**STATEMENT OF WORK**

**Procedures for Acquisition, Preparation, Solubility Testing, and
Distribution of Test Chemicals for a Validation Study for *In Vitro*
Basal Cytotoxicity Testing**

**April 26, 2002**

**Revision 1: May 8, 2002**

**Revision 2: June 21, 2002**

**Revision 3: September 17, 2002**

**Revision 4: October 11, 2002**

Prepared by

The National Toxicology Program (NTP) Interagency Center for the
Evaluation of Alternative Toxicological Methods (NICEATM)

National Institute of Environmental Health Sciences (NIEHS)

National Institutes of Health (NIH)

U.S. Public Health Service

Department of Health and Human Services

Table of Contents

# 

**1.0 project OBJECTIVEs and general requirements
[64](#project-objectives-and-general-requirements)**

1.1 Project Objectives [64](#project-objectives)

1.2 Response to the Statement of Work
[64](#response-to-the-statement-of-work)

1.2.1 General Capabilities [65](#general-capabilities)

1.3 Guidelines [65](#guidelines)

1.4 Definitions [65](#definitions)

2.0 ORGANIZATION [65](#organization)

2.1 Validation Study Sponsors [66](#validation-study-sponsors)

2.2 Management Team [66](#management-team)

2.2.1 Project Management and Chemical Distribution Team
[66](#project-management-and-chemical-distribution-team)

2.2.2 Contract Management [66](#contract-management)

2.2.3 Study Management Team [66](#study-management-team)

2.2.3.1 NIEHS/NICEATM [66](#niehsniceatm)

2.2.3.2 ECVAM [66](#ecvam)

2.2.4 Testing Facilities [67](#testing-facilities)

3.0 CONTRACTOR AND KEY PERSONNEL [67](#contractor-and-key-personnel)

3.1 Contractor [67](#contractor)

3.1.1 Personnel [67](#personnel)

3.1.1.1 Facility Management [67](#facility-management)

3.1.1.2 Study Director [67](#study-director)

3.1.1.3 Quality Assurance (QA) Director
[68](#quality-assurance-qa-director)

3.1.1.4 Scientific Advisor(s) [68](#scientific-advisors)

3.1.1.5 Laboratory Technician(s) [68](#laboratory-technicians)

3.1.1.6 Safety Officer [68](#safety-officer)

3.1.2 Facilities, Equipment, and Supplies
[68](#facilities-equipment-and-supplies)

3.1.2.1 Laboratory [68](#laboratory)

3.1.2.2 Equipment [68](#equipment)

3.1.2.3 Supplies [69](#supplies)

3.1.3 Health and Safety [69](#health-and-safety)

3.1.4 Quality Assurance [69](#quality-assurance)

4.0 TEST PHASES AND SCHEDULE [70](#test-phases-and-schedule)

4.1 Study Timeline [70](#study-timeline)

4.2 Deliverables [70](#deliverables)

4.3 *In Vitro* Validation Study Phases
[71](#in-vitro-validation-study-phases)

4.4 Report Submission Timelines [71](#report-submission-timelines)

4.4.1 Draft Reports [71](#draft-reports)

4.4.2 Final Report [72](#final-report)

5.0 Acquisition, Preparation, and Distribution of Test Chemicals
[72](#acquisition-preparation-and-distribution-of-test-chemicals)

5.1 Test Chemicals [72](#test-chemicals)

5.1.1 Range of Toxicities [72](#range-of-toxicities)

5.1.2 Procurement of Test Chemicals [72](#procurement-of-test-chemicals)

5.1.3 Dispensing Chemicals [72](#dispensing-chemicals)

5.1.4 Shipment of Chemicals [72](#shipment-of-chemicals)

5.1.5 Receipt of Chemicals by the Testing Facilities
[73](#receipt-of-chemicals-by-the-testing-facilities)

5.1.6 Test Chemical Information for the Study Director
[73](#test-chemical-information-for-the-study-director)

5.2 Handling of Test Chemicals [74](#handling-of-test-chemicals)

5.3 Determination of Purity, Composition, and Stability of Test
Chemicals
[74](#determination-of-purity-composition-and-stability-of-test-chemicals)

6.0 solubility determination OF TEST CHEMICALs
[74](#solubility-determination-of-test-chemicals)

6.1 Cell Culture Media and Control Material
[74](#cell-culture-media-and-control-material)

6.1.1 Test Chemical Medium Solvents [74](#test-chemical-medium-solvents)

6.1.1.1 ~~Treatment~~ Chemical Dilution[^1] Medium (BALB/c 3T3 NRU)
[74](#chemical-dilution-medium-balbc-3t3-nru)

6.1.1.2 Routine Culture Medium (NHK NRU)
[74](#routine-culture-medium-nhk-nru)

6.1.2 Positive Control (PC) [75](#positive-control-pc)

6.2 Preparation of Test Chemical [75](#preparation-of-test-chemical)

6.2.1 Dissolving the Test Chemical [75](#dissolving-the-test-chemical)

6.2.1.1 ~~Treatment~~ Chemical Dilution^3^ Medium/Routine Culture
Medium) [78](#treatment-mediumroutine-culture-medium)

6.2.1.2 DMSO [78](#dmso-1)

6.2.1.3 Ethanol [78](#ethanol-1)

6.2.2 pH of Solutions [78](#ph-of-solutions)

7.0 DATA COLLECTION [78](#data-collection)

7.1 Nature of Data to be Collected [78](#nature-of-data-to-be-collected)

7.1.1 Solubility Studies [78](#solubility-studies)

7.1.2 Chemical Information [79](#chemical-information)

7.2 Type of Media Used for Data Storage
[79](#type-of-media-used-for-data-storage)

7.3 Documentation [79](#documentation)

8.0 DRAFT AND FINAL REPORTs [79](#draft-and-final-reports)

9.0 RECORDS AND ARCHIVES [80](#records-and-archives)

10.0 ALTERATIONS OF THE statement of work
[80](#alterations-of-the-statement-of-work)

11.0 REFERENCES [80](#references)

12.0 APPROVAL OF Statement of work [82](#approval-of-statement-of-work)

ADDENDUM I [83](#addendum-i)

ADDENDUM II [89](#addendum-ii)

ADDENDUM III [90](#addendum-iii)

ADDENDUM IV [93](#addendum-iv)

ADDENDUM V [95](#addendum-v)

#  

**STATEMENT OF WORK**

**Procedures for Acquisition, Preparation, Solubility Testing, and
Distribution of Test Chemicals for a Validation Study for *In Vitro*
Basal Cytotoxicity Testing**

### 

# 1.0 project OBJECTIVEs and general requirements 

## 1.1 Project Objectives

This Statement of Work outlines and supports the procedures that the
Contractor will initiate for the acquisition, preparation, solubility
testing, and distribution of the test chemicals needed to perform two
*in vitro* basal cytotoxicity assays (the BALB/c 3T3 Neutral Red Uptake
\[NRU\] assay and the Normal Human Keratinocyte \[NHK\] Neutral Red
Uptake \[NRU\] assay) for a multi-laboratory Validation Study. These
assays, recommended in *Guidance Document On Using In Vitro Data To
Estimate In Vivo Starting Doses For Acute Toxicity* (ICCVAM, 2001), use
mammalian cell culture techniques to assess the basal cytotoxicity of
chemicals.

A primary goal of this Validation Study is to evaluate the usefulness of
the BALB/c 3T3 Neutral Red Uptake (NRU) and the Normal Human
Keratinocyte (NHK) NRU assays for reducing and refining animal use for
acute oral toxicity determinations of chemicals by predicting starting
doses for *in vivo* rodent acute lethality assays.

> The proposed Validation Study will determine IC~20~, IC~50~, and
> IC~80~ values for a test set of 72 chemicals with varying degrees of
> toxicity. This set of chemicals was selected separate and prior to
> this Statement of Work by the Study Management Team. The basis for
> selection of this test set is discussed in the Study Design document
> prepared by the Study Management Team.
>
> The Contractor shall perform the following activities:

-   Acquire 73 high quality and high purity (99% or greater when
    economically feasible) chemicals from reputable commercial sources

-   Perform solubility tests on all chemicals using solvents and
    procedures that have been recommended to the test laboratories

-   Repackage chemicals into multiple smaller units

-   Code chemicals with a unique identification number so that chemicals
    can be provided to testing laboratories in a blinded fashion

-   Distribute chemicals and health and safety information to the
    Testing Facilities

-   Provide draft and final reports of these activities.

## Response to the Statement of Work 

> Proposals submitted in response to this Statement of Work shall
> include:

a)  A Work Plan

b)  A timetable for project milestones

c)  A cost estimate based on chemical acquisition, performance of
    solubility tests for all test chemicals, chemical coding,
    repackaging, and distribution to two U. S labs and one U. K. lab.

### 1.2.1 General Capabilities 

### 

> The Contractor shall be capable of performing the following:
>
> a\) Prepare/provide Standard Operating Procedures (SOPs) for the
> performance of the activities outlined in **Section 1.1** (see
> **Section 1.4** -- Definitions - SOPs)
>
> b\) Perform all aspects of the Test Chemical Preparation in accordance
> with Good Laboratory Practices (GLP).
>
> c\) Adhere to this Statement of Work throughout the Validation Study.

## 1.3 Guidelines

> The Project Officer and/or her/his representatives (e.g., Study
> Management Team) may inspect and audit the Contractor to ensure that
> the Project Officer's minimum requirements and guidelines are being
> followed.

## 1.4 Definitions

> **Blinded/Coded Chemicals:** Test chemicals supplied to the Testing
> Facilities that are coded and distributed by the Contractor such that
> only the Project Officer, Management Team, and the Contractor have
> knowledge of the contents of each test chemical vessel. The test
> chemicals will be purchased, aliquoted, coded, and distributed by the
> Contractor under the guidance of the NIEHS/NTP Project Officer and the
> Management Team.
>
> **Contractor:** Facility that will initiate the acquisition,
> preparation, solubility testing, and distribution of the test
> chemicals needed to perform two *in vitro* basal cytotoxicity assays
> for a multi-laboratory *in vitro* Validation Study.
>
> **Good Laboratory Practices (GLPs):** Regulations governing the
> conduct, procedures, and operations of toxicology laboratories;
> regulations to assure the quality and integrity of the data and to
> address such matters as organization and personnel, facilities,
> equipment, facility operations, test chemicals, and study protocol
> (Statement of Work) and conduct (U.S. Food and Drug Administration,
> Title 21 CFR Part 58; Environmental Protection Agency, Title 40 CFR
> Part 160).
>
> **Standard Operating Procedures (SOPs):** Written documents that
> describe, in great detail, the routine procedures to be followed for a
> specific operation, analysis, or action; consistent use of an approved
> SOP ensures conformance with organizational practices, reduced work
> effort, reduction in error occurrences, and improved data
> comparability, credibility, and defensibility; SOPs also serve as
> resources for training and for ready reference and documentation of
> proper procedures;
>
> **Statement of Work:** A description of test chemical preparation
> required for the *in vitro* Validation Study; defines all phases of
> the Validation Study and the purpose of the procedures; provides the
> details of test chemical acquisition, preparation, solubility testing,
> and distribution; provides guidance for the preparation of reports
>
> **Testing Facility:** A laboratory that has been designated to
> participate in the *In Vitro* Validation Study; facilities identified
> in **Section 2.2.4**.

