It's that cheese again

Fd Cosmet. Toxicol. Vol. 3, pp. 839-854. PergamonPress 1965. Printed in Great Britain

ARTICLES OF GENERAL INTEREST IT'S THAT CHEESE AGAIN Over the last few years we have continually drawn attention to the adverse effects of eating amine-containing foodstuffs on the blood pressure of patients taking monoamine-oxidase (MAO) inhibitors for the treatment of hypertension and depression (Cited in F.C.T. 1964, 2, 227, 281,383 & 488; ibm 1965, 3, 508). Cheese is the commonest food incriminated in these hypertensive crises and this has been attributed to the high tyramine (I) content of some cheeses, I exerting its effects mainly by releasing the pressor amines adrenaline and noradrenaline. Patients eating broad beans react similarly and the active agent here (present chiefly in the pod) is 3,4-dihydroxyphenylalanine(dopa), this being converted in the body to dopamine, a precursor of the above catecholamines. Confirmatory work on the content of I in various cheeses has been published by Blackwell & Mabbitt (Lancet 1965 i, 938). They emphasize the great variability in the amount of I present in different samples of the same cheese and report values of 0-953 t~g I/g in 14 samples of Cheddar cheese, which is implicated in most clinical reports. Two diamines, cadaverine and putrescine, known to have depressor actions were also found present, but unlike histamine and many other diamines do not serve as substrates for MAO. No other amines likely to have pressor activity were detected in a sample which provoked a hypertensive crisis although small amounts of histamine may have been present. The Lancet (1965, i, 945) in a recent review on this subject draws atterltion to a report by Horwitz et al. (J. Am. med. Ass. 1964, 188, 1108) that cheese on account of its conten~ of I may also be potentially dangerous in two other clinical conditions. First, severe flushing attacks in patients with the carcinoid syndrome have been induced by I (Levine & Sjoerdsma, Ann. intern. Med. 1953, 58, 818) and indeed by ingestion of cheese (Waldenstr/Sm et al. Acta reed. scand. 1956, 156, 73). Second, patients with phaeochromocytoma have also been shown to have a greatly enhanced pressor response to small amounts of I because of the excessive release of catecholamines from the tumour. It now seems probable that yet another amine may be implicated in hypertensive reactions. Following reports of adverse effects of yeast extract ('Marmite') in patients taking MAO inhibitors (Cited in F.C.T. 1964, 2, 488), Black~ell et al. (Lancet 1965, i, 940) have now found 'Marmite' to contain up to 1.5 mg/g and in addition as much as 2 mg histamine (II)/g, the latter arising from the decarboxylation of histidine in yeast extracts. Since I is a pressor agent and II lowers the blood pressure, the effects of yeast extracts are likely to be variable. The possible participation of II in the hypertensive crises depends upon the following facts. In the first place II can release pressor catecholamines in the body. Secondly histaminase and the methylating enzyme, involved in the metabolism of II, are inhibited by MAO inhibitors and in addition II is a substrate of MAO. It is thus suggested that following prolonged use of MAO inhibitors, II may exert a pressor response and potentiate the activity of I. However, there is as yet no clinical evidence that this occurs although BlackweU et al. (Ioc. cit.) have found that in animals the absorption of both I and II from the intestine was facilitated by MAO inhibitors and their action potentiated. This suggested that foods 839

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such as 'Marmite' which contain II might initiate or aggravate allergic conditions in patients treated with MAO inhibitors, but no clinical evidence of this was forthcoming in a later study (Blackwell et al. Lancet 1965, i, 1166). Little new has been published on the antidepressant activity of MAO inhibitors which is probably attributable to increased brain concentrations of various monoamines, notably 5-hydroxytryptamine and noradrenaline (Cited in F.C.T. 1965, 3, 509). In reviewing the use of MAO inhibitors in the treatment of depression, Pare (Lancet 1965, i, 923) reports a notable decrease in the incidence of hypertensive reactions in patients receiving these drugs after deleting cheese from the diet. It is probable that these drugs may have actions other than the inhibition of MAO and recent experiments in dogs and rabbits (Panisset et al. Nature, Lond. 1965, 206, 31 I) indicate that tranylcypromine may in fact enhance the pressor activity of I by releasing catecholamines. However tranylcypromine had the opposite effect on the pressor activity of I in cats. That additional potential hazards may be connected with long-term treatment with MAO inhibitors in conjunction with the eating of amine-containing foods is perhaps indicated by the results of experiments in which the effects of large unphysiological concentrations of I and tryptamine (III) were studied in pregnant rats (Quigley, Fedn Proc. Fedn Am. Socs exp. Biol. 1965, 249 157). In animals receiving daily intraperitoneal injections of 40-160 mg I/kg from day 6 of gestation, deliveries were normal but multiple foci of muscle necrosis and infiltration by histiocytes and lymphocytes were found in the hearts of 3 animals, compatible with severe constriction of tile small coronary vessels presumably by released catecholamines. However even larger doses of I administered in the diet produced no heart lesions probably as a result of the protective action of intestinal MAO (Cited in F.C.T. 1965, 3, 508). In contrast, 20-40 mg III/kg/day given intraperitoneally caused a high mortality at delivery and produced intra-uterine haemorrhage and deposition of fibrin in the splenic red pulp. All foetuses in the group given I survived but 32% of those given 11I died in utero. The practical importance of all these findings obviously lies in their prevention, by warning patients on MAO inhibitors to avoid cheese, game, wines such as Chianti, broad beans and yeast extracts (Cited in F.C.T. 1964, 2, 488; ibid 1965, 3, 508). Since cheese is the chief offender it seems appropriate to end with a quotation from Hippocrates (Greek Medicine): "It is not enough to know that cheese is a bad article of f o o d . . , for cheese does not harm all people equally. If it had been harmful to the human constitution in general everyone would have suffered. Now, whoever knows these facts will not suffer."

