SOLANINE POISONING

BRITISH MEDICAL JOURNAL 8 DECEMBER 1979 1459 Potatoes are such a common feature of the Western diet that most people are surprised to learn that they are the produce of a poisonous plant. In fact potato stems and leaves contain a series of alkaloidal glycosides, termed solanines, which are highly toxic. The normal tuber contains only small amounts of solanines in the peel and none in the flesh. Poisoning due to feeding the leaves and stems to domestic animals is well recognised, and one instance of poisoning in man was traced to the use of leaves and young shoots as a boiled green vegetable.' The main hazard, however, comes from eating "greened" potatoes. Greening and sprouting occur when potato tubers are exposed to light or are stored in adverse conditions, and these processes are associated with the production of the alkaloids. Initially this occurs at the sites of increased metabolic activity, such as the "eyes"; but eventually solanines can be detected in the flesh of the tuber, and the normal, high concentration-gradient between the peel and the flesh is lost. Fortunately, few people cook greened or sprouted potatoes because of their appearance and their bitter, unpleasant taste; so that in practice solanine poisoning appears to be rare except in times of food shortage. A few outbreaks, however, have been due to catering errors or unusual conditions.2 Such an error caused an outbreak of poisoning that affected 78 schoolboys in South London, recently reported in carefully documented detail by McMillan and Thompson.3 The onset of symptoms occurred some four to 14 hours after the boys had eaten boiled potatoes. Vomiting and diarrhoea were predominant symptoms, preceded or accompanied by abdominal pain. Fever was not invariable and was often only slight, tending to subside early in the illness. Depression of the central nervous system occurred in the more serious cases, and several patients were comatose with episodes of convulsive twitching. These boys also showed signs of peripheral circulatory collapse, even when dehydration was only slight. Little blood was lost in the stools or vomitus, even though symptoms continued for up to six days. Death has occurred in previous outbreaks, usually within 24 hours4 5; but those cases were mainly in undernourished patients who may not have received adequate treatment. In the recent London episode all patients recovered fully, though some were confused and hallucinated for several days. The treatment for solanine poisoning is replacement of fluid and electrolyte losses; anticonvulsants (diazepam or paraldehyde) may also be needed. Avoiding inappropriate treatment (for example, for supposed bacterial enteritis or acute appendicitis) is, however, no less important; this means speedy diagnosis based on the history and symptoms, backed by negative laboratory tests for infection. The diagnosis can then be confirmed by examining the remaining potatoes or potato waste. Possibly unrecognised mild solanine poisoning may be the cause of many mild episodes of "gastroenteritis." Perhaps greater awareness of this possibility will lead to further reports. 1 Willimott, S G, The Analyst, 1933, 58, 431. 2 Wilson, G S, Monthly Bulletin of the Ministry of Health Public Health Laboratory Service, 1959, 18, 207. 3 McMillan, M, and Thompson, J C, Quarterly J'ournal of Medicine, 1979, 48, 227. 4 Pfuhl, E, Deutsche Medicinische Wochenschrift, 1899, 25, 753. 5 Hansen, A A, Science, 1925, No 1578, 340.

SOLANINE AND CHACONINE

EXPLANATION The common potato, Solanum tuberosum, contains toxic steroidal glycoalkaloids derived biosynthetically from cholesterol (Sharma & Salunkhe, 1989). In older literature (before 1954) these have been referred to only as 'solanine' or as total glycoalkaloids (TGA). The potato glycoalkaloids have not been evaluated previously by the Joint FAO/WHO Expert Committee. Potatoes that have been exposed to light in the field or during storage may become green, due to an accumulation of chlorophyll. This greening may affect only the surface (peel) or it may extend into the flesh of the potato. Exposure to light is only one of the stress factors affecting potatoes. Other pre- or post-harvest stress factors are mechanical damage, improper storage conditions, either as a tuber or after partial food processing, and sprouting (Sharma & Salunkhe, 1989). As a result of any of the above stress factors, there can be a rapid increase in the concentration of TGA, notably, alpha-solanine and alpha-chaconine, which gives the potatoes a bitter taste. These natural toxicants (stress metabolites) have insecticidal and fungicidal properties; each of the two major glycoalkaloids is normally present in all tubers in small amounts (< 5 mg/100 g of tuber fresh weight) (Table 1). The glycoalkaloids are formed in the parenchyma cells of the periderm and cortex of tubers, and in areas of high metabolic activity such as the eye regions. The glycoalkaloids are unevenly distributed throughout the potato, with a large part concentrated under the skin (Table 1). Some cultivars are more prone to develop elevated levels of TGA than others. Growing conditions may also affect the level of glycoalkaloids. None of cooking, baking, frying nor microwaving destroys the glycoalkaloids (Bushway & Ponnampalam, 1981). Table 1. Normal Levels of TGA in various tuber tissues TGA1mg/100g FW 7.5 (4.3-9.7) 1.2-5 30-60 15-30 25-80 150-220
1

whole tuber flesh skin 2-3% of tuber peel 10-15% of tuber bitter tuber peel from bitter tuber

Wood & Young, 1974

In commercially available potato tubers destined for human consumption, as much as 95% of the TGA fraction consists of alpha-solanine and alpha-chaconine (Fig. 1) There is usually slightly more alphachaconine than alpha-solanine. These compounds are derivatives of the aglycone solanidine, each containing three sugar moieties. Solanidine itself may also be present in potato tubers. The remainder of the TGA fraction may consist of other glycoalkaloids or their aglycones (Sharma & Salunkhe, 1989). Other aglycones include demissidine, tomatidenol and 5-solanidan-3alpha-ol. Alpha- And -solamarine are examples of glycoalkaloids derived from tomatidenol found in potatoes. Through plant breeding using wild potatoes other glycoalkaloids, such as commersonine and demissine, both derived from demissidine, and various leptines, derived from leptinidine, may be introduced (Sharma & Salunkhe, 1989). The most extensive review on Solanum and solanine is by Jadhav et al. (1981); other reviews are by Maga (1980), Dalvi & Bowie (1983), Morris & Lee, (1984), Morgan & Coxon (1987) and Sharma &

Salunkhe (1989). Most of the toxicity data deals with alpha-chaconine and alpha-solanine. A Nordic view and assessment of the health risks from glycoalkaloids in potatoes was recently compiled (Slanina, 1990a,b).

2. BIOLOGICAL DATA 2.1 Biochemical aspects 2.1.1 Absorption, distribution, and excretion 2.1.1.1 Mice Groups of 4 female Swiss-Webster mice, weighing 20 g, were orally administered alpha-chaconine once at a dose of 10 mg/kg bw. Animals were sacrificed by exsanguination at 3, 6, 14, 72 and 120 h after

dosing. Blood was obtained (0.1 ml/time point) by multiple incisions into the tail vein. Absorption was slow, with peak values in blood (0.82 g/ml) obtained after 14 h. Decrease in blood levels was slow, with 0.31 g/ml present at 120 h. The peak level in the liver (2.97 g/g) was reached 6 h after dosing. A second, but lower, peak of radioactivity was seen in the liver at 120 h, suggesting enterohepatic recycling. Cell fractionation studies showed that within liver cells, there was no preferential location for alpha-chaconine, and there was no binding of alkaloid to isolated RNA or DNA fractions (Sharma et al., 1983). 2.1.1.2 Rats Male Fischer rats (180-250 g) were orally administered alpha-solanine, tritiated at the carbon atoms adjacent to the nitrogen atom and the double bond (Fig. 1), at a dose of 5 mg/kg bw. Blood samples were taken from the abdominal aorta at 1, 3, 6, 12, 24, 48, 72, and 96 h (2 animals per time point). Radioactivity in the gastrointestinal tract started to disappear from 3 to 6 h after dosing. During the first 24 h interval the total urinary and faecal excretion was 78% of the dose, with most in the faeces. Maximum concentrations of radio-activity occurred near 12 h for all tissues, with the largest concentration in the kidneys, spleen, liver, and lungs, and the lowest concentration in the blood. By 24 h only 10% of the administered dose remained, and 12% was unaccounted for (presumed to be associated with other organs and the carcass). At that time the amount of tritium in the liver represented 1.54% of the dose, and for blood this was 0.375% (based on a total blood volume of 64.1 ml/kg bw). By 4 days 84% of the dose had been excreted by the faecal route, and urinary excretion accounted for 10% of the dose (Nishie et al., 1971). Male Sprague-Dawley rats (200-300 g) were orally administered alpha-chaconine, tritiated at the carbon atoms adjacent to the nitrogen atom and the double bond (Fig. 1), at a dose of 5 mg/kg bw. Blood samples were taken from the abdominal aorta at 1, 3, 6, 12, 24, 48, 72, and 96 h (2 animals per time point). alphaChaconine was poorly absorbed since faecal elimination accounted for 60% of the dose within 12 h, and 80% of the dose within 24 h. Urinary excretion of tritium was 5% of the dose 3 to 6 h after dosing, and reached a plateau of 10% of the dose between 12 and 24 h. Maximum concentrations of radioactivity occurred between 6 to 12 h for all tissues, with the largest concentration in the liver. Intermediate concentrations were seen in the kidneys, spleen, and lungs, and the lowest concentrations were seen in the blood, brain and abdominal fat. At 24 h after dosing the amount of tritium associated with the liver represented 1.29% of the dose, and that with the blood was 0.17% of the dose (based on a total blood volume of 64.1 ml/kg bw) (Norred et al., 1976). 2.1.1.3 Hamsters Golden hamsters (130-150 g) were orally administered randomly tritiated alpha-chaconine at a dose of 10 mg/kg bw. At specified times (3, 12, 24, 72, and 168 h) hamsters (3 animals per time point) were exsanguinated by cardiac puncture. (The reviewers noted an error in reporting and have adjusted the results of the original report by changing ng to g). At 3 h after dosing, the highest concentration of alpha-chaconine was seen in the intestines, including intestinal contents (125 g/g), and this represented 63% of the administered dose. By 24 h these values were 75 g/g or 44% of the administered dose, and by 168 h they had declined to 1.73 g/g or 0.92% of the administered dose. Peak blood (1.74 g/ml) and peak tissue levels (liver = 27.2 g/g) of alpha-chaconine for most tissues were seen by 12 h, and for the heart and kidneys by 24 h. By 168 h after dosing, blood levels had declined to 0.29 g/ml. The ratio of liver concentration to blood concentration at 72 h was greater than at 24 h, indicating the possibility of enterohepatic recycling. Only small amounts of radioactivity were recovered from the faeces in the elimination phase (non-detectable at 3 h, 0.15% at 24 h, to 0.24% of the administered dose by 168 h). In the urine these values increased from non-detectable at 3 h to 0.25% at 12 h, and to 21% by 168 h. These results suggest that most of the alpha-chaconine was absorbed, but that absorption from the

gastrointestinal tract was slow. Much of the radioactivity appeared in various tissues in bound form (Alozie et al., 1979a). 2.1.1.4 Humans Tritiated solanidine (dose not given, but expressed as radioactivity) was administered to 3 human volunteers (2 males, 1 female) by iv injection. Blood and urine samples were collected at various times up to 150 h. Ninety per cent of tritium had disappeared from the blood within 20 minutes of injection. Presuming that radioactivity represented solanidine or its metabolites, three phases of elimination were identified in plasma with half-lives of 2 to 3.7 min, 2 to 5 h, and 72 to 104 h, respectively. Within minutes of injection, the concentration of tritium in erythrocytes exceeded that in plasma. Erythrocytes were found to be a mobile reserve of solanidine, thereby delaying transfer of solanidine from vascular to extravascular compartments. Low rates of excretion were seen in urine and faeces, and together accounted for about 5% of the administered dose during the first 24 h. Thus a fraction in excess of 90% of the dose was sequestered somewhere in the body 24 h after dosing. After this time, the rate of elimination from the body was low, about 1-2% per day, corresponding to an overall half-life of 34 to 68 days. The authors calculated that if absorption of solanidine were 1 mg/day, then with a fractional rate of excretion of 0.02, the body burden would be 50 mg. The authors suggested that mobilization from various storage loci could occur during times of 'metabolic stress', including pregnancy (Claringbold et al., 1982). Mean levels of 1.56 1.17 (7 males) and 1.20 0.93 (27 females) ng/ml solanidine were found, using radioimmunoassay, in human plasma samples obtained by a hospital clinic in the UK, collected in the morning before lunch (Matthew et al., 1983). Thirty healthy males, aged 18-44 years, and 27 healthy females, aged 16-62 years, participated in a study in the UK designed to measure levels of serum solanidine in persons eating their usual diet (during the winter). Intake of the type of potato product (i.e., French fried, boiled or baked, and whether the skin was included) was recorded daily for one month, with arbitrary units, corresponding to approximate levels of TGA in those products, assigned to each product; the weight of product ingested was not measured. Serum samples were collected before the midday meal, and were analyzed by radioimmunoassay (detection limit 0.5 ng/ml). In males the mean level of solanidine was 10.8 5.4 ng/ml (range 2.1-22.5 ng/ml), whereas in females the respective values were 7.9 4.3 (range 1.6-18.5). For both genders there was a significant correlation between serum solanidine levels and the alkaloid intake (expressed in units as indicated above) during the month (R = 0.878 and R = 0.703, respectively). In two male subjects serum solanidine levels dropped to 0.5 ng/ml 2 to 3 weeks after they had been on a potato avoidance diet, indicating a relatively long serum half-life for solanidine. It was suggested by the authors that solanidine may be bound to blood constituents such as free sterols (Harvey et al., 1985a). Eighteen healthy males, aged 20-45 years, and 15 healthy females, aged 19-63 years, from the London area in the UK, participated in a study designed to measure levels of total serum alkaloids (alpha-solanine + alpha-chaconine + solanidine) and solanidine in persons eating their usual diet (during the summer). For comparison, 5 males, aged 31-41 years, and 5 females, aged 31-67 years, from the Uppsala area in Sweden also participated in this study. In Sweden, 2 of the males and 1 female consumed 200-300 g potatoes of 2 varieties high in TGA, including the skin, for 1 week (mean 24 mg TGA/100 g), giving an intake of approximately 60 mg/person or 1 mg/kg bw/day. Blood samples were collected before the midday meal, and were analyzed by radio-immunoassay (detection limits for total alkaloids and solanidine were 0.4 and 0.5 ng/ml serum, respectively). The mean levels of serum solanidine were, respectively, 3.5 and 4.0 ng/ml in the UK and Swedish subjects eating their usual diets, whereas in those three Swedes consuming potatoes with a higher TGA content the mean serum solanidine level was 31 ng/ml (range 27.8-35.5). The respective serum total alkaloid levels were 12.0, 16.9 and 50 ng/ml. The mean serum total alkaloid concentration was about 2.7 times the solanidine concentration, which,

according to the authors, suggests considerable metabolism in man of the glycoalkaloids alpha-chaconine and alpha-solanine (they represent the major proportion of alkaloids in potatoes) through hydrolysis of the sugar residues. It was suggested that hydrolysis could take place in the acid medium of the stomach, or at the site of absorption, or the ratio could reflect the preferential absorption of the more lipophilic solanidine. Alternatively, alpha-solanine and alpha-chaconine might be absorbed unchanged and metabolized within the body (Harvey et al., 1985b). Blood serum levels of alpha-solanine, alpha-chaconine, and solanidine resulting from a single meal of mashed potatoes (equivalent to 1 mg TGA/kg bw/day) were monitored in 8 healthy subjects (HPLC, detection limit 1 ng/ml). Peak concentrations were achieved after 4-8 h; these were 3-11 ng/ml for alphasolanine and 6-21 ng/ml for alpha-chaconine. The 1:2 ratio was maintained for the duration of the experiment. After longer time intervals the level of solanidine was < 4 ng/ml. The serum half-lives for alpha-solanine and alpha-chaconine were 11 and 19 h, respectively (unpublished data by K.E. Hellens, cited by Slanina, 1990b). 2.1.2 Biotransformation 2.1.2.1 Rats Male Fischer rats (180-250 g) were orally administered 5 mg/kg bw solanine, tritiated at the carbon atoms adjacent to the nitrogen atom and the double bond (Fig. 1). Approximately 65% of the radioactivity in the faeces was identified as solanidine. In urine 72% of radioactivity was present as basic compounds of which 6% was identified as solanidine. Two other compounds, present at 80% and 13%, possessed intermediate polarity with respect to solanine and solanidine (Nishie et al., 1971). Male Sprague-Dawley rats (200-300 g) were orally administered alpha-chaconine, tritiated at the carbon atoms adjacent to the nitrogen atom and the double bond (Fig. 1) at a dose of 5 mg/kg bw. Urine and faecal samples were collected 24 h later. The major constituent in both faeces and urine was presumed to be solanidine because it showed the same Rf. Similarly, 25% of the radioactivity in the faeces was attributed to unchanged alpha-chaconine. In addition, 2 minor compounds, possessing intermediate polarity between solanidine and alpha-chaconine and representing 1-5% of total activity, were found in faecal and urine extracts. The authors concluded that the absorption and metabolism of alpha-chaconine was similar to alpha-solanine (Norred et al., 1976). 2.1.2.2 Hamsters Golden hamsters (130-150 g) were orally administered randomly tritiated alpha-chaconine at a dose of 10 mg/kg bw. At specified times (3, 12, 24, 72, and 168 h), hamsters were exsanguinated by cardiac puncture (3 animals per time point) (see above Alozie et al., 1979a). Thin-layer chromatographic separation was performed on the chloroform soluble fractions from urine and faeces collected at various time intervals after dosing. In urine, over half of the eliminated radioactivity during the initial 24 h was due to unaltered alpha-chaconine. A major urinary metabolite was solanidine, which was the major peak by 72 h. In addition, 4 other unidentified metabolites were present at various concentrations. Two of these were the major peaks by 168 h after dosing. In faeces, much of the eliminated radioactivity was due to alphachaconine, and a major metabolite was solanidine. There were 2 additional unidentified metabolites present in about the same concentration as solanidine (Alozie et al., 1979b). 2.1.3 Effects on enzymes and other biochemical parameters Groups of male Sprague-Dawley rats (5/group) were fasted overnight and then given alpha-solanine by gavage at 0 and 250 mg/kg bw or i.p. at 0 and 20 mg/kg bw. In the orally dosed animals serum glutamic

