Pre-clinical toxicological evaluation of potent substances should have complied with OECD Guidelines. This PDF file includes general considerations for toxicity evaluation, animal selection criteria and further biochemical parameters, and additional tests specified for the intended use of the drug under investigation.

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Toxicity evaluation of drugs in
animal models- OECD Guidelines
Prepared By: Vaidehi Vadhvana,Ph.D.Scholar,ITRA Jamnagar
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Introduction:
Herbal drugs have made a great contribution to maintaining human health. The
majority of the world’s population in developing countries still relies on herbal
drugs. WHO supports the use of herbal drugs and encourages remedies that have
been proven to be safe and effective. A few herbal drugs have withstood scientific
testing, but others are used simply for traditional reasons to protect, restore or
improve health. No evidence to answer the question of safety and efficacy about
most of the herbal drugs now in use. It still needs to be validated scientifically.
Toxicity evaluation of herbal drugs to document toxicological profile and boost the
export of herbal drugs manufacturing in India.
Drug (D & C Act 1940):
All medicines for internal or external use of human beings or animals and all
substances intended to be used for or in the diagnosis, treatment, mitigation or
prevention of any disease or disorder in human beings or animals, including
preparations applied on the human body for the purpose of repelling insects like
mosquitoes. • The objective of toxicity testing in the laboratory is to elucidate the
toxic properties of drugs.

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OECD Guidelines:
The OECD Guidelines are a unique tool for assessing the potential effects of
chemicals on human health and the environment.
Accepted internationally as standard methods for safety testing.
Used by professionals in industry, academia and government involved in the testing
and assessment of chemicals (industrial chemicals, pesticides, personal care
products, etc.).
These Guidelines are regularly updated with the assistance of thousands of
national experts from OECD member countries.
Covered by the Mutual Acceptance of Data, implying that data generated in the
testing of chemicals in an OECD member country, or a partner country having
adhered to the Decision, in accordance with OECD Test Guidelines and Principles of
Good Laboratory Practice (GLP), be accepted in other OECD countries and partner
counties having adhered to the Decision, for the purposes of assessment and other
uses relating to the protection of human health and the environment.
The Globally Harmonized System (GHS),defines TOXICITY as "those adverse effects
occurring following oral or dermal administration of a single dose of a substance, or
multiple doses given within 24 hours, or an inhalation exposure of 4 hours". The
preferred species for oral and inhalation testing is the rat, and for dermal testing,
the rat or rabbit. Oral administration is the most common form of acute systemic
toxicity testing.
Pre-Clinical Studies:
OBJECTIVES:
Ø To determine whether such studies support clinical use of a herbal drug.
Ø To characterize the range of pharmacological action of herbal drug.
Ø To evaluate the pharmacologically active natural products and to find their
mechanism or actions.

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Ø Toxicological study:
1. Assess the safety of herbal drugs
2. Define the possible toxicity from short-term and long term use.
CPCSEA: Committee for the Purpose of Control and Supervision on
Experiments on Animals.
IAEC: Institutional Animal Ethics Committee
Members of IAEC:
1. One biological scientist
2. Two scientists from different biological discipline
3. Veterinarian
4. Scientist in-charge of animal facility
5. Scientist from outside the institute
6. Sociologist and CPCSEA Nominee
Toxicity study:
Paracelsus (1493-1541) stated that “All substances are poisons; The right dose
differentiates a poison and remedy” this concept is the fundamental principle of
toxicology. WHO- guidelines have given the important criteria to establish the safety
profile of the drugs. Toxicological study results play an important safety assessment
for Herbal Drugs, Pharmaceuticals, food additives , pesticides and other chemicals.
It is essential for any compounds having biological activity.
Important OECD guidelines:
420 Acute oral Toxicity- fixed dose method
423 Acute oral Toxicity –acute toxic class method

