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PRIVILEGED & CONFIDENTIAL ATTORNEY WORK PRODUCT Covington & Burllng Draft May 20~ 19@2 SUMMARY OF DATA ON POTASSIUM CITRATE Abstract. Potassium citrate is used as a flavor enhancer. It has been recognized as GRAS by the FDA for multiple uses in food. Potassium citrate has been proposed as a cigarette coating or as an additive to cigarette paper. However, there is no information available concerning the pyrolysis of potassium citrate. Potassium citrate is metabolized by the body much like citric acid. Excessive amounts of potassium citrate result in abnormal cardiac behavior in humans, as well as gastrointestinal discomfort. There were no significant treatment related effects in two chronic feeding studies using rats. ; Inlsubchronic studies, potassium citrate effected the growth rate of rats, but it does not appear to be mutagenic or teratogenic. Sodium citrate accelerated mortality in mice infected with tuberculosis. I. Background. Potassium citrate (C~H~K~07; CAS No. 008~6-84-2), also known as the tripotassium salt of'2- ~ hydroxy-propane-l,2,3-tri-carbo{ic acid, is a natural constituent and common metabolite of animals and plants. ~itricacid, acid and its 2- hydroxy- !, 2,3-propanetricarboxylic salts are also natural constituents and common metabolites of animals and plants~ Citric acid is an intermedlal-y compound in the Krebs cycle. In humans, the concentration of citrate is about 20 mg/! in whole blood, and 0.~/'0 g of citrate is excreted daily in the urine (Ostberg, 1931; SJSstrSm, iS37i. Citrate occurs in particularly high concentrations in fruits,

including citrus fruits and their juices (Thunberg, 1953; Clements, 1964; Johnston and Hammill, 1968). Typical concen- trations, fresh weight, are about i percent in orange juice and up to 8 percent in unripe lemon juice, as compared to 0.i percent or less in peas, corn, cabbage, or human milk. Potassium citrate is used as a food additive and therapeutic agent. In foods, it is used most frequently for pH control and as a flavor enhancer (GRAS llst, 1972). The metal ion complexing properties of citrate make it useful as a sequestrant, antioxidant and preservative. For the age group 2 to 65+ years, the average intake of potassium citrate resulting from processed foods was calculated to be 280 mg/day (GRAS list, 1972). The level of addition was usually below 0.5 percent, expressed as a weighted mean. For the 0 to 5 month age group, the estimated average daily intake of added citrate is 560 mg. Potassium citrate is used therapeutically as a diaphoretic and antipyretic in rheumatism, gout, bronchitis, and malaria. For these purposes, potassium is given in multiple daily doses of 1-2 g (Hagers Handbuch, 1976). Potassium citrate (i0 mmol/l) also has been found to be useful in treating acidosis, diarrhea, and associated hypokalaemia (Islam, 1985~, because the citrate anion can be oxidized to co= (Hagers Handbuch, 1976). Potassium citrate has been used in the oral treatment of ~ne~rolithiasis (Lake and Brown, IS85; Fak &% al~n--~S85 a--Z~.-~--B~s~aiew,-~1989), which can -- 2

result from chronic hypocitraturia by renal tubular acidosis, high intake of animal protein, or chronic diarrhea (Am. PharTn., 1984). Citrate helps prevent the formation of kidney stones through its ability to increase urinar-f pH'by forming a complex with calcium. The chemical complex interferes with the urinar-f c~fstallization of calcium salts, reducing urinary saturation of calcium oxalate. Citrate also acts directly as an inhibitor of calcium phosphate and calcium oxalate crystal growth in the urine. Alkalinization increases the solubility of uric acid and reduces the formation of uric acid stones. Potassium induced cardioplegia (intracorona~f injection of a buffered, isotonic solution of potassium citrate containing 26 mEq. potassium/l) was shown to prolong the duration of cardiac tolerance to ischemia and to facilitate cardiac surgerl~ (Eff!er ena!., 1957; Lam e__~t a_~l., 1957; Sones, 1958; Gay and Ebert, 1973; Tyers e__%t a__~l., 1975). Potassium citrate has been used to support sulfonamide therapy. It preven<s cr-ysta!luria by alkalization of the urine (Hagers Handhuch, 1976). Potassium citrate also is used as gastric antacid (Merck Index, 1983) and to protect acid labile penicillin and other drugs against stomach acid (Hagers Handbuch, 1976). P~zassium citrate(has also been use¢ therapeutically to assist persons in smoking cessation (Faust, 1976). In veterinary therapy, potassium citrate is used as a diuretic (Merck Index, 1983). The chemical also serves as a - 3 -

