OP In Vitro Inhibition Program - PowerPoint Presentation

1. OP In Vitro Inhibition ProgramAMVAC Chemical CorporationFMC CorporationGowan CompanySeptember 15-18, 2020

2. Outline of PresentationIntroduction to Testing Program (Rick Reiss) – 15 minutesExperimental Procedures and Results (Jan Chambers) – 20 minutesStatistical Analysis of Data (Kelly Higgins) – 15 minutesResults of Supplemental Variability Study (Rick Reiss and Kristin Lennox) – 15 minutesBiological Understanding of Interspecies and Intraspecies Variability (Rudy Richardson) – 20 minutesWrap-Up (Rick Reiss) – 5 minutes2

3. Introduction to Testing Program

4. BackgroundOPs share a common toxic mechanism of action, acetylcholinesterase (AChE) inhibitionAll registrants have a rich database of in vivo rat AChE inhibition dataBetter understanding of human/rat differences important for risk assessment and in the spirit of Tox21An in vitro testing program was developed at Mississippi State University to understand potential rat and human differences in AChE inhibition was developed4

5. Pharmacodynamics vs. PharmacokineticsPharmacokinetics: processes leading to target tissue concentrationsPharmacodynamics: target tissue response to toxicantAn in vitro system can isolate pharmacodynamics for AChE inhibition by:Isolating AChE enzyme in systemEliminating metabolizing enzymes, most of which are in the liverUsing the active inhibiting form of the OP5

6. Estimation of Data-Derived Extrapolation FactorsEPA guidance issued in 2014 recommends in vitro testing, where possible, to estimate Data Derived Extrapolation Factors (DDEFs)AChE inhibition is ideally suited for testing in vitro Easily separated from pharmacokinetics6https://www.epa.gov/risk/guidance-applying-quantitative-data-develop-data-derived-extrapolation-factors-interspecies-and

7. Acetylcholinesterase Inhibition for OPs Most sensitive endpoint for risk assessment for decadesEPA generally regulates OPs based on 10% AChE inhibition in the most sensitive of the RBC or brain compartmentsThe fundamental value driving the pharmacodynamics of inhibition is the bimolecular rate constant between the OP or its active moiety and AChEThis value can be readily measuredLimited literature measurements of bimolecular rate constant; this program vastly expands the available data7

8. DDEF EstimationInterspecies pharmacodynamics:Uses AChE derived from rat and human sources Intraspecies pharmacodynamics:Use AChE derived from a range of samples from human sources (e.g., differences in age, gender, race, etc,)Supplementary literature ReviewReview available literature on interspecies and intraspecies variability8

9. OPs in ProgramAcephate (Methamidophos)Chlorethoxyfos (oxon)DDVPEthopropFenamiphosNaledPhorate (sulfoxide and sulfone)Terbufos (sulfoxide and sulfone)Tebupirimphos (oxon)TribufosDicrotophosDimethoate (oxon)Malathion (oxon)Bensulide (oxon)Phosmet (oxon)9

10. DDEF Guidance Criteria for Use of In Vitro SystemCriteriaResponseWas the toxicologically active form of the agent studied?Yes. OPs or their active forms that bind to AChE were used.How directly was the measured response linked to the adverse effect?Adverse effect is the result of the reaction for which we are measuring rateAre the biological samples used in the assays derived from equivalent organs, tissues, cell types,age, stage of development, and sex of the animals/humans in which the target organ toxicity was identified?Yes. We are using ghosts derived from RBCs, which contain AChE. Samples from different sex and ages were usedWhat is the range of variability (e.g., diverse human populations and life stages) that thebiological materials cover?AChE derived from cord blood, juvenile and adult subjects of different sexes and ethnicities10CriteriaResponseWas the toxicologically active form of the agent studied?Yes. OPs or their active forms that bind to AChE were used.How directly was the measured response linked to the adverse effect?Yes. Adverse effect is the result of the reaction for which we are measuring rateAre the biological samples used in the assays derived from equivalent organs, tissues, cell types,age, stage of development, and sex of the animals/humans in which the target organ toxicity was identified?Yes. We are using ghosts derived from RBCs, which contain AChE. Samples from different sex and ages were usedWhat is the range of variability (e.g., diverse human populations and life stages) that thebiological materials cover?Yes. AChE derived from cord blood, juvenile and adult subjects of different sexes and ethnicities