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425 Acute oral Toxicity-Up and Down method
402 Acute dermal Toxicity
404 Acute dermal Irritation/ Corrosion
403 Acute inhalation Toxicity
405 Acute Eye Irritation /Corrosion
406 Skin sensitization
407 28 days repeated oral Toxicity studies in rodents
408 90 days repeated oral Toxicity studies in rodents
409 90 days repeated oral Toxicity studies in non rodents
410 90 days repeated Dermal Toxicity
411 90 days inhalation Toxicity study
412 28/14 days repeated dose inhalation Toxicity study
413 90 days repeated dose inhalation Toxicity study
414 Prenatal Developmental Toxicity study
421 Reproduction /Development toxicity screening test

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422 Combined Repeated dose toxicity study with
Reproduction/Developmental Toxicity screening test
422 Neurotoxicity study in rodents
451 Carcinogenicity studies
452 Chronic Toxicity studies
453 Combined chronic toxicity/carcinogenic studies
OECD 420: Acute Oral Toxicity –Fixed dose procedure
Principle:
Groups of animals of a single sex are dosed in a stepwise procedure using the fixed
doses of 5, 50, 300 and 2000 mg/kg (exceptionally an additional fixed dose of 5000
mg/kg may be considered). The initial dose level is selected on the basis of a
sighting study as the dose is expected to produce some signs of toxicity without
causing severe toxic effects or mortality. The preferred rodent species is the rat,
although other rodent species may be used. Normally females are used. This is
because literature surveys of conventional LD50 tests show that usually there is
little difference in sensitivity between the sexes
The initial dose selected from the sighting study is the dose expected to produce
some sign of toxicity without causing severe toxic effects. Further groups of animals
may be dose at higher or lower fixed dose depending on the presence or absence
of toxicity.
Limit tests perform at 2000 mg/kg bw, when information indicating test material is
likely to be nontoxic or toxicity above regulatory limit dose.
Animal: Rat of female sex preferable and 10-12 weeks old.
Number: Total of 5 animals used for each dose level.

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Dose: Single dose Fasting: For Rats: overnight fasting; For Mice: 3-4 hours fasting
Time interval between dosing: Period of 3 or 4 days between dosing at each dose
level is recommended.
Observations:
Animals are observed individually after dosing at least once during the first 30
minutes, periodically during the first 24 hours, with special attention given during
the first 4 hours, and daily thereafter, for a total of 14 days, except where they need
to be removed from the study and humanely killed for animal welfare reasons or
are found dead. However, the duration of observation should not be fixed rigidly.
OECD 423 (Acute toxic class method)
Principle:
It is based on a stepwise procedure with the use of a minimum number of animals
per step, sufficient information is obtained on the acute toxicity of the test
substance to enable its classification. The substance is administered orally to a
group of experimental animals at one of the defined doses. The substance is tested
using a stepwise procedure, each step using three animals of a single sex (normally
females). Absence or presence of compound-related mortality of the animals dosed
at one step will determine the next step. No further testing is needed, Dosing of
three additional animals, with the same dose Dosing of three additional animals at
the next higher or the next lower dose level.
Preparation of the animals:
Randomly select the animals and keep them in their cages for at least 5 days prior
to start of the test to allow for acclimatization of the laboratory conditions.
Preparation of dose:
If vehicles other than water than toxic characteristics of the vehicle should be
known (gum acacia, SCMC, tween 20 & 80, honey, vegetable oils, palm jaggery etc.)
Procedure:
3 animals in each step.

