The Slides present guidelines for Acute oral testing of drugs as well as other OECD guidelines in short.

1. Organisation for Economic Co-operation
and Development Guidelines for Toxicity
Testings
Dr Urmila M. Aswar,
Poona College of Pharmacy,
BVDU, PUNE, INDIA

2. Drug: Safe and Effective
• 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 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.

3. What are 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.

4. Purpose
• Hundreds of new chemicals, such as industrial chemicals,
pesticides, food additives, biotechnology products and
pharmaceuticals, flood the world market each year and may
require safety testing in most parts of the world.
Purpose:
• To enhance the validity and international acceptance of test
data;
• Make the best use of available resources in both
governments and industry;
• Avoid the unnecessary use of laboratory animals;
• Minimise non-tariff trade barriers.

5. Website

6. Common considerations
• The Globally Harmonized System (GHS),defines TOXICITY as
"those adverse effects occurring following oral or dermal administrati
on 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 toxici
ty testing.

7. Toxicological investigation
• Acute toxicity study
• Repeated dose toxicity study
• Dermal toxicity test
• Mutagenicity test
• Carcinogenicity test
• Reproductive and development toxicity tests

8. Toxicological investigation
 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.

9. Toxicity study
 Essential for any compounds having biological activity.
 Acute toxicity: Adverse effects occurring within a short time following
administration of single dose.
 Sub- acute toxicity
 Chronic toxicity- Period of 90 days or more.
 Special toxicity studies- Period of 2 years or more
 Carcinogenicity
 Mutagenicity
 Teratogenicity
 Reproductive toxicity

10. Initial Considerations
• The testing laboratory should consider all available
information on the test substance prior to conducting
the study.
• The identity and chemical structure of the substance
• Its physico‐chemical properties
• The results of any other in vitro or in vivo toxicity tests
on the substance
• Toxicological data on structurally related substances;
• The anticipated use(s) of the substance

11. Cont…
Animal species: Rat or Mice
Age : Nine weeks old.
Housing condition
• Temperature: 22 ± 3 ° 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/100gm for suspension and 2ml/100gm b.w for
aqueous.

12. Necessities of Toxicological Studies
• Benefit –risk ratio can be calculated
• Prediction of therapeutic index
• Therapeutic index= Maximum tolerated dose/
Minimum curative dose
• Smaller ratio, better safety of the drug.

13. Important OECD guideline
420 Acute oral Toxicity- fixed dose method
423 Acute oral Toxicity –acute toxic class method
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

14. Important OECD guidelines
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
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

15. Test Guidelines- 402, 403, 420, 423, and 425
• .
• Fixed Dose Procedure-OECD 420,
• Acute Toxic Class method -OECD 423,
• Up‐and‐Down Procedure -OECD 425,
• 28 days repeated oral Toxicity studies in rodents- OECD 407
• 90 days repeated oral Toxicity studies in rodents OECD 408
• Acute Dermal Toxicity-OECD 402,
• Acute inhalation toxicity-OECD 403.

16. OECD- 420: Acute Oral Toxicity - Fixed Dose
Procedure
• 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
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

17. Sighting study(1rat)- 420

18. Cont..

19. Main Study(5 rats)- 420

20. AOT 420- Important points

21. 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.

22. Acute Oral Toxicity 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.

23. Acute Oral Toxicity 423 – Acute Toxic Class
Method

24. Cont…

25. Acute Oral Toxicity 425- Up-and-Down
The first animal dose below best preliminary estimate of LD50.
If the animal survives. The dose for second animal is increased by (a factor of)
3.2 times the original dose. If the first animal dies or appears morbid than dose for
second animal is decrease by (a factor of) 3.2 times the original dose.
3.2 is default factor corresponding to a dose progression of one half log unit.
Dosing would be select 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 available regarding lethality of drug than dosing start
from 175 mg/kg, bw.

