The sampling time points ( 5) from the media are at time 0 ( after a 1 hour equilibration) and 1, 2, 3, and 4 hours post equilibration. Each sample will be analyzed for testosterone in duplicate. In addition, at the end of the incubation period, a piece of the tissue fragment will be collected and snap frozen for 3$ HSD staining and microscopic examination. Thus, the overall total number of testosterone samples for analysis is 540 samples/ laboratory [ 54 runs x 5 time points x 2 ( duplicate) analyses]. The overall total number of tissue samples for microscopic examination are 54/ laboratory [ 54 runs x 1 time point].

5.7 MULTICHEMICAL EXPERIMENTS This experiment will be conducted by the lead laboratory only.

The purposes of this study are:

° to determine the response of the assay when challenged with putative positive test chemicals. The test chemicals will differ in their mode( s) of action, which will include altering signal transduction, cholesterol transport into the mitochondria, and the series of enzymatic reactions that lead to the production of testosterone in the gonadal steroidogenic pathway. ° to determine the response of the assay when challenged with putative negative test chemicals. DRAFT PROTOCOL [ Name and address of lab] Page 25 of 37 ° to select test chemicals and concentrations that would be used for validation of the assay. ° to estimate interlaboratory variability based on the results from the assay using the selected test chemicals.

The test chemicals and their concentrations that will be tested are summarized in the following table:

Test Chemical Mode of Actiona Test Concentrationsb Reference Aminoglutethimide ( positive control) P450SCC, aromatase 10, 100, and 1000 µ M Powlin et al., 1998; Johnston, 1997; Uzgiris et al., 1977 Diethylumbelliferyl phosphate cAMP stimulated accumulation of StAR TBD Choi et al., 1995 Dimethoate StAR protein TBD Walsh et al., 2000 Ketoconazole P450SCC, P450c17 0.1, 1, and 10 µ M Powlin et al., 1998; Kan et al., 1985; Albertson et al., 1988; DeCoster et al., 1989; Chaudhary/ Stocco, 1989; Malozowski et al., 1985, 1986 Trilostanec 3$ HSD TBD Takahashi et al., 1990 Genistein or epostane 3$ HSD TBD Ohno et al., 2002; Tanaka et al., 1992 Flutamide P450c17 10, 100, and 1000 µ M Powlin et al., 1998; Ayub and Levelll, 1987 Finasterided or MK 434 5" reductase 10, 100, and 1000 µ M ( MK 434  TBD) Morris, 1996 Fenarimol aromatase TBD Vinggaard et al., 2000 Vinclozolin ( negative chemical) antiandrogen TBD

Bisphenol A cAMP TBD

Lindane cAMP TBD  a. Site of action that leads to a decrease in Testosterone concentration for those chemicals with inhibitory activity. Negative test chemicals would have no effect on the testosterone concentration. b. Final concentrations in the incubation mixture. c. Requires synthesis by commercial laboratory. Availability may be difficult due to proprietary claim. d. Commercial source may limit availability. DRAFT PROTOCOL [ Name and address of lab] Page 26 of 37 The experimental design of this study for any two test chemicals is summarized in the following table:

Sample Type hCG Number of Incubation s ( Runs) Testis Fragment( s)

Media Vehicle control no 3 1  3 Media Vehicle control yes 3 4  6 Positive control  Aminogluthethimide @ 1 conc yes 3 7  9 Cytotoxicant control  TBD @ 1 conc yes 3 10  12 Media + Test Chemical A ( low) yes 3 13  15 Media + Test Chemical A ( mid) yes 3 16  18 Media + Test Chemical A ( high) yes 3 19  21 Media + Test Chemical A ( high) no 3 22  24 Media + Test Chemical B ( low) yes 3 25  27 Media + Test Chemical B ( mid) yes 3 28  30 Media + Test Chemical B ( high) yes 3 31  33 Media + Test Chemical B ( high) no 3 34  36 The information summarized in the table is one replicate of the experiment. The nonstimulated incubations ( non hCG) were excluded from the study since the affect of AG on the non stimulated tissue fragments would not be expected to reduce the baseline levels to any great extent. However, a high concentration test chemical with non stimulated tissue fragment was retained in the experimental design to confirm this response. In addition, in the case when the assay would be used to evaluate a chemical with an unknown effect on the testosterone concentration, it would be important to test the non stimulated fragment at the high concentration to determine whether the test chemical had a stimulatory effect on the testosterone concentration. A total of three independent replicates will be conducted. The overall study will use two rats/ replicate study and four testes total/ replicate study. The overall total number of individual fragments or incubations used is 72.