# 2.0 ORGANIZATION

## 2.1 Validation Study Sponsors

## 

-   National Institute of Environmental Health Sciences (NIEHS)

-   The National Toxicology Program (NTP) Interagency Center for the
    Evaluation of Alternative Toxicological Methods (NICEATM)

-   U.S. Environmental Protection Agency (U.S. EPA)

-   The European Centre for the Validation of Alternative Methods
    (ECVAM).

## 2.2 Management Team

### 2.2.1 Project Management and Chemical Distribution Team 

> Ms. Molly Vallant (NIEHS) -- NIEHS Project Officer for BioReliance,
> Inc.
>
> NIEHS
>
> MD E1-03
>
> P.O. BOX 12233
>
> RTP, NC 27709
>
> Dr. Martin L. Wenk (BioReliance, Inc.) -- Chemical acquisition,
> preparation,
>
> solubility testing, and distribution
>
> BioReliance Corporation
>
> 14920 Broschart Road
>
> Rockville, Maryland 20850-3349

### 

### 2.2.2 Contract Management 

### 

> Ms. Jackie Osgood (NIEHS) -- Contracting Officer
>
> Mr. Don Gula (NIEHS) -- Contracting Officer

### 

### 2.2.3 Study Management Team 

#### 2.2.3.1 NIEHS/NICEATM

> Dr. William S. Stokes (NICEATM/NIEHS) -- Co-chair -- Study Management
> Team
>
> Dr. Judy Strickland (NICEATM/ILS) -- Project Coordinator
>
> Mr. Michael Paris (NICEATM/ILS) -- Assistant Project Coordinator
>
> Dr. Ray Tice (NICEATM/ILS) -- Technical Advisor
>
> NICEATM
>
> 79 T.W. Alexander Drive
>
> Bldg. 4401, MD-EC-17
>
> 3^rd^ Floor, Room 3126
>
> P.O. Box 12233
>
> Research Triangle Park, NC 27709

#### 2.2.3.2 ECVAM

> Professor Michael Balls -- Co-chair -- Study Management Team
>
> Dr. Silvia Casati
>
> Dr. Andrew Worth
>
> European Commission
>
> Joint Research Centre
>
> Institute for Health and Consumer Protection
>
> Management Support Unit - TP 202
>
> I-21020 Ispra (VA) - Italy

### 2.2.4 Testing Facilities 

> [XXX, Safety Officer]{.mark}
>
> Institute for *In Vitro* Sciences (IIVS)
>
> 21 Firstfield Road
>
> Suite 220
>
> Gaithersburg, MD 20878
>
> []{.mark}
>
> []{.mark}
>
> Bill Cappuccio, Safety Officer
>
> 5183 Blackhawk Rd
>
> E3330/Room 278
>
> Aberdeen Proving Ground-EA, MD 21010
>
> 410-436-7462
>
> Rodger Dainty, Safety Officer
>
> School of Biomedical Sciences
>
> University of Nottingham Medical School
>
> Queen\'s Medical Centre
>
> Nottingham, NG7 2UH UK
>
> []{.mark}
>
> []{.mark}

## []{.mark}

# 3.0 CONTRACTOR AND KEY PERSONNEL 

## 

## 3.1 Contractor

> The Contractor shall have competence in chemical acquisition,
> preparation, solubility testing, and distribution and shall provide
> competent personnel, adequate facilities, equipment, supplies, proper
> health and safety guidelines, and satisfactory quality assurance
> procedures.

### 3.1.1 Personnel

#### 3.1.1.1 Facility Management

> The facility management is responsible for establishing scientific
> guidelines and procedures, training and supervision of professional
> and technical staff, and evaluation of results and performance within
> their discipline area relative to the Project Officer's stated
> requirements. The manager must maintain records of the qualifications,
> training and experience, and a job description for each professional
> and technical individual involved in test chemical acquisition,
> preparation, solubility testing, and distribution.

## 

#### 3.1.1.2 Study Director

> A scientist or other professional of appropriate education, training,
> and experience in chemical acquisition, preparation, solubility
> testing, and distribution, or combination thereof, shall be the Study
> Director. The Study Director has the overall responsibility for the
> technical conduct of chemical acquisition, preparation, solubility
> testing, and distribution for the Validation Study (e.g., GLP
> adherence) and shall be responsible for determining test acceptance.
> The Study Director shall be responsible for providing SOPs that
> incorporate pertinent information obtained from the Statement of Work.
> Other duties include the interpretation and analysis of test chemical
> solubility data, documentation of all study aspects (including
> maintenance of a Study Workbook), and production of all draft and
> final written reports.

#### 3.1.1.3 Quality Assurance (QA) Director

> The Quality Assurance Director shall **monitor** all tasks and assure
> conformance with GLP requirements (i.e., facilities, equipment,
> personnel, methods, practices, records, controls, transference of data
> into software, SOPs). Quality Assurance Director or unit can be any
> person or organizational element, except the Study Director,
> designated by Contractor management to perform the duties relating to
> quality assurance of the studies and tasks. The Quality Assurance
> duties are not a substitute for the Study Director duties.

#### 3.1.1.4 Scientific Advisor(s)

> Scientists or other professionals of appropriate education, training,
> and experience in chemical acquisition, preparation, solubility
> testing, and distribution who provide scientific guidance to the Study
> Director and other laboratory personnel.

#### 3.1.1.5 Laboratory Technician(s)

> Each individual engaged in the conduct of or responsible for the
> supervision of a study shall have education, training, and experience,
> or combination thereof, to enable that individual to perform the
> assigned duties. The individuals must be trained in GLP requirements
> and technical ability must be documented as per GLP requirements.

#### 3.1.1.6 Safety Officer

> The Contractor shall designate a Safety Officer who will provide a
> sealed health and safety information package that will accompany the
> test chemicals to the Test Facilities. A duplicate package will be
> provided to the Project Officer and Management Team.

### 

### 3.1.2 Facilities, Equipment, and Supplies

#### 3.1.2.1 Laboratory

> The Contractor must provide a designated laboratory/area to ensure
> that test chemical preparation and solubility testing can be performed
> under clean conditions. Potential for cross-contamination of chemicals
> should be minimal.

#### 3.1.2.2 Equipment

> The Contractor must provide at a minimum the following equipment:

a)  Water bath (37^o^C)

b)  Sonication unit

c)  Vortex unit

d)  Pippettors (micropipettors,)

e)  Computer (for data transformation and analysis)

f)  Balance

g)  pH meter

> All equipment maintenance and calibration shall be routinely performed
> and documented as per GLP guidelines and Contractor procedures

#### 3.1.2.3 Supplies

> All cell culture reagents must be labeled so as to indicate source,
> identity, concentration, stability, preparation and expiration dates,
> and storage conditions.

a)  Dulbecco's Modification of Eagle's Medium (DMEM) without
    L-Glutamine; should have Hanks' salts and high glucose \[4.5gm/l\]
    (e.g., ICN-Flow Cat. No. 12-332-54)

b)  L-Glutamine 200 mM (e.g., ICN-Flow \# 16-801-49)

c)  New Born Calf Serum (NBCS) (e.g., Biochrom \# SO 125)

d)  Dimethyl sulfoxide (DMSO), U.S.P. analytical grade. DMSO shall be
    stored under nitrogen at --20^o^C.

e)  Ethanol (ETOH), U.S.P. analytical grade (100%, non-denatured)

f)  Keratinocyte Basal Medium without Ca^++^ (KBM, Clonetics CC-3104)
    that is completed by adding the KBM SingleQuots [^2] (Clonetics
    CC-4131) to achieve the proper concentrations of epidermal growth
    factor, insulin, hydrocortisone, antimicrobial agents, bovine
    pituitary extract, and calcium (e.g., Clonetics Calcium
    SingleQuots, CC-4202)\*.

g)  Penicillin/streptomycin solution (e.g. ICN-Flow \# 16-700-49)

> *\** BioWhittaker, 8830 Biggs Ford Road, Walkersville, MD 21793
> ([[http://www.cambrex.com/subsidiaries/s%2Dbw%5Finc/s%2Dbiowhittaker%2Dinc%2Dcontact2.htm]{.underline}](http://www.cambrex.com/redirect/))

### 3.1.3 Health and Safety

> The Contractor shall conform to all local, state, and federal statutes
> in effect at the time of this study.

### 3.1.4 Quality Assurance

> The Contractor shall conduct the acquisition, preparation, solubility
> testing, and distribution of test chemicals in compliance with Good
> Laboratory Practice (GLP) Standards (U.S. Food and Drug
> Administration, Title 21 CFR Part 58; Environmental Protection Agency,
> Title 40 CFR Part 160). The appropriate QA unit (as per GLPs) shall
> audit the procedures and final report.
>
> The Final Report shall be audited by the Quality Assurance unit of the
> Contractor for GLP compliance and a QA Statement shall be provided by
> the Contractor. The Final Report shall identify: 1) the phases and
> data inspected, 2) dates of inspection, and 3) dates findings were
> reported to the Study Director and Contractor management. The QA
> Statement shall identify whether the methods and results described in
> the Final Report accurately reflect the raw data produced during the
> study.

# 4.0 TEST PHASES AND SCHEDULE

## 4.1 Study Timeline 

> The following timeline is for the **laboratory testing aspect** of the
> *In Vitro* Validation Study. The Contractor shall provide the required
> chemicals in a timely fashion so that each phase of the study can
> start on the appointed date.