11. DDEF Guidance Criteria for Use of In Vitro SystemCriteriaResponseIf the effect occurs or can be measured in several tissues, is the studied tissues or tissuepreparation an appropriate surrogate? Or, in situations where the effect is not localized, is theeffect consistent across tissues?Yes. The effect can be measured in RBC and brain. We used RBCs because they are readily available and brain and RBC AChE are identicalDoes the design of the study allow for statistically valid comparisons based on such factors asreplicate and sample size?Yes. See upcoming statistical analysis, plus supplementary variability studyWas chemical uptake considered when the chemical was applied to the samples so as to givecomparable intracellular concentrations across tissuesYes. Care was taken to avoid non-specific binding and no additional proteins were added.Were similar tissues or samples evaluated across species?Yes. AChE was derived from rats and human blood samples11

12. DDEF Guidance Criteria for Use of In Vitro SystemCriteriaResponseDo the concentrations in the in vitro studies allow for comparison with in vivo conditions?Yes. The bimolecular rate constant is the measured value and is not dependent on in vivo concentrations12

13. Experimental Procedures and Results

14. Experiments to Obtain PD Parameters for Use in PBPK Modeling: Inhibition KineticsMississippi State UniversityCenter for Environmental Health SciencesCollege of Veterinary MedicineJanice Chambers, PhD, DABT, ATS, Edward Meek, PhD, and Allen Crow, MD/PhD14

15. Experimental ObjectivesObtain inhibition kinetic parameters for AChE inhibition by OPs in RBC “ghosts” derived from blood of humans or rats.A main experiment and a supplemental variability study will be described:Main experiment included 18 human subjects and six pooled rat samplesSupplemental study included replicate measurements in 5 human subjects to better understand experimental variability15

16. Inhibition EquationsInhibition Equation Simplified Inhibition Equation ki = bimolecular rate constant 16

17. Inhibition Kinetics MethodDetermine experimentally the inhibition kinetics constants (KI, KA, and kp) for OPs.Target enzyme: acetylcholinesterase (AChE) assayed in “ghost” preparations (i.e., erythrocyte cell membranes separated from hemoglobin and other cytoplasmic constituents) obtained from human and rat erythrocytes.Because the AChE of erythrocytes is the same gene product as neural AChE, the information obtained from the erythrocytes is relevant to the neural target AChE. 88% homology between human and rat AChE, active site identical.17

18. Method: Materials16 OPs were supplied by sponsor or purchased (purity 84-99%).Paraoxon served to validate the consistency of the assay.Human blood: cord blood (n = 4) and youth (ages 10-13; n = 5) (from BioreclamationIVT) or adult (ages 16-65; n = 9) (from Innovative Research); total age range: 0-65 years; both sexes, any ethnicity.Blood shipped overnight.Blood screened for common pathogens. Rat blood: pooled from 5 adult rats (n = 6; 3 male and 3 female) and refrigerated.“Ghost” prep conducted 2 days after blood draw.18

19. Human Blood DonorsSample #Age (years)EthnicityGender110CaucasianFemale210CaucasianMale3Cord blood, 39 weeks, 4 daysCaucasianMale430African-AmericanFemale516CaucasianFemale627CaucasianFemale7Cord blood, 41 weeksCaucasianFemale813CaucasianMale911CaucasianFemale1046CaucasianMale1131African-AmericanMale1210CaucasianFemale13Cord blood, 38 weeks, 5 daysCaucasianMale1460CaucasianMale1523HispanicMale1651CaucasianFemale17Cord blood, 39 weeks, 1 dayAfrican-AmericanFemale1835HispanicFemale19

20. Method: RBC Ghost PreparationCentrifugation of whole blood anticoagulated with K2EDTA (3000 g for 10 min)Plasma removed; packed erythrocytes washed 3 times (500 g centrifugation) with 100mM phosphate buffer (pH 7.4). Erythrocytes lysed with hypotonic phosphate buffer (6.7mM, pH 7.4), 10 min on ice.Centrifugation at 50,000 g for 30 min; pellet washed 3 times with 100mM phosphate buffer and resuspended to original volume in 100mM phosphate buffer. Final ghost preparation showed minimal evidence of hemoglobin. Ghost preparation adjusted to similar activity level, aliquoted and stored at -80°C until assay. 20

21. Method: AChE AssayContinuous spectrophotometric assay (modification of Ellman et al., 1961; Chambers et al., 1988).Acetylthiocholine (ATCh) as the substrate.5,5’-dithiobis(nitrobenzoic acid) (DTNB) as the chromogen.21

