UNCONTROLLED COPY

SAM 204.06

Page 1 of 9

United States Department of Agriculture

Center for Veterinary Biologics

Testing Protocol

SAM 204

Supplemental Assay Method for Potency Testing Clostridium perfringens

Type D Epsilon Antitoxins

Date: December 13, 2017

Number: SAM 204.06

Supersedes: SAM 204.05, April 1, 2014

Standard Requirement: 9 CFR 113.455

Contact: Janet M. Wilson, (515) 337-7245

Approvals: /s/Larry R. Ludemann Date: 18Dec17

Larry R. Ludemann, Section Leader

Bacteriology

/s/Paul J. Hauer Date: 22Dec17

Paul J. Hauer, Director

Policy, Evaluation, and Licensing

Center for Veterinary Biologics

United States Department of Agriculture

Animal and Plant Health Inspection Service

P. O. Box 844

Ames, IA 50010

Mention of trademark or proprietary product does not constitute a guarantee or warranty of the product by USDA 

and does not imply its approval to the exclusion of other products that may be suitable.

Entered into CVB Quality Management System

by: /s/Linda S. Snavely 26Dec17 

 Linda S. Snavely Date

 Quality Management Program Assistant
Center for Veterinary Biologics SAM 204.06

Testing Protocol Page 2 of 9

Supplemental Assay Method for Potency Testing Clostridium perfringens Type D Epsilon Antitoxins

UNCONTROLLED COPY

Table of Contents

1. Introduction

2. Materials

2.1 Equipment/instrumentation

2.2 Reagents/supplies

2.3 Test animals

3. Preparation for the Test

3.1 Personnel qualifications/training

3.2 Preparation of equipment

3.3 Preparation of reagents

4. Performance of the Test

4.1 Toxin neutralization

4.2 Inoculation of mice

5. Interpretation of the Test Results

5.1 Criteria for a valid test

5.2 Interpretation of serial results

6. Report of Test Results

7. References

8. Summary of Revisions
Center for Veterinary Biologics SAM 204.06

Testing Protocol Page 3 of 9

Supplemental Assay Method for Potency Testing Clostridium perfringens Type D Epsilon Antitoxins

UNCONTROLLED COPY

1. Introduction

This Supplemental Assay Method (SAM) describes the method used to determine the epsilon 

antitoxin content of Clostridium perfringens type D antitoxin as prescribed by title 9, Code of

Federal Regulations (9 CFR), section 113.455. The epsilon antitoxin is titrated by a toxinantitoxin neutralization test, using mice as an indicator.

2. Materials

2.1 Equipment/instrumentation

Equivalent equipment or instrumentation may be substituted for any brand name listed 

below.

2.1.1 Vortex mixer

2.1.2 Autoclave

2.1.3 Freezer, -70oC

2.1.4 Refrigerator, 2o- 7oC

2.1.5 Micropipettes, 100-μL and 1000-μL

2.2 Reagents/supplies

Equivalent reagents or supplies may be substituted for any brand name listed below.

2.2.1 C. perfringens type D (epsilon) standard antitoxin IRP 249

2.2.2 C. perfringens type D (epsilon) standard toxin IRP 632

2.2.3 Peptone diluent

2.2.4 Glass screw-top tubes, 13 x 100-mm with caps

2.2.5 Pipettes, 1-mL, 5-mL, 10-mL, 25-mL

2.2.6 Syringes, 1-cc

2.2.7 Needles, 25- to 27-gauge x 1/2- to 1 1/4-inch

2.2.8 Screw-top Erlenmeyer flask, 500-mL with cap
Center for Veterinary Biologics SAM 204.06

Testing Protocol Page 4 of 9

Supplemental Assay Method for Potency Testing Clostridium perfringens Type D Epsilon Antitoxins

UNCONTROLLED COPY

2.2.9 Glass dilution bottles, 160-mL

2.2.10 Polypropylene conical screw-cap tubes, 50-mL

2.2.11 Polystyrene snap-top tubes, 17 x 100-mm with caps

2.2.12 Water, distilled or deionized, or water of equivalent purity

2.2.13 Tips for micropipettes

2.3 Test animals

White Swiss non-pregnant female mice, 16-20 g (Five mice are required for each toxinantitoxin mixture.)

3. Preparation for the Test

3.1 Personnel qualifications/training

Technical personnel need a working knowledge of the use of general laboratory 

chemicals, equipment, and glassware; and have specific training and experience in the 

safe handling of clostridial toxins. Personnel should also have specific training in the care 

and handling of laboratory mice.

3.2 Preparation of equipment

All equipment is operated according to the manufacturer’s instructions.

