Vol.:(0123456789) Archives of Toxicology (2024) 98:3337–3350 https://doi.org/10.1007/s00204-024-03830-2 MOLECULAR TOXICOLOGY Interpreting mono‑ and poly‑SCRA intoxications from an activity‑based point of view: JWH‑018 equivalents in serum as a comparative measure Liesl K. Janssens1   · Michaela J. Sommer2,3,4 · Katharina Elisabeth Grafinger2,5   · Maren Hermanns‑Clausen3,6   · Volker Auwärter2,3   · Christophe P. Stove1  Received: 1 May 2024 / Accepted: 25 July 2024 / Published online: 8 August 2024 © The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2024 Abstract Synthetic cannabinoid receptor agonists (SCRAs) are a class of synthetic drugs that mimic and greatly surpass the effect of recreational cannabis. Acute SCRA intoxications are in general difficult to assess due to the large number of compounds involved, differing widely in both chemical structure and pharmacological properties. The rapid pace of emergence of unknown SCRAs hampers on one hand the timely availability of methods for identification and quantification to confirm and estimate the extent of the SCRA intoxication. On the other hand, lack of knowledge about the harm potential of emerging SCRAs hampers adequate interpretation of serum concentrations in intoxication cases. In the present study, a novel compara- tive measure for SCRA intoxications was evaluated, focusing on the cannabinoid activity (versus serum concentrations), which can be measured in serum extracts with an untargeted bioassay assessing ex vivo CB1 activity. Application of this principle to a series of SCRA intoxication cases (n = 48) allowed for the determination of activity equivalents, practically entailing a conversion from different SCRA serum concentrations to a JWH-018 equivalent. This allowed for the interpreta- tion of both mono- (n = 34) and poly-SCRA (n = 14) intoxications, based on the intrinsic potential of the present serum levels to exert cannabinoid activity (cf. pharmacological/toxicological properties). A non-distinctive toxidrome was confirmed, showing no relation to CB1 activity. The JWH-018 equivalent was partly related to the poison severity score (PSS) and causality of the clinical intoxication elicited by the SCRA. Altogether, this equivalent concept allows to comparatively and timely interpret (poly-)SCRA intoxications based on CB1 activity. Keywords  Bioassay · Biological matrices · Serum · Patient · Cannabinoid · CB1 Introduction SCRAs are chemically synthesized drugs that interact with the cannabinoid receptors CB1 and CB2, mimicking (and often greatly exceeding) the cannabimimetic effects of Δ9- tetrahydrocannabinol (Δ9-THC), the primary psychoactive component in cannabis (Winstock and Barratt 2013; Casta- neto et al. 2014; De Oliveira et al. 2023). These synthetic variants were initially developed in the 1970s to study the endocannabinoid system and to explore potential thera- peutics (Castaneto et al. 2014; De Luca and Fattore 2018; Sholler et al. 2020). However, many SCRAs have found their way into the recreational drug market, spearheaded by JWH-018, a preclinical compound which was confirmed in an unregulated recreational drug product in Germany in 2008 (Auwärter et al. 2009). JWH-018 and CP47,497-C8 became the first SCRAs identified in these herbal blends and * Christophe P. Stove christophe.stove@ugent.be 1 Laboratory of Toxicology, Department of Bioanalysis, Faculty of Pharmaceutical Sciences, Ghent University, Ghent, Belgium 2 Institute of Forensic Medicine, Forensic Toxicology, Medical Center, University of Freiburg, Freiburg, Germany 3 Faculty of Medicine, University of Freiburg, Freiburg, Germany 4 Hermann Staudinger Graduate School, University of Freiburg, Freiburg, Germany 5 Institute of Chemistry and Bioanalytics, University of Applied Sciences and Arts Northwestern Switzerland, Muttenz, Switzerland 6 Department of General Pediatrics, Adolescent Medicine and Neonatology, Poisons Information Center, Center for Pediatrics, Medical Center, University of Freiburg, Freiburg, Germany http://orcid.org/0000-0001-9740-5036 http://orcid.org/0000-0002-3647-7455 http://orcid.org/0000-0002-9803-7324 http://orcid.org/0000-0002-1883-2804 http://orcid.org/0000-0001-7126-348X http://crossmark.crossref.org/dialog/?doi=10.1007/s00204-024-03830-2&domain=pdf 3338 Archives of Toxicology (2024) 98:3337–3350 were soon classified as new psychoactive substances (NPS) (European Monitoring Centre for Drugs and Drug Addic- tion 2009; Auwärter et al. 2009). Over fifteen years later, SCRAs are the largest class of NPS, comprising of more than 245 substances monitored by the European Monitoring Centre for Drugs and Drugs Addiction (EMCDDA), creating a highly dynamic drug market with compounds disappearing and emerging continuously (European Monitoring Centre for Drugs and Drug Addiction 2023). SCRAs are generally more active at the cannabinoid receptors than Δ9-THC (Banister and Connor 2018). This entails a greater risk of toxicity by increasing the chance of side effects of SCRAs in comparison to Δ9-THC (De Oliveira et al. 2023). SCRAs have appealed to both can- nabis and polydrug users as they are perceived to be safer than other drugs of abuse, easily accessible over the inter- net and may avoid positive cannabinoid urine tests (Van- drey et al. 2012; Castaneto et al. 2014; De Oliveira et al. 2023). The worldwide use and the greater toxicity associ- ated with SCRAs is evident from the numerous case reports and observational studies, reporting on various toxicities for different SCRAs, as recently reviewed by De Oliveira et al. (De Oliveira et al. 2023). Many different SCRAs have been listed in intoxication reports, including AB-CHMINACA, ADB-CHMINACA, AB-FUBINACA, MDMB-CHMICA, 5F-ADB, JWH-018 and 5F-AKB-48 (or 5F-APINACA), which have been associated with a wide range of toxico- logical effects (De Oliveira et al. 2023). Importantly, SCRAs encompass a large number of highly diverse compounds, both in terms of chemical structure as well as in terms of pharmacological properties (Banister and Connor 2018; Banister et al. 2019; Cannaert et al. 2020b; Grafinger et al. 2021b). On one hand, the rapid pace of struc- tural evolution hampers the effective monitoring, analysis, and characterization of SCRAs. On the other hand, the evi- dent wide range in pharmacological properties of SCRAs, including highly potent and highly efficacious compounds, impedes clinical understanding of SCRA intoxications. Substantial efforts are being made with structure–activity relationship studies to understand (and predict) the cor- responding changes in pharmacology induced by (small) differences in chemical structure (Wiley et al. 1998; Noble et al. 2019; Sparkes et al. 2021; Grafinger et al. 2021a, b; Pike et al. 