**DRAFT ICCVAM TEST METHOD RECOMMENDATIONS**

# **In Vitro Pyrogenicity Test Methods**

**December 1, 2006**

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TABLE OF CONTENTS

Page Number

1.0 ICCVAM Draft Recommendations for *In Vitro* Pyrogenicity Test
Methods 7

1.1 Draft Recommended Test Method Uses 7

1.2 Draft Recommended Future Studies 9

Appendix A Draft Performance Standards for *In Vitro*

Pyrogenicity Test Methods 10

1.0 Purpose and Background of Performance Standards 12

1.1 Introduction 12

1.2 Elements of ICCVAM Performance Standards 13

1.3 ICCVAM Process for the Development of Performance Standards 13

1.4 ICCVAM Development of Performance Standards for *In Vitro*

Pyrogenicity Test Methods 14

1.4.1 Current Regulatory Testing Requirements for Pyrogenicity 15

1.4.2 Test Methods for Assessing Pyrogenicity 15

1.4.3 Intended Regulatory Uses for *In Vitro* Pyrogenicity Test

Methods 16

1.4.4 Similarities and Differences in the Endpoints of *In Vitro*

Pyrogenicity Test Methods and Currently Recognized

Pyrogenicity Test Methods 16

2.0 *In Vitro* Pyrogenicity Test Methods 17

2.1 Background 17

2.2 Principles of *In Vitro* Pyrogenicity Test Methods 17

2.3 Essential Test Method Components for *In Vitro* Pyrogenicity

Test Methods 18

2.3.1 *In Vitro* Cell Culture Conditions 19

2.3.2 Application of the Test Substances 20

2.3.2.1 Test Substance Preparation 20

2.3.2.2 Test Substance Application and Sample Collection 20

2.3.3 Control Substances 21

2.3.3.1 Negative Control 21

2.3.3.2 Positive Control 21

2.3.3.3 Benchmark Controls 21

2.3.4 Cytokine Measurements 22

2.3.5 Interpretation of Results 22

2.3.6 Test Report 22

2.4 Reference Substances for *In vitro* Pyrogenicity Test Methods 24

2.5 Accuracy and Reliability 25

2.5.1 Accuracy 25

2.5.2 Reliability 26

2.5.2.1 Intralaboratory Repeatability 27

2.5.2.2 Intralaboratory Reproducibility 27

2.5.2.3 Interlaboratory Reproducibility 29

**3.0 References** 31

**Appendix B Proposed *In Vitro* Pyrogenicity Test Method Protocols**
B-1

B1 Cryopreserved Whole Blood (Cryo WB)/Interleukin-1 (IL-1β) *In Vitro*
Pyrogen Test B1-1

B2 Mono Mac 6 (MM6)/Interleukin-6 (IL-6) *In Vitro* Pyrogen Test B2-1

B3 Peripheral Blood Mononuclear Cell (PBMC)/Interleukin-6 (IL-6)

*In Vitro* Pyrogen Test B3-1

B4 Whole Blood (WB)/Interleukin-1β (IL-1β) *In Vitro* Pyrogen Test B4-1

B5 Whole Blood (WB)/Interleukin -6 (IL-6) *In Vitro* Pyrogen Test B5-1

**LIST OF TABLES**

Page Number

Table 1 Performance Statistics for *In Vitro* Pyrogenicity Test Methods
8

Table 2 Test Substances (Parenteral Drugs) Used in the Validation
Studies

for Determining Test Method Performance 8

Table 1-1 Summary of U.S. and European Legislation and Statutory
Protocol Requirements for Pyrogenicity Testing 16

Table 2-1 Recommended Reference Substances for the *In Vitro*
Pyrogenicity

Test Methods 26

Table 2-2 Performance Statistics for *In Vitro* Pyrogenicity Test
Methods 27

Table 2-3 Intralaboratory Reproducibility of *In Vitro* Pyrogenicity

Test Methods 29

Table 2-4 Interlaboratory Reproducibility of *In Vitro* Pyrogenicity

Test Methods 30

Table 2-5 Interlaboratory Reproducibility of *In Vitro* Pyrogenicity

Test Methods 31

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**1.0 ICCVAM DRAFT RECOMMENDATIONS FOR *IN VITRO* PYROGENICITY TEST
METHODS**

**1.1 Draft Recommended Test Method Uses**

ICCVAM has evaluated the validation status of the following *in vitro*
test methods proposed as replacements for the *in vivo* rabbit pyrogen
test (RPT) only (i.e., not for the bacterial endotoxin test \[BET\]):

-   cryo WB/IL-1 (The Human Whole Blood/IL-1 *In Vitro* Pyrogen Test:
    Application of cryopreserved human whole blood)

-   MM6/IL6 (An Alternative *In Vitro* Pyrogen Test Using the Human
    Monocytoid Cell Line MONO MAC-6 \[MM6\])

-   PBMC/IL-6 (The Human Peripheral Blood Mononuclear Cell \[PBMC\]/IL-6
    *In Vitro* Pyrogen Test)

-   WB/IL-1 (The Human Whole Blood \[WB\]/IL-1 *In Vitro* Pyrogen Test)

-   WB/IL-6 (The Human Whole Blood/IL-6 *In Vitro* Pyrogen Test)

There is sufficient information (see **Table 1**), based on validation
studies with a limited number of pharmaceuticals (see **Table 2**), to
substantiate the use of these test methods (PBMC/IL-6, cryo WB/IL-1 \[96
well plate method\], WB/IL-6, and MM6/IL-6) for the detection of
pyrogenicity mediated by Gram-negative endotoxin in materials that are
currently tested in the RPT, subject to product-specific validation to
demonstrate equivalency[^1]^,^[^2]. While the scientific basis of these
test methods suggests that they have the capability to detect
pyrogenicity produced by a wider range of pyrogens (i.e., those mediated
by non-endotoxin sources), there is insufficient data to support this
broader application.