+------------------------------+-------------+-------------------------+
| **TASK**                     | **WEEK**    | **ESTIMATED DATE**      |
+------------------------------+-------------+-------------------------+
| Statement of Work issued by  | 0           | March 29, 2002          |
| NIEHS                        |             |                         |
|                              |             |                         |
| to the Testing Facility      |             |                         |
+------------------------------+-------------+-------------------------+
| Response /Proposal received  | 6           | May 10, 2002            |
| from                         |             |                         |
|                              |             |                         |
| the Testing Facility         |             |                         |
+------------------------------+-------------+-------------------------+
| ^2^                          | ^2^         | ^2^                     |
+------------------------------+-------------+-------------------------+
| Submission of Study          | 11          | June 12, 2002           |
| Protocol, CVs of Key         |             |                         |
| Personnel, SOPs[^3]          |             |                         |
+------------------------------+-------------+-------------------------+
| Award of Contracts^2^        | 13^2^       | June 28, 2002^2^        |
+------------------------------+-------------+-------------------------+
| Start Testing -- Phase I     | 18^2^       | July 29^2^, 2002        |
| (Phase Ia)                   |             |                         |
+------------------------------+-------------+-------------------------+
| End Phase Ia                 | 22^2^       | August 26^2^, 2002      |
+------------------------------+-------------+-------------------------+
| Begin Phase Ib               | 26^2^       | September 26^2^, 2002   |
+------------------------------+-------------+-------------------------+
| End Phase Ib                 | 31^2^       | October 29^2^, 2002     |
+------------------------------+-------------+-------------------------+
| Begin Phase II               | 36^2^       | December 2^2^, 2002     |
+------------------------------+-------------+-------------------------+
| End Phase II                 | 46^2^       | February 10^2^, 2003    |
+------------------------------+-------------+-------------------------+
| Begin Phase III              | 52^2^       | March^2^ 26, 2003       |
+------------------------------+-------------+-------------------------+
| Final Report (Phase III) to  | 89^2^       | December 9^2^, 2003     |
| SMT                          |             |                         |
+------------------------------+-------------+-------------------------+

## 

## 4.2 Deliverables 

> The following schedule of deliverables is for **the acquisition,
> preparation, solubility testing and distribution of test chemicals**.

+---------------+------------+------------+------------+-------------+
|               | *          |            |            |             |
|               | *ESTIMATED |            |            |             |
|               | DUE DATES  |            |            |             |
|               | (to        |            |            |             |
|               | Project    |            |            |             |
|               | Officer)** |            |            |             |
+---------------+------------+------------+------------+-------------+
| Submission of | **Week     |            | **June 12, |             |
| SOPs for      | 11**       |            | 2002**     |             |
| **Section     |            |            |            |             |
| 1.1**         |            |            |            |             |
| activities    |            |            |            |             |
+---------------+------------+------------+------------+-------------+
| **REPORTS**   | **PHASE    | **PHASE    | **PHASE    | **PHASE     |
|               | Ia**       | Ib**       | II**       | III**       |
+---------------+------------+------------+------------+-------------+
| Biweekly      | a          | a          | a          | a           |
| Reports       |            |            |            |             |
+---------------+------------+------------+------------+-------------+
| Draft Phase   | Week 17    |            | Week 33    | Week 48     |
| Reports       |            |            |            |             |
|               | July       |            | Nov.       | Feb. 26^2^, |
|               | 24^2^,     |            | 13^2^,     | 2003 ^b^    |
|               | 2002 ^b^   |            | 2002 ^b^   |             |
+---------------+------------+------------+------------+-------------+
| Draft Final   | Week 52    |            |            |             |
| Report        |            |            |            |             |
|               | March^2^   |            |            |             |
| (all phases   | 26, 2003   |            |            |             |
| combined)     | ^c^        |            |            |             |
+---------------+------------+------------+------------+-------------+
| Final Report  | Week 54    |            |            |             |
|               |            |            |            |             |
| (all phases   | April      |            |            |             |
| combined)     | 9^2^, 2003 |            |            |             |
|               | ^d^        |            |            |             |
+---------------+------------+------------+------------+-------------+

> *a* Biweekly reports shall begin at the time of implementation of the
> contracts and continue until the final report is submitted.
>
> *b* Draft Phase Reports shall be submitted to the Project Officer no
> later than the dates provided (at least two weeks before shipment of
> chemicals to the Test Facilities).
>
> *c* Draft Final Report shall be submitted to the Project Officer no
> later than the date provided (at the most one month after final
> shipment of chemicals to the Test Facilities).
>
> *d* Final Report shall be submitted to the Project Officer no later
> than the date provided (at the most one month after the Project
> Officer receives the Draft Final Report.
>
> The following schedule is for the **distribution of test chemicals**
> to the Testing Facilities.

+---------------+------------+------------+------------+-------------+
|               | *          |            |            |             |
|               | *ESTIMATED |            |            |             |
|               | DUE DATES  |            |            |             |
|               | (to        |            |            |             |
|               | Testing    |            |            |             |
|               | Fac        |            |            |             |
|               | ilities)** |            |            |             |
+---------------+------------+------------+------------+-------------+
| **CHEMICAL    | **PHASE    | **PHASE    | **PHASE    | **PHASE     |
| SHIPPING TO   | Ia**       | Ib**       | II**       | III**       |
| TESTING       |            |            |            |             |
| FA            |            |            |            |             |
| CILITIES^a^** |            |            |            |             |
+---------------+------------+------------+------------+-------------+
| Positive      | Before     | \-\--      | \-\--      | \-\--       |
| Control       |            |            |            |             |
|               | July       |            |            |             |
| (SLS)         | 29[^4],    |            |            |             |
|               | 2002       |            |            |             |
+---------------+------------+------------+------------+-------------+
| Phase Ib      | \-\--      | Before     | \-\--      | \-\--       |
|               |            |            |            |             |
| (3 chemicals) |            | September  |            |             |
|               |            | 26^2^,     |            |             |
|               |            | 2002       |            |             |
+---------------+------------+------------+------------+-------------+
| Phase II      | \-\--      | \-\--      | Before     | \-\--       |
|               |            |            |            |             |
| (9 chemicals) |            |            | December   |             |
|               |            |            | 2^2^, 2002 |             |
+---------------+------------+------------+------------+-------------+
| Phase III     | \-\--      | \-\--      | \-\--      | Before      |
|               |            |            |            |             |
| (60           |            |            |            | March^2^    |
| chemicals)    |            |            |            | 26, 2003    |
+---------------+------------+------------+------------+-------------+

> *a* Dates for chemical shipments are to ensure that the Testing
> Facilities receive Test Chemicals prior to the start dates of each lab
> testing phase. Phase III chemicals shall be shipped as one group of 60
> chemicals. Chemicals for each phase are identified in Addendum IV.

## 4.3 *In Vitro* Validation Study Phases 

> ***[Phase I:]{.underline}*** The training phase for laboratory
> personnel. This phase includes developing a positive control database
> (Phase Ia) and testing three unknown chemicals (Phase Ib).
>
> ***[Phase II:]{.underline}*** The qualification phase. This phase
> requires testing nine blinded/coded chemicals in the same *in vitro*
> cytotoxicity assays and in the same concentration-response fashion as
> in Phase Ib.
>
> ***[Phase III:]{.underline}*** Testing 60 blinded/coded chemicals in
> the same manner as in Phases I and II.

## 4.4 Report Submission Timelines

### 4.4.1 Draft Reports

> Draft reports for each phase shall be submitted to the Project Officer
> as per **Section 4.2**.

### 4.4.2 Final Report

> The Final report shall be submitted to the Project Officer as per
> **Section 4.2**.

# 

# 5.0 Acquisition, Preparation, and Distribution of Test Chemicals

#### 

## 5.1 Test Chemicals

### 5.1.1 Range of Toxicities

> The chemicals proposed for the Validation Study are representative of
> a range of toxicities and are relevant with regard to human exposure
> potential. The test chemicals will represent each of the Globally
> Harmonized System (GHS) classification groups for rat oral LD50s: ≤ 5
> mg/kg, \>5 ≤ 50 mg/kg, \>50 ≤ 300 mg/kg, \>300 ≤ 2000 mg/kg, \>2000 ≤
> 5000 mg/kg, and \>5000 mg/kg (OECD, 2001). Addenda III and IV provide
> the list of test chemicals for the *In Vitro* Validation Study.

## 5.1.2 *Procurement of Test Chemicals*

> The Contractor shall purchase 73 chemicals specified in Addenda III
> and IV (72 "test chemicals" and one "positive control") from
> commercial manufacturers. Chemical purity shall be 99% or greater when
> economically feasible. Chemical information from the manufacturers
> shall be collected as specified in **Section 7.1.2** and reported as
> indicated in Addendum I. Chemicals shall be stored as recommended by
> the manufacturer.

### 5.1.3 Dispensing Chemicals

### 

> While preparing the purchased chemicals for distribution to the
> Testing Facilities, only one bulk substance shall be dispensed at any
> time. All test samples shall be sealed and labeled before dispensing
> the next substance. Once test samples have been dispensed into
> aliquots, they shall be returned to appropriate storage conditions
> until they are dispatched.
>
> During dispensing, all test chemicals, with the exception of the
> positive control, will be randomly blinded/coded so that testing by
> the Testing Facilities will be conducted on chemicals with a masked
> identity. Each chemical shall have a code that is unique for each
> Testing Facility (i.e., no chemical shall have the same code in any
> Testing Facility). The Contractor shall dispense 4 g of test
> chemical/Testing Facility (see Addendum V for assumptions used to
> determine the amount of chemical/Testing Facility) into clean, sterile
> containers, and assign unique code identifiers, and archive two
> additional samples. About 100 g of the positive control shall be
> distributed to each lab and one additional sample shall be archived.

### 5.1.4 Shipment of Chemicals

> After dispensing and labeling chemical aliquots with unique codes, the
> Contractor shall ship a set of the test chemicals, including the
> positive control, to the each of three Testing Facilities. Two
> Facilities will be in the US and one will be in the United Kingdom.
> The Contractor will package test chemicals so as to minimize damage
> during transit and will ship them to each Testing Facility according
> to proper regulatory procedures. Except for the positive control in
> Phase Ia, chemicals are to be packaged and shipped so as to conceal
> their identities. Test chemicals shall be shipped under conditions
> that will preserve the integrity of the chemicals. The Contractor
> shall notify the Testing Facilities (and the Project Officer) when the
> test chemicals are shipped so as to prepare for receipt.
>
> The Contractor will retain the archived chemicals, which may be
> required for retesting or purity analysis, until the completion of the
> Validation Study.
>
> **5.1.4.1 *[Distribution Phases]{.underline}***
>
> ***Phase Ia***: For Phase I, the positive control chemical identified
> in Addendum III shall be distributed to all three Testing Facilities.
>
> ***Phase Ia***: For Phase Ib, the three (3) blinded/coded chemicals
> identified in Addendum III shall be distributed to all three Testing
> Facilities.
>
> ***Phase II***: Nine (9) blinded/coded chemicals identified in
> Addendum III shall be distributed to all three Testing Facilities.
>
> ***Phase III:*** Sixty (60) blinded/coded chemicals identified in
> Addendum III shall be distributed to the Test Facilities. Chemicals
> will be shipped --as a group of 60 chemicals.

