Immunalysis Ethyl Alcohol Enzyme Assay

K181553 · Immunalysis Corporation · DIC · Oct 31, 2018 · Clinical Toxicology

Device Facts

• Record ID: K181553
• Device Name: Immunalysis Ethyl Alcohol Enzyme Assay
• Applicant: Immunalysis Corporation
• Product Code: DIC · Clinical Toxicology
• Decision Date: Oct 31, 2018
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 862.3040
• Device Class: Class 2

Intended Use

The Immunalysis Ethyl Alcohol Enzyme Assay is an in vitro diagnostic device for the quantitative analysis of ethyl alcohol (ethanol) in human urine, serum or plasma with automated clinical chemistry analyzers. The measurement of ethanol is used for the diagnosis and treatment of alcohol intoxication and poisoning. This assay is calibrated against ethyl alcohol. This device is intended for prescription use only.

Device Story

The Immunalysis Ethyl Alcohol Enzyme Assay is an in vitro diagnostic reagent kit used on automated clinical chemistry analyzers in laboratory settings. It measures ethanol concentration in human urine, serum, or plasma samples. The assay utilizes an enzymatic reaction where alcohol dehydrogenase (ADH) oxidizes ethyl alcohol to acetaldehyde in the presence of nicotinamide adenine dinucleotide (NAD), which is reduced to NADH. The resulting change in absorbance is measured spectrophotometrically at 340 nm; the absorbance change is directly proportional to the ethanol concentration in the sample. Healthcare providers use the quantitative output to diagnose and treat patients for alcohol intoxication or poisoning. The device provides rapid, automated results, facilitating clinical decision-making in emergency or toxicology contexts.

Clinical Evidence

Bench testing only. Precision studies (n=80 per concentration) evaluated repeatability and within-lab reproducibility across urine, serum, and plasma. Linearity/recovery studies confirmed a measuring range of 3-550 mg/dL (r² ≥ 0.999). Analytical specificity testing demonstrated no significant interference (bias <10%) from numerous drugs, endogenous compounds, or anticoagulants. Method comparison against the predicate device (n=80 urine, 76 serum, 64 plasma) showed strong correlation (r² ≥ 0.993). No clinical studies were performed.

Technological Characteristics

Enzymatic assay using alcohol dehydrogenase (ADH) and NAD. Reagents stored at 2-8°C. Quantitative spectrophotometric measurement at 340 nm. Compatible with automated clinical chemistry analyzers. No specific materials of construction or software algorithm class (e.g., ML) specified; relies on standard clinical chemistry photometric analysis.

Indications for Use

Indicated for quantitative analysis of ethyl alcohol (ethanol) in human urine, serum, or plasma to aid in the diagnosis and treatment of alcohol intoxication and poisoning. For prescription use only.

Regulatory Classification

Identification

An alcohol test system is a device intented to measure alcohol (e.g., ethanol, methanol, isopropanol, etc.) in human body fluids (e.g., serum, whole blood, and urine). Measurements obtained by this device are used in the diagnosis and treatment of alcohol intoxication and poisoning.

Predicate Devices

• Lin-Zhi Ethyl Alcohol Enzymatic Assay (k032461)

Submission Summary (Full Text)

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October 31, 2018

Immunalysis Corporation Wenying (Jessica) Zhu Regulatory Affairs Specialist III 829 Towne Center Drive Pomona, CA 91767

Re: k181553

Trade/Device Name: Immunalysis Ethyl Alcohol Enzyme Assay Regulation Number: 21 CFR 862.3040 Regulation Name: Alcohol test system Regulatory Class: Class II Product Code: DIC Dated: September 19, 2018 Received: September 20, 2018

Dear Wenying (Jessica) Zhu:

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. Although this letter refers to your product as a device, please be aware that some cleared products may instead be combination products. The 510(k) Premarket Notification Database located at https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfpmn/pmn.cfm identifies combination product submissions. 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 Part

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801 and Part 809); medical device reporting of medical device-related adverse events) (21 CFR 803) for devices or postmarketing safety reporting (21 CFR 4, Subpart B) for combination products (see https://www.fda.gov/CombinationProducts/GuidanceRegulatoryInformation/ucm597488.htm); good manufacturing practice requirements as set forth in the quality systems (OS) regulation (21 CFR Part 820) for devices or current good manufacturing practices (21 CFR 4, Subpart A) for combination products; and, if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR 1000-1050.

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 comprehensive regulatory information about medical devices and radiation-emitting products, including information about labeling regulations, please see Device Advice

(https://www.fda.gov/MedicalDevices/DeviceRegulationandGuidance/) and CDRH Learn (http://www.fda.gov/Training/CDRHLearn). Additionally, you may contact the Division of Industry and Consumer Education (DICE) to ask a question about a specific regulatory topic. See the DICE website (http://www.fda.gov/DICE) for more information or contact DICE by email ([e-mail]) or phone [phone] or 301-796-7100).

