Positive Doping Test Linked to Crumb Rubber From Artificial Football Pitch: Case Report

1,3-DMBA was detected in urine samples of eight athletes following a doping control, resulting in one AAF and levels below WADA's MRL in seven teammates. Investigation revealed contamination from rubber infill used on the indoor artificial turf, with analysis confirming 1,3-DMBA in the turf's crumb rubber originating from recycled tires. There is broad agreement across the sporting world that the use of substances and methods to illegally enhance athletic performance, also known as doping, poses a threat to fair play and the health of athletes. To prevent, detect and deter doping, the World Anti-Doping Agency (WADA) is responsible for developing and harmonizing global anti-doping regulations through the World Anti-Doping Program [1]. An essential part of the World Anti-Doping Program is the Prohibited List (PL), which describes substances and methods prohibited for use by athletes [2]. While doping is often associated with deliberate and strategic use of prohibited substances or methods to gain a competitive edge, there are also examples where athletes inadvertently or without deliberate intent violate anti-doping rules, so called unintentional doping. Lately, unintentional ingestion or exposure to substances on the PL resulting in Adverse Analytical Findings (AAF) in an athlete's biological sample are an increasing concern among anti-doping professionals and athletes alike [3]. There are several ways how an athlete may inadvertently get a prohibited substance in their body [4]. These include, but are not limited to using high-risk dietary supplements [5], consuming certain foods [6], using pharmaceuticals containing substances on the PL [3] or by intimate contact [7]. It is less well known how environmental exposure to prohibited substances may trigger a positive doping test. This report describes a case in which eight post-match urine samples from an elite women's football match played on an indoor artificial turf pitch revealed one AAF finding for the prohibited stimulant 1,3-dimethylbutylamine (1,3-DMBA; 4-methylpentan-2-amine), whereas the other samples contained detectable but sub-threshold levels of the same substance. During a regular, in-competition doping control in the Norwegian premier female football division on April 22nd, 2025, eight players (four from each team) were selected for testing. All eight players provided urine samples. The football match was played on an indoor pitch using crumb rubber (styrene-butadiene rubber [SBR]) as infill on the artificial turf (Figure 1). The urine samples were subsequently sent for analysis to the WADA accredited Norwegian Doping Control Laboratory in Oslo. Out of the eight samples, one sample was returned as an AAF positive for the substance 1,3-DMBA, above the WADA determined Minimum Reporting Limit (MRL) for the substance of 50 ng/mL. In addition, the urine samples from the other seven tested athletes all had confirmed findings of the same substance, but with concentrations below the MRL, resulting in no AAF. It is not common nor mandatory practice that WADA accredited doping laboratories report analytical results below the WADA MRL. However, due to the unusual results from this doping control, with the presence of the same prohibited substance found in all collected samples from the respective control, the laboratory informed Anti-Doping Norway (ADNO) directly about the findings. Upon receiving the notification from the laboratory, several investigative and analytical steps were taken to shed light on the case and to identify the source of 1,3-DMBA. The urine samples were analyzed by liquid chromatography–tandem mass spectrometry (LC-MS/MS) using routine methods for doping substances established in the Norwegian Doping Control Laboratory. Briefly, a dilute-and-shoot method was applied where urine aliquots were diluted with water and 1,3-DMBA-d5 as internal standard. The diluted samples were analyzed using an Agilent 1290 Infinity liquid chromatograph coupled with Agilent 6490 triple quadrupole mass spectrometer (Agilent, Santa Clara, USA) in positive ionization mode. More detailed description of analysis of urine and forensic samples, including method development and results, is presented in a separate, analytical report (Gundersen et al., in preparation). Analytical results are presented in absolute values, and as means with standard deviation (SD). ADNO initiated an extensive investigation in line with the WADA International Standard for Testing and Investigations (ISTI) [8]. The forensic investigation was conducted by ADNO's intelligence officer with support from anti-doping personnel from testing, science and medicine departments. The investigation was aimed at gaining clarity into the following hypotheses: (a) Did the player(s) intentionally ingest the prohibited substance, (b) was the intake of 1,3-DMBA in any way organized, cf. findings in all players who were tested after the match, and (c) had the player(s) unintentionally been exposed to the prohibited substance. Finding the source of 1,3-DMBA was also an essential part of the investigation. The investigation included conversations with involved players and support personnel from the two teams, staff involved in catering and pitch maintenance at the indoor stadium, relevant national and local public bodies, the Norwegian Doping Control Laboratory, and the National Football Federation. The initial testing procedure (ITP) resulted in detection of 1,3-DMBA in all eight urine samples. Due to this unusual finding, all eight samples were forwarded to the confirmation procedure (CP). The CP resulted in one sample with an estimated concentration of 67 ng/mL, above the MRL (50 ng/mL) for 1,3-DMBA, and was reported as an AAF. The seven remaining samples contained detectable levels below the MRL, with estimated concentrations ranging from 12 to 45 ng/mL (28.5 ± 18.6), and hence were all reported as negative according to TDMRPL2022. Such clustered findings are unusual for this substance. Following the addition of 1,3-DMBA to the PL in 2018, a total of 37 AAFs have been reported