SCCS Scientific Opinion on Acid Yellow 3 (submission II) - SCCS/1631/21.

Opinion to be cited as: SCCS (Scientific Committee on Consumer Safety), Opinion on Acid Yellow 3 - C054 (CAS Number 8004-92-0, EC No 305-897-5), submission II, preliminary version of 7 May 2021, final version of 23 July 2021, SCCS/1631/21.

SCCS scientific opinion on Butylated hydroxytoluene (BHT) - SCCS/1636/21.

OPINION TO BE CITED AS: SCCS (Scientific Committee on Consumer Safety), scientific opinion on Butylated hydroxytoluene (BHT), preliminary version of September 27, 2021, final version of December 2, 2021, SCCS/1636/21.

The SCCS scientific advice on the safety of nanomaterials in cosmetics.

The Cosmetic Regulation (EC) No 1223/2009 specifically covers the risk of nanomaterials used in cosmetic products. If there are concerns regarding the safety of a nanomaterial, the European Commission refers it to the SCCS for a scientific opinion. The Commission mandated the SCCS to identify the scientific basis for safety concerns that could be used as a basis for identifying and prioritising nanomaterials for safety assessment, and to revisit previous inconclusive SCCS opinions on nanomaterials to identify any concerns for potential risks to the consumer health. The SCCS Scientific Advice identified the key general aspects of nanomaterials that should raise a safety concern for a safety assessor/manager, so that the nanomaterial(s) in question could be subjected to safety assessment to establish safety to the consumer. The Advice also developed a list of the nanomaterials notified to the Commission for use in cosmetics in an order of priority for safety assessment, and revisited three previous inconclusive opinions on nanomaterials to highlight concerns over consumer safety that merited further safety assessment.

The way forward for assessing the human health safety of cosmetics in the EU - Workshop proceedings.

Although the need for non-animal alternatives has been well recognised for the human health hazard assessment of chemicals in general, it has become especially pressing for cosmetic ingredients due to the full implementation of testing and marketing bans on animal testing under the European Cosmetics Regulation. This means that for the safety assessment of cosmetics, the necessary safety data for both the ingredients and the finished product can be drawn from validated (or scientifically-valid), so-called "Replacement methods". In view of the challenges for safety assessment without recourse to animal test data, the Methodology Working Group of the Scientific Committee on Consumer Safety organised a workshop in February 2019 to discuss the key issues in regard to the use of animal-free alternative methods for the safety evaluation of cosmetic ingredients. This perspective article summarises the outcomes of this workshop and reflects on the state-of-the-art and possible way forward for the safety assessment of cosmetic ingredients for which no experimental animal data exist. The use and optimisation of "New Approach Methodology" that could be useful tools in the context of the "Next Generation Risk Assessment" and the strategic framework for safety assessment of cosmetics were discussed in depth.

Opinion of the Scientific Committee on Consumer Safety (SCCS) - Revision of the Opinion on hydroxyapatite (nano) in cosmetic products.

In response to the concerns of the European Commission about potential absorption and entry of nanoparticles of hydroxyapatite into the cells when used in oral cosmetic products, the Scientific Committee on Consumer Safety (SCCS) was requested to provide a safety assessment of hydroxyapatite (nano). After making a detailed evaluation of the data provided in the submissions and scientific literature, the SCCS considered needle-shaped hydroxyapatite (nano) to be of concern due to its potential toxic effects, and stated that it should not be used in cosmetic products. In terms of other shapes of hydroxyapatite (nano), the available evidence was insufficient to allow drawing a conclusion on the safety of hydroxyapatite (nano) when used in oral cosmetic products up to a concentration of 10%.

