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Appropriate Use of Drug Testing in Clinical Addiction Medicine

Jarvis, Margaret MD, DFASAM; Williams, Jessica MPH; Hurford, Matthew MD; Lindsay, Dawn PhD; Lincoln, Piper MS; Giles, Leila BS; Luongo, Peter PhD; Safarian, Taleen BA

American Society of Addiction Medicine, Rockville, MD (MJ, TS); Institute for Research, Education and Training in Addiction (JW, DL, P Lincoln, LG, P Luongo); and Community Care Behavioral Health Organization, Pittsburgh, PA (MH).

Send correspondence and reprint requests to American Society of Addiction Medicine (ASAM), 11400 Rockville Pike Suite 200, Rockville, MD 20852. E-mail: [email protected]

Received 14 February, 2017

Accepted 16 April, 2017

The authors report no conflicts of interest.

Supplemental digital content is available for this article. Direct URL citation appears in the printed text and is provided in the HTML and PDF versions of this article on the journal's Web site ( www.journaladdictionmedicine.com ).

This article is the summary of a supplement to this issue titled The Appropriate Use of Drug Testing in Clinical Addiction Medicine .

Biological drug testing is a tool that provides information about an individual's recent substance use. Like any tool, its value depends on using it correctly; that is, on selecting the right test for the right person at the right time. This document is intended to clarify appropriate clinical use of drug testing in addiction medicine and aid providers in their decisions about drug testing for the identification, diagnosis, treatment, and recovery of patients with, or at risk for, addiction. The RAND Corporation (RAND)/University of California, Los Angeles (UCLA) Appropriateness Method (RAM) process for combining scientific evidence with the collective judgment of experts was used to identify appropriate clinical practices and highlight areas where research is needed. Although consensus panels and expert groups have offered guidance on the use of drug testing for patients with addiction, very few addressed considerations for patients across settings and in different levels of care. This document will focus primarily on patients in addiction treatment and recovery, where drug testing is used to assess patients for a substance use disorder, monitor the effectiveness of a treatment plan, and support recovery. Inasmuch as the scope includes the recognition of addiction, which often occurs in general healthcare settings, selected special populations at risk for addiction visiting these settings are briefly included.

The purpose of the American Society of Addiction Medicine (ASAM) document Appropriate Use of Drug Testing in Clinical Addiction Medicine is to provide guidance about the effective use of drug testing in the identification, diagnosis, treatment, and promotion of recovery for patients with, or at risk for, addiction. This document draws on existing empirical evidence and clinical judgment on drug testing with the goal of improving the quality of care that people with addiction receive.

Drug testing uses a biological sample to detect the presence of a specific drug (or drugs) as well as drug metabolites that remain in the body following use for a window of time. No universal standards exist today in clinical drug testing for addiction identification, treatment, medication monitoring, or recovery. Relatedly, there is very limited empirical evidence about whether the use of drug testing in addiction treatment settings leads to improved clinical outcomes.

DOCUMENT FOCUS

This document focuses on when, where and how often it is appropriate to perform drug testing in the identification, treatment and recovery of patients with, or at risk for, addiction. These recommendations are not meant to be clinical practice guidelines, which typically focus on either more generalized or disease-specific recommendations. ASAM recognizes that drug testing is used in other contexts (eg, criminal justice, workplace and pain management settings). ASAM's intent with this document, however, is to focus primarily on patients in addiction treatment and recovery, where drug testing is used to assess the patient for a substance use disorder (SUD), monitor the effectiveness of their treatment plan and support recovery, and to also focus on selected special populations at risk for addiction in general healthcare settings. Although ASAM acknowledges that these recommendations may be applied to other settings where drug testing is utilized, note that the materials reviewed and methodology used were restricted to the populations and settings described.

TARGET POPULATION

This appropriateness document is intended for addiction specialists and for all providers utilizing drug testing in the context of the identification, treatment and monitoring of patients with, or at risk for, addiction. This document will also be useful for physicians and other providers concerned about the possibility of addiction in their patient population.

RECOMMENDATION DEVELOPMENT PROCESS

The RAND Corporation (RAND)/University of California, Los Angeles (UCLA) Appropriateness Method (RAM) provides a specific process for combining the best available scientific evidence with the collective clinical judgment of field experts to arrive at recommended practices ( Fitch et al., 2001 ). The RAM is ideal for the identification of underuse or overuse of specific clinical procedures or tests, as well as in situations where rigorous clinical trials are lacking. The use of the RAM produced a set of appropriateness statements regarding the use of drug testing in the identification, diagnosis, treatment, and promotion of recovery for patients with, or at risk for, addiction.

ASAM's Quality Improvement Council (QIC) was the oversight committee for the development of the appropriateness document. The QIC appointed a 10-member expert panel to participate throughout the development process, rate treatment scenarios, and review the draft document. In selecting the panel members, the QIC made every effort to avoid actual, potential, or perceived conflicts of interest that may arise as a result of relationships with industry and other entities among members of the expert panel. All QIC members, expert panel members, and external reviewers of the document were required to disclose all current related relationships, which are presented in the Supplemental Digital Content, https://links.lww.com/JAM/A56 .

The expert panel was comprised experts and researchers from multiple disciplines, medical specialties, and subspecialties, including academic research, internal medicine, adolescent medicine, pain medicine, emergency medicine, medical toxicology, anesthesiology, psychiatry, and obstetrics/gynecology. Physicians with both allopathic and osteopathic training were represented. Furthermore, the panel members represented a range of practice settings including opioid treatment programs (OTPs), physician health programs, private practice, and academic medical centers. The expert panel was assisted by a technical team from the Institute for Research, Education and Training in Addictions (IRETA). The expert panel moderator and medical advisor was selected by the IRETA project team and approved by the QIC.

EVIDENCE REVIEW AND GRADING

Existing clinical guidelines offering guidance on the use of drug testing for patients with, or at risk for, addiction were located and reviewed. Overall, the review of existing guidelines revealed that numerous consensus panels and expert groups have offered guidance on the use drug testing for patients with addiction. However, with the notable exceptions of the Substance Abuse and Mental Health Services Administration's (SAMHSA) Treatment Improvement Protocols (TIP) 40 and 43 ( CSAT, 2007; CSAT, 2012 ), very few of these guidelines address drug testing in the context of patient levels of care. Publications by authoritative professional societies, including the American Society of Addiction Medicine (ASAM), the American Academy of Pediatrics (AAP), and the American College of Obstetrics and Gynecologists (ACOG) were also consulted. Although not typically evidence-based, a representative sample of payer policies was also consulted for information about the patient populations and types and frequency of drug testing that are currently reimbursed in clinical care. See the Supplemental Digital Content, https://links.lww.com/JAM/A56 for a complete list of clinical guidelines reviewed.

A review of empirical evidence regarding the use of drug testing in the identification, treatment, and monitoring of patients with, or at risk for, addiction was conducted. Relevant research was identified via a PubMed MeSH term search for Substance-related Disorders and Substance Abuse Detection articles published in the previous 10 years, capturing the most up-to-date findings for a field defined by rapidly advancing technological innovations. Important earlier articles were identified through reverse citation search. Given the relative paucity of research directly examining drug testing in SUD populations and settings, the review was not limited to randomized controlled trials or similarly rigorous methodologies; it included cohort studies and case studies. Of the 866 articles identified, 113 were retained following a title and abstract review for relevance to the topic of biological detection of addictive substances in an appropriate population or setting. See the Supplemental Digital Content, https://links.lww.com/JAM/A56 for a complete list of articles reviewed.

Overall, the literature review revealed that drug testing has rarely been examined for its value as a clinical intervention or as a differential source of information. Many research studies include drug testing as an outcome measure of treatment adherence or progress, but few examined whether and how drug testing itself works to improve outcomes for patients with, or at risk for, addiction.

RAND/UCLA Appropriateness Method

Statements pertaining to the appropriate use of drug testing in the identification, treatment, and monitoring of patients with, or at risk for, addiction were derived from the review of existing guideline publications, payer policies, and literature. There were some clinical areas identified by the project team and medical advisor relevant to addiction treatment settings where existing clinical recommendations or adequate empirical evidence were not found (eg, certain levels of care). In these situations, appropriateness statements were generated in conjunction with the medical advisor and the lack of the existing evidence was clearly documented.

Each appropriateness statement was rated by the project team on degree of clinical consensus from previous guidelines and quality of empirical evidence. A high clinical consensus rating was reserved for statements supported by multiple sources. A high empirical evidence rating was reserved for statements emerging from multiple studies using rigorous study methodology (eg, randomized controlled trials). The statements and supporting evidence ratings were organized into a table, which served as the foundation for rating by the expert panel. A background article discussing each appropriateness statement and relevant clinical or empirical evidence was also developed and provided to the expert panel members.

Each panel member rated the appropriateness of each statement on a 9-point scale where 1 = extremely inappropriate and 9 = extremely appropriate. Appropriateness refers to whether the expected benefit of following the guidance offered by a statement outweighs any anticipated risks, irrespective of cost. The experts were asked to use their own best clinical judgment (rather than perception of what other experts might say) of appropriateness for an average patient presenting to an average provider who performs drug testing in an average setting that provides care for patients with addiction.

Statements with median scores in the 1 to 3 range were classified as inappropriate, those in the 4 to 6 range as uncertain, and those in the 7 to 9 range as appropriate. Consensus was defined as a statement that received no more than 2 ratings outside of the median score range. This cutoff for disagreement is commonly used for panel sizes of 8 to 10 members.

Expert Panel Meeting

The 10-member expert panel came together for a 2-day meeting to discuss their ratings, focusing on statements that were rated uncertain or about which they disagreed. The goal of the discussion was to discern whether uncertain and divergent ratings were due to real clinical disagreement or “artefactual” disagreement, such as fatigue while completing the rating instrument or misunderstanding of the statements. The expert panel was encouraged to modify statements for clarity and suggest additional statements during the discussion.

After the meeting, each expert rated the appropriateness of the subset of previously uncertain and disagreed upon statements, as well as the new statements that were constructed, on a 9-point scale, where 1 = extremely inappropriate and 9 = extremely appropriate. A table of the statements, their final ratings and associated evidence ratings is included in the Supplemental Digital Content, https://links.lww.com/JAM/A56 .

COMMENTS AND MODIFICATION

The first draft of the appropriateness document was created and sent to the expert panel and ASAM staff. During a subsequent teleconference held in January 2017, ASAM shared feedback with the project team regarding the document's organization, and a revised version was provided. ASAM directed an external review of the appropriateness document, which consisted of input from ASAM members and stakeholders including experts from the addiction treatment community, professional societies, and the public. The external review period was conducted from February 3, 2017, to February 28, 2017. Further edits to the appropriateness document were made on the basis of this feedback.

CLINICAL RECOMMENDATIONS

The clinical recommendations generated by the RAM and external review process are listed below. Additional discussion and references are included in the Supplemental Digital Content, https://links.lww.com/JAM/A56 .

PART 1: PRINCIPLES OF DRUG TESTING IN ADDICTION TREATMENT

Clinical value of drug testing, principles of biological detection of substance use.

Providers should understand that drug tests are designed to measure whether a substance has been used within a particular window of time.

Drug Testing and Self-Reported Substance Use

Drug testing should be used in combination with a patient's self-reported information about substance use.

Drug testing is an important supplement to self-report because patients may be unaware of the composition of the substances(s) they have used.

Drug testing is particularly appropriate for patients facing negative consequences if substance use is detected, who are therefore less likely to provide accurate self-reported substance use information.

Discrepancy between self-report and drug tests results can be a point of engagement for the provider.

Drug Testing and Patient Outcomes

Because evidence suggests that drug testing assists with monitoring adherence and abstinence in treatment and can improve patient outcomes, drug testing should be used widely in addiction treatment settings.

Drug Testing and Evidence-Based Therapy

Contingency management is the most extensively researched behavioral therapy used in conjunction with drug testing. When utilizing contingency management therapy to encourage abstinence, providers should consider incorporating drug testing.

Clinical Use of Drug Testing

Therapeutic tool.

Drug testing is recommended as a therapeutic tool as part of evidence-based addiction treatment.

Providers should utilize drug testing to explore denial, motivation, and actual substance use behaviors with patients.

If drug-testing results contradict self-reports of use, therapeutic discussions should take place.

Providers should present drug testing to patients as a way of providing motivation and reinforcement for abstinence.

Providers should educate patients as to the therapeutic purpose of drug testing. To the extent possible, persuade patients that drug testing is therapeutic rather than punitive to avoid an “us versus them” mentality.

If a patient refuses a drug test, the refusal itself should be an area of focus in the patient's treatment plan.

Treatment providers should include drug testing at intake to assist in a patient's initial assessment and treatment planning.

Results of a medical and psychosocial assessment should guide the process of choosing the type of drug test and matrix to use for assessment purposes.

Drug test results should not be used as the sole determinant in assessment for SUD. They should always be combined with patient history, psychosocial assessment, and a physical examination.

Drug testing may be used to help determine optimal placement in a level of care.

Drug testing can serve as an objective means of verifying a patient's substance use history.

Drug testing can demonstrate a discrepancy between a patient's self-report of substance use and the substances detected in testing.

For a patient presenting with altered mental status, a negative drug test result may support differentiation between intoxication and presence of an underlying psychiatric and/or medical condition that should be addressed in treatment planning.

Drug testing can be helpful if a provider is required to document a patient's current substance use.

Drug testing should be used to monitor recent substance use in all addiction treatment settings.

Drug testing should be only 1 of several methods of detecting substance use or monitoring treatment; test results should be interpreted in the context of collateral and self-report and other indicators.

PART 2: PROCESS OF DRUG TESTING IN ADDICTION TREATMENT

Choosing a test, clinical necessity and value.

Before choosing the type of test and matrix, providers should determine the questions they are seeking to answer and familiarize themselves with the benefits and limitations of each test and matrix.

Test selections should be individualized based on specific patients and clinical scenarios.

Patients’ self-reported substance use can help guide test selection.

Identifying Substance(s) of Interest

Drug testing panels should be based on the patient's drug(s) of choice and prescribed medications, and drugs commonly used in the patient's geographic location and peer group.

Addiction treatment programs/providers should establish a routine immunoassay panel.

Providers should not rely on the National Institute on Drug Abuse 5 (also known as the SAMHSA 5) as a routine drug panel.

Test panels should be regularly updated based on changes in local and national substance use trends. Providers should collaborate with the testing laboratory when determining the preferred test selections to obtain information about local and demographic trends in substance use.

Matrix Advantages and Disadvantages

Providers should understand the advantages and disadvantages of each matrix before considering rotational strategies.

If a particular specimen cannot be collected (eg, due to baldness, dry mouth, shy bladder), providers should consider collecting an alternative specimen.

If a given sample is likely to be prone to confounds, providers should choose an alternative matrix. For example, heavily chemically treated hair is not appropriate for drug testing.

Presumptive and Definitive Tests

Presumptive testing should be a routine part of initial and ongoing patient assessment.

Presumptive testing should be used when it is a priority to have more immediate (although less accurate) results.

Providers should know the cutoff threshold concentrations that their laboratory uses when interpreting a report of “no drug present.”

Federal cutoff threshold concentrations used for occupational testing are not appropriate for clinical use because they are calibrated for workplace testing.

Definitive testing techniques should be used whenever a provider wants to detect specific substances not identified by presumptive methods, quantify levels of the substance present, and refine the accuracy of the results.

Definitive testing should be used when the results inform clinical decisions with major clinical or non-clinical implications for the patient (eg, treatment transition, changes in medication therapies, changes in legal status).

If a patient disputes the findings of a presumptive test, a definitive test should be done.

When ordering a definitive test, providers should advise the testing laboratory if the presence of any particular substance or group of substances is suspected or expected.

Because not all laboratories automatically perform a definitive test of positive presumptive results (the common term for this is “reflex” testing), providers should be aware that laboratories may require a specific order for definitive testing.

Providers should always consider cost both to patients and insurers when utilizing drug testing.

Responding to Test Results

Providers should attach a meaningful therapeutic response to test results, both positive and negative, and deliver it to patients as quickly as possible.

Providers should not take a confrontational approach to discussing positive test results with patients.

Providers should be aware that immediate abstinence may not be a realistic goal for patients early in treatment.

When making patient care decisions, providers should consider all relevant factors surrounding a case rather than make a decision based solely on the results of a drug test.

Considering all relevant factors is particularly important when using drug test results to help make irreversible patient care decisions.

Unclear Test Results

Providers should contact the testing laboratory if they have any questions about interpreting a test result or to request information about the laboratory procedures that were used.

Providers may consult with a medical toxicologist or a certified Medical Review Officer (MRO) for assistance in interpreting drug test results.

If the provider suspects the test results are inaccurate, he or she should consider repeating the test, changing the test method, changing/adding to the test panel, adding specimen validity testing, or using a different matrix.

If tampering is suspected, samples should not be discarded. Rather, further testing should be performed to help identify whether and how tampering occurred.

Providers should consider samples that have been tampered with to be presumptive positive.

Presumptive Test Results

Positive presumptive test results should be viewed as “presumptive positive” results until confirmed by an independent chemical technique such as Gas Chromatography-Mass Spectrometry (GC-MS) or Liquid Chromatography-Mass Spectrometry (LC-MS).

An appropriate response to positive presumptive test results includes speaking with the patient.

Providers should review all medications, herbal products, foods, and other potential causes of positive results with the patient.

