Breast cancer -ONCO IN MEDICAL AND SURGICAL NURSING.pptx
OECD Guidline on acute and chronic toxicity
2. CONTENTS
2
INTRODUCTION TO TOXICOLOGY
OECD GUIDELINE FOR ACUTE ORAL TOXICITY
LD50
LD50/LC50
Methods to calculate LD50
Limitation of LD50
How OECD GUIDELINES More Humane?
Alternatives to use of Animal in AOT
Description of Whole AOT Guidelines along with
Its Sighting Study (Guidelines no. 401,420,423,425)
OECD GUIDELINES FOR CARCINOGENICITY (451)
(Principle ,Housing –Feeding,Objectives,
Procedure,Result,DiscussIon)
OECD GUIDELINES FOR CHRONIC TOXICITY (452)
(Principle ,Housing –Feeding,Objectives,
Procedure,Result,DiscussIon)
3. WHAT IS TOXICOLOGY?
Toxicology is the scientific
study of adverse effects that
occur in living organisms due
to chemicals. It involves
observing and reporting
symptoms, mechanisms,
detection and treatments of
toxic substances, in
particular relation to the
poisoning of humans.
3
4. ADVERSE DRUG EFFECTS
Any undesirable &/ or unintended effects
of drug,
1. Predictable (type A reactions)
2. Non-predictable (type B reactions)
SIDE EFFECTS
Unwanted but
often
unavoidable
effects at
therapeutic
doses.
SECONDARY
EFFECTS
- Indirect
consequences
of primary
action of drug.
TOXIC EFFECTS –
Are results of
excessive
pharmacological
effect of drug
due to over
dosage or
prolonged use.
4
(Barar,2005; Ghosh, 2005)
5. ANIMAL TOXICITY TESTS
Acute toxicity 14 days
Sub-acute (repeated doses)
toxicity 28 days
Sub-chronic toxicity 3 months
Chronic toxicity 6 months to 2
Special toxicity e.g. Carcinogenicity
5
(Curtis,2003;Ghosh,2005)
7. ACUTE TOXICITY SYMPTOMS
Tremor
Salivation
Muscle
spasm
Convulsion
Lacrima
tion
Weight
loss
Diarrhoea
Altered
Respiration
Loss of
righting
reflex
7
(Ghosh., 2005)
8. WHAT IS LD50?
LD50 represents the individual dose required to kill 50
percent of a population of test animals. It is an index
determination of medicine and poison’s virulence. lower
the LD50 dose, the more toxic the pesticide.
WHAT IS LC50?
The concentrations of the chemical in air that kills 50%
of the test animals during the observation period is the
LC50 value. Other durations of exposure (versus the
traditional 4 hours) may apply depending on specific
laws.
8
(P.A. Botham et al. 2004)
10. METHODS TO DETERMINE LD50
Karber’s method
Miller and Tainter method
Lorke’s method
Fixed dose method
UP and down method
10
OECD,1987; Ghosh, 2005)
11. KARBER`S METHOD
This method is also known as ARITHMATIC
method.
Simplest & rapid.
It does not involve any plotting of dose-response
curve.
It is useful method particularly when number of
animals is small.
The interval mean of the no. of dead in each group
of animals was used as well as the differences
between doses for the same interval.
The number of animal in every group must be equal.
11
12. LD50 CALCULATION BY KARBER`S METHOD
Group Dose
Mg/kg
No. of
animal
Dose
difference
(a)
dead Mean
mortality
(b)
Product
(a.b)
1 64 10 - 0 - -
2 71 10 7 2 1 7
3 81 10 10 4 3 30
4 90 10 9 9 6.5 58.5
5 100 10 10 10 9.5 95
LD50 =
100 – {€a.b/n}
n is the
number
of animals
12
13. MILLER AND TAINTER METHOD
Also called as Graphical method Simple & accurate
enough.
The observed % mortality is converted into probit (
deviation from the mean) by referring to the table.
Percentage dead for 0% & 100% are corrected.
The probit value thus obtained are plotted against
log dose.
And then dose corresponding to probit 5 (50%) is
found out.
The LD50 may be determined from the graph if the
line is straight enough.
13
14. LD50 CALCULATION BY GRAPHICAL
METHOD
Group Dose
Mg/kg
Log
dose
Dead/
total
%
Dead
correct
ed %
dead
probit
1 64 1.81 0/10 0 2.5 -
2 71 1.85 2/10 20 20 4.16
3 81 1.91 4/10 40 40 4.75
4 90 1.95 9/10 90 90 6.28
5 100 2.00 10/10 100 97.5 -
corrected
formula
for
0 % = 100(0.25/n)
corrected
formula
for 100% =
100{(n –0.25) /n}
14
17. LORKE‘S METHOD
Phase 1: 3 groups of mice are prepared .one
dose was given to each group i.p The treated
mice were monitored for 24hr for mortality and
general behavior .
