November 4, 2010
TO: All Regional Food and Drug Directors
Attn: Regional Milk Specialists
FROM: Milk Safety Branch (HFS-626)
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 3 SL3 Beta-lactam Test Kit for raw, commingled bovine milk.
The FDA evaluation of the data, presented by Charm Sciences, Inc., indicates that the performance of this test kit meets the standards established to determine the acceptance of a test kit for use in raw, commingled bovine milk. The acceptance of the test kit for raw, commingled bovine milk represents a claim for amoxicillin, ampicillin, cephapirin, ceftiofur, cloxacillin and penicillin G. This data has been evaluated in accordance with the standards established for the acceptance of screening tests for monitoring raw, commingled milk in accordance with the provisions of Appendix N-Drug Residue Testing and Farm Surveillance of the Grade “A” Pasteurized Milk Ordinance (PMO).
The NCIMS Executive Board during a conference call held September 29, 2010 accepted the use of this test kit when used as labeled. Attached is the memorandum of acceptance from FDA's CVM.
An electronic version of this memorandum is available for distribution to Regional Milk Specialists, State Milk Regulatory Agencies, State Laboratory Evaluation Officers and State Rating Officers in your region. The electronic version 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.
CAPT Robert F. Hennes
FDA, Milk Safety Team
Date: October 6, 2010
From: Philip James Kijak, Ph.D.
Subject: Charm Sciences, Inc. Charm 3 SL3 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 3 SL3 Beta-Lactam Test for amoxicillin, ampicillin, ceftiofur, cephapirin, cloxacillin, and penicillin G in raw cow milk. These data have been evaluated in accordance with the standards established for the acceptance of screening tests for raw, commingled bovine milk to monitor milk in accordance to the provisions of appendix N of the Pasteurized Milk Ordnance (PMO).
The 90/95 percent detection levels (ppb) and drug concentration responses for the six beta-lactam drugs are as follows:
Penicillin G 3.8
|Concentration ppb (part per billion)||Amoxicillin||Ampicillin||Ceftiofur*||Cephapirin||Cloxacillin||Penicillin G|
* 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.
Charm Sciences Inc. has indicated that with the acceptance of the Charm 3 SL3 test, the Charm ROSA SL3 will no longer be manufactured. The use of the ROSA SL3 test will be acceptable until the expiration date of the last manufactured lot.
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 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. We also recommend that the next revision of M-a-85 and M-I-96-10 reflect the acceptance of this test.
Philip James Kijak, Ph.D.
Director, Division of Residue Chemistry
CVM Office of Research
TE Graham HFS-450
SL3 Beta-lactam Test for Amoxicillin, Ampicillin, Ceftiofur, Cephapirin,
Cloxacillin and Penicillin G Validated for Raw, Commingled Cow Milk
The SL3 Beta-lactam Test is a receptor assay utilizing Charm 3 lateral flow technology. The Charm 3 SL3 Beta-lactam Test detects all six 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-INC4-3-56D) 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, Disposable Supplies, and Order Codes 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 (part per billion)||Amoxicillin||Ampicillin||Ceftiofur*||Cephapirin||Cloxacillin||Penicillin G|
|Safe Level/ Tolerance (ppb)||10||10||100||20||10||5|
|Calculated 90/95 Detection Level(ppb)¤§||8.4||8.0||79||20.0||8.6||3.8|
¤ 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.
Results of sensitivity evaluations are presented in Table 1 and show concentration- response information — (# Positive per 30 Tests, Displayed as % Positive).None of these drugs 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 five studies and 2 positives of 60 in one 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, enrofloxacin, florfenicol, nitrofurazone metabolites AOZ and AMOZ, furosemide, trichlormethiazide, chlorothiazide, thiabendazole, oxytocin, phenylbutazone, dexamethasone, flunixin, PABA and dipyrone. Table 2 lists some other cross reactive beta-lactam (intra-family) drugs.
|Cefacetrile||50 ppb||Cefquinome||50 ppb||Nafcillin||150 ppb|
|Cefalonium||7 ppb||Cefuroxime||200 ppb||Oxacillin||15 ppb|
|Cefazolin||30 ppb||Dicloxacillin||7 ppb||Ticarcillin||50 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 email@example.com.
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.
- If Milk being tested for NCIMS purposes may not be frozen at any time during testing process. Testing of milk samples must be completed within 72 hours of sampling. Completed includes the time required to finish the initial testing, any necessary re-testing of the initial positive, confirmation, and/or producer trace back testing.
- High fat samples (greater than 6.5%) may cause invalid results.
- Keep equipment clean.
- Keep dust and milk off exterior of test strips.
- ROSA® Incubator must be clean, 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.
Daily Performance Check
- Obtain negative raw milk and use as a Negative Control and prepare Positive Control (sections below).
- Check ROSA Pearl 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 SL3 Beta-lactam Test users.
