An engaged research study to assess the effect of a ‘real-world’ dietary intervention on urinary bisphenol A (BPA) levels in teenagers

Objective Bisphenol A (BPA) has been associated with adverse human health outcomes and exposure to this compound is near-ubiquitous in the Western world. We aimed to examine whether self-moderation of BPA exposure is possible by altering diet in a real-world setting. Design An Engaged Research dietary intervention study designed, implemented and analysed by healthy teenagers from six schools and undertaken in their own homes. Participants A total of 94 students aged between 17 and 19 years from schools in the South West of the UK provided diet diaries and urine samples for analysis. Intervention Researcher participants designed a set of literature-informed guidelines for the reduction of dietary BPA to be followed for 7 days. Main outcome measures Creatinine-adjusted urinary BPA levels were taken before and after the intervention. Information on packaging and food/drink ingested was used to calculate a BPA risk score for anticipated exposure. A qualitative analysis was carried out to identify themes addressing long-term sustainability of the diet. Results BPA was detected in urine of 86% of participants at baseline at a median value of 1.22 ng/mL (IQR 1.99). No effect of the intervention diet on BPA levels was identified overall (P=0.25), but there was a positive association in those participants who showed a drop in urinary BPA concentration postintervention and their initial BPA level (P=0.003). Qualitative analysis identified themes around feelings of lifestyle restriction and the inadequacy of current labelling practices. Conclusions We found no evidence in this self-administered intervention study that it was possible to moderate BPA exposure by diet in a real-world setting. Furthermore, our study participants indicated that they would be unlikely to sustain such a diet long term, due to the difficulty in identifying BPA-free foods.

Conclusions 55 We found no evidence in this self-administered intervention study that it was possible to 56 moderate BPA exposure by diet in a real world setting. Furthermore, our study participants 57 indicated that they would be unlikely to sustain such a diet long term, due to the difficulty in 58 identifying BPA-free foods. • This study represents the largest assessment to date of the potential for moderating 65 one's own BPA exposure through diet 66 • The study was carried out in a real-world setting rather than a regulated, controlled 67 environment.

68
• The study was carried out in teenagers, the demographic with amongst the highest 69 exposure.

70
• Qualitative analysis reveals challenges with sustaining such a diet.

71
Limitations of the study 72 • Calculation of a risk score is challenging due to the pervasive nature of BPA

What is already known on this topic
• Bisphenol A (BPA) is an endocrine disrupting chemical ubiquitously present in our diet and environment. It has been associated with a variety of adverse human health outcomes in animal models, in human populations and in human in-vitro work.
• Previous studies have shown that it may be possible to reduce BPA exposure by following a researcher-supplied very controlled diet for a period of 3 days.

What this study adds
• Our study suggests that although it may be possible to reduce dietary BPA in a controlled setting, it is not possible to do so in a community-based 'real world' setting.
• Qualitative research suggests that even if it were possible to reduce BPA exposure by dietary means, the restrictions placed on lifestyle and the current inadequacies in labelling of BPA containing foods means that such a diet would be impossible to follow long-term, in the real world.
• Improved labelling of foods and packaging of foodstuffs suspected to contain BPA may allow consumers an informed choice on their own exposure.    creatinine (p = 0.27). We found no association between ∆BPA and BPA risk score when 227 considering only the exposure on the day prior to testing, taking into account the short half-228 life of BPA (p = 0.16 and p = 0.33 for adjusted and unadjusted data respectively). We 229 conclude that the 'real world' diet designed to reduce BPA exposure had no effect on   Although levels of urinary BPA in our study cohort were slightly lower at the outset of the 281 study in our cohort than in others [13], measureable levels were present in the vast majority 282 of our participants. Participants were unable to achieve a reduction in their urinary BPA 283 levels over the 7 day trial period, despite good compliance to supplied guidelines. Avoidance 284 of BPA was not easily achieved on an individual level in our study population, with 285 qualitative analysis indicating that participants experienced feelings of restriction and 286 difficulties in sourcing BPA-free food due to inadequate labelling of foods and food 287 packaging. This suggests that the intervention would be difficult to sustain in the longer term.

General instructions
The purpose of this dietary intervention trial is to follow a diet designed to minimise routes of exposure to the food packaging chemical bisphenol A (BPA). For the dietary intervention period, please follow as closely as possible the instruction given below. Try to maintain your diet during the intervention period to be as closely similar to your normal diet as possible, in terms of the content, amount and calorific value of the food you eat. Please record details of each meal and the drinks and snacks you consume on the forms provided. Below are some general cooking and eating tips and an indication of which foods are best to avoid and those that are considered a low source of BPA.
Cooking and eating tips for the intervention period.
The general approach is to replace any food items that fall into the 'avoidance' category with an alternative, chosen to minimise exposure to BPA  Switch to stainless steel and glass food storage and drink containers.
 Move foods to ceramic or glass food containers before microwaving.
 Consider a coffee filter or percolator for coffee -home coffee makers (Such as Nespresso TM ) may have polycarbonate-based water tanks and phthalate-based tubing.
 Eat out less, especially at restaurants that do not use fresh ingredients.
 Avoid canned food consumption. Where possible, replace with fresh produce or cardboard or tetrapack packaged alternatives.
 Choose fresh fruits and vegetables when possible, and frozen if not.