Phase 2: After 24 hr 3-4 groups of one mouse
were given doses based on the findings of phase
1. I.p . The mice were again monitored for 24 hrs
.The geographic mean of least dose that killed
mice and the highest dose that did not kill mice
was taken as the median lethal dose
17
18. ALTERNATIVE METHOD
(1) FIXED DOSE PROCEDURE
This method does not use death as an end points
instead it uses the observation of clear signs of
toxicity developed at one of a series of fixed dose
levels to estimate the LD50
(2) UP AND DOWN METHOD (Staircase method)
Two mice were injected with a particular dose and
observed for a period of 24 hrs for any mortality .
18
19. The subsequent doses were then increased by
a factor 3.2 if the dose was tolerated ,or if not
then decreased by a factor of 0.5. The max.
Non lethal and the min lethal doses were thus
determined using only 15 mice.
A final and more reliable LD50 assay was
planned using at least 3 or 4 dose levels within
the range of max non lethal and min doses
19
20. LD50 LIMITATIONS
LD50 is somewhat unreliable and results may vary greatly
between testing facilities due to factors such as the genetic
characteristics of the sample population, animal species
tested, environmental factors and mode of administration
There can be wide variability between species as well; what
is relatively safe for rats may very well be extremely toxic
for humans (cf. paracetamol toxicity), and vice versa. For
example, chocolate, comparatively harmless to humans, is
known to be toxic to many animals. When used to
test venom from venomous creatures, such as snakes,
LD50 results may be misleading due to the physiological
differences between mice, rats, and humans.
20
21. HOW OECD GUIDELINES FOR
ACUTE TOXICITY MORE HUMANE ?
A humane endpoint can be defined as the earliest
indicator in an animal experiment of severe pain, severe
distress, suffering, or impending death.
OECD Test Guidelines do not require death as an
endpoint. Animals humanely killed during the test will
be regarded as dosage-dependent deaths.
Three alternative test methods (Guidelines 420, 423,
and 425) to the traditional acute oral toxicity
test have been adopted by the OECD. One of these, the
Fixed Dose Procedure (Guideline 420).
21
22. CONTINUE…
Refinement of the traditional acute oral test in that it
requires fewer, but fixed, dosage groups to be tested, and
thus fewer animals. It also employs non-lethal endpoints to
determine the toxicity of the test substance.
Two other methods, the Acute Toxic Class Method
(Guideline 423) and the Up-and-Down Procedure (Guideline
425), use impending death as the only endpoint.
Series on Testing and Assessment: Testing for Human
Health in that guidline no. 19 contains Guidance document
on the recognition, assessment and use of clinical signs as
humane endpoints
22
23. CONTINUE…
GUIDLINES NUMBER OF
ANIMAL PER
TEST
NUMBER OF
DEATH PER
TEST
AIM
401 Up to 25 Up to 12 -
420 5-7 1
Endpoint-
evident
toxicity
423 Average 7 2-3 -
425 6-9 2-3
Stopping
criteria to
limit number
of
animals used
Animal welfare considerations,
All three alternatives contain requirement to
follow OECD Document on Humane Endpoints.
23
24. SOME ALTERNATIVES TO THE
USE OF ANIMALS IN TESTING
in vitro (test tube) test methods and models based on
human cell and tissue cultures.
computerized patient-drug databases and virtual drug
trials.
computer models and simulations.
stem cell and genetic testing methods.
non-invasive imaging techniques such as MRIs and CT
Scans.
microdosing (in which humans are given very low
quantities of a drug to test the effects on the body on
the cellular level, without affecting the whole body
system).
24
25. ICCVAM
The need for alternatives to the traditional use of animals in
toxicity testing was officially recognized by the U.S. government
in 1993 with passage of the NIH Reauthorization Act.
Requirements under the Act lead to the establishment of
committee called the Interagency Coordinating Committee for
the Validation of Alternative Methods (ICCVAM).
To Providing guidance to test method developers
To Induce use of alternative tests that encourage the reduction,
refinement, or replacement of animal test methods
Evaluating recommendations from expert peer reviews of
alternative toxicological test methods
25
26. OECD GUIDELINES FOR ACUTE
ORAL TOXICITY
Number Title Original
adoption
Number of
updates
Most recently
updated
401
Acute oral
toxicity-
conventional
acute
toxicity test
12 may 1981 1
Date of
deletion: 20
December
2002
420
Acute oral
toxicity- fixed
dose
procedure
17 July 1992 1
17 December
2001
423
Acute oral
toxicity-
acute toxic
class method
22 march
1996
1
17 December
2001
425
Acute oral
toxicity- up
and down
procedure
21
September
1998
2
23 March
2006
26
27. DESIGN OF ACUTE TOXICITY
14 days study.
