Review of Lung Particle Overload, Rat Lung Cancer, and the Conclusions of the Edinburgh Expert Panel-It's Time to Revisit Cancer Hazard Classifications for Titanium Dioxide and Carbon Black.

Chronic inhalation of titanium dioxide or carbon black by rats at concentrations which overload lung particle clearance can result in lung cancer. Based on this rat lung response, IARC, NIOSH, and ECHA classified titanium dioxide, and IARC classified carbon black, as potential human carcinogens. These classifications have been questioned based on an extensive data base demonstrating: the rat lung cancer occurred only under conditions of extreme lung particle overload; the lung cancer response in rats has not been seen in other animal species; and studies in titanium dioxide and carbon black exposed human populations have not shown an increased incidence of cancer. In 2019 an international panel of science and regulatory experts was convened to document the state of the science on lung particle overload and rat lung cancer after exposure to poorly soluble low toxicity particles. Regarding hazard identification, the expert panel concluded, in the absence of supporting data from other species, lung particle overload-associated rat lung cancer does not imply a cancer hazard for humans. Regarding high to low dose extrapolation, the expert panel concluded rat lung tumors occurring only under conditions of lung particle overload are not relevant to humans exposed under non-overloading conditions. The conclusions of the Edinburgh Expert Panel directly conflict with IARC, ECHA and NIOSH's extrapolation of lung particle overload associated rat lung cancer to hazard for humans. The hazard classifications for titanium dioxide and carbon black inhalation should be assessed considering the state-of-the-science on lung particle overload and rat lung cancer.

Comparative pulmonary toxicities of lunar dusts and terrestrial dusts (TiO2 & SiO2) in rats and an assessment of the impact of particle-generated oxidants on the dusts' toxicities.

Humans will set foot on the Moon again soon. The lunar dust (LD) is potentially reactive and could pose an inhalation hazard to lunar explorers. We elucidated LD toxicity and investigated the toxicological impact of particle surface reactivity (SR) using three LDs, quartz, and TiO2. We first isolated the respirable-size-fraction of an Apollo-14 regolith and ground two coarser samples to produce fine LDs with increased SR. SR measurements of these five respirable-sized dusts, determined by their in-vitro ability to generate hydroxyl radicals (•OH), showed that ground LDs > unground LD ≥ TiO2 ≥ quartz. Rats were each intratracheally instilled with 0, 1, 2.5, or 7.5 mg of a test dust. Toxicity biomarkers and histopathology were assessed up to 13 weeks after the bolus instillation. All dusts caused dose-dependent-increases in pulmonary lesions and toxicity biomarkers. The three LDs, which possessed mineral compositions/properties similar to Arizona volcanic ash, were moderately toxic. Despite a 14-fold •OH difference among these three LDs, their toxicities were indistinguishable. Quartz produced the lowest •OH amount but showed the greatest toxicity. Our results showed no correlation between the toxicity of mineral dusts and their ability to generate free radicals. We also showed that the amounts of oxidants per neutrophil increased with doses, time and the cytotoxicity of the dusts in the lung, which supports our postulation that dust-elicited neutrophilia is the major persistent source of oxidative stress. These results and the discussion of the crucial roles of the short-lived, continuously replenished neutrophils in dust-induced pathogenesis are presented.

Comment on Saber et al. (2019), "Commentary: the chronic inhalation study in rats for assessing lung cancer risk may be better than its reputation".

In their Commentary Saber et al. (Part Fibre Toxicol 16: 44, 2019) argue that chronic inhalation studies in rats can be used for assessing the lung cancer risk of insoluble nanomaterials. The authors make several significant errors in their interpretation and representation of the underlying science. In this Letter to the Editor we discuss these inaccuracies to correct the scientific record. When the science is recounted accurately it does not support Saber et al's statements and conclusions.

Viscosupplementation may preserve tibial cartilage and collagen in osteoarthritis: findings from a preclinical model of osteoarthritis.