An appropriate response to positive presumptive test results may include speaking with the laboratory for assistance in interpreting the test results.

Because presumptive tests may use cutoff values, a negative presumptive test result should not be over-interpreted. It does not rule out substance use or SUD, as the latter is a clinical diagnosis.

It is appropriate to consider ordering a definitive test if presumptive test results are negative, but the patient exhibits signs of relapse.

Definitive Test Results

In the event of a positive definitive test result, consider intensifying treatment or adding adjunctive treatments.

An appropriate response to positive definitive test results may include speaking with the laboratory for assistance in interpretation.

Providers should use caution when using drug test results to interpret a patient's amount or frequency of substance use. Individual metabolism and variability in absorption should be considered.

Providers should not over-interpret a negative definitive test result. It does not rule out substance use or SUD, as the latter is a clinical diagnosis.

Test Scheduling

Test frequency.

For people in addiction treatment, frequency of testing should be dictated by patient acuity and level of care.

Providers should look to tests’ detection capabilities and windows of detection to determine the frequency of testing.

Providers should understand that increasing the frequency of testing increases the likelihood of detection of substance use, but there is insufficient evidence that increasing the frequency of drug testing has an effect on substance use itself.

Drug testing should be scheduled more frequently at the beginning of treatment; test frequency should be decreased as recovery progresses.

During the initial phase of treatment, drug testing should be done at least weekly. When possible, testing should occur on a random schedule.

When a patient is stable in treatment, drug testing should be done at least monthly. Individual consideration may be given for less frequent testing if a patient is in stable recovery. When possible, testing should occur on a random schedule.

Random Testing

Random unannounced drug tests are preferred to scheduled drug tests.

A random-interval schedule is preferable to a fixed-interval schedule because it eliminates known non-testing periods (eg, if Monday is randomly selected from a week interval, the patient knows they will not be tested Tuesday-Saturday) and it is preferable to a truly random schedule because it limits the maximum number of days between tests.

PART 3: ADDITIONAL KEY ELEMENTS OF A TESTING PROGRAM

Documentation and confidentiality.

Addiction treatment programs should provide written drug testing procedures to patients. Procedures should be reviewed with the patient at the start of his or her treatment.

Providers should document the rationale for the drug tests they order and the clinical decisions that are based upon drug test results.

Providers should ask patients about and document potential sources of cross-reactivity, including various foods and current medications.

Particular characteristics of a sample with the potential to lead to problems with interpretation (eg, hair that has been chemically treated) should be documented at the time of collection.

Test results should be documented.

Test results should be kept confidential to the extent permitted by law. Providers should thoroughly explain to patients all rules regarding confidentiality, consent, and sharing test results with outside entities.

In general, providers should use caution when sharing test results with outside entities such as justice settings or employers. When sharing test results with outside entities, it is optimal that positive results be verified with a definitive test.

Practitioner Education and Expertise

Knowledge and proficiency.

Providers responsible for ordering tests should be familiar with the limitations of presumptive and definitive testing.

Providers responsible for ordering tests should be familiar with the potential for cross-reactivity in drug testing.

Providers responsible for ordering tests should consider the possible impact of tampering on test results. Providers should note that tampering is more likely in settings where consequences for substance use are severe, such as discharge from treatment.

Providers responsible for ordering tests should understand the potential benefits of alternative matrices to urine (eg, oral fluid, hair, etc).

Providers responsible for ordering tests should be aware of the costs of different test methods.

If the provider responsible for making clinical decisions based on test results does not have training in toxicology, he or she should collaborate with a medical toxicologist, a toxicologist from the testing laboratory, or an individual with MRO certification, as needed.

Language and Attitude

Providers should communicate with patients about drug testing using non-stigmatizing language. For example, results should be discussed as “positive” or “negative” as opposed to “clean” or “dirty.”

Providers should exhibit a consistent and positive attitude toward drug testing. Ambivalent attitudes toward drug testing among staff can be a barrier to its effective use.

Test Facilities and Devices

Point of care tests.

Staff training and demonstrated proficiency is particularly important for organizations that use point of care tests (POCTs).

Providers performing POCTs should be evaluated for their proficiency. POCTs should be performed only by providers who demonstrate adequate proficiency with the drug test in question. Facilities using POCTs should periodically evaluate the accuracy of their system in comparison to a qualified laboratory.

They need to understand the statistical and analytical sensitivity of the device.

They need to understand the spectrum of analytes (drugs and metabolites) detected by the device.

They need to understand any known interferences from drugs or metabolites that could affect interpretation of results.

They need to understand the nomenclature of the device.

Users of POCTs should refer to the POCT package insert and/or the manufacturer to determine the device's capabilities.

Cost issues should be considered when deciding to initiate a POCT protocol. These include costs associated with additional staff time and training, space to perform testing, quality assurance procedures, and documentation of POCT results.

Choosing a Laboratory

Providers should seek to work with a laboratory that has expertise in drug testing in addiction treatment settings.

When selecting a laboratory, providers should investigate whether state law requires a specific certification.

It is important to work with a laboratory qualified to perform accurate tests and assist in the interpretation of results.

Providers should work to create a collaborative relationship with the laboratory; important areas for collaboration are test panel selection, detecting sample tampering, interpreting test results, and identifying regional drug use trends.

When selecting a laboratory, providers should contact the toxicology director or a medical toxicologist at the laboratory to discuss panels, types of drug tests, testing procedures, and technical assistance.

Because drug testing should be individualized, laboratories should allow providers to order specific tests for each patient.

PART 4: BIOLOGICAL MATRICES

Use of urine drug testing in addiction treatment.

Urine should be considered the most well-established and well-supported biological matrix for presumptive detection of substance use in a clinical setting.

Urine should be considered the best established matrix for POCTs.

If tampering is of high concern or appropriate measures to reduce the likelihood of tampering cannot be taken, providers should consider using an alternative specimen type.

Urine Sample Integrity

Urine should be considered the matrix most prone to sample tampering through dilution, adulteration and substitution.

Providers should choose collection methods that protect patients’ dignity and privacy while minimizing opportunities for tampering.

Observed sample collection can deter urine sample tampering; if there are concerns about tampering, collection should be observed by a same-gender staff member.

Observed urine sample collection does not completely prevent sample tampering; providers should consider other strategies to mitigate urine sample tampering.

Providers should consider the use of an unobtrusive sample collection method for patients with a history of psychological trauma, especially sexual trauma.

• Ensure that potential adulterants, such as soap, ammonia, or bleach are not readily available in the collection area.
• Consider placing blue dye in the toilet and turn off the water source to the collection area during collection.

If a provider suspects that a patient has engaged in substance use but continues to produce negative urine test results, sample collection should be observed and specimen validity testing should be conducted.

If a sample is suspected of having been tampered with, it should be tested for specimen validity including creatinine concentration, pH level, specific gravity, and adulterants.

All samples undergoing definitive testing should be tested for creatinine concentration, pH level, and specific gravity (if creatinine is low).

Signs of Urine Sample Tampering

Temperature outside expected range of 90 to 100 degrees within 4 minutes of production (This can be checked using a heat sensitive strip).

Unusual color or smell, soapy appearance, cloudiness or particles floating in the liquid.

If a urine sample exhibits unusual specimen characteristics, the sample should undergo specimen validity testing to help identify whether and how tampering occurred.

Responding to Specimen Validity Test Results

For patients with past incidences of dilute urine samples, it is advisable to collect samples in the morning or request that patients decrease water intake prior to sample collection.

For patients with past incidences of dilute urine samples, use creative solutions, such as collecting before work, on days off, or use an alternative matrix.

Urine Testing for Specific Substances

Ethanol-containing products, including hand sanitizers and mouthwash, should be avoided before an ethyl glucuronide test.

Urine testing is helpful when assessing amphetamine use. Particular caution should be paid to the interpretation of amphetamine immunoassays due to known limitations in specificity.

Particular caution should be paid to the interpretation of benzodiazepine immunoassays due to known limitations in specificity.

Immunoassay results should be used cautiously when monitoring a patient's adherence to prescribed benzodiazepines. If a patient reports that he or she is taking the drug but a urine drug screen is negative, further analysis using definitive testing should be considered.

Particular caution should be paid to the interpretation of opiate immunoassays due to known limitations in specificity.

Patients should be instructed to avoid the consumption of food items that contain poppy seeds because they can result in a positive opiate test.

Urine testing is helpful when assessing cannabis use, although it is difficult to determine the timing or cessation of consumption in chronic users due to extended windows of detection for tetrahydrocannabinol.

The relevance of blood testing in addiction treatment is limited mostly to emergency situations where there is a need to assess intoxication or impairment.

No statements about the appropriateness of breath testing were endorsed by the expert panel.

Oral fluid testing is appropriate for presumptive detection of substance use in addiction treatment settings.

Oral fluid collection with a device that facilitates saliva collection is preferable to expectoration.

The creation of a sample for oral fluid testing should be observed.

It is recommended that patients abstain from eating for 15 to 60 minutes prior to oral fluid sample collection.

If a patient recently took a drug by mouth (ingestion or inhalation), it is recommended to wait at least 2 hours before collecting an oral fluid sample.

There is insufficient evidence to support the use of sweat testing in addiction treatment. More research is needed before sweat testing can be recommended over urine testing in clinical settings.

Hair testing in addiction treatment can detect long-term patterns of use. Routine use of hair testing is not appropriate for addiction treatment.

PART 5: SETTINGS

Outpatient services (level of care 1.0) and intensive outpatient/partial hospitalization services (2.0).

Because the opportunity for substance use is greater in outpatient treatment than in more intensive levels of care, drug testing has a particularly important role in monitoring substance use.

Providers should implement a random unannounced schedule of testing in outpatient services whenever possible, because the patient's opportunity for substance use is greater relative to residential treatment.

Drug testing should be scheduled on days following weekends, holidays, and paydays when feasible. Providers should communicate with patients about plans for additional drug tests around events/special occasions.

Additional drug testing should be considered if a patient is experiencing stressful psychological events.

Residential/Inpatient Services (3.0) and Medically Managed Intensive Inpatient Services (4.0)

Drug testing plays an important role in maintaining a drug-free therapeutic environment in residential treatment.

When residents leave the treatment program on passes, they should be asked to provide a sample for drug testing shortly after their return. Providers should communicate with patients about plans for additional drug testing following their return.

Opioid Treatment Services

The primary purposes of drug testing in the context of opioid treatment services (OTS) are (a) detecting substance use that could complicate treatment response and patient management, (b) monitoring adherence with the prescribed medication, and (c) monitoring possible diversion.

Drug testing can be an important tool for detecting the use of substances that can be lethal in combination with a prescribed opioid agonist medication (eg, benzodiazepines).

Drug testing has potential application across all stages of OTS including pre-induction assessment and treatment planning, active treatment, and during maintenance and recovery. Providers should utilize drug testing during the assessment phase and throughout treatment.

Providers should utilize drug testing as an aspect of contingency management in OTS.

Provider education should include knowledge of the metabolic pathways of commonly prescribed opioids.

Testing Schedule

Drug testing frequency is determined by stage of treatment as well as other patient factors and should be individualized.

Testing should be more frequent during the stabilization period and less frequent during the maintenance period.

Drug testing during and after tapering from methadone or buprenorphine continues to be an important way to support a patient's recovery; providers may want to consider increasing drug testing frequency during tapering and in the period after tapering.

Expected drug test results (ie, positive for prescribed medication and negative for unexpected substances) should be praised and responded to with tangible contingencies such as take-home doses of medication.

High concentration of a parent drug in the absence of its metabolites is consistent with sample tampering in the form of post-collection addition of the drug to the sample and potential diversion. In this case, a follow-up assessment should be conducted with the patient.

A test that is negative for the prescribed medication (eg, negative for buprenorphine in a patient prescribed buprenorphine) should not be used on its own to determine that diversion is occurring.

Unexpected drug test results could indicate the need for 1 or more of the following responses: (a) a higher level of care; (b) a higher dose of medication; (c) a different schedule of testing, such as random rather than scheduled and/or more frequent; and/or (d) increased education for the patient.

Considerations for Opioid Treatment Service Settings

For patients in OTP settings, the federally mandated 8 tests per year should be seen as a minimum, and it is often appropriate to perform testing more frequently than 8 times per year; determinations about testing frequency and duration should be made with consideration of individual patients, as noted above.

For patients in OTP settings, provider responses to unexpected test results can include discontinuation or reduction of take home doses of medication, more frequent or random schedule of drug testing, and increased counseling and peer group sessions.

Considerations for Office-Based Opioid Treatment Settings

For patients in office-based opioid treatment settings, the drug test panel should include the therapeutic drug and/or its metabolites.

In addition to drug testing, diversion can be reduced or prevented by frequent office visits, Prescription Monitoring Programs, observed dosing, and medication counts.

In order to provide buprenorphine or naltrexone treatment, providers must have access to drug testing laboratories.

Frequency of drug testing in buprenorphine treatment should be at least monthly, unless otherwise clinically indicated (eg, patients who have become stable in recovery may require less frequent testing).

Drug testing (and a negative test result for opioids) is indicated before starting treatment of opioid use disorder using naltrexone. Drug testing also is indicated throughout treatment using naltrexone.

Frequency of drug testing in treatment of opioid use disorder using naltrexone should be at least monthly, unless otherwise clinically indicated.

Recovery Residences

Weekly random drug testing is appropriate in a recovery residence.

Any patient expelled from a recovery residence should be able to continue an ongoing therapeutic relationship with his or her outpatient addiction treatment provider.

PART 6: SPECIAL POPULATIONS

Adolescents.

Use drug testing to assist in early identification of substance use in high-risk populations of adolescents including but not limited to those with known past substance use and those in treatment for mental health disorders.

Drug testing to monitor adolescents in addiction treatment or recovery from an SUD can be performed by providers in primary care.

When an adult observes symptoms characteristic of substance use in an adolescent, providers should use drug testing as part of an assessment for a possible addiction.

Adolescents and Self-Reported Substance Use

Even if an adolescent reports substance use, providers should consider drug testing for additional information because adolescents are less likely to self-report accurately.

Adolescents and Home Testing Kits

Because of a variety of limitations with home drug testing process and interpretation, providers should not encourage the use of home drug testing for adolescents.

Adolescent Consent

Before beginning the drug testing process with an adolescent, providers should explain drug testing protocols in full.

Drug testing an adolescent without his or her consent is not appropriate, except in emergency situations (eg, accidents, suicide attempts, and seizures).

Providers should acquire consent before drug testing an adolescent with symptoms such as school failure, fatigue, or excessive moodiness. Because these are not emergency situations, they are not hazardous enough to warrant skipping this step.

If an adolescent refuses to consent to a drug test, the provider should clearly document refusal and continue to evaluate the possibility of SUD through other methods and refer the patient to a specialist with additional mental health or substance use expertise.

Adolescent Confidentiality

Before beginning the drug testing process, providers should ask the adolescent for permission to share the results with parents/guardians and discuss confidentiality with parents/guardians in order to encourage parental involvement.

If an adolescent declines to share drug test results, the provider should not share them unless there is an acute risk of harm to the patient or others.

Test Choice

Drug test panels for adolescents should include the substances most used by the demographic.

Responding to Positive Test Results

If a positive definitive drug test result indicates that an adolescent is engaging in high-risk substance use, the provider should assist the patient and his or her parent or guardian in developing a plan for monitoring and treatment.

Pregnant Patients

Consequences and confidentiality.

Providers should be aware of the adverse legal and social consequences of detecting substance use among pregnant women. They should familiarize themselves with local and state reporting requirements before conducting a drug test and relay this information to each patient before conducting a drug test.

Screening, Assessment, and Monitoring

Comprehensive substance use assessment, which may include drug testing, is part of obstetrical best practices. Providers working with this population should learn about and appropriately use clinical laboratory tests.

For a pregnant patient with a history of addiction, providers should be aware that the postpartum period is a time of increased vulnerability. Therefore, assessment for relapse, which may include drug testing, should be part of the postpartum visit.

Providers should keep drug test results and associated diagnoses confidential to the extent permitted by law.

Patient-Provider Relationship

When speaking with patients, providers should emphasize the therapeutic reasons for drug testing to avoid stigmatization.

Test Considerations

In a prenatal care setting, routine Screening and Brief Intervention for alcohol use should be conducted, but laboratory testing is not recommended except in cases of suspected or known risk factors for Alcohol Use Disorder.

As pregnant women who use substances are less willing to disclose the use of opioids and benzodiazepines than other substances, testing for opioids and benzodiazepines helps identify an often underreported behavior.

Urine is an appropriate matrix for drug testing women who are pregnant.

Test Results

As a follow up to a presumptive positive test result, providers should use definitive tests to clearly identify individual drugs.

Responses to positive drug test results can include: patient education, referral to treatment, and the creation of a treatment plan.

Providers should be familiar with local treatment resources and programs for pregnant women.

People in Recovery

It is appropriate to conduct drug testing for a minimum of 5 years in healthcare settings for most patients in stable recovery. The frequency of drug testing for patients in stable recovery should depend on the severity and chronicity of the patient's addiction.

It is appropriate for patients in stable recovery to receive periodic Recovery Management Checkups that include a drug testing component.

Immediate evaluation for treatment or treatment intensification as a response to a positive drug test result is appropriate for most patients in stable recovery.

Health and Other Professionals

Drug testing is especially useful in supporting recovery of individuals who have increased access to psychoactive substances, including healthcare professionals and professionals in safety sensitive positions. Additional testing should be considered for those in recovery who have significant occupational exposure to addictive substances.

