An investigation into sources of contamination of cattle with the veterinary
drug phenylbutazone
Steven Crooks,
Veterinary Sciences Division,
Agri-Food and Biosciences Institute,
Belfast, UK
What is Phenylbutazone?
• A non-steroidal anti-inflammatory drug (NSAID)
• Abbreviated to PBZ; often called “Bute”
• Introduced to human medicine in 1949 for the treatment of
rheumatoid and arthritic disorders
• Found to induce disorders of the blood including aplastic
anaemia, leukopenia, granulocytosis and thrombocytopenia, in
some cases leading to death
• Consequently, its use in human medicine became limited with
the licence for use in man revoked in the U.K. in 1984
N
N
O
O
CH3
Phenylbutazone ……………….
• The principal metabolites are oxyphenylbutazone and γ-hydroxyphenylbutazone
• They possess analgesic/anti-inflammatory properties and thus
contribute (probably small-moderate degree) to the
pharmacological actions of phenylbutazone
• For both metabolites, urine concentrations are much higher
than those in plasma
• Suxibuzone is a pro-drug of PBZ, designed to reduce gastro-
intestinal disturbances. Also banned in food producing animals
• Alternative NSAIDs are available which do not have the safety
issues associated with PBZ i.e. they are MRL compounds
Use in horses
• Choice of NSAID for equines since its introduction to veterinary
medicine in the 1950s
• Substantial clinical history of efficacy and safety in horses
accumulated over both short and long treatment periods
• Problem is that horse meat is consumed in many countries
• The CVMP assessed PBZ in 1997:
• Health risks are blood dyscrasias and the genotoxic & carcinogenic potential
• No thresholds identified so maximum residue limits could not be established
• Therefore PBZ is not permitted for use in any food producing animals
• Findings confirmed by EFSA and EMA 2013
So residues of phenylbutazone are a horse problem??
From 2005-2013, 1.6% of equines tested in EU were non-compliant
However………
EFSA report 2012 and 2013 indicate 0.1% non compliance in bovines in UK
So ……..it’s a horse and cow problem!!
In Northern Ireland, 2008 – present, 0.77% of bovines tested were non
compliant
Represents significant non compliance for an unauthorised drug
EFSA 2013 report highlights 41 suspect bovine samples non compliant in
Belgium/Germany
On farm investigation of two non compliant samples
Farm 1
• Plasma sample taken from single animal at slaughter
• PBZ confirmed by LC-MS/MS at 0.4 ng/ml
• Intentionally fed to bulls due to issues with lameness
• Animal sampled was a cow!
Farm 2
• Plasma sample taken from single animal at slaughter
• PBZ confirmed by LC-MS/MS at 0.32ng/ml
• No intentional misuse
• Horse on farm fed PBZ in diet – however housed separately
from cattle
• The dirty bucket theory ………….
Is it possible that the bovine problem could be largely due
to cross contamination from misuse or from horses?
Investigation of potential contamination sources
1. The dirty bucket – could sharing of feeding vessels provide a
bovine with sufficient PBZ to give rise to detectable residues
of the drug?
2. Treated animals – could detectable residues be due to
contamination from an animal (horse/cow) which had been
treated
3. Contaminated pasture – could pasture be sufficiently
contaminated to give rise to detectable PBZ concentrations
So how much is a detectable concentration of PBZ?
Plasma sample analysed (0.5ml)
Supported Liquid Extraction columns
Elution in hexane:dichloromethane
Evaporate to dryness & reconstitute
Analysis using UHPLC-MS/MS
CCα phenylbutazone 0.28 µg/L-1
Study 1 – The dirty bucket
• A bovine (T1) was treated with PBZ orally, via meal in a bucket
(Pro-Dynam® Oral Powder)
• Dosage regime was that recommended for treatment of a horse
(similar to that being utilized on the treated bulls)
• A second bovine (B1) was fed non-medicated meal from the
same bucket each time
Can bovine plasma contain detectable PBZ if fed from a contaminated bucket?
Day 1: 4.4 mg/kg bw twice (two sachets twice daily)
Days 2-4: 2.2 mg/kg bw twice (one sachet twice daily)
Day 5: 2.2 mg/kg bw (one sachet)
Study 2 – Treated animal contamination
• When treatment was complete, T1 was moved into a house
with three other steers (H1, H2 & H3)
Study 3 – Pasture contamination
• A second bovine (T2) was treated with PBZ in the same way as
T1
• After treatment T2 was moved onto a paddock to graze for
four days
• A group of steers (P1, P2 & P3) were allowed to graze on the
paddock for three days, after removal of T2
• A second group (E1, E2 & E3) were allowed to graze on new
grass in the same paddock 20 days after removal of P1-P3
The dirty bucket………..
