Anne-Marie Duguet

Emmanuelle Rial-Sebbag

Aurelie Mahalachtimy

Mou Li

Anne Cambon- Thomsen

Ethical and legal frameworks for embryonic stem-cell based research in France and in Europe: a challenge for biotechnology

Keywords:

Developing research based on the use of biological material raises the question of the sources of the cells. Should we use human stem cells or embryonic stem cells? For human stem cells, since 1994, the French law considers the human body is inviolable and cannot be subject of property (Art 16-1 of the Civil Code) that means no trade and no patent. Focusing on embryos, the Oviedo convention (Council of Europe) on Human rights and biomedicine (1997) says in Art 18: it is up to each country to authorize or not the embryo research. There are differences among countries in Europe, from a total ban to an approval of the research with nuclear transfer. In France, several opinions of the French National Ethics committee (CCNE) claimed for the protection of the embryo. Regarding commercialization of cells and patentability, the European Directive 98/44.CE on legal protection of biological inventions says: Biotechnology inventions are essential for the development of the community; only adequate protection can make them profitable and facilitate trade. The national patent law remains the reference. In France, in 2006 research on embryo and embryonic stem cells was organized by the decree of April 27 th , 2006 under the control of the French National biomedicine Agency. Even if the research on embryo was not permitted it could have been conducted under exceptions. . In 2010 the French National Ethics committee changed his position in the opinion 112 and the Act 2013-715 of August 6,

Introduction

The use of biological material for biotechnology applications and research raises the question of sources. Should we use human stem cells or embryonic stem cells? For human stem cells, since 1994, the French law considers the human body is inviolable and cannot be subject of property (Art 16-1 of the Civil Code) that means no trade and no patent.

Regarding the use of embryonic stem cells in research, one of the major ethical issues is relying to the destruction of the embryo, the impossibility to ensure its development, and the ethical consequences regarding the principle of respect for the beginning of life.

In France (A), several opinions of the French National Ethics committee (CCNE) claim for the protection of the embryo. From a formal opposition to use embryonic stem cells for research, to an authorization under specific conditions.

In the meanwhile, research on embryo and stem cells was organized by the decree of April 27 th , 2006 under the control of the French National biomedicine Agency. Even if the research on embryo was not allowed it could have been conducted under exceptions. The regime has been modified since the adoption of the Act 2013-715 of August 6, 2013 allowing human embryonic stem-cells (hESC) research under conditions.

A quite different evolution can be seen at the European level (B) where the institutions were less keen to frame this issue on research on embryo through hard law instruments.

A-Evolution of the ethical and legal frameworks in France for using human embryonic stem cells in research.

For a long time the ethical issues dominated the debate on the authorization of research on embryos and since 1984, the National Consultative Ethics Committee (CCNE) has produced 8 opinions. The last one in 2010, opinion112 synthesizes the previous reflection. Meanwhile the legislator in various laws and regulations organized the conditions of access and use of embryonic cells, ending by a change of legal regime in 2013 after deep discussions occurred in the CCNE.

1) The ethical values at a glance: the opinions of the National Consultative Ethics Committee CCNE

The first opinion adopted by the CCNE in 1984 was on "sampling of dead human embryonic and fetal tissue for therapeutic, diagnostic, and scientific purposes". [1] In this opinion the embryo was considered as a potential human being. The opinion followed the first in vitro fertilization carried out in France. The development of these new techniques questioned the new possibilities to separate reproduction from sexuality while also posing challenges to the legislative ability to use tissues that were not previously available for research (in particular for technical reasons).

In Opinion No. 8 (1986) [2] on research on supernumerary embryos, there were large discrepancies among the members of the Committee on the statute of the embryo and on the moral ability to use them (or their cells) for research. Following the progresses on IVF and on the technical possibilities to extract and to store hESC, the Committee supported the possibility to make some research on supernumerary embryos in opinion 53 (1997) [3] and 67 (2001) [4] but only under restricted conditions. Regarding reproductive cloning, in its opinion 54 (1997) [5] the CCNE was clearly against and this position was comforted by the adoption of the Law: Art 16-4 al. 3 of the Civil Code stating "Any intervention seeking to raise a child genetically identical to another, living or deceased, is prohibited.". So, two reactions have developed in parallel, on the one hand the doctrine of the National Ethics Committee on Ethics regarding the possibility of using embryos and embryonic stem cells in research, and secondly a legislative framework to strictly regulate these practices.

The ethical questions which came out from the committee were relating to the nature of the embryo and the cells: [START_REF] Morris | Substance ontology cannot determine the moral status of embryos[END_REF] [START_REF] Lee | The ontological status of embryos: a reply to Jason Morris[END_REF] Is the embryo a person? A bundle of cells ? The opinion No. 93 of the Committee adopted on the 13 December 2006 on the Commercialization of human cell lines and stem cells [START_REF]Opinion n° 93 on the Commercialisation of human stem cells and other cell lines (22/06/06[END_REF], questioned mainly the ethical values that can be balanced as to the distribution of cells. Two principles were identified and used as a framework for the analysis made by the committee: "the nature and the limits of acceptable commercialization of human cell" and "the parents 'consent" to research. Even this opinion is not only covering embryonic stem cells but all types of cells on; the embryo had been largely targeted because of its ontological nature.

After noticing that the majority of the States in the world was considering the human body "out of market", the CCNE admitted, however, that this issue should be reconsidered in light of new practices that tended to develop. Indeed, the human body elements which can currently be completely detached from the body, can also be submitted to large transformations and therefore can potentially be turned into products that could be freely commercialized on national territories as goods. At the same time, the French bioethics law applies a common principle to all elements of the body in article 16-1 of the Civil Code: "The human body, its elements and products cannot be the subject of proprietary law ". This provision totally banned the cells and stem cells from being commercialized in France. Nevertheless, the CCNE applied this principle according a certain degree of flexibility. It insisted on the fact that this principle of non-commercialization should fully apply to the links between the donor and the researcher (no remuneration of the donor, no commercialization), whereas compensation of costs or commercial uses could apply to "sufficiently" or "radically" transformed elements of the body. These exemptions could also be applicable in case of transformation of embryonic stem cells. The criterion of "transformation" has been discussed in the 93 opinion and has been defined as human manipulations leading to the change of the ontological status of the cells, in other words transform human cell to a cell product. This argument has not been shared by all the members of the Committee as some of them provided divergent thoughts. Indeed, some members argued that the representation of the embryo as laboratory material, underlying the possible commercialization of cell lines, could lead to "crossing a very important line into a trivialization of embryo research". The other issue raised by the commercialization of stem cells, including embryonic stem cells, was relating to patents [9]. The question posed was linked to the nature of the cells which are coming from the human body: should these natural elements be patentable?

2) Research on embryo and on embryonic stem cells: legal overview

In France, research on embryos and hESC is organized by the Decree 2006-121 [10]. The embryos that may be devoted to research are supernumerary embryos that are no longer subject to a parental project, or embryos not subject to transfer. At the end of the parental project, a prior informed consent specific for embryo research is necessary before starting the research. The written consent of the couple (or of the surviving member), is compulsory after informing on the other possibilities either to destroy the embryo or to make it available for a transfer for another couple. The embryos which are not subject to transfer in utero because of their quality (developmental abnormalities) rejected after preimplantion diagnosis when an abnormality is detected, may be used for research. In all cases, the consent is revocable at any time.

The authorization for conducting research on embryo and embryonic stem cells is delivered by the Biomedicine Agency for 5 years to the establishments holding an authorization for conservation (L.2151-3-1 CSP). The biomedicine Agency verifies the feasibility of the protocol, the sustainability of the organization and the research team; the conditions of premises and equipment; the means to ensure the quality, the safety, the traceability of embryonic cells. The Agency holds a national register of embryos and cells (coded).

When research is performed on embryo and embryonic stem cells without respecting the law, punitive sanctions are defined by the criminal code. Sanction for cloning is five years in prison and 7 500 000 euros fine (Article 214-2 of the Penal Code). The article 511-1 PC punishes anyone who lends itself to gamete donation for cloning, even abroad. Regarding embryo research, the Article 511-19 PC punishes research without consent and without authorization of seven years' imprisonment.

