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Title 9Chapter ISubchapter EPart 113 → Subject Group


Title 9: Animals and Animal Products
PART 113—STANDARD REQUIREMENTS


Live Virus Vaccines

§113.300   General requirements for live virus vaccines.

When prescribed in an applicable Standard Requirement or in the filed Outline of Production, a live virus vaccine shall meet the applicable requirements in this section.

(a) Purity tests—(1) Bacteria and fungi. Final container samples of completed product and comparable samples of each lot of Master Seed Virus shall be tested for bacteria and fungi in accordance with the test provided in §113.27.

(2) Mycoplasma. Final container samples of completed product and comparable samples of each lot of Master Seed Virus shall be tested for mycoplasma in accordance with the test provided in §113.28.

(3) Avian Origin Vaccine. Samples of each lot of Master Seed Virus and bulk pooled material or final container samples from each serial shall also be tested for:

(i) Salmonella contamination as prescribed in §113.30; and

(ii) Lymphoid leukosis virus contamination as prescribed in §113.31; and

(iii) Hemagglutinating viruses as prescribed in §113.34.

(4) Extraneous viruses. Each lot of Master Seed Virus used to prepare live virus vaccine recommended for animals other than poultry shall meet the requirements for extraneous viruses as prescribed in §113.55

(b) Safety tests. Samples of each lot of Master Seed Virus and final container samples of completed product from each serial or first subserial of live virus vaccine recommended for animals other than poultry shall be tested for safety in at least one species for which the vaccine is intended using methods prescribed in §§113.39, 113.40, 113.41, 113.44, and 113.45 or in a filed Outline of Production. The mouse safety test prescribed in §113.33(a) shall also be conducted unless the virus or agent in the vaccine is inherently lethal for mice.

(c) Virus identity test. At least one of the virus identity tests provided in this paragraph or a suitable identity test prescribed in the filed Outline of Production shall be conducted on the Master Seed Virus and final container samples from each serial or first subserial of biological product.

(1) Fluorescent antibody test. The fluorescent antibody test shall be conducted using virus inoculated cells and uninoculated control cells. Cells shall be stained with fluorochrome conjugated specific antiserum. Fluorescence typical of the virus concerned shall be demonstrated in the inoculated cells. The control cells shall remain free of such fluorescence.

(2) Serum neutralization test. The serum neutralization test shall be conducted using the constant serum-decreasing virus method with specific antiserum. For positive identification, at least 100 ID50 of vaccine virus shall be neutralized by the antiserum.

(d) Cell Culture Requirements. If cell cultures are used in the preparation of Master Seed Virus or of the vaccine, primary cells shall meet the requirements prescribed in §113.51, cell lines shall meet the requirements prescribed in §113.52, and ingredients of animal origin shall meet the applicable requirements in §113.53.

(e) Moisture content. (1) The maximum moisture content in desiccated vaccines must be stated in the filed Outline of Production.

(2) Final container samples of completed product from each serial or subserial must be tested for moisture content in accordance with the test prescribed in §113.29.

[39 FR 27430, July 29, 1974, as amended at 43 FR 49528, Oct. 24, 1978; 50 FR 1042, Jan. 9, 1985; 54 FR 19352, May 5, 1989. Redesignated at 55 FR 35562, Aug. 31, 1990; 60 FR 24549, May 9, 1995; 68 FR 57608, Oct. 6, 2003]

§113.301   Ovine Ecthyma Vaccine.

Ovine Ecthyma Vaccine shall be prepared from tissue culture fluids or virus-bearing tissues obtained from sheep that have developed ovine ecthyma following inoculation with virulent ovine ecthyma virus. Ovine Ecthyma Vaccine is exempt from the requirements prescribed in §§113.27 and 113.300(a), (b), and (c). Each serial shall meet the moisture requirements in §113.300(e) and the special requirements prescribed in this section. Any serial found unsatisfactory by a prescribed test shall not be released.

(a) Safety tests. (1) Bulk or final container samples of completed product from each serial shall be tested for safety as prescribed in §113.38.

(2) The prechallenge period of the potency test shall constitute a safety test. If unfavorable reactions attributable to the vaccine occur in either of the vaccinates during the observation period, the serial is unsatisfactory.

(b) Potency test. Final container samples of completed product from each serial and each subserial shall be tested for potency using susceptible lambs. The vaccine shall be prepared as recommended for use on the label.

(1) Each of two lambs (vaccinates) shall be vaccinated by application of the vaccine to a scarified area on the medial surface of the thigh and observed each day for 14 days.

(2) The immunity of the two vaccinates and one or more unvaccinated lambs (controls) shall be challenged in the same manner as for vaccination, using the opposite thigh.

(3) If typical signs of ovine ecthyma, such as hyperemia, vesicles, and pustules do not develop on the controls during the first 2 weeks following challenge and persist for approximately 30 days, the test is a No Test and may be repeated.

(4) If the vaccinates do not show a typical immune reaction, the serial is unsatisfactory: Provided, That, an initial active reaction with hyperemia which resolves progressively and disappears within 2 weeks, may be characterized as a typical immune reaction.

[39 FR 27430, July 29, 1974. Redesignated at 55 FR 35562, Aug. 31, 1990, as amended at 56 FR 66786, Dec. 26, 1991]

§113.302   Distemper Vaccine—Mink.

Distemper Vaccine—Mink 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 for preparing the production seed virus for vaccine production. All serials of vaccine 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 and the requirements prescribed in this section.

(b) The lot of Master Seed Virus shall be tested for extraneous viruses as follows:

(1) To detect virulent canine distemper virus, each of two distemper susceptible mink or ferrets shall be inoculated with 1 ml of the Master Seed Virus and observed each day for 21 days. If undesirable reactions occur in either test animal, the lot of Master Seed Virus is unsatisfactory.

(2) Master Seed Virus propagated in chicken embryos shall be tested for pathogens by the chicken embryo test prescribed in §113.37 except lesions typical of distemper virus may be disregarded. If found unsatisfactory, the Master Seed Virus shall not be used.

(c) Each lot of Master Seed Virus used for vaccine production shall be tested for immunogenicity. The selected virus dose from the lot of Master Seed Virus shall be established as follows:

(1) At least 25 distemper susceptible mink shall be used as test animals. Blood samples shall be drawn from these animals and individual serum samples tested. The mink shall be considered susceptible if the results are negative at a 1:2 final serum dilution in a varying serum-constant virus neutralization test with less than 500 ID50 of canine distemper virus. Other means of insuring susceptibility may be used if prior approval from Animal and Plant Health Inspection Service is received.

(2) 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. At least 20 mink shall be vaccinated with a predetermined quantity of vaccine virus and at least 5 additional mink shall be held as unvaccinated controls. To confirm the dosage calculations, five replicate virus titrations shall be conducted on a sample of the vaccine virus dilution used.

(3) At least twenty-one days post-injection, the immunity of each of the vaccinates and the controls shall be challenged with the same size dose of virulent distemper virus and observed each day for 21 days.

(i) If at least 80 percent of the controls do not die or show severe signs of distemper, the test is a No Test and may be repeated.

(ii) If at least 19 of 20, 27 of 30, or 36 of 40 of the vaccinates do not survive without showing clinical signs of distemper during the observation period, the Master Seed Virus is unsatisfactory.

(4) An Outline of Production change shall be made before authority for use of a new lot of Master Seed Virus shall be authorized by Animal and Plant Health Inspection Service.

(d) Test requirements for release: Each serial and subserial shall meet the general requirements prescribed in §113.300 and the requirements 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) Mink safety test. Each of 2 mink shall be vaccinated with the equivalent of 10 doses of vaccine rehydrated with sterile diluent and administered in the manner recommended on the label. The mink shall be observed each day for 21 days. If unfavorable reactions attributable to the product occur in either of the mink during the observation period, the serial or subserial is unsatisfactory. If unfavorable reactions which are not attributable to the product occur, the test shall be declared a No Test and may be repeated: Provided, That if the test is not repeated, the serial or subserial shall be declared unsatisfactory.

(2) Potency Test. An in vitro potency test shall be conducted. To be eligible for release, each serial and subserial shall have a virus titer sufficiently greater than the titer of vaccine virus used in the immunogenicity test prescribed in paragraph (c) of this section to assure that, when tested at any time within the expiration period, each serial and subserial shall have a virus titer 100.7 greater than that used in such immunogenicity test when tested by the method used in paragraph (c)(2) of this section.

[40 FR 53000, Nov. 14, 1975, as amended at 48 FR 33471, July 22, 1983. Redesignated at 55 FR 35562, Aug. 31, 1990, as amended at 56 FR 66784, 66786, Dec. 26, 1991; 72 FR 72564, Dec. 21, 2007]

§113.303   Bluetongue Vaccine.

Bluetongue Vaccine shall be prepared from virus-bearing cell culture fluids. Only Master Seed which has been established as pure, safe, and immunogenic shall be used for preparing the seeds for vaccine production. All serials of vaccine shall be prepared from the first through the tenth passage from the Master Seed.

(a) The Master Seed shall meet the applicable general requirements prescribed in §113.300 and the requirements in this section.

(b) Each lot of Master Seed shall be tested for transmissibility and reversion to virulence in sheep using a method acceptable to Animal and Plant Health Inspection Service. If reversion to virulence is demonstrated, the Master Seed is unsatisfactory.

(c) Each lot of Master Seed used for vaccine production shall be tested for immunogenicity. The selected virus dose from the lot of Master Seed shall be established as follows:

(1) Twenty-five lambs, susceptible to the bluetongue virus serotype contained in the vaccine, shall be used as test animals (20 vaccinates and 5 controls). Blood samples shall be drawn from these animals and individual serums tested. A lamb shall be considered susceptible if there is no neutralization at a 1:2 final serum dilution in a constant virus varying serum neutralization test with 60 to 300 TCID50 of bluetongue virus or another method acceptable to Animal and Plant Health Inspection Service.

(2) A geometric mean titer of the vaccine produced from the highest passage from the Master Seed shall be established before the immunogenicity test is conducted. The 20 lambs to be used as vaccinates shall be administered a predetermined quantity of vaccine virus by the method recommended on the label. To confirm the virus dosage administered, five replicate virus titrations shall be conducted on a sample of the vaccine used.

(3) At least once during the period of 14 to 18 days postvaccination, individual serum samples shall be collected from each of the vaccinates and tested for virus neutralizing antibody using the 60 to 300 TCID50 of bluetongue virus.

(4) Twenty-one to twenty-eight days postvaccination the vaccinates and the controls shall each be challenged with virulent bluetongue virus and observed for 14 days. The rectal temperature of each animal shall be taken and recorded for 17 consecutive days beginning 3 days prechallenge. The presence or absence of lesions or other clinical signs of bluetongue noted and recorded on each of 14 consecutive days postchallenge.

(i) If at least four of the five controls do not show clinical signs of bluetongue and a temperature rise of 3 °F or higher over the prechallenge mean temperature, the test shall be considered a No Test and may be repeated.

(ii) If at least 19 of the 20 vaccinates tested as prescribed in paragraph (c)(3) of this section do not have bluetongue neutralizing antibody titers of 1:4 final serum dilution or higher, or if more than one of the vaccinates shows a temperature rise of 3 °F or higher than its prechallenge mean temperature for 2 or more days, or if more than one of the vaccinates exhibits clinical signs of bluetongue, the Master Seed is unsatisfactory.

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

(d) Test requirements for release. Each serial and subserial shall meet the applicable general requirements prescribed in §113.300 and the requirements 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) Safety test. The mouse safety test prescribed in §113.33(a) and the lamb safety test prescribed in §113.45 shall be conducted.

(2) Virus titer requirements. Final container samples of completed product shall be tested for virus titer using the titration method used in paragraph (c)(2) of this section. To be eligible for release, each serial and subserial shall have a virus titer sufficiently greater than the titer of vaccine virus used in the immunogenicity test prescribed in paragraph (c) 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 100.7 greater than that used in such immunogenicity test.

[50 FR 23796, June 6, 1985. Redesignated at 55 FR 35562, Aug. 31, 1990, as amended at 56 FR 66784, 66786, Dec. 26, 1991; 72 FR 72564, Dec. 21, 2007]

§113.304   Feline Panleukopenia Vaccine.

Feline Panleukopenia 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 for preparing the production seed virus for vaccine production. All serials of vaccine shall be prepared from the first through the fifth passage from the Master Seed Virus.

(a) The Master Seed Virus shall meet the applicable general requirements prescribed in §113.300 and the requirements prescribed in this section.

(b) The lot of Master Seed Virus shall be tested for other agents as follows:

(1) To detect virulent feline panleukopenia virus or virulent mink enteritis virus, each of two feline panleukopenia susceptible cats, as determined by the criteria prescribed in paragraph (c)(1) of this section, shall be injected subcutaneously with the equivalent of one cat dose each and the cats observed each day for 21 days. If either or both cats show signs of disease or reduced white blood cell counts below 50 percent of the normal level established by an average of three or more counts taken prior to injection, the Master Seed Virus is unsatisfactory.

(2) To detect chlamydial agents, the Master Seed Virus shall be tested as prescribed in §113.43.

(c) Each lot of Master Seed Virus used for vaccine production shall be tested for immunogenicity. The selected virus dose from the lot of Master Seed Virus shall be established as follows:

(1) Twenty-five feline panleukopenia susceptible cats shall be used as test animals (20 vaccinates and 5 controls). Blood samples drawn from each cat shall be individually tested for neutralizing antibody against feline panleukopenia virus to determine susceptibility.

(i) A constant virus-carrying serum neutralization test in tissue culture using 100 to 300 TCID50 of virus shall be used.

