Classes of organic pigments meet tentative PSLT criteria and lack toxicity in short-term inhalation studies.

Here, we present a non-animal testing battery to identify PSLT (poorly soluble, low toxicity) substances based on their solubility in phagolysosomal lung fluid simulant, surface reactivity and effects on alveolar macrophages in vitro. This is exemplified by eleven organic pigments belonging to five chemical classes that cover a significant share of the European market. Three of the pigments were tested as both, nanoform and non-nanoform. The results obtained in this integrated non-animal testing battery qualified two pigments as non PSLT, one pigment as poorly soluble and eight pigments as poorly soluble and low toxicity in vitro. The low toxic potency of the eight PSLT and the one poorly soluble pigment was corroborated by short-term inhalation studies with rats. These pigments did not elicit apparent toxic effects at 10 mg/m3 (systemic and in the respiratory tract). One of the pigments, Diarylide Pigment Yellow 83 transparent, however, caused minimal infiltration of neutrophils; hence its low toxicity is ambiguous and needs further verification or falsification. The present test battery provides an opportunity to identify PSLT-properties of test substances to prioritise particles for further development. Thus, it can help to reduce animal testing and steer product development towards safe applications.

Indicators for lack of systemic availability of organic pigments.

Experimental data of all 143 organic pigments registered with the European Chemicals Agency, of which 88 were listed in a nanomaterial inventory, was retrieved from the registered substance fact sheets. Availability of the data was 93% for solubility, 82% for bacterial mutagenicity, 79% for acute oral toxicity, 75% for irritation, 59% for skin sensitisation, 36% for repeated dose toxicity and 34% for each clastogenicity and mutagenicity in mammalian cells and 23% for toxicity to reproduction. Pigments mostly had a water and octanol solubility of significantly below 0.1 mg/L, but fourteen were found to be of higher solubility. None were irritating to skin and eyes. Except for the metal salt and the ß-naphthol pigments, none of the insoluble pigments showed adverse effects up to limit doses indicating that poor solubility prevents systemic uptake of toxicologically relevant amounts. The few available toxicokinetic data shows absence of metabolism or significant uptake and is in support of this. Occasional effects observed on bacterial mutagenicity and skin sensitisation are attributed to impurities. There is no indication that for organic pigments other particle characteristics such as surface area or morphology have an impact on the investigated toxicological endpoints.

Refinement of the acute inhalation limit test for inert, nano-sized dusts by an in silico dosimetry-based evaluation: case study for the dissolution of a regulatory dilemma.

This case study aims to describe the dilemma faced when exposing rats to very high concentrations of fine, pulverulent materials for acute inhalation studies and to address the regulatory question of whether the effects seen here are relevant to humans and the subject of classification according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS). Many powders match the definition of nanomaterials in the EU; therefore, information on acute inhalation testing of powders up to the GHS cutoff of 5 mg/L is required. However, testing rats at such a high aerosol concentration can cause physical obstruction of the airways and even mortality by suffocation. Therefore, to evaluate whether the physical effects on airway obstruction in rats exposed to 5 mg/L for 4 hours and alternative exposures to 1 and 2 mg/L are relevant for humans, an in silico evaluation of aerosol deposition was conducted using the multiple-path particle dosimetry (MPPD) model. For this evaluation, actual exposure conditions for an organic, nano-sized pigment which produced 100% lethality in rats at 5 mg/L, but not at 1 mg/L, were used to assess the potential for airway obstruction in rats and accordingly in humans. As an indicator of the potential for airway obstruction, the ratio of the diameter of the deposited, aggregated aerosol to airway diameter was calculated for each exposure condition. For rats exposed to 5 mg/L for 4 h, approximately 75% of tracheobronchial and 22% of pulmonary/alveolar airways were considered vulnerable to significant or complete obstruction (ratios >0.5). In humans, an equivalent exposure resulted in just over 96% of human tracheobronchial airways that received deposited mass to airway diameter ratios between 0.3 and 0.4 (nasal) or 0.4 and 0.5 (oral), with no airways with ratios >0.5. For the pulmonary/alveolar region, ∼88% of the airways following nasal or oral breathing were predicted to have deposited aerosol diameter to airway diameter ratios <0.1, with no airways with ratios >0.5. Thus, the in silico results obtained for rats are in line with the pathological findings of the animal test. The predicted results in humans, however, affirm the hypothesis of a rat-specific high dose effect which does not justify a classification according to GHS.

Possibilities to group nanomaterials across different substances - A case study on organic pigments.

Grouping concepts to reduce the testing of NFs have been developed for regulatory purposes for different forms of the same substance. Here we explore possibilities to group nanomaterials across different substances for non-regulatory applications, using the example of 16 organic pigments from six chemical classes. Organic pigments are particles consisting of low-molar-mass organic molecules, and rank by tonnage among the most important substances manufactured in nanoform (NF). Tiered testing strategies relevant to the inhalation route included Tier 1 (deposition, dissolution, reactivity, inflammation) and if available Tier 3 data (in vivo). A similarity assessment of the pigment NF data was conducted in a quantitative (Tier 1 and Tier 3 in vivo potency) or qualitative (Tier 3 in vivo effects) manner. We observed that chemical similarity of organic pigments was predictive for their similarity of reactivity and dissolution, but that additional NF descriptors such as surface area or size, modulate the similarity in inflammation or cytotoxicity. We applied the concept of biologically relevant ranges to crop the values of the Tier 1 data matrix before applying similarity algorithms. The Tier 3 assessment by in vivo inhalation confirmed the IATA methodical choices and IATA assessment criteria as consistent and conservative. We suggested limits of acceptable similarity for Tier 1 data and demonstrated their application to support the grouping of some candidate NFs (subsequently confirmed by Tier 3 data). Four candidate NFs exceeded the limits of acceptability for Tier 1 and were escalated from Tier 1 to Tier 3, but were then included in the group, demonstrating the conservative Tier 1 criteria. The resulting group of low-solubility, low-reactivity materials included both NFs and non-NFs of various substances, and could find use for risk management purposes in the occupational handling of pigment powders.

Pleiotropic role of Rac in mast cell activation revealed by a cell permeable Bordetella dermonecrotic fusion toxin.

To activate the GTPase Rac in rat basophilic leukemia (RBL) cells and mouse bone marrow-derived mast cells (BMMC) a TAT fusion toxin of Bordetella dermonecrotic toxin (DNT-TAT) was constructed. The fusion toxin activated Rac1 and RhoA in vitro but only Rac in RBL cells and BMMC. DNT-TAT caused an increase in inositol phosphate formation, calcium mobilization, ERK activation and degranulation of mast cells. All these effects were inhibited by the Rho GTPase-inactivating Clostridium difficile toxin B and Clostridium sordellii lethal toxin. Also the calcium ionophore A23187 caused mast cell activation, including ERK phosphorylation, by processes involving an activation of Rac. The data indicate pleiotropic functions of Rac in mast cell activation.