Drug-Facilitated Sexual Assaults (DFSA): a Serious Underestimated Issue

European Review for Medical and Pharmacological Sciences 2019; 23: 10577-10587 Drug-facilitated sexual assaults (DFSA): a serious underestimated issue

F.P. BUSARDÒ1, M.R. VARÌ2, A. DI TRANA1, S. MALACA1, J. CARLIER3, N.M. DI LUCA3

1Department of Excellence of Biomedical Sciences and Public Health, University “Politecnica delle Marche”, Ancona, Italy 2National Centre on Addiction and Doping, Istituto Superiore di Sanità, Rome, Italy 3Department of Anatomical, Histological, Forensic and Orthopedic Sciences, Sapienza University, Rome, Italy

Abstract. – OBJECTIVE: Drug-facilitated sex- Key Words: ual assault (DFSA) is a nonconsensual sexual Drug-facilitated sexual assault, Urine and hair anal- act in which the victim is incapacitated or un- ysis, Epidemiology. conscious due to the effects of alcohol, a drug and/or other intoxicating substances. Dozens of drugs (including ethanol) can potentially be used to commit sexual assaults, but γ-hydroxy- Introduction butyric acid (GHB) and flunitrazepam are the most common “date rape drugs”. MATERIALS AND METHODS: Multidisciplinary Drug-facilitated sexual assault (DFSA) is a databases were browsed using the following form of sexual violence against an individual search terms: “drug-facilitated sexual assault”, incapacitated by a mind-altering substance, “chemical submission”, “date rape”, “rape drugs”, such as alcohol or “date rape drugs”. It is es- and “drink-spiking”. Moreover, a search for re- timated that 75% of all acquaintance rapes in- ports was conducted on Institutional websites to volve alcohol and/or drugs. Drugs, when used identify documentation published by international with alcohol, can result in a loss of conscious- agencies or institutions. Articles and reports were independently evaluated by each author. ness and a loss of the ability to consent to sexu- 1 RESULTS: There are no accurate estimates al intercourse . Most DFSA victims are women of the number of DFSA occurring each year, al- and perpetrators are men, although men can though assaults are increasingly reported. Ma- also be the victims. DFSA occur in three spe- ny DFSA, however, are still not reported. Vic- cific circumstances: (1) when the victim invol- tims are reluctant to report incidents because untarily ingests an intoxicating substance, (2) of embarrassment, guilt or perceived responsibility, or because they do not clearly remember when the victim ingests both voluntarily and the assault. Moreover, most of the drugs typical- involuntarily an intoxicating substance, and (3) ly used in sexual assaults are rapidly metabo- when the victim voluntarily ingests an intoxi- lized, making them undetectable in routine drug cating substance2. The psychoactive substance screenings. most commonly associated with DFSA is alco- CONCLUSIONS: Most of the substances in- hol, but levels of recreational drug use are ris- volved in DFSA, with the exception of alco- ing and drug markets are expanding, providing hol, are under international control and sched- uled under the United Nations Single Conven- faster and cheaper ways to incapacitate a vic- tion on Narcotic Drugs of 1961 and the Conven- tim. Using drugs for nonconsensual sexual action on Psychotropic Substances of 1971. How- tivity has led to the introduction of terms, such ever, several psychotropic substances and anti- as “date rape drugs” and “drink spiking”. Ac- histamines used in sexual assaults are still not curate data on the incidence of sexual assaults under international control, allowing for traffick- is not available, and reports to police are likely ing, often via the Internet and courier. The ab- sence of international control makes it difficult to significantly understate the actual numbers. to obtain accurate data on the nature and the ex- The low reporting rate may be due to many fac- tent of the problem. tors, including psychological barriers (shame