22. Method: OptimizationDetermination of an amount of ghost preparation that yields 0.5-0.6AU (adjusted) in 1 min.Determination of concentrations of OP in EtOH that yield 10-90% inhibition.22

23. Method: Conditions 96 well plates warmed to 37°C. 158µl of each preparation added into each well. No additional proteins added for enzyme stabilization to avoid any additional potential binding sites for the test compounds. 60µM eserine sulfate used to correct for non-enzymatic hydrolysis in the blank samples to correct for non-AChE hydrolysis of substrate. Non-enzymatic hydrolysis of ATCh was <10%. 23

24. Method: AChE InhibitionInhibition reaction initiated by pre-incubation of ghost prep with ethanol vehicle or eight concentrations of each oxon predetermined to give 10-90% AChE inhibition. OP’s dissolved in EtOH at concentrations 100-fold higher than the desired final concentration, and added at 2 µl per well.Six pre-incubation periods used: 0-5 mins at 1 min intervals.24

25. Method: AChE ReactionAfter pre-incubation, inhibition reaction terminated by addition of substrate ATCh (being at least 3 orders of magnitude greater in concentration than the inhibitors, and out-compete the OP). The AChE reaction initiated by 40µl mixture of 5mM substrate ATCh For the 0-min time point, the substrate/chromogen mixture added at the same time as OP. Reactions monitored by recording the absorbance at 412nm for 8 min at 50 second intervals (10 readings)Velocity of each of 6 pre-incubation times obtained for each of 8 OP concentrations and control. 25

26. Methods: CalculationsTwo analyses: double reciprocal and hyperbolicFollowed method of Kitz and Wilson (1962) to calculate ki, KI, and kpGenerally hyperbolic gave lines fitting data betterMalaoxon will be used as an example in the following slidesOver 20,000 data points26

27. Inhibition curves for eight concentrations of malaoxon over six incubation periods.27

28. Kinetic Curves for Malaoxonmalaoxon AChE inhibition double reciprocal (Lineweaver-Burke)malaoxon AChE inhibition hyperbolic curve28

29. Inhibition kinetic constants for malaoxon in erythrocyte “ghosts” from humans (hyperbolic analysis).29

30. Inhibition kinetic constants for malaoxon in erythrocyte “ghosts” adult rats (hyperbolic analysis).30

31. Inhibition Concentration RangesOPInhibition Concentration Range (M)Paraoxon (positive control)10-7.5-10-5.5 DDVP10-6.25-10-4.5Naled10-8.5-10-6.75Dicrotophos10-6.5-10-3.75Tribufos10-5.5-10-3Phorate oxon sulfone10-7-10-5.25Phorate oxon sulfoxide10-6.75-10-5Ethoprop10-4.75-10-3Methamidophos10-5.25-10-3.5Fenamiphos10-4.25-10-0.5Terbufos oxon sulfone10-7-10-5.25Terbufos oxon sulfoxide10-7-10-5.25Chlorethoxyfos oxon10-9.5-10-7.75Tebupirimphos oxon10-8-10-6.25Malaoxon10-7.5-10-4.75Omethoate10-5-10-3.25Phosmet oxon10-7.75-10-5Bensulide oxon10-5.75-10-331

32. Bimolecular Rate Constants32

33. Statistical Analysis of Data

34. MethodsEPA requested additional statistical analysis using nonlinear mixed effects modelsInhibition kinetics, including the bimolecular rate constant (ki), phosphorylation constant (kp), and disassociation constant (KI), were measured for the 16 OPs. RBC AChE were derived from 6 pooled Sprague Dawley rat sources (3 male and 3 female) and 18 human sources, including subjects of various age, sex, and race/ethnicity. 8 male samples and 10 female samples14 adult samples (≥ 10 years of age) and 4 infant samples (including samples from in utero cord blood).13 Caucasian samples and 5 non-Caucasian samples34

35. Methods - Nonlinear Mixed Effects Models Three nonlinear mixed effects models were used for consideration of the interspecies and intraspecies DDEF for pharmacodynamics for each OP.Model 1. No random effectsModel 2. Same random effects for rat and humanModel 3. Different random effects between speciesGeneral equation to estimate the interspecies factorAkaike Information Criterion (AIC) was used to compare model fit.kapp = Apparent rate of AChEE phosphorylation (mol/L/min)A = Phosphorylation Constant (kp) (min-1)B = Dissociation Constant (K1) (mol/L)Conc = Concentrationkapp =[(HA * Human_Indicator + RA * Animal_Indicator) x Conc](HB * Human_Indicator + RB * Animal_Indicator + Conc)35

