FINAL REPORT
VIRUCIDAL EFFECTIVENESS TEST
Coronavirus
Test Agents: Oreganol P73 Extra Strength Formula
and
Oregacyn
Data Requirements
Research Development
Author
M. Khalid Ijaz, DVM, Ph.D.
Performing Laboratory
MICROBIOTEST, INC.
105B Carpenter Drive
Sterling, Virginia 20164
Laboratory Project Identification Number
514-101
Submitted to: North American Herb and Spice
9812 East Karen Drive
Scottsdale, AZ 85260
Page 1 of 10
Final Report: VET - Coronavirus Project 514-101 Page 2 of 10
QUALITY ASSURANCE UNIT STATEMENT
Title of Study: Virucidal Effectiveness Test – Coronavirus
The Quality Assurance Unit of MICROBIOTEST has inspected the final report for Project
Number 514-101 for accuracy in data transcription.
The dates that inspections were made and the dates that findings were reported to
management and to the study director are listed below.
PHASE DATE OF DATE REPORTED TO DATE REPORTED
INSPECTED INSPECTION STUDY DIRECTOR TO MANAGEMENT
Final Report 08/11/03 08/11/03 08/14/03
________________________________________ ______
Nathan S. Jones Date
Quality Assurance Unit
COMPLIANCE STATEMENT
This study was conducted under my direction according to the protocol and extant SOPs.
The following technical personnel participated in this study:
Zheng Chen, Samina S. Raja
Study Director: MICROBIOTEST, INC.
______________________________ _______________
M. Khalid Ijaz, DVM, Ph.D. Date
MICROBIOTEST, INC.
Final Report: VET - Coronavirus Project 514-101 Page 3 of 10
TABLE OF CONTENTS
FINAL REPORT - COVER PAGE ................................................................................... 1
QUALITY ASSURANCE UNIT STATEMENT.................................................................. 2
COMPLIANCE STATEMENT .......................................................................................... 2
TABLE OF CONTENTS .................................................................................................. 3
TEST SUMMARY............................................................................................................ 4
TEST CONDITIONS..................................................................................................... 5-6
STUDY DATES AND FACILITIES................................................................................... 6
RECORDS TO BE MAINTAINED.................................................................................... 6
RESULTS................................................................................................................... 7-10
CONCLUSIONS ............................................................................................................ 10
APPENDIX I ......................................................................................................................
APPENDIX II .....................................................................................................................
MICROBIOTEST, INC.
Final Report: VET - Coronavirus Project 514-101 Page 4 of 10
TEST SUMMARY
TITLE: Virucidal Effectiveness Test – Coronavirus
STUDY DESIGN: This study was performed according to the signed protocol and project
sheets issued by the Study Director.
See Project Sheets (Appendix I)
See signed protocol (Appendix II)
TEST MATERIALS SUPPLIED BY THE SPONSOR OF THE STUDY:
1. Oreganol P73 Extra Strength Formula, received at
MICROBIOTEST on 06/23/03 and assigned DS No. 6287.
2. Oregacyn, Lot No. 111102, received at MICROBIOTEST on
06/23/03 and assigned DS No. 6288.
SPONSOR: North American Herb and Spice
9812 East Karen Drive
Scottsdale, AZ 85260
MICROBIOTEST, INC.
Final Report: VET - Coronavirus Project 514-101 Page 5 of 10
TEST CONDITIONS
Challenge virus:
Human Coronavirus (HCoV), ATCC VR-740
Host:
MRC-5 cells, Diagnostic Hybrids, Inc., Athens, OH
Active ingredient in test product:
Wild Oregano Oil & Olive Oil Base (Oreganol P73 Extra Strength Formula)
Wild Oregano, Wild Cumin, Wild Sage, Wild Cinnamon (Oregacyn)
Neutralizer:
N/A
Exposure time:
2, 5, 10, 15, and 20 minutes
Exposure temperature:
Ambient room temperature (21C)
Diluent:
Earle’s Balanced Salt Solution (EBSS)
Dilution:
1:1000 (Oreganol P73 Extra Strength Formula) and 1:10000 (Oregacyn)
Organic load:
Viral stock contained at least 5% fetal bovine serum
MICROBIOTEST, INC.
Final Report: VET - Coronavirus Project 514-101 Page 6 of 10
TEST CONDITIONS (continued)
Media and reagents:
Minimum Essential Medium Eagle containing 10% fetal bovine serum (CCM)
Phosphate Buffered Saline (PBS)
Earle’s Balanced Salt Solution (EBSS)
Test Agent Application:
For both test agents tested at the appropriate concentration: 1mL of viral
inoculum was added to 9mL of diluted test agent and held for the specified
contact times. At the completion of the 2, 5, 10, 15, and 20 minute contact
times, the virus-test agent mixture was serially diluted tenfold in EBSS.
Selected dilutions were inoculated into 4 wells of MRC-5 monolayers.
STUDY DATES AND FACILITIES
The laboratory phase of this test was performed at MICROBIOTEST, INC., 105B Carpenter
Drive, Sterling, VA 20164, from 07/17/03 to 07/29/03. The study director signed the
protocol 07/17/03. The study completion date is the date the study director signed the final
report.
