E160b(i), Annatto, Bixin is a red-orange dye listed on the list of food additives and derived from the seeds of the achiote tree, native to tropical regions of the Americas, the plant Bixa orellana L. native to the Amazon. It is commonly used in cosmetics and food for its coloring properties. 

Chemical name

• 6,6'-Diapo-psi.,psi.-carotenedioic acid

Industrial Production Process

• Preparation of reagents. The main raw materials include Bixa orellana (annatto) seeds and solvents such as water or vegetable oil.
• Harvesting and cleaning. Bixa orellana seeds are harvested and cleaned to remove impurities and debris.
• Drying. The cleaned seeds are dried at controlled temperatures to reduce moisture content and facilitate color extraction.
• Extraction. The dried seeds are treated with a suitable solvent (water or vegetable oil) to extract the coloring compounds, mainly bixin and norbixin. Extraction can be done through maceration, infusion, or solvent extraction.
• Filtration. The extracted mixture is filtered to remove solid residues and obtain a clear coloring solution.
• Concentration. The coloring solution is concentrated through vacuum evaporation to reduce the volume and increase the concentration of the coloring compounds.
• Purification. The concentrated solution may be further purified to remove unwanted impurities and improve the quality of the color.
• Stabilization. The purified coloring is stabilized to ensure its stability during transportation and storage, preventing oxidation and degradation.
• Drying. The coloring can be dried at controlled temperatures to obtain a fine annatto powder.
• Quality control. The Annatto, Bixin undergoes rigorous quality testing to ensure it meets standards for purity, color intensity, and safety. These tests include chemical analysis, spectroscopy, and microbiological testing.

What it is used for and where

Cosmetics

Restricted cosmetic ingredient as IV/109  a Relevant Item in the Annexes of the European Cosmetics Regulation 1223/2009. Substance or ingredient reported:

• Annatto. Purity criteria as set out in Commission Directive 95/45/EC (E 160b)

Cosmetics - INCI Functions

Colorant. This ingredient has the function of colouring the solution in which it is inserted in a temporary, semi-permanent or permanent manner, either alone or in the presence of the complementary components added for colouring.

Cosmetic Applications

• Natural Colorant. Annatto is used to impart an orange-red hue to various cosmetic products, including lipsticks, tinted creams, and body products.
• Antioxidant Properties. It contains compounds such as carotenoids, which act as antioxidants to protect the skin from damage caused by free radicals.
• Protective Effect. Thanks to its antioxidants, it can help protect the skin from premature aging and harmful environmental exposure.
• Versatile Applications. It can be used in a variety of cosmetic formulations to add color and antioxidant benefits.

Other Applications

• Food Industry. Annatto is widely used as a food coloring in cheese, butter, cereals, sauces, and baked goods, where it provides a bright yellow-orange color.
• Pharmaceutical Products. Used to color capsules and oral liquids in pharmacology, enhancing the appearance of medications.
• Textile Industry. Traditionally used to dye fabrics with shades of yellow and orange, utilizing its natural coloring properties.

Studies

According to the Regulation (EU) No 2020/0771,  Annatto, Bixin, Norbixin is deleted from the Union List and is replaced by two separate Food Additives namely Annatto bixin (E 160b(i)) and Annato norbixin (E 160bii))

The seeds of annatto have been used as a traditional medicine to cure infection as they contain tocotrienol, very close to vitamin E in the δ form (δT3).

The aim of this study was to determine whether annatto T3 is effective in attenuating NLRP3 inflammasome activation in macrophages. Results showed that annatto δT3 significantly attenuated NLRP3 inflammasome by decreasing IL-1β reporter activity, IL-1β secretion, and caspase-1 cleavage against lipopolysaccharide (LPS) followed by nigericin stimulation. With regard to mechanism, annatto δT3 1) reduced LPS-mediated priming of the inflammasome and dampened reactive oxygen species production, the second signal required for assembly of the NLRP3 inflammasome in macrophages. This work suggests that annatto δT3 may hold therapeutic potential for delaying the onset of NLRP3 inflammasome–associated chronic metabolic diseases (1).

Annatto-derived tocotrienol up to 600 mg per day for 12 weeks appeared to be safe in postmenopausal osteopenic women, particularly in terms of liver and kidney functions. Tocotrienol supplementation for 12 weeks did not affect body composition, physical activity, quality of life, or intake of macro- and micro-nutrients in these subjects (2).

