Uncertainty in the results from oral repeated dose toxicity tests: Impact on regulatory classifications.

The Lowest Observed (Adverse) Effect Level (LO(A)EL) values are point-of-departure (PoD) values that quantify repeat dose toxicity (RDT). Here, the uncertainty in the regulatory classification of these PoDs is investigated. In the application stage, the dose-response was approximated for a large set of series, giving an account of the possible presence of a hormesis zone. The minimal effect dose (MED) or dose was computed, and the ratio MED/LO(A)EL was used to represent the two components of the experimental uncertainty. The uncertainty estimations were calculated for any combination of gender and reported examination item. Subsequently, how this uncertainty affects the possible classifications was analyzed, and the percentage of the chemicals receiving ambiguous classification was determined. It was shown that more than 40% of the investigated chemicals cannot be classified unambiguously in the Globally Harmonized System (GHS) classification scheme and bear a potential for misclassification when a regulatory decision is based on a single LO(A)EL value. A table containing grey zones for different risk levels and a table with GHS classification distributions for various LO(A)EL values were prepared to facilitate the use of the RDT uncertainty in the practice.

Six-well plate-based colony-forming efficacy assay and Co-Culture application to assess toxicity of metal oxide nanoparticles.

The development of a universal, label-free, and reliable in vitro toxicity testing method for nanoparticles is urgent because most nanoparticles can interfere with toxicity assays. In this regard, the colony-forming efficacy (CFE) assay has been suggested as a suitable in vitro toxicity assay for testing nanoparticles without such interference. Recently, the Organisation for Economic Co-operation and Development (OECD) developed a 60 × 15 mm Petri dish-based CFE assay for testing nanoparticles in MDCK-1 cells. However, further investigations are needed, including testing with other cell types, at a smaller scale for greater efficiency, and the application of the co-culture technique. In this study, we selected TiO2, CuO, CeO2, and SiO2 as test nanoparticles and successfully developed a 6-well plate-based CFE assay using HepG2 and A549 cells and a co-culture assay for combinations of HepG2 cells and THP-1 macrophages or A549 cells and THP-1 monocytes. The results suggest that the 6-wellplate-based CFE assay for HepG2 and A549 cells can be applied to nanoparticles, but the co-culture CFE assay has limitations in that it is not different from the single culture study, and it inhibits colony-formation by A549 cells in the presence of macrophages; this warrant further study.

Integrating toxicokinetics into toxicology studies and the human health risk assessment process for chemicals: Reduced uncertainty, better health protection.

The database of practical examples where toxicokinetic (TK) data has benefitted all stages of the human health risk assessment process are increasingly being published and accepted. This review aimed to highlight and summarise notable examples and to describe the "state of the art" in this field. The overall recommendation is that for any in vivo animal study conducted, measurements of TK should be very carefully considered for inclusion as the numerous benefits this brings continues to grow, particularly during the current march towards animal free toxicology testing and ambitions to eventually conduct human health risk assessments entirely based upon non-animal methods.

Opportunities and challenges related to saturation of toxicokinetic processes: Implications for risk assessment.

Top dose selection for repeated dose animal studies has generally focused on identification of apical endpoints, use of the limit dose, or determination of a maximum tolerated dose (MTD). The intent is to optimize the ability of toxicity tests performed in a small number of animals to detect effects for hazard identification. An alternative approach, the kinetically derived maximum dose (KMD), has been proposed as a mechanism to integrate toxicokinetic (TK) data into the dose selection process. The approach refers to the dose above which the systemic exposures depart from being proportional to external doses. This non-linear external-internal dose relationship arises from saturation or limitation of TK process(es), such as absorption or metabolism. The importance of TK information is widely acknowledged when assessing human health risks arising from exposures to environmental chemicals, as TK determines the amount of chemical at potential sites of toxicological responses. However, there have been differing opinions and interpretations within the scientific and regulatory communities related to the validity and application of the KMD concept. A multi-stakeholder working group, led by the Health and Environmental Sciences Institute (HESI), was formed to provide an opportunity for impacted stakeholders to address commonly raised scientific and technical issues related to this topic and, more specifically, a weight of evidence approach is recommended to inform design and dose selection for repeated dose animal studies. Commonly raised challenges related to the use of TK data for dose selection are discussed, recommendations are provided, and illustrative case examples are provided to address these challenges or refute misconceptions.

