Use of Supramolecular Solvents in the Identification and Quantification of Substances of Very High Concern and Endocrine Disruptors Released From Michigan-type Occlusal Splints in Saliva: in Vitro and in Vivo Pilot Study.
Conditions: Bruxism
Sponsor: Universidad Europea de Madrid

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RESEARCH PROJECT 
 
 
TITLE: Use of supramolecular solvents in the identification, detection and 
quantification of substances of particular concern or endocrine disruptors released from 
Michigan-type occlusal splints in saliva: an in vitro and in vivo pilot study. 
 
PROMOTOR: Universidad Europea de Madrid PRINCIPAL 
INVESTIGATOR: Susana David Fernández CENTRE: 
Policlínica Universidad Europea de Madrid 
1 INTRODUCTION 
1.1 PROBLEM DEFINITION 
The use of plastics in medical specialties, dentistry being no exception, has been a 
singular advance in the prevention of the spread of diseases. However, some of them 
contain substances considered endocrine disruptors that mimic or antagonise the action 
of endogenous hormones, alter their metabolism and synthesis, and modulate the levels 
of their receptors. Given their impact on health and well-being, the evolution in this 
respect has been remarkable: in 1993 Colborn et al. coined the concept "endocrine 
disruption"; Weybridge in 1996 defined endocrine disruptor (ED) as: "an exogenous 
substance or mixture that alters the functions of the endocrine system, causing adverse 
health effects in an intact organism, its progeny or (sub)populations". In 2002, the 
World Health Organisation (WHO) and the International Programme on Chemical 
Safety (IPCS) clarified what can cause such alterations (endocrine disrupting potential). 
In 1999, the European Commission published the communiqué: Community strategy on 
endocrine disrupters, a series of substances suspected of interfering with the hormone 
systems of humans and wildlife. On 1 June 2007, the REACH (Registration, Evaluation, 
Authorisation and Restriction of Chemicals) regulation comes into force to collect and 
evaluate information on the properties and hazards of substances. The European 
Chemicals Agency (ECHA) also considers substances of very high concern (SVHC). 
These are substances that may have serious and often irreversible effects on human 
health and the environment, and are listed in Annex XV of the REACH regulation. It is 
established at the level of 

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European a guideline to follow: Identify to assess and monitor. In 2019, the European 
Commission created a "roadmap" for the review of compliance with the "Fitness Check" 
for endocrine disrupters, which aims to protect health and the environment. 
 
Numerous organisations, objectives, and laws are shaped. Among these provisions, 
those concerning dental devices or restorations are shown in section 10.4 of Annex 1 of 
the European Medical Devices Regulation (MDR) Regulation (EU) 2017/745, focusing 
on action on the identification of substances considered carcinogenic, mutagenic or 
toxic for reproduction (CMR) and/or endocrine disruptors. 
 
In dentistry, the use of polymers, acrylics or resin-based composites (RBC) ranges from 
dental cements, fissure sealants, temporary acrylic materials, definitive and aesthetic 
restorations, partial dentures, to splints for the treatment of bruxism. Efforts are being 
made to develop materials that not only aesthetically but also biomechanically mimic 
the teeth and are easily repairable. This is why plastic materials are becoming 
increasingly popular in all dental specialties, from paediatric dentistry to gerontology. 
However, it is striking to find in their composition monomers such as bisphenol A 
(BPA), triethylene glycol dimethacrylate (TEGMA), methacrylate urethane, BisGMA 
copolymer (UDMA), or hydroxyethyl methacrylate (HEMA), substitutes for bisphenols: 
Bisphenol S (BPS), Bisphenol F (BPF), among others; whose purpose is to provide 
malleability, adhesion and strength, but which are not inert, by their nature. The possible 
hormonal, endocrine, neuronal, carcinogenic, mutagenic, etc. effects of the most 
common endocrine disruptors such as bisphenols and phthalates are well known, but 
even their substitutes have undesirable physiological effects. The effects of endocrine 
disruptors (EDs) and substances of particular concern (SVHCs) also alter dental genesis, 
as well as the microbiome, facilitating the proliferation of series with higher 
parodontogenic or cariogenic potential, favouring the two most common pathologies 
such as caries and periodontitis, and even malignant lesions and alterations in tooth 
formation (Bisphenol A and Incisive Molar Hypomineralisation:: Topic Review - Dialnet, n.d.; 
Chimenos-Küstner et al, 2019; Chimenos-Küstner & EM, n.d.; Da & Tavares, n.d.; Jacomossi, 
2019; Piedrahita Sánchez et al., 2021; Tejada, 2021) . 

