Question

The DG III has asked the Scientific Committee for Medicinal Products and Medicinal Devices (SCMPMD) to express its opinion on the suitability / safety of the "colours permitted for certain uses only" listed in Annex IV of EEC 94/36 [in particular: E 123 (Amaranth); E 127 (Erythrosin); E 161 (Canthaxanthine); 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 fo od additives by the Scientific Committee for Food (SCF), which established the following limits for acceptable daily intake (ADI): amaranth, 0.8 mg/kg body weight (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 in the present Opinion regards the following colourant : E 127 Erythrosin (FD & C Red N° 3)

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 is 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 erythrosin that are already on the European market, the content of the colourant ranges from 0.0017 mg to 0.96 mg per capsule/tablet/sugar-coated pill, and from 0.009 to 0.8 mg / mL in liquid preparations (drops, syrups, etc). For a 70-kg man, these doses represent 0.0002-0.013 mg/kg bw per capsule, tablet or pill or millilitre liquid preparation ingested.

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 (160 mg/kg bw/day: Butterworth et al., 1976) is ~ 12,000 times greater than the dose (0.013 mg/kg bw) ingested with one maximum dosing unit with pharmaceuticals. The single pharmaceutical dose/kg bw of oral liquid preparations, capsules, tablets, or sugar-coated pills is 7.6 lower than the Acceptable Daily Intake (ADI) [100 ?g/kg bw SCF, 1987; up to 100 ?g/kg bw (Martindale Extra Pharmacopoiea 1996)]. However, the ADI might be reached with a daily dose of 5-7 pills, capsules or tablets containing erythrosin, or 5-7 ml of a liquid preparation / day.

Full opinion

Terms of Reference:

The 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 pharmaceutical products sold in European states that contain the coloring 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)

Capsules 0.3 mg / capsule

Liquid oral preparations (drops, syrups, suspensions, etc.) - 0.01-0.6 mg/mL

E 127 (erythrosin)

Capsules / tablets/ sugar-coated pills 0.00017-0.96 mg / capsule or tablet

Liquid oral preparations (drops, syrups, suspensions, etc.) - 0.009-0.8 mg/mL

E 161 (canthaxanthine)

Capsules /Tablets - 0.0049-0.042 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 bw to 0.013 mg / kg bw.

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 only (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 mg pro kg bw 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, Annex 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-127 Erythrosin

E 127 - Erythrosin

Erythrosin is employed as a colouring agent in foods and cosmetics and as a dental-plaque-disclosing agent (Martindale Extra Pharmacopoiea, 1996).

Toxicity

The LD50 values for orally administered erythrosin reported by Yankell and Loux (1977) were 2558 ± 1.35 mg/kg bw in mice and 2891 ± 1.02 mg/kg bw in rats. These values are close to those found by Lu and Lavablée (1964) and Hansen et al. (1973b). Higher LD50 values have been reported by Butterwoth et al. (1976) (7-day LD50 6.7-7.4 g/kg bw in both sexes of rats and mice).

Depression of growth was observed by Hansen et al. (1973 b) in rats given graded dietary levels of 0.5% erythrosin (roughly 400 mg/kg bw/day). In another study, erythrosin was administered to rats either by intubation (at doses of up to 1500 mg/kg bw twice weekly for 85 weeks) or in the diet (at levels of up to 4% for 86 weeks). The major finding was an increase in serum protein bound iodine, which was attributed to the iodine released from erythrosin. The values had returned to normal 16 weeks after cessation of treatment. Thyroid levels of iodine were not affected by erythrosin, and there was no gross or microscopic pathology that could be attributed to the administration of colourant (roughly 180 mg/kg bw/day).

Administered to growing rats at dietary concentrations of 0.25-2% (roughly 180-1400 mg/kg bw/day) for 13 weeks, erythrosin had no effect on weight gain, food intake, haematological parameters, blood chemistry or renal function indices. However, caecal distension, increased thyroid weight and pigmentation of kidney tubules were observed for all dose levels. The no-untoward effect level was 0.25% of the diet, which is roughly equivalent to a dose of 160-170 mg/kg bw/day (Butterworth et al., 1976a).

No toxicity was evident in male or female rats maintained on a diet supplemented with 2% erythrosin. The gross and macroscopic findings of main organs and the results of clinical tests were all within normal ranges (Sekigawa et al., 1979).

When erythrosin was added to the diet of rats at doses of 49-251-567-2464 mg/kg bw/day approximately 30 months, no adverse effects were observed in terms of clinical pathology, laboratory values or survival. The highest dose used was associated with decreased weight gain in females, and, in both sexes, increased thyroid weight and higher incidences of follicular hypertrophy, hyperplasia and thyroid adenoma, which might have been due to the compound's inhibition of peripheral conversion of thyroxin (T4) to triiodothyronin (T3) and increased release of TSH (Borzelleca et al., 1987). Regarding these findings, it is important to recall that erythrosin is an iodinated compound.

