Question

The DG III has asked the Scientific Committee for Medicinal Products and Medical Devices (SCMPMD) to express its opinion on the suitability / safety of the "colours permitted for certain use only" listed in Annex IV of EEC 94/36 [in particular: E 123 (Amaranth); E 127 (Erythrosin); E 161 (Canthaxanthin); E 173 (Aluminium); E 174 (Silver); E 175 (Gold)] for use in pharmaceutical products and the question of whether the use of these agents might represent a consumer health/safety concern.

The Committee has noted that these colouring agents have been evaluated as potential food additives by the Scientific Committee for Food (SCF), which established the following limits for acceptable daily intake (ADI): amaranth, 0.8 mg/kg bw (SCF, 1983); erythrosin, 0.1 mg/kg bw (SCF, 1987) and canthaxanthine, 0.03 mg/kg bw (SCF, 1997). The Committee is unaware of any recent information that would necessitate revision of these ADIs.

The question to be examined by the Scientific Committee for Medicinal Products and Medical Devices (SCMPMD) regards the following colourant:

E123 Amaranth (FD & C Red No. 2)

Answer

Given the quantities of the colourant allowed in certain foods, which can be consumed without any restriction whatsoever, it seems paradoxical to prohibit its use at levels that are absolutely negligible in pharmaceutical products, the sale and consumption of which are regulated by law or in any case limited.

Main elements of the scientific justification of the answer

In almost all of the pharmaceutical products containing amaranth that are already on the European market, the content of the colourant ranges from 0.1 mg/mL of syrup to 0.03-0.6 mg per mL of drops and 0.3 mg per capsule. For a 70-kg man, these doses represent 0.0014-0.008 mg/kg.

The pro kg bw and pro die doses of this dye that have been perfectly tolerated by various animal species are much higher than those that can reasonably be expected to be ingested in pharmaceutical products. On a pro kg basis, even the lowest dose tolerated by rats (50 mg/kg bw/day: Clode et al. 1987) is approximately 6250 times higher than the maximum one dosing unit ingested with pharmaceuticals, i.e., 0.008 mg/kg. The single maximum pharmaceutical dose/kg bw of the above preparations is 100 times lower than the ADI established in 1983 (0.8 mg/kg bw: SCF, 1987) and -62.5 times lower than that of 1986 (0.5 mg/kg bw: Martindale Extra Pharmacopoiea, 1996).

Full opinion

Terms of ReferenceThe Committee has been asked to respond to the following question:Would use of the colourants listed in Annex IV ("colours permitted for certain uses only") of Directive 94/36 (in particular: E123 Amaranth, E127 Erythrosin, E161 Canthaxanthine, E173 Aluminium, E174 Silver and E175 (Gold) in medicinal products represent a consumer health/safety concern?

Context of the question

EEC Directive 78/25, which deals with colouring agents that can be used in medicinal products, makes reference to the Directive issued on 23 October 1962 dealing with colouring agents in food (OJL 115 f 11.11.1962 p. 2645). However, the EEC policy on food-colouring agents has been updated since then by Directive 94/36. Of particular interest in the latter document are Annex I, which lists all substances approved as food colourants, and Annex IV, which contains 10 agents whose use is restricted to certain foods.

The pharmaceutical industry is questioning the scientific justification for excluding the use of Annex-IV colourants in medicinal products, citing in particular the clause in EEC Directive 78/25 that states, "Experience has shown that on health grounds there is no reason why the colouring matters authorized for use in foodstuffs intended for human consumption should not also be authorized for use in medicinal products."

Assessment

The question requires an evaluation of the toxicological characteristics of said colourants in relation to: 1) the maximum quantities and concentrations / unit of weight allowed in foodstuffs, 2) those currently found in pharmaceutical products, and 3) the pro kg amounts that have been well tolerated in the various in vivo toxicity tests.

