First Sign Drug of Abuse Dip Card Test (MDMA, EDDP, Nortriptyline), First Sign Drug of Abuse Cup Test (MDMA, EDDP, Nortriptyline)

K160793 · W.H.P.M., Inc. · DJR · Aug 17, 2016 · Clinical Toxicology

Device Facts

• Record ID: K160793
• Device Name: First Sign Drug of Abuse Dip Card Test (MDMA, EDDP, Nortriptyline), First Sign Drug of Abuse Cup Test (MDMA, EDDP, Nortriptyline)
• Applicant: W.H.P.M., Inc.
• Product Code: DJR · Clinical Toxicology
• Decision Date: Aug 17, 2016
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 862.3620
• Device Class: Class 2

Intended Use

First Sign Drug of Abuse MDMA Cup Test First Sign Drug of Abuse MDMA Dip Card Test First Sign Drug of Abuse MDMA Tests are immunoassay tests. The test can detect MDMA in human urine. The cut-off value is 500 ng/mL.. The tests are available in a Cup format and a Dip Card format. The tests provide only preliminary test results. If you want to get a confirmed result, you must use a more specific chemical method. Gas Chromatography/Mass Spectrometry is the preferred confirmatory method. Clinical consideration and professional judgment should be used when you get any drug of abuse test result. It should be used particularly when the preliminary result is positive. For in vitro diagnostic use only. The tests are intended for over-the-counter and for prescription use. First Sign Drug of Abuse EDDP Cup Test First Sign Drug of Abuse EDDP Dip Card Test First Sign Drug of Abuse EDDP Tests are immunoassay tests. The test can detect EDDP in human urine. The cut-off value is 300 ng/mL. The tests are available in a Cup format and a Dip Card format. The tests provide only preliminary test results. If you want to get a confirmed result, you must use a more specific chemical method. Gas Chromatography/Mass Spectrometry is the preferred confirmatory method. Clinical consideration and professional judgment should be used when you get any drug of abuse test result. It should be used particularly when the preliminary result is positive. For in vitro diagnostic use only. The tests are intended for over-the-counter and for prescription use. First Sign Drug of Abuse Nortriptyline Cup Test First Sign Drug of Abuse Nortriptyline Dip Card Test First Sign Drug of Abuse Nortriptyline Tests are immunoassay tests. The test can detect Nortriptyline in human urine. The cut-off value is 1,000 ng/mL. The tests are available in a Cup format and a Dip Card format. The tests provide only preliminary test results. If you want to get a confirmed result, you must use a more specific chemical method. Gas Chromatography/Mass Spectrometry is the preferred confirmatory method. Clinical consideration and professional judgment should be used when you get any drug of abuse test result. It should be used particularly when the preliminary result is positive. For in vitro diagnostic use only. The tests are intended for over-the-counter and for prescription use.

Device Story

Lateral flow immunochromatographic assays for qualitative detection of MDMA, EDDP, or Nortriptyline in human urine; available in cup or dip card formats. Principle: competitive binding; target drug in urine competes with drug-conjugate for limited antibody binding sites on particles; absence of test line indicates positive result (drug concentration above cut-off); presence of test line indicates negative result. Control line confirms proper flow. Used in point-of-care or home settings by lay users or clinicians. Provides preliminary results; requires professional clinical judgment and confirmatory testing (GC/MS) for positive results. Benefits include rapid, accessible drug screening.

Clinical Evidence

No clinical trials; performance established via analytical bench testing (precision, specificity, interference, stability) and a lay-user study (n=280 per analyte). Lay-user study confirmed correct results across various concentrations relative to cut-offs. Precision studies performed over 25 days confirmed consistency across lots and formats.

Technological Characteristics

Lateral flow immunochromatographic assay; competitive binding principle; monoclonal mouse antibody; cup or dip card form factor; single-use; qualitative output; visual interpretation. Stable at 4-30°C for 24 months.

Indications for Use

Indicated for qualitative detection of MDMA (500 ng/mL), EDDP (300 ng/mL), or Nortriptyline (1,000 ng/mL) in human urine. Intended for over-the-counter and prescription use as a preliminary screening test. Requires confirmatory testing via GC/MS.

Regulatory Classification

Identification

A methadone test system is a device intended to measure methadone, an addictive narcotic pain-relieving drug, in serum and urine. Measurements obtained by this device are used in the diagnosis and treatment of methadone use or overdose and to determine compliance with regulations in methadone maintenance treatment.

Special Controls

*Classification.* Class II (special controls). A methadone test system is not exempt if it is intended for any use other than employment or insurance testing or is intended for Federal drug testing programs. The device is exempt from the premarket notification procedures in subpart E of part 807 of this chapter subject to the limitations in § 862.9, provided the test system is intended for employment and insurance testing and includes a statement in the labeling that the device is intended solely for use in employment and insurance testing, and does not include devices intended for Federal drug testing programs (*e.g.,* programs run by the Substance Abuse and Mental Health Services Administration (SAMHSA), the Department of Transportation (DOT), and the U.S. military).

Predicate Devices

• Co-Innovation Rapid Single/Multi Drug Test (K142800)
• Co-Innovation One Step Single/Multi Drug Test (K140748)

Submission Summary (Full Text)

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Image /page/0/Picture/1 description: The image shows the logo for the U.S. Department of Health & Human Services. The logo consists of a circular seal with the text "DEPARTMENT OF HEALTH & HUMAN SERVICES - USA" around the perimeter. Inside the circle is a stylized image of three human profiles facing to the right, with flowing lines representing hair or movement.

Auqust 17,2016

Food and Drug Administration 10903 New Hampshire Avenue Document Control Center - WO66-G609 Silver Spring, MD 20993-0002

W.H.P.M., INC. C/O JOE SHIA LSI CONSULTING 504 EAST DIAMOND AVE., SUITE I GAITHERSBURG MD 20877

Re: k160793

Trade/Device Name: First Sign Drug of Abuse MDMA Cup Test: First Sign Drug of Abuse MDMA Dip Card Test; First Sign Drug of Abuse EDDP Cup Test; First Sign Drug of Abuse EDDP Dip Card Test; First Sign Drug of Abuse Nortriptyline Cup Test; First Sign Drug of Abuse Nortriptyline Dip Card Test Regulation Number: 21 CFR 862.3620 Regulation Name: Methadone test system Regulatory Class: II Product Code: DJR, LAF, LFG Dated: June 29, 2016 Received: July 7, 2016

Dear Joe Shia:

We have reviewed your Section 510(k) premarket notification of intent to market the device referenced above and have determined the device is substantially equivalent (for the indications for use stated in the enclosure) to legally marketed predicate devices marketed in interstate commerce prior to May 28, 1976, the enactment date of the Medical Device Amendments, or to devices that have been reclassified in accordance with the provisions of the Federal Food. Drug, and Cosmetic Act (Act) that do not require approval of a premarket approval application (PMA). You may, therefore, market the device, subject to the general controls provisions of the Act. The general controls provisions of the Act include requirements for annual registration, listing of devices, good manufacturing practice, labeling, and prohibitions against misbranding and adulteration. Please note: CDRH does not evaluate information related to contract liability warranties. We remind you, however, that device labeling must be truthful and not misleading.