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Their dose-response curve is not monotonic, they alter biogenesis, being up to 
childbearing age, the window of greatest risk (Kakonyi et al., 2021). If we take into 
account that our activity reverts at any time in the life cycle of the population, the time 
that the restorations or devices remain in the mouth, the oral conditions (pH, occlusion, 
temperature...) that facilitate the wear and release of particles, and clinical protocols 
with levels of evidence that can be improved, it would be desirable to know the current 
status of dental materials of a plastic nature, in accordance with European regulations on 
identification and prevention (de Angelis et al., 2022; de Nys, D., 2022), 2022; de Nys, 
Duca, et al., 2021; Mulligan et al., 2022; van Landuyt et al., 2011; Vervliet et al., 2022). 
 
 
 
 
1.2 BACKGROUND 
Despite the fact that the dose-effect curve is not monotonic, in 2015 the European Food 
Safety Authority (EFSA) published a new global limit for BPA exposure and toxicity at 
4 µg/kg body weight/day, compared to the 50 µg/kg/day estimated by the US 
Environmental Protection Agency (USEPA) in 1993 ("Hormones and Endocrine- 
Disrupting Chemicals: Low-Dose Effects and Nonmonotonic Dose Responses", 
published in March 2012 in Endocrine Reviews). On 15 December 2021, a draft proposal 
was made to reduce the limit by 0.04ng/kg/day, in view of the evidence shown in the 
literature from 2015-2018 (publication and testing of the risk assessment protocol in 2017 
and EFSA https://www.efsa.europa.eu/en in 2019). With regard to BPA substitutes, it 
is observed that some show even more genotoxicity, such as BPF; alterations in 
testosterone levels such as bisphenol B (BPB); or obesogenic activity such as BPS 
((Alharbi et al., 2022; Chen et al., 2023; Karrer et al., 2018; Lu et al., 2022; Moreno-
Gómez-Toledano et al., 2022, vansecal II, Tolerable Dose Intake). 
 
Despite all this, an alternative to plastic materials is not foreseen in the short to medium 
term, on the contrary: an increase of 6.9 to 13% of aligners is estimated in France, Italy 
and Germany (Koletsi et al., 2022), and the compound annual growth rate (CAGR) is 
estimated to be 13.5% between 2020 and 2025 (Mordor Intelligence, industries reports). 
This same upward trend extends to relaxation splints, made of acrylic materials, which 
remain in the mouth for at least 6h/day for years. These are part of the treatment of 
bruxism. The 'White Paper 2023: population-based survey of oral health in post- 
pandemic Spain 2023', shows an increase in the number of patients with bruxism. 

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increase from 6% in 2019, to 23% at present, in Spain ((Carrillo-Diaz et al., 2022; Dias et 
al., 2022; List of Results: BRUXISM INCREASE AFTER COVID: EBSCOhost, n.d.; Osses-Anguita et 
al., 2023). This is because its aetiology is closely associated with stress. One of the 
consequences of bruxism is the fracture of teeth, whether or not they are devitalised. As 
an alternative, the restoration of lost tooth tissue is indicated with inlays made of 
ceromers (among others). These materials fall into the group of dental composites 
(divided into reinforced, universal, flowable and other composites for everyday clinical 
use), whose global market was valued at US$ 24.7 Mn in 2021. It is estimated to grow 
at a CAGR of 9.0% from 2021 to 2031, and by the end of 2031, the global dental 
composites market is expected to reach a value of US$ 59.3 Mn. This trend extends to 
all fields of dentistry, due to its characteristics, as described above. The dental materials 
industry is also focusing on improvements in the manufacturing process of the devices 
(injection moulded, subtraction or milled, addition or horizontal/vertical impression) 
achieving a sustainable, environmentally friendly, economical practice, and releasing 
fewer additives (Alghazzawi, 2016; Aretxabaleta et al, 2021; Carneiro Pereira et al., 
2021; de Oliveira Limírio et al., 2021; Mourouzis et al., 2020, 2022; Wei et al., 2022). 
In particular, 3D printing is more environmentally friendly than milling because it does 
not use discs, which are more costly and complex to reuse. 
 
