Maximum levels of cross‐contamination for 24 antimicrobial active substances in non‐target feed. Part 13: Diaminopyrimidines: trimethoprim

Abstract The specific concentrations of trimethoprim in non‐target feed for food‐producing animals below which there would not be an effect on the emergence of, and/or selection for, resistance in bacteria relevant for human and animal health, as well as the specific antimicrobial concentrations in feed which have an effect in terms of growth promotion/increased yield were assessed by EFSA in collaboration with EMA. Details of the methodology used for this assessment, associated data gaps and uncertainties, are presented in a separate document. To address antimicrobial resistance, the Feed Antimicrobial Resistance Selection Concentration (FARSC) model developed specifically for the assessment was applied. The FARSC for trimethoprim was estimated. Uncertainties and data gaps associated to the levels reported were addressed. To address growth promotion, data from scientific publications obtained from an extensive literature review were used. No suitable data for the assessment were available. It was recommended to perform further studies to supply more diverse and complete data related to the requirements for calculation of the FARSC for trimethoprim.


Introduction
The European Commission requested the European Food Safety Authority (EFSA) to assess, in collaboration with the European Medicines Agency (EMA), (i) the specific concentrations of antimicrobials resulting from cross-contamination in non-target feed for food-producing animals, below which there would not be an effect on the emergence of, and/or selection for, resistance in microbial agents relevant for human and animal health (term of reference 1, ToR1), and (ii) the levels of the antimicrobials which have a growth promotion/increase yield effect (ToR2). The assessment was requested to be conducted for 24 antimicrobial active substances specified in the mandate. 1 For the different substances (grouped by class if applicable) 1 , separate scientific opinions included within the 'Maximum levels of cross-contamination for 24 antimicrobial active substances in non-target feed' series (Scientific Opinions Part 2 -Part 13, EFSA BIOHAZ Panel, 2021b-lsee also the Virtual Issue; for practical reasons, they will be referred as 'scientific opinion Part X' throughout the current document) were drafted. They present the results of the assessments performed to answer the following questions: Assessment Question 1 (AQ1), which are the specific antimicrobial concentrations in non-target feed below which there would not be emergence of, or selection for, resistance in the large intestines/rumen, and AQ2: which are the specific antimicrobial concentrations in feed of foodproducing animals that have an effect in terms of growth promotion/increased yield. The assessments were performed following the methodology described in Section 2 of the Scientific Opinion 'Part 1: Methodology, general data gaps and uncertainties' (EFSA BIOHAZ Panel, 2021a, see also the Virtual Issue). The present document reports the results of the assessment for trimethoprim.

1.1.
Background and Terms of Reference as provided by the requestor The background and ToRs provided by the European Commission for the present document are reported in Section 1.1 of the Scientific Opinion "Part 1: Methodology, general data gaps and uncertainties" (see also the Virtual Issue).

Interpretation of the Terms of Reference
The interpretation of the ToRs, to be followed for the assessment is in Section 1.2 of the Scientific Opinion 'Part 1: Methodology, general data gaps and uncertainties' (see also the Virtual Issue).

1.3.
Additional information 1.3.1. Short description of the class/substance Trimethoprim is a folate pathway antagonist discovered in 1956(Anderson et al., 2012. It is a synthetic antimicrobial that can be produced through various routes (Anderson et al., 2012). Trimethoprim enters bacterial cells by passive diffusion due to its sufficiently small size and lipophilic properties. Once inside the bacterial cell, it inhibits dihydrofolate reductase (DHFR), which reduces dihydrofolate to tetrahydrofolate (O'Grady, 1975). This blocks the synthesis of folate which is an essential co-factor in the biosynthesis of thymidine and thus in DNA synthesis (Anderson et al., 2012). As bacteria cannot take up folate from the environment, disruption of this metabolic pathway results in inhibition of bacterial growth. Over the years, trimethoprim has been mainly administered in combination with sulfamethoxazole and many other sulphonamides in veterinary medicine as the main hypothesis was that sulphonamides potentiate the action of trimethoprim (Minato et al., 2018). More recently, it has been determined that: (i) trimethoprim-sulfamethoxazole synergy is driven by mutual potentiation of the action of each drug by the other (Minato et al., 2018) and (ii) although there are several clinical indications supporting administration of trimethoprim alone, this practice is unusual in veterinary medicine, especially for feed administration, and is nearly exclusively applied in human medicine in selected geographical areas (Anderson et al., 2012;Gigu ere et al., 2013).