CONSENT STATEMENTS
Thank you for reading this leaflet. If you wish to participate in this study, you will be asked to agree to the consent statements below in the presence of a member of the research team. 3. I agree to participate in this study as a research subject. This means that you agree to participate in a one-week diet and to provide two blood and urine samples.
4. I understand that my anonymised blood and urine samples and linked anonymous questionnaire data will be sent to University of Exeter Medical School, Royal Devon & Exeter Hospital and my urine sample only will be sent to the Rolvaltain laboratory, a specialist BPA analysis company. This means that laboratory staff will not know that samples belong to you, but dedicated staff at the University of Exeter, with training and experience in data protection ,will be able to link your sample data to your questionnaire data.

5.
I understand that RNA (genetic material) will be extracted from my blood and will be stored anonymously. This means that Professor Harries' team will use our RNA to provide data that you will help analyse but may also do further research on the samples to identify reasons for any changes seen.
6. I understand that data relating to my participation in the study will be returned anonymously to my school to be used for educational purposes. This means that although you will get to analyse data from your samples there is no way you will know which data relates to your samples and which to other participants.

Complaints:
If

Involvement & Engagement
The aim of this year-long project is to involve teenagers in a research study that is relevant to them, by allowing them to help design a research project, analyse non-identifiable participant data and help to present and publish the outcomes.

Participation
Students will be asked to undertake a one week diet to reduce their intake of BPA, a chemical found in plastics. They will be asked to provide urine and blood samples before and after their diet.  1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46 F o r p e e r r e v i e w o n l y

What will I need to do?
This project is being run as a student-involvement project to answer two specific questions:

1.
Can we see the effects of dietary BPA on our genes?

2.
Can we effectively reduce BPA in our diet?
In the past, small-scale experiments have shown that BPA levels in the human body can be reduced by rigid dietary interventions but these interventions would be difficult to implement in the "real world". In this study a one-week dietary intervention designed by teenagers will be used by them to determine whether BPA levels, and the activity of BPA-responsive genes can be effectively reduced in young people by avoiding food packaging that contains this chemical.

What is BPA?
BPA (Bisphenol A) is a chemical used in the manufacture of plastics. Plastics containing BPA are found in a wide range of products including food and drink containers. BPA in these products can be ingested and there are concerns that high BPA levels in the blood could possibly affect human health. Research is therefore needed to understand its effects on the human body and how we can reduce its consumption by minor changes to our diet. The diet will exclude sources of BPA as much as possible but will be nutritionally and calorifically similar to your usual diet.  You will be asked to complete a food diary and answer a questionnaire about how easy it was to follow this diet.

Day 8
 Provide a nurse with a 2.5ml blood sample and a urine sample.
We recommend that you discuss the project with your family and involve them in planning what you eat and how you will prepare it.
Are there any risks in taking part?
Blood samples will be taken by fully qualified and insured NHS personnel. Any potential discomfort or side-effects will be equivalent to that experienced giving a blood sample to your GP. All data will be fully anonymised before analysis. This means that you will not find out anything about your blood or urine samples. Following the diet may minimally increase the cost of your groceries for the week, but since fresh foods are usually less expensive than pre-packaged foods, we do not expect this to be an issue.

What will happen to my samples and data?
When you participate in the study you will be allocated with a numerical study ID. Your samples and data will be labelled with this number so that we can match your 'before' and 'after' diet samples with your food diary data. Once all data has been collated and coded it will be further anonymised by a person external to the project so that no data can be linked to any of the participants.
Urine samples from before and after the diet will be sent to the Royal Devon & Exeter NHS Foundation Trust for creatinine analysis and to the Rolvaltain laboratory for BPA analysis. RNA will be extracted from blood samples at the Royal Devon & Exeter Molecular Genetics Laboratory and the expression levels of two BPA-responsive genes will be measured in the samples taken before and after the diet. These anonymised RNA samples will be stored and used only by Professor Harries team for further research on the mechanisms behind our findings.
What will happen to the results of the research study?
You will be given the opportunity to help analyse anonymised data from this project and to help disseminate the outcomes of this research. It is hoped that the findings will be published in peer-reviewed journals and the wider media.

Who is organising this research?
The research is organised by Professors Lorna Harries & Tamara Galloway of the University of Exeter as part of their research program into BPA and part of the University's outreach program to involve schools in academic research.

What are the benefits of taking part?
This project will help you to understand how you might be able to reduce BPA in your diet and your involvement in the design will give you an excellent insight into clinical research, community outreach and scientific practise. Your role as a participant is unlikely to have any direct health benefits.

