Validation and results: VPHP bio-decontamination of FFP masks

DeconBox™ is a Vapour-Phase-Hydrogen-Peroxide bio-decontamination system that allows FFP2 and FFP3 masks to be reused. Our R&D dept. explains the process of validating the mobile, purpose-built system for use in controlled settings.

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This article presents the test methodology and the results behind our guaranteed mask decontamination system.


As the Covid-19 virus spread around the world causing critical mask shortages, Comecer decided to study a solution which would put to good use its vast experience decontaminating the isolators it manufactures for pharmaceutical companies.

The result is DeconBox™, a mobile, purpose-built, VPHP bio-decontamination system that allows FFP2 and FFP3 masks to be reused. The system is suited for hospitals and other organizations that must provide masks to their employees.
Using Deconbox does not just eliminate supply issues, it also reduces the environmental impact of discarding a large number of masks daily.
DeconBox is also cost-effective as you start saving when an organization uses more than a total of 200 masks per day (try the DeconBox Convenience Calculator).

Methods and Results

As a starting point for our internal test, we considered a generic operative process flow for Filtering FacePiece  (FFP) mask regeneration (Figure 1).

Process flow for FFP mask regeneration

Figure 1

In that context, DeconBox takes a role in the step dedicated to the microbial decontaminating process.
However, this treatment could lead to several consequences impacting the success of the quality controls.

For this reason, our aim was to develop a Vapor-Phase-Hydrogen-Peroxide (VPHP) cycle associated to the DeconBox, compliant with the following three conditions:

Mask eligibility to the decontaminating procedure

Previous studies showed how blood and other organic soils could impair the efficacy of VPHP-driven bioburden reduction in tissues and surfaces1.
We collected used FFP masks and only those without any trace of organic soils were selected for our internal test.
Two different types of FFP mask were analyzed: 3M™ Aura™ 9332+ and Honeywell 2211 as models for FFP3 and FFP2, respectively (Figure 2.)

2 FFP mask types analyzed

Figure 2

Construction of customized biological indicator

The Tier classification system was introduced by the FDA to define the level of bioburden reduction required during the microbial decontamination process of FFP mask and respirators (
Tier 1 level claims the verified ≥ 6-log spore reduction of the most resistant spore for the proposed process.
Due to the great Comecer experience in VPHP bio-decontamination gained over the years, our ultimate goal was to develop a VPHP-based decontaminating process for FFP masks able to assure a Tier 1 level.

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Spores of the strain ATCC® 7953™ of Geobacillus stearothermophilus are widely exploited in validation processes of pharmaceutical and industrial VPHP bio-decontamination cycles due to their proven resistance to hydrogen peroxide.
We selected this specific strain of spores as a biological indicator (BI) for the bioburden reduction level required for Tier 1 class.
Commercially available BIs take advantage of a monolayer of a certified amount of spores spotted on disks of common industrial materials, such as 316 stainless steel and polyethylene.
However, due to the 3D structural complexity of FFP masks given by the multi-layer tissue, BIs able to simulate its interactions with microbial agents and sterilant are not currently available on the market.
Thus, we designed a customized BI for both the FFP masks under analysis with the purpose to address the achievement of the log-6 decontamination of the previously mentioned ATCC® 7953™ spores through VPHP.

Scheme 1

Mask pieces were cut, sterilized and then an average of 1.5*10^6 spores (target loading of 1.5×106 CFU per 1 cm2) (Mesalab) was spotted on each piece (Scheme 1).

This system avoids potential cross-contamination during the handling of BIs at the end of the decontamination cycle.
The treated BIs were subsequently incubated in Tryptic Soy Broth (TSB) modified with pH indicator at 57.5 °C, allowing the rapid discrimination between active spores and inactivated spores directly from the colour variation of the broth (Figure 3).


Figure 3

VPHP bio-decontamination cycle

Based on tests performed on the DeconBox (Figure 4) and results obtained with the previously described BI, we selected a unique VPHP cycle appropriate for most of the FFP masks present on the market.

DeconBox™ Fast Decontamination of Respirator Masks and Other Devices

Figure 4

The VPHP bio-decontamination cycle is characterized by the following parameters (Table 1):

Phase Duration (min) Injection rate (g/min)
Conditioning 5 /
Injection 25 3.0
Dwell 60 1.3
Aeration 1 120 /
Aeration 2 240 /

The overall cycle lasts 7.5 hours, with a total INTEROX® H2O2 35% consumption of 150 g.

Moreover, the decontaminating efficacy of the cycle was proven at different external temperatures, ranging from 23°C to 32°C.

Assessment of the bioburden reduction

To officially validate the VPHP cycle from a microbiological point of view, we established a collaboration with the microbiology department at the Università di Modena.
Besides the preparation of the same BI containing the ATCC® 7953™ of Geobacillus stearothermophilus spores, the microbiological laboratory team also prepared BIs containing spotted titres of Cox B5  and HumanCoronaVirus-OC43 (HCoV – OC43) viruses, respectively selected for the high resistance to VPHP and the phylogenetic proximity to the well-known SARS-CoV-2 responsible for the Covid-19 pandemic.             
All the prepared BIs were subjected to the VPHP cycle reported in Table 1 and their viability has been analysed.
Once BIs have been collected at the end of the VPHP cycle, they were incubated in Tryptic Soy Broth and their growth analysed by the OD600 spectrometric technique.
The impact of the VPHP on the Geobacillus stearothermophilus spores was further addressed by comparing the bacterial viability after the treatment (referred to as # BI) with respect to an untreated sample (ctrl +). Colony Forming Unit (CFU) is a unit commonly used in the field of microbiology to estimate the number of viable bacteria in a sample; in this case, CFU is relative to the non-inactivated spores able to give rise viable colonies. Thus, we used the concentration of CFU within the culture broth to directly quantify the effect of the VPHP bio-decontamination cycle. Since the starting population of spores was 1.5*10^5, Figure 5 shows how a log-6 reduction was obtained through the decontaminating cycle performed with the DeconBox.

