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Corrigendum to "Biological impact of metal nanomaterials in relation to their physicochemical characteristics" [Toxicol in Vitro. 2019 Jan 29;56:172-183]. [Toxicol In Vitro. 2019]. Toxicol In Vitro. 2019 Feb 16. pii: S0887-2333(19)30110-9. doi:10.1016/j.tiv.2019.02.001. [Epub ahead of print]. Disponível em: https://www.sciencedirect.com/science/article/pii/S0887233319301109?via%3Dihub ; Several metal and metal oxide nanomaterials (NMs), e.g., cerium dioxide NMs(CeO2), barium sulphate NMs(BaSO4) and titanium dioxide NMs(TiO2), display advantageous properties over the bulk materials and have a broad range of innovative applications in food, industry and consumer products. Whether these materials are hazardous and impact on human health or the environment remains an issue that needs to be addressed by reliable studies focused on nano-bio interactions. To contribute to the comprehensive investigation of the toxicological effects of metal NMs, we have assessed the cytotoxic and genotoxic effects of benchmark NMs in human respiratory cells, concomitantly with the analysis of their secondary properties in the cellular moiety. This study shows no effects of BaSO4, while some, but not all, of the other metal-related NMs analyzed have adverse effects. Human respiratory cells were prone to CeO2 cytotoxicity and to DNA damage induction following exposure to anatase TiO2 (NM-100, NM-101 and NM-102), but not rutile TiO2. No clastogenic/aneugenic effects were ascribed to any of the tested NMs. Using correlation analysis, this work also suggests that among these TiO2, the size in the cellular moiety may be the most relevant secondary feature that determines their biological consequences. ; This work was financed by national funds through the FCT - Foundation for Science and Technology, I.P., under the project PTDC/ SAU-PUB/29481/2017. Research co-funded by European Union Seventh Framework Programme (FP7/2007e2013) under the project NANoREG (A common European approach to the regulatory testing of NMs), grant agreement 310584 and by UID/BIM/00009/2013 (Centre for Toxicogenomics and Human Health – ToxOmics, Foundation for Science and Technology). ; info:eu-repo/semantics/publishedVersion

To contribute with scientific evidence to the grouping strategy for the safety assessment of multi-walled carbon nanotubes (MWCNTs), this work describes the investigation of the cytotoxic and genotoxic effects of four benchmark MWCNTs in relation to their physicochemical characteristics, using two types of human respiratory cells. The cytotoxic effects were analysed using the clonogenic assay and replication index determination. A 48h-exposure of cells revealed that NM-401 was the only cytotoxic MWCNT in both cell lines, but after 8-days exposure, the clonogenic assay in A549 cells showed cytotoxic effects for all the tested MWCNTs. Correlation analysis suggested an association between the MWCNTs size in cell culture medium and cytotoxicity. No induction of DNA damage was observed after any MWCNTs in any cell line by the comet assay, while the micronucleus assay revealed that both NM-401 and NM-402 were genotoxic in A549 cells. NM-401 and NM-402 are the two longest MWCNTs analyzed in this work, suggesting that length may be determinant for genotoxicity. No induction of micronuclei was observed in Beas-2B cell line and the different effect in both cell lines is explained in view of the size-distribution of MWCNTs in the cell culture medium, rather than cell's specificities. ; The research leading to these results was co-funded by European Union Seventh Framework Programme (FP7/2007e2013) under the project NANoREG (A common European approach to the regulatory testing of nanomaterials), grant agreement 310584, by NANOGENOTOX Joint Action, in the framework of the Health Programme under grant agreement 2009 21 and by UID/BIM/00009/2013 (Centre for Toxicogenomics and Human Health – ToxOmics, Foundation for Science and Technology). The authors also acknowledge Dr. José Catita (Paralab, Portugal) for the support with the DLS analysis.

