Relationships between urea dilution measurements and body weight and composition of lactating dairy cows.

The objective of the present study was to investigate the potential of the urea dilution technique, coupled with live animal measures to predict the body components of dairy cattle. The study involved 104 lactating Holstein-Friesian cows offered grass silage-based diets. Urea space volume (USV) was calculated from 2 collection periods of blood samples following infusion of urea at 12 (USV12, kg) and 30 (USV30, kg) min after infusion, and then as a proportion of live weight (LW) or empty body weight (EBW). All cows were slaughtered within 2 d of the USV trials. Large ranges existed in EBW and empty body concentrations of water, crude protein (CP), lipid, ash, and gross energy (GE). The USV12 and USV30 were both positively related to LW, EBW, and empty body component weights. The r2 values for USV12 were greater than USV30. The r2 values in the relationships of EBW and empty body composition with USV, however, were smaller than those with LW. Nevertheless, the relationships were improved when both USV and LW were used as predictors, rather than using either alone. Adding milk yield and body condition score as supporting predictors to prediction equations using USV and LW data for EBW, lipid, and GE contents further improved the relationships (r2 = 0.93, 0.66, and 0.77, respectively). Internal evaluation of one-third of the present data using equations developed from two-thirds of the present data indicated that using USV, live weight, and other live animal variables as predictors, rather than using USV alone, considerably improved the prediction accuracy. It was concluded that USV can be used to predict body composition, but the relationships with USV were poorer than those with LW. The USV can only be used as a supporting variable to live weight for prediction of body components in lactating dairy cows.

Contamination of animal feedingstuffs with nicarbazin: investigations in a feed mill.

Some mechanisms of nicarbazin contamination were investigated in a feed mill. Three sequential 3-tonne batches of nicarbazin-free feed were produced directly after a batch of nicarbazin-containing feed (125 mg kg(-1)). Sampling of the nicarbazin-free feed took place at two points before pelleting and at one point post-pelleting. The study was repeated on two further occasions, i.e. three separate nicarbazin-containing feeds and 27 tonnes of 'flushing' feeds were manufactured and sampled in total. Pre-pelleting, the highest nicarbazin concentrations (3.4+/- 0.26 mg kg(-1)) were observed in the first tonne milled after the nicarbazin containing ration. Thereafter, concentrations steadily declined in successive batches. Post-pelleting samples contained much higher concentrations of the drug. After 8 tonnes had passed through, the concentrations (7.2+/- 1.29 mg kg(-1)) were between 10 and 20 times greater than the corresponding concentrations detected post-mixing. These concentrations are sufficient to cause violative residues in eggs and broiler liver. The practice of returning post-press sieved material to the pre-press bins was identified as the cause of the problem. Re-routing of sieved material along with better segregation of nicarbazin-containing and nicarbazin-free feedingstuffs markedly reduced the incidence of feed contamination with this compound.

Accelerated solvent extraction of animal feedingstuffs for microbial growth inhibition screening for the presence of antimicrobial feed additives.

Three plate systems (combinations of indicator organism and growth medium) were evaluated for the detection of analytical standards of the banned feed additives avoparcin, bacitracin zinc, spiramycin, tylosin and virginiamycin. When authorized in the EU, the previously recommended minimum inclusion rate (MIR) for each compound was 5 mg kg(-1). One of the plate systems (Micrococcus luteus ATCC 10240, nutrient agar) detected all five additives. This plate was used in a further study that evaluated the suitability of accelerated solvent extraction (ASE) as a first step in the development of a rapid single-plate screening assay. A drug-free (negative control) feedingstuff was fortified with the compounds (0-50 mg kg(-1)), extracted by ASE and the extracts applied to the plate at each of three pH ranges - unadjusted extract (pH 5.7-5.9), pH 6.5 and 8.0. At pH 6.5, sub-MIR concentrations of virginiamycin and tylosin were detectable. Avoparcin was detectable at 6.3 mg kg(-1). The detection of zinc bacitracin was#10; pH-independent (10 mg kg(-1)). At pH 8.0, spiramycin was detectable at 5.4 mg kg(-1). Mean +/- SD analytical recoveries from fortified feedingstuffs (n = 10) ranged from 57 +/- 1.5% for avoparcin to 96 +/- 4% for virginiamycin. The five additives were also detectable following ASE extraction from a range of different feedingstuffs fortified with each of the drugs. A further 24 compounds permitted for use in animal feeds were tested. Of these, nine were detectable at their recommended MIR. It is concluded that ASE is a versatile technique suitable for the automated extraction of a range of antimicrobials from animal feedingstuffs. Employing ASE with this single-plate detection system permits the rapid antimicrobial screening of animal feedingstuffs and allows the detection of the banned additives. Whilst the method is applicable as a screening test, more specific postscreening methods would be necessary for subsequent identification (and quantification) of antimicrobials in screening-positive samples.

