V. Naidoo and others Toxicity of ketoprofen to Gyps vultures

Ketoprofen Toxicity - Supplementary Information 2

Estimating MLE and analysis of cattle tissue residues and blood parameters

The estimation of Maximum Levels of Exposure (MLE) was based upon the methods outlined in Appendix II of Swan et al. (2006) estimated for a 4.75 kg Oriental white-backed vulture (G. bengalensis) consuming a 1.023 kg meal (sufficient to provide a birds’ energetic requirements for three days; Swan et al 2006) of kidney tissues from cattle slaughtered 1 hour after a three-day course of ketoprofen. The choice of slaughter at 1 hour was based on the rapid elimination of ketoprofen in cattle (plasma half-life of 0.42 to 0.49 hours; De Graves et al 1996; Landoli et al 1995) and information on ketoprofen tissue residues indicating highest levels were in kidney tissues (EMEA 1995). Following the protocol of Swan et al (2006) and Swarup et al (2007) for meloxicam, ketoprofen was administered to cattle at 6.0 mg kg-1, which is twice the normal veterinary dose of 3.0 mg kg-1. Higher than recommended doses were administered as the analysis of livestock carcass samples available to vultures in India indicates that animals are sometimes administered doses of NSAIDs far in excess of the recommended guidelines (Taggart et al 2007). Five cattle (Bos taurus) were dosed by attending veterinarians at the Faculty of Veterinary Science, University of Pretoria and were slaughtered 1 hour after the final injection at the Veterinary Pathology Department by means of captive bolt to the brain and subsequent pithing. After slaughter, three samples of each kidney and two of liver, each composed of two sub-samples from different areas of the organ (e.g. 12 (since 2 kidneys) and 4 samples in total respectively) were collected from each animal and placed in sealed glass vials and frozen at -20 C. Frozen tissue samples were couriered on dry ice to the University of Aberdeen and remained frozen throughout transportation. Analysis of ketoprofen residues was initially undertaken by LC-MS at the University of Pretoria Medical School. Samples were homogenised and extracted in to acetonitrile and analysed following a standard methodology. The estimated residue levels from this analysis resulted in an estimated MLE of 5.11 mg kg-1 and a dose of 5.0 mg kg-1 was therefore administered in Phase 3. A subsequent analysis of ketoprofen residues in cattle tissues was undertaken using a validated LC-ESI/MS methodology (Taggart et al., 2009). Samples of tissue (0.5g) were weighed to an accuracy of ± 0.001g, homogenised for 45 seconds with 2ml of HPLC grade acetonitrile, and then centrifuged at 1000 g for five minutes. Quantification was undertaken at m/z of 253.1 in negative ion mode (with confirmation at m/z of 197.1) following compound separation on an Agilent ZORBAX 300SB-C18 column (4.6mm x 150mm, 5μm). Limits of quantification were equivalent to 12 ug kg-1 in liver tissue (see Taggart et al. 2009 for a full description of the methodology).Repeat analysis of additional tissue samples and known dilutions of ketoprofen dosing solutions (analysed blind) at both Aberdeen and Pretoria revealed that the initial estimation of residue levels at Pretoria was erroneous and too high. Based on the re-analysis of 30 kidney samples and 20 liver samples (6 kidney and 4 replicate liver samples per cow) the LC-ESI/MS analysis indicated that 1 hour after the final dose average tissue concentrations were 7.13 mg kg-1 in kidney tissues and 0.16 mg kg-1in liver tissues. Based on these validated results the correct MLE was 1.54 mg kg-1 and a dose of 1.5 mg kg-1 was administered in Phase 4.

Plasma levels of blood parameters were analysed with standard laboratory procedures. Uric acid concentration was measured using an ACETMUric Acid reagent, albumin concentration using the NExTTMAlbumin reagent, ALT activity using the Alfa Wasserman ALT reagent, and CK using the Alfa Wasserman CK reagent e ACETMclinical chemistry system (Alfa Wassermann, Bayer Health). The analyses were performed using the ACETMand NExTTM Clinical Chemistry Systems (Alfa Wassermann, Bayer Health Care, SA). The electrolytes sodium (Na+),potassium (K+) and calcium (Ca2+) were measured with a blood gas analyser (Rapidlab 34E Chiron diagnostics, Bayer SA).

De Graves, F.J., Riddell M.G. & Schumacher J. 1996 Ketoprofen concentrations in plasma and milk after intravenous administration in dairy cattle. American Journal of Veterinary Research 57, 1031-1033.

EMEA 1995 Committee for Veterinary Medicinal Products: Ketoprofen Summary Report. EMEA/MRL/020/95. Accessed online at: /002095en.pdf

Landoli, M.F., Cunningham, F.M. & Lees, P. 1995 Pharmacokinetics and pharmacodynamics of ketoprofen in calves applying PK/PD modelling. J. Vet. Pharm. Thera. 18, 315-324.

Swan, G., Naidoo, V., Cuthbert, R., Green, R., Pain, D., Swarup, D., Prakash, V., Taggart, M.A., Bekker, L., Das, D., Diekmann, J., Diekmann, M., Killian, E., Meharg, Patel, R., Mohini, S. & Wolter, K. 2006 Removing the Threat of Diclofenac to Critically Endangered Asian Vultures. PLoS Biology4, 395-402.

Taggart, M.A., Senacha, K., Green, R.E., Cuthbert, R., Pain, D.J. Randagan, B., Jhala, Y., Rahmani, A., Meharg, A.A. 2007 Diclofenac residues in carcasses of domestic ungulates available to vultures in India. Environment International 33, 759-765.

Taggart, M.A., Senacha, K., Green, R.E., Cuthbert, R., Jhala, Y., Rahmani, A., Meharg, A.A. Mateo, R. & Pain, D.J. 2009 Analysis of nine NSAIDs in ungulate tissues available to Critically Endangered vultures in India. Environ. Sci. Technol.In press