Regulation of Sun Protection Products in the EU.

Unlike more "traditional" cosmetic products, sunscreens do not sit inertly on the skin, providing a simple decorative effect. Their recognized and important contribution to public health has led many regions in the world to treat them as drugs or special cosmetics. Against the trend at that time, in 1976, the EU legislator already took a conscious decision to treat and regulate sunscreens as fast-moving consumer products. Since then, the EU Cosmetics Directive/Regulation balances the need for strict safety and efficacy requirements, with need for rapid innovation and easy consumer availability. Whilst the EU Regulation considers that "all cosmetic products are equal," sunscreens are clearly "more equal." In several areas of the legislation, specific requirements or guidance for sunscreen products have been introduced over the years. Whilst staying in the overall spirit of the legislation, these requirements take into account the specificity of sunscreens with regard to ingredient safety (positive list for UV filters), product safety assessment (photostability, deliberate exposure to UV light), minimum efficacy (UVA/UVB), efficacy testing (standardized test methods) and labelling (clear use instructions, non-misleading information to consumers). The article presents the history of the EU Cosmetics Regulation, its main requirements, where applicable, and specific considerations relating to sunscreens are highlighted and explained.

Minimum reporting standards based on a comprehensive review of the zebrafish embryo teratogenicity assay.

Reproductive toxicity chemical safety assessment involves extensive use of vertebrate animals for regulatory testing purposes. Although alternative methods such as the zebrafish embryo teratogenicity assay (identified in the present manuscript by the acronym ZETA) are promising for replacing tests with mammals, challenges to regulatory application involve lack of standardization and incomplete validation. To identify key protocol aspects and ultimately support improving this situation, a comprehensive review of the literature on the level of harmonization/standardization and validation status of the ZETA has been conducted. The gaps and needed advances of the available ZETA protocols were evaluated and discussed with respect to its applicability as an alternative approach for teratogenicity assessment. Based on the review outcomes, a set of minimum reporting standards for the experimental protocol is proposed. Together with other initiatives towards implementation of alternative approaches at the screening and regulatory levels, the application of minimum reporting requirements is anticipated to support future method standardization and validation, as well as identifying potential improvement aspects. Present findings are expected to ultimately support advancing the ongoing validation initiatives towards the regulatory acceptance of the ZETA.

A Review of the Main Considerations for Formulation Development in Preclinical Toxicology Studies.

The main considerations for the development of a formulation for preclinical safety assessment testing are explored. Intravenous, inhalation, oral and dermal dosing are given focus and although different dose routes do present their own individual challenges there are common themes that emerge. In each case it is necessary to maximise exposure to achieve high doses to satisfy regulatory requirements for safety assessment testing. This often involves producing formulations that are at the limits of solubility and maximum volumes possible for administration to different test species by the chosen route. It is concluded that for all routes it is important to thoroughly explore the stability of the test item in the proposed formulation matrix well ahead of dosing any animals, giving careful consideration to which excipients are used and what their underlying toxicity profile may be for the relevant preclinical species. In addition, determining the maximum achievable concentrations and weighing that against the maximum volumes that can be given by the chosen route in all the test species at an early stage will also give a read on whether it would be theoretically possible to achieve suitably high enough doses to support clinical work. Not doing so can cause delays in the development programme and may have ethical repercussions.

Current Trends of Practices in Nonclinical Toxicology: An Industry Survey.

