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

Indications for 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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# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY ASSAY ONLY TEMPLATE

A. 510(k) Number:
k133303

B. Purpose for Submission:
New device

C. Measurand:
Testosterone in human saliva

D. Type of Test:
Quantitative, Enzyme Immunoassay (EIA)

E. Applicant:
Bio-analysis, Inc.

F. Proprietary and Established Names:
Pantex Salivary Direct Testosterone Enzyme Immunoassay (EIA) Kit
Pantex Sample Collection Device

G. Regulatory Information:

| Product Code | Classification | Regulation Section | Panel |
| --- | --- | --- | --- |
| JKA, Tubes, Vials, Systems, Serum Separators, Blood Collection | Class II | 21 CFR 862.1675; Blood specimen collection device | Clinical Chemistry (75) |
| CDZ, Radioimmunoassay, Testosterones and Dihydrotestosterone | Class I, reserved | 21 CFR 862.1680; Testosterone test system | Clinical Chemistry (75) |

H. Intended Use:

1. Intended use(s):
Refer to Indications for use below.

2. Indication(s) for use:
The Pantex Salivary Direct Testosterone EIA Kit is an Enzyme Immunoassay

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(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.

3. Special conditions for use statement(s):

For Prescription Use only.

Oral fluid for this test should be collected using the Pantex Sample Collection Device.

4. Special instrument requirements:

Microplate colorimeter reader capable of reading at 450 nm with 4 parameter data reduction or comparable software.

I. Device Description:

The kit consists of the following components:

- A 96 well GARGG (goat anti-rabbit gamma globulin) coated microplate (12x8 breakable strip wells) placed in a re-sealable foil bag with desiccant. One 96 well kit is sufficient for 39 duplicate patient measurements.
- Assay buffer (zero calibrator), one bottle (20 mL).
- Concentrated stock testosterone (synthetic) calibrator (62,500 pg/mL in BSA buffer), one bottle (250 µL).
- The concentrated stock testosterone calibrator solution in BSA and the assay buffer are used to prepare seven salivary testosterone working calibrators at 625.0, 250.0, 100.0, 40.0, 16.0, 6.4 and 0 pg/mL testosterone concentrations.
- Concentrated stock testosterone (synthetic) control (40,000 pg/ml), one bottle (250 µL).
- The concentrated stock testosterone control and assay buffer are used to prepare the working control #2 at 200 pg/mL testosterone concentration. The working control #2 solution is used to prepare working control #1 at 20 pg/mL using the assay buffer as a diluent.
- Anti-Testosterone rabbit monoclonal antibody, one bottle (6 mL).
- Concentrated (20X) testosterone-horse radish peroxidase (HRP), one amber bottle, 350 µL. Testosterone derivative is conjugated to HRP. The solution is yellow and light sensitive.
- A testosterone-HRP conjugate buffer, one bottle, 6 mL of phosphate buffered saline, pH 7.4. To be used to prepare the testosterone-HRP working reagent only.

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- The testosterone-HRP working reagent is prepared by diluting testosterone-HRP concentrate 1:20 with the conjugate buffer.
- The color development reagent contains the substrate (tetramethylbenzidine (TMB) plus hydrogen peroxide), one amber plastic bottle (15 mL), light sensitive.
- The stop reaction solution contains a mixture of diluted sulfuric and hydrochloric acid, one bottle (15 mL).
- Concentrated (10X) wash solution, one bottle containing 50 mL of phosphate buffered saline, pH 7.4. Prior to use, it should be diluted 1:10 with deionized water.

The Pantex Sample Collection Device (cat # PCD 602) for saliva collection is required, but not supplied with the kit. It is sold separately by the sponsor. This device is a 10 mL non-sterile, plain polypropylene capped tube with no preservatives or other active ingredients. It is sold as a package of 50 units. The expiration date is 48 months from the day of manufacture.

## J. Substantial Equivalence Information:

1. Predicate device name(s):

IBL Testosterone Luminescence Immunoassay (LIA)

2. Predicate 510(k) number(s):

k033786

3. Comparison with predicate:

| Items | Pantex Salivary Direct Testosterone EIA Kit (k133303; Candidate Device) | IBL Testosterone LIA (k033786; Predicate Device) |
| --- | --- | --- |
| Intended Use | Immunoassay for the quantitative measurement of testosterone in saliva. | Same |
| Analyte | Testosterone | Same |
| Sample Type | Oral Fluid (Saliva) | Same |
| Methodology | Enzyme Immunoassay | Luminescence Immunoassay |
| Detection Instrument | Microplate Colorimeter Reader | Microplate Luminometer Reader |
| Test Principle | Competition principle. | Same |
| Quality Controls | Use of two controls (20 and 200 pg/mL) recommended with each run. | Use of two controls (Level I and II) recommended with each run. |
| Calibrators | Multiple (seven) calibrators used for generating a standard curve. | Same |
| Calculations | Quantitative determination using | Same |

