Evaluation of the EMA log kow trigger for fish BCF testing based on data for several human pharmaceuticals.

In the European Medicines Agency (EMA) "Guideline for Environmental Risk Assessment of Medicinal Products for Human Use," a fish bioconcentration factor (BCF) study is triggered in Phase I for pharmaceuticals having log Kow >4.5, to support Persistence, Bioaccumulation and Toxicity (PBT) screening, and in Phase II to assess secondary poisoning and bioaccumulation ('B') potential when log Kow ≥3. The standard sampling schedule outlined in OECD Test Guideline 305 (TG305) may require assessment of approximately 200 fish following exposure to low- and high-test concentrations and a negative control. We report experimental log Kow and BCF values for 64 human pharmaceuticals that were used to evaluate the current BCF testing trigger of log Kow ≥3, and whether a single BCF exposure concentration allows accurate classification of bioaccumulation potential. Our data support raising the BCF testing trigger to log Kow ≥4, and use of a single test concentration. The resulting reduction in the use of fish is consistent with the 3 R s principle and did not adversely affect classification accuracy. An assessment of potential risk of secondary poisoning was also conducted for three drugs classified as either B or vB, and no risks were identified.

Human Health Risk Assessment (HHRA) for environmental development and transfer of antibiotic resistance.

BACKGROUND: Only recently has the environment been clearly implicated in the risk of antibiotic resistance to clinical outcome, but to date there have been few documented approaches to formally assess these risks. OBJECTIVE: We examined possible approaches and sought to identify research needs to enable human health risk assessments (HHRA) that focus on the role of the environment in the failure of antibiotic treatment caused by antibiotic-resistant pathogens. METHODS: The authors participated in a workshop held 4-8 March 2012 in Québec, Canada, to define the scope and objectives of an environmental assessment of antibiotic-resistance risks to human health. We focused on key elements of environmental-resistance-development "hot spots," exposure assessment (unrelated to food), and dose response to characterize risks that may improve antibiotic-resistance management options. DISCUSSION: Various novel aspects to traditional risk assessments were identified to enable an assessment of environmental antibiotic resistance. These include a) accounting for an added selective pressure on the environmental resistome that, over time, allows for development of antibiotic-resistant bacteria (ARB); b) identifying and describing rates of horizontal gene transfer (HGT) in the relevant environmental "hot spot" compartments; and c) modifying traditional dose-response approaches to address doses of ARB for various health outcomes and pathways. CONCLUSIONS: We propose that environmental aspects of antibiotic-resistance development be included in the processes of any HHRA addressing ARB. Because of limited available data, a multicriteria decision analysis approach would be a useful way to undertake an HHRA of environmental antibiotic resistance that informs risk managers.

Segregation of experimental autoimmune glomerulonephritis as a complex genetic trait and exclusion of Col4a3 as a candidate gene.

Experimental autoimmune glomerulonephritis (EAG), an animal model of Goodpasture's disease, can be induced in Wistar-Kyoto (WKY) rats (RT1-l) by immunization with rat glomerular basement membrane (GBM) in adjuvant. The model in this rat strain is characterized by anti-GBM antibody production accompanied by focal necrotizing glomerulonephritis with crescent formation. The main autoantigen in humans and rats has been identified as the non-collagenous domain of the alpha3 chain of type IV collagen (alpha3(IV)NC1). By contrast, Lewis (LEW) rats with the same MHC background (RT1-l), immunized with the same antigen, develop similar levels of circulating anti-GBM antibodies, but no histological evidence of nephritis. In order to investigate the genetic basis of susceptibility to EAG, we examined the response of both F1 (WKY x LEW) and backcross (BC1; WKY x F1) rats to immunization with rat GBM. F1 animals were completely resistant to the development of EAG, while BC1 animals showed a range of responses from severe crescentic glomerulonephritis to no histological evidence of disease. The results indicate that EAG is inherited as a complex trait under the control of WKY genes unlinked to the MHC. cDNA sequence analysis of alpha3(IV)NC1 in the two parental strains was identical, indicating no predicted amino acid sequence variation in the alpha3(IV)NC1 domain between these strains. Radiation hybrid mapping, using two separate PCR amplicons from rat alpha3(IV)NC1, localized rat Col4a3 to a region of chromosome 9. Since Col4a3 (encoding the autoantigen) is a candidate for susceptibility to EAG, we screened the region of rat chromosome 9 where Col4a3 is localized, using polymorphic microsatellite markers in segregating BC1 progeny. No significant linkage was detected. These results exclude Col4a3 as a recessive susceptibility gene for EAG in the BC1 progeny.

In Goodpasture's disease, CD4(+) T cells escape thymic deletion and are reactive with the autoantigen alpha3(IV)NC1.

Goodpasture's disease is characterized by rapidly progressive glomerulonephritis and pulmonary hemorrhage, in association with circulating and deposited anti-glomerular basement membrane antibodies that recognize the alpha3 chain of type IV collagen [alpha3(IV)NC1] (known as the Goodpasture antigen). Unlike many other autoimmune diseases, recurrences are rare. In experimental models and human studies, both humoral and cellular mechanisms have been demonstrated to be involved in disease pathogenesis. However, there are few data on the characteristics of the autoreactive T cells or the mechanisms of tolerance to the autoantigen in human patients. It was demonstrated, using immunohistochemical analyses and reverse transcription-PCR, that the Goodpasture antigen is expressed in normal human thymus. Using limiting dilution analyses, the frequencies of circulating autoreactive T cells in patients and control subjects were assessed. During acute disease, there were increased frequencies of CD4(+) T cells reactive with alpha3(IV)NC1 (ranging from 1:6300 to 1:65,000), which decreased with time. There was a significant difference between patients during their acute disease phase and control subjects with respect to the frequency index for alpha3(IV)NC1-specific CD4(+) T cells (P < 0.05, Mann Whitney U test). The decrease in autoreactive CD4(+) T-cell numbers during recovery may be the reason why recurrences are infrequent and may explain the loss of pathogenic autoantibodies with time, because of a lack of T-cell help.