Metabolic syndrome (MetS) is associated with osteoporosis due to the underlying inflammatory and hormonal changes. Annatto tocotrienol has been shown to improve medical complications associated with MetS or bone loss in animal studies. This study aimed to investigate the effects of annatto tocotrienol as a single treatment for MetS and osteoporosis in high-carbohydrate high-fat (HCHF) diet-induced MetS animals. Three-month-old male Wistar rats were randomly divided into five groups. The baseline group was euthanized at the onset of the study. The normal group received standard rat chow and tap water. The remaining groups received HCHF diet and treated with three different regimens orally daily: (a) tocopherol-stripped corn oil (the vehicle of tocotrienol), (b) 60 mg/kg annatto tocotrienol, and (c) 100 mg/kg annatto tocotrienol. At the end of the study, measurements of MetS parameters, body compositions, and bone mineral density were performed in animals before sacrifice. Upon euthanasia, blood and femur of the rats were harvested for the evaluations of bone microstructure, biomechanical strength, remodelling activities, hormonal changes, and inflammatory response. Treatment with annatto tocotrienol improved all MetS parameters (except abdominal obesity), trabecular bone microstructure, bone strength, increased osteoclast number, normalized hormonal changes and inflammatory response in the HCHF animals. In conclusion, annatto tocotrienol is a potential agent for managing MetS and osteoporosis concurrently. The beneficial effects of annatto tocotrienol may be attributed to its ability to prevent the hormonal changes and pro-inflammatory state in animals with MetS (3).

Following a request from the European Commission to EFSA, the EFSA Panel on Food Additives and Nutrient Sources added to Food (ANS) was asked to re‐evaluate the safety of annatto extracts, bixin‐based and norbixin‐based, when used as a food additive and to evaluate the safety of aqueous‐processed bixin (Annatto E); solvent‐extracted bixin (Annatto B); alkali‐processed norbixin, acid‐precipitated (Annatto F); alkali‐processed norbixin, not acid‐precipitated (Annatto G) and solvent‐extracted norbixin (Annatto C) with the view to replace the currently authorised annatto extracts (E 160b). Given: (a) that read‐across among the five bixin‐ and norbixin‐based annatto extracts was feasible; (b) the availability of adequate 90‐day toxicity studies with Annatto B, C, E and F; (c) the absence of concern for mutagenicity, carcinogenicity, reproductive and developmental toxicity of Annatto B, C, F and G, whereas the mutagenicity of Annatto E is equivocal, the Panel concluded that the safety of the currently authorised solvent‐extracted bixin and norbixin (E 160b(i)), alkali‐extracted annatto (E 160b(ii)) and oil‐extracted annatto (E 160b(iii)), with the specifications defined in Commission Regulation (EU) No 231/2012, could not be assessed due to the lack of data, both in terms of identification and toxicological studies; solvent‐extracted bixin (Annatto B), solvent‐extracted norbixin (Annatto C), alkali‐processed, acid‐precipitated norbixin (Annatto F) and alkali‐processed, not acid‐precipitated norbixin (Annatto G and its norbixin salts) should comply with the specifications as recommended by the Panel. The toxicological database is sufficient to derive an acceptable daily intake (ADI) of 6 mg bixin/kg body weight (bw) per day and an ADI of 0.3 mg norbixin/kg bw per day. Exposure estimates for bixin were below the ADI for all population groups and for all refined exposure scenarios, including for the extension of use. For norbixin, exceedance was observed for the extension of use at the 95th percentile for some population groups (4).

Molecular Formula: C₂₄H₂₈O₄    C₂₅H₃₀O₄

Molecular Weight  380.484

CAS: 626-76-6  6983-79-5  

UNII    MH1WZE9GBD

EC Number 215-735-4  208-810-8

CHEMBL1420783

DTXSID60274023

Synonyms :

Annatto  -  alpha-Norbixin  -  AN-49143  -  Annatto coloring dye - Annatto extract  -  Annatto pigment  -  Annotta extract  -  Bixa orellana extract  -  CCRIS 3651  -  CHEMBL1420783  -  CI 75120  -  cis-Norbixin  -  DTXSID60274023  -  EINECS 215-735-4  -  FEMA No. 2103  -  FEMA No. 2104  -  HSDB 7976  -  LS-2208  -  MH1WZE9GBD  -  Natural orange 4  -  NCGC00091523-01  -  SC-23249  -  SCHEMBL340220  -  UNII-6PQP1V1B6O  -  UNII-MH1WZE9GBD  -  W-108218  -  ZINC8582047

References_____________________________________________________________________

(1) Buckner T, Fan R, Kim Y, Kim J, Chung S. Annatto Tocotrienol Attenuates NLRP3 Inflammasome Activation in Macrophages.  Curr Dev Nutr. 2017 May 31;1(6):e000760. doi: 10.3945/cdn.117.000760. eCollection 2017 Jun.

(2) Shen CL, Wang S, Yang S, Tomison MD, Abbasi M, Hao L, Scott S, Khan MS, Romero AW, Felton CK, Mo H. A 12-week evaluation of annatto tocotrienol supplementation for postmenopausal women: safety, quality of life, body composition, physical activity, and nutrient intake.  BMC Complement Altern Med. 2018 Jun 28;18(1):198. doi: 10.1186/s12906-018-2263-0.

(3) The safety of annatto extracts (E 160b) as a food additive - EFSA Journal 2016;14(8):4544 [87 pp.].