The Feedingstuffs (Zootechnical Products) Regulations 1999

Regulation 2(1)

SCHEDULE 1 EC REGULATIONS DELETING ADDITIVES FROM ANNEX B OF DIRECTIVE 70/524/EEC

1.  Commission Regulation (EC) No. 2788/98 (OJ No. L347, 23.12.98, p. 31) amending Council Directive 70/524/EEC concerning additives in feedingstuffs as regards the withdrawal of authorisation for certain growth promoters.

2.  Council Regulation (EC) No. 2821/98 (OJ No. L351, 29.12.98, p. 4) amending, as regards withdrawal of the authorisation of certain antibiotics, Directive 70/524/EEC concerning additives in feedingstuffs.

3.  Commission Regulation (EC) No. 45/1999 (OJ No. L6, 12.1.1999, p. 3) amending Council Directive 70/524/EEC concerning additives in feedingstuffs as regards withdrawal of the authorisation of certain additives belonging to the group of coccidiostats and other medicinal substances.

Regulation 2(2), (3) and (4)

SCHEDULE 2 SUPPLEMENTARY PROVISIONS RELATING TO INTERPRETATION

PART I Expressions having the same meaning as in Directive 70/524/EEC

• animal feeding

• antibiotics

• coccidiostats and other medicinal substances

• compositional characteristics

• country of destination

• growth promoters

• market

• use

PART II Expressions having the same meaning as in Directive 70/524/EEC as amended by Directive 96/51/EC

• Community authorisation

• delivered

• identification note

• incorporate

• last stage of circulation

• monograph

• originate

• period of authorisation

• reference sample

• representative within the Community

• standard sample

• supply

• toxicological tests on vertebrates

• unforeseen undesirable interaction

• veterinary medicine

PART III Expressions having the same meaning as in Directive 95/69/EC

• cease

• demonstrate

• essential condition

• exclusive requirements

• facilities

• holding

• located

• manufacture

• on the spot verification

• package

• produce

• reasonable time

• register

• store

• update

• wrap

Regulations 6(8)(b), 9(1), 15(1) and 23(1)

SCHEDULE 3 FEES

PART I Fees payable in relation to the submission of dossiers

Application	Fee (£) per dossier
Application under regulation 5(3)	25,000
Application under regulation 6(1)	25,000
Application under regulation 7(1)(a)	25,000
Application under regulation 7(1)(b)	10,000
Application under regulation 8(1)	2,500

PART II Fees payable in relation to the approval of establishments

Application	Fee (£)
Application under regulation 10(1)(a) or 12 for approval of an establishment to manufacture a zootechnical additive	405
Application under regulation 10(1)(b) or 12 for approval of an establishment to manufacture a zootechnical premixture	405
Application under regulation 10(1)(c) or 12 for approval of an establishment to manufacture a zootechnical compound feedingstuff	113
Application under regulation 10(1)(d) or 12 for approval of an establishment to produce a zootechnical compound feedingstuff for the exclusive requirements of the applicant’s holding	113
Application under regulation 10(1)(e) or 12 for approval of an establishment to manufacture a zootechnical compound feedingstuff using a minimum proportion of 0.05% by weight of a premixture	405

PART III Fees payable in relation to the approval of intermediaries

Application	Fee (£)
Application for approval under regulation 18 or 20 to exercise an intermediary activity	151

Regulations 80 and 82(2)(b)

SCHEDULE 4 FORM OF CERTIFICATE OF ANALYSIS

CERTIFICATE OF ANALYSIS OF SAMPLE OF PRODUCT ANALYSED PURSUANT TO THE FEEDINGSTUFFS (ZOOTECHNICAL PRODUCTS) REGULATIONS 1999 (1)

Regulation 84(1), (2) and (3)

