30 Jan 2006 Page 1 FOCUS Kinetics training workshop Chapter 7 Recommended Procedures to Derive Endpoints for Parent Compounds Ralph L. Warren, Ph.D. DuPont Crop Protection Delaware, USA

Page 2 FOCUS Kinetics training workshop Objectives of this part of the training: Description of the procedures to follow for a parent compound to derive kinetic fitting endpoints a) best fit values (compared to triggers for additional work in EU) b) inputs for environmental exposure models Assessment of kinetic model fits to the observed data using visual and statistical techniques. Selection of the appropriate kinetic model and endpoints for the case of triggers and exposure modeling in the EU. 30 Jan 2006

Page 3 FOCUS Kinetics training workshop Why the distinction between fitting for trigger endpoints versus exposure modeling endpoints? Current EU regulatory environmental exposure models are based on SFO kinetics. Therefore, an endpoint (i.e. DT 50 ) calculated using a non-SFO kinetic model will not appropriately represent the observed behavior when input into a SFO-based exposure model. A SFO endpoint, if appropriate, or a conservative estimate or a work around must be used. EU regulatory triggers are based on DT 50 and DT 90 values which are not constrained to any kinetic model form. The model that most appropriately describes the observed data should be used to generate the endpoint values. 30 Jan 2006

Page 4 FOCUS Kinetics training workshop The same DT 50 does not mean the same pattern of decline when calculated using different kinetic models 30 Jan 2006

Page 5 FOCUS Kinetics training workshop EU regulatory trigger examples Annex II to Directive 91/414/EEC Field dissipation studies are required when DT 50lab > 60 days at 20 C or 90 days at 10 C Annex III to Directive 91/414/EEC Testing for effects on soil micro-organisms required when DT 90field > 100 days Draft Guidance Doc. Terrestrial Ecotoxicology (SANCO/10329/2002 rev. 2 final) Sub-lethal earthworm tests required depending on number of applications and DT 90field Guidance Doc. Aquatic Ecotoxicology (SANCO/3268/2001 rev. 4 final) Chronic study on daphnids required when DT 50 in water > 2 days 30 Jan 2006

Page 6 FOCUS Kinetics training workshop So whats involved in the fitting procedure? 30 Jan 2006 Run SFO and FOMC as a first step Check visual fit and calculate error percentage at which 2 test passed Check parameter uncertainty If FOMC better than SFO, test other bi-phasic models Use best fit model Run SFO as a first step Check visual fit and calculate error percentage at which 2 test passed Check parameter uncertainty If error % < 15% and visual fit acceptable, use SFO DT 50 If error % > 15% and visual fit not acceptable, run bi-phasic model If 10% of initial reached in study period then calculate DT 50 as FOMC DT 90 /3.32 If 10% of initial not reached in study period then use longer DT 50 from slow phase of HS or DFOP

Page 7 FOCUS Kinetics training workshop Chi-square ( 2 ) test statistic – test of association where C= calculated value O= observed value = mean of observed (element of scale) err= measurement error (element of proportionality) 30 Jan 2006 If calculated 2 > tabulated 2 then the model is not appropriate at the chosen level of significance (5%) Error percentage unknown Calculate error level at which 2 test is passed

Page 8 FOCUS Kinetics training workshop Visual Assessment Subjective, yet powerful tool for assessing goodness of fit. Keeps common sense in the assessment process. Two recommended plots 30 Jan 2006 Observed and predicted through time Residuals (predicted - observed)

Page 9 FOCUS Kinetics training workshop Parameter uncertainty Confidence intervals or t-tests may be used. The t-test is shown below, which assumes normally distributed parameters. where = estimate of parameter i = standard error of parameter i The probability (p-value) for the calculated t-value can be read from statistical tables or calculated with Excel TDIST(t caclulated,df,1) If P is < 0.05 then the parameter is considered significantly different than zero. If P is between 0.05 and 0.1 then weight of evidence should be considered. The t-test is most applicable to degradation rates (k), not necessarily other parameters such as or for FOMC. 30 Jan 2006

Page 10 FOCUS Kinetics training workshop Parent only flow chart for deriving trigger endpoints (zoom to view) Triggers flowchart 30 Jan 2006

Page 11 FOCUS Kinetics training workshop Parent only flow chart for deriving exposure modeling endpoints (zoom to view) Modeling flowchart 30 Jan 2006

Page 12 FOCUS Kinetics training workshop Lets look at an example for the triggers flowchart… Time (days) % of applied radioactivity Laboratory degradation of a compound in aerobic soil 30 Jan 2006

Page 13 FOCUS Kinetics training workshop 30 Jan error (%) = 19.0 DT 50 (d) = 18.1 DT 90 (d) = error (%) = 6.69 DT 50 (d) = 10.6 DT 90 (d) = 158 SFOFOMC

Page 14 FOCUS Kinetics training workshop 30 Jan error (%) = 6.69 DT 50 (d) = 10.6 DT 90 (d) = 158 FOMC 2 error (%) = 1.36 DT 50 (d) = 10.6 d DT 90 (d) = 481 d DFOP

