EXPERT OPINION OF BLANCA LAFFON, PhD

In the Matter of an Arbitration under the Rules of the

United Nations Commission on International Trade Law

Chevron Corporation and Texaco Petroleum Company vs.

The Republic of Ecuador, PCA Case No. 2009-23

November 7, 2014

Prepared for Winston & Strawn LLP

1700 K Street N.W. Washington DC 20006-3817

Prepared by

Prof. Blanca Laffon, PhD

Location A Coruña, Spain

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EXPERT OPINION OF BLANCA LAFFON, PhD

CONTENTS

1. Executive Summary ............................................................................................................... 1 

1.1. Personal qualifications and experience ............................................................................ 1 

1. 2. Summary of scope of retention ....................................................................................... 2 

1.3. Summary of opinions ....................................................................................................... 2 

2. Bases of opinions ................................................................................................................... 3 

2.1. Background information regarding the presence of contamination in El Oriente region 3 

2.2. Similarity between exposure in the Concession Area and exposure to oil spills ............ 4 

2.3. Acute health effects reported in populations exposed to oil spills .................................. 5 

2.4. Alterations in the genetic material are in the origin of cancer development ................... 7 

2.5. Genotoxicity tests and cancer risk ................................................................................... 9 

2.6. Genotoxicity studies in people exposed to oil spills ...................................................... 13 

2.7. Immune and endocrine toxicity studies in people exposed to oil spills ........................ 16 

2.8. Miscellanea: IARC classification of crude oil ............................................................... 17 

3. References ............................................................................................................................ 19 

Appendix A – Epidemiological studies on acute toxic effects related to exposure to oil spills

Appendix B – Epidemiological studies on genotoxicity, immunotoxicity and endocrine toxicity, and studies on potential toxicological assessment, related to exposure to oil spills

Appendix C – Curriculum vitae

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1. Executive Summary

1.1. Personal qualifications and experience

I am an Associate Professor of Psychobiology at the University of A Coruña (Spain), and I have been accredited by ANECA (Spanish National Agency for Evaluation of Quality and Accreditation) as full Professor (meaning that I have earned enough merits for that category) since February 2014.

I obtained my B.S. in Pharmacy from the University of Santiago de Compostela, Spain, with honors and extraordinary award, in 1996, and my Ph.D. in Pharmacy from the same University, also with honors and extraordinary award, in 2001. Following several postgraduate university fellowships, including 23 months at the Portugal National Institute of Health (Department of Environmental Health), I became an Associate Professor at the University of A Coruña in December 2008. After completing my doctorate, I conducted additional postgraduate studies in the fields of genomics, proteomics and bioinformatics (2002), and genetic and molecular epidemiology (2006).

My research interest is focused on the effects of pollutants on organisms, especially at the molecular and cytogenetic levels, by conducting in vitro, in vivo and human epidemiological studies aimed to evaluate the genotoxicity and cytotoxicity associated with exposure to environmental or occupational contaminants. Genotoxicity studies adverse effects on genetic material. Cytotoxicity studies adverse effects at the cellular level, specifically on the cell cycle and viability.

In this context, I have conducted (with my research group) a complete biomonitoring study of people exposed to the Prestige oil tanker spill, which occurred off the coast of Galicia (Northwest of Spain) in November 2002. The primary objective was to evaluate the possible damage to the genetic material (genotoxicity) in people exposed to this oil as a consequence of participating in the cleanup operations. We also determined other markers of endocrinologic toxicity (hormones indicating psychophysiological stress), and of immunologic toxicity (several parameters indicating alterations in the immune system, which is closely connected to the endocrine and nervous systems). In these studies we identified significant alterations that my team and I detailed in our published articles.

We completed a follow-up assessment of the alterations observed seven years after the workers were initially exposed. As a result of these studies, and the related papers published in international journals, the Institute of Medicine of the National Academies invited me to participate as an advisor in the Workshop assessing the human health effects of the Gulf of Mexico oil spill, held in New Orleans (LA-USA) in June 2010 (2 months after the Deepwater Horizon BP platform accident).

I have led or participated in over a dozen research projects, supported by grants from the Galician and Spanish Ministries of Science and the European Commission. The results of these investigations were published in over 90 scientific articles and book chapters I authored or co-authored (more than 1,000 citations received, h-index = 20 according to Scopus

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database), and a great part of them focused on genotoxic effects associated with exposures to potentially toxic agents (in vitro and human population studies). Six of my research works were awarded scientific prizes from different private and public entities. Additionally, I am Associate Editor of several indexed peer-reviewed scientific journals, and I serve as a journal peer-reviewer for over thirty journals and as a research project peer-reviewer for public institutions from different countries.

My academic activities include teaching Genetic Toxicology, Environmental Toxicology and Public Health, Psychopharmacology, and Congenital Alterations of Language in different Degrees and Masters, and supervising research works. In the past 12 years, I have supervised 8 Ph.D. theses, 3 Master theses and 7 Honors Degrees. I have provided a detailed CV in Appendix C.

1.2. Summary of scope of retention

I was retained in August 2014 by Winston & Strawn LLP to provide my expert opinion regarding the health impacts of petroleum in conjunction with the Bilateral Investment Treaty (BIT) arbitration between Chevron Corp. and the Republic of Ecuador.

1.3. Summary of opinions

1. The closest exposure situations to the one present in the Concession Area that have been the subject of genotoxicity research are those experienced as a consequence of major marine spills of crude oils or fuel oils by residents and workers who participated in the cleanup tasks.

2. Most investigations carried out after oil spill accidents are cross-sectional epidemiological studies that analyze acute physical effects or psychological consequences in the exposed population: cleanup workers or residents. Data obtained in these studies indicate that people exposed to oil spills experience acute physical consequences, including upper respiratory tract illnesses, headaches, nausea, vomiting, and more.

3. Although classical epidemiological studies are very useful for establishing causal relationships between an exposure and an adverse health outcome, molecular epidemiology studies using genotoxicity biomarkers (indicative of damages in the genetic material) are an important tool by which to assess cancer risk in people exposed to occupational or environmental carcinogens. Genotoxicity biomarkers provide early and reliable warning signals of cancer risk.

4. My molecular epidemiology studies of the genotoxic effects in people exposed to the Prestige oil spill as a consequence of their participation in the cleanup operations indicate that this exposure induced DNA damage. That damage became fixed as chromosome alterations, thus increasing the risk of cancer development, after only several months of exposure.

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Additional studies carried out two years after the exposure to Prestige oil detected that a higher proportion of exposed participants had structural chromosomal alterations, which seemed to increase with intensity of exposure.

The most recent Prestige study to be carried out examined individuals seven years after they were exposed to the oil for a mean of 9 months (range 2-10 months). While this study suggests that a prolonged period of non-exposure to oil might lead to the removal of DNA damage induced by the exposure, the plasma cortisol levels and percentage of natural killer cells continued to be significantly altered in the population that was previously exposed, notwithstanding that this population had been free from exposure for seven years. These alterations to the exposed population’s immunological and endocrine systems lead to an increased risk for developing cancer and/or other diseases. Accordingly, the study recommended periodic health monitoring for those people who were exposed to the Prestige oil spill.

5. Taken together these studies show that the exposed population in El Oriente is at risk for developing health problems, including in particular, cancer. Unlike the populations who were exposed to marine oil spills for mere months, the Ecuadorians living in the Concession Area have been exposed to oil for decades and continue to be exposed even today. Additionally, the people who engaged in the cleanup of the oil spills usually wore protective gear, meaning their exposure pathways were limited mostly to inhalation. In contrast, the people living in El Oriente have been exposed to oil through various pathways and they do not wear protective clothing.

These opinions are given to a reasonable degree of scientific probability. They are based on my education, training, experience, information and data available in the scientific literature, and information and data about this lawsuit made available to me at the time these opinions were formulated. If additional information becomes available, I may supplement my opinion to reflect such additional information.

The bases for these opinions are provided in this report. The documents I relied upon to reach these opinions are cited in the document and listed in the references section at the end of the report.

2. Bases of opinions

2.1. Background information regarding the presence of contamination in El Oriente region

Oil extraction and production operations in the Ecuadorian Concession Area involved petroleum exploration surveys, drilling exploration and production wells, processing crude oil at the wellhead or production facility, maintenance activities, and transporting oil via pipelines. According to the Louis Berger Group (LBG) report, “these operations resulted in the uncontrolled release of waste materials and byproducts into the air, surface water, stream sediment groundwater, and soil. Materials released included crude oil, drilling mud, formation

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(produced) water, cleaning solvents, diesel fuel, sanitary wastes, burned and unburned flare gases, and diesel exhaust”, containing hazardous and toxic chemicals (December 2013 LBG Expert Report at 47). Many of these chemicals are persistent in the environment.

Extensive data collected by Dr. Harlee Strauss in her opinions show that, as a consequence of oil extraction and production operations, adults and children residing in the Concession Area have been exposed to toxic and hazardous contaminants via multiple exposure pathways (ingestion and dermal exposures) through diverse activities. (December 2013 Strauss Report at 5). These exposures to contaminated environmental media have been nearly continuous during the time that individuals lived in the vicinity of the facilities, often counted in many years. Unlike occupational exposures, there have been no recovery periods from the exposures (nights/weekends/vacations), and vulnerable groups such as the very young, fetuses, elderly, and the infirm are also part of the exposed population. (December 2013 Strauss Report at 5). In this regard, as pointed out by Goldstein in regards to the Gulf of Mexico oil spill, “children are at particular risk for effects from environmental exposures” since, “[a]s compared with adults, they breathe in more air per unit of body mass, their bodies detoxify many chemicals less effectively, and they explore more adventurously” (Goldstein, 2011 at 1339). In addition, “[t]here is inadequate information about the potential reproductive and developmental effects of crude-oil components”, thus “[p]regnant women should particularly avoid dermal contact with oil and should avoid areas with visible oil contamination or odors” (Goldstein, 2011 at 1339).

As demonstrated in the LBG rejoinder report, such contamination in the Concession Area is still present and widespread; even some of the sites included in Texaco Petroleum’s remediation plan continue to be a persistent source of environmental contamination. Therefore, some exposure is on-going.

2.2. Similarity between exposure in the Concession Area and exposure to oil spills

As already stated by Dr. Grandjean, perhaps the closest exposure situations to the one present in the Concession Area are those experienced as a consequence of major marine oil spills by residents and workers who participated in the cleanup tasks (Grandjean Report (Nov. 22, 2013) at 5). For some of these accidents (9 out of 40 major oil spills), studies on effects of exposure to diverse aspects of human health have been performed. In 6 of these accidents (Exxon Valdez, MV Braer, Sea Empress, Tasman Spirit and Hebei Spirit tankers, and Deepwater Horizon platform), the spill consisted of crude oil; in the 3 other cases, the spill was caused by fuel oil No. 6 (also named bunker C) (Nakhodka, Erika and Prestige). I conducted several studies following the Prestige spill, detailed below in this report.

Crude oil is a complex combination of hydrocarbons consisting predominantly of paraffinic (straight and branched-chain alkanes), naphthenic (cycloalkanes or cycloparaffins), and aromatic hydrocarbons (API, 2011 at 5). Sulfur, oxygen and nitrogen compounds, organometallic complexes notably of nickel and vanadium, and dissolved gases, such as

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hydrogen sulfide, are also found in crude oil. Similar hydrocarbons, heterocyclics, metals and other constituents, e.g., hydrogen sulfide, are present in all crude oils but their proportions vary depending on the crude source (API, 2011 at 5). Fuel oils are produced from crude petroleum by different refining processes, depending on their intended use, and are composed of complex and variable mixtures of aliphatic (alkanes, alkenes, cycloalkanes) and aromatic hydrocarbons, containing low percentages of sulfur, nitrogen, and oxygen compounds (Laffon, 2014 at 667). The exact chemical composition of each of the fuel oils may vary somewhat, depending on the source, the refinery involved, the presence of additives or modifiers, and other factors (Laffon, 2014 at 667).

Dr. Jeffrey Short has compared the crude oil produced in the Oriente to the Prestige fuel oil and has concluded that the two share similar suites of toxic compounds. (Short Expert Report (Nov. 7, 2014) at Section 4.6).

2.3. Acute health effects reported in populations exposed to oil spills

Most investigations carried out in human populations after oil spill accidents are cross-sectional epidemiological studies that analyze acute physical effects or psychological consequences in the exposed people: cleanup workers or residents. Data obtained in these studies, reviewed in Aguilera et al. (2010) and discussed in Dr. Strauss’ first report (Strauss Report (Feb. 18, 2013) at 28-31), indicate that people exposed to oil spills experience acute physical consequences, including upper respiratory tract illnesses, throat and eye irritation, headaches, dizziness, nausea, and vomiting. These studies concluded that, although respiratory symptoms are long-lasting, these consequences generally diminish with time once exposure has ceased (Aguilera, 2010 at 297-98).

Additional studies published recently, and therefore not included in the Strauss and Aguilera reviews, also support these general conclusions, the results of which are described briefly in the following paragraphs. Summarized data of all studies published so far on acute toxic effects in people exposed to oil spills are presented in Appendix A.

New studies regarding Prestige oil-exposed populations showed persistent respiratory symptoms in fishermen two years after the exposure (Rodríguez-Trigo et al., 2010 at 489-90), and a higher prevalence of lower respiratory tract symptoms five years after cleanup in the exposed fishermen than in the controls (Zock et al., 2012 at 508). Six years after exposure, the Zock data indicated the persistence of objectively measured indices of respiratory health impairment in cleanup workers1 (Zock et al., 2014).

After the Tasman Spirit disaster, Meo et al. (2009a) noted higher rates of health complaints like eye irritations, respiratory problems, headaches, nauseas, and general illness in oil-exposed individuals. A significant reduction in lung function parameters was observed in those subjects exposed for more than 15 days (Meo et al., 2009b).                                                             1 The authors recognized that they could not formally demonstrate that this persistence was due to exposure because of limitations in the study design (mainly related to the selection of the control population). (Zock et al., 2014).

