Predicate Devices

Reference Devices

Not Found

AI/ML (Artificial Intelligence / Machine Learning)

No
The device description and performance studies focus on a traditional immunoassay method for detecting hemoglobin. There is no mention of AI, ML, image processing, or any computational analysis of the test results beyond visual interpretation.

Therapeutic

No.
The README states that the device is "used as an aid in the diagnosis of gastrointestinal(GI) bleeding" and is for "in vitro diagnostic use only." It detects occult blood, which is a diagnostic indicator, not a treatment.

Diagnostic

Yes.

Explanation: The "Intended Use / Indications for Use" section explicitly states, "It is used as an aid in the diagnosis of gastrointestinal(GI) bleeding." This directly indicates its role as a diagnostic device. Also stated is, "For in vitro diagnostic use only."

SaMD (Software as a Medical Device)

No

The device description clearly outlines physical components like test devices (cassettes), collection tubes with buffer solution, and collection papers. This indicates it is a hardware-based in vitro diagnostic device, not a software-only medical device.

IVD (In Vitro Diagnostic)

Yes, this device is an IVD (In Vitro Diagnostic).

Here's why:

Explicit Statement: The "Intended Use / Indications for Use" section clearly states: "For in vitro diagnostic use only."
Nature of the Test: The device is designed to detect a substance (human occult blood) in a biological sample (feces) outside of the body to aid in the diagnosis of a medical condition (gastrointestinal bleeding). This is the core definition of an in vitro diagnostic test.
Device Description: The components described (test devices, collection tubes with buffer, etc.) are typical of an IVD kit.
Performance Studies: The detailed performance studies (precision, linearity, stability, interference, specificity, method comparison) are standard evaluations for IVD devices to demonstrate their analytical and clinical performance.
Predicate Device: The mention of a predicate device (K110309; Orient Gene Biotech One Step Rapid FOB Test) indicates that this device is being compared to an already cleared IVD.

PCCP (Predetermined Change Control Plan) Authorized

N/A

Overview

Intended Use / Indications for Use

Wondfo One Step Fecal Occult Blood Test is a rapid test for the qualitative detection of human occult blood in feces. It is used as an aid in the diagnosis of gastrointestinal(GI) bleeding. The device is suitable for use in laboratories and physician's offices as well as for over the counter use. For in vitro diagnostic use only. For prescription use and over the counter use.

Product codes (comma separated list FDA assigned to the subject device)

KHE

Device Description

The Wondfo One Step Fecal Occult Blood Test utilizes double antibodies sandwich immunoassay for the detection of hemoglobin in test samples. The test kit consists of:
a. Test devices, one test in one pouch. One pouch contains a test cassette and a desiccant. The desiccant is for storage purposes only and is not used in the test procedures.
b. Collection tubes with 1.5mL extraction buffer solution.
c. Clean collection papers.
d. Instructions for use.

Mentions image processing

Not Found

Mentions AI, DNN, or ML

Not Found

Input Imaging Modality

Not Found

Anatomical Site

feces (gastrointestinal bleeding)

Indicated Patient Age Range

Not Found

Intended User / Care Setting

laboratories and physician's offices as well as for over the counter use.

Description of the training set, sample size, data source, and annotation protocol

Not Found

Description of the test set, sample size, data source, and annotation protocol

Not Found

Summary of Performance Studies (study type, sample size, AUC, MRMC, standalone performance, key results)

Analytical Performance
a. Precision/Reproducibility

Repeatability: Evaluated in-house, using one test kit lot and one operator. 21 replicates of fecal test samples (Hb-free stool spiked with hemoglobin at 7 concentrations: 0, 1.2, 1.35, 1.5, 1.8, 2.25, and 60 ug/g stool). Overall Percent Agreement: 99.3%. Positive Percent Agreement: 100.0% (96.2% – 100.0%). Negative Percent Agreement: 98.1% (89.9% – 99.7%).
Reproducibility (Lot-to-Lot, Between-run, Between-Device, Between-site, Combined): Conducted at three point-of-care (POC) sites in the U.S. using three test kit lots (one per site), three operators per site, and one run per day for five non-consecutive days. 21 replicates of fecal test samples (Hb-free stool spiked with human blood at 7 concentrations: 0, 1.2, 1.35, 1.8, 1.8, 60 ug/g stool).
- Lot-to-Lot: Overall PA: 99.2%, Positive PA: 99.2%, Negative PA: 99.2%
- Between-run: Overall PA: 98.9%, Positive PA: 99.2%, Negative PA: 98.5%
- Between-Device: Overall PA: 99.3%, Positive PA: 99.4%, Negative PA: 99.2%
- Between-site: Overall PA: 98.9%, Positive PA: 99.2%, Negative PA: 98.5%
- Combined: Overall PA: 99.1%, Positive PA: 99.2%, Negative PA: 99.0%

b. Linearity (Prozone/Hook effect)

Evaluated using three test kit lots, three operators, and Hb-free stool specimens spiked with high concentration human blood (7 concentrations: 60000, 6000, 600, 30, 15, 7.5 ug/g stool).
Result: Wondfo One Step Fecal Occult Blood (FOB) Test was not found susceptible to prozone/hook effect as it would display positive test results with fecal samples containing a hemoglobin concentration up to 200,000 ng/mL.

c. Stability

Fecal samples stability: Tested at room temperature (15 days), 2-8°C (30 days), and -20°C (3 months) using spiked samples (7 concentrations). Stool samples collected in Wondfo One Step Fecal Occult Blood (FOB) Test sampling tubes are stable up to 3 months when stored -20℃, 30 days when stored at 2 to 8°C, and stable up to 15 days when stored at room temperature.
In-use Stability: 1 lot, 21 aliquots of spiked stool samples (7 concentrations). Tested at 0.5, 1, 1.5, and 2 hours at 25℃, 50% RH; 40℃, 50% RH; and 25℃, 90% RH.
Test Kit Stability (Accelerated): 3 lots, 21 aliquots of spiked stool samples (7 concentrations). Stores at 50°C and tested at various intervals. Estimated stability: 24 months at 4-30°C.
Test Kit Real Time Stability: 3 lots, 21 aliquots of spiked stool samples (7 concentrations). Stored at 4, 10, 20, and 30°C and tested at various monthly intervals.
Test Kit Transport Simulation Test: 3 lots, 21 aliquots of spiked stool samples (7 concentrations). Simulated extreme shipping temperatures (-20°C and 40°C). Stable up to 35 transport days.