SCHEDULE 5 METHODS OF ANALYSIS

PART I COMMUNITY METHODS OF ANALYSIS

Column (1)	Column (2)
Substance	Community provision
Amprolium	Part 1 of Annex II to Directive 74/203/EEC(1) as corrected by a corrigendum published on 1st May 1974(2)
Avoparcin	Part 1 of the Annex to Directive 81/715/EEC(3)
Carbadox	Part C of the Annex to Directive 1999/27/EC(4)
Copper	Part 3 of the Annex to Directive 78/633/EEC(5)
Diclazuril	Part B of the Annex to Directive 1999/27/EC(d)
Dinitolmide (DOT)	Part 3 of Annex II to Directive 74/203/EEC(a)
Ethopabate	Part 2 of Annex II to Directive 74/203/EEC(a)
Flavophospholipol	Part 2 of the Annex to Directive 78/633/EEC(e)
Halofuginone	The Annex to Directive 93/70/EEC(6)
Menadione (vitamin K3)	Part 5 of Annex II to Directive 74/203/EEC(a)
Methyl benzoquate	Part 2 of the Annex to Directive 93/117/EC(7)
Monensin sodium	Part 2 of the Annex to Directive 81/715/EEC(c)
Nicarbazin	Part 4 of Annex II to Directive 74/203/EEC(a)
Olaquindox	Part C of the Annex to Directive 98/64/EC(8)
Robenidine	Part 1 of the Annex to Directive 93/117/EC(g)
Spiramycin	The Annex to Directive 84/425/EEC(9)
Tylosin	Part 4 of Annex II to Directive 72/199/EEC(10)
Virginiamycin	Part 5 of Annex II of Directive 72/199/EEC(j) as replaced by Article 2 of, and Annex II to, Directive 84/4/EEC(11)
Vitamin A	Part 1 of Annex II to Directive 73/46/EC(12)
Zinc bacitracin	Part 1 of the Annex to Directive 78/633/EEC(e) as replaced by Article 3 of, and Annex III to, Directive 84/4/EEC(k)

PART II DETERMINATION OF METICLORPINDOL (3,5-dichloro-2,6- dimethylpyridine-4-ol)

1.  SCOPE AND FIELD OF APPLICATION

• The method is for the determination of the quantity of meticlorpindol in complete feeding stuffs, protein concentrates and feed supplements. The lower limit of the determination is 60mg/kg.

2.  PRINCIPLE

• Meticlorpindol is extracted from the feed with methanolic ammonia solution, and a portion of the extract is passed through a column of aluminium oxide onto a column of ion-exchange resin. The meticlorpindol is retained on the resin and interfering substances are removed by washing with 80% methanol. The meticlorpindol is eluted from the resin with 40% acetic acid and the absorbance is measured at 267nm.

3.  REAGENTS

• Note: The suitability of a batch of aluminium oxide and of the other reagents should be tested before use by analysing a blank feed to which a known amount of meticlorpindol has been added.

  3.1.

  Aluminium oxide for column chromatography, 100 to 250 mesh, alkaline, Brockman activity 1.

  3.2.

  Ammonia (density 0.88g per ml).

  3.3.

  Anion exchange resin, AG1-X8 or Dowex 1-X8, 100 to 200 mesh—To convert Dowex resin in the chloride form to the acetate form add 1 litre of 6M hydrochloric acid to 350g of resin in a 3 litre beaker, and heat the mixture on a steam bath for 2 to 3 hours. Pour the slurry into a glass Buchner funnel, and wash the resin with water until the washings are free from chloride (about 6 litres of water are required). Transfer the resin to a 50mm diameter glass column having a coarse sintered-glass disc at the bottom end, and wash with sodium acetate solution (5g sodium acetate, anhydrous, dissolved in water and diluted to 100ml) until the column effluent gives only a cloudy solution on addition of silver nitrate solution. Return the resin to the glass Buchner funnel, and wash with water. Transfer the resin to a 3 litre beaker, add 1 litre of 40% v/v acetic acid solution (3.4) and heat on a steam bath for 3 hours or longer. Filter, and wash the resin again with water until the washings are free from chloride. Store the resin in water.

