1. Nondefault Uncertainty Factor Values John Lipscomb, Ph.D., DABT, ATS US Environmental Protection Agency Office of Research and Development National Center for Environmental Assessment Cincinnati, Ohio 513-569-7217 Lipscomb.John@epa.gov Methodologies in Human Health Risk Assessment Society of Toxicology March 23, 2014 Phoenix, AZ The views expressed in this presentation are those of the author and do not necessarily represent the views or policies of the U.S. Environmental Protection Agency.

2. 2 Goals Think quantitatively when developing risk values Consider the impact of toxicokinetics on the adverse health outcome Relate the development of the adverse health outcome to the tissue dose

3. 3 Lecture Organization Dose response IPCS CSAF Guidance Data evaluation Interspecies differences in toxicokinetics (AK AF ) Interspecies differences in toxicodynamics (AD AF )

4. Glossary BMD: An estimate of the dose or concentration that produces a predetermined change in response rate BMDL: 95% lower-bound confidence limit on the BMD BMR: A predetermined response level based on which a BMD or BMDL is calculated NOAEL: No Observed Adverse Effect Level LOAEL: Low Observed Adverse Effect Level POD: a point of departure used in estimate risk values when divided by an uncertainty factor UF: Factors used in risk assessment to account for uncertainty in the data or extrapolations to human no-effect levels CSAF: Chemical Specific Adjustment Factors PBPK: Physiologically based pharmacokinetic AUC: area under the curve Cl: clearance ED: effective dose

5. Exposure-Dose-Response Continuum External dose Internal dose Target organ responses Toxic Response Target organ metabolism Target organ dose External dose Toxic Response External dose Internal dose Toxic Response External dose Internal dose Toxic Response Target organ dose External dose Internal dose Toxic Response Target organ metabolism Target organ dose Toxicokinetics Toxicodynamics

6. Reference Dose, Reference Concentration NOAEL, LOAEL or BMD L RfD or RfC = UF - Or - RfD or RfC = POD UF

7. Uncertainty Factor Default Values Factor*Extrapolation H10Average Human to Sensitive Human A10 or 3Animal to Human S10 Sub-chronic to Chronic Exposure L10 LOAEL to NOAEL D10 or 3Minimum to Complete Database * These factors are as used by the U.S. EPA. Other health organizations use similar factors.

8. 8 Uncertainty and Variability Variability = heterogeneity in time, space (pharmacokinetics, pharmacodynamics)  Variability is inherent to the population  Can quantify variability, but more data doesn’t decrease variability  Interspecies differences  Interindividual variability Uncertainty = lack of knowledge, can be decreased by additional data  Subchronic to chronic  LOAEL to NOAEL  Database

9. 9

10. 10 Mode of Action An understanding of how a chemical induces adverse effects, including a consideration of target tissue, dose metric and time normalization regarding both:  Toxicokinetics: disposition  Toxicodynamics: sensitivity IPCS/ILSI Human Relevance Framework  Has a MOA been established in animals?  Can it be ruled out on the basis of qualitative species differences?  Can it be ruled out on the basis of quantitative species differences?

11. 11 Goals of the IPCS’ CSAF Guidance Chemical Specific Adjustment Factors, 2005 Communicate the framework for inclusion of quantitative data. Guide a critical evaluation of the available data. Harmonize international efforts at data evaluation and application. Stimulate research to develop quantitatively valuable data.

12. IPCS’ Default Value Framework 100-fold uncertainty factor Interspecies Differences 10-fold Interindividual Variability 10-fold Toxicokinetics AK 10 E0.6 (4.0) Toxicodynamics HD 10 E0.5 (3.16) Toxicokinetics HK 10 E0.5 (3.16) Toxicodynamics AD 10 E0.4 (2.5)

13. 13 (Toxico)kinetics vs. Dynamics To a PBPK modeler:  Kinetics = what the body does to the chemical (movement in body, distribution, metabolism - ADME)  Dynamics = what the chemical does to the body (DNA binding, receptor binding, denaturation, etc., up to effect at target) In the IPCS (1994) framework:  Kinetics = uptake, movement of chemical in body, biotransformation, distribution and elimination of substance and metabolites from the body (disposition)  Dynamics = Interaction of chemical with target sites and subsequent interactions leading to adverse effects (sensitivity). Includes metabolism in target tissue

