Charm Sciences, Inc. Charm Flunixin and Beta-lactam Test
February 3, 2012
|TO:||All Regional Food and Drug Directors |
Attn: Regional Milk Specialists
|FROM:||Dairy and Egg Branch (HFS-316)|
|SUBJECT:||Charm Sciences, Inc. Charm 3 SL3 Beta-lactam Test Kit|
The Food and Drug Administration's (FDA) Center for Veterinary Medicine (CVM) has evaluated data supporting the use of the Charm Sciences, Inc. Charm Flunixin and Beta-lactam Test for raw, commingled cow milk.
The FDA evaluation of the data, presented by the Charm Sciences, Inc., indicates that the performance of the test meets the standards established to determine the acceptance of a test for use in raw, commingled cow milk. The acceptance of the test for raw, commingled cow milk represents a claim for the non-steroid anti-inflammatory drug (NSAID) Flunixin and the Beta–lactam drugs Amoxicillin, Ampicillin, Ceftiofur, Cephapirin, and Penicillin G. These data have been evaluated in accordance with the standards established for the acceptance of screening tests for monitoring raw, commingled cow milk in accordance with the provisions of Appendix N-Drug Residue Testing and Farm Surveillance of the Grade “A” Pasteurized Milk Ordinance (PMO).
Please note that the Charm Flunixin and Beta-lactam test is the first test kit approved for PMO testing capable of detecting Flunixin residues in raw, commingled cow milk. In accordance with Appendix N of the PMO: “One (1) year after test(s) have been evaluated by FDA and accepted by the NCIMS for a particular drug or drug family, other unevaluated tests are not acceptable for screening milk. The acceptance of evaluated tests does not mandate any additional screening by industry with the evaluated method.”
Therefore, one (1) year from the issuance of this M-I (February 3, 2013), the use of unapproved tests to screen for Flunixin residues in raw, commingled cow milk will not be acceptable for PMO testing.
This action was taken in response to Proposal 219 as passed at the 2011 NCIMS Conference and was taken in compliance with Appendix N of the PMO.
Attached is the letter of acceptance from FDA's CVM.
Copies of this memorandum are enclosed for distribution to Regional Milk Specialists, State Milk Regulatory Agencies, State Laboratory Evaluation Officers and State Milk Sanitation Rating Officers in your region. This memorandum should be widely distributed to representatives of the dairy industry, State Veterinarians, State Veterinary and Pharmacy Boards, Veterinarian Professional Organizations and other interested parties and will also be available on the FDA Web Site at http://www.fda.gov at a later date.
If you would like an electronic version of this document prior to it being available on the FDA Web Site, please e-mail your request to Robert.Hennes@fda.hhs.gov.
Robert F. Hennes, RS, MPH
CAPT, U.S. Public Health Service
Dairy and Egg Branch
Attachment: FDA CVM Memorandum addressing the acceptance of the test kit
Charm Flunixin and Beta-lactam Test Label Insert
DEPARTMENT OF HEALTH & HUMAN SERVICES
Food and Drug Administration College Park, MD 20740
|Date||February 3, 2012|
|From||Philip James Kijak, Ph.D.|
|Subject||Charm Sciences, Inc. Charm Flunixin and Beta-lactam Test|
|To||Robert F. Hennes, CFSAN, Dairy and Egg Branch/Milk Safety Team|
Charm Sciences, Inc. has provided data supporting the use of the Charm Flunixin and Beta-lactam Test for Flunixin, Amoxicillin, Ampicillin, Ceftiofur, Cephapirin, and Penicillin G for raw, commingled cow milk. These data has been evaluated in accordance with the standards established for the acceptance of screening tests for raw, commingled cow milk to monitor milk in accordance to the provisions of Appendix N-Drug Residue Testing and Farm Surveillance of the Grade “A” Pasteurized Milk Ordinance (PMO).
The 90/95 percent detection levels and drug concentration responses for flunixin and the five (5) beta-lactam drugs are as follows:
Drug Concentration Response (Displayed as Percent Positive)
Concentration ppb (part per billion)
* The ceftiofur tolerance is based on measuring the sum of ceftiofur and desfuroylceftiofur related metabolites in milk as desfuroylceftiofur. The screening test detection concentrations for ceftiofur were evaluated using milk containing ceftiofur and desfuroylceftiofur related metabolites from treated animals.
