Computational framework for identifying and evaluating mutagenic and xenoestrogenic potential of food additives.

Food additives are chemicals incorporated in food to enhance its flavor, color and prevent spoilage. Some of these are associated with substantial health hazards, including developmental disorders, increase cancer risk, and hormone disruption. Hence, this study aimed to comprehend the in-silico toxicology framework for evaluating mutagenic and xenoestrogenic potential of food additives and their association with breast cancer. A total of 2885 food additives were screened for toxicity based on Threshold of Toxicological Concern (TTC), mutagenicity endpoint prediction, and mutagenic structural alerts/toxicophores identification. Ten food additives were identified as having mutagenic potential based on toxicity screening. Furthermore, Protein-Protein Interaction (PPI) analysis identified ESR1, as a key hub gene in breast cancer. KEGG pathway analysis verified that ESR1 plays a significant role in breast cancer pathogenesis. Additionally, competitive interaction studies of the predicted potential mutagenic food additives with the estrogen receptor-α were evaluated at agonist and antagonist binding sites. Indole, Dichloromethane, Trichloroethylene, Quinoline, 6-methyl quinoline, Ethyl nitrite, and 4-methyl quinoline could act as agonists, and Paraldehyde, Azodicarbonamide, and 2-acetylfuranmay as antagonists. The systematic risk assessment framework reported in this study enables the exploration of mutagenic and xenoestrogenic potential associated with food additives for hazard identification and management.

Commentary: Value of information case study strongly supports use of the Threshold of Toxicological Concern (TTC).

A Value of Information (VOI) analysis can play a key role in decision-making for adopting new approach methodologies (NAMs). We applied EPA's recently developed VOI framework to the Threshold of Toxicological Concern (TTC). Obtaining/deriving a TTC value for use as a toxicity reference value (TRV) for substances with limited toxicity data was shown to provide equivalent or greater health protection, immense return on investment (ROI), greater net benefit, and substantially lower costs of delay (CoD) compared with TRVs derived from either traditional human health assessment (THHA) chronic toxicity testing in lab animals or the 5-day in vivo EPA Transcriptomic Assessment Product (ETAP). For all nine exposure scenarios examined, the TTC was more economical terms of CoD and ROI than the ETAP or the THHA; expected net benefit was similar for the TTC and ETAP with both of these more economical than the THHA The TTC ROI was immensely greater (5,000,000-fold on average) than the ROI for THHA and the ETAP ROI (100,000-fold on average). These results support the use of the TTC for substances within its domain of applicability to waive requiring certain in vivo tests, or at a minimum, as an initial screening step before conducting either the ETAP or THHA in vivo studies.

Application of the threshold of toxicological concern (TTC) in the evaluation of drinking water contact chemicals.

The Threshold of Toxicological Concern (TTC) is an approach for assessing the safety of chemicals with low levels of exposure for which limited toxicology data are available. The original TTC criteria were derived for oral exposures from a distributional analysis of a dataset of 613 chemicals that identified 5th percentile no observed effect level (NOEL) values grouped within three tiers of compounds having specific structural functional groups and/or toxic potencies known as Cramer I, II and III classifications. Subsequent assessments of the TTC approach have established current thresholds to be scientifically robust. While the TTC has gained acknowledgment and acceptance by many regulatory agencies and organizations, use of the TTC approach in evaluating drinking water chemicals has been limited. To apply the TTC concept to drinking water chemicals, an exposure-based approach that incorporates the current weight of evidence for the target chemical is presented. Such an approach provides a comparative point of departure to the 5th percentile TTC NOEL using existing data, while conserving the allocation of toxicological resources for quantitative risk assessment to chemicals with greater exposure or toxicity. This approach will be considered for incorporation into NSF/ANSI/CAN 600, a health effects standard used in the safety evaluation of chemicals present in drinking water from drinking water contact additives and materials certified to NSF/ANSI/CAN 60 and 61, respectively.

Evaluating the applicability of the Ames test for cosmetic packaging assessment by comparing carcinogenic risk levels of migrants from plastics with biological detection limits.

