The fate of 2-chlorobenzylidene malononitrile (CS) in rats

1. The fate of 3H-ring labelled, 14C-cyanide labelled and (14C = C) side chain labelled 2-chlorobenzylidene malononitrile (CS), 2-chlorobenzyl alcohol and 2-chlorobenzyl malononitrile (dihydro CS) in rats and isolated rat liver cells has been examined. 2. CS was administered both i.v. and i.g. to rats at doses from 0.08 to 159 mumol/kg and in most cases the greatest proportion of the dose was eliminated in the urine (44-100%). The principal urinary metabolites were 2-chlorohippuric acid, 1-O-(2-chlorobenzyl) glucuronic acid, 2-chlorobenzyl cysteine and 2-chlorobenzoic acid. Lesser amounts of 2-chlorophenyl acetyl glycine, 2-chlorobenzyl alcohol and 2-chlorophenyl 2-cyano propionate were identified. 3. The major urinary metabolite from 2-chlorobenzyl alcohol was 2-chlorohippuric acid (43%), 2-chlorobenzyl cysteine, 2-chlorobenzoic acid and 2-chlorobenzyl glucuronic acid were identified. 4. The products of dihydro-CS metabolism were 2-chlorophenyl acetyl glycine, 2-chlorophenyl 2-cyanopropionate and 2-chlorophenyl proprionamide. 5. Urinary thiocyanate levels increased with the dose of CS up to 159 mumol/kg. At 212 mumol/kg there was a large increase in the amount of thiocyanate produced (molar conversion: 21.5-29.9%). Similarly malononitrile, the hydrolysis product of CS, gave a dose related increase in urinary thiocyanate levels. However at a higher dose (212 mumol/kg) the molar conversion was greater than 60%. 6. The metabolism of CS by isolated rat liver cells confirmed the results in vivo but demonstrated a marked limitation of this preparation to form conjugates. 7. It is concluded that CS in vivo is hydrolysed mainly to 2-chlorobenzaldehyde which is then either oxidized to 2-chlorobenzoic acid for subsequent glycine conjugation, or reduced to 2-chlorobenzyl alcohol for ultimate excretion as 2-chlorobenzyl acetyl cysteine or 1-O-(2-chlorobenzyl) glucuronic acid. Malononitrile is converted to thiocyanate via the formation of cyanide.