Mutagenic MOA Carcinogens How High is the Burden
Mutagenic MOA Carcinogens: How High is the Burden of Proof ? RASS Telecom 09/10/08 Rita Schoeny, Ph. D. Senior Science Advisor Office of Water, U. S. EPA 1
Disclaimer u The views expressed in this presentation are those of the author and do not represent the policy of the U. S. EPA. Some of this is EPA policy 2
Risk Assessment is constantly evolving u Science and Judgment – Describe all defaults – Ensure they are health protective, documented, departures are warranted u Cancer Guidelines 2005 – Use data before defaults u Rather than determine how much data needed to depart from default u Including default procedures such as linear low dose risk 3
Mode of Action and Cancer Assessment u MOA is the keystone to all aspects of the assessment process True for other endpoints and is the major factor in harmonization among risk assessments 4
Why Do You Care about MOA ? u MOA is key in Hazard Identification – Helps describe circumstances under which agent is carcinogenic (High dose? Route? ) – Relevance of data for humans u MOA determines choice of Low Dose Extrapolation u Life stage risk 5
Mode of Action u “. . . a sequence of key events and processes, starting with interaction of an agent with a cell, proceeding through operational and anatomical changes, and resulting in cancer formation. . . Mode of action is contrasted with “mechanism of action, ” which implies a more detailed understanding and description of events, often at the molecular level, than is meant by mode of action” Exposure Key event Toxicity 6
Mode of Action Frameworks IPCS U. S. EPA u u Hypothesized MOA: summary description and identification of key events Experimental support: – Strength, consistency, specificity of association – Dose-response concordance – Temporal relationship – Biological plausibility and coherence Consideration of the possibility of other MOAs Relevance to humans u u u u u Postulated mode of action (theory of the case) Key events Concordance of doseresponse relationships Temporal association Strength, consistency and specificity of association of tumour response with key events Biological plausibility and coherence Other modes of action Uncertainties, Inconsistencies, and Data Gaps Assessment of postulated mode of action 7
MOA/Human Relevancy ILSI/IPCS Is the weight of evidence sufficient to establish a mode of action (MOA) in animals? NO Proceed with risk assessment YES MOA not Relevant YES Can human relevancy of the MOA be reasonably excluded on the basis of fundamental, qualitative differences in key events between animals and humans? NO MOA not Relevant YES Can human relevancy of the MOA be reasonably excluded on the basis of quantitative differences in either kinetic or dynamic factors between animals and humans? NO Proceed with Risk assessment 8
Key Event u. A “key event” is an empirically observable precursor step that is itself a necessary element of the mode of action or is a biologically based marker for such an element. Key event is necessary, but not sufficient If a key event doesn’t occur, there is no cancer If one key event occurs, there may or may not be cancer 9
Postulated Mode Of Action 1 Metabolism E 2 P Y C e v idati Ox Chloroform Phos ge ne Sustained Toxicity Regenerative Cell Proliferation Key Events Tumor Development 10
MOA and Kids u Supplemental Guidance for Assessing Susceptibility from Early-Life Exposure to Carcinogens – Effects observed in childhood – Early life exposures that contribute to later life effects – MOA determines whether quantitative adjustment is made 11
Supplemental Guidance Use age-specific values for exposure and potency u When data permit, develop separate potency estimates for childhood exposure u In risk characterization, mutagenic MOA risk is increased by age-dependent adjustment factor (used with exposure info for age group) u u <2 yrs old, 10 fold u 2 to < 16 yrs, 3 fold u No MOA, linear extrapolation without ADAF; non-linear MOA, no ADAF 12
Framework for Determining • Public Comment completed 12/07 a Mutagenic Mode of • External peer review Action for Carcinogenicity completed 05/08 Using EPA’s 2005 Cancer Guidelines and Supplemental Guidance for Assessing www. epa. gov/ Susceptibility from Early-Life osa/mmoaframework/ Exposure to Carcinogens pdfs/MMOA-ERD-FINAL -83007. pdf 13
Framework on Default MOA u “ It should also be noted that there is no ‘default MOA. ’ The Cancer Guidelines offer some default procedures to use when no MOA can be determined. ” • MOA determinations follow Cancer Guidelines Framework • If insufficient data to support MOA, use low dose linear extrapolation and no ADAF Determination of mutagenic MOA is as scientifically rigorous as any other MOA 14
What is a mutagen? u A chemical that induces biologically relevant mutations in any one of a number of validated mutation assays u Mutation assays detect the induction of mutants u Mutants are cells with genetic alterations that can be passed to viable daughter and granddaughter cells -heritable 15