RESEARCH RECOMMENDATIONS

This document is intended to provide guidance about the effective use of drug testing in the identification, diagnosis, treatment, and promotion of recovery for patients with, or at risk for, addiction. There were areas with insufficient evidence to make a recommendation, and/or a recommendation was not rated with agreement by the expert panel members. These areas were translated into the research recommendations below.

• Further research is needed on whether and how drug testing can be used to determine efficacy of and adjustments to treatment plans.
• Additional research is needed on the relationship between drug testing and functional status and other addiction treatment outcomes. Further research should include mediators and moderators of the relationship.
• More research is needed on the utility of clinical drug testing in populations where SUD is often identified, including primary care, emergency room, and pain management patients.
• Significantly more research is needed on optimal testing frequency as well as the relationship between specific frequency and duration of drug testing and treatment monitoring and outcomes.
• Additional research is needed on how to utilize drug testing to detect novel and synthetic drugs (eg, cannabinoids, cathinones).
• Although evidence suggests that random testing schedules are more effective than testing on a predictable timeline, further study is needed to determine whether there are situations where non-random testing is sufficient.
• Further and ongoing research is needed on which drugs should be included in drug test panels.
• Further research is needed on determinations of when a definitive test as follow-up or in place of a presumptive test should occur.
• Additionally, more research is needed on the benefits of forgoing presumptive testing and beginning with definitive testing, and on discerning the roles of different kinds of definitive testing.

PART 3: ADDITIONAL CONSIDERATIONS FOR DRUG TESTING IN ADDICTION TREATMENT

• More research on effective personnel training to increase the reliability of drug testing conducted at the point of care is needed.
• The development of appropriate cutoffs for POCT needs more research. Though manufacturer recommended cutoffs are generally more appropriate for workplace rather than clinical drug testing, producing guidelines for a clinical setting requires more information.
• Further research is needed on the effects of conducting onsite testing and interpretation versus routinely sending tests to a laboratory for results.
• Further research on the impact of insurer regulations and restrictions on drug testing, addiction treatment, and overall healthcare costs would be useful.
• Further research is needed to develop a protocol for evaluating sample tampering in urine drug testing. Further research is also needed to clarify what methods should be employed to verify specimen validity in alternative matrices.
• Additional study is required to determine the detectability of cannabis use in multiple matrices, namely oral fluid and hair.
• Research is lacking on which substances’ metabolites can be helpfully detected through hair testing. More information on false positives, environmental adulterants, and detection windows would be beneficial.
• More research is needed on whether hair and nail testing is clinically useful in ascertaining substance use patterns and history.
• More research is needed on the utility of sweat testing in addiction treatment settings.
• Additional research is needed on oral fluid, including which specific drugs/metabolites oral fluid testing might best detect.
• Further research on tobacco testing in the context of addiction treatment would be useful.
• Further research is needed on the role of drug testing for identification of potential issues in primary care or other settings outside of addiction treatment such as mental health settings.
• Before making any specific recommendations of frequency or duration specific to level of care, further research should occur.
• Further research will be required to offer complete information regarding appropriate drug testing panels in OTS. The same applies to the role of drug testing in determining optimal dosing in the context of OTS.
• In the context of OTS, further research is needed on frequency of drug testing and on response to drug testing results.
• Further research is needed to determine whether testing frequency should vary between full agonists, partial agonists, and antagonists when treating addiction involving opioid use.
• Although it is agreed that instances exist where an adolescent ought to be drug tested regardless of their own desires, the exact circumstances would benefit from further refinement.
• Further research is needed to determine what, if any, clinical benefit there is to routinely utilizing drug testing with pregnant women.
• Additional research is needed on what methods might be utilized to test for identification of alcohol use during pregnancy.
• Further research is needed on how widely the drug testing standards developed for Primary Health Providers could be applied to other addiction treatment programs.

APPLICABILITY AND IMPLEMENTATION ISSUES

This document is intended to aid providers in their clinical decision-making and patient management. The document strives to identify and define clinical decision-making junctures that meet the needs of most patients in most circumstances. Clinical decision-making should involve consideration of the quality and availability of expertise and services in the community wherein care is provided. In circumstances in which the document is being used as the basis for regulatory or payer decisions, improvement in quality of care should be the goal. Because lack of patient understanding and adherence may adversely affect outcomes, providers should make every effort to promote the patient's understanding of, and adherence to, prescribed and recommended pharmacological and psychosocial treatments and any associated testing. Patients should be informed of the risks, benefits, and alternatives to a particular treatment or test, and should be an active party to shared decision-making whenever feasible. Recommendations in this document do not supersede any federal or state regulation.

CONCLUSIONS

Drug testing should be a routine part of initial and ongoing patient assessment of recent substance use in all addiction treatment settings. Drug test results should be not be used as the sole determinant when making patient care decisions; instead, they should be used in conjunction with patients’ substance use self-reports, treatment history, psychosocial assessment, and physical examination. Drug testing should be included at intake to assist in a patient's initial assessment and treatment planning and as a routine part of ongoing assessment for substance use that could complicate treatment response and patient management. Test selections should be individualized based a patient's drug of choice, prescribed medications, and drugs commonly used in the patient's geographic location and peer group. Treatment setting factors such as opportunity for substance use, the need to maintain a drug-free therapeutic environment, ensuring adherence with prescribed medications and monitoring for possible diversion also play a role in test selection. Frequency of testing should be dictated by patient acuity and level of care and tests’ detection capabilities and windows of detection.

APPROPRIATENESS DOCUMENT GROUP MEMBERS

Louis Baxter, MD, DFASAM

Lawrence Brown, MD, MPH, DFASAM

Matthew Hurford, MD, Expert Panel Moderator

William Jacobs, MD

Kurt Kleinschmidt, MD

Marla Kushner, DO, FASAM

Lewis Nelson, MD

Michael Sprintz, DO, FASAM

Mishka Terplan, MD, MPH, FASAM

Elizabeth Warner, MD

Timothy Wiegand, MD, FACMT, FAACT

John Femino, MD, DFASAM

Kenneth Freedman, MD, MSA, MBA, DFASAM

Barbara Herbert, MD, DFASAM

Margaret A. Jarvis, MD, DFASAM, Chair

Margaret Kotz, DO, DFASAM

David Pating, MD, FASAM

Sandrine Pirard, MD, PhD, MPH, FAPA, FASAM

Robert Roose, MD, MPH, FASAM

Brendan McEntee, ASAM Staff

Taleen Safarian, ASAM Staff

Penny S. Mills, MBA, e Executive Vice President

Peter Cohen, MD, Medical Advisor

Leila Giles, BS

Piper Lincoln, MS

Dawn Lindsay, PhD

Peter Luongo, PhD

Jessica Williams, MPH

addiction identification; addiction treatment; American Society of Addiction Medicine; drug testing; medication monitoring; opioid treatment services; substance use disorder

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• Open access
• Published: 31 July 2017

An overview of forensic drug testing methods and their suitability for harm reduction point-of-care services

• Lane Harper 1 ,
• Jeff Powell 2 &
• Em M. Pijl 1  

Harm Reduction Journal volume  14 , Article number:  52 ( 2017 ) Cite this article

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Given the current opioid crisis around the world, harm reduction agencies are seeking to help people who use drugs to do so more safely. Many harm reduction agencies are exploring techniques to test illicit drugs to identify and, where possible, quantify their constituents allowing their users to make informed decisions. While these technologies have been used for years in Europe (Nightlife Empowerment & Well-being Implementation Project, Drug Checking Service: Good Practice Standards; Trans European Drugs Information (TEDI) Workgroup, Factsheet on Drug Checking in Europe, 2011; European Monitoring Centre for Drugs and Drug Addiction, An Inventory of On-site Pill-Testing Interventions in the EU: Fact Files, 2001), they are only now starting to be utilized in this context in North America. The goal of this paper is to describe the most common methods for testing illicit substances and then, based on this broad, encompassing review, recommend the most appropriate methods for testing at point of care.

Based on our review, the best methods for point-of-care drug testing are handheld infrared spectroscopy, Raman spectroscopy, and ion mobility spectrometry; mass spectrometry is the current gold standard in forensic drug analysis. It would be prudent for agencies or clinics that can obtain the funding to contact the companies who produce these devices to discuss possible usage in a harm reduction setting. Lower tech options, such as spot/color tests and immunoassays, are limited in their use but affordable and easy to use.

Given the current opioid crisis in Canada [ 1 , 2 , 3 ] and around the world [ 4 ], harm reduction agencies are seeking to help people who use drugs to do so more safely. Harm reduction sites and/or clinics are increasing in number and service provision across the world, making it crucial to provide point-of-care workers with the tools and knowledge necessary to provide proper care for people who use drugs. Drug, pill, and substance testing are increasingly being used as a harm reduction strategy throughout the world [ 5 , 6 , 7 , 8 ] to decrease the risk of adverse effects. Indeed, various approaches to drug testing have been around, even in North America, for decades [ 9 , 10 , 11 ]. More recently, in Canada, drug testing is becoming more common at music festivals [ 12 ]. In Canada, the Standing Committee on Health [ 13 ] recommended that the Government of Canada grant exemptions under the Controlled Drugs and Substances Act so that drug testing could occur at designated sites. While there are certainly legal hurdles to overcome when it comes to drug testing [ 6 ], there are three primary advantages to testing drugs before they are consumed: short- and long-term adverse effects (including overdose and fatality) can be avoided by the person using the substance; other institutions (such as hospitals) and public health authorities can be made aware when a lethal or novel substance begins to circulate; and, a global picture of drugs in circulation can be generated [ 5 , 14 , 15 , 16 ]. The goal of this paper is to describe the most common methods of testing chemical substances in both laboratory and point-of-care settings. We will conclude with recommendations for point-of-care testing of illicit substances. In this paper, we use the term “drug testing” to refer to the forensic testing of illicit substances in their intended consumption form. Please note that the legal issues surrounding, and the service models of, drug testing are beyond the scope of this paper.

Introduction to substance testing methods

The following methods have been validated by the Scientific Working Group for the Analysis of Seized Drugs (SWGDRUG). Scientific Working Groups consist of scientific subject-matter experts who collaborate to determine best practices and develop consensus standards. As such, these methods have been proven to be effective in the analysis of unknown (forensic) examination of illicit substances and are therefore also the best methods to use in identifying unknown substances. Not all of these methods are easily accessible in a point-of-care framework, as some require high technical knowledge and/or a laboratory setting. Therefore, any of the following methods may be suitable on a case-by-case basis. This is due to the fact that some clinics may be able to easily access more discriminatory methods, through direct funding or industry partnership, whereas some clinics may have to rely on less precise testing methodologies and equipment due to lack of funding or support.

More discriminatory methods carry a much larger price tag to invest in the proper equipment. This may require community partnerships or a serious cost-benefit analysis or both. To keep the information precise and to attempt to interpret some of the associated technical details, the methods have been broken down into subheadings. Each method has three subheadings: “How does it work?” (a brief discussion of the theory behind the method), “What substances can be detected and how accurately?”, and “How easy is it to use?” The methods have also been broadly assigned into two larger categories: most discriminatory, or methods that will accurately identify a substance/mixture and that also have the potential to quantify the amount of substance, and least discriminatory, or methods that presumptively identify a substance and/or mixture without quantification. At the end of the paper, there will be a recommendation section that will focus strictly on the best methods/devices considering only point-of-care situations. The methods are summarized in Table 1 .

Most discriminatory

Mass spectrometry

How does it work?

Mass spectrometry (MS) is the most discriminatory of the drug testing techniques. Mass spectrometry measures the precise molecular mass of ions as determined by their mass to charge ratio ( m / z ) and is the current gold standard in forensic drug analysis [ 17 ]. In general, mass spectrometry requires separation, ionization, and finally detection. Separation can be accomplished through gas chromatography (GC), liquid chromatography (LC), or capillary electrophoresis (CE). There are various ionization methods. The most commonly used in analysis of illicit substances are electron ionization (EI), atmospheric pressure chemical ionization (APCI), electrospray ionization (ESI), matrix-assisted laser desorption ionization (MALDI), atmospheric pressure photoionization (APPI), fast atom bombardment (FAB), and more recently direct analysis in real time (DART). Ionization methods can be grouped into hard or soft techniques.

Hard techniques like EI, FAB, and APCI cause molecules to fragment generating complex mass spectra. Fragmentation is useful in analysis because molecules have known fragmentation patterns. A spectral database allows for a computer to quickly match spectra and determine the molecular species. Hard techniques are limited to detecting small molecules. Most illicit drugs are small molecules with the exception of drugs of a biological nature being consumed in their raw form.

Soft ionization techniques such as MALDI and ESI minimize fragmentation and allow for the molecules being analyzed to remain intact. Soft ionization techniques are useful for large biomolecules such as proteins.

DART is of particular interest as it allows non-destructive testing, is fast, and can quickly quantify when used with an internal standard. A pill can be held in front of the gas stream and within seconds determine the molecular species present. DART does not require separation of each molecular species prior to analysis allowing untrained personnel to collect data [ 18 ].

What substances can be detected and how accurately?

Virtually, any substance can be identified using MS in combination with a separation (chromatographic) technique. Sensitivity of current mass spectrometers allows for detection of analytes at concentration in the attomolar range (10 −18 ) [ 19 ]. MS has increased sensitivity over some other analytical techniques as the analyzer, a mass-charge filter, reduces background interference (i.e., a clearer reading/analyte fingerprint can be produced). It demonstrates excellent specificity due to characteristic fragmentation patterns, high resolution, and unique filtering abilities available especially in tandem or higher order mass spectrometry [ 20 ].

MS provides information about molecular mass and isotopic abundance of elements and temporally resolved chemical data, allowing for highly accurate identification. Newer devices are easier to utilize and much smaller than older versions. Interfacing with computers allows for refined database searches, making the drug identification process easier.

A major drawback of MS is that the tested sample taken from the supply is destroyed by the testing process (DART being an exception). Only a very small sample size (milligrams) is required. There are also continuing costs due to consumable materials required, and some of these consumables are poisonous/hazardous. Complex mixtures must be separated with a chromatographic technique (either gas or liquid chromatography) to correctly identify each constituent (unless using DART).

How easy is it to use?

The expertise required to utilize this technology is intermediate to expert (for definitions of terms in context with this paper please, see Table 1 ). Individuals should have some theoretical knowledge of how the technology and specific instrument work and specialized training from an expert. The cost of a mass spectrometer can vary from US$5000 to US$1,000,000. While an older used mass spectrometer may be less expensive upfront, it is not necessarily suitable for point-of-care drug testing. There are also considerable ongoing operational costs, such as chromatography (separation) reagents, gas consumables (nitrogen, helium, etc.), sample preparation items, and routine maintenance and service. Some labs offer MS services with costs between US$5 and US$100 per sample.

Ion mobility spectrometry

Ion mobility spectrometry (IMS) separates and identifies ions based on their speed through a carrier gas. Ion mobility is dependent on three molecular characteristics: the charge, reduced mass, and the collision cross section of the ion. IMS requires ionization before samples are passed into the instrument. This can be accomplished by ESI, MALDI, APPI, and coronal discharge or by using radioactive sources such as nickel-63.

There are many designs for ion mobility spectrometers including drift tube, ion trap, traveling wave, high-field asymmetric waveform, and differential mobility types. Drift tube IMS determines the ion mobility based on the amount of time it takes for ions to reach the detector. Many modern instruments use a drift tube for analysis.

Of interest is the field asymmetric subtype of the high-field asymmetric waveform IMS. A field asymmetric ion mobility spectrometer (FAIMS) uses a high (strong) electric field to control the movement of the ions through a physical filter. A pulsing electric field can then be applied to select for ions with specific ion mobility. Only ions with the specifically selected mobility will be able to maintain a stable trajectory through the filter. The others will crash into the side walls and not reach the detector.

Any small molecule of illicit substance can be detected very quickly and accurately. FAIMS sensitivity is based on multiple characteristics of both the ion of interest and the physical environment. IMS can detect one molecule in a billion (ppb) and is very selective. IMS selectivity can be further enhanced when using FAIMS. FAIMS is able to operate in environments with high levels of interference with minimal adjustment to operating conditions [ 21 ]. IMS is non-destructive and only requires a very small sample if a quantitative method calls for destructive testing. Determination is very quick and can be accomplished in a few seconds even for a complex sample.

IMS instruments do not require a trained operator. They can be used to quickly analyze a sample. Identification does require a database of known molecules to compare the sample against. The process of building a database would require a trained chemist using another technique or a standard. Once built, a database could be referenced from any instrument without additional technical help [ 22 ]. Quantification is possible when using internal standards or prebuilt methods. IMS is regularly used by law enforcement agencies at airports to detect narcotics and explosives. Minimal maintenance, ease of use by non-technical personnel, low cost, fast and accurate determination, minimal cost of consumables, and robust methodologies make IMS one the best choices for drug identification.