Until today, there is no jurisprudence of criminal courts for violating the law. Nevertheless there is a decision against an authorization to import stem cells. In 2003 the "Tribunal Administratif "of Paris has received a request from an association defending the 'Right to live" for cancellation of the authorization to import stem cells. The association argued that the research on stem cells is a destruction of the embryo. The Tribunal considered that these stem cells could not be regarded as embryos. The decision was confirmed on appeal by the Administrative Court of appeal [11].

3/ Latest evolution of the ethical and legal frameworks in France.

France has undergone profound changes in recent years at the ethical and the legal level. While research on stem cells advances, including embryonic stem cell research, the CCNE doctrine and the legal framework had to evolve to frame the new practices. French law has modified the legal regime for embryonic stem cells research in 2013 as a result of a dual influence: on one hand the ethical assessment made by CCNE in 2012 on research on the embryo [START_REF] Ccne | Ethical reflection concerning research on human embryonic cells and on human embryos in vitro[END_REF] and the other hand one court decision.

As for the opinion, the committee starts its argumentation by emphasizing on where the majority of States have agreed "There is one true interdict, shared by everyone: the integrity of embryos in vitro cannot be jeopardized as long as they are included in the concerned couple's plans to-have a child.". Insisting, therefore, on parental project as the foundation for the embryo integrity protection (this excludes de facto Human Dignity as a principle to ensure embryo protection), the Committee draws consequences to legitimate research on embryos, even though in 2012 we were still under a legal regime of prohibition of research with exceptions. CCNE admitted, according to his previous doctrine, that the "issue of the exact nature of the embryo" is an enigma which cannot be resolved by consensus. The only consensus which can be reached is on the need of a parental project as a source of protection of the embryo, the end of the parental project is considered the only moral condition to make the embryo available for research. Thus, the first condition to be fulfilled to morally allow research on embryos is the expression of consent from the couple to waive their parental project on their spare embryos. This condition obviously assumes that the embryos were originally designed as part of a medically assisted procreation project which thereby prohibits creating embryos for research directly. Thus, as already stated in previous opinions, the Committee legitimates the use of embryos for research purposes in the framework set by the law and since the parental project (which only establishes the link "potential human being") no longer exists. Recall that in 2012 the legal regime prevailing in France was a ban of the research on embryos and embryonic stem cells with exemptions.

This framework has been also modified on a different basis with the adoption of a court decision in 2012. [13] In this case a Foundation was appealing against a decision of the Biomedicine Agency which was authorizing research using embryonic stem cells. Interpreting the 2011 bioethics law [14] and the requirements for research posed by the law, the Court has considered that two of the conditions required by law were not fulfilled: research should allow "major therapeutic progress" and cannot be carried out "by an alternative method of comparable effectiveness ". The Foundation formed an appeal arguing the authorization adopted by the Biomedecine Agency respects neither the one nor the other of these conditions. The Foundation was successful since the Court noted that "the Biomedicine Agency, by merely opposing defense to the lack of scientific consensus on the substitutability of the two techniques, does not establish that an alternative method did not exist at the date of the contested decision." Thus, the judge clearly places the burden of the proof concerning the lack of comparable methods, on the Agency. Indeed, secondly, the lack of demonstration, created ambiguity about the possible "major therapeutic progress", making, therefore, the second condition of the law inoperative.

Since that decision other appeals have been formed but with a limited impact because of the adoption of the law in 2013 [15] which now defines new conditions for research in France.

Since 2013 the legal balance has changed in France. Now, research on embryos and human embryonic stem cells is no longer subject to a prohibition regime with exemption but an authorized activity under conditions. One could interpret this legal regime as a move towards liberalization of research [START_REF] Bioy | L'évolution de la recherche sur l'embryon, une question de principes, Les Petites affiches[END_REF] but it can also be felt as a response to a future market [17]. This change in the regulation framing the research on embryos was also the occasion for the French legislator to soften the legal requirements to realize a research protocol [START_REF] Bioy | L'évolution de la recherche sur l'embryon, une question de principes, Les Petites affiches[END_REF]. Since the 2013 law the following conditions have to be demonstrated prior the commencement of the research: the scientific relevance of the research project as well as the medical purpose (this condition is interpreted largely as it includes basic research) ; the law maintains the principle of subsidiarity but in a flexible formulation; ethical principles should be respected but the incentive to promote alternative research is deleted; the consent of the partners is required in the same manner as under the 2011 law, but now parents will not be informed of the nature of the research that will be considered. From this last provision one can say that the law is validating a form of blanked consent from the parents. In fact, as parents are no longer informed on the nature of the future research on the embryos, their consent could be considered valid for any research protocol. This position could have been problematic as it is the first time in the French law where a legal provision legitimates blanked consent. This issue has been partially solved by the adoption of the implementing decree in 2015 [18] where it is required from the practitioner to deliver any available information on the research to be conducted in the future. In addition, the new law and implementing decree further specifies procedural requirements for the evaluation, approval and implementation of research protocols.

B -European dimension: ethics and law

For a long time in Europe, ethics committees focused their reflection on embryo research, taking into account the different historical and cultural sensitivities.

1) The European Group on Ethics

The European Group on Ethics in Science and New Technologies (EGE) [19] is a neutral forum, independent, pluralist and multidisciplinary; it is composed of fifteen experts appointed by the European Commission for their expertise and personal qualities. The Group's mission is to examine the ethical issues related to Science and New Technologies and on the basis of their work, to submit opinions to the European Commission as part of the development of legislation and the establishment of Community policies

The opinion 9 (1997) [START_REF]Ethical aspects of cloning techniques[END_REF] focuses on the ethical aspects of cloning and opposes reimplantation into the uterus. Opinion 12 (1998) [21] allows research on embryos within a maximum period of 14 days after fertilization, with parental consent, but forbids the human / animal hybrids. In 2000 an opinion on stem cell specifies that the egg donation should not increase the constraints on women (Opinion 15, 22).

In 2002, an opinion was issued on the patentability of stem cells. Indeed in 2002 over 2000 patent applications were filed worldwide for human and non-human stem cells among them, 25% for embryonic cells. The patent may cover either process: isolation, enrichment, culture, genetic differentiation modification processing of somatic cells into stem cells; either product: the cells themselves, the lines, the genetically modified cells. The EGE concludes with the need not to ban patent on cells or cell lines that would be contrary to the public interest and in the interest of patients. The EGE distinguished according to the nature of stem cell, the lines that have not undergone any changes that do not meet the conditions for patentability, and cells modified by treatment in vitro or by genetic engineering which can fulfill the conditions for patentability (opinion 16, 23).

2-The Council of Europe : The Oviedo Convention

The member States of the Council of Europe, have signed on April 4 1997 the Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine, so called the Oviedo Convention [START_REF]Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine[END_REF]. The convention takes into account all the previous declarations or conventions on human rights (WHO, European social charter, Convention for data protection, rights of the child…..).

The preamble recalls the following points. The aim of the Council of Europe is the achievement of a greater unity between its members and that one of the methods by which that aim is to be pursued is the maintenance and further realization of human rights and fundamental freedoms. The accelerating developments in biology and medicine needs international co-operation so that all humanity may enjoy the benefits of biology and medicine. The Council is conscious that the misuse of biology and medicine may lead to acts endangering human dignity and affirms that progress in biology and medicine should be used for the benefit of present and future generations. The Council considers that measures as are necessary to safeguard human dignity and the fundamental rights and freedoms of the individual with regard to the application of biology and medicine.

The general provisions of the convention are the protection of dignity rights and fundamental freedoms. In article 1, parties to this Convention shall protect the dignity and identity of all human beings and guarantee everyone, without discrimination, respect for their integrity and other rights and fundamental freedoms with regard to the application of biology and medicine. Each Party shall take in its internal law the necessary measures to give effect to the provisions of this Convention. The primacy of the human being is recalled in article 2 : the interests and welfare of the human being shall prevail over the sole interest of society or science.

Regarding the research on embryos in vitro, the convention says in article 18 : Where the law allows research on embryos in vitro, it shall ensure adequate protection of the embryo. The creation of human embryos for research purposes is prohibited. We can conclude from this analysis that there is no consensus at the Council of Europe level on the research on embryo. This question is still regulated under national laws and no European framework have been proposed and adopted as a common rule. This position is demonstrating the impossibility, for some very few area, to reach a European position due to cultural and legal traditions.