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Regarding the Hebei Spirit spill, significantly increased risks of several physical symptoms like headache, nausea, dizziness, fatigue, tingling of limb, hot flushing, sore throat, cough, runny nose, shortness of breath, itchy skin, rash, and sore eyes were observed in residents from the heavy and moderately oil soaked areas as compared with residents from light soaked areas (Lee et al., 2010 at 170). Children who lived closest to the oil spill area showed higher respiratory effects (Jung et al., 2013 at 367-68). In a questionnaire study, the scientists observed that more frequent and greater exposure in people engaged in cleanup was strongly associated with a higher occurrence of acute symptoms (Sim et al, 2010 at 51). Another study reported a similar result, showing associations in residents between physical symptoms and exposure levels by evaluating urinary metabolites of volatile organic compounds (“VOC”), polycyclic aromatic hydrocarbons (“PAH”) and heavy metals (Cheong et al., 2011 at 3-5). Furthermore, longer cleanup work in volunteers was also associated with an increase in symptoms such as visual disturbance, nasal and bronchus irritation, headaches, heart palpitations, fatigue and fever, memory and cognitive disturbance, and abdominal pain (Ha et al., 2012 at 169). The first study to quantify the burden of disease (BOD) due to an oil spill, which is “necessary to assess the scale of health damage at the population level as well as the associated compensation costs,” (Kim et al., 2013 at 2) found that the BOD for 1 year for the residents living near contaminated coastal areas was significant and related to proximity to the spill (Kim et al., 2013 at 2). The Kim study also found that for persons who participated in cleanup efforts, asthma and post-traumatic stress disorder comprised the most prominent disease burden in the contaminated areas. One year after the Hebei Spirit accident, eye symptoms, headaches, skin symptoms, and neurovestibular symptoms had a longer duration in people involved with the cleanup efforts than did back pain or respiratory symptoms (Na et al., 2012 at 1251).

Many studies focused on the health effects of oil on human populations following the Deepwater Horizon platform disaster in the Gulf of Mexico. Over one-third of children experienced either physical symptoms or mental health distress, as reported by their parents (Abramson, 2010 at 4). Additionally, significant alterations were observed in several clinical parameters such as platelet counts, hemoglobin levels, hematocrit, and a number of liver enzymes in subjects participating in the cleanup activity when compared to the controls (D’Andrea and Reddy, 2013 at 967). Data obtained in the same exposed population indicated that they are at risk of developing alterations in their hematological profile and liver function (D’Andrea and Reddy, 2014 at 866e.12).

Dr. Moolgavkar in his May 2013 expert report for Chevron (pages 17-18) manifested his concern that “many of the oil spill studies that Dr. Strauss cites (page 29 of her report) lack appropriate comparison populations to determine whether the observed health symptoms are in excess of expectation. Nearly all of these studies evaluated nonspecific self-reported health symptoms, with a high probability of recall bias.” Nevertheless, the newly published studies on health effects related to exposure to oil spills often include a comparison group: either control groups (Rodríguez-Trigo et al., 2010; Zock et al., 2012 and 2014; Meo et al., 2009a and b; Cheong et al., 2011; D’Andrea and Reddy, 2013), lightly exposed individuals

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(Lee et al., 2010), or the same individuals before the exposure started (Ha et al., 2012). Moreover, although some of these studies evaluated self-reported health symptoms, others were based on analysis of objective and specific clinical parameters, such as respiratory parameters (e.g., forced spirometry, methacoline challenge, markers of oxidative stress, airway inflammation and growth factor activity in exhaled breath condensate, and the skin prick test for common inhalant allergens) (Rodríguez-Trigo et al., 2010; Zock et al., 2014; Meo et al., 2009b; Jung et al., 2013), or hematological parameters (e.g., white blood cell and platelets counts, hemoglobin, hematocrit, blood urea nitrogen, creatinine, liver enzymes) (D’Andrea and Reddy, 2013 and 2014).

Thus, previous and recently published studies provide evidence sufficient to establish the relationship between exposure to oil spills and the development of acute physical effects in the exposed individuals. As pointed out by Levy and Nassetta in their review article, “these studies found that cleanup workers and community residents who were exposed more intensively and/or for longer periods of time tended to have a higher frequency of acute symptoms” (Levy and Nasetta, 2011 at 162).

The health effects reported in all of the studies discussed above in populations exposed to oil spills are similar to the ones reported in the Concession Area communities, extensively reviewed in Dr. Strauss’ report. They also provide further support to her opinion that symptoms and pathologies described in individuals exposed to crude oil and residues from El Oriente extraction and production activities are consistent with effects reported for exposure to oil spills.

2.4. Alterations in the genetic material are in the origin of cancer development

There are several epidemiological studies referenced in the expert reports filed in this litigation that discuss the likelihood of a causal connection between exposure to oil and cancer (Grandjean, Strauss, Moolgavkar). Cancer is one of the most complex diseases affecting humans; it remains a major chronic health problem associated with toxicological substances (Barret, 1993). The cause-effect relationship that represents the basis of the pathological investigation is not easy to apply to the process of human carcinogenesis. Most of the population is exposed to a variety of human carcinogens in their daily life, yet only a small fraction of exposed individuals actually develop cancer (Carbone and Pass, 2004 at 400). Since fewer than 10% of all cancers are hereditary, and cancers caused by infection are thought to constitute some 15% of the non-hereditary cancers, the 70% to 80% remaining are called “sporadic,” because they are essentially of unknown etiology (Brucher and Jamall, 2014 at 2). They are probably related to exposure to chemical and physical agents with carcinogenic potential. Agents present in food, tobacco smoke, occupational environments, alcohol, urban pollution, medicine and medical procedures, and industrial products have been under investigation for at least three decades (Doll and Peto, 1981), and evidence of their carcinogenesis has now been obtained for many of these agents (reviewed in Clapp et al., 2008; Irigay et al., 2007).

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Cancer is today recognized as a highly heterogeneous disease: more than 100 distinct types of human cancer have been described, and various tumor subtypes can be found within specific organs (Grizzi and Chiriva-Internati, 2004). Because all cancers share the properties of uncontrolled growth, invasion, and metastasis, a common mechanism for their origin has often been suggested (Couch, 1996 at 136).

The association between genetic alterations and human cancer was first observed decades ago and explained in Theodor Boveri’s somatic mutation theory of cancer (Balmain, 2001 at 77), which states that a tumor can arise by self-proliferation from a cell that has been transformed by acquired modification of its genetic material. A causal association between genetic alterations and cancer is supported by extensive experimental and epidemiological data (Dixon and Kopras, 2004 at 441), proving that Boveri’s theory is as sound and correct as any scientific theory ever can be (Heim, 2014 at 138). Thus, somatic gene mutations are widely accepted as the basic event in the conversion of a normal cell into a cancer cell: carcinogens interact with DNA resulting in irreversible changes, which predispose the cells to malignant transformation.

It is generally accepted that chemical carcinogenesis is a multistep process, each step corresponding to a genetic event in a cell which provides the cell with a selective advantage in terms of survival and/or proliferation (Monier, 2000 at 603-604). The final risk of cancer development is a function of the combined probabilities of relatively rare events occurring in each stage (Franco et al., 2004 at 415). Extensive experimental observations in chemical carcinogenesis have demonstrated this process can be separated operationally into three general stages, i.e., initiation, promotion, and progression, through which a normal cell evolves into a cancer cell as the result of heritable changes in multiple, independent genes (Vincent and Gatenby, 2008 at 729).

Initiation follows exposure to mutagens and involves the induction of a permanent and irreversible change in a cell’s genome, which provides it with a growth advantage over its neighbors, although little or no observable changes in the cellular or tissue morphology can be observed. Promotion is the experimentally defined process by which the initiated cell expands by self-proliferation into a visible tumor, often a benign lesion. During progression benign tumors are transformed into malignant cancers, involving the acquisition of one or more qualitative changes in the precursor cells. When chronic exposure is involved, few chemicals, if any, will affect only one stage in the multistep carcinogenic process (Barret and Wiseman, 1987 at 65). In fact, most chemical carcinogens operate via a combination of mechanisms (they are not mutually exclusive; rather, they probably work in conjunction to result in neoplastic development), and even their primary mechanism of action may vary depending on the target tissue/cells (Barret, 1993 at 9).

The vast majority of chemical carcinogens are ‘genotoxic’ in their carcinogenic mode of action, which means that they (or their metabolites) are capable of interacting with the genetic material, thereby inducing DNA damage. There is, however, a smaller group of carcinogens that induce cancer via ‘non-genotoxic’ mechanisms. Hernández et al. (2009)

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reviewed these possible mechanisms including endocrine modification, tumor promotion, tissue-specific toxicity and inflammation, cytotoxicity and immune suppression, and inhibition of gap-junction intercellular communications, among others.

The correlation between the ability to induce changes in DNA and tumorigenesis is well established for most chemical initiating agents (Couch, 1996 at 136). Indeed, most initiating agents are genotoxic. For instance, PAHs are mutagenic agents that act as tumor initiators. A single exposure to these agents does not typically give rise to a tumor, but may produce latent damage that can result in tumor formation following a subsequent insult (Couch, 1996 at 136).

A very important aspect of the chemical carcinogens dose-effect relationship is the eventual determination of a threshold. As indicated by Monier, “[f]or a non-genotoxic carcinogen, a threshold [in the dose–effect relationship] can be safely assumed. For genotoxic drugs [chemicals], however, it is usually difficult to prove or disprove that a threshold does exist, and the tendency is to accept linear no-threshold relationships in determining permissible levels of exposures” (Monier, 2000 at 604). Thus, Goldstein et al. (2011) suggest that “[r]egulatory prudence has led to the use of ‘one-hit models’ for mutagenic end points, particularly cancer, in which every molecule of a carcinogen is presumed to pose a risk”. In other words, the safety threshold for genotoxic carcinogens is effectively zero, with the presumption that any exposure increases risk, or there is no dose free of risk.

Since a malignant cell needs to acquire multiple, heritable alterations at independent genetic locations, chemical carcinogenesis development involves a long delay (long latency period) between the causal event and the clinical manifestation of disease (Couch, 1996 at 134). In the case of solid tumors there is a 20 to 40-year interval from the time of exposure of an individual to a chemical or viral carcinogen until the clinical detection of a tumor (Wogan et al., 2004 at 482).

2.5. Genotoxicity tests and cancer risk

The traditional epidemiological technique has always been the hallmark approach to demonstrate associations between exposure to hazardous substances and the development of disease such as cancer (Bonassi and Au, 2002 at 73). As expressed in his expert opinion of May 2014 (page 3), Dr. Moolkgavkar contends that epidemiological studies, in contrast to risk assessment, “are necessary to reach a conclusion that an exposure resulted in adverse health outcomes”, since they “evaluate what actually did happen.” I agree that epidemiological studies are very useful when the conditions are appropriate to carry them out. However, epidemiological methods – the study of the factors that control the patterns of incidence of disease – normally require large numbers of subjects and/or long periods of time, because what is measured (the occurrence of disease) is a rare event (Collins, 1998 at 360).

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Not surprisingly, therefore, few epidemiologic studies on cancer incidence or mortality related to exposure to oil have been performed to date. Nonetheless, data useful for assessing causal associations between oil and cancer risk may be obtained by other scientifically reliable methodologies. Specifically, molecular epidemiology has developed to attempt to integrate traditional epidemiological investigation of cancer risk factors with the substantial expansion of knowledge of the molecular mechanisms of cellular processes (Shields and Harris, 1991). This approach has a great potential in monitoring cancer risk in people exposed to occupational or environmental carcinogens, especially when waiting for large scale studies conducted over decades of time will not sufficiently protect the health of those exposed.

The essential feature of molecular epidemiology is the use of biomarkers, with clear advantages of economy, speed and precision, to measure in individuals such things as exposure to agents implicated in the etiology of a particular disease, pre-clinical manifestations of disease, or features of the disease itself. Biomarkers are measurable biological parameters (something that can be measured in human subjects) that reflect, in some way, an individual's risk of disease, because they indicate exposure to a causative agent, or because they represent an early stage in the development of the disease. Therefore, the ultimate goal of using biomarkers in molecular epidemiological studies is to provide valuable information to be able to predict health risks (Collins, 1998 at 360). Thus, biomarkers are used as meaningful and indispensable tools for investigation into environmental mutagenesis and cancer risk assessment, since they provide early and reliable warning signals of cancer risk (Au, 2007 at 241).

In the context of carcinogenicity, biomarkers can mean proof of exposure to a carcinogen, detection of a reaction product or an indication that a preliminary genotoxic event or actual DNA damage has occurred (Committee on Carcinogenicity, 2013 at 2). The following describes biomarkers used in studies on genotoxic effects in oil-exposed populations, which are frequently employed in cancer molecular epidemiology, and their association with risk of cancer estimation:

Chromosomal alterations: In normal circumstances, when a DNA insult is produced, most of the damage is repaired within hours if not minutes. Importantly, however, some of the DNA damage may not be repaired. The amount of unrepaired damage depends on the extent of the damage not only to the DNA, itself, but also to the system that functions to repair DNA2. Some of the unrepaired damage can result in microscopically visible changes in chromosomes, which are cytogenetically detected as micronuclei or chromosome aberrations.

Micronuclei (MN): MN represent whole chromosomes or chromosome fragments that are excluded from the re-forming nucleus at the end of nuclear division and remain in

                                                            2 Physical and chemical agents that are able to react with DNA and proteins (e.g., DNA repair enzymes) might at low doses interfere with cellular DNA repair processes. Furthermore, the individual DNA repair capacity is influenced by the possible presence of numerous polymorphisms in DNA repair genes which may modify the activity of the encoded proteins.

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the cytoplasm forming a small nuclear body (a micronucleus). The use of MN as a measure of early genotoxic effects has become a standard assay in human biomonitoring studies (Mateuca et al., 2012 at 317). Regarding populations occupationally exposed to PAHs, a recent meta-analysis showed that frequencies of MN in lymphocytes may be indicators of early genetic change in these individuals (Wang et al., 2012 at 22). MN assessment is a relevant biomarker because MN represent irreversible biological alterations that can lead to the development of cancer (Au, 2007 at 241). Thus, MN are considered to be biomarkers of early carcinogenic effects (through genotoxic mechanisms). Indeed, an analysis performed within the framework of the HUMN project (HUman MicroNucleus international collaborative project, http://humn.org) indicates that an increased frequency of MN in peripheral blood lymphocytes predicts cancer risk in humans (Bonassi et al., 2007 at 625). The existing evidence linking MN frequencies with cancer risk was also substantiated by a recent meta-analysis of 37 publications, which clearly showed a 45% increase (28%-64%, 95% confidence interval) in the baseline MN level of untreated cancer patients compared to cancer-free referents (Iarmarcovai et al., 2008 at 274). A recent review on this topic (Bonassi et al., 2011 at 94) concluded that “the presence of association between MN formation in the leukocytes of healthy individuals and subsequent risk of cancer is supported not only by theoretical considerations but also by a large range of experimental findings”.