oxalacetic transaminase (SGOT) and serum glutamic pyruvic transaminase (SGPT) were increased, and cholinesterase activity was decreased, but the differences were not statistically significant. With the i.p.dosed animals statistically significant increases of 29 and 63% in SGOT and SGPT, respectively, and a 27% decrease in cholinesterase activity were observed. In addition, a significant inhibition of liver benzphetamine N-demethylase activity and a decrease in liver cytochrome P-450 were observed after i.p. dosing, whereas after oral dosing these differences were statistically insignificant (Dalvi, 1985). 2.2 Toxicological studies 2.2.1 Acute toxicity studies Oral LD50 values for solanine in rodents are considerably higher than LD50 values determined after intraperitoneal administration (Table 2), probably because these species do not absorb much of the solanine. Post-mortem examination failed to reveal the cause of death in rats that had been dosed orally (stomach tube). The oral LD50 values in rodents were 300 to > 500 times the toxic dose of about 2 mg/kg bw and a lethal dose of 3 to 6 mg/kg bw estimated for humans (see Section 2.3.1). Table 2. LD50 values in mg/kg bw alpha-solanine i.p. 32.31 42.02 32.35 30.06 753 674 <201,2 406 <407 alpha-chaconine i.p. 19.25 27.56 solanidine i.p. >5002

Mice

p.o. >10002

Rat Rabbit Rhesus Monkey

1 2

590

506

Patil et al. (1972) Nishie et al. (1971) 3 Gull et al. (1970) 4 Chaube & Swinyard (1976) 5 Sharma et al. (1979) 6 Nishie et al. (1975) 7 Swinyard & Chaube (1973) Two rhesus monkeys died 48 h after an i.p. injection of 40 mg/kg bw of total glycoalkaloids; one other died 2 h after having been dosed i.p. twice (24 h apart) with 20 mg solanine/kg bw (Swinyard & Chaube, 1973). 2.2.2 Short term studies 2.2.2.1 Rabbits One group of 4 rabbits, each weighing about 950 g (strain not given), was fed normal potatoes (TGA content 7.5 mg/100 g) and 4 more rabbits were fed greened potatoes (TGA content 20.4 mg/100 g) for a period of 20 days. After 4 to 6 days the latter group, consuming 49-53 mg TGA/kg bw/day, became dull

and inactive; after 10 days, diarrhoea, hair loss and weight loss occurred, which was followed by watering eyes, body rigidity and dullness. Protein digestibility (amount of protein from potatoes ingested less amount of protein excreted expressed as a percentage of protein from ingested potatoes) decreased by 45% from day 1. This was accompanied by a significant decrease in body weight, resulting in an average body weight of about 650 g for treated and 1150 g for 'control' rabbits at 25 days after cessation of feeding the experimental diets. One of the 4 treated rabbits died within 10 to 20 days. The control animals which consumed 20 to 23 mg TGA/kg bw/day were unaffected (Azim et al., 1983). The same authors similarly fed 2 groups of 5 rabbits each (weight and strain not given) a control potato diet (7.5 mg TGA/100 g) and a high TGA potato diet (29.75 mg TGA/100 g) for 45 days. Daily intake of TGA was 16.8 to 17.9 mg/kg bw in the control diet, and 73.9 to 75.0 mg/kg bw in the high TGA diet. Blood samples were collected from the ear vein every 15 days. RBC counts and haemoglobin concentrations were determined. A significant decrease in RBC counts was seen throughout feeding the high TGA diet, and this was 27.5% by 45 days. This compared to a decrease of 12.5% seen by 45 days in the control diet. Decreases in haemoglobin concentrations paralleled the findings with RBC counts. The authors suggested that these results indicate that the rabbits developed haemolytic anaemia. This could be explained by the metabolite solanidine increasing the permeability and fragility of RBC membranes (Azim et al., 1984). 2.2.2.2 Monkeys Four time-mated rhesus monkeys were fed ad libitum a diet of diced potatoes of the B5141-6 variety (since withdrawn from the market), containing on average 26 mg solanine per 100 g tuber for 25 consecutive days during days 0-42 following mating. It subsequently became apparent that the monkeys were not pregnant. The monkeys ingested the equivalent of 0.77 to 1.02 mg/kg bw/day alpha-solanine or 3.08 to 4.07 mg/kg bw/day TGA. No adverse effects were observed (Swinyard & Chaube, 1973). 2.2.3 Long-term/carcinogenicity studies No studies available. 2.2.4 Reproduction studies 2.2.4.1 Rats Groups of Holzman rats, approximately 4 months of age, were mated, one male to 3 females. Increase in weight was taken as an indication of pregnancy, and afterwards the females were individually caged and given a basal diet of (I) ground lab chow; (II) ground lab chow to which was added 10% ground frozen potato sprouts; (III) 30 mg/kg diet solanine (commercial); (IV) 40 mg/kg diet solanine (commercial); and (V) 30 mg/kg diet solanine that was isolated from the frozen sprouts. The time on the test diets was variable, since increase in weight is not a sensitive indicator of pregnancy, and some dams dropped their litters within a few days on the test diet. They were then kept on the test diet until they had a second litter. No food consumption records were kept, but the rats readily ate the diets. With diets II to V, many of the pups died within 3 days of birth, evidently from starvation as indicated by an absence of milk in their stomachs. The percentage of pups successfully weaned was 82.6, 50.6, 31.0, 31.1, and 19.5 for diets I to V, respectively. All of the pups in 18/33 litters born of the rats eating the test diets died before reaching weaning age, whereas only one of 11 control litters was lost. The authors concluded that the toxicity of the potato sprout diet was due to 'solanine'. It was speculated by the authors that solanine may exert an anti-hormonal effect and prevent lactation in some sensitive dams. The reviewers feel that further studies to examine these effects and other aspects of reproduction are necessary (Kline et al., 1961).

2.2.5 Special studies on embryotoxicity/teratogenicity 2.2.5.1 Rats Potential teratogenicity of alpha-solanine and alpha-chaconine was investigated in four different experiments with Wistar rats weighing 175-200 g. In the first experiment, three groups of rats (910/group) were gavaged with alpha-solanine at dose levels of 0.3, or 3.0 mg/kg bw/day, from days 6 to 15 of gestation. In the second experiment, a group of 9 rats was given alpha-solanine by gavage at a dose level of 6 mg/kg bw/day, from days 7 to 10 of gestation. In the third experiment, groups of rats (34/group) received alpha-solanine at dose levels of 2, 10 or 25 mg/kg bw/day from days 8 to 11 of gestation. In the fourth experiment, a group of 4 rats received alpha-chaconine by gavage at a dose level of 1.5 mg/kg bw/day, from days 6 to 15 of gestation. In the first three experiments, concurrent control groups composed of 2 to 10 rats/group were included. On day 22 of gestation, all females were sacrificed and the following parameters were investigated: corpora lutea, resorption sites, litter size, litter weight, and gross, visceral and skeletal fetal anomalies. The only adverse effect was observed in the first experiment. One fetus with craniorachischisis and exopthalmos (1/117), from the group receiving 3 mg/kg bw/day, and another with twisted pelvic limbs and absent tail (1/108), from the 0.3 mg/kg bw/day group were observed. No maternal toxicity was reported. The authors concluded that the observed effects were not treatment-related (Ruddick et al., 1974). A group of 14 Wistar rats, weighing 175-200 g, was fed a diet containing about 73% of cooked and freeze-dried, visibly blighted parts of potato tubers, from days 1 to 22 of gestation. The intake of blighted potatoes was approximately 70 g/kg bw/day. The control group of 13 females was fed a diet containing the same amount of freeze-dried potatoes inoculated with heat-killed Phytophthora infestans. The content of glycoalkaloids in the diets was not determined. All dams were sacrificed on day 22 of gestation and the standard parameters (corpora lutea, resorption sites, litter size, litter weight, and gross, visceral and skeletal anomalies) were investigated. There was no evidence of maternal toxicity, fetal toxicity nor teratogenicity (Ruddick et al., 1974). 2.2.5.2 Hamsters Groups of hamsters (12-15/group), weighing about 100 g, were fed from days 5 to 10 of gestation diets of commercial hamster ration containing 50% freeze-dried, unblighted potato concentrate (group 1); 50% Phytophthora infestans infected freeze-dried, blighted potato concentrate (group 2); or 50% Alternaria solani infected freeze-dried, blighted potato concentrate (group 3). A group of 13 hamsters was fed commercial hamster ration only, throughout gestation (group 4). The content of glycoalkaloids in the diets was not determined. Food and water were provided ad libitum. On day 15 of gestation, the dams were sacrificed, and fetuses were examined for gross, visceral and skeletal anomalies. Feed consumption, maternal body weight gain, litter size, number of resorptions and fetal weight were not affected by the treatment. The most frequent gross anomaly was haemorrhagic necrosis of the central nervous system, but the frequency of this effect was not treatment-related (group 1 - 1/114; group 2 - 3/153; group 3 - 0/135; group 4 - 11/99) (Sharma et al., 1978). Groups of Syrian hamsters (body weights and age not given) were gavaged on day 8 of gestation with alpha-chaconine, isolated from Arran Pilot potato sprouts, at levels of 165 mg/kg bw/day (23/group) or 180 mg/kg bw/day (14/group) and alpha-solanine, isolated from Arran Pilot potato sprouts, at a level of 200 mg/kg bw/day (37/group). Females of the vehicle control group (37/group) received vehicle material alone (2% ethanol at pH 5-6 in 1% carboxymethyl cellulose or in water). Animals were individually caged in a room maintained at 20-26 C and received food and water ad libitum. Maternal toxicity was monitored by daily weighing and clinical observation. On day 15 of gestation, the dams were sacrificed and necropsied. The uteri were exposed and the number of resorptions and live fetuses were determined.

The corpora lutea were counted and all fetuses were examined for gross anomalies. Maternal mortality was observed in all treated dose groups; 4 and 6 dams died in the 165 and 180 mg/kg bw/day alphachaconine dose groups, respectively, and 3 dams died in the 200 mg/kg bw/day alpha-solanine dose group. The days of gestation on which the dams had died were not indicated. No maternal mortality was observed in the vehicle control group. A high incidence of neural tube defects such as interparietal encephalocoele (11-13%) and exencephaly (5-12%) was observed in fetuses, of which the mothers were exposed to either alpha-chaconine or alpha-solanine. In the vehicle control group only 1 fetus of 393 examined exhibited exencephaly. The authors concluded that the observed teratogenic effects were treatment-related. They also indicated that a number of fetuses exhibited CNS malformations without apparent toxicity or weight loss in the dam, making it unlikely that the malformations were secondary to maternal toxicity. Occasional short tail and minor digital anomalies were noted in fetuses from all experimental groups, but those effects were not treatment-related (Renwick et al., 1984). 2.2.5.3 Rabbits Groups of New Zealand rabbits (2-6/group), weighing on average 4 kg, were fed, throughout gestation, diets containing 50% freeze-dried, unblighted, potato concentrate, 50% Phytophthora infestans infected freeze-dried, blighted, potato concentrate, or 50% Alternaria solani infected freeze-dried, blighted, potato concentrate. The content of glycoalkaloids in the diets was not determined. Prior to parturition (day not defined), all dams were sacrificed and fetuses removed and examined for gross, visceral and skeletal anomalies. During fetal examination particular attention was paid to any malformations of brain and spinal cord. Among 21 fetuses examined in the Phytophthora infestans blighted potato group, three fetuses (from three litters) exhibited incomplete closure of the caudal vertebral column, and two other fetuses were very small and had shortened appendages. Among 28 fetuses examined in the Alternaria solani blighted potato group, two fetuses exhibited incomplete closure of the caudal vertebral column, one fetus had a very small brain (nearly half the normal size) and the cranial cavity was filled with fluid, and two other fetuses were abnormally small in size. All six abnormal fetuses were from different litters. None of the nine fetuses (two litters) from the unblighted potato group were affected. The authors concluded that feeding pregnant rabbits potatoes blighted with either of the fungi, at high concentrations in the diet, can produce a low incidence of the caudal vertebral column malformation. This result must be considered with caution since the small number of control litters examined does not permit an adequate estimate of the spontaneous incidence of this malformation in rabbits (Sharma et al., 1978). 2.2.5.4 Miniature swine Groups of female miniature swine (2/group), weighing about 39 kg, were fed laboratory diets containing 50% freeze-dried, unblighted, potato concentrate (group 1); 50% Phytophthora infestans infected freezedried, blighted, potato concentrate (group 2); or 50% Alternaria solani infected freeze-dried, blighted, potato concentrate (group 3), during the first half of gestation (about the first 57 days of gestation). The content of glycoalkaloids in the diets was not determined. At the end of gestation (the day was not indicated), all dams were sacrificed and necropsied. All fetuses were removed and examined for gross and visceral malformations. Depressed weight gain was observed in sows of group 3. One fetus of 15 examined from group 2 exhibited anencephaly with extensive internal hydrocephaly. Other fetuses from this and other groups were not affected. The authors concluded thatfeeding potatoes blighted with Phytophthora infestans may be a causative factor in the production of anencephaly in miniature swine. However, the small sample size makes definite conclusions difficult (Sharma et al., 1978). 2.2.5.5 Marmosets A group of 6 female marmosets (Callithrix jacchus), five years of age, weighing about 375 g, previously producing normal offspring, was fed a diet containing freeze-dried concentrate of blighted potatoes

