The document outlines the course ZOL-610 in Environmental Toxicology, detailing its objectives, content, and the methodology for toxicity studies, including acute, subacute, and chronic toxicity assessments for various test substances. It emphasizes the importance of understanding the effects of environmental toxicants and the need for specific animal studies to evaluate toxicity and safety in potential drug development. The course also covers the evaluation of reproductive and local toxicity, as well as the procedures and parameters to be monitored throughout the testing process.

1. COURSE TITLE ENVIRONMENTAL TOXICOLOGY
COURSE CODE ZOL- 610
CREDIT HOURS 3 (2-1)
SEMESTER BS ZOOLOGY 8TH
M-A, E-A, E-B
DEPARTMENT OF ZOOLOGY

2. COURSE CONTENTS
ENVIRONMENTAL TOXICOLOGY ZOL 610 3(2-1)
Toxicology: History , Terms and definitions, principles of Toxicology, Development
and present scope of environmental toxicology, Framework of environmental
toxicology, toxicological evaluation, Sources of environmental toxicants, /
Pollutants: Gaseous chemicals and heavy metals, toxicity testing,
Characteristics of exposure, spectrum of Toxic effects, Indices of toxicity, toxico-
dynamics, toxico-kinetics (absorption, distribution and elimination of toxic agents)
Biotransformation, Detoxification and biodegradation. Pollution and remediation,
ecological risk assessment

3. Toxicity Studies

4. Source, Synthesis &
chemistry
Preclinical Studies &
Toxicology Studies
Animal models,
Efficacy and Safety
Clinical Trials
Drug Development

5. Preclinical Safety & Toxicity Testing
 Aims at discovering complications or sequelae
arising from the pharmacological actions of
drug and any unexpected side-effects.

6. Primary Goals
Estimate safe starting dose for clinical studies &
subsequent dose escalation schemes in humans.
Identify potential target organs for toxicity and study
whether such toxicity is reversible.
Assess dose dependence & relationship to exposure
Assess hazards that cannot be evaluated in clinical trials
(e.g. carcinogenicity and teratogenicity)
Identify hazards and estimate safe dose range

7. Necessity of toxicity studies
 Completely novel compound of unique action under
consideration
 Any chemical modification made to a known drug
 Formulations of a compound are considered
( Separately as well as formulated form)
 Novel combination of drugs made available as a single
formulation
 Veterinary drugs → if treatment is given to animals
intended for human food

8. Requirements
 Chemical Criteria
 Substance used in toxicity studies should be as pure as
material to be given to man

9. Materials: Animals
 Species:
 Species difference → single largest difficulty in
interpretation of toxicity studies
 Species →health, behaviour, endemic disease and
reaction to well studied toxic agent is familiar
 Two different species(Rodent & Non Rodent) used
 Should differ phylogenetically as widely as possible

10. Materials: Animals
 Species:
 Species in which test material is pharmacologically active
due to presence of receptor should be used
Ex. Avoid dogs for studying toxic effects of
sulphonamides, monkeys are better
 Strains:
 Use either animals derived from random breeding in
a closed colony or hybrids of two inbred lines

11. Materials: Animals
 Number, age and sex:
 Should be sufficient
 Long term experiments i.e. Carcinogen testing, large
number may be necessary to detect relatively low
incidence of the reactions
 Young immature animals are preferred generally → rapid
growth, but for detection of actions on endocrine and
reproductive systems, sexually mature animals are
required
 Should include groups of both males and females

12. Route / Frequency of administration
 Two routes to be used, one should be which is
intended for clinical use
 Aim should be to maintain the appropriate blood and
tissue level of the compound under consideration for
test duration

13. Duration
 Depends on type of the test (acute, sub-acute, chronic)
 All the important toxic effects of test substances can
probably predicted from experiments lasting no more
than three months
 In the absence of any rational basis for choosing a period,
a duration of twice the maximum exposure likely for
the human beings is convenient guide. 2 yrs. max

