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 The findings have not been peer reviewed and were not evaluated in accordance with the levels of evidence criteria established by NTP in March 2009.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 is a natural coloring material extracted from the outer coatings of seeds of the annatto tree (Bixa orellana L.). There are two main types of purified annatto carotenoid pigments from the annatto dye: bixin (C25H30O4; oil soluble) and norbixin (C24H28O4; alkaline water soluble). Bixin (mainly the diapoicaroteniod 9'cis-bixin), is a carotenoid with two carboxylic acid groups. Bixin is widely used in butter, margarine, cheese, smoked fish, snack foods, beverages, and cereals. Reported adverse reactions associated with annatto dye ingestion include urticaria and angioedema. However, dermal contact with annatto dye is likely to be one of the exposure routes both occupationally and non-occupationally. There were no reported studies on bixin when the exposure occurred by dermal route except for a case report in which annatto at 1:1000 dilution was tested positive by the skin prick test in a 62 year old patient who had anaphylactic reaction following ingestion of annatto-containing food.
Our previous studies have demonstrated that annatto dye can induce contact sensitization in BALB/c mice. The objective of this study was to determine what components of annatto dye may be sensitizers, therefore the potential irritant or contact sensitization effect of cis-bixin (BIX), a purified annatto carotenoid pigment, was evaluated when applied dermally to female BALB/c mice. Measurement of the contact hypersensitivity response was initially accomplished using the combined local lymph node assay and irritancy assay. Solubility tests indicated that the highest achievable concentration of cis-bixin in acetone: olive oil (4:1) was 12.5%. Accordingly, the concentrations of cis-bixin used for the combined LLNA and IRR assays were 0.05%, 0.1%, 0.25%, 0.5%, 1%, 2.5%, 5%, and 12.5%. In the LLNA, cis-bixin at concentrations of 0.05-1% produced significant increases in lymph node cell proliferation (DPM/mouse) when compared to the vehicle control (p < 0.01), with the treatment groups of 0.25%, 0.5%, and 1% exceeding the three-fold threshold of the vehicle DPM value. However, cis-bixin was not an irritant at concentrations up to 12.5%, as demonstrated by no changes in ear thickness at 24 hours following the last dermal exposure.
To further confirm the positive LLNA response, the Mouse Ear Swelling Test was performed following exposure to cis-bixin. In the sensitization phase, two concentrations of cis-bixin (0.05% and 0.5%) were used, and a concentration of 0.5% was applied during the challenge phase. cis-Bixin at the sensitization concentration of 0.5% significantly increased mouse ear thickness at 48 hours post challenge.
Overall, the results from these studies have demonstrated that cis-bixin at concentrations of 0.05%-1% produced significant increases in lymph node cell proliferation in the LLNA with the treatment groups of 0.25%, 0.5% and 1% exceeding the three-fold threshold of the vehicle control value in BALB/c mice. Furthermore, cis-bixin also induced a significant increase in the ear thickness as evaluated using MEST, which is consistent with the LLNA results. However, cis-bixin did not produce any significant changes in the irritancy responses.