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Doses: Starting dose from- 5, 50, 300, 2000 mg/kg b/w. If no information is available
regarding lethality of drug than dosing start from 300 mg/kg, bw.
Administration of Doses:
Rodents: Volume not to exceed 1 ml/100 gm b.w. however for aqueous solution 2
ml/100g b.w. can be considered.
Dose: Single dose Fasting: For Rats: overnight fasting; For Mice: 3-4 hours fasting
but free access to drinking water. After administration of drugs, food may be
withdrawn for 3-4 hrs in rats and 2 hours in mice.
Limit test:
When information suggests that mortality is unlikely at the higher starting dose
level 2000 mg/kg, orally, then conduct the limit test. Limit test carryout at a dose of
2000 mg/kg body weight with six animals (three animals per step) Exceptionally an
additional dose of 5000 mg/kg can be considered when justified by specific
regulatory need.
Observation:
Observe for 14 days. Closely observe for the first four hours after the dosing and
then at least once daily thereafter up to 14 days.
Parameters:
Ø Mortality, Changes in skin, eyes, mucus.
Ø Behavioural pattern changes
Ø Respiratory, Circulatory, ANS, CNS (stimulants or depressant)
Ø Observation of tremors, convulsions, salivation, diarrhea, sleep and coma
Ø Body weight – At least once in a week
Ø Pathology – Gross necropsy and microscopic examination of organs
showing any changes.

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OECD 425: Acute Oral Toxicity –up and down method
Principle:
This is a sequential test using a maximum of 5 animals. Dose one animal at the test
dose.When animal dies, conduct the main test to determine the LD50 ..If animal
survives, dose four additional animals If the animal survives. The dose for the
second animal is increased by (a factor of) 3.2 times the original dose. If the first
animal dies or appears morbid then the dose for the second animal decreases by (a
factor of) 3.2 times the original dose. 3.2 is the default factor corresponding to a
dose progression of one half log unit. Dosing would be selected from the sequence
dose 1.75, 5.5, 17.5, 55, 175, 550, 2000, (or 1.75, 5.5, 17.5, 55, 175, 550, 1750 and
5000 for specific regulatory needs). If no information is available regarding lethality
of drug than dosing start from 175 mg/kg, bw, and sequentially observe animals for
14 days.
Main Test:
The main test consists of single ordered dose progression in which single animals
are dose in sequence usually at 48 hour intervals (dosing is determined by the
onset, duration and severity of toxic signs).
The first animal dose below the best preliminary estimate of LD50.
If the animal survives. The dose for the second animal is increased by (a factor of)
3.2 times the original dose. If the first animal dies or appears morbid then the dose
for the second animal decreases by (a factor of) 3.2 times the original dose.
3.2 is the default factor corresponding to a dose progression of one half log unit.
Dosing would be selected from the sequence dose 1.75, 5.5, 17.5, 55, 175, 550,
2000, (or 1.75, 5.5, 17.5, 55, 175, 550, 1750 and 5000 for specific regulatory needs).
If no information is available regarding lethality of drug then dosing starts from 175
mg/kg, bw.
Limit test carryout at 2000 mg/kg, orally (Exceptionally 5000 mg/kg) when
information indicating test material is likely to be nontoxic or toxicity above
regulatory limit dose.

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The LD50 less than the test dose (2000 mg/kg) when 3 or more animals die. LD50 is
greater than the test dose (2000 mg/kg) when 3 or more animal survive.
Observation: 14 days same as OECD 423.
OECD-407 (28 day repeated dose oral Toxicity study)
Animal species: Rat or Mice Age: Nine weeks old.
Housing condition
Temperature: 220
c ± 30
c
Humidity: 50-60 %
Lighting: 12 hr light 12 hr dark light
Feed : laboratory feed with water ad libitum
House: individually, small groups in same sex, not more than five/cage
Preparation of doses: Administration by gavages or via the diet or drinking water
in volume of 1ml/100 gm for suspension and 2ml/100gm b.w for aqueous. If
vehicles use other than water than toxic properties of the vehicle must be known.
Number of animals: 10 animals per group (5 female and 5 male)
Dosage: At least three test groups and the control group should be use.
Limit test: 1000 mg/kg/day for 28 days daily
Observation: 28 days
❖ General clinical observation at least once a day.
❖ Morbidity and mortality – at least twice daily.
❖ Changes in skin, eyes, mucous membrane, secretion and excretion.
❖ ANS activity- lacrimation, piloerection, pupil size, respiratory pattern, grip
strength, motor activity assessment.
❖ Body weight: weekly.
❖ Food consumption: weekly
❖ Water consumption:weekly
❖ Hematology parameters: Haematocrit, Hb, RBC, WBC, DC, platelet, clotting
time.
❖ Clinical Biochemistry: Liver and kidney function test
Plasma or serum. Na ,K, glucose, cholesterol, urea, creatinine, SGOT, SGPT,