Opinion of the Scientific Committee on Consumer Safety (SCCS) - Revision of the opinion on o-Phenylphenol, Sodium o-phenylphenate and Potassium o-phenylphenate (OPP), in cosmetic products.

o-Phenylphenol, Sodium o-phenylphenate, Potassium o-phenylphenate, CAS n. 90-43-7, 132-27-4, 13707-65-8 as preservatives are regulated in Annex V/7 of the Cosmetics Regulation (EC) n. 1223/2009 at a maximum concentration of 0.2% (as phenol). In February 2013, the Commission received a risk assessment submitted by the French Agency ANSM (Agence nationale de sécurité des médicaments et des produits de santé) which rose concerns about the use of o-Phenylphenol as preservatives in cosmetic products. In the context of the ANSM report (Evaluation du risque lié à l'utilisation de l'orthophénylphénol CAS n. 90-43-7 dans les produits cosmétiques) o-Phenylphenol has been identified as likely to be an endocrine disruptor. The report concludes that the maximum authorised concentration (currently of 0.2%) of o-Phenylphenol for use as a preservative should be revised due to low margin of safety. In January 2014, in response to a call for data on o-Phenylphenol by the Commission, Industry submitted a safety dossier in order to defend the current use of o-Phenylphenol, Sodium o-phenylphenate, Potassium o-phenylphenate, CAS n. 90-43-7, 132-27-4, 13707- 65-8 as preservatives in cosmetic formulations at a maximum concentration of 0.2% (as phenol). o-Phenylphenol as preservative with a maximum concentration of 0.2% in leave-on cosmetic products is not safe. Also, in view of further exposures including noncosmetic uses (see Anses, 2014), the maximum concentration of o-Phenylphenol in leave-on cosmetic products should be lowered. However, the proposed maximum use concentration of up to 0.15% by the applicant can be considered safe. The use of o-Phenylphenol as preservative with a maximum concentration of 0.2% in rinse-off cosmetic products is considered safe. Based on the information provided, no conclusions of safe use can be drawn for Sodium o-phenylphenate and Potassium o-phenylphenate. In vitro data indicate an absent or very weak binding affinity of OPP to the oestrogen receptor, in line with limited stimulation of proliferation in oestrogen responsive cells. No information is available on androgenic and anti-androgenic effects of OPP in vitro. Agonistic or antagonistic effects on thyroid hormones were not observed with OPP. There might be a potential of injury to the vision system attributable to OPP. Aggregate exposure to OPP should be considered.

Opinion of the scientific committee on consumer safety (SCCS)--2nd Revision of the safety of the use of poly(hexamethylene) biguanide hydrochloride or polyaminopropyl biguanide (PHMB) in cosmetic products.

Conclusion of the opinion: On the basis of the data available, the SCCS concludes that Polyaminopropyl Biguanide (PHMB) is not safe for consumers when used as a preservative in cosmetic spray formulations and in all cosmetic products up to the maximum concentration of 0.3%. The safe use could be based on a lower use concentration and/or restrictions with regard to cosmetic products' categories. Dermal absorption studies on additional representative cosmetic formulations are needed. PHMB is used in a variety of applications other than cosmetics. General exposure data from sources others than cosmetics should be submitted for the assessment of the aggregate exposure of PHMB.

Extrahepatic metabolism at the body's internal-external interfaces.

In general, xenobiotic metabolizing enzymes (XMEs) are expressed in lower levels in the extrahepatic tissues than in the liver, making the former less relevant for the clearance of xenobiotics. Local metabolism, however, may lead to tissue-specific adverse responses, e.g. organ toxicities, allergies or cancer. This review summarizes the knowledge on the expression of phase I and phase II XMEs and transporters in extrahepatic tissues at the body's internal-external interfaces. In the lung, CYPs of families 1, 2, 3 and 4 and epoxide hydrolases are important phase I enzymes, while conjugation is less relevant. In skin, phase I-related enzymatic reactions are considered less relevant. Predominant skin XMEs are phase II enzymes, whereby glucuronosyltransferases (UGT) 1, glutathione-S-transferase (GST) and N-acetyltransferase (NAT) 1 are important for detoxification. The intestinal epithelium expresses many transporters and phase I XME with high levels of CYP3A4 and CYP3A5 and phase II metabolism is mainly related to UGT, NAT and Sulfotransferases (SULT). In the kidney, conjugation reactions and transporters play a major role for excretion processes. In the bladder, CYPs are relevant and among the phase II enzymes, NAT1 is involved in the activation of bladder carcinogens. Expression of XMEs is regulated by several mechanisms (nuclear receptors, epigenetic mechanisms, microRNAs). However, the understanding why XMEs are differently expressed in the various tissues is fragmentary. In contrast to the liver - where for most XMEs lower expression is demonstrated in early life - the XME ontogeny in the extrahepatic tissues remains to be investigated.

