Adopted on 25 January 2007

Transcription

1 The EFSA Journal (2007) 447, 1-51 OPINION OF THE SCIENTIFIC PANEL ON CONTAMINANTS IN THE FOOD CHAIN ON A REQUEST FROM THE EUROPEAN COMMISSION RELATED TO PYRROLIZIDINE ALKALOIDS AS UNDESIRABLE SUBSTANCES IN ANIMAL FEED (Question N EFSA-Q ) Adopted on 25 January 2007 SUMMARY The term pyrrolizidine alkaloids (PAs) describes a group of more than 350 individual compounds that share as a basic structure one of the four necine bases platynecine, retronecine, heliotridine, or otonecine. PAs are produced as secondary metabolites of more than 6000 plant species, belonging to the families of Boraginaceae, Compositae (Asteraceae) and Leguminosae (Fabaceae) and occur world-wide. The pattern of PAs in plants varies largely, depending on the plant variety, climatic conditions, period of sampling and part of the plant analysed. Basic alkaloids seem to accumulate in the seeds, whereas the respective N-oxides dominate in the green parts of a plant. It is assumed that PAs are among the most widely distributed natural toxins affecting wildlife and livestock. In farm animals, however, acute intoxications caused by PAs are rare, as animals avoid PA containing plants if other feed is available. However, this recognition fails in preserved forages such as silage and hay. Acute intoxications caused by PAs are characterized by hepatotoxicity and hemorrhagic liver necrosis. Long-term exposure causes hepatic megalocytosis, veno-occlusion in liver and to a lesser extent in the lungs, proliferation of the biliary tract epithelium, fatty liver degeneration and liver cirrhosis. European Food Safety Authority, 2007 page 1 of 51

2 The onset of clinical symptoms often occurs with delay, and exposure is recognized only in cases in which significant alteration in the liver occur. The progressive hepatotoxicity is related to the metabolic activation of the parent alkaloid into toxic dehydropyrrole alkaloids that are highly reactive alkylating agents. In contrast, the transformation into N-oxides represents a common detoxification pathway. Structural characteristics such as the degree of esterification and the nature of the ester groups determine the degree of bioactivation towards the toxic pyrroles, and differences in the expression of enzymes involved in the biotransformation seem to explain the typical species differences in the sensitivity toward PAs. At present, the data available for farm animal species do not allow tolerance levels to be set for individual PAs in feed materials. In humans, PAs cause primarily hepatic veno-occlusive disease (VOD). Although VOD was endemic in the past century in certain geographic regions of South America, epidemiological evidence for PA-induced cancers in humans is lacking. Toxicological concerns about the potential human exposure to PAs were based on the results of extensive rodent studies indicating a carcinogenic potential of this class of compounds, and on the in vitro investigations that convincingly demonstrated that the dehydropyrrolic metabolites of PAs can form DNA-adducts, DNA-cross links and DNA-protein cross links, and result in genotoxicitiy and mutagenicity in a variety of bioassays conducted in rodent models. Studies devoted to the carry over of PAs from feed into edible tissues of farm animals did show that PAs are excreted with milk of dairy cows (and lactating sheep) albeit at a low rate, varying between 0.04 and 0.08 % of the ingested dose. Experimental evidence for the rate of disposition of PAs in eggs is lacking, but market analyses in Australia indicated the presence of certain PAs in eggs. No residues have been found in other animal tissues. The contribution of the residues in animal derived tissues to human exposure is low; however, honey, in which PA residues are regularly found, deserves special attention. KEYWORDS: Pyrrolizidine alkaloids, Senecio alkaloids, animal feed, toxicity, analysis, occurrence, animal health, intoxications, carry-over, human health. Page 2 of 51

3 TABLE OF CONTENTS BACKGROUND General background Specific background...5 TERMS OF REFERENCE...6 ASSESSMENT Introduction Chemistry of pyrrolizidine alkaloids Methods of analysis Extraction and analysis of plant materials Analysis of biological samples Statutory limits for pyrrolizidine alkaloids in feed materials Occurrence of pyrrolizidine alkaloids in feed materials Estimating the intake by farm livestock Toxicity of pyrrolizidine alkaloids Mechanisms involved in the toxicity of pyrrolizidine alkaloids Mutagenicity, genotoxicity, and carcinogenicity Adverse effects on livestock Adverse effects in pigs Adverse effects in poultry Adverse effects in ruminants Adverse effects in horses Adverse effects in other animal species Toxicokinetics, metabolism and tissue distribution Absorption Metabolism Distribution and elimination Carry-over and residues Human dietary exposure...25 CONCLUSIONS...27 RECOMMENDATIONS...28 REFERENCES...30 SCIENTIFIC PANEL MEMBERS...38 ACKNOWLEDGEMENT...38 ANNEX Page 3 of 51