chelating agent for divalent cations in biological research and technology, e.a., i0 mmol PTC/I can block the-growth of cultured plant cells (Polacco, 1977). Other uses of potassium citrate include formulating photographic gelatin emulsions (Roempps, 1983), scrubbing sulfur dioxide from flue gases s Jnd (Patent: Koro electroplating with copper (Patent: Sumitomo Metal Mining Co., Ltd.). The FDA has recognized potassium citrate as a GRAS substance (21 CFR ~ 182.1625 (1991)) for use as a sequestrant and multiple purpose food substance. FDA standards of identity provide for the addition of potassium citrate as an optional ingredient in certain cheeses [21 CFR ~ 133], ice cream [21CFR S 135], jellies and preserves [21 CFR S 150], canned vegetables [21 CFR ~ 155, 156], and dressings [21 CFR ~ 169]. Food grade specifications for potassium citrate limit the concentrations of arsenic to 3 ppm and heavy metals (such as lead) to i0 ppm. The Select Committee on GRAS substances of the U.S. Life Sciences Research Office concluded in 1977 that, "It]here is no evidence in the available information on potassium citrate that demonstrates, or suggests reasonable grounds to suspect, a hazard to the public when used at levels that are now current or that might reasonably be expected in the future." Use in Tobacco. Function.

Potassium citrate has been proposed as a coating for cigarettes or additive to cigarette paper to produce a "self- exting~g cigarette" (Patents: Mathews e__!n a!.; Guess; Reynolds)|. Information on the proposed amounts of the chemical required for these purposes is unavailable. B. Use Levsl. An Industry maximum use level for potassium citrate has not been established. III. Chemistry & Pyrolysis. Potassium citrate is synthesized from a concentrated solution of citric acid and potassium hydrogen carbonate. The dried product is crystallized from 60 percent ethanol (Hagers Handbuch, 1976) and appears as white cr~ystals, granules or hygroscopic powder. The relative molecular mass of potassium citrate is 324.341 the density is 1.98, and the melting point is 230°C (pyrolysis). The solubility of potassium citrate is i g/0.65 ml in water, 1 g/2.5 ml in glycerol, and the chemical is practically insoluble in alcohol. The pH of potassium citrate is 8.5 in an aqueous solution. The chemical has no odor, a weakly basic/salty taste, and the monohydrate loses its water at 180°C. Although unknown, it can be assumed that the fate of potassium citrate in a burning cigarette would be much like that of citric acid. Based on this assumption, and based on knowledge of the fate of citric acid in a burning cigarette, there are several conclusions that can be drawn concerning the - 5 -

vitamin B~2 deficiency only in persons already having marginal deficiency attributable to other causes. In one 37-year-old man, who reportedly was allergic to several organic acids and had developed canker sores, headaches, general lassitude, and irritability from eating foods containing citric acid, direct application of potassium citrate crystals to the oral mucosa produced no effect (Tuft and Ettelson, 1956). C. Chronic Animal Studies. Chronic toxicity studies involving citric acid have been performed on rats. In studies by Bonting (1952) and Bonting and Jansen (1956), three successive generations of albino Wistar rats were fed citric acid in the diet at concentrations of 0.15, 0.45, and 1.20 percent, providing an average intake of i00, 300 and 800 mg/kg/day, respectively. After a feeding period of up to 12 month~, no adverse effects were observed in the treatment groups i~~ growth, reproduction, mortality or blood components. The teeth were not harmed by the acid diets. Metabolic analyses of female rats found no abnormalities in nitrogen balance, mineral balance, acid-base balance or the gross and microscopic appearance of the tissues. On the other hand, the analysis found a decrease in ash and an increase in calcium ,,~antent of the tibia, a slight increase in muscle calcium, a,' .decrease in muscle sodium.j~nd tgtal muscle phosphorus, and a decrease ±n l±ver sod±um: The invest±gators d±d not consider

