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M-I-96-1: Milk Monitoring with Antimicrobial Drug Screening Tests

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HHS:PHS:FDA:CFSAN:OC:DCP:MSB

200 C Street, SW
Washington, DC 20204

M-I-96-1

January 25, 1996

TO: All Regional Food and Drug Directors
Attn: Regional Milk Specialists

FROM: Chief, Milk Safety Branch, HFS-626

SUBJECT: Milk Monitoring with Antimicrobial Drug Screening Tests

The FDA Center for Veterinary Medicine has issued a CVM Update "Milk Monitoring with Antimicrobial Drug Screening Tests". A copy is provided for your information and use in responding to questions about drug screening test methods used for monitoring raw milk as required by Appendix N of the Grade A Pasteurized Milk Ordinance.

Copies of this memorandum are provided for distribution to State Milk Sanitation Regulatory Agencies, State Milk Sanitation Rating Officers, State Laboratory Evaluation Officers and District Milk Specialists in your region. It should be made readily available to the dairy industry and to veterinarians in dairy practices.

Joseph M. Smucker


M-I-96-1

January 25, 1996

CVM UPDATE

(301) 594-1735
FAX (301) 594-1831
Center for Veterinary Medicine


MILK MONITORING WITH ANTIMICROBIAL DRUG SCREENING TESTS

Under the Public Health Service Act, the Food and Drug Administration (FDA) and the States administer the Interstate Milk Shippers Program, a voluntary Federal/State program established to ensure the safety and wholesomeness of fresh milk in the United States. Under this program, the FDA publishes the Grade A Pasteurized Milk Ordinance (PMO), a model regulation used in voluntary, cooperative interstate milk safety programs in which all 50 States, the District of Columbia, and Puerto Rico participate. The PMO specifically requires that all bulk milk pickup tankers be tested for the presence of beta-lactam drug residues.

Prior to 1991, the PMO recognized only one official test method for detecting drug residues in milk, the Bacillus stearothermophilus Disc Assay (BSDA). Changes to the PMO in 1991 required intensified testing of milk for beta-lactam residues and created the need for additional rapid, reliable screening tests that "have been evaluated through AOAC and accepted by FDA." (AOAC International, formerly known as the Association of Official Analytical Chemists, is a scientific organization whose primary objective is to validate and improve analytical methods.) As a result of this change to the PMO, 17 screening tests for beta-lactam antibiotics, one test for chloramphenicol, and one test for sulfonamide drugs have been evaluated and accepted by FDA. These tests are accepted for the monitoring of truck tanker loads of raw, commingled, bovine milk in accordance with Appendix N of the PMO and from bulk tank producer samples in accordance with Section 6 of the PMO.

The reliability of these tests to monitor the nation's milk supply has been questioned by some individuals. This CVM UPDATE addresses these issues and clarifies the role of screening tests for monitoring raw milk.

The accepted screening tests have met a standard for a low incidence of false positive and false negative findings. Combined with these standards, there are important principals of use which must be considered. These are:

  1. A positive result from a screening test is a presumptive indication that an analyte is present in the milk sample.

  2. The screening test does not necessarily identify the specific analyte causing the test to be positive nor does it measure the quantity.

  3. All the accepted tests may produce a positive result when the drug concentration is below the tolerance/safe level. This is a false violative result, not a false positive result.

  4. A chemical analysis is required to determine whether or not a given milk sample contains antimicrobial drug residues above the tolerance/safe level (violative).

  5. Despite their limitations, the accepted tests represent the "state of the art" in rapid detection of drug residues in milk.

The percentage of truck tankers found positive in 1994 (National Milk Drug Residue Third Party Data Base) was 0.063 percent. This low incidence of positive truck tankers supports our standard for selectivity (false positive test result). Further, this low incidence also demonstrates that the majority of the milk producers are using drugs in a responsible manner. The FDA has found no evidence which would indicate that the use of approved beta-lactam drugs in accordance with label directions will cause a violative or non-violative residue in a truck tanker. The FDA has concluded that despite the inherent limitations of screening tests, the issue remains one of proper drug use. The FDA believes the use of the accepted tests under the provisions of Appendix N, PMO, has reduced the amount of positive milk entering the food supply.