Commentary: dermal penetration of bisphenol A--consequences for risk assessment.

With this comment we would raise awareness for applying appropriate procedures in route-to-route extrapolation. The paper of Demierre et al. (2012) prompted us to comment on the simple approach for route-to-route extrapolation and to explain some short comings. For the risk assessment of exposures resulting from a non-oral route, route-to-route extrapolation is often done by correcting the non-oral route exposure by the route specific absorption into the systemic circulation and comparing the result with the (oral) threshold value. Making use of this procedure means that an internal dose obtained from the non-oral route is compared with an external dose of the oral route. This procedure would be appropriate only if the absorption on the oral route is 100%. If the absorption on the oral route is less than 100% the procedure may underestimate the risk of the exposure of the non-oral route. For some chemicals with a high first pass metabolism in the liver, e.g. BPA, the situation is even more complex and in addition, the target organ for toxicity has to be taken into consideration.

Toxicokinetics as a key to the integrated toxicity risk assessment based primarily on non-animal approaches.

Toxicokinetics (TK) is the endpoint that informs about the penetration into and fate within the body of a toxic substance, including the possible emergence of metabolites. Traditionally, the data needed to understand those phenomena have been obtained in vivo. Currently, with a drive towards non-animal testing approaches, TK has been identified as a key element to integrate the results from in silico, in vitro and already available in vivo studies. TK is needed to estimate the range of target organ doses that can be expected from realistic human external exposure scenarios. This information is crucial for determining the dose/concentration range that should be used for in vitro testing. Vice versa, TK is necessary to convert the in vitro results, generated at tissue/cell or sub-cellular level, into dose response or potency information relating to the entire target organism, i.e. the human body (in vitro-in vivo extrapolation, IVIVE). Physiologically based toxicokinetic modelling (PBTK) is currently regarded as the most adequate approach to simulate human TK and extrapolate between in vitro and in vivo contexts. The fact that PBTK models are mechanism-based which allows them to be 'generic' to a certain extent (various extrapolations possible) has been critical for their success so far. The need for high-quality in vitro and in silico data on absorption, distribution, metabolism as well as excretion (ADME) as input for PBTK models to predict human dose-response curves is currently a bottleneck for integrative risk assessment.

Alternative (non-animal) methods for cosmetics testing: current status and future prospects-2010.

The 7th amendment to the EU Cosmetics Directive prohibits to put animal-tested cosmetics on the market in Europe after 2013. In that context, the European Commission invited stakeholder bodies (industry, non-governmental organisations, EU Member States, and the Commission's Scientific Committee on Consumer Safety) to identify scientific experts in five toxicological areas, i.e. toxicokinetics, repeated dose toxicity, carcinogenicity, skin sensitisation, and reproductive toxicity for which the Directive foresees that the 2013 deadline could be further extended in case alternative and validated methods would not be available in time. The selected experts were asked to analyse the status and prospects of alternative methods and to provide a scientifically sound estimate of the time necessary to achieve full replacement of animal testing. In summary, the experts confirmed that it will take at least another 7-9 years for the replacement of the current in vivo animal tests used for the safety assessment of cosmetic ingredients for skin sensitisation. However, the experts were also of the opinion that alternative methods may be able to give hazard information, i.e. to differentiate between sensitisers and non-sensitisers, ahead of 2017. This would, however, not provide the complete picture of what is a safe exposure because the relative potency of a sensitiser would not be known. For toxicokinetics, the timeframe was 5-7 years to develop the models still lacking to predict lung absorption and renal/biliary excretion, and even longer to integrate the methods to fully replace the animal toxicokinetic models. For the systemic toxicological endpoints of repeated dose toxicity, carcinogenicity and reproductive toxicity, the time horizon for full replacement could not be estimated.

