8.0 3T3 AND NHK NRU TEST METHOD DATA QUALITY 8-3

8.1 Adherence to Good Laboratory Practice Guidelines 8-3

> 8.1.1 Guidelines Followed for *In Vitro* NRU Cytotoxicity Testing 8-3
>
> 8.1.2 Quality Assurance (QA) for *In Vitro* NRU Cytotoxicity Test Data
> 8-5
>
> 8.1.3 Guidelines Followed for *In Vivo* Rodent Oral LD~50~ Data
> Collection 8-7

8.2 Results of Data Quality Audits 8-8

> 8.2.1 QA Statements 8-8
>
> 8.2.2 QA Statements from the Laboratories 8-9

8.3 Impact of Deviations from GLPs/Non-compliance 8-12

> 8.3.1 Laboratory Error Rates 8-12
>
> 8.3.2 Test Failure Rates for Definitive Tests and PC Tests 8-13
>
> 8.3.3 Intralaboratory Reproducibility 8-15
>
> 8.3.4 Globally Harmonized System Toxicity Category Predictions 8-15

8.4 Availability of Laboratory Notebooks 8-16

8.5 Summary 8-17

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

**8.0 3T3 AND NHK NRU** **TEST METHOD DATA QUALITY**

This section of the BRD presents the extent of adherence to national and
international GLP guidelines during for generation of the NICEATM/ECVAM
validation study data. Data quality is described along with any
deviations from the guidelines and the impact of any noncompliance.
Statistical results are provided to show comparison of data generation,
collection, and reporting of the two GLP adherent cytotoxicity testing
laboratories and the one non-GLP adherent cytotoxicity testing
laboratory as well as the GLP laboratory that distributed the reference
substances and performed solubility studies. Discussions of various
quality assurance aspects of the study are included.

**8.1 Adherence to Good Laboratory Practice Guidelines**

8.1.1 [Guidelines Followed for *In Vitro* NRU Cytotoxicity
Testing]{.underline}

*Good Laboratory Practices*

The SOW provided the following definition of U.S. Regulatory agency GLPs
to each laboratory:

> *"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 and
> control articles, and validation study protocol, and conduct (U.S.
> Food and Drug Administration, Title 21 CFR Part 58; U.S. Environmental
> Protection Agency, Title 40 CFR Part 160)."*

IIVS, ECBC, and BioReliance performed testing under all GLP guidelines.
The details of GLP compliance and training are addressed in **Section
11**.

*Spirit of GLP*

The SMT determined a definition for "spirit of GLP" and provided the
following verbiage to the laboratories:

> *"Laboratories that are non GLP-compliant shall adhere to GLP
> principles and other method parameters as put forth in this Statement
> of Work and the Test Method Protocols (provided by NIEHS/NICEATM);
> documentation and accountability shall be equal to GLP requirements;
> laboratories must make assurances that they are equal in performance
> criteria and that there is parity amongst the laboratories."*

FAL performed testing in the "spirit of GLP" (see **Section 11.2.2**) by
following the international GLP standards referenced in the ECVAM
Workshop 37 Report (Cooper-Hannan 1999) and the OECD Principles of GLP
(OECD 1998). The laboratory did not have data and test method procedures
reviewed by an independent quality assurance (QA) auditor. At a minimum,
the SOW directed FAL to routinely document the following laboratory
tasks (e.g., equipment monitoring) and record keeping (see **Table
8-1**) and to archive the documents. The FAL laboratory already had most
of the following procedures and guidelines in place for routine
laboratory procedures before initiation of this study. The various
general laboratory-related activities were documented in workbooks and
logbooks and the information was made available to the SMT.

