Critical assessment of the endocrine potential of Linalool and Linalyl acetate: proactive testing strategy assessing estrogenic and androgenic activity of Lavender oil main components.

The acyclic linear monoterpenes Linalool (Lin) and Linalyl acetate (LinAc) occur in nature as major constituents of various essential oils such as lavender oils. A potential endocrine activity of these compounds was discussed in literature including premature thelarche and prepubertal gynecomastia due to lavender product use. This study aims to follow-up on these critical findings reported by testing Lin and LinAc in several studies in line with current guidance and regulatory framework. No relevant anti-/ER and AR-mediated activity was observed in recombinant yeast cell-based screening tests and guideline reporter gene in vitro assays in mammalian cells. Findings in the screening test suggested an anti-androgenic activity, which could not be confirmed in the respective mammalian cell guideline assay. Mechanistic guideline in vivo studies (Uterotrophic and Hershberger assays) with Lin did not show significant dose related changes in estrogen or androgen sensitive organ weights and a guideline reproductive toxicity screening study did not reveal evident effects on sex steroid hormone sensitive organ weights, associated histopathological findings and altered sperm parameters. Estrous cycling and mating/fertility indices were not affected and no evident Lin-related steroid hormone dependent effects were found in the offspring. Overall, the initial concerns from literature were not confirmed. Findings in the yeast screening test were aberrant from follow-up guideline in vitro and in vivo studies, which underlines the need to apply careful interpretation of single in vitro test results to support a respective line of evidence and to establish a biologically plausible link to an adverse outcome.

Avoiding a reproducibility crisis in regulatory toxicology-on the fundamental role of ring trials.

The ongoing transition from chemical hazard and risk assessment based on animal studies to assessment relying mostly on non-animal data, requires a multitude of novel experimental methods, and this means that guidance on the validation and standardisation of test methods intended for international applicability and acceptance, needs to be updated. These so-called new approach methodologies (NAMs) must be applicable to the chemical regulatory domain and provide reliable data which are relevant to hazard and risk assessment. Confidence in and use of NAMs will depend on their reliability and relevance, and both are thoroughly assessed by validation. Validation is, however, a time- and resource-demanding process. As updates on validation guidance are conducted, the valuable components must be kept: Reliable data are and will remain fundamental. In 2016, the scientific community was made aware of the general crisis in scientific reproducibility-validated methods must not fall into this. In this commentary, we emphasize the central importance of ring trials in the validation of experimental methods. Ring trials are sometimes considered to be a major hold-up with little value added to the validation. Here, we clarify that ring trials are indispensable to demonstrate the robustness and reproducibility of a new method. Further, that methods do fail in method transfer and ring trials due to different stumbling blocks, but these provide learnings to ensure the robustness of new methods. At the same time, we identify what it would take to perform ring trials more efficiently, and how ring trials fit into the much-needed update to the guidance on the validation of NAMs.

Retrospective evaluation of the eye irritation potential of agrochemical formulations.

Multiple in vitro eye irritation methods have been developed and adopted as OECD health effects test guidelines. However, for predicting the ocular irritation/damage potential of agrochemical formulations there is an applicability domain knowledge gap for most of the methods. To overcome this gap, a retrospective evaluation of 192 agrochemical formulations with in vivo (OECD TG 405) and in vitro (OECD TG 437, 438, and/or 492) data was conducted to determine if the in vitro methods could accurately assign United Nations Globally Harmonized System for Classification and Labelling of Chemicals (GHS) eye irritation hazard classifications. In addition, for each formulation the eye irritation classification was derived from the classification of the contained hazardous ingredients and their respective concentration in the product using the GHS concentration threshold (CT) approach. The results herein suggest that the three in vitro methods and the GHS CT approach were highly predictive of formulations that would not require GHS classification for eye irritation. Given most agrochemical formulations fall into this category, methods that accurately identify non-classified agrochemical formulations could significantly reduce the use of animals for this endpoint.

Report of the First ONTOX Stakeholder Network Meeting: Digging Under the Surface of ONTOX Together With the Stakeholders.