Chromosome aberrations (CAs): CAs include breaks, deletions, duplications, circularisation, dicentrics (i.e., two centromeres on one chromosome) and translocations. Lymphocytes, when stimulated to proliferate in vitro, may reveal the effects of accumulated, unrepaired damage as chromosome aberrations at the first cell division. Many aberrations lead to loss of chromosomal material in one of the daughter cells, or may even disrupt division itself resulting in a high probability of cellular dysfunction or death (Collins, 1998 at 372). However, translocation of a segment of one chromosome to a site on another chromosome tends not to involve significant loss of genetic material, and translocations tend to be stably transmitted through generations of cells. They have a potential clinical importance; although genes are not lost, the regulation of their expression may be altered in the new chromosomal context (Collins, 1998 at 372). Some CAs are typically found in particular types of cancer. For instance, the characteristic ‘Philadelphia chromosome’ is present in the leukemic cells of almost all patients with chronic myelocytic leukemia. It typically results from a balanced reciprocal translocation, which transposes the abl proto-oncogene (found on chromosome 9) to a region on chromosome 22. As a result, an abnormal fusion protein with oncogenic properties is produced (Jabbour and Kanterjian, 2014 at 548). Other B and T cell lymphomas and leukemias are also accompanied by specific translocations. CAs have been demonstrated to be an early predictor of cancer risk. The extensive use of this assay has resulted in the accumulation of valuable data in many laboratories. This has enabled the examination of the potential association between previously measured CA frequency and subsequent cancer outcome. An association between high CA frequency and increased

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cancer incidence was originally detected in a collaborative project of 10 Nordic cytogenetic laboratories (Hagmar et al., 1994 at 2921). An independent study among 10 laboratories in Italy, based on cancer mortality data, arrived at the same conclusion (Bonassi et al., 1995 at 133). The two cohorts were afterwards updated and examined together; the results supported the findings that CAs are predictive of cancer risk (Hagmar et al., 1998 at 2921). Furthermore, a case-control study nested within the two cohorts indicated that this association is not merely a reflection of smoking or occupational exposure to carcinogens, but is similarly seen in apparently unexposed subjects (Bonassi et al., 2000 at 1619).

Sister chromatid exchanges (SCE): SCEs are reciprocal DNA exchanges occurring during replication of the genetic material, just before cell division, between the two sister chromatids of a duplicated chromosome (Mateuca et al., 2012 at 306). It is thought that SCEs reflect a disruption of the normal replication process by the presence of DNA lesions (Collins, 1998 at 374). Since SCEs are the manifestation of damage to DNA, i.e., they may involve errors and therefore possible mutations, they are direct indicators of the adverse effects of exposure to DNA damaging agents (Tsongas, 1984 at 988).

Measurements of primary DNA damage: This includes DNA breaks, altered bases or adducts. Two of the most common methods to determine this kind of DNA damage are the evaluation of DNA adducts and the comet assay. DNA adducts are formed by the chemical reaction of DNA with a variety of classes of DNA-damaging agents. The comet assay measures breaks in the DNA strands or lesions which give rise to breaks; it is commonly used in investigations evaluating populations potentially exposed to genotoxicants. Although exposures to non-genotoxic carcinogens will not be detected using these assays, they are considered to be valuable methods for detection of genotoxic exposure in humans. However, the DNA damage measured by the comet assay (and also by evaluation of DNA adducts) identifies hazard rather than risk, and its value for predicting cancer is not yet known because it has not been investigated in prospective cohort studies (Albertini et al., 2000 at 129).

Mutations in marker genes: Mutations are exceedingly rare events. The mutagenic potential associated with a given exposure is evaluated by determining mutations induced in several well-established marker genes. One of them, and probably the most frequently used in biomonitoring studies, is the hprt gene, commonly studied in lymphocytes. Although the implications of elevated frequency of hprt mutations for cancer risk have not been assessed in prospective human studies, molecular analyses of in vivo derived hprt mutations have shown types of mutations similar to mutagenic changes seen in cancer-related genes or genomic regions associated with cancer (Albertini and Hayes, 1997; Cole and Skopek, 1994).

Regarding the relationship between genotoxicity biomarkers and risk of cancer, it is noteworthy that, in evaluating the carcinogenic potential of chemicals, the International Agency for Research on Cancer (IARC) reviews data from genotoxicity studies (including

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DNA damage, gene mutation, SCEs, MN formation, CAs and aneuploidy) in view of the relevance of these processes to carcinogenesis (IARC, 2006 at 10-12).

The described biomarkers are usually assessed in peripheral blood leukocytes. In human trials, only a limited range of biological material can be obtained without ethically unacceptable intrusion. For this reason, to estimate events occurring at the target organs and to provide early warning signals for health risk, assessment of genotoxicity is normally carried out in readily available surrogate cells (Mateuca et al., 2012 at 306). The most frequently used surrogate cells in human studies are the peripheral blood leukocytes (reviewed in Salama et al., 1999 at 99). The major motive for using leukocytes is that these cells circulate throughout the body and that they have reasonably long life-span if a suitable cell type is considered (e.g., T-lymphocytes); therefore, they can be damaged in any tissue/organ-specific toxic environment (Au, 2007 at 241).

2.6. Genotoxicity studies in people exposed to oil spills

Given the relationship between genotoxicity parameters and cancer risk, several studies have aimed to evaluate genotoxic effects in people exposed to oil spills (MV Braer and Prestige). The details on the design and results of these studies are presented in Appendix B.

The two studies corresponding to the Prestige oil spill were carried out by a research group of which I was a part. The first study included people involved in autopsies and cleanup of oil-contaminated birds (Laffon et al., 2006), and the second one, partially published in several different papers (Pérez-Cadahía et al., 2006, 2007, 2008a, 2008b, 2008c) analyzed volunteers and workers who participated in the cleanup of beaches and rocks. Results obtained in the group of volunteers handling oil-contaminated birds showed significant increase, when compared to the control group, in DNA damage (evaluated by means of the comet assay), related to the duration of exposure, and also in the chromosomal damage (MN test), although in this last case significance was not reached.

Exposed individuals included in the second study were divided into three groups: volunteers who cleaned up oil on the beaches for 5 days; workers who collected oil manually on the beaches for 3 months (MW), and workers who used high-pressure water jets to clean rocks on or near the beach for 4 months (HPW). Significant increases in DNA damage over the control individuals were observed in all exposed groups. Significant increases were also detected for the MN test in MW, and for SCE test in HPW. It is generally considered that, for chronic exposures, cytogenetic techniques (such as MN and SCE tests) express cumulative events, while the comet assay provides information about recent repairable exposure levels (Maluf and Erdtmann, 2000 at 26). Hence, the results obtained indicate that exposure to Prestige oil induced DNA damage, and this damage became fixed as chromosome alterations, thus increasing the risk of cancer development, after only several months of exposure. Additionally, a cell proliferation index, indicative of toxicity to the cell cycle, was also

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evaluated, and again significant effects for this index were observed only in those subjects exposed for months.

Afterwards, as further confirmation of the results obtained in these two epidemiologic studies, an in vivo study using a rat model of subchronic exposure to a fuel oil with similar characteristics to that spilled by the Prestige tanker was carried out by our research group (Valdiglesias et al., 2012 at 756), in order to determine potential genotoxic effects under strictly controlled exposure conditions. Results obtained showed that inhalation oil exposure induced DNA damage in the rats, and also alterations in the DNA repair response, although the sensitivity to oil substances varied depending on the rat strain. These data supported the previously described genotoxic effects in humans exposed to Prestige oil during cleanup tasks.

Regarding the design of the abovementioned epidemiologic studies, Dr. Moolgavkar in his report (page 5) expressed concerns about epidemiologic studies which are ecologic in design (not including individual-level data on exposure), and about the lack of control for potential confounders in these studies, in order to use their results to establish causal associations. Unlike the studies criticized by Dr. Moolgavkar, the Prestige studies were not ecologic in design. The oil-exposed subjects in fact reported individual-level data on exposure — excepting the one Prestige study wherein an automated sampler was used to analyze environmental levels of VOC in the working room for individuals handling oil-contaminated birds — and all of the studies reported data on several potential confounders (age, gender and smoking habits, etc.) known to influence genotoxicity assays results. Moreover, since these studies were based on experimental laboratory analyses, they were free of recall bias (in contrast with studies that rely on self-reported health symptoms). Finally, the analyses were made and the results were analyzed ‘blindly’, i.e., the persons responsible for these tasks did not have information on the exposure status of the subjects.

Another study, carried out two years after the exposure to Prestige oil in highly exposed fishermen, detected that a higher proportion of exposed participants had structural chromosomal alterations, in comparison with the control group, and the risk seemed to increase with intensity of exposure (Rodríguez-Trigo et al., 2010 at 489). A more thorough analysis of the chromosomal locations revealed three chromosomal bands commonly involved in hematological cancer as the most affected by acute oil exposure, and significantly higher dysfunction in DNA repair mechanisms, expressed as chromosomal damage, in oil-exposed participants than in those not exposed (Monyarch et al., 2013 at e81726).

The only study that found no relationship between oil exposure and genotoxic damage was a simple longitudinal study conducted after the MVBraer oil spill. That study was carried out to assess the primary damage in the DNA (DNA adducts) and the frequency of mutations in the hprt gene in the peripheral leukocytes of residents in the Shetland Islands polluted area and controls (who lived about 40 miles [72 km] north of Sumborough Head) at 3 sampling times (10 days, 10 weeks and 1 year after the accident) (Cole et al., 1997 at 98). These authors did not obtain any evidence of genotoxicity in DNA adducts or the hprt gene. However, the

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size of the two groups analyzed was extremely small, especially the control group, thus precluding the possibility of producing any statistically reliable conclusions.3 Additionally, participation of the exposed individuals in the cleanup tasks was not specified by the authors of the study (so it can be assumed to be null). Indeed, for the study participants, only their status as residents in the polluted area was mentioned but no indication of the absolute or relative level of their exposure was provided. It may well be that those who participated in the cleanup work were more exposed to the oil compounds than that those who lived near the spill but who did not carry out cleanup tasks.

There is only one molecular epidemiology study that analyzed genotoxicity parameters in people from the parish of San Carlos located in Sachas, Orellana province (Paz y Miño et al., 2008). That study focused on individuals who were exposed to oil whilst working at the Sacha South production station. DNA primary damage was evaluated by the comet assay and chromosomal alterations by the CA test. Results obtained showed a greater percentage of DNA damage and CA in the exposed individuals than in the controls. These results are in line with the ones reported for Prestige oil exposed individuals, further supporting the increase in genotoxic risk (and consequently cancer risk) associated with exposure to oil.

To determine the persistence of the genotoxic alterations observed beyond a two year period, as was studied in one of the Prestige oil studies (Rodríguez-Trigo et al., 2010), a follow-up study was carried out seven years later in individuals exposed to Prestige oil for a mean of 9 months (range 2-10 months). This study reported no significant differences between the exposed population and the controls in the genotoxicity parameters (Laffon et al., 2014 at 10). These results suggest that bone marrow hematopoietic stem cells, which produce leukocytes (the surrogate cells in which genotoxicity was evaluated), do not necessarily have permanent damage in their DNA, so long as the subjects remain exposure free for a prolonged period of time.

A potential toxicological risk assessment was also carried out after decontamination of beaches polluted by the Erika oil spill (Dor et al., 2003). Seven different scenarios of exposure for people using the beaches were contemplated, selecting the most conservative available toxicological values for computing risks. Like the 2014 Prestige oil study, the results obtained in this study indicated that risks were low, both in the long-term and short-term, for people on holiday and for people working at these cleaned beaches during the summer period following the oil spill. Cancer risks in decontaminated beaches did not differ

                                                            3 For determination of DNA adducts only 20 exposed and 7 non-exposed individuals were analyzed immediately after the accident, and the number of controls was further reduced to 4 in the samples taken 1 year later (no samples of 10 weeks were analyzed for DNA adducts). Moreover, the authors described methodological problems during the analyses, e.g., poor quality of the thin layer chromatography plates. For the hprt mutation assay, the number of exposed and control samples evaluated was 21 vs. 7, 24 vs. 9, and 20 vs. 5, respectively in the 3 sampling times. In the experimental design discussion, the authors cite the Robinson et al. (1994) study, as recommending “that, given the variability in mutant hprt frequency, a minimum of 30-50 individuals per donor group would be necessary to have a 90% chance of detecting a 1.5-fold increase in mutant frequency over the control level.” (Cole, 1997 at 106). Thus, sample size of Cole et al. study was clearly too small to detect any significant effect (Robinson et al., 1994 at 109 (recognizing the limitations of their own study)).

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substantially from those estimated for control beaches, except when decontamination work was not completed, as observed in some rocky areas. Consequently, the authors hypothesized that risks of cancer at beaches not cleaned yet, or recently spoiled by fuel deposits, would be of concern and would justify temporarily closing the beaches.

The results obtained in the abovementioned studies suggest that carrying out a properly executed remediation in the Concession Area would be beneficial for the residents, since according to the current data the risk of long-term adverse effects for their health (at least the risk of cancer) would decrease and likely reach unexposed levels. It is nevertheless important to note that we have no data to date that show how long the reversal process takes. In other words, we do not know how long it would it take for people who have been exposed for much longer than several months (individuals analyzed were at most 10 months exposed to Prestige oil) to return to the control level. Here, the residents in El Oriente region have been substantially exposed through mutually reinforcing media for decades.

In addition, the main exposure pathway for individuals exposed to Prestige oil was by inhalation, and to a lesser extent through dermal contact. Most people involved in the cleanup tasks wore boots, protective clothing, gloves, and often but not always face masks. And ingestion of oil, if it occurred at all, was only accidental. These exposure conditions are drastically less pronounced than those present in the Concession Area, where no personal protective devices are used and, as set forth by Dr. Strauss in her report, multiple pathways are involved.

2.7. Immune and endocrine toxicity studies in people exposed to oil spills

The studies our group carried out in Prestige oil-exposed individuals also included other parameters reflecting longer-term physiological changes. At the moment of exposure, decreases in the hormones prolactin and cortisol, both markers of psychophsysiological stress, were observed in the exposed individuals as compared to the controls, indicating alterations in the normal endocrine function in the individuals (Pérez-Cadahía et al., 2007, 2008a). Another study, performed by a group we collaborated with in the study of the same groups of exposed individuals, analyzed several immunological parameters. This study showed that individuals exposed for several months to oil had significant modifications in some lymphocyte subpopulations (increases in %T lymphocytes and %T-helper lymphocytes, and decrease in %T-cytotoxic lymphocytes), as well as in concentrations of plasma cytokines (increases in interleukin-2, interleukin-4, interleukin-10 and interferon gamma), but no effects were detected in the group of short-term exposed volunteers (Gestal et al., 2004). All these effects indicate that exposure to the oil induced significant changes in the endocrine and immune systems.

