Subjects: The study included 14 nonsmoking subjects (6 women and 8 men with a median age of 37 years, range 24–63 years) who had been suffering from grass pollen-induced allergic rhinoconjunctivitis for at least two years. In a prick test, the subjects developed a wheal measuring ≥ 3 mm in diameter (grass mix, 30 HEP/ml; LETI Pharma GmbH, 85737 Ismaning, Germany), and they achieved a Total Symptom Score of at least 6 points in previous provocation tests with grass pollen in the allergen exposure chamber (AEC). None of the subjects was undergoing medical treatment or using inhaled medication for bronchial asthma.
Furthermore, none of the subjects had received allergen-specific immunotherapy in the last 5 years; none of them used systemic or local antiallergic drugs for at least 7 days before the first (V1) and until the last exposure (V5) in the chamber.
The subjects were informed about the study verbally and in writing. Approval for this study was based on a positive vote obtained from the ethics commission at Charité (Ethics Commission, Charité Campus Mitte, no. EA1/406/20).
Exposure in the allergen exposure chamber (AEC): The study was carried out in the exposure chamber of ECARF from 16 March 2021 to 15 April 2021. The methodology  and evaluation  of the study are described.
Exposure procedure: The exposure tests were conducted with grass pollen (grass pollen raw material, 38 ± 2 µm, Phleum pratense; Allergon HB, Angelhom, Sweden) with 4000 pollen/m3 according to the preliminary tests for the validation of the AEC. All tests are performed at 21 °C and 55% relative humidity. Before starting the test, the subjects are acclimatised for 20 min without any exposure. The subsequent exposure period for the subjects was 120 min.
The subjects were seated in the AEC according to a random number and retained the same seat for all exposure phases.
On V0, the subjects wore no face masks; on V3, they wore a FFP2 mask (particle filtering half mask, manufacturer: Shengquan, CE2834, EN: EN149:2001 + A1:2009) and on V5 a medical (surgical) mask (manufacturer: MERSUII GB: GB/T 32610-2016).
The intervals between the three exposure phases were seven days for all of the subjects.
Recording of symptoms: The subjects used tablets to document 13 symptoms every 10 min: four eye symptoms (itching, foreign body sensation, lacrimation, redness), four nasal symptoms (itching, sneezing, runny nose, stuffy nose), three bronchial symptoms (wheezing, coughing, shortness of breath) and other symptoms (itchy skin and itchy palate).
The severity of each symptom is rated on a scale of 0 to 3 as follows: 0 = no symptoms, 1 = mild symptoms (clearly present but only very mild, causing a minimal degree of discomfort or none at all), 2 = moderate symptoms (definite presence of symptoms that cause discomfort but are still tolerable) and 3 = severe symptoms (symptom is difficult to tolerate, interferes with the subject’s daily life).
The symptom severity scores are added together to obtain the Total Eye Symptom Score (TESS), Total Nasal Symptom Score (TNSS), Total Bronchial Symptom Score (TBSS), and Total Other Symptom Score (TOSS). The Total Symptom Score (TSS) was calculated as the sum of TESS, TNSS, TBSS and TOSS with a maximum of 39 points.
Measurements (according to SOPs in the AEC)
Spirometry using EasyOne™ Spirometer (ndd, Medizintechnik AG, Zurich, Switzerland) before V1 and after V5.
Peak flow values using peak flow meters (PersonalBest, Philips GmbH, Herrsching, Germany) before exposure on V1, V3 and V5 and after 30, 60, 90 and 120 min inside the AEC. For all measurements, the highest value of two measurements each was documented.
Peak nasal inspiratory flow (PNIF, Clement Clarke International Ltd., Harlow, Essex, United Kingdom): The PNIF was determined prior to exposure on V1, V3 and V5 and after 30, 60, 90 and 120 min inside the AEC. For all measurements, the highest value of two measurements each was documented.
Nasal secretion: Each subject was given a resealable bag with a packet of tissues for the duration of the exposure. To determine the amount of nasal discharge, the bags are weighed before and after exposure and the weight is documented.
Overall well-being (VAS): The subjects rated their general well-being before, every 30 min during and after allergen exposure in the AEC on a visual analogue scale of 10 cm, which represents the severity level from 0 cm ‘very good’ to 10 cm ‘very poor.’
Follow-up calls (safety calls): Around 24 h after each exposure, the subjects were called as a safety measure; they were asked whether they had any symptoms of a late reaction to the allergen exposure, and any findings were documented. The subjects were asked: “From the moment you left the exposure chamber yesterday until now, have you experienced any allergy or other symptoms?”
Data entry and data protection: The participants’ data were anonymised and entered into a database. All data are managed in accordance with the Berlin Data Protection Act (BlnDSG) and the EU General Data Protection Regulation (GDPR).
Statistical evaluation: The data are evaluated according to protocol including all subjects who started the first exposure and were shown to react to exposure to grass pollen (TSS of ≥ 6). The primary endpoint was the change in TSS, TNSS, TESS and TBSS values over 120 min during exposure. These values were then subjected to a two-sample t‑test on dependent samples (V1/V3, V1/V5 and V3/V5). In addition, all parameters were also evaluated in a descriptive analysis.
COVID-19 hygiene measures: Compliance with a comprehensive set of hygiene and safety measures was ensured throughout the study, from the start of the appointment scheduling until the end of the study. These included patient information, disinfection of all surfaces, writing instruments and medical devices, compulsory mask wearing, gloves and physical distancing in the AEC.
Before beginning the study, the subjects and study staff (study nurse/study doctor) were required to complete a questionnaire about COVID-19 and indicate any symptoms they may have had.