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OECD GUIDELINE and GENOTOXICITY

Presentation by : Sandhya TallaDepartment of PharmacologyM.Pharm

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INTRODUCTION OECD

HISTORY

OBJECTIVES

TOXICITY TESTING IN INDIA

GENERAL CLASSIFICATION OF GUIDELINES

GENETIC TOXICITY GUIDELINES

CONTENT

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Organization for Economic Co-Operation and Development

An intergovernmental organization.

A forum in which governments work together to address the economic, social and environmental challenges of interdependence and globalization

A provider of comparative data, analysis and forecasts to underpin multilateral co-operation

Representatives of 34 industrialized countries in North America, Europe, Pacific and the European Commission.

INTRODUCTION www.oecd.org

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Born after World War II as the Organization for European Economic Co-operation to coordinate the Marshall Plan

Transformed in 1961 into the Organization for Economic Co-operation and Development with trans-Atlantic and then global reach

Today the OECD has 34 member countries

More than 70 developing and transition economies are engaged in working relationships with the OECD

HISTORY www.oecd.org

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HOW WAS OECD ESTABLISHED ?

The marshall planThis idea of a US supported European recovery program(ERP)

Recipient of Marshall plan aid sign a convention

Establishment of development assistance group (DAG ) on 13 Jan For consultation among aid donors on assistance to less developed countries

DAG in its fifth and final meeting discusses US proposal to set up OECD development centre OECD comes into operation in sept 1961

1961

1960

1948

1947

www.oecd.org

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The OECD budget for 2015 is EUR 363 million

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THE OECD SECRETARIAT

Staff members are

international civil servants

Works in two official

languages: English and

French

2500 staff at Paris

headquarters No quota system for

national representation

www.oecd.org

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OECD WAYS OF WORKING

Data Collection

Analysis

Discussion

Implementation Peer reviews,multilateral surveillance

www.oecd.org

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OECD TG Development Process

National Coordinators of the TG Programme

OECD Considers Animal Welfare in the Development of TG

OECD Validation

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OECD TG Development Processwww.oecd.org

Member CountriesTUAC

BIAC

NGOs

Int’l Sci. Societies

Secretariat

Int’l Orgs.

Proposal for Test Guideline (TG)

Development

WNT

Draft Proposal for TG

Meetings

CommentingRound(s)

Revised DraftTG Proposal

Final TG ProposalWNT

JM EPOC Council Final TestGuideline Implementation

Publication

Small ad hocExpert Group

SPSFEssential CriteriaDesirable Criteria

Draft Proposal for TG

Meetings

CommentingRound(s)

Revised DraftTG Proposal

Final TG ProposalWNT

JM EPOC Council Final TestGuideline Implementation

Publication

Small ad hocExpert Group

Partner Organisations:

• EC/ECB• ICH• IOMC• ISO• others

Industry Organisations:

• BIAC• ICAPO• others

National Position Paper(responsibility of the

National Co-ordinator)Organisation’sPosition Paper

Industry’sPosition Paper

Secretariat

OECD Secretariat’s Document

National Co-ordinators

Academia Government Industry

Test Guidelines ProgrammeExpert Review Of Draft Documents

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National Coordinators of the TG Programmewww.oecd.org

Argentina

Australia Austria Belgium Brazil Canada Czech Republic Denmark Estonia European Commissi

on

• Finland

• France

• Germany

• Greece

• Hungary

• India

• Ireland

• Israel

• Italy

• Japan

• Korea

• Luxembourg

• Malaysia

• The Netherlands

• New Zealand

• Norway

• Poland

• Portugal

• Singapore

• Sloval Republic

• Slovenia

• South Africa

• Spain

• Sweden

• Switzerland

• Thailand

• Turkey

• United Kingdom

• United States

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Over 25 years ago, an OECD High Level Meeting on Chemicals recognized the need to protect animals particularly those used in testing for chemical safety.