The FDA recognizes the economic losses to the milk producer which would result from false violative and false positive test results. With this issue in mind, the NCIMS and the FDA agreed to retest all original positive truck tanker samples using the same test when the initial test is conducted by an industry analyst. Only after the results from retesting indicate a positive finding is a truck tanker rejected. Retesting increases the probability of acceptance of a non-violative milk tanker and decreases the number of non-violative truck tanker rejections. The FDA must also be concerned with the incidence of false negative results to ensure the public health.

The low incidence of positive truck tanker results do not appear to be caused by unreliable tests. Based on reports from the States, the FDA has concluded that misuse of animal drugs is the cause of most positive test results from truck tanker testing even when residue concentrations are below the tolerance/safe level. The FDA has found no evidence which indicates that treating lactating cows in accordance with labeled directions will cause a positive truck tanker. The follow-up by the State regulatory agencies on positive truck tankers indicates that the positive test results are primarily the result of misuse of animal drugs.

It has been suggested by some individuals that testing under the provisions of Appendix N, PMO, with the FDA and NCIMS accepted screening tests be discontinued. The FDA takes the position that discontinuing tanker truck testing is not consistent with a commitment to a safe milk supply, and therefore would be unacceptable to the Agency. Prior to the implementation of Appendix N, PMO, the Government accounting Office concluded that there was no comprehensive strategy to ensure the safety of the milk supply. The State regulatory agencies and FDA are committed to maintaining a safe milk supply and have developed a comprehensive strategy for ensuring a safe milk supply. The strategy adopted by FDA and NCIMS includes monitoring of truck tankers in accordance with Appendix N, PMO, monitoring producer bulk tanks in accordance with Section 6, PMO, participation in the ten point Milk and Dairy Beef Quality Assurance Program in the event of a violation, monitoring the use and labeling of drugs through the PMO Farm Inspection Program, and individual cow testing.

Although research indicates that some screening tests may produce false positive test results in milk from individual cows, the FDA is not aware of any data which supports the conclusion that unique factors in the milk from individual cows produce false positive findings in truck tanker milk samples. The FDA maintains the view that the misuse of animal drugs causes a majority of screening test positives at the truck tanker. Despite the inherent limitations of screening tests, the issue remains one of proper drug use.

No screening test has been evaluated by the Center for Veterinary Medicine or the AOAC International Research Institute for use on milk from individual cows. Nine of the currently accepted tests for testing truck tanker milk are being evaluated for this use.

FDA has prepared a document entitled "Evaluation and Use of Milk and Antimicrobial Drug Screening Tests" which provides a detailed discussion of the evaluation and use of the screening tests as well as related issues regarding the monitoring for animal drug residues in milk. Copies of this document are available by calling CVM's Communications and Education Branch on (301) 594-1755 or by writing to Communications and Education Branch, HFV-12, CVM/FDA, 7500 Standish Place, Rockville, MD 20855. Comments or questions on this issue may be addressed to Dr. Norris E. Alderson, HFV-500, CVM/FDA, 7500 Standish Place, Rockville, MD 20855. Phone 301-594-1702; email alderson@a1.cvm.fda.gov.

FOOD AND DRUG ADMINISTRATION

EVALUATION AND USE OF MILK
ANTIMICROBIAL DRUG SCREENING TESTS

  1. BACKGROUND

    The use, in accordance with label directions, of the approximately 40 animal drugs approved for use in lactating dairy cows presents consumers with a negligible risk from residues in milk 1. The eleven approved drugs used to treat mastitis and respiratory infections -- penicillin, ampicillin, cephapirin, hetacillin 2, amoxicillin, ceftiofur, oxytetracycline, chlortetracycline, novobiocin, erythromycin, and sulfadimethoxine -- are among the most widely used drugs to treat dairy cows. The first five can cause hypersensitivity reactions in some people. Because of this, residues of beta-lactam type antibiotics are of greatest concern from the standpoint of human food safety.

