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Toxicity Effects

CAS Registry Number: 1393-63-1

Selected toxicity information from HSDB, one of the National Library of Medicine's databases. 2.

Names 1

  • Annatta Extract
  • Annatto
  • Annatto Extract Acid Proof
  • Annotta
  • Arnatta
  • Chebi:3136
  • Ci 75120
  • Ci Natural Orange 4
  • Methyl (9-Cis)-Hydrogen-6,6'-Diapo-Psi,Psi-Carotenedioate

Human Toxicity Excerpts

  • CASE REPORTS: An anaphylactic reaction attributed to annatto was reported in a 62-year-old man who had consumed /a breakfast cereal/ for the first time. This cereal product contains wheat bran, corn bran, aspartame, corn syrup, vitamins A, C, D, B6, B12, thiamine and annatto extract. Within minutes of eating this product, the patient developed symptoms characteristic of anaphylactic shock (generalized pruritis, generalized urticaria, angioedema of the eyes and lips, undetectable blood pressure and loss of consciousness). Skin prick tests (1:10 000) to milk, corn and wheat and annatto extract were all negative, but a positive reaction to 1:1000 annatto extract was obtained and the patient's serum was positive for the presence of annatto-specific IgE. The IgE was shown to recognize a protein (relative molecular mass, 50) in the annatto extract, thought by the authors to be a contaminant from the pericarp extraction process. Annatto extract is widely present in food, and although it has been implicated in this case, an anaphylactic reaction of this type must be regarded as a very rare event.[WHO/FAO; Joint Expert Committee on Food Additives (JECFA): Food Additive Series 52: Annatto Extracts. Available from, As of June 22, 2011: http://www.inchem.org/pages/jecfa.html] **PEER REVIEWED**
  • GENOTOXICITY: Annatto (AN), a natural food colorant rich in carotenoids, has been reported as being an effective antioxidant, but little is known about its potential chemopreventive properties. In this study, /investigators/ evaluated the ability of AN to protect human hepatoma cells (HepG2) from micronucleus (MN) induction against three different mutagens: benzo(a)pyrene (B(a)P), doxorubicin (DXR), and methyl methanesulfonate (MMS). In an attempt to clarify the possible mechanism of antimutagenicity of AN, three protocols of treatment were applied (pretreatment; simultaneous treatment, and post-treatment with AN following treatment with the mutagens). Also, cells exposed only to AN were assayed for cytotoxicity and mutagenicity. A dosage up to 10 ug/mL of AN was devoid of mutagenic activity. Protective effects were seen on micronuclei induced by B(a)P and DXR using /pre-treatment/ and simultaneous treatment, but AN had no significant effect on MN induction by MMS in any of the protocols. ... /The/ results also show that exposure of cells to concentrations of AN higher than 10 ug/mL decreased cell viability. Taken together /these/ findings indicate that AN presents antimutagenic activity in vitro, but its protective effect is dependent on the mutagen and on type of treatment suggesting its potential use as a chemopreventive agent.[Barcelos GR; Environ Mol Mutagen 50 (9): 808-814 (2009)] **PEER REVIEWED** PubMed Abstract
  • HUMAN EXPOSURE STUDIES: ... 56 patients who had previously suffered from chronic urticaria and angioedema were given a gelatin capsule containing 25 uL of an extract of annatto (the amount of annatto contained in 25 g of butter), 27% of the patients revealed symptoms of urticaria and angioedema. This trial however was not double-blind, did not include placebo controls, and some patients had symptoms on entering the study.[WHO/FAO; Joint Expert Committee on Food Additives (JECFA): Food Additive Series 52: Annatto Extracts. Available from, As of June 22, 2011: http://www.inchem.org/pages/jecfa.html] **PEER REVIEWED**
  • HUMAN EXPOSURE STUDIES: ...132 subjects underwent high or low dose challenges with a range of food additives, administered (either alone or in combination) in opaque gelatin capsules tinted with iron oxide and titanium dioxide. Placebo capsules contained lactose powder. Annatto was administered at doses of 1 mg or 10 mg. Eighty-one patients completed the study. It was concluded that the prevalence of reactions to annatto in the population was estimated to be between 0.01% (lower limit) and 0.07% (upper limit) with a confidence interval of 95%.[WHO/FAO; Joint Expert Committee on Food Additives (JECFA): Food Additive Series 52: Annatto Extracts. Available from, As of June 22, 2011: http://www.inchem.org/pages/jecfa.html] **PEER REVIEWED**
  • HUMAN EXPOSURE STUDIES: ...335 children underwent open oral challenges; 23 had positive reactions and of these 16 underwent double-blind placebo-controlled challenges in their own homes. Two patients experienced positive reactions to the natural food coloring (atopic dermatitis in one and urticarial symptoms in the other) ... which contained turmeric (2.5 mg/100 mL), annatto (1.6 mg/100 mL), beta-carotene (6.0 mg/100 mL), canthaxanthine (1.0 mg/100 mL) and beet coloring (5.5 mg/100 mL)...[WHO/FAO; Joint Expert Committee on Food Additives (JECFA): Food Additive Series 52: Annatto Extracts. Available from, As of June 22, 2011: http://www.inchem.org/pages/jecfa.html] **PEER REVIEWED**
  • HUMAN EXPOSURE STUDIES: A study in Denmark in which 271 children (98 controls and 173 with atopic symptoms) underwent open oral challenges to a variety of food additives prepared in a lemonade solution, 17 children had positive reactions. The additives included preservatives, natural colors, synthetic colors, flavorings and acids. Twelve of the 17 children then underwent double-blind placebo-controlled challenges to the additives prepared in gelatine capsules. Of the 12, five reacted to the synthetic food colorings and one reacted positively to citric acid, but none reacted positively to the natural food colorings capsule which contained turmeric (2.5 mg/100 mL), annatto (1.6 mg/100 mL), beta-carotene (6.0 mg/100 mL), canthaxanthine (1.0 mg/100 mL) and beet coloring (5.5 mg/100 mL).[WHO/FAO; Joint Expert Committee on Food Additives (JECFA): Food Additive Series 52: Annatto Extracts. Available from, As of June 22, 2011: http://www.inchem.org/pages/jecfa.html] **PEER REVIEWED**
  • HUMAN EXPOSURE STUDIES: In /a/ double-blind placebo-controlled study of the effects of oral challenges in adults, 101 patients (25 men and 76 women) suffering from eczema were studied. After a standard elimination diet, the subjects received capsules containing a placebo control or one of four different mixtures of five food additives: Sodium benzoate; Sodium propionate; Sorbic acid; A mixture of food colorings (10 mg annatto, 10 mg erythrosine, 10 mg Ponceau 4R, 10 mg tartrazine, 10 mg patent blue V, 10 mg sunset yellow, 10 mg betanine, 10 mg curcumin, 10 mg quinoline; Placebo. Twenty-five patients reacted to the food colorings while 76 did not. However, since 16 patients reacted to the placebo, reaction to the food colorings was not a statistically significant effect. Of the patients who reacted to the first oral challenge, only one-third reacted when challenged again.[WHO/FAO; Joint Expert Committee on Food Additives (JECFA): Food Additive Series 52: Annatto Extracts. Available from, As of June 22, 2011: http://www.inchem.org/pages/jecfa.html] **PEER REVIEWED**
  • HUMAN EXPOSURE STUDIES: In a study of 330 patients (121 men, 209 women) suffering from urticaria and angioedema, various potentially allergenic substances were administered in an attempt to diagnose the cause of these conditions. There were no placebo controls and the study was not double-blind. One hundred and twelve patients received annatto extract (5 mg or 10 mg); of these, 10% showed a positive reaction, 14% showed an uncertain reaction, while the remainder showed negative reactions.[WHO/FAO; Joint Expert Committee on Food Additives (JECFA): Food Additive Series 52: Annatto Extracts. Available from, As of June 22, 2011: http://www.inchem.org/pages/jecfa.html] **PEER REVIEWED**
  • OTHER TOXICITY INFORMATION: A critical evaluation of the available information demonstrates that reactions to natural color additives are rare... In rare cases, annatto dye may provoke a severe, adverse reaction in individuals with an uncommon hypersensitivity, and may aggravate the symptoms of patients suffering from recurrent urticaria. In its long history of use, there has been only one reported case of anaphylaxis resulting from the ingestion of annatto. Studies designed to investigate the role of annatto in recurrent urticaria sufferers were limited due to the absence of double-blind challenge and placebo controls... Despite their widespread use in food products, few reports of allergic reactions following ingestion have been reported for the majority of natural color additives. It is concluded that the ingestion of natural color additives presents a very low risk of provoking adverse reactions.[Lucas CD et al; Adv Food Nutr Res 43: 195-216 (2001)] **PEER REVIEWED** PubMed Abstract
  • SIGNS AND SYMPTOMS: Annatto has been implicated as a cause of allergic reactions, these reactions generally taking the form of angioedema, urticaria or eczema, although one case of anaphylaxis has been documented.[WHO/FAO; Joint Expert Committee on Food Additives (JECFA): Food Additive Series 52: Annatto Extracts. Available from, As of June 22, 2011: http://www.inchem.org/pages/jecfa.html] **PEER REVIEWED**

