Evaluating Scientific Studies for Carcinogen Classification under GHS

Walden Dalbey, MA, PhD, DABT

DalbeyTox, LLCWest Chester, PA

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• Mixtures and bridging principles

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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)

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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)

Mutation: Unrepaired change in DNA sequence that is stable enough to be passed on during cell division. Mutations can be in genes, structure of chromosomes, or number of chromosomes.

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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.

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Carcinogenicity Studies: 2-year Rodent Bioassay

Typically two species, each with 50 animals/sex/ dose, three doses plus controls

High dose often Maximum Tolerated Dose (MTD)• 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 MTD

Mainstay for many years, but being questioned now

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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)

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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, MutaMouse, 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+/-, TgrasH2, and XPA-/-

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Short-Term Tests (Faster, Less Expensive, Mechanistic)Use based on assumption that mutations might lead to carcinogenicity.

Many 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 EPA’s Office of Chemical Safety and Pollution Prevention (OCSPP) - Harmonized Test

Guidelines for pesticides and toxic substances (Health Effects Test Guidelines, Series 870) available at www.epa.gov/ocspp/pubs/frs/home/guidelin.htm.

• OECD Test Guidelines for chemicals and chemical preparations. Section 4 guidelines on health effects are at oecd.org/env/ehs/Testing.

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AssayOCSPP

Harmonized Test Guideline

OECD 2013 Test Guideline Endpoint

Chronic in vivo studiesCarcinogenicity study over major portion of

life span of animals 870.4200 451 Carcinogenicity in mammals

Combined chronic toxicity/carcinogenicity studies 870.4300 453 Carcinogenicity in mammals

Assays for gene mutations

Bacterial reverse mutation test 870.5100 471 Point mutations in bacteria

Gene Mutation in Aspergillus nidulans 870.5140 Forward and reverse mutations in eukaryotic fungus

Mouse Biochemical Specific Locus Test 870.5195 Mutations in offspring of treated mice detected biochemically

Mouse Visible Specific Locus Test 870.5200 Mutations in offspring of treated mice observed visually

Gene Mutation in Neurospora crassa 870.5250 Forward and reverse mutations in eukaryotic fungus

Sex-linked recessive lethal test in Drosophila melanogaster 870.5275 477 Point mutations and small deletions in germ line of

fruit flies

In vitro mammalian cell gene mutation test 870.5300 476Mutations in cultured cells, e.g., mouse lymphoma

cells, TK6 human lymphoblastoid cells, and Chinese hamster cells (CHO, AS52, or V79)

Saccharomyces cerevisiae, gene mutation assay 480 Base substitution and frameshift mutations gene

mutations in eukaryotic yeast

Mouse spot test 484 Spots of altered color in hair of mice exposed to chemical while in uterus early in fetal development

Transgenic rodent somatic and germ cell gene mutation assay 488 Frequency of mutations in selected tissues of

transgenic rodents exposed in vivo

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AssayOCSPP

Harmonized Test Guideline

OECD 2013 Test Guideline Endpoint

Assays for chromosomal aberrations

In vitro mammalian chromosome aberration test 870.5375 473 Chromosomal aberrations in cultured mammalian cells

Mammalian spermatogonial chromosome aberration test 870.5380 483 Chromosomal aberrations in vivo in germ line

leading to sperm cellsMammalian bone marrow chromosome

aberration test 870.5385 475 Various chromosomal aberrations in vivo

Mammalian erythrocyte micronucleus test 870.5395 474Micronuclei containing lagging chromosome fragments or whole chromosomes in RBCs in

vivo

Rodent dominant lethal test 870.5450 478Chromosomal damage or gene mutations in

parental germ cells in vivo leading to embryonic or fetal death

Mouse heritable translocation assay 870.5460 485 Chromosomal damage (reciprocal translocations) in treated parental males transmitted to progeny

In vitro mammalian cell micronucleus test 487Micronuclei containing lagging chromosome fragments or whole chromosomes in daughter

cells of cells exposed in vitro

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AssayOCSPP

Harmonized Test Guideline

OECD 2013 Test Guideline Endpoint

Assays for effects on DNA

Bacterial DNA Damage or Repair Tests 870.5500

Unscheduled DNA synthesis in mammalian cells in vitro 870.5550 482 UDS in mammalian cells in vitro as measure of

repair of DNA damageUnscheduled DNA synthesis in mammalian

liver cells in vivo 486 UDS in liver cells of treated animals as measure of repair of DNA damage

Saccharomyces cerevisiae, mitotic recombination assay 870.5575 481 Conversion of alleles from inactive to wild-type

by mutations in yeast

In vitro Sister Chromatid Exchange Assay in mammalian calls 870.5900 479

Exchange of DNA between two sister chromatids of a duplicating chromosome in

vitro

In vivo Sister Chromatid Exchange Assay 870.5915Exchange of DNA between two sister

chromatids of a duplicating chromosome in vivo, as in lymphocytes or bone marrow

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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

Mammalian > 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.