Infrared spectrometry

Infrared (IR) spectroscopy is another highly discriminatory method and is based on the measurement of the amount of IR radiation which is absorbed or emitted by a sample as a function of wavelength. A spectrum is obtained by passing infrared radiation through a sample and determining the amount of the incident radiation (radiation that actually hits the molecule rather than passing through) that is absorbed at each IR frequency [ 23 ]. Interpretation of the spectra allows for determination of molecular functional groups. The IR spectra of a pure molecular compound provides a distinctive fingerprint which can be easily differentiated from the IR absorption pattern of other compounds, including compounds with the same chemical formula, but a different arrangement of atoms in the molecule (known as isomers) [ 23 ]. An advantage of IR techniques is that virtually, all compounds have IR active vibrational modes and can therefore be investigated both qualitatively and quantitatively. However, quantitative analysis can pose a problem with unknown samples and mixtures. The spectroscopic expertise required to forensically analyze and quantify a substance may be difficult or impossible to find in harm reduction clinics. Most papers that describe relatively simple quantification methods are carried out in pharmaceutical research with controlled standards, methodologies, and standards. While quantification of unknown substances is technically possible, it really comes down to a case-by-case basis and is generally a laborious process undertaken by advanced to expert level technicians and chemists in forensic laboratories. It is highly unlikely that quantification would be viable using this technology in this kind of setting. Recent advances in IR technology have allowed for the development of portable IR devices.

When reference spectra are available, most compounds can be unambiguously identified based on their IR spectra. Drugs can be identified through a searchable database (such as http://webbook.nist.gov /). IR cannot distinguish enantiomers (similar to MS) [ 24 ]. According to the SWGDRUG [ 24 ], IR can produce structural information that will provide sufficient selectivity that generates the highest discriminating capability. IR can discriminate between diastereomers (such as pseudoephedrine and ephedrine) and free base/acid and salt forms. Free base/acid and salt forms refer to differences in physical properties that can alter the application of the substance. Free base is usually more volatile and normally has a lower boiling point, allowing the substance to be smoked. The salt form is usually more stable and tends to be crystalline and dissolvable in water, allowing for ingestion, insufflation (inhaling through the nose), or injection. A common example is crack cocaine (free base) and cocaine (salt); they are in fact the same drug (cocaine), and the actual effect on the body is the same, but due to different absorption and dosages based on method of use, it is possible to observe a spectrum of differing responses to each of the drugs. One of the notable benefits of IR spectroscopy is that it does not destroy the sample provided—an important consideration when working with drugs and the people who use them. As well, it requires only a very small sample size in the range of milligrams or less. Additionally, samples can be studied in virtually any physical state (primarily solid or liquid). Interference is very common and causes difficulty in identification.

The level of expertise required to use this technology varies depending on the device. There are portable IR devices on the market that have been optimized for basic to intermediate knowledge base, such as by outreach workers. These devices can analyze the obtained spectrum and search internal databases to display the identified substance or substances in a mixture (to a certain concentration, based on the specifications of a given device). This is considered presumptive or qualitative testing, in that it may only give an accurate breakdown of the constituents of a substance or mixture and sometimes offer a semi-quantitative analysis (i.e., rank-ordered most to least in a mixture). For quantification (as percent mass by total mixture weight), Sorak et al. have shown that some portable IR devices may be used for low error quantitative analysis [ 25 ]; although in order to interpret the obtained spectrum in the these devices in a quantitative manner, advanced to expert level knowledge is required as the devices do not perform this task for the user. Many other IR devices also require at least an intermediate level understanding of the procedures and some require advanced to expert knowledge to correctly analyze and quantify the substances (including operation of the equipment and database searching). Costs of IR devices can be anywhere from the low thousands to US$60,000 and above.

Raman spectroscopy

Raman spectroscopy is an optical technique based on the inelastic scattering of radiation after it interacts with matter. The interaction of incident radiation with the molecules of the substance gives spectral vibrational information [ 26 ]. The technique involves shining a laser on a sample and detecting the scattered light. A small amount of the scattered light is shifted in energy from the laser frequency due to electromagnetic and molecular interactions in the sample [ 26 ]. Plotting the intensity of the shifted light versus frequency gives a Raman spectrum of the sample. An exciting breakthrough in this technology is the development of handheld, portable Raman spectrometers. Many of these devices, most notably the TruNarc device by Thermo Fisher Scientific, have been optimized for drugs of abuse detection with simple “point and shoot” action. These devices also search databases in real time at a device level and give a clear readout of what substance(s) were detected.

Virtually, any drug can be identified with Raman spectroscopy. It can be used to determine active pharmaceutical ingredients (APIs) as well as molecules with the same chemical formula but different molecular arrangement and polymorphs. This is important as many of the novel psychoactive substances that have been emerging are isomers, derivatives, and analogues of many of the classical drugs of abuse. Being able to differentiate between small differences in physical or chemical structure aids greatly in unambiguous identification. Portable Raman spectroscopy has even been reported to be able to detect the date-rape drug rohypnol (flunitrazepam) in spiked beverages [ 27 ].

Raman spectroscopy may have difficulty in identifying substances that exhibit strong fluorescence. These substances tend to be plant-based narcotics such as heroin. However, with proper sample preparation, it is possible to analyze even these substances. The TruNarc Raman spectroscopy device has been shown to have a very high level of agreement with laboratory results (MS) for cocaine, heroin, and methamphetamine; inconclusive results are generally related to illicit substances that are present at extremely low percentages of the total mixture. Some studies have indicated that cocaine can be detected at concentrations as low as 5% when the cocaine was cut with sorbitol [ 28 ]. Others have detected amphetamine residues (milli- to micrograms) on paper currency using Raman spectroscopy [ 29 ]. It must be stressed that the particular technology discussed (TruNarc by Thermo Fisher Scientific) does not offer quantitative data in its “point and shoot” identification action, although it does offer highly accurate and extremely easy-to-use qualitative testing. The Raman technique as a whole is able to identify and quantify (depending on the device) a wide range of illicit drugs, even in the presence of contaminants and adulterants [ 26 ]. Given that there are many substances used to “cut” illicit drugs, this feature is an important one.

RS is rapid and non-destructive, does not require chemical reagents, can detect separate substances in mixtures, is not subject to interference from water or moisture, and importantly, can detect substances through transparent packaging (such as plastic bags and glass containers). Little or no sample preparation is required, although some sample preparation is required for substances that exhibit high fluorescence (including some cutting agents). RS is ideal for both organic and inorganic species and can be used for both qualitative and quantitative analysis. Due to the similarity to IR (detecting forms of molecular movement to identify), Raman has similar issues with quantitative analysis. While quantitative analysis can absolutely be done with Raman spectroscopy, it can be a much more difficult process that may not be possible in a harm reduction setting. Due to the difficulty of quickly and easily performing quantitative analysis on many unknown samples, an important consideration for outreach is that portable handheld devices specifically designed to detect drugs of abuse are available. Qualitative results can be obtained in a fraction of seconds to several minutes.

The cost of a RS unit can vary widely (in the low thousands of dollars to US$50,000 and above). Like all of the previous devices, care must absolutely be taken in selecting the appropriate tool. Advanced knowledge is required for devices that are not optimized for drug testing.

The level of expertise required to use this technology varies depending on the device, similar to IR. Some Raman spectrometers have been optimized for “point and shoot” action, giving a clear interpretation/reread of the substance(s) analyzed, and thus require merely basic to intermediate expertise for presumptive analysis. The requirements for quantitative analysis for portable “point and shoot” Raman spectrometers are similar to IR. Sorak et al. have also shown that some portable Raman spectrometers can offer quantitative analysis to a high degree of precision [ 25 ], although it must be stressed that this comes with the exact same considerations as the portable IR, as stated above. Other bench top or lab specific devices are most often not as simple and may require some database searching and interpretation of results. This can push the level of expertise required to intermediate, advanced, or expert, depending on the chosen device.

X-ray diffractometry

In X-ray diffractometry (X-ray D), the drug sample is bombarded with high-energy X-ray radiation and crystalline atoms in the substance cause incident X-ray beams to diffract in various directions [ 30 ]. This allows for the determination of the spatial structure of molecules by measurement of how X-ray radiation is scattered by the molecular crystal lattice structure. By measuring the angles and intensities of the diffracted X-rays, it is possible to produce a three-dimensional picture of the density of electrons in the crystal, and, from this, it is possible to determine the positions of the atoms in the crystal as well as their chemical bonds and other structural information [ 30 ].

Any crystalline or partially crystalline substance (i.e., substances that are solid and usually either evidently crystalline or powder or pill, such as methamphetamine, ketamine, and cocaine) including those in mixtures and compounds with currently unidentified structure can be identified [ 31 , 32 ]. This method is generally restricted to solid substances. X-ray D is used to identify precise chemical forms but not to quantify them. It can be used to identify diluents or adulterants [ 31 ]. This method is sensitive to both polymorphs and contaminants (common in illicit drugs). X-ray diffractometry determines structural information of the substance, so the substance can be identified with a very high degree of accuracy. This method is specific because substances have unique diffraction lines or an “X-ray fingerprint.” It is also sensitive in that drug concentrations and any additional agents used in cutting can be discerned through the obtained data. Studies have shown that this method can be used to identify a specific drug at only 5% of the total pharmaceutical formulation [ 33 ].

One benefit of X-ray D is that it requires no sample preparation and does not destroy the substance being tested. As well, only a very small sample size is needed (milligrams to micrograms) [ 31 ]. While it is the most reliable structural determination method and can determine the structure of currently unknown molecules, it is not suitable outside of a laboratory environment.

X-rays are highly radioactive and very damaging to organic cells/DNA. Thus, this method requires a high level of training and safety procedures and is restricted to laboratory environments. The skill level involved in operation is advanced to expert.

Least discriminatory

Microcrystalline tests.

These chemical tests result in the formation of unique microcrystals of a given analyte when a specific reagent is applied. The unique crystal formation is compared to a reference standard/control using a common light microscope. Microcrystals are compared based on shape, size, color, and spatial arrangement [ 34 ].

Several commonly abused substances can be identified, including cocaine, heroin, methadone, GHB (gamma hydroxybutyrate ) , ketamine, phencyclidine, amphetamines, and methamphetamine [ 34 ]. With test reagents chosen to induce development of specific microcrystals with the analyte and a reference/control standard available, these tests can be highly specific as the crystals formed are a direct consequence of choice of reagent and analyte and are unique under these circumstances. This is provided that other substances do not react in a similar way, if at all, with the reagent, and provided that impurities, dilutents, and adulterants do not prevent or mask the formation of characteristic microcrystals for the drug tested. In these cases, a microcrystalline test can be considered highly characteristic but non-specific enough for a confirmatory test. Thus, this method is best suited to pure and/or separated samples. Sensitivity is high as samples require only micrograms of substance.

The benefit of microcrystalline tests is their relatively low cost. Minute amounts of reagents are required. Instrumentation is simple; however, this method does not quantify how much of a substance is present. Unfortunately, the sample that is tested is destroyed in the process, which may be less than ideal for people who are bringing the samples for identification.

The expertise required is intermediate to advanced and requires adept interpretation of results.

Thin-layer chromatography

Thin-layer chromatography (TLC) is a technique in which a sample is placed onto a planar stationary phase then a liquid mobile phase resulting in capillary action. The analyte is either adsorbed to the stationary phase or is in the mobile phase, and the time spent on the stationary phase or time spent in the mobile phase determines its retention time. Components of the sample travel at differing rates depending on the component’s size and affinity for the mobile phase [ 35 ]. The result is a plate of spots (separated components of the mixture) that have moved various distances on the stationary phase.

TLC can detect barbiturates, benzodiazepines, GHB, heroin, morphine, opium, oxycodone, and other opiates, amphetamines, cocaine, methamphetamine, MDMA (methylenedioxymethamphetamine or Ecstasy), ketamine, LSD, marijuana, mescaline, synthetic cannabinoids, and cathinones (commonly referred to as “bath salts”). Using TLC, it may be difficult to separate and identify novel psychoactive substances [ 36 ]. TLC performs fairly poorly at separating complex mixtures. Sensitivity is in the micro-nanogram range. Specificity can range from intermediate to high depending on the mixture, and measured retention factors can be used to make a preliminary identification of a substance but are not specific to a single compound [ 35 ]. In order to increase specificity in cases of similar retention factors, it must be used in conjunction with another technique such as Raman spectroscopy or colorimetric testing or in the case of UV active species, UV.

TLC is a relatively low-cost way to test substances and demonstrates good sensitivity and speed of separation. It can be used as a presumptive test with a fairly high degree of accuracy depending on sample purity. While TLC can identify some known substances in provided samples, it does not indicate (quantify) how much of a substance is present in the sample. TLC is best used in conjunction with a more discriminating technique such as Raman spectroscopy, MS, or IR.

TLC is relatively simple to use and interpret and is thus suitable for basic to advanced skill level. This means that someone with basic skill may be able to perform a test following instructions but have trouble interpreting the results, whereas someone with intermediate to advanced skill level would have greater ability to interpret a test and could supervise basic skill level users.

Spot/color tests

Spot/color tests offer presumptive testing based on chemical reactions between analytes and indicators. There are many possible indicator tests such as cobalt thiocyanate, Dille-Koppanyi, Duquenois-Levine, Mandelin, Marquis, nitric acid, para-dimethylaminobenzaldehyde, ferric chloride, Froehde, Mecke, Zwikker, and Simon’s (nitroprusside) [ 37 ]. The indicator chemically reacts with the analyte and causes a reaction that creates a certain color staining depending on the analyte tested. Spots are then compared visually with reference charts, the current standard being the Munsell color charts. There is a method that bypasses the human eye and its subjectivity by using a simple smartphone app to identify colors with high precision and accompanying software that matches the results in a searchable database [ 38 ]. This allows for a more precise quantitation of the color and therefore higher accuracy identification.

What substances can be detected, and how accurately?

Colorimetric tests exist for most drugs of abuse, including cocaine, various pharmaceutical opioids, amphetamines, LSD (lysergic acid diethylamide), cathinones (bath salts), heroin, and fentanyl. There may be other novel psychoactive substances that do not (yet) have any associated colorimetric tests. Each specific named test will have information on what analytes it can be used with. Unfortunately, the test also destroys the sample provided. That said, color tests do not require much sample: if it can be seen, it can be tested.

Colorimetric tests can be quite sensitive, with limits of detection in the microgram range depending on the spot test utilized and the analyte [ 37 ]. Multiple tests with multiple reagents can be used if a mixture of drugs is suspected, though each test requires in the low milligram range of substance and destroys the substance in testing. With the proper standards, these tests can be quite specific, although multiple analyses may be required for high specificity. Some knowledge about what the substance is supposed to be and about general appearance of certain substances can increase specificity. Colorimetric tests are considered presumptive, in that they can only identify presence or non-presence of a particular substance based on the test administered. A single test/reagent will only test for the presence or absence of a drug or class of drugs. A typical test is not sufficient for a suspected mixture or even an unsuspected mixture if there is any reason at all to have suspicion of the substance. An example battery test protocol for considerations of how to test a suspected mixture is included below.

Actual color results may vary depending on the concentration, whether the drug is in salt or free base form, additional diluents, or contaminants; positive result may indicate a specific drug or class of drugs present, but not always specific for a single drug or class. Colorimetric tests rely on simple chemical reactions and produce visible results that can be interpreted with the naked eye.

Reagents and laboratory materials needed are inexpensive and readily available and can be performed with minimal training. Because each individual perceives color uniquely and because lighting conditions are not always optimal in non-lab settings, accuracy can be greatly enhanced with the use of smartphone apps to report color test results quantitatively [ 38 ]. Overall skill level required is basic to intermediate. A basic user can run the simple test and obtain results, whereas an intermediate user would run a standard protocol. An example of an intermediate protocol would be to run a battery of tests based on how much sample can be obtained without objection from the user. The tests should be based on an educated guess system, narrowing down possibilities through analysis and questions. Potential questions would be as follows: What did the user think it was or was told it was? What are recent novel substances that have been appearing in the clinic or on the street lately? What is the most dangerous substances worth testing for (smallest window of dosage)? Is there any knowledge of common mixtures, such as opioid mixtures?

The tests should be interpreted within a maximal 10-min window. The tests can be analyzed via smartphone or at least under good lighting if using the naked eye in order to most accurately determine color. The tests can then be matched against a database if a computer or the internet is available. From a system such as this, a presumptive test can then become a much more powerful tool.

Immunoassay

Immunoassay involves the binding of an antibody that is selective for the drug or drug group of interest (antigen) and a label that will be part of the antibody-antigen complex that can be detected using some means (such as fluorescence). Antigen-antibody binding is based on a typical immune system response in which antibodies in biological tissue bind to antigens in order to neutralize or remove them. This technique is rarely used in drug analysis because these methods were originally designed for analysis in biological materials (primarily metabolites in urine). Thus, traditionally, immunoassay provides important patient information for clinicians but does not provide a determination of the type or amount of a drug prior to its ingestion/injection. ELISA can, however, be used to perform other types of biochemical assays in the detection of an analyte in a liquid sample. Very little scholarly information is easily accessible about which specific drugs ELISA can detect outside of biological samples (post ingestion/metabolization).

Various opioids and cocaine can be detected rapidly and somewhat effectively using immunoassay technology. There are problems with specificity regarding immunoassays, and there have been many instances of false positives due to similarity in drug structures or metabolites. Sensitivity is quite high with detection in the microgram range as antibody-antigen interactions occur on a molecular level [ 39 ].

Immunoassay is fast and relatively inexpensive and in most instances, does not require high-level scientific knowledge to perform and interpret. Running such tests can require intermediate skill level. However, there is very little information available that has been scientifically published or available for public access on the usage of immunoassays for whole drug analysis. Immunoassay is most often employed to detect drug usage after the fact, such as in urine drug screens.