In the follow-up study carried out seven years later (Laffon et al., 2013), significant endocrine and immunological alterations were observed in the exposed subjects, namely increase in cortisol concentration and decrease in the percentage of natural killer (NK) cells. The increase in cortisol in the exposed subjects, contrasting with the decrease initially detected, suggests an alteration in the endocrine system. Significantly higher levels of plasma

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cortisol were also reported in outdoor workers chronically exposed to urban pollution, which shares several compounds with oil (Rosati et al., 2011; Tomei et al., 2003), and it has been established that a chronic increase in cortisol, subsequent to the increase in hypothalamic pituitary-adrenal axis activity, is associated with negative health outcomes (Rosati et al., 2011). Additionally, the NK cells are effector lymphocytes of the innate immune system that control several types of tumors and microbial infections by limiting their spread and subsequent tissue damage (reviewed in Vivier et al., 2008). Since cortisol suppresses the immune response, it may be that the overall decrease of NK cells observed in the exposed group was an indirect consequence of the increase in cortisol in those individuals. Because NK cells are the major cell type involved in immune surveillance against cancer cells, the decrease in NK cells is presumed to increase cancer risk.

The persistence of immunological alterations seven years after the exposure supports Dr. Strauss’ opinion (page 54 of her initial report) that “the risk of delayed impacts [in Concession Area inhabitants] is on-going even if there is no additional exposure”, and “the risk of delayed impacts continues to increase if the exposure remains”. Again, it is necessary to consider that these immediate and delayed immunological alterations were observed in people exposed to oil for only several months; the effects on subjects exposed for years, or even decades (such as El Oriente residents) is for now unknown.

The immunological alterations described in oil-exposed individuals, both at the time of exposure and also seven years later, provide further support for Dr. Strauss’ opinion (page 48 of her rejoinder report) that exposure to crude oil has immunosuppressive effect, causing a reduction in the body’s defense against infection and in the immunosurveillance against cancer cells.

In summary, genotoxic, immunotoxic and endocrine toxicity results obtained in Prestige oil-exposed individuals refute Dr. Moolgavkar’s conclusion (page 23 of his report) that “the available epidemiologic evidence does not support a causal effect of environmental exposure to petroleum from oil exploration and production activities on cancer or other health outcomes in residents of surrounding communities, either in general or specifically in the Texpet Concession Area”. The increased risk for developing cancer and/or other diseases related to dysfunction of the immunological and endocrine systems lead to the recommendation of periodic health monitoring for those people who were exposed to the Prestige oil spill. To facilitate the early detection of possible health problems, the recommendation focused on the determination of cancer biomarkers, clinical immunological parameters and cortisol levels.

2.8. Miscellanea: IARC classification of crude oil

IARC evaluated the carcinogenic risk of crude oil and included it in group 3 as “not classifiable as to its carcinogenicity in humans” (IARC, 1989), on the basis of “inadequate evidence for the carcinogenicity in humans” and “limited evidence for the carcinogenicity in experimental animals.” As IARC sets forth in the Preamble document for the IARC Monographs on the Evaluation of Carcinogenic Risks to Humans (IARC, 2006), “agents that

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do not fall into any other group are also placed in this category” (group 3), and “an evaluation in group 3 is not a determination of non-carcinogenicity or overall safety”. Instead, “it often means that further research is needed, especially when exposures are widespread” (as is the case with crude oils) “or the cancer data are consistent with differing interpretations”. We now have an additional 25 years of research since the 1989 IARC classification of crude oil, which was based on only a few studies. In light of the accumulating evidence over the past two decades showing the genotoxicity of crude oil and its relationship to cancer in humans, a new review is necessary to include evidence obtained from more recently published studies.

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3. References

Expert opinions

Expert Opinion of Kenneth J. Goldstein, M.A., CGWP and Jeffrey W. Short, PhD, regarding the Environmental Contamination From Texpet’s E&P Activities in the Former Napo Concession Area Oriente Region, Ecuador. February 2013.

Expert Opinion of Harlee S. Strauss, PhD, regarding human health‐related aspects of the environmental contamination from Texpet’s E&P activities in the former Napo concession area Oriente region, Ecuador. February, 2013.

Expert Report of Suresh H. Moolgavkar, MD, PhD. May 31, 2013.

Rejoinder Opinion of Harlee Strauss, PhD, regarding human health risks, health impacts, and drinking water contamination caused by crude oil contamination in the former

Petroecuador‐Texaco concession, Oriente Region, Ecuador. December, 2013.

Opinion of Philippe Grandjean, MD. November, 2013.

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1

APPENDIX A

Epidemiological studies on acute toxic effects related to exposure to oil spills (ordered by the chronology of the spills). Black letter: from Aguilera et al. (2010); blue letter: studies not reviewed before by any expert in this arbitration process.

Accident – Reference

Study characteristics Methods Results

MV Braer – Campbell et al. (1993)

Cross-sectional. Initial acute effects in residents (N=420) and controls (N=92)

Questionnaires of acute symptoms, peak expiratory flow, hematology, liver and renal function tests, blood and urine toxicology

Principal health effects arose on days 1 and 2 (headaches, itchy eyes, and throat irritation). No significant differences between exposed and controls were found for any of the biological markers. Toxicological studies did not show any exposure that are known to affect human health

MV Braer – Campbell et al. (1994)

Cross-sectional. Follow up after 6 months of acute effects in residents (N=344) and controls (N=77)

General health questionnaire. Peak expiratory flow, urine analysis, hematology, and liver and renal function tests

The mean general health questionnaire score of exposed was significantly greater than that of controls. Exposed had greater overall scores for somatic symptoms, anxiety and insomnia, but not for personal dysfunction and severe depression

MV Braer – Crum (1993)

Cross-sectional. Affectation of respiratory tract in children living close to Braer shipwreck (N= 44 at 3 days and 56 at 9-12 days after oil spill)

Peak expiratory flow rate Peak expiratory flow rates were within the normal range in both parts of the study, and no deterioration was seen over the study period

Sea Empress – Lyons et al. (1999)

Cross-sectional. Acute health and psychological effects in exposed (N=539) and controls (N=550)

Questionnaires of acute symptoms. HAD and SF-36 scores

Exposed showed significantly higher anxiety and depression scores, worse mental health, and self-reported headache and sore eyes and throat

Sea Empress – Gallacher et al. (2007)

Cross-sectional. Acute symptomathology attributable to psychological exposure in exposed (N=794) and controls (N=791)

Questionnaires of acute toxic and non-toxic symptoms and Hospital Anxiety and Depression Scale

Perceived risk was associated with raised anxiety and non-toxicologically related symptom reporting. Toxic symptom reporting was associated with oil exposure and with raised perceived risk

Nakhodka – Morita et al. (1999)

Cross-sectional. Acute health problems in exposed (N=282)

Questionnaires of acute and toxic symptoms. Personal air samplers to assess carcinogenic benzene, toluene and xylene. Metabolites of benzene, toluene and xylene in urine

Levels of hydrocarbons in air were far below the occupational acceptable limit. The principal complaints of symptoms were low back pain, headache, and symptoms of eyes and throat

Erika – Schvoerer et al. (2000)

Cross-sectional. Acute health effects in volunteers and workers who participated in the cleanup (N=1,465)

Self-questionnaires sent by postal mail

The more recurrent health disorders were lower back pains, headaches and skin irritations. Duration of the cleanup activity was identified as risk factor for the health problems that occurred

2

Prestige – Suarez et al. (2005)

Cross-sectional. Acute health problems among subjects involved in the cleanup operation after the spill (N=800)

Questionnaire on exposure conditions, acute health problems, and use of protective material

Bird cleaners accounted for the highest prevalence of injuries. Working more than 20 days in highly polluted areas was associated with increased risk of injury in all workers. Toxic effects were higher among seamen. No severe disorders were identified.

Prestige – Carrasco et al. (2006)

Cross-sectional. Association between health information, use of protective devices and occurrence of acute health problems in exposed (N=799)

Questionnaire on exposure conditions, acute health problems, use of protective material and health-protection information received

Health-protection briefing was associated with use of protective devices and clothing. Uninformed subjects registered a significant excess risk of itchy eyes, nausea/vomiting/dizziness, headaches and throat and respiratory problems. Seamen, the most exposed group, were the worst informed and registered the highest frequency of toxicological problems

Prestige – Zock et al. (2007)

Longitudinal 12-24 months after the spill. Association between participation in cleanup work and respiratory symptoms in exposed (N= 6,780)

Questionnaires with qualitative and quantitative information on cleanup activities and respiratory symptoms

The risk of LRTS increased with the number of exposed days, exposed hours per day, and number of activities. The excess risk of LRTS decreased when more time had elapsed since last exposure

Prestige – Rodríguez-Trigo et al. (2010)

Cross-sectional, two years after the exposure. Respiratory effects in fishermen highly exposed (N=501) and not exposed (N=177)

Respiratory symptoms, forced spirometry, methacholine challenge, markers of oxidative stress, airway inflammation, and growth factor activity in exhaled breath condensate

Participation in clean-up was associated with persistent respiratory symptoms and elevated markers of airway injury in breath condensate. The risk for elevated levels of exhaled 8-isoprostane, vascular endothelial growth factor, and basic fibroblast growth factor seemed to increase with intensity of exposure to clean-up work

Prestige – Zock et al. (2012)

Cross-sectional, five years after cleanup. Persistence of respiratory symptoms in exposed fishermen (N=466) and non-exposed individuals (N=156)

Questionnaire on upper and lower respiratory tract symptoms, allergic conditions, anxiety and beliefs about the effects of the oil spill on the participant’s own health

The prevalence of lower respiratory tract symptoms had slightly decreased in both groups, but remained higher among the exposed. The risk of having persistent respiratory symptoms increased with the degree of exposure for moderately and highly exposed, when compared with those without any symptoms. Findings for nasal symptoms and for respiratory medication usage were similar

Prestige – Zock et al. (2014)

Cross-sectional. Four-year follow-up, six years after cleanup work, and comparison with previous evaluation (Rodríguez-Trigo et al., 2010). Persistence of functional and biological respiratory health effects in never-smoking fishermen exposed (N=158) and non-exposed (N=57) to the oil

Respiratory symptoms, forced spirometry, methacholine challenge, markers of oxidative stress, airway inflammation and growth factor activity in exhaled breath condensate

During the four-year follow-up period lung function, bronchial hyperresponsiveness and the levels of respiratory biomarkers of oxidative stress and growth factors had deteriorated notably more among non-exposed than among exposed. At follow-up, respiratory health indices were similar or better in cleanup workers than in non-exposed. No clear differences between highly exposed and moderately exposed cleanup workers were found

3

Tasman Spirit – Janjua et al. (2006)

Cross-sectional. Acute health effects in exposed residents (N=216) and controls living 2 Km (N=83) and 20 Km (N=101) far from the coastline

Questionnaires on acute health symptoms and on perception about the role of oil spill in producing ill health, and anxiousness about the effect of oil spill on health

Data showed moderate-to-strong associations between the exposed group and the symptoms. There was a trend of decreasing symptom-specific prevalence odds ratios with increase in distance from the spill site

Tasman Spirit – Khurshid et al. (2008)

Cross sectional. Health parameters of people working/living in the vicinity of an oil-polluted beach (N=100)

Hydrocarbon/organic content in seawater and sand samples. Hematological and biochemical parameters. Liver and renal function tests

Seawater had no traces of hydrocarbon content. Lymphocyte and eosinophil levels were slightly increased. About 11 people had raised SGPT, but this was not significant

Tasman Spirit – Meo et al. (2008)

Cross sectional. Lung function in exposed (N=20) and controls (N= 31)

Spirometry Significant reduction in FVC, FEV1, FEF25%-75% and MVV in exposed. Lung function parameters were improved when the subjects were withdrawn from polluted air environment

Tasman Spirit – Meo et al. (2009a)

Cross sectional. Health complaints among males involved in cleanup operations (N=50) and controls (N=50)

Standardized questionnaire on respiratory and general health complaints

The subjects involved in oil cleanup operations had significantly higher rates of health complaints including cough, runny nose, eye irritation/redness, sore throat, headache, nausea and general illness, compared to their matched controls

Tasman Spirit – Meo et al. (2009b)

Cross sectional. Lung function in subjects exposed to crude oil spill into sea water (N=31) and controls (N=31)

Spirometry Subjects exposed to polluted air for periods longer than 15 days showed a significant reduction in FVC, FEV1, FEF25–75% and MVV

Hebei Spirit – Lee et al. (2009)

Cross-sectional. Protective effects of wearing protective devices on exposure and symptoms among the residents (N=288) and volunteers (N=724) who participated in the cleanup

Questionnaires about symptoms, use of protective devices and potential confounding variables. Analysis of VOCs, PAHs and heavy metals in urine

Levels of fatigue and fever were higher among residents not wearing masks than among those who did wear masks. Urinary mercury levels were found to be significantly higher among residents not wearing work clothes or boots

Hebei Spirit – Lee et al. (2010)

Cross-sectional. Acute health effects in residents from seashore villages of a heavy and moderately oil soaked area and a lightly oil soaked area (10 villages from each area, 10 male and female adults from each village)

Questionnaire on the characteristics of residents, the cleanup activities, the perception of oil hazard, depression and anxiety, and the physical symptoms

The more highly contaminated the area, the more likely it was for residents to be engaged in cleanup activities and have a greater chance of exposure to oil. The indexes of anxiety and depression were higher in the heavy and moderately oil soaked areas. Significantly increased risks of several physical symptoms was obtained

Hebei Spirit – Sim et al. (2010)

Acute health problems in people engaged in the cleanup (N=846)

Questionnaire on demographic information, operation and exposure to oil, and health status

Residents and volunteers experienced acute health problems. More frequent and greater exposure (including lack of protective suit and mask) was strongly associated with a higher occurrence of symptoms

4

Hebei Spirit – Cheong et al. (2011)

Cross-sectional. Physical symptoms in residents participating in cleanup work (N=288) and controls (N=39)

Questionnaire regarding subjective physical symptoms, sociodemographic characteristics and cleanup activities. Urinary metabolites of VOCs, PAHs and heavy metals

Exposed residents showed associations between physical symptoms and the exposure levels

Hebei Spirit – Ha et al. (2012)

Cross-sectional. Exposure status and acute health effects on volunteers that participated in the cleanup (N=565)

Questionnaire regarding physical symptoms. Urinary metabolites of VOCs and PAHs before and after exposure

Volunteers that participated for longer cleanup work reported an increase in physical symptoms (visual disturbance, nasal and bronchus irritation, headaches, heart palpitations, fatigue and fever, memory and cognitive disturbance, and abdominal pain). The levels of t,t-muconic acid, mandelic acid, and 1-hydroxypyrene were significantly higher in samples after cleanup than those measured before participation

Hebei Spirit – Na et al. (2012)

Cross-sectional, one year after the accident. Health problems of people involved with cleanup efforts (N=442)

Questionnaire on demographic information, risk factors and the continuation and duration of any health symptoms

Eye symptoms, headaches, skin symptoms, and neurovestibular symptoms had a longer duration than did back pain or respiratory symptoms

Hebei Spirit – Jung et al. (2013)

Cross-sectional. Respiratory effects on children who lived along the Yellow Coast (N=436)

Modified International Study of Asthma and Allergies in Childhood questionnaire. Health examination (skin prick test, pulmonary function test, and MBPT),

The children who lived close to the oil spill area showed a significantly lower FEV1, an increased prevalence of ‘asthma ever’ (based on a questionnaire), and ‘airway hyperresponsiveness’ (based on the MBPT) than those who lived far from the oil spill area. Male sex, family history of asthma, and residence near the oil spill area were significant risk factors for asthma

Hebei Spirit – Kim et al. (2013)

Cross-sectional, 1.5 years after the spill. Burden of disease (BOD), including physical and mental diseases, of the residents living in contaminated coastal area (N=10,171)

Questionnaires on exposure and medical problems, and to assess psychological health and asthma, and physical and laboratory examinations of respiratory, cardiovascular, neurological and psychological systems

The YLD of mental diseases including PTSD and depression for men were higher than that for women. The YLD for women was higher in asthma and allergies (rhinitis, dermatitis, conjunctivitis) than that for men. The effects of asthma and allergies were the greatest for people in their 40s, with the burden of mental illness being the greatest for those in their 20s. Proximity to the spill site was associated with increased BOD.