  3.4.

  Acetic acid solution 40% v/v.

  3.5.

  Methanol.

  3.6.

  Methanol solution 80% v/v.

  3.7.

  Ammoniacal methanol solution: dilute 1 volume of ammonia (3.2) with 19 volumes of methanol (3.5).

  3.8.

  Meticlorpindol standard solution: weigh, to the nearest 0.1mg, 125mg of meticlorpindol into a beaker, add 25ml of sodium hydroxide solution (2g sodium hydroxide dissolved in water and diluted to 100ml) to dissolve the meticlorpindol, transfer the solution to a 500ml graduated flask, and dilute to the mark with water. This solution contains 250μg per ml meticlorpindol.

4.  APPARATUS

4.1 Aluminium oxide column: constructed as indicated in the diagram included in this method of analysis.

4.2 Ion exchange column: constructed as indicated in the diagram included in this method of analysis.

4.3 Spectrophotometer, recording, with 10mm silica cells.

5.  PROCEDURE

5.1 Extraction of meticlorpindol

• Weigh, to the nearest 0.001g, approximately 50g of the finely divided and mixed sample, or a suitable amount expected to contain about 12mg of meticlorpindol, transfer to a 500ml graduated flask, and add 400ml of ammoniacal methanol solution (3.7). Place a magnetic stirring bar in the flask and stir the mixture on a magnetic stirrer for 20 minutes. Remove the stirring bar from the flask, dilute to the mark with ammoniacal methanol solution (3.7), mix the contents well, and set aside for 20 to 30 minutes.

5.2 Purification

5.2.1 Aluminium oxide column: For each column required weigh approximately 25g of aluminium oxide (3.1) into an aluminium foil dish and place in an oven at 105±5°C for 1 hour. Remove the dish from the oven and cool to room temperature in a desiccator. Make a slurry of the aluminium oxide with 25ml of ammoniacal methanol solution (3.7) and filter on a Buchner funnel. Wash the aluminium oxide with methanol (3.5) until the washings are neutral. Form a slurry of the aluminium oxide with 50ml of methanol (3.5) and pour the slurry into the column (4.1). Allow the methanol to drip through the column. Place a plug of glass wool lightly on top of the aluminium oxide and then wash with 25ml of methanol (3.5). Do not allow the liquid in the column to fall below the top of the aluminium oxide. Discard the eluate.

5.2.2 Anion exchange column: Form a slurry in acetic acid (3.4) of sufficient resin (3.3) to fill the columns required. Filter on a Buchner funnel, wash the resin with twice its own volume of acetic acid (3.4) and then with aqueous methanol (3.6) until the washings are neutral. Form a slurry of a resin with aqueous methanol (3.6) and add sufficient to a column (4.2) to give a resin bed 20 to 30 mm deep after settling. Place a small plug of glass wool on top of the resin and wash the column with two 13ml portions of aqueous methanol (3.6). Do not allow the liquid level in the column to fall below the top of the resin. Discard the eluate.

5.2.3 Chromatographic procedure: By pipette transfer 10.0ml of the extract of the feed sample (5.1) directly onto an aluminium oxide column and at the same time transfer the same volume of ammoniacal methanol solution (3.7) directly onto a second aluminium oxide column (reagent blank). Allow the solutions to drain to the top of the aluminium oxide and then wash each column with three 12ml portions of aqueous methanol (3.6), allowing the liquid to drain to the top of the aluminium oxide each time. Let all the eluate from each column drain directly into separate ion-exchange columns, and then remove the aluminium oxide columns. Allow the liquid to drain to the top of the ion-exchange resin and then wash each column with four 13ml portions of aqueous methanol (3.6). Discard the eluates.