Corresponding Author: Francesco Paolo Busardò, MD, MSc, DipFMS, Ph.D; e-mail: [email protected] 10577 F.P. Busardò, M.R. Varì, A. di Trana, S. Malaca, J. Carlier, N.M. di Luca and embarrassment), cultural beliefs, fear of sexual assault cases through “drink spiking”, stigmatization, and lack of confidence in the the manufacturer (Roche Pharmaceuticals, criminal justice system. Basel, Switzerland) modified the product for- The drugs the most commonly involved in sex- mulation and added a blue dye fizzing in liq- ual assaults are central nervous system (CNS) de- uids. The colorant, however, is not present in pressants. These substances can alter a victim’s flunitrazepam from illicit sources. In France, behaviour, ranging from loss of inhibition to loss additional manufacturing restrictions have of consciousness, and are often associated with been implemented. Benzodiazepines can in- anterograde amnesia. Dozens of drugs (including duce confusion, impaired thinking, and mem- ethanol) can be used in DFSA. γ-hydroxybutyric ory loss, drowsiness, sleepiness and fatigue, acid (GHB) and flunitrazepam (Rohypnol®) are impaired coordination and dizziness3. Benzo- the most common “date rape drugs”. However, diazepines are found in forms of tablets, cap- scientific reports demonstrated that numerous sules, and injectables4. They are lipophilic and CNS depressants and over-the-counter, prescrip- therefore less soluble in polar solvents, such tion, and illegal drugs are also involved. Typical as water and ethanol. However, a higher lipo- drugs used in DFSA include: philicity ensures a faster onset of action. • GHB is a prescription CNS depressant in parts • Benzodiazepines (i.e., Valium®, Xanax® or Ro- of Europe and in the United States, where it is hypnol®); marketed as an anaesthetic and a treatment for • Antidepressants (i.e., Elavil® or Zoloft®); alcohol withdrawal symptoms. In 2005, the • Muscle relaxants (i.e., Soma® or Flexeril®); European Medicines Agency (EMA) also au- • Antihistamines (i.e., Benadryl®); thorized GHB use as a medicine to treat narco- • Over-the-counter sleep aids (i.e., Unisom®); lepsy with cataplexy in adults. In March 2001, • Hallucinogens (i.e., ecstasy, marijuana, or ket- GHB was listed as a Schedule IV drug in the amine); 1971 United Nations (UN) Convention on Psy- • Opioids (i.e., Vicodin® or Oxycontin®). chotropic Substances, thereby compelling Euro- pean Union (EU) Member States to control the DFSA drugs are typically found at raves, substance under their own national legislation. dance clubs, and bars, but they are also sold GHB induces sedation and anaesthesia and has in schools, on college campuses and at private a generally steep dose-response curve with a parties. Many drugs can also be purchased via high interindividual variability. A small dose Internet while others, such as prescription ben- increase can cause loss of physical control and zodiazepines, are often available at home. The consciousness. It also increases sex drive, lowers most commonly used substance is ethanol, as inhibitions, and induces memory lapses, drowsi- it is legal, inexpensive, and there is usually no ness, and dizziness. Degenhardt et al5 highlight- need to force consumption. ed the potential risk of GHB, reporting that over 50% of individuals who had voluntarily used the • Ethanol is the most common CNS depressant drug for recreational purposes had experienced in DFSA. It can impair judgement and reduce unconsciousness following use. GHB is used in inhibition and, in larger quantities, can cause amnesia treatment with a 50 mg/kg dose, and loss of physical control and consciousness. The as an analgesic at 10-20 mg/kg. Intoxication can effects of most drugs with anxiolytic, sedative occur above 15 mg/kg and doses higher than 50 or hypnotic properties may be significantly in- mg/kg are toxic, while a 4-g dose is lethal6. GHB creased when taken with alcohol. is quickly metabolized with a half-life varying • Benzodiazepines also are CNS depressants, between 20 min and 1 h6. although most are controlled under drug or • GHB is easily manufactured from its precur- medicine legislation. Flunitrazepam (wide- sors γ-butyrolactone (GBL) and 1,4-butanediol ly known as “roofies”) is the most common (1,4-BD), which are widely used solvents in in- benzodiazepine in DFSA. Flunitrazepam is dustry. GBL and 1,4-BD are rapidly converted marketed under the trade name of Rohyp- to GHB upon ingestion. GBL and 1,4-BD are nol® in many European countries as a pow- not controlled in most countries, but Sweden, erful sedative/hypnotic prescription drug. It Italy, and Latvia enforced controls similar to is tasteless, odourless, and dissolves in liquid. those for GHB. In the United Kingdom, restric- When flunitrazepam began being involved in tive measures are being currently examined.