(Kerr's Pink variety), at a level of 4.7 g/kg bw/day (equivalent to 0.9 mg/kg bw/day of glyco-alkaloids), for 50 days, during either days 0-50 or 20-70 of gestation. A control group of 6 pregnant marmosets received a standard unsupplemented diet. Marmosets were sacrificed between days 80-120 of gestation and fetuses were examined for developmental anomalies. Four of 11 fetuses in the blighted potato groups exhibited gross abnormalities, described as cranial osseous defects. Histological examination revealed replacement of bone by a collagenous membrane in the occipital area. Brain examination of affected fetuses revealed enlargement of the lateral ventricle. Eleven fetuses of the control group showed no gross abnormalities in any system. The investigators indicated that, during the 2 years of existence of the marmoset colony, similar defects had occurred once spontaneously in twin fetuses (spontaneously aborted) among 104 live births. The authors concluded that the occurrence of cranial dysplasia in 4 of 11 fetuses, in the experimental group, is suggestive of teratogenicity of blighted potatoes in marmosets (Poswillo et al., 1972, 1973). Three experiments were conducted to investigate possible teratogenic effects of different varieties of unblemished or blemished potatoes in 5 year-old, pregnant marmosets (Callithrix jacchus), previously producing normal offspring. In the first experiment, a group of 6 female marmosets were fed freeze-dried concentrate of unblemished 'domestic potatoes' (Cornish White and King Edward varieties), at a level of about 4.7 g/kg bw/day, equivalent to 0.56 mg/kg bw/day of glycoalkaloids. In the second experiment a group of 7 female marmosets were fed freeze-dried concentrate of blemished 'industry rejected potatoes' (King Edward, Cornish White varieties), at a level of about 4.7 g/kg bw/day, equivalent to 0.78 mg/kg bw/day of glycoalkaloids. In the third experiment a group of 5 female marmosets were fed freeze-dried concentrate of King Edward variety potatoes, infected with Erwinia carotovera (a bacterial pathogen responsible for 'blackleg'), at a level of about 4.7 g/kg bw/day, equivalent to 0.07 mg/kg bw/day of glycoalkaloids. Feeding trials were commenced 10 days postpartum of the previous litter, to cover the period of the expected postpartum oestrous and were carried throughout an undefined period of gestation. Female marmosets fed both the 'domestic potatoes' and 'industry rejected potatoes' diets were allowed to proceed with pregnancy to term, and the offspring was grossly examined at birth and at regular intervals up to 6 months of age. Females fed 'infected potatoes' (with Erwinia carotovora) diet were sacrificed between days 90 to 110 of gestation, and fetuses were examined grossly and radiographically for abnormalities. Behavioural anomalies such as continuous clinging to parents or siblings, and prolonged weaning time, were observed in three sets of twins, born to dams of the second experiment. No anatomical abnormalities were observed in any experimental groups. The authors concluded that the significance of the behavioural abnormalities observed in this study cannot be determined at this stage but further observation of growth and development to sexual maturity may throw more light on this phenomenon (Poswillo et al., 1973). 2.2.5.6 Chicken embryos Fertile chicken eggs (White Leghorn) were injected either with pure solanine, mixed glycoalkaloids or an ethanol extract (obtained from potatoes infected with Phytophthora infestans) into the yolk sac, at levels ranging from 0.13 to 0.26 mg/egg, between 0 and 26 h of incubation. A high incidence of embryo mortality (20-27%) and increased incidence of abnormalities (16-25%) such as cranioschisis, celosoma, cardiac septal defects, rumplessness (absence of tail) and trunklessness (absence of trunk below the wing bud) were observed in treated embryos. The most frequent defect was rumplessness and trunklessness. In controls injected with chick Ringer or HCl solvent, the percentage of abnormal embryos was 9-10% and the mortality was 1-8% (Jelinek et al., 1976; Mun et al., 1975). 2.2.6 Special studies on cholinesterase inhibition The inhibitory effect of alpha-solanine and solanidine, as well as an extract from potatoes, were studied using a 1:100 dilution of sera from 21 human individuals. These persons had been previously phenotyped

as 'usual' (95% of population in Great Britain), 'intermediate' (3-4% of the population), and 'atypical' (uncommon), using the acetylcholinesterase inhibitor dibucaine. At a concentration of 2.88 M and 3.14 M, respectively, alpha-solanine and solanidine were about equally effective causing 86.2 1.2 % and 80.0 1.4 % inhibition in the 'usual' phenotype, and parallel effects to dibucaine in the other two phenotypes. The results with the potato extract were similar. The authors noted that it is not clear to what extent the toxic effects of solanine can be attributed to the inhibition of serum cholinesterase, but if it plays a role then individuals with the 'atypical' phenotype, would presumably be less susceptible (Harris & Whittaker, 1962). Male and female New Zealand rabbits (2 of each sex) were given a single i.p. dose of 20 or 30 mg solanine/kg body weight. These doses resulted in severe depression, with difficult breathing and prostration, and were lethal in 3 of the rabbits within 24 h. One rabbit survived. Blood samples were obtained at 15 to 225 min post-dosing; plasma and erythrocyte acetylcholinesterase activities were measured and compared to control samples taken from the same rabbit before dosing. Solanine was a weak to moderate inhibitor of both specific and non-specific cholinesterase. Maximum inhibition of plasma cholinesterase was seen at 80 min after injection, with the activity decreasing to about 45% of the control value; inhibition of erythrocyte cholinesterase was somewhat lower, and was maximally reduced to 68.6% at 85 min after injection. The same authors injected i.v. 5 doses of 6 mg/kg body weight, 10 min apart, in one anaesthetized male dog (15 kg bw). Quick inhibition of serum cholinesterase was followed by rapid recovery. Erythrocyte cholinesterase was not inhibited (Patil et al., 1972). Male Sprague-Dawley rats (3 per group) were injected i.p. with 0, 10, 30 or 60 mg/kg bw of alphachaconine and sacrificed 3 h after dosing. All rats administered alpha-chaconine showed initial signs of depression, as well as other signs of poisoning by an anticholinesterase agent, such as respiratory depression. Following electrophoresis in acrylamide slabs, homogenates of brain (diluted 1:6) showed 3 zones of acetylcholinesterase isoenzyme activity with a dose-related decrease in peak heights. Overall acetylcholinesterase activity, using a colorimetric method, was reduced to 79, 55, and 18% of the control value for the 3 respective dose groups. Heart acetylcholinesterase activity was reduced to about 40% of the control value in all treatment groups; plasma cholinesterase activity in controls was about 30% of that seen in brain homogenates, and was reduced to 50% in the 10 mg/kg bw dosage group, with no further reduction in rats given 30 mg/kg bw. The authors concluded that alpha-chaconine is a fairly potent inhibitor of cholinesterases (Alozie et al., 1978). In an in vitro assay the anticholinesterase activity of several glycoalkaloids was compared, using highly purified acetyl cholinesterase isolated from human and bovine erythrocytes (Sigma). Alpha-solanine and alpha-chaconine were equally effective, 100 M caused about 80% inhibition of both human and bovine enzymes. Tomatine was less effective, causing 40% and 50% inhibition of bovine and human enzymes, respectively. Solasine, solamargine and the aglycones solanidine, tomatidine and solasidine were ineffective. Over a range of Ph 5 to pH 8, pH of the medium was not very important. These results show that the nature of the aglycone moiety is important (Roddick, 1989). 2.2.7 Special studies on genotoxicity Pure alpha-solanine (Sigma) at 0.01 to 0.05 mg/plate and extracts from potatoes were negative in the Ames test both with strains TA98 and TA100, and in the presence or absence of activation by S9 fraction from PCB-induced rat liver (Ness et al., 1984). Alpha-solanine (25 and 250 M) tested negative in a DNA-cell-binding assay using Ehrlich ascites cells and Escherichia coli cells mixed with 32P-labelled nucleic acids (Kubinski et al., 1981).

2.2.8 Special studies on mitotic index Cultured human fibroblasts were treated up to 40 h with 0, 4.1, 8.3, 16.6, 33.3, and 66.6 g alphasolanine/ml. At the highest dose there was an inhibition of growth, whereas at lower dose levels there was a stimulation of growth, as evidenced by an increase in the mitotic index from 1.7% in controls to 2.4% at the 4.1 g/ml dose level, which according to the authors was similar to the sex-hormonal type of effect exerted by estrogens on target tissues. Using pulse labelling with tritiated thymidine, it was shown that at 5 g alpha-solanine/ml the mean cell cycle time decreased from 42.5 1.87 h in controls to 28.5 0.29 h in treated cells. This was however accompanied by a 4 h increase in the period of DNA synthesis (S phase) in treated cells, and a decrease to virtually zero for the G1 phase. The authors concluded that if alpha-solanine reached the fetus, the observed types of effects could be hazardous to it, and could lead to malformations (Kirk & Mittwoch, 1975). 2.2.9 Special studies on calcium transport Alpha-solanine (100 M at pH 7.4) caused a 90% inhibition of active calcium transport in rat duodenum when added to everted intestinal sacs (8 replicates) in vitro. A Dixon plot revealed that the inhibition by alpha-solanine was non-competitive, and the inhibition constant was 25 M. The inhibition of active calcium transport was accompanied by a 40% decrease in oxygen consumption (Michalska et al., 1985). When alpha-solanine was given to 12 male and female Wistar albino rats (5-6 weeks old) in their drinking water (5 mM, pH 6.4) for 12 days, calcium transport in duodenal sacs was reduced to about one-third of the control value, but oxygen consumption was not significantly reduced (Michalska et al., 1985). 2.3 Observations in humans 2.3.1 Gastrointestinal and neurotoxic effects There have been many reported cases of human poisonings (sometimes fatal) due to the ingestion of greened or otherwise damaged potatoes. The symptoms of low grade solanine poisoning are acute gastrointestinal upset with diarrhoea, vomiting and severe abdominal pain. In more severe cases, neurological symptoms, including drowsiness and apathy, confusion, weakness, and vision disturbances, followed by unconsciousness and, in some cases, death have also been reported. The vital signs include fever, rapid and weak pulse, low blood pressure and rapid respiration. Onset of symptoms has ranged from minutes to 2 days after ingestion of toxic potatoes, with longer incubation periods generally associated with the more severe cases. As is usual with case histories of this nature, the available data are not complete. Over the years, various analytical methods or assays have been used to determine the concentration of 'solanine' in cases of suspected poisonings. With most of the older data, the estimate for solanine included the other glycoalkaloids, such as alpha-chaconine. McMillan and Thompson (1979) showed that gravimetric methods gave higher values than colorimetric methods. A few case reports, for which the reviewers have estimated the dose ingested, are given below. Additional reports were compiled by Morris and Lee (1984), who indicated that more than 2000 cases with about 30 deaths have been reported in the literature. Not all of these reports were available to the reviewers. Fifty-six German soldiers suffered typical 'solanine' poisoning after eating 1 to 1.5 kg cooked peeled potatoes containing 24 mg TGA/100 g (whole uncooked tubers contained 38 mg TGA/100 g). In a few cases jaundice and partial paralysis were also observed. If one assumes a body weight of 70 kg, the intake of 'solanine' was 3.4 to 5.1 mg/kg bw (Pfuhl, 1899).

In 18 separate households in Scotland, 61 persons suffered typical 'solanine' poisoning soon to several hours after eating potatoes. Persons not eating potatoes were not ill. One 5-year old died. The potatoes in that household contained 41 mg 'solanine'/100 g. Assuming the child ate 200 g potatoes and had a bw of 18 kg, the lethal dose was estimated at 4.5 mg/kg bw. Assuming adults ate 500 g potatoes and had a bw of 60 kg, their intake of 'solanine' would have been 3.4 mg/kg bw (Harris & Cockburn, 1918). A small outbreak of solanine poisoning affected a family of four adults on three consecutive Sunday evenings in Great Britain, about 8 h after they had eaten 1 to 3 baked potatoes in their jackets (weight of potatoes not given). A 5th person who only ate the flesh of the potatoes was not affected. The severity of symptoms was related to the number of potatoes ingested, and consisted of abdominal pain, diarrhoea, and general malaise. Patients recovered within 24 h. The level of solanine was 50 mg/100 g tuber, as determined chemically and by cholinesterase inhibition. Assuming a weight of 150 g per potato, and body weights of 60 and 70 kg, the dose was estimated at 1.25 to 3.2 mg 'solanine'/kg bw (Wilson,1959). Seventy-eight junior schoolboys in Great Britain became ill from solanine poisoning 7 to 9 h after eating two small boiled peeled potatoes each (weight of potatoes not given) as part of their lunch, and 17 were admitted to hospital. Symptoms included vomiting, diarrhoea, and general abdominal pain. Most of the boys developed a fever, suffered from headache, dizziness, mental confusion, hallucinations and their vision was affected. Three boys were comatose and stuporose on admission, with peripheral circulatory collapse. All were discharged 6-11 days following admission, and 4-5 weeks later there were no sequelae. Tests for the presence of biocides, such as nicotine, organophosphorus or organochloride pesticides were negative. Six days after eating the meal, plasma pseudocholinesterase levels in 10 out of 17 schoolboys was subnormal (about 25% below the normal range for this age group). Red blood cell cholinesterase levels were normal. The source of toxic potatoes was traced to a bag of old potatoes that had been condemned for consumption because of their appearance, but that had inadvertently been cooked (peeling of the potatoes had been done by an automatic peeling machine). Insufficient potatoes were left over after the meal for direct chemical analysis. Solanine levels in the boiled peeled potatoes were therefore estimated from the in vitro reduction in pseudocholinesterase activity in human plasma, using acetylcholine as a substrate, and were equivalent to 25-30 mg/100 g tuber of alpha-solanine. Assuming an intake of 200 g potatoes and a bw of 40 kg (age = 11-14 years), the reviewers estimate that the intake of 'solanine' by the schoolboys would therefore have been approximately 1.4-1.6 mg/kg bw. Because of the small margin of safety between normal potatoes and toxic potatoes, the authors speculated that in toxic potatoes other toxic steroids besides glycoalkaloids may be synthesized, such as sapogenins and saponins, which might enhance the toxicity of solanine alkaloids by promoting gastro-intestinal absorption or other means (McMillan & Thompson, 1979). In a recent (1983) poisoning associated with a school lunch programme, 61 of 109 school children and staff in Alberta, Canada, became ill, most within 5 minutes, after eating baked potato (weight of potato not given) containing 49.4 mg 'solanine' per 100 g (analytical method not indicated). Test results showed that there was no evidence that the illness occurred due to the presence of viruses, bacteria, moulds, pesticides or other chemicals in the food items or their containers. The potatoes had a slight tinge of green and had a bitter or unusual taste (noted by 44% of those affected), causing a burning sensation in the throat of 18% of those affected. The predominant symptoms in order of frequency were nausea (69%), abdominal cramps (43%), headache (33%), vomiting (11%), fever and diarrhoea (8%). The children recovered in about 3 h. The reviewers estimate that, assuming the children ingested 200 g, and had a bw of 40 kg, the dose was about 2.5 mg 'solanine'/kg bw (Anon, 1984). Based on the available human data (Table 3), an intake of 3-6 mg TGA/kg bw is considered a potentially lethal dose for humans, and >1 to 3 mg TGA/kg bw is considered a toxic dose for humans.Children may

be more sensitive than adults. Other factors may be present in suspect potatoes and modulate the toxicity of the steroidal glycoalkaloids. No signs of acute toxicity were noted in 3 Swedish adult volunteers who ingested for 1 week a diet estimated to give an intake of 1 mg/kg bw TGA (Harvey et al., 1985b). Table 3. Summary of published reports of solanine poisoning in humans Affected Potato type Quantity Consume d 1-1.5 kg 500 g? 200 g? ? ? ? Concentration of TGA mg/kg bw 24 (38) 41 Estimated Toxic Dose mg/kg bw 3.4-5.1 3.4 4.5 ? ? 2.8 Outcome Reference

56(soldiers) 60 adults 1 child (5 yr-old) 7 (family) 50-60 (Cyprus) Prisoners

peeled, cooked (whole uncooked) potatoes

recovered Recovered 1 fatal 2 fatal 1 fatal recovered

greened potatoes shoots, leaves experimental

? 27 49 ?

Child

potato berries

1 fatal

4 (family adults) 78(schoolbo ys)

baked potatoes with skin old potatoes

1-3 potatoes 150-450 g 2 small potatoes 200 g 200 g

50

1.2-3.2

dose-related, recovered 3 comatose, all recovered young boys more affected recovered

Pfuhl, 1899 Harris & Cockburn 1918 Hansen, 1925 Willimot, 1933 Report cited by Ruhl 1951 Report cited by Ruhl 1951 Wilson, 1959 McMillan & Thompso n 1979 Anon. 1984

25-30

1-4-1.6

61(schoolchildren) Alberta

baked potato

49

2.5

? Data not available.

