Virucidal efficacy of peracetic acid for instrument disinfection
Becker et al. Antimicrobial Resistance and Infection Control
Virucidal efficacy of peracetic acid for instrument disinfection
Florian H. H. Brill
Daniel Todt 0
Eike Steinmann 0
0 , DE-28259 Bremen , Germany
1 Partner GmbH Institute for Hygiene and Microbiology , Norderoog
Background: Various peracetic-acid (PAA)-based products for processing flexible endoscopes on the market are often based on a two-component system including a cleaning step before the addition of PAA as disinfectant. The peracetic acid concentrations in these formulations from different manufacturers are ranging from 400 to 1500 ppm (part per million). These products are used at temperatures between 20 °C and 37 °C. Since information on the virus-inactivating properties of peracetic acid at different concentrations and temperature is missing, it was the aim of the study to evaluate peracetic acid solutions against test viruses using the quantitative suspension test, EN 14476. In addition, further studies were performed with the recently established European pre norm (prEN 17111:2017) describing a carrier assay for simulating practical conditions using frosted glass. Methods: In the first step of examination, different PAA solutions between 400 and 1500 ppm were tested at 20 °C, 25 °C, and 35 °C with three test viruses (adenovirus, murine norovirus and poliovirus) necessary for creating a virucidal action according to the European Norm, EN 14476. A second step for simulating practical conditions based on prEN 17111:2017 followed by spreading a test virus together with soil load onto a glass carrier which was immerged into a peracetic acid solution. A fixed exposure time of five minutes was used in all experiments. Results: In the quantitative suspension test 1500 ppm PAA solution was needed at 35 °C for five minutes for the inactivation of poliovirus, whereas only 400 ppm at 20 °C for adeno- and murine norovirus were necessary. In the carrier assay 400 ppm peracetic acid at 20 °C were sufficient for adenovirus inactivation, whereas 600 ppm PAA were needed at 25 °C and 35 °C and 1000 ppm at 20 °C for murine norovirus. A PAA solution with 1000 ppm at 35 °C was required for complete inactivation of poliovirus. However, a dramatically decrease of titer after the drying and immerging could be observed. In consequence, a four log reduction of poliovirus titer could not be achieved in the carrier test. Conclusion: In summary, 1500 ppm PAA at 35 °C was necessary for a virucidal action in the quantitative suspension test. After passing the requirements of the suspension test, additional examinations with adeno- and murine norovirus on glass carriers based on prEN 17111:2017 will not additionally contribute to the final claim of an instrument disinfectant for virucidal efficacy. This is due to the great stability of poliovirus in the preceded quantitative suspension test and the fact that poliovirus could not serve as test virus in the following carrier assay.
Peracetic acid; Virucidal efficacy; Instrument disinfection
Peracetic acid (PAA) is often incorporated as active
ingredient of instrument disinfectants for reprocessing
flexible endoscopes in manual and automatic
procedures. Such instrument disinfectants are often used
between room temperature and 40 °C with short exposure
times. By introducing PAA as active ingredient, a broad
range of virucidal efficacy for instrument disinfectants
can be achieved, as requested by the Commission for
Hospital Hygiene and Infection Prevention (Kommission
für Krankenhaushygiene und Infektionsprävention,
]. There is only a minor temperature stress
for the instruments when using short exposure times
with PAA and only aldehydes are able to demonstrate a
comparative range of efficacy against viruses. But for
aldehydes, higher temperatures are necessary in general
for reaching a sufficient virucidal action resulting in a
claim of these chemicals against enveloped and
The virus-inactivating properties of PAA had been
demonstrated earlier in detail by the group of Sprößig
]. Later it was questioned whether
peracetic-acidbased formulations are suited for the cleaning step when
reprocessing flexible endoscopes due to the fixation
potential of PAA . Current formulations on the
market are always based on a two-step procedure including
a cleaning step before the addition of PAA.
The concentrations of PAA in the products for
reprocessing endoscopes differ and there are only few
data on the behaviour of PAA in test methods
developed as European Norms (EN). Therefore, we
evaluated the virucidal activity of PAA solutions in clean
conditions according to a quantitative suspension test
(phase 2/step 1) which is described as EN 14476 with
a short exposure time [
]. This was followed by a
phase 2/step 2 carrier test, simulating practical
conditions recently established as prEN 17111:2017 for
instrument disinfectants in Europe [
For the examination of the virucidal efficacy of different
concentrations of PAA a quantitative suspension test
according to the European Guideline EN 14476 with
poliovirus (PV), adenovirus (AdV) and murine norovirus
(MNV) as surrogate of human norovirus was used [
Subsequently, a quantitative carrier assay using frosted glass
based on prEN 17111:2017 [
] was run with identical
conditions regarding exposure time and test temperature with
AdV and MNV and PV [
]. For all tests, clean conditions
(0.3 g/L bovine serum albumin) and a fixed exposure time
of five minutes were used.