Urine dipstick test

This method has recently come under attention as a relatively cheap, easy-to-use presumptive test for fentanyl [ 40 ]. A sample of the drug sample is dissolved in water, and if the drug contains fentanyl in a concentration above the cut-off levels, an indicator on the strip will appear. The methodology works via chromatographic immunoassay, and in the presence of an appropriate analyte, a strip on the indicator stick appears/changes color.

To date, fentanyl is the only drug for which this method of drug checking has been reported being used [ 25 ], and there is little published data about this methodology. There is no scientific data on sensitivity, although the strips have been developed to detect fentanyl in urine and are therefore specific to testing for fentanyl and/or fentanyl metabolites.

The provided sample is destroyed in the testing process. Urine dipsticks are very easy to use, quick to check, specific for fentanyl, proven in urine test situations, and recently been proven efficacious in testing unknown drug mixtures for the presence of fentanyl. However, dipsticks were designed for drug detection in urine, and therefore, due to low specific weight in other mediums, it may be possible that false positives occur.

Another potential concern with this method is that many retailers will only sell to health professionals, and thus, these items may be difficult to procure for harm reduction agencies unless they are affiliated with a health clinic. Some medical device companies may object to such a test being used in a harm reduction setting, even in the presence of qualified health professionals for liability reasons.

Ultraviolet spectroscopy

This method is based on the absorption of light energy in the ultraviolet (UV) wavelength range. Light in this range can raise the energy levels of the electrons within a molecule from ground state to higher energy levels. Each transition to a higher energy level requires a given amount of energy, provided by light of a particular wavelength. Using a particular wavelength of light, a characteristic UV absorption spectrum can be obtained based on the electronic structure of the whole molecule as this structure will determine what wavelength(s) are absorbed versus which pass through a sample. UV-vis (ultraviolet visible) spectrophotometers measure the intensity of light passing through a sample and compare it to the intensity of light before it passes through the sample and capture this information to create a characteristic spectrum.

Drugs with similar structures may provide the same UV spectra. UV-vis has been used to identify MDMA, ketamine hydrochloride, cocaine hydrochloride, diazepam, phenobarbital, and barbital concentrations in the microgram range, as well as specifically identify six different compounds and for the first time, accurately discriminate some mixtures [ 41 ]. Other substances may be identifiable although literature is sparse on confirmatory usage for a broad spectrum of illegal drugs. UV spectrometry can be used on solid samples and therefore can be non-destructive in nature, although some samples may need preparation that can make them unsuitable for use afterwards. UV can be used quantitatively (amounts) and qualitatively (identification) and yields rough structural information providing modest selectivity to allow for some discriminating capability [ 24 ].

UV can be combined with chromatographic techniques for greater selectivity and specificity. It is not suitable for detection of several drugs in a mixture. Samples must be diluted or the technique can yield saturated spectra. Compounds lacking suitable chromophore provide no signal (for example, GHB has a low wavelength chromophore which makes analysis by UV-vis much more difficult without further sample preparation), although most drugs of abuse have a suitable chromophore due to aromatic ring structures in their chemical structures. Additionally, UV spectrum can vary depending upon the pH of the sample solution, and it is possible for chemical composition to change during the analysis. The level of expertise involved in UV is basic to advanced. The technique may be easily taught to someone with little to know theoretical knowledge of the technique, although interpretation of results would require intermediate to advanced knowledge.

There are many variables to consider when selecting technology for drug checking on the front lines of harm reduction. Harm reduction agencies, if pursuing the addition of drug testing services, will need to consider not only the quantitative capabilities of the tests but whether the agency can afford the human and fiscal resources to support the use of the technology. Thus, the recommendations include a strong bias to cost-benefit and beg the important question of whether some of the less discriminatory interventions are better than no intervention at all. With these considerations in mind, the following recommendations will summarize the methods for drug testing at a point-of-care level.

The techniques that are the strongest candidates based on all considerations are IMS, IR, Raman spectroscopy, and spot/color tests, although these too have some associated drawbacks. Spot/color tests are purely presumptive. In most cases, quantitation is contingent on expert interpretation. In some cases, the therapeutic index is so small and such miniscule quantities can be used as an additive to mixtures that only the highest discriminatory techniques mentioned above are capable of proving unequivocally that the quantity present would fall in therapeutic index (i.e., would produce a high but not be fatal, barring extraneous circumstances).

In our review, the best methods for point-of-care drug testing are handheld IR or Raman spectroscopy. From a cost-to-benefit analysis, these methods (specifically the portable/handheld units) are superior in almost every way to every other method. Manufacturers have simply made these technologies extremely easy to use and effective at identification of unknown analytes. The major downsides of this technology are that quantitation may require advanced expertise and that these units are still fairly expensive. To use these units qualitatively usually requires very little technical expertise or training. Intended for use in the field, these units are small and portable and tend to be fairly rugged, while also being able to have near-lab identification ability [ 25 ]. While many of these devices are only currently in use in law/drug enforcement settings, use in harm reduction settings would be worth exploring.

IMS spectrometers are very robust and require minimal maintenance. They are routinely used in airports worldwide for narcotics detection. Training is easy and quick, and sensitivity and selectivity are very high. Consumables are cheap and have long lives. Sampling is non-destructive and quantification is possible without expert level understanding. Analysis is quick and accurate. IMS is the best option available for clinics with a moderate level of funding. Some gas analyzers allow online updating; rapid sample analysis of liquid, solid, and gas; and discrimination of multiple interfering species in a complex matrix. The capability to update online allows methodologies and new molecular species to be shared instantly among clinics enabling point-of-care testing to remain current.

Other methods worth considering for point-of-care drug testing are MS, TLC, and UV spectroscopy. MS is considered the current gold standard in forensic drug analysis. Since MS units have been in use for a long time, it is actually possible to obtain one for a decent price (low-to-mid thousands) in the used market. However, in order to obtain a newer device optimized for drug testing or for testing extremely low concentrations, it would come with a higher price tag, usually in the hundreds of thousands of dollars. This presents a difficulty of its own because of the wide range of machines available, it would take some considerable research at clinic level to determine the cost-benefit analysis of a new or used machine to ensure acquisition of a machine that is suitable for its intended purpose. Additionally, operation and maintenance of MS machines is still complex, so a clinic would have to assess training, operation, maintenance, and associated ongoing costs which may place such a device beyond the time and/or monetary costs to the clinic compared to the benefits provided.

UV spectroscopy and TLC are more affordable options, but also much less discriminatory. Both of these methods tend to be less technical in operation, maintenance, and interpretation of results, but also do not offer quantification at the same level of the more discriminatory methods. They are also less expensive than all of the more discriminatory techniques. However, when used in conjunction, TLC and UV can be quite powerful in identification of a wide variety of substances (including mixtures) and offer a more rudimentary quantification than the more discriminatory techniques.

A lower technology option is the spot/color tests, which are purely presumptive in nature, although they can be fairly specific at identification of a compound and/or mixture when utilizing a standardized procedure utilizing a battery of tests (as described above). Information about optimal technique can be easily accessed via the internet. Color tests are cost effective, fast to complete, and very easy to perform. The use of a smartphone app can aid in identifying the exact color profile. This can then be used in conjunction with a searchable database to perform the most accurate identifications. The fact that this technology is so cost-effective, easy to perform, and requires a very minute amount of substance makes it really stand out from many of the other presumptive methods [ 16 ]. This type of test is widely used in Europe [ 16 ]. These tests are not perfect and can be performed incorrectly. A proper standardization of technique should be implemented at the clinic level to maximize the accuracy of these tests.

Drug testing methods that are less suited to point-of-care drug testing situations include immunoassay, microcrystalline testing, and X-ray diffractometry. Immunoassays are traditionally designed for usage in biological samples as they work based on antibody-antigen interactions and as such are best suited for testing excreted metabolites (such as in urine). At best, an immunoassay can indicate the presence of drug(s), and at worst, they can give a high proportion of false positives. This may result in people using the substances anyways or serve to give the clinic a poor reputation, and users may soon stop going to the site for drug testing. That said, they are affordable and portable and can detect potentially fatal drugs like fentanyl.

Microcrystalline testing is a highly limited method as the drug needs to be mostly (or completely) pure. This testing has no quantification capabilities at all and requires high skills and knowledge to identify drugs based purely on crystal structure. X-ray diffractometry is a highly discriminating testing method; however, this method basically requires partnership with a specialized lab/institution. X-ray diffractometers are incredibly expensive (mid-to-high tens of thousands), difficult to maintain and operate, and have the added factor of using radioactivity which may present health and safety concerns.

There is a wide variety of techniques that have been validated for drug identification and/or quantification. Each of these techniques has a variety of associated pros and cons that must be considered. With this in mind, this review is not meant to be an in-depth rigorous scientific treatment of each of these methods, but a guide for the practical consideration of usage and recommendations for point-of-care harm reduction purposes. It is sincerely expected that this document will help to narrow down consideration of each of these techniques and that each clinic would then determine a smaller subset of techniques to consider implementing. It would be prudent for clinics that can obtain the funding to contact the companies who produce and design these devices and discuss possible usage in a harm reduction setting as many of the devices are only currently in use in law enforcement and research.

Abbreviations

Active pharmaceutical ingredients

Field asymmetric ion mobility spectrometer

Gas chromatography

Gamma hydroxybutyrate

Liquid chromatography

Lysergic acid diethylamide

Methylenedioxymethamphetamine or Ecstasy

Scientific Working Group for the Analysis of Seized Drugs

Ultraviolet

Ultraviolet visible

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LH analyzed the extant literature, creating the basis for the paper. JP offered technical analysis and editorial support. EP worked with LH to make the text suitable to a non-technical audience. All authors read and approved the final manuscript.

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LH holds a Bachelors of Engineering, majoring in Biomedical Engineering and minoring in Biotechnology obtained from the University of Guelph in 2016. He is currently enrolled in a second degree program and participating in research in Biochemistry at the University of Lethbridge. Lane is also interested in the politics of sensible drug policies and associated programs, including, but not limited to, the implementation of harm reduction best practices in Canada.

JP received a Bachelors of Science in Chemistry from Carleton University. He currently works on automation and sensing technology.

EP holds degrees in nursing and is an assistant professor in the Faculty of Health Sciences at the University of Lethbridge. EP has a clinical background in outreach nursing and harm reduction and conducts research and evaluation studies with local harm reduction agencies.

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Harper, L., Powell, J. & Pijl, E.M. An overview of forensic drug testing methods and their suitability for harm reduction point-of-care services. Harm Reduct J 14 , 52 (2017). https://doi.org/10.1186/s12954-017-0179-5

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Drug Testing

• Drug testing looks for the presence or absence of specific drugs in a biological sample, such as urine, blood, or hair. Drug testing cannot diagnose a substance use disorder. As a tool in substance use treatment programs , drug testing can monitor a patient’s progress and inform their treatment. Substance use treatment programs should not use drug testing results alone to discharge patients from treatment. Drug testing is also used in workplace and justice settings.
• While multiple test options are available, urine drug screening is most common. An initial urine drug screen can deliver rapid results but can be affected by factors, such as certain medications, that can cause incorrect results (called a false positive or a false negative) . If an initial test is positive, health care providers can order a sensitive and specific confirmatory test. For all testing methods, accurate interpretation may require consultations with specialists such as Medical Review Officers (MRO) and medical toxicologists.
• NIDA supports and conducts research to improve drug testing by investigating more accurate and accessible technologies and applying drug testing in new ways to support individual and public health. NIDA does not administer drug testing programs, assist in interpreting drug test results , or manufacture, regulate, or distribute drug screening products. Learn more about drug testing regulation from the U.S. Food and Drug Administration (FDA) and about workplace drug testing from the Substance Abuse and Mental Health Services Administration (SAMSHA) .

What is a drug test?

A drug test looks for the presence or absence of a drug in a biological sample, such as urine, blood, or hair. Drug tests may also look for drug metabolites in the sample. A drug metabolite is a substance made or used when the body breaks down (or “metabolizes”) a drug.

Drug tests target only specific drugs or drug classes above a predetermined cutoff level. 1  A cutoff level is a point of measurement at or above which a result is considered positive and below which a result is considered negative. For example, in workplace drug testing the federal cutoff level for a cannabis drug test in urine is 50ng/mL. A test result below 50ng/mL will be reported as negative even if the result is above 0. This cutoff level helps to limit false positive s. 2

Drug testing is different than “ drug checking ,” which helps people who use drugs determine which chemicals are found in the substance they intend to take. Drug checking is a type of harm reduction .

What is the difference between a drug screen and a confirmatory drug test?

Usually, drug testing involves a two-step process: an initial drug screen and a confirmatory test.

• Initial drug screens or presumptive drug tests are used to identify possible use of a drug or drug class. These tools are also called point-of-care testing and are useful because they can produce rapid results. Initial urine drug screens use the immunoassay method for analysis, which uses antibodies to detect drugs at the molecular level.
• Confirmatory or definitive tests either verify or refute the result of an initial screen. These tests are more specific, more sensitive, and results take longer because they are sent to a laboratory. These tests use methods called gas chromatography/mass spectrometry or liquid chromatography/mass spectrometry to analyze samples. The results can indicate specific drugs and provide more exact information about how much of a drug is present. 1,3

If an initial drug screen is positive, a second round of more precise confirmatory testing is done to confirm or rule out that positive result.

What substances do drug tests detect?

Drug tests are commonly used to detect five categories of drugs as defined by the federally mandated workplace drug testing guidelines, although health care providers can order additional tests, if needed. 2,3 This list may also change as new drugs enter the drug supply. 4

These drug tests are usually urine tests, though other biological samples can also be used: 2

• Amphetamines, including methamphetamine
• Cannabis (marijuana), which tests for cannabinoid metabolites, including THC metabolites
• Cocaine , which tests for benzoylecgonine, a cocaine metabolite
• Opioids , a class of drugs that includes heroin, synthetic opioids such as fentanyl, and pain relievers like oxycodone (OxyContin ® ), hydrocodone (Vicodin ® ), codeine, and morphine. This category will have separate tests depending on which opioid is being tested. There is currently no drug test that tests for all opioids.
• Phencyclidine (PCP)

Drug tests can also detect additional categories of drugs:

• Barbiturates
• Benzodiazepines
• MDMA/MDA (Ecstasy/Molly)

How is NIDA advancing research on drug testing?

NIDA supports and conducts research to improve drug testing methods by investigating more accurate and accessible technologies and applying drug testing in new ways to support individual and public health. This research includes efforts to develop new highly specific and sensitive tests for urine, breath, and sweat. It also includes the development of innovative technologies such as wearable sensors that can test for drugs in real time.

NIDA also supports the National Drug Early Warning System , which helps collect and share information on emerging drugs that may inform the development of drug tests.

NIDA does not administer drug testing programs, assist in interpreting drug test results , or manufacture, regulate, or distribute drug screening products. Learn more about drug testing regulation from the U.S. Food and Drug Administration (FDA) and about workplace drug testing from the Substance Abuse and Mental Health Services Administration (SAMSHA) .

What is drug testing used for?

Drug testing is used to find out whether a person has used a substance in the recent past. Drug testing can sometimes also detect passive exposure to drugs, such as secondhand smoke or prenatal exposure. The length of time following exposure that a drug can be detected during testing can vary.

Drug testing cannot diagnose a substance use disorder. 2

A drug test may be used for different reasons, including:

• Supporting the clinical aims of substance use disorder treatment .
• A medical assessment , such as during emergency department visits for unintentional poisoning, attempts at self-harm, or environmental exposures. In these cases, point-of-care testing is used to help diagnose and manage patients whose symptoms may be related to drug use. 5,6 Newborns can also be tested for possible prenatal exposure to illicit drugs using urine, blood, meconium (an infant’s first bowl movement), hair, or umbilical cord samples. 7
• Preventing prescription drug misuse. Some health care providers will schedule or order random urine drug testing while prescribing controlled substances, such as opioids, stimulants, and depressants. However, this practice is not standardized. 8,9
• Employment. Employers may require drug testing as part of a drug-free workplace program. Drug testing is required by federal law in some workplaces, including safety and security-sensitive industries like transportation, law enforcement, and national security.
• Legal evidence. Drug testing may be part of a criminal or motor vehicle accident investigation or ordered as part of a court case.
• Athletics. Professional and other athletes are tested for drugs that are used to improve performance (sometimes referred to as “doping”). The U.S. Anti-Doping Agency conducts this testing.
• Recovery residences. Communities of people in recovery from substance use disorders may use drug testing to monitor abstinence of residents. 10  

How are drug tests performed?

Urine is the preferred and most used biological sample for drug testing, as it is available in large amounts, contains higher concentrations of drugs and metabolites than blood, and does not require needles. 11 Urine drug tests are also available during point-of-care, or outside the laboratory (e.g., doctor’s office, hospital, ambulance, at home). 12

Less commonly, drug testing may use blood or serum, oral fluid (saliva), breath, sweat, hair, or fingernails. 1

There are FDA-approved at-home drug tests (urine or saliva) readily available at pharmacies. It is important to follow specific instructions and send a urine sample to a laboratory for confirmation.

How is drug testing used during treatment for substance use disorders?

