Prevalidation Study Plan for Sliced Testes Assay Gary Timm Presented to EDMVS August 20, 2003

Sliced Testes Assay Prevalidation/Validation Study Plan June EDMVS Meeting Discussed: –Objectives of validation study –Data interpretation procedure –Basic sliced testes protocol –Prevalidation study design –Reference chemicals –Laboratory selection –Validation study design –Measurements of reliability –Data analysis and reporting

June Proposal for Prevalidation Studies Conduct prevalidation studies in two laboratories –Baseline study –Pilot study –Multichemical study

June Proposal for Prevalidation Studies (2) Baseline study –Run optimized protocol –3 runs without hCG –3 runs with hCG challenge –Measure testosterone formation and LDH –No test chemical –Three replicate studies

June Proposal for Prevalidation Studies (3) Pilot studies of positive controls –Aminoglutethimide (positive control) –Ethane dimethanesulfonate (Leydig cell toxicant) –Two labs –Three replicates

June Proposal for Prevalidation Studies (4) Multichemical studies –9 chemicals –2 laboratories –2 replicate studies Validation in 6 laboratories would begin after the successful conclusion of these studies

EDMVS Responses to Questions 1.Does the EDMVS agree with the stated objectives and data interpretation in the proposed Validation Study Plan? Yes, but: Concerns expressed about the assay –Accuracy and sensitivity of sliced testes assay –Potentially better assays on the horizon; don’t invest too heavily in sliced testes assay –Need a reliable means to detect Leydig cell toxicity

EDMVS Responses to Questions 2.Does the EDMVS agree with the structure of the prevalidation and validation Program? Yes, agreed that two laboratories was a reasonable choice for prevalidation. Concern that program should be more efficient –Do we need to look at data from multiple time points or is end of assay OK? –Use data from prevalidation to pick number of labs for validation, do not rely on literature values

EDMVS Responses to Questions Concerns about positive control chemicals –Aminoglutethimide may not be a good positive control chemical –EDS may not be a good cytotoxicant reference chemical Concerns about reference chemicals –Should not attempt to cover every known mode of action, use phamacologic agents of known mode of action –Ideally should choose only chemicals with a single mode of action, however selectivity seems to be dose dependent. –Should include several cytotoxicants during prevalidation

EDMVS Responses to Questions 3.Have we selected appropriate measures of reliability? –Yes, but the endpoints being used for the power calculations need to be clearly stated –Need to verify linearity in baseline Testosterone production curve –Should estimate potency of test chemicals by calculating an EC 10 or EC 50.

EDMVS Responses to Questions 4.Are the number of replicates taken over both prevalidation and validation sufficient to generate robust statistics? –Yes, three replicates seems sufficient in prevalidation. If the variability is small, consider reducing to 2 in validation –Need to add more negative chemicals –Negatives should include evaluation of influence of pH and osmolality

EDMVS Responses to Questions 5. Should dosing solutions be prepared centrally or on site? –The stock solution should be prepared centrally –Dilutions should be made on site with instructions provided by the lead lab or chemical repository

EDMVS Responses to Questions 6.Do doses need to be confirmed by analytical chemistry? –The identity and purity of the neat test substance should be checked by the chemical repository –The suitability and solubility of the test substance should be checked and the concentration of stock solution should be confirmed by the repository –Labs should save aliquots of dosing solutions should for analysis; analysis should be performed only “for cause”

EDMVS Responses to Questions 6. Naïve labs/trained labs issue –No naïve labs! Training is necessary to minimize interlaboratory difference in techniques –Competency of labs should be demonstrated by conducting a positive control prior to full validation effort –Incompetent laboratories should be excluded

Issue: Leydig cell toxicity Concerns: –Don’t want false positive by artificially high in vitro dosing that kills cells –Don’t want general cytotoxicants to trigger positive, 2 gen is wrong follow-up for general cytotoxicants –May be “red herring.” If cytotoxic to Leydig cells at biologically relevant concentration levels, isn’t this a legitimate positive?

