The Cozart Bioscience EIA Cotinine Urine Kit is intended for laboratory based testing in clinical and analytical laboratories and for insurance assessment. It provides qualitative screening results for Cotinine in human urine at a cut-off concentration of 500ng/ml. The Cozart EIA Cotinine Urine Kit acts as an aid in the detection of cotinine after use of tobacco products or other products containing nicotine.

This assay is for professional use only and provides only a preliminary analytical test result. Clinical consideration and professional judgement must be applied to any drug of abuse test result, particularly in evaluating a preliminary positive result. In order to obtain a more confirmed analytical result a more specific alternative chemical method is needed. Gas Chromatography/Mass Spectrometry (GC/MS) is the preferred confirmatory method.

Device Description

The Cozart EIA Cotinine Urine Kit test is a competitive ELISA for the detection of Cotinine in human urine. The kit supplies the following reagents - a microtitre plate coated with antibody, enzyme conjugate reagent, wash buffer, substrate solution, stop solution and four calibrators (0, 50, 500 and 5000ng/ml Cotinine in human urine).

AI/ML Overview

Here's a breakdown of the acceptance criteria and the study details for the Cozart EIA Cotinine Urine Kit, based on the provided text:

Acceptance Criteria and Device Performance

Parameter	Acceptance Criteria (Implied by Predicate/Performance Claims)	Reported Device Performance (Cozart EIA Cotinine Urine Kit)
Intended Use	Qualitative test for Cotinine in human urine with a 500ng/ml cutoff. Recommendation for GC/MS confirmation.	Qualitative test for Cotinine in human urine with a 500ng/ml cutoff. Recommendation for GC/MS confirmation.
Method Comparison	High agreement with GC/MS for positive and negative samples.	98% agreement for positive results (56/57), 100% agreement for negative results (38/38) with GC/MS (based on 95 samples).
Precision	CV (%) for the assay should be acceptable for qualitative ELISA (e.g., <10% for internal controls, <11% for total precision).	CVs less than 10% for internal controls; total precision CVs less than 11%.
Sensitivity	A low detection limit for cotinine.	1.2 ng/ml
Specificity	Minimal cross-reactivity with common interferents.	Only nicotine cross-reacted (at 10,000 ng/ml) out of 28 tested substances. No interference observed with pH, specific gravity, ascorbic acid, or protein.
Cutoff Concentration	Proper differentiation around the 500ng/mL cutoff.	Successfully differentiated between 375ng/mL (higher absorbance) and 625ng/mL (lower absorbance) compared to the 500ng/mL cutoff calibrator.
Sample Stability	Samples should remain stable for a reasonable period under various storage conditions.	Stable for 21 days at 2-8°C, 25°C, and 37°C. For longer storage, freeze at -20°C.
Stopped Assay Stability	Finished assay should be stable for a practical reading window.	Must be read within 15 minutes at 450nm.
Assay Drift	Minimal drift with variation in sample addition time.	Little change observed when samples added within 25 minutes.

Study Details

Sample Size Used for the Test Set and Data Provenance:
- Sample Size: 95 urine specimens.
- Data Provenance: The samples were collected from drug dependency units in Coventry and Warwick, UK. The data is retrospective, as the samples were collected and then tested.
Number of Experts Used to Establish the Ground Truth for the Test Set and Their Qualifications:
- The ground truth was established by Gas Chromatography/Mass Spectrometry (GC/MS). While GC/MS is an analytical method rather than an "expert," the results were performed by the Analytical Services Laboratory at Cozart Bioscience Ltd. The qualifications of the personnel performing these analyses are not explicitly stated but are presumed to be qualified laboratory staff.
Adjudication Method for the Test Set:
- Not applicable in the traditional sense, as the ground truth was established by a single, definitive analytical method (GC/MS). The Cozart EIA result was compared directly against the GC/MS result for agreement. There was no "expert consensus" or "adjudication" between multiple interpretations of the ground truth.
If a Multi Reader Multi Case (MRMC) Comparative Effectiveness Study was done:
- No, an MRMC comparative effectiveness study was not done. The study focused on the performance of the Cozart EIA Cotinine Urine Kit against a gold standard analytical method (GC/MS) rather than comparing different human readers or human readers with and without AI assistance.
If a Standalone Study (algorithm only without human-in-the-loop performance) was done:
- Yes, a standalone study was performed. The Cozart EIA Cotinine Urine Kit is a laboratory-based immunoassay kit, and its performance was evaluated intrinsically as a diagnostic device, without human interpretation influencing the primary result generated by the kit itself. The results are read spectrophotometrically, which is an objective measurement.
The Type of Ground Truth Used:
- The ground truth used was Gas Chromatography/Mass Spectrometry (GC/MS), which is considered a highly specific and sensitive reference method for cotinine detection (often referred to as the "gold standard" in toxicology).
The Sample Size for the Training Set:
- The document does not explicitly mention a separate "training set" for the Cozart EIA Cotinine Urine Kit. For an immunoassay kit like this, the development process involves reagent formulation and optimization, calibration, and validation. The "training" in this context would typically refer to the internal development and optimization of the assay parameters, not a separate data set for a machine learning algorithm. The provided "Method Comparison" data is for the test set (validation of the final product).
How the Ground Truth for the Training Set Was Established:
- As no explicit "training set" is mentioned in the context of typical machine learning models, the concept of establishing ground truth for it doesn't directly apply here. For the development and optimization of the assay itself, known concentrations of cotinine (calibrators) and controls would have been used. The GC/MS method would likely have also been used during the R&D phase to define the kit's performance characteristics and optimize its components (e.g., antibody binding, enzyme conjugates, cutoff values).