### 5.1.5 Receipt of Chemicals by the Testing Facilities

> With the exception of the positive control shipment, which shall be
> shipped directly to the Study Director, the chemical shipments shall
> be addressed to the Testing Facility Safety Officers and accompanied
> by a sealed information packet containing the appropriate health and
> safety procedures for use (i.e., Material Safety Data Sheets (MSDS) or
> equivalent documentation with proper protection, procedures for
> accidental ingestion or contact with skin or eyes, and procedures for
> containing and recovering spills) and a disclosure key for identifying
> test chemicals by code. The shipment shall include instructions for
> the Testing Facility Safety Officer to:

1)  Immediately notify the Contractor and Study Project Coordinator upon
    receipt of chemicals,

2)  Retain the health and safety package and pass the test chemicals to
    the Study Director without revealing the identities of the test
    chemicals,

3)  Notify the Management Team if Test Facility personnel open the
    health and safety packet at any time during the Validation Study,
    and

> 4\) Return the unopened health and safety package to the Contractor
> after testing is complete. The Contractor shall immediately notify the
> Project Officer regarding chemical receipt.
>
> If regulatory transportation requirements dictate that each package
> must display a list of the chemicals it contains on the outside of the
> package, the Contractor shall direct the Testing Facility Safety
> Officer to remove it prior to passing the chemicals to the Study
> Director.

### 5.1.6 Test Chemical Information for the Study Director

> The Contractor shall supply, with each test chemical, data sheets
> giving a minimum of essential information, including color, odor,
> physical state, weight or volume of sample, specific density for
> liquid test chemicals, and storage instructions. The Study Director
> shall receive this information from the Safety Officer.

## 

## 5.2 Handling of Test Chemicals

> Appropriate routine safety procedures shall be followed in handling
> the test chemicals. The Contractor shall include instructions to the
> Test Facilities to treat all blinded/coded test chemicals as ***very
> hazardous and potentially carcinogenic.*** After the studies are
> completed, the remaining test chemicals will be returned by the
> Testing Facilities to the Contractor.

## 5.3 Determination of Purity, Composition, and Stability of Test Chemicals

As indicated in **Section 7.1.2**, the Contractor will be directly
responsible for collecting information (from manufacturer and supplier
documentation) on the analytical purity, composition, and stability of
the test chemicals and the positive control material, and their
homogeneity (via Contractor solubility studies) in the vehicle.

# 6.0 solubility determination OF TEST CHEMICALs

The Contractor shall determine solubility of the test chemicals in the
same manner as recommended to the Testing Facilities (i.e., by following
the hierarchy below).

## 

## 6.1 Cell Culture Media and Control Material

### 6.1.1 Test Chemical Medium Solvents

#### 6.1.1.1 Chemical Dilution[^5] Medium (BALB/c 3T3 NRU) 

> Serum-free^3^ Dulbecco's Modification of Eagle's Medium (DMEM) \[see
> **Section 3.1.2.3.a**\] buffered with sodium bicarbonate and
> supplemented with (final concentrations in DMEM are quoted):
>
> ^3^
>
> 4 mM Glutamine
>
> 200 IU/mL^3^ Penicillin
>
> 200 µg/ml^3^ Streptomycin
>
> This serum-free^3^ medium is used in the assay for dissolving^3^ test
> chemicals prior to application^3^ to the 3T3 cells.

#### 6.1.1.2 Routine Culture Medium (NHK NRU)

> KBM (Clonetics CC-3104) supplemented with KBM SingleQuots
> (Clonetics CC-4131) and Clonetics Calcium SingleQuots (CC-4202) to
> make 500ml of medium. Final concentration of supplements in medium
> are:[^6]
>
> 0.0001 ng/ml^2^ Human recombinant epidermal growth factor
>
> 5 μg/ml^2^ Insulin
>
> 0.5 g/ml^2^ Hydrocortisone
>
> 30 μg/ml^2^ Gentamicin
>
> 15 ng/ml^2^ Amphotericin B
>
> 0.10 mM Calcium
>
> 30 μg/ml^2^ Bovine pituitary extract.
>
> This medium is used in the assay as the routine culture medium and for
> application of test chemicals to the NHK cells.
>
> *Complete media should be kept at 4°C and stored for no longer than
> two weeks*.^2^
>
> NOTE:
>
> KBM SingleQuots contain the following stock concentrations and
> volumes:^2^
>
> 0.1 ng/ml hEGF 0.5 ml^2^
>
> 5.0 mg/ml Insulin 0.5 ml^2^
>
> 0.5 mg/ml Hydrocortisone 0.5 ml^2^
>
> 30 mg/ml Gentamicin, 15 ug/ml Amphotericin-B 0.5 ml^2^
>
> 7.5 mg/ml Bovine Pituitary Extract (BPE) 2.0 ml^2^
>
> Clonetics Calcium SingleQuots are 2 ml of 300mM concentration of
> calcium. ^2^
>
> 165 ul of solution per 500 ml calcium-free medium equals 0.10 mM
> calcium in the medium.^2^

### 6.1.2 Positive Control (PC)

> Sodium Lauryl Sulfate (\[SLS\], CAS \# 151-21-3) will be the positive
> control material for the *In Vitro* Validation Study.

## 6.2 Preparation of Test Chemical

> All chemicals (including the positive control \[SLS\]) shall be
> weighed on a calibrated balance (including liquid test chemicals) and
> added to the appropriate solvent (**Section 6.2.1**). Test chemicals
> must be at room temperature before dissolving. Preparation under red
> light or yellow light may be necessary, if rapid photodegradation is
> likely to occur. The solutions must not be cloudy nor have noticeable
> precipitate.

### 6.2.1 Dissolving the Test Chemical[^7]

> The hierarchy specified in **Sections 6.2.1.1 to 6.2.1.3** (i.e.,
> culture medium, DMSO, ethanol) shall be followed for dissolving the
> test chemicals and positive control. Both assay-specific culture media
> specified in **Section 6.1.1** (i.e., Chemical Dilution Medium for 3T3
> cells and Routine Culture Medium for NHK cells) must be tested.
> Approximately 100 mg (100,000 μg) of the test chemical will be weighed
> into a glass tube and the weight will be documented. Assay-specific
> media will be added to the vessel so that the concentration is 200,000
> μg/ml (200 mg/mL) (i.e., approximately 0.5 mL). The solution is mixed
> as specified in Section 6.2.1.1. If complete solubility is achieved,
> then additional solubility procedures are not needed. If only partial
> solubility is achieved, follow the test chemical dissolving steps in
> Table 1, derived from EPA (1998), to add additional medium in steps
> until the concentration is a minimum of 2,000 μg/mL (2 mg/mL). If
> complete solubility at 2,000 μg/mL in medium can't be attained, then
> repeat the solubility steps using the other solvent(s) in the
> solubility hierarchy. Test chemicals that are only soluble in DMSO or
> ethanol will be prepared at 500,000 μg/mL as the highest concentration
> of stock solution.
>
> ***Table 1: Determination of Solubility in Media***

+---------------------+---------+--------+---------+--------+--------+
| # Step              | **1**   | **2**  | **3**   | **4**  | **5**  |
+---------------------+---------+--------+---------+--------+--------+
| Total Volume of     | 0.5 mL  | 2.5 mL | 5.0 mL  | 2.0 mL | 10.0   |
| Medium              |         |        |         |        | mL     |
+---------------------+---------+--------+---------+--------+--------+
| Concentration of    | 200,000 | 40,000 | 20,000  |        |        |
| Test Chemical (Add  | μg/mL   | μg/mL  | μg/mL   |        |        |
| 100 mg to a tube.   |         |        |         |        |        |
| Add the first       | (200    | (40    | (20     |        |        |
| volume of medium.   | mg/mL)  | mg/mL) | mg/mL)  |        |        |
| Dilute with         |         |        |         |        |        |
| subsequent volumes  |         |        |         |        |        |
| if necessary.)      |         |        |         |        |        |
+---------------------+---------+--------+---------+--------+--------+
| Concentration of    |         |        |         | 10,000 | 2,000  |
| Test Chemical (Add  |         |        |         | μg/mL  | μg/mL  |
| 20 mg to a large    |         |        |         |        |        |
| tube. Add the first |         |        |         | (10    | (2.0   |
| volume of medium.   |         |        |         | mg/mL) | mg/mL) |
| Dilute with         |         |        |         |        |        |
| subsequent volume   |         |        |         |        |        |
| if necessary.)      |         |        |         |        |        |
+---------------------+---------+--------+---------+--------+--------+
| If test chemical is |         |        |         |        |        |
| insoluble in medium |         |        |         |        |        |
| at 2000 μg/mL, then |         |        |         |        |        |
| attempt to dissolve |         |        |         |        |        |
| chemical in DMSO.   |         |        |         |        |        |
| Actual volume of    |         |        |         |        |        |
| solution can be     |         |        |         |        |        |
| determined after    |         |        |         |        |        |
| test chemical is    |         |        |         |        |        |
| dissolved and       |         |        |         |        |        |
| solution is         |         |        |         |        |        |
| measured using a    |         |        |         |        |        |
| calibrated          |         |        |         |        |        |
| instrument (e.g.,   |         |        |         |        |        |
| micropipettor, or   |         |        |         |        |        |
| serological         |         |        |         |        |        |
| pipette). The       |         |        |         |        |        |
| actual stock        |         |        |         |        |        |
| concentration can   |         |        |         |        |        |
| be calculated       |         |        |         |        |        |
| accordingly.        |         |        |         |        |        |
+---------------------+---------+--------+---------+--------+--------+

Example: If complete solubility is not achieved in 0.5 mL medium
(Step 1) using the mixing procedures specified in **Section 6.2.1.1,
b-d,** then 2.0 mL must be added to obtain a total volume of 2.5 mL
(Step 2). Chemical and medium are again mixed as prescribed in **Section
6.2.1.1** in an attempt to dissolve. If solubility is not achieved at
Step 2, then 2.5 mL medium is added in Step 3. Chemical and medium are
again mixed as prescribed in Section **6.2.1.1** in an attempt to
dissolve. No additional weighing of the chemical is required until Step
4.

#### 

#### 6.2.1.1 Chemical Dilution Medium/Routine Culture Medium)

a)  Dissolve test chemical in Chemical Dilution Medium and Routine
    Culture Medium as in Step 1 of **Table 1**.

b)  Gently mix. Vortex for 1-2 minutes.

c)  If test chemical hasn't dissolved, use sonication for up to five
    minutes.

d)  If sonication doesn't work, then warm solution to 37°C.

e)  Proceed to Step 2 (and Steps 3-5, if necessary) of **Table 1** and
    repeat procedures b-d.