Sincerely,

# Kellie B. Kelm -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) k181553

Device Name Immunalysis Ethyl Alcohol Enzyme Assay

#### Indications for Use (Describe)

The Immunalysis Ethyl Alcohol Enzyme Assay is an in vitro diagnostic device for the quantitative analysis of ethyl alcohol (ethanol) in human urine, serum or plasma with automated clinical chemistry analyzers. The measurement of ethanol is used for the diagnosis and treatment of alcohol intoxication and poisoning. This assay is calibrated against ethyl alcohol. This device is intended for prescription use only.

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

| <span> <span style="font-size: 16px;">☑</span> Prescription Use (Part 21 CFR 801 Subpart D) </span> |
|-------------------------------------------------------------------------------------------------------|
| <span> <span style="font-size: 16px;">☐</span> Over-The-Counter Use (21 CFR 801 Subpart C) </span> |

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# k181553 510(k) SUMMARY

# A. GENERAL INFORMATION

| Applicant Name: | Immunalysis Corporation<br>829 Towne Center Drive<br>Pomona, CA 91767<br>Establishment # 2020952 |
|------------------|--------------------------------------------------------------------------------------------------------------------|
| Company Contact: | Wenying (Jessica) Zhu<br>Regulatory Affairs Specialist III<br>Phone: [phone]<br>Email: [e-mail] |
| Date Prepared: | September 19, 2018 |

# B. DEVICE IDENTIFICATION

| Trade or Proprietary Names: | Immunalysis Ethyl Alcohol Enzyme Assay |
|-----------------------------|----------------------------------------|
| Common Name: | Ethyl Alcohol Enzyme Assay |

# C. REGULATORY INFORMATION

| Device Classification Name: | Alcohol Test System |
|-----------------------------|-------------------------------------------------|
| Product Codes: | DIC |
| Regulatory Class: | II |
| Classification Regulation: | 21 CFR 862.3040, Alcohol Test System |
| Panel: | Toxicology (91) |
| Predicate Device: | Lin-Zhi Ethyl Alcohol Enzymatic Assay [K032461] |

# D. DEVICE DESCRIPTION

The Immunalysis Ethyl Alcohol Assay is based on the oxidation of ethyl alcohol to acetaldehyde by alcohol dehydrogenase (ADH) and nicotinamide adenine dinucleotide (NAD) reduced to NADH resulting in an absorbance change measured spectrophotometrically at 340nm. The concentration of ethanol in the sample is directly proportional to the ADH activity.

## E. INTENDED USE

The Immunalysis Ethyl Alcohol Enzyme Assay is an in vitro diagnostic device for the quantitative analysis of ethyl alcohol (ethanol) in human urine, serum or plasma with automated clinical chemistry analyzers. The measurement of ethanol is used for the diagnosis and treatment of alcohol intoxication and poisoning. This assay is calibrated against ethyl alcohol. This device is intended for prescription use only.

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| Attribute | Predicate Device<br>Lin-Zhi Ethyl Alcohol Enzymatic<br>Assay [k032461] | Candidate Device<br>Immunalysis Ethyl Alcohol Enzyme<br>Assay |
|-------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------------------------------------------------------|
| Intended Use | For quantitative analyses of ethyl<br>alcohol in human urine, serum, or<br>plasma. | Same |
| Sample Matrix | Urine, serum or plasma | Same |
| Measured Analytes | Ethyl Alcohol (EtOH) | Same |
| Test Principle | Enzymatic assay | Same |
| User Environment | For use in laboratories | Same |
| Test Methodology | In the presence of nicotinamide<br>adenine dinucleotide (NAD), ADH<br>converts ethyl alcohol to<br>acetaldehyde and reduces NAD to<br>NADH. The ethyl alcohol<br>concentration is then directly<br>proportional to the ADH activity.<br>The enzyme activity is measured at<br>340 nm wavelength. | Same |
| Instrumentation | Performed on clinical chemistry<br>analyzers | Same |
| Reagent Storage | 2-8°C until expiration date | Same |
| Assay Range | 3-600 mg/dL | Same |

# F. COMPARISON WITH PREDICATE

# G. PERFORMANCE CHARACTERISTICS

The following laboratory performance studies were performed to determine substantial equivalence of the Immunalysis Ethyl Alcohol Enzyme Assay to the predicate device. Assay performance was established using the Olympus AU400e analyzer.

## 1. Limit of Blank (LoB)/Limit of Detection (LoD)

Sixty (60) blank measurements were taken from unaltered drug free negative human urine, serum and plasma to calculate Limit of blank (LoB). Four samples of each matrix containing concentrations in the range LoB to 4xLoB were tested in replicates of five on two reagent lots over three days to calculate Limit of Detection (LoD). LoB of the assay is 0.481 mg/dL for urine and 0.668 mg/dL for serum and 0.652 mg/dL for plasma. LoD of the assay is 1.3 mg/dL for urine, 1.7 mg/dL for serum and 1.7 mg/dL for plasma.

## 2. Limit of Quantitation (LoQ)

Limit of Quantitation (LoQ) is selected at a level equal to or higher than LoD. Four independent samples of each matrix spiked at concentration of LoQ were tested on two reagent lots to verify LoQ. Results demonstrated that drug concentration equal to or above LoQ, 2.9 mg/dL, can be quantitatively determined with suitable accuracy.