ALL QUIET ON THE FLUORIDE FRONT Several investigations on fluoride (F) have been published since our last article on this subject (Cited in F.C.T. 1965, 3, 343), some dealing with familiar topics and others with aspects which have not been discussed previously. First the familiar one of water fluoridation (Citedin F.C.T. 1964, 2, 132, 226, 318 & 319). Results obtained in the USA concerning the safety and beneficial effects of water fluoridation since this measure was introduced in 1945 have been very lucidly summarized by Dunning (New Engl. J. Med. 1965, 272, 30 & 84). Controlled fluoridation of water (1 ppm F), now affecting nearly 46 million Americans, has produced a 50-70% reduction in the incidence of dental caries in young people. No adverse effects have been reported and it

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appears probable that this practice may even be beneficial to older people and, by improving calcium (Ca) balance, may alleviate bone disorders suchas osteoporosis. Alternative methods of supplying F, including its addition to milk, salt, dentifrices, incorporation into pills and topical application (Cited in F.C.T. 1964, 2, 226 & 320; ibid 1965, 3, 344), besides being more costly and less practical, are in general less effective than water fluoridation. There is, however, one recent method which appears to afford as good a protection against dental caries as fluoridated water, namely the topical application of a fluoride-phosphate solution at low pH. In a 2-yr study on school children, single annual topical applications have been claimed to reduce the incidence of caries by 70% (Brudevold et al. Archs. oral Biol. 1963, 8, 167; Wellock & Brudevold, ibid 1963, 8, 179). Sweden and W. Germany in 1952 followed by the Netherlands a year later were the first to introduce water fluoridation in Europe while 1955 marked Britain's entry. The fluoridation programme is much less extensive in Europe than in the USA and by 1962 had affected only 18 communities covering about one million people. However, beneficial effects of the same order as experienced in the USA have been reported from all these areas and fluoridation schemes are rapidly expanding and will soon include some 26 communities and some 4 million people (Auermann & Lingelbach, Am. J. publ. Hlth. 1964, 54, 1545). Although fluoridation of water leads to no adverse effect on man, possible effects on animals cannot necessarily be excluded. The question has recently arisen as to whether the continuous discharge of sewage effluents into rivers in districts where the public water supply is fluoridated might be harmful to fish (Herbert & Shurben, Wat. Waste Treat. J. 1964, 10, 141).These authors cite the work of Neuhold & Sigler (Trans. Am. Fish. Soc. 1960, 89, 358) who showed that the eggs of both carp and rainbow trout were more resistant to F than adult fish and that rainbow trout were more susceptible than carp. The LDsos for carp and rainbow trout exposed for 20 days to soft water were 75-91 and 3.6 ppm F, respectively. Since the maximum F content of sewage effluents would be that of the water supply (1 ppm), it was considered probable that not more than 0.I ~'o of carp and 3-4% of rainbow trout would be likely to die under these conditions. In practice, of course, effluents are usually diluted with river water so that toxic concentrations of F are very unlikely to occur in polluted waters. However, since the fish in these experiments were kept in the same solution of F for 20 days, the toxicity of F might have been modified by the accumulation of metabolic products in the water and for this reason Herbert & Shurben (loc. cit.) have reinvestigated the toxicity of F to rainbow trout, exposing yearling fish for 21 days to F-containing waters of varying degrees of hardness, and changing the water daily. In soft water, 4 ppm F produced a 5% mortality after 3 wk and the mortality curves indicated that this incidence was unlikely to be increased even after 3 months' exposure. Not more than 0-01% mortality was deemed likely at 1 ppm and since F was even less toxic in hard waters it was concluded, in confirmation of previous work, that there would be virtually no hazard to fisheries preserved in rivers which receive sewage effluents containing F derived from public water supplies. Hoogstratten et al. (J. Am. reed. Ass. 1965, 192, 112) have provided valuable data on the long-term effects of F on the haematopoietic system of cattle. Four groups of 8 heifers, 3-4 months old, were fed diets containing 10-100 ppm F for 7.5 yr. The lowest level represents the concentration of F normally present in the diet of dairy animals. Detailed blood studies revealed that 25 and 50 ppm F produced no significant change in serum levels of Ca, phosphorus, vitamin B~2 and folic acid, in several protein fractions or in leucocyte alkaline phosphatase activity. No evidence was found of anaemia, abnormalities of the bone marrow or indeed of any adverse effect on the haematopoietic system. There

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were also no gross, histological or functional effects on the liver or on the thyroid gland even at the highest level. The only outstanding signs of toxicity were severe bone and tooth defects confined to animals on 100 ppm F. However the increased eosinophil count, which might have been an early toxic manifestation, and also a slightly reduced serum folic acid level, probably due to reduced food intake, were the only other effects observed at the highest level. The premature death of three animals at 100 ppm F may indicate a decreased resistance to infection. This valuable long-term study demonstrates that cattle can consume diets containing at least five times the normal content of F (i.e. 50 ppm) for many years without any obvious adverse effects. Finally, from intact animals to another instalment of "Fluoride Can Kill Human Cells". It will be recalled that the Oxford workers Berry & Trillwood (Cited in F.C.T. 1964, 2, 227) obtained inhibition of tumour cell growth in vitro in media containing as little as 0.045 ppm F whereas Armstrong et al. (Cited in F.C.T. 1965, 3, 344) found that about 15 ppm F was necessary to inhibit growth of human endothelial cells in vitro. Berry & Trillwood (Br. med. J. 1965, i, 793) now contend that in the experiments of Armstrong et al. (loc. cit.) the cells did not multiply at a rate sufficient to reveal the inhibitory effect of F. Far from accepting this criticism, Armstrong et al. (Br. reed. J. 1965, i, 1435) have accepted the challenge by carrying out additional experiments using the same type of turnout cells multiplying at approximately the same rate as that of the Oxford workers. Again, much higher concentrations of F ( > 1 0 ppm) were found to be necessary to inhibit cell growth than those (0.0454.5 ppm F) reported by Berry & Trillwood (Cited in F.C.T. 1964, 2, 227). Although these differences have not been resolved, the recent admission by the Oxford workers that they have never bothered to interpret their results in relation to the safety or hazard of water fluoridation but have merely reported their observations will we hope terminate this unfortunate episode.

