A Review of Studies submitted to CVM Assessing the
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1 A review of studies submitted to CVM assessing the effects of sub-therapeutic use of 2 antimicrobial drugs on the Salmonella reservoir in food producing animals 3 4 Concern regarding the impact of antimicrobial drug use in animals on the potential 5 development of antimicrobial resistant zoonotic foodborne bacterial pathogens and 6 subsequent transmission to humans as food contaminants was first raised in the 1960’s. In the 7 1970s, the U.S. Food and Drug Administration (FDA) questioned the effect of feeding sub- 8 therapeutic levels of antibiotics to a large number of animals to promote growth. Prominent 9 scientists expected drug resistance to develop as a result of feeding antibiotics to animals 10 under this condition, and expected that these resistant organisms could cause human disease. 11 Scientists were also concerned that the use of certain antibiotics in food-producing animals 12 would promote an increase in the animal reservoir of Salmonella spp. through promotion of 13 cross colonization and infection, prolongation of the carrier state, and relapse of disease. The 14 1970 Antibiotic in Animal Feed Task Force (task force) was formed to address the safety and 15 efficacy issues associated with antibiotics administered in animal feed. The task force report 16 considered the issues of target animal safety, drug effectiveness, and potential public health 17 consequences. 18 19 The task force concluded that therapeutic use presented a small risk to public health and 20 therefore no pre-approval study requirement was imposed on products intended for such use. 21 The conclusion that therapeutic use of antibiotics is safe was based on the following points: 22 23 1. Therapeutic antibiotics used to relieve animal disease were believed to present a small 24 risk 25 because they are typically used at high dose for a short duration in young animals; 26 2. Benefits to the animal outweigh the potential risks to humans; 558.15 History Page 2 27 3. It is necessary to use antibiotics in animals to relieve pain and suffering; 28 4. FDA recognized that healthy animals result in a safer food supply. 29 Scientists of the day argued that sub-therapeutic use created more potential for adverse 30 effects than therapeutic use because of the larger proportion of animals treated, and because 31 of how sub-therapeutic drugs are delivered to animals. A majority of food-producing animals 32 at that time were being fed antibiotics for sub-therapeutic uses such as growth promotion. 33 The antibiotics were given to the animals at levels that exposed bacteria to sub-lethal doses 34 for prolonged periods of time (therapeutic doses are generally high enough to inhibit or kill 35 most bacteria). The task force concluded that a significant effect of therapeutic antibiotic use 36 on pathogen load or antibiotic resistance among pathogens present at time of slaughter would 37 be a low probability event. Additionally, the task force suggested that the benefit to animal 38 health from therapeutic use of antibiotics outweighed the limited risk of adverse 39 microbiological effects on humans that such use might create. Therefore, pre-approval 40 studies were to be required to support the microbiological safety of antibiotics in food- 41 producing animals intended for sub-therapeutic uses only, including growth promotion and 42 feed efficiency. 43 44 21 CFR 558.15 studies 45 As a consequence of the 1970 task force report, requirements for data to address 46 microbiological safety concerns were outlined in the Code of Federal Regulations (21 CFR 47 558.15). Sponsors of antibiotic products were required to submit study results demonstrating 48 that their product did not promote bacterial drug resistance only if their product was intended 49 to be administered for greater than 14 days, for non-prescription use in animals. Sponsors 50 were required to submit all information to the Agency on the impact of their drug(s) on the 558.15 History Page 3 51 “salmonella reservoir” in animals by specific dates depending on the class of drug. As 52 outlined in 21 CFR 558.15 (b)(1) – (b)(3), the following was to be accomplished or submitted 53 to the Agency: 54 1) By July 19, 1973 – records and reports of completed, ongoing, or planned studies, 55 including protocols, on the tetracyclines, streptomycin, dihydrostreptomycin, penicillin, 56 and the sulfonamides; 57 2) By October 17, 1973 - records and reports of completed, ongoing, or planned studies, 58 including protocols, on all other antibiotics; 59 3) By March 4, 1974 - records and reports of completed, ongoing, or planned studies, 60 including protocols, on nitrofurans; 61 4) By April 20, 1974 – data from completed studies on the tetracyclines, streptomycin, 62 dihydrostreptomycin, the sulfonamides, and penicillin assessing the effect of the 63 subtherapeutic use of these drugs in the feed on the salmonella reservoir in the target 64 animal as compared to that in nonmedicated controls; 65 5) By April 20, 1975 – data satisfying all other specified criteria for safety and effectiveness, 66 including the effect on the salmonella reservoir for any antibiotic or sulfonamide drug 67 approved for sub-therapeutic use in animal feeds; 68 6) By September 5, 1975 – data satisfying all other specified criteria for safety and 69 effectiveness, including the effect on the salmonella reservoir for the nitrofuran drugs 70 approved for sub-therapeutic use in animal feeds. 