    Prior to 1991, the only required monitoring of raw milk for animal drug residues was described in Section 6 of the Grade A Pasteurized Milk Ordinance (PMO). It states, "...drug tests on each producer's milk shall be conducted by State regulatory agencies at least four times during any consecutive 6 months." The tests accepted at that time by the National Conference on Interstate Milk Shipments (NCIMS) for Section 6, PMO, testing were the Difco Bacillus stearothermophilus Disc Assay (BSDA), the generic BSDA described in Appendix G of the PMO, the Charm I and II Liquid Tests, and the Delvo Test P.

  2. DEFINITIONS

    The following definitions are important to the understanding of the evaluation and use of milk antimicrobial drug screening tests:

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    1. Additional animal drugs may be given to lactating cattle by veterinarians under the Center for Veterinary Medicine's Extra Label Drug Use Policy.
    2. Residues of hetacillin are almost immediately and completely metabolized to ampicillin following administration to animals, and for this reason, are not normally considered in drug residue monitoring programs.
      1. A truly positive test result is a positive test result on a milk sample in which the actual drug concentration is at or above the established tolerance or safe level.
      2. A false violative test result is a positive test result on a milk sample in which the actual drug concentration is at or above the detectable concentration, but below the established tolerance or safe level.
      3. A false positive test result is a positive test result on a milk sample which actually contains no drug residue at a detectable concentration.
      4. A false negative test result is a negative test result on a milk sample which contains drug residue at a detectable concentration.
      5. The sensitivity rate is the percentage of truly positive samples that are found by the test to be positive.
      6. The selectivity rate is the percentage of truly negative samples that are found by the test to be negative.
      7. The estimated minimum ninety percent sensitivity level is the estimated lowest drug concentration which gives, with 95 percent confidence, a positive result with the test at least 90 percent of the time on samples which are truly positive.
  3. APPENDIX N, PASTEURIZED MILK ORDINANCE

    At their 1991 biennial meeting, the National Conference on Interstate Milk Shipments (NCIMS) passed a resolution modifying the PMO to require the contents of every bulk milk pick-up truck tanker to be tested for beta-lactam residues prior to entering the food supply. This change in the PMO, which was identified as Appendix N, created a need for rapid, reliable screening tests that could detect residues of beta-lactam antibiotics in milk when present at or above the concentrations established by the U.S. Food and Drug Administration (FDA) to be safe.

    In part because Appendix N, PMO, required methods used in monitoring programs to "have been evaluated through AOAC and accepted by FDA," the Agency began the evaluation program in cooperation with the AOAC International-Research Institute (AOACI-RI) and the A. D. Little, Inc. (A. D. Little, Inc. is no longer participating in the evaluation program). These firms served as independent third parties in the initial evaluation.

    A protocol was developed for the evaluation of the tests for detecting antibiotics in raw, bulk, commingled milk. This protocol was developed with cooperation and input of representatives of all interests of the milk industry. Since the NCIMS (Appendix N, PMO) required monitoring at the truck tanker, the protocol was only for raw, bulk, commingled milk and not for milk from individual cows.

    The selectivity and sensitivity data developed by the test sponsors were verified in an independent laboratory. The selectivity standard used requires tests to correctly identify, with 95 percent confidence, milk containing no drug residues in 90 percent of the samples (90/95). The initial sensitivity standard required the tests to detect milk containing residues of claimed drugs at their tolerance/safe levels 90 percent of the time with 95 percent confidence. A test could meet these standards by correctly identifying 30 of 30 zero control samples and 30 of 30 samples containing each claimed drug at its tolerance/safe level. With this selectivity standard for acceptance, the probability is low that an accepted screening test would produce a positive result on a milk sample that does not contain any of the drug it is designed to detect.

    Although the only standards FDA established for acceptance of tests were those for selectivity rate and sensitivity rate, other parameters of test performance were also evaluated in the joint program, and the sensitivity was validated with incurred residues in milk. Parameters studied include: effect of the presence in milk of PMO-permitted levels of bacteria and somatic cells; possible interference of other classes of animal drugs which might be present as residues in milk; the percent of positive results to be expected from a test when milk containing residue levels below a drug's tolerance are assayed (dose response)(false violative test results); and ruggedness testing provided for the evaluation of variation in times, volumes, temperatures, etc. in the directions for operating the test that would impact the performance of each test. In requiring the incurred residue parameter, milk from animals receiving the different drugs are evaluated using the tests. This requires that the tests detect the incurred residues at the tolerance/safe level with the concentration in the milk being quantitatively determined by a chemical test. The objective of including these parameters of test performance is to insure that manufacturers of FDA accepted tests provide potential users with as much information as possible about the performance of a given test. This enables individual users to make informed decisions about which test is the best one for them to use and also to encourage test manufacturers to develop improved tests.