If your device is classified (see above) into either class II (Special Controls) or class III (PMA), it may be subject to additional controls. Existing major regulations affecting your device can be found in the Code of Federal Regulations, Title 21, Parts 800 to 898. In addition, FDA may publish further announcements concerning your device in the Federal Register.

Please be advised that FDA's issuance of a substantial equivalence determination does not mean that FDA has made a determination that your device complies with other requirements of the Act or any Federal statutes and regulations administered by other Federal agencies. You must comply with all the Act's requirements, including, but not limited to: registration and listing (21 CFR Part 807); labeling (21 CFR Parts 801 and 809); medical device reporting of medical device-related adverse events) (21 CFR 803); good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820); and if applicable, the

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electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR 1000-1050.

If you desire specific advice for your device on our labeling regulations (21 CFR Parts 801 and 809), please contact the Division of Industry and Consumer Education at its toll-free number [phone] or [phone] or at its Internet address

http://www.fda.gov/MedicalDevices/Resourcesfor You/Industry/default.htm. Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21 CFR Part 807.97). For questions regarding the reporting of adverse events under the MDR regulation (21 CFR Part 803), please go to

http://www.fda.gov/MedicalDevices/Safety/ReportaProblem/default.htm for the CDRH's Office of Surveillance and Biometrics/Division of Postmarket Surveillance.

You may obtain other general information on your responsibilities under the Act from the Division of Industry and Consumer Education at its toll-free number [phone] or [phone] or at its Internet address

http://www.fda.gov/MedicalDevices/ResourcesforYou/Industry/default.htm.

Sincerely yours,

Katherine Serrano -S

For: Courtney H. Lias, Ph.D.

Director Division of Chemistry and Toxicology Devices Office of In Vitro Diagnostics and Radiological Health Center for Devices and Radiological Health

Enclosure

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## Indications for Use

510(k) Number (if known) k160793

#### Device Name

First Sign Drug of Abuse MDMA Cup Test; First Sign Drug of Abuse MDMA Dip Card Test First Sign Drug of Abuse EDDP Cup Test; First Sign Drug of Abuse EDDP Dip Card Test First Sign Drug of Abuse Nortriptyline Cup Test; First Sign Drug of Abuse Nortriptyline Dip Card Test

Indications for Use (Describe)

First Sign Drug of Abuse MDMA Cup Test

First Sign Drug of Abuse MDMA Dip Card Test

First Sign Drug of Abuse MDMA Tests are immunoassay tests. The test can detect MDMA in human urine. The cut-off value is 500 ng/mL.. The tests are available in a Cup format and a Dip Card format.

The tests provide only preliminary test results. If you want to get a confirmed result, you must use a more specific chemical method. Gas Chromatography/Mass Spectrometry is the preferred confirmatory method. Clinical consideration and professional judgment should be used when you get any drug of abuse test result. It should be used particularly when the preliminary result is positive.

For in vitro diagnostic use only. The tests are intended for over-the-counter and for prescription use.

First Sign Drug of Abuse EDDP Cup Test First Sign Drug of Abuse EDDP Dip Card Test First Sign Drug of Abuse EDDP Tests are immunoassay tests. The test can detect EDDP in human urine. The cut-off value is 300 ng/mL. The tests are available in a Cup format and a Dip Card format.

The tests provide only preliminary test results. If you want to get a confirmed result, you must use a more specific chemical method. Gas Chromatography/Mass Spectrometry is the preferred confirmatory method. Clinical consideration and professional judgment should be used when you get any drug of abuse test result. It should be used particularly when the preliminary result is positive.

For in vitro diagnostic use only. The tests are intended for over-the-counter and for prescription use.

First Sign Drug of Abuse Nortriptyline Cup Test

First Sign Drug of Abuse Nortriptyline Dip Card Test

First Sign Drug of Abuse Nortriptyline Tests are immunoassay tests. The test can detect Nortriptyline in human urine. The cut-off value is 1,000 ng/mL. The tests are available in a Cup format and a Dip Card format.

The tests provide only preliminary test results. If you want to get a confirmed result, you must use a more specific chemical method. Gas Chromatography/Mass Spectrometry is the preferred confirmatory method. Clinical consideration and professional judgment should be used when you get any drug of abuse test result. It should be used particularly when the preliminary result is positive.

For in vitro diagnostic use only. The tests are intended for over-the-counter and for prescription use.

Type of Use (Select one or both, as applicable)

X Prescription Use (Part 21 CFR 801 Subpart D)

Z Over-The-Counter Use (21 CFR 801 Subpart C)

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- 1. Date: March 15, 2016
- 2. Submitter W.H.P.M., Inc. 5358 Irwindale Ave. Irwindale, CA 91706
- 3. Contact person: John Wan W.H.P.M., Inc. 5358 Irwindale Ave. Irwindale, CA 91706 Telephone: [phone] Fax: [phone] Email: [e-mail]
- 4. Device Name: First Sign® Drug of Abuse MDMA Cup Test First Sign® Drug of Abuse MDMA Dip Card Test First Sign® Drug of Abuse EDDP Cup Test First Sign® Drug of Abuse EDDP Dip Card Test First Sign® Drug of Abuse Nortriptyline Cup Test First Sign® Drug of Abuse Nortriptyline Dip Card Test
- MDMA Urine Test Common Name: EDDP Urine Test Nortriptyline Urine Test

| Product Code | Class | CFR # | Panel |
|--------------|----------|----------------------------------------------------------------|------------|
| LAF | Class II | 21 CFR, 862.3610 Methamphetamine Test System | Toxicology |
| DJR | Class II | 21 CFR, 862.3620 Methadone Test System | Toxicology |
| LFG | Class II | 21 CFR, 862.3910 Tricyclic Antidepressant Drugs<br>Test System | Toxicology |

- 5. Predicate Devices:
K142800, Co-Innovation Rapid Single/Multi Drug Test K140748, Co-Innovation One Step Single/Multi Drug Test

- 6. Intended Use First Sign® Drug of Abuse MDMA Cup Test First Sign® Drug of Abuse MDMA Dip Card Test

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First Sign® Drug of Abuse MDMA Tests are immunoassay tests. The test can detect MDMA in human urine. The cut-off value is 500 ng/mL. The tests are available in a Cup format and a Dip Card format.

The tests provide only preliminary test results. If you want to get a confirmed result, you must use a more specific chemical method. Gas Chromatography/Mass Spectrometry is the preferred confirmatory method. Clinical consideration and professional judgment should be used when you get any drug of abuse test result. It should be used particularly when the preliminary result is positive. For in vitro diagnostic use only. The tests are intended for over-the-counter and for prescription use.

First Sign® Drug of Abuse EDDP Cup Test First Sign® Drug of Abuse EDDP Dip Card Test First Sign® Drug of Abuse EDDP Tests are immunoassay tests. The test can detect EDDP in human urine. The cut-off value is 300 ng/mL. The tests are available in a Cup format and a Dip Card format.

The tests provide only preliminary test results. If you want to get a confirmed result, you must use a more specific chemical method. Gas Chromatography/Mass Spectrometry is the preferred confirmatory method. Clinical consideration and professional judgment should be used when you get any drug of abuse test result. It should be used particularly when the preliminary result is positive. For in vitro diagnostic use only. The tests are intended for over-the-counter and for prescription use.

