Protective role of SARS-CoV-2 anti-S IgG against breakthrough infections among European healthcare workers during pre and post-Omicron surge-ORCHESTRA project.

PURPOSE: Anti SARS-CoV-2 vaccination initially showed high effectiveness in preventing COVID-19. However, after the surge of variants of concern, the effectiveness dropped. Several studies investigated if this was related to the decrease of the humoral response over time; however, this issue is still unclear. The aim of this study was to understand whether SARS-CoV-2 anti-S IgG levels can be used to predict breakthrough infection risk and define the timing for further booster doses administration. METHOD: Within the framework of the ORCHESTRA Project, over 20,000 health workers from 11 European centers were enrolled since December 2020. We performed two Cox proportional hazards survival analyses regarding pre-Omicron (from January to July 2021) and Omicron (December 2021-May 2022) periods. The serological response was classified as high (above the 75th percentile), medium (25th-75th), or low (< 25th). RESULTS: Seventy-four (0.33%) and 2122 (20%) health workers were infected during the first and second periods, respectively. Both Cox analyses showed that having high anti-S titer was linked to a significantly lower risk of infection as compared to having medium serological response [HR of high vs medium anti-S titer = 0.27 (95% CI 0.11-0.66) during the first phase, HR = 0.76 (95% CI 0.62-0.93) during the second phase]. CONCLUSION: Vaccine effectiveness wanes significantly after new variants surge, making anti-S titer unsuitable to predict optimal timing for further booster dose administration. Studies on other immunological indicators, such as cellular immunity, are therefore needed to better understand the mechanisms and duration of protection against breakthrough infection risk.

The impact of SARS-CoV-2 on healthcare workers of a large University Hospital in the Veneto Region: risk of infection and clinical presentation in relation to different pandemic phases and some relevant determinants.

Aim: The aim of this study is to evaluate the incidence of SARS-CoV-2 infection and the prevalence of COVID-19-related symptoms in relation to pandemic phases and some relevant variables in a cohort of 8,029 HCWs from one of the largest Italian University Hospitals. Methods: A single-center retrospective study was performed on data collected during SARS-CoV-2 infection surveillance of HCWs. Cox's multiple regression was performed to estimate hazard ratios of SARS-CoV-2 infection. Logistic multivariate regression was used to assess the risk of asymptomatic infections and the onset of the most frequent symptoms. All analyses were adjusted for sociodemographic and occupational factors, pandemic phases, vaccination status, and previous infections. Results: A total of 3,760 HCWs resulted positive (2.0%-18.6% across five study phases). The total incidence rate of SARS-CoV-2 infection was 7.31 cases per 10,000 person-days, significantly lower in phase 1 and higher in phases 4 and 5, compared to phase 3. Younger HCWs, healthcare personnel, and unvaccinated subjects showed a higher risk of infection. Overall, 24.5% were asymptomatic infections, with a higher probability for men, physicians, and HCWs tested for screening, fully vaccinated, and those with previous infection. The clinical presentation changed over the phases in relation to vaccination status and the emergence of new variants. Conclusion: The screening activities of HCWs allowed for the early detection of asymptomatic cases, limiting the epidemic clusters inside the hospital wards. SARS-CoV-2 vaccination reduced infections and symptomatic cases, demonstrating again its paramount value as a preventive tool for occupational and public health.

Comparison of Adverse Effects of Two SARS-CoV-2 Vaccines Administered in Workers of the University of Padova.

Introduction: In Italy, on December 2020, workers in the education sector were identified as a priority population to be vaccinated against COVID-19. The first authorised vaccines were the Pfizer-BioNTech mRNA (BNT162b2) and the Oxford-AstraZeneca adenovirus vectored (ChAdOx1 nCoV-19) vaccines. Aim: To investigate the adverse effects of two SARS-CoV-2 vaccines in a real-life preventive setting at the University of Padova. Methods: Vaccination was offered to 10116 people. Vaccinated workers were asked to voluntarily report symptoms via online questionnaires sent to them 3 weeks after the first and the second shot. Results: 7482 subjects adhered to the vaccination campaign and 6681 subjects were vaccinated with ChAdOx1 nCoV-19 vaccine and 137 (fragile subjects) with the BNT162b2 vaccine. The response rate for both questionnaires was high (i.e., >75%). After the first shot, the ChAdOx1 nCoV-19 vaccine caused more fatigue (p < 0.001), headache (p < 0.001), myalgia (p < 0.001), tingles (p = 0.046), fever (p < 0.001), chills (p < 0.001), and insomnia (p = 0.016) than the BNT162b2 vaccine. After the second dose of the BNT162b2 vaccine, more myalgia (p = 0.033), tingles (p = 0.022), and shivers (p < 0.001) than the ChAdOx1 nCoV-19 vaccine were elicited. The side effects were nearly always transient. Severe adverse effects were rare and mostly reported after the first dose of the ChAdOx1 nCoV-19 vaccine. They were dyspnoea (2.3%), blurred vision (2.1%), urticaria (1.3%), and angioedema (0.4%). Conclusions: The adverse effects of both vaccines were transient and, overall, mild in severity.

