Evaluating Scientific Studies for Carcinogen Classification under - PowerPoint PPT Presentation

Evaluating Scientific Studies for Carcinogen Classification under GHS Walden Dalbey, M A, PhD, DABT DalbeyTox, LLC West Chester, P A

Outline of Presentation Goal: To provide background on carcinogenicity and discuss approaches to decisions regarding classification based on data from laboratory studies. Topics: Introduction to carcinogens and mutagens • • Tests for carcinogens and mutagens Criteria for classification under GHS • Examples of test results and classification under GHS • M ixtures and bridging principles • Dalbey SCHC 2014 2

What is a Carcinogen? A “substance or a mixture which induces cancer or increases its incidence.” • “ Substances and mixtures which have induced benign and malignant tumours in well performed experimental studies on animals are considered also to be presumed or suspected human carcinogens unless there is strong evidence that the mechanism of tumour formation is not relevant for humans.” (GHS Rev. 5, Section 3.6.1) A physical or chemical agent that causes or induces neoplasia (new or autonomous growth of tissue). (Derived from Casarett & Doull’s Toxicology ) Dalbey SCHC 2014 3

What is… DNA: Deoxyribonucleic acid, the hereditary material with genetic code based on sequence of paired bases along the length of these long, thin structures (~3 billion base pairs in people) Gene: A portion of DNA with code for a particular protein (~20,000-25,000 genes in people) Chromosome: Thread-like structures in nucleus containing DNA and protein (23 pairs in people) M utation: Unrepaired change in DNA sequence that is stable enough to be passed on during cell division. M utations can be in genes, structure of chromosomes, or number of chromosomes. Dalbey SCHC 2014 4

Genotoxic vs Nongenotoxic Carcinogens Genotoxic carcinogens Interact with DNA or alter its structure and/or function, • resulting in mutation No theoretical threshold in response at low doses of • genotoxic carcinogens Nongenotoxic carcinogens Nonmutagenic. No direct damage to DNA • Threshold exists in response at low doses. • Dalbey SCHC 2014 5

Carcinogenicity Studies: 2-year Rodent Bioassay Typically two species, each with 50 animals/sex/ dose, three doses plus controls High dose often M aximum Tolerated Dose (M TD) • Exaggerated doses are used to see if effects occur in a small population of lab animals. Incidence of specific tumors, time to develop, and types of tumors evaluated statistically. Criticisms: Time, cost ($1M /species), number of animals, false positives, confounding effects at M TD M ainstay for many years, but being questioned now Dalbey SCHC 2014 6

Carcinogenicity Studies: Organ-Specific Tests, e.g. Rodent liver, a common target organ for chemical carcinogens Use of preneoplastic lesions rather tumors as endpoint in • order to shorten the assay Skin-painting in mice Accelerated development of lung tumors in strain A mice (genetically susceptible strain) Dalbey SCHC 2014 7

Transgenic Animals Animals into which one or more DNA sequences from another species have been introduced. Rapidly expanding area. Used in research and more recently in hazard characterization. Some models designed with reporter genes to detect mutations (Big Blue, M utaM ouse, etc.), but expense high for routine screening Some models used as screening assays for carcinogenicity or, combined with data from other bioassays, genotoxicity, reactivity with DNA, etc., as more definitive model for carcinogenicity Such as mouse models Tg.AC, p53 +/ - , T gras H2, and XPA -/ - • Dalbey SCHC 2014 8

Short-Term Tests (Faster, Less Expensive, M echanistic) Use based on assumption that mutations might lead to carcinogenicity. M any short-term tests for mutagenicity are available to screen for mutagenic carcinogens. Tests for structural damage to chromosomes are also used since • many carcinogens cause this too. Which short-term tests can be used to support assessment of possible carcinogenicity and how much weight should be given to them? These screening tests were designed to test for genotoxic • substances, not to detect carcinogens independently. Guidelines on conduct of some relevant tests: US EP A’s Office of Chemical Safety and Pollution Prevention (OCSPP) - Harmonized T est • Guidelines for pesticides and toxic substances (Health Effects T est Guidelines, Series 870) available at www.epa.gov/ocspp/ pubs/ frs/ home/guidelin.htm. OECD T est Guidelines for chemicals and chemical preparations. Section 4 guidelines on health • effects are at oecd.org/env/ehs/ T esting. Dalbey SCHC 2014 9