Table 8-1 SMT-Recommended Documentation for FAL Laboratory

+----------------------+-----------------------+-----------------------+
| **Daily**            | **Per Use**           | **Periodic**          |
+----------------------+-----------------------+-----------------------+
| [Tempe               | [Cryogenic Storage    | [Laboratory           |
| ratures]{.underline} | Unit]{.underline}     | Sup                   |
|                      |                       | plies^1^]{.underline} |
| Laboratory,          | Liquid N~2~ volume    |                       |
| incubators, water    |                       | Lot numbers and       |
| baths,               |                       | expiration dates for  |
| refrigerators,       |                       | stock media           |
| freezers             |                       | formulations and      |
|                      |                       | components, NRU       |
|                      |                       | reagents, tissue      |
|                      |                       | culture plasticware   |
+----------------------+-----------------------+-----------------------+
| [Humidit             | [Equipment            | [Cells]{.underline}   |
| y/CO~2~]{.underline} | Cal                   |                       |
|                      | ibration]{.underline} | Quantity and          |
| Cell culture         |                       | cryogenic storage     |
| incubators           | Balances, pH meters,  | conditions for 3T3    |
|                      | and cell counters     | and NHK cells         |
+----------------------+-----------------------+-----------------------+
| [Visual              | [                     | [Equipment            |
| Obser                | Reagents]{.underline} | Cal                   |
| vations]{.underline} |                       | ibration]{.underline} |
|                      | Lot numbers and       |                       |
| Cell Culture Growth  | expiration dates of   | Incubators, laminar   |
|                      | medium/supplements    | flow hoods,           |
|                      |                       | autoclaves,           |
|                      |                       | micropipettors,       |
|                      |                       | spectrophotometer     |
|                      |                       | plate readers,        |
|                      |                       | computers (software)  |
+----------------------+-----------------------+-----------------------+

^1^Periodic documentation for laboratory supplies occurs when supplies
are purchased and received in the laboratory

*Good Cell Culture Practices (GCCP)*

The SMT provided guidance in the SOW for implementing GLPs in a cell
culture laboratory environment. The initial assumption by the SMT was
that each laboratory had the basic cell culture skills and knowledge
(e.g., as described in Freshney 2000) to perform the NRU cytotoxicity
test methods in a reliable manner. Reviews of historical documents and
scientific and professional exchanges with the laboratory personnel
assured the SMT that each laboratory had demonstrated, through previous
validation studies and other scientific endeavors, that personnel were
capable of providing quality scientific data through the use of good
cell culture practices. A comparison of the SOW and the *in vitro* NRU
cytotoxicity protocols to the ECVAM Good Cell Culture Practices (GCCP)
Reports (Hartung 2002; Coecke et al. 2005) and the OECD document on GLPs
and *in vitro* studies (OECD 2004a) showed that the guidelines in place
for the NICEATM/ECVAM study were harmonious with the ECVAM and OECD
guidelines.

8.1.2 [Quality Assurance (QA) for *In Vitro* NRU Cytotoxicity Test
Data]{.underline}

*Coded Reference Substances*

BioReliance acquired 73 high purity chemicals (72 reference substances
and one positive control chemical at 99% or greater purity when
economically feasible) from reputable commercial sources according to
the SOW provided by the SMT (see **Appendix G**). Seven reference
substances were less than 99% pure (three less than 98% pure; lactic
acid had the lowest purity \[89%\]). The substances were coded with
unique identification numbers and provided to the testing laboratories
in a blinded fashion*.* Preparation of substances for distribution was
performed under GLP guidelines. **Section 3.6** provides detailed
information concerning acquisition and distribution of reference
substances.

*Solubility Testing* *and Data Review*

All laboratories performed solubility tests on all reference substances
using the solvents and procedures specified by the protocols provided by
the SMT and submitted solubility data as hard copy printouts and
electronic worksheets. The laboratories also maintained solubility data
in their workbooks. The Study Directors reviewed all laboratory
procedures and all data produced at their respective laboratories. The
QA designee reviewed all data in the GLP-adherent laboratories. The SMT
Project Coordinators served as informal QA reviewers for the FAL (i.e.,
reviewed all raw data sheets). Detection of errors and omissions were
reported to FAL and corrections were requested. The SMT reviewed all
solubility data and all NRU assay data produced by all laboratories for
this study.