36. Methods - ki Ratio EstimationEstimates of human ki, rat ki, and the human ki : rat ki ratio were calculated for the interspecies analysisSimilar ki ratios were calculated for the intraspecies analysis models to test for differences in ki with the human samples across subpopulations including:males vs. femalesadults vs. infantsCaucasians vs. other racesHuman : Rat ki ratio =[HA/HB][RA/RB]36

37. Methods - Coverage Ratio for Intrahuman Variability The individual random effects for A and B parameters were estimated for each subject using the final NLMIXED model, and were subsequently used to estimate the individual ki.The associated coverage ratios for the distributions of the ki values were derived for each chemical. 37

38. Interspecies Factor Analysis 38

39. Intraspecies Factor Analysis - Male vs. Female39

40. Intraspecies Factor Analysis – Adult vs Infant**Adult samples from subjects ≥ 10 years of age; infant samples from in utero cord blood. 40

41. Intraspecies Factor Analysis - Caucasian vs. Other Race41

42. Coverage Ratio for Intrahuman VariabilityChemicalLognormal distribution assumptionModel fit issue/ model fit concernGMGSDP90/ GMP95/ GMP97.5/ GMP99/ GMBensulide oxon373.91.301.401.541.671.84No Chlorethoxyfos24540802.51.171.231.301.371.45DDVP50877.61.181.241.311.381.47Ethoprop1545.21.181.241.321.391.48Fenamiphos145.31.231.311.411.501.62Methamidophos1145.81.231.311.411.511.63Phorate oxon sulfoxide52168.61.121.151.201.241.29Phosmet oxon100625.91.211.271.361.451.55Terbufos oxon sulfone384135.81.561.772.082.392.82Dicrotophos5280.31.221.291.381.471.58Model fit concernMalaoxon109441.22.493.224.485.978.33Naled3129880.62.162.693.564.546.02Omethoate658.51.711.992.412.863.48Phorate oxon sulfone77304.11.391.531.721.912.16Tebupirimphos oxon662191.61.732.022.472.943.59Terbufos oxon sulfoxide11260.01.321.431.581.721.91Model fit issueCells highlighted in green are values >3. GM, geometric mean; GSD, geometric standard deviation; P, percentile42

43. LimitationsPresence of outliers.Due to the small sample sizes of the subpopulations, the results of these intraspecies analyses should be interpreted with caution.Consortium of companies sponsored additional work at MSU to better characterize intrahuman variabilityModel warnings were generated in some cases due to a full rank final Hessian matrix. Cholesky root reparameterization of the covariance matrices of the random effects resolve these warnings (ICF, 2020).43

44. ConclusionDifferences in human and rat ki values observed for 9 of 16 OPs. Intrahuman variability analysis showed very limited evidence for effects among different human subpopulations. Attempts to estimate intrahuman variability were hampered by high variability and a small number of subsamples.The coverage ratio of the ki distribution provides a ceiling on the intrahuman variability because it represents both experimental and intrahuman variability.The coverage ratio is less than 3 for most chemicals, the intrahuman pharmacodynamic default (PD). These coverage ratios could be used to modify the intrahuman PD. The coverage ratio was greater than 3 for a few chemicals. It would be inappropriate to use these values for the intrahuman PD factor, because the values include contributions from both experimental and intrahuman variability. 44

45. Supplementary Variability Study

46. IntroductionTo better understand experimental vs. true variability, a supplemental study was performedGoals of the study were to:Assess experimental variability by performing replicate measurements on the same subjectsAssess potential variability from preparation of AChE ghostsSeparate experimental and intrahuman variability46

47. Observations on Variability in the Original StudyVariability concluded to be mostly related to experimental variability:Humans and rat had similar variability despite rats AChE being from pooled samples of rat strain genetically bred to minimize variationSubsamples (or replicates) had similar variability as the overall experimentNo clear differences with age, sex, or ethnicityLittle covariance between values for subject and chemicalsThough limited pattern in some results may be explained by clotting of a few samples47