Environmental tobacco smoke (ETS) is recognized as one of the most common indoor pollutants worldwide. Portuguese legislation prohibits smoking in most indoor public spaces. However, in some restaurants/bars smoking is still allowed, representing a potential risk factor for the workers health, particularly for chronic respiratory diseases and cancer onset.The aim of this study was to characterize early signs of ETS-associated adverse systemic effects in workers from restaurants with smoking permission in comparison with workers from smoke-free spaces, considering the modulating effects of genetic susceptibility.The ETS-exposed workers did not display differences in the frequencies of SCEs, cells with high frequency of SCEs (HFCs), MN or DNA strand breaks, as assessed by the comet assay, when compared to non-ETS workers. Smoking workers presented a significantly increased level of HFCs as compared to non-smokers. Interestingly, the ex vivo challenge of leukocytes with EMS resulted in a lower level of DNA breaks in ETS-exposed as compared to non-exposed workers (P<0,0001), suggesting an increased DNA repair capacity associated to ETS-exposure. Regarding the genetic polymorphisms studied, GSTM1 null genotype carriers presented increased frequencies of SCEs and HFCs associated with ETS exposure, suggesting an increased susceptibility to this environmental stressor. On the contrary, XRCC1-399 wild type carriers presented a lower level of MN than the variant allele carriers, in response to ETS exposure. Finally, among the ETS-exposed subjects, those carrying the hOGG1 variant alleles presented a lower level of ex vivo EMS-induced DNA damage comparatively to the wild type subjects, suggesting a higher DNA repair capacity.The study of ETS exposure in an occupational setting in Lisbon restaurants revealed that the exposed workers did not display systemic genetic effects detectable by the biomarkers analysed. However, the results of the ex vivo challenge comet assay, suggests that ETS may have a more subtle modulating effect on the DNA repair response of blood cells to a genotoxic insult. In addition, the association between some genetic polymorphisms and increased genotoxic effects in subsets of individuals, highlights the possibility of increased health risks in susceptible individuals exposed to ETS, that should be further investigated.

Portuguese legislation prohibits smoking in most indoor public spaces. However, some restaurants/bars remain, in which smoking is still allowed, representing a potential risk for employees, particularly for chronic respiratory diseases and cancer development. The objectives of this project were to compare the indoor air quality of some restaurants with and without smoking permission and, in their workers, to search for associations between respiratory dysfunctions and biomarkers of exposure, biological effects (DNA and proteome alterations) and susceptibility. Herein, we present data on DNA damage and genetic susceptibility in 37 workers occupationally exposed to environmental tobacco smoke (ETS) and from 44 non-exposed workers. DNA damage was assessed by the comet assay in blood leukocytes and by micronucleus (MN) analysis in buccal cells. DNA repair capacity was evaluated by a challenge of blood cells with EMS (32 µM), preceding the comet assay. Polymorphisms in metabolism (GSTP1, GSTM and GSTT) and DNA-repair genes (hOGG1,XRCC1, XRCC3, and NBS1) were analysed by PCR/RFLP. No significant differences in the MN frequency and in level of DNA damage was observed between ETS and non-ETS groups. However, challenge with EMS resulted in a significantly lower level of DNA breaks in ETS-exposed as compared to non-exposed workers (P<0.0001). Concerning polymorphisms analysis, GSTP1 variant allele carriers showed a decreased frequency of MN in buccal cells, which was not influenced by ETS exposure. No association related to the other polymorphisms was detected. In conclusion, the present data show that blood cells from ETS-exposed workers display a lower mutagenic response to EMS challenge, suggestive of an adaptive response elicited by the previous exposure to low levels of ETS. ; Fundação Calouste Gulbenkian, ACSS and FCT/Pluriannual funding