The sampling time points ( 5) from the media are at time 0 ( after a 1 hour equilibration) and 1, 2, 3, and 4 hours post equilibration. Each sample will be analyzed for testosterone in duplicate. In addition, at the end of the incubation period, a piece of the tissue fragment will be DRAFT PROTOCOL [ Name and address of lab] Page 27 of 37 collected and snap frozen for 3$ HSD staining and microscopic examination. Thus, the overall total number of testosterone samples for analysis is 720 samples [ 72 runs x 5 time points x 2 ( duplicate) analyses]. The overall total number of tissue samples for microscopic examination are 72 [ 72 runs x 1 time point].

6.0 CHEMISTRY The test chemicals and AG ( positive control) will be procurred, characterized, formulated, and analyzed as described below. A central chemistry laboratory will perform these activities. However, the formulations prepared by the central chemistry laboratory will be limited to the stock solution, which will be shipped to the assay testing laboratories, where the appropriate dilutions will be made.

6.1 CHEMICAL PROCUREMENT AND CHARACTERIZATION A single lot of each test chemical will be procurred so that all prevalidation experiments are performed using the same lot. The lot obtained will have a purity > 95  98 percent. Upon receipt of each test chemical, the identity will be confirmed by running an IR spectrum and comparing it to a reference spectrum from the manufacturer or from the literature. Within 30 days of using a test chemical in the assay, the purity will be determined using one chromatographic method and one additional or complementary method.

6.2 FORMULATION PREPARATIONS Prior to testing, the solubility of the test chemical in the media at a concentration that will result in the desired final concentration in the incubation mixture will need to be determined. Formulation development will be based on the highest concentration needed for a given test chemical. In addition, a formulation will be developed using a) media only or b) a vehicle that can be used to dissolve the test chemical prior to mixing it with the media.

Only select solvents will be used as vehicles. The vehicles that will be used and their concentrations were determined during the optimization studies. Based on these results, selected concentrations of ethanol, DMSO, or Tween 20 will be used as vehicles if the chemical is not soluble in the media.

It will be important to confirm that the formulation is a solution. This will be done by analyzing the formulation before and after filtering since the results of the two analyses will be similar if the formulation is a solution. In addition, since the media is gassed in the assay, the solubility of gassed formulation will be tested. DRAFT PROTOCOL [ Name and address of lab] Page 28 of 37 Formulation development will seek to prepare a solution of the test chemical in media, with and without hCG, so that a given aliquot of the formulation, when added to the incubation mixture, will result in the desired final concentration of test chemical to be tested, as well as the final total incubation volume. Also, if a vehicle is needed to make the formulation, then the vehicle concentration would be formulated at an acceptable level.

When formulations for multiple laboratories are required, a formulation will be prepared in a sufficient batch size that will allow the same formulation to be dispensed to all laboratories. If different concentrations are to be tested for a given test chemical, then the central laboratory will prepare a stock solution, which will be shipped to the laboratories. Each laboratory will then use the stock solution to prepare the various concentrations to be tested.

Formulations, if solutions, will be prepared to within 5 percent of target.

6.3 FORMULATION ANALYSIS For each chemical to be tested, a method for analysis will be developed for the test chemical in media/ vehicle over the concentration range to be tested.

Prior to shipment of a formulation to the laboratory( ies), a sample of the stock solution formulation to be shipped will be taken and analyzed. Samples will be analyzed in duplicate. If the determined concentration is not within 5 percent of the target concentration, then the formulation will be re prepared.

After the laboratory( ies) have used the diluted formulations, a sample will be shipped back to the central laboratory and archived for possible analysis at a later date if there is some question about the results of the assay.

6.4 FORMULATION STABILITY Prior to being used in the assay, the formulations will be tested for stability. Stability testing will be performed at the concentration of the stock solution formulation. Stability will be determined at time 0 ( day of preparation) and at weekly intervals for a period of at least 6 weeks and, thereafter, at 9 and 12 weeks. Stability will be tested at room temperature and at refrigerated temperatures. Stability will be tested using amber glass bottles with Teflon lined lids.

