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SAM 213.03

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United States Department of Agriculture

Center for Veterinary Biologics

Testing Protocol

SAM 213

Supplemental Assay Method for Potency Testing of 

Clostridium botulinum Type C Bacterin-Toxoids

Date: May 17, 2016

Number: SAM 213.03

Supersedes: SAM 213.02, December 28, 2009

Standard Requirement: 9 CFR 113.110

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

Approvals: /s/Larry R. Ludemann Date: 01Jun16

Larry R. Ludemann, Section Leader

Bacteriology

/s/Paul J. Hauer Date: 08Jun16

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 13Jun16

 Linda S. Snavely Date

 Quality Management Program Assistant
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Supplemental Assay Method for Potency Testing of Clostridium botulinum Type C Bacterin-Toxoids

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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 Selection and handling of test animals

3.3 Preparation of supplies

3.4 Preparation of reagents

4. Performance of the Test

4.1 Vaccination

4.2 Challenge of immunity

5. Interpretation of the Test Results

6. Report of Test Results

7. References

8. Summary of Revisions

Appendix
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Supplemental Assay Method for Potency Testing of Clostridium botulinum Type C Bacterin-Toxoids

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1. Introduction

This Supplemental Assay Method (SAM) describes procedures for potency testing biological 

products containing Clostridium botulinum Type C antigen as prescribed in title 9, Code of 

Federal Regulations (9 CFR), part 113.110. Mink are vaccinated subcutaneously then challenged 

intraperitoneally 21 to 28 days later with a standard dose of C. botulinum Type C toxin.

2. Materials

2.1 Equipment/instrumentation

2.1.1 Vortex-type mixer

2.1.2 Refrigerator, 2- 7C

2.1.3 Freezer, -70C or lower

2.2 Reagents/supplies

2.2.1 C. botulinum Type C toxin, IRP 583

2.2.2 Pipettes, 1-mL, 2-mL, 5-mL, 10-mL, and 25-mL

2.2.3 Syringes, needle-locking, 3-mL, and 5-mL

2.2.4 Needles, 23-gauge x 1-inch

2.2.5 Serum vials, 50-mL, with caps and seals

2.2.6 M/15 phosphate buffered physiological saline with 0.2% gelatin

2.3 Test animals

2.3.1 Mink, young adult, from the same source and similar age. Vaccinate a 

group of 5 mink for each product tested. Three additional mink are held as 

unvaccinated controls.

2.3.2 Mice, 16-20 g, fifty mice are required to determine the LD50 of the 

challenge inoculum. All mice must be from the same source colony.
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3. Preparation for the Test

3.1 Personnel qualifications/training

Technical personnel need to have 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 need specific training in the care and 

handling of laboratory animals.

3.2 Selection and handling of test animals

3.2.1 Healthy, young adult mink are caged individually for the potency test.

3.2.2 Mink of either sex or any color may be selected.

3.2.3 Susceptible mink are required. They must not have been vaccinated with 

any C. botulinum Type C-containing product.

3.3 Preparation of supplies

3.3.1 Sterilize all glassware before use.

3.3.2 Use only sterile supplies (pipettes, syringes, needles, etc.).

3.3.3 Operate all equipment according to the manufacturers’ instructions.

3.4 Preparation of reagents

3.4.1 M/15 phosphate buffered saline with 0.2% gelatin

NaCl 8.5 g

Gelatin 2.0 g

M/15 phosphate buffer at pH 7.4 1000.0 mL

Stock solution of M/15 KH2PO4 (Solution A):

Dissolve 9.072 grams of KH2PO4 in one liter of distilled water.

Stock solution of M/15 Na2HPO4 (Solution B):

Dissolve 9.465 grams of Na2HPO4 in one liter of distilled water.

