HIGH SENSITIVITY CORTISOL ENZYME IMMUNOASSAY

K031348 · Salimetrics, LLC · NHG · Jun 10, 2003 · Clinical Chemistry

Device Facts

• Record ID: K031348
• Device Name: HIGH SENSITIVITY CORTISOL ENZYME IMMUNOASSAY
• Applicant: Salimetrics, LLC
• Product Code: NHG · Clinical Chemistry
• Decision Date: Jun 10, 2003
• Decision: SESE
• Submission Type: Special
• Regulation: 21 CFR 862.1205
• Device Class: Class 2

Intended Use

Salimetrics HS-Cortisol kit is a competitive immunoassay specifically designed for the quantitative in vitro diagnostic measurement of salivary cortisol. This kit may be used to measure adrenal cortical function and as a screen for Cushing's and Addison's disease. Saliva cortisol accurately reflects the amount of serum cortisol in the circulation. This kit is not intended for use with serum or plasma samples.

Device Story

Competitive enzyme immunoassay (EIA) for quantitative measurement of cortisol in human saliva. Input: saliva sample; Principle: cortisol in sample competes with horseradish peroxidase-linked cortisol for rabbit antibody binding sites on microtitre plate; Output: blue color intensity measured via standard plate reader at 450 nm after TMB substrate reaction; inversely proportional to cortisol concentration. Used in clinical laboratory settings by trained personnel. Results assist clinicians in evaluating adrenal function and screening for Cushing's or Addison's disease. Benefits include non-invasive monitoring of cortisol levels compared to serum testing.

Clinical Evidence

Bench testing only. Evaluated precision (intra-assay CV <6%, inter-assay CV <11%), sensitivity (<0.007 µg/dL), linearity (84-105% recovery), and specificity (cross-reactivity tested against 15 compounds). Clinical correlation study (n=68) showed significant correlation between saliva and serum cortisol (r=0.89, p<0.0001).

Technological Characteristics

Competitive solid-phase immunoassay. Components: rabbit antibody-coated 96-well microtitre plate, HRP-linked cortisol tracer, TMB substrate, sulfuric acid stop solution. Instrumentation: standard plate reader (450 nm). Connectivity: none. Sterilization: N/A (reagent kit).

Indications for Use

Indicated for quantitative in vitro diagnostic measurement of salivary cortisol to assess adrenal cortical function and screen for Cushing's and Addison's disease. Not for use with serum or plasma.

Regulatory Classification

Identification

A cortisol (hydrocortisone and hydroxycorticosterone) test system is a device intended to measure the cortisol hormones secreted by the adrenal gland in plasma and urine. Measurements of cortisol are used in the diagnosis and treatment of disorders of the adrenal gland.

Predicate Devices

• Diagnostic Systems Laboratory Active Cortisol EIA (K850141)

Submission Summary (Full Text)

{0}------------------------------------------------

K031348

JUN 1 0 2003

Image /page/0/Picture/2 description: The image shows the logo for "SALIMETRICS LLC". The logo is black with white text. To the left of the text is a grid of nine dots, arranged in a 3x3 pattern.

PO Box 395 • State College PA 16801-0395 • 814.234.7748 • Fax [phone] www.salimetrics.com

# Summary of Safety and Effectiveness

This summary of 510(k) safety and effectiveness information is being submitted in accordance with the requirements of SMDA 1990 and 21 CFR 807.92.

The assigned 510(k) number is K011323.

#### I. General Information

### Submitter:

Eve Schwartz Salimetrics, LCC 101 Innovation Blvd. Suite 302 State College, Pa. 16803 P [phone] F [phone]

## Date of 510(k) preparation:

| Device Trade Name: | HS(high sensitivity) Salivary Cortisol Enzyme<br>Immunoassay |
|--------------------|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Generic Name: | Immunoassay for the in vitro quantitative determination<br>of cortisol in saliva |
| Predicate Device: | The HS Salivary Cortisol EIA is substantially<br>equivalent to the Diagnostic Systems Laboratory Active<br>Cortisol EIA, which was approved by FDA (K850141)<br>for IVD use. |

{1}------------------------------------------------

### VI. Intended Use

Salimetrics HS-Cortisol kit is a competitive immunoassay specifically designed for the quantitative in vitro diagnostic measurement of salivary cortisol. Saliva cortisol accurately reflects the amount of serum cortisol in the circulation. Saliva values may be converted into serum values using the equation y(serum ug/dL)= 5.177+15.132x(saliva ug/dL). This kit is not intended for use with serum or plasma samples.

