PRINCIPLES FOR SAFETY ASSESSMENT OF

ADDITIVES IN FOOD- TOXICOLOGICAL

TESTING PROCEDURES
 Safety of Food Additives

• Rapid changes in diet and nutrition - With increase in processed and

packaged food intake, there has been a growing concern regarding
excessive use of food additives in food production- as many of these
additives may have adverse effects on human health if consumed in
excess
• Food additives- used safely - thoroughly studied, including extensive
toxicological testing, before they are approved for use in food.

• Testing includes short-term and long-term toxicity studies, including

carcinogenicity studies with a built in safety factor to account for
uncertainties.
 JECFA

• Joint FAO/WHO Expert Committee on Food Additives- International expert

scientific committee that is administered jointly by the Food and Agriculture
Organization of the United Nations (FAO) and the World Health Organization
(WHO)
• Global body that performs toxicological evaluations (risk assessment and exposure
assessment)- Meet once a year to review food additive safety data submitted
• Works in conjunction with Codex Committee on Food Additives (CCFA) - Has
evaluated more than 2,500 food additives
• JECFA Tasks:
1. Elaborate principles for evaluating the safety of food additives
2. Undertake toxicological evaluations of certain food additives
3. Review and prepare specifications for certain food additives
 Safety evaluation of food additives

• Two-stage process-

1st stage- Involves the collection of relevant data including the

results of studies on experimental animals and, where possible,
observations in man.

2nd stage - Involves the assessment of data to determine the

acceptability of the substance as a food additive.
Concept of periodic review:
JECFA indicated- may be necessary to carry out a periodic re- evaluation of substances
previously assessed by the Committee.
In addition to the continuing evaluation of food additives, that there would be a re-
evaluation process associated with the programme on food additive safety
assessment
Periodic review of past decisions on safety is made necessary by one or more of the
following developments
(a) New manufacturing process for the food additive.
(b) New specification.
(c) New data on the biological properties of the compound.
(d) Advances in scientific knowledge - nature or mode of action of food additives.
(e) Changes in consumption patterns or level of use of a food additive.
 CRITERIA FOR TESTING AND EVALUATION

•
Establishment of principles- for safety evaluation of food chemicals/additives –
1. Exposure data and
2. Potential toxicity (both should be considered)
Exposure Analysis:
• Exposure- defined as the “total intake of a chemical substance by human beings.

• Exposure to food additives present in foods is determined in three steps:

1. Estimating quantitatively the presence of an additive in individual foods and diets;

2. Estimating the intake patterns of the individual foods containing the relevant food
additives
3. Integration of the chance that consumers eating large amounts of the given foods and the
relevant food additive being present in these foods at high levels.
• Number of additives in use- High-estimating population exposure can be a
tedious and expensive exercise.
• CAC - prioritize the additives that can pose a greater risk.
• Priority should be given to-
- Authorized at high levels in highly consumed foodstuffs
- Authorized in highly consumed foodstuffs

- Having a low ADI

Types of Dietary Survey Methods

JECFA estimates exposure to food additives using three general types of approaches:
1. per capita estimates,
2. Estimates from dietary food intake surveys, and

3. Analytical values from total-diet surveys

per capita approach:
• 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.

Dietary Food intake survey:

• Surveys are performed on food- stuffs consumed by a representative group of individuals,
within a national population, over a short period of time, e.g., 1 - 14 days.
• 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.

Food sources of specific additives- identified

3. Duplicate portion study : requires the cooperation of a group of randomly
selected individuals, often from a group suspected to be at risk for consuming high
levels of selected food additives usually children and pregnant or lactating
women.

• Each participant is asked to collect a duplicate portion of all foods and beverages
consumed over a 24-hour period in a utensil provided by the investigator (several
consecutive 24 hour period considered)- homogenized and analysed .

• In addition, the participants are asked to make a written record of their daily food
intake to ascertain the foods and quantities consumed.