It addressed this ethical issue and adopted the following statement at that time:

"The welfare of laboratory animals is important; it will continue to be an important factor influencing the work in the OECD Chemicals Programme. The progress in OECD on the harmonization of chemicals control, in particular the agreement on Mutual Acceptance of Data, by reducing duplicative testing, will do much to reduce the number of animals used in testing. Such testing cannot be eliminated at present, but every effort should be made to discover, develop and validate alternative testing systems".

In the meantime OECD has made major efforts to develop and implement tools which will reduce or replace animal testing in chemical safety

OECD Considers Animal Welfare in the Development of TG

www.oecd.orgCOMMITMENT TO ANIMAL WELFARE

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APPLY INTEGRATED APPROACHES TO TESTING AND ASSESSMENT

AVOID DUPLICATION OF TESTING

ESTIMATE PROPERTIES WITH (Q)SARs

ESTIMATE PROPERTIES FOR GROUPS OF CHEMICALS

SCREEN LARGE NUMBERS OF CHEMICALS WITH MOLECULAR SCREENING ASSAYS

AND TOXICOGENOMICS

DEVELOP ALTERNATIVE TEST METHODS

Main Objectiveswww.oecd.org

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Regulatory purposes OECD member countries recognize international harmonization of test methods(GLP).

To introduce economics in test costs and time If individual countries can confidently rely on test data in other country duplicate testing can be avoided .

OECD lays down Toxicity testing guidelines for chemicals For Eg. Toxicity test for Acute, Chronic Toxicity, Teratogenicity

WHY PHARMA INDUSTRY REQUIRE OECD ?

WE NEED YOU!!

www.oecd.org

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To achieve sustainable economic growth and employment and rising standards of living

To maintain financial stability

To assist sound economic expansion

To contribute to growth in world trade on a multilateral, non-discriminatory basis

OBECJTIVES www.oecd.org

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Paracelsus (Father of Toxicology) "All substances are poisons; there is none which is not a poison. The right dose differentiates a poison and a remedy”.

Determined specific chemicals responsible for the toxicity of plants and animals (dose-response relationship).

Mathieu Orfila, determined the relationship between poisons and their biological. He is referred to as the father of modern toxicology

History of Toxicity Studies

Paracelsus (1493-1541)

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Toxicity Testing in India

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The Humane Society of the United States proposed a ‘big biology’ project known as “The Human Toxicology Project”

It formed affiliations with Humane Society International and Humane Society Legislative Fund

HSI is currently actively working to implement a landmark vision of the “21st century toxicology”

Its implementation in India starts with the understanding of the Indian safety regulatory system

Toxicity Testing in India

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OBJECTIVES

a. Identify private companies, contract research organizations, government and academic institutions in India that do toxicity testing work.

b. Identify toxicity testing requirements for regulatory approval in India of Biologicals, Drugs, Foods, Pesticides, Cosmetics and Industrial Chemicals in the country.

c. Identify key individuals and agencies involved in chemical safety regulation and in toxicity testing.

d. Identify progressive Indian scientists who are considered to be visionaries as regards the development and application of new technologies and who are at the forefront of promoting alternate methods to animal testing, particularly in toxicology.

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Role of International standard(Sengupta, Alokparna (2012) Toxicity testing in India: An animal welfare perspective).

The U.S Food and Drug administration (USFDA) guidelines for pre-clinical testing.

OECD guidelines for evaluating toxicological effects of chemicals.

The International Conference on Harmonisation

ISO standards are another set of standard guidelines which are used extensively by Indian pharmaceutical and other chemical companies to standardize their products internationally.

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Why study toxicology??? Benefit –risk ratio can be calculated

Prediction of therapeutic index

Therapeutic index= Maximum tolerated dose Minimum curative dose

Smaller ratio, better safety of the drug

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GUIDELINES

Section I-Blue pages(101-121) Physico-chemical properties Eg: M.P.,B.P., Vapour pressure, Adsorption, Viscosity, Dissociation constant, pH, partion coefficient,etc.

Section II-Green pages(201-222) Biotic systems Eg: Microbial growth inhibition, Aquatic & avian toxicity, Soil microrganisms toxicity,etc.

Section III-Yellow pages(301-312) Degradation and accumulation Eg:.Biodegradation in soil due to aerobic and anaerobic microbes,etc.

