Emerging toxicity of 5,6-methylenedioxy-2-aminoindane (MDAI): Pharmacokinetics, behaviour, thermoregulation and LD50 in rats

    Emerging toxicity of 5,6-methylenedioxy-2-aminoindane (MDAI): Pharmacokinetics, behaviour, thermoregulation and LD50 in rats ˇ ıdkov´a, Marie Bal´ıkov´a, Martin Tom´asˇ P´alen´ıcˇ ek, Eva Lhotkov´a, Monika Z´ ˇ Kuchaˇr, Michal Himl, Petra Mikˇsa´ tkov´a, Martin Cegan, Karel Valeˇs, Filip Tylˇs, Rachel R. Horsley PII: DOI: Reference:

S0278-5846(16)30051-3 doi: 10.1016/j.pnpbp.2016.04.004 PNP 8909

To appear in:

Progress in Neuropsychopharmacology & Biological Psychiatry

Received date: Revised date: Accepted date:

12 January 2016 9 April 2016 9 April 2016

ˇ ıdkov´ Please cite this article as: P´ alen´ıˇcek Tom´aˇs, Lhotkov´ a Eva, Z´ a Monika, ˇ Bal´ıkov´ a Marie, Kuchaˇr Martin, Himl Michal, Mikˇs´atkov´ a Petra, Cegan Martin, Valeˇs Karel, Tylˇs Filip, Horsley Rachel R., Emerging toxicity of 5,6-methylenedioxy2-aminoindane (MDAI): Pharmacokinetics, behaviour, thermoregulation and LD50 in rats, Progress in Neuropsychopharmacology & Biological Psychiatry (2016), doi: 10.1016/j.pnpbp.2016.04.004

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ACCEPTED MANUSCRIPT Emerging toxicity of 5,6-Methylenedioxy-2-aminoindane pharmacokinetics, behaviour, thermoregulation and LD50 in rats

(MDAI):

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Tomáš Páleníčeka,c, Eva Lhotkováa, Monika Žídkováb, Marie Balíkováb, Martin Kuchařa,d, Michal Himld, Petra Mikšátkováa,d, Martin Čegane, Karel Valeša, Filip Tylša,c, Rachel R. Horsleya

National Institute of Mental Health, Topolová 748, 250 67 Klecany, Czech republic

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Institute of Forensic Medicine and Toxicology, Charles University in Prague, Studničkova 4, 128 21 Prague 2, Czech Republic 3rd Medical faculty, Charles University in Prague, Ruská 87, 110 00 Prague 10, Czech Republic

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Faculty of Food and Biochemical Technology & Faculty of Chemical Technology, University of Chemistry and Technology Prague, Technická 5, 166 28 Prague 6, Czech Republic e

Corresponding author:

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Masaryk hospital in Ústí nad Labem, Sociální péče 3316 /12A, 401 13 Ústí nad Labem, Czech Republic

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Tomáš Páleníček, MD, PhD.

National Institute of Mental Health in the Czech Republic Topolová 748,

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250 67 Klecany, Czech Republic

Telephone: +420 283 088454 Email: [email protected]

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ACCEPTED MANUSCRIPT Abstract

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MDAI (5,6-Methylenedioxy-2-aminoindane) has a reputation of a non-neurotoxic ecstasy replacement amongst recreational users, however the drug has been implicated in some severe and lethal intoxications. Due to this, and the fact that that the drug is almost unexplored scientifically we investigated a broad range of effects of acute MDAI administration: pharmacokinetics (in sera, brain, liver and lung); behaviour (open field; prepulse inhibition, PPI); acute effects on thermoregulation (in group-/individually-housed rats); and systemic toxicity (median lethal dose, LD50) in Wistar rats.

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Pharmacokinetics of MDAI was rapid, maximum median concentration in serum and brain was attained 30 minutes and almost returned to zero 6 hours after subcutaneous (sc.) administration of 10 mg/kg MDAI; brain/serum ratio was ~4. MDAI particularly accumulated in lung tissue. In the open field, MDAI (5, 10, 20 and 40 mg/kg sc.) increased exploratory activity induced signs of behavioural serotonin syndrome and reduced locomotor habituation, although by 60 min effects had diminished. All doses of MDAI significantly disrupted PPI and the effect was present during the onset of its action as well as 60 min after treatment. Unexpectedly, 40 mg/kg MDAI killed 90% of animals in the first behavioural test, hence LD50 tests were conducted which yielded 28.33 mg/kg sc. and 35 mg/kg intravenous but was not established up to 40 mg/kg after gastric administration. Disseminated intravascular coagulopathy (DIC) with brain oedema was concluded as a direct cause of death in sc. treated animals. Finally, MDAI (10, 20 mg/kg sc.) caused hyperthermia and perspiration in grouphoused rats.

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In conclusion, the drug had fast pharmacokinetics and accumulated in lipohilic tissues. Behavioural findings were consistent with mild, transient stimulation with anxiolysis and disruption of sensorimotor processing. Together with hyperthermia, the drug had a similar profile to related entactogens, especially 3,4-metyhlenedioxymethamphetamine (MDMA, ecstasy) and paramethoxymethamphetamine (PMMA). Surprisingly subcutaneous MDAI appears to be more lethal than previously thought and its serotonergic toxicity is likely exacerbated by group housing conditions. MDAI therefore poses greater risks to physical and mental health than recognised hitherto.

Keywords: 5,6-Methylenedioxy-2-aminoindane; MDAI; Body temperature; Locomotion; Median lethal dose; Open field; Pharmacokinetics; Prepulse inhibition; Sensory gating; Toxicology; Wistar rat.

List of abbreviations: ASR, Acoustic startle response; DAT, dopamine transporter; EMCDDA, European Monitoring Centre for Drugs and Drug Addiction; HPLC, High performance liquid chromatography; ISI, Inter-trial interval; ISI, Inter-stimulus interval; iv., intravenous; LC/MS, Liquid chromatography with mass spectrometry; LC/HRMS liquid chromatography with high resolution mass spectrometry; LD50, Median lethal dose; MDA, 3,4methylenedioxyamphetamine; MDAI, 5,6Methylenedioxy-2-aminoindane; MDMA, 3,4-MethylenedioxyN-methylamphetamine; MMAI, 5methoxy-6-methyl-2-aminoindane; NPS, Novel psychoactive substances; PMMA, methyl-MA,4methoxy-N-methylamphetamine; 2C-B, 4-bromo-2,5-dimethoxyphenethylamine; po., per oral; PPI, prepulse inhibition; sc., Subcutaneous; SERT, serotonin transporter; s.e.m., Standard error of the mean; U/HPLC, Ultra high pressure liquid chromatography; 2-AI, 2-aminotetralin; HE, haematoxylineosin; DIC, disseminated intravascular coagulopathy .

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ACCEPTED MANUSCRIPT Introduction

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Numerous novel psychoactive substances (NPS) have been designed to substitute for 3,4metyhlendioxymethamphetamine (MDMA, ‘ecstasy’) including the serotonin-releasing aminoindanes 5,6-Methylenedioxy-2-aminoindane (MDAI), 2-aminoindane (2-AI) and 5-iodo-2-aminoindane (5-IAI), which are readily available for purchase over the internet. MDAI was previously the fifth most abundant for sale (European Monitoring Centre for Drugs and Drug Addiction, 2012; EMCDDA) and the most likely novel compound to replace recreational MDMA and the popular cathinone mephedrone (Leach, 2010; Townsend, 2010). Worryingly, several severe toxic reactions and deaths after MDAI have been reported, with serotonin syndrome as a characteristic feature (Corkery et al., 2013). MDAI was derived from 3,4-methylenedioxyamphetamine (MDA) during pharmacological investigations of MDMA (Nichols et al., 1990; Oberlender and Nichols, 1990); therefore it is structurally and pharmacologically related to both MDA and MDMA (Oberlender and Nichols, 1991). The MDAI doses used by humans range typically from 70–300mg, with an average dose of 150200mg (Corkery et al., 2013). This is approximately twice as much as a typical recreational MDMA dose (75-125 mg) reported as common for most of the people on Erowid.org (Erowid, 2015). Desirable subjective effects of MDAI in humans are similar to MDMA, i.e., empathy, sexual arousal and intensification of sensory experience, mild euphoria and sociability (Gallagher et al., 2012; Nichols et al., 1990), which is further supported in pre-clinical models, where MDAI substitutes for MDMA in drug discrimination studies (Oberlender and Nichols, 1988). Its robust monoaminereleasing properties are comparable to that of MDMA, although MDAI has greater selectivity for serotonin reuptake (Johnson et al., 1991a; Johnson et al., 1991b; Sprague et al., 1996). Since MDAI has very little effect on dopamine (its dopamine transporter (DAT)/serotonin transporter (SERT) inhibition ratio is 0.21), its stimulatory effects are modest, and there have even been reports of mild sedation (Sainsbury et al., 2011). Owing to this overall psychopharmacological profile, MDAI represents a typical entactogen*(Nichols, 1986). Despite its availability, reported use and acute poisoning, little is known regarding its acute pharmacokinetics, metabolism, behavioural activity and toxicity. This study therefore was designed to describe in detail the effects of MDAI in the Wistar rat.

