(a) Propagation of mycobacteria. The medium used for production of mycobacteria shall not contain ingredients known to be capable of producing allergenic effects in human subjects.

(b) Tests for viable mycobacteria. The culture filtrate from each strain in its most concentrated form shall be shown to be free of viable mycobacteria by the following tests:

(1) Animal test. A 1.0 ml. sample of the filtrate shall be injected intraperitoneally into each of at least three healthy guinea pigs weighing between 300 and 400 gm. At least two-thirds of the animals must survive an observation period of at least 6 weeks and must show a normal weight gain. After the observation period the animals shall be necropsied and examined for signs indicative of tuberculosis except that animals that die during the observation period shall be necropsied and examined as soon as feasible after death. The filtrate is satisfactory for Tuberculin manufacture if none of the animals in the test show evidence of tuberculosis infection.

(2) Culture test. A 2.0 ml. sample of the filtrate shall be inoculated onto Löwenstein-Jensen's egg medium or other media demonstrated to be equally capable of supporting growth. A control test on the culture medium shall be conducted simultaneously with the sample under test and shall be shown to be capable of supporting the growth of small numbers of the production strain(s). All the test vessels shall be incubated at a suitable temperature for a period of 6 weeks under conditions that will prevent drying of the medium, after which the cultures shall be examined for evidence of mycobacterial colonies. The filtrate is satisfactory for Tuberculin manufacture if the test shows no evidence of mycobacteria.

§ 73.613 Potency test.

The potency of each lot of Tuberculin shall be estimated from a comparison of the responses obtained by the intradermal injection into sensitized guinea pigs weighing over 500 gm. of a sample of the lot under test and of the appro

priate standard preparation. The U.S. Standard Tuberculin, Old, shall be used in determining the potency of tuberculins made from the concentrated filtrate of the soluble products of the growth of the mycobacteria. The U.S. Standard Tuberculin, Purified Protein Derivative, shall be used in determining the potency of tuberculins made from protein fraction of the soluble products of the growth of the mycobacteria. The test shall be performed as follows:

(a) Sensitization of test animals. At least four white guinea pigs shall be sensitized with M. tuberculosis or M. bovis. The degree of sensitivity shall be such that an intradermal injection of one U.S. unit of the appropriate standard preparation will produce in each test animal an erythematous reaction approximately 100 mm2 within 18-24 hours.

(b) Test Procedure. The hair shall be removed from both sides of the sensitized test animals without producing abrasions of the skin. Dilutions of the standard containing 0.5, 1, 2, and 4 U.S. units in the test dose of 0.1 ml. and four comparable levels of activity of the lot under test shall be injected intradermally into opposite and parallel sites of each animal. Only three dilutions need be used when the initial concentration of the lot under test does not contain four units in 0.1 ml. Within 18-24 hours following injection, measurements of the greater and lesser diameters of erythema measured to the closest millimeter shall be made at each site. The mean value of the product of the diameters for each dilution shall be calculated. The number of U.S. units in the lot under test shall be estimated from its relationship to the reactivity of the appropriate standard preparation.

(c) Potency. The potency of the lot is satisfactory if the test results are within limits, as follows:

(1) Products for Mantoux testing. +20 percent of the labeled U.S. units.

(2) Liquid products for multiple puncture testing. ±20 percent of the U.S. units claimed by the manufacturer in the license application.

(3) Products dried on multiple puncture devices. ±50 percent of the U.S. units claimed by the manufacturer in the license application. § 73.614

General requirements.

(a) General safety. Each lot of Tuberculin shall be tested for safety as pre

scribed in § 73.72, except that the sample of tuberculin from multiple puncture devices shall be obtained by removing the tuberculin in a manner that will permit the injection of material from at least five devices into each of two guinea pigs and from at least two devices into each of two mice.

(b) Labeling. In addition to complying with all other applicable labeling provisions of this part, the package label shall state the following:

(1) For Tuberculin for Mantoux testing, the number of U.S. units (TU) per dose.

(2) For Tuberculin for multiple puncture testing, a statement indicating that the activity per test is comparable to a stated number of U.S. units (TU) administered by the Mantoux method.

(3) The applicable type of Tuberculin placed immediately following and of no less prominence than the proper name, as follows:

(i) "Old," or

(ii) "Purified Protein Derivative" or "PPD."

(c) Samples; protocols; official release. For each lot of Tuberculin the following shall be submitted to the Director, Division of Biologics Standards, National Institutes of Health, Bethesda, Md. 20014:

(1) A protocol which consists of a summary of the history of manufacture of each lot including all results of each test for which test results are requested by the Director, Division of Biologics Standards.

(2) Tuberculin distributed on a multiple puncture device, as follows:

(i) A total of no less than 100 devices, (ii) A total of no less than 20 ml. of bulk tuberculin.

(3) A total of no less than 20 ml. of liquid tuberculin.

(4) Sufficient dried tuberculin in final containers so that upon reconstitution as recommended in labeling it will yield at least 20 ml.

