Harmonized 3Rs-based non-mutagenic impurity qualification study designs developed using the results of an IQ consortium survey.

As per the ICH Q3A(R2) and Q3B(R2) regulatory guidelines, safety studies may be needed when an impurity in new drug substances or products is above the qualification threshold, and such qualification studies should be conducted in one nonclinical species for a duration of 14-90 days. However, the guidelines do not specify details about species selection, recommended study design, and the exact study duration that would support clinical use of a specific duration. This lack of guidance leads to ambiguity and sponsors have used various study designs to qualify impurities. In 2018, the European Medicines Agency provided a draft reflection paper encouraging the incorporation of 3Rs (Replacement, Reduction, and Refinement) principles for animal use into impurity qualification. As a response, the IQ DruSafe Impurity Working Group (WG) surveyed the IQ member companies to capture the current practices for impurity qualification, and evaluate study designs for a potential reduction in animal testing. This article summarizes the results and learnings from the survey. Additionally, the WG leveraged the survey learnings and provided harmonized study design considerations aimed towards achieving the study objectives, while supporting the 3Rs initiative in reducing the total number of animals used (up to 90%) for impurity qualification.

Lung antioxidant and cytokine responses to coarse and fine particulate matter from the great California wildfires of 2008.

The authors have previously demonstrated that wildfire-derived coarse or fine particulate matter (PM) intratracheally instilled into lungs of mice induce a strong inflammatory response. In the current study, the authors demonstrate that wildfire PM simultaneously cause major increases in oxidative stress in the mouse lungs as measured by decreased antioxidant content of the lung lavage supernatant fluid 6 and 24 h after PM administration. Concentrations of neutrophil chemokines/cytokines and of tumor necrosis factor (TNF)-alpha were elevated in the lung lavage fluid obtained 6 and 24 h after PM instillation, consistent with the strong neutrophilic inflammatory response observed in the lungs 24 h after PM administration, suggesting a relationship between the proinflammatory activity of the PM and the measured level of antioxidant capacity in the lung lavage fluid. Chemical analysis shows relatively low levels of polycyclic aromatic hydrocarbons compared to published results from typical urban PM. Coarse PM fraction is more active (proinflammatory activity and oxidative stress) on an equal-dose basis than the fine PM despite its lower content of polycyclic aromatic hydrocarbons. There does not seem to be any correlation between the content of any specific polycyclic aromatic hydrocarbon (or of total polycyclic aromatic hydrocarbon content) in the PM fraction and its toxicity. However, the concentrations of the oxidation products of phenanthrene and anthracene, phenanthraquinone and anthraquinone, were several-fold higher in the coarse PM than the fine fraction, suggesting a significant role for atmospheric photochemistry in the formation of secondary pollutants in the wildfire PM and the possibility that such secondary pollutants could be significant sources of toxicity in the wildfire PM.

Mouse lung inflammation after instillation of particulate matter collected from a working dairy barn.

Coarse and fine particulate matter (PM(2.5-10) and PM(2.5), respectively) are regulated ambient air pollutants thought to have major adverse health effects in exposed humans. The role of endotoxin and other bioaerosol components in the toxicity of PM from ambient air is controversial. This study evaluated the inflammatory lung response in mice instilled intratracheally with PM(2.5-10) and PM(2.5) emitted from a working dairy barn, a source presumed to have elevated concentrations of endotoxin. PM(2.5-10) was more pro-inflammatory on an equal weight basis than was PM(2.5); both fractions elicited a predominantly neutrophilic response. The inflammatory response was reversible, with a peak response to PM(2.5-10) observed at 24 h after instillation, and a return to control values by 72 h after instillation. The major active pro-inflammatory component in whole PM(2.5-10), but not in whole PM(2.5), is heat-labile, consistent with it being endotoxin. A heat treatment protocol for the gradual inactivation of biological materials in the PM fractions over a measurable time course was developed and optimized in this study using pure lipopolysaccharide (LPS) as a model system. The time course of heat inactivation of pure LPS and of endotoxin activity in PM(2.5-10) as measured by Limulus bioassay is identical. The active material in both PM(2.5-10) and PM(2.5) remained in the insoluble fraction when the whole PM samples were extracted with physiological saline solution. Histological analysis of lung sections from mice instilled with PM(2.5-10) or PM(2.5) showed evidence of inflammation consistent with the cellular responses observed in lung lavage fluid. The major pro-inflammatory components present in endotoxin-rich PM were found in the insoluble fraction of PM(2.5-10); however, in contrast with PM(2.5-10) isolated from ambient air in the Central Valley of California, the active components in the insoluble fraction were heat-labile.

