PANTEX SALIVARY DIRECT TESTOSTERONE EIA KIT, Pantex Sample Collection Device

K133303 · Bio-Analysis, Inc. · JKA · Nov 19, 2014 · Clinical Chemistry

Device Facts

• Record ID: K133303
• Device Name: PANTEX SALIVARY DIRECT TESTOSTERONE EIA KIT, Pantex Sample Collection Device
• Applicant: Bio-Analysis, Inc.
• Product Code: JKA · Clinical Chemistry
• Decision Date: Nov 19, 2014
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 862.1675
• Device Class: Class 2

Intended Use

The Pantex Salivary Direct Testosterone EIA Kit is an Enzyme Immunoassay (EIA) for the quantitative measurement of testosterone in human saliva collected with the Pantex Sample Collection Device. The measurement of testosterone is used in the diagnosis and treatment of disorders involving the male sex hormones (androgens), including primary and secondary hypogonadism, impotence in males, and in females, hirsutism (excessive hair) and virilization (masculinization) due to tumors, polycystic ovaries and adrenogenital syndromes.

Device Story

Quantitative enzyme immunoassay (EIA) for testosterone in human saliva; utilizes competitive binding principle. Input: saliva sample collected in Pantex Sample Collection Device (polypropylene tube). Process: testosterone in sample competes with HRP-conjugated testosterone for binding sites on rabbit monoclonal antibody-coated microplate wells; enzyme substrate (TMB/H2O2) added; reaction stopped with acid; optical density measured at 450 nm via microplate colorimeter. Output: testosterone concentration inversely proportional to optical density. Used in clinical laboratories; operated by trained personnel. Results assist clinicians in diagnosing/treating androgen-related disorders (hypogonadism, impotence, hirsutism, virilization).

Clinical Evidence

No clinical diagnostic studies performed. Analytical performance validated via bench testing: precision (intra/inter-assay, inter-lot), linearity (5.5-650 pg/mL), and method comparison (n=106, y=0.9035x+5.81, R^2=0.98) against predicate. Reference ranges established using 480 healthy subjects (240 male, 240 female).

Technological Characteristics

Microplate-based EIA; GARGG-coated 96-well plates; HRP-conjugated testosterone; TMB substrate; acid stop solution. Requires microplate colorimeter (450 nm). Calibrators/controls traceable to USP reference standards. Sample collection: 10 mL polypropylene tube. Software: 4-parameter data reduction required for curve fitting.

Indications for Use

Indicated for quantitative measurement of testosterone in human saliva for diagnosis and treatment of androgen-related disorders. Patient population includes males with primary/secondary hypogonadism or impotence, and females with hirsutism or virilization due to tumors, polycystic ovaries, or adrenogenital syndromes. Age range 20-70 years.

Regulatory Classification

Identification

A blood specimen collection device is a device intended for medical purposes to collect and to handle blood specimens and to separate serum from nonserum (cellular) components prior to further testing. This generic type device may include blood collection tubes, vials, systems, serum separators, blood collection trays, or vacuum sample tubes.

Predicate Devices

• Testosterone Luminescence Immunoassay IBL International GMBH (K033786)

Submission Summary (Full Text)

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Image /page/0/Picture/1 description: The image shows the seal of the Department of Health & Human Services - USA. The seal is circular, with the text "DEPARTMENT OF HEALTH & HUMAN SERVICES - USA" arranged around the perimeter. In the center of the seal is a stylized design of three human profiles facing to the right, stacked on top of each other.

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

November 19, 2014

BIO-ANALYSIS, INC. C/O REGINA O'MEARA PRINCIPAL 27735 TAMARA DRIVE YORBA LINDA CA 92887

Re: K133303 Trade/Device Name: Pantex Salivary Direct Testosterone Enzyme Immunoassay (EIA) Kit, Pantex Sample Collection Device Regulation Number: 21 CFR 862.1675 Regulation Name: Blood specimen collection device Regulatory Class: II Product Code: JKA, CDZ Dated: November 17, 2014 Received: November 19, 2014

Dear Ms. Regina O'Meara:

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

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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/ResourcesforYou/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,

# Courtney H. Lias -S

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) k133303

Device Name Pantex Salivary Direct Testosterone EIA Kit Pantex Sample Collection Device

#### Indications for Use (Describe)

The Pantex Salivary Direct Testosterone EIA Kit is an Enzyme Immunoassay (EIA) for the quantitative measurement of testosterone in human saliva collected with the Pantex Sample Collection Device. The measurement of testosterone is used in the diagnosis and treatment of disorders involving the male sex hormones (androgens), including primary and secondary hypogonadism, impotence in males hirsutism (excessive hair) and virilization (masculinization) due to tumors, polycystic ovaries and andrenogential syndromes.