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Non-Human Toxicity Excerpts

  • ALTERNATIVE and IN VITRO TESTS: ... Little is known about the CYP1A1-inducing properties of bixin and annatto. The present study was performed to determine the effects of an annatto extract (28% bixin) and bixin (95% pure) on rat liver monooxygenases. Adult female Wistar rats were treated by gavage with daily doses of annatto (250 mg/kg body weight, which contains approximately 70 mg bixin/kg body weight), bixin (250 mg/kg body weight) or the vehicle only (corn oil, 3.75 g/kg body weight) for 5 consecutive days, or were not treated (untreated control). The activities of aniline-4-hydroxylase (A4H), ethoxycoumarin-O-deethylase (ECOD), ethoxy- (EROD), methoxy- (MROD), pentoxy- (PROD) and benzyloxy- (BROD) resorufin-O-dealkylases were measured in liver microsomes. Annatto (250 mg/kg containing 70 mg bixin/kg) induced EROD (3.8x), MROD (4.2x), BROD (3.3x) and PROD (2.4x). Bixin (250 mg/kg) was a weaker inducer of EROD (2.7x), MROD (2.3x) and BROD (1.9x) and did not alter PROD, A4H or ECOD activities. These results suggest that constituents of the extract other than bixin play an important role in the induction of CYP1A and CYP2B observed with annatto food colorings.[de-Oliveira AC et al; Braz J Med Biol Res 36 (1): 113-8 (2003)] **PEER REVIEWED** PubMed Abstract
  • ALTERNATIVE and IN VITRO TESTS: All three annatto extracts induced the expression of CYP4A (primarily CYP4A2 and CYP4A3), annatto F having the most potent effect. There was an appreciable difference between the sexes, females exhibiting a much weaker response, consistent with published data. Hepatic microsomal CYP4A was constitutively expressed in both male and female rats, levels being four to five-fold higher in males. Annatto F caused a dose-dependent increase in the expression of CYP4A in males. There was no increase in the expression of CYP4A in the females receiving low doses of annatto F, but in the females receiving high doses, there was a five to six-fold increase in expression. Similarly, annatto B caused a dose-dependent increase in the expression of CYP4A in male rats, while any increase in females was very modest. The effects of annatto E were less consistent, with the expression of CYP4A in males increasing by approximately three-fold, apparent only in the group receiving the higher dose. In females, the response was also not as consistent, but increased expression of CYP4A was observed in groups receiving both the low dose (approximately three-fold) and the high dose (approximately two-fold). The positive control, clofibric acid, produced the anticipated increase in the expression of CYP4A. The increases in expression of CYP4A in male rats after treatment with high doses of the annatto extracts, particularly annatto F, were similar to those observed after treatment with clofibric acid.[WHO/FAO; Joint Expert Committee on Food Additives (JECFA): Food Additive Series 52: Annatto Extracts. Available from, As of June 22, 2011: http://www.inchem.org/pages/jecfa.html] **PEER REVIEWED**
  • ALTERNATIVE and IN VITRO TESTS: Bixin has also been investigated for its effect on drug-metabolizing enzymes. A variety of carotenoids were administered to male Wistar rats at a concentration of 300 mg/kg of diet over a period of 16 days and the activities of a range of enzymes in the liver, lung, kidney and small intestine were assessed. Control animals received diet only or the enzyme-inducing agent, 3-methylcholanthrene. CYP enzyme activity, glutathione-S-transferase and reduced glutathione status, and carotenoid uptake into the tissues were all assessed. Bixin was shown to induce expression of the CYP1A1 iso-enzyme in the liver, lung and kidney and, to a lesser extent, the CYP2B1/2 enzyme in the liver. None of the carotenoids had detectable effects on intestinal enzymes or on glutathione status. /Bixin/[WHO/FAO; Joint Expert Committee on Food Additives (JECFA): Food Additive Series 52: Annatto Extracts. Available from, As of June 22, 2011: http://www.inchem.org/pages/jecfa.html] **PEER REVIEWED**
  • ALTERNATIVE and IN VITRO TESTS: In view of the increase in absolute and/or relative liver weight observed in the 90-day studies of toxicity with annatto extract, it was decided to assay samples of the liver for CYP (cytochrome P450) enzymes, to determine whether induction of CYP enymes might account for the increased liver weight. At the termination of the 90-day studies of toxicity with annatto extracts B, E, and F conducted and reported later, liver samples were taken from 10 males and 10 females in the groups with the highest numbers of animals. Microsomes prepared from these samples were frozen, transported, and analyzed for the expression of CYP apoproteins using highly specialized Western blotting techniques by which levels of specific apoproteins can be assessed. Total CYP content and the levels of several of the CYP apoproteins were sexually dimorphic. In all cases, the data were consistent with those reported in previous publications. The annatto extracts did not cause an increase in total CYP content, with the possible exception of annatto E when administered at a high dose to females; total CYP content in females receiving annatto E at a dose of 1000 mg/kg bw was 1.42 nmol/mg microsomal protein compared with the control value of 0.96 nmol/mg microsomal protein. Annatto B and E induced expression of CYP1A2, but not of CYP1A1, suggesting that induction was via an aryl hydrocarbon-independent mechanism. None of the extracts induced CYP2B1 or CYP2B2, and hence none acted via a phenobarbital-like mechanism. None of the extracts induced CYP2E1 expression. CYP3A1 was induced by two to threefold in some groups, whilst CYP3A2 expression was not affected consistently. In comparison with the positive control (pregnanolone 16alpha-acarbonitrile, PCN), any PCN-type induction by the annatto extracts was extremely modest.[WHO/FAO; Joint Expert Committee on Food Additives (JECFA): Food Additive Series 52: Annatto Extracts. Available from, As of June 22, 2011: http://www.inchem.org/pages/jecfa.html] **PEER REVIEWED**
  • GENOTOXICITY: ... /The/ present study... /evaluated/ the activity of annatto (bixa orellana l.), a natural food colorant rich in carotenoid, on the formation of aberrant crypt foci (ACF) induced by dimethylhydrazine (DMH) in rat colon. Further, /the authors/ investigate, the effect of annatto on DMH-induced DNA damage, by the comet assay. Male Wistar rats were given sc. injections of DMH (40 mg/kg body wt) twice a week for 2 weeks to induce ACF. They also received experimental diets containing annatto at 20, 200 or 1000 ppm for five 5 weeks before (pre-treatment), or 10 weeks after (post-treatment) DMH treatment. In both protocols the rats were sacrificed on week 15th. For the comet assay, the animals were fed with the same experimental diets for 2 weeks. Four hours before the sacrifice, the animals received an sc injection of DMH (40 mg/kg body wt.). Under such conditions, dietary administration of 1000 ppm annatto neither induce DNA damage in blood and colon cells nor aberrant crypt foci in rat distal colon. Conversely, annatto was successful in inhibiting the number of crypts/colon (animal), but not in the incidence of DMH-induced ACF, mainly when administered after DMH. However, no antigenotoxic effect was observed in colon cells. These findings suggest possible chemopreventive effects of annatto through their modulation of the cryptal cell proliferation but not at the initiation stage of colon carcinogenesis.[Agner AR et al; Mutat Res 582 (1-2): 146-54 (2005)] **PEER REVIEWED** PubMed Abstract
  • GENOTOXICITY: ...In the present study, the radical-scavenger activity of the food additive norbixin, a water-soluble carotenoid extracted from Bixa orellana seeds and commercialized as annatto, was evaluated under conditions of DNA damage induced by reactive oxygen species, particularly by hydroxyl radicals. The cell-free scavenger activity of norbixin was evaluated using plasmid DNA as target molecule and Sn(II) or Fe(II) as oxidant. The addition of H2O2 enhanced DNA breakage induced by metal ions, particularly Fe(II). Under these conditions, norbixin started to protect plasmid DNA against single- and double-strand breakage at a metal:norbixin ratio of 1:1 (Sn(II)) and 1:10 (Fe(II)). However, at lower ratios to Sn(II), norbixin enhanced Sn(II)-induced DNA breakage (P < 0.05). The ability of norbixin to protect genomic DNA against oxidative damage was assessed in murine fibroblasts submitted to H2O2-induced oxidative stress and the results were evaluated by the comet assay. Under low serum conditions (2 % fetal bovine serum (FBS)), a protective effect of norbixin against H2O2-induced DNA breakage was inversely related to its concentration, a protection ranging from 41 % (10 um) to 21 % (50 um). At higher concentrations of norbixin, however, oxidative DNA breakage was still enhanced, even in the presence of a high serum concentration (10 % FBS). Under normal conditions, norbixin per se has no detectable genotoxic or cytotoxic effects on murine fibroblasts. The antimutagenic potential of norbixin against oxidative mutagens was also evaluated by the Salmonella typhimurium assay, with a maximum inhibition of 87 % against the mutagenicity induced by H2O2. ...[Kovary K et al; Br J Nutr 85 (4): 431-40 (2001)] **PEER REVIEWED**
