Articles of general interest--Fd Cosmer. Toxicol. Vol. 18. no. 4 435

arises from the widely different routes, levels and dur- ations of exposure used.

Styrene did not increase the mutation rate of Sal- monella ryphimurium strain TA1535 at concentrations ranging from I x 10e3 to IO~mol/plate either with or without S-9 mix (Loprieno er al. lot. cit.). Survival ranged from 0.8 to 100°? under these conditions. Sty- rene oxide was mutagenic both with and without the S-9 mix at levels of l-100 pmol/plate and the response was dose-related except at the highest dose-level which was extremely toxic. These results support those of Stoltz et al. (Bull. enuir. Contam. Toxicol. 1977. 17, 739; Cired in F.C.T. 1978, 16, 397) but con- tradict those of De Meester et al. (Mutation Res. 1977, 56, 147; Cited in F.C.T. 1979, 17, 298) who found that styrene itself was mutagenic towards this strain when a metabolic activating system was used.

In cultured Chinese hamster bone-marrow cells there was no increase in the mean number of sister- chromatid exchanges in any of the styrene oxide inha- lation exposure groups used by Norppa et al. (lot. cit.). However the group given 500 mg styrene oxide/kg by ip injection showed a marginally signifi- cant (P < 0.05) increase in the number of sister- chromatid exchanges, but the increase was only seen in one out of six animals. Styrene oxide was pre- viously shown to be a potent inducer of sister-

chromatid exchanges in oitro in Chinese hamster ovary cells (Cited in F.C.T. 1979, 17, 298).

Fabry et al. (lot. cit.) found no evidence of muta- genicity when polychromatic erythrocytes from 8-wk-old mice were examined 30 hr after a single ip injection of 250 mg-styrene oxide/kg. They also obtained negative results of the meiotic chromosomes of treated male mice and in dominant lethal tests per- formed 1-3 wk after the injection. DNA repair in cul- tured human (EUE) cells was stimulated by styrene oxide at a level of 4.4 mM, but styrene did not increase DNA repair even in the presence of a metabolic acti- vation system (Loprieno et al. lot. cit.). These authors also found that styrene oxide, but not styrene, pro- duced point mutations in the Chinese hamster cell line V-79. They consider that the lack of mutagenic activity of styrene itself may be due to rapid conver- sion of its active metabolite. styrene oxide, to phenyl- ethylene glycol. The latter conversion, which is acti- vated by microsomal epoxide hydratase, occurs at a faster rate than that of styrene to styrene oxide. More- over, it has been shown (Cited in F.C.T. 1978, 16, 300) that epoxide-hydratase activity is markedly increased by the treatment of rats with styrene but is largely unaffected by similar treatment with styrene oxide.

[P. Cooper-BIBRA]

THE VARYING FATE OF VINYLIDENE CHLORIDE

Vinylidene chloride (l,l-dichloroethylene; VDC) is used as a monomer in plastics manufacture, particu- larly for the production of wrapping films. Acute inhalation of VDC has been shown to decrease gluta- thione stores in the rat ,liver (Jaeger er al. Expl mol. Path. 1974, 20, 187) and inhalation of 25 ppm VDC for 52wk has been associated with the development of malignant tumours of the kidney in male mice, although 200 ppm had no such effect in rats (Maltoni et al. Medna Lau. 1977,&3,241). Several recent studies of VDC have highlighted the ways in which its metab- olism differs in mice and rats as well as the effects of inadequate food intake and of various routes of ad- ministration on its fate in rats. In other work, the species differences have been reflected in the results of short-term mutagenecity tests on VDC.

In the study by Jaeger er al. (lot. cit.). depletion of hepatic glutathione stores by VDC and the ac- companying hepatotoxicity were found to be exacer- bated by a period of fasting prior to VDC exposure. Later McKenna rt al. (Toxic. appl. Pharmac. 1978. 45, 599 & 821) studied the pharmacokinetics of VDC ad- ministered by inhalation or orally to both fed and fasted rats. In the inhalation study (p. 599). groups of male rats fed normally or fasted for 18 hr before treat- ment were exposed to 10 or 200 ppm [14C]VDC vapour for 6 hr. and the elimination of 14C activity was studied for the following 72 hr.