Non-sugar sweeteners and cancer: Toxicological and epidemiological evidence.

Several toxicological and epidemiological studies were published during the last five decades on non-sugar sweeteners (NSS) and cancer. Despite the large amount of research, the issue still continues to be of interest. In this review, we provided a comprehensive quantitative review of the toxicological and epidemiological evidence on the possible relation between NSS and cancer. The toxicological section includes the evaluation of genotoxicity and carcinogenicity data for acesulfame K, advantame, aspartame, cyclamates, saccharin, steviol glycosides and sucralose. The epidemiological section includes the results of a systematic search of cohort and case-control studies. The majority of the 22 cohort studies and 46 case-control studies showed no associations. Some risks for bladder, pancreas and hematopoietic cancers found in a few studies were not confirmed in other studies. Based on the review of both the experimental data on genotoxicity or carcinogenicity of the specific NSS evaluated, and the epidemiological studies it can be concluded that there is no evidence of cancer risk associated to NSS consumption.

Persistent Increase of Sympathetic Activity in Post-Acute COVID-19 of Paucisymptomatic Healthcare Workers.

Healthcare workers (HCWs) represent a population with a significant burden of paucisymptomatic COVID-19, as the general population. We evaluated autonomic nervous system activity by means of heart rate variability (HRV) in HCWs during health surveillance visits. Short-term electrocardiogram (ECG) recordings were obtained 30 days (IQR 5.25-55.75) after a negative naso-pharyngeal swab for SARS-CoV-2 in 44 cases and compared with ECGs of 44 controls with similar age and sex distribution. Time and frequency domain HRV were evaluated. HCWs who used drugs, had comorbidities that affected HRV, or were hospitalized with severe COVID-19 were excluded. Frequency domain HRV analysis showed a significantly higher low/high-frequency power ratio (LF/HF) in the case study compared with controls (t = 2.84, p = 0.006). In time domain HRV analysis, mean standard deviation of normal-to-normal intervals (SDNN) and root mean square of successive RR interval differences (RMSSD) were significantly lower for cases compared with controls (t = -2.64, p = 0.01 and t = -3.27, p = 0.002, respectively). In the post-acute phase of infection, SARS-CoV-2 produces an autonomic imbalance mirrored by a reduction in HRV. These results are consistent with epidemiological data that suggest a higher risk of acute cardiovascular complications in the first 30 days after COVID-19 infection.

Moderate alcohol consumption during pregnancy increases potency of two different drugs (the antifungal fluconazole and the antiepileptic valproate) in inducing craniofacial defects: prediction by the in vitro rat whole embryo culture.

The prenatal exposure to ethanol (Eth), fluconazole (FLUCO) and sodium valproate (VPA) is related to effects on development, producing characteristic syndromic pictures. Among embryotoxic effects described for the three molecules, the alteration on craniofacial morphogenesis is a common feature in humans and animal models, including rodent embryos developed in vitro. The aim of the present work is to evaluate the developmental effects of low Eth serum concentration (17 mM, corresponding to the legal limit to drive in UK, USA, Canada, and many other countries) in mixture with increasing realistic concentrations of the antifungal drug FLUCO (62.5-500 µM) or with increasing realistic concentrations of the antiepileptic drug VPA (31.25-250 µM). Groups exposed to Eth alone (17-127.5 mM), FLUCO alone (62.5-500 µM) or VPA alone (31.25-750 µM) were also included. The chosen alternative animal model was the post-implantation rat whole embryo culture (WEC). E9.5 embryos were exposed in vitro to the test molecules during the whole test period (48 h, corresponding to the developmental stages characteristics of any vertebrate, for human embryos post-fertilization days 23-31). Data were statistically analyzed and processed for modelling applying the benchmark dose (BMD) and relative potency factor (RPF) approaches. Concentration-related effects on facial outcomes were observed in all experimental groups, with a significant enhancement in the groups co-exposed with Eth in comparison to the single exposures. Data obtained by the present work suggest an additional alert for the assumption of even low levels of alcohol in pregnant women during FLUCO or VPA therapy.

