Assessment of thimerosal effects in sublethal concentrations on zebrafish (Danio rerio) embryos exploring NMR-based metabolomics profile.

Thimerosal, a preservative commonly used in the pharmaceutical and cosmetic industry, has raised concerns regarding its potentially toxic effects as an organic mercury compound. Within this context, using an NMR-based metabolomics profile and chemometric analysis, zebrafish embryos were used as an in vivo model to study the effects of thimerosal in metabolic profiles after exposure to sublethal concentrations of the mercury compound. The thimerosal concentrations of 40 and 80 nM were employed, corresponding to 40% and 80% of the LC50, respectively, for zebrafish embryos. The most significant alterations in the metabolic profile included changes in carbohydrates, amino acids, nucleotides, trimethylamine-N-oxide, ethanolamine, betaine, and ethanol. Furthermore, thimerosal exposure affects various metabolic pathways, impairing the nervous system, disrupting protein metabolism, and potentially causing oxidative damage. Therefore, adopting a metabolomics approach in this investigation provided insights into the potentially implicated metabolic pathways contributing to the deleterious effects of thimerosal in biological systems.

In vitro assessment of Thimerosal cytotoxicity and antimicrobial activity.

BACKGROUND: Thimerosal (Merthiolate) is a well-known preservative used in pharmaceutical products, the safety of which was a matter of controversy for decades. Thimerosal is a mercury compound, and there is a debate as to whether Thimerosal exposure from vaccination can contribute to the incidence of mercury-driven disorders. To date, there is no consensus on Thimerosal safety in Vaccines. In 1977, a maximum safe dose of 200 µg/ml (0.5 mM) was recommended for Thimerosal by the WHO experts committee on biological standardization. Up-to-date guidelines, however, urge national control authorities to establish their own standards for the concentration of vaccine preservatives. We believe such safety limits must be studied at the cellular level first. The present study seeks a safe yet efficient dose of Thimerosal exposure for human and animal cells and control microorganism strains. METHODS: The safety of Thimerosal exposure on cells was analyzed through an MTT cell toxicity assay. The viability of four cell types, including HepG2, C2C12, Vero Cells, and Peripheral blood mononuclear cells (PBMCs), was examined in the presence of different Thimerosal concentrations and the maximum tolerable dose (MTD) and the half maximal inhibitory concentration (IC50) values for each cell line were determined. The antimicrobial effectiveness of Thimerosal was evaluated on four control strains, including Pseudomonas aeruginosa, Staphylococcus aureus, Candida albicans, and Aspergillus brasiliensis, to obtain the minimum inhibitory concentration (MIC) of Thimerosal. The MIC test was performed in culture media and under optimal growth conditions of microorganisms in the presence of different Thimerosal concentrations. RESULTS: The viability of all examined cell lines was suppressed entirely in the presence of 4.6 µg/ml (12.5 µM) of Thimerosal. The MTD for HepG2, C2C12, PBMC, and Vero cells was 2, 1.6, 1, and 0.29 µg/ml (5.5, 4.3, 2.7 and 0.8 µM), respectively. The IC50 of Thimerosal exposure for HepG2, C2C12, PBMC, and Vero cells was 2.62, 3.17, 1.27, and 0.86 µg/ml (7.1, 8.5, 3.5 and 2.4 µM), respectively. As for antimicrobial effectiveness, the growth capability of Candida albicans and Staphylococcus aureus was suppressed entirely in the presence of 6.25 µg/ml (17 µM) Thimerosal. The complete growth inhibition of Pseudomonas aeruginosa in culture media was achieved in 100 µg/ml (250 µM) Thimerosal concentration. This value was 12.5 µg/ml (30 µM) for Aspergillus brasiliensis. CONCLUSION: According to our results Thimerosal should be present in culture media at 100 µg/ml (250 µM) concentration to achieve an effective antimicrobial activity. We showed that this amount of Thimerosal is toxic for human and animal cells in vitro since the viability of all examined cell lines was suppressed in the presence of less than 5 µg/ml (12.5 µM) of Thimerosal. Overall, our study revealed Thimerosal was 333-fold more cytotoxic to human and animal cells as compared to bacterial and fungal cells. Our results promote more study on Thimerosal toxicity and its antimicrobial effectiveness to obtain more safe concentrations in biopharmaceuticals.