R A D I A T I O N PRESERVATION HAS COME TO STAY There is little need to dwell on the value of ionizing radiation as a means of preserving food nor will we attempt to compare its merits with those of other methods currently employed in food preservation. Our interest primarily lies in ensuring that consumption of irradiated food in man is not injurious to health. A most convenient starting point is made possible by the appearance of what could be the most important publication so far emanating from this country on this subject. The Working Party set up in 1962 by the Committee on Medical and Nutritional Aspects of Food Policy is to be congratulated for a thorough appraisal of all facets--technological and toxicological ----of radiation preservation of food (Report of the Working Party on Irradiation of Food, Ministry of Health, HMSO, London, p. 85). Another major task facing the Working Party was to decide whether control was necessary or desirable and if so in which form it should be applied. Dealing first with this aspect the Report favours the use of radiation in food preservation but proposes to introduce control by means of prohibition, from which exemption could be obtained in specific cases. Exemptions would at first be confined to individual foods and only later on, after experience had been gained, would it be possible to consider general exemption for groups of similar foods or food products treated in similar ways. Applications for an exemption would have to be supported by evidence of the following: (1) Reasons for proposed irradiation and nature of consumers envisaged; (2) source, energy and dose of

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ionizing radiation used; (3) nature of the food containers used during irradiation and storage; (4) conditions and duration of storage or irradiated food; (5) nature and results of tests for wholesomeness after storage and cooking if necessary; (6) nature and results of microbiological tests on the irradiated food before and after storage. Testing for wholesomeness would necessitate investigations into the possible destruction of nutrients, the possible formation of toxic substances especially mutagens and carcinogens, the possible presence of induced radioactivity and finally the possible influence on the chemical stability of food additives present in irradiated food. The Report recommends the establishment of a body to scrutinize applications for exemptions from the prohibiting control and to offer advice on the conditions of acceptance or rejection. It is also recommended that irradiated foods intended for animal consumption should not ipso facto be exempted from the provision of the prohibiting control. The extensive bibliography containing no less than 352 references bears witness to the comprehensiveness with which this campaign has been conducted. All in all, this Report is an education in itself and represents a landmark in the current attitude of this country towards irradiated foods. In contrast, a relatively modest but nevertheless important contribution in the form of a report of original work has come our way. So far toxicity studies on a variety of foods irradiated under different conditions have revealed only one important toxicological finding. This has been the induction of haemorrhagic diathesis in male rats fed irradiated beef which was attributed to a deletion of the essential amino acid methionine in the beef (Cited in F.C.T. 1964, 2, 72). Along come Malhotra et al. (Toxic. appl. Pharmac. 1965, 7, 402) with the argument that the loss of methionine is not an important factor in the production of haemorrhagic diathesis and that this condition is instead brought about by a vitamin K deficiency, thus lending support to the views of Johnson et al. (Fedn Proc. Fedn Am. Socs exp. Biol. 1960, 19, 1038) and of Mameesh et al. (J. Nutr. 1962, 77, 165). Malhotra et al. (loc. eit.) reached their conclusion after studying the effects of methionine and vitamin K 3 on haemorrhagic diathesis induced in male rats placed on diets containing irradiated beef for up to 14 weeks. They found that oral doses of vitamin K 3 (0-4/~g/rat) given thrice weekly were far more effective in reducing mortality and restoring prothrombin rate to normal than supplementary methionine (0-1.37% of the diet). Two important meetings reflect the rapidly growing interest in radiation preservation of food: The Second Scandinavian Meeting on Food Preservation by Ionizing Radiation, arranged by the Royal Swedish Academy of Engineering Sciences and held in Stockholm in September 1963 (Radiation Preservation of Foodstuffs, edited by P.-O. Kinell & Vera Runnstr6m-Reio, IVA Meddelande NR 138); and the international conference organized by the US Atomic Energy Commission and held in Boston, September 1964 (Radiation Preservation of Foods, National Academy of Sciences--National Research Council Publ. 1273, 1965, pp. 425). Of the Scandinavian countries interest has been especially high in Denmark but progress has also been made in Norway and Finland. The experience gained since the first Scandinavian Meeting of 1960 with regard to experimental techniques, application to special foodstuffs and the fundamental problems at stake formed the subject of the 1963 Meeting. One contribution on the wholesomeness of y-irradiated potatoes merits special mention. Jaarma & Henricson (Radiation Preservation of Foodstuffs (loe. tit.), p. 46) carried out studies in pigs fed on potatoes irradiated with about 15 krad. Preliminary work has so far indicated

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the absence of any toxic effect and no loss in nutritional adequacy. A full report of this work is to be published in Acta veterinaria Scandinavica. The American conference paid detailed attention to all major trends in radiation preservation-technological and microbiological aspects, sources of irradiation, and wholesomeness and food legislation, and it is hoped to review the proceedings of this Conference in a future issue of this Journal.

DRAIZE UNDER FIRE The 'Draize',test (Citedin F.C.T. 1963, 1,137) for eye irritation is used in the safety assessment of cosmetics, toiletries, perfumes, hair-sprays and their ingredients. In the test, 0-1 ml of the material is instilled into one eye of each of 9 rabbits, of which three are rinsed at 2 sec, 3 at 4 sec and the remainder not rinsed. This has been the standard procedure for a number of years but was recently modified to use only 6 animals, with all the eyes remaining unwashed (Cited in F.C.T. 1964, 2, 46). The method has, however, been under fire because of the variability of the results and difficulty in interpreting them. Juggling with the scoring system (Cited in F. C.T. 1963, 1, 137) is only a palliative measure. What the test needs is a great deal of radical improvement ! For example, Buehler & Newmann (Cited in F.C.T. 1965, 3, 540) showed that much of the variation could be reduced by the use of a special applicator which allowed direct contact of the test material with the cornea only. Recently Battista & McSweeney (J. Soc. cosmet. Chem'. 1965, 16, 119) have considered further factors which could cause variable results, especially the rate of clearance from the eye, affected by wiping with the eyelids, stimulation of lachrymal flow and the viscosity of the test sample. Exposure of the cornea only, by the use of an applicator, allowed accurately-timed exposures and the plotting of effect against time and thus the estimation of the no-effect duration of exposure or the time needed to cause a predetermined degree of effect. Compared with animals exposed under the 'classical' conditions, the corneal reaction was more severe but more consistent, especially eliminating the patchy nature of opacity often seen. The authors suggest, therefore, that only the degree and not the area of effect should be scored, since variations in area are probably due to uneven exposure. Using the applicator, it was found that for a given compound (0.25, 5.0 and 10% solutions of sodium hydroxide at low and high viscosities, cellulose thickener being employed to obtain the latter) the irritation did not increase with viscosity. This indicates that any effects of viscosity are probably only exercized through the rate of clearance from the eye. The authors also question the inclusion of chemosis (conjunctival swelling) in the score as this may result from either chemical effect or bacterial contamination. The practice of summing the scores for various parts of the eye is criticized as, especially with low scores, the same score in two animals may represent two very different types of reaction. Weltman et al. (Toxic. appl. Pharmac. 1965, 7, 308) have investigated other factors affecting the scores resulting from the application of the standard Draize scoring system. The possibility of scorer bias, particularly in the case of borderline effects, was shown not to be important by comparing the results calculated from either the high or low possible scores for such effects. This does not, however, eliminate the possibility of considerable interlaboratory differences in the interpretation of lesions. Using groups varying from 8-24