Deepwater Horizon-Abramson (2010)

Cross-sectional. Short and potential long-term impact of the Deepwater Horizon disaster on coastal residents (children and families) (N=1,203)

Telephone interviews on exposure, physical and mental health, and decisions related to oil spill on a daily basis

Over one-third of parents reported that their children had experienced either physical symptoms or mental health distress as a consequence of the oil spill. One in five households has seen their income decrease as a result of the oil spill and 8% have lost jobs. Over 25% of coastal residents think they may have to move from the area because of the oil spill

5

Deepwater Horizon – D’Andrea and Reddy (2013)

Cross-sectional. Adverse health effects in subjects participating in the cleanup activity (N=117) and controls (N=130)

Clinical data (white blood cell and platelets counts, hemoglobin, hematocrit, blood urea nitrogen, creatinine, ALP, AST, ALT) and somatic symptom complaints

Platelet counts were significantly decreased, and hemoglobin and hematocrit levels were significantly increased, among oil spill-exposed subjects. Similarly, oil spill-exposed subjects had significantly higher levels of ALP, AST, and ALT compared with the unexposed subjects

Deepwater Horizon – D’Andrea and Reddy (2014)

Cross-sectional. Hematological and liver function indices in subjects who participated in the cleanup operations (N=117)

White blood cell and platelets counts, hemoglobin, hematocrit, blood urea nitrogen, creatinine, ALP, AST, ALT), and urinary phenol. Values were compared with the standardized normal range reference values

Data obtained indicate that people exposed are at risk of developing alterations in hematological profile and liver function. Results support the earlier study (D’Andrea and Reddy, 2013) findings

ALT, alanine amino transferase; ALP, alkaline phosphatase; AST, aspartate amino transferase; BOD, burden of disease; FEF25%-75%, forced expiratory flow; FEV1, forced expiratory volume in first second; FVC, forced vital capacity; LRTS, low respiratory tract symptomathology; MBPT, methacholine bronchial provocation test; MVV, maximum voluntary ventilation; PAH, polycyclic aromatic hydrocarbons; PTSD, post-traumatic stress disorder; SF-36, short form-36; SGPT, serum glutamic pyruvic transaminase; VOC, volatile organic compounds; YLD, years lived with disability.

1

APPENDIX B

Epidemiological studies on genotoxicity, immunotoxicity and endocrine toxicity, and studies on potential toxicological risk assessment, related to exposure to oil spills (ordered by the chronology of the spills). Black letter: from Aguilera et al. (2010); blue letter: not included in Aguilera et al. (2010).

Accident – Reference

Study characteristics Methods Results

Braer – Cole et al. (1997)

Longitudinal. Genotoxicity in residents (N=26) and controls (N=9) at 3 sampling times (10 days, 10 weeks and 1 year after the accident)

DNA adducts in the mononuclear cell fraction and frequency of hprt mutations in T lymphocytes

No evidence of genotoxicity was obtained for either end point

Erika – Baars (2002)

Potential toxicological risk assessment for people involved in cleaning activities and for tourists

Risk characterizations on the basis of suppositions of the potential exposure during cleaning and tourist activities

The risk for the general people was limited. Increased risk for developing skin irritation and dermatitis, and very limited risk for developing skin tumors, were described for people who had been in bare-handed contact with the oil

Erika – Dor et al. (2003)

Potential toxicological risk assessment after decontamination of 36 beaches polluted by the Erika oil spill and 7 control beaches

Determination of the 16 PAHs selected by the U.S.EPA in sand, water and surface of rocks. Seven scenarios of exposure for people using the beaches were contemplated, and the most conservative available toxicological values were selected for computing risks

The sand and water were slightly polluted, with values similar to those found in the control beaches. The rocky areas were still highly polluted. No lethal risk was found for a young child who had accidentally ingested small ball of fuel. The life-long excess risks for skin cancer and for all other cancers were about 10-5 in scenarios including contact with the polluted rocks. The hazard quotient for teratogenic effects was very small, except in scenarios where pregnant women would walk among rocks containing high pollution levels.

Prestige – Laffon et al. (2006)

Cross-sectional. Genotoxicity in individuals performing autopsies and cleaning of oil-contaminated birds (N=34) and controls (N=35)

Environmental VOCs. Comet assay and MN test. DNA repair genetic polymorphisms (XRCC1, XRCC3, APE1)

Significant increase in the comet assay, but not in the MN test, related to the time of exposure. Exposed individuals carrying XRCC1-399Gln or APE1-148Glu alleles showed increased DNA damage.

Prestige – Pérez-Cadahía et al. (2006)

Cross-sectional. Genotoxicity in volunteers and hired workers participating in the cleanup (N=68) and controls (N=42).

Environmental VOCs. Comet assay, SCE, MN test

Highest VOC levels in the volunteer’s environment. Significant increase in the comet assay in exposed individuals. Influence of sex, age and tobacco smoking on the genotoxicity variables. No effect of using protective mask during cleanup labors

2

Prestige – Pérez-Cadahía et al. (2007)

Cross-sectional. Gentoxicity and endocrine alterations in volunteers and hired workers participating in the cleanup (N=68) and controls (N=42).

Environmental VOCs. Heavy metals in blood (Al, Cd, Ni, Pb, Zn). SCEs. Prolactin and cortisol. Metabolic genetic polymorphisms (GSTM1, GSTT1, GSTP1)

Highest VOC levels in the volunteer’s environment. Significant increase in the levels of Al, Ni and Pb, and decrease of Zn, in exposed individuals. Significant increase in SCE rate in exposed, influenced by age, sex, smoking and GSTM1 polymorphism. Significant decrease in prolactin and cortisol levels in exposed subjects.

Prestige – Pérez-Cadahía et al. (2008a)

Cross-sectional. Gentoxicity and endocrine alterations in volunteers and hired workers participating in the cleanup (N=180) and controls (N= 60)

Heavy metals in blood (Al, Cd, Ni, Pb, Zn). Comet assay. Prolactin and cortisol. Metabolic genetic polymorphisms (CYP 1A1, EPHX1, GSTM1, GSTT1, GSTP1)

Significant increase in the levels of Al, Ni and Pb, and decrease of Zn, in exposed individuals. Significant increase in comet assay and decrease in cortisol levels in exposed individuals. Higher DNA damage was related to CYP 1A1 and EPHX1 variant alleles, and lower DNA damage to GSTM1 and GSTT1 null genotypes

Prestige – Pérez-Cadahía et al. (2008b)

Cross-sectional. Gentoxicity in volunteers and hired workers participating in the cleanup (N=159) and controls (N=60)

MN test. Metabolic genetic polymorphisms (CYP 1A1, CYP 1B1, EPHX1, GSTM1, GSTT1, GSTP1). DNA repair genetic polymorphisms (XRCC1, XRCC3, XPD)

General increases in MN frequency and decreases in the proliferation index were observed in individuals with longer time of exposure. All the polymorphisms analyzed, excepting for CYP 1B1 and XRCC1, influenced cytogenetic damage levels

Prestige – Pérez-Cadahía et al. (2008c)

Cross-sectional. Relationship between blood concentrations of heavy metals and cytogenetic and endocrine parameters in exposed by means of univariate statistics (general linear model) (N=179)

Heavy metals in blood (Al, Cd, Ni, Pb, Zn). Comet assay, SCEs, MN test. Prolactin and cortisol

Pb was related to the comet assay. Cortisol plasma concentration was influenced by Al and Ni inversely and by Cd positively. In women there was a strong association between Cd and prolactin levels

Prestige – Rodríguez-Trigo et al. (2010)

Cross-sectional, two years after the spill. Chromosomal damage in fishermen highly exposed (N=501) and not exposed (N=177)

Chromosomal lesions and structural alterations in circulating lymphocytes

A higher proportion of exposed participants had structural chromosomal alterations, predominantly unbalanced alterations. The risk for structural chromosomal alterations seemed to increase with intensity of exposure

Prestige – Laffon et al. (2013)

Cross-sectional, follow-up seven years after the exposure. Endocrine and immunological alterations in individuals exposed for at least two months (N=54) and controls (N=50)

Prolactin and cortisol plasma concentrations, percentages of lymphocyte subsets, plasma levels of circulating cytokines, and serum concentrations of neopterin, tryptophan and kynurenine

Significant differences in exposed individuals vs. referents were observed in cortisol (increase), kynurenine and %CD16+56+ lymphocytes (both decrease). Effect of using protective mask was observed on neopterin, %CD8+ cells, CD4+/CD8+ ratio and interleukin 4

3

Prestige – Monyarch et al. (2013)

Cross-sectional, two years after the spill. Analysis of chromosomal lesion locations in fishermen highly exposed (N=91) and not exposed (N=46), and DNA repair efficiency in exposed (N=14) and not exposed (N=14) individuals

Chromosomal aberrations in banded preparations, and DNA repair efficiency with aphidicolin

The chromosomal bands 2q21, 3q27 and 5q31, commonly involved in hematological cancer, were most affected by acute oil exposure. The dysfunction in DNA repair mechanisms, expressed as chromosomal damage, was significantly higher in exposed-oil participants than in those not exposed

Prestige – Laffon et al. (2014)

Cross-sectional, follow-up seven years after the exposure. Genotoxic effects in individuals exposed for at least two months (N=54) and controls (N=50)

Comet assay, TCR mutation assay, MN test (both cytokinesis-block test and flow cytometry)

No significant differences were obtained between the exposed and the controls in the comet assay, the TCR mutation assay and the cytokinesis-block MN test

MN, micronucleus test; SCE, sister chromatid exchanges; TCR, T-cell receptor; VOC, volatile organic compounds.

Appendix C – Page 1

APPENDIX C – CV

Date: 10/04/2014

Name: Blanca Laffon, Ph.D. Current position: Associate Professor, University of A Coruña, spain. National identity card: 32814183-Z. Passport: AC712624. Address: Ramón del Cueto 1, 11º A izda. 15002-A Coruña, Spain. Email: blaffon@udc.es Date & place of birth: march 22, 1973. Ferrol, Spain.

EDUCATION

1996 B.S. in Pharmacy (honors and extraordinary award)

University of Santiago de Compostela, Spain

1996 Honors Degree in Pharmacy University of Santiago de Compostela, Spain

2001 Ph.D. in Pharmacy (honors and extraordinary award)

University of Santiago de Compostela, Spain

2002 Postgraduate in Genomics, Proteomics and Bioinformatics

University of Barcelona, Spain

2006 Postgraduate in Genetics and Molecular Epidemiology

Autonomous University of Barcelona, Spain

PREVIOUS AND CURRENT SCIENTIFIC OR PROFESSIONAL ACTIVITIES

1997-2000 Predoctoral fellow (Spanish Ministry of Education and Science)

Dept. Cell and Molecular Biology, University of A Coruña, Spain

2001 Predoctoral fellow (University of A Coruña)

Dept. Cell and Molecular Biology, University of A Coruña, Spain

2001-2002 Postdoctoral researcher (A Coruña county council)

Dept. Cell and Molecular Biology, University of A Coruña

2002-2003 Postdoctoral researcher (Galicia Ministry of Science)

Dept. Environmental Health, National Institute of Health, Portugal

2003 Postdoctoral researcher (University of A Coruña)

Dept. Psychology, University of A Coruña, Spain

2003-2008 Assistant Professor (tenure-track position)

Dept. Psychology, University of A Coruña, Spain

2008-2010 Associate Professor (not a civil servant) Dept. Psychology, University of A Coruña, Spain

2010-present Associate Professor (civil servant) Dept. Psychology, University of A Coruña, Spain

Appendix C – Page 2

2012 Invited researcher Clinical and Molecular Epidemiology Unit, IRCCS San Raffaele Pisana, Rome, Italy

2013 Invited Professor (EU Erasmus Program) Gazi University, Ankara, Turkey

ACADEMIC AND SCIENTIFIC PRIZES AND AWARDS

1997 Spanish Award of B.Sc. in Pharmacy Spanish Ministry of Education and Science

1997 Galicia Award of B.Sc. in Pharmacy Xunta de Galicia

1997 Award to the best Galicia Health Sciences Degree student

Caixa Galicia Foundation: Claudio Sanmartín

1997 Juan Abelló Research Award Spanish Royal Academy of Pharmacy

1997 Dolores Trigo Research Award Association of Former Students - Faculty of Pharmacy, University of Santiago de Compostela

2002 Alcalíber Research Award Spanish Royal Academy of Pharmacy

2003 Dolores Trigo Research Award Association of Former Students - Faculty of Pharmacy, University of Santiago de Compostela

2008 Research Award Galician Royal Academy of Sciences

2013 Dolores Trigo Research Award Association of Former Students - Faculty of Pharmacy, University of Santiago de Compostela

RESEARCH SUPPORT 1. Title: Cytogenetic, molecular and neurophysiological evaluation of individuals exposed to

styrene. Financial entity: Galicia Ministry of Science (XUGA 10605B98). Date: 1998-1999. Principal investigator: Eduardo Pásaro

2. Title: Differential quantification of genetic expression of p53, growing factors and Bcl-2 group, in the kidney epithelium depending on time of ischemia and kidney section. Financial entity: Galicia Ministry of Science (PGIDT01PXI1062PR). Date: 2002-2004. Principal investigator: Eduardo Pásaro.