14. 14 Key Steps in Developing a CSAF Determine the active chemical species Choose the dose metric Evaluate relevancy of data  Population  Exposure route  Dose / Exposure  Number of subjects/samples

15. BLOOD CONCENTRATION DOSE Parent Metabolite Determine the Active Chemical Species DOSE RESPONSE DOSE RESPONSE Inhibition studies and Knockout strains also valuable

16. 16 Determine the Active Chemical Species If the data are insufficient to identify the active species, the default UF approach is used, unless –  The interspecies differences in the TK of parent or metabolite(s) would lead to a CSAF greater than the default, consider using the higher value, even in the absence of data clearly defining the active chemical species. The default may not be sufficiently protective. Mechanistic and metabolic data In vitro or in vivo studies with parent and metabolite(s)

17. Choose the Dose Metric Blood concentrations are acceptable Cmax: Acute or short term effects AUC: Longer term or chronic effects Clearance: In lieu of AUC  Glomerular filtration rate  Not intrinsic clearance (Vmax/Km)  Not half-life 17

18. 18 Chemicals partition between air and blood; and between blood and solid tissues Blood concentration data may serve as a surrogate for tissue concentration data, especially when tissue partitioning has been measured. Air Blood Fat Kidney Liver Why Blood Concentrations? Species differences in partitioning between blood and solid tissues may be minimal.

19. 19 Relevance of Experimental Data Population  Good quality data; animal mean versus human mean  Representative samples & sensitive populations Route  Human exposures versus animal studies  Route to route extrapolation possible Dose  Animal point of departure vs anticipated human exposure  Evaluate nonlinearities Number of Samples/Subjects  Reliable estimate of central tendency  Without data assume normal or lognormal distribution

20. Interspecies TK Variability Determine species-dependent differences in tissue exposure External dose Internal dose Target organ responses Toxic Response Target organ metabolism Target organ dose External dose Toxic Response External dose Internal dose Toxic Response External dose Internal dose Toxic Response Target organ dose External dose Internal dose Toxic Response Target organ metabolism Target organ dose

21. 21 TD TK AD AF AK AF 2.5X 4.0X

22. 22 TOXICOKINETIC PARADIGM Animal Dose Human Dose Target Tissue Concentration Default 4.0 fold Chemical Specific Chemical Specific AK AF = AUC A or Cmax A or Cl H AUC H or Cmax H or Cl A

23. 23 Example - AK AF Aliphatic hydroxylamine, sparingly soluble in water Testicular toxicity in rats, NOAEL is 10 mg/kg-day, LOAEL of 20 mg/kg-day Present in drinking water, daily intake for humans estimated at 0.25 mg/kg-day. Toxicity correlates with the parent compound Wistar rats excrete 20% of dose as hydroxy metabolite; DA rats excrete 2% as the hydroxy metabolite; they have comparable sensitivity

24. Oral Toxicokinetic Data in Rats Species Dose (mg/kg body weight) C max (µg/mL) T max (h) AUC [(µg/mL)- hr] Eliminati on Half-life (hr) CL (mL/min per kg body weight) Rat (Wistar) N=5 10.4910.46236 100.9014.52.237 201.49312.42.927 502.7434.83.324 N =number of animals studied at each time point (animals were killed and blood and testes were taken for analysis). C max =maximum observed plasma concentration. T max =time of C max. AUC = area under the plasma concentration-time curve (extrapolated to infinity). CL = total plasma clearance (calculated assuming bioavailability = 1) (= dose/AUC).

25. Oral Toxicokinetic Data in Humans (n=12) Dose (mg/kg body weight) C max (µg/mL) T max (hr) Elimination Half-life (hr) CL a (mL/min) CL b (mL/min per kg body weight) 0.250.081.23.5 ± 0.3805 ± 409.9 1.00.31.24.8 ± 0.6540 ± 506.6 a p < 0.05 between high and low doses. b The CL adjusted to body weight (mL/min per kg body weight) was not reported, and the values have been calculated as mean CL (mL/min) divided by mean body weight reported in the study

26. 26 Key Points - AK AF Active species: parent – all TK data are for parent No information on choice of TK parameter; Cl is conservative, as would be AUC Testes equilibrate rapidly with plasma, considered part of central compartment Route for animal data: relevant Dose: have TK data at NOAEL - 10 mg/kg-day Numbers: adequate sample size, have good estimate of mean Quantify animal TK near the ___________.