- Negative Control must be RESULT NEGATIVE and with a READING less than (more negative than) -600 on the reader.
- Positive Control must be RESULT POSITIVE and with a READING greater than +400 on reader. Re-make/re-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 beta-lactam negative raw, commingled milk as a Negative Control for performance monitoring and to reconstitute Positive Tablet for Positive Control.
- Qualified raw milk should give a reading less than (more negative than) -600.
- The Charm 3 SL3 Beta-lactam Test uses a 5 ppb (0.0089 IU/ml) Penicillin G Positive Tablet for the Positive Control for performance monitoring.
- Store tablets refrigerated, closed in the supplied moisture resistant foil bag.
- Using a clean vial reconstitute 1 tablet with 5.0 ml Negative Control (qualified raw milk) as per label and shake well. Refrigerate for 5 minutes and shake before use. When re-using supplied vial, wash thoroughly with soap and water, rinse well, then dry between uses.
- Negative Control may be used for up to 72 hours refrigerated at 0 to 4.4°C. Reconstituted Positive Control may be used for up to 48 hours refrigerated.
- For longer storage of controls, freeze 1 ml aliquots within 6 hours of reconstitution. Frozen aliquots may be stored up to 2 months at -15°C or below. To use frozen controls, thaw slowly overnight in refrigerator or with cool tap water and shake well. Thawed milk sample may be tested without additional treatment. Noticeable protein precipitation indicates a poor sample quality and an unsuitable sample for testing.
- Thawed positive control is stable for24 hours refrigerated.
- Discard remainingthawed controls. Do not refreeze.
Charm 3 SL3 Beta-lactam Test Milk – Operating Instructions
Check that ROSA Incubator temperature is 56 ± 1°C.
Use Charm 3 SL3 test strips. Re-shape dented sample compartments to fit into ROSA Incubator.
|Step 1||Shake milk sample(s) thoroughly before testing. Refer to 2400 form Cultural Procedures – General Requirements form.
|Step 2||Label test strip(s) with sample identification(s).
|Step 3||Place test strip in ROSA Incubator.|
|Step 4||Holding test strip flat in ROSA Incubator, use tab to expose sample compartment by peeling tape to “Peel to Here” line.
|Step 5||Using 300 µl pipet, draw up sample, avoiding foam and bubbles.Note: ROSA-Pipets are available for screening testing only.|
|Step 6||Holding pipet vertically, slowly pipet 300 µl (± 15 µl) sample into sample compartment at ROSA Incubator etched indicator line (as shown).|
|Step 7||Reseal tape over sample compartment.
|Step 8||Close lid on ROSA Incubator and latch. Timer starts and red light illuminates.|
|Step 9||Incubate for 3 minutes, but not more than 3.5 minutes.
|Step 10||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.
Inspect C (Control) line for even development before placing strip in ROSA Pearl Reader.
The test is INVALID if any of the following are observed:
DO NOT PUT INVALID TEST STRIPS IN READER. IF INVALID THE SAMPLE MUST BE RETESTED.
Within 3 minutes of completing the test, insert the strip, with valid C line, into ROSA Pearl Reader. Slide completely into slot.
SLBL channel displayed in upper left of reader screen should display steadily and not blink. Use the down arrow to enter other optional information such as SAMPLE or OPERATOR ID. If strip is not fully inserted, the reader will display an error message; re-insert strip and read again. Press ENTER. A numerical READINGand a RESULT are displayed on screen in 8 seconds.
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) -600 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 “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 1-4 to confirm as “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: firstname.lastname@example.org (monitored during 8:30 AM – 5:30 PM EST).
Additional Test Information
|PIP-300UL-1STOP-M||300 μl fixed volume pipet, single stop||LF-ROSAPIPET-100||100 disposable ROSA-Pipets, 300 μl|
|1-MLT-X1||200 disposable pipet tips, 200-1000 μl||LF-ROSAPIPET-500||500 disposable ROSA-Pipets, 300 μl|
|1-MLT-X4||1000 disposable pipet tips, 200-1000 μl||LF-ROSAPIPET-5000||5000 disposable ROSA-Pipets, 300 μl|
|PRN-THERMAL-SYS||Thermal printer for ROSA Reader, with RS232 adapter and cable||UNKSTD-LF||4 Blind Coded Proficiency Samples: freeze-dried negative and positive samples, 8-10 ml reconstitution|
Post-Testing Sample and Test Strip Storage
To preserve samples or test strips for diagnostic or record keeping purposes (these are not NCIMS accepted practices):
- Samples can be preserved after testing for up to two months 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 SL3 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 SL3 test.
- After incubation test strip 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. Freezing test strips will slow additional line development and is the best method of preserving a strip test result.
|Negative - T (Test) line is same as or darker/more than C (Control) line.|
|Positive - T line is clearly lighter/less than C line, or T line is absent, or partially or unevenly colored. Sample should be retested. See Retest of Initial Positive.|
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.