Diethylhexyl phthalate as a factor in blood tranfusion and haemodialysis

Toxicology, 9 (1978} 319--329 © Elsevier/North-Holland Scientific Publishers Ltd.

D I E T H Y L H E X Y L P H T H A L A T E AS A F A C T O R IN BLOOD T R A N S F U S I O N AND HAEMODIALYSIS R.W.R. BAKER

Department of Chemical Pathology, Guy's Hospital Medical School, London, S.E.19 RT. (Great Britain) (Received June 21st, 1977) (Revision received October 26th, 1977) (Accepted November 21st, 1977)

SUMMARY

Di-2-ethylhexyl phthalate (DEHP), the most frequently occurring plasticiser in medical equipment manufactured from polymers of vinyl chloride, forms a b o u t 40% w / w of tubes and containers used for storing blood and for haemodialysis. The plasticiser leaches o u t into liquids with lipid contents, although it is very sparingly soluble in purely aqueous solutions. On infusion of 2--3 1 of stored blood, up to 200 mg DEHP may be transferred to the patient, while much higher quantities may be given during dialysis,which is moreover often repeated frequently over long periods. The acute toxicity of DEHP is very low (> 20 g/kg as LDs0 in rats), and the ester is rapidly metabolised to products which are excreted in the urine and bile; chronic toxicity from the levels of dosage obtaining is thus very improbable. Carcenogenicity has never been demonstrable in animals, while teratological effects are of a very low order. Serious acute results observed after transfusion of neonates have n o t been proved to be caused by DEHP, and might be ascribable to accompanying foreign substances. Atheroma in chronic dialysis subjects is still unexplained, b u t hepatitis probably caused by diethylphthalate from plastic was resolved when apparatus plasticised by DEHP alone was substituted. The benefits of DEHP appear vastly to outweigh any risks. The status of DEHP as environmental contaminant is noted.

Disposable plastics have largely replaced glass and rubber in medical equipment used for infusions and for other widely applied treatments. Nevertheless, after more than a decade in which literally millions of plastic units have been employed, various doubts concerning the possibility of harmful affects Abbreviation : DEHP, di-2~ethylhexyl phthalate.

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continue to be expressed. The most extensively used plastic material is based on polyvinyl chloride, which of necessity is found in combination with other substances, the chief one being a plasticiser. Many plasticisers are used, and nearly all have been examined for toxic affects, but by far the most c o m m o n are esters of phthalic acid, especially the diester formed with 2-ethylhexanol. This substance, di-2~ethylhexyl phthalate (DEHP), is produced in quantities far exceeding 106 tons/year throughout the world, and is perhaps the plasticiser most frequently found in medical applications such as the storage of human blood, fractions from blood or solutions for infusion. It is used widely also in tubing and in apparatus for dialysis in patients. Thus, tons of DEHP are brought each year into intimate contact with fluids transfused into human beings. Plasticised polymers are physical mixtures of variable compositions so there is no chemical bonding to prevent elution of plasticiser from the composition, and this process is well known to occur. Extraction of plasticiser is favoured especially when there is exposure to liquids having lipid content, as found in the case of blood or its components. Since DEHP is almost insoluble in water, extraction of this substance by saline or other aqueous media is very much less pronounced. The present review concerns the extraction of DEHP from plastic equipment by liquids to be administered to human patients, the metabolism of the plasticiser and toxic effects on living organisms. Most of the evidence on toxicity is derived from experiments with animals of various species, but interpretation in human terms has to be attempted. It will be seen that with few exceptions there is little cause for anxiety, although technological advances should be capable of providing added assurance. Medical usage of DEHP accounts for only a small proportion of the total consumption in industry of an ester more extensively found not only in plastics generally, but also as a solvent and in food-packaging materials. Indeed, it is recognised as being now an almost ubiquitous constituent of the human environment. Its introduction into patients undergoing treatment must therefore be seen not as an isolated incident, but as an increment over background intake. This latter can n o t be estimated reliably, but is likely for the present to be of an order of magnitude below the levels now under review. It has been established for some years that blood or blood-fractions can extract materials from plastic containers or tubing, and this effect with DEHP has received considerable attention. Thus, Valeri et al. [1] found that whole blood t o o k up plasticiser to a significant extent, and that the material was found almost entirely in the plasma, confirming earlier results from Marcel and Noel [2] and Jaeger and Rubin [3]. These last authors [4] later reported that blood stored at 4°C took up 0.25 mg/100 ml/day, and that platelets were particularly avid in this respect. Thus, a 70 kg patient given 5 bags of whole blood which had been stored for 4 weeks would receive about 175 mg of DEHP, a dosage of 2.5 mg/kg. The uptake of the plasticiser by plasma from transfusion-bags was investigated in Stockholm by Vessman