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animals, they found that the variability of the results was considerably reduced with the larger group size. Surprisingly, early release of the animals from restraint, and the subsequent pawing of the treated eyes appeared to decrease the severity of the lesions (observations on small groups only). Histological examination showed the main effects to be erosion of the corneal epithelium and infiltration of the main substance of the cornea by white blood cells. There was also a distinct relation between the severity of effect and onset of repair at 5-7 days, as seen microscopically and as judged from the scores. A further difficulty with the Draize test is the difference in response between rabbits and man. Gaunt & Harper (Cited in F.C.T. 1964, 2, 212) found that 10 shampoos readily available to the public and used apparently with little untoward effect could all be classed as eye irritants when tested in the rabbit eye. Bonfield & Scala (Proc. scient. Sect. Toilet Goods Ass. 1965, No. 43, p. 34) have carried out a similar study using 13 brands of shampoo. They found corneal and conjunctival damage in all cases, after mild iriditis. On the basis of these tests the materials would all be classified as eye irritants and yet do not apparently cause trouble in normal use. It is becoming increasingly evident that to evaluate fully the irritant potential of materials to the eye a far more sophisticated test is required than is at present used and some attempt must be made to correlate the results with experience in man.

H A Z A R D S AT H O M E A N D AT W O R K Living, especially in an over-motorized society, is always a perilous business. Some of the everyday risks are unavoidable, but only the arrogantly eccentric would challenge the Fates by stepping out directly in front of an omnibus. Some, metaphorically it seems, do just that. The development of aplastic anaemia following pentachlorophenol (PCP) poisoning has been reported already (Cited in F.C.T. 1964, 2, 263). Now, two papers by Bergner et al. (Can. med. Ass. J. 1965, 92, 448) and Hernberg & Pessi (Arch. Gewerbepath. Gewerbehyg. 1964, 21, 23) record the aftermath of ignoring warning notices or avoiding commonsense precautions. Both reports deal with poisoning arising from the percutaneous absorption or inhalation of vapour of a wood preservative containing PCP. The affected persons were working in the vicinity of vats containing the preservative or came into direct contact with it by dipping wood. Gloves had been provided but were not worn, and warning notices posted but ignored. The management was also guilty of neglecting to install proper ventilation and to provide adequate supervision for the workers, many of whom were of low mental ability. One worker died and others suffered extreme prostration with, in one case, total paralysis of a limb accompanied by areas of sensory loss. Percutaneous poisoning by PCP is also reported by Chapman & Robson (Lancet 1965, i, 1266), this time occurring in the home. The case concerned a child who had been bathed in water contaminated by wood preservative which had been sprayed on the attic rafters and had thus found its way into the domestic water tank. The wood preservative mixture contained PCP, 2-naphthol and dieldrin. All the family, who had been washing and bathing in the contaminated water for 13 days, had symptoms resembling hay-fever, with mild nasal stuffiness; a boy who had played in the attic had swollen, painful eyes. In another house similarly treated, a patient suffered from nasal stuffiness and persistent lachrymation.

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The clinical signs and symptoms of PCP poisoning arise from uncoupling of oxidative phosphorylation processes leading to an increased basal metabolic rate and heat retention, thus resembling the effects of dinitrophenols (Cann & Verlust, Am. J. Dis. Child. 1960, 100, 947). A report by Anderson et al. (Can. med. Ass. J. 1965, 92, 809) underlines the importance of vigilance in tracking down the causative agent in the poisoning of two children with signs indicative of anticholinesterase poisoning. On treatment with atropine, the signs cleared but soon recurred. The cause was traced to flannelette sheets which had become accidentally contaminated with parathion during transit by ship. While the symptoms of accidents of this nature appear relatively rapidly after exposure to the causative agent, in the case of other exposures, such as asbestosis, it may be many years before the full consequences are revealed. Enticknap & Smither (Br. J. ind. Med. 1964, 21, 20) have shown that up to 20 yr may elapse between exposure to asbestos dust and the development of mesothelial tumours. This association had been indicated already by Wagner et aL (ibid 1960, 17, 260) in 47 cases of diffuse pleural mesothelioma. In all but one case there was a remote history of previous exposure to asbestos, although only 8 showed frank asbestosis of the lungs. Selikoff et al. (New Engl. J. Med. 1965, 272, 560) in an exhaustive study examined the significance of diffuse pleural mesothelioma in 307 consecutive deaths among asbestos workers. They conclude that this tumour is an important cause of death following chronic exposure to asbestos, joining lung cancer and cancer of stomach and colon in this respect. In an Editorial, the New England Journal of Medicine (1965, 272, 590) emphasizes the relationship between remote exposures to asbestos dust and development of chest tumour. It is stressed that although a history of asbestosis is not always forthcoming and, indeed, exposure may have been extremely light, tumours from this primary cause may subsequently develop. Clearly, as the Editorial states, the conditions in asbestos works should give cause for concern and many obviously dangerous operations be eliminated. A Leading Article (Br. med. J. 1964, ii, 202) is more emphatic, urging: "Until we know the answers to some of the questions posed by the recent findings all exposure to asbestos dust should be considered as hazardous, and supervision should be extended to insulation workers in ships, factories, and domestic buildings who may be intermittently but nevertheless heavily exposed to asbestos dust." With the benefit of hindsight, it is clear that many of the above cases might have been avoided. However, it is not always possible to know where danger lurks, as exemplified by a cautionary note from the Times of 12 June 1965. This records the death from lung cancer of a mother who, over a period of years, had been washing her daughters' protective overalls, hats and masks with commendable devotion, unaware of the fact that heavy impregnation with asbestos dust was a source of danger to her. We cannot avoid all the buses all the time.