First Sign® Drug of Abuse Nortriptyline Cup Test

First Sign® Drug of Abuse Nortriptyline Dip Card Test

First Sign® Drug of Abuse Nortriptyline Tests are immunoassay tests. The test can detect Nortriptyline in human urine. The cut-off value is 1,000 ng/mL. The tests are available in a Cup format and a Dip Card format.

The tests provide only preliminary test results. If you want to get a confirmed result, you must use a more specific chemical method. Gas Chromatography/Mass Spectrometry is the preferred confirmatory method. Clinical consideration and professional judgment should be used when you get any drug of abuse test result. It should be used particularly when the preliminary result is positive. For in vitro diagnostic use only. The tests are intended for over-the-counter and for prescription use.

## 7. Device Description

First Sign™ Drug of Abuse MDMA Test, First Sign™ Drug of Abuse EDDP Test and First Sign™ Drug of Abuse Nortriptyline Test are immunochromatographic assays. Each assay test is a lateral flow system for the qualitative detection of MDMA, or EDDP or Nortriptyline in human urine. The products are single-use in vitro diagnostic devices, which come in the formats of Dip Cards or Cups. Each test kit contains a Test Device (in one of the two formats), a package insert and a urine cup for sample collection. Each test device is sealed with a desiccant in an aluminum pouch.

## 8. Substantial Equivalence Information

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A summary comparison of features of the candidate device and the predicate device is provided in Table 1, Table 2 & Table 3.

| Table 1: Features Comparison of First Sign® Drug of Abuse MDMA Test and the Predicate | |
|---------------------------------------------------------------------------------------|--|
| Device | |

| Item | Candidate Device<br>First Sign® Drug of Abuse MDMA Test | Predicate Device<br>(k142800)<br>Co-Innovation<br>Multi Drug Test |
|--------------------------|-------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------|
| Indication(s)<br>for Use | For the qualitative determination of<br>MDMA in human urine. | Same |
| Calibrator | MDMA | Same |
| Methodology | Competitive binding, lateral flow<br>immunochromatographic assays based on<br>the principle of antigen antibody<br>immunochemistry. | Same |
| Specimen Type | Human urine | Same |
| Cut-Off Values | 500 ng/mL | Same |
| Intended<br>Population | For over-the-counter and prescription<br>uses. | Same |
| Configurations | Cup, Dip Card | Same |

| Table 2: Features Comparison of First Sign® Drug of Abuse EDDP Test and the Predicate | | |
|---------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------|
| Device | | |
| Item | Candidate Device<br>First Sign® Drug of Abuse EDDP Test | Predicate Device<br>(k140748)<br>Co-Innovation<br>Multi Drug Test |
| Indication(s)<br>for Use | For the qualitative determination of<br>EDDP in human urine. | Same |
| Calibrator | EDDP | Same |
| Methodology | Competitive binding, lateral flow<br>immunochromatographic assays based on<br>the principle of antigen antibody<br>immunochemistry. | Same |
| Specimen Type | Human urine | Same |
| Cut-Off Values | 300 ng/mL | Same |
| Intended<br>Population | For over-the-counter and prescription<br>uses. | Same |
| Configurations | Cup, Dip Card | Same |

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## Table 3: Features Comparison of First Sign® Drug of Abuse Nortriptyline Test and the Predicate Device

| Item | Candidate Device<br>First Sign® Drug of Abuse Nortriptyline<br>Test | Predicate Device<br>(k140748)<br>Co-Innovation<br>Multi Drug Test |
|--------------------------|-------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------|
| Indication(s)<br>for Use | For the qualitative determination of<br>Nortriptyline in human urine. | Same |
| Calibrator | Nortriptyline | Same |
| Methodology | Competitive binding, lateral flow<br>immunochromatographic assays based on<br>the principle of antigen antibody<br>immunochemistry. | Same |
| Specimen Type | Human Urine | Same |
| Cut-Off Values | 1000 ng/mL | Same |
| Intended<br>Population | For over-the-counter and prescription<br>uses. | Same |
| Configurations | Cup, Dip Card | Same |

## 9. Test Principle

Each assay test is a lateral flow chromatographic immunoassay. During testing, a urine specimen migrates upward by capillary action. If target drugs are present in the urine specimen below its cut-off concentration, it will not saturate the binding sites of its specific antibody (monoclonal mouse antibody) coated on the particles. The antibody-coated particles will then be captured by immobilized drugconjugate and a visible colored line will show up in the test line region. The will not form in the test line region if the target drug level exceeds its cut-off concentration because it will saturate all the binding sites of the antibody coated on the particles. A band should form in the control region of the devices regardless of the presence of drug or metabolite in the sample.

## 10. Performance Characteristics

## First Sign® Drug of Abuse MDMA Test

Analytical Performance

## a. Precision

Precision studies were carried out for samples with concentrations of -100% cut-off, -50% cut-off, -25% cut-off, at the cut-off, +25% cut-off, +75% cut-off, +75% cut-off and +100% cut-off. These samples were prepared by spiking drug in negative urine samples. Each drug concentration was confirmed by GC/MS. All sample aliquots were blind-labeled and randomized

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by the person who prepared samples and did not take part in the sample testing. For each concentration, tests were performed two runs per day for 25 days by three different operators for each format of devices. Different set of operators tested each format. The results obtained are summarized in the following tables:

| | Result | -100%<br>Cut-off | -75%<br>Cut-off | -50%<br>Cut-off | -25%<br>Cut-off | Cut-off | +25%<br>Cut-off | +50%<br>Cut-off | +75%<br>Cut-off | +100%<br>Cut-off |
|------|--------|------------------|-----------------|-----------------|-----------------|---------|-----------------|-----------------|-----------------|------------------|
| Drug | Lot 1 | 50-/0+ | 50-/0+ | 50-/0+ | 50-/0+ | 2-/48+ | 50+/0- | 50+/0- | 50+/0- | 50+/0- |
| Drug | Lot 2 | 50-/0+ | 50-/0+ | 50-/0+ | 50-/0+ | 3-/47+ | 50+/0- | 50+/0- | 50+/0- | 50+/0- |
| Drug | Lot 3 | 50-/0+ | 50-/0+ | 50-/0+ | 50-/0+ | 3-/47+ | 50+/0- | 50+/0- | 50+/0- | 50+/0- |

50-/0+

50-/0+

50-/0+

3-/47+

2-/48+

2-/48+

50+/0-

50+/0-

50+/0-

50+/0-

50+/0-

50+/0-

50+/0-

50+/0-

50+/0-

50+/0-

50+/0-

50+/0-

## MDMA Dip Card Format

# b. Linearity

Not applicable.

Lot 4

Lot 5

Lot 6

50-/0+

50-/0+

50-/0+

50-/0+

50-/0+

50-/0+

50-/0+

50-/0+

50-/0+

## c. Stability

The devices are stable at 39-86ºF (4-30ºC) for 24 months based on the accelerated stability study at 50°C. Control materials are not provided with the device. The labeling provides information on how to obtain control materials.