• Limitations :participants may alter their food consumption patterns during the
study. In addition, this the approach can prove to be very costly
Output of Exposure analysis:
EDI- Estimated Daily intake (mg/kg bw/week)

= Usage(mg/kg) * Consumption (kg/week)/ Body

weight(kg)
 Toxicological studies

1. Introduction to toxicology
2. Methods of toxicological evaluation:

a) Epidemiological studies
b) Animal studies

c) Cell culture studies

d) Acute Toxicity studies, Sub Chronic and Chronic toxicity
studies
e) Computational studies
Animal studies:
General principles of animal studies includes

1. For the risk assessment and safety evaluation of a new chemical

substances before human tests

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
2. Care, Maintenance and Housing
3. Selection of species, strains and sex:
• Rodents (rats) and Non-rodents (dogs)- both male and female category
• Healthy and should not been subjected to previous experimental procedures
• Important to consider test animals’ general sensitivity and responsiveness
of particular organs and tissues of test animals to toxic chemicals
• Selection of inbreed, outbreed or hybrid rodent strains for toxicity tests
should be based upon scientific reasons.
• Test animals comes from well-characterised and healthy colonies- survivability
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:

• Age of an individual- very important factor in the susceptibility to the effect of

toxicants—in the case of both test animals and humans. The reasons are the age-
related differences in the functioning of the nervous, respiratory, endocrinal, and
cardiovascular systems, gastrointestinal tract, and different membranous barriers. The
aging of an organism is accompanied by an alteration of the rates of substance
distribution, metabolism, detoxication, and excretion- young individuals are more
sensitive to toxic effects than adults

• Testing should be performed on young animals with dosing beginning as soon as

possible after weaning and following acclimation period of at least 5 days.

• Dosing of rodents should begin no later than 6 to 8 weeks of age, Dogs- Dosing
should begin no latter than 4 to 6 months of age.
5. Number and sex:
• 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.
• These recommendations will help to ensure that number of animals that
survive until end of the study – to ensure that surviving animals will be
sufficient to persist meaningful evaluation of toxicological effects.
6. Infected animals:
• It is not possible to treat animals for infection during course of study
without risking interaction between compound used for treatment and test
substance
(Interaction may complicate interpretation of study results)
7. Animal identification:

• Test animals should be characterisized by reference to their species, strain,

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.
• “Extrapolation” – used to describe how data obtained from animal studies
reliably can be used to apply to humans.
• Relationship between animal and human toxicity can be estimated most
accurately by using the principles of “interspecies extrapolation”
Why extrapolation?
• Though both anatomical and physiological similarities outnumber the
dissimilarities between humans and laboratory animals (rat) , thus
justifying the use of animals in the toxicological studies.
• At the same time, there are numerous qualitative as well as quantitative
interspecies differences that cannot be ignored at the extrapolation of the
results of the toxicological tests
• 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 response of humans to a chemical compound can differ from the response of an

animal both qualitatively and quantitatively.

• It is necessary to be extremely careful when interpreting the results of toxicity

studies on laboratory animals and extrapolation to humans.- design the experiment

to duplicate the potential exposure of humans as closely as possible.

• 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
where K is a species-specific factor

Calculation of Human equivalent dose

Human equivalent dose = animal dose in mg/kg * (animal weight in
kg/human weight in kg)^ 0.33
 Acute toxicity testing

• Commonly used in vivo tests for toxicity assessment

• 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.
• Study involves administration of single dose (at different levels) of
investigational product and determination of no. of deaths after 14 days
• In addition to mortality and weight, periodic examination of test animals-
conducted for signs of intoxication : Lethargy, behavioral modifications
• Outcome of Acute toxicity testing- LD50 Parameter
LD50 – Median Lethal Dose

• LD- stands for Lethal dose- amount of investigational product, given all at once,
which causes death of 50% of a group test animals.
• In terms of signs or symptoms, LD50 is redefined as the dose active for
producing a certain mark in 50% of the experimental animals
Clinical manifestations to be observed:
Structure/Function affected Possible Effects
Respiratory Changes in rate/depth of breathing
Motor activities Changes in frequency and nature of
movement

Reflex Response to external stimuli

Ocular Tearing
Cardiovascular Changes in heart rate
Gastrointestinal Vomiting, cramps
Dermal Swelling/redness
• LD50- index of toxicity – Lower the lethal dose, more toxic is substance
• Expressed as amount of substances (mg) administered per 100 gm (for
smaller animals) or per kg (for biggest test animals) of body weight of
test animal
• LD50- can be found for any route of entry or administration but dermal
or oral route administration- most common
Purpose of LD50 study:
• To compare toxic potency or intensity of different chemicals
• To measure how much of chemical is required to cause death in test
animals
Methods to determine LD50 :
• Karber’s method
• Up and Down procedure
• Fixed dose method
• Reed munech method
• Miller and Tinter method
• Lorke method
Karber’s method:
• Dose must be increased with geometric proportion series
• Number of animals in every group must be equal
Arithmetic Karber’s method:
• The interval mean of the no. dead in each group of animals was used as

well as the difference between the doses for the same interval.