YES / NO
3. I agree to participate in this study as a research subject. This means that you agree to participate in a one-week diet and to provide two blood and urine samples.

YES / NO
4. I understand that my anonymised blood and urine samples and linked anonymous questionnaire data will be sent to University of Exeter Medical School, Royal Devon & Hospital and my urine sample only will be sent to the Rolvaltain laboratory, a specialist BPA analysis company. This means that laboratory staff will not know that samples belong to you, but dedicated staff at the University of Exeter, with training and experience in data protection, will be able to link your sample data to your questionnaire data.

YES / NO
5. I understand that RNA (genetic material) will be extracted from my blood and will be stored anonymously. This means that Professor Harries' team will use our RNA to provide data that you will help analyse but may also do further research on the samples to identify reasons for any changes seen.

General instructions
The purpose of this dietary intervention trial is to follow a diet designed to minimise routes of exposure to the food packaging chemical bisphenol A (BPA). For the dietary intervention period, please follow as closely as possible the instruction given below. Try to maintain your diet during the intervention period to be as closely similar to your normal diet as possible, in terms of the content, amount and calorific value of the food you eat. Please record details of each meal and the drinks and snacks you consume on the forms provided. Below are some general cooking and eating tips and an indication of which foods are best to avoid and those that are considered a low source of BPA.
Cooking and eating tips for the intervention period.
The general approach is to replace any food items that fall into the 'avoidance' category with an alternative, chosen to minimise exposure to BPA  Switch to stainless steel and glass food storage and drink containers.
 Move foods to ceramic or glass food containers before microwaving.
 Consider a coffee filter or percolator for coffee -home coffee makers (Such as Nespresso TM ) may have polycarbonate-based water tanks and phthalate-based tubing.
 Eat out less, especially at restaurants that do not use fresh ingredients.
 Avoid canned food consumption. Where possible, replace with fresh produce or cardboard or tetrapack packaged alternatives.
 Choose fresh fruits and vegetables when possible, and frozen if not.
 Soak dried beans for cooking rather than tinned. Carbonated/fizzy drinks and juices in cans. Avoid carbonated drinks in cans and drinks stored for prolonged periods in reusable sports bottles, unless they are labelled 'BPA free' (many commercial sports bottles are).
Fast food from commercial outlets. Most processed food has passed through numerous processes, and each additional processing step provides an opportunity for BPA to enter through packaging or tubing. Try to replace fast and processed foods with a freshly prepared and cooked alternative.
Packaged fruit and vegetables. Replace these where possible with unpackaged, loose fruit and vegetable items as far as possible.
Convenience/ready meals. Plastics types considered safest in terms of chemical migration are recycling numbers 2 and 5. Avoid food prepared in packaging with recycling number 7, which includes many different types of polymer and mixed polymers, including polycarbonate, a source of BPA. Try to avoid foods that are designed to be heated in the microwave in their packaging.
Chocolate and ice cream. Individuals who report eating chocolate bars and ice cream on a regular basis have been reported to have higher than average BPA exposure. Try to avoid excessive consumption.
Non-food or food packaging routes of exposure Although plastics found in consumer goods such as DVDs, CDs, computer goods and sunglasses do contain BPA, this is not an important route of exposure.
Till receipts often contain high levels of BPA, so wash your hands before eating or drinking if you have been handling them.
Dental sealants may contain BPA, so avoid any pre-planned dental work

CONSENT STATEMENTS
Thank you for reading this leaflet. If you wish to participate in this study, you will be asked to agree to the consent statements below in the presence of a member of the research team. 3. I agree to participate in this study as a research subject. This means that you agree to participate in a one-week diet and to provide two blood and urine samples.
4. I understand that my anonymised blood and urine samples and linked anonymous questionnaire data will be sent to University of Exeter Medical School, Royal Devon & Exeter Hospital and my urine sample only will be sent to the Rolvaltain laboratory, a specialist BPA analysis company. This means that laboratory staff will not know that samples belong to you, but dedicated staff at the University of Exeter, with training and experience in data protection ,will be able to link your sample data to your questionnaire data.

5.
I understand that RNA (genetic material) will be extracted from my blood and will be stored anonymously. This means that Professor Harries' team will use our RNA to provide data that you will help analyse but may also do further research on the samples to identify reasons for any changes seen.
6. I understand that data relating to my participation in the study will be returned anonymously to my school to be used for educational purposes. This means that although you will get to analyse data from your samples there is no way you will know which data relates to your samples and which to other participants.

Complaints:
If

Involvement & Engagement
The aim of this year-long project is to involve teenagers in a research study that is relevant to them, by allowing them to help design a research project, analyse non-identifiable participant data and help to present and publish the outcomes.