Detection of streptomycin and dihydrostreptomycin residues in milk, honey and meat samples using an optical biosensor.

Immuno-biosensor inhibition assays for the detection of streptomycin and dihydrostreptomycin residues in whole cows' milk, honey, pig kidney and pig muscle are reported. The antibody showed high cross-reactivity with dihydrostreptomycin in various foodstuffs (buffer 103%, milk 96%, honey 84%, kidney extract 129% and muscle extract 98%). There was no significant cross-reaction with other aminoglycosides or commonly used antibiotics. A streptomycin derivative was used to prepare a stable, reusable sensor chip surface. The assay allowed the direct analysis of bovine whole milk (fat content approximately 3.5%). Honey samples required dilution with buffer, while kidney and muscle samples from pigs were homogenized in an aqueous extraction buffer and clarified by centrifugation. The limit of detection for each assay was determined from known streptomycin-free samples (n = 20; mean - (3 x standard deviation)) and the results were as follows: milk 30 microg kg(-1), honey 15 microg kg(-1), kidney 50 microg kg(-1) and muscle 70 microg kg(-1). Repeatability (or relative standard deviation) between runs were calculated (n = 3) at the respective Community maximum residue limits (MRL) and 0.5 x MRL with the exception of honey since no European MRL exists at present. Results were determined as 4.3% (200 microg kg(-1)) and 2.8% (100 microg kg(-1)) in milk, 13.3% (40 microg kg(-1)) and 9.5% (20 microg kg(-1)) in honey, 7.1% (1000 microg kg(-1)) and 7.6% (500 microg kg(-1)) in kidney and 7.1% (500 microg kg(-1)) and 11% (250 microg kg(-1)) in muscle.

Development of clenbuterol reference materials: lyophilized bovine eye samples free of clenbuterol (CRM 673) and containing clenbuterol (CRM 674). Part 1. Preparation, homogeneity and stability.

Within the EU Standards, Measurement and Testing Program (SMT) two clenbuterol reference materials (RMs) were developed. Since clenbuterol readily accumulates and is slowly depleted from pigmented tissues such as the retina, homogenized eye liquid content is the most sensitive tissue for the detection of clenbuterol misuse. Therefore, both of the RMs were produced from bovine eye matrix: a negative control--RM 673 eye reference material, clenbuterol free (<0.50 microg/kg eye matrix) and a positive--RM 674 eye reference material containing clenbuterol (approximately 10 microg/kg eye matrix). Eyes were sampled from 103 German Simmental cattle and the inner liquid content was homogenized to a wet homogenized liquid content (HLC). This clenbuterol negative pool was divided into two sub-pools, one of which was spiked with clenbuterol to a final concentration of 10 microg clenbuterol/kg HLC. Of each pool exactly 2.0 +/- 0.01 g (+/- 0.5%) portions were weighed into 790 containers. Lyophilization of the 1,580 containers was performed in one batch. Parameters for the filling of containers, dry matter content, and residual moisture were in accordance with EU requirements. A three-year stability study and two homogeneity studies at various storage temperatures (-60 degrees C, -20 degrees C, +4 degrees C, +20 degrees C, and +37 degrees C) were performed. Low variation was observed within all of the homogeneity studies, proving that each of the RMs were homogeneous and that this was independent of storage temperature and storage time. In the stability studies, measured clenbuterol concentrations remained constant for RM 673 under the detection limit at 0.15 +/- 0.01 microg clenbuterol equivalent/kg HLC (n = 110) and were also constant for RM 674 at 11.21 +/- 0.15 microg clenbuterol/kg HLC (n=150; measured as duplicates). These studies demonstrate that clenbuterol-containing and clenbuterol-free RMs in bovine eye matrix can be successfully produced. Based on the results described above, it is concluded that both RMs may be suitable as candidates for certification.