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In pharmacokinetic experiments serum, brain, liver and lung levels of MDAI were analysed. Although the reported elimination of its analogue 2-AI is rapid (half-life in the rat brain is 1-2 hours: (Fuller et al., 1977) here, kinetics were measured for six hours after drug administration. Sera and brain levels of MDAI were analysed to determine relative rapidity of influx into brain tissue and to complement interpretation of psychopharmacological effects in the behavioural experiments. Inclusion of liver and lung levels in the analysis was motivated by previous findings with congeners 4-bromo2,5dimethoxyfenylethylamine (2C-B) and paramethoxymethamphetamine (PMMA) which have been shown to accumulate in lungs, and to identify MDAI’s potential degree of liver metabolism. To detect early and delayed behavioural effects of the drug on locomotor activity (open field) test and prepulse inhibition (PPI) of the acoustic startle response (ASR), two schemes of MDAI administration were used (Palenicek et al., 2007a; Palenicek et al., 2008; Palenicek et al., 2011): animals were tested shortly after drug administration, or after estimated peak effects. Open field locomotor activity (and its temporo-spatial characteristics in the open field) is sensitive to stimulatory/sedative effects of centrally acting drugs, as well as to locomotor habituation, and approach-avoidance conflict (exploration and anxiety). Since MDAI has been reported to mimic the effects of MDMA, modest hyperlocomotion (but see (Sainsbury et al., 2011), as well as attenuation of locomotor habituation were expected, with stereotypy and/or initial motor ataxia as a possibility at higher doses (Palenicek et al., 2007b; Palenicek et al., 2011). PPI is a behavioural operationalisation of sensorimotor gating that reflects pre-attentional filtering of redundant information (Swerdlow and 3

ACCEPTED MANUSCRIPT Geyer, 1998). PPI is useful in assessing the psychomimetic properties of psychedelic/serotonergic and stimulant /dopaminergic drugs and is a behavioural endophenotype of psychosis (Geyer et al., 2001). Owing to the resemblance of MDAI with MDMA-like compounds which typically disinhibit PPI (Bubenikova et al., 2005; Palenicek et al., 2011), a similar disruption was predicted.

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It is well-established that the use of MDMA and related entactogens can result in serious complications related to (most notably) hyperthermia which is a cardinal sign of serotonin syndrome (Liechti, 2003; Liechti et al., 2005; Simmler et al., 2011). Moreover, environmental factors affecting ambient temperature (such as crowded conditions) can interact with serotonergic compounds and exacerbate hyperthermic reactions (Green et al., 2004a; Green et al., 2004b; Palenicek et al., 2011). Since MDAI fatalities have indicated the possible presence of serotonin syndrome (and to simulate the typical environmental conditions in which human use often occurs), we investigated MDAI under group (crowded) versus individual home-cage conditions with the expectation that hyperthermia would be exacerbated in group-housed animals. Finally, due to the fact that unexpected lethality was observed during behavioural tasks in group housed animals, we collected preliminary data on median lethal dose (LD50) and the cause of death in this condition.

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* The term “entactogen” has been proposed for drugs such as MDMA that do not have pure hallucinogenic or stimulant properties. It has a phenomenological connotation of producing a ‘touching within’(Nichols, 1986).

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ACCEPTED MANUSCRIPT Methods

Animals

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Drugs and chemicals

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Male outbred Wistar rats (Velaz, Czech Republic) weighing 180g –250g were housed in pairs at 22±2°C on a 12h light/dark cycle and with ad libitum water and standard diet. Rats were acclimatised for 7–10 days prior to testing, during which they were weighed twice and handled four times. All tests were conducted under standard conditions: humidity 30-70% and temperature 22±2 °C. Naïve rats were used for each experiment with the exception that rats from behavioural experiments were used for subsequent pharmacokinetic sampling (to reduce animal use). Data from vehicle control animals for PPI, open field and temperature studies were collected twice per annum as part of a series of NPS studies undertaken in our laboratory, rather than per NPS studied. Control data used for analyses were selected based on proximity in time to the NPS (here MDAI) data collection. In consequence, the same vehicle control data may appear in more than one published article. We argue this is justifiable as ethical (in terms of minimising animal use) as well scientifically acceptable: analysis of historic control data showed no significant differences between saline vehicle treated animals collected at different time points. One-way ANOVAs showed no differences between control data and two other previous sets of control data, maximum F(2,26) =1.07, p =0.38. All experiments respected the Guidelines of the European Union (86/609/EU) and the National Committee for the Care and Use of Laboratory Animals (Czech Republic) and were approved by the committee under the number: MEYSCR-27527/2012-31.

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5,6-Methylenedioxy-2-aminoindane (MDAI) was purchased via the internet and subsequently purified and converted to a hydrochloride by Alfarma s.r.o. (Czech Republic). The resulting MDAI was certified to be 99.18% purity (analysed by infrared spectroscopy) and also served as a reference standard for pharmacokinetic analyses using liquid chromatography. In all cases (with the exception of LD50), MDAI was dissolved in physiological saline at doses 5, 10, 20 and 40 mg/kg in a volume of 2 ml/kg for sc. administration. For LD50, MDAI was administered sc., po. or iv. at doses 20, 25, 30, 35, and 40 mg/kg in 1ml/kg phosphate buffered saline (to reduce the administration volume for iv. administration and to maintain blood pH). Internal standard for quantitative liquid chromatography / mass spectrometry (LC/MS) assays was MDA-D2.HCL with the purity 99.7%, (Lipomed Inc. Switzerland). Reference standards for confirmation of metabolites in urine by LC/HRMS (high resolution mass spectrometry) and gas chromatography/mass spectrometry (GC/MS) were synthesized with purity within 97.5 – 89.3 % (Institute of Chemical Technology, Department of Organic Chemistry, Prague). B-Glucuronidase type HP-2 from Helix Pomatia, EC 3.2.1.31 (184 973 units/ml) was purchased from Sigma-Aldrich, Prague. Extraction columns Bond Elut Certify 50mg/3 mL were supplied by Labio s.r.o., Olomouc. Other chemicals used for laboratory purposes were of analytical grade purity.

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ACCEPTED MANUSCRIPT Dosage

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The MDAI doses used in the present study were estimated according to the reported usage by humans and according to our previous studies with entactogens MDMA, PMMA and 2C-B. Doses were selected to range from those that: 1) at the lower end are close to those used by humans to 2) higher doses that might produce significant acute non-lethal toxicity and 3) with respect to our previous analogous experiments with MDMA, PMMA and 2C-B (Bubenikova et al., 2005; Palenicek et al., 2007a; Palenicek et al., 2005; Palenicek et al., 2011). Our treatment range was set to be 5, 10, 20 and 40 mg/kg for behavioural experiments. Doses for pharmacokinetic and temperature experiments (10 and 20 mg/kg) were selected from this range according to effectiveness in behavioural tasks and the inherent sensitivity of the procedure utilised. MDAI was administered subcutaneously (sc. for comparability with previous studies) in all cases, with the exception of LD50, where doses were titrated at 5 mg/kg intervals from 20-40 mg/kg (orally, po.; intravenously, iv.; or sc.).

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For the pharmacokinetic study a single bolus of MDAI at 10 mg/kg or vehicle was administered sc., and subsequently animals were decapitated after 30, 60, 120, 240 or 480 minutes (n=8 per group). Separated sera and whole brains, livers and lungs were kept at −20°C until the toxicological analyses.

Determination of MDAI levels in serum and tissue samples using LC/HRMS: Serum pre-

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treatment: 0.2 ml of rat serum was fortified with the internal standard MDA-D2 in methanolic solution (in amount with respect to the level of MDAI in assayed samples) and 0.5 ml of a 0.1 M phosphate buffer (pH 6) in a labelled tube.