Participation
Students will be asked to undertake a one week diet to reduce their intake of BPA, a chemical found in plastics. They will be asked to provide urine and blood samples before and after their diet. F o r p e e r r e v i e w o n l y

What will I need to do?
This project is being run as a student-involvement project to answer two specific questions:

1.
Can we see the effects of dietary BPA on our genes?

2.
Can we effectively reduce BPA in our diet?
In the past, small-scale experiments have shown that BPA levels in the human body can be reduced by rigid dietary interventions but these interventions would be difficult to implement in the "real world". In this study a one-week dietary intervention designed by teenagers will be used by them to determine whether BPA levels, and the activity of BPA-responsive genes can be effectively reduced in young people by avoiding food packaging that contains this chemical.

What is BPA?
BPA (Bisphenol A) is a chemical used in the manufacture of plastics. Plastics containing BPA are found in a wide range of products including food and drink containers. BPA in these products can be ingested and there are concerns that high BPA levels in the blood could possibly affect human health. Research is therefore needed to understand its effects on the human body and how we can reduce its consumption by minor changes to our diet. The diet will exclude sources of BPA as much as possible but will be nutritionally and calorifically similar to your usual diet.  You will be asked to complete a food diary and answer a questionnaire about how easy it was to follow this diet.
Day 8  Provide a nurse with a 2.5ml blood sample and a urine sample.
We recommend that you discuss the project with your family and involve them in planning what you eat and how you will prepare it.
Are there any risks in taking part?
Blood samples will be taken by fully qualified and insured NHS personnel. Any potential discomfort or side-effects will be equivalent to that experienced giving a blood sample to your GP. All data will be fully anonymised before analysis. This means that you will not find out anything about your blood or urine samples. Following the diet may minimally increase the cost of your groceries for the week, but since fresh foods are usually less expensive than pre-packaged foods, we do not expect this to be an issue.

What will happen to my samples and data?
When you participate in the study you will be allocated with a numerical study ID. Your samples and data will be labelled with this number so that we can match your 'before' and 'after' diet samples with your food diary data. Once all data has been collated and coded it will be further anonymised by a person external to the project so that no data can be linked to any of the participants.
Urine samples from before and after the diet will be sent to the Royal Devon & Exeter NHS Foundation Trust for creatinine analysis and to the Rolvaltain laboratory for BPA analysis. RNA will be extracted from blood samples at the Royal Devon & Exeter Molecular Genetics Laboratory and the expression levels of two BPA-responsive genes will be measured in the samples taken before and after the diet. These anonymised RNA samples will be stored and used only by Professor Harries team for further research on the mechanisms behind our findings.

What will happen to the results of the research study?
You will be given the opportunity to help analyse anonymised data from this project and to help disseminate the outcomes of this research. It is hoped that the findings will be published in peer-reviewed journals and the wider media.

Who is organising this research?
The research is organised by Professors Lorna Harries & Tamara Galloway of the University of Exeter as part of their research program into BPA and part of the University's outreach program to involve schools in academic research.

What are the benefits of taking part?
This project will help you to understand how you might be able to reduce BPA in your diet and your involvement in the design will give you an excellent insight into clinical research, community outreach and scientific practise. Your role as a participant is unlikely to have any direct health benefits.

YES / NO
3. I agree to participate in this study as a research subject. This means that you agree to participate in a one-week diet and to provide two blood and urine samples.

YES / NO
4. I understand that my anonymised blood and urine samples and linked anonymous questionnaire data will be sent to University of Exeter Medical School, Royal Devon & Hospital and my urine sample only will be sent to the Rolvaltain laboratory, a specialist BPA analysis company. This means that laboratory staff will not know that samples belong to you, but dedicated staff at the University of Exeter, with training and experience in data protection, will be able to link your sample data to your questionnaire data.

YES / NO
5. I understand that RNA (genetic material) will be extracted from my blood and will be stored anonymously. This means that Professor Harries' team will use our RNA to provide data that you will help analyse but may also do further research on the samples to identify reasons for any changes seen.

YES / NO
6. I understand that data relating to my participation in the study will be returned anonymously to my school to be used for educational purposes. This means that although you will get to analyse data from your samples there is no way you will know which data relates to your samples and which to other participants. reclassified BPA as a chemical of very high concern due to its endocrine disrupting properties  Students were asked to minimise their intake of known sources of BPA according to a set of 156 guidelines that had been co-designed with them based on the known literature. We requested 157 that calorific intake was maintained as near to their usual diet as possible and recorded details 158 of their daily diet including all food and drink, and its associated packaging, in a self-reported 159 food diary (Supplementary information file 2). Adherence was assessed using a 'BPA risk 160 score'; each individual dietary item potentially containing BPA was given a score of 1.

161
Heavily processed items were also scored 1 per item. These scores were collated at the end of        samples were due to non-attendance of participants or non-provision of a suitable sample.