2021; Janssens et al. 2023). Timely characteriza- tion is of utmost importance to identify harmful compounds, aid clinical interpretation and guide prioritization of legisla- tive efforts. In addition, pro-active analysis (e.g., untargeted screening with activity-based assays/high resolution mass spectrometry (HRMS)) and timely development of identifi- cation and quantification methods (mass spectrometry) for SCRAs are invaluable to get a hold of upcoming/ongoing public health threats and aid clinical diagnosis (confirma- tion) (Cannaert et al. 2018, 2019; Janssens et al. 2022a). Smaller doses of SCRAs are linked to higher degrees of toxicity (as compared to Δ9-THC), however, the danger (extent of toxicity and harmful dose) is hypothesized to depend highly on the individual SCRA and its pharma- cological profile (De Oliveira et al. 2023). Acute SCRA intoxications are, therefore, difficult to identify/interpret for multiple reasons: (i) challenging clinical diagnosis because of unexpected effects and absence of a distinct toxidrome to identify SCRA intoxications, (ii) rapid pace of emergence of unknown SCRAs and the (possibly) delayed availability of identification/quantification methods to confirm and estimate the extent of the SCRA intoxication, (iii) (possible) lack of knowledge on the SCRA pharmacology and harm potential, including toxic doses, (iv) challenging interpretation of (fre- quent) poly-SCRA intoxications. SCRA concentrations in blood/plasma/serum do not pro- vide a full picture of the extent of a SCRA intoxication, since the latter also requires insight into the SCRAs’ harm potential and the combined effect of multiple SCRAs. In a recent study involving mono-SCRA intoxications with 5F-MDMB-PICA, we linked the concentrations in serum to the ex vivo cannabinoid activity, as measured with a func- tional assay applied to serum sample extracts and predicted by pharmacological profiling of 5F-MDMB-PICA (Janssens et al. 2022b). In the present study, we took this one step further and aimed at comparatively evaluating recent SCRA intoxications involving different SCRAs and including both mono- and poly-SCRA intoxications. To aid the (clinical) interpretation of individual intoxica- tion cases, we propose in this study the concept of activity equivalents as a means to estimate and compare the extents of SCRA intoxications from a cannabinoid activity point of view. For the present study, a unique data set of SCRA intoxications from the Poisons Information Center Freiburg was used, combined with serum SCRA concentrations and assessment of ex vivo CB1 activation in serum with an in vitro cell-based assay. JWH-018 was used as the ‘refer- ence SCRA’ for normalization to provide a comparative and comprehensible measure to express the cannabinoid activ- ity (i.e., JWH-018 equivalents), since this firstly identified SCRA is widely used as a reference compound in SCRA research. This activity equivalent concept considers a com- bination of contributing toxicity factors (SCRA concentra- tions, receptor activation potential, etc.) for the estimation of the extent of an intoxication. We validated the relation- ship between the measured JWH-018 equivalentMEAS and the SCRA concentrations and SCRAs’ intrinsic pharmacol- ogy, using a mathematical approach to derive a calculated JWH-018 equivalentCALC. We hypothesize that the use of this concept should facilitate the comparative evaluation of intoxications involving one or multiple SCRAs of various origins and with different pharmacological properties, with- out requiring prior pharmacology investigation. 3339Archives of Toxicology (2024) 98:3337–3350 Materials and methods Materials Dulbecco’s Modified Eagle’s Medium (DMEM Glu- taMAX™), Opti-MEM I Reduced Serum Medium (Opti- MEM I), penicillin–streptomycin (5 000 U/mL) and amphotericin B (250 μg/mL) were supplied by Thermo Fisher Scientific (Merelbeke, Belgium). JWH-018 (1-pen- tyl-3-(1-naphthoyl)-indole) was purchased from Cayman Chemical Company (Ann Arbor, MI, US). Deionized water was prepared in-house using a Medica® Pro deion- izer from ELGA (Celle, Germany) in Freiburg (Germany). Fetal bovine serum (FBS), sodium bicarbonate and poly- D-lysine were obtained from Sigma Aldrich (Overijse, Belgium). The Nano-Glo® Live Cell reagent was procured from Promega (Madison, WI, US). For sample preparation in Belgium, hexane and ethyl acetate were purchased from CHEM-LAB NV (Zedelgem, Belgium) and sodium car- bonate (≥ 99.5%, anhydrous) was purchased from Merck (Hoeilaart, Belgium). Methanol (MeOH) (HiPerSolv CHROMANORM®) and acetonitrile (ACN) (HiPerSolv CHROMANORM®) were purchased from VWR Chemi- cals (Leuven, Belgium). For the analytical analysis in Freiburg, ethyl acetate (p.a.) was obtained from Honeywell Riedel-de Häen® (Seelze, Germany), sodium hydrogen carbonate (≥ 99.5%, anhydrous) and formic acid (p.a.) were from Carl Roth GmbH (Karlsruhe, Germany) and ammonium formate was obtained from Sigma–Aldrich (Steinheim, Germany). Intoxication cases This study included intoxication cases from a prospective observational study of patients treated in the emergency departments after the consumption of an NPS, as previ- ously described (Hermanns-Clausen et al. 2018; Sommer et al. 2022). Roughly 250 German emergency departments (mainly in southern Germany) reported to the Poisons Information Center Freiburg about NPS intoxications. Study inclusion criteria for the observational study were the following: patients treated in an emergency department after reported or suspected NPS intake and whose treat- ing physician contacted the Poisons Information Center. Patient recruitment was conducted with informed consent and for patients aged under 18, the consent of the caregiv- ers was obtained. Physicians recorded clinical symptoms and follow-up information through a structured ques- tionnaire. Collected serum samples and completed ques- tionnaires were sent to the Poisons Information Center Freiburg. Serum samples were analyzed by the Institute of Forensic Medicine Freiburg via liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS). The study was conducted in accordance with the Declaration of Helsinki and approved by the regional ethics committee of the University of Freiburg (No. 235/13_130683). Intoxica- tion cases that were included in this specific study are the cases for which SCRA use was confirmed via LC–MS/MS and for which the sample volume allowed for additional investigation with the bioassay. LC–MS/MS analysis of serum samples Sample preparation of the serum samples was performed using liquid–liquid extraction (LLE). Briefly, 200 µL of serum or blood was extracted with 200 µL ammonium formate and 1 mL ACN. Part of the supernatant (800 µL) was evaporated to dryness (N2, 40 °C) and reconstituted in mobile phase. Quantification of the samples was per- formed by LC–MS/MS analysis using an Ultimate 3000RS UHPLC (Dionex, Sunnyvale, USA) coupled to a QTRAP® 6500 triple quadrupole-linear ion trap instrument (SCIEX, Darmstadt, Germany) with positive electrospray ionization (ESI). A Kinetex® C18 column (2.6 μm, 100 Å, 100 × 2.1 mm; Phenomenex, Aschaffenburg, Germany) was used for chromatographic separation. The mobile phase consisted of 1% v/v ACN, 0.1% v/v HCOOH, 2 mM ammonium formate in water (mobile phase A) and 0.1% v/v HCOOH, 2 mM ammonium formate in ACN (mobile phase B), which were prepared freshly prior to analysis. The total LC run time was 12 min, using the following gradient: mobile phase B starting at 20%, linearly increased to 60% in 1.5 min, further increased to 65% in 1.5 min, held at 65% for 1.5 min, further increased to 90% in 2.5 min, held at 90% for 2 min, and then decreased to starting conditions in 0.1 min and held for 1.9 min for re-equilibration. The autosampler was constantly kept at 10 °C and the column oven temperature was 40 °C. The injection volume was 10 µL. The mass spectrometer was operated with positive electrospray ionization in multiple reaction monitoring (MRM) mode. The scheduled multiple reaction monitoring (sMRM) method included two tran- sitions for each analyte and one transition for the internal standard. MRM transitions were recorded in a time window of ± 22.5s around the expected retention time. For each com- pound, the declustering potential (DP), the entrance poten- tial (EP), the collision energy (CE), and cell exit potential (CXP) were optimized. Sample preparation for bioassay Serum samples (130 to 200 µL) were mixed with 500 µL of carbonate buffer (pH 10) in a glass tube (15.5 × 100 mm). Addition of 3 mL of N-hexane:ethyl acetate mix- ture (99:1 V/V), followed by extensive mixing (1 min) 3340 Archives of Toxicology (2024) 98:3337–3350 and centrifugation (10 min at 2500 rpm), allowed LLE of the SCRAs. The organic phase was transferred to another glass tube (16 × 100 mm) and evaporated to dryness at 40 °C under a stream of nitrogen (Zymark Turbovap, Zymark Ltd., Chesire, UK). Calibration standards were prepared from blank serum, extracted as described above and post-extraction spiked with stock solutions of JWH-018 (1:1 MeOH/Opti-MEM I) to yield extract concentrations of 5, 10, 25, 50, 100, 500, 1000, 5000, 10 000 ng/mL. Cell culture and cannabinoid reporter assay A previously reported live cell-based reporter assay that monitors protein − protein interactions (recruitment of truncated β-arrestin 2 to the cannabinoid 1 receptor) via the NanoLuc® Binary Technology was used to assess the can- nabinoid activity in the biological samples (Cannaert et al. 2018). Cells, stably expressing the CB1 reporter system, were routinely maintained at 37 °C, 5% CO2, under humidi- fied atmosphere in DMEM (high glucose) supplemented with 10% heat-inactivated FBS, 100 IU/mL of penicillin, 100 g/mL of streptomycin, and 0.25 g/mL of amphotericin B (Cannaert et al. 2018). For experiments, cells were plated on poly-D-lysine coated 96-well plates at 5 × 104 cells/well and incubated overnight. The cells were washed twice with Opti-MEM I to remove any remaining FBS, and 100 µL of Opti-MEM I was added. The Nano-Glo Live Cell reagent (Promega, Madison, USA), a nonlytic detection reagent containing the cell-permeable furimazine substrate, was prepared by diluting the Nano-Glo Live Cell substrate 1:20 in Nano-Glo LCS Dilution buffer and 25 µL was added to the wells. Subsequently, luminescence was measured in a Tristar2 LB 942 Multimode Microplate Reader (Berthold, Technologies GmbH & Co., Bad Wildbad, Germany) to establish equilibration of the signal (10–15 min). For the evaluation of biological extracts, evaporated extracts were reconstituted in 100 µL of 1:1 MeOH/Opti-MEM I, of which 10 µL was added per well. Luminescence was monitored continuously over a period of 120 min. In every experi- ment, two calibration curves (JWH-018, 5–10,000 ng/mL) and reconstituted blank extracts were taken along. The final concentration of methanol (3.7%) did not interfere with the viability of the cells in the short term or with the readout of the bioassay. Data analysis All samples and calibrators were run in duplicate. Curve fit- ting of the calibration curves was performed using GraphPad Prism 9 software (San Diego, CA, US) via nonlinear regres- sion (four parameter logistic fit; 4PL). The data are repre- sented as mean areas under the curve (AUC) ± standard error of means (SEM) with two replicates for each data point. The AUC values are corrected for interwell variability and solvent control. Curve fitting allowed to use the JWH-018 calibration curve to calculate JWH-018 equivalentMEAS in the biological samples (including correction for the sample enrichment) based on the detected CB1 activity, using the following formula (Eq. 1): In this formula a is the minimal value, b is the hill’s slope, c is the point of inflection, d is the maximal value, x is the concentration, and y is the AUC. Kruskal–Wallis analysis was used for comparison of the hill’s slope and the point of inflection over different runs. For the determination of JWH- 018 equivalentsMEAS, numerical values were only assigned for signals within the range of the calibration curve. Sam- ples with mean AUC values above or below the range of the fitted calibration curve were classified as either too high or too low to be calculated by the bioassay, respectively. Depending on the signal obtained, samples were assigned the label > 5000 ng/mL (expressed as serum concentration, calculated assuming a starting serum volume of 200 µL) when the signal exceeded that of the highest calibrator and as ‘non-quantifiable’ (NQ, i.e., activity detected, but below the lowest point of the calibration curve) if the signal lay below the bottom of the sigmoidal calibration curve or if the calculated concentration was below the lowest calibrator (5 ng/mL in extract), corresponding to a serum concentration of 2.5 ng/mL (assuming a starting serum volume of 200 µL). Those samples for which the signals were not distin- guishable from blanks, were labeled as ‘no activity detected’ (ND). The determination ‘ND’ or ‘NQ’ was based on visual interpretation of the CB1 activation profile, objectified by use of a decision tree (Janssens et al. 2022a). Results LC–MS/MS analysis Identified compounds and serum concentrations are listed for the individual samples in Supplementary Information. In total, twelve different SCRAs were quantified: 5F-ADB (n = 14, 0.4–9.7 ng/mL), MDMB-CHMICA (n = 12, 0.04–10.0 ng/mL), MDMB-4en-PINACA (n = 10, 0.1–0.56 ng/mL), ADB-CHMINACA (n = 8, 0.23–7.80 ng/mL), 5F-MDMB-PICA (n = 6, 0.1–45 ng/mL), AB-CHMINACA (n = 5, 7.3–23 ng/mL), AB-FUBINACA (n = 3, 0.63–4.5 ng/mL), AMB-CHMICA (n = 1, 0.41 ng/mL), 5F-AKB-48 (n = 1, 2.48 ng/mL), CUMYL-PEGACLONE (n = 1, 1.2 ng/ mL), 4F-MDMB-BICA (n = 1, 5.2 ng/mL) and AB-PINACA (1)x = c ( a − d y − d − 1 )1∕b . 