The first Stakeholder Network Meeting of the EU Horizon 2020-funded ONTOX project was held on 13-14 March 2023, in Brussels, Belgium. The discussion centred around identifying specific challenges, barriers and drivers in relation to the implementation of non-animal new approach methodologies (NAMs) and probabilistic risk assessment (PRA), in order to help address the issues and rank them according to their associated level of difficulty. ONTOX aims to advance the assessment of chemical risk to humans, without the use of animal testing, by developing non-animal NAMs and PRA in line with 21st century toxicity testing principles. Stakeholder groups (regulatory authorities, companies, academia, non-governmental organisations) were identified and invited to participate in a meeting and a survey, by which their current position in relation to the implementation of NAMs and PRA was ascertained, as well as specific challenges and drivers highlighted. The survey analysis revealed areas of agreement and disagreement among stakeholders on topics such as capacity building, sustainability, regulatory acceptance, validation of adverse outcome pathways, acceptance of artificial intelligence (AI) in risk assessment, and guaranteeing consumer safety. The stakeholder network meeting resulted in the identification of barriers, drivers and specific challenges that need to be addressed. Breakout groups discussed topics such as hazard versus risk assessment, future reliance on AI and machine learning, regulatory requirements for industry and sustainability of the ONTOX Hub platform. The outputs from these discussions provided insights for overcoming barriers and leveraging drivers for implementing NAMs and PRA. It was concluded that there is a continued need for stakeholder engagement, including the organisation of a 'hackathon' to tackle challenges, to ensure the successful implementation of NAMs and PRA in chemical risk assessment.

Plant extracts, polymers and new approach methods: Practical experience with skin sensitization assessment.

Over the last decade, research into methodologies to identify skin sensitization hazards has led to the adoption of several non-animal methods as OECD test guidelines. However, predictive accuracy beyond the chemical domains of the individual validation studies remains largely untested. In the present study, skin sensitization test results from in vitro and in chemico methods for 12 plant extracts and 15 polymeric materials are reported and compared to available in vivo skin sensitization data. Eight plant extracts were tested in the DPRA and h-CLAT, with the 2 out of 3 approach resulting in a balanced accuracy of 50%. The balanced accuracy for the 11 plant extracts assessed in the SENS-IS was 88%. Excluding 5 polymers inconclusive in vitro, the remainder, assessed using the 2 out of 3 approach, resulted in 63% balanced accuracy. The SENS-IS method, excluding one polymeric material due to technical inapplicability, showed 68% balanced accuracy. Although based on limited numbers, the results presented here indicate that some substance subgroups may not be in the applicability domains of the method used and careful analysis is required before positive or negative results can be accepted.

Accounting for Precision Uncertainty of Toxicity Testing: Methods to Define Borderline Ranges and Implications for Hazard Assessment of Chemicals.

For hazard classifications of chemicals, continuous data from animal- or nonanimal testing methods are often dichotomized into binary positive/negative outcomes by defining classification thresholds (CT). Experimental data are, however, subject to biological and technical variability. Each test method's precision is limited resulting in uncertainty of the positive/negative outcome if the experimental result is close to the CT. Borderline ranges (BR) around the CT were suggested, which represent ranges in which the study result is ambiguous, that is, positive or negative results are equally likely. The BR reflects a method's precision uncertainty. This article explores and compares different approaches to quantify the BR. Besides using the pooled standard deviation, we determine the BR by means of the median absolute deviation (MAD), with a sequential combination of both methods, and by using nonparametric bootstrapping. Furthermore, we quantify the BR for different hazardous effects, including nonanimal tests for skin corrosion, eye irritation, skin irritation, and skin sensitization as well as for an animal test on skin sensitization (the local lymph node assay, LLNA). Additionally, for one method (direct peptide reactivity assay) the BR was determined experimentally and compared to calculated BRs. Our results demonstrate that (i) the precision of the methods is determining the size of their BRs, (ii) there is no "perfect" method to derive a BR, alas, (iii) a consensus on BR is needed to account for the limited precision of testing methods.

Human-Derived In Vitro Models Used for Skin Toxicity Testing Under REACh.