(ii) Cats shall be considered susceptible if there is no neutralization at a 1:2 final serum dilution.

(2) 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 cats used as vaccinates shall be injected with a predetermined quantity of vaccine virus and the remaining five cats 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.

(3) Fourteen days post-injection, the vaccinates and the controls shall be challenged with virulent feline panleukopenia virus furnished by Animal and Plant Health Inspection Service and the cats observed each day for 14 days.

(i) If at least 80 percent of the controls do not show clinical signs of feline panleukopenia during the observation period, the test is a No Test and may be repeated. Clinical signs of feline panleukopenia shall include a pronounced leukopenia wherein the white cell count drops to 4,000 or less per cubic mm, or the white cell count drops to less than 25 percent of the normal level established by an average of three or more counts taken prior to challenge.

(ii) If at least 19 of the 20 vaccinates do not survive the observation period without showing clinical signs of feline panleukopenia as described in paragraph (c)(3)(i) of this section, the Master Seed Virus is unsatisfactory.

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

(d) 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) Safety test. The mouse safety test prescribed in §113.33(a) and the cat safety test prescribed in §113.39 shall be conducted.

(i) Each of two healthy cats shall be injected with 10 cat doses by the method recommended on the label and the cats observed each day for 14 days.

(ii) If unfavorable reactions attributable to the biological product occur during the observation period, the serial is unsatisfactory. If unfavorable reactions occur which are not attributable to the product, the test shall be declared a No Test and repeated: Provided, That, if not repeated, the serial shall be unsatisfactory.

(2) Virus titer requirements. Final container samples of completed product shall be tested for virus titer using the titration method used in paragraph (c)(2) 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 prescribed in paragraph (c) 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 100.7 greater than that used in such immunogenicity test but not less than 102.5 TCID50 per dose.

[39 FR 44716, Dec. 27, 1974, as amended at 40 FR 53378, Nov. 18, 1975; 43 FR 25078, June 9, 1978; 43 FR 41186, Sept. 15, 1978; 44 FR 58900, Oct. 12, 1979; 48 FR 33471, July 22, 1983. Redesignated at 55 FR 35562, Aug. 31, 1990, as amended at 56 FR 66784, 66786, Dec. 26, 1991; 72 FR 72564, Dec. 21, 2007]

§113.305   Canine Hepatitis and Canine Adenovirus Type 2 Vaccine.

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 100.7 greater than that used in such immunogenicity test(s) but not less than 102.5 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]

[60 FR 14361, Mar. 17, 1995, as amended at 72 FR 72564, Dec. 21, 2007]

§113.306   Canine Distemper Vaccine.

Canine Distemper Vaccine shall be prepared from virus-bearing cell culture fluids or embryonated chicken eggs. Only Master Seed Virus which has been established as pure, safe, and immunogenic shall be used for preparing the production seed virus for vaccine production. All serials of vaccine shall be prepared from the first through the fifth passage from the Master Seed Virus.

(a) Master Seed Virus. The Master Seed Virus shall meet the applicable requirements prescribed in §113.300 and the requirements prescribed in this section.

(1) To detect ferret virulent canine distemper virus, each of five canine distemper susceptible ferrets shall be injected with a sample of the Master Seed Virus equivalent to the amount of virus to be used in one dog dose and observed each day for 21 days. If undesirable reactions are observed during the observation period, the lot of Master Seed is unsatisfactory.

(2) Master Seed Virus propagated in tissues or cells of avian origin shall be tested for pathogens by the chicken embryo test prescribed in §113.37. If found unsatisfactory, the Master Seed Virus shall not be used.

(b) Each lot of Master Seed Virus used for vaccine production shall be tested for immunogenicity. The selected virus dose from the lot of Master Seed Virus shall be established as follows:

(1) Twenty-five canine distemper 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 distemper virus.

(2) 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 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.

(3) At least 14 days post-injection, the vaccinates and the controls shall each be challenged intracerebrally with virulent canine distemper virus furnished or approved by the Animal and Plant Health Inspection Service and observed each day for 21 days.

(i) If at least four of the five controls do not die and the survivor, if any, does not show clinical signs of canine distemper the test is a No Test and may be repeated.

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

(4) 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. Except for §113.300(a)(3)(ii), 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) The test for pathogens prescribed in §113.37 shall be conducted on each serial or one subserial of avian origin vaccine.

(2) Virus titer requirements. Final container samples of completed product shall be tested for virus titer using the titration method used in paragraph (b)(2) of this section. To be eligible for release, each serial and subserial shall have a virus titer sufficiently greater than the titer of vaccine virus used in the immunogenicity test 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 100.7 greater than that used in such immunogenicity test but not less than 102.5 TCID50 per dose.

[60 FR 14362, Mar. 17, 1995, as amended at 72 FR 72564, Dec. 21, 2007]

§113.308   Encephalomyelitis Vaccine, Venezuelan.

Encephalomyelitis Vaccine, Venezuelan, shall be prepared from virus-bearing cell culture fluids. Only Master Seed which has been established as pure, safe, and immunogenic shall be used for preparing seeds for vaccine production. All serials of vaccine shall be prepared from the first through the fifth passage from the Master Seed.

(a) The Master Seed shall meet the applicable general requirements prescribed in §113.300 except (b), and the requirements prescribed in this section.

(b) Each lot of Master Seed shall be tested for immunogenicity. The selected virus dose from the lot of Master Seed shall be established as follows:

(1) Tests conducted by the Department have established that horses having Venezuelan equine encephalomyelitis antibody titers of 1:20 by the hemagglutination-inhibition (HI) method or 1:40 by the serum neutralization (SN) method were immune to challenge with virulent virus. The immunogenicity test is based on the demonstration of a serological response of at least that magnitude following vaccination of serologically negative horses.

(2) At least 22 horses (20 vaccinates and 2 controls), susceptible to Venezuelan equine encephalomyelitis, shall be used as test animals. Blood samples shall be taken from each horse and the serums individually tested for neutralizing antibody. Horses shall be considered susceptible if there is no neutralization at a 1:2 final serum dilution in a constant virus-varying serum neutralization test using 60 to 300 TCID50 of Venezuelan equine encephalomyelitis virus.

(3) A geometric mean titer of the vaccine produced from the highest passage of the Master Seed shall be established using a method acceptable to Veterinary Services before the immunogenicity test is conducted. The 20 horses used as vaccinates shall be injected with a predetermined quantity of vaccine virus by the method to be recommended on the label. To confirm the dosage administered, five replicate virus titrations shall be conducted on a sample of the vaccine virus dilution used.

(4) Twenty-one to twenty-eight days postvaccination, blood samples shall be drawn from all test animals. For a valid test, the controls shall remain seronegative at 1:2 final serum dilution. In a valid test, if at least 19 of 20 vaccinates do not have antibody titers of at least 1:20 in a hemagglutination-inhibition test or at least 1:40 in a serum neutralization test, the Master Seed is unsatisfactory.

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

(c) Test requirements for release. Each serial and subserial shall meet the applicable general requirements prescribed in §113.300 and special requirements in this paragraph. Any serial or subserial found unsatisfactory by a prescribed test shall not be released.

(1) Safety test. The mouse safety test prescribed in §113.33(b) shall be conducted.

(2) Virus titer requirements. Final container samples of completed product shall be tested for virus titer using the method in paragraph (b)(3) of this section. To be eligible for release, each serial and subserial shall have a virus titer sufficiently greater than the titer of the vaccine used in the immunogenicity test 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 100.7 greater than that used in the immunogenicity test, but not less than 102.5 TCID50 per dose.

[50 FR 23797, June 6, 1985. Redesignated at 55 FR 35562, Aug. 31, 1990, as amended at 56 FR 66784, 66786, Dec. 26, 1991; 72 FR 72564, Dec. 21, 2007]

§113.309   Bovine Parainfluenza3 Vaccine.

Bovine Parainfluenza3 Vaccine shall be produced from virus-bearing cell culture fluids. Only Master Seed Virus which has been established as pure, safe, and immunogenic shall be used for preparing the production seed virus for vaccine production. All serials of vaccine shall be prepared from the first through the tenth passage from the Master Seed Virus.

(a) The Master Seed Virus shall meet the applicable general requirements prescribed in §113.300.

(b) Each lot of Master Seed Virus shall meet the special requirements prescribed in this section.

(c) Each lot of Master Seed Virus used for vaccine production shall be tested for immunogenicity. The selected virus dose from the lot of Master Seed Virus shall be established as follows:

(1) Twenty-five bovine parainfluenza, susceptible calves shall be used as test animals (20 vaccinates and five controls). Blood samples shall be drawn from these animals and individual serums tested. Also, nasal specimens shall be collected for virus isolation attempts. The calves shall be considered susceptible if:

(i) The results are negative at a 1:2 final serum dilution in a varying serum constant virus neutralization test with less than 500 TCID50 of bovine parainfluenza3 virus; and

(ii) Shall be negative to bovine parainfluenza3 virus isolation attempts from the nasal specimens on the day of injection.

(2) 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 calves to be used as vaccinates shall be injected with a predetermined quantity of vaccine virus and the remaining five calves held as uninjected controls. To confirm the dosage calculation, five replicate virus titrations shall be conducted on a sample of the vaccine virus dilution used.

(3) The vaccinates and controls shall be examined for clinical signs of respiratory disease and the body temperature taken and recorded on each of the first 14 consecutive days post-injection. The vaccinates shall be bled on day 6 ±2 days post-injection.

(4) Three to four weeks post-vaccination, all calves shall be bled for serum antibodies and nasal specimens shall be collected for PI3 virus isolation. On the same day, all vaccinates and controls shall be given acceptable challenge PI3 virus titrating at least 107.0 TCID50 per ml and the animals observed for 14 days. Two ml of the challenge virus shall be instilled in each nostril or shall be inhaled as an aerosol suspension. Upon request, challenge virus and instructions shall be furnished by Animal and Plant Health Inspection Service.

(5) Each animal shall be examined for clinical signs of respiratory disease and the body temperature recorded on each of the 14 consecutive days of the post-challenge observation period. Each day for at least the first 10 days post-challenge, nasal specimens for virus isolation attempts shall be taken. All animals shall be bled on day 6 ±2 days post-challenge, and all animals shall be bled at least once 14 to 28 days post-challenge for serum antibody studies.

(6) Satisfactory Test Criteria:

(i) All virus isolations attempts shall be by culture and at least one subculture in PI3 susceptible cells for a total of at least 14 days.

(ii) Two to four weeks post-vaccination, at least 19 of the 20 vaccinates shall have PI3 neutralizing antibody titers of at least 1:4 and all five controls shall be negative at 1:2 dilution. None of the post-vaccination serums collected from the vaccinates on day 6 ±2 days shall reveal serum neutralization antibody titers of 1:32 or greater based upon final dilution.

(iii) Satisfactory resistance to challenge by vaccinates shall be determined by a significant difference between virus isolation rates from vaccinates and controls. The virus neutralization titers of post-challenge serums and respiratory symptoms and temperatures from all animals shall be considered in the evaluation of the test validity.

(7) Designated animal alternates for test animals showing anamnestic antibody responses (titers 1:32 or greater) on day 6 serums may be included in the study under the following provisions:

(i) No more than five alternates shall be allowed for the vaccinates and no more than two for the controls.

(ii) Alternates shall be subject to all requirements outlined for the animals for which they are alternates.

(iii) Antibody values from alternate animals may be used only to replace values from up to and including five vaccinates which develop antibody of 1:32 or greater by day 6 ±2 days post-vaccination or up to and including two controls which develop antibody titers of 1:32 or greater by day 6 ±2 days post-challenge.

(8) A sequential test procedure may be used in lieu of the 20 calf requirement. A beta value of .05 and a tolerance level of .78 shall be required.

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

(d) Test requirements for release: Each serial and subserial shall meet the applicable general requirements prescribed in §113.300 and the requirements in this paragraph. Final container samples of completed product shall be tested except as prescribed in paragraph (d)(1) of this section. Any serial or subserial found unsatisfactory by a prescribed test shall not be released.

(1) Purity test. The test for Brucella contamination prescribed in §113.32 shall be conducted on each batch of primary cells intended for production use.

(2) Safety test. The mouse safety test prescribed in §113.33(a) and the calf safety test prescribed in §113.41 shall be conducted.

(3) Virus titer requirements. Final container samples of completed product shall be tested for virus titer using the titration method used in paragraph (c)(2) of this section. To be eligible for release, each serial and each subserial shall have a virus titer per dose sufficiently greater than the titer of vaccine virus used in the immunogenicity test prescribed in paragraph (c) 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 100.7 greater than that used in the immunogenicity test but not less than 102.5 TCID50 per dose.

[39 FR 44719, Dec. 27, 1974, as amended at 40 FR 41089, Sept. 5, 1975; 43 FR 49529, Oct. 24, 1978; 48 FR 33472, July 22, 1983. Redesignated at 55 FR 35562, Aug. 31, 1990, as amended at 56 FR 66784, 66786, Dec. 26, 1991; 60 FR 14357, Mar. 17, 1995; 72 FR 72564, Dec. 21, 2007]

§113.310   Bovine Rhinotracheitis Vaccine.

Bovine Rhinotracheitis 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 for preparing the production seed virus for vaccine production. All serials of vaccine shall be prepared from the first through the tenth passage from the Master Seed Virus.

(a) The Master Seed Virus shall meet the applicable general requirements prescribed in §113.300.

(b) Each lot of Master Seed Virus shall meet the special requirements prescribed in this section.