10578 Drug-facilitated sexual assaults (DFSA): a serious underestimated issue

GHB and GBL onset of action is 20-60 min Materials and Methods and 10-30 min, respectively7. • Ketamine, a Schedule III short-acting anesthet- Reports from 256 female patients who were ic for humans and animals, is another common admitted (voluntarily or accompanied by their drug in DFSA. It is a general anesthetic induc- parents if under 18 years of age) to the Sexual ing a feeling of depersonalization and dereal- Assaults Centre of Careggi University Hospital ization. The victim can be aware of the assault, in Florence, Italy, between January 2010 and Ju- but unable to move or fight back. Ketamine can ly 2018 were examined. The centre received vic- also cause amnesia. tims from the “Area Vasta Firenze”, including 4 cities and more than 1,500,000 inhabitants. The DFSA drugs are typically odorless, colorless, procedure for identification and data and sample and tasteless when mixed in a drink. GBL is an collection was described in an agreement proto- exception due to its bitter taste, although it is eas- col between the Sexual Assault Centre and the ily concealed with flavorsome drinks. Within 30 Unit of Forensic Toxicology of the University of minutes of ingestion, the victim may struggle to Florence, approved by the Medical Ethical Com- talk or move and may lose consciousness. Be- mittee10. cause of the drug, the victim may have little to no Other epidemiological data were retrieved from recollection of the events, and many assaults are PubMed, PsycINFO, and Scopus databases using not reported. the search terms “drug-facilitated sexual assault”, Not everyone is similarly affected by drugs and “chemical submission”, “date rape”, “rape drugs”, it is difficult to accurately predict the effects of and “drink-spiking” to identify relevant studies any drug on a particular individual. The effects to include in the review. Moreover, reports were may vary depending on the drug, the dose in- retrieved from the websites of international agen- gested, and whether the drug is mixed with al- cies or institutions, including the United Nations cohol or other drugs. Other influencing factors Office on Drugs and Crime (UNODC), the World are the victim’s weight, gender and metabolism, Health Organization (WHO), and the European and technical considerations, such as how rapidly Monitoring Centre for Drugs and Drug Addic- medical assistance is provided. Depending on the tion (EMCDDA). Articles and reports were inde- substance, the initial effects of a drug can be un- pendently evaluated by each investigator involved noticed or become quickly apparent: in the review. This study focused on victims of alleged DFSA aged 16 years and over, when toxi- • Nausea; cology results were reported. • Loss of bowel or bladder control; • Breathing difficulties; • Lightheadedness with little to no alcohol con- Results sumption; • Sudden increase in dizziness, disorientation or Most studies were published in the United blurred vision; States, followed by the United Kingdom and Eu- • Sudden body temperature change that can be rope with only one study in Australia and Africa. signaled by sweating or chattering teeth; Alcohol was the most commonly detected sub- • Waking up with little to no memory of recent stance and co-exposure to other drugs was com- events. mon. Cannabinoids and benzodiazepines were frequently detected. Many of these drugs are quickly eliminated from the body, from 12 to 72 hours8. Rohypnol® is eliminated within 36-72 hours and GHB within Discussion 10-12 hours. GBL leaves the urinary system within 6 hours and the bloodstream within 24 hours9. Epidemiology The aim of this review is to provide an updated It is difficult to estimate the number of DFSA outline of drugs involved in sexual assaults. The occurring every year considering the low report- authors also provide epidemiological data of the ing rates11, although assaults are increasingly phenomenon and discuss the analytical strategies reported. Victims are often reluctant to report and methods currently available to identify these incidents because of embarrassment, guilt or substances. perceived responsibility, or because they do not

10579 F.P. Busardò, M.R. Varì, A. di Trana, S. Malaca, J. Carlier, N.M. di Luca clearly remember the assault. Moreover, most of predominantly aged between 20 and 39 with the drugs used during sexual assaults are rapidly two peaks of 22%, although a 17% peak was absorbed by the body and metabolized, making observed for males under 15. Voluntary alcohol difficult the detection of these drugs in routine ingestion was reported in 46% subjects over 18 urine and blood drug screenings. or old enough to use another non-therapeutic Over the past ten years, the number of DF- substance (435 cases over the age of 12 years), SA reports increased in the United States12, the while this information was unavailable for 132 United Kingdom13, France14, the Netherlands15, cases (30%). Cannabis use was reported by and Australia16, where serious concerns were ex- 16% victims21. pressed about the incidence of alcohol and drug UK: Over a 3-year period from 2000-2002, the use in nonconsensual sexual activity. Forensic Science Service (FSS) studied 1014 cases of alleged DFSA22. Exposure to alcohol, Italy/scientific literature: Only few cases common drugs, and usual drugs of abuse was were reported in the literature17,18. Barbiturates, analytically documented. Most of blood and/or morphine, and GHB were involved in isolated urine samples (81%) contained alcohol. Nearly cases19. 50% cases were tested positive for alcohol and/ Italy/Careggi admissions: A total of 95 patients or other incapacitating drugs. Illicit drugs were (37.1% of n = 256) were tested positive for at detected in 35% cases, cannabis being the most least one substance. Alcohol was the most de- commonly detected substance (26%) followed tected substance (n = 57), followed by cannabis by cocaine (11%). In only 2% of cases (n = 521), (n = 19), cocaine (n = 15), and opiates/meth- a sedative or a disinhibiting drug was detected, adone (heroin: n = 5, morphine: n = 1, meth- although its presence could not be explained by adone: n = 6); benzodiazepines and amphet- the victim. These drugs were: benzodiazepines amine were found in 13 and 2 cases, respec- (n = 512), MDMA (n = 53), antihistamines (n = tively. Only one case of GHB exposure was 52), GHB (n = 52), zopiclone (n = 51), and mir- observed, while new psychoactive substances tazapine (n = 51). Similar results were reported were not detected. Sedative drugs were found by Beynon et al23 a few years later. Papers from in patients suspecting a DFSA attempt10. the UK after 2006 all cite Operation Matisse24, France: Biological samples from 35 DFSA vic- a 12-month study on DFSA from 2004-2005 tims (19 males and 16 females) were collected and including 120 victims of alleged DFSA. In over an 8-month period in 1995. Initial immu- this study, sexual assaults were reported within noassay screening detected benzodiazepines 72 hours. Controlled or prescription drugs were in 48.5% urine samples, followed by cannabis detected in 48% of cases, cannabis (20%), and (detection of tetrahydrocannabinol) (17.1%), al- cocaine (17%) being the most common sub- cohol (11.0%), phenothiazines (8.6%), opiates stances. GHB was detected in two cases, but (8.6%), tricyclic antidepressants (5.7%), and flunitrazepam was not identified. barbiturates (2.9%)20. Northern Ireland: Data from the Forensic Sci- France: Over a 9-year period from 2003 to the ence Northern Ireland (FSNI) database from end of 2011, 473 cases of probable chemical 1999 to 2005 were examined. Blood and/or submission or DFSA were identified. The study urine were analyzed, but the methods were not differenciated chemical submission, character- detailed. In 2005, alcohol was involved in 65% ized by a premeditated choice of substance by cases, and contained alcohol, drugs or both the offender, and forced ingestion, when the of- were involved in 78% cases. Drugs were iden- fender acted alone or as part of a group. Forced tified in 20 of 51 cases (39%). Analgesic drugs ingestion represented 8% of cases (29 women were the common substances (n = 511). Recre- and 11 men). Victims were predominantly fe- ational drugs (cannabis, MDMA) were found male (n = 295, 62%) with a 0.6 sex ratio, and in eight samples. Six samples were tested pos- were aged between 2 months and 90 years. itive for benzodiazepines. More than one drug Mean age was 29.8 years for the 452 victims was involved in almost half of the cases25. over age 1, when age was reported. Sex and age Netherlands: Data from the Netherlands Foren- distribution showed that 15 to 39-year-old fe- sic Institute (NFI) were published by Bosman males were more frequently victims of drug-fa- et al26 in 2011. Forensic cases were reported cilitated crimes (DFC), with a 36% peak for over a 3-year period (January 2004 to Decem- 20 to 29-year-old females. Male victims were ber 2006), including sexual assault cases with