3) The legal framework in Europe for research and patent

There are major differences regarding research on the embryo among the EU member states and foreign countries, from a ban in Italy and Germany, to an approval of the research and nuclear transfer in UK, Belgium, Sweden, Canada, USA, Japan, China, Singapore, South Korea. Some countries authorize research, but prohibit the nuclear transfer: Brazil, Australia, France, Denmark, Greece, Finland, Estonia, Latvia, Slovenia and Switzerland.

In the EU each country defines the organization of research as the EU is claiming not to have competency in that field. However, the UE has funded research on stem cells in the 6 th and 7 th framework research and development program, under the condition of compliance with ethical and legal framework of the country. The EU recognizes the great economic potential of new technologies that is necessary to promote and protect.

Through the Directive 98/44/EC, on biotechnology inventions [25], we can find arguments on the position of the EU on the status of the human body and its elements. The preamble of the Directive states that biotechnology inventions are essential for the development of community. Only adequate protection can make them profitable and facilitate trade. The national patent law remains the reference. The patent is an industrial property that allows especially the limits of use by third parties of the patented invention. To be patentable an invention must: being new, Have an inventive step and industrial application Regarding the patentability of human body elements, the European Directive 98/44.CE says in Article 5: "The human body … Including the partial sequence of a gene, cannot constitute patentable inventions …An element isolated from the human body or otherwise, produced by a technical process ,may constitute a patentable invention ... the partial application of a sequence or partial sequence of a gene must be described in the patent application".

Inventions shall be considered non patentable where their commercial exploitation would be contrary to public order or morality; however, exploitation shall not be deemed to be so contrary merely because it is prohibited by law or regulation (article 6). The following, in particular, shall be considered non patentable: processes for cloning human beings; for modifying the germ line genetic identity of human beings; the uses of human embryos for industrial or commercial purposes and processes for modifying the genetic identity of animals which are likely to cause them suffering without any substantial medical benefit to man or animal, and also animals resulting from such processes.

Considering 16 asserts the principle that the human body, at any stage in its formation or development, including germ cells, and the simple discovery of one of its elements or one of its products, including the sequence or partial sequence of a human gene, cannot be patented.

There is a European consensus that says: the person to whom the item is taken must have given consent under national law.

In France, transposition of the Directive was delayed because of a degree of reluctance. The matter was referred to the European Court of Justice in the course of an action for failure to fulfil obligations, which led to the ECJ's ruling against France on July 1, 2004, (Aff. C-448/03)."By not adopting the laws, regulations and administrative provisions necessary to comply with the Directive 98/44 / EC of the European Parliament and of the Council of 6 July 1998 on the legal protection of biotechnological inventions the French Republic has failed to fulfill its obligations under Article 15 of that Directive. The French Republic shall pay the costs".

The provisions of the Law on Bioethics voted on August 6, 2004 as a result of the transposition of Directive 98/44 were a compromise solution and attempted to temper the scope of patents. CPI Article L. 611-18 transposes differently Article 5 of the Directive by stipulating that "only an invention constituting the technical application of a function of an element of the human body can be patent-protected". Nevertheless, significant divergence between the formulation of the above text (and that of CPI Article L. 613-2-1) and Article 5 of the Directive dated July 6, 1998, is problematic.

In conclusion, the French law has evolved in a sense to open the conditions for conducting research on embryos and stem cells in France. This position was the one claimed by the French researchers notably because of their willing to contribute to the international "race" which the previous legislation was impairing. However, even though we can support this vision, the French law and its interpretation will have to ensure a high level of protection of the parents and of the embryo, opening the research should not have to be detrimental to the respect of fundamental rights. At the European level, we can conclude that the European institutions do not want to go further on solving the issue from a regulatory point of view. As it is considered as a very problematic issue, only the European Group on Ethics has decided to propose some values and rules about embryo research. In a sense, these values can be considered as common roots for considering embryo research acceptable in Europe.

[1] CCNE, Opinion n°1 on sampling of dead human embryonic and fetal tissue for therapeutic, diagnostic, and scientific purposes (22/05/1984), http://www.ccneethique.fr/en/publications/opinion-sampling-dead-human-embryonic-and-fetaltissue.html#.VMZJcS5ec4A

[2] CCNE, Opinion n° 8 on research and use of in vitro human embryos for scientific and medical purposes, (15/12/86), http://www.ccne-ethique.fr/en/publications/opinion-researchand-use-vitro-human-embryos-scientific-and-medical-purposes#.WAYehSRnBek

[ 3 ]

 3 CCNE, Opinion n°53 on the establishment of collections of human embryo cells and their use for therapeutic or scientific purposes (11/03/97), http://www.ccneethique.fr/en/publications/opinion-establishment-collections-human-embryo-cells-and-theiruse-therapeutic-or#.WAYftiRnBek

2013 is now allowing research on embryo and on embryonic Stem cells under conditions. This work is part of the results of the bilateral collaboration between France (It has been partially supported by the EUcelLEX project (FP7, GA n°601806).


Nicholas Moore
email: nicholas.moore@u-bordeaux.fr

Stephanie Duret

Adeline Grolleau

Regis Lassalle

Vanessa Barbet

Mai Duong

Nicolas Thurin

Cécile Droz-Perroteau

Sinem Ezgi Gulmez

Previous drug exposure in patients hospitalized for acute liver injury: a case-population study in the French national healthcare data system

Keywords: Acute liver injury, case-population, drug exposure, claims database, pharmacoepidemiology

Liver Injury and in particular Acute Liver Injury (ALI) is an important source of druginduced regulatory action, drug-induced hospital admissions and burden of care.

• studies based on clinical identification of hospitalized cases rarely include more than a few hundred cases.

• The EPIHAM study provides information on the drugs associated with the greatest burden of ALI, and drugs with the highest individual associated risk, over the whole French population over 5 years. Most of these drugs are well known to be able to cause liver injury.

Introduction

Liver Injury and in particular Acute Liver Injury (ALI) associated with medicines (druginduced liver injury (DILI)) is a major source of regulatory action, of hospital admissions and burden of care [START_REF] Gulmez | Liver transplant associated with paracetamol overdose: results from the seven-country SALT study[END_REF][START_REF] Gulmez | Transplantation for acute liver failure in patients exposed to NSAIDs or paracetamol (acetaminophen): the multinational casepopulation SALT study[END_REF][START_REF] Gulmez | Methodology for a multinational case-population study on liver toxicity risks with NSAIDs: the Study of Acute Liver Transplant (SALT)[END_REF][START_REF] Keisu | Drug-induced liver injury in humans: the case of ximelagatran[END_REF][START_REF] Reuben | Drug-induced acute liver failure: results of a U.S. multicenter, prospective study[END_REF][START_REF] Lee | Drug-induced hepatotoxicity[END_REF][START_REF] Bernal | Acute liver failure[END_REF]. Many drugs have been shown to cause or are suspected of causing liver injury, such as paracetamol, anti-mycobacterial drugs, erythromycin, or Valproic acid [START_REF] Zimmerman | Hepatotoxicity:The Adverse Effects of Drugs and Other Chemicals on the Liver[END_REF][START_REF] David | Drug-induced Liver Injury[END_REF][START_REF] Navarro | Drug-related hepatotoxicity[END_REF].

DILIs can range from simple elevation of liver enzymes, to acute liver failure leading to death or liver transplantation [START_REF] Zimmerman | The spectrum of hepatotoxicity[END_REF][START_REF] Zimmerman | Drug-induced liver disease[END_REF]. A previous European study (SALT) of acute liver failure leading to liver transplantation (ALFT) in 7 countries over 3 years and confirmed that many drugs are involved in ALFT [START_REF] Gulmez | Liver transplant associated with paracetamol overdose: results from the seven-country SALT study[END_REF][START_REF] Gulmez | Transplantation for acute liver failure in patients exposed to NSAIDs or paracetamol (acetaminophen): the multinational casepopulation SALT study[END_REF][START_REF] Gulmez | Methodology for a multinational case-population study on liver toxicity risks with NSAIDs: the Study of Acute Liver Transplant (SALT)[END_REF][START_REF] Gulmez | Administrative complexities for a European observational study despite directives harmonising requirements[END_REF]. The majority of the DILIs are not just acute liver failure or necrosis resulting in transplantation, but result in hospital admissions for ALI. [START_REF] David | Drug-induced Liver Injury[END_REF].

