Incorporating lines of evidence from New Approach Methodologies (NAMs) to reduce uncertainties in a category based read-across: A case study for repeated dose toxicity.

A group of triazole compounds was selected to investigate the confidence that may be associated with read-across of a complex data gap: repeated dose toxicity. The read-across was evaluated using Assessment Elements (AEs) from the European Chemicals Agency's (ECHA's) Read-Across Assessment Framework (RAAF), alongside appraisal of associated uncertainties. Following an initial read-across based on chemical structure and properties, uncertainties were reduced by the integration of data streams such as those from New Approach Methodologies (NAM) and other existing data. In addition, addressing the findings of the ECHA RAAF framework, complemented with specific questions concerning uncertainties, increased the confidence that can be placed in read-across. Although a data rich group of compounds with a strong mechanistic basis was analysed, it was clearly demonstrated that NAM data available from publicly available resources could be applied to support read-across. It is acknowledged that most read-across studies will not be so data rich or mechanistically robust, therefore some targeted experimentation may be required to fill the data gaps. In this sense, NAMs should constitute new experimental tests performed with the specific goal of reducing the uncertainties and demonstrating the read-across hypothesis.

Evaluating potential refinements to existing Threshold of Toxicological Concern (TTC) values for environmentally-relevant compounds.

The Toxic Substances Control Act (TSCA) mandates the US EPA perform risk-based prioritisation of chemicals in commerce and then, for high-priority substances, develop risk evaluations that integrate toxicity data with exposure information. One approach being considered for data poor chemicals is the Threshold of Toxicological Concern (TTC). Here, TTC values derived using oral (sub)chronic No Observable (Adverse) Effect Level (NO(A)EL) data from the EPA's Toxicity Values database (ToxValDB) were compared with published TTC values from Munro et al. (1996). A total of 4554 chemicals with structures present in ToxValDB were assigned into their respective TTC categories using the Toxtree software tool, of which toxicity data was available for 1304 substances. The TTC values derived from ToxValDB were similar, but not identical to the Munro TTC values: Cramer I ((ToxValDB) 37.3 c. f. (Munro) 30 µg/kg-day), Cramer II (34.6 c. f. 9.1 µg/kg-day) and Cramer III (3.9 c. f. 1.5 µg/kg-day). Cramer III 5th percentile values were found to be statistically different. Chemical features of the two Cramer III datasets were evaluated to account for the differences. TTC values derived from this expanded dataset substantiated the original TTC values, reaffirming the utility of TTC as a promising tool in a risk-based prioritisation approach.

Pulmonary toxicity in rats following inhalation exposure to poorly soluble particles: The issue of impaired clearance and the relevance for human health hazard and risk assessment.

Intensive discussions are ongoing about the interpretation of pulmonary effects observed in rats exposed to poorly soluble particles. Alveolar clearance differs between rats and humans and becomes impaired in rats at higher exposure concentrations. Some have doubted the human relevance of toxic effects observed in rats under impaired clearance conditions and have suggested that experimental exposures should stay below concentrations inducing impaired clearance. However, for regulatory purposes, insight in potential health effects at relatively high concentrations is needed to fully understand the hazard. Many aspects of impaired particle clearance remain unclear, hampering human health hazard and risk assessment. For an adequate evaluation of the impact of impaired clearance on pulmonary toxicity, a clear definition of alveolar clearance is needed that enables to quantitatively relate the level of impairment to the induction of adverse pulmonary health effects. Also, information is needed on the mechanism of action and the appropriate dose metric for the pulmonary effects observed. In absence of these data, human hazard and risk assessment can only be performed in a pragmatic way. Unless available data clearly point out otherwise, rat pulmonary toxicity including lung inflammation and tumour formation, needs to be considered relevant for human hazard and risk assessment.

Acute and subchronic oral toxicity studies in rats with nanoscale and pigment grade titanium dioxide particles.