71 72 73 Study design 558.15 History Page 4 74 The studies were conducted as a set that included a bacterial shedding study and a 75 bacterial resistance study. Protocols for these experiments were clearly defined and 76 controlled to avoid introducing bias. Both studies included a negative control group, and a 77 treated group. The negative control and treated groups were inoculated with a laboratory 78 strain of Salmonella typhimurium that possessed an identifying characteristic, commonly 79 nalidixic acid resistance. Additionally, the laboratory strain of S. typhimurium needed to be 80 free of transferable resistance elements. 81 82 Fecal and tissue Salmonella spp. were enumerated and susceptibility tested in the 83 shedding studies, and fecal Escherichia coli were tested for susceptibility in the resistance 84 studies. The studies generally lasted 8 weeks. The test animal was generally the same 85 species as that intended for the marketed product and the test animals were not required to be 86 near normal market or slaughter age. 87 88 The parameters used to determine whether antimicrobial drugs increased consumer 89 exposure to bacterial resistance or pathogen load were 1) drug effect on pathogen quantity, 90 prevalence, and duration of infection, and 2) drug effect on Salmonella spp. antibiotic 91 susceptibility. A second study was conducted to determine drug effect on resident E. coli 92 resistance. These parameters are described in the Center for Veterinary Medicine (CVM) 93 guidelines 18 and 19 titled Human Health Safety Criteria, and Animal Health Safety Criteria, 94 respectively. These guidelines are the product of the task force effort, and can be accessed 95 via the CVM internet site ( www.fda.gov/cvm ). 96 The determination of passing or failing was generally based upon statistical 97 differences 558.15 History Page 5 98 between the treated group and the control group. Aside from measuring the drug effects on 99 shedding, tissue invasion, and resistance, other measurements and/or information were 100 collected to ensure the integrity of the study. These measurements included information 101 relevant to the following questions: 102 103 1. Did the product have antibiotic properties? 104 2. Did the study address cross-contamination and did it occur? 105 3. Were there sufficient numbers of animals in the shedding and resistance studies? 106 4. Did the animals consume enough drug to test the highest intended dose? 107 5. Was any other drug intentionally or unintentionally given to the animals during the study? 108 6. Was a natural strain of Salmonella present in animals, water, or feed to confound the test? 109 7. Did the test strain of Salmonella carry a distinct stable marker? 110 8. Was the strain capable of receiving resistance factors, and could it colonize in the animal? 111 9. Was the microbiology methodology appropriate and standardized? 112 10. What tissues were examined for Salmonella spp.? 113 11. How often were samples taken? 114 12. Were the studies performed long enough to detect an effect if an effect occurred? 115 13. What level of resistance in coliforms was present in the animals in the resistance study 116 prior to starting the study? 117 118 These integrity measurements were the basis for study rejection rather than failure. Minor 119 faults in the integrity measurements were usually justified as not affecting the outcome. 120 Results from pre-approval studies intended to address the 558.15 requirements 558.15 History Page 6 121 CVM accumulated data from the shedding and resistance studies described above over 122 a 20-year period. A retrospective analysis of all the submitted data follows. The following 123 table describes results from the analysis according to the following pharmacological 124 categories. Since the results of individual products are proprietary information, the results are 125 summarized according to broad drug classes. 126 127 Drug class Total Drug:Animalb Passc Repeat Fail Reject Macrolides/Lincosamides 9 4:3 4 3 2 3 Ionophores 13 7:3 8 3 2 4 Unclassified Gram+ drug 15 6:3 7 5 4 4 Streptogramins 1 1:1 1 0 0 0 Glycopeptides 2 2:1 1 0 0 1 Bambermycins 2 1:2 2 0 0 0 Broad spectrum drug 2 2:2 1 0 1 0 TOTALa 44 24 11 9 12 a 128 Total refers to total number of studies submitted b 129 Drug:animal refers to the number of drugs tested in a drug class: number of animals species in which the drug 130 was tested c 131 Pass, repeat, or fail refers to how FDA classified their conclusion of the study results 132 133 A total of 44 sets of resistance and shedding studies were submitted. Eleven of the 134 studies were repeats of previously submitted studies. In nine of the sets, one of the two 135 studies in the set failed to satisfy the safety criteria (statistically significant difference 136 between treatment and control groups with respect to numbers of bacteria shed). Two of the 558.15 History Page 7 137 nine studies failed because of increased resistance in the resistance study, and the remainder 138 failed because of increased shedding in the shedding study. 139 140 Of the three studies where the spectrum of the drug matched the test organism's Gram 141 stain response, two of them failed because of increased resistance (one of the drugs was a 142 broad spectrum product tested in coliforms; the other drug was a Gm+ product, tested against 143 a Gm+ organism). 144 145 Two of the applications were never approved based on failure to meet the microbiological 146 safety criteria. Seven succeeded in satisfying the criteria by altering the conditions of use: 147 148 • One application was approved after the duration of treatment was shortened to 2 weeks. 149 • One application was approved after successfully repeating the study using a lower dose 150 (the application was approved with the lower dose). 