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and Rietz [ 5 ] , who studied extensively the analytical problems involved; they also described the partition of DEHP over various plasma fractions. Typically, they give 10 mg/100 ml as the concentration of phthalate ester in plasma stored at 4°C for 5 weeks. The rate of uptake declined curvflinearly, which could be due to diffusion of the ester to the inner surface of the bag to replace the more easily accessible plasticiser immediately in contact with liquid; such a biphasic elution by methanol is described by Kim et al. [6]. From the Swedish data, 5 bags of plasma would contain 250 mg DEHP, equivalent to 3.6 mg/kg if infused into a 70 kg patient over a short period. Much smaller doses would be involved if specific protein fractions were used. The uptake of DEHP by plasma was significantly correlated with the concentration of triglycerides, but not related with the a m o u n t of cholesterol present [2]. It might be t h o u g h t that if the use of stored blood or plasma were restricted to samples kept only for a day or two, then transfer of adventitious material to patients might be severly minimised, but this is not so since extraction of plasticiser is maximal in the first few hours, when the liquid is at higher temperatures and is moved over new plastic surfaces in handling, all factors which increase the elution process. The bags used for storing blood, with the associated tubing, contain about 10 g of DEHP per 500 ml unit, so the mg quantities of ester eluted account for only a microscopic proportion, and certainly do not represent saturation of the liquid. Stored blood is n o t generally used after 21 days at 4°C, so the estimates above of dosages exceed the c o m m o n m a x i m u m by a factor of about × 2, even when 5 units are infused. Plasma may be kept longer, but can be lyophilised: In the haemodialysis of patients with kidney failure there is again exposure of blood to plastic, and this aspect was investigated by Easterling, Johnson and Napier [7], who measured concentrations of DEHP, in plasma circulated through plastic tubes connected to dialysis equipment. They give 10 mg as the weight of DEHP that would enter a patient in typical dialysis, i.e., about 0.14 mg/kg for a 70 kg subject. Somewhat similar work was undertaken by Ono et al. [8], using whole blood in which both DEHP and free phthalic acid were measured, and working both with patients under dialysis and with an artificial model. In 3 h of circulation in the model, 1 1 of blood dissolved 0.5 mg DEHP. The uptake of plasticiser by their patients is difficult to interpret since metabolic elimination occurs as discussed below, but the authors stress that in 100--150 treatments in a year, a patient can receive significant quantities of the ester. More recently, it was estimated by Gibson et al. [9] that as much as 150 mg DEHP could be transferred to a patient undergoing dialysis for 5 h. This is much greater than the upper limit (15 mg) inferred from the results of Fayz et al. [10] who tested 3 types of tubing plasticised with DEHP in a model system they devised as an improvement on those referred to above. They found also t h a t the adipate ester in other types of tubing was transferred more extensively than was the phthalate. There is ample evidence that DEHP is rapidly eliminated by intact animals,