2.3.2 Teratogenic effects In 1972, Renwick showed that areas with an increased incidence of neural tube defects (NTD) (anencephaly and spina bifida) were associated with areas where potato consumption was higher, and where potato blight was more common. The worldwide incidence of NTD varies from < 1 to about 7 per 1000 total births. He postulated that this disease was due to toxic factors in potatoes, such as alphachaconine and alpha-solanine. These antifungal compounds offer resistance to potato blight and increase in amount in blightedpotatoes, infected with the fungus Phytophthora infestans. Several studies have

been conducted to prove or disprove this theory (Renwick, 1972). The same author more recently suggested that a long half-life of potato glycoalkaloids could lead to their retention in the body, and possible release early during pregnancy (Renwick, 1982). In a prospective study with women who had previously borne a child with NTD, 27 women did not handle or eat potatoes or potato containing foods after deciding on a future pregnancy, and throughout gestation; another 61 women, attending the same clinic, did not avoid potatoes. The allocation to the two groups was non-random, but voluntary. The groups did not differ significantly with respect to age distribution, social class, parity or history of outcome of previous pregnancies. The incidence of NTD was 8.7% in the group of women avoiding potatoes, and 3.6% in the group eating potatoes (p=0.58). This study failed to support the Renwick hypothesis, but the authors pointed out that the size of the groups was small (Nevin & Merrett, 1975). Although there is a geographical similarity between neural tube defect occurrence and potato blight in Canada, no annual or seasonal associations were demonstrated. The author concluded that socioeconomic factors were probably more important as a risk factor for NTD, but suggested that better exposure assessment to factors present in potatoes, at the level of the individual, would be necessary to resolve this question. Such prospective studies should also assess the significance of other risk factors (Elwood, 1976). An epidemiological study (prospective study) was conducted in Great Britain, whereby human serum specimens from 380 patients, who were being screened for NTD by measuring their serum alphafetoprotein at 15-22 weeks of gestation (most at 16 weeks), were also analyzed for potato glycoalkaloids, using a sensitive radioimmunoassay. The samples were analyzed blind, regardless of the outcome of pregnancy, which resulted in 210 NTD cases and 170 normal offspring. In most of the 9 centres studied, serum TGA and serum solanidine levels were higher (p <0.05 in 2 centres) in the women with a normal fetus than in those with a fetus affected by NTD. Although closure of the neural tube normally takes place at 4-5 weeks of gestation, the authors felt that measurements at the later date might reflect glycoalkaloid exposure earlier during gestation. The results of this study are therefore the opposite of what one would expect if the ingestions of potatoes contributed to the etiology of NTD. Instead the authors suggested that avoidance of potatoes might contribute to a vitamin deficiency thereby increasing rather than decreasing the incidence of NTD (Harvey et al., 1986). 3. COMMENTS Numerous studies performed on a variety of experimental animal species to elucidate the toxicological properties of glycoalkaloids, including teratogenicity, have been evaluated. Cranial abnormalities have been observed in some teratogenicity studies with laboratory animals, particularly with the hamster at levels of 165-200 mg glycoalkaloids/kg bw/day. However, the suggested association of the consumption of blighted potatoes during pregnancy with increased incidences of spina bifida and anencephaly has not been substantiated. In a limited study in humans, the daily consumption of potato tubers containing approximately 24 mg glycolkaloids/100 g did not result in any signs of acute toxicity. However, human poisonings have been associated with the consumption of poor-quality potato tubers with elevated levels of glycoalkaloids. The signs of low-grade glycoalkaloid poisoning are acute gastrointestinal upset with diarrhoea, vomiting, and severe abdominal pain. In more severe cases, neurological symptoms, including drowsiness, apathy, confusion, weakness, and vision disturbances followed by unconsciousness, have also been reported. 4. EVALUATION

The Committee considered that, despite the long history of human consumption of plants containing glycoalkaloids, the available epidemiological and experimental data from human and laboratory animal studies did not permit the determination of a safe level of intake. The Committee recognized that the development of empirical data to support such a level would require considerable effort. Nevertheless, it felt that the large body of experience with the consumption of potatoes, frequently on a daily basis, indicated that normal glycoalkaloid levels (20-100 mg/kg) found in properly grown and handled tubers were not of concern. To support the continued safe use of potato tubers, those developing new cultivars, and others growing, harvesting, storing, processing, and consuming potatoes, should be aware of the possibility of inadvertently increasing the content of glfycoalkaloids to potentially toxic levels. 5. REFERENCES ALOZIE, S.O., SHARMA, R.P. & SALUNKHE, D.K. (1978). Inhibition of rat cholinesterase isoenzymes in vitro and in vivo by the potato alkaloid, alpha-chaconine. J. Food Biochem., 2: 259-276. ALOZIE, S.O., SHARMA, R.P. & SALUNKHE, D.K. (1979a). Physiological disposition, subcellular distribution and tissue binding of alpha-chaconine (3H). J. Food Safety, 1: 257-273. ALOZIE, S.O., SHARMA, R.P. & SALUNKHE, D.K. (1979b). Excretion of alpha-chaconine-3H, a steroidal glycoalkaloid from Solanum-tuberosum L. and its metabolites in hamsters. Pharmacol. Res. Commun., 11: 483-490. ANON. (1979). Solanine poisoning [editorial]. Br. Med. J., 2: 1458-1459. ANON. (1984). Solanine food poisoning associated with a school lunch program - Alberta. Canada Diseases Weekly Report, Health and Welfare Canada, 10-18: 71. AZIM, A., SHAIKH, H.A. & AHMAD, R. (1983). Toxic effects of high glycoalkaloid feeding on the protein digestibility and growth of rabbits. J. Pharm. Univ. Karachi., 2: 15-24. AZIM, A., SHAIKH, H.A. & AHMAD, R. (1984). Toxic effects of high glycoalkaloid feeding on the red blood cell counts and haemoglobin concentration of rabbit blood. J. Pharm. Univ. Karachi, 3: 43-49. BMER, A. & MATTIS, H. (1924). [Solanine content of potatoes] Der Solaningehalt der Kartoffeln. Z. Nahr. Genussm., 47: 97-127. BUSHWAY, R.J. & PONNAMPALAM, R. (1981). alpha-chaconine and alpha-solanine content of potato products and their stability during several modes of cooking. J. Agric. Food Chem., 29: 814-817. CHAUBE, S. & SWINYARD, C.A. (1976). Teratological and toxicological studies of alkaloidal and phenolic compounds from Solanum tuberosum L. Toxicol. Appl. Pharmacol., 36: 227-237. CLARINGBOLD, W.D.B., FEW, J.D. & RENWICK, J.H. (1982). Kinetics and retention of solanidine in man. Xenobiotica, 12: 293-302. DALVI, R.R. & BOWIE, W.C. (1983). Toxicology of solanine: an overview. Vet. Hum. Toxicol., 25: 1315.

DALVI, R.R. (1985). Comparative assessment of the effect of solanine administered orally and intraperitoneally on hepatic dysfunction in male rats. Jpn. J. Vet. Sci., 47: 657-659. ELWOOD, J.M. (1976). Anencephalus, spina bifida and potato blight in Canada. Can. J. Public Health, 67: 122-126. GULL, S.D., ISENBERG, F.M. & BRYAN, H.H. (1970). Alkaloid toxicology of Solanum-tuberosum. Hort. Science, 5: 316. HANSEN, A.A. (1925). Two fatal cases of potato poisoning. Science, 61: 340-341. HARRIS, F.W. & COCKBURN, T. (1918). Alleged poisoning by potatoes. Am. J. Pharm., 90: 722-726. HARRIS, H. & WHITTAKER, M. (1962). Differential inhibition of the serum cholinesterase phenotypes by solanine and solanidine. Ann. Hum. Genet., 26: 71-76. HARVEY, M.H., McMILLAN, M., MORGAN, M.R.A. & CHAN, H.W.-S. (1985a). Solanidine is present in sera of healthy individuals and in amounts dependent on their dietary potato consumption. Hum. Toxicol., 4: 187-194. HARVEY, M.H., MORRIS, B.A., McMILLAN, M. & MARKS, V. (1985b).Measurement of potato steroidal alkaloids in human serum and saliva by radioimmunoassay. Hum. Toxicol., 4: 503-512. HARVEY, M.H., MORRIS, B.A., McMILLAN, M. & MARKS, V. (1986). Potato steroidal alkaloids and neural tube defects: serum concentrations fail to demonstrate a causal relation. Hum. Toxicol., 5: 249253. JADHAV, S.J., SHARMA, R.P. & SALUNKHE, D.K. (1981). Naturally occurring toxic alkaloids in foods. Crit. Rev. Toxicol., 9: 21-104. JELINEK, R., KYZLINK, V. & BLATTNY, C., Jr. (1976). An evaluation of the embryotoxic effects of blighted potatoes on chicken embryos. Teratology, 14: 335-342. KIRK, D. & MITTWOCH, U. (1975). Changes in the mitotic cycle induced by alpha-solanine. Humangenetik, 26: 105-111. KLINE, B.E., VON ELBE, H., DAHLE, N.A. & KUPCHAN, S.M. (1961). Toxic effects of potato sprouts and of solanine fed to pregnant rats. Proc. Soc. Exp. Biol. Med., 107: 807-809. KUBINSKI, H., GUTZKE, G.E. & KUBINSKI, Z.O. (1981). DNA-cell-binding (DCB) assay for suspected carcinogens and mutagens. Mutat. Res., 89: 95-136. MAGA, J.A. (1980). Potato glycoalkaloids. Crit. Rev. Food Sci. Nutr., 12: 371-405. MATTHEW, J.A., MORGAN, M.R.A., McNERNEY, R., CHAN, H.W.-S. & COXON, D.T. (1983). Determination of solanidine in human plasma by radioimmunoassay. Food Chem. Toxicol., 21: 637-640. McMILLAN, M. & THOMPSON, J.C. (1979). An outbreak of suspected solanine poisoning in schoolboys: examination of criteria of solanine poisoning. Q. J. Med., 48: 227-243.

MICHALSKA, L., NAGEL, G., SWINIARSKI, E. & ZYDOWO, M.M. (1985). The effect of alphasolanine on the active calcium transport in rat intestine. Gen. Pharmacol., 16: 69-70. MORGAN, M.R.A. & COXON, D.T. (1987). Tolerances: glycoalkaloids in potatoes. Ch. 7. In: Watson, D.H. (ed.). Ellis Horwood series in food science and technology: Natural toxicants in food: progress and prospects, Ellis Horwood, Chichester, England, pp. 221-230. MORRIS, S.C. & LEE, T.H. (1984). The toxicity and teratogenicity of Solanaceae glycoalkaloids particularly those of the potato (Solanum tuberosum): a review. Food Technol. Aust., 36: 118-124. MUN, A.M., BARDEN, E.S., WILSON, J.M. & HOGAN, J.M. (1975). Teratogenic effects in early chick embryos of solanine and glycoalkaloids from potatoes infected with late-blight, Phytophthora infestans. Teratology, 11: 73-77. NESS, E., JONER, P.E. & DAHLE, H.K. (1984). Alpha-solanine tested for mutagenicity with the Ames test. Acta Vet. Scand., 25: 145-147. NEVIN, N.C. & MERRETT, J.D. (1975) Potato avoidance during pregnancy in women with a previous infant with either anencephaly and/or spina bifida. Br. J. Prev. Soc. Med., 29: 111-115. NISHIE, K., GUMBMANN, M.R. & KEYL, A.C. (1971). Pharmacology of solanine. Toxicol. Appl. Pharmacol., 19: 81-92. NISHIE, K., NORRED, W.P. & SWAIN, A.P. (1975). Pharmacology and toxicology of chaconine and tomatine. Res. Commun. Chem. Pathol. Pharmacol., 12: 657-668. NORRED, W.P., NISHIE, K. & OSMAN, S.F. (1976). Excretion, distribution and metabolic fate of 3Halpha-chaconine. Res. Commun. Chem. Pathol. Pharmacol., 13: 161-171. PATIL, B.C., SHARMA, R.P., SALUNKHE, D.K. & SALUNKHE, K. (1972).Evaluation of solanine toxicity. Food Cosmet. Toxicol., 10: 395-398. PFUHL, E. (1899). [Regarding an outbreak of illness due to poisoning by solanine in potatoes] ber eine Massenerkrankung durch Vergiftung mit stark solaninhaltigen Kartoffeln. Deutsch. Med. Wochenschr.,25: 753-754. POSWILLO, D.E., SOPHER, D. & MITCHELL, S.J. (1972) Experimental induction of fetal malformation with "blighted" potato: a preliminary report. Nature, 239: 462-464. POSWILLO, D.E., SOPHER, D., MITCHELL, S.J., COXON, D.T., CURTIS, R.F. & PRICE, K.R. (1973). Investigations into the teratogenic potential of imperfect potatoes. Teratology, 8: 339-347. RENWICK, J.H. (1972). Hypothesis: anencephaly and spina bifida are usually preventable by avoidance of a specific but unidentified substance present in certain potato tubers. Br. J. Prev. Soc. Med., 26: 6788. RENWICK, J.H. (1982). Food and malformation. Practitioner, 226: 1947-1953.

RENWICK, J.H., CLARINGBOLD, W.D.B., EARTHY, M.E., FEW, J.D. & McLEAN, A.C.S. (1984). Neural-tube defects produced in Syrian hamsters by potato glycoalkaloids. Teratology, 30: 371-381. RODDICK, J.G. (1989). The acetylcholinesterase-inhibitory activity of steroidal glycoalkaloids and their aglycones. Phytochemistry, 28: 2631-2634. RUDDICK, J.A., HARWIG, J. & SCOTT, P.M. (1974). Nonteratogenicity in rats of blighted potatoes and compounds contained in them. Teratology, 9: 165-168. RHL, R. (1951). [Contribution on the pathology and toxicology of solanine] Beitrag zur Pathologie und Toxikologie des Solanins. Arch. Pharm., 284: 67-74. SHARMA, R.P., WILLHITE, C.C., WU, M.T. & SALUNKHE, D.K. (1978). Teratogenic potential of blighted potato concentrate in rabbits, hamsters, and miniature swine. Teratology, 18: 55-61. SHARMA, R.P., WILLHITE, C.C., SHUPE, J.L. & SALUNKHE, D.K. (1979). Acute toxicity and histopathological effects of certain glycoalkaloids and extracts of Alternaria solani or Phytophthora infestans in mice. Toxicol. Lett., 3: 349-355. SHARMA, R.P., TAYLOR, M.J. & BOURCIER, D.R. (1983). Subcellular distribution of alpha-chaconine in mouse hepatocytes. Drug Chem. Toxicol., 6: 219-234. SHARMA, R.P. & SALUNKHE, D.K. (1989). Solanum glycoalkaloids. In: Cheeke, P. R. (ed.). Toxicants of Plant Origin, Vol. 1 Alkaloids, CRC Press, Boca Raton, Florida. pp. 179-236. SLANINA, P. (1990a). Assessment of health-risks related to glycoalkaloids ("solanine") in potatoes: a Nordic view. Report from the Nordic working group on food toxicology and risk assessment. Vr Fda, 43: 1-14. SLANINA, P. (1990b). Solanine (glycoalkaloids) in potatoes: toxicological evaluation. Food Chem. Toxicol., 28: 759-761. SWINYARD, C.A. & CHAUBE, S. (1973). Are potatoes teratogenic for experimental animals? Teratology, 8: 349-357. WILLIMOTT, S.G. (1933). An investigation of solanine poisoning. Analyst, 58: 431. WILSON, G.S. (1959). A small outbreak of solanine poisoning. Monthly Bulletin, Ministry of Health (London), 18: 207-210. WILSON, A.M., McGANN, D.F. & BUSHWAY, R.J. (1983). Effect of growth, location and length of storage on glycoalkaloid content of roadside stand potatoes as stored by consumers. J. Food Prot., 46: 119-121. WOOD, F.A. & YOUNG, D.A. (1974). TGA in potatoes. Agric. Can. Publ. 1533: pp. 1-2.