Please note that the Charm Flunixin and Beta-lactam test is the first test kit approved for PMO testing capable of detecting Flunixin residues in raw commingled cow milk. In accordance with Appendix N of the PMO: “One (1) year after test(s) have been evaluated by FDA and accepted by the NCIMS for a particular drug or drug family, other unevaluated tests are not acceptable for screening milk. The acceptance of evaluated tests does not mandate any additional screening by industry with the evaluated method.”
Our evaluation of the data presented by Charm Sciences, Inc. indicates that the performance of this test meets the standards established for acceptance of screening tests for monitoring raw, commingled cow milk in accordance with the provisions of Appendix N of the PMO. We recommend that the appropriate announcement be issued to the State Regulatory Agencies and the milk industry advising of the Agency’s concurrence with the use of this test as labeled. A revision of M-a-85 and M-I-96-10 should be issued to reflect the acceptance of this test.
Philip James Kijak, Ph.D.
Director, Division of Residue Chemistry
CVM Office of Research
TE Graham HFS-450
Charm® Flunixin and Beta-lactam Test for Flunixin, Amoxicillin, Ampicillin, Ceftiofur, Cephapirin, and Penicillin G for Raw, Commingled Cow Milk
The Charm Flunixin and Beta-lactam Test is a combination receptor assay and antibody assay utilizing ROSA® (Rapid One Step Assay) lateral flow technology. The Charm Flunixin and Beta-lactam Test detects flunixin, a non-steroidal anti-inflammatory drug, and five U.S. approved beta-lactam drugs at or below the U.S. Safe Level/Tolerance, see Table 1.
Equipment and Supplies Required But Not Included With Kits (order codes bolded)
The ROSA 56°C Incubator (LF-INC4-8-56D) and ROSA Pearl Reader (LF-ROSA-PEARL-X-NB) are available only from Charm Sciences. A daily hard copy print out is required for NCIMS testing and a printer (PRN-THERMAL-SYS) is available that interfaces with the reader RS-232 connector. A 300 µl pipet and a timer (the timer may be part of the incubator) are also required.
Pipet options include a fixed 300 µl pipet (PIP-300UL-1STOP-M) with disposable 200 -1000 µl Pipet Tips (1-MLT-X4) ROSA-Pipets (LF-ROSA-PIPET-500) are available for use by NCIMS screening labs only. Refer to Optional Materials and Disposable Supplies for more order options and information.
Reagent Use and Storage Requirements
- Strips are required to be shipped refrigerated.
- Store refrigerated at 0 to 4.4 °C, tightly sealed in the supplied container.
- To open, remove and save the plastic lid.
- Lift foil tab on canister and peel the foil-seal off the container. Discard foil seal.
- Inspect desiccant indicator. Beads inside desiccant packets should be blue. Do not use test strips if blue beads have turned white or pink.
- In high humidity, limit condensation by opening container after it is warmed to room temperature (20 to 30 minutes from the time the container is removed from refrigerator).
- Remove from container the number of strips to be used in one day and immediately re-seal container and return it to refrigeration. Removed strips may be kept at room temperature during daily use for up to 12 hours. Unused tests must be discarded.
- Desiccant should remain in tightly closed container at all times. Discard strips if blue beads inside desiccant packets turn white or pink.
- Always, tightly seal the container with the supplied plastic foil-lined-lid and return to refrigerated storage.
Concentration ppb (parts per billion)
Safe Level/ Tolerance (ppb)
Calculated 90/95 Detection Level(ppb)¤
¤ The 90% sensitivity with 95% confidence level is calculated based on FDA-CVM statistical analysis of independent lab blind study results. Results with the exception of ceftiofur are based on fortified
raw milk samples.
§ The test sensitivity for ceftiofur was evaluated using milk containing ceftiofur and desfuroylceftiofur (DFC) related metabolites from treated animals. The ceftiofur tolerance is based on measuring the sum of ceftiofur and desfuroylceftiofur related metabolites in milk as desfuroylceftiofur.
† 5-hydroxyflunixin is the major metabolic form of flunixin and is the chemical marker of flunixin in milk.
Results of sensitivity evaluations are presented in Table 1 and show concentration- response information — (# Positive per 30 Tests, Displayed as % Positive).