Data generated using standardized testing protocols for toxicity studies generally provide reproducible and reliable results for establishing safe levels and formulating risk assessments. The findings of three OECD guideline-type oral toxicity studies of different duration in rats are summarized in this publication; each study evaluated different titanium dioxide (TiO2) particles of varying sizes and surface coatings. Moreover, each study finding demonstrated an absence of any TiO2 -related hazards. To briefly summarize the findings: 1) In a subchronic 90-day study (OECD TG 408), groups of young adult male and female rats were dosed with rutile-type, surface-coated pigment-grade TiO2 test particles (d50 = 145 nm - 21% nanoparticles by particle number criteria) by oral gavage for 90 days. The no-adverse-effect level (NOAEL) for both male and female rats in this study was 1000 mg/kg bw/day, the highest dose tested. The NOAEL was determined based on a lack of TiO2 particle-related adverse effects on any in-life, clinical pathology, or anatomic/microscopic pathology parameters; 2) In a 28-day repeated-dose oral toxicity study (OECD TG 407), groups of young adult male rats were administered daily doses of two rutile-type, uncoated, pigment-grade TiO2 test particles (d50 = 173 nm by number) by daily oral gavage at a dose of 24,000 mg/kg bw/day. There were no adverse effects measured during or following the end of the exposure period; and the NOAEL was determined to be 24,000 mg/kg bw/day; 3) In an acute oral toxicity study (OECD TG 425), female rats were administered a single oral exposure of surface-treated rutile/anatase nanoscale TiO2 particles (d50 = 73 nm by number) with doses up to 5000 mg/kg and evaluated over a 14-day post-exposure period. Under the conditions of this study, the oral LD50 for the test substance was >5000 mg/kg bw. In summary, the results from these three toxicity studies - each with different TiO2 particulate-types, demonstrated an absence of adverse toxicological effects. Apart from reporting the findings of these three studies, this publication also focuses on additional critical issues associated with particle and nanotoxicology studies. First, describing the detailed methodology requirements and rigor upon which the standardized OECD 408 guideline subchronic oral toxicity studies are conducted. Moreover, an attempt is made to reconcile the complex issue of particle size distribution as it relates to measurements of nanoscale and pigment-grade TiO2 particles. Clearly this has been a confusing issue and often misrepresented in the media and the scientific literature. It is clear that the particle-size distribution for pigment-grade TiO2, contains a small ("tail") component of nanoscale particles (i.e., 21% by particle number and <1% by weight in the test material used in the 90-day study). However, this robust particle characterization finding should not be confused with mislabeling the test materials as exclusively in the nanoscale range. Moreover, based upon the findings presented herein, there appears to be no significant oral toxicity impact contributed by the nanoscale component of the TiO2 Test Material sample in the 90-day study. Finally, it seems reasonable to conclude that the study findings should be considered for read-across purposes to food-grade TiO2 particles (e.g., E171), as the physicochemical characteristics are quite similar.

Evaluación de la seguridad de una arcilla modificada y su extracto de migración en bazo de ratas Wistar expuestas de forma subcrónica / Influence of a subchronical exposure to a modified clay and its migration extract in the spleen of Wistar rats

Las ventajas tecnológicas de la incorporación de arcillas modificadas en polímeros para el envasado de alimentos son bien conocidas, pero aún quedan muchas incertidumbres sobre la seguridad de estos materiales. El Instituto Tecnológico del Embalaje, Transporte y Logística ha desarrollado una arcilla, Clay1, modificando una montmorillonita con una sal de amonio cuaternario. Esta organoarcilla, incorporada al polímero (ácido poliláctico), da lugar a un material nanocompuesto, reforzándose así el material de partida. El principal objetivo de este estudio es evaluar la actividad de biomarcadores de estrés oxidativo en bazo de ratas expuestas durante 90 días a Clay1 (40 mg/kg/día) y al extracto de migración obtenido a partir del material nanocompuesto resultante. Los parámetros evaluados fueron la peroxidación lipídica y las actividades enzimáticas superóxido dismutasa y catalasa. Además, se realizó un análisis del contenido en bazo de los metales más característicos que componen la organoarcilla (Al, Ca, Fe, Mg, Si) para comprobar su posible acumulación. En dicho estudio se trabajó con tres grupos de ratas Wistar (n=10): control (comida estándar + agua como bebida), Clay1 (comida estándar mezclada con 40mg/kg/día de arcilla + agua) y extracto de Clay1 (comida estándar + extracto como bebida). Tras el tiempo de exposición los animales se sacrificaron y se extrajo el bazo. De forma general, no se observaron diferencias significativas en ninguno de los parámetros evaluados con respecto al grupo control, por lo que Clay1 muestra un buen perfil toxicológico respecto a los biomarcadores ensayados con vistas a su uso en la industria alimentaria (AU)

