r Environmental Risk Assessment Summary Trimethoprim Introduction The publication of environmental risk assessment summaries is part of Roche s engagement on developing a better understanding of issues regarding pharmaceuticals in the environment (PIE). New pharmaceutical substances are investigated for biodegradability and initial ecotoxicity during their development. For registration, a full state-of-the-art environmental risk assessment is developed based on chronic environmental effects and advanced environmental fate data, as required by the pertinent regulations. While not a regulatory requirement, Roche also investigates older pharmaceutical substances, normally at a simpler scale, in order to assess their environmental risks. For pharmaceutical substances, the potential environmental risk is calculated from the ratio between the Predicted Environmental Concentration (PEC) of the substance in the aquatic environment based on a conservative emission scenario and the Predicted No Effect Concentration (PNEC), a concentration below which no adverse effects on the environment have to be expected. If Measured Environmental Concentrations (MECs) are available, these can also be compared with the PNEC. Summary Trimethoprim is a diaminopyrimidine antimicrobial agent which is effective against a wide range of Gram-positive and Gram-negative microorganisms. It is normally used in combination with a sulphonamide and prevents the conversion of folic acid into folinic acid in the bacterial or protozoal cell, by inhibiting the enzyme dihydrofolate reductase and consequently affecting DNA synthesis [11]. Trimethoprim is the active pharmaceutical ingredient used in combination with Sulfamethoxazole in the Roche product Bactrim. In humans, Trimethoprim was rapidly and almost completely absorbed (more than 95%) after oral administration. Trimethoprim was excreted chiefly by glomerular filtration and renal tubular secretion. 70 90% of an orally administered dose was recovered from the urine within 24 h and 92 102% within 3 d. Some hepatic transformation occurred with a small proportion of the dose excreted in the bile. 80% of the administered dose was excreted unmetabolised. Five metabolites were identified in the remaining 20% [11]. Trimethoprim is neither readily nor inherently biodegradable in standard OECD tests over 28 days. However, significant co-metabolic degradation (>50%) was observed in certain studies. The average and the median removal rates for full-scale working sewage treatment plants (STP) with 107 recorded removal rates, representing at least 63 STPs are 25% and 30%, respectively [26]. F. Hoffmann-La Roche Ltd, Group SHE (LSO), ANH, 30.10.2018 page 1/7
The PEC/ PNEC SW ratio for ecotoxicology is 0.0023. With reference to the Guideline on the Environmental Risk Assessment on Medicinal Products for Human Use of the European Medicines Agency [10], a PEC/PNEC SW ratio of 1 means that Trimethoprim and/or its metabolites are unlikely to represent a risk to the aquatic environment. Also when considering a provisional PEC/PNEC R ratio of 0.46 (i.e. 1) for antimicrobial resistance selection, Trimethoprim and/or its metabolites are unlikely to represent a risk to the aquatic environment. Predicted Environmental Concentration (PEC) The PEC is based on the following data: PEC (μg/l) = (A x 10 9 x (1-R)) / (365 x P x V x D) A Total patient consumption of Trimethoprim in the European country with the highest yearly per capita use in the period 2013 2017 (data from IQVIA [21]) R Removal rate in sewage treatment plant (STP): 0.25 (average removal rate for full-scale working STP of 25% [26] P Number of inhabitants in the country with the highest per capita use in the respective year of the period 2013 2017 [12]; resulting in a consumption of 225 mg/inhabitant V Volume of wastewater per inhabitant and day (default value) = 200 L day -1 [10] D Dilution factor of wastewater by surface water flow (default value) = 10 [10] PEC = 0.231 μg/l Note: Trimethoprim is metabolised in the body to an extent of about 20%. Since little is known about the ecotoxicity of these metabolites, it is assumed as a worst case that they have the same ecotoxicological relevance as Trimethoprim. Measured Environmental Concentration (MEC) Trimethoprim has been measured in European surface waters at least since the mid-1990s and today very many MECs can be located. In total, data representing at least 1899 single MECs have been collated [26] The values were collated into one single distribution. The calculated 50th and 95th percentiles (MEC 50 and MEC 95, respectively) were 0.012 μg/l and 0.129 μg/l [26]. The MEC 95 of 0.129 μg/l is in agreement with the calculated PEC of 0.231 μg/l. F. Hoffmann-La Roche Ltd, Group SHE (LSO), ANH, 30.10.2018 page 2/7
Predicted No Effect Concentration for Aquatic Ecosystem Function (PNEC SW ) Acute and chronic data for species from three trophic levels, partially assessed based on OECD Test Guidelines [24], but also taken from the literature, have been used to calculate the PNEC SW The lowest No Observed Effect Concentration (NOEC) or chronic effective concentration (EC 10 ) is 1.00 mg/l (1000 μg/l) of the 72 h NOEC growth with the diatom blue-green alga Anabaena flos-aquae [19], performed according to Guideline OECD 201. Applying an assessment factor of 10 according to the EMA Guideline [10], this results in a PNEC SW of 100 μg/l. PNEC SW = 1000 μg/l / 10 = 100 μg/l Provisional Predicted No Effect Concentration for Antimicrobial Resistance Development (PNEC R ) In the discussion about antimicrobial resistance (AMR) development, provisional PNEC R are derived. An approach is to use the 1 st percentile of the minimal inhibitory concentrations (MIC) for different bacteria genera, corresponding to the value containing the bottom 1% of the MIC values and by applying a safety factor of 10. With this approach a provisional PNEC R of 0.5 μg/l was derived [2][5]. PNEC R = 0.5 μg/l PEC/PNEC ratios PEC = 0.231 μg/l PNEC SW = 100 μg/l PNEC R = 0.5 μg/l (provisional) PEC/PNEC SW = 0.0023 PEC/PNEC R = 0.46 (provisional) With reference to the Guideline on the Environmental Risk Assessment on Medicinal Products for Human Use of the European Medicines Agency [10], a PEC/PNEC SW ratio of 0.0023 (i.e. 1) means that Trimethoprim and/or its metabolites are unlikely to represent a risk to the aquatic environment. Also when considering a provisional PEC/PNEC R ratio of 0.46 (i.e. 1) for AMR selection, Trimethoprim and/or its metabolites are unlikely to represent a risk to the aquatic environment. F. Hoffmann-La Roche Ltd, Group SHE (LSO), ANH, 30.10.2018 page 3/7
Aquatic Toxicity Data for Trimethoprim Study Guideline Results Ref. green alga Raphidocelis subcapitata OECD 201 OECD 201 OECD 201 72 h EC50 = 40 mg/l 72 h NOEC = 16 mg/l 72 h EC50 (growth) = 98 mg/l 72 h EC50 (biomass) = 70 mg/l 72 h NOEC = 32 mg/l 96 h NOEC = 63.4 mg/l [27] [22] [19] cyanobacteria Anabaena variabilis cyanobacteria Anabaena flos-aquae cyanobacteria Anabaena flos-aquae diatom Navicula pelliculosa marine diatom Phaeodactylum tricornutum duckweed Lemna minor not specified 6 d MIC = 50 mg/l 6 d EC50 = 11 mg/l 6 d NOEC = 3.1 mg/l [3] OECD 201 96 h EC10 = 18.3 mg/l [19] 96 h NOEC = 13.6 mg/l OECD 201 72 h EC10 = 3.3 mg/l [2] 72 h NOEC = 1.0 mg/l OECD 201 96 h EC10 = 1.32 mg/l [19] 96 h NOEC = 1.20 mg/l ISO 10253 72 h EC10 = 2.4 mg/l [8] OECD 221 7 d EC50 (growth) = 215 mg/l 7 d EC50 (yield) = 133 mg/l 7 d NOEC = 53.5 mg/l [6] OECD 202 48 h EC50 >100 mg/l [23] 48 h NOEC = 100 mg/l OECD 203 72 h NOEC = 100 mg/l [20] Acute immobilisation test with Daphnia magna Acute Toxicity to Zebrafish (Danio rerio) Daphnia magna, reproduction test OECD 211 21 d NOEC (overall) = 6 mg/l [25] Fish, early-life stage toxicity