10580 Drug-facilitated sexual assaults (DFSA): a serious underestimated issue

or without blood and/or urine collection. A to- 264 of 730 patients for toxicological analyses. tal of 134 cases of alleged DFSA were screened Fifty-seven (22%) of the 264 victims suspected for drugs of abuse, prescription drugs, and a proactive DFSA. Twenty-two of the 57 pa- GHB, and 108 were screened for alcohol. Most tients (38%) were positive for alcohol only; 13 victims were women (94%). Twenty-seven per- (22%) were positive for at least one drug oth- cent of the cases were negative. Alcohol alone er than alcohol. Five patients were positive for or together with other drugs was the most com- benzodiazepines (diazepam and/or oxazepam, mon finding (51 of 108 cases). A wide range clonazepam), one was positive for morphine of drugs was detected: cocaine (14%), benzo- and oxycodone, 2 were positive for cannabis diazepines (10%), MDMA, and 3,4-methylene and 4 were positive for amphetamines; sever- dioxyamphetamine (MDA) (10%), cannabis al patients tested positive for more than one (10%), amphetamine (4%), GHB (detected in drug29. only two cases), and ketamine (detected in one Poland: A 15-fold increase in DFSA cases was case). Sedative therapeutic drugs were also reported from 2000-2002 to 2003-2004. The found in single cases: amitriptyline, codeine, most common substances detected in blood methadone, and zolpidem. A combination of and/or urine were amphetamine and cannabis, drugs was often found. while alcohol, MDMA, benzodiazepines, pro- Germany: From 1995 to 1998, the Munich De- pranolol, and lidocaine were detected in few partment of Forensic Medicine (Bavaria) reg- cases only. No precise data were reported30. istered 92 DFC cases. From 1997 to 2006, the Belgium: No comprehensive data on DFSA were Bonn Department of Forensic Medicine (North currently published in Belgium19. Rhine-Westphalia) reported a 10-fold increase Spain: The National Institute of Toxicology and in the number of investigations on alleged DF- Forensic Sciences of Madrid conducted a de- SA, currently reaching 40 to 50 cases per year. scriptive and retrospective study on alleged Madea and Musshoff27, the authors reported DFSA cases reported from 2010-2013. A to- that benzodiazepines were the most common- tal of 152 of 445 sexual assault cases were ly used substances, followed by other hypnotic examined. Biological specimens collected for agents (zopiclone, GHB >10 mg/mL in urine), toxicological analysis included blood (28.9%), antihistamines (diphenhydramine), sedating urine (15.8%) or both (53.9%) and were most- antidepressants, and other illegal drugs (MD- ly collected between 6 and 12 h after the in- MA). cidents (40.33% of documented cases). Toxico- Denmark: A total of 167 blood samples from logical analyses were positive in 85.5% cases, sexual assault victims in the Aarhus area were with ethanol (76.9%), pharmaceuticals (36.1%, studied over a 2.5-year period. Twenty (12%) mainly benzodiazepines), and illicit drugs victims suspected an exposure to a “date rape (29.2%, mainly cocaine), either alone or in drug”. Seventeen of the 20 victims reported combination31. alcohol consumption before the assault, but United States: From 1996 to 2000, specimens only four (20%) were tested positive for alco- from 3303 victims of suspected DFSA were hol. Benzodiazepines were identified in 25% collected32. A total of 61% victims tested pos- cases, either alone or in combination with other itive for at least one substance and 39% sam- drugs. Meprobamate, phenobarbital, oxyco- ples were negative. A single drug was detect- done, methylphenidate, and amphetamine were ed in 64% positive specimens; two drugs were each detected once. Tetrahydrocannabinol detected in 22% specimens; and three or more and/or 11-nor-9-carboxy-tetrahydrocannabinol were detected in 14% specimens. Alcohol, ei- were detected in three cases. The authors noted ther alone (44%) or in combination with oth- that a high amount of alcohol had been ingested er substances (23%), was the most common in several cases in which no sedative drugs had substance, present in 67% positive samples. been detected. As observed in other studies, al- Cannabis was the second most prevalent drug cohol was the most prominent substance, and as cannabinoids were detected in 19% of all therefore is a high-risk factor of DFSA27,28. samples and 30% of positive samples. Benzo- Norway: In Norway, 730 female patients were ad- diazepines accounted for 15% of all positive mitted to the Sexual Assault Center in Trond- samples. Cocaine was detected in 14%, am- heim from July 2003 to December 2010. Blood phetamines in 11%, GHB in 5%, and opiates and/or urine specimens were obtained from (morphine, codeine and heroin, and their me-