The technological advantages of the incorporation of modified clays into polymers for food packaging are well known. However, there are still many uncertainties about the safety of these materials. The Technological Institute of Packaging, Transport and Logistic has developed Clay1, a modified clay with a quaternary ammonium salt. This organoclay is incorporated into the polymer (polylactic acid), giving a nanocomposite material and reinforcing the bulk material. The aim of this study is to evaluate the activity of several oxidative stress biomarkers in the spleen of rats exposed for 90 days to Clay1 (40 mg/kg/day) and its migration extract obtained from the resultant nanocomposite material. The parameters evaluated were lipid peroxidation and superoxide dismutase and catalase activities. Moreover, the characteristic metallic components of the organoclay (Al, Ca, Fe, Mg, Si) were also analyzed to test the possible accumulation. In this study, three groups of Wistar rats (n=10) were used: control (standard food + water), Clay1 (food with Clay1+water) and Clay1 extract (standard food+ Clay1 extract as water). After the exposure the spleen was removed. In general, no significant differences were observed in any of the parameters evaluated compared to the control group, therefore Clay1 showed a good toxicologic profile regarding the biomarkers assayed for its use in the food industry (AU)

Calcium lignosulphonate: re-evaluation of relevant endpoints to re-confirm validity and NOAEL of a 90-day feeding study in rats.

A 90-day feeding study in Han/Wistar rats with calcium lignosulphonate was evaluated by the EFSA. The study was considered to be inadequate due to potentially impaired health status of the animals based upon a high incidence of minimal lymphoid hyperplasia in mesenteric/mandibular lymph nodes and Peyer's patches, and minimal lymphoid cell infiltration in the liver in all animals. The EFSA Panel further disagreed with the conclusion that the treatment-related observation of foamy histiocytosis in mesenteric lymph nodes was non-adverse and asked whether this observation would progress to something more adverse over time. A PWG was convened to assess the sections of lymph nodes, Peyer's patches and liver. In addition, all lymphoid tissues were re-examined. The clinical pathology and animal colony health screening data were re-evaluated. The question whether the foamy histiocytosis could progress to an adverse finding with increasing exposure duration was addressed by read-across. In conclusion, the animals on the 90-day feeding study were in good health, the study was adequate for safety evaluation, and the foamy histiocytes in the mesenteric lymph nodes were not considered adverse, but rather an adaptive response that was considered unlikely to progress to an adverse condition with time. The NOAEL was re-affirmed to be 2000 mg/kgbw/d.

Assessment of menstrual cycle length in cynomolgus monkeys as a female fertility endpoint of a biopharmaceutical in a 6 month toxicity study.

According to ICH S6(R1), mating studies are not practical for assessing effects on female fertility of biopharmaceuticals that are pharmacologically active only in non-human primates (NHPs). Instead, fertility should be assessed by evaluating histopathology and organ weights of the reproductive tract in studies of at least 3 months dosing duration using sexually mature NHPs. An assessment of the menstrual cycle in females can be included if there is cause for concern based on pharmacological mode of action or relevant findings in previous studies. However, many factors unrelated to the molecule under evaluation can impact cycle length and thus affect data interpretation. Assessment of a monoclonal antibody in a 6 month repeat dose toxicity study is used as an example in this manuscript to review potential sources of background variability, identify strategies to minimize its impact and recommend optimal ways to collect, present and analyze menstrual cycle data. Experimental variables include the amount of time required for menses to normalize following the transport of animals to the testing facility, stress-related effects on the cycle length due to socialization issues with new cagemates, and the normal background irregularity of cycle length in NHPs. Study related procedures (i.e., animal handling for dosing, blood draws, body weights, ECGs, etc.) did not affect cycle lengths in this study. We show that introducing a number of key experimental control procedures to minimize cycle variability can enable a robust assessment of the effects of a biotherapeutic on menstrual cycling within a chronic toxicity study in NHPs.

Standardized methods for acute and semichronic toxicity tests with the copepod Acartia tonsa.

The availability of standardized protocols for both organism culture and bioassay with ecologically relevant species is of great concern in ecotoxicology. Acartia tonsa represents an important, often dominant, member of zooplankton communities and meets all the practical criteria suggested for model species. New standardized procedures for laboratory culturing of the copepod A. tonsa and standardized methods for acute (24- and 48-h) and semichronic (7-d, static-renewal) toxicity tests with the nauplius stage are described. In both cases, eggs are the starting stage, and nauplius immobilization is the endpoint. The methods were the object of an intercomparison test involving nine laboratories, and nickel was the reference toxicant. Relative reproducibility was 24, 25, and 34% for 24-h, 48-h, and 7-d tests, respectively.