237
Samples below the limit of quantification were scored as 0.07 ng/ml (LoQ/√2).   Participants indicated that following the diet had no significant cost implications on family 271 finances, with 50% of participants reporting that it had cost more, and 50% reporting that

General instructions
The purpose of this dietary intervention trial is to follow a diet designed to minimise routes of exposure to the food packaging chemical bisphenol A (BPA). For the dietary intervention period, please follow as closely as possible the instruction given below. Try to maintain your diet during the intervention period to be as closely similar to your normal diet as possible, in terms of the content, amount and calorific value of the food you eat. Please record details of each meal and the drinks and snacks you consume on the forms provided. Below are some general cooking and eating tips and an indication of which foods are best to avoid and those that are considered a low source of BPA.
Cooking and eating tips for the intervention period.
The general approach is to replace any food items that fall into the 'avoidance' category with an alternative, chosen to minimise exposure to BPA  Switch to stainless steel and glass food storage and drink containers.
 Move foods to ceramic or glass food containers before microwaving.

Consider a coffee filter or percolator for coffee -home coffee makers (Such as Nespresso TM ) may have polycarbonate-based water tanks and phthalate-based tubing.
 Eat out less, especially at restaurants that do not use fresh ingredients.
 Avoid canned food consumption. Where possible, replace with fresh produce or cardboard or tetrapack packaged alternatives.

Choose fresh fruits and vegetables when possible, and frozen if not.
 Soak dried beans for cooking rather than tinned. Carbonated/fizzy drinks and juices in cans. Avoid carbonated drinks in cans and drinks stored for prolonged periods in reusable sports bottles, unless they are labelled 'BPA free' (many commercial sports bottles are).

Fast food from commercial outlets.
Most processed food has passed through numerous processes, and each additional processing step provides an opportunity for BPA to enter through packaging or tubing. Try to replace fast and processed foods with a freshly prepared and cooked alternative.
Packaged fruit and vegetables. Replace these where possible with unpackaged, loose fruit and vegetable items as far as possible.
Convenience/ready meals. Plastics types considered safest in terms of chemical migration are recycling numbers 2 and 5. Avoid food prepared in packaging with recycling number 7, which includes many different types of polymer and mixed polymers, including polycarbonate, a source of BPA. Try to avoid foods that are designed to be heated in the microwave in their packaging.
Chocolate and ice cream. Individuals who report eating chocolate bars and ice cream on a regular basis have been reported to have higher than average BPA exposure. Try to avoid excessive consumption.
Non-food or food packaging routes of exposure Although plastics found in consumer goods such as DVDs, CDs, computer goods and sunglasses do contain BPA, this is not an important route of exposure.
Till receipts often contain high levels of BPA, so wash your hands before eating or drinking if you have been handling them.
Dental sealants may contain BPA, so avoid any pre-planned dental work

If you heated your food in a microwave, what was the food in? Tick any which apply and give indication of frequency.
A food storage container or bowl known or suspected to contain BPA  times

When you or your family drank water, where did your water come from? Tick any which apply and give indication of frequency.
Plastic filter jug known or suspected to contain BPA  times Individual water bottle known or suspected to contain BPA  times Larger water container known or suspected to contain BPA  times 4. How many times during the week did you eat food that had been stored or transported in plastic containers known or suspected to contain BPA? 5. How many times during the week did you eat tinned food or drink from cans?

CONSENT STATEMENTS
Thank you for reading this leaflet. If you wish to participate in this study, you will be asked to agree to the consent statements below in the presence of a member of the research team. 3. I agree to participate in this study as a research subject. This means that you agree to participate in a one-week diet and to provide two blood and urine samples.

I understand that my anonymised blood and urine samples and linked
anonymous questionnaire data will be sent to University of Exeter Medical School, Royal Devon & Exeter Hospital and my urine sample only will be sent to the Rolvaltain laboratory, a specialist BPA analysis company. This means that laboratory staff will not know that samples belong to you, but dedicated staff at the University of Exeter, with training and experience in data protection ,will be able to link your sample data to your questionnaire data.

5.
I understand that RNA (genetic material) will be extracted from my blood and will be stored anonymously. This means that Professor Harries' team will use our RNA to provide data that you will help analyse but may also do further research on the samples to identify reasons for any changes seen.
6. I understand that data relating to my participation in the study will be returned anonymously to my school to be used for educational purposes. This means that although you will get to analyse data from your samples there is no way you will know which data relates to your samples and which to other participants.

Complaints:
If you have any complaints about the way in which this study has been carried out please contact the Chair of the

Involvement & Engagement
The aim of this year-long project is to involve teenagers in a research study that is relevant to them, by allowing them to help design a research project, analyse non-identifiable participant data and help to present and publish the outcomes.

Participation
Students will be asked to undertake a one week diet to reduce their intake of BPA, a chemical found in plastics. They will be asked to provide urine and blood samples before and after their diet.