10581 F.P. Busardò, M.R. Varì, A. di Trana, S. Malaca, J. Carlier, N.M. di Luca

tabolites) in 4% of positive cases. Propoxy- Massachusetts (n = 66), and Mississippi (n = phene and barbiturates were detected in 2% of 54). The top five compounds detected in urine positive cases. were cannabinoids, ethanol, amphetamine/ United States: Alleged DFSA cases were report- methamphetamine, cocaine/metabolites, and ed from four states (Texas, California, Minne- clonazepam, while the top five compounds sota, and Washington) between January 2002 detected in blood were ethanol, cannabinoids, and March 200433. Biological specimens from amphetamine/methamphetamine, clonazepam, 144 cases (16.8%) were tested. All victims were and alprazolam. Regarding polysubstance use, female and over 18; 60.4% were 25 or young- 276 cases were positive for one substance, er. A total of 45.8% victims self-reported alco- while 220 cases were positive for two substanc- hol use, but only 9.7% samples were positive es, 109 cases for three substances, 63 cases for for alcohol. Alcohol was followed by cannabis four substances, 37 cases for five substances (9.0% of self-report, confirmed by the detection and 79 cases for six or more substances35. of tetrahydrocannabinol and/or 11-nor-9-car- Canada: Demographic data were retrospectively boxy-delta-9-tetrahydrocannabinol in 32.6% studied over an extended period (1993-2002) in samples), cocaine (5.6% of self-report, con- British Columbia, although no data were pro- firmed in 18.1% samples), and amphetamines vided regarding alcohol and drug use in DFC/ (3.5% of self-report, confirmed in 6.9% of sam- DFSA. The authors showed that the incidence ples). All victims denied voluntary use of opi- of hospital reported DFSA has steadily in- ates and benzodiazepines, but these substances creased since 1999 and that young women in were detected in 6.9% and 3.5% cases, respec- their teens are particularly vulnerable to this tively. form of sexual assault. Du Mont et al36 also North America: Among 45 alleged DFSA cas- conducted a prospective cohort study in seven es in 2015, 58% tested positive for cannabis, hospital-based sexual assault treatment centers whilst 43% were positive for alcohol, 26% for in Ontario (2005-2007). In 178 cases of sus- cocaine, 13% for amphetamines, 11% for ben- pected DFSA, reported between June 2005 and zodiazepines and opiates, 5% for methamphet- March 2007, 96.2% victims were female, and amines, and 1% for methadone. “Other” or the mean age was 25.8 years36. A total of 85% undetermined drugs were identified in 33% of victims reported alcohol use before the alleged cases34. assault; 25.6% reported use of over-the-counter United States: One thousand cases from 38 medications; 29.4% reported use of prescription American states and territories, from March medications; and 25.5% reported illicit drug 2015 until June 2016, were examined. When use. Alcohol (30.9% of urine samples) was the gender was indicated (n = 613), most of the most commonly detected substance, followed victims (91.7%) were females, and the mean by cannabinoids (33.7%), cocaine (21.4%), an- age was 26.8 years. Blood and/or urine sam- tidepressants (16.4%; citalopram was the most ples were tested. A total of 21.6% cases were commonly detected [6.7%]), benzodiazepines negative for intoxicating substances (n = 216). (11.3%; lorazepam was the most commonly A hundred and one different substances were detected [6.2%]), amphetamines (7.3%), MD- detected. Ethanol was the most prevalent sub- MA (7.3%), codeine (4.5%), morphine (3.9%), stance, detected in 30.9% cases (n = 309), fol- antipsychotics (3.4%), methadone (1.1), GHB lowed by cannabinoids (tetrahydrocannabinol, (1.1%), and ketamine (1.1%). Flunitrazepam 11-nor-9-carboxy-delta-9-tetrahydrocannab- (Rohypnol®) was not detected. inol, and/or 11-hydroxy-tetrahydrocannabi- South Africa: A number of 107 victims of sus- nol) in 28.8% cases (n = 288), amphetamine/ pected DFSA from the Victoria Hospital Clin- methamphetamine (16.5%, n = 165), cocaine/ ical Forensic Unit in Cape Town were exam- metabolites (10.4%, n = 104), and clonazepam/ ined between October 2013 and 30 June 2016. metabolite (7.6%, n = 76). Mean, median and Biological specimens were screened for drugs range concentrations of ethanol in blood (n = of abuse. Most of the patients were female (n 309) were 98.6 mg/dL, 82.0 mg/dL, and 9.2-366 = 104, 97%), aged 18 to 25 (n = 54, 50%), and mg/dL, respectively. Ethanol and cannabinoids were admitted to the Clinical Forensic Unit were the most frequent combination. Califor- within 24 h of the assault (n = 78, 73%). Alto- nia was the state with the highest number of gether, 30 patients (28%) reported a history of cases (n = 260), followed by Maryland (n = 79), mental health issues, drugs and/or alcohol use