Studies based on clinical identification of hospitalized cases often concern only a few hundred cases [START_REF] Donati | Risk of acute and serious liver injury associated to nimesulide and other NSAIDs: data from drug-induced liver injury case-control study in Italy[END_REF][START_REF] Douros | Drug-induced liver injury: results from the hospital-based Berlin Case-Control Surveillance Study[END_REF][START_REF] Sabate | Risk of acute liver injury associated with the use of drugs: a multicentre population survey[END_REF]. A study conducted in Thailand in the nationwide hospital admission database identified 6515 patients hospitalized with drug-induced liver injury, or drug intoxications (using the ICD-10 codes K71 and T36-T65), among which 33.5% were acute hepatitis [START_REF] Sobhonslidsuk | The incidence, presentation, outcomes, risk of mortality and economic data of drug-induced liver injury from a national database in Thailand: a population-base study[END_REF].

This study confirmed well-known associations but also some unexpected ones. A preliminary study in the French Claims database 1/97 th sample EGB confirmed the feasibility of such a study, but also the need of accessing the full Système National des Données de Santé (SNDS) database that includes 99% of the French population [START_REF] Gulmez | Risk of hospital admission for liver injury in users of NSAIDs and nonoverdose paracetamol: Preliminary results from the EPIHAM study[END_REF][START_REF] Bezin | The national healthcare system claims databases in France, SNIIRAM and EGB: Powerful tools for pharmacoepidemiology[END_REF]. EPIHAM (epidemiology of acute liver injury from medicines), the study described here, identified all hospital admissions for ALI in SNDS over 5 years, not related to acute drug overdose or other clinical causes. Exposures identified from drug dispensing in these patients were compared to drug utilisation in EGB, using the case-population approach [START_REF] Gulmez | Transplantation for acute liver failure in patients exposed to NSAIDs or paracetamol (acetaminophen): the multinational casepopulation SALT study[END_REF][START_REF] Moore | Choice of the denominator in case population studies: event rates for registration for liver transplantation after exposure to NSAIDs in the SALT study in France[END_REF]. From these data, we identified the drugs most often associated with ALI, and those with the highest apparent risk.

Material and Methods

This was a case-population study in the French National Healthcare System Claims Database.

Data sources

This study was done in the Système National des Données de Santé, (SNDS), and its random permanent representative 1/97 th sample, Echantillon Généraliste de Bénéficiaires (EGB). SNDS currently includes 66.6 million people (99% of the French population) [START_REF] Bezin | The national healthcare system claims databases in France, SNIIRAM and EGB: Powerful tools for pharmacoepidemiology[END_REF]. SNDS (and its sample, EGB) contains information on (i) general demographics (gender, year of birth, region of residence, date of death but not cause of death), (ii) outpatient healthcare claims, with dates and codes for medical and other healthcare professionals visits, medical procedures, lab tests and other exams (but not results), drugs and medical devices, (iii) registration for a list of 30 long-term diseases (LTD) and their associated International Classification of Diseases 10 th revision (ICD-10) codes allowing patients to receive full reimbursement for expenditure related to LTD, and (iv) hospital discharge data, which include start and end dates for all medical, obstetric and surgery hospitalizations, medical procedures, cost coding system, and the medical unit data when the patient is hospitalized successively in several medical units. The hospital discharge summary includes ICD-10 codes for main, related and associated diagnoses for all hospitalizations. The main diagnosis is the health problem that motivated the admission in the hospital, determined at hospital discharge. The related diagnoses exist if the main diagnosis is a procedure for a chronic or LTD disease and indicates the disease at the origin of the procedure. The associated diagnoses represent specific healthcare status modifiers, mainly underlying chronic diseases [START_REF] Bezin | Choice of ICD-10 codes for the identification of acute coronary syndrome in the French hospitalization database[END_REF].

Hospital discharge ICD-10 codes are based on definitions. They are routinely coded by the physicians in charge of the patient, based on clinical data, assisted by trained professional medical information technicians who verify the codes to ensure consistency, with national coding instructions and manuals. The medical information departments in the hospitals that are in charge of the quality of the coding conduct random validation checks on the diagnostic codes that are used, in addition to training programmes. This is part of the National accreditation and certification process for the Hospitals that includes the completeness and exactitude of the coding process. [START_REF] Gilleron | Certification des comptes: Une incitation à la traçabilité des processus de contrôle[END_REF] The national Healthcare insurance system, which pays the hospitals and clinics based on disease or diagnosis-related groups (DRG) regularly audits these codes and may levy large fines in the case of discrepancies. Certain individual diagnoses have also been subject to validation studies, such as heart failure, stroke, coronary heart disease, or other diseases, which have found positive predictive values around 70-90% [START_REF] Bezin | Choice of ICD-10 codes for the identification of acute coronary syndrome in the French hospitalization database[END_REF][START_REF] Aladjidi | Reliable assessment of the incidence of childhood autoimmune hemolytic anemia[END_REF][START_REF] Bosco-Lévy | Diagnostic accuracy of the international classification of disease 10th revision codes of heart failure in administrative database[END_REF][START_REF] Daveluy | Identification of abuse and dependence cases through a hospital database[END_REF][START_REF] Gouverneur | Agreement between hospital discharge diagnosis codes and medical records to identify metastatic colorectal cancer and associated comorbidities in elderly patients[END_REF], consistent with the national effort at coding quality.

Study design

The EPIHAM study was a case-population study in a claims database [START_REF] Bezin | The national healthcare system claims databases in France, SNIIRAM and EGB: Powerful tools for pharmacoepidemiology[END_REF]. This approach consists in the comparison of exposure rates to a given drug in subjects presenting an event of interest (cases) with the exposure rate in the source population (reference population), as previously described [START_REF] Moore | Choice of the denominator in case population studies: event rates for registration for liver transplantation after exposure to NSAIDs in the SALT study in France[END_REF][START_REF] Theophile | The case-population study design: an analysis of its application in pharmacovigilance[END_REF][START_REF] Capella | Case-population studies in pharmacoepidemiology[END_REF][START_REF] Moore | Relative risks from casepopulation data[END_REF].

Definition of cases

Cases of ALI were identified and extracted over 5 years from SNDS, defined as patients aged Patients with at least one of the following criteria were also excluded from the analysis:

-Patients without health care usage, or not affiliated to the national healthcare insurance system during the year before hospitalization for ALI, -Patients with no history or less than 60 days of history in the database before the first hospitalization for ALI, -Patients with previous hospital stay for chronic liver disease (alcoholic cirrhosis, chronic hepatitis, alcoholics or malignant neoplasm (pancreatic or hepatobiliary)), -Patients with a medical procedure including endoscopies, diagnostic procedures, or related to HIV disease during the first hospitalization for ALI, -Patients with hospitalization (whatever the main diagnosis) ending within the previous 30 days, or starting more than 7 days before the first hospitalization for ALI, because the database does not contain information or the use of routine drugs in hospital, so that exposure in such cases could not be ascertained.

Definition of reference populations

The reference population was identified in the EGB database as all subjects aged 18 years and over. The total exposed population for each drug of interest was estimated from the number of patients dispensed that drug at least once during the study period. That number was extrapolated to the general population based on the age and gender structure in France at each considered year according to data published by the French national institute of statistics (Institut National de la Statistique et des Etudes Economiques, INSEE) [START_REF]Pyramides des âges (Population totale par sexe et âge -France et France métropolitaine)[END_REF].

In this situation, EGB is a convenience sample that is readily accessible, and is representative of the contents of the whole SNDS database. EGB has been used similarly for the casepopulation study of liver transplantation [START_REF] Gulmez | Transplantation for acute liver failure in patients exposed to NSAIDs or paracetamol (acetaminophen): the multinational casepopulation SALT study[END_REF][START_REF] Moore | Choice of the denominator in case population studies: event rates for registration for liver transplantation after exposure to NSAIDs in the SALT study in France[END_REF][START_REF] Moore | Relative risks from casepopulation data[END_REF], for the feasibility study of EPIHAM [START_REF] Gulmez | Risk of hospital admission for liver injury in users of NSAIDs and nonoverdose paracetamol: Preliminary results from the EPIHAM study[END_REF], for other drug utilisation studies [START_REF] Duong | Usage patterns of paracetamol in France[END_REF][START_REF] Duong | Usage patterns of 'overthe-counter' vs. prescription-strength nonsteroidal anti-inflammatory drugs in France[END_REF], as well as for many other studies. In the present design it was clearly not feasible nor useful to ask for drug utilisation data in 66 million persons for each of the 200+ drugs identified.

