WADA - WADA ISL Technical Letter TL27– Sample Tampering

WADA ISL Technical Letter TL27– Sample Tampering Document number: ISL TL27 Version number: 1.0

Written by: Reviewed by:

WADA Science / EAAS Working Group WADA Laboratory Expert Advisory Group Approved by: WADA Executive Committee Date: 17 March 2026 Effective date: 01 January 2027 TL27 Tampering – Version 1.0 – 01 January 2027 Page 1 / 5 Urine Sample Tampering or Attempted Tampering 1.0 Introduction WADA wishes to draw the attention of the Laboratories, Anti-Doping Organizations (ADOs) and Delegated Third Parties (DTP) in charge of Doping Control activities on behalf of ADOs to the following observations and instructions regarding circumstances where a urine Sample is suspected to have been subjected to a possible Tampering or Attempted Tampering. Whenever a urine Sample is suspected of Tampering or Attempted Tampering , the Laboratory shall perform a series of analytical assessments to determine the authenticity and validity of the Sample. These evaluations are intended to identify the absence of typical urinary constituents, the presence of atypical compounds, and/or indicators of Sample adulteration [1]. 2.0 Sample Tampering or Attempted Tampering Analytical Assessment 2.1 Initial Assessment a) Atypical Sample Appearance Upon inspection of the Sample at the time of “A” Sample aliquoting, the Laboratory shall document as part of the Sample’s records any significant Sample differences from physiological urinary characteristics (e.g., color, odor, turbidity, presence of particulates not typically found in human urine) that may indicate a possible Sample Tampering or Attempted Tampering. If necessary, the Laboratory shall inform the Testing Authority (TA) and seek instructions on the performance of Analytical Testing on the Sample, in writing, as soon as possible.

[Comment to Article 2.1 a): As per the International Standard for Laboratories (ISL) Article 5.3.2.1 [2], the TA shall inform the Laboratory, in writing within seven (7) days, whether a urine Sample with the noted Sample appearance irregularity(- ies) shall be analyzed or not, and/or of any further measures to be taken (e.g., forensic analysis, DNA analysis), or that the Sample should be stored for Further Analysis. The communication between the Laboratory and the TA shall be recorded as part of the Sample’s documentation. In the absence of a timely reply (within seven (7) days) by the TA, the Laboratory should report the Sample as “Not Analyzed” in ADAMS or, at the Laboratory’s discretion, analyze the Sample for other indicators of Sample Tampering or Attempted Tampering as described below.] Differences between the “A” and “B” Samples shall be handled as per ISL TL14 [3]. It is recommended that pictures of both the “A” and “B” Samples be taken wherever relevant. b) Specific Gravity and pH The Laboratory’s initial assessment of the Specific Gravity ( SG) and pH values outside the physiological range, i.e., - SG < 1.001 [4,5] and pH < 4.0 or > 9.0 [6]. [Comment to Article 2. 1 b): Differences between SG and pH values obtained from Sample collection and Laboratory measurements may be observed and not necessarily indicate possible Tampering or Attempted Tampering]WADA ISL Technical Letter TL27– Sample Tampering Document number: ISL TL27 Version number: 1.0

Written by: Reviewed by:

WADA Science / EAAS Working Group WADA Laboratory Expert Advisory Group Approved by: WADA Executive Committee Date: 17 March 2026 Effective date: 01 January 2027

TL27 Tampering – Version 1.0 – 01 January 2027 Page 2 / 5 c) Evaluation of the Urinary Steroid Markers The absence or suppression of the urinary steroid Markers [7] may suggest non-urine matrices or