Main use 2
Trimethoprim belongs to the class of diaminopyrimidines and is commonly used in combination with sulfonamides in veterinary medicine in Europe (EMEA/CVMP, 1997). The combinations trimethoprimsulfonamides are used in most animal species. The spectrum is broad, including Gram-negative and Gram-positive bacteria (including Enterobacterales, Pasteurella spp., Staphylococcus spp., Streptococcus pneumoniae) and many protozoans (e.g. Histoplasma, Toxoplasma and coccidia) for both constituents, so many bacterial respiratory infections, urinary tract and soft tissues infections and protozoan intestinal infections can be treated by this antimicrobial. The main indications for trimethoprim in combination with sulfonamides in food-producing animals are for the curative and metaphylactic treatment of respiratory (e.g. Pasteurella multocida, Actinobacillus pleuropneumoniae, Streptococcus spp. and Haemophilus parasuis) and gastrointestinal (e.g. Escherichia coli) infections in cattle, swine and poultry. Other treated infections include atrophic rhinitis when associated with Bordetella bronchiseptica, streptococcal meningitis caused by Streptococcus suis and coryza caused by Avibacterium paragallinarum.

Main pharmacokinetic data
Trimethoprim, by the oral route, has a bioavailability of 67% in horses (Van Duijkeren et al., 1996) higher than 90% in pigs (Nielsen and Gyrd-Hansen, 1994), 80% in broilers (Baert et al., 2003) and 100% in Atlantic salmon at 10°C (Horsberg et al., 1997). No value for the bioavailability of trimethoprim by oral route is available for rabbits. In accordance with this high bioavailability in monogastric species, a study of Peeters et al. (2016) showed that the administration of 2.5 mg of trimethoprim per kg feed to pigs led to intestinal and faecal concentrations lower than the limit of quantification (LOQ) of 0.016 mg/kg (Peeters et al., 2016). In another study from the same group, the administration of a far higher dose of 2.5 mg/kg body weight (bw) to pigs (standard dose) by oral route also led to concentrations of trimethoprim below the LOQ of 0.025 mg/kg in the rectum and faeces. The trimethoprim concentrations decreased from proximal to distal intestines with the maximum concentrations of around 1 mg/kg in the jejunum and concentrations between the LOQ and 0.5 mg/kg in ileum and caecum (De Smet et al., 2017). These data also suggested a low level of secretion through the epithelium (De Smet et al., 2017).
In calves, the absorption of trimethoprim decreased with age. In an old study using drug quantification by microbiological assay, trimethoprim was not detectable in serum (concentrations lower than 0.1 lg/mL) after oral administration of a combination of trimethoprim and sulfadiazine to 6 or 12 week-old calves fed with grain-fibre-based feed (Shoaf et al., 1987). For ruminants, it was also shown that trimethoprim is extensively degraded by ruminal microbiota (partly explaining the very low bioavailability in these animals despite the high lipophilicity of the antimicrobial) (Nielsen and Dalgaard, 1978;Shoaf et al., 1987).

Main resistance mechanisms
Resistance to trimethoprim in bacteria can be mediated by different mechanisms including alteration, overproduction or replacement of the target and impaired cell permeability (Eliopoulos and Huovinen, 2001). Trimethoprim resistance is widespread in Gram-positive and Gram-negative bacteria. Replacement of the target by acquisition of exogenous genes encoding trimethoprim-resistant DHFR is the most common mechanism observed in Gram-negatives. More than 100 genes/gene variants have been described to date, generally in associations with various transposons and plasmids. This resistance mechanism also occurs in Gram-positive bacteria, but at a lower frequency compared to chromosomal mutations leading to amino acid changes and thus alteration of DHFR (Anderson et al., 2012). Notably, several anaerobic bacteria including species of the gut microbiome are intrinsically resistant to trimethoprim, mainly due to insensitive or absent DHFR (Huovinen, 1987) and in Campylobacter jejuni absence of folA, coding for dihydrofolate reductase, so they do not offer any target for the antifolate, conferring intrinsic resistance to this antimicrobial (Myllykallio et al., 2003).