Drugs shown with * are detected more than 25% lower than their Safe Level/Tolerance. Sole reliance on the use of this test kit may result in rejection of milk which the FDA considers safe for human consumption.
60 negative milk samples were evaluated in six separate blind studies; 0 of 60 samples tested positive in four fortified drug studies and 1 of 60 samples tests positive in the ceftiofur incurred study and one drug fortified study. The test meets 90% selectivity with 95% confidence requirements.
There are no known interferences from the following non-beta-lactam animal drugs at 100 ppb: sulfadiazine, sulfanilamide, sulfathiazole, sulfamethazine, sulfapyridine, sulfadimethoxine, tetracycline, oxytetracycline, chlortetracycline, doxycycline, gentamicin, neomycin, streptomycin, ivermectin, erythromycin, pirlimycin, tilmicosin, novobiocin, furosemide, trichlormethiazide, chlorothiazide, thiabendazole, oxytocin, phenylbutazone, dexamethasone, enrofloxacin, florfenicol, AOZ, AMOZ, PABA, and dipyrone. Table 2 lists some other cross reactive beta-lactam (intra-family) drugs.
|Cefacetrile||30 ppb||Cefoperazone||9 ppb||Dicloxacillin||60 ppb|
|Cefadroxil||30 ppb||Cefquinome||75 ppb||Nafcillin||200 ppb|
|Cefalexin||50 ppb||Cefuroxime||20 ppb||Oxacillin||100 ppb|
|Cefalonium||5 ppb||Cloxacillin||75 ppb||Ticarcillin||100 ppb|
- Equipment setup and use is simple and can be self-taught from the manual. In addition, training materials and/or video is available for review of procedure, equipment use, or maintenance.
- Proficiency samples (blind coded positive and negative samples) are available for retraining and procedural validation (see Additional Test Information).
- For questions contact your local representative or Charm Sciences at +1.978.687.9200 or firstname.lastname@example.org.
Sample and Test Information
- Raw, commingled milk must be cooled and refrigerated. Refrigerated samples should be well agitated and mixed prior to testing.
- Test at ambient temperatures of 10 to 35°C in naturally circulating air.
- Milk being tested for NCIMS purposes may not be frozen at any time during testing process. Milk test must be completed within 72 hours of sampling.
- High fat samples (greater than 6.5%) may cause invalid results. Do not read invalid test strips in the ROSA Reader.
- Debris on test strips may alter the ROSA Reader optics. Keep equipment clean; wipe dust and milk off test strips before reading.
- ROSA incubator must be clean, level, and the temperature indicator green (indicating 56 ± 1°C). Keep ROSA Incubator lid lowered, but not latched, unless performing procedure. A daily temperature check is required. ROSA Incubators may take more than 10 minutes to reach proper temperature, depending on ambient temperature.
Daily Performance Check
- Obtain negative raw milk and use as a Negative Control and prepare Positive Control (sections below).
Check ROSA Reader calibration strips and verify reader performance. High and Low Calibrator strips must read within range printed on strips.
- Verify performance of test strips and equipment each day prior to testing actual samples. Rotate performance among all Flunixin and Beta-lactam Test users.
- Negative Control must be RESULT NEGATIVE and with a READING less than (more negative than) -400 on the reader.
- Positive Control must be RESULT POSITIVE and with a READING greater than +400 on reader.
Remake and test control if it tests out of range. Do not proceed and contact Charm Sciences, Inc. if still out of range.
- If calibrator strips or controls are not in specifications, do not proceed with testing. Note in official laboratory record, notify state LEO, and contact technical support at Charm Sciences.
- Use flunixin and beta-lactam negative raw, commingled milk as a Negative Control for performance monitoring and to reconstitute Standard for Positive Control.
- Qualified raw milk should give a READING less than (more negative than) -400.
- This 2 ppb Flunixin and 5 ppb Penicillin G Standard is used as the Positive Control for performance monitoring.
- Store Flunixin and Penicillin G Standards refrigerated.
- Add 10.0 ml Negative Control milk (qualified raw milk) to 2 ppb Flunixin Standard bottle. Mix well and allow to stand refrigerated or on ice for 10 minutes. Mix well again.