4 BACKGROUND 1. General background Directive 2002/32/EC of the European Parliament and of the Council of 7 May 2002 on undesirable substances in animal feed 1 replaces since 1 August 2003 Council Directive 1999/29/EC of 22 April 1999 on the undesirable substances and products in animal nutrition 2. The main modifications can be summarised as follows: - extension of the scope of the Directive to include the possibility of establishing maximum limits for undesirable substances in feed additives. - deletion of the existing possibility to dilute contaminated feed materials instead of decontamination or destruction (introduction of the principle of non-dilution). - deletion of the possibility for derogation of the maximum limits for particular local reasons. - introduction the possibility of the establishment of an action threshold triggering an investigation to identify the source of contamination ( early warning system ) and to take measures to reduce or eliminate the contamination ( pro-active approach ). In particular the introduction of the principle of non-dilution is an important and far-reaching measure. In order to protect public and animal health, it is important that the overall contamination of the food and feed chain is reduced to a level as low as reasonably achievable providing a high level of public and animal health protection. The deletion of the possibility of dilution is a powerful means to stimulate all operators throughout the chain to apply the necessary prevention measures to avoid contamination as much as possible. The prohibition of dilution accompanied with the necessary control measures will effectively contribute to safer feed. During the discussions in view of the adoption of Directive 2002/32/EC the Commission made the commitment to review the provisions laid down in Annex I on the basis of updated scientific risk assessments and taking into account the prohibition of any dilution of contaminated noncomplying products intended for animal feed. The Commission has therefore requested the Scientific Committee on Animal Nutrition (SCAN) in March 2001 to provide these updated 1 OJ L140, , p OJ L 115, , p Page 4 of 51

5 scientific risk assessments in order to enable the Commission to finalise this review as soon as possible (Question 121 on undesirable substances in feed) 3. The opinion on undesirable substances in feed, adopted by SCAN on 20 February 2003 and updated on 25 April provides a comprehensive overview on the possible risks for animal and public health as the consequence of the presence of undesirable substances in animal feed. It was nevertheless acknowledged by SCAN itself and by the Standing Committee on the Food Chain and Animal Health that for several undesirable substances additional detailed risk assessments are necessary to enable a complete review of the provisions in the Annex. 2. Specific background Many toxicants are alkaloids. However alkaloids represent structurally-diverse groups of plant secondary metabolites. SCAN 5 indicated that the more toxic alkaloids occurring as potential contaminants are limited to a few specific groups such as the tropane or pyrrolizidine alkaloids (PAs) for which established analytical schemes exist and for which statutory limits could be developed. Tropane alkaloids are the subject of a separate request to the European Food Safety Authority (EFSA) for scientific opinion. PAs are not listed as such as undesirable substance in the Annex of Directive 2002/32/EC. However several plant species or specific parts of these plant species, containing PAs, are explicitly listed as undesirable substance such as Crotalaria spp., Lolium temulentum, and Lolium remotum. SCAN concluded 6 that risk assessments could be made for some of the compounds presumed responsible for their toxicity and consequently maximum limits for botanical contaminants of particular concern should be set on the basis of their known toxicants. The ease of microscopic detection of botanical contaminants is inversely related to the degree of processing, particularly contamination, of feedingstuffs. Therefore SCAN recommended that it 3 Summary record of the 135 th SCAN Plenary meeting, Brussels, March 2001, point 8 New questions ( 4 Opinion of the Scientific Committee on Animal Nutrition on Undesirable Substances in Feed, adopted on 20 February 2003, updated on 25 April 2003 ( 5 Opinion of the Scientific Committee on Animal Nutrition on Undesirable Substances in Feed, point 9.3. Relationship of botanical contaminants with the natural plant products listed in the annex.. 6 Opinion of the Scientific Committee on Animal Nutrition on Undesirable Substances in Feed, point 9.5. Conclusion and point 9.6 Recommendations. Page 5 of 51

6 would be advantageous if the physical detection of the presence of a specific PA could be supported or replaced by a quantitative chemical analysis of the specific compound(s) presumed responsible for their toxicity and maximum limits set accordingly based on a risk assessment of the toxic compound. TERMS OF REFERENCE In accordance with Article 29 (1) (a) of Regulation (EC) No 178/2002 the European Commission asks the European Food Safety Authority to provide a scientific opinion on the presence of PAs and on botanical impurities as sources of pyrrolizidine alkaloids in animal feed. This scientific opinion should determine whether the PAs should be assessed as a group or individually where relevant for animal or public health determine the toxic daily exposure levels of the PAs (as a group or for relevant individual alkaloids) for the different animal species of relevance (difference in sensitivity between animal species) above which signs of toxicity can be observed (impact on animal health) the level of transfer/carry over of these undesirable substances from the feed to the products of animal origin results in unacceptable levels of these undesirable substances or possibly their toxic metabolites in the products of animal origin in view of providing a high level of public health protection. confirm that pyrrolizidine alkaloids are the substances responsible for the toxicity of Crotalaria spp., Lolium temulentum and Lolium remotum in animal feed and identify other botanical impurities which could possibly contribute significantly to the presence of PAs in animal feed. The relative importance of all identified botanical impurities should be determined. identify feed materials which could be considered as sources of contamination by these undesirable substances (PAs or botanical impurities as sources of PAs) and the characterisation, insofar as possible, of the distribution of levels of contamination. assess the contribution of the different identified feed materials as sources of contamination by these undesirable substances. to the overall exposure of the different relevant animal species to these undesirable substances, to the impact on animal health, Page 6 of 51