14. Toxicity Studies
 Systemic Toxicity studies
 Reproductive toxicology studies
 Local Toxicity studies
 Allergenicity/ Hypersensetivity studies
 Genotoxicity studies
 Carcinogenicity studies

15. Systemic
Toxicity Studies

16. Single dose
(Acute)
Short term
repeated
dose
(Subacute)
Repeated dose:
10% life-span
(Subchronic)
Repeated
dose: >10%
life span
(Chronic)
Systemic Toxicity Studies

17. Single-dose (Acute) Toxicity Studies
 Dose – toxicity relationship
 Specific toxic effects
 Mode of toxic action
 Median Lethal dose (LD 50)
 Route dependent toxicity
 Sex dependent toxicity

18. Single-dose (Acute) Toxicity Studies
 Rodent species (mice and rats)
 Use the same route as intended for humans
 Use at least one more route in one of the species. This
 Ensures systemic absorption of the drug
(unless the intended route in humans is only intravenous)
 Recommended limit for oral dosing : Higher value of
either
 2000 mg/kg or
 10 times the normal dose that is intended in humans

19. Single-dose (Acute) Toxicity Studies
 At least 5 animals / sex / group
 At least 4 graded doses (4 dose-groups)
 Observation for toxic effects (if any) : 14 days
 Obsevation for mortality :
 7 days [parenteral administration]
 14 days [oral administration]

20.  Reduction of the number of animals, use of single sex
 Preliminary studies, followed by main test (Limit test)
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Modifications
(OECD guidelines: 420, 423, 425; yr 2000)

21. Other parameters :
 Symptoms, signs and mode of death
 LD 10 and LD 50 values, preferably with 95 % CI
Genetic effects if any
Establish the:
 Minimum lethal dose (MLD)
 Maximum tolerated dose (MTD)
 Target organ of toxicity (if possible)

22. Cytotoxic anticancer agents
 MTD determined first in Mice
 Findings in Rat confirmed to establish a linear
relationship between toxicity and body surface area
 MTD can be established in non-rodent species, if:
 Predictability is known to be poor in Rodents
(e.g. Antifolates)
 Drug has a novel mechanism of action

23. Toxicokinetic studies
Generation of pharmacokinetic data (ADME):
• A part of non-clinical toxicity studies or
• A separate supportive study
Purpose:
• Assessment of systemic exposure of test substance
• Relationship of toxicology to dose and time course
• Choose species & regimen in subsequent toxicity studies
• Designing of subsequent non-clinical toxicity studies

24. Rodents
• One rodent species (preferably rat)
• At least 4 graded doses including control
• Minimum of 5 animals of each sex
• Proposed clinical route of administration
• Test substance given daily for 10 consecutive days
Dose-ranging study
 Establishment of MTD for repeated dose studies
 Identification of Target organ of toxicity
Non-Rodents
• One male and one female (Dogs > Primates)
• Starting dose (3 or 5 x extrapolated effective dose) or
MTD
(whichever is less)
• Dose escalation every 3rd day, lowered if toxicity seen
• Then test substance given daily for 10 consecutive days

25. Repeated-dose Toxicity Studies
 Duration depends on proposed clinical trial
 At least 2 species, one non-rodent
 Species-specific pharmacokinetics, if any, should
preferably resemble human beings
 Route intended for human clinical use

26. Repeated-dose Toxicity Studies
 Wherever applicable, include a Control group
 3 other groups are formed, as:
 Highest dose
 Lowest dose
 Intermediate dose
Observable toxicity [MTD]
No observable toxicity
[NOAEL]
intended therapeutic dose or
multiple of it
Some symptoms ; not gross
toxicity or death placed
logarithmically betn doses

