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43, 577) measured the in uiuo incorporation of thymidine into DNA and the activity of pulmonary thymidine kinase after a BHT injectih in mice pretreated with inducers or inhibitors of microsomal enzymes. or exposed after the BHT injection to loOo/, oxygen, which would allow for non-enzymic formation of metabolites. Incorporation measurements, using BHT labelled with 14C at the toluene methyl group, demonstrated that maximum concentrations of BHT were present in the lung between 4 and 8 hours after the injection. Pretreatment with phenobarbital diminished the toxic effect of BHT, but 3-methylcholanthrene and a-naphthylisothiocyanate had no effect on its toxicity. Similarly the oxygen inhalation was without effect. Although they point out that the evidence is circumstantial, the authors conclude that the results indicate that metabolic activation is not a prerequisite for BHT toxicity in the lung. The phenobarbital reduction of BHT toxicity was due in all probability to its known capacity for increasing biliary excretion. Moreover, it was not possible to prevent cell death by suppressing protein synthesis in the target organ by pretreatment with cycloheximide, a procedure that has been used to abolish the toxic effects of some other compounds requiring metabolic activation. The mechanism of BHT toxicity, however, still remains unclear. The susceptibility of the squamous

epithelium to injury is similar to that seen after oxygen exposure, radiation and treatment with bloodborne bleomycin, but the recovery pattern is quite different. Thus, for example, the repair cell profile is not typical and even at high BHT dosage, capillary destruction was always found to be secondary to that of the type I epithelium. Cellular regeneration as reflected by DNA synthesis is also different. In both casesthere is early endothelial injury followed by type I cell necrosis but the peak of DNA synthesis in the epithelium precedes that in the endothelium following BHT toxicity whereas the reverse is true after oxygen poisoning. Adamson rt al. (lot. cit.) suggest that this may be related to the severity of damage; after oxygen poisoning there is early endothelial necrosis, while after BHT the changes appear to be related rather to increased permeability. BHT is thought to cause cell lysis and death as a result of interaction with the cell membrane and Williamson et al. (lot. cit.) comment that a similar mechanism may account for the early disruption of type I alveolar cell membranes. However, the reason has yet to be found why these are the first target cells; the capillary endothelial cells have earlier contact with BHT but only become damaged 4-6 days later when BHT has virtually disappeared from the circulation. [R. Hawkins-BIBRA]

STYRENE BEFORE THE FRAY Paranoia may become an occupational hazard in the plastics industry. Findings confirming vinyl chloride’s carcinogenicity were quickly followed by those implicating acrylonitrile. The preliminary report from a long-term inhalation study (conducted at Dow Chemical’s Toxicology Laboratory, Midland, MI) indicating that styrene may be carcinogenic in the rat must have given members of the industry a feeling of dPja vu. The 2-year inhalation study, conducted on behalf of the Manufacturing Chemists’ Association, will provide the first opportunity for assessing the carcinogenicity of styrene, previous animal studies having used much shorter treatment periods. Chronic oral toxicity data will also be available soon. Longterm studies are being conducted in the rat and mouse by the IARC at Lyon, and a similar programme is being sponsored by the National Cancer Institute at Litton Bionetics. Mutagenicity

Styrene oxide, a possible alkylating agent, may provide the clue to styrenes carcinogenic activity. The oxide induced gene conversion in Schizosaccharomyces cerevisiae and forward mutations in S. pombe and hamster cells, whereas styrene was inactive in ail three systemseven in the presence of a weak metabolizing system (Cited in F.C.T. 1978, 16, 300). In hostmediated assays,both styrene and its oxide induced gene conversion in S. cerevisiae, but had no effect on S. pombe (ibid 1978, 16, 300). In the more established Salmonella typhimurium system. styrene has given conflicting results. In one study