PAA was supplied by AppliChem GmbH (order
number 143495, 15% solution) (Ottoweg 4, DE-64291
Darmstadt). Dilutions of PAA were prepared with hard
water according to the European norms immediately
before the inactivation tests started.
PV type 1 strain LSc-2ab (Chiron-Behring) was
obtained from PD Dr. O. Thraenhart, Eurovir, DE-14943
Luckenwalde. AdV type 5 strain Adenoid 75 (ATCC
VR5) from PD Dr. A. Heim, Institute of Medical Virology,
Hannover Medical School, DE-30625 Hannover. MNV
S99 was obtained from PD Dr. E. Schreier at the Robert
Koch-Institute (RKI) in DE-13302 Berlin (now available
at the Friedrich-Loeffler-Institute
Bundesforschungsinstitut für Tiergesundheit, Ile of Riems).
The test virus suspensions were prepared by
infecting monolayers of the respective cell lines. The virus
titers of these suspensions ranged from 108 to 109
TCID50/mL (tissue culture infectious dose 50). PV
type 1 was propagated in BGM cells (buffalo green
monkey kidney cell line; supplied by Prof. Dr. Lindl,
Institute for Applied Cell Culture, DE-81669
München) in Dulbecco’s Modified Eagle’s Medium
(DMEM) with 1 g/L glucose. AdV type 5 replication
was performed in A549 cells (human lung epithelial
carcinoma cells). The A549 cells originated from the
Institute of Medical Virology, Hannover Medical
School, DE-30625 Hannover and were cultivated in
Eagle’s Minimum Essential Medium with Earle’s BSS
(EMEM). MNV strain S99 was propagated in RAW
264.7 cells (a macrophage-like, Abelson leukemia
virus transformed cell line derived from BALB/c mice,
ATCC TIB-71) in DMEM with 1 g/L glucose.
Tests according to EN 14476 were run with PV,
AdV and MNV as test viruses of the EN 14476 in
clean conditions with a fixed exposure time of five
]. 20 °C, 25 °C and 35 °C were used as test
temperatures. Hard water was added as a control
instead of PAA and cytotoxicity was additionally
determined by addition of hard water instead of virus
suspension. Infectivity was stopped by immediate
serial dilution with ice-cold medium according to the
standards of the European Committee for
]. Of each dilution, 100 μL were placed in
eight wells of a sterile polystyrene flat bottomed
96well microtiter plate containing 100 μL cell
suspension. Cultures were observed for cytopathic effects
(CPE) after 4–10 days of inoculation depending on
the cell culture system used.
The virus titers were determined using the
Spearman and Kaerber method [
] and expressed
as log10TCID50/mL with 95% confidence interval (CI).
Titer reduction caused by the biocide is presented as
the difference between the virus titer after defined
contact time with the water control and the
disinfectant and defined as reduction factor (RF). A reduction
of infectivity of ≥4 log10 steps (inactivation ≥99.99%,
RF = 4) is regarded as evidence of virucidal activity.
The quantitative carrier test based on prEN 17111:2017
was performed in clean conditions with AdV and MNV
and additionally with PV [
]. The surface sandblasted
frosted glass carriers (15 mm × 60 mm × 1 mm,
manufacturer: Zell Quarzglas und Technische Keramik
Technologie GmbH, DE-21502 Geesthacht) were prepared as
described in the prEN 17111:2017. One volume of
interfering substance was mixed with nine volumes of test
virus suspension (virus inoculum); 50 μL of this virus
inoculum were pipetted on the inoculation square of the
carrier followed by drying [
Ten mL of the different PAA solutions in a cylindrical
screw tube were placed in a water bath at the chosen test
temperature. After the drying process had been finished,
the inoculated carrier was immersed in the prepared PAA
solution (or hard water as control). Immediately at the
end of the exposure time the carrier was transferred into a
second screw tube with medium and glass beads and
mixed for 60 s. After five minutes a second mixture was
started for 60 s. Virus titer was determined by end point
dilution titration in microtiter plates. Of each dilution
100 μL were placed in eight wells of a sterile polystyrene
flat bottomed 96-well microtiter plate containing 100 μL
cell suspension. Cultures were observed for cytopathic
effects (CPE) after 4–10 days of inoculation depending on
the cell culture system.
