30 Jan 2006

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

2. FOCUS Kinetics training workshop • Objectives of this part of the training: • Description of the procedures to follow for a parentcompound 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 2

3. FOCUS Kinetics training workshop Why the distinction between fitting for trigger endpoints versus exposure modeling endpoints? • EU regulatory triggers are based on DT50 and DT90 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. • Current EU regulatory environmental exposure models are based on SFO kinetics. Therefore, an endpoint (i.e. DT50) 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. 30 Jan 2006 Page 3

4. FOCUS Kinetics training workshop The same DT50 does not mean the same pattern of decline when calculated using different kinetic models 30 Jan 2006 Page 4

5. FOCUS Kinetics training workshop • EU regulatory trigger examplesAnnex II to Directive 91/414/EEC • 7.1.1.2.2. Field dissipation studies are required when DT50lab > 60 days at 20C or 90 days at 10 C • Annex III to Directive 91/414/EEC • 10.7.1 Testing for effects on soil micro-organisms required when DT90field > 100 days • Draft Guidance Doc. Terrestrial Ecotoxicology (SANCO/10329/2002 rev. 2 final) • Sub-lethal earthworm tests required depending on number of applications and DT90field • Guidance Doc. Aquatic Ecotoxicology (SANCO/3268/2001 rev. 4 final) • Chronic study on daphnids required when DT50 in water > 2 days 30 Jan 2006 Page 5

6. FOCUS Kinetics training workshop So what’s involved in the fitting procedure? • Run SFO as a first step • 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 • Check visual fit and calculate error percentage at which 2 test passed • Check parameter uncertainty • If error % < 15% and visual fit acceptable, use SFO DT50 • If error % > 15% and visual fit not acceptable, run bi-phasic model • Use best fit model • If 10% of initial reached in study period then calculate DT50 as FOMC DT90/3.32 • If 10% of initial not reached in study period then use longer DT50 from slow phase of HS or DFOP 30 Jan 2006 Page 6

7. FOCUS Kinetics training workshop Chi-square (2) test statistic – test of association whereC = calculated valueO = observed value = mean of observed (element of scale)err = measurement error (element of proportionality) 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 30 Jan 2006 Page 7

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 Observed and predicted through time Residuals(predicted - observed) 30 Jan 2006 Page 8

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(tcaclulated,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 9

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

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

12. FOCUS Kinetics training workshop Let’s look at an example for the triggers flowchart… Laboratory degradation of a compound in aerobic soil 30 Jan 2006 Page 12

13. FOCUS Kinetics training workshop SFO FOMC 2 error (%) = 19.0DT50 (d) = 18.1DT90 (d) = 60.1 2 error (%) = 6.69DT50(d) = 10.6DT90 (d) = 158 30 Jan 2006 Page 13

14. FOCUS Kinetics training workshop FOMC DFOP 2 error (%) = 6.69DT50(d) = 10.6DT90 (d) = 158 2 error (%) = 1.36DT50(d) = 10.6 dDT90 (d) = 481 d 30 Jan 2006 Page 14

15. FOCUS Kinetics training workshop Parameter uncertainty -- = not applicable 30 Jan 2006 Page 15

16. FOCUS Kinetics training workshop Possible conclusions for this data set for the triggers flowchart • Use DFOP with associated endpoints > DT50 = 10.6 d, DT90 = 481 d > Relax t-test criteria for k2 based on visual fit and 2. > Check if other aerobic soil deg and fate studies support this DT90. • Use DFOP. Fix k2 to a conservative value (e.g. 1000 d) > 2 and visual fits equivalent to above. > DT50 = 10.7 d, DT90 = 962 d > Check if other aerobic soil deg and fate studies support this DT90. • For comparison with EU regulatory DT50 triggers, the result is the same. • For comparison with EU regulatory DT90 triggers, the result is the same. 30 Jan 2006 Page 16

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

18. FOCUS Kinetics training workshop SFO FOMC 2 error (%) = 19.0DT50 (d) = 18.1DT90 (d) = 60.1 2 error (%) = 6.69DT50(d) = 10.6DT90 (d) = 158 30 Jan 2006 Page 18

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 DT90/3.32 to derive a conservative estimate of SFO DT50 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 DT50 for modeling.We did not reach 10% of initial in this example so further analysis is required. 30 Jan 2006 Page 19

20. FOCUS Kinetics training workshop FOMC DT90/3.32 example (assume last point did reach 10%) SFO DT50 = 18.1 d DT90 = 60.1 dFOMC DT50 = 10.6 d DT90 = 158 dFOMC DT90/3.32 = 47.6 d (SFO) FOMC DT90/3.32 is a conservative option where parent only exposure modeling is desired (can’t link to metabolites!) 30 Jan 2006 Page 20

21. FOCUS Kinetics training workshop DFOP DFOS (HS) 2 error (%) = 1.36DT50(d) = 10.6 dDT90 (d) = 481 d 2 error (%) = 2.59DT50(d) = 10.7DT90 (d) = 244 30 Jan 2006 Page 21

22. FOCUS Kinetics training workshop Parameter uncertainty -- = not applicable 30 Jan 2006 Page 22

23. FOCUS Kinetics training workshop DFOPfast phase, k1, DT50 = ln(2)/0.0931 = 7.45 d slow phase, k2, DT50 = ln(2)/0.0015 = 462 d DFOS (HS)fast phase, k1, DT50 = ln(2)/0.0646 = 10.7 d slow phase, k2, DT50 = ln(2)/0.0040 = 173 d 30 Jan 2006 Page 23

24. FOCUS Kinetics training workshop Possible conclusions for this data set for the modeling flowchart • Use longest phase of DFOS (HS) to derive conservative value of DT50 > 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. • Conduct higher-tier modeling using conservative value for DFOP slow phase DT50 (e.g. 1000 d). 30 Jan 2006 Page 24

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 25

26. FOCUS Kinetics training workshop Questions? 30 Jan 2006 Page 26

27. FOCUS Kinetics training workshop Now it’s your turn to work through the flowcharts using some other real data sets… 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. 30 Jan 2006 Page 27