Risk assessments for cosmetic packaging are required according to the EU Cosmetics Regulation (EC) No. 1223/2009, however, the assessment method is well-established for food packaging but limited for cosmetic packaging. In food packaging assessments, Cramer class III TTC (90 µg/day) is applied as the threshold for systemic toxicity when the Ames test including the process of sample concentration steps provides the negative results. However, the human health risks of mutagenic and carcinogenic migrants at exposure levels where the Ames test with the concentrated samples cannot detect are unclear. In the present study, to confirm the applicability of the Ames test for cosmetic packaging assessments, the toxicological data on 37 candidate migrants with Ames test-positive results was collected. For these migrants, the carcinogenic risk levels through cosmetics use were compared to the detection levels of the Ames test for concentrated samples. Regarding at least 32 migrants, the case study showed the negative result from the Ames test incorporating the sample concentration process would indicate negligible mutagenic and carcinogenic risks of packaging extracts. Therefore, application of the Ames test to cosmetic packaging assessments would be helpful to ensure the safety for mutagenicity and carcinogenicity as well as use Cramer-TTC for systemic toxicity.

Evaluating the utility of the Threshold of Toxicological Concern (TTC) and its exclusions in the biocompatibility assessment of extractable chemical substances from medical devices.

The Threshold of Toxicological Concern (TTC) is a pragmatic approach used to establish safe thresholds below which there can be no appreciable risk to human health. Here, a large inventory of ~45,000 substances (referred to as the LRI dataset) was profiled through the Kroes TTC decision module within Toxtree v3.1 to assign substances into their respective TTC categories. Four thousand and two substances were found to be not applicable for the TTC approach. However, closer examination of these substances uncovered several implementation issues: substances represented in their salt forms were automatically assigned as not appropriate for TTC when many of these contained essential metals as counter ions which would render them TTC applicable. High Potency Carcinogens and dioxin-like substances were not fully captured based on the rules currently implemented in the software. Phosphorus containing substances were considered exclusions when many of them would be appropriate for TTC. Refinements were proposed to address the limitations in the current software implementation. A second component of the study explored a set of substances representative of those released from medical devices and compared them to the LRI dataset as well as other toxicity datasets to investigate their structural similarity. A third component of the study sought to extend the exclusion rules to address application to substances released from medical devices that lack toxicity data. The refined rules were then applied to this dataset and the TTC assignments were compared. This case study demonstrated the importance of evaluating the software implementation of an established TTC workflow, identified certain limitations and explored potential refinements when applying these concepts to medical devices.

Estimating provisional margins of exposure for data-poor chemicals using high-throughput computational methods.

Current computational technologies hold promise for prioritizing the testing of the thousands of chemicals in commerce. Here, a case study is presented demonstrating comparative risk-prioritization approaches based on the ratio of surrogate hazard and exposure data, called margins of exposure (MoEs). Exposures were estimated using a U.S. EPA's ExpoCast predictive model (SEEM3) results and estimates of bioactivity were predicted using: 1) Oral equivalent doses (OEDs) derived from U.S. EPA's ToxCast high-throughput screening program, together with in vitro to in vivo extrapolation and 2) thresholds of toxicological concern (TTCs) determined using a structure-based decision-tree using the Toxtree open source software. To ground-truth these computational approaches, we compared the MoEs based on predicted noncancer TTC and OED values to those derived using the traditional method of deriving points of departure from no-observed adverse effect levels (NOAELs) from in vivo oral exposures in rodents. TTC-based MoEs were lower than NOAEL-based MoEs for 520 out of 522 (99.6%) compounds in this smaller overlapping dataset, but were relatively well correlated with the same (r 2 = 0.59). TTC-based MoEs were also lower than OED-based MoEs for 590 (83.2%) of the 709 evaluated chemicals, indicating that TTCs may serve as a conservative surrogate in the absence of chemical-specific experimental data. The TTC-based MoE prioritization process was then applied to over 45,000 curated environmental chemical structures as a proof-of-concept for high-throughput prioritization using TTC-based MoEs. This study demonstrates the utility of exploiting existing computational methods at the pre-assessment phase of a tiered risk-based approach to quickly, and conservatively, prioritize thousands of untested chemicals for further study.