A longitudinal cohort study of the relationship between Thimerosal-containing hepatitis B vaccination and specific delays in development in the United States: Assessment of attributable risk and lifetime care costs.

Epidemiological evidence suggests a link between mercury (Hg) exposure from Thimerosal-containing vaccines and specific delays in development. A hypothesis-testing longitudinal cohort study (n=49,835) using medical records in the Vaccine Safety Datalink (VSD) was undertaken to evaluate the relationship between exposure to Hg from Thimerosal-containing hepatitis B vaccines (T-HBVs) administered at specific intervals in the first 6months of life and specific delays in development [International Classification of Disease, 9th revision (ICD-9): 315.xx] among children born between 1991 and 1994 and continuously enrolled from birth for at least 5.81years. Infants receiving increased Hg doses from T-HBVs administered within the first month, the first 2months, and the first 6months of life were significantly more likely to be diagnosed with specific delays in development than infants receiving no Hg doses from T-HBVs. During the decade in which T-HBVs were routinely recommended and administered to US infants (1991-2001), an estimated 0.5-1million additional US children were diagnosed with specific delays in development as a consequence of 25µg or 37.5µg organic Hg from T-HBVs administered within the first 6months of life. The resulting lifetime costs to the United States may exceed $1 trillion.

Sociological Environmental Causes are Insufficient to Explain Autism Changepoints of Incidence.

The Environmental Protection Agency (EPA) recently published a study analyzing time trends in the cumulative incidence of autistic disorder (AD) in the U.S., Denmark, and worldwide. A birth year changepoint (CP) around 1988 was identified. It has been argued that the epidemic rise in autism over the past three decades is partly due to a combination of sociologic factors along with the potential contribution of thimerosal containing vaccines. Our work conducted an expanded analysis of AD changepoints in CA and U.S., and determined whether changepoints in time trends of AD rates temporally coincide with changepoints for the proposed causative sociologic and environmental factors. Birth year changepoints were identified for 1980.9 [95% CI, 1978.6-1983.1], 1988.4 [95% CI, 1987.8-1989.0] and 1995.6 [95% CI, 1994.6-1996.6] for CA and U.S. data, confirming and expanding the EPA results. AD birth year changepoints significantly precede the changepoints calculated for indicators of increased social awareness of AD. Furthermore, the 1981 and 1996 AD birth year changepoints don't coincide with any predicted changepoints based on altered thimerosal content in vaccines nor on revised editions of the Diagnostic and Statistical Manual of Mental Disorders (DSM).

Determination of thimerosal in pharmaceutical industry effluents and river waters by HPLC coupled to atomic fluorescence spectrometry through post-column UV-assisted vapor generation.

A high performance liquid chromatography coupled with atomic fluorescence spectrometry method for the determination of thimerosal (sodium ethylmercury thiosalicylate, C9H9HgNaO2S), ethylmercury, and inorganic mercury is proposed. Mercury vapor is generated by the post-column reduction of mercury species in formic acid media using UV-radiation. Thimerosal is quantitatively converted to Hg(II) followed by the reduction of Hg(II) to Hg(0). This method is applied to the determination of thimerosal (THM), ethylmercury (EtHg) and inorganic Hg in samples of a pharmaceutical industry effluent, and in waters of the San Luis River situated in the west side of San Luis city (Middle West, Argentine) where the effluents are dumped. The limit of detections, calculated on the basis of the 3σ criterion, where 0.09, 0.09 and 0.07 µg L(-1) for THM, EtHg(II) and for Hg(II), respectively. Linearity was attained from levels close to the detection limit up to at least 100 µg L(-1).

Commentary--Controversies surrounding mercury in vaccines: autism denial as impediment to universal immunisation.

