(c) If, at any time after a certificate is received by the person to whom it is issued, any color from the batch covered by such certificate becomes changed in composition in any manner whatever, such certificate shall expire with respect to such changed color. After such expiration such color shall be considered to be from a batch that has not been certified in accordance with this part; except that such color shall not be so considered for the purpose of certifying a batch of a mixture in which such color was used as an ingredient, if such change resulted solely from such use.

(d) A certificate shall expire with respect to any coal-tar color covered thereby if the package in which such color was closed for shipment or delivery is opened, unless opened solely for repacking by the person to whom such certificate was issued. After such expiration such color shall be considered to be from a batch that has not been certified in accordance with this part; except that such color shall not be so considered:

(1) When such color is used, subject to the restrictions prescribed by paragraphs (f), (g), and (h) of this section, in coloring a food, drug, or cosmetic;

(2) For the purpose of certifying a batch made by repacking such color; or

(3) For the purpose of certifying a batch of a mixture in which such color is used as an ingredient.

(e) A certificate shall not be effective with respect to a package of coal-tar color, and such color shall be considered to be from a batch that has not been certified in accordance with this part, if such package is shipped or delivered under a label which does not bear all words, statements, and other information required by § 135.11 to appear thereon.

(f) If a mixture from a batch containing a diluent permitted by § 135.6 (a) (3) only because such diluent does not become a component of a food colored by such mixture is used in coloring another food, such mixture so used shall be considered to be from a batch that has not been certified in accordance with this part.

(g) If coal-tar color from a batch which was certified for use in drugs and cosmetics only is used in coloring a food; such color so used shall be considered

to be from a batch that has not been certified in accordance with this part.

(h) If a coal-tar color from a batch which was certified for use only in externally applied drugs and cosmetics is used in food or in other drugs or other cosmetics, such color so used shall be considered to be from a batch that has not been certified in accordance with this part.

(i) If a coal-tar color from a batch containing any lake listed in § 135.3 is used in coloring any food except shell eggs, such color so used shall be considered to be from a batch that has not been certified in accordance with the regulations in this part.

[4 F. R. 1935, as amended at 5 F. R. 1141]

§ 135.11 Labeling. (a) The label on each package of coal-tar color from a batch that has been certified in accordance with this part shall bear, in addition to other words, statements, and information required by or under authority of the act to appear on such label: (1) The name of such color;

(2) The lot number of such batch unless, in the case of any mixture for household use which contains not more than 15 percent of pure dye and which, if it is liquid, is in packages containing not more than 2 fluid ounces, or, if it is solid, semisolid, or viscous, is in packages containing not more than 2 avoirdupois ounces, such lot number appears on the labeling or on each invoice accompanying shipments or deliveries of such mixture, or unless in the case of such mixture there appears on the label, a code number which the manufacturer has identified with the lot number by giving to the Food and Drug Administration written notice that such code number will be used in lieu of the lot number; (3) The percentage of pure dye in such color as provided in paragraph (b) of this section; and,

(4) In case such batch was certified for use only in drugs and cosmetics, the statement "Not for use in coloring food"; or in case such batch was certified for use only in externally applied drugs and cosmetics, the statement "Not for use in coloring food, or in coloring any drug or cosmetic used internally or on the lips or any mucous membrane"; or in case such batch is a mixture containing a diluent permitted by § 135.6 (a) (3) only because such diluent does not become a

(the blank to be filled in by the name of such food); or in case such batch contains any lake listed in § 135.3 or any diluent permitted by § 135.6 (a) (3) only because such batch is for use in coloring shell eggs, the statement "Not for use in coloring any food except shell eggs."

(b) The statement of the percentage of pure dye in a coal-tar color shall express:

(1) The percentage of pure dye shown in the certificate covering such color; or (2) The minimum percentage by weight of pure dye in such color. Where the statement expresses the minimum percentage, no variation below the stated minimum shall be permitted, and variations above shall not be unreasonably large.