3. Title: Collection and cleanup of Prestige oil. Evaluation of exposure and health damages in volunteers and workers. Financial entity: Arao Foundation. Date: 2003-2004. Principal investigator: Juan Jesús Gestal.

4. Title: Evaluation of genetic damage associated to occupational exposure to hydrocarbons in refineries and petrol stations

Appendix C – Page 3

Financial entity: Spanish Ministry of Work and Social Affairs (04-12752-UCO-4). Date: 2004-2006. Principal investigator: Eduardo Pásaro.

5. Title: Evaluation of human populations exposed to potential carcinogens or mutagens. Financial entity: Galicia Ministry of Science (PGIDT04PXIB10602PR). Dates: 2004-2006. Principal investigator: Eduardo Pásaro.

6. Title: Follow up and new evaluation of health in volunteers and workers involved in Prestige oil cleanup. Financial entity: Galicia Ministry of Science (INCITE08PXIB106155PR). Date: 2008-2011. Principal investigator: Eduardo Pásaro.

7. Title: Consolidation and structuring of a research network in Applied Psychology Financial entity: Galicia Ministry of Science Date: 2009-2010. Principal investigator: Manuel J. Blanco-Rial.

8. Title: Occupational exposure to formaldehyde. Genotoxic damage and susceptibility in pathology anatomy laboratory workers. Financial entity: Portuguese Science and Technology Foundation (PTDC/SAU-ESA/102367/2008) Date: 2010-2012. Principal investigator: Joao Paulo Teixeira.

9. Title: Nanotoxicology Link between India and European Nations Financial entity: European Research Area (ERA-NET) – New INDIGO Program (PIM2010ENI-00632). Date: 2010-2012. Principal investigator: Blanca Laffon.

10. Title: Study of AR, ERBETA and CYP19 gene polymorphisms, and rearrangements of X and Y chromosomes in two population of individuals with gender identity disorder. Financial entity: Spanish Ministry of Science and Innovation (PSI2010-15115). Date: 2011-2013. Principal investigator: Eduardo Pásaro.

11. Title: Risk related to exposure to metal oxide nanoparticles: cellular and molecular effects in neuronal cells Financial entity: Mapfre Foundation Date: 2012. Principal investigator: Blanca Laffon.

12. Title: Consolidation of a research network in Experimental Psychology Financial entity: Galicia Ministry of Science (CN2012/170) Date: 2012-2013. Principal investigator: Miguel Fernández del Olmo.

13. Title: Evaluation of possible neurotoxicity of superparamagnetic iron oxide nanoparticles (SPION) with biomedical applications. Financial entity: Galicia Ministry of Science (EM 2012/079). Date: 2012-2015. Principal investigator: Blanca Laffon.

Appendix C – Page 4

PUBLICATIONS Papers in international peer-reviewed journals

Scopus h-index = 20, total No. of citations = 1054 (PDF files available for personal use at my lab's website http://dicomosa.org)

1. Laffon Lage, B.; García Falcón, S.; González Amigo, S.; Lage Yusty, M. A.; Simal Lozano, J. (1997) Comparison of supercritical fluid extraction and liquid-solid extraction for the analysis of Benzo(a)pyrene in water-soluble smoke. Food Additives and Contaminants 14: 469-474.

2. García Falcón, M. S.; González Amigo, S.; Lage Yusty, M. A.; Laffon Lage, B.; Simal Lozano, J. (1999) Application of the effects of solvent and dissolved oxygen on the determination of benzo[a]pyrene by constant-wavelength synchronous spectrofluorimetry in smoke-flavouring. Talanta 48: 377-384.

3. Laffon, B.; Pásaro, E.; Méndez, J. (2001) Effects of styrene-7,8-oxide over p53, p21, bcl-2 and bax expression in human lymphocyte cultures. Mutagenesis 16: 127-132.

4. Laffon, B.; Lema, M.; Méndez, J. (2001) Simultaneous high-performance liquid chromatographic determination of urinary mandelic and phenylglyoxylic acids as indirect evaluation of styrene exposure. Journal of Chromatography B 753: 385-393.

5. Laffon, B.; Pásaro, E.; Méndez, J. (2001) Genotoxic effects of styrene-7,8-oxide in human leukocytes: comet assay in relation to the induction of sister-chromatid exchanges and micronuclei. Mutation Research 491: 163-172.

6. Laffon, B.; Pásaro, E.; Méndez, J. (2002) DNA damage and repair in human leukocytes exposed to styrene-7,8-oxide measured by the comet assay. Toxicology Letters 126: 61-68.

7. Laffon, B.; Pásaro, E.; Méndez, J. (2002) Evaluation of genotoxic effects in a group of workers exposed to low levels of styrene. Toxicology 171: 175-186.

8. Laffon, B.; Pérez-Cadahía, B.; Pásaro, E.; Méndez, J. (2003) Individual sensitivity to DNA damage induced by styrene in vitro: influence of cytochrome P450, epoxide hydrolase and glutathione S-transferase genotypes. Toxicology 186: 131-141.

9. Laffon, B.; Pérez-Cadahía, B.; Pásaro, E.; Méndez, J. (2003) Role of epoxide hydrolase and glutathione S-transferase genotypes on styrene-7,8-oxide induced micronuclei and DNA damage. Mutation Research 536: 49-59.

10. Teixeira, J.P.; Gaspar, J.; Silva, S.; Torres, J.; Silva, S.N.; Azevedo, M.C.; Neves, P.; Laffon, B.; Méndez, J.; Gonçalves, C.; Mayan, O.; Farmer, P.; Rueff, J. (2004) Occupational exposure to styrene: modulation of cytogenetic damage and levels of urinary metabolites of styrene by polymorphisms in genes CYP2E1, EPHX1, GSTM1, GSTT1 and GSTP1. Toxicology 195: 231-242.

11. Pérez-Cadahía, B.; Laffon, B.; Pásaro, E.; Méndez, J.(2004) Evaluation of PAH bioaccumulation and DNA damage in mussels (Mytilus galloprovincialis) exposed to spilled Prestige crude oil. Comparative Biochemistry and Physiology 138: 453-460.

12. Laffon, B.; Teixeira, J.P.; Silva, S.; Loureiro, J.; Torres, J.; Pásaro, E.; Méndez, J.; Mayan, O. (2005) Genotoxic effects in a population of nurses handling antineoplastic drugs, and relationship with genetic polymorphisms in DNA repair enzymes. American Journal of Industrial Medicine 48: 128-136.

Appendix C – Page 5

13. Laffon, B.; Rábade, T.; Pásaro, E.; Méndez, J. (2006) Monitoring of the impact of Prestige oil spill on Mytilus galloprovincialis from Galician coast. Environment International 32: 342-348.

14. Roma-Torres, J.; Teixeira, J.P.; Silva, S.; Laffon, B.; Cunha, L.; Méndez, J.; Mayan, O. (2006) Evaluation of genotoxicity in a group of workers from a petroleum refinery aromatics plant. Mutation Research 604: 19-27.

15. Kirsch-Volders, M.; Tremp, A.; Bonassi, S.; Roelants, M.; Holland, N.; Chang, W.P.; Zeiger, E.; Aka, P.; Godderis, L.; Ishikawa, H.; Laffon, B.; Leopardi, P.; Lucero, L.; Mateuca, R.; Migliore, L.; Norppa, H.; Pitarque, M.; Teixeira, J.P.; Fenech, M. (2006) The effects of GSTM1 and GSTT1 polymorphisms on micronucleus frequencies in human lymphocytes in vivo. Cancer Epidemiology Biomarkers and Prevention 15: 1038-1042.

16. Laffon, B.; Aldao, I.; Pérez-Cadahía, B; Pásaro, E.; Méndez, J. (2006) First step in the evaluation of the effects of Prestige oil on the shore environment: availability, bioaccumulation and DNA damage. Ciencias Marinas 32: 389-399.

17. Laffon, B.; Fraga-Iriso, R.; Pérez-Cadahía, B.; Méndez, J. (2006) Genotoxicity associated to exposure to Prestige oil during autopsies and cleaning of oil-contaminated birds. Food and Chemical Toxicology 44: 1714-1723.

18. Laffon, B.; Teixeira, J.P.; Silva, S.; Roma-Torres, J.; Pérez-Cadahía, B.; Méndez, J.; Pásaro, E.; Mayan, O. (2006) Assessment of occupational genotoxic risk in the production of rubber tyres. Annals of Occupational Hygiene 50: 583-592.

19. Pérez-Cadahía, B.; Laffon, B.; Pásaro, E.; Méndez, J. (2006) Genetic damage induced by accidental environmental pollutants. TheScientificWorldJournal 6: 1221-1237.

20. Costa, C.; Teixeira, J.P.; Silva, S.; Roma-Torres, J.; Coelho, P.; Gaspar, J.; Alves, M.; Laffon, B.; Rueff, J.; Mayan, O. (2006) Cytogenetic and molecular biomonitoring of a Portuguese population exposed to pesticides. Mutagenesis 21: 343-350.

21. Pérez-Cadahía, B.; Lafuente, A.; Cabaleiro, T.; Pásaro, E.; Méndez, J.; Laffon, B. (2007) Initial study on the effects of Prestige oil on human health. Environment International 33: 176-185.

22. Pérez-Cadahía, B.; Valdiglesias, V.; Pásaro, E.; Méndez, J.; Laffon, B. (2007) Genetic polymorphism in cytochrome P450 1B1 in a Spanish population. Basic and Clinical Pharmacology and Toxicology 101: 70-72.

23. Valdes, F.; Pasaro, E.; Diaz, I.; Centeno, A.; Lopez, E.; Garcia-Doval, S.; Gonzalez-Roces, S.; Alba, A.; Laffon, B. (2008) Segmental heterogeneity in bcl-2, bcl-xl and bax expression in rat tubular epithelium after ischemia-reperfusion. Nephrology 13: 294-301.

24. Pérez-Cadahía, B.; Méndez, J.; Pásaro, E.; Lafuente, A.; Cabaleiro, T.; Laffon, B. (2008) Biomonitoring of human exposure to Prestige oil: Effects on DNA and endocrine parameters. Environmental Health Insights 2: 83-92.

25. Perez-Cadahia, B.; Laffon, B.; Porta, M.; Lafuente, A.; Cabaleiro, T.; Lopez, T.; Caride, A.; Pumarega, J.; Romero, A.; Pasaro, E.; Mendez, J. (2008) Relationship between blood concentrations of heavy metals and cytogenetic and endocrine parameters among subjects involved in cleaning coastal areas affected by the `Prestige' tanker oil spill. Chemosphere 71: 447-455.

26. Pérez-Cadahía, B.; Laffon, B.; Valdiglesias, V.; Pásaro, E.; Méndez, J. (2008) Cytogenetic effects induced by Prestige oil on human populations: The role of

Appendix C – Page 6

polymorphisms in genes involved in metabolism and DNA repair. Mutation Research 653: 117-123.

27. Pérez-Cadahía, B.; Méndez, J.; Pásaro, E.; Valdiglesias, V.; Laffon, B. (2009) DNA damage related to exposure to oil spills: the Prestige experience. International Journal of Medical and Biological Frontiers 15: 1-31.

28. Laffon, B.; Valdiglesias, V.; Pásaro, E.; Méndez, J. (2010) The organic selenium compound selenomethionine modulates bleomycin-induced DNA damage and repair. Biological Trace Element Research 133: 12-19.

29. García-Lestón, J.; Méndez, J.; Pásaro, E.; Laffon, B. (2010) Genotoxicity evaluation of exposure to lead. International Journal of Medical and Biological Frontiers 16: 201-330.

30. Valdiglesias, V.; Pásaro, E.; Méndez, J.; Laffon, B. (2010) In vitro evaluation of selenium genotoxic, cytotoxic and protective effects – A review. Archives of Toxicology 84: 337-351.

31. Aguilera, F.; Méndez, J.; Pásaro, E.; Laffon, B. (2010) Review on the effects of the exposure to spilled oils on human health. Journal of Applied Toxicology 30: 291-301.

32. Valdiglesias, V.; Méndez, J.; Pásaro, E.; Laffon, B. (2010) The importance of the in vitro genotoxicity evaluation of food components: the selenium. International Journal of Medical and Biological Frontiers 16: 339-378.

33. García-Lestón, J.; Méndez, J.; Pásaro, E.; Laffon, B. (2010) Genotoxic effects of lead: an updated review. Environment International 36: 623-636.

34. Valdiglesias, V.; Méndez, J.; Pásaro, E.; Cemeli, E.; Anderson, D.; Laffon, B. (2010) Assessment of okadaic acid effects on cytotoxicity, DNA damage and DNA repair in human cells. Mutation Research 689: 74-79.

35. Teixeira, J.P.; Gaspar, J.; Coelho, P.; Moreira, A.; Costa, C.; Pinho-Silva, S.; Costa, S.; da Silva, S.; Laffon, B.; Rueff, J.; Farmer, P. (2010) Cytogenetic and DNA damage in workers exposed to styrene. Mutagenesis 25: 617-621.

36. Valdiglesias, V.; Laffon, B.; Pásaro, E.; Méndez, J. (2011) Evaluation of okadaic acid-induced genotoxicity in human cells by micronucleus test and H2AX analysis. Journal of Toxicology and Environmental Health, part A 74: 980-992.

37. García-Lestón, J.; Roma-Torres, J.; Vilares, M.; Pinto, R.; Cunha, L.M.; Prista, J.; Teixeira, J.P.; Mayan, O.; Pásaro, E.; Méndez, J.; Laffon, B. (2011) Biomonitoring of a population of Portuguese workers exposed to lead. Mutation Research 721: 81-88.