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although isolated perfused rat liver, which metabolised glycolybutyl phthalate, appeared to take up DEHP without change [ 1 1 ] . Dillingham and Autian [12] showed that 0.5 of a dose of [14C]DEHP introduced into mice intraperitoneally, or b y intravenous injection of a saturated solution in saline, was excreted in the urine within 4 days with evidence of conversion to metabolites. In rats, plasticiser labelled with 14C administered b y injection or orally seems to be cleared b y 2 processes, one giving a half-life of under 10 min, the second with a b o u t 30 min [13,14] b u t these rates depend to some extent on the size of the dose, and also on the degree of dispersion of the ester [ 1 5 ] . The distribution anatomically and the excretion of 14C labelled DEHP b y rats is described also by Tanaka et al. [ 1 6 ] , who demonstrated rapid elimination as metabolites o f both orally and intravenously administered doses. With human subjects there is little published knowledge, but reports by Shaffer et al. [17] on the results of ingestion and by Jaeger and Rubin [4] and Rubin and Schiffer [18] on the fate of infused DEHP support the contention that laboratory animals are reliable models for human behaviour in that speedy elimination occurs. When emulsified DEHP is used, there is obvious transfer of a significant proportion to the lungs and spleen, whereas the truly dissolved ester is carried to the spleen, liver and kidney, from which it disappears over the course of a day or so [19,20]. The first stage of removal from the organism appears to reside in partial hydrolysis to the mono-ester, which can be effected by various tissue lipases from several species as described b y Albro and Thomas [21]. The same authors found that pancreas was especially active, as proved by Daniel and Bratt [ 1 9 ] , while Lake et al. [22] have shown very recently that microsomal fractions from liver, as well as mucosal cells from the intestine, mediate this hydrolysis. Tissues from a lower primate {baboon) showed greater activity than those from rats. The hemi-ester and free alcohol then undergo oxidative changes with formation of various end-products, including glucuronides, which are found in the urine [ 2 3 ] . Daniel and Bratt [19] found 14 metabolites, using 2-dimensional thin-layer chromatography in conjunction with autoradiography: they characterised 5 of these, one being phthalic acid. No DEHP was excreted unchanged. There was ~2-oxidation both of ethyl sidechains and of the main chain to produce alcohols, while ~2-1 oxidation t o o k place with formation of the secondary alcohols and further of the ketones. Albro [24] also described in detail the metabolic fate in rats of 2-ethylhexanol, and states that no markedly toxic product was involved. It is possible that a small proportion of DEHP is eliminated in the bile in cases where injected plasticiser is studied; with oral administration to rats and dogs, this route is much more prominent, accounting for 80% of the elimination by dogs [ 1 4 ] . Aquatic animals also concentrate and metabolise DEHP, and in the case of the rainbow trout, detoxication leads to excretion through the bile of conjugated mono-2-ethylhexyl phthalate as the glucuronide [ 2 5 ] . This extends an earlier report b y Mayer et al. [26] w h o recorded the concentration of DEHP b y aquatic creatures from their environment without obvious disadvantage. L o w toxicities were also observed b y Mayer and

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Sanders [ 2 7 ] , while Sanders et al. [ 2 8 ] , working with 5 species o f invertebrates, recorded low toxicities together with a reversible b u t highly active ability to concentrate the phthalic ester from the environment. There was evidence for metabolic change o f the DEHP and reproductive capacity was reduced. Evidence that metabolism does in fact occur is given in a further communication [ 2 9 ] . Curiously, appreciable concentrations of DEHP were found in ox-heart mitochondria by Nazir et al. [ 3 0 ] , which raises the intriguing question as to whether DEHP forms part of the natural environment as a cell constituent. Smaller quantities were found in subcellular fractions from only the cardiovascular systems in dogs, rats and rabbits [ 3 1 ] . The very low concentrations (0.10--0.06 ppm) of DEHP in human placenta, measured by Poole and Wibberley [ 3 2 ] , are considered b y the authors to have been derived from the exterior environment. Although it would seem reasonable to conclude that in normal patients there is biochemically active, speedy and complete elimination of DEHP, this inference does not necessarily apply to persons with renal deficiency. Fortunately, a second route exists while liver-function remains intact, and elimination can occur, probably more slowly, b y way of the bile duct. In considering the toxicity of DEHP it is necessary to take into account not only the quantities involved, but also t h e form in which the substance is administered. As with all substances sparingly soluble in aqueous solutions, there is the possibility of the formation of emulsions, but to become concerned with the physiological effect o f these is scarcely relevant to clinical situations. Suspended particulate matter can produce pathological manifestations which result from mechanical rather than chemical properties. Experiments have indeed been carried out with the introduction into animals of DEHP suspended in micro-particulate form, and the resulting changes were mostly marked in the lungs. The administration has been effected b o t h by stomach-tube and b y injection in various ways, taking DEHP dissolved in oil or emulsified b y detergents in aqueous solution. Much more appropriate experimental designs, as described below, where realised by Miripol et al. w h o used plasma brought to predetermined high concentrations of DEHP b y soaking strips o f plastic in it. For all practical purposes, no published experiences with human adults or results from experiments with healthy intact animals give any indication of the existence of acute toxic properties of DEHP in conditions that could arise in routine medical treatment. In such a c o n t e x t it would seem to be merely an academic curiosity to q u o t e LDs0 values of the order of 20 g/kg [33] or 38 g/kg [34] for mice treated intraperitoneally and for rats by the same and by intravenous and b y oral routes [ 1 4 , 3 5 ] . There is however an important exception to this happily negative state o f affairs, for in the case of babies there is definite suspicion that fatal intestinal perforation which has been observed after transfusion of blood [36] might be ascribable to the use o f plastics, while Jaeger and Rubin [4] consider that DEHP may be implicated in cases of peritonitis. In fact, acute deaths have been suspected