Development of clenbuterol reference materials: lyophilized bovine eye samples free of clenbuterol (CRM 673) and containing clenbuterol (CRM 674). Part 2: certification.

The certification by inter-laboratory testing of two candidate reference materials (RMs) for the mass concentration of the anabolic agent clenbuterol in bovine eye material is described: RM 674 with ca 10 microg clenbuterol per kg of eye matrix and RM 673 clenbuterol-free eye matrix as the negative control (<0.50 microg kg(-1)). Both candidate RMs were certified by eleven EU laboratories, and sixty-six accepted replicate measurements were included in the "Certification Study". The precision of the measurement process was assessed by calculation of the standard variation determined within each laboratory during the certification step. The study was performed according to the "Guidelines for the production and certification of BCR reference materials" and to "ISO guide 31, 33, and 35". The certified clenbuterol mass concentration for clenbuterol-free eye material CRM 673 (calculated on the basis of clenbuterol as the free base) was <0.50 microg kg(-1). The corresponding concentration for clenbuterol-containing eye material CRM 674 was 9.42 +/- 0.88 microg kg(-1). These certified values are very close to the desired target concentration of <0.5 microg kg(-1) and ca 10 microg kg(-1). This study has demonstrated that successful certification of clenbuterol-containing and clenbuterol-free bovine eye materials is possible.

Transfer of chlortetracycline from contaminated feedingstuff to cows' milk.

Three groups of four Friesian cows in mid-lactation were fed a compound feedingstuff contaminated with 2, 10 or 300 mg chlortetracycline/kg for 21 days, and were then fed an uncontaminated diet for seven days. A fourth group of four cows was fed an uncontaminated diet throughout the study. Daily pooled milk samples from each cow were analysed by high performance liquid chromatography (HPLC) with a detection limit of 50 microg chlortetracycline/litre. Chlortetracycline was detected in only two milk samples taken from one of the animals fed feed containing 300 mg 300 mg chlortetracycline/kg, and both contained less than the maximum residue limit (MRL) specified by the European Union (100 microg/litre). All the milk samples were also analysed by the Delvotest SP microbiological assay, which has a detection limit of 300 microg chlortetracycline/litre. During the treatment period, this method gave four presumptive false-positive results, because they were not confirmed by HPLC. Selected daily pooled samples from each treatment group were also analysed by the semi-quantitative Charm II radioreceptor assay with a detection limit of 10 microg chlortetracycline/litre. Immunoreactive chlortetracycline was detected only in the animals fed feed containing 300 mg chlortetracycline/kg and several of the results exceeded the EU MRL during the treatment period. No significant treatment effects on animal performance were observed. However, there was a trend towards a higher milk fat concentration (P<0.09) and a lower milk protein concentration (P<0.07) with increasing concentration of chlortetracycline in the diet.

Measurements for certification of chlortetracycline reference materials within the European Union Standards, Measurements and Testing programme.