1978, 16, 397) it was not mutagenic at levels up to I mg/plate against strains TA1535, 1537, 1538. 98 and 100 in the presence of a liver metabolizing system obtained from rats and hamsters pretreated with polychlorinated biphenyls. However, according to the results of the Ames test conducted by De Meester et al. (Mutation Res. 1977, 56, 147) who used the same metabolizing system, styrene was mutagenic in strain TA1535 although there was no evidence of mutagenicity in the absence of the metabolizing system. At concentrations from 1 to 11 pmol (1 mg)/plate the number of revertants increased with dose; an appreciable dose-related toxic effect was also observed. Both in the absence and the presence of a metabolic activating system, styrene oxide was shown to be mutagenic towards the TAl535 and TA 100 strains. A number of recent studies have indicated that styrene is mutagenic in mammalian systems. In an in vitro study (de Raat, Chemico-Biol. Interactions 1978, 20, 163) the monomer induced sister chromatid exchanges in Chinese hamster ovary cells. Styrene’s mutagenic activity was only observed, however, in the presence of both a metabolic activation system (an Sg fraction from phenobarbitone-treated rats) and the epoxide-hydrase inhibitor cyclohexene oxide. Styrene oxide was a potent inducer of sister chromatid exchanges even in the absence of the activation systern. Nevertheless when the Sg fraction was present.’ the mutagenicity by styrene oxide was increased by the addition of cyclohexene oxide. The lack of mutagenic activity of styrene even in the presence of the metabolic activation system can be ascribed to a very

(ibid

Articles of general interest--f{/ rapid decomposition of styrene oxide, the decomposition being inhibited by the cyclohexene oxide. Meretoja er al. have shown styrene to be mutagenic iri riro (To.uico/oyy Lett. 1978. 1, 3 1.5). Male rats inhaling 300 ppm styrene for 6 hours/day, 5 days/week, for up to 11 weeks first exhibited an increased rate of chromosomal aberrations, almost totally chromosome-type breaks, in their bone-marrow cells at week 9. This increased rate, the aberrant cell incidence ranging from 8 to 12:; in the exposed group compared with I to 6:; in the control group, was maintained until the end of the experiment. Styrene exposure was also associated with an increase in polyploid cells. The same group has studied styrene’s mutagenic activity in man. The chromosomes in cultured lymphocytes from ten men occupationally exposed to the monomer were compared with those from five control individuals (Meretoja et al. Mutation Res. 1977, 56, 193). Styrene exposure was associated with a significant increase in the rate of chromosomal aberration. a control incidence of 3% or less being increased into the I l-26% range. The men, aged from 20 to 41 years. had been employed in the lamination industry for up to 85 years and all felt they were in good health, Extensive investigations into the effects of styrene on man have not been attempted although limited epidemiological data are available. Western contributions in this sphere include a study by Zielhuis et cd. (Fourteenth

Intrrrlatiorlal

Corlgress

011 Occupatiorud

Hrulrh. Madrid 1963. 3, 1092) who found no significant difference between the health of workers exposed to styrene (at an average concentration below 100 ppm) and that of a control population. apart from an increased occurrence of subjective symptoms, such as irritation of the mucosae and headache. In the study of Wink (Ann. occup. Hyg. 1972. 15, 21 I), styrene exposure was associated with a decrease in urinary l7-oxosteroid levels but no other objective adverse effects. In a small group of Swiss workers, high exposures to styrene were shown to result in conjunctivitis (Cirrd in F.C.T. ‘1976. 14. 201). Four out of the nine workers who underwent haematological examination had a lymphocytosis. Scientists at the Institute of Occupational Health. Helsinki. have investigated the psychological and neurophysiological consequences of working with styrene. A group of about a hundred workers employed for an average of 5 years in the manufacture of reinforced plastics were compared from the psychological angle with a group of 43 workers in the concrete industry (Lindstrom er a/. Scud J. Work Eurlir. H/t/l 1976. 3, 129). Exposure to styrene was assessed by means of the urinary mandelic acid levels; these varied from 7 to 4715 mg/dm’ with an average value of 808 mg/dm’. There were statistically significant differences between the two groups in visuomotor accuracy and psychomotor performance. Disturbances in visuomotor accuracy and psychomotor activity were related to urinary mandelic acid level. whilst performance in tests measuring visuomotor speed and visual memory was related to the length of service in the styrene industry. Since at least 20 hours had elapsed between the last workshift and the examination. the