SARS-CoV-2 Breakthrough Infections: Incidence and Risk Factors in a Large European Multicentric Cohort of Health Workers.

BACKGROUND: The research aimed to investigate the incidence of SARS-CoV-2 breakthrough infections and their determinants in a large European cohort of more than 60,000 health workers. METHODS: A multicentric retrospective cohort study, involving 12 European centers, was carried out within the ORCHESTRA project, collecting data up to 18 November 2021 on fully vaccinated health workers. The cumulative incidence of SARS-CoV-2 breakthrough infections was investigated with its association with occupational and social-demographic characteristics (age, sex, job title, previous SARS-CoV-2 infection, antibody titer levels, and time from the vaccination course completion). RESULTS: Among 64,172 health workers from 12 European health centers, 797 breakthrough infections were observed (cumulative incidence of 1.2%). The primary analysis using individual data on 8 out of 12 centers showed that age and previous infection significantly modified breakthrough infection rates. In the meta-analysis of aggregated data from all centers, previous SARS-CoV-2 infection and the standardized antibody titer were inversely related to the risk of breakthrough infection (p = 0.008 and p = 0.007, respectively). CONCLUSION: The inverse correlation of antibody titer with the risk of breakthrough infection supports the evidence that vaccination plays a primary role in infection prevention, especially in health workers. Cellular immunity, previous clinical conditions, and vaccination timing should be further investigated.

A new approach to the classification of carcinogenicity.

Concern over substances that may cause cancer has led to various classification schemes to recognize carcinogenic threats and provide a basis to manage those threats. The least useful schemes have a binary choice that declares a substance carcinogenic or not. This overly simplistic approach ignores the complexity of cancer causation by considering neither how the substance causes cancer, nor the potency of that mode of action. Consequently, substances are classified simply as "carcinogenic", compromising the opportunity to properly manage these kinds of substances. It will likely be very difficult, if not impossible, to incorporate New Approach Methodologies (NAMs) into binary schemes. In this paper we propose a new approach cancer classification scheme that segregates substances by both mode of action and potency into three categories and, as a consequence, provides useful guidance in the regulation and management of substances with carcinogenic potential. Examples are given, including aflatoxin (category A), trichlorethylene (category B), and titanium dioxide (category C), which demonstrate the clear differentiation among these substances that generate appropriate levels of concern and management options.

The University of Padua salivary-based SARS-CoV-2 surveillance program minimized viral transmission during the second and third pandemic wave.

BACKGROUND: The active surveillance of students is proposed as an effective strategy to contain SARS-CoV-2 spread and prevent schools' closure. Saliva for molecular testing is as sensitive as naso-pharyngeal swab (NPS), self-collected and well accepted by participants. This prospective study aimed to verify whether the active surveillance of the Padua University employees by molecular testing of self-collected saliva is an effective and affordable strategy for limiting SARS-CoV-2 spread. METHODS: A surveillance program based on self-collection of saliva every 2 weeks (October 2020-June 2021) was conducted. Among 8183 employees of the Padua University, a total of 6284 subjects voluntarily took part in the program. Eight collection points guaranteed the daily distribution and collection of barcoded salivary collection devices, which were delivered to the laboratory by a transport service for molecular testing. Quarantine of positive cases and contact tracing were promptly activated. RESULTS: Among 6284 subjects, 206 individuals were SARS-CoV-2 positive (99 by salivary testing; 107 by NPS performed for contact tracing or symptoms). The cumulative SARS-CoV-2 incidence in this cohort was 3.1%, significantly lower than that of employees not in surveillance (8.0%), in Padua (7.1%) and in the Veneto region (7.2%). Employees with positive saliva results were asymptomatic or had mild symptoms. The levels of serum antibodies after 3 months from the infection were correlated with age and Ct values, being higher in older subjects with greater viral loads. CONCLUSIONS: Salivary-based surveillance with contact tracing effectively allowed to limit SARS-CoV-2 contagion, also in a population with a high incidence.

Occupational Exposure to Halogenated Anaesthetic Gases in Hospitals: A Systematic Review of Methods and Techniques to Assess Air Concentration Levels.

