INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY WORLD HEALTH ORGANIZATION SUMMARY OF TOXICOLOGICAL DATA OF CERTAIN FOOD ADDITIVES WHO FOOD ADDITIVES SERIES NO. 12 The data contained in this document were examined by the Joint FAO/WHO Expert Committee on Food Additives* Geneva, 18-27 April 1977 Food and Agriculture Organization of the United Nations World Health Organization * Twenty-first Report of the Joint FAO/WHO Expert Committee on Food Additives, Geneva, 1977, WHO Technical Report Series No. 617 SODIUM AND CALCIUM CYCLAMATES Sodium and calcium cyclamates were previously evaluated by the Joint FAO/WHO Expert Committee on Food Additives for the Fourteenth (FAO/WHO, 1970) and Eighteenth (FAO/WHO, 1974) Reports. Since the previous evaluation, additional data have become available. Feeding studies Kroes et al. (1975) designed a study to examine the short-term (reproductive, perinatal, teratogenic) and long-term (carcinogenic) toxicological effects of sodium cyclamate and acid saccharin, through 6 generations of Swiss-SPF derived outbred mice. The following dietary treated groups were used: 1. Control 2. 5% sodium cyclamate 3. 2% sodium cyclamate 4. 5% sodium cyclamate + 0.5% acid saccharin 5. 2% sodium cyclamate + 0.2% acid saccharin 6. 0.5% acid saccharin 7. 0.2% acid saccharin The P-generation consisted of 50 males and 50 females per group. Five weeks after the start of the study, 20 females and 10 males per group were selected to produce the F1 litter. The litter size was inadequate and the remaining 30 females were mated with 15 males to produce the F1a' generation. Subsequently, 20 females and 10 males were chosen to produce litters for each succeeding generation. The P, F3b and F6a generation consisted of 50 males and 50 females and were used to study carcinogenicity. The remaining generations consisted of 10 males and 20 females. Reproduction was studied in generations F1a',, F2a, F3a, F4a and F5a. Perinatal studies were conducted with the F1a', F2b, F4b and F5b generations. A teratogenic study was undertaken with the F6b generation. Although the authors did not attempt to explain the F1a results, no consistent effect upon the various reproductive indices was observed due to treatment. Similarly no consistent toxic, embryotoxic, teratogenic or carcinogenic effects were observed. A total of 7 bladder tumours were observed during the study in a total of 2100 animals. The tumours were scattered among the different groups including controls. Testicular atrophy studies - Cyclamate Mouse Brantom et al. (1972) fed mice diets containing up to 7% sodium cyclamate and no testicular changes were observed. Rat In a 12-month feeding study with rats, Nees and Derse (1967) fed calcium cyclamate at dietary levels up to 10%. The absolute and relative testicular weights were similar to controls. Histopathological examination of the testes was not carried out. In a 3-generation reproduction study Ferrando and Huchet (1968) reported that calcium cyclamates at dietary levels of 3% caused atrophy of the testes. Following the administration of graded doses of a 10:1 cyclamate/saccharin mixture to rats for 24 months, Oser et al. (1970) found testicular atrophy in rats treated with 2500 mg/kg body weight/day. These animals also had significantly lower body weights than controls. Similar results were also reported by Taylor (1973) when calcium cyclamate was fed at a dietary level of 5% for 24 months. In another long-term study (28 months), Miyaji (1973) also found a marked testicular atrophy associated with a reduction in body weight of rats treated with sodium cyclamate or a 10:1 cyclamate/saccharin mixture at a dietary level of 5%. Friedman et al. (1972) fed rats diets containing up to 10% sodium or calcium cyclamate for 101 weeks and Schmahl (1973) fed 5% sodium cyclamate diets to rats for their entire lifespan. No testicular atrophy was observed in either study. METABOLISM Animal studies When various laboratory animals were fed diets containing cyclamate, there was generally an initial slow increase of cyclohcxylamine (CHA) excretion for the first 4 days after commencement of treatment. Thereafter, the urinary levels of CHA fluctuate markedly, often on a day-to-day basis. For example, Saunders and Wiegand (1967) reported that one rat fed a 5% cyclamate diet excreted a maximum amount of 81 mg of CHA in the