The SMT reviews of submitted data in Phases Ia and Ib revealed that even
after data review by the Study Directors, data files contained an
unacceptable high frequency of errors (see **Section 2.6.3**). The
laboratories were alerted to the problem and personnel from all the
laboratories attended a weeklong training session to enhance
harmonization among the laboratories. After the training, errors were
still found in data files submitted for Phase III, albeit less
frequently; such errors generally occurred due to the rapid submission
of data files to the SMT shortly after the conclusion of each test. The
formal QA review of the files occurred later in each phase of the study.

Errors included typographical mistakes, transcriptional and data entry
errors in the Microsoft^®^ EXCEL^®^ and the GraphPad PRISM^®^ 3.0
templates, and incorrect labeling of files. The SMT reviewed every
electronic file and hard copy printout throughout the study and alerted
the Study Directors when errors were found. All data files were checked
for consistency within the documents and for compliance with the
protocols. The SMT also documented errors on the hard copy printouts as
handwritten notations and included these notations in the electronic
data summary files compiled for data management. Files that were revised
and/or corrected by the Study Director were resubmitted to the SMT and
noted as corrected files.

*In Vitro NRU Cytotoxicity Test Tallies*

Periodically, the laboratories received individualized test tallies from
NICEATM that detailed:

-   the number of range finder tests performed

-   the number of definitive tests performed and the pass/fail status of
    each test

-   the number of positive control assays performed and the pass/fail
    status of each test

-   the number of acceptable tests completed per the SMT and protocol
    requirements

-   the status of test completion for each substance (i.e., whether one
    range finder test and three acceptable definitive tests had been
    completed for the substance)

The laboratories compared the NICEATM tallies to their own records to
verify consistency and accuracy. Discrepancies were resolved through
direct communication between the Study Director and the SMT.

8.1.3 [Guidelines Followed for *In Vivo* Rodent Oral LD~50~ Data
Collection]{.underline}

The *in vitro* NRU cytotoxicity test methods are proposed as methods to
predict starting doses for acute oral lethality *in vivo* (specifically,
rat) assays and not as replacement tests for an *in vivo* reference
method. No *in vivo* tests were performed for this validation study. All
*in vivo* data (i.e., rodent \[rat and mouse\] LD~50~ values) were
collected by NICEATM through reviews of the literature. All data and
pertinent information were gathered and stored in a spreadsheet
database.

*Rodent Acute Oral LD~50~ Values Used in the Registry of Cytotoxicity
(RC)*

The RC rodent (rat and mouse) acute oral LD~50~ values came largely from
the 1983/84 RTECS database (compiled by NIOSH). The RC is a database of
acute oral LD~50~ values for rats and mice obtained from RTECS^®^ and
IC~50~ values from *in vitro* cytotoxicity assays using multiple cell
lines and cytotoxicity endpoints for chemicals with known molecular
weights (Halle 1998). Collection and reporting methods used for
generating the data were not a part of any data collection hierarchy
employed by the NIOSH. The data in the RTECS database were not evaluated
for quality and accuracy by NIOSH. Many sources of the values come from
secondary references with no citation for the original report. GLP
guidelines for acute oral toxicity testing were not part of any criteria
for determining acceptable data for the database. The only criterion the
NIOSH used for reporting acute oral toxicity data in RTECS was that the
LD~50~ value was the most toxic LD~50~ value for a chemical that could
be found in the literature.

*Rodent Acute Oral LD~50~ Values Collected by NICEATM*

One critical aspect of the study design was the establishment of a rat
acute oral LD~50~ reference value for each of the 72 reference
substances (see **Section 4**). These reference values were used to
evaluate the extent to which the two *in vitro* test methods can predict
rat acute oral LD~50~ values. Primary rat acute oral LD~50~ studies were
located through searching electronic databases, published literature,
and secondary references. Rat data were not available for three of the
reference substances and, for these, mouse acute oral LD~50~ values were
collected. Very little data collected from the literature were produced
under GLP guidelines; in fact, only seven of the 455 LD~50~ values
collected were obtained under GLP conditions.