OCSPP OECD 2013 Assay Harmonized Endpoint Test Guideline Test Guideline Chronic in vivo studies Carcinogenicity study over major portion of 870.4200 451 Carcinogenicity in mammals life span of animals Combined chronic toxicity/carcinogenicity 870.4300 453 Carcinogenicity in mammals studies Assays for gene mutations Bacterial reverse mutation test 870.5100 471 Point mutations in bacteria Forward and reverse mutations in eukaryotic Gene Mutation in Aspergillus nidulans 870.5140 fungus Mutations in offspring of treated mice detected Mouse Biochemical Specific Locus Test 870.5195 biochemically Mutations in offspring of treated mice observed Mouse Visible Specific Locus Test 870.5200 visually Forward and reverse mutations in eukaryotic Gene Mutation in Neurospora crassa 870.5250 fungus Sex-linked recessive lethal test in Drosophila Point mutations and small deletions in germ line of 870.5275 477 melanogaster fruit flies Mutations in cultured cells, e.g., mouse lymphoma In vitro mammalian cell gene mutation test 870.5300 476 cells, TK6 human lymphoblastoid cells, and Chinese hamster cells (CHO, AS52, or V79) Saccharomyces cerevisiae, gene mutation Base substitution and frameshift mutations gene 480 assay mutations in eukaryotic yeast Spots of altered color in hair of mice exposed to Mouse spot test 484 chemical while in uterus early in fetal development Transgenic rodent somatic and germ cell gene Frequency of mutations in selected tissues of 488 mutation assay transgenic rodents exposed in vivo Dalbey SCHC 2014 10

OCSPP OECD 2013 Test Assay Harmonized Test Endpoint Guideline Guideline Assays for chromosomal aberrations Chromosomal aberrations in cultured mammalian In vitro mammalian chromosome aberration test 870.5375 473 cells Mammalian spermatogonial chromosome Chromosomal aberrations in vivo in germ line 870.5380 483 aberration test leading to sperm cells Mammalian bone marrow chromosome 870.5385 475 Various chromosomal aberrations in vivo aberration test Micronuclei containing lagging chromosome Mammalian erythrocyte micronucleus test 870.5395 474 fragments or whole chromosomes in RBCs in vivo Chromosomal damage or gene mutations in Rodent dominant lethal test 870.5450 478 parental germ cells in vivo leading to embryonic or fetal death Chromosomal damage (reciprocal translocations) Mouse heritable translocation assay 870.5460 485 in treated parental males transmitted to progeny Micronuclei containing lagging chromosome In vitro mammalian cell micronucleus test 487 fragments or whole chromosomes in daughter cells of cells exposed in vitro Dalbey SCHC 2014 11

OCSPP OECD 2013 Test Assay Harmonized Test Endpoint Guideline Guideline Assays for effects on DNA Bacterial DNA Damage or Repair Tests 870.5500 Unscheduled DNA synthesis in mammalian UDS in mammalian cells in vitro as measure of 870.5550 482 cells in vitro repair of DNA damage Unscheduled DNA synthesis in mammalian UDS in liver cells of treated animals as measure 486 liver cells in vivo of repair of DNA damage Saccharomyces cerevisiae, mitotic Conversion of alleles from inactive to wild-type 870.5575 481 recombination assay by mutations in yeast Exchange of DNA between two sister In vitro Sister Chromatid Exchange Assay in 870.5900 479 chromatids of a duplicating chromosome in mammalian calls vitro Exchange of DNA between two sister In vivo Sister Chromatid Exchange Assay 870.5915 chromatids of a duplicating chromosome in vivo, as in lymphocytes or bone marrow Dalbey SCHC 2014 12

Weight Given to Different Short-Term Tests In vivo > in vitro In living organism or normal setting > “ in glass” • Organisms with cell nucleus > those without cell nucleus M ammalian > non-mammalian Assays of DNA damage (e.g., SCE or UDS) indicate interaction with DNA but not necessarily mutation. Results are useful supplemental data. No one test is sufficient, so batteries of tests are often used. Short-term assays provide useful qualitative information, but should be used with care in decisions of carcinogenicity. Dalbey SCHC 2014 13

GHS Categories of Carcinogens (GHS Rev. 5, Section 3.6.2.1) Category 1 : Known or presumed carcinogen Category 1A: Known to have carcinogenic potential for humans… • largely based on human evidence Category 1B: Presumed to have carcinogenic potential for humans… • largely based on animal evidence Category 2: Suspected human carcinogen… on the basis of evidence obtained from human and/ or animal • studies, but which is not sufficiently convincing to place the substance in Category 1… Such evidence may be from either limited evidence of carcinogenicity in human studies or… animal studies. Not Classified: A conclusion based on sufficient data rather than a stated category Dalbey SCHC 2014 14