TL27 Tampering – Version 1.0 – 01 January 2027 Page 2 / 5 c) Evaluation of the Urinary Steroid Markers The absence or suppression of the urinary steroid Markers [7] may suggest non-urine matrices or a diluted Sample and, therefore, may further support the possibility of Sample Tampering or Attempted Tampering. Wherever applicable, the Laboratory should inform the relevant TA or Results Management Authority (RMA, if different) or the Passport Custodian and recommend that the associated Athlete Passport Management Unit (APMU) investigate the Athlete’s steroidal Passport with particular attention to the suspected Sample. [Comment to Article 2.1.c: the substitution of an Athlete’s urine Sample with the urine of another individual (urine exchange) can also be uncovered using the steroidal Passport and confirmed by DNA analysis across multiple Samples, as described in the ISL TD APMU [8].] d) Evaluation of Other Endogenous Substances In addition, depending on the analytical capability of the Laboratory, the evaluation of other endogenous substances typically excreted in urine, such as 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), cortisol and cortisone , should be performed. Moreover, other relevant endogenous substances that may be evaluated include pregnanediol (PD), pregnanetriol (PT), 5α-androst-16-en-3α-ol (16 -en), 11β -hydroxy-androsterone (11 -OH-A), and 11 -oxoetiocholanolone (11-oxo-Etio). The absence or relatively low concentrations of these additional endogenous substances should be assessed in conjunction with other physiological parameters to determine the consistency of the Sample with normal human urine . If GC/C/IRMS [9] analysis of any endogenous substances listed above has been performed, and the carbon isotope ratio values are incompatible with human reference ranges, the data may also be used to support the investigation of Tampering or Attempted Tampering.

the Sample with normal human urine . If GC/C/IRMS [9] analysis of any endogenous substances listed above has been performed, and the carbon isotope ratio values are incompatible with human reference ranges, the data may also be used to support the investigation of Tampering or Attempted Tampering. e) Evaluation of Proteolytic Activity The Laboratory may assess the presence and activity of proteolytic enzymes (proteases), which can degrade endogenous proteins normally present in urine Samples. The Laboratory may analyze the integrity of specific endogenous proteins commonly found in urine, such as endogenous erythropoietin (EPO) [10], albumin, and others. The d etection of abnormal fragmentation patterns or the absence of intact proteins , particularly when corresponding unexpected degradation products (peptides or fragments) are present , may provide evidence of protease activity. In addition, proteolytic enzymes may be detected directly using enzyme activity assays or mass spectrometry-based proteomics targeting known protease Markers. The presence of active proteases in properly stored urine Samples is unusual and may indicate Tampering or Attempted Tampering.WADA ISL Technical Letter TL27– Sample Tampering Document number: ISL TL27 Version number: 1.0

Written by: Reviewed by:

WADA Science / EAAS Working Group WADA Laboratory Expert Advisory Group Approved by: WADA Executive Committee Date: 17 March 2026 Effective date: 01 January 2027

TL27 Tampering – Version 1.0 – 01 January 2027 Page 3 / 5 2.2 Further Assessment The Laboratory shall conduct the further assessments when the initial evaluations described in Article 2.1 produce abnormal results. If the Laboratory lacks the analytical capacity to perform the required additional analyses, the Laboratory may, in consultation with the relevant TA (or RMA, if different), subcontract the analysis to another Laboratory or WADA-authorized laboratory that has the procedure included in its ISO/IEC 17025 [11] Scope of Accreditation.

additional analyses, the Laboratory may, in consultation with the relevant TA (or RMA, if different), subcontract the analysis to another Laboratory or WADA-authorized laboratory that has the procedure included in its ISO/IEC 17025 [11] Scope of Accreditation. a) Concentrations of creatinine, electrical conductivity, and other physiological constituents of urine The Laboratory is not required to have Analytical Testing Procedures (ATPs) [12] for the measurement of creatinine, urinary electrical conductivity, or any other physiological constituents of urine (e.g., glucose/sugars, electrolytes, uric acid, etc. ) that may be used as corroborating evidence to determine the origin or authenticity of the Sample; however, assessing these parameters may help identify a possible case of Tampering or Attempted Tampering. Therefore, the Laboratory may consider implementing or subcontracting [13] such analysis. [Comment to Article 2.2.a): All procedures used in these evaluations shall be conducted using validated Test Methods. If the Laboratory subcontracts the analysis, the subcontracted laboratory shall be accredited to the ISO/IEC 17025 [11] (or ISO 15189 [14]) for the applied analytical procedures.] Concentrations significantly different from normal ranges for [6]: - Creatinine < 20 mg/dL (i.e., < 200 mg/L). - Urinary electrical conductivity < 1mS/cm [15]. These suggested values may be used to indicate that the Sample’s urinary parameters are significantly different from normal ranges and may therefore not be representative of authentic urine. A complete record of all results, interpretations, and justifications shall be documented and maintained as part of the Sample’s records. b) Detection of Atypical Compounds The Laboratory may analyze and evaluate results for non-physiological compounds that are not typically found in urine Samples or that are found in concentrations inconsistent with normal excretion patterns [16, 1 7]. This includes substances commonly associated with commercial beverages (e.g., high levels of caffeine, artificial sweeteners, or colorants) , industrial products