**8.2 Results of Data Quality Audits**

The QA unit or designee of each GLP laboratory provided a systematic and
critical comparison of the data provided in the study report to the raw
data in the laboratory records. The SOW provided to each laboratory
contained the following guidance on QA statements:

> *"The Final Reports for all phases of the Validation Study shall be
> audited by the Quality Assurance unit of the Testing Facility for GLP
> compliance and a QA Statement shall be provided by the Testing
> Facility. Each 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 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
> Validation Study."*

8.2.1 [QA Statements]{.underline}

The QA statements from the GLP-compliant laboratories noted the QA
reviews of:

-   protocols

-   laboratory standard operating procedures (SOPs)

-   laboratory operations

-   3T3 and NHK NRU experiment data

-   final report

The QA statements report that the test methods described in the
protocols are the methods that the laboratory personnel used and that
the data reported to the SMT is an accurate reflection of the raw data
obtained by the laboratory. See **Section 8.2.2** for information about
the QA statements for the non-GLP laboratory.

8.2.2 [QA Statements from the Laboratories]{.underline}

*BioReliance QA Statements*

The Study Director/Laboratory Director provided the following statement
in all of the final reports from BioReliance:

> *"The solubility studies, acquisition, preparation, and distribution
> of the test chemicals were conducted in compliance with GLP. Although
> not audited (per SOW), the work described in this report for Phase X
> (i.e., Ia, Ib, and II) fully and accurately reflects to the best of my
> knowledge the raw data generated in the study."*

*FAL QA Statements*

The Study Director for the FAL laboratory performed the final review of
all data and reports before sending to the SMT and provided two
statements in the final reports (provided to the SMT).

-   *"The laboratory worked under the principles of GLP whilst not being
    a GLP-compliant laboratory."*

-   *"The report accurately reflects the work undertaken and the results
    obtained at the FRAME Alternatives Laboratory."*

Since the SMT performed QA reviews of the FAL as an informal reviewer,
formal QA statements were not provided to FAL.

*ECBC QA Statements*

The QA statements reported what particular study phase and which
laboratory procedures were examined for compliance with GLP guidelines.
In addition, the statement reiterated that the scope of work, associated
protocols, and quality control acceptance criteria were updated/changed
during the study which made it more difficult to assess the procedures
and data for conformance to the protocols. However, during the review of
SOPs and the observance of operations, the requirements and intent of
GLP guidelines were continually assessed. The QA reviews found the ECBC
protocols to be in compliance with the NICEATM/ECVAM study protocols.
The phases of the studies inspected by the QA designee were as follows:

-   review of protocols and laboratory SOPs

-   review of waste handling

-   review of laboratory operations

-   certification of new personnel

-   review of data

-   review of the final report for each phase

The QA designee also observed preparation of reference substances,
96-well plate configuration, application of reference substance,
annotation to the workbook, and appropriate sterile technique while
performing the testing. The number of inspections of laboratory
operations were reduced in the latter phases of the validation study
since the same personnel conducted the testing throughout the entire
study.

[ECBC Review Dates of Various Aspects of the Study]{.underline}

-   Phase Ia: July 2002 through May 2003

-   Phase Ib: July 2002 through January 2003

-   Phase II: May 2003 through February 2004

-   Phase III: November 2003 through March 2005

*IIVS QA Statements*

Because the IIVS QA unit is small, it carried out reviews in phases. The
IIVS QA Statement reads:

> "This study has been divided into a series of in-process phases. Using
> a random sampling approach, Quality Assurance monitors each of these
> phases over a series of studies. Procedures, documentation, equipment
> records, etc., are examined to assure that the study is performed in
> accordance with the U.S. FDA Good Laboratory Practice regulations (21
> CFR 58), the U.S. EPA GLP Standards (40 CFR 792 and 40 CFR 160) and
> the OECD Principles of Good Laboratory Practice and to assure that the
> study is conducted according to the protocol and relevant Standard
> Operating Procedures."