The experimental design of a material certification programme is described. The matrix reference materials (RMs) comprised chlortetracycline (CTC)-containing and CTC-free lyophilised porcine liver, kidney and muscle produced under the European Commission's Standards Measurements and Testing (SMT) programme. The aim of the certification programme was to determine accurately and precisely the concentration of CTC and 4-epi-chlortetracycline (epi-CTC) contained in the RMs. A multi-laboratory approach was used to certify analyte concentrations. Participants (n = 19) were instructed to strictly adhere to previously established guidelines. Following the examination of analytical performance criteria, statistical manipulation of results submitted by 13 laboratories, (6 withdrew) allowed an estimate to be made of the true value of the analyte content. The Nalimov test was used for detection of outlying results. The Cochran and Bartlett tests were employed for testing the homogeneity of variances. The normality of results distribution was tested according to the Kolmogorov-Smirnov-Lilliefors test. One-way analysis of variance (ANOVA) was employed to calculate the within and between-laboratory standard deviations, the overall mean and confidence interval for the CTC and epi-CTC content of each of the RMs. Certified values were within or very close to the target concentration ranges specified in the SMT contract. These studies have demonstrated the successful production and certification of CTC-containing and CTC-free porcine RMs.

Bilevel non-invasive ventilation in malignant large airways obstruction during chemotherapy and radiotherapy.

A case is described of acute respiratory failure secondary to variable intrathoracic large airway obstruction due to a lung neoplasm. Successful ventilation was achieved with facemask bilevel non-invasive ventilatory assistance allowing radiotherapy and chemotherapy to be undertaken.

Transfer of sulphamethazine from contaminated dairy feed to cows' milk.

Four groups of four healthy mid-lactation Friesian cows were fed a compound feeding stuff containing either 2, 10 or 250 mg sulphamethazine/kg, corresponding to 0, 2, 10 and 250 per cent of the therapeutic inclusion rate in rations for pigs, at a flat rate of 3 kg twice daily for 21 days, followed by a seven-day withdrawal period. The cows were machine-milked twice daily and pooled milk samples from each cow were analysed by a commercially available microbiological assay with a sensitivity of 100 micrograms/litre and by a high performance liquid chromatography (HPLC) procedure with a limit of detection of 10 micrograms/litre. No sulphamethazine was detected by HPLC in the milk samples taken from any of the cows fed the concentrate containing 2 or 10 mg/kg. The milk samples from all four cows fed the highest concentration of sulphamethazine contained from 21 to 120 micrograms/litre while they were being fed the contaminated concentrate. The cow with the highest concentrations of sulphamethazine was the only one which repeatedly tested positive by the microbiological assay. The concentration of sulphamethazine declined rapidly during the withdrawal period and the drug was not detectable by either method in samples taken from two days after the contaminated feed was withdrawn.

Nortestosterone is not a naturally occurring compound in male cattle.

Nortestosterone (beta-NT) is a hormonal growth promoter banned from livestock production in the EU. Following injection, the major metabolite in cattle is the 17 alpha-epimer (alpha-NT). However, this also occurs naturally in pregnant cattle. It is not known whether alpha-NT is also endogenous to intact or castrated male cattle. Three surveys were undertaken to assess whether alpha-NT is naturally produced in this subset of the population. Bile samples from a total of 1,281 cattle (73 bulls and 1,208 steers) from 366 herds were collected at slaughter and initially screened by using a semi-automated EIA with multi-analyte immunoaffinity chromatography (IAC) clean-up. Bile samples from a further 38 male cattle (10 bulls and 28 steers) were analysed by high-resolution gas chromatography-mass spectrometry (GC-MS) with IAC pretreatment. Only samples containing more than 2 ng/ml alpha-NT were subjected to GC-MS. With 2 ng/ml alpha-NT as a threshold for confirmatory testing, the false positive rate of the screening EIA was 1.8%. Bulls (n = 16) and steers (n = 179) from government farms (n = 2) and which were not treated with exogenous beta-NT, did not have measurable concentrations of alpha-NT in their bile. Bulls (n = 35) and steers (n = 606) taken from herds (n = 204) which had no previous history of illegal growth promoter abuse also did not have alpha-NT in their bile. Of 32 bulls and 451 steers of unknown treatment history sampled from herds (n = 160), 56 steers from 19 herds contained GC-MS confirmed concentrations of alpha-NT higher than the limit of quantification of the assay LOQ (0.7 ng/ml). Of these animals, two had beta-NT-containing injection sites and five had residues of the beta-agonists clenbuterol and mabuterol. Examination of the animal movement and ownership histories of the 56 confirmed positive animals strongly suggested that exogenous beta-NT had been administered at the presenting farm. It is concluded that alpha-NT is not endogenous to this subset of the cattle population and that detection of this hormone in bile from bulls and steers constitutes evidence of abuse.

