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2586. Another check on ethylene oxide sterilization
916
PROCESSING AND PACKAGING CONTAMINANTS
faeces, while after iv injection the proportion appearing in the urine was greater (Schulz & Rubin, Phthalic Acid Esters Conference, NIEHS, Pinehurst, N.C., 6-7 September 1972). A study is now reported in which rats were given two intubated doses of 0.2 ml [14C]DEHP 24 hr apart. Urine was collected for 48 hr after the first dose and was subjected to thin-layer chromatography and, after treatment with diazomethane, to thin-layer and gasliquid chromatography. The metabolites isolated in this way were characterized by infra-red and nuclear magnetic resonance spectroscopy and mass spectrometry. It appeared that the DEHP metabolites were not conjugated prior to excretion. Free phthalic acid accounted for less than 3 ~o of the urinary metabolites, but was the only detectable 14C-labelled product after alkaline hydrolysis of either the total ether extract of the urine or the individual metabolites. No mono-2-ethylhexyl phthalate (MEHP) was present in the urine, but the five metabolites identified suggested that hydrolysis of the administered DEHP to MEHP was followed by oJ-oxidation and ( c o - 1)-oxidation, probably in the liver, with the alcohol intermediates being further oxidized to the level of ketone after (oJ -- l)-oxidation or acid after oJ - oxidation and the resulting acid metabolite then undergoing fl-oxidation. It thus appears that, in the rat, DEHP is handled like a fatty acid, oJ-oxidation being the main metabolic pathway since a- and E-oxidation are initially impossible.
2586. Another check on ethylene oxide sterilization
Lawrence, W. H., Dillingham, E. O., Turner, J. E. & Autian, J. (1972). Toxicity profile of chloroacetaldehyde. J. pharm. Sci. 61, 19. Mantz, J. M., Tempe, J. D., Jaeger, A. et Vidal, S. (1972). St6noses trach~ales et st6rilisation des canules de trach~otomie par l'oxyde d'~thyl6ne. Sem. H6p. Paris 48, 3367. Ethylene chlorohydrin (EC) is a reaction product occurring in plastics, spices and foods containing chloride and sterilized by exposure to ethylene oxide (EO) (Cited in F.C.T. 1972, 10, 592). One metabolite of EC in rats has been found to be chloroacetaldehyde (CA), so that a clear picture of the potential hazard of CA should be of assistance in any assessment of the possible results of inadequate removal of EO from sterilized products. The LDso of CA by ip injection was found to be 6/zl/kg in mice, 6--8 tzl/kg in rats, 2 tzl/kg in guinea-pigs and 5/~l/kg in rabbits. When administered orally by intubation, its LDsos in mice and rats were 69 and 75-86 tzl/kg, respectively, while its dermal LDso in rabbits was 224 t~l/kg. When mice were exposed to a CA-air mixture in an inhalation chamber, the time required to kill 5070 of the mice (LTso) was 2.57 min, at which time the chamber atmosphere had reached approximately 45 70 of equilibrium with the incoming mixture, produced by bubbling air through 3070 aqueous CA. CA was extremely toxic to mouse fibroblast cultures, inducing 50 70 inhibition of protein synthesis at 5.62 × 10- 5 M concentration. It also proved very irritant to the skin and eyes of rabbits. Plastics implants previously treated with CA produced muscle necrosis in rabbits. Pretreatment of mice with CA, either by inhalation or ip injection, resulted in a dose-related prolongation of pentobarbitone sleeping time, an effect which had no apparent association with hepatic necrosis. When injected iv, CA reduced the blood pressure of anaesthetized rabbits, had a variable effect on their respiration and, in large doses, inhibited neuromuscular transmission. Repeated ip injection of 1.6 or 3.2/zl/kg thrice weekly for 12 wk induced chronic focal bronchopneumonia and associated morphological alterations of the respiratory epithelium suggestive of a premalignant condition.
PROCESSING AND PACKAGING CONTAMINANTS
917
Comparison of these results with others already obtained for EC (ibid 1972, 10, 589) indicates that while the two compounds are similar in their acute oral toxicity in rats and mice, CA is some 10-30 times more toxic than EC when injected ip. Conversely EC is more toxic than CA following application to the skin. CA is by far the more irritant of the two compounds, however. The second paper cited above reports an abrupt increase during 1969 in the incidence of tracheal stenosis in patients subjected to tracheotomy and prolonged artificial respiration, and attributes this to the inefficient removal of residues from cannulae sterilized with EO 48 hr before use. When the storage period between EO sterilization and use was increased to 15 days, the incidence of tracheal stenosis fell again to the levels recorded before EO sterilization was instituted as a routine measure. Storage of sterilized equipment of this kind for at least 8 days after exposure to EO is recommended as a safety precaution.