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as being related to the use of plastics in treatments of gravely ill babies, and Hillman et al. [37], who analysed post-mortem tissues, found that DEHP was present in heart-tissue to about nine-fold the control concentrations. They did not however conclude that there was any causative correlation, and indeed the a m o u n t of plasticiser transferred to a patient may well parallel the administration of other materials leached from plastic apparatus. Further, mortality was greatly reduced by treatment of otherwise moribund patients. Transitory effects of DEHP on the energy-providing metabolism of fatty acids by heart tissue [59], mentioned below, must however be considered to be relevant. Of much greater pertinence, perhaps, is the assessment of chronic effects, since patients treated by haemodialysis, or given infusions of blood at frequent intervals, are sure to receive over a long term a series of low quantities of plasticiser and probably of other substances extractable from plastics. Experiments conducted so as to reproduce this condition are described by Miripol et al. [ 14], who injected into young rats, twice weekly over 63 days, plasma containing DEHP leached from plastic strips. Doses from 20 ml plasma/kg gave 1.0 or 3.7 mg DEHP/kg. The rats continued to grown like the control animals. There was no impairment of reticulo-endothelial or liver functions; haematocrits and the 17 constituents of blood that were measured were all the same as in control rats. No gross or microscopic pathology could be discerned, and the weights of organs were normal. Furthermore, no accumulation of DEHP in any tissue could be detected. The findings confirm and extend those of Carpenter et al. [38], who saw no effect when dogs were fed 0.06 ml DEHP/kg/day over extended periods. Nevertheless, as cited by Jaeger and Rubin [3], a hepatitis-like effect in long-term haemodialysis subjects has been ascribed speculatively as being associated with plastics by Neergaard et al. [39], who demonstrated, however, that diethyl phthalate, and not DEHP was responsible for the serious effects seen. In the same class of patients, an abnormally high incidence of atheroma accounted for 14 deaths out of 23 as reported by Lindner et al. [40], who make no mention of the aetiology. The effects of DEHP on fertility and on foetal development have been subject to relatively few reports, although studies in long-term toxicology with rats over 3 generations on maintainence doses of DEHP failed to evince any marked effects. With all esters of phthalic acid (review, Peakall, 41), high doses had to be used before any reactions in embryos were produced. Results with DEHP [42,43] were more pronounced than with diethylhexyl adipate [44,45]. Their experimental design ensured that any results were truly teratological, for the esters were administered (in high doses) to male mice which were later mated with untreated females. A minor depression of male fertility was observed, as noted by other investigators [17,46]. The DEHP caused significant foetal resorption, as was confirmed by Dillingham and Autian [12]. In contrast, Carpenter et al. [38] had earlier found DEHP to be w i t h o u t effect on rat litters, while repeated infections into pregnant rats of plasma containing 185 ~g DEHP/ml by Garvin, Lewandowski and