worldwide, the majority occurring in 2018–2019, with only four cases reported in the period 2020–2024 [9]. There are limited or no data on the pharmacology, toxicology, and effects in humans on 1,3-DMBA [10]. Given both the rarity of 1,3-DMBA findings globally and the lack of toxicological data, it was necessary to evaluate whether the results could reflect systematic use among the players involved. To assess this possibility, all eight players who were tested on April 22nd were without advance notice re-tested during two following matches played on two other stadiums on May 24th and 25th. At that time (i.e., May 24th and 25th), no players were familiar with the test results from the April 22nd match. None of the urine samples collected during the May matches showed traces of 1,3-DMBA. This helped determine whether the April findings reflected ongoing use or an isolated exposure linked to the match environment. On May 26th, ADNO informed the player with the AAF (the index player) about a suspected violation of the anti-doping regulations. Conversations were conducted with the player in question on May 26th and May 27th. In the following days, information was collected from the seven other players tested after the match April 22nd. Stimulants, found in PL group S6 [2], are commonly found in contaminated supplements [5]. Amongst them, 1,3-DMBA has been found in several dietary supplements [11]. The chemical structure of 1,3-DMBA closely resembles that of methylhexanamine, and it was likely formulated to circumvent the regulatory restrictions associated with methylhexanamine [12]. The US Food and Drug Administration (FDA) considers dietary supplements containing 1,3-DMBA as adulterated [13]. Collecting details on all the players' nutritional intake on match day was thus an essential part in the early stages of the investigation. The initial investigation revealed no common denominators among the tested players, neither regarding food intake before the competition, drinks, pharmaceuticals, dietary supplements, social gatherings, nor for other possible sources. Dietary supplements and water bottles used by the involved players were analyzed, but none contained 1,3-DMBA. Athlete support personnel from both teams were questioned, including the medical officer and sports manager from one of the teams, as well as the medical officer, sports manager, equipment manager, and groundsman from the other team. These conversations did not reveal any relevant information. During the initial interviews, it was mentioned that both teams were served fruits in the locker rooms before and after the match. As this was a common factor for both teams, the fruit supply was examined as a possible contamination source. Staff at the purchasing supermarket confirmed that the fruit was not touched directly, bananas were unpeeled, and grapes were stored in closed plastic containers. Staff in the supermarket involved in handling the fruit did not use any medicine or supplements that could contaminate the fruit with 1,3-DMBA. Based on an overall assessment, fruit was excluded as a plausible source of 1,3-DMBA. The Norwegian Institute for Air Research (NILU), the Norwegian Food Safety Authority, and the local water and sewerage company were contacted for possible assistance in tracing the source. NILU had a suggestion that the 1,3-DMBA could originate from the crumbed rubber as they had done extensive research on car tires. As the initial investigations failed to identify a plausible source of the 1,3-DMBA, the investigation was expanded to the arena where the match was played, including the turf and the locker rooms. Water samples from faucets in the locker rooms and from faucets surrounding the pitch used to sprinkle the turf (not used for drinking water by the players) were collected and analyzed. In a sample from one sprinkling faucet, small crumbs of rubber granules came out of the tap along with the water, possibly contaminating the water (Figure 2). Analysis of water samples from the locker rooms was negative for 1,3-DMBA. The water sample from a sprinkling faucet that was contaminated with rubber granules was filtrated and consecutive analysis was positive for 1,3-DMBA. Investigations confirmed that the contamination of 1,3-DMBA in the water was local and not arising from the external water supply. An immediate hypothesis was that the contamination could have arisen from a local water reservoir inside the stadium, but no such reservoir existed on site. Another hypothesis was that the substance could have originated from rubber hoses used to sprinkle the pitch. To determine if these hoses were the source of 1,3-DMBA in the water sample, a piece of the hose was cut and analyzed, but no traces of the prohibited substance were found (Figure 3). This indicated that the contamination originated from loose granules entering the water line rather than from the hose material itself. Following the positive water sample, additional samples were taken from two other water taps around the pitch, in addition to samples of the artificial turfs (i.e., plastic grass) and rubber granules obtained from several locations inside the football stadium. The crumbed rubber, the artificial grass itself and the water from the water faucet used to water the pitch were all positive for 1,3-DMBA. To interpret these findings, it was necessary to consider known degradation pathways of tire-derived rubber compounds. 1,3-DMBA is described as a major hydrolysis product of the antioxidant N-(1,3-dimethylbutyl)-N′-phenyl-p-phenylenediamine (6PPD), amongst others in a report from OSPAR in 2006 [14]. 6PPD is widely used in tire rubber to protect polymer matrices from oxidative and ozone-induced degradation [14]. Its primary transformation product, 6PPD-quinone (6PPDq), has attracted considerable attention due to its high toxicity, notable to coho salmon [15]. However, compared with the rapidly expanding literature on 6PPDq, 1,3-DMBA has to date been far less studied regarding environmental occurrence, toxicology and human exposure. 