Ethyl ester of β-apo-8’- carotenoic acid - 2a160f

In the current application authorisation is sought under articles 4(1) and 10(2) for ethyl ester of beta-apo-8'-carotenoic acid under the category/functional group 2(a) "sensory additives"/"colourants", subgroup (ii) "substances which, when fed to animals, add colours to food of animal origin", according to the classification system of Annex I of Regulation (EC) No 1831/2003. According to the Applicant, the active substance of the feed additive is ethyl ester of beta-apo-8'-carotenoic acid (apoester) with a minimum purity of 90 % (expressed as total carotenoids). Specifically, authorisation is sought for the use of the feed additive for poultry for fattening and laying (table eggs and liquid eggs for processed food). The Applicant proposed the following maximum levels of the feed additive in the feedingstuffs: 
- 8 mg/kg for poultry for laying for table eggs; - 15 mg/kg for poultry for fattening and ‑ 80 mg/kg for poultry for laying for liquid eggs (for processed food). Furthermore, the Applicant proposed the following Maximum Residue Limits (MRL): - 2.5 mg/kg for fat and skin; - 6 mg/kg for liver; - 25 mg/kg for egg yolk in table eggs and - 250 mg/kg for egg yolk in eggs for industrial processing. The limits proposed for feedingstuffs and target tissue and egg yolk refer to the sum of the all-trans and cis isomers of the active substance, expressed as total carotenoids.

For the identification and quantification of apoester in the feed additive, the Applicant proposed the internationally recognised FAO JECFA monograph for Beta-apo-8'-carotenoic acid ethyl ester in food additives, based on spectrophotometry at 449 nm. Even though no performance characteristics are provided, the EURL recommends for official control the JECFA monograph based on spectrophotometry for the quantification of the apoester in the feed additive.

For the quantification of apoester in premixtures, feedingstuffs and water, the Applicant submitted a single laboratory validated and further verified method, based on High Performance Liquid Chromatography (HPLC) with VIS detection at 446 nm. The following performance characteristics were reported:

for premixtures: - a precision (repeatability and intermediate precision) ranging from 0.9 to 3.4 %; and - a recovery rate (R_Rec) ranging from 100 to 106 %; 

for feedingstuffs: - a precision ranging from 2.7 to 10.2 %; - R_Rec ranging from 77 to 107 %; and a limit of quantification (LOQ) of 0.5 mg/kg feedingstuffs; and

for water: LOQ of 0.1 mg/L water.

Based on the performance characteristics presented, the EURL recommends for official control, the single laboratory validated and further verified method, based on High Performance Liquid Chromatography (HPLC) with VIS detection, submitted by the Applicant, to determine apoester in premixtures, feedingstuffs and water.

For the quantification of apoester in poultry tissues (liver, skin, fat) the Applicant proposed a single laboratory validated and further verified method, based on High Performance Liquid Chromatography (HPLC) coupled to Diode Array Detector (HPLC-DAD) measuring at 446 nm. For the quantification of apoester in egg yolk the Applicant proposed single laboratory validated and further verified method, based on HPLC with VIS detection at 446 nm. The methods were validated and verified at the content of apoester ranging from 0.5 to 250 mg/kg tissues and 0.3 to 250 mg/L egg yolk.

The following performance characteristics were reported:

for liver: - a precision ranging from 1.9 to 8.9 %; and - R_Rec ranging from 88 to 108 %;

for skin: - a precision ranging from 2.2 to 4.4 %; and - R_Rec ranging from 91 to 101 %; 

for fat: - a precision ranging from 1 to 5.1 %; and - R_Rec ranging from 95 to 104 %; and

for egg yolk: - a precision ranging from 1.8 to 7.8 %; and - R_Rec ranging from 95 to 109 %; and 

LOQ ranging from 0.1 to 0.3 mg/kg poultry tissues and egg yolk.

Based on the performance characteristics presented, the EURL recommends for official control, the single laboratory validated and further verified method, based on HPLC-DAD and HPLC-VIS, submitted by the Applicant, to determine apoester in poultry tissues and egg yolk.

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.

Recommended Method

as of:

- Commission Implementing Regulation (EU) 2020/1400

For the quantification of ethyl ester of β-apo-8’-carotenoic acid in the feed additive preparation: 

- Spectrophotometry at 446 nm

For the quantification of ethyl ester of β-apo-8’-carotenoic acid in the premixtures and feedingstuffs: 

- Normal Phase High Performance Liquid Chromatography coupled to visible detection (NP-HPLC-VIS, 446 nm)