Review ; A number of human biomonitoring (HBM) studies have presented data on exposure to hexavalent chromium [Cr(VI)] and cadmium (Cd), but comparatively few include results on effect biomarkers. The latter are needed to identify associations between exposure and adverse outcomes (AOs) in order to assess public health implications. To support improved derivation of EU regulation and policy making, it is of great importance to identify the most reliable effect biomarkers for these heavy metals that can be used in HBM studies. In the framework of the Human Biomonitoring for Europe (HBM4EU) initiative, our study aim was to identify effect biomarkers linking Cr(VI) and Cd exposure to selected AOs including cancer, immunotoxicity, oxidative stress, and omics/epigenetics. A comprehensive PubMed search identified recent HBM studies, in which effect biomarkers were examined. Validity and applicability of the markers in HBM studies are discussed. The most frequently analysed effect biomarkers regarding Cr(VI) exposure and its association with cancer were those indicating oxidative stress (e.g., 8-hydroxy-2'-deoxyguanosine (8-OHdG), malondialdehyde (MDA), glutathione (GSH)) and DNA or chromosomal damage (comet and micronucleus assays). With respect to Cd and to some extent Cr, β-2-microglobulin (B2-MG) and N-acetyl-β-D-glucosaminidase (NAG) are well-established, sensitive, and the most common effect biomarkers to relate Cd or Cr exposure to renal tubular dysfunction. Neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule (KIM)-1 could serve as sensitive biomarkers of acute kidney injury in response to both metals, but need further investigation in HBM studies. Omics-based biomarkers, i.e., changes in the (epi-)genome, transcriptome, proteome, and metabolome associated with Cr and/or Cd exposure, are promising effect biomarkers, but more HBM data are needed to confirm their significance. The combination of established effect markers and omics biomarkers may represent the strongest approach, especially if based on knowledge of mechanistic principles. To this aim, also mechanistic data were collected to provide guidance on the use of more sensitive and specific effect biomarkers. This also led to the identification of knowledge gaps relevant to the direction of future research. ; This research was supported by funding from the European Union´s Horizon 2020 research and innovation Programme under grant agreement No 733032 HBM4EU ; info:eu-repo/semantics/publishedVersion

Abstract
In the scope of the European Union (EU) human biomonitoring initiative, a multicentric study on different occupational settings from several European countries was performed, to provide information on occupational exposure to hexavalent chromium [Cr(VI)], a known lung carcinogen. Biomonitoring approaches were used to obtain exposure data to support the implementation of new risk management measures and policy actions at the national and European levels. This work describes the Portuguese contribution to the study, which aimed to assess workers' exposure to Cr, by using exposure biomarkers (urinary chromium [U-Cr]), and industrial hygiene samples (air and hand wipes) and to link exposure to potential long-term health effects by using effect biomarkers. Exposure determinants influencing exposure were explored from the contextual information and human biomonitoring data. The ultimate goal of the study was to appraise the risk management measures contributing to minimize exposure and protect workers' health. Several occupational settings and activities were considered, including plating, welding, and painting. A control group from the Portuguese general population was also included. Data on age, sex, and smoking habits from both groups were considered in the statistical analysis. Information on the risk management measures available for workers was collected and used to identify the ones that mainly contributed to reduce exposure. Environmental monitoring and human biomonitoring revealed that painters were the highest exposed group. The use of respiratory protection equipment showed an influence on total U-Cr levels for workers involved in painting activities. Concerning early health effects, the painters presented also a significantly higher level of DNA and chromosomal damage in peripheral blood cells, as compared to the control group, suggesting a plausible association between exposure to Cr(VI) and early genotoxic effects. The results showed that workers are exposed to Cr(VI) in those occupational settings. These findings point to the need to improve the prevention and risk management measures and the implementation and enforcement of new regulatory actions at the national level.

