This document outlines the toxicological approach to drug development. It discusses the importance of conducting various toxicity studies at different stages of drug development to ensure safety. These include single dose, repeated dose, fertility, reproductive, developmental and genotoxicity studies in animals. It describes the typical safety program involving staged approach and discusses factors to consider in designing toxicity studies. The goal is to obtain sufficient non-clinical safety data to support clinical trials and assess safety for human use.

1. Toxicological Approach To
Drug Development
Prepared by: Dr. C. Suhas Reddy
Dept. of Pharmacy Practice

2. Introduction
Toxicology is defined as the study of adverse effects of xenobiotic.
Toxicology has evolved into three professional areas.
Mechanistic toxicology deals with identifying and understanding mechanisms
associated with toxic effects.
Descriptive toxicology is toxicology testing to provide information for safety
assessment & regulatory requirement.
Regulatory toxicology deals with risk assessment based on the data generated
by descriptive and mechanistic toxicity studies.

3. Once a compound has evolved to lead status, its safety has to be evaluated in
suitable in-vitro and in-vivo models.
Toxicologists in pharmaceutical developments must properly design the toxicity
studies to characterise the toxicity of a compound.
It requires the highest possible dose be given for an adequate period of time to
elicit a toxic response.
The purpose of the toxicity studies in pharmaceutical development is to provide
data in animals that can be used to assess the safety of a compound for human
use.
To accomplish this requires knowledge of toxic effects of a compound.

4. OUTLINE OF TYPICAL SAFETY
PROGRAM
A typical non-clinical safety program requires a ‘staged approached’.
With the staged approach, non clinical toxicity studies required to support the
next clinical phase are conducted and the data made available prior to initiation
of that clinical phase, the so-called “just in time” approach to clinical
development.

5. For the 1st dose in human study, the non clinical safety program generally
consists of single dose toxicity studies (two species), repeated-dose toxicity
studies (two species: one rodent &one non rodent), local tolerance (generally
part of the repeated-dose toxicity studies), and Genotoxicity ( Ames test: bacterial
mutation assay and in vitro chromosomal damage with mammalian cells or in
vitro mouse lymphoma assay) studies.
These studies usually provide sufficient information to allow dose for 1st phase
1 study.

6. Systemic Toxicity Studies
a) Single dose study (Acute toxicity studies)
b) Repeated-dose systemic toxicity studies
c) Male Fertility Studies
d) Female Reproduction and Developmental Toxicity Studies
e) Teratogenicity Study
f) Perinatal Study
g) Local Toxicity
h) Genotoxicity (description for all this studies available @ end of slides)

7. a) Chronic toxicity studies
b) Carcinogenicity
c) special toxicity study

8. As the data becomes available from the initial toxicity studies, the toxicologist
assesses the findings, reassesses the clinical development plan, and prepares for
the next toxicity studies.
These studies involve the conduct of long term repeated-dose toxicity,
reproductive toxicity, and in vivo Genotoxicity studies.
The duration of the repeated-dose studies is dependent upon the duration of the
clinical studies and the phase of clinical development.

9. While the longer term repeated-dose toxicity studies are being conducted,
reproduction and additional Genotoxicity studies are initiated.
In all regions, male and female fertility studies, embryo-foetal development, and
pre/postnatal development studies are required prior to marketing approval.

10. Also concurrent with the conduct of long-term repeated-doses toxicity and
reproduction toxicity studies, the remaining study in the standard battery of
Genotoxicity studies, the in-vivo test for chromosomal damage using rodent
hematopoietic cells, should be conducted.
This is an in-vivo rodent micronucleus assay, which should be completed prior
to initiation of phase 2 clinical studies.

11. The last studies to be conducted are chronic toxicity studies and the
carcinogenicity studies. However, these studies are not always required.
Chronic toxicity studies consist of 6-month studies in rodents and 9 or 12
month toxicity studies in non-rodents.
This is required only when the duration of clinical use of drug is greater than 3
months.
Carcinogenicity studies are required for drugs intended for continuous use for
at least 6 months and /or there is a cause for concern.

12. Causes for concern include:
Evidence of carcinogenicity in the compound class
SAR suggesting carcinogenic risk
Evidence of pre-neoplastic lesions in repeated-dose toxicity studies
Long-term tissue retention of parent compound and/or metabolites resulting in
local tissue reactions.

13. Doses for carcinogenicity studies may
be based one of the following;
Comparison of AUC in animals and man
Saturation of absorption
Pharmacodynamics
Maximum feasible dose
Maximum tolerated dose or
Limit dose.
The final type of toxicity is the special toxicity study.
These studies are generally conducted when an issue arises and there is a need to better
understand findings in other toxicity studies.
A special toxicity study examining the toxicity of the metabolite alone may be conducted by
administration of the metabolite to rats.