The product shall not be issued by the manufacturer until notification of official release of the lot is received from the Director, Division of Biologics Standards. § 73.615 Equivalent methods.

Modification of any particular method or process or the conditions under which it is conducted as set forth in the additional standards relating to Tuberculin, shall be permitted whenever the manu

(a) As used in this part, all terms not defined herein shall have the meaning given them in the Act.

(b) "Accredited institution" means an institution accredited by an agency or organization recognized for such purpose by the U.S. Commissioner of Education.

(c) "Accredited laboratory" means a laboratory, or a laboratory in a hospital, accredited by an approved accreditation body.

(d) "Act" means the Public Health Service Act, as amended, 42 U.S.C. 201, et seq.

(e) "Approved accreditation body" means, with respect to hospitals, Joint Commission on the Accreditation of Hospitals, or American Osteopathic Association, and with respect to laboratories, Commission on Inspection and Accreditation of the College of American Pathologists; or any other national accreditation body which has been approved by the Secretary as provided in section 353 of the Act.

(f) "Director" means the Director of the National Communicable Disease Center, Health Services and Mental Health Administration, Department of Health, Education, and Welfare.

(g) "Health insurance program" means the program created under Title XVIII of the Social Security Act, pursuant to which individuals are entitled to have payments made on their behalf for services performed by independent laboratories as provided in Title 20, Code of Federal Regulations, Part 405, Subpart M.

(h) A "physician" is a person licensed to practice medicine or osteopathy in any state or possession of the United States.

(i) "Radiobioassay" includes (1) an examination to identify radionuclides or determine and quantitate body levels of radionuclides which are taken in by chronic or acute absorption, ingestion or inhalation; or, (2) following the adminIstration of a radioactive material to a patient, the subsequent analysis of a body fluid or excreta in order to evaluate body function; or, (3) the use of a radioactive tracer in an in vitro assay of a nonradioactive biological substance of clinical significance.

(j) A "referee laboratory" is a laboratory designated by the Secretary to examine specimens or other materials for purposes of proficiency testing using the same time schedule allowed for licensed laboratories and under conditions similar to those under which licensed laboratories examine materials.

(k) A "reference laboratory" is a laboratory designated by the Secretary to authenticate the identification, content, and titer of samples and other materials used or to be used in proficiency testing.

(1) "Secretary" means the Secretary of Health, Education, and Welfare or his designee.

(m) "Specimen" means any material derived from the human body for examination or other procedure for the purpose of providing information for the diagnosis, prevention, or treatment of any disease or impairment of, or the assessment of the health of, man.

§ 74.2 Applicability.

(a) Except as otherwise provided herein, the regulations in this part apply to laboratories engaged in the laboratory examination of, or other laboratory procedures relating to, specimens solicited or accepted in interstate commerce directly or indirectly, for the purpose of providing information for the diagnosis, prevention, or treatment of any disease or impairment, or the assessment of the health, of man.

(b) The regulations in this part do not apply to the following:1

4.4 1 The coverage of services of independent laboratories under section 1861 of the Social Security Act, as amended, 42 U.S.C. 1395x, is subject to the provisions of Title 20, Code of Federal Regulations, Part 405, and the regulation of biological products under section 351 of the Public Health Service Act, as amended, 42 U.S.C. 262, is subject to the provisions of Title 42, Code of Federal Regulations, Part 73.

(1) Any laboratory with respect to any category in which it accepts no more than 100 specimens during any calendar year. For purposes of this paragraph, a category shall be one of the following: (i) Microbiology and serology; (ii) clinical chemistry; (iii) immune-hematology; (iv) hematology; (v) pathology; (vi) radiobioassay.

(2) Pursuant to section 353 (i), any clinical laboratory operated by a licensed physician, osteopath, dentist, or podiatrist, or group thereof, who performs or perform laboratory tests or procedures solely as an adjunct to the treatment of his or their patients.

(3) Pursuant to section 353 (i), any laboratory with respect to tests or other procedures made by it for any person engaged in the business of insurance if made solely for the purposes of determining whether to write an insurance contract or of determining eligibility or continued eligibility for payments thereunder.

(4) Clinical laboratories exempted by the Secretary pursuant to section 353 (1) of the Act.

(c) The requirements of this part for the issuance and renewal of licenses do not apply to an accredited laboratory: Provided, That the Secretary finds that the standards applied by the accrediting body are equal to or more stringent than the provisions of the Act and of this part and there is adequate provision for assuring that such standards continue to be met by such hospital and such laboratory: Provided further, That the laboratory holds an unrevoked and unsuspended letter of exemption issued pursuant to section 74.46.

Subpart B-License: Applications and

(a) An application for the issuance or renewal of a license for a laboratory applicable to one or more laboratory procedures or categories of laboratory procedures for which standards are set out herein may be made to the Director by the owner, operator or authorized representative of such laboratory.

(b) The application shall be made on a form or forms prescribed by the Secretary, signed by the owner, operator, or by an authorized representative, and shall contain or be accompanied by such information, agreements and data as the Secretary may require, including an

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agreement that the applicant will operate the laboratory in accordance with standards which have been prescribed by the Secretary to carry out the purposes of the Act.