(c) Each lot of Master Seed Virus used for vaccine production shall be tested for immunogenicity. The selected virus dose from the lot of Master Seed Virus shall be established as follows:

(1) Twenty-five infectious bovine rhinotracheitis susceptible calves shall be used as test animals (20 vaccinates and five controls). Blood samples shall be drawn from these animals and individual serums tested. The calves shall be considered susceptible if the results are negative at a 1:2 final serum dilution by the virus plaque reduction method.

(2) 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 calves to be used as vaccinates shall be injected with a predetermined quantity of vaccine virus and the remaining five calves 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.

(3) At least once during a period of 14 to 28 days post-vaccination, individual serum samples shall be collected for virus-neutralization tests from each of the vaccinates. The test virus shall be 100 to 500 TCID50 bovine rhinotracheitis virus. Results shall be used in making a determination as prescribed in paragraph (c)(6) of this section.

(4) The vaccinates and the controls shall each be challenged with virulent infectious bovine rhinotracheitis virus and observed for 14 days. The rectal temperature of each animal shall be taken and the presence or absence of respiratory or other clinical signs of bovine rhinotracheitis noted and recorded on each of the 14 consecutive days.

(5) If at least four of the five controls do not show clinical signs of infectious bovine rhinotracheitis and a marked temperature rise to 104.5 °F. or higher post-challenge, the test shall be considered a No Test and may be repeated.

(6) If less than 19 of the post-injection serum samples tested as prescribed in paragraph (c)(3) of this section show neutralization in all tubes of the 1:2 final serum dilution, or if more than one of the vaccinates show a temperature of 103.5 °F. or higher for 2 or more days, or if more than one of the vaccinates exhibit respiratory or other clinical signs of infectious bovine rhinotracheitis, or both, the Master Seed Virus is unsatisfactory.

(7) A sequential test procedure may be used in lieu of the 20 calf requirement. A beta value of .05 and a tolerance level of .78 shall be required.

(8) An outline of Production change shall be made before authority for use of a new lot of Master Seed Virus shall be granted by Animal and Plant Health Inspection Service.

(d) Test requirements for release: Each serial and subserial shall meet the applicable general requirements prescribed in §113.300 and the requirements in this paragraph. Final container samples of completed product shall be tested except as prescribed in paragraph (d)(1) of this section. Any serial or subserial found unsatisfactory by a prescribed test shall not be released.

(1) Purity test. The test for Brucella contamination prescribed in §113.32 shall be conducted on each batch of primary cells intended for production use.

(2) Safety test. The mouse safety test prescribed in §113.33(a) and the calf safety test prescribed in §113.41 shall be conducted.

(3) Virus titer requirements. Final container samples of completed product shall be tested for virus titer using the titration method used in paragraph (c)(2) of this section. To be eligible for release, each serial and each subserial shall have a virus titer per dose sufficiently greater than the titer of vaccine virus used in the immunogenicity test prescribed in paragraph (c) 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 100.7 greater than that used in the immunogenicity test but not less than 102.5 TCID50 per dose.

[39 FR 44720, Dec. 27, 1974, as amended at 40 FR 20067, May 8, 1975; 40 FR 23989, June 4, 1975; 40 FR 41089, Sept. 5, 1975; 43 FR 49529, Oct. 24, 1978; 48 FR 33472, July 22, 1983. Redesignated at 55 FR 35562, Aug. 31, 1990, as amended at 56 FR 66784, 66786, Dec. 26, 1991; 72 FR 72564, Dec. 21, 2007]

§113.311   Bovine Virus Diarrhea Vaccine.

Bovine Virus Diarrhea 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 for preparing the production seed virus for vaccine production. All serials of vaccine shall be prepared from the first through the tenth passage from the Master Seed Virus.

(a) The Master Seed Virus shall meet the applicable general requirements prescribed in §113.300.

(b) Each lot of Master Seed Virus shall meet the special requirements prescribed in this section.

(c) Each lot of Master Seed Virus used for vaccine production shall be tested for immunogenicity. The selected virus dose from the lot of Master Seed Virus shall be established as follows:

(1) Twenty-five bovine virus diarrhea susceptible calves shall be used as test animals (20 vaccinates and five controls). Blood samples shall be drawn from these animals and individuals serum samples tested. The calves shall be considered susceptible to bovine virus diarrhea virus infection if the results are negative at a 1:2 final serum dilution in a varying serum-constant virus neutralization test with less than 500 TCID50 of bovine virus diarrhea virus.

(2) 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 calves to be used as vaccinates shall be injected with a predetermined quantity of vaccine virus and the remaining five calves 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.

(3) At least once during a period 14 to 28 days post-vaccination, individual serum samples shall be collected for virus-neutralization tests from each of the vaccinates. The test virus shall be less than 500 TCID50 of bovine virus diarrhea virus. The white cell count for all vaccinates and controls shall be established at least 3 days just before challenge. Results shall be used in making a determination as prescribed in paragraph (c)(5) of this section.

(4) The vaccinates and the controls shall each be challenged with virulent bovine virus diarrhea virus and observed for 14 consecutive days. The white cell count shall be determined daily on each animal from the second through the eighth day post-challenge. If leukopenia does not develop in at least four of the five controls as compared with the vaccinates, the test shall be considered a No Test and may be repeated.

(5) If less than 19 of the post-injection serum samples, tested as prescribed in paragraph (c)(3) of this section, show neutralization in all tubes of the 1:8 dilution; or if more than one of the vaccinates exhibits respiratory or other clinical signs of bovine virus diarrhea post-challenge; or both, the Master Seed Virus is unsatisfactory.

(6) A sequential test procedure may be used in lieu of the 20 calf requirement. A beta value of .05 and a tolerance level of .78 shall be required.

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

(d) Test requirements for release: Each serial and subserial shall meet the applicable general requirements prescribed in §113.300 and the requirements in this paragraph. Final container samples of completed product shall be tested except as prescribed in paragraph (d)(1) of this section. Any serial or subserial found unsatisfactory by a prescribed test shall not be released.

(1) Purity test. The test for Brucella contamination prescribed in §113.32 shall be conducted on each batch of primary cells intended for production use.

(2) Safety test. The mouse safety test prescribed in §113.33(a) and the calf safety test prescribed in §113.41 shall be conducted.

(3) Virus titer requirements. Final container samples of completed product shall be tested for virus titer using the titration method used in paragraph (c)(2) of this section. To be eligible for release, each serial and each subserial shall have a virus titer per dose sufficiently greater than the titer of vaccine virus used in the immunogenicity test prescribed in paragraph (c) of this section to assure that when tested at any time within the expiration period, each serial and subserial shall have virus titer of 100.7 greater than that used in the immunogenicity test but not less than 102.5 TCID 50 per dose.

[39 FR 44721, Dec. 27, 1974, as amended at 40 FR 20067, May 8, 1975; 40 FR 41089, Sept. 5, 1975; 43 FR 49529, Oct. 24, 1978; 48 FR 33472, July 22, 1983. Redesignated at 55 FR 35562, Aug. 31, 1990, as amended at 56 FR 66784, 66786, Dec. 26, 1991; 72 FR 72564, Dec. 21, 2007]

§113.312   Rabies Vaccine, Live Virus.

Rabies Vaccine shall be prepared from virus-bearing cell cultures or embryonated chicken eggs. Only Master Seed Virus which has been established as pure, safe and immunogenic shall be used for preparing the production seed virus for vaccine production. All serials of vaccine shall be prepared from the first through the fifth passage from the Master Seed Virus.

(a) The Master Seed Virus shall meet the applicable general requirements prescribed in §113.300.

(1) Each lot of Master Seed Virus shall meet the special requirements prescribed in this section.

(2) Each lot of Master Seed Virus propagated in tissues or cells of avian origin shall be tested for pathogens by procedures prescribed in §113.37.

(3) Each lot of Master Seed Virus propagated in primary cell cultures of mouse or hamster origin or brain tissues of mouse origin shall be tested for lymphocytic choriomeningitis (LCM) virus by the procedure prescribed in §113.42. If LCM virus is detected, the Master Seed Virus is unsatisfactory.

(4) The Master Seed Virus shall be studied in each species of carnivore or domesticated wild animal for which the vaccine is specifically recommended to attempt to determine the fate of the vaccine virus. Results shall be considered in evaluating safety of vaccine virus.

(i) Obtain at least 10 unvaccinated animals, negative at 1:2 final serum dilution, of each species in which tests will be conducted. Divide each species into two groups of five animals.

(ii) For each species of animal, inject one group of five animals intramuscularly. Infiltrate a major nerve and the surrounding tissue in each of the five animals in the other group. Use 1.0 ml of high titer virus for each method of administration.

(iii) Observe all animals for signs of rabies until scheduled time to sacrifice. If animals show definite symptoms, sacrifice and check regional lymph nodes, brain, salivary glands, and kidney for rabies virus by injection of suckling mice (not more than 7 days of age). Tissues may be held frozen at −70 °C. until suckling mice are available. Inject each mouse in one litter intracerebrally with 0.02 ml of a ground tissue suspension from each organ. Observe mice each day for 21 days. If any mice die, determine if the deaths were due to rabies virus in the brain by a fluorescent antibody test.

(iv) Sacrifice animals that do not show signs of rabies according to the following schedule and check regional lymph nodes, brain, salivary glands, and kidney in suckling mice.

Route of injectionDays after injectionNumber of animals
Intramuscularly15, 20, 25, 30, 351 each day.
Intraneurally3, 6, 9, 15, 301 each day.

(5) Each lot of Master Seed Virus shall be tested for safety in at least 10 unvaccinated serologically negative animals of each domestic species for which the vaccine is recommended.

(i) Each group of 10 animals shall be divided into 2 groups of 5 animals. For each species, inject one group intramuscularly with 10 doses of high titer virus.

(ii) Infiltrate a major nerve of each of the animals in the other group of 5 with 10 doses of the same high titer virus. For all species except dogs and cats, multiple injections along the cervical spine in the proximity to the nerve trunks emerging from the spinal cord may be used: Provided, That a 1-dose volume shall be injected into each of four or more sites bilaterally.

(iii) Observe all animals each day for 90 days.

(iv) If any animals show clinical signs of rabies, sacrifice the animal and check appropriate brain tissue for rabies virus by the fluorescent antibody test and by mouse injection.

(v) If rabies is confirmed, the lot of Master Seed Virus is unsatisfactory.

(b) The immunogenicity of vaccine prepared with virus at the highest passage of the Master Seed shall be established in each species for which the vaccine is recommended. Tests shall be conducted in accordance with a protocol filed with Animal and Plant Health Inspection Service before initiation of the tests. The vaccine shall be prepared using methods prescribed in the Outline of Production. If Rabies Vaccine is to be in combination with other fractions, the product tested shall include all fractions to be recommended.

(1) A geometric mean virus titer of the dried vaccine produced from the highest passage of the Master Seed Virus shall be established before the immunogenicity test is conducted. To confirm the dosage calculations, five replicate virus titrations shall be conducted on a sample of the vaccine virus dilution used.

(2) The dose of vaccine to be used in the immunogenicity test shall be no more than the amount of rehydrated vaccine which, on the basis of previous titrations, has been diluted to the proposed minimum acceptable virus titer.

(3) Test animals shall be uniform and have no neutralizing antibodies to rabies as determined by serum-neutralization (SN) tests.

(i) Twenty-five or more animals shall be used as vaccinates. Each shall be injected intramuscularly at one site in the thigh with a dose of vaccine at the proposed minimum virus titer as specified in the filed Outline of Production.

(ii) Ten or more additional animals shall be held as controls.

(iii) On or about days 30, 90, 180, 270, and 365 postvaccination, all animals shall be bled and individual serums tested for neutralizing antibodies to rabies virus.

(iv) All surviving test animals of each species shall be challenged intramuscularly with virulent rabies virus furnished or approved by Animal and Plant Health Inspection Service 1 year after vaccination, except as provided in paragraphs (b)(4), (b)(5), and (b)(6) of this section. The challenged animals shall be observed each day for 90 days as prescribed in §113.5(b). The brain of each test animal that dies following challenge shall be examined for rabies by the fluorescent antibody test or other method acceptable to Animal and Plant Health Inspection Service.

(v) Requirements for acceptance in challenge tests shall be death due to rabies in at least 80 percent of controls while at least 22 of 25 or 26 of 30 or a statistically equivalent number of the vaccinates remain well for a period of 90 days.

(4) An alternative to challenging all surviving test animals in accordance with paragraph (b)(3)(iv) of this section may be used when the test animals are of species other than carnivores. Vaccinates shall be challenged at 1 year postvaccination. These shall include five vaccinates with the lowest SN titers at the 270th-day bleeding, five vaccinates with the lowest SN titers at the 365th-day bleeding, and all vaccinates with SN titers below 1:10 by the mouse SN test or below 1:16 by the rapid-fluorescent-focus-inhibition test at any bleeding. At least five SN-negative controls of each species shall be challenged at the same time as the vaccinates. All SN titers shall be iterated to an endpoint. All of the challenged vaccinates must remain well for a period of 90 days, and at least 80 percent of the controls must die of rabies for a satisfactory test without further challenge. If one or more of the vaccinates die from rabies, all the remaining vaccinates, regardless of titer, along with the five controls shall be challenged. The cumulative results from the two challenges shall be evaluated for acceptance as specified in paragraph (b)(3)(v) of this section.

(5) An outline of Production change shall be made before authority for use of a new lot of Master Virus shall be granted by Animal and Plant Health Inspection Service.

(c) If more than 1 year duration of immunity is to be claimed, a duration of immunity test for the additional time shall be conducted and interpreted as prescribed in paragraph (b) of this section for the 1 year test. The test animals shall be monitored serologically at least every 180 days. The time of challenge may be adjusted accordingly.

