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TUBERCULIN PURIFIED PROTEIN DERIVATIVE

Tuberculin PPD

Category Diagnostic preparation.

          Tuberculin Purified Protein Derivative (Tuberculin PPD) for human use is a sterile liquid or freeze-dried preparation obtained by precipitation from the heated products of the culture and lysis of Mycobacterium bovis and/or Mycobacterium tuberculosis and capable of demonstrating a delayed hypersensitivity in an animal sensitized to micro-organisms of the same species.

Description Liquid Tuberculin PPD is a clear colourless or pale-yellow liquid.

Freeze-dried Tuberculin PPD, when reconstituted, becomes a colourless or pale-yellow liquid.

Strength available It contains the PPD equivalent to 5 IU of PPD-S¹ per 0.1 ml.

Dose Intradermal, 0.1 ml.

Contra-indication It is not for intravenous, intramuscular or subcutaneous administration.

Warning

          1. It should not be administered to persons with severe blistering tuberculin reactions in the past, persons with documented active tuberculosis or a clear history of treatment for tuberculosis infection or disease, and persons with extensive burns or eczema.

2. It may cause vesiculation, ulceration, necrosis, pain, or pruritus at the test site in some tuberculinsensitive individuals.

3. Nausea, headache, dizziness, malaise, rash, urticaria, edema, and pyrexia may occur.

Additional information To ensure accurate results, Tuberculin PPD should be administered before, simultaneously with, or 4 to 6 weeks or longer after administration of live viral vaccines.

Expiration date When stored under the prescribed conditions, the expiration date is not later than 6 months for the liquid form, and not later than 2 years for the freeze-dried form, from the date of the last satisfactory test for potency.

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 ¹ The first international standard preparation for Purified Protein Derivative (PPD) of M. tuberculosis (human strain) tuberculin, established in 1951 for WHO, by the Henry Phipps Institute, Philadelphia, Pa., USA.

Packaging and storage Tuberculin PPD shall be stored at a temperature of 2º to 8º, protected from light; avoid freezing.

Labelling Complies with the “General Information for Biological Products”, p. 177. In addition, the label on the container states (1) the number of International Units or its equivalent per container; (2) the species of mycobacteria used to prepare the product; (3) the name andquantity of any antimicrobial preservative or other substances added to the preparation; (4) for freeze-dried products, a statement that the product is to be reconstituted using the liquid provided by the manufacturer. (Note If the package does not contain a leaflet warning that the inhalation of concentrated tuberculin PPD may produce toxic effects, this warning must be shown on the label on the container together with a statement that the powder must be handled with care.)

Identification The assay may serve as identification.

pH 6.5 to 7.5 (Appendix 4.11).

Sterility Complies with the “Sterility Test” (Appendix 10.1).

Live mycobacteria This test may be omitted if it has been carried out on concentrated bulk.

Carry out the following methods for live mycobacteria.

          Animal inoculation method: Inject 5.0 ml intraperitoneally or subcutaneously into each of two guinea-pigs weighing 300 to 400 g. Observe the animals for not less than 42 days. Kill the animals and carry out an autopsy. If no guinea-pig shows sign of infection with mycobacteria, the preparation complies with the test.

          Culture method: If the sample to be examined may be contaminated by micro-organisms other than mycobacteria, treat it with a suitable decontamination solution, such as acetylcysteine-sodium hydroxide solution or sodium laurylsulfate solution. Inoculate 0.2 ml of the sample in triplicate onto each of two suitable solid media (Löwenstein-Jensen medium and Middlebrook 7H 10 medium are considered suitable). Inoculate 0.5 ml in triplicate into a suitable liquid medium. Incubate all media at 37º for 56 days.

          The growth promotion of the media in the presence of the preparation shall be examined by inoculation of a suitable strain of Mycobacterium sp. and if necessary use a suitable neutralizing substance.

If contaminating micro-organisms develop during the first 8 days of incubation, repeat the test and carry out at the same time a bacteriological sterility test.

If at the end of the incubation time no growth of mycobacteria occurs in any of the test media, the preparation complies with the test.

Antimicrobial preservative Where applicable, determine the amount of antimicrobial preservative by a suitable chemical or physico-chemical method. The content is not less than the minimum amount shown to be effective and not more than 115 per cent of the amount stated on the label. If phenol has been used in the preparation, the concentration is not more than 0.5 per cent w/v when determined by the test described under the “Determination of Phenol”, p. 179.

Sensitizing effect of PPD This test may be omitted if it has been carried out on concentrated bulk. Use three guinea-pigs that have not been subjected to any treatment likely to interfere with the test. On three occasions at intervals of 5 days, inject intradermally into each guinea-pig about 500 IU of the preparation to be examined in a volume of 0.1 ml. Two to three weeks after the third injection, administer the same dose intradermally to the same animals and to a control group of three guinea-pigs of the same weight that have not previously received injections of tuberculin. After 24 to 72 hours, the reactions in the two groups of animals are not substantially different.

Assay The potency of tuberculin PPD is determined by comparing the reactions produced by the intradermal injection of increasing doses of the preparation to be examined into sensitized guinea-pigs with the reactions produced by known concentrations of the reference preparation.

Test animals Sensitize not less than six white or albino guinea-pigs weighing not less than 300 g.

          Methods of sensitization Inject intradermally, four sites at one time, each of 0.1 ml of suspension containing a suitable amount (0.1 to 0.4 mg per ml) of heatkilled, dried tubercle bacilli in liquid paraffin. Alternatively, 0.1 ml of an aqueous suspension of 0.5 mg of BCG vaccine per ml can be used instead of heat-killed, dried tubercle bacilli.

Carry out the test after the period of time required for optimal sensitization which is usually 4 to 8 weeks, but not later than 6 months after sensitization.

          Procedure Depilate the flanks of the animals so that it is possible to make at least three injections on each side but not more than a total of 12 injection points per animal.

          Prepare dilutions of the preparation to be examined and of the reference preparation using phosphate-buffered saline (pH 6.5 to 7.5) containing 50 mg per litre of polysorbate 80. If the preparation to be examined is freezedried and does not contain a stabilizer, reconstitute it using the liquid described above. Use at least three different doses of the reference preparation and at least three different doses of the preparation to be examined. For both preparations, use doses such that the highest dose is about 10 times the lowest dose. Choose the doses such that when they are injected the lesions produced have a diameter of not less than 8 mm and not more than 25 mm. In any given test, the order of the dilutions injected at each point is chosen at random in a Latin square design. Inject each dose intradermally in a constant volume of 0.1 ml or 0.2 ml. Measure the diameters of the lesions 24 to 48 hours later and calculate the results of the test by the statistical methods, assuming that the diameters of the lesions are directly proportional to the logarithm of the concentration of the preparation.

          The estimated potency is not less than 80 per cent and not more than 125 per cent of the stated potency. The confidence limits (P = 0.95) are not less than 64 per cent and not more than 156 per cent of the stated potency.

          If the preparation fails to meet the requirement for potency, it shall be discarded, or the assay shall be repeated by the same method using a different group of sensitized animals. The estimates of potency and confidence limits shall be made using the results of all the assays. The preparation is of acceptance potency if the combined results of the assays meet the criteria specified above.