7 insofar relevant, to the contamination of food of animal origin (the impact on public health), taking into account dietary variations and carry over rates. identify possible gaps in the available data which need to be filled in order to complete the evaluation. ASSESSMENT 1. Introduction The term alkaloid was introduced by W. Meisner at the beginning of the nineteenth century to designate natural substances reacting like bases, in other words like alkalis (from the Arabic al kaly, soda, and from the Greek eidos, appearance). Alkaloids are very common plant metabolites and are grouped according to typical structural characteristics, resulting in large families of molecules as for example tropane alkaloids and PAs. PAs, formerly also called Senecio alkaloids, are widely distributed among plant species, and it is assumed that more than 6000 plant species belonging to the families of Boraginaceae, Compositae (Asteraceae) and Leguminosae (Fabaceae) contain PAs at different levels and in different patterns. In turn it has been estimated that about 3 % of all flowering plants contain one or more of the more than 350 toxic PAs. A typical example is sunn hemp (Crotalaria juncea L) being the most widely grown legume in the tropics where it is used as green manure, animal fodder crop and fibre source. This plant contains the alkaloids junceine and trichodesmine, albeit at rather low concentrations (Ji et al., 2005). In addition, animals are exposed to various toxic PAs following the ingestion of toxinogenic plants, or PA containing seeds that contaminated the grains used in their diet. Hence it appears that PAs are among the most widely distributed natural toxins affecting wildlife and livestock (Röder, 2000). Human exposure originates from PA containing herbs, teas and dietary supplements. Various herbal medicines and dietary supplements containing comfrey (Symphytum officinale) have been widely used for more than 200 years in traditional medicine, and it was only in 2001 that the US- FDA requested all manufacturers to withdraw these products from the market (with the exception of products intended for external use) 7. Moreover, more that 50 different plants that have been 7 a comprehensive review of data related to comfrey is available under Page 7 of 51

8 used for centuries in the traditional Chinese medicine are found to contain significant amounts of PAs and hence may cause liver injury (Yu and Li, 2005). Acute intoxications caused by PAs are characterized by hepatotoxicity and hemorrhagic liver necrosis. Long-term exposure causes hepatic megalocytosis, veno-occlusion in liver and to a lesser extent in the lungs, proliferation of the biliary tract epithelium, fatty liver degeneration and liver cirrhosis. Moreover, pyrrolic metabolites of PAs can form DNA-adducts and DNA-cross links and DNA-protein cross links, and many PAs are genotoxic and carcinogenic in rodents. In humans, PAs cause primarily hepatic veno-occlusive disease (VOD), also referred to as sinusoidal obstruction syndrome (SOS). As yet, there is no epidemiological evidence for PA-induced cancers in humans, despite the endemic occurrence of VOD in certain geographic regions in the last century. The toxicity and human risk associated with the ingestion of PA containing products have been previously summarized by the WHO-IPCS (1988). More recently the Australian New Zealand Food Authority presented a technical report on PAs in Foods (ANZFA, 2001). In this assessment it is stated that there is accumulating epidemiological evidence for the involvement of PAs 8 in the pathogenesis of hepatocellular injury, liver cirrhosis and VOD, but evidence for PA-induced cancers is lacking in humans, as mentioned above. Therefore, VOD was considered to be the most sensitive toxicological endpoint in humans and a NOEL for VOD of 10 µg/kg b.w. has been derived from the available epidemiological data. Subsequently the Australian Food Safety Authority proposed a PTDI of 1 µg/kg b.w. per day Chemistry of pyrrolizidine alkaloids PAs are heterocyclic compounds and most of them are derived from four necine bases: platynecine, retronecine, heliotridine and otonecine. Retronecine and heliotridine are enantiomers at the C7 position. Most of the naturally occurring PAs in plants are esterified necines or alkaloid N-oxides (except for the otonecine-type alkaloids), whereas unesterified PAs hardly occur in plants (for a comprehensive overview see Mattocks, 1986). The esters can be divided in monoesters, non-macrocyclic diesters and macrocyclic diesters of a necine base. Figure 1 shows the basic structure of the four necine bases forming toxic PAs. 8 The assessment refers to Heliotropium europaeum, Echium plantagineum, Symphytum spp. and Crotalaria retusa Page 8 of 51

9 a. b. c. d. Figure 1. Basic structure of the four necine bases forming toxic PAs; a. platynecine, b. retronecine, c. heliotridine, d. otonecine. Hepatotoxic PAs have an unsaturated necine base (with a 1,2-double bond) in their structure, whereas in the non-hepatotoxic PAs the necine base is saturated. Other structural requirements for toxicity are according to the WHO (1988): the presence of a 1-hydroxylmethoxylpyrrolizidine ring system unsaturated in the 1:2 position, with preferably a second hydroxyl group in the 7- position. At least one of the hydroxyls needs to be esterified, but toxicity increases if both hydroxyls are esterified. One ester group should contain a branched chain in its acid moiety that is resistant to metabolism. It has been shown that monoesters are the least toxic group, nonmacrocyclic diesters have an intermediate toxicity, being about four times as toxic as their respective monoesters, and macrocyclic diesters are the most toxic compounds within the group of PAs (Cheeke, 1988). The degree of toxicity is to a large extent associated with the hepatic metabolism of individual PAs, as outlined below. Examples of the different PA types are given in Table A1 of the Annex. Most plants produce mixtures of PAs in varying concentrations ranging from less than % to 5 % in certain plant seeds (examples are presented in Table A2 of the Annex). 2. Methods of analysis PAs have been analysed both in plant material and feed materials, as well as in biological fluids (blood, urine, milk, honey) and tissues in which the parent compounds as well as the major metabolites can be detected (Mattocks, 1986; WHO-IPCS, 1988) Extraction and analysis of plant materials Most methods of extraction are based on the method originally described by Koekemoer and Warren (1951) in which plant material is extracted in cold or hot alcohol (methanol or ethanol). After evaporation of the alcohol, the alkaloids are dissolved in diluted aqueous acids to which zinc to reduce the N-oxides is added. This step might be omitted in the analysis of seeds that seldom Page 9 of 51

