National Toxicology Program

National Toxicology Program

Abstract from Report IMM20505 on Norbixin

Report on the Assessment of Contact Hypersensitivity of Norbixin in Female BALB/c Mice (CAS No. 542-40-5)

Report Date: May 2009

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 The findings have not been peer reviewed and were not evaluated in accordance with the levels of evidence criteria established by NTP in March 2009.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.


Norbixin (C24H28O4) is a water-soluble carotenoid extracted from the outer coating of seeds of the annatto tree (Bixa orellana L.). Norbixin is prepared by hydrolytic removal of a methyl ester group of bixin. Annatto is one of the most highly consumed colorants in the U.S. food supply, and reported hypersensitivity reactions associated with annatto dye ingestion include urticaria and angioedema. While there were no reported studies of norbixin following dermal exposure, there was one case report in which annatto at a 1:1000 dilution tested positive in the skin prick test in a 62 year old patient who had an anaphylactic reaction following ingestion of annatto-containing food. However, dermal contact with annatto dye is one of the exposure routes which occurs both occupationally and non-occupationally.

The following studies have been undertaken to determine the potential irritant and contact sensitization effects of norbixin when applied dermally to female BALB/c mice. The local lymph node assay was initially performed to measure sensitization potential at norbixin concentrations of 1%, 2.5%, 5%, 10%, and 20% (w/v) in an acetone vehicle (norbixin was found to have greater solubility in acetone than in 4:1 acetone:olive oil). These concentrations were selected because the maximal norbixin solubility in acetone was found to be 20%. After taking into consideration factors such as the volume applied and animal body weight, norbixin at 20% approximately produced a dose level of 500 mg/kg/day. Currently, the allowed daily intake for cis-norbixin is less than 0.4 mg/kg body weight. At the concentrations utilized in these studies, norbixin did not induce an increase in the lymph node cell proliferation (DPM/mouse) in BALB/c mice as compared to the control animals in the LLNA, although a significant dose-related trend was observed. The Mouse Ear Swelling Test was subsequently performed to further evaluate the contact hypersensitivity potential. Two sensitization concentrations, 10% and 20% were used. In both sensitization conditions, a 20% challenge concentration was utilized. No significant change in percent ear swelling was observed in animals exposed to norbixin when compared to background control in the Mouse Ear Swelling Test. However, a significant dose-related trend in percent ear swelling at 48 hrs after challenge was observed. An additional study was undertaken by combining the LLNA and Irritancy Assay to confirm these findings. Consistent with the previous results, norbixin did not induce any changes in either the draining lymph node cell proliferation or the irritancy response. No statistically significant effects were observed in the assays performed, and the stimulation index in the LLNA was less than 3-fold at all concentrations tested.

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