Principle considerations for the risk assessment of sprayed consumer products.

In recent years, the official regulation of chemicals and chemical products has been intensified. Explicitly for spray products enhanced requirements to assess the consumers'/professionals' exposure to such product type have been introduced. In this regard the Aerosol-Dispensers-Directive (75/324/EEC) with obligation for marketing aerosol dispensers, and the Cosmetic-Products-Regulation (1223/2009/EC) which obliges the insurance of a safety assessment, have to be mentioned. Both enactments, similar to the REACH regulation (1907/2006/EC), require a robust chemical safety assessment. From such assessment, appropriate risk management measures may be identified to adequately control the risk of these chemicals/products to human health and the environment when used. Currently, the above-mentioned regulations lack the guidance on which data are needed for preparing a proper hazard analysis and safety assessment of spray products. Mandatory in the process of inhalation risk and safety assessment is the determination and quantification of the actual exposure to the spray product and more specifically, its ingredients. In this respect the current article, prepared by the European Aerosol Federation (FEA, Brussels) task force "Inhalation Toxicology", intends to introduce toxicological principles and the state of the art in currently available exposure models adapted for typical application scenarios. This review on current methodologies is intended to guide safety assessors to better estimate inhalation exposure by using the most relevant data.

Diverging binding capacities of natural LD78beta isoforms of macrophage inflammatory protein-1alpha to the CC chemokine receptors 1, 3 and 5 affect their anti-HIV-1 activity and chemotactic potencies for neutrophils and eosinophils.

Recently, the LD78beta isoform of the CC chemokine macrophage inflammatory protein (MIP)-1alpha was shown to efficiently chemoattract lymphocytes and monocytes and to inhibit infection of mononuclear cells by R5 HIV-1 strains. We have now demonstrated that after cleavage of the NH2-terminal Ala-Pro dipeptide by CD26, LD78beta(3 - 70) became the most potent chemokine blocking HIV-1. LD78beta(3 - 70) competed tenfold more efficiently than LD78beta(1 - 70) with [125I] RANTES for binding to the CC chemokine receptors CCR5 and CCR1. Contrary to LD78alpha, LD78beta(1 - 70) at 30 ng/ml efficiently competed with [125I] RANTES for binding to CCR3 and mobilized calcium in CCR3 transfectants, whereas LD78beta(3 - 70) showed a 30-fold decrease in CCR3 affinity compared to LD78beta(1 - 70). This demonstrates the importance of the penultimate proline in LD78beta(1 - 70) for CCR3 recognition. Both LD78beta isoforms efficiently chemoattracted eosinophils from responsive donors. In contrast, only the CCR3 agonist LD78beta(1 - 70) and not LD78beta(3 - 70), induced calcium increases in eosinophils with low levels of CCR1. In responder neutrophils, LD78beta(3 - 70) elicited calcium fluxes at a 30-fold lower dose (10 ng/ml) compared to intact LD78beta and LD78alpha, whereas the three MIP-1alpha isoforms were equipotent neutrophil chemoattractants. Taken together, both LD78beta isoforms are potent HIV-1 inhibitors (CCR5) and activators for neutrophils (CCR1) and eosinophils (CCR1, CCR3), affecting infection and inflammation.

Biochemical and biological characterization of neutrophil chemotactic protein, a novel rabbit CXC chemokine from alveolar macrophages.