10 contain PA N-oxides. The zinc is removed by filtration and the ph adjusted before the alkaloids are extracted using organic solvents, such as chloroform, acetonitrile and butan-1-ol (N-oxides). Certain pyrrolizidine amino alcohols such as retronecine and heliotridine are too polar to be extracted with organic solvents and should be extracted with saturated potassium carbonate solutions. The further sample clean-up may include a passage over florisil (magnesium silicate) columns that retain alkaloids which subsequently can be eluted using methanol, with or without added ammonia. Other authors described the use of preparative TLC in silica plates prior to the application of chromatographic techniques such as gas chromatography (GC) or high-pressure liquid chromatography (HPLC). More recently, cation-exchange solid-phase extraction using LiChrolut SCX cartridges filled with polymeric strong cation-exchangers have been found to be very effective and to improve the sensitivity of HPLC analysis towards detection limits of 0.06 µg/ml and 0.2 µg/ml for senecionine and senkirkine, respectively. The first quantitative HPLC analysis was developed for PAs from Senecio species, using a Bondapak CN column and a mixture of tetrahydrofuran (THF) and 0.01 M ammoniumcarbonate adjusted to ph 7.8 as mobile phase (Qualls and Segall, 1978). Subsequently, reverse phase systems (C18 columns) were introduced and solvent mixtures containing methanol and 0.01 M potassium phosphate buffers at ph 6.3 were introduced, but later replaced by acetonitril in 0.1 M ammonium acetate solution used under heterocratic (gradient) conditions. At present, the combination of HPLC with mass-spectrometry is the most frequently applied method (Colegate et al., 2005). For example, Mroczek et al. (2004) recently described a procedure comprising gradient HPLC with diode array and thermobeam electron impact mass spectrometry (EI/MS) for the analysis of dried plant material from nine different plant species, known to contain PAs. While this method elegantly provides insight into the PA composition in individual plants, the limit of detection (for minor PAs) needs to be defined. GC and GC-MS with fused silica columns have been considered for a long time as the most sensitive methods of detection. However, this method is not suitable for the detection of N-oxides as the latter are unstable under GC conditions (Mroczek et al., 2002). Hence, a reduction of the N- oxides prior to analysis is required in the application of these GC methods resulting in the necessity of multiple analyses of the same sample (Beales et al., 2004). In contrast, HPLC/EI/MS allows the simultaneous analysis of PAs and their N-oxides without a need for prior reduction or derivatisation steps. The limit of detection for full scan HPLC/EI/MS was determined to remain above 2 ng on column, whilst with the application of HPLC/EI/MS/MS a ten-fold lower sensitivity with a limit of detection of 0.2 ng on column can be achieved, which is comparable to the sensitivity of GC methods (Than et al., 2005). Specifically for the analysis of plants used in traditional Chinese medicine, a dynamic ph junction sweeping Capillary Electrophoresis for online pre-concentration of PAs has been developed, Page 10 of 51

11 which enhanced the limit of detection by factors between 20 and 90-fold, hence reaching a sensitivity of about 30 ppb (LOD) (Yu and Li, 2005). Capillary Electrophoresis has been used also for the isolation of different PAs from plant materials (Yu et al., 2005). In particular, micellar electrokinetic chromatography (MEKC) has been found to be very effective in the isolation of different PAs. In the past, Ehrlich reagent had been used as a screening method for the detection of unsaturated PAs and their N-oxides (Mattocks, 1986). Toxic PAs that have an unsaturated basic moiety can be oxidized to the respective N-oxide, which subsequently in the presence of acetic anhydride (or acetyl chlorine) is converted to the dehydrogenated pyrrole that reacts with the Ehrlich reagent (4- dimethyl-aminobenzaldehyde). This colour reaction can be combined with spectrophotometric methods for quantification. The detection of separated alkaloids occurs normally with UV light ( nm), but this method is limited to PAs having a chromophore in their structure. Additionally, ELSD (evaporative light scattering detector) can be used to quantify for example heliotrine, which is not detectable under UV light (Schaneberg et al., 2004). At present, immunological detection methods such as ELISA techniques, based on polyclonal antibodies raised against one group of closely related PAs have been developed as rapid, and costsaving screening methods. For example, the Australian CSIRO Plant Toxin Research Group developed 2 separate antisera against typical Echium and Heliotropium alkaloids as well as a general antiserum that reacts with a large number of PAs including those of Senecio and Crotalaria species. These antisera have been applied in ELISA kits and tested for their suitability in the analysis of feed samples as they reach a sensitivity of approximately 10 pg/well (Cavallaro et al., 2004; Than et al., 2005). However, all developed antisera are unable to detect N-oxides of PAs and (like GC methods) require an additional reduction step prior to analysis that may impair the accuracy in the detection of other PAs. In conclusion, various analytical techniques, particularly chromatographic methods in conjunction with mass spectrometry can be used to detect PAs in plants or plant derived products (Altamirano et al., 2005; Colegate et al., 2005; Ji et al., 2005). None of these methods, however, has been validated for the analysis of (mixed) feed samples Analysis of biological samples Analyses of biological samples, such as milk, eggs and animal tissues, have to address the parent molecules, and the species-specific metabolites originating from hepatic metabolism of the animal. For the detection of PAs in milk, Goeger et al. used gas chromatography followed by mass spectrometry (Goeger et al., 1982; Deinzer et al., 1982). Methods for the analysis of PAs from blood plasma have been described by Jago et al. (1969) and McComish et al. (1980). Mattocks (1986), Jago et al. (1969) and Evans et al. (1979) describe the extraction and analysis of PAs from urine and bile fluid (Jago et al., 1969; Lafranconi et al. 1985). None of the above Page 11 of 51