Comparison of the effects of injections of nortestosterone phenylpropionate at single and multiple sites in cattle on the detection of its residues in plasma, urine and bile.

The synthetic androgen 17 beta-19-nortestosterone (beta-NT) has been used illegally as a growth promoter in cattle production in the European Union. The elimination of beta-NT and its metabolites in plasma and urine was studied in cattle which had received intramuscular injections of its phenylpropionate ester (NTPP) at either single or multiple sites at a dose rate of 1 mg/kg bodyweight. In both groups, the plasma concentrations of beta-NT, measured by enzyme immunoassay, were consistently greater than the assay's limit of quantification (0.24 ng/ml) during days 1 to 7 of the study. The mean (sd) maximum plasma concentration (Cmax) was significantly greater in the multiply injected animals (4.4 [0.48] v 2.7 [0.15] ng/ml), but other plasma pharmacokinetic parameters, AUC, CL, T1/2 beta, Tmax and MRT, were not significantly different in the two groups. The equivalent urinary concentrations exceeded the limit of quantification of the assay (4.5 ng/ml) for up to 24 days after injection. In a second study, the biliary concentrations of beta-NT and its 17 alpha-epimer (alpha-NT) were measured by gas chromatography-high resolution mass spectrometry after cattle were injected intramuscularly at either single or multiple sites with NTPP. Only alpha-NT was detected in bile for up to 62 days after injection at concentrations above the limit of quantification of the assay (0.7 ng/ml). It is concluded that in some animals, intramuscular injections of NTPP at several sites may decrease the period after injection during which free beta-NT and its metabolites are detectable in plasma and urine. After the injection of NTPP, alpha-NT was detected in bile for longer than it was detected in plasma or urine.

Evaluation of a single plate microbiological growth inhibition assay as a screening test for the presence of antimicrobial agents in compound animal feedingstuffs at therapeutic and contaminating concentrations.

The Inhibitory Substance Test (IST), a microbiological growth inhibition test, is used for screening animal feedingstuffs for the presence of (contaminating) antimicrobial compounds. The effectiveness of the IST was established for 33 compounds that may be incorporated in feedingstuffs. Minimum detectable concentrations (MDCs) for standard solutions were established and compared with those obtained following solvent extraction of an antimicrobial-free compound feedingstuff spiked with each compound at 0-20 mg/kg. Of the 33 standard solutions examined, the test organism was not sensitive to 11 and the MDC for one was greater than its maximum inclusion rate in complete feedingstuffs. Following routine extraction (25% acetone-phosphate buffer) of feedingstuffs spiked with each of the 22 compounds to which the organism was sensitive, 10 were not detected, 15 were detectable at both minimum and maximum feed-inclusion rates and four were only detectable at their maximum feed-inclusion rates. Extraction with methanol (25%) had a deleterious effect with 12 compounds not detected, nine detectable at both minimum and maximum feed-inclusion rates and five detectable at their maximum feed-inclusion rates. Increasing acetone and methanol concentrations to 40 and 55% respectively resulted in larger inhibitory zones for antibiotic-free feedingstuff (25.3 + 2.43 mm vs 21.1 + 1.02 mm) compared with both 25% acetone (11.3 + 0.22 mm) and 25% methanol (11.2 + 0.22 mm), requiring the establishment of greater threshold zone diameters and negating any advantage in increasing the solvent concentration under these test conditions. It is concluded that the IST may be particularly useful for detection of a number of the zootechnical feed-additives recently banned in the EU, which, if used illegally, may be present at sufficiently high inclusion rates to facilitate detection. Further alteration of extraction conditions may improve the scope of the assay.

Plasma, urinary and biliary residues in cattle following intramuscular injection of nortestosterone laurate.