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total protein, albumin, ALP, bilirubin, Gamma glutamyl transpeptidase, Lipid
profile.
❖ Urine analysis
❖ Last week of the study: Volume, appearance , specific gravity, pH , protein
glucose and blood cells.
❖ Metabolic profiles: Calcium, phosphate and triglycerides.
❖ Pathology: Liver, kidney, adrenals, testes, thymus, spleen, brain, stomach,
intestine, breast, lungs, urinary bladder, peripheral nerve, bone marrow etc.
OECD -408: 90 day repeated dose oral Toxicity study
Animal species: Rat or Mice
Age: Nine weeks old.
Housing condition:
Temperature: 220
c ± 30
c
Humidity: 50-60 %
Lighting: 12 hr light 12 hr dark light
Feed : laboratory diet with water ad libitum
House: Individually, small groups of same sex, not more than five/cage
Preparation of doses: Administration by gavages or via the diet or drinking water
in volume of 1ml/100 gm for suspension and 2ml/100gm b.w for aqueous. If
Vehicles use other than water than toxic properties of the vehicle must be known.
Number of animals: 10 animals per group (5 female and 5 male).
Dosage: At least three test groups and the control group should be used.
Limit test: 1000 mg/kg/day for 90 days daily
Observation: Same as OECD 407

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OECD -410: 21/28 –day Repeated dose dermal Toxicity study
Prerequisites:
● Solid or liquid
● Purity
● Solubility
● pH
● Stability, including stability in vehicle
● Melting point/ boiling point
Principle:
The test substances apply daily to the skin in graduate doses to several
groups of experimental animals, one dose per group for 21/28 days.
Animals: Adult rat - 200-300 gm; Rabbits- 2-3 kg; Guinea pigs - 350-450 gm.
Number and sex: 10 animals (5 female and 5 male).
Housing Conditions:
Individual housing
Temperature 220
c (±30
c)
Relative humidity 30- 70% preferable 50 to 60 %.
Lighting 12 hr light: 12 hr dark
Laboratory animal feed + ad libitum supply of drinking water
Dose level: At least three dose level with control and vehicle control if necessary
Limit test: One dose level at least 1 gm/kg ,b.w, dermal.
Application:
Test drugs apply not less than 10% of the body surface area uniformly for 6 hours
for a day.
Observation:
❖ Sign and toxicity
❖ ANS

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❖ CNS
❖ Behavior pattern
❖ Food consumption weekly
❖ Weight variation weekly
❖ Clinical examination:
❖ Haematology parameters: Haemocrit, Hb, RBC, WBC, DC, clotting time,
prothrombin time, platelet count, MCH, MCHC etc.
❖ Clinical biochemistry: Liver and Kidney function test, Lipid profile, protein,
albumin, Na, K, Cl, Ca, glucose etc.
❖ Pathology: Gross necropsy.
❖ Histopathology of organs showing gross changes.
OECD-402: Acute Dermal Toxicity Study
Prerequisites:
● Solid or liquid test substance
● Chemical identification of test substance
● Purity (impurities) of test substance
● Solubility characteristics
● Melting point/boiling point
● pH
Acute dermal toxicity is the adverse effects occurring within a short time of dermal
application of a single dose of a test substance.
Principle:
The test substance is applied to the skin in graduated doses to several groups of
experimental animals, one dose being used per group.
Preparations:
24 hours before the test, fur should be removed from the dorsal area of the trunk
of the test animals by clipping or shaving. Care must be taken to avoid abrading the
skin, which could alter its permeability. Not less than 10 percent of the body surface
area should be clear for the application of the test substance. The weight of the
animal should be taken into account when deciding on the area to be cleared and
on the dimensions of the covering.