In another study, treatment of rats for 21 days with 68 or 136 mg/kg bw/day was associated with signs of testicular toxicity (Abdel Aziz et al., 1997).

Gerbils treated for 2 years with erythrosin (added to the diet at levels of 1-2-4% or administered by intubation at levels of 200-750 or 900 mg/kg bw/day) presented weight loss, enlargement of thyroid follicles with increased storage of colloid, consistent foci of very small follicles (and in few animals focal hyperplasia and intraluminal and intertestitial leucocyte infiltration) – all reminiscent of human nodular goitre, and depression of haematological values. The alterations were mild in the group fed 1% (roughly 700 mg/kg bw/day), and marked in the group receiving 4% diet (roughly 2800 mg/kg bw/day). In animals receiving 750-900 mg/kg bw/day by gavage, thyroid changes were mild or absent. A reduction of granulomatosis, a disease of undetermined etiology, was observed in gerbils treated with erythrosin 1% and 2% compared with untreated controls (Collins e Long, 1976)..

Dogs receiving dietary levels of 0.5-1-2% erythrosin for 2 years did not present any changes that could be attributed to the treatment (Hansen et al., 1973b).

Erythrosin given orally at doses of 167-500-1500 mg/kg bw/day had no significant effects on weight gain, haematological findings, urine parameters or histopathological findings in male or female pigs. However, increases in the weight of thyroid glands and decreased serum levels of thyroxine were found at all treatment levels (Butterworth et al., 1976).

Genotoxicity

In the Ames test erythrosin is negative against different strains of Salmonella typhimurium at concentrations ranging from 2 mg / plate (Lakdwalls and Nerrawal, 1988) to 10 mg / plate (Lin and Brusik, 1986). Concentrations of up to 10 mg/ml were also negative in mitotic recombination assays with Saccharomycescerevisiae strain D5 (with and without S9) (Lin e Brusik, 1986). In V79 Chinese hamster lung cells, negative results were obtained with doses up to 300 ?g/ml at two gene loci and on sister chromatid exchanges (all experiments were conducted +/- S9). The highest dose produced an increase in the frequency of micronuclei only in the absence of S9 (Rogers et al., 1988). A dose-related increase in the frequency of micronuclei was observed in murine erythrocytes (only 3 mice/group used) after intraperitoneal (i.p.) injection of erythrosin 100-200-300 mg/kg (Godbole and Laidya, 1980).

In a study employing three cytogenetic endpoints (sister chromatid exchange in peripheral blood lymphocytes, micronuclei frequency in bone marrow polychromatic erythrocytes and micronuclei frequency in peripheral blood reticulocytes), no sign of genotoxicity was produced by 2 doses (administered 24 hr apart) of erythrosin (59-100-200 mg/kg) (Zijno et al., 1994). Erythrosin was negative in the mouse lymphoma assay at concentrations of up to 600 ?g/ml without activation and up to 350 ?g/ml with S9 activation (Lin and Brusik, 1986). It was also negative in the micronuclei test when doses of up to 240 mg/kg were administered i.p. to CD-1 mice (Lin and Brusik, 1986).

Cytotoxicity

The xanthene dye, phloxin (10-4 M), which is almost the same concentration as used in food, produces a slight reduction in cell growth. Erythrosin, which is chemically related to this dye, presents a similar profile (Saks et al., 1980).

Carcinogenicity

In CD-1 mice maintained for 24 months on diets containing 0.3% -1.0% - 3.0% erythrosin (equivalent to a daily dose of 424-1474-4759 mg/kg bw /day in males and 507-1824-5779 mg/kg in females), there was no increase in the incidence of tumours, and survival was not affected.. Weight gain was reduced in both males and females treated with the highest doses and in females of the intermediate-dose group (Borzelleca e Hallaghan, 1987).

Rats raised a diet containing 4% erythrosin (roughly 3800 mg/kg bw) for 30 months beginning in utero have an increased incidence of thyroid adenomas and adenocarcinomas (Jennings et al., 1990).

Erythrosin used at 4% in the basal diet and administered for 19 weeks significantly promoted the development of thyroid tumors in partially thyroidectomized rats given N-bis (2-hydroxypropyl) nitrosamine, but had no significant effects in non-thyroidectomized rats (Hiasa et al., 1988).

When erythrosin was added to the diet of CD rats at doses of 49-251-567-2464 mg/kg/ die for approximately 30 months, males receiving the highest dose used presented increased thyroid weight and higher incidences of follicular hypertrophy, hyperplasia and thyroid adenoma, along with some equivocal evidence for an increased rate of thyroid carcinoma. These changes may have been due to the compound's inhibition, at the peripheral and thyrotroph cell levels, of conversion of thyroxin to triiodothyronine and increased release of TSH with consequent disruption of the pituitary-thyroid axis (Borzelleca et al., 1987; Capen, 1992; Hiasa et al., 1988; Jennings et al., 1990).