PURPOSE: Intraarticular (IA) hyaluronic acid (HA) injection is used to reduce pain and improve mobility in knee osteoarthritis (OA). Little is known about histopathological changes underlying HA efficacy. This study investigated dose-related effects of 1% sodium hyaluronate (BioHA) on knee joint histopathology and pain responses in a medial meniscal tear (MMT) rat model of OA. METHODS: Following MMT surgery, rats were randomized into treatment groups: single IA injection of vehicle, BioHA, or an avian-derived hyaluronic acid (hylan G-F 20) on Day 7; or 3 weekly injections of vehicle or BioHA on Days 7, 14, and 21. On Day 35, joints were evaluated by microscopic histopathology for cartilage degeneration, collagen degeneration, synovitis, and cytokine expression (tumor necrosis factor α, transforming growth factor ß). RESULTS: Joint pathology for control animals was consistent with that expected for the MMT model. Rats treated with 3 injections of IA-BioHA had significantly reduced collagen degeneration (21%) relative to control animals. No significant change in collagen degeneration was observed for rats given a single injection of hylan G-F 20 or IA-BioHA compared to control animals. HA treatment did not affect cytokine expression. CONCLUSIONS: IA-BioHA viscosupplementation in a rat MMT model of OA showed preservation of joint cartilage and collagen. This effect was most pronounced on tibial surfaces having less severe injury, suggesting that treatment should be initiated early in the disease process. A comparison of responses to IA-BioHA or hylan G-F 20 in the MMT rat OA model suggest IA-BioHA may be more effective in preserving joint connective tissue.

Expert workshop on the hazards and risks of poorly soluble low toxicity particles.

'Lung particle overload' refers to the impaired lung particle clearance and increased particle retention occurring with high lung doses of poorly soluble low toxicity (PSLT) particles. In rats, lung particle overload is associated with inflammation, epithelial hyperplasia, and, in extreme cases, lung cancer. While the human relevance of rat lung tumors occurring under overload has been questioned, recent regulatory decisions have considered these outcomes evidence of possible human hazard. To better understand the state-of-the-science on PSLT toxicology, an Expert Workshop was held to document agreements and differences amongst a panel of highly experienced scientists and regulators. Key outcomes included: a functional definition of PSLTs; agreement the rat is a sensitive model for PSLT inhalation toxicology; identifying lung inflammation as a critical endpoint for PSLT risk assessment; and, agreement rat lung cancer occurring only under conditions of lung particle overload does not imply a cancer hazard for humans under non-overloading exposures. Moreover, when asked - should PSLTs be considered as human lung carcinogens based on rat data alone (and no supporting data from other species), the expert consensus was: 'No. However, the experts noted the current default regulatory position on rat lung overload data alone would be the suspicion of human carcinogen hazard.' The many areas of the expert agreement provide guidance for design, interpretation, and extrapolating PSLT inhalation toxicology studies. Considering the workshop outcomes, the authors recommend guidelines for evaluation and classification of PSLT be reassessed; and, prior decisions on PSLT hazard classification be revisited to determine if they remain appropriate.

The hazards and risks of inhaled poorly soluble particles - where do we stand after 30 years of research?

BACKGROUND: In 2006, titanium dioxide and carbon black were classified by IARC as "possibly carcinogenic to humans" and in 2017 the European Chemicals Agency's (ECHA) Committee for Risk Assessment concluded titanium dioxide meets the criteria to be classified as suspected of causing cancer (category 2, through the inhalation route). These classifications were based primarily on the occurrence of lung cancer in rats exposed chronically to high concentrations of these materials, as no such responses have been observed in other animal species similarly exposed. After the EU classification of titanium dioxide, it was suggested that Poorly Soluble particles of Low Toxicity (PSLTs) can be evaluated as a group. MAIN BODY: To better understand the current state of scientific opinion, we sought perspective from several international experts on topics relevant to the classification of carbon black; titanium dioxide; and, the potential future classification of PSLTs. Areas discussed included: grouping of PSLTs; the relevance of rat lung cancer responses to high concentrations of PSLTs; and, clearance overload and implications for interpretation of inhalation toxicology studies. We found there were several areas where a large majority of experts, including ourselves, agreed. These included concerns on the grouping of PSLT and the definition of clearance overload. Regarding the extrapolation of PSLT associated lung cancer in rats there were some strongly held differences, although most experts questioned the relevance when excessive exposures which overwhelm lung clearance were required. SHORT CONCLUSION: Given the ongoing discussion on PSLT classification and safety, we believe it is important to re-activate the public debate including experts and stakeholders. Such an open discussion would serve to formally document where scientific consensus and differences exist. This could form the basis for design of future safety programs and safety assessments.

Toxicity of lunar dust assessed in inhalation-exposed rats.