typically found in urine Samples or that are found in concentrations inconsistent with normal excretion patterns [16, 1 7]. This includes substances commonly associated with commercial beverages (e.g., high levels of caffeine, artificial sweeteners, or colorants) , industrial products (protease reagents), or even related to the preparation of synthetic urine . While some of these compounds may be present in urine following dietary intake or supplementation, unusually h igh concentrations and/or unexpected combinations may indicate Sample Tampering or Attempted Tampering. [Comment to Article 2.2.b: An ATP applied by the Laboratory for the evaluation of the presence of non-prohibited atypical compounds is not required to be ISO/IEC 17025 accredited [11], and such compounds may be detected inWADA ISL Technical Letter TL27– Sample Tampering Document number: ISL TL27 Version number: 1.0

Written by: Reviewed by:

WADA Science / EAAS Working Group WADA Laboratory Expert Advisory Group Approved by: WADA Executive Committee Date: 17 March 2026 Effective date: 01 January 2027

TL27 Tampering – Version 1.0 – 01 January 2027 Page 4 / 5 Samples without the need for identification in compliance with the ISL TD IDCR [18]. Nevertheless, the Laboratory shall have established Quality Assurance requirements for the procedure applied.] 2.3 Urine Substitution: DNA Profiling If deemed necessary, the Laboratory may perform DNA profiling to confirm whether the Sample contains human DNA and, if so, whether the DNA profile matches that of the Athlete. If necessary, a reference Sample of the Athlete shall be obtained in consultation with the TA (or RMA, if different) or WADA. [Comment 1 to Article 2.3: For the avoidance of doubt, the absence of DNA is not conclusive evidence that the Sample is non-human or that it has been substituted, as DNA may be present in insufficient quantities in urine for profiling.]

WADA. [Comment 1 to Article 2.3: For the avoidance of doubt, the absence of DNA is not conclusive evidence that the Sample is non-human or that it has been substituted, as DNA may be present in insufficient quantities in urine for profiling.] [Comment 2 to Article 2.3: The Laboratory shall inform WADA when DNA profiling analysis needs to be performed. WADA will then verify whether another Sample belonging to the same Athlete has been previously collected and is available to be used as a reference Sample.] If the Laboratory lacks the analytical capacity to perform the DNA analysis, the Laboratory may, in consultation with the relevant TA (or RMA, if different) , subcontract the DNA analysis to another Laboratory or WADA-authorized laboratory that has the procedure included in its ISO/IEC 17025 [13] Scope of Accreditation. 3.0 Reporting of Sample Tampering or Attempted Tampering 3.1 Adverse Analytical Finding The Laboratory shall report the finding as an Adverse Analytical Finding (AAF) for Tampering or Attempted Tampering if the Laboratory can determine that the general nature/type of the adulterated Sample is not consistent with human urine, i.e., when: a) The assessments described in Article 2.1 show irregular results together with anomalous results obtained with the assessments described in Article 2.2, and/or b) The DNA profiling performed according to Article 2.3 concludes that the Athlete’s urine has been substituted. 3.2 Atypical Finding The Laboratory shall report the finding as an Atypical Finding (ATF) for Tampering or Attempted Tampering if the assessments described in Article 2.1 show irregular results without the observation of anomalous results in the assessments described in Articles 2.2 and/or 2.3. The Laboratory shall also include a comment in ADAMS advising the TA (or RMA, if different) to perform further investigations (e.g., additional analyses on the Sample, Target Testing the Athlete, or

of anomalous results in the assessments described in Articles 2.2 and/or 2.3. The Laboratory shall also include a comment in ADAMS advising the TA (or RMA, if different) to perform further investigations (e.g., additional analyses on the Sample, Target Testing the Athlete, or placing the Sample in long-term storage for Further Analysis).WADA ISL Technical Letter TL27– Sample Tampering Document number: ISL TL27 Version number: 1.0

