The Committee did not undertake a general re-evaluation of beta-carotene for use as a colouring agent but focused its assessment on the production and analytical characteristics of beta-carotene from Blakeslea trispora.

beta-Carotene is obtained from B. trispora by co-fermentation of the two sexual types of the fungus in specific proportions. Both types are stable in cultures maintained under conditions consistent with good manufacturing practice. These source organisms are neither pathogenic nor toxinogenic. The compound is isolated from the fungal biomass by solvent extraction and crystallized. The main articles of commerce are suspensions in food-grade vegetable or plant oil and water-dispersible powders. These formulations are made for ease of use and in order to improve stability, as carotenes easily oxidize.

As in synthetic beta-carotene, the colouring principle of beta-carotene from B. trispora consists predominantly of all-trans beta-carotene. The content of total colouring matter is not less than 96% (expressed as beta-carotene). beta-Carotene from B. trispora may also contain other carotenoids, of which gamma-carotene accounts for the major part, at concentrations up to 3%. These molecules occur naturally in carotenoid-containing vegetables.

Two male and two female Wistar rats (age not stated) were given a single oral dose of 2000 mg/kg of beta-carotene biomass dissolved in propylene glycol (method of administration not stated) and were observed and weighed daily for 8 days. There was minimal weight loss and no deaths, and gross macroscopy showed no adverse effects (Kluifhooft, 2000). The Committee noted that only means and standard deviations were provided, which, in view of the small size of the study, appeared inappropriate.

Two male and two female Wistar rats (age not stated) were given a single dermal application of beta-carotene biomass at a concentration of 10 ml/kg bw (2000 mg/kg bw). The material was placed under a dressing which was removed after 24 h. The animals were weighed and inspected grossly, and yellow and orange staining were recorded. There were no other findings (Kluifhooft, 2000).

One New Zealand white male rabbit received 5 g of beta-carotene biomass on the shaved skin of the abdomen on a dressing which was held in place by an elastic bandage. The dressing was removed after 4 h, and the remaining material was removed with wet tissue. The area was reshaved 3 h before recording observations. Well-defined erythema was observed after 1 h and slight erythema after 24 h. The skin was recorded as normal (with yellow–orange staining) after 48 and 72 h (Kluifhooft, 2000).

Approximately 1 ml (47 mg) of biomass beta-carotene was instilled into the conjunctival sac of one eye of a New Zealand white rabbit, and the lids were held together for approximately 1 s. The other eye was used as the untreated control. The only finding was slight reddening after 1 h (Kluifhooft, 2000).

Groups of five male and five female Wistar rats (age not stated) were fed powdered diet containing beta-carotene derived from B. trispora at 0.2, 1, or 5% for 28 days. A further group was fed the powdered diet only, another group was fed a different sample of beta-carotene from B. trispora, and a further group was fed beta-carotene from a commercial supplier (source not specified). The animals were weighed, and their dietary intake and haematological parameters were measured. The biochemical measurements included the activities of alkaline phosphatase, alanine and aspartate aminotransferases, and gamma-glutamyl transpeptidase, total protein, albumin, the albumin:globulin ratio, urea, creatinine, bilirubin, cholesterol, triglycerides, phospho-lipids, calcium, sodium, potassium, chloride, and inorganic phosphate. The animals were killed on day 28, and the adrenals, brain, heart, liver, spleen, testes, and all gross lesions were examined histologically. No significant adverse findings were recorded (Kluifhooft, 2000).

The procedure described by Buehler and Griffith was used to determine contact sensitivity in seven guinea-pigs (age not stated). beta-Carotene biomass was dissolved in propylene glycol and applied at a concentration of 50% for 6 h once a week for 3 weeks. Three control guinea-pigs were treated similarly with propylene glycol. Two weeks later, the animals were challenged with 50% beta-carotene biomass or with propylene glycol. The reactions were assessed 24 and 48 h after removal of the bandages. No indication of sensitization was observed (Kluifhooft, 2000).

beta-Carotene derived from B. trispora was tested for mutagenic activity in Salmonella typhimurium TA1535, TA1537, TA98, and TA100, with and without an exogenous metabolic activation system from rat liver, in two independent studies with five concentrations of the test substance. Negative controls were tested with dimethyl sulfoxide and positive controls with sodium azide, 9-aminoacridine, benzo[a]pyrene, 2-nitrofluorene, and 2-aminoanthracene. No mutagenic activity was observed (Kluifhooft, 2000).

beta-Carotene derived from B. trispora was tested for its ability to induce chromosomal aberrations in cultured Chinese hamster ovary cells in two independent assays with and without exogenous metabolic activation. The material was dissolved in dimethyl sulfoxide and tested in serial dilutions from 25 to 0.1 mg/ml. A vehicle control and a positive control with cyclophosphamide were included. No clastogenic effects were recorded (Kluifhooft, 2000).

The Committee concluded that, on the basis of the source organisms, the production process, and its composition characteristics, beta-carotene from B. trispora does not raise specific concerns and from a toxicological point of view should be considered equivalent to chemically synthesized beta-carotene, for which an ADI of 0–5 mg/kg bw was established by the Committee at its eighteenth meeting (Annex 1, reference 35). This opinion was supported by the negative results in two tests for genotoxicity (mutagenesis and chromosomal aberration) considered at the present meeting.

The Committee established a group ADI of 0–5 mg/kg bw for synthetic beta-carotene and beta-carotene derived from B. trispora. This ADI applies to use of beta-carotene as a colouring agent and not to its use as a food supplement.

Kluifthoof, J.D. (2001) Unpublished data submitted to WHO by DSM Food Specialties.

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       Toxicological Abbreviations