**Table 1 Performance Statistics for *In Vitro* Pyrogenicity Test
Methods^1^**

  ----------- -------------------- ----------------- ----------------- ------------ -----------
  **Test      **Concordance^2^**   **Sensitivity**   **Specificity**   **False      **False
  Method**                                                             Negative     Positive
                                                                       Rate**       Rate**

  PBMC/IL-6   93.3%\               92.2%\            95.0%\            7.8%\        5.0%\
              (140/150)            (83/90)           (57/60)           (7/90)       (3/60)

  cryo        91.7%\               97.4%\            81.4%\            2.6%\        18.6%\
  WB/IL-1     (110/120)            (75/77)           (35/43)           (2/77)       (8/43)

  WB/IL-6     91.9%\               88.8%\            96.6%\            11.2%\       3.4%\
              (136/148)            (79/89)           (57/59)           (10/89)      (2/59)

  MM6/IL-6    93.2%\               95.5%\            89.8%\            4.5%\        10.2%\
              (138/148)            (85/89)           (53/59)           (4/89)       (6/59)

  WB/IL-1     92.0%\               98.8%\            83.6%\            1.2%\        16.4%\
  (plate      (129/139)            (83/84)           (46/55)           (1/84)       (9/55)
  method)                                                                           
  ----------- -------------------- ----------------- ----------------- ------------ -----------

^1^Based on combined results of 10 different parenteral drugs tested in
each of three different laboratories; samples of each drug were tested
with or without being spiked with a Gram-negative endotoxin standard (0,
0.25, 0.5, 0.5, and 1.0 EU/mL).

^2^Percentage (Number of correct runs/total number of runs)

**Table 2 Test Substances (Parenteral Drugs) Used in the Validation
Studies for Determining Test Method Performance^1^**

  ----------------- ------------ ------------------ ---------------- -----------
  **Test            **Source**   **Active           **Indication**   **MVD
  Substance^2^**                 Ingredient**                        (-fold)**

  Beloc            Astra Zeneca Metoprolol         Heart            140
                                 tartrate           dysfunction      

  Binotal          Aventis      Ampicillin         Antibiotic       140

  Ethanol 13% (w/w) B. Braun     Ethanol            Diluent          35

  Fenistil         Novartis     Dimetindenmaleat   Antiallergic     175

  Glucose 5% (w/v)  Eifel        Glucose            Nutrition        70

  MCP              Hexal        Metoclopramid      Antiemetic       350

  Orasthin         Aventis      Oxytocin           Initiation of    700
                                                    Delivery         

  Sostril          GSK          Ranitidine         Antiacidic       140

  Drug A - 0.9%     \-           0.9% NaCl          \-               35
  NaCl                                                               

  Drug B - 0.9%     \-           0.9% NaCl          \-               70
  NaCl                                                               
  ----------------- ------------ ------------------ ---------------- -----------

^1^Each substance was tested in all five *in vitro* pyrogenicity test
methods.

^2^Each test substance was spiked with 0, 0.25, 0.5, 0.5, 1.0 EU/mL of
endotoxin (WHO-LPS 94/580 \[*E. coli* O113:H10:K-\]). Each sample
contained the appropriate spike concentration when tested at its Maximum
Valid Dilution (MVD).

Users should be aware that the performance characteristics for these *in
vitro* pyrogenicity test methods could be revised as additional data
become available. Therefore, test method users should consult the
ICCVAM/NICEATM website (<http://iccvam.niehs.nih.gov/>) and other
sources to obtain the most current information relevant to the current
performance and usefulness and limitations of these test methods.

**1.2 Draft Recommended Future Studies**

To further the use of these five test methods (cryo WB/IL-1, MM6/IL-6,
PBMC/IL-6, WB/IL-1 \[plate method\], and WB/IL-6) as potential
replacements for the RPT for detecting non-endotoxin pyrogens,
additional studies that include a broader range of pyrogenic materials
are recommended. For a direct comparison between the *in vitro* pyrogen
test(s) and the RPT, such studies should include parallel RPT
testing[^3].

The hazards associated with human blood products should be carefully
considered and all technical staff must be trained to observe all
necessary safety precautions.

**Appendix A** provides Draft Performance Standards for *In Vitro*
Pyrogenicity Test Methods that are based on ICCVAM guidelines (ICCVAM
2003[^4]). **Appendix B** provides five draft proposed *in vitro*
pyrogenicity test method protocols that are based on those used in the
ECVAM validation study. **Appendix B1** is the *Proposed Test Method
Protocol for the Human Whole Blood/IL-1 In Vitro Pyrogen Test:
Application of Cryopreserved Human Whole Blood.* **Appendix B2** is the
*Proposed Test Method Protocol for the In Vitro Pyrogen Test Using the
Human Monocytoid Cell Line MONO MAC-6 (MM6).* **Appendix B3** is the
*Proposed Test Method Protocol for the Human PBMC/IL-6 In Vitro Pyrogen
Test*. **Appendix B4** is the *Proposed* *Test Method Protocol for the
Human Whole Blood/IL-1 In Vitro Pyrogen Test*. **Appendix B5** is the
*Proposed Test Method Protocol for the Human Whole Blood/IL-6 In Vitro
Pyrogen Test*.

# 

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**Appendix A**

# **Draft Performance Standards for In Vitro Pyrogenicity Test Methods** 

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**1.0 PURPOSE AND BACKGROUND OF PERFORMANCE STANDARDS**

**1.1 Introduction**

Prior to the acceptance of a new test method for regulatory testing
applications, validation studies are conducted to assess its reliability
(i.e., the extent of intra- and inter-laboratory reproducibility) and
its relevance (i.e., the ability of the test method to correctly predict
or measure the biological effect of interest) (OECD 1996, 2002; ICCVAM
1997, 2003). The purpose of performance standards is to communicate the
basis by which new proprietary (i.e., copyrighted, trademarked,
registered) and nonproprietary test methods have been determined to have
sufficient relevance and reliability for specific testing purposes.
These performance standards, based on test methods accepted by
regulatory agencies, can be used to evaluate the reliability and
relevance of other test methods that are based on similar scientific
principles and measure or predict the same biological or toxic effect.
Five *in vitro* pyrogenicity test methods, cryo WB/IL-1 (The Human Whole
Blood/IL-1 *In Vitro* Pyrogen Test: Application of cryopreserved human
whole blood), MM6/IL6 (An Alternative *In Vitro* Pyrogen Test Using the
Human Monocytoid Cell Line MONO MAC-6 \[MM6\]), PBMC/IL-6 (The Human
PBMC/IL-6 *In Vitro* Pyrogen Test, WB/IL-1 (The Human Whole Blood/IL-1
*In Vitro* Pyrogen Test), and WB/IL-6 (The Human Whole Blood/IL-6 *In
Vitro* Pyrogen Test) were included in a validation study to evaluate the
correlation between *in vitro* cytokine release and the *in vivo* rabbit
fever response and the feasibility of using *in vitro* cytokine assays
to predict a pyrogenic response.

