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Another try at BHT metabolism in man
TOXICOLOGY:
BIBRA
FLAVOURINGS,
ABSTRACTS SOLVENTS
Relatives of cyclamate Spillane, W. J. & Benson, G. A. (1978). Metabolic studies of the non-nutritive sweeteners cyclopentylmethylsulfamate and cyclopentylsulfamate: determination of metabolites in rat urine. J. pharm. Sci. 67, 226. Cyclopentylmethylsulphamate (CPMS), cyclopentylsulphamate (CPS) and cycloheptylsulphamate (CHS) are non-nutritive sweeteners chemically related to cyclamate. The authors cited above have previously described studies in which single oral doses of CPS and CHS were administered to rats and rabbits. Some breakdown of the two sulphamates to their amine, alcohol and ketone metabolites occurred in both species (Benson & Spillane, J. pharm. Sci. 1976, 65, 1841 : i&m, ihid 1977, 66, 881). In the study cited above. similar investigations were carried out into the effects of administering a single dose of CPMS to rats and of extended feeding of CPS. Sodium CPMS in aqueous solution was administered to five female Wistar rats in a single oral dose of of 1450 mg/kg. Urine was collected for 3 days after dosing. The urine samples were analysed for cyclopentylmethylamine and cyclopentylmethanol by gasliquid chromatography (GLC). A spectrophotometric method was used to analyse for sulphamates. Neither of the sulphamates was converted to its amine, alcohol or ketone derivatives during storage in solution in urine for 3 days under refrigeration.
AND
AND COMMENTS SWEETENERS
Three of the rats fed CPMS excreted cyclopentylmethylamine and cyclopentylmethanol. The mean conversions of the sulphamate to its amine and alcohol metabolites were 0,011 and 0.012°Z;. In previous studies the mean conversions of CPS and CHS to cyclopentylamine and cycloheptylamine were 0.57 and 0.064”:, respectively (Benson & Spillane. 1976 Kc 1977 lot. cit.). Therefore CPMS appears more stable than either of these other sulphamates. However. levels of cyclamate (cyclohexylsulphamate) conversion to cyclohexylamine in rats not pretreated with cyclamate are generally reported to be lower (Renwick & Williams, Biochem. J. 1972, 129, 869; Suenaga c’t a/. Chem. phurm. Bu/l., Tokyo 1972, 20, 1357). The mean level of excretion of unchanged CPMS in the urine was 15.4”“. For CPS and CHS. the amounts of :I single dose recovered unchanged in the urine were reported to be I5 and 35’?,,, respectively (Benson & Spillane, 1976 & 1977 lot. cit.). Some metabolism of CPS occurred in all rats fed the compound over 9 days. Following administration of 200mg on days l--5 and on days 8 and 9 to fi\e females. cyclopentylamine was excreted in the urine of all rats on day 1, but on the following 6 days there were considerable differences between individual animals in the levels of cyclopentylamine, cyclopentanol and cyclopentanone excreted. When feeding w/as resumed on days 8 and 9 only traces of metabolites were detected in the urine of all the rats.
ANTIOXIDANTS and 200 mg respectively. Urine was collected for 72 hr after dosing. The major urinary metabolite was shown to have a mass spectrum comparable to the dicarboxylic acid aldehyde reported earlier (Cited in F.C.T. 1968, 6, 533). However analysis by proton and r3C-nuclear magnetic resonance spectroscopy indicated that the metabolite was in fact a dicarboxylic acid hemiacetal. 5-carboxy-7-(l-carboxy-l-methylethyl)-3,3-dimethyl2-hydroxy-2,3_dihydrobenzofuran. The acetylated dimethyl derivative of the hemiacetal proved amenable to quantification by gas chromatography (GC). Over a period of 72 hr, 21.4:; of the radioactive dose of [i3C]BHT was excreted in the urine as the hemiacetal. This metabolite was present in the urine samples collected during the first 3 hr after dosing. and excretion was at a maximum in the period 3 6 hr after BHT administration. Although the GC detection limit for BHT and its metabolites was of the order 01 1 ng/ml of urine or whole blood extracted, the oni! other urinary metabolite identified was a small
Another try at BHT metabolism in man Wiebe. L. I., Mercer. J. R. & Ryan, A. J. (1978). Urinary metabolites of 3,5-di-(l-[‘3C]methyl-l-methylethyl)-4-hydroxytoluene (BHT-13C) in man. Drug Met&.