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authors considered that the findings were indicative of more than an acute change. In the neurophysiological investigation (Seppllainen & Horkonen. ihid 1976. 3. 140). abnormalities in the electro-encephalograms of workers illustrated the toxic effect of styrene on the central nervous system. Abnormalities were found in 24”i, of the workers exposed to styrene. a significant increase over the control incidence. Lowlevel exposure to styrene did not increase the prevalence of abnormal electro-encephalograms, but almost one third of those subjected to higher exposures (again judged from the urinary mandelic acid level) were adversely affected. Neurotoxic effects of styrene have been investigated by Lilis et al. (Ewir. Res. 1978. 15, 133) in workers involved in its production and polymerization. As expected, prenarcotic symptoms such as light-headedness. were found in 13% of the 488 workers, and these symptoms were seen more frequently in those classified as having a high exposure to the monomer. Distal hypo-aesthesia of the lower extremities occurred in 85% of the I80 workers with over 20 years experience in the industry, but in cnly 4.1% of the I21 subjects with less than 7 years exposure. As no control group was included in the study it is uncertain whether the difference between the two groups was merely a consequence of increased age. In a further examination of 80 of the workers, reductions in the conduction velocity of the radial nerves ( < 55 m/second) and peroneal nerve ( ~40 m/second) were seen in 18.8 and 16.4%. respectively. of those tested. Although the authors noted that there was a steady decrease in the ,peroneal nerve velocity with duration of styrene exposure. the small number of subjects in each exposure subgroup largely undermine the significance of this result. By contrast, there was no evidence from the study of Seppalainen & Hiirkonen (lot. cir.) that peripheral nerves are susceptible to styrene exposure: nerve conduction velocities were comparable in an exposed and control group. A single case of retrobulbar neuritis tentatively attributed to styrene exposure was reported in 1964 (Cited irl F.C.T. 1965. 3. 534). Ocular examination of 345 workers exposed to styrene produced no confirmatory evidence of styrenes activity in this respect (Kohn. Aria. J. Ophtl7trl. 1978. 85, 569). Conjunctival irritation affected 223, of this workforce and this irritation was correlated with intensity of exposure. Some attempt has been made to assess possible consumer exposure to styrene. A report from the Canadian Health Protection Branch (Withey & Collins. Bit//. rrn. corvm,]. & Torid. (U.S.) 1978. 19, 861 has shown that styrene levels from as low as 698 ppm up to 3285 ppm occur in samples of polystyrcnc packaging material. No clear relationship between the monomer content of the packaging and the amount leached into the foodstuffs was identified. although for food of approximately similar composition. the extent to which migration occurred was proportional to the original monomer content. Relatively large amounts of styrene migrated during the short period immediately after the food was placed in the container but after this initial effect there was a regular increase in monomer contamination of the food with

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Articlesof general interest-Fd Cosmer.Toxicol. Vol. 17, No. 3

time. The maximum level of styrene in any of the foods tested was 246ppb (in sour cream), with a more usual level of the order of 20 b. blished so far provide The epidemiological data PB” no direct evidence that styrene is carcinogenic to man. However carcinogens typically have latent periods of 20 years or more and few of the workers studied had an occupational history of this order. The data currently available do suggest, however, that workers in a number of industries are exposed to styrene concentrations that produce subjective complaints such as eye irritation and headache and evidence of neurotoxicity. This does not necessarily indicate that the

present TLV of 100 ppm is too high; comphance with the limit may be the problem. Styrene’s carcinogenic status in animals will presumably be resolved in the near future. Any evidence of a positive response in animals will need to be answered by adequate epidemiological data. If styrene does prove carcinogenic in animals, there is likely to be an immediate call for polystyrene food-packaging materials to contain “zero’* monomer, while even the suspicion of carcinogenicity may. provide enough impetus to lower the present TLV. [J. Hopkins-BIBRA]