As in the suspension assay the method of Spearman and
] was used for calculating virus titers. These
were expressed as log10TCID50/mL with 95% CI. Titer
reduction caused by the biocide is also presented as the
difference between the virus titer after defined contact time
with the water control and the disinfectant. As in the
suspension test a reduction of infectivity of ≥4 log10 steps
(inactivation ≥99.99%, RF = 4) is regarded as virucidal
Linear regression analyses and statistical testing of
differences between slopes and Y-intercepts were performed
using GraphPad Prism v7.03. For individual linear
regression per temperature, infectivity values between 400 and
1500 ppm PAA were taken into account (****P < 0.0001,
**P < 0.01). Slopes and Y-intercepts are depicted in
separate plots with 95% CI indicated by vertical lines.
The PAA solutions between 400 ppm to 1500 ppm were
examined in the suspension test according to the
European Standard EN 14476 [
]. A four log10 reduction of
the titer of PV was only achieved with 1500 ppm PAA at
35 °C when the initial titer of 8.38 log10TCID50/mL
dropped to ≤3.63 log10TCID50/mL (RF = ≥ 4.75 ± 0.64)
as depicted in Fig. 1. Lower concentrations (between
400 ppm and 1200 ppm) and lower temperatures (20 °C
and 25 °C) were not successful in inactivating this
respective test virus. These results were also visualized by a linear
regression analysis and statistical testing of differences
between slopes and Y-intercepts (Fig. 1). For inactivation
of AdV, in nearly all cases no residual virus could be
detected. The initial virus titre of 7.63 log10TCID50/mL at all
temperatures tested decreased to ≤2.50 log10TCID50/mL
(lower detection limit) resulting in a maximum RF of
≥5.13 ± 0.25. Likewise for MNV, 400 ppm PAA was able
to inactivate the test virus at 20 °C. After five minutes
exposure the titer was ≤3.50 log10TCID50/ml (initial virus
titre 8.00 log10TCID50/mL, RF = ≥ 4.50 ± 0.52). In contrast
to AdV, for all concentrations tested residual MNV could
be detected except with 1500 ppm (Fig. 1).
In the test simulating practical conditions based on
the prEN 17111:2017, the initial titer of PV dropped
from 7.63 to 3.63 log10TCID50/mL at 20 °C, to 3.88
log10TCID50/mL at 25 °C and to 3.19 log10TCID50/mL
at 35 °C in the virus controls, respectively, during the
drying process and the additional incubation of the
carrier in hard water (Fig. 2). Therefore, it was impossible
with such a virus inoculum to demonstrate a four log10
reduction due to the virus loss. Nevertheless, no residual
PV could be detected with 1000 ppm PAA at 35 °C. The
initial virus titer of 3.19 ± 0.17 log10TCID50/mL dropped
to ≤0.50 log10TCID50/mL (max. RF = ≥ 2.69 ± 0.17). For
AdV 400 ppm PAA at 20 °C was sufficient for a four log
reduction (Fig. 2). MNV was more stable than AdV in
the carrier test requiring 1000 ppm PAA at 20 °C (RF
= ≥ 4.19 ± 0.52) and 600 ppm PAA at 25 °C (RF = 4.13 ±
0.35) and 37 °C (RF = ≥ 4.87 ± 0.50) (Fig. 2).
There are some peracetic-acid-based products on the
European market as recently listed by Kampf et al. [
with different PAA concentrations and different
exposure temperatures. They are used for instrument
disinfection with a virucidal action presumably based on
quantitative suspension tests. Currently, a European
standardized test simulating practical conditions for
reaching a virus inactivation is only being drafted with
AdV and MNV as presumed test viruses [
]. PV is not
included in this European normalisation assay due to
problems of virus loss during drying.
In Europe, an instrument disinfectant has to pass
first the quantitative suspension test followed by the
carrier test. Therefore we used both test methods for
evaluating the virus-inactivating properties of PAA.
Kline and Hull already demonstrated in 1960 for the first
time, the strong virus-inactivating properties of PAA [
They showed that a 400 ppm PAA solution was able to
produce a 7.5 log10 step reduction of PV after five minutes
exposure time without any soil loading in a suspension test.
Interestingly, they pointed out that formaldehyde showed
an identical activity as a 5% solution after 20 min [
Additional data were published on solutions of PAA often
in alcohol by Sprößig and Mücke [
]. Furthermore, they
introduced PAA as a disinfectant in human medicine .
