A Cautionary tale for using read-across for cancer hazard classification: Case study of isoeugenol and methyl eugenol.

Chemical grouping and read-across are frequently used non-animal alternatives for filling toxicological data gaps. When grouping chemicals, it is critical to define the applicability domain because minor differences in chemical structure can lead to significant differences in toxicity. Here, we present a case study on isoeugenol and methyl eugenol, which are scheduled for review by IARC in June 2023, to illustrate that structural similarity alone may not be sufficient to group chemicals for hazard classification. Isoeugenol and methyl eugenol are plant-derived phenylpropenes that share similar physicochemical properties. The major metabolic pathway for isoeugenol includes conjugation of the phenolic hydroxyl group with sulfate and glucuronic acid as an efficient detoxification process, whereas the major metabolic pathway for methyl eugenol involves benzylic hydroxylation and formation of the 1'-sulfoxymethyleugenol which leads to carbocation formation. The carbocation can form DNA adducts and induce genotoxicity and carcinogenicity. Consistently, genotoxicity and carcinogenicity alerts are identified from in silico prediction tools for methyl eugenol but not isoeugenol. Moreover, the available toxicogenomic, genotoxicity, and carcinogenicity studies confirm that these chemicals have significantly different bioactivities. Data on other structurally similar chemicals further supports our conclusion that it is not appropriate to group these two chemicals for cancer hazard classification.

Assessing chemical carcinogenicity: hazard identification, classification, and risk assessment. Insight from a Toxicology Forum state-of-the-science workshop.

The Toxicology Forum convened an international state-of-the-science workshop Assessing Chemical Carcinogenicity: Hazard Identification, Classification, and Risk Assessment in December 2020. Challenges related to assessing chemical carcinogenicity were organized under the topics of (1) problem formulation; (2) modes-of-action; (3) dose-response assessment; and (4) the use of new approach methodologies (NAMs). Key topics included the mechanisms of genotoxic and non-genotoxic carcinogenicity and how these in conjunction with consideration of exposure conditions might inform dose-response assessments and an overall risk assessment; approaches to evaluate the human relevance of modes-of-action observed in rodent studies; and the characterization of uncertainties. While the scientific limitations of the traditional rodent chronic bioassay were widely acknowledged, knowledge gaps that need to be overcome to facilitate the further development and uptake of NAMs were also identified. Since one single NAM is unlikely to replace the bioassay, activities to combine NAMs into integrated approaches for testing and assessment, or preferably into defined approaches for testing and assessment that include data interpretation procedures, were identified as urgent research needs. In addition, adverse outcome pathway networks can provide a framework for organizing the available evidence/data for assessing chemical carcinogenicity. Since a formally accepted decision tree to guide use of the best and most current science to advance carcinogenicity risk assessment is currently unavailable, a Decision Matrix for carcinogenicity assessment could be useful. The workshop organizers developed and presented a decision matrix to be considered within a carcinogenicity hazard and risk assessment that is offered in tabular form.

Butylated hydroxyanisole: Carcinogenic food additive to be avoided or harmless antioxidant important to protect food supply?

Tumor data from rodent bioassays are used for cancer hazard classification with wide-ranging consequences. This paper presents a case study of the synthetic antioxidant butylated hydroxyanisole (BHA), which IARC classified as Group 2B ("possibly carcinogenic to humans") on the basis of forestomach tumors in rodents following chronic dietary exposure to high levels. IARC later determined that the mechanism by which BHA induces forestomach tumors is not relevant to humans; however, the classification has not been revoked. BHA was listed on California Proposition 65 as a direct consequence of the IARC classification, and there is widespread concern among consumers regarding the safety of BHA driven by the perception that it is a carcinogen. While many regulatory agencies have established safe exposure limits for BHA, the IARC classification and Proposition 65 listing resulted in the addition of BHA to lists of substances banned from children's products and products seeking credentials such as EPA's Safer Choice program, as well as mandatory product labeling. Classifications have consequences that many times pre-empt the ability to conduct an exposure-based risk-based assessment., It is imperative to consider human relevance of both the endpoint and exposure conditions as fundamental to hazard identification.

Hazard identification, classification, and risk assessment of carcinogens: too much or too little? - Report of an ECETOC workshop.