## d. Cut-off

A total of 150 samples equally distributed at concentrations of -50% cut-off; cutoff; +25% cut-off; +50% cut-off were tested using three different lots of each device by three different operators. Results were all positive at and above +25% cut-off and all negative at and below -25% cut-off for MDMA. The following cut-off value for the test devices have been verified.

| Test | Calibrator | Cut-off (ng/mL) |
|-------------------------------------|------------|-----------------|
| First Sign® Drug of Abuse MDMA Test | MDMA | 500 |

## e. Interference

Potential interfering substances found in human urine of physiological conditions were added to drug-free urine and to urine containing target drugs at 25% below and 25% above cut-off levels. These urine samples were tested using three lots of each device for all formats.

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Compounds that showed no interference at a concentration of 100µg/mL are summarized in the following tables. There were no differences observed for different formats.

| 4-Acetamidophenol | (L) - Epinephrine | Pentobarbital |
|-------------------------|---------------------------|----------------------------------------------|
| Acetophenetidin | Erythromycin | Perphenazine |
| N-Acetylprocainamide | β-Estradiol | Phencyclidine |
| Acetylsalicylic acid | Estrone-3-sulfate | Phenelzine |
| Aminopyrine | Ethyl-p-aminobenzoate | Phenobarbital |
| Amitryptyline | Fenoprofen | Phentermine |
| Amobarbital | Furosemide | Trans-2-phenylcyclopropylamine hydrochloride |
| Amoxicillin | Gentisic acid | L-Phenylephrine |
| Ampicillin | Hemoglobin | β-Phenylethylamine |
| L-Ascorbic acid | Hydralazine | Phenylpropanolamine |
| Apomorphine | Hydrochlorothiazide | Prednisolone |
| Aspartame | Hydrocodone | Prednisone |
| Atropine | Hydrocortisone | Procaine |
| Benzilic acid | O-Hydroxyhippuric acid | Promazine |
| Benzoic acid | 3-Hydroxytyramine | Promethazine |
| Benzoylecgonine | Ibuprofen | DL-Propranolol |
| Bilirubin | Imipramine | D-Propoxyphene |
| (±) - Brompheniramine | Iproniazid | D-Pseudoephedrine |
| Buspiron | (±) - Isoproterenol | Quinacrine |
| Caffeine | Isoxsuprine | Quinidine |
| Cannabidiol | Ketamine | Quinine |
| Cannabinol | Ketoprofen | Ranitidine |
| Chloralhydrate | Labetalol | Salicylic acid |
| Chloramphenicol | Levorphanol | Secobarbital |
| Chlordiazepoxide | Loperamide | Serotonin (5- Hydroxytyramine) |
| Chlorothiazide | Maprotiline | Sulfamethazine |
| (±) - Chlorpheniramine | Meperidine | Sulindac |
| Chlorpromazine | Meprobamate | Sustiva |
| Chloroquine | Methadone | Temazepam |
| Cholesterol | Morphine-3-β-Dglucuronide | Tetracycline |
| Clomipramine | Morphine sulfate | Tetrahydrocortisone 3-(β-Dglucuronide) |
| Clonidine | Nalidixic acid | Tetrahydrozoline |
| Cocaethylene | Naloxone | Thebaine |
| Cocaine hydrochloride | Naltrexone | Theophynine |
| Codeine | Naproxen | Thiamine |
| Cortisone | Niacinamide | Thioridazine |
| (-) Cotinine | Nifedipine | Tolbutamide |
| Creatinine | Nimesulidate | Trazodone |
| Deoxycorticosterone | Norcodein | Triamterene |
| Dextromethorphan | Norethindrone | DL-Tyrosine |
| Diclofenac | D-Norpropoxyphene | Trifluoperazine |
| Diazepam | Noscapine | Trimethoprim |
| Diflunisal | D,L-Octopamine | Trimipramine |
| Digoxin | Oxalic acid | Tryptamine |
| Dicylomine | Oxazepam | D L-Tryptophan |
| Diphenhydramine | Oxolinic acid | Tyramine |
| 5,5 - Diphenylhydantoin | Oxycodone | Uric acid |
| Doxylamine | Oxymetazoline | Verapamil |
| Ecgonine hydrochloride | Papaverine | Zomepirac |
| Ecgonine methylester | Penicillin-G | |
| [1R,2S](-) Ephedrine | Pentazocinehydrochloride | |

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#### f. Specificity

To test the specificity, drug metabolites and other components that are likely to interfere in urine samples were tested using three lots of each device for all formats. The obtained lowest detectable concentration was used to calculate the cross-reactivity. There were no differences observed for different formats.

| Drug | Concentration (ng/ml) | % Cross-Reactivity |
|-------------------------------------------|-----------------------|--------------------|
| Methylenedioxymethamphetamine (MDMA) | 500 | 100% |
| 3,4-Methylenedioxyamphetamine (MDA) | 8000 | 6.3% |
| 3,4-Methylenedioxyethylamphetamine (MDEA) | 1000 | 50% |
| Ephedrine | 40000 | 1.3% |
| d-methamphetamine | Negative at 100000 | Not Detected |
| d-amphetamine | Negative at 100000 | Not Detected |
| l-amphetamine | Negative at 100000 | Not Detected |
| l-methamphetamine | Negative at 100000 | Not Detected |

## g. Effect of Urine Specific Gravity and Urine pH

To investigate the effect of urine specific gravity and urine pH, urine samples with a range of 1.000 to 1.035 specific gravity or urine samples with a range of pH 4 to 9 were spiked with target drugs at 25% below and 25% above cut-off levels. These samples were tested using three lots of each

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device for all formats. Results were all positive for samples at and above +25% Cut-Off and all negative for samples at and below -25% Cut-Off. There were no differences observed for different formats.

- h. Comparison Studies
The method comparison studies for the First Sign® Drug of Abuse MDMA Test was performed in-house with three different laboratory assistants for each format of the device. Operators ran 80 (40 negative and 40 positive) unaltered clinical samples were blind labeled and compared to GC/MS results. The results are presented in the tables below:

| Dip Card<br>format | | Negative | Low<br>Negative<br>by GC/MS<br>(less than -<br>50%) | Near Cutoff<br>Negative by<br>GC/MS<br>(Between -<br>50% and<br>cut-off) | Near<br>Cutoff<br>Positive by<br>GC/MS<br>(Between<br>the cut-off<br>and +50%) | High<br>Positive by<br>GC/MS<br>(greater<br>than +50%) |
|--------------------|----------|----------|-----------------------------------------------------|--------------------------------------------------------------------------|--------------------------------------------------------------------------------|--------------------------------------------------------|
| Viewer A | Positive | 0 | 0 | 0 | 13 | 26 |
| | Negative | 10 | 10 | 20 | 1 | 0 |
| Viewer B | Positive | 0 | 0 | 1 | 14 | 26 |
| | Negative | 10 | 10 | 19 | 0 | 0 |
| Viewer C | Positive | 0 | 0 | 1 | 13 | 26 |
| | Negative | 10 | 10 | 19 | 1 | 0 |

