Toxicological studies

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

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

Toxicity Tests ConcernLevelLow(I)

ConcernLevel

Intermediate(II)

ConcernLevelHigh(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

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

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 µg 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 ORb) (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


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 AnimalsA. Care, Maintenance and HousingB. Selection of Rodent Species, Strains and SexC. AgeD. Number and SexE. Infected AnimalsF. Animal IdentificationH. DietI. Assignment of Control and Compound Treated AnimalsJ. MortalityK. AutolysisL. Necropsy

Test SubstanceA. IdentityB. Composition/PurityC. Conditions of StorageD. Expiration Date

Experimental DesignA. Duration of Testing (7d/week, 4 weeks)B. Route of AdministrationC. Dose groupsD. Computerized systems

Observations and Clinical TestsA. Observations of Test Animals:B. Body Weight and Feed Intake DataC. Clinical Testing

Necropsy and Microscopic ExaminationA. Gross NecropsyB. Organ WeightC. Preparation of Tissues for Microscopic ExaminationD. Microscopic EvaluationE. Histopathology of Lymphoid Organs


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)



Experimental DesignA. Duration of Testing (7d/week, 90 days = 3 months)B. Route of AdministrationC. Dose groupsD. Computerized systems




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)



Experimental DesignA. Duration of Testing (7d/week, 90 days= 3 months)B. Route of AdministrationC. Dose groupsD. Computerized systems




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)



Experimental DesignA. Duration of Testing (7d/week,52 weeks = 1 year)B. Route of AdministrationC. Dose groupsD. Computerized systems




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 (I I I )

Test AnimalsA. Care, Maintenance and HousingB. Selection of Rodent Species, and Strains.

C. Age (start of dosing):

D. Number and SexE. Infected AnimalsF. Animal IdentificationG. CagingH. DietI. Assignment of Control and Compound Treated AnimalsJ. MortalityK. AutolysisL. Necropsy


Experimental DesignA. Duration of Testing B. Route of AdministrationC. Dose groupsD. Computerized systems




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 (I I I )

Test AnimalsA. Care, Maintenance and HousingB. Selection of Rodent Species, and Strains.

C. Age (start of dosing):

D. Number and SexE. Infected AnimalsF. Animal IdentificationG. CagingH. DietI. Assignment of Control and Compound Treated AnimalsJ. MortalityK. AutolysisL. Necropsy


Experimental DesignA. Duration of Testing B. Route of AdministrationC. Dose groupsD. Computerized systems




Guidelines for Reproduct ion 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 (I I ), CL(I I I )

Dose Range-Finding Study- should preferably, but not necessarily, be done in pregnant animals.

Main StudyA. Experimental Animals, Species and Strain Selection and HousingB. Number, Sex, and AgeC. Assignment to Dose GroupsD. Dose SelectionE. Control Group(s)F. Duration of TestingG. Substance AdministrationH. Mating ProceduresI. Standardizing the Number of Pups per LitterJ. Selection of Parental Animals for Next GenerationK. Optional Third GenerationL. Optional Second MatingM. Optional Teratology PhaseN. Clinical ObservationsO. Growth of OffspringP. Optional Neurotoxicity ScreeningQ. Optional Immunotoxicity ScreeningR. Gross Necropsy and Microscopic Examination

End Points of Female Reproductive ToxicityA. Female Fertility IndexB. Gestation IndexC. Live-born IndexD. Weaning IndexE. Sex Ratio and Percentage by SexF. Viability Indices

End Points of Male Reproductive ToxicityA. Evaluation of Testicular Spermatid NumbersB. Sperm Evaluation for Motility, Morphology and Numbers

Analysis of Data

Reporting the Results of Reproduction Studies


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.


Dose Range-Finding Study- should preferably, but not necessarily, be done in pregnant animals.

Main StudyA. Experimental Animals, Species and Strain Selection and HousingB. Animal HusbandryC. Number, Sex, and AgeC. Duration of TestingD. Route AdministrationG. Mating ProceduresI. Control and Dosed GroupsJ. Maternal Toxicity and its SignificanceK. Clinical Observation and Examination of Dams and FetusesL. HistopathologyM. End Points MeasuredN. Analysis of Data

Reporting the Results of Developmental Toxicity Studies


Metabolism and Pharmacokinet ic

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.


be a dministered daily throughout the treatment period.


1. Considerat ions in the Design of,

Analysis of, and Use of Data from

Metabolic and

Pharmacokinet ic Studies:

a. Design and Analysis of Metabolic

and Pharmacokinet ic Studies:

i. Test Compoundii. Animalsiii. Route of Administrationiv. Dosage Regimenv. Samplingvi. In Vitro studiesvii. Analysis of Data

b. Use of Data from Metabolism and

Pharmacokinet ic Studies

2. Recommended Metabolism and

Pharmacokinet ic Studies

3. Addit ional Studies


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.


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

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).

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.

Macro-Additives-Chapter VII Bcommon 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.

THANK YOU

Presented by:

Rabe'ah Adam Rabe'ah Adam Rabe'ah Adam Rabe'ah Adam

(MK 121004)(MK 121004)(MK 121004)(MK 121004)

Sharifah Norzi Syed Sharifah Norzi Syed Sharifah Norzi Syed Sharifah Norzi Syed Hassan Hassan Hassan Hassan

(MK 121042)(MK 121042)(MK 121042)(MK 121042)

Toxicological studies

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