CYANOGENIC GLYCOSIDES
EXPLANATION Cyanogenic glycosides are phytotoxins which occur in at least 2000 plant species, of which a number of species are used as food in some areas of the world. Cassava and sorghum are especially important staple foods containing cyanogenic glycosides (Conn, 1979a,b; Nartey, 1980; Oke,1979, 1980; Vennesland et al., 1982; Rosling, 1987). There are approximately 25 cyanogenic glycosides known. The major cyanogenic glycosides found in the edible parts of plants used for human or animal consumption are summarized in Table The potential toxicity of a cyanogenic plant depends primarily on the potential that its consumption will produce a concentration of HCN that is toxic to exposed animals or humans (see Table 2). Several factors are important in this toxicity: The first aspect is the processing of plant products containing cyanogenic glycosides. When the edible parts of the plants are macerated, the catabolic intracellular enzyme -glucosidase can be released, coming into contact with the glycosides. This enzyme hydrolyzes the cyanogenic glycosides to produce hydrogen cyanide and glucose and ketones or benzaldehyde. The hydrogen cyanide is the major toxic compound causing the toxic effects. Plant products (notably cassava), if not adequately detoxified during the processing or preparation of the food, are toxic because of the release of this preformed hydrogen cyanide. The second aspect is the direct consumption of the cyanogenic plant. Maceration of edible parts of the plants as they are eaten can release -glucosidase. The -glucosidase is then active until the low pH in the stomach deactivates the enzyme. Additionally, it is possible that part of the enzyme fraction can become reactivated in the alkaline environment of the gut. At least part of the potential hydrogen cyanide is released, and may be responsible for all or part of the toxic effect of cyanogenic glycosides in the cases of some foods. Table 1: The occurrence of cyanogenic glycosides in major edible plants (Conn, 1979a,b) Plant species Latin name Prunus amygdalus. Sorghum album, Sorghum bicolor. Manihot esculenta, M. carthaginensis Phaseolus lunatus Manihot carthaginensis Phaseolus lunatus Prunus species e.g., P. avium, P. padus, P. persica, P. macrophylla. Bambusa vulgaris

Cyanogenic glycosides Amygdalin Dhurrin Linamarin Lotaustralin Prunasin Taxiphyllin

Common name almonds sorghum cassava lima beans cassava lima beans stone fruits bamboo shoots

The third aspect is that the cyanogenic glycosides taken up intact with the food are (partly) hydrolyzed by the -glucosidase activity of the bacteria of the gut flora of animals or humans (Conn, 1979a,b; Oke, 1979, 1980; Nartey, 1980; Rosling, 1987; Gonzales & Sabatini, 1989). Cyanide, released from a cyanogenic glycoside in food by -glucosidase either of plant or from gut microflora origin and taken up, follows the known cyanide metabolic pathway and toxicokinetics both for animals and man. Cyanide is detoxified by the enzyme rhodanese, forming thiocyanate, which is excreted

by urine (Conn, 1979a,b; Oke, 1979, 1980). Due to several factors influencing hydrolysis of cyanogenic glycosides and the confounding influence of nutritional status (such as riboflavin, vit. B12, sodium, methionine intake) human case studies and epidemiological studies of the chronic toxicological effects have shown very variable results and were not conclusive. In addition, the data in these studies are rarely of a quantitative character (Conn, 1979a,b; Oke, 1979, 1980; Nartey, 1980; Rosling, 1987). In several studies both in animals and man the toxicity of cyanogenic glycosides is often expressed as mg releasable cyanide. Table 2: Concentration of cyanide in some tropical foodstuffs (Summarized in Nartey, 1980). Plant/tissue Cassava(bitter)/dried root cortex Cassava(bitter)/leaves Cassava(bitter)/whole tubers Cassava(sweet)/leaves Cassava(sweet)/whole tubers Sorghum/whole immature plant Bamboo/immature shoot tip Lima beans from Java (coloured) Lima beans from Puerta Rico (black) Lima beans from Burma (white) mg HCN/kg 2450 310 395 468 462 2500 8000 3120 3000 2100

Because this monograph first discusses the toxicity of cyanide as a basis for understanding that of cyanogenic glycosides, a modified form of the general monograph format has been used, presenting first biological data for cyanide, then that for cyanogenic glycosides. CYANIDE 2. BIOLOGICAL DATA 2.1 Biochemical aspects 2.1.1.1 Absorption, distribution, and excretion Hydrogen cyanide after oral administration is readily absorbed (it is also readily absorbed after inhalation exposure and through skin and eyes). After absorption, cyanide is rapidly distributed in the body through the blood. The concentration of cyanide is higher in erythrocytes than in plasma. It is known to combine with iron in both methaemoglobin and haemoglobin present in erythrocytes. The cyanide level in different human tissues in a fatal case of HCN poisoning has been reported: gastric content; 0.03, blood; 0.50, liver; 0.03, kidney; 0.11, brain; 0.07, and urine; 0.20 (mg/100 g) (EPA, 1990). The pharmacokinetics of 14CN- and S14CN- in rats exposed to these agents in diet for 3 weeks was investigated. All tissues contained radioactivity 9 h after intraperitoneal injection of 14CN-; highest radioactivity was found in the stomach (18%). Eighty per cent of this activity was in the form of thiocyanate. At this point 25% of the dose had already been eliminated in the urine and 4% in the expired air. When S14CN- was given per os to rats with elevated plasma thiocyanate levels due to chronic oral exposure to cyanide, most of the activity was eliminated in the urine and only small amounts were found

in the faeces. This indicated the existence of a gastrointestinal circulation of thiocyanate (Okoh & Pitt, 1981). The excretion of an acute oral dose of 14C-labelled cyanide in urine, faeces and expired air was studied in rats (12 animals/group) pretreated orally for 6 weeks with unlabelled KCN or a control diet. Urinary excretion was the main route of elimination of 14C-labelled cyanide in these rats, accounting for 83% of the total excreted radioactivity at 12 h and 89% of the total excreted radioactivity at 24 h. The major metabolite of cyanide excreted in urine was thiocyanate, and this metabolite accounted for 71% and 79% of the total urinary activity at 12 h and 24 h, respectively. Only 4% of the mean total activity excreted was found in expired air after 12 h, and this value did not change after 24 h. Of the total activity in expired air in 24 h, 90% was present as carbon dioxide and 9% as cyanide. When these results were compared with those observed for control rats, it was clear that the mode of elimination of cyanide carbon was altered in neither urine nor breath by the chronic intake of cyanide (Okoh, 1983). Golden hamsters exposed to cyanide by subcutaneous infusion appeared to excrete only a relatively low percentage (10-15%) of the dose as thiocyanate in the urine (Doherty et al., 1982). The major defence of the body to counter the toxic effects of cyanide is its conversion to thiocyanate mediated by the enzyme rhodanese. The conversion of cyanide to the less toxic thiocyanate by rhodanese was discovered by Lang (1933). Thiosulfate and 3-mercapto-pyruvate can act as sulfur donors, but neither free cystine nor cysteine can. The enzyme contains an active disulfide group which reacts with the thiosulfate and cyanide. The trivial name rhodanese is more widely used than that assigned by the Enzyme Commission [thiosulfate-cyanide sulfur transferase, EC. 1.8.1.1]; it has been inappropriately called rhodanase in several reports. The rhodanese-catalyzed irreversible conversion of cyanide to thiocyanate, in the presence of thiosulfate, provides a means for the treatment of cyanide poisoning. Since the enzyme, which is usually localized in the mitochondria in different tissues, is relatively abundant, but in sites which are not readily accessible to thiosulfate, the limiting factor for the conversion of cyanide is thus thiosulfate (EPA, 1990). The overall rate of in vivo detoxification of cyanide may be influenced by several minor reactions. Cystine may directly react with cyanide to form 2-imino-thiazolidine-4-carboxylic acid which is excreted in saliva and urine. Traces of hydrogen cyanide may be found in expired air, saliva, sweat and urine. A minor amount may be converted into formic acid which may be excreted in urine or participate in the metabolism of one carbon compound. One minor detoxification route is the combination of cyanide with hydroxycobalamine (vitamin B12) to form cyanocobalamine which is excreted in urine and bile. It may be reabsorbed by the intrinsic factor mechanism at the level of the ileum allowing effective recirculation of vitamin B12. Methaemoglobin effectively competes with cytochrome oxidase for cyanide and its formation from haemoglobin, effected by sodium nitrite or amylnitrite, is exploited in the treatment of cyanide (EPA, 1990). It has been reported that other species have lower rhodanese activity than the rat and hence the rat may be able to convert cyanide to thiocyanate more easily than other species (Himwich & Saunders, 1948). 2.1.2 Biotransformation No information available. 2.1.3 Effects on enzymes and other biochemical parameters

Cyanide causes a decrease in the utilization of oxygen in the tissues, producing a state of histotoxic anoxia. This occurs through inactivation of tissue cytochrome oxidase by cyanide, which combines with Fe3+/Fe2+ contained in the enzyme. The enzyme-cyanide complex dissociation constant has been found to be 1 * 10-6 and 1 10-4 (moles/l) for the oxidized and reduced form of the enzyme, respectively. Thus, the affinity of cyanide for the oxidized form of the enzyme is two orders of magnitude higher than for the reduced form. However, the rate of reaction of cyanide with the reduced enzyme is twice the rate of reaction with the oxidized form. Cyanide can inhibit several other metalloenzymes most of which contain iron, copper or molybdenum (e.g., alkaline phosphatase, carbonic anhydrase), as well as enzymes containing Schiff base inter-mediates (e.g., 2-keto-4-hydroxyglutarate aldolase). The effect of sublethal doses of cyanide on the metabolism of glucose in mice has been studied using radiorespirometric techniques (Solomonson, 1981). Cyanide causes an increase in blood glucose and lactic acid levels and a decrease in the ATP/ADP ratio indicating a shift from aerobic to anaerobic metabolism. Cyanide apparently activates glycogenolysis and shunts glucose to the pentose phosphate pathway decreasing the rate of glycolysis and inhibiting the tricarboxylic acid cycle (EPA, 1990). 2.2 Toxicological studies 2.2.1 Acute toxicity studies Lethal doses of HCN in mg/kg bw were reported for mouse, 3.7; dog, 4.0; cat, 2.0 and for cattle and sheep 2.0 (Summarized by Conn, 1979a). Table 3. Acute toxicity of cyanide Species Mouse Rat Guinea-pig Rabbit Cat Dog Monkey Mouse Rat Dog Rabbit Guinea-pig Dog Mouse Rat . * as summarized in Route i.v. i.v. i.v. i.v. i.v. i.v. i.v. s.c. i.v oral i.v. oral s.c. s.c. i.v. oral i.v oral i.p. LD50 (mg/kg bw) 0.99 (HCN) 0.81 (HCN) 1.43 (HCN) 0.66 (HCN) 0.81 (HCN) 1.34 (HCN) 1.30 (HCN) 6.0 (KCN) 2.5 (KCN) 10-15 (KCN) 2.5 (KCN) 5.3 (KCN) 2.2 (NaCN) 5.8 (NaCN) 2.8 (NaCN) 598 (NaSCN) 484 (NaSCN) 765 (NaSCN) 540 (NaSCN) References EPA 1990* EPA 1990* EPA 1990* EPA 1990* EPA 1990* EPA 1990* EPA 1990* WHO, 1965* WHO, 1965* WHO, 1965* WHO, 1965* WHO, 1965* WHO, 1965* WHO, 1965* WHO, 1965* WHO, 1965* WHO, 1965* WHO, 1965* WHO, 1965*

2.2.2 Short-term toxicity studies

2.2.2.1 Rats In a 13-week toxicity study male Sprague-Dawley rats (approximately 30 rats/group) were administered KCN in the drinking water. The dose levels were 40, 80 and 160/140 mg KCN/kg bw/24 h. Three control groups were used, respectively a normal drinking water ad libitum, a "paired drinking" group (parallel to the high dose level KCN) and a group receiving drinking water with 10% ethyl alcohol. In addition, one group received drinking water with KCN (80 mg/kg bw) and 10% alcohol. Behaviour, external appearance, body weight, food consumption (daily) and drinking water consumption (twice weekly) were recorded frequently. Extensive haematological, clinical chemical (in serum) and urine analyses were carried out in 5 animals per group in week 6 and week 13. Autopsy and macroscopy were performed after 13 weeks (approximately 20 animals/group) and 11 organs were weighed. Histopathological examination was performed in brain, kidneys, heart, liver and testes of these animals. In addition thyroids of the control, the "pair drinking" control and the high-dose group (160/140 mg/kg bw/day) were examined. There was a clear indication that reduced food consumption and body weight in the KCN groups were caused by a decrease in water consumption due to decreased palatibility. Urinalyses revealed a higher level of protein in the animals receiving KCN. The amounts of protein determined showed a clear correlation to the increasing doses of KCN, as did the drinking water. Several changes in absolute organ weights were seen in the 160/140 mg KCN/kg bw/day group. Relative weights of organs were very slightly increased in the 40, slightly increased in the 80 mg and clearly increased in the 160/140 mg KCN/kg bw groups. The thymus weight was, however, reduced in the high-dose group. Histopathological examination revealed no indication of damage to the brain, heart, liver, testes, thyroids nor kidneys due to treatment with KCN (Leuschner et al., 1989b). 2.2.3 Long-term carcinogenicity studies No data on the carcinogenicity of hydrogen cyanide have been published. However, anticarcinogenic effects of cyanide have been reported. Longevity of mice with transplanted Ehrlich ascites tumours and Sarcoma 180 was increased 20 to 70% on i.p. injection of sodium cyanide in the dose range 0.75 to 2.0 mg/kg bw (EPA, 1990). 2.2.4 Reproduction studies 2.2.4.1 Rats A short-term reproductive study (49 day study in adults and 28 day study in pups) was performed to evaluate the cumulative effects of adding 500 mg KCN/kg to cassava root flour-based diet in pregnant rats. This meal was prepared from a low-HCN cassava variety (21 mg HCN/kg feed). High dietary level of KCN did not have any marked effect in gestation and lactation performance of female rats. No carry-over effect of high cyanide-containing diet fed during gestation was observed on lactation performance. The high cyanide-containing diet, however, significantly reduced feed consumption and daily growth rate of the offspring when fed during post-weaning period. Protein efficiency ratio was not only reduced by the cyanide diet during post-weaning growth phase but there was an additional carry-over effect from gestation. Serum thiocyanate was significantly increased in lactating rats and their offspring during lactation and in the postweaning growth phase of the pups. No apparent carry-over effect was noticed on this parameter. Rhodanese activity in liver and kidneys was unaffected by feeding the high cyanide diet during gestation, lactation, nor during postweaning growth (Tewe & Maner, 1981b).

2.2.5 Special studies on embryotoxicity and teratogenicity 2.2.5.1 Hamsters Pregnant golden hamsters were exposed to sodium cyanide on days 6-9 of gestation by infusion via subcutaneously implanted osmotic minipumps. Cyanide (0.126-0.1295 mmol/kg/h) induced high incidences of resorptions and malformations in the offspring. The most common abnormalities observed were neural tube defects (Doherty et al., 1982). 2.2.6 Special studies on the thyroid gland 2.2.6.1 Rats A group of 10 male rats was fed a 10% casein diet containing added methionine, vitamin B12, iodine and potassium cyanide (1500 mg/kg feed) for nearly one year. Compared to a control group not receiving cyanide, depression of body-weight was observed throughout the study period, but there were no deaths nor clinical signs of toxicity. Depression of both plasma thyroxine and thyroxine secretion rate suggestive of depressed thyroid function were evident at 4 months but less so after 1 year. At autopsy the animals were found to have enlarged thyroids and this may have been the mechanism of adaptation. Some differences in the histopathology of the spinal cord, notably the white matter, were also found between controls and cyanide-treated animals (Philbrick et al., 1979). 2.2.6.2 Pigs Performance and metabolic and pathological changes were evaluated in 48 growing pigs fed different levels of dietary protein (9 and 16%), cyanide, and iodine (0 and 0.36 mg iodine/kg feed) during 56 days. Protein deficiency reduced urinary iodine excretion and the concentrations of protein, protein-bound iodine (PBI) and thiocyanate in serum. It also reduced liver rhodanese activity and caused a decrease in urinary thiocyanate excretion which was not significant. Dietary cyanide increased urinary thiocyanate and iodine excretion and serum PBI. Pathological studies showed that cyanide treatment had no marked effect on the microanatomy of the tissues examined. Dietary iodine deficiency caused histological changes in the thyroid gland and bone which suggested a decline in metabolic activity. Iodine deficiency caused hyperplastic goitre in the experimental animals (Tewe & Maner, 1980). 2.2.7 Genotoxicity Two negative and one marginally positive genotoxicity studies for cyanide have been reported. Potassium cyanide was not mutagenic in Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98 nor TA100 with or without S-9 liver microsomes. Cyanide was also negative in recombinant-assay in Bacillus subtilis. One study reported marginally mutagenic activity of HCN to Salmonella typhimurium strain TA100 in the absence of S-9 mix. In the same study no mutagenic activity in strain TA98 with or without S-9 mix was observed (EPA, 1990). An in vitro Ames test with HCN in Salmonella strains TA1537, TA1538 and TA98 for detection of frame shift mutation and TA1535 and TA100 for base-pair substitutions was performed with and without metabolic activation of a S-9 microsome mixture. There was no indication of mutagenic properties under these conditions (Leuschner et al., 1983a). An in vivo mutagenicity study in Chinese hamsters detecting chromosomal aberrations with HCN orally administered to Chinese hamsters was carried out. Preparations of metaphase cells were studied for structural chromosome aberrations after 6, 24 and 48 h after oral administration of 0.4 mg HCN/kg bw.

