This study proposes a randomized, double-blind, placebo-controlled crossover trial. Participants need to spend at least 3 consecutive hours in an environment where loud music is played (>95 dB(A)) after taking either a combination of antioxidants (NAC and Mg2+) or a placebo, 1 hour prior to noise exposure. Each participant needs to repeat this procedure four times (interval between trials at least 4 days) and each participant will receive twice the antioxidants as well as the placebo. This study aims to investigate the combined effects of NAC and Mg2+ by use of four repeated treatments on the hearing of noise-exposed young adults.
We are recruiting students by advertisements sent by email and distributing information sheets around the University of Antwerp (Antwerp, Belgium). The aim is to always investigate groups (at least two individuals) of peers going out together and therefore attending the clinic within the time range of 7 hours after noise exposure. It is assumed that the pressure of peers also participating in the study will reduce the risk of drop-out. In addition, at the stage of enrolment it will be determined which occasions the subjects will attend and test moments will be scheduled. The aim is to plan all treatments within the range of 3 months in order to reduce the risk of drop-out.
Participants will receive an antioxidant combination of NAC and Mg2+, or a placebo drug (sugar pill). The antioxidant packets contain two pills of each 300 mg NAC and 100 mg Mg2+. Both pills, providing a total dose of 600 mg NAC and 200 mg Mg2+, should be taken orally 1 hour before noise exposure with a large glass of water. In 50%, the packets contain a placebo with an identical appearance and weight as the antioxidants. All participants (as well as the researchers) will be blinded to the order of antioxidants and placebo. The effects of NAC and Mg2+ at the respective doses of 600 mg and 200 mg will be analyzed as well as the effects of the combined product, for which no significant adverse effects have been reported in the past. Adverse events are defined as symptoms occurring after the administration of antioxidants or placebo that were not necessarily related to the intervention. Participants will receive a telephone number to report adverse events during the study and will be urged to come to the clinic in case of serious adverse events.
Permitted and prohibited concomitant treatments
The use of alcohol and tobacco is limited to a maximum of two alcohol consumptions and no to two cigarettes. Other psychoactive drugs are strictly prohibited.
The inclusion criteria are as follows:
Age between 18 and 28 years old (males as well as females).
Experience of temporary tinnitus after leisure noise exposure in the past scoring at least 5 or more on a visual analogue scale (VAS) for loudness.
Willing to attend an indoor/outdoor music event with mean noise levels ≥95 dB(A) LAeq, 60 min for at least 3 consecutive hours.
The exclusion criteria are as follows:
Middle ear pathologies such as otitis media and the perforation of the tympanic membrane and history of such pathologies.
Known allergies for NAC or Mg2+.
Adolescents using hearing protection.
Recreational noise exposure within 4 days prior to a study trial.
The production of the antioxidant and placebo packets as well as the randomization of the ABAB protocol will be performed by an independent pharmacist. The packets of antioxidants and placebos will be labeled with a number corresponding with the numbers on the forms. The randomization file will be put into a sealed envelope and retained in a safe at the pharmacy. The randomization table will not be available for assessment by anyone else involved in the study. At the end of the study the envelope will be requested.
Sample size calculation and statistical analyses
A power calculation was performed in order to make an estimation of the sample size needed to detect significant differences in the VAS between the experimental and control group by use of the software G-Power. A two-sided paired t-test for mean differences was performed in which an α-level of 0.05 and a nominal power of 80% were used. Assuming a minimal difference of clinical effect of 0.5 between the pre- and post-noise measurements on the VAS, a non-parametric calculation showed a requirement of 21 participants.
All data in this trial will be assessed with SPSS Statistics version 20.0 (SPSS, Chicago, IL, USA) and SAS version 9.1.3 (SAS, Cary, NC, USA). Because multiple correlated measurements will be performed in the same participants, a mixed effects model will be applied. This analysis is preferable over more traditional approaches such as repeated measures analysis of variance (ANOVA) because of the advantages to deal with missing values. The main analysis will be fully specified before unmasking.
The primary outcome measure will be the score on the VAS for tinnitus loudness, which will be measured pre-noise exposure (at the time of antioxidant intake) and 1 hour after noise exposure. The assumed expectation is a drop of 50% of the tinnitus loudness in the antioxidant group compared to the placebo group.
