teratogenicity studies

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Presented by NEGARISH ZIA
SubjecT : P’COLOGICAL &TOXICOLOGICAL
SCREENING
Department of pharmacy
Integral university lucknow
 Table of content

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 Introduction
 Initial consideration
 Principle of the test
 Preparation of the test
 Procedure
 Data and reporting
 Prenatal Developmental Toxicity Study
414
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Developmental toxicology
 The study of adverse effects on the developing organism that may result
from exposure prior to conception, during prenatal development, the time
of sexual maturation.
 The major manifestations of developmental toxicity include 1) death of the
organism, 2) structural abnormality, 3) altered growth, and 4) functional
deficiency. Developmental toxicology was formerly often referred to as
teratology
 In June 1995, an OECD Working Group on Reproduction and
Developmental Toxicity discussed the need to update existing OECD Test
Guidelines for reproduction and developmental toxicity and the
development of new Guidelines for endpoints not yet covered.
 INITIAL CONSIDERATIONS

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 This guideline for developmental toxicity testing
is designed to provide general information
concerning the effects of prenatal exposure on
the pregnant test animal and on the developing
organism.
 This may include assessment of maternal effects
as well as death, structural abnormalities, or
altered growth in the foetus.
 PRINCIPLE OF THE TEST

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 The test substance is administered to pregnant animals at least
from implantation to one day prior to the day of scheduled kill.
 The guideline is not intended to examine solely the period of
organogenesis, (e.g. days 5-15 in the rodent, and days 6-18 in
the rabbit) but also effects from pre- implantation, when
appropriate, through the entire period of gestation to the day
before caesarean section.
 Shortly before caesarean section, the females are killed, the
uterine contents are examined, and the foetuses are evaluated
for soft tissue and skeletal changes.
 PREPARATION FOR THE TEST
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Selection of animal species
 It is recommended that testing be performed in the most
relevant species, and that laboratory species and strains
which are commonly used in prenatal developmental
toxicity testing be employed.
 The preferred rodent species is the rat and the preferred
non-rodent species is the rabbit.
 Justification should be provided if another species is used.
 CONTD…

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Housing, and feeding conditions
 The temperature in the experimental animal room should be
22 (±3 °C) for rodents and 18 (±3 °C) rabbits.
 The relative humidity should be at least 30% and preferably
not exceed 70% .
 Lighting should be artificial, the sequence being 12 hours
light, 12 hours dark. For feeding, conventional laboratory diets
may be used with an unlimited supply of drinking water.
 Mating procedures should be carried out in cages suitable for
the purpose.
CONTD….

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Preparation of the animals
 Healthy animals, which have been acclimated to laboratory
conditions for at least 5 days and have not been subjected to
previous experimental procedures, should be used.
 The test animals should be characterized as to species, strain,
source, sex, weight or age.
 Young adult nulliparous female animals should be used at each
dose level.
 The females should be mated with males of the same species
and strain, and the mating of siblings should be avoided.
contd….

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 For rodents day 0 of gestation is the day on which a
vaginal plug and/or sperm are observed; for rabbits
day 0 is usually the day of coitus or of artificial
insemination, if this technique is used.
 Each animal should be assigned a unique
identification number.
 Mated females should be assigned in an unbiased
manner to the control and treatment groups, and if
the females are mated in batches, the animals in each
batch should be evenly distributed across the groups.
PROCEDURE

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Number and sex of animals
 Each test and control group should contain
approximately 20 female animals with implantation
sites at necropsy.
 Groups with fewer than 16 animals with implantation
sites may be inappropriate.
 Maternal mortality does not necessarily invalidate the
study providing it does not exceed approximately 10
percent.
CONTD…

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Preparation of doses
 Effects on the absorption, distribution, metabolism, and
retention or excretion of the test substance
 Effects on the chemical properties of the test substance
which may alter its toxic characteristics
 Effects on the food or water consumption or the
nutritional status of the animals.
 The vehicle should neither be developmentally toxic nor
have effects on reproduction.
CONTD…

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Dosage
 The test substance should be administered daily from implantation
(e.g., day 5 post mating) to the day prior to scheduled caesarean
section.
 It is well known that inappropriate handling or stress during
pregnancy can result in prenatal loss.
 At least three dose levels and a concurrent control should be used.
 Unless limited by the physical/chemical nature or biological
properties of the test substance, the highest dose should be chosen
with the aim to induce some developmental and/or maternal toxicity
but not death or severe suffering.
 contD…

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 At least one intermediate dose level should produce minimal
observable toxic effects.
 The lowest dose level should not produce any evidence of
either maternal or developmental toxicity.
 Two- to four-fold intervals are frequently optimal for setting the
descending dose levels, and the addition of a fourth test group is
often preferable to using very large intervals between dosages.
 Although establishment of a maternal NOAEL is the goal,
studies which do not establish such a level may also be
acceptable .
 