• Elute each column with two 10ml and then one 4ml portions of acetic acid (3.4). Collect the eluates from each column in separate 25ml graduated flasks and dilute the contents of each to the mark with acetic acid (3.4).

5.3 Determination

• Record the absorption spectrum of the sample extract between 350 and 245nm in 10mm silica cells with the reagent blank solution (5.2.3) as reference. Measure the absorbance of the sample extract at 267nm above a baseline obtained by drawing a line through the absorbance at 327 and 297nm and extending it through 267nm.

• (Note: Background absorption due to the feed approaches a linear function that can be described by the points on the curve at 296 and 327nm. Occasionally this is not the case, as can be detected by absorption peaks in the region between 350 and 297nm).

• Determine the concentration of meticlorpindol in the sample by reference to the calibration curve (5.4).

5.4 Calibration curve

• By pipette transfer 1, 5, 7.5, 10, 12.5 and 15ml portions of meticlorpindol standard solution (3.8) to separate 250ml graduated flasks. Dilute the contents of each flask to the mark with acetic acid (3.4). Record the absorption spectra of these solutions in 10mm silica cells between 350 and 245nm with acetic acid (3.4) as reference. Construct a calibration curve using the absorbances at 267nm as ordinates and the corresponding concentrations of meticlorpindol in μg per ml as abscissae.

6.  CALCULATION OF RESULTS

• The meticlorpindol contents in mg/kg of sample is given by the formula:

• 

• in which:

  • C=concentration of meticlorpindol, in μg per ml, read from the calibration curve equivalent to the absorbance of the test solution;

  • 23.23=a factor that makes allowance for the volume of the feed sample in the flask;

• and

  • M=mass of test portion in g.

• Absorbance at 327 and 297nm should not differ by more than 0.05 units and both points should be below 0.2. Results should be satisfactory as long as these criteria are kept in mind along with any obvious distortion in the appearance of the curve. No maximum other than that of meticlorpindol should be present.

PART III DETERMINATION OF NIFURSOL [3,5-dinitro-2-(5-nitrofurfuryli dene)salicylohydrazide]

1.  SCOPE AND FIELD OF APPLICATION

The method is for the determination of the quantity of nifursol in complete feeding stuffs, protein concentrates and feed supplements. Other substances that will provide a nitro group under the conditions of the method, e.g. nitrofurazone and furazolidone, will interfere. The lower limit of the determination is 20mg/kg.

2.  PRINCIPLE

The sample is extracted with dimethylformamide and the extract is purified on a column of aluminium oxide. A portion of the purified extract containing the nifursol is treated with phenylhydrazine hydrochloride and the resulting phenylhydrazone extracted into toluene. The addition of methylbenzethonium hydroxide to the toluene solution produces a blue colour, the absorbance of which is measured as 515nm.

3.  REAGENTS

3.1 Toluene.

3.2 Aluminium oxide for column chromatography, 80 to 200 mesh, alkaline, Brockman activity 1. To 100 parts of the aluminium oxide add 6 parts of powdered magnesium hydroxide. Shake in a screw-cap bottle to mix, add 8 parts of water, and mix until free from lumps.

3.3 Sand; acid washed.

3.4 Dimethylformamide solution, 95% v/v.

3.5 Dimethylformamide solution, 50% v/v.

3.6 Phenylhydrazine, hydrochloride solution: shake 0.25±0.005g of phenylhydrazine hydrochloride in 25ml of water, add 25ml of concentrated hydrochloric acid, and shake to dissolve the solid, filtering if necessary. Prepare this reagent immediately before use.

3.7 Methylbenzethonium hydroxide solution: about 10% in methanol.

3.8 Nifursol standard solution: weigh, to the nearest 0.1mg, 25mg of pure nifursol into a 100ml graduated flask, add 5ml of 95% v/v dimethylformamide solution (3.4), and mix until all the solid has dissolved. Dilute to the mark with methanol. Prepare this solution freshly each day.