Toxicological assessment compilation of selected examples of raw materials for homeopathic and anthroposophic medicinal products - Part 2.

The present successor article comprises more than 180 substances representing a continuative compilation of toxicologically evaluated starting materials prompted by the wide use and high number of homeopathic and anthroposophic medicinal products (HMP) on the market together with the broad spectrum of active substances of botanical, mineral, chemical or animal origin contained therein, and by the equally important requirement of applying adequate safety principles as with conventional human medicinal products in line with the European regulatory framework. The February 2019 issue of the Regulatory Toxicology and Pharmacology journal includes the antecedent article bearing the same title and entailing safety evaluations of more than 170 raw materials processed in HMP. This part 2 article highlights scientific evaluation following recognized methods used in toxicology with a view to drug-regulatory authority's assessment principles and practice in the context of HMP, and offers useful systematic, scientifically substantiated and simultaneously pragmatic approaches in differentiated HMP risk assessment. As a unique feature, both articles provide the most extensive publicly available systematic compilation of a considerable number of substances processed in HMP as a transparent resource for applicants, pharmaceutical manufacturers, the scientific community and healthcare authorities to actively support regulatory decision making in practice.

Updating the Dermal Sensitisation Thresholds using an expanded dataset and an in silico expert system.

The Dermal Sensitisation Thresholds (DST) are Thresholds of Toxicological Concern, which can be used to justify exposure-based waiving when conducting a skin sensitisation risk assessment. This study aimed to update the published DST values by expanding the size of the Local Lymph Node Assay dataset upon which they are based, whilst assigning chemical reactivity using an in silico expert system (Derek Nexus). The potency values within the expanded dataset fitted a similar gamma distribution to that observed for the original dataset. Derek Nexus was used to classify the sensitisation activity of the 1152 chemicals in the expanded dataset and to predict which chemicals belonged to a High Potency Category (HPC). This two-step classification led to three updated thresholds: a non-reactive DST of 710 µg/cm2 (based on 79 sensitisers), a reactive (non-HPC) DST of 73 µg/cm2 (based on 331 sensitisers) and an HPC DST of 1.0 µg/cm2 (based on 146 sensitisers). Despite the dataset containing twice as many sensitisers, these values are similar to the previously published thresholds, highlighting their robustness and increasing confidence in their use. By classifying reactivity in silico the updated DSTs can be applied within a skin sensitisation risk assessment in a reproducible, scalable and accessible manner.

SpheraCosmolife: a new tool for the risk assessment of cosmetic products.

A new, freely available software for cosmetic products has been designed that considers the regulatory framework for cosmetics. The software allows an overall toxicological evaluation of cosmetic ingredients without the need for additional testing and, depending on the product type, it applies defined exposure scenarios to derive risk for consumers. It takes regulatory thresholds into account and uses either experimental values, if available, or predictions. Based on the exper­imental or predicted no observed adverse effect level (NOAEL), the software can define a point of departure (POD), which is used to calculate the margin of safety (MoS) of the query chemicals. The software also provides other toxico­logical properties, such as mutagenicity, skin sensitization, and the threshold of toxicological concern (TTC) to provide an overall evaluation of the potential chemical hazard. Predictions are calculated using in silico models implemented within the VEGA software. The full list of ingredients of a cosmetic product can be processed at the same time, at the effective concentrations in the product as given by the user. SpheraCosmolife is designed as a support tool for safety assessors of cosmetic products and can be used to prioritize the cosmetic ingredients or formulations according to their potential risk to consumers. The major novelty of the tool is that it wraps a series of models (some of them new) into a single, user-friendly software system.

The Threshold of Toxicological Concern (TTC) is a pragmatic tool for the safety assessment: Case studies of cosmetic ingredients with low consumer exposure.

