Mutagenic rubber accelerators

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Information section--Fd Chem. Toxic. Vol. 22, No. 4

aldehyde. A high proportion (50%) of deaths in the study group occurred before age 65. This was probably because the subjects were identified from files in the local government department concerned with licensing and data for embalmers who died after retiring were likely to be incomplete. Finally, the actual periods of exposure to formaldehyde were unknown. After 1947 the Bureau issued one licence which covered both embalming and funeral directing, and so among the group of persons licensed after this

date the degree of exposure to formaldehyde is likely to have been even more variable. These two epidemiological studies seem to cast further doubt on the degree to which the findings in the CIIT experiments in rats ( C I T T Activities 1982, 2 (3), 1) may be relevant to man. However, both reports do raise some questions that may be answered if the further studies called for by both Marsh (Ioe. cir.) and Walrath & Fraumeni (lot. cir.) are indeed carried out.

GUT FEELINGS ON ACRYLONITRILE Acrylonitrile (ACN) is widely used in the manufacture of synthetic fibers, plastics and rubbers, and is also an important intermediate in the chemical industry. Recent interest in ACN's toxicological profile has tended to centre on its carcinogenic potential (Food Chemical News 1981, 22 (45), 19: ibid 1981, 22 (52), 39; ibid 1982, 24 (18), 3; Cited in £ C . T . 1982, 20, 488). However, Ghanayem & Ahmed (Toxic. appl. Pharmac. 1983, 68, 290) have recently explored a different aspect of ACN's biological activity--its ability to induce gastro-intestinal (GI) haemorrhage. Ghanayem & Ahmed (lot. tit.) report that GI haemorrhage was induced in rats after administration of 50 mg ACN/kg body weight, either orally or by sc injection. The total amount of G1 blood loss did not vary according to the route of ACN administration, suggesting that the bleeding was not a result of a direct irritant action on the Gl tract. A time-course study revealed that the amount of blood recovered from the stomach contents was significantly higher than control values at 1, 2 and 3 hr after administration of 50 mg ACN/kg (by sc injection). However, the amount of blood recovered from the intestinal contents did not become significantly higher than control values until after 2 hr. This time discrepancy could indicate that the bleeding occurred primarily in the stomach and the blood sequentially passed to the intestine, or that the intestinal bleeding was simply delayed compared with that in the stomach. The authors were able to identify a dose-response relationship for the effect: 30 mg/kg injected sc did not induce GI bleeding whereas 40 70 mg/kg produced a statistically significant response, which was maximal at 50 mg/kg. In an attempt to determine the primary hae-

morrhagic agent, different metabolic modulators were administered to the rats prior to sc injection of 50 mg ACN/kg and G1 bleeding was measured 3 hr later. Pretreatment of the rats with sodium phenobarbital or Aroclor 1254, both inducers of cytochrome P-450, decreased total GI bleeding in the former case and increased it in the latter. However, pretreatment with either of two cytochrome P-450 inhibitors (cobalt chloride or SKF 525A) decreased GI bleeding. Glutathione depletion (induced by pretreatment with diethyl maleate) had no significant effect on ACN-induced GI blood loss. Finally, as it had been previously shown that ACN is metabolized to cyanide (Gut et al. Archs Toxicol. 1975, 33, 151: Kopecky et al. ibid 1980, Suppl. 4, 322), the effects of a sublethal sc dose of potassium cyanide (6 mg/kg) were examined. The cyanide did not exert any haemorrhagic action on the gut. The authors conclude that a prerequisite for ACN's induction of GI haemorrhage is its activation by cytochrome P-450-dependent enzymes to a reactive metabolite other than inorganic cyanide. They also postulate that the differing effects of sodium phenobarbital and Aroclor 1254 pretreatments were due either to these compounds" differing specificities in inducing particular forms of cytochrome P-450, and/or to sodium phenobarbital's ability to influence neuronal activity, which could interfere with ACN's haemorrhagic actions. A third possibility is that sodium phenobarbital may be competing with ACN for metabolism by cytochrome P-450-dependent enzymes. The epoxide 2-cyanoethylene oxide, a proposed cytochrome P-450-mediated metabolite of ACN (Ghanayem & Ahmed, Archs Toxicol. 1982, 50, 175), is suggested as the possible primary haemorrhagic agent.

MUTAGENIC RUBBER ACCELERATORS Bladder cancer mortalities in the British rubber industry were once unusually frequent, but since the withdrawal from use of recognized bladder carcinogens (principally /~-naphthylamine) in 1949, it appears that bladder cancer is no longer the occupational hazard it once was. Now, it is the

unusually high incidence of lung and stomach cancers that concerns those in the rubber industry (Rubber: Health and Safety 1976 80, Health & Safety Executive, HMSO London, 1981; Parkes et al. Br. J. ind. Med. 1982, 39, 209; I A R C Monographs on the Evaluation o f the Carcinogenic Risk 0 / Chemicals to