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Wallin [pers. comm.] did not effect dams or litters. The resilience of the developing embryo towards insult through the male or more directly through the female stands significantly in contrast to the observations noted below t h a t DEHP even in low concentrations is toxic to tissues cultured in isolation. It would seem t h a t the plasticiser is subjected to rapid biochemical detoxication in intact animals, so that the developing young are protected from all but very low concentrations. Only with dosage-levels far in excess of any that could be found in h u m a n subjects was DEHP seen to have an influence on the reproductive system of animals. After 3 months on diets containing 3% w/w of the ester, rats exhibited signs of testicular degeneration [17] as found also [46] with ferrets, but fertility in female rats was n o t affected [44]. In this connection it is interesting to note t h a t dibutyl phthalate, administered orally to rats, at a daily rate of 2 g/kg, appeared to promote depletion of zinc from the testes, with simultaneous appearance of atrophy [47]. As already mentioned, DEHP in noticeably unfavourable to cultures of isolated tissue. Solutions used for culture acquire a strongly marked toxicity to chick embryonic heart cells if stored in plastic containers [48]. A similar effect on perfused rat hearts had been reported by Meyler et al. [49]. Investigations by O'Leary and Guess [50] showed that polyvinyl chloride plastics incorporating DEHP or other phthalate esters were toxic to mouse fibroblasts after sterilisation with ethylene oxide, whereas no marked toxicity was reported from control experiments with material not so sterilised. Gesler [51], reviewing this topic, concludes that DEHP is not cytotoxic, but the balance of evidence does not now support such an opinion. With human cells, Jones et al. [48] found toxic effects. The mechanism of the adverse action of DEHP has n o t been elucidated, but details of cell damage have been described (e.g., 48, 53). The last authors cited found that in true solutions of DEHP at concentrations exceeding 10 pg/ml, chick embryo cells exhibited vacuoles and abbreviated cell-processes. This stands in contrast to the findings of Fishbein and Albro [54] that emulsions of DEHP were nontoxic to chick embryo cells and to mouse fibroblasts. In spite of the large a m o u n t of experimental long-term work that has been carried out with animals involving administration of DEHP, there is as y e t no evidence that the ester has any carcinogenic or co-carcinogenic properties [55]. One specific effect attributed to DEHP is a low-grade stimulation of the central nervous system, described by Fishbein and Albro [54], who found that sleeping-time induced by hexobarbital in mice was reduced from 46 to 36 min by the phthalate diester. Interpretation as a direct action must however be reconsidered, since an indirect mechanism could be implicated, as discussed by Daniel and Bratt [19], who obtained similar results from oral doses in rats, but found a reverse effect after intravenous administration. A perhaps more significant specific result concerns the reaction of bloodvessels to hypoxia. This was found by Duke and Vane [56] to be inhibited reversibly by extracts of plastics. Although the plastics used did not contain