3341Archives of Toxicology (2024) 98:3337–3350 (n = 1, 0.33 ng/mL). MDMB-4en-PINACA and 5F-MDMB- PICA were also detected below their lower limit of quanti- fication (LLOQ: < 0.1 ng/mL, n = 7 and n = 1, respectively). In these cases, the LLOQ was used as quantitative result for further data analysis. Determination of JWH‑018 equivalents An in vitro, cell-based bioassay was used to assess CB1 acti- vation by the SCRAs in the extracts from serum samples from individual intoxication cases (n = 48) alongside cali- bration curves of a reference SCRA, JWH-018 (Cannaert et al. 2020a). This bioassay measures cannabinoid activity via monitoring the recruitment of β-arrestin2 (coupled to one part of a split-nanoluciferase) to activated CB1 (cou- pled to the complementing part of this split-nanoluciferase) (Cannaert et al. 2016, 2018). Functional complementation of the coupled split-nanoluciferase enables the generation of a bioluminescent signal in the presence of a substrate (furima- zine), which is a direct measurement of the extent of receptor activation during the 2 h assay (Dixon et al. 2016; Cannaert et al. 2016, 2018). The intoxication samples were measured in different runs and in every run two calibration curves were included for the reference SCRA JWH-018 (extract concentrations ranging from 5 to 10,000 ng/mL). Via nor- malization of the AUC signals (cumulative luminescence over 2 h) to the maximal receptor activation of JWH-018 in each range, confirmation of overlap between calibration curves was established (Fig. 1). Statistical analysis did not reveal any significant difference between the hill’s slopes (b, Eq. 1) and the points of inflection (c, Eq. 1) of the calibration curves from the different runs. This confirms the robustness of the bioassay towards detection of cannabinoid activity in serum extracts. As reported before, this sigmoidal calibra- tion model enables estimation of drug concentrations with the applied bioassay (Cannaert et al. 2020a). The calibration model allows to derive a novel com- parative measure for cannabinoid activity in the biological samples, through the determination of activity equivalents. Based on the bioassay signal that was obtained for the extracts of the serum samples (involving various SCRAs and metabolites) we derived “JWH-018 equivalentsMEAS” (Table 1), using the sigmoidal calibration model (Eq. 1). Essentially, this JWH-018 equivalentMEAS entails that the cannabinoid activity, which was measured in the serum extracts, relates to a certain serum concentration (x ng/ mL) if the present SCRA would have been JWH-018. In other words, this JWH-018 equivalentMEAS would result in the same cannabinoid activity in the bioassay as the (con- centration of) SCRA(s) present in the serum extract. Such JWH-018 equivalentsMEAS could be determined in 30 sam- ples, covering a theoretical JWH-018 range of 3.6–1048 ng/ mL. Two samples showing pronounced CB1 activity, with signals exceeding that of the fitted calibration curve, were semi-quantified as > 5000 ng/mL. In 11 samples CB1 activity was detected, yet no numerical value could be derived as the obtained signals, though distinguishable from blanks, were below the lowest point of the JWH-018 calibration curve, with 2.5 ng/mL as corresponding JWH-018 equivalentMEAS (assuming a serum volume of 200 µL) (Janssens et  al. Fig. 1   Overlap of all JWH-018 calibration curves from three inde- pendent experiments, each encompassing two independently prepared sets of calibrators. Data are normalized to the maximal JWH-018 receptor activation within each separate calibration curve Table 1   Measured JWH-018 equivalentsMEAS (equiv., ng/mL), in samples for individual intoxication cases. Intoxication cases were assigned an arbitrary number (#) based on increasing JWH-018 equivalent (NQ, not quantified; ND, no activity detected) # Equiv., ng/mL # Equiv., ng/mL # Equiv., ng/mL # Equiv., ng/mL # Equiv., ng/mL # Equiv., ng/mL 1 ND 9 NQ 17 3.6 25 7.6 33 16.8 41 37.3 2 ND 10 NQ 18 3.8 26 7.8 34 17.3 42 37.5 3 ND 11 NQ 19 4.6 27 8.3 35 23.0 43 47.2 4 ND 12 NQ 20 4.7 28 10.9 36 23.7 44 69.3 5 ND 13 NQ 21 4.9 29 11.4 37 24.5 45 78.3 6 NQ 14 NQ 22 5.8 30 11.5 38 25.1 46 1048 7 NQ 15 NQ 23 6.3 31 14.8 39 33.0 47  > 5000 8 NQ 16 NQ 24 7.2 32 16.2 40 36.1 48  > 5000 3342 Archives of Toxicology (2024) 98:3337–3350 2022a). These samples were assigned an arbitrary JWH-018 equivalentMEAS of 1.25 ng/mL (0.5 times the lowest calibra- tion point). In five samples no activity was detected—these were arbitrarily assigned 0 ng/mL (Janssens et al. 2022a). It should be noted that the same extraction efficiency (i.e., 100%) was assumed for all SCRAs in the determination of the JWH-018 equivalentsMEAS—inherently no internal stand- ards can be taken along in the bioassays, as these would also activate CB1. Hence, the assigned values are to be consid- ered as ‘minimal equivalents’, since recoveries will most likely not be 100% for all analytes. In addition, the activity that was measured in the extracts represent the combined activities of all analytes—essentially (different) main com- pounds and metabolites, which likely also entail different extraction efficiencies. Measured JWH‑018 equivalentMEAS—what is it related to? While the JWH-018 equivalentsMEAS represents an equiva- lent concentration of one (reference) SCRA as a comparative measure from an activity-based point of view (also referred to as activity equivalent), the measured ex vivo activity in the sample extracts represents the combined activity of all active compounds in the bioassay. In multiple intoxication cases, the samples included multiple/different SCRAs (n = 14) and corresponding (or other) SCRA hydrolysis products (n = 48) (Supplementary Information, (other) SCRA metabolites were not targeted by the MS-method). Depending on the receptor activation potential of each individual compound present in the sample extract, these can all contribute to the ex vivo activity that is detected with the bioassay. This study included intoxications involving 12 different SCRAs that inherently attain various capacities of activating CB1. Pharmacological characterization of SCRAs is the sub- ject of published and (ever-)ongoing research, given the constant emergence of new compounds and their—at the time—unknown harm potential (Noble et al. 2019; Cannaert et al. 2020b; Grafinger et al. 2021b; Deventer et al. 2022; European Monitoring Centre for Drugs and Drug Addiction 2023; Janssens et al. 2023). The