27. • Rodent
• Non-rodent
: 6-10/sex/group
: 2-3/sex/group
14-28 Day repeated-dose
(Subacute toxicity
studies)
• Rodent
• Non-rodent
: 15-30/sex/group
: 4-6/sex/group
90-Day repeated-dose
(Subchronic toxicity
studies)
• Rodent
• Non-rodent
: 15-30/sex/group
: 4-6/sex/group
180-Day repeated-dose
(9 months for non
rodents?)
(Chronic toxicity studies)

28. In all cases:
 Behavioural : General appearance, activity,
behaviour
 Physiological : Body weight, food intake
 Biochemical : Hematology, serum and urine
analysis
 Pathological : Organ weights, gross & microscopic
study of viscera & tissues
Parameters to be monitored
 Injection site : Gross and Microscopic
examination
•In non-rodent species:
 Electrocardiogram : Initial and Final
 Fundus examination
•If parenteral drug administration:

29. Reproductive
Toxicology

30. Reproductive Toxicity
 Male Fertility
 Female Reproduction & Developmental toxicity
 Female Fertility
 Teratogenicity
 Perinatal development

31. Male Fertility Study
 One rodent species (preferably rat)
 3 dose groups and a control group :
 Dose selection : results of previous 14 or 28-day toxicity
study
 Highest dose : showing minimal toxicity in systemic
studies
 6 adult male animals per group

32. Test substance by intended route of use
for :
• minimum 28 days
• maximum 70 days
Paired with female animals of proven
fertility in a ratio of 1:2
Drug treatment of male animals continues
during pairing
Pairing continued till detection of Sperm
in vagina or 10 days, whichever is earlier

33. Pregnant females examined after day 13
of gestation
Males sacrificed at the end of the study
Weights of each testis and epididymis
recorded
Sperms from one epididymis examined
for motility and morphology
Other epididymis and both testes
examined for histology

34. Female Reproduction and
Developmental Toxicity Studies
 For all drugs proposed for women of child bearing age
• Segment I : Female fertility
• Segment II : Teratogenicity
• Segment III : Perinatal development
 Segment I, II and III studies in albino mice or rats, and
 Segment II study also in albino rabbits as a second test
species

35. Female Fertility Study (Segment I)
 One rodent species (rat preferred)
 3 graded doses
 Highest dose : doesn’t affect general health of parent
animals
(usually the MTD from previous systemic studies)
 At least 15 males and 15 females per dose group
 Route of administration same as intended for therapeutic
use

36. Pups : Physiology, Behaviour, Pathology (sex-wise distribution
noted)
Body weight
Food intake
Clinical signs
of intoxication
Reproduction
and
Parturition
Pathology
Gross &
Micro
Drug treatment continued during mating and gestation period
Females allowed to litter,
medication continued till weaning of pups
Drug administration : 28 days (males) & 14 days (females) before
mating

37. Teratogenicity Study (Segment II)
 Rodent (preferably rat) and Non-rodent (rabbit)
 Drug administered throughout organogenesis
 3 dose levels and a Control group:
 Highest dose : minimum maternal toxicity
 Lowest dose : as proposed clinical dose or its multiple
 Route of administration : same as intended for humans
 At least 20 pregnant rats (or mice) and 12 rabbits, for
each dose

38. Observation parameters
Females
General
Signs of
intoxication
Body weight
Food
intake
Reproductive
Uterus,
Ovaries
Products of
conception
Foetuses
General
Number
Gender
Length
Weight
Pathology
Gross (all)
Visceral (half)
Skeletal (half)

39. Perinatal Study (Segment III)
 Specially if
→ Drug to pregnant / nursing mothers for long periods
→ Indications of possible adverse effects on foetus
 One rodent species (preferably rat)
 Dosing comparable to multiples of human dose and route
 At least 4 groups (including control), 15 females / group
 Drug throughout last trimester (from day 15 of gestation)
 Dose causing low foetal loss continued throughout weaning

40. F1 litter
1 male and 1 female from each group
Test substance given
Throughout growth to sexual life
Mating performance and fertility of F1 :
F2 generation
F2 generation
growth parameters monitored till
weaning