The European Centre for Ecotoxicology and Toxicology of Chemicals (ECETOC) organized a workshop "Hazard Identification, Classification and Risk Assessment of Carcinogens: Too Much or Too Little?" to explore the scientific limitations of the current binary carcinogenicity classification scheme that classifies substances as either carcinogenic or not. Classification is often based upon the rodent 2-year bioassay, which has scientific limitations and is not necessary to predict whether substances are likely human carcinogens. By contrast, tiered testing strategies founded on new approach methodologies (NAMs) followed by subchronic toxicity testing, as necessary, are useful to determine if a substance is likely carcinogenic, by which mode-of-action effects would occur and, for non-genotoxic carcinogens, the dose levels below which the key events leading to carcinogenicity are not affected. Importantly, the objective is not for NAMs to mimic high-dose effects recorded in vivo, as these are not relevant to human risk assessment. Carcinogenicity testing at the "maximum tolerated dose" does not reflect human exposure conditions, but causes major disturbances of homeostasis, which are very unlikely to occur at relevant human exposure levels. The evaluation of findings should consider biological relevance and not just statistical significance. Using this approach, safe exposures to non-genotoxic substances can be established.

How the 62-year old Delaney Clause continues to thwart science: Case study of the flavor substance ß-myrcene.

The Delaney Clause is a provision of the 1958 Food Additive Amendment to the Food, Drug and Cosmetic Act of 1938 which stipulates that if a substance is found by the Food and Drug Administration to be carcinogenic in any species of animal or in humans, then it cannot be used as a food additive. This paper presents a case study of ß-myrcene, one of seven synthetic substances that was challenged under the Delaney Clause, ultimately resulting in revocation of its regulatory approval as a food additive despite a lack of safety concern. While it is listed as a synthetic flavor in 21 CFR 172.515, ß-myrcene is also a substance naturally occurring in a number of dietary plants. The exposure level to naturally-occurring ß-myrcene is orders of magnitude higher (estimated to be 16,500 times greater) than the exposure via ß-myrcene added to food as a flavoring substance. The National Toxicology Program conducted genotoxicity testing (negative), a 13-week range-finding study, and a two-year cancer bioassay in B6C3F1 mice and F344/N rats. An increase in liver tumors was seen in male mice and kidney tumors in male rats, ultimately resulting in ß-myrcene being classified by IARC as a Class 2B carcinogen and being listed on California Proposition 65; in contrast, ß-myrcene is not classified as a carcinogen by any other regulatory authority. The doses administered in the NTP bioassay were five-six orders of magnitude higher than human exposures, and the FDA concluded after a thorough evaluation that there was no safety concern associated with the use of ß-myrcene as a flavor substance at the current use level. The Delaney Clause, however, does not consider the exposure potential or the human health relevance of effects observed in animals. The lack of options available to the US FDA led to the 2018 decision to remove ß-myrcene from the list of approved food additives. This revocation has contributed to the ongoing erosion of trust in regulatory agencies (and industry), which has both economic implications for food manufacturers and consumers alike, and implications for consumer perception of safety of the US food supply. It is time for us to reconsider the rationale behind any legislation that relies on classification alone, and whether there is, in fact, a reason to still classify nongenotoxic carcinogens at all.

Bolstering the existing database supporting the non-cancer Threshold of Toxicological Concern values with toxicity data on fragrance-related materials.

The use of threshold of toxicological concern (TTC) supports the safety assessment of exposure to low levels of chemicals when toxicity data are limited. The Research Institute for Fragrance Materials (RIFM) delivers safety assessments for fragrance materials that result in safe products for consumer use. A major goal for the RIFM safety assessment program is to invest in alternative methods to animal testing for use in assessment of fragrance materials. This includes use of TTC, which provides a pragmatic approach for safety evaluation of fragrance materials in the absence of chemical-specific toxicity data and reduces the need to generate new animal data. To bolster the TTC approach for support of fragrance materials and specifically to strengthen the Cramer class II threshold, the RIFM database was reviewed with a goal of identifying fragrance materials with data that can be added to the existing TTC databases. The RIFM database identified a total of 476 chemicals that were added to the existing TTC databases. The chemicals were then individually assigned a Cramer class and 238, 76 and 162 chemicals in Cramer class I, II and III respectively were identified. The RIFM-TTC dataset was then combined with the COSMOS-Federated TTC dataset for a total of 421, 111 and 795 chemicals in Cramer class I, II and III respectively. The combined dataset further expands the chemical space thereby providing more robust 5th percentile thresholds. Moreover, the combined dataset bolsters the threshold for Cramer class II to include a total of 111 chemicals which is an improvement over the original (Munro) TTC dataset which only included 28 chemicals in Cramer Class II and the COSMOS Federated dataset which had 40 chemicals. This allows for a more reliable and robust 5th percentile NOAEL value for Cramer class II chemicals of 1.27 mg/kg bw/day. The 5th percentile NOAELs for Cramer class I, II and III from the combined dataset are 4.91, 1.27 and 0.29 mg/kg bw/day, which supports the threshold values derived from the original Munro dataset. This work confirms the adequacy of the existing TTC values and provides further support for the use of TTC as a tool to conduct safety assessments for fragrance materials. It further opens the future possibility of updating the existing values with more robust TTC values for fragrance and cosmetic materials.

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.

Allergic contact dermatitis: Adequacy of the default 10X assessment factor for human variability to protect infants and children.

When evaluating consumer products for safety, one must consider the heterogeneity of the population that might use those products, including the potential for different sensitivity based on factors such as age, gender, and genetics. For both systemic endpoints and allergic contact dermatitis (ACD), quantitative safety evaluations typically include a default 10-fold uncertainty/assessment factor to account for inter-individual variability. While this factor is intended to include age, the adequacy of the default 10-fold factor has been questioned for infants, for whom a precautionary assumption is often made that they are more sensitive. In-depth evaluations of the adequacy of the 10-fold factor have been published for systemic endpoints, but relatively little has been published to substantiate this for ACD. This paper reviews the state of the science regarding the etiology of ACD and factors that suggest an overall decreased sensitivity associated with early life exposures, thus confirming the sufficiency of the 10-fold inter-individual factor to provide protection for children and infants. While it remains prudent for all age groups to avoid contact with sensitizers, it is concluded that the quantitative methods used in safety evaluation to prevent the induction of skin sensitization are protective for infants, including neonates and premature infants.

Human relevance of rodent liver tumors: Key insights from a Toxicology Forum workshop on nongenotoxic modes of action.

The Toxicology Forum sponsored a workshop in October 2016, on the human relevance of rodent liver tumors occurring via nongenotoxic modes of action (MOAs). The workshop focused on two nuclear receptor-mediated MOAs (Constitutive Androstane Receptor (CAR) and Peroxisome Proliferator Activated Receptor-alpha (PPARα), and on cytotoxicity. The goal of the meeting was to review the state of the science to (1) identify areas of consensus and differences, data gaps and research needs; (2) identify reasons for inconsistencies in current regulatory positions; and (3) consider what data are needed to demonstrate a specific MOA, and when additional research is needed to rule out alternative possibilities. Implications for quantitative risk assessment approaches were discussed, as were implications of not considering MOA and dose in hazard characterization and labeling schemes. Most, but not all, participants considered the CAR and PPARα MOAs as not relevant to humans based on quantitative and qualitative differences. In contrast, cytotoxicity is clearly relevant to humans, but a threshold applies. Questions remain for all three MOAs concerning what data are necessary to determine the MOA and to what extent it is necessary to exclude other MOAs.

Safety evaluation for ingredients used in baby care products: Consideration of diaper rash.

Diaper rash can adversely impact the barrier properties of skin, with potential implications for increased absorption of chemicals through the skin, and this should be accounted for in any exposure assessment used in the safety evaluation of consumer products used in the diaper ("nappy") area. In the absence of a quantitative evaluation of the potential impact of diaper rash, a default assumption of 100% dermal penetration is often made for substances applied in the diaper area. We consider here the extent, duration and severity of diaper rash and make a recommendation for conservative assumptions to incorporate into exposure assessments. Using a time-weighted average, the potential impact of diaper rash is illustrated for substances that have varying degrees of absorption through healthy skin. Results confirm that for assessments that already assume dermal absorption of 50% or higher, there is no impact on the overall exposure assessment. For substances that have a very low degree of dermal penetration (1%) through healthy skin, the impact of rash is expected to be less-than four-fold. This can be refined with additional data as there are many examples of poorly absorbed compounds for which dermal penetration is still low even for compromised skin.

Assessment of health risks resulting from early-life exposures: Are current chemical toxicity testing protocols and risk assessment methods adequate?

Abstract Over the last couple of decades, the awareness of the potential health impacts associated with early-life exposures has increased. Global regulatory approaches to chemical risk assessment are intended to be protective for the diverse human population including all life stages. However, questions persist as to whether the current testing approaches and risk assessment methodologies are adequately protective for infants and children. Here, we review physiological and developmental differences that may result in differential sensitivity associated with early-life exposures. It is clear that sensitivity to chemical exposures during early-life can be similar, higher, or lower than that of adults, and can change quickly within a short developmental timeframe. Moreover, age-related exposure differences provide an important consideration for overall susceptibility. Differential sensitivity associated with a life stage can reflect the toxicokinetic handling of a xenobiotic exposure, the toxicodynamic response, or both. Each of these is illustrated with chemical-specific examples. The adequacy of current testing protocols, proposed new tools, and risk assessment methods for systemic noncancer endpoints are reviewed in light of the potential for differential risk to infants and young children.

Correlation of chemical structure with reproductive and developmental toxicity as it relates to the use of the threshold of toxicological concern.

In the absence of toxicological data on a chemical, the threshold of toxicological concern (TTC) approach provides a system to estimate a conservative exposure below which there is a low probability of risk for adverse health effects. The original toxicology dataset underlying the TTC was based on NOELs from repeat dose studies. Subsequently there have been several efforts to assess whether or not these limits are also protective for reproductive/developmental effects. This work expands the database of chemicals with reproductive and developmental data, presents these data in a comprehensive and transparent format and groups the chemicals according to the TTC "Cramer Class" rules. Distributions of NOAELs from each of these classes were used to assess whether the previously proposed TTC values based on repeat dose data are protective for reproductive/developmental toxicity endpoints as well. The present analysis indicates that, for each Cramer Class, the reproductive and developmental endpoints would be protected at the corresponding general TTC tiers derived by Munro et al. (1996).

Critical analysis of literature on low-dose synergy for use in screening chemical mixtures for risk assessment.

There is increasing interest in the use of tiered approaches in risk assessment of mixtures or co-exposures to chemicals for prioritization. One possible screening-level risk assessment approach is the threshold of toxicological concern (TTC). To date, default assumptions of dose or response additivity have been used to characterize the toxicity of chemical mixtures. Before a screening-level approach could be used, it is essential to know whether synergistic interactions can occur at low, environmentally relevant exposure levels. Studies demonstrating synergism in mammalian test systems were identified from the literature, with emphasis on studies performed at doses close to the points of departure (PODs) for individual chemicals. This search identified 90 studies on mixtures. Few included quantitative estimates of low-dose synergy; calculations of the magnitude of interaction were included in only 11 papers. Quantitative methodology varied across studies in terms of the null hypothesis, response measured, POD used to test for synergy, and consideration of the slope of the dose-response curve. It was concluded that consistent approaches should be applied for quantification of synergy, including that synergy be defined in terms of departure from dose additivity; uniform procedures be developed for assessing synergy at low exposures; and the method for determining the POD for calculating synergy be standardized. After evaluation of the six studies that provided useful quantitative estimates of synergy, the magnitude of synergy at low doses did not exceed the levels predicted by additive models by more than a factor of 4.

A proposed framework for assessing risk from less-than-lifetime exposures to carcinogens.

Quantitative methods for estimation of cancer risk have been developed for daily, lifetime human exposures. There are a variety of studies or methodologies available to address less-than-lifetime exposures. However, a common framework for evaluating risk from less-than-lifetime exposures (including short-term and/or intermittent exposures) does not exist, which could result in inconsistencies in risk assessment practice. To address this risk assessment need, a committee of the International Life Sciences Institute (ILSI) Health and Environmental Sciences Institute conducted a multisector workshop in late 2009 to discuss available literature, different methodologies, and a proposed framework. The proposed framework provides a decision tree and guidance for cancer risk assessments for less-than-lifetime exposures based on current knowledge of mode of action and dose-response. Available data from rodent studies and epidemiological studies involving less-than-lifetime exposures are considered, in addition to statistical approaches described in the literature for evaluating the impact of changing the dose rate and exposure duration for exposure to carcinogens. The decision tree also provides for scenarios in which an assumption of potential carcinogenicity is appropriate (e.g., based on structural alerts or genotoxicity data), but bioassay or other data are lacking from which a chemical-specific cancer potency can be determined. This paper presents an overview of the rationale for the workshop, reviews historical background, describes the proposed framework for assessing less-than-lifetime exposures to potential human carcinogens, and suggests next steps.

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.

Case studies to test: A framework for using structural, reactivity, metabolic and physicochemical similarity to evaluate the suitability of analogs for SAR-based toxicological assessments.

A process for evaluating analogs for use in SAR (Structure-Activity Relationship) assessments was previously published (Wu et al. 2010). Subsequently, this process has been updated to include a decision tree for estrogen binding (from US EPA) and flags for developmental and reproductive toxicity (DART). This paper presents the results of blinded case studies designed to test this updated framework. The results of these case studies support the conclusion that the process outlined by Wu et al. (2010) can be successfully applied to develop surrogate values for risk assessment. The read across results generated by the process were shown to be protective when compared to the actual toxicity data. Successful application of the approach requires significant expertise as well as discipline to not overstep the boundaries of the defined analogs and the rating system. The end result of this rigor can be the inability to read across all endpoints for all chemicals resulting in data gaps that cannot be filled using read across, however, this reflects the current state of the science and is preferable to making non-protective decisions. Future work will be targeted towards expanding read across capabilities. Two examples of a broader category approach are also shown.

Safety evaluation of disposable baby diapers using principles of quantitative risk assessment.

Baby diapers are complex products consisting of multiple layers of materials, most of which are not in direct contact with the skin. The safety profile of a diaper is determined by the biological properties of individual components and the extent to which the baby is exposed to each component during use. Rigorous evaluation of the toxicological profile and realistic exposure conditions of each material is important to ensure the overall safety of the diaper under normal and foreseeable use conditions. Quantitative risk assessment (QRA) principles may be applied to the safety assessment of diapers and similar products. Exposure to component materials is determined by (1) considering the conditions of product use, (2) the degree to which individual layers of the product are in contact with the skin during use, and (3) the extent to which some components may be extracted by urine and delivered to skin. This assessment of potential exposure is then combined with data from standard safety assessments of components to determine the margin of safety (MOS). This study examined the application of QRA to the safety evaluation of baby diapers, including risk assessments for some diaper ingredient chemicals for which establishment of acceptable and safe exposure levels were demonstrated.

Utilizing Threshold of Toxicological Concern (TTC) with High Throughput Exposure Predictions (HTE) as a Risk-Based Prioritization Approach for thousands of chemicals.

Regulatory agencies across the world are facing the challenge of performing risk-based prioritization of thousands of chemicals in commerce. Here, we present an approach using the Threshold of Toxicological Concern (TTC) combined with heuristic high-throughput exposure (HTE) modelling to rank order chemicals for further evaluation. Accordingly, for risk-based prioritization, chemicals with exposures > TTC would be ranked as higher priority for further evaluation whereas substances with exposures < TTC would be ranked as lower priority. An initial proof of concept, using a dataset of 7986 substances with previously modeled median and upper 95% credible interval (UCI) total daily median exposure rates showed fewer than 5% of substances had UCI exposures > the Cramer Class III TTC (1.5 µg/kg-day). We extended the analysis by profiling the same dataset through the TTC workflow published by Kroes et al (2004) which accounts for known exclusions to the TTC as well as structural alerts. UCI exposures were then compared to the appropriate class-specific TTC. None of the substances categorized as Cramer Class I or Cramer Class II exceeded their respective TTC values and no more than 2% of substances categorized as Cramer Class III or acetylcholinesterase inhibitors exceeded their respective TTC values. The modeled UCI exposures for the majority of the 1853 chemicals with genotoxicity structural alerts did exceed the TTC of 0.0025 µg/kg-day, but only 79 substances exceeded this TTC if median exposure values were used. For substances for which UCI exposures exceeded relevant TTC values, we highlight possible approaches for consideration to refine the HTE : TTC approach. Overall, coupling TTC with HTE offers promise as a pragmatic first step in ranking substances as part of a risk-based prioritization approach.