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Tissue pre-treatment: 250 mg of tissue (brain, lung, liver) was homogenized with 5 ml methanol and the internal standard MDA-D2 (in amount with respect to the MDAI levels in samples). The specimen was then ultrasonicated for 20 minutes and after supernatant separation by centrifugation, the supernatant was transferred into a clean labelled tube and evaporated to dry. The residue was reconstituted in a 0.1 M phosphate buffer (pH 6). Solid phase extraction (SPE) of MDAI in pre-treated samples: A pre-treated sample of serum or tissue with the buffer and internal standard was loaded onto a Bond Elut Certify cartridge previously conditioned with 0.5 ml of a 0.1 M phosphate buffer (pH 6). After application of a pre-treated sample, the cartridge was washed with 0.5 ml of distilled water, 0.5 ml of 0.1 M HCl and 0.5 ml of CH3OH/H2O (1/1, v/v) and then dried by air for 5 minutes. The analytes were eluted three times with 0.5 ml of a freshly prepared mixture of dichloromethane/2-propanol/ammonium hydroxide (25 %), 80/20/4, v/v/v. The eluate was gently evaporated to dryness under a stream of air at 40°C and then dissolved into mobile phase for LC/HRMS analysis. LC/HRMS conditions: The analyses were performed using Dionex Ultimate 3000 UHPLC coupled to an Exactive Plus-Orbitrap MS (ThermoFisher Scientific, Bremen, Germany) equipped with a HESI-II source. The chromatographic analyses of serum and tissue samples were performed using a Kinetex PFP 100A (50 x 2.1 mm, 2.6 mm) and Security Guard Cartridge PFP 4 x 2.0 mm (Phenomenex) with a flow rate of 400 ml/minutes, gradient elution with 10 mM ammonium formate in 0.1 % of formic acid as the mobile phase B. Gradient 0 min 5% B, 4 min 45% B, 5-6 min hold at 95%. The MS conditions were: full MS, positive scan mode from 50 to 500 m/z, resolution 70.000 FWHM (scan speed 3 Hz), spray voltage 3 kV, and ion transfer capillary temperature 320° C. 6

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Design and statistics

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For open field, PPI, and temperature analyses, factorial designs (as already described) were used for later analysis of variance (ANOVA). Analyses were conducted using IBM SPSS version 22. Unless stated otherwise, default alpha was set at p=0.05, two tailed. Significant main effects and interactions involving MDAI were followed up with a priori pairwise comparisons using t-tests; unplanned post-hoc comparisons used t-tests with Bonferroni correction for multiple tests (alpha/number of comparisons). For independent t-tests, where Levene’s test for equality of variance was significant, corrected statistics were used. For repeated measured ANOVA, where Mauchly’s test of sphericity was significant Greenhouse-Geisser corrections were used.

Behaviour: Open field and PPI

Open field: A square black plastic open field arena (68×68×30cm) was situated in a sound-proof

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room that was diffusely lit with low-intensity reflected light. The arena was virtually divided into 5×5 identical square zones with 16 located around the periphery and 9 centrally. Rats (n=10 per cell) were placed individually into the centre of the arena, and behaviour was video-recorded for 30 minutes. Ethovision Colour-Pro version 3.1.1, (Noldus, Netherlands) was used for behavioural capture and pre-processing. A mixed factorial design was used, with drug treatment (vehicle or 5, 10, 20 mg/kg MDAI, and in initial tests, 40 mg/kg) and testing onset (15 minutes or 60 minutes post drug administration) as independent factors. Trajectory length was measured in cm (corrected for deviations of <3cm)) over the 30 minute session course, and was subsequently divided into 6x5 minute time blocks for inclusion as a repeated measures factor in analyses. Frequency (f) of line crossings into different zones of the arena was used to calculate thigmotaxis (= fperipheral zones / fall zones) which indicates the probability of appearance in peripheral zones. Time spent in the centre of the arena (Tcentre) was calculated timecentral zones (Palenicek et al., 2011).

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PPI: Two ventilated startle chambers (SR-LAB, San Diego Instruments, California, USA) each comprising a sound-attenuated, evenly-lit enclosure containing a high frequency loudspeaker (which produced the discrete broadband acoustic pulses and prepulses) mounted 24 cm above a Plexiglas stabilimeter (8.7cm inner diameter). A dynamic calibration system ensured equivalent stabilimeter sensitivity between chambers. A piezoelectric accelerometer detected the average startle amplitudes (AVG) which were digitised and used for subsequent analyses. Sound levels were measured using a RadioShack sound level meter. Two days before test, rats were acclimatized to the startle chamber with a drug-free 5 minute pretraining procedure consisting of 5 presentations of pulse alone stimuli (115 dB/20ms) over background white noise (75 dB). Startle data were not recorded for acclimatisation. On the test day, 15 minutes or 60 minutes prior to PPI/ASR testing sc. MDAI at 5, 10 or 20 mg/kg versus saline vehicle, was administered. There were 72 trials in all with an inter-trial interval (ITI) of 4-20s (mean ITI: 12.27s). Rats (n =10 per experimental group) were acclimatised for 5 minutes to the startle chamber in which a 75 dB background white noise was continuously presented. Six 125 dB / 40 ms duration pulse alone trials were then delivered to establish baseline ASR. Following this, 60 trials of the following were presented in a pseudorandom order: (A) pulse alone: 40 ms 125dB; (B) prepulsepulse: 20 ms 83 dB or 91 dB prepulse, a variable (30, 60 or 120 ms) inter-stimulus interval (ISI: mean 70ms), then 40 ms 125 dB pulse; (C) 60 ms no stimulus. Finally, six pulse alone trials were delivered. 7

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Habituation was calculated by the percentage reduction in ASR from the initial six baseline trials, to the final six trials. PPI was calculated as: [100 – (mean prepulse–pulse trials/mean pulse alone trials)*100]. Mean ASR was derived from pulse alone trials. PPI analyses used a factorial design, with drug treatment and testing onset as between subjects factors.

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Body temperature and preliminary LD50

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Rectal temperature and perspiration: To test interactive effects of MDAI and environmental crowding on body temperature, rats (N = 40) were housed singly versus five to a home-cage. Two MDAI doses (10 mg/kg, 20 mg/kg) were selected based on the general drug effects observed in PPI and open field. Rectal temperature and perspiration were measured in rats housed individually versus housed in groups of five (home-cage condition).

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Temperature and perspiration were observed in the animal keeping room (previously described) during the light phase at 13 time points: the first three measurements were drug-free, and were measured hourly, 07:00h-09:00h (inclusive) whereupon MDAI or vehicle was administered. Thereafter, temperature was recorded at 0.5h intervals 09.30h-11.00h, before resuming hourly measurements 12.00h-17.00h.

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For each time point, rectal temperature (°C) was measured by a digital thermometer (10 s), whilst rats were briefly immobilized in a Plexiglas tube. To quantify perspiration, experimenter observations of percentage perspiration (0%; 1-10%; 11-20%; 21-50% and >50% of the body) were made at each time point. These nominal categorisations were transformed as follows: 0 = 0% body surface wet; 1 = 1%-10% of body surface wet; 2 = 11% –20%, of the body surface wet); 3 = 21%–50% of the body surface wet); 4 = >51% of the body surface wet). Mean perspiration was then calculated across the 13 time-point, thus yielding a continuous variable suitable for parametric analysis with one-sample ttests.

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Analysis of rectal temperature used a mixed factorial design, with drug treatment and home-cage condition as between subjects factors (n = 10 per experimental group) and time-course as a repeated measures factor.

Preliminary LD50: According to the fatal effects of 40 mg/kg in the open field testing (see below in results), we conducted additional LD50 tests within the range of 20 - 40 mg/kg. A day before the drug administration pre-treatment weights (N = 60) were recorded. Survivors were weighed daily for seven subsequent days. Route of administration (sc., po., iv.) and MDAI dose (20, 25, 30, 35 and 40 mg/kg) yielded experimental group sizes of n = 6. Prior to MDAI administration, rats were briefly anesthetized with isoflurane, in order to enable non-stressful iv. and po. administration. Subsequently animals were weighed again for precision calculation of MDAI dose, which was then administered by sc. injection into the scruff; by oral gavage; or by iv. injection into the tail vein. After administration, rats were placed back into the home-cage in groups of 6 animals. Animals were observed for approximately 120 minutes (c.f. maximum interval noted here for 40 mg/kg in open field) and the visible effects of the drug were noted. LD50 was calculated as mortality occurring within 24 hours of drug administration (Litchfield and WILCOXON, 1949) and were expressed as mg/kg for the hydrochloride salt. Organs from one dead experimental animal (kidney, lung, brain, heart and liver) fixed in 4 % buffered formaldehyde solution were kept for pathologic-anatomical analysis. Tissue was prepared and histologic samples were performed by classical histological technics embedded in paraffin blocks. 8

ACCEPTED MANUSCRIPT Sections of 4 micrometer thickness was made and stained by basic haematoxylin-eosin (HE) method, no special staining method was required. Then microscopic analysis was done.

