Study Title

Product Identity

Test Microorganism

Study Identification Number


Study Completion Date

Testing Facility 

Study Sponsor

Virucidal Efficacy of a Test Substance For Use on Inanimate, Nonporous Surfaces

Bioneat (BIO-1001)

Human coronavirus, Strain 229E, ATCC VR-740


Emily Cox, B.S.


Microchem Laboratory 1304 W. Industrial Blvd. Round Rock, Texas 78681

United Food Products Jeff Kaufman 11555 Heron Bay Blvd., Ste 200 Parkland, FL 33076 


General Study Information

Study Title: 

ASTM E1053 Method Virucidal Efficacy of a Test Substance For Use on Inanimate, Nonporous Surfaces 

Study Identification Number:


Test System Test Microorganism:

Human coronavirus, Strain 229E, ATCC VR-740

Host Cell:

MRC-5 (CCL-171)

Test Substance:

Bioneat (BIO-1001)

Test Substance Receipt Date:


Test Parameters Test Substance Dilution:

Ready to use liquid test substance

Test Substance Application:

2.0 ml delivered by serological pipette

Organic Soil Load:

No additional soil load incorporated into the inoculum

Number of Replicates Per Lot:


Contact Time

10 minutes

Exposure Temperature:

Ambient room temperature (23.4 – 23.5°C) and 42% Relative Humidity (RH) 

Neutralization Method:

Sephadex LH-20 gel filtration columns

Study Dates Experimental Start Date/Time:

17JUL2020 / 1135

Experimental Termination Date/Time:

24JUL2020 / 1545

Study Completion Date: 




  • Stock virus was thawed and was not supplemented with an organic soil load.
  • Sterile glass Petri dish carriers (100 x 15 mm) were inoculated with a volume of virus suspension. A sufficient number of test and control carriers were prepared.
  • Inoculated carriers were dried at room temperature under laminar flow conditions.
  • The test substance was prepared according to the Study Sponsor’s instructions as requested, and applied to the test carriers using a serological pipette.
  • The treated carriers were held for the predetermined contact time(s), and then neutralized in a manner appropriate for the test substance (e.g. dilution and/or gel filtration).
  • The control carrier was held covered for the contact time then harvested and neutralized in the same manner as the test.
  • Following neutralization of test and control carriers, the viral suspensions were quantified to determine the levels of infectious virus using standard cell culture (e.g. TCID50) or plaque assay techniques.
  • Assay trays/plates were incubated for the period most suitable for the virus-host cell system (e.g. 7 days).
  •  After the incubation period, the assay was scored for the presence/absence of test virus and cytotoxic effects. The appropriate calculations were performed (e.g. Spearman-Karber) to determine viral titers and levels of test substance cytotoxicity, where applicable.
  • Log10 and percent reductions were calculated for viral films exposed to the test product relative to the titer obtained for the study control carrier(s), and reported to the Study Sponsor


The following measures are met to ensure the acceptability of virucidal efficacy data:

  •  A minimum of 4.80 log10 infective units/control carrier is recovered from each plate recovery control film(s).
  • The virus titer control demonstrate obvious and or typical cytopathic effects on the monolayers unless a detection method other than cytopathic effect is used.
  • Neutralization of the test substance with a low titer (e.g. 1000-5000 infective units) of the test virus is demonstrated.
  • Quantification of the test and control parameters are conducted at a minimum of four determinations per dilution.

The product performance criteria follows:

  • In the presence or absence of cytotoxicity, the product should demonstrate a ≥3.00 log10 reduction in viral titer on each surface.
  • If cytotoxicity is present, the virus control titer should be increased if necessary to demonstrate a ≥3.00 log10 reduction in viral titer on each surface beyond the cytotoxicity level.


The TCID50 (Tissue Culture Infectivity Dose) represents the endpoint dilution where 50% of the cell cultures exhibit cytopathic effects due to infection by the test virus. The endpoint dilution at which 50% of the host cell monolayers exhibit cytotoxicity is termed the Tissue Culture Dose (TCD50). The TCID50, and TCD50 was determined using the SpearmanKärber method and calculated as follows:

Negative logarithm of endpoint titer = 

– Log of first dilution inoculated] – [((sum of % mortality at each dilution/100) – 0.5) x Logarithm of dilution]

The result of this calculation is expressed as TCID50/0.1 ml (or volume of dilution inoculated) for the test, virus control, and neutralization control and TCD50/0.1 ml (or volume of dilution inoculated) for the cytotoxicity control.

Calculation of the Log Reduction

The log reduction in viral titer was calculated as follows:

Plate Recovery Control Log10 TCID50 – Virus-Test Substance Log10 TCID50

Calculation of the Percent Reduction

The percent reduction in viral titer was calculated as follows:

Percent Reduction = 1- (C/B) x 100, where:
B = Average TCID50 of virus in control suspensions.
C = Average TCID50 of virus in virus-test suspensions.

The presence of any test substance cytotoxicity were taken into account when calculating the log and percent reductions in viral titer.

If multiple virus control and test replicates were performed, the average TCID50 of each parameter was calculated and the average result used to calculate the log reductions in viral titer.



The purpose of the study was to determine the virucidal efficacy of Bioneat (BIO-1001) against Human coronavirus Strain 229E, with no additional soil load incorporated into the inoculum, at a contact time of 10 minutes, and at an exposure temperature of room temperature (23.4 – 23.5°C) and 42% RH.

The Plate Recovery Control demonstrated an average viral titer of 4.80 Log10 TCID50 per carrier. 

Test Substance cytotoxicity was detected in the lot of test substance assayed at 1.50 Log10.

The Test Substance Neutralization Control demonstrated that the test substance was neutralized at 1.50 Log10 for the lot assayed.

Taking the cytotoxicity and neutralization control results into consideration, the evaluated test substance, Bioneat (BIO-1001), demonstrated an average ≥3.00 Log10 reduction in viral titer (≥99.90%) at a contact time of 10 minutes.  

We are working on the lab test on the Corona Patients