The mechanism of PAA on viruses is characterised by
disruption of the capsid and a RNA fragmentation as shown
with PV type 1 [
Our data show that among the test viruses of the
European norm EN 14476, the PV was much more
stable than adenovirus and MNV that miss the strong
hydrophilic character of PV. In contrast to the older data
of Kline and Hull [
], higher concentrations were
required but the experimental design between their study
and our experiments is difficult to compare mainly
related to the ratio of biocide to virus suspension and the
Sauerbrei et al. found in comparative studies with
PV type 1 and echovirus type 1 that 0.05% PAA was
not active in clean and dirty conditions against PV,
whereas 0.5% was virucidal within 10–30 min
exposure time, thus showing similar data in comparison to
our study [
]. For AdV type 5, Sauerbrei et al. found
that 0.1% and 0.2% PAA were necessary to inactivate
the AdV after 15 and 5 min, respectively [
these tests were run with a higher soil load (10%
foetal calf serum) according to the DVV Guideline in
contrast to clean conditions.
In our suspension tests, AdV and MNV showed a
similar behaviour. There might be a difference in
stability between both viruses at concentrations lower that
400 ppm, However, we did not use lower concentrations
because the concentration of PAA in the instrument
disinfectant on the market in general is higher.
The greater stability of PV in contrast to AdV and MNV
was also found performing test simulated practical
conditions. Due to a high loss of virus titer for PV during drying
and immerging, a four log10 reduction could not be
observed with this virus. It can only be mentioned that
35 °C and at least 1000 ppm PAA were necessary to detect
no residual PV. Here, in the test simulating practical
conditions MNV was more stable than AdV. At 20 °C the
required concentration of PAA for MNV was 1000 ppm
in comparison to 400 ppm for AdV.
An identical procedure as shown here with frosted
glass carriers was performed with PAA testing vaccinia
virus strain Elstree and polyomavirus SV40 strain 777 by
Strohhäcker und Eggers [
]. They found that even
0.05% PAA was sufficient for virus inactivation of both
viruses within five minutes in clean and dirty conditions.
Following the procedure of virucidal testing in Europe,
first the requirements of the suspension test have to be
fulfilled. Then the phase 2/step 2 test must follow. According
to our data with PAA and PV, it is much more difficult to
reach an inactivation with PV, AdV and MNV in the
quantitative suspension test due to the great stability of PV than
to be successful with AdV and MNV in the carrier test
based on prEN 17111:2017 simulating practical conditions.
Therefore, it should be put into consideration in the future
in Europe to include the stable murine parvovirus as a test
virus in the phase 2/step 2 procedure even when using
temperature < 40 °C. At the moment murine parvovirus is
only used as sole test virus for instrument disinfectants at
temperature ≥ 40 °C.
In summary, 1500 ppm peracetic acid at 35 °C was
necessary for a virucidal action in the quantitative
suspension test. After passing the requirements of the
suspension test, additional examinations with adeno- and
murine norovirus on glass carriers based on prEN 17111:2017
will not additionally contribute to the final claim of an
instrument disinfectant to have sufficient virucidal efficacy.
This is due to the great stability of poliovirus in the
preceded quantitative suspension test and the fact that
poliovirus could not serve as test virus in the carrier assay.
AdV: Adenovirus; CI: Confidence interval; CPE: Cytopathic effect;
DMEM: Dulbecco’s modified Eagle medium; EN: European Norm;
KRINKO: Kommission für Krankenhaushygiene und Infektionsprävention;
MNV: Murine norovirus; n.d.: Not determined; PAA: Peracetic acid; ppm: Part
per million; prEN: European pre norm; PV: Poliovirus; RF: Reduction factor;
RKI: Robert Koch-Institute; TCID50: Tissue culture infectious dose 50
This study was supported by Chemische Fabrik Dr. Weigert GmbH & Co.KG,
Germany. E. S. was supported by the Helmholtz Centre for Infection
Research, Hannover Germany.
Availability of data and materials
All data generated during this study are included in the published article.
JS and JL together with FB and ES formulated the study questions and
designed the study. BB, DP and BB were responsible performing all
experimental data. DT was responsible for data evaluation. All authors read
and approved the final manuscript.
Ethics approval and consent to participate
Consent for publication
JL is employee of Chemische Fabrik Dr. Weigert GmbH & Co.KG. The other
authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in
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1Dr. Brill + Partner GmbH Institute for Hygiene and Microbiology, Norderoog
2, DE-28259 Bremen, Germany. 2Institute for Experimental Virology,
TWINCORE Centre for Experimental and Clinical Infection Research; a joint
venture between the Medical School Hannover (MHH) and the Helmholtz
Centre for Infection Research (HZI), Hannover, Germany. 3Chemische Fabrik
Dr. Weigert GmbH & Co.KG, Hamburg, Germany.
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