    To date, 17 screening tests for beta-lactam residues, one test for chloramphenicol, and one test for sulfonamide residues in milk have been accepted by FDA, certified as "performance tested" by AOACI-RI, and/or accepted by A.D. Little, Inc. These tests are also accepted by the NCIMS for Appendix N, PMO, testing. The attached Chart contains the tolerance/safe levels for approved beta-lactam drugs and the 90/95 percent sensitivity levels for accepted tests.3

  4. BACILLUS STEAROTHERMOPHILUS DISC ASSAY(BSDA)

    Appendix G of the PMO presently recognizes a generic BSDA. The BSDA is a microbiological test based on growth inhibition to detect antibiotic presence. Performance of the BSDA was evaluated in 1982 in an AOAC collaborative study using only penicillin. A 16 mm zone was accepted as the standard for a violative result. A positive test result with this test indicates that an inhibitor is present in the milk. It does not provide the identity nor quantity of the inhibitor. In addition to penicillin, this BSDA procedure has been demonstrated to detect residues of ampicillin, cephapirin, and amoxicillin at or below safe levels. The test also detects residues of ceftiofur at slightly above its level of concern for misuse.

    Charm Sciences and Difco Laboratories market a complete BSDA kit. The Charm Sciences' test has been evaluated and accepted for Appendix N monitoring. Difco recently decided to have its test evaluated. At this time, the Difco BSDA is not accepted for Appendix N, PMO, testing.

    At the 1995 meeting of the NCIMS, all the tests accepted for milk monitoring under the provisions of Appendix N, PMO, were also accepted

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    1. The BSDA described in Appendix G and discussed above as having been evaluated earlier by AOAC, was widely used in the years prior to 1991. The test was available as a complete kit from Difco Laboratories, but some testing facilities used a generic BSDA made in-house from agar, spores, standards, etc. obtained from Difco or from other sources. The BSDA listed in the attached Chart is a complete kit manufactured by CharmSciences, Inc. Neither the Difco BSDA nor any generic BSDA was submitted for inclusion in the Agency's initial evaluation of the BSDA test. Under the provisions of Appendix N, any version of the BSDA that has not been accepted by FDA should no longer be used to monitor tanker truck milk.

    for Section 6, PMO, testing (producer bulk tank). The Difco BSDA, the Delvo Test P 5 Pack, the generic BSDA described in Appendix G, PMO, and the Charm I and II Liquid tests are accepted for use on farm bulk tank samples but not Appendix N, PMO, tanker truck samples. These tests have not passed the performance standards of the Appendix N, PMO, tests, and their use in regulatory decisions is an issue that is expected to be an item for consideration by the NCIMS. (Note: The Charm I and II Liquid tests are no longer manufactured

  5. SENSITIVITY STANDARD

    Since the screening tests give positive results at drug concentrations below the tolerance/safe level, there have been a number of questions regarding the acceptance of the tests. These comments have inferred that tests should not have been accepted by the NCIMS as they were to have been evaluated exactly "at the safe level or tolerance" (Appendix N, PMO).

    The original evaluation required a determination of a 90/95 percent concentration at or below the tolerance/safe level. All the evaluations have included the tolerance/safe level. Had FDA not accepted tests which give a positive result at drug concentrations below the tolerance/safe level, none of the tests would have been accepted (including the current Section 6, PMO, tests). The current state of screening test technology is inadequate for this level of precision.