3.3 Preparation of reagents

3.3.1 Peptone diluent

Peptone (Difco) 8 g

NaCl, reagent grade 2 g

Water q.s. to 800 mL

Dissolve peptone and sodium chloride in water. Adjust pH to 7.2 with 1N sodium 

hydroxide. Fill a 500-mL Erlenmeyer flask no more than 3/4 full with diluent. 
Center for Veterinary Biologics SAM 204.06

Testing Protocol Page 5 of 9

Supplemental Assay Method for Potency Testing Clostridium perfringens Type D Epsilon Antitoxins

UNCONTROLLED COPY

Autoclave with caps loosened at ≥121°C for 25 to 30 minutes. Cool flasks in ice

water and tighten caps. Store at 2°- 7°C for up to 3 months.

3.3.2 Preparation of C. perfringens type D standard epsilon antitoxin

1. C. perfringens type D (epsilon) antitoxin IRP 249 contains 50 

International Units of antitoxin per mL (AU/mL) and has been 

standardized against the World Health Organization C. perfringens (C. 

welchii) type D International antitoxin. Each vial contains 3.4 mL of 

antitoxin.

2. Prepare a dilution of antitoxin that contains 1.0 AU epsilon antitoxin 

per mL by adding 2.0 mL of IRP 249 to 98.0 mL of peptone diluent in a 

160-mL glass dilution bottle. Mix well. Dispense in 2.5-mL amounts into 

13 x 100-mm tubes. Store at -70 5C until used.

3.3.3 Preparation of C. perfringens type D standard epsilon toxin

1. Prepare a 1:10 dilution of C. perfringens type D epsilon toxin by 

adding 0.5 mL of IRP 632 to 4.5 mL of peptone diluent in a screw-cap 

tube. Mix well. Dispense diluted epsilon toxin in 1.5-mL amounts into 13 

x 100-mm tubes. IRP 632, diluted 1:10, is stable when stored at -60°C or 

lower.

2. Further dilute the epsilon toxin to 1:150 by adding 1.0 mL of diluted 

(1:10) epsilon toxin to 14.0 mL of peptone diluent in a 17 x 100-mm snaptop tube. For the purpose of this test, the 1:150 dilution of IRP 632 is 

referred to as the standard epsilon toxin. 

Note: A volume of 0.6 mL of standard epsilon toxin and 0.4 mL of 

peptone diluent represents 10 Lo doses. A volume of 0.8 mL of the 

standard epsilon toxin and 0.2 mL of peptone diluent represents 10 L+

doses (see Sections 4.1.1 and 4.1.2). For the purposes of this SAM, 

10 Lo dose is defined as the greatest amount of toxin that, when mixed 

with 1.0 AU, results in 100% survival of all mice inoculated 

intravenously (IV) with 0.2 mL of this mixture. The 10 L+ dose is 

defined as the least amount of toxin that, when mixed with 1.0 AU,

results in the death of 80%-100% of all mice inoculated IV with 

0.2 mL of this mixture.
Center for Veterinary Biologics SAM 204.06

Testing Protocol Page 6 of 9

Supplemental Assay Method for Potency Testing Clostridium perfringens Type D Epsilon Antitoxins

UNCONTROLLED COPY

4. Performance of the Test

4.1 Toxin neutralization

4.1.1 Product and standard epsilon toxin

1. Mix a sufficient volume of standard epsilon toxin and peptone diluent 

(0.6 mL of standard toxin and 0.4 ml peptone diluent (10 Lo doses)) for 

each product antitoxin dilution and the Lo control.

2. Mix each product sample (C. perfringens type D antitoxin or 

C. perfringens types C & D antitoxin) by shaking thoroughly.

3. Dilute each product sample according to the table below using 50-mL

conical tubes. Add 1 mL of each antitoxin dilution to 1 mL of the standard 

epsilon toxin-peptone diluent mixture (10 Lo doses) in 17 x 100-mm snaptop tubes. Mix each tube with a vortex-type mixer.

4. Let the mixtures sit at 22°- 26°C (room temperature) for 1 hour before 

placing tubes in ice.

4.1.2 Standard epsilon toxin and standard epsilon antitoxin controls

1. Add 1.0 mL of standard epsilon antitoxin containing 1.0 AU/mL to 

1 mL of the standard epsilon toxin-peptone diluent mixture (10 Lo doses) 

in a 17 x 100-mm snap-top tube. Mix well with a vortex mixer.

2. Add 1.0 mL of standard epsilon antitoxin containing 1.0 AU/mL to a 

17 x 100-mm snap-top tube containing 0.8 mL of epsilon toxin and 0.2

mL of diluent (10 L+ doses). Mix well with a vortex mixer.