(c) A separate application must be filed for each laboratory location.

(d) An application for the issuance or renewal of a license shall be accompanied by the appropriate fee. The amount of the fee shall be $25 per annum for each of the categories enumerated in § 74.2(b) (1): Provided, That such fee of $25 per annum shall be imposed without regard to the number of procedures within each such category to which the license is applicable: Provided further, That the maximum fee required for each laboratory shall not exceed $125 per

annum.

(e) Licenses shall be issued to be applicable to one or more of the following procedures or categories of procedures except upon application for a limited license approved by the Secretary:

(1) Microbiology and serology:
(i) Bacteriology.
(ii) Mycology.
(iii) Parasitology.
(iv) Virology.

(v) Serology (syphilis).
(vi) Serology (nonsyphilis).
(2) Clinical chemistry:

(i) Blood and cerebrospinal fluid chemistry.

(ii) Endocrinology.
(iii) Toxicology.
(iv) Urinalysis.

(3) Immuno-hematology.
(4) Hematology.

(5) Pathology:

(i) Exfoliative cytology.
(ii) Histopathology.
(iii) Oral pathology.
(6) Radiobioassay.

(f) An application for the renewal of a license shall be submitted not less than 30 days nor more than 60 days prior to the expiration of the period for which the license was issued.

§ 74.11 Issuance or renewal of license.

(a) If, after a review of the application and such additional information as the Secretary may require and an onsite inspection of the laboratory premises with respect to which the license is sought, the Secretary is satisfied that such laboratory will be operated in accordance with the standards and other requirements of the Act and of this part and will provide consistent performance

of accurate laboratory procedures and services, he shall issue an initial or renewal license with respect to such laboratory applicable to laboratory procedures or categories of procedures as specified therein.

(b) Such initial or renewal license shall be issued for a term of 1 year.

(c) If the Secretary determines that the application for the issuance or renewal of a license shall not be granted in whole or in part, he will, prior to denial of such application, give reasonable notice and opportunity for a hearing as provided herein to the applicant and a statement of the grounds on which it is proposed to deny the application or any part thereof.

§ 74.12 Repetitious applications.

Where an application for a license has been denied in whole or in part, or a license has been revoked or limited, an application for a license to be applicable to procedures or categories of procedures affected by such adverse action made by or on behalf of the same applicant or licensee, shall not be accepted for consideration until after 1 year from the effective date of the adverse action: Provided, That upon good cause found, the Secretary may waive the application of this section.

Subpart C-Quality Control

§ 74.20 General.

Quality controls imposed and practiced by the laboratory must provide for and

assure:

(a) Preventive maintenance, periodic inspection, or testing for proper operation of equipment and instruments; validation of methods, evaluation of reagents and volumetric equipment, surveillance of results; and remedial action to be taken in response to detected defects.

(b) Adequacy of facilities, equipment, instruments, and methods for performance of the procedures or categories of procedures for which a license application is filed or granted; proper lighting for accuracy and precision; convenient location of essential utilities; monitoring of temperature controlled spaces and equipment, including water baths, incubators, sterilizers, and refrigerators, to assure proper performance; evaluation of analytical measuring devices, such as photometers and radioactivity counting equipment, with respect to all critical operating characteristics.

(c) Labeling of all reagents and solutions to indicate identity, and when significant, titer, strength, or concentration, recommended storage and preparation or expiration date, and other pertinent information. Materials of substandard reactivity and deteriorated materials may not be used.

(d) The availability at all times, in the immediate bench area of personnel engaged in examining specimens and performing related procedures within a category, e.g., clinical chemistry, hematology, bacteriology, of current laboratory manuals or other complete written descriptions and instructions relating to the analytical methods used by those personnel, properly designated and dated to reflect the most recent supervisory reviews, and of reagents, control and calibration procedures, and pertinent literature references. Text books may be used as supplements to such written descriptions but may not be used in lieu thereof.

(e) Written approval by the director or supervisor of all changes in laboratory procedures.

(f) Maintenance and availability to laboratory personnel and to the Secretary of records reflecting dates, and where appropriate the nature, of inspection, validation, remedial action, monitoring, evaluation, and changes and dates of changes in laboratory procedures.

(g) Solicitation designed to provide for collection, preservation, and transportation of specimens sufficiently stable to provide accurate and precise results suitable for clinical interpretation. § 74.21 Microbiology.

Chemical and biological solutions, reagents, and antisera shall be tested and inspected each day of use for reactivity and deterioration.

(a) Bacteriology and mycology. Staining materials shall be tested for intended reactivity by concurrent application to smears or microorganisms with predictable staining characteristics. Each batch of medium shall be tested before or concurrently with use with selected organisms to confirm required growth characteristics, selectivity, enrichment, and biochemical response.

(b) Parasitology. A reference collection of slides, photographs, or gross specimens of identified parasites shall be available in the laboratory for appropriate comparison with diagnostic specimens. A calibrated ocular micrometer