Reproductive and developmental toxicity

Erythrosin given to female rats (0.25-1% of the diet) throughout gestation and lactation produced no reductions in parental or offspring weight or food consumption. There was no evidence of developmental or psychotoxicity (Uorhees et al., 1983).

Chronic oral administration of erythrosin (1 mg/kg bw/day) to rat pups during their first month of postnatal life failed to elicit any significant effect on the activity or cognitive performance (Golderning et al., 1981).

In studies on the possible reproductive toxicity of erythrosin (Collins et al., 1993; Borzelleca et al., 1987), CD rats received up to 2464 mg/kg/ die for 2 months prior to mating and throughout gestation and lactation. These studies failed to reveal any adverse effects in terms of the course of gestation or the offspring. Collins et al. (1993a) treated female Osborne Mendel rats with erythrosin added to drinking water (64-121-248-472 mg/kg/ die) or administered by gastric gavage (15-30-300-200-400-800 mg/kg/ die) (Collins et al., 1993b) through the 20th day of pregnancy, and there were no dose-related changes in maternal clinical findings, implantation, fetal size, weight, length, viability, visceral or skeletal development and no sign of dose-related teratogenesis. These investigators (Collins et al., 1993a; 1993b) consider erythrosin neither foetotoxic nor teratogenic at the doses tested. Similar doses have also been reported to be nonteratogenic in rabbits (Burnett et al., 1974).

Immunotoxicity

Erythrosin can increase the release of serotonin by leukaemic basophils in vitro, which means it might increase the intensity of immediate-type allergic reactions (Tanaka et al., 1995). This finding is quite insignificant.

Adverse reactions in humans

Safety problems have never been encountered with E 127 erythrosin. In those cases in which adverse reactions to the product have been reported, including allergic reactions to products containing erythrosin (E 127), it has not been possible to determine whether the reaction was provoked by the colouring agent or the active ingredient of the product (EMEA report, 1998). Erythrosin is considered to be an uncommon cause of clinically severe bronchoconstriction in perennial asthmatics (Weber et al. 1979).

Opinion

Given the quantities of the E 127 erythrosin 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 is regulated by law or in any case limited.

The maximum amounts of erythrosin found in pharmaceutical products currently marketed in Europe are: 0.0017 – 0.96 mg / capsule or tablet or sugar-coated pill, and 0.009-0.8 mg/mL for oral liquid preparations.

The maximum quantity of erythrosin that can be ingested with a single tablet, capsule or sugar-coated pill, or in 1 ml of a liquid preparation, represents a pro Kg dose for a 70-Kg adult male of 0.013 mg / kg.

The pro kg and pro die doses of erythrosin 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 (160 mg/kg bw/day) is ~12,000 times higher than doses which can be ingested with one dosing unit in medicinal products. The ADI for erythrosin is up to 100 ?g/kg (SCF 1987; Martindale Extra Pharmacopoiea 1996), which is 7.6 times higher than the dose that might be consumed with a single capsule, tablet, or pill, or with 1 millilitre of a liquid preparations from one medicinal product. The ADI may be reached only with a daily dose of 5-7 pills, capsules or tablets or 5-7 ml of liquid oral preparations a day.

Erythrosin has been delisted in the United States since 1990, following studies in rats that suggested that is was potentially carcinogenic for the thyroid gland (Borzelleca et al., 1987). This move was a result of the Delaney Clause, which restricts the use of any colourant shown to induce cancer in humans or animals, regardless of the amount (Hogan et al., 1994).

However, erythrosin was not regarded as being an immediate hazard to health. The FDA applied the Delaney clause when it outlawed use of the colourant in cosmetics and externally applied drugs, as well as all uses of the colour's non-water-soluble "lakes". Though the FDA viewed the thyroid-cancer risk associated with erythrosin as small –- about 1 in 100,000 over a 70–year lifetime – the agency was forced to ban provisional listings because of the Delaney directives. At the same time, erythrosin has "permanent" listings for food and drug uses that are still allowed, although the agency has announced plans to propose revoking these uses as well. For now erythrosin can be used in foods and oral medications. Products such as maraschino cherries, bubble gum, baked goods, all sorts of snack food and candy may contain erythrosin. In fact according to information received from the US Mission to the European Union – Commercial Service, FD&C Red No. 3 (E161 Erythrosin) figures in the list of Color Additives Approved for Use in Drugs (part 74, support B: Color additives subject to batch certification 21 CFR Section 74 1303).

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