#### 6.2.1.2 DMSO 

> If the test chemical doesn't dissolve in the Chemical Dilution Medium
> or Routine Culture Medium, then follow the dilution steps in **Table
> 1A** and mixing steps a) through e) in **Section 6.2.1.1** using DMSO
> instead of Chemical Dilution Medium/Routine Culture Medium.

#### 6.2.1.3 Ethanol 

> If the test chemical doesn't dissolve in DMSO, then follow the
> dilution steps in **Table 1A** and mixing steps a) through e) in
> **Section 6.2.1.1** using ethanol instead of DMSO.
>
> ***Table 1A: Determination of Solubility in DMSO and Ethanol***

+--------------+---------+---------+--------+--------+-------+-------+
| **Steps**    | **1**   | **2**   | **3**  | **4**  | **5** | **6** |
+--------------+---------+---------+--------+--------+-------+-------+
| Total Volume | 0.2 mL  | 0.5 mL  | 2.5 mL | 5.0 mL | 2.0   | 10.0  |
| of DMSO or   |         |         |        |        | mL    | mL    |
| Ethanol      |         |         |        |        |       |       |
+--------------+---------+---------+--------+--------+-------+-------+
| C            | 500,000 | 200,000 | 40,000 | 20,000 |       |       |
| oncentration | μg/mL   | μg/mL   | μg/mL  | μg/mL  |       |       |
| of Test      |         |         |        |        |       |       |
| Chemical     | (500    | (200    | (40    | (20    |       |       |
| (Add 100 mg  | mg/mL)  | mg/mL)  | mg/mL) | mg/mL) |       |       |
| to a tube.   |         |         |        |        |       |       |
| Add the      |         |         |        |        |       |       |
| first volume |         |         |        |        |       |       |
| of solvent.  |         |         |        |        |       |       |
| Dilute with  |         |         |        |        |       |       |
| subsequent   |         |         |        |        |       |       |
| volumes if   |         |         |        |        |       |       |
| necessary.)  |         |         |        |        |       |       |
+--------------+---------+---------+--------+--------+-------+-------+
| C            |         |         |        |        | 1     | 2,000 |
| oncentration |         |         |        |        | 0,000 | μg/mL |
| of Test      |         |         |        |        | μg/mL |       |
| Chemical     |         |         |        |        |       | (2.0  |
| (Add 20 mg   |         |         |        |        | (10   | m     |
| to a tube.   |         |         |        |        | m     | g/mL) |
| Add the      |         |         |        |        | g/mL) |       |
| first volume |         |         |        |        |       |       |
| of solvent.  |         |         |        |        |       |       |
| . Dilute     |         |         |        |        |       |       |
| with         |         |         |        |        |       |       |
| subsequent   |         |         |        |        |       |       |
| volume if    |         |         |        |        |       |       |
| necessary.)  |         |         |        |        |       |       |
+--------------+---------+---------+--------+--------+-------+-------+
| If test      |         |         |        |        |       |       |
| chemical is  |         |         |        |        |       |       |
| insoluble in |         |         |        |        |       |       |
| DMSO at 2000 |         |         |        |        |       |       |
| μg/mL, then  |         |         |        |        |       |       |
| attempt to   |         |         |        |        |       |       |
| dissolve     |         |         |        |        |       |       |
| chemical in  |         |         |        |        |       |       |
| ethanol.     |         |         |        |        |       |       |
| Actual       |         |         |        |        |       |       |
| volume of    |         |         |        |        |       |       |
| solution can |         |         |        |        |       |       |
| be           |         |         |        |        |       |       |
| determined   |         |         |        |        |       |       |
| after test   |         |         |        |        |       |       |
| chemical is  |         |         |        |        |       |       |
| dissolved    |         |         |        |        |       |       |
| and solution |         |         |        |        |       |       |
| is measured  |         |         |        |        |       |       |
| using a      |         |         |        |        |       |       |
| calibrated   |         |         |        |        |       |       |
| instrument   |         |         |        |        |       |       |
| (e.g.,       |         |         |        |        |       |       |
| mi           |         |         |        |        |       |       |
| cropipettor, |         |         |        |        |       |       |
| or           |         |         |        |        |       |       |
| serological  |         |         |        |        |       |       |
| pipette).    |         |         |        |        |       |       |
| The actual   |         |         |        |        |       |       |
| stock        |         |         |        |        |       |       |
| c            |         |         |        |        |       |       |
| oncentration |         |         |        |        |       |       |
| can be       |         |         |        |        |       |       |
| calculated   |         |         |        |        |       |       |
| accordingly. |         |         |        |        |       |       |
+--------------+---------+---------+--------+--------+-------+-------+

> If the test chemical does not dissolve in Chemical Dilution
> Medium/Routine Culture Medium, DMSO, or ethanol, at 2 mg/mL, then
> repeat the entire solubility procedure with each solvent (in the order
> of Chemical Dilution Medium/Routine Culture Medium, DMSO, and ethanol)
> using the dilution steps in **Table 1B** and mixing steps a) through
> e) in **Section 6.2.1.1**.[^8]

Table 1B: Further Determination of Solubility in Chemical Dilution
Medium/Routine Culture Medium, DMSO, or Ethanol^4^

+---------------------+---------+--------+---------+--------+--------+
| # Step              | 6       | 7      | 8       | 9      | 10     |
+---------------------+---------+--------+---------+--------+--------+
| Total Volume of     | 5 mL    | 10 mL  | 20 mL   | 40 mL  | 100 mL |
| Solvent             |         |        |         |        |        |
+---------------------+---------+--------+---------+--------+--------+
| Concentration of    | 1,000   | 500    | 250     | 125    | 50     |
| Test Chemical (Add  | μg/mL   | μg/mL  | μg/mL   | μg/mL  | μg/mL  |
| 5 mg to a tube. Add |         |        |         |        |        |
| the first volume of | (1      | (0.5   | (0.25   | (0.125 | (0.05  |
| solvent. Dilute     | mg/mL)  | mg/mL) | mg/mL)  | mg/mL) | mg/mL) |
| with subsequent     |         |        |         |        |        |
| volumes if          |         |        |         |        |        |
| necessary.)         |         |        |         |        |        |
+---------------------+---------+--------+---------+--------+--------+
| If test chemical is |         |        |         |        |        |
| insoluble in medium |         |        |         |        |        |
| at 50 μg/mL, then   |         |        |         |        |        |
| attempt to dissolve |         |        |         |        |        |
| chemical in DMSO    |         |        |         |        |        |
| and then ethanol.   |         |        |         |        |        |
| Actual volume of    |         |        |         |        |        |
| solution can be     |         |        |         |        |        |
| determined after    |         |        |         |        |        |
| test chemical is    |         |        |         |        |        |
| dissolved and       |         |        |         |        |        |
| solution is         |         |        |         |        |        |
| measured using a    |         |        |         |        |        |
| calibrated          |         |        |         |        |        |
| instrument. The     |         |        |         |        |        |
| concentration can   |         |        |         |        |        |
| be calculated       |         |        |         |        |        |
| accordingly.        |         |        |         |        |        |
+---------------------+---------+--------+---------+--------+--------+

> ~~Approximately~~ ~~200~~ ~~mg (200,000 μg)~~[^9] ~~of the test
> chemical will be weighed into a glass tube and the weight will be
> documented. Assay-specific culture media will be added to the vessel
> so that the concentration is~~ ~~2,000,000 μg/ml (2000~~ ~~mg/ml)~~^2^
> ~~(i.e., approximately 0.1 ml). If complete solubility is achieved,
> then additional solubility procedures are not needed. If only partial
> solubility is achieved, follow the test chemical dissolving steps in
> Table 1, derived from EPA (1998), to add additional medium in steps
> until the concentration is a minimum of~~ ~~200,000 μg/ml (200~~
> ~~mg/ml)~~^2^~~. If complete solubility at 100,000 μg/ml in culture
> medium can't be attained, then repeat the solubility steps using the
> other solvent(s) in the solubility hierarchy. Test chemicals that are
> only soluble in DMSO or ethanol will be prepared at~~ ~~2,000,000
> μg/ml~~^2^ ~~as the highest concentration of stock solution.~~
>
> ***~~Table 1: Determination of Solubility~~***

+------------------------------------+----------+----------+----------+
| **~~Solubility Data~~**            | **~~Step | **~~Step | **~~Step |
|                                    | 1~~**    | 2~~**    | 3~~**    |
+------------------------------------+----------+----------+----------+
| ~~Total volume of medium added     | ~~0.1~~  | ~~0.5~~  | ~~1.0~~  |
| (ml)~~                             |          |          |          |
|                                    | ~~0.1~~  | ~~0.     | ~~       |
| ~~Total volume of DMSO or ethanol  |          | 5~~[^10] | 1.0~~^2^ |
| added (ml)~~                       | ~~≥~~    |          |          |
|                                    | ~~2,000, | ~~400,   | ~~200,   |
| ~~Approximate solubility (μg/ml)~~ | 000~~^2^ | 000~~^2^ | 000~~^2^ |
+------------------------------------+----------+----------+----------+

#### 

#### ~~6.2.1.1 Treatment Medium/Routine Culture Medium)~~

f)  ~~Dissolve test chemical in Treatment Medium and Routine Culture
    Medium~~

g)  ~~Gently mix. Vortex for~~ ~~1-2 minutes.~~^2^

h)  ~~If test chemical hasn't dissolved, use sonication (up to five
    minutes).~~

i)  ~~If sonication doesn't work, then warm solution to 37°C.~~

#### ~~6.2.1.2 DMSO~~ 

> ~~If the test chemical doesn't dissolve in the Treatment
> Medium/Routine Culture Medium, then follow steps a) through d) in
> **Section 6.2.1.1** using DMSO instead of Treatment Medium/Routine
> Culture Medium.~~

#### ~~6.2.1.3 Ethanol~~ 

> ~~If the test chemical doesn't dissolve in DMSO, then follow steps a)
> through d) in **Section 6.2.1.1** using ethanol instead of DMSO.~~

### 6.2.2 pH of Solutions

> Measure the pH (using pH paper) of the highest concentration of test
> chemical dissolved in the culture media. Document the pH and note the
> color of each test chemical concentration in medium.