Data and methodologies
The data sources and methodology used for this opinion are described in a dedicated document, the Scientific Opinion 'Part 1: Methodology, general data gaps and uncertainties' (see also the Virtual Issue).

Introduction
As indicated in the Scientific Opinion 'Part 1: Methodology, general data gaps and uncertainties' (see also the Virtual Issue), exposure to low concentrations of antimicrobials (including sub-minimum inhibitory concentrations, sub-MIC) may have different effects on bacterial antimicrobial resistance evolution, properties of bacteria and in animal growth promotion. Some examples including emergence of, and selection for, antimicrobial resistance, mutagenesis, virulence and/or horizontal gene transfer (HGT), etc. for the antimicrobials under assessment are shown below.

Resistance development/spread due to sub-MIC concentrations of trimethoprim: examples
Trimethoprim has been used as an antimicrobial in human and veterinary medicine since the late 1960s, with rapid and extensive spread of resistance genes and/or resistant bacteria from diverse environments.

Effects of sub-MIC concentrations on selection for resistance and mutagenesis
• Several publications report a correlation between trimethoprim use and the emergence of resistance in humans, with earlier studies after trimethoprim initial use demonstrating a clear increase in trimethoprim-resistant E. coli and Shigella spp. (Huovinen et al., 1995). The complexity of factors accounting for the success of an acquired antimicrobial resistance complicates the current extraction of evidences for different bacterial groups.
• Minimal selective concentration (MSC) has been determined for trimethoprim in one study using competitions between susceptible and trimethoprim resistant (due to the presence of a dfr gene) (Gullberg et al., 2014). MSC was 0.002 mg/L and wild type had a MIC of trimethoprim of 0.2 mg/L, showing that the MSC was 100-fold lower than the MIC.
• Sulfonamide + trimethoprim administration via liquid feeding pipelines for pigs selected for sulfonamide + trimethoprim-resistant bacteria in the biofilm lining the pipelines. This may cause contamination of liquid feed for extended periods and represent a source of resistance genes for the gastrointestinal microbiota of the pigs (Heller et al., 2017).
• No statistically significant correlation between trimethoprim usage and resistance was reported in a large European study involving multiple food animal species (Ceccarelli et al., 2020).

Effects of sub-MIC concentrations on horizontal gene transfer and virulence
• Data on concentrations of trimethoprim having secondary effects on, e.g. HGT frequencies or induction of virulence are very limited. One study (Jutkina et al., 2018) found that trimethoprim had no effect on HGT rates at sub-MIC concentrations (up to 0.25 mg/L). Another study found that trimethoprim at 20 mg/L increased the expression levels of genes involved in the conjugative transfer of a trimethoprim, sulphonamide and tetracycline resistance plasmid from Aeromonas hydrophila, which is expected to result in increased conjugation frequency (Cantas et al., 2012).