The incidence of aberrations or gaps was within the spontaneous range. Neither multiple aberrations nor pulverised metaphases were found. There was no indication of mutagenic properties relative to structural chromatid or chromosome damage (Leuschner et al., 1983b). A gene mutation assay in cultured Chinese hamster cells, V79 (genetic marker HGPRT) both in the presence and absence of metabolic activation system was carried out with KCN. The duration of the exposure with the test substance was 24 h in the experiments without S-9 mix and 2 h in the experiments with S-9 mix. The test compound dose levels employed were chosen following a preliminary toxicity experiment. The dose-levels for the main study were 400, 800, 1000, 2000 and 3000 g/ml without S9 mix and 1000, 2000, 3000, 4000, 6000, 8000 and 10 000 g/ml with S9 mix. KCN was tested up to a high cytotoxicity in the absence and presence of metabolic activation. Under the present test conditions KCN was negative in the V79 mammalian cell mutagenicity test (Leuschner et al., 1989a). 2.2.8 Special studies on nervous system A special study on the behavioural effects of chronic sublethal dietary cyanide (KCN; 0.4, 0.7 and 1.2 CN-/kg bw) in juvenile swine, mimicked the situation of free CN- intake in Liberia due to eating cassavabased foods. There were two clear behavioural trends: increasing ambivalence and slower response time in reacting to various stimuli and 2) an energy conservation gradient influencing which specific behaviours would be modified in treated animals. Serum SCN- was positively correlated with daily CNintakes. CN- treatment diminished T3 and T4 levels but elevated fasting blood glucose values (CollierJackson, 1988). Neuronal lesions in several animal species have been produced by chronic cyanide intoxication either by injection of unbuffered alkaline cyanide salts or by inhalation of hydrogen cyanide. The neuropathological changes include areas of focal necrosis especially around the centrum ovale, corpus striatum, corpus callosum, substantia nigra, anterior horn cells, and patchy demyelination in the periventricular region. In some species, the earliest effects may be on the oligodendroglia and hence myelin lesions may precede neuronal damage. Bass (1968) showed that in rats chronic cyanide intoxication produces myelin loss by its primary effect on glial cells followed by breakdown of myelin. Brierly et al. (1977) reported myelin damage and changes in the oligodendroglia in cyanide poisoning in rats. Clark (1936) described fatty degeneration of the liver and secretory tubules of the kidney in rats subjected to chronic cyanide intoxication. In these animal experiments relatively large doses of cyanide were given, often sufficient to cause partial asphyxia. Therefore it was doubtful whether the neuropathological effects demonstrated were due to asphyxia or chronic cyanide intoxication so they did not appear to have an obvious parallel to human exposure at lower levels. It is noteworthy, however, that changes in optic nerves and tracts occur consistently only in primates (Ferraro, 1933; Hurst, 1940; Lessell, 1971). In a carefully controlled experiment in which small weekly doses of cyanide were administered over several months to rats, neuronal degeneration and demyelination were reported (Smith et al., 1963). Williams and Osuntokun (1969) found that the demyelination of peripheral nerves induced in rodents by cyanide injection bore a striking resemblance to the lesions found in biopsy specimen of peripheral nerves of Nigerian patients who suffer from tropical neuropathy (reviewed by Osuntokun, 1981). 2.3 Observations in humans 2.3.1 Acute toxicity studies in humans The acute oral lethal dose of HCN for human beings is reported to be 0.5-3.5 mg/kg bw corresponding to 1.0-7.0 mg/kg bw of KCN. The clinical signs are well described (Montgomery, 1969; Gosselin et al., 1976) and include headache, dizziness, mental confusion, stupor, cyanosis with twitching and convulsions, followed by terminal coma (Conn, 1979a).

The acute oral lethal dose of HCN for man was reported to be 60 mg (Sinclair & Jeliffe, 1961). For man the acute oral dose of HCN is usually given as 50-90 mg and for potassium cyanide as 200 mg, corresponding to 81 and 110 mg HCN respectively (Lehman, 1959). Data on the oral lethal dose of cyanide for man in four cases of suicide, calculated from the amount of HCN absorbed in the body at the time of death, and from the amount of HCN found in the digestive tract, differed considerably ((calculated as mg HCN: 1450 (62.5 kg bw), 556.5 (74.5 kg), 296.7 (50.7 kg) and 29.8 (51 kg)) (Geitler & Baine, 1938). This corresponds to doses varying from 0.58-22 mg/kg bw (in WHO, 1965). CYANOGENIC GLYCOSIDES 2. Biological data 2.1 Biochemical aspects 2.1.1 Absorption, distribution, and excretion Wistar rats (4 animals/sex/group) were given 50 mg amygdalin/rat respectively, intravenously and orally after an overnight fast. During the experiment and the night before they were kept in metabolic cages which allowed collection of urine without faeces and minimized coprophagy. After intravenous and oral administration fractions of the dose excreted by the rat as unchanged amygdalin were 70% and 0.8%, respectively. The fraction excreted as prunasin after intravenous administration was 6.6%, whereas it was 39% of the total dose after oral administration (Rauws et al., 1982). In a toxicokinetic study 10 male rats (100-120 g) were given 50 mg pure linamarin (dissolved in water, volume 0.5 ml) and 6 male rats were given water alone by stomach tube. Seven rats dosed with 50 mg linamarin died within 4 h. In a second trial 6 male rats were given 30 mg linamarin. Following dosing, urine and faeces were collected after 24, 48 and 72 h, and heparinized blood samples were taken from the optic vein or lateral tail vein. Blood was taken after 30, 40, 60, 80 and 100 min and at 2, 4, 8, 24 and 48 h. No intact linamarin was detected in faeces nor blood of the rats dosed with 30 mg (300 mg/kg bw). No linamarin in the faeces of the three surviving rats dosed with 50 mg (500 mg/kg bw) was detected, however blood and urine were not examined in these animals. Linamarin was excreted in the urine at a level of 5.65 mg (cumulated after 72 h) along with 0.823 mg of thiocyanate. These findings indicate that linamarin was absorbed intact in considerable proportion and was partially hydrolyzed (Barrett et al., 1977). In a toxicokinetic study beagle dogs (4 animals/sex/group) were administered 500 mg amygdalin in 10 ml solution respectively, intravenously and orally after overnight fast. Blood was sampled from jugular vein and urine was collected by a funnel. Faeces were removed from the funnel. The major part of the dose (71%) was recovered in the urines collected during 6 h following intravenous amygdalin administration. The fraction of the dose excreted by glomerular filtration was calculated using the ratio of diatrizoate (which was administered simultaneously) clearance to amygdalin clearance, showing that 97% of the amount of amygdalin to be expected was recovered from the urine. The result of the experiments after intravenous

administration were analyzed assuming a two-compartment model. The distribution T alpha was 0.10 and the elimination T 0.57. No prunasin was detected in urine (detection limit = 0.2% of dose). After oral administration of amygdalin a very low maximal plasma level is found after approximately 0.75 h. Only 2.3% of the amygdalin was systematically available (absolute bioavailablity). Prunasin was found in plasma and urine of dogs. In the urine collected during 6 h following amygdalin administration only about 1% of the dose was recovered unchanged and 21% of the dose was identified as prunasin (Rauws et al., 1982). In a toxicokinetic study prunasin was administered intravenously and orally as 100 mg doses to female dogs (2/group; 10 kg) after an overnight fast. Blood was sampled from jugular vein and urine was collected by a funnel. The results of the experiment after intravenous administration were analyzed assuming a two-compartment model. The prunasin results were compared to those obtained with amygdalin in a earlier experiment. The distribution T alpha was 0.08 h and elimination T was approximately 0.64 h. Prunasin was absorbed to a large extent after oral administration. The absolute bioavailability after oral administration was 50% of the administered dose. The volume of distribution (0.34 L/kg) and the clearance (0.55 L/kg h) are larger than those of amygdalin (respectively 0.19 L/kg and 0.39 L/kg h). The oral bioavailability of prunasin is considerably greater, whereas amygdalin is only slightly (2.3%) absorbed (Rauws et al., 1983). In a toxicokinetic study pure linamarin, at a dose level of 300 mg/kg bw, was administered in food to a group of Wistar rats maintained on vitamin B2-deficient, -sufficient, and -excess diets for 5 weeks and to another group of kwashiorkor rats. Free and total cyanide, intact linamarin and thiocyanate levels were estimated in urine and faeces obtained at 0, 24, 48 and 72 h periods and in blood samples obtained 72 h after the compound had been administered. There was no detectable cyanide nor intact linamarin in the faecal samples. Rats on vitamin B2-sufficient and B2-excess diets excreted higher total and free cyanide in urine than the respective vitamin B2-deficient groups. Most of the linamarin was degraded after 24 h. The rate of breakdown of the glycoside within the first 24 h was slowest for zero and half normal vitamin B2 status rats as evidenced by appearance of the glycoside in large quantities in the urine. The kwashiorkor rats, on the other hand, excreted less thiocyanate than the controls. In addition, their control group excreted most of the thiocyanate in the first 24 h, whilst the kwashiorkor rats excreted most of the thiocyanate in the first 48 h. Dietary protein

deficiency prolongs the time of metabolism and hence increases the toxicity of cyanogenic glycosides in the body (Umoh et al., 1986). 2.1.2 Biotransformation Strained ruminal fluid was collected from cattle fed five diets (concentrate diet, freshly harvested alfalfa, cubed alfalfa, alfalfa hay and orchard grass) to determine in vitro rates of cyanogenesis from the glycosides amygdalin, prunasin and linamarin. Rates of dissociation for the corresponding aglycones, benzaldehyde cyanohydrin and acetone cyanohydrin were also determined. Hydrogen cyanide (HCN) in ruminal fluid was determined with a modified method of HCN analysis that independently measured the overall rate of cyanogenesis and the non-enzymatic dissociation of cyanohydrins, the intermediate products in the degradation of cyanogenic glycosides to HCN. Rate of dissociation of cyanohydrins in ruminal fluid was pH-dependent, with high rates of dissociation (as expressed by the rate constant or half-life of reaction) occurring at pH >6 and slower rates at pH 5 to 6. Cyanohydrin dissociation was most rapid when cattle were fasted for 24 to 48 h and ruminal pH was high; rate of dissociation was much slower during feeding and digestion. When the glycosides were examined, highest rates of cyanogenesis (mg HCN/L/s) were observed after a 24 h postprandial period. The rates were highest after feeding hay: 0.019 for amygdalin, 0.033 for linamarin and 0.048 for prunasin. Hence cattle are most susceptible to poisoning by cyanogenic plants when the pH of ruminal fluid is elevated, leading to rapid dissociation, and also when the activity of -glucosidase is adequate for rapid hydrolysis of glycosidic bonds. Rates of cyanogenesis were higher when ruminal inocula were from cattle fed fresh alfalfa or cubed alfalfa hay rather than from those fed grain or long hay. Rates of HCN production were lowest using inocula from cattle fed grain; rates for the three glycosides were negligible at the 3 and 6 h postprandial sampling times. In agreement with previous studies, prunasin was degraded in ruminal fluid much more rapidly than linamarin or amygdalin (Majak et al., 1989). In a comparative metabolism study rates of cyanide liberation resulting from hydrolysis of the cyanogenic glycosides linamarin, amygdalin and prunasin by a crude -glucosidase prepared from hamster caecum were studied in vitro. In addition, hamster blood cyanide and thiocyanate concentrations were determined at 0.5, 1, 2, 3, and 4 h after oral dose of 0.44 mmol linamarin or amydalin/kg bw. Plots of cyanide liberated versus time for linamarin and prunasin yielded straight lines, whereas for amygdalin the plot was curvilinear; the rate of cyanide release increased with time. At 10-3 M substrate concentrations, the averaged rates of hydrolysis of prunasin, amygdalin and linamarin were 1.39, 0.57 and 0.13 nmol/min/mg protein, respectively. Lineweaver-Burk plots

yielded apparent Km and Vmax values of 3.63 * 10-5 M and 0.13 nmol/min/mg protein, respectively for amygdalin, and 7.33 * 10-3 M and 1.04 nmol/min/mg protein, respectively for linamarin. Blood cyanide concentrations following amygdalin treatment of the hamster reached their highest level (130 nmol/ml) 1 h after dosing and remained elevated until 3 h after treatment. Blood cyanide concentrations following linamarin treatment reached their highest level (116 nmol/ml) after 3 h and then declined immediately. Area under the blood cyanide concentration-time curve was 395 nmol h/ml for amygdalin and 318 nmol h/ml for linamarin. The results suggest a faster rate of enzymatic hydrolysis and cyanide absorption for amygdalin than for linamarin (Frakes & Sharma, 1986). In humans, pharmacological studies have shown that amygdalin is broken down to HCN, benzaldehyde and glucose by enzymes found in gut bacteria, but not intracellularly in humans. Animal and human tissues contain no significant concentrations of -glucosidase, the only known activating enzyme of hydrolysis of cyanogenic glycosides in vivo (Dorr & Paxinas, 1978). Cyanogenic glycosides are hydrolyzed by -glucosidase produced by intestinal bacteria to glucose, HCN and benzaldehyde or acetone. Benzaldehyde is oxidized to benzoic acid (and subsequently to hippuric acid) or salicylic acid isomers. Thiocyanate is present in body fluids; blood, urine, saliva, sweat and tears (Oke, 1979). The cyanide-yielding capacity of insufficiently processed cassava probably occurs as linamarin, or an intermediate break-down product, from which cyanide may be yielded in the gut by action of microbial enzymes. Significant amounts of linamarin are observed in the urine after consumption of insufficiently processed cassava as well as after consumption of other plants containing linamarin. These results indicate that linamarin, if not metabolized in the gut, will be absorbed and excreted in the urine without causing exposure to HCN. About 80% of ingested cyanide will be turned into thiocyanate and is excreted in the urine after a short period (Rosling, 1987). 2.1.3 Effects on enzymes and other biochemical parameters No information available. 2.2 Toxicological studies 2.2.1 Acute toxicity studies Table 4. Acute toxicity studies of cyanogenic glycosides or cyanogenic plant tissues Acute toxicity

Species Mouse Rat Rat

Sex ? ? ?

Route i.p. i.v oral

LD50(mg/kg bw) 0.1 mmole amygdalin/kg 20 000 linamarin 450 linamarin

References Solomonson, 1981 Oke, 1979 Oke, 1979

A dose of 25 mg linamarin (250 mg/kg bw) fed to rats (100-120 g bw) caused clinical signs of toxicity, including apnoea, ataxia and paresis. These symptoms were very marked in the absence of methionine supplementation, 50% of these rats died within 4 h. In the presence of adequate methionine supplementation, 10% of rats died and about 40% showed no signs of toxicity. The activity of Na+K+-dependent ATPase was reduced in much the same way as it was by the glycoside, digitalis (reviewed by Oke, 1980). In a toxicokinetic study 7 out 10 rats (100 g bw) died after administration of 50 mg linamarin by stomach tube (Barrett et al., 1977). Oral doses of 100, 120 and 140 mg linamarin/kg bw given by stomach tube to hamsters (90 g bw) produced signs of cyanide intoxication in a large percentage. The signs appeared within 1 h after dosing included dyspnoea, hyperpnoea, ataxia, tremors and hypothermia. Two animals dosed with 140 mg/kg bw and one animal dosed with 120 mg/kg bw died within 2 h of dosing. The signs of poisoning were greatly reduced or gone within 3 h after treatment in the surviving animals. No relationship between length of intoxication and dose was observed (Frakes et al., 1985). Hamsters have been reported to be more susceptible than rats to the acute toxic effects of orally administered amygdalin and prunasin (Willhite, 1982). A species difference in reaction to cyanogenic glycosides is observed due to a difference of detoxifying ability due to anatomical structure. Ruminants, e.g., cattle and sheep are supposed to be more susceptible to the acute toxic effects because of their larger flora of microorganisms and considerable quantities of the enzyme emulsine which hydrolyzes the glycoside (Oke, 1979). 2.2.2 Short-term toxicity studies 2.2.2.1 Rats Albino female rats (10 animals/group) were fed ad libitum one of the following diets, A; a normal laboratory diet (control), B; a 50% gari diet (Nigerian preparation of cassava), C; a raw cassava diet, D; a diet containing 5 g KCN/100 g and E; a diet containing 10 g KCN/100 g during a 14-day period. The 50% gari diet caused no significant biochemical nor haematological changes in the female

rats, whereas for both the raw cassava diet and the KCN diets a decrease of Hb, PCV, total serum protein concentration and T4 concentration was observed. In the 50% gari diet group, and to a greater extent, in the other treatment groups, the serum thiocyanate levels were increased. The body weight gain was not significantly decreased in the 50% gari group, whereas the other treatment groups showed instead of gain a loss of body weight (Olusi et al., 1979). 2.2.2.2 Guinea-pigs In a 24-day toxicity experiment guinea-pigs (8 animals/group) were dosed daily with, respectively, laetrile (10 mg amygdalin) and 8 mg KCN/kg bw with and without ascorbic acid (100 mg). No significant effect on the body weight nor liver weight was observed. However, treatment with laetrile alone for 4, 16 or 24 days, respectively, resulted in a significant increase in urinary levels of thiocyanate. The increase was less in animals treated with vitamin C. In guinea-pigs treated with 8 mg KCN/kg bw, toxic effects were seen as evidenced in slight tremors in 3 of the 8 animals, which recovered within 5 min. All animals in the KCN group which were supplemented with ascorbate showed severe tremors, motor ataxia, bizarre neuromuscular manifestations and rhythmic head movements. The toxicity of KCN increased with elevation of vitamin C, whereas urinary excretion of thiocyanate decreased (Basu, 1983). 2.2.2.3 Chickens In two feeding experiments (respectively 63 and 56 days) one day old broiler chickens (male and female) were fed a diet containing 0, 10, 20 or 30% cassava, respectively. The animals were studied for haematological and histopathological effects. The cassava diet studied in the 1st experiment consisted of a high-cyanide-containing cassava root meal (CRM) supplying 300 mg of total cyanide/kg, most of it in the form of cyanogenic glycosides. The cassava diet in the 2nd experiment also contained cassava foliage meal (CFM) supplying 156 mg total cyanide/kg. In the 1st experiment 26 chickens per group were used and in the 2nd experiment 160 chickens were used for the cassava groups and 80 for the control group. No changes in the haematological parameters due to cassava were seen. Addition of up to 30% CRM failed to adversely affect broiler survival, performance nor feed efficiency, but the inclusion of CFM in the experimental diets increased mortality, decreased weight gain and decreased feed efficiency. In both experiments, increased quantities of dietary cassava cyanate were associated with increased (P < 0.05) blood serum thiocyanate concentrations. Histopathological examination of thyroid, liver and kidney revealed no appreciable alterations due to the cassava feeding, however there was no conclusive evidence of cyanide or thiocyanate effects on