A number of human biomonitoring (HBM) studies have presented data on exposure to hexavalent chromium [Cr(VI)] and cadmium (Cd), but comparatively few include results on effect biomarkers. The latter are needed to identify associations between exposure and adverse outcomes (AOs) in order to assess public health implications. To support improved derivation of EU regulation and policy making, it is of great importance to identify the most reliable effect biomarkers for these heavy metals that can be used in HBM studies. In the framework of the Human Biomonitoring for Europe (HBM4EU) initiative, our study aim was to identify effect biomarkers linking Cr(VI) and Cd exposure to selected AOs including cancer, immunotoxicity, oxidative stress, and omics/epigenetics. A comprehensive PubMed search identified recent HBM studies, in which effect biomarkers were examined. Validity and applicability of the markers in HBM studies are discussed. The most frequently analysed effect biomarkers regarding Cr(VI) exposure and its association with cancer were those indicating oxidative stress (e.g., 8-hydroxy-2-deoxyguanosine (8-OHdG), malondialdehyde (MDA), glutathione (GSH)) and DNA or chromosomal damage (comet and micronucleus assays). With respect to Cd and to some extent Cr, -2-microglobulin (B2-MG) and N-acetyl--D-glucosaminidase (NAG) are well-established, sensitive, and the most common effect biomarkers to relate Cd or Cr exposure to renal tubular dysfunction. Neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule (KIM)-1 could serve as sensitive biomarkers of acute kidney injury in response to both metals, but need further investigation in HBM studies. Omics-based biomarkers, i.e., changes in the (epi-)genome, transcriptome, proteome, and metabolome associated with Cr and/or Cd exposure, are promising effect biomarkers, but more HBM data are needed to confirm their significance. The combination of established effect markers and omics biomarkers may represent the strongest approach, especially if based on knowledge of mechanistic principles. To this aim, also mechanistic data were collected to provide guidance on the use of more sensitive and specific effect biomarkers. This also led to the identification of knowledge gaps relevant to the direction of future research. ; (VLID)6012056

This deliverable was produced under Task 6.3 of Work Package 6 on "Longer-term needs and expectations of stakeholders (2021-2030)". The aim of this task was to gather information on the needs and expectations of both the National Hubs (NH) and a broad range of stakeholders regarding a long-term Human Biomonitoring (HBM) programme for Europe, to be establish in follow up to the current project, HBM4EU. Firstly and with a focus on options for financing a future initiative, information was collected on a range of available funding mechanisms at national, regional and international level. In terms of national funding, the results presented rely on responses to a survey with the NH. Unfortunately, the response from NHs was very limited, and as such the report only captures funding mechanisms from a limited number of countries, including Portugal, Spain, France, Germany and Cyprus. In order to gain insight into the status of the National Hubs and their capacities for HBM, a survey was conducted. The results provide an overview of the current situation across the NHs with regards to a range of aspects, including the level of activity of the NH, status of political support, availability of funding, ongoing HBM studies and willingness to align studies with HBM4EU. The results suggest that HBM4EU has raised the political profile of HBM in partner countries. Regarding ongoing HBM studies, only six countries have national HBM programmes, with most countries having only hot spot studies. Despite this, there was support for the alignment of studies to achieve European geographical coverage. Regarding funding, access to European funding is seen as important to leverage funds at national level. A second survey was targeted at a wide range of stakeholders, aiming to better understand their expectations for a long-term sustainable HBM initiative. The survey was followed by a workshop, where a more limited number of stakeholders had the opportunity to discuss the survey results and address such aspects as the scope of a future initiative, how to achieve financial sustainability, how to involve and how a future initiative might contribute to chemical policies. Concerning the needs and expectations of stakeholders, there is a strong interest in and support for a future HBM initiative at European level. The involvement of a European Union (EU) institution as part of a future steering committee was seen as crucial. According to this stakeholders' consultation, the initiative should focus on protecting human health and the environment in Europe from hazardous chemical exposures by producing harmonised, high quality, transparent and inclusive data for effective risk assessment and management. ; HBM4EU - Grant agreement 733032 HORIZON 2020 Programme ; info:eu-repo/semantics/publishedVersion