7.0 ENDPOINT MEASUREMENTS The sliced testis assay incubation media will be sampled at selected time points and the media analyzed for testosterone concentration using an RIA method. In addition, at the conclusion of the incubation period, the testis fragment will be sampled and processed for DRAFT PROTOCOL [ Name and address of lab] Page 29 of 37 Leydig cell identification using a staining procedure specific for 3$ HSD.

7.1 TESTOSTERONE RIA PROCEDURE A radioimmunoassay ( RIA) commercial kit ( Diagnostic Products Corporation, Los Angeles, CA), that utilizes 125I testosterone and a testosterone specific antibody affixed to polypropylene culture tubes, will be used to measure testosterone.

Testosterone ( Sigma, St. Louis, MO; T 1500) to be used for preparing the standard curve will be stored desiccated at room temperature. A standard will be prepared in ethanol ( 0.1 mg/ ml). Up to an 8 point standard curve but not less than a 4 point standard curve will be prepared using standards with concentrations of 0.07, 0.16, 0.41, 1.02 , 2.56, 6.4, 16, and 40 ng/ ml in PBS Gel Buffer ( 0.1 M phosphate buffered saline with 0.1% ( w/ v) sodium azide and 0.1% ( w/ v) gelatin, pH 7.4). In addition, procedural controls will be included in each run. These procedural controls will include low or high serum testosterone samples and two reagent blanks. The standard curve points and the procedural controls will be prepared in quadruplicate; the bioassay unknowns and the internal standards ( see below) will be prepared in duplicate. The volume of all standards and controls ( including bioassay unknowns) will be adjusted to 50 : L by adding the PBS Gel Buffer. Next, 1 ml 125I testosterone will be added to each antibody coated tube and mixed ( Vortex). The tubes will be incubated in a 37/ C water bath for three hours, during which time testosterone, whether labeled or unlabeled, will compete for testosterone specific antibody binding sites. At the end of the incubation period, the free ( unbound) testosterone, in the supernatant fluid of all tubes will be aspirated and wiped clean of fluid. The bound testosterone will be counted in a gamma counter for 1 minute. The concentration of testosterone will be estimated against the standard curve. Values will be reported as a mean concentration ( ng/ mL) of duplicate analyses.

Verification of the testosterone assay will involve preparation of internal standards ( no less than three) using spiked media with concentrations ranging from 12.5 to 500 ng/ mL. Each concentration will be run at each of three volumes  10, 25, and 50 µ L, to check for parallellism, and each sample will be adjusted to 50 µ L by adding the PBS Gel Buffer. The low and high standards will be analyzed in at least duplicate. Verification will be based on results determined for accuracy, precision, specificity, and linearity. Accuracy will be expressed as the relative error, which will be determined by comparing the measured to the target concentration. Relative errors within 10 percent will be acceptable. Precision will be expressed as the relative standard deviate ( RSD) or coefficient of variance ( CV), which will be determined by calculating the mean and standard deviation ( sd) of the low and high standards. A RSD or CV within 10 percent will be acceptable. The sensitivity will be acceptable if the means of the blanks and low standards are significantly different at the 5 percent significance level. Linear determinations of the standard curve line will be made and a correlation coefficient ( r) calculated. An r of 0.90 or greater will be considered acceptable. DRAFT PROTOCOL [ Name and address of lab] Page 30 of 37 Inter and intra assay variability will be determined. The intra assay variability will be determined from the precision results calculated from the results obtained by measuring the low and high standards in triplicate. The inter assay variability will be determined by repeating analysis of the standards by generating a standard curve on three different days.

7.2 3$ HSD LEYDIG CELL STAINING PROCEDURE Analysis results will be reported as Leydig cell density ( cells/ cm2).