Days after last feeding from
contaminated vessel
Plasma (B1)
(µg/L-1)
(pre-bleed) <0.28
6 990
9 758
17 42.94
20 22.44
25 8.20
28 16.65
31 12.70
39 3.44
46 0.62
49 <0.28
53 <0.28
62 <0.28
68 <0.28
77 <0.28
Contaminated feeding vessels present a significant risk of residues in an “untreated animal” for an extended period.
T1
B1
Risk by association with treated animals
T1
H1
H2 H3
T1
Days after T1 was introduced to
the pen
Containing H1-H3
Plasma
µg/L-1
H1 H2 H3
0 <0.28 <0.28 <0.281 0.39 2.65 0.412 0.97 2.85 0.823 1.39 2.67 1.474 1.39 2.22 1.655 1.44 2.43 2.126 1.55 2.11 1.937 1.41 1.83 1.519 0.71 0.85 0.96
11 0.41 0.57 0.6014 <0.28 0.29 0.3420 <0.28 <0.28 <0.2828 <0.28 <0.28 <0.28
Housing treated and non treated animals together after completion of medication may result in detectable PBZ residues
The Risk from Contaminated PastureT2
Graze for 4 days
Days after P1-P3 were introduced
to the grass paddock
Plasma
µg/L-1
P1 P2 P3
0 <0.28 <0.28 <0.281 8.98 0.29 <0.282 11.95 0.68 0.483 8.78 0.85 0.704 5.50 0.69 0.585 4.19 0.47 0.317 1.85 <0.28 <0.28
10 0.55 <0.28 <0.2816 <0.28 <0.28 <0.2824 <0.28 <0.28 <0.28
T2
P1 P2 P3
Graze for 4 days immediately after T2 was removed
Grazed 20 days after P1-P3 were removed
E1 E2 E3Days after E1-E3 introduction to
the grass paddock
Plasma
µg/L-1
E1 E2 E3
0 <0.28 <0.28 <0.285 0.73 1.36 0.677 1.10 1.69 1.05
Contaminated pasture may result in detectable PBZ residues
Is contaminated pasture really a risk……………??
• Studies unusual in that untreated animals very quickly follow treated ontopasture
• Study more closely mimicking normal farming practice
• 5 animals housed over the winter period
• Extended treatment with PBZ
• Manure stored as per normal
• Spread onto field mid February
• Field remained empty for 70 days
• Five animals allowed onto grass
Days after introduction
to field
Plasma
µg/L-1
W1 W2 W3 W4 W5
0 <0.28 <0.28 <0.28 <0.28 <0.285 2.54 5.32 4.24 6.46 2.428 3.42 5.21 9.91 10.34 4.85
12 4.46 3.68 10.67 7.28 4.6715 1.46 3.34 9.42 4.65 2.3319 2.7 4.27 4.99 3.52 3.3322 1.69 4.34 6.03 2.61 2.5927 1.81 4.26 6.37 3.02 2.60
Is contaminated pasture really a risk……………??
Normal farming practice may also give rise to PBZ residues
Is contaminated pasture really a risk……………??
• Trial stopped after 27 days due to lack of grass
• Field remained empty for 23 days to permit re-growth
• Introduced five new cattle
• Sampled at various intervals over 44 days
• Only one bovine sample showed detectable concentrations
• 0.29 µg L-1 PBZ
Contamination shown to be a risk…………………
• Studies clearly demonstrated the risks of contamination
• How much PBZ is required to provide a detectable concentration?
• Normal dose is ~ 2.2mg/kg bodyweight
• Four bovines given a single dose of PBZ
• Plasma sample taken 1 day after treatment
30ppb sufficient to give rise to detectable residues of PBZ
PBZ
incorporated in
1 kg of feed (µg)
PBZ Conc. in
plasma (µg/L-1)
Therapeutic 1,100,000 --------
C1 100,000 1279
C2 1000 13.80
C3 30 0.290
C4 20 <0.28
Equates to 1/36,666 of therapeutic dose!!
Conclusions
• Residues are a significant possibility as a result of contamination fromlegal use of PBZ
• PBZ is used therapeutically at high concentrations yet extremely smallamounts can give rise to detectable concentrations in plasma
• Environmental contamination is clearly an issue
• Is PBZ contamination surface contamination only?
• Difficult to limit contamination risk
• Alternative NSAIDs available for use
• Is the ongoing authorisation for use of PBZ in horses justified?
Thanks
• Colleagues from AFBI: Terence Fodey, Wesley Smyth, Paul Barnes, Imelda Traynor & Glenn Kennedy for their
assistance in this study
• Organising Committee and Scientific Committee for
providing the opportunity to present this work
• You for your attention
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