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# 3. Linearity/Recovery

A linearity study in the quantitative mode was conducted by spiking a drug free urine, serum and plasma pool with a high concentration of EtOH at upper limit of claimed measuring range. Additional pools were made by serially diluting the high concentration specimen with drug free urine, serum and plasma to achieve concentrations ranging from 3 mg/dL to 600 mg/dL. The high value specimen was evaluated with the predicate device to determine initial high-level value. The 0 mg/dL specimen was made from drug free urine, serum and plasma. Each pool was tested in triplicate to calculate the mean concentration values that were used to calculated drug recovery. Linearity test results are presented in Table 1 to Table 3.

| Expected Concentration<br>(mg/dL) | Mean Observed<br>Concentration<br>(mg/dL) | Recovery (%) | |
|-----------------------------------|-------------------------------------------|--------------|--|
| 0.0 | 0.0 | N/A | |
| 3.0 | 3.1 | 102.2 | |
| 12.0 | 12.0 | 100.3 | |
| 72.0 | 73.9 | 102.6 | |
| 132.0 | 132.2 | 100.2 | |
| 192.0 | 195.4 | 101.8 | |
| 252.0 | 250.3 | 99.3 | |
| 312.0 | 308.0 | 98.7 | |
| 372.0 | 363.0 | 97.6 | |
| 432.0 | 421.3 | 97.5 | |
| 492.0 | 484.4 | 98.5 | |
| 552.0 | 565.8 | 102.5 | |
| Slope | | 0.996 | |
| Intercept | | -0.172 | |
| r2 | | 0.999 | |

## Table 1. Linearity/Recovery Test Results - Urine

## Table 2. Linearity/Recovery Test Results - Serum

| Expected Concentration<br>(mg/dL) | Mean Observed<br>Concentration<br>(mg/dL) | Recovery (%) |
|-----------------------------------|-------------------------------------------|--------------|
| 0.0 | 0.0 | N/A |
| 3.0 | 3.0 | 101.1 |
| 23.2 | 23.1 | 99.4 |
| 83.2 | 87.2 | 104.8 |
| 143.2 | 146.1 | 102.0 |
| 203.2 | 199.8 | 98.3 |
| 263.2 | 262.2 | 99.6 |
| 323.2 | 317.8 | 98.3 |
| 383.2 | 385.9 | 100.7 |

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| Expected Concentration<br>(mg/dL) | Mean Observed<br>Concentration<br>(mg/dL) | Recovery (%) |
|-----------------------------------|-------------------------------------------|--------------|
| 443.2 | 441.4 | 99.6 |
| 503.2 | 493.6 | 98.1 |
| 563.2 | 544.3 | 96.6 |
| Slope | 0.978 | |
| Intercept | 2.869 | |
| r² | 0.999 | |

### Table 3. Linearity/Recovery Test Results - Plasma

| Expected Concentration<br>(mg/dL) | Mean Observed<br>Concentration<br>(mg/dL) | Recovery (%) |
|-----------------------------------|-------------------------------------------|--------------|
| 0.0 | 0.0 | N/A |
| 3.0 | 3.0 | 98.9 |
| 9.9 | 10.5 | 106.1 |
| 69.9 | 69.7 | 99.8 |
| 129.9 | 127.5 | 98.2 |
| 189.9 | 188.4 | 99.2 |
| 249.9 | 242.3 | 96.9 |
| 309.9 | 299.9 | 96.8 |
| 369.9 | 351.9 | 95.1 |
| 429.9 | 410.0 | 95.4 |
| 489.9 | 470.4 | 96.0 |
| 549.9 | 547.0 | 99.5 |
| Slope | 0.970 | |
| Intercept | 0.307 | |
| r2 | 0.999 | |

## 4. Precision

Precision study was performed for 20 days, 2 runs per day in duplicate on drug free urine, serum and plasma (N=80 for each matrix) spiked with EtOH to concentrations of 25 mg/dL, 75 mg/dL, 125 mg/dL, 150 mg/dL, 175 mg/dL and 200 mg/dL and calibrators and controls at concentrations of 50 mg/dL, 100 mg/dL and 300 mg/dL. The spiked concentrations were confirmed by Gas Chromatography - Flame Ionization Detector (GC-FID). The study established assay reproducibility and verified the precision performance in the claimed measuring range. Precision test results are presented in Table 4 to Table 6.