12 described advanced methods of detection have been validated for its suitability to analyse these biological samples as yet. For the extraction and isolation of PAs from honey, a method of solid-phase extraction and liquid chromatography-mass spectrometry is described (Beales et al., 2004). An improvement of this method is described by Betteridge et al. (2005), but the accuracy of these methods is still impaired by a rather low extractability (recovery rate) for PAs present in honey. 3. Statutory limits for pyrrolizidine alkaloids in feed materials The current EU maximum levels for PAs in feed materials are given in Tableo1. It should be noted that these data refer to weed seeds or uncrushed feeds, which can be detected by microscopic examination, but do neither differentiate between individual plants, with the exception of the three mentioned species, nor provide limits for the amount of individual or groups of PAs. Table 1. EU legislation on PA containing plant materials used as feed. Undesirable substances (or plants) Weed seeds and unground and uncrushed fruits containing alkaloids, glucosides or other toxic substances separately or in combination including Product intended for animal feed (a) Lolium temulentum L., 1000 (b) Lolium remotum Schrank, 1000 Crotalaria spp. All feedingstuffs Maximum content in mg/kg relative to a feedingstuff with a moisture content of 12 % The inclusion of Lolium species in this regulation remains unclear. Both mentioned Lolium species are seldom found in Europe, and are not used as pasture grasses, in contrast to other Lolium species, such as Lolium perenne, which is regularly used in grass mixtures. Moreover, Lolium species have not been reported to be major PA producers. Lolium temulentum (Darnel ryegrass) is known to produce Corynetoxin, a toxic glycopeptide, not belonging to the group of PAs. Lolium temulentum might also contain small amounts of lolin and lolinin. Recent Page 12 of 51

13 evidence suggests that these compounds do not originate from plant metabolism, but are associated with the invasion of the grasses by endophytes (Neotyphodium spp) that also account for the production of lolitrem B, a neurotoxic mycotoxin (for review see Fink-Gremmels, 2005). Hence it is recommended to omit these plant names under this heading in the forthcoming regulations. 4. Occurrence of pyrrolizidine alkaloids in feed materials Plants containing PAs are common throughout the world. The major PA-containing plants of importance in Europe are Cynoglossum spp., Echium spp., Heliotropium spp. (e.g., common heliotrope), and Senecio spp. (e.g., ragwort, groundsel). These plants are usually unpalatable to livestock and are therefore avoided when better quality grazing is available. Hence, most cases of PA toxicosis with fresh plant material occur when pastures are overgrazed and/or there is a limited supply of green forage. Model experiments indicated that the PA concentrations in plants do not decrease significantly during common feed preservation methods, implying that toxicity can result also from contaminated hay, silage (although some reports claim a reduction of the PA content in silage), or grains and hence may occur at any time of the year (Talcott, 2003). The main risk of exposure therefore results from feeds contaminated with weeds, either as the whole plant or as seeds. The weeds responsible for the known outbreaks were Heliotropium, Trichodesma, Senecio, and Crotalaria species, and mortality in such outbreaks has been reported to be high. 5. Estimating the intake by farm livestock The pattern of PAs in plants varies largely, depending on the plant variety, climatic conditions, period of sampling (season) and part of the plant analysed. For example, in Crotalaria retusa, basic alkaloids are accumulating in the seeds, whereas the respective N-oxides dominate in the green parts of the plant. Moreover, the ratio between free bases and N-oxides may be altered during analysis, depending on the extraction conditions and due to the fact that many alkaloids can be present also in the acetylated from, escaping organic solvent extraction procedures (Colegate et al., 2005). During fodder preservation, the alkaloid content can also change. Bull et al. (1968) reported in some cases -but not in all- a loss of total alkaloid content by % in Crotalaria and Heliotopium species. Pedersen (1975) observed a change in the ratio of free necine bases to N- Page 13 of 51

14 oxides during drying of leaves from Boraginaceae, suggesting that enzymatic oxidation or reduction can take place during the post-harvest stage. Candrian et al. (1984) describe that PAs from Senecio alpinus are stable during drying (i.e. in hay) but are rapidly decreasing in silage. In addition, it needs to be considered that for the majority of non-ruminant livestock (pigs and poultry as well as farmed fish) in the EU, feed is provided as compounded feed, consisting of a mixture of individual feed components, to which additives and/or mineral supplements are added. As various ingredients of such a diet can be incidentally contaminated, the average exposure rate cannot be calculated exactly. For cattle, goats and sheep, the daily ration usually consists of forage (or mixture of forages), either fresh or conserved, together with complementary feeds or individual feed materials necessary to achieve the required level of production (growth rate, milk yield). Exposure may result from the forage fraction (local produce) or from contamination of concentrates with seeds of PA producing plants. In conclusion, the exact level of exposure of livestock cannot be estimated due to the high variability in the content of alkaloids in plants, as well as the variability in animal diets. Moreover, systematic analyses of feed materials have not been conducted in the EU Member States as yet. It should be reiterated, however, that due to the increasing awareness of the toxicity of certain plant species (like Senecio), the increasing weed control on farms with intensive animal production, and the current techniques applied in the post-harvest cleaning of grains, the risk livestock seems to be effectively reduced. This is reflected by a continuing decrease in the number of cases of intoxications reported worldwide. In contrast, the re-introduction of certain plant species in nature reserves (in Europe) may increase the incidence of accidental intoxications in wildlife and grazing animals like horses and cattle. 6. Toxicity of pyrrolizidine alkaloids As mentioned already in the introduction, there are some hundreds of naturally occurring PAs and a comprehensive list of individual PAs grouped according to the producing plant species is presented in the WHO report (WHO-IPCS, 1988, Appendix I). As an indication of the differences in the toxicity of individual PAs, the available LD 50 -values in the rat after intraperitoneal injection are listed in Table 2. Page 14 of 51

