Clostebol detection after transdermal and transmucosal contact. A systematic review

Introduction

Clostebol (4-chloro-testosterone; 4-chloro-4-androsten-17β-ol-3-one) is the 4-chloro derivative of testosterone. It is a synthetic androgenic steroid with anabolic effect (AAS). The approved medical use of clostebol in humans is limited to topical administration, primarily clostebol acetate in dermatological drugs (Veterabol^® or Trofodermin^® cream or spray containing 0.5 % clostebol acetate, equivalent to 5 mg of clostebol acetate each application), as well as ophthalmological and gynecological preparations. These pharmaceutical formulations are available in several countries such as Italy, where it can be obtained in the pharmacy without a medical prescription, Brazil, Peru, Central America, and Pakistan. Clostebol, as other AAS or anabolic agents, if taken for long periods, can boost physical performance, for example increase strength, support muscle growth and accelerate recovery [1]. Clostebol is listed as a prohibited substance by the World Anti-Doping Agency (WADA). An increased number of adverse analytical findings (AAFs) involving clostebol were reported in the last years. Some cases where athletes claimed an inadvertent uptake were famous. Before the Rio de Janeiro 2016 Summer Olympic Games, some athletes tested positive to clostebol. The case of the skier Therese Johaug and a very recent case involved a top tennis athlete were subjects of controversial discussions. Several metabolites have been detected in the urine of subjects exposed to clostebol, including glucurono- and sulforo-conjugated metabolites [2] using a gas chromatography–mass spectrometry (GC-MS) or liquid chromatography–mass spectrometry (LC-MS) [3], 4]. It is not clear if the detection of clostebol metabolites in urine, and in particular the main clostebol metabolite (4-chloro-androst-4-en-3α-ol-17-one, M1) generally used at the screening level, can be reconcilable with a transdermal or transmucosal direct or indirect transfer, nor at which concentration, even in the case of a single intake of clostebol, whether or not intentional or inadvertent [5], 6]. Therefore, we performed a systematic review (SR) regarding the correlation between the presence of detectable amounts of clostebol metabolites in urine and the transdermal or transmucosal contact of clostebol. Moreover, we want to analyze if the urine concentration of clostebol metabolites derived from transdermal or transmucosal contact with clostebol is lower than therapeutic or recreative concentration (for example to enhance muscular performance).

Methods

Results

The extensive search of the literature followed by a careful manual screening of retrieved articles led to the identification of 7 papers pertinent to our questions. The search algorithm is detailed in Figure 1. According to our aims, all retrieved studies have been included for data extraction regardless of their methodological quality. In detail, we found three case reports [7], [8], [9] one experimental study [10] and three case reports with an experimental section [11], [12], [13]. Overall, 32 cases of determination of urine clostebol metabolite after TD or TM exposure to the drug were found (Table 1): 7 athletes (2 males, 2 females and 3 unspecified) and 25 healthy volunteers (19 males, 5 females and 1 unspecified). In 19 cases the exposure of clostebol was direct: 8 subjects self-applied cream (5 TM and 3 TD), 4 subjects applied cream to another person (TD) and 7 subjects received application of cream (TD). In 13 cases the exposure of clostebol was indirect: 3 subjects had sexual intercourse (TM), 9 subjects had contact with the hand of a volunteer self-applying cream (TD) and in one case the subject touched her dog, which she applied spray to. The dose of clostebol to which the subjects was exposed varied from 0.5 to 200 mg, whilst it was not specified in the case reports. The time of exposure varied in the different studies, but only in the study by de la Torre et al. in 8 subjects (24 %) it could be defined as a fast contact (<1 min). In 26 cases (79 %) GC-MS was performed within 24 h from the exposure to clostebol. The median concentration of M1 in the overall population was 2.6 ng/mL (range 0.086–36.6 ng/mL; 25 %–75 % IQ: 0.805–10.625 ng/mL). The median concentration of M1 was 0.5 ng/mL (range 0.086–4.000 ng/mL; 25 %–75 % IQ: 0.5–0.9 ng/mL) and 8.1 ng/mL (range 1.0–36.6 ng/mL; 25 %–75 % IQ: 2.8–22.0 ng/mL), in subjects with indirect and direct exposure of clostebol, respectively (Table 2). These values were significantly different (p=0.005). The overall rate of M1 concentration<2 ng/mL was 50 %: 85 % (11/13) and 26 % (5/19) in subjects indirectly exposed to clostebol and in those directly exposed to clostebol, respectively (p=0.004). Moreover, M1 was<5 ng/mL in all subjects indirectly exposed to clostebol.

