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Metabolic studies with radiolabelled chlorhexidine in animals and man
Cosmetics, toiletries and household products
587
Cultures ofS. atra, grown for 4 wk at 20--22°C on oats, produced five compounds which were toxic to the larvae of the brine shrimp (Artemia salina). The compounds were designated satratoxins C, D, F, G and H, according to their distribution on thin-layer chromatograms. From 2 kg oats were isolated 2 mg satratoxin C, 3 mg D, 2 mg F, 8 mg G and 12 mg H. Satratoxin D was found to be identical with the known fungal metabolite roridin E, a macrocyclic sesquiterpcne. Satratoxins G and H, with molecular weights of 544 ( C 2 8 H 3 2 O t l ) and 528 (C29H3609) , respectively, both yielded verrucarol on alkaline hydrolysis in methanol. In this respect, they resembled the roridins and verrucarins. The ~ltratoxins thus appear to be members of the 12,13-epoxy-Ag-trichothecene group, closely rcl~ttcd to the toxins produced by various species of Myrothecium.
2737. Another fungal toxin Hutchison, R. D., Steyn, P. S. & van Rensburg, S. J. (1973). Viridicatum-toxin, a new mycotoxin from Penicillium t:iridicatunt Westling. Toxic. appl. Pharmac. 24, 507. One of the relatively recent additions to the seemingly endless list of toxigenic fungi is Pe~licillium viridicatum, which is known to cause tubular degeneration in the pig kidney and liver lesions in rats and guinea-pigs (Cited in F.C.T. 1971, 9, 759). It has also been shown to have some carcinogenic effect (Zwicker et al. Fd Cosmet. Toxicol. 1973, 11,989). A substance which was apparently the sole acutely toxic component of maize cultures of this fungus has now been characterized. Cultures of a toxigenic P. viridicatum strain were subjected to several chloroform and methanol extractions and the final methanol-soluble product was fractionated by column chromatography on formamide-impregnated cellulose powder. The toxin so obtained was reported to be a yellow pigment with a molecular formula C30H31NOI0. The acute oral LDso in male rats was 122-4 mg/kg. Deaths were generally sudden, with no preceding clinical signs, and the time of occurrence was dose related, the first deaths occurring within 24 hr in the group given the highest dose (500 mg/kg) and the last on day 7 among rats given the lowest dose (50mg/kg). Post-mortem examination revealed hyaline-droplet degeneration of the myocardium in all treated animals, renal tubular necrosis in those that dicd and a frequent incidence of hepatocyte changes and splenic atrophy.
COSMETICS, TOILETRIES AND H O U S E H O L D PRODUCTS
2738. Investigations into the fate of chiorhexidine Winrow, M. J. (1973). Metabolic studies with radiolabelled chlorhexidine in animals and man. J. periodont. Res. 8, (Suppl. 12), 45. Although chlorhexidine has been used for some 20 yr as a topically applied bactericide, interest in its metabolism, absorption and excretion has come to the fore only recently, prompted no doubt by its increasing use in oral hygiene, by its potential as a substitute for hexachlorophene and, on the practical side, by the increasing availability of radiolabelled compounds. We reported last month (Cited in F.C.T. 1974, 12, 433) on an autora-
588
Methods for assessing toxicity
diographic study of the tissue distribution of [t4C]chlorhexidine given orally to mice, and the paper cited above reports further on the fate of the compound in several species. Chlorhexidine labelled with 14C in either the aromatic ring or central hexamethylene chain was administered orally, as the digluconate salt, in varying amounts to the rat, mouse, dog, marmoset and rhesus monkey. Urinary excretion of chlorhexidinc was extremely low in all species, over 90~o of the administered dose being recovered, mainly from the faeces, within 72 hr. Except in the mouse (in which the figure was 3-7~o), less than 1% of the administered dose was recovered in the urine over a period of about 7 days. Less than 0.25% of the administered dose was found in the liver and kidney of the rodents, while the brain, lungs, heart, spleen, fat and muscle were devoid of radioactivity (the limit of detection being 0"01/~g chlorhexidine/'g). Similar results were obtained in the other animal species examined. In the single human voluntecr studied, only 82% of the dose of 0-07 mg/kg, administered as a solution of the digluconatc in a gelatin capsule, was recovered in the 6 days of the experiment. It was thought, however, that quantitative