(d) Test requirements for release: Each serial and each subserial shall meet the general requirements prescribed in §113.300 and special requirements in this paragraph.

(1) Purity and safety tests. Final container samples of completed product from each serial or one subserial shall be tested.

(i) The test for pathogens, prescribed in §113.37 shall be conducted on each serial or one subserial of avian origin. If necessary, neutralize the rabies virus with specific rabies antiserum.

(ii) A test for safety in three young seronegative animals of the most susceptible species for which the vaccine is recommended shall be conducted. Each shall be injected intramuscularly with 10 recommended doses of vaccine. If unfavorable reactions attributable to the product occur during a 28 day observation period, the serial is unsatisfactory.

(iii) If primary cell cultures of hamster origin or of mouse origin are used vaccine production, they shall be tested for LCM virus as prescribed in §113.42. The cells shall be disrupted and undiluted cell fluids from each lot shall be tested.

(2) Virus titrations. Final container samples of completed product shall be tested for virus titer using the titration method used in paragraph (b)(1) of this section. To be eligible for release, each serial and each subserial shall have a virus titer sufficiently higher than the titer of the vaccine virus used 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 equal to or greater than that used in the immunogenicity test.

(3) Young adult mice, each weighing 14 to 16 grams, shall be used as test animals when the virus in vaccine prepared with a low egg passage Flury Strain or high cell passage Street Alabama Dufferin Strain (HCP SAD) of rabies virus is titrated. At least 10 mice for each dilution shall be used.

(i) At least 10 mice shall be used for each dilution. Each shall be injected intracerebrally with 0.03 ml.

(ii) The injected young adult mice shall be observed each day for 14 days except when testing vaccines made with HCP SAD strain of rabies virus, in which case, the mice shall be observed each day for 21 days. Deaths and paralysis occurring subsequent to the fourth day post-injection shall be noted and the LD50 titer calculated by the Reed and Muench Method.

(iii) Virus titer requirements for release and at expiration date shall be determined for each vaccine on the basis of data available: Provided, That, the lowest titer permitted at expiration date when determined by this test shall be 103.0 LD50 per 0.03 ml.

(4) Suckling mice, 6 days of age or younger, shall be used as test animals when virus in vaccine prepared with a high egg passage Flury Strain of rabies virus is titrated.

(i) Six to twelve mice shall be used for each dilution. Each shall be injected intracerebrally with 0.02 ml.

(ii) The injected suckling mice shall be observed each day for 21 days. Deaths and paralysis occurring subsequent to the fourth day post-injection shall be noted and the LD50 titer calculated by the Reed and Muench Method; and

(iii) Virus titer requirements for release and at expiration date shall be determined for each vaccine on the basis of data available: Provided, That, the lowest titer permitted at expiration date when determined by this test shall be 103.0 LD50 per 0.02 ml.

[39 FR 44721, Dec. 27, 1974, as amended at 40 FR 20067, May 8, 1975; 42 FR 6795, Feb. 4, 1977; 43 FR 49529, Oct. 24, 1978; 50 FR 20090, May 14, 1985; 50 FR 23797, June 6, 1985. Redesignated at 55 FR 35562, Aug. 31, 1990, as amended at 56 FR 66784, 66786, Dec. 26, 1991; 61 FR 31823, June 21, 1996; 72 FR 72564, Dec. 21, 2007]

§113.313   Measles Vaccine.

Measles 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 for preparing the production seed virus for vaccine production. All serials of vaccine shall be prepared from the first through the fifth passage from the Master Seed Virus.

(a) The Master Seed Virus shall meet the applicable general requirements prescribed in §113.300. Each lot of Master Seed Virus shall meet the special requirements prescribed in this section.

(b) To detect virulent canine distemper virus, each of two canine distemper susceptible ferrets shall be injected with a sample of the Master Seed Virus equivalent to the amount of virus to be used in one dog dose and observed each day for 21 days. If undesirable reactions occur in either ferret, the lot of Master Seed Virus is unsatisfactory.

(c) Each lot of Master Seed Virus used for vaccine production shall be tested for immunogenicity. The selected virus dose from the lot of Master Seed Virus shall be established as follows:

(1) Twenty-five dogs, less than 12 weeks of age and free of measles antibody, shall be used as test animals (20 vaccinates and five 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 with less than 500 ID50 of measles virus.

(2) 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. Twenty dogs shall be vaccinated with a predetermined quantity of vaccine virus and the remaining five dogs held as unvaccinated controls. To confirm the dosage calculations, five replicate virus titrations shall be conducted on a sample of the vaccine virus dilution used.

(3) On the day of challenge, serum samples shall be obtained from each vaccinate and individually tested for antibody against canine distemper virus. For a valid test, each vaccinate shall be negative at a 1:4 final serum dilution in varying serum-constant virus neutralization test using less than 500 ID50 of canine distemper virus.

(4) At least 21 days postinoculation, the immunity of the vaccinates and controls shall be challenged by exposure to a uniform dose of aerosolized virulent canine distemper virus. All test dogs shall be observed daily for 21 days postchallenge.

(i) If at least 4 of the 5 controls do not die or show signs of distemper, including a temperature of 104.0 °F. or higher and at least 15 percent weight loss, the test is a No Test and may be repeated.

(ii) If at least 19 of the 20 vaccinates do not survive without showing a temperature of 104.0 °F. or higher and a weight loss exceeding 15 percent after day 8 postchallenge, the Master Seed Virus is unsatisfactory.

(5) When approved in advance by Animal and Plant Health Inspection Service, a sequential test procedure may be used in lieu of the 20 dog requirement. A beta value of 0.05 and a tolerance level of 0.78 shall be required.

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

(d) Test requirements for release: Each serial and subserial shall meet the general requirements prescribed in §113.300 and the requirements 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) Safety tests. The dog safety test prescribed in §113.40 and the mouse safety test prescribed in §113.33(a) shall be conducted.

(2) Virus titer requirements. Final container samples of completed product shall be tested for virus titer using the titration method used in paragraph (c)(2) of this section. To be eligible for release, each serial and each subserial shall have a virus titer sufficiently greater than the titer of the vaccine virus used in the immunogenicity test prescribed in paragraph (c) 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 100.7 greater than that used in the immunogenicity test but not less than 102.5 ID50 per dose.

[40 FR 53001, Nov. 14, 1975, as amended at 43 FR 49529, Oct. 24, 1978; 48 FR 33472, July 22, 1983. Redesignated at 55 FR 35562, Aug. 31, 1990, as amended at 56 FR 66784, 66786, Dec. 26, 1991; 72 FR 72564, Dec. 21, 2007]

§113.314   Feline Calicivirus Vaccine.

Feline Calicivirus 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 for preparing the production seed virus for vaccine production. All serials of vaccine shall be prepared from the first through the fifth passage from the Master Seed Virus.

(a) The Master Seed Virus shall meet the applicable general requirements prescribed in §113.300.

(b) The Master Seed Virus shall be tested for chlamydial agents as prescribed in §113.43.

(c) Each lot of Master Seed Virus used for vaccine production shall be tested for immunogenicity. The selected virus dose from the lot of Master Seed Virus shall be established as follows:

(1) Thirty feline calicivirus susceptible cats shall be used as test animals (20 vaccinates and 10 controls). Throat swabs shall be collected from each cat and individually tested on susceptible cell cultures for the presence of feline calicivirus. Blood samples shall be drawn and individual serum samples tested. The cats shall be considered suitable for use if all swabs are negative for virus isolation and if all serums are negative for calicivirus antibody at the 1:2 final dilution in a 50 percent plaque reduction test or other SN test of equal sensitivity.

(2) 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 cats used as vaccinates shall be administered a predetermined quantity of vaccine virus by the method to be recommended on the label and the remaining 10 cats shall be held as controls. To confirm the dosage calculations, five replicate virus titrations shall be conducted on a sample of the vaccine virus dilution used. If two doses are used, five replicate confirming titrations shall be conducted on each dose.

(3) Twenty-one or more days after the final dose of vaccine, the vaccinates and controls shall each be challenged intranasally with a minimum of 100,000 TCID50 or plaque forming units of virulent feline calicivirus furnished or approved by 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 or absence of clinical signs, particularly lesions on the oral mucosa, noted and recorded each day.

(i) If less than 8 of 10 controls show clinical signs of feline calicivirus infection other than fever, the test is a No Test and may be repeated.

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

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

(d) 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) Safety test. The mouse safety test prescribed in §113.33(a) and the cat safety test prescribed in §113.39(b) shall be conducted.

(2) Virus titer requirements. Final container samples of completed product shall be tested for virus titer using the titration method used in paragraph (c)(2) 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 prescribed in paragraph (c) 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 100.7 greater than that used in the immunogenicity test but not less than 102.5 TCID50 or plaque forming units per dose.

[44 FR 58899, Oct. 12, 1979; 44 FR 63083, Nov. 2, 1979, as amended at 48 FR 33472, July 22, 1983. Redesignated at 55 FR 35562, Aug. 31, 1990, as amended at 56 FR 66784, 66786, Dec. 26, 1991; 72 FR 72564, Dec. 21, 2007]

§113.315   Feline Rhinotracheitis Vaccine.

Feline Rhinotracheitis 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 for preparing the production seed virus for vaccine production. All serials of vaccine shall be prepared from the first through the fifth passage from the Master Seed Virus.

(a) The Master Seed Virus shall meet the applicable general requirements prescribed in §113.300.

(b) The Master Seed Virus shall be tested for chlamydial agents as prescribed in §113.43.

(c) Each lot of Master Seed Virus used for vaccine production shall be tested for immunogenicity. The selected virus dose from the lot of Master Seed Virus shall be established as follows:

(1) Thirty feline rhinotracheitis susceptible cats shall be used as test animals (20 vaccinates and 10 controls). Throat swabs shall be collected from each cat and individually tested on susceptible cell cultures for the presence of feline rhinotracheitis virus. Blood samples shall be drawn and individual serum samples tested. The cats shall be considered suitable for use if all swabs are negative for virus isolation and if all serums are negative for feline rhinotracheitis virus antibody at the 1:2 final dilution in a 50 percent plaque reduction test or other SN test of equal sensitivity.

(2) 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 cats used as vaccinates shall be administered a predetermined quantity of vaccine virus by the method to be recommended on the label and the remaining 10 cats shall be held as controls. To confirm the dosage calculations, five replicate virus titrations shall be conducted on a sample of the vaccine virus dilution used. If two doses are used, five replicate confirming titrations shall be conducted on each dose.

(3) Twenty-one or more days after the final dose of vaccine, the vaccinates and controls shall each be challenged intranasally with a minimum of 100,000 TCID50 or plaque forming units of virulent feline rhinotracheitis virus furnished or approved by Animal and Plant Health Inspection Service and observed each day for 14 days post-challenge. The rectal temperature of each animal shall be taken and the presence of respiratory or other clinical signs of feline rhinotracheitis noted and recorded each day.

(i) If less than 8 of 10 controls show clinical signs of feline rhinotracheitis infection other than fever, the test is a No Test and may be repeated.

(ii) If a significant difference in clinical signs cannot be demonstrated between vaccinates and controls using a scoring system approved by Veterinary Services and prescribed in the Outline of Production, the Master Seed Virus is unsatisfactory.

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

(d) 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) Safety test. The mouse safety test prescribed in §113.33(a) and the cat safety test prescribed in §113.39(b) shall be conducted.

(2) Virus titer requirements. Final container samples of completed product shall be tested for virus titer using the titration method used in paragraph (c)(2) 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 prescribed in paragraph (c) 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 100.7 greater than that used in the immunogenicity test but not less than 102.5 TCID50 or plaque forming units per dose.

[44 FR 58899, Oct. 12, 1979, as amended at 48 FR 33472, July 22, 1983. Redesignated at 55 FR 35562, Aug. 31, 1990, as amended at 56 FR 66784, 66786, Dec. 26, 1991; 72 FR 72564, Dec. 21, 2007]

§113.316   Canine Parainfluenza Vaccine.

Canine Parainfluenza Vaccine shall be prepared from virus-bearing cell culture fluids. Only Master Seed which has been established as pure, safe, and immunogenic shall be used for preparing seeds for vaccine production. All serials of vaccine shall be prepared from the first through the fifth passage from the Master Seed.

(a) The Master Seed shall meet the applicable general requirements prescribed in §113.300 and the requirements in this section.

(b) Each lot of Master Seed shall be tested for immunogenicity. The selected virus dose shall be established as follows:

(1) Twenty-five canine parainfluenza susceptible dogs (20 vaccinates and 5 controls) shall be used as test animals. Nasal swabs shall be collected from each dog on the day the first dose of vaccine is administered and individually tested on susceptible cell cultures for the presence of canine parainfluenza virus. Blood samples shall also be drawn and individual serum samples tested for neutralizing antibody. Dogs shall be considered susceptible if all swabs are negative for virus isolation and if all serums are negative for canine parainfluenza antibody at a 1:2 final dilution in a constant virus-varying serum neutralization test using 50 to 300 TCID50 of canine parainfluenza virus.

(2) A geometric mean titer of vaccine produced at the highest passage from the Master Seed shall be established before the immunogenicity test is conducted. The 20 dogs used as vaccinates shall be administered a predetermined quantity of vaccine virus. Five replicate virus titrations shall be conducted on a sample of the vaccine virus dilution used to confirm the dosage administered. If two doses are used, five replicate confirming titrations shall be conducted on each dose.