Summary

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K021944

MAY 1 4 2003

PREMARKET NOTIFICATION [510(k)] SUMMARY

Submitter Cozart Bioscience Ltd 45 Milton Park Abingdon Oxfordshire OX14 4RU UK Tel No: 01235 861483 Fax No: 01235 835607
Contact Person Dr Roberto Liddi Quality Assurance & Regulatory Affairs Manager

Date 6th December 2002

Device Name

Trade Name: Classification Name: Cozart EIA Cotinine Urine Kit Cotinine Test System

Classification

Class I Code of Federal Regulations Title 21 Food and Drugs Part 862 Clinical Chemistry and Clinical Toxicology Devices Subpart D Clinical Toxicology Test Systems Section 862.3220 Cotinine Test System

Establishment Registration No

3002336046

Performance Standards

BS EN ISO 9001:1994; EN 46001:1996

Substantial Equivalence

The Cozart EIA Cotinine Urine Kit is substantially equivalent to the Auto-Lyte Cotinine EIA (K972481) manufactured by Orasure Technologies, Inc.

See below for table of comparison for substantial equivalence.

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Parameter	Cozart Cotinine Urine EIA	Auto-Lyte Cotinine EIA
Intended Use	Qualitative test for Cotinine inhuman urine with a 500ng/mlcutoff. Recommendconfirmation of positive resultsby GC/MS.	Qualitative and Semi-quantitative test for Cotinine inhuman urine with a 500ng/mlcutoff. Recommendconfirmation of positive resultsby GC/MS.
Target Population	Clinical and forensic samplesand insurance assessment.	Insurance risk assessment.
Design	Competitive ELISA	Competitive EIA for use onclinical chemistry analysers.
Enzyme	Horse Radish Peroxidase	Glucose-6-phosphatedehydrogenase.
Substrate	Tetramethylbenzidine (TMB)	Glucose-6-phosphate
Results	Read spectrophotometrically at450nm.	Measure production of NADHspectrophotometrically at 340nm.
Calibrators	0, 100, 300 and 1000ng/ml.	0, 500 and 5000ng/ml.
Controls	None supplied but Cozartrecommends using externalcontrols.	None supplied.
MethodComparison	95 Urine Specimens were tested,57 screened positive of which 56were confirmed by GC/MS. 38samples screened negative andall 38 were confirmed negativeby GC/MS.	218 Urine specimens were tested,160 from self reported smokers,and 58 from non-smokers. Allsamples were confirmed byGC/MS
Precision	CV (%) of 2-11%	Precision of less than 2%.
Sensitivity	1.2ng/ml	Unknown
Specificity	Twenty-Eight potentialinterferents tested - none cross-reacted.	48 potential interferents tested -none cross-reacted.

Introduction

The Cozart EIA Cotinine Urine Kit is a laboratory based test for the detection of Cotinine in human urine using a cutoff of 500ng/mL. The device detailed above was compared to Gas Chromatography/Mass Spectrometry (GC/MS).

Cozart Bioscience Ltd is the manufacturer of the Cotinine Urine Kit. We have not purchased this device from another manufacturer and the device is not marketed under another product name.

