The document discusses guidelines for toxicity testing from the Organisation for Economic Co-operation and Development (OECD). It provides an overview of OECD guidelines for acute oral toxicity testing, including the fixed dose procedure (Guideline 420), acute toxic class method (Guideline 423), and up-and-down procedure (Guideline 425). It also discusses guidelines for acute dermal toxicity testing (Guideline 402) and repeated dose toxicity studies lasting 28 days (Guideline 407) or 90 days (Guideline 408). The purpose of the OECD guidelines is to enhance the validity and international acceptance of toxicity test data.

1. Organisation for Economic Co-operation
and Development
Guidelines for Toxicity Testings
Dr Urmila M. Aswar,
Department of
Pharmacology, PCP, BVDU

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 elu
cidate 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 it as "those adverse
effects occurring following oral or dermal administration of a single d
ose of a substance, or multiple doses given within 24 hours, or an i
nhalation 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 info
rmation on the test substance prior to conducting the st
udy.
• 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. 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.

12. Important OECD guideline
Organisation for Economic Co-operation and Development (OECD)
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

13. 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

14. 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,
• Acute Dermal Toxicity-OECD 402,
• Acute inhalation toxicity-OECD 403.

15. 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

16. Sighting study-

17. Cont..

18. Main Study- 420

19. AOT 420- Important points

20. 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.

21. 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.

22. Acute Oral Toxicity 423 – Acute Toxic Class
Method

23. Cont…

24. Acute Oral Toxicity 425- Up-and-Down
Procedure
• The Limit Test is a sequential test that uses a maximum of 5
animals. A test dose of 2000, or exceptionally 5000 mg/kg,
may be used. The procedures for testing at 2000 and 5000
mg/kg
• The main test consists of a single ordered dose progression
in which animals are dosed, one at a time, at a minimum of
48-hour intervals. The first animal receives a dose a step
below the level of the best estimate of the LD50. If the animal
survives, the dose for the next animal is increased by [a
factor of] 3.2 times the original dose; if it dies, the dose for
the next animal is decreased by a similar dose progression.
(Note: 3.2 is the default factor corresponding to a dose
progression of one half log unit.

25. Procedure
• The first animal is dosed a step below the best preliminary
estimate of the LD50. If the animal survives, the second animal
receives a higher dose. If the first animal dies or appears
moribund, the second animal receives a lower dose. The dose
progression factor should be chosen to be the antilog of 1/(the
estimated slope of the dose-response curve) and should remain
constant throughout testing (a progression of 3.2 corresponds to a
slope of 2). When there is no information on the slope of the
substance to be tested, a dose progression factor of 3.2 is used.
Using the default progression factor, doses would be selected
from the sequence 1.75, 5.5, 17.5, 55, 175, 550, 2000 (or 1.75,
5.5, 17.5, 55, 175, 550, 1750, 5000 for specific regulatory needs).
If no estimate of the substance’s lethality is available, dosing
should be initiated at 175 mg/kg.

26. OECD 425- MAIN TEST
The first animal dose below best preliminary estimate of LD50.
If the animal survives. The dose for second animal is increase 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.

27. 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

28. 28 day repeated dose oral Toxicity study
(OECD-407)
Animal species: Rat or Mice
Age : Nine weeks old.
Housing condition
Temperature: 220 c ± 30c
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

29. Cont..
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.
Vehicles
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

30. 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

31. 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.

32. 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.

33. 90 day repeated dose oral Toxicity study
(OECD-408)
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 diet with water ad libitum
House: Individually, small groups of same sex, not more than five/cage

34. 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 is 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

35. 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

36. 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.

37. 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

38. Reproductive toxicity
421, 422
Dr Urmila M. Aswar

39. 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.

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

41. 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

42. 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.

43. 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 oestrous cycle, each female
that is cycling normally should be in oestrus four or
five times during a 21-day mating period.

44. 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.

45. 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.

46. • 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.

47. 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.

48. 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.

49. 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.

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

51. 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.

52. Endpoints

53. Mating Index =
Pregnancy Index =

58. Genotoxicity-
Micronucleus test-OECD 474
• A micronucleus test is a test used in toxicological
screening for potential genotoxic and carcinogenic
compounds.
• 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,
usually rodents.
• There are two major versions of this test, one in
vivo and the other in vitro.

59. • 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

60. • This mammalian in vivo micronucleus test is
especially relevant to assessing mutagenic hazard
in that it allows consideration of factors of in vivo
metabolism, pharmacokinetics and DNA repair

61. Procedure
• In vivo: Animals are exposed to the test substance by an
appropriate route. If bone marrow is used, the animals are
sacrificed at appropriate times after treatment, the bone
marrow extracted, and preparations made and stained.
• In vitro: When peripheral blood is used, the blood is
collected at appropriate times after treatment and smear
preparations are made and stained. For studies with
peripheral blood, as little time as possible should elapse
between the last exposure and cell harvest. Preparations
are analyzed for the presence of micronuclei.

62. • Mice or rat, with spleen not able to remove the
micronucleus RBC.
• Positive control: Ethyl methanesulphonate, Ethyl
nitrosourea , Mitomycin C
• Negative Control:

63. • No standard treatment schedule (i.e. 1, 2, or more
treatments at 24 h intervals) can be recommended.
• Two types
• Animals are treated with the test substance once. Samples
of bone marrow are taken at least twice, Sampling done
between 24 to 48 hours after treatment, but not extending
beyond 48 hours after treatment with appropriate
interval(s) between samples.

64. • If 2 or more daily treatments are used
• Treatment at 24 hrs interval.
• samples should be collected once between 18 and
24 hours and then between 36 and 48 hours
following the final treatment for the peripheral blood

65. • If there is toxicity, three dose levels are used for the
first sampling time. These dose levels should cover
a range from the maximum to little or no toxicity.
• At the later sampling time only the highest dose
needs to be used. The highest dose is defined as
the dose producing signs of toxicity such that higher
dose levels, based on the same dosing regimen,
would be expected to produce lethality.

66. 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.

69. 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.

70. 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.

71. Test substance
• Test substance: - identification data and CAS no., if
known;
• - physical nature and purity;
• - physicochemical properties relevant to the conduct of
the study;
• - stability of the test substance, if known.
Solvent/Vehicle:
• - justification for choice of solvent/vehicle.
• - solubility and stability of the test substance in
solvent/vehicle, if known.

72. Cells
• Cells: - type and source of cells - karyotype features
and suitability of the cell type used;
• - absence of mycoplasma, if applicable
• - for cell lines, information on cell cycle length, doubling
time or proliferation index
• - sex of blood donors, whole blood or separated
lymphocytes, mitogen used
• - methods for maintenance of cell cultures if applicable

73. Conc used
• At least three analysable test concentrations from
duplicate cultures should be evaluated.
• For substances demonstrating little or no toxicity:
concentration intervals of approximately 2 to 3 fold
will usually be appropriate.
• Moderate toxicity: it will be necessary to use more
closely spaced concentrations.
• Dose response relationship: more than three
concentrations will be needed

74. • Positive control: Mitomycin C,
• 4-Nitroquinoline-N-Oxide
• Metabolic activation: Benzo(a)pyrene,
Cyclophosphamide