California wildfires of 2008: coarse and fine particulate matter toxicity.

BACKGROUND: During the last week of June 2008, central and northern California experienced thousands of forest and brush fires, giving rise to a week of severe fire-related particulate air pollution throughout the region. California experienced PM(10-2.5) (particulate matter with mass median aerodynamic diameter > 2.5 mum to < 10 mum; coarse ) and PM(2.5) (particulate matter with mass median aerodynamic diameter < 2.5 mum; fine) concentrations greatly in excess of the air quality standards and among the highest values reported at these stations since data have been collected. OBJECTIVES: These observations prompt a number of questions about the health impact of exposure to elevated levels of PM(10-2.5) and PM(2.5) and about the specific toxicity of PM arising from wildfires in this region. METHODS: Toxicity of PM(10-2.5) and PM(2.5) obtained during the time of peak concentrations of smoke in the air was determined with a mouse bioassay and compared with PM samples collected under normal conditions from the region during the month of June 2007. RESULTS: Concentrations of PM were not only higher during the wildfire episodes, but the PM was much more toxic to the lung on an equal weight basis than was PM collected from normal ambient air in the region. Toxicity was manifested as increased neutrophils and protein in lung lavage and by histologic indicators of increased cell influx and edema in the lung. CONCLUSIONS: We conclude that the wildfire PM contains chemical components toxic to the lung, especially to alveolar macrophages, and they are more toxic to the lung than equal doses of PM collected from ambient air from the same region during a comparable season.

Lung response to coarse PM: bioassay in mice.

Particulate matter (PM) elicits inflammatory and toxic responses in the lung specific to its constituents, which can vary by region, time, and particle size. To identify the mechanism of toxicity in PM collected in a rural area in the San Joaquin Valley of Central California, we studied coarse particles of 2.5-10 mum diameter (PM(2.5)-PM(10)). Potential pro-inflammatory and toxic effects of PM(2.5)-PM(10) in the lung were investigated using intratracheally instilled mice. We determined total and differential cell profiles and inflammatory chemokines in lung lavage fluid, and biomarkers of toxicity resulting from coarse PM exposure. Responses of the mice were readily observed with total doses of 25-50 mug of PM per mouse. Changes in pro-inflammatory cellular profiles and chemokines showed both dose and time responses; peak responses were observed 24 h after PM instillation, with recovery as early as 48 h. Furthermore, macrophage inflammatory protein (MIP-2) profiles following PM exposures were correlated to levels of measured macrophages and neutrophils recovered from lung lavage fluid of PM-treated animals. Our data suggest that pro-inflammatory effects observed from coarse PM collected during the summer months from California's hot and dry Central Valley are driven largely by the insoluble components of the PM mixture, and are not caused by endotoxin.

In vivo effects of ozone exposure on protein adduct formation by 1-nitronaphthalene in rat lung.

The incidence of serious photochemical smog events is steadily growing in urban environments around the world. The electrophilic metabolites of 1-nitronaphthalene (1-NN), a common air pollutant in urban areas, have been shown to bind covalently to proteins. 1-NN specifically targets the airway epithelium, and the toxicity is synergized by prior long-term ozone exposure in rat. In this study we investigated the formation of 1-NN protein adducts in the rat airway epithelium in vivo and examined how prior long-term ozone exposure affects adduct formation. Eight adducted proteins, several involved in cellular antioxidant defense, were identified. The extent of adduction of each protein was calculated, and two proteins, peroxiredoxin 6 and biliverdin reductase, were adducted at high specific activities (0.36-0.70 and 1.0 nmol adduct/nmol protein). Furthermore, the N-terminal region of calreticulin, known as vasostatin, was adducted only in ozone-exposed animals. Although vasostatin was adducted at relatively low specific activity (0.01 nmol adduct/nmol protein), the adduction only in ozone-exposed animals makes it a candidate protein for elucidating the synergistic toxicity between ozone and 1-NN. These studies identified in vivo protein targets for reactive 1-NN metabolites that are potentially associated with the mechanism of 1-NN toxicity and the synergistic effects of ozone.