A Leydig cell specific staining method and microscopic evaluation will be used to evaluate the viability of these cells. At the end of the incubation period, a piece will be taken from each fragment for staining. The media vehicle control fragment will be used to evaluate the result of incubation with the test chemical treatment. The staining procedure is specific for cells containing 3  HSD, a steroidogenic enzyme specific to the Leydig cells when examining testis tissue. The general staining procedure [ Ref.: Payne et al., ( 1980). Endocrinology 106: 1424; Klinefelter et al., ( 1993). In: Methods in Toxicology, Vol. 3, pp. 166 181.] is described as follows:

° after the last sample collection, snap freeze the tissue, section it (~ 15 um), and mount it on slides. ° stain the slide using the following solutions and procedure:

a) etiocholanolone stock solution ( 1 mg/ ml in DMSO), b) 2 mg Nitroblue Tetrazolium in 0.6 ml Etiocholanolone stock, c) 10 mg NAD+ dissolved in 9.5 ml warm Dulbeccos Phosphate buffered saline ( DPBS), and d) 10 mg NAD+ dissolved in 9.5 ml warm Dulbeccos Phosphate buffered saline ( DPBS). ° Mix solutions b and c. ° Cover section tissue on slide with staining solution for 1  2 hours. ° Rinse in deionized water. ° Fix in 10% formalin in DPBS with 5% sucrose. ° Coverslip with glycerol: DPBS ( 1: 1) and seal with nail polish.

8.0 DATA ANALYSIS The results for each analysis will be reported individually, with sufficient identifying information to determine which results correspond to duplicate analyses, to different time points within one vial, to different vials within the same replicate, and to different replicates. The lead and participating laboratories will maintain a database to include all data generated during the study. The databases will have uniform structure, formatting, and variable naming across DRAFT PROTOCOL [ Name and address of lab] Page 31 of 37 laboratories. Test conditions, background environmental conditions, and results for each analysis for each sample at each time point will be reported.

Analysis results to be reported are testosterone ( T) concentration ( ng T/ mg tissue/ hour) and Leydig cell density ( cells/ cm2). Detection limits and indications of inability to detect will be reported, as will confirmation of the acceptability or non acceptability of each individual value.

9.0 STATISTICAL ANALYSIS 9.1 CONTRALATERAL TESTIS FRAGMENT EXPERIMENT The principal endpoint is testosterone ( T) concentration. T analyses will be carried out in duplicate. Results of the duplicate T analyses will be averaged. Statistical analysis will be carried out on the averages the duplicates.

The statistical analysis will be carried out by the lead laboratory. Mixed effects repeated measures models will be fitted to the data to describe trends in concentration vs. time and to estimate animal to animal, testis to testis within animal, and fragment to fragment within testis components of variation. For the T concentration analyses fixed effects terms in the models will describe linear and non linear trends in T for hCG stimulated testis fragments and for nonstimulated testis fragments. Random effects terms in the model will estimate variance components that account for variation among fragments within testes, testes within animals, animals within replicates, and variation among replicates, primarily for the hCG stimulated testis fragments. A correlation structure will be fitted to describe the relation among the responses based on the same vial ( i. e. the same fragment) at the various times. The testis to testis variation within animals and the fragment to fragment variation within testes will be determined only for the hCG stimulated testis fragments.

The results of the model fit will be parametric functions that describe the time trends and variance components that describe the within and among replicate variation about the time trends.

Residuals from the model fits will be examined to determine goodness of fit to the model assumptions, to assess the nature of the random variation about the model, and to search for outlying observations. Outliers may be individual responses that deviate from the trend within their vial, individual vials that deviate from the average across vials within their replicate, and individual replicates that deviate from the average across replicates. Each of these types of residuals will be examined.

Based on the model fits multiple endpoints will be reported. Endpoints include DRAFT PROTOCOL [ Name and address of lab] Page 32 of 37 ° T concentrations at 1, 2, 3, 4 hours following equilibration, with and without hCG stimulation ° Ratios of T concentrations with and without hCG stimulation, at 1, 2, 3, 4 hours following equilibration ° Response trends, with and without hCG stimulation ° Ratios of trends with and without hCG stimulation. ° Average Leydig cell densities after two hours of incubation and after four hours, with and without hCG stimulation For each response the average value across runs, the standard error of estimate, and confidence intervals about the average will be reported. Estimates of run to run variation within replicates and replicate to replicate variation will be reported. Separate variance components will be estimated for responses with hCG stimulation and for responses without hCG stimulation.

Random effects terms in the model will describe variance components that account for animal to animal variation, testis to testis variation within animal, and fragment to fragment variation within testis. These components of variation will be estimated for hCG stimulated testis fragments at two hours and at four hours following equilibration. A correlation structure will be fitted to describe the relation among the responses at the various times based on the same vial ( i. e. the same fragment). The results of the model fit will be parametric functions that describe the time trends and estimates of the variance components.

