Canine Hepatitis Vaccine and Canine Adenovirus Type 2 Vaccine shall be prepared from virus-bearing cell culture fluids. Only Master Seed Virus which has been established as pure, safe, and immunogenic shall be used in preparing the production seed virus for vaccine production. All serials shall be prepared from the first through the fifth passage from the Master Seed Virus.

(a) The Master Seed Virus shall meet the applicable requirements prescribed in § 113.300 except that the dog safety test prescribed in § 113.40(a) shall be conducted by the intravenous route.

(b) Each lot of Master Seed Virus used for vaccine production shall be tested for immunogenicity by one or both of the following methods:

(1) Immunogenicity for canine hepatitis. Twenty-five canine hepatitis susceptible dogs shall be used as test animals (20 vaccinates and 5 controls). Blood samples shall be drawn from these animals and individual serum samples tested. The dogs shall be considered susceptible if the results are negative at a 1:2 final serum dilution in a varying serum-constant virus neutralization test using 50 to 300 TCID50 of canine adenovirus.

(i) A geometric mean titer of the dried vaccine produced from the highest passage of the Master Seed Virus shall be established before the immunogenicity test is conducted. The 20 dogs to be used as vaccinates shall be injected with a predetermined quantity of vaccine virus and the remaining five dogs held as uninjected controls. To confirm the dosage calculations, five replicate virus titrations shall be conducted on a sample of the vaccine virus dilution used.

(ii) Not less than 14 days postinjection, the vaccinates and the controls shall each be challenged intravenously with virulent infectious canine hepatitis virus furnished or approved by the Animal and Plant Health Inspection Service and observed each day for 14 days.

(A) If at least four of the five controls do not show severe clinical signs of canine hepatitis, the test is a No Test and may be repeated.

(B) If at least 19 of the 20 vaccinates do not survive without showing clinical signs of infectious canine hepatitis during the observation period, the Master Seed Virus is unsatisfactory.

(2) Immunogenicity for canine adenovirus Type 2. Thirty canine adenovirus type 2 susceptible dogs shall be used as test animals (20 vaccinates and 10 controls). Blood samples shall be drawn from these animals and individual serum samples tested. The dogs shall be considered susceptible if the results are negative at a 1:2 final serum dilution in a varying serum-constant virus neutralization test using 50 to 300 TCID50 of canine adenovirus.

(i) A geometric mean titer of the dried vaccine produced from the highest passage of the Master Seed Virus shall be established before the immunogenicity test is conducted. The 20 dogs to be used as vaccinates shall be injected with a predetermined quantity of vaccine virus and the remaining 10 dogs held as uninjected controls. To confirm the dosage calculations, five replicate virus titrations shall be conducted on a sample of the vaccine virus dilution used.

(ii) Not less than 14 days postinjection, the vaccinates and the controls shall be challenged by exposure to a nebulized aerosol of virulent canine adenovirus type 2 furnished or approved by the Animal and Plant Health Inspection Service and observed each day for 14 days postchallenge. The rectal temperature of each animal shall be taken and the presence of respiratory or other clinical signs of canine adenovirus type 2 noted and recorded each day.

(A) If at least 6 of 10 controls do not show clinical signs of canine adenovirus type 2 infection other than fever, the test is a No Test and may be repeated.

(B) If a significant difference in clinical signs in a valid test cannot be demonstrated between vaccinates and controls using a scoring system approved by the Animal and Plant Health Inspection Service, the Master Seed Virus is unsatisfactory.

(iii) An Outline of Production change shall be made before authorization for use of a new lot of Master Seed Virus shall be granted by the Animal and Plant Health Inspection Service.

(c) Test requirements for release. Each serial and subserial shall meet the requirements prescribed in § 113.300 and in this paragraph. Final container samples of completed product shall be tested. Any serial or subserial found unsatisfactory by a prescribed test shall not be released.

(1) Virus titer requirements. Final container samples of completed product shall be tested for virus titer using the titration method used in paragraph (b)(1)(i) and/or (b)(2)(i) of this section. To be eligible for release, each serial and each subserial shall have a virus titer sufficiently greater than the titer of vaccine virus used in the immunogenicity test(s) prescribed in paragraph (b) of this section to assure that when tested at any time within the expiration period, each serial and subserial shall have a virus titer of 10 0.7 greater than that used in such immunogenicity test(s) but not less than 10 2.5 TCID50 dose. If both immunogenicity tests in paragraph (b) of this section are conducted and a different amount of virus is used in each test, the virus titer requirements shall be based on the higher of the two amounts.

(2) [Reserved]