Definition of exposure

Drug exposures in cases were defined as at least one recorded dispensing within the 7-60 days period before the index date. The 7-day lag time was chosen to minimise protopathic (drug given for the symptoms of hepatic injury) and indication bias (drugs given for a disease that causes hepatic injury). The first would typically be antiemetic agents early before the onset of clinical hepatitis, the latter the use of loop diuretics in acute heart failure resulting in "cardiac liver".

Drug exposures in the reference population were defined according to two different metrics; -As number of subjects with at least one drug dispensing during the study period, as might be recommended for idiosyncratic liver injury, which occurs in susceptible individuals early in the treatment [START_REF] Moore | Choice of the denominator in case population studies: event rates for registration for liver transplantation after exposure to NSAIDs in the SALT study in France[END_REF] [START_REF] Moride | Evidence of the depletion of susceptibles effect in non-experimental pharmacoepidemiologic research[END_REF] -As total number of Defined Daily Doses (DDD) dispensed over the study period. This corresponds to the more classical person-time approach [START_REF] Theophile | The case-population study design: an analysis of its application in pharmacovigilance[END_REF], which would be appropriate to drugs with a direct, cumulative toxicity, but which probably overestimates risks for drugs with mostly short-term use [START_REF] Moore | Choice of the denominator in case population studies: event rates for registration for liver transplantation after exposure to NSAIDs in the SALT study in France[END_REF].

Drugs of interest were identified by their anatomical therapeutic chemical (ATC) codes at the various levels, from drug classes (3 digit code, e.g., A02; drugs for acid related disorders), to the 4 digit chemical class, (e.g. M01A NSAIDs) to the 7-digit individual product code (eg, N02BE01 paracetamol). Classes, families and drugs of interest were identified by the frequency in the cases.

Statistical analyses

ALI cases were described, using standard statistical metrics for quantitative and qualitative variables.

Event rates of ALI for each drug, family or class of interest were estimated as the total number of cases exposed to the drug within 7-60 days before ALI to the total number of patients exposed during the study period, with their 95% Poisson confidence intervals, and to the total number of DDD dispensed over the study period with their 95% Poisson confidence intervals Drugs, families or classes with fewer than 5 exposed cases were not analysed.

For descriptive purposes, the instant and cumulative hazards were determined for the typical hepatotoxic drugs valproic acid and erythromycin as representative of different toxic mechanisms, using duration of exposure previous to index date for cases, and distribution of exposure durations from the reference population. Cumulative incidences were assessed using the Kaplan-Meier method.

All Statistical analyses were carried out using SAS software versus 9.1 (SAS Institute, Cary, NC).

Regulatory aspects

In accordance with the French regulations, this study received authorization from the Institute of Health Data (Institut des Données de Santé) and from the French data protection commission (Commission Nationale de l'Informatique et des Libertés). This study was registered with ENCEPP (www.encepp.eu), at the European Medicines Agency (EMA, London, UK) with the registration number ENCEPP/SDPP/7549. This study was supported by a grant from the French national Research Institute in Public Health ((IRESP), Grants # AAP-2014-05 and AAP-2014-06)

Results

Characteristics of the cases

Over the study timeframe, 4,807 adult cases of hospitalized ALI were identified (Figure 1), 61.4% with acute toxic liver injury and 38.6% with hepatic failure (K72.0). More than half of the cases were women (58.7%). The mean age was 54.5 years (SD ± 19.8), compared to an average age of the French population of 40.4 years. Among these cases, 47.8% had at least one registered chronic disease. The most common (≥ 5%) were diabetes (10.6%), chronic psychiatric disorders (9.1%), and malignant tumours, malignant lymphatic or hematopoietic tissue diseases (6.7%). Nearly all cases (97.6%) had at least one consultation or medical visit within 12 months before the index date, and most of them had seen their general practitioner (83.4%). The average number of medical visits within 12 months before the index date was 11.9 (SD ± 10.8), the mean duration of ALI hospitalization was 6.9 days (SD ± 7.1) and 79.8% of the cases had one or more associated diagnoses at index date (Table 1).

Exposures

There were 249 different drugs of interest with at least one dispensing within 7-60 days before index date in 3,633 ALI cases (75.6%), with at least 5 cases exposed per drug (Figure 2). The most frequent ATC classes or families were analgesics (N02, 40.6%), drugs for acid related disorders (A02, 31.6%), anxiolytics or hypnotics (at least one N05B or N05C, 23.6%), antibacterials for systemic use (J01, 23.1%), agents acting on the renin-angiotensin system (C09, 20.0%), lipid modifying agents (C10, 18.7%), antithrombotic agents (B01, 18.2%), NSAIDs (M01A, 17.0%), psychoanaleptics (N06, 16.6%) and drugs for functional gastrointestinal disorders (A03, 15.3%).

Most common exposures

The drugs (ATC with 7 digit code) most frequently dispensed within 7-60 days period before ALI were paracetamol with 1,495 cases (31.1% of all cases), esomeprazole (10.4%), omeprazole (8.5%), phloroglucinol (6.5%), domperidone (6.2%), amoxicillin in combination with enzyme inhibitors (6.1%), furosemide (5.9%), atorvastatin (5.5%), pantoprazole (5.1%) and zolpidem (5.1%) (Table 2). Some drugs were those widely dispensed both in cases and in the general population such as furosemide (284 cases), atorvastatin (263 cases) and bisoprolol (210 cases), with relatively high event rates around one case for 15,000 exposed patients.

Drugs with highest event rates per patient

The drugs with the highest event rates (exposed cases per exposed population) of ALI were antimycobacterials (J04). The combination of rifampicin, pyrazinamide and isoniazid was the highest ranked drug with one hospital admission for ALI in 578 exposed patients, followed by pyrazinamide alone (one case for 770 exposed patients), ethambutol (one case for 825 exposed patients), isoniazid (one case for 1,281 exposed patients) and rifampicin-isoniazid combination (one case for 1,928 exposed patients) (Table 3). The 25 highest ranked drugs also included colestyramine (one case of ALI for 5,312 exposed patients), erythromycin (one case for 5,374 exposed patients), cibenzoline (one case for 6,452 exposed patients) or methyldopa (one case for 6,719 exposed patients). Among these drugs one also finds antiepileptics (oxcarbazepine, phenobarbital, carbamazepine, valproic acid, and lamotrigine) with 1 case for 8 to 10 000 patients treated; anti-Alzheimer drugs rivastigmine, memantine, galantamine with 1/11000 to 1/12000 patients, and anticoagulants acenocoumarol and warfarin (around 1/11000 patients), and a number of drugs used to treat heart failure, such as isosorbide mononitrate, carvedilol, betaxolol, captopril, which could suggest miscoding of heart-failure associated hepatic injury, or heart failure as a risk factor for drug-related ALI.

These top 25 drugs were among the drugs with the lowest numbers of exposed cases, 71 or fewer.

Drugs with the highest event rate per patient-time

A common measure of exposure is patient-time rather than patients as presented in table 4.

The drugs with the highest rate of ALI per DDD are the same as those per patient, i.e. antituberculosis drugs. 21 out of the 25 drugs with high per-DDD event rate were antibacterial agents, with event rates below 1 case per million DDD. These are drugs with short treatment duration: in this case the number of patients per DDD is highest, which could explain such high per DDD risk if the event is not dose/duration dependent but patient dependent (idiosyncratic) [START_REF] Moore | Choice of the denominator in case population studies: event rates for registration for liver transplantation after exposure to NSAIDs in the SALT study in France[END_REF].

3.6: Hazard functions

Comparing drug utilisation patterns in cases and the general user population may give indications on hazard functions and possible mechanisms of drug toxicity. As examples, we chose to consider erythromycin and valproic acid: the former is a typical type B (idiosyncratic) reaction, with a risk that is maximal at 10 days of treatment, and decreases thereafter (Figure 3). The initial zero-risk period between the date of dispensing and the index date for ALI is related to the 7-day exclusion period: none of the dispensings during these 7 days are considered (zero-exposure bias, akin to immortal time bias).