recovery would have been obtained if sampling had continued longer. No radioactivity was found in the blood or saliva. Urine samples, including those from the volunteer, were examined by thin-layer chromatography (TLC), particularly for p-chloroaniline, an established chemical breakdown product of chlorhexidine known to be rapidly excreted by the kidney after oral administration. No p-chloroanilinc was detected in any of the samples examined. Ringand chain-labelled chlorhexidine produced essentially the same radioactivity distribution on the TLC plates, showing that metabolic cleavage was minimal. Only unchanged chlorhexidine was detected in the faecal extracts. The contention that the high faecal content of chlorhexidine was duc to extremely low absorption rather than to absorption followed by biliary excretion was supported by the finding that in rats with implanted biliary cannulae, 50-70~,~ of an iv dose was excreted in the bile within 48 hr, while only 0'2°41 was recovered in that time after oral dosing. TLC examination of the bile of iv-dosed rats revealed only polar metabolites and a small amount of chlorhexidinc. A mouth-rinse study, using 10 ml of a 0.2o/o [ t4C]chlorhexidine solution, was conducted on a volunteer to investigate uptake and retention in the human mouth. After rinsing with the solution for 1 min, the subject removed about 67% of the radioactivity by ejecting the solution and subsequently rinsing his mouth with water. Levels of ~4C in plaque and saliva were then monitored for 24 hr. The amount in saliva fell steadily, while plaque levels remained more constant.
METHODS FOR ASSESSING TOXICITY 2739. Serum urocanase in toxicity testing Trip, J. A. J., van Dam. J., Eibergen, R. & Que, G. S. (1973). Investigations on correlations between serum enzymes and histological findings in liver disease. With special reference to transaminases and urocanase. Acta reed. scand. 193, 113. The value of serum enzyme studies in toxicity testing has been mentioned in these pages recently (Cited in F.C.T. 1973, i I, 930). It was reported that although microscopic methods
587
Cultures ofS. atra, grown for 4 wk at 20--22°C on oats, produced five compounds which were toxic to the larvae of the brine shrimp (Artemia salina). The compounds were designated satratoxins C, D, F, G and H, according to their distribution on thin-layer chromatograms. From 2 kg oats were isolated 2 mg satratoxin C, 3 mg D, 2 mg F, 8 mg G and 12 mg H. Satratoxin D was found to be identical with the known fungal metabolite roridin E, a macrocyclic sesquiterpcne. Satratoxins G and H, with molecular weights of 544 ( C 2 8 H 3 2 O t l ) and 528 (C29H3609) , respectively, both yielded verrucarol on alkaline hydrolysis in methanol. In this respect, they resembled the roridins and verrucarins. The ~ltratoxins thus appear to be members of the 12,13-epoxy-Ag-trichothecene group, closely rcl~ttcd to the toxins produced by various species of Myrothecium.
2737. Another fungal toxin Hutchison, R. D., Steyn, P. S. & van Rensburg, S. J. (1973). Viridicatum-toxin, a new mycotoxin from Penicillium t:iridicatunt Westling. Toxic. appl. Pharmac. 24, 507. One of the relatively recent additions to the seemingly endless list of toxigenic fungi is Pe~licillium viridicatum, which is known to cause tubular degeneration in the pig kidney and liver lesions in rats and guinea-pigs (Cited in F.C.T. 1971, 9, 759). It has also been shown to have some carcinogenic effect (Zwicker et al. Fd Cosmet. Toxicol. 1973, 11,989). A substance which was apparently the sole acutely toxic component of maize cultures of this fungus has now been characterized. Cultures of a toxigenic P. viridicatum strain were subjected to several chloroform and methanol extractions and the final methanol-soluble product was fractionated by column chromatography on formamide-impregnated cellulose powder. The toxin so obtained was reported to be a yellow pigment with a molecular formula C30H31NOI0. The acute oral LDso in male rats was 122-4 mg/kg. Deaths were generally sudden, with no preceding clinical signs, and the time of occurrence was dose related, the first deaths occurring within 24 hr in the group given the highest dose (500 mg/kg) and the last on day 7 among rats given the lowest dose (50mg/kg). Post-mortem examination revealed hyaline-droplet degeneration of the myocardium in all treated animals, renal tubular necrosis in those that dicd and a frequent incidence of hepatocyte changes and splenic atrophy.
