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Presentation by : Ashwini Patil

Department Of Pharmacology

M.Pharm -2nd –Sem R.C.P.I.P.E.R,Shirpur.

GENOTOXICITY GUIDELINES

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GENOTOXICITY GUIDELINES

Presentation by:- Ashwini Patil Department Of Pharmacology

M.Pharm -2nd Sem R.C.P.I.P.E.R,Shirpur.

HISTORY OF TOXICITY STUDIES

GENERAL CLASSIFICATION OF GUIDELINES

GENETIC TOXICITY GUIDELINES

CONTENT2

PHASES OF CELL CYCLE

TEST GUIDELINES ON GENOTOXICITYTEST GUIDELINES

REFERNCES

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

0 5 / 0 2 / 2 0 2 3 3History of Toxicity Studies

Paracelsus (1493-1541)

Mathieu Orfila(24 April 1787 – 12 March 1853)

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

4AIM

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 (Deleted on 15 Jan 2015)

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

assayPrimary DNA damage test TG 486: Unscheduled DNA synthesis test

with mammalian liver cells TG 489: Mammalian alkaline comet assay

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PHASES OF CELL CYCLE 14

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For the detection of damage induced by the test substance to the chromosomes.

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

7TG 474: Mammalian erythrocyte micronucleus Test(OECD 474)

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PRINCIPLE

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9PROCEDURE

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.

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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: Structural and Numerical

11TG 475: Mammalian bone marrow chromosomal aberration test (OECD 475)

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COLCHICINE,COLECIMID

(Metaphase arresting agent)

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.

Cell microscopy

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

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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.

FISH Staining technique have been used to increase the senstivity of CA ,allowing each chromosome to be differentially stained.

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Dominant lethal (DL) effects cause embryonic or fetal 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.

14TG 478: Rodent dominant lethal assay) (OECD 478)IN

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PROCEDURE 15

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

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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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

17TG 485: Mouse heritable translocation assay (OECD 485)

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The test is usually performed by analysis of male first

generation progeny.

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

A single administration of the test substance or the

administration of the test substance on 7 days/week for 35

days, are possible.

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

About 500 first generation males per

dose level are required.

PROCEDURE 18

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

Requires large number of animals and rarely used.

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LIMITATION

PARAMETER

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 `

0 5 / 0 2 / 2 0 2 3 20TG 486: Unscheduled DNA synthesis (UDS) test with mammalian liver cells (OECD 486)

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

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

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.

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LIMITATION

PARAMETER

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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.

23TG 489: In vivo Mammalian alkaline comet assay (OECD 489)

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

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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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CASPLAB SOFTWARE

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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

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

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