This section describes the three elements of performance standards
identified by ICCVAM (2003) and the ICCVAM process used to develop
performance standards during a test method evaluation. These test method
performance standards are proposed as standards that can be used to
evaluate future *in vitro* pyrogenicity test methods. If other *in
vitro* pyrogenicity test methods are adequately validated and
demonstrate significantly improved performance, then the test method
performance standards may be revised accordingly.

**1.2 Elements of ICCVAM Performance Standards**

Performance standards are standards based on a validated test method
that provide a basis for evaluating the comparability of a proposed test
method that is mechanistically and functionally similar (ICCVAM 2003).
The three elements of performance standards are:

-   **Essential test method components:** These consist of essential
    structural, functional, and procedural elements of a validated test
    method that should be included in the protocol of a proposed,
    mechanistically and functionally similar test method. Essential test
    method components include unique characteristics of the test method,
    critical procedural details, and quality control measures.

-   **A minimum list of reference substances:** Reference substances are
    used to assess the accuracy and reliability of a proposed,
    mechanistically and functionally similar test method. These
    substances are a representative subset of those used to demonstrate
    the reliability and the accuracy of the validated test method, and
    are the minimum number that should be used to evaluate the
    performance of a proposed, mechanistically and functionally similar
    test method.

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-   **Accuracy and reliability values:** These are the accuracy and
    reliability characteristics that the proposed test method should be
    comparable to or exceed when evaluated using the minimum list of
    reference chemicals.

**1.3 ICCVAM Process for the Development of Performance Standards**

The process followed by ICCVAM for developing performance standards for
new test methods is as follows:

-   NICEATM and the ICCVAM Pyrogenicity Working Group (PWG) develop
    proposed performance standards for consideration during the ICCVAM
    evaluation process. If performance standards are proposed by a test
    method sponsor, they will be considered by ICCVAM at this stage.
    Generally, the proposed performance standards are based on the
    information and data provided in the test method submission or on
    other available applicable data.

-   The ICCVAM/NICEATM Peer Review Panel evaluates the proposed
    performance standards for completeness and appropriateness during
    its evaluation of the validation status of the proposed test method.
    The proposed performance standards, as well as the test method
    submission, are made available to the public for comment prior to
    and during the Peer Review Panel meeting.

-   The PWG, with the assistance of NICEATM, prepares the final
    performance standards for ICCVAM approval, taking into consideration
    the recommendations of the Peer Review Panel and public comments.

Performance standards recommended by ICCVAM are incorporated into ICCVAM
test method evaluation reports, which are then provided to U.S. Federal
agencies and made available to the public. Regulatory authorities can
then reference the performance standards in the ICCVAM report when they
communicate their acceptance of a new test method. In addition,
performance standards adopted by U.S. Federal regulatory authorities can
be provided in guidelines issued for new test methods. Availability of
ICCVAM test method evaluation reports are announced routinely in the
*Federal Register*, in NTP Newsletters, and by e-mail to ICCVAM/NICEATM
listserve groups.

**1.4 ICCVAM Development of Performance Standards for *In Vitro*
Pyrogenicity Test Methods**

1.4.1 [Current Regulatory Testing Requirements for
Pyrogenicity]{.underline}

The major regulatory requirement for pyrogenicity testing is for end
product release of human and animal parenteral drugs, medical devices,
and human biological products. Results from pyrogenicity testing are
used to limit to an acceptable level the risks of febrile reaction in
the patient to injection and/or implantation of the product of concern.
The current U.S. legislation requiring the use of pyrogenicity testing
is stated in the Federal Food, Drug, and Cosmetic Act (U.S.C., Title 21,
Chapter 9). In addition, the U.S. Pharmacopeia (USP) maintains sterility
requirements for pharmaceuticals that include pyrogenicity testing. As
detailed in **Table 1-1**, the U.S. Food and Drug Administration (FDA)
is the principal U.S. regulatory agency that requires pyrogenicity
testing, with different Centers within the FDA regulating the affected
products (i.e., human and animal parenteral drugs, biological products,
and medical devices). **Table 1-1** also shows the statutory protocol
requirements used by each FDA Center, along with the comparable
international standards presently required by European Union member
nations.

Table 1-1 Summary of U.S. and European Legislation and Statutory
Protocol Requirements for Pyrogenicity Testing

  ------------- ------------------ ------------- -------------- ------------------
  Agency        Regulated Products Legislation   Statutory      Non-Governmental
                                                 Protocol       Standards
                                                 Requirements   

  United States                                                 

  FDA-CBER      Biological         \- Federal    \- 21 CFR      \- USP28
                products           Food, Drug,   610.13         NF23\<85\>\
                                   and Cosmetic                 - USP28
                                   Act (U.S.C.                  NF23\<151\>\
                                   Title 21,                    - ISO 10993-11
                                   Chapter 9)                   

  FDA-CDER      Human parenteral                                
                pharmaceuticals                                 

  FDA-CDRH      Medical devices                                 

  FDA-CVM       Veterinary                                      
                pharmaceuticals                                 

  Europe                                                        

  EDQM          Human/veterinary   \- Council    \- EP5.0       \- ISO 10993-11
                parenteral         Regulation    2.6.8\         
                pharmaceuticals,   (EEC)         - EP5.0 2.6.14 
                biological         230/9/93\                    
                products, medical  - Council                    
                devices            Directive                    
                                   93/39/EEC\                   
                                   - Council                    
                                   Directive                    
                                   93/40/EEC                    

  EMEA                                                          

  Regulatory                                                    
  Authorities                                                   
  for                                                           
  Individual EU                                                 
  Countries                                                     
  ------------- ------------------ ------------- -------------- ------------------

Abbreviations: CBER = Center for Biologics Evaluation and Research; CDER
= Center for Drug Evaluation and Research; CDRH = Center for Devices and
Radiological Health; CFR = Code of Federal Regulations; CVM = Center for
Veterinary Medicine; EDQM = European Directorate for the Quality of
Medicines; EMEA = European Medicines Agency; EP = European
Pharmacopoeia; EU = European Union; FDA = U.S. Food and Drug
Administration; US =: U.S. Pharmacopeia