The phases of the studies inspected by the QA designee were as follows:

-   protocol and initial paperwork

-   reading of the plates (definitive assay)

-   dilution of the test articles (definitive assay)

-   termination of treatment and addition of the NR dye (definitive
    assay)

-   cell concentration determination and seeding of the plates (third
    definitive)

-   termination of treatment and addition of the NR dye

-   washing the cells

-   treatment of the cells

-   draft report and data

-   final report

[IIVS Review Dates of Various Aspects of the Study]{.underline}

-   Phase Ia: August 2002 Final Report Review: October 2005

-   Phase Ib: January 2003 Final Report Review: October 2005

-   Phase II: July-August 2003 Final Report Review: October 2005

-   Phase III: January-November 2004 Final Report Review: October 2005

*Other QA Information*

Data generated by the laboratories and reviewed by their respective
Study Directors were provided directly to the SMT. Often, the data were
provided electronically within days of the end of testing. The SMT was
very active as a secondary QA reviewer concerning all information
provided by the Study Directors. If the SMT found discrepancies, then
the Project Coordinators corresponded with the appropriate Study
Director to rectify the mistake. The Study Director made
corrections/adjustments to any discrepancies in data reporting and
presented any changes to the SMT. The SMT did not initiate any external
data quality audits.

The quality of the reference substances was assured in the form of
certificates of analysis provided by the chemical manufacturer to
BioReliance at the time of purchase. The SMT and the laboratories
obtained certificates of analysis from CAMBREX specifically for
Clonetics^®^ NHK culture medium and supplements. In addition, the SMT
obtained quality control data directly from CAMBREX technical
departments for determining the NHK medium's ability to support
keratinocyte growth.

3.  **Impact of Deviations from GLPs/Non-compliance**

Several error rates were determined by the SMT in regard to
documentation, testing methods, and data manipulation by the
laboratories. Many errors (particularly in Phases Ia and Ib) were minor
mistakes (e.g., typographical, mislabeling) and did not affect the
quality of the data.

8.3.1 [Laboratory Error Rates]{.underline}

During Phases Ia and Ib, the SMT was concerned about the number of
errors in documentation and testing methods and compiled the number of
detected errors from each laboratory. The types of errors noted and
compiled included errors in documentation (e.g., reference substance
identification did not match on all associated data sheets, IC~20~ and
IC~80~ values were switched in the EXCEL^®^ template, a test acceptance
criterion flag in data sheet was incorrect, etc.) and in testing (e.g.,
wrong dilution scheme was used for the PC, wrong SLS IC~50~ was used as
the PC IC~50~, etc.). Error rates were compiled as number of tests with
errors per total number of tests. As shown in **Table 2-3**, FAL had the
highest error rates: 93% for the 3T3 assay and 41% for the NHK assay.
The highest error rates of the other laboratories were 10% for the 3T3
assay and 23% for the NHK assay (both ECBC).

There were very few errors detected in the Phase III data files. The SMT
did not compile typographical and transcriptional errors but reported
the errors directly to the appropriate Study Director so that the data
sheets could be immediately rectified. The SMT did not detect errors in
the raw optical density data from the 96-well plates provided in each
data file. The laboratories and the SMT corrected any typographical and
transcriptional errors (e.g., incorrect logIC~50~ value entered) in the
EXCEL^®^ templates. The template formulas calculated the correct values
for the statistical analyses and the quality of the data was not
compromised.