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| | a standard curve. | |
| --- | --- | --- |
| Saliva Collection Device | Pantex Sample Collection Device ) is a 10 mL, non-sterile, plain polypropylene tube with a screw cap. | IBL Saliva Sampling Set (conical polypropylene tubes with 2.0 mL capacity). |
| Measuring Range | 6.4 pg/mL to 500 pg/mL. | 2 pg/mL to 500 pg/mL. |

K. Standard/Guidance Document Referenced (if applicable):

CLSI EP5-A2 Evaluation of Precision Performance of Quantitative Measurement Methods (August 2004).

CLSI EP6-A Evaluation of the Linearity of Quantitative Measurement Procedures: A Statistical Approach (April 2003).

CLSI EP9-A2IR Method Comparison and Bias Estimation using Patient Samples (July 2010).

CLSI EP17-A Protocols for Determination of Limits of Detection and Limits of Quantitation (October 2004).

CLSI C28-A3 Defining, Establishing, and Verifying Reference Intervals in the Clinical Laboratory (November 2008).

CLSI EP7-A2 Interference Testing in Clinical Chemistry (November 2005).

CLSI EP25-A Evaluation of Stability of In-Vitro Diagnostic Reagents (September 2009).

L. Test Principle:

The Pantex Salivary Direct Testosterone EIA kit 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.

M. Performance Characteristics (if/when applicable):

1. Analytical performance:

a. Precision/Reproducibility:

Precision studies were performed at one site by one operator over 25 days with two runs per day and duplicate testing per run using three lots of reagents

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N=100). Two levels of controls and three pools of saliva testosterone samples at low, medium and high concentrations were tested. During each assay run, calibrators and two controls were tested in duplicate. The imprecision results for each reagent lot and combined three reagent lots are as shown below:

Reagents Lot 1 (N=36)

| Sample Testosterone Concentration | Mean (pg/mL) | Repeatability | | Total Precision | |
| --- | --- | --- | --- | --- | --- |
| | | SD (pg/mL) | % CV | SD (pg/mL) | % CV |
| Low | 19.4 | 0.8 | 3.9 | 1.0 | 5.0 |
| Medium | 155.0 | 2.3 | 1.6 | 7.1 | 4.7 |
| High | 275.3 | 5.7 | 1.9 | 12.3 | 4.1 |
| Control 1 | 19.2 | 0.9 | 4.5 | 1.1 | 5.7 |
| Control 2 | 202.9 | 3.8 | 1.9 | 4.5 | 2.2 |

Reagents Lot 2 (N=32)

| Sample Testosterone Concentration | Mean (pg/mL) | Repeatability | | Total Precision | |
| --- | --- | --- | --- | --- | --- |
| | | SD (pg/mL) | % CV | SD (pg/mL) | % CV |
| Low | 19.3 | 0.9 | 4.5 | 1.0 | 5.0 |
| Medium | 151.2 | 4.7 | 3.2 | 8.0 | 5.3 |
| High | 272.2 | 7.3 | 2.4 | 11.3 | 3.8 |
| Control 1 | 19.0 | 1.0 | 4.9 | 1.1 | 5.5 |
| Control 2 | 198.7 | 3.5 | 1.7 | 9.3 | 4.5 |

Reagents Lot 3 (N=32)

| Sample Testosterone Concentration | Mean (pg/mL) | Repeatability | | Total Precision | |
| --- | --- | --- | --- | --- | --- |
| | | SD (pg/mL) | % CV | SD (pg/mL) | % CV |
| Low | 19.5 | 0.5 | 2.6 | 1.3 | 6.7 |
| Medium | 159.85 | 3.86 | 2.6 | 6.51 | 4.3 |
| High | 279.3 | 8.92 | 3.0 | 11.79 | 3.9 |
| Control 1 | 19.7 | 0.9 | 4.5 | 1.3 | 6.6 |
| Control 2 | 202.4 | 4.1 | 2.0 | 10.2 | 4.9 |

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Combined Reagents Lots 1, 2 and 3 (N=100)