  • GENOTOXICITY: ...The aims of the present study were to evaluate the mutagenicity and antimutagenicity of annatto, a natural pigment ... The micronucleus test was performed in bone marrow cells from Swiss male mice treated with one of the three concentrations of annatto (1330, 5330 and 10,670 ppm), incorporated into the diet. The animals were fed with the diets for 7 days and sacrificed 24 hr after the last treatment. For the evaluation of the antimutagenic potential of annatto, at day 7, the animals received an intraperitoneal injection of cyclophosphamide (50 mg/kg body weight). Under the concentrations tested annatto did not present mutagenic or antimutagenic activities on the mice bone marrow cells. However, an increased frequency of micronucleated cells was observed when the highest concentration (10,670 ppm) was administered simultaneously with cyclophosphamide. In conclusion, the data indicate that annatto color, for the conditions used, is neither mutagenic nor an inhibitor of induced mutations, although it should be used carefully since high doses may increase the effect of a mutagen.[Alves de Lima RO et al; Food Chem Toxicol 41 (2): 189-92 (2003)] **PEER REVIEWED** PubMed Abstract
  • GENOTOXICITY: ...The present study... /investigated/ the carcinogenic and anticarcinogenic effects of dietary annatto in Wistar rat liver using the preneoplastic glutathione s-transferase (gst-p) foci and DNA damage biomarkers. Annatto, containing 5% bixin, was administered in the diet at concentrations of 20, 200, and 1000 ppm (0.07; 0.80 and 4.23 bixin/kg body wt/day, respectively), continuously during 2 weeks before, or 8 weeks after diethylnitrosamine (DEN) treatment (200 mg/kg body wt, ip), to evaluate its effect on the liver-carcinogenesis medium-term bioassay. The comet assay was used to investigate the modifying potential of annatto on DEN (20 mg/kg body wt)-induced DNA damage. The results showed that annatto was neither genotoxic nor carcinogenic at the highest concentration tested (1000 ppm). No protective effects were also observed in both gst-p foci development and comet assays. In conclusion, in such experimental conditions, annatto shows no hepatocarcinogenic effect or modifying potential against DEN-induced DNA damage and preneoplastic foci in the rat liver. Diethylnitrosamine[Agner AR et al; Food Chem Toxicol 42 (10): 1687-1693 (2004)] **PEER REVIEWED** PubMed Abstract
  • GENOTOXICITY: Annatto extract (30% bixin) was studied for its possible mutagenicity and antimutagenicity in a test for formation of micronuclei in mice. No increase in frequencies of micronucleated polychromatic erythrocytes was seen when mice were given diets containing annatto extract at a concentration of 1330, 5330 or 10 670 ppm (90, 360 and 470 mg/kg bw respectively) for 7 days. When annatto extract was given for 7 days at these concentrations, together with cyclophosphamide on day 7, no interference with the mutagenic action of cyclophosphamide was seen at the two lower concentrations. At the highest dose, however, the frequency of micronucleated polychromatic erythrocytes was increased. The authors suggested that at the highest dose, annatto extract might enhance the formation of the active metabolite of cyclophosphamide.[WHO/FAO; Joint Expert Committee on Food Additives (JECFA): Food Additive Series 52: Annatto Extracts. Available from, As of June 22, 2011: http://www.inchem.org/pages/jecfa.html] **PEER REVIEWED**
  • GENOTOXICITY: Annatto extract (bixin content not specified) was investigated for ability to induce DNA damage in an E. coli rec-assay. Induction of reverse mutations in E. coli trp uvr A and Salmonella typhimurium TA 1538 his rfa uvrB was investigated in fluctuation assays. Both types of assays were conducted with and without metabolic activation using cecal extracts and liver microsomes from rats. Annatto extract did not induce detectable genotoxicity in these test systems.[WHO/FAO; Joint Expert Committee on Food Additives (JECFA): Food Additive Series 17: Annatto Extracts. Available from, As of June 22, 2011: http://www.inchem.org/pages/jecfa.html] **PEER REVIEWED**
  • GENOTOXICITY: In a model designed to investigate the antigenotoxic potential of norbixin, /investigators/ exposed Balb/c fibroblasts to H2O2 and evaluated DNA strand breakage. Prior incubation with norbixin showed firstly that norbixin alone was not genotoxic, secondly, that in concentrations of up to 50 mmol/L, norbixin reduced oxidative DNA damage in a manner that was inversely related to concentration and, finally, at higher concentrations it enhanced the damage induced by H2O2. /Norbixin/[WHO/FAO; Joint Expert Committee on Food Additives (JECFA): Food Additive Series 52: Annatto Extracts. Available from, As of June 22, 2011: http://www.inchem.org/pages/jecfa.html] **PEER REVIEWED**
  • LABORATORY ANIMALS: Acute Exposure: ... The present studies were carried out to examine a possible contact allergenic effect of two purified carotenoid pigments from annatto extract: bixin (BIX) and norbixin (NOR). The experiments were conducted in female Balb/c mice by applying BIX (1-25% w/v) and NOR (1-20% w/v) in acetone topically, and assays performed including the local lymph node assay (LLNA), the irritation assay, the mouse ear swelling test (MEST) and flow cytometric analysis. There was no treatment-related adverse effect on the body weights. However, a three-fold increase in the proliferation of auricular lymph node cells was observed at bix concentrations of 5%-25% (p < or = 0.01). A significant increase in percent ear swelling was also produced in mice treated with bix (5-10%) at 24 hr after challenge as measured in MEST assay. Further study indicated that BIX was not an irritant at the concentrations tested. Additional study demonstrated that exposure to BIX induced a significant increase in the percentage of /subset of B cells having immunoglobulins on their surface/ in the draining lymph node. In contrast, the results from studies on NOR showed that exposure to NOR at concentration levels of 1-20% did not alter the lymph node cell proliferation in the LLNA assay. Taken together, these results suggested that in annatto extract bixin but not norbixin is a contact sensitizer in female Balb/c mice. /Bixin and Norbixin/[Auttachoat W et al; Toxicol Sci 84 (1-s): 248 (2005)] **PEER REVIEWED**
  • LABORATORY ANIMALS: Acute Exposure: A number of food colors, including bixin, have been shown to inhibit the production in vitro of immunoglobulin E (IgE) by rat spleen lymphocytes, at doses of 10 and 1 mmol/L. Variable effects were seen on the production of immunoglobulin G (IgG) and immunoglobulin M (IgM), depending on the dose, but water-soluble colorings enhanced the production of IgM at concentrations as low as 1 mmol/L. The authors concluded that these food colors may regulate the production of immunoglobulins. /Bixin/[WHO/FAO; Joint Expert Committee on Food Additives (JECFA): Food Additive Series 52: Annatto Extracts. Available from, As of June 22, 2011: http://www.inchem.org/pages/jecfa.html] **PEER REVIEWED**
  • LABORATORY ANIMALS: Acute Exposure: The effects of annatto on cellular enzymes and metabolites in the rat have also been investigated as part of a wider investigation of synthetic and natural food colors, in an attempt to provide a simple diagnostic test for toxicological damage. Four groups of eight male albino rats were used for the part of the study concerning annatto. Group 1 served as a control, group 2 was given annatto in a single dose of 800 mg/kg of diet, equivalent to approximately 80 mg/kg bw, and groups 3 and 4 were given 400 mg/kg of diet, equivalent to approximately 40 mg/kg bw, for 3 weeks. Animals in groups 2 and 3 were killed by decapitation 24 hr after the last dose, while the animals in group 4 were killed 1 week after the cessation of treatment. Brain, liver and kidneys were removed from all animals, and cytoplasm and mitochondria were prepared for measurement of the activities of glucose-6-phosphate dehydrogenase (G-6-PD) and 6-phosphogluconate dehydrogenase (6-PGD). These enzymes were selected because of their key role in DNA and RNA synthesis. Annatto produced a minimal rise in the activity of these enzymes, mainly in the liver, and consistently less than that produced by synthetic food colors such as tartrazine, carmoisine and sunset yellow.[WHO/FAO; Joint Expert Committee on Food Additives (JECFA): Food Additive Series 52: Annatto Extracts. Available from, As of June 22, 2011: http://www.inchem.org/pages/jecfa.html] **PEER REVIEWED**