For each response estimates of variance components will be reported.

9.2 BASELINE EXPERIMENT The statistical analysis will be divided into intra laboratory and inter laboratory components. The intra laboratory analyses will be carried out by each laboratory individually, based on a common analysis plan. Mixed effects repeated measures models will be fitted to the data to describe trends in concentration vs. time. Fixed effects terms in the models will describe linear and non linear trends in T time trends for hCG stimulated testis fragments and for nonstimulated testis fragments. Random effects terms in the model will describe variance components that account for variation among the repeat vials within replicates and variation among replicates. A correlation structure will be fitted to describe the relation among the responses at the various times based on the same vial ( i. e. the same fragment). The results of the model fit will be parametric functions that describe the time trends and variance components that describe the within and among replicate variation about the time trends.

Residuals from the model fits will be examined to determine goodness of fit to the model assumptions, to assess the nature of the random variation about the model, and to search for DRAFT PROTOCOL [ Name and address of lab] Page 33 of 37 outlying observations. Outliers may be individual responses that deviate from the trend within their vial, individual vials that deviate from the average across vials within their replicate, and individual replicates that deviate from the average across replicates. Each of these types of residuals will be examined.

Based on the model fits, multiple endpoints will be reported. Possible endpoints include T concentrations at 1, 2, 3, 4 hours following equilibration with and without hCG stimulation, ratios of T concentrations with and without hCG stimulation at 1, 2, 3, 4 hours following equilibration, response trends with and without hCG stimulation, and ratios of trends. For each response the average value across runs, the standard error of estimate, and confidence intervals about the average will be reported. Estimates of run to run variation within replicates and replicate to replicate variation will be reported.

The inter laboratory analysis will be carried out by the DCC. The objective of the interlaboratory analysis is to assess the extent of variation across laboratories with respect to average response and variability of response. For each endpoint the average response and associated standard error, the variation among runs within replicates, and the variation among replicates will be determined for each laboratory. Comparisons of within laboratory variance components will be made among the participating laboratories based on control charts to assess homogeneity of variance. The reference for comparisons will be either the lead laboratory results or the average results across all laboratories. If there is no evidence of lab to lab variation the within laboratory variance components will be pooled among laboratories. Otherwise separate within laboratory variance components will be specified for each laboratory.

The average responses will be compared across laboratories based on random effects one way analysis of variance. The among laboratories variance in average response will be added to the within laboratory variance, resulting in the total assay variability. Estimates and confidence intervals will be reported for each laboratory based on the total assay variability. Estimates and confidence intervals will be reported on the differences between the participating laboratory results and the lead laboratory results and the differences between each laboratory's results and the average of the laboratory results based on the total assay variability.

9.3 POSITIVE CONTROL EXPERIMENT Regarding data analysis and statistical evaluation, the statistical analysis will be divided into intra laboratory and inter laboratory components. The intra laboratory analyses will be carried out by each laboratory individually, based on a common analysis plan. Mixed effects repeated measures models will be fitted to the data to describe trends in T concentration vs. time and trends in AG concentration. Fixed effects terms in the models will describe linear and nonlinear T time trends and AG concentration trends for hCG stimulated testis and differences relative to the media vehicle control. Random effects terms in the model will describe variance DRAFT PROTOCOL [ Name and address of lab] Page 34 of 37 components that account for variation among the repeat vials within replicates and variation among replicates. A correlation structure will be fitted to describe the relation among the responses at the various times based on the same vial ( i. e. the same fragment) and at the various AG concentrations, based on fragments taken from the same testis. The results of the model fit will be parametric functions that describe the time trends and concentration trends and variance components that describe the within and among replicate variation about the time trends and concentration trends.

Residuals from the model fits will be examined to determine goodness of fit to the model assumptions, to assess the nature of the random variation about the model, and to search for outlying observations. Outliers may be individual responses that deviate from the time trend within their vial or from the concentration trend across vials, individual vials that deviate from the average across vials with the same conditions within their replicate, and individual replicates that deviate from the average across replicates. Each of these types of residuals will be examined.