3.2.
ToR1. Estimation of the antimicrobial levels in non-target feed that would not result in the selection of resistance: Feed Antimicrobial Resistance Selection Concentration (FARSC) As explained in the Methodology Section (2.2.1.3) of the Scientific Opinion 'Part 1: Methodology, general data gaps and uncertainties' (see also the Virtual Issue), the estimation of this value for trimethoprim for different animal species followed a two-step approach as described below.
The first step was the calculation of the predicted minimal selective concentration (PMSC) for tetracycline as indicated in Table 1.
The minimal selective concentration (MSC) has been determined for trimethoprim in one study (Gullberg et al., 2014) and was 0.002 mg/L (wild type MIC 0.2 mg/L). Accordingly, the ratio MIC test / MSC test was 100 ( Table 1).
The PMSC for trimethoprim, calculated using the lowest MIC value available in the EUCAST MIC distribution database (MIC lowest ) as described in Bengtsson-Palme and Larsson (2016), multiplied by the MIC test /MSC test factor (as described in Section 2.2.3.2 of the Scientific Opinion Part 1), was 0.00016 mg/L (Table 1).
From the PMSC for trimethoprim, the FARSC (FARSC intestine and FARSC rumen ) corresponding to the maximal concentrations in feed was calculated for each species from the equations below (for details, see Section 2.2.1.3.2 of the Scientific Opinion Part 1; see also the Virtual Issue) by including specific values for trimethoprim.
With daily faeces being the daily fresh faecal output in kg, I the inactive fraction, F the fraction available, GE the fraction of the antimicrobial that is secreted back into the intestinal tract for elimination, after initially being absorbed into the bloodstream, and daily feed intake being the daily dry-matter feed intake expressed in kg.
From the study of Van Duijkeren et al. (1996) in horses, I was set to 0.45 for monogastric animals. A worse scenario was also simulated with I equal to 0.3. For ruminants, due to extensive degradation by ruminal microbiota (Nielsen and Dalgaard, 1978;Shoaf et al., 1987), I was set to 0.8. Due to the lack of quantitative data, other simulations were performed with I equal to 0.45 and 0.9.
According to the literature, the bioavailability (F) of trimethoprim was set to 0.9 for pigs (Nielsen and Gyrd-Hansen, 1994), 0.8 for broilers (Baert et al., 2003) and 0.7 for horses (Van Duijkeren et al., 1996). From the low secretion through the epithelium demonstrated by De Smet et al. (2017), GE was set to 0.1. Since no quantitative data were reported in the previous study, other simulations were performed with GE equal to 0 and 0.2 for monogastric animals.
The bioavailability was described as very low for ruminants after weaning. The potential sources for this low bioavailability such as the absence of absorption or extensive hepatic first-pass effect were not further investigated, and no quantitative data are available. Thus, the bioavailability of trimethoprim was set to 0 for ruminants (sheep, goats and cattle). A slightly higher value of 0.1 was also used for the calculations.
The different values of the parameters used for the calculations are summarised in Table 2 and the estimated FARSC values are reported in Table 3. There is no value for the bioavailability in veal calves and rabbits. The first set of values (scenario 1) corresponds to the average of published values while scenario 2 corresponds to scenario that would lead to lower FARSC and scenario 3 to scenario that would lead to higher FARSC. The lowest FARSC (scenario 2) were obtained from lowest published values of I (lower inactivation of the drug resulting in higher activity on bacteria), lowest published values of F (lower absorption resulting in more drug in the intestines) and highest values of GE (higher elimination in intestines resulting in more drug in the intestines). Gastrointestinal elimination (GE) is the fraction of the antimicrobial that is secreted back into the intestinal tract for elimination, after initially being absorbed into the bloodstream. The fraction remaining in the digestive tract and that could be available for the bacteria is equal to (1 -F + F 9 GE), thus (1 -F) in Scenario 3. The values of FARSC intestine , for the species with available data, ranged in the first scenario using averaged published values from 0.64 9 10 À3 mg/kg feed in turkeys for fattening to 11.73 9 10 À3 mg/kg feed in dairy cows. From other simulations (scenario 2 and scenario 3) made with a wider range of values for the data used in the calculation, FARSC intestine would range from 0.32 to 0.90 9 10 À3 mg/kg feed in turkeys for fattening, from 0.93 to 32.97 9 10 À3 mg/kg feed in salmon and from 2.25 to 44.57 9 10 À3 mg/kg feed in dairy cows. In general, for the different species, the FARSC intestine for trimethoprim ranged from 0.32 to 44.57 9 10 À3 mg/kg feed.
For the estimation of Feed Antimicrobial Resistance Selection Concentration of trimethoprim in rumen (FARSC rumen ), I was also set to 0.8 due to extensive degradation by ruminal microbiota (Nielsen and Dalgaard, 1978;Shoaf et al., 1987). However, due to the lack of quantitative data, other simulations were performed with I equal to 0 and 0.9.
The different values of the parameters used for the calculations are summarised in Table 4 and the estimated FARSC rumen values are reported in Table 5.
The values of FARSC rumen ranged from 3.6 to 12 9 10 À3 mg/kg feed with the first scenario. By considering that the trimethoprim is totally active in the rumen (I = 0), the values of FARSC rumen ranged from 0.72 to 2.4 9 10 À3 mg/kg feed whereas inactivation of 90% of the trimethoprim would led to values of FARSC rumen from 7.2 to 24 9 10 À3 mg/kg feed. In general, for the different species, the FARSC rumen for trimethoprim ranged from 1.20 to 24.00 9 10 À3 mg/kg feed.

