toxicological studies

1. Introduction Background
summary of recommendations for
the minimum toxicity tests to be
performed for safety evaluation
of direct food additives and color
additives used in food based on
their levels of concern
Safety evaluation for a direct
food additive or color additive used in
food involves assigning the additive
to a Concern Level
low (I),
intermediate (II) or
high (III)) based on
Chem structure
Recommended Toxicological Testing for Additives Used
in Food

2. Background
Safety evaluation for a direct food additive or color additive used in food
involves assigning the additive to a Concern Level:
(i.e., low (I), intermediate (II) or high (III)) based on information on the
additive's toxicological potential predicted from its chemical structure:
(i.e., low (A), intermediate (B), or high (C)) and an estimation of cumulative
human exposure.(Please :Refer to journal as attached)
For additional details, see chapter III from the draft 1993 Redbook II.
Frequently, exposure information has more weight than structure alert
information in assigning additives to a Concern Level.
If available, other information may be considered when setting the concern
level for a food or color additive, and final safety decisions are made on a
case-by-case basis

3. Toxicity Tests Concern
Level
Low
(I)
Concern
Level
Intermediate
(II)
Concern
Level
High
(III)
Genetic Toxicity Tests X X X
Short-term toxicity tests with rodents Xc
Xa,c
Xa,c
Subchronic toxicity studies with rodents Xc
Xa,c
Subchronic toxicity studies with non-rodents Xc
Xa,c
One-year toxicity studies with non-rodents Xc
Chronic toxicity or Combined chronic
toxicity/carcinogenicity studies with rodents
Xc
Carcinogenicity studies with rodents X
Reproduction studies Xc
Xc
Developmental toxicity studies Xb,c
Xb,c
Metabolism and Pharmacokinetic studies Xb
Xb
Human studies Xb
Recommended Toxicological Testing Summary Table for Additives
Used in Food
(a)If needed as preliminary to further study, (b)If indicated by available data or information
(c) Including screens for neurotoxicity and immunotoxicity

4. Concern Levels (CL) as Related to Human Exposure
and Chemical Structure

5. Toxicity Tests Recommended Test/Requirement
Genetic Toxicity Tests
Purpose:to detect the induction of
chromosomal aberrations are
performed using cells exposed to
chemicals in vitro or in vivo.
(daily intake exceeds 50 ppb in the
diet (150 g per person per day).
Level: (CL I), (CL II), (CL III)
1) a test for gene mutations in bacteria (2-3 days)
The bacterial reverse mutation test uses amino acid-requiring
strains of Salmonella typhimurium (S. typhimurium) and
Escherichia coli (E. coli) to detect point
mutations/substitution/addition/deletion -DNA
2) a) an in vitro test with cytogenetic evaluation of chromosomal
damage using mammalian cells (2-3 days)
to identify agents that cause structural chromosomal aberrations
in cultured mammalian cells OR
b) (i) an in vitro mouse lymphoma thymidine kinase+/- gene
mutation assay(the mouse lymphoma)assay is preferred)
(1-2 days)
to detect gene alterations induced by chemical substances. AND
(ii) an in vivo test for chromosomal damage using
mammalian hematopoietic cells. (4 weeks)
To detect of damage induced by the test substance to the
chromosomes or the mitotic apparatus of erythroblasts by
analysis of erythrocytes as sampled in bone marrow and/or
peripheral blood cells of animals, usually rodents.
Recommended Toxicological Testing Summary Table
for Additives Used in Food

6. Toxicity Tests Recommended Test/Requirement
Short-term toxicity tests with
rodents
(GLP)
Purpose:
1) can help predict appropriate doses of the
test substance for future subchronic or
chronic toxicity studies,
(2) can be used to determine NOELs for
some toxicology endpoints, and
(3) allow future studies in rodents to be
designed with special emphasis on
identified target organs.
Duration of Testing (7d/week, 4 weeks)
Level: (CL I), (CL II), (CL III)
Test Animals
A. Care, Maintenance and Housing
B. Selection of Rodent Species, Strains and Sex
C. Age
D. Number and Sex
E. Infected Animals
F. Animal Identification
H. Diet
I. Assignment of Control and Compound Treated Animals
J. Mortality
K. Autolysis
L. Necropsy
Test Substance
A. Identity
B. Composition/Purity
C. Conditions of Storage
D. Expiration Date
Experimental Design
A. Duration of Testing (7d/week, 4 weeks)
B. Route of Administration
C. Dose groups
D. Computerized systems
Observations and Clinical Tests
A. Observations of Test Animals:
B. Body Weight and Feed Intake Data
C. Clinical Testing
Necropsy and Microscopic Examination
A. Gross Necropsy
B. Organ Weight
C. Preparation of Tissues for Microscopic Examination
D. Microscopic Evaluation
E. Histopathology of Lymphoid Organs