## Discordant Results

| Viewer | Sample Number | GC/MS (ng/mL) Result | Dipcard Format Viewer Results |
|----------|---------------|----------------------|-------------------------------|
| Viewer A | 2014111947 | 561 | Negative |
| Viewer B | 2014102405 | 468 | Positive |
| Viewer C | 2014111901 | 474 | Positive |
| Viewer C | 2014102302 | 544 | Negative |

| Cup<br>format | | Negative | Low<br>Negative<br>by GC/MS<br>(less than -<br>50%) | Near Cutoff<br>Negative by<br>GC/MS<br>(Between -<br>50% and<br>cut-off) | Near<br>Cutoff<br>Positive by<br>GC/MS<br>(Between<br>the cut-off<br>and +50%) | High<br>Positive by<br>GC/MS<br>(greater<br>than +50%) |
|---------------|----------|----------|-----------------------------------------------------|--------------------------------------------------------------------------|--------------------------------------------------------------------------------|--------------------------------------------------------|
| Viewer A | Positive | 0 | 0 | 1 | 14 | 26 |
| | Negative | 10 | 10 | 19 | 0 | 0 |
| Viewer B | Positive | 0 | 0 | 0 | 13 | 26 |
| | Negative | 10 | 10 | 20 | 1 | 0 |

{12}------------------------------------------------

| Viewerし | Positive | | | C<br>60 |
|---------|----------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----|---------|
| | Negative | 1 C<br>ﻟﺘﻌﻠﻴﻘﺎﺕ ﺍﻟﻤﺘﺤﺪﺓ ﺍﻟﻤﺘﺤﺪﺓ ﺍﻟﻤﺘﺤﺪﺓ ﺍﻟﻤﺘﺤﺪﺓ ﺍﻟﻤﺘﺤﺪﺓ ﺍﻟﻤﺘﺤﺪﺓ ﺍﻟﻤﺘﺤﺪﺓ ﺍﻟﻤﺘﺤﺪﺓ ﺍﻟﻤﺘﺤﺪﺓ ﺍﻟﻤﺘﺤﺪﺓ ﺍﻟﻤﺘﺤﺪﺓ ﺍﻟﻤﺘﺤﺪﺓ ﺍﻟﻤﺘﺤﺪﺓ ﺍﻟﻤﺘﺤﺪﺓ ﺍﻟﻤﺘﺤﺪﺓ ﺍﻟﻤﺘﺤﺪﺓ ﺍﻟﻤﺘﺤﺪﺓ ﺍﻟﻤﺘﺤﺪﺓ ﺍﻟﻤﺘﺤﺪﺓ ﺍﻟﻤﺘﺤﺪﺓ ﺍﻟﻤﺘ | 1 C | |

Discordant Results

| Viewer | Sample Number | GC/MS (ng/mL)<br>Result | Cup Format<br>Viewer Results |
|----------|---------------|-------------------------|------------------------------|
| Viewer A | 2014102306 | 477 | Positive |
| Viewer B | 2014111950 | 517 | Negative |
| Viewer C | 2014102315 | 470 | Positive |

#### i. Lay-user study

A lay user study was performed at three intended user sites with 280 lay persons testing the MDMA devices. They had diverse educational and professional backgrounds and ranged in age from 21 to > 50 years. Urine samples were prepared at the following concentrations; negative, +/-75%, +/-50%, +/-25% of the cutoff by spiking drugs into drug free-pooled urine specimens. The concentrations of the samples were confirmed by GC/MS. Each sample was aliquoted into individual containers and blind-labeled. Each participant was provided with the package insert, 1 blind labeled sample and a device. The results are summarized below.

| % of Cutoff | Number of<br>samples | MDMA Concentration by<br>GC/MS<br>(ng/mL) | Lay person results<br>No. of<br>Positive | Lay person results<br>No. of<br>Negative | The<br>percentage of<br>correct results<br>(%) |
|--------------|----------------------|-------------------------------------------|------------------------------------------|------------------------------------------|------------------------------------------------|
| -100% Cutoff | 20 | 0 | 0 | 20 | 100% |
| -75% Cutoff | 20 | 115 | 0 | 20 | 100% |
| -50% Cutoff | 20 | 237 | 0 | 20 | 100% |
| -25% Cutoff | 20 | 358 | 0 | 20 | 100% |
| +25% Cutoff | 20 | 598 | 19 | 1 | 95% |
| +50% Cutoff | 20 | 755 | 20 | 0 | 100% |
| +75% Cutoff | 20 | 912 | 20 | 0 | 100% |

Comparison between GC/MS and Lay Person Results (MDMA DipCard)

Comparison between GC/MS and Lay Person Results (MDMA Cup)

| | Number | MDMA Concentration by | Lay person results | | The |
|--------------|---------------|-----------------------|--------------------|--------------------|-----------------------------------------|
| % of Cutoff | of<br>samples | GC/MS<br>(ng/mL) | No. of<br>Positive | No. of<br>Negative | percentage of<br>correct results<br>(%) |
| -100% Cutoff | 20 | 0 | 0 | 20 | 100% |
| -75% Cutoff | 20 | 115 | 0 | 20 | 100% |
| -50% Cutoff | 20 | 237 | 0 | 20 | 100% |
| -25% Cutoff | 20 | 358 | 1 | 19 | 95% |
| +25% Cutoff | 20 | 598 | 20 | 0 | 100% |
| +50% Cutoff | 20 | 755 | 20 | 0 | 100% |
| +75% Cutoff | 20 | 912 | 20 | 0 | 100% |

{13}------------------------------------------------

Lay-users were also given surveys on the ease of understanding the package insert instructions. All lay users indicated that the device instructions can be easily followed. A Flesch-Kincaid reading analysis was performed on each package insert and the scores revealed a reading Grade Level of 7.

- j. Clinical Studies
Not applicable.

## First Sign® Drug of Abuse EDDP Test

Analytical Performance

- a. Precision
Precision studies were carried out for samples with concentrations of -100% cut-off, -50% cut-off, -25% cut-off, at the cut-off, +25% cut-off, +75% cut-off, +75% cut-off and +100% cut-off. These samples were prepared by spiking drug in negative urine samples. Each drug concentration was confirmed by GC/MS. All sample aliquots were blind-labeled and randomized by the person who prepared samples and did not take part in the sample testing. For each concentration, tests were performed two runs per day for 25 days by three different operators for each format of devices. Different set of operators tested each format. The results obtained are summarized in the following tables:

| Result | -100%<br>Cut-off | -75%<br>Cut-off | -50%<br>Cut-off | -25%<br>Cut-off | Cut-off | +25%<br>Cut-off | +50%<br>Cut-off | +75%<br>Cut-off | +100%<br>Cut-off |
|--------|------------------|-----------------|-----------------|-----------------|---------|-----------------|-----------------|-----------------|------------------|
| Drug | | | | | | | | | |
| Lot 1 | 50-/0+ | 50-/0+ | 50-/0+ | 50-/0+ | 3-/47+ | 50+/0- | 50+/0- | 50+/0- | 50+/0- |
| Lot 2 | 50-/0+ | 50-/0+ | 50-/0+ | 50-/0+ | 2-/48+ | 50+/0- | 50+/0- | 50+/0- | 50+/0- |
| Lot 3 | 50-/0+ | 50-/0+ | 50-/0+ | 50-/0+ | 2-/48+ | 50+/0- | 50+/0- | 50+/0- | 50+/0- |

# EDDP Dip Card Format

- b. Linearity
Not applicable.

- c. Stability

{14}------------------------------------------------

The devices are stable at 39-86ºF (4-30ºC) for 24 months based on the accelerated stability study at 50°C. Control materials are not provided with the device. The labeling provides information on how to obtain control materials.