In regulatory toxicology, in vivo studies are still prevailing, and human-derived in vitro models are mostly used in testing for local toxicity to the skin and the eyes. A single in vitro model may be limited to address one or few molecular or cellular events leading to adverse outcomes. Hence, in many instances their regulatory use involves the combination of several in vitro models to assess the hazard potential of test substance. A so-called defined approach combines different testing methods and a 'data interpretation procedure' to obtain a comprehensive overall assessment which is used for the regulatory hazard classification of the test substance.Validation is a prerequisite of regulatory acceptance of new testing methods: This chapter provides an overview of the method development from an experimental method to a test guideline via application of GIVIMP (good in vitro method practice), standardization, validation to the regulatory adoption as an OECD test guidelines. Quandaries associated with the validation towards reference data from in vivo animal studies with limited accuracy and limited human relevance are discussed, as well as uncertainty and limitations arising from restricted applicability and technical and biological variance of the in vitro methods.This chapter provides an overview of human-derived in vitro models currently adopted as OECD test guidelines: From the first skin corrosion tests utilizing reconstructed human epidermis models (RhE), to models to test for skin irritation, phototoxicity, eye irritation, and skin sensitization. The latter is using a battery of different methods and defined approaches which are still under discussion for their regulatory adoption. They will be a vanguard of future applications of human-derived models in regulatory toxicology. RhEs for testing of genotoxicity and of dermal penetration and absorption, have been developed, underwent validation studies and may soon be adopted for regulatory use; these are included in this chapter.

Replacing the refinement for skin sensitization testing: Considerations to the implementation of adverse outcome pathway (AOP)-based defined approaches (DA) in OECD guidelines.

While single non-animal methods have been adopted in OECD test guidelines, combinations of methods (so called defined approaches, DA) are not. Hardly any animal study can be replaced by a single non-animal method, rather DA are needed. The OECD published the Adverse Outcome Pathway (AOP) on skin sensitization in 2012 and is currently discussing the implementation of DA into a guideline. Obviously, it takes thorough considerations and evaluations to validate such DA. Currently we see four preconditions for a proper and expedient implementation of DA in a guideline: (i) The reference data should be selected to allow meaningful evaluations and must not replicate the limitations of the murine local lymph node assay (LLNA) (ii) Methods and prediction models should be validated before they are used in an OECD-adopted DA, (iii) An OECD-adopted DA should follow the respective AOP and (iv) acknowledge regulatory needs and successful toxicological practice. These points still need to be considered in the current discussion at the OECD. A guideline for skin sensitization DA is setting the scene for regulatory acceptance of all new approaches (for any toxicological endpoint) in the future. In this commentary, we are expounding these preconditions to allow a scientifically valid and sustainable application of modern (no-animal) approaches in regulatory toxicology.

A review of substances found positive in 1 of 3 in vitro tests for skin sensitization.

There has been significant progress in recent years in the development and application of alternative methods for assessing the skin sensitization potential of chemicals. The pathways involved in skin sensitization have been described in an OECD adverse outcome pathway (AOP). To date, a single non-animal test method is not sufficient to address this AOP so numerous approaches involving the use of 2 or more assays are being evaluated for their performance. The 2 out of 3 approach is a simple approach that has demonstrated very good sensitivity, specificity and overall accuracy numbers for predicting the skin sensitization potential of chemicals. Chemicals with at least two positive results in tests addressing Key events 1-3 are predicted sensitizers, while chemicals with none or only one positive outcome are predicted non-sensitizers. In this report we have thoroughly reviewed the discordant results of 29 chemicals with 1 out of 3 positive results to understand better what led to the results observed and how this information might impact our hazard assessments of these chemicals. We initially categorized each chemical using a weight of evidence approach as positive, negative or indeterminate based on review of available human and animal data as well as what skin sensitization alerts were triggered using two versions of OECD Toolbox and DEREK Nexus. We determined that 4 of the 29 chemicals should be classified as indeterminate and not included in analysis of method performance based on insufficient, borderline and/or conflicting data to confidently categorized the chemicals as allergens or non-allergens. Of the 29 chemicals included in this analysis, 17 were classified as negative and would be correctly identified using a 2 out of 3 approach while 8 chemicals were classified as positive in vivo and would be false-negative with this approach. For some of these chemicals, the outcomes observed can be explained by in vitro borderline results (13 chemicals) or in some instances there is mechanistic understanding of why a chemical is positive or negative in a particular assay (9 chemicals). Thus, when comparing the performance of different defined approaches, one should attempt to only include chemicals which demonstrate clear evidence to be categorize as allergens or non-allergens. Finally, when interpreting the results obtained for an individual unknown chemical it is critical that the in vitro skin sensitization data is reviewed critically and there is a good understanding of the variance and applicability domain limitations for each assay being used.