41. Local
Toxicity

42. Local Toxicity
 Dermal Toxicity
 Ocular Toxicity
 Vaginal Toxicity
 Rectal tolerance test
 Parenteral Drugs Tolerance
 Inhalation toxicity

43. Local Toxicity
 If intended for special route (other than oral) in humans
 Appropriate site (e.g., skin or vaginal mucous membrane)
in a suitable species
 Preferably use of 2 species
 3 dose levels and untreated and / or vehicle Control
 If the drug is absorbed systemically, appropriate systemic
toxicity studies also required

44. Dermal Toxicity
 As cutaneous contamination is always a possibility
 Rabbit and Rat
 Applied on shaved skin
 Concentrations 7 fold higher than the clinical doses
 Period of application : 7 to 90 days
 Evaluation
› Local signs (erythema, oedema and eschar formation)
› Histological examination of sites of application

45. Ocular toxicity studies
 2 species, including an albino rabbit with a large
conjunctival sac
 Initial single dose application:
To decide exposure concentrations for repeated-dose
studies
Repeated dose study :
 Duration subject to clinical use (Maximum of 90 days)
 2 different concentrations exceeding human dose
 In acute studies, one eye kept as control. A separate control
group should be included in repeated-dose studies.

46. Ocular toxicity studies
 Evaluation
 Slit-lamp examination
 To detect the changes in cornea, iris and aqueous
humor.
 Fluorescent dyes (sodium fluorescein, 0.25 to 1.0%)
 To detect defects in surface epithelium
 Intra-ocular tension monitored by a tonometer
 Histological examination (fixation in Davidson’s or
Zenker’s fluid)

47. Vaginal Toxicity Test
 Rabbit or Dog
 Topical application (vaginal mucosa) as pessary, cream or
ointment
 6-10 animals per dose group
 Higher concentrations / several daily applications (in
multiples of daily human dose)
 7-30 days, as per clinical use
 Observation parameters :
 General : swelling, closure of introitus
 Histopathology of vaginal wall

48. Rectal Tolerance Test
 Preparations meant for rectal administration
 In rabbits or dogs
 6-10 animals per dose group
 Volume comparable to human dose (or the maximum possible
volume) to achieve administration of multiples of daily human dose
 7-30 days, as per clinical use
 Observation parameters
 Clinical signs :- signs of pain, blood and/or mucus in faeces,
condition of anal region/sphincter
 Gross examination and (if required)
 Histological examination of rectal mucosa

49. Parenteral Drugs
 Intravenous/ intramuscular/ subcutaneous/ intradermal
inj.
 Injection sites in systemic toxicity studies examined
grossly and microscopically
 If needed, reversibility of adverse effects may be
determined on a case to case basis

50. Inhalation toxicity studies
 1 rodent and 1 non-rodent species
 Acute, subacute and chronic toxicity studies according to
the intended duration of human exposure
 Gases and vapors given in whole body exposure
chambers; aerosols are given by nose-only method
 Dose (limit dose of 5mg/l) in multiples of human
exposure
 Particle size of 4 micron (especially for aerosols) with not
less that 25% being 1 micron

51. Inhalation toxicity studies
 3 dose groups and a control
 Duration of exposure : maximum of 6 hr/day & 5
days/week
Evaluation :
 Respiratory rate, BALF examination, histological
examination of respiratory passages and lung tissue
 Regular parameters of systemic toxicity studies or
assessment of margin of safety

52. Allergenicity /
Hypersensitivity

53. Allergenicity / Hypersensitivity
Any one of the standard tests:
 Guinea pig :
 Maximization test (GPMT)
 Mouse :
 Local lymph node assay (LLNA)

54. Guinea Pig Maximization Test
Challenge
(skin reaction
appears)
Induction : Minimum irritant dose (intradermal
injection)
Challenge : Maximum nonirritant dose (topical
application)
Doses are determined by a preliminary study
Induction
(immune response
develops)