What is “Mutagenic”? EPA does not have a standard definition of “mutagenic” u Operationally for use in “mutagenic MOA for cancer” u – “. . . capacity of either the carcinogen or its metabolite to react with or bind to DNA in a manner that causes mutations. In this context, mutagens usually (though not always) produce positive effects in multiple test systems for different genetic endpoints, particularly gene mutations and structural chromosome aberrations, both in vitro and in vivo. ” – The peer reviewers hated it 16
Framework: Multi-step Process u Risk assessment is an iterative process u Visualize the Framework as series of linear steps u Step 1 is assemble relevant data – Genetic toxicity testing, tumor data, pk, SAR, etc. – Framework describes test batteries 17
Step 2: Evaluate Data Quality u Look at primary papers u Judge against current acceptability criteria – e. g. were tests done at cytotoxic levels u Cites publications for evaluating quality (e. g. Cimino 2006, OECD, ICH, IWGT, DHHS 2006) u Keep, but weigh 18
Gene- tox Tests Measure Different Events Genotoxicity Assays Type of Damage Mouse Lymphoma Chromosome Aberrations CHO cells Ames Bacterial Mutagenicity Point mutation Yes No Yes Oligonucleotide Yes insertion or deletion No Yes Allele Loss Yes No No Small Chromosome alteration Yes ? No Large Chromosome alteration Yes No Aneuploidy ? Yes No TERA’s Dose-Response Assessment Boot Camp Adapted from M. Moore (2004) 19
Step 3: WOE for Mutagenic Activity -- 1 Evaluation requires someone expert in gene-tox (all tests don’t measure same things) u Categorize data – suggest use of our table in Appendix A. u – Put in all data with notes on quality – Use consistent terms for assay types or endpoints: positive, negative, inconclusive, contradictory – Present summary of database 20
WOE for Mutagenic Activity -- 2 – Conclusions across endpoints: some endpoints carry more weight than others u e. g. No Checklist Sperm head morphology may be caused by modification of protein structure u Morphologic cell transformation does not measure mutation – Hierarchy of data utility interaction ≠DNA damage ≠mutation No Minimum Data Set u DNA u e. g. most useful are mutations in relevant genes in humans – WOE for mutagenic activity: negative, data are inadequate, data are of questionable quality, data are equivocal, data are positive 21
How to Weigh the Evidence as to Whether a Chemical Causes Specific Tumors by a Mutagenic Mode of Action (Mutation is THE Key Event) (Listed in decreasing order of relevance/importance) 1. Cancer relevant oncogene/tumor suppressor gene mutations can be detected in the target tissue following chemical exposure 2. Surrogate gene mutations can be detected in the target tissue following chemical exposure 3. DNA adducts (known to be mutagenic adducts) can be detected in the target tissue following chemical exposure 4. Primary DNA damage can be detected in the target tissue following chemical exposure 5. Gene mutations and/or DNA adducts or other measures of primary DNA damage can be detected in vivo. 6. Evidence that the chemical can induce mutations, cytogenetic damage, DNA adducts and/or primary DNA damage in vitro. 22
Not Finished yet u Mutagenicity + carcinogenicity ≠ Mutagenic MOA Apply MOA Framework Step 4 l. Hypothesized MOA l. Experimental support: –Dose-response concordance –Strength, consistency, specificity of association –Temporal relationship –Biological plausibility and coherence l. Consideration of the possibility of other MOAs l. Relevance to humans 23
Key Events DNA changes resulting in mutation u “ For a chemical to act by a mutagenic MOA, either the chemical or its direct metabolite is the agent inducing the mutations that initiate cancer. ” u u “This is contrasted with a MOA wherein mutagenicity occurs as an indirect effect of another key event in carcinogenesis. ” u Properties for mutagenicity as the key event u Long list in Guidelines: early tumor response, initiator, target tissue is exposed to DNA-reactive chemical, mutation is early event, mutation in oncogenes, etc 24
Tumor Induction: Time-related Accumulation of Events Mutagenic Carcinogen Initiating Multiple events Tumor Mutation Nonmutagenic Carcinogen Toxicity Altered Gene Expression Cell Proliferation Initiating Mutation Multiple events Tumor 25
Applying the MOA Framework u Types of data supporting WOE – Consistency across assays – Induction of ≥ 1 type of effect – Effects in vivo – Mutation in absence of cytotoxicity – Belongs to a class of compounds with established mutagenic MOA u Including the “Supplemental Guidance 12” 26
Cyclophosphamide Cytotoxic, alkylating Alkylating Cytotoxic 27
Postulated Mode Of Action CP Metabolism Cyt p 450 s Phosp horam ide mu Acrole stard, in PAM Tumor Development DNA damage Mutations 28
