Testing Status of Agents at NTP

Executive Summary Bixin



Bixin (Annatto)
6983-79-5 (1393-63-1)

Prepared by Technical Resources International, Inc. under Contract No. NO2-CB-50511


Table of Contents


  1. Basis of Nomination to the CSWG


  2. Selection Status


  3. Input from Government Agencies/Industry
  4. Chemical Identification


  5. Exposure Information


    Table 1. Annatto Products and Their Uses


  6. Evidence for possible carcinogenic activity

    Table 2. In Vitro Genotoxicity of Annatto and Its Components

    Table 3. Summary of Information on Annatto and b-Carotene


  7. References







The nomination of bixin to the CSWG is basedon the high production volume of annatto, which is consumed byvirtually every person in the United States. According to dataprovided by the Food and Drug Administration (FDA), annatto isone of the most highly consumed colorants in the US food supply.Bixin is the ingredient that contributes this color.

Despite annatto's status as an unregulatedcolor additive, surprisingly little is known about the toxicityof bixin or norbixin, which are intentionally concentrated inannatto extracts and oils. Chronic toxicity studies have beenconducted on annatto; at first glance, they suggest that annattois nontoxic. However, these studies do not hold up well underscrutiny. In these studies, the exact concentrations of bixinor norbixin were not reported; these may vary anywhere from 2to 50%. Also, no attempt has been made to characterize other ingredientsin annatto that might have biological activity. Concern aboutthe potential toxicity of bixin is increased because a few short-termtests for genotoxicity of annatto have been described as positive.

Subchronic studies of purified bixin and short-termtests of bixin, norbixin, and technical grade annatto productswould alleviate concerns about the inferior data underpinningthe widespread use of annatto. Also, mechanistic and metabolismstudies would extend scientific knowledge about an important andubiquitous class of chemicals, the carotenoids. Testing by theNTP would be especially appropriate since the producers of annattoare farmers in third world countries; the revenues from annattoare insufficient for these producers to finance toxicity testingfor this product.



ACTION BY CSWG: 12/10/97
Studies requested: Subchronic study
In vitro cytogenetics
In vivomicronucleus
Lung adenomas in Strain-A mouse model
Priority: Moderately high
Rationale/Remarks: Widespread human exposure
Unknown potential for adverse health effects
Reconsider for carcinogenicity after subchronicstudy is completed



Dr. Dan Benz, Center for Food Safety and AppliedNutrition (CFSPAN), FDA, and Dr. Ed Matthews, formerly with CFSPAN,provided information on annatto from FDA's Priority-based Assessmentof Food Additives (PAFA) database.

Ms. Joellen Putnam, Scientific Project Manager,Flavor and Extract Manufacturers' Association (FEMA), provideda copy of the British Industry Biological Research Association(BIBRA) profile on annatto.


CAS Registry No.: Annatto 1393-63-1
Bixin (cis) 6983-79-5
Bixin (trans) 39937-23-0
Norbixin (cis) 626-76-6
Norbixin (trans) 542-40-5
Chemical Abstract Service Names: Annatto: None

Bixin: methyl (9-cis)-hydrogen.6,6'-diapo-.psi.,psi-carotenoidioate

Norbixin: 6,6'-Diapo-,psi., psi., carotenedioicacid

Synonyms and Trade Names: Achiote, Achiotl, Achote, Urucu, Beni-No-Ki, Bija, Onota, Orleanstrauch,Roucou, Roucouyer, Roucoyer, Uruku (The Raintree Group, 1997);Annotta, Arnatta, Arnatto, Arnotta, CI Natural Orange 4, CI 75120.
Structural Class: Unsaturated aliphatic hydrocarbon; carboxylic acid derivative;carotenoid
Structure, Molecular Formula, and MolecularWeight: chemical structure
Bixin: Norbixin:
Bixin: R=COOCH3; Norbixin: R=COOH
Molecular formula: (C25H30O4) Molecular formula: (C24H28O4)
Molecular weight: 394.5 Molecular weight: 380.5


Chemical and Physical Properties (bixin):

Description:Orange crystals (Lewis, 1993)

Melting Point:198°C (Lide, 1995); Decomposes at 217°C (Lewis, 1993)

Solubility:Insoluble in water; soluble in ethyl alcohol and acetone, slightlysoluble in ether (Lide, 1995)

Technical Products and Impurities:Annatto is an orange-red colorant obtained through extractionof the seeds of the fruit of the Bixa orellana tree. Bixinand norbixin are the principle coloring constituents of annatto(Hallagan et al., 1995)

Annatto seeds contain 40-45% cellulose, 3.5-5.5%sucrose, 0.3-0.9% essential oil, 3% fixed oil, 4.5-5.5% pigments(comprised of 70-80% bixin), 13-16% protein, and other constituents.Annatto seeds also contain tannins, ethereal oils, saponins, mustardoil-like substances and mono- and sesquiterpenes (The RaintreeGroup, 1997; BIBRA, 1986).

Annatto oil solutions contain bixin. Bixinis present mainly in the cis form, but trans bixin, a thermaldegradation product, is also present. Water-soluble annatto solutionscontain the alkali salts of norbixin, which results when bixinis saponified and the methylethyl group is removed (TJP MarketDevelopment, 1997; BIBRA, 1986; Noonan, 1975).

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