55. Main test
 A minimum of 6 male and 6 female animals per group
 One test group
 One control group
 One positive control group (preferable)
 If no response : re-challenge 7-30 days after primary
challenge

56. Induction (Day 0)
3 pairs of intradermal injections on either shoulders :
• 0.1 ml Freund’s adjuvant alone
• 0.1 ml test material (lowest irritant dose)
• 0.1 ml test material in Freund’s adjuvant
Day 7 : Topical patch at prepared shoulders (lowest irritant dose)
Challenge (Day 21)
Topical patch at prepared flanks (highest non-irritant dose)
• Left side: Test agent
• Right side: Vehicle
Evaluation [Edema and Erythema] after 48 hr.

57. Local Lymph Node Assay
 Mice of the same sex, either only males or only females
 Drug treatment given on ear skin
 3 graded doses (the highest being maximum nonirritant
dose) plus vehicle control
 A minimum of 6 mice per group
Test material applied on ear skin on 3 consecutive days
On day 5, i.v. 3H-thymidine / bromo-deoxy-uridine (BrdU)
Draining auricular lymph nodes dissected after 5 hrs
Evaluation : Increase in 3H-thymidine or BrdU
incorporation

58. Genotoxicity

59.  Genotoxic compounds are presumed to be trans-species
carcinogens, need not require long-term carcinogenicity
studies
 If intended for chronic administration, a chronic toxicity
study (up to one year) to detect early tumorigenic effects
ICH Standard Tests are generally conducted
• In vitro test for gene mutation in bacteria
• In vitro cytogenetic evaluation of chromosomal
damage :
• Mammalian cells or
• Mouse lymphoma tk assay
• In vivo test for chromosomal damage using rodent
hematopoietic cells

60. Ames’ Test
( Bacterial Reverse Mutation Assay )
 S. typhimurium tester strains TA98, TA100, TA102,
TA1535, TA97 or
 Escherichia coli WP2 uvrA or Escherichia coli WP2 uvrA
 In-vitro exposure at a minimum of 5 log dose levels
 “Solvent” and “positive control”
 2.5 fold (or more) increase in number of revertants in
comparison to spontaneous revertants are considered
positive

61. In-vitro cytogenetic assay
 Performed in CHO cells or on human lymphocyte in
culture
 In-vitro exposure using a minimum of 3 log doses
 “Solvent” and “positive control”
 [Positive control : Cyclophosphamide / Mitomycin C
gives a reproducible and detectable increase in
clastogenic effect]
 > 50% inhibition of cells is considered significant

62. Mammalian cell test systems
Division stimulated with phytohemagglutin
Division arrested in metaphase using a spindle inhibitor
Evaluation by light microscopy
Increased number of aberrations in metaphase chromosomes

63. In Vitro Mouse Lymphoma TK Assay
Mouse TK lymphoma cells
Thymidine kinase (TK) enzyme involved in salvage pathway
for incorporation of thymidine into cells via phosphorylation
Trifluorothymidine (TFT) also phosphorylated by TK
Cells containing TK are sensitive to toxic effects of TFT
Forward mutations from TK+ to TK- result in loss of TK
activity
Quntifiication of mutant cells : Cells with TFT resistance

64. In Vivo Tests for Chromosomal Damage
Mammalian Bone
Marrow
Chromosomal
Aberration Assay
Rodent
Erythrocyte
Micronucleus
Assay

65. In-vivo cytogenetic assay
• One rodent species (preferably rat)
• Route of administration same as intended for humans
• 5 animals/sex/dose groups
• 3 dose levels, “solvent” and “positive” control (Cyclophosphamide)
Dosing on day 1 followed by i.p. colchicine administration at 22
hours
Sacrificed 2 hours after colchicine administration
Bone marrow : Giemsa staining
Clastogenic & Aneugenic effects in metaphase chromosomes (min
100)