What will I need to do?
This project is being run as a student-involvement project to answer two specific questions:

1.
Can we see the effects of dietary BPA on our genes?

Can we effectively reduce BPA in our diet?
In the past, small-scale experiments have shown that BPA levels in the human body can be reduced by rigid dietary interventions but these interventions would be difficult to implement in the "real world". In this study a one-week dietary intervention designed by teenagers will be used by them to determine whether BPA levels, and the activity of BPA-responsive genes can be effectively reduced in young people by avoiding food packaging that contains this chemical.

What is BPA?
BPA (Bisphenol A) is a chemical used in the manufacture of plastics. Plastics containing BPA are found in a wide range of products including food and drink containers. BPA in these products can be ingested and there are concerns that high BPA levels in the blood could possibly affect human health. Research is therefore needed to understand its effects on the human body and how we can reduce its consumption by minor changes to our diet. The diet will exclude sources of BPA as much as possible but will be nutritionally and calorifically similar to your usual diet.  You will be asked to complete a food diary and answer a questionnaire about how easy it was to follow this diet.

Day 8
 Provide a nurse with a 2.5ml blood sample and a urine sample.
We recommend that you discuss the project with your family and involve them in planning what you eat and how you will prepare it.

Are there any risks in taking part?
Blood samples will be taken by fully qualified and insured NHS personnel. Any potential discomfort or side-effects will be equivalent to that experienced giving a blood sample to your GP. All data will be fully anonymised before analysis. This means that you will not find out anything about your blood or urine samples. Following the diet may minimally increase the cost of your groceries for the week, but since fresh foods are usually less expensive than pre-packaged foods, we do not expect this to be an issue.

What will happen to my samples and data?
When you participate in the study you will be allocated with a numerical study ID. Your samples and data will be labelled with this number so that we can match your 'before' and 'after' diet samples with your food diary data. Once all data has been collated and coded it will be further anonymised by a person external to the project so that no data can be linked to any of the participants.
Urine samples from before and after the diet will be sent to the Royal Devon & Exeter NHS Foundation Trust for creatinine analysis and to the Rolvaltain laboratory for BPA analysis. RNA will be extracted from blood samples at the Royal Devon & Exeter Molecular Genetics Laboratory and the expression levels of two BPA-responsive genes will be measured in the samples taken before and after the diet. These anonymised RNA samples will be stored and used only by Professor Harries team for further research on the mechanisms behind our findings.

What will happen to the results of the research study?
You will be given the opportunity to help analyse anonymised data from this project and to help disseminate the outcomes of this research. It is hoped that the findings will be published in peer-reviewed journals and the wider media.

Who is organising this research?
The research is organised by Professors Lorna Harries & Tamara Galloway of the University of Exeter as part of their research program into BPA and part of the University's outreach program to involve schools in academic research.

What are the benefits of taking part?
This project will help you to understand how you might be able to reduce BPA in your diet and your involvement in the design will give you an excellent insight into clinical research, community outreach and scientific practise. Your role as a participant is unlikely to have any direct health benefits.

YES / NO
2. I understand that my participation is voluntary and that I am free to withdraw at any time without giving any reason, without right to participate in the rest of the study being affected. This means that even if you helped design this study you do not have to be a participant and you should not feel under any pressure to participate.

YES / NO
3. I agree to participate in this study as a research subject. This means that you agree to participate in a one-week diet and to provide two blood and urine samples.

YES / NO
4. I understand that my anonymised blood and urine samples and linked anonymous questionnaire data will be sent to University of Exeter Medical School, Royal Devon & Hospital and my urine sample only will be sent to the Rolvaltain laboratory, a specialist BPA analysis company. This means that laboratory staff will not know that samples belong to you, but dedicated staff at the University of Exeter, with training and experience in data protection, will be able to link your sample data to your questionnaire data.

YES / NO
5. I understand that RNA (genetic material) will be extracted from my blood and will be stored anonymously. This means that Professor Harries' team will use our RNA to provide data that you will help analyse but may also do further research on the samples to identify reasons for any changes seen.

Results
Participants 13* (a) Report numbers of individuals at each stage of study-eg numbers potentially eligible, examined for eligibility, confirmed eligible, included in the study, completing follow-up, and analysed Table 1 (b) Give reasons for non-participation at each stage  (c) Cohort study-Summarise follow-up time (eg, average and total amount)

48
Information on packaging and food/drink ingested was used to calculate a BPA risk score for 49 anticipated exposure. A qualitative analysis was carried out to identify themes addressing 50 long term sustainability of the diet.  1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46  47  48  49  50  51  52  53  54  55  56  57  58  59  We found no evidence in this self-administered intervention study that it was possible to 60 moderate BPA exposure by diet in a real world setting. Furthermore, our study participants 61 indicated that they would be unlikely to sustain such a diet long term, due to the difficulty in 62 identifying BPA-free foods. Students were asked to minimise their intake of known sources of BPA according to a set of 156 guidelines that had been co-designed with them based on the known literature. We requested 157 that calorific intake was maintained as near to their usual diet as possible and recorded details 158 of their daily diet including all food and drink, and its associated packaging, in a self-reported 159 food diary (Supplementary information file 2). Adherence was assessed using a 'BPA risk 160 score'; each individual dietary item potentially containing BPA was given a score of 1.