This article belongs to the Special Issue 2nd Edition: Human Biomonitoring of Environmental and Occupational Exposures ; Study Protocol ; Workers involved in the processing of electronic waste (e-waste) are potentially exposed to toxic chemicals. If exposure occurs, this may result in uptake and potential adverse health effects. Thus, exposure surveillance is an important requirement for health risk management and prevention of occupational disease. Human biomonitoring by measurement of specific biomarkers in body fluids is considered as an effective method of exposure surveillance. The aim of this study is to investigate the internal exposure of workers processing e-waste using a human biomonitoring approach, which will stimulate improved work practices and contribute to raising awareness of potential hazards. This exploratory study in occupational exposures in e-waste processing is part of the European Human Biomonitoring Initiative (HBM4EU). Here we present a study protocol using a cross sectional survey design to study worker's exposures and compare these to the exposure of subjects preferably employed in the same company but with no known exposure to industrial recycling of e-waste. The present study protocol will be applied in six to eight European countries to ensure standardised data collection. The target population size is 300 exposed and 150 controls. Biomarkers of exposure for the following chemicals will be used: chromium, cadmium and lead in blood and urine; brominated flame retardants and polychlorobiphenyls in blood; mercury, organophosphate flame retardants and phthalates in urine, and chromium, cadmium, lead and mercury in hair. In addition, the following effect biomarkers will be studied: micronuclei, epigenetic, oxidative stress, inflammatory markers and telomere length in blood and metabolomics in urine. Occupational hygiene sampling methods (airborne and settled dust, silicon wristbands and handwipes) and contextual information will be collected to facilitate the interpretation of the biomarker results and discuss exposure mitigating interventions to further reduce exposures if needed. This study protocol can be adapted to future European-wide occupational studies. ; This work has received external funding from the European Union's Horizon 2020 research and innovation program under grant agreement No. 733032 and received co-funding from the author's organizations and/or Ministries. ; info:eu-repo/semantics/publishedVersion

WP5 had two objectives: - To generate in vitro genotoxicity data on manufactured MNs (specific objective #3). - To perform a round robin test on in vitro genotoxicity testing of MNs (specific objective #4) WP5 addressed the basic questions of in vitro genotoxicity testing of manufactured nanomaterials (MNs): how well in vitro assays can be used for revealing the genotoxic potential of MNs, which assays are suitable for this task, and which modifications are needed in standard tests when MNs are studied. WP5 aimed at establishing robust methodology to screen the in vitro genotoxicity of MNs in pulmonary, oral, and epidermal cell systems. These assays were to be applied to all MNs included in the project. On the basis of the results obtained, a ring test was to be performed using the most promising approach. Finally, the results were to be evaluated for correlation with in vivo results and used, together with other genotoxicity data available and the kinetic results, to formulate a strategy for genotoxicity testing of MNs (this deliverable). ; This document arises from the NANOGENOTOX Joint Action which has received funding from the European Union, in the framework of the Health Programme under Grant Agreement n°2009 21.

Multicenter Study ; The EU human biomonitoring initiative, HBM4EU, aims to co-ordinate and advance human biomonitoring (HBM) across Europe. As part of HBM4EU, we presented a protocol for a multicentre study to characterize occupational exposure to hexavalent chromium (Cr(VI)) in nine European countries (HBM4EU chromates study). This study intended to collect data on current occupational exposure and to test new indicators for chromium (Cr) biomonitoring (Cr(VI) in exhaled breath condensate and Cr in red blood cells), in addition to traditional urinary total Cr analyses. Also, data from occupational hygiene samples and biomarkers of early biological effects, including genetic and epigenetic effects, was obtained, complementing the biomonitoring information. Data collection and analysis was completed, with the project findings being made separately available. As HBM4EU prepares to embark on further European wide biomonitoring studies, we considered it important to reflect on the experiences gained through our harmonised approach. Several practical aspects are highlighted for improvement in future studies, e.g., more thorough/earlier training on the implementation of standard operating procedures for field researchers, training on the use of the data entry template, as well as improved company communications. The HBM4EU chromates study team considered that the study had successfully demonstrated the feasibility of conducting a harmonised multicentre investigation able to achieve the research aims and objectives. This was largely attributable to the engaged multidisciplinary network, committed to deliver clearly understood goals. Such networks take time and investment to develop, but are priceless in terms of their ability to deliver and facilitate knowledge sharing and collaboration. ; Highlights: Feasibility of conducting harmonised Pan-European biomonitoring study on occupational exposure demonstrated; Developing a successful network and implementation of systematic methodology takes significant dedication from all involved; Methodological improvements were identified which will benefit future large-scale occupational biomonitoring campaigns; Developed multicentre network allows and promotes further opportunities for future research, knowledge sharing and collaboration; Data produced supports science to policy interface in the scope of REACH and occupational safety and health regulations. ; This project has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No 733032 and received co-funding from the author's organizations and/or Ministries. ; info:eu-repo/semantics/publishedVersion