Dispos.
6, 296.
It has been reported that the major urinary metabolite of BHT in man is 4-carboxy-2-( l-carboxy-lmethylethyl)-6-(1-formyl-1-methylethyl)phenol (Cited in F.C.T. 1968, 6, 533). However, other workers have been unable to detect this compound, but have reported the presence of 3,5-di-tert-butyl-4-hydroxybenzoic acid (BHT-acid) in human urine (ibid 1971, 9, 296; Ryan, Fd Cosmet. Toxicol. 1971, 9, 769). Further work on BHT metabolism is described below. On three occasions, separated by intervals of 2 months, a single volunteer was given an oral dose of BHT suspended in olive oil. In one experiment 201.7 mg [i3C]BHT was taken, and in the other two, unlabellcd material was administered at doses of 100 551
552
Miscellaneous
direct additives--Fd
Cosnw~. Touicol. Vol. 17. no, 5
amount of the BHT-acid (0.3% of the administered dose over 72 hr), and traces of S-(3,5-di-l-methyl-lmethylethyl-4-hydroxybenzyl)-hJ-acetylcysteine (mercapturic acid) were indicated in one fraction. Trace quantities of unchanged BHT, but no free metabolites, were found in serum samples taken at 15-min intervals up to 90 min after administration of 100 mg BHT. The total blood content of unchanged BHT (estimated on 4 litres of whole blood) ranged from 0.3 to 3.0 /Lg. The low recovery of the adminstered dose of radioactivity indicated that urinary elimination is not the only route of elimination for BHT in man-a finding
MISCELLANEOUS
at variance with an earlier tracer study (Daniel et LI/. Fd Comet. TGxicol. 1967, 5, 475twhile the rapid disappearance of BHT from the blood suggested that hepatobiliary excretion may be an important factor. [This preliminary study may provide the much needed spur for further investigation of BHT’s complex pharmacokinetics in both man and animals. The need for further studies was underlined by a FASEB Report to the FDA (Fd them. News 1977, 19 (1 l), 17) which called for metabolic studies on the antioxidant to find a suitable species for evaluating the possible hazards of BHT to man.]
DIRECT
A talc-on-rice survey Stemmermann, G. N. & Kolonel, L. N. (1978). Talccoated rice as a risk factor for stomach cancer. Am. J. c/in. Nutr. 31. 2017. The coating of rice with talc is an accepted way to slow down spoilage and prolong shelf life, and consumers seem to prefer to buy rice that has a sheen (the rice is actually coated with glucose syrup and talc). In Hawaii, 8%90% of the rice sold is talc-coated. On the other hand, the sale of coated rice has been prohibited in Japan since 1947. These two areas have thus provided some opportunity for investigation of the hypothesis that the high incidence of stomach cancer among Japanese may be associated with the consumption of talc-coated rice, or more specifically of rice coated with asbestos-contaminated talc. Alreadyreported studies of the considerable Japanese population living in Hawaii (Haenszel et al. J. n&n. Cancer Inst. 1972, 49, 969) and of indigenous Japanese, in Miyagi and Okayama (Waterhouse et ul. Cancer Incirlerlcv in Five Continents, Vol. III, p. 498; IARC, Lyon,
AGRICULTURAL The renal effects of diphenylamine Evan, A. P., Hong, S. K., Gardner, K., Jr., Park, Y. S. & Itagaki, R. (1978). Evolution of the collecting tubular lesion in diphenylamine-induced renal disease. Lab. Invest. 38, 244. Cystic dilation of the renal tubules, invariably accompanied by chronic interstitial nephritis, was found in rats fed diphenylamine (DPA) at dietary levels of @lx or more (Cited in F.C.T. 1968,6, 289). A 2-yr-old sample of DPA had far more severe renal effects than a freshly-manufactured sample, and a contaminant of the former was identified as the causative agent (ibid 1973, 11, 334). The DPA-induced development of functional and structural changes in the rat kidney has now been followed in detail. When rats were fed 1% dietary DPA for up to