GHS additivity formula: can it predict the acute systemic toxicity of agrochemical formulations that contain acutely toxic ingredients?

In vivo acute systemic testing is a regulatory requirement for agrochemical formulations. GHS specifies an alternative computational approach (GHS additivity formula) for calculating the acute toxicity of mixtures. We collected acute systemic toxicity data from formulations that contained one of several acutely-toxic active ingredients. The resulting acute data set includes 210 formulations tested for oral toxicity, 128 formulations tested for inhalation toxicity and 31 formulations tested for dermal toxicity. The GHS additivity formula was applied to each of these formulations and compared with the experimental in vivo result. In the acute oral assay, the GHS additivity formula misclassified 110 formulations using the GHS classification criteria (48% accuracy) and 119 formulations using the USEPA classification criteria (43% accuracy). With acute inhalation, the GHS additivity formula misclassified 50 formulations using the GHS classification criteria (61% accuracy) and 34 formulations using the USEPA classification criteria (73% accuracy). For acute dermal toxicity, the GHS additivity formula misclassified 16 formulations using the GHS classification criteria (48% accuracy) and 20 formulations using the USEPA classification criteria (36% accuracy). This data indicates the acute systemic toxicity of many formulations is not the sum of the ingredients' toxicity (additivity); but rather, ingredients in a formulation can interact to result in lower or higher toxicity than predicted by the GHS additivity formula.

Regulatory accepted but out of domain: In vitro skin irritation tests for agrochemical formulations.

Several in vitro methods have gained regulatory acceptance for the prediction of skin irritation and corrosion. However, the test guidelines for the majority of in vitro methods do not state whether they are applicable to agrochemical formulations. Hence, we would like to share the results from our routine skin corrosion and irritation testing of agrochemical formulations in which both in vitro (according to OECD TG 431 and OECD TG 439) and in vivo (according to OECD TG 404) tests were conducted as regulatory requirements. The in vitro skin irritation test did not correlate well with the CLP classification by in vivo results (44% sensitivity, 60% specificity, and 54% accuracy, based on 65 data pairs). This indicates a lack of applicability of the current protocol of the in vitro skin irritation test for agrochemical formulations. The data set did not contain formulations which were skin corrosive in vivo and hence its applicability could not be assessed. The correlation of in vitro skin corrosion testing to formulations which were not corrosive in vivo was, however, high (95% specificity based on 81 data pairs).

Lacking applicability of in vitro eye irritation methods to identify seriously eye irritating agrochemical formulations: Results of bovine cornea opacity and permeability assay, isolated chicken eye test and the EpiOcular™ ET-50 method to classify according to UN GHS.

In vitro methods have gained regulatory acceptance for the prediction of serious eye damage (UN GHS Cat 1). However, the majority of in vitro methods do not state whether they are applicable to agrochemical formulations. This manuscript presents a study of up to 27 agrochemical formulations tested in three in vitro assays (three versions of the bovine corneal opacity and permeability test (BCOP, OECD TG 437) assay, the isolated chicken eye test (ICE, OECD TG 438) and the EpiOcular™ ET-50 assay). The results were compared with already-available in vivo data. In the BCOP only one of the four, one of five in the ICE and six of eleven tested formulations in the EpiOcular™ ET-50 Neat Protocol resulted in the correct UN GHS Cat 1 prediction. Overpredictions occurred in all assays. These data indicate a lack of applicability of the three in vitro methods to reliably predict UN GHS Cat 1 of agrochemical formulations. In order to ensure animal-free identification of seriously eye damaging agrochemical formulations testing protocols and/or prediction models need to be modified or classification rules should be tailored to in vitro testing rather than using in vivo Draize data as a standard.

Assessment of skin sensitization under REACH: A case report on vehicle choice in the LLNA and its crucial role preventing false positive results.