test OECD 210 35 d NOEC (overall) = 100 mg/l [9] with zebrafish (Danio rerio) Activated sludge respiration OECD 209 3 h EC50 = 17.8 mg/l [20] inhibition test 3 h EC50 >200 mg/l [7] 3 h EC10 = 0.435 mg/l Nitrification inhibition test ISO 9509 4 h EC10 >96 mg/l [7] 4 h NOEC = 96 mg/l Not specified NOEC = 0.05 mg/l [16] Anaerobic inhibition OECD 224 NOEC = 100 mg/l [14] Minimal inhibitory concentration EUCAST MIC = 16 μg/l [5] EC50 ECx EUCAST MIC NOEC concentration of the test substance that results in 50% effect concentration of the test substance that results in x% effect European Committee on Antimicrobial Susceptibility Testing Minimal inhibitory concentration No Observed Effect Concentration F. Hoffmann-La Roche Ltd, Group SHE (LSO), ANH, 30.10.2018 page 4/7
Environmental Fate Data for Trimethoprim Study Guideline Results Ref. Ready biodegradability test OECD 301 F OECD 301 D 0% after 28 days with respect to BOD 4% after 28 days with respect to BOD [20] [1] 27% after 28 days with respect to BOD (with co-metabolism) not readily biodegradable Inherent biodegradability test OECD 302 B 0% after 28 days with respect to TOC (mineralization) 16% after 28 days with respect to TOC (DOC elimination) not inherently biodegradable [15] Inherent biodegradability tests (in reactors) Sludge adsorption coefficient Half-life (total system) = 22 41 d Elimination = ~70% Half-life (total system) = 96 h Average and standard deviation Kd = 208 ± 49 L/kg Grab samples Kd = 157 L/kg Kd = 375 L/kg [20] [4] [18] 1) BOD Biochemical oxygen demand TOC Total organic carbon 1) Sorption to activated sewage sludge was found to be of minor importance In general, compounds with a Kd of <500 L/kg are eliminated by less than 10% through sorption on to activated sludge [17]. Physical Chemical Data for Trimethoprim Study Guideline Results Ref. Water solubility 300 mg/l (20 C) [13] n-octanol/water Partition Coefficient log P OW = 0.64 [13] F. Hoffmann-La Roche Ltd, Group SHE (LSO), ANH, 30.10.2018 page 5/7
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[17] Göbel A, McArdell CS, Joss A, Siegrist H, Giger W. 2007. Fate of sulfonamides, macrolides, and trimethoprim in different wastewater treatment technologies. Sci Total Environ. 372(2-3):361-371 [18] Göbel A, Thomsen A, McArdell CS, Joss A, Giger W. 2005. Occurrence and sorption behavior of sulfonamides, macrolides, and trimethoprim in activated sludge treatment. Environ Sci Technol. 39(11):3981-3989 [19] Guo J, Selby K, Boxall AB. 2016. Comparing the sensitivity of chlorophytes, cyanobacteria, and diatoms to major-use antibiotics. Environ Toxicol Chem. 35(10):2587-2596 [20] Halling-Sørensen B, Lützhøft HC, Andersen HR, Ingerslev F. 2000. Environmental risk assessment of antibiotics: comparison of mecillinam, trimethoprim and ciprofloxacin. J Antimicrob Chemother. 46 Suppl 1:53-8 [21] IQVIA MIDAS Quantum, Q1 2018 [22] NOTOX B.V., on behalf of F. Hoffmann-La Roche Ltd, Basel, Switzerland (1996). Fresh Water Algal Growth Inhibition Test with Trimethoprim. NOTOX study no. 180089 [23] NOTOX B.V., on behalf of F. Hoffmann-La Roche Ltd, Basel, Switzerland (1996). Acute Toxicity Study in Daphnia magna with Trimethoprim. NOTOX study no. 180001 [24] Organisation for Economic Co-operation and Development (OECD). OECD Guidelines for the Testing of Chemicals [25] Park S, Choi K. 2008. Hazard assessment of commonly used agricultural antibiotics on aquatic ecosystems. Ecotoxicology. 17(6):526-38 [26] Straub JO. 2013. An Environmental Risk Assessment for Human-Use Trimethoprim in European Surface Waters. Antibiotics, 2, 115-162 [27] Yang LH, Ying GG, Su HC, Stauber JL, Adams MS, Binet MT. 2008. Growth-inhibiting effects of 12 antibacterial agents and their mixtures on the freshwater microalga Pseudokirchneriella subcapitata. Environ Toxicol Chem. 27(5):1201-8 F. Hoffmann-La Roche Ltd, Group SHE (LSO), ANH, 30.10.2018 page 7/7