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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

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Criteria for Classification of Carcinogens under GHS

“The guidance provides an approach to analysis rather than hard and fast rules… The weight of evidence called for in GHS is an integrative approach which considers important factors in determining carcinogenic potential along with the strength of evidence analysis.” (GHS Rev. 5, section 3.6.5.3.2)

• Strength of evidence “involves the enumeration of tumours in human and animal studies”. (GHS Rev. 5, Section 3.6.2.4)

• Weight of evidence - A “number of other factors should be considered that influence the overall likelihood that an agent may pose a carcinogenic hazard to humans.” (GHS Rev. 5, Section 3.6.2.5)

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Criteria for Classification: Strength of EvidenceDerived from GHS Sections 3.6.2.1, 3.6.2.4 and 3.6.5.3.1, and IARC

Carcinogenicity in Humans

Sufficient evidence

Causal relation between human exposure and development of cancer is established. Chance, bias, or confounding can be ruled out with reasonable confidence.

Limited evidence

Positive association is shown between exposure and cancer for which a causal relation seems credible, but chance, bias, or confounding cannot be ruled out with reasonable confidence.

Carcinogenicity in Laboratory Animals

Sufficient evidence

Causal relation between exposure and increased incidence of malignant neoplasms or an appropriate combination of benign and malignant neoplasms in >2 species or >2 independent studies in 1 species done at different times or different labs. (Unusually definite data from one species in one study can be considered.)

Limited evidence

Data suggest carcinogenic effect, but are less than sufficient because (1) data come from one study, (2) questions remain on design, conduct, or interpretation of the study, or (3) increased incidence is seen only in benign neoplasms, lesions of uncertain neoplastic potential, or neoplasm that occur spontaneously in high incidence.

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Criteria for Classification: Weight of Evidence (Sections 3.6.5.2-6 and 3.6.5.3.2.1-5)Factors to consider in evaluation of data from long-term in vivo studies include:

Relevance of route of exposure Statistical significance for specific tumorsReduced latency (time-to-tumor) Progression of lesions to malignancyOccurrence of unusual tumors Historical range of tumors in controlsWere differences in one or multiple species and/or sex?Were tumors only at doses with severe toxicity (less weight of evidence)?

“Evidence of mutagenic activity in vivo may indicate that a substance has a potential for carcinogenic effects.”

“A substance that has not been tested for carcinogenicity may in certain instances be classified in Category 1 or Category 2 based on tumour data from a structural analogue together with substantial support from… other factors”.

Also consider if substance is absorbed by a given route, only local tumors occurred at site of dosing, or testing by other routes showed lack of carcinogenicity.

Data on absorption, distribution, metabolism (activation and detoxification), and elimination

Information on chemical analogues

Information on mode of action and relevance to humansDalbey SCHC 2014 17

Example #1 of Test Results and Classification under GHSStrength of Evidence

Data Other GHS 2 GHS 1B GHS 1A

Clearly positive chronic study in 2 rodent species

Positive HPRT assay for gene mutation in cultured hamster cells (in vitro)

Positive micronucleus assay in mice in vivo (chromosomal damage)

Structural analogs: none

Kinetics and metabolism similar in humans and rodents

Weight of Evidence: Guideline studies with no confounding factors. Substance was carcinogenic in two species in rodent bioassay. Additional evidence was chromosomal damage in mammals in vivo and data supporting extrapolation from rodents to humans. Supporting evidence was mutagenicity in mammalian cells in vitro.

Possible Conclusion: Category 1B, Presumed human carcinogen, depending on assessment of relevance to humans

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Example #2 of Test Results and Classification under GHSStrength of Evidence

Data Other GHS 2 GHS 1B GHS 1A

Clearly positive results in rats and marginal in mice in chronic tumor study.

Positive HPRT assay for gene mutation in cultured hamster cells in vitro

Negative micronucleus assay in mice in vivo (chromosomal damage)

Structural analog is Category 2, suspected carcinogen based on limited evidence. Kinetics and metabolism very similar to

test substance

Kinetics and metabolism similar in humans and rodents

Weight of Evidence: Guideline studies with no confounding factors. Tumorigenicity in chronic study was limited mainly to one of two species, but analog is Category 2 and metabolism and kinetics of tested substance and analog support extrapolation to humans. Mutagenicity in mammalian cells in vitro was supporting evidence. Lack of chromosomal damage is not a significant factor. Relevance of mode of action to humans should be evaluated.

Possible Conclusion: Category 2, Suspected carcinogen based on limited evidenceDalbey SCHC 2014 19

Example #3 of Test Results and Classification under GHSStrength of Evidence

Data Other GHS 2 GHS 1B GHS 1A

Clearly positive tumor study in transgenic mice. ???Positive HPRT assay for gene mutation in cultured hamster

cells in vitroStructural analog is Category 2, suspected carcinogen based

on limited evidence. Kinetics and metabolism are very similar to test substance.