Section IV-Pink pages(401-486) Health effects Eg:Oral toxicity, eyes & skin irritation, acute & chronic toxicity, carcinogenicity, Genetic toxicity, Neurotoxicity.

www.oecd.org

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GENETIC TOXICITY TEST GUIDELINES

ESTABLISHED IN 1983 AND PUBLISHED IN 1987

Global update of the Genetic Toxicology TGscompleted in 2015

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To identify substances that causes genetic alterations and thus use this information in regulatory decisions

Measuring direct, irreversible damage to the DNA that is transmissible to the next cell generation.

Those measuring early, potentially reversible effects to DNA or mechanism involved in the preservation of integrity of genome

AIM

Genetic Toxicology Endpoints

Genetic toxicology guidance document,2015

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In vitro Genetic Toxicology In vivo Genetic ToxicologyTests for gene mutation TG 471: Bacterial reverse mutation test TG 476: Mammalian cell gene mutation test

using Hprt / Xprt / Thymidine kinase gene

Tests for gene mutation TG 488: Transgenic rodent somatic and germ

cell gene mutation assay

Test for chromosomal abnormalities TG 473: Mammalian chromosomal aberration

test TG 487: Mammalian cell micronucleus test

Test for chromosomal abnormalities TG 474: Mammalian erythrocyte micronucleus

test TG 475: Mammalian bone marrow chromosomal

aberration test TG 478: Rodent dominant lethal assay TG 483: Mammalian spermatogonial

chromosomal aberration test TG 485: Mouse heritable translocation assay

Primary DNA damage test TG 486: Unscheduled DNA synthesis test with

mammalian liver cells TG 489: Mammalian alkaline comet assay

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Mitosis: M phase (nuclear division)

prophase

metaphase

anaphase

telophase

Chromatids condense by folding chromatin fibers attached to chromosome

Chromatids alignat the center ofthe spindle and Nuclear envelopedeforms Chromatids

seggregate i.e.Separate andMove to the poles of spindle

Nuclear envelopereforms around segregated Chromatids at two ends and cytokinesis.

Interphase: The cell prepare itself for division.

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Identifies substances that induce gene mutations by base substitutions or frameshifts

Two species of bacteria Salmonella typhimurium and Escherichia coli with identified mutations in an amino acid i.e His or Trp as the reporter locus

It detects mutations which revert mutations present in the test strains and restore the functional capability of the bacteria to synthesize an essential amino acid.

TG 471: Bacterial Reverse Mutation Test IN

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PRINCIPLE

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PROCEDURE

Maximum test concentration is 5 mg/plate or 5 ml/plate.

Two methods: The plate incorporation method and The pre-incubation method

For both techniques incubate at 37°C for two or three days

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PARAMETER

Large number of cell can be exposed

There have been developments to

automate the test and minimize the use of

test substances

Highest induced mutant frequency can be

detected

ADVANTAGES

Relativetly sensitive and reliable detection of

compound

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In the cell lines the genetic endpoints measure mutation at thymidine kinase (TK) and hypoxanthine-guanine phosphoribosyl transferase (HPRT) and a transgene of xanthineguanine phosphoribosyl transferase (XPRT).

Hprt is present on X-chromosome, various cell lines of Hprt are CHO, CHL, V79 of Chinese hamster cells, etc.

Xprt coded by gpt transgene, AS52 cell lines are used.

TK: Mouse lymphoma assay and TK6 assay

TG 476: Mammalian cell gene mutation test Using Hprt or Xprt / TG 490: TK locus

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Cells in suspension culture are exposed to at least four analyzable concentrations of the test substance, both with and without metabolic

activation, for a suitable period of time.

They are sub-cultured to determine cytotoxicity and to allow phenotypic expression prior to mutant selection.

The treated cultures are maintained in growth medium for a sufficient period of time to allow near-optimal phenotypic expression of induced mutations.

Mutant frequency is determined by seeding known numbers of cells in medium containing the selective agent to detect mutant cells, and in medium

without selective agent to determine the cloning efficiency (viability).