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

| Prescription Use (Part 21 CFR 801 Subpart D) |
|----------------------------------------------|
| Over-The-Counter Use (21 CFR 801 Subpart C) |

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# 510(k) Summary Pantex Salivary Direct Testosterone EIA Kit

| Submitted by: | Pantex, Division of Bio-Analysis, Inc.<br>1701 Berkeley Street<br>Santa Monica, CA 90404<br>USA<br>[phone] |
|----------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Company Contact: | Romulo Garza, Ph.D., President & Senior Scientist |
| Date: | 10/27/14 |
| Trade Name:<br>Common Name:<br>Regulation Number and Panel:<br>Classification Product Code:<br>Classification: | Pantex Sample Collection Device<br>Blood Specimen Collection Device<br>862.1675 / Clinical Chemistry (75)<br>JKA<br>Class II |
| Trade Name:<br>Common Name:<br>Regulation Number and Panel:<br>Classification Product Code:<br>Classification: | Pantex Salivary Direct Testosterone EIA Kit<br>Enzyme immunoassay, Testosterone, salivary<br>862.1608 / Clinical Chemistry (75)<br>CDZ<br>Class I, Reserved |

#### Predicate Device:

Device Name: Company: 510(k) Reference:

Testosterone Luminescence Immunoassay IBL International GMBH K033786

#### 1. Device Description:

#### a. Test principle

The Pantex Salivary Direct Testosterone EIA kit, Cat # 635 is based on the competition principal and microplate separation. Testosterone calibrators of known concentration, unknown amounts of testosterone in saliva samples and a fixed amount of testosterone (analog) conjugated to horse radish peroxidase (Testosterone-HRP) compete for binding sites with a rabbit monoclonal antiserum bound to GARGG (goat anti-rabbit gamma globulin) coated wells of a microplate. After incubation, unbound components are washed away, enzyme substrate solution is added and a blue color formed. This reaction is stopped with an acid solution to produce a yellow color. The optical density is then read at 450 nm. The amount of Testosterone-HRP detected is inversely proportional to the amount of testosterone in a sample.

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### b. Pantex Sample Collection Device

The Pantex collection device is a 10 mL, non-sterile, plain polypropylene tube with a screw cap. It is provided as 50 units per package. The expiration date of the Pantex Sample Collection Device is 48 months from the day of manufacture.

#### Kit Contents ﻦ

The kit consists of a 96 well GARGG (goat anti-rabbit gamma globulin) coated microplate (12x8 breakable strip wells), one concentrated stock testosterone calibrator (62,500 pg/ml), one stock testosterone control (40,000 pg/ml), both traceable to the USP, Anti-Testosterone (rabbit monoclonal), 20X concentrated testosterone-peroxidase (analog), substrate solution, stop reaction solution and 10X concentrated wash solution.

### d. Intended Use

The Pantex Salivary Direct Testosterone ELA Kit is an Enzyme Immunoassay (EIA) for the quantitative measurement of testosterone in human saliva collected with the Pantex Sample Collection Device. The measurement of testosterone is used in the diagnosis and treatment of disorders involving the male sex hormones (androgens), including primary and secondary hypogonadism, impotence in males, and females hirsutism (excessive hair) and virilization (masculinization) due to tumors, polycystic ovaries and andrenogential syndromes.

