Absence of phototoxicity/photoirritation potential of bergamottin determined In Vitro using OECD TG 432.

The phototoxic potential of a number of furocoumarins is well established. On the other hand, studies have shown that bergamottin, a furocoumarin containing a bulky, hydrophobic side chain, has significantly less or is even absent of phototoxicity potential. The OECD Test Guideline 432 3T3/Neutral Red Uptake (NRU) in vitro phototoxicity test has shown to be a highly predictive test for identifying compounds that exhibit no phototoxicological potential. In this study using OECD 432, the established phototoxic furocoumarin 5-methoxypsoralen (5-MOP), 8-methoxypsoralen (8-MOP) and psoralen were phototoxic, whereas bergamottin showed no phototoxic potential. When compared to 5-MOP, 8-MOP and psoralen, bergamottin was clearly negative at molar-adjusted concentrations that were more than 9 times higher than those that produced phototoxicity in 8-MOP; nearly 16 times than those for psoralen and more than 36 times higher than those for 5-MOP. These data using in vitro 3T3 NRU Phototoxicity Test (OECD 432) are supportive of earlier studies showing bergamottin does not exhibit phototoxicological properties. The detection and quantification of bergamottin should therefore not contribute to the potential marker furocoumarins for risk management interventions intended to reduce the phototoxicity of natural furocoumarin containing preparations.

Assessment of 8-methosypsoralen, lomefloxacin, sparfloxacin, and Pirfenidone phototoxicity in Long-Evans rats.

Phototoxicity has a strong impact on drug development. Although several animal models have been developed to quantitatively assess human risks, none have been validated for standardized use. In this study, we validated an in vivo phototoxicity model using Long-Evans (LE) rats treated with 4 well-known phototoxic drugs, namely 8-methoxypsoralen, lomefloxacin, sparfloxacin, and pirfenidone. Daily macroscopic observations of skin and eyes, ophthalmological examinations 4 days after dosing, and blood sampling for toxicokinetics (TKs) were performed after exposure of treated animals to ultraviolet, and dose-dependent eye and/or skin reactions were noted for all compounds. Margins of safety were calculated when possible and correlated well with known relative phototoxicity of the 4 compounds. We conclude that the present in vivo phototoxicity assay using LE rats with TK analysis can be used to quantitatively predict the risk of pharmaceutical phototoxicity in humans.

Update of the toxicological assessment of furanocoumarins in foodstuffs (Update of the SKLM statement of 23/24 September 2004)--Opinion of the Senate Commission on Food Safety (SKLM) of the German Research Foundation (DFG).

The DFG Senate Commission on Food Safety (SKLM) has discussed the toxicological assessment of furanocoumarins in foodstuffs and adopted an opinion on 23/24 September 2004 [SKLM, English version: Toxicological assessment of furanocumarins in foodstuffs, 2006; Mol. Nutr. Food Res. 2007, 51, 367-373]. At that time, no analytical data were available on the occurrence and content of furanocoumarins in citrus oils, especially in lime oil and the foodstuffs produced from it. According to the SKLM, the highest levels were likely to be found in products containing lime or bergamot oil. Distilled and cold pressed oils differ in their levels of furanocoumarins; in distilled oils, no furanocoumarins were found. The original estimate of the average daily intake of furanocoumarins in Germany made by the SKLM is based on the assumption that flavoured foods contain cold-pressed citrus oils exclusively (worst case scenario). Recent data, however, indicate that distilled citrus oils are mainly used in flavoured soft drinks. The SKLM has therefore decided to update the assessment of the average intake of furanocoumarins from flavoured food. The following opinion was released in German on 25 January 2010, the English version was agreed on 27/28 September 2010.

Assessment of safety in neonates for transfusion of platelets and plasma prepared with amotosalen photochemical pathogen inactivation treatment by a 1-month intravenous toxicity study in neonatal rats.