Page 15 FOCUS Kinetics training workshop Parameter uncertainty ModelParameterOptimized value Standard error Different than zero? (P<0.05) SFOM0 (%) k (d -1 ) Yes FOMCM0 (%) DFOPM0 (%) g k 1 (d -1 ) k 2 (d -1 ) Yes No (P=0.225) 30 Jan = not applicable

Page 16 FOCUS Kinetics training workshop Use DFOP with associated endpoints > DT 50 = 10.6 d, DT 90 = 481 d > Relax t-test criteria for k 2 based on visual fit and 2. > Check if other aerobic soil deg and fate studies support this DT 90. Use DFOP. Fix k 2 to a conservative value (e.g d) > 2 and visual fits equivalent to above. > DT 50 = 10.7 d, DT 90 = 962 d > Check if other aerobic soil deg and fate studies support this DT 90. Possible conclusions for this data set for the triggers flowchart For comparison with EU regulatory DT 50 triggers, the result is the same. For comparison with EU regulatory DT 90 triggers, the result is the same. 30 Jan 2006

Page 17 FOCUS Kinetics training workshop Continuing with the same data, now lets look at it using the modeling flowchart… 30 Jan 2006

Page 18 FOCUS Kinetics training workshop 30 Jan error (%) = 19.0 DT 50 (d) = 18.1 DT 90 (d) = error (%) = 6.69 DT 50 (d) = 10.6 DT 90 (d) = 158 SFOFOMC

Page 19 FOCUS Kinetics training workshop Assuming no artifacts, the data is clearly bi-phasic. FOMC fit to the data is superior based on visual assessments and 2 error. If aim of modeling is to link parent with metabolites, then the guidance in Chapter 8 should be followed (covered later). If the aim is to model parent fate only then check to see if 10% of the initially measured value was reached during the study period. > If yes, then use FOMC DT 90 /3.32 to derive a conservative estimate of SFO DT 50 for modeling (i.e. 158 d/3.32 = 47.6 d). > If no, then use slower k from DFOS (HS) or slower k from DFOP to derive a conservative estimate of DT 50 for modeling. We did not reach 10% of initial in this example so further analysis is required. 30 Jan 2006

Page 20 FOCUS Kinetics training workshop SFODT 50 = 18.1 d DT 90 = 60.1 d FOMCDT 50 = 10.6 d DT 90 = 158 d FOMC DT 90 /3.32 = 47.6 d (SFO) FOMC DT 90 /3.32 is a conservative option where parent only exposure modeling is desired (cant link to metabolites!) FOMC DT 90 /3.32 example (assume last point did reach 10%) 30 Jan 2006

Page 21 FOCUS Kinetics training workshop 30 Jan error (%) = 2.59 DT 50 (d) = 10.7 DT 90 (d) = 244 DFOS (HS) 2 error (%) = 1.36 DT 50 (d) = 10.6 d DT 90 (d) = 481 d DFOP

Page 22 FOCUS Kinetics training workshop Parameter uncertainty ModelParameterOptimized value Standard error Different than zero? (t-test) DFOPM0 (%) g k 1 (d -1 ) k 2 (d -1 ) Yes No (P=0.225) DFOS (HS)M0 (%) tb (d) k 1 ( d -1 ) k 2 (d -1 ) Yes Yes 30 Jan = not applicable

Page 23 FOCUS Kinetics training workshop 30 Jan 2006 DFOP fast phase, k 1, DT 50 = ln(2)/ = 7.45 d slow phase, k 2, DT 50 = ln(2)/ = 462 d DFOS (HS) fast phase, k 1, DT 50 = ln(2)/ = 10.7 d slow phase, k 2, DT 50 = ln(2)/ = 173 d

Page 24 FOCUS Kinetics training workshop Use longest phase of DFOS (HS) to derive conservative value of DT 50 > 10% of initial not reached, so DFOS (HS) and DFOP were assessed. > Longest k from DFOP is not different than zero so it is unreliable. Possible conclusions for this data set for the modeling flowchart Conduct higher-tier modeling using conservative value for DFOP slow phase DT 50 (e.g d). 30 Jan 2006

Page 25 FOCUS Kinetics training workshop Summary Standardized procedures (flow charts) can be readily followed for deriving parent only endpoints Two flow charts are provided, one for determination of best fit kinetic parameters, the other for deriving inputs for use with SFO environmental exposure models Statistical and visual methods described provide a consistent way to assess kinetic model fits There is still room for judgment and discussion in the fitting and endpoint selection process, but the procedures described here should lead to greater consistency and transparency 30 Jan 2006

Page 26 FOCUS Kinetics training workshop Questions? 30 Jan 2006

Page 27 FOCUS Kinetics training workshop Now its your turn to work through the flowcharts using some other real data sets… 30 Jan 2006 If you finish the exercise and have additional time, you might try duplicating the fitting (SFO, FOMC, DFOS, DFOP) of the example data given in this presentation.