27. 27 Key Points - AK AF Human population: relevant – in males Route: relevant – oral Dose: Data for 0.25 vs 1 mg/kg doses suggest saturation kinetics. Choose lower dose; higher dose is 10% of animal NOAEL, and higher than expected exposure Numbers: adequate, limited variability. SE = SD/√n = 40/ √12 = 11.5. This is 1.4% of the mean clearance (805 mL/min) that is <20% of the mean. Quantify human TK near the ___________________.

28. Oral Toxicokinetic Data in Rats Species Dose (mg/kg body weight) C max (µg/mL) T max (h) AUC [(µg/mL)-hr] Elimination Half-life (hr) CL (mL/min per kg body weight) Rat (Wistar) N=5 10.4910.46236 100.9014.52.237 201.49312.42.927 502.7434.83.324 N =number of animals studied at each time point (animals were killed and blood and testes were taken for analysis). C max =maximum observed plasma concentration. T max =time of C max. AUC = area under the plasma concentration-time curve (extrapolated to infinity). CL = total plasma clearance (calculated assuming bioavailability = 1) (= dose/AUC).

29. Oral Toxicokinetic Data in Humans (n=12) Dose (mg/kg body weight) C max (µg/mL) T max (hr) Elimination Half-life (hr) CL a (mL/min) CL c (mL/min per kg body weight) 0.250.081.23.5 ± 0.3805 ± 409.9 1.00.31.24.8 ± 0.6540 ± 506.6 a p < 0.05 between high and low doses. b Renal CL = renal clearance calculated from amount excreted in urine and plasma concentrations c The CL adjusted to body weight (mL/min per kg body weight) was not reported, and the values have been calculated as mean CL (mL/min) divided by mean body weight reported in the study

30. 30 Calculation of AK AF Cl is normalized by body weight Clearance in rats: 37 mL/min – kg bw at NOAEL of 10 mg/kg Clearance in humans: 9.9 mL/min-kg bw at 0.25 mg/kg AK AF = 37/9.9 = 3.7 This is close to default AK AF = AUC A or Cmax A or Cl H AUC H or Cmax H or Cl A

31. 31 AK AF = AUC A or Cmax A or Cl H AUC H or Cmax H or Cl A IF… Human AUC were 0.4 ug/ml-hr @ 0.25 mg/kg, then… AUC H = 0.4 ug/ml-hr / 0.25 mg/kg = 1.6 ug/ml-hr/(mg/kg), and AUC A = 4.5 ug/ml-hr / 10 mg/kg = 0.45 ug/ml-hr / (mg/kg), and AK AF = AUC H / AUC A = 1.6 / 0.45 = 3.6

32. 32 AK AF = AUC A or Cmax A or Cl H AUC H or Cmax H or Cl A IF… Human AUC were 0.05 ug/ml-hr @ 0.25 mg/kg, then… AUC H = 0.05 ug/ml-hr / 0.25 mg/kg = 0.2 ug/ml-hr/(mg/kg), and AUC A = 4.5 ug/ml-hr / 10 mg/kg = 0.45 ug/ml-hr / (mg/kg), and AK AF = AUC H / AUC A = 0.2 / 0.45 = 0.4

33. SUMMARY Use Toxicity data to guide TK evaluations (dose metric) Focus on active chemical species Similarity in dose route must be accounted-for Dose normalization forces knowledge of linearity of kinetic processes Avoid or account for nonlinearities Compare TK measurements in range of species-relevant doses Do not use half-life data for calculations Assume AUC for non-acute effects

34. Interspecies TD Variability Determine species-dependent differences in concentrations producing the response External dose Internal dose Target organ responses Toxic Response Target organ metabolism Target organ dose External dose Toxic Response External dose Internal dose Toxic Response External dose Internal dose Toxic Response Target organ dose External dose Internal dose Toxic Response Target organ metabolism Target organ dose

35. 35 TD TK AD AF AK AF 2.5X 4.0X

36. 36 TOXICODYNAMIC PARADIGM Animal Dose Human Dose Response Level Default 2.5 fold Chemical Specific Chemical Specific AD AF = ED H ED A