Cyclophosphamide GAP 29
CP In Vivo Tests: Animals u Gene Mutation Assays – Positive Mouse Spot Test (2. 5 -10 mg/kg) – Positive Muta Mouse (lac. Z) 100 mg/kg x 5 days in bone marrow – B 6 C 3 F 1 mouse (lac. I) 100 mg/kg MF increased in lungs and urinary bladder – No transgenic studies in rats 30
CP In Vivo Tests: Humans – Micronuclei peripheral blood lymphocytes (PBL) & buccal epithelials 26/26 nurses handling CP – Structural chromosome aberrations & SCE, gene mutations or DNA damage (Comet assay) in PBL or bone marrow, patients – Structural chromosome aberrations in children – Mutation of p 53 in bladder tumors (cumulative doses of 6 -125 mg/kg) – 6 -Thioguanine-resistant T lymphocytes from multiple sclerosis patients (750 mg/m 2) 31
So CP Is Mutagenic u And it’s carcinogenic u Apply MOA Framework 32
Dose Resp Concordance u Mutation is key event – Expect mutations and / or DNA interaction at lower dose than tumors u Mutation is not the key event – Expect increased mutation at doses higher than those required for tumor induction (the increase in mutations likely results as a secondary effect of cytotoxicity or cell proliferation) 33
CP Dose / Resp Concordance u Rodents – Lowest effective dose [induction of SCE in rat bone marrow (0. 62 mg/kg)] – Consistent with data showing significant tumor formation in the urinary bladder of male rats at 1. 25 and 2. 5 mg/kg/day (488 mg total) u Humans – Chromosome aberrations & SCEs 2 hrs after dosing 33 -40 mg/kg – p 53 mutations at a cumulative dose of 6 g – Cohort of 6171 survivors of non-Hodgkin's lymphoma; 48 developed cancer of the urinary tract – those receiving a total dose of 20 g had a 2. 4 -fold risk of bladder cancer; 2049 g, a 6 -fold risk 34
Temporality: Evaluate time-to-mutation Mutant Frequency Mutagenic carcinogens would be expected to show a positive mutation response after relatively short treatment periods Time in Weeks Nonmutagenic carcinogens would be expected to be negative after long chronic treatment, or show a positive response only after long chronic treatment 35
CP TEMPORAL ASSOCIATIONS u u u SCE bone marrow of Fischer 344 rats dosed with 20 mg/kg (ip) CP after 30 min. (1 hr after 5 or 10 mg/kg) Chromosomal aberration & micronuclei in human bone marrow 24 hrs post therapeutic dose of 40 mg/kg (iv) Cytotoxicity & regenerative proliferation in the rat also occur early: – Bladder damage (ulceration of mucosa, necrosis of bladder epithelium)— 1 day – Regeneration of bladder epithelia – 36 hrs – Hyperplasia of bladder epithelia – 48 hrs – Malignant bladder tumors — 40 -60 weeks 36
CP Database Plausibility & Coherence Qualitative & quantitative data for key events leading to tumors u Concordance of most key events in animal models & humans u No stop/recovery studies found, but there is evidence suggesting that CP-associated cancers may occur up to several years after drug treatment has ceased. u Gaps in human data (e. g. , DNA adducts & cell proliferation) do not compromise the analysis 37 u
MOA Relevance Rats PAM generation DNA adducts Mutagenicity Bladder cytotoxicity Epithelial regeneration Hyperplasia Bladder tumors Humans Yes Yes Plausible Yes Yes 38
Postulated Mode Of Action 1 Metabolism E 2 P Y C e v idati Ox Chloroform Phos ge ne Sustained Toxicity Regenerative Cell Proliferation Key Events Tumor Development 39
CCl 3 Genetic Activity Profile 40
Mutagenicity: Lines of Evidence Negative in vitro u Conflicting evidence in vivo u Initiation-Promotion Studies u – CCl 3 is not an initiator u Molecular Based Approaches – Negative for tumors in p 53 +/- transgenic mouse cancer bioassay – Negative for mutations in Lac. I transgenic B 6 C 3 F 1 mice – Negative for mutation in rat GST transfected bacteria 41
Mutagenicity CCl 3: Conclusions u Weight of Evidence ØMutagenicity is not a component of chloroform induced neoplasia 42
Metabolism: Conclusions Predominate pathway – P 450 (CYP 2 E 1)-mediated oxidative pathway Ø Phosgene key reactive metabolite l. The following play little, if any role in chloroform induced tumors-–Reductive P 450 metabolism & free radical production –GST catalyzed conjugation u 43
MOA Conclusions for Chloroform Hypothesized MOA Well Supported Other MOAs NOT Well Supported Human Relevance Presumed (also epidemiological data on chlorinated water) Applies to Children (but not more susceptible) Consistent with Nonlinear Dose Response Risk Approach Based on Protection Against Sustained Toxicity/Proliferation 44
Consider u What data are available? – Screening genetox data, batteries of test designed for hazard identification u What data are optimal? – Real, live MOA data (e. g. time course studies in relevant human genes) u What data are practical? – Something less than what was available for cyclophosphamide – Requires some strategic thought in test design. 45
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