PEOPLE, PLANTS A N D P O L L U T I O N Whatever action we direct against the forces which are polluting the atmosphere we and the plants have to breathe, it has to be a compromise. Haagen-Smit (Chem. Engng News 1965, 43 (I 5), 99) remarks that to wait for proof of all the supposed ill effects of air pollution would be to apply the remedy too late to save the patient. While the results are trickling in, too much control will be better than too little. The present obstacles in the path of more effective control are neither scientific nor technical, but political. Quoting from Haagen-Smit

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(Ioc. cit.): "An intelligent development of living areas, mass transportation, and zoning of industry is an essential part of our task in keeping the air fit to breathe". That is for thelongrange planners. Nearer home: "Small gains can be made in many ways by voluntary action of citizens as well as by intelligent smog-orientated city f a t h e r s . . . " . Already the cleaning of coal and oil is carried out to the point of economic defensibility (Technical Committee of the National Society for Clean Air, Sulphur Dioxide. An Examination of Sulphur Dioxide as an Air Pollutant, National Society for Clean Air, London, 1964, p. 32). Industrial sulphur dioxide discharge is carried out at high levels; sulphur absorption processes can only be applied economically to large plants such as monster power stations. One shining example from industry is Concawe*, an international study group which considers clean air and water conservation in Western Europe. It was set up in 1963 and is now financed by international oil companies. Its objectives are to collate and disseminate to interested parties information concerning air, water and soil pollution attributable to oil production, to promote active co-operation between its participants, and to provide funds to support special studies in research institutions (Chem. Engng News 1965, 43, (18), 66). The crux of the matter is indeed cost. Reforms are always expensive, being directed against momentum, and it is necessary to convince those who will have to pay of the righteousness of the cause. Parker, in a presidential address to the Clean Air Conference (Proc. Clean Air Conf. Harrogate, 1964, National Society for Clean Air, London, 1965, p. 11), pointed out that although there has been an increase in sulphur dioxide emission recently, it has been at a high atmospheric level, mainly from newer power stations, and has not been accompanied by measurable change in the near-ground concentration. On the other hand, three quarters of the low-level discharge of smoke into the U K atmosphere during 1963 came from domestic chimneys. Lord Sherfield's bon mot, when opening the same conference, was: " I f you want to have cheap power with clean air, go nuclear". The two statements are not quite reconcilable, and we must remain healthily suspicious of the efficacy of industrial safeguards while we go about convincing the common citizen of his responsibility for our lethal smogs. To convince either an industrialist or the man in the street that he must shoulder his share of the burden calls for an unequivocal statement of facts as regards both the genesis of pollutants and the hazard posed by any specific pollutant. It is such facts which are being reaped in research institutions and which are slowly falling into the pattern of a formidable indictment. To revert to the summary of the obstacles propounded by Haagen-Smit (loc. cit.), we must determine the level at which a pollutant becomes harmless to humans of widely varying susceptibility, and the limit which plant life in general will tolerate. The second is easier, and important because plant life is intricately interwoven with human activities. Limits of human tolerance for chemical agents used for limited hours in industrial processes are not a useful guide in the outside world, where men breathe for 24 hr a day. It is easier to transfer a man to a process to which he is not unduly sensitive than it is to transplant a family from a city into the wide open spaces. In general, limits of tolerance will be far lower than in industry, but they will have to be determined and protected by regulations. This is the first step. The second is to agree upon a norm for health which is universally applicable, so that defections from it can be recognized and correlated with a pollutant. After this will come a study of long-term effects which might affect future generations. Meaningful results are hard to deduce from the meagre samples of population available *In full, "The Oil Refining Companies' International Study Group for Clean Air and Water Conservation in

Western Europe".

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for direct research. The fact that air pollution by a single agent alone does not occur in practice hampers the epidemiologist who wants to draw conclusions; respiratory diseases are aggravated by polluted air, agreed, but which agent is primarily responsible ? Even the prime agent in the deliberate and calculated pollution of an individual's respiratory tract by tobacco smoke eludes search, and we have to fall back upon the consideration of multiple agents. Such is the importance of recent changes in man's environment that their effects on health were considered by a WHO Expert Committee (Environmental Change and Resulting Impacts on Health, Wld Hlth Org. techn. Rep. Ser. 1964, 292). Five major aspects were discussed: ionizing radiation, air pollution, drinking water supplies and waste water, solid waste and food hygiene. On air pollution, the Committee had this to say: "Air pollution, while a serious threat to susceptible individuals in the high-density urban or industrial al eas where it occu/rs, is not, relative to other health problems, a major one affecting the whole modern world. Continuing efforts must, of course, be made to avoid creating fresh problems in the developing countries and, as money and priorities permit, to abate existing problems". It is a relief to turn from such general considerations to findings in specific areas of research, even though they are fragmentary and cannot yet be fitted logically together. Wynder & Hoffmann (J. Air Pollut. Control Ass. 1965, 15, 155) have examined polluted air samples from New York City and Detroit, in which the concentration of aromatic polycyclic hydrocarbons rose in traffic-congested areas and was higher by day than by night and in winter than in summer. Although 1,2-benzanthracene and 3,4-benzopyrene were both more concentrated by day than by night, the ratio of the first to the second rose after dark: an effect attributable to the gre,ater photosensitivity of 1,2-benzanthracene. Cleary & Sullivan (Med. J. Aust. 1965, i, 758) have provided the first ever report on atmospheric pollution by polycyclic aromatic hydrocarbons in an Australian city. Pyrene, fluoranthene, 1,2-benzanthracene, chrysene, 1,2- and 3,4-benzopyrene, 1,12-benzoperylene and coronene were identified and estimated in the air of Sydney. As in the American cities, there was a marked winter increase in the levels of these hydrocarbons. The small seasonal variation in fuel usage and domestic coal consumption could not account for this finding. In general there was good correlation between smoke density and hydrocarbon levels. Atmospheric levels in Sydney are 10 times lower than in towns in the UK but are of the same order as those found in American cities. The organic extract from air samples obtained in New York and Detroit produced mutiple papillomas and carcinomas when applied to the skin of mice (Hoffmann & Wynder, (Proc. Am. Ass. Cancer Res. 1965, 6, 29) and fractionation showed a positively carcinogenic neutral fraction with a number of acidic and other fractions which promoted tumour growth. Wynder & Hoffmann (loc. cit.) suggest that the aromatic polycyclic hydrocarbons act as turnout initiators, which are then backed up by the acidic promoters. They consider, however, that general air pollution contributes no more than a minor fraction to the total of lung cancer in cities, particularly for non-smokers. Multiple factors are indicated by the epidemiological studies, which show more lung cancer in city cigarette smokers than in country ones. The mere fact that city air contains agents which poison the ciliated lining of the nasal cavities, and others which can initiate and promote the growth of tumours, nevertheless argues the case for preventing pollution. Carcinogenic components in motor oil additives are liable to pass as aerosols into the exhaust. Baldwin et al. (Br. J. Cancer 1964, 18, 503) have made further investigations of an