d. Interference

Vegetable extracts, vitamin supplements (Vitamin C), iron (FeCl3+6H2O) and horseradish peroxidase: Tested with spiked stool samples (7 Hb concentrations) and various concentrations of interferents.
Result: Did not show significant interference with any of the vegetable extracts, Vitamin C, iron or horseradish peroxidase up to 250 ug /ml, 2000 ug/ml and 20000 ug/ml respectively.
Toilet Cleaners and regular toilet water: Tested with spiked stool samples (7 Hb concentrations) and Clorox (1%), Frosch (1%), Mr. Muscle (1%), and regular toilet water (1%).
Result: Did not show significant interference from regular toilet water. However, there were false negative results when 1% toilet cleaner was added to the water.

e. Specificity

Specificity to human hemoglobin variant: Tested detection of hemoglobin A (HbA), hemoglobin-S (HbS), and hemoglobin-C (HbC) using spiked stool samples (7 Hb concentrations).
Result: Equivalently recognized variants of hemoglobin HbA, HbS and HbC.
Cross-Reactivity with non-human hemoglobin and meat extracts: Tested various non-human hemoglobins (beef, pig, fish, horse, goat, rabbit, sheep, chicken Hb at 500 ug/mL) and meat extracts (beef, pork, fish, horse, goat, rabbit, sheep, chicken meat extract at 1 mL/g feces) with spiked Hb-free stool (7 Hb concentrations).
Result: Did not show significant interference from any of the non-human hemoglobin or animal meat extracts tested.

f. Cut-off

Evaluated in-house using spiked stool samples (7 Hb concentrations) and compared side-by-side with the predicate.
Cut-off was determined to be 1.35 µg hemoglobin/g stool or 45 ng/mL (hemoglobin in fecal sample mixed with detection buffer).
Agreement with predicate: Overall PA: 99.3%, Positive PA: 100%, Negative PA: 98.1%.

Comparison Studies

Method comparison: Compared with the predicate device (Orient Gene Biotech One Step Rapid FOB Test) using 407 patient samples, 18 stool samples around the cut-off (purchased), and 100 spiked samples. Three lots of subject device, one lot of predicate device. Performed at three POC testing sites by three different operators at each site.
- Combined Sites results showed overall agreement of 99.6%, positive agreement of 99.2%, and negative agreement of 99.7%.
Lay-user study: Performed at three intended user sites with 100 lay users testing their own stool sample and spiked samples. Comparison between lay-users and professional users.
- Overall agreement between lay-users and professional users for own stool samples: 100%.
- Overall agreement between lay-users and professional users for spiked samples: 98%. Two discrepant results for samples at 37.5 ng/mL.
- Lay-users found device instructions easy to follow.

Test Kit Reaction Time:

Evaluated using spiked stool samples (7 Hb concentrations) at various time points (3, 5, 8, 10, 20, 30, 40, 60 minutes).
Result: Positivity rate of 0 ng/mL was 0%, 40 ng/mL was 4.8%, 45 ng/mL was 47.6%, and 50 ng/mL was 100% at 10 minutes. The appropriate reaction time was demonstrated as 10 minutes.

Operator Intensity Reading:

Evaluated using spiked stool samples (7 Hb concentrations) by five different readers at one intended use site. Test line intensity graded from 10 levels.
Result: Intensity increased with Hb concentration. No statistical significance in analyses by operators.

Specimen Collection Verification:

Performed by five lay users using five positive and five negative clinical samples per lay user. Weighed sampling stick before and after stool collection.
Result: Average sample volume collected was 0.0517g with standard deviation of 0.0027, demonstrating consistency. Lay users collected adequate and consistent amounts.

Key Metrics (Sensitivity, Specificity, PPV, NPV, etc.)

See "Summary of Performance Studies" for Overall Percent Agreement, Positive Percent Agreement, and Negative Percent Agreement values from reproducibility, cut-off, and comparison studies.

Predicate Device(s): If the device was cleared using the 510(k) pathway, identify the Predicate Device(s) K/DEN number used to claim substantial equivalence and list them here in a comma separated list exactly as they appear in the text. List the primary predicate first in the list.

K110309

Reference Device(s): Identify the Reference Device(s) K/DEN number and list them here in a comma separated list exactly as they appear in the text.

Not Found

Predetermined Change Control Plan (PCCP) - All Relevant Information for the subject device only (e.g. presence / absence, what scope was granted / cleared under the PCCP, any restrictions, etc).

Not Found

Regulation Number and Section

§ 864.6550 Occult blood test.

(a)
Identification. An occult blood test is a device used to detect occult blood in urine or feces. (Occult blood is blood present in such small quantities that it can be detected only by chemical tests of suspected material, or by microscopic or spectroscopic examination.)(b)
Classification. Class II (special controls). A control intended for use with an occult blood test is exempt from the premarket notification procedures in subpart E of part 807 of this chapter subject to the limitations in § 864.9.

Summary

0

Image /page/0/Picture/1 description: The image shows the logo for the U.S. Department of Health & Human Services. The logo consists of a circular seal with the text "DEPARTMENT OF HEALTH & HUMAN SERVICES - USA" around the perimeter. Inside the circle is an emblem featuring three stylized human profiles facing to the right, representing health and human services.

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

May 14, 2017

Guangzhou Wondfo Biotech Co., Ltd. c/o Joe Shia LSI International Inc. 504 East Diamond Ave., Suite I Gaithersburg, MD 20877

Re: K162333

Trade/Device Name: Wondfo One Step Fecal Occult Blood Test Regulation Number: 21 CFR 864.6550 Regulation Name: Occult blood test Regulatory Class: Class II Product Code: KHE Dated: April 11, 2017 Received: April 14, 2017

Dear Mr. Enomoto:

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 Part 801); medical device reporting (reporting of medical device-related adverse events) (21 CFR 803); good manufacturing practice requirements as set

1

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.