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Additional confirmation of the origin of perspiration, of toxicity and further behavioural observations:

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In order to confirm the origin of the perspiration, otherwise very atypical/unlikely reaction observed in group housed animals (see results section), we have decided to make several additional tests. Initially we have identified three theoretical sources that might cause/contaminate/or contribute: sweat, urine and salivation. To differentiate those careful visual observations and liquid chromatography LC/MS were used. Sweat and urine have very comparable matrix from analytical point of view (Mosher, 1933), therefore fingerprinting as well as chromatographic profile were employed. Since urea and creatinine should be present in much higher concentrations in urine (Mosher, 1933), quantitative analysis of these was also performed in all samples.

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To collect the samples, three additional male Wistar rats were given 20mg/kg MDAI sc. while being housed together. Semi-structured behavioural observation was made by an experienced observer for the following 100 min (in 10 min bins). Behaviour frequencies coded for were: rears, sniffing, licking & grooming, rotational behaviour, forelimb padding/stepping behaviour (animal alternates forelimbs in the air, or on the cage floor/walls forelimbs in the air), flat body posture, salivation, number of faecal boli and urination. Any behaviours not pre-coded for were noted, and included in the remainder of the observation. Ratings were also made for general activity (subjective scale 0-9, where 0 is sleeping, and 9 is extremely hyperactive), salivation (0-5, where 0 is no salivation, and 5 is profuse) and for perspiration (in the same way as described above).

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Sample collection: Biological samples for fingerprinting and chromatographic analyses were taken as follows: fur was sampled into vials prior to drug injections and again at 60 min after administration during the highest perspiration expected (from the top of the head; from the back; from the belly, close to the genitals). Samples were stored at -20°C until further chromatographic examination. Urine in the home cage was collected on blotting paper which was removed every 10 min, placed into a vial and frozen at -20°C for later analysis. When pink/redness was observed exuding from the mouth and nose (in two of three rats), this was collected on a swab, and placed into a physiological saline and stored at 3-8°C until analysis. Samples of blank rat saliva were also collected and kept at 38°C until analysis. After 100 minutes, animals were killed by cervical dislocation, brain, liver and kidneys were removed and placed in 4% formalin for further pathologic-anatomical examination. Organs were analysed in the same way as described above in LD50 study, only the smears on the swab were stained by Giemsa. Sample preparation and analysis: Fur and blotting paper were mixed with 1.5 and 2 ml respectively of pure water and stirred for 30 min. Diluted rat urine samples (100µl of urine + 900µl of water) collected as blanks for metabolic studies were also used as blanks in UHPLC-MS/MS analysis. UHPLC 1290 Infinity coupled with 6460 Triple quadrupole mass detector (Agilent Technologies, Santa Clara, USA) were used for the analyses. Methanol and 5mM ammonium acetate were used as the mobile phases. Two methods were used for: 1) Fingerprinting: direct injection without chromatographic separation with mobile phase composition 65% methanol + 35% ammonium acetate and 2) Chromatographic separation and detection of urea and creatinine: separation was made at chromatographic column: Eclipse Plus C18 RRHD, 1,8 µm, 2,1 x 50 mm; Agilent. Followed gradient elution was used (all steps linear): 0min – 10% MeOH; 4min – 100% MeOH; 5min – 100% MeOH; 5.2min - 10% MeOH; 7min- 10% MeOH. Injected volume: 2µl 9

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In each case, electrospray ionization in positive mode and detection in scan mode in m/z 30-1000 range was used.

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ACCEPTED MANUSCRIPT Results

Pharmacokinetics and metabolism

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10 mg/kg MDAI time profile in serum and brain: Maximum median MDAI serum concentration

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4,3 mg/L (SD 0.8 mg/L) was attained 30 minutes after the 10 mg/kg sc. dose with estimated elimination half-life 0.8 h. Influx into the brain tissue was not delayed; the maximum median concentration in the brain 18.2 mg/L (SD 1.4 mg/L) was reached 30 minutes after the dose. The brain to serum ratio was close to 4 through the whole temporal observation. Finally, the drug robustly accumulated in lungs; on the contrary the liver had slightly higher levels compared to serum. See Figure 1.

Figure 1: Median (ng/g) MDAI in serum (ng/ml), brain (ng/mg), lung (ng/mg) and liver (ng/mg) over 6 hours after 10 mg/kg MDAI sc.

Behaviour: Open field and PPI After commencing open field testing, we discovered 40 mg/kg MDAI to produce 90% mortality and so this dosage was excluded from further tests. Most animals died within 45 - 120 minutes of drug administration. Stereotyped circling around the home-cage, body perspiration, and salivation were observed followed by seizures and/or sudden death. Animals exuded dark red/pink discharge from the nose and mouth (at this time we did not know whether it was blood or porphyrin (Hanson, 2006; Segal et al., 2003)) and their tails and limbs were pale. After the open field testing, animals were placed back in their cages in pairs, therefore were in an aggregated condition when they died. We have only partial data (open field, 15 minute testing onset) from animals administered 40 mg/kg MDAI therefore it is not possible to include the 40 mg/kg dose in full factorial analysis as intended 11

ACCEPTED MANUSCRIPT instead these are presented in the open field figures and compared with vehicle treated rats with separate independent t-tests.

Open field: Trajectory length was analysed using 4 x 2 x 6 mixed factorial ANOVAs with drug

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There were no main effects of drug treatment or testing onset, maximum F(3,72) = 1.49, p = 0.23, but there was a significant interaction between drug treatment and testing onset, F(3,72) = 3.41, MSe = 2712044.21, p = 0.02. We observed a significant overall effect of blocks which interacted significantly with drug treatment, testing onset and with the drug treatment x testing onset interaction, minimum F(15,360) = 5.43, MSe = 122540.33, p = 0.00. Figure 2A shows that within the 15 minute testing onset group, the vehicle treated rats showed progressively diminishing locomotor activity over the course of the six blocks of the test session. By contrast, 5 mg/kg MDAI treated rats showed steady activity over the session, shown as significantly lower activity in block 1 , t(18) = 2.09, p = 0.05 and significantly greater activity in blocks 3-6, minimum t (18) = 2.44, p = 0.03. Similarly, 10 and 20 mg/kg showed significantly less activity in block 1 (minimum t(13.35) = 2.74, p = 0.02, but in blocks 3-6 their activity progressively increased compared to vehicle, minimum t(14.83) = 2.73, 0.02. The locomotor activity of animals treated with 40 mg/kg progressively increased as with 10 and 20 mg/kg and the total distance travelled was more than 50% greater compared to 20 mg/kg.

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In the 60 minute testing onset group (Figure 2B), vehicle treated rats again showed progressively diminishing locomotor activity over the course of the test session. Both 5 and 10 mg/kg MDAI showed a similar pattern of decreasing locomotion, however activity was significantly reduced compared to vehicle (5 mg/kg in blocks 2-5, and 10 mg/kg in blocks 1-3, minimum t(18) = 2.36, p = 0.03. 20 mg/kg MDAI dose produced consistent locomotion over the session, manifested statistically as significantly lower activity than vehicle in block 1, t(11.97) = 2.89, p = 0.01; although means then suggest higher activity in later blocks, there were no further significant differences between 20 mg/kg and vehicle, maximum t (18) = 1.77.

Figure 2 A/B: Mean total trajectory length over 30 minutes (cm/5 minute block) by testing onset (15 minutes (A) or 60 minutes (B)) after sc. MDAI (5 mg/kg, 10 mg/kg, 20 mg/kg or vehicle). Error bars show +/- 1 standard error of the mean (+/-1 s.e.m.). Timings showed on the X axis indicate time blocks since the placement into the testing arena. Inserts show characteristic trajectories.