15 Table 2. LD 50 -values of several PAs after intraperitoneal injection (WHO-IPCS, 1988). PA Retrorsine 34 Senecionine 50 Seneciphylline 77 Lasiocarpine 77 Symphytine 130 Monocrotaline 175 Senkirkine 220 Retrorsine N-oxide 250 Lasiocarpine N-oxide 547 LD 50 (mg/kg) In recognition of the potential carcinogenicity more than 50 plant extracts of purified alkaloids have been tested in rodent bioassays for their potential to induce tumours (see NTP Technical Program). In contrast, studies on the mechanisms involved in the toxicity, including metabolic conversion (biotransformation) have been conducted only for a few compounds, including monocrotaline, lasiocarpine, ridelliine and heliotropine Mechanisms involved in the toxicity of pyrrolizidine alkaloids The parent alkaloids are chemically non-reactive. Following ingestion, PAs undergo hepatic metabolism, as explained in detail in Chapter 8. The critical step is the formation of the reactive bifunctional pyrrolic derivate, the 6,7-dihydro-7-hydroxy-1-hydroxy-methyl-5H pyrrolizidine (DHP). DHP reacts with cellular macromolecules including proteins and DNA and accounts for organ- and cell specific toxicity, as well as for the genotoxiciy and mutagenicity as it is able to form DNA-adducts, DNA cross links and DNA-protein cross links (for review see Prakash et al., 1999; Fu et al., 2002). The pyrrolic metabolites play a key role in liver toxicity, which is characterized by megalocytosis of parenchymal cells, followed by necrosis and finally liver cirrhosis. The acute hepatotoxicity in rats is characterized by a loss of central venous and sinusoidal endothelial cells, dilated and congested sinusoids, and finally centrilobular parenchymal cell necrosis (Hanumegowda et al., 2003). The progression of acute PA-intoxications is described by Copple et al. (2002, 2003): in the early acute phase, different cell populations including sinusoidal endothelial cells (SECs), central venular endothelial cells (CVECs) and hepatic parenchymal cells (HPCs) are injured. Damage to SECs results in microvasculary changes. These changes precede parenchymal cell Page 15 of 51

16 injury (coagulative hepatocellular necrosis). Therefore it was proposed that damage to SECs leads to ischemic parenchymal injury. Studies devoted to the mechanisms resulting in hepatic parenchymal cell death after PA-exposure revealed extensive apoptosis, for example following exposure of hepatocytes to retrorsine and monocrotaline (Gordon et al., 2000; Copple et al., 2004; Picard et al., 2004). In vivo, however, a competition between apoptosis (panlobular) and oncosis (centrilobular) takes place, depending on the composition of PAs in a given plant and the concentration ingested (Copple et al., 2004). Chronic intoxications result in megalocytosis of hepatocytes, sometimes accompanied by bile-duct proliferation, fibrosis and vascular damage (Picard et al., 2003) and ultimately in tumour formation in rodents (WHO-IPCS, 1988). Secondary to liver toxicity, which prevails in all clinical cases, pneumotoxicity (pulmonary hypertensions) and cardiac right ventricular hypertrophy have been described, as well as a secondary neurotoxic syndrome related to hyperammonaemia resulting from acute liver failure (Mattocks and Driver, 1983; Mattocks, 1986; WHO-IPCS, 1988). Trichoderma alkaloids may induce also direct neurotoxicity in the form of hind limb paresis and convulsions (Ismailov et al., 1970). Renal toxicity is characterized by glomerular necrosis and degenerative changes in the epithelial and mesangial cells, and tubular megalocytosis. These alteration are generally seen only following very high (experimental) exposure rates, or in cases of fatal acute intoxication (Hooper, 1974). The teratogenic potential of various PAs has been investigated in a few experimental studies only. It is assumed that certain PAs and/or their metabolites pass the placental barrier. Teratogenic and foetotoxic effects were observed only in rodents after high (experimental) doses, and have been incidentally reported in cases of acute poisoning in farm animals (Mattocks, 1986). In humans, hepatic VOD, characterized by epigastric pain and ascites, prevails. Endothelial cell damage leads to activation of the coagulation cascade and fibrin deposition resulting in the subacute phase in VOD, also termed sinusoidal obstruction syndrome (SOS), which is often accompanied by haemorrhage in centrilobular regions. In the final stage fibrotic occlusion of the central and sublobular veins and sinusoidal fibrosis and centrilobular parenchymal necrosis occurs (Copple et al., 2002, 2003). Inflammatory changes can contribute to the development of toxic liver injury (Copple et al., 2003; Yee et al., 2003). There is one case report on an incidence of VOD in a human fetus caused by PAs present in a herbal mixture used for cooking in the household of the mother (Rasenack et al., 2003). Page 16 of 51