66. In-vivo micronucleus assay
• 1 rodent species (preferably mouse) needed
• Route of administration of test substance same as humans
• 5 animals / sex / dose groups
• At least 3 dose levels, plus “solvent” and “positive” control
• Positive control : mitomycin C or cyclophosphamide
Dosing : day 1 and 2 of study
Sacrifice of animals 6 hours after the last injection
Bone marrow : Smeared with May Gruenwald / Giemsa stain
↑micronuclei in polychromatic erythrocytes [M-PCE] (min 1000)

67. Carcinogenicity

68. Carcinogenicity
 For expected clinical use more than 6 months
 For drugs used frequently in an intermittent manner
 If concern about the carcinogenic potential due to :
 Previous demonstration in the product class
 Structure-activity relationship suggests carcinogenic
risk
 Preneoplastic lesions in repeated dose toxicity studies
 Long-term tissue retention results in reactions

69. Carcinogenicity
 Where life-expectancy in the indicated population is
short (i.e., less
than 2 - 3 years) - no long-term carcinogenicity studies
 Where therapy is generally successful, there may be later
concerns regarding secondary cancers. So
carcinogenicity studies needed
 A rodent species (preferably rat). Mouse only if justified
 Strain should not have normal incidence of spontaneous
tumors

70. Carcinogenicity
 At least 3 dose levels :
 Highest dose : sub-lethal, should not reduce life span
of animals by more than 10% of expected normal
 Lowest dose : should be comparable to the intended
human therapeutic dose or a multiple of it, e.g. 2.5x; to
make allowance for the sensitivity of the species
 Intermediate dose : placed logarithmically between the
other 2
 Untreated control and (if indicated) a Vehicle control
group

71. Carcinogenicity
 Life span comparable to human’s over which drug use is
intended
 Generally, 24 months for rats and 18 months for mice
 Atleast 50 animals of each sex
 Observation parameters
 Physiology, Behaviour, Biochemistry, Pathology
 Comprehensive descriptions of benign and malignant
tumour development, time of their detection, site,
dimensions, histological typing etc

72. Carcinogenicity

73. Requirements of Clinical
Trials and Marketing

74. Clinical trials & marketing- Requirements
Systemic Toxicity Studies -oral/parenteral/
transdermal route
Duration of
proposed
human
administration
Human phases
for which
study is
proposed to be
conducted
Species Long term
toxicity
requirement
Upto 1 week I, II, III 2 2 weeks
1-2 weeks ” ” 4 weeks
2-4 weeks ” ” 12 weeks
> 1 month ” ” 24 weeks

75. Systemic toxicity studies – Inhalational route
Duration of
proposed
human
administration
Human phases for
which study is
proposed to be
conducted
Species Long term
toxicity
requirments
Upto 2 wk I, II, III 2 1 month (exposure
time 3hr/d, 5d/wk)
Upto 4 wk I, II, III 2 3 months (exposure
time 6hr/d, 5d/wk)
> 4 wks I, II, III 2 6 months (exposure
time 6hr/d, 5d/wk)

76. Local Toxicity Studies
Route Duration
of Clinical
trial
Phase Species Duration of
Toxicity
study
Dermal
Ocular
Upto 2
weeks
I, II 1 4 wk
Otic
Nasal
Vaginal
Rectal
> 2 weeks
III 2 4 wk
I,II,III 2 12 wk

77. REFRENCES
• Hughes, W. William. 2005. Essentials of environmental toxicology: The Effects of
Environmentally Hazardous Substances on Human Health. Taylor & Francis, 325
Chestnut St., Suite 800, Philadelphia, Pa 19106. pp. 71-78
• Shaw, I. and J. Chadwick. 2002. Principles of Environmental Toxicology. Taylor &
Francis Ltd, 1 Gunpowder Square, London EC4A 3DF.
• Yu, Ming-Ho. 2005. Environmental toxicology: biological and health effects of
pollutants. 2nd edition. CRC Press LLC, 2000 N.W. Corporate Blvd., Boca Raton,
Florida 33431