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Tcentre and thigmotaxis were analysed with 4 x 2 independent ANOVAs with drug treatment and testing onset as between subjects factors. Analysis of thigmotaxis produced significant main effects for drug treatment, testing onset and a significant interaction, minimum F(3,72) = 6.21, MSe = 0.01, p = 0.00, see Figure 3C. Rats were less likely (than vehicle) to appear in the periphery under 10 mg/kg, and 20 mg/kg MDAI (but not 5 mg/kg, t(18) = 0.89 p = 0.39) in the 15 minute testing onset group minimum t(10.44) = 2.37, p = 0.02. In the 60 minute testing onset condition, all doses MDAI increased likelihood of appearance in the periphery, minimum t(13.94) = 2.60, p = 0.02.

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Analysis of Tcentre produced significant effects for drug treatment, testing onset and a significant interaction between the two, minimum F(3,72) = 12.16, MSe = 31935.56, p = 0.00, see Figure 3D. In the 15 minute testing onset group, rats spent more time in the central zones (than vehicle) under 10 mg/kg and 20 mg/kg MDAI, (but not 5 mg/kg, t(18) = 1.53, p = 0.14) minimum t(18) = 4.56, p = 0.00. In the 60 minute testing onset condition, MDAI did not significantly affect time spent in the central zone, although the effect of 5 mg/kg was marginal, maximum t(18) = 2.03, p = 0.06.

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Separate independent t-tests comparing 40 mg/kg MDAI to vehicle at 15 minute testing onset induced similar effects (to 10 and 20 mg/kg) on locomotion, time in the centre and on thigmotaxis; that is, 40 mg/kg MDAI animals had a significantly lower baseline in block 1, followed by progressive increases in activity, significant hyperactivity in blocks 3-6, minimum t(13.59) = 2.59, p = 0.02, but not block 2, t(11.81) = 1.23, p = 0.23. 40 mg/kg treated rats were significantly less likely to appear in the periphery, and spent significantly longer in central zone, minimum t (12.23) = 7.33, p = 0.00.

Figure 3 A/B: Mean thigmotaxis (A) and mean Tcentre (B) over 30 minutes by testing onset (15 minutes or 60 minutes) after sc. MDAI (5 mg/kg, 10 mg/kg, 20 mg/kg or vehicle). Error bars show +/-1 s.e.m. Asterisks indicate significant differences from vehicle at minimum p<0.05.

Prepulse inhibition: Habituation, ASR and PPI data were analysed with 4 (drug treatment) x 2 (testing onset) independent ANOVAs. ASR data showed a significant main effect of testing onset, F(1,72) = 5.42, MSe = 6194.10, p = 0.02, manifested as higher startle values when testing was 60 minutes (mean = 154.80; s.e.m. = 27.07) versus 15 minutes (mean = 113.82; s.e.m. = 21.05) post drug administration. The drug treatment x

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There was a main effect of drug treatment on habituation, F(3,72) = 4.12, MSe 1285.38, p = 0.00, mean (s.e.m.) per drug group as follows: Veh = 61.59 (5.36); 5 mg/kg = 30.26 (11.50); 10 mg/kg = 26.75 (8.54); 20 mg/kg = 30.73 (6.55). Neither the drug treatment x testing onset interaction nor the main effect for testing onset were significant, maximum F(1,72) = 3.80, p = 0.06. Independent t-tests for the main effect of drug treatment showed that all doses of MDAI significantly reduced habituation, minimum t(18) = 2.47, p = 0.02. MDAI drug treated rats did not differ from one another, maximum t(18) = 2.45, p = 0.81.

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Since there was a significant main effect of drug treatment on habituation, this was included as a covariate in analysis of PPI data. With habituation as a covariate, neither the drug treatment x testing onset, nor the main effect for testing onset were significant, maximum F(1,71) = 2.45, p = 0.12. MDAI treatment significantly disrupted PPI, shown as a main effect of drug treatment, F(3,71) = 13.34, MSe 361.75, p = 0.00; mean (s.e.m.) per drug group as follows: Veh = 42.23 (4.52); 5 mg/kg = 22.85 (4.28); 10 mg/kg = 5.10 (4.30); 20 mg/kg = 9.94 (4.27). Independent t-tests on the main effect showed that 10 and 20 mg and 5 mg doses of MDAI significantly disrupted responding compared to vehicle (irrespective of testing onset), minimum t (38) = 2.56, p = 0.02. 10 mg/kg and 20 mg/kg produced a significantly greater disruption of PPI than 5 mg/kg MDAI, minimum t(38) = 2.01, p = 0.05, but did not differ from one another, t(38) = 0.38, p = 0.47. The drug x testing onset interaction was not significant, however descriptive statistics showed a noticeable difference between 15 and 60 min for the 5 mg/kg dose, further, by 60 min, the difference between 5 mg/kg and vehicle was no longer present (Figure 4). ANOVA can be insensitive when small group sizes are used (as is necessarily the case with animal research); therefore despite the lack of statistical significance in our interaction, additional t-tests were conducted comparing 5 mg/15 min and 5 mg/60 min which shows that the effect of 5mg/kg MDAI had diminished by 60 min, t(14.51) = 2.22, p = 0.04. Moreover at 15 min, 5 mg/kg MDAI disrupted PPI compared to vehicle, t(18) = 3.68, p = 0.00, and by 60 min, it did not, t(18) = 0.43, p = 0.67. All other doses of MDAI disrupted PPI (compared to vehicle) at both testing onsets, minimum t(18) = 2.95, p = 0.01. The disruptive main effect of MDAI on PPI is clear; furthermore our results tentatively suggest that by 60 minutes the disruptive effect of 5 mg/kg MDAI on PPI may diminish.

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ACCEPTED MANUSCRIPT Figure 4: Mean percentage prepulse inhibition (AVG amplitude) 15 min and 60 min after sc. MDAI (5 mg/kg, 10 mg/kg, 20 mg/kg or vehicle). Error bars show +/-1 s.e.m. Asterisks indicate significant differences from corresponding vehicle at minimum p<0.05.

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Toxicity: Body temperature and preliminary LD50

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Rectal temperature and perspiration: 3 x 2 x 13 factorial ANOVA showed significant main effects

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of drug treatment, F(2,648) = 5.53, MSe = 3.42, p = 0.00); home-cage condition, F(1,648) = 48.22, MSe = 29.82, p = 0.00; and time, F(12,648) = 33.27, MSe = 5.21, p = 0.00, on rectal temperature. All interactions were significant, minimum F(2,54) =, 5.51, MSe = 3.41, p = 0.00. Means for the significant three-way drug x home-cage x time interaction suggested that for rats in group-housed conditions, MDAI elevated temperature compared to vehicle controls, F(24,648) = 5.66, MSe = 0.89, p = 0.00 (Figures 5A and 5B).

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Independent t-tests showed that under group-housed conditions, MDAI produced an elevation in temperature first evident at 09.30h, which was maintained for both MDAI doses until 12.00h in 10 and 20 mg/kg groups, extending to 13.00h in the case of (minimum t(18) = 2.19, p = 0.05.

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In rats housed individually, (despite a significantly lower baseline temperature than vehicle at 09.00h) MDAI, 10 mg/kg significantly increased body temperature from 10.30h – 14.00h, minimum t(18) = 2.22, p = 0.04. At the 20 mg/kg dose, baseline temperature was significantly lower than vehicle at 09.00h, and remained significantly lower until 10.00h. Between 12.00h - 14.00h 20g/kg MDAI rats had significantly higher temperatures than vehicle, minimum t(18) = 2.17, p = 0.04.

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Perspiration (11% to >51% body) was observed in all 20 mg/kg MDAI treated group-housed rats at all time points from 09.30h – 11.00h (inclusive). Within the same time period, all 10 mg/kg MDAI treated group-housed rats showed perspiration (11% to 50% body), albeit neither so persistent nor profound (only 80% of rats were wet at 10.30 and only 40 % at 11.00). Only one MDAI treated rat (20 mg/kg) in the individually-housed condition was observed to become wet (11-20% body). None of the vehicle treated rats became wet, irrespective of housing condition. Since perspiration was not observed in all conditions, the full factorial analysis intended was not possible. Instead, single-sample t-tests were conducted to test whether mean perspiration (calculated as average perspiration over 13 measurement time-points) in groups where it was observed was significantly different from zero. Significant perspiration was observed under MDAI at 10 mg/kg (mean = 0.62, s.e.m. = 0.1), and 20 mg/kg (mean = 0.90, s.e.m. = 0.1) in the group housed conditions minimum t(9) = 8.34, p = 0.00. Perspiration (mean = 0.12, s.e.m. = 0.1) was observed under MDAI at 20 mg/kg in individually housed rats (just one animal) within the same time period, but this was not significant t(9) = 1.15, p = 0.28 (see Figure 5A and 5B).