(3) Three to 4 weeks after the final dose of vaccine, all dogs shall be bled for serum antibodies and nasal swabs shall be collected for canine parainfluenza virus isolation. On the same day, all vaccinates and controls shall be challenged with canine parainfluenza virus furnished or approved by Animal and Plant Health Inspection Service.

(4) The rectal temperature of each dog shall be taken and the presence of respiratory or other clinical signs of canine parainfluenza virus infection noted and recorded each day for 14 consecutive days postchallenge. Nasal swabs shall be collected from each dog each day for at least 10 consecutive days postchallenge. Individual swabs shall be tested for virus isolation by culture in canine parainfluenza virus susceptible cells for at least 7 days. Results shall be evaluated according to the following criteria:

(i) If five of five controls have not remained seronegative at a final serum dilution of 1:2 during the prechallenge period, the test is a No Test and may be repeated.

(ii) If more than one vaccinate shows febrile response, respiratory or other clinical signs of canine parainfluenza virus infection; or, if less than 19 of 20 vaccinates show serum neutralization titers of 1:4 or greater; or, if there is not a significant reduction in virus isolation rate in vaccinates when compared with controls, the Master Seed is unsatisfactory.

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

(c) Test requirements for release. Each serial and subserial shall meet the applicable general requirements prescribed in §113.300 and the requirements in this paragraph. 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)(2) 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 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 at least 100.7 greater than that used in the immunogenicity test but not less than 102.5 TCID50 per dose.

(2) [Reserved]

[50 FR 436, Jan. 4, 1985. Redesignated at 55 FR 35562, Aug. 31, 1990, as amended at 56 FR 66784, 66786, Dec. 26, 1991; 72 FR 72564, Dec. 21, 2007]

§113.317   Parvovirus Vaccine (Canine).

Parvovirus Vaccine recommended for use in dogs shall be prepared from virus-bearing cell culture fluids. Only Master Seed which has been established as pure, safe, and immunogenic shall be used for preparing seeds for vaccine production. All serials of vaccine shall be prepared from the first through the fifth passage from the Master Seed.

(a) The Master Seed shall meet the applicable general requirements prescribed in §113.300 and the requirements in this section.

(b) The Master Seed shall be tested for reversion to virulence in dogs using a method acceptable to Animal and Plant Health Inspection Service. If a significant increase in virulence is seen within five backpassages, the Master Seed is unsatisfactory.

(c) Each lot of Master Seed shall be tested for immunogenicity. The selected virus dose shall be established as follows:

(1) Twenty-five canine parvovirus susceptible dogs (20 vaccinates and 5 controls) shall be used as test animals. Blood samples drawn from each dog shall be individually tested for neutralizing antibody against canine parvovirus to determine susceptibility. Dogs shall be considered susceptible if there is no neutralization at a 1:2 final serum dilution in a constant virus-varying serum neutralization test in cell culture using 50 to 300 TCID50 of canine parvovirus.

(2) A geometric mean titer of the vaccine produced at the highest passage from the Master Seed shall be established before the immunogenicity test is conducted. The 20 dogs used as vaccinates shall be administered a predetermined quantity of vaccine virus by the method recommended on the label. To confirm the dosage calculations, five replicate virus titrations shall be conducted on a sample of the vaccine virus dilution used. If two doses are used, five replicate confirming titrations shall be conducted on each dose.

(3) Fourteen days or more after the final dose of vaccine the vaccinates and the controls shall be challenged with virulent canine parvovirus furnished or approved by Animal and Plant Health Inspection Service and the dogs observed each day for 14 days. Rectal temperature, blood lymphocyte count, and feces for viral detection shall be taken from each dog each day for at least 10 days postchallenge and the presence or absence of clinical signs noted and recorded each day.

(i) The immunogenicity of the Master Seed shall be evaluated on the following criteria of infection: temperature ≥103.4 °F; lymphopenia of ≥50 percent of prechallenge normal; clinical signs such as diarrhea, mucus in feces, or blood in feces; and viral hemagglutinins at a level of ≥1:64 in a 1:5 dilution of feces or a test of equal sensitivity. If at least 80 percent of the controls do not show at least three of the four criteria of infection during the observation period, the test is a No Test and may be repeated.

(ii) If at least 19 of the 20 vaccinates do not survive the observation period without showing more than one criterion of infection described in paragraph (c)(3)(i), of this section, the Master Seed is unsatisfactory.

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

(d) Test requirements for release. Each serial and subserial shall meet the applicable general requirements prescribed in §113.300 and the requirements in this paragraph. 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 (c)(2) 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 used in the immunogenicity test in paragraph (c) 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 100.7 greater than that used in the immunogenicity test, but not less than 102.5 ID50 per dose.

[50 FR 436, Jan. 4, 1985. Redesignated at 55 FR 35562, Aug. 31, 1990, as amended at 56 FR 66784, 66786, Dec. 26, 1991; 72 FR 72564, Dec. 21, 2007]

§113.318   Pseudorabies Vaccine.

Pseudorabies Vaccine shall be prepared from virus-bearing cell culture fluids. Only Master Seed which has been established as pure, safe, and immunogenic shall be used for preparing seeds for vaccine production. All serials of vaccine shall be prepared from the first through the fifth passage from the Master Seed.

(a) The Master Seed shall meet the applicable general requirements prescribed in §113.300 and the requirements in this section.

(b) Each lot of Master Seed shall be tested for immunogenicity. The selected virus dose shall be established as follows:

(1) Twenty-five pseudorabies susceptible pigs (20 vaccinates and 5 controls) of the youngest age for which the vaccine is recommended, shall be used as test animals. Blood samples shall be taken from each pig and the serums inactivated and individually tested for neutralizing antibody against pseudorabies virus. Pigs shall be considered susceptible if there is no neutralization at a 1:2 final serum dilution in a constant virus-varying serum neutralization test using 50 to 300 TCID50 pseudorabies virus.

(2) A geometric mean titer of the vaccine produced at the highest passage from the Master Seed shall be established before the immunogenicity test is conducted. The 20 pigs used as vaccinates shall be administered a predetermined quantity of vaccine virus by the method recommended on the label. To confirm the dosage administered, five replicate virus titrations shall be conducted on a sample of the vaccine virus dilution used.

(3) Fourteen to 28 days postvaccination, the vaccinates and controls shall be challenged with virulent pseudorabies virus furnished or approved by Animal and Plant Health Inspection Service and observed each day for 14 days.

(i) If at least four of the five controls do not develop severe central nervous system signs or die, the test is a No Test and may be repeated.

(ii) If at least 19 of the 20 vaccinates in a valid test do not remain free of signs of pseudorabies, the Master Seed is unsatisfactory.

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

(c) Test requirements for release. Each serial and subserial shall meet the applicable general requirements prescribed in §113.300 and the requirements in this paragraph.

(2) Virus titer requirements. Final container samples of completed product shall be titrated by the method used in paragraph (b)(2) of this section. To be eligible for release, each serial and subserial shall have a virus titer sufficiently greater than the titer of the vaccine used in the immunogenicity test 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 at least 10.0.7 greater than that used in the immunogenicity test, but not less than 102.5 TCID50 per dose.

[50 FR 437, Jan. 4, 1985. Redesignated at 55 FR 35562, Aug. 31, 1990, as amended at 56 FR 66784, 66786, Dec. 26, 1991; 72 FR 72564, Dec. 21, 2007]

§§113.319-113.324   [Reserved]

§113.325   Avian Encephalomyelitis Vaccine.

Avian Encephalomyelitis Vaccine shall be prepared from virus-bearing tissues or fluids from embryonated chicken eggs. Only Master Seed Virus which has been established as pure, safe, and immunogenic in accordance with the requirements in paragraphs (a), (b), and (c) of this section shall be used for 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 and the requirements prescribed in this section.

(b) Each lot of Master Seed Virus shall be tested for pathogens by the chicken embryo inoculation test prescribed in §113.37, except that, if the test is a No Test because of a vaccine virus override, the test may be repeated and if the repeat test is inconclusive for the same reason, the chicken inoculation test prescribed in §113.36 may be conducted and the virus judged accordingly.

(c) Each lot of Master Seed Virus shall be tested for immunogenicity and the selected virus dose to be used shall be established as follows:

(1) Avian encephalomyelitis susceptible chickens, all of the same age (eight weeks or older) and from the same source, shall be used. Twenty or more chickens shall be used as vaccinates for each method of administration recommended on the label. Ten additional chickens of the same age and from the same source shall be held as unvaccinated controls.

(2) A geometric mean titer of the vaccine produced from the highest passage of the Master Seed Virus shall be established before the immunogenicity test is conducted. Each vaccinate shall receive a predetermined quantity of vaccine virus. Five replicate virus titrations shall be conducted on an aliquot of the vaccine virus to confirm the amount of virus administered to each chicken used in the test. At least three appropriate (not to exceed tenfold) dilutions shall be used and the test conducted as follows:

(i) For each dilution, inoculate at least 10 embryos, 5 or 6 days old, in the yolk sac with 0.2 ml each. Twenty similar embryos obtained from the same source shall be kept as uninoculated negative controls. Disregard all deaths during the first 48 hours post-inoculation.

(ii) Eggs for each dilution shall be kept in separate containers and allowed to hatch. Sufficient precaution shall be taken to assure that chickens from each dilution remain separated. To be a valid test, at least 75 percent of the uninoculated eggs shall hatch.

(iii) On the third day after normal hatching time, count all unhatched eggs and all dead, paralyzed and ataxic chickens as positive evidence of viral infection.

(iv) A satisfactory titration shall have at least one dilution with between 50 and 100 percent positives and at least one dilution with between 50 and 0 percent positives.

(v) Calculate the EID50 by the Spearman-Karber or Reed-Muench method.

(3) At least 21 days post-vaccination, the vaccinates and the controls shall be challenged intracerebrally with a virulent avian encephalomyelitis virus and observed each day for 21 days.

(4) If at least 80 percent of the controls do not show signs of avian encephalomyelitis or die, the test is a No Test and may be repeated. If at least 19 of 20, or 27 of 30, or 36 of 40 of the vaccinates in each group do not remain free from clinical signs of avian encephalomyelitis during the observation period, the Master Seed Virus is unsatisfactory.

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

(d) After a lot of Master Seed Virus has been established as prescribed in paragraphs (a), (b), and (c) of this section, each serial and subserial shall meet the applicable requirements in §113.300 and the requirements prescribed in this paragraph.

(1) Final container samples from each serial shall be tested for pathogens by the chicken embryo inoculation test prescribed in §113.37, except that, if the test is a No Test because of a vaccine virus override, the chicken inoculation test prescribed in §113.36 may be conducted and the vaccine judged accordingly.

(2) Safety test. Final container samples of completed product shall be tested for safety as follows:

(i) At least 25 AE susceptible birds (6 to 10 weeks of age) shall be vaccinated with the equivalent of 10 doses by each of all routes recommended on the label and be observed each day for 21 days.

(ii) If unfavorable reactions attributable to the biological product occur during the observation period, the serial is unsatisfactory. If unfavorable reactions occur which are not attributable to the product, the test shall be declared a No Test and repeated, except that, if the test is not repeated, the serial shall be unsatisfactory.

(3) Virus titer requirements. Final container samples of completed product shall be tested for virus titer using the titration method used in paragraph (c)(2) 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 prescribed in paragraph (c) 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 100.7 greater than that used in such immunogenicity test but not less than 10.5 EID50 per dose.

[39 FR 44723, Dec. 27, 1974, as amended at 40 FR 18405, Apr. 28, 1975; 40 FR 41089, Sept. 5, 1975; 42 FR 43617, Aug. 30, 1977; 48 FR 33473, July 22, 1983. Redesignated at 55 FR 35562, Aug. 31, 1990, as amended at 56 FR 66784, 66786, Dec. 26, 1991; 72 FR 72564, Dec. 21, 2007; 79 FR 55969, Sept. 18, 2014]

§113.326   Avian Pox Vaccine.

Fowl Pox Vaccine and Pigeon Pox Vaccine shall be prepared from virus-bearing cell culture fluids or embryonated chicken eggs. Only Master Seed Virus which has been established as pure, safe, and immunogenic in accordance with the requirements in paragraphs (a), (b), and (c) of this section shall be used for 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 paragraph (c) of this section and shall meet the requirements prescribed in this section.

(b) Each lot of Master Seed Virus shall be tested for pathogens by the chicken inoculation test prescribed in §113.36.

(c) Each lot of Master Seed Virus shall be tested for immunogenicity and the selected virus dose to be used shall be established as follows:

(1) Fowl pox susceptible birds all of the same age and from the same source, shall be used as test birds. Twenty or more birds shall be used as vaccinates for each method of administration recommended on the label. Ten additional birds of the same age and from the same source as the vaccinates shall be held as unvaccinated controls.

(2) 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. Each vaccinate shall receive a predetermined quantity of vaccine virus. Five replicate virus titrations shall be conducted on an aliquot of the vaccine virus to confirm the amount of virus administered to each bird used in the test. At least three appropriate (not to exceed tenfold) dilutions shall be used and the test conducted as follows:

(i) For each dilution, inoculate at least five embryos, 9 to 11 days old, on the chorioallantoic membrane with at least 0.2 ml each. Disregard all deaths during the first 24 hours post-inoculation. To be a valid test, at least four embryos in each dilution shall remain viable beyond 24 hours.

(ii) Examine the surviving embryos for evidence of infection 5 to 7 days post-inoculation.

(iii) A satisfactory titration shall have at least one dilution with between 50 and 100 percent positives and at least one dilution with between 50 and 0 percent positives.

(iv) Calculate the EID50 by the Spearman-Karber or Reed-Muench method.