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

An inter-assay precision study was conducted by testing pooled saliva samples at low, medium and high testosterone concentrations in duplicate in twelve separate assays using one lot of reagents. The mean of the average of duplicate measurements of twelve separate assays was used to calculate the SD and $\% \mathrm{CV}$ . The results are as shown below:

| Sample | N | Mean (pg/mL) | SD (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 intra-assay precision was determined from testing twenty measurements of low, medium and high pools with the same assay using one lot of reagents. The results are as shown below:

| Sample | N | Mean (pg/mL) | SD (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-lot precision was determined from testing in duplicate measurements of five saliva samples and two levels of control using three reagents lots. The results are as shown below:

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| Saliva Samples ID | Reagents Lot 1 mean (pg/mL) | Reagents Lot 2 mean (pg/mL) | Reagents Lot 3 mean (pg/mL) | Inter-lot mean (pg/mL) | Inter-lot SD (pg/mL) | Inter-lot %CV (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 |

b. Linearity/assay reportable range:

A linearity study was performed in accordance with the CLSI EP6-A guideline. Ten samples were prepared by serial dilution using a low saliva testosterone sample and a high spiked saliva testosterone sample. The samples ranging in concentration from $5.5\mathrm{pg / mL}$ to $650.0~\mathrm{pg / mL}$ were tested in duplicate. The mean observed values (y-axis) were plotted against the calculated target values (x-axis). The polynomial regression analysis of the data suggested the best fit with the first order model, resulting in the linear regression equation $\mathrm{y} = 0.9597\mathrm{x} + 3.335$ , $\mathrm{R}^2 = 0.99685$ . Additionally, the percent recovery between mean calculated target values and the observed values ranged from $91.6\%$ to $106.5\%$ . The linearity study results suggest that the candidate device is linear from 5.5 to $650.0~\mathrm{pg / mL}$ of testosterone.

The data support the sponsor's claimed measuring range of $6.4\mathrm{pg / mL}$ to $500.0~\mathrm{pg / mL}$ .

Since the testosterone used to prepare calibrators and controls is synthesized, it is also important to show that the synthetic testosterone is read accurately by the test system when spiked into native saliva samples. Therefore, a Spiking Recovery Study was performed to evaluate whether added testosterone to native saliva sample can be accurately recovered over the claimed measuring range of the candidate device. Seven saliva samples were collected in the Pantex Sample Collection Device. The samples were aliquoted and stored frozen. The frozen aliquots of the seven samples were thawed, centrifuged to remove mucins, equilibrated to room temperature and assayed with the candidate device to measure the testosterone concentration. The samples were then spiked to approximate expected saliva testosterone levels (17.8 pg/mL to 353.7 pg/mL) within the claimed measuring range of the candidate device, and assayed with the candidate device. The percent recoveries between the observed value and the expected value for the seven samples were calculated and ranged from 92.6% to 108.9%.

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A manual dilution study was conducted to show accuracy of the test results obtained by 1:10 dilution of unknown high testosterone samples. The percent recovery obtained on the six 1:10 diluted high testosterone samples (229.5, 274.3, 346.7, 633.1, 850 and 1125.8 pg/mL) were calculated and ranged from 96.0% to 109.5%. The sponsor has included the following in the device labeling, “Samples with testosterone values greater than 500pg/mL should be diluted 1:10 with zero (0) calibrator and re-run for accuracy. Obtain the final testosterone concentration by multiplying the diluted sample by 10.”

c. Traceability, Stability, Expected values (controls, calibrators, or methods):

The concentrations of all testosterone calibrators and controls are traceable to the internal standard, which is traceable to the referenced material, US Pharmacopeia (USP) reference standard.

The stability study protocols to determine the shelf-life (close vial) and the stability of in-use (open vial) kit components were reviewed and found to be acceptable. Based on the real time stability studies, the shelf life of the reagent kit is 4 months when stored at 2 to 8 °C. Based on the open vial stability study, the in-use kit components/reagents, and the working solutions were determined to be stable for 31 days when stored at 2 to 8 °C.

Value assignments for calibrators and controls: The testosterone stock calibrator and the testosterone stock control are traceable to the US Pharmacopeia (USP) Cat. No. 1646009, Lot J0G360, and are prepared gravimetrically and are actual to the stated testosterone concentration on its label. Sponsor states that prior to release, the values of the calibrator and control are verified by assaying repeatedly on the Pantex Salivary Direct Testosterone EIA Kit.

