WSA (14 mg/kg) produced a blood level of 12 μg/mL after 2.25 h, which corresponds to 6% of the dose. E160d. Testing for mutagenic activity with Salmonella typhimurium TA1535, TA1537, TA98, TA100, TA102, TA104 and Escherichia coli WP2uvrA. The applicant stated that, like many natural products, annatto seeds originate from many different countries and are extracted for commercial use by a variety of procedures, involving oil, water, alkali, heat, etc. The solvent‐extracted norbixin (Annatto C) did not increase the number of revertants at any concentration in any strain, whereas the alkali‐processed, acid‐precipitated norbixin (Annatto F) was positive against S. Typhimurium TA100, with and without activation, inducing at the highest tested dose approximately twofold and fourfold increases in revertant counts in the presence and absence of S9, respectively. The only exceedance observed for norbixin was at the 95th percentile in the brand‐loyal scenario for infants (in one country), toddlers (in three countries) and children (in one country). At the 61st JECFA Meeting (2003), the Committee noted that, whereas the reproductive toxicity of bixin has been studied, such data for a norbixin‐rich extract were not available. The amphoteric nature of norbixin makes it suitable in products where the pigment is absorbed by protein and/or starch, stabilising it thereby and making it less soluble in water. E numbers are codes for substances that can be used as food additives for use within the European Union[1] and Switzerland. The authors noted that similar conversion occurred with other carotenoids, such as β‐carotene and lycopene. and sacrificed 4 h later, along with the corresponding control groups. Norbixin‐based annatto extracts have traditionally been used for the colouring of cheese, but are now used in many other applications. Orange discolouration of the mesentery was reported in a few animals which had received 20,000/50,000 mg/kg diet (Group 5), due to the intense orange colour of the annatto extract. Reduced food conversion efficiency values were noted in the Group 4 males in weeks 3 and 4 (30,000 mg/kg diet), but these were not affected in females. Call for scientific data on food colours to support re‐evaluation of all food colours authorised under the EU legislation. Studies have been reported in the literature in the mouse, rat and dog, and the results for all the three species were discussed in previous evaluations of the SCF and JECFA. Since then, a few observations have been made on the effects of annatto extracts (obtained from the seeds of B. orellana) on the physiological and biochemical functioning of the body, following administration to animals. Most food additives in the EU are authorised at a specific MPL. If Animal 6M was excluded from the data analysis, five out of six animals treated at 1,000 mg/kg per day and five out of six animals treated at 2,000 mg/kg per day exceeded the range of tail intensity values seen in the vehicle control group. Studies by Fernandes et al. The authors indicated that the reduced body weight gain in the absence of any effect on food conversion efficiency observed in the females fed 10,000 mg Annatto E/kg diet was most likely due to a palatability effect on food intake. There were no dose‐related effects of treatment on body weight gain, food intake and organ weights. Halal if used as 100% dry color. Positive results were also reported from a mouse dominant lethal test using the same test materials (CE and BE) (Aranez and Bayot, 1997). Chisté et al. Increase in liver weight and centrilobular hepatocellular hypertrophy are common findings in rodents which have been given high levels of a xenobiotic, and are generally considered to be an adaptive response and not a toxic effect of treatment. HPLC‐PDA provided superior qualitative and quantitative data, particularly with respect to the determination of coloured degradation compounds. For the non‐brand‐loyal scenario, the main food categories were fine bakery wares for all population groups together with flavoured fermented milk products for infants, toddlers, children and adolescents. Polyploidy was 2.5%, while 7% of cells had structural aberrations, compared to ≤ 3% in untreated and solvent‐treated cultures. The solvent‐extracted norbixin (Annatto C) is derived from the solvent‐extracted bixin (Annatto B); they share the small quantity of non‐pigment material. The Panel noted that complete information on the methods of analysis (e.g. non‐compliance controls). Four groups of 20 male and 20 female (Crl:CD (SD)IGS BR VAF/Plus) rats, 40–44 days of age at the start of the treatment, were used for the study. provided to EFSA n.3), several systematic reviews on annatto have been published (Lim, 2012; Ulbricht et al., 2012; Vilar et al., 2014). The uptake of bixin isomers in liver and bone marrow was examined, and