## III. Device Description

Test Principle- A microtitre plate is coated with rabbit antibodies to cortisol. Cortisol in standards and unknowns compete with cortisol linked to horseradish peroxidase for the antibody binding sites. After incubation, unbound components are washed away. Bound cortisol peroxidase is measured by the reaction of the peroxidase enzyme on the substrate tetramethylbenzidine (TMB). This reaction produces a blue color. A vellow color is formed after stopping the reaction with sulfuric acid. Optical density is read on a standard plate reader at 450 nm. The amount of cortisol peroxidase detected is inversely proportional to the amount of cortisol present. The optical density readings of the calibrators are used to form a standard curve to which the optical densities of the controls and samples are compared.

Kit Description- The kit consists of an antibody coated 96 well plate, six calibrators at a concentration of 1.800, 0.600, 0.200, 0.067. 0.022, and 0.007 µg/dL of NIST(National Institute of Standards and Technology) cortisol, two controls representing a high (1.00 ug/dL) and low (0.100 ug/dL) level of salivary cortisol, the enzyme conjugate (1600X concentrate), assay diluent containing a pH indicator, wash buffer(10X concentrate), tetramethylbenzidine substrate solution, stop solution, and kit insert.

### IV. Prior Marketing History

The HS Salivary Cortisol EIA has been sold as a research tool since November of 1998. The device was cleared for diagnostic use in December of 2001 and afterwards marketed as such. There have been no withdrawals of this device from any country related to safety and effectiveness.

### VII. Contraindications, Warnings and Precautions

- . This kit is designed to measure cortisol levels in saliva and should not be used to measure serum or plasma levels.
- . A pH value should be obtained on samples that appear vellow or purple after assay diluent is added and the plate is mixed. Samples with pH values > than 9 or < 3.5 should be recollected.
- Particulate matter may affect antibody binding. Pipette only clear saliva into wells. . Saliva may be filtered through cotton to eliminate particulate matter.

{2}------------------------------------------------

- Saliva samples contaminated with blood may give false results.
- Saliva collection and handling recommendations are presented in the kit insert. .

### VIII. Comparison to Predicate Device

### Correlation with Serum and Relationship to Predicate Device

The correlation between saliva and serum is highly significant, r (64) = . 89, p < . 0001. The relationship between the HS Cortisol Salivary EIA and the serum cortisol predicate device, determined by linear regression is y(serum µg/dL)= 5.177+15.132x(saliva µg/dL).

List of similarities and differences between the HS salivary Cortisol EIA and the predicate device

| Device<br>Characteristic | HS Salivary Cortisol EIA | Predicate Device |
|--------------------------|----------------------------------------------|----------------------------------------------|
| Basic Principal | Competitive Solid Phase<br>Immunoassay | Competitive Solid Phase<br>Immunoassay |
| Calibrator matrix | Saliva-like matrix | Serum matrix |
| Tracer | Horseradish Peroxidase linked to<br>cortisol | Horseradish Peroxidase linked to<br>cortisol |
| Substrate | Tetramethylbenzidine(TMB) | Tetramethylbenzidine(TMB) |
| Primary Antibody | goat anti rabbit | goat anti rabbit |
| Calibrator Range | 0.007 - 1.800 μg/dL | 0.5 - 60 μg/dL |
| Sample Type | saliva | serum or plasma |
| Instrumentation | standard plate reader | standard plate reader |
| Intra-assay Precision | Coefficient of variation < 6% | Coefficient of variation < 11% |
| Inter-assay Precision | Coefficient of variation < 11% | Coefficient of variation <12% |
| Sensitivity | <0.007 μg/dL | 0.1 μg/dL |
| Linearity | 84-105 % recovery | 81-119% recovery |
| Recovery | 84-115% | 93-124 |
| Control Levels | 1.000 μg/dL and 0.100 μg/dL | 20 μg/dL and 4 μg/dL |

### Table 1 List of assay Characteristics

### VI. Conclusion

The data presented in the 510(k) demonstrates that cortisol can be measured in the HS Salivary Cortisol EIA with comparable accuracy in saliva when compared to the serum predicate device. Saliva cortisol values measured with the HS Salivary Cortisol ELA correlate well with serum cortisol values. There is also a large amount of supporting literature that indicates cortisol can be measured accurately in saliva when samples are collected and stored in an appropriate manner.