According to Annex IV of Directive 94/36, the colourants in question may not exceed the following concentrations in foods and beverages:

(*Used in external sugar-based coatings; for decoration of cakes and pastries; coating for chocolates and candies; liquors.)

A brief note from the EMEA (completed with data obtained from the Drug Department Italian Ministry of Health) provides data on the products sold in European states that contain the colouring agents in question.

Many of these products have been on the market since the 1960s.

The maximum amounts of amaranth, erythrosin and canthaxanthine found in pharmaceutical products currently marketed in Europe are:

E 123 (amaranth)

Syrups 0.1 mg/mL

Drops 0.03-0.6 mg/mL

Capsules 0.3 mg / capsule

E 127 (erythrosin)

Capsules / tablets/sugar-coated pills 0.017-0.96 mg / capsule, tablet or pill

Liquid oral preparations (drops, syrup, suspension) - 0.009-0.8 mg/mL

E 161 (canthaxanthine)

Capsules/Tablets 0.0049 - 0.0042 mg / capsule or tablet

The quantity pro Kg for a 70-Kg adult male for one dosing unit of the above agents ranges from 0.00002 mg / kg to 0.013 mg / kg.

E 173 (aluminium powder) and E 174 (silver) are found in very few pharmaceutical products in Europe (E 173 in Denmark, Germany and Spain; E 174 in Germany only). E 175 is used in one medicinal product (in Germany).

The following table shows the lowest estimates available of the quantities of these three colouring agents likely to be ingested with foods and beverages. The figures in parentheses are the amount in mg pro kg doses for a 70-kg adult male.

E 173 (Aluminium), E 174 (Silver) and E 175 (Gold) are used to decorate cakes, candies and other sweets. For all three of these agents, Amex IV of Directive 94/36 allows unlimited use ( quantum satis) since there do not seem to be any toxicological problems at all associated with these substances.

Attention will now be focused specifically on E 123 Amaranth

E 123 - Amaranth

Toxicity

Rats fed 20 mg/day for 78 weeks showed 58% mortality compared with 13% in controls; vascular dystrophy, with eventual fatty degeneration of the liver cells, was observed (Galea et al., 1972). Liver function and histology impairment was observed with amaranth in other papers by Galea et al. (1971).

A diet containing 5% amaranth (roughly 3500 mg/kg/ die) inhibits growth in rats (Takeda e Kiriyama, 1991; Takeda et al., 1992). This effect is thought to be due to a reduced availability of nutrients caused by the rapid transit of amaranth-containing chyme through the upper segments of the gastrointestinal tract (Aritsuka et al., 1989; Ershoff and Thurston, 1974; Takeda et al., 1992) and to colourant's inhibitory effects on the processes of digestion and absorption (Takeda et al., 1992). The physical effects of large amounts of unabsorbed dye in the gut may contribute to these effects. According to Kimura et al. (1983), the toxicity of dietary amaranth is due to the exfoliating or dissolving effects it exerts at high doses on the brush border membrane of the small intestine. In fact, only 10-20% of the colourant is absorbed after azoreduction in the gut, 75-85% being excreted in the faeces. The gastrointestinal and toxic effects can be attenuated by fibre obtained from edible burdock ( Arctium lappa) (Takeda e Kiriyama, 1991; Takeda et al., 1992) and buckwheat protein extract (Kayashita et al., 1996), but the most significant protective effects in rats have been achieved with beet dietary fiber (BDT), citrus dietary fiber (CDF) (Yoshida et al., 1988) and hemicellulose (Aritsuka et al., 1989)

In Sprague-Dawley rats receiving cholesterol-supplemented diets providing approximately 8% dietary fiber from pectin, cellulose, oat bran, amaranth or a fiber-free control diet, amaranth behaved like soluble fibers in lowering serum cholesterol, but like insoluble fibers in terms of its action on colon (Dana and Lupton, 1992).

No growth changes have been observed in chickens fed a diet containing approximately 6% amaranth (roughly 5000 mg/kg/ die), which is associated with delayed growth only when added to a starch-based diet. The mechanisms underlying amaranth toxicity in this species appear to be different from those in rats since burdock-root fibre has no protective effects (Takeda e Kiriyama, 1991).

