Concentration of Bisphenol A in Saliva and Urine Before and After Treatment With Polymer-based Dental Filling Materials
Conditions: Dental Polymer Based Filling Material
Sponsor: NORCE Norwegian Research Centre AS

«Concentration of bisphenol A in saliva and urine after filling therapy with polymer-based dental restorative materials» Rev. 06/22/16 
RESEARCH PROTOCOL (Rev 22.06.16) 
Concentration of bisphenol A in saliva and 
urine after treatment with polymer-based 
dental restorative materials 
Project summary 
Bisphenol A (BPA) is an estrogen-mimicking substance, which even at low concentrations, has been 
shown to cause abnormalities in sex development and lead to reproductive damage in fish and 
animals. In population-based studies, BPA has been detected in concentrations that indicate levels 
that could cause health effects. 
The majority of dental plastic filling materials contain methacrylates which are synthesized from, 
among other substances, BPA. It has not been sufficiently investigated whether BPA is released in 
vivo from polymer-based dental fillings. 
The purpose of the project is to gain knowledge about the BPA concentration in saliva and urine, and 
whether it is affected by newly placed polymer-based fillings. We will measure and compare the 
saliva and urine concentrations of BPA before and after filling therapy in individuals who receive their 
first composite filling. 
Project Manager 
Dentist Trine Lise Lundekvam Berge, TK-Vest Hordaland and Årstad Dental Clinic, Public Dental 
Health Service in Hordaland and Dental Biomaterials Adverse Reaction Group, Uni Research Health. 
Project staff 
Professor Lars Björkman, PhD, Dental Biomaterials Adverse Reaction Group, Uni Research Health and 
Department of Clinical Dentistry, University of Bergen. 
Researcher II Gunvor Bentung Lygre, PhD, Dental Biomaterials Adverse Reaction Group, Uni Research 
Health. 
Responsible research institution 
Uni Research AS will be responsible research institution. Owners of data will be the Dental Health 
Service in Hordaland and Uni Research AS. 
This document is translated from the Norwegian version. 
Bisphenol A in Saliva and Urine Related to Placement of Dental Composites (NCT02575118)

«Concentration of bisphenol A in saliva and urine after filling therapy with polymer-based dental restorative materials» Rev. 06/22/16 
Collaborators 
The project is organized as a collaborative project between the Dental Health Service, Hordaland 
County and the Dental Biomaterials Adverse Reaction Group (Uni Research Health). 
The Public Dental Health Service, Hordaland County, contributes with 
• Access to project participants 
• Access to journal archive. Working hours to perform clinical examination, sampling and filling 
therapy on the subjects, as well as design of necessary information and questionnaire (TK-Vest 
Hordaland) 
• Dental office 
The Dental Biomaterials Adverse Reaction Group (Uni Research Health) contributes with 
• Working time 
• Guidance 
 
Background 
Bisphenol A (CAS No. 80-05-7) is a chemical substance that has been shown to be weakly hormone-
mimicking, so-called estrogen-mimicking. Excess estrogens can lead to reduced development of male 
sex characteristics and reduced fertility. 
The concentration of bisphenol A measured in human blood and urine cannot be explained by 
previously described routes of exposure and known half-lives [1]. This indicates a higher intake of 
bisphenol A than what has been calculated so far and in addition, bioaccumulation of bisphenol A 
after exposure. A study of bisphenol A concentrations in urine in relation to food intake [2] confirms 
this. This study showed that concentrations of bisphenol A in urine decreased more slowly than 
might be expected based on previously calculated half-lives. The authors conclude that there is a 
significant "nonfood exposure" and / or bioaccumulation. 
The majority of polymer-based dental filling materials contain methacrylates which are produced 
from, among other substances, bisphenol A. Although bisphenol A is not added as an ingredient, 
contaminated raw materials may contain low concentrations. 
It has been shown that treatment with some fissure sealing materials containing bis-DMA (bisphenol 
A dimethacrylate) may result in a short-term exposure to bisphenol A immediately after treatment 
[3]. 
Another study [4] has observed that filling therapy with plastic-based filling material is associated 
with a higher concentration of bisphenol A in saliva and urine immediately after treatment. 
Differences in sampling methods, examined materials, follow-up time and analysis methods and 
detection levels give variation in the results. 
Results from a recent study [5] showed that it could not be ruled out that individuals with plastic-
based dental fillings have a higher concentration of bisphenol A in saliva compared to individuals 
without plastic-based dental fillings. More research on the subject is warranted. 
 