(3) Fourteen to twenty-one days post-vaccination, all vaccinates and controls shall be challenged by the wing web method and observed each day for 10 days. If the wing web method was used for vaccination, the opposite wing shall be used for challenge. Challenge virus shall be provided or approved by Animal and Plant Health Inspection Service.

(4) If at least 90 percent of the controls do not develop fowl pox during the observation period, the test is a No Test and may be repeated. If at least 19 of 20, or 27 of 30, or 36 of 40 of the vaccinates in each group do not remain free from clinical signs of fowl pox during the observation period, the Master Seed Virus is unsatisfactory.

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

(d) After a lot of Master Seed Virus has been established as prescribed in paragraphs (a), (b), and (c) of this section, each serial and subserial shall meet the requirements in §113.36, in §113.300 except paragraph (c), and in this paragraph.

(1) Safety test. Final container samples of completed product from each serial shall be tested. Vaccines recommended for use in birds 10 days of age or younger shall be tested in accordance with paragraphs (d)(1)(i), (ii), and (iii) of this section.

(i) Each of 25 susceptible birds 5 days of age or younger, properly identified and obtained from the same source and hatch, shall be vaccinated with the equivalent of 10 doses of vaccine by each of all routes recommended on the label and observed each day for 14 days. Severe clinical signs or death shall be counted as failures. Two-stage sequential testing may be conducted if the first test (which then becomes stage one) has three failures.

(ii) The results shall be evaluated according to the following table:

Cumulative Totals

StageNumber of birdsFailures for satisfactory serialsFailures for unsatisfactory serials
1252 or less4 or more.
2505 or less6 or more.

(iii) If unfavorable reactions occur which are not attributable to the product, the test shall be declared a No Test and may be repeated or, in lieu thereof, the serial declared unsatisfactory.

(iv) Vaccines not recommended for use in birds 10 days of age or younger shall be tested for safety as follows: Each of twenty-five 3- to 5-week-old, fowl-pox susceptible birds shall be vaccinated with the equivalent of 10 doses of vaccine by each of all routes recommended on the label and observed each day for 14 days. If any of the birds show severe clinical signs of disease or death during the observation period due to causes attributable to the product, the serial is unsatisfactory. If unfavorable reactions occur which are not attributable to the product, the test shall be declared a No Test and may be repeated or, in lieu thereof, the serial declared unsatisfactory.

(2) Virus titer requirements. Final container samples of completed product shall be tested for virus titer using the titration method used in paragraph (c)(2) 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 prescribed in paragraph (c) 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 100.7 greater than that used in such immunogenicity test but not less than 102.0 EID50 per dose.

[39 FR 44724, Dec. 27, 1974, as amended at 40 FR 18406, Apr. 28, 1975; 40 FR 41089, Sept. 5, 1975; 44 FR 33051, June 8, 1979; 48 FR 33473, July 22, 1983. Redesignated at 55 FR 35562, Aug. 31, 1990, as amended at 56 FR 66784, 66786, Dec. 26, 1991; 72 FR 72564, Dec. 21, 2007]

§113.327   Bronchitis Vaccine.

Bronchitis Vaccine shall be prepared from virus-bearing cell culture fluids or embryonated chicken eggs. Only Master Seed Virus which has been established as pure, safe, and immunogenic in accordance with the requirements in paragraphs (a), (b), and (c) of this section shall be used for 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 and the requirements prescribed in this section.

(b) Each lot of Master Seed Virus shall be tested for pathogens by the chicken embryo inoculation test prescribed in §113.37, except that, if the test is a No Test because of a vaccine virus override, the test may be repeated and if the repeat test is a No Test for the same reason, the chicken inoculation test prescribed in §113.36 may be conducted and the virus judged accordingly.

(c) Each lot of Master Seed Virus used for vaccine production shall be tested for immunogenicity and the selected virus dose to be used shall be established as follows:

(1) Bronchitis susceptible chickens, all of the same age and from the same source, shall be used in the virus-recovery test. For each method of administration recommended on the label for each serotype against which protection is claimed, twenty or more chickens shall be used as vaccinates. Ten additional chickens for each serotype against which protection is claimed shall be held as unvaccinated controls.

(2) A geometric mean titer of the dried vaccine produced from the highest passage of the Master Seed Virus shall be established before the immunogenicity tests are conducted. Each vaccinate shall receive a predetermined quantity of vaccine virus. Five replicate virus titrations shall be conducted on an aliquot of the vaccine virus to confirm the amount of virus administered to each chicken used in such tests. At least three approved (not to exceed tenfold) dilutions shall be used and the test conducted as follows;

(i) For each dilution, inject at least five embryos, 9 to 11 days old, in the allantoic cavity with 0.1 ml each. Deaths occurring during the first 24 hours shall be disregarded, but at least four viable embyros in each dilution shall survive beyond 24 hours of a valid test. After 5 to 8 days incubation, examine the surviving embryos for evidence of infection.

(ii) A satisfactory titration shall have at least one dilution with between 50 and 100 percent positives and at least one dilution with between 50 and 0 percent positives.

(iii) Calculate the EID50 by the Spearman-Karber or Reed-Muench method.

(3) Twenty-one to twenty-eight days post-vaccination, all vaccinates and controls shall be challenged by eye-drop with virulent bronchitis virus. A separate set of vaccinates and controls shall be used for each serotype against which protection is claimed. Each challenge virus shall be approved or provided by Animal and Plant Health Inspection Service and shall titer at least 104.0 EID50 per ml.

(i) Tracheal swabs shall be taken once, 5 days post-challenge, from each control and vaccinate. Each swab shall be placed in a test tube containing 3 ml of tryptose phosphate broth and antibiotics. The tube and swab shall be swirled thoroughly and if they are to be stored, be immediately frozen and be stored at below −40 °C. pending egg evaluation. For each chicken swab, at least five chicken embryos 9 to 11 days old shall be inoculated in the allantoic cavity with 0.2 ml each of broth from each tube.

(ii) All embryos surviving the third day post-inoculation shall be used in the evaluation, except that, if a swab is not represented by at least four embryos, the test of that swab is invalid and the results a No Test. A tracheal swab shall be positive for virus recovery when any of the embryos in a valid test show typical infectious bronchitis virus lesions, such as but not limited to, stunting, curling, kidney urates, clubbed down, or death during the 4 to 7 day post-inoculation period. If less than 20 percent of the embryos which survive the third day post-inoculation die during the 4 to 7 day post-inoculation period and show no gross lesions typical of infectious bronchitis, they may be disregarded.

(iii) If less than 90 percent of the controls are positive for virus recovery, the test is a No Test and may be repeated.

(iv) If less than 90 percent of the vaccinates are negative for virus recovery, the Master Seed Virus is unsatisfactory.

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

(d) After a lot of Master Seed Virus has been established as prescribed in paragraphs (a), (b), and (c) of this section, each serial and subserial shall meet the applicable requirements in §113.300 and the requirements prescribed in this paragraph, except that, if the vaccine contains more than one virus type, bulk samples taken from each type prior to mixing shall be used in the virus identity tests prescribed in §113.300(c). The additional requirements in this paragraph shall also be met.

(1) Final container samples from each serial shall be tested for pathogens by the chicken embryo inoculation test prescribed in §113.37, except that, if the test is a No Test because of a vaccine virus override, the chicken inoculation test prescribed in §113.36 may be conducted and the vaccine judged accordingly.

(2) Safety test. Final container samples of completed product shall be tested to determine safety for use in bronchitis susceptible young chickens.

(i) Twenty-five susceptible chickens, 5 days of age or younger, properly identified and obtained from the same source and hatch, shall be vaccinated by the eye-drop method with the equivalent of 10 doses of vaccine and observed each day for 21 days post-vaccination. Severe respiratory signs or death shall be counted as failures. Two-stage sequential testing may be conducted if the first test (which then becomes stage one) has three failures.

(ii) The results shall be evaluated according to the following table:

Cumulative Totals

StageNumber of chickensFailures for satisfactory serialsFailures for unsatisfactory serials
1252 or less4 or more.
2505 or less6 or more.

If unfavorable reactions occur which are not attributable to the product, the test shall be declared a No Test and repeated or, in lieu thereof, the serial declared unsatisfactory.

(3) Virus titer requirements. Final container samples of completed product shall be tested for virus titer using the procedure prescribed in paragraph (c)(2) of this section and in this paragraph.

(i) The Newcastle disease virus fraction of combined Newcastle-Bronchitis Vaccines shall be neutralized prior to titration of the bronchitis virus fraction. Equal parts of heat-inactivated Newcastle disease antiserum shall be mixed with each appropriate serial ten-fold dilution of the vaccine. After inactivation, embryos shall be injected with 0.2 ml each and results calculated as a 0.1 ml dose to allow for serum dilution of the vaccine. The allantoic fluids, tested as prescribed in §113.34 shall not show hemagglutinating activity in the lowest dilution used in the titration.

(ii) Each bronchitis virus type shall be harvested separately and a sample of bulk harvested material shall be collected prior to mixing with the other virus type(s). Each sample shall contain not less than the minimum virus titer stated in the filed Outline of Production.

(iii) 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 prescribed in paragraph (c) 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 100.7 greater than that used in such immunogenicity test but not less than 102.0 EID50 per dose.

[39 FR 44724, Dec. 27, 1974, as amended at 40 FR 18406, Apr. 28, 1975; 40 FR 41089, Sept. 5, 1975; 42 FR 43617, Aug. 30, 1977; 48 FR 33473, July 22, 1983. Redesignated at 55 FR 35562, Aug. 31, 1990, as amended at 56 FR 66784, 66786, Dec. 26, 1991; 64 FR 43045, Aug. 9, 1999; 72 FR 72564, Dec. 21, 2007]

§113.328   Fowl Laryngotracheitis Vaccine.

Fowl Laryngotracheitis Vaccine shall be prepared from virus-bearing cell culture fluids or embryonated chicken eggs. Only Master Seed Virus which has been established as pure, safe, and immunogenic in accordance with the requirements in paragraphs (a), (b), and (c) of this section shall be used for 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 and the requirements prescribed in this section.

(b) Each lot of Master Seed Virus shall be tested for pathogens by the chicken embryo inoculation test prescribed in §113.37, except that, if the test is a No Test because of vaccine virus override, the test may be repeated and if the repeat test is a No Test for the same reason, the chicken inoculation test prescribed in §113.36 may be conducted and the virus judged accordingly. Each lot shall also be tested for safety as follows:

(1) Each of at least ten 3 to 4 week old susceptible chickens obtained from the same source and hatch as those used in the immunogenicity test prescribed in paragraph (c) of this section shall be injected intratracheally with 0.2 ml of the virus as used in the vaccine and the chickens observed each day for 14 days.

(2) If more than 20 percent of the chickens die during the observation period, the virus is unsatisfactory.

(c) Each lot of Master Seed Virus used for vaccine production shall be tested for immunogenicity and the selected virus dose to be used shall be established as follows:

(1) Fowl laryngotracheitis susceptible chickens all of the same age and from the same source shall be used. Twenty or more chickens shall be used as vaccinates for each method of administration recommended on the label. Ten additional chickens of the same age and from the same source shall be held as unvaccinated controls.

(2) 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. Each vaccinate shall receive a predetermined quantity of vaccine virus. Five replicate virus titrations shall be conducted on an aliquot of the vaccine virus to confirm the amount of virus administered to each chicken used in the test. At least three appropriate (not to exceed tenfold) dilutions shall be used for vaccine of chicken embryo origin and the test conducted as follows:

(i) For each dilution, inject at least five embryos, 9 to 11 days old, on the chorioallantoic membrane with 0.2 ml each. Disregard all deaths during the first 24 hours post-injection. To be a valid test, at least four embryos in each dilution shall remain viable beyond 24 hours.

(ii) Examine the surviving embryos for evidence of infection 5 to 8 days post-injection.

(iii) A satisfactory titration shall have at least one dilution with between 50 and 100 percent positives and at least one dilution with between 50 and 0 percent positives.

(iv) Calculate the EID50 by the Spearman-Karber or Reed-Muench method.

(3) Tissue culture origin vaccine may be titrated by a tissue culture method approved by Animal and Plant Health Inspection Service and written into the filed Outline of Productions.

(4) Ten to fourteen days post-vaccination, all vaccinates and controls shall be challenged intratracheally or in the orbital sinus with infectious fowl laryngotracheitis virus and observed each day for 10 days. Challenge virus shall be provided or approved by Animal and Plant Health Inspection Service.

(5) If at least 80 percent of the controls do not die or show clinical signs of fowl laryngotracheitis during the observation period, the test is a No Test and may be repeated. If at least 19 of 20, 27 of 30, or 36 of 40 of the vaccinates in each group do not remain free of clinical signs of fowl laryngotracheitis during the observation period, the Master Seed Virus is unsatisfactory.

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

(d) After a lot of Master Seed Virus has been established as prescribed in paragraphs (a), (b), and (c) of this section, each serial and subserial shall meet the applicable requirements in §113.300 and the requirements prescribed in this paragraph.

(1) Final container samples from each serial shall be tested for pathogens by the chicken embryo inoculation test prescribed in §113.37, except that, if the test is a No Test because of a vaccine virus override, the chicken inoculation test prescribed in §113.36 may be conducted and the vaccine judged accordingly.

(2) Safety test. Final container samples of completed product from each serial of modified live virus vaccine shall be tested for safety as provided in this paragraph. Live virus vaccine not prepared with modified live virus shall be tested for safety as provided in the filed Outline of Production.

(i) Twenty-five 3 to 4 week old laryngotracheitis susceptible chickens shall be injected intratracheally with 0.2 ml of vaccine rehydrated at the rate of 30 ml for 1,000 doses. Chickens shall be observed each day for 14 days. Deaths shall be counted as failures. Two-stage sequential testing may be conducted if the first test (which then becomes stage one) has five, six, or seven failures.