38. Fernández-Tajes, J.; Rábade, T.; Laffon, B.; Méndez, J. (2011) Monitoring follow up of two areas affected by the Prestige oil four years after the spillage. Journal of Toxicology and Environmental Health, part A 74: 1067-1075.

39. Valdiglesias, V.; Laffon, B.; Pásaro, E.; Cemeli, E.; Anderson, D.; Méndez, J. (2011) Induction of oxidative damage by the marine toxin okadaic acid depends on cell type. Toxicon 57: 882-888.

40. Pereira, S.; Fernández-Tajes, J.; Rábade, T.; Flórez-Barrós, F.; Laffon, B.; Méndez, J. (2011) Comparison between two bivalve species as tools for the assessment of the pollution level in an estuary environment. Journal of Toxicology and Environmental Health, part A 74: 1020-1029.

Appendix C – Page 7

41. Mendes, A.; Madureira, J.; Neves, P.; Carvalhais, C.; Laffon, B.; Teixeira, J.P. (2011) Chemical exposure and occupational symptoms among Portuguese hairdressers. Journal of Toxicology and Environmental Health, part A 74: 993-1000.

42. Fernández-Tajes, J.; Flórez, F.; Pereira, S.; Rábade, T.; Laffon, B.; Méndez, J. (2011) Use of three bivalve species for biomonitoring a polluted estuarine environment. Environmental Monitoring and Assessment 177: 289-300.

43. Valdiglesias, V.; Pásaro, E.; Mendez, J.; Laffon, B. (2011) Assays to determine the DNA repair ability. Journal of Toxicology and Environmental Health, part A 74: 1094-1109.

44. Valdiglesias, V.; Laffon, B.; Pásaro, E.; Méndez, J. (2011) Okadaic acid induces morphological changes, apoptosis and cell cycle alterations in different human cell types. Journal of Environmental Monitoring 13: 1831-1840.

45. Costa, C.; Silva, S.; Neves, J.; Coelho, P.; Costa, S.; Laffon, B.; Snawder, J.; Teixeira, J.P. (2011) Micronuclei in reticulocytes and micronuclei in lymphocytes: Biomonitoring of a pesticide exposed population. Journal of Toxicology and Environmental Health, part A 74: 960-970.

46. Dhawan, A.; Shanker, R.; Laffon, B.; Fernández-Tajes, J.; Fuchs, D.; van der Laan, G.; van Broekhuizen, P.; Becker, H.; Moriske, H.-J.; Teixeira, J.P.; Carriere, M.; Herlin-Boime, N.; Engin, A.B.; Coskun, E.; Karahalil, B. (2011) NanoLINEN: Nanotoxicology link between India and European Nations. Journal of Biomedical Nanotechnology 7: 203-204.

47. Coelho, P.; García-Lestón, J.; Silva, S.; Costa, C.; Costa, S.; Coelho, M.I.; Laffon, B.; Pásaro, E.; Teixeira, J.P. (2011) Geno- and immunotoxic effects on populations living near a mine - case study: Panasqueira mine. Journal of Toxicology and Environmental Health, part A 74: 1076-1086.

48. Costa, S.; Pina, C.; Coelho, P.; Costa, C.; Silva, S.; Vasconcelos, M.B.; Laffon, B.; Teixeira, J.P. (2011) Occupational exposure to formaldehyde: genotoxic risk evaluation by comet assay and micronucleus test. Journal of Toxicology and Environmental Health, part A 74: 1040-1051.

49. Valdiglesias, V.; Fernández-Tajes, J.; Pásaro, E.; Méndez, J. Laffon, B. (2012) Identification of differentially expressed genes in SHSY5Y cells exposed to okadaic acid by suppression subtractive hybridization. BMC Genomics 13: 46.

50. García-Lestón, J.; Roma-Torres, J.; Vilares, M.; Pinto, R.; Prista, J.; Teixeira, J.P.; Mayan, O.; Conde, J.; Pingarilho, M.; Gaspar, J.F.; Pásaro, E.; Méndez, J.; Laffon, B. (2012) Genotoxic effects of occupational exposure to lead and influence of polymorphisms in genes involved in lead toxicokinetics and in DNA repair. Environment International 43: 29-36.

51. Costa, C.; Costa, S.; Silva, S.; Coelho, P.; Botelho, M.; Gaspar, J.; Rueff, J.; Laffon, B.; Teixeira, J.P. (2012) DNA damage and susceptibility assessment in industrial workers exposed to styrene. Journal of Toxicology and Environmental Health, part A 75: 735-746).

52. Valdiglesias, V., Kiliç G., Costa, C.; Amor-Carro, O., Mariñas-Pardo, L., Ramos-Barbón, D., Méndez, J., Pásaro, E., Laffon, B. (2012) In vivo genotoxicity assessment in rats exposed to Prestige-like oil by inhalation. Journal of Toxicology and Environmental Health, part A 75: 756-764.

Appendix C – Page 8

53. Forchhammer, L., Ersson, C., Loft, S., Möller, L., Godschalk, R.W.L., Jones, G.D.D., Higgins, J.A., Cooke, M., Karbaschi, M., Collins, A.R., Azqueta, A., Phillips, D.H., Sozeri, O., Routledge, M.N., Nelson-Smith, K., Riso, P., Porrini, M., Matullo, G., Allione, A., Stepnik, M., Komorowska, M., Teixeira, J.P., Costa, S., Corcuera, L.A. López de Cerain, A., Laffon, B., Valdiglesias., V., Møller P. (2012) Reduction of inter-laboratory variation in DNA damage measured by a reference comet assay protocol. Mutagenesis 27: 665-672.

54. Valdiglesias, V.; Fernández-Tajes, J.; Costa, C.; Pásaro, E.; Méndez, J. Laffon, B. (2012) Alterations in metabolism-related genes induced in SHSY5Y cells by okadaic acid exposure. Journal of Toxicology and Environmental Health, part A 75: 844-856.

55. Moreira, A.O.; Almeida, A.; Costa, S.; Laffon, B.; García-Lestón, J.; Pásaro, E.; Méndez, J.; Teixeira, J.P. (2012) Genotyping an ALAD polymorphism with real-time PCR in two populations from the Iberian Peninsula. Biochemical Genetics 50: 560-564.

56. Coelho, P.; Costa, S.; Silva, S.; Walter, A.; Ranville, J.F., Sousa, A.C.A.; Costa, C.; Coelho, M.I.; García-Lestón, J.; Pastorinho, M.R.; Laffon, B.; Pásaro, E.; Harrington, C.; Taylor, A.; Teixeira, J.P. (2012) Metal(loid)s levels in biological matrices from human populations exposed to mining contamination - Panasqueira Mine area. Journal of Toxicology and Environmental Health, part A 75: 893-908.

57. García-Lestón, J.; Roma-Torres, J.; Mayan, O.; Oliveira, A.O.; Teixeira, J.P.; Schroecksnadel, S.; Fuchs, D.; Pásaro, E.; Méndez, J.; Laffon, B. (2012) Assessment of immunotoxicity parameters in individuals occupationally exposed to lead. Journal of Toxicology and Environmental Health, part A 75: 807-818.

58. Valdiglesias, V.; Fernández-Tajes, J.; Pásaro, E.; Méndez, J.; Laffon, B. (2013) Okadaic acid induces alterations in the expression level of cancer-related genes. Ecotoxicology and Environmental Safety 92: 303-311.

59. Ersson, C., Møller P. Forchhammer, L., Loft, S., Azqueta, A., Godschalk, R.W.L., van Schooten, F., Jones, G.D.D., Higgins, J.A., Cooke, M., Mistry, V., Karbaschi, M., Phillips, D.H., Sozeri, O., Routledge, M.N., Nelson-Smith, K., Riso, P., Porrini, M., Matullo, G., Allione, A., Stepnik, M., Komorowska, M., Teixeira, J.P., Costa, S., Corcuera, L.A. López de Cerain, A., Laffon, B., Valdiglesias., V., Collins, A., Möller, L. (2013) An ECVAG inter-laboratory validation study of the comet assay: inter-laboratory and intra-laboratory variation of DNA strand breaks and FPG-sensitive sites in human mononuclear cells. Mutagenesis 28: 279-286.

60. Valdiglesias, V.; Costa, C.; Sharma, V.; Kiliç, G.; Pásaro, E.; Teixeira, J.P.; Dhawan, A.; Laffon, B. (2013) Comparative study on effects of two different types of titanium dioxide nanoparticles on human neuronal cells. Food and Chemical Toxicology 57: 352-361.

61. Valdiglesias, V.; Costa, C.; Kiliç, G.; Costa, S.; Pásaro, E.; Laffon, B.; Teixeira, J.P. (2013) Neuronal cytotoxicity and genotoxicity induced by zinc oxide nanoparticles. Environment International 55: 92-100.

62. Costa S., García-Lestón J., Coelho M., Coelho P., Costa C., Silva S., Porto B., Laffon B., Teixeira J.P. (2013) Cytogenetic and immunological effects of formaldehyde in a group of exposed workers. Journal of Toxicology and Environmental Health 76: 217-229.

63. Laffon, B.; Aguilera, F.; Ríos-Vázquez, J.; García-Lestón, J.; Fuchs, D.; Valdiglesias, V.; Pásaro, E. (2013) Endocrine and immunological parameters in individuals involved in Prestige spill cleanup tasks seven years after the exposure. Environment International 59: 103-111.

Appendix C – Page 9

64. Valdiglesias, V.; Prego-Faraldo, V.; Pásaro, E.; Méndez, J.; Laffon, B. (2013) Okadaic acid: much more than a diarrheic toxin. Marine Drugs 11: 4328-4349.

65. Coelho P.; García-Lestón J.; Costa S.; Costa C.; Silva S.; Dall'Armi V.; Zoffoli R.; Bonassi S.; Pereira de Lima J.; Gaspar J.F.; Pásaro E.; Laffon B.; Teixeira J.P. (2013) Genotoxic effect of environmental and occupational exposure to metal(loid)s. A molecular epidemiology survey of populations living and working in the Panasqueira Mine area, Portugal. Environment International 60: 163-170.

66. Coelho P.; Costa S.; Costa C.; Silva S.; Walter A.; Ranville J.; Pastorinho M.R.; Harrington C.; Taylor A.; Dall'Armi V.; Zoffoli R.; Candeias C.; Ferreira da Silva E.; Bonassi S.; Laffon B.; Teixeira J.P. (2014) Impact of Panasqueira mine activities on populations environmentally and occupationally exposed – quantification of several metal(loid)s in different biological matrices. Environmental Geochemistry and Health 36: 255-269.

67. Laffon, B.; Aguilera, F.; Ríos-Vázquez, J.; Valdiglesias, V.; Pásaro, E. (2014) Follow-up study of genotoxic effects in individuals exposed to Prestige oil seven years later. Mutation Research 760: 10-16.

68. Rodríguez-Lombardero, S.; Rodríguez-Belmonte, E.; González-Siso, M.I.; Vizoso-Vázquez, A.; Valdiglesias, V.; Laffon, B.; Cerdán, M.E. (2014) Proteomic analyses reveal that Sky1 modulates apoptosis and mitophagy in Saccharomyces cerevisiae cells exposed to cisplatin. International Journal of Molecular Sciences 15: 12573-12590.

69. Godschalk, R.W.L.; Ersson, C.; Stepnik, M.; Ferlinska, M.; Palus, J.; Teixeira, J.P.; Costa, S.; Jones, G.D.D.; Higgins, J.A.; Kain, J.; Moller, L.; Forchhammer, L.; Loft, S.; Lorenzo, Y.; Collins, A.R.; van Schooten, F.-J.; Laffon, B.; Valdiglesias, V.; Cooke, M.; Mistry, V.; Karbaschi, M.; Phillips, D.H.; Sozeri, O.; Routledge, M.N.; Nelson-Smith, K.; Riso, P.; Porrini, M.; López de Cerain, A.; Azqueta, A.; Matullo, G.; Allione, A.; Moller, P. (2014) Variation of DNA damage levels in peripheral blood mononuclear cells isolated in different laboratories. Mutagenesis 29: 241-249.

70. Coelho, P.C.; García-Lestón, J.; Costa, S.; Costa, C.; Silva, S.; Fuchs, D.; Geisler, S.; Dall'Armi, V.; Zoffoli, R.; Bonassi, S.; Pásaro, E.; Laffon, B.; Teixeira, J.P. (2014) Immunological alterations in individuals exposed to metal(loid)s in the Panasqueira mining area, Central Portugal. Science of the Total Environment 475: 1-7.

71. Costa, C.; García-Lestón, J.; Costa, S.; Coelho, P.C.; Silva, S.; Pingarilho, M.; Valdiglesias, V.; Mattei, F.; Dall’Armi, V.; Bonassi, S.; Laffon, B.; Snawder, J.; Teixeira, J.P. (2014) Is organic farming safer to farmers' health? A comparison between organic and traditional farming. Toxicology Letters (in press, DOI: 10.1016/j.toxlet.2014.02.011).

72. Valdiglesias, V.; Kiliç, G.; Costa, C.; Fernández-Bertólez, N.; Pásaro, E.; Teixeira, J.P.; Laffon, B. (2014) Effects of iron oxide nanoparticles: cytotoxicty, genotoxicity, developmental toxicity and neurotoxicity. Environmental and Molecular Mutagenesis (in press, DOI: 10.1002/em).

Papers in national peer-reviewed journals 1. Laffon-Lage, B.; Pérez Cadahía, B.; Méndez Felpeto, J. (2004) Influencia de

determinados polimorfismos de enzimas metabólicas en la genotoxicidad del estireno [Influence of certain polymorphims in metabolic enzymes on styrene genotoxicity, in Spanish]. Anales de la Real Academia Nacional de Farmacia 70: 95-123.

Appendix C – Page 10

2. Laffon, B.; Pérez-Cadahía, B.; Loureiro, J.; Méndez, J.; Pásaro, E. (2004) Papel de los polimorfismos genéticos para enzimas de reparación en el daño en el ADN inducido por el estireno y estireno-7,8-óxido [Role of genetic polymorphisms in DNA repair enzymes on DNA damage induced by styrene and styrene-7,8-oxide, in Spanish]. Revista de Toxicología 21: 92-97.

3. Pérez Cadahía, B.; Laffon-Lage, B.; Méndez Felpeto, J. (2007) Biomonitorización de la exposición ocupacional a hidrocarburos aromáticos presentes en el ambiente de refinerías [Biomonitoring of occupational exposure to aromatic hydrocarbons in refineries, in Spanish]. Mapfre Seguridad 106: 18-26.