10582 Drug-facilitated sexual assaults (DFSA): a serious underestimated issue

or before sexual abuse. Samples included blood urine and blood specimens are not available, hair (n = 40, 37%), urine (n = 96, 90%) and/or hair should be tested within four weeks of the reported (n = 2), as well as breath for ethanol measure- assault. Urine is preferred to blood since it allows ments (58 prospective cases, 54%). Specimens for a longer window of detection of drugs and me- were positive for drugs and/or ethanol in 72 pa- tabolites and the sampling is easier and less inva- tients (67%), with drugs other than ethanol be- sive. Many drugs are quickly absorbed by the body ing detected in 60 patients (56%). Samples were and metabolized; the sooner the urine sample is positive for drugs and/or ethanol in 72 patients collected the higher the chance of detection41. A (67%), with multiple drugs detected in 35 cas- positive urine sample usually is a sufficient proof es and a single drug detected in 37 cases. The of exposure. On the contrary, a negative result of- most common substances detected were stim- ten requires further investigation. Nevertheless, ulants (methamphetamine, n = 30; cocaine, n = the period of detection depends on the pharma- 10) and sedative hypnotics (methaqualone, n = cological profile of the specific substances test- 15; doxylamine or diphenhydramine, n = 13). ed41. Positive blood samples can document drug Combinations of methaqualone, diphenhydr- exposure within a shorter period of time when amine, and methamphetamine were identified compared to urine. Blood concentrations give in- in 15 cases. Benzodiazepines were detected in formation on drug pharmacological effects and 4 patients, although benzodiazepine use was pharmacokinetics and may corroborate the vic- not self-reported. Among the 32 negative cases, tim’s symptoms. In case of whole blood analysis, 22 patients reported ethanol use (69%), and 3 the parent drugs are targeted, and the detection of reported use of recreational drugs (9%)37. metabolites is possible in only few cases. Australia: A total of 76 alleged DFSA cases Interestingly, several “date rape drugs”, such as among 434 cases of sexual assault (17.5%) were GHB are also endogenous substances produced identified over a one-year period (2002-2003)38. by the human body. In this concern, the analysis Most victims were females, and the average of multiple matrices can be performed to obtain age was 25.6 years. The median delay from al- complementary information to differentiate en- leged incident to time of examination was 20 h. dogenous production from exogenous adminis- Alcohol consumption was reported in 77% cas- tration41,42. Since hair can retain xenobiotics for es prior to the assault. Alcohol was detected in a large period, it is the matrix of choice in case 37% cases, with an average blood alcohol con- of late DFSA reporting. Segmental analysis of centration of 0.11% at the time of examination. hair provides chronological information about the Forty-nine percent reported using prescription suspected drug consumption, differentiating oc- medications and 26% reported using recre- casional, and chronic consumption. Hair analysis ational drugs. Non-self-reported drugs were allows to identify endogenous GHB concentra- detected in 15 cases. The drugs detected in- tions (under 12 ng/mg)42. Exogenous GHB can be cluded cannabis (n = 4), antidepressants39, am- identified in hair samples 7 days after exposure43. phetamines40, benzodiazepines39, and opiates40. Nevertheless, GHB can be detected as a metabolite of the prodrug GBL, which can be administered instead of GHB since it is cheaper and easier Analytical Strategies to Detect Drugs to purchase44. Involved in DFSA In more complex cases in which blood and urine tests are not enough to prove exposure, the Matrices investigation of alternative matrices can be con- When a DFSA is reported, the choice of an sidered. For instance, oral fluid (OF) and nails appropriate specimen is crucial for toxicologi- have been recently evaluated as potential bio- cal investigations. Depending on the time that logical specimens to document drug exposure in has passed since the sexual assault was reported, DFSA. OF consists of saliva and other debris and there are different approaches on how to choose food products in the oral cavity. Hydrophilic xe- the biological matrix to be analyzed. nobiotics can enter OF from the bloodstream by When different samples of high volume and passive diffusion and drugs can be detected as allowing longer windows of detection are avail- glucuronidated metabolites. Several factors influ- able, the matrix of choice is urine (up to five days). ence the elimination of drugs in OF, such as the Parent compounds and metabolites can be detect- pKa of molecules, the pH of oral cavity and sys- ed in blood for a shorter time (up to two days). If temic diseases. Although OF drug concentrations