151 • Two studies were repeated and the results were acceptable on the second attempt. 152 • Three studies were approved based on further information submitted by the sponsor. 153 154 Twelve of the studies were rejected for one or more of the following critical reasons (the 155 numbers in parentheses refers to the number of times this problem was identified): 156 157 • Salmonella susceptibility test results were not submitted (2); 158 • Coliform susceptibility test results were not submitted (3); 159 • Susceptibility test quality control was inadequate (3); 160 • Shedding lasted too long to measure prevalence/duration (2); 558.15 History Page 8 161 • Environmental control animals were contaminated (4); 162 • Environmental control animals were not included (1); 163 • Animals used in the study failed to meet the 20% resistance baseline for inclusion (1); 164 • The data were too disorganized to interpret(2); 165 • The study contained too few animals(1). 166 167 Problems identified 168 Although information gained over 20 years experience was useful for determining how 169 many of the drugs tested impacted pathogen shedding, problems were identified during the 170 review of the studies, and included: 171 1. Of the studies submitted, 42 out of 44 were for Gm+ spectrum drugs. The spectrum of the 172 test drug matched Salmonella spp. and coliform bacteria in two study sets (they are Gm- 173 and as such are inherently resistant to the test drugs). For similar reasons, the study 174 design prevented the detection of transfer of resistance from E. coli to Salmonella spp. 175 Most studies demonstrated the transfer of resistance to test Salmonella from the enteric 176 flora, but the transfer occurred equally often in the control group without drug pressure. 177 None of the studies showed transfer of resistance to Salmonella due to a drug effect. 178 CVM believes this drug effect was not detected because Salmonella was inherently 179 resistant to the drugs being tested, i.e. there was no selective advantage to acquired 180 resistance. 181 2. Susceptibility test results provided limited information regarding a drug’s effect on the 182 naturally-occurring intestinal flora. Susceptibility testing was performed on isolated 183 species and may not have reflected changes in the susceptibility of the majority of 184 organisms in the gut population. Studies like these should ultimately seek to capture a 558.15 History Page 9 185 picture of changes in the entire population regarding antibiotic resistance, before, during 186 and after a study for a particular antibiotic. By selecting a few isolates of a species for 187 testing there is a chance for “selective” reporting of desired MIC results, while ignoring 188 trends in changing susceptibility to the tested drug(s). 189 3. In order to measure drug effects, artificially high inocula were used. Differences detected 190 in the results may be artifacts created by the high numbers of organisms placed in the test 191 animals. 192 4. A laboratory strain of bacteria may not be representative of how naturally-occurring 193 Salmonella spp. respond when challenged by an antimicrobial drug. 194 5. Strict research conditions may not represent the actual “drug:bug” interaction that would 195 occur under normal animal management conditions. Animals in the studies are unusually 196 stressed because they must be maintained in isolation to avoid cross contamination, and 197 stress is recognized as an important factor in the response to disease challenge. 198 6. The studies typically tested a small number of animals, particularly in comparison with 199 the large numbers of animals in studies that assess effectiveness. The studies have too 200 few animals to be sure that the effect seen is really due to the drug, or even more 201 importantly, that an effect that is not seen is not one that will eventually have a substantial 202 public health impact. 203 7. The small size of the study creates environmental biases. Drug effects can be masked by 204 cross contamination when the sample size is small, and the animals in these studies were 205 housed separately in small groups to prevent cross-contamination. Consequently, 206 differences noted between treatment groups reflect environmental effects as well as drug 207 effects. 558.15 History Page 10 208 8. Retrospective analysis suggests that antibiotic drugs affect pathogen load more than 209 resistance. Recall that 7/9 studies that failed did so because of increased shedding. 210 211 Summary 212 Twenty years of experience reviewing the “558.15 studies” described above revealed 213 that in most cases, failures could be attributed to apparent pathogen shedding effects. The 214 studies conducted on some drugs clearly demonstrated an increase in the shedding of bacteria 215 of public health concern. The retrospective analysis of these studies also served to highlight 216 the various problems associated with the design and conduct of such studies and the 217 intepretation of their results. Although questions have arisen regarding these studies, they 218 were designed and conducted within the constraints of policy and regulation at the time of 219 inception, and did yield some useful information. A consideration of the potential 220 microbiological effects of antimicrobial new animal drugs has been identified as a significant 221 component of the animal drug safety evaluation process. The lessons learned from the 222 “558.15 studies” along with 20 years of advancement in scientific knowledge, are important 223 factors for helping to develop appropriate methodologies for evaluating the relevant 224 microbiological effects associated with the use of antimicrobial drugs in food-producing 225 animals.