  • LABORATORY ANIMALS: Acute Exposure: The effects of synthetic and natural food colorants on subcellular fraction enzymes, involving the pentose phosphate pathway were studied in rats that received carmoisine, FD&C Yellow No. 6 (sunset yellow) and tartrazine as the synthetic source, and curcumin, annatto and chlorophyll-a as the natural source. Glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities in cytoplasm and mitochondria of the brain, liver and kidney were increased. The increase was greater for synthetic food colorants than for natural sources. This effect continued for one week after termination of the synthetic colorant, while enzyme activities returned to the normal control values when the natural colorant was stopped.[Hamama AA; J Drug Res 17 (1-2): 231-238 (1987)] **PEER REVIEWED**
  • LABORATORY ANIMALS: Chronic Exposure or Carcinogenicity: Four groups of 3 male and 3 female beagles were fed in their diet 0%, 5% and 10% aqueous extract of annatto seed for 1 year. The fourth group received 20% aqueous extract for 16 weeks in their diet and then half of the extract in the diet and half in gelatine capsules for 36 weeks. Controls received 0.48% potassium chloride. Growth inhibition and reduced food intake occurred at the 20% dietary level. Mortality rate, liver and kidney function tests, hematology and histopathology of all major tissues showed no abnormalities attributable to the test substance.[WHO/FAO; Joint Expert Committee on Food Additives (JECFA): Food Additive Series 17: Annatto Extracts. Available from, As of June 22, 2011: http://www.inchem.org/pages/jecfa.html] **PEER REVIEWED**
  • LABORATORY ANIMALS: Chronic Exposure or Carcinogenicity: In an unpublished bioassay for medium term liver carcinogenesis in rats, diets containing annatto C (87.3% norbixin) at a concentration of 0, 300, 1000 or 3000 ppm (0, 20, 66 or 200 mg/kg bw, respectively) to male F344/DuCrj rats that had been treated previously with a single dose of N-nitrosodiethylamine (DEN) of 200 mg/kg bw, given intraperitoneally. As a positive control, phenobarbital sodium was given at a concentration of 500 ppm in the diet. Three groups of animals given diets containing annatto C at a concentration of 0, or 3000 ppm, or phenobarbital sodium at 500 ppm were not treated with DEN and served as controls. All groups were given the treated diet for 6 weeks. Partial hepatectomy was performed during week 3. The experiment was ended after 8 weeks. Body weights and food and water consumption in animals treated with annatto C did not differ from those of controls. Absolute and relative liver weights were significantly increased in the animals receiving annatto C at 1000 and 3000 ppm. Treatment with annatto C did not increase the quantitative values measured for liver cell foci observed to be positive for glutathione S-transferase placental form (GSTP) after DEN initiation, in contrast to treatment with phenobarbital sodium in the group of positive controls. These results indicate that annatto C lacks modifying potential for liver carcinogenesis in this test system.[WHO/FAO; Joint Expert Committee on Food Additives (JECFA): Food Additive Series 52: Annatto Extracts. Available from, As of June 22, 2011: http://www.inchem.org/pages/jecfa.html] **PEER REVIEWED**
  • LABORATORY ANIMALS: Chronic Exposure or Carcinogenicity: Modifying potential of annatto extract (norbixin) on liver carcinogenesis was investigated in male F344/ducrj rats initially treated with n-nitrosodiethylamine (DEN). Two weeks after a single dose of DEN (200 mg/kg, intraperitoneally), rats were given annatto extract at dietary levels of 0, 0.03, 0.1 and 0.3%, or phenobarbital sodium at 0.05% as a positive control for 6 weeks. All animals were subjected to partial hepatectomy at week 3, and were killed at week 8. There were no deaths related to annatto extract ingestion, and the treatment had no effects on body weights, or food and water consumption. Statistically significant increases of absolute and relative liver weights were apparent in the 0.1 and 0.3% groups. However, annatto extract did not significantly increase the quantitative values for glutathione S-transferase placental form positive liver cell foci observed after DEN initiation, in clear contrast to the positive control case. The results thus demonstrate that annatto extract at a dietary level of 0.3% (200 mg/kg/day) lacks modifying potential for liver carcinogenesis in our medium-term bioassay system.[Hagiwara A et al; Cancer Lett 199 (1): 9-17 (2003)] **PEER REVIEWED** PubMed Abstract
  • LABORATORY ANIMALS: Chronic Exposure or Carcinogenicity: Seventy male and 30 female mice were injected s.c. with 0.1 mL annatto. Occasionally a sarcoma was produced at the site of injection. No definite effect was seen on distant tumor development either as regards time of appearance or number.[WHO/FAO; Joint Expert Committee on Food Additives (JECFA): Food Additive Series 17: Annatto Extracts. Available from, As of June 22, 2011: http://www.inchem.org/pages/jecfa.html] **PEER REVIEWED**
  • LABORATORY ANIMALS: Chronic Exposure or Carcinogenicity: Three groups of 10 male and 10 female rats received corn oil with 0, 0.05% fat-soluble annatto and 0.5% water-soluble annatto for their life span. Those extracts varied in total bixin content from 0.2 to 2.6%. Two daughter generations were bred, each being fed similar diets for 7 and 8-1/2 months. No deleterious effect was observed on growth and reproduction. No teratogenic effects were seen. No consistent effect on mortality was noted in the 3 generations. Organ weights and tumor incidence were comparable in all groups.[WHO/FAO; Joint Expert Committee on Food Additives (JECFA): Food Additive Series 17: Annatto Extracts. Available from, As of June 22, 2011: http://www.inchem.org/pages/jecfa.html] **PEER REVIEWED**
  • LABORATORY ANIMALS: Chronic Exposure or Carcinogenicity: Two groups of 3 male and 3 female beagles were fed 0 or 2.7% in the diet of fat-soluble extract of annatto seed for 9 weeks, then fed normal diet for 5 weeks and then fed only 1.35% in the diet of fat- soluble extract in capsules for 38 weeks. No abnormalities were found as regards growth, food intake, mortality, liver and kidney function, hematology or histopathology. One female dog died in the test group. The liver of this animal showed hepatocellular degeneration.[WHO/FAO; Joint Expert Committee on Food Additives (JECFA): Food Additive Series 17: Annatto Extracts. Available from, As of June 22, 2011: http://www.inchem.org/pages/jecfa.html] **PEER REVIEWED**
  • LABORATORY ANIMALS: Developmental or Reproductive Toxicity: In a study of developmental toxicity in rats, an annatto extract with a bixin content of 28% was administered by gavage to groups of 16-27 female Wistar rats at a dose of 0, 31.2, 62.5, 125, 250 or 500 mg/kg bw per day on days 6-15 of gestation. The annatto extract was neither maternally toxic or embryotoxic. Cesarean sections were performed on day 21; implantations, living and dead fetuses and resorptions were recorded. Fetuses were weighed and examined for externally visible abnormalities. One-third of the fetuses in each litter were examined for visceral abnormalities using a microsectioning technique. The remaining fetuses were cleared and stained with alizarin red S for skeletal evaluation. No adverse effects of the annatto extract on the dams were noted; there was no increase in embryolethality and no reduction in fetal body weight. Furthermore, the annatto extract did not induce any increase in the incidence of externally visible visceral, or skeletal abnormalities in the exposed offspring. The NOAEL for annatto extract in this study was 500 mg/kg bw per day, the highest dose tested, equivalent to an intake of bixin of 140 mg/kg bw per day.[WHO/FAO; Joint Expert Committee on Food Additives (JECFA): Food Additive Series 52: Annatto Extracts. Available from, As of June 22, 2011: http://www.inchem.org/pages/jecfa.html] **PEER REVIEWED**
  • LABORATORY ANIMALS: Subchronic or Prechronic Exposure: Fifty male and 50 female mice were painted twice a week for 3 months at the interscapular region with 0.05 mL 50% annatto in benzene. No skin papillomas or other tumors were encountered.[WHO/FAO; Joint Expert Committee on Food Additives (JECFA): Food Additive Series 17: Annatto Extracts. Available from, As of June 22, 2011: http://www.inchem.org/pages/jecfa.html] **PEER REVIEWED**
  • LABORATORY ANIMALS: Subchronic or Prechronic Exposure: Five groups of six male and six female Wistar rats received diets containing three annatto preparations, either singly or in combination, as follows: Control- Purified diet (0% bixin); OSB- 0.1% solution of annatto in vegetable oil (0.22% bixin); R10- 0.02% suspension of annatto in vegetable oil (1.84% bixin); WSA- 0.1% water soluble preparation of annatto (0.27% norbixin); Combined- 0.1% OSB, 0.02% R10 and 0.1% WSA (2.33% norbixin/bixin). The rats were killed during weeks 48-52 of treatment and were fed with control diet during the 24 hr before death. Liver, kidneys and abdominal adipose tissue were removed at postmortem, weighed, examined (gross appearance only) and subjected to an extraction procedure to identify the presence of annatto pigments. Body weights and food intakes were similar in all treatment groups and not significantly different from those of the control group. The appearances and weights of organs and tissues showed that the animals were in good health, with no differences between the groups. Analysis of the extracts of liver, kidney, adipose tissue and carcass failed to reveal the presence of annatto pigments.[WHO/FAO; Joint Expert Committee on Food Additives (JECFA): Food Additive Series 52: Annatto Extracts. Available from, As of June 22, 2011: http://www.inchem.org/pages/jecfa.html] **PEER REVIEWED**