In female beagle dogs fed 20,000 mg amaranth in the diet, no histopathological or other abnormalities were reported (FAO/WHO, 1966). Amaranth 500 and 750 mg/kg bw/day administered for 1-21 and 22-days respectively to weaned pigs had no effect on hematological, clinical or blood-chemistry parameters or on the histological features of main organs (Sondergaard et al., 1997).

Genotoxicity

Amaranth has proved to be nongenotoxic in the Ames test (Auletta et al., 1977; Brown et al., 1978; Al-Mossawi, 1983; Lecointe e Lesca, 1978; Ishidate et al., 1984; Izbirak et al., 1990), the mouse dominant lethal assay (Arnold et al., 1976), the cell transformation assay with Syrian hamster embryo cells (Casto, 1983), in Saccharomyces cerevisiae (Parry, 1977; Sankaranarayanan e Murthy, 1979) and in somatic and germ-line cells of Drosophila (Tripathy et al., 1995). Urine samples from Sprague-Dawley rats given intraperitoneal (i.p.) amaranth (100 mg or 2 x 100 mg/animal or 200 mg/kg bw) were also negative in the Ames test (Munzes et al., 1979). However, in the fluctuation test, the colourant was found to enhance both forward and reverse mutation in cultures of auxotrophs of Escherichia coli and Salmonella typhimurium that contain drug-resistant plasmids (Al-Mossawi, 1983). Amaranth has been associated with positivity in the chromosome aberration test with Chinese hamster fibroblasts (Ishidate et al., 1984) and in the Ames test when ether extracts of aqueous solutions of the dye were used in the standard plate assay (Prival et al., 1988). In the thymidine kinase heterozygous (TK+-) mouse lymphoma assay, a dose-related increase in mutation frequency (compared with values for negative controls) was observed for R-amino salt, a metabolite of amaranth. In contrast, in the dominant lethal test, the effect of amaranth (1200 mg/kg bw) and its R-amino salt was not statistically significant (Palmer et al., 1979). The mutagenic activity of -naphtylamine (NA) is lost in presence of 9.99 mg amaranth (0.1% -NA in amaranth), and similar results have been obtained with -NA in amaranth. It is possible that interaction between the colouring agent and naphtylamines hinders the conversion of NA to an active mutagen or destroys the active mutagen before it can react with an appropriate molecular site (Stoltz et al., 1979).

Carcinogenicity

Sarcomas of the peritoneum and intestine were observed in 11/18 rats receiving amaranth paste (65-75% pure chemical in dry paste) in the diet at a concentration of 20,000-40,000 mg/kg diet, each rat receiving roughly 1 g/kg bw paste and a total dose of 245 g (Baigusheva, 1968). Fifteen malignant tumours were observed in 13/48 noninbred rats treated with a diet containing 20,000 mg/kg diet pure amaranth for 33 months. Surprisingly, there were no tumours at all in the untreated control group (Andrianova, 1970). In mice and rats given amaranth in the diet or by gavage at doses of 300-40,000 mg per kg of diet for more than 1-2 years, there were no tumours that could be attributed to the colourant (Mannel et al., 1958; Willheim and Ivy, 1953). In older studies (Cook, 1940), no treatment-related tumors were observed in mice (sex and strain unspecified) treated for life (about 15 months) with 15-20 mg amaranth given over 5 days a week, as aqueous solution added to brown bread.

An IARC monograph published in 1975 provides uncertain data on the question of amaranth carcinogenicity. The working group of the IARC reviewed ten studies on amaranth tumorigenicity, nine of which yielded negative results. The conclusion was withheld owing to the paucity of data given in most of these reports. Weekly application for 18 months of 0.1 ml of a 1% solution of amaranth to the backs of Swiss Webster mice (18 males, 20 females) had no effect on survival, weight gain or incidence of tumours (Carson, 1984).