ADDITIVES
1976), like more recent studies carried out elsewhere (Cuello et al. J. nutn. Cancer Inst. 1976, 57, 1015), provided little support for this hypothesis. Further light was thrown on the question when the Epidemiology Unit at the University of Hawaii conducted a nutritional survey among the various races in Hawaii on the basis of an annual random sample of 3” of the Hawaiian state population (Stemmermann & Kolonel, cited above). Questioning about weekly rice consumption revealed that the highest consumers of rice were the Filipinos, who had the lowest incidence of gastric cancer, and the next were the Japanese, who showed the highest incidence of this type of tumour. The survey thus emphasized the earlier lack of support for the suggestion that talccoated rice might be an important factor in the aetiology of gastric cancer. It is interesting to note that the high rate of gastric cancer among indigenous Japanese seemed to persist at a fairly stable level between the early 1960s and 197Os, during which period there was some decrease in the occurrence of this tumour among the Hawaiian Japanese.
CHEMICALS 78 wk, a decrease in urinary osmolality was evident after 2 wk, and was statistically significant at wk 6 and 20. The decrease in urinary osmolality was primarily caused by a decrease in the urinary concentration of urea and was accompanied by an increase in urine flow. Giomerular filtration, measured by endogenous creatinine clearance, was similar to that of the controls. After 5 wk, the medullary collecting tubules showed hyperplasia, with multilayering of cells, an increase in dark cells, and an increased number of [3H]thymidine-labelled nuclei. By wk 10 some collecting ducts were dilated. with focal areas of cellular necrosis, and a few contained small amounts of cast material. These changes were far more frequent after 15-20 wk, and by wk 24 there were frank cysts in the cortex and medulla. After 52-78 wk such cysts were found in almost every segment of the nephron and the collecting tubules con-
BIBRA
FLAVOURINGS,
ABSTRACTS SOLVENTS
Relatives of cyclamate Spillane, W. J. & Benson, G. A. (1978). Metabolic studies of the non-nutritive sweeteners cyclopentylmethylsulfamate and cyclopentylsulfamate: determination of metabolites in rat urine. J. pharm. Sci. 67, 226. Cyclopentylmethylsulphamate (CPMS), cyclopentylsulphamate (CPS) and cycloheptylsulphamate (CHS) are non-nutritive sweeteners chemically related to cyclamate. The authors cited above have previously described studies in which single oral doses of CPS and CHS were administered to rats and rabbits. Some breakdown of the two sulphamates to their amine, alcohol and ketone metabolites occurred in both species (Benson & Spillane, J. pharm. Sci. 1976, 65, 1841 : i&m, ihid 1977, 66, 881). In the study cited above. similar investigations were carried out into the effects of administering a single dose of CPMS to rats and of extended feeding of CPS. Sodium CPMS in aqueous solution was administered to five female Wistar rats in a single oral dose of of 1450 mg/kg. Urine was collected for 3 days after dosing. The urine samples were analysed for cyclopentylmethylamine and cyclopentylmethanol by gasliquid chromatography (GLC). A spectrophotometric method was used to analyse for sulphamates. Neither of the sulphamates was converted to its amine, alcohol or ketone derivatives during storage in solution in urine for 3 days under refrigeration.