The growth in drug development over the past years reflects significant advancements in basic sciences and a greater understanding of molecular pathways of disease. Benchmarking industry practices has been important to enable a critical reflection on the path to evolve pharmaceutical testing, and the outcome of past industry surveys has had some impact on best practices in testing. A survey was provided to members of SPS, ACT, and STP. The survey consisted of 37 questions and was provided to 2550 participants with a response rate of 24%. Most respondents (∼75%) came from the US and Europe. The survey encompassed multiple topics encountered in nonclinical testing of pharmaceuticals. The most frequent target indications were oncology (69%), inflammation (55%), neurology/psychiatry/pain (46%), cardiovascular (44%), and metabolic diseases (39%). The most frequent drug-induced toxicology issues confronted were hepatic, hematopoietic, and gastrointestinal. Toxicological effects that impacted the no observed adverse effect level (NOAEL) were most frequently based on histopathology findings. The survey comprised topics encountered in the use of biomarkers in nonclinical safety assessment, most commonly those used to assess inflammation, cardiac/vascular, renal, and hepatic toxicity as well as common practices related to the assessment of endocrine effects, carcinogenicity, genotoxicity, juvenile and male-mediated developmental and female reproductive toxicity. The survey explored the impact of regulatory meetings on program design, application of the 3 Rs, and reasons for program delays. Overall, the survey results provide a broad perspective of current practices based on the experience of the scientific community engaged in nonclinical safety assessment.

Graphene-Based Materials In Vitro Toxicity and Their Structure-Activity Relationships: A Systematic Literature Review.

The unique properties of graphene-based materials (GBMs) placed them among the most exciting nanomaterials of the past decade. Scientists and industry are looking forward to working with not only efficient but also safe, sustainable GBMs. Designing a safer-by-design GBM implies to acquire the knowledge of which physicochemical characteristics (PCCs) can increase toxicity. In this systematic review, we extracted data from the literature to provide the available information about the structure-activity relationship of GBMs. 93 papers studying a total of 185 GBMs are included. Graphene oxides (GOs) and few-layer graphenes (FLGs) are the most studied GBMs. While reduced graphene oxides were often classified as poorly oxidant and weakly cytotoxic, graphene quantum dots were mostly moderately or highly cytotoxic. FLGs demonstrated relationships between median size and oxidative stress, between lateral size and both cytotoxicity and oxidative stress, and between thickness and cytotoxicity. We also underline relationships between median size, lateral size, and thickness of GOs and oxidative stress. However, it appears difficult to highlight clear structure-activity relationships for most PCCs and biological end points because despite a large amount of available data, the GBMs are often too poorly characterized in terms of PCCs descriptors and the biological end points investigation is not standardized enough. There is an urgent need for a better standardization of the experimental investigation of both PCCs and biological end points to allow research teams to play a part in the collaborative work toward the construction of a safer-by-design GBM through a better understanding of their key toxicity drivers.

The safety of a Kluyveromyces lactis strain lineage for enzyme production.

Kluyveromyces lactis is broadly considered as a safe yeast in food and a suitable organism for the production of food enzymes. The K. lactis enzyme production strains of DSM are used to produce a variety of enzymes, for example beta-galactosidase (lactase), chymosin and esterase. All of these production strains are derived from the same lineage, meaning they all originate from the same ancestor strain after classical mutagenesis and/or genetic engineering. Four different enzyme preparations produced with strains within this lineage were toxicologically tested. These enzyme preparations were nontoxic in repeated-dose oral toxicity studies performed in rats and were non-genotoxic in vitro. These studies confirm the safety of the DSM K. lactis strains as a production platform for food enzymes, as well as the safety of the genetic modifications made to these strains through genetic engineering or classical mutagenesis. The outcome of the toxicity studies can be extended to other enzyme preparations produced by any strain from this lineage through read across. Therefore, no new toxicity studies are required for the safety evaluation, as long as the modifications made do not raise safety concerns. Consequently, this approach is in line with the public ambition to reduce animal toxicity studies.

Safety evaluation of food enzymes produced by a safe strain lineage of Bacillussubtilis.