After exposure to IOppm, about 98”~; of the ab- sorbed VDC was converted to non-volatile metab- elites both in normal and fasted rats. However. after exposure to 200ppm. the proportion of VDC metabo-

lized was significantly lower and showed a significant difference between the fed and fasted rats, at 96 and 92% respectively of the total amount absorbed. There was also a significant difference between the total amounts absorbed by the fed and by the fasted rats, the total body burdens in the fasted rats being lower at both exposure levels. Despite the markedly higher pulmonary elimination of unchanged [‘4C]VDC by fasted rats exposed to 2OOppm, compared with rats exposed to 10 ppm, the former also showed a higher level of retention of 14C activity. Retention in the fed rats exposed to 200 ppm, however, differed little from that in the IO-ppm groups. The main sites of VDC retention in all the groups were the liver, kidneys and lungs. Liver and kidney lesions developed after 200-ppm exposure of the fasted but not the fed rats. No such effect resulted in any rats treated with IOppm VDC. The affected livers showed centrilobu- lar necrosis and the kidneys marked degeneration of the proximal tubular epithelium. The liver lesions were associated with a higher level of covalently bound 14C activity than was seen in fed rats and the levels in the latter were in turn considerably higher than those in normally fed rats exposed to IOppm VDC.

Similar results were obtained in rats given an oral dose of 1 or 50mg [14C]VDC/kg (McKenna er al. lot. cit. p. 82 I ). In normally fed rats the proportions of the lower and higher dose exhaled unchanged were 1-3 and 19”:,. respectively, and the metabolism of the larger dose was further reduced (29’4 being exhaled unchanged) in rats fasted for 18 hr before treatment.

436 Articles of general interest-F‘d C’osrner. T&co/. Vol. 18, no. 4

Elimination of non-volatile metabolites was some- what greater in the fed than in the fasting rats given the higher dose and, again aa was found in the rats inhaling VDC vapour, fasting increased the concen- tration of covalently-bound 14C activity in the liver after ingestion of 50 mg [ ‘%Y] VDC/kg.

In both studies, two of the major metabolites of VDC in the urine were identified as N-acetyl-S-(2- hydroxyethyl)cysteine and thiodiglycolhc acid. Conju- gation with liver glutathione therefore seems to be a major pathway for the detoxication of VDC in the rat, but the evidence points also to some metabolism of VDC to a more reactive compound which binds to subcellular components. It seems possible that when a high dose of VDC saturates the detoxifying capacity of liver glutathione stores depleted by fasting the covalent binding of VDC metabolite(s) to tissue con- stituents is enhanced, with the resulting development of overt toxicity.

A comparison of the patterns of VDC elimination in rats following administration of similar doses by different routes was made by Jones & Hathway (Chemico-Biol. Interactions 1978, 20, 27). Male rats were given [‘4C]VDC in single doses of 0.5 or 350mg/kg by the intragastric or ip route and of 05 mg/kg iv, and 14C activity was measured in the urine, faeces and exhaled air over 72 hr. The change in excretion patterns with dose was clearly demonstrated by the use of these widely differing doses. At the higher level, a high proportion of the administered 14C (68 and 92% after intragastric and ip dosing. re- spectively) was eliminated via the lungs, mainly as unchanged VDC (67 and 91% of the dose), with uri- nary excretion in these groups accounting for 30 and 8% respectively. In contrast, the lower dose was excreted mainly in the urine, which accounted for 80% of the intragastric and 69”/, of the ip dose. At this level, about 5% of the intragastric dose was exhaled as CO2 and less than 1% as unchanged VDC. with about 8% in the faeces, while after ip dosing some 4% was exhaled as CO*, 12”/, was unmetabolized and about 16% was in the faeces.