(ii) The results shall be evaluated according to the following table:

Cumulative Totals

StageNumber of chickensFailures for satisfactory serialsFailures for unsatisfactory serials
1254 or less8 or more.
25010 or less11 or more.

(iii) If unfavorable reactions occur which are not attributable to the product, the test shall be declared a No Test and repeated or in lieu thereof, the serial declared unsatisfactory.

(3) Virus titer requirements. Final container samples of completed product shall be tested for virus titer using the titration method provided in paragraphs (c)(2) or (3) 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 prescribed in paragraph (c) 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 100.7 greater than that used in such immunogenicity test but not less than 102.5 EID50 per dose for chicken embryo origin vaccine and 102.0 EID50 or 102.5 TCID50 per dose for tissue culture origin vaccine.

[39 FR 44726, Dec. 27, 1974, as amended at 40 FR 18407, Apr. 28, 1975; 40 FR 41089, Sept. 5, 1975; 41 FR 44359, Oct. 8, 1976; 42 FR 43617, Aug. 30, 1977; 48 FR 33473, July 22, 1983. Redesignated at 55 FR 35562, Aug. 31, 1990, as amended at 56 FR 66784, 66786, Dec. 26, 1991; 72 FR 72564, Dec. 21, 2007]

§113.329   Newcastle Disease Vaccine.

Newcastle Disease Vaccine shall be prepared from virus-bearing cell culture fluids or embryonated chicken eggs. Only Master Seed Virus which has been established as pure, safe, and immunogenic in accordance with the requirements in paragraphs (a), (b), and (c) of this section shall be used for 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 §113.34, and the requirements prescribed in this section.

(b) Each lot of Master Seed Virus shall be tested for pathogens by the chicken embryo inoculation test prescribed in §113.37, except that, if the test is a No Test because of a vaccine virus override, the test may be repeated and if the repeat test is a No Test for the same reason, the chicken inoculation test prescribed in §113.36 may be conducted and the virus judged accordingly.

(c) Each lot of Master Seed Virus used for vaccine production shall be tested for immunogenicity and the selected virus dose to be used shall be established as follows:

(1) Newcastle Disease susceptible chickens, all of the same age and from the same source, shall be used. Twenty or more chickens shall be used as vaccinates for each method of administration recommended on the label. Ten additional chickens of the same age and from the same source shall be held as unvaccinated controls.

(2) 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. Each vaccinate shall receive a predetermined quantity of vaccine virus. Five replicate virus titrations shall be conducted on an aliquot of the vaccine virus to confirm the amount of virus administered to each chicken used in the test. At least three appropriate (not to exceed tenfold) dilutions shall be used and the test conducted as follows:

(i) For each dilution, inject at least five embryos, 9 to 11 days old, in the allantoic cavity with at least 0.1 ml each. Disregard all deaths during the first 24 hours post-injection. To be a valid test, at least four embryos in each dilution shall remain viable beyond 24 hours.

(ii) Examine the surviving embryos for evidence of infection 5 to 7 days post-injection.

(iii) A satisfactory titration shall have at least one dilution with between 50 and 100 percent positives and at least one dilution with between 50 and 0 percent positives.

(iv) Calculate the EID50 by the Spearman-Karber or Reed-Muench method.

(3) Twenty to twenty-eight days postvaccination, all vaccinates and controls shall be challenged intramuscularly with at least 104.0 EID50 of virus per chicken and observed each day for 14 days. Challenge virus shall be provided or approved by Animal and Plant Health Inspection Service.

(4) If at least 90 percent of the controls do not develop clinical signs of Newcastle disease during the observation period, the test is a No Test and may be repeated. If at least 19 of 20, or 27 of 30, or 36 of 40 of the vaccinates in each group do not remain free from clinical signs of Newcastle disease during the observation period, the Master Seed Virus is unsatisfactory.

(5) A strain identity test acceptable to Animal and Plant Health Inspection Service shall be conducted.

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

(d) After a lot of Master Seed Virus has been established as prescribed in paragraphs (a), (b), and (c) of this section, each serial and subserial shall meet the applicable requirements in §113.300, except §113.34, and the requirements prescribed in this paragraph.

(1) Final container samples from each serial shall be tested for pathogens by the chicken embryo inoculation test prescribed in §113.37, except that, if the test is a No Test because of a vaccine virus override, the chicken inoculation test prescribed in §113.36 may be conducted and the vaccine judged accordingly.

(2) Safety test: Final container samples of completed product from each serial shall be tested to determine whether the vaccine is safe for use in susceptible young chickens. Vaccines recommended for use in chickens 10 days of age or younger shall be tested in accordance with paragraphs (d)(2)(i), (ii), and (iii) of this section.

(i) Twenty-five susceptible chickens, 5 days of age or younger, properly identified and obtained from the same source and hatch, shall be vaccinated by the eye drop method with the equivalent of 10 doses of vaccine and the chickens observed each day for 21 days. Severe respiratory signs or death shall be counted as failures. Two-stage sequential testing may be conducted if the first test (which then becomes stage one) has 3 failures.

(ii) The results shall be evaluated according to the following table:

Cumulative Totals

StageNumber of chickensFailures for satisfactory serialsFailures for unsatisfactory serials
1252 or less4 or more.
2505 or less6 or more.

(iii) If unfavorable reactions occur which are not attributable to the product, the test shall be declared a No Test and may be repeated.

(iv) Vaccines not recommended for use in chickens 10 days of age or younger shall be tested for safety as follows:

Each of twenty-five 3 to 5 week old Newcastle disease susceptible chickens shall be vaccinated as recommended on the label with the equivalent of ten doses and observed each day for 21 days. If any of the birds show severe clinical signs of disease or death during the observation period due to causes attributable to the product, the serial is unsatisfactory.

(3) Virus titer requirements. Final container samples of completed product shall be tested for virus titer using the titration method used in paragraph (c)(2) of this section. To be eligible for release, each serial and each subserial shall have a virus titer per dose sufficiently greater than the titer of vaccine virus used in the immunogenicity test prescribed in paragraph (c) 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 100.7 greater than that used in the immunogenicity test but not less than 105.5 EID50 per dose.

[39 FR 44727, Dec. 27, 1974, as amended at 40 FR 18407, Apr. 28, 1975; 40 FR 23721, June 2, 1975; 40 FR 41090, Sept. 5, 1975; 42 FR 43618, Aug. 30, 1977; 48 FR 33473, July 22, 1983. Redesignated at 55 FR 35562, Aug. 31, 1990, as amended at 56 FR 66784, 66786, Dec. 26, 1991; 72 FR 72564, Dec. 21, 2007]

§113.330   Marek's Disease Vaccines.

Marek's disease vaccine shall be prepared from virus-bearing tissue culture cells. Only Master Seed Virus which has been established as pure, safe, and immunogenic shall be used for preparing the production seed virus for vaccine production.

(a) The Master Seed Virus shall meet the applicable requirements prescribed in §113.300, and the requirements prescribed in this section. The identity test required in §113.300(c) shall be conducted in a serotype-specific manner by a method acceptable to APHIS. Each lot of Master Seed Virus shall also be tested for pathogens by the chicken embryo inoculation test prescribed in §113.37, except that, if the test is a No Test because of a vaccine virus override, the chicken inoculation test prescribed in §113.36 may be conducted and the virus judged accordingly.

(b) Safety test. The Master Seed Virus shall be nonpathogenic for chickens as determined by the following procedure:

(1) Specific pathogen free chickens or embryos, negative for Marek's disease virus antibodies, and from the same source, shall be isolated into the following groups:

(i) Group 1. At least 50 test subjects shall be inoculated with 10 times as much viable virus as will be contained in one dose of vaccine, by the route recommended for vaccination.

(ii) Group 2. At least 50 test subjects shall be injected with a very virulent Marek's disease virus provided or approved by APHIS, at a dosage level that will cause gross lesions of Marek's disease in at least 80 per cent of the chickens within 50 days.

(iii) Group 3. Fifty uninoculated controls. For in ovo studies, this group should receive a sham inoculation of diluent.

(iv) Group 4. For studies evaluating Serotype 1 Master Seed Viruses, a group of 50 uninoculated control chickens shall be housed in contact with the group 1 vaccinated chickens.

(2) At least 40 chickens in each group shall survive to 5 days of age. All chickens that die shall be necropsied and examined for lesions of Marek's disease and cause of death. The test shall be judged according to the following criteria:

(i) At 50 days of age, the remaining chickens in group 2 shall be killed and examined for gross lesions of Marek's disease. If at least 80 percent of this group do not develop Marek's disease, the test is a No Test and may be repeated.

(ii) At 120 days of age, the remaining chickens in groups 1, 3, and 4 shall be weighed, killed, and necropsied. If less than 30 of the chickens in group 3 survive the 120 day period, or if any of the chickens in group 3 have gross lesions of Marek's disease at necropsy, the test is declared a No Test. If less than 30 chickens in groups 1 and 4 survive the 120 day period; or if any of the chickens in groups 1 and 4 have gross lesions of Marek's disease at necropsy; or if the average body weight of the chickens in groups 1 or 4 is significantly (statistically) different from the average in group 3 at the end of the 120 days, the lot of Master Seed Virus is unsatisfactory.

(3) For tests involving in ovo inoculation, hatchability results shall also be reported for each group.

(c) Immunogenicity. Each lot of Master Seed Virus used for vaccine production shall be tested for immunogenicity at the highest passage level allowed for the product, and the virus dose to be used shall be established as follows:

(1) Specific pathogen free chickens or embryos, negative for Marek's disease antibodies, and from the same source, shall be isolated into the following groups:

(i) Group 1. A minimum of 35 test subjects shall be inoculated with the vaccine, using the recommended route, at 1 day of age for chicks or 18 days of embryonation for embryos. The dose used shall be established by 5 replicate virus titrations conducted by a cell culture system or other titration method acceptable to APHIS.

(ii) Group 2. A minimum of 35 nonvaccinated test subjects shall be held as challenge controls.

(iii) Group 3. A minimum of 25 nonvaccinated test subjects shall be held as nonchallenge controls.

(iv) Group 4. Except for studies evaluating vaccines which contain only a Serotype 3 virus as the Marek's disease fraction, a minimum of 35 chicks shall be vaccinated at 1 day of age with a licensed Serotype 3 vaccine, in order to document the severity of the very virulent challenge.

(2) At least 30 chickens in groups 1, 2, and 4, and at least 20 chickens in group 3, shall survive to 5 days of age. All chickens in groups 1, 2, and 4 shall be challenged at 5 days of age in the following manner:

(i) For studies evaluating vaccines which contain only a Serotype 3 virus as the Marek's disease fraction, groups 1 and 2 shall be inoculated with a standard virulent challenge virus provided or approved by APHIS.

(ii) For all other Marek's disease vaccines, groups 1, 2, and 4 shall be inoculated with a very virulent challenge virus provided or approved by APHIS.

(3) All chickens shall be observed until 7 weeks of age, necropsied, and examined for grossly observable lesions consistent with Marek's disease. All chickens dying before the end of the 7 week observation period shall be necropsied and evaluated for gross lesions of Marek's disease. Any chickens not so examined shall be scored as positive for Marek's disease.

(4) For a valid test, at least 80 percent of the chickens in group 2 must develop grossly observable lesions, none of the chickens in group 3 shall develop grossly observable lesions, and (when included) greater than 20 percent of the chickens in group 4 must develop grossly observable lesions.

(5) For a valid test to be considered satisfactory, at least 80 percent of the chickens in group 1 must remain free of grossly observable lesions. The appropriate product claim resulting from a satisfactory test would be to aid in the prevention of Marek's disease, for vaccines containing only a Serotype 3 virus as the Marek's disease fraction, or to aid in the prevention of very virulent Marek's disease, for all other vaccines.

(d) Test requirements for release. Each serial and subserial shall meet the applicable requirements prescribed in §113.300. The identity test required in §113.300(c) shall be conducted in a serotype-specific manner by a method acceptable to APHIS. Final container samples of completed product shall also meet the requirements in paragraphs (d) (1), (2), and (3) of this section. Any serial or subserial found unsatisfactory by a prescribed test shall not be released.

(1) Purity test. The chicken embryo inoculation test prescribed in §113.37 shall be conducted, except that, if the test is a No Test because of a vaccine virus override, the chicken inoculation test prescribed in §113.36 may be conducted and the virus judged accordingly.

(2) Safety test. At least 25 one-day-old, specific pathogen free chickens shall be injected, by the subcutaneous route, with the equivalent of 10 chicken doses of virus (vaccine concentrated 10X). The chickens shall be observed each day for 21 days. Chickens dying during the period shall be examined, cause of death determined, and the results recorded.

(i) If at least 20 chickens do not survive the observation period, the test is a No Test.

(ii) If lesions of any disease or cause of death are directly attributable to the vaccine, the serial is unsatisfactory.

(iii) If less than 20 chicks survive the observation period and there are no deaths or lesions attributable to the vaccine, the test may be repeated one time, Provided, that if the test is not repeated, the serial shall be declared unsatisfactory.

(3) Potency test. The samples shall be titrated using a cell culture system or other titration method acceptable to APHIS. For vaccines composed of more than one Marek's disease virus serotype, each fraction shall be titrated in a serotype-specific manner.

(i) Samples of desiccated vaccine shall be incubated at 37 °C for 3 days before preparation for use in the potency test. Samples of desiccated or frozen vaccine shall be reconstituted in diluent according to the label recommendations, and held in an ice bath at 0 °C to 4 °C for 2 hours prior to use in the potency test.