If you desire specific advice for your device on our labeling regulation (21 CFR Part 801), please contact the Division of Industry and Consumer Education at its toll-free number (800) 638-2041 or (301) 796-7100 or at its Internet address

http://www.fda.gov/MedicalDevices/Resourcesfor You/Industry/default.htm. Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21CFR 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 (800) 638-2041 or (301) 796-7100 or at its Internet address

http://www.fda.gov/MedicalDevices/ResourcesforYou/Industry/default.htm.

Sincerely yours, Kelly Oliner -S

Kelly Oliner, Ph.D. Deputy Director Division of Immunology and Hematology Devices Office of In Vitro Diagnostics and Radiological Health Center for Devices and Radiological Health

For

Leonthena R. Carrington, MS. MBA, MT(ASCP) Director Division of Immunology and Hematology Devices Office of In Vitro Diagnostics and Radiological Health Center for Devices and Radiological Health

Enclosure

2

Indications for Use

510(k) Number (if known) K162333

Device Name

Wondfo One Step Fecal Occult Blood Test

Indications for Use (Describe)

Wondfo One Step Fecal Occult Blood Test is a rapid test for the qualitative detection of human occult blood in feces. It is used as an aid in the diagnosis of gastrointestinal(GI) bleeding. The device is suitable for use in laboratories and physician's offices as well as for over the counter use.

For in vitro diagnostic use only. For prescription use and over the counter use.

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)
---------------------------------------------------------------------	--------------------------------------------------------------------

CONTINUE ON A SEPARATE PAGE IF NEEDED.

This section applies only to requirements of the Paperwork Reduction Act of 1995.

DO NOT SEND YOUR COMPLETED FORM TO THE PRA STAFF EMAIL ADDRESS BELOW.

The burden time for this collection of information is estimated to average 79 hours per response, including the time to review instructions, search existing data sources, gather and maintain the data needed and complete and review the collection of information. Send comments regarding this burden estimate or any other aspect of this information collection, including suggestions for reducing this burden, to:

Department of Health and Human Services Food and Drug Administration Office of Chief Information Officer Paperwork Reduction Act (PRA) Staff PRAStaff@fda.hhs.gov

"An agency may not conduct or sponsor, and a person is not required to respond to, a collection of information unless it displays a currently valid OMB number."

3

510(k) SUMMARY

1. Date:	May 9, 2017
2. Submitter:	Guangzhou Wondfo Biotech Co., Ltd.
No.8 Lizhishan Road, Science City,
Luogang District, Guangzhou, Guangdong, P.R. China 510663
3. Contact person:	Joe Shia
LSI International Inc.
504 East Diamond Ave., Suite I
Gaithersburg, MD 20877
Telephone: 240-505-7880
Fax: 301-916-6213
Email:shiajl@yahoo.com

1. Device Name: Wondfo One Step Fecal Occult Blood Test

Product Code	Classification	Regulation Section	Panel
KHE	Class II	21 CFR 864.6550
Occult Blood Test	Hematology

5. Predicate Devices:

K110309

Orient Gene Biotech One Step Rapid FOB Test

1. Intended Use
  Wondfo One Step Fecal Occult Blood Test is a rapid test for the qualitative detection of human occult blood in feces. It is used as an aid in the diagnosis of gastrointestinal(GI) bleeding. The device is suitable for use in laboratories and physician's offices as well as for over the counter use. For in vitro diagnostic use only. For prescription use and over the counter use.
1. Device Description
  The Wondfo One Step Fecal Occult Blood Test utilizes double antibodies sandwich immunoassay for the detection of hemoglobin in test samples. The test kit consists of:

Test devices, one test in one pouch. One pouch contains a test cassette and a desiccant. The a. desiccant is for storage purposes only and is not used in the test procedures.

Collection tubes with 1.5mL extraction buffer solution. b.
Clean collection papers. C.
Instructions for use. d.

4

1. Substantial Equivalence Information
  A summary comparison of features of the WONDFO One Step Fecal Occult Blood Test and the predicate device is provided in Table 1.

	Table 1: Features Comparison of WONDFO One Step Fecal Occult Blood Test and the Predicate
Device

Device
Similarities
Item	Device
Wondfo One Step Fecal Occult
Blood Test, K162333	Predicate
Orient Gene Biotech One Step
Rapid FOB Test, K110309
Intended/Indications
for Use	Wondfo One Step Fecal Occult
Blood Test is a rapid test for the
qualitative detection of human occult
blood in feces. It is used as an aid in
the diagnosis of gastrointestinal
bleeding. The device is suitable for
use in laboratories and physician's
offices as well as for over the counter
use.	The FOB One Step Rapid Test is a
rapid chromatographic
immunoassay for the qualitative
detection of human occult blood in
human fecal specimens as an aid in
the diagnosis of gastrointestinal
disorders. The device is suitable
for use in laboratories and
physician's offices as well as for
over the counter use.
Specimen Type	Fecal	Same
Test Principle	Qualitative test system intended for
immunochemical detection of fecal
occult blood in feces.	Same
Detection Method	Lateral flow chromatographic
immunoassay	Same
Result Format	Visible pink line in control region
and test region	Same

Differences
	Device	Predicate
Item	Wondfo One Step Fecal Occult
Blood Test, K162333	Orient Gene Biotech One Step
Rapid FOB Test, K110309
Assay Cut-off	45 ng/mL (Human hemoglobin in
human fecal sample mixed with
detection buffer)	50 ng/mL (Human hemoglobin
in human fecal sample mixed
with detection buffer)
Reaction time	Read the test results after 10
minutes. Some positive results
may be seen earlier. Do not read	Read test results between 5-10
minutes. Test results read earlier
than 5 minutes and later than 10

5

Differences
Item	Device	Predicate
	Wondfo One Step Fecal Occult
Blood Test, K162333
results after 30 minutes.	Orient Gene Biotech One Step
Rapid FOB Test, K110309
minutes are not valid.
Storage	4-30°C	2-30°C

9. Test Principle

Wondfo One Step Fecal Occult Blood Test is a qualitative test designed for the immunochemical detection of human hemoglobin (hHb) in stool specimens. When the extracted specimen is introduced to the sample well of the testing cassette, the specimen is absorbed into the device by capillary action, mixes with the hemoglobin specific antibody-dye conjugate, and flows across the pre-coated membrane. When the hemoglobin antigen levels in specimens are at or above the target cutoff, the antigens in the specimen bind to the antibody-dye conjugate and are captured by antibodies immobilized in the test region (T) of the device. This produces a colored test band and indicates a positive result. When the hemoglobin antigen levels in specimens are zero or below the target cut off, there is no visible colored band in the test region (T) of the device, indicating a negative result.