resulting pharmacological profile is typically expressed in literature as potency (EC50) and efficacy (Emax) values, obtained with a certain bioas- say. To link the measured JWH-018 equivalentMEAS with the pharmacological profiles of the individual SCRAs, we used previously published potency and efficacy estimates, deter- mined with the same assay format (CB1-β-arr2 recruitment assay) as applied in this study (compiled in Supplementary Table 2, including references). Of note, these values may show some biological—and other—variation over different studies, performed at different moments in time with differ- ent batches of cell freezings and by different operators. In case of multiple studies reporting data on the same SCRA in the CB1-β-arr2 recruitment assay, the data compiled in Sup- plementary Table 2 and used for calculations in this study, were (randomly) chosen, with a preference for those studies with a direct comparison to JWH-018 as a reference SCRA. An overview of the intoxication cases is presented in Fig. 2 (analytical investigation on the top side, bioassay investigation on the lower side), showing which SCRA(s) was (were) present in each sample, and at what (molar) concentration(s). The samples are ordered based on increas- ing JWH-018 equivalents, illustrated by the heatmap below. This increase in JWH-018 equivalentMEAS corresponds with a general trend of increasing concentrations of SCRAs that were found. Additionally, the SCRAs were ordered based on increasing potency (color-coded in the legend) as this is hypothesized to be a (first) important intrinsic characteris- tic of SCRAs that influences the estimation of a JWH-018 equivalentMEAS. The potency of a compound will typically show a strong inverse correlation with a typical recreational dose of that compound. Compounds with a high potency only require low doses (and, hence, corresponding low blood concentrations) to already result in a pronounced activity (and, hence, high JWH-018 equivalentsMEAS). Based on different studies in literature, the potency decreases in the following order: CUMYL-PEGACLONE (Janssens et al. 2020) > ADB-CHMINACA (Wouters et al. 2019) > MAB- CHMINACA (Wouters et  al. 2019) > 5F-ADB (Wout- ers et al. 2019) > MDMB-4en-PINACA (Grafinger et al. 2021b) > MDMB-CHMICA (Wouters et al. 2020) > AMB- CHMICA (Wouters et al. 2020) > 5F-MDMB-PICA (Noble et al. 2019) > 5F-AKB-48 (Wouters et al. 2020) > AB-CHMI- NACA (Wouters et al. 2019) > AB-FUBINACA (Noble et al. 2019) > JWH-018 (Grafinger et al. 2021b) > AB-PINACA (Noble et al. 2019) > 4F-MDMB-BICA (Cannaert et al. 2020b) (Supplementary Table 2). Indeed, in Fig. 2 increas- ing JWH-018 equivalentsMEAS mostly correspond to (i) higher concentrations of present SCRAs (e.g., sample 20 versus sample 30); or (ii) the presence of (lower concentra- tions of) more potent SCRAs (e.g., sample 40 versus sample 44). However, these patterns are not ever-present throughout the entire dataset, e.g., sample 45 represents a lower con- centration of a less potent SCRA compared to sample 44, whereas the JWH-018 equivalentMEAS was somewhat higher (78 ng/mL versus 69 ng/mL). The pharmacology of SCRAs is characterized by on the one hand the potency, which is related to the concentrations at which SCRAs can exert effects, and on the other hand, the efficacy, which corresponds to the extent of cannabinoid activity that can intrinsically be achieved by the SCRA. This intrinsic maximal activation potential will also influence the estimation of JWH-018 equivalentsMEAS, as many SCRAs have higher efficacies than JWH-018. A higher efficacy entails greater cannabinoid activity at concentrations equal to the EC50. Generally, the estimated JWH-018 equivalents 3343Archives of Toxicology (2024) 98:3337–3350 are related to the concentration of the SCRA parent com- pound, which was detected, and its intrinsic relative activity (Rai) compared to JWH-018 (Fig. 3; Supplementary Table 2, determined by Eq. 2 based on data of individual studies). This Rai-value represents both the potency and the efficacy of the compounds relative to a reference compound—in this case JWH-018—that is used to provide a comparative meas- ure. A Rai value > 1, entails that the present SCRA is intrin- sically more active than JWH-018, while a Rai value < 1 means that the SCRA is less active than JWH-018. When focusing on mono-SCRA intoxications, mostly higher JWH-018 equivalentsMEAS are estimated in com- parison to the concentrations of the parent SCRAs found in serum (Fig. 3). These concentrations are expressed as molar concentrations for adequate comparison (calculated by conversion with the corresponding molecular weight). The former observation corresponds to the fact that all SCRAs involved in these mono-SCRA intoxications are charac- terized by a Rai > 1, relative to JWH-018. Generally, for compounds with a Rai > 1, a lower concentration is needed (2)Rai = Emaxi.EC50REF EC50i.Emax REF . to yield the same activity at CB1 as JWH-018, while com- pounds with a Rai < 1 would require higher concentrations than JWH-018 to yield the same effect. For the mono-SCRA intoxications in this study, higher JWH-018 equivalents were related to either high concentrations of a present SCRA, to a high Rai value, or the combination of both. Calculated JWH‑018 equivalentCALC – validation of relationship to pharmacological parameters Figure  3 suggests that the JWH-018 equivalentMEAS is related to the concentrations of the SCRA and the intrin- sic relative activity. To verify this relationship, we have calculated the JWH-018 equivalentCALC based on the ana- lytically determined serum concentrations and the pharma- cological parameters described in literature (potency and efficacy). In case of poly-SCRA intoxications, these multiple SCRAs are present at different concentrations and will all entail a different (relative) pharmacology (Rai). To estimate the intrinsic contribution of the individual SCRAs to the overall ex vivo cannabinoid activity (and estimated JWH- 018 equivalentMEAS), convergence of the relative intrinsic activity and the present (molar) concentrations of SCRAs is required. We, therefore, introduce a new concept of Fig. 2   Comparison of the molar serum SCRA concentrations and the JWH-018 equivalentMEAS. A Molar concentrations of parent SCRAs in the intoxication serum samples. The legend and the color code are ordered based on (reported) potencies: dark red is most potent (CUMYL-PEGACLONE), light purple is least potent (4F-MDMB- BICA). B Heatmap of the JWH-018 equivalentsMEAS. The lower bar shows the legend for interpretation of the colors in the heatmap, cov- ering a JWH-018 equivalentMEAS range of 1–5000 ng/mL 3344 Archives of Toxicology (2024) 98:3337–3350 ‘Estimated Intrinsic