All changes or revisions of the protocol were documented, signed by the study director,
dated and maintained with the protocol.
RECORDS TO BE MAINTAINED
All testing data, protocol, protocol modifications, test material records, the final report, and
correspondence between MICROBIOTEST and the sponsor will be stored in the archives at
MICROBIOTEST, INC., 105B Carpenter Drive, Sterling, VA 20164, or at a controlled facility
off site.
MICROBIOTEST, INC.
Final Report: VET - Coronavirus Project 514-101 Page 7 of 10
RESULTS
Results are presented in Tables 1 – 7. A titration was performed to determine the titer of
the viral stock. The 50% cell culture infectious unit does per mL (CCID50/mL) was
determined from the test and pertinent control data using the method of Reed and Muench,
Am. J. of Hyg. 1938, 27:493. The cell viability control demonstrated MRC-5 cell viability
and media sterility. Virus was not recovered in the cell viability control.
Table 1 – Cytotoxicity 7/11/03
Cytotoxicity
Oreganol P73 Extra
Oregacyn Oregacyn
Strength Formula
(5%) (1%)
(10%)
CCCC CCCC CCCC
Table 2– Cytotoxicity 7/14/03
Cytotoxicity
Oreganol P73 Extra
Dilution Oregacyn Oregacyn
Strength Formula
(log10) (5%) (1%)
(10%)
-1 CCCC CCCC CCCC
-2 0000 CCCC 0000
-3 0000 0000 0000
Table 3– Cytotoxicity 7/16/03
Cytotoxicity
Oreganol P73 Extra
Dilution Oregacyn
Strength Formula
(log10) (1%)
(10%)
-2 0000* 0000
*20% cytotoxicity observed
Key: C = cytotoxicity observed
0 = no cytotoxicity observed
MICROBIOTEST, INC.
Final Report: VET - Coronavirus Project 514-101 Page 8 of 10
RESULTS (continued)
Table 4 - Test Results
Human Coronavirus titer (CCID50/mL)
Oreganol P73 Extra Strength Formula
Dilutions
(log 10) CONTACT TIME
2min 5 min 10 min 15 min 20 min
-1 ++++ ++++ ++++ ++++ ++++
-2 ++++ +++- ++-+ +--- ----
-3 -+-+ +--- ---- ---- ----
-4 ---- ---- ---- ---- ----
-5 ---- ---- ---- ---- ----
-6 ---- ---- ---- ---- ----
CCID50/mL 103.00 102.50 102.33 101.67 101.50
Key: + = HCoV infected cells detected by cytopathic effects observed
- = no HCoV infected cells detected by lack of cytopathic effects
observed
MICROBIOTEST, INC.
Final Report: VET - Coronavirus Project 514-101 Page 9 of 10
RESULTS (continued)
Table 5 - Test Results
Human Coronavirus titer (CCID50/mL)
Oregacyn
Dilutions Lot No. 111102
(log 10) CONTACT TIME
2min 5min 10 min 15 min 20 min
-1 ++++ ++++ ++++ +-++ ----
-2 ++-+ -+-- ---- ---- ----
-3 ---- ---- ---- ---- ----
-4 ---- ---- ---- ---- ----
-5 ---- ---- ---- ---- ----
-6 ---- ---- ---- ---- ----
CCID50/mL 102.33 101.67 101.50 101.33 ≤101.50
Table 6 - Cytotoxicity Control
Cytotoxicity Control
Dilutions
(log10) Oreganol P73 Extra Strength Oregacyn
Formula Lot No. 111102
-1 0000 0000
-2 0000 0000
-3 0000 0000
Key: + = HCoV infected cells detected by cytopathic effects observed
- = no HCoV infected cells detected by lack of cytopathic effects
observed
0 = no cytotoxicity observed
MICROBIOTEST, INC.
Final Report: VET - Coronavirus Project 514-101 Page 10 of 10
RESULTS (continued)
Table 7 – Input Virus Control and Virus Stock Titer
Human Coronavirus titer (CCID50/mL)
Dilutions
(log 10) Input Virus Control
Virus Stock Titer
-1 PNS ++++
-2 ++++ ++++
-3 ++++ ++++
-4 ++++ ++++
-5 +-+- ++++
-6 ++-- ++--
-7 ---- --+-
-8 ND ----
CCID50/mL 105.50 106.23
Key: + = HCoV infected cells detected by cytopathic effects observed
- = HCoV cells not detected by a lack of cytopathic effects
observed
ND = not determined
PNS = post-neutralized sample
CONCLUSIONS
When tested as described, Oreganol P73 Extra Strength Formula and Oregacyn at final
testing concentrations of 0.1% and 0.01% respectively, inactivated HCoV in direct
proportion to exposure time when HCoV was exposed to each test agent for 2, 5, 10,
15, and 20 minutes at ambient room temperature. All of the controls met the criteria
established for a valid test. These conclusions are based on observed data.
MICROBIOTEST, INC.