Human CYP2E1 mediates the formation of glycidamide from acrylamide.

Regarding the cancer risk assessment of acrylamide (AA) it is of basic interest to know, as to what amount of the absorbed AA is metabolized to glycidamide (GA) in humans, compared to what has been observed in laboratory animals. GA is suspected of being the ultimate carcinogenic metabolite of AA. From experiments with CYP2E1-deficient mice it can be concluded that AA is metabolized to GA primarily by CYP2E1. We therefore examined whether CYP2E1 is involved in GA formation in non-rodent species with the focus on humans by using human CYP2E1 supersomes, marmoset and human liver microsomes and in addition, genetically engineered V79 cells expressing human CYP2E1 (V79h2E1 cells). Special emphasis was placed on the analytical detection of GA, which was performed by gas chromatography/mass spectrometry. The results show that AA is metabolized to GA in human CYP2E1 supersomes, in marmoset and human liver microsomes as well as in V79h2E1 cells. The activity of GA formation is highest in supersomes; in human liver it is somewhat higher than in marmoset liver. A monoclonal CYP2E1 human selective antibody (MAB-2E1) and diethyldithiocarbamate (DDC) were used as specific inhibitors of CYP2E1. The generation of GA could be inhibited by MAB-2E1 to about 80% in V79h2E1 cells and to about 90% in human and marmoset liver microsomes. Also DDC led to an inhibition of about 95%. In conclusion, AA is metabolized to GA by human CYP2E1. Overall, the present work describes (1) the application and refinement of a sensitive methodology in order to determine low amounts of GA, (2) the applicability of genetically modified V79 cell lines in order to investigate specific questions concerning metabolism and (3) the involvement, for the first time, of human CYP2E1 in the formation of GA from AA. Further studies will compare the activities of GA formation in genetically engineered V79 cells expressing CYP2E1 from different species.

Exposure-triggered reproductive toxicity testing under the REACH legislation: a proposal to define significant/relevant exposure.

Under the new REACH legislation, toxicological testing is required in relation to annual tonnages produced or imported. Requirements for toxicological information increase when production volume increases. The respective information requirements are laid down in the REACH Annexes VII-X. Concerning human toxicology, certain toxicological tests may be waived under specific conditions. Aside from waiving criteria such as technical feasibility, exposure plays a decisive role in the waiving process with the consequence that toxicological testing will not be required in case of "no relevant exposure", "limited exposure", "no exposure" or "no significant exposure" (as expressed in the documents). However, up to now criteria are lacking which precisely define these terms. Attempts have been made to establish cut-off criteria between "non-relevant" and "relevant" (detrimental) exposure based on external exposure concentrations and the threshold of toxicological concern (TTC) principle. In this paper we make a proposal and describe a strategy how to define the currently insufficiently described terms "relevant/significant" exposure. We propose to define relevant/significant exposure based on an endpoint-specific TTC approach, starting from a comparison of the tentative external exposure to the specific TTC. This can be followed by a refinement of exposure estimates and may culminate in the experimental determination of internal and target tissue exposure. This strategy enables a well-founded assessment of what "no relevant exposure" is and safeguards an appropriate level of protection of the general population. The feasibility of the approach is demonstrated for reproductive toxicity endpoints.

Characterisation of the xenobiotic-metabolizing Cytochrome P450 expression pattern in human lung tissue by immunochemical and activity determination.

