Proposal for regulatory risk mitigation measures for human pharmaceutical residues in the environment.

Environmental risks of human pharmaceutical products should be made transparent and mitigated as far as possible. We propose to apply a risk mitigation scheme to the marketing authorisation of human medicinal products which is pragmatic and tailored, and thus will not increase the burden to regulators and industry too much. This scheme takes into account increasing knowledge and accuracy of the environmental risk estimates, applying preliminary risk mitigation when risks are determined based on model estimates, and definitive, more strict and far-reaching risk mitigation when risks are based on actual measured environmental concentrations. Risk mitigation measures should be designed to be effective, proportional, easy to implement, and in line with current (other) legislation, as well as not being a burden to the patient/health care professionals. Furthermore, individual risk mitigation measures are proposed for products showing environmental risks, while general risk mitigation measures can be applied to all products to reduce the overall burden of pharmaceuticals in the environment. In order to effectively mitigate risk, linking marketing authorisation legislation to environmental legislation is essential.

Improving the regulatory environmental risk assessment of human pharmaceuticals: Required changes in the new legislation.

One of the flagship actions of the Pharmaceutical Strategy for Europe is to address environmental challenges associated with pharmaceutical use. This includes strengthening the Environmental Risk Assessment (ERA) at marketing authorisation (MA) of pharmaceuticals, and revision of the pharmaceutical legislation where needed. The overall aim of an ERA should be to enable comprehensive and effective identification and management of environmental risks of pharmaceuticals without affecting the availability of pharmaceuticals to patients. As experts in the evaluation of ERAs of human medicinal products submitted by pharmaceutical industries (Applicants), we have summarized the current status of the ERA and suggest legislative changes to improve environmental protection without affecting availability. Six regulatory goals were defined and discussed, including possible ways forward: 1) mandatory ERAs in accordance to the EMA guideline at the time of the MA, 2) enforcement of risk mitigation measures including re-evaluation of the ERA, 3) facilitated exchange of environmental data between pharmaceutical and environmental legislations, 4) substance-based assessments, 5) transparency of data, and 6) a catching-up procedure for active pharmaceutical ingredients that lack an ERA. These legislative proposals can be considered as prerequisites for a harmonised assessment and effective management of environmental risks and hazards of human pharmaceuticals.

Permitted Daily Exposure for Diisopropyl Ether as a Residual Solvent in Pharmaceuticals.

Solvents can be used in the manufacture of medicinal products provided their residual levels in the final product comply with the acceptable limits based on safety data. At worldwide level, these limits are set by the "Guideline Q3C (R6) on impurities: guideline for residual solvents" issued by the ICH. Diisopropyl ether (DIPE) is a widely used solvent but the possibility of using it in the pharmaceutical manufacture is uncertain because the ICH Q3C guideline includes it in the group of solvents for which "no adequate toxicological data on which to base a Permitted Daily Exposure (PDE) was found". We performed a risk assessment of DIPE based on available toxicological data, after carefully assessing their reliability using the Klimisch score approach. We found sufficiently reliable studies investigating subchronic, developmental, neurological toxicity and carcinogenicity in rats and genotoxicity in vitro. Recent studies also investigated a wide array of toxic effects of gasoline/DIPE mixtures as compared to gasoline alone, thus allowing identifying the effects of DIPE itself. These data allowed a comprehensive toxicological evaluation of DIPE. The main target organs of DIPE toxicity were liver and kidney. DIPE was not teratogen and had no genotoxic effects, either in vitro or in vivo. However, it appeared to increase the number of malignant tumors in rats. Therefore, DIPE could be considered as a non-genotoxic animal carcinogen and a PDE of 0.98 mg/day was calculated based on the lowest No Observed Effect Level (NOEL) value of 356 mg/m3 (corresponding to 49 mg/kg/day) for maternal toxicity in developmental rat toxicity study. In a worst-case scenario, using an exceedingly high daily dose of 10 g/day, allowed DIPE concentration in pharmaceutical substances would be 98 ppm, which is in the range of concentration limits for ICH Q3C guideline class 2 solvents. This result might be considered for regulatory decisions.