48. Study Design5 human blood samples were obtained, including six OPs:malaoxon, omethoate, naled, tebupirimphos oxon, phosmet oxon, and bensulide oxonFor one human subject, the blood was aliquoted into three parts and AChE ghosts were preppedThree replicates were done for each of the prepsFor the other four human subjects, a single prep was performed and five replicates were donePreliminary data just became available last week; detailed study report is forthcoming48

49. Efforts to Reduce Experimental VariabilityVendor instructed to draw sample near shipping timeQuicker preparation of ghosts following arrival of blood at labAvoided multiple samples coming in at the timeA full unit of blood instead of smaller 10 mL samples were obtained, which helps in reducing coagulation and provides a more consistent preparation49

50. Intrahuman Coefficients of Variation in Original and Supplemental Variability StudyChemicalCV in Original StudyCV in Supplemental StudyBensulide oxon31%13%Phosmet oxon31%4%Tebupirimphos oxon33%31%Naled48%19%Omethoate 74%12%Malaoxon58%14%50

51. Statistical Analysis of Preparation EffectTo test for effect of preparation of the AChE, the following linear mixed effect model was applied:where is the ki measurement for the kth measurement from the jth preparation replicate, is the expected value for all ki measurements, is the preparation effect for the jth replicate, and is a normally distributed random error term. 51

52. Variability Due to AChE Preparation52

53. Results of Sample Preparation Effect AnalysisChemicalP-valueBensulide oxon0.43Omethoate0.57Naled0.39Malaoxon0.96Phosmet oxon0.95Tebupirimphos oxon0.7453

54. Statistical Analysis of Intrahuman VariabilityRandom effects models of the following form were fit for each chemical:where is the ki measurement for the kth measurement from the jth subject, is the expected value for all ki measurements, Bj is a normally distributed random effect associated with the jth subject, and is a normally distributed random error associated with the kth measurement of the jth subject.  54

55. Statistical Analysis of Intrahuman VariabilityThree of the chemicals (phosmet oxon, bensulide oxon, and tebupirmiphos oxon) produced singular fits with the estimate of equal to 0. In these cases, an alternative fixed effect model was used to assess the significance of a subject effect. This model had the form: where is a normally distributed random error term, as before, but the subject effect for the jth subject is represented by a constant, . 55

56. Intrahuman Variability56

57. Result of Intrahuman Statistical AnalysisChemical/+)P-valueBensulide oxon0.2140.15Omethoate0.2040.29Naled0.3730.06Malaoxon0.0600.98Phosmet oxon 0.87Tebupirimphos oxon 0.0440.27ChemicalP-valueBensulide oxon0.2140.15Omethoate0.2040.29Naled0.3730.06Malaoxon0.0600.98Phosmet oxon 0.87Tebupirimphos oxon 0.0440.2757

58. Conclusions for Supplemental StudyGenerally, overall variability was substantially less than in original studyNo evidence for an effect of sample preparationLimited evidence for intrahuman variabilityIntrahuman variability is likely to be low; most variability is experimental variabilityHighest intrahuman variability was for naled at about 30% of total variability, thought this result is not statistically significantFuture analyses will apply EPA’s recommended statistical methods58

59. Biological Understanding of Interspecies Differences and Intraspecies Variability

60. Response to the US EPA Comments on the Exponent White Paper (MRID 50773505)Rudy J. Richardson, ScD, DABTUniversity of MichiganAnn Arbor, Michigan 48109-2029rjrich@umich.edu60

61. 1. Similar amino acid sequences and 3D structures.Comment: “… similar 3D structures lead to similar interactions with AChE inhibitors. … assumes that the structural similarities would therefore be reflected by similar PD parameters.”Molecular structure is a primary determinant of function.Proteins: amino acid sequence determines 3D structure.3D structure determines function, e.g., PD parameters of enzymes.61

62. Comment: “… concentrates on the structure and function of the catalytic site; however, there was no discussion of additional aspects of AChE structure that may have the potential to affect its function and activity. For example, binding of compounds to the peripheral anionic site....”The catalytic domain = 543 amino acid residues of the catalytic subunit of AChE from human brain or RBCs or rat brain or RBCs.The catalytic domain includes the catalytic active site (CAS), peripheral anionic site (PAS), and gorge (tunnel) that connects these regions.Inhibitors can bind to any of these sites; however, OP insecticides ultimately bind covalently to the active site Ser203 residue in the CAS.62