The lung represents an important target for the toxic effects of chemicals. Many of the chemicals require enzymatic activation to exert their adverse effects, which is mostly catalysed by Cytochrome P450 (CYP) enzymes. Although there is considerable evidence that individual members of the xenobiotic-metabolizing P450 family are expressed in human lung tissue at the mRNA level, there is conflicting evidence concerning the following issues: (I) the qualitative expression pattern of CYP isoenzymes; (II) CYP expression at the protein and/or activity level; and (III) interindividual variability of CYP enzymes in human lung. The latter can be the basis for individual susceptibility towards the adverse effects of lung toxicants. In preparing for studying factors to explain interindividual variability of CYP expression in lung tissue, we investigated the qualitative pulmonary expression pattern of xenobiotic-metabolizing CYP enzymes and elaborated the optimal conditions for quantification at the protein and activity level. By using either individual human lung samples or pooled microsomes from different individuals, immunoreactive bands specific for the following CYP enzymes could be determined by Western blotting: CYP1A1, CYP1A2, CYP2E1 and CYP3A5. Western blotting experiments were also supportive of the presence of CYP2A, CYP2B6, CYP2D6 and CYP3A4 in human lung. By using antibodies specific for CYP2C enzymes and CYP1B1, respectively, immunoreactive bands, which differed slightly in mobility from corresponding standards, were detectable. In addition, we measured methoxy- and ethoxyresorufin dealkylase activities and chlorzoxazone (CLX)-hydroxylase activity in human lung and confirmed the specifities of the latter two activities by inhibition experiments. In summary, we have established methodologies to quantify a panel of CYP enzymes in human lung samples among which there are CYP enzymes whose expression at the protein and activity level has not been evidenced so far.

The use of in vitro data in risk assessment.

Describing the toxicological profile of a substance is the first step required for risk assessment. Although a wide range of in vitro methods are widely used to characterise toxicological properties including toxicokinetics, regulatory acceptance is mainly confined to in vitro tests which investigate genotoxic end-points. In vitro tests have been proposed for the endpoints acute toxicity, repeated dose toxicity and toxicity to reproduction which encompass the minimum requirements in the OECD SIDS programme. However, until now, limitations of the proposed tests preclude their application in a regulatory framework. Presently, in vitro tests play a major role in obtaining information on mechanism of toxicity with the perspective to be able to identify pathways of toxic responses by applying toxicogenomics techniques. Physiologically based toxicokinetic modelling is using data from in vitro studies to build up the model for a specific compound. Information from both areas is incorporated into the risk assessment to derive compound-specific safety factors, which account for species differences and for the variability among the human population, including possible sensitive subpopulations. Future developments to further enhance the use of in vitro methods in regulatory toxicology include the development of (Q)SAR approaches supplemented by mechanisms of toxicity, which can be addressed by developing methods of molecular toxicology.

Immunochemical analysis of cytochrome P450 variability in human leukapheresed samples and its consequences for the risk assessment process.

Xenobiotic metabolizing cytochrome P450 (P450) enzymes were investigated in leukapheresed samples from 50 human individuals. It was the aim of the study (a). to get insight into the extent of extrahepatic P450 variability, (b). to investigate whether and to which extent P450 expression and variability as it is seen in the liver corresponds to P450 expression at extrahepatic sites, and (c). to contribute to the replacement of traditionally used default factors (usually 10 for interindividual variability) by data-derived factors in the risk assessment process. P450 enzymes were determined by Western Blotting. Immunoquantification was performed for P450 1A, 1B1, 2C, 2D6, 2E1, and 3A which were-with the exception of the polymorphically expressed CYP2D6-detectable in all samples investigated. Amounts of P450 enzymes in leukapheresed samples were (except CYP1B1) lower compared to those reported for the liver. The P450 variabilities were expressed by the ratios between the 95th and the 5th percentiles. They displayed 7-(CYP1A), 4-(CYP1B1), 6-(CYP2C), 30-(CYP2D6), 3-(CYP2E1), and 4-(CYP3A) fold variability in specific protein content. The results show (a). qualitative and quantitative differences in the expression of P450 proteins in leukapheresed samples from 50 individuals compared to liver, (b). a different extent of variability depending on the P450 enzyme, and (c). in cases where polymorphically distributed P450 enzymes are involved, the traditionally used factor of 10 might be too low to account for interindividual variability in both toxicokinetics and toxicodynamics.