26. OECD 425- Limit test
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.
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

27. 28 day repeated dose oral Toxicity study
(OECD- 407)
Number of animal: 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 28 days daily

28. 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

29. Cont..
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, 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.

30. Cont..
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.

31. 90 day repeated dose oral Toxicity study
(OECD- 408)
Preparation of doses: Administration by gavages or via the diet or drinking water in
volume of 1ml/100gm 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 animal: 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 90 days daily
Observation: Same as OECD 407

32. Acute Dermal Toxicity Study OECD-402
Prerequisites
•Solid or liquid test substance
•Chemical identification of test substance
•Purity (impurities) of test substance
•Solubility characteristics
•Melting point/boiling point
•PH

33. Cont..
C.S.M.D.R.I.A.S
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.

34. Cont..
C.S.M.D.R.I.A.S
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 per cent 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

35. Reproductive toxicity
421, 422

36. 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.

37. • Effects on onset of puberty,
• Gamete production and transport,
• Reproductive cycle normality,
• Sexual behavior,
• Fertility, gestation,
• Parturition, lactation, developmental toxicity
• Premature reproductive senescence

38. 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
include
• (1) death of the developing organism,
• (2) structural abnormality,
• (3) altered growth, and
• (4) functional deficiency

39. 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.

40. 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.

41. Cont…
• 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.

42. 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.

43. • The single-generation reproduction test evaluates effects
of subchronic exposure of peripubertal and adult animals.
• In the multigeneration reproduction protocol, F1 and F2
offspring are exposed continuously in utero from
conception until birth and during the preweaning 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.

44. 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.

45. Two-generation reproduction test
• 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.

46. 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.

47. Cont..
• Maturation and behavioural testing may also be
performed on the pups.

48. 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.

49. Endpoints

50. Mating Index =
Pregnancy Index =

55. Genotoxicity-
Micronucleus test-OECD 474
• A micronucleus test is a test used in toxicological
screening for potential genotoxic.
• The mammalian in vivo micronucleus test is used for
the detection of damage induced by the test substance
to the chromosomes or the mitotic apparatus of
erythroblasts by analysis of erythrocytes as sampled in
bone marrow and/or peripheral blood cells of animals,
by evaluating the presence of micronuclei.
• There are two major versions of this test, one in
vivo and the other in vitro.

56. • The in vivo test normally uses mouse bone marrow or mouse
peripheral blood.
• When a bone marrow erythroblast develops into a
polychromatic erythrocyte, the main nucleus is extruded;
• any micronucleus that has been formed may remain.
• Visualisation of micronuclei is facilitated in these cells
because they lack a main nucleus.
• An increase in the frequency of
micronucleated polychromatic erythrocytes in treated animals
is an indication of induced chromosome damage

57. Procedure
• In vivo (0ECD 474): Animals are
exposed to the test substance by an
appropriate route. If bone marrow is
used, the animals are sacrificed at
appropriate times (24 h) after treatment,
the bone marrow extracted, and
preparations made and stained.

58. • In vitro (OECD 487): The test can be performed in primary human
peripheral blood lymphocytes (HPBLs) or established cell lines.
• Cultures are incubated with several concentrations of the test
compound for three to four hours in the presence and absence of
metabolic activation (S9) and for 21 to 24 hours in the absence of
S9.
• A positive outcome is characterized by a statistically significant,
dose-dependent increase in micronucleated cells that exceeds
historical negative control limits.

59. In Vitro Mammalian Chromosome Aberration
Test
• The purpose of the in vitro chromosome aberration
test is to identify agents that cause structural
chromosome aberrations in cultured mammalian
cells.

60. Consequence
• Chromosome aberrations and related events are
the cause of many human genetic diseases and
there is substantial evidence that chromosome
mutations and related events causing alterations in
oncogenes and tumour suppressor genes of
somatic cells are involved in cancer induction in
humans and experimental animals.

61. Principle behind
• Cell cultures are exposed to the test substance both
with and without an exogenous source of metabolic
activation unless cells with an adequate metabolizing
capability are used.
• At predetermined intervals after the start of exposure of
cell cultures to the test substance, they are treated with
a metaphase-arresting substance (e.g. Colcemid® or
colchicine), harvested, stained and metaphase cells
are analysed microscopically for the presence of
chromosome aberrations.