    All the tests have a characteristic sigmoid response curve. This means that as drug concentration increases, there is also a corresponding increase in percent positives until you reach a concentration plateau after which all samples will be positive. In the original evaluations, only a minimal number of assays were required at the lower concentrations. The Food and Drug Administration originally established a sensitivity rate standard requiring a test to give 90 percent positive results with 95 percent confidence on milk samples containing drug concentrations at or below the tolerance/safe level. All the tests met this standard for each drug claimed on the label. When the data were presented to the NCIMS, they requested that the test sponsors provide data which define the dose response at the lower drug concentrations with equal confidence as the 90/95 percent level. These data provide the means to determine the estimated minimal 90 percent level, with 95 percent confidence. The development of these data is nearing completion and will be reflected on revised labeling for each test. The FDA remains convinced that the dose response information is important to the user in deciding on the test to use. This information is on the label of each accepted test.

    Ideally, the accepted tests should give no positive results when there are drug residues below the tolerance/safe level (false violative test result). Equally desired is for the tests to give a positive result 100 percent of the time when the drug concentration is exactly at or above the tolerance/safe level (no false negative test results). The screening tests evaluated to date do not perform in this manner. They do not "turn on and off" precisely at the tolerance/safe level.
     

  6. QUESTIONS/ISSUES ON PERFORMANCE OF THE CURRENTLY ACCEPTED TESTS

    There is no ideal screening test currently available for detecting antimicrobial drug residues in milk (see attached Chart). None of the beta-lactam tests detect all the beta-lactam drugs. Some have the 90/95 percent sensitivity level for specific drugs below the tolerance/safe level, and all give false violative test results. These multi-residue tests are developed to be sensitive to at least four of the beta-lactam drugs at their tolerance/safe level. Each multi-residue test, including the BSDA, detects one or more drugs at levels below their tolerance, and each test fails to detect one or more approved beta-lactam drugs at their tolerances. There is a consumer concern when a test is used which does not detect all the beta-lactam drugs because milk might contain residue that has not been shown to be safe. The above is also true for the sulfonamide test.

    The rejection of safe milk is an economic issue for the milk producer. Currently the NCIMS accepted screening tests, including the BSDA, produce positive results on a certain proportion of samples which contain below tolerance/safe level concentrations of residues (false violative test results). For example, a test that demonstrates a 90/95 percent sensitivity of 10 ppb for residues of ampicillin, might produce a positive result on 40 percent of the samples with 4 ppb, etc. Of course, the number of truck tankers that would be rejected because of this detection characteristic of screening tests is a function of the number of truck tankers that arrive at a processor containing 4 ppb of ampicillin. Our best estimates of this number is that it is low. To help test users select the most appropriate test for their situation and minimize the number of false violative test results, the Agency requires concentration-response data to be placed on the labels of all accepted screening tests.

    Furthermore, it is obvious from Chart that different tests demonstrate different sensitivities toward the six approved beta-lactams. It is not surprising therefore, that the action of replacing the single official BSDA (Appendix G, PMO) with 17 different, more reliable, and some more sensitive, accepted beta-lactam tests has resulted in some confusion. For example, the same sample of milk could produce different results when tested by different receiving stations, processors, or State regulatory agencies, that do not employ the same test.

    Recognizing the economic importance of false violative test results to the milk producer, the Agency and the NCIMS agreed to a procedure whereby all truck tankers found to have a positive test result in their initial screening test must be confirmed using a procedure to reduce the incidence of false positive and false violative test results. This is actually a retesting of the positive sample using the same test when the initial test is conducted by the industry analyst. This retesting includes the following:

    1. Positive control sample -- Ensures that truly positive milk will test positive.
    2. Negative control sample -- Ensures that truly negative milk will test negative.
    3. Two truck tanker samples -- For the truck tanker to be rejected, one of the retests must be positive.

    In the example of the test described above, the follow-up testing procedure would reduce the chance of finding a positive truck tanker with milk containing 4 ppb of ampicillin from 40 to 26 percent. Some comments have criticized this procedure because of use of the term "confirm." The purpose of the retesting is to reduce the number of false violatives.