PROCEDURE

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The cell density in culture plate should be limited in order to avoid metabolic co-operation between mutant and non-mutant cells which would alter mutant selection.

This is particularly important for cells growing monolayer rather than suspension.

PARAMETER

LIMITATIONS

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Tests for Chromosomal Aberrations

Chromosomal Aberrations

Endpoints

Micronuclei

Cells are not viable and aberrations are not transmitted to daughter cells.

Micronuclei are visualized in cells following the first cell division

Visualized under a microscope.

Nonviable chromosomal aberrations arethe basis for dominant lethal mutations resulting in fetal loss

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o Cell lines: CHO, CHL, V79, TK6

Structural aberrations may be of two types: chromosome or chromatid

Observed only in metaphase of 1st or 2nd mitotic division after treatment

G-2 active substance induces chromatid aberration at 1st mitosis but many of its events get converted into chromosome aberration in 2nd mitosis

Damage induced pre-S-phase ---------- chromosome aberration Damage induced post-S-phase ---------- chromatid aberration

TG 473: Mammalian Chromosomal Aberration Test

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Test may employ cultures of established cell lines, cell strains or primary cell cultures.

Cell cultures are exposed to the test substance (liquid or solid) both with and without metabolic activation during about 1.5 normal cell cycle

lengths

At least three analyzable concentrations of the test substance should be used.

At each concentration duplicate cultures should normally be used.

At predetermined intervals after exposure of cell cultures to the test substance, the cells are treated with a metaphase-arresting substance,

harvested, stained

PROCEDURE

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Detection of polyploidy including endoreduplication which indicates cell cycle perturbation.

This test is not optimized for detection of aneuploidy

PARAMETER

LIMITATION

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Cell lines: CHO, V79, CHL, L5178Y, TK6 or primary human or other mammalian peripheral blood lymphocyte.

For the detection of micronuclei in the cytoplasm of interphase cells.

Micronuclei may originate from acentric chromosome fragments (i.e. lacking a centromere), or whole chromosomes that are unable to migrate to the poles during the anaphase stage of cell division.

The assay detects the activity of clastogenic and aneugenic test substances in cells that have undergoes cell division during or after exposure to the test substance.

TG 487: Mammalian cell micronucleus test IN

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PROCEDURE

Cytochalasin B used to block cyotokinesis and generate binucleate cells during or after test substance exposure.

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PARAMETER

1. Automated system that measures micronucleated cell frequencies2. Sensitive

1. Does not allow identification of translocation and other complex chromosomal rearrangement

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The transgenes contain reporter genes for the detection of various types of mutations or chromosomal deletion and rearrangements resulting in DNA size changes induced by test substances

Transgenic rats or mice that contain multiple copies of chromosomally integrated plasmid or phage shuttle vectors are used.

TG 488: Transgenic rodent somatic and germ cell gene mutation assays

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Genomic DNA extracted from tissue

Transgenes are recovered from genomic DNA and then transfected into a bacterial host deficient for reporter gene

A -ve control group and a min 3 treatment groups of transgenic animals are treated for 28 consecutive days.

Administration is usually followed by a 3-day period of time, prior to sacrifice, during which the agent is not administered and during which

unrepaired DNA lesions are fixed into stable mutations.

At the end of this 3-day period, the animals are sacrificed; genomic DNA is isolated from the tissue(s) of interest and purified

PROCEDURE

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Mutant frequency is calculated by dividing the number of plaques/plasmids containing mutations in the transgene by the total number of plaques/plasmids recovered from the same DNA sample.

PARAMETER

Target genes are functionally neutral thus increases sensitivity for detection of mutation

Estimates the amount of clonal expansion of

originally mutated cells

These transgene respond to the treatment

in same way as endogenous gene and

with similar genetic background

ADVANTAGES

DNA sequencing of mutants confirms

mutational spectra or type of mutation

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For the detection of damage induced by the test substance to the chromosomes or the mitotic apparatus of erythroblasts (rodents)

Identifies micronuclei containing lagging chromosome fragments or whole chromosomes.