7. Toxicity Tests Recommended Test/Requirement
Subchronic toxicity studies
with rodents
(GLP)
Purpose:
(1) can help predict appropriate doses of
the test substance for future chronic
toxicity studies,
(2) can be used to determine NOELs for
some toxicology endpoints, and
(3) allow future long-term toxicity studies
in rodents and non-rodents to be designed
with special emphasis on identified target
organs.
Duration of Testing (7d/week, 90 days = 3
months)
Level: (CL II), (CL III)
Test Animals
A. Care, Maintenance and Housing
B. Selection of Rodent Species, Strains and Sex
C. Age
D. Number and Sex
E. Infected Animals
F. Animal Identification
H. Diet
I. Assignment of Control and Compound Treated Animals
J. Mortality
K. Autolysis
L. Necropsy
Test Substance
A. Identity
B. Composition/Purity
C. Conditions of Storage
D. Expiration Date
Experimental Design
A. Duration of Testing (7d/week, 90 days = 3 months)
B. Route of Administration
C. Dose groups
D. Computerized systems
Observations and Clinical Tests
A. Observations of Test Animals:
B. Body Weight and Feed Intake Data
C. Clinical Testing
Necropsy and Microscopic Examination
A. Gross Necropsy
B. Organ Weight
C. Preparation of Tissues for Microscopic Examination
D. Microscopic Evaluation
E. Histopathology of Lymphoid Organs

8. Toxicity Tests Recommended Test/Requirement
Subchronic toxicity studies
with no rodents
(GLP)
Purpose:
(1) can help predict appropriate doses of
the test substance for future chronic
toxicity studies,
(2) can be used to determine NOELs for
some toxicology endpoints, and
(3) allow future long-term toxicity studies
in rodents and non-rodents to be designed
with special emphasis on identified target
organs.
Duration of Testing (7d/week, 90 days = 3
months)
Level: (CL II), (CL III)
Test Animals
A. Care, Maintenance and Housing
B. Selection of Rodent Species, Strains and Sex
C. Age
D. Number and Sex
E. Infected Animals
F. Animal Identification
H. Diet
I. Assignment of Control and Compound Treated Animals
J. Mortality
K. Autolysis
L. Necropsy
Test Substance
A. Identity
B. Composition/Purity
C. Conditions of Storage
D. Expiration Date
Experimental Design
A. Duration of Testing (7d/week, 90 days= 3 months)
B. Route of Administration
C. Dose groups
D. Computerized systems
Observations and Clinical Tests
A. Observations of Test Animals:
B. Body Weight and Feed Intake Data
C. Clinical Testing
Necropsy and Microscopic Examination
A. Gross Necropsy
B. Organ Weight
C. Preparation of Tissues for Microscopic Examination
D. Microscopic Evaluation
E. Histopathology of Lymphoid Organs

9. Toxicity Tests Recommended Test/Requirement
One-year toxicity studies with
non-rodents
(GLP)
Purpose:
(1) characterize the toxicity of the test
substance in non-rodents and
(2) determine the dose of the test substance
that produces no observed adverse effects
(NOEL or NOAEL) for some toxicological
endpoints
Duration of Testing (7d/week,52 weeks = 1
year)
Level: (CL III)
Test Animals
A. Care, Maintenance and Housing
B. Selection of Rodent Species, Strains and Sex
C. Age
D. Number and Sex
E. Infected Animals
F. Animal Identification
H. Diet
I. Assignment of Control and Compound Treated Animals
J. Mortality
K. Autolysis
L. Necropsy
Test Substance
A. Identity
B. Composition/Purity
C. Conditions of Storage
D. Expiration Date
Experimental Design
A. Duration of Testing (7d/week,52 weeks = 1 year)
B. Route of Administration
C. Dose groups
D. Computerized systems
Observations and Clinical Tests
A. Observations of Test Animals:
B. Body Weight and Feed Intake Data
C. Clinical Testing
Necropsy and Microscopic Examination
A. Gross Necropsy
B. Organ Weight
C. Preparation of Tissues for Microscopic Examination
D. Microscopic Evaluation
E. Histopathology of Lymphoid Organs