14. Duration of toxicity studies:
For clinical studies of 2 weeks in duration, rodent toxicity studies of 2-4 weeks are
required.
For 2-4 week human trials, animal studies should be 1 month duration.
For clinical trials of 1-3 months, 3 month toxicity studies is required
The amount of planning, compound synthesis, and tissue assessment required for an in
vivo toxicological evaluation can take from 3-6 months for sub acute toxicology.
Chronic toxicity studies require 6-12 months of exposure.
From these studies, a No Adverse Event Level (NOAL) can be determined.
An NCE in clinical trials will also be evaluated over a 2 yr period for reproductive
effects, teratogenicity and immunologic and behavioural toxicity both in adult mammal
and their offspring.

15. Duration of Repeat Dose Toxicology
Studies to Support Clinical Trials

18. FACTORS IN DESIGNING
STUDIES
Several factors must be considered when designing toxicity studies. These
include the use of main study or satellite animals ( In rodents, Toxicokinetics is
commonly assessed in a so-called satellite group of animals which run in parallel
to the main group devoted to toxicological evaluation.
Satellite animals are extra animals dosed as per protocol but not subjected to
toxicological and pathological observations and tests) for the collection of
pharmacokinetic data, the total blood volume that can be extracted from animals
during a given period of time, as well as the amount of blood that can be
collected at each time point, route of exposure, sampling time points, number of
animals per time point, number of sampling days and bio analytical support

19. One of the 1st decisions is whether the matrix (generally plasma) will be
obtained from animals being used for collection of toxicity data or from satellite
animals.
The next question is what matrix will be measured.
Drug concentrations may be measured in a wide variety of matrices, but not
limited to plasma, serum, tissue homogenates, urine and so on.
For large animals (e.g., dogs) the samples are collected from main study
animals, while for smaller animals (e.g., rodents) a satellite group is commonly
used. Generally, 3 or more animals per time point are used.

20. Sufficient sample times should be employed to allow calculation of an area
under the curve and plasma concentrations should be determined on a sufficient
number of days to establish the level of exposure achieved during the course of
the study.
In rodents, blood is not only required for the plasma concentration
determination, but also for toxicity endpoints. Therefore, a spate satellite set of
animals are used for the toxicity endpoint data.

21. Blood volumes that will not alter physiological parameters vary depending upon
the frequency of collection and the volume collected each time.
Different route of exposure will require different study designs with respect to
sampling for plasma concentration determination.
Oral, intravenous, and subcutaneous administrations will all result in different
plasma concentration vs. time curves. Therefore selection of time points for
sampling will need to be adjusted accordingly.

22. COMPOUND LIBRARIES
If the lead compound is found to be toxic it is kept in the compound library for
future use.
A compound library is a collection of compounds.
Compound libraries from past projects are kept and may be screened for the
biological activity you are looking for in a new project.
New compounds may also be made “in-house” but nowadays specialist
chemical companies are often contracted to simply make NCEs for big
pharmaceutical companies.
A disadvantage of synthetic libraries is that they are often limited.

23. Systemic Toxicity Studies
a) Single dose study (Acute toxicity studies)
b) Repeated-dose systemic toxicity studies
c) Male Fertility Studies
d) Female Reproduction and Developmental Toxicity Studies
e) Teratogenicity Study
f) Perinatal Study
g) Local Toxicity
h) Genotoxicity

24. a) Chronic toxicity studies
b) Carcinogenicity
c) special toxicity study

25. a) Single dose study (Acute toxicity
studies)
Single dose studies in animals are essential for any pharmaceutical product
intended for human use.
The information obtained from these studies is useful in choosing doses for
repeat dose studies, providing preliminary identification of target organs of
toxicity and occasionally, revealing delayed toxicity.
Acute toxicity studies may also aid in the selection of starting doses for phase I
human studies, and provide information relevant to acute overdosing in humans.

26. Acute toxicity studies should be carried out in at least two species, usually mice
and rats using the same route as intended for humans.
In addition, at least two more route should be used to ensure systemic
absorption of the drug, this route may depend on the nature of the drug.
Mortality should be looked for up to 72 hours after parentral administration and
up to 7 days after oral administration.
Symptoms, signs and mode of death should be reported, with appropriate
macroscopic and microscopic findings where necessary.

27. b) Repeated-dose systemic toxicity
studies
The primary goal of repeated dose toxicity studies is to characterize the
toxicological profile of the test compound following repeated administration.
This includes identification of potential target organs of toxicity and
exposure/response relationships, and may include the potential reversibility of
toxic effects.
This information should be part of the safety assessment to support the conduct
of human clinical trials and the approval of a marketing authorization.

28. The decision whether a developmental toxicity study needs to be performed
should be made on a case-by-case basis taking into consideration;
Historical use,
Product features,
Intended target population
Intended clinical use.

29. c) Male Fertility Studies
Male fertility studies are designed to provide general information concerning
the effects of a test substance on male reproductive system such as gonadal
function.

30. d) Female Reproduction and
Developmental Toxicity Studies
Female fertility studies are designed to provide general information concerning
the effects of a test substance on female reproductive system such as ovary
function and lactation.
These studies need to be carried out for all drugs proposed to be studied or used
in women of childbearing age.