The role of interleukin-8 (IL-8) and related CXC chemokines has been demonstrated in many human diseases. However, more profound studies, e.g., by blocking the effect of these inflammatory mediators, request animal models and hence the identification of all human counterparts for commonly used laboratory animals. In this study, we describe the identification of a novel neutrophil chemotactic protein (NCP) of the rabbit. Intact and NH(2)-terminally truncated NCP forms and IL-8 were isolated from LPS-stimulated rabbit alveolar macrophages and purified to homogeneity by a four-step purification procedure. Determination of the complete primary structure of NCP by mass spectrometry and NH(2)-terminal sequencing of natural protein revealed high structural homology with human epithelial cell-derived neutrophil attractant-78 (ENA-78) and granulocyte chemotactic protein-2 (GCP-2), two related ELR(+)CXC chemokines. Intact NCP(1-76) was found to be 10-fold less potent than truncated NCP(7, 8-76) at inducing neutrophil chemotaxis. NCP(7,8-76) was equally potent as intact rabbit IL-8 at chemoattracting human neutrophils and at inducing calcium fluxes in rabbit neutrophils, 1 ng/mL being the minimal effective concentration. However, like IL-8, NCP failed to induce monocyte or eosinophil migration at 300-fold higher concentrations. IL-8 desensitized the calcium increase induced by NCP and vice versa. Finally, intradermal injection of NCP induced a dose-dependent and significant infiltration of neutrophils in mice skin. It can be concluded that NCP is a novel rabbit CXC chemokine that is, like IL-8, implicated in animal models used to study various human disorders in which neutrophils play an important role.

In vivo neutrophil recruitment by granulocyte chemotactic protein-2 is assisted by gelatinase B/MMP-9 in the mouse.

Granulocyte chemotactic protein-2 (GCP-2) of the mouse is a potent neutrophil chemotactic and activating factor in vitro and in vivo. Gelatinase B/matrix metalloproteinase-9 is released from neutrophils within 1 h after stimulation with GCP-2. In vitro neutrophil chemotaxis by GCP-2 was not impaired by specific inhibitory monoclonal antibodies (mAb) against gelatinase B, indicating that gelatinase B is not involved in chemotaxis of neutrophils through polycarbonate filters. To investigate if gelatinase B degranulation is involved in in vivo cell migration toward GCP-2, experiments were performed with gelatinase B knockout mice. When mouse GCP-2 was injected intradermally in mice, a dose-dependent neutrophil chemotactic response was observed, and this cell migration was significantly impaired in young mice by genetic gelatinase B knockout. In adult vs. young gelatinase B-deficient mice, such compensatory mechanisms as higher basal neutrophil counts and less impairment of chemotaxis toward local GCP-2 injection were observed. These experiments prove the concept that gelatinase B release under pressure of GCP-2 is a relevant, but not exclusive, effector mechanism of neutrophil chemotaxis in vivo and that known mechanisms, other than the release of gelatinase B, allow for a full-blown chemotactic response and compensate for gelatinase B deficiency in adult life in the mouse.

NH2- and COOH-terminal truncations of murine granulocyte chemotactic protein-2 augment the in vitro and in vivo neutrophil chemotactic potency.

Chemokines are important mediators of leukocyte migration during the inflammatory response. Post-translational modifications affect the biological potency of chemokines. In addition to previously identified NH2-terminally truncated forms, COOH-terminally truncated forms of the CXC chemokine murine granulocyte chemotactic protein-2 (GCP-2) were purified from conditioned medium of stimulated fibroblasts. The truncations generated 28 natural murine GCP-2 isoforms containing 69-92 residues, including most intermediate forms. Both NH2- and COOH-terminal truncations of GCP-2 resulted in enhanced chemotactic potency for human and murine neutrophils in vitro. The truncated isoform GCP-2(9-78) was 30-fold more potent than intact GCP-2(1-92)/LPS-induced CXC chemokine (LIX) at inducing an intracellular calcium increase in human neutrophils. After intradermal injection in mice, GCP-2(9-78) was also more effective than GCP-2(1-92)/LIX at inducing neutrophil infiltration. Similar to human IL-8 and GCP-2, murine GCP-2(9-78) and macrophage inflammatory protein-2 (MIP-2) induced calcium increases in both CXCR1 and CXCR2 transfectants. Murine GCP-2(9-78) could desensitize the calcium response induced by MIP-2 in human neutrophils and vice versa. Furthermore, MIP-2 and truncated GCP-2(9-78), but not intact GCP-2(1-92)/LIX, partially desensitized the calcium response to human IL-8 in human neutrophils. Taken together, these findings point to an important role of post-translationally modified GCP-2 to replace IL-8 in the mouse.