the isomers present in these tissues were consistent with those seen in the plasma at 4 and 12 h for each extract; however, the assay method was not validated for these tissues, therefore quantitative conclusions cannot be drawn. For the regulatory maximum level exposure assessment scenario, the main food categories contributing to the total mean exposure to annatto (E 160b) were fine bakery wares, flavoured fermented milk products, and fats and oils, and for infants, fine bakery wares and flavoured fermented milk products for toddlers, children and adolescents, and fine bakery wares for adults and the elderly. Annatto is the name of the crude extract, whereas bixin is the fat-soluble colour and norbixin the water-soluble colour. The "E" stands for "Europe". It represents FDA's best advice for a uniform system of provisions that address the safety and protection of food offered at retail and in food service. As discussed in Section 2.2.3, the composition of the alkali‐processed norbixin, not acid‐precipitated (Annatto G) is similar to that of the alkali‐processed, acid‐precipitated norbixin (Annatto F), when compared on a calculated dry weight basis, as the liquid Annatto G contains 90% water. For this reason, a number of extracts (rather than bixin or norbixin) have been used for the toxicological investigations. (abbreviation) In summaries of a Japanese co‐operative programme on short‐term assays for carcinogenicity, it was reported by Kawachi et al. In a first study, as reported by JECFA ‘groups of four male and four female rats were fed 0% or 5% of OSB, R10 and WSA annatto extracts for a 4‐week period. Dietary concentrations used in this study were based on the results from the preliminary 4‐week range‐finding study discussed in the previous subsection (HLS, 2001b). Some 9′‐cis‐norbixin was present in the urine of male rats following dosing with Annatto E at 1,000 mg/kg bw, but this accounted for less than 0.1% of the dose. The Panel agreed with this conclusion. Table 2 presents the annatto extracts evaluated in the present opinion. Via Carlo Magno 1A, 43126 The food categories in which the use of annatto extracts (E 160b) is authorised were selected from the nomenclature of the EFSA Comprehensive Database (FoodEx classification system food codes) at the most detailed level possible (up to FoodEx Level 4) (EFSA, 2011b). The Panel agreed with this conclusion. Hepatocellular hypertrophy was found in all rats fed 9,000 mg Annatto C/kg diet, and six males and five females in the 3,000 mg/kg diet group; one male and one female in the top‐dose group had focal necrosis in the liver. Mechanical abrasion using food grade vegetable oil … The patient's serum immunoglobulin E (IgE) recognised a 50‐kDa protein in the annatto extract, considered by the authors to be a contaminant from the pericarp of the seed in the preparation of the annatto extract. Considering the low purity of these annatto preparations used by Van Esch et al. A-1. The aqueous solution is filtered and acidified to precipitate the norbixin. (pKM101), Cytogenetic effects of 60 chemicals on cultured human and Chinese hamster cells, Reports of the Scientific Committee for Food, Reports of the Scientific Committee on Food, Characterisation of the coloured thermal degradation products of bixin from annatto and a revised mechanism for their formation, The chemistry and analysis of annatto food colouring – a review, Method development and analysis of retail foods for annatto food colouring material, Studies on the mass balance of annatto extracts – annatto types B, F and G, Annatto extracts – chemical and technical assessment report, An evidence‐based systematic review of annatto (, Über die chronishe Verträglichkeit von Annattofarbstoff, Traditional uses, chemical constituents, and biological activities of, The prevalence of reaction to food additives in a survey population. Findings reported at necropsy comprised orange discolouration of the tongue and the gastrointestinal tract in the majority of animals from Groups 3, 4 and 5, and in several animals from Group 2. The rats and mice, when treated in this way, demonstrated hyperinsulinaemia and hypoinsulinaemia, respectively, indicating that the pancreatic β‐cells were functional. The mutagenicity of Annatto B, Annatto E and Annatto F in mammalian cells was evaluated in the mouse lymphoma L5178Y cell tk+/− forward mutation assay (Riach, 2001a,b,c). As colours were among the first additives to be evaluated, these food additives should be re‐evaluated with the highest priority. Decreased overall body weight gains were noted for males receiving 30,000 mg Annatto E/kg diet and in females receiving 30,000 and 10,000 mg Annatto E/kg diet. 89957‐43‐7 and 289‐561‐2, respectively ( Doc, 1975, 1982 ) other available data the FCS categories... 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