An increase in the frequency of micronucleated polychromatic erythrocytes in treated animals is an indication of induced chromosome damage because they lack main nucleus

TG 474: Mammalian erythrocyte micronucleus test

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Animals are exposed to the test substance by an appropriate route.

Bone marrow and/or blood cells are collected, prepared and stained.

Preparations are analyzed for the presence of micronuclei.

Each treated and control group must include at least 5 analyzable animals per sex.

Administration of the treatments consists of a single dose or two daily doses (or more).

The limit dose is 2000 mg/kg/body weight/day for treatment up to 14 days, and 1000 mg/kg/body weight/day for treatment longer than 14 days.

PROCEDURE

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PARAMETER

1. Automated system that measures micronucleated erythrocyte frequencies

1. Cells must be proliferated before tissue collection and must be properly sampled2. Restricts only for detection of organ specific genotoxic substances

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For the detection of structural chromosome aberrations induced by test compounds only in bone marrow cells of animals (rodents).

Structural chromosome aberrations may be of two types: chromosome or chromatid.

TG 475: Mammalian bone marrow chromosomal aberration test

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Animals are exposed to the test substance by an appropriate route

Then animals are treated with a metaphase-arresting agent.

Chromosome preparations are then made from the bone marrow cells and stained, and metaphase cells are analyzed for chromosome aberrations.

Each treated and control group must include at least 5 analyzable animals per sex.

The limit dose is 2000 mg/kg/body weight/day for treatment up to 14 days, and 1000 mg/kg/body weight/day for treatment longer than 14 days.

PROCEDURE

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Various types of chromosomal aberrations is visualized in individual cell using microscopy.

PARAMETER

1. Increased incidences of polyploidy can be seen

1. Standard design is not optimized for detection of aneuploidy.2. Long exposure decreases the sensitivity of this test.

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Dominant lethal (DL) effects cause embryonic or foetal death.

Induction of a dominant lethal event after exposure to a test substance indicates that the substance has affected germinal tissue of the test species.

Dominant lethals are generally accepted to be the result of chromosomal aberrations (structural and numerical anomalies)

Chemicals that causes Dominant lethality also causes F1 congenital malformations.

TG 478: Rodent dominant lethal assay IN

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Male animals are exposed to the test substance and mated to untreated virgin females

The most widely used is the single administration of the test substance by oral or by intraperitoneal injection.

Normally, three dose levels should be used.

The various germ cell stages can be tested separately by the use of sequential mating intervals.

The females are sacrificed after an appropriate period of time

PROCEDURE

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PARAMETER

The calculation of the dominant lethal effect is based on comparison of the live implants per female in the treated group to the live implants per female in the control group

Chemicals that are positive in Dominant lethal test also are +ve in translocation test and specific locus test.

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Measures chromosome events in spermatogonial germ cells and is, therefore, expected to be predictive of induction of inheritable mutations in germ cells.

Chromosomal aberration in spermatogonial cells observed in metaphase of 1st or 2nd mitotic cell division of spermatogenesis.

Male Chinese hamsters and mice are commonly used.

TG 483: Mammalian spermatogonial chromosomal aberration test

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Each treated and control group must include at least five analyzable males.

Animals are exposed to the test substance once or twice a day by an appropriate route

Then animals are treated with a metaphase-arresting agent.

Chromosome preparations are then made from germ cells and stained, and metaphase cells are analyzed for chromosome aberrations.

A limit test may be performed if no effects would be expected at a dose of 2000 mg/kg/d

PROCEDURE

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PARAMETER Cytotoxicity is measured as the ratio between spermatogonial metaphases to either meiotic

metaphases or interphases cells. Positive results indicate that a substance induces chromosome aberrations in the germ cells

of the species tested

1. Increased incidences of polyploidy can be seen2. FISH gives additional information about translocation and other rearrangements.

1. Standard design is not optimized for detection of aneuploidy.2. Long exposure decreases the sensitivity of this test.

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Detects structural and numerical chromosome changes in mammalian germ cells

The types of chromosome changes detected in this test system are reciprocal translocations.

Carriers of translocations and XO-females show reduced fertility which is used to select first generation progeny for cytogenetic analysis.