The secondary outcome measure is the evaluation of a significant difference in test results of the audiological testing (audiometry, otoacoustic emissions and speech-in-noise tests) when comparing the placebo results to the antioxidant trials.
A series of audiological tests (described in the following paragraphs) will be performed prior to and within 7 hours after each noise exposure.
Main outcome measure
Visual analogue scale (VAS)
Tinnitus loudness will be scored on a VAS from 0 to 10 (0, no tinnitus at all; 10, extremely loud tinnitus, could not possibly be louder), once at the time of antioxidant (or placebo) intake (1 hour prior to exposure) and once 1 hour after noise exposure. At both occasions the VAS will be undertaken in a quiet room.
Secondary outcome measures
Pure tone audiometry
Pure tone liminar audiometry will be performed according to the current clinical standards (International Organization for Standardization (ISO) 8253-1:2010) using a two-channel AC40 audiometer (Interacoustics, Assens, Denmark) in a silent room. Air conduction thresholds will be measured under headphones at 125 Hz, 250 Hz, 500 Hz, 1 kHz, 2 kHz, 3 kHz, 4 kHz, 6 kHz and 8 kHz. In addition, high frequency air conduction thresholds will be performed including 9 kHz, 10 kHz, 11,200 Hz, 12,500 Hz, 14 kHz and 16 kHz. When air conduction thresholds between 250 Hz and 4 kHz exceed normality levels of 20 dB HL, the bone conduction threshold will be measured on 250 Hz, 500 Hz, 1 kHz, 2 kHz, 3 kHz and 4 kHz in order to make a distinction between conductive and sensorineural hearing loss.
Transient-evoked otoacoustic emissions (TEOAEs) as well as distortion-product otoacoustic emissions (DPOAEs) will be performed. TEOAEs are elicited with click sounds presented at an intensity level of 80 dB SPL and recorded over a frequency range of 500 to 4,000 Hz. DPOAEs are elicited by use of a set of two pure tone frequencies (f1 and f2) closely spaced and presented simultaneously at a level of 55 dB SPL for f1 and 65 dB SPL for f2. The largest and most robust distortion product is 2f1-f2 and can be detected in almost all normal ears. The amplitude of 2f1-f2 is optimized by use of the frequency ratio f2/f1 = 1.22. DPOAEs will be recorded in the frequency region from 1 kHz to 6 kHz.
Speech-in-noise testing will be performed using the Leuven intelligibility sentence test (LIST)  in an adaptive procedure with noise at a fixed level of 65 dB SPL. Each list consists of ten sentences. Both ears will be tested separately by use of headphones. The procedure starts at a signal-to-noise ratio (SNR) of 0 dB meaning that speech and noise are presented with an equal loudness (65 dB SPL). Subsequently, the intensity level within a list of sentences varies in steps of 2 dB adaptively in a 1-down (when the keywords in the sentence are correctly repeated), 1-up procedure to determine the 50% correct identification point, which is called the speech reception threshold (SRT), expressed in dB SNR. Three conditions will be tested for both ears: sentences in steady-state noise, sentences in amplitude-modulated noise by a 10 Hz modulation and sentences in amplitude-modulated noise by a 15 Hz modulation.
Controlling the noise levels
In order to be able to respect the inclusion criterion of the minimal noise levels of 95 dB(A) LAeq, 60 min for 3 consecutive hours, all participants will be equipped with portable decibel meters (CEL350, Casella, Bedford, UK). The decibel meter can be fixed onto a shirt or trousers but participants must be aware that the microphone entrance should remain free from overhanging clothing.
Written consent will be obtained from each participant. This study protocol was approved by the ethical committee at Antwerp University Hospital in June 2012 with protocol number 12/18/172.
According to the Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT) guidelines, the authors declare that data that break the blind will not be presented prior to the release of mainline results. The breaking of the blind will occur at the end of the study. A clinical article will be written on the primary (including also secondary) outcomes of the study and results will be disseminated regardless of the magnitude or direction of effect. The present trial is not industry-initiated. As such, there are no publication restrictions imposed by sponsors.
In addition, a full study report, anonymized participant-level dataset and statistical code for generating the results will be made publicly available no later than 3 years after the termination of the study for sharing purposes.