AND
AND COMMENTS SWEETENERS
Three of the rats fed CPMS excreted cyclopentylmethylamine and cyclopentylmethanol. The mean conversions of the sulphamate to its amine and alcohol metabolites were 0,011 and 0.012°Z;. In previous studies the mean conversions of CPS and CHS to cyclopentylamine and cycloheptylamine were 0.57 and 0.064”:, respectively (Benson & Spillane. 1976 Kc 1977 lot. cit.). Therefore CPMS appears more stable than either of these other sulphamates. However. levels of cyclamate (cyclohexylsulphamate) conversion to cyclohexylamine in rats not pretreated with cyclamate are generally reported to be lower (Renwick & Williams, Biochem. J. 1972, 129, 869; Suenaga c’t a/. Chem. phurm. Bu/l., Tokyo 1972, 20, 1357). The mean level of excretion of unchanged CPMS in the urine was 15.4”“. For CPS and CHS. the amounts of :I single dose recovered unchanged in the urine were reported to be I5 and 35’?,,, respectively (Benson & Spillane, 1976 & 1977 lot. cit.). Some metabolism of CPS occurred in all rats fed the compound over 9 days. Following administration of 200mg on days l--5 and on days 8 and 9 to fi\e females. cyclopentylamine was excreted in the urine of all rats on day 1, but on the following 6 days there were considerable differences between individual animals in the levels of cyclopentylamine, cyclopentanol and cyclopentanone excreted. When feeding w/as resumed on days 8 and 9 only traces of metabolites were detected in the urine of all the rats.
ANTIOXIDANTS and 200 mg respectively. Urine was collected for 72 hr after dosing. The major urinary metabolite was shown to have a mass spectrum comparable to the dicarboxylic acid aldehyde reported earlier (Cited in F.C.T. 1968, 6, 533). However analysis by proton and r3C-nuclear magnetic resonance spectroscopy indicated that the metabolite was in fact a dicarboxylic acid hemiacetal. 5-carboxy-7-(l-carboxy-l-methylethyl)-3,3-dimethyl2-hydroxy-2,3_dihydrobenzofuran. The acetylated dimethyl derivative of the hemiacetal proved amenable to quantification by gas chromatography (GC). Over a period of 72 hr, 21.4:; of the radioactive dose of [i3C]BHT was excreted in the urine as the hemiacetal. This metabolite was present in the urine samples collected during the first 3 hr after dosing. and excretion was at a maximum in the period 3 6 hr after BHT administration. Although the GC detection limit for BHT and its metabolites was of the order 01 1 ng/ml of urine or whole blood extracted, the oni! other urinary metabolite identified was a small
Another try at BHT metabolism in man Wiebe. L. I., Mercer. J. R. & Ryan, A. J. (1978). Urinary metabolites of 3,5-di-(l-[‘3C]methyl-l-methylethyl)-4-hydroxytoluene (BHT-13C) in man. Drug Met&.
Dispos.
6, 296.
It has been reported that the major urinary metabolite of BHT in man is 4-carboxy-2-( l-carboxy-lmethylethyl)-6-(1-formyl-1-methylethyl)phenol (Cited in F.C.T. 1968, 6, 533). However, other workers have been unable to detect this compound, but have reported the presence of 3,5-di-tert-butyl-4-hydroxybenzoic acid (BHT-acid) in human urine (ibid 1971, 9, 296; Ryan, Fd Cosmet. Toxicol. 1971, 9, 769). Further work on BHT metabolism is described below. On three occasions, separated by intervals of 2 months, a single volunteer was given an oral dose of BHT suspended in olive oil. In one experiment 201.7 mg [i3C]BHT was taken, and in the other two, unlabellcd material was administered at doses of 100 551
552
Miscellaneous
direct additives--Fd
Cosnw~. Touicol. Vol. 17. no, 5
amount of the BHT-acid (0.3% of the administered dose over 72 hr), and traces of S-(3,5-di-l-methyl-lmethylethyl-4-hydroxybenzyl)-hJ-acetylcysteine (mercapturic acid) were indicated in one fraction. Trace quantities of unchanged BHT, but no free metabolites, were found in serum samples taken at 15-min intervals up to 90 min after administration of 100 mg BHT. The total blood content of unchanged BHT (estimated on 4 litres of whole blood) ranged from 0.3 to 3.0 /Lg. The low recovery of the adminstered dose of radioactivity indicated that urinary elimination is not the only route of elimination for BHT in man-a finding
MISCELLANEOUS
at variance with an earlier tracer study (Daniel et LI/. Fd Comet. TGxicol. 1967, 5, 475twhile the rapid disappearance of BHT from the blood suggested that hepatobiliary excretion may be an important factor. [This preliminary study may provide the much needed spur for further investigation of BHT’s complex pharmacokinetics in both man and animals. The need for further studies was underlined by a FASEB Report to the FDA (Fd them. News 1977, 19 (1 l), 17) which called for metabolic studies on the antioxidant to find a suitable species for evaluating the possible hazards of BHT to man.]