Objective During the induction of gaseous anaesthesia, waste anaesthetic gases (WAGs) can be released into workplace air. Occupational exposure to high levels of halogenated WAGs may lead to adverse health effects; hence, it is important to measure WAGs concentration levels to perform risk assessment and for health protection purposes. Methods A systematic review of the scientific literature was conducted on two different scientific databases (Scopus and PubMed). A total of 101 studies, focused on sevoflurane, desflurane and isoflurane exposures in hospitals, were included in this review. Key information was extracted to provide (1) a description of the study designs (e.g., monitoring methods, investigated occupational settings, anaesthetic gases in use); (2) an evaluation of time trends in the measured concentrations of considered WAGs; (3) a critical evaluation of the sampling strategies, monitoring methods and instruments used. Results Environmental monitoring was prevalent (68%) and mainly used for occupational exposure assessment during adult anaesthesia (84% of cases). Real-time techniques such as photoacoustic spectroscopy and infrared spectrophotometry were used in 58% of the studies, while off-line approaches such as active or passive sampling followed by GC-MS analysis were used less frequently (39%). Conclusions The combination of different instrumental techniques allowing the collection of data with different time resolutions was quite scarce (3%) despite the fact that this would give the opportunity to obtain reliable data for testing the compliance with 8 h occupational exposure limit values and at the same time to evaluate short-term exposures.

Modified Xenopus laevis approach (R-FETAX) as an alternative test for the evaluation of foetal valproate spectrum disorder.

In compliance to animal welfare 3Rs principle there is a great demand for refined tests alternative to classical mammal teratogenicity tests. We propose a refined alternative amphibian method (R-FETAX) to evaluate chemical induced embryotoxicity. The human foetal valproate spectrum disorder (FVSD) characteristics are morphological defects (including cranio-facial, neural tube defects) and behavioural alterations due to valproate (VPA) exposure in pregnancy. Vertebrate assays to evaluate FVSD include classical and alternative mammal (implying adult sacrifice), and non-mammal developmental models (zebrafish, amphibians, chick). Among these latter only zebrafish assays report in the same test both morphological and behavioural examinations. Compared to zebrafish, the amphibian Xenopus laevis excels having a more comparable organ development and morphology to mammalian systems. We used X. laevis embryos exposed during developmental specific windows to VPA therapeutic concentrations. Different VPA effects were observed depending on the exposure window: concentration-related embryo-lethal and teratogenic effects (neural tube, facial, tail defects) were observed in groups exposed at the organogenetic phylotypic stages. Neurobehavioral deficits were described using a functional swimming test at the highest VPA concentration exposure during the phylotypic stages and at any concentration during neurocognitive competent stages. Malformations were compared to those obtained in a mammalian assay (the rat post-implantation whole embryo culture method, WEC), that we used in the past to evaluate VPA teratogenicity. R-FETAX and WEC data were modelled and their relative sensitivity was calculated. We suggest the amphibian R-FETAX as a refined windowed alternative test for the evaluation of chemicals inducing both morphological and behavioural anomalies, including VPA.

The codification of hazard and its impact on the hazard versus risk controversy.

The long running controversy about the relative merits of hazard-based versus risk-based approaches has been investigated. There are three levels of hazard codification: level 1 divides chemicals into dichotomous bands of hazardous and non-hazardous; level 2 divides chemicals into bands of hazard based on severity and/or potency; and level 3 places each chemical on a continuum of hazard based on severity and/or potency. Any system which imposes compartments onto a continuum will give rise to issues at the boundaries, especially with only two compartments. Level 1 schemes are only justifiable if there is no variation in severity, or potency or if there is no threshold. This is the assumption implicit in GHS/EU classification for carcinogenicity, reproductive toxicity and mutagenicity. However, this assumption has been challenged. Codification level 2 hazard assessments offer a range of choices and reduce the built-in conflict inherent in the level 1 process. Level 3 assessments allow a full range of choices between the extremes and reduce the built-in conflict even more. The underlying reason for the controversy between hazard and risk is the use of level 1 hazard codification schemes in situations where there are ranges of severity and potency which require the use of level 2 or level 3 hazard codification. There is not a major difference between level 2 and level 3 codification, and they can both be used to select appropriate risk management options. Existing level 1 codification schemes should be reviewed and developed into level 2 schemes where appropriate.

Use of Plant Protection Products in Lombardy, Italy and the Health Risk for the Ingestion of Contaminated Water.