BACKGROUND: It is estimated that approximately 300,000 neonates undergo transfusions annually. The neonatal immune system is immature, making such patients more susceptible to the effects associated with transfusion-transmitted bacteria, viruses, protozoa, and white blood cells (WBCs). The INTERCEPT Blood System is a photochemical process (PCT) utilizing amotosalen and long-wavelength ultraviolet to inactivate pathogens and WBCs in both platelet (PLT) and plasma components for transfusion. A series of clinical studies has shown PCT PLTs and PCT plasma to be safe and effective for transfusion in adults and pediatric patients. Because clinical studies in neonates are technically difficult and ethically challenging, preclinical toxicologic studies were conducted in neonatal rats to evaluate the safety of PCT blood components for neonates. STUDY DESIGN AND METHODS: This study examined daily intravenous administration to neonatal rats of amotosalen in 35 percent:65 percent plasma:InterSol from 1 microg per kg per day (representing 1-unit transfusion) to 457 microg per kg per day (representing multiple transfusions) from Postnatal Day 4 (PND4) to PND31. Rats were observed for viability, clinical signs, and body weights until PND31 and then subjected to pathology evaluation. Hematology, clinical chemistry, and urinalysis data were also collected on PND31. Toxicokinetic parameters were evaluated on PND4 and PND31. RESULTS: There were no amotosalen-related effects on clinical signs, body weight, hematology, clinical chemistry, urinalysis, gross pathology, or histopathology, despite the exposure of neonatal rats to amotosalen concentrations as high as approximately 48 times the standard exposure in adult patients. CONCLUSION: This study demonstrates the safety of PCT for transfusion in neonatal rats and augments data from other studies and clinical use supporting the use of PCT in neonatal patients.

The pathogen reduction treatment of platelets with S-59 HCl (Amotosalen) plus ultraviolet A light: genotoxicity profile and hazard assessment.

Despite restrictive donor criteria and screening procedures, infections resulting from the transfusion of bacterially contaminated platelet products continue to occur. Pathogen reduction technologies targeting nucleic acids have been developed. However, concerns about the safety of these procedures exist; the main concern being the possible mutagenic and carcinogenic effects of the pathogen-inactivated preparation in the recipient. This report reviews the genotoxicity profile of the S-59 (Amotosalen) plus long wavelength ultraviolet light (UVA) pathogen reduction technology, and assesses the mutagenic and carcinogenic hazards in recipients of treated platelets. S-59, a synthetic heterocyclic psoralen, non-covalently intercalates into the nucleic acids of pathogens and forms crosslinks when UVA photoactivated. Before clinical use, the levels of residual S-59 and free photoproducts are greatly reduced using a 'compound adsorption device' (CAD). In vitro, S-59 is mutagenic in Salmonella typhimurium and mouse lymphoma L5178Y TK(+/-) cells, and is clastogenic in CHO cells. There is reduced activity (Salmonella, CHO cells) or no activity (mouse lymphoma cells) with metabolic activation (S9 mix). When tested up to toxic dose levels, S-59 was negative in the mouse bone marrow micronucleus assay and the rat hepatocyte unscheduled DNA synthesis (UDS) test. Based on comparative studies conducted with S-59 plus UVA-treated platelets (up to 25 times without CAD), any genotoxic effects can be attributed to residual S-59. Considering (1) the known genotoxic mechanism of action for S-59, (2) the negative in vivo studies for S-59 at multiples >40,000x over clinical peak plasma levels, and (3) the fact that the positive in vitro genotoxicity effects for the end product seem due to residual S-59, any mutagenic hazard to a recipient of S-59 plus UVA-treated platelets is negligible and there is no concern about a carcinogenic potential as a consequence of a mutagenic activity. This conclusion is supported by a negative p53(+/-) mouse carcinogenicity study.

Assessment of diphenylcyclopropenone for photochemically induced mutagenicity in the Ames assay.

The photochemical conversion of diphenylcyclopropenone to diphenylacetylene has recently been reported. Diphenylcyclopropenone is used in the treatment of alopecia areata and is nonmutagenic in a limited Ames assay. We examined diphenylcyclopropenone and diphenylacetylene, as well as synthetic precursors of diphenylcyclopropenone--dibenzylketone and alpha,alpha'-dibromodibenzylketone--for mutagenicity against TA100, TA98, TA102, UTH8413, and UTH8414. All compounds were nonmutagenic except alpha,alpha'-dibromodibenzylketone, which was a potent mutagen in TA100 with and without S-9 activation. The effect of photochemical activation of diphenylcyclopropenone in the presence of bacteria demonstrated mutagenicity in UTH8413 (two times background) at 10 micrograms/plate with S-9 microsomal activation. 8-Methoxypsoralen produces a mutagenic response in TA102 at 0.1 microgram/plate with 60 seconds of exposure to 350 nm light. In vitro photochemically activated Ames assay with S-9 microsomal fraction may enhance the trapping of short-lived photochemically produced high-energy mutagenic intermediates. This technique offers exciting opportunities to trap high-energy intermediates that may play an important role in mutagenesis. This method can be applied to a variety of topically applied dermatologic agents, potentially subjected to photochemical changes in normal use.