Relative activity’ (EIRa, calculated by Eq. 3, Fig. 4) for straightforward prediction of the individual contributions of each SCRA. This concept is to be under- stood as the conversion of one SCRA concentration to the concentration of a reference SCRA based on their relative intrinsic potential to exert cannabinoid activity. In this study, the EIRa corresponds to the molar concentration (nM) of an individual SCRA that is converted to a JWH-018 con- centration based on its CB1 activation potential (using the bioassay employed here), relative to JWH-018, allowing the estimation of a theoretical contribution to the combined CB1 activity that is measured in serum. A comparison of the predicted contributions (EIRa) and the measured JWH-018 equivalentsMEAS is presented in Fig. 5. For mono-SCRA intoxications, the EIRa can be considered a calculated activity equivalent (JWH-018 equivalentCALC). For poly-SCRA intoxications the calculated JWH-018 equivalentCALC consists of a combination of the individual contributions (EIRa). For simplicity, an additive effect is assumed in this study and contributions of co-pre- sent SCRAs are depicted as stacked bars for visual interpre- tation (SCRA1, SCRA2, SCRA3, ordered based on decreas- ing estimated EIRa). In most cases the (sum of the) predicted contribution(s) (EIRa) was higher than the correspond- ing JWH-018 equivalentMEAS, as measured in the extract (Fig. 5). For intoxication case 4, no EIRa could be calculated due to the lack of parent compound that was analytically found in the confirmation analysis. For nine mono-SCRA intoxication cases (6, 17, 19, 24, 33, 39, 45, 47 and 48), the JWH-018 equivalentMEAS exceeded the predicted EIRa. These cases included intoxication with MDMB-CHMICA (6, 17, 19, 24, 39, 45, 48), MDMB-4en-PINACA (33) and ADB-CHMINACA (47). Whereas calculation of the EIRa allows prediction of a JWH-018 equivalentCALC based on the SCRA concentration and the SCRA’s potency and efficacy, relative to the reference that was used to provide activity (3)EIRa = Rai × Concentration molar. Fig. 3   Correlation of the JWH-018 equivalentMEAS to the serum con- centration and the intrinsic relative activity (Rai) of the parent com- pound in mono-SCRA intoxications. All SCRAs involved in mono- SCRA intoxications show a higher intrinsic activity (Rai > 1) relative to reference compound JWH-018. Samples are color-coded for the SCRA detected in serum Fig. 4   Method to calculate the JWH-018 equivalentCALC as validation approach for the correlation with serum concentrations and intrin- sic pharmacology. This method includes the intrinsic relative activ- ity (Rai) of each present SCRA in the intoxication sample to account for the SCRA pharmacology. Convergence of the Rai with the serum concentrations leads to the estimated intrinsic relative activity (EIRa). This EIRa is used as the JWH-018 equivalentCALC in case of mono- SCRA intoxications. For poly-SCRA intoxications, additive effects are assumed to derive the JWH-018 equivalentCALC by adding the individual contributions (EIRa) of the different SCRAs 3345Archives of Toxicology (2024) 98:3337–3350 equivalents, it does not account for differential hill-slopes in sigmoidal pharmacological profiling, differential recov- ery, differential binding affinity, possible competition and/or matrix effects. Hence, these factors, in addition to metabolite activity, might explain the discrepancies. Clinical intoxication in relation to JWH‑018 equivalentsMEAS A variety of toxicological effects was observed in the intoxicated patients at the hospital, including effects on car- diovascular functions, the central nervous system, motoric functioning, respiratory functions, glycemic status, etc. Interestingly, opposite effects were observed in different patients such as breathing pauses (temporary apnoea) or bradypnoea versus hyperventilation, mydriasis versus mio- sis, hyperglycemia versus hypoglycemia, tachycardia versus bradycardia and hypertension versus hypotension. Symp- toms that were most observed (≥ 20% of the cases) were tachycardia (n = 16), nausea and/or vomiting (n = 14), epi- leptic seizures (n = 12), agitation (n = 10) and disorientation (n = 10). The clinical presentation of the intoxicated patients was not clear-cut, as was observed before for mono- SCRA intoxications with 5F-MDMB-PICA (Janssens et al. 2022b). No patterns could be observed for frequently occurring symptoms in relation to increasing JWH-018 equivalentsMEAS in serum (Fig. 6). However, in all but four cases polydrug use was observed, which obscures interpretation and hampers the causality assessment of presented symptoms to the specific intoxication with SCRAs. Other drugs that were detected include THC and its metabolites (n = 18), various first-aid drugs (n = 8; e.g., lidocaine, paracetamol, rocuronium), antipsychotics (n = 9; e.g., quetiapine, risperidone), antidepressants (n = 4; e.g., venlafaxine, paroxetine, citalopram), benzodiazepines (n = 12; e.g., midazolam, temazepam, oxazepam), anti-epi- leptics (n = 3; e.g., levetiracetam, pregabalin, gabapentin), cathinones (n = 9; e.g., 3-MMC, pentylone), opioids (n = 3; e.g., morphine, fentanyl), MDMA (n = 6), and ampheta- mine (n = 5). Additionally, a withdrawal syndrome was the suspected diagnosis in five cases, which also hampers the assignment to toxicity of SCRAs. Some symptoms were only documented in single cases. In Fig. 6 these symptoms were ordered within each category (i.e., clinical or labora- tory) based on the (increasing) corresponding JWH-018 equivalentMEAS that was measured in serum: epistaxis, cyanosis, somnolence, euphoria, aphasia, rhabdomyoly- sis, hypoglycemia, miosis (pinpoint pupils), leukocytosis, exsiccosis, double vision and (initially) increased potas- sium levels. The latter two observations were only noted in two intoxicated patients with very high serum activity equivalents (> 5000 ng/mL JWH-018). These latter two cases also had the highest number of documented symp- toms (8 different symptoms). Only for one other intoxica- tion case (sample 30, corresponding with an activity of 11.5 ng/mL JWH-018) seven different toxicological effects were observed. Fig. 5   Comparison of the JWH-018 equivalentCALC and the JWH-018 equivalentMEAS in mono- and poly-SCRA intoxications. The contri- bution of each SCRA is calculated as the estimated intrinsic relative activity (EIRa), which is plotted for the different SCRAs. This EIRa (or the sum of the EIRa of different SCRAs, in case of poly-SCRA intoxications) forms the JWH-018 equivalentCALC. For poly-SCRA intoxications, the SCRAs were ordered based on estimated contribu- tion (EIRa SCRA 1 > EIRa SCRA 2 > EIRa SCRA 3; do note the log- arithmic scale). JWH-018 equivalentsMEAS (otherwise reported