Pesticides used to protect agricultural crops may contaminate groundwater. This work aimed to identify the pesticides used in Lombardy, Italy, in 2016, their concentration in the groundwater and the risk for health associated with the intake of drinkable water in the adult population. The risk was evaluated for the presence of single and multiple active substances in the groundwater, calculating the hazard quotient (HQ) and the hazard index (HI), respectively. Lombardy utilises an agricultural area of 980,112 h, which is mainly cultivated with cereals (74%). Approximately 2354 pesticides (about 1.3 × 107 kg), containing 410 active substances (about 4.5 × 106 kg) were sold. There were groundwater contamination measurements in 158 monitoring points, which were investigated twice a year for 31 active substances, and a total of 9152 determinations. Only 17 currently used active substance were measured in the groundwater, among which three belonged to the 10 best-sold pesticides. The exceedance of the environmental quality standard was observed for about 1.5% determinations. The intake of contaminated water in the adult population resulted in a HQ typically ranging between 10-3 and 10-4 and a HI of about 10-3. Although the number of pesticides sold in 2016 in Lombardy was big, only a small fraction of active substances was monitored in the groundwater. Considering these monitored substances, the intake of contaminated groundwater in the adult general population posed an irrelevant risk for health.

Response to Vaccination against Mumps in Medical Students: Two Doses Are Needed.

Mumps is a vaccine-preventable infectious disease diffuse worldwide. The implementation of mumps vaccination reduced largely the spread of infection. On 11,327 Medical School students the prevalence of mumps positive antibodies was evaluated according to dose/doses of vaccine, year of birth and sex. Compliance to mumps vaccine was low in students born before 1990 but increased consistently after this year, above all compliance to two doses, due to the implementation of the vaccine offer. Positivity of mumps antibodies is significantly (p < 0.0001) lower in students vaccinated once (71.2%) compared to those vaccinated twice (85.4%). In addition, students born after 1995, largely vaccinated twice, showed a seropositivity near to 90%. Further, females had a significantly (p < 0.0001) higher proportion of positive antibodies after vaccination than males, both one (74.6% vs. 64.7%) and two doses (86.8% vs. 82.9%). Finally, seropositivity after two vaccine doses remains high (86.1%) even 15 years after the second dose. In conclusion, the research highlighted that vaccination against mumps reaches a good level of coverage only after two doses of vaccine persisting at high levels over 15 years and induces a more significant response in females.

An adverse outcome pathway on the disruption of retinoic acid metabolism leading to developmental craniofacial defects.

Adverse outcome pathway (AOP) is a conceptual framework that links a molecular initiating event (MIE) via intermediate key events (KEs) with adverse effects (adverse outcomes, AO) relevant for risk assessment, through defined KE relationships (KERs). The aim of the present work is to describe a linear AOP, supported by experimental data, for skeletal craniofacial defects as the AO. This AO was selected in view of its relative high incidence in humans and the suspected relation to chemical exposure. We focused on inhibition of CYP26, a retinoic acid (RA) metabolizing enzyme, as MIE, based on robust previously published data. Conazoles were selected as representative stressors. Intermediate KEs are RA disbalance, aberrant HOX gene expression, disrupted specification, migration, and differentiation of neural crest cells, and branchial arch dysmorphology. We described the biological basis of the postulated events and conducted weight of evidence (WoE) assessments. The biological plausibility and the overall empirical evidence were assessed as high and moderate, respectively, the latter taking into consideration the moderate evidence for concordance of dose-response and temporal relationships. Finally, the essentiality assessment of the KEs, considered as high, supported the robustness of the presented AOP. This AOP, which appears of relevance to humans, thus contributes to mechanistic underpinning of selected test methods, thereby supporting their application in integrated new approach test methodologies and strategies and application in a regulatory context.

Vaccination and Immunity toward Measles: A Serosurvey in Future Healthcare Workers.