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| Concentration<br>(mg/dL) | # of<br>Sample | Mean<br>Conc.<br>(mg/dL) | Repeatability | | Between Run | | Within-<br>Laboratory | |
|--------------------------|----------------|--------------------------|---------------|-----|-------------|-----|-----------------------|-----|
| | Sample | (mg/dL) | SD | CV% | SD | CV% | SD | CV% |
| 0 | 80 | 0.0 | 0.1 | N/A | 0.0 | N/A | 0.1 | N/A |
| 25 | 80 | 24.5 | 0.3 | 1.2 | 0.3 | 1.1 | 0.5 | 1.8 |
| 50 | 80 | 49.0 | 0.4 | 0.9 | 0.6 | 1.2 | 0.8 | 1.5 |
| 75 | 80 | 74.0 | 0.7 | 0.9 | 1.0 | 1.3 | 1.1 | 1.5 |
| 100 | 80 | 99.6 | 0.8 | 0.8 | 1.6 | 1.6 | 1.6 | 1.6 |
| 125 | 80 | 121.3 | 1.2 | 1.0 | 1.5 | 1.2 | 1.8 | 1.5 |
| 150 | 80 | 146.1 | 1.3 | 0.9 | 1.9 | 1.3 | 2.4 | 1.7 |
| 175 | 80 | 169.1 | 1.4 | 0.9 | 2.0 | 1.1 | 2.7 | 1.6 |
| 200 | 80 | 193.9 | 1.8 | 0.9 | 2.6 | 1.3 | 3.4 | 1.7 |
| 300 | 80 | 292.0 | 2.2 | 0.8 | 3.8 | 1.3 | 4.6 | 1.6 |

# Table 4. Precision - Urine

# Table 5. Precision – Serum

| Concentration<br>(mg/dL) | # of<br>Sample | Mean<br>Conc.<br>(mg/dL) | Repeatability | | Between Run | | Within-<br>Laboratory | |
|--------------------------|----------------|--------------------------|---------------|-----|-------------|-----|-----------------------|-----|
| | | | SD | CV% | SD | CV% | SD | CV% |
| 0 | 80 | 0.4 | 0.2 | N/A | 0.1 | N/A | 0.2 | N/A |
| 25 | 80 | 25.8 | 0.2 | 0.9 | 0.5 | 2.0 | 0.8 | 3.2 |
| 50 | 80 | 49.3 | 0.3 | 0.7 | 1.0 | 2.0 | 1.0 | 2.0 |
| 75 | 80 | 75.7 | 0.4 | 0.5 | 1.4 | 1.8 | 2.2 | 3.0 |
| 100 | 80 | 99.6 | 0.5 | 0.5 | 2.0 | 2.1 | 2.1 | 2.1 |
| 125 | 80 | 125.9 | 0.8 | 0.6 | 2.4 | 1.9 | 3.9 | 3.1 |
| 150 | 80 | 148.4 | 1.0 | 0.6 | 2.6 | 1.7 | 3.8 | 2.6 |
| 175 | 80 | 172.3 | 1.0 | 0.6 | 3.1 | 1.8 | 5.1 | 3.0 |
| 200 | 80 | 195.2 | 1.3 | 0.7 | 3.4 | 1.8 | 4.9 | 2.5 |
| 300 | 80 | 290.4 | 1.4 | 0.5 | 5.5 | 1.9 | 6.0 | 2.1 |

# Table 6. Precision - Plasma

| Concentration<br>(mg/dL) | # of<br>Sample | Mean<br>Conc.<br>(mg/dL) | Repeatability | | Between Run | | Within-<br>Laboratory | |
|--------------------------|----------------|--------------------------|---------------|-----|-------------|-----|-----------------------|-----|
| | | | SD | CV% | SD | CV% | SD | CV% |
| 0 | 80 | 0.2 | 0.1 | N/A | 0.1 | N/A | 0.1 | N/A |
| 25 | 80 | 24.3 | 0.2 | 0.7 | 0.2 | 1.0 | 1.2 | 4.9 |
| 50 | 80 | 49.9 | 0.3 | 0.6 | 0.5 | 0.9 | 0.7 | 1.5 |
| 75 | 80 | 69.8 | 0.4 | 0.5 | 0.7 | 1.0 | 1.8 | 2.6 |

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| Concentration<br>(mg/dL) | # of<br>Sample | Mean<br>Conc.<br>(mg/dL) | Repeatability | | Between Run | | Within-<br>Laboratory | |
|--------------------------|----------------|--------------------------|---------------|-----|-------------|-----|-----------------------|-----|
| 100 | 80 | 100.5 | 0.5 | 0.5 | 1.0 | 1.0 | 1.6 | 1.6 |
| 125 | 80 | 118.3 | 0.6 | 0.5 | 1.0 | 0.9 | 2.7 | 2.2 |
| 150 | 80 | 155.8 | 0.9 | 0.6 | 1.2 | 0.8 | 4.1 | 2.6 |
| 175 | 80 | 170.6 | 0.9 | 0.5 | 1.5 | 0.9 | 3.2 | 1.9 |
| 200 | 80 | 197.6 | 1.1 | 0.6 | 1.4 | 0.7 | 3.9 | 2.0 |
| 300 | 80 | 292.7 | 2.0 | 0.7 | 2.7 | 0.9 | 5.2 | 1.8 |

## 5. Specificity and Cross-Reactivity

Structurally and functionally similar compounds were spiked into drug free urine, serum and plasma at levels that will yield a result that is equivalent to the concentration of 10 mg/dL and 100 mg/dL. The study verified assay performance relative to the ability of the device to exclusively determine certain drugs, in quantitative mode. Cross-reactivity test results are presented in Table 7 to Table 9.