The authors relate these findings not only to the possible saturation of the metabolizing capacity but also to an efficient arterial-alveolar transfer, which rapidly reduces the concentration of unchanged VDC in systemic blood, thus leaving little available for transformation in successive passes through the liver. Support for this contention comes from their finding that more than 60% of the small iv dose was exhaled unchanged within 5 min of injection and 80% within 60min. The change in pulmonary excretion of unchanged VDC over a range of intragastric doses between 0.5 and 650mg/kg suggested a rapid release of VDC which had not been absorbed by the tissues or which exceeded the saturation of the body pool and a subsequent slower release from the tissues (probably adipose tissue and liver). Metabolites appeared in the urine over 3 days and this relatively slow rate of excretion was consistent with the view that at least part of the urinary radioactivity had been transported in the bile. Like McKenna et a/. (lot. cit. p. 599). Jones & Hathway (lot. cit.) identified thiodi- glycollic acid as the ultimate detoxication product of VDC. Another major product was an N-acetyl-S- cysteinyl acetyl derivative, and they also demon-

strated substantial amounts of chloroacetic acid, di- thioglycollic acid and thioglycollic acid. It is probable that chloroacetic acid, which may be derived from VDC by migration of one chlorine atom and loss of the other, is on the main metabolic pathway for VDC, since the two compounds have several metabolites in common. The VDC-derived CO2 may be the product of a minor oxidative pathway for chloroacetic acid or possibly of the action of epoxide hydratase on l.l-dichloroethylene oxide. The reaction of l.l-dichloroethylene oxide with glutathione S-epoxide transferase probably accounts for the N-acetyl-S- cysteinyl acetyl derivative.

These authors also demonstrated some differences in the metabolism of VDC in rats and mice (Jones & Hathway, Br. J. Cancer 1978, 37, 411). After an oral dose of 50mg [‘4C]VDC/kg mice and rats, respect- ively, excreted 6 and 287, of the dose as unchanged VDC in exhaled air, and 3 and 22% as thiodiglycollic acid, 23 and Sq/, as dithioglycollic acid and 50 and 280/; as the N-actyl-S-cysteinyl acetyl derivative in the urine. Mice excreted no chloroacetic acid and rats no N-acetyl-S-(2-carboxymethyI)cysteine. At this dose level, mice metabolized over 20% more of the VDC dose than did rats. These findings are in accordance with known differences in certain enzyme activities, in particular the higher levels of P-450 activity that have been demonstrated in mice, as well as with the differ- ence in the toxic effects of VDC in the two species. Not only is VDC more readily metabolized in mice but it also shows a much higher degree of acute tox- icity in that species and. as previously mentioned. has induced kidney tumours in mice but not in rats. The authors of this paper suggest that the murine carcino- genicity of VDC may reflect the greater availability of the reactive metabolite 1,1-dichloroethylene oxide and its rearrangement product chloroacetyl chloride, re- sultingfrom therelativelyhighlevelofcytochromeP-450 activity, as well as a greater likelihood that these products will react with DNA than is the case in the rat.

Some support for this contention was obtained by Jones & Hathway (Cancer LRrr. 1978, 5, 1) from a short-term mutagenicity study. Exposure of Salrm- nella typhimuriurn strain TA 1535 to VDC vapour indi- cated that VDC was weakly mutagenic in the pres- ence of a post-mitochondrial supernatant (S-9 mix) from normal mouse kidney or liver and was strongly mutagenic in the presence of S-9 mix from Aroclor 1254-induced murine organs. With S-9 mixes derived from rats, however, only the liver preparation from Aroclor-induced animals was associated with a posi- tive bacterial response to VDC, and this was rela- tively weak. VDC showed no significant mutagenic effect in the presence of kidney or liver preparations from rats that had not been pretreated with Aroclor. Similar results were obtained with S. ryphirnurium strain TAJOO. Mention is made in this paper of some very limited data from Ames tests in which S-9 mixes derived from human or marmoset livers were used. These suggested that primates may respond more like rats than mice as far as the generation of alkylating metabolites of VDC and their reaction with bacterial DNA are concerned, but clearly a lot more work will be required before any conclusion on that aspect can be drawn.

[P. Cooper-BIBRA]