The main problem when studying the presence of additives released by dental materials 
in the organism is the extraordinary difficulty in associating them explicitly to this type 
of device, given that they are commonly found in our surroundings. Their multiple 
origins: environmental, nutritional, hygienic sources, etc., make it extremely difficult to 
associate the origin with the substance identified. For this reason, in vitro studies are a 
widely used method. However, the protocols developed to date show a certain lack of 
uniformity: in some, discs are analysed without considering the inhibited layer, in others 
their polishing does not correspond to the clinical protocol, or there is a lack of uniform 
extraction ratio, or the solvents are different, or salivary turnover is not taken into 
account (the possible alterations in its composition, pH, viscosity, or flow that may 
occur in the same subject, and its role in the chemical erosion of the material). On the 
other hand, in some of these tests, occlusal wear is not considered. They provide data on 
quantification and identification of the substances in saliva, mainly bisphenols, 
substitutes for 

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and phthalates, in different materials, depending on their manufacturing technique 
(milled, printed, injected), and their curing (thermo-, self-, photopolymerised). Clinical 
studies focus on paired samples, or on epidemiological, retrospective descriptive, 
comparative, retrospective and prospective studies where saliva, blood and urine 
samples are analysed to determine their assimilation. Among the methodologies used, 
the FDA (Food and Drug Administration) recommends High Performance Liquid 
Chromatography (HPLC) or Gas Chomatography (GC). Authors such as Hampe 
Tristan, advise Mass Spectrometry (MS), or the HPLC/MS tandem, establishing as ideal 
the combination of High Resolution Mass Spectrometry (HRMS) and MS, since it does 
not detect products such as Bis GMA, - EMA and -UDMA (De Angelis et al., 2022; 
Hampe et al., 2022; Kessler et al., 2020; Tichy et al., 2021). (Francisco et al., 2022; Van 
Landuyt et al., 2011a, 2011b; Vervliet et al., 2022; Wedekind et al., 2021). 
 
The literature reports values within the limits (4µg/kg/day); however, some studies 
report higher concentrations in groups with associated pathologies than in those without. 
This is the case of the study carried out with patients with squamous cell carcinoma vs. 
control group, the results showed higher concentrations of BPA and phthalate 
metabolites (Hu et al., 2021; Wei et al., 2022; Zheng et al., 2017). 
 
1.3 JUSTIFICATION 
This study is framed within the European Community framework on substances that can 
have an impact on the health and well-being of the population, focusing on their 
identification, quantification, monitoring and prevention. The population susceptible to 
treatment of this kind is almost universal. Companies are investing in improvements in 
this respect, incorporating substitutes to bisphenols, with effects yet to be confirmed, 
generating a very dynamic and growing market. The literature demands more solid 
results and improvements in protocols. The Supramolecular Analytical Chemistry (SAC) 
research group at the University of Cordoba has developed an innovative, sustainable, 
specific and more economical method based on the use of supramolecular solvents 
(SUPRAS) combined with high performance liquid chromatography-high performance 
mass spectrometry (HPLC-HRMS/MS). This method allows the identification of 
compounds (known and unknown) by covering a wider range of substances present in the 
materials under study than solvents. 

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organic waste cannot be dealt with. Given that this method has not yet been 
implemented in dentistry, despite being amply tested, this pilot study has been 
developed to lay the foundations of a protocol, and to have a more exact and extensive 
knowledge of our treatments. One of the most widely used, especially after the 
pandemic, is the Michigan type acrylic splint, as a treatment for bruxism. It is also a 
removable device, which is used at least 6-8 hours a day, and does not involve the use of 
additional plastic materials that could be other sources of additives, such as cements, 
sealants or dental temporaries, as is the case with fixed prostheses. The population 
eligible for such treatment must have completed their bone and tooth growth (18 years 
of age or older). In the present study, two forms of manufacture were chosen: 
conventional and printed. The reason for not choosing the milled ones is because the 
latter generate  more  waste,  and  their  mechanical response  as  well  as  the  patient's 
preference are not better. Therefore, identifying the substances released by the acrylics, 
using SUPRAS and HPLC-HRMS/MS, by contrasting the in vitro and in vivo results, 
may shed some light on this issue. 
 