31. Reproductive studies done on rats

32. e) Teratogenicity Study
The drug should be administered throughout the period of organogenesis in
animals if the test drug is intended for women of childbearing age and if women
of childbearing age are to be included as subjects in the clinical trial stage, Using
three dose levels.
One of the doses should cause minimum maternal toxicity and one should be
the proposed dose for clinical use in humans and other one will be multiple of it.
The route of administration should be the same as for human therapeutic use.

33. f) Perinatal Study
This study is specially recommended if the drug is to be given to pregnant or
nursing mothers for long periods or where there are indications of possible
adverse effects on foetal development.
The drug should be administered throughout the last third of pregnancy and then
through lactation and weaning.
The control of each treated group should have at least 12 pregnant females and
the dose which causes low foetal loss should be continued throughout lactation
weaning.

34. Animals should be sacrificed and observations should include macroscopic
autopsy and where necessary, histopathology

35. g) Local Toxicity
These studies are required when the new drug is proposed to be used by some
special route (other than oral) in humans.
The drug should be applied to an appropriate site (e.g. Skin, Ocular or Vaginal
mucous membrane) to determine local effects in a suitable species.
If the drug is absorbed from the site of application, appropriate systemic toxicity
studies will also be required.
Examples:- of local toxicity are Dermal Toxicity Study, Vaginal Toxicity Study,
Photoallergy, Rectal Tolerance Test, Ocular Toxicity Studies, Inhalational
Toxicity Studies, And Hypersensitivity.

36. The control and the treated groups should consist of at least 20 pregnant females
in case of non-rodents, on each dose used.
Observations should include the
a) number of implantation sites, restorations if any;
b) number foetuses with their Gender, weights and malformations if any.

37. Depicts rabbits kept ready for ocular tests

38. h) Genotoxicity
Genotoxicity refers to potentially harmful effects on genetic material (DNA)
which may occur directly through the induction of permanent transmissible
changes (mutations) in the amount or structure of the DNA within cells.
In vitro (artificial environment) and in vivo (in living organisms) genotoxicity
tests are conducted to detect compounds which induce genetic damage directly or
indirectly.
These tests should enable hazard identification with respect to damage to DNA
and its fixation.

39. Damage to DNA can occur at three levels:
1. Point mutations ( where a single nucleotide base is changed, inserted or deleted
from a sequence of DNA or RNA inside the cell).
2. Chromosomal mutations (changes involving whole chromosomes or parts of
chromosomes).
3. Genomic mutations (permanent alteration in the DNA sequence that makes up a
gene, such that the sequence differs from what is found in most people).

40. The following standard test battery is generally expected to be conducted:
a) A test for gene mutation in bacteria (Ames test)
b) An in vitro test with cytogenetic evaluation of chromosomal damage with
mammalian cells or an in vitro mouse lymphoma assay
c) An in vivo test for chromosomal damage using rodent hematopoietic cells.

41. i) Chronic toxicity studies
Studies should be performed for all drugs that are expected to be clinically used
for six months or more than six months as well as for drugs used frequently in an
intermittent manner in the treatment of chronic or recurrent condition

42. Depicts carcinogenicity test done on
mice
Lab mice showing one with a tumor, the other
treated with toxin cancer drug

43. Fertility studies:
The drug should be administered to both males and females, beginning a
sufficient number of days before mating.
In females the medication should be continued after mating and the pregnant
one should be treated throughout pregnancy.
The highest dose used should not affect general health or growth of the animals.
 The route of administration should be the same as for therapeutic use in
humans.

44. The control and the treated group should be of similar size and large enough to
give at least 20 pregnant animals in the control group of rodents and at least 8
pregnant animals in the control group of non-rodents.
Observations should include total examination of the litters from both the
groups, including spontaneous abortions, if any.

45. Carcinogenicity
These studies are required to be carried out if the drug or its metabolite is
related to a known carcinogen or when the nature and action of the drug is such
as to suggest a carcinogenic/mutagenic potential. For carcinogenicity studies, at
least two species should be used.
These species should not have high incidence of spontaneous tumours and
should preferably be known to metabolize the drug in the same manner as
humans.

46. At least three does levels should be used;
a) the highest does should be sub-lethal but cause observable toxicity;
b) the lowest does should be comparable to the intended human therapeutic does
or a multiple of it, example 2.5x; to make allowance for the sensitivity of the
species;
c) the intermediate does to be placed logarithmically between the other two
doses.

47. A control group should always be included.
The drug should be administered 7 days a week or a fraction of the life span
comparable to the fraction of human life span over which the drug is likely to be
used therapeutically.
Observations should include macroscopic changes observed at autopsy and
detailed histopathology.

48. Reference
Chapter 1- DRUG DISCOVERY AND CLINICAL RESEARCH - SK Guptha
https://pdfs.semanticscholar.org/presentation/5c92/0271fc9f009c486ec3ab91d0
1e811bd58e60.pdf
Pharmacokinetics in drug development : Regulatory and developmental
paradigms. By Peter Bonate, Danny Howard, Pg no: 87-94.
New drug development : Design methodology and analysis. By J. Rick Turner.

49. THAN ‘Q’