2587. Testing of plastics for pharmaceutical use Lefaux, R. (1972). Valeur et limites d'un contr61e analytique des mati6res plastiques /l usage pharmaceutique et m6dical. Annls pharm, fr. 30, 673. Pentelow, J. E. (1973). Practical problems in the examination and control of plastics for pharmaceutical use. Proc. Soc. analyt. Chem. 10, 41. The increasing use of plastics containers for pharmaceutical products raises the problem of the analytical testing of these materials. Two authors, one French and one English, have recently presented reviews on this subject and show some degree of accord in their general approach. The test methods for the assessment of plastics reviewed by Lefaux (first paper cited above) fall into three general categories covering tests on the plastics material itself, tests on extracts prepared by autoclaving the plastics, usually in contact with distilled water, and biological testing. The first category covers the physico-chemical properties of the plastics material (permeability to light, ultraviolet rays, air, water vapour, bacteria and viruses and absorption properties), and when these have been checked the polymer itself can be examined by a variety of methods involving extraction by various solvents, chromatographic separation and analysis by ultraviolet, infra-red, mass spectrometric and nuclear magnetic resonance methods. The polymer should be subject to routine analytical control on the basis of its infra-red spectrum and the extraction of additives. The autoclaved extract is tested for organic materials, heavy metals, tin, zinc and chromium, and can be subjected to spectrophotometric analysis. These tests may give an adequate analysis of the polymer itself, but biological testing is still very important and the author points out that there is room for improvement in current methods. He stresses the potential value of adding more sensitive procedures, such as cytotoxicity and haemolysis tests, to the acute toxicity tests in mice. The risk of accumulation of substances that are not metabolized is probably a more frequent problem than that of acute toxicity. The second paper cited above again reviews existing test procedures, laying particular stress on the testing of the final container. The testing of the physico-chemical properties of the plastics and the monitoring of extracts are described, with reference to the methods approved by those authorities who have produced monographs or guidelines on this subject. On the same basis, the author restricts his attention to the biological tests already recommended, without speculating on possible advantageous additions to the list. Consistent quality control of the plastics is again advocated.
PROCESSING AND PACKAGING CONTAMINANTS
faeces, while after iv injection the proportion appearing in the urine was greater (Schulz & Rubin, Phthalic Acid Esters Conference, NIEHS, Pinehurst, N.C., 6-7 September 1972). A study is now reported in which rats were given two intubated doses of 0.2 ml [14C]DEHP 24 hr apart. Urine was collected for 48 hr after the first dose and was subjected to thin-layer chromatography and, after treatment with diazomethane, to thin-layer and gasliquid chromatography. The metabolites isolated in this way were characterized by infra-red and nuclear magnetic resonance spectroscopy and mass spectrometry. It appeared that the DEHP metabolites were not conjugated prior to excretion. Free phthalic acid accounted for less than 3 ~o of the urinary metabolites, but was the only detectable 14C-labelled product after alkaline hydrolysis of either the total ether extract of the urine or the individual metabolites. No mono-2-ethylhexyl phthalate (MEHP) was present in the urine, but the five metabolites identified suggested that hydrolysis of the administered DEHP to MEHP was followed by oJ-oxidation and ( c o - 1)-oxidation, probably in the liver, with the alcohol intermediates being further oxidized to the level of ketone after (oJ -- l)-oxidation or acid after oJ - oxidation and the resulting acid metabolite then undergoing fl-oxidation. It thus appears that, in the rat, DEHP is handled like a fatty acid, oJ-oxidation being the main metabolic pathway since a- and E-oxidation are initially impossible.
