Following a request from the European Commission, the EFSA Panel on Contaminants in the Food Chain (CONTAM Panel) was asked to deliver a scientific opinion on the risks to human and animal health related to the presence of tropane alkaloids (TA) in food and feed.
TAs are secondary metabolites which naturally occur in plants of several families including Brassicaceae, Solanaceae (e.g. mandrake, henbane, deadly nightshade, Jimson weed) and Erythroxylaceae (including coca). The TAs are found in all parts of the plants and are responsible for the toxic effects of some of these plants. TAs contain an azabicyclo[3.2.1]octane ring structure. The common structural element is the tropane skeleton, (1R,5S)-8-methyl-8-azabicyclo[3.2.1]octane. The group of TAs comprises more than 200 compounds and the wide range of compounds occurring especially in the Solanaceae family arises from the esterification of tropine with a variety of acids, such as acetic acid, propanoic acid, isobutyric acid, isovaleric acid, 2-methylbutyric acid, tiglic acid, (+)-α-hydroxy-β-phenylpropionic acid, tropic acid, and atropic acid.
Although more than 200 different TAs were so far identified in various plants, respective data on their occurrence in food and feed and on toxicity are limited. The most studied TAs are (-)-hyoscyamine and (-)-scopolamine, which in contrast to the (+)-enantiomers are formed naturally. The racemic mixture of (-)-hyoscyamine and (+)-hyoscyamine is called atropine. Cocaine is also a prominent member of the group of TAs. However, since almost no data are available concerning the occurrence of cocaine in food and feed, it was not further considered in this opinion. Besides data on toxicity, some information on occurrence in feed and food were only available for (-)-hyoscyamine and (-)-scopolamine. Therefore, the CONTAM Panel could only perform a risk assessment on these compounds.
Plant extracts containing TAs have been used for centuries in human medicine and are still used, as are atropine, (-)-hyoscyamine and (-)-scopolamine. These uses include for example the treatment of wounds, gout and sleeplessness, and pre-anaesthesia. Furthermore, extracts from deadly nightshade (Atropa belladonna) were used to dilate pupils for cosmetic reasons and to facilitate ophthalmological examination. The genus Datura is long known for its content of TAs. In India, the root and leaves of Datura stramonium, commonly called thorn apple or Jimson weed, were burned and the smoke inhaled to treat asthma. This plant is widely distributed in temperate and tropical regions of the world. For this reason, seeds of this plant have been found as impurities in important agricultural crops such as linseed, soybean, millet, sunflower and buckwheat and products thereof. The consumption of a few berries from henbane (Hyoscyamus niger) or from Atropa belladonna has caused severe intoxication, including deaths in young children.
Atropine/(-)-hyoscyamine and (-)-scopolamine are readily absorbed from the gastrointestinal tract, quickly and extensively distributed into tissues, and excreted predominantly in the urine. N-demethylation and Phase II conjugation of atropine, (-)-hyoscyamine and (-)-scopolamine are known metabolic pathways in humans. The toxicological effects of (-)-hyoscyamine and (-)-scopolamine relate to their pharmacological effects, which are mediated by inhibition of muscarinic acetylcholine receptors in the central nervous system (CNS) and the autonomic nervous system (ANS). Inhibition of these receptors in the ANS results in decreased secretions from the salivary, bronchial and sweat glands, dilation of the pupils, paralysis of accommodation, change in heart rate, inhibition of micturition, reduction in gastrointestinal tone and inhibition of gastric acid secretion. (-)-Hyoscyamine and (-)-scopolamine differ in their ability to affect the CNS, with (-)-scopolamine having more prominent depressing central effects at therapeutic doses. The pharmacological effects of (-)-hyoscyamine and (-)-scopolamine occur within a short time after administration, and therefore the CONTAM Panel concluded that it was appropriate to establish an Acute Reference Dose (ARfD) for these substances. Since they are not bioaccumulative, or genotoxic and do not exhibit chronic toxicity, the ARfD would also protect against effects of long-term exposure. Due to the common mode of action through receptor interaction, the CONTAM Panel considered it appropriate to establish a group ARfD for (-)-hyoscyamine and (-)-scopolamine assuming equivalent potency.
From the results of a study in which human volunteers were given a relevant mixture of (-)-hyoscyamine and (-)-scopolamine in food, the CONTAM Panel identified a no–observed-adverseeffect level (NOAEL) of 0.16 μg/kg body weight (b.w.), expressed as the sum of (-)-hyoscyamine and (-)-scopolamine as the basis for establishing a group ARfD. The Panel noted that the next higher dose in the human volunteer study of 0.48 μg/kg b.w. resulted in a transient statistically significant lowering of the heart rate, which is not adverse in healthy individuals but could be in more susceptible individuals, such as those with bradycardia. The Panel decided to apply an uncertainty factor of 10 for interindividual differences to allow for the fact that this was a small study in young healthy male volunteers. The Panel divided the NOAEL of 0.16 μg/kg b.w. by the uncertainty factor of 10 and established a group ARfD of 0.016 μg/kg b.w. expressed as the sum of (-)-hyoscyamine and (-)-scopolamine, assuming equivalent potency. The group ARfD is approximately two orders of magnitude lower than the lowest single doses of (-)-hyoscyamine and (-)-scopolamine used therapeutically.