To serve as a procedure control, a colored line will appear at the control region (C), if the test has been performed properly.

10. Performance Characteristics

1. Analytical Performance
a. Precision/Reproducibility

The repeatability of Wondfo One Step Fecal Occult Blood (FOB) Test was evaluated in house, using one test kit lot and one operator. The repeatability study was performed using 21 replicates of fecal test samples collected in Wondfo One Step Fecal Occult Blood (FOB) Test sampling collection tubes with extraction buffer from Hb-free stool specimens spiked with hemoglobin to obtain test samples with seven different concentrations of human hemoglobin: 0 µg/g stool, 1.2 µg/g stool, 1.35 µg/g stool, 1.5 µg/g stool, 1.8 µg/g stool, 2.25 µg/g stool, and 60 µg/g stool, that are equivalent to 0 ng/mL, 40 ng/mL, 45 ng/mL, 50 ng/mL, 60 ng/mL, 75 ng/mL and 2000 ng/mL, respectively.

Reproducibility studies were conducted at three point of care (POC) sites in the U.S. using three test kit lots (one lot of test kit per site), three operators per site, and one run per day of test samples for five non-consecutive days. The reproducibility of Wondfo One Step Fecal Occult Blood (FOB) Test was evaluated by testing 21 replicates of fecal test samples collected in Wondfo One Step Fecal Occult Blood (FOB) Test sampling collection tubes with extraction buffer from Hb-free stool specimens spiked with human blood to obtain test samples containing seven different concentrations of human

6

hemoglobin: 0 µg/g stool, 1.2 µg/g stool, 1.35 µg/g stool, 1.8 µg/g stool, 1.8 µg/g stool, and 60 ug/g stool, that are equivalent to 0 ng/mL, 40 ng/mL, 50 ng/mL, 60 ng/mL, 75 ng/mL and 2000 ng/mL, respectively.

Wondfo One Step Fecal Occult Blood (FOB) Test repeatability and reproducibility testing was carried out in a random and blinded manner. No invalid or indeterminate results were obtained throughout the precision studies. FOBT-CHECK 'Negative and Positive Controls' were also tested daily to ensure and confirm the validity of the test results.

| Type of
Precision Study | Actual Results Percent
Agreement | Positive Percent
Agreement
(95% CI) | Negative Percent
Agreement
(95% CI) |
|-----------------------------------|-------------- | | Wondfo (FOB) Test | | | Positive
Results | Negative
Results | Total
Results | | | Repeatability | Positive Results | | Negative Results | | Total Results | Lot-to-Lot
Reproducibility | Positive Results | | Negative Results | | Total Results | Between-run
Reproducibility | Positive Results | | Negative Results | | Total Results | Between-Device
Reproducibility | Positive Results | | Negative Results | | Total Results | Between-site
Reproducibility | Positive Results | | Negative Results | | Total Results | Combined
Reproducibility | Positive Results | | Negative Results | | Total Results | | | Expected Results | | | Overall
-----------------|--|--|---------------------|---------------------|------------------|---------------------------------|-------------------------------------------|-------------------------------------------|
One Step
| |
| | | 95 | 1 | 96 | 99.3% | 100.0% | 98.1% |
| | | 0 | 51 | 51 | | (96.2% – 100.0%) | (89.9% – 99.7%) |
| | | 95 | 52 | 147 | | | |
| | | 471 | 2 | 473 | 99.2% | 99.2% | 99.2% |
| | | 4 | 258 | 262 | | (97.8% – 99.7%) | (97.2% – 99.8%) |
| | | 475 | 260 | 735 | | | |
| | | 471 | 4 | 475 | 98.9% | 99.2% | 98.5% |
| | | 4 | 256 | 260 | | (97.8% – 99.7%) | (96.1% – 99.4%) |
| | | 475 | 260 | 735 | | | |
| | | 472 | 2 | 474 | 99.3% | 99.4% | 99.2% |
| | | 3 | 258 | 261 | | (98.2% – 99.8%) | (97.2% – 99.8%) |
| | | 475 | 260 | 735 | | | |
| | | 471 | 4 | 475 | 98.9% | 99.2% | 98.5% |
| | | 4 | 256 | 260 | | (97.8% – 99.7%) | (96.1% – 99.4%) |
| | | 475 | 260 | 735 | | | |
| | | 1414 | 8 | 1422 | 99.1% | 99.2% | 99.0% |
| | | 11 | 772 | 783 | | (98.6% – 99.6%) | (98.0% – 99.5%) |
| | | 1425 | 780 | 2205 | | | |

Statistical analysis of repeatability and reproducibility studies of Wondfo One Step Fecal Occult Blood (FOB) Test

b. Linearity

Prozone (hook effect)

Susceptibility of Wondfo One Step Fecal Occult Blood (FOB) Test to prozone effect was evaluated by using three test kit lots, three operators and testing Hb-free stool specimens spiked with high concentration human blood of know hemoglobin levels so as to obtain fecal test samples with seven different Hb concentrations: 60000 µg/g stool, 6000 µg/g stool, 600 µg/g stool, 30 µg/g

7

stool, 15 µg/g stool and 7.5 µg/g stool, that are equivalent to 2000000 ng/mL, 200000 ng/mL, 20000 ng/mL, 2000 ng/mL, 1000 ng/mL, 500 ng/mL and 250 ng/mL respectively. Five aliquots of each sample concentration were mixed with extraction buffer in the specimen collection tubes and tested in a randomized order.