(c) The name by which a straight color is designated on its label shall be the name by which such color is listed in § 135.3, § 135.4, or § 135.5.

(d) No batch of a mixture shall be certified under this part if:

(1) The name of such mixture is false or misleading in any particular; or

(2) The name of such mixture is the same as or simulates the name of a previously certified batch of a mixture containing a different substance, or a different percentage of a pure dye; but this provision shall not apply if the person who requests certification of such batch is the owner of such name and has given 3 months' written notice to the Food and Drug Administration specifying the change to be made in the composition of such mixture.

[4 F. R. 1935, as amended at 5 F. R. 1141]

(a)

§ 135.12 Records of distribution. The person to whom a certificate is issued shall keep complete records showing the disposal of all the coal-tar color from the batch covered by such certificate. Upon the request of any officer or employee of the Food and Drug Administration, or of any other officer or employee of the United States acting on behalf of the Federal Security Administrator, such person, at all reasonable hours until at least 1 year after disposal of all such color, shall make such records available to any such officer or employee, and shall accord to such officer or employee full opportunity to make inventory

of stocks of such color on hand and otherwise to check the correctness of such records.

(b) The records required to be kept by paragraph (a) of this section shall show:

(1) Each quantity used by such person from such batch and the date and kind of such use;

(2) The date and quantity of each shipment or delivery from such batch, and the name and post office address of the person to whom such shipment or delivery was made.

§ 135.13 Authority to refuse certification service. When it appears to the Federal Security Administrator that a person has:

(1) Obtained a certificate through fraud or misrepresentation of a material fact;

(2) Falsified the records required to be kept by § 135.12; or

(3) Failed to keep such records, or to make them available, or to accord full opportunity to make inventory of stocks on hand or otherwise to check the correctness of such records, as required by such section,

the Administrator may immediately suspend service to such person under this part, and may continue such suspension unless and until such person shows adequate cause why such suspension should be terminated.

§ 135.14 Procedure for admitting colors to listing. (a) An application for the admission of a coal-tar color to listing in § 135.3, § 135.4, or § 135.5 shall be accompanied by:

(1) Full reports of investigations which are adequate to show whether or not such color is harmless and suitable for use in food, drugs, and cosmetics, or in drugs and cosmetics, or in externally applied drugs and cosmetics, as the case may be;

(2) A full statement of the percentages and compositions of the pure dye and all intermediates and other impurities contained in such color;

(3) A full statement showing the identity, purity, and quantity or proportion of each intermediate and other article used as a component of such color, and all steps in the process used for the manufacture of such color;

(4) A full description of practical and accurate methods of analysis for the

quantitative determination of the pure dye and of all intermediates and other impurities contained in such color;

(5) A full description of practical and accurate methods for the identification of such dye in food, drugs, and cosmetics colored therewith, or in drugs and cosmetics colored therewith;

(6) A 5-pound sample of such color (unless the Food and Drug Administration authorizes or requires submission of other quantity suitable to the need for investigation) taken from a batch produced under practical manufacturing conditions, and accurately representative of such batch; and

(7) The advance deposit prescribed by 135.15 (b) (2).

(b) If the Administrator determines, after such investigation as he considers to be necessary, that:

(1) Such color is harmless and suitable for use in foods, drugs, and cosmetics, or in drugs and cosmetics, or in externally applied drugs and cosmetics;

(2) Practical and accurate methods of analysis exists for the quantitative determination of the pure dye and all intermediates and other impurities contained in such color; and

(3) Practical and accurate methods exist for the identification of such dye in food, drugs, and cosmetics colored therewith, or in drugs and cosmetics colored therewith, the Administrator, for the purpose of listing such color in § 135.3, § 135.4, or § 135.5, shall proceed with a proposed amendment to this part as prescribed by section 701 (e) of the act.