1.4.2 [Test Methods for Assessing Pyrogenicity]{.underline}

The currently recognized test methods for evaluating pyrogenicity are
the *in vivo* rabbit pyrogen test (RPT) and the bacterial endotoxin test
(BET). The RPT (USP28 NF23\<151\>, EP5.0 2.6.8) involves measuring the
rise in body temperature evoked in rabbits by the intravenous injection
of a test solution. The RPT is a sequential test, using the response of
the first three rabbits tested to determine the need for additional
testing. The BET (USP28 NF23\<85\>, EP5.0 2.6.14) is used to detect or
quantify the presence of gram-negative bacterial endotoxins using
amoebocyte lysate from the horseshoe crab (*Limulus polyphemus*). There
are three different BET techniques: the gel-clot technique (based on gel
formation due to clotting of *Limulus* amoebocyte lysate \[LAL\] in the
presence of endotoxin); the turbidometric technique (based on the
development of turbidity after cleavage of an endogenous substrate); and
the chromogenic technique (based on color development resulting from
cleavage of a synthetic peptide-chromagen complex).

1.4.3 [Intended Regulatory Uses for *In Vitro* Pyrogenicity Test
Methods]{.underline}

The *in vitro* pyrogenicity test methods are not intended as
replacements for the BET. However, five of these methods (cryo WB/IL-1,
MM6/IL-6, PBMC/IL-6, WB/IL-1\[plate method\], and WB/IL-6) may be
considered for the detection of the presence of Gram-negative endotoxin
in materials that are currently tested in the RPT, subject to
product-specific validation to demonstrate equivalency.

1.4.4 [Similarities and Differences in the Endpoints of In Vitro
Pyrogenicity Test Methods and Currently Recognized Pyrogenicity Test
Methods]{.underline}

The endpoint measured in the *in vitro* pyrogenicity test methods is
cytokine release, either IL-1β or IL-6, depending on the test method
employed. The RPT involves measuring the rise in body temperature evoked
in rabbits by the intravenous injection of a test solution. While there
is not a direct association between the endpoints measured in these
assays, cytokine release is involved in the development of an
inflammatory response, which can result in an increase in temperature.
Therefore, the *in vitro* release of pro-inflammatory cytokines, such as
IL-1β and IL-6, is intended to predict the onset of such a response. The
cell types used for the various *in vitro* methods presumably are those
that would be directly associated with an inflammatory response. Both
the *in vitro* and *in vivo* tests provide quantitative endpoints.

There are different endpoints for the BET, depending on the technique
used. The gel-clot technique is based on the observation of gel
formation due to clotting of LAL in the presence of endotoxin. The
turbidometric technique evaluates the development of turbity after
cleavage of an endogenous substrate. Finally, the chromogenic technique
measures color development resulting from cleavage of a synthetic
peptide-chromogen complex. Clearly, there are no biological similarities
between the endpoints measured in the *in vitro* test methods and the
various BET techniques. However, like the *in vitro* test methods, the
turbidometric and chromogenic techniques provide quantitative
measurements, while the gel-clot technique is qualitative.

**2.0 *IN VITRO* PYROGENICITY TEST METHODS**

**2.1 Background**

Pre-validation and validation studies have been completed to evaluate
the ability of the five *in vitro* pyrogenicity test methods to be used
as alternatives to the RPT. This section briefly describes the
principles of *in vitro* pyrogenicity test methods followed by the
recommended performance standards that would be used to evaluate test
methods that are functionally and mechanistically similar to these
methods. The performance standards consist of 1) essential test method
components, 2) reference substances, and 3) the comparable accuracy and
reliability that should be achieved.

**2.2 Principles of *In Vitro* Pyrogenicity Test Methods**

Although there are differences among the *in vitro* pyrogenicity test
methods based predominantly on the cell type used, there are some basic
steps that are consistent across all methods as follows:

-   The test substance is applied to the specific human-derived cells
    used in the *in vitro* test method (i.e., mixed with a suspension of
    cells).

-   The test substance is incubated with the cells for a specified
    period of time.

-   The concentration of pro-inflammatory cytokines (e.g., IL-1β, IL-6)
    is quantified via a cytokine-specific enzyme-linked immunosorbent
    assay (ELISA) by comparison to a standard curve.

-   Using an endotoxin standard curve, the endotoxin content of the
    product is calculated.

-   A product "passes" (i.e., is considered negative for endotoxin) if
    the endotoxin content is \< 0.5 endotoxin units (EU)/mL.

While it is possible that comparable cell types derived from other
species would also be capable of detecting the presence of Gram-negative
endotoxin, one of the strengths of these test methods is that they are
derived from human tissues, and thus avoid the potential uncertainty
associated with cross-species interpretation. Therefore, test method
developers are encouraged to focus on human cell-based systems.

Investigators using an *in vitro* pyrogenicity test method for detecting
the presence of Gram-negative endotoxin must be able to demonstrate that
the assay is valid for its intended use. This includes demonstrating
that any modification to the existing validated reference test method
does not adversely affect its performance characteristics. *In vitro*
pyrogenicity test methods may be used to test pharmaceuticals,
biological products, and medical devices. Interference testing must be
included to demonstrate that the properties of the test substance do not
impede the release and detection of proinflammatory cytokines.

**2.3 Essential Test Method Components for *In Vitro* Pyrogenicity Test
Methods**

Essential test method components consist of essential structural,
functional, and procedural elements of a validated test method that
should be included in the protocol of a mechanistically and functionally
similar proposed test method. These components include unique
characteristics of the test method, critical procedural details, and
quality control measures. Adherence to these components will help assure
that a proposed test method is based on the same concepts as the
corresponding validated test method.

The following is a description of the essential test method components
for *in vitro* pyrogenicity test methods.

2.3.1 [*In Vitro* Cell Culture Conditions]{.underline}

-   A mammalian cell line, primary cells, or heparinized whole blood (as
    described above, either would preferably be of human origin) is
    used. Cryopreserved cells/whole blood may be used, where it has been
    demonstrated that cryopreservation is not detrimental to the test
    method.

