Information section--Fd

The urine and faeces of two SPF rats of each sex given 5 g NP/kg by gavage and kept in individual metabolism cages were collected for 4 days. One male and one female were killed and the other two kept for a further 10-day observation period. Again no deaths or toxic effects were reported. Examination of tissue samples from all four rats revealed no differences between treated and untreated animals. Except for the urine of one female rat, no free NP or AP was detected in dichloromethane extracts of the urine during the first 4 days after treatment. However, small amounts of AP were released when the urine samples of all rats were treated with glucuronidase or sulphatase indicating the presence of N-conjugates. Analysis of urine collected by manually restraining rats over a receptacle while they urinated confirmed that this finding was not related to faecal con- tamination of the samples. Quantitative data derived from the urine of one male rat revealed that only a small fraction of the dose, 4-10 #g/daily sample, was present in conjugated form. Faecal extracts (meth- anol) contained both NP and AP, determined by thin-layer chromatography. Quantitative analysis of the faeces from the same male rat showed that more than 70% of the dose was eliminated as unchanged

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NP during the first 4 days after treatment, 80% of this being excreted in the first 24 hr. Free AP, equivalent to about 1% of the original dose, was excreted in the faeces on each of the first 2 days after dosing. This was thought to result from NP reduction by gut microflora. Excretion of methanol-soluble metabo- lites in lhe faeces was essentially complete within 48 hr. A total of 28% of the dose was not accounted for and may have been excreted in conjugated or bound forms in the faeces and/or as undetected metabolites in the urine.

Although the mutagenicity and genotoxicity of NP were confirmed, the results of these studies suggest that the potential hazard of this compound may not be as high as predicted from bacterial assays. The acute oral toxicity appeared to be low which may have been related to low absorption. Nevertheless, the presence of N-conjugates in the urine indicates that at least some of the dose was absorbed. Further metabolism of NP, probably by gut micro-organisms, produced small quantities of the mutagenic reduction product AP. The fate of about one third of the dose remains a mystery. Clarification of the carcinogenic risk and metabolic fate of NP is required.

D I N I T R O T O L U E N E G E N O T O X I C I T Y A N D T H E G U T F L O R A

Dinitrotoluene (DNT) is an important inter- mediate in the production of polyurethanes and is also used in the manufacture of dyes. Technical grade DNT is a mixture composed predominantly of 2,4- and 2,6-DNT with lesser amounts of other isomers. Recent work at CIIT ( C H T Activities 1981, 1 (66), 5) on DNT isomers attempted to explain why male rats are more sensitive than females to DNT's hepato- carcinogenic effect. The CIIT studies showed that absorbed 2,4- and 2,6-DNT are converted in the liver to the corresponding dinitrobenzyl alcohol glucuron- ide, which in the male is preferentially excreted in the bile, but in the female is preferentially returned to the general circulation and excreted in the urine. From the bile, the glucuronide reaches the small intestine where it is further metabolized with the help of the gut microflora to metabolites that are reabsorbed and that may bind to hepatic macromolecules.

Various in vitro and in vivo short-term tests using different genotoxic endpoints have produced equi- vocal results (Wai Chiu et al. Mutation Res. 1978, 58, 11; Couch et al. ibid 1981, 90, 373; Abernethy & Couch, ibid 1982, 103, 53; Chemical Industry Institute o f Toxicology I981 Annual Report and Scientific Review: Science in the Public Interest). For example, various DNT isomers were shown not to induce unscheduled DNA synthesis (UDS) in primary male rat hepatocytes in vitro (Bermudez et al. Envir. Mu- tagen. 1979, 1, 391). However, using the in vivo-in

vitro hepatocyte DNA-repair assay (which measures chemically-induced UDS in hepatocytes isolated from rats treated by gavage), DNT was found to be a potent UDS inducer in male rats (Mirsalis & Butterworth, Carcinogenesis 1982, 3, 241). A possible explanation for the discrepancy between the in vivo and in vitro findings, which ties in with the results on hepatocarcinogenicity, is that metabolism by the gut flora may be required for the formation of genotoxic metabolites from DNT.

Marsalis et al. (Nature, Lond. 1982, 295, 322) have investigated this possibility by comparing the extent of DNT-induced DNA repair in male rats having the normal complement of gut flora with repair in rats having no gut flora. The rats were given 100rag sterile DNT/kg body weight by gavage in sterile corn oil. Primary hepatocyte cultures were prepared after 12hr and cultured in the presence of 3H-thymidine and UDS was subsequently measured by quantitative autoradiography. Rats which had the normal com- plement of gut flora showed a marked increase in UDS after DNT treatment, whereas axenic rats showed no such increase in UDS. The use of N- nitrosodimethylamine as a positive control confirmed that the axenic animals could carry out DNA repair after exposure to a genotoxic agent. Thus, the study clearly suggests that DNT must be metabolized by intestinal flora in order to become genotoxic in the rat.