thyroid activity. Aflatoxin contamination appeared to have contributed to the high mortality rate associated with CFM diets. The results showed that broilers were tolerant of relatively high levels of dietary cyanogenic glycosides (Gomez et al., 1988). 2.2.3 Long-term/carcinogenicity studies No information available. 2.2.4 Reproduction studies 2.2.4.1 Rats In a one-generation reproduction study albino female rats (10 rats/group) were fed ad libitum one of the following diets: A, a normal laboratory diet, B, a 50% gari diet (Nigerian preparation of cassava), C, a raw cassava diet, D, a diet containing 5 g KCN/100 g and E, a diet containing 10 g KCN/100 g. After 2 weeks rats in each group were mated with 5 adult males fed normal diet. Pregnant rats from each group were maintained on their respective diets. After littering, the newborn rats were studied for postnatal development. After 21 days F1 rats were put for another 4 weeks on their respective diets. The offspring of the rats fed the 50% gari diet had significantly lower birth weights and brain weights and never attained the same adult weights as those of the controls. The adult female rats fed a diet consisting entirely of raw cassava had significantly reduced haematological and biochemical parameters (Hb, PCV, serum protein and T4 concentration). This diet also caused an increased incidence of cannibalism and a significant reduction in the frequency of pregnancy, the average number of pups per litter and birth weights among these pups. In addition there was an increased incidence of neonatal deaths among the offspring which also had poor development, reduced brain weights and an increased tendency of aggression towards their litter mates. Adult female rats fed diets containing 5 and 10 g KCN/100 g laboratory diet survived for more than three months but never became pregnant. They developed enlarged thyroid glands and tumours of the large intestine. The usual content of cyanide in cassava varies from 70 to 500 mg/kg which is much less than the levels used in these experiments; thus the rats were able to cope with the 50% gari diet and detoxify the glycoside present (Olusi et al., 1979). 2.2.4.2 Pigs General toxicity and reproductive effects were studied for cassava in combination with added cyanide. In a 110-day feeding experiment 18 pregnant Yorkshire gilts were allocated to three equal groups and fed fresh cassava (containing 40.2 HCN/kg) supplemented

with 0, 250, and 500 mg cyanide (KCN) per kg of fresh cassava offered. Serum thiocyanate concentration was slightly but not significantly increased in the 500 mg KCN/kg group and serum protein bound iodine decreased during gestation in all groups. Fetal serum thiocyanate concentration was significantly (p <0.05) higher in the group fed 500 mg KCN/kg. A small increase in maternal thyroid weight with increasing levels of cyanide was observed. Pathological studies showed proliferation of glomerular cells of the kidneys in gilts of all groups and reduced activity of the thyroid gland in gilts fed 500 mg KCN/kg group. Cyanide fed during gestation did not affect performance during lactation. Milk thiocyanate and colostrum iodine concentrations were significantly higher in the group fed 500 mg KCN/kg feed. No effects of cyanide were reported on indices of reproduction performance (Tewe & Maner, 1981a). 2.2.5 Special studies on embryotoxicity and/or teratogenicity In a teratogenicity study pregnant hamsters received oral doses of 70, 100, 120 or 140 mg linamarin/kg bw or an equivalent volume (0.5 ml/100 g) of isotonic saline during the early primitive streak stage of gestation (day 8 of gestation). The hamsters were killed on the morning of day 15 of pregnancy. Fetuses were removed by caesarian section and the numbers of resorption sites, dead fetuses, and living fetuses were recorded. Living fetuses were examined for gross external malformations and by means of histopathological methods for internal malformations. A dose of 120 or 140 mg linamarin/kg bw was associated with an increased incidence of vertebral and rib anomalies as well as the production of encephaloceles in the offspring. These larger doses of linamarin also resulted in obvious maternal toxicity (dyspnoea, hyperpnoea, ataxia, tremors and hypothermia). Two animals dosed with 140 mg and one animal dosed 120 mg/kg bw died. In surviving animals the signs of poisoning were greatly reduced or gone within 3 h after treatment. Linamarin treatment had no effect on fetal body weight, ossification of skeletons, embryonic mortality, nor litter size. Although ingestion of the cyanogenic glycoside was associated with a significant teratogenic response, the effects occurred only at doses that elicited signs of maternal intoxication (Frakes et al., 1985). In a teratogenic study groups of pregnant hamsters (8 dams/group) were fed diets consisting of cassava meal:laboratory chow (80:20) during days 3-14 of gestation. One low cyanide (sweet) cassava meal and one high cyanide (bitter) cassava meal were studied. An additional group was fed a diet which resembled cassava in nutrtional value, but which lacked cyanogenic glycosides. Thiocyanate concentrations in the urine and blood of dams fed cassava diets increased significantly. Increased tissue thiocyanate

concentrations were observed in fetuses recovered from cassava-fed dams. Cassava-fed dams gained significantly less weight than did control animals and their offspring showed evidence of fetotoxicity. Reduced fetal body weight and reduced ossification of sacrocaudal vertebrae, metatarsals and sternebrae were associated with cassava diets. High cyanide cassava diets were also associated with a significant increase in the numbers of runts compared to litters from dams fed either low protein or laboratory stock diets (Frakes et al., 1986). 2.2.6 Special studies on the thyroid gland In a study cited by Oke (1980), the influence of a 100% cassava diet on the thyroid in a 7-day experiment with rats. A significant decrease in glandular stores of stable iodine, significantly higher thyroid weight and higher thyroidal 131I uptake were observed. Each effect is due to a synthetic block in the conversion of monoiodothyronine to diiodothyronine. 2.3 Observations in humans 2.3.1 Acute toxicity studies in humans One to 10 g of amygdalin have been given parenterally in humans, apparently without acute toxicity. This indirectly suggests that there is no significant metabolism of the intact injected glycoside. The cyanide-containing breakdown products possess well-defined toxicities, and 50 mg of hydrogen cyanide can be fatal. With oral dosing of amygdalin, a toxic potential is manifest. -Glucosidase is present in the gastrointestinal lumen, a contribution of intestinal microflora. According to Eyerly (1976) oral laetrile (amygdalin) could be 40 times more toxic than parenterally administered doses. This is probably due to the free HCN released by the -glucosidase enzyme present in the gut (Dorr & Paxinos, 1978). The lethal dose of amygdalin for man when ingested is reported to be in the range of 0.02-0.13 mmol/kg bw (Solomonson, 1981). If it is assumed that about 100-2000 mg HCN is the lethal dose for man, as much as 10-20 kg of Lafun cassava (10-20 mg cyanide/kg) will have to be consumed at a sitting to produce toxicity (Oke, 1980). Well-nourished individuals have ingested 1000 mg or more of pure amygdalin every day without any evidence of "side effects" (Oke, 1979). In a case study an 11-month-old girl was reported accidentally to have ingested 1-5 amygdalin tablets (500 mg). The patient became

listless within one half hour of ingestion and vomited. Breathing became irregular and her state of consciousness became altered. An hour after ingestion she was in shock and died approximately 72 h following ingestion in spite of hospital treatment (Humbert et al., 1977). In a case-study a 17-year-old girl suffering from cancer made a practice of taking, instead of radiotherapy, four ampoules of laetrile (3 g amygdalin) intravenously. One day she swallowed three 1/2 ampoules of laetrile. Shortly after ingestion, a severe headache and dizziness developed, and she collapsed. Laboured breathing developed, her pupils became dilated, and she became comatose. All symptoms occurred within 8-10 minutes after ingestion. She died 24 h after ingestion (Sadoff et al., 1978). In Anatolia (Turkey) 9 cases of cyanide intoxication of children due to the ingestion of wild apricot seeds (217 mg HCN/100g) were reported. The victims had probably eaten more than 10 seeds. Also in studies of Jeanin et al. (1961) and Pijoun (1942) poisoning after consuming a relative large amount of peach seeds or bitter almonds are reported. Quantitative figures on cyanogenic glycoside or cyanide intake are not given (Sayre & Kaymakcalavu, 1964). The toxicity of cassava and cassava processing products was until recently assumed to be associated with free cyanide, this was 50-60 mg which constitutes a lethal dose for an adult man. The cyanogenic glycosides were at first thought to be of little consequence to mammals if cassava hydrolytic enzymes have been inactivated. The possibility of hydrolysis during digestion, however, is also important (Cooke & Coursey, 1981). In a case-study a 67-year-old woman collapsed after ingestion of a slurry of 12 bitter almonds ground up and mixed with water. She recovered after treatment in the hospital. The average cyanide content was 6.2 mg HCN/bitter almond (Shragg et al., 1982). The consumption of 60 bitter almonds is deadly for an adult. For young children, however 5-10 almonds or 10 droplets of bitter almond oil are fatal (Askar & Moral, 1983). 2.3.2 Long-term toxicity studies in humans with cyanogenic glycosides and cyanides A study to evaluate the possible association of high cyanide and low sulfur intake in cassava-induced spastic paraparesis was performed. The north-eastern part of Mozambique suffered a severe drought in 1981: the only crop available was the most toxic variety of cassava and, due to lack of food during the harvest period, the roots were eaten after only a few days of sun drying. A field survey revealed 1102 cases of spastic paraparesis. In 1982 urine was

collected from 30 apparently healthy children (age 8.1 years). As reference 17 Swedish children (age 8.6 years) were used. In a second stage urine was sampled in 1983 (when the nutritional situation was improved but still unsatisfactory) from 31 children (9.0 years) in the same village and 30 schoolchildren (8.1 years) in a nearby district where no cases of paraparesis were seen in 1981 and from 28 children (7.1 years) of the city who ate virtually no cassava. The children from the village had increased thiocyanate and decreased inorganic sulfate excretion, indicating high cyanide and low sulfur-containing amino acid intake. Children from a neighbouring cassava-eating area, where no cases of spastic paraparesis had occurred, had lower thiocyanate excretion but higher organic sulfate excretion. These results support the hypothesis that the epidemic was due to the combined effects of high dietary cyanide exposure and sulfur deficiency (Cliff et al., 1985). Several studies were performed, including epidemiological studies, on the role of chronic cyanide intoxication caused by the consumption of cassava diet in the etiology of tropical (ataxia) neuropathy (TAN) in Nigerian populations. As described in over 400 Nigerian patients the essential neurological components of the disease are myelopathy, bilateral optic atrophy, bilateral perceptive deafness and polyneuropathy. The initial and most common symptoms consist of various forms of paraesthesia and dysaesthesia usually starting in the distal part of the lower limbs. The next most common finding is blurring or loss of vision. Other common symptoms in order of frequency are ataxia, tinnitus, deafness, weakness and thinning of the legs. In about a third of the patients stomatoglossitis is present, additionally motor neurone disease, Parkinson's disease, cerebellar degeneration, psychosis and dementia have been associated with the disease. TAN affects males and females in all age groups equally, but occurs only rarely in children under 10 years. Patients usually give histories of almost total dependence on a monotonous diet of cassava derivatives; occasional dietary supplements include yam, maize, rice, vegetables and animal protein. Analysis of family relationships among patients showed no evidence of a genetically determined predisposition. The families were usually poor and members lived communally. Clinical evidence of malnutrition was frequently absent. The occurrence of cyanide intoxication in Nigerian patients was indicated by the significantly higher cyanide and thiocyanate plasma levels and higher excretion of thiocyanate than controls. Hepatic rhodanese activity was not different from that in controls and histology of liver biopsy specimens showed no abnormality. Total plasma vitamin B12 levels are normal or high in patients and healthy Nigerians but plasma concentration of cyanocobalamin was highly significantly raised in patients. A small proportion of

cyanocobalamin was found in the liver of patients. Methylmalonic acid excretion was normal in patients, indicating that there was physiological vitamin B12 adequacy at tissue or cellular level in these patients. In Nigeria, endemic foci of the disease (in epidemiological studies) recognized since the early 1930's, correspond with the areas where cassava is intensively cultivated and consumed as the major or the sole dietary source of carbohydrate. There was evidence of increased exposure to cyanide in members of families where multiple cases were found. In biochemical studies no biochemical evidence of protein-calorie malnutrition was seen and serum transferrin, said to be sensitive index of protein nutritional status, was normal. No haematological abnormalities were seen in patients. Investigation of patient nutritional status with regard to the water-soluble vitamins showed no abnormality, except in riboflavin intake. Plasma concentrations of calcium, phosphate, sodium, potassium, chloride, bicarbonate, and cholesterol, and tests of thyroid, hepatic, and renal functions were normal. Amino acids and porphyrobilinogen and other porphyrins were absent in urine. There was no biochemical nor other evidence of malabsorption. Glucose tolerance tests were normal. Histamin-fast achlorhydria was very rare. Serologic tests for syphilis, typhoid, typhus, brucellosis and screening for prevalent viral infections gave negative or insignificant results. There was no increased prevalence of malaria and urinary tract infections were not encountered in patients. ECG's were normal. Diminished urinary excretion and riboflavin (vitamin B2) and low serum riboflavin and caeruloplasmin levels in patients compared with healthy persons were the only significant abnormalities found. Riboflavin deficiency is, however, widespread in many parts of Nigeria and especially in areas endemic for TAN, probably because cassava is a poor source of the vitamin. The daily intake of HCN from cassava derivatives in areas in Nigeria endemic for TAN may be as high as 50 mg, which is nearly an acutely sublethal amount, and can conceivably produce cyanide intoxication. This is particularly plausible as cassava root is a major item of food, often the main source of carbohydrate, and is poor in sulfur-containing amino acids which are essential for detoxification of cyanide. A high prevalence (2%) of goitre in populations with a high incidence of TAN is seen; this appears to be related to cassava diet and high plasma thiocyanate. The effects of riboflavin insufficiency may combine with those of chronic cyanide intoxication in the etiology of TAN (Osuntokun, 1981). Several epidemiological and experimental studies revealed that TAN resulted from chronic cyanide poisoning due to the release of HCN from cyanogenic glycosides present in certain cassava food products. Since TAN invariably occurs among poorly nourished people, the condition may result from unidentified nutritional deficiencies or excesses as well as cyanide toxicity. Among patients showing the

TAN syndrome the frequency of goitre was increased (Conn, 1979a,b). From both experimental and epidemiological studies there is strong, but not conclusive, evidence that cassava toxicity is a causative factor in some neurological disorders like TAN and endemic spastic paraparesis. As well, different deficiencies such as deficiency of sulfur-containing amino acids may play a role. The geographical distribution of malnutrition-related diabetes coincides with that of cassava consumption. Dietary exposure to cyanide has been proposed as a possible cause, but has not been proven. The poor nutritional quality of cassava seems a very likely causative factor (Rosling, 1987). Workers in certain occupations are exposed to HCN additional to sources encountered by the general public. These individuals have been studied to determine whether chronic cyanide poisoning is a clinical entity. Symptoms reported were headache, vertigo, tinnitus, nausea, vomiting, and tremors. Although these symptoms are sufficiently documented and characteristic, they are transitory in that exposure to fresh air causes recovery. These do not seem to produce the outward symptoms of TAN, the pathological condition that has been attributed in part to exposure to cyanide or cyanogenic glycosides in certain preparations of cassava (Conn, 1979b). Neurological disorders such as ataxic neuropathy and cretinism have been associated directly with the intake of cyanogenic glycoside-contaminated diets. There are grounds to suspect that cyanogenic glycoside-contaminated foodstuffs such as cassava and pulses are directly implicated in acute and chronic cyanide toxicity in the tropics. Although correlation between dietary cyanogen-contaminants and disease such as TAN, goitre, cretinism and mental retardation exists and is based on experimental evidence, the mechanism of action of cyanide and thiocyanate on the cellular level is not well understood (Nartey, 1980). Based on several studies in humans consuming as their main food cassava or other food products rich in cyanogenic glycosides, it seems that chronic cyanide intoxication in combination with deficient intake of riboflavin and/or a poor quality of protein and hence methionine deficiency is/are responsible, to a large extent, for the etiology of TAN in cassava-eating areas, whereas chronic cyanide intoxication in combination with a deficient iodine intake is responsible for goitre (Oke, 1979, 1980). Epidemiological and experimental studies show that cyanogenic glycosides in food products play a important role in the development of goitre. Thiocyanate, the detoxification product from the HCN derived from cyanogenic products, is responsible for interference with thyroid function. Studies on endemic goitre in Africa have identified iodine deficiency and an antithyroid activity of cassava diets as major etiological factors of the disease (Conn, 1979a,b).