The synthetic androgen 19-nortestosterone (beta-NT) has been used illegally as a growth promoter in cattle production in the European Union. Elimination of beta-NT and its metabolites in plasma, urine and bile was studied in three cattle with cannulated gallbladders following intramuscular injection at a single site of 500 mg of the laurate ester (NTL) containing 300.5 mg beta-NT. Using enzyme immunoassay quantification, plasma Cmax of free beta-NT was 0.5 +/- 0.15 microgram/L (mean +/- SEM). Concentrations of free beta-NT in plasma were consistently greater than the assay limit of quantification (0.12 microgram/L) for 32.7 +/- 13.42 days. Mean residence time for the beta-NT in plasma was 68.5 +/- 20.75 days. Following sample preparation by immunoaffinity chromatography, high-resolution GC-MS was used to quantify beta-NT and alpha-NT in urine and bile. beta-NT was detected irregularly in urine from two of the three animals post injection. The principal metabolite present in the urine, alpha-NT, was detected for 160.3 +/- 22.67 days post injection. Cmax for alpha-NT in urine was 13.7 +/- 5.14 micrograms/L. Mean urinary AUC0-183 days for alpha-NT was 845.7 +/- 400.90 (microgram h)/L. In bile, alpha-NT was the only metabolite detected for 174.3 +/- 8.67 days post treatment. Cmax for alpha-NT in bile was 40.8 +/- 12.70 micrograms/L and mean biliary AUC0-183 days for alpha-NT was 1982.6 +/- 373.81 (microgram h)/L. Concentrations of alpha-NT in bile samples were greater than those in urine samples taken at the same time. The mean ratio of biliary:urinary AUC0-183 days was 3.0 +/- 0.72. It is concluded that bile is a superior fluid for detection of alpha-NT following injection of NTL, owing to the longer period during which residues may be detected after administration.

Biliary elimination of endogenous nortestosterone by pregnant cows.

A temporal study of the biliary elimination of endogenous 19-nortestosterone during two successive pregnancies was made in three cows with cannulated gall bladders. Bile samples were analysed for 17 beta-19-nortestosterone (beta-NT) and the 17 alpha-epimer (alpha-NT) by using high resolution gas chromatography and mass spectroscopy. No beta-NT was detected in any of the samples analysed. However, alpha-NT was detected from around 120 days of gestation in each of the cows. Peak concentrations were observed in the last week before calving and ranged from 9.5 to 36.7 ng/ml. After parturtion, the concentrations of alpha-NT declined rapidly and were undetectable by seven days after calving, and it was not detected again until after 120 days of gestation. The biliary concentrations of alpha-NT detected subsequently were similar to those observed in cattle several weeks after an exogenous injection of the synthetic ester beta-NT phenylpropionate.

Zeranol is formed from Fusarium spp. toxins in cattle in vivo.

Zeranol, a semi-synthetic oestrogenic growth promoter, was banned in the EU in 1988. The ability of Member States to police the ban on zeranol has been hampered by suggestions from New Zealand and from this laboratory that zeranol may be formed by the in vivo metabolism of naturally occurring Fusarium spp. toxins. The present study demonstrates that zeranol is formed from alpha-zearalenol and zearalenone in vivo and is detected in bovine bile following the oral administration of these compounds. However, it is not detected following administration of beta-zearalenol. These data suggest that hydrogenation of alpha-zearalenol, probably in the rumen, is responsible for the appearance of zeranol. The present study shows that environmental contamination with Fusarium spp. toxins is widespread in Northern Ireland. Fusarium spp. toxins were present in 32% (n = 422) of all bovine bile samples tested for zeranol during 1995. Zeranol itself was confirmed in 6.6% (n = 28) of the samples. However, the mean alpha-zearalenol and beta-zearalenol concentrations in the bile of zeranol-positive animals were 12 and 9 times higher, respectively, than those in the zeranol-negative animals. The alpha-zearalenol concentration always exceeded the zeranol concentration by at least 5:1. This may, in the future, permit differentiation between zeranol abuse and natural contamination.

Contamination of animal feedingstuffs with undeclared antimicrobial additives.