During valproic acid exposure, on the other hand, the risk of ALI was constant over time, so that the cumulative risk increases with duration of exposure (Figure 4).

Discussion

Main results

This population-based study provides a countrywide assessment of patients admitted for toxic acute liver injury, and the drugs dispensed during a period that would provide exposure that is compatible with a causality of the drug in the event (RUCAM) [START_REF] Benichou | Causality assessment of adverse reactions to drugs--II. An original model for validation of drug causality assessment methods: case reports with positive rechallenge[END_REF][START_REF] Danan | Causality assessment of adverse reactions to drugs--I. A novel method based on the conclusions of international consensus meetings: application to drug-induced liver injuries[END_REF]. In this initial report we describe individual drugs most concerned and a general overview of the possible use of such an approach for alert generation purposes, including the overall population burden of ALI for individual drugs by the absolute number of cases that were exposed to the drug of interest, and for the individual risk by the number of patients dispensed the drug for each case of ALI.

The diagnostic criteria we chose for ALI were rather restrictive: all cases with indications of a possible other cause were excluded, based on main, associated or secondary hospitalisation diagnosis or chronic disease ICD10 codes indicative of for instance alcoholic liver disease, other toxins, viral diseases or other clinically defined causes for liver injury. For legal privacy reasons we are not allowed to return from the recorded diagnoses to the individual patient files so that we have to rely on the quality of hospital diagnoses and coding.

Though errors may of course be possible and certainly occurred, it may be thought unlikely that they would be affected by drug exposure, though certainly exposure to known hepatotoxic drugs may have resulted in wrongly attributing the ALI to the exposure rather than to another cause, for instance viral. This is unlikely: viral tests are systematic at admission for ALI. On the other hand, drugs not thought to be associated with ALI, and that may not be cited in clinical files or spontaneous reports, will be identified from dispensing data.

Drugs whose first dispensing was within 0-7 days before the date of hospital admission were excluded. Hepatic lab tests before index date were done within 7 days before hospital admission, which could indicate that the hepatic symptoms started before that date. Drugs with a first dispensing after that day could have been prescribed because of ALI symptoms (protopathic bias). Indeed, drugs most often first dispensed within 7 days before index date were symptomatic medications (analgesics, antiemetics, propulsives) that could be related to the symptoms of hepatic injury (abdominal pain, nausea or vomiting, dyspepsia). This does not formally exclude that they may have been involved in the occurrence of ALI, but the chance of bias is such that we preferred to exclude them in a first analysis. This does not concern drugs that may have been renewed within the 0-7 days timeframe, but had already been prescribed before.

Our results are generally consistent with expectations [START_REF] Sabate | Risk of acute liver injury associated with the use of drugs: a multicentre population survey[END_REF][START_REF] Sgro | Incidence of drug-induced hepatic injuries: a French population-based study[END_REF]: the drug most commonly dispensed between 7 and 60 days before admission for ALI was paracetamol with 1,495 cases, the drug by far the most dispensed in France [START_REF] Duong | Usage patterns of paracetamol in France[END_REF]. Paracetamol was present in more than 30% of all ALI cases, with one case per 35,000 persons dispensed paracetamol at least once in 5 years. Other drugs often found before ALI were the proton pump inhibitor omeprazole (or esomeprazole, its active isomer), for 910 cases altogether (19%) and one case per 35 000 users of either. Phloroglucinol, and domperidone are drugs used for symptoms of the digestive sphere that may be associated with ALI, and for which further analyses will be needed to elucidate the exact chronology and likelihood of protopathic bias, also called reverse causality, a drug being prescribed for the event studied rather than causing it. Further down the list, drugs are found that are known to cause known liver injury and probably escape protopathic bias such as amoxicillin+clavulanic acid, furosemide or atorvastatin. Of course the absolute number of cases is not indicative of increased hepatotoxic risk per se, just that the drugs may be widely used. These drugs are among the most frequently used, such as paracetamol (52 million exposed adult patients over 5 years, i.e., most adult population), coamoxiclav (19 million users), ibuprofen (26 million users), desloratadine (13 million users) or ketoprofen (19 million users). They did not head the ranking for event rates, with fewer than 7 cases per 100,000 exposed patients, or 1 case for 14,000 exposed patients. The large numbers of hepatic events may however impact overall medical resource utilisation and should be considered in the overall risk-benefit ratio of similar drugs.

To have an indication of the actual risk with individual drugs, one needs to turn to per-user event rates. Of course this is only indicative, and any attempt to compare drugs should be extremely cautious, using the appropriate adjustment methods and a good knowledge of the disease states the drugs are prescribed in. Different drugs are prescribed to different patients with different susceptibilities to liver injury related to patient backgrounds and to the diseases treated. However, within drug families, which share common indications and are used in similar patients, there may be no major differences in the patients or only few confounders.

Different event rates may raise signals of possible concern, and may lead to further explorations. For instance, the first drugs for events per patient are the antimycobacterial drugs, where ALI occurs in one out of 600 to 2,000 exposed patients. Antimycobacterial drugs are known to be hepatotoxic. Rifampicin alone may be less toxic than the others. The next drug by importance of risk is colestyramine, one in 5000 users. Of course colestyramine is given for pruritus in chronic jaundice, which might also suggest protopathic bias or at least a risk factor for acute injury. This will certainly warrant further exploration to test what other drugs may have been associated with colestyramine in these cases. It has been reported as an antidote to paracetamol, [START_REF] Rosa | Paracetamol hepatic necrosis and its prevention by cholestyramine[END_REF][START_REF] Siegers | Cholestyramine as an antidote against paracetamol-induced hepato-and nephrotoxicity in the rat[END_REF] and is used to facilitate the elimination of toxins. However increased transaminases have also been reported, but without overt liver injury. [START_REF] Singhal | Benign elevations in serum aminotransferases and biomarkers of hepatotoxicity in healthy volunteers treated with cholestyramine[END_REF] Among the well-known hepatotoxic agents one finds methyldopa (1/6,700), the antiepileptic drugs carbamazepine, oxcarbazepine, phenobarbital, valproic acid, and lamotrigine, all with around one case in 7-9 000 patients. Drugs used in heart failure (isosorbide mononitrate, carvedilol, or betaxolol) with 1/10-11,000 patients suggest potential confounding by indication, and the possibility of misdiagnosed heart failure and cardiac liver disease.

Warfarine and acenocoumarol have the same event rate (1/11,400), but fluindione and the direct acting anticoagulants do not appear in this list. The anti-Alzheimer drugs memantine, rivastigmine, galantamine also appear together with around one case in 12 000 exposed patients, a toxicity also previously described with tacrine [START_REF] Watkins | Hepatotoxic effects of tacrine administration in patients with Alzheimer's disease[END_REF].

Erythromycine, with one case in 5,400 patients, is the only macrolide in this list. The first descriptions of erythromycin hepatotoxicity date back to the early 1960's [START_REF] Johnson | Allergic hepatitis caused by propionyl erythromycin ester of lauryl sulfate[END_REF]. Hepatotoxicity seems to have been associated with all its esters or salts [START_REF] Pessayre | Hepatotoxicity of erythromycin derivatives[END_REF]. There have been recent metaanalyses of the hepatic risks of antibiotics or macrolides within the European IMI-Protect project, but not comparing the individual drugs [START_REF] Brauer | Risk of acute liver injury associated with use of antibiotics. Comparative cohort and nested case-control studies using two primary care databases in Europe[END_REF][START_REF] Ferrer | Systematic Review and Meta-Analysis: Macrolides-and Amoxicillin/Clavulanate-induced Acute Liver Injury[END_REF]. Further analyses will be needed to verify whether the apparent risk we found is related to uses of erythromycin in patients at high risk of ALI, or whether it is a specific risk of the drug, different from the other macrolides, in which case the real benefits of this ancient drug might need to be reconsidered.

Strengths

The main strength of our study is its power: studying 5 years of hospital admissions for ALI in a whole country database yields a much greater number of cases than previous studies, enabling the study of over 200 different drugs.