DIRECT
A talc-on-rice survey Stemmermann, G. N. & Kolonel, L. N. (1978). Talccoated rice as a risk factor for stomach cancer. Am. J. c/in. Nutr. 31. 2017. The coating of rice with talc is an accepted way to slow down spoilage and prolong shelf life, and consumers seem to prefer to buy rice that has a sheen (the rice is actually coated with glucose syrup and talc). In Hawaii, 8%90% of the rice sold is talc-coated. On the other hand, the sale of coated rice has been prohibited in Japan since 1947. These two areas have thus provided some opportunity for investigation of the hypothesis that the high incidence of stomach cancer among Japanese may be associated with the consumption of talc-coated rice, or more specifically of rice coated with asbestos-contaminated talc. Alreadyreported studies of the considerable Japanese population living in Hawaii (Haenszel et al. J. n&n. Cancer Inst. 1972, 49, 969) and of indigenous Japanese, in Miyagi and Okayama (Waterhouse et ul. Cancer Incirlerlcv in Five Continents, Vol. III, p. 498; IARC, Lyon,
AGRICULTURAL The renal effects of diphenylamine Evan, A. P., Hong, S. K., Gardner, K., Jr., Park, Y. S. & Itagaki, R. (1978). Evolution of the collecting tubular lesion in diphenylamine-induced renal disease. Lab. Invest. 38, 244. Cystic dilation of the renal tubules, invariably accompanied by chronic interstitial nephritis, was found in rats fed diphenylamine (DPA) at dietary levels of @lx or more (Cited in F.C.T. 1968,6, 289). A 2-yr-old sample of DPA had far more severe renal effects than a freshly-manufactured sample, and a contaminant of the former was identified as the causative agent (ibid 1973, 11, 334). The DPA-induced development of functional and structural changes in the rat kidney has now been followed in detail. When rats were fed 1% dietary DPA for up to
ADDITIVES
1976), like more recent studies carried out elsewhere (Cuello et al. J. nutn. Cancer Inst. 1976, 57, 1015), provided little support for this hypothesis. Further light was thrown on the question when the Epidemiology Unit at the University of Hawaii conducted a nutritional survey among the various races in Hawaii on the basis of an annual random sample of 3” of the Hawaiian state population (Stemmermann & Kolonel, cited above). Questioning about weekly rice consumption revealed that the highest consumers of rice were the Filipinos, who had the lowest incidence of gastric cancer, and the next were the Japanese, who showed the highest incidence of this type of tumour. The survey thus emphasized the earlier lack of support for the suggestion that talccoated rice might be an important factor in the aetiology of gastric cancer. It is interesting to note that the high rate of gastric cancer among indigenous Japanese seemed to persist at a fairly stable level between the early 1960s and 197Os, during which period there was some decrease in the occurrence of this tumour among the Hawaiian Japanese.
CHEMICALS 78 wk, a decrease in urinary osmolality was evident after 2 wk, and was statistically significant at wk 6 and 20. The decrease in urinary osmolality was primarily caused by a decrease in the urinary concentration of urea and was accompanied by an increase in urine flow. Giomerular filtration, measured by endogenous creatinine clearance, was similar to that of the controls. After 5 wk, the medullary collecting tubules showed hyperplasia, with multilayering of cells, an increase in dark cells, and an increased number of [3H]thymidine-labelled nuclei. By wk 10 some collecting ducts were dilated. with focal areas of cellular necrosis, and a few contained small amounts of cast material. These changes were far more frequent after 15-20 wk, and by wk 24 there were frank cysts in the cortex and medulla. After 52-78 wk such cysts were found in almost every segment of the nephron and the collecting tubules con-