{3}------------------------------------------------

Image /page/3/Picture/0 description: The image shows the logo for Salimetrics LLC. The logo is black with white text. To the left of the text is a grid of nine white dots.

# Comparison Between the Performance Data of the HS Salivary Cortisol EIA and the Predicate Device

### Intra-assay Precision Table 2

The intra-assay precision was determined from the mean of 12 replicates.

| SAMPLE | N | MEAN (ug/dL) | STANDARD<br>DEVIATION (ug/dL) | COEFFICIENT OF<br>VARIATION (%) |
|---------------------------|----|--------------|-------------------------------|---------------------------------|
| HS cortisol<br>Saliva EIA | | | | |
| Level 1 | 12 | 1.591 | 0.068 | 4.3 |
| Level 2 | 12 | 0.702 | 0.035 | 5.0 |
| Level 3 | 12 | 0.188 | 0.011 | 5.9 |
| Level 4 | 12 | 0.115 | 0.006 | 5.2 |
| Predicate<br>(Serum EIA) | | | | |
| Level 1 | 12 | 29.2 | 3.0 | 10.3 |
| Level 2 | 12 | 15.9 | 0.8 | 5.0 |
| Level 3 | 12 | 8.4 | 0.2 | 2.4 |

#### Table 3 Inter-assay Precision

The inter-assay precision was determined from the mean of average duplicates for 12 separate runs.

| SAMPLE | N | MEAN (µg/dL) | STANDARD DEVIATION (µg/dL) | COEFFICIENT OF VARIATION (µg/dL) |
|---------------------------|----|--------------|----------------------------|----------------------------------|
| HS cortisol<br>Saliva EIA | | | | |
| Level 1 | 12 | 1.436 | 0.098 | 6.9 |
| Level 2 | 12 | 1.000 | 0.086 | 8.6 |
| Level 3 | 12 | 0.194 | 0.013 | 6.7 |
| Level 4 | 12 | 0.023 | 0.002 | 10.6 |
| Predicate<br>(Serum EIA) | | | | |
| H | 12 | 15.0 | 1.2 | 8.0 |
| M | 12 | 11.4 | 0.7 | 6.1 |
| L | 12 | 5.0 | 0.6 | 12.0 |

{4}------------------------------------------------

### Sensitivity

HS Cortisol Saliva EIA: The lower limit of sensitivity was determined by interpolating the mean minus 2 standard deviations for 10 sets of duplicates at 0 ug/dL standard. The minimal concentration of cortisol that can be distinguished from 0 is <.007 µg/dL.

Predicate (Serum EIA): The theoretical sensitivity or minimum detection limit, calculated by the interpolation of the mean minus two standard deviations of ten replicates of the 0 µg/dL Cortisol standard, is 0.1 µg/dL.

#### Linearity of Dilution Table 4

Two saliva samples were diluted with assay diluent and assayed.

| SAMPLE | DILUTION<br>FACTOR | EXPECTED (µg/dL) | OBSERVED (µg/dL) | RECOVERY (%) |
|---------------------------|--------------------|------------------|------------------|--------------|
| HS cortisol<br>Saliva EIA | | IX. | X. | XI. |
| Sample 1 | | --- | 0.161 | --- |
| | 1:2 | 0.081 | 0.079 | 97.5 |
| | 1:4 | 0.040 | 0.042 | 105.0 |
| | 1:8 | 0.020 | 0.019 | 95.0 |
| | 1:16 | 0.010 | 0.010 | 100.0 |
| Sample 2 | | --- | 0.523 | --- |
| | 1:2 | 0.262 | 0.263 | 100.4 |
| | 1:4 | 0.131 | 0.110 | 84.0 |
| | 1:8 | 0.065 | 0.056 | 86.2 |
| | 1:16 | 0.033 | 0.033 | 100.0 |
| Sample 3 | | --- | 1.708 | --- |
| | 1:2 | 0.854 | 0.782 | 91.6 |
| | 1:4 | 0.427 | 0.389 | 91.1 |
| | 1:8 | 0.214 | 0.210 | 98.1 |
| | 1:16 | 0.107 | 0.106 | 99.1 |
| Predicate<br>(Serum EIA) | | | | |
| Sample 1 | | --- | 13.0 | --- |
| | 1:2 | 6.5 | 6.9 | 106 |
| | 1:4 | 3.2 | 3.8 | 119 |
| | 1:8 | 1.6 | 1.8 | 113 |
| | 1:16 | --- | --- | --- |
| Sample 2 | | --- | 27.2 | --- |
| | 1:2 | 13.6 | 12.4 | 91 |
| | 1:4 | 6.8 | 5.5 | 81 |
| | 1:8 | 3.4 | 3.4 | 100 |
| | 1:16 | 1.7 | 2.0 | 118 |