A study was carried out on 397 feedingstuffs and 11 premixes on which 161 (39.5%) were declared free of medication and 247 (60.5%) were medicated. These were subjected to analysis for the presence of antibiotics. Of the 247 medicated feeds 87 (35.2%) contained undeclared antimicrobials of which 59 (23.9%) were at a concentration sufficient to allow quantification by HPLC. Of the 161 unmedicated feeds 71 (44.1%) were shown to contain detectable antimicrobials of which 42 (26.1%) contained concentrations which could be quantified by HPLC. The most frequently identified contaminating antimicrobials were chlortetracycline (CTC) (15.2%), sulphonamides (6.9%), penicillin (3.4%) and ionophores (3.4%). Four samples (ionophores--3, sulphadimidine--1) contained therapeutic concentrations and one sample a supra-therapeutic concentration (monensin). The remainder were sub-therapeutic. All the contaminating concentrations of sulphadimidine detected were sufficient to cause violative tissue residues if fed to animals immediately prior to slaughter.

Metabolism of chlortetracycline: drug accumulation and excretion in the hen's egg.

Chlortetracycline (CTC) is one of the few antibiotics that can be used without any withdrawal period in chickens laying eggs intended for human consumption. 6-Iso-CTC and 4-epi-6-iso-CTC have recently been identified as the principal metabolites of CTC in eggs. Although not covered by the European Union (EU) maximum residue limit (MRL) for CTC, these compounds, taken together, accumulate in the eggs of birds treated therapeutically with CTC to a mean concentration equivalent to more than twice the EU MRL (200 micrograms kg-1) in eggs. Plateau concentrations in eggs were achieved after approximately 3 d of medication. Following withdrawal of medication, mean egg concentrations of these compounds were maintained for 48 h, before falling below a level equivalent to the MRL after 5 d. Feeds containing typical sub-therapeutic contamination concentrations of CTC did not produce mean concentrations of 6-iso-CTC plus 4-epi-6-iso-CTC, combined, greater than 200 micrograms kg-1. It is not known whether these compounds are formed as a result of metabolism or of chemical degradation. However, analysis of ovules pre-lay showed that all of the CTC present in this matrix was in the form of 6-iso-CTC and 4-epi-6-iso-CTC, and not as the parent drug. Although microbiologically inactive, the toxicological properties of 6-iso-CTC and 4-epi-6-iso-CTC are not known.

Residues of nortestosterone esters at injection sites. Part 1. Oral bioavailability.

Injectable esters of 17 beta-19-nortestosterone (beta-NT) have been used illegally for growth promotion in European Union (EU) cattle production. There are no data on oral bioavailability of free beta-NT from beta-NT esters. Adult non-pregnant female Landrace pigs (n = 4) were fitted with jugular vein cannulae and were treated as follows with an appropriate 'flushing out' period between each treatment: an intravenous bolus of beta-NT at 0.1 mg kg-1 body weight (NTi.v.), 300 mg nortestosterone phenylpropionate (NTPP) in oil of arachis administered per os (NTPPoil) and 300 mg NTPP in aqueous suspension administered per os (NTPPaq). Blood samples were taken for up to 24 h and analysed for free beta-NT by enzyme immunoassay. Calculation of the area under the plasma time concentration curve (AUC), allowed absolute bioavailability estimations for both oral treatments. Mean bioavailability of beta-NT following NTPPaq was 0.35% (range 0.25-0.41%) compared to 2.25% (range 0.86-2.85%) for NTPPoil. Mean +/- standard error of mean time of maximum plasma concentration (Tmas) of free beta-NT occurred earlier (2.3 +/- 0.6 h) with NTPPoil compared to NTPPaq (10.3 +/- 1.03 h) and the maximum plasma concentration (Cmax) was also greater for NTPPoil compared to NTPPaq (36.1 +/- 6.49 vs. 3.2 +/- 0.31 micrograms l-1). It is concluded that the presence of arachis oil enhances the absorption of NTPP. Notwithstanding the possible effects that cooking and other food processing may have on such injection site residues, extrapolation of these results to man suggests that consumption of NTPP-containing injection sites may result in peak plasma concentrations of free beta-NT which are significantly greater than those observed following injection of NT esters.