2586. Another check on ethylene oxide sterilization
Lawrence, W. H., Dillingham, E. O., Turner, J. E. & Autian, J. (1972). Toxicity profile of chloroacetaldehyde. J. pharm. Sci. 61, 19. Mantz, J. M., Tempe, J. D., Jaeger, A. et Vidal, S. (1972). St6noses trach~ales et st6rilisation des canules de trach~otomie par l'oxyde d'~thyl6ne. Sem. H6p. Paris 48, 3367. Ethylene chlorohydrin (EC) is a reaction product occurring in plastics, spices and foods containing chloride and sterilized by exposure to ethylene oxide (EO) (Cited in F.C.T. 1972, 10, 592). One metabolite of EC in rats has been found to be chloroacetaldehyde (CA), so that a clear picture of the potential hazard of CA should be of assistance in any assessment of the possible results of inadequate removal of EO from sterilized products. The LDso of CA by ip injection was found to be 6/zl/kg in mice, 6--8 tzl/kg in rats, 2 tzl/kg in guinea-pigs and 5/~l/kg in rabbits. When administered orally by intubation, its LDsos in mice and rats were 69 and 75-86 tzl/kg, respectively, while its dermal LDso in rabbits was 224 t~l/kg. When mice were exposed to a CA-air mixture in an inhalation chamber, the time required to kill 5070 of the mice (LTso) was 2.57 min, at which time the chamber atmosphere had reached approximately 45 70 of equilibrium with the incoming mixture, produced by bubbling air through 3070 aqueous CA. CA was extremely toxic to mouse fibroblast cultures, inducing 50 70 inhibition of protein synthesis at 5.62 × 10- 5 M concentration. It also proved very irritant to the skin and eyes of rabbits. Plastics implants previously treated with CA produced muscle necrosis in rabbits. Pretreatment of mice with CA, either by inhalation or ip injection, resulted in a dose-related prolongation of pentobarbitone sleeping time, an effect which had no apparent association with hepatic necrosis. When injected iv, CA reduced the blood pressure of anaesthetized rabbits, had a variable effect on their respiration and, in large doses, inhibited neuromuscular transmission. Repeated ip injection of 1.6 or 3.2/zl/kg thrice weekly for 12 wk induced chronic focal bronchopneumonia and associated morphological alterations of the respiratory epithelium suggestive of a premalignant condition.
PROCESSING AND PACKAGING CONTAMINANTS
917
Comparison of these results with others already obtained for EC (ibid 1972, 10, 589) indicates that while the two compounds are similar in their acute oral toxicity in rats and mice, CA is some 10-30 times more toxic than EC when injected ip. Conversely EC is more toxic than CA following application to the skin. CA is by far the more irritant of the two compounds, however. The second paper cited above reports an abrupt increase during 1969 in the incidence of tracheal stenosis in patients subjected to tracheotomy and prolonged artificial respiration, and attributes this to the inefficient removal of residues from cannulae sterilized with EO 48 hr before use. When the storage period between EO sterilization and use was increased to 15 days, the incidence of tracheal stenosis fell again to the levels recorded before EO sterilization was instituted as a routine measure. Storage of sterilized equipment of this kind for at least 8 days after exposure to EO is recommended as a safety precaution.
2587. Testing of plastics for pharmaceutical use Lefaux, R. (1972). Valeur et limites d'un contr61e analytique des mati6res plastiques /l usage pharmaceutique et m6dical. Annls pharm, fr. 30, 673. Pentelow, J. E. (1973). Practical problems in the examination and control of plastics for pharmaceutical use. Proc. Soc. analyt. Chem. 10, 41. The increasing use of plastics containers for pharmaceutical products raises the problem of the analytical testing of these materials. Two authors, one French and one English, have recently presented reviews on this subject and show some degree of accord in their general approach. The test methods for the assessment of plastics reviewed by Lefaux (first paper cited above) fall into three general categories covering tests on the plastics material itself, tests on extracts prepared by autoclaving the plastics, usually in contact with distilled water, and biological testing. The first category covers the physico-chemical properties of the plastics material (permeability to light, ultraviolet rays, air, water vapour, bacteria and viruses and absorption properties), and when these have been checked the polymer itself can be examined by a variety of methods involving extraction by various solvents, chromatographic separation and analysis by ultraviolet, infra-red, mass spectrometric and nuclear magnetic resonance methods. The polymer should be subject to routine analytical control on the basis of its infra-red spectrum and the extraction of additives. The autoclaved extract is tested for organic materials, heavy metals, tin, zinc and chromium, and can be subjected to spectrophotometric analysis. These tests may give an adequate analysis of the polymer itself, but biological testing is still very important and the author points out that there is room for improvement in current methods. He stresses the potential value of adding more sensitive procedures, such as cytotoxicity and haemolysis tests, to the acute toxicity tests in mice. The risk of accumulation of substances that are not metabolized is probably a more frequent problem than that of acute toxicity. The second paper cited above again reviews existing test procedures, laying particular stress on the testing of the final container. The testing of the physico-chemical properties of the plastics and the monitoring of extracts are described, with reference to the methods approved by those authorities who have produced monographs or guidelines on this subject. On the same basis, the author restricts his attention to the biological tests already recommended, without speculating on possible advantageous additions to the list. Consistent quality control of the plastics is again advocated.