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Figure 5 A/B: Mean temperature (°C) over 12 hours in rats (n = 10) housed individually (A) or in groups of five (B). MDAI sc. (10 mg/kg, 20 mg/kg or vehicle) was administered at 09.00 am (dotted vertical line). Error bars show +/-1 s.e.m. The perspiration is marked in the B part of the figure perspiration indicating a percentage of body surface wet for MDAI treated rats under group housed conditions (+ = 11-20%, ++ 21-50%, +++ = >51%; when in BOLD = all animals perspired, when NORMAL = only some animals became wet). No notable perspiration was observed in vehicle treated or individually housed animals (one animal administered with 20 mg/kg perspired little bit).

Preliminary LD50: MDAI administered sc. yielded LD50 value of 28.33 mg/kg. Mortality typically

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occurred 2-24 hours after the injection (Table 1). Rats showed hyperventilation, signs of serotonin syndrome, especially flat body posture and intense perspiration (most pronounced in the areas of injection), copious salivation, and seizures. Weight observations of survivors showed modest initial reductions in percentage weight gain at higher doses (25 and 35 mg/kg). Percentage weight gain in these animals was then at the same rate as po. treated animals, although absolute weights remained lower than other doses. All animals treated with 40 mg/kg. sc. died shortly after administration so it is not possible to comment on trends in weight.

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Rats treated with po. MDAI (at all doses) survived the full seven day observation period; no toxicity was observed, therefore LD50 could not be calculated. However ataxia and flat body posture were noted with increasing dose (20 mg/kg = 1 animal, 25 mg/kg = 2 animals, 30 mg/kg = 5 animals) and were present in all rats treated with 35 and 40 mg/kg MDAI po. Weight observations showed steady percentage weight gain over the subsequent seven days. MDAI administered iv. produced LD50 of 35 mg/kg. Mortality was typically within 10 minutes and was associated with similar symptoms as sc. administration, in particular seizures but no perspiration. Notably, where animals survived the 35 mg/kg and 40 mg/kg doses, seizures were observed. Percentage weight gain in surviving rats was steady over the subsequent seven days, almost indistinguishable from po. treated rats.

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20 25 30 35 40

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Table 1: Number of animals whom died/survived per group (n = 6) during LD50 test of sc./po./iv. MDAI at 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg or 40 mg/kg.

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Pathological examination of organs of a dead animal: Microscopy showed hyperaemia in all organs. In brain, kidney and myocardium we found hyaline thrombi in microvessels with predominant occurrence in the brain. In myocardium was found an early ischaemic changes; the brain shows stamps of pericellular oedema and the in kidneys was found hyaline thrombi in the capillaries of Bowman bodies. Histological findings of the lung were completely negative, whilst the liver exhibited dystrophic changes indicative of multiorgan failure. Image of histological changes was closed as disseminated intravascular coagulopathy (Galera-Davidson et al., 1989) with predominant changes in the brain, with heavy oedema as the likely cause of death (see online supplemental material Fig. 6AD).

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Fig.6. Pathological–anatomical slides, HE staining: (A) Myocardium - hyaline thrombi in microvessels, magnification 400 x, (B) Myocardium - early ischaemic changes, magnification 200 x, (C) Kidney hyaline thrombi in capillaries of glomerulus, magnification 400 x, (D) Brain - hyaline thrombi in microvessels, magnification 400 x

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Additional confirmation of the origin of perspiration, of toxicity and further behavioural observations:

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Systematic observation results: First 10 min observation, the rats were active, exploring the cage, showing reasonably normal behaviour. Behavioural activity was moderately increased starting at 10 min with an overall rating of 7, declining slowly to 2 or 1 (almost immobile), by 100 min. Two of the rats produced between 0 – 16 rears per each bin up until 60 min, with just 1 rear recorded for Rat 1, thereafter. Rat 3 reared a little, just 12 rears altogether. Sniffing appeared not altered; no signs of stereotyped sniffing were present. Rats produced almost no licking behaviour or grooming (therefore they did not spread saliva on themselves, or spread sweat/urine during grooming). Four instances of stepping/padding air (<3 sec each) were observed, none after 60 min. Also observed was front paw clapping (Rat 3), and all rats ‘paddled’ the floor, and as the session progressed they padded at the walls. At 100 minutes animals were no longer producing much behaviour.

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Some atypical behavioural patterns, characteristic for serotonin syndrome were also observed in all animals, especially backward walking and flat body posture (start before 10 min, and remained throughout the observation), wet dogs shakes and head weaving (around 50-60 min ). Each rat exhibited brief loss of tone, and loss of control over their hind-quarters, some evidence of collapse, lying with head and paws halfway up the wall but body flat on floor, lying flat on back, collapsing onto side. This ‘collapsing’ behaviour commenced around 30 min, and grew worse towards the end 100 min, particularly in rat 3. It seemed to be accompanied by paw clapping/rubbing or paw cycling. Rat 3 hopped backwards on its hind legs, clapping, with a rigid posture. There was some rotational behaviour before 60 min with maximum around 40 min (most in the rat 3). To begin with, all rats also trembled for up to 30 min, and all showed very marked ‘ear-swivelling/ear-wiggling’ and ‘vibrating’ ears. Defecation was more evident in the first 40 min (11 boli in total and none after), and overall urination did not seemed to be robustly altered. This is also supported by the fact that when we collected fur from the belly at 60 min, it was quite dry, whilst fur on the head/neck and back was evidently wet. Salivation commenced around 20-30 min and was moderate – profuse. Although we continued to record moderate-profuse salivation in later bins, it was not possible to tell if rats had salivated much more, or whether they remained wet from before. Since rats did not lick or groom much, saliva was not transferred to fur of the body, but large amounts of saliva were observed on the cage wall, and the rats had close contact with one another at times, and some saliva may have been transferred. All rats became wet, moderately to profusely (delayed in rat 3). The perspiration appeared initially around the back area above the tail and then crescent to back area of the neck and then diffusely so rats were almost completely wet (more than 50% of the body surface). By 60 min, the perspiration appeared to be drying slightly (fur hardening into points) - although we continued to record sweating after 60 min, it is not clear if animals produced much more sweat. The animals had dried noticeably by 100 min.

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Two of the three rats exhibited bright red exudate from nose and mouth at 50 min. This was present for 10-20 min, but did not change/increase during this time. Microscopic evaluation confirmed presence of erythrocyte membranes, epithelium cells and coccus bacteria. Also noted was that rats had very bright red paws (especially between the digits) from 30 min onwards, appearing bloody, but they were not.

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At 60 min, it was noticed that two of the three rats had a large pea-sized firm lump of pale white/cream exudate from the penis. Most likely it was a spermatic proteinaceous penile plug (Goswami et al., 2014; Lejnieks, 2007) which is made from a build-up of sperm mixed with exfoliated urothelial cells and cellular debris. It is believed that these plugs result from abnormal (spontaneous) ejaculation, and the secretions from the male accessory sex glands (Mohr et al., 1992).

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Pathologic-anatomical examination of organs revealed hyperaemia of all organs (brain, liver and kidney) with commencing shock necrosis in the liver. As stated above, erythrocytes were confirmed in the smears of red exudate with cocci bacteria and epithelial cells (see online supplemental material fig. 7).

Fig. 7. Giemsa staining – erythrocyte in the middle and cocci bacteria in the smear of the redness exudate

Chromatographic analyses: Sample fingerprinting & chromatographic separation: There were minor differences between the fingerprints of fur collected before or during maximal perspiration (see online supplemental material Fig. 8A-C).

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Fig. 8. Example of fingerprint of dry fur from the neck before drug administration (A) and wet fur from the neck of the same animal 60 min after drug administration (B) and saliva from an untreated rat (C).

