Clostebol and sport: about controversies involving contamination vs. doping offence

Clostebol, the 4-chloro derivative of testosterone, is available as Over The Counter product in pharmacies and drugstores from several countries, including Italy and Brazil. It is exclusively commercialized as a cream or a spray, most often under the trade name Trofodermin^®. The active ingredient is the ester form clostebol acetate and in Trofodermin^® preparation, clostebol acetate is always associated with neomycin. The tube contains 30 g of cream at 5 mg/g of clostebol acetate for a total of 150 mg of clostebol acetate, while the spray contains 5 mg/mL of clostebol acetate in 30 mL. Because of the therapeutic properties of this anabolic steroid, Trofodermin^® is recommended to heal over small skin injuries (e.g. cut, skin abrasion or lesion) or gynaecologic injuries. Differently, no tablet, capsule, injectable or other pharmaceutical preparation of clostebol is approved for human use. Illegal preparations can be found on the black market, and it seems that some have been used in the past for doping purposes [1].

Several metabolites have been detected in the urine of subjects exposed to clostebol, including glucuronide and sulfate metabolites 2], [3], [4. In doping control, the key metabolite is the M1 metabolite, or 4-chloro-androst-4-en-3α-ol-17-one. This compound is not easy to purchase, thus limiting private laboratories which may offer testing to cheating athletes trying to have their urine controlled out of official anti-doping programs. However, to determine whether an athlete has ingested, been exposed or been administered clostebol, the World Anti-Doping Agency (WADA) laboratories can elect to test for any known clostebol metabolite.

As other anabolic steroids, clostebol is listed on the WADA list [5] as a prohibited substance at all times (Class S1.1, exogenous androgenous anabolic steroids). All administration routes are banned, including the topical one. The use of Trofodermin^® is therefore fully banned for athletes, having a “doping” label on its box, but not on the tube or the spray. Starting from 2018, the WADA has indicated in its testing figures a slight increase in the number of adverse analytical findings (AAFs) involving clostebol, with 25 positives cases over 4,117 AFFs (0.61 %) in 2018 to 25 positives cases over 2,680 AAFs (0.93 %) in 2022. Specifically, according to the WADA statistics, more than half of the total AAFs involving clostebol are observed in Italy with concentration of metabolite M1 lower than 2 μg/L (ng/mL) in 77 % of the cases [4].

Although clostebol tablets for oral ingestion or clostebol esters injectable forms do exist (caproate or propionate) in some part of the world, almost all cases reported in the literature are dealing with misuse of Trofodermin^® via topical administration. In an old publication in 1992, it was also reported that a clostebol metabolite (4-chloro-delta-4-androstene-3-α-ol-17-one) could be detected after consumption of contaminated meat but this has never been observed later, probably due to the European regulations on meat consumption for humans [6].

Topical administration is a route of drug delivery in which medications are applied directly to the skin. Clostebol acetate is a lipophilic drug. It tends to slowly cross through the epidermis and dermis layers. Once in the hypodermis, it essentially diffuses in this layer where it remains for a prolonged time. Clostebol acetate is therefore applied straight to the site of the injury and can rapidly produce its effects. This route of administration can also result in significant absorption of the drug by the lower layers of the skin and subsequent transfer into the systemic circulation, resulting in distribution in the whole body. Finally, the drug is metabolized and eliminated via the urine, where it can be detected by anti-doping or toxicological analyses.

It has been published that controlled transdermal application of clostebol acetate can produce detectable amounts of metabolites in urine, even after a single exposure [4, 7], [8], [9. Depending on the protocols, metabolite M1 was detectable up to 30–40 μg/L at the peak for more than 10 days. In other studies [4, 7], [8], [9, transfer of clostebol from one subject to another during hand shaking or sexual intercourse was demonstrated. Even transfer during treating a dog with Trofodermin^®, also purchased in Italy, was reported and recognized as possible by the sport authorities 10], [11], [12], [13.