Study on at least two species.
One rodent –mice/rat.
One non rodent –usually rabbit.
Dose administered orally & parenterally.
Various dose levels to groups of both sexes.
Dose selection such that causing less than 50% but not
0% and more than 50% but not 100% mortality.
27
(Ghosh,2005)
28. ACUTE ORAL TOXICITY
OECD GUIDELINES NO.401
(CONVENTIONAL ACUTE TOXICITY METHOD )
In a study of toxic characteristics of substance, acute
oral toxicity testing is initial step.
Gives information on health hazards.
Test substance administered orally, in graduated
doses to several groups of experimental animals.
One dose used per group.
28
(OECD,1987)
29. CONTINUE…
At least 5 rodents at each dose level of same
sex are used.
Observations for effects & death are made.
After completion of study in one sex, study in
another sex is carried out.
Studies suggested in rodents but can be adopted
for studies in non-rodents.
29
30. ACUTE ORAL TOXICITY
GUIDLINE NO. 420 (FIXED DOSE
PROCEDURE)
New approach in 1984 by British toxicology society
based on administration of series of fixed dose
levels.
Instead of death, clear signs of toxicity to animals
as end point.
Adopted as 1st alternative to conventional acute
toxicity test.
Testing in 1 sex usually females is considered
sufficient.
Uses fewer animals.
30
(OECD, 2001)
31. CONTINUE…
Reproducible procedure.
Causes less suffering to the animals.
Uses only moderately toxic doses, doses expected to be
lethal should be avoided.
31
37. ACUTE ORAL TOXICITY
GUIDLINE NO.423 (ACUTE TOXIC
CLASS METHOD)
No sighting study.
3 animals of single sex per step.
On avg. 2-4 steps may be necessary to allow judgment
on the acute toxicity of the test substance.
Not intended to allow the calculation of precise LD50 .
Death of a proportion of animals as the major end
point (response).
Ld50 cut off values are indicated .
37
(OECD, 2001)
38. PRINCIPLE
Stepwise procedure with the use of minimum no.
of animals per step.
Substance administered orally to 2 groups of
animals at defined doses .
3 animals per step of single sex (normally
females).
Compound related mortality determines the next
step.
Report
38
42. ACUTE ORAL TOXICITY
GUIDLINE NO.425 (UP AND DOWN
METHOD)
Up and down testing approach was 1st described by
Dixon and Mood.
Bruce in 1985 proposed to use it for acute toxicity
determination of chemicals .
Estimates confidence intervals for LD50 .
42
(OECD2006)
43. CONTINUE …
In procedure (main test ) 1-animal dosed at a
time, at minimum of 48 hrs interval.
Suggested starting dose is 175 mg/kg or can
be selected from 1.75, 5.5, 17.5,
55,175,550,2000mg/kg .
Animal receives 1st dose a step below the
level of the best estimate of LD50 .
43
44. CONTINUE …
Depending upon the outcome for the previous
animal, the dose for the next animal is adjusted
up or down.
5 reversal in 6 consecutive animals when
obtained test is terminated.
No. of animals limited to 15.
Report
AOT 425 START PROG.
44
45. COMPARISON OF GUIDELINES OF LD50
No 401 420 423 425
Sighting study Absent Present Absent Absent
Preferred dose 2 to 5000mg/kg
per animal
5 to 5000 mg/kg
per animal
5 to 5000 mg/kg
per animal
1st animal dose
less than
estimated Ld 50
outcome by
factor 3.2
Drug effect Dose causing
high mortality
Dose causing
evident toxicity
Dose causing
mortality
Stopped if no
reversal occur
by reaching
selected upper
limit
Grouping of
animal
5 animals per
group
Group of 5
animal of 1 sex
3 animals per
group
1 animal per
group
Estimation of
end point
Up to 12 animal
expected to die
1 animal
expected to die
2,3 animal
expected to die
2,3 animal
expected to die
Limitation It used high
animal mortality
as endpoint, at
high dose, so
this method
require
reduction or
refinement.
It used evident
toxicity as
endpoint instead
of death dose
level close to
lethal dose not
always obtained
Finding of a
delayed death
may require
additional lower
dose level to be
used
No limitation
45
(R. L. LIPNICK et al.1995)
46. LIMITATION OF METHODS
USED FOR TOXICITY STUDY
Indicated that all are likely to perform poorly for
chemicals with shallow dose-response slopes
Because Guideline 420 uses evident toxicity as an
endpoint instead of death.