- d. Cut-off
A total of 150 samples equally distributed at concentrations of -50% cut-off; cutoff; +25% cut-off; +50% cut-off were tested using three different lots of each device by three different operators. Results were all positive at and above +25% cut-off and all negative at and below -25% cut-off for EDDP. The following cut-off value for the test devices have been verified.

| Test | Calibrator | Cut-off (ng/mL) |
|----------------------------------------|------------|-----------------|
| First Sign® Drug of Abuse EDDP<br>Test | EDDP | 300 |

- e. Interference
Potential interfering substances found in human urine of physiological or pathological conditions were added to drug-free urine and to urine containing target drugs at 25% below and 25% above cut-off levels. These urine samples were tested using three lots of each device for all formats.

Compounds that showed no interference at a concentration of 100µg/mL are summarized in the following tables. There were no differences observed for different formats.

| Acetaminophen | Ecgonine hydrochloride | O-Hydroxyhippuric acid |
|------------------------|--------------------------|------------------------|
| Acetophenetidin | Ecgonine methylester | Oxalic acid |
| Acetylsalicylic acid | (IR,2S)(-)Ephedrine | Oxazepam |
| Amobarbital | Erythromycin | Oxolinic acid |
| Aminopyrine | β-Estradiol | Oxycodone |
| Amitryptyline | Estrone-3-sulfate | Oxymetazoline |
| Amoxicillin | Ethyl-p-aminobenzoate | Papaverine |
| DL-Amphetamine sulfate | Fenoprofen | Penicillin-G |
| Ampicillin | Furosemide | Pentazocine |
| Apomorphine | Gentisic acid | Pentobarbital |
| Ascorbic acid | Hemoglobin | Perphenazine |
| Aspartame | Hydralazine | Phencyclidine |
| Atropine | Hydrochlorothiazide | Phenelzine |
| Benzilic acid | Hydrocodone | Phenobarbital |
| Benzoic acid | Hydrocortisone | Phentermine |
| Benzoylecgonine | p-Hydroxyamphetamine | β-Phenylethylamine |
| Bilirubin | p-Hydroxymethamphetamine | Phenylpropanolamine |
| Brompheniramine | 3-Hydroxytyramine | Prednisolone |

{15}------------------------------------------------

| Caffeine | Ibuprofen | Prednisone |
|------------------------|----------------------------------------------------------|------------------------------------------|
| Cannabidiol | Imipramine | Procaine |
| Cannabinol | (-) Isoproterenol | Promazine |
| Chloralhydrate | Isoxsuprine | Promethazine |
| Chloramphenicol | Ketamine | Quinidine |
| Chlorothiazide | Ketoprofen | Quinine |
| (±) - Chlorpheniramine | Labetalol | Ranitidine |
| Chlorpromazine | Levorphanol | Salicylic acid |
| Chloroquine | Loperamide | Secobarbital |
| Cholesterol | L-Phenylephrine | Serotonin |
| Clomipramine | Maprotiline | Sulfamethazine |
| Clonidine | Meperidine | Sulindac |
| Cocaine hydrochloride | Meprobamate | Temazepam |
| Codeine | Methamphetamine | Tetracycline |
| (-) Cotinine | Methoxyphenamine | Tetrahydrocortisone 3- (β-D-glucuronide) |
| Cortisone | (±) - 3,4-Methylenedioxy-<br>amphetamine hydrochloride | Tetrahydrozoline |
| Creatinine | (±)-3,4-Methylenedioxy-<br>methamphetamine hydrochloride | Thebaine |
| Deoxycorticosterone | Morphine Sulfate | Thiamine |
| Dextromethorphan | Morphine-3-β-D glucuronide | Thioridazine |
| Diazepam | N-Acetylprocainamide | Triamterene |
| Diclofenac | Nalidixic acid | Trifluoperazine |
| Diflunisal | Naloxone | Trimethoprim |
| Digoxin | Naltrexone | Trimipramine |
| Diphenhydramine | Naproxen | Tryptamine |
| D-Norpropoxyphene | Niacinamide | DL-Tryptophan |
| D-Propoxyphene | Nifedipine | Tyramine |
| D,L-Tyrosine | Norcodein | Uric acid |
| DL-Octopamine | Norethindrone | Verapamil |
| DL-Propranolol | Noscapine | Zomepirac |

## f. Specificity

To test the specificity, drug metabolites and other components that are likely to interfere in urine samples were tested using three lots of each device for all formats. The obtained lowest detectable concentration was used to calculate the cross-reactivity. There were no differences observed for different formats.

| Drug | Concentration (ng/ml) | % Cross-Reactivity |
|------|-----------------------|--------------------|
|------|-----------------------|--------------------|

{16}------------------------------------------------

| EDDP (2-ethylidene-1,5-dimethyl-3,3-<br>diphenylpyrrolidine) | 300 | 100% |
|--------------------------------------------------------------|--------------------|--------------|
| EMDP (2-Ethyl-5-methyl-3,3-<br>diphenylpyrroline) | Negative at 100000 | Not Detected |
| Disopyramide | Negative at 100000 | Not Detected |
| Methadone | Negative at 100000 | Not Detected |
| LAAM (Levo-alpha-acetylmethadol) | Negative at 100000 | Not Detected |
| Alpha Methadol | Negative at 100000 | Not Detected |
| Doxylamine | Negative at 100000 | Not Detected |

- g. Effect of Urine Specific Gravity and Urine pH
To investigate the effect of urine specific gravity and urine pH, urine samples with a range of 1.000 to 1.035 specific gravity or urine samples with a range of pH 4 to 9 were spiked with target drugs at 25% below and 25% above cut-off levels. These samples were tested using three lots of each device for all formats. Results were all positive for samples at and above +25% Cut-Off and all negative for samples at and below -25% Cut-Off. There were no differences observed for different formats.

- h. Comparison Studies
The method comparison studies for the First Sign® Drug of Abuse EDDP Test was performed in-house with three different laboratory assistants for each format of the device. Operators ran 80 (40 negative and 40 positive) unaltered clinical samples were blind labeled and compared to GC/MS results. The results are presented in the tables below:

| Dip Card<br>format | | Negative | Low<br>Negative<br>by GC/MS<br>(less than -<br>50%) | Near Cutoff<br>Negative by<br>GC/MS<br>(Between -<br>50% and<br>cut-off) | Near<br>Cutoff<br>Positive by<br>GC/MS<br>(Between<br>the cut-off<br>and +50%) | High<br>Positive by<br>GC/MS<br>(greater<br>than +50%) |
|--------------------|----------|----------|-----------------------------------------------------|--------------------------------------------------------------------------|--------------------------------------------------------------------------------|--------------------------------------------------------|
| Viewer A | Positive | 0 | 0 | 1 | 13 | 26 |
| | Negative | 10 | 10 | 19 | 1 | 0 |
| Viewer B | Positive | 0 | 0 | 1 | 14 | 26 |
| | Negative | 10 | 10 | 19 | 0 | 0 |
| Viewer C | Positive | 0 | 0 | 1 | 13 | 26 |
| | Negative | 10 | 10 | 19 | 1 | 0 |