urine on the 29th test day while on day 34 only 0.1 mg of CHA was excreted. Oser et al. (1968) classified rats as either high, low or zero converters of cyclamate to CHA, based on urinary levels of CHA collected during week 27 of the study. Subsequent urinary samples from these animals collected 2.5 months later showed marked changes in cyclamate to CHA conversion. One animal previously classified as a zero converter was found to be a high converter. Similarly with pigs fed diets containing 0.1% cyclamate, Collings (1971) found that high urinary CHA levels were often preceded by very low levels of CHA excretion, despite continued cyclamate administration. Cyclohexylamine has also been found in the urine of the dog, guinea-pig, rabbit and monkey following the administration of cyclamate (Renwick and Williams, 1972; Goldberg et al., 1969; Asahina et al., 1972; Ichibagase et al., 1972; Parekh et al., 1970). Clinical studies The metabolic conversion of cyclamate to cyclohexylamine has been extensively studied in over 1000 human subjects. The urinary excretion of cyclohexylamine was found to be extremely variable from individual to individual and fluctuated greatly from day to day. The incidence of subjects that convert cyclamate to CHA would appear to be 10-30% of the test population. The majority of these convert < 0.1% to approximately 8% of the ingested cyclamate to CHA. A relatively few individuals convert up to 60% of the ingested cyclamate to CHA. Several studies indicate the conversion of cyclamate to CHA was inversely related to the cyclamate dose. When 5.0 g of cyclamate was ingested 0.6% of it was converted to CHA, while 13.4% of a 1 g dose of cyclamate was converted to CHA. For more specific details see Asahina et al., 1971, 1972; Collings, 1971; Davis et al., 1969; Golberg et al., 1969; Ichibagase et al., 1972; Leahy et al., 1966, 1967; Litchfield and Swan, 1971; Oser et al., 1968; Pawan, 1970; Saunders, 1967, 1968; Saunders and Wiegand, 1967, 1968a, 1968b; Saunders et al., 1967; Renwick and Williams, 1972; Williams, 1971; Wills et al., 1968. CYCLOHEXYLAMINE Cyclohexylamine (CHA), a bacterial metabolite of cyclamate produced in the gastrointestinal tract, was previously evaluated by the Joint FAO/WHO Expert Committee on Food Additives for the Fourteenth (FAO/WHO, 1970) and Eighteenth (FAO/WHO, 1974) Reports. Since the previous evaluation, additional data have become available. Feeding studies Kroes et al. (1975) designed a study to examine the short-term (reproduction, perinatal, teratogenic) and long-term (carcinogenic) toxicological effects of cyclohexylamine through 6 generations using Swiss-SPF derived outbred mice. The dietary groups consisted of 0, or 0.5% CHA. The P-generation consisted of 50 male and 50 female group. Five weeks after the start of the study, 20 females and 10 male group were selected to produce the F1a litter. The litter size was inadequate and the remaining 30 females were mated with 15 males to produce the F1a' generation. The P, F3b and F6a generations consisted of 50 males and 50 females and were used to investigate potential carcinogenicity. The remaining generation consisted of 10 males and 20 females. Reproduction was studied in generations F1a', F2a, F3a, F4a, F5a. Perinatal studies were conducted with the F1a', F2b, F4b and F5b generations and a teratogenicity study was undertaken with the F6b generation. The authors did not explain the F1a results. The CHA treatment resulted in a slight decrease in litter size, weight and survival of the pups in all generations. Maternal body weight and feed consumption were reduced due to CHA treatment and this may have contributed to the embryo-toxic effects. The perinatal study indicated that CHA significantly reduced the number of implantation sites in all but one generation. The teratogenicity study showed that CHA had a slight tendency to delay ossification. No bladder tumours were observed in the 300 CHA-treated animals, while one bladder tumour was observed in controls. The incidence of testicular atrophy in the CHA-treated males was similar to the incidence in control males. Testicular atrophy studies -Cyclohexylamine Mouse When CHA was administered at dietary levels of 0, 0.03, 0.1 or 0.3% to mice for 80 weeks, no testicular changes were observed (Crampton, 1973). Rat Collings and Kirkby (1974) fed rats diets containing 0, 0.01, 0.05, 0.1, 0.2, 0.5 or 1% CHA for 13 weeks. Decreased absolute testicular weights and body weight gains occurred at the two highest dosage levels. A dose-related decrement in food consumption occurred in the groups treated with 0.2, 0.5 and 1.0% CHA. A significantly increased incidence of histological changes in the testes (tubular degeneration) occurred only at the 1% dietary level. The no-effect level for testicular change was 0.5% (approv. 