  • LABORATORY ANIMALS: Subchronic or Prechronic Exposure: Four groups of /twenty/ rats (Crl: CD (SD) IGS BR VAF/Plus) received diets containing annatto E at a concentration of 0, 3000, 10 000 or 30 000 ppm for 90 days. Group mean achieved actual dosages over the period of treatment were 224, 734 or 2204 mg/kg bw per day for males receiving 3000, 10 000 or 30 000 ppm, respectively, and 238, 801 or 2398 mg/kg bw per day for the corresponding groups of females. Since annatto E contains 26% bixin, the corresponding doses of bixin were 58, 191 and 573 mg/kg bw for males and 62, 208 and 623 mg/kg bw for females. The administration of annatto E produced a number of treatment-related changes, particularly in the groups receiving the highest dose. At the intermediate dose, a reduced body-weight gain in the absence of any effect on food conversion efficiency in the females was most likely to have been caused by an effect of the palatability of the diet on food intake. Increased liver weights seen in many of the treated animals, with the exception of females at 3000 ppm, were associated with slight adaptive centrilobular hepatocyte hypertrophy; no clear signs of liver pathology were noticed. Thyroid follicular cell hypertrophy was present in some animals receiving annatto E at a concentration of 30 000 ppm. Raised plasma concentrations of creatinine were seen in all females treated with annatto E and in males at 30 000 ppm. A slight increase in the weight of the kidney relative to body weight was seen in the females at the highest concentration. Raised plasma concentrations of phosphorus seen in males and females at the highest concentration of annatto E may indicate a reduction in the rate of glomerular filtration. The no-observed-adverse-effect level (NOAEL) for both sexes can be considered to be the intermediate concentration of 10 000 ppm in the diet. This is equivalent to an intake of annatto E of 734 mg/kg bw per day for males and 801 mg/kg bw per day for females. As annatto E contains 26% bixin, these NOAELs correspond to a bixin intake of 191 and 201 mg/kg bw per day in males and females respectively.[WHO/FAO; Joint Expert Committee on Food Additives (JECFA): Food Additive Series 52: Annatto Extracts. Available from, As of June 22, 2011: http://www.inchem.org/pages/jecfa.html] **PEER REVIEWED**
  • LABORATORY ANIMALS: Subchronic or Prechronic Exposure: Four groups of 10 male and 10 female (Crj: CD (SD) IGS) rats were given diets containing annatto C at a concentration of 0, 1000, 3000 or 9000 ppm for 90 days. The overall intakes of annatto C were 69, 204 or 598 mg/kg bw for males and 76, 242 or 735 mg/kg bw for females, corresponding to an intake of norbixin of 63, 187 or 548 mg/kg bw per day for males, and 70, 222 or 673 mg/kg bw per day for females in the groups receiving low, intermediate and high doses respectively. There was no effect on body weight or food intake, and no changes in hematology parameters. Changes in clinical chemistry parameters were observed in animals at the highest dose and, to a minor degree, at the intermediate dose. Increased absolute and relative liver weights were observed in animals of each sex in the groups receiving the two higher doses, and this was accompanied by hepatocyte hypertrophy in animals in these groups and focal necrosis in one male and one female in the groups receiving the highest dose. A small increase in kidney weight in each sex at the highest dose was not accompanied by any histopathological changes and may reflect a physiological response to the extra metabolic load imposed by the annatto extract. The lowest concentration of 1000 ppm, equivalent to an intake of annatto C of 69 and 76 mg/kg bw per day, or an intake of norbixin of 63 and 70 mg/kg bw, for males and females, respectively, can be considered to be the NOEL.[WHO/FAO; Joint Expert Committee on Food Additives (JECFA): Food Additive Series 52: Annatto Extracts. Available from, As of June 22, 2011: http://www.inchem.org/pages/jecfa.html] **PEER REVIEWED**
  • LABORATORY ANIMALS: Subchronic or Prechronic Exposure: Four groups of 20 male and 20 female rats (Crl: CD (SD) IGS BR VAF/Plus), aged 5 weeks received diets containing annatto B at a concentration of 0, 5000, 16 000 or 50 000 ppm for 90 days. Group mean achieved actual dosages over period of treatment were 407, 1311 or 4201 mg/kg bw per day for males in each group, respectively, and 449, 1446 or 4507 mg/kg bw per day for the equivalent groups of females. Since annatto B contained 92% bixin, these achieved doses of annatto B correspond to doses of bixin of 374, 1206 or 3865 mg/kg bw for males and 413, 1330 or 4146 mg/kg bw for females. There was no overall significant effect on body-weight gain in either the males or females. Neither was there any effect of treatment on food consumption (which was within 3% of control values overall) or on food conversion efficiency. The functional observational battery tests revealed no treatment-related effects on behavioral, motor or neurological activity. Ophthalmoscopic examinations revealed no abnormalities. Hematological investigations revealed no findings that were clearly related to treatment. Raised lymphocyte counts (9.28 +/- 1.64 compared with 7.30 +/- 1.72 x 10 +9/L in controls) were noted for males receiving annatto B at 50 000 ppm; however, although the change was statistically significant, the change was minor, was not seen in females, and no dose-response relationship was observed. When compared with the controls, statistically but not biologically significant elevations were seen in plasma concentrations of phosphorus (2.17 compared with 1.97 mmol/L) in males at 50 000 ppm, in plasma concentrations of potassium in females at 16 000 or 50 000 ppm, and in plasma concentrations of glucose in females at 50 000 ppm. The activities of alanine and aspartate amino-transferase were significantly raised in 2/10 and 3/10 females receiving annatto B at a concentration of 16 000 or 50 000 ppm, respectively. This effect was not seen in males. These findings were not accompanied by histopathological evidence of liver damage and were considered by the author to be of only minor importance in the evaluation of the toxicity of annatto B. Minor statistically significant variations in albumin:globulin ratios were observed, but these were not dose-related, and were not considered to be toxicologically significant. Significantly raised concentrations of protein were recorded in urine samples obtained from males receiving annatto B at a concentration of 50 000 ppm. Microscopic examination of the urine sediment revealed the presence of "crystals with a red sediment on top" for one female at 5000 ppm and two males at 16 000 ppm. In another female at 5000 ppm and in three males and four females at 50 000 ppm, patches of red staining were seen on the microscope slides. These presumably reflect the dark color of the annatto extract. Analysis of the data on organ weights revealed a slight but significant increase in relative (but not absolute) liver weights for males (9% increase) and females (6% increase) receiving annatto B at a concentration of 50 000 ppm, when compared with controls. These increases were not associated with any histopathological findings. No other effects on absolute or relative organ weights were observed. Microscopic examination revealed no findings that were related to treatment with annatto B. The NOEL for annatto B was 16 ppm, equivalent to an overall achieved dosage of 1311 mg/kg bw per day (1206 mg/kg bw per day expressed as bixin) in the males, and 1446 mg/kg bw per day (1330 mg/kg bw per day expressed as bixin) in the females.[WHO/FAO; Joint Expert Committee on Food Additives (JECFA): Food Additive Series 52: Annatto Extracts. Available from, As of June 22, 2011: http://www.inchem.org/pages/jecfa.html] **PEER REVIEWED**