{5}------------------------------------------------

### Table 5 Recovery

| SAMPLE | ENDOGENOUS<br>(µg/dL) | ADDED<br>(µg/dL) | EXPECTED<br>(µg/dL) | OBSERVED<br>(µg/dL) | RECOVERY<br>(%) |
|---------------------------|-----------------------|------------------|---------------------|---------------------|-----------------|
| HS Cortisol<br>Saliva EIA | | | | | |
| 1 | 0.632 | 0.180 | 0.812 | 0.816 | 100.5 |
| 2 | 0.093 | 0.180 | 0.273 | 0.308 | 112.8 |
| | | 0.020 | 0.113 | 0.095 | 84.1 |
| 3 | 0.700 | 0.500 | 1.200 | 1.290 | 107.5 |
| | | 0.180 | 0.88 | 0.891 | 101.3 |
| 4 | 0.696 | 0.020 | 0.716 | 0.745 | 104.1 |
| 5 | 0.039 | 1.00 | 1.039 | 1.060 | 102.0 |
| 6 | 0.137 | 1.00 | 1.137 | 1.310 | 115.2 |
| 7 | 0.289 | 0.020 | 0.309 | 0.307 | 99.4 |
| Predicate<br>(serum EIA) | | | | | |
| 1 | 5.1 | 5.5 | 10.6 | 9.8 | 93 |
| 2 | 13.3 | 1.8 | 15.1 | 18.7 | 124 |
| 3 | 20.4 | 3.6 | 24.0 | 24.9 | 104 |

Seven saliva samples containing different levels of endogenous cortisol were spiked with known quantities of cortisol and assayed. Compare to serum results listed below.

#### Table 6 Specificity

Percent cross-reactivity in the predicate device is expressed as the ratio of cortisol concentration to the concentration of the reacting compound at 50% binding of the 0 ug/dL cortisol standard.

In the HS Salivary Cortisol EIA, the following compounds were tested at concentrations up to 66,000 ng/mL for cross-reactivity:

| Compound | % Cross-<br>reactivity<br>in predicate | Spiked<br>Concentration<br>(ng/mL) | % Cross-reactivity<br>in HS Salivary Cortisol EIA |
|----------------------------|----------------------------------------|------------------------------------|---------------------------------------------------|
| Prednisolone | 58.3 | 100 | 9.530 |
| Prednisone | 10.9 | 1000 | 0.421 |
| Cortisone | 7.0 | 1000 | 0.31 |
| 11-Deoxycortisol | 5.7 | 500 | 3.116 |
| 21-Deoxycortisol | 1.9 | 1000 | 0.745 |
| 17-a Hydroxy- progesterone | 0.9 | 1000 | 0.611 |
| Dexamethasone | 0.9 | 1000 | 1.277 |
| Tiamcinolone | 0.4 | 1000 | 0.430 |
| Corticosterone | ND | 10,000 | 0.093 |
| Progesterone | ND | 1000 | 0.060 |
| 17-\$\beta\$ estradiol | No data | 10 | ND |
| DHEA | ND | 10,000 | ND |
| Testosterone | No data | 10,000 | 0.007 |
| Transferrin | No data | 66,000 | ND |
| Aldosterone | No data | 10,000 | ND |

ND = non-detectable (<0.004)

{6}------------------------------------------------

### Salivary Cortisol Expected Ranges

Each laboratory should establish its own range of expected values. The following values have been reported for salivary cortisol.