GENETIC EFFECTS OF FORMALDEHYDE Formaldehyde is widely used in chemical syntheses and in the manufacture of textiles, papers and synthetic resins. It is also used in agricultural chemicals, particularly in insecticides, fungicides and disinfectants and under some circumstances is present at very low levels in certain foods. Formaldehyde has been shown to be a mutagen in Drosophila (Cited in F.C.T. 1966. 4, 99). but this effect has been detected only in male larvae whose early spermatocytes appear to be sensitive (Auerbach et al. Mutation Res. 1977, 39, 317). Moreover, it has been argued that, because formaldehyde is able to damage DNA in DNA polymerase-deficient strains of Escherichia co/i, it may have significant carcinogenic potential (Cited in F.C.T. 1973, 11,923). There is, however, no experimental evidence for this, carcinogenicity studies in mammals having produced, at most, only equivocal results, while indications of mutagenicity in bacteria and other simple organisms remain of very questionable relevance to man. Nevertheless, studies of the type of genetic damage induced in such organisms by formaldehyde are of considerable interest. Auerbach et al. (lot. cit.) considered in 1976 that the widespread use of formaldehyde and related compounds in industry called for a keen assessment of their potential hazard and produced an extensive review (citing 189 papers) of available data. In biological materials, the reaction of formaldehyde with amino groups is likely to be of prime importance. With amino acids and polypeptide chains, the first step in this reaction is the formation of unstable methyl01 derivatives, while the second produces stable condensation products with methylene cross-links. With nucleosides, nucleotides and nucleic acids a similar progression may occur, and it appears that with aminopurines the methylene bridges can actually link the purine groups. The primary lesions in the genetic effects of formaldehyde are likely to be the formation of cross-links between two amino acids in a protein. between two nucleic acid bases or between an amino acid and a nucleic acid base (Auerbach et

its reaction with glycine (Poverenny ef al. Mutation Res. 1975, 27, 123) cell survival was depressed to a similar degree by the two additions. However, both treatments resulted in a much lower survival of the mutant strain, deficient in excision-repair capacity, than in the wild strain, the mutant strain showing a large number of single-stranded breaks in DNA, which could be repaired in the wild-type cells. Spectrophotometric studies of the reaction between nucleotides and formaldehyde in the presence of varying concentrations of glycine showed that a threefold molar excessof the amino acid, such as was used in these incubations of the two compounds with E. coli, almost completely bound the formaldehyde present. This study indicated, therefore, that the action of formaldehyde on bacterial DNA was exerted not directly by formaldehyde itself but by the products of its reaction with amino-containing compounds, either the glycine added with it or free amino acids present in the bacterial cells. The possibility of excision repair of formaldehydeinduced lesions has also been demonstrated in strains of yeast (Saccharomyces cerevisiae), a eukaryotic organism (Chanet et al. ibid 1976, 35, 29). This group (idem, ibid 1975,33, 179) also showed that, in contrast to yeast cells exposed to radiations, cells in the stationary phase were more resistant to the action of formaldehyde than were exponentially growing cells. In synchronized populations. the lag and G, phases of the cell cycle were the most resistant to both the lethal effect and the recombinant-inducing action of formaldehyde, while the end of the GL phase and mitosis were the most sensitive stages. Discussing the possible reasons for this fluctuation in sensitivity to formaldehyde, the authors put forward the hypothesis that this may be related to the variations in the cellular pool of free amino acids. The nitrogen pool is known to reach a maximum roughly in mid-cycle, and this could yield a maximum level of formaldehyde-amino acid reaction products, resulting in more DNA damage and a higher level of cell inactivation al. lot. cit.). and recombination. In micro-organisms, formaldehyde-induced genetic More recent work on haploid strains of 5. cererieffects have been found to be subject to excision siae, including two ultraviolet-sensitive mutants, has repair, which is dependent on the activity of DNA provided further information on the lesions induced polymerase I. When wild strains of Escherichia co/i in DNA by formaldehyde (Magaiia-Schwencke et trl. and a strain deficient in this DNA polymerase were ibid 1978, 50, 181; Magafta-Schwencke & Ekert, ibid incubated with formaldehyde or with the product of 1978, 51, 11). The technique used for much of this