Extensive studies in Zaire have established that goitre and cretinism due to iodine deficiency can be considerably aggravated by a continuous dietary cyanide exposure from insufficiently processed cassava. This effect is caused by thiocyanate. Thiocyanate has a similar size to the iodine molecule and interferes with the iodine uptake in the thyroid gland. Thiocyanate levels which can occur after exposure to cyanide from cassava can only affect the gland when the iodine intake is below 100 micrograms/day, which is regarded minimal for normal function. Several studies have shown that populations with considerable cyanide exposure from inadequately processed cassava are free from goitre as long as their iodine intake is sufficient. Populations in northern Zaire with very low iodine intake and in addition high thiocynate levels resulting from the consumption of cassava products, show very severe endemic goitre. When the population was given iodine supplementation, the goitre decreased (reviewed by Rosling, 1987). In persons who ingested cassava a decreased 131I-uptake by the thyroids was seen, confirming the goitrogenous nature of cassava (De Lange, 1973 as cited by Oke, 1980). In one study more than 50% of workers in a processing factory revealed thyromegaly and an increased 131I-uptake. All workers had increased blood haemoglobin concentration as well as increased lymphocyte counts. Two of the employees developed psychosis, although it is difficult to know whether this was cause or effect. Abnormal thyroid function has also been found in workers from a photographic plant in which a cyanide extracting process was used to recover silver from X-ray films (El Ghawabi et al., 1975). Alterations in vitamin B12 and folate metabolism have also been noted to play a role, although the significance of these observations are not known. Short of having an elevated whole blood cyanide level, the laboratory findings of cyanide poisoning are fairly non-specific. Perhaps the most valuable clue is the finding of a severe metabolic acidosis with a high anion gap. In clinical medicine there have been reported only 7 causes of anion gap metabolic acidosis. In most of these disorders lactic acidosis is present owing to severe tissue underperfusing and hypoxia. In the patient with cyanide poisoning tissue hypoxia is universally present (Gonzales & Sabatini, 1989). 3. COMMENTS AND EVALUATION The potential toxicity of a cyanogenic plant depends primarily on its capacity to produce a concentration of hydrogen cyanide toxic to animals and humans. The release of hydrogen cyanide can occur either following maceration of the plant material - this activates the intracellular -glucosidase which in turn hydrolyses glycoside -

or by hydrolysis of glycoside by the -glucosidase produced by the microflora of the gut. The level of -glucosidase activity in the gut depends on the pH and the bacterial composition. The cyanogenic glycoside content of a foodstuff, when known, is usually expressed in terms of the amount of cyanide released by acid hydrolysis; exact figures for the concentration of the glycosides themselves are very rarely given. Hydrogen cyanide absorbed from the gut can be detoxified by metabolic conversion to thiocyanate; this depends on the presence of nutritional factors, such as sulfur-containing amino acids and vitamin B12. Acute toxicity results when the rate of absorption of hydrogen cyanide is such that the metabolic detoxification capacity of the body is exceeded. Available reports of toxicological studies lack information on the level of intake of cyanogenic glycosides or on the amount of hydrogen cyanide potentially released. No long-term toxicity or carcinogenicity studies were available to the Committee. However, in vitro and in vivo genotoxicity were negative. Teratogenic and adverse reproductive effects attributable to linamarin (cassava) and hydrogen cyanide were seen only at doses that also caused maternal toxicity. The toxic effects of cyanide on the thyroid (via its metabolite thiocyanate) depend on the iodine status of the test animals, as indicated in the twenty-fifth report of the Committee (Annex 1, reference 56). On the basis of epidemiological observations, associations have been made between chronic exposure to cyanogenic glycosides and diseases such as spastic paraparesis, tropical ataxic neuopathy, and goitre. However, these observations were confounded by nutritional deficiencies, and causal relationships have not been definitely established. Traditional users of foods containing cyanogenic glycosides usually have a basic understanding of the treatment required to render them safe for consumption. However, some products are sold commercially and are consumed by people who may not be familiar with such procedures. The Committee therefore recommended that guidelines be developed to provide reliable and sensitive methods for the analysis of these foodstuffs for hydrogen cyanide releasable from cyanogenic glycosides, in order to ensure that amounts in foods as consumed do not present a hazard. Because of a lack of quantitative toxicological and epidemiological information, a safe level of intake of cyanogenic glycosides could not be estimated. However, the Committee concluded that a level of up to 10 mg/kg hydrogen cyanide in the Codex

Standard for Cassava Flour (CAC, 1991) is not associated with acute toxicity. 4. REFERENCES ASKAR, A., & MORAD, M.M. (1983). Lebensmittelvergiftigung 1. Toxine in natrlichen Lebensmittel. Alimentia, 19: 59-66. BARRETT, M.D., HILL, D.C., ALEXANDER, J.C. & ZITNAK, A. (1977). Fate of orally dosed linamarin in the rat. Can. J. Physiol. Pharmacol., 55: 134- 136. BASS, N.H. (1968). Pathogenesis of myelin lesions in experimental cyanide poisoning: a microchemical study. Neurology, 18: 167-177, as cited in Osuntokun, 1981. BASU, T.K. (1983). High-dose ascorbic acid decreases detoxification of cyanide derived from amygdalin (laetrile): Studies in guinea-pigs. Can. J. Physiol. Pharmacol., 61: 1426-1430. BRIERLEY, J.B., PRIOR, P.F., CALVERLEY, J. & BROWN, A.W. (1977). Cyanide intoxication in Macaca mulatta: Physiological and neurological aspects. J. Neurol. Sci., 31: 133-157, as cited in Osuntokun, 1981. CAC (1991). Codex Alimentarius, Vol. XII, Suppl 4. Codex Standard for Edible Cassava Flour (African Regional Standard). Rome, Food and Agriculture Organization of the United Nations (CODEX STAN 176). CLARK, A. (1936). Report on effects of certain poisons contained in food plants of West Africa upon health of native races. J. Trop. Med. Hyg., 39: 285-295, as cited in Osuntokun, 1981. CLIFF, I., LUNDQUIST, P., MRTENSSON, I., ROSLING, H. & SRBO, B. (1985). Association of high cyanide and low sulfur intake in cassava-induced spastic paraparesis. Lancet, II: 1211-1213 COLLIER JACKSON, J. (1988). Behavioral effects of chronic sublethal dietary cyanide in an animal model: implications for humans consuming cassava (Manihot esculenta). Journal of the Society for the Study of Human Biology, 60: 597-614. CONN, E.E. (1979a). Cyanide and cyanogenic glycosides. In Rosenthal, G.A. & Janzen, D.H. (eds.), Herbivores: Their interaction with secondary plant metabolites, Academic Press, Inc., New York-London, pp 387-412. CONN, E.E. (1979b). Cyanogenic glycosides. International review of biochemistry. In Biochemistry and Nutrition 1A, Neuberger, A., & Jukes, T.H. (eds), University Park Press, Baltimore, 27: 21-43.

COOKE, R.D. & COURSEY, D.G. (1981). Cassava: A major cyanide-containing food crop. In Vennesland, B., Conn, E.E., Knowles, W, Westley, J. & Wissing, F. (Eds). Cyanide in Biology. Academic Press, London. DOHERTY, P.A., FERN, V.H. & SMITH, R.P. (1982). Congenital malformations induced by infusion of sodium cyanide in the Golden hamster. Toxicology and Applied Pharmacology, 64: 456-464. DORR, R.T. & PAXINOS, I. (1978). The current status of laetrile. Annals of Internal Medicine, 89: 389-397. EPA (1990). Summary Review of Health Effects Associated with Hydrogen Cyanide, Health Issue Assessment Environmental Criteria and Assessment Office, Office of Health and Environmental Assessment Office of Research and Development, US Environmental Protection Agency Research Triangle Park, North Carolina, USA. EYERLY, R.C. (1976). Laetrik; focus on the the facts. Cancer, 26: 50-54. FERRARO, A. (1933). Experimental toxic encephalomyelopathy (diffuse sclerosis following subcutaneous injection of postassium cyanide). Archs. Neurol. Psychiat., 29: 1364-1367, as cited in Osuntokun, 1981. FRAKES, R.A., SHARMA, R.P. & WILLHITE, C.C. (1985). Development toxicity of the cyanogenic glycoside linamarin in the golden hamster. Teratology, 31: 241- 246. FRAKES, R.A., SHARMA, R.P., WILLHITE, C.C. & GOMEZ, G. (1986). Effect of cyanogenic glycosides and protein content in cassava diets on hamster prenatal development. Fundamental and Applied Toxicology, 7: 191-198. FRAKES, R.A., SHARMA, R.P. & WILLHITE, C.C. (1986). Comparative metabolism of linamarin and amygdalin in hamsters. Food Chemical Toxicology, 24: 417-420. GEITLER, A.O. & BAINE, J.O. (1983). The toxicity of cyanide. Am. J. Med. Sci., 195: 182-198. GOMEZ, G., APARICIO, M.A. & WILLHITE, C.C. (1988). Relationship between dietary cassava cyanide levels and Brailer performance. Nutrition Reports International, 37: 63-75. GONZALES, I. & SABATINI, S. (1989). Cyanide poisoning: pathophysiology and current approaches to therapy. The Int. I. of Artificial Organs, 12(6): 347-355. HIMWICH, W.A. & SAUNDERS, I.P. (1984). Enzymatic conversion of

cyanide to thiocyanate. American Journal of Physiology, 58: 348. HUMBERT, I.R., TRESS, I.H. & BRAICO, K.T. (1977). Fatal cyanide poisoning: accidental ingestion of amygdalin. JAMA, 238: 482. HURST, E.W. (1940). Experimental demyelination of the central nervous system. Aust. J. Exp. Biol. Med. Sci., 18: 201-223, as cited in Osuntokun, 1981. LANG, K. (1933) Die Rhodanbildung im Tierkrper. Biochem Z., 259: 243-256. LESSELL, S. (1971) Experimental cyanide optic neuropathy. Archs. Ophtal., 84: 194-204, as cited in Osuntokun, 1981. LEUSCHNER, F., NEUMANN, B.W., & LIEBSCH, M. (1983a). Mutagenicity study of hydrocyanic acid in the Ames Salmonella/microsome plate test (in vitro). Unpublished study, Laboratory of Pharmacology and Toxicology, Hamburg, August 1983, submitted to the WHO by Detia Freyberg GmbH. LEUSCHNER, F., NEUMANN, B.W., & LIEBSCH, M. (1983b). Mutagenicity study of hydrocyanic acid in Chinese hamster (chromosome aberration) by oral administration. Unpublished study, Laboratory of Pharmacology and Toxicology, Hamburg, August 1983, submitted by Detia Freyberg GmbH. LEUSCHNER, F. & NEUMANN, B.W. (1989a). In vitro mutation assay of KCN in Chinese hamster cells. Unpublished study, Laboratory of Pharmacology and Toxicology, July 1989, submitted by Detia Freyberg GmbH. LEUSCHNER, F., NEUMANN, B.W., OTTO, H. & MLLER, E. (1989b). 13-Week toxicity study of potassium cyanide administered to Sprague-Dawley rats in the drinking water. Unpublished study, Laboratory of Pharmacology and Toxicology, July 1989, submitted by Detia Freyberg GmbH. MAJAK, W., MCDIARMID, R.E., JAKOBER, K. & CHENG, K.I. (1989). Diurnal changes in rates of degradation of cyanogenic glycosides in bovine rumen fluid. Toxicon., 27: 61. NARTEY, F. (1980). Toxicological aspects of cyanogenesis in tropical foodstuffs in Toxicology in the Tropics. Editors R.L. Smith and E.A. Bababumni, Taylor & Francis Ltd, London, 53-73. OKE, O.L. (1979). Some aspects of the role of cyanogenic glycosides in nutrition. Wld. Rev. Nutr. Diet, 33: 70-103. OKE, O.L. (1980). Toxicity of cyanogenic glycosides. Food Chemistry, 6: 97-109.

OKOH, P.N. & PITT, G.A.J. (1981). The metabolism of cyanide and the gastrointestinal circulation of the resulting thiocyanate under conditions of chronic cyanide intake in the rat. Can. J. Physiol. Pharmacol., 60: 381-385. OKOH, P.N. (1983). Excretion of 14C-labeled cyanide in rats exposed to chronic intake of potassium cyanide. Toxicology and Applied Pharmacology, 70: 335-339. OLUSI, S.O., OKE, O.L. & ODUSOTE, A.C. (1979). Effects of cyanogenic agents on reproduction and neonatal development in rats. Biol. Neonate, 36: 233-293. OSUNTOKUN, B.O. (1981). Cassava diet, chronic cyanide intoxication and neuropathy in the Nigerian Africans. Wld. Rev. Nutr. Diet, 36: 141-173. PHILBRICK, D.I., HOPKINS, I.B., HILL, D.C., ALEXANDER, I.C. & THOMSON, R.G. (1979). Effects of prolonged cyanide and thiocyanate feeding in rats. Journal of Toxicology and Environmental Health, 5: 579-592. RAUWS, A.G., OLLING, M. & TIMMERMAN, A. (1982). The pharmacokinetics of amygdalin. Arch. Toxicol., 49: 311-319. RAUWS, A.G., OLLING, M. & TIMMERMAN (1983). The pharmacokinetics of prunasin, a metabolite of amygdalin. J. Toxicol. Clin. Toxicol., 19: 851-856. ROSLING, H. (1987). Cassava toxicity and food security. Ed. Rosling. Tryck Kontakt, Uppsala, Sweden, 3-40. SADOFF, L., FUCHS, K. & HOLLANDER, I. (1978). Rapid death associated with laetrile ingestion. JAMA, 239: 1532. SAYRE, I.W. & KAYMAKCALAVU, S. (1964). Cyanide poisoning from apricot seeds among children in Central Turkey. New. Engl. J. Med., 270: 1113-1118. SHRAGG, T.A., ALBERTSON, T.E. & FISHER, C.J. (1982). Cyanide poisoning after bitter almond ingestion. The Western Journal of Medicine, 136: 65-69. SMITH, A.D.M., DUCKETT, S. & WATERS, A.H. (1963) Neuropathological changes in chronic cyanide intoxication. Nature, 200: 179-181, as cited in Osuntokun, 1981. SOLOMONSON, L.P. (1981). Cyanide as a metabolic inhibitor. In Cyanide in Biology, Vennesland, B., Conn, E.E., Knowles, C.J., Westley, J & Wissing, F. Academic Press, London, New York, Toronto,

11-18. TEWE, O.O. & MANER, I.H. (1980). Cyanide, protein and iodine interactions in the performance, metabolism and pathology of pigs. Research in Veterinary Science, 29: 271-276. TEWE, O.O. & MANER, I.H. (1981a). Performance and patholophysiological changes in pregnant pigs fed cassava diets containing different levels of cyanide. Research in Veterinary Science, 30: 147-151. TEWE, O.O. & MANER, I.H. (1981b). Long-term and carry-over effect of dietary inorganic cyanide (KCN) in the life cycle performance and metabolism of rats. Toxicological and Applied Pharmacology, 58: 1-7. UMOH, L.B., MADUAGWA, E.N. & AMOLE, A.A. (1986). Fate of ingested linamarin in malnourished rats. Food Chemistry, 20: 1-9. VENNESLAND, B., CASTRIC, P.A., CONN, E.E., SOLOMONSON, L.P., VOLINI, M. & WESTLEY, I. (1982). Cyanide metabolism. Fed. Proc., 41(10): 2639-2648. WILLHITE, C.C. (1982). Congenital malformations induced by laetrile. Science, 215: 1513-1515. WILLIAMS, A.O. & OSUNTOKUN, B.O. (1969). Light and electron microscopy of peripheral nerves in tropical ataxic neuropathy. Archs. Neurol., 21: 475-492, as cited in Osuntokun, 1981. WHO (1965). Evaluation of the hazards to consumers resulting from the use of fumigants in the protection of food. Report of the second Joint Meeting of FAO Committee on Pesticides in Agriculture and the WHO Expert Committee on Pesticides Residues, FAO Meeting Report No Pl/1965/10/2: WHO Food series 28.65.