Urine drug screening can be an important tool for substance use disorder treatment. 13 Health care providers can use urine drug screens to follow a patient’s progress. Test results are used to determine whether dosing adjustments or other treatment interventions are needed. After unexpected results , patients and health care providers can speak openly about treatment and progress to better tailor the treatment to the patient’s needs. 10,13

Federal guidelines for Opioid Treatment Programs require drug testing. Urine drug tests are often administered as part of the intake process to confirm substance use history and as a routine part of therapy. 14

Understanding the limits of urine drug screening and other toxicology testing is an important part of making treatment decisions. Drug testing is never the sole determinant when making patient care decisions. 10

Contingency management is a behavioral therapy that uses motivational incentives including tangible rewards for drug-negative urine specimens. Contingency management has been demonstrated to be highly effective in the treatment of substance use disorders including addiction to stimulants. 15  

What happens if a drug test result is positive during substance use disorder treatment?

If a drug test result is positive during substance use disorder treatment, health care providers may prescribe additional or alternative treatments. Drug test results should not be used as the sole factor when making patient care decisions, including discharge decisions. 10,13 It is best practice for addiction treatment providers to avoid responding punitively to a positive drug test or using it as the basis for expelling someone from treatment. However, actual consequences of a positive drug test during substance use treatment may depend on state laws and the individual program.

Recovery residences (e.g., sober living homes) may also use drug testing to monitor the abstinence of residents, and residents may be expelled on the basis of positive drug tests. However, it is important that expulsion should not prevent or interfere with the individual continuing to receive outpatient addiction treatment. 10

What are the limitations of drug testing?

Urine drug tests do not provide information regarding the length of time since last ingestion, overall duration of use, or state of intoxication. 16

Sometimes urine can be difficult to obtain due to dehydration, urinary retention (the person is unable to empty their bladder), or other reasons. 17

Drug testing can be a useful tool, but it should not be the only tool for making decisions. Drug testing results should be considered alongside a patient’s self-reports, treatment history, psychosocial assessment, physical examination, and a practitioner’s clinical judgment. 2,18

Drug testing can also produce false positives and false negatives.

How accurate are drug tests? Can a drug test result in a false positive or false negative?

All tests have limitations, and false positives or false negatives can occur. 1

A false positive is when a drug test shows the presence of a substance that isn’t there. This can happen during the initial urine drug screening, which uses the immunoassay method (antibodies to detect drugs at the molecular level). Immunoassays are the most commonly available method of testing for drugs in urine. 2 Immunoassays rely on a chemical reaction between an antibody and a drug the test is designed to identify. Sometimes the antibodies can react to other chemicals that are similar to the drug—called cross-reactivity. Cross-reactivity can occur with some over-the-counter medicines, prescription medicines, and certain foods, like poppy seeds. For example, some cough and cold medicines, antidepressants, and antibiotics can cause false positive results. 19,3

A false negative is when a drug test does not show the presence of a substance that is there. This can happen during the initial urine drug screening. A false negative result can happen when the cutoff level used was set too high, so small amounts of the drug or drug metabolites were missed. 2  False negatives can also happen when contaminants are deliberately ingested or added to urine to interfere with a test’s ability to detect a drug’s presence. 20

Laboratory errors can also result in false positives or false negatives.

A confirmatory test can be performed to confirm the initial screening test results. A medical review officer can also interview the patient and review the lab results to help resolve any discrepancies. 1,14

How are drug test results confirmed?

An essential component of any drug testing program is a comprehensive final review of laboratory results. 18 In federally mandated drug testing programs, this role is often filled by a medical review officer, who will review, verify, and interpret positive test results. Medical review officers provide quality assurance and evaluate medical explanations for certain drug test results. The medical review officer should be a licensed physician with a knowledge of substance use disorders. 21

To avoid misinterpreting drug test results, health care providers can use experts in the field. This includes clinical chemists or medical toxicologists at hospitals, clinics, or poison control centers. Expert assistance with toxicology interpretations can improve the accuracy of drug test results. 

Can NIDA assist me with interpreting or disputing the results of a drug screen?

NIDA is a biomedical research organization and does not provide personal medical advice, legal consultation, or medical review services to the public. While NIDA-supported research may inform the development and validation of drug-screening technologies, NIDA does not manufacture, regulate, or distribute laboratory or at-home drug screening products. The U.S. Food and Drug Administration (FDA ) regulates most of these products in the United States. Those with concerns about drug screening results may consider reaching out to the drug-screening program or a qualified health care professional. For more information on workplace drug screening, please visit the Substance Abuse and Mental Health Services Administration (SAMSHA) Division of Workplace Programs website.

Latest from NIDA

What do drug tests really tell us?

NIH and FDA leaders call for more research, lower barriers to improve and implement drug- checking tools amid overdose epidemic

NIH launches harm reduction research network to prevent overdose fatalities

Find more resources on drug testing.

• Learn more about drug test regulation from the U.S. Food and Drug Administration (FDA) .
• Learn more about workplace drug-testing programs from the Substance Abuse and Mental Health Services Administration (SAMHSA) .
• Find basic drug testing information from MedlinePlus , a service of NIH’s National Library of Medicine (NLM).
• Find information about drug testing in child welfare from the National Center on Substance Abuse and Child Welfare . 
• McNeil SE, Chen RJ, Cogburn M. Drug testing . In: StatPearls . StatPearls Publishing; January 16, 2023.
• Center for Substance Abuse Treatment. Clinical drug testing in primary care . Substance Abuse and Mental Health Services Administration. Technical Assistance Publication Series , No. 32 2012. Accessed May 3, 2023
• Moeller KE, Lee KC, Kissack JC. Urine drug screening: practical guide for clinicians . Mayo Clin Proc . 2008;83(1):66-76. doi:10.4065/83.1.66
• Gerona RR, French D. Drug testing in the era of new psychoactive substances . Adv Clin Chem . 2022; 111:217-263. doi:10.1016/bs.acc.2022.08.001
• Mukherji P, Azhar Y, Sharma S. Toxicology screening . StatPearls . Updated August 8, 2022. Accessed May 3, 2023.
• Bhalla A. Bedside point of care toxicology screens in the ED: Utility and pitfalls . Int J Crit Illn Inj Sci . 2014;4(3):257-260. doi:10.4103/2229-5151.141476
• Farst KJ, Valentine JL, Hall RW. Drug testing for newborn exposure to illicit substances in pregnancy: pitfalls and pearls . Int J Pediatr . 2011;2011:951616. doi:10.1155/2011/951616
• Chakravarthy K, Goel A, Jeha GM, Kaye AD, Christo PJ. Review of the Current State of Urine Drug Testing in Chronic Pain: Still Effective as a Clinical Tool and Curbing Abuse, or an Arcane Test? . Curr Pain Headache Rep . 2021;25(2):12. Published 2021 Feb 17. doi:10.1007/s11916-020-00918-z
• Raouf M, Bettinger JJ, Fudin J. A practical guide to urine drug monitoring . Fed Pract . 2018;35(4):38-44.
• Jarvis M, Williams J, Hurford M, et al. Appropriate use of drug testing in clinical addiction medicine . J Addict Med . 2017;11(3):163-173. doi:10.1097/ADM.0000000000000323
• Kapur BM. Drug-testing methods and clinical interpretations of test results . Bull Narc . 1993;45(2):115-154.
• Hadland SE, Levy S. Objective Testing: Urine and Other Drug Tests . Child Adolesc Psychiatr Clin N Am . 2016;25(3):549-565. doi:10.1016/j.chc.2016.02.005
• Center for Substance Abuse Treatment. Substance abuse: Clinical issues in intensive outpatient treatment . Substance Abuse and Mental Health Services Administration. Treatment Improvement Protocol (TIP) Series , No. 47 2006. Accessed May 3, 2023.
• Substance Abuse and Mental Health Services Administration . Federal guidelines for opioid treatment programs . Published January 2015. Accessed May 3, 2023.
• Petry NM. Contingency management: what it is and why psychiatrists should want to use it . Psychiatrist . 2011;35(5):161-163. doi:10.1192/pb.bp.110.031831
• Dobrek L. Lower Urinary Tract Disorders as Adverse Drug Reactions-A Literature Review . Pharmaceuticals (Basel). 2023;16(7):1031. Published 2023 Jul 20. doi:10.3390/ph16071031
• Chua I, Petrides AK, Schiff GD, et al. Provider Misinterpretation, Documentation, and Follow-Up of Definitive Urine Drug Testing Results . J Gen Intern Med . 2020;35(1):283-290. doi:10.1007/s11606-019-05514-5
• Reisfield GM, Teitelbaum SA, Jones JT. Poppy seed consumption may be associated with codeine-only urine drug test results . J Anal Toxicol . 2023;47(2):107-113. doi:10.1093/jat/bkac079
• Kale N. Urine Drug Tests: Ordering and interpreting results . Am Fam Physician . 2019;99(1):33-39.
• Center for Substance Abuse Treatment. Division of Workplace Programs. Medical review officer guidance manual for federal workplace drug testing programs . Substance Abuse and Mental Health Services Administration . 2020. Accessed April 20, 2023.

Drug Testing and the Right to Privacy: Arguing the Ethics of Workplace Drug Testing

• Published: December 1998
• Volume 17 , pages 1805–1815, ( 1998 )

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• Michael Cranford  

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As drug testing has become increasingly used to maximize corporate profits by minimizing the economic impact of employee substance abuse, numerous arguments have been advanced which draw the ethical justification for such testing into question, including the position that testing amounts to a violation of employee privacy by attempting to regulate an employee's behavior in her own home, outside the employer's legitimate sphere of control. This article first proposes that an employee's right to privacy is violated when personal information is collected or used by the employer in a way which is irrelevant to the terms of employment. This article then argues that drug testing is relevant and therefore ethically justified within the terms of the employment agreement, and therefore does not amount to a violation of an employee's right to privacy. Arguments to the contrary, including the aforementioned appeal to the employer's limited sphere of control, do not account for reasonable constraints on employee privacy which are intrinsic to the demands of the workplace and implicit in the terms of the employment contract.

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Cranford, M. Drug Testing and the Right to Privacy: Arguing the Ethics of Workplace Drug Testing. Journal of Business Ethics 17 , 1805–1815 (1998). https://doi.org/10.1023/A:1005742923601

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Current practice and experience in drug and alcohol testing in the workplace

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• 1 Injury Prevention Research Center, College of Medicine, University of Iowa, Iowa City.
• PMID: 7920540

The present paper presents a review of the current practice and experience in drug and alcohol testing in the workplace, focusing primarily on the United States of America. After reviewing the history of workplace drug screening, the author describes the growth and impact of the drug-testing industry. He outlines the four most common rationales for workplace drug testing: safety, productivity, decreasing drug use and legislative/regulatory requirements. He summarizes the best studies on the prevalence of workplace drug testing in the United States and describes employer attitudes in that country. The author reviews in some detail the association between drugs, alcohol, occupational injuries and other adverse employment outcomes. He then reviews the literature on cost-benefit analysis of workplace drug testing. The author concludes that relatively little is known about the role of alcohol and drugs in the workplace. The important association between substance abuse and occupational injury has not been established. If there is such an association, it is much weaker than previously believed. The contradictory findings in different studies suggest that substance abuse may well play different roles in different occupational and cultural settings. Thus, caution should be exercised in transposing results from one setting to another. Finally, the review of cost-benefit analyses suggests that any economic analysis of workplace drug screening is likely to be greatly influenced by the prevalence of drugs in the population screened.

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“What’s That Drug? Fast Screening of Seized Drugs"

In forensic laboratories, samples are often screened for drugs through so-called color tests, designed to change color in the presence of drugs. Although these tests are quick and inexpensive, they are limited because they are non-specific, meaning they do not identify which drug may be present, and can be inconclusive, requiring follow-up testing. A recently completed National Institute of Justice-funded study proposed to test the use of powerful electrochemical techniques to detect emerging drugs, such as fentanyl and novel psychoactive substances, in seized drug cases. These techniques are widely used in biomedicine, chemistry, environmental sciences, and many other industrial applications. The researchers confirmed their findings using Raman spectroscopy, a non-destructive technique that reveals the structural fingerprint of an unknown substance. They propose that using these methods in tandem could improve current drug screening procedures.

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• v.9(2); 2020 Apr

Toxicity testing is evolving!

Ida fischer.

Institution of Medical Sciences, University of Aberdeen, Aberdeen AB25 2ZD, UK

Catherine Milton

Heather wallace.

The efficient management of the continuously increasing number of chemical substances used in today’s society is assuming greater importance than ever before. Toxicity testing plays a key role in the regulatory decisions of agencies and governments that aim to protect the public and the environment from the potentially harmful or adverse effects of these multitudinous chemicals. Therefore, there is a critical need for reliable toxicity-testing methods to identify, assess and interpret the hazardous properties of any substance. Traditionally, toxicity-testing approaches have been based on studies in experimental animals. However, in the last 20 years, there has been increasing concern regarding the sustainability of these methodologies. This has created a real need for the development of new approach methodologies (NAMs) that satisfy the regulatory requirements and are acceptable and affordable to society. Numerous initiatives have been launched worldwide in attempts to address this critical need. However, although the science to support this is now available, the legislation and the pace of NAMs acceptance is lagging behind. This review will consider some of the various initiatives in Europe to identify NAMs to replace or refine the current toxicity-testing methods for pharmaceuticals. This paper also presents a novel systematic approach to support the desired toxicity-testing methodologies that the 21st century deserves.

Introduction

In the 21st century, the ever-increasing population and the constant demand for new and improved technologies have set a huge challenge for regulatory toxicology (RT), as many of these innovations are based upon chemical substances. The search for novel chemical entities and the repurposing of existing chemicals are at the heart of the needs of everyday society. Major industries, such as pharmaceutical, food, cosmetics and agriculture, are continuously creating and remodelling chemicals. With this creativity comes the risk that these substances may have harmful effects on consumers and the environment [ 1 ]. Therefore, the effective management of the use and safety of such chemicals is crucial to the well-being of all.

Today’s RT is relatively new, resulting mainly from the infamous thalidomide incident in the early 1960’s. However, toxicity testing, one of the main pillars of RT, dates back many centuries. The history starts with Paracelsus (1493–1541), the father of toxicology, who demonstrated the dose–response relationship of numerous known remedies and toxins of the time, proving that ‘All substances are poisons; there is none which is not a poison. The right dose differentiates a poison and a remedy’ [ 2 ]. Finding the dose that has no observable adverse effect is crucial, and it has been one of the main aims of toxicity studies of pharmaceutical innovations [ 3 ]. It has been the responsibility of RT, including but not limited, to approve the right dose and control of the safe marketing of these pharmaceutical innovations [ 4 ].

In the 1920s, there was a marked increase with the use of animals for toxicity testing especially after the introduction of the LD 50 (50% lethal dose) test by J. W. Trevan [ 5 ]. The increasing recognition of the need to quantify regulatory concepts, such as the acceptable daily intake (ADI) and no observable (adverse) effect level (NOEL or NOAEL), subsequently led to statistical innovations, which were mainly influenced by pharmacological evaluations [ 6 ]. For example, Bliss (1934) introduced first the applications of probit regression analysis to fit a dose–response model, which could help to calculate the LD 50 [ 6 ]. Several other testing methods were developed through the 20th century, which required the extensive use of animals and were neither time nor cost effective [ 7 ]. Many of these are still used today, including the 2-year carcinogenicity bioassay and developmental/embryotoxicity studies. Recently, there has been some significant progress in replacing animal models and improving the toxicity-testing approaches, such as the replacement of the Draize Test with in vitro skin irritation testing [ 8 ].

However, toxicological risk assessment methodologies have remained relatively unchanged for more than 40 years, containing many well-known, well-used yet imperfect models [ 9 ]. Even today, standard toxicity testing mainly uses high dose, mostly chronic, exposure in animals from which, using linear extrapolations and apical endpoints, a specific substance is determined as potentially toxic in humans. Failure of these animal-based toxicity studies to provide reproducible data, due to the various endpoints in multiple testing, can lead to a high number of false-positive and false-negative results, and thereby statistical and biological inaccuracy [ 10 ]. This can lead to flawed toxicology-based decision-making by regulatory authorities, industries and governments despite these organizations doing everything they can to prevent it. For example, in case of the selection of NOAEL, the failure to reject a null hypothesis of there being, no difference between doses does not necessarily mean no difference at all in reality [ 6 ]. Or in case of thalidomide, the low significance of dose-dependent foetal anomalies in rat and mice compared to the white rabbit did not mean no teratogenic effect in human [ 11 ]. Furthermore, the incidence of adverse drug reactions (ADRs) from approved medications can result in withdrawal from the market. In Europe, approximately 3.6% of all hospital admissions and nearly 0.5% of deaths are due to ADRs, resulting not only in extra expense for the pharmaceutical industry, but also for the national healthcare systems [ 12 ].

The aim of the toxicological regulations of pharmaceuticals is the research, development and production of new and effective therapeutics for the market, while protecting consumers from unsafe products. However, regulation may seem an obstacle in drug development, as it requires high levels of evidence of safety and efficiency of the product before approval for use [ 13 ]. This can be due to a cautious approach trying to ensure there is no approval of an ineffective drug, which may even cause ADRs, and/or failing to approve a useful agent. Nevertheless, one drug can be a poison for an individual while being a lifesaver for another. For example, propulsid, a medication developed for heartburn was removed from the market in March 2000, following the death of eight people due to irregular heartbeat caused by its active component, cisapride [ 14 ]. However, patients with cerebral palsy can use this agent successfully in order to be able to digest food painlessly [ 15 ].