«Concentration of bisphenol A in saliva and urine after filling therapy with polymer-based dental restorative materials» Rev. 06/22/16 
Aim 
The aim of the project is to gain knowledge about the concentration of bisphenol A in saliva and 
urine in individuals before and after treatment with a polymer-based dental restorative material. 
We want to test a hypothesis (Hₒ) that the bisphenol A concentration in saliva and urine is 
unchanged or lower after filling therapy with a polymer-based dental restorative material. The 
alternative hypothesis (H1) is that bisphenol A concentration in saliva and urine increases after 
treatment. 
 
Materials and methods 
Study design 
20 healthy and voluntary individuals (10 women and 10 men) who need a minimum of two tooth 
surfaces treated with a polymer-based dental restorative material are recruited from dental clinics in 
Sentrum Dental Health District, Hordaland County and from the Student Association's dental clinic in 
Bergen. 
Inclusion criteria: 
Need for filling therapy that involves at least two surfaces with a polymer-based dental restorative 
material. 
Healthy patient (No known chronic diseases) 
Age: 16 - 40 years 
Written informed consent 
 
Exclusion criteria: 
Substance abuse 
Smoking 
Use of (‘snus’) snuff 
Treated with polymer-based dental restorative material the last 3 months 
Removable prosthetic dentures or orthodontic appliances 
Use of dental splint 
Work at a dental clinic 
 
• Informed consent will be obtained according to current rules. 
• All subjects answer questions related to dietary habits and living habits. (See attached 
questionnaires 1, 2 and 3) 
 
Recruitment 
Patients attend an ordinary examination at their regular dental clinic. If the dentist/dental hygienist 
considers that the patient needs filling therapy with polymer-based dental restorative material and is 
otherwise suitable for the study (inclusion/exclusion criteria), the patient receives oral information 
about the project and is asked if he/she wishes to participate. If the patient agrees, the name and 
contact information are entered on a recruitment list (see appendix). At the same time, the patient is 
given an information letter with a thorough description of the study and a consent form (see 
appendix). It will be emphasized that participation is voluntary. The patient will have the opportunity 
to read through the information letter at home before he/she is contacted and possibly given an 
appointment with the project manager. 

«Concentration of bisphenol A in saliva and urine after filling therapy with polymer-based dental restorative materials» Rev. 06/22/16 
 
Day 1 with project manager 
• Project participant meets in the morning with written consent. 
• The participant must be fasting, i.e. not eating or drinking for the last 10 hours. Only tap water is 
allowed. The bladder must be emptied of morning urine at home, preferably as soon as possible after 
waking up. The time of emptying is noted by the participant. Avoid lip balm/lipstick. 
• Dental status is mapped according to ordinary clinical procedure. If polymer-based dental 
restorative material is detected in the oral cavity, the individual must be excluded. 
• Project participant answers questions from questionnaire 1. 
• Saliva and urine samples are collected according to specified methods (See appendix). 
Filling therapy is performed according to the ordinary procedure. 
 
Day 2 with project manager 
• The patient meets fasting, i.e. has avoided food and drink for the last 10 hours. Only tap water is 
allowed. The bladder must be emptied of morning urine at home, preferably as soon as possible after 
waking up. The time of emptying is noted by the participant. Avoid lip balm/lipstick. 
• Answers questionnaire 2. 
Saliva and urine samples are collected from the subjects according to specified methods 
 
Day 8 with project manager 
• The patient meets fasting, i.e. has avoided food and drink for the last 10 hours. Only tap water is 
allowed. The bladder must be emptied of morning urine at home, preferably as soon as possible after 
waking up. The time of emptying is noted by the participant. Avoid lip balm / pins. 
• Answers questionnaire 3. 
Saliva and urine samples are collected from the subjects according to specified methods 
 
Work protocols are stored at the clinic (see appendix). 
 
Filling materials 
As an adhesive Optibond FL will be used and as a filling material Tetric Evo Ceram (capsules from 
Ivoclar Vivadent AB). 
The materials are chosen because they are used routinely at the relevant dental clinic. 
 
Saliva samples and urine samples 
Saliva and urine samples are collected from the subjects before and after filling therapy according to 
the specified method. 