Reproductive toxicity

Male rats treated with 1.5-15 mg/kg bw/day for 12 months demonstrated a reduced resistance of spermatozoids. Female rats likewise treated showed depression of the oestral cycle. In both sexes a heightened gonadotropic function of the pituitary was observed. The same dosages given to pregnant rats throughout gestation resulted in a greater post-implantation lethality of embryos (Schienberg and Gavrilenko, 1972).

abnormalities were seen in the experimental animals. There was no sex-related foetal effect (Collins and McLaughlin, 1972). Multiple resorptions followed administration of amaranth metabolites R-amino salt (100-200 mg/kg) and sodium naphtionate (200 mg/kg). Sodium naphtionate produced a significant increase in the percentage of foetuses with sternebral abnormalities, and R-amino salt (200 mg/kg) increased the incidence of litters with one or more foetuses with skeletal abnormalities. The R-amino salt did not affect, sternebral or soft-tissue development.

Male and female Wistar rats were given amaranth in their diets at doses of 50-250-1250 mg/kg for 60 days before mating, throughout pregnancy and during nursing. Treatment was continued in the pups for 111 (males) and 112 (females) weeks after being weaned. No changes were noted in the latter animals as far as hematological parameters, serum chemistry, fertility or incidence of tumours was concerned. At the high doses, however, the faeces and the fur were red in colour, and the faecal pellets were poorly formed. After 18 months of treatment, the older females presented epithelial hyperplasia of the renal pelvis, which was interpreted as a senile change (Clode et al., 1987).

Doses up to 264 mg/kg/ die given prior to mating and/or during pregnancy had no teratogenic effects or adverse effects on embryo-foetal development in mice (Larson, 1975), in rats (Khera et al., 1974; Piersma et al., 1995), in cats (Khera et al., 1976) or in the Fetax test (Bantle et al., 1990).

Amaranth had no adverse effects in either the rat limb-bud micromass assays or in rat embryo culture systems (Amacher et al., 1996). In a collaborative study performed by three US government-industry laboratories, different rat strains received amaranth (200 mg/kg bw) by gavage or in drinking water on days 0-19, 6-15, 7-9 of gestation: there was no increase in abnormalities or other indications of embryo-toxicity (Collins et al., 1976).

Drake (1977) underlines that in 17 or more studies on amaranth that have been reported to date have rarely involved more than scattered indications of an increased absorption rate. Embryotoxicity is usually indicated by combinations of increased reabsorption, decreased foetal weight and an increased incidence of skeletal variants. Studies on the teratology of amaranth have failed to reveal any adverse events that were both biologically sound and reproducible.

Dietary intake of 30-300-3000 or 30,000 ppm of amaranth were ingested by Osborne-Mendel rats in a 3-generation reproduction and teratology study without affecting fertility, average litter size, average number of liveborn animals, viability or survival of offspring (Collins et al., 1975a).

In rats treated with dietary intake up to 30,000 ppm of amaranth, no dose-related increases were seen in F1A resorption, but there was a non-dose-related decrease in the mean foetal weight of the progeny. None of the implantation and foetal survival parameters analyzed for F2B were different from control values. No specific skeletal or soft tissue abnormality could be correlated with dose levels of the dye in either F1A or F3B (Collins et al., 1975b).

Mice were treated with diets containing 0.03-0.09-0.27% amaranth (roughly 50-90-270 mg/kg bw/day) 4 weeks before mating, during the 5-week mating period and throughout gestation. The F1 generation was treated until 9 weeks of age. In another experiment mice were given 0.025 - 0.075 0.225 % amaranth in the diet (approximately 25-75-225 mg/kg/ die). No negative effects were noted in terms of litter size, pup weight or litter weight. However, the body weight of the pups during the lactation period in the treatment group increased less significantly, and the survival index at postnatal day (PND) 21 of the amaranth 0.025 - 0.27% group was reduced. Neurobehavioural developmental parameters, surface righting at PND 4 in female offspring, direction of the swimming on PND 4 in male pups and olfactory orientation in both sexes were significantly reduced (Tanaka, 1993) in the 0.075% treatment group (Tanaka, 1992; 1993; 1995). The dose levels of amaranth used in these studies produced almost no adverse effects on behavioural development in mice.