Leydig cell toxicity (cont) EPA Options: 1.Use LDH assay for cell viability 2.Use 3β-HSD staining specific for Leydig cell viability instead of the more general LDH assay

Issue: Choice of reference chemicals EPA confirmed by search of literature that selectivity is not unique but is dose dependent. EPA will select reference chemicals that work at key stages of steroidogenesis EPA will run 4 known cytotoxicants to select the best positive control for cytotoxicity

EPA Listened and Redesigned Prevalidation Program Focus only on prevalidation; validation will be a separate work assignment. Redesigned the prevalidation program: lead lab and 3 participating labs Lead lab: –Baseline, testes variability study –Test of positive control –Cytotoxicity studies –Multichemical studies –Training of participating laboratories’ personnel

Redesigned Prevalidation Program Participating Laboratories (3) and lead lab –Baseline studies in triplicate measure testosterone with and without hCG no test chemical –Positive control studies Positive control (aminoglutethimide) Reference cytotoxicant

Baseline and Testes Variability Study (Lead Lab) Purpose: –To demonstrate competence of lead lab using optimized assay –To obtain testosterone production data as a function of time in the absence of inhibitors –To estimate Leydig cell density after 4 hr incubation –To evaluate the variability in contralateral testes fragments to decide on future experimental design: Randomized fragments Block design using single testes

Baseline and Testes Variability Study (Lead Lab) Study Design: –5 animals –24 runs (a run corresponds to an incubation vial containing 1 testes fragment) –Incubate for 4 hours with and without hCG –3 replicate studies with measurements (samples) at 5 time points for runs 1-6. (A replicate study is an independent study.) –2 replicate studies with measurements at 2 and 4 hrs (incubation times) for fragments 7-24

Baseline and Testes Variability Study Sample time hCGAnimalTestisFragment number Run 0-5N1-3A1 0-5Y1-3A24-6 2,4Y1B1,27,8 2,4Y2A3,49,10 2,4Y2B1,211,12 2,4Y3A3,413,14 2,4Y3B1,215,16 2,4Y4A1,217,18 2,4Y4B1,219,20 2,4Y5A1,221,22 2,4Y5B1,223,24

Baseline Study Design Sample Type hCGNumber runs Testes fragment MediaN31-3 MediaY34-6

Sample typehCGNumber runsTestes fragment Media-vehicle control N31-3 Media-vehicle control Y34-6 Media +AG (low) Y37-9 Media +AG (med) Y Media +AG (high) Y Positive Control Study Design

Cytotoxicity Study Design Sample typehCGNumber runsTestes fragment Media control N31-3 Media control Y34-6 Positive control Y37-9 Toxicant A- low Y Toxicant A- med Y Toxicant A- high Y Toxicant B- low Y Toxicant B- med Y Toxicant B- high Y325-27

Multichemical Study Design Sample typehCGNumber runsTestes fragment Media controlN31-3 Media controlY34-6 Positive controlY37-9 Cytotox controlY Chemical A-lowY Chem A- medY Chem A- highY Chem A- highN Chemical B- lowY Chem B- medY Chem B- highY Chem B- highN334-36

Reference Chemicals by Mode of Action Mode of ActionChemicalMode 2 P450 SCC AminoglutethimideAromatase P450 SCC KetoconazoleP450c17, arom cAMP inhibitorBisphenol AER binder cAMP inhibitorLindane StARDimethoate

Reference Chemicals by Mode of Action Mode of ActionChemicalMode 2 3β-HSDGenesteinER binder 3β-HSDEpostane P450c17FlutamideAR antagonist P450c17EconazoleAromatase

Reference Chemicals by Mode of Action Mode of ActionChemicalMode 2 AromataseProchloraz 5α-reductaseFinasteride Leydig cell toxicantEthane dimethanesulfonate Negative chemicalVinclozolinAR binder

Summary Lead lab: –Baseline, testes variability study –Test of positive control –Cytotoxicity studies –Multichemical studies –Training of participating laboratories’ personnel Participating Laboratories (3) and lead lab –Baseline studies in triplicate –Positive control studies in triplicate Estimated completion