## Table 7. Cross-Reactivity - Urine

| | 10 mg/dL | | 100 mg/dL | |
|------------------|---------------------------|-------------------------|---------------------------|-------------------------|
| Compound | Compound Conc.<br>(mg/dL) | Cross-Reactivity<br>(%) | Compound Conc.<br>(mg/dL) | Cross-Reactivity<br>(%) |
| Lot #1 | | | | |
| Acetaldehyde | 3,000 | <0.3 | 3,000 | <3.3 |
| Acetone | 3,000 | <0.3 | 3,000 | <3.3 |
| n-Butanol | 500 | 2.0 | 3,000 | <3.3 |
| Ethylene Glycol | 3,000 | <0.3 | 3,000 | <3.3 |
| Isopropanol | 3,000 | <0.3 | 3,000 | <3.3 |
| Methanol | 3,000 | <0.3 | 3,000 | <3.3 |
| n-Propanol | 60 | 16.7 | 1,025 | 9.8 |
| Propylene Glycol | 3,000 | <0.3 | 3,000 | <3.3 |
| Lot #2 | | | | |
| Acetaldehyde | 3,000 | <0.3 | 3,000 | <3.3 |
| Acetone | 3,000 | <0.3 | 3,000 | <3.3 |
| n-Butanol | 500 | 2.0 | 3,000 | <3.3 |
| Ethylene Glycol | 3,000 | <0.3 | 3,000 | <3.3 |
| Isopropanol | 3,000 | <0.3 | 3,000 | <3.3 |
| Methanol | 3,000 | <0.3 | 3,000 | <3.3 |
| n-Propanol | 60 | 16.7 | 1,025 | 9.8 |
| Propylene Glycol | 3,000 | <0.3 | 3,000 | <3.3 |
| Lot #3 | | | | |

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| Compound | 10 mg/dL | | 100 mg/dL | |
|------------------|----------|------|-----------|------|
| Acetaldehyde | 3,000 | <0.3 | 3,000 | <3.3 |
| Acetone | 3,000 | <0.3 | 3,000 | <3.3 |
| n-Butanol | 500 | 2.0 | 3,000 | <3.3 |
| Ethylene Glycol | 3,000 | <0.3 | 3,000 | <3.3 |
| Isopropanol | 3,000 | <0.3 | 3,000 | <3.3 |
| Methanol | 3,000 | <0.3 | 3,000 | <3.3 |
| n-Propanol | 60 | 16.7 | 1,025 | 9.8 |
| Propylene Glycol | 3,000 | <0.3 | 3,000 | <3.3 |

## Table 8. Cross-Reactivity - Serum

| Compound | 10 mg/dL | | 100 mg/dL | |
|------------------|---------------------------|-------------------------|---------------------------|-------------------------|
| | Compound Conc.<br>(mg/dL) | Cross-Reactivity<br>(%) | Compound Conc.<br>(mg/dL) | Cross-Reactivity<br>(%) |
| Acetaldehyde | 3,000 | <0.3 | 3,000 | <3.3 |
| Acetone | 3,000 | <0.3 | 3,000 | <3.3 |
| n-Butanol | 500 | 2.0 | 3,000 | <3.3 |
| Ethylene Glycol | 3,000 | <0.3 | 3,000 | <3.3 |
| Isopropanol | 3,000 | <0.3 | 3,000 | <3.3 |
| Methanol | 3,000 | <0.3 | 3,000 | <3.3 |
| n-Propanol | 90 | 11.1 | 900 | 11.1 |
| Propylene Glycol | 3,000 | <0.3 | 3,000 | <3.3 |

## Table 9. Cross-Reactivity - Plasma

| Compound | 10 mg/dL | | 100 mg/dL | |
|------------------|---------------------------|-------------------------|---------------------------|-------------------------|
| | Compound Conc.<br>(mg/dL) | Cross-Reactivity<br>(%) | Compound Conc.<br>(mg/dL) | Cross-Reactivity<br>(%) |
| Acetaldehyde | 3,000 | <0.3 | 3,000 | <3.3 |
| Acetone | 3,000 | <0.3 | 3,000 | <3.3 |
| n-Butanol | 500 | 2.0 | 3,000 | <3.3 |
| Ethylene Glycol | 3,000 | <0.3 | 3,000 | <3.3 |
| Isopropanol | 3,000 | <0.3 | 3,000 | <3.3 |
| Methanol | 3,000 | <0.3 | 3,000 | <3.3 |
| n-Propanol | 90 | 11.1 | 900 | 11.1 |
| Propylene Glycol | 3,000 | <0.3 | 3,000 | <3.3 |

# 6. Interference – Structurally Unrelated Compounds in Urine

Structurally unrelated compounds were evaluated in quantitative mode by spiking the potential interferent into drug free urine containing EtOH at 10 mg/dL and 100 mg/dL. All potential interferents analyzed verified that assay performance is unaffected by externally ingested

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compounds. The levels of structurally unrelated compounds that did not interfere in the assay are presented in Table 10.