The study population was exhaustive and representative of the French population, through data sources that cover nearly all the country's population. Because this database was not set up for this study, neither selection bias nor recall bias should be an issue. The case-population design has been proven feasible, adequate, and efficient by the SALT study, even on a wider scale, for ALFT, a very rare event with 100% ascertainment rates [START_REF] Gulmez | Transplantation for acute liver failure in patients exposed to NSAIDs or paracetamol (acetaminophen): the multinational casepopulation SALT study[END_REF][START_REF] Gulmez | Methodology for a multinational case-population study on liver toxicity risks with NSAIDs: the Study of Acute Liver Transplant (SALT)[END_REF][START_REF] Moore | Choice of the denominator in case population studies: event rates for registration for liver transplantation after exposure to NSAIDs in the SALT study in France[END_REF]. Selection bias was also reduced by the choice of inclusion and exclusion criteria that provided an accurate definition of acute hepatotoxicity from medicines. The defined exposure period reduced protopathic bias, as drugs dispensed in the 7 days before hospital admission for ALI could have been prescribed to relieve the symptoms of liver failure.

Since the drug utilisation patterns can be described in cases and the reference populations, it is feasible to compare the drug dispensing patterns in cases compared to the general population and derive hazard function curves.

Limitations

As all claims and hospital admissions databases, there are limitations.

The first concerns the diagnosis of acute liver injury. Because we cannot identify patients from the database, we cannot directly validate the diagnoses, and have to rely on hospital coder proficiency and professionalism. The basic coding is generated by hospital personnel, usually for statistical and billing purposes. In recent years, professional coders have been deployed in some hospitals, mostly to ensure the quality and consistency of coding and the proper coding of associated diagnoses. There are national coding instructions for various diseases, to ensure homogeneous coding over the thousands of hospitals concerned.

The algorithms we used excluded other causes of acute liver injury, such as viral diseases, non-drug toxicity (chemical, plants), cardiac disorders, Budd-chiari syndrome and other vascular diseases, as well as acute drug intoxications, especially paracetamol. In addition to the codes used for the main diagnosis, other information such as secondary codes, or indication of chronic disease or alcohol related disorders were used to exclude cases. Hospital stays for whatever reason within the 7-60 days before ALI were also a reason for exclusion, since during the hospital period there is no indication on drug exposures, so that potential exposures of interest could be missed.

Exposure was measured by drug dispensing, which is one step closer to actual exposure than prescriptions or electronic health records. Ideally one would want to have direct contact with the patient, knowing that even that is sometimes uncertain [START_REF] Moore | What medicines do patients really take? A comparison of free form vs oriented questionnaires[END_REF][START_REF] Moore | Comparison of patient questionnaires, medical records, and plasma assays in assessing exposure to benzodiazepines in elderly subjects[END_REF]. Though most claims databases cannot identify OTC drugs, in France over 84% of the paracetamol and 70% of analgesic strength ibuprofen is prescribed and identified in the database [START_REF] Duong | Usage patterns of paracetamol in France[END_REF][START_REF] Duong | Usage patterns of 'overthe-counter' vs. prescription-strength nonsteroidal anti-inflammatory drugs in France[END_REF][START_REF] Duong | Coronary Events After Dispensing of Ibuprofen: A Propensity Score-Matched Cohort Study Versus Paracetamol in the French Nationwide Claims Database Sample[END_REF]. Drug dispensing is specific and precise, in that only commercial preparations are dispensed, indicating the exact number of tablets, and their individual strength, so that the total quantity of drug that is dispensed in known.

Further analyses will consider individual drug classes (e.g., NSAIDs, antidepressants, antiepileptic drugs or antibiotics, as well as different analytical strategies, beyond the casepopulation approach used here, such as case-control, case crossover or self-controlled case series. These will be used to compare event rates or odds ratios of different drugs within the same indications or drug families.

Future developments

Such a resource may be used to test known hepatotoxic drugs, compare marketed drugs within drug families, and confirm the validity of the method. A potential use could also be for early recognition of drug alerts of hepatotoxicity, once the background rates of hepatotoxicity have been established for drug classes or clinical situations. We voluntarily restricted the analysis to only the drugs with at least 5 cases exposed over 5 years. This would be too restrictive for alert generation where even one case for instance in the first years of marketing may be of interest. Early surveillance of new cases in the SNDS exposed to newly marketed drugs, may alert to the hepatotoxic potential of such new drugs. The main restriction is the data delay: the hospital data (cases) are available for a given year in the third quarter of the following year year. This may be enough for routine surveillance of new drugs, may too long for emerging acute drug alerts, and needs to be compared to the performance of the spontaneous reporting system.

Conclusion

To conclude, this first analysis of the collection of all toxic ALI admitted to hospital in France over 5 years already provides information on the drugs associated with the greatest burden of ALI, and drugs with the highest apparent individual risk. Paracetamol is by far the drug most often associated with hospitalisation for ALI, but it is also by far the drug most often prescribed in France, at least once in 5 years to 80% of the population. At the other end of the spectrum, antituberculosis antibiotics were by far the most hepatotoxic drugs, with the highest individual risk of hepatotoxicity, whether per patient or per DDD.

This study provides a rich source of information on suspected hepatotoxicity, which will be explored using different analytical methods. Individual drugs and drugs families will be further explored, as will be the use of this method to generate alerts. Interested researchers are invited to contribute and participate in this research.

l'évaluation et des statistiques (MiRe-DREES) of Caisse Nationale d'Assurance Maladie des Travailleurs Salariés (CNAMTS), Régime Social Indépendants (RSI) and Caisse Nationale de

Solidarité́ pour l'Autonomie (CNSA), as part of the general call for projects by IReSP (Appel à Projets, Institut de Recherche en Santé Publique), n° 2013-29. It was designed, conducted and analysed independently by the Bordeaux PharmacoEpi Research Platform, CIC Bordeaux CIC1401.

Ethical approval.

In accordance with the French regulations, this study received authorization from the Institute of Health Data (Institut des Données de Santé) and from the French Data Protection Agency (Commission Nationale de l'Informatique et des Libertés). This study was registered with ENCEPP (www.encepp.eu), at the European Medicines Agency (EMA, London, UK) with the registration number ENCEPP/SDPP/7549.

Patient consent

Not applicable. ALI: acute liver injury; DDD: Defined daily dose; * Case-population ratio: ratio of exposure in cases compared to whole population, in DDD (person-time)

  18 years and over with a first hospital admission for Acute Liver Injury (ALI) between 1 January 2010 and 31 December 2014. The date of first admission for ALI was considered as the index date. Hospitalization for ALI was defined as hospitalization with a main diagnosis ICD-10 code for acute toxic liver injury (K71.1, K71.2, K71.6, K71.9) or hepatic failure (K72.0), without previous hospitalization for ALI. If a patient had several hospitalisations for ALI over the study period, only the first was considered. Exclusion criteria included: -Any diagnosis code for non-drug-related liver disease or potential liver injury during the hospitalization of interest or in the previous 60 days as identified by the ICD-10 codes for associated, related or main diagnosis: chronic viral hepatitis (B18), malignant neoplasms (all C codes), mental and behavioural disorders due to use of alcohol (F10), other degenerative diseases of nervous system, not elsewhere classified (G31), heart failure (I50), portal vein thrombosis (I81), oesophageal varices (I85), alcoholic liver disease (K70), fibrosis and cirrhosis of liver (K74), other diseases of liver (K76), cholelithiasis (K80), other diseases of biliary tract (K83), ascites (R18), presence of cardiac and vascular implants and grafts (Z95), liver transplant failure and rejection (T86.4)) -Hospitalization with a diagnosis of liver transplant status in the previous 60 days (ICD-10 code for associated, related or main diagnosis: Z94.4); -A diagnosis of poisoning during the hospitalization of interest and the concomitant stays (ICD-10 code for associated, related or main diagnosis: from T36 to T50).

Figure 1 .

 1 Figure 1. Flow chart of adult ALI cases identification between 2010-2014. ALI: Acute liver injury; DRG: Diagnosis related group; ICD: International Classification of Diseases 10th revision; MD: main diagnosis, SNDS: Système National des Données de Santé

Figure 2 :Figure 3 .

 23 Figure 2: selection of hospitalisations for acute liver injury ALI: Acute liver injury; ID: Index date

Figure 4 .