In 2004, the US Center for Disease Control (CDC) published a paper showing that there is no link between the age at which a child is vaccinated with MMR and the vaccinated children's risk of a subsequent diagnosis of autism. One of the authors, William Thompson, has now revealed that statistically significant information was deliberately omitted from the paper. Thompson first told Dr S Hooker, a researcher on autism, about the manipulation of the data. Hooker analysed the raw data from the CDC study afresh. He confirmed that the risk of autism among African American children vaccinated before the age of 2 years was 340% that of those vaccinated later.

Thimerosal as discrimination: vaccine disparity in the UN Minamata Convention on mercury.

When addressing toxins, one unmistakable parallel exists between biology and politics: developing children and developing nations are those most vulnerable to toxic exposures. This disturbing parallel is the subject of this critical review, which examines the use and distribution of the mercury (Hg)-based compound, thimerosal, in vaccines. Developed in 1927, thimerosal is 49.55% Hg by weight and breaks down in the body into ethyl-Hg chloride, ethyl-Hg hydroxide and sodium thiosalicylate. Since the early 1930s, there has been evidence indicating that thimerosal poses a hazard to the health of human beings and is ineffective as an antimicrobial agent. While children in the developed and predominantly western nations receive doses of mostly no-thimerosal and reduced-thimerosal vaccines, children in the developing nations receive many doses of several unreduced thimerosal-containing vaccines (TCVs). Thus, thimerosal has continued to be a part of the global vaccine supply and its acceptability as a component of vaccine formulations remained unchallenged until 2010, when the United Nations (UN), through the UN Environment Programme, began negotiations to write the global, legally binding Minamata Convention on Hg. During the negotiations, TCVs were dropped from the list of Hg-containing products to be regulated. Consequently, a double standard in vaccine safety, which previously existed due to ignorance and economic reasons, has now been institutionalised as global policy. Ultimately, the Minamata Convention on Hg has sanctioned the inequitable distribution of thimerosal by specifically exempting TCVs from regulation, condoning a two-tier standard of vaccine safety: a predominantly no-thimerosal and reduced-thimerosal standard for developed nations and a predominantly thimerosal-containing one for developing nations. This disparity must now be evaluated urgently as a potential form of institutionalised discrimination.

Parents' preferences for seasonal influenza vaccine for their children in Japan.

In Japan, trivalent inactivated influenza vaccine is the only approved influenza vaccine. It is typically administrated by hypodermic injection, and children under 13 years of age are recommended to be vaccinated two times during each winter season. Live-attenuated influenza vaccine (LAIV) is administered by a thimerosal-free nasal spray. If LAIV is approved in the future in Japan, parents will have an alternative type of influenza vaccine for their children. This study investigated parents' preference for the type of seasonal influenza vaccine for their children if alternatives are available. The marginal willingness to pay for vaccine benefits was also evaluated. We conducted a discrete choice experiment, a quantitative approach that is often used in healthcare studies, in January 2013. Respondents were recruited from a registered online survey panel, and parents with at least one child under 13 years of age were offered questionnaires. This study showed that for seasonal influenza vaccines for their children, parents are more likely to value safety, including thimerosal-free vaccines and those with a lower risk of adverse events, instead of avoiding the momentary pain from an injection. If LAIV is released in Japan, the fact that it is thimerosal-free could be an advantage. However, for parents to choose LAIV, they would need to accept the slightly higher risk of minor adverse events from LAIV.

O uso de Timerosal em vacinas, as implicações para o Programa Nacional de Imunizações Brasileiro e a nova Convenção de Minamata sobre o mercúrio / The use of Thimerosal in vaccines, the implications for the Brazilian National Immunization Program and the new Minamata Convention on Mercury

Este trabalho teve por objetivo contextualizar a discussão em relação ao uso de Timerosal em Vacinas e suas implicações para a saúde das pessoas, tendo em vista a discussão no Conselho de Ministros do Programa das Nações Unidas para o Meio Ambiente (PNUMA), onde se montou um Grupo de Trabalho para o estabelecimento de uma Convenção com o objetivo de banir o metal mercúrio e seus subprodutos do uso cotidiano, como forma de reduzir a exposição antrópica e a toxidade desse elemento em todas as cadeias humanas de produção. Nesse processo de discussão, com a participação dos Países Partes do PNUMA, da sociedade civil internacional,da academia, de outros Órgãos das Nações Unidas - a exemplo da Organização Mundial da Saúde (OMS) - e da indústria, foi concretizada a Convenção de Minamata para o Mercúrio. Esse trabalho reflete as participações do pesquisador nos encontros entre os Países e inúmeras outras reuniões entre os grupos de contato, onde participou como delegado do Ministério da Saúde.Trabalhos de pesquisa, onde o pesquisador é ator participante da discussão e de seus ensinamentos, permitem uma visão do contexto da criação da normatização que tem utilidade no contexto de Políticas Públicas para o Setor Saúde.