The food contaminant semicarbazide acts as an endocrine disrupter: Evidence from an integrated in vivo/in vitro approach.

Semicarbazide (SEM) is a by-product of the blowing agent azodicarbonamide, present in glass jar-sealed foodstuffs mainly baby foods. The pleiotropic in vivo SEM toxicological effects suggested to explore its possible role as endocrine modulator. Endocrine effects of SEM were assessed in vivo in male and female rats after oral administration for 28 days at 0, 40, 75, 140mg/kg bw pro die during the juvenile period. Vaginal opening and preputial separation were recorded. Concentration of sex steroid in blood, the ex vivo hepatic aromatase activity and testosterone catabolism were detected. The in vitro approach to test SEM role as (anti)estrogen or N-methyl-d-aspartate receptors (NMDARs)-(anti)agonist included different assays: yeast estrogenicity, MCF-7 proliferation, stimulation of the alkaline phosphatase activity in Ishikawa cells and LNCaP-based NMDAR interference assay. In vivo SEM-treated female rats showed delayed vaginal opening at all tested doses, whereas in males preputial separation was anticipated at SEM 40 and 75mg/kg and delayed at 140mg/kg, the latter effect probably due to the significantly decreased body weight gain seen at the higher dose in both sexes. Serum estrogen levels were dose-dependently reduced in treated females, whereas dehydrotestosterone serum levels were also decreased but a clear dose-response was not evidenced. Testosterone catabolism was altered in a gender-related way, aromatase activity was increased in treated males at 75 and 140mg/kg and in females in all dose groups. In the three estradiol-competitive assays, SEM showed a weak anti-estrogenic activity, whereas in the LNCaP-based NMDAR interference assay SEM activity resembled MK-801 antagonist effect. SEM appeared to act as an endocrine disrupter showing multiple and gender specific mechanisms of action(s). A possible cascade-mechanism of SEM on reproductive signalling pathways may be hypothesized. Such in vivo-in vitro approach appeared to be an useful tool to highlight SEM activity on endocrine homeostasis.

Antimutagenic and mutagenic activities of some terpenes in the bacterial reverse mutation assay.

The mutagenic and antimutagenic effects of linalool, linalyl acetate and beta-caryophyllene were evaluated by the bacterial reverse mutation assay on Salmonella typhimurium TA 98 and TA 100, and on Escherichia coli WP2uvrA strains. Neither linalool nor beta-caryophyllene showed mutagenicity, but linalyl acetate induced a statistically significant increase in the number of revertant colonies in WP2uvrA, both with and without S9 mixture. Linalool was devoid of antimutagenic activity against 2-nitrofluorene (2NF), sodium azide (SA), methyl methane sulfonate (MMS) and 2-aminoanthracene (2AA). In contrast, beta-caryophyllene showed a strong antimutagenic activity against 2NF: at the maximum concentration tested (6.40mg/plate) the number of 2NF-induced revertant colonies was reduced by 83.9%. beta-Caryophyllene also showed to counteract the mutagenicity of SA (in TA 100), MMS and 2AA (in WP2uvrA): the effect was weak against SA (inhibition lower than 25%) and moderate against MMS and 2AA (up to 30.5%). The antimutagenic activity of beta-caryophyllene observed here suggests further studies to evaluate its possible chemopreventive properties.

Quercetin reduces chromosome aberrations induced by atrazine in the Allium cepa test.

Quercetin is a widely distributed plant flavonoid possessing a variety of chemical and biological activities, including chelation, free-radical scavenging, and antioxidant activity. Atrazine is a selective triazine herbicide that has been the subject of an international revision program for human and ecological health risks because of its persistence in the environment. In a previous study, we demonstrated that atrazine was clastogenic in the Allium cepa test. In this present study, we investigated whether quercetin affords protection from the chromosome breaks induced by atrazine. In a preliminary assay, 0.1-20 microg/ml quercetin produced no toxicity or clastogenic activity in the Allium cepa test. Subsequently, we evaluated the effects of 0.5 and 5 microg/ml quercetin on the clastogenicity of 2.5, 5.0, and 7.5 microg/l atrazine. Quercetin (0.5 microg/ml) significantly reduced the frequency of total aberrations induced by 7.5 microg/l atrazine, while both concentrations of quercetin significantly decreased the frequency of fragments induced by 7.5 microg/l atrazine. The results of this study indicate that plant flavonoids such as quercetin may protect against the genotoxic effects of atrazine. Efforts to understand the extent to which plant flavonoids influence the biological activities of genotoxicants and the mechanisms involved in the interactions could help to better discern the advantages and disadvantages of their use and to clarify their possible protective role against pollutants.