Figure 1:

Flow diagram of the study selection process.

Only in 14 cases (43 %) a control value of M1 concentration after 15–288 h was available (Table 2).

Discussion

To the best of our knowledge, this is the first-ever SR including adults with detection of clostebol urine metabolites after transdermal or transmucosal contact.

As expected, we did not find observational studies, but only case reports of athletes and results of experiments conducted on healthy volunteers. We decided to manage data from all subjects together because the biological characteristics of the athletes cited in the case reports can be considered very similar to those of the volunteers. Moreover, the same urine metabolite M1 was reported, and GC-MS was used in all the cases, although in the works of Gessner [13] and Kintz [8], 10] LC-MS was also performed. Both GC-MS and LC-MS are the most reported systems in literature [3], 4] and, interestingly, in the last decade laboratories have developed methods lowering the limit of detection, which is now established at 0.1 ng/mL [12].

We found that the detection of clostebol metabolites in urine can be reconcilable with a transdermal or transmucosal direct or indirect exposure to clostebol, even in case of a single or fast contact. The concentration of urine M1 was significantly higher in the case of direct exposure (median 33.5 ng/mL) than in the case of indirect exposure (median 0.5 ng/mL). The dose of clostebol to which the subjects were directly or indirectly exposed was similar in all the studies, but two: it was higher (200 mg) in two of the volunteers in the study by Pereira [11] and in one (50 mg) of the volunteers in the study by Kintz [10]. In these three cases the concentration of M1 was higher than in the others, confirming a direct correlation between administered doses and urinary metabolites’ levels. We found that time of direct or indirect exposure to clostebol and time from exposure to urine exam correlated with M1, too. The values of M1 were higher after intermediate/prolonged exposure and if the urine exams were performed more than 24 h after the exposure (only in 5/31 cases, 16 %). In our opinion, it would be very interesting to observe at what time and at what concentration urine clostebol metabolites remain detectable, but only a few studies reported a control value of urine M1, which was lower than the first one and almost always<0.1 ng/mL [8], 12], 13].

Other factors influencing the detection of clostebol urine metabolites are individual metabolism, such as genetic and ethnical variations [14], creatinine concentration, but the latter was studied only by Gessner et al. [13], confirming the correlation between dilution of urine samples and the detectability of substances’ metabolites. In the case of transdermal contact, skin thickness could be a key factor influencing the detection of clostebol urine metabolites. Moreover, hand washing could increase removal of the substance from the palm of the hands. Therefore, interpretations of the data must be considered carefully.

Indeed, the case described by Pokrywka [9] appeared unprecedented because it involves the use of clostebol in spray form and its absorption both trough hand-to-dog’s paw contact, and via product inhalation.

Finally, a low urine concentration can also be interpreted as the residue of a drug voluntarily used to enhance physical performance. Illegal preparations, such as clostebol caproate or propionate tablets or injectable clostebol esters can be found on the black market [15], and their use seems to correlate to clostebol urine metabolites exceeding 2–5 ng/mL. This finding seems to be confirmed in our SR. In fact, we found that the urine concentrations of M1 derived from transdermal or transmucosal indirect contact of clostebol were slower than 5 ng/mv in all the cases and also lower than 2 ng/mL in 86 % of cases. On the other hand, after direct exposure to clostebol only one in five subjects had M1 concentration lower than 5 (and also<2) ng/mL.

Finally, the identification of clostebol in hair has been seldom reported [7], [8], [9]. Although WADA does not recognize the use of hair as routine doping control matrix, it can support athletes by demonstrating the contamination scenario.

Conclusions

In conclusion, our SR identified some flaws in the available literature. However, we found consistent data that the detection of M1 in urine can be reconcilable with a transdermal or transmucosal direct or indirect transfer of clostebol. In the cases of indirect exposure to clostebol, the urine concentrations of clostebol metabolites seem to be far lower than the concentrations found in case of therapeutic or recreative use of clostebol.