- Use 8.0 ml of this 2 ppb Flunixin Standard to reconstitute the 2 ppb Flunixin and 5 ppb Penicillin G Standard and shake well. Refrigerate for 5 minutes and shake before use.
- Positive Control should give a READING greater than +400.
- Negative Control may be used for up to 72 hours refrigerated. Reconstituted Positive Control may be used for up to 48 hours refrigerated.
For long term storage of controls, mix sample and freeze (at least 0.5 ml) aliquots in clean vials. Controls should be frozen within 6 hours of preparation. Freeze aliquots at -15°C or below for up to 3 weeks. Thaw slowly (overnight in refrigerator or with cool water) and shake well. Thawed sample is stable for 24 hours refrigerated. Noticeable protein precipitation indicates an unsuitable sample. Discard thawed sample. Do not refreeze.
Charm Flunixin and Beta-lactam Test for Milk – Procedure
Check that ROSA Incubator temperature is 56 ± 1°C. Use test strips with lot code FLUSLBL.
Re-shape dented sample compartments to fit into ROSA Incubator
Mix all samples well before testing. Shake 25 times in 1 foot movement for seven seconds. Controls may be vortexed.
Label test strip(s) with sample identification; test strip(s) may be placed in incubator to avoid crushing sample compartment.
Place test strip in ROSA Incubator.
Holding test strip flat in ROSA Incubator, use tab to expose sample compartment by peeling tape to “Peel to Here” line.
Using 300 µl pipet, draw up sample, avoiding foam and bubbles.
Holding pipet vertically, slowly pipet 300 µl (± 15 µl) sample into sample compartment at ROSA Incubator etched indicator line (as shown).
Reseal tape over sample compartment.
Close lid on ROSA Incubator and latch. Timer starts and red light illuminates.
Incubate for 8 minutes, but not more than 9 minutes. At 8 minutes, a beeper and alternating yellow and red blinking lights start.
Remove test strip(s) from ROSA Incubator. Do not squeeze sample compartment. Hold test strip with sample compartment in the down position until interpreted.
Inspection and Interpretation of Results
Hold the test strip vertically with sample compartment in the down position. Do not squeeze the sample compartment. Wipe foreign matter (dust, etc.) off test strips.
The test is INVALID if any of the following are observed:
Hold the test strip vertically with sample compartment in the down position. Do not squeeze the sample compartment. Wipe foreign matter (dust, etc.) off test strips.
Insert the clean and visually valid test strip into ROSA Reader as shown. Slide completely into slot.
Read results on SLBL channel of ROSA Reader (use 3-line reading mode; SLBL channel displayed in upper left of reader screen should rapidly blink). If desired, enter SAMPLE and/or OPERATOR and press ENTER. A numerical value (READING) and an interpretation (RESULT) are displayed on screen.Reader results including lot number and time/date are stored in memory for later review, download or printing, refer to ROSA Reader Manual. NCIMS policy requires a hard copy of data be printed daily.
Negative - If READING is a negative number or zero, the ROSA Reader will display RESULT NEGATIVE. Report as “Not Found”.
Positive - If READING is a positive number, the ROSA Reader will display RESULT POSITIVE. Sample should be retested; see Retest of Initial Positive.
Retest of Initial Positive
- Test “Initial Positive” samples in duplicate along with one Positive Control and one Negative Control.
- Negative Control must be RESULT NEGATIVE with a READING less than (more negative than) -400 on Reader.
- Positive Control must be RESULT POSITIVE with a READING greater than +400 on Reader.
- If either control is not within range then rerun assay. If either control is still not within range do not continue testing. Contact appropriate regulatory agency to determine milk status. Contact Charm Sciences for technical assistance with test performance.
- If both controls are within range and either or both of the retested samples are RESULT POSITIVE, sample is a “Flunixin or Beta-lactam Presumptive Positive Test”. In accordance with appendix N of the PMO, report all presumptive positives to appropriate regulatory agency. If acting as a confirmation lab, repeat Steps 1 through 4 to confirm as “Flunixin and Beta-lactam Screening Test Positive” sample.
- Call 1-800-343-2170 hours 8:30 AM - 5:30 PM EST.
- For emergency 24-hour 365-day per year service call 1-978-687-9200 and follow prompts.
- E-mail: email@example.com (monitored during 8:30 AM – 5:30 PM EST).