| Compound | Compound Conc. (mg/dL) |
|--------------------------------------|------------------------|
| Acetaminophen | 500,000 |
| 6-Acetylcodeine | 100,000 |
| 6-Acetylmorphine | 100,000 |
| Acetylsalicylic Acid | 500,000 |
| Alphenal | 100,000 |
| Alprazolam | 100,000 |
| 7-Aminoclonazepam | 40,000 |
| 7-Aminoflunitrazepam | 100,000 |
| 7-Aminonitrazepam | 100,000 |
| Amitriptyline | 100,000 |
| Amobarbital | 100,000 |
| S-(+)-Amphetamine | 100,000 |
| Aprobarbital | 100,000 |
| Barbital | 100,000 |
| Benzoylecgonine | 100,000 |
| Benzylpiperazine | 100,000 |
| Bromazepam | 100,000 |
| 4-bromo 2-5, dimethoxyphenethylamine | 100,000 |
| Buprenorphine | 50,000 |
| Bupropion | 100,000 |
| Butabarbital | 100,000 |
| Butalbital | 100,000 |
| Caffeine | 500,000 |
| Cannabidiol | 100,000 |
| Cannabinol | 100,000 |
| Carbamazepine | 100,000 |
| Carisoprodol | 100,000 |
| Chlordiazepoxide | 100,000 |
| Chlorpromazine | 100,000 |
| Clobazam | 100,000 |
| Clomipramine | 100,000 |
| Clonazepam | 100,000 |
| Clozapine | 100,000 |
| Cocaine | 100,000 |
| Codeine | 100,000 |
| Compound | Compound Conc. (mg/dL) |
| Cotinine | 100,000 |
| Cyclobenzaprine | 100,000 |
| Cyclopentobarbital | 100,000 |
| Demoxepam | 100,000 |
| Desalkylflurazepam | 100,000 |
| Desipramine | 100,000 |
| Dextromethorphan | 100,000 |
| Diazepam | 100,000 |
| Digoxin | 100,000 |
| Dihydrocodeine | 100,000 |
| Diphenhydramine | 500,000 |
| Dehydronorketamine | 40,000 |
| delta9 THC | 100,000 |
| Doxepin | 100,000 |
| Doxylamine | 100,000 |
| ecgonine | 100,000 |
| ecgonine methyl ester | 100,000 |
| EDDP | 100,000 |
| EMDP | 100,000 |
| 1R,2S Ephedrine | 100,000 |
| 1S,2R Ephedrine | 100,000 |
| Ethyl-β-D-Glucuronide | 50,000 |
| Ethylmorphine | 100,000 |
| Fenfluramine | 100,000 |
| Fentanyl | 100,000 |
| Flunitrazepam | 100,000 |
| Fluoxetine | 100,000 |
| Flurazepam | 100,000 |
| Haloperidol | 100,000 |
| Heroin | 100,000 |
| Hexobarbital | 100,000 |
| hydrocodone | 100,000 |
| hydromorphone | 100,000 |
| 11-hydroxy-delta9 THC | 100,000 |
| Ibuprofen | 100,000 |
| Imipramine | 100,000 |
| Ketamine | 100,000 |
| Lamotrigine | 100,000 |
| Levorphanol Tartrate | 100,000 |
| Compound | Compound Conc. (mg/dL) |
| Lidocaine | 100,000 |
| Lorazepam | 100,000 |
| Lorazepam Glucuronide | 50,000 |
| Lormetazepam | 100,000 |
| LSD | 100,000 |
| Maprotiline | 100,000 |
| MDA | 100,000 |
| MDEA | 100,000 |
| MDMA | 100,000 |
| Meperidine | 100,000 |
| Meprobamate | 100,000 |
| Methamphetamine | 100,000 |
| Methadone | 500,000 |
| Methaqualone | 100,000 |
| Methoxetamine | 100,000 |
| Methylone | 100,000 |
| Methylphenidate | 100,000 |
| Midazolam | 100,000 |
| Morphine | 100,000 |
| Morphine-3-glucuronide | 100,000 |
| Morphine-6-glucuronide | 100,000 |
| N-Desmethyltapentadol | 100,000 |
| Nalorphine | 100,000 |
| Naloxone | 100,000 |
| Naltrexone | 100,000 |
| Naproxen | 100,000 |
| Nitrazepam | 100,000 |
| 11-nor-9-carboxy-delta9-THC | 100,000 |
| Norbuprenorphine | 50,000 |
| Norcodeine | 100,000 |
| Nordiazepam | 100,000 |
| Norketamine | 100,000 |
| Normorphine | 100,000 |
| Norproxyphene | 100,000 |
| Norpseudoephedrine | 50,000 |
| Nortriptyline | 100,000 |
| N-desmethyltramadol | 100,000 |
| N-desmethylvenlafaxine | 100,000 |
| O-desmethyltramadol | 100,000 |
| Compound | Compound Conc. (mg/dL) |
| O-desmethylvenlafaxine | 100,000 |
| Olanzapine | 100,000 |
| Oxycodone | 100,000 |
| Oxymorphone | 100,000 |
| Oxazepam | 100,000 |
| PCP | 100,000 |
| Pentazocine | 100,000 |
| Pentobarbital | 100,000 |
| Phenazepam | 100,000 |
| Phenobarbital | 100,000 |
| Phentermine | 100,000 |
| Phenylephrine | 100,000 |
| Phenylpropanolamine | 100,000 |
| Phenytoin | 100,000 |
| PMA | 100,000 |
| Prazepam | 100,000 |
| Propranolol | 100,000 |
| Propoxyphene | 100,000 |
| Protriptyline | 100,000 |
| R,R Pseudoephedrine | 100,000 |
| S,S Pseudoephedrine | 100,000 |
| Ritalinic Acid | 100,000 |
| Salicylic Acid | 100,000 |
| Sertraline | 100,000 |
| Sufentanil Citrate | 50,000 |
| Talbutal | 50,000 |
| Tapentadol | 100,000 |
| Temazepam | 100,000 |
| Theophylline | 100,000 |
| Thiopental | 100,000 |
| Thioridazine | 100,000 |
| Tramadol | 100,000 |
| Trazadone | 100,000 |
| Triazolam | 100,000 |
| Trifluoromethylphenyl-piperazine | 100,000 |
| Trimipramine | 100,000 |
| Venlafaxine | 100,000 |
| Verapamil | 100,000 |
| Zolpidem Tartrate | 100,000 |