as ng/ mL) are presented in nanomolar concentrations for comparison 3346 Archives of Toxicology (2024) 98:3337–3350 Toxicity assessment in relation to JWH‑018 equivalentsMEAS Even though polydrug use was frequently observed in this case series, causality of the intoxication symptoms and SCRA intake was in most cases probable or certain. The severity of the poisoning was also evaluated by assigning a poisoning severity score (PSS) to every intoxication, rang- ing from 1 (minor), 2 (moderate) to 3 (severe) (Persson et al. 1998). No fatal intoxications (PSS = 4) were included in this study. The correlation of the PSS grade with the JWH-018 equivalentsMEAS was analyzed by plotting the JWH-018 equivalentsMEAS of the intoxication cases per PSS score (Fig. 7). In addition, we indicated the causality (not assessable, probable or certain) on the plot. On one hand, this allowed us to derive tentative thresholds: a JWH- 018 equivalentMEAS > 24 ng/mL always resulted in PSS > 1, whereas a JWH-018 equivalentMEAS > 47 ng/mL always cor- responded to certain causality. On the other hand, a wide range of JWH-018 equivalentsMEAS, including both low and high equivalents (1.25–5000 ng/mL), corresponded to PSS of 2 or 3 and certain causality assessment for the present SCRAs. Hence, whereas a low JWH-018 equivalentMEAS wasn’t necessarily associated with a low PSS, a high JWH-018 equivalentMEAS (above the observed thresholds) always resulted in more severe poisoning and it was asso- ciated with a certain causality of the SCRA to the intoxi- cation. The tentative thresholds could, therefore, be seen Fig. 6   Symptoms (or observa- tions) in intoxicated patients with confirmed SCRA use, with the patients ranked based on the corresponding JWH-018 equivalentsMEAS in serum. The JWH-018 equivalentsMEAS are indicated in the upper heatmap. Clinical and laboratory-based symptoms and observations are ordered from top to bottom by the frequency of occur- rence (indicated by the boxes). The total number of observed symptoms per patient is indi- cated in the lower heatmap. The term ‘respiratory symptoms’ subsumes breathlessness (n = 4), bradypnoea (n = 1), short episodes of apnoea (n = 1) and hyperventilation (n = 1) 3347Archives of Toxicology (2024) 98:3337–3350 as upper limits for, respectively, minor poison severity and the SCRA being associated to the symptoms with probable causality. It is important though to remark that the JWH- 018 equivalentsMEAS were not homogenously distributed throughout the dataset, with 12 samples exceeding the JWH- 018 equivalentMEAS cut-off of 24 ng/mL (3 of which were above 1000 ng/mL), 11 samples with an arbitrarily assigned JWH-018 equivalentMEAS of 1.25 ng/mL and 5 samples for which no ex vivo cannabinoid activity was detected (arbi- trarily assigned as 0 ng/mL). While this imbalance might reflect the real-life situation (i.e., less intoxications with a very high ex vivo cannabinoid activity), it could also skew the data, rendering the proposed thresholds as ‘tentative’. Discussion Forty-eight SCRA intoxication cases were investigated from three points of view: (i) serum levels of SCRAs were analyti- cally determined with LC–MS/MS; (ii) the ex vivo cannabi- noid activity of serum extracts was assessed via a CB1 bio- assay to determine JWH-018 equivalentsMEAS; and (iii) the clinical presentation of the patients reported by the treating physicians, followed by a grading of the reported symptoms according to PSS as well as a causality assessment, was eval- uated by the consulted physicians in the Poisons Information Center. In total, 12 different SCRAs were detected, at con- centrations that were generally in line with those reported in literature for non-fatal intoxications (Adamowicz and Gieroń 2016; Allibe et al. 2017; Mohr et al. 2022; Janssens et al. 2022b; De Oliveira et al. 2023). A variety of symptoms were observed in the patients, corresponding to a non-distinctive toxidrome of SCRAs, as reported before (De Oliveira et al. 2023). Opposing symptoms were observed amongst patients and similarities with the clinical syndrome of other drug classes could be observed, such as reduced respiratory rate (cf. opioid intoxication) (De Oliveira et al. 2023). Impor- tantly, polydrug use was largely present amongst the patient cohort and should be considered as a confounder in the clini- cal assessment. The concept of activity equivalents was put forward after demonstrating the ability of the bioassay used here to (semi-)quantify concentrations based on sigmoidal calibra- tion models (Cannaert et al. 2020a; Janssens et al. 2022b). In this study, we used the prototypical SCRA JWH-018 as a reference to express the CB1 activating potential of serum extracts (containing a variety of SCRAs) in a com- prehensible and uniform manner, by referring to the extent of cannabinoid activity as JWH-018 equivalentsMEAS. Hence, all ex vivo cannabinoid activities that were meas- ured in the serum extracts could be expressed as a JWH-018 equivalentMEAS, as long as they were within the sigmoidal window of the calibration model. The ex vivo cannabinoid activity measured in the bio- assay is hypothesized to depend on (i) the concentrations of individual compounds present in the samples, (ii) the intrinsic CB1 receptor activation potential of these com- pounds, and (iii) possibly competition between co-present compounds based on differential binding affinity. Whereas the Rai concept is usually used as a means to evaluate and express signaling bias, it allowed us to put the pharmacolog- ical profile (potency and efficacy combined) of the SCRAs in perspective relative to JWH-018, the reference compound used in this study (Wouters et al. 2020). Interestingly, all but one (4F-MDMB-BICA) of the SCRAs involved in the intoxication series of the current study are reported to be more active than JWH-018 (i.e., Rai > 1) (Noble et al. 2019; Wouters et al. 2019, 2020; Janssens et al. 2020; Cannaert et al. 2020b; Grafinger et al. 2021b). This is an important explanation as to why the JWH-018 equivalentsMEAS are higher molar concentrations than the molar SCRA con- centrations as derived through LC–MS/MS analysis of the serum samples. We next combined the SCRAs’ calculated relative activity with their (molar) concentrations, to estab- lish the new concept of Estimated Intrinsic Relative Activity (EIRa). By doing this for the individual SCRAs and combin- ing the calculated theoretical contributions, a preliminary prediction of the combined activity of all SCRAs contained in a sample could be obtained, expressed as a JWH-018 equivalentCALC. This newly introduced EIRa is related to the Estimated Intrinsic Potency/Efficacy (EIP/EIE) as pro- posed by Antonides et al., which was previously used to