  • LABORATORY ANIMALS: Subchronic or Prechronic Exposure: Four groups of the same strain of rats (n= 20) received diets containing annatto F at a concentration of 0, 1000, 3000 or 9000 ppm for 90 days. Group mean achieved actual dosages were 79, 240 or 753 mg/kg bw per day for the groups of males receiving 1000, 3000 or 9000 ppm, respectively, and 86, 275 or 816 mg/kg bw per day for the equivalent groups of females. Since annatto F contains 38.4% norbixin, these dosages correspond to an intake of norbixin of 30, 92 or 289 mg/kg bw for males and 33, 106 or 313 mg/kg bw for females. The administration of annatto F did not result in any treatment-related deaths. Transitory reductions in body weight and food consumption were noticed in males receiving the highest dose of annatto F in the first weeks of the experiment. The functional observational battery tests revealed no evidence of behavioral, motor or neurotoxicity, and ophthalmoscopic examination did not indicate the presence of any treatment-related lesions. Slightly (but statistically significant) low erythrocyte volume fractions, concentrations of hemoglobin, red blood cell counts and mean cell volumes were apparent for females at 3000 or 9000 ppm, suggesting slight anemia caused by annatto F. In the animals receiving annatto F at a concentration of 9000 ppm, significantly increased activity of alkaline phosphatase was reported in males, with activity of alanine amino-transferase also being increased in two of these males. Concentrations of urea, creatinine, glucose, triglyceride and albumin were raised in males and females at 9000 ppm. Concentrations of total protein were raised in females. Reduced concentrations of alpha-1-globulin and beta-globulin were noticed in males, but concentrations of alpha-1-globulin were raised in females. The albumin:globulin ratio in males was increased. At 3000 ppm, total protein and concentrations of albumin were significantly raised in females, whereas the concentration of beta-globulin and the albumin:globulin ratio were increased in males. The plasma obtained from all treated animals had a definite yellow coloration, which was presumably norbixin. A very marked increase in absolute and relative liver weights were seen in males treated with annatto F at a concentration of 9000 ppm and in females at 3000 and 9000 ppm. In females at 1000 ppm, only slight, although statistically significant, increases were noted. No changes were found microscopically, except in animals receiving a concentration of 1000 ppm, in which these changes were associated with centrilobular hepatocyte hypertrophy. The hypertrophy observed after 90 days of treatment was no greater than that observed after 28 days of treatment in the study on annatto F reported above. The weight of the kidney was slightly increased in males and females at 9000 ppm and in males at 3000 ppm. Plasma concentrations of creatinine were raised in animals receiving annatto F at a concentration of 3000 or 9000 ppm, and concentrations of urea were raised in males and females at 9000 ppm. The NOAEL for annatto F was 1000 ppm, this corresponds to an overall achieved dosage of 79 mg/kg bw per day for males and 86 mg/kg bw per day for females, corresponding to an intake of norbixin of 30 mg/kg bw for males and 33 mg/kg bw for females.[WHO/FAO; Joint Expert Committee on Food Additives (JECFA): Food Additive Series 52: Annatto Extracts. Available from, As of June 22, 2011: http://www.inchem.org/pages/jecfa.html] **PEER REVIEWED**
  • LABORATORY ANIMALS: Subchronic or Prechronic Exposure: In a 28-day study of palatability, groups of five males and five females were fed diets containing varying amounts of annatto extract. Body weights were recorded twice weekly and food intakes were measured weekly. After 28 days of treatment, the animals were killed and subjected to full postmortem examination, major organs were weighed and tissues were fixed for histological examination. It was concluded that annatto B was well tolerated in the diet at concentrations of up to 50 000 ppm; no dose-related absolute or relative organ weight changes were observed, but macroscopic examination revealed orange coloration of the gastrointestinal tract. At the highest dose, orange coloration of the mesentery was observed in a few animals. Annatto D was well tolerated in the diet at concentrations of up to 11 000 ppm. Increases in concentration up to 22 000 ppm were less well accepted and there was a suggestion that gradual increase in dosage was better tolerated than abrupt introduction of a high dose. An increase in relative liver weights was recorded for all treated males compared with their respective controls, but only in females treated with up to 22 000 ppm. Microscopic examination of the livers of rats in the control group and of males treated with annatto D at up to 16 500 ppm revealed minor periacinar hepatocyte hypertrophy in 4/5 treated males. Macroscopic examination again revealed orange coloration of the tongue and gastrointestinal tract in the majority of the treated animals. In addition, the mesentery tissue was stained orange in several of the treated animals. Annatto E at concentrations of up to and including 40 000 ppm was found to be acceptable to the animals. However, increased relative liver weights were seen in all treated animals. Microscopic examination of the livers revealed diffuse and periacinar hepatocyte hypertrophy in rats treated with 20 000-30 000 ppm or 20 000-40 000 ppm over the period of study. Again, orange staining of the tongue and gastrointestinal tract was reported for all animals receiving annatto E. The majority of the treated animals had orange staining of the mesentery. Treatment with annatto F resulted in orange coloration of the tongue and gastrointestinal tract in the majority of the animals and of the mesentery in a few animals. The most significant finding in this study was the increased relative liver weight seen in all groups treated with annatto F (at concentrations ranging from 7000 to 20 000 ppm). Animals treated with annatto G showed similar orange coloration of the tongue and gastrointestinal tract to that in animals treated with annatto F. Increased liver weights, accompanied by periacinar to diffuse hepatocytic hypertrophy, were noticed at all concentrations (7000-30 000 ppm) and concentrations of >15 000 ppm were less well tolerated.[WHO/FAO; Joint Expert Committee on Food Additives (JECFA): Food Additive Series 52: Annatto Extracts. Available from, As of June 22, 2011: http://www.inchem.org/pages/jecfa.html] **PEER REVIEWED**
  • LABORATORY ANIMALS: Subchronic or Prechronic Exposure: Male Crj: CD (SD) IGS rats received diets containing annatto C at a concentration of 0 (one animal) and 9000 ppm (three animals) for 2 weeks. Absolute and relative liver weights were increased and mild hypertrophy and mild necrosis of hepatocytes were noticed on histopathological examination of rats treated with annatto C. Electron microscopic examination revealed an abundance of mitochondria in the cytoplasm of hepatocytes of the treated animals. It was concluded that the hypertrophy of hepatocytes was linked to the increase of cytoplasmic mitochondria.[WHO/FAO; Joint Expert Committee on Food Additives (JECFA): Food Additive Series 52: Annatto Extracts. Available from, As of June 22, 2011: http://www.inchem.org/pages/jecfa.html] **PEER REVIEWED**
  • LABORATORY ANIMALS: Subchronic or Prechronic Exposure: Three groups of 10 male and 10 female rats were fed 0 and 2% of fat-soluble annatto and 2% water-soluble annatto for 13 weeks. Food intake, growth, hematological examination, organ weights and histopathology of major organs showed no abnormalities.[WHO/FAO; Joint Expert Committee on Food Additives (JECFA): Food Additive Series 17: Annatto Extracts. Available from, As of June 22, 2011: http://www.inchem.org/pages/jecfa.html] **PEER REVIEWED**
  • LABORATORY ANIMALS: Subchronic or Prechronic Exposure: Three groups of 2 male and 1 female pigs were fed 0 and 1% fat- soluble annatto and 1% water-soluble annatto for 21 weeks. One animal in the test group died from a cause unrelated to the test substance. Food intake, growth, hematology, organ weights and histopathology of all major tissues were normal.[WHO/FAO; Joint Expert Committee on Food Additives (JECFA): Food Additive Series 17: Annatto Extracts. Available from, As of June 22, 2011: http://www.inchem.org/pages/jecfa.html] **PEER REVIEWED**
  • LABORATORY ANIMALS: Subchronic or Prechronic Exposure:...The toxic effects of annatto powder (bixin 27%) have been assessed following administration of a subacute regimen (4 weeks, 20 doses) in Wistar male and female rats. A full study with three dose levels was considered unnecessary since no sign of toxicity had been noted in a preliminary experiment with 1000 mg/kg body weight/day as was recommended by the OECD guideline. In this study, annatto administered by gavage at a dose level of 2000 mg/kg/day decreased male body weight gain, but had no effect on either food intake or food conversion efficiency. Hematological and plasma biochemical examination as well necropsy performed at the end of administration (29th day) and observation (43rd day) periods revealed no alterations related with annatto administration. Kidney apoptosis occurred in 20% treated female rats in restricted areas without proliferation or tubular segments modification. The precise nature of apoptosis was not investigated in the present study. These findings suggest that annatto was not toxic to the rat.[Bautista AR et al; Food Chem Toxicol 42 (4): 625-629 (2004)] **PEER REVIEWED** PubMed Abstract