| Group | Number | AM range in µg/dL | PM range in µg/dL |
|------------------------|--------|-------------------|-------------------|
| Children ages 2.5-5.5 | 112 | 0.060-0.700 | 0.08-0.660 |
| Children ages 8-11 | 285 | 0.112-0.904 | ND-0.249 |
| Adolescents ages 12-18 | 403 | 0.046-0.950 | ND-0.296 |
| Males ages 21-30 | 26 | 0.112-0.743 | ND-0.308 |
| Females ages 21-30 | 20 | 0.272-1.348 | ND-0.105 |
| Males ages 31-50 | 67 | 0.122-1.551 | ND-0.359 |
| Females ages 31-50 | 31 | 0.094-1.515 | ND-0.181 |
| Males ages 51-70 | 28 | 0.112-0.812 | ND-0.228 |
| Females ages 51-70 | 23 | 0.149-0.739 | 0.02-0.254 |
| All adults | 192 | 0.094-1.551 | ND-0.359 |

### CORRELATION WITH SERUM:

The correlation between serum and saliva cortisol was determined by assaying 68 matched samples from presumed normal adults using the predicate device for serum and the Salimetrics HS salivary cortisol EIA. The correlation between saliva and serum was highly significant, r (64) = . 89, p < . 0001. The relationship between the HS Cortisol Salivary EIA and the serum cortisol predicate device, determined by linear regression is y(serum ug/dL)= 5.177+15.132x(saliva ug/dL).

Saliva explained 79.2% of the variance in serum, F(1,63) = 240.528, p < 0.0001. The intercept is 5.177 (SE = 0.450, and is significantly above zero, t = 11.512, p < 0.0001. The slope of saliva is 15.132 (SE = 0.976), and is also significant, t = 15.509, p<0.0001. Scores with residuals > 3 and dfbeta > 1 (N = 3) were removed to minimize influential observations.

{7}------------------------------------------------

Since both serum (skew = 1.325) and saliva (skew = 1.793) are skewed, after taking a log transformation, the R2 was only slightly reduced, with logged saliva explaining 74.7% of the variance in logged serum, E(1,63) = 185.530, p < 0.0001. The new constant is 2.947 (SE = 0.054), and is significant, t = 54.544, p < 0.0001, and the slope of the saliva is also significant, t = 13.621, p < 0.001 (B = 0.538, SE = 0.040).

{8}------------------------------------------------

Image /page/8/Picture/1 description: The image shows the logo for the U.S. Department of Health & Human Services. The logo consists of a stylized eagle or bird-like figure with three curved lines representing its body and wings. The logo is encircled by the text "DEPARTMENT OF HEALTH & HUMAN SERVICES - USA" in a circular arrangement. The text is in all capital letters and is evenly spaced around the circle.

Food and Drug Administration 2098 Gaither Road Rockville MD 20850

JUN 1 0 2003

Ms. Eve Schwartz VP of Operations Salimetrics, LLC P.O. Box 395 State College, PA 16804-0395

Re: k031348

> Trade/Device Name: HS Salivary Cortisol Enzyme Immunoassay Kit Regulation Number: 21 CFR 862.1205 Regulation Name: Cortisol (hydrocortisone and hydroxycorticosterone) test system Regulatory Class: Class II Product Code: NHG Dated: May 28, 2003 Received: May 30, 2003

Dear Ms. Schwartz:

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).

{9}------------------------------------------------

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.

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 [phone]. 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 [phone] or [phone] or at its Internet address http://www.fda.gov/cdrh/dsma/dsmamain.html.

Sincerely yours,

Steven Butman

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

{10}------------------------------------------------

Image /page/10/Picture/0 description: The image shows the logo for Salimetrics LLC. The logo is black with white text. The text is "SALIMETRICS LLC" in all caps. There are white dots on the left side of the logo.

PO Rox 395 · State College PA 16801-0395 814 234 7748 · Fax [phone]

### INDICATIONS FOR USE STATEMENT

510(k) number: K011323

Device Name: _ HS Salivary Cortisol Enzyme Immunoassay Kit

### Indications for Use:

Salimetrics HS-Cortisol kit is a competitive immunoassay specifically designed for the quantitative in vitro diagnostic measurement of salivary cortisol. This kit may be used to measure adrenal cortical function and as a screen for Cushing's and Addison's disease. Saliva cortisol accurately reflects the amount of serum cortisol in the circulation. This kit is not intended for use with serum or plasma samples.

Stan Cooper
Division Sign-Off

Office of In Vitro Diagnostic Device Evaluation and Safety

510(k) K031348

Concurrence of CDRH, Office of Device Evaluation (ODE)

Prescription Use √

Over-the-Counter Use