37. 37 RESPONSE DOSE AD AF > 1.0 HUMAN ANIMAL ED H ED A ANIMAL HUMAN ED A ED H AD AF < 1.0 AD AF = ED H ED A

38. 38 Choice of Endpoint Critical effect or a key event.  The key event is intimately linked to the critical toxic effect based on the MOA  Observable, measurable & quantifiable  Dose-response and temporal relationships for key event should be similar to that of critical effect  No guidance in document for identifying, but see EPA’s cancer guidelines and IPCS framework (modified Hill criteria) In vitro studies are a useful

39. 39 Adequacy of Dose-Response The experimental methods should be comparable. Where the animal and human dose-response curves are parallel (same mathematical model), can use any response between 10 and 90%. Where the dose-response curves are not parallel in these experiments, use the lowest point on the curve that provides reliable information without extrapolation below the experimental data. This is usually the ED 10 The default is to use ED 10A /ED 10H.

40. 40 Example - AD AF Compound C The critical effects of compound C:  Cholinergic toxicity (accumulation of ACh) Route of exposure: oral Toxicity from parent compound Rat is the most sensitive animal species Rats and humans have similar toxicokinetics

41. stimulationAcetylcholine (ACh) Central nervous system Peripheral nervous system Ach-Esterase Chemical C Mode of Action X ACh destruction Chemical C Toxicity

42. 42 MOA Active species: parent compound Inhibit acetylcholinesterase (AChE) Accumulation of acetylcholine results in cholinergic toxicity Central and peripheral nervous system toxicity

43. Calculation of AD AF via BMD Rat RBC (mg/kg) Human RBC (mg/kg) Ratio (CSAF – AD AF ) BMD 10 0.0310.0161.9 BMDL 10 0.0200.0131.5 AD AF = ED H ED A

44. 44 Key Points - AD AF Compare the same endpoint (AChE inhibition in RBC as a surrogate as CNS) Identify a response (same magnitude) that is covered by dose-response data from both humans and animals (10% AChE inhibition). Estimate effective dose/concentration(s) causing that response (BMD 10 ). (Recall-we are comparing doses, not levels of response.) Calculate AD AF = ED animal / ED human

45. 45 Calculation of AD AF AD AF = ED animal / ED human = 0.031 (mg/kg) / 0.016 (mg/kg) = 1.9

46. SUMMARY Compare doses or concentrations resulting in the same level of response Compare central tendency measures, not variability When D-R curves are not parallel, avoid complications by making comparison at lowest reliable point Relate the observed, measured response to toxicity In vitro studies may be particularly useful (avoids TK influence on dose) The experimental methods should be similar between species

47. 47 References IPCS final guidance for CSAF http://www.who.int/ipcs/methods/harmonization/areas/uncertainty/en/index.html IPCS guidance on evaluation of mode of action Sonich-Mullin, C., R. Fielder, J. Wiltse, K. Baetcke, J. Dempsey, P. Fenner-Crisp, D. Grant, M. Hartley, A. Knaap, D. Kroese, I. Mangelsdorf, E. Meek, J.M. Rice, and M. Younes. 2001. IPCS conceptual framework for evaluating a mode of action for chemical carcinogenesis. Reg. Tox. Pharm. 34: 146-152 ILSI extension to human relevance of cancers Meek, ME, Bucher, JR, Cohen, SM, DeMarco, V, Hill, RN, Lehman- McKeeman, LD, Longfellow, DG, Pastoor, T, Seed, J, and Patton, DE (2003). A framework for human relevance analysis of information on carcinogenic modes of action. Crit Rev Toxicol 33:591-654 ILSI extension to noncancer endpoints Seed, J, Carney, E, Corley, R, Crofton, KM, DeSesso, JM, Foster, PMD, Kavlock, R, Kimmel, G, Klaunig, J, Meek, ME, Preston, RJ, Slikker, W, Tabacova, S, Williams, GM, Wiltse, J, Zoeller, RT, Fenner-Crisp, P, and Patton, DE. (2005). Overview: Using mode of action and life stage information to evaluate the human relevance of animal toxicity data. Crit Rev Toxicol 35:664-672. U.S. EPA. 2005. Guidelines for Carcinogen Risk Assessment. EPA/630/P- 03/001B, March 2005. http://www.epa.gov/iris/backgrd.html