Based on the model fits multiple endpoints will be reported. Possible endpoints will be include T concentrations at 1, 2, 3, 4 hours following equilibration for the media vehicle control and graded AG concentrations, with hCG stimulation, ratios of T concentrations from the same groups with hCG stimulation at 1, 2, 3, 4 hours following equilibration, response trends in time or in AG concentration with hCG stimulation, and ratios of trends. For each response the average value across runs, the standard error of estimate, and confidence intervals about the average will be reported. Estimates of run to run variation within replicates and replicate toreplicate variation will be reported. Differences between the T concentrations in the presence of AG and the T concentrations in the media vehicle control group will also be analyzed.

The inter laboratory analysis will be carried out by the DCC. The objective of the interlaboratory analysis is to assess the extent of variation across laboratories with respect to average response and variability of response. For each endpoint developed in the intra laboratory analysis the average response and associated standard error, the variation among repetitions within replicates, and the variation among replicates will be calculated for each laboratory. Comparisons of within laboratory variance components will be made among the participating laboratories based on control charts to assess homogeneity of variance. The reference for comparisons will be either the lead laboratory results or the average results across all laboratories. If there is no evidence of lab to lab variation the within laboratory variance components will be pooled among laboratories. Otherwise separate within laboratory variance components will be specified for each laboratory.

The average responses will be compared across laboratories based on random effects one way analysis of variance. The among laboratories variance in average response will be added to the within laboratory variance, resulting in the total assay variability. Estimates and DRAFT PROTOCOL [ Name and address of lab] Page 35 of 37 confidence intervals will be reported for each laboratory based on the total assay variability. Estimates and confidence intervals will be reported on the differences between the participating laboratory results and the lead laboratory results and between each laboratory's results and the average of the laboratory results based on the total assay variability.

The responses included in the intra laboratory and inter laboratory analyses will be categorized as primary or secondary endpoints. The primary endpoints will be restricted to a small number ( two to five) of the most important responses for comparisons. Power calculations to assess inference sensitivity versus sample size will be based on the primary endpoints. The studies will be powered to attain desired sensitivity for comparisons of the primary endpoints within or among laboratories. The secondary endpoints are the ( relatively large number of) remaining responses that are included in the statistical analyses. The studies will not be powered for comparisons among the secondary endpoints.

For purposes of assessing sample size versus inference sensitivity for the validation tests, two responses have been selected as primary endpoints. These are:

° differences between T concentrations associated with the high aminoglutethimide concentration and the media vehicle control at three hours past equilibration, with hCG stimulation.

° Sum of absolute differences between T concentrations associated with the high aminoglutethimide concentration and the media vehicle control at 1, 2, 3, and 4 hours past equilibration, with hCG stimulation.

The second response approximates the area between the T concentration versus time curves for these two conditions.

Sensitivity analyses will be carried out based on response variability estimates obtained from the analyses. Numbers of replicates per laboratory necessary to detect heterogeneity of replicate to replicate variability within laboratories will be assessed. Numbers of laboratories and numbers of replicates per laboratory necessary to detect various levels of coefficient of variation across laboratories with high power or to detect various levels of ratio of between laboratory standard deviation to within laboratory standard deviation with high power will be determined.

9.4 MULTICHEMICAL EXPERIMENT Separate statistical analyses will be carried out for each test chemical. Consideration will be given to pooling the media vehicle control groups across those chemical tests that use the same vehicle. The statistical analysis considerations are the same as those described above in the DRAFT PROTOCOL [ Name and address of lab] Page 36 of 37 previous section, except that there are two replicates per chemical rather than three. Extent of replicate to replicate variation can be determined from these tests only in a qualitative manner.

10.0 RETENTION OF SPECIMENS AND RECORDS All specimens and records that remain the responsibility of the testing laboratory will be retained in the testing laboratory's archives for the life of the contract.

11.0 QUALITY CONTROL/ QUALITY ASSURANCE PROCEDURES This study will be conducted according to Good Laboratory Practice Guidelines. Quality control ( QC) and quality assurance ( QA) procedures will follow those outlined in the Quality Assurance Project Plan ( QAPP) that will be prepared for this study.

12.0 REPORTING Interim data sets will be submitted from the lead laboratory to the EPA and Battelle upon summarization of the raw data and completion of a technical review for the experiments listed below:

° baseline/ contralateral tissue fragment and positive control ° cytotoxicity ° training and hands on results of participating laboratories This interim data submission will not be audited by the Quality Assurance Unit and will be stamped as " Preliminary Data". The data sets will be reviewed promptly by the EPA so that a decision can be reached to determine whether to proceed to the next experiment.