In cases of drug transfer between two humans, as expected, urine concentrations of the metabolite M1 were lower than in cases of direct ingestion, generally not exceeding 1–5 μg/L. Transfer of a drug to an athlete during intimate moments is one of the major concerns when dealing with cross contamination between two subjects and subject to more controversies as it concerns the private life [14]. The WADA is perfectly aware about possible skin transfer, as shown in the statement [15] that it made in 2021 in response to a documentary published by German broadcaster ARD. WADA specifically mentions the “very limited” risk of contamination through the skin and therefore an anti-doping rule violation, for example, for sabotage. Testing for other metabolites, such as the sulfate one, does not allow to definitively discriminate the route of administration of clostebol, i.e. topical vs. oral or intramuscular ones [4].

Due to remarkable progresses in the sensitivity and the specificity of the analytical instruments currently available (e.g. high-resolution mass spectrometry), it is not unusual to see reported urine concentrations of a doping agent in the low ng/L range. When requesting the laboratory documentation package (i.e. the document that contains the whole traceability of the testing) from the WADA accredited laboratory, the criteria of identification of the drug, including its retention time and ratios between transitions, are always met, even at very low concentrations. This fact unambiguously demonstrates that the drug or its metabolite are present in the specimen. According to the WADA code, the pharmacological (or toxicological) significance of such a low concentration has not to be discussed and the athlete is considered having violated the anti-doping rules. However, it can be argued that the simple identification of some ng/L (pg/mL) of a doping agent is enough to qualify for a 4-years suspension without further investigation.

Indeed, a low urine concentration can be interpreted in two different ways: 1. it can be the tail end of a drug voluntarily used to enhance performance; or 2. it is the direct consequence of a contamination. By contamination, one can include laced supplements, either voluntarily or by lack of hygiene measures; contaminated meat by growth promoters; poor quality pharmaceuticals with chemical residues, and finally drug transfer during intimate moments or various other scenarios [16]. This has been summarized by Thevis et al. under the concept of “athlete exposome” [17].

To claim for the contamination scenario, in 2021, Kintz proposed [18] to consider the following strategy: 1. the concentration in the urine of the athlete must be low or far from a therapeutic or recreative concentration; 2. a hair test of the athlete must demonstrate incidental exposure; 3. the source of contamination must be identified; and 4. an independent pharmacologist or toxicologist must verify the claims of the athlete, particularly to demonstrate that the exposure was unintentional and that the source of contamination was not known by the athlete.

Although WADA does not recognize the use of hair or nail clippings as routine doping control matrix, these specimens can offer an additional piece of evidence for result management. In that way, the analysis of biological keratinous matrices is a complement and not an alternative to standard anti-doping practices. It is widely accepted by scientists that a negative hair test cannot overrule a positive urine finding, particularly because this can be interpreted as a lack of sensitivity of the test (i.e. the hair test is not able to detect the low amount that entered in the body of the subject or the incorporation of the drug of interest in hair is very limited due to its chemical structure). This has been observed when challenging an anti-doping violation involving anabolic steroids, SARMs such as ostarine or ligandrol or diuretics. Testing for drugs in hair to document exposure has been recognized since the early 80s, taking advantage of a longer detection window when compared to blood and urine, less embarrassing conditions of collection and storage/shipping at ambient temperature. Hair tests are useful to verify drug exposure in any situation in which a pattern of drug use must be established. For this, segmental analysis can be achieved by cutting the hair into 1 or 2 cm segments to measure drug use during shorter periods of time. Given an average growth rate of 1 cm per month, each cm of hair in the vertex region represents what was present in the body during the corresponding month. Almost all doping agents but hormones are detectable in hair [19].