Chart of Log-6 spores decontamination

Figure 5

Similar results were achieved with the BIs containing the viruses, as shown in Figure 6. The plaque assay was set up to experimentally calculate the magnitude of decontamination expressed in log. In both viruses, we observed a 100% reduction of the initial viral titre.

Log 6.5 Cox B5 decontamination

Figure 6

The results derived from the experimental test highlight how the DeconBox VPHP bio-decontamination cycle is suitable for obtaining a log 6 reduction in Geobacillus stearothermophilus spores, compliant with the FDA’s Tier 1 level and for a significant log reduction also in two viruses used as models for their biological properties.

Measurement of VPHP residues in regenerated masks

A crucial point in the overall process of FFP mask regeneration is represented by the removal of potential hydrogen peroxide residual molecules stuck in the mask matrix.
In fact, according to what is defined by the Occupational Safety and Health Administration (OSHA), the threshold limit value (TLV) for an operator exposed for 8 hours to VPHP is represented by 1 part-per-million (ppm), or the equivalent 1.4 mg/m3.
Thus, we designed an experimental apparatus able to generate a modulable air flux and, in parallel, measure the VPHP residues in ppm released by masks (Figure 7).

experimental apparatus for testing mask decontamination

Figure 7

The resulting air flux was directed from the outer to the inner side of the mask to simulate the most realistic condition of H2O2 inhalation by operators and it was set at 10 l/min; this value aims to simulate the mean air flux of human breaths at rest and represents the most stressful experimental condition.

Taking advantage of the experimental apparatus, we measured the VPHP residues released by the mask matrix under the 10 l/min air flux after 2, 3 and 4 hours of aeration 2 (see Table 1), respectively.
We found that 4 hours represent the safety duration of the aeration 2 required for decreasing the H2O2 residues far below the 1 ppm TLV for both FFP3 (Figure 8) and FFP2 (Figure 9) mask models.

VPHP residues over aeration time for FFP3 mask

Figure 8

VPHP residues over aeration time for FFP2 mask

Figure 9

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Conservation of the filtering performance

The third and last point essential for successfully concluding the overall process of FFP mask regeneration is assuring that the native filtering performances of the mask are not impaired at all by the decontamination treatment.
To demonstrate the non-invasiveness of our process, we commissioned two different tests on the tested FFP3 mask model, as governed by the EN 149:2001 + A1:2009 legislation.
The first test is based on the measurement of the penetration yield of an emulsion of paraffin oil fluxed through the mask. Since the testing oil emulsion is characterized by droplets with an average of 0,3 µm in diameter and the filtration performances of the mask are claimed on that specific particle size, the percentage of penetration of the oil across the mask tissue represents an indirect valuable measurement of the filtration efficiency. In Figure 11, the table shows the experimental parameters, in accordance with the EN 149:2001 legislation.
Moreover, the VPHP-regeneration treatment does not significantly impair the filtration performance of the analyzed FFP3 masks; in fact, the mean penetration percentage of the treated mask is statistically comparable with the untreated control and far below the 1% threshold of paraffin oil penetration (Figure 10).

Test results

Figure 10

The second test aims to analyse the resistance that the FFP mask physically opposes to the air fluxes related to breath.
Concerning the analysis of the exhalation resistance, as shown in Figure 11, the VPHP-treated masks are characterized by comparable air pressure drops with respect to the untreated controls.

Test results - exhalation resistance

Figure 11

The same result has been obtained also for the test of inhalation resistance, performed at both 30 l/min and 95 l/min air fluxes (Figure 12).

Inhalation resistance

Figure 12


DeconBox is born as a mobile bio-decontamination system that allows FFP2 and FFP3 masks regeneration.
We demonstrated how DeconBox is able to regenerate FFP mask exploiting the VPHP-decontaminating technology by log-6-reducing the bio-burden nested in the FFP matrix. The bio-burden reduction has been experimentally demonstrated on VPHP resistant spores and viruses.
Moreover, The VPHP cycle associated to the DeconBox assures that at the end of the decontaminating cycle, the potential VPHP residues stuck within the FFP tissue are much lower with the official safety threshold of 1 ppm.
Finally, the regenerating process does not affect the filter and respiratory performance of the mask, allowing FFP masks to be reused in completely safe conditions.

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  1. Wood, J. P. et al. Evaluating the Environmental Persistence and Inactivation of MS2 Bacteriophage and the Presumed Ebola Virus Surrogate Phi6 Using Low Concentration Hydrogen Peroxide Vapor. (2020) doi:10.1021/acs.est.9b06034.


Riccardo Volta, R&D Comecer Specialist

Giacomo Nicolini, Comecer Product Specialist

In addition

Watch the on-demand webinar on the results of our test campaign on DeconBox™.

In the webinar, our R&D dept. presents the results, demonstrating how the VPHP decontamination process associated with our product is able to bio-decontaminate FFP mask by a log-6 factor and at the same time release a safe product.

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