Since, the 1970s, the number of novel medications reaching the market has decreased, but the cost of R&D has increased [ 16 ], especially, in the regions of Europe, USA and Japan [ 16 ]. Current procedures used in preclinical or non-clinical phases of drug development are not totally reliable, thus finding alternative approaches for a more predictive assessment is desirable [ 78 ]. Overall, failure rate of candidate drugs in the clinical phase is >90%, which means for every 20 compounds reaching clinical trials, only 1 achieves a marketing authorization, a situation which has not changed in the last decade [ 17 ]. Mostly, preclinical data are based on animal models; however, translation between species does not always work; therefore, many adverse effects are only found in clinical phases or post-marketing [ 12 ]. In the last 10 years or so, delineation of the pathway of toxicity and development of new reliable in vitro and in silico screening methods have provided useful tools for screening molecules at an early stage, but these methods have limitations [ 18 ] ( Table 1 ).

General advantages and disadvantages of study designs, including in vivo, in vitro and in silico

Clearly, there is a need for new approach methodologies (NAMs), which include new technologies and alternative strategies and ideally predictive models that can enable improved development of new and better medicines . Classical in vitro and in silico toxicological approaches along with new non-test applications, including (quantitative) structure–activity relationships ((Q)SARs), read-across, pharmacokinetic/pharmacodynamic and uncertainty factor models, are all collectively referred to as NAMs. In vitro toxicology with its cellular and organotypic models and assays aims to predict toxicity responses to different substances, including drug candidates, in human. The goal of in silico toxicology is to complement in vitro and in vivo toxicity tests. It has many different computational tools, including huge databases for storing toxicological properties of chemicals, software for simulation of biological systems and molecular dynamics, and modelling software for predicting toxicity [ 18 ]. Additionally, the impressive benefits of (Q)SARs, low cost, speed and potential to reduce animal-based toxicity testing, make it a useful piece of NAMs [ 19 ]. It demonstrates with mathematical/statistical relationship models the connection between the quantitatively measured chemical structure(s) and biological, including toxic activity of chemicals [ 19 ]. In addition, the read-across method, which predicts unknown toxicity of chemicals based on known toxicity of similar chemicals, is gaining attention as another member of NAMs as in terms of reproducibility outperforms animal testing [ 20 ].

The 3Rs (reduction, refinement and replacement of animals used in scientific research) provides the foundation for NAMs. Reduction of animal use in toxicity testing can be done with improvements to study design, including the use of historical in vivo data or using control groups for multiple experiments. Refinement of animal studies aims to reduce the pain and suffering animals may experience; this is most commonly achieved by improving the living conditions of the animals. These improvements can reduce stress and improve behaviours of the animals, which leads to more accurate results [ 21 ]. Replacement of animal testing remains a more challenging task. The paradigm change in toxicity testing moves towards increasing application of in silico models as it does not require animals or samples of tissue to be harvested [ 21 ]. Many areas have been developing in silico models, for example cancer research has various genetic models (cBioPortal, EPA ToxCast screening library, Gene Ontology) to help with prediction, diagnostic and prognostic markers or to characterize entire biological processes [ 22 , 46 ].

However, the validation process of NAMs is not completely harmonized and has been slower than expected [ 23 ]. In theory, NAMs are accepted when they are considered qualified for a specific concept of use. However, there is no specification for how much evidence is required [ 24 ]. Also, the new methods are mostly validated against previous results of the chemical with animal models (retrospective); but what if the animal model was biased? Currently, the general rule is that with the introduction of new methods, there is only addition of extra knowledge to the drug candidate’s toxicity profile without eliminating the need for the old data gathering and testing methods [ 25 ]. However, there are great examples for implementing the vision of faster and cheaper toxicity testing, which are more relevant to humans and requires minimal number of tested animals. The Integrated Testing Strategy (ITS), which merges data from testing and non-testing methods, and the Integrated Approaches to Testing and Assessment (IATA) are brilliant examples of the many approaches that are working towards a complete paradigm shift in regulatory toxicity testing.

How then has toxicity testing been changing to realize the vision of more reliable, more rapid and less expensive approaches? Can the gap between research and regulation be completely eliminated? What successes have been achieved in drug development by revolutionizing toxicity-testing techniques? What is next for RT of pharmaceuticals? This paper aims to address these questions using some of the past, present and future European and a few milestones in American NAMs.

A systematic strategy for searching and collecting data was specified at the outset. Subject-specific professional books and chapters, journals, articles, reports and websites were used as a source of information, and they provided the foundation for data collection and analyses. The most suitable terms, used for search, were carefully selected. Table 2 indicates how key terms and suitable synonyms were categorized and used.

Searching parameters, defined key terms used in research

Search was conducted by using key terms in selected databases and information sources.

The collected book chapters were included if they were directly related to regulatory toxicology or toxicity testing for pharmaceutical medicines, and their date of publication was after 2014. Inclusion criteria for websites were that the website must be either an official website of a medicinal regulatory authority or it must contain the latest information related to various toxicity-testing methods in drug regulation or to drug regulation itself. Articles had less strict inclusion criteria as the time period for their publication was not specified. However, key terms or part of the key terms had to be mentioned or referred to either in the title or in the abstract (where abstract was available) of the selected articles.

Analysis of systematically collected books, book chapters, websites and articles involved the categorization of selected documents according to their relevance in the predetermined main structure of this paper. Identified information and data were used to prepare the appropriate figures and tables, as well as to serve as evidence for the discussion and the conclusion reached.

The principle of the spiral cycle and research onion processes described by Saunders et al. (2007) served as a foundation for the research methodology scheme created and used to collect the relevant information required ( Fig. 1 ).

Research methodology scheme. Subject-specific books, journals articles and websites were mainly used as a source of information. Before conduction of research inclusion/exclusion criteria, findings and key terms were identified. When collected information was found to be irrelevant or inadequate, parameters and key terms were refined, and new search was conducted. When all requirements met, data were recorded; however, findings were revised continuously.

Imperfect models

The ultimate goal of toxicity testing for novel human medicines or any chemical is the identification of any hazardous properties the substance may possess. The traditional methods rely mainly on toxicity endpoints (adverse effects) in the animal models. The adverse effects can be described as quantitative (such as the aforementioned LD 50 ) or qualitative, including binary (toxic or non-toxic) and ordinary (low and high toxicity) outcomes. The complexity of these tests is that the adverse effect depends on various factors. Not only on the chemical properties of a substance, but also on the route of exposure (oral, dermal, inhalation, etc.), the dose (acute or multiple), frequency (single, multiple), duration (24 hours or 24 months), as well as on the biological (gender, age, etc.) and ADME properties [ 18 ].

There are numerous testing methods for screening potential toxic effects of drug candidates ( Table 3 ), including, the current gold standard for carcinogenicity testing, the 2-year bioassay, which is carried out in two genetically distinct rodent species. During and after exposure to the chemical being tested, the animals are observed, and any tumour development is noted. If there are no signs of tumours, then the assay can be ended at 18 months for mice or 24 months for rats. The animals are then sacrificed, and histopathological studies carried out on all tissues [ 26 ].

The main properties of the carcinogenicity and the genotoxicity-testing methods

Representation of the main properties and limitations of selected study designs used for toxicity testing of drug candidates. The table was tabulated based on the safety guidelines of ICH.

The significant time and resources used during the 2-year bioassay make it a very expensive procedure [ 27 ]. However, the ability of this bioassay to predict human carcinogenicity is questionable, which has made the usefulness of this assay debatable [ 28 ]. There are also assumptions in the 2-year bioassay that are not always met. For example, the effects of a chemical in rodents will produce the same effects in humans. Rodents do not grow spontaneous tumours in the same tissues as human, consider the lung, the skin, the liver and the colon [ 29 ]. Also, many anticancer drugs increase the risk of secondary cancers 10–30 years after treatment, which could never be predicted by a 2-year bioassay [ 30 ].

Currently, genotoxicity uses both in vitro and in vivo tests. One of the main problems with the range of genotoxicity tests available is the high rate of false positives due to tests having low specificity ( Table 3 ). This is best shown in the Ames test, which is only about 60% specific and lacks some of the eukaryotic properties found in humans due to using bacteria [ 31 ]. This problem of specificity was first officially raised in 2007 during a European Centre for the Validation of Alternative Methods (ECVAM) workshop, it was suggested that more mechanistic data on humans were required as well as clearer guidance on the use of positive results that have no human relevance [ 28 ]. To give an example, it is estimated that to detect a 1/10 000 Drug Induced Liver Injury (DILI) related to idiosyncrasies, 30 000 patients would be required during clinical trials [ 32 ]. The onset of DILI can happen weeks after treatment; this might be because the liver can repair damage up to a point; however, there are many different mechanisms that can induce hepatotoxicity. One study conducted a survey finding around 45% of hepatotoxicity tests done in both a rodent and non-rodent species failed to predict DILI in human trials [ 33 ]. Thereby, highlighting the need for novel models with improved predictability for hepatotoxicity. Arguably, the validation process is one of the major huddles faced by both in vitro and in silico tests as their outcomes are measured against the data of old tests. Validation is a five-step process ( Fig. 2 ) where sensitivity, specificity, accuracy and reproducibility of a test are assessed.

The five stages to regulatory acceptance. Test is optimized during the first two stages. Data are gathered during the third stage from prospective studies. Data are then peer reviewed by internal and external examiners to reduce bias and submitted to a regulatory body to be accepted or rejected.

However, the current practice is that novel technologies and methods do not replace the old ones. Rather they are added as extra steps to provide supporting information for toxicological risk assessments [ 25 ]. Differences in viewpoint between toxicologists and regulators can also lead to complications. The former wants to find the truth and explain the natural mechanisms through its hypothesis-based investigations, while the latter wants to solve problems through decisions based on available data, sometimes where the quality of data is less than ideal. In essentially, the science is advancing rapidly while the regulations are changing more slowly resulting in a disconnection that is hampering progress.

There have been many critiques of the current validation processes, especially regarding in vitro tests, the main concern being the time taken to approve a new test.

The time for change Tox21 and Tox21c

The toxicology in the 21st Century Consortium (Tox21) is a federal research collaboration between the US Environmental Protection Agency (EPA) and the US National Toxicology Program (NTP). It was started in 2004 and initially was composed of 22 experts in various fields of toxicology, risk assessment and animal welfare. The experts of Tox21 developed and published a milestone report in 2007, entitled ‘Toxicity Testing in the 21st Century – A Vision and a strategy’ (Tox21c). The main aim was to induce a paradigm shift in toxicity testing; the report focuses on developing testing methods to allow rapid and effective evaluation of the safety of medical products, food additives, commercial chemicals and pesticides [ 34 ]. The key concept of Tox21c is to identify and delineate the finite number of pathways to toxicity (PoT) [ 9 ]. By determining the connection between the chemical–biological interaction of a toxicant with its adverse outcome in molecular and cellular terms, our understanding of how chemical substances can be harmful to human health and the environment will be much improved [ 35 ].

The impact of Tox21c in finding better and more reliable alternatives to current model systems has been enormous. Determination of PoT using in vitro and in silico models to uncover toxic effects of chemicals based on their mechanism of action (MoA) rather than using animal models with toxicity endpoints (such as carcinogenicity or genotoxicity) is a more logical and favourable approach for toxicologists [ 37 ]. In vitro and in silico models can provide a faster, cheaper and, in some cases, more reliable toxicity assessment using advanced high-throughput screening (HTS) and computational resources (i.e. software, algorithms, databases, analysis methods, etc.). Over the last two decades and following the publication of Tox21c, derived initiatives aimed at improving collaboration between academia, industries and regulatory bodies have increased markedly. These collaborations have been searching for and identified the major technical advancements that can be used to improve risk assessment [ 1 ]. They have been also working towards the accelerated regulatory acceptance of alternative methods by facilitating the sharing of information and knowledge.

Tox21 takes advantage of technological advances to enable regulatory toxicity testing to move away from the traditional system, which is based on apical endpoints in vertebrate animal models, to one that is based on mechanistic endpoints in human-relevant in vitro models [ 36 ]. This goal is shared by many other organizations. In October 2018, Tox21 expanded its focus. In addition to its predominant research on developing and applying HTS methods for toxicity testing, it now aims to create and refine alternative test systems and will address limitations of current in vitro tests over the next 5 years [ 34 ].

Current chemical risk assessment is not capable of keeping up with the constantly increasing number of chemicals that require testing. It is for this reason that organizations are starting to take advantage of in silico technologies, alongside in vivo and in vitro methods, to better understand PoT and the development of adverse outcome pathways (AOP). To this end, the Organization for Economic Cooperation and Development (OECD) developed IATA. IATA integrates data from multiple methodologies (i.e. in silico, in vivo, in vitro , etc.) to better define the hazardous characteristics of chemicals, which can be used to aid regulatory decision-making [ 38 ]. The idea of integrated approaches is not a novel concept; with talks of IATA dating back to 2007 and with the ever-advancing computer technologies and NAMs, there has been an increased drive towards these approaches [ 39 ]. The process of IATA can be broadly summarized in three steps ( Fig. 3 ), (1) gathering relevant existing data from multiple methodologies, (2) assessing if the weight of evidence (WoE) of gathered data is to a satisfactory level to make a regulatory decision or if further evidence is required and (3) generating new data to reach satisfactory level. IATA can utilize AOPs to help gather the existing data or to help generate further data when needed. A prime example of the use of IATA in the cosmetic industry, where the use of animals has been banned in Europe [ 40 ]. This has led impressive advancements in NAMs in this area. Due to this, there are IATA that are used in regulatory decision-making for acute toxicity endpoints such as skin irritation, corrosion and sensitization [ 41 ].

Overview of steps for IATA and AOPs are used to support IATA (diagram adapted from OECD [ 38 ]).

The purpose of IATA is to allow flexibility and expert input in the gathering and organization of data from several methodologies. Despite the flexibility, there are some areas that have to be standardized and expert input removed, known as defined approaches (DAs). DAs are developed as our understanding of the mechanism leading to an adverse outcome progresses [ 41 , 42 ]. However, as IATA using WoE and requiring expert input, it cannot currently be used for more complex processes or areas that do not have a large number of NAMs. This is due to the lack of understanding of the underlying mechanistic detail in these areas that therefore make an IATA difficult to develop.

While currently IATA is not commonly used, their potential is promising with the increasing advancements in NAMs and as our understanding of the underlying mechanisms improves. The prospect of using IATA as a regulatory tool seems very possible.

Development of NAMs

The need to develop human relevant testing strategies for toxicological risk assessments that are based only on reliable NAMs originates from the principles of the 3Rs, coined by Russell and Burch in 1959 [ 43 ]. While the refinement and reduction of animal use has been addressed and, in many cases obtained, the complete replacement of animal models has still a long way to go.

In general, the main goals of the various NAMs developing programmes are the development and standardization of novel alternative testing methods by utilizing many advanced technologies, and acceleration of the regulatory acceptance for full exploitation of these novel techniques for regulatory purposes. In recent years, there has been an increase of in vitro- and in silico -based initiatives ( Fig. 4 ).

Timeline of highlighted initiatives for NAMs. Chronological order and brief summary of NAMs are listed in this paper. Initiatives with shaded backgrounds are the American ones, while all listed with white background are located in Europe.

Toxicity Forecaster

Toxicity Forecaster (ToxCast) is part of the EPA’s Computational Toxicology Research Program (CompTox), which provides public access to computational toxicology research data via online databases or resources [ 44 , 79 ]. These online data sources include Aggregated Computational Toxicology Resource (ACToR), Distributed Structure-Searchable Toxicity Database Network (DSSTox), Toxicity Reference Database (ToxRefDB), Exposure-Based Chemical Prioritization Database (ExpoCastDB) and Toxicity Forecaster Database (ToxCastDB) [ 44 , 45 ]. These all contribute to provide better knowledge of, and access to, animal chemical toxicity studies and other exposure or toxicity data of chemicals based on their chemical structure, using quantitative models (both in vitro and in silico ) for predictive toxicology. ToxCast was launched in three phases in 2007. The first phase was the ‘Proof of Concept’ phase, followed by the second phase between 2009 and 2015, and by the third phase, which was completed in 2018. The second two phases helped expand the starting database of the first phase. Since its launch in 2007, ToxCast has evaluated more than 4500 chemicals using over 700 different HTS assays [ 44 ]. These chemicals include bioactive small molecules that are listed in the NIH Chemical Genomics Centre (NCGC) Pharmaceutical Collection and being found useful for repurposing applications [ 47 ]. Despite the above programmes normally patent their innovative ideas, the EPA programme supports the transparency and sharing of data and knowledge as far as possible, which all contributes towards the paradigm shift in toxicity testing [ 48 , 49 ].

By helping prioritize more and more chemicals based on their potential human risk, these and similar future projects can help enormously to repurpose small-molecule drugs approved for human use in a more consistent manner in order to use them in diseases, other than their initial application.

Fund for the Replacement of Animals in Medical Experiments

The Fund for the Replacement of Animals in Medical Experiments (FRAME) was founded in London in 1969 by Dorothy Hegarty. Her aim was to validate reliable and reproducible alternative methods for predicting adverse effects in humans [ 50 ].

The first toxicity committee of FRAME was established in 1979 and presented its first report on alternative toxicity-testing methods in 1982 [ 50 ]. It contributed greatly to the 1986 Animals Act (UK government), which complies with the European Union’s Directive 2010/63/EU regarding the protection of animals used for scientific purposes [ 50 , 51 ].