Separation on column revealed that the chromatograms of fur had two main peaks around 0.6 and 1.4 min which were more pronounced after the treatment in a wet fur and in addition fur after administration contained another peak at around 3.8 min. Urine samples were characterised by a peak at 0.5-0.6 min followed by another at 0.7-0.8 min and no specific peaks further, saliva in contrast showed characteristic peaks at approximately 0.55 and 0.7peaks (see online supplemental material Fig. 9. I.-IV.). Comparison of m/z values in the early peaks in fur and saliva revealed different spectrum of compounds with different molecular masses (data not shown)

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Fig. 9. Examples of chromatograms. I. & II. fur from the neck area of two animals before (A) and after (B) treatment. III. chromatograms of blotter papers 20 min (A) and 90 min (B) after administration and blank urine sample (C). IV. chromatograms of 3 samples of blank saliva. 21

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Chromatographic detection of urea and creatinine: There were minimal amounts of urea in the fur, which slightly increased after treatment, however in samples from blotted papers as well as from blank urine clear peaks at retention times tr=0.7 min was obvious with slightly smaller peak in urine compared to blotter. Creatinine had a clear peak in fur (tr=0.86); no large difference was observed before and after administration (sweated fur). The peak was several times more pronounced in blank urine as well as on blotters with no big differences between the two. There were almost none urea and creatinine present in the blank saliva samples. For comparison of chromatograms see online supplemental material Fig. 10.

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Fig. 10. Creatinine peaks in examples of analysed samples: (A) blotter paper 90 min after treatment, (B) blank urine, (C) fur before treatment, (D) fur after treatment, (E) saliva

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To summarise, in behavioural tests MDAI generally resulted in: 1) a modest transitory stimulatory effect on locomotion, and attenuated habituation to the novel environment of the open field; 2) reduced habituation to auditory stimuli and disrupted PPI, consistent with persistent psychomimetic effects, and 3) behavioural serotonin syndrome. MDAI rapidly penetrated the blood-brain barrier, and maximal behavioural effects corresponded well to peak brain and serum levels of the drug. MDAI induced hyperthermia and profuse perspiration especially in group-housed animals. Preliminary LD50 values were established for sc. (28.33 mg/kg) and iv. (35 mg/kg) administration with a disseminated intravascular coagulopathy (DIC) and brain oedema a as a cause of death; gastric administration showed no acute toxicity up to doses of 40 mg/kg, on the contrary 20 mg/kg sc. already induced liver toxicity.

Pharmacokinetics

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With an elimination half-life of 0.8 h MDAI showed slightly faster kinetic profile than its reported congener 2-AI (1-2 hours: (Fuller et al., 1977) and was markedly faster than after sc. MDMA (2.2-2.5 hours) in rats (Fitzgerald et al., 1990) and oral MDMA (3.6-5.8 hours in humans (Fallon et al., 1999). It is of note that compared to our and Fuller´s experiments the study done by Fitzgerald et al. was performed on Sprague-Dawley rats. Since there are strain related differences in pharmacokinetics and pharmacodynamics of serotonergic drugs this has to be considered with caution while making such direct comparisons further on (Fernandez et al., 2003; Wright, Jr. et al., 2012). MDAI, like PMMA and 2C-B (Palenicek et al., 2007a; Palenicek et al., 2011) significantly accumulated in brain and lungs which might be associated with toxicity of these drugs as has been described for human fatal MDMA overdoses (De Letter et al., 2004). The high influx into the brain tissue and especially lungs was rapid and un-delayed relative to sera suggesting high lipid solubility which is most likely attributable to the presence of amino group on the pentylic cycle of the molecule. Its tissue distribution profile is similar to the aforementioned compounds (Palenicek et al., 2011; Rohanova et al., 2008). The time-course of these kinetic observations are consistent with behavioural effects (discussed below) and also with human user reports of the onset of subjective effects (after oral dosage) at 10-12 minutes with peak subjective effects lasting 30-45 minutes (Corkery et al., 2013).

Thermoregulation, LD50 and acute systemic toxicity As expected, MDAI increased body temperature robustly (up to 2°C) and furthermore produced profuse perspiration in group-housed animals (temperature increases after MDAI in individuallyhoused animals were within a similar range to vehicle treated rats), therefore crowded environmental conditions exacerbates the hyperthermic potential of MDAI, and increases the risk of harm. We have finally observationally and analytically confirmed the origin of profuse perspiration even though it is highly improbable in rats under normal circumstances. Interestingly the increase of temperature persisted for relatively long period of time (3 hours) compared to the estimated elimination half-life which indicates possible prolonged toxicity. The underlying mechanism is most likely related the effects of MDAI on 5-HT release, since hyperthermia is one of the core symptoms of serotonin syndrome (Boyer and Shannon, 2005). The animals exposed to the highest doses showed also other typical signs of this syndrome and toxicity: hyperventilation, flat body posture, head waving, wet dog shakes, walking backwards , rotational behaviours, salivation and convulsions. In addition, limb anaemia and dark red/pink exudate with a presence of erythrocytes from nose and mouth was observed, most likely as a result of DIC (Boyer and Shannon, 2005; Palenicek et al., 2011). 23

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This also supports the fact that the exudate is not related to the porphyrin secretion from Harderian gland (Hanson, 2006; Segal et al., 2003). The accumulation of MDAI in liver (mean liver/serum ratio of 1.9) might also contribute to observed liver toxicity. Altogether, symptoms of acute liver dysfunction / failure, metabolic acidosis, rhabdomyolysis, renal failure and DIC are typical causes of death associated with hyperthermia during serotonin syndrome (Boyer and Shannon, 2005; Isbister et al., 2007). MDAI po. did not induce signs of severe toxicity except mild behavioural symptoms of serotonin syndrome (flat body posture and ataxia) which indicates that the first pass metabolism or lower / slower absorption seem to have protective effect. Surprisingly, the LD50 for iv. administration was slightly higher (35 mg/kg) than in the sc. experiments. One possibility that might account for such effect is that higher doses were affected by incomplete solubility in the 1 ml volume used for LD50 testing, which may have affected pharmacokinetics differentially, according to route of administration. Since there were no problems with solubility at doses below 30 mg/kg, the sc. LD50 value is likely accurate. Death was rapid (within minutes) after iv. MDAI which could also indicate a mechanical reasons caused by the suspension e.g., acute embolia. Since MDAI accumulated in lung tissue, acute pulmonary or cardiac toxicity, e.g., due to exaggerated sympathomimetic activity could also contribute to rapid lethality. However pathological examination confirmed hyperaemia and signs of toxicity (ischemia, necrosis and microthrombi) in all organs except lungs of a dead animal. Hyperaemia was also observed in organs of survived animals with signs of initiating shock necrosis of the liver parenchyma indicating that already lower doses might produce substantial liver and organ toxicity.

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In line with the kinetic findings, the overall locomotor stimulant activity of MDAI (at all doses) was more pronounced at 15 minutes post-administration; however, when tested 60 minutes after administration, 5 mg/kg and 10 mg/kg resulted in hypolocomotion (40 mg/kg was not tested). It is unlikely that this reflects a sedative effect at 60 minutes; rather the overall picture is that MDAI has a mild stimulatory effect on locomotion that tends to diminish 60-90 minutes post-administration particularly at lower doses. The 20 mg/kg dose (60 minutes post-administration) seemed to produce a residual stimulatory effect, although statistically this was not significant.

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Locomotor habituation in the open field was clearly reduced by MDAI over time as indicated by initial inhibition/ataxia, followed by progressive increases in locomotion at 15 minutes (at all doses) and steady state locomotor effects at 60 minutes (10 and 20 mg/kg) which contrasted the very typical progressive decline in activity (i.e., locomotor habituation) in controls. Ataxia, flat body posture, falls etc. were also confirmed by our additional observations for 20 mg sc. therefore being the most likely cause of the locomotor inhibition present with the highest dose. At 60 minutes post-administration the 5 mg/kg dose appeared to increase, rather than reduce locomotor habituation compared to controls. The increased time spent in the centre of arena with concomitant reduction in the likelihood of appearing in the peripheral zones at 15 minutes post-administration of all doses (except 5 mg/kg) might reflect increases in exploration and anxiolysis. Non-specific modest locomotor stimulant effects do not likely account for observed effects, on the contrary one might speculate that inhibition/ataxia might contribute: even so, this could have only a minor influence since this only affected activity initially. By 60 minutes post administration MDAI did not affect time in the central zones and increased thigmotaxis, therefore anxiolytic effects have diminished. According to MDAI users, the peak effects of the drug are followed by ‘moderate serotonin burnout or mild comedown’ (Corkery et al., 2013; Townsend, 2010). Therefore hypolocomotion observed with the two lower doses at 60 minutes post-administration coupled with increased thigmotaxis might be consistent 24

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with negative affect (e.g., increased anxiety) as a result of the ‘come down’. Given that PPI remained disrupted at 60 minutes post-administration (see discussion, below), indicating the continued presence of psychomimetic effects (whilst effects on locomotion and exploration have already subsided), and that brain and serum levels of MDAI peaked at 30 minutes and diminished rapidly thereafter, this seems plausible. Again, a non-specific motor effect does not account for increased thigmotaxis at the two lower doses, since rats did not show any signs of locomotor stimulation. The trajectories of 20 mg/kg treated rats (60 minutes post-administration) suggested stereotyped circling of the arena and this group showed some evidence of hyperlocomotion at 60 minutes postadministration, however, statistically, this was non-significant. Overall, open field findings suggest that MDAI, especially at higher doses, acts as a mild locomotor stimulant, with mild anxiolytic effects that produce exploratory behaviour, and reduce locomotor habituation. It seems that such effects are not sustained; being greatly reduced 60-90 minutes post-administration. Furthermore, the pattern of MDAI effects in the in the open field suggests that at 60 minutes, residual effects of higher doses resemble effects of lower doses seen at 15 minutes, hence behaviour is related to dosage and time in an orderly and predictable manner.