-   Fresh whole blood may be stored at room temperature, but should be
    used within four hours of collection. Blood donors should be in good
    health (i.e., not suffering from bacterial or viral infections for
    at least one week prior to donation), and not taking any medications
    known to influence cytokine production (e.g., immunosuppressant or
    anti-inflammatory drugs). As an additional measure, assay acceptance
    criteria allow for the identification of low or high responders such
    that results with Blood/PBMCs from a compromised donor are omitted.

-   Where necessary, cells are propagated in sterile tissue culture
    flasks and then subcultured to sterile 96-well plates for use in
    testing. Initial cell seeding should be done at a density that
    allows rapid growth throughout the exposure period. However, cell
    density should not reach confluency by the end of the test exposure
    period.

-   Appropriate cell culture growth conditions (e.g., 37°C ± 1°C, 90% ±
    10% humidity, 5.0% ± 1% CO~2~ in ambient air) should be maintained
    throughout the testing period. The cell cultures should be free of
    contamination with bacteria, mycoplasma, or fungi.

Cell culture media should be prequalified by the testing laboratory via
a standardized protocol before initiating the test to guarantee that the
media provide cells with appropriate nutrients to meet the growth
criteria needed for the test method.

2.3.2. [Application of the Test Substances]{.underline}

2.3.2.1 *Test Substance Preparation*

-   All disposables (e.g., pipette tips, pipettes, culture ware, etc.)
    should be labeled sterile, pyrogen free.

-   Test substances (i.e., pharmaceuticals, biological products) should
    be diluted to their respective maximum valid dilution (MVD) in
    sterile, pyrogen-free 0.9% NaCl.

-   Medical devices can be directly incubated with to the cells in
    suspension. Alternatively, and if necessary, eluates/extracts from
    medical devices may be prepared with a volume of pyrogen-free water
    appropriate to their use and, where applicable, to the surface area
    that comes in contact with body tissues or fluids.

-   Each test should contain a range of concentrations of either the
    international reference standard endotoxin (i.e., WHO LPS 94/580),
    or an LPS standard that has been calibrated against this standard,
    with which to generate a standard curve. *NOTE: In the ECVAM
    validation study, concentrations of 0, 0.25, 0.5, and 1.0 endotoxin
    units (EU)/mL were used to establish the decision criteria for a
    pyrogenic response based on the in vivo rabbit threshold fever
    concentration (i.e., the concentration at which an increase in
    temperature was recorded in 50% animals tested) (see **Section
    2.3.5**).*

-   Test substances should be fully solubilized (i.e., no visual
    observation of test substance in the dosing solution).

2.3.2.2 *Test Substance Application and Sample Collection*

-   Whole blood samples may be dosed in either 96-well plates or
    microcentrifuge tubes.

-   The cells should be exposed for from 16 to 24 hours

-   Each substance should be tested in a minimum of three replicates.

-   At the end of the exposure period, supernatants may be collected
    either directly from each well, or following centrifugation for
    microcentrifuge tubes.

2.3.3 [Control Substances]{.underline}

*2.3.3.1 Negative Control*

To ensure that the test system is functioning properly and that the
specific test is valid, the negative control (i.e., 0.9% NaCl) should
not induce a significant increase in IL-1β or IL-6 release.

*2.3.3.2 Positive Control*

The purpose of a positive control chemical is to demonstrate that the
cell system is responding with adequate sensitivity to a pyrogenic
substance for which the magnitude of the pyrogenic response is well
characterized. Each test should generate a response that is comparable
to the historical range generated by the laboratory. Therefore, the
positive control should be the international reference standard
endotoxin (i.e., WHO-LPS 94/580 \[*E. coli* 0113:h10:K-\]), or an
endotoxin standard that has been calibrated against this standard. A
laboratory should perform a minimum of 10 *in vitro* pyrogenicity tests
using the positive control over a number of days to develop a minimum
historical database of cyokine data. Typically, for biologically based
test methods, suggested acceptable ranges for the positive control
response are within two to three standard deviations of the historical
mean response, but developers of proprietary test methods may establish
tighter ranges. The positive control chemical should be tested
concurrently with (and independent of) the test substance. Test
substances spiked with known quantities of the positive control should
be used for interference testing.

*2.3.3.3 Benchmark Controls*

Benchmark controls may be useful to demonstrate that the test method is
functioning properly for detecting the pyrogenic potential of chemicals
(e.g., parenterals or medical device eluates) of a specific chemical
class or a specific range of responses, or for evaluating the relative
pyrogenic potential of a test substance. Appropriate benchmark controls
should have the following properties:

-   structural and functional similarity to the class of the substance
    being tested

-   known physical/chemical characteristics

-   supporting data on known effects in animal models

-   known potency in the range of response

2.3.4 [Cytokine Measurements]{.underline}

Only standardized, quantitative methods should be used to measure
cytokine release (e.g., an enzyme immunoassay with a species-specific
antibody for the relevant proinflammatory cytokine). Each assay should
contain a range of concentrations of the relevant cytokine standard
(e.g., IL-1β, IL-6) in order to generate a standard curve for the
analytical assay. The protocol should be compatible with analytical
laboratory equipment (e.g., spectrophotometer) that allows a quick and
precise measurement of the endpoint. Colorimetric, fluorometric, or
luminometric endpoints should have the optical density (OD) measured at
the appropriate wavelength, and OD values for blanks should be
subtracted from all measurements. Each supernatant should be assayed
with a minimum of three replicates.

2.3.5 [Interpretation of Results]{.underline}

The endpoint values obtained for each test sample can be used to
calculate the level of cytokine release relative to the positive control
samples (i.e., the endotoxin standard curve). A sample is considered
positive for a pyrogenic response if the level of cytokine release is
greater than or equal to that induced by the 0.5 EU/mL endotoxin
standard, the reported threshold fever concentration for the *in vivo*
rabbit test (see **Section 2.3.2.1**).

2.3.6 [Test Report]{.underline}

The test report should include the following information, if relevant to
the conduct of the study:

*Test Substances and Control Substances*

-   Name of pharmaceutical, biological product, medical device eluate,
    etc.