FOBT-CHECK 'Negative and Positive Controls' were also tested to ensure and confirm the validity of the test results of the test prozone/hook effect study. Wondfo One Step Fecal Occult Blood (FOB) Test was not found susceptible to prozone/hook effect as it would display positive test results with fecal samples containing a hemoglobin concentration up to 200,000 ng/mL.

c. Stability

Stability of fecal samples

Samples for stability testing were prepared by spiking stool samples with human blood (of known hemoglobin level) to obtain fecal samples containing seven different hemoglobin concentrations: 0 ug/g stool, 1.2 ug/g stool, 1.35 ug/g stool, 1.8 ug/g stool, 2.25 ug/g stool, 2.25 ug/g stool, and 60 ug/g stool, that are equivalent to 0 ng/mL, 40 ng/mL, 45 ng/mL, 60 ng/mL, 75 ng/mL, 75 ng/mL and 2000 ng/mL, respectively.

To claim 15 days of sample stability at room temperature each sample was tested with Wondfo One Step Fecal Occult Blood (FOB) Test and stored at 10, 20, and 30°C at time points of 1, 5, 10, 15, and 16 days from the start of storage.

To claim 30 days of sample stability at 2 to 8°C each sample was tested with Wondfo One Step Fecal Occult Blood (FOB) Test and stored at 2, 4, 8, and 10°C at time points of 3, 10, 25, 30, and 32 days from the start of storage. Wondfo One Step Fecal Occult Blood (FOB) Test sampling tubes must be brought back to room temperature before testing.

To claim 3 months of sample stability at -20°C each sample was tested with Wondfo One Step Fecal Occult Blood (FOB) Test and stored at 2, 4, 8, and 10°C at time points of 1, 30, 60, 90, and 92 days from the start of storage. Wondfo One Step Fecal Occult Blood (FOB) Test sampling tubes must be brought back to room temperature before testing.

FOBT-CHECK 'Negative and Positive Controls' were also tested daily to ensure and confirm the validity of the test results of the stability study. Stool samples collected in Wondfo One Step Fecal Occult Blood (FOB) Test sampling tubes are stable up to 3 months when stored -20℃, 30 days when stored at 2 to 8°C, and stable up to 15 days when stored at room temperature.

In-use Stability

In-use stability study was conducted with 1 lot of Wondfo One Step Fecal Occult Blood (FOB) Test kit (test cassette and sampling tubes). Testing was performed by preparing 21 aliquots of spiked stool test samples collected in extraction buffer in Wondfo One Step Fecal Occult Blood (FOB) Test sample

8

collection tubes. The Hb-free stool specimens were spiked with human blood (of known hemoglobin level) to obtain fecal samples containing seven different hemoglobin concentrations: 0 µg/g stool, 1.2 µg/g stool, 1.35 µg/g stool, 1.5 µg/g stool, 1.8 µg/g stool, 2.25 µg/g stool, and 60 µg/g stool, that are equivalent to 0 ng/mL, 40 ng/mL, 45 ng/mL, 60 ng/mL, 75 ng/mL and 2000 ng/mL, respectively. To claim 1 hour of in-use stability, each sample was tested with Wondfo One Step Fecal Occult Blood (FOB) Test at time points 0.5, 1, 1.5, and 2 hours intervals under the following conditions:

Condition 1 (regular temperature and humidity): 25℃, 50% RH Condition 2 (high temperature): 40℃, 50% RH Condition 3 (high humidity): 25℃, 90% RH

FOBT-CHECK 'Negative and Positive Controls' were also tested daily to ensure and confirm the validity of the test results of the stability study.

Test Kit Stability (Accelerated)

Accelerated stability study was conducted with 3 lots of Wondfo One Step Fecal Occult Blood (FOB) Test kit (test cassette and sampling tubes). Test kit stability testing was performed by preparing 21 aliquots of spiked stool test samples collected in extraction buffer in Wondfo One Step Fecal Occult Blood (FOB) Test sample collection tubes. The Hb-free stool specimens were spiked with human blood (of known hemoglobin level) to obtain fecal samples containing seven different hemoglobin concentrations: 0 µg/g stool, 1.35 µg/g stool, 1.5 µg/g stool, 1.8 µg/g stool, 1.8 µg/g stool, 2.25 µg/g stool, and 60 µg/g stool, that are equivalent to 0 ng/mL, 45 ng/mL, 50 ng/mL, 60 ng/mL, 75 ng/mL and 2000 ng/mL, respectively. Three operators tested the three lots stored at 50°C at 1°, 30°, 60th, 70th, 80th, 85th and 90th day intervals.

FOBT-CHECK 'Negative and Positive Controls' were also tested daily to ensure and confirm the validity of the test results of the stability study. The results showed that the test kit was stable for an estimated period of 24 months at 4-30°C.

Test Kit Real Time Stability

Real time stability was conducted with three lots of Wondfo One Step Fecal Occult Blood (FOB) Test kit (test cassette and sampling tubes). Test kit stability testing was performed by preparing 21 aliquots of spiked stool test samples collected in extraction buffer in Wondfo One Step Fecal Occult Blood (FOB) Test sample collection tubes. The Hb-free stool specimens were spiked with human blood (of known hemoglobin level) to obtain fecal samples containing seven different hemoglobin concentrations: 0 µg/g stool, 1.2 µg/g stool, 1.5 µg/g stool, 1.8 µg/g stool, 1.8 µg/g stool, 2.25 µg/g stool, and 60 µg/g stool, that are equivalent to 0 ng/mL, 45 ng/mL, 50 ng/mL, 60 ng/mL, 75 ng/mL and 2000 ng/mL, respectively. Three operators tested the three lots stored at 4, 10, 20, and 30°C at time points of 1, 5, 10, 15, 20, 22, 24, 25, 26, and 27 months intervals.

