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Toxicological Studies
Toxicological Studies
Toxicological Studies
• Global body that performs toxicological evaluations (risk assessment and exposure
assessment)- Meet once a year to review food additive safety data submitted
• JECFA Tasks:
• Two-stage process-
JECFA estimates exposure to food additives using three general types of approaches:
• Using this approach, it is assumed that the intake of a food additive is equally distributed
across the population.
• For example, per capita intake for a nation may be calculated by dividing the total yearly
production volume, corrected for imports and exports, of an additive used in food, within a
nation, by the national population.
• Intake of an additive per food type, can be calculated by multiplying the usual additive
level in each type of food by the dietary intake of the food- intakes per food type can then
be summed to derive a total additive or contaminant intake.
• Advantage of the dietary survey approach - additive intakes for selected subpopulations,
such as different age groups or high-frequency consumers of certain foodstuffs, may be
computed
Total diet survey:
1. Market basket study - approach where food items that are part of the average
diet of the selected age and sex group of interest are purchased from retail
outlets of the country, on one or more occasions per year- analyzed - estimating
the actual level of additive or contaminant in a selected total diet.
2. Individual food items - approach where a list of the most commonly consumed
food items is compiled from national food consumption surveys.
Samples of each of these food items are then collected, sometimes more than once
a year, from major cities of the identified country- analyzed in central labs.
1. Introduction to toxicology
a) Epidemiological studies
b) Animal studies
e) Computational studies
Animal studies:
2. For the estimation of the NOAEL values of food additives before taking
into use
3. For the risk assessment of various chemicals used in the industry and in
the environment
General guidelines for designing and conducting toxicity studies in
animals
1.Good laboratory practice
• Rodents (rats) and Non-rodents (dogs)- both male and female category
problems exist for some strains of rats- it should be selected that are likely to
achieve recommended duration of study
• FDA encourages petitioners and notifiers to consult with agency scientist before
toxicity testing is began- to ensure appropriateness of particular species, strains or
sub-strains
4. Age:
• Equal number of males and females of each species and strains should be
used for the study.
• For sub chronic toxicity studies – experimental and control groups should
have at least 20 rodents per sex per group (or) at least 4 dogs per sex per
group
• For study that involves finding the range (or) when long term studies are
anticipated – Ten rodents/sex/group may be acceptable.
7. Animal identification:
sex, age and weight- Each animal must be assigned unique identification
number
Extrapolation of Animal models to Humans:
• When experimental results have been generated in animal model, they have
to be validated with respect to their applicability to target species , which is
normally humans.
• One of the most commonly used species in toxicology research, rat, differs
substantially from humans: it lacks gall bladder, very effective biliary
excretor, displays less efficient plasma binding of proteins, Nocturnal,
different location of gut flora, different skin characteristics.
• The sensitivity of different animal species, including Homo sapiens, to one and the
same toxicant can be very different. Such variability can be caused by differences in
the absorption, metabolism, and excretion of the compound and its metabolites. The
• Route of exposure must be the same, the age of animals should relate to that of
humans, for most routine tests both genders are used, dose levels are usually
selected so as to determine the threshold doses as well as dose–response
relationship.
INTERSPECIES COMPARISONS BASED ON BODY WEIGHT
• First step in the search for a common denominator upon which to base
interspecies comparisons - body weight (normalizing factor)
• Numerous biological parameters- liver weight, water intake, creatinine
clearance, total nitrogen output, and hemoglobin synthesis - can be
expressed as a mathematical function of body weight
• But physiologic and metabolic processes such as renal function, metabolic
rate, and cardiac function are not directly proportional to body weight.
• Unfortunately, direct conversion of animal toxicity to humans on the basis
of body weight is common- same dosage in mg/kg bw serves as the basis
for this extrapolation. For human safety reasons, it is considered that
humans are 10 times more sensitive than animals.
• Another basis may be the body area and the unit mg/m2. For the
calculation of the body area, the following equation is used:
m2 = K × w/100
• Simplest and most commonly applied toxicity test- Single exposure study with
death as criteria for toxicity
• Recommended- carried out with two different animal species- Rodents and non-
rodents- most commonly used- mice, rats, rabbits and dog.
• LD- stands for Lethal dose- amount of investigational product, given all at once,
which causes death of 50% of a group test animals.
• LD50- can be found for any route of entry or administration but dermal
or oral route administration- most common
• Lorke method
Karber’s method:
well as the difference between the doses for the same interval.
• The sum of the product was divided by number of animals in the group &
the resulting quotient was subtracted from the Ldy in order to obtain LD 50
value.
Study model- Determination of LD50 for Tartrazine and carmoisine
• Male white mice were randomly divided into tartrazine group & carmoisine
group.
• Observation period- 3 days - signs and symptoms of toxicity as well as the death
rate of each group were recorded
Limitations of Karber’s method:
Too many animals are sacrificed to determine LD50 value.
Calculate LD50 value for following table by Karber’s method
1 1000 10 2
2 1100 10 3
3 1200 10 4
4 1300 10 6
5 1400 10 8
MILLER AND TAINTER METHOD
• Obtained probit values are plotted against log-doses- to get Log dose probit
graph
• Test is carried out with death of an animal as criteria for toxicity- involves two phases
Phase I – Nine mice divided into three groups of three mice each and are given 10, 100 and 1000
mg/kg body weight of test substance
• After administration of test substance- observation is made at regular interval to check for
onset of adverse effect, time to death {Period of observation in phase I- 24 hours}
Phase II – involves use of three animals divided into three groups
• Dose level used in this phase – either step up or step down depending on outcome of the result
obtained from phase I
• Animals are administered higher dose of 1600, 2900 and 5000 mg/kg of body weight
• Toxic symptoms – observed for 24 hours as well as delayed toxic symptoms for 7-14 days
Lethal dose = Geometric mean of {Lower dose that does not produce
mortality and Higher dose that produces mortality}
Alternate method- UP and Down Procedure
• Test consist of single ordered dose progression in which animals are dosed, one
at a time, at a minimum of 24 hour interval.
• For selecting the starting dose, all available information should be used, When
the information suggests that mortality is unlikely then a limit test should be
conducted. When there is no information on the substance to be tested, for
animal welfare reasons it is recommended to use the starting dose of 200 or 500
mg/kg body weight.
• If animal survives, dose for the next animal is increased by a factor (3.2 times of
that of original dose)
Doses should not exceed 2000 mg/kg which is considered the upper limit dose. When the
first animal is dosed with the upper limit dose and survives, the second animal receives
the same dose. When a total of three animals have been dosed with the limit dose and
no deaths have occurred, then three animals of the other sex should be tested at the limit
dose level. If there is again no lethality, the test can be terminated.
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- observations includes mortality and
clinical signs
All animals, including those which die during the test or are killed for animal welfare
reasons during the test and those that survive at day 14, are subjected to pathological
examination
Data:
• Method not only uses death but also clinical signs as end point of toxicity
• Stepwise approach with the use of minimum number of animals per step, sufficient
information is obtained on acute toxicity of test substance to enable its
classification.
• Three animals of one sex are used for each step. Normally, females are used
(considered to be slightly more sensitive)
• Initial dose- selected from one of four fixed dose levels- 5mg, 50, 300 and 5000
mg/kg of body weight.
• Substance is administered orally to group of experimental animals at one of the
defined doses
• Absence or presence of compound related mortality of animals dosed at one step
will determine next step
Either:
Limitations: