In vitro to in vivo extrapolation to derive a metabolism factor for estimating the aggregate exposure to salicylic acid after dermal exposure of its esters.

As part of the safety assessment of salicylate esters in cosmetics, we developed a metabolism factor based on in vitro to in vivo extrapolation (IVIVE) to provide a better estimation of the aggregate internal exposure to the common metabolite, salicylic acid. Optimal incubation conditions using human liver S9 were identified before measuring salicylic acid formation from 31 substances. Four control substances, not defined as salicylic esters but which could be mistaken as such due to their nomenclature, did not form salicylic acid. For the remaining substances, higher in vitro intrinsic clearance (CLint, in vitro) values generally correlated with lower LogP values. A "High-Throughput Pharmacokinetic" (HTPK) model was used to extrapolate CLint, in vitro values to human in vivo clearance and half-lives. The latter were used to calculate the percentage of substance metabolised to salicylic acid in 24 h in vivo following human exposure to the ester, i.e. the "metabolism factor". The IVIVE model correctly reproduced the observed elimination rate of 3 substances using in silico or in vitro input parameters. For other substances, in silico only-based predictions generally resulted in lower metabolism factors than when in vitro values for plasma binding and liver S9 CLint, in vitro were used. Therefore, in vitro data input provides the more conservative metabolism factors compared to those derived using on in silico input. In conclusion, these results indicate that not all substances contribute equally (or at all) to the systemic exposure to salicylic acid. Therefore, we propose a realistic metabolism correction factor by which the potential contribution of salicylate esters to the aggregate consumer exposure to salicylic acid from cosmetic use can be estimated.

Proposals for new transfer coefficient (TC) values for worker re-entry activities in grape vineyards.

New transfer coefficient (TC) values were derived for vineyard workers handling treated grapevines during harvesting and crop maintenance activities. Re-entry exposure and dislodgeable foliar residue (DFR) studies were performed in Europe, covering hand harvesting, pruning/training, pruning/tying and pruning/shoot lifting. Foliar applications of fungicides (iprovalicarb, dimethomorph, dithianon, pyrimethanil and fenbuconazole) were made and 73 workers at 16 sites were monitored over one working day. Exposure was measured on inner and outer dosimeters, face/neck wipes and hand washes. In concurrent DFR studies, leaf punches were taken at each site during the time of worker re-entry. Potential exposure values correlated well with DFR values. TC values were derived for various re-entry activities for potential and actual exposure, with and without gloves. The harvesting task resulted in lower TC values than the other crop maintenance tasks. Additional TC values reflecting the use of protective gloves can be derived from the results. The TC values are much lower than current European Food Safety Authority (EFSA) default values. This project addresses a data gap identified by EFSA for specific EU TC values to permit more realistic and reliable re-entry worker exposure estimates for grapes.

Proposals for new spray drift exposure values in orchards and vineyards for residents and bystanders.

In the EU, predicted exposure to spray drift for residents and bystanders from applications in orchards and vineyards is based on data from one study published in 1987, where one downwind distance (8 m) was considered. CropLife Europe conducted sixteen new GLP compliant studies in 4 EU countries, 8 in orchards, 8 in vineyards with early and late season applications, using adult and child mannequins located 5, 10 and 15 m downwind from the last row to measure dermal and inhalation exposures. The resulting "Bystander Resident Orchard Vineyard (BROV)" database comprises 288 observations and offers a more comprehensive option for exposure prediction. There were differences between adult and child, crop type, leaf cover and distance from the sprayer, supporting the derivation of mean, median, 75th and 95th percentile exposures for each subset. Exposures did not generally correlate with wind speed, wind direction, sprayer type, spray quality, spray concentration or amount applied. Dermal and inhalation exposure were lower in vineyards than in orchards and further analysis is required to understand why.

Ambient air concentrations of plant protection products: Data collection for the combined air concentration database and associated risk assessment.

CropLife Europe collected literature values from monitoring studies measuring air concentrations of Plant Protection Products (PPPs) that may be inhaled by humans located in rural areas but not immediately adjacent to PPP applications. The resulting "Combined Air Concentration Database" (CACD) was used to determine whether air concentrations of PPPs reported by the French "Agency for Food, Environmental and Occupational Health & Safety" (ANSES) are consistent with those measured by others to increase confidence in values of exposure to humans. The results were put into risk assessment context. Results show that 25-90% of samples do not contain measurable PPP concentrations. Measured respirable fractions were below EU default air concentrations used for risk assessment for resident exposure by the European Food Safety Authority. All measured exposures in the CACD were also below established toxicological endpoints, even when considering the highest maximum average reported concentrations and very conservative inhalation rates. The highest recorded air concentration was for prosulfocarb (0.696 µg/m³ measured over 48 h) which is below the EFSA default limit of 1 µg/m³ for low volatility substances. In conclusion, based on the CACD, measured air concentrations of PPPs are significantly lower than EFSA default limits and relevant toxicological reference values.

Safety assessment for nail cosmetics: Framework for the estimation of systemic exposure through the nail plate.

All cosmetics products, including nail care products, must be evaluated for their safety. The assessment of systemic exposure is a key component of the safety assessment. However, data on the exposure, especially via ungual route (nail plate) are limited. Based on the physicochemical properties of human nails and permeability data of topical onychomycosis drugs, the nail plate is considered a good barrier to chemicals. We examine factors impacting penetration of nail care ingredients through the nail plate, including properties of the nails of the ingredients and formulations. The molecular weight, vapor pressure, logP, water solubility, and keratin binding, as well as formulations properties e.g., polymerization of acrylate monomers are considered important factors affecting penetration. To estimate systemic exposure of nail care ingredients through the nail plate, a standardized framework is applied that quantifies the impacts of these properties on penetration with an adjustment factor for each of these influencing properties. All the adjustment factors are then consolidated to derive an integrated adjustment factor which can be used for calculation of the systemic exposure dose for the ingredient. Several case studies are presented to reflect how this framework can be used in the exposure assessment for nail cosmetic products.

Suitability of different reconstructed human skin models in the skin and liver Chip2 microphysiological model to investigate the kinetics and first-pass skin metabolism of the hair dye, 4-amino-2-hydroxytoluene.

The HUMIMIC skin-liver Chip2 microphysiological systems model using the epidermal model, EpiDerm™, was reported previously to mimic application route-dependent metabolism of the hair dye, 4-amino-2-hydroxytoluene (AHT). Therefore, we evaluated the use of alternative skin models-SkinEthic™, EpiDermFT™ and PhenionFT™-for the same purpose. In static incubations, AHT permeation was similar using SkinEthic™ and EpiDerm™ models. Older Day 21 (D21) SkinEthic™ models with a thicker stratum corneum did not exhibit a greater barrier to AHT (overall permeation was the same in D17 and D21 models). All epidermal models metabolised AHT, with the EpiDerm™ exhibiting higher N-acetylation than SkinEthic™ models. AHT metabolism by D21 SkinEthic™ models was lower than that by D17 SkinEthic™ and EpiDerm™ models, thus a thicker stratum corneum was associated with fewer viable cells and a lower metabolic activity. AHT permeation was much slower using PhenionFT™ compared to epidermal models and better reflected permeation of AHT through native human skin. This model also extensively metabolised AHT to N-acetyl-AHT. After a single topical or systemic application of AHT to Chip2 model with PhenionFT™, medium was analysed for parent and metabolites over 5 days. The first-pass metabolism of AHT was demonstrated, and the introduction of a wash step after 30 min decreased the exposure to AHT and its metabolites by 33% and 40%-43%, respectively. In conclusion, epidermal and FT skin models used in the Chip2 can mimic the first-pass skin metabolism of AHT. This highlights the flexibility of the Chip2 to incorporate different skin models according to the purpose.

Application of a skin and liver Chip2 microphysiological model to investigate the route-dependent toxicokinetics and toxicodynamics of consumer-relevant doses of genistein.

The HUMMIC skin-liver Chip2 microphysiological system using EpiDerm™ and HepaRG and stellate liver spheroids was used to evaluate the route-specific metabolism and toxicodynamic effects of genistein. Human-relevant exposure levels were compared: 60 nM representing the plasma concentration expected after topical application of a cosmetic product and 1 µM representing measured plasma concentrations after ingesting soya products. Genistein was applied as single and repeated topical and/or systemic doses. The kinetics of genistein and its metabolites were measured over 5 days. Toxicodynamic effects were measured using transcriptional analyses of skin and liver organoids harvested on Days 2 and 5. Route-specific differences in genistein's bioavailability were observed, with first-pass metabolism (sulfation) occurring in the skin after topical application. Only repeated application of 1 µM, resembling daily oral intake of soya products, induced statistically significant changes in gene expression in liver organoids only. This was concomitant with a much higher systemic concentration of genistein which was not reached in any other dosing scenario. This suggests that single or low doses of genistein are rapidly metabolised which limits its toxicodynamic effects on the liver and skin. Therefore, by facilitating longer and/or repeated applications, the Chip2 can support safety assessments by linking relevant gene modulation with systemically available parent or metabolite(s). The rate of metabolism was in accordance with the short half-life observed in in vivo in humans, thus supporting the relevance of the findings. In conclusion, the skin-liver Chip2 provides route-specific information on metabolic fate and toxicodynamics that may be relevant to safety assessment.

Effect of vehicle on the in vitro penetration and metabolism of genistein and daidzein in ex vivo skin explants and the Phenion full-thickness skin model.

In a read-across assessment of the safety of genistein and daidzein in cosmetic products, additional information was required to account for differences in their systemic exposure after topical application in a typical body lotion formulation. Therefore, we measured the penetration and metabolism of two doses (3 and 30 nmoles/cm2) of genistein and daidzein applied in ethanol and in a body formulation to fresh pig skin, fresh and frozen human skin, and PhenionFT models. Both chemicals readily penetrated all skin models when applied in ethanol. The same sulfate and glucuronide metabolites were formed in fresh pig skin, fresh human skin, and PhenionFT models, which also all demonstrated that (a) these pathways could be saturated between 3 and 30 nmoles/cm2 and (b) the extent of metabolism of daidzein was lower than genistein. Although the relative amounts of radiolabeled chemical in human skin and medium compartments were altered by freezing, their overall bioavailability was not affected. The greatest impact on the bioavailability and distribution of both chemicals was observed when they were applied in the formulation. Most of the dose applied in the formulation was retained on the skin surface, especially at 30 nmoles/cm2 (60%-90%), resulting in much lower amounts in the medium and/or skin. In conclusion, all four skin models demonstrated first-pass metabolism of genistein and daidzein and a marked alteration in their disposition by applying them in a body lotion formulation. This supports the use of fresh pig skin and PhenionFT models as alternatives to human skin for investigating skin metabolism and formulation effects for these two chemicals. The results were used to develop the dermal module of a PBPK model and dose setting for organ-on-chip experiments. They could also be used to refine internal exposure estimates in regulatory safety assessments.

Characterization of the in vitro penetration and first-pass metabolism of genistein and daidzein using human and pig skin explants and Phenion full-thickness skin models.

OECD test guideline compliant skin penetration studies, which also comply with the SCCS basic criteria, are lacking for genistein and daidzein. Therefore, we have measured their penetration and metabolism using ex vivo explants of fresh (i.e., metabolically viable) pig skin, fresh and frozen human skin, and Phenion full-thickness (FT) models. Preliminary studies using fresh pig skin helped to define the optimal experimental conditions. The dermal absorption of 10 nmoles/cm2 genistein and daidzein in ethanol was comparable in all four models. A first-pass metabolism in skin to glucuronide and sulfate metabolites was demonstrated for both chemicals in all models except frozen human skin. The main difference between fresh skin models was the overall extent of metabolism and the relative ratio of each metabolite, for example, much lower sulfate conjugates were formed in pig skin incubations. The extent of parent chemical metabolized and the contribution of the glucuronide pathway were relatively lower in PhenionFT models than in fresh human skin, possibly due to a higher penetration rate in this model and differences in the expression of functional metabolizing enzymes. When metabolism in human skin was abolished by freezing, more radiolabelled chemical remained in the skin tissue but the overall dermal absorption was unchanged. In conclusion, this initial characterization study showed that all models tested indicated that genistein and daidzein extensively penetrated the skin when applied to skin in ethanol. All fresh skin models produced the same metabolites, with the known species difference in the sulfation pathway demonstrated in pig skin.

Practical application of the interim internal threshold of toxicological concern (iTTC): a case study based on clinical data.

We present a case study that provides a practical step-by-step example of how the internal Threshold of Toxicological Concern (iTTC) can be used as a tool to refine a TTC-based assessment for dermal exposures to consumer products. The case study uses a theoretical scenario where there are no systemic toxicity data for the case study chemicals (avobenzone, oxybenzone, octocrylene, homosalate, octisalate, octinoxate, and ecamsule). Human dermal pharmacokinetic data following single and repeat dermal exposure to products containing the case study chemicals were obtained from data published by the US FDA. The clinical studies utilized an application procedure that followed maximal use conditions (product applied as 2 mg/cm2 to 75% of the body surface area, 4 times a day). The case study chemicals were first reviewed to determine if they were in the applicability domain of the iTTC, and then, the human plasma concentrations were compared to an iTTC limit of 1 µM. When assessed under maximum usage, the external exposure of all chemicals exceeded the external dose TTC limits. By contrast, the internal exposure to all chemicals, except oxybenzone, was an order of magnitude lower than the 1 µM interim iTTC threshold. This work highlights the importance of understanding internal exposure relative to external dose and how the iTTC can be a valuable tool for assessing low-level internal exposures; additionally, the work demonstrates how to use an iTTC, and highlights considerations and refinement opportunities for the approach.

Workshop report: Challenges faced in developing inhalation thresholds of Toxicological Concern (TTC) - State of the science and next steps.

A challenging step in human risk assessment of chemicals is the derivation of safe thresholds. The Threshold of Toxicological Concern (TTC) concept is one option which can be used for the safety evaluation of substances with a limited toxicity dataset, but for which exposure is sufficiently low. The application of the TTC is generally accepted for orally or dermally exposed cosmetic ingredients; however, these values cannot directly be applied to the inhalation route because of differences in exposure route versus oral and dermal. Various approaches of an inhalation TTC concept have been developed over recent years to address this. A virtual workshop organized by Cosmetics Europe, held in November 2020, shared the current state of the science regarding the applicability of existing inhalation TTC approaches to cosmetic ingredients. Key discussion points included the need for an inhalation TTC for local respiratory tract effects in addition to a systemic inhalation TTC, dose metrics, database building and quality of studies, definition of the chemical space and applicability domain, and classification of chemicals with different potencies. The progress made to date in deriving inhalation TTCs was highlighted, as well as the next steps envisaged to develop them further for regulatory acceptance and use.

Measured air concentrations of pesticides for the estimation of exposure to vapour in European risk assessments.

There is an identified need to revise the default air concentration values and assumptions applied in assessing vapour exposure in the risk assessment of bystanders and residents to plant protection products. To address this, we evaluated inhalation exposure via vapour using previously unpublished data from 29 field and wind tunnel studies. The database comprises 35 trials with 11 active ingredients covering a wide range of scenarios with respect to vapour pressure, crops, application rates and European regions. Of the 961 individual measurements, 634 were below the Limit of Detection (LOD), 282 were between the LOD and Limit of Quantification (LOQ) and only 45 (4.7%) were quantifiable. Ten individual non-normalized samples exceeded 0.1 µg/m³. Of the 81 first-day measurements after the application, 36 were

Use of in vitro metabolism and biokinetics assays to refine predicted in vivo and in vitro internal exposure to the cosmetic ingredient, phenoxyethanol, for use in risk assessment.

A novel approach was developed to help characterize the biokinetics of the cosmetic ingredient, phenoxyethanol, to help assess the safety of the parent and its major stable metabolite. In the first step of this non-animal tiered approach, primary human hepatocytes were used to confirm or refute in silico predicted metabolites, and elucidate the intrinsic clearance of phenoxyethanol. A key result was the identification of the major metabolite, phenoxyacetic acid (PAA), the exposure to which in the kidney was subsequently predicted to far exceed that of phenoxyethanol in blood or other tissues. Therefore, a novel aspect of this approach was to measure in the subsequent step the formation of PAA in the cells dosed with phenoxyethanol that were used to provide points of departure (PoDs) and express the intracellular exposure as the Cmax and AUC24. This enabled the calculation of the intracellular concentrations of parent and metabolite at the PoD in the cells used to derive this value. These concentrations can be compared with in vivo tissue levels to conclude on the safety margin. The lessons from this case study will help to inform the design of other non-animal safety assessments.

A 10-step framework for use of read-across (RAX) in next generation risk assessment (NGRA) for cosmetics safety assessment.

This paper presents a 10-step read-across (RAX) framework for use in cases where a threshold of toxicological concern (TTC) approach to cosmetics safety assessment is not possible. RAX builds on established approaches that have existed for more than two decades using chemical properties and in silico toxicology predictions, by further substantiating hypotheses on toxicological similarity of substances, and integrating new approach methodologies (NAM) in the biological and kinetic domains. NAM include new types of data on biological observations from, for example, in vitro assays, toxicogenomics, metabolomics, receptor binding screens and uses physiologically-based kinetic (PBK) modelling to inform about systemic exposure. NAM data can help to substantiate a mode/mechanism of action (MoA), and if similar chemicals can be shown to work by a similar MoA, a next generation risk assessment (NGRA) may be performed with acceptable confidence for a data-poor target substance with no or inadequate safety data, based on RAX approaches using data-rich analogue(s), and taking account of potency or kinetic/dynamic differences.

Read-across and new approach methodologies applied in a 10-step framework for cosmetics safety assessment - A case study with parabens.

Parabens are esters of para-hydroxybenzoic acid that have been used as preservatives in many types of products for decades including agrochemicals, pharmaceuticals, food and cosmetics. This illustrative case study with propylparaben (PP) demonstrates a 10-step read-across (RAX) framework in practice. It aims at establishing a proof-of-concept for the value added by new approach methodologies (NAMs) in read-across (RAX) for use in a next-generation risk assessment (NGRA) in order to assess consumer safety after exposure to PP-containing cosmetics. In addition to structural and physico-chemical properties, in silico information, toxicogenomics, in vitro toxicodynamic, toxicokinetic data from PBK models, and bioactivity data are used to provide evidence of the chemical and biological similarity of PP and analogues and to establish potency trends for observed effects in vitro. The chemical category under consideration is short (C1-C4) linear chain n-alkyl parabens: methylparaben, ethylparaben, propylparaben and butylparaben. The goal of this case study is to illustrate how a practical framework for RAX can be used to fill a hypothetical data gap for reproductive toxicity of the target chemical PP.

Validation of the 3D reconstructed human skin Comet assay, an animal-free alternative for following-up positive results from standard in vitro genotoxicity assays.

As part of the safety assessment process, all industrial sectors employ genotoxicity test batteries, starting with well-established in vitro assays. However, these batteries have limited predictive capacity for the in vivo situation, which may result in unnecessary follow-up in vivo testing or the loss of promising substances where animal tests are prohibited or not desired. To address this, a project involving regulators, academia and industry was established to develop and validate in vitro human skin-based genotoxicity assays for topically exposed substances, such as cosmetics ingredients. Here, we describe the validation of the 3D reconstructed skin (RS) Comet assay. In this multicenter study, chemicals were applied topically three times to the skin over 48 h. Isolated keratinocytes and fibroblasts were transferred to slides before electrophoresis and the resulting comet formation was recorded as % tail DNA. Before decoding, results of the validation exercise for 32 substances were evaluated by an independent statistician. There was a high predictive capacity of this assay when compared to in vivo outcomes, with a sensitivity of 77 (80)%, a specificity of 88 (97)% and an overall accuracy of 83 (92)%. The numbers reflect the calls of the performing laboratories in the coded phase, whereas those in parenthesis reflect calls according to the agreed evaluation criteria. Intra- and inter-laboratory reproducibility was also very good, with a concordance of 93 and 88%, respectively. These results generated with the Phenion® Full-Thickness skin model demonstrate its suitability for this assay, with reproducibly low background DNA damage and sufficient metabolic capacity to activate pro-mutagens. The validation outcome supports the use of the RS Comet assay to follow up positive results from standard in vitro genotoxicity assays when the expected route of exposure is dermal. Based on the available data, the assay was accepted recently into the OECD test guideline development program.

Validation of the 3D reconstructed human skin micronucleus (RSMN) assay: an animal-free alternative for following-up positive results from standard in vitro genotoxicity assays.

In vitro test batteries have become the standard approach to determine the genotoxic potential of substances of interest across industry sectors. While useful for hazard identification, standard in vitro genotoxicity assays in 2D cell cultures have limited capability to predict in vivo outcomes and may trigger unnecessary follow-up animal studies or the loss of promising substances where animal tests are prohibited or not desired. To address this problem, a team of regulatory, academia and industry scientists was established to develop and validate 3D in vitro human skin-based genotoxicity assays for use in testing substances with primarily topical exposure. Validation of the reconstructed human skin micronucleus (RSMN) assay in MatTek Epi-200™ skin models involved testing 43 coded chemicals selected by independent experts, in four US/European laboratories. The results were analysed by an independent statistician according to predefined criteria. The RSMN assay showed a reproducibly low background micronucleus frequency and exhibited sufficient capacity to metabolise pro-mutagens. The overall RSMN accuracy when compared to in vivo genotoxicity outcomes was 80%, with a sensitivity of 75% and a specificity of 84%, and the between- and within-laboratory reproducibility was 77 and 84%, respectively. A protocol involving a 72-h exposure showed increased sensitivity in detecting true positive chemicals compared to a 48-h exposure. An analysis of a test strategy using the RSMN assay as a follow-up test for substances positive in standard in vitro clastogenicity/aneugenicity assays and a reconstructed skin Comet assay for substances with positive results in standard gene mutation assays results in a sensitivity of 89%. Based on these results, the RSMN assay is considered sufficiently validated to establish it as a 'tier 2' assay for dermally exposed compounds and was recently accepted into the OECD's test guideline development program.

New framework for a non-animal approach adequately assures the safety of cosmetic ingredients - A case study on caffeine.

This case study on the model substance caffeine demonstrates the viability of a 10-step read-across (RAX) framework in practice. New approach methodologies (NAM), including RAX and physiologically-based kinetic (PBK) modelling were used to assess the consumer safety of caffeine. Appropriate animal systemic toxicity data were used from the most relevant RAX analogue while assuming that no suitable animal toxicity data were available for caffeine. Based on structural similarities, three primary metabolites of the target chemical caffeine (theophylline, theobromine and paraxanthine) were selected as its most relevant analogues, to estimate a point of departure in order to support a next generation risk assessment (NGRA). On the basis of the pivotal mode of action (MOA) of caffeine and other methylxanthines, theophylline appeared to be the most potent and suitable analogue. A worst-case aggregate exposure assessment determined consumer exposure to caffeine from different sources, such as cosmetics and food/drinks. Using a PBK model to estimate human blood concentrations following exposure to caffeine, an acceptable Margin of Internal Exposure (MOIE) of 27-fold was derived on the basis of a RAX using theophylline animal data, which suggests that the NGRA approach for caffeine is sufficiently conservative to protect human health.

Demonstration of the first-pass metabolism in the skin of the hair dye, 4-amino-2-hydroxytoluene, using the Chip2 skin-liver microphysiological model.

We used TissUse's HUMIMIC Chip2 microfluidic model, incorporating reconstructed skin models and liver spheroids, to investigate the impact of consumer-relevant application scenarios on the metabolic fate of the hair dye, 4-amino-2-hydroxytoluene (AHT). After a single topical or systemic application of AHT to Chip2 models, medium was analysed for parent and metabolites over 5 days. The metabolic profile of a high dose (resulting in a circuit concentration of 100 µM based on 100% bioavailability) of AHT was the same after systemic and topical application to 96-well EpiDerm™ models. Additional experiments indicated that metabolic capacity of EpiDerm™ models were saturated at this dose. At 2.5 µM, concentrations of AHT and several of its metabolites differed between application routes. Topical application resulted in a higher Cmax and a 327% higher area under the curve (AUC) of N-acetyl-AHT, indicating a first-pass effect in the EpiDerm™ models. In accordance with in vivo observations, there was a concomitant decrease in the Cmax and AUC of AHT-O-sulphate after topical, compared with systemic application. A similar alteration in metabolite ratios was observed using a 24-well full-thickness skin model, EpiDermFT™, indicating that a first-pass effect was also possible to detect in a more complex model. In addition, washing the EpiDermFT™ after 30 min, thus reflecting consumer use, decreased the systemic exposure to AHT and its metabolites. In conclusion, the skin-liver Chip2 model can be used to (a) recapitulate the first-pass effect of the skin and alterations in the metabolite profile of AHT observed in vivo and (b) provide consumer-relevant data regarding leave-on/rinse-off products.

A comprehensive view on mechanistic approaches for cancer risk assessment of non-genotoxic agrochemicals.

Currently the only methods for non-genotoxic carcinogenic hazard assessment accepted by most regulatory authorities are lifetime carcinogenicity studies. However, these involve the use of large numbers of animals and the relevance of their predictive power and results has been scientifically challenged. With increased availability of innovative test methods and enhanced understanding of carcinogenic processes, it is believed that tumour formation can now be better predicted using mechanistic information. A workshop organised by the European Partnership on Alternative Approaches to Animal Testing brought together experts to discuss an alternative, mechanism-based approach for cancer risk assessment of agrochemicals. Data from a toolbox of test methods for detecting modes of action (MOAs) underlying non-genotoxic carcinogenicity are combined with information from subchronic toxicity studies in a weight-of-evidence approach to identify carcinogenic potential of a test substance. The workshop included interactive sessions to discuss the approach using case studies. These showed that fine-tuning is needed, to build confidence in the proposed approach, to ensure scientific correctness, and to address different regulatory needs. This novel approach was considered realistic, and its regulatory acceptance and implementation can be facilitated in the coming years through continued dialogue between all stakeholders and building confidence in alternative approaches.