(c) If the Administrator, after such investigation as he considers to be necessary, determines that such color does not comply with the requirements laid down by paragraph (b) of this section with respect to procedure for the purpose of listing such color in § 135.3, § 135.4, or § 135.5, the Administrator shall give notice thereof to the applicant, stating the respects in which such color does not so comply.

§ 135.15 Fees. (a) (1) The fee for the service provided by the regulations in this part, in the case of each request for certification submitted in accordance with § 135.8 (b), shall be 6 cents per pound of the batch covered by such request; but no such fee shall be less than $9 nor more than $75.

(2) The fee for the service provided by the regulations in this part, in the case of each request for certification submitted in accordance with § 135.8 (c) or (d), shall be 6 cents per pound of the batch covered by such request; but no such fee shall be less than $3 nor more than $25.

(b) (1) The fee for the service provided by this part, in the case of each application for the admission of a coaltar color in listing in § 135.3, § 135.4, or § 135.5, shall be the cost incurred by the Agency in making the investigations contemplated by § 135.14 (b) and (c).

(2) Such application shall be accompanied by an advance deposit of $500 to cover such fee, and thereafter advance deposits of the same amount (unless the Food and Drug Administration authorizes deposit in other amount) shall be made whenever necessary to prevent arrears in the payment of such fee. Any excess advance deposit so made shall be returned to the applicant after the close of such investigation;

(c) All fees required by this part should be paid by money-order, bank draft or certified check, drawn to the order of the Treasurer of the United States, collectible at par, at Washington, D. C.

(d) All earned fees shall be deposited in the Treasury of the United States to the credit of Miscellaneous Receipts, Federal Security Agency.

[4 F. R. 1935, as amended at 5 F. R. 1141, 11 F. R. 6970]

tency-(a) Cylinders (cups). Use stainless steel cylinders with an outside diameter of 8 mm. (±0.1 mm.), an inside diameter of 6 mm. (±0.1 mm.), and a length of 10 mm. (±0.1 mm.).

(b) Culture media. Use ingredients that conform to the standards prescribed by the U. S. P. or N. F. (1) Make nutrient agar for the seed layer and for carrying the test organism as follows:

Crystalline penicillin tablets.

Penicillin bougies.

Crystalline penicillin and epineph

Aluminum penicillin.

Aluminum penicillin in oil.

Penicillin for inhalation therapy.

Procaine penicillin for aqueous injection.

141.30 Ephedrine penicillin.

141.31 141.32

141.33 141.101

Ephedrine penicillin tablets.

Procaine penicillin and buffered crystalline penicillin for aqueous injection.

Buffered penicillin powder. Streptomycin sulphate, streptomycin hydrochloride, streptomycin phosphate, streptomycin trihydrochloride calcium chloride; potency.

141.102 Streptomycin sulphate, streptomycin hydrochloride, streptomycin phosphate, streptomycin trihydrochloride calcium chloride; sterility.

141.103 Streptomycin sulphate, streptomycin hydrochloride, streptomycin phosphate, streptomycin trihydrochloride calcium chloride; toxicity.

141.104 Streptomycin sulphate, streptomycin hydrochloride, streptomycin phosphate, streptomycin trihydrochloride calcium chloride; pyrogens.

141.105 Streptomycin sulphate, streptomycin hydrochloride, streptomycin phosphate, streptomycin trihydrochloride calcium chloride;

In lieu of preparing the media from the individual ingredients specified in paragraph (b) (1), (2), and (3) of this section, they may be made from a dehydrated mixture which, when reconstituted with distilled water, has the same composition as such media. Minor modification of the individual ingredients specified in paragraph (b) (1), (2), and (3) of this section are permissible if the resulting media possess growth promoting properties at least equal to the media described.

(c) Working standard. Keep the working standard (obtained from the Food and Drug Administration) at room temperature in tightly stoppered vials, which in turn are kept in larger stoppered tubes containing a suitable desiccant. Weigh out carefully in an atmosphere of 50 percent relative humidity or less between 4 and 5 mg. of the working standard and dilute with sterile 1 per

cent phosphate buffer (pH 6.0) to make a stock solution of any convenient concentration. Keep this solution at a temperature of about 10° C., and use for one day only. From this stock solution make appropriate working dilutions.