The Threshold of Toxicological Concern (TTC) is an internationally accepted pragmatic and conservative tool for the safety assessment of substances, which is used in a wide range of regulatory contexts. The TTC approach produces human exposure threshold values (TTC values) originally derived by Munro from oral toxicity data on cancer and non-cancer toxicity endpoints. This database has been recently substantially enlarged by the COSMOS database, an enhanced oral non-cancer TTC dataset on a larger chemical domain, thereby resulting in a new, transparent and public TTC database also including 552 cosmetics-related chemicals. The 5th percentile point of departure value for each Cramer Class was determined, from which human exposure TTC values have been derived. The combined COSMOS/Munro dataset provided TTC values of 46, 6.2 and 2.3 µg/kg bw/day for Cramer Classes I, II or III, respectively. In order to demonstrate the diverse scope and successful application of the TTC concept to cosmetic ingredients including hair dyes, fragrances and plant-derived ingredients, Cosmetics Europe has prepared several case studies. Overall, the TTC concept is not only useful to replace animal testing but can also successfully be applied to the safety evaluation of cosmetic ingredients in the marketed formulas with low human exposure.

[Study of No Observed Adverse Effect Level of Nickel and Its Preliminary Evaluation Biocompatibility].

The purpose of this study was to investigate the NOAEL of the nickel ion and provide with basic data for the biological evaluation of those medical devices containing nickel. Five groups SD rats were repeatedly exposed during 14 d respectively to nickel at first stage doses of 4.9, 3.7, 2.5 mg/(kg.d), and the second stage doses of 1.2, 0.25 mg/(kg.d) by the intravenous route. The results showed that the NOAEL of nickel ion is 0.25 mg/(kg.d) for SD rats, and the result was verified by subchronic systemic toxicity test of nickel alloy. The threshold of toxicological concern (TTC) of nickel is 150 µg/d (based on application of 100-fold uncertainty factor and a body weight of 60 kg)deduced by these data.

Derivation of New Threshold of Toxicological Concern Values for Exposure via Inhalation for Environmentally-Relevant Chemicals.

The requirements of amended Toxic Substances Control Act (TSCA) stipulates that the US Environmental Protection Agency (US EPA) evaluate existing chemicals and make risk based assessments. There are ~33,000 substances that are active in commerce on the TSCA public non-confidential inventory, many of which lack available toxicity and exposure information to inform risk-based decision making. One approach to facilitate the assessment of these substances being considered is the Threshold of Toxicological Concern (TTC). TTC values are intended to identify safe levels of exposure for data poor substances. TTC values derived based on non-cancer data notably by Munro et al. (1996) are well-established and are in routine use for food additive applications however far less attention has been focused on developing TTC values where inhalation is the route of exposure. Here, an effort was made to derive new inhalation TTC values using the EPA's Toxicity Values database, ToxValDB. A total of 4,703 substances captured in ToxValDB were assigned into their respective TTC categories using the Kroes module within the Toxtree software tool and custom profilers developed in Nelms et al. (2019) and Patlewicz et al. (2018). For the substances assigned into the 3 Cramer classes, the 5th percentiles were calculated from the empirical cumulative distributions of No observed (adverse) effect level (concentration) values. The 5th percentiles were converted to their respective TTC values and compared with published values reported by Escher et al. (2010) and Carthew et al. (2009). The TTC values derived from ToxValDB were orders of magnitude more conservative, further, Cramer classification was not found to be effective at discriminating potencies. Instead, use of aquatic toxicity modes of action such as Verhaar et al. (1992) were found to be effective at separating substances in terms of their potencies and new TTC thresholds were derived.

Utility of In Vitro Bioactivity as a Lower Bound Estimate of In Vivo Adverse Effect Levels and in Risk-Based Prioritization.

Use of high-throughput, in vitro bioactivity data in setting a point-of-departure (POD) has the potential to accelerate the pace of human health safety evaluation by informing screening-level assessments. The primary objective of this work was to compare PODs based on high-throughput predictions of bioactivity, exposure predictions, and traditional hazard information for 448 chemicals. PODs derived from new approach methodologies (NAMs) were obtained for this comparison using the 50th (PODNAM, 50) and the 95th (PODNAM, 95) percentile credible interval estimates for the steady-state plasma concentration used in in vitro to in vivo extrapolation of administered equivalent doses. Of the 448 substances, 89% had a PODNAM, 95 that was less than the traditional POD (PODtraditional) value. For the 48 substances for which PODtraditional < PODNAM, 95, the PODNAM and PODtraditional were typically within a factor of 10 of each other, and there was an enrichment of chemical structural features associated with organophosphate and carbamate insecticides. When PODtraditional < PODNAM, 95, it did not appear to result from an enrichment of PODtraditional based on a particular study type (eg, developmental, reproductive, and chronic studies). Bioactivity:exposure ratios, useful for identification of substances with potential priority, demonstrated that high-throughput exposure predictions were greater than the PODNAM, 95 for 11 substances. When compared with threshold of toxicological concern (TTC) values, the PODNAM, 95 was greater than the corresponding TTC value 90% of the time. This work demonstrates the feasibility, and continuing challenges, of using in vitro bioactivity as a protective estimate of POD in screening-level assessments via a case study.

Study of No Observed Adverse Effect Level of Nickel and Its Preliminary Evaluation Biocompatibility / 中国医疗器械杂志

The purpose of this study was to investigate the NOAEL of the nickel ion and provide with basic data for the biological evaluation of those medical devices containing nickel. Five groups SD rats were repeatedly exposed during 14 d respectively to nickel at first stage doses of 4.9, 3.7, 2.5 mg/(kg.d), and the second stage doses of 1.2, 0.25 mg/(kg.d) by the intravenous route. The results showed that the NOAEL of nickel ion is 0.25 mg/(kg.d) for SD rats, and the result was verified by subchronic systemic toxicity test of nickel alloy. The threshold of toxicological concern (TTC) of nickel is 150 μg/d (based on application of 100-fold uncertainty factor and a body weight of 60 kg)deduced by these data.

Finding synergies for the 3Rs - Repeated Dose Toxicity testing: Report from an EPAA Partners' Forum.

The European Partnership for Alternative Approaches to Animal Testing (EPAA) convened a Partners' Forum on repeated dose toxicity (RDT) testing to identify synergies between industrial sectors and stakeholders along with opportunities to progress these in existing research frameworks. Although RTD testing is not performed across all industrial sectors, the OECD accepted tests can provide a rich source of information and play a pivotal role for safety decisions relating to the use of chemicals. Currently there are no validated alternatives to repeated dose testing and a direct one-to-one replacement is not appropriate. However, there are many projects and initiatives at the international level which aim to implement various aspects of replacement, reduction and refinement (the 3Rs) in RDT testing. Improved definition of use, through better problem formulation, aligned to harmonisation of regulations is a key area, as is the more rapid implementation of alternatives into the legislative framework. Existing test designs can be optimised to reduce animal use and increase information content. Greater use of exposure-led decisions and improvements in dose selection will be beneficial. In addition, EPAA facilitates sharing of case studies demonstrating the use of Next Generation Risk Assessment applying various New Approach Methodologies to assess RDT.

[Application of the Threshold of Toxicological Concern (TTC) to Biological Evaluation of Medical Devices].

The threshold of toxicological concern (TTC), a risk estimation method based on compound structurally-related toxicity data, has been widely used by many countries and regions for the safety risk assessment of food packaging materials and additives etc. Toxicological risk estimation is of importance in the biological evaluation of medical devices. Application of the TTC approach to leachable from medical devices may reduce or replace some unnecessary biocompatibility tests, but consideration should be taken for contact duration and route differences, which could affect the applicability of TTC. We herein focused on analyzing the eligibility of TTC for its further application in biological evaluation of medical devices.

Toxicological assessment compilation of selected examples of raw materials for homeopathic medicinal products.