Limit test
 If a test at one dose level of at least 1000 mg/kg body
weight/day by oral administration
Administration of doses
 The test substance or vehicle is usually administered orally by
intubation. If another route of administration is used, the tester
should provide justification and reasoning for its selection.
 Caution should be exercised when adjusting the dose during
the last trimester of pregnancy.
contD…

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 Accurate dose are selected to prevent excess maternal toxicity.
 If excess toxicity is noted in the treated dams, those animals
should be humanely killed.
 If several pregnant animals show signs of excess toxicity,
consideration should be given to terminating that dose group.
 When the substance is administered by gavages, this should
preferably be given as a single dose to the animals using a
stomach tube or a suitable intubation canula.
 The maximum volume of liquid that can be administered at
one time depends on the size of the test animal.
CONTD…

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Observations of the dams
 The condition of the animals should be recorded including mortality,
morbidity, pertinent behavioral changes, and all signs of overt toxicity.
Post-mortem examination
 Females should be killed one day prior to the expected day of delivery.
 Females showing signs of abortion or premature delivery prior to
scheduled kill should be killed and subjected to a thorough macroscopic
examination.
 At the time of termination or death during the study, the dam should be
examined macroscopically for any structural abnormalities or pathological
changes.
CONTD…

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Examination of uterine contents
 Immediately after termination or as soon as possible after death, the
uteri should be removed and the pregnancy status of the animals
ascertained.
 Uteri that appear non-gravid should be further examined to confirm
the non-pregnant status
 Gravid uteri including the cervix should be weighed.
 The number of corpora lutea should be determined for pregnant
animals.
 The uterine contents should be examined for numbers of embryonic or
foetal deaths and viable foetuses.
CONTD…

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Examination of foetuses
 The sex and body weight of each fetus should be determined.
 Each foetus should be examined for external alterations.
 Foetuses should be examined for skeletal and soft tissue
alterations (e.g. variations and malformations ).
 For rodents, approximately one-half of each litter should be
prepared and examined for skeletal alterations.
 The remainder should be prepared and examined for soft
tissue alterations, using accepted or appropriate serial
sectioning methods or careful gross dissection techniques.
 cont.. .

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 For non-rodents, e.g. rabbits, all foetuses should be
examined for both soft tissue and skeletal alterations.
 The heads of one-half of the foetuses examined in
this manner should be removed and processed for
evaluation of soft tissue alterations (including eyes,
brain, nasal passages and tongue), using standard
serial sectioning methods.
 DATA AND REPORTING

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Data
 Number of animals at the start of the test
 The number of animals found dead during the test or killed for
humane reasons,
 The time of any death or humane kill,
 The number of pregnant females.
 The number of animals showing signs of toxicity,
 Signs of toxicity observed, including time of onset, duration, and
severity of any toxic effects,
 The types of foetal observations, and all relevant litter data.
CONTD…

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Evaluation of Results
 The evaluation will include the following information:
 Maternal and fetal test results, including an evaluation of the
relationship thereof, between the exposure of the animals to the test
substance and the incidence and severity of all findings.
 Criteria used for categorizing foetal external, soft tissue, and skeletal
alterations if categorization has been done.
 Adequate statistical analysis of the study findings, when appropriate,
which should include sufficient information on the method of
analysis, so that an independent reviewer can re-evaluate and
reconstruct the analysis.
CONTD…

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Interpretation of Results
 A prenatal developmental toxicity study will provide information
on the effects of repeated oral exposure to a substance during
pregnancy.
 The results of the study should be interpreted in conjunction with
the findings of sub chronic, reproduction, toxicokinetic and other
studies.
 Since emphasis is placed on both general toxicity and
developmental toxicity endpoints, the results of the study will
allow for the discrimination between developmental effects
occurring in the absence of general toxicity and those which are
only expressed at levels that are also toxic to the maternal animal.
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