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additive previously found to be carcinogenic. The base oil employed was highly toxic, causing inflammatory changes in the skin of mice, and repeated painting induced a tumour incidence of 66% ; mostly benign papillomas, but 17~/osquamous cell carcinomas. The whole additive induced a similar crop of tumours of which 51% were carcinomatous. Although the lead naphthenate fraction was not demonstrably tumorigenic, it induced kidney damage and tubular adenomas. A second sample of additive lacked the direct irritancy of the first, but induced tumours in 48% of mice, 19% being squamous cell carcinomas. A rather more restricted hazard posed by the fumes from heated petroleum asphalt has been studied by Simmers (Archs envir. Hlth 1964, 9, 727), who exposed mice to aerosols of emulsified hot asphalt in water and to smoke from boiling asphalt for short periods daily for many months. I n both groups of animals the respiratory tract showed congestion, inflammation of the lungs and bronchi, stretching of the bronchioles and some infiltration of round cells around the bronchi. The degenerative changes were much more pronounced after inhalation of the smoke than of the aerosol. Despite the covering of the exposure chamber and its contents with a yellow oily deposit smelling of petroleum, the animals ate well and developed no tumours of the alimentary tract. There were rare suspicious squamous cell developments in the respiratory system, but on the whole the picture was one of progressive loss of functional respiratory tissue. Some animals in each group proved abnormally resistant to the irritation. The results suggest that exposure to such smoke may be a non-specific irritation which does not depend upon the precise nature of the inhaled particles. Quite apart from any carcinogenic hazards, the mushrooming of the motoring habit has obliged us to breathe products of fuel combustion and partial combustion emitted at low level into the atmosphere and thereafter subjected to a complex of photochemical reactions whose precise nature varies with the climate. Incomplete combustion of motor fuel follows inevitably from the inherent weaknesses of engine design (Haagen-Smit, loc. cir.). The ratio of air to fuel which gives maximum flame propagation (12:1) differs from that which would ensure complete combustion (15:1), and there are considerable losses round pistons and carburettors. California, the classic home of the engine-exhaust pollution hazard, has already gone far in passing legislation and devising equipment to deal with this menace. But there remain the products of combustion themselves--in particular the aldehydes and the oxides of nitrogen, which take part in complicated and still largely obscure photochemical reactions. A review of the photochemistry of air pollution by Altshuller & Bufalini (Photochem. Photobiol. 1965, 4, 97) of the US Public Health Service points out that the products of the solar irradiation of automobile exhaust fumes resemble qualitatively those obtained experimentally by the photo-oxidation of hydrocarbon and nitric oxide (NO) mixtures, concerning which a considerable volume of research has been published. The dissociation of NO2 to NO + O takes place with wavelengths below the threshold value of 404.7-435.8 mtz. NO2 also reacts with atomic oxygen to yield NO and ozone, which remain in a steady state until the advent of a hydrocarbon upsets the balance, so that both ozone and NO2 build up. Atomic oxygen and ozone react with hydrocarbons to produce phenols and aldehydes, which in turn may yield alkyl nitrates and later peroxyacyl nitrates, which are important pollutants. The olefine content of a motor fuel may determine the rates of formation of ozone and organic oxidants, while high-olefine fuels encourage the production of high concentrations of peroxyacyl nitrates. Thus, a certain amount of pollutant control is possible by the careful formulation of a fuel. Even without irradiation effects, motor exhaust gases contain aldehydes and phenols,

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with formaldehyde constituting half the aldehyde content and acrolein 10% of the aliphatic aldehydes. By trapping exhaust gases in alkali, acidifying and extracting with chloroform, Barber et al. (Analyt. Chem. 1964, 36, 2442) obtained a phenolic mixture from which paper and gas chromatography separated and identified salicylaldehyde, phenol, 5-ethylphenol, cresols and xylenols, with acetophenone and several unidentifiable compounds. The irradiation of petroleum exhaust and nitrogen oxides moreover produces light-scattering aerosols composed of compounds of the carbonyl, hydroxyl and nitrate ester type (Altshuller & Bufalini, loc. cit.). Sulphur dioxide usually enters this unholy alliance to produce a fog which not only impairs visibility but contains sulphuric acid to irritate the eyes and the respiratory tract. Such aerosol formation is marked with high-olefine fuels, but becomes insignificant with paraffin fuels. What of the animals and plants which are exposed to this formidable barrage of pollutants? One i~nmediate human reaction is eye irritation, a reversible manifestation which may be measured by its degree, by the delay in its onset after exposure commences, or by the proportion of subjects who suffer it over a given period (Altshuller & Bufalini, loc. cit.). In this field, formaldehyde, acrolein and peroxyacyl nitrates, are important primary irritants. About 55% of their combined activity is contributed by ethylene and propylene in fuels, 16% by the terminal olefines, 18% by the internal olefines and 11% by the di-olefines. It may be presumed that irritation of the respiratory tract, though not so immediate or so obvious, goes hand in hand with eye irritation. Murphy et al. (Toxic. appl. Pharmac. 1964, 6, 520) have shown that rats exposed to ozone, nitrogen dioxide, sulphur dioxide, formaldehyde or acrolein, either by inhalation or by intraperitoneal injection in solution, develop a marked increase in liver alkaline phosphatase activity which becomes maximal after 24 hr. It is associated with an increase in weight of both liver and adrenals. Prolonged exposure to very low concentrations of these compounds produces this apparently non-specific reaction, which appears to be part of the overall stress reaction in animals. An effect upon isolated enzyme systems has been reported by Estes & Pan (Archs envir. Hlth 1965, 10, 207) who subjected bacterial and mammalian glutamic dehydrogenase and lactic dehydrogenase preparations to the irradiation product of nitrogen dioxide and but-l-ene and to a number of aldehydes. The irradiation product inhibited the conversion of glutamate to a-ketoglutarate, with little effect on the reverse reaction (though the mammalian-derived enzyme was inhibited markedly in the ketoglutarate-,glutamate direction). Propionaldehyde and butyraldehyde among the constituent aldehydes had little effect, but formaldehyde and acetaldehyde strongly inhibited glutamic dehydrogenase. All four aldehydes were relatively weak inhibitors of lactic dehydrogenase. In plants, widespread damage to foliage has been reported after exposure to ozone, peroxyacyl nitrates and unidentified intermediates in the olefine-ozone reaction, and growth has been stunted even where no lesions were evident (Altshuller & Bufalini, loc. cit.). Fumigation with irradiated motor exhaust may reduce photosynthesis. Ozone in a concentration of 0.1-1.0 ppm produces necrosis and bleaching of leaves; peroxyacyl nitrates at less thin 0.01 ppm causing glazing, bronzing or silvering of the lower surfaces. Mature leaves are relatively resistant, but younger, expanding ones are peculiarly subject to such injury. Ordin (Archs envir. Hlth 1965, 10, 189) has shown that cell wall metabolism, and particularly galacturonic acid metabolism, in plants is inhibited by ozone and by peroxyacetyl nitrate, which probably attack the sulphydryl groups of enzymes. Growth in the presence of indoleacetic acid was greatly reduced by both compounds. Peroxyacetyl nitrate inhibited glucose absorption by oat seedlings, though ozone did not. The main effect seems to be on