1.4 
OBJECTIVES 
 
There is no evidence of release of substances of particular concern, or endocrine 
disruptors released from Michigan type acrylic occlusal splints. 
 
General objectives: To describe and quantify the values of substances of very high 
concern (SVHC) or endocrine disruptors (ED) released from printed or injected acrylic 
Michigan type splints. 
 
Specific objectives: 
 
1. To identify the SEPs and EDs released by Michigan-type splints made by injection 
or in vitro impression technique, simulating oral environment and wear. 
2. Identify SEPs and EDs in saliva released from printed or saliva-injected acrylic 
Michigan type splints before, during and after insertion in the mouth. 
3. To quantify the levels of SEP and ED in saliva released from printed or injected 
acrylic Michigan type splints in saliva before, during and after insertion in the 
mouth. 
4. Contrast the results obtained in the laboratory test with those recorded in the 
saliva samples of the in vivo study. 

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5. Relate the results to the manufacturing process, stage of exposure, time, habits 
and individual characteristics of each volunteer. 
 
2 METHODOLOGY OF THE STUDY 
 
 
2.1 DESIGN 
This is a prospective analytical observational experimental pilot study that aims to 
analyse the substances of particular concern and endocrine disruptors detected in saliva, 
released by conventional dental devices, before and after the intervention. They will 
therefore be paired samples, where the control group will be the same subject, 
minimising the independent variables: saliva pH, microbiome, salivary flow, habits, 
occlusal wear, erosion by external agents and saliva. 
 
Saliva sampling shall be carried out in accordance with the conventional protocol RD 
1716/2011. The saliva containers shall comply with the requirements of the ISO 
6717:2021 standard. They shall be packaged and transported according to the protocol 
Order JUS/1291/2010 of 13 May. All samples shall be identified with the number 
assigned to the volunteer, time, material and type of manufacture (processed, machined 
or printed), and shall be sent to the Chemical Institute for Energy and Environment 
(IQUEMA). University of Cordoba, where they will be analysed by the SAC group, for 
subsequent statistical treatment and interpretation. 
 
 
 
2.2 LOCATION 
It will take place in the facilities of the University Polyclinic of the European University 
of Madrid (UEM) located in Plaza de Francisco Morano s/n, registered in the Register 
of Health Centres of the Community of Madrid with the number CS5720. 
 
2.3 RESPONSIBLE TEAM 
2.3.1 PRINCIPAL DIRECTORS: 
- 
Dr. Víctor Díaz-Flores García: Associate Professor in the Preclinical Department 
of the Faculty of Dentistry at the European University of Madrid. PI of the 
research group on Patient Safety in Dentistry. 
- 
Dr. Noelia Caballero Casero: postdoctoral researcher at the Chemical Institute 
for Energy and Environment (IQUEMA). University of Cordoba. Member of the 
research group FQM-186 (Supramolecular Analytical Chemistry). 
- 
Dr. Adriana Ivette Ávila Álvarez: Dean of the Faculty of A Coruña. Research 
Unit in Occupational Therapy in Non-pharmacological Interventions. 
 
2.3.2 OPERATOR RESPONSIBLE FOR DATA AND SAMPLE COLLECTION. 
- 
Dr. Susana David Fernández: Associate Professor in the Clinical Department of the 
Faculty of  Dentistry at  the  European University  of Madrid. PI  of the Sustainable 
Dentistry and Sport research group. 

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2.3.3 SUPERVISOR OF COLLECTION, 
TREATMENT 
Y 
ANALYSIS 
SAMPLE 
Dr. Adriana Ivette Ávila Álvarez: Dean of the Faculty of A Coruña. Research 
Unit in Occupational Therapy in Non-pharmacological Interventions. 
- 
Dr. Noelia Caballero Casero: postdoctoral researcher at the Chemical Institute 
for Energy and Environment (IQUEMA). University of Cordoba. Member of the 
research group FQM-186 (Supramolecular Analytical Chemistry). 
- 
Celia Sánchez Vallejo PhD student at the Chemical Institute for Energy and 
Environment (IQUEMA). University of Cordoba. And member of the research 
group FQM-186 (Supramolecular Analytical Chemistry). 
 