The safety of microbially-derived food enzymes must be carefully assessed before market introduction. The production strain's safety is central to the assessment. In this paper, we have determined that DSM's Bacillus subtilis strain lineage can be considered safe for food enzyme production. The mutations introduced into this non-pathogenic and non-toxigenic microorganism do not lead to any safety concerns, as ensured by a thorough characterization of the strain lineage. The safety of both targeted and randomly introduced changes into the production strain's genome is confirmed by validating the absence of vector sequences and antibiotic resistance genes in all relevant production strains, and by demonstrating absence of cytotoxic peptide production. Furthermore, three food enzyme preparations produced by strains within this lineage did not show genotoxic potential. 90-day oral toxicity studies performed with the same enzyme preparations did not reveal toxicologically significant adverse effects. These results demonstrate absence of safety concerns from the introduced genetic modifications. Based on the establishment of this safe strain lineage, we postulate that future enzymes produced by current and new strains derived from the lineage can be safely developed without additional genotoxicity and systemic toxicity studies, allowing for a reduction of animal testing without compromising on product safety.

Species selection for nonclinical safety assessment of drug candidates: Examples of current industry practice.

In drug development, nonclinical safety assessment is pivotal for human risk assessment and support of clinical development. Selecting the relevant/appropriate animal species for toxicity testing increases the likelihood of detecting potential effects in humans, and although recent regulatory guidelines state the need to justify or dis-qualify animal species for toxicity testing, individual companies have developed decision-processes most appropriate for their molecules, experience and 3Rs policies. These generally revolve around similarity of metabolic profiles between toxicology species/humans and relevant pharmacological activity in at least one species for New Chemical Entities (NCEs), whilst for large molecules (biologics) the key aspect is similarity/presence of the intended human target epitope. To explore current industry practice, a questionnaire was developed to capture relevant information around process, documentation and tools/factors used for species selection. Collated results from 14 companies (Contract Research Organisations and pharmaceutical companies) are presented, along with some case-examples or over-riding principles from individual companies. As the process and justification of species selection is expected to be a topic for continued emphasis, this information could be adapted towards a harmonized approach or best practice for industry consideration.

Calculating qualified non-mutagenic impurity levels: Harmonization of approaches.

The presence of impurities in drugs is unavoidable. As impurities offer no direct benefit to the patient, it is critical that impurities do not compromise patient safety. Current guidelines on the derivation of acceptable impurity levels leave aspects of calculations open for interpretation, resulting in inconsistencies across industry and regulators. To understand current impurity qualification practices from a safety standpoint, regulatory expectations and the safety risk that impurities pose, the IQ DruSafe Impurities Working Group (WG) conducted a pharmaceutical industry-wide survey. Survey results highlighted areas that could benefit from harmonization, including nonclinical species/sex selection and the application of adjustment factors (i.e., body surface area). Recommendations for alignment on these topics is included in this publication. Additionally, the WG collated repeat-dose toxicity information for 181 starting materials and intermediates, reflective of pharmaceutical impurities, to understand the toxicological risks they generally pose in relation to the drug substance (DS) and the assumptions surrounding the calculation of qualified impurity levels. An evaluation of this dataset and the survey were used to harmonize how to calculate a safe limit for an impurity based on toxicology testing of the impurity when present within the DS.

Perspectives on microalgae as model organisms toward the standardization of soil algal toxicity test methods.

When considering test species for soil ecotoxicity, the development of new model organisms is often suggested to increase the reliability of ecological risk assessments. Ubiquitous soil algae could offer potential test species for assessing various soil pollution levels. Currently, there are few reviews offering comprehensive perspectives on stressors-based toxicological studies using microalgae in soil media, with the majority of scholarly attention paid to the toxicological effects of freshwater algae or marine algae in aquatic ecosystems. In this review, we focus on current toxicological studies of microalgae assessed in soil-related media and suggest considerations for using microalgae in soil toxicity tests based on 22 publications (1998-2021). In addition, we analyzed characteristics of soil algae based on criteria for selecting test species and suggest that future research should be directed toward the standardization of soil algal toxicity test methods. This review discusses a promising method using soil algae as new test species for soil toxicity assessment as cost-effective and environmentally sound soil quality bioindicators. The review also addresses the lack of understanding behind how soil algae can serve as important test species for soil ecotoxicity.