# 7.0 DATA COLLECTION

###### 

## 7.1 Nature of Data to be Collected

#### 7.1.1 Solubility Studies

> The Contractor shall record all information pertinent to the
> solubility of the test chemical;

a)  Approximate t^3^est chemical solubility in all solvents tested
    (i.e., media, DMSO, and/or ethanol) in weight per unit volume (i.e.
    mg/mL) estimated by following the step-wise solubility protocol
    ~~culture medium at a minimum of~~ ~~200,000~~[^11] ~~μg/ml~~[^12]

b)  pH of test chemical in culture medium; color of culture medium

c)  ~~Test chemical solubility in DMSO or ethanol at~~ ~~2,000,000~~^2^
    ~~μg/ml~~^3^

d)  Need of vortexing, sonication, and/or heating

> The Contractor shall provide this information to the Study Management
> Team via the Project Officer by the avenues described in Section 8.
> **This information shall NOT be provided to the Testing Facilities.**
> Information to be provided to the Testing Facilities is specified in
> Sections 5.1.5 and 5.1.6.

#### 7.1.2 Chemical Information

> The Contractor shall supply at a minimum the following information
> about each test chemical and report as specified in Addendum I.

a)  Purity

b)  CAS \#

c)  Supplier

d)  Specification sheets

e)  Certificates of analysis

f)  Material Safety Data Sheet (MSDS)

g)  Color

h)  Odor

i)  Physical state

j)  Weight or volume of sample distributed to the Testing Facility

k)  Specific density for liquid test chemicals

l)  Storage instructions

m)  Chemical hazards

n)  Special handling instructions

o)  Amount of material archived

> \[Note: Much of the information will be in the MSDS.\]

## 7.2 Type of Media Used for Data Storage

> Originals of the raw data (the Study Workbook) and copies of other raw
> data such as instrument logs shall be collected and archived at the
> end of the study (under the direction of the Study Director),
> according to GLP-compliant procedures. Data that are stored
> electronically shall be periodically copied, and backup files shall be
> produced and maintained.

## 7.3 Documentation

> Original raw data that shall be collected shall include but are not
> limited to the following:

-   Data recorded in the Study Workbook, which shall consist of all
    recordings of all activities related to acquisition, preparation,
    solubility testing, and distribution of the test chemicals;

-   Other data collected as part of GLP compliance

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-   Equipment logs

-   Equipment calibration records

# 8.0 DRAFT AND FINAL REPORTs

***[Biweekly Reports]{.underline}***: The Contractor will provide a
biweekly progress report to the Project Officer and copied to the
Project Coordinators of the Study Management Team (See **Section 4.2**
and Addendum I). These reports will include raw and interim data as the
study progresses. These reports will be in electronic format (i.e.,
email with Microsoft^^ Word (or equivalent) or Excel attachments).

> []{.mark}

***[Draft Reports]{.underline}***: A draft report shall be submitted to
the Project Officer for each Validation Study phase (See **Section 4.2**
and Addendum I). A Draft Final Report detailing the Contractor's
involvement in all phases of the Validation Study shall be prepared by
the Contractor, signed by the Study Director, and provided to the
Project Officer. The submitted results shall accurately describe all
methods used for generation and analysis of the data, provide a complete
record of the preparation of test chemicals, and present any relevant
data necessary for the assessment of the results (See Addendum I).

***[Final Report]{.underline}***: The Draft Final Report shall be
revised according to comments from the Project Officer and submitted as
the Final Report (**See Section 4.2** and Addendum I).

# 9.0 RECORDS AND ARCHIVES

At the conclusion of the Contractor's participation in the distribution
of chemicals for the Validation Study, the original raw and derived
data, as well as copies of other raw data not exclusive to this
Validation Study (instrument logs, calibration records, facility logs,
etc.), shall be submitted to NIEHS/NICEATM (via the Project Officer) for
storing and archiving according to the facility\'s SOP and in compliance
with GLP Standards.

Originals of all raw and derived data, or copies where applicable, shall
be stored and archived at NIEHS/NICEATM.

# 10.0 ALTERATIONS OF THE statement of work

No changes in the Statement of Work shall be made without the consent of
the Project Officer and Study Management Team. A Statement of Work
Amendment detailing any change(s) and the basis for the change(s) shall
be approved and prepared by the Study Director, and the amendment shall
be signed and dated by the Study Director and the NIEHS representative.
The amendment shall be retained with the original Statement of Work.

# 11.0 REFERENCES

Clonetics Normal Human Keratinocyte Systems Instructions for Use,
AA-1000-4-Rev.03/00. (http://www.clonetics.com).

EPA Product Properties Test Guidelines. OPPTS 830.7840. 1998. Water
Solubility: Column Elution Method; Shake Flask Method. United States
Environmental Protection Agency. Prevention, Pesticides and Toxic
Substances (7101). EPA 712-C-98-041. March 1998.

National Toxicological Program, September 2000, Attachment 2 revised.
Specifications for the Conduct of Studies to Evaluate the Toxic and
Carcinogenic Potential of Chemical, Biological and Physical Agents in
Laboratory Animals for the National Toxicology Program (NTP).

NICEATM (The National Toxicology Program \[NTP\] Interagency Center for
the Evaluation of Alternative Toxicological Methods). 2001. Test Method
Protocol for the BALB/c 3T3 Neutral Red Uptake Cytotoxicity Test. A Test
for Basal Cytotoxicity for an *In Vitro* Validation Study.

NICEATM (The National Toxicology Program \[NTP\] Interagency Center for
the Evaluation of Alternative Toxicological Methods). 2001. Test Method
Protocol for the Normal Human Keratinocyte \[NHK\] Neutral Red Uptake
Cytotoxicity Test. A Test for Basal Cytotoxicity for an *In Vitro*
Validation Study.

ICCVAM (Interagency Coordinating Committee on the Validation of
Alternative Methods). 2001. Guidance document on using *in vitro* data
to estimate *in vivo* starting doses for acute toxicity NIH publication
01-4500. NIEHS, Research Triangle Park, North Carolina.

OECD (Organisation for Economic Co-operation and Development). 2001.
Harmonised Integrated Classification System for Human Health and
Environmental Hazards of Chemical Substances and Mixtures as Endorsed by
the 28^th^ Joint Meeting of the Chemicals Committee and the Working
Party on Chemicals in November 1998, Part 2, p. 21. OECD, Paris.
<http://www.oecd.org/ehs/class/HCL6htm>.

# 12.0 APPROVAL OF Statement of work

**\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_
\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_**

> **Sponsor Representative Date**

**\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_
\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_**

> **Testing Facility Management Date**

# ADDENDUM I

Suggested Report Format

**[TITLE PAGE]{.underline}**

-   **Study Title**

> Draft Report 1: *Acquisition, Preparation, Solubility Testing, and
> Distribution of Test Chemicals: Phase I of the In Vitro Validation
> Study*
>
> Draft Report 2: *Acquisition, Preparation, Solubility Testing, and
> Distribution of Test Chemicals: Phase II of the In Vitro Validation
> Study*
>
> Draft Report 3: *Acquisition, Preparation, Solubility Testing, and
> Distribution of Test Chemicals: Phase III of the In Vitro Validation
> Study*
>
> Draft/Final Report: *Acquisition, Preparation, Solubility Testing, and
> Distribution of Test Chemicals: Final Report for the In Vitro
> Validation Study*

-   **Test Articles**

> Draft Report 1: Identify the positive control chemical of Phase Ia and
> the three (3) test chemicals of Phase Ib

Draft Report 2: Identify the nine (9) test chemicals of Phase II

Draft Report 3: Identify the sixty (60) test chemicals of Phase III

Draft/Final Report: Identify all seventy-two (72) test chemicals and
positive control of the *In Vitro* Validation Studies

-   **Authors**

-   **Study Completion Date**

-   **Contract Facility**

-   **Study Number/Identification**

**[SIGNATURE PAGE]{.underline}**

-   **Study Initiation Date:** Date Statement of Work was signed

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-   **Initiation Date of Laboratory Studies:** Actual laboratory start
    date

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-   **Study Completion Date:** Date report signed by Study Director

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-   **Sponsor Representative:**

> Ms. Molly Vallant -- Project Officer
>
> The National Institute of Environmental Health Sciences (NIEHS)

-   **Study Management Team Representatives**

Judy Strickland, Ph.D. (Project Coordinator)

Michael Paris (Assistant Project Coordinator)

-   **Contractor Facility:** Name and address

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```
-   **Archive Location:** Name and address

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```
-   **Study Director:** Name and signature and date

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```
-   **Key Personnel:** Laboratory technicians, QA Director, Safety
    Officer

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```
-   **Facility Management:** Name

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```
-   **Scientific Advisor:** Name

**ADDENDUM I (cont.)**

**DRAFT REPORT 1**

*Acquisition, Preparation, Solubility Testing, and Distribution of Test
Chemicals: Phase I of the In Vitro Validation Study*

-   **[Table of Contents]{.underline}**

-   **[Objectives]{.underline} :**The report shall provide specific
    objectives

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```
-   **[Summary of the Findings]{.underline}:** Referenced to the raw
    data where appropriate; Include all information for the positive
    control (SLS) and the three (3) Phase Ib chemicals.

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```
-   **[Narrative Description of the Solubility Studies]{.underline}:**
    Describe any problems that were encountered and how such problems
    were solved. Justifications for solvents used for each test chemical
    will be included in the description. Provide the information
    requested in **Section 7.1.1**. Deviations from the protocols, SOPs,
    and/or the Statement of Work shall be addressed in this section.
    Copies of appropriate sections of the Study Workbook shall be
    included with the report (as attachments). The draft report will
    include unaudited Study Workbook pages. The final report will
    include a copy of the audited Study Workbook with a statement
    (signed and dated by the Study Director) on the front of it stating
    that it is an exact copy of the original audited workbook.

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```
-   **[Statement Signed by the Study Director]{.underline}:** Confirm
    that the solubility studies, acquisition, preparation, and
    distribution of the test chemicals were conducted in compliance with
    GLP (or indicating where the Study deviated from GLP). Confirm that
    the report fully and accurately reflects the raw data generated in
    the Study.

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```
-   **[Other Information]{.underline}:** (All copies of documents will
    be noted as exact duplicates of the data.)

-   Information requested in **Section 7.1.2**

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```
-   Deviations to the protocols, SOPs, and Statement of Work

-   Revisions/amendments to the protocols, SOPs, and Statement of Work

**DRAFT REPORT 2**

*Acquisition, Preparation, Solubility Testing,* *and Distribution of
Test Chemicals: Phase II of the In Vitro Validation Study*

-   **[Table of Contents]{.underline}**

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```
-   **[Objectives]{.underline}:** The report shall provide specific
    objectives

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```
-   **[Summary of the Findings]{.underline}:** Referenced to the raw
    data where appropriate; Include all information for the nine (9)
    Phase II chemicals.

```{=html}
<!-- -->
```
-   **[Narrative Description of the Solubility Studies]{.underline}:**
    Describe any problems that were encountered and how such problems
    were solved. Justifications for solvents used for each test chemical
    shall be included in the description. Provide the information
    requested in **Section 7.1.1**. Deviations from the protocols, SOPs,
    and/or the Statement of Work shall be addressed in this section.
    Copies of appropriate sections of the Study Workbook shall be
    included with the report (as attachments). The draft report will
    include unaudited Study Workbook pages. The final report will
    include a copy of the audited Study Workbook with a statement
    (signed and dated by the Study Director) on the front of it stating
    that it is an exact copy of the original audited workbook.