 
 
2.3.4   STATISTICAL DATA CONTROLLER 
Dr. Israel John Thuissard Vasallo (Methodological and Statistical Advisory 
Group, Department of Medicine, Faculty of Biomedical and Health Sciences), 
European University of Madrid. 
 
2.4 POPULATION AND CALCULATION OF THE SAMPLE. 
The sample will be obtained from patients who are registered in the database of the 
Polyclinic of the European University of Madrid, who request a Michigan type splint for 
the treatment of bruxism, made of acrylic, made in two of the usual ways: printed or 
injected, in accordance with the inclusion and exclusion criteria shown in Appendix I. 
As the samples were treated with an innovative methodology in the field of dentistry, 
such as super solvents, this was a pilot study in which two groups of 15 participants 
each were formed, depending on the manufacturing process of the splint: injected or 
printed, totaling 30 volunteers. 
 
2.5 PERIOD OF STUDY 
Recruitment is expected to be completed in January 2024, and sample collection is 
expected to be completed in September 2024. 
 
 
 
2.6 DESCRIPTION OF THE STUDY 
2.6.1   VOLUNTEER RECRUITMENT: 
The study groups will be formed in accordance with the requirements and inclusion 
criteria. Each of the subjects will be informed of the purpose and methodology of the 
project by means of an information document for the volunteer (Appendix II) and must 
sign the informed consent form provided for this purpose (Appendix III); they may 
withdraw from participation at any time. The data of the participants will be stored in a 
clinical history with an individual key number, in accordance with the regulations 
contained in the current Organic Law 3/2018, of 5 December, on the Protection of 
Personal Data. 

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Appointments will be made as follows: 
 
APPOINTMENT 1: A conventional computerised clinical history is taken of the patient, 
in which data is collected on general health, orofacial and nutritional habits and hygiene. 
We explain in detail what the treatment consists of: Michigan type relaxation splint 
(device to prevent tooth wear due to bruxism). Each intervention is governed by scientific 
and deontological criteria, respecting all the ethical rules of research. The following 
is a detailed description of the study, its duration and methodology. The four moments 
of saliva collection will be explained: before, during, and 3 and 6 months after placement, 
as follows: 
 
-Time 0: the day of the appointment, before the intervention. 
 
-Step 1: On the day of the appointment, device placement. 
 
-Time 2: 3 months after device placement. 
 
-Time 3: 6 months after treatment. 
 
Information and informed consent will be provided. 
 
APPOINTMENT 2: Preparation and taking of models for the Michigan type splint 
according to the conventional protocol for this treatment. 
 
APPOINTMENT 3: The models will be assembled in the articulator to send and request 
the making of the splint, specifying whether it is injected or printed. 
 
APPOINTMENT 4: A saliva sample is taken in the device for this purpose. After 
checking for proper fit and retention, the occlusal adjustments will be made with a 
handpiece and conventional acrylic and polishing burs. After completion of the 
procedure, a new saliva sample shall be taken. The samples shall be identified with the 
patient's assigned code and stored according to conventional standards. An appointment 
will be made after 6 months to continue with the study and in accordance with the 
check-up times for correct oral health. 
 
APPOINTMENT 5: The intraoral check-up will be carried out, checking the intraoral 
health and the state of the splint, with the pertinent adjustments, according to 
conventional protocol. A new saliva sample will be taken. The patient will be seen after 
3 months. 
 
APPOINTMENT 6: The intraoral check-up will be carried out, checking the intraoral 
and splint health status, with its pertinent adjustments, according to conventional 
protocol. A new saliva sample will be taken. The sample collection period for this 
subject will end. 
 