161
Heavily processed items were also scored 1 per item. These scores were collated at the end of     samples were due to non-attendance of participants or non-provision of a suitable sample.

General instructions
The purpose of this dietary intervention trial is to follow a diet designed to minimise routes of exposure to the food packaging chemical bisphenol A (BPA). For the dietary intervention period, please follow as closely as possible the instruction given below. Try to maintain your diet during the intervention period to be as closely similar to your normal diet as possible, in terms of the content, amount and calorific value of the food you eat. Please record details of each meal and the drinks and snacks you consume on the forms provided. Below are some general cooking and eating tips and an indication of which foods are best to avoid and those that are considered a low source of BPA.
Cooking and eating tips for the intervention period.
The general approach is to replace any food items that fall into the 'avoidance' category with an alternative, chosen to minimise exposure to BPA  Switch to stainless steel and glass food storage and drink containers.
 Move foods to ceramic or glass food containers before microwaving.
 Consider a coffee filter or percolator for coffee -home coffee makers (Such as Nespresso TM ) may have polycarbonate-based water tanks and phthalate-based tubing.
 Eat out less, especially at restaurants that do not use fresh ingredients.
 Avoid canned food consumption. Where possible, replace with fresh produce or cardboard or tetrapack packaged alternatives.
 Choose fresh fruits and vegetables when possible, and frozen if not.
 Soak dried beans for cooking rather than tinned. Carbonated/fizzy drinks and juices in cans. Avoid carbonated drinks in cans and drinks stored for prolonged periods in reusable sports bottles, unless they are labelled 'BPA free' (many commercial sports bottles are).
Fast food from commercial outlets. Most processed food has passed through numerous processes, and each additional processing step provides an opportunity for BPA to enter through packaging or tubing. Try to replace fast and processed foods with a freshly prepared and cooked alternative.
Packaged fruit and vegetables. Replace these where possible with unpackaged, loose fruit and vegetable items as far as possible.
Convenience/ready meals. Plastics types considered safest in terms of chemical migration are recycling numbers 2 and 5. Avoid food prepared in packaging with recycling number 7, which includes many different types of polymer and mixed polymers, including polycarbonate, a source of BPA. Try to avoid foods that are designed to be heated in the microwave in their packaging.
Chocolate and ice cream. Individuals who report eating chocolate bars and ice cream on a regular basis have been reported to have higher than average BPA exposure. Try to avoid excessive consumption.
Non-food or food packaging routes of exposure Although plastics found in consumer goods such as DVDs, CDs, computer goods and sunglasses do contain BPA, this is not an important route of exposure.
Till receipts often contain high levels of BPA, so wash your hands before eating or drinking if you have been handling them.
Dental sealants may contain BPA, so avoid any pre-planned dental work A score of 1 is given to each item containing suspected to contain BPA or be packaged in BPA-containing materials. Highly processed foods are also scored as 1, due to uncertainties in the processing procedures. The daily totals are summed to produce a BPA risk score for the 7 day intervention. A food storage container or bowl known or suspected to contain BPA  times 3. When you or your family drank water, where did your water come from? Tick any which apply and give indication of frequency.

Item
Plastic filter jug known or suspected to contain BPA  times Individual water bottle known or suspected to contain BPA  times Larger water container known or suspected to contain BPA  times 4. How many times during the week did you eat food that had been stored or transported in plastic containers known or suspected to contain BPA? 5. How many times during the week did you eat tinned food or drink from cans? 6. Did the BPA reduced diet affect How much you spent on shopping?

CONSENT STATEMENTS
Thank you for reading this leaflet. If you wish to participate in this study, you will be asked to agree to the consent statements below in the presence of a member of the research team. 3. I agree to participate in this study as a research subject. This means that you agree to participate in a one-week diet and to provide two blood and urine samples.
4. I understand that my anonymised blood and urine samples and linked anonymous questionnaire data will be sent to University of Exeter Medical School, Royal Devon & Exeter Hospital and my urine sample only will be sent to the Rolvaltain laboratory, a specialist BPA analysis company. This means that laboratory staff will not know that samples belong to you, but dedicated staff at the University of Exeter, with training and experience in data protection ,will be able to link your sample data to your questionnaire data.

5.
I understand that RNA (genetic material) will be extracted from my blood and will be stored anonymously. This means that Professor Harries' team will use our RNA to provide data that you will help analyse but may also do further research on the samples to identify reasons for any changes seen.
6. I understand that data relating to my participation in the study will be returned anonymously to my school to be used for educational purposes. This means that although you will get to analyse data from your samples there is no way you will know which data relates to your samples and which to other participants.

Complaints:
If you have any complaints about the way in which this study has been carried out please contact the Chair of the

Involvement & Engagement
The aim of this year-long project is to involve teenagers in a research study that is relevant to them, by allowing them to help design a research project, analyse non-identifiable participant data and help to present and publish the outcomes.