Translocations are cytogenetically observed in meiotic cells at diakinesis metaphase I

TG 485: Mouse heritable translocation assay IN

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The test is usually performed by analysis of male first generation progeny.

About 500 first generation males per dose level are required.

One dose level is tested, usually the highest dose associated with the production of minimal toxic effects, and administered by oral intubation or intraperitoneal injection

A single administration of the test substance or the administration of the test substance on 7 days/week for 35 days, are possible.

Translocation are cytogenetically observed as quadrivalent which are compromised of two sets of homologous chromosome in meiotic cells at diakinesis of meiosis of F1 male

progeny

PROCEDURE

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PARAMETER

Monitoring of litter size of F1 generation indicates that Dominant lethality is occuring.

Requires large number of animals and rarely used.

LIMITATION

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To identify substances that induce DNA repair after excision and removal of a stretch of DNA containing a region of damage induced by chemical substances or physical agents in the liver.

The test is usually based on the incorporation of tritium-labelled thymidine, 3H-TdR, (during 3-8 hours) into the DNA of liver cells

The uptake of 3H-TdR is usually determined by autoradiography

TG 486: Unscheduled DNA synthesis (UDS) test with mammalian liver cells

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Rats are commonly used

Each treated and control group must include at least 3 analysable animals per group.

At least two dose levels are used.

A limit test may be performed if no effects would be expected at a dose of 2000 mg/kg bw/d.

Test substances are generally administered as a single treatment.

Liver cells are prepared from treated animals 12-16 hours after dosing of animal.

After autoradiography, normally 100 cells are scored from each animal

PROCEDURE

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PARAMETER

Response is dependent on number of DNA bases excised and replaced at the site of origin.

A positive result indicates that a substance induces DNA damage in mammalian liver cells in vivo that can be repaired by unscheduled DNA synthesis in vitro.

This test is not considered as surrogate mutation test, thus less reliable than other primary DNA damage test.

LIMITATION

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Measures the DNA strand breaks in eukaryotic cells.

These strand breaks may be: 1) repaired, 2) lethal to the cell, 3) fixed as mutation resulting in permanent heritable change.

Alternate name: Alkaline single-cell gel electrophoresis assay.

Under alkaline condition (>13) this assay can detect single and double strand breaks. For eg: direct interactions with DNA alkali liable sites or consequences of transient DNA strand discontinuities resulting from DNA excision repair.

TG 489: In vivo Mammalian alkaline comet assay IN

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PROCEDURE

Administration of the treatment consists of daily doses over duration of 2 days or more, ensuring the test chemical reaches the target tissue

Hacettepe University, Faculty of Pharmacy, Department of Toxicology, Ankara

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PARAMETER

Based on their size DNA fragments migrate away from the head to the tail, and the intensity of the comet tail relative to the total intensity (head plus tail) reflects the amount of DNA breakage.

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1. Cell division is not required.2. Detecting exposure to target tissue3. Small amount of test substances.4. Less time consuming.

1. Does not detect aneuploidy.2. Structural chromosomal damage or mutation is not detected directly.3. Long exposure: decreased sensitivity.4. Difficult to distinguish between genotoxicity and apoptosis after electrophoresis. 5. Not able to detect small DNA fragment

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Sengupta , Alokparna (2012) Toxicity testing in India: An animal welfare perspective. Federation of Indian Animal Protection Organizations and Humane Society International April 2012

Combes RD, Gaunt, I, Balls M (2004). A Scientific and Animal Welfare Assessment of the OECD Health Effects Test Guidelines for the Safety Testing of Chemicals under the European Union REACH System. ATLA 32, 163-208.

NRC (2007). Toxicity Testing in the 21st Century: A Vision and a Strategy. Washington, DC: The

National Academies Press. www.oecd.org Genetic toxicology guidance document,2015 Hacettepe University, Faculty of Pharmacy, Department of Toxicology, Ankara Department of Environmental Biotechnology, University of Warmia and Mazury in

Olsztyn, ul. Sloneczna 45G, 10–712 Olsztyn, Poland

REFERENCES

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