Astaxanthin (2a161j)

Recommended Method

as of:

Commission Implementing Regulation (EU) 2015/1415

For the quantification of Astaxanthin in the feed additive preparation:

- spectrophotometry at 431 nm

For the quantification of Astaxanthin in the premixtures and feedingstuffs:

- Normal Phase High Performance Liquid Chromatography coupled to visible detection (NP-HPLC-VIS, 470 nm)

Executive Summary

Astaxanthin is a feed additive for which authorisation is sought under the category "sensory additives", functional group 2(a) "colourants", sub-classification (ii) "substances which, when fed to animals, add colours to food of animal origin", (iii) "substances which favourably affect the colour of ornamental fish or birds" according to Annex I of Regulation (EC) No 1831/2003. In the current application submitted according to Article 4(1) (new use in water) and Article 10(2) (re-evaluation of additives already authorised under Directive 70/524/EC) of Regulation (EC) No 1831/2003, authorisation is requested for salmon and trout, ornamental fish and birds, crustaceans and other fish. The active ingredient and the additive for registration is Astaxanthin, produced by a synthetic process and marketed in a stabilised form, e.g. with a spray-dried coating material (i.e. carbohydrates, protein). The applicant proposed a maximum Astaxanthin concentration of 100 mg/kg feedingstuffs for salmon and trout and for minor species (crustaceans and other fish). However, no maximum content was proposed for pets (ornamental fish and birds). No minimum contents were proposed by the applicant. The Astaxanthin concentration in premixtures and in feedingstuffs corresponds to the sum of geometrical Astaxanthin isomers detected, namely (1) all-E Astaxanthin, (2) 9Z Astaxanthin, (3) 13Z Astaxanthin and (4) other non-identified Z isomer(s). Here the E/Z-isomers notation is used instead of the terms trans/cis. Furthermore, the presence of Canthaxanthin with Astaxanthin in completed feedingstuffs is allowed for salmon and trout with a maximum concentration of the sum of both substances of 100 mg/kg.

For the determination of the purity of the crystalline Astaxanthin (feed additive), the applicant proposed a spectrophotometric method measuring at 486-487 nm, and a Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC) using a visible detector with a wavelength measuring at 474 nm. The CRL considers the two methods submitted by the applicant suitable for intended purposes.

For the determination of Astaxanthin in the premixtures and feedingstuffs, the applicant proposed a ring-trial validated chromatographic method, based on Normal-Phase High-Performance Liquid Chromatography (NP-HPLC) using a visible detector at 470 nm. The following performance characteristics were reported:

For premixtures containing Astaxanthin at 4500 mg/kg:

• a relative standard deviation for reproducibility (RSD_R) of 10.2 %.

For feedingstuffs, in the concentration ranging of Astaxanthin from 20 to 80 mg/kg:

• a relative standard deviation for repeatability (RSD_r) ranging from 1.5 to 3.2 %,
• a relative standard deviation for reproducibility (RSD_R) ranging from 3.5 to 12.6 %
• a recovery rate (R_Rec) ranging from 98 to 102 %
• a limit of detection (LOD) and quantification (LOQ) of 0.005 and 0.01 mg/kg feedingstuffs, respectively.

Based on the acceptable performance characteristics presented, the CRL recommends for official control - in the frame of this authorisation - the ring-trial validated methods submitted by the applicant for the determination of Astaxanthin in premixtures and feedingstuffs.

Upon request of CRL, the applicant submitted experimental data proving that the ring-trial validated spectrophotometry method for the determination of Astaxanthin in the powdery or water dispersible formulations is also applicable for the determination of Astaxanthin in water. The target values were ranging from 30 to 100 mg/kg of Astaxanthin in drinking water. The following performance characteristics were reported: - RSD_r ranging from 0.45 to 1.10 %; - RSD_R ranging from 1.0 to 3.3 %; - R_Rec = 99.9 %.

Based on the acceptable performance characteristics presented, the CRL recommends for official control - in the frame of this authorisation - the ring-trial validated methods submitted by the applicant for the determination of Astaxanthin in water.

For the determination of Canthaxanthin in feedingstuffs, the applicant submitted, upon request from the CRL, a single laboratory validated method, similar to the chromatographic method for Astaxanthin in premixtures and feedingstuffs, with slight modification of chromatographic conditions. The following performance characteristics for feedingstuffs, in the concentration ranging from 5 to 1000 mg/kg, were reported: - RSD_r ranging from 1 to 7 %; - R_Rec ranging from 101 to 103 %; and – LOD = 0.003 mg/kg.

Based on the acceptable performance characteristics presented, the CRL recommends for official control - in the frame of this authorisation - the ring-trial validated methods submitted by the applicant for the determination of Canthaxanthin in feedingstuffs.

Further testing or validation of the methods to be performed through the consortium of National Reference Laboratories as specified by article 10 (Commission Regulation (EC) No 378/2005) is not considered necessary.