| | Predicate Device: | New Device: |
|------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| | IBL Testosterone LIA<br>(k033786) | Pantex Salivary Direct Testosterone EIA Kit<br>(k133303) |
| Indications<br>for use | Luminescence Immunoassay (LIA) for the in-vitro diagnostic quantitative measurement of testosterone in saliva.<br>Measurement of testosterone (a male sex hormone) is used in the diagnosis of treatment of disorders involving the male sex hormones (androgens), including primary and secondary hypogonadism, impotence in males and in females, hirsutism (excessive hair) and virilization (masculinization) due to tumors, polycystic ovaries and adrenogenital syndromes. | The Pantex Salivary Direct Testosterone EIA Kit is an Enzyme Immunoassay (EIA) for the quantitative measurement of testosterone in human saliva collected with the Pantex Sample Collection Device. The measurement of testosterone is used in the diagnosis and treatment of disorders involving the male sex hormones (androgens), including primary and secondary hypogonadism, impotence in males and in females, hirsutism (excessive hair) and virilization (masculinization) due to tumors, polycystic ovaries and adrenogenital syndromes. |
| Analyte | Free Testosterone | Free Testosterone |
| Sample<br>Type | Saliva | Saliva |
| Method | Luminescence Immunoassay | Enzyme Immunoassay |

#### 2. Technology Comparison

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| Detection<br>Method | Microplate Luminometer reader | Microplate Colormetric reader | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | |
|-------------------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--|--|--|--|--|--|--|--|--|--|--|--|--|--|--|--|--|--|--|--|--|--|--|--|--|--|--|--|--|--|--|--|--|--|--|--|--|--|--|--|--|--|--|--|--|--|--|--|--|--|--|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--|--|--|--|--|--|--|--|--|--|--|--|--|--|--|--|--|--|--|--|--|--|--|--|--|--|--|--|--|--|--|--|--|--|--|
| Test<br>Principle | Luminescence immunoassays (LIA) are based on the competition principle. An unknown amount of antigen present in the sample and a fixed amount of enzyme labeled antigen compete for the binding sites of the antibodies coated onto the wells of a microplate. After incubation the wells are washed to stop the competition reaction. After addition of the luminescence substrate solution, the intensity of the luminescence measured is inversely proportional to the amount of the antigen in the sample. Results of samples can be determined directly using the standard curve. | Enzyme Immunoassays (EIA) are based on the competition principle. An unknown amount of antigen in the sample and a fixed amount of enzyme labeled antigen compete for the binding sites of antibodies coated onto a GARGG coated microplate. After incubation, the microplate is washed to stop the competition reaction and remove unbound components. After addition of the enzyme substrate (TMB) solution, color is formed and the optical density read. The intensity of the color measured is inversely proportional to the amount of the antigen present in the sample. Results of samples can be determined directly using the standard curve. | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | |
| Calculations | Quantitative determination with standard curve | Quantitative determination with standard curve | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | |
| Quality<br>Control | Use of reference controls is recommended | Use of reference controls is recommended | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | |
| Analytical<br>Measuring<br>Range<br>(AMR) | 2.0 pg/mL – 500 pg/mL | 6.4 pg/mL – 500 pg/mL | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | |
| Expected<br>Values<br>(Normal<br>range) | Female Age Median Range<br>5-95% N 20-29 19.0 5.5-49.0 40 30-39 17.3 5.2-49.0 39 40-49 13.8 4.5-49.0 47 50-59 13.2 3.6-49.0 53 60-69 15.8 2.9-38.8 33 Male 20-29 78.8 41.4-<br>142.5 55 30-39 58.8 31.8-<br>100.4 35 40-49 54.4 30.1-97.8 48 50-59 54.8 30.0-92.0 64 60-69 42.9 23.2-86.9 63 | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | Female Age Median<br>(pg/mL) 95% ref.<br>limits 90% CI<br>(pg/mL) N 20-49 17.20 3.91-40.9 3.70-50.0 120 50-70 14.75 4.51-34.17 4.2-35.1 120 Male 20-49 86.6 41.12-<br>142.16 33.6-<br>157.9 120 50-70 60.15 24.03-<br>119.52 22.0-<br>131.9 120 | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | |
| | Limits of<br>Detection | Analytical Sensitivity = 1.8 pg/mL | Limit of Blank (LoB)<br>1.8 pg/mL<br>Limit of Detection (LoD)<br>2.1 pg/mL<br>Limit of Quantitation (LoQ)<br>3.9 pg/mL | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | |
| | Saliva<br>Collection<br>Device | IBL Saliva Sampling Set (conical polypropylene tubes with 2.0 mL capacity). | Pantex sample collection device, Cat # PCD602. The Pantex collection device is a 10 mL, non-sterile, plain polypropylene tube with a screw cap. The expiration date of the device is 48 months from the day of | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | |

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| | | manufacture. |
|---------------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Interference | Did not test for interferences in<br>salivary testosterone measurement<br>when testing with caffeine, food,<br>nicotine, alcohol. or chewing gum. | An in-vivo study with caffeine, food,<br>nicotine, alcohol and chewing gum<br>not reveal significant interference in the<br>measurement of testosterone in saliva<br>using the Pantex Salivary Direct<br>Testosterone EIA Kit, Cat #635.<br>Table Continues |
| Stability and<br>Storage of<br>Kit reagents | Stated as stable at 2-8°C until kits<br>expiration date | 4 months at 2-8°C. The stability study is<br>still on-going. |

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#### 3. Test Summary

#### Performance Characteristics

The performance characteristics of the Pantex Salivary Direct Testosterone EIA Kit were based on evaluations by the following analytical performance tests. All saliva samples used in the performance testing were collected and processed through the Pantex Sample Collection Device (Cat # PCD602).

#### a. Precision/Reproducibility

The intra-assay precision was determined from 20 replicates of low, medium and high saliva pools.

| Sample | N | Mean (pg/mL) | Standard<br>Deviation<br>(pg/mL) | %CV |
|--------|----|--------------|----------------------------------|-----|
| Low | 20 | 21.0 | 1.063 | 5.1 |
| Medium | 20 | 174.7 | 3.641 | 2.1 |
| High | 20 | 318.7 | 13.517 | 4.2 |

The inter-assay precision was determined from the mean of the average of duplicates for 12 separate assays with low, medium and high saliva pools.

| Sample | N | Mean (pg/mL) | Standard<br>Deviation<br>(pg/mL) | %CV |
|--------|----|--------------|----------------------------------|-----|
| Low | 12 | 19.1 | 1.232 | 6.4 |
| Medium | 12 | 155.6 | 7.106 | 4.6 |
| High | 12 | 285.5 | 8.847 | 3.1 |

The inter-lot precision was determined by duplicate measurements of five (5) saliva samples and two levels of one (1) spiked control in saliva like matrix, using three (3) different reagent lots. The results of intra-assay, inter-assay and inter-lot variation concluded a %CV of ≤10% for each sample tested.

| Saliva<br>Samples<br>ID | Lot # 007<br>mean<br>(pg/ml) | Lot # 008<br>mean<br>(pg/ml) | Lot # 009<br>mean<br>(pg/ml) | Inter-lot<br>mean<br>(pg/ml) | Inter-lot<br>Std. Dev.<br>(pg/ml) | Inter-lot<br>%CV<br>(pg/ml) |
|-------------------------|------------------------------|------------------------------|------------------------------|------------------------------|-----------------------------------|-----------------------------|
| 1 | 314.6 | 324.0 | 320.1 | 319.6 | 4.723 | 1.5 |
| 2 | 101.8 | 97.4 | 99.3 | 99.5 | 2.207 | 2.2 |
| 3 | 69.2 | 69.4 | 61.0 | 66.5 | 4.793 | 7.2 |
| 4 | 8.8 | 9.7 | 9.9 | 9.5 | 0.586 | 6.2 |
| 5 | 23.8 | 21.7 | 21.3 | 22.3 | 1.343 | 6.0 |
| C1 | 21.1 | 19.5 | 20.5 | 20.4 | 0.808 | 4.0 |
| C2 | 210.2 | 196.3 | 212.2 | 206.2 | 8.660 | 4.2 |

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#### Repeatability

This study was conducted during 5 days of a familiarization period and 20 days of testing. Two assays were performed daily with a minimum of 1 hour between assays. Three (3) different reagents lots and three (3) saliva pools were used for the study (low, medium and high concentration). The pools were aliquoted and frozen until the day of assay.

| Sample<br>Concentration | N | Mean<br>(pg/mL) | Repeatability | | Total Precision | |
|-------------------------|-----|-----------------|---------------|-----|-----------------|-----|
| Low | 100 | 19.4 | 0.8 | 3.8 | 1.1 | 5.6 |
| Medium | 100 | 155.3 | 3.7 | 2.5 | 7.9 | 5.3 |
| High | 100 | 275.6 | 7.4 | 2.5 | 11.9 | 4.0 |
| Control 1 | 100 | 19.3 | 0.9 | 4.6 | 1.2 | 5.8 |
| Control 2 | 100 | 201.4 | 3.8 | 1.8 | 8.2 | 4.0 |

#### Precision Study Data Summary

#### b. Linearity

Ten (10) sample concentrations that span the assay measuring range were prepared and assayed per CLSI EP6-A, Evaluation of the Linearity of Quantitative Measurement Procedures.