Prior to this, the substances released by the specific material used to make the devices 
will be identified in the laboratory. The reason for this is the highly dynamic nature of 
the dental materials market. For this purpose, the solutions will be analysed by liquid 
chromatography coupled to a mass spectrometer (LC-HRMS). The identification of the 
compounds present will be based on a non-directed suspect screening approach, 
comparing the results obtained in the analyses with those already existing for the 

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compounds included in the 

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list of suspects. This list shall be compiled on the basis of information gathered during the literature 
review, among other sources. Thus, it shall include from compounds whose presence is expected 
such as those indicated by the manufacturer, to emerging compounds that could be present due 
to storage conditions, use, etc. For the identification, statistical and chemometric techniques will 
be used to establish the correlation between the analytical signals obtained in the LC-HRMS and 
the information (e.g. masses, structure, mass spectra, etc.) available in bibliography and publicly 
available spectrophotometric libraries (e.g. Mass Bank). In a second phase, the compounds identified 
in the first stage will be prioritised according to their level of identification, frequency of 
detection, toxicity (if known) and availability of analytical standards. Finally, quantification of these 
compounds will be carried out (figure 1). 
 
 
 
 
Figure 1: Timeline In vitro study of acrylic-based dental materials: Resting and weathering phase leaching, identification and quantification 
of released substances EDs and SEP. SAC innovative research group at the University of Córdoba (Spain) that belongs to the Institute 
of Fine Chemistry and Nanochemistry (IUNAN). 
 
Thus, knowing the possible elements identified, the saliva samples can be treated. This is carried 
out with a volatile supramolecular solvent spontaneously synthesised by adding hexanol and 
tetrahydrofuran. The methodology to be followed is the same as previously described in the in 
vitro phase. 
Dependent variables: substances of very high concern and endocrine disruptors. Independent 
variables: usual residence, type of manufacture (printed or injected), time, 
age, type of polymerisation. 
 
After identification and quantification of the detected substances, the data will be collected in 
a database with the volunteer's information (ANNEX IV). 
 

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Finally, the results will be analysed and correlated with those obtained in the experimental 
laboratory phase, and the possible participating variables: age, type of material, type of 
manufacture and habits of the participant (figure 2). 
 
 
 
 
 
Figure 2. Timeline of in vivo pilot study on substances released from injected or printed acrylic occlusal splints. 
University Polyclinic of the European University of Madrid. 
 
 
 
2.7 
ETHICAL AND CONFIDENTIALITY ISSUES 
2.7.1 
RIGHT TO BE INFORMED: 
Each volunteer will be provided with an information document (Annex II), and informed consent 
(Annex III), which, once understood, must be voluntarily signed and returned. 
 
2.7.2 COLLECTION AND USE OF BIOLOGICAL SAMPLES 
Sample collection will be carried out in accordance with the ethical and legal requirements set 
out in RD 1716/2011. The containers will meet the requirements according to ISO 6717:2021, 
and will be packaged and transported according to the protocol Order JUS/1291/2010, of 13 
May. 
 
2.7.3 RISKS AND INCONVENIENCE FOR THE PARTICIPANT 
The present study does not involve any risk for the patient, as these are procedures and 
treatments used in everyday dental practice, with a long clinical and scientific history. The 
discomforts are those inherent to these processes, without any additional discomfort. 
 
2.7.4 ADVERSE EVENTS 
It does not imply any additional consideration to those that such treatments may entail. 
 
2.7.5 POTENTIAL BENEFITS 
The benefits are those inherent to the treatments indicated: muscular improvements, 
avoidance of further wear and tear and more severe damage to dental tissue, loss of 
teeth and mucosal lesions. As an indirect benefit, knowledge of the possible substances 
detected will provide a better approach to the prevention of possible adverse effects. 
 
2.8 STATISTICAL ANALYSIS 
For the descriptive analysis of the variables collected, the mean ± standard deviation or 
median [interquartile range] will be used once the distribution has been checked, in the 
case of the variables collected. 

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quantitative variables. To describe qualitative variables, absolute (n) and relative (%) 
frequencies will be used and their 95% confidence interval (95% CI) will be calculated. 
 
Chi-square tests (or Fisher's exact tests) will be used to study differences between 
qualitative variables. In case of quantitative variables, Student's t-test or Man-Whitney 
U-test will be used, depending on whether they follow parametric or non-parametric 
behaviour. Binary logistic regression models will be used to determine the Odds ratio 
(OR) of seroprevalence and its 95% confidence interval (95% CI) to analyse the effect 
of each variable on the results obtained. 
 
All statistical calculations were performed using SPSS statistical software (version 25.0 
IBM Corp.; USA). Statistically significant differences were assumed to exist if the p- 
value obtained was less than or equal to 0.05. 
 