Written by: Reviewed by:

WADA Science / EAAS Working Group WADA Laboratory Expert Advisory Group Approved by: WADA Executive Committee Date: 17 March 2026 Effective date: 01 January 2027

TL27 Tampering – Version 1.0 – 01 January 2027 Page 5 / 5 4.0 References [1] Piper, T., et al. "When is a sample a urine sample? Markers for urine sample authenticity assessment in sports drug testing." Bioanalysis (2025): 1-9. [2] The World Anti-Doping Code International Standard for Laboratories (ISL). [3] WADA Technical Letter ISL TL14 – Difference in “A” and “B” Sample Urine Characteristics. [4] Edgell, Kenneth, et al. "The defined HHS/DOT substituted urine criteria validated through a controlled hydration study." Journal of Analytical Toxicology 26.7 (2002): 419-423. [5] Athanasiadou, I., et al. "The effect of athletes hyperhydration on the urinary ‘steroid profile’ markers in doping control analysis." Drug Testing and Analysis 10.9 (2018): 1458-1468. [6] Department of Health and Human Services. “Mandatory guidelines for federal workplace drug testing programs." Substance Abuse and Mental Health Services Administration, Fed Reg (2023): 70814-50. [7] WADA Technical Document ISL TD USM: Analytical and Reporting Requirements for the Urinary Markers of the Steroidal Module of the Athlete Biological Passport.

Substance Abuse and Mental Health Services Administration, Fed Reg (2023): 70814-50. [7] WADA Technical Document ISL TD USM: Analytical and Reporting Requirements for the Urinary Markers of the Steroidal Module of the Athlete Biological Passport. [8] WADA Technical Document ISL TD APMU: Athlete Passport Management Unit – Requirements and Procedures. [9] WADA Technical Document ISL TD IRMS: Detection of Synthetic Forms of Prohibited Substances by GC/C/IRMS. [10] WADA Technical Document ISL TD EPO: Harmonization of Analysis and Reporting of Erythropoietin (EPO) and other EPO-Receptor Agonists (ERAs) by Polyacrylamide Gel Electrophoretic (PAGE) Analytical Methods. [11] ISO/IEC 17025:2017 - General Requirements for the Competence of Testing and Calibration Laboratories. [12] WADA Technical Document ISL TD ATP: Analytical Testing Procedures [13] WADA Laboratory Guidelines : Conducting and Reporting Subcontracted Analysis and Further Analysis for Doping Control. [14] ISO 15189:2022 - Medical Laboratories — Requirements for Quality and Competence. [15] Fazil Marickar, Y.M. Electrical conductivity and total dissolved solids in urine. Urol Res 38, 233–235 (2010). [16] Segura, Jordi, et al. "If you play with fire, you may get burned." Drug Testing and Analysis 12.5 (2020): 582-587. [17] Alaejos, Ana Rubio, et al. "Evaluation of doping control samples to determine the prevalence of nicotine use by German elite athletes." Manfred Donike Workshop on Dope Analysis: Cologne Workshop on Dope Analysis . Sportverlag Strauß, 2021. [18] WADA Technical Document ISL TD IDCR: Minimum Criteria for Chromatographic -Mass Spectrometric Confirmation of the Identity of Analytes for Doping Control Purposes.

Sportverlag Strauß, 2021. [18] WADA Technical Document ISL TD IDCR: Minimum Criteria for Chromatographic -Mass Spectrometric Confirmation of the Identity of Analytes for Doping Control Purposes.

[Comment to Article 4.0: Current versions of WADA’s ISL, Technical Documents, Technical Letters and Laboratory Guidelines may be found at https://www.wada-ama.org/en/what-we-do/science-medical/laboratories]