comparatively evaluate the CB1 activity contained in seized drug samples and infused paper involving different SCRAs at different concentrations (Antonides et al. 2019, 2021). Fig. 7   Toxicity appraisal of SCRAs in intoxication cases (PSS, cau- sality), in relation to the JWH-018 activity equivalentsMEAS in serum. The determined thresholds of JWH-018 equivalentsMEAS correspond- ing to the upper limits for PSS = 1 (black, 24 ng/mL) or probable cau- sality (orange, 47 ng/mL) are illustrated by a dotted and a dashed line, respectively 3348 Archives of Toxicology (2024) 98:3337–3350 The EIRa is essentially a combination of both EIP and EIE as it combines information on a substance’s potency and efficacy. Using this EIRa to estimate the contributions of the quantified SCRAs and mathematically derive a JWH- 018 equivalentCALC largely led to an overestimation (i.e., calculated JWH-018 equivalentCALC > measured JWH-018 equivalentMEAS). A logical explanation for this lies in an incomplete recovery during the sample preparation and a matrix effect of the serum in the bioassay, as was previ- ously shown when assessing the ex vivo cannabinoid activ- ity in serum from mono-intoxications involving 5F-MDMB- PICA (Janssens et al. 2022b). In poly-SCRA intoxications, the presence of multiple SCRAs can additionally lead to competition between compounds with potentially different binding affinities, hence not leading to additive effects, as assumed here (Janssens et al. 2022b). In 9 cases the meas- ured JWH-018 equivalentMEAS exceeded the EIRa. Besides limitations of the utilized concept, this could also be related to the co-presence of high concentrations of active metab- olites that were not quantified in this study or to the co- presence of other SCRAs, which were not contained in the applied method (Cannaert et al. 2016; Gamage et al. 2019; Cabanlong et al. 2022). Of note, the identified metabolites and/or degradation products in this study were hydrolysis products, which have been shown to generally retain much lower CB1 activation potential in comparison to the parent compound (Noble et al. 2019; Wouters et al. 2019). It is hypothesized that correction for incomplete recovery and for matrix effects could improve the concurrence between the predicted SCRA contributions as EIRa and the JWH-018 equivalentMEAS, as was shown before (Janssens et al. 2022b). However, determination of the recovery of each individual SCRA in this study for the bioassay sample preparation method was outside the scope of the analytical investiga- tion and not enough sample volume was available to study the matrix effects in the bioassay. The JWH-018 equivalentMEAS is determined by running sample extracts alongside a calibration model. In essence, this allows to kill two birds with one stone to keep up with the dynamic NPS market. First, the application of serum on the bioassay allows a universal screening for the involvement of SCRAs in the intoxication, irrespective of the compound structure and mass spectral libraries (Cannaert et al. 2019; Janssens et al. 2022a). Secondly, the co-presence of the cali- bration model allows to interpret the extent of the intoxica- tion from a CB1 activity-based point of view, potentially even prior to identifying the SCRA involved and without any knowledge on its harm potential—presuming the latter is linked to CB1 activation. The JWH-018 equivalentMEAS showed no clear relationship with the clinical presenta- tion in terms of symptoms that were observed, apart from the fact that those intoxications with the highest JWH-018 equivalentsMEAS presented with the highest number of symptoms. This lack of relationship between the JWH-018 equivalentMEAS—determined based on CB1 activity—and clinical features, could be related to CB1-unrelated phar- macological effects of the different SCRAs. Further insight into the pharmacology of SCRAs at other molecular targets could shed further light on this. The toxicity assessment was partly related to the measured JWH-018 equivalentMEAS, as upper limits for probable causality and minor intoxication symptoms could be observed, yet no lower limit for severe intoxications with a certain causality could be concluded. While these tentative thresholds support the link of high JWH-018 equivalentsMEAS being linked to more severe intoxications, the inverse cannot be concluded, i.e., low JWH-018 equivalentsMEAS were not necessarily associated to minor symptoms in intoxications—obviously, the con- comitant presence of other drugs in most intoxication cases, as well as a possible delay between clinical presentation and sample collection are only some of the obfuscating factors that may explain this apparent discrepancy. In conclusion, JWH-018 equivalents are proposed as a comparative measure to study mono-SCRA and poly-SCRA intoxications from a cannabinoid activity point of view. The measured JWH-018 equivalentsMEAS were shown to be linked to both the concentration of the parent compound and the intrinsic CB1 activity (in terms of potency and efficacy) relative to the reference JWH-018. This correlation was verified by comparing a mathematically derived JWH-018 equivalentCALC with the measured JWH-018 equivalentMEAS. The JWH-018 equivalentMEAS showed to be related (in part) to the severity of symptoms according to PSS and the causal relationship of the SCRA to the acute drug intoxication. The clinical presentation of intoxicated patients confirmed a non-distinctive toxidrome of SCRAs with a high variety of toxicological symptoms and no evident relationship to the CB1 activity in serum. Activity equivalent determination can aid in the identification of SCRA intoxications (untargeted screening), while simultaneously allowing evaluation of the extent of an intoxication (based on cannabinoid activity), without prior knowledge on the individual SCRA’s pharma- cological profile or harm potential. In those cases, exceeding a certain activity threshold it may guide the interpretation. In addition, the proposed concept allows (better) compari- son of different SCRA intoxications potentially involving newly emerging SCRAs using a known reference SCRA as a comparative measure. Supplementary Information  The online version contains supplemen- tary material available at https://​doi.​org/​10.​1007/​s00204-​024-​03830-2. Acknowledgements  L.K. Janssens is supported by The Special Research Fund (BOF) of Ghent University (grant number BOF20/ DOC/051). K. E. 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Calculated JWH-018 equivalentCALC – validation of relationship to pharmacological parameters Clinical intoxication in relation to JWH-018 equivalentsMEAS Toxicity assessment in relation to JWH-018 equivalentsMEAS Discussion Acknowledgements References