Drug safety research is frequently faced with the challenge of the selection of appropriate vehicles for use in in vivo non-clinical safety assessment studies. Reported here are the results of blend Labrasol, Labrafil and Transcutol, [L/L/T, (4/4/2, v/v/v)], excipients used as bioavailability enhancer and solubilizer for poorly water-soluble compounds and tested daily for 4 weeks by oral route in Wistar rats (10/sex/group) at dose volumes of 5, 10 or 20 mL/kg/day and compared to controls given 20 mL/kg/day of 1% (w/v) hydroxyethylcellulose in purified water. L/L/T was broadly well tolerated at 5 mL/kg/day and lethal at 20 mL/kg/day in 1 of 20 rats treated at this level. Changes in appearance and behaviour were observed from 10 mL/kg/day with volume-related incidence, severity and duration. Reduced feed intake observed from 5 (females) or 10 mL/kg/day (males) resulted in low bodyweights for high volume males only (-11% of controls). There was a volume-related induction of hepatic CYP 1A1/2, 2B1/2 and/or 2E1 subfamilies from 5 mL/kg/day, with high liver weight, centrilobular hepatocellular hypertrophy and high ALT, triglyceride and cholesterol serum values at 20 mL/kg/day. Renal tubular dilation in medulla, cortical cell degeneration/necrosis with granular material in adjacent glomerular spaces, crystal deposits in the inner medulla, papilla and/or renal pelvis, and tubular mineralization, associated with proteinuria and calcium oxalate crystalluria, were observed at 20 mL/kg/day as well as vacuolation in the adrenal cortex, with a sex-dependant localization. According to these results, 5 mL/kg/day was considered as an acceptable volume for further use of L/L/T (4/4/2, v/v/v) blend as a vehicle for poorly water soluble drugs in Wistar rat toxicity studies.