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DEHP, the results are interesting because the material, a medical grade of tubing, comprised organo-tin or other metals as well as "epoxidised soybean oil", a type of additive c o m m o n l y incorporated into plastic formulations. Referring back to the atheroma of dialysis subjects already mentioned, it may be observed t h a t the biochemistry of such persons may well be disturbed in ways that could account for the deposition of arterial plaques, but, although the authors do not specify any aetiology, adventitious invasion by DEHP might be involved [9]. Since metabolism of DEHP occurs largely in the liver, it is not surprising that this organ has been studied in experiments on chronic intoxication. Thus, some morphological changes were reported by Carpenter et al. [38] who used dogs fed for a year on diets containing DEHP. Only with large doses can DEHP be found to have any effect on the liver biochemistry of rats [ 5 7 ] . Following intravenous dosing, the activities of succinic dehydrogenase and of adenosine triphosphatase were decreased but that of alkaline phosphatase was enhanced to nearly 4 times that found in control animals. Hepatic effects in rats of DEHP administered orally were studied extensively by Lake et al. [58], who gave 2000 mg/kg daily over 21 days; this is equivalent in man to about 140 g/day. The livers increased in weight to more than twice that of controls, while cytochrome P-450 and alcohol dehydrogenase rose to 150% of the normal activities, in contrast, there was marked decrease in aniline~4-dehydroxylase, succinic dehydrogenase and glucose~6-phosphatase, the last change comparing strangely with the prominent increase in alkaline phosphatase reported by Srivastava et al. [57]. On ultramicroscopic examination, some morphological changes were seen, as were alterations in the distribution of enzymes. Hepatomegaly in rats was evident also when reduced doses of 100 mg/kg were given. The effects evinced by DEHP were produced also by its known metabolites 2-ethylhexyl alcohol and its phthalate monoester. Lake et al. [46] found much the same results from a 14-month study of ferrets; decreased body weight was also observed. The authors concluded that similar metabolic pathways occur in both species. The study in detail of effects in the liver and heart of low doses of DEHP given in the diet of rats, rabbits and pigs is beginning to show that the phthalic ester may have influence, both chronic and acute, on the metabolism of fatty acids. Bell and Gillies [59] isolated mitochondria from the organs of the 3 species after feeding as little as 100 mg DEHP/day to rats, and measured changes in the oxidation of palmityl CoA. Liver activity increased up to treble the control values; the enhancement was not extended with greater daffy intake. In heart mitochondria, there was, with rats at least, a transient pronounced fall of about 30% in fatty acid metabolism, maximal at 4 days and evident only for an equal period. The authors point out the relevance of their findings to the heavy reliance of heart muscle on fatty acids as a source of energy. It is not inconceivable that changes in the liver induced by DEHP may reflect adaptive responses, possibly reversible, to biochemical stress imposed by high doses of phthalate ester. The organ might well cope, without modi-

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fication, with lower intakes of the unnatural substrate. Threshold dosages of DEHP, below which morphological and enzymatic changes are undetectable have not been established, even in the more c o m m o n laboratory animals. There is a wide range of dose-levels between that giving no reaction [14] and those cited above as producing marked effects. Human intakes as discussed here are well below those usual in pharmacological research. The surface reaction at plastic interfaces with blood components, including the physiologically important platelets, was investigated b y Kim et al. [6] and was seen to depend on the t y p e of bag used. Survival and function of platelets were found b y Valeri et al. [1] to be unaffected by DEHP. Although the present discussion is confined to consideration of DEHP, which constitutes a b o u t 30--40% of the weight of the plastic, it is obvious that fluids may elute other materials. In fact, over 20 different substances are used as plasticisers, either alone or in combination. The phthalate ester treated here is perhaps the most commonly found, but phthalic esters of other alcohols, as well as various esters of adipic acid are also widely employed. Stabilisers, mould lubricants, dyes and other additives can in general occur, but are seldom found in plastics for medical use. Polyvinyl chloride itself is not to be expected in eluates, since only traces of low polymers are present, but it has been measured [ 6 0 ] . To summarise, it might be said that such toxicity as may be attributed to DEHP incorporated in plastics is of a very low order, and may well be truly negligible. Certainly, from the cost-benefit point of view, the use of such materials confers enormous advantages. It is probably justifiable to discount the risks of teratological and other long-term effects in the episodal treatment of adult patients. However, there are 2 aspects of the use of plastics in which firm conclusions are much more difficult of attainment. The treatment by infusion of the new-born [61] is an emergency procedure with hazards extending b e y o n d the scope of this review. The second aspect concerns the long-term haemodialysis subject, whose own pathology might contribute to some of the effects observed and whose biochemical defence against agents normally inoccuous might be gravely impaired. Such impairment as the result of disease could be pleaded more generally, and must be considered to modify the acceptance of results with healthy experimental animals as applying directly to the human patient. With multi-wall containers now being used in food-packaging [ 6 2 ] , the development of special materials for use in medically applied plastics should not be b e y o n d the capabilities of the plastics industry. For the majority of transfusion work, however, existing materials seem to be perfectly acceptable, for n o t only is every patient protected to aremarkable degree by his own metabolic system, b u t also it must be remembered that environmental contamination may introduce chronically more DEHP than a single transfusion. REFERENCES 1 C.R. Valeri, T.J. Contreras, H. Feingold, R.H. Sheibley and R.J. Jaeger, Environ Health Perspec., 3 (1973) 103.

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