-   Purity and composition of the substance or preparation

-   Physicochemical properties (e.g., physical state, water solubility)
    relevant to the conduct of the study

-   Treatment of the test/control substances prior to testing, if
    applicable (e.g., vortexing, sonication, warming; resuspension
    solvent)

*Justification of the In Vitro Test Method and Protocol Used*

*Test Method Integrity*

-   The procedure used to ensure the integrity (i.e., accuracy and
    reliability) of the test method over time

-   If the test method employs proprietary components, documentation on
    the procedure used to ensure their integrity from "lot-to-lot" and
    over time

-   The procedures that the user may employ to verify the integrity of
    the proprietary components

*Criteria for an Acceptable Test*

-   Acceptable concurrent positive control ranges based on historical
    data

-   Acceptable negative control data

*Test Conditions*

-   Cell system used

-   Calibration information for the spectrophotometer used to read the
    ELISA

-   Details of test procedure

-   Description of any modifications of the test procedure

-   Reference to historical data of the model

-   Description of evaluation criteria used

*Results*

-   Tabulation of data from individual test samples

*Description of Other Effects Observed*

*Discussion of the Results*

*Conclusion*

*A Quality Assurance Statement for Good Laboratory Practice
(GLP)-Compliant Studies*

-   This statement should indicate all inspections made during the study
    and the dates any results were reported to the Study Director. This
    statement should also confirm that the final report reflects the raw
    data

If GLP-compliant studies are performed, then additional reporting
requirements provided in the relevant guidelines (e.g., OECD 1998; EPA
2003a, 2003b; FDA 2003) should be followed.

**2.4 Reference Substances for *In Vitro* Pyrogenicity Test Methods**

Reference substances are used to assess the accuracy and reliability of
a proposed, mechanistically and functionally similar test method and are
a representative subset of those used to demonstrate the reliability and
the accuracy of the validated test method. These substances are:

-   representative of the range of responses that the validated test
    method is capable of measuring or predicting

-   have produced consistent results in the validated test method

-   reflect the accuracy of the validated test method

-   have well-defined chemical structures

-   are readily available

-   are not associated with excessive hazard or prohibitive disposal
    costs

To demonstrate technical proficiency with the validated test method, the
user should evaluate his/her ability to identify Gram-negative endotoxin
that has been spiked into each of the reference substances listed in
**Table 2-1.** These eight substances are marketed pharmaceuticals that
were tested in the ECVAM *in vitro* pyrogenicity test methods validation
study. Only released clinical lots that have been labeled as having no
detectable pyrogens should be used as reference substances to be spiked
with Gram-negative endotoxin. As indicated in **Section 2.3.3**, the
spike should be either the international reference standard endotoxin
(WHO-LPS 94/580 \[*E. coli* 0113:h10:K-\]) or an endotoxin standard that
has been calibrated against this standard. Each reference substance
should be tested clean (i.e., unspiked) and spiked with endotoxin (0.5
EU/mL).

**Table 2-1 Recommended Reference Substances for *In Vitro* Pyrogenicity
Test Methods**

  ------------------ ---------------- ----------------------- ------------
  **Test             **Source**       **Active Ingredient**   **MVD
  Substance^1^**                                              (-fold)**

  Beloc             Astra Zeneca     Metoprolol tartrate     140

  Binotal           Aventis          Ampicillin              140

  Ethanol 13% (w/w)  B. Braun         Ethanol                 35

  Fenistil          Novartis         Dimetindenmaleat        175

  Glucose 5% (w/v)   Eifel            Glucose                 70

  MCP               Hexal            Metoclopramid           350

  Orasthin          Aventis          Oxytocin                700

  Sostril           GSK              Ranitidine              140
  ------------------ ---------------- ----------------------- ------------

^1^Each reference substance should be spiked with 0.5 EU/mL of endotoxin
(WHO-LPS 94/580 \[*E. coli* O113:H10:K-\]). Each sample should contain
the appropriate spike concentration when tested at its Maximum Valid
Dilution (MVD). MVD = (endotoxin limit concentration)/(detection limit
of the assay)\
Positive Control: 0.9% NaCl spiked with 0.5 EU/mL

Negative Control: 0.9% NaCl

**2.5 Accuracy and Reliability**

The third element of the performance standards is the determination of
accuracy (also known as relevance) and reliability values. A proposed
test method, functionally and mechanistically similar to the *in vitro*
pyrogenicity test methods described above, will use selected reference
substances to assess accuracy and reliability.

2.5.1 [Accuracy]{.underline}

When evaluated using the minimum list of recommended reference
substances (**Table 2-1**), the proposed test method should have
performance characteristics that are comparable to the performance of
the validated *in vitro* pyrogenicity test methods. Accuracy is defined
as the closeness of agreement between a test method result and an
accepted reference value (ICCVAM 2003). The substances tested in the
ECVAM validation studies are included so that the performance of the
proposed test method can be determined and compared to that of the
validated reference test methods.

The accuracy of these assays to identify a pyrogenic concentration of
Gram-negative endotoxin was evaluated. This accuracy evaluation
characterizes the extent that additional test methods will be necessary
to achieve accurate *in vitro* predictions of contamination by
Gram-negative endotoxin for labeling and lot release purposes. **Table
2-2** shows that overall accuracy among the test methods is comparable
(91.7% to 93.3%), with false negative rates ranging from 1.2% to 11.2%,
and false positive rates ranging from 3.4% to 18.6%.

**Table 2-2 Performance Statistics for *In Vitro* Pyrogenicity Test
Methods^1^**

  ----------- ----------------- ----------------- ----------------- ------------ -----------
  **Test      **Accuracy^2^**   **Sensitivity**   **Specificity**   **False      **False
  Method**                                                          Negative     Positive
                                                                    Rate**       Rate**

  PBMC/IL-6   93.3%\            92.2%\            95.0%\            7.8%\        5.0%\
              (140/150)         (83/90)           (57/60)           (7/90)       (3/60)

  cryo        91.7%\            97.4%\            81.4%\            2.6%\        18.6%\
  WB/IL-1     (110/120)         (75/77)           (35/43)           (2/77)       (8/43)

  WB/IL-6     91.9%\            88.8%\            96.6%\            11.2%\       3.4%\
              (136/148)         (79/89)           (57/59)           (10/89)      (2/59)

  MM6/IL-6    93.2%\            95.5%\            89.8%\            4.5%\        10.2%\
              (138/148)         (85/89)           (53/59)           (4/89)       (6/59)