In 1989, FRAME established the INVITTOX database for the collection of protocols of in vitro methods in toxicity testing, which today is part of the Scientific Information Service of the ECVAM (a.k.a. EURL ECVAM). ECVAM has been supported by the European Union Network of Laboratories for the Validation of Alternative Methods (NETVAL) since 2013 [ 52–55 ]. The work of FRAME has contributed to regulatory approval of newer alternative toxicity-testing methods, including the Direct Peptide Reactivity Assay (DPRA) for in vitro skin sensitization test of chemicals [ 50 ].

Current projects in the FRAME Alternatives Laboratory include development of a cell-based liver toxicology model using 3D printing, which has the potential to be used for HTS of drug candidates [ 56 ]. If this technology reaches the accuracy in predicting toxicity in human, then it has the potential to replace animals in hepatotoxicity testing.

National Centre for Replacement, Refinement and Reduction of Animals in Research and CRACK IT

Another important UK-based organization dedicated to the complete implementation of the principles of the 3Rs by facilitating collaboration among academic institutions, pharmaceutical companies, chemical and consumer product industries, regulatory bodies and other research founders, is the National Centre for Replacement, Refinement and Reduction of Animals in Research (NC3Rs). It was established in 2004, and it works through its open innovation programme CRACK IT [ 57 ]. This has been developed in two parts [1] CRACK IT Challenges, which funds collaborations between academia and industry for the benefits of both, and [2] CRACK IT Solutions, which aims to maximize scientific and commercial benefits from accelerating development and validation of novel technologies with potential 3Rs impact [ 57 ].

The main objectives of a recent workshop of NC3Rs, jointly hosted with Unilever in London in 2018, were the application of in vitro and in silico approaches for decision-making in safety assessments particularly within regulatory setting, the identification of scientific gaps that still need to be addressed, and the encouragement of the collaboration among members of the sector, including academia, industries and regulatory agencies [ 57 , 82 ]. In addition, funded by Royal Dutch Shell PLC through CRACK IT Challenges, KREKATiS has developed a skin/eye (Q)SAR model, iSafeRabbit, for predicting skin/eye irritation and corrosivity potential of petrochemical substances, to replace animal studies [ 58 ]. iSafeRabbit is appropriate for regulatory purposes as it satisfies the five recommended OECD principles for QSARs. KREKATiS is expanding its scope to other chemicals, including pharmaceuticals [ 58 ].

European Partnership for Alternative Approaches to Animal Testing

The European Partnership for Alternative Approaches to Animal Testing (EPAA), launched in 2005, is a voluntary collaboration between the European Commissioners Verheugen and Potocnik, European trade associations, and companies from seven industry sectors, including the pharmaceutical industry [ 53 , 54 ]. The aim of EPAA is to pool and share knowledge and resources in a more general context to accelerate the development, validation and acceptance of alternative methods to currently used animal models, thereby the application of the 3Rs principle, in toxicity testing [ 53 , 54 ]. In addition, many companies of the European Federation of Pharmaceutical Industries and Associations (EFPIA) are members of this collaboration, which has had many fruitful projects. For instance, in 2009, data sharing was led by AstraZeneca and facilitated by NC3Rs, leading to the removal of the regulatory requirements for conventional single dose acute toxicity test for any human pharmaceuticals [ 53 , 54 ]. This regulatory change was a historic landmark change in toxicity-testing requirements of drug development.

Innovate Medicines Initiative

The Innovate Medicines Initiative (IMI) is the largest public–private initiative and partnership in Europe between the European Commission and the European pharmaceutical industries (represented by the members of EFPIA) [ 59 ]. It supports 113 collaborative projects with 2225 participants for addressing issues, including antimicrobial resistance, diabetes, immune and brain disorders and the challenges of regulatory safety testing designs for human medicines [ 59 , 84 ]. IMI was launched in 2008 with an overall budget of €5.3 billion [ 59 ].

Even though the IMI mainly focuses on the discovery of advanced medicines, the 3Rs principle plays key role in that and therefore, in many IMI projects, including eTOX, eTRANSAFE, MARCAR and VAC2VAC. eTOX and the MARCAR are two successfully finished IMI projects [ 60 , 61 ]. eTOX integrates bioinformatics and chemo-informatics approaches for the development of expert systems to allow in silico prediction of toxicities, while MARCAR classifies biomarkers and molecular tumour for non-genotoxic carcinogenesis. Both VAC2VAC and eTRANSAFE projects are the ongoing ones, ending in 2021 and 2022, respectively [ 62 , 63 ]. eTRANSAFE enhances translational safety assessment through integrative knowledge management while VAC2VAC compares vaccine LOT to vaccine LOT by consistency testing. Based on the success of IMI programme, the European Union’s research and innovation programme, Horizon 2020, which is a continuation of European Commission’s 7th Framework Programme (FP7), is funding the IMI2 with a budget of €1.65 billion from 2014 to 2020 [ 54 ]. This includes projects like the aforementioned eTRANSAFE and VAC2VAC [ 62 , 63 ].

Safety Evaluation Ultimately Replacing Animal Testing

On the long road to transitioning from animal-testing-derived information approach of the regulatory field to a revolutionary way to identify and characterize toxicological hazards of chemical substances and predict safety, there is a significant role of the Safety Evaluation Ultimately Replacing Animal Testing (SEURAT) research initiative. It has been sponsored by the FP7, along with several other programs [ 64 ]. For instance, the Virtual Physiological Human (VPH) project of the VPH Institute in Belgium for the full realization of ‘ in silico medicine’ is still ongoing. Another example is the EXERA on the development of 3D in vitro models of oestrogen-reporter mouse tissues for the pharmaco-toxicological analysis of nuclear receptor-interacting compounds from 2006 to 2009 in Italy [ 65 , 66 ].

The first phase of SEURAT initiative is SEURAT-1, launched on 1 January 2011 with an overall budget of €50 million [ 64 ]. The goal of SEURAT-1 was the replacement of repeated dose systemic toxicity testing. This includes the development of innovative testing methods to support regulatory safety assessment, the introduction of a toxicological mode-of-action strategy to describe how any substance can affect AOPs, and the demonstration of proof-of-concept of novel regulatory paradigms at multiple levels (theoretical, systems and application) through the formulation of case studies [ 64 ]. The initiative is composed of six complementary research projects and one coordination and support project. Despite the complexity and efforts of SEURAT-1, more steps must be taken to reach the ultimate goal of overcoming the obstacle of predicting toxicity of drug candidates in complex biological systems.

Joint ad hoc expert group on the application of the 3Rs in Regulatory Testing of Medicinal Products

In 2010, the European Medicines Agency (EMA) set up the Joint ad hoc expert group on the application of the 3Rs in Regulatory Testing of Medicinal Products (JEG 3Rs) to provide advice and recommendations to committees, including the Committee for Medicinal Products for Human Use (CHMP), on all matters relating to the use of animals in the testing of medicines for regulatory purposes [ 67 ]. The group cooperates with both the EURL ECVAM and the European Directorate for the Quality of Medicines and Healthcare (EDQM) to improve and promote the application of 3Rs in the regulatory testing of pharmaceutical products. JEG 3Rs developed and proposed EMA guidelines, in collaboration with the Scientific Advice Working Party (SAWP) of CHMP, regarding regulatory acceptance processes of novel 3R testing approaches (EMA/CHMP/CVMP/JEG-3Rs/450091/2012) [ 67 ].

JEG 3Rs via EMA also proposed several suggestions towards changes in some International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) safety guidelines. For instance, since 2016, there have been suggestions for the carcinogenicity testing of the ICH S1 guidelines. However, the debate on the necessity of this 2-year bioassay in adding substantial value to the drug development program still remains [ 68 ].

Integrated European ‘Flagship’ Programme Driving Mechanism-based Toxicity Testing and Risk Assessment

The Integrated European ‘Flagship’ Programme Driving Mechanism-based Toxicity Testing and Risk Assessment (EU-ToxRisk) for the 21st century project is another exemplary European collaboration project funded by Horizon 2020, with a budget over €30 million [ 69 ]. It was launched on 1 January 2016, following on from SEURAT-1, and will end in 2022. It integrates advancements in cell biology, ‘-omics’ technologies, systems biology and computational modelling to define complex PoTs. Furthermore, it provides proof of concept for mechanism-based safety testing strategies and guidance for their universal application. It focuses on repeated dose systemic toxicity and developmental and reproductive toxicity.

Over the last 10–15 years, many initiatives have been launched. The achievements are significant and provide a solid foundation for future developments.

What is next?

Enormous improvements in in vitro human-derived cell culture methodologies, for instance development of micro-physiological systems (MPS) like organ-on-a-chip (OOC), are leading the focused efforts to find more suitable, reproducible and predictive alternative systems for toxicity-testing purposes [ 70 ]. OOC is a type of artificial organ that simulates the activities, mechanics and physiological response of entire organs [ 70 ]. Mainly, it utilizes human-induced pluripotent stem cells (hiPSCs). Currently, this multi-channel 3D microfluidic cell culture chip is receiving a lot of attention from pharmaceutical companies and regulatory authorities worldwide as a means of modelling sequential metabolism and identifying adverse side and/or off target effects [ 59 ]. The hepatic microfluidic bioreactor reactor of the SEURAT-1’s HeMiBio project is an excellent example, with its innovative culture system that integrate hepatocytes and non-parenchymal liver cells, which are derived from hiPSCs, into MPS [ 71 ]. The generated co-culture allows induction and maintenance of various types of mature hepatic cell function in a bioreactor that can provide clinically relevant information on drug and chemical clearance toxicity.

There are several projects aiming to create and analytically validate individual-organ-chip models into single platforms. For example, the multiple tissue chip testing centres (TCTCs) that are funded through the tissue chip for drug screening program by the NCATS in the USA [ 72 ]. Despite the fact that the concept of human-on-a-chip and the stem cell field itself are still fairly new, huge strides have been made in the last decade towards creating reliable and biologically relevant platforms, which can be applied not just to pre-clinical but also to clinical phases of drug development and in precision medicine (‘you-on-a-chip’) in the future [ 59 ].

The International Consortium for Innovation and Quality in Pharmaceutical Development (IQ) is mainly made up of pharmaceutical representatives to address some of the biggest problems in drug development, including the already mentioned DILI [ 73 ]. The IQ-DILI initiative focuses on eliminating the existing gaps in current regulatory guidelines on detecting, monitoring, managing and preventing DILI in clinical phases of drug development. This is another outstanding example to show, in addition to the many previously listed and described initiatives, how synergistic intentions and interests of regulators, industry and academia can lead the technological evolution of toxicity testing forward. However, is the validation of modern strategies for regulatory safety assessment possible in either the pharmaceutical industry or other industries? Can the traditional approaches, which largely rely on animal safety test data, be abandoned to eliminate or at least reduce the risk of flawed data from these animal tests? [ 81 ]

The relatively new principle that finite number of PoTs exist provides an excellent modern approach in drug development as toxicological pathways can be pharmacological targets or even pathways for efficacy. Previously, drug targets were identified through the analysis of pathways leading to the disease, which is the basic of concept of AOPs. Additionally, the pharmaceutical industry has already been using the sophisticated ITS approach in forms of serial target-specific mechanism-based tests, including various in vitro and in silico methods. Thereby, if one test fails to detect all possible MoA of a given compound, another test or NAM can be applied, thus decreasing the number of bad candidates reaching the regulatory testing. Despite both the ITS and PoT approaches being implemented by many recent initiatives and pharmaceutical companies, the gap between research and regulatory acceptance still remains. The human predictivity value of alternative methods is validated against the predictivity value of animal models, which probably prevents the abandoning of earlier components of regulatory safety assessments.

What could be done?

The regulations to validate toxicity tests may need to be completely overhauled to be able to keep up with scientific innovation. Although, there are examples of the acceptance of a few alternative testing methods, including the DPRA or the 3 T3 Neutral Red Uptake (3 T3 NRU) phototoxicity tests that made it to regulatory acceptance, further gradual introduction to facilitate paradigm shift is necessary [ 74 ]. The continuous evaluation of novel toxicity-testing methods, preferably without simply accumulating the new ones alongside the old ones, needs to be more specific and strategies for its implementation must be defined. Novel technologies should be fully exploited for regulatory purposes [ 75 , 76 ]. For example, ‘-omics’ technologies that allow concurrent monitoring of many thousands of macromolecules, thereby revealing any functional disturbances. These technologies can give the necessary tools to examine and identify the differences in various cellular molecules, including DNA, RNA and proteins. However, the extensive biological interpretation of the role that these macromolecules and their related pathways play in physiological processes still needs to be fully delineated and understood.

Nevertheless, there are challenges to face before ‘-omics’, such as toxicogenomics can be used in RT. These challenges include how the data produced by these relatively new technologies can be fully incorporated in regulatory decision-making. In addition, computational modelling that allows analysis, visualization, simulation and prediction of toxicity, and any other mechanistic approaches should be more utilized in regulatory context.

From a pharmacological perspective, today’s toxicity-testing strategies are good but not efficient enough, a situation which needs to be change. The time, as well as the many requirements, it takes to validate NAMs and reach a complete regulatory acceptance for use further complicates this situation. The current complex process of drug development and the continued demand for more evidence by regulators on the safety, efficacy and quality of new drug candidates may be decreasing the number of valuable drugs that can reach the public. Nevertheless, many potential human medicines fail even before entering clinical trials due to demonstrating a toxic or adverse effect in animal models during the preclinical phase. The increasing concerns of regulatory agencies who, reasonably, fear authorizing unsafe medications do not help the paradigm shift needed. Therefore, the predictive value of toxicity testing needs to be further increased to prevent harm to the reputation of industry, regulation and academia.

From the brilliant examples currently ongoing the extensive changes in RT and specifically in toxicity testing can be clearly seen. There have been and are exemplary projects for replacing the conservative toxicity-testing methods. For example, the Horizon Europe, which is the next innovation framework programme of the European Commission, has five mission areas. One of them is cancer, which can provide an excellent opportunity for the emerging of further NAMs, providing more predictive and faster solution than the 2-year bioassay [ 83 ]. However, more needs to be done.

As a strategic step to promote the complete paradigm shift in toxicity testing, the development of a novel categorization system would be beneficial and valuable ( Fig. 5 ). This pre-validation system of already developed and currently developing NAMs could use the principles of International Organization for Standardization (ISO). NAMs should gain a certificate and a specific number, which categorize them mainly on the bases of producing a human- or animal-relevant, alternative toxicity-testing method. The two main categories could be subdivided into in vivo , in vitro and in silico methods, which could be sub-subdivided into pathway-based, systems biology, computational biology, simulation studies and non-test approaches. All sub-subdivision can be further divided and divided again if necessary. This alternative ISO system could feed into an international database, which would provide worldwide access to the location, participants, validation requirements, time, descriptions and other specifications for all NAMs. This database could use ICH and other regulatory guidelines to further categorize and support developers of NAMs.

Schematic representation of the suggested AISNAMs and IDBNAMs. The categorization system could feed into the international database (TT: toxicity testing).

Both the alternative ISO system for NAMs (AISNAMs) and the international database for NAMs (IDBNAMs) could promote and help the information flow of NAM initiatives and projects to the regulatory bodies as well as to the initiatives. They would provide transparency, while protecting the interest and benefit of each member. AISNAMs could help initiatives to know what regulations they must follow to gain regulatory approval and find investors. The transparency that the combination of this classification system and IDBNAMs could offer may help each member to better realize what is still needed and eliminate the gaps between research and regulatory acceptance. This could support the vision of a faster, cheaper and more relevant toxicity testing for not only pharmaceuticals but also for other chemical entities.

In addition, since the ban of animal testing in cosmetics in Europe, there has been a significant reduction in the number of registrations for new chemical approval [ 77 ]. Despite the impressive advancements in NAMs and their regulatory acceptance, there is still a heavy reliance on chemical data from previous animal tests, which may not allow the complete abandoning of the conservative paradigm. In this and similar situations, the recommended combined AISNAMs and IDBNAMs system could help to overcome this reliance on animal data.

Conclusions

Evolutional changes in the scientific community and in scientific developments are clearly reshaping and revolutionizing today’s ‘old’ toxicity testing for the better. Many examples in this paper demonstrate that great efforts had been made to realize the vision of more reliable, more rapid and less expensive approaches. Excellent technological and intellectual innovations are contributing to the elimination of existing gaps between toxicology and regulations.

However, at the time this paper has been written, the complete abandoning of traditional testing methods, for example in case of pharmaceuticals, is not possible. Probably, this is due to either the desired technology not existing yet or that it does exist but not in a ready-to-be-validated form. Despite the complete paradigm shift not a reality yet, it is highly possible, and seemingly it is the close future of toxicity testing.

The increasing application of NAMs at earlier phases of drug development, the complete shift of cosmetics towards NAMs and the continuous attempts of other areas to attain the desired toxicity-testing approaches are supporting the vision of both industries and RT. In this work, the authors have presented a new internationally shared scheme that combines a qualification/categorization and a database system. This approach may be applied to help further the transparency of NAMs and to support validation by regulatory bodies. A possible improvement of this approach consists of a complete global harmonization in all guidelines related to toxicity testing of not only new pharmaceutical but also other chemical entities. As a long-term effect, this novel scheme could eliminate those remaining gaps between research and regulations.

The author’s systematic review of the current structure and the techniques used has highlighted some areas that are changing and/or should be changed to allow the evolution of the regulatory toxicity testing that meets the needs of the 21st century.

Conflict of interest

There are no conflicts to declare.