Steven Crooks Euro Res 2016 PBZ presentation FINAL

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

Contamination Investigation

Fodey, T. L., Smyth, W.G., Barnes, P., Traynor, I.M., Kennedy, D.G., Crooks, S.R.H. (2015)

Investigation into sources of contamination of cattle with phenylbutazone. Veterinary

Record 176(3) 74-76