3. Let the mixtures stand at 22°- 26°C (room temperature) for 1 hour.

4. Place tubes in ice.

Int’l AU 

tested

Unknown Antitoxin 10 Lo doses

Std Toxin Diluent

34 1.0 mL diluted 1:34

(1 mL product + 33 mL diluent)

0.6 mL 0.4 mL

39 1.0 mL diluted 1:39

(1 mL product + 38 mL diluent)

0.6 mL 0.4 mL
Center for Veterinary Biologics SAM 204.06

Testing Protocol Page 7 of 9

Supplemental Assay Method for Potency Testing Clostridium perfringens Type D Epsilon Antitoxins

UNCONTROLLED COPY

4.2 Inoculation of mice

4.2.1 Inject 0.2 mL of each standard epsilon toxin-product antitoxin mixture 

into each of 5 mice.

4.2.2 Inject 0.2 mL of each standard epsilon toxin-standard epsilon antitoxin 

mixture into each of 5 mice.

4.2.3 Inoculate all mice intravenously into a lateral tail vein. Use 1-cc syringes 

fitted with 25- to 27-gauge x 1/2- to 1 1/4-inch.

4.2.4 Always inoculate the mice receiving the standard epsilon toxin-standard 

epsilon antitoxin mixtures (controls) last.

4.2.5 Mouse inoculations should be completed within 1 hour of placing the 

toxin-antitoxin mixtures in the ice.

4.2.6 The test is concluded 24 hours after the mice are inoculated.

5. Interpretation of the Test Results

5.1 Criteria for a valid test

5.1.1 All 5 mice inoculated with the standard 10 Lo/1.0 AU control mixture 

must survive.

5.1.2 At least 4 out of 5 mice inoculated with the standard 10 L+/1.0 AU control 

mixture must die.

Note: Moribund animals exhibiting clinical signs consistent with the expected 

disease pathogenesis that are unable to rise or move under their own power 

may be humanely euthanized and considered as deaths as outlined in 

9 CFR 117.4.

5.2 Interpretation of serial results

5.2.1 The product contains at least 34 International Units of epsilon antitoxin 

per ml if 5 out of 5 mice inoculated with the 1:34 dilution of product-standard 

epsilon toxin mixture survive.

5.2.2 The product contains at least 39 International Units of epsilon antitoxin 

per mL if 5 out of 5 mice inoculated with the 1:39 dilution of product-standard 

epsilon toxin mixture survive.
Center for Veterinary Biologics SAM 204.06

Testing Protocol Page 8 of 9

Supplemental Assay Method for Potency Testing Clostridium perfringens Type D Epsilon Antitoxins

UNCONTROLLED COPY

5.2.3 The product is considered satisfactory if it contains at least 34 

International Units of epsilon antitoxin per mL.

5.2.4 The product is considered unsatisfactory if it contains less than 34 

International Units of epsilon antitoxin per mL. (If any mice inoculated with the 

1:34 dilution and 10 Lo doses of standard epsilon toxin die, the product is 

considered to contain less than 34 International Units/mL.)

6. Report of Test Results

Report results of the test(s) as described by the standard operating procedures.

7. References

7.1 Title 9, Code of Federal Regulations, section 113.455, U.S. Government Printing 

Office, Washington, DC.

7.2 History of toxin: C. perfringens type D (epsilon) culture CN3688, used to produce 

IRP 632, was obtained from Coopers Animal Health, Inc., 1201 Douglas Avenue, Kansas 

City, Kansas 66103-1438, on January 5, 1976. The number of passages is unknown.

7.3 History of antitoxin: C. perfringens type D (epsilon) antitoxin, IRP 249, was 

produced in 1981 at the Center for Veterinary Biologics (CVB) (then part of the National 

Veterinary Services Laboratories (NVSL)), Ames, Iowa. The antitoxin is of equine 

origin. 

8. Summary of Revisions

Version .06

 Toxin lot IRP 632 replaces IRP 450 throughout the document.

 The Director was updated on the cover page.

Version .05

 The Bacteriology Section Leader was updated.

 Minor word changes for clarification of procedures.
Center for Veterinary Biologics SAM 204.06

Testing Protocol Page 9 of 9

Supplemental Assay Method for Potency Testing Clostridium perfringens Type D Epsilon Antitoxins

UNCONTROLLED COPY

Version .04

 The Contact information has been updated.

Version .03

 The document number has been changed from BBSAM0204 to SAM 204.

 5.2: Clarification of a satisfactory product has been added.

Version .02

This document was revised to clarify the practices currently in use at the Center for Veterinary 

Biologics and to provide additional detail. While no significant changes were made that impact 

the outcome of the test, the following changes were made to the document:

 IRP 410 has changed to IRP 450 throughout the document.

 4.1 The format and content have been modified to clarify the LO and L+ levels of the 

Toxin Neutralization process.

 Humane endpoint language has been added.

 Dilution/holding vessel sizes have been added for clarification.

 The contact person has been changed to Janet M. Wilson.