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Human Toxicity Values

  • None found

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Non-Human Toxicity Values

  • LD50 Mouse ip 700 mg/kg bw /Water soluble Annatto/[WHO/FAO; Joint Expert Committee on Food Additives (JECFA): Food Additive Series 17: Annatto Extracts. Available from, As of June 22, 2011: http://www.inchem.org/pages/jecfa.html] **PEER REVIEWED**
  • LD50 Rat oral >25 mL /Fat soluble Annatto/[WHO/FAO; Joint Expert Committee on Food Additives (JECFA): Food Additive Series 17: Annatto Extracts. Available from, As of June 22, 2011: http://www.inchem.org/pages/jecfa.html] **PEER REVIEWED**
  • LD50 Rat oral >35 mL /Water soluble Annatto/[WHO/FAO; Joint Expert Committee on Food Additives (JECFA): Food Additive Series 17: Annatto Extracts. Available from, As of June 22, 2011: http://www.inchem.org/pages/jecfa.html] **PEER REVIEWED**
  • LD50 Rat oral >50 mL /Fat soluble Annatto/[WHO/FAO; Joint Expert Committee on Food Additives (JECFA): Food Additive Series 17: Annatto Extracts. Available from, As of June 22, 2011: http://www.inchem.org/pages/jecfa.html] **PEER REVIEWED**