For Phase III, assessment of error rates was performed specifically for
Phase III for one particular clerical error -- the transfer of
statistical results (e.g., ICx values) from the GraphPad PRISM^®^ 3.0
template to the Microsoft^®^ EXCEL^®^ template. It was often necessary
for the SMT to revise the Microsoft^®^ EXCEL^®^ data files provided by
the laboratories because the incorrect values had been transferred to
the template. The SMT revised files (using the data in the PRISM^®^ 3.0
template) due to this error and reports as follows as the number of
errors/total number of definitive tests:

Table 8-2 Error Rates

  ----------------- ----------------- ----------------- -----------------
  **Laboratory**    **Number of       **Number of       **Percentage of
                    Errors            Definitive        Tests with
                    Detected^1^**     Tests**           Detected Errors**

  ECBC              49                402               12

  FAL               171               513               33

  IIVS              25                419               6
  ----------------- ----------------- ----------------- -----------------

^1^Clerical error -- transfer of statistical results from PRISM^®^ to
EXCEL^®^

8.3.2 [Test Failure Rates for Definitive Tests and PC Tests]{.underline}

**Table 8-3** illustrates the test failure rates experienced for Phase
III of the validation study. Approximately 25% of all 3T3 definitive
tests and 18% of all NHK definitive tests failed (i.e., did not meet
test acceptance criteria). If a definitive test (see **Section 2.2.2**
for the definition of a definitive test) failed, then the laboratory
repeated the test and attempted to reach the goal of three acceptable
definitive tests for each reference substance and each cell type (see
**Section 2.5** for criteria for repeating tests). PC failure occurred 0
-- 18% of the time with an overall average failure rate of 8% combined
for both assays. FAL had the highest individual laboratory test failure
rates for 3T3 definitive tests (30%), NHK definitive tests (32%), and
NHK PC tests (18%). ECBC had the highest failure rate for 3T3 PC tests
(11%).

Phase III guidelines called for each laboratory to provide three
acceptable definitive tests for each substance for both cell types (3 x
60 x 2 = 360 definitive tests). PC tests were run concurrently with the
definitive tests and generally more than one reference substance was
tested in conjunction with one PC test plate. Due to test failures, each
laboratory performed additional testing to attempt to obtain the three
acceptable definitive tests requested for each substance.

Table 8-3 Definitive Test and Positive Control (PC) Test Failure Rates

<table>
<colgroup>
<col style="width: 30%" />
<col style="width: 8%" />
<col style="width: 7%" />
<col style="width: 7%" />
<col style="width: 7%" />
<col style="width: 8%" />
<col style="width: 6%" />
<col style="width: 7%" />
<col style="width: 7%" />
<col style="width: 7%" />
</colgroup>
<tbody>
<tr class="odd">
<td rowspan="2"><h5 id="test-type">Test Type</h5></td>
<td colspan="4"><strong>3T3 NRU Test Method</strong></td>
<td colspan="4"><strong>NHK NRU Test Method</strong></td>
<td rowspan="2"><strong>Total</strong></td>
</tr>
<tr class="even">
<td><strong>ECBC</strong></td>
<td><strong>FAL</strong></td>
<td><strong>IIVS</strong></td>
<td><strong>Total</strong></td>
<td><strong>ECBC</strong></td>
<td><strong>FAL</strong></td>
<td><strong>IIVS</strong></td>
<td><strong>Total</strong></td>
</tr>
<tr class="odd">
<td>Definitive Tests - Acceptable</td>
<td>169</td>
<td>177</td>
<td>176</td>
<td>522</td>
<td>173</td>
<td>175</td>
<td>174</td>
<td>522</td>
<td>1044</td>
</tr>
<tr class="even">
<td>Definitive Tests - Total</td>
<td>215</td>
<td>257</td>
<td>225</td>
<td>697</td>
<td>187</td>
<td>256</td>
<td>194</td>
<td>637</td>
<td>1334</td>
</tr>
<tr class="odd">
<td>% Definitive Tests Failed</td>
<td>21</td>
<td>30</td>
<td>22</td>
<td>25</td>
<td>8</td>
<td>32</td>
<td>10</td>
<td>18</td>
<td>22</td>
</tr>
<tr class="even">
<td>PC Tests - Acceptable</td>
<td>66</td>
<td>40</td>
<td>16</td>
<td>122</td>
<td>58</td>
<td>37</td>
<td>20</td>
<td>115</td>
<td>237</td>
</tr>
<tr class="odd">
<td>PC Tests - Total</td>
<td>74</td>
<td>42</td>
<td>17</td>
<td>133</td>
<td>59</td>
<td>45</td>
<td>20</td>
<td>124</td>
<td>257</td>
</tr>
<tr class="even">
<td>% PC Tests Failed</td>
<td>11</td>
<td>5</td>
<td>6</td>
<td>8</td>
<td>2</td>
<td>18</td>
<td>0</td>
<td>7</td>
<td>8</td>
</tr>
<tr class="odd">
<td><p>Definitive Tests Failed Only</p>
<p>Because PC Tests Failed</p></td>
<td>14</td>
<td>6</td>
<td>14</td>
<td>34</td>
<td>0</td>
<td>22</td>
<td>0</td>
<td>22</td>
<td>56</td>
</tr>
<tr class="even">
<td>% Definitive Tests Failed Only Because PC Tests Failed</td>
<td>7</td>
<td>2</td>
<td>6</td>
<td>5</td>
<td>0</td>
<td>9</td>
<td>0</td>
<td>4</td>
<td>4</td>
</tr>
</tbody>
</table>