Each participating laboratory will prepare a draft final report upon completion of the baseline and positive control experiments. A single draft final report that includes both experiments will be prepared. The draft final reports will be submitted to the lead laboratory, Battelle, and the EPA. Review comments from the lead laboratory and the EPA will be collected by Battelle and submitted to the respective participating laboratory for inclusion of the changes prior to their submission of a final report.

The lead laboratory will prepare a draft final report that will include the results of all experiments, including the final reports submitted by the participating laboratories. This overall draft final report will include the statistical analyses that evaluates inter and intra laboratory variability. Upon review of the draft final report by the EPA, the lead laboratory will submit a final report that incorporates any comments from the EPA's review of the draft final report. DRAFT PROTOCOL [ Name and address of lab] Page 37 of 37 The report format used by the laboratories will have a standardized format. This format will be provided to the laboratories prior to initiating report preparation. All reports will include introduction, methods and materials, results, discussion, and conclusion sections. Any difficulties in executing the studies should be noted and there should be data summaries as well as the raw data tables.

13.0 PERSONNEL Personnel:

Study Director: Principal Investigator: Animal Research Facility Vet.: Animal Research Facility Manager: LORET Laboratory Supervisor: Research Toxicologist: Research Biologists: Biologists: Biological Laboratory Assistants: Statistical Analyses: Quality Assurance Unit ( QAU):

14.0 STUDY RECORDS TO BE MAINTAINED ° Chemical Receipt, Storage and Use Records ° Animal Receipt Records ° Quarantine Animal Health Surveillance Records ° Balance Check Sheets ° Temperature and Relative Humidity Records ° Room Log Sheets ° Feed and Water Analyses ° All records that document the conduct of the laboratory experiments and results obtained, as well as the equipment and chemicals used ° Protocol and any Amendments ° List of any Protocol Deviations ° List of Standard Operating Procedures ° Computer records: all records of data sets and statistical analyses Battelle Report B 1 August, 2003 APPENDIX B Data Coordination and Distribution/ Report Submission Battelle Report B 2 August, 2003 DATA COORDINATION AND DISTRIBUTION/ REPORT SUBMISSION The Data Coordination Center ( DCC) is the central coordinator of all information being generated under the EDSP. For the sliced testis assay prevalidation, the DCC will foster movement of data from the laboratories to the EPA. Critical data coordination and distribution points are anticipated to occur in between the completion of various experiments. To ensure accurate and timely coordination and analysis of interim data, as well as compilation of data for final report submission, the following procedures will be followed and serve as guidelines to the laboratories.

In general, there are two levels of deliverables. The first level consists of data that are generated upon completion of a given individual experiment and are used to make a decision to proceed to the next experiment. Such data sets will be referred to as interim data. The second level consists of all the data from all the prevalidation experiments. This data set is designated as final data and will be used for submission of the final report. Further descriptions of how each of these data sets will be handled and distributed are described in the following subsections.

INTERIM DATA REVIEW There are several different decision points that will require submission of interim data for review. The interim data sets to be submitted for review will be generated after completion of the following experiments:

° Lead Laboratory's conduct of the baseline/ contralateral tissue fragment and positive control experiments ° Lead Laboratory's conduct of the cytotoxicity experiments ° Training and Hands on experience results from the participating laboratories conduct of the baseline and positive control experiments using the assay The Lead laboratory will generate data upon completion of these experiments that will be used to make decisions to proceed to the next phase of prevalidation. In order for the decision to proceed to be made promptly, the lead laboratory will need to process its data rapidly. It is proposed that the laboratory will provide only a technical review of the data for accuracy before making the data available for outside review. This interim data review would proceed without the data going through a formal quality assurance audit. As such, the interim data sets would be stamped as unaudited interim data. In this way, the data generated after each phase could be more quickly transferred to the DCC for collation and statistical analysis of all data sets, thereby providing the EPA and Lead Laboratory with complete data sets and summarized data analysis for them to make decisions to proceed. The flow of interim data that is critical for making a decision to proceed with the next phase is illustrated in Figure B 1. Battelle Report B 3 August, 2003 Lead Laboratory Participating Laboratories DCC EPA Lead Laboratory Decision to Proceed to Next Phase Technically Reviewed Data Cholate Data,