This study aimed to contextualize the discussion regarding the use of Thimerosal in vaccines and their implications for human health, in view of the discussion in the Council of Ministers of the United Nations Environment Programme (UNEP), where a Working Group was set for the establishment of a Convention with the purpose of banning mercury and by products of every day use, as a way to reduce anthropogenic exposure and toxicity of this element in all human production chains. The Minamata Convention on Mercury is the result of a process of discussion, with the participation of UNEP Country Parties, international civil society, academia, other United Nations bodies - such as the World Health Organization (WHO) - and industry. This work reflects the participation of the researcher, as delegate of the Ministry of Health, to the meetings of the Working Groups, and all other ones held by contact groups, and its learning, which made possible to be inside the context of the creation of norms, which is useful to a better understanding of Public Policies for the Health Sector.

Timerosal en las vacunas: ¿blanco, negro, o más bien todo lo contrario? / Thimerosal in vaccines: white, black, or quite the opposite?

El presente artículo tiene por objetivo analizar la controversia ocurrida en Chile, especialmente durante los últimos meses, en relación a un proyecto de ley que busca prohibir la fabricación, importación, comercialización o distribución de vacunas que contengan dentro de sus compuestos, en cualquier nivel de concentración, timerosal o compuestos organomercúricos. Sin constituir una síntesis formal de toda la investigación existente, se analiza la evidencia científica que los distintos actores han utilizado, las razones de la controversia y las anomalías en el proceso de toma de decisión sanitaria.

This article analyzes the recent controversy regarding the introduction of a bill to Chilean Congress that aims to ban thiomersal and/or any trace of organomercurial compounds from vaccines in the country. Rather than providing a formal overview of all available evidence, this analysis focuses on the reasons behind the controversy, the scientific evidence invoked by both sides in the debate, and the anomalies in the healthcare decision-making process.

J'accuse desde la bioética: análisis de la discusión sobre el timerosal en Chile / A bioethical j’accuse: analysis of the discussion around thiomersal in Chile

El Poder Legislativo chileno propone una ley que elimine el timerosal como preservante de las vacunas parenterales del Programa Nacional de Inmunizaciones, proyecto que el Poder Ejecutivo se ha propuesto vetar. El mundo científico informa mayoritariamente que la sospecha de neurotoxicidad atribuida al timerosal es infundada. Pese a ello, las autoridades médicas han oscilado entre sostener que la precaución sugiere apoyar la ley y en otros momentos han manifestando que es más precautorio mantener los programas de vacunación actualmente vigentes. Estas contradicciones y oposiciones ilustran que materias que conciernen a la ciudadanía, requieren una reflexión bioética acabada sobre las políticas públicas sanitarias. Han quedado claro las deficiencias de la deliberación política y la falta de participación social en decisiones que, dado el grado de incertidumbre involucrada en temas como inmunización, requieren no sólo la inclusión de la ciudadanía sino el respeto de la autonomía individual para aceptar o rechazar la inclusión en los programas de vacunación propuestos por las políticas sanitarias. La participación ciudadana en nuestro país se ve severamente limitada por la falta de instrumentos sociales como el plebiscito, el ombudsman y, especialmente, la desidia en crear la Comisión Nacional de Bioética exigida por la Ley 20.120 de 2006, una de cuyas funciones más importantes es mediar deliberativamente entre legos, expertos y políticos en la generación de políticas sanitarias legitimadas por la participación ciudadana.