these small changes in tissue composition to be.evidence of adverse effects. In a study by Horn e__!t a__~l. (1957), citric acid was fed to a group of 20 young male albino Cal-worth rats for 2 years at concentrations of 3 and 5 percent of the diet (average daily intake was 1.2 and 2.0 g/kg, respectively). Both experimental groups grew more slowly than the controls, but survival rates did not decrease. At the time of the sacrifice (2 years), there were no differences in the organ weights of the control and experimental groups. Results of microscopic examinations of the thyroid, lungs, heart, liver, spleen, kidneys, adrenal gland, stomach, small and large intestines, pancreas, bone marrow, and testes were within normal limits. No published studies covering the carcinogenicity of potassium citrate or citrate were found. D. Acute & Subchronic Animal Studies. The acute oral LD~0 of citric acid (produced by Candida sp fermentation of normal paraffin) in mice was approximately 5 g/kg body weight and 12 g/kg body weight in rats (Yokotani et al., 1971). The signs of acute toxicity from orally administered citric acid in mice and rats were similar to those seen in organic acidosis and calcium deficiency. Animals given lethal doses of citric acid orally showed hemorrhaging of the gastric mucosa at necropsy. - i0 -

Six young rats weighing about 75 g were fed a diet supplemented with 2.5 percent citric acid (about 2 g/kg body weight) for nine days (de Albuquerque and Henriques, 1970). The experimental group showed loss or no appreciable gain in weight during the first few days and then returned to the expected weight gain pattern. Yokotani et al. (197!) fed groups of ten SD-JCL male rats (9~-112 g body weight) citric acid (a refined product of yeast fermentation) for six weeks at 1.2, 2.4, and 4.8 percent of the diet; mean intakes of citric acid were 1.15, 2.26, and 4.67 g/ kg/day, respectively. Food intake decreased compared to the control group by 0.7, 2.6, and 4 percent, respectively. Growth rate was slightly reduced at all dose levels. The total plasma protein concentration was significantly less than that of the controls only at the 2.4 percent dieta~f level; slight decreases in blood cell counts and hemoglobin were not statistically significant. At the highest dietary level, plasma cholesterol concentration decreased, serum glutamic oxalacetic transaminase activity increased, the thymus weights were lower, and slight atrophy of the thymus and splenic follicles was found at necropsy. Daily oral citric acid administration of 600 mg/kg (1.2 percent in the diet) to rats for more than 90 days ~ produced no abnormalities in body weight gain, blood, histopathology of the viscera, or reproduction (Krop and Gold, i~45). Also, daily oral administration of citric acid to - i! -

dogs, 1.38 g/kg, for 112-120 days was shown to produce no behavioral, biochemical, or histopathological abnormalities. There are no available acute or subchronic animal studies for potassium citrate itself. Information on dermal, subcutaneous, intravenous, or intraperitoneal acute toxicity was not found in the literature. E. Genotoxicity & MutaqenicitV. No evidence of potassium citrate mutagenicity has been reported in microbial assays involving ~. cerevisiae D4 and ~. typhimurium (strains TA1535, TA1537, TA1538), both with and without the addition of mammalian metabolic activation preparations (Litton Bionetics, The gen4~-i-e~toxicity of potassium citrate has been evaluated in one Japanese study .inuesti~d, apparently using a cytogenic recombinanion assay. The results of the study are not readily accessible, because the publication is in Japanese (Ishizaki and Ueno, 1989). F. Reproductive Toxicity & Teratolo~. Studies evaluating the teratological potential of citric acid in pregnant mice (~ 241 mg/kg administered on days 6 through 15 of gestation), rats (s 295 mg/kg administered on days through 15 of gestation), hamsters (g 272 mg/kg administered on days ~ through i0 of gestation), and rabbits (S 425 mg/kg administered on days 6 through 18 of gestation) found no - 12 -

adverse effects on nidation, maternal or fetal survival, or the number of abnormalities occurring in either soft or skeletal tissues. (Food and Drug Research Laboratories, Inc., 1973). In an evaluation conducted in the developing chicken embryo (Verrett, 1976), potassium citrate had no teratogenic effects. In studies of chicken emb~ros performed by Landauer and Rhodes (1952) and Beaudoin (1968), citric acid was found to decrease t~e.teratogenic effect of insulin and of t~pan blue. I Incubation of transplantable tumors (Walker carcinoma and Pliss lympho-sarcoma) in sodium citrate inhibited tumor at concentrations above 30 mg/kg (Prizhivoit, 1969).~~ growth G. InununotoxicitV. Fatal experimental tuberculosis progressed more rapidly in mice given a diet containing 8 to i0 percent sodium citrate (approximately 5.5 g/kg body weight~ than in control mice (Dubos, 1955). Adding 2 percent to the drinking water also caused an accelerated rate of mortality in the animals. The investigator noted that addition of sodium citrate to the diets of control mice markedly reduced their rate of weight gain. There are no available studies concerning the immunotoxicity of potassium citrate. - 13 -