10. Toxicity Tests Recommended Test/Requirement
Chronic Toxicity Studies with
Rodents:
(Chapter IV.C.5.a. )
i) Purpose :
1) to characterize the toxicity of a food
ingredient following prolonged and
repeated exposure, and
2) to determine toxicological dose-
response relationships needed to
establish the maximum dose that
produces no adverse effects .
(i.e., NOEL or NOAEL).
iii) Duration :
12 months (one-year)
iii) Level : CL (III)
Test Animals
A. Care, Maintenance and Housing
B. Selection of Rodent Species, and Strains.
C. Age (start of dosing):
D. Number and Sex
E. Infected Animals
F. Animal Identification
G. Caging
H. Diet
I. Assignment of Control and Compound Treated Animals
J. Mortality
K. Autolysis
L. Necropsy
Test Substance
A. Identity
B. Composition/Purity
C. Conditions of Storage
D. Expiration Date
Experimental Design
A. Duration of Testing
B. Route of Administration
C. Dose groups
D. Computerized systems
Observations and Clinical Tests
A. Observations of Test Animals:
B. Body Weight and Feed Intake Data
C. Clinical Testing
Necropsy and Microscopic Examination
A. Gross Necropsy
B. Organ Weight
C. Preparation of Tissues for Microscopic Examination
D. Microscopic Evaluation
E. Histopathology of Lymphoid Organs

11. Toxicity Tests Recommended Test/Requirement
Carcinogenicity Studies w ith
Rodents:
(Chapter IV.C.6.)
i) Purpose :
1) to determine whether a food ingredient
possesses carcinogenic activity when
administered to rodents in regularly
repeated oral doses for the "lifetime" of
the test animal.
ii) Duration :
7 days per week for two years, or for the
life span of the animal.
iii) Level : CL (III)
Test Animals
A. Care, Maintenance and Housing
B. Selection of Rodent Species, and Strains.
C. Age (start of dosing):
D. Number and Sex
E. Infected Animals
F. Animal Identification
G. Caging
H. Diet
I. Assignment of Control and Compound Treated Animals
J. Mortality
K. Autolysis
L. Necropsy
Test Substance
A. Identity
B. Composition/Purity
C. Conditions of Storage
D. Expiration Date
Experimental Design
A. Duration of Testing
B. Route of Administration
C. Dose groups
D. Computerized systems
Observations and Clinical Tests
A. Observations of Test Animals:
B. Body Weight and Feed Intake Data
C. Clinical Testing
Necropsy and Microscopic Examination
A. Gross Necropsy
B. Organ Weight
C. Preparation of Tissues for Microscopic Examination
D. Microscopic Evaluation
E. Histopathology of Lymphoid Organs

12. Toxicity Tests Recommended Test/Requirement
Guidelines for Reproduction Studies:
(Chapter IV.C.9.a.)
i) Purpose :
1) to evaluate the effects of a test
substance on the reproductive systems of
both males and females, the postnatal
maturation and reproductive capacity of
offspring, and possible cumulative effects
through several generations.
2) to provide information concerning the
effects of a substance on gonadal function,
estrous cycles, mating behavior,
conception, parturition, neonatal
morbidity, mortality, lactation, weaning,
growth and development of the offspring,
and target organs in the offspring.
iii) Duration :
The test substance should be
administered daily throughout the
treatment period.
Level : CL (II), CL(III)
Dose Range-Finding Study
- should preferably, but not necessarily, be done in pregnant animals.
Main Study
A. Experimental Animals, Species and Strain Selection and Housing
B. Number, Sex, and Age
C. Assignment to Dose Groups
D. Dose Selection
E. Control Group(s)
F. Duration of Testing
G. Substance Administration
H. Mating Procedures
I. Standardizing the Number of Pups per Litter
J. Selection of Parental Animals for Next Generation
K. Optional Third Generation
L. Optional Second Mating
M. Optional Teratology Phase
N. Clinical Observations
O. Growth of Offspring
P. Optional Neurotoxicity Screening
Q. Optional Immunotoxicity Screening
R. Gross Necropsy and Microscopic Examination
End Points of Female Reproductive Toxicity
A. Female Fertility Index
B. Gestation Index
C. Live-born Index
D. Weaning Index
E. Sex Ratio and Percentage by Sex
F. Viability Indices
End Points of Male Reproductive Toxicity
A. Evaluation of Testicular Spermatid Numbers
B. Sperm Evaluation for Motility, Morphology and Numbers
Analysis of Data
Reporting the Results of Reproduction Studies