COSMETICS, TOILETRIES AND H O U S E H O L D PRODUCTS
2738. Investigations into the fate of chiorhexidine Winrow, M. J. (1973). Metabolic studies with radiolabelled chlorhexidine in animals and man. J. periodont. Res. 8, (Suppl. 12), 45. Although chlorhexidine has been used for some 20 yr as a topically applied bactericide, interest in its metabolism, absorption and excretion has come to the fore only recently, prompted no doubt by its increasing use in oral hygiene, by its potential as a substitute for hexachlorophene and, on the practical side, by the increasing availability of radiolabelled compounds. We reported last month (Cited in F.C.T. 1974, 12, 433) on an autora-
588
Methods for assessing toxicity
diographic study of the tissue distribution of [t4C]chlorhexidine given orally to mice, and the paper cited above reports further on the fate of the compound in several species. Chlorhexidine labelled with 14C in either the aromatic ring or central hexamethylene chain was administered orally, as the digluconate salt, in varying amounts to the rat, mouse, dog, marmoset and rhesus monkey. Urinary excretion of chlorhexidinc was extremely low in all species, over 90~o of the administered dose being recovered, mainly from the faeces, within 72 hr. Except in the mouse (in which the figure was 3-7~o), less than 1% of the administered dose was recovered in the urine over a period of about 7 days. Less than 0.25% of the administered dose was found in the liver and kidney of the rodents, while the brain, lungs, heart, spleen, fat and muscle were devoid of radioactivity (the limit of detection being 0"01/~g chlorhexidine/'g). Similar results were obtained in the other animal species examined. In the single human voluntecr studied, only 82% of the dose of 0-07 mg/kg, administered as a solution of the digluconatc in a gelatin capsule, was recovered in the 6 days of the experiment. It was thought, however, that quantitative recovery would have been obtained if sampling had continued longer. No radioactivity was found in the blood or saliva. Urine samples, including those from the volunteer, were examined by thin-layer chromatography (TLC), particularly for p-chloroaniline, an established chemical breakdown product of chlorhexidine known to be rapidly excreted by the kidney after oral administration. No p-chloroanilinc was detected in any of the samples examined. Ringand chain-labelled chlorhexidine produced essentially the same radioactivity distribution on the TLC plates, showing that metabolic cleavage was minimal. Only unchanged chlorhexidine was detected in the faecal extracts. The contention that the high faecal content of chlorhexidine was duc to extremely low absorption rather than to absorption followed by biliary excretion was supported by the finding that in rats with implanted biliary cannulae, 50-70~,~ of an iv dose was excreted in the bile within 48 hr, while only 0'2°41 was recovered in that time after oral dosing. TLC examination of the bile of iv-dosed rats revealed only polar metabolites and a small amount of chlorhexidinc. A mouth-rinse study, using 10 ml of a 0.2o/o [ t4C]chlorhexidine solution, was conducted on a volunteer to investigate uptake and retention in the human mouth. After rinsing with the solution for 1 min, the subject removed about 67% of the radioactivity by ejecting the solution and subsequently rinsing his mouth with water. Levels of ~4C in plaque and saliva were then monitored for 24 hr. The amount in saliva fell steadily, while plaque levels remained more constant.
METHODS FOR ASSESSING TOXICITY 2739. Serum urocanase in toxicity testing Trip, J. A. J., van Dam. J., Eibergen, R. & Que, G. S. (1973). Investigations on correlations between serum enzymes and histological findings in liver disease. With special reference to transaminases and urocanase. Acta reed. scand. 193, 113. The value of serum enzyme studies in toxicity testing has been mentioned in these pages recently (Cited in F.C.T. 1973, i I, 930). It was reported that although microscopic methods