1,3-DMBA was recently reported for the first time in environmental samples as an ozonation product of 6PPD from tire wear [16], and has subsequently been detected in crumb rubber and various elastomeric consumer products [17]. In a recent Spanish survey of eight artificial turf football pitches, 1,3-DMBA and 6PPD were detected and quantified in all crumb rubber samples, with the highest concentration found in newer installations [18]. The concentrations of 1,3-DMBA ranged from 0.21 to 73 μg/g. The highest and the third highest concentrations originated from the newer pitches, suggesting that 1,3-DMBA levels may be elevated following recent replacement of turf and crumb rubber. Concurrent with forensic and supplementary analytical assessments, laboratory results from urine samples of football players previously collected by ADNO under environmental conditions consistent with those described in this case were identified and re-examined by the Norwegian Doping Control Laboratory. Between March 2019 and April 2025, a total of 38 in-competition urine samples were obtained following football matches played on indoor pitches utilizing SBR as infill (Table 1). Of these samples, 22 (58%) contained 1,3-DMBA in concentrations from 12 to 67 ng/mL (26.4 ± 15.9 nm/mL). Beside the AAF which is discussed in this case report, one other sample collected in 2019 contained 1,3-DMBA above the MRL of 50 ng/mL. Both samples leading to AAFs had a concentration of 1,3-DMBA of 67 ng/mL. The AAF from 2019 was collected after a match on a different indoor football stadium (identified as arena 2 in Table 1) than the ones presented in this case report (arena 1 in Table 1). At the time of the AAF in 2019, no possible association between the crumb rubber on the pitch and the positive finding was identified. Because the athlete's urine concentration exceeded the MRL of 1,3-DMBA, the case was recorded and adjudicated as an AAF, resulting in a 2-year period of ineligibility for the athlete. Considering the new information that has emerged following the 2025 case, ADNO is in the process of assessing the potential reopening of the matter. Results from the investigation, together with published data in the research literature, point toward crumb rubber used in the artificial grass to be the probable source of 1,3-DMBA in the players' urine samples. However, the specific exposure route remains undetermined. Although literature explicitly documenting the presence of 1,3-DMBA in the human body is lacking, studies have confirmed that related tire-derived compounds such as 6PPD and 6PPD-quinone can enter the body via ingestion, inhalation of particulate matter, and dermal contact [15]. Recent experimental work has also identified 1,3-DMBA in an in vitro gastrointestinal model simulating human digestion of crumb rubber, demonstrating its oral bioaccessibility [19]. Moreover, the exposure routes documented for structurally related tire-derived compounds, including 6PPD and 6PPDq, indicate that inhalation of particulate matter and dermal contact represent plausible pathways for systemic uptake of 1,3-DMBA. Taken together, these findings suggest that oral ingestion of rubber particles, inhalation of dust or vapor from artificial turf surfaces, and dermal uptake should all be considered plausible pathways for systemic exposure, although their relative contributions cannot currently be determined [20]. Following completion of the investigation, the case was reviewed by ADNO's independent adjudication panel. Considering the documented environmental contamination associated with the indoor artificial-turf pitch, the panel determined that the circumstances did not warrant the imposition of a sanction on the athlete, based on that the athlete had acted with no fault or negligence. Given that the anti-doping framework is premised on strict liability on the part of the athlete upon the detection of a prohibited substance in the athlete's body, it was nevertheless determined that the regulations had been violated. The panel further noted that this conclusion does not establish a general precedent for future cases involving positive findings of 1,3-DMBA following play on indoor artificial turf surfaces. Rather, it emphasized that a risk of environmentally derived findings may persist on pitches containing similar infill material, and that, under the applicable anti-doping regulations, the responsibility for ensuring compliance ultimately rests with the athlete. In April 2025, eight female football players were tested by ADNO following a match played at an indoor stadium. One urine sample contained 1,3-DMBA at a concentration above the WADA MRL, constituting an AAF. The remaining seven samples contained detectable traces of 1,3-DMBA, but with values below the MRL and therefore did not meet reporting criteria for an AAF. An extensive investigation combining forensic methods, literature review and chemical analysis of turf infill identified rubber infill made from recycled car tires as the likely source of 1,3-DMBA detected in the athletes' samples. The case was subsequently assessed by ADNO's independent adjudication panel, which accepted the hypothesis regarding the source of the substance and determined that the athlete bore no fault or negligence in accordance with Article 10.5 of the WADC [1], as the exposure resulted from environmental contamination of the indoor artificial-turf pitch. The exact uptake route (i.e., orally, transdermal, or through air), and exposure levels leading to the observed urinary concentrations of 1,3-DMBA above 50 ng/mL remain unknown, and ongoing studies may help clarify these aspects. To the best of our knowledge, this is the first documented doping case linking an AAF in a urine sample to crumb rubber from a football pitch. The findings highlight the potential for artificial turfs to act as a source of contamination leading to inadvertent antidoping rule violations, and they represent another example of unintentional exposure to substances included on the WADA Prohibited list. Microsoft Copilot was used sporadically during the preparation of the manuscript to improve language and readability. The authors declare no conflicts of interest. The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.