Currently, only methods with mass spectrometric (MS) detection allow analysis of TAs at trace levels in food and feed. Basically, two MS based approaches are applied, either in combination with gas chromatography (GC) or predominantly with high performance liquid chromatography (HPLC).
Following a continuous call by EFSA in July 2010 for data in food and feed on a list of chemical contaminants, including plant toxins such as TAs, analytical data on TAs in 124 food samples and 611 feed samples were available in the EFSA database by the end of February 2013. The samples were collected in two Member States (the Netherlands and Germany) and all analysed and reported by the Netherlands. The data refer to atropine and (-)-scopolamine. When atropine was reported, the CONTAM Panel used these data as (-)-hyoscyamine. As the biosynthesis of TAs leads to (-)-hyoscyamine and (-)-scopolamine, any analytical results where no stereoselective separation is achieved are thus regarded in this opinion as 100 % (-)-hyoscyamine or (-)-scopolamine. Most of the food samples (83 %) were left-censored (below limit of detection/limit of quantification). Almost all food data with quantified TA concentrations belonged to the food category for infants and young children ―Simple cereals that are or have to be reconstituted with milk or other appropriate nutritious liquids‖. The ingredients in these samples included wheat, maize, rye, oats and rice, indicating the possibility of contamination of different cereals. Risk characterisation was only possible for the toddlers‘ age class because a reliable exposure assessment was not possible for other age classes.
For comparison with the ARfD, it is necessary to consider estimates of acute exposure. The estimates of dietary exposure were based on the two available dietary surveys for toddlers reporting consumption of the selected food group, which are from Germany and Finland, and not necessarily representative of all European countries. Although the data represent only the food group for infants and young children ‗Simple cereals which are or have to be reconstituted with milk or other appropriate nutritious liquids‘, these are the foods for toddlers most likely to contain TAs, and taking into account that other food samples did not contain detectable concentrations, the total exposure from all food sources is unlikely to be much higher.
The CONTAM Panel performed estimates of acute dietary exposure using both a deterministic and a probabilistic approach. Based on the limited available information, the dietary exposure of toddler consumers could be up to seven times the group ARfD (deterministic approach), and exceeded the ARfD in approximately 11 to 18 % of the consumption days (probabilistic approach).
Most feed data (91 %) were left-censored. More than half of the quantified samples were reported for compound feed. The highest levels of TAs were reported in samples of millet grains. Plants containing TAs are generally unpalatable, and will be avoided by most livestock unless other feed is unavailable. Therefore, animal exposure to the sum of (-)-hyoscyamine and (-)-scopolamine is primarily from consuming feed contaminated with TA-containing plant material. Except for rabbits with an estimated upper bound (UB) exposure of 2.5 μg/kg b.w., the estimated UB exposures for lactating and fattening ruminants, piglets, fattening pigs, sows, poultry, cats, dogs, horses and fish were all below 0.35 μg/kg b.w.
TA toxicosis in livestock and companion animals is relatively rare because TA-containing plant products appear to be unpalatable and animals try to avoid them where possible. Furthermore, compared to other livestock, poultry, rabbits and certain breeds of small ruminants are considerably less sensitive to TAs due to the expression of specific hydrolysing enzymes that inactivate the alkaloids. A NOAEL has been proposed for ruminants and a lowest-observed-adverse-effect level (LOAEL) for pigs, but these are significantly higher than estimated exposure.
The European Agency for the Evaluation of Medicinal Products (EMEA; now the European Medicines Agency (EMA)) and the European Food Safety Authority (EFSA) concluded in their evaluations in 1997 and 2008 respectively, that residues of TAs in edible tissues (milk, meat or eggs) were unlikely to constitute a risk for consumers following the legal use of Atropa belladonna and atropine as authorised veterinary medicines and no information has subsequently been published to alter these conclusions.
The CONTAM Panel recommended to better characterise TAs occurring in food and feed either naturally or as contaminants and that analytical data on the occurrence of TAs in cereals and oilseeds should be collected, including TAs not considered in this opinion occurring in food and feed commodities. Moreover, there is a need for: i) investigations into the agricultural conditions under which TAs occur in cereals and oilseeds; ii) defined performance criteria for the analysis of TAs in food and feed; iii) for certified reference materials containing TAs at levels of interest as well as for proficiency tests; iv) information on stability of TAs during food and feed processing and the identity and toxicity of potential degradation products; v) data on relative potency of (-)-hyoscyamine and (-)-scopolamine and for data on endogenous formation of (+)-hyoscyamine and its biological relevance; vi) toxicity data for TAs which are relevant in food and feed commodities other than those covered in this opinion.