(d) Preparation of sample. Dissolve aseptically, in sterile distilled water, the sample to be tested to make an appropriate stock solution.

(e) Preparation of plates. Add 21 ml. of agar to each Petri dish (20 x 100 mm.). Distribute the agar evenly in the plates and allow it to harden. Use the plates the same day they are prepared. The test organism is Staphylococcus aureus (FDA 209-P) or (9144) American Type Culture Collection. Maintain the test organism on agar slants and transfer to a fresh agar slant about once a week. Prepare an inoculum for the plates by transferring the culture from the agar slant into broth and incubate at 37° C. From 16 to 24 hours thereafter add 2.0 ml. of this broth culture to each 100 ml. of agar which has been melted and cooled to 48° C. Mix the culture and agar thoroughly and add 4 ml. to each of the plates containing the 21 ml. of the uninoculated agar. Tilt the plates back and forth to spread the inoculated agar evenly over the surface. Porcelain covers glazed on the outside are used. Place four cylinders on the agar surface so that they are at approximately 90° intervals on a 2.8 cm. radius. In so placing the cylinders drop them from a height of 2 inch, using a mechanical guide or device. A suspension of the test organism may be used in place of the broth culture described above in preparing the inoculum for the seeding of plates. Prepare such suspension as follows: Wash the organisms from an agar slant which has been incubated for 24 hours at 37° C. and stored for 24 hours at room temperature with 2.0 ml. of sterile physiological saline onto a large agar surface such as that provided by a Roux bottle containing 300 ml. of agar. Spread the suspension of organisms over the entire agar surface with the aid of sterile glass beads. Incubate 24 hours at 37° C. and store for 24 hours at room temperature. Wash the resulting growth from the agar surface with about 50 ml. of sterile physiological saline. Standardize this suspension by determining the dilution which will permit 20 percent light transmission through a filter at 6500 Ångstrom units in a photoelectric colorimeter.

Add 1.5 to 2.0 ml. of this resulting dilution to each 100 ml. of agar which has been melted and cooled to 48° C. to prepare the inoculum for the plates. The suspension may be used for one week.

(f) Assay. Use four plates for each sample. Fill one cylinder on each plate with a 1.0 unit per ml. dilution, and one with a 0.25 unit per ml. dilution, of the working standard. Add the estimated dilutions of 1.0 unit per ml. and 0.25 unit per ml. of the sample under test to the remaining two cylinders on each plate. Carefully place the plates in racks and incubate 16 to 18 hours at 37° C. After incubation measure the diameter of each circle of inhibition to the nearest 0.5 mm. using a colony counter with a mm. scale etched into the supporting glass over the light source. Other measuring devices of equal accuracy may be used.

(g) Estimation of potency and error. (1) Use the accompanying chart (Chart 1) and nomograph (Chart 2) for estimating the potency and its error. Το use the chart for estimating potency two values, namely, V and W, are required. For each plate calculate two values

where SH and si are the diameters of the zones of inhibition in mm. of the 1.0 unit and 0.25 unit dilutions of the standard, respectively, and uн and u refer similarly to the corresponding dilutions of the sample under test. The value V is the sum of the v values for all plates and W is the sum of the w values for all plates. To estimate the potency locate the point on the chart corresponding to the values of V and W, and the potency can be read from the radial lines on the chart.

(2) The error of the assay is estimated by using the nomograph which requires five values, namely, the potency, V, W, Rv, and Rw. Rv (the range of the v's) is the highest value of v minus the lowest value of v obtained from the individual plates. Similarly, Rw is the difference between the highest and lowest w values. After obtaining these five values, connect with a straight edge the points corresponding to v and w on the respective scales on the right side of the nomograph. Mark with a pin or sharp-pointed pencil the intersection of the straightedge and the diagonal line of the nomograph.