63. Linear Domain Structure of Human Brain AChE63

64. Overall Identity 86%Similarity 91%Catalytic domainIdentity 89%Similarity 94%Sequence Alignment of Human and Rat RBC AChE64

65. Human AChE65

66. RatAChE66

67. 3D AlignmentRMSD = 0.74 ÅTM-Score = 0.9867

68. Comment: “… to fully understand the interaction of AChE with an inhibitor or substrate, molecular dynamic simulations should be conducted. … the acetylcholinesterase molecule is not a static molecule ….”Agreed! “The conformational dynamics of protein molecules is encoded in their structures and is often a critical element in their function” – Martin Karplus68

69. MD Simulation: Human AChE69

70. Human AChE Molecular DynamicsNormal Mode Slow #170

71. Rat AChEMDNormal ModeSlow #871

72. Correlated motion analysisHuman and Rat AChE Correlated Motions = 98% similar72

73. 2. PD parameters for inhibition of RBC as a surrogate for brain AChEComment: ... “If additional studies are identified in the literature or additional data are generated (preferably with tissues taken and tested from the same animals/subjects), the weight of evidence to either support or refute this hypothesis would be further strengthened.”There are additional studies that can be reviewed, including investigations of compounds other than paraoxon.Suggest that ki or IC50 data be expressed as logki or pIC50 after converting to molar units.73

74. 3. Differences in PD parameters for human and rat RBC AChEComment: “The studies conducted and submitted to the Agency to measure AChE inhibition kinetics following exposure to several OP pesticides (MRID 50773501-50773503) were performed to quantify potential differences in PD parameters, if any.”Noted as the purpose of the in vitro inhibition kinetics studies.74

75. 4. Differences in PD parameters across populationsComment: “… analysis … primarily in the 3D structure and sequence homology at the AChE catalytic site.”Analysis focused on entire sequence. Emphasis on catalytic domain (includes CAS, PAS, gorge, and other regions)..75

76. Comment: “… it appears that Velan and coworkers demonstrated that, in some cases, the mutant:wild type IC50 ratio for three different inhibitors may change 2-7.5 times, meaning that preventing some patterns of post-translational modification has the potential to change the PD properties of AChE.”The study by Velan et al. (1993) involved comparisons of wild type and mutant forms of human AChE. The mutants were not naturally occurring but were created in vitro via site-directed mutagenesis.76

77. Comment: “With respect to mutations, Exponent’s supplemental document only considered mutations of the catalytic site. It has been shown in studies of insect mutants resistant to OP pesticides that although the site of the mutation conferring resistance (i.e., changing PD parameters) usually occurs in the active site gorge, mutations influencing PD parameters may also occur elsewhere on the molecule (Mutero et al., 1994; Casida and Durkin, 2013) and, therefore, need to be considered.”The entire AChE molecule was considered. The “catalytic site” refers to the immediate area of the catalytic triad (Ser203, Glu334, His447).It is well known that AChE mutations occur in insects, and some of these mutations are associated with insecticide resistance owing to modification of PD parameters. However, corresponding mutations have not been reported in the human population (Lockridge et al., 2016).77

78. Comment: … [Human AChE] “… mutations of concern ….”Uniprot: 4 natural variants: Arg34Gln, Pro135Ala, Val333Glu (no indication of clinical significance); and His353Asn (Yt blood group antigen; does not affect AChE function).Lockridge et al., (2016) have reviewed genomic data for 60,706 unrelated individuals comprising subjects from European, American, African, and Asian populations. Among these were 137 missense mutations and 141 synonymous mutations in the ACHE gene. Synonymous mutations in the gene do not result in amino acid substitutions in the AChE protein. Of the 137 missense mutations, 3 were loss of function, but there were no homozygous loss of function mutations. The most abundant missense mutation was His353Asn (the Yt blood group antigen), but no alteration in AChE activity or catalytic properties.Consequently, there should not be any significant intraspecies human variability in susceptibility to OP insecticides arising from variations in AChE.78

79. Final Thoughts

80. Final ConclusionsBimolecular rate constants between rats and humans are substantially similar, despite very large differences among chemicalsRobust estimates for the human/rat ki values form an adequate basis for DDEFs for interspecies pharmacodynamicsBimolecular rate constants among humans are substantially similarMost of the variability in the experiment is due to experimental error; thus, these data can be used to modify intraspecies DDEFBoth of these conclusions are supported by sound biological evidence80