2.9 DISSEMINATION OF RESULTS 
The members of the study's research team are committed to disseminating the results in 
scientific media, supports and publications. 
 
2.10 EXPENSES AND FINANCIAL COMPENSATION 
This study is exempt from financial compensation to participants. 
 
 
 
ANNEX I 
 
 
 
INCLUSION CRITERIA FOR THE FORMATION OF THE "MICHIGAN SPLINTS" 
GROUP 
 
• 
Be of legal age 
 
• 
To be able to attend Polyclinic for 6 months 
 
• 
Require a Michigan type acrylic relaxation splint. 
 
• 
Not pregnant 
 
• 
Not be breastfeeding 
 
 
 
 
EXCLUSION CRITERIA: 

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• 
Being a minor 
 
 
• 
Allergy to any type of plastic material, acrylic or resins used in dentistry. 
 
ANNEX II 
 
 
 
 
VOLUNTEER INFORMATION SHEET FORMAT 
 
 
 
Information document for volunteers on disruptors and substances of 
particular concern in critical materials in dentistry. 
 
Study Name: Use of supramolecular solvents in the identification, detection and 
quantification of substances of very high concern and endocrine 
disruptors released from Michigan-type occlusal splints in saliva: an 
in vitro and in vivo pilot study. 
 
Principal Investigator:Dr. Susana David Fernández 
 
Place of research: University Polyclinic of the Universidad Europea. Plaza Francisco 
Morano s/n. 28005. Madrid. Spain (registered in the Registry of the Community of 
Madrid under No. CS5720). 
 
OBJECTIVE OF THE STUDY: 
 
To analyse the presence of additives in saliva before, during and after common clinical 
procedures in dentistry. 
 
STUDY PROCEDURE: 
 
This study will be carried out in accordance with the Code of Ethics and Professional 
Deontology drawn up by the Ethics and Deontology Commissions of the Spanish Dental 
and Stomatology Associations. 
 
The conventional protocol will be carried out (clinical history, treatment plan, budget 
and consents) that any dental treatment entails. Once you have decided to start your 
dental treatment, on the day your splint is to be given to you, saliva samples will be 
taken, explaining the procedure at the previous appointment. Subsequently, you will be 
given an appointment every 3 months to take a saliva sample, according to the detailed 
explanations that you will be given beforehand. The study lasts 9 months from the first 
appointment to the last. 
 
All the data obtained from the volunteers will be stored in a medical record with an 
individual key number that will be filed in a space designated for this purpose, in the 
University Polyclinic of the Universidad Europea following the following regulations 

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contained in the current Organic Law 3/2018, of 5 December, on the Protection of 
Personal Data. 
 
 
 
REVIEW 
 
Volunteers will be notified of successive scheduled appointments (4 times in total). 
 
 
 
RISK TO THE PARTICIPANT: 
 
All the materials used in this study follow the sterilisation and disinfection procedure 
contained in ORDER 288/2010, of 28 May, which regulates the general and specific 
technical requirements for health centres and services without hospitalisation, and for 
health services integrated in non-health organisations in the Community of Madrid. 
 
As this is a non-invasive test in which only saliva samples are taken, there is no risk to 
the volunteer. 
 
The examiner has more than 25 years of clinical experience. 
 
 
 
BENEFITS: 
 
There is no direct benefit to participating in this study as a volunteer. 
 
 
 
ALTERNATIVES: 
 
As this is a non-invasive observational study, there is no alternative to it. 
 
 
 
VOLUNTARY PARTICIPATION AND WAIVER 
 
Your participation in this study is voluntary. You may withdraw from the study at any 
time during the study. 
 
Investigators may terminate their participation in the study for any of the following 
reasons: 
 
1. Development of medical conditions that prevent you from participating in the study. 
2. If, during the study, the prosthesis is damaged or lost in such a way that it cannot 
be followed up. 
3. If you become pregnant between the recruitment period and the start of the 
study. 
4. If you fail to keep scheduled appointments 
5. If the study is cancelled. 

