SOLANINE AND CHACONINE First draft prepared by Dr T. Kuiper-Goodman and Dr P.S. Nawrot Bureau of Chemical Safety Health and Welfare Canada Ottawa, Ontario, Canada 1. 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 TGA1 mg/100g FW whole tuber 7.5 (4.3-9.7) flesh 1.2-5 skin 2-3% of tuber 30-60 peel 10-15% of tuber 15-30 bitter tuber 25-80 peel from bitter tuber 150-220 1 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 alpha-chaconine 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 5ß-solanidan-3alpha-ol. Alpha- And ß-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 184.108.40.206 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 µg/ml) obtained after 14 h. Decrease in blood levels was slow, with 0.31 µg/ml present at 120 h. The peak level in the liver (2.97 µg/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). 220.127.116.11 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). alpha-Chaconine 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). 18.104.22.168 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 µg). At 3 h after dosing, the highest concentration of alpha-chaconine was seen in the intestines, including intestinal contents (125 µg/g), and this represented 63% of the administered dose. By 24 h these values were 75 µg/g or 44% of the administered dose, and by 168 h they had declined to 1.73 µg/g or 0.92% of the administered dose. Peak blood (1.74 µg/ml) and peak tissue levels (liver = 27.2 µg/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 µg/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). 22.214.171.124 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 alpha-solanine 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. Hellenäs, cited by Slanina, 1990b). 2.1.2 Biotransformation 126.96.36.199 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). 188.8.131.52 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 alpha-chaconine, 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 alpha-chaconine solanidine i.p. p.o. i.p. i.p. Mice 32.31 >10002 19.25 >5002 42.02 27.56 32.35 30.06 Rat 753 5903 674 Rabbit <201,2 506 406 Rhesus <407 Monkey 1 Patil et al. (1972) 2 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 184.108.40.206 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). 220.127.116.11 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 18.104.22.168 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 22.214.171.124 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 (9-10/group) were gavaged with alpha-solanine at dose levels of 0.3, 1.0 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 (3-4/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). 126.96.36.199 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 alpha-chaconine 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). 188.8.131.52 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). 184.108.40.206 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 freeze-dried, 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 that feeding 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). 220.127.116.11 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). 18.104.22.168 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 µM and 3.14 µM, 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 alpha-chaconine 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 µM 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 µM) 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 µg alpha-solanine/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 µg/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 µg 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 µM 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 µM. 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 Concentration Estimated Outcome Reference Consumed of TGA Toxic Dose mg/kg bw mg/kg bw 56 peeled, 1-1.5 kg 24 3.4-5.1 recovered Pfuhl, (soldiers) cooked (38) 1899 (whole uncooked) 60 adults potatoes 500 g ? 41 3.4 recovered Harris & 1 child 200 g ? 4.5 1 fatal Cockburn, (lethal) (5 yr-old) 1918 7 (family) greened ? ? ? 2 fatal Hansen, 1925 potatoes 50-60 shoots, ? 27 ? 1 fatal Willimot, (Cyprus) leaves 49 1933 Prisoners experimental ? ? 2.8 recovered Report cited by Ruhl, 1951 Child potato ? ? ? 1 fatal Report cited berries by Ruhl, 1951 4 (family baked 1-3 50 1.2-3.2 dose-related, Wilson, 1959 adults) potatoes potatoes recovered with skin 150-450 g Table 3 (continued) Affected Potato type Quantity Concentration Estimated Outcome Reference Consumed of TGA Toxic Dose mg/kg bw mg/kg bw 78 old potatoes 2 small 25-30 1-4-1.6 3 comatose, McMillan & (schoolboys) potatoes all Thompson, 200 g recovered; 1979 young boys, more affected 61 baked potato 200 g 49 2.5 recovered Anon. 1984 (school-children) Alberta ? 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 alpha-chaconine and alpha-solanine. These antifungal compounds offer resistance to potato blight and increase in amount in blighted potatoes, 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 alpha-fetoprotein 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). 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See Also: Toxicological Abbreviations