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```
-   **[Statement Signed by the Study Director]{.underline}**: Confirm
    that the solubility studies, acquisition, preparation, and
    distribution of the test chemicals were conducted in compliance with
    GLP (or indicating where the Study deviated from GLP). Confirm that
    the report fully and accurately reflects the raw data generated in
    the Study.

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<!-- -->
```
-   **[Other Information]{.underline}:** (All copies of printouts,
    documents, and spreadsheets shall be noted as exact duplicates of
    the data.)

-   Information requested in **Section 7.1.2**

```{=html}
<!-- -->
```
-   Deviations to the protocols, SOPs, and Statement of Work

-   Revisions/amendments to the protocols, SOPs, and Statement of Work

**ADDENDUM I (cont.)**

**DRAFT REPORT 3**

*Acquisition, Preparation, Solubility Testing,* *and Distribution of
Test Chemicals: Phase III of the In Vitro Validation Study*

-   **[Table of Contents]{.underline}**

-   **[Objectives]{.underline}:** The report shall provide specific
    objectives

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```
-   **[Summary of the Findings]{.underline}:** Referenced to the raw
    data where appropriate; Include all information for sixty (60) Phase
    III chemicals.

```{=html}
<!-- -->
```
-   **[Narrative Description of the Solubility Studies]{.underline}:**
    Describe any problems that were encountered and how such problems
    were solved. Justifications for solvents used for each test chemical
    will be included in the description. Provide the information
    requested in **Section 7.1.1**. Deviations from the protocols, SOPs,
    and/or the Statement of Work shall be addressed in this section.
    Copies of appropriate sections of the Study Workbook shall be
    included with the report (as attachments). The draft report will
    include unaudited Study Workbook pages. The final report will
    include a copy of the audited Study Workbook with a statement
    (signed and dated by the Study Director) on the front of it stating
    that it is an exact copy of the original audited workbook.

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```
-   **[Statement Signed by the Study Director]{.underline}:** Confirm
    that the solubility studies, acquisition, preparation, and
    distribution of the test chemicals were conducted in compliance with
    GLP (or indicating where the Study deviated from GLP). Confirm that
    the report fully and accurately reflects the raw data generated in
    the Study.

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```
-   **[Other Information]{.underline}:** (All copies of printouts,
    documents, and spreadsheets shall be noted as exact duplicates of
    the data.)

-   Information requested in **Section 7.1.2**

```{=html}
<!-- -->
```
-   Deviations to the protocols, SOPs, and Statement of Work

-   Revisions/amendments to the protocols, SOPs, and Statement of Work

**DRAFT/FINAL REPORT**

*Acquisition, Preparation, Solubility Testing,* *and Distribution of
Test Chemicals: Draft/Final Report for the In Vitro Validation Study*

-   **[Table of Contents]{.underline}**

-   **[Objectives]{.underline}:** The draft/final report shall provide
    specific objectives

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```
-   **[Summary of the Findings]{.underline}:** Referenced to the raw
    data where appropriate; Include all information for the
    seventy-two (72) test chemicals and the positive control (SLS).

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```
-   **[Narrative Description of the Solubility Studies]{.underline}:**
    Describe any problems that were encountered and how such problems
    were solved. Justifications for solvents used for each test chemical
    shall be included in the description. Provide the information
    requested in **Section 10.1.1**. Deviations from the protocols,
    SOPs, and/or the Statement of Work shall be addressed in this
    section. Copies of appropriate sections of the Study Workbook shall
    be included with the report (as attachments). The draft report will
    include unaudited Study Workbook pages. The final report will
    include a copy of the audited Study Workbook with a statement
    (signed and dated by the Study Director) on the front of it stating
    that it is an exact copy of the original audited workbook.

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<!-- -->
```
-   **[Statement Signed by the Study Director]{.underline}:** Confirm
    that the acquisition, preparation, solubility studies, and
    distribution of the test chemicals were conducted in compliance with
    GLP (or indicating where the Study deviated from GLP). Confirm that
    the report fully and accurately reflects the raw data generated in
    the Study.

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```
-   **[Quality Assurance Statement: (For Final Report
    only)]{.underline}**

> QA Statement identifying: 1) the phases and data inspected, 2) dates
> of inspection, and 3) dates findings were reported to the Study
> Director and Testing Facility management. The QA Statement shall
> identify whether the methods and results described in the Final Report
> accurately reflect the raw data produced during the Study.

-   **[Other Information]{.underline}:** (All copies of printouts,
    documents, and spreadsheets shall be noted as exact duplicates of
    the data.)

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```
-   Deviations to the protocols, SOPs, and Statement of Work

-   A list of all SOPs used by the laboratory (SOP title and laboratory
    identification code)

-   The Statement of Work

**ADDENDUM I (cont.)**

**BIWEEKLY REPORTS**

**Contract Facility:**

**Chemicals Acquired:**

**Chemicals Tested for Solubility:**

**Results of Solubility Tests:**

**Chemicals Shipped to Testing Facilities:**

**Date of Shipping:**

**Problems Encountered/Resolutions:**

**Projected Shipping Schedule:**

# ADDENDUM II

**SUGGESTED Standard Test Reporting Template for Study WORKBOOK**

[^13]SOLUBILITY TESTING

**Test Chemicals for the *In Vitro* Validation Study**

Study No.\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

Test Chemical\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ Test Chemical
Code\_\_\_\_\_\_\_\_\_\_ CAS #\_\_\_\_\_\_\_\_\_\_\_\_

Physical
Description\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_
Liquid Density\_\_\_\_\_\_\_\_\_

Solubility Determined
by\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_
Date\_\_\_\_\_\_\_\_\_\_\_\_\_\_

+--------+------+------+-----+------+-----------+-------------------+
| S      | Am   | Vo   | To  | pH   | Vortex    | Comments          |
| olvent | ount | lume | tal | and  | (V)       |                   |
|        | of   | A    | Vol | me   |           |                   |
|        | Test | dded | ume | dium | S         |                   |
|        | Chem |      |     | c    | onication |                   |
|        | ical |      |     | olor | (S)       |                   |
|        |      |      |     |      |           |                   |
|        |      |      |     |      | Heati     |                   |
|        |      |      |     |      | ng-37^o^C |                   |
|        |      |      |     |      | (H)       |                   |
+--------+------+------+-----+------+-----------+-------------------+
| Tre    |      | 0    |     |      |           |                   |
| atment |      | .1ml |     |      |           |                   |
| Medium |      |      |     |      |           |                   |
|        |      |      |     |      |           |                   |
| (3T3   |      |      |     |      |           |                   |
| NRU)   |      |      |     |      |           |                   |
+--------+------+------+-----+------+-----------+-------------------+
|        |      | 0    |     |      |           |                   |
|        |      | .5ml |     |      |           |                   |
+--------+------+------+-----+------+-----------+-------------------+
|        |      | 1    |     |      |           |                   |
|        |      | .0ml |     |      |           |                   |
+--------+------+------+-----+------+-----------+-------------------+
| R      |      | 0    |     |      |           |                   |
| outine |      | .1ml |     |      |           |                   |
| C      |      |      |     |      |           |                   |
| ulture |      |      |     |      |           |                   |
| Medium |      |      |     |      |           |                   |
|        |      |      |     |      |           |                   |
| (NHK   |      |      |     |      |           |                   |
| NRU)   |      |      |     |      |           |                   |
+--------+------+------+-----+------+-----------+-------------------+
|        |      | 0    |     |      |           |                   |
|        |      | .5ml |     |      |           |                   |
+--------+------+------+-----+------+-----------+-------------------+
|        |      | 1    |     |      |           |                   |
|        |      | .0ml |     |      |           |                   |
+--------+------+------+-----+------+-----------+-------------------+
| DMSO   |      | 0    |     |      |           |                   |
|        |      | .1ml |     |      |           |                   |
+--------+------+------+-----+------+-----------+-------------------+
|        |      |      |     |      |           |                   |
+--------+------+------+-----+------+-----------+-------------------+
|        |      |      |     |      |           |                   |
+--------+------+------+-----+------+-----------+-------------------+
| E      |      | 0    |     |      |           |                   |
| thanol |      | .1ml |     |      |           |                   |
+--------+------+------+-----+------+-----------+-------------------+
|        |      |      |     |      |           |                   |
+--------+------+------+-----+------+-----------+-------------------+
|        |      |      |     |      |           |                   |
+--------+------+------+-----+------+-----------+-------------------+

Reference Color of Treatment
Medium\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

Reference Color of Routine Culture
Medium\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

Balance I.D.\_\_\_\_\_\_\_\_\_\_\_\_\_\_

Treatment Medium and Routine Culture Medium: minimum concentration of
100mg/ml.

DMSO and Ethanol: minimum concentration of 1000mg/ml.