[Establishment and International Harmonization of Photosafety Testing Strategy].

Considerable attention has been drawn to predict a photosafety hazard on new chemicals. A number of phototoxins tend to generate reactive oxygen species (ROS) via energy transfer mechanisms following UV/VIS excitation, including superoxide and singlet oxygen. Then, ROS assay has been designed to assess photoreactivity of pharmaceuticals, of which the principle is to monitor types I and II photochemical reactions of the test chemicals when exposed to simulated sunlight. This simple analytical test could be used to screen potential chemical scaffolds, leads, and candidate drugs to identify and/or select away from those having phototoxic potential. The validation study for the ROS assay has been being carried out by the Japan Pharmaceutical Manufacturers Association (JPMA), supervised by the Japanese Center for the Validation of Alternative Methods (JaCVAM). Although several false positives appeared, the ROS assay on 42 coded chemicals has provided no false negative predictions. The validation study tentatively indicates satisfactory outcomes in terms of transferability, intra- and inter-laboratory variability, and predictive capacity. Thus, a negative result in this ROS assay would indicate a very low probability of phototoxicity, whereas a positive result would be a flag for follow-up assessment. Upon international harmonization activities supported by several agencies and industrial groups, ROS assay was successfully adopted as International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) S10 guideline (2014) and Organisation for Economic Co-operation and Development (OECD) test guideline 495 (2019).

Refinement of decision tree to assess the consequences of increased serum ALP in dogs: Additional analysis on toxicity studies of pesticides evaluated recently in Japan.

Recently we provided a new interpretation that increased serum ALP in dogs is not adverse if no hepatotoxic finding coexists in the analysis of toxicity studies of over 200 pesticides evaluated in Japan (Yokoyama et al., 2019). We also proposed a decision tree to evaluate the adversity of the increased ALP. The present analysis was conducted to validate the reliability of this interpretation with 129 pesticides more recently evaluated. Before applying, the decision tree was revised to be consistent in all steps. The pesticides showed similar characterization of increased ALP to the previous analysis in that the increase was more frequent than in rats and that liver hypertrophy and hepatotoxicity commonly coexisted with an increase in ALP in dogs. When short- and long-term studies of 58 pesticides inducing ALP activity in dogs were applied to the revised tree, the increased ALP in 8 pesticides was judged not adverse in either study. The revision of the tree did not affect the NOAEL judgment of these pesticides; however, the revised routes contributed to the judgment more robustly. This study showed the reliability of our interpretation and applicability of the decision tree to evaluate the adversity of increased ALP in dogs.

Beyond dermal exposure: The respiratory tract as a target organ in hazard assessments of cosmetic ingredients.

Dermal contact is the main route of exposure for most cosmetics; however, inhalation exposure could be significant for some formulations (e.g., aerosols, powders). Current cosmetic regulations do not require specific tests addressing respiratory irritation and sensitisation, and despite the prohibition of animal testing for cosmetics, no alternative methods have been validated to assess these endpoints to date. Inhalation hazard is mainly determined based on existing human and animal evidence, read-across, and extrapolation of data from different target organs or tissues, such as the skin. However, because of mechanistic differences, effects on the skin cannot predict effects on the respiratory tract, which indicates a substantial need for the development of new approach methodologies addressing respiratory endpoints for inhalable chemicals in general. Cosmetics might present a particularly significant need for risk assessments of inhalation exposure to provide a more accurate toxicological evaluation and ensure consumer safety. This review describes the differences in the mechanisms of irritation and sensitisation between the skin and the respiratory tract, the progress that has already been made, and what still needs to be done to fill the gap in the inhalation risk assessment of cosmetic ingredients.