Immunotoxicity

Amaranth showed clear immunosuppressive effects in vitro at a concentration that had proved to be non-cytotoxic in neutral red uptake and thiazolyl blue tetrazoliun bromide (MTT) assays (Koutsogeorgopoulou et al., 1998). The dye reduces the release of serotonin by basophilic and might therefore be expected to exert a protective effect against immediate-type hypersensitivity (Tanaka, 1995).

Molecular toxicology

In vitro mitochondrial respiration was inhibited in rat liver and kidney by organic synthetic food colours (including amaranth) but only at the improbable concentration of 0.1 mg food colour per mitochondrial protein (Reyes et al. 1996).

In vitro changes in the activities of certain enzymes (ornithine carbamoyl transferase, glutamic pyruvic transaminase, glutamic oxalacetic transaminase) have been observed by Valeanu et al. (1969). According to Galea et al. (1972), the vitamin A content of the liver decreases after administration of amaranth in rats. Holmberg (1978) giving amaranth by gavage (85 mg/kg bw/day) reduced acid phosphatase in the hepatic liver cytosol. Amaranth also caused a significant decrease in the activity of rat liver lysosomal acid phosphatase. Hence both primary lysosomes and phagosomes are influenced by amaranth in vitro.

A 2-3-5 fold stimulation of RNA synthesis after administration of amaranth (300 mg/kg bw/day for 30 days) (Yoshimoto et al., 1984a) and in vitro (Yoshimoto et al., 1984b) might be related to changes in the liver (liver weight, protein and nuclear RNA with dissociation of heterochromatin revealed in isolated rat liver nuclei ) (Yoshimoto et al., 1984b).

Adverse reactions in humans

Safety problems have never been encountered with E 123 amaranth. Only one case of an adverse reaction to amaranth has been reported in the UK (EMEA report, 1998). In reported cases of adverse reactions, including allergic reactions, to products containing amaranth, it has not been possible to determine whether the reaction was provoked by the colouring agent or the active ingredients of the product.

The available toxicological data on amaranth indicated no serious toxic responses.

Opinion

Given the quantities of the E 123 Amaranth allowed in certain foods [30 mg/L (wines) -30 mg/kg (caviar)], which can be consumed without any restriction whatsoever, it seems paradoxical to prohibit its use at levels that are absolutely negligible in pharmaceutical products, the sale and consumption of which are regulated by law or in any case limited.

The maximum content of amaranth in pharmaceutical products that are already on the European market is 0.1 mg/ mL of syrup, 0.6 mg /mL of drops and 0.3 mg per capsule. For a 70-kg man, these doses represent 0.0014-0.008 mg/kg bw.

The pro kg bw and pro die doses of amaranth that have been perfectly tolerated by various animal species are much higher than those that can reasonably be expected to be ingested in pharmaceutical products. On a pro kg bw basis, even the lowest tolerated dose (50 mg / kg bw / die) is 6250 times higher than the dose which can be ingested with the maximum one dosing unit in medicinal products (i.e., 0.008 mg / kg bw). The ADIs for amaranth established in 1983 and 1996 were respectively 0.8 (Scientific Food Committee, 1983) and 0.5 mg/kg bw (Martindale Extra Pharmacopoiea, 1996). The dose that might be consumed with a single dose of syrups, drops or capsules is 100-62.5 times lower than these ADIs.

Since more than one capsule or 1 mL of syrups or drops may be consumed in a day, the actual margin remains elevated, i.e., the dose pro kg consumed with 5 capsules or 5 mL of syrups or drops would be roughly 20-12 times lower than the proposed ADIs.

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