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OECD 421 & 422: Reproductive toxicity
Introduction:
The occurrence of biologically adverse effects on the reproductive systems of
females or males that may result from exposure to environmental agents. The
toxicity may be expressed as alterations to the female or male reproductive organs,
the related endocrine system, or pregnancy outcomes.
● Effects on onset of puberty
● Gamete production and transport
● Reproductive cycle normality
● Sexual behavior
● Fertility, gestation
● Parturition, lactation, developmental toxicity
● Premature reproductive senescence
Developmental toxicity
Exposure prior to conception (either parent), during prenatal development, or
postnatally to the time of sexual maturation. The major manifestations of
developmental toxicity includes;
● Death of the developing organism
● Structural abnormality
● Altered growth
● Functional deficiency
Experiment:
● Adequate dosing
● For example, damage to spermatogonial stem cells will not appear in
samples from the cauda epididymis or in ejaculates for 8 to 14 weeks.
● Chlordimeform develops the compensatory mechanism.
● Knowledge of the relevant pharmacokinetic and pharmacodynamic data can
facilitate selection of dose levels and treatment duration. • Proper timing of
examination of treated animals relative to initiation and termination of
exposure to the agent.

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Length of Mating Period:
● Traditionally, pairs of rats or mice are allowed to cohabit for periods ranging
from several days to 3 weeks.
● Given a 4- or 5-day estrous cycle, each female that is cycling normally should
be in estrus four or five times during a 21-day mating period.
● Reproductive toxicity studies in laboratory animals generally involve
continuous exposure to a test substance for one or more generations. The
objective is to detect effects on the integrated reproductive process as well
as to study effects on the individual reproductive organs.
Number of animals: {Use of 20 males and enough females to produce at least 20
pregnancies for each dose group in each generation in the multigeneration
reproduction test.}
● 20 pregnancies may have been achieved by mating two females with each
male and using fewer than 20 males per treatment group.
● The single-generation reproduction test evaluates effects of subchronic
exposure of peripubertal and adult animals.
● In the multigeneration reproductive protocol, F1 and F2 offspring are
exposed continuously in utero from conception until birth and during the pre
weaning period.
● Animals producing the first generation of offspring should be considered the
parental (P) generation, and all subsequent generations should be
designated filial generations (e.g., F1, F2).
● Only the P generation is mated in a single-generation test, while both the P
and F1 generations are mated in a two generation reproduction test.
Single generation testing:
In the P generation, both females and males are treated prior to and during mating,
with treatment usually beginning around puberty.
Cohabitation can be allowed for up to 3 weeks, females are monitored for evidence
of mating. • Females continue to be exposed during gestation and lactation.
Two-generation reproduction test:

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Randomly selected F1 male and female offspring continue to be exposed after
weaning (day 21) and through the mating period.
Treatment of mated F1 females is continued throughout gestation and lactation.
Parameters evaluated:
● Visual examination of reproductive animals.
● Weights of the testes, epididymides, and accessory sex glands from males,
and histopathology of these organs.
● Histopathology of the vagina, uterus, cervix, ovaries, and mammary glands
from females.
● In addition, litters (individual pups) are weighed at birth and examined for
number of live and dead offspring, gender, gross abnormalities, and growth
and survival to weaning
● Maturation and behavioral testing may also be performed on the pups.
Results:
If adverse effects are observed on litters in a study using one of these protocols:
Male/ Female parent.
Therefore, male (histopathology or sperm evaluations) and female (ovarian and
reproductive tract histology or changes in estrous cycle) evaluation parameters will
help to decide.