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Intended Use

The Cozart Bioscience EIA Cotinine Urine Kit is intended for laboratory based testing in clinical and forensic laboratories and for insurance assessment. It provides qualitative screening results for Cotinine in human urine at a cutoff concentration of 500ng/mL. The Cozart EIA Cotinine Urine Kit acts as an aid in the detection of cotinine after use of tobacco products or other products containing nicotine.

This assay is for professional use only and provides only a preliminary analytical test result. Clinical consideration and professional judgement must be applied to any drug of abuse test result, particularly in evaluating a preliminary positive result. In order to obtain a more confirmed analytical result a more specific alternative chemical method Gas Chromatography/Mass Spectrometry (GC/MS) is the preferred is needed. confirmatory method.

Target Population

The target population for the Cozart EIA Cotinine Urine Kit is clinical and forensic samples.

Where Used

The Cozart EIA Cotinine Urine Kit is designed for use in clinical and forensic laboratories and for insurance risk assessment. To be used by trained laboratory personnel only.

Design

As can be seen from the Principle of the Test section in the pack insert, the Cozart EIA Cotinine Urine Kit test is a competitive ELISA for the detection of Cotinine in human urine.

Materials

The Cozart EIA Cotinine Urine kit supplies the following reagents - a microtitre plate coated with antibody, enzyme conjugate reagent, wash buffer, substrate solution, stop solution and four calibrators (0, 50, 500 and 5000ng/ml Cotinine in human urine).

Performance

Method Comparison

The Cozart EIA Cotinine Urine Kit was compared to Gas Chromatography/Mass Spectrometry. All the samples were tested through the Cozart EIA Cotinine Urine Kit according to the pack insert enclosed. Forty-four samples were positive by Cozart Cotinine Microplate EIA and forty-four were confirmed

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positive by GC/NPD. Of these forty-four samples, three were in the range 625 -500ng/mL (between +25% cutoff and the cutoff).

All the samples were tested with the Cozart EIA Cotinine Urine Kit according to the pack insert enclosed. Ninety-five samples were tested through the Cozart EIA Cotinine Urine Kit, of which 57 screened positive and 56 were confirmed positive by GC/MS subject to cut offs of 500ng/mL. Thirty-eight samples screened negative for cotinine and all 38 were confirmed by GC/MS.

Of the 95 samples tested 9 were in the range 375 - 625g/mL (between -25% cutoff and +25% cutoff).

New Device	GC/MS Negs	Near Cutoff GC/MS Negs *	Near Cutoff GC/MS Pos **	GC/MS Pos***	Percent Agreement with GC/MS
Pos	57	1	0	3	56	98
Neg	38	38	6	0	0	100

Between -25% Cutoff and the Cutoff.

** Between +25% Cutoff and the Cutoff.

*** Total number of positives (includes near cutoff samples).

Precision

The precision obtained for the Cozart EIA Cotinine Urine Kit produced CVs less than 10%. The total precision for the Kit produced CVs less than 11%. The Cozart EIA Cotinine Urine Kit is a qualitative manual ELISA assay and CVs of less than 10% are acceptable for this assay type.

Sensitivity

The sensitivity of the Cozart EIA Cotinine Urine Kit is 1.2ng/ml.

Specificity

Twenty-eight potentially interfering substances were tested for cross reactivity in the Cozart EIA Cotinine Urine Kit and only nicotine was found to cross react at 10,000ng/mL.

Cutoff Concentration

Testing samples at the cutoff concentration, 25% above and 25% below was carried out to validate the cutoff concentration. The absorbances for the 375ng/mL sample were all higher than the 500ng/mL cutoff calibrator. Similarly the absorbances obtained for the 625ng/ml sample were all lower than the 500ng/ml cutoff calibrator.

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Interference Studies

A range of parameters including pH, specific gravity, ascorbic acid and protein were tested for potential interference in the Cozart EIA Cotinine Urine Kit. No interference was observed with any of the parameters.

Sample Stability

Sample stability was carried out at 2-8°C, room temperature and 37°C. Each sample was tested on day 0, 4, 7, 14 and 21. Urine samples are stable for 21 days stored at 2-8°C, 25°C and 37°C when tested in the Cozart EIA Cotinine Urine Kit. For longer storage urine samples must be stored frozen (-20℃).

Stopped Assay Stability

The stability of the stopped assay was investigated by reading the absorbance at 450nm at time 0, 5, 10, 15, 30, 45 and 60 minutes. The Cozart EIA Cotinine Urine Kit must be read within 15 minutes at 450nm.

