Are poultry and eggs a source of minute amounts of clomiphene in doping control samples

Code: 19C02MT

The anti-estrogen clomiphene is prohibited at all times in sport and since 2011 a continuous trend in increasing numbers of adverse analytical findings is noted. Recent studies have outlines a particularly long detection window for clomiphene in human urine; further, few studies have demonstrated a significantly enhanced egg production if layig hens are treated with clomiphene. Hence, concerns have ben raised whether trace amounts of clomiphene are present in eggs or poultry due to a potential use of clomiphene in the farming industry, and whether such trace amounts could lead to adverse analytical findings in doping controls. In order to protect the athletic community, a controlled administration study is planeed, where clomiphene is administered to laying hens, and both the produced aggs as well as the edible tissue will be tested for residues of clomiphene. Fruther, eggs and edible tissue will be consumed by study volunteers, and urine sample will be subjected to routine doping control analytical assays to prove for th presence of the prohibited substance. The information gained from this study is vital to fair result managment and decision-making processes in case of clomiphene findings in sports drug testing programs. If the results prove the possibility of clomiphene contamination in dietary products such as eggs of poultry, athletes and anti-doping organizations must be warned and informed.

Main Findings: 

Athletes are permanently at risk of inadvertent ingestion of prohibited substances, as shown so prominently across countries by the clenbuterol scandal. In consideration of the growing number of AAFs related to clomiphene, it cannot be excluded that food contamination is one reason of this phenomenon. In this study, the possible contamination of eggs and chicken meat with clomiphene was investigated since it had been shown that clomiphene administration can lead to an increased egg production rate in hens.

When a drug is administed to a chicken, it can accumulate in certain parts of the body or even in the eggs. This distribution depends on physical and chemical properties such as the diffusivity of trhe substances, the molecular mass, lipophilicity and on other properties such as the ability to bind to plasma proteins. All this has an influence on if clomiphene accumulates in the meat and eggs of hens fed with the drug and if yes, how fast, how long and how much of it can be found.

The results of this study reinforce the concern that clomiphene given orally to laying hens migrates to and is stored in eggs. The determined clomiphene levels result in absolute amounts of 10 to 20 µg per egg. These amounts are more than 1000-fold below the daily therapeutic dose, so no effect would be expected with the consumption of a contaminated egg. However, many athletes have a high-protein diet and may consume clomiphene-contaminated eggs in larger quantities. This could subsequently lead to clomiphene residues in urine, leading to adverse analytical findings. This was simulated in the second part of the study where healthy male volunteers consumed the eggs produced in the animal administration study. A single consumption of two clomiphene-containing eggs led to maximum hydroxy-clomiphene concentrations between 82 and 266 pg/mL in the tested urine. Discrimination of the isomers already took place in eggs. Therefore, it was not surprising that almost exclusively (Z)-clomiphene and its metabolites were detected in urine. The (Z)-form of clomiphene has a longer half-life and is mostly found as the only isomer in urine long after clomiphene therapy. Hence, the findings indicated that it is possible to generate an adverse analytical finding by consuming clomiphene-containing eggs. Consequently, a more detailed study of the metabolites was conducted.

The intake of the substance differed between the both studies: While the microdose study volunteers took a preparation containing 42 % (Z)- and 58 % (E)-clomiphene, the egg consumers took almost only (Z)-clomiphene. Additionally, the egg consumers took 10-20 % of the clomiphene amount as hydroxy clomiphene. As (Z) 4 HC was already present in eggs, it was suggested that this had an influence on the formation of the metabolites. By analyzing the phase-II metabolites, it was found that the preferably formed hydroxy metabolite must differ between egg consumers and the comparator group, since the glucuronide peaks differed in retention time and product ion mass spectra. This was confirmed when analyzing the samples with the method for differentiation of the hydroxy metabolites of clomiphene by derivatization with dansyl chloride to achieve chromatographic separation. After consumption of clomiphene-containing eggs, over 90 % of the hydroxy metabolites was (Z)-4-HC, which was found neither in samples from the microdose study nor in the re-analyzed samples with clomiphene AAFs. 

In conclusion, a method was developed to assist in distinguishing between the consumption of eggs containing clomiphene and ingestion of the drug.