# ADDENDUM III

**Test chemicals for the *in vitro* validation study**

**alphabetical**

***[\[Note: testing facilities MUST not see this list of
chemicals\]]{.underline}***

  ----------------------------------- -----------------------------------
  **CHEMICAL**                        **CAS NO.**

  1,1,1-Trichloroethane               71-55-6

  2-Propanol                          67-63-0

  5-Aminosalicylic acid               89-57-6

  Acetaminophen                       103-90-2

  Acetonitrile                        75-05-8

  Acetylsalicylic acid                50-78-2

  [^14]                               

  Aminopterin                         54-62-6

  Amitriptyline HCl[^15]              549-18-8^3^

  Arsenic III trioxide                1327-53-3

  Atropine sulfate monohydrate^3^     73791-47-6^3^

  Boric aid                           10043-35-3

  Busulphan                           55-98-1

  Cadmium II chloride                 10108-64-2

  Caffeine                            58-08-2

  Carbamazepine                       298-46-4

  Carbon tetrachloride                56-23-5

  Chloral hydrate                     302-17-0

  Chloramphenicol                     56-75-7

  Citric Acid                         77-92-9

  Colchicine                          64-86-8

  Cupric sulfate \* 5 H2O             7758-99-8

  Cycloheximide                       66-81-9

  Dibutylphthalate                    84-74-2

  Dichlorvos (DDVP)                   62-73-7

  Diethyl phthalate                   84-66-2

  Digoxin                             20830-75-5

  Dimethylformamide                   68-12-2

  Diquat                              2764-72-9

  Disulfoton                          298-04-4

  Endosulfan                          115-29-7

  Epinephrine bitartrate              51-42-3

  Ethanol                             64-17-5

  Ethylene glycol                     107-21-1

  Fenpropathrin                       39515-41-8

  Gibberellic acid                    77-06-5

  Glutethimide                        77-21-4

  Glycerol                            56-81-5

  Haloperidol                         52-86-8

  Hexachlorophene                     70-30-4

  Lactic acid                         50-21-5

  Lindane                             58-89-9
  ----------------------------------- -----------------------------------

**ADDENDUM III (cont.)**

  ----------------------------------- -------------------------------------------------------------------------
  **CHEMICAL**                        **CAS NO.**

  Lithium I carbonate[^16]            554-13-2^3^

  Meprobamate                         57-53-4

  Mercury II chloride                 7487-94-7

  Methanol                            67-56-1

  Nicotine                            54-11-5

  Paraquat                            1910-42-5,
                                      (*3765-78-4,57593-74-5,65982-50-5,136338-65-3,205105-68-6,247050-57-3*)

  Parathion                           56-38-2

  Phenobarbital                       50-06-6

  Phenol                              108-95-2

  Phenylthiourea                      103-85-5

  Physostigmine[^17]                  57-47-6^1^

  Potassium cyanide                   151-50-8

  Potassium I chloride                7447-40-7

  Procainamide HCl^3^                 614-39-1^3^

  Propranolol HCl                     318-98-9, (*3506-09-0, 146874-86-4*)

  Propylparaben                       94-13-3

  Sodium arsenite                     7784-46-5

  Sodium chloride                     7647-14-5

  Sodium dichromate dihydrate         7789-12-0

  Sodium hypochlorite                 8007-59-8, (*7681-52-9*)

  Sodium I fluoride                   7681-49-4

  Sodium oxalate                      62-76-0

  Sodium selenate^1^                  13410-01-0^1^

  Strychnine                          57-24-9

  Thallium I sulfate                  7446-18-6

  Trichloroacetic acid                76-03-9

  Triethylene melamine                51-18-3

  Triphenyltin hydroxide              76-87-9

  Valproic acid                       99-66-1

  Verapamil HCl                       152-11-4

  Xylene                              1330-20-7
  ----------------------------------- -------------------------------------------------------------------------

# ADDENDUM IV

**Test chemicals for the *in vitro* validation study**

**by STUDY phase**

**Phase ia**

  ----------------------------------- -----------------------------------
  Sodium laurel sulfate               151-21-3

  ----------------------------------- -----------------------------------

**Phase ib**

  ----------------------------------- -----------------------------------
  Arsenic III trioxide                1327-53-3

  Ethylene glycol                     107-21-1

  Propranolol HCl                     318-98-9, (*3506-09-0,
                                      146874-86-4*)
  ----------------------------------- -----------------------------------

**Phase iI**

  ----------------------------------- -----------------------------------
  Aminopterin                         54-62-6

  Chloramphenicol                     56-75-7

  Colchicine                          64-86-8

  Cupric sulfate \* 5 H2O             7758-99-8

  Lithium I carbonate[^18]            554-13-2^3^

  Potassium I chloride                7447-40-7

  2-Propanol                          67-63-0

  Sodium I fluoride                   7681-49-4

  Sodium selenate[^19]                13410-01-0^1^
  ----------------------------------- -----------------------------------

**Phase iII**

  ----------------------------------- -----------------------------------
  1,1,1-Trichloroethane               71-55-6

  5-Aminosalicylic acid               89-57-6

  Acetaminophen                       103-90-2

  Acetonitrile                        75-05-8

  Acetylsalicylic acid                50-78-2

  ^1^                                 

  Amitriptyline HCl^3^                549-18-8^3^

  Atropine sulfate monohydrate^3^     73791-47-6^3^

  Boric aid                           10043-35-3

  Busulphan                           55-98-1

  Cadmium II chloride                 10108-64-2

  Caffeine                            58-08-2

  Carbamazepine                       298-46-4

  Carbon tetrachloride                56-23-5

  Chloral hydrate                     302-17-0

  Citric Acid                         77-92-9

  Cycloheximide                       66-81-9

  Dibutylphthalate                    84-74-2

  Dichlorvos (DDVP)                   62-73-7

  Diethyl phthalate                   84-66-2

  Digoxin                             20830-75-5

  Dimethylformamide                   68-12-2

  Diquat                              2764-72-9

  Disulfoton                          298-04-4

  Endosulfan                          115-29-7

  Epinephrine bitartrate              51-42-3
  ----------------------------------- -----------------------------------

**ADDENDUM IV (cont.)**

PHASE III (cont.)

  ----------------------------------- -------------------------------------------------------------------------
  Ethanol                             64-17-5

  Fenpropathrin                       39515-41-8

  Gibberellic acid                    77-06-5

  Glutethimide                        77-21-4

  Glycerol                            56-81-5

  Haloperidol                         52-86-8

  Hexachlorophene                     70-30-4

  Lactic acid                         50-21-5

  Lindane                             58-89-9

  Meprobamate                         57-53-4

  Mercury II chloride                 7487-94-7

  Methanol                            67-56-1

  Nicotine                            54-11-5

  Paraquat                            1910-42-5,
                                      (*3765-78-4,57593-74-5,65982-50-5,136338-65-3,205105-68-6,247050-57-3*)

  Parathion                           56-38-2

  Phenobarbital                       50-06-6

  Phenol                              108-95-2

  Physostigmine[^20]                  57-47-6^1^

  Phenylthiourea                      103-85-5

  Potassium cyanide                   151-50-8

  Procainamide HCl[^21]               614-39-1^3^

  Propylparaben                       94-13-3

  Sodium arsenite                     7784-46-5

  Sodium chloride                     7647-14-5

  Sodium dichromate dihydrate         7789-12-0

  Sodium hypochlorite                 8007-59-8, (*7681-52-9*)

  Sodium oxalate                      62-76-0

  Strychnine                          57-24-9

  Thallium I sulfate                  7446-18-6

  Trichloroacetic acid                76-03-9

  Triethylene melamine                51-18-3

  Triphenyltin hydroxide              76-87-9

  Valproic acid                       99-66-1

  Verapamil HCl                       152-11-4

  Xylene                              1330-20-7
  ----------------------------------- -------------------------------------------------------------------------

# ADDENDUM V

**Assumptions for Calculation of Amount of TEST MATERIAL NEEDED FOR EACH
testing Facility**

  ---------------- ------- ------- ------------ ------- ---------------- ------- ------- -------
                                   **Chemical           **Assumption**                   
                                   Amount**                                              

  **Phase I**                                                                            

  Test in 3                        300          mg      Chemical must be                 
  solvents                                              tested in all 3                  
                                                        solvents                         

  Test in 3                        300                  3 replicate                      
  replicate assays                                      assays must be                   
                                                        performed                        

  Repeat 3 times                   300                  3 replicate                      
                                                        assays must be                   
                                                        repeated 3 times                 

  **Phase I                        900          mg                                       
  Amount/Testing                                                                         
  Facility**                                                                             

                                                                                         

  x 3 Testing                      2700                 Assumes 3 labs                   
  Facilities                                            participate in                   
                                                        study                            

  2 Archive                        1200                 Archive samples                  
  samples (3                                            use same amount                  
  solubility + 3                                        of chemical as                   
  assays)                                               testing sample                   

  **Total Phase I                  3900         mg                                       
  Amount**                                                                               

                                                                                         

  **Phase II**                                                                           

  Test in 3                        300          mg      Chemical must be                 
  solvents                                              tested in all 3                  
                                                        solvents                         

  Test in 3                        300                  3 replicate                      
  replicate assays                                      assays must be                   
                                                        performed                        

  Repeat 2 times                   200                  2 replicate                      
                                                        assays must be                   
                                                        repeated 3 times                 

  **Phase II                       800          mg                                       
  Amount/Testing                                                                         
  Facility**                                                                             

                                                                                         

  x 3 Testing                      2400                 Assumes 3 labs                   
  Facilities                                            participate in                   
                                                        study                            

  2 Archive                        1200                 Archive samples                  
  samples (3                                            use same amount                  
  solubility + 3                                        of chemical as                   
  assays)                                               testing sample                   

  **Total Phase II                 3600         mg                                       
  Amount**                                                                               

                                                                                         

  **Phase III**                                                                          

  Test in 3                        300          mg      Chemical must be                 
  solvents                                              tested in all 3                  
                                                        solvents                         

  Test in 3                        300                  3 replicate                      
  replicate assays                                      assays must be                   
                                                        performed                        

  **Phase III                      600          mg                                       
  Amount/Testing                                                                         
  Facility**                                                                             

                                                                                         

  x 3 Testing                      1800                 Assumes 3 labs                   
  Facilities                                            participate in                   
                                                        study                            

  2 Archive                        1200                 Archive samples                  
  samples (3                                            use same amount                  
  solubility + 3                                        of chemical as                   
  assays)                                               testing sample                   

  **Total Phase                    3000         mg                                       
  III Amount**                                                                           
  ---------------- ------- ------- ------------ ------- ---------------- ------- ------- -------

Specification of 4 g of chemical per Testing Facility in **Section
5.1.3** was chosen to allow a generous amount of error (in the direction
of the Testing Facilities being provided with more chemical than
necessary) in the calculations and assumptions made here.

***\[This Page Intentionally Left Blank\]***

[^1]: ^3^ Revised 9/17/02

[^2]: ^2^ Revised 6/21/02

[^3]: ^2^ Revised 6/21/02

[^4]: ^2^ Revised 6/21/02

[^5]: ^3^ Revised 9/17/02

[^6]: ^2^ Revised 6/21/02

[^7]: ^3^ Section 6.2.1 replaced 9/17/02

[^8]: ^4^ Added 10/11/02

[^9]: ^2^ Revised 6/21/02

[^10]: ^2^ Revised 6/21/02

[^11]: ^2^ Revised 6/21/02

[^12]: ^3^ Revised 9/17/02

[^13]: Adaptation of Institute of In Vitro Sciences (IIVS) form -- 350
    \[2/2002\]

[^14]: ^1^ Revised 5/23/02

[^15]: ^3^ Revised 9/17/02

[^16]: ^3^ Revised 9/17/02

[^17]: ^1^ Revised 5/23/02

[^18]: ^3^ Revised 9/17/02

[^19]: ^1^ Revised 5/23/02

[^20]: ^1^ Revised 5/23/02

[^21]: ^3^ Revised 9/17/02