Project Report

Testing of the efficacy of

Didecyldimethyl Ammonium Chloride
(DDAC) against African swine fever
virus.
By:

Prof. R.R. Bragg B.Sc., B.Sc. (Hons), M.Sc., PhD.

Veterinary Biotechnology
Dept of Microbiology & Biochemistry
University of the Orange Free State
P.O. Box 339
Bloemfontein
9300
Tel ++ 27 51 401 2676
E-mail : Braggrr@ufs.ac.za

Report date:

28 January 2020

Department of Microbial, Biochemical & Food Biotechnology

P.O. Box 339 / 205 Nelson Mandela Drive
9301 Bloemfontein, South Africa
Tel: +27(0)51 4012676 Fax: +27(0)51 401 9376 E-mail: braggrr@ufs.ac.za
Website: www.ufs.ac.za/biotech
Aims :

To test the in vitro efficacy of various dilutions of Didecyldimethyl Ammonium

Chloride (DDAC) against the Lisbon strain of African Swine fever virus at different
dilutions and different contact times.

Methods :

Test methods:

Tests performed by J. Shirai, T. Kanno, Y. Tsuchiya, S. Mitsubayashi &

R. Seki of the Department of Exotic Diseases, National Institute of Animal
Health, Tokyo, Japan
Test results published in Journal of Veterinary Medical Science, 62, 85 –
92.

The Lisbon strain of African Swine fever virus was used in this
experiment. The virus under test was titrated in Vero cells containing
2% bovine serum before and after exposure to the different dilutions of
disinfectant under test. Results are presented as 50% tissue culture
infectious dose (TCID50) per 0.1ml virus solution.

The disinfectant used in this experiment was a 10% solution of DDAC. Virukill
contains 12% DDAC that has been modified to increase efficacy.

The 10% solution of DDAC used in this experiment was diluted as follows:

Dilution Dilution % dilution % DDAC

factor
1 1/400 0.25% 0.025%
2 1/800 0.125% 0.0125%
3 1/1600 0.0625% 0.00625%
4 1/3200 0.03125% 0.003125%
5 1/6400 0.016% 0.0016%
6 1/12800 0.008% 0.0008%
7 1/25600 0.004% 0.0004%

The experiment was performed by mixing equal quantities of the virus sample
with the dilution of disinfectant under test. This was incubated at room
temperature for a contact time of 30 mins.

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 A virus control was also mixed with an equal volume of sterile distilled water
and incubated at room temperature for 30 mins.

After the incubation period, all samples were immediately mixed with tissue
culture medium containing 5% calf serum and diluted for performing titrations.
The 5% calf serum added to the sample will neutralise any residual DDAC in
the sample.

Results

The titre of the Lisbon strain of (ASF) treated according to the methods
discussed about was found to be a TCID50 of 104.5 per 0.1 ml virus sample.

No cytotoxic effects were noted with DDAC when the 10% concentration of the
DDAC based product was diluted by more than 1 in 200. Thus, no cell damage
as a result of the toxic action of DDAC on the Vero cells, at the dilutions above
a 1/200, which were used in this experiment were recorded.

The calculated TCID50 per 0.1 ml of the sample of African swine fever virus after
exposure to the different dilutions of DDAC with a contact time of 30 min at room
temperature, can be seen in Table 1 below.

Table 1. TCID50 per 0.1 ml virus sample recorded after exposure to different
dilutions of DDAC with a 30 min contact time and incubated at room
temperature.

Dilution Dilution % DDAC TCID50 per 0.1 ml

factor
1 1/400 0.025% 0 (no CPE recorded
in any dilutions)
2 1/800 0.0125% 0 (no CPE recorded
in any dilution)
3 1/1600 0.00625% 0 (no CPE recorded
in any dilution)
4 1/3200 0.003125% 0 (no CPE recorded
in any dilution)
5 1/6400 0.0016% TCID50 2.5
6 1/12800 0.0008% TCID50 4.0

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Discussion

It can be seen from the results presented in Table 1 that DDAC is highly effective
against the strain of African swine fever virus used in this experiment. The toxicity
level of DDAC is very low. No cell damage was recorded in the Vero cells at a
dilution of the starting product (a 10% DDAC solution) of greater than 1/200
(0.5% dilution of the product or 0.05% DDAC). As such, it is possible to state that
the disinfectant completely neutralised the virus at the dilutions were no CPE
were recorded. There is therefore, 100% inactivation of the virus.

From this experiment it was established that the MIC for DDAC against African
swine fever virus is 0.003%.. A dose dependent effect of DDAC can be seen at
dilutions greater than 0.003%, as some virus was found to survive at more diluted
samples of DDAC.

The recommended dose of Virukill (which is a 12% DDAC solution) is a 1%

dilution for all disinfection of hard surfaces. This dilution is substantially above
the calculated MIC for DDAC, thus indicating that Virukill will be highly effective
for the disinfection of hard surfaces for the removal of African swine fever virus.

It must be stressed that these experiments were not performed under condition
of hard water or in the presence of organic load. These conditions can have a
significant influence on the efficacy of a disinfectant. Based on extensive testing
on the efficacy of Virukill with and without organic load and hard water against a
wide variety of pathogens, both bacteria and virus, it has been demonstrated that
these conditions can greatly influence the MIC value. The effects of hard water
and organic load normally influence the MIC in the region of a two dilution factor
difference. Based on this, the hypothesized MIC under hard water conditions with
organic load would be in the region of 0.0125% DDAC. This would translate into
a Virukill dilution of 0.01% under conditions of hard water and high organic load.

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Conclusions

Based on this experimental data, it can be seen that Virukill, which contains a
12% DDAC solution will be highly effective against African swine fever virus at
the recommended application rate of a 1% Virukill solution at 600 ml/m2 surface
area to be disinfected.

Signed this 28th day of January 2020.

Prof R.R. Bragg