{17}------------------------------------------------

| Viewer | Sample Number | GC/MS (ng/mL)<br>Result | DipCard Format<br>Viewer Results |
|----------|---------------|-------------------------|----------------------------------|
| Viewer A | 94911951 | 340 | Negative |
| Viewer A | 94910121 | 276 | Positive |
| Viewer B | 94911312 | 269 | Positive |
| Viewer C | 94911296 | 344 | Negative |
| Viewer C | 94911562 | 260 | Positive |

| Cup<br>format | | Negative | Low<br>Negative<br>by GC/MS<br>(less than -<br>50%) | Near Cutoff<br>Negative by<br>GC/MS<br>(Between -<br>50% and<br>cut-off) | Near<br>Cutoff<br>Positive by<br>GC/MS<br>(Between<br>the cut-off<br>and +50%) | High<br>Positive by<br>GC/MS<br>(greater<br>than +50%) |
|---------------|----------|----------|-----------------------------------------------------|--------------------------------------------------------------------------|--------------------------------------------------------------------------------|--------------------------------------------------------|
| Viewer A | Positive | 0 | 0 | 1 | 13 | 26 |
| | Negative | 10 | 10 | 19 | 1 | 0 |
| Viewer B | Positive | 0 | 0 | 0 | 13 | 26 |
| | Negative | 10 | 10 | 20 | 1 | 0 |
| Viewer C | Positive | 0 | 0 | 1 | 13 | 26 |
| | Negative | 10 | 10 | 19 | 1 | 0 |

#### Discordant Results

| Viewer | Sample Number | GC/MS (ng/mL) Result | Cup Format Viewer Results |
|----------|---------------|----------------------|---------------------------|
| Viewer A | 94911296 | 344 | Negative |
| Viewer A | 94910742 | 269 | Positive |
| Viewer B | 94911951 | 340 | Negative |
| Viewer C | 94911928 | 342 | Negative |
| Viewer C | 94910755 | 266 | Positive |

- i. Lay-user study
A lay user study was performed at three intended user sites with 280 lay persons testing the EDDP devices. They had diverse educational and professional backgrounds and ranged in age from 21 to > 50 years. Urine samples were prepared at the following concentrations; negative, +/-75%, +/-50%, +/-25% of the cutoff by spiking drugs into drug free-pooled urine specimens. The concentrations of the samples were confirmed by GC/MS. Each sample was aliquoted into individual containers and blind-labeled. Each participant was provided with the package insert, 1 blind labeled sample and a device. The results are summarized below.

## Comparison between GC/MS and Lay Person Results (EDDP DipCard)

{18}------------------------------------------------

| % of Cutoff | Number of samples | EDDP Concentration by GC/MS (ng/mL) | Lay person results | | The percentage of correct results (%) |
|--------------|-------------------|-------------------------------------|--------------------|-----------------|---------------------------------------|
| -100% Cutoff | 20 | 0 | No. of Positive | No. of Negative | 100% |
| -75% Cutoff | 20 | 81 | 0 | 20 | 100% |
| -50% Cutoff | 20 | 157 | 0 | 20 | 100% |
| -25% Cutoff | 20 | 235 | 2 | 18 | 90% |
| +25% Cutoff | 20 | 410 | 20 | 0 | 100% |
| +50% Cutoff | 20 | 485 | 20 | 0 | 100% |
| +75% Cutoff | 20 | 566 | 20 | 0 | 100% |

Comparison between GC/MS and Lay Person Results (EDDP Cup)

| % of Cutoff | Number of samples | EDDP Concentration by GC/MS (ng/mL) | Lay person results | | The percentage of correct results (%) |
|--------------|-------------------|-------------------------------------|--------------------|-----------------|---------------------------------------|
| | | | No. of Positive | No. of Negative | |
| -100% Cutoff | 20 | 0 | 0 | 20 | 100% |
| -75% Cutoff | 20 | 81 | 0 | 20 | 100% |
| -50% Cutoff | 20 | 157 | 0 | 20 | 100% |
| -25% Cutoff | 20 | 235 | 1 | 19 | 95% |
| +25% Cutoff | 20 | 410 | 20 | 0 | 100% |
| +50% Cutoff | 20 | 485 | 20 | 0 | 100% |
| +75% Cutoff | 20 | 566 | 20 | 0 | 100% |

Lay-users were also given surveys on the ease of understanding the package insert instructions. All lay users indicated that the device instructions can be easily followed. A Flesch-Kincaid reading analysis was performed on each package insert and the scores revealed a reading Grade Level of 7.

- j. Clinical Studies
Not applicable.

## First Sign® Drug of Abuse Nortriptyline Test

Analytical Performance

- a. Precision
Precision studies were carried out for samples with concentrations of -100% cut-off, -50% cut-off, -25% cut-off, at the cut-off, +25% cut-off, +75% cut-off, +75% cut-off and +100% cut-off. These samples were prepared by spiking drug in negative urine samples. Each drug concentration was confirmed by GC/MS. All sample aliquots were blind-labeled and randomized by the person who prepared samples and did not take part in the sample testing. For each concentration, tests were performed two runs per day for 25 days by three different operators for

{19}------------------------------------------------

each format of devices. Different set of operators tested each format. The results obtained are summarized in the following tables:

| | Result | -100%<br>Cut-off | -75%<br>Cut-off | -50%<br>Cut-off | -25%<br>Cut-off | Cut-off | +25%<br>Cut-off | +50%<br>Cut-off | +75%<br>Cut-off | +100%<br>Cut-off |
|-------|--------|------------------|-----------------|-----------------|-----------------|---------|-----------------|-----------------|-----------------|------------------|
| Drug | | | | | | | | | | |
| Lot 1 | | 50-/0+ | 50-/0+ | 50-/0+ | 50-/0+ | 3-/47+ | 50+/0- | 50+/0- | 50+/0- | 50+/0- |
| Lot 2 | | 50-/0+ | 50-/0+ | 50-/0+ | 50-/0+ | 2-/48+ | 50+/0- | 50+/0- | 50+/0- | 50+/0- |
| Lot 3 | | 50-/0+ | 50-/0+ | 50-/0+ | 50-/0+ | 2-/48+ | 50+/0- | 50+/0- | 50+/0- | 50+/0- |

## Nortriptyline Dip Card Format

## Nortriptyline Cup Format

| | Result | -100%<br>Cut-off | -75%<br>Cut-off | -50%<br>Cut-off | -25%<br>Cut-off | Cut-off | +25%<br>Cut-off | +50%<br>Cut-off | +75%<br>Cut-off | +100%<br>Cut-off |
|-------|--------|------------------|-----------------|-----------------|-----------------|---------|-----------------|-----------------|-----------------|------------------|
| Drug | | | | | | | | | | |
| Lot 4 | | 50-/0+ | 50-/0+ | 50-/0+ | 50-/0+ | 2-/48+ | 50+/0- | 50+/0- | 50+/0- | 50+/0- |
| Lot 5 | | 50-/0+ | 50-/0+ | 50-/0+ | 50-/0+ | 3-/47+ | 50+/0- | 50+/0- | 50+/0- | 50+/0- |
| Lot 6 | | 50-/0+ | 50-/0+ | 50-/0+ | 50-/0+ | 3-/47+ | 50+/0- | 50+/0- | 50+/0- | 50+/0- |

## b. Linearity

Not applicable.