Case analysis of kinetics investigations in toxicity studies of pesticides to identify the nonlinearity of internal exposure and the influences of nonlinearity on the toxicological interpretation of pesticide residue.

The nonlinearity of internal exposure to 8 pesticides was investigated in toxicity studies using kinetics to identify nonlinearity visually and to investigate the influence of nonlinearity on toxicological evaluation. Data were obtained from risk assessment reports published by the Food Safety Commission (FSCJ). Nonlinearity was defined using 2 indicators: the lowest visual inflection point (LVIP) and the second lowest visual inflection point (SVIP) of kinetics by drawing a linear distribution chart. The area under the curve and 24-h urine concentrations were stable parameters used to identify the LVIP/SVIP. The sampling timing affected the blood concentrations, and the LVIP/SVIP was detected for 6 pesticides using the parent compounds or their metabolites as analytes. The subproportional nonlinearity was significant for these pesticides. The LVIP/SVIP values were consistent in the same species up to a 1-year period, but the values showed species-specific differences in several compounds. In all compounds found to be nonlinear, apical outcomes were observed at the SVIP or above. The presence of nonlinearity was recognized by the FSCJ. The recognition influenced their judgment of no-observed-adverse-effect levels (NOAELs) for carcinogenicity or health-based guidance values, indicating the importance of appropriate kinetics to identify the nonlinearity for toxicological evaluation of pesticide residue.

Quality assurance for nanomaterial inhalation toxicity testing.

The recent revision of OECD inhalation toxicology test guidelines 412 and 413 presents new challenges for both the study director (SD) and quality assurance (QA) personnel when conducting GLP (good laboratory practice) studies. In the case of nanomaterial inhalation exposure studies, GLP has rarely been applied, yet the new revisions are applicable to soluble and insoluble nanomaterials, as well as conventional chemicals. For example, the new guidelines require an additional bronchoalveolar lavage (BAL) fluid assay and lung burden measurement during the post-exposure observation (PEO) period, plus nanomaterial physicochemical characterization before and after nano-aerosol generation when exposing experimental animals. Implementing these revised guidelines will prove especially challenging for QA measures related to the physicochemical characterization and aerosolization of test nanomaterials. Therefore, this review examines the key elements involved in nanomaterial inhalation GLP testing under the revised OECD guidelines, suggests an alternative to the increased animal numbers, in consideration of animal welfare and with scientific merits, and discusses the limitation of toxicokinetic estimation using the new testing guidelines.

Enhancing between-facility reproducibility of the SH test as an in vitro skin sensitization test by the improved test method.

There has been an increased demand to eliminate animal experiments and to replace the experiments with alternative tests for assessing the safety of cosmetics. The SH test is an in vitro skin sensitization test that evaluates the protein binding abilities of a test substance. Skin sensitization must be evaluated by multiple test methods. The SH test uses the same cell line and measuring instruments as the human Cell-Line Activation Test (h-CLAT), which is one of the test methods used to evaluate different key events and is listed in the OECD test guidelines. There are cost advantages to usher the SH test into facilities that are already running the h-CLAT. The SH test is conducted only at a facility that has developed the SH test because studies on the between-facility reproducibility and validity have not been performed. Therefore, to verify the transferability of the SH test and the between-facilities reproducibility, we evaluated the reproducibility of the SH test results at three facilities, including the development facility. After an initial round of testing, the protocol was refined as follows to improve reproducibility among the three facilities: i) determine the optimum pH range, ii) change the maximum applicable concentration of water-soluble substances, and iii) define the appropriate dispersion conditions for evaluating hydrophobic substances. These refinements markedly enhanced the between-facility reproducibility (from 76.0% to 96.0%) for the 25 substances evaluated in this study. This study confirmed that the SH test is an effective skin sensitization test method with high technical transferability and between-facility reproducibility.