Information section--Fd Chem. Toxic. Vol. 22, No. 4 1982, 28, 227).). Clearly, this concern should not only be focused on measures in the workplace designed to reduce exposure to potentially harmful dusts and fumes, but also be directed towards identifying the chemicals that may be responsible for the high rate of the cancers. Accelerators, one of the many classes of chemicals involved in rubber manufacture, are highly reactive compounds used to increase the rate and efficiency of vulcanization. Hedenstedt et al. (Mutation Res. 1979, 68, 313) found that various thiuram and dithiocarbamate accelerators were mutagenic in Salntonella o,phimurium strains TA1535 and TA100. Now, a report has been published on the mutagenic evaluation of some sulphenamide and disulphide accelerators in a battery of in vitro tests (Hinderer et al. Ent, ir. Mutagen. 1983, 5, 193) which may provide some useful guidance in the epic task of trying to locate carcinogenic agents among the multitudinous reagents and by-products involved in rubber manufacture. Four widely used rubber accelerators, N-oxydiethylene thiocarbamyl-N-oxydiethylene sulphenamide (OTOS; a purified sample and two commercial samples, OTOS-I and OTOS-2), N-oxydiethylene-2benzothiazole sulphenamide (OBTS), 2-mercaptobenzothiazole disulphide (MBTS) and N-butyl-2benzothiazole sulphenamide (BBTS), were tested. The tests used were Ames assays in S. typhimurium TA1535, TA1537, TA1538, TA98 and TAI00, Escherichia coli plate assays in E. coli WP2 uvrA , DNA repair assays (plate and suspension) in E. coli W3110 (pol A +) and P3078 (pol A ), mouse lymphoma assays with the L5178Y TK+/ cell line, cell transformation assays in BALB/3T3 mouse cells and chromosome aberration assays in Chinese hamster ovary cells. All of the tests were performed with and without metabolic activation by a rat-liver S-9 mix, except for the cell transformation assay, in which no S-9 was used. None of the accelerators showed a positive mutagenic effect in any S. typhimurium strains or in E. coli WP2 uvr A . Purified OTOS and OTOS-2 (which contained 0.44 ppm N-nitrosomorpholine, a known animal carcinogen) produced zones of inhibition in the repair-deficient E. coli strain in the DNA repair plate assay (with or without activation) but OBTS, MBTS and BBTS did not produce any inhibition in either strain (OTOS-1 not tested). This suggested that low solubility might be responsible for the generally negative results in this assay and perhaps in the two bacterial reverse-mutation assays also. To investigate this, OBTS only was tested in the DNA repair suspension assay and was found to be mutagenic: the survival index (a measure of the preferential killing of the repair-deficient strain) decreased with increasing OBTS dose, both with and without activation. This Humans,

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supports the suggestion that poor solubility was responsible for the lack of activity in the plate assay, though it might have been even more convincing had MBTS and BBTS also been re-tested in the suspension assay. Solubility or cell-wall permeability may have been a factor in the Ames and E. coli WP2 uvrA tests, and the authors make it clear that studies with modifications of these tests are necessary to determine whether the lack of response in them was due to an experimental artefact or whether it was a "true indication of the absence ofmutagenic activity". In the mouse lymphoma assay, all of the accelerators were mutagenic with S-9 activation, but only OBTS and purified OTOS were mutagenic without S-9. However, mutagenic activity was generally seen only at doses that were considerably toxic to the cells. In the tests with S-9, MBTS was mutagenic with the least degree of cell toxicity, but the relative mutagenic potency of the accelerators followed the order: OTOS (purified) > OTOS-2 > MBTS > OBTS > BBTS. The authors believe that because purified OTOS was mutagenic without S-9, while the commercial samples were not, purification of OTOS may remove cytotoxic components, permitting exposure to higher, mutagenic amounts of OTOS. In the cell transformation assay, all of the accelerators except M BTS and OTOS-2 were active. Although quantitalive comparisons are difficult for this assay, the authors determined that the relative transformation responses followed the order: OTOS (purified) > OTOS-1 > OBTS > BBTS. In the chromosome aberration assays, only OTOS-2 (without S-9) and purified OTOS (with or without S-9) were unequivocally mutagenic. The main aberrations were simple chromatid and chromosome breaks, though quadriradials, triradials and ring chromosomes were also seen. Although OTOS-2 produced a higher frequency of aberrations than did the purified OTOS (suggesting interference from genotoxic contaminants in the commercial sample), the point is made that OTOS remained clastogenic after about half of the contaminants (by weight) were removed. OBTS and MBTS showed no significant effects in this assay, and although one dose of BBTS (with S-9) produced a significant increase in chromosome damage, the aberration frequency was not significantly higher than historical control levels and the pattern of responses to BBTS was not dose related. So, these four rubber accelerators have been shown to be mutagenic to varying degrees in various #~ vitro tests. Although the results in the bacterial reversemutation tests were uniformly negative, the results in the assay for point mutation in a mammalian cell system (the mouse lymphoma cell line) were uniformly positive in the presence of metabolic activation. These rubber accelerators would seem to be worthy of further investigation.

T H R E S H O L D DOSE FOR C A R C I N O G E N S ? Is there or is there not a threshold dose for a carcinogen that directly attacks DNA? Although the theory says there is not--any dose, no matter how

small, will be associated with some increase in risk-the toxicologists, with all their megamouse ambition, find it difficult to generate the necessary convincing