10583 F.P. Busardò, M.R. Varì, A. di Trana, S. Malaca, J. Carlier, N.M. di Luca are not clearly correlated to blood concentrations mentary matrix to urine, preferably within 48 h and there is currently no proper biomarker to nor- of the alleged DFSA. Collection of blood sam- malize OF drug concentrations, OF testing can ples should be perfomed by trained specialized provide qualitative information on drug exposure. personnel using appropriate disposable syring- Nails is a keratinized matrix accumulating drugs es and sterile tubes. A gas syringe is recom- over time. Drugs and metabolites are incorporat- mended for the analysis of volatile xenobiotics, ed during the bidirectional growth of nails, but such as alcohol or chloroform. A preservative, they are also incorporated through occupational such as 2.5 g/L sodium fluoride or 2 g/L po- exposure. Like OF, the correlation between blood tassium oxalate, should be added to the test and nail concentrations is not clearly understood, tubes to prevent degradation. Skin disinfection but nail analysis can provide useful information with ethanol or other volatile solvents should in cases of late DFSA reporting. be avoided to not interfere with ethanol quanti- fication in blood. Like urine, at least two 5-mL Sampling samples should be collected and immediately The systematic collection of biological spec- stored at 2-8°C, or at -18°C if the analysis can- imens is the first phase of DFSA investigations. not be conducted within 24 h41. Incorrect sampling procedure can affect the re- Hair: Hair specimens should be collected at least sults of the analysis due to the loss of target com- 4 weeks after the reported assault. Pubic, ax- pounds. According to the UNODC guideline41, illary, torso or leg hair may be collected in- biological evidence should be collected as soon as stead of head hair when the subject presents possible, ideally before any medication is admin- alopecia, is shaved or applied massive cosmetic istered to the subject. Custody chain is highly rec- treatment that can invalidate analytical results. ommended. Timing of sampling is crucial, specif- International guidelines recommend collecting ically when faced to substances undergoing fast at least 100-150 mg hair, cutting the samples metabolism, such as GHB. In case of suspected as close as possible to the scalp due to the ki- GHB administration, whole blood samples should netics of incorporation in hair. It is important be collected within eight hours and urine samples to tightly attach hair strands to not lose their should be collected in less than twelve hours af- orientation for segmental analysis. Due to the ter exposure. Endogenous levels of GHB can be stability of the matrix, samples can be stored detected in urine (less than 1 mg/dL) and blood at room temperature in antistatic envelopes41. (less than 4 mg/mL) after twelve hours following Oral fluid and nails: Since OF and nails are intake43. Each matrix investigated in DFSA cases considered as alternative matrices, there are requires particular care on sampling and storage: currently no guidelines on sampling. Howev- er, several recommendations can be found in Urine: Urine should be collected as soon as pos- the literature. OF can be collected by passive sible after the suspected DFSA. Although in- drooling, expectoration, and salivary stimula- ternational guidelines suggest collecting urine tion or via a collection device. Different types specimens within 5 days after drug adminis- of devices are commercially available and are tration, the pharmacokinetic of the substances generally preferred to other sampling tech- should be considered for the interpretation of niques. Several sampling kits do not require analytical results. Since urine is the matrix of particular care for the storage of the samples45. choice for drug screening, a minimum volume Nails can be collected with disposable sterile of 50 mL should be collected and splitted into nailclippers to avoid sample contamination and two sterile plastic containers with screw cap. cut as close as possible to the nail bed. Due to Preservatives are not required, but they may the stability of the matrix, nail samples can be be useful in case of Candida albicans contam- stored in sealed bags at room temperature46. ination. The first sample should be used for screening and confirmatory tests, and the sec- Analytical Strategies ond sample should be stored at -18°C if further Many drugs used in DFSA may induce similar testing is needed. If the analysis cannot be con- clinical symptoms. It is not possible to conclude ducted within 24 h after collection, all samples that symptoms are due to drug exposure without should be stored at -18°C41. clear analytical evidence. Moreover, a negative Blood: According to international guidelines, toxicological result does not exclude drug expo- whole blood should be collected as a comple- sure, as the drug and its metabolites may have