The considerable number of homeopathic medicinal products (HMP) on the German market and the staggering breadth of active substances of various origin along with the specific legal requirements of adequate safety principles posed the need to compile data on toxicologically evaluated raw materials. In line with the European regulatory framework, HMP applications must consider appropriate safety standards in analogy to conventional human medicinal products. This review presents an option for a systematic and scientifically substantiated approach for regulatory use. Furthermore, this paper provides a multitude of data for selected raw materials processed in HMP with up to now rather scarce knowledge and, thus, aims at filling data gaps on acceptable amounts per day (AAD). The inclusion of raw materials into the compilation was determined considering the frequencies of their occurrence in HMP in Germany along with the availability of appropriate safety assessments. This safety evaluation compilation represents a practical, fairly comprehensive and systematic set of more than 170 raw materials. It is designed to both effectively support regulatory decision making and to be recognized and exploited by applicants, stakeholders and the scientific community.

Application of the Threshold of Toxicological Concern (TTC) to Biological Evaluation of Medical Devices / 中国医疗器械杂志

The threshold of toxicological concern (TTC), a risk estimation method based on compound structurally-related toxicity data, has been widely used by many countries and regions for the safety risk assessment of food packaging materials and additives etc. Toxicological risk estimation is of importance in the biological evaluation of medical devices. Application of the TTC approach to leachable from medical devices may reduce or replace some unnecessary biocompatibility tests, but consideration should be taken for contact duration and route differences, which could affect the applicability of TTC. We herein focused on analyzing the eligibility of TTC for its further application in biological evaluation of medical devices.

Finding synergies for 3Rs - Toxicokinetics and read-across: Report from an EPAA partners' Forum.

The European Partnership for Alternative Approaches to Animal Testing (EPAA) convened a Partners' Forum Toxicokinetics and Read-Across to provide an overview on research activities to develop in vitro toxicokinetics methods and physiologically-based kinetic (PBK) models and to find synergies to enhance use of toxicokinetic data to strengthen read-across. Currently, lacking toxicokinetic data often prevent the application of read-across. Preferably, toxicokinetic data should be generated using in vitro and in silico tools and anchored towards human relevance. In certain sectors, PBK modelling is being used for risk assessment, but less so in others. Specific activities were identified to facilitate the use of in vitro and in silico toxicokinetic data to support read-across: The collation of available tools indicating the parameters and applicability domains covered; endpoint-specific guidance on toxicokinetics parameters required for read-across; case studies exemplifying how toxicokinetic data help support read-across. Activities to enhance the scientific robustness of read-across include the further user-friendly combination of read-across tools and formal guidance by the authorities specifying the minimum information requirements to justify read-across for a given toxicity endpoint. The EPAA was invited to continue dissemination activities and to explore possibilities to collate a contemporaneous list of open toxicokinetics tools that assist risk assessment.

Potential impurities in drug substances: Compound-specific toxicology limits for 20 synthetic reagents and by-products, and a class-specific toxicology limit for alkyl bromides.

This paper provides compound-specific toxicology limits for 20 widely used synthetic reagents and common by-products that are potential impurities in drug substances. In addition, a 15 µg/day class-specific limit was developed for monofunctional alkyl bromides, aligning this with the class-specific limit previously defined for monofunctional alkyl chlorides. Both the compound- and class-specific toxicology limits assume a lifetime chronic exposure for the general population (including sensitive subpopulations) by all routes of exposure for pharmaceuticals. Inhalation-specific toxicology limits were also derived for acrolein, formaldehyde, and methyl bromide because of their localized toxicity via that route. Mode of action was an important consideration for a compound-specific toxicology limit. Acceptable intake (AI) calculations for certain mutagenic carcinogens assumed a linear dose-response for tumor induction, and permissible daily exposure (PDE) determination assumed a non-linear dose-response. Several compounds evaluated have been previously incorrectly assumed to be mutagenic, or to be mutagenic carcinogens, but the evidence reported here for such compounds indicates a lack of mutagenicity, and a non-mutagenic mode of action for tumor induction. For non-mutagens with insufficient data to develop a toxicology limit, the ICH Q3A qualification thresholds are recommended. The compound- and class-specific toxicology limits described here may be adjusted for an individual drug substance based on treatment duration, dosing schedule, severity of the disease and therapeutic indication.