«Concentration of bisphenol A in saliva and urine after filling therapy with polymer-based dental restorative materials» Rev. 06/22/16 
 
 
Just before 
treatment 
Just after 
treatment 
1 hour after 
treatment 
24 hours after 
treatment 
1 week after 
treatment 
 
Saliva test no. 
0 
1 
2 
3 
4 
Urine sample 
no. 
0 
 
1 
2 
3 
 
Before sampling, the sampling chain is checked so that the risk of contamination is minimized. The 
samples are immediately placed in a refrigerator and frozen as soon as possible at -80 ° C before 
analysis. 
Do not use equipment that can cause contamination from plastic products/bisphenol A-containing 
products. The effect of the curing lamp is checked and documented weekly for as long as the project 
lasts. Previous filling therapy and points are registered in a separate diagram. 
 
Filling therapy 
• Detect current tooth surfaces that need filling 
• Filling therapy according to normal clinical routines 
• Any rubber-dam use is registered 
• Estimate the amount of composite/surface area (Weigh the composite ampoules before and after 
filling therapy). 
• Type of curing lamp and curing time are registered. 
 
Analyzes 
Analysis of bisphenol A in saliva and urine is performed as a commissioned analysis at the 
Department of Occupational and Environmental Medicine (AMM), Lund University, Sweden. A 
method of liquid chromatography (LC) and mass spectrometry (MS) (LC-MS) which we have used in a 
previous project [5] will be used. 
 
Possible advantages and disadvantages for the participants 
Participation in the project will mean that the patient must give five saliva samples (just before filling 
therapy, just after filling therapy, after 1 hour, after 24 hours and after 1 week) and four urine 
samples, which entails extra visits to the dental clinic. As a small appreciation, all participants will be 
given two cinema tickets and two lottery tickets (“Flax”) after passing the last urine test. Paying 
patients (persons over 18 years of age) will not be charged financially for the treatment in question. 
 
Outcome 
Concentration of bisphenol A in saliva and urine. 
 

«Concentration of bisphenol A in saliva and urine after filling therapy with polymer-based dental restorative materials» Rev. 06/22/16 
Time plan 
August 2014 
Assessment by local Research Ethics Committee 
 
September 2015 - April 2017 
Recruitment of and filling therapy on project participants 
 
May - June 2017 
The saliva and urine samples are analyzed 
 
August - October 2017 
Data analyses 
 
October - December 2017 
Publication of poster and manuscript 
 
 
Statistical analysis 
The primary outcome measure is the concentration of bisphenol A in saliva. Data from an 
unpublished study [5] showed that the concentration of bisphenol A in saliva is very low and in many 
samples below the detection limit (0.1 ng / ml saliva). Therefore, non-parametric tests are primarily 
used for the statistical analysis of the saliva data. Values below the detection limit are given ½ the 
value of the detection limit in the statistical analyzes. 
Concentration of bisphenol A in urine is a secondary outcome measure. Concentrations of bisphenol 
A in urine in individuals without plastic fillings are probably above the detection limit in most cases, 
and data are probably not normally distributed. Data on bisphenol A in urine are therefore primarily 
analyzed also with non-parametric tests (Friedman test and Wilcoxon Signed Rank Test for pairwise 
testing of data). 
For analysis of observations before treatment (t0), respectively after treatment (t10 min = t4-7 hrs = t24 hrs 
= t7 days) Friedman test is used, which tests the hypothesis (H0), that the values at all times are equal 
(i.e. t0 = t10 min = t4-7 hrs = t24 hrs = t7 days). 
For the analysis of observations divided into the groups "<detection limit" and "≥ detection limit", 
respectively, before and after treatment, McNemar's test is used, which compares observations from 
two times at a time. SPSS will be used for the statistical analyzes (Version 22). 
 
Power / sample size 
Reliable data that can be used to calculate sample size is missing. Therefore, the following 
assumptions are made for saliva data (see Figure 1): 
1. The proportion of individuals with values below the detection limit before treatment and above 
(or equal to) the detection limit after treatment is 0.7 (i.e. 70%). 
2. The proportion of individuals with values above (or equal to) the detection limit before treatment 
and below the detection limit after treatment is 0.1 (i.e. 10%). 

«Concentration of bisphenol A in saliva and urine after filling therapy with polymer-based dental restorative materials» Rev. 06/22/16 
3. The proportion of persons who show no change, i.e. they have values below the detection limit 
both before and after treatment 0.1 (10%) or values above (or equal to) the detection limit both 
before and after treatment 0.1 (10%). 
 