Measles is a very contagious infectious disease, and vaccination is the only medical aid to counter the spread of the infection. The aim of this study was to evaluate the influence of vaccination schedule and type of vaccine, number of doses, and sex on the immune response. In a population of Italian medical students (8497 individuals born after 1980 with certificate of vaccination and quantitative measurement of antibodies against measles), the prevalence of positive antibodies to measles and antibody titer was measured. Vaccination schedule such as number of doses and vaccine type (measles alone or combined as measles, mumps and rubella (MMR)) and sex were the variables considered to influence the immune response. The vaccination schedule depends on the year of birth: students born before 1990 were prevalently vaccinated once and with measles vaccine alone (not as MMR). One dose of vaccine induces a significantly (p < 0.0001) higher positive response and antibody titer than two doses, in particular when measles alone is used (p < 0.0001). Females have a significantly higher percentage of positive response (p = 0.0001) than males but only when the MMR formulation was used. Multiple linear regression confirms that sex significantly influences antibody titer when only MMR is used, after one (p = 0.0002) or two (p = 0.0060) doses. In conclusion, vaccination schedule and, partially, sex influence immune response to measles vaccination. Most notably, the measles vaccine alone (one dose) is more effective than one and two doses of MMR.

A generic PBTK model implemented in the MCRA platform: Predictive performance and uses in risk assessment of chemicals.

Physiologically-based toxicokinetic (PBTK) models are important tools for in vitro to in vivo or inter-species extrapolations in health risk assessment of foodborne and non-foodborne chemicals. Here we present a generic PBTK model implemented in the EuroMix toolbox, MCRA 9 and predict internal kinetics of nine chemicals (three endocrine disrupters, three liver steatosis inducers, and three developmental toxicants), in data-rich and data-poor conditions, when increasingly complex levels of parametrization are applied. At the first stage, only QSAR models were used to determine substance-specific parameters, then some parameter values were refined by estimates from substance-specific or high-throughput in vitro experiments. At the last stage, elimination or absorption parameters were calibrated based on available in vivo kinetic data. The results illustrate that parametrization plays a capital role in the output of the PBTK model, as it can change how chemicals are prioritized based on internal concentration factors. In data-poor situations, estimates can be far from observed values. In many cases of chronic exposure, the PBTK model can be summarized by an external to internal dose factor, and interspecies concentration factors can be used to perform interspecies extrapolation. We finally discuss the implementation and use of the model in the MCRA risk assessment platform.

Cumulative dietary risk assessment overarching different regulatory silos using a margin of exposure approach: A case study with three chemical silos.

Risk assessment of chemicals occurring in our diet is commonly performed for single chemicals without considering exposure to other chemicals. We performed a case study on risk assessment of combined dietary exposure to chemicals from different regulatory silos, i.e. pesticides (PPRs), persistent organic pollutants (POPs) and food additives (FAs). Chemicals were grouped into the cumulative assessment group (CAG) liver steatosis using a component-based approach. Based on literature, the CAG included 144 PPRs, 49 POPS and 7 FAs for which concentration data were available. For each silo, chronic combined dietary exposure was assessed for adults and children of nine European countries following the most commonly used exposure methodologies in Europe and by using a relative potency factor approach. For risk characterization, a Margin of Exposure (MOE) was calculated. To overarch the risk across silos, a normalised combined margin of exposure (nMOET) approach was proposed. This case study demonstrated that risk assessment of combined exposure to chemicals can be performed within regulatory silos. It also highlighted important differences in the conservatism of exposure scenarios, the derivation of point of departures and the subsequent acceptable MOEs between the silos. To overarch the risk despite these differences, a nMOET approach can be used.

Development of an adverse outcome pathway for cranio-facial malformations: A contribution from in silico simulations and in vitro data.

Mixtures of substances sharing the same molecular initiating event (MIE) are supposed to induce additive effects. The proposed MIE for azole fungicides is CYP26 inhibition with retinoic acid (RA) local increase, triggering key events leading to craniofacial defects. Valproic acid (VPA) is supposed to imbalance RA-regulated gene expression trough histone deacetylases (HDACs) inhibition. The aim was to evaluate effects of molecules sharing the same MIE (azoles) and of such having (hypothetically) different MIEs but which are eventually involved in the same adverse outcome pathway (AOP). An in silico approach (molecular docking) investigated the suggested MIEs. Teratogenicity was evaluated in vitro (WEC). Abnormalities were modelled by PROAST software. The common target was the branchial apparatus. In silico results confirmed azole-related CYP26 inhibition and a weak general VPA inhibition on the tested HDACs. Unexpectedly, VPA showed also a weak, but not marginal, capability to enter the CYP 26A1 and CYP 26C1 catalytic sites, suggesting a possible role of VPA in decreasing RA catabolism, acting as an additional MIE. Our findings suggest a new more complex picture. Consequently two different AOPs, leading to the same AO, can be described. VPA MIEs (HDAC and CYP26 inhibition) impinge on the two converging AOPs.