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Absorption, Distribution And Excretion

  • A technique /was developed/ using reversed-phase high-performance liquid chromatography (HPLC) to determine concentrations of bixin and norbixin in human plasma, to a sensitivity of 5 ug/L. After a normal breakfast, seven male and female volunteers each ingested a single dose of 1 mL of a commercial annatto food color containing 16 mg of cis-bixin and approximately 0.5 mg of cis-norbixin in soya bean oil, followed by a glass of milk. Blood samples were taken 0, 2, 4, 6 and 8 hr after ingestion; in some subjects, additional samples were taken after 24 and 48 hr. No control of food intake was made after 6 hr . The average values and range of concentrations of bixin and norbixin in the plasma of the subjects are shown in the table provided.
    Plasma concentrations of bixin and norbixin in volunteers given a single dose of commercial annatto food color

    Time after ingestion (hr) Average concentration of bixin (ug/L) (range) Average concentration of norbixin (ug/L) (range)
    0 2.9 (0-11) 10.5 (0-32)
    2 11.6 (0-18) 48.0 (3-144)
    4 10.1 (2-24) 57.8 (3-144)
    6 2.8 (0-9) 53.2 (38-74)
    8 0.0 (0-3) 28.7 (7-41)
    24 0.0 16.1 (17-20)
    [WHO/FAO; Joint Expert Committee on Food Additives (JECFA): Food Additive Series 52: Annatto Extracts. Available from, As of June 22, 2011: http://www.inchem.org/pages/jecfa.html] **PEER REVIEWED**
  • After dosing orally with annatto B at 100 or 1000 mg/kg bw, the following chemical species were present in the plasma of male and female rats: 9'cisbixin, trans-bixin (retention time, 26 min), 9'cis-norbixin, trans-norbixin, di cis-norbixin and a norbixin isomer with retention time, 6.8 min. After dosing with annatto E (at both concentrations), the above isomers plus an additional trans-bixin species with retention time of 267 min were detected in the plasma of both sexes. In contrast, after administration of annatto F (at both doses), only the norbixin isomers (9'cis-norbixin, trans-norbixin, di cis-norbixin and the isomer with a retention time 6.8 minutes) were present in the plasma of male and female rats. Plasma concentrations of 9'cis-norbixin were higher than those of 9'cis-bixin after the administration of annatto B, E or F, despite the fact that annatto B and E contain >90% 9'cis-bixin. The Tmax for the major component in plasma for each extract in males and females at both doses was 2-4 hr; by 12 hr, only trace amounts of bixin remained, although concentrations of norbixin were still measurable at 24 hr. When the oral dose was raised from 100 mg/kg bw to 1000 mg/kg bw, the plasma concentrations of the major components of annatto B (9'cis-bixin), annatto E (9'cis-bixin) and annatto F (9'cis-norbixin) were increased.[WHO/FAO; Joint Expert Committee on Food Additives (JECFA): Food Additive Series 52: Annatto Extracts. Available from, As of June 22, 2011: http://www.inchem.org/pages/jecfa.html] **PEER REVIEWED**
  • In a 4-week study, groups of four male and four female Wistar rats were fed diets containing 0% or 5% of a solution containing 0.1% annatto (0.22% bixin) and thermal degradation products, in vegetable oil (OSB), a suspension containing 0.02% annatto (mainly bixin, 1.84%), in vegetable oil (R10) or a water-soluble preparation containing 0.1% annatto (mainly norbixin, 0.27%) (WSA). Animals received either: (1) diet containing annatto extract during the first 2 weeks and normal diet for the second 2 weeks; or (2) normal diet for 2 weeks followed by the diet containing annatto extract for 2 weeks. In the animals that were killed immediately after receiving annatto extract for 2 weeks, measurable amounts of yellow pigment were observed in the blood, but in animals that were killed 2 weeks after treatment with annatto extract had stopped, only trace amounts were detected. Yellow pigment was also found in the adipose tissue of animals treated with OSB and R10, but not in animals receiving WSA. Chromatographic analysis of these pigments confirmed that they were not major annatto pigments (bixin or norbixin). There was also a clear difference in the degree of discoloration of the fat in animals killed immediately after cessation of treatment, compared with that in animals killed 2 weeks after treatment had stopped, indicating that clearance of the pigment was rapid. Feces were collected during the second week of treatment and analyzed for pigment content. About 20% of the administered dose of OSB and WSA and about 55% of R10 was recovered unchanged from the feces.[WHO/FAO; Joint Expert Committee on Food Additives (JECFA): Food Additive Series 52: Annatto Extracts. Available from, As of June 22, 2011: http://www.inchem.org/pages/jecfa.html] **PEER REVIEWED**
  • Single oral doses of /solution containing 0.1% annatto (0.22% bixin) and thermal degradation products, in vegetable oil/ OSB (7 mg/kg), /suspension of 0.2% annatto (mainly bixin, 1.84%), in vegetable oil) R10 (7 mg/kg) and a water-soluble preparation of 0.1% annatto (mainly norbixin, 0.27%) WSA (14 mg/kg) were given to adult male /volunteers/ and the blood and excreta were analyzed for annatto pigments. Blood samples were taken between 2-12 hours after treatment, urine was collected during 7 hours after the dose and feces over the 2 days following the day of treatment. WSA (14 mg/kg) produced a blood level of 12 ug/mL after 2-1/4 hours which corresponds to 6% of the dose. OSB (7 mg/kg) produced a blood level of 2.4 ug/mL after 3 hours which corresponds to 2.4% of the dose. R10 (7 mg/kg) produced a blood level of 0.44 ul/mL after 3-1/4 hours which corresponds to 0.32% of the dose. Blood levels had returned to zero 6 hours after WSA (14 mg/kg), OSB (7 mg/kg) and R10 (7 mg/kg) respectively. No annatto pigments were detected in the urine samples and none were detected in feces samples collected the next day. The feces collected the second day after treatment contained 0.17 mg R10 (0.03% of the dose) and 0.44 mg WSA (0.06% of the dose) but no pigments associated with the consumption of OSB were detected. Thus, as in the rat, the annatto pigments were absorbed and rapidly cleared from the blood.[WHO/FAO; Joint Expert Committee on Food Additives (JECFA): Food Additive Series 17: Annatto Extracts. Available from, As of June 22, 2011: http://www.inchem.org/pages/jecfa.html] **PEER REVIEWED**

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Metabolism/Metabolites

  • None found

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Tsca Test Submissions

  • None found

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Footnotes

1 Source: the NTP's CEBS database.

2 Source: the National Library of Medicine's Hazardous Substance Database, 02/28/2017.

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