 
 
                                                         
 
 
                                
Figure 1. Proportions with values below and above the detection limit (d.l.) before and after 
treatment. 
 
Power calculation 
If a one-sided test (Mc Nemar test of paired proportions) is used, a study with 10 subjects then has a 
power of 77.5% to be able to detect a significant difference before and after treatment with 
significance level alpha set to 0.05 . If the number of subjects increases to 20, a power of 96% is 
obtained (Figure 2). 
 
 
Figure 2. Test strength (power) as a function of sample size (one-sided test). (From SPSS Sample 
Power; Mc Nemar test of paired proportions). 
                                                         Before treatment 
 
 
       After treatment  
 
< d.l. 
 ≥ d.l. 
< d.l. 
0,1 
0,1 
≥ d.l. 
0,7 
0,1 

«Concentration of bisphenol A in saliva and urine after filling therapy with polymer-based dental restorative materials» Rev. 06/22/16 
 
Handling of possible adverse events 
As we see it, there is no particular risk that the project will lead to an increased risk of adverse 
events. Should complications occur, these will be handled according to the dental clinic's usual 
procedures. 
 
Assessment of research ethics 
The project will be assessed by the Regional Committee for Medical and Health Research Ethics. 
In our opinion, the benefits of carrying out the project are significantly greater than the 
disadvantages. Participation in the project does not pose a significant risk the subjects, beyond what 
ordinary dental treatment may entail. 
Previous studies have shown that bisphenol A can be detected in saliva and urine. The source of this 
is unclear and more knowledge is needed. Increased knowledge of potential risks with dental filling 
materials is important for choosing safe dental restorative materials. 
If it turns out that bisphenol A cannot be detected in saliva and urine after filling therapy, the 
probability that dental filling materials are a source of exposure to bisphenol A is low. 
The participants are made aware that participation is voluntary and that they can withdraw from the 
project at any time. 
All subjects must sign informed consent, approving that the samples can be used for analysis. Patient 
data will be pseudonymized and processed properly after approval. 
All information and samples from the participants will be processed without name and date of birth. 
Each participant will be linked to the project by a code corresponding to a list of names. Only 
personnel directly connected to the project have access to the name list. It will not be possible to 
identify the participants in the project when the results are published. 
After the last urine test, the participants will be given two cinema tickets and two lottery tickets 
(“Flax”) as an appreciation for the participation. All patients, including people over the age of 18, will 
receive the actual dental filling free of charge. In our opinion, the total value of this compensation 
does not exceed what can be considered reasonable for participation. 
 
Publication - dissemination plan 
The project started in the autumn of 2015 and is planned to be completed by the end of 2017. This 
means that there will be a poster for presentation at a scientific meeting and a manuscript for 
submission to a scientific journal. 
 
Budget (omitted) 
 

«Concentration of bisphenol A in saliva and urine after filling therapy with polymer-based dental restorative materials» Rev. 06/22/16 
Significance 
The results could provide important information to assess whether filling therapy with polymer-
based dental restorative materials is associated with increased concentration of bisphenol A in saliva. 
We also have an opportunity to look at the concentrations in urine. 
If the results from the study show that there is an association between exposure to dental polymer-
based dental restorative materials and bisphenol A in biological media, this motivates follow-up 
studies. 
 
References 
1. Welshons, W.V., S.C. Nagel, and F.S. vom Saal, Large effects from small exposures. III. Endocrine mechanisms 
mediating effects of bisphenol A at levels of human exposure. Endocrinology, 2006. 147 (6 Suppl): pp. S56-69. 
2. Stahlhut, R.W., W.V. Welshons, and S.H. Swan, Bisphenol A data in NHANES suggest longer than expected 
half-life, substantial nonfood exposure, or both. Environ Health Perspect, 2009. 117 (5): pp. 784-9. 
3. Arenholt-Bindslev, D., et al., Time-related bisphenol-A content and estrogenic activity in saliva samples 
collected in relation to placement of fissure sealants. Clin Oral Investig, 1999. 3 (3): pp. 120-5. 
4. Kingman, A., et al., Bisphenol A and other compounds in human saliva and urine associated with the 
placement of composite restorations. J Am Dent Assoc, 2012. 143 (12): pp. 1292-302. 
5. Berge, T.L.L., G.B. Lygre, and L. Björkman, Bisphenol a concentration in human saliva related to dental 
composites. Unpublished data. 2014.