  WB/IL-1     92.8%\            98.8%\            83.6%\            1.2%\        16.4%\
  (plate      (129/139)         (83/84)           (46/55)           (1/84)       (9/55)
  method)                                                                        
  ----------- ----------------- ----------------- ----------------- ------------ -----------

^1^Based on combined results of 10 different substances tested in three
different laboratories

^2^Percentage (Number of correct runs/total number of runs)

2.5.2 [Reliability]{.underline}

Test method reliability (intralaboratory repeatability and intra- and
inter-laboratory reproducibility) is the degree to which a test method
can be performed reproducibly within and among laboratories over time
(ICCVAM 2003). Repeatability refers to the closeness of agreement
between test results obtained within a single laboratory when the
procedure is performed on the same substance under identical conditions
within a given time period. Intralaboratory reproducibility refers to
the determination of the extent to which qualified personnel within the
same laboratory can replicate results using a specific test protocol at
different times. Interlaboratory reproducibility refers to the
determination of the extent to which different laboratories can
replicate results using the same protocol and test chemicals, and
indicates the extent to which a test method can be transferred
successfully among laboratories.

The reliability of the proposed test method for the reference substances
should be comparable to or better than that of the validated *in vitro*
pyrogenicity test methods. The following sections provide these
reference statistics.

2.5.2.1 *Intralaboratory Repeatability*

In the ECVAM validation study, intralaboratory repeatability was
evaluated in each test method by testing saline and various endotoxin
spikes (0.06 to 0.5 EU/mL) in saline and evaluating the closeness of
agreement between optical density readings for cytokine measurements for
each concentration. Up to 20 replicates per concentration were tested
and results indicated that variability in measurements increased with
endotoxin concentration, but that the 0.5 EU/mL spike concentration
(i.e., the threshold for pyrogenicity for each test method) was clearly
distinguishable from lower concentrations.

2.5.2.2 *Intralaboratory Reproducibility*

Intralaboratory reproducibility was evaluated with three marketed
pharmaceuticals spiked with various concentrations of endotoxin.
Reproducibility was assessed from three identical, independent runs
conducted in each of the three testing laboratories (with the exception
of the cryo WB/IL-1 test method[^5]). From these results, agreement
between different runs was determined for each substance in three
laboratories. As shown in **Table 2-3**, the agreement across three runs
in an individual lab ranged from 75% to 100%.

**Table 2-3 Intralaboratory Reproducibility of *In Vitro*** Pyrogenicity
Test Methods

  ----------------- ------------- ------- ------- ----------- ------- ------- ------------- ------- ------- --------------- ------- ------- -------------- ------- -------
  **Run             **WB/IL-1**                   **Cryo                      **WB/IL-6**                   **PBMC/IL-6**                   **MM6/IL-6**           
  Comparison^1^**                                 WB/IL-1**                                                                                                        

                    **Lab 1**     **Lab   **Lab   **Lab 1**   **Lab   **Lab   **Lab 1**     **Lab   **Lab   **Lab 1**       **Lab   **Lab   **Lab 1**      **Lab   **Lab
                                  2**     3**                 2**     3**                   2**     3**                     2**     3**                    2**     3**

  1 vs 2            92%           100%    100%    ND          ND      ND      75%           92%     100%    92%             100%    100%    100%           92%     100%

  1 vs 3            83%           88%     92%     ND          ND      ND      100%          92%     100%    100%            100%    92%     100%           92%     92%

  2 vs 3            92%           NA      92%     ND          ND      ND      75%           100%    100%    92%             100%    92%     100%           100%    92%

  Mean              89%           \-      94%     ND          ND      ND      83%           94%     100%    94%             100%    94%     100%           94%     94%

  Agreement across  83%           \-      92%     ND          ND      ND      75%           92%     100%    92%             100%    94%     100%           92%     92%
  3 runs                                                                                                                                                           
  ----------------- ------------- ------- ------- ----------- ------- ------- ------------- ------- ------- --------------- ------- ------- -------------- ------- -------

**NA**: Not assessed due to lack of sufficient data. The sensitivity
criteria were not met for 1/3 substances in run 2, and 1/3 substances in
run 3; **ND**: Not done. The cryo WB/IL-1 test method BRD states that an
assessment of intralaboratory reproducibility was performed in the WB
IL-1 (fresh blood) test method, and it is assumed that variability is
not affected by the change to cryopreserved blood assayed in 96-well
plates.

^1^Comparison between 3 individual runs within each laboratory

2.5.2.3 *Interlaboratory Reproducibility*

Interlaboratory reproducibility was evaluated in two different studies.
In both studies, each run from one laboratory was compared with all
other runs of another laboratory. The proportion of equally classified
samples provides a measure of reproducibility. In the first study, like
the intralaboratory reproducibility evaluation, interlaboratory
reproducibility was evaluated with the three marketed pharmaceuticals
spiked with endotoxin, and tested three times in three different
laboratories. As shown in **Table 2-4**, the agreement across three
laboratories for each test method (where three runs per laboratory were
conducted) ranged from 72% to 86%, depending on the test method
considered. In comparison, the agreement across three laboratories for
the WB/IL-1 (96-well plate method) and cryo WB/IL-1 test methods, for
which only one run per laboratory was conducted, was 83% and 92%,
respectively.

**Table 2-4 Interlaboratory Reproducibility of *In Vitro*** Pyrogenicity
Test Methods

  ----------------- ------------------- ------------ -------------- --------------- --------------
  **Lab             **Agreement Between                                             
  Comparison^1^**   Laboratories^1^**                                               

                    **WB/IL-1**         **Cryo       **WB/IL-6**    **PBMC/IL-6**   **MM6/IL-6**
                                        WB/IL-1**                                   

  1 vs 2            92%\                92%\         72%\           81%\            97%\
                    (11/12)             (11/12)      (78/108)^2^    (87/108)        (105/108)

  1 vs 3            83%\                92%\         75%\           86%\            89%\
                    (10/12)             (11/12)      (81/108)^2^    (93/108)        (96/108)

  2 vs 3            92%\                92%\         97%\           89%\            86%\
                    (11/12)             (11/12)      (105/108)^2^   (96/108)        (93/108)

  Mean              89%                 92%          81%            85%             90%

  Agreement across  83%\                92%\         72%\           78%\            86%\
  3 labs^3^         (10/12)             (11/12)      (234/324)^2^   (252/324)       (279/324)
  ----------------- ------------------- ------------ -------------- --------------- --------------

^1^Data from three substances (see **Table 2-3**) spiked with endotoxin
(WHO-LPS 94/580 \[*E. coli* O113:H10:K-\]) at 0, 0, 0.5 and 1.0 EU/mL
tested three times in three different laboratories, with the exception
of the WB/IL-1 (96-well plate method) and the cryo WB/IL-1 (only the
preliminary run from each laboratory used for analysis)

^2^Some of the runs did not meet the assay acceptance criteria and
therefore were excluded from the analysis.