Additional Test Information
Optional Materials, Disposable Supplies, and Order Codes
300 μl fixed volume pipet, single stop
100 disposable ROSA-Pipets, 300 μl
200 disposable pipet tips, 200-1000 μl
500 disposable ROSA-Pipets, 300 μl
1000 disposable pipet tips, 200-1000 μl
5000 disposable ROSA-Pipets, 300 μl
Thermal printer for ROSA Reader, with RS232 adapter and cable
4 Blind Coded Proficiency Samples:
freeze-dried negative and positive samples, 8-10 ml reconstitution
Use of ROSA-Pipet (for use in NCIMS screening labs only)
Determination of Flunixin or Beta-lactam Contamination
To determine if a positive test result is caused by flunixin or beta-lactam contamination, test the sample with an approved beta-lactam test such as the Charm SL Beta-lactam Test. If the test result is negative for beta-lactam, then the sample contains flunixin. If the beta-lactam test result is positive, then the sample contains beta-lactam but could also contain flunixin. Visually compare F line to C line to determine if sample might contain flunixin. If F line is lighter than C line the sample could contain flunixin.
Post Testing - Sample and Test Strip Storage
To preserve test strips or samples for diagnostic or record keeping purposes (these are not NCIMS accepted practices):
- Samples can be preserved after testing for up to 3 weeks by freezing at -15°C or below.
- Frozen samples must be thawed and cooled before testing. Mix well to make sample homogeneous. Noticeable protein precipitation indicates an unsuitable sample for FLUSLBL lateral flow testing. Protein precipitation can be removed by centrifugation for 3 minutes at 1200 ± 200 g, and cooled before testing. Thawed/skim samples have not been validated using FLUSLBL test.
- After incubation test strips results are stable at room temperature for up to 3 minutes. Over several hours positive results gradually become more positive while negative results remain stable. If refrigerated, results are stable up to 1 hour. If frozen (-15°C or below) results are stable up to 1 week.
Visual Interpretation of Test Strip
Negative - BL (beta-lactam) line and F (flunixin) line are same as or darker/more than C (Control) line.
Positive - BL line or F line is clearly lighter/less than C line, or either BL or F line is absent, or partially or unevenly colored. Sample should be retested. See Retest of Initial Positive.
F line lighter than C line indicates likely flunixin positive sample. BL line lighter than C line indicates likely beta-lactam positive sample.
Visual interpretation is not accepted in NCIMS Milk Programs.
Charm Sciences, Inc. (“Charm”) warrants each reagent product, including but not limited to test kits, to be free from defects in materials and workmanship and to be free from deviations from the specifications and descriptions of Charm’s reagent products appearing in Charm’s product literature, when stored under appropriate conditions and given normal, proper and intended usage, until the expiration of such reagent product’s stated shelf life, or, if none is stated, for one year from the date of delivery of such reagent product to the end-user purchaser. THIS WARRANTY IS IN LIEU OF ALL OTHER WARRANTIES, WHETHER STATUTORY, EXPRESS, IMPLIED (INCLUDING WARRANTIES OF TITLE, NON-INFRINGEMENT, MERCHANTABILITY AND FITNESS FOR A PARTICULAR PURPOSE AND ALL WARRANTIES ARISING FROM COURSE OF DEALING OR USAGE OF TRADE). The warranty provided herein may not be altered except by express written agreement signed by an officer of Charm. Representations, oral or written, which are inconsistent with this warranty are not authorized and if given, should not be relied upon. In the event of a breach of the foregoing warranty, Charm’s sole obligation shall be to replace any reagent product or part thereof that proves defective in materials or workmanship within the warranty period, provided the customer notifies Charm promptly of any such defect prior to the expiration of said warranty period. The exclusive remedy provided herein shall not be deemed to have failed of its essential purpose so long as Charm is willing to replace any nonconforming reagent product or part. Charm shall not be liable for consequential, incidental, special or any other indirect damages resulting from economic loss or property damages sustained by any customer from the use of its reagent products. Except for Charm’s obligation set forth above to replace any reagent product that proves defective within the warranty period, Charm shall not be liable for any damages of any kind arising out of or caused by any incorrect or erroneous test results obtained while using any such reagent product, whether or not caused by a defect in such reagent product.