Evaluation of brominated diphenyl ether-99 toxicity with Raphidocelis subcapitata and Daphnia magna.

Brominated flame retardants belong to a new class of environmental contaminants. To obtain new information regarding the effects of 2,2',4,4',5-brominated diphenyl ether (BDE-99), one of the most frequently reported congeners in freshwater biota, the inhibition of algal growth of Raphidocelis subcapitara (also known as Selenastrum capricornutum) and acute toxicity to Daphnia magna were examined. The experimental design also involved a comparison with the polychlorinated biphenyl (PCB) Aroclor 1254. The uptake of BDE-99 by R. subcapitata and the transfer to D. magna (i.e., a higher level of aquatic biota in the food chain) was also assessed to verify the bioaccumulation phenomenon. After 24 h, BDE-99 appeared to be less toxic than Aroclor 1254 to D. magna, but the two compounds showed a similar toxicity at 48 h. In contrast to Aroclor 1254, BDE-99 was nontoxic to R. subcapitata at up to 100 microM, the highest tested concentration. However, the dose-dependent decrease in survival and impairment of reproduction of D. magna fed with BDE-99-treated algal culture demonstrate uptake by R. subcapitata. Because of the high persistence and bioconcentration, polybrominated diphenyl ethers as well as PCBs might be of environmental concern for years.

Tachykinin-independent activity of capsaicin on in-vitro lamb detrusor.

The capsicum alkaloid capsaicin is an afferent fibre exciter. In the vesical bladder, capsaicin acts by releasing peptides stored in afferent fibres. The aim of this work was to verify the activity of capsaicin on in-vitro lamb urinary bladder and to ascertain whether this alkaloid evokes peptide release. Capsaicin relaxed about 80% of the lamb detrusor muscle preparations tested and contracted about 20%. Whereas neurokinin A and substance P antagonists, administered alone or together, left the contractile responses to capsaicin unchanged, atropine and tetrodotoxin totally inhibited contraction. Ruthenium red and indometacin abolished contractions and relaxation. The substance P and neurokinin A antagonists and the NO-synthesis inhibitor N omega-nitro-L-arginine methyl ester (L-NAME) left relaxation unchanged; conversely, the calcitonin gene-related peptide antagonist alpha h-CGRP (8-37) abolished this response. These results suggest that capsaicin relaxes lamb detrusor muscle not through tachykinins but by releasing CGRP from afferent fibres. Our observation that indometacin blocks the capsaicin response in in-vitro lamb urinary bladder also suggests a role of prostanoids.

The controversial efficacy of vitamin E for human male infertility.

Vitamin E (VE) is major lipophilic chain-breaking antioxidant which protects tissue polyunsaturated fatty acids (PUFA) against peroxidation, a property that could be beneficial in the male reproductive physiology because the membranes of germ cells and spermatozoa are very sensitive to oxidation because of their high content of PUFA. Some of the available data on the efficacy of VE as an oral drug for male infertility or as an additive during in vitro manipulations of spermatozoa were reviewed here, observing that they are often contradictory, possibly because: (1) antioxidant therapy could be ineffective in certain studies not concentrated on men in whom oxidative stress is implicated as an infertility factor, and (2) the VE antioxidant therapy is a double-edged sword strictly depending on the dosage or the in vitro concentration of the vitamin. Thus, further laboratory and clinical studies with better-defined experimental conditions should be performed to establish the in vitro and in vivo efficacy of VE for human male infertility.