**Table 8-4** illustrates the success rates of the testing for each
laboratory and for the combined laboratories.

Table 8-4 Definitive Test and PC Test Success Rates for 3T3 and NHK NRU
Test Methods (Combined Total Tests)

  ---------------------- ------------ ------------ ------------ ------------
  **Test Type**          **ECBC**     **FAL**      **IIVS**     **Total**

  Acceptable Definitive  342/402      352/513      350/419      1044/1334
  Tests/ Total                                                  
  Definitive Tests                                              

  \% Acceptable          85%          69%          84%          78%
  Definitive Tests                                              

  Acceptable PC          124/133      77/87        36/37        237/257
  Tests/Total PC Tests                                          

  \% Acceptable PC Tests 93%          89%          97%          92%
  ---------------------- ------------ ------------ ------------ ------------

8.3.3 [Intralaboratory Reproducibility]{.underline}

CV values for each reference substance were determined for each
laboratory using the IC~50~ values from the acceptable definitive tests
as described in **Section 5.3.1**. **Table 8-5** illustrates the average
CV values for the substances tested in each of the phases and for the
entire study.

Table 8-5 Coefficients of Variation

+-------+---------+--------+-------+--------+-------+--------+-------+
| *     | *       | **     |       | *      |       | **All  |       |
| *Cell | *Labs** | Phases |       | *Phase |       | Ph     |       |
| T     |         | I &    |       | III**  |       | ases** |       |
| ype** |         | II**   |       |        |       |        |       |
+-------+---------+--------+-------+--------+-------+--------+-------+
|       |         | Number | Av    | Number | Av    | Number | Av    |
|       |         | of     | erage | of     | erage | of     | erage |
|       |         | Ref    |       | Ref    |       | Ref    |       |
|       |         | erence | \% CV | erence | \% CV | erence | \% CV |
|       |         | Subs   |       | Subs   |       | Subs   |       |
|       |         | tances |       | tances |       | tances |       |
+-------+---------+--------+-------+--------+-------+--------+-------+
| **    | ECBC    | 12     | 17    | 57     | 24    | 69     | 23    |
| 3T3** |         |        |       |        |       |        |       |
+-------+---------+--------+-------+--------+-------+--------+-------+
|       | FAL     | 11     | 28    | 55     | 33    | 66     | 33    |
+-------+---------+--------+-------+--------+-------+--------+-------+
|       | IIVS    | 11     | 20    | 56     | 22    | 68     | 21    |
+-------+---------+--------+-------+--------+-------+--------+-------+
|       |         |        |       |        |       |        |       |
+-------+---------+--------+-------+--------+-------+--------+-------+
| **    | ECBC    | 12     | 24    | 57     | 22    | 69     | 23    |
| NHK** |         |        |       |        |       |        |       |
+-------+---------+--------+-------+--------+-------+--------+-------+
|       | FAL     | 12     | 31    | 57     | 45    | 69     | 42    |
+-------+---------+--------+-------+--------+-------+--------+-------+
|       | IIVS    | 12     | 14    | 58     | 14    | 70     | 14    |
+-------+---------+--------+-------+--------+-------+--------+-------+