Equivocal interspecies comparison of kinetics and metabolism

Weight of Evidence: Guideline studies with no confounding factors. Results in transgenic mice might meet criteria for Category 2 (limited evidence in lab animals) and are supported by read-across from carcinogenic analog. However, data on interspecies extrapolation weremarginal. Mutagenicity in mammalian cells in vitro provides supporting data.

Possible Conclusion: Dependent of strength of the results and validation of the assay in transgenic mice.

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Example #4 of Test Results and Classification under GHSStrength of Evidence

Data Other GHS 2 GHS 1B GHS 1A

Positive mouse spot test for mutagenicity in mammals in vivoPositive test in mammalian bone marrow for chromosomal

aberration in vivoPositive Ames test for mutations in bacteria in vitro

Structural analog considered mutagenic, but no decision on carcinogenicity

No data on similarity of kinetics and metabolism between humans and lab animals

Weight of Evidence: Guideline studies with no confounding factors. No direct data on carcinogenicity in vivo. Substance was mutagenic in mammals in vivo and caused chromosomal aberrations in mammals in vivo. Positive test for mutagenicity in bacteria is supporting, but other supporting information is too weak to make a classification as a carcinogen.

Possible Conclusion: Classification not possible. Data warrant further investigation before making a final conclusion.

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Classification of Mixtures: Step 1

Are data available on the complete mixture? If so, apply section 3.6.3.1. If data on the complete mixture are not available or are not conclusive, go to section 3.6.3.2 (bridging principles).

Section 3.6.3.1:• “Classification of mixtures will be based on the available test data of

the individual ingredients of the mixture using cut-off values/ concentration limits for those ingredients.” Values for cut-offs are in Table 3.6.1 (slide 25 here).

• “The classification may be modified on a case-by-case basis based on the available test data for the mixture as a whole”, provided that results for the mixture are conclusive and factors such as dose, duration of study, observations, statistical analyses, and sensitivity of the carcinogenicity test system are taken into account.

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Classification of Mixtures: Step 2If a mixture is untested, “but there are sufficient data on both the individual ingredients and similar tested mixtures to adequately characterize the hazards of the mixture, these data will be used in accordance with the following agreed bridging principles.” (GHS Section 3.6.3.2)

Dilution: “If a tested mixture is diluted with a diluent that is not expected to affect the carcinogenicity of other ingredients, then the new diluted mixture may be classified as equivalent to the original tested mixture.” Original classification is retained and no further testing is required.

Batching: “The carcinogenic potential of a tested production batch of a mixture can be assumed to be substantially equivalent to that of another untested production batch of the same commercial product, when produced by or under the control of the same manufacturer unless there is reason to believe there is a significant variation in composition such that the carcinogenic potential of the untested batch has changed.”

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Classification of Mixtures: Bridging Principles Continued

Substantially similar mixtures (one tested and the other not tested):Mixture 1: Ingredient A+ Carcinogen B (tested)Mixture 2: Ingredient C + Carcinogen B (untested)If: 1) concentration of carcinogen B is the same in both mixtures,

2) concentrations of ingredients A and C are equal,3) toxicity of respective ingredients is substantially equivalent, and4) ingredients will not affect carcinogenicity,

Then the untested mixture can be classified the same as the tested mixture.

Ingredients A and C can be mixtures themselves.

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Classification of Mixtures – Step 3

If bridging principles are not used, go to section 3.6.3.3. The mixture will be classified as a carcinogen based on the concentration of at least one carcinogen in it using the following cut-offs in section 3.6.3.3.

a) With a Category 2 carcinogen between 0.1 and 1.0%, regulatory authorities require information on the SDS for a product. Warning on label would be optional; authorities might or might not require a label.

b) SDS and label are generally expected with Category 2 carcinogen >1%.Dalbey SCHC 2014 25

Cut-offs for Classification of a Mixture as CategoryCategory of ingredient 1A 1B 2

1A >0.1%1B >0.1%

2 >0.1%a

>1.0%b

Classification of Mixtures: A Cautionary NoteGHS Section 3.6.3.1: Classification can be modified on a case-by-case basis using data on the whole mixture.

Imagine a carcinogen that caused a small, but statistically significant, increase in tumors in lab animals at a concentration of 0.5%.

A mixture with that carcinogen at <0.09% was tested and caused a trend for increased tumors (not statistically significant).

Should the total mixture with <0.09% be considered…A. Not carcinogenic, based on the data with the whole mixture.B. Carcinogenic, because a more obvious carcinogenic effect

probably would be apparent if more animals had been in the group treated with the mixture.

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Classification of Mixtures: Another Cautionary Note

Consider a mixture with two Category 2 carcinogens, each at 0.075% in the mixture.

Should they be considered additive (combined concentration of 0.15%) or not additive (each <0.1%)?

Do the carcinogens act via same mechanism, affect the same target organs, have the same kinetics, etc. ?

orIs there substantial reason to expect that they would not act additively?

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Classification Can Be a Head-Scratcher.

Judgment is involved with classification for carcinogenicity and conclusions will vary among people. Establish your rationale. Follow it, but modify it as warranted.

Hopefully this talk gives some additional framework to help you proceed.

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