Calibrator’s target value is 62,500 pg/mL

Control’s target value is 40,000 pg/mL

Saliva sample stability studies were performed to evaluate whether the testosterone in the sample is stable when the sample is exposed to different temperature conditions over several days. Two saliva samples, one from male (spiked to obtain a high testosterone sample at 222.2pg/mL) and one from female (low testosterone sample at 14.2pg/mL), were collected in the Pantex Sample Collection Device. The samples were aliquoted and stored frozen. For the stability study testing, the aliquots were thawed, centrifuged to remove mucins, and exposed to the following storage conditions viz. at 2 to 8 °C, 20 to 28 °C, 37 °C, ≤ -15 °C (for long term sample storage), and at ≤ -15 °C for testing seven freeze/thaw cycles. The samples were assayed once every day with the candidate device for seven days. Based on the calculated percent recoveries (ranged from 91.7% to 100%) between the observed value and the zero day value, the saliva samples are stable for seven days when stored at 2 to 8 °C, 20 to 28 °C, 37 °C, for up to 180 days at ≤ -15 °C, and stable to seven freeze/thaw cycles up to seven days at ≤ -15 °C.

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A sample shipping and handling study was conducted to evaluate whether the stability of testosterone in saliva is affected by shipping, handling and storage conditions. Five saliva samples at different testosterone concentrations (15.4, 28.2, 44.8, 106.4 and 221.9 pg/mL) were collected in the Pantex Sample Collection Device. The samples were aliquoted and stored frozen. For the study testing, the aliquots were thawed, centrifuged to remove mucins, and tested on the day of shipping (Day zero read prior to shipping). The samples were shipped out and remained in transit for 9 days at 19 to 24 °C (Site A), 12 to 28 °C (Site B) and 24 to 41 °C (Site C). On receiving the samples, the samples were frozen until the assay date. On the day of the assay, the samples were thawed and centrifuge to remove mucins. The aliquots of the five samples were then stored the following storage conditions at 2 to 8 °C, 20 to 28 °C, 37 °C and ≤ -15 °C for seven days. The samples were tested with the candidate device on day three and seven. The samples were shown to be stable (recovery ranged from 91.1% to 99.8%) in transit for 9 days at 2 to 8 °C and 20 to 28 °C, followed by storage at 2 to 8 °C, 20 to 28 °C, 37 °C and ≤ -15 °C for seven days.

d. Detection limit:

The limits of detection were determined in accordance with the CLSI EP17-A guideline.

The limit of blank (LoB) study was conducted using four low or non-detectable testosterone saliva samples. The four testosterone saliva samples were tested with the candidate device 96 times over three days using two reagent lots (16 testings per day per reagent lot; 48 testing per lot). LoB was calculated using the nonparametric option of LoB calculation. LoB was determined to be 1.8 pg/mL.

The limit of detection (LoD) was determined using four low salivary sample containing testosterone concentration at approximately ≤ 3.0 pg/mL. Each low sample was tested with the candidate device in quadruplicates, two tests per day for a period of three days using two reagent lots to generate 12 testosterone measurements over three days per reagent lot (4 testing per day per reagent lot). Using the calculated standard deviation in the formula LoD = LoB + (1.645*SD), the LoD was determined to be 2.1 pg/mL.

The limit of quantitation (LoQ) study was conducted using four low testosterone saliva samples at concentrations > 3.0 pg/mL and < 6.4 pg/mL. Each sample was tested with the candidate device in quadruplicates, two tests per day for a period of three days using two reagent lots to generate 12 testosterone measurements over three days per reagent lot. The LoQ was estimated as 3.9 pg/mL.

The sponsor’s claimed measuring range is 6.4 pg/mL to 500 pg/mL.

e. Analytical specificity:

Interference studies were performed following the CLSI EP7-A2 guideline to evaluate whether commonly orally consumed substances like food, coffee,

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wine, chewing gum and cigarette smoking interfere with the quantitation of saliva testosterone using the candidate device. Four individuals were enrolled in the study for five days. Each day at the same time (8:00 AM) a control saliva sample was collected, followed by exposure to one of the interferents in the mouth for 3 minutes. The interferent is then removed from the mouth by spitting or blowing out, and after 12 minutes a second saliva sample was collected. The samples were assayed for testosterone level using the candidate device. Sponsor defines non-significant interference as ≤ 10% bias between the tested sample and the control sample.

## Results summary:

The percent recovery between the control and test sample ranged from 93.3% to 105.2%, indicating no significant interference observed from the presence of food, coffee, wine, chewing gum and cigarette smoking.

However, significant interference was observed with whole blood (bias ranged from -14.8% to 148.4%), sodium azide (bias ranged from -5.4% to 179.3%) and thimerosal (bias ranged from -12.8% to 62.2%). The sponsor has included the following limitations in the labeling, "Avoid the use of samples with blood contamination, sodium azide and thimerosal as it may lead to false results."