250 mg/kg bw) CHA in the diet. In a 13-week study (Gaunt et al., 1974) and a 24-month study (Crampton, 1973) in which CHA was fed at dietary levels of 0, 0.06, 0.2 and 0.6% in the diet of rats, significantly decreased absolute testicular weights were observed in the 0.2 and 0.6% groups; these groups also exhibited significantly decreased body weight gains and food consumption. An increased incidence of testicular changes was observed with 0.6% CHA in both studies, with a questionable increase in the incidence at 0.2% CHA in the 13-week study. Five rats treated with 0.6% CHA in diet for 10 months exhibited normal fertility indices when tested for reproductive capacity. A no-effect level of 0.2% (approv. 100 mg/kg bw) was suggested. Crampton (1975) fed CHA at dietary levels of 0, 0.06, 0.1, 0.2 and 0.6% to two strains of rats. The study included a pair-fed and a paired-weight control. In both strains, a significant increase in testicular effects (decreased spermatogenesis) was observed at 0.6%. A no-effect level of 0.2% (‰ 100 mg/kg bw) was suggested. CHA was administered to rats at dosage levels of 0, 15, 50, 100 and 150 mg/kg body weight/day for 24 months (Oser et al., 1972). Trends towards dose-related decreases in food intake, body weight gain, absolute testes weight and relative testicular weights were observed, but testicular weights were not significantly different from controls. The histopathological changes in the testes from treated animals were not significantly different from controls. Calandra (1969) fed CHA for 24 months at dosage levels of 0, 0.15, 1.5 and 15 mg/kg body weight/day to rats and reported that food intake, body weight gain, testes weights and testes histopathology of the treated animals were comparable to controls. Reproduction study Rat In a 5-generation reproduction study by Oser et al. (1972), CHA was fed at dietary levels of 0, 15, 50, 100 and 150 mg/kg bw/day. An increased number of resorption sites, lower number of pups cast alive and lower body weights of pups at weaning were found for the groups receiving 100 and 150 mg/kg. However, these effects were considered secondary, due to the decreased food consumption and decreased body weight gains of the pregnant dams in these groups. Calculation of an ADI The committee noted an unresolved problem in the toxicological evaluation of cyclamates due to its conversion to CHA by gut flora and the committee is also aware that additional studies concerning this problem are under way. CHA is known to produce testicular atrophy in rats. It was considered that this may be a species specific effect and it was not known whether this effect had ever occurred in human males. Data available from clinical studies of cyclamate metabolism indicates that about 40% of dietary cyclamate was absorbed and thus unavailable for conversion by gut flora into CHA. The inter- and intra-subject variability in the conversion of cyclamate to CHA was considered to be related to changes in the gut flora. It was estimated that for safety evaluation purposes an average figure of 30% conversion of the unabsorbed cyclamate could be accepted. Thus taking into consideration all these factors a temporary ADI was calculated as follows: No-effect level of cyclohexylamine (free base) in rat = 74 mg/kg bw/day. Temporary ADI (200-fold safety factor) for man for cyclohexylamine = 0.37 mg/kg bw. Converting cyclohexylamine to cyclamic acid Molecular weight cyclamic acid = 2 Molecular weight cyclohexylamine Thus if all cyclamate was converted to cyclohexylamine, the ADI for cyclamate would be 0.74 mg/kg bw. Since only 60% of ingested cyclamate is available for conversion, and only 30% of cyclamate is converted to cyclohexylamine, the temporary ADI for cyclamate is: 0.74 × 1.67 × 3.33 approv. 4 mg/kg bw.* * Expressed as cyclamic acid. 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See Also: Toxicological Abbreviations