APPARATUS

(A) Spectrophotometer (Cary 118 or equivalent).

(B) Separatory funnels-one 1000 mL and one 500 mL.

REAGENTS

NOTE: Use distilled water when water is required.

(A) Glacial Acetic Acid (ACS grade). (B) Diethyl ether (Anhydrous)-Note and follow safety precautions on container.

(C) 8 N HCl-Pour 165 mL H2O into a 500 mL graduate. Place the graduate in hood, then add HCl conc. to bring to volume. Carefully pour this solution into a 500 mL Erlenmeyer flask, Stopper and shake. Label the flask.

(D) 2% (w/w) NaOH-Pour ca 190 mL H2O into a 250 mL mixing graduate. Add 8 g. (5.23 mL) of 50% (w/w) NaOH, bring to 200 mL volume with water, stopper and mix. Pour this solution into a glass bottle, label and stopper with a polytetrafluoroethylene top. (E) Ethanol (95%).

PROCEDURE

Weigh a 250 mL beaker to tenths of a mg and add 100 mg of dye. Record weight to tenths of a mg.

NOTE: The following work must be performed in the hood.

Add 75 mL of 8 N HCl and 100 mL of glacial acetic acid to the beaker and stir.

Place the beaker on a hot plate and heat with stirring, until all of the dye is in solution.

Remove the beaker from the hot plate, cover with a watch glass and allow to cool to room temperature (1-2 hrs).

When the dye solution is at room temperature, transfer the solution to a 1000 mL separatory funnel.

Rinse the beaker three times with 50 mL portions of H2O, transferring each rinse to the 1000 mL funnel.

Add 150 mL of ether to the funnel, stopper and shake for 10 seconds, then invert funnel and open stopcock to remove gas buildup.

Shake the funnel for one minute, opening the stopcock a few times while the funnel is inverted to remove gas buildup. (Use this shake procedure throughout method.)

Allow the funnel to stand until the layers have separated.

Transfer the bottom (aqueous) layer to a 500 mL separatory funnel, add 100 mL of ether, stopper and shake for one minute.

When the layers have separated, drain off the bottom layer into a waste beaker.

Pour the ether layer in the 500 mL separatory funnel into the 1000 mL separatory funnel.

Rinse the 500 mL sep. funnel with 100 mL H2 O, then transfer it to the 1000 mL sep. funnel, stopper and shake for one minute.

When the layers have separated, drain off the bottom aqueous layer into the waste beaker.

Rinse the 500 mL funnel at least three times (total) and repeat the 100 mL water washes until no color is present in the aqueous layer. Discard the bottom aqueous layer to the waste beaker after each separation.

Shake the ether layer twice more with 100 mL portions of H2 O, discarding the bottom aqueous layer after each separation.

Remove the unsulfonated subsidiary color from the ether by shaking the ether layer for one minute with 20 mL of 2% (w/w) NaOH. Appropriately label a 100 mL beaker. After the layers separate, drain the aqueous alkaline layer into the beaker and save for the determination of 3-hydroxy-4-[(4-methylphenyl) azo]-2-naphthalenecarboxylic acid, sodium

salt.

If there is any color left in the ether, shake for one minute with another 20 mL portion of 2% (w/w) NaOH. After the layers have separated, drain off the aqueous alkaline layer into the 100 mL beaker.

If color remains in the ether layer, repeat the above step for a total of three washes of the ether with 2% (w/w) NaOH. Note: Three washes is usually sufficient to remove the unsulfonated subsidiary.

With the stopper removed, gently swirl the ether layer in the sep. funnel twice to separate the remaining aqueous base. Drain this into the 100 mL beaker.

Appropriately label a 250 mL beaker. Pour the ether layer into the beaker. Allow the ether to evaporate to near dryness. Cool to room temperature. Add ca 8 mL ethanol (95%). Swirl beaker to mix contents. Quantitatively transfer to a 25 mL graduate using ethanol (95%) rinses. Add ethanol (95%) to bring volume to 15 mL.

SPECTROPHOTOMETRIC ANALYSIS

Spectrophotometer Parameters:
Scan Range: 400-700 nm
Scan: 50 nm/in; 5.0 nm/sec.
Absorbance Range: 0-1 AUFS

Cell length: 1 cm (Note: Reference and Sample cells)

(1) Record the visible spectrum of a blank. Fill the reference cell with distilled water and the sample cell with ethanol (95%).

(2) Rinse the sample cell with 2-3 mL of the ether soluble material (in ethanol solution); then fill the cell. Record the visible spectrum of the ether soluble material.

(3) Compare the spectra obtained to the spectra attached. The attached spectra represents 150% of the absorbance at each wavelength for similarly analyzed D&C Red No. 6 Lot AA5169.