9

FOBT-CHECK 'Negative and Positive Controls' were also tested daily to ensure and confirm the validity of the test results of the stability study.

Test Kit Transport Simulation Test

Three lots of Wondfo One Step Fecal Occult Blood (FOB) Test kits were evaluated during the transport simulation test. Test kit transport stability testing was performed by preparing 21 aliquots of spiked Hb-free stool specimens with human blood (of known hemoglobin level) to obtain fecal samples containing seven different hemoglobin concentrations: 0 µg/g stool, 1.2 µg/g stool, 1.35 µg/g stool, 1.5 µg/g stool, 1.8 µg/g stool, 2.25 µg/g stool, and 60 µg/g stool, that are equivalent to 0 ng/mL, 40 ng/mL, 45 ng/mL, 50 ng/mL, 60 ng/mL, 75 ng/mL and 2000 ng/mL, respectively. Extreme shipping temperatures were simulated at -20°C and 40°C. Twenty one aliquots of the 7 Hb concentrations stool samples/test kit stored at -20°C were brought to room temperature before testing. Three operators tested the three lots performance in 18, 7th, 28th, 33th and 35th day intervals. Wondfo One Step Fecal Occult Blood (FOB) Test kits are stable up to 35 transport days when stored at -20°C and 40°C.

FOBT-CHECK 'Negative and Positive Controls' were also tested daily to ensure and confirm the validity of the test results of the stability study.

d. Interference

Susceptibility of Wondfo One Step Fecal Occult Blood (FOB) Test to interference from vegetable extracts, vitamin supplements Vitamin C (ascorbic acid), iron (FeCl3+6H2O) and horseradish peroxidase was evaluated using one kit lot and one operator. Test samples were prepared by spiking stool samples containing seven different hemoglobin concentrations: 0 µg/g stool, 1.35 ug/g stool, 1.5 µg/g stool, 1.8 µg/g stool, 2.25 µg/g stool, and 60 µg/g stool, that are equivalent to 0 ng/mL, 40 ng/mL, 45 ng/mL, 50 ng/mL, 60 ng /mL, 75 ng/mL and 2000 ng/mL, respectively.

Twenty one aliquots of hemoglobin-negative stool spiked listed above were spiked with the intended level of respective vegetable extracts: broccoli extracts (1 mL/g feces), cantaloupe extract (1 mL/g feces), cauliflower extract (1 mL/g feces), horseradish extract (1 mL/g feces), parsnip extract (1 mL/g feces), red radish extract (1 mL/g feces), and raw turnip extract (1 mL/g feces).

Twenty one aliquots of hemoglobin-negative stool spiked listed above were also spiked with the intended level of respective Vitamin C (250µg /ml), iron (2000µg/ml) and horseradish peroxidase (20000 µg/ml).

All samples were mixed with extraction buffer in Wondfo One Step Fecal Occult Blood (FOB) Test sample collection tubes and tested in a randomized order. Wondfo One Step Fecal Occult Blood (FOB) Test did not show significant interference with any of the vegetable extracts, Vitamin C, iron or horseradish peroxidase up to 250 ug /ml, 2000 ug/ml and 20000 ug/ml respectively.

Interference from Toilet Cleaners

10

Susceptibility Wondfo One Step Fecal Occult Blood (FOB) Test to interference from toilet cleaners and regular toilet water was evaluated using one kit lot and one operator. Twenty one aliquots of test samples were prepared by spiking Hb-free stool specimen with known levels of human Hb solution to obtain fecal samples with seven different Hb concentrations: 0 µg/g stool, 1.2 µg/g stool, 1.35 µg/g stool, 1.5 µg/g stool, 1.8 µg/g stool, 2.25 µg/g stool, and 60 µg/g stool, that are equivalent to 0 ng/mL, 40 ng/mL, 45 ng/mL, 50 ng/mL, 60 ng/mL, 75 ng/mL and 2000 ng/mL, respectively. The 21 aliquots of spiked hemoglobin-negative stool concentrations were spiked with the following cleaners: Clorox (1%), Frosch (1%), Mr. Muscle (1%), and regular toilet water (1%). All samples were mixed with extraction buffer in Wondfo One Step Fecal Occult Blood (FOB) Test sample collection tubes and tested in a randomized order. Wondfo One Step Fecal Occult Blood (FOB) Test did not show significant interference from regular toilet water. However, there were false negative results when 1% toilet cleaner was added to the water.

e. Specificity

Specificity to human hemoglobin variant

The ability of Wondfo One Step Fecal Occult Blood (FOB) Test to detect human hemoglobin variants was determined using one kit lot and one operator testing hemoglobin A (HbA), hemoglobin-S (HbS) and hemoglobin-C (HbC) by preparing spiked stool samples containing seven different hemoglobin concentrations: 0 µg/g stool, 1.2 µg/g stool, 1.35 µg/g stool, 1.8 µg/g stool, 1.8 µg/g stool, 2.25 µg/g stool, and 60 µg/g stool, that are equivalent to 0 ng/mL, 45 ng/mL, 50 ng/mL, 60 ng/mL, 75 ng/mL and 2000 ng/mL, respectively. Twenty one aliquots of each of the seven concentrations for each hemoglobin variant of spiked stool test samples were mixed with extraction buffer in Wondfo One Step Fecal Occult Blood (FOB) Test sample collection tubes. Samples were tested in a randomized order. Results showed Wondfo One Step Fecal Occult Blood (FOB) Test equivalently recognized variants of hemoglobin HbA, HbS and HbC.

Cross-Reactivity

Cross-reactivity of Wondfo One Step Fecal Occult Blood (FOB) Test with non-human hemoglobin and meat extracts was evaluated by using one kit lot and one operator. Test samples were prepared by spiking Hb-free stool specimen with known levels of human Hb solution to obtain fecal samples with seven different Hb concentrations: 0 µg/g stool, 1.35 µg/g stool, 1.35 µg/g stool, 1.5 µg/g stool, 1.8 µg/g stool, 2.25 ug/g stool, and 60 µg/g stool, that are equivalent to 0 ng/mL, 40 ng/mL, 50 ng/mL, 60 ng /mL, 75 ng/mL and 2000 ng/mL, respectively.