13. Toxicity Tests Recommended Test/Requirement
Guidelines for Developmental
Toxicity Studies:
(Chapter IV.C.9.b.)
i) Purpose :
1) to evaluate the effects of test
substances on developing fetuses that
result from exposure of either parent prior
to conception or to mothers during
prenatal development.
iii) Duration : The test substance should
be
administered daily throughout the
treatment period.
Level : CL (II), CL(III)
Dose Range-Finding Study
- should preferably, but not necessarily, be done in
pregnant animals.
Main Study
A. Experimental Animals, Species and Strain Selection
and Housing
B. Animal Husbandry
C. Number, Sex, and Age
C. Duration of Testing
D. Route Administration
G. Mating Procedures
I. Control and Dosed Groups
J. Maternal Toxicity and its Significance
K. Clinical Observation and Examination of Dams and
Fetuses
L. Histopathology
M. End Points Measured
N. Analysis of Data
Reporting the Results of Developmental Toxicity
Studies

14. Toxicity Tests Recommended Test/Requirement
Metabolism and Pharmacokinetic
Studies:
i) Purpose :
1) to determine dose-response
characteristics for any effects
observed
in the eva luation of the safety of food
and color additive s.
iii) Duration : The test substance should
be a dministered daily throughout the
treatment period.
Level : CL (II), CL(III)
1. Considerations in the Design of,
Analysis of, and Use of Data from
Metabolic and
Pharmacokinetic Studies:
a. Design and Analysis of Metabolic
and Pharmacokinetic Studies:
i. Test Compound
ii. Animals
iii. Route of Administration
iv. Dosage Regimen
v. Sampling
vi. In Vitro studies
vii. Analysis of Data
b. Use of Data from Metabolism and
Pharmacokinetic Studies
2. Recommended Metabolism and
Pharmacokinetic Studies
3. Additional Studies

15. Toxicity Tests Recommended Test/Requirement
Human Studies:
i) Purpose :
1) to assess aspects of safety that
cannot be addressed adequately by non-
human studies or by existing data on
population exposure.
iii) Duration : The test substance should
be administered throughout the
treatment period.
v) Level : CL (III)
1. Clinical Evaluation of Foods and
Food Additives
a. General Considerations for
Clinical Studies of Foods and Food
Additives.
c. Specific Considerations for
Clinical Studies
of Foods and Food Additives
i. Protocol Design
ii. The Study Population
iii. Statistical Analyses
c. Sequence of Clinical Studies for
Foods and
Food Additives
iv. Early Clinical Studies
v. Further Clinical Studies

16. Human studies
The Agency does not require petitioners to conduct human clinical
studies to support the safety of direct food additives and color
additives used in food.
However, petitioners may elect to perform such studies in certain
circumstances, such as when the proposed additive will be
consumed by humans at relatively high levels (see Chapter VII B).

17. Macro-Additives-Chapter VII B
Macro-additives are a class of food additives that are intended to be
replacements for conventional macro-nutrients such as fats, proteins, and
carbohydrates and are intended for use at relatively high levels in food.
Macro-additives may be nutritive or non-nutritive;
they may be reasonably pure, well characterized chemicals or they may be
complex mixtures whose complete characterization is not feasible;
they may be well absorbed from the gastrointestinal tract or poorly
absorbed;
they may be manufactured from unusual or novel food sources or obtained
by chemical synthesis.

18. Macro-Additives-Chapter VII B
common characteristic of macro-additives is that they will be consumed
in large quantities compared to conventional food additives and, as a
consequence, they will present testing problems that require "customized"
approaches.
e.g.,-it may not be feasible to calculate safety factors in the
conventional way, that is, as a fraction of the highest oral dose that has no
adverse effects in animals.
Other means of providing margins of safety for macro-additives will have to
be used; these may include information derived from metabolic,
pharmacokinetic, and human clinical studies.

19. THANK YOU
Presented by:
Rabe'ah AdamRabe'ah AdamRabe'ah AdamRabe'ah Adam
(MK 121004)(MK 121004)(MK 121004)(MK 121004)
Sharifah Norzi SyedSharifah Norzi SyedSharifah Norzi SyedSharifah Norzi Syed
HassanHassanHassanHassan
(MK 121042)(MK 121042)(MK 121042)(MK 121042)