(ii) For a serial or subserial to be eligible for release, each serotype contained in the vaccine shall have a virus titer per dose which is at least 3 times greater than the number of plaque forming units (pfu) used in the immunogenicity test prescribed in paragraph (c) of this section, but not less than 1000 pfu per dose.

(iii) When tested (without the pretest incubation of desiccated products) at any time within the expiration period, each serotype contained in the vaccine shall have a virus titer per dose which is at least 2 times the number of pfu used in the immunogenicity test, but not less than 750 pfu per dose.

[61 FR 33841, July 1, 1996]

§113.331   Bursal Disease Vaccine.

Bursal Disease Vaccine shall be prepared from virus-bearing cell culture fluids or embryonated chicken eggs. Only Master Seed Virus which has been established as pure, safe, and immunogenic in accordance with the requirements in paragraphs (a), (b), and (c) of this section shall be used for 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 and the requirements prescribed in this section.

(b) Each lot of Master Seed Virus shall be tested for pathogens by the chicken embryo inoculation test prescribed in §113.37, except that, if the test is a No Test because of a vaccine virus override, the chicken inoculation test prescribed in §113.36 may be conducted and the virus judged accordingly. Each lot of Master Seed Virus used in the preparation of modified live virus vaccines shall also be nonpathogenic to chickens as determined by the following procedures:

(1) Each of twenty-five 1-day-old bursal disease susceptible chickens (vaccinates) shall be injected subcutaneously with 10 times the recommended dose of vaccine virus and observed for 21 days. Fifteen chickens of the same source and hatch shall be kept isolated as controls.

(i) Seventeen days postvaccination, each of five controls shall be administered at least 102.0 EID50 of a virulent bursal disease virus by eye-drop, isolated, and used as positive controls. The remaining controls shall be used as negative controls.

(ii) If the vaccinates do not remain free of clinical signs of bursal disease, the Master Seed Virus is unsatisfactory. If unfavorable reactions which are not attributable to the Master Seed Virus occur in more than two of the vaccinates, the test shall be declared a No Test and may be repeated.

(iii) Twenty-one days postvaccination, the vaccinates and the controls shall be necropsied and examined for gross lesions of bursal disease. If more than two of the vaccinates have such lesions, the Master Seed Virus is unsatisfactory, except that, if any of the negative controls or less than four of the positive controls have such lesions, the test is a No Test and may be repeated. For purposes of this test, gross lesions shall include obvious pathological processes and/or obvious reduction in size of the bursa from normal.

(2) Each of thirty-five 3- to 4-week-old bursal disease susceptible chickens (vaccinates) shall be vaccinated with approximately one minimum protective dose of vaccine virus as determined in paragraph (c) of this section. Each of 10 chickens of the same source and hatch shall be administered at least 102.0 EID50 of a virulent bursal disease virus by eye-drop, isolated, and used as positive controls. Also, each of 20 additional chickens of the same source and hatch shall be isolated and held as negative controls.

(i) Three or four days postvaccination, 10 of the vaccinates, the 10 positive controls, and 10 of the negative controls shall be necropsied and examined for gross lesions of bursal disease. If any of the vaccinates have such lesions, the Master Seed Virus is unsatisfactory, except that, if any of the negative controls or less than 8 of the positive controls have such lesions, the test is a No Test and may be repeated. For purposes of this test, gross lesions shall include peri-bursal edema and/or edema and/or macroscopic hemorrhage in the bursal tissue.

(ii) Fourteen days post-vaccination, the remaining vaccinates and negative controls shall be necropsied and examined for obvious bursal atrophy. If any of the vaccinates have such atrophy, the Master Seed Virus is unsatisfactory, except that, if any of the negative controls have such atrophy, the test is a No Test and may be repeated.

(c) Each lot of Master Seed Virus shall be tested for immunogenicity and the selected virus dose to be used shall be established as follows:

(1) Bursal Disease susceptible chickens, all of the same age (3 weeks or younger) and from the same source, shall be used. Twenty or more chickens shall be used as vaccinates for each method of administration recommended on the label. Ten additional chickens of the same age and from the same source shall be held as unvaccinated controls.

(2) A geometric mean titer of the vaccine produced from the highest passage of the Master Seed Virus shall be established before the immunogenicity test is conducted. Each vaccinate shall receive a predetermined quantity of vaccine virus. Five replicate virus titrations shall be conducted on an aliquot of the vaccine virus to confirm the amount of virus administered to each chicken used in the test. At least three appropriate (not to exceed tenfold) dilutions shall be used to conduct the titrations by a method acceptable to Animal and Plant Health Inspection Service.

(3) When the test chickens are 28 to 35 days of age but not less than 14 days postvaccination, each vaccinate and each control shall be challenged by eye-drop with a virulent bursal disease virus provided or approved by Animal and Plant Health Inspection Service.

(i) Three to five days postchallenge, all vaccinates and controls shall be necropsied and examined for gross lesions of bursal disease as described in paragraph (b)(2)(i) of this section.

(ii) If at least 19 of 20, or 27 of 30, or 36 of 40 vaccinates in each group are not free from such lesions, the Master Seed Virus is unsatisfactory, except that, if less than 90 percent of the controls have such lesions, the test is a No Test and may be repeated.

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

(d) After a lot of Master Seed Virus has been established as prescribed in paragraphs (a), (b), and (c) of this section, each serial and subserial shall meet the applicable requirements in §113.300 and the requirements prescribed in this paragraph.

(1) Tests for pathogens. Final container samples from each serial shall be tested for pathogens by the chicken embryo inoculation test prescribed in §113.37, except that, if the test is a No Test because of a vaccine virus override, the chicken inoculation test prescribed in §113.36 may be conducted and the serial judged accordingly.

(2) Safety tests. (i) Final container samples of completed product from each serial shall be tested to determine whether the vaccine is safe as follows:

(A) For vaccines intended for parenteral administration, each of twenty-five 1-day-old bursal disease susceptible chickens shall be vaccinated with the equivalent of 10 doses by subcutaneous injection.

(B) For vaccines intended for drinking water administration, each of twenty-five 4- to 5-week-old bursal disease susceptible chickens shall be vaccinated orally with the equivalent of 10 doses.

(C) Ten chickens of the same source and hatch shall be maintained in isolation as negative controls. The vaccinates and controls shall be observed each day for 21 days.

(ii) If unfavorable reactions which are attributable to the biological product occur during the observation period, the serial is unsatisfactory. If unfavorable reactions occur in more than one of the controls or if unfavorable reactions which are not attributable to the biological product occur in more than two of the vaccinates, the test shall be declared a No Test and repeated, except that, if the test is not repeated, the serial shall be unsatisfactory.

(3) Virus titer requirements. Final container samples of completed product shall be tested for virus titer using the titration method used in paragraph (c)(2) 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 prescribed in paragraph (c) 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 100.7 times greater than that used in such immunogenicity test, but not less than 102.0 titration units (PFU or ID50's) per dose.

[44 FR 60263, Oct. 19, 1979, as amended at 44 FR 67087, Nov. 23, 1979; 48 FR 33473, July 22, 1983. Redesignated at 55 FR 35562, Aug. 31, 1990, as amended at 56 FR 66784, 66786, Dec. 26, 1991; 64 FR 43045, Aug. 9, 1999; 72 FR 72564, Dec. 21, 2007]

§113.332   Tenosynovitis Vaccine.

Tenosynovitis Vaccine shall be prepared from virus-bearing cell culture fluids or embryonated chicken eggs.

Only Master Seed which has been established as pure, safe, and immunogenic shall be used for preparing seeds for vaccine production. All serials of vaccine shall be prepared from the first through the fifth passage from the Master Seed.

(a) The Master Seed shall meet the applicable general requirements prescribed in §113.300, except (a)(3)(ii) and (c), and the special requirements in this section.

(b) Each lot of Master Seed shall be tested for:

(1) Pathogens by the chicken inoculation test prescribed in §113.36.

(2) Lymphoid leukosis virus contamination as follows:

(i) Each of at least 10 3-week-old or older lymphoid leukosis free chickens from the same source and hatch shall be injected intra-muscularly with an amount of Master Seed equal to 100 label doses of vaccine. At least 15 chickens of the same source and hatch shall be used as controls; 5 or more shall be unvaccinated and serve as negative controls; 5 or more shall be injected with subgroup A lymphoid leukosis virus; and 5 or more with subgroup B lymphoid leukosis virus. Each group of control chickens shall be held isolated from each other and from the vaccinates.

(ii) Twenty-one to 28 days postinoculation, blood samples shall be taken from each chicken and the serum separated using a technique conducive to virus preservation. These serums shall be used as inocula in the complement fixation for avian lymphoid leukosis (COFAL) test prescribed in §113.31.

(iii) Serums from the vaccinates shall be tested separately, but serums within each control group may be pooled. A valid test shall have positive COFAL reactions from each virus inoculated group and negative reactions from the uninoculated controls. If any of the chickens injected with the Master Seed have positive COFAL test reactions in a valid test, the Master Seed is unsatisfactory.

(3) Identity using the following agar gel immunodiffusion test. The undiluted Master Seed may be used as test antigen or the Master Seed may be inoculated onto the chorioallantoic membrane (CAM) of fully susceptible chicken embryos and the infected CAMs ground and used as antigen. A known tenosynovitis antiserum and a known tenosynovitis antigen shall be used in the test. A precipitin line shall form between the test antigen and the known antiserum in the center well which shows identity with the line formed between the antiserum and the known antigen, or the Master Seed is unsatisfactory.

(4) Safety using the following chicken test:

(i) For vaccines intended for use in chickens less than 14 days of age, Master Seed equal to 10 label doses shall be administered subcutaneously to each of 25 1-day-old tenosynovitis susceptible chickens.

(ii) For vaccines intended for use only in chickens 14 days of age or older, Master Seed equal to 10 label doses shall be administered subcutaneously to each of 25 4-week-old or older tenosynovitis susceptible chickens.

(iii) The vaccinates shall be observed each day for 21 days. If unfavorable reactions occur which are attributable to the vaccine, the Master Seed is unsatisfactory. If unfavorable reactions occur which are not attributable to the vaccine, the test is a No Test and may be repeated.

(c) Each lot of Master Seed shall be tested for immunogenicity. The selected virus dose shall be established as follows:

(1) Tenosynovitis susceptible chickens, of the same age and from the same source shall be used as test birds. Vaccines intended for use in very young chickens shall be administered to chickens of the youngest age for which the vaccine is recommended. Vaccines intended for use in older chickens shall be administered to 4-week-old or older chickens. Twenty or more vaccinates shall be used for each method of administration recommended on the label. Ten or more chickens shall be held as unvaccinated controls.

(2) A geometric mean titer of the vaccine produced at the highest passage from the Master Seed shall be established using a method acceptable to Animal and Plant Health Inspection Service before the immunogenicity test is conducted. A predetermined quantity of vaccine virus shall be administered to each vaccinate. Five replicate virus titrations shall be conducted on an aliquot of the vaccine virus to confirm the dose.

(3) Twenty-one to 28 days postvaccination, each vaccinate and control shall be challenged by injecting virulent virus furnished or approved by Animal and Plant Health Inspection Service into one foot pad. The vaccinates and controls shall be observed each day for 14 days. If at least 90 percent of the controls do not develop swelling and discoloration in the phalangeal joint area of the injected foot pad typical of infection with tenosynovitis virus, the test is a No Test and may be repeated. If at least 19 of 20, 27 of 30, or 36 of 40 vaccinates do not remain free from these signs, disregarding transient swelling which subsides within 5 days postchallenge, the Master Seed is unsatisfactory.

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

(d) Test requirements for release. Each serial and subserial shall meet the applicable general requirements prescribed in §113.300, except (c), and the requirements in this paragraph.

(1) Purity. Final container samples of completed product from each serial shall be tested for pathogens by the chicken inoculation test prescribed in §113.36.

(2) Safety. (i) Final container samples of completed product from each serial shall be safety tested as follows:

(A) For vaccines intended for use in very young chickens, each of 25 1-day-old tenosynovitis susceptible chickens shall be vaccinated with the equivalent of 10 doses by one method recommended on the label.

(B) For vaccines intended for use in older chickens, each of 25 4-week-old or older tenosynovitis susceptible chickens shall be vaccinated with the equivalent of 10 doses by one method recommended on the label.

(ii) The vaccinates shall be observed each day for 21 days. If unfavorable reactions occur which are attributable to the product, the serial is unsatisfactory. If unfavorable reactions occur in more than two vaccinates which are not attributable to the product, the test is a No Test and may be repeated. If the test is not repeated, the serial is unsatisfactory.

(3) Virus titer requirements. Final container samples of completed product shall be titrated by the method used in paragraph (c)(2) of this section. To be eligible for release, each serial and subserial shall have a virus titer sufficiently greater than the titer of the vaccine virus used in the immunogenicity test prescribed in paragraph (c) of this section to assure that, when tested at any time within the expiration period, each serial and subserial shall have a virus titer 100.7 times greater than that used in the immunogenicity test, but not less than 102.0 titration units (PFU or ID50) per dose.

(4) Identity. Bulk or final container samples of completed product from each serial shall be tested for identity as prescribed in paragraph (b)(3) of this section and shall meet the criteria stated therein.

[50 FR 438, Jan. 4, 1985. Redesignated at 55 FR 35562, Aug. 31, 1990, as amended at 56 FR 66784, 66786, Dec. 26, 1991; 64 FR 43045, Aug. 9, 1999; 72 FR 72564, Dec. 21, 2007]

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