S=10 samples (dilutions)

Concentration = (C1*V1 + C10*V10)/(V1+V10)

| | C1 | V1 | C10 | V10 | Calculated<br>Concentration | Observed<br>Concentration | Recovery |
|----|-------|-------|-------|-------|-----------------------------|---------------------------|----------|
| | pg/ml | ml | pg/ml | ml | pg/ml | pg/ml | % |
| 1 | | | | * | 5.5 | 5.2 | 94.5 |
| 2 | 5.2 | 0.889 | 631.5 | 0.111 | 74.7 | 76.3 | 102.1 |
| 3 | 5.2 | 0.778 | 631.5 | 0.222 | 144.2 | 153.6 | 106.5 |
| 4 | 5.2 | 0.667 | 631.5 | 0.333 | 213.8 | 216.1 | 101.1 |
| 5 | 5.2 | 0.556 | 631.5 | 0.444 | 283.3 | 275.3 | 97.2 |
| 6 | 5.2 | 0.444 | 631.5 | 0.556 | 353.4 | 323.6 | 91.6 |
| 7 | 5.2 | 0.333 | 631.5 | 0.667 | 422.9 | 401.8 | 95.0 |
| 8 | 5.2 | 0.222 | 631.5 | 0.778 | 492.5 | 461.7 | 93.8 |
| 9 | 5.2 | 0.111 | 631.5 | 0.889 | 562.0 | 561.4 | 99.9 |
| 10 | | | | * | 650.0 | 631.5 | 97.2 |

* Targets of low and high sample concentrations.

The results demonstrate that the assay is linear from 5.5 - 650.0 pg/mL. (Y=0.9597x + 3.335, R2 = 0.99685)

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#### c. Recovery

| Seven (7) saliva samples containing different levels of endogenous testosterone were spiked with known | | |
|--------------------------------------------------------------------------------------------------------|--|--|
| quantities of testosterone and assayed. | | |

| Sample | Endogenous<br>(pg/ml) | Added<br>(pg/ml) | Expected<br>(pg/ml) | Observed<br>(pg/ml) | Recovery<br>(%) |
|--------|-----------------------|------------------|---------------------|---------------------|-----------------|
| 1 | 10.000 | 7.813 | 17.813 | 19.400 | 108.9 |
| 2 | 19.000 | 15.625 | 34.625 | 36.300 | 104.8 |
| 3 | 27.600 | 31.250 | 58.850 | 54.500 | 92.6 |
| 4 | 28.200 | 62.500 | 90.700 | 95.100 | 104.9 |
| 5 | 9.800 | 125.000 | 134.800 | 127.900 | 94.9 |
| 6 | 103.700 | 250.000 | 353.700 | 346.800 | 98.0 |
| 7 | 38.600 | 500.000 | 538.600 | 497.600 | 92.4 |

#### d. Manual Dilution

A 1:10 sample manual dilution study was performed utilizing six (6) samples to verify the accuracy of the dilution procedure with a dilution factor of 10. The following table indicates the summary of results:

| Subject | Observed<br>Endogenous<br>Value<br>(pg/mL) | Testosterone<br>Spiked<br>(pg/mL) | Expected<br>Value<br>(pg/mL) | Observed<br>1:10 diluted<br>Value<br>(pg/mL) | Applied<br>Factor 10<br>(pg/mL) | Recovery<br>(%) |
|---------|--------------------------------------------|-----------------------------------|------------------------------|----------------------------------------------|---------------------------------|-----------------|
| AL-209 | 346.7 | 0 | 346.7 | 33.3 | 333 | 96.0 |
| AL-210 | 274.3 | 0 | 274.3 | 26.5 | 265 | 96.6 |
| *AL-225 | 133.1 | 500 | 633.1 | 67.2 | 672 | 106.1 |
| AL-227 | 229.5 | 0 | 229.5 | 22.7 | 227 | 98.9 |
| *AL-228 | 100 | 750 | 850 | 93.1 | 931 | 109.5 |
| *AL-247 | 125.8 | 1000 | 1125.8 | 121.3 | 1213 | 107.7 |

* Spiked samples.