4.  APPARATUS

4.1 Chromatographic column—A glass column, internal diameter: 20 to 25mm; length: 100 to 150mm plugged at the lower end with glass wool.

4.2 Spectrophotometer, with 10mm cells.

5.  PROCEDURE

5.1 Extraction

• Weigh to the nearest 0.001g, approximately 5g of the finely divided and mixed sample, or a suitable amount expected to contain about 350μg of nifursol and transfer to a 125ml conical flask. Add 50.0ml of 95% v/v dimethylformamide solution (3.4), insert a stopper loosely, and place the flask in a water-bath at 60°C±5°C for 30 minutes. Swirl the contents of the flask occasionally during this period. Shake the flask on a mechanical shaker for 30 minutes and then filter the contents through a rapid filter-paper, preferably under reduced pressure on a Buchner funnel. Transfer 40.0ml of water, and stir. Set the beaker aside, protected from light, for 30 minutes.

5.2 Purification

• Pack the chromatographic column (4.1) to a depth of 70mm with the prepared aluminium oxide (3.2) and on top of the aluminium oxide add a layer of sand (3.3) 15mm deep. Wash the column with 50ml of 50% v/v dimethylformamide solution (3.5) and then pass the dimethylformamide extract of the test sample through the column; reject the first 45ml of eluate and collect the next 17ml.

5.3 Determination

• Pipette 5.0ml of the eluate to a 20ml centrifuge tube, add 5ml of phenylhydrazine hydrochloride solution (3.6), mix, and place the tube in a water-bath at 40°C±2°C for 20 minutes. Remove the tube from the water-bath and cool it in running water for 5 minutes. Add 5.0ml of toluene (3.1) to the contents of the tube, insert a glass or plastic stopper (a rubber stopper must not be used), and shake vigorously 40 times. Centrifuge for 5 minutes to clear the toluene layer, and transfer 3.0ml of the toluene layer to a 10mm spectrophotometer cell. Add 0.2ml of methylbenzethonium hydroxide solution (3.7), mix immediately, and measure the absorbance of the solution within one minute at 515nm with toluene as reference. Determine the quantity of nifursol by reference to the calibration curve (5.4).

5.4 Calibration curve

• Pipette 5.0ml of nifursol standard solution (3.8) to a 200ml graduated flask, add 100ml of 95% v/v dimethylformamide solution (3.4), dilute to the mark with water and mix. Into separate 20ml centrifuge tubes transfer by pipette 1, 2, 3, 4 and 5ml portions of this solution and dilute the contents of each tube to 5ml with 50% v/v dimethylformamide solution (3.5).

• Treat the contents of each tube as described under “Determination” (5.3) beginning at “… add 5ml of phenylhydrazine hydrochloride solution (3.6) …”. Plot the calibration curve using the absorbance as the ordinates and the corresponding quantities of nifursol in μg as abscissae.

6.  CALCULATING THE RESULTS

• The nifursol content in mg/kg of sample is given by the formula:

• 

• in which:

  • A=μg of nifursol read from the calibration curve; and

  • M=mass of the test portion in g.

(1)

OJ No. L108, 22.4.74, p. 7.

(2)

OJ No. L121, 3.5.74, p. 56.

(3)

OJ No. L257, 10.9.81, p. 38.

(4)

OJ No. L118, 6.5.99, p. 36.

(5)

OJ No. L206, 29.7.78, p. 43.

(6)

OJ No. L234, 17.9.93, p. 17.

(7)

OJ No. L329, 30.12.93, p. 54.

(8)

OJ No. L257, 19.9.98, p. 14.

(9)

OJ No. L238, 6.9.84, p. 34.

(10)

OJ No. L123, 29.5.72, p. 6 (OJ/SE 1966—72, p. 74).

(11)

OJ No. L15, 18.1.84, p. 28.

(12)

OJ No. L83, 30.3.73, p. 21.