    The following are other issues for consideration in forming an opinion regarding the use of milk residue screening tests:

    1. Milk producers rarely, if ever, treat their entire herd with an antibiotic. Rather, in most instances, a few cows are treated at the same time. Following the milk discard time, the milk from a few treated cows is commingled in the farm bulk tank with a greater amount of milk from untreated animals. Farm bulk tank milk is normally further diluted with milk from several farms while being transported to a milk processing plant where it is tested. The effect of this dilution virtually eliminates the possibility that truly positive truck tanker milk will occur when drugs are used in an approved manner. For a test to give a positive result, even if it is a false violative test result, the drug generally must have been misused in the cow(s) that contributed the milk containing residues at a high enough concentration to contaminate the truck tanker. A positive test result on a truck tanker sample is an indication that the producer whose milk caused the positive truck tanker test offered milk for sale which was above the recommended tolerance/safe level (regulatory action level). Follow-up testing on producer samples and on-farm investigations confirm this conclusion. The proper use of approved animal drugs in individual dairy cows does not cause the accepted screening tests to produce a truly positive truck tanker result as some may suggest.

      In accordance with the provisions of Appendix N, PMO, all positive test results must be investigated. As a false violative test result is a possible outcome from the monitoring of milk with screening tests and based on a case by case review of each investigation, additional testing and/or investigation may be justified.

    2. An objective of the National Drug Residue Milk Monitoring Program (NDRMMP) is to estimate the incidence of non-beta-lactam drug residues. This objective is being accomplished through use of screening tests for other animal drugs which have neither been evaluated nor accepted for use in Appendix N type monitoring programs.

      Some have misinterpreted data from FDA's NDRMMP to conclude that the screening tests used in this program produced an 85.6 percent false positive rate. This is a misrepresentation of the data. These quarterly reports include any screening test samples submitted by the states to FDA for confirmation as well as the NDRMMP samples. Those samples previously found to be positive by screening tests are reported as violative as determined by chemical tests only when the drug concentration is found to be at or above the tolerance/safe level. The majority of the screening test positive samples (presumptive positive result) have been found to contain detectable concentrations of drug; these are false violative test results. This misleading report of a 85.6 percent false positive results includes those samples found to contain drug concentrations below the tolerance/safe level but not reported as violative. Except for the beta/lactam samples, all confirmed positive test results are based on a chemical test.

    3. The FDA funds a contract under which State milk regulatory agencies report the results of milk monitoring in their states. The proportion of reported positive loads throughout the country has been very low. In 1994, there was 3.2 million truck tanker loads tested with 0.063 percent found positive (63 of each 100,000 truck tankers). The milk rejected for human food totaled approximately 68 million pounds which is approximately 0.044 percent of the milk supply. These numbers represent the maximum percent of positive test results at or above the tolerance/safe level plus false violative and false positive test results.
    4. Several reports in the scientific literature describe the presence of "natural defense secretions" in milk from mastitic cows that will produce positive screening test results on milk from cows that have not been treated with any animal drug. These data indicate that certain screening tests produce false positive test results in milk from individual cows secreting these natural inhibitors. Some authors have attempted to apply the false positive rates obtained in milk from individual cows to samples from truck tankers containing commingled milk derived from many cows. The FDA believes these are invalid comparisons as the data reported would not support a conclusion that false positive milk from one or several cows produces a false positive test result in truck tanker milk. Using the level of somatic cells as an indicator of the presence of natural inhibitors, none of the research and test evaluations conducted at FDA has revealed an effect on test performance from natural inhibitors unless the milk has a somatic cell count that exceeds several million - many times the level of these cells permitted under the PMO.
  7. TRULY POSITIVE, FALSE POSITIVE, OR FALSE VIOLATIVE TEST RESULT

    Milk discard time is the number of hours after treatment that are required for residues in milk to reach the tolerance/safe level. The milk discard time is not the point at which residues can no longer be detected with the chemical tests or some screening tests. Currently, milk discard times are established using a chemical test; the accepted milk screening tests do not have the required analytical characteristics to establish official milk discard times.

  8. EVALUATION OF SCREENING TESTS FOR USE IN MILK FROM INDIVIDUAL COWS

    The FDA announced in 1993 its interest in developing a protocol for the evaluation of screening tests for milk from individual cows. A number of meetings were held involving representatives of the industry, including the National Mastitis Council. A protocol was designed using the same administrative procedures as for the accepted tests for commingled milk with the test manufacturers paying for the evaluation. As there is no regulatory requirement for the testing of milk from individual cows, the manufacturers saw no economic incentive to have their tests evaluated; and consequently, no tests have been evaluated under this protocol.