The sponsor also indicates the following instructions for sample collection in the labeling:

"Avoid food consumption, drinking coffee or alcohol, smoking or chewing gum 15 minutes prior to sample collection. Rinse mouth thoroughly with water 15 minutes prior to collection."

Cross-reactivity studies were performed to evaluate the interference from structurally related compounds. The potential cross-reactants were tested up to a concentration of 10,000 pg/mL and 20,000 pg/mL in saliva samples containing low, medium and high concentration of testosterone (approximately 15, 50 and 225 pg/mL of testosterone). The percent cross-reactivity mean values calculated between the sample spiked with 10,000 pg/mL and 20,000 pg/mL of the cross-reactant is used as the representative value for each corresponding low, medium and high testosterone saliva sample and its spiked compound. The results are shown in the table below:

| Compounds | Percent Cross-Reactivity | | |
| --- | --- | --- | --- |
| | Low Testosterone Sample (~15pg/mL) | Medium Testosterone Sample (~52pg/mL) | High Testosterone Sample (~225pg/mL) |
| Testosterone | 100 | 100 | 100 |
| 11β-OH Testosterone | 0.407 | 0.486 | 0.242 |

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| 11α-OH Testosterone | 0.869 | 1.033 | 0.601 |
| --- | --- | --- | --- |
| 5α-Dihydrotestosterone | 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 |
| 17β-Estradiol | 0.175 | 0.173 | 0.135 |
| 17α-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-3β, 17β-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 |
| Testosterone propionate | 0.068 | 0.065 | 0.134 |
| 5 α-androstane-3β, 17β-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 |

f. Assay cut-off:

Not applicable.

2. Comparison studies:

a. Method comparison with predicate device:

Method comparisons between the candidate device, Pantex Salivary Direct Testosterone EIA and the predicate device, IBL Testosterone LIA were performed according to the CLSI EP9-A guidelines. A total of 106 samples of which 97 samples were real patient's saliva samples and 9 samples were spiked samples, ranging in testosterone concentration from $6.45\mathrm{pg / mL}$ to $458.35\mathrm{pg / mL}$ were used in the study. The candidate device results (Y-axis)

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were compared to the corresponding predicate device results (X-axis). Linear regression analysis resulted in the linear regression equation: $y = 0.9035x + 5.81$ ; $R^2 = 0.98$

The results correlate well around the clinically relevant range (<150 pg/mL). However, the results near the upper end of the measuring range demonstrate some scatter leading to the regression statistics noted above. However, the scatter observed in the upper section of the assay range is clinically insignificant.

b. Matrix comparison:

Not applicable. Oral fluid is the only claimed matrix.

3. Clinical studies:

a. Clinical Sensitivity:

Not applicable.

b. Clinical specificity:

Not applicable.

c. Other clinical supportive data (when a. and b. are not applicable):

Not applicable.

4. Clinical cut-off:

Not applicable.

5. Expected values/Reference range:

Reference range studies were performed following the CLSI C28-A3 guideline. Oral fluid samples were collected in the morning from 240 normal healthy females and 240 normal healthy males ranging in age from 20 to 70 years. The inclusion criteria used for recruitment of normal healthy subjects included adults of all ages, all genders, all ethnicity, not undergoing medical treatment or drug therapy and free of any illness on the day of sample collection. The exclusion criteria used for recruitment of normal healthy subjects included those on hormone therapy or taking oral contraceptives, history of thyroid or other autoimmune disease, history of Cushing's Syndrome or Addison's disease, and pregnant or lactating women. The results are summarized in the tables below:

Reference Values for Females:

| Subjects (Number) | Age (years) | Median (pg/mL) | 95% Reference values (pg/mL) |
| --- | --- | --- | --- |
| 120 | 20 to 49 | 17.2 | 3.91 to 40.99 |
| 120 | 50 to 70 | 14.75 | 4.51 to 34.17 |

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Reference Values for Males:

| Subjects (Number) | Age (years) | Median (pg/mL) | 95% Reference values (pg/mL) |
| --- | --- | --- | --- |
| 120 | 20 to 49 | 86.6 | 41.12 to 142.16 |
| 120 | 50 to 70 | 60.15 | 24.03 to 119.52 |

N. Proposed Labeling:
The labeling is sufficient and it satisfies the requirements of 21 CFR Part 809.10.

O. Conclusion:
The submitted information in this premarket notification is complete and supports a substantial equivalence decision.

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