One hundred ninety-two (192) milliliters of Solution A are mixed with 808 mL of 

Solution B to provide 1000 mL of M/15 phosphate buffer at pH 7.4.
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Eight and one-half (8.5) grams of NaCl and 2 grams of gelatin are dissolved in the 

1000 mL of M/15 phosphate buffer (pH 7.4). It is then autoclaved at ≥ 121°C for 

30 minutes. Store at 2°- 7°C for up to 6 months.

3.4.2 C. botulinum Type C toxin 

C. botulinum Type C challenge toxin, IRP 583, is supplied by the Center for 

Veterinary Biologics (CVB). Each vial contains 1.3 mL. Vials may be combined 

to make sufficient quantity for each challenge. Store toxin vials at -70°C or 

colder.

4. Performance of the Test

4.1 Vaccination

Vaccinate the mink subcutaneously with the mink dose recommended on the label of the 

test serial final container using 5-mL sterile, disposable syringes with 23-gauge x 1-inch 

disposable needles.

4.2 Challenge of immunity

4.2.1 Challenge both vaccinated mink and unvaccinated control mink 21 to 28 

days after the vaccination.

4.2.2 Dilute C. botulinum Type C toxin, IRP 583, 1:10 in M/15 phosphate 

buffered saline with 0.2% gelatin. Prepare the dilution by adding 1 mL IRP 583 to 

9 mL diluent. IRP 583 diluted 1:10 contains 104.0 mouse lethal dose fifty (LD50) 

per 0.5 mL.

4.2.3 Inoculate the mink intraperitoneally with 0.5 mL of diluted IRP 583 using 

a 3-mL disposable syringe and 23-gauge x 1-inch needle.

4.2.4 Immediately following the mink inoculations, the 1:10 toxin dilution is 

further diluted to the final toxin dilutions of 1:100,000, 1:200,000, 1:400,000, and 

1:800,000 in M/15 phosphate buffered physiological saline with 0.2% gelatin (see 

Appendix). Inoculate intraperitoneally each of 10 mice with 0.5 mL of each final 

toxin dilution. In most tests, at least 9 of 10 mice inoculated with the 1:100,000 

dilution will die within 7 days and at least 9 of 10 mice inoculated with the 

1:800,000 dilution will survive for 7 days.
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5. Interpretation of the Test Results

Observe the mink for 7 days after challenge for signs of botulism and note deaths. For a valid 

test, the unvaccinated controls must die. If the test is valid and at least 80% of the vaccinated 

mink do not remain free of botulism, the serial is unsatisfactory.

Note: Moribund animals observed exhibiting clinical signs consistent with the expected

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

euthanized and considered as deaths as outlined in 9 CFR 117.4.

6. Report of Test Results

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

7. References

Title 9, Code of Federal Regulations, part 113.110, U.S. Government Printing Office, 

Washington, DC.

8. Summary of Revisions

Version .03

 Section Leader and Director information have been updated.

 Toxin lot number and corresponding use dilutions have been updated throughout the 

document.

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:

 The document number has been changed from BBSAM0213 to SAM 213.

 The Standard Requirement has been changed from 9 CFR 113.95 to 9 CFR 113.110.

 The Contact information has been updated.
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 Section 4.3 information has been moved to Section 5.

 Toxin lot numbers and corresponding use dilutions have been updated throughout the 

document.
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IRP 583,

undiluted

Appendix: Mink Challenge and LD50 Dilution Preparation

1 mL

9 mL

1 mL

1 mL 2 mL 9 mL 9 mL

9 mL 

diluent 9 mL

diluent

9 mL 

diluent

9 mL 

diluent

18 mL 

diluent

9 mL 

diluent

9 mL

diluent

Dilution = 1:10 1:100 1:1,000 1:10,000 1:100,000 1:200,000 1:800,000

 Dilutions used in mouse titration

*Diluent = M/15 phosphate buffered saline with 0.2% gelatin.

Shake each dilution vigorously before each transfer and before filling syringe. All dilutions are 

held in an ice bath.

9 mL 

diluent

1:400,000

1 mL