints are assayed:

the inhibition of D3 cell differentiation,

the inhibition of D3 cell growth,

the inhibition of 3T3 cell growth.



ContdContd«.«.

Three values are derived:

(i) the concentration of substance at which there is 50% inhibition of D3 differentiation

(ID50),

(ii) the concentration at which there is 50% inhibition of D3 growth (IC50 D3),

(iii) the concentration at which there is 50% inhibition of 3T3 growth (IC50 3T3.

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;

--not embryotoxic,

--w eak embryotoxic, And

--strong embryotoxic .

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Whole Embryo Culture (WEC)

y Teratogen screening systems using whole mouse , rat and rabbit embryoscultured for short periods during the phase from fertilization to the end of

organogenesis have been described.

y These methods involve the dissection of embryos at the head-fold or early

somite stage away from maternal tissue, parietal yolk sac and Reichert·smembrane, leaving the visceral yolk sac and ectoplacental cone intact.

y The conceptus is then cultured in an appropriate high-serum medium for 24-48

hours, during which time the test substance is added.

y Metabolic activation systems may also be included, such as S9 or microsomalfractions from liver, or co-culture with hepatocytes

y In terms of appropriate end-points and to correctly classify the embryotoxicpotentials of test chemicals, adverse effects on yolk sac development, embryonic

growth and differentiation are assayed in addition to dysmorphogenesis.

y Two prediction models were developed (PM1 and PM2)

y PM1 involves two end-points; IC50 for malformation (the concentration of test

substance at which 50% of embryos are malformed), and ICNOEC for TMS (the

maximum concentration that has no observable effect on the total

morphological score (TMS).

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Other test includeOther test include--

The chick embryotoxicity screening test (CHEST)- here the Intraamnioticinjection eliminates the problem of continuous exposure of the embryo because

the test substance is readily distributed to the extraembryonic compartments.

Growth retardation, malformation, and death as well as dose-response and

stage-response relationships and malformation spectra are easily determined.

One general problem with CHEST has been the inability to distinguish general

toxicity from specific developmental effects.

The production of a direct effect on the developing organism depends on theconcentration/ time relationship of the chemical and/or its active

metabolite(s) in the target cells. Therefore, 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 (47,48).In vivo target concentrations are dependent on maternal

absorption of the compound, its distribution, metabolism, and excretion, and its

placental transfer and distribution in the embryo.

Toxicokinetic studies are also important in vitro. The presence of the compound

and its stability in the culture medium must be verified, along with an

assessment of its transport to, and uptake by, the tissues and cells

in culture, its metabolic activation, and its cellular distribution. 21



The US FDA has a categorization of drugs, depending on their safety of The US FDA has a categorization of drugs, depending on their safety of

use during pregnancy.use during pregnancy.

CategoryA- Controlled studies in women fail to demonstrate a risk to the

foetus , and the possibility of foetal harm appears remote.

EXAMPLES

folic acid,

vitamin B6,

simethicone

terbutaline

azithromycin

Category B- Either animal-reproduction studies have not demonstrated a foetal

risk but there are no controlled studies in pregnant women, or animal-

reproduction studies have shown an adverse effect (other than a decrease in

fertility) that was not confirmed in controlled studies in women.

EXAMPLES: Acetaminophen

Aspartame

Famotidine

Prednisone

Insulin 22



ContdContd««

Category C- Either studies in animals have revealed adverse effects on thefoetus (teratogenic or embryocidal or other) and there are no controlled studies

in women, 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.

EXAMPLES:

Prochlorperzaine,

Sudafed

Fluconazole

Ciprofloxacin

Diclofenac

Rifampicin

CategoryD- There is positive evidence of human foetal risk, but the benefits

from use in pregnant women may be acceptable despite the risk (e.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).

EXAMPLES:

Lithium Phenytoin

Alcohol

Chemotherapy drugs to treat cancer. 23



ContdContd««

Category X- Studies in animals or human beings have demonstrated foetalabnormalities, 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. The drug is contraindicated in women who are or may

become pregnant.

EXAMPLES:

Accutane

Diethylstilbestrol

Thalidomide

Tegison or Soriatane

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y Whole embryo culture appears to be an excellent method to screen chemicals for teratogenic

hazard. Compared to in vivo testing it is cheap and rapid and does not involve experimentation on

live adult animals.

y whole embryo culture offers distinct advantages over in vivo teratogenicity testing.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 inthe human.A similar comparison is not possible after in vivo testing because for most compounds

there are major pharmacokinetic differences between humans and experimental animals. In vivo

testing is also limited by the possibility that metabolites that occur in the human do not occur in the

test animal. 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. There is only one major

disadvantage to in vitro testing and that is the limited period of embryogenesis. 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.

y 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. This large

number of in vivo false-positive cannot easily be distinguished from true-positives. In this respect in

vivo testing is severely deficient. The embryo culture testing system would also be expected to

produce many false-positives; but by comparing effective drug concentrations with human

therapeutic concentrations they can be differentiated from true-positives.

y Thalidomide remains an important index chemical because it is not teratogenic in rats or mice butis teratogenic in the rabbit and human. 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.

y 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.