The spectra of the current sample must not exceed the attached spectra at any wavelength in order to pass test.

80.38 Treatment of batch after certification. 80.39 Records of distribution.

AUTHORITY: 21 U.S.C. 371, 379e.

SOURCE: 42 FR 15662, Mar. 22, 1977, unless otherwise noted.

Subpart A-General Provisions 880.10 Fees for certification services.

(a) Fees for straight colors including lakes. The fee for the services provided by the regulations in this part in the case of each request for certification submitted in accordance with §80.21(j)(1) and (j)(2) shall be $0.35 per pound of the batch covered by such requests, but no such fee shall be less than $224.

(b) Fees for repacks of certified color additives and color additive mixtures. The fees for the services provided under the regulations in this part in the case of each request for certification submitted in accordance with §80.21(j)(3) and (j)(4) shall be:

(1) 100 pounds or less-$35.

(2) Over 100 pounds but not over 1,000 pounds-$35 plus $0.05 for each pound over 100 pounds.

(3) Over 1,000 pounds-$89 plus $0.02 for each pound over 1,000 pounds.

(c) Advance deposits. Any person regularly requesting certification services may deposit funds in advance of requests as prepayment of fees required by this section.

(d) Method of payment. All deposits and fees required by this section shall be paid by money order, bank draft, or certified check, drawn to the order of

the Food and Drug Administration, collectable at par at Washington, DC. All such deposits and fees shall be forwarded to the Center for Food Safety and Applied Nutrition (HFS-100), Food and Drug Administration, 5100 Paint Branch Pkwy., College Park, MD 20740, whereupon after making appropriate records thereof, they will be transmitted to the Treasurer of the United States for deposit to the special account "Salaries and Expenses, Certification, Inspection, and Other Services, Food and Drug Administration."

(e) Refunds from advance deposits. Whenever in the judgment of the Commissioner the ratio between fees collected (which are based upon experience and the best estimate of costs and the best estimate of earnings) and the costs of providing the service during an elapsed period of time, in the light of all circumstances and contingencies, warrants a refund from the fund collected during such period, he shall make ratable refunds to those persons to whom the services were rendered and charged, except that no refund shall be made where the computed ratable amount for the elapsed period is less than $5.00.

[42 FR 15662, Mar. 22, 1977, as amended at 47 FR 24692, June 8, 1982; 54 FR 24890, June 12, 1989; 59 FR 60899, Nov. 29, 1994; 61 FR 3572, Feb. 1, 1996; 61 FR 14479, Apr. 2, 1996; 66 FR 56035, Nov. 6, 2001; 70 FR 15756, Mar. 29, 2005]

Subpart B-Certification Procedures

§ 80.21 Request for certification.

A request for certification of a batch of color additive shall:

(a) Be addressed to the Commissioner of Food and Drugs.

(b) Be prepared in the manner set forth in paragraph (j) of this section. (c) Be submitted in duplicate. (d) Be signed by a responsible officer of the person requesting certification of the batch. In the case of a foreign manufacturer, the request for certification must be signed by a responsible officer of such firm, and, by his agent who resides in the United States.

(e) Show the name and post office address of the actual manufacturer in

(f) Be accompanied by the fee prescribed in §80.10 unless the person has established with the Food and Drug Administration an advanced deposit to be used for prepayment of such fees. In no case shall the Commissioner consider a request for certification of a batch of color additive if the fee accompanying such request is less than that required by § 80.10 or if such fee exIceeds the amount held in the advance deposit account of the manufacturer submitting such request for certification.

(g) Be accompanied by the sample prescribed in § 80.22 consisting of: (1) Four ounces in the straight colors and lakes.

case of

(2) Two ounces in the case of repacks and mixtures.

A sample accompanying a request for certification must be submitted under separate cover and should be addressed to the Color Certification Branch.

(h) The name of a color additive shall be given in the following manner:

(1) The name of a straight color shall be the name of the color as listed in parts 74 and 81 of this chapter.

(2) The name of a lake shall be the name derived in the manner described in part 82 of this chapter.

(3) The name of a mixture shall be the name given to such mixture by the person requesting certification.

(4) The name of a repack shall be the name described in paragraph (h)(1), (2), or (3) of this section, whichever is applicable.

(i) The information and samples enumerated in paragraphs (a) to (h), inclusive, of this section are the minimum required. Additional information and samples shall be submitted at the request of the Food and Drug Administration when such additional information and samples are necessary to determine compliance with the requirements of §80.31 for the issuance of a certificate.

(j) The form for submission of the application shall be one of the following, depending upon whether the color additive is a straight color, a lake, a repack of a previously certified color additive, or a color additive mixture.

(1) Request for certification of a batch of straight color additive.

Date