Table 10. Non-Interfering Structurally Unrelated Compounds in Urine

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## 7. Interference - Endogenous Compounds and Urine Preservatives

Endogenous compounds and urine preservatives were evaluated in quantitative mode by spiking the potential interferent into drug free urine containing EtOH at concentration of 10 mg/dL and 100 mg/dL. Assay performance is unaffected by all the internally existing physiological conditions tested. The levels of endogenous compounds and urine preservatives that did not interfere in the assay are presented in Table 11 to Table 12.

| Compound | 10 mg/dL and 100 mg/dL<br>Compound Conc. |
|---------------------|------------------------------------------|
| Ascorbic Acid | 1.5 g/dL |
| Bilirubin | 0.02 g/dL |
| Creatinine | 0.5 g/dL |
| Galactose | 0.01 g/dL |
| γ-Globulin | 0.5 g/dL |
| Glucose | 2.0 g/dL |
| Hemoglobin | 0.3 g/dL |
| Human Serum Albumin | 0.5 g/dL |
| Oxalic Acid | 0.1 g/dL |
| Riboflavin | 0.0075 g/dL |
| Sodium Chloride | 6.0 g/dL |
| Urea | 6.0 g/dL |

Table 11. Non-interfering Endogenous Compounds - Urine

## Table 12. Non-interfering Urine Preservatives - Urine

| Compound | 10 mg/dL and 100 mg/dL | |
|-----------------------|------------------------|----------------|
| | Compound Conc. | |
| Boric Acid | 1% w/v | |
| Sodium Azide | 1% w/v | |
| Sodium Fluoride | 1% w/v | |
| Compound | 10 mg/dL | 100 mg/dL |
| | Compound Conc. | Compound Conc. |
| Acetaminophen | 20 mg/dL | 20 mg/dL |
| Amikacin | 8 mg/dL | 8 mg/dL |
| Ampicillin | 5.3 mg/dL | 5.3 mg/dL |
| Ascorbic Acid | 6 mg/dL | 6 mg/dL |
| Bilirubin total | 60 mg/dL | 80 mg/dL |
| Bilirubin direct | 60 mg/dL | 80 mg/dL |
| Caffeine | 6 mg/dL | 6 mg/dL |
| Carbamazepine | 3 mg/dL | 3 mg/dL |
| Chloramphenicol | 5 mg/dL | 5 mg/dL |
| Chlordiazepoxide | 1 mg/dL | 1 mg/dL |
| Chlorpromazine | 0.2 mg/dL | 0.2 mg/dL |
| Cholesterol | 30 mg/dL | 503 mg/dL |
| Cimetidine | 2 mg/dL | 2 mg/dL |
| Creatinine | 30 mg/dL | 30 mg/dL |
| Dextran | 6000 mg/dL | 6000 mg/dL |
| Diazepam | 0.51 mg/dL | 0.51 mg/dL |
| Digoxin | 6.1 mg/dL | 6.1 mg/dL |
| Erythromycin | 6 mg/dL | 6 mg/dL |
| Ethosuximide | 25 mg/dL | 25 mg/dL |
| Furosemide | 6 mg/dL | 6 mg/dL |
| Gentamicin | 1 mg/dL | 1 mg/dL |
| Hemoglobin | 62.5 mg/dL | 1,000 mg/dL |
| Heparin | 3 U/mL | 3 U/mL |
| Ibuprofen | 50 mg/dL | 50 mg/dL |
| IgG | 5,000 mg/dL | 5,000 mg/dL |
| Intralipid | 187.5 mg/dL | 750 mg/dL |
| Lactate Dehydrogenase | 1,000 U/L | 237,500 U/L |
| Lactate | 150 mg/dL | 901 mg/dL |
| Lidocaine | 1.2 mg/dL | 1.2 mg/dL |
| Lithium | 2.2 mg/dL | 2.2 mg/dL |
| Mannitol | 500 mg/dL | 500 mg/dL |
| Penicillin | 25 U/mL | 25 U/mL |
| Pentobarbital | 8 mg/dL | 8 mg/dL |
| Phenobarbital | 10 mg/dL | 10 mg/dL |
| Phenytoin | 5 mg/dL | 5 mg/dL |
| Primidone | 4 mg/dL | 4 mg/dL |
| Propoxyphene | 0.16 mg/dL | 0.16 mg/dL |
| HSA (albumin) | 37.5 mg/dL | 1,250 mg/dL |
| Compound | 10 mg/dL | 100 mg/dL |
| | Compound Conc. | Compound Conc. |
| Protein (total) | 50 mg/dL | 1,500 mg/dL |
| Salicylic Acid | 60 mg/dL | 60 mg/dL |
| Theophylline | 4 mg/dL | 4 mg/dL |
| Triglycerides | 500 mg/dL | 500 mg/dL |
| Urea | 500 mg/dL | 500 mg/dL |
| Uric Acid | 20 mg/dL | 20 mg/dL |
| Valproic Acid | 50 mg/dL | 50 mg/dL |

