Canthaxanthin (2a161g)

Recommended Method

as of:

- Commission Implementing Regulation (EU) 2015/1486

For the quantification of canthaxanthin in the feed additive:

- spectrophotometry at 426 nm

For the quantification of canthaxanthin in the premixtures and feedingstuffs:

- Normal Phase High Performance Liquid Chromatography coupled to visible detection (NP-HPLC-VIS, 466 nm)

Executive Summary

In the current application, authorisation is sought under articles 4(1) and 10(2) for Canthaxanthin, under the category/functional group 2(a) "sensory additives"/"colourants" according to the classification system of Annex I of Regulation (EC) No 1831/2003. According to the Applicant, the active substance in the formulation is the 10 % of the complete preparation (with a level of at least 96 % of colouring matters expressed as sum of all trans/cis isomers of Canthaxanthin and a maximum of 5 % of carotenoids other than Canthaxanthin). Specifically, authorisation is sought for the use of the feed additive for chickens for fattening, chickens reared for laying, laying hens, other poultry, salmon, trout, other fish, pets and other non-food producing animals. The Applicant proposed the following maximum levels of the feed additive in the feedingstuffs: - 8 mg/kg for chickens reared for laying and laying hens; - 25 mg/kg for chickens for fattening, other poultry, salmon, trout and other fish.

Furthermore, the Applicant proposed the following Maximum Residue Limits (MRLs) for Canthaxanthin (sum all trans/cis isomers): 2.5 mg/kg for poultry fat and skin; 5 mg/kg for trout flesh; 10 mg/kg for salmon flesh; 15 mg/kg for poultry liver; and 30 mg/kg for egg yolk.

For the determination of the active substance, Canthaxanthin, in the feed additive, the Applicant submitted a ring trial validated method based on spectrophotometry. The following performance characteristics were reported:

- a standard deviation for repeatability (RSD_r) ranging from 0.2 to 0.8 %;

- a standard deviation for reproducibility (RSD_R) ranging from 1.3 to 4.0 %, and

- a recovery rate (R_Rec) ranging from 96.2 to 105 %.

Based on the performance characteristics presented the EURL recommends for official control the ring trial validated spectrophotometric method, submitted by the Applicant, to determine Canthaxanthin, in the feed additive.

For the determination of Canthaxanthin in premixtures and feedingstuffs the Applicant submitted a single laboratory validated and further verified method based on Normal Phase High-Performance Liquid Chromatography coupled to VIS detection (NP-HPLC-VIS). The following performance characteristics were reported:

- RSD_r ranging from 1.4 to 15 %;

- a standard deviation for intermediate precision (RSD_ip) ranging from 2.1 to 14.8 %;

- R_Rec ranging from 85.5 to 107 %, and

- a limit of quantification (LOQ) of 1 mg/kg in feedingstuff.

Based on the performance characteristics presented the EURL recommends for official control the single laboratory validated and further verified NP-HPLC-VIS method, submitted by the Applicant, to determine Canthaxanthin in premixtures and feedingstuffs.

For the determination of Canthaxanthin in water, the Applicant submitted the above mentioned NP-HPLC-VIS method. However no experimental data have been provided. Therefore the EURL could not evaluate nor recommend a method for official control to determine Canthaxanthin in water.

For the quantification of Canthaxanthin in poultry tissues (liver, skin, fat) and egg yolk the Applicant proposed a single laboratory validated and further verified method, based on NP-HPLC coupled to VIS detection measuring at 466 nm. For the quantification of Canthaxanthin in fish flesh (salmon and trout) the Applicant proposed the CEN/TS 16233-1:2011 analytical method (further ring trial validated by six laboratories in a collaborative study organised by the University of Trondheim - HIST on behalf of the Norwegian Seafood Federation - FHL), based on NP-HPLC coupled to VIS detection measuring at 470 nm. These methods were validated and verified at the content of Canthaxanthin ranging from 0.7 to 114 mg/kg in poultry tissues, 2.5 to 40 mg/kg for egg yolk and applicable for a rage above 0.02 mg/kg for fish flesh. The following performance characteristics were reported: for liver, skin, fat and egg yolk: RSD_r and RSD_ip ranging from 1 to 9.5 %; R_Rec ranging from 93 to 110 %; LOQ of 0.3 mg/kg and for egg yolk of 0.5 mg/kg; for fish flesh: RSD_r and RSD_R ranging from 4.1 to 9.2 %; R_Rec of 95.8% and LOQ of 0.01 mg/kg.

Based on the performance characteristics presented, the EURL recommends for official control, the single laboratory validated and further verified methods, based on NP-HPLC-VIS, submitted by the Applicant, to determine Canthaxanthin in poultry tissues, egg yolk and the CEN/TS 16233-1:2011 method, based on NP-HPLC-VIS to determine Canthaxanthin in fish flesh.

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.