In the EU, chemicals with a production or import volume in quantities of one metric ton per year or more have to be tested for skin sensitizing properties under the REACH regulation. The murine Local Lymph Node Assay (LLNA) and its modifications are widely used to fulfil the data requirement, as it is currently considered the first-choice method for in vivo testing to cover this endpoint. This manuscript describes a case study highlighting the importance of understanding the chemistry of the test material during testing for 'skin sensitization' of MCDA (mixture of 2,4- and 2,6-diamino-methylcyclohexane) with particular focus on the vehicle used. While the BrdU-ELISA modification of the LLNA using acetone/olive oil (AOO) as vehicle revealed expectable positive results. However, the concentration control analysis unexpectedly revealed an instability of MCDA in the vehicle AOO. Further studies on the reactivity showed MCDA to rapidly react with AOO under formation of various imine structures, which might have caused the positive LLNA result. The repetition of the LLNA using propylene glycol (PG) as vehicle did not confirm the positive results of the LLNA using AOO. Finally, a classification of MCDA as skin sensitizer according to the Globally Harmonized System (GHS) was not justified.

Assessment of Pre- and Pro-haptens Using Nonanimal Test Methods for Skin Sensitization.

Because of ethical and regulatory reasons, several nonanimal test methods to assess the skin sensitization potential of chemicals have been developed and validated. In contrast to in vivo methods, they lack or provide limited metabolic capacity. For this reason, identification of pro-haptens but also pre-haptens, which require molecular transformations to gain peptide reactivity, is a challenge for these methods. In this study, 27 pre- and pro-haptens were tested using nonanimal test methods. Of these, 18 provided true positive results in the direct peptide reactivity assay (DPRA; sensitivity of 67%), although lacking structural alerts for direct peptide reactivity. The reaction mechanisms leading to peptide depletion in the DPRA were therefore elucidated using mass spectrometry. Hapten-peptide adducts were identified for 13 of the 18 chemicals indicating that these pre-haptens were activated and that peptide binding occurred. Positive results for five of the 18 chemicals can be explained by dipeptide formations or the oxidation of the sulfhydryl group of the peptide. Nine of the 27 chemicals were tested negative in the DPRA. Of these, four yielded true positive results in the keratinocyte and dendritic cell based assays. Likewise, 16 of the 18 chemicals tested positive in the DPRA were also positive in either one or both of the cell-based assays. A combination of DPRA, KeratinoSens, and h-CLAT used in a 2 out of 3 weight of evidence (WoE) approach identified 22 of the 27 pre- and pro-haptens correctly (sensitivity of 81%), exhibiting a similar sensitivity as for directly acting haptens. This analysis shows that the combination of in chemico and in vitro test methods is suitable to identify pre-haptens and the majority of pro-haptens.

Eye irritation testing of nanomaterials using the EpiOcular™ eye irritation test and the bovine corneal opacity and permeability assay.

BACKGROUND: Assessment of eye irritation hazard has long been a core requirement in any chemical legislation. Nevertheless, publications focussing on the eye damaging potential of nanomaterials are scarce. Traditionally, eye irritation testing was performed using rabbits. The OECD Test Guideline 437 Bovine Corneal Opacity and Permeability (BCOP) test method allows determining severely irritating substances without animals, and the recently adopted OECD Test Guideline 492 Reconstructed human cornea-like epithelium test method allows identifying chemicals that neither induce eye irritation nor serious eye damage. For substances applicable to these tests, huge progress has been made in replacing animal testing. METHODS: The in vitro eye irritation potential of 20 nanosized and 3 non-nanosized materials was investigated in a 2-tier EpiOcular™ Eye Irritation Test (EpiOcular™-EIT) and BCOP testing strategy including histopathology of the bovine corneas. Furthermore, applicability of the testing strategy for nanomaterials was assessed. Test materials encompassed OECD representative nanomaterials (metals (Ag), metal oxides (ZnO, TiO2, CeO2), amorphous SiO2 and MWCNTs), three organic pigments, quartz, and talc. RESULTS: None of the dry-powder nanomaterials elicited eye irritation in either the EpiOcular™-EIT or the BCOP assay. Likewise, an amorphous SiO2 nanomaterial that was supplied as suspension was tested negative in both assays. By contrast, in the EpiOcular™-EIT, the silver nanomaterial that was supplied as dispersion was tested positive, whereas its surfactant-containing dispersant was borderline to negative. In the BCOP assay, the silver nanomaterial elicited highly variable results and dark-brown patches remained on the corneal surface, whereas the results for its dispersant alone were borderline to positive, which was assessed as inconclusive due to high inter-assay variability. CONCLUSION: The present study points to the low eye irritation potential of a spectrum of nanomaterials, which is consistent with available in vivo data for the same test materials or for nanosized or bulk materials of the same composition.