Statistical Analysis, and Inter laboratory Analysis Figure B 1. Interim Data Flow Diagram FINAL REPORT SUBMISSION Final Reports will be submitted by all laboratories. The participating laboratories will submit a final report for the baseline and positive control experimental results. Their reports will be written after the positive control experiment is completed since the participating laboratories are not involved in the remaining prevalidation experiment ( multi chemical testing). By contrast, the lead laboratory is assigned the task of putting together the overall study report. It is expected that the lead laboratory will need the participating laboratories' reports, as well as the DCC's summary of the inter laboratory analysis to complete the report. These reports will need to be prepared and sent to the lead laboratory, such that the overall study report can be completed immediately upon the completion of the multi chemical test experiment. In general, the flow of information during the course of this study should proceed as illustrated in Figure B 2.

A unified format for data and report submission will be implemented. Prior to the start the training phase, the DCC staff will meet with the lead laboratory staff to discuss the format of the results and statistical analysis files for the data. Based on this discussion, the DCC will put together a draft template and provide it to the lead laboratory and the EPA for review. Comments will be collected via a conference call with the EPA and the lead laboratory staff. Following the conference call, a final template will be developed. The lead laboratory will distribute this information to the participating laboratories at the training session. These steps are recommended to ensure that data is presented in the same format from all laboratories in order to foster the collating process and the inter laboratory analysis. By being proactive at the beginning of the study, this should minimize time for completing the inter laboratory analysis, as well as problems with interpretation of data from each laboratory. Battelle Report B 4 August, 2003 Participating Laboratories Lead Laboratory Statistical Analysis DCC Lead Laboratory Inter laboratory Analysis Overall Study Report Participating Lab Reports Inter laboratory analysis report Statistical analysis data Participating Laboratory Final Report Figure B 2. Data Flow for Final Report Submission It is anticipated that the participating laboratories and the lead laboratory need to provide data to the DCC at interim points for two reasons:

1) The results of the inter laboratory analysis at the end of the baseline and positive control experiments may delay submission of the final report.

2) EPA desires to receive data soon after generation for purposes of reviewing the data for additional information based on their experience and the goals of the EDSP.


[Federal Register: October 23, 2006 (Volume 71, Number 204)]
[Notices]
[Page 62103]
[DOCID:fr23oc06-40]

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DEPARTMENT OF ENERGY

Federal Energy Regulatory Commission

[Docket No. CP05-74-001]

Maritimes & Northeast Pipeline, L.L.C.; Notice of Compliance
Filing

October 16, 2006.
    Take notice that on September 29, 2006, Maritimes & Northeast
Pipeline, L.L.C., (Maritimes) tendered for filing as part of its FERC
Gas Tariff, First Revised Volume No. 1, Eighth Revised Sheet No. 14,
with an effective date of November 1, 2006.
    Maritimes states that the filing is being made in compliance with
the Commission's order issued on July 27, 2005 in the above-captioned
proceeding.
    Any person desiring to protest this filing must file in accordance
with Rule 211 of the Commission's Rules of Practice and Procedure (18
CFR 385.211). Protests to this filing will be considered by the
Commission in determining the appropriate action to be taken, but will
not serve to make protestants parties to the proceeding. Such protests
must be filed on or before the date as indicated below. Anyone filing a
protest must serve a copy of that document on all the parties to the
proceeding.
    The Commission encourages electronic submission of protests in lieu
of paper using the ``eFiling'' link at http://www.ferc.gov. Persons

unable to file electronically should submit an original and 14 copies
of the protest to the Federal Energy Regulatory Commission, 888 First
Street, NE., Washington, DC 20426.
    This filing is accessible online at http://www.ferc.gov, using the

``eLibrary'' link and is available for review in the Commission's
Public Reference Room in Washington, DC. There is an ``eSubscription''
link on the Web site that enables subscribers to receive e-mail
notification when a document is added to a subscribed docket(s). For
assistance with any FERC Online service, please e-mail
<EMAIL_ADDRESS>or call<PHONE_NUMBER> (toll free). For

TTY, call<PHONE_NUMBER>.
    Comment Date: 5 p.m. Eastern Time on October 18, 2006.

Magalie R. Salas,
Secretary.
 [FR Doc. E6-17639 Filed 10-20-06; 8:45 am]

BILLING CODE 6717-01-P