Identification of clostebol in hair has been seldom reported and the literature lacks controlled studies. Today, the minimal detectable dose of clostebol in hair is unknown. However, to produce an anabolic effect, administration of clostebol must be repeated preferably on a daily basis over several weeks, so to facilitate the molecule incorporation into hair. In hair, the target compound is clostebol acetate as clostebol itself was never reported. On the opposite to urine, where gas chromatography after derivatization is the technique of choice to measure the M1 metabolite, liquid chromatography is preferred in hair. Salomone et al. [20] have measured clostebol acetate with concentrations in the range 3–21 pg/mg in four positive subjects. After drug transfer from her dog by spraying Trofodermin^® on the dog’s three injured paws and by sharing the same bedding, Pokrywka et al. [10] identified clostebol acetate at 52 and 78 pg/mg in two consecutive hair segments of an athlete challenging her violation. The hair of the dog, collected close to the injuries tested positive for clostebol acetate at 980 pg/mg. In this last case, the Court of Arbitration for Sports Anti-Doping Division found that the athlete committed an anti-doping rule violation, but under circumstances that amounted to “no fault”.

A recent case involving a top athlete has caused numerous discussions in the scientific community [21]. The subject is a male athlete, who returned an AAF with a urine concentration for the M1 metabolite below 0.1 μg/L [21]. The athlete claimed that the AAF was due to a contamination caused by drug transfer from his masseur who used a spray of Trofodermin^® to treat a hand cut. Instead of a provisional suspension over months (or years) and consequent negative impact on the athlete reputation, an independent tribunal of the International Tennis Integrity Agency (ITIA) stealthily ruled “no fault or negligence”, a situation that created unequal treatment with athletes sanctioned in the past with several months or even years of suspension. According to the ITIA decision [21], it seems that no hair test was performed.

Although the purpose of the authors is not to discuss the ITIA decision, there is an interest in discussing the scientific aspects of the case. Based on the details provided in the ITIA decision, a similar protocol of contamination was reproduced by the authors and the urine of the subject receiving the massage was tested 21 h after the last massage performed by a masseur who sprayed Trofodermin^® on one of his fingers to mimic a cut treatment. The presence of the metabolite M1 was confirmed in the urine specimen of the contaminated subject at 0.52 μg/L, confirming the possibility of cross contamination [9].

In the last years, some of the authors have dealt with more than 20 doping cases of clostebol, in which the athletes were able to identify the source of accidental exposure in Trofodermin^® or Veterabol^® use. In cases when the exposure was considered to be indirect (namely when Trofodermin^® was not used by the athlete but instead by someone which somehow interacted with the athlete), the urine concentration of M1 measured by the WADA laboratory was always below 1 μg/L.

To avoid tedious discussion about possible contamination, it is not unreasonable to establish a reporting level for M1. The authors suggest to WADA medical commission to evaluate the possibility of reporting the result of the urine test as an atypical finding as it is done for clenbuterol [22], pending the concentration does not exceed 1 or 2 μg/L.

The authors have a long-time experience with hair analysis, which has often shed light on the origin and frequence of use of licit and illicit substances. In case of clostebol exposure from skin contamination, the hair concentration is expected to be in the low pg/mg range, or even undetectable [20]. Instead, the hair collected from the person who contaminated the athlete after having used Trofodermin^® will likely result in a higher concentration, as it was reported in the case involving contamination after treating an injured dog [10].

In conclusion, the presence of a prohibited substance in the urine of an athlete may not be only related to intentional doping. However, unknowingly being exposed to a doping agent can have very negative consequences, including financial ones, as the consequent period of suspension can be up to four years. Consequently, it is important that athletes are warned against the personal or accidental/unintentional use/exposure of dermal preparations containing doping agents, as in the specific case of Trofodermin^®. In addition, athletes must be aware of the medical treatment, including substances listed as doping agents by their relatives, trainers, health staff and partners, which can result as a minimal amount in athlete urine and can be considered as a doping offense. Since athletes are responsible of what entered in their bodies, it can be difficult to prove innocence. In case of clostebol AAF, urine concentration should be assessed to rule out recent or massive ingestion. If also a hair test is performed to support the contamination scenario, then the athlete has a chance to obtain a reduced suspension or acquittal.