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the metabolism of cellulose and the glucan components of the more soluble hemicelluloses. As a tailpiece we refer to a study by Nusbaum et al. (ibid 1965, 10, 227) of the uptake o f 14 elements, and lead (Pb) in particular, in the bones of late residents of Los Angeles. Postmortem samples of ribs and skull calvaria showed no correlation between Pb concentration and the age at death or the length of residence of the owner in Los Angeles. On the other hand, there was more Pb in men than in women (perhaps an occupational bias), and the ribs and lungs of men who had smoked were richer in Pb than those of women who had smoked or men who had not. The significance of thesefindings is not clear; but they indicate another route of approach to the pollution problem.

TIN STABILIZATION OF PVC: T H E 'INSIDE' STORY The degradation of vinyl chloride polymers (PVC) by heat and light involves liberation of hydrochloric acid (HCI) and production of coloured and oxidizable polyenes, and is accompanied by loss of strength and impact resistance and impairment of electrical and surface properties. The readiness with which these changes occur has led to the development of a wide range of stabilizers, which include salts of lead (Pb), salts and soaps of barium (Ba), cadmium (Cd) and zinc (Zn), organotin compounds, epoxidized oils, phenyl phosphites, ~-phenylindole and diphenylthiourea. As a result, what promised in the early days to be a major problem has, from a technical point of view, been satisfactorily overcome. It is an unfortunate fact, however, that some of the most efficient stabilizers are toxicologically suspect and therefore present particular problems when PVC is to be used for food packaging, for conveying drinking water or beverages and for those applications, such as children's toys and certain items of household equipment, where the plastics material may come in contact with the mouth. Thus Pb salts and soluble Cd compounds are highly toxic, while Zn salts and certain epoxidized oils, whose toxicity is relatively low, are ineffective stabilizers when used alone. Some of the newer organic stabilizers, however, such as ~-phenylindole and some alkyl phenylphosphites and dioctyltin compounds, have been subjected to feeding tests and found to be suitable for use in many applications within certain extractability limits. It must be remembered that such toxicity assessments are generally based on the biological activity of the free stabilizer. Consideration of the oral toxicity of the initial product, whether organic or inorganic, does not necessarily give a true picture of the situation which exists when the stabilizer has been incorporated into a polymeric composition. The important factor is not what is put into the composition, but how much may migrate out and in what form it may appear. Thus, if a plastics water pipe contains a Pb stabilizer so firmly bound that, after preliminary washings, virtually none will migrate out, no hazard is likely to arise from its use. It must be remembered in this connexion that normal mixing procedures often necessitate the use of an excess of stabilizer over the theoretical requirement and that much of the material migrating out is likely to be in the form of derivatives that may be more or less toxic than the original additive. The fate of the stabilizers added to PVC is therefore of prime importance in assessing their suitability for an application such as food packaging. Some light is thrown on this question and specifically on the nature of the bonds between stabilizer and polymer, by a series of papers describing a meticulous study made by a group of US workers, first on Cd, Ba and Zn soaps and later on a group of organotin compounds.