Unusually test substances may cause delayed
deaths (5 days or more after test substance
administration) mostly in case of guidline no.425
However, both in Guideline 420 and 423, the finding
of a delayed death may require additional lower
dose levels to be used or a study to be repeated.
46
47. CARCINOGENICITY STUDY
The test substance is administered daily in graduated doses
Observed closely for signs of toxicity and for the development of
neoplastic lesions.
Died or are killed animals are necropsied and at the conclusion
surviving animals are also killed and necropsied.
PRINCIPLE
ROUTES OF
ADMINISTRATION
451 451
47
www.OECD.org
48. OBJECTIVES
carcinogenic
properties of
chemicals
target organ(s) of
carcinogenicity
NOAEL or point of
departure for
establishment of a
BMD
extrapolation of
carcinogenic effects
to low dose human
exposure levels
provision of data to
test hypotheses
regarding mode of
action
OBJECTIVES
48
49. HOUSING AND FEEDING
housed individually or in a group of 3
Maintain temperature at 22˚C (± 3˚C).
relative humidity should be at least 30% (50-60%)
artificial lighting, 12 hours light and 12 hours dark cycle
conventional laboratory diets + unlimited supply of drinking water
Analytical information on the contaminant levels should be generated
periodically
49
50. ANIMAL TO BE USED,
Rodents/non-rodents used
12 months study for rodents
90 days study for non-rodents
Mostly mice are used due to short life-span and
susceptibility
Healthy animals, acclimated to laboratory conditions
for 7 days and not been subjected to previous
experimental procedures
the weight variation of animals used should not
exceed ± 20 % of the mean weight
Animals should be randomly assigned
50
51. DOSE GROUP AND DOSAGE
51
3 Dose levels
short-term repeated dose
Toxicological existing
data
Toxicokinetics existing
data
Range finding studies
Highest dose level to identify the principal target
organs and toxic effects (avoid severe toxicity,
morbidity, or death)
STUDY DURATION
RATS
• 24 MONTHS
HAMSTERS
• 18 MONTHS
MICE
• 18 MONTHS
51
52. Nature, severity,
and duration of
clinical
observations
(whether
transitory or
permanent)
signs of toxicity Incidence of any
abnormality
52
54. RESULT
Survival data Toxicokinetic
data
Body weight
changes
General
Food
consumption,
calculations of
food efficiency,
water
consumption
54
55. body weights and
organ weights
Tumour
incidences
Statistical
results
feed consumption
(or water
consumption)
Dose
response
curve
Histori
cal
control
data
Mode
of
action
of drug
Relevan
ce for
humans
DISCUSSION
55
56. CHRONIC TOXICITY STUDY
The test substance is administered daily in graduated doses to
several groups of experimental animals for a period of 12
months
During the period of administration the animals are observed
closely for signs of toxicity. Animals which die or are killed
during the test are necropsied and, at the conclusion of the
test, surviving animals are also killed and necropsied.
452 452PRINCIPLE
ROUTES OF
ADMINISTRATION
56
www.OECD.org
57. PREPARATION OF DOSE AND
DURATION OF STUDY
The test substance is normally administered orally, by
gavage or via the diet or drinking water.
When the test substance is administered by gavage to the
animals this should be done using a stomach tube or a
suitable intubation cannula, at similar times each day.
Normally the volume should be kept as low as practical,
and should not exceed 1 ml/100g body weight, except in
the case of aqueous solutions where 2 ml/100g body weight
may be used.
While this Test Guideline primarily is designed as a 12
month chronic toxicity study, the study design,
also allows for and can be applied to either shorter
(e.g. 6 or 9 months) or longer (e.g., 18 or 24 months)
57
59. parameters
Body weight,
food/water
consumption
and food
efficiency.
(At least
once a week
for the first 13
weeks)
Haematology
and clinical
biochemistry
(10 females
and 10 males)
Total erythrocyte
,leucocytes and
platelets count,
erythrocytes
morphology
,glucose ,urea ,
creatinine
albumine ,total
protiens count
,total cholesterol
count, total bile
level , presence of
enzymes
Appearance,Volume,
Specific gravity,pH,
Bilirubine,Glucose,
Occult blood
Urine
analysis
59
60. Survival data; urinalysis tests
Outcome of any investigations of neurotoxicity or
immunotoxicity
Terminal body weight; organ weights
Necropsy findings; A detailed description of all
treatment-related histopathological findings
Absorption data if available
Body weight/body weight changes
Toxic response data by sex and dose level,
including signs of toxicity
Duration of clinical observations
Ophthalmological examination
Haematological tests
Clinical biochemistry tests
RESULT
60