17 6.2. Mutagenicity, genotoxicity, and carcinogenicity The WHO report of 1988 summarizes the numerous studies regarding mutagenicity testing (bacterial assays, mutagenicity in Drosophila, sister chromatid exchange, and micronucleus assay). Many of these tests have been conducted in the 1970s and 1980s and describe the effects of crude plant extracts. These studies are difficult to interpret, but various compounds such as clivorine, heliotrine, lasiocarpine, ligularidine, monocrotaline, petasitenine, retrorsine, senecionine, seneciophylline, senkirkine and retronecine showed a clear positive response for example in the AMES test with a metabolic system (S9 mix). Several of these compounds are also genotoxic in cell culture assays. There is no obvious relation between the hepatotoxicity and the genotoxic potency of individual PAs. More recent investigations focus on the mechanisms involved in DNA damage caused by PAs. As mentioned above, dehydropyrrolizidines (DHP) forms at nearly equal proportions DNA interstrand and DNA-protein cross-links. A linear correlation between the cross-links and the cytotoxic, antimitotic and megalocytic activity could be established in in vitro studies. Analyses of the DNA-protein cross-links following DNAse treatment revealed that actin was the major protein involved in the cross-linking with DNA in the presence of PAs (here dehydrosenecionine and dehydromonocrotaline (Kim et al., 1995). The involvement of actin in DPCs (DNA-protein-cross links) is also documented for cisplatin and mitomycin C. DNA adduct formation measured by 32 P-post labelling/hplc analysis was described for monocrotaline by Wang et al. (2005a), and confirmed the in vivo experiments. The levels of DHP- DNA adducts in the liver were, however, lower than that of reddilliine treated rats (Chou et al., 2003a). Reddelliine was found to induce liver hemangiosarcomas in male and female F344 rats. Complementary experiments identified 8 different dehydroretronecine-derived DNA adducts in the liver of rats treated with reddilliine. Further analysis showed that the adduct level in the endothelial cells is significantly higher than that in parenchymal cells, which correlates with the preferential induction of liver hemangiosarcomas (Chou et al., 2003a). DNA adducts were formed also by the dehydro metabolites (DHPs) of clivorine, a representative of the otonecine type of PAs (Xia et al., 2004) and by lasiocarpine, a prototype of the heliotrine type of PAs (Xia et al., 2006). Taken together, these results indicate that three of the four major classes of PAs, including retronecines, heliotrines and otonecines are genotoxic, whereas platynecive type PAs, that do not contain a double bond in the base, are not. The pyrrolic metabolites formed by the three mentioned classes form DNA-adducts, DNA cross-links and DNA-protein cross-links, which forms the basis for the observed mutagenicity and carcinogenicity. Further structural analyses suggested that the individual bio-potency of PAs is associated with the steric form of the (allyl) ester group that may favour or hinder easy hydrolysis (for review see Fu et al., 2002). Page 17 of 51

18 In the rodent bioassays, most of the liver tumours observed following exposure to PAs are described as hepatomas, hepatocellular carcinomas, haemangiogenic and cholangiogenic tumours or hepatocellular tumours. Some authors refer to pre-neoplastic lesions, neoplastic nodules or nodular hyperplasia, but these expressions are used inconsistently as well. 7. Adverse effects on livestock All livestock species are susceptible to PA toxicity, albeit with different susceptibility (Galey, 2002). Historically, PA toxicity has been predominantly a problem in ruminant livestock production. However, improvements in herd management practices have minimised the risk in most areas. Cases of PA poisoning of non-ruminants have also been reported, mainly as a result of feeding cereal grains contaminated with seeds from PA-containing weeds. In 1993, there was a serious outbreak of poisoning in South Australia, when 4000 pigs died over a 3-month period, because the wheat in their ration was contaminated with potato weed (H. europaeum) (Gaul et al., 1994). Contamination of grain with the seeds from the same plant resulted in poisoning of chickens and ducks (Pass et al., 1979). Hepatotoxicity in animals consuming certain plants of the genera Senecio, Crotalaria and Heliotropium has been recognised already for more than 100 years and has been associated with considerable losses of livestock. In addition, alkaloids from Amsinckia spp and Echium spp are found to induce hepatotoxicity in animals and man (WHO-IPCS, 1988). These intoxications have been reported from all continents, reflecting the worldwide distribution of the above mentioned plant families (Cheeke, 1991) and PA intoxications are considered as a major problem worldwide (Eloff et al., 2003). An intoxication with PAs can be acute or chronic (Fu et al., 2004). The time that elapses between uptake of the toxic compound and the onset of clinical disease can range from several days to several months, depending on the animal species, ingested total amount of PAs and the individual resistance of the affected animal (general health, age, metabolic capacity). The sensitivity of species to PA-toxicosis differs between the species. Horses, pigs, poultry and cattle are generally considered to be sensitive to PA-intoxication, whereas small ruminants and certain minor species such as rabbits are among the species that are more resistant to this intoxication (Cheeke, 1988). Detailed reports on PA-toxicosis have been published for agricultural animal species, including pigs (Hooper and Scanlan, 1977), poultry (Hooper and Scanlan, 1977; Eröksüz et al., 2003), cattle (Dickinson et al., 1976; Harper et al., 1985; Noble et al., 1994; Vos et al., 2002), sheep (Seaman, 1985; Nobre et al., 2004, 2005) and horses (Giles, 1983; Lessard et al., 1986; Creeper et al., 1999, and others). Page 18 of 51