Abstract
Citric acid esters of mono‐ and diglycerides of fatty acids (E 472c) was re‐evaluated in 2020 by the Food Additives and Flavourings Panel (FAF Panel) along with acetic acid, lactic acid, tartaric acid, mono‐ and diacetyltartaric acid, mixed acetic and tartaric acid esters of mono‐ and diglycerides of fatty acids (E 472a,b,d,e,f). As a follow‐up to this assessment, the FAF Panel was requested to assess the safety of citric acid esters of mono‐ and diglycerides of fatty acids (E 472c) for its use as food additive in food for infants below 16 weeks of age belonging to food categories (FCs) 13.1.1 (Infant formulae as defined by Directive 2006/141/EC) and 13.1.5.1 (Dietary foods for infants for special medical purposes and special formulae for infants). In addition, the FAF Panel was requested to address the recommendation of the re‐evaluation of E 472c as a food additive to update the EU specifications in Commission Regulation (EU) No 231/2012. For this, a call for data was published to allow interested partied to provide the requested information for a risk assessment. The Panel concluded that the technical data provided by the interested business operators support an amendment of the EU specifications for E 472c. Regarding the safety of the use of E 472c in food for infants below 16 weeks of age, the Panel concluded that there is no safety concern from its use at the reported use levels and at the maximum permitted levels in food for infants below 16 weeks of age (FCs 13.1.1 and 13.1.5.1).

Abstract
The EFSA Panel on Food Additives and Flavourings (FAF Panel) evaluated the safety of the extension of uses of quillaia extract (E 999) as a food additive in food supplements supplied in a solid or liquid form, excluding food supplements for infants and young children. Quillaia extract (E 999) was re‐evaluated in 2019 by the EFSA FAF Panel, which derived an acceptable daily intake (ADI) of 3 mg saponins/kg bw per day for E 999, while in 2024 a follow‐up of the re‐evaluation was published by the FAF Panel, recommending some modifications of the existing EU specifications for quillaia extract (E 999). Currently, quillaia extract (E 999) is authorised in two food categories (FCs) i.e. FC 4.1.4 'Flavoured drinks' and FC 14.2.3 'Cider and perry' (excluding cidre bouché, cydr jakościowy, perry jakościowe, cydr lodowy, perry lodowe). A 'food supplements consumers only' scenario was calculated for this opinion considering the proposed extension of uses, together with the current authorised uses at both the maximum permitted level (MPLs) and the typical reported use levels of quillaia extract (E 999) at the time of the 2019 re‐evaluation. The Panel concluded that the exposure estimates using the typical reported use levels for the currently authorised food categories and considering the proposed extension of uses for E 999 in FC 17.1 'Food supplements supplied in a solid form, excluding food supplement for infants and young children' and FC 17.2 'Food supplements supplied in a liquid form, excluding food supplement for infants and young children', if authorised, would not result in an exceedance of the ADI in any population group.

Abstract
The Panel on Food Additives and Flavourings (FAF) provides a scientific opinion re‐evaluating the safety of the two food additives argon (E 938) and helium (E 939). Argon (Ar) and helium (He) are two noble gases, highly stable single atoms. Their chemical inertness is well known. Their physicochemical properties have served as a basis for their previous evaluations by SCF and JECFA, which have considered the use of these food additives safe even in the absence of a toxicological evaluation. No business operator or other interested party provided information in response to the call for data published by EFSA to support the re‐evaluation of these two food additives with respect to their identity and specifications, manufacturing process (including the identification and quantification of potential impurities) and how they are applied to food to exert their technological function. One business operator replied to the call for data issued by EFSA reporting use levels of E 938 as a packaging gas in one food category. Based on their physicochemical properties, both gases are considered by the Panel to be of low toxicological concern when used as food additives. No information was available on the potential presence of impurities of toxicological concern resulting from the manufacturing process(es) applied to the production of the food additives E 938 and E 939. The Panel however noted that a minimum purity of 99.0% is required to comply with existing specifications. The Panel concluded that the use of argon (E 938) and helium (E 939) as food additives does not raise a safety concern. The Panel recommended an amendment of the existing EU specifications to introduce the respective CAS numbers.