#### 4. Reagent Stability/Sample Stability/Expected Values

The long term stability study for reagents stored at 2 - 8° C is currently on-going; however, at the time of this executive summary report the expiration date supported by the results obtained on the stability study is 4 months from the manufacturing date when the reagents are stored at 2 - 8° C. As the stability study progresses, the expiration date will be upgraded accordingly.

#### a. Open Vial Stability

| Condition | Stability | Storage Temperature |
|-----------------------------------------------|-----------|---------------------|
| Open vial stability | 31 days | 2 - 8°C |
| Working Testosterone-HRP Conjugate solution | 31 days | 2 - 8°C |
| Diluted working testosterone stock calibrator | 31 days | 2 - 8°C |
| Diluted working testosterone stock control | 31 days | 2 - 8°C |

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### b.Sample Stability

| Storage | 20-28°C | 37°C | 2-8°C | ≤-15°C<br>(7 freeze/thaw cycles) | ≤-15°C<br>(Long term) |
|-----------|--------------|--------------|--------------|----------------------------------|-----------------------|
| Stability | Up to 7 days | Up to 7 days | Up to 7 days | Up to 7 days | Up to 180 days |

#### Sample Shipping, Handling and Storage Conditions Stability C.

The samples were shipped to a location outside the testing site on 7/2/14 and returned to the testing site on 7/11/14 representing a total of 9 days in transit; thereafter, the samples were stored at 2-8℃, 20-28°C, 37°C and ≤-15°C for 7 days and tested after 3 days and 7 days. Based on the results obtained, it appears that the samples retained their physical integrity and testosterone levels remained unaffected for 9 days in transit and up to 7 days when stored at the stated temperatures.

| Storage | 2-8°C and<br>room<br>temperature<br>during transit | 20-28°C | 37°C | 2-8°C | <-15°C | |
|------------------------------------------------------------------------|----------------------------------------------------|---------|--------------|--------------|--------------|-----------------|
| In transit<br>stability<br>upon<br>return to<br>the<br>testing<br>site | Up to 9 days | | | | | |
| | After<br>returning<br>stability | | Up to 7 days | Up to 7 days | Up to 7 days | Up to 7<br>days |

#### 5. Expected Reference Values:

The reference range was established by testing 120 male saliva samples and 120 female saliva samples to have an equal number of male samples. The reference range and median were calculated using CLSI C28-A3 as a guide. The following tables indicate the summary of the results.

#### Female Expected Values:

| Subjects<br>(Number) | Age<br>(Age) | Median (pg/mL) | 95% Reference<br>Limits (pg/mL) | 90% CI (pg/mL) |
|----------------------|--------------|----------------|---------------------------------|----------------|
| 120 | 20-49 | 17.20 | 3.91 - 40.99 | 3.70 - 50.0 |
| 120 | 50-70 | 14.75 | 4.51 - 34.17 | 4.2 - 35.1 |

#### Male Expected Values:

| Subjects<br>(Number) | Age<br>(Age) | Median (pg/mL) | 95% Reference<br>Limits (pg/mL) | 90% CI (pg/mL) |
|----------------------|--------------|----------------|---------------------------------|----------------|
| 120 | 20-49 | 86.6 | 41.12 - 142.16 | 33.6 - 157.9 |
| 120 | 50-70 | 60.15 | 24.03 - 119.52 | 22.0 - 131.9 |

{11}------------------------------------------------

#### 6. Detection Limits:

The Detection Limit Study for determining the limit of the blank (LoB), limit of detection (LoD) and the limit of quantitation (LoQ) for the Pantex Salivary Direct Testosterone EIA Kit, Cat #635 was performed using several low testosterone samples and two different reagent lot numbers that were assayed twice per day over a period of 3 days. (Reference, CLSI EP 17-A, protocols for Determination of Limits of Detection and Limits of Quantitation).

| Limit of the Blank<br>(LoB)<br>pg/mL | Limit of Detection<br>(LoD)<br>pg/mL | Limit of Quantitation<br>(LoQ)<br>pg/mL |
|--------------------------------------|--------------------------------------|-----------------------------------------|
| 1.8 | 2.1 | 3.9 |

## 7. Analytical Specificity/Cross Reactivity

The cross reactivity of the antiserum was determined by spiking three (3) saliva pools (low, medium and high) with two (2) concentrations (10,000 pg/ml and 20,0000 pg/mL) of each potential cross reactant. The percent cross reaction was calculated using the following equation as per CLSI (EPT-A2) guidelines.