Twenty one aliquots of the spiked hemoglobin-negative stool concentrations listed above were spiked with the intended level of respective non-human hemoglobin: beef Hb (500 µg/mL), pig Hb (500 ug/mL), fish Hb (500 µg/mL), horse Hb (500 µg/mL), goat Hb (500 µg/mL), rabbit Hb (500 µg/mL), sheep (500 µg/mL), and chicken Hb (500 µg/mL).

Twenty one aliquots of the spiked hemoglobin-negative stool concentrations listed above were also spiked with the intended level of respective animal meat extracts: beef meat extract (1 mL/g feces),

11

pork meat extract (1 mL/g feces), fish meat extract (1 mL/g feces), horse meat extract (1 mL/g feces), goat meat extract (1 mL/g feces), rabbit meat extract (1 mL/g feces), sheep meat extract (1 mL/g feces), and chicken meat extract (1 mL/g feces).

All samples were mixed with extraction buffer in Wondfo One Step Fecal Occult Blood (FOB) Test sample collection tubes and tested in a randomized order. Wondfo One Step Fecal Occult Blood (FOB) Test did not show significant interference from any of the non-human hemoglobin or animal meat extracts tested.

f. Cut-off
The cutoff value study of Wondfo One Step Fecal Occult Blood (FOB) Test was evaluated in house. Fecal test samples for cut-off testing were prepared by spiking stool samples with human blood (of known hemoglobin level) to obtain fecal samples containing seven different hemoglobin concentrations: 0 µg/g stool, 1.2 µg/g stool, 1.35 µg/g stool, 1.8 µg/g stool, 1.8 µg/g stool, 2.25 µg/g stool, and 60 ug/g stool, that are equivalent to 0 ng/mL, 45 ng/mL, 50 ng/mL, 60 ng/mL, 60 ng/mL, 75 ng/mL and 2000 ng/mL, respectively. Twenty one aliquots of each of the seven concentrations of spiked stool test samples were mixed with extraction buffer in Wondfo One Step Fecal Occult Blood (FOB) Test sample collection tubes and twenty one aliquots of each of the seven concentrations of spiked stool test samples were mixed with the predicate (FOB One Step Rapid Test) sample collection tubes. Samples were tested in randomized order. Testing was performed side-by-side with the predicate by comparing the test results of the device with that of the predicate. Cut-off was determined to be 1.35 µg hemoglobin/g stool or 45 ng/mL (hemoglobin in fecal sample mixed with detection buffer).

| Cut-off study | Agreement | Expected results | | | Overall Percent
Agreement | Positive Percent
Agreement
(95% CI) | Negative
Percent
Agreement
(95% CI) |
|-----------------------------------------------------|---------------------|---------------------|---------------------|------------------|------------------------------|-------------------------------------------|----------------------------------------------|
| | | Positive
Results | Negative
Results | Total
Results | | | |
| FOB One Step
Rapid Test
(Predicate) | Positive
Results | 95 | 1 | 96 | 99.3% | 100%
(96.1%-100%) | 98.1%
(89.9%-99.7%) |
| | Negative
Results | 0 | 51 | 51 | | | |
| | Total
Results | 95 | 52 | 147 | | | |
| Wondfo One Step
Fecal Occult
Blood (FOB) Test | Positive
Results | 95 | 1 | 96 | 99.3% | 100%
(96.1%-100%) | 98.1%
(89.9%-99.7%) |
| | Negative
Results | 0 | 51 | 51 | | | |

Cut-off Study Summary

12

| Cut-off study | Agreement | Expected results | | | Overall Percent
Agreement | Positive Percent
Agreement
(95% CI) | Negative
Percent
Agreement
(95% CI) |
|---------------|------------------|---------------------|---------------------|------------------|------------------------------|-------------------------------------------|----------------------------------------------|
| | | Positive
Results | Negative
Results | Total
Results | | | |
| | Total
Results | 95 | 52 | 147 | | | |

2. Comparison Studies

A method comparison of Wondfo One Step Fecal Occult Blood (FOB) Test with the predicate test, Orient Gene Biotech One Step Rapid FOB Test, was conducted by assessing 407 patient samples, 18 stool samples around the cut-off (purchased), and 100 spiked samples using three different lots of the proposed device and one lot of the predicate device. The method comparison study was performed at three POC testing sites by three different operators at each site. The FOBT-CHEK external controls (positive and negative) were run prior to testing. Statistical analysis of site-wide test results as well as combined results showed that Wondfo One Step Fecal Occult Blood (FOB) test results have acceptable overall percent agreement as well as positive percent agreement and negative percent agreement with Orient Gene Biotech One Step Rapid FOB Test. The method comparison study demonstrated that the analytical performance of the Wondfo One Step Fecal Occult Blood (FOB) Test is substantially equivalent to the predicate device.

| Study
Site | New Test | | Predicate Test | | | Overall
Percent
Agreement | Positive
Agreement
Percent
(95% CI) | Negative
Agreement
Percent
(95% CI) |
|---------------------|--------------------------------|-------------------------|---------------------|---------------------|------------------|---------------------------------|----------------------------------------------|----------------------------------------------|
| | Wondfo
One Step FOB
Test | FOB One Step Rapid Test | Positive
Results | Negative
Results | Total
Results | | | |
| Study
POC Site 1 | Positive
Results | 35 | 35 | 0 | 35 | 100% | 100%
(90.1%-100%) | 100%
(96.3%-100%) |
| | Negative
Results | 0 | 0 | 101 | 101 | | | |
| | Total
Results | 35 | 35 | 101 | 136 | | | |
| Study
POC Site 2 | Positive
Results | 45 | 45 | 0 | 45 | 99.3% | 97.8%
(88.7%-99.6%) | 100%
(96.3%-100%) |
| | Negative
Results | 1 | 1 | 101 | 102 | | | |
| | Total
Results | 46 | 46 | 101 | 147 | | | |