## 8. Interference - Interferents in Serum and Plasma

Potential interfering substances in serum and plasma matrices were evaluated in quantitative mode by spiking the compound into drug free serum and plasma containing EtOH at concentration of 10 mg/dL and 100 mg/dL, respectively. Additional anticoagulants were evaluated by spiking into drug free plasma containing EtOH at concentration of 10 mg/dL and 100 mg/dL. Assay performance is unaffected by all the internally existing physiological conditions tested. The levels of substances that did not interfere in the assay are presented in Table 13 and Table 14.

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# Table 13. Non-interfering Substances – Serum and Plasma

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#### Table 14. Non-interfering Anticoagulants -Plasma

| Compound | 10 mg/dL<br>Compound Conc. | 100 mg/dL<br>Compound Conc. |
|-----------------|----------------------------|-----------------------------|
| Heparin | 3 U/mL | 3 U/mL |
| EDTA | 2 mg/mL | 2 mg/mL |
| Oxalic Acid | 2 mg/mL | 2 mg/mL |
| Sodium Citrate | 3.8 g/dL | 3.8 g/dL |
| Sodium Fluoride | 2 mg/mL | 2 mg/mL |

#### 9. Interference - pH

To evaluate potential interference from the effect of urine pH, device performance in the quantitative mode was tested using a range of urine pH values. All test samples were prepared in drug-free urine containing EtOH at concentration of 10 mg/dL and 100 mg/dL. No interference was observed at urine pH values ranging from 3.0 to 11.0.

#### 10. Interference - Specific Gravity

To evaluate potential interference from the specific gravity of urine, device performance in the quantitative mode was tested using a range of physiologically relevant urine specific gravity values. All test samples were prepared in drug free urine containing EtOH at concentration of 10 mg/dL and 100 mg/dL. No interference was observed at urine specific gravity values ranging from 1.000 to 1.030.

#### 11. Calibration Duration

Drug free negative urine spiked with EtOH at concentration of 10 mg/dL, 100 mg/dL and 300 mg/dL were tested in quantitative mode at time points up to 14 days. At the initial time point, a two-point calibration curve was established. This calibration was used through the duration of this study. The test results met acceptance criteria at each time point. The recommended frequency of calibration is 14 days.

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## 12. Specimen Stability

Drug free negative urine, serum and plasma spiked with EtOH at concentration of 100 mg/dL and 300 mg/dL were stored in industry standard collection containers and tested by assay kit at each time point at 30°C and at 2°C - 8°C. Test results indicated that urine samples containing EtOH are stable for up to 14 days stored at ambient temperature up to 30℃, up to 6 months stored at 2°C - 8°C; serum samples containing EtOH are stable for up to 10 days stored at ambient temperature up to 30°C, up to 14 days stored at 2°C - 8°C; plasma samples containing EtOH are stable for up to 3 day stored at ambient temperature up to 30°C, up to 6 days stored at 2°C - 8°C.

## 13. Method Comparison

Eighty (80) de-identified, unaltered leftover clinical urine samples, seventy-six (76) de-identified, unaltered leftover clinical serum samples and sixty-four (64) de-identified, unaltered leftover clinical plasma samples obtained from clinical testing laboratories were analyzed for EtOH with the Immunalysis Ethyl Alcohol Enzyme Assay in quantitative mode and compared to results of predicate device. The instruments used were an Olympus AU 400e. Method comparison test results are presented in Table 15.

## Table 15. Method Comparison Test Results

| Matrix | n | Slope | Intercept | r² | Candidate<br>Device Range<br>(mg/dL) | Predicate<br>Device<br>Range<br>(mg/dL) |
|--------|----|--------|-----------|--------|--------------------------------------|-----------------------------------------|
| Urine | 80 | 1.0284 | -2.7848 | 0.9964 | 3-600 | 3-600 |
| Serum | 76 | 1.0067 | -5.8802 | 0.9948 | 3-600 | 3-600 |
| Plasma | 64 | 1.0595 | -7.3902 | 0.9932 | 3-600 | 3-600 |

# CONCLUSION

The information provided in this pre-market notification demonstrates that the Immunalysis Ethyl Alcohol Enzyme Assay is substantially equivalent to the legally marketed predicate device for its intended use.