National Toxicology Program

National Toxicology Program

Abstract from Report IMM20405 on Annatto

Report on the Assessment of Contact Hypersensitivity of Annatto in Female BALB/c Mice (Dermal Studies) (CAS No. 1393-63-1)

Report Date: June 2008

The following abstract presents results of a study conducted by a contract laboratory for the National Toxicology Program. The findings have not been peer reviewed and were not evaluated in accordance with the levels of evidence criteria established by NTP in March 2009. The findings and conclusions for this study should not be construed to represent the views of the NTP or the U.S. Government.


Annatto dye, a natural carotenoid, is an orange-yellow food coloring extracted from the outer coatings of seeds of the Annatto tree (Bixa orellana L.). It has been used to color butter, margarine, cheese, smoked fish, snack foods, beverages, and cereals. The seeds, leaves, and roots of annatto have also been used in Brazilian folk medicine to prepare aphrodisiac potions as well as remedies to treat fevers, inflammatory conditions, and parasitic diseases.4 The extractable forms of annatto include bixin (C25H30O4: oil soluble) and norbixin (C24H28O4: alkaline water soluble; hydrolyzed derivative). Reported adverse reactions associated with annatto dye ingestion include urticaria and angioedema.

The objective of this study was to determine the sensitizing potential for ANT when applied dermally to female BALB/c mice. Measurement of the contact hypersensitivity response was initially accomplished using the local lymph node assay. Solubility testing indicated that ANT had better solubility in acetone than in acetone:olive oil (4:1). The maximal solubility of ANT in acetone was approximately 25%. Accordingly, the highest concentration of ANT used for LLNA was 25% in acetone. For the LLNA, ANT at the concentrations of 5-25% produced a significant increase in lymph node cell proliferation (DPM/mouse) when compared to the vehicle control (p < 0.01).

The irritant potential of ANT was examined using the irritancy assay. ANT was not an irritant at concentrations up to 25%, as demonstrated by no changes in the ear thickness 24 hour following the last exposure.

To further confirm the positive LLNA response, the Mouse Ear Swelling Test was performed to investigate any potential contact hypersensitivity effects of ANT. In the sensitization phase, two concentrations of ANT (5% and 10%) were used, and the concentration of 10% was applied during the challenge phase. Consistent with the observation in the LLNA, ANT at both concentrations significantly increased the ear thickness at 24 hour post challenge; however, the increases of percent ear swelling were not statistically significant at 48 hour post challenge.

To the best of our knowledge, no significant deviations occurred that affected the quality of the data or the ability to interpret the data with respect to the allergic potential of ANT.

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