Assay Drift

Sample addition at time 0, 2.5, 5, 7.5, 10, 12.5, 15, 17.5, 20, 22.5 and 25 minutes was investigated. Little change was observed across the plate and therefore sample addition to a Cozart EIA Cotinine Urine Kit must take place within 25 minutes.

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Method Comparison

The method comparison study was performed at Cozart Bioscience Ltd by Cozart Bioscience staff. The GC/MS was performed by the Analytical Services Laboratory at Cozart Bioscience Ltd.

The samples were collected from drug dependency units in Coventry and Warwick, UK. They were selected for testing due to the assumption that a large proportion of these people would be smokers.

The Cozart EIA Cotinine Urine Kit was compared to Gas Chromatography/Mass Spectrometry (GC/MS). All the samples were tested with the Cozart EIA Cotinine Urine Kit according to the pack insert enclosed. Ninety-five samples were tested through the Cozart EIA Cotinine Urine Kit, of which 57 screened positive and 56 were confirmed positive by GC/MS subject to cut offs of 500ng/mL. Thirty-eight samples screened negative for cotinine and all 38 were confirmed by GC/MS.

Of the 95 samples tested 9 were in the range 375 - 625g/mL (between -25% cutoff and +25% cutoff).

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Image /page/6/Picture/1 description: The image shows the logo for the U.S. Department of Health & Human Services. The logo is circular and contains the text "DEPARTMENT OF HEALTH & HUMAN SERVICES • USA" around the perimeter. In the center of the circle is an abstract symbol that resembles an eagle or a stylized human profile.

Food and Drug Administration 2098 Gaither Road Rockville MD 20850

Dr. Roberto Liddi Quality Assurance/Regulatory Affairs Manager Cozart Bioscience Limited 45 Milton Park Abingdon Oxfordshire, OX14 4RU United Kingdom

MAY 1 4 2003

Re: K021944

Trade/Device Name: Cozart EIA Cotinine Urine Test Regulation Number: 21 CFR § 862.3220 Regulation Name: Carbon Monoxide Test System Regulatory Class: I Product Code: MKU Dated: April 14, 2003 Received: April 18, 2003

Dear Dr. Liddi:

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.

If your device is classified (see above) into either class II (Special Controls) or class III (PMA), it may be subject to such additional controls. Existing major regulations affecting your device can be found in Title 21, Code of Federal Regulations (CFR), Parts 800 to 895. 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); and good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820). This letter will allow you to begin marketing your device as described in your Section 510(k) premarket notification. The FDA finding of substantial equivalence of your device to a legally marketed predicate device results in a classification for your device and thus, permits your device to proceed to the market.

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Page 2 -

If you desire specific information about the application of labeling requirements to your device, or questions on the promotion and advertising of your device, please contact the Office of In Vitro Diagnostic Device Evaluation and Safety at (301) 594-3084. Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21CFR Part 807.97). You may obtain other general information on your responsibilities under the Act from the Division of Small Manufacturers, International and Consumer Assistance at its toll-free number (800) 638-2041 or (301) 443-6597 or at its Internet address http://www.fda.gov/cdrh/dsma/dsmamain.html.

Sincerely yours.

steven Sutman

Steven I. Gutman, M.D., M.B.A. Director Office of In Vitro Diagnostic Device Evaluation and Safety Center for Devices and Radiological Health

Enclosure

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Page I of

K021944 510(k) Number (if known):

Cozart EIA Cotinine Urine Kit Device Name:_

Indications For Use:

Jan
Copper

(Division Sign-Off)
Division of Clinical Laboratory Devices
510(k) Number K021944

(PLEASE DO NOT WRITE BELOW THIS LINE-CONTINUE ON ANOTHER PAGE IF NEEDED)

Concurrence of CDRH, Office of Device Evaluation (ODE)

Division Sign-Off

Office of In Vitro Diagnostic Device Evaluation and Safety

(Optional Format 3-10-98)

510(k)
prescription

OTC

Regulation Number and Section

§ 862.3220 Carbon monoxide test system.

(a)
Identification. A carbon monoxide test system is a device intended to measure carbon monoxide or carboxyhemoglobin (carbon monoxide bound to the hemoglobin in the blood) in blood. Measurements obtained by this device are used in the diagnosis and treatment of or confirmation of carbon monoxide poisoning.(b)
Classification. Class I (general controls). 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.