Chilean legislators have voted to ban vaccines preserved with thiomersal, an initiative that the Executive has vetoed. Most scientific evidence has dismissed the alleged toxicity of this substance, in accordance with the formal and publicly expressed opinion of local experts, and yet, medical authorities have issued contradictory statements. Some have argued that the principle of precaution suggests eliminating thiomersal preserved vaccines; others have declared that current vaccines should be maintained to protect the population. From the perspective of bioethics, this polemic is another example of the shortcoming of the deliberation process leading to controversial laws in lieu of including citizens in the discussion of regulations that harbor uncertainties, and respect for individual autonomy to accept or reject public immunization programs. The Chilean legal system has been unwilling to implement participatory democratic procedures like plebiscites or institutions such as the ombudsman. In 2006 a law was enacted that creates a National Commission of Bioethics, but successive governments have failed to create such a commission, which is an efficient social instrument to conduct deliberation on bioethical issues that require a balanced participation of the public, experts, and politicians.

Mercury disposition in suckling rats: comparative assessment following parenteral exposure to thiomersal and mercuric chloride.

Due to the facts that thiomersal-containing vaccine is still in use in many developing countries, and all forms of mercury have recognised neurotoxic, nephrotoxic, and other toxic effects, studies on disposition of ethylmercury and other mercury forms are still justified, especially at young age. Our investigation aimed at comparing mercury distribution and rate of excretion in the early period of life following exposure to either thiomersal (TM) or mercuric chloride (HgCl2) in suckling rats. Three experimental groups were studied: control, TM, and HgCl2, with 12 to18 pups in each. Both forms of mercury were administered subcutaneously in equimolar quantities (0.81 µmol/kg b.w.) three times during the suckling period (on the days of birth 7, 9, and 11) to mimic the vaccination regimen in infants. After the last administration of TM or HgCl2, total mercury retention and excretion was assessed during following six days. In TM-exposed group mercury retention was higher in the brain, enteral excretion was similar, and urinary excretion was much lower compared to HgCl2-exposed sucklings. More research is still needed to elucidate all aspects of toxicokinetics and most harmful neurotoxic potential of various forms of mercury, especially in the earliest period of life.

Addressing the vaccine confidence gap.

Vaccines--often lauded as one of the greatest public health interventions--are losing public confidence. Some vaccine experts have referred to this decline in confidence as a crisis. We discuss some of the characteristics of the changing global environment that are contributing to increased public questioning of vaccines, and outline some of the specific determinants of public trust. Public decision making related to vaccine acceptance is neither driven by scientific nor economic evidence alone, but is also driven by a mix of psychological, sociocultural, and political factors, all of which need to be understood and taken into account by policy and other decision makers. Public trust in vaccines is highly variable and building trust depends on understanding perceptions of vaccines and vaccine risks, historical experiences, religious or political affiliations, and socioeconomic status. Although provision of accurate, scientifically based evidence on the risk-benefit ratios of vaccines is crucial, it is not enough to redress the gap between current levels of public confidence in vaccines and levels of trust needed to ensure adequate and sustained vaccine coverage. We call for more research not just on individual determinants of public trust, but on what mix of factors are most likely to sustain public trust. The vaccine community demands rigorous evidence on vaccine efficacy and safety and technical and operational feasibility when introducing a new vaccine, but has been negligent in demanding equally rigorous research to understand the psychological, social, and political factors that affect public trust in vaccines.

Determinants of influenza vaccine purchasing decision in the US: a conjoint analysis.

We explore the determinants of influenza vaccine purchasing decision in the US via a nationwide survey of 251 medical office managers and physicians on preferences for seven vaccine presentation attributes: price, presence of thimerosal, contamination risk, storage space requirement, number of preparation steps, dosing errors and speed. The findings show that thimerosal, contamination risk, and dosing errors were the most important attributes. For pediatricians, thimerosal's absence was shown to be the most valuable attribute. Participants would be willing to spend the following additional amounts per dose of influenza vaccine to acquire products as follows: $5.06 for the absence of thimerosal, $5.23 for a lower contamination risk, $4.94 for lower chance of dosing errors. They would pay $1.08 more for influenza vaccines that were faster to administer, $1.27 more for vaccines that were easier to store, and $1.76 more for vaccines that had fewer steps to administer.