19 7.1. Adverse effects in pigs Hooper and Scanlan (1977) described a field outbreak of Crotalaria retusa poisoning in pigs. In plants of the genus Crotalaria, monocrotaline is the predominant PA. The initial clinical signs observed were inappetance and growth retardation. In the course of the intoxication, the pigs became severely ill and lethargic with signs of pneumonia, laboured respiration and mucous nasal discharge. Upon pathologic examination, the main lesions were found in the liver, kidneys and lungs of affected animals. In the field outbreak, the main syndrome was severe nephrosis and uraemia. The kidneys were firm and pale, with petechiae. Microscopic examination showed megalocytosis, mainly of the cells of the proximal tubules. The livers of these animals showed minor macroscopic alterations, but clear microscopic lesions, including megalocytosis, mainly in the portal areas. Some livers showed periacinar congestion and necrosis. In the lungs of most of the animals a severe interstitial pneumonia was observed and microscopic examination revealed oedema and fibrosis, leukocyte infiltration and thickened alveolar septa caused by alveolar cell enlargement and cellular infiltration. In a subsequent controlled feeding experiment, Crotalaria retusa seeds were added in amounts of 0.1 % of the diet for 21 days, followed by 0.05 % for seven days. This seed contained % of monocrotaline. At this level of exposure, severe symptoms of PA-toxicosis were observed resulting in a high mortality rate. Clinical signs became apparent about six weeks after exposure to the toxic PAs. The feeding experiment furthermore indicated that even lower levels of toxic PAs in the feed can be detrimental to pigs, as another group fed C. retusa seeds in amounts of 0.02 % of the diet (or at higher inclusion rates) had to be euthanized in a moribund state after an average time period of 81 ± 11 days. At lower concentrations ( % C. retusa seeds in the diet) severe megalocytosis was detected in the liver and kidneys (Hooper and Scanlan, 1977). In rats monocrotaline primarily affected the respiratory system by causing pulmonary vasculitis and hypertension (Wang et al., 2005b; Wilson et al., 1992; Roth and Reindel, 1991), while other PAs, like senecionine, one of the predominant PAs from plants of the genus Senecio (Macel et al., 2004), mainly causes hepatic alterations (Williams et al., 1989). It remains to be investigated whether or not in pigs intoxications with Senecio spp. induce also primary liver lesions Adverse effects in poultry Hooper and Scanlan (1977) described hepatic disease, with clear lesions in the liver, as the predominant feature of intoxication with Crotalaria retusa also in chickens. In a controlled feeding experiment, chickens (2 weeks of age) were exposed to levels of 0, 0.005, 0.01, 0.05, 0.1 and 0.5 % of C. retusa seeds (containing % monocrotaline) in their diet. Exposure to levels exceeding 0.05 % C. retusa seeds led to severe clinical symptoms of both acute and chronic intoxication and a high mortality rate (Hooper and Scanlan, 1977). Almost all of the animals given 0.1 and 0.5 % of C. retusa seeds died between 12 and 56 days after onset of the experiment, and Page 19 of 51

20 one out of four animals died in the group fed 0.05 %. In contrast, no mortality was seen in the groups, fed 0.01 and %. Acute toxicity was characterized in these animals by enlarged livers, covered with fibrin and enlarged bile bladders. Microscopic examination revealed acute hepatocellular necrosis and moderate microscopic changes in the kidneys. Chronic toxicity was also associated with clear liver changes and liver lobes appeared irregular in size and shape. Moderate megalocytosis was the predominant feature upon microscopic examination of these livers, with necrosis becoming more apparent in the late stages of the intoxication. The spleens of animals were significantly enlarged. These microscopic lesions could be also detected in the group given the lowest level of 0.005h% C. retusa seeds, and hence a no-effect level could not be derived from this study. Eröksüz et al. (2003), exposed 13-week-old broiler chickens to a diet supplemented with 0.0, 0.5, 2.0 or 4.0 % ground S. vernalis for a period of 210 days. The total PA content of the plant material was 0.14 % and senecionine was the main PA present. No mortality was observed in any of the experimental groups. The animals that were fed 2 % or 4 % PA containing plant material showed a significant decrease in body weight, feed intake and feed efficiency, and a significant reduction in egg production. Moreover, serum γ-gt was elevated and serum albumin and total protein were significantly lower in these animals. In addition, the 4h% group also had a significant increase in the level of total bilirubin. Pathologic examination revealed changes in the 2 and 4 % groups: mild to moderate chronic liver changes, characterized by fibrosis in the periportal regions, megalocytosis, bile duct hyperplasia and early signs of regeneration Adverse effects in ruminants There are a number of reports on PA-toxicosis in cattle (Seawright et al., 1991a; Odriozola et al., 1994; Noble et al., 1994; Craig et al., 1991; Vos et al., 2002). The main clinical signs in cattle associated with PA intoxications include loss of appetite, weight loss, aggressiveness, incoordination, compulsive movements, diarrhoea and persistent tenesmus progressing occasionally into prolapsus recti. Some authors also mention oedema and ascites/hydrothorax and jaundice (Odriozola et al., 1994; Craig et al., 1991). Photosensitivity, especially of the nonpigmented skin, can also occur (Noble et al., 1994). During the course of PA toxicosis, changes in liver enzymes can be detected. The levels of these diagnostic enzymes vary during the course of the intoxication (Craig et al., 1991). Moreover, total protein, albumin and total bilirubin have been suggested as diagnostic markers, but their levels depend also on the stage of intoxication (Vos et al., 2002). During pathologic examination of exposed animals enlarged, firm livers are observed. Microscopic examination shows intranuclear vacuolisation as an early sign of PA toxicosis. These changes are followed by proliferation of bile ducts, and fibrosis in the portal areas and around the Page 20 of 51