^3^All possible combinations of runs among the 3 laboratories were
compared (with the exception of cryo WB/IL-1, which was only tested once
in each laboratory, resulting in only one possible combination per
substance).

In a second study, reproducibility was evaluated with the same ten
substances used for evaluating accuracy. In this study, the ten
substances were spiked with five concentrations of endotoxin and tested
once in each of three laboratories. As indicated in **Table 2-5**, the
agreement across three laboratories for each test method ranged from 79%
to 88%, depending on the test method considered.

**Table 2-5 Interlaboratory Reproducibility of *In Vitro*** Pyrogenicity
Test Methods

  ----------------- ------------------- ------------ ------------- --------------- --------------
  **Lab             **Agreement Between                                            
  Comparison^1^**   Laboratories^1^**                                              

                    **WB/IL-1**         **Cryo       **WB/IL-6**   **PBMC/IL-6**   **MM6/IL-6**
                                        WB/IL-1**                                  

  1 vs 2            88%\                84%\         85%\          84%\            90%\
                    (37/42)             (38/45)      (41/48)       (42/50)         (45/50)

  1 vs 3            90%\                88%\         85%\          86%\            90%\
                    (35/39)             (21/24)      (41/48)       (43/50)         (43/48)

  2 vs 3            92%\                100%\        88%\          90%\            83%\
                    (43/47)             (25/25)      (44/50)       (45/50)         (40/48)

  Mean              90%                 91%          86%           87%             88%

  Agreement across  85%\                88%\         79%\          80%\            81%\
  3 labs            (33/39)             (21/24)      (38/48)       (40/50)         (39/48)
  ----------------- ------------------- ------------ ------------- --------------- --------------

^1^Data from 10 substances spiked with endotoxin (WHO-LPS 94/580 \[*E.
coli* O113:H10:K-\]) at 0, 0.25, 0.5, 0.5, and 1.0 EU/mL tested once in
three different laboratories

**3.0 REFERENCES**

ICCVAM. 1997. Validation and Regulatory Acceptance of Toxicological Test
Methods. A Report of the *ad hoc* Interagency Coordinating Committee on
the Validation of Alternative Methods. NIH Publication No. 97-3981.
Research Triangle Park, NC. National Institute of Environmental Health
Sciences. Available:
<http://iccvam.niehs.nih.gov/docs/guidelines/validate.pdf> \[accessed 20
July 2006\].

ICCVAM. 2003. ICCVAM Guidelines for the Nomination and Submission of
New, Revised, and Alternative Test Methods. NIH Publication No. 03-4508.
National Institute for Environmental Health Sciences, Research Triangle
Park, NC. Available: <http://iccvam.niehs.nih.gov/>. \[accessed 20 July
2006\].

OECD. 1996. Final Report of the OECD Workshop on Harmonisation of
Validation and Acceptance Criteria for Alternative Toxicological
Methods. (Solna Report) Paris: Organisation for Economic Co-operation
and Development. Available:
[http://www.oecd.org](http://www.oecd.org/EN/home/0,,EN-home-8-nodirectorate-no-no--8,00.htmlhttp://www.oecd.org)
\[accessed 20 July 2006\].

OECD. 2002. Report of the Stockholm Conference on Validation and
Regulatory Acceptance of New and Updated Methods in Hazard Assessment.
Paris: Organisation for Economic Co-operation and Development.
Available: [[http://www.oecd.org]{.underline}](http://www.oecd.org/)
\[accessed 20 July 2006\]

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

.

**Appendix B**

**Individual BRDs Submitted by ECVAM on Five *In Vitro* Pyrogenicity
Test Methods**

**B1 The Human Whole Blood (WB)/IL-1 *In Vitro* Pyrogen Test:**

**Application of Cyropreserved Human WB B1-1**

**B2 An Alternative *In Vitro* Pyrogenicity Test Using the Monocytoid**

**Cell Line Mono Mac 6 (MM6)/IL-6 B2-1**

**B3 The Human Peripheral Blood Mononuclear Cell (PBMC)/IL-6**

***In Vitro* Pyrogen Test B3-1**

**B4 The Human WB/IL-1 *In Vitro* Pyrogen Test B4-1**

**B5 The Human WB/IL-6 *In Vitro* Pyrogen Test B5-1**

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

[^1]: Equivalent methods can be regulated under 21 CFR 610.9 as
    alternatives to the currently accepted test method(s).

[^2]: There are substances other than endotoxin that may induce the
    cellular release of IL-1β and/or IL-6. For this reason, users of
    these test methods should be aware of the potential for a false
    positive result, suggesting that endotoxin is present, which
    actually is due to the presence of another pyrogenic material.

[^3]: In order to demonstrate the utility of these test methods for the
    detection of non-endotoxin pyrogens, either an international
    standard is needed (as is available for endotoxin \[i.e., WHO-LPS
    94/580 *E. coli* O113:H10:K-\]) or, when a positive
    non-endotoxin-mediated RPT result is encountered, this same sample
    should be subsequently tested *in vitro*.

[^4]: ICCVAM. 2003. ICCVAM Guidelines for the Nomination and Submission
    of New, Revised, and Alternative Test Methods. NIH Publication No.
    03-4508. National Institute for Environmental Health Sciences,
    Research Triangle Park, NC. Available:
    <http://iccvam.niehs.nih.gov/>. \[accessed 2 June 2005\].

[^5]: The cryo WB/IL-1 test method BRD states that an assessment of
    intralaboratory reproducibility was performed in the WB IL-1 (fresh
    blood) test method, and it is assumed that variability is not
    affected by the change to cryopreserved blood assayed in 96-well
    plates.