8.3.4 [Globally Harmonized System Toxicity Category
Predictions]{.underline}

Predicted LD~50~ values were compared to the GHS *in vivo* acute oral
toxicity categories to determine category match (i.e., accuracy) or
toxicity underprediction or overprediction for the reference substances
(see **Table 8-6**). Predicted LD~50~ values were determined for the
reference substances by using the mean IC~50~ values from the
laboratories in the RC regression. The reference GHS *in vivo* acute
oral toxicity category presented in **Table 8-6** was the initial LD~50~
value used to select the substances (see **Table 3-1**). The
laboratories were generally in agreement with each other in the
predictions. Although FAL had the highest error rates and CV values,
their predictions of GHS toxicity category using these NRU methods were
consistent with the other laboratories. (See **Appendix J** for
additional laboratory comparisons for the other *in vitro -- in vivo*
regressions evaluated in **Section 6**.)

**Table 8-6 GHS Toxicity Category Predictions by Laboratory^1^**

  --------- ----------- -------------- ------------ ----------------- ------------------
            **Labs**    **Total        **Category   **Toxicity        **Toxicity
                        Reference      Match**      Overpredicted**   Underpredicted**
                        Substances**                                  

  **3T3**   ECBC        69             29%          41%               30%

            FAL         67             28%          43%               28%

            IIVS        69             28%          41%               32%

                                                                      

  **NHK**   ECBC        69             28%          42%               30%

            FAL         69             28%          41%               32%

            IIVS        70             29%          40%               31%
  --------- ----------- -------------- ------------ ----------------- ------------------

> ^1^GHS-Globally Harmonized System categories of acute oral toxicity
> with LD~50~ in mg/kg (UN 2003). 3T3 and NHK NRU test method IC50 data
> (geometric mean of within laboratory replicates) used with the RC
> regression: log(LD~50~ mmol/kg) = 0.425 x log(IC~50~ mM) +0.625.

**8.4 Availability of Laboratory Notebooks**

All laboratories maintained laboratory notebooks patterned after a
template provided by IIVS and provided copies of them to the SMT
(archived at NICEATM) after each phase. The workbooks contained
information from all aspects of testing including but not limited to:

-   environmental conditions

-   reagent identification

-   preparation of 96-well plates

-   preparation of reference substances

-   treatment of cell cultures

-   visual observations of cell cultures

-   NRU assays

-   data analysis

    5.  **Summary**

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-   Various determinations of test method and data collection errors
    consistently showed that FAL had the highest error level; however,
    the laboratory's GHS acute oral toxicity category predictions were
    comparable to the other laboratories' results. Data were not
    adversely affected by general transcriptional errors.

-   The laboratories reported no significant deviations from the test
    method protocols and deviations that did occur during the testing
    phases were generally quickly acknowledged and addressed by the
    Study Directors. If a deviation occurred that would affect data
    (e.g., improper concentration of DMSO solvent), then that Study
    Director would reject the test, notify the SMT, and perform an
    additional test. Improper transfer of data to either the EXCEL^®^ or
    PRISM^®^ templates, which would affect the data, were recognized,
    documented, and rectified by the Study Director and/or the SMT.

-   The SMT was diligent in reviewing all data sheets to ensure that
    data were not inadvertently attributed to the incorrect data summary
    files and that the correct data were used in all statistical
    analyses.

An electronic copy of all data for this validation study can be obtained
upon request from NICEATM.

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