 4 Figure 4. Cumulative incidences for valproic acid according to the number of DDD dispensed in cases and in the extrapolated reference population between 2010-2014. DDD: Defined daily dose

Table 1 .

 1 Characteristics of cases of hospital admissions for acute liver injury cases identified in the French national health data SNDS from 2010 to 2014

	Cases
	n = 4,807

Table 2 .

 2 Top 25 drugs dispensed within 7-60 days before hospital admission for ALI between 2010 and 2014, by number of exposed ALI.

		Number of ALI	Number of DDD	Number of users	Number of cases per	Number of cases per	Number of
		n (%)	dispensed	over 5 years	million DDD	10 000 users	users/ case
		n = 4,807	n=119,310,075,800	n = 65,375,384	[95% CI]	[95% CI]
	N02BE01 -Paracetamol	1,495 (31.1)	5,219,032,555	52,020,957	0.29 [0.26 ; 0.32] 0.29 [0.26 ; 0.32]	34,797
	A02BC05 -Esomeprazole	502 (10.4)	2,982,803,352	14,161,092	0.17 [0.15 ; 0.19] 0.35 [0.31 ; 0.40]	28,209
	A02BC01 -Omeprazole	408 (8.5)	2,260,375,155	17,494,549	0.18 [0.16 ; 0.20] 0.23 [0.21 ; 0.26]	42,879
	A03AX12 -Phloroglucinol	311 (6.5)	568,912,425	20,033,756	0.55 [0.48 ; 0.62] 0.16 [0.14 ; 0.18]	64,417
	A03FA03 -Domperidone	298 (6.2)	850,851,417	13,555,192	0.35 [0.31 ; 0.40] 0.22 [0.19 ; 0.25]	45,487
	J01CR02 -Amoxicillin and enzyme inhibitor	293 (6.1)	724,464,594	19,059,269	0.40 [0.35 ; 0.46] 0.15 [0.13 ; 0.18]	65,049
	C03CA01 -Furosemide	284 (5.9)	3,432,035,850	4,284,346	0.08 [0.07 ; 0.09] 0.66 [0.58 ; 0.76]	15,086
	C10AA05 -Atorvastatin	263 (5.5)	3,839,738,298	4,140,201	0.07 [0.06 ; 0.08] 0.64 [0.55 ; 0.73]	15,742
	A02BC02 -Pantoprazole	245 (5.1)	1,130,527,478	8,622,136	0.22 [0.19 ; 0.25] 0.28 [0.25 ; 0.33]	35,192
	N05CF02 -Zolpidem	244 (5.1)	1,623,764,111	6,741,764	0.15 [0.13 ; 0.17] 0.36 [0.31 ; 0.42]	27,630
	N02AA59 -Codeine combinations	236 (4.9)	480,605,654	15,435,944	0.49 [0.42 ; 0.56] 0.15 [0.13 ; 0.18]	65,407
	M01AE01 -Ibuprofen	228 (4.7)	658,447,116	25,768,233	0.35 [0.30 ; 0.40] 0.09 [0.08 ; 0.10]	113,019
	J01CA04 -Amoxicillin	223 (4.6)	924,921,578	27,387,752	0.24 [0.21 ; 0.28] 0.08 [0.07 ; 0.09]	122,815
	N02AX52 -Tramadol, combinations	217 (4.5)	476,468,850	14,487,560	0.46 [0.39 ; 0.53] 0.15 [0.13 ; 0.17]	66,763
	N05BA12 -Alprazolam	215 (4.5)	1,054,354,871	7,742,811	0.20 [0.18 ; 0.24] 0.28 [0.24 ; 0.32]	36,013
	C07AB07 -Bisoprolol	210 (4.4)	1,483,228,124	2,977,061	0.14 [0.12 ; 0.16] 0.71 [0.61; 0.82]	14,176
	N05BA08 -Bromazepam	189 (3.9)	1,092,243,431	6,312,999	0.17 [0.15 ; 0.20] 0.30 [0.26 ; 0.35]	33,402
	C10AA07 -Rosuvastatin	182 (3.8)	2,294,431,995	3,705,341	0.08 [0.07 ; 0.09] 0.49 [0.42 ; 0.57]	20,359
	R06AX27 -Desloratadine	182 (3.8)	1,354,186,403	12,676,021	0.13 [0.11 ; 0.16] 0.14 [0.12 ; 0.17]	69,648
	N05BB01 -Hydroxyzine	166 (3.5)	458,934,834	6,194,431	0.36 [0.31 ; 0.42] 0.27 [0.23 ; 0.31]	37,316
	N05CF01 -Zopiclone	163 (3.4)	1,110,657,434	4,717,977	0.15 [0.12 ; 0.17] 0.34 [0.29 ; 0.40]	28,945
	N02AX02 -Tramadol	162 (3.4)	648,184,200	8,737,883	0.25 [0.21 ; 0.29] 0.19 [0.16 ; 0.22]	53,938
	A02BC03 -Lansoprazole	161 (3.3)	900,407,668	6,889,923	0.18 [0.15 ; 0.21] 0.23 [0.20 ; 0.27]	42,795
	A02BX13 -Alginic acid	154 (3.2)	299,064,119	7,628,487	0.51 [0.43 ; 0.61] 0.20 [0.17 ; 0.24]	49,536
	M01AE03 -Ketoprofen	153 (3.2)	813,655,782	18,897,000	0.19 [0.16 ; 0.22] 0.08 [0.07 ; 0.10]	123,510
	ALI, Acute Liver Injury

Table 3 .

 3 Top 25 drugs (ATC code, ingredients) dispensed within 7-60 days before hospital admission for ALI with the highest ratios of exposed cases to exposed population between 2010 and 2014 Acute Liver Injury ATC: anatomical chemical therapeutic classification of medicines. Case-population ratio: ratio of exposure in cases to exposure in the reference population in number of patients exposed.

	Number of	Number of patients	Number of cases	Number of	Case-population
	ALI		per 10 000 users [95% CI]	users per case	ratio (users)

Table 4 .

 4 Top 25 drugs (ATC Code, ingredients) dispensed within 7-60 days before hospital admission for ALI with the highest ratio of exposed cases to patient exposure (in DDD) between 2010 and 2014

	Number	Number of DDD	Average treatment	Number of cases per	Million DDD	Case-population
	of ALI	dispensed	duration (days)	million DDD (for 5 years)	per case	ratio (DDD)*

Authors contributions

The present paper is a product of the EPIHAM Project that was submitted for public funding by the Bordeaux PharmacoEpi research Team, and served as support for the Eu2P PhD project of Prof Sinem Ezgi Gulmez. Prof Nicholas Moore had the original idea and supervised the project. Cécile Droz-Perroteau was the chief operating officer, discussed the study background and coordinated study logistics. Adeline Grolleau was the operational study manager, and collaborated to draft the manuscript. Régis Lassalle was the chief statistician and data manager. Vanessa Barbet and Mai Duong provided data-management and statistical analyses. Nicolas Thurin is a scientific adviser, and he contributed to the selection algorithm of the cases. Stéphanie Duret developed this specific analysis, collaborated to draft the manuscript.

All authors contributed comments and approved the final version of this paper.

Acknowledgements

The authors wish to thank ADERA, the non-profit organization that provides legal, administrative and human resources support to Bordeaux PharmacoEpi;

Compliance with ethical standards.

The corresponding author has full access to all of the data in the study, and takes responsibility for the integrity of the data and accuracy of the data analysis.

Conflicts of interest

Nicholas Moore, Stephanie Duret, Adeline Grolleau, Regis Lassalle, Vanessa Barbet, Mai Duong, Nicolas Thurin, Cécile Droz-Perroteau, Sinem Ezgi Gulmez have no conflicts of interest that are directly relevant to the content of this study All authors have completed the Unified Competing Interest form at www.icmje.org/coi_disclosure.pdf (available on request from the corresponding author) and declare: no support from any organisation for the submitted work, no other relationships or activities that could appear to have influenced the submitted work. Bordeaux PharmacoEpi receives funding from various pharmaceutical companies and governmental bodies to conduct research on marketed drugs at the request of regulatory authorities, or as a consequence of research grant applications. None of these concern the subject at hand.

Funding

This study was supported by an unconditional public joint grant from Direction Générale de la Santé (DGS), from Mission recherche de la Direction de la recherche, des etudes, de