## c. Stability

The devices are stable at 39-86ºF (4-30ºC) for 24 months based on the accelerated stability study at 50°C. Control materials are not provided with the device. The labeling provides information on how to obtain control materials.

# d. Cut-off

A total of 150 samples equally distributed at concentrations of -50% cut-off; cutoff; +25% cut-off; +50% cut-off were tested using three different lots of each device by three different operators. Results were all positive at and above +25% cut-off and all negative at and below -25% cut-off for Nortriptyline. The following cut-off value for the test devices have been verified.

| Test | Calibrator | Cut-off (ng/mL) |
|-------------------------------------------------|---------------|-----------------|
| First Sign® Drug of Abuse Nortriptyline<br>Test | Nortriptyline | 1000 |

# e. Interference

Potential interfering substances found in human urine of physiological conditions were added to drug-free urine and to urine containing target drugs at 25% below and 25% above cut-off levels. These urine samples were tested using three lots of each device for all formats.

{20}------------------------------------------------

Compounds that showed no interference at a concentration of 100μg/mL are summarized in the following tables. There were no differences observed for different formats.

| 4-Acetamidophenol | Erythromycin | Oxycodone |
|------------------------|----------------------------------------------------|----------------------------------------------|
| Acetophenetidin | β-Estradiol | Oxymetazoline |
| N-Acetylprocainamide | Estrone-3-sulfate | Papaverine |
| Acetylsalicylic acid | Ethyl-p-aminobenzoate | Penicillin-G |
| Aminopyrine | Fenoprofen | Pentazocine hydrochloride |
| Amobarbital | Furosemide | Pentobarbital |
| Amoxicillin | Gentisic acid | Perphenazine |
| Ampicillin | Hemoglobin | Phencyclidine |
| L-ascorbic acid | Hydralazine | Phenelzine |
| DL-Amphetamine sulfate | Hydrochlorothiazide | Phenobarbital |
| Apomorphine | Hydrocodone | Phentermine |
| Aspartame | Hydrocortisone | β-Phenylethylamine |
| Atropine | O-Hydroxyhippuric acid | Trans-2-phenylcyclopropylamine hydrochloride |
| Benzilic acid | p-Hydroxyamphetamine | L-Phenylephrine |
| Benzoic acid | p-Hydroxy- methamphetamine | Phenylpropanolamine |
| Benzoylecgonine | 3-Hydroxytyramine | Prednisolone |
| Benzphetamine | Ibuprofen | Prednisone |
| Bilirubin | Iproniazid | Procaine |
| (±) - Brompheniramine | (±) - Isoproterenol | DL-Propranolol |
| Caffeine | Isoxsuprine | D-Propoxyphene |
| Cannabidiol | Ketamine | D-Pseudoephedrine |
| Cannabinol | Ketoprofen | Quinacrine |
| Chloralhydrate | Labetalol | Quinidine |
| Chloramphenicol | Loperamide | Quinine |
| Chlorothiazide | MDE | Ranitidine |
| (±) Chlorpheniramine | Meperidine | Salicylic acid |
| Chlorpromazine | Meprobamate | Secobarbital |
| Chloroquine | Methadone | Serotonin |
| Cholesterol | (L)Methamphetamine | Sulfamethazine |
| Clonidine | Methoxyphenamine | Sulindac |
| Cocaethylene | (±)-3,4-Methylenedioxyamphetamine hydrochloride | Tetracycline |
| Cocaine hydrochloride | (+)3,4-Methylenedioxymethamphetamine hydrochloride | Tetrahydrocortisone 3-(β-D-glucuronide) |
| Codeine | Morphine 3-β-Dglucuronide | Tetrahydrozoline |
| Cortisone | Morphine sulfate | Thiamine |
| (-) Cotinine | Nalidixic acid | Thioridazine |
| Creatinine | Naloxone | DL-Tyrosine |
| Deoxycorticosterone | Naltrexone | Tolbutamide |
| Dextromethorphan | Naproxen | Triamterene |
| Diclofenac | Niacinamide | Trifluoperazine |
| Diflunisal | Nifedipine | Trimethoprim |
| Digoxin | Norcodeine | Tryptamine |
| Diphenhydramine | Norethindrone | DL-Tryptophan |
| Doxylamine | D-Norpropoxyphene | Tyramine |
| Ecgonine | Noscapine | Uric acid |
| hydrochloride | | |
| Ecgonine methylester | Oxalic acid | Verapamil |
| Ephedrine | Oxazepam | Zomepirac |
| (L) - Epinephrine | Oxolinic acid | |

{21}------------------------------------------------

#### f. Specificity

To test the specificity, drug metabolites and other components that are likely to interfere in urine samples were tested using three lots of each device for all formats. The obtained lowest detectable concentration was used to calculate the cross-reactivity. There were no differences observed for different formats.

| Drug | Concentration (ng/ml) | % Cross-Reactivity |
|-----------------|-----------------------|--------------------|
| Nortriptyline | 1000 | 100% |
| Amitriptyline | 1500 | 67% |
| Clomipramine | 15000 | 6.7% |
| Desipramine | 1000 | 100% |
| Doxepine | 2000 | 50% |
| Imipramine | 600 | 167% |
| Nordoxepin | 1000 | 100% |
| Promazine | 24000 | 4% |
| Trimipramine | 4000 | 25% |
| Cyclobenzaprine | 1500 | 67% |
| Maprotiline | Negative at 100000 | Not Detected |
| Promethazine | Negative at 100000 | Not Detected |
| Norclomipramine | Negative at 100000 | Not Detected |

## g. Effect of Urine Specific Gravity and Urine pH

To investigate the effect of urine specific gravity and urine pH, urine samples with a range of 1.000 to 1.035 specific gravity or urine samples with a range of pH 4 to 9 were spiked with target drugs at 25% below and 25% above cut-off levels. These samples were tested using three lots of each

{22}------------------------------------------------

device for all formats. Results were all positive for samples at and above +25% Cut-Off and all negative for samples at and below -25% Cut-Off. There were no differences observed for different formats.

- h. Comparison Studies
The method comparison studies for the First Sign® Drug of Abuse Nortriptyline Test was performed in-house with three different laboratory assistants for each format of the device. Operators ran 80 (40 negative and 40 positive) unaltered clinical. The samples were blind labeled and compared to GC/MS results. The results are presented in the tables below:

| DipCard format | | Negative | Low<br>Negative<br>by GC/MS<br>(less than -<br>50%) | Near Cutoff<br>Negative by<br>GC/MS<br>(Between -<br>50% and cut-<br>off) | Near Cutoff<br>Positive by<br>GC/MS<br>(Between<br>the cut-off<br>and +50%) | High<br>Positive by<br>GC/MS<br>(greater<br>than<br>+50%) |
|----------------|----------|----------|-----------------------------------------------------|---------------------------------------------------------------------------|-----------------------------------------------------------------------------|-----------------------------------------------------------|
| Viewer A | Positive | 0 | 0 | 1 | 14 | 26 |
| | Negative | 10 | 10 | 19 | 0 | 0 |
| Viewer B | Positive | 0 | 0 | 1 | 13 | 26 |
| | Negative | 10 | 10 | 19 | 1 | 0 |
| Viewer C | Positive | 0 | 0 | 1 | 13 | 26 |
| | Negative | 10 | 10 | 19 | 1 |…