    Given FDA's view of the importance of having reliable, independently evaluated tests available for use by the producer/practitioner in milk from individual cows, the Agency decided to fund an evaluation of the tests. Recognizing the screening tests which had already been evaluated for commingled milk and the parameters included in that evaluation, FDA developed a different protocol using the selectivity and mastitis concerns as parameters for additional evaluation. This revised protocol was also reviewed by representative of the National Mastitis Council. The protocol addresses the issues of false positive test results in milk from individual cows which are healthy and have not been treated with drugs and also a mastitis model to address the issue of cows recovering from mastitis. In the mastitis model study, the tests must give the correct result on a milk sample from healthy cows and also on visually normal milk following recovery from an endotoxin challenge in the udder. The tests must also give the correct response on the visually normal milk when claimed drugs are added. The Agricultural Research Service at the United States Department of Agriculture, Beltsville, MD is conducting the studies under an Interagency Agreement with the Center for Veterinary Medicine. Test manufacturers requested that nine of the accepted beta-lactam tests be evaluated. The selectivity study section of the protocol has been completed. The mastitis model study was completed in 1995. The FDA expects to issue a report on these studies in the spring of 1996.

    As screening tests can detect drug concentrations below the tolerance/safe level, a positive test result is possible with a screening test on a milk sample collected from cows after the labeled milk discard time. For this reason, FDA does not recommend the use of screening tests on cows which have been individually treated in accordance with label directions. It should be noted, however, that these lower concentrations will not cause a violative or non-violative positive truck tanker except in those exceptions described previously.

    If it is desirable to have no detectable residue in milk from individual cows, then testing with a screening test until the sample is negative is a viable option. To preclude the possibility of a truck tanker positive result, screening tests may also be used in milk from individual cows treated in an extra-label manner.

  9. STATE OF THE ART

    The FDA recognizes that screening tests for detecting antimicrobial drugs in milk are neither drug specific nor quantitative in their performance. A positive test result means that something in the milk caused this outcome. The evaluation protocol used provides ample assurance that a positive test will result when the drug claimed on the label is in the sample at concentrations at or above the tolerance/safe level. Similarly, FDA is confident that the test result will be negative when there is no drug in the sample. The residue concentration between no detectable concentration and the 90/95 percent concentration (false violative test result) is a concern, particularly for the producer, as these concentrations may cause a positive test result and the truck tanker of milk deemed unacceptable for human food. From a human safety perspective, FDA is also concerned for the false negative potential of the tests.

    It is important to understand that under Section 6 of the PMO, producer bulk tank milk has been monitored for a number of years, before implementation of Appendix N, PMO, using some of the accepted Appendix N, PMO, tests. These screening tests have the same limitations whether used as a Section 6, PMO, test or an Appendix N, PMO, test. The difference is that now the PMO requires the monitoring of each tanker truck for beta-lactam residues before entry into the food chain.

  10. SUMMARY

    Some have inaccurately reported results of milk monitoring to advance the proposition that the use of the accepted milk residue screening tests be discontinued until more accurate tests are available. The percentage of truck tankers found positive in 1994 (National Milk Drug Residue Third Party Data Base) was 0.063 percent. This low incidence of positive truck tankers supports our standard for selectivity (false positive test result). Further, this low incidence of positive truck tankers also demonstrates that the majority of the milk producers are using drugs in a responsible manner. The FDA has found no evidence which indicates that the use of approved drugs in accordance with label directions will cause a positive test result in a truck tanker with the possible exception when chlortetracycline is administered in feed on a herd basis. The FDA has concluded that despite the inherent limitations of screening tests, the issue remains one of proper drug use. The FDA believes the use of the accepted tests under the provisions of Appendix N, PMO, has reduced the amount of milk containing antimicrobial drugs entering the food supply.

    The low incidence of positive truck tankers and the rejection of those truck tanker loads for human food is recognition of the milk industry's focus on and commitment to the human safety of milk. This low incidence and Appendix N, PMO, monitoring is a significant factor for the milk industry in maintaining consumer confidence in the safety of milk. The reliability of the accepted tests to detect drug residues adds credibility to the testing program, and the small percentage of positive tankers confirms the validity of the acceptance standards for the tests.