The effect of communication and implicit associations on consuming insects: An experiment in Denmark and Italy.

It has been widely noted that the introduction of insects in Westerns' diet might be a promising path towards a more sustainable food consumption. However, Westerns' are almost disgusted and sceptical about the eating of insects. In the current paper we report the results of an experiment conducted in two European countries-Denmark and Italy-different for food culture and familiarity with the topic of eating insects. We investigated the possibility to foster people's willingness to eat insect-based food through communication, also comparing messages based on individual vs. societal benefits of the eating of insects. Communication proved to be effective on intention and behaviour, and the societal message appeared to be more robust over time. The communication effect is significant across nation, gender, and previous knowledge about the topic. In addition, we investigated the impact of non-conscious negative associations with insects on the choice to eat vs. not eat insect-based food. Implicit attitudes proved to be a powerful factor in relation to behaviour, yet they did not impede the effectiveness of communication.

Local tolerance testing under REACH: Accepted non-animal methods are not on equal footing with animal tests.

In general, no single non-animal method can cover the complexity of any given animal test. Therefore, fixed sets of in vitro (and in chemico) methods have been combined into testing strategies for skin and eye irritation and skin sensitisation testing, with pre-defined prediction models for substance classification. Many of these methods have been adopted as OECD test guidelines. Various testing strategies have been successfully validated in extensive in-house and inter-laboratory studies, but they have not yet received formal acceptance for substance classification. Therefore, under the European REACH Regulation, data from testing strategies can, in general, only be used in so-called weight-of-evidence approaches. While animal testing data generated under the specific REACH information requirements are per se sufficient, the sufficiency of weight-of-evidence approaches can be questioned under the REACH system, and further animal testing can be required. This constitutes an imbalance between the regulatory acceptance of data from approved non-animal methods and animal tests that is not justified on scientific grounds. To ensure that testing strategies for local tolerance testing truly serve to replace animal testing for the REACH registration 2018 deadline (when the majority of existing chemicals have to be registered), clarity on their regulatory acceptance as complete replacements is urgently required.

Assessing skin sensitization hazard in mice and men using non-animal test methods.

Sensitization, the prerequisite event in the development of allergic contact dermatitis, is a key parameter in both hazard and risk assessments. The pathways involved have recently been formally described in the OECD adverse outcome pathway (AOP) for skin sensitization. One single non-animal test method will not be sufficient to fully address this AOP and in many cases the use of a battery of tests will be necessary. A number of methods are now fully developed and validated. In order to facilitate acceptance of these methods by both the regulatory and scientific communities, results of the single test methods (DPRA, KeratinoSens, LuSens, h-CLAT, (m)MUSST) as well for a the simple '2 out of 3' ITS for 213 substances have been compiled and qualitatively compared to both animal and human data. The dataset was also used to define different mechanistic domains by probable protein-binding mechanisms. In general, the non-animal test methods exhibited good predictivities when compared to local lymph node assay (LLNA) data and even better predictivities when compared to human data. The '2 out of 3' prediction model achieved accuracies of 90% or 79% when compared to human or LLNA data, respectively and thereby even slightly exceeded that of the LLNA.

Immunophenotyping does not improve predictivity of the local lymph node assay in mice.

The local lymph node assay (LLNA) is a regulatory accepted test for the identification of skin sensitizing substances by measuring radioactive thymidine incorporation into the lymph node. However, there is evidence that LLNA is overestimating the sensitization potential of certain substance classes in particular those exerting skin irritation. Some reports describe the additional use of flow cytometry-based immunophenotyping to better discriminate irritants from sensitizing irritants in LLNA. In the present study, the 22 performance standards plus 8 surfactants were assessed using the radioactive LLNA method. In addition, lymph node cells were immunophenotyped to evaluate the specificity of the lymph node response using cell surface markers such as B220 or CD19, CD3, CD4, CD8, I-A(κ) and CD69 with the aim to allow a better discrimination above all between irritants and sensitizers, but also non-irritating sensitizers and non-sensitizers. However, the markers assessed in this study do not sufficiently differentiate between irritants and irritant sensitizers and therefore did not improve the predictive capacity of the LLNA.