The % Cross-reactivity = 100* [measured value - true value]

[concentration of interferant ]

Where, Measured value = is the spiked measured value = is the un-spiked obtained value and concentration of interferant = is the amount of the compound spiked

| Compound | Low Testosterone<br>sample (% Cross<br>Reactivity) | Medium Testosterone<br>Sample (% Cross<br>Reactivity) | High Testosterone<br>sample (% Cross<br>Reactivity) |
|--------------------------------|----------------------------------------------------|-------------------------------------------------------|-----------------------------------------------------|
| Testosterone | 100 | 100 | 100 |
| 11 ß-OH Testosterone | 0.407 | 0.486 | 0.242 |
| 11 a-OH Testosterone | 0.869 | 1.033 | 0.601 |
| 5 α-Dihydro-Testosterone | 5.540 | 5.474 | 5.347 |
| Androstenedione | 0.718 | 0.876 | 0.604 |
| Methyl Testosterone | 1.410 | 1.597 | 0.889 |
| Testosterone SO4 | 0.005 | 0.010 | 0.067 |
| DHEA SO4 | 0.001 | 0.001 | 0.006 |
| DHEA | 0.001 | 0.003 | 0.006 |
| 7-Keto DHEA | 0.003 | 0.004 | 0.008 |
| Progesterone | 0.243 | 0.276 | 0.179 |
| Cortisol | 0.005 | 0.002 | 0.005 |
| 17 ß-Estradiol | 0.175 | 0.173 | 0.135 |
| 17 a-Estradiol | 0.007 | 0.002 | 0.008 |
| Cortisone | 0.014 | 0.013 | 0.013 |
| Danazol | 0.011 | 0.018 | 0.063 |
| Dexamethasone | 0.007 | 0.025 | 0.051 |
| D-5-Androstene-3 ß, 17 ß -diol | 0.691 | 0.827 | 0.497 |
| Estrone | 0.015 | 0.003 | 0.012 |
| Ethisterone | 0.037 | 0.050 | 0.084 |
| Norgestrel | 0.033 | 0.020 | 0.071 |

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

| Testosterone propionate | 0.068 | 0.065 | 0.134 |
|-------------------------------|-------|-------|-------|
| 5 α-Androstane-3 β, 17 β-diol | 2.497 | 2.745 | 2.571 |
| 11-Keto Testosterone | 0.137 | 0.158 | 0.079 |
| Prednisone | 0.038 | 0.009 | 0.018 |
| Prednisolone | 0.023 | 0.007 | 0.028 |

### 8. Method Comparison Studies

A comparative study was performed between the Pantex Salivary Direct Testosterone EIA Kit Cat #635 and a FDA cleared predicate device. A total of 106 samples were used for the study (range 6.45-458.35 pg/ml) of which 9 samples were spiked representing 8.5 % (range 252.1 - 424.2 pg/mL) of the total number of samples. The results show the following regression and correlation statistics.

| Linear Regression equation | $Y = 0.9035X + 5.81$ |
|----------------------------|----------------------|
| Correlation | $R^2 = 0.98$ |

### Traceability

The synthetic Testosterone used in the manufacturing of the Testosterone Stock Calibrator and the Testosterone Stock Control is traceable to the U.S. Pharmacopeia (USP) catalog number 1646009, Lot number J0G360.

### 9. Interference Studies

Using CLSI-A2 Interference Testing in Clinical Chemistry as a guide, an in-vivo simulated experiment was performed after collecting saliva samples from subjects before (control) and after contact (exposed) with five (5) commonly consumed products (coffee, food, gum, alcohol and cigarette smoke). The control and exposed samples were tested with the Pantex Salivary Direct Testosterone EIA Kit, Cat #635. The results indicated no significant differences in salivary testosterone values between the controls and exposed samples.