10584 Drug-facilitated sexual assaults (DFSA): a serious underestimated issue been fully metabolized and eliminated at the time Conclusions of sample collection. In addition, due to drug adulteration, several drug may be administered si- DFSA is an underestimated issue. Many drugs multaneously47, acting with a synergistic effect48. are involved and many of them are easily obtain- The final result also depends on the screening and able. To combat this phenomenon, two different confirmation methods. As such, they should be strategies must be developed and implemented. sensitive and reliable enough to guarantee the vi- The first priority is to increase public awareness, ability of those results41. to help victims recognize the effects of “date rape Concerning urine and blood analysis, sever- drugs” and to urge them to reach emergency ser- al techniques are recommended. Volatile com- vices for a correct diagnosis and better care. The pounds testing can be performed using head- second priority is to inform toxicologists on the space-gas chromatography (HS-GC) with mass best analytical strategies and most informative bi- spectrometry (HS-GC-MS)48 or flame ioniza- ological matrices to document DFSA. tion (HS-GC-FID) detectors. As an alternative to HS, solid-phase microextraction (SPME) can be used41. For drug screening, metabolites, and non-volatile compounds, instruments should op- Conflict of Interest erate in full-scan acquisition in MS and spec- The Authors declare that they have no conflict of interests. trophotometry UV-Visible, and spectra should be further compared to references to confirm drug detection41. When available, urine samples References should be screened for a predefined selection of substances likely to be used in DFSA, such as 1) West Virginia Foundation for Rape Information & Ser- vices ethanol, drugs of abuse, and pharmaceuticals, . Drug facilitated sexual assault. Available at: 49 http://www.fris.org/SexualViolence/DrugFacilitat- including GHB, flunitrazepam, and ketamine . ed.html. (Accessed: 27th November 2019). Gas chromatography coupled to mass spectrom- 2) LeBeau MA, Moyazani A. Drug facilitated sexual etry (GC-MS) and liquid chromatography cou- assault: a forensic handbook. Academic Press, pled to tandem mass spectrometry (LC-MS/MS) 2001. or diode array detector (LC-DAD) are the most 3) Alcohol and Drug Foundation. Drug Facts. Avail- recommended technologies for DFSA, with op- able at: https://adf.org.au/drug-facts/. 2017. (Ac- timized analytical methods to specifically target cessed: 27th November 2019). “date rape drugs” and their metabolites to detect 4) European Monitoring Centre for Drugs and Drug Addiction (EMCDDA low concentrations50-52. In general screening pro- ). Drug profiles. Available at: http://www.emcdda.europa.eu/drug-profiles. 2014. cedures, GC-MS tests are performed after sample (Accessed: 27th November 2019). derivatization. Unfortunately, in routine practice, 5) Degenhardt L, Darke S, Dillon P. GHB use among most samples are delivered to testing laboratories Australians: characteristics, use patterns and as- a long time after collection, jeopardizing the de- sociated harm. Drug Alcohol Depend 2002; 67: tection of low drug concentrations. To improve 89-94. detection, it is recommended to use MS/MS de- 6) Moffat AC, Osselton MD, Widdop B, Watts J. tection, such as LC-MS/MS or GC-MS/MS, due Clarke’s analysis of drugs and poisons: In phar- to high sensitivity and selectivity41. For etha- maceuticals, body fluids and postmortem materi- al. Pharmaceutical Press, 2011. nol detection, gas chromatography coupled to a 7) TripSit. TripSit Factsheets. Available at: http:// flame ionization detector with (GC-HS-FID) or drugs.tripsit.me. (Accessed: 27th November withouth (GC-FID) head-space sampler is recom- 2019). mended. Metabolites biomarkers of ethanol, such 8) Kent State University. Alcohol, drugs, and sexual as ethylglucuronide (EtG) and ethyl sulfate (EtS) assault. Available at: https://www.kent.edu/srvss/ are often targeted as they are detectable in GC- alcohol-drugs-and-sexual-assault. (Accessed: MS or LC-MS/MS. 27th November 2019). In hair, confirmatory methodologies can be used 9) Rape, Abuse & Incest National Network (RAINN). for identifying GHB and cannabinoids, with GC- Drug-facilitated sexual assault. Available at: 41 https://www.rainn.org/articles/drug-facilitat- MS/MS or LC-MS/MS detection . Illicit and pre- ed-sexual-assault. (Accessed: 27th November scribed drugs can be detected by GC-MS, GC-MS/ 2019). 41,53 MS, and LC-MS/MS , and benzodiazepines and 10) Bertola E, Di Milia MG, Fioravanti A, Maria F, Palum- hypnotics can be detected by LC-MS/MS41,52-55. bo D, Pascali JP, Vaiano F. Proactive drugs in DF-

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