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IS THERE SPECIFICITY OFACTION IN EXPERIMENTALIS THERE SPECIFICITY OFACTION IN EXPERIMENTAL

TERATOGENESIS?TERATOGENESIS?

y T ime-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.These time-specific effects arerelated to definite stages or events in embryonic development which might

be regarded as periods of special susceptibility. Excessive doses tend to

obscure time-specificityby causing teratogenic effects at times other than

during periodsof special susceptibility.

y Recent experiments have indicated that, irrespective of time, many

teratogenic agents seem to produce distinctive patterns of anomalies whichdiffer qualitatively and quantitatively from those caused by other agents. The

associationof a particular group of malformations with a particular agent maybe termed agent-specificity.

y each agent acts by interfering with a particular metabolic process in a specific

way in the differentiating and growing embryo. Such action can be localized,

generalized or selectively distributed, depending on the distribution within

the embryo of the process concerned.

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Species Sensitivities and Prediction of Species Sensitivities and Prediction of TetratogenicTetratogenic PotentialPotential

Many chemicals shown to be teratogenic in laboratory animals are not known to be teratogenic in

humans.However, it remains to be determined if the unresponsiveness of humans is due to lessened

sensitivity, to generally subteratogenic exposure levels, or to the lack of an appropriate means of identifying

human teratogens. On the other hand, with the exception of the coumarin anticoagulant drugs, those

agents well accepted as human teratogens have been shown to be teratogenic in one or more laboratory

species. Yet, no single species has clearly distinguished itself as being more advantageous in the detection

of human teratogens over any other.Among the species used for testing, the rat and mouse most successfully

model the human reaction, but the rabbit is less likely than other species to give a false positive

finding.Among species less commonly used for testing, primates offered a higher level of predicability

than others. Regarding concordance of target malformations, the mouse and rat produced the greatest

number of concordant defects, but they also were responsible for the most noncorcordant responses as

well. Since no other species is clearly more predictive of the human response, it is concluded that safety

decisions should be based on all reproductive and developmental toxicity data in light of the agent's known

pharmacokinetic, 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. it is seen that the predictive value

of animal teratogenicity tests in extrapolating results in terms of human safety isimperfect. A number of factors relate to the inability to predict accurately and the

impreciseness in extrapolating from one species to another, and include genetic

heterogeneity (affecting absorption, metabolism and excretion of a given chemical), and

variability in diet, size, developmental patterns, intercurrent disease processes,

placental transfer, etc. It seems likely that variations in metabolic pathways are a major

cause of species differences 28



While the laboratory rat has been the most frequently used rodent species, the

susceptibility of this species to putative teratogens has been variable, and

y certain teratogens such as cortisone (4,9), thalidomide (5,6), trimethadione (10), and

lithium carbonate (11) have elicited a poor teratogenic response. Mice have also

been used frequently in teratology studies despite the marked variability of

responses observed with different strains. The rat and rabbit are less prone to

stressinduced teratogenicity. Recently it has been suggested that hamsters & guinea

pig may serve as an appropriate rodent species for teratogenicity testing.

Rabbits have been used routinely as the nonrodent species required by mostregulatory agencies. the maternal-placental-embryonic relationship as characterized

in mammals is essential. In addition, metabolic rates as well as the pathways of

xenobiotic metabolism should be comparable to those of man. Parent compounds

and their respective intermediates should undergo distribution, including transplacental

crossing, in a manner similar to that in human beings.Also, the patterns of

embryonic and fetal structural and metabolic development should parallel those inman. Finally, the ideal animal model should be able to be easily bred, have

a short gestation, produce large litters, and be economically housed and easily handled.

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Species Selection by RegulatoryAgenciesSpecies Selection by RegulatoryAgencies

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y Assessment of the safety of drugs and other chemicals regarding teratogenic

potential must take into account the following points. The greater the numberof species with positive results, the greater the likelihood of an adverse effect

in humans.All reproductive and developmental data should be used to predict

safety, not just data on malformations. The relevancy of the route of exposure

and existence of a dose-response relationship are important for all species. Data

from any species must be used in the context of the total data base for

the agent, including pharmacologic, disposition, and toxicologic data.

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