Participation
Students will be asked to undertake a one week diet to reduce their intake of BPA, a chemical found in plastics. They will be asked to provide urine and blood samples before and after their diet.  1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46 F o r p e e r r e v i e w o n l y

What will I need to do?
This project is being run as a student-involvement project to answer two specific questions:

1.
Can we see the effects of dietary BPA on our genes?

2.
Can we effectively reduce BPA in our diet?
In the past, small-scale experiments have shown that BPA levels in the human body can be reduced by rigid dietary interventions but these interventions would be difficult to implement in the "real world". In this study a one-week dietary intervention designed by teenagers will be used by them to determine whether BPA levels, and the activity of BPA-responsive genes can be effectively reduced in young people by avoiding food packaging that contains this chemical.

What is BPA?
BPA (Bisphenol A) is a chemical used in the manufacture of plastics. Plastics containing BPA are found in a wide range of products including food and drink containers. BPA in these products can be ingested and there are concerns that high BPA levels in the blood could possibly affect human health. Research is therefore needed to understand its effects on the human body and how we can reduce its consumption by minor changes to our diet. The diet will exclude sources of BPA as much as possible but will be nutritionally and calorifically similar to your usual diet.  You will be asked to complete a food diary and answer a questionnaire about how easy it was to follow this diet.
Day 8  Provide a nurse with a 2.5ml blood sample and a urine sample.
We recommend that you discuss the project with your family and involve them in planning what you eat and how you will prepare it.
Are there any risks in taking part?
Blood samples will be taken by fully qualified and insured NHS personnel. Any potential discomfort or side-effects will be equivalent to that experienced giving a blood sample to your GP. All data will be fully anonymised before analysis. This means that you will not find out anything about your blood or urine samples. Following the diet may minimally increase the cost of your groceries for the week, but since fresh foods are usually less expensive than pre-packaged foods, we do not expect this to be an issue.

What will happen to my samples and data?
When you participate in the study you will be allocated with a numerical study ID. Your samples and data will be labelled with this number so that we can match your 'before' and 'after' diet samples with your food diary data. Once all data has been collated and coded it will be further anonymised by a person external to the project so that no data can be linked to any of the participants.
Urine samples from before and after the diet will be sent to the Royal Devon & Exeter NHS Foundation Trust for creatinine analysis and to the Rolvaltain laboratory for BPA analysis. RNA will be extracted from blood samples at the Royal Devon & Exeter Molecular Genetics Laboratory and the expression levels of two BPA-responsive genes will be measured in the samples taken before and after the diet. These anonymised RNA samples will be stored and used only by Professor Harries team for further research on the mechanisms behind our findings.

What will happen to the results of the research study?
You will be given the opportunity to help analyse anonymised data from this project and to help disseminate the outcomes of this research. It is hoped that the findings will be published in peer-reviewed journals and the wider media.

Who is organising this research?
The research is organised by Professors Lorna Harries & Tamara Galloway of the University of Exeter as part of their research program into BPA and part of the University's outreach program to involve schools in academic research.

What are the benefits of taking part?
This project will help you to understand how you might be able to reduce BPA in your diet and your involvement in the design will give you an excellent insight into clinical research, community outreach and scientific practise. Your role as a participant is unlikely to have any direct health benefits.

YES / NO
3. I agree to participate in this study as a research subject. This means that you agree to participate in a one-week diet and to provide two blood and urine samples.

YES / NO
4. I understand that my anonymised blood and urine samples and linked anonymous questionnaire data will be sent to University of Exeter Medical School, Royal Devon & Hospital and my urine sample only will be sent to the Rolvaltain laboratory, a specialist BPA analysis company. This means that laboratory staff will not know that samples belong to you, but dedicated staff at the University of Exeter, with training and experience in data protection, will be able to link your sample data to your questionnaire data.

YES / NO
5. I understand that RNA (genetic material) will be extracted from my blood and will be stored anonymously. This means that Professor Harries' team will use our RNA to provide data that you will help analyse but may also do further research on the samples to identify reasons for any changes seen.

YES / NO
6. I understand that data relating to my participation in the study will be returned anonymously to my school to be used for educational purposes. This means that although you will get to analyse data from your samples there is no way you will know which data relates to your samples and which to other participants.  1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46  47  48  49  50  51  52  53  54  55  56  57  58  59  Case-control study-Give the eligibility criteria, and the sources and methods of case ascertainment and control selection. Give the rationale for the choice of cases and controls

YES / NO
Cross-sectional study-Give the eligibility criteria, and the sources and methods of selection of participants

Results
Participants 13* (a) Report numbers of individuals at each stage of study-eg numbers potentially eligible, examined for eligibility, confirmed eligible, included in the study, completing follow-up, and analysed (c) Cohort study-Summarise follow-up time (eg, average and total amount)