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An interesting radioactive tracer technique was used involving labelled stabilizers and measurement of the activity retained by the polymer after controlled heat treatments. Supplementary evidence was obtained from infrared (IR) studies. To determine the radiation retention, a sample of PVC was milled with a labelled stabilizer and then converted to film which was subjected to various heat treatments of controlled duration and severity. After this the treated polymer was dissolved in tetrahydrofuran and reprecipitated with methanol, and this sequence was repeated a number of times, the radioactivity of the precipitated resin being measured each time. If a sample was found to retain an effectively constant amount of radioactivity through a series of dissolution-precipitation cycles, it was concluded that the labelled part of the stabilizer molecule had become linked to the polymer by a strong chemical bond. This technique was used to investigate the behaviour of Ba, Cd and Zn salts of 2-ethylhexanoic acid (EH) labelled with carbon-14 (1-14C) and the results were compared with those of earlier IR studies (Frye & Horst, J. Polym. Sci. 1960, 45, 1 ; ibid 1959, 40, 419). In the latter, films containing salts of EH had been found to exhibit a band at 5-75/z which was absent both in "pure" PVC and in the salts themselves. This band was identified as being due to art esterifying displacement in the polymer chain, whereby labile chlorine (CI) atoms were replaced by more stable carboxylate groups. The radioactive tracer studies supported this concept, since the retention of radioactivity varied with the duration and temperature of heat treatment in a way which paralleled the variation of intensity of the 5.75-/z band. Moreover, analysis of the precipitated resins by emission spectroscopy showed that no traces of heavy metal remained in the polymer. Prolonged heat treatment eventually leads to aloss of radioactivity, because the free carboxylic acid is liberated from the chain, leaving an olefinic bond. This observation too was supported by IR studies. Visual inspection of the films after the heat treatments showed that only the Cd salt was an effective stabilizer when used alone. The others, particularly the Zn salt, appeared to promote deterioration, because of the deleterious action of the heavy metal chlorides freed by the carboxylate displacement. Only the Cd salt was a sufficiently efficient stabilizer to compensate adequately for this adverse effect, which was demonstrated by visual comparison of "pure" PVC and PVC milled with the chlorides of Zn, Cd or Ba and subjected to controlled heat experiments. The Zn salt showed the worst effect, the Cd salt the least. This action of heavy metal chlorides is frequently counteracted in commercial stabilizers by the addition of organic phosphites. Of perhaps greater interest, on account both of the complexity of the problem and of current technological trends, is the more recent work on dialkyltin stabilizers. This was designed to demonstrate which part of the molecule is chemically bound to the polymer and whether the reaction is one of displacement and/or addition. The compounds were accordingly labelled with ~4C in the C~ position of the alkyl (butyl)* groups or acid radical or with tin-ll3 ("3Sn) (Frye & Horst, Int. J. appl. Radiat. Isotopes 1964, 15, 169). Tests were made on compounds of the general class R2SnY2, where Y was monomethyl maleate (MM), oxooctyl thioglycolate (OT), fl-mercaptopropanoate (MP) or EH. It was found that each_ retention curve rose to a maximum with increase in the duration of heat treatment and then declined. The fall coincided with blackening of the film, but the height of the curve could not be correlated with the effectiveness of the stabilizers. The existence of a relatively weak associative link formed during milling and heat treatment between the butyl group and the polymer was indicated by the fact that for a given heat #Tracer studies with labelled alkyl groups (Frye et al. J. Polym. Sci. (Part A) 1964, 2, 1765) were limited to dibutyl (DB) compounds.

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treatment, the radioactivity of the resin decreased with each successive dissolution-precipitation cycle, but the retention was still greater than that in a resin precipitated from a tetrahydrofuran solution to which labelled stabilizer had been added. The authors show that only a very minor proportion of the retained radioactivity is due to actual butylation of the resin, as was suggested by Kenyon (Natn. Bur. Stand. (US), Circ. No. 525, 1953, p. 91) and that this is unlikely to contribute much to the stabilizing effect. The weak associative link is probably a coordinate organotin complex which is partially ruptured by successive solvent treatments. When stabilizers labelled with 11SSn were used, the variation in retained radioactivity with the extent of heat treatment and the nature of the Y group closely paralleled that found with the labelled butyl groups, suggesting that little cleavage of the alkyl-Sn bond occurs (Frye et al. J. Polym. Sci. (Part A) 1964, 2, 1785). A comparison of results with D B - M P and dioctylMP is of interest in view of conflicting opinions on the relative merits of these compounds. While dibutyltin stabilizers tend to be more widely used industrially because they are cheaper, the dioctyltins are sometimes considered to have a slight toxicological advantage, if only because they have been more widely tested, and their stabilizing action is possibly superior. It was in fact found that retention of radioactivity was markedly higher with the dioctyl than with the DB compounds, but only when heat treatment was fairly severe (at least 2 hr at 175°C). Some confirmation for the coordinate linkage hypothesis was provided by the almost complete loss of Sn from a radioactive resin treated with anhydrous HCI. Such treatment had already been shown to cause little or no C-Sn bond cleavage, so the implication is that ,.n.~CHz - - C H

CH 2 - - C H

--CHzun.r~

--

CHzva.rv

~

CHz -

CH - -

CH z ,.rv'u

I

Ck +

Bu

Y

Bu

~Sn J Bu

"I+ ~Y

Bu

I

CH - -

/

I

y

./

s0 Bu

CL

cl. ua.n~ CHz -

s.

CHzu'v~

I CH 2 - c H u-Lr~ C H z - -

/ Y

CL --

CHz~

CH - -

./v~CH 2 -

t

CH - -

CH2~/~

CHz v'V'~

I Bu

Y ~Sn

Bu~

CL /

+ HY

+~ct CL

,.,'v~ CH 2

FIG. I.

Mechanism

I

CH--

CH2 u-v~

of organotin stabilization.

cleavage occurs between the Sn atoms and certain donor atoms covalently bonding the stabilizer to the polymer. Finally the question was studied with labelled acid radicals (Frye et al. J. Polym. Sci. (Part A) 1964, 2, 1801) and here part of the radioactivity was found to become firmly

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associated with the resin. In a series of 6 dissolution-precipitation cycles, the radioactivity of the stabilized resin remained constant within the limits of experimental error, although again it varied with the duration of heat treatment and the nature of the Y-group. Surprisingly, retention was relatively low in PVC treated with D B - M M , although it was greater with this than with other stabilizing systems when labelling was located on the alkyl group or Sn atom. This may have been due to cleavage of the half-ester, promoted by eliminated HCI. The retention pattern for D B - E H indicated a displacement of CI by the carboxylate group in a manner analogous to that described above for heavy metal salts. The similarity did not extend to a marked 5"75-t~ band in IR studies, but this is tentatively explained by a lower degree of esterification. Similar displacement of labile CI by a more stable carboxylate group may occur with D B - M M and DB-MP. To explain the very high level of radioactivity retained by resin treated with DB-OT, the authors postulate a replacement of C1 in the main polymer chain by thioether groups, a process which may lead to crosslinking between chains. It is clear that much work still remains to be done on this subject. Nevertheless it has been established that a finn chemical bond exists between the polymer and the acid radical of the stabilizer. In the organotin stabilizers a subsidiary coordinate link through the alkyltin moiety may retard HCI elimination and act as a precursor for the displacement of labile halogen by the acid radicals (Fig. 1). [Would that these labelled resins could be used to settle the numerous burning issues concerned with the materials extracted by food or food-simulating solvents from tinstabilized PVC. In the meantime, it is clear from the fine work described here that the dialkyltin dichloride is the compound to which first consideration must be given in the course of safety evaluation of all organotin stabilizers used in food-packaging applications.]