Associated data gaps and uncertainties
With regard to the uncertainties and data gaps described in the Scientific Opinion Part 1 (Sections 3.1 and 3.3; see also the Virtual Issue) we identified the following for trimethoprim to perform the assessment: i) MSC data: MSC is available only for E. coli (Gullberg et al., 2014). ii) Impact of complexity on determined MSC: only available in single species experiment (Gullberg et al., 2014). iii) Inactive fraction: for monogastric species, the data only come from one study conducted in horses. For ruminants, the degradation was described as extensive. However, this observation is only derived from two cows studied in 1978.  A detailed analysis of the associated uncertainties for trimethoprim is included in Appendix A ( Table A.1) of the current, and the Section 3.3 of the Scientific Opinion Part 1.

Concluding remarks
The FARSC for trimethoprim (for large intestine and/or rumen in the case of adult ruminants after weaning) ranges, for the different species, from 0.32 to 44.57 9 10 À3 mg/kg feed. No FARSC was determined for veal calves and rabbits.

3.3.
ToR2. Specific antimicrobials concentrations in feed which have an effect in terms of growth promotion/increased yield 3.3.1. Trimethoprim

Literature search results
The literature search, conducted according to the methodology described in Section 2.2.2.1 of the Scientific Opinion 'Part 1: Methodology, general data gaps and uncertainties' (see also the Virtual Issue), resulted in 1598 papers mentioning trimethoprim and any of the food-producing animal species considered 3 and any of the performance parameters identified as relevant for the assessment of the possible growth-promoting effects of trimethoprim. 4 After removing the reports not matching the eligibility criteria, 39 publications were identified.

Evaluation of the studies
The 39 publications identified in the literature search were appraised for suitability for the assessment of the effects of trimethoprim on growth or yield of food-producing animals; this appraisal was performed by checking each study against a series of pre-defined exclusion criteria (see Section 2.2.2.2.1 of the Scientific Opinion 'Part 1: Methodology, general data gaps and uncertainties'; see also the Virtual Issue). 5 None of the publications was considered suitable for the assessment because of several shortcomings identified in their designs or in the reporting of the results. The list of excluded publications and their shortcomings are presented in Appendix A (Table A.1).

Concluding remarks
Owing to the lack of suitable data, levels of trimethoprim in feed which may have a growth promotion/production yield effect in any food-producing animal species could not be identified.

Conclusions
ToR1: to assess the specific concentrations of antimicrobials resulting from crosscontamination in non-target feed for food-producing animals, below which there would not be an effect on the emergence of, and/or selection for, resistance in microbial agents relevant for human and animal health.
AQ1. Which are the specific concentrations of trimethoprim in non-target feed below which there would not be emergence of, and/or selection for, resistance in the large intestines/rumen?
• The Feed Antimicrobial Resistance Selection Concentration (FARSC, for large intestine and/or rumen in the case of adult ruminants after weaning) corresponding to the concentrations of trimethoprim in feed below which there would not be expected to be an effect on the emergence of, and/or selection for, resistance in microbial agents relevant for human and animal health ranges, for the different species, from 0.32 to 44.57 9 10 À3 mg/kg feed. No FARSC was determined for veal calves and rabbits.
ToR2: to assess which levels of the antimicrobials have a growth promotion/increase yield effect.
AQ2. Which are the specific concentrations of trimethoprim in feed of food-producing animals that have an effect in terms of growth promotion/increased yield?
• Owing to the lack of suitable data, levels of trimethoprim in feed which may have a growth promotion/production yield effect in any food-producing animal species could not be identified.
The results from these assessments for the different animal species are summarised in Annex F (Tables F.1 and F.2) of EFSA BIOHAZ Panel, 2021a -Scientific Opinion 'Part 1: Methodology, general data gaps and uncertainties' (see also the Virtual Issue).

Recommendations
To perform further studies to supply more diverse and complete data to reduce uncertainties around the calculation of the FARSC for trimethoprim.