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

| Sample ID | Interference<br>Substance | Collection Time | Observed value<br>(pg/mL) | Recovery from<br>Control |
|-----------|---------------------------|-----------------|---------------------------|--------------------------|
| JGA-Day1 | No food | 8:00 AM | 23.1 | |
| JGB-Day1 | Food | 8:15 AM | 22.5 | 97.4 |
| JGA-Day 2 | No coffee | 8:00 AM | 25.7 | |
| JGB-Day 2 | Coffee | 8:15 AM | 26.2 | 101.9 |
| JGA-Day3 | No wine | 8:00 AM | 21.8 | |
| | Wine | 8:15 AM | 22.0 | 100.9 |
| JGA-Day 4 | No gum | 8:00 AM | 15.4 | |
| | Gum | 8:15 AM | 16.2 | 105.2 |
| JGA-Day 5 | No smoke | 8:00 AM | 25.5 | |
| | Smoke | 8:15 AM | 25.3 | 99.2 |
| MGA-Day1 | No food | 8:00 AM | 42.0 | |
| MGB-Day1 | Food | 8:15 AM | 39.2 | 93.3 |
| MGA-Day 2 | No coffee | 8:00 AM | 41.1 | |
| MGB-Day 2 | Coffee | 8:15 AM | 39.9 | 97.1 |
| MGA-Day3 | No wine | 8:00 AM | 28.4 | |
| MGB-Day 3 | Wine | 8:15 AM | 29.0 | 102.1 |
| MGA-Day 4 | No gum | 8:00 AM | 47.1 | |
| MGB-Day 4 | Gum | 8:15 AM | 46.8 | 99.4 |
| MGA-Day 5 | No smoke | 8:00 AM | 40.0 | |
| MGB-Day 5 | Smoke | 8:15 AM | 38.7 | 96.8 |
| DGA-Day1 | No food | 8:00 AM | 86.1 | |
| DGB-Day1 | Food | 8:15 AM | 87.9 | 102.1 |
| DGA-Day 2 | No coffee | 8:00 AM | 63.1 | |
| DGB-Day 2 | Coffee | 8:15 AM | 65.5 | 103.8 |
| DGA-Day3 | No wine | 8:00 AM | 123.9 | |
| DGB-Day 3 | Wine | 8:15 AM | 118.7 | 95.8 |
| DGA-Day 4 | No gum | 8:00 AM | 99.1 | |
| DGB-Day 4 | Gum | 8:15 AM | 101.0 | 101.9 |
| DGA-Day 5 | No smoke | 8:00 AM | 70.5 | |
| DGB-Day 5 | Smoke | 8:15 AM | 71.7 | 101.7 |

| | In-vivo simulated experiment results | |
|--|--------------------------------------|--|
| | | |

Table Continues

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

| Sample ID | Interference<br>Substance | Collection Time | Observed value<br>(pg/mL) | Recovery from<br>Control |
|-----------|---------------------------|-----------------|---------------------------|--------------------------|
| RGA-Day1 | No food | 8:00 AM | 43.7 | |
| RGB-Day1 | Food | 8:15 AM | 41.3 | 94.5 |
| RGA-Day 2 | No coffee | 8:00 AM | 41.7 | |
| RGB-Day 2 | Coffee | 8:15 AM | 43.8 | 105.0 |
| RGA-Day3 | No wine | 8:00 AM | 33.1 | |
| RGB-Day 3 | Wine | 8:15 AM | 32.6 | 98.5 |
| RGA-Day 4 | No gum | 8:00 AM | 36.8 | |
| RGB-Day 4 | Gum | 8:15 AM | 37.3 | 101.4 |
| RGA-Day 5 | No smoke | 8:00 AM | 35.4 | |
| RGB-Day 5 | Smoke | 8:15 AM | 36.9 | 104.2 |

Using CLSI-A2 as a guide, an interference study was conducted using three (3) potential interferants whole blood, sodium azide and thimerosal to determine their effects on salivary testosterone samples. The interferants were tested using the following concentrations 0.05%, 0.10%, 0.25% and 0.5%. Based on the results, it was determined that whole blood, thimerosal and sodium azide affect salivary testosterone samples by either increasing or suppressing testosterone values. Saliva samples containing any of the three interferants in question should be avoided when using the Pantex Salivary Direct Testosterone EIA Kit, Cat # 635.

#### 10. Conclusion:

Taken together, the performance characteristics, comparison studies with a predicate device and acceptable statistical performance studies in this 510(k) Class I Reserved submission demonstrates that the Pantex Salivary Direct Testosterone EIA Kit, Cat #635, is safe and effective for its intended use and is substantially equivalent to the predicate device