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PARTICIPATION RIGHTS/AVAILABILITY OF INFORMATION 
 
If you have any questions before or during the study contact the principal investigator 
(Dr. 
Susana 
David 
Fernández, 
913858800, 
susana.david@universidadeuropea.es) 
 
                               ANNEX III 
 
 
INFORMED CONSENT 
 
 
 
Informed Consent to Participate in a Clinical Study on Disruptors and 
Substances of Very High Concern in Acrylics in Dentistry. 
 
Study Name: Use of supramolecular solvents in the identification, detection and 
quantification of substances of very high concern and endocrine 
disruptors released from Michigan-type occlusal splints in saliva: an 
in vitro and in vivo pilot study. 
 
Project Code: 23.282 
 
 
 
Principal Investigator; Dr. Susana David Fernández 
 
 
 
Place of research: Polyclinic of the Universidad Europea. Plaza Francisco Morano s/n. 
28005. Madrid. Spain (registered in the Registry of the Community of Madrid under 
No. CS5720). 
 
 
 
INTRODUCTION: 
 
You and 29 other volunteers, who are patients of the European University, have been 
invited to participate in this research study on the presence of additives in dental 
materials of recognised quality and guarantees, used daily in dentistry. Please review the 
contents of this informed consent form and make sure that all your questions are 
answered before you decide to participate in this study. The investigators will explain 
all aspects of the study to you and will be available to answer any questions you may 
have throughout the duration of the study. 
 
 
 
CONFIDENTIALITY CLAUSE: 
 
The data obtained by participating in this study will be processed in accordance with 
Organic Law 3/2018, of 5 December, on the Protection of Personal Data, with the 
participating volunteer reserving the right to oppose, access, rectify or delete the data. 

RESEARCH PROTOCOL 
O.V. JUNE 2023 
 
cancellation of your data in accordance with the procedure described in the Regulations 
for the development of Organic Law 3/2018, of 5 December, on the protection of 
personal data. 
 
The data  may  be used  for  statistical and  academic  purposes, while  respecting  the 
confidentiality of the data and the personal image of the volunteer. Any publication or 
presentation in which the data obtained may be used will not identify the volunteer. 
 
 
 
PARTICIPATION RIGHTS/AVAILABILITY OF INFORMATION 
 
 
 
If you have any questions before or during the study contact the principal investigator 
(Dr. 
Susana 
David 
Fernández, 
913858800, 
susana.david@universidadeuropea.es). 
 
 
 
 
 
 
 
 
CONSENT TO PARTICIPATE: 
 
By signing this consent form, I declare that I am 18 years of age or older. I confirm that 
I have read this informed consent, and that I have been given sufficient opportunity to 
ask any questions about this consent or about the study. I also confirm that I understand 
the purpose of my participation in this study, and that all my questions have been 
answered satisfactorily. I sign this consent form voluntarily, agreeing to participate in 
this study. I understand that I am not giving up any of my rights by signing this consent 
or participating in this study and that I will receive a copy of this consent. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Name, surname, ID number and signature of volunteer                            Date 

RESEARCH PROTOCOL 
O.V. JUNE 2023 
 
 
 
ANNEX IV 
 
Voluntary code 
CONCENTRATIONS 
CONCENTRATIONS 
CONCENTRATIONS 
CONCENTRATIONS 
 
 
SUBSTANCES 
M .Saliva 0 
M. Saliva 1 
M. Saliva 2 
M. Saliva 3 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Name, surname, ID number and signature of the researcher 
Date 
 
 
 
 
 
 
 
 
 
 
Sample collection table for each volunteer 
 
 
 
 
VARIABLES 
Volunteer 1 
Volunteer 2 
Volunteer 3 
Volunteer 4 
Volunteer 5 
 
AGE 
- 
GENDER (MALE, FEMALE, OTHER) 
MATERIAL (ACRYLIC) 
MANUFACTURING(INJECTED or PRINTED) 
- 
USUAL RESIDENCE (URBAN, RURAL) 
- 
EXPOSURE TIME (HOURS) BRUXIST 
- 
(YES/NO) 
REFLUX (YES/NO) 
ANTIDEPRESSANTS 
(YES/NO) ANTIHISTAMINES 
(YES/NO) 
DECONGESTANTS (YES/NO) 

RESEARCH PROTOCOL 
O.V. JUNE 2023 
 
Variable collection table 
 
 
 
 
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