13

| Study
POC Site 3 | Positive
Results | 52 | 1 | 53 | 99.6% | 100%
(93.1%-100%) | 99.5%
(97.1%-99.9%) |
|---------------------|---------------------|-----|-----|-----|-------|------------------------|------------------------|
| | Negative
Results | 0 | 189 | 189 | | | |
| | Total
Results | 52 | 190 | 242 | | | |
| Combined
Sites | Positive
Results | 132 | 1 | 133 | 99.6% | 99.2%
(95.9%-99.9%) | 99.7%
(98.6%-100%) |
| | Negative
Results | 1 | 391 | 392 | | | |
| | Total
Results | 133 | 392 | 525 | | | |

Lay-user study

A lay user study was performed at three intended user sites with 100 lay users testing his/her own stool sample using the device according to the package insert. The lay users also provided a sample for professional testing. The overall agreement between the results obtained by lay-users and professional users was 100%. The lay users also tested spiked samples. Human Negative stool samples in collection buffer tubes (50mg feces in 1.5mL FOB buffer solution) were spiked with hemoglobin at concentrations of 0, 37.5, 50, 62.5, and 2000ng/ml. A total of twenty spiked samples were made at each concentration. Each lay user tested only one spiked sample. All results are compared with that obtained by professionals. Two discrepant results were found for two samples at 37.5ng/mL concentration. The overall agreement between the results obtained by lay-users and professional users was 98%. Lay-users were also given surveys on the ease of understanding the package insert instructions. All lay users indicated that the device instructions can be easily followed. All data was analyzed based on 45 ng/mL as the cutoff. The data analysis for the lay user study below shows that all of the lay users performed the test correctly and the results show acceptable accuracy when compared to the professional test results.

		Professional Test Results		Total Results
Lay User Test
Results	Results	Positive	Negative
	Positive	12	0	12
	Negative	0	88	88
Total Results		12	88	100

Results of lay users testing their own specimens are shown in the following table

			Results of lav user testing spiked specimens are shown in the following table.

| Hemoglobin
Concentration
(ng/mL) | Number
of
Samples | Lay User
Results | | Professional
Results | | %
Negative
agreement | %
Positive
agreement |
|----------------------------------------|-------------------------|---------------------|-----|-------------------------|-----|----------------------------|----------------------------|
| 0 | 20 | - 20 | + 0 | - 20 | + 0 | 100% | |

14

37.5	20	2	0
50	20	20	20	100%
62.5	20	20	20	100%
2000	20	20	20	100%

Test Kit Reaction Time:

The Wondfo One Step Fecal Occult Blood (FOB) Test reaction time was demonstrated by using seven stool concentrations at eight different time points. Reaction time test samples were prepared by spiking Hb-free stool specimen with known levels of human Hb solution to obtain fecal samples with seven different Hb concentrations: 0 µg/g stool, 1.2 µg/g stool, 1.5 µg/g stool, 1.5 µg/g stool, 2.25 ug/g stool, and 60 ug/g stool, that are equivalent to 0, 40, 45, 50, 60, 75 and 2000 ng/mL, respectively. Twenty one replicates of each sample were collected with Wondfo One Step Fecal Occult Blood (FOB) Test sample collection tubes. All specimens were tested with one test kit lot in a randomized and blinded manner by one operator. The test results were read and recorded at 3, 5, 8, 10, 20, 30, 40 and 60 minutes. Positivity rate of 0 ng/mL was 0%, positivity rate of 40 ng/mL was 4.8%, positivity rate of 45 ng/mL was 47.6%, and the positivity rate of 50 ng/mL was 100% at 10 minutes of reaction. The appropriate reaction time of Wondfo One Step Fecal Occult Blood (FOB) Test was demonstrated as 10 minutes.

Operator Intensity Reading:

The operator intensity reading test was performed by utilizing test samples prepared by spiking Hb-free stool specimen with known levels of human Hb solution to obtain fecal samples with seven different Hb concentrations: 0 µg/g stool, 1.35 µg/g stool, 1.5 µg/g stool, 1.8 µg/g stool, 1.8 µg/g stool, and 60 ug/g stool, that are equivalent to 0, 40 , 45, 50, 60, 75 and 2000 ng/mL, respectively. Twenty-five replicates were prepared for each concentration. All specimens were tested with one test kit lot in a randomized and blinded manner by five different readers at one intended use site. The study participants recorded the results and graded the intensity of the test line from 10 levels: (B (no test line), C9 (faint test line), C8, C7, C6, C5, C4, C3, C2 and C1 (deepest test line). The intensity of the test line increased in correlation with the Hb concentration of stool test samples. There was no statistical significance in the analyses of the test samples performed by the operators for the intensity reading test study.

Specimen Collection Verification:

Verification that the applicator device for the Wondfo One Step Fecal Occult Blood (FOB) Test consistently delivers the specified amount of stool required for optimal test performance was performed by five lay users using five positive and five negative clinical samples per lay user. The study was performed at one intended use site. Five positive and 5 negative clinical samples results were confirmed using the predicate device and the proposed device. A professional operator used an electronic balance and weighed the sampling stick first so that this weight could be offset. The lay users then inserted the sampling stick into test stool sample at six different sites to collect samples and placed the sampling stick back in to the sample collection tube (without buffer). After, the professional operator used the electronic balance to weigh the sample stick with stool sample.

15

The lay users next used three sample collection tubes (with buffer) to collect the sample again and used three test cassettes of Wondfo One Step Fecal Occult Blood (FOB) Test to test the result according to the test procedure directions of the package insert. The average value for sample volume collected was 0.0517g and the standard deviation was 0.0027, which demonstrated the consistency of the sampling. The total positive and negative results obtained by the lay users are consistent with the expected results for the test samples. The lay users collected adequate consistent amounts of stool samples when following the package insert directions to perform the Wondfo One Step Fecal Occult Blood (FOB) Test.

1. Clinical Studies
  Not applicable.
11.Conclusion
Based on the test principle and acceptable performance characteristics including precision, interference, specificity and method comparison of the devices, it is concluded that the Wondfo One Step Fecal Occult Blood Test device is substantially equivalent to the predicate.