Humans will again set foot on the moon. The moon is covered by a layer of fine dust, which can pose a respiratory hazard. We investigated the pulmonary toxicity of lunar dust in rats exposed to 0, 2.1, 6.8, 20.8 and 60.6 mg/m(3) of respirable-size lunar dust for 4 weeks (6 h/day, 5 days/week); the aerosols in the nose-only exposure chambers were generated from a jet-mill ground preparation of a lunar soil collected during the Apollo 14 mission. After 4 weeks of exposure to air or lunar dust, groups of five rats were euthanized 1 day, 1 week, 4 weeks or 13 weeks after the last exposure for assessment of pulmonary toxicity. Biomarkers of toxicity assessed in bronchoalveolar fluids showed concentration-dependent changes; biomarkers that showed treatment effects were total cell and neutrophil counts, total protein concentrations and cellular enzymes (lactate dehydrogenase, glutamyl transferase and aspartate transaminase). No statistically significant differences in these biomarkers were detected between rats exposed to air and those exposed to the two low concentrations of lunar dust. Dose-dependent histopathology, including inflammation, septal thickening, fibrosis and granulomas, in the lung was observed at the two higher exposure concentrations. No lesions were detected in rats exposed to ≤6.8 mg/m(3). This 4-week exposure study in rats showed that 6.8 mg/m(3) was the highest no-observable-adverse-effect level (NOAEL). These results will be useful for assessing the health risk to humans of exposure to lunar dust, establishing human exposure limits and guiding the design of dust mitigation systems in lunar landers or habitats.

A comparative dose-related response of several key pro- and antiinflammatory mediators in the lungs of rats, mice, and hamsters after subchronic inhalation of carbon black.

OBJECTIVE: The objective of this study was to investigate mechanisms underlying species specificity in particle-induced lung inflammation. METHODS: Rats, mice, and hamsters exposed to air, 1, 7, or 50 mg/m3 of carbon black for 13 weeks were killed at 1 day, 3 months, and 11 months after exposure. Bronchoalveolar lavage was performed and characterized for cell number, cell type, reactive oxygen and nitrogen species, and cytokine levels. Ex vivo mutational analysis of inflammatory cells was evaluated by coincubating with lung epithelial cells. Lung tissue was evaluated for gene expression of various antiinflammatory mediators. RESULTS: There was a dose- and time-related effect with all the parameters. Rats demonstrated greater propensity for generating a proinflammatory response, whereas mice and hamsters demonstrated an increased antiinflammatory response. CONCLUSIONS: These differences in pro- and antiinflammatory responses may contribute to the apparent species differences in inflammation and tumorigenesis.

Effects of subchronically inhaled carbon black in three species. I. Retention kinetics, lung inflammation, and histopathology.

Exposure to high concentrations of carbon black (Cb) produces lung tumors in rats, but not mice or hamsters, presumably due to secondary genotoxic mechanisms involving persistent lung inflammation and injury. We hypothesized that the lung inflammation and injury induced by subchronic inhalation of Cb are more pronounced in rats than in mice and hamsters. Particle retention kinetics, inflammation, and histopathology were examined in female rats, mice, and hamsters exposed for 13 weeks to high surface area Cb (HSCb) at doses chosen to span a no observable adverse effects level (NOAEL) to particle overload (0, 1, 7, 50 mg/m(3), nominal concentrations). Rats were also exposed to low surface area Cb (50 mg/m(3), nominal; LSCb). Retention and effects measurements were performed immediately after exposure and 3 and 11 months post-exposure; retention was also evaluated after 5 weeks of exposure. Significant decreases in body weight during exposure occurred only in hamsters exposed to high-dose HSCb. Lung weights were increased in high-dose Cb-exposed animals, but this persisted only in rats and mice up to the end of the study period. Equivalent or similar mass burdens were achieved in rats exposed to high-dose HSCb and LSCb, whereas surface area burdens were equivalent for mid-dose HSCb and LSCb. Prolonged retention was found in rats exposed to mid- and high-dose HSCb and to LSCb, but LSCb was cleared faster than HSCb. Retention was also prolonged in mice exposed to mid- and high-dose HSCb, and in hamsters exposed to high-dose HSCb. Lung inflammation and histopathology were more severe and prolonged in rats than in mice and hamsters, and both were similar in rats exposed to mid-dose HSCb and LSCb. The results show that hamsters have the most efficient clearance mechanisms and least severe responses of the three species. The results from rats also show that particle surface area is an important determinant of target tissue dose and, therefore, effects. From these results, a subchronic NOAEL of 1 mg/m(3) respirable HSCb (Printex 90) can be assigned to female rats, mice, and hamsters.