• The product of interval mean and dose difference was obtained.

• 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.
• Each group divided to 6 subgroups of 6 animals were received orally 5 different
dosages of each dye alone as following (1250 mg/kg ,2500 mg/kg ,3750 mg/kg ,
5000 mg/kg , 6250 mg/kg)
• Mice in the control group received distilled water .
• 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
Group Dose (mg/kg) Number of mice No of dead
in group

1 1000 10 2

2 1100 10 3

3 1200 10 4

4 1300 10 6

5 1400 10 8
 MILLER AND TAINTER METHOD

• Involves the Conversion of percentage mortality into probability units (probits)

by referring to appropriate tables.
• Obtained probit values are plotted against log-doses- to get Log dose probit
graph
• Probit 5 (equivalent to 50% mortality) on Y-axis is interpolated to X-axis to get
log LD50 value, antilog of which gives LD50
 LORKE’S METHOD

• 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

• Classical or traditional method requires 15-30 animals per group- to

calculate single LD50- because method relies on analysis of group
responses.
• Alternate methods- developed- estimation of LD50 by analyzing
response of individual animals
• Up and down procedure- Single animal exposed per dosage, but
subsequent dosage were adjusted up and down by constant factor
depending on outcome of previous dosage.
Principle:
• 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)
• If it dies, dose for the next is decreased by similar dose progression.
• Decision will be based on 24 hour survival pattern of an animal.
Limit test:

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:
Individual animal data should be provided. Additionally, all data should be
summarised in tabular form, showing for each test concentration the
number of animals used, the number of animals displaying signs of
toxicity, the number of animals found dead during the test or killed for
humane reasons, time of death of individual animals, a description and the
time course of toxic effects and reversibility, and necropsy findings

LD50 – calculated using Software

 FIXED DOSE PROCEDURE
• Purpose: Minimizing the number of animals required to estimate acute oral
toxicity of chemical- does not use death as end point- involves observation of
clear signs of toxicity developed at one of series of fixed doses.
Procedure:
• Test substance is given at one of four dose levels (5mg, 50, 500, and 2000
mg/kg )- exceptionally an additional dose of 5000 mg/kg may also be
considered, to five male and five female rats.
• Initial dose level selected as dose expected to produce some signs of toxicity
without causing severe toxic effects or mortality based on in vivo or in vitro
data (if no information exists, then the starting dose will be 300 mg/kg)
• Objective- to identify a dose that produces clear signs of toxicity but no
mortality.
• Depending on results from first test: Either no further testing is needed (or)
Higher or lower dose is tested
• If mortality occurs at chosen dose- retesting at lower dose level is
necessary (except if the original dose chosen is 5 mg/kg)
• If no signs of toxicity occurs at initial dose level – it is necessary to retest
at higher dose levels.
• Based on results- possible to assign chemical to one of OECD
classification of chemicals.
 ACUTE TOXIC CLASS METHOD

• 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:
1. No further testing is required

2. Dosing of three additional animals, with the same dose

3. Dosing of three additional animals at next higher or next lower dose
level.
• LD50 for food additives- limited value, because

Single dose experiment- no meaning in case of food additives - other than

studying distribution and metabolism- any food additive- low toxic
potential- excessive amounts have to be administered before observation

Limitations:
• Interpretation of experiments involving use of massive doses – difficult
• Information from acute toxicity studies on food additives – demonstrates
large difference in toxicity following oral and parenteral administration
(intestinal mucosa or liver may have barrier function)
Reference:
http://sphinxsai.com/2016/ph_vol9_no4/2/(364-367)V9N4PT
.pdf
http://www.authorstream.com/Presentation/patelcharmi91-
1826002-methods-determine-ld50/
https://books.google.co.in/books?
id=l0UpBY6thl8C&pg=PA50&lpg=PA50&dq=Determination+
of+LD50+from+Acute+toxicity+studies+food+additives&sou
rce=bl&ots=j8kQwslBMq&sig=EfhTnEuoPWy8S_XeYjVsm_v
RO_Y&hl=en&sa=X&ved=0ahUKEwjIhtea0qTOAhVFqo8KHS
LIAHoQ6AEIMzAD#v=onepage&q=Determination%20of
%20LD50%20from%20Acute%20toxicity%20studies
%20food%20additives&f=false
Sub chronic and Chronic toxicity studies

1. Sub chronic and Chronic Toxicity studies -

Refer Google books (From URL)

2. Dose response curve- Refer PDF

3. Role of FSSAI in food additive regulation-

Refer PDF