4. García-Lestón, J.; Laffon, B.; Roma-Torres, J.; Teixeira, J.P.; Monteiro, S.; Pásaro, E.; Prista, J.; Mayan, O.; Méndez, J. (2008) Efectos genotóxicos e inmunotóxicos de la exposición laboral al plomo [Genotoxic and immunotoxic effects of occupational exposure to lead, in Spanish]. Archivos de Prevención de Riesgos Laborales. 11: 124-130.

5. Valdiglesias, V.; Laffon, B.; Pásaro, E.; Méndez, J. Evaluación del efecto de la selenometionina sobre la reparación del daño en el ADN [Assessment of selenomethionine effect on DNA damage repair, in Spanish]. (2010) Revista Real Academia Gallega de Ciencias XXVII: 41-94.

6. Valdiglesias, V.; Costa, C.; Kiliç, G.; Costa, S.; Teixeira, J.P.; Pásaro, E.; Laffon, B. (2013). Toxicidad celular inducida por nanopartículas de óxidos metálicos en células neuronales humanas [Cell toxicity induced by metal oxide nanoparticles on human neuronal cells, in Spanish]. Seguridad y Medio Ambiente 130: 34-45.

Book chapters 1. Laffon, B.; Pérez-Cadahía, B.; Pásaro, E.; Méndez, J. (2007) Human health: The great

forgotten after accidental oil spills. In: Environmental research advances. Clarkson, P.A. (Ed.). New York: Nova Science Publishers. Pp. 1-3.

2. Pérez-Cadahía, B.; Méndez, J.; Pásaro, E.; Valdiglesias, V.; Laffon, B. (2008) DNA damage related to exposure to oil spills: the Prestige experience. In: Progress in DNA damage research. Miura, S.; Nakano, S. (Eds.). New York: Nova Science Publishers. Pp: 165-198.

3. García-Lestón, J.; Méndez, J.; Pásaro, E.; Laffon, B. (2009) Genotoxicity evaluation of exposure to lead. In: Genotoxicity: evaluation, testing and prediction. Kocsis, A.; Molnar, H. (Eds.). New York: Nova Science Publishers. Pp: 75-105.

4. Valdiglesias, V.; Méndez, J.; Pásaro, E.; Laffon, B. (2009) The importance of the in vitro genotoxicity evaluation of food components: the selenium. In: Genotoxicity: evaluation, testing and prediction. Kocsis, A.; Molnar, H. (Eds.). New York: Nova Science Publishers. Pp: 1-38.

5. Fernández-Tajes, J.; Laffon, B.; Méndez, J. (2010) The use of biomarkers in bivalve molluscs for the evaluation of marine environment pollution. In: Impact, monitoring and management of environmental pollution. El Nemr, A. (Ed.). New York: Nova Science Publishers. Pp: 409-429.

6. Laffon, B. (2010) Research on the human health effects of exposure after oil spills: the Prestige experience. In: Assessing the effects of the Gulf of Mexico oil spill on human health. Washington, D.C.: The National Academies Press. Pp: 22-24.

Appendix C – Page 11

7. Laffon, B. (2014) Fuel oils. In: Encyclopedia of Toxicolgy, (3rd Edition, volume 2). Wexler, P. (Ed.). New York: Elsevier. Pp: 667-670.

8. Valdiglesias, V.; Laffon, B., Fernández-Tajes, J.; Méndez, J. (2014) Okadaic acid. In: Encyclopedia of Toxicolgy, (3rd Edition, volume 3). Wexler, P. (Ed.). New York: Elsevier. Pp: 682-683.

9. Valdiglesias, V.; Sánchez-Flores, M.; Cemeli, E.; Anderson, D.; Pásaro, E.; Méndez, J.; Laffon, B. (2014) Assessment of cellular and molecular toxicity by in vitro tests: the okadaic acid toxin case. In: Shellfish. Human consumption, health implications and conservation concerns. Hay, R.M. (Ed.). New York: Nova Science Publishers. Pp: 89-139.

Books Laffon-Lage, B.; Lage Yusty, M. A.; Simal Lozano, J.; García Falcón, M. S.; González

Amigo, S. (1997) Application of supercritical fluids to benzo(a)pyrene extraction [in Spanish]. Santiago de Compostela: Santiago University Press.

Laffon, B.; Pásaro, E. (2012) Congenital bases for language alterations [in Spanish]. Madrid: Bubok.

Laffon, B.; Valdiglesias, V.; Pásaro, E. (2014) Gene therapy [in Spanish]. Madrid: La Catarata-CSIC (in press).

PARTICIPATION IN RESEARCH / EXPERTS NETWORKS

Member of working groups of the following European COST (Cooperation in Science and Technology) Actions:

TD1204 Modelling of nanomaterials.

MP1106 Smart green interfaces.

IS1402 Ageism – A multi-national, interdisciplinary perspective

Member of the World Health Organization (WHO) Chemical Risk Assessment Network.

SUPERVISING EXPERIENCE

PhD Thesis Supervisor

1. "Toxicological evaluation of human populations exposed to Prestige oil". Beatriz Pérez-Cadahía. University of A Coruña. 2006. Qualification: with honors and extraordinary award.

2. "Biomarkers of pollution in the marine environment: cytogenetic and molecular tools". Tamara Rábade. University of A Coruña. 2010. Qualification: with honors.

3. "Evaluation of cytogenetic and molecular effects of okadaic acid in human cell lines". Vanessa Valdiglesias. University of A Coruña. 2010. Qualification: with honors and extraordinary award.

4. "Occupational exposure to lead: genotoxic and immunotoxic effects". Julia García-Lestón. University of A Coruña. 2012. Qualification: with honors and extraordinary award.

Appendix C – Page 12

5. “Follow up of health effects in people involved in Prestige tanker oil spill cleanup seven years after the accident”. Julia Ríos Vázquez. University of A Coruña. 2013. Qualification: with honors.

6. “Effects of different types of nanoparticles in human cell lines”. Gözde Kiliç. University of A Coruña. In progress since 2011.

7. “Development of new clinic and molecular tools for frailty identification in the elderly”. María Sánchez-Flores. University of A Coruña. In progress since 2013.

8. “Cellular and molecular biomarkers associated with frailty in the elderly”. Diego Marcos. University of A Coruña. In progress since 2014.

Master Thesis supervisor

1. “Experimental genotoxicity study in rats exposed to a Prestige-like oil. Gözde Kiliç. University of a Coruña. 2011. Qualification: Excellent A.

2. “Assessment of genotoxicity related to exposure to metal oxide nanoparticles in neuronal cells”. Aida Castelo. 2014. Qualification: Excellent A.

3. “Study of cellular and molecular effects of iron oxide nanoparticles on human neuronal cells”. Natalia Fernández-Bertólez. In progress since 2014.

Honors Degree supervisor

“Influence of several genetic polymorphisms in metabolic enzymes on styrene genotoxicity". Beatriz Pérez-Cadahía. University of A Coruña. 2002. Qualification: Excellent A.

“Evaluation of genotoxic damage in nurses exposed to antineoplastic drugs". Jesús Loureiro. University of A Coruña. 2003. Qualification: Excellent A.

“Evaluation of genotoxic effects in volunteers exposed to Prestige oil during autopsies and cleaning of oil-contaminated birds". Rebeca Fraga-Iriso. University of A Coruña. 2005. Qualification: Excellent A.

“Experimental study on the effects of Prestige oil on the mussel Mytilus galloprovincialis". Iria Aldao. University of A Coruña. 2005. Qualification: Excellent A.

“Evaluation of genotoxic damage in Mytilus galloprovincialis of A Coruña shore". Tamara Rábade. University of A Coruña. 2005. Qualification: Excellent A.

“Evaluation of the effect of selenomethionine on DNA damage repair". Vanessa Valdiglesias. University of A Coruña. 2007. Qualification: Excellent A.

“Evaluation of the toxic effects associated with occupational exposure to lead". Julia García-Lestón. University of A Coruña. 2007. Qualification: Excellent A.

TEACHING EXPERIENCE

University of A Coruña (Spain)

B.S. in Biology

1997-2007 Teaching Assistant: Cytogenetics 2004-2008 Co-lecturer: Environmental toxicology and public health

Appendix C – Page 13

2008-2011 Lecturer: Environmental toxicology and public health

B.S. in Speech Therapy

2004-present Co-lecturer: Congenital alterations of language

M.S. in Molecular Biology and Genetics

2009-present Lecturer: Genetic toxicology

M.S. in Applied Psychology

2009-present Lecturer: Psychopharmacology

M.A. in Dependency and Social Services Administration

2003-present Lecturer: Psychopharmacology in dependence

Ph.D. Program in Genetics, Biochemistry and Biotechnology

2003-2009 Co-lecturer: Use of biomarkers in human population studies

Ph.D. Program in Health Promotion

2007-2008 Lecturer: Toxic response and assessment of toxicity

Other seminars and courses

Seminar on “Tools in Genetic Toxicology”. Continuous formation course on Pneumological markers, organized by the Pneumology Department, A Coruña University Hospital (Spain). 2008.

Seminar on “Genotoxicity biomarkers”. Advanced Toxicology Course, organized by the European Society of Toxicology (EUROTOX). 2012.

Seminar on “Molecular Toxicology”. Course on Introduction to biomedical research applied to respiratory pathology, organized by the Pneumology Department, A Coruña University Hospital (Spain). 2013.

Seminar on “Biomonitoring health effects of exposure to oil spills”. Biomarkers 2014 – Spring school on human population studies with genetic biomarkers, organized by the Public Health Institute, University of Porto (Portugal). 2014

PARTICIPATION IN SCIENTIFIC COMMITTEES

XIX Meeting of the Spanish Mutagen Society. A Coruña (Spain). October 2010.

International Conference on Occupational and Environmental Health (ICOEH2011). Porto (Portugal). October 2011.

Nanosafety Congress. Antalya (Turkey). April 2012.

International Congress on Environmental Health (ICEH 2012). Lisbon (Portugal). May 2012.

2nd Iberoamerican Meeting on Toxicology and Environmental Health (IBAMTOX 2013). Sao Paulo (Brasil). June 2013.

2nd International Conference on Occupational and Environmental Toxicology (ICOETox 2013). Porto (Portugal). September 2013.

Appendix C – Page 14

10th International Comet Assay Workshop (ICAW 2013). Porto (Portugal). September 2013.

EDITORIAL SERVICE

Biomed Research International (Public Health area). Associate Editor (2013-present).

Environmental and Analytical Toxicology. Associate Editor (2013-present).

FABAD Journal of Pharmaceutical Sciences. Associate Editor (2011-present).

Journal of Environmental Medicine. Associate Editor (2013-present).

Journal of Toxicology and Environmental Health, part A. Guest Editor for Vol. 74, nº 15-16 (2011), and Vol. 75, nº 13-14 (2012).

Journal of Translational Toxicology. Associate Editor (2012-present).

Marine Drugs. Guest Editor for the special issue “Cytogenetic and Molecular Effects of Marine Compounds” (2014).

RESEARCH PROJECTS REVIEWER

Executive Agency for Higher Education, Research, Development and Innovation Funding, Romania (2012-present).

Spanish Agency for Normalization and Certification (AENOR) (2012).

UK Medical Research Council (2013).

National Centre of Science and Technology Evaluation, Kazakhstan (2013-present).

JOURNAL REVIEWER

American Journal of Industrial Medicine; Carcinogenesis; Chemosphere; Comparative Biochemistry and Physiology; Environment International; Environmental and Molecular Mutagenesis; Environmental Health Perspectives; Environmental Research; Environmental Toxicology and Pharmacology; Food and Chemical Toxicology; Journal of Food Science; Nephrology; Journal of Nanomaterials & Molecular Nanotechnology; Journal of Occupational Health; Journal of Toxicology and Environmental Health; Journal of Translational Toxicology; Molecular Biology Reports; Gene; Mutation Research; Cancer Therapy; Nanotoxicology; Occupational and Environmental Medicine; Pteridines; Science of the Total Environment; Toxicology and Applied Pharmacology; Toxicology and Industrial Health; Toxicology in Vitro; Toxicology; Toxins; Urban Climate.

PROFESSIONAL MEMBERSHIPS

Spanish Society of Genetics (SEG)

Spanish Environmental Mutagen Society (SEMA)

European Environmental Mutagen Society (EEMS)

INVITED TALKS (most relevant)

2007 Biomonitoring human populations after oil spills: the Prestige experience. 4th Sea

Appendix C – Page 15

Alarm Conference. Ostende (Belgium). October.

2009 Biomonitoring of individuals exposed to Prestige oil. ECNICS workshop on biomarkers and cancer. Porto (Portugal). September.

2010 The compelling need to understand the effects of oil spills on human health. IOM Workshop on Assessing the human health effects of the Gulf of Mexico oil spill. New Orleans (LA-USA). June.

2011 Application of flow cytometry MN test to the evaluation of nanomaterials genotoxicity. International Symposium on the Safe Use of Nanomaterials. Lucknow (India). February.

2011 Follow up study of genotoxic effects in individuals exposed to Prestige oil. 41st Annual Meeting of the European Environmental Mutagen Society. Barcelona (Spain). July.

2011 Human health effects in individuals exposed to spilled oils. International Conference on Occupational and Environmental Health. Porto (Portugal) October.

2012 Human genotoxicity evaluation seven years after Prestige oil spill. Nanosafety Congress Turkey – Workshop on Genotoxicity tests to assess human toxicity. Antalya (Turkey). April.

2012 New biomonitoring of Prestige oil exposure seven years after the spill. European Congress of Epidemiology (EUROEPI 2012). Porto (Portugal). September.

2012 Human health effects of exposure to oil spills. Maritime Safety Conference. 10 years after Prestige sinking. Biarritz (France). November.

2013 Endocrine and immmunological biomonitoring of individuals exposed to Prestige oil seven years later. 2nd International Conference on Occupational and Environmental Toxicology (ICOETox 2013). Porto (Portugal). September.

2014 In vitro analysis of neuronal DNA damage induced by magnetite nanoparticles. International Conference on Environmental Health (ICEH2014). Porto (Portugal). September.

SCIENTIFIC CONFERENCES ORGANIZED

XIX Meeting of the Spanish Mutagen Society (SEMA2010). President of the Organizing Committee. A Coruña (Spain). October 2010.

International Symposium on the Safe Use of Nanomaterials and Workshop on Nanomaterial Safety: Status, Procedures, Policy and Ethical Concerns (SUN2011). Member of the International Organizing Committee. Lucknow (India). February 2011.

International Conference on Occupational and Environmental Health (ICOEH2011). Co-president of the Organizing Committee. Porto (Portugal). October 2011.