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In PPI, the disruptive effect of MDAI (at all doses, irrespective of testing onset) on sensorimotor gating was clearly evident and, together with reduced habituation, indicates robust alteration in general sensorimotor function. When habituation was included in analyses as a covariate, PPI remained disrupted, suggesting that MDAI exerts effects on sensorimotor gating that are dissociable from general effects on sensorimotor function. ASR was unaffected by MDAI (or tended to be decreased, rather than increased with the lowest 5mg/kg dose used): statistically this effect was marginal and so the nonspecific effects on the startle reaction can be excluded.

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In the open field when tested 60 minutes after MDAI administration, locomotor, habituation and exploratory/anxiolytic effects had diminished greatly, however in PPI, the psychomimetic effects of even the lowest dose of MDAI seem to persist. Taken together, this suggests that anxiolytic effects diminish before effects on locomotor activity do, and that altered sensorimotor gating/ psychomimetic effects are sustained at least for 90 minutes even at low levels of MDAI (<5 mg/kg). This suggests that even when (other) peak psychopharmacological effects (e.g., subjective intoxication) have diminished, marked attentional deficits may be present for a prolonged period of time, which has obvious implications for and situations where intact cognitive function is required, e.g. driving a car.

Comparison of behavioural and toxicological findings to MDMA and other compounds Behavioural effects in the open field after MDAI closely resembled those observed after the entactogen MDMA as well as PMMA , albeit of shorter duration (Palenicek et al., 2005; Palenicek et al., 2007b; Palenicek et al., 2011). They are, however, clearly distinct from those produced by classical serotonergic hallucinogens (psilocin, LSD, mescaline, which typically result in either inhibition of locomotion or a biphasic effect with initial inhibition followed by late onset increase in ambulation (Palenicek et al., 2007a; Palenicek et al., 2008; Palenicek et al., 2010; Rambousek et al., 2014; Tyls et al., 2015). Effects of MDAI on locomotion were much milder than those of amphetamines by comparison (Fukushima et al., 2007; Hall et al., 2008; Kehne et al., 1992) which is unsurprising given MDAI’s DAT/SERT ratio (Liechti, 2015). Overall, these findings suggest that pattern and magnitude of behavioural effects in the open field can distinguish between stimulant, hallucinogenic and entactogenic classes of drugs. In turn, if purported entactogenic compounds produce a distinct behavioural profile in the open field, this supports the notion of entactogens as a unique drug class. On the other hand, related aminoindanes 2-AI and 5-methoxy-6-methyl-225

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aminoindane (MMAI) do not increase motor activity in rats or mice (Marona-Lewicka and Nichols, 1994; Mrongovius et al., 1978), however these compounds have a different profile of monoamine release: 2-AI has a greater effect on norepinephrine release (and, unlike MDAI here, is reported to have very little psychotomimetic effect in humans) whereas MMAI is a more highly selective serotonin-releaser (Marona-Lewicka and Nichols, 1994). MDAI by contrast, induces both serotonin release (primarily) with more modest effects on dopamine release, and little effect on norepinephrine (Simmler et al., 2014). Effects of MDAI on thermoregulation (elevated temperature, profuse sweating) are similar to PMMA and MDMA suggesting a common serotonergic mechanism (Palenicek et al., 2011). PMMA and MDMA also show the same effects in PPI paradigms, i.e., attenuated habituation to startle (Kehne et al., 1992; Liechti et al., 2001; Martinez and Geyer, 1997; Palenicek et al., 2011) and persistent deficits in PPI. Concomitant serotonin and dopamine activation may therefore underpin the unique profile of behavioural activity associated with entactogenic drugs (including MDAI), distinct from dominant dopamine release in stimulants or 5-HT2A receptor agonism characteristic of hallucinogens.

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As already noted, several deaths have been reported associated with MDAI use. Its relatively mild and transient effects on locomotion and anxiolysis is in line with that MDAI is thought to be less potent and safer than MDMA by recreational users, and that doses are often double those taken of MDMA. Within-session re-dosing, and combination with other drugs is commonplace, all of which (along with environmental conditions typical at the time of use) significantly increase acute systemic toxicity. However most subjects whom died after MDAI ingestion had symptoms of serotonin syndrome congruent with our findings (Corkery et al., 2013) with the very first case reported whom survived overdose with 5g of the substance showed hyperthermia, psychotic reactions, multiorgan failure and DIC (George et al., 2011). Although the 40 mg/kg sc. was deliberately chosen as a high dose of MDAI for behavioural studies that might produce some acute systemic toxicity, 90% fatality at this dose was unexpected since the same dose and route had been used previously in SpragueDawley rats that survived at least one week (Nichols et al., 1990). MDMA in doses up to 40 mg/kg sc. has been used safely in some cases, although has been fatal in others (Dornan et al., 1991; Hardman et al., 1973; Kehne et al., 1992; Martinez and Geyer, 1997; Soto-Montenegro et al., 2007). The LD50 of MDMA is 49 mg/kg intraperitoneally (Hardman et al., 1973) and 325 mg/kg orally in rats (Goad, 1985). PMMA sc. has been shown to have LD50 in rats between 80-100 mg/kg. Variation in route of administration across different compounds makes like-for-like comparison difficult; nevertheless our preliminary LD50 findings suggest that sc. MDAI may be potentially more toxic than either MDMA, or PMMA. There is ample evidence that environmental manipulations such as group-housing of rodents can increase the toxicity of serotonin releasing drugs, in some cases quite dramatically, (Davis and Borne, 1984; Glennon et al., 1988; Hardman et al., 1973; Steele et al., 1992). Hence, LD50 studies that test under ‘crowded’ animal housing conditions would be particularly useful given the environmental conditions in which typical human use occurs.

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ACCEPTED MANUSCRIPT Conclusions

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MDAI had mild stimulatory effect, disrupted sensorimotor gating, induced signs of behavioural serotonin syndrome and robust increase of body temperature followed by profuse perspiration in group-housed condition. Our findings are generally consistent with previous estimations of effects of MDAI that it is an entactogen showing serotonergic profile comparable to MDMA but not to hallucinogens or stimulants. However, our findings are at odds with MDAI’s reputation amongst recreational users as non-(neuro) toxic compound with mild effects. Here, we present evidence that MDAI can produce acute systemic toxicity in rats that seems to be fatal at much lower doses than MDMA, or even PMMA. To date, the number of human fatalities reported due to MDAI use are fortunately few (Corkery et al., 2013). Nevertheless, our findings are alarming, and their implications for harm reduction are obvious.

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ACCEPTED MANUSCRIPT Acknowledgements

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This work was supported by projects MICR VG20122015075, VG20122015080, NT/13897, PRVOUKP34, MH CZ - DRO (NIMH-CZ, 00023752) and ED2.1.00/03.0078.

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Research highlights MDAI induces locomotor stimulant effects and behavioural serotonin syndrome MDAI disrupts sensorimotor processing MDAI accumulates in the brain and liver compared to serum MDAI increases body temperature and induces profuse perspiration under aggregated conditions 5) LD50 after subcutaneous administration in aggregated condition is 28.33 mg/kg with multiorgan failure, disseminated intravascular coagulopathy (DIC) and brain oedema as a cause of death

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1) 2) 3) 4)

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