    The FDA recognizes the limitations of the screening tests. These are:

    1. The tests cannot identify the drug residue nor the concentration of the residue. A safety determination cannot be determined from a positive result.
    2. The screening tests produce some positive results when the drug concentration is below the tolerance/safe level. Under the provisions of Appendix N, PMO, this limitation provides for a few positive truck tanker loads of milk to be rejected for human consumption when the milk is safe.

    While these accepted tests have their limitations in drug identification and quantification, they work well in detecting violative drug residues in milk. The low probability of a false positive and a false negative finding is inherent in the acceptability standards of the evaluation. The FDA has found no evidence which would indicate that the accepted tests are not performing in this manner.

    The FDA recognizes the economic losses to the milk producer which would result from false violative and false positive test results. With this issue in mind, the NCIMS and the FDA agreed to retest all original positive truck tanker samples using the same test when tested by an industry analyst. Only after the results from retesting indicate a positive finding is a truck tanker rejected. Retesting increases the probability of acceptance of a non-violative milk tanker and decreases the number of non-violative truck tanker rejections. The FDA must also be concerned with the incidence of false negative results to ensure the public health.

    Based on reports from the states, the FDA has concluded that misuse of animal drugs is the cause of most positive test results from truck tanker testing even when residue concentrations are below the tolerance/safe level. The FDA has found no evidence which indicates that treating lactating cows in accordance with labeled directions will cause a positive truck tanker. The low incidence of positive truck tanker results do not appear to be caused by unreliable tests. The follow-up by the State regulatory agencies on positive truck tankers indicates that the positive test results are primarily the result of misuse of animal drugs.

Office of Science, HFV-500
CVM/FDA
7500 Standish Place
Rockville, MD 20855

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MILK DRUG RESIDUE TEST DETECTION LEVELSa

A. SCREENING TESTS FOR BETA-LACTAM DRUGS

DRUG PEN AMP AMOX CLOX CEPH CEFT
Tolerance/Safe
Level PPB
5 10 10 10 20 50
TEST            
Charm I/Cowside
II Test for
Cloxacillin
NDb NDb NDb 10 NDb NDb
Charm II Tablet
Competitive Assay
4.8 9 10 70c 4.5 23
Charm Farm Test 5 10 10 40 20 25
Charm II Tablet
Sequential Assay
4.8 8 10 50c 4.5 23
Charm II Tablet
Transit Test
4.8 8 10 80c 4.5 13
Charm Rapid
Inhibition Test
3 4.5 4.5 25c 16 50
Charm I/Charm II
Tablet Test
4.8 10 10 50c 8 40
Charm B.
Stearothermophilus
Disk Assay
5 6.5 10 48c 11 75c
Delvotest P 3 10 8 30c 8 50
Delvo-X-PRESS 5 10 10 50 10 10
Lactek B-L 5 10 8 8 16 NDb
Lactek CEF NDb NDb NDb NDb NDb 50
Penzyme III Test 5 10 8 80c 8 80
Penzyme Milk Test 5 10 8 80 8 80
SNAP Test 5 10 10 50c 8 50

B. SCREENING TESTS FOR OTHER DRUGS

DRUG CAP SMZ SDZ SDM STZ
Tolerance/Safe
Level PPB
1 10 10 10 10
TEST          
Charm II
Chlororamphenicol
Test
1 NDb NDb NDb NDb
Charm II
Chlororamphenicol
Test
NDb 9.6 5.8 3.6 7.5

a - PPB which can be detected by test 90 percent of time with 95 percent confidence.

b - ND indicates not detected at 100 ppb.

c - Precise 90/95% levels were not determined for these sensitivities that are significantly above the tolerance/safe level.

ABBREVIATIONS

PEN = Penicillin
AMP = Ampicillin
AMOX = Amoxicillin
CLOX = Cloxacillin
CEPH = Cephapirin
CEFT = Ceftiofur
CAP = Chloramphenicol
SMZ = Sulfamethazine
SDM = Sulfadimethoxine
STZ = Sulfathiazole

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