PMF 005484 C 0002

April 28, 2000

Meg Oeller, D.V.M.
FDA Liaison to the NRSP-7
HFV-130
Center for Veterinary Medicine
7500 Standish Place
Rockville, Maryland 20855

Dear Dr. Oeller:

We refer to your submission to the public master file (PMF 005484) dated September 17, 1999, for the use of ivermectin pour-on for the treatment and control of the gastrointestinal nematode, Ostertagia ostertagi, in the American bison. Included in this submission are a Target Animal Safety study, a Tissue Residue Depletion study, and a Freedom of Information Summary. You requested acceptance of this public master file and a notice of the availability of the data to be published in the FEDERAL REGISTER.

We have reviewed your submission and have the following comments.

EFFECTIVENESS – Complete

This section was acceptable as transmitted in the CVM letter dated February 9, 1995, in response to the C-0002 submission.

TARGET ANIMAL SAFETY - Complete

HUMAN FOOD SAFETY - Incomplete

In-Life Study:

We conclude that the in-life portion of the tissue residue study was conducted so as to provide the types of data needed to evaluate the depletion of ivermectin residues in bison tissues. We have the following comments:

The laboratory notebook for the in-life portion of the study identifies one protocol deviation associated with the collection of the tissue residue samples. For several of the animals there was only a limited amount of treatment site adipose tissue. As such, the goal of collecting 50 g of treatment site adipose tissue was not met for several animals. No adipose tissue was collected from animals #8, 20, and 22.

We note that animal #20 is a control animal and we would anticipate no residues of ivermectin in any of the collected tissues. Therefore, the omission of treatment site adipose tissue for this animal is not considered a significant protocol deviation. We note, however, that residue values are provided for treatment site adipose tissue for animals #8 and 22. As these animals are treated animals from which the laboratory notebook indicated fat samples were not collected, you should explain how residue values were available from non-collected samples.

The laboratory notebook indicates that one animal would not enter the trailer catch pen at the time of sacrifice. This animal was euthanized using a pistol shot to the head rather than the captive bolt method used on the other animals. Although we anticipate that this deviation is unlikely to have adversely affected the interpretation of the study results, the information is contained only in the laboratory notebook. You should revise the final study report and Freedom of Information (FOI) Summary to reflect this information.

We note that several entries in the laboratory notebook were made after the events they describe (i.e., entry dated 02/26/96 describes the weighing, examining, tagging, pretreatment bleeding and treatment of the bison on 02/21/96). Although we believe that this procedure is unlikely to have adversely affected the reporting of the study events, we remind you that entries should be made coincident with the events they report.

We note that both control animals were sacrificed at the beginning of the necropsy schedule. Our records do not indicate that the protocol for this study was reviewed prior to its initiation. Had the study protocol been reviewed, we would have indicated our preference that the control animals be interspersed within the sacrifice schedule. Although we believe that this procedure is unlikely to have adversely affected the interpretation of the study results, we remind you that it is preferable for control animals to be interspersed throughout the sacrifice schedule.

We note that the physical examination sheets provide for data entry regarding the weight and body temperature for each animal. For both weight and temperature, the examination sheets call for the examiner to write in the numerical value for each parameter and circle the appropriate units, either metric or English (i.e., kg or lbs for body weight and °C or °F for body temperature). None of the examination sheets include appropriate annotations for body weight or body temperature units. On the basis of the information included in the study report, the magnitude of the entered numbers (i.e., body temperatures above 100 degrees can only be °F), as well as the ivermectin dose subsequently calculated for each animal, we conclude that both weight and body temperature were recorded in English units (i.e., body weight in lb and body temperature in °F).

We note that although the Quality Assurance unit inspected the animal facility, animal dosing and necropsy, as well as the final study report, several errors in the submitted materials went undetected:

1. The failure to include units for body weight and temperature was not identified as a study deviation.
2. The QA unit also failed to note that the technical report incorrectly identifies both the location of the NRSP-7 sponsor and the storage site for the raw data and final study report as the University of Michigan. We believe the correct designation for the NRSP-7 sponsor is Michigan State University and we anticipate that this is also the storage site for the raw data and final study report.
3. The QA statements indicate that the QA inspections were February 20-22, although the laboratory notebook does not indicate that the site was visited on February 20, 1996, a Tuesday.

Analytical Study:

We note that none of the analytical phase of the study was QA inspected.

With regard to the study time line, we note that although samples were immediately frozen following collection (<1 hr), the same cannot be said for the residue analysis schedule. We calculate an elapsed time of 14 (liver) to 21 (muscle) months from the collection of the samples to their analyses. There is no storage stability data provided for these samples. You should provide storage stability data for samples retained for more than 1 week between collection and analysis.

The analytical report contains individual animal/tissue data. However, there are no chromatograms or chromatographic data printouts (i.e., uV response) to allow us to confirm the reported concentrations. Since these data are also missing for the standard curves, it is not possible to confirm either the equations for the standard curves or to pair standard curves with the sample analyses in which they were used. You should provide 10% of the chromatograms/uV response printouts so that the individual animal data can be cross-referenced to specific chromatographic data. You should annotate and date the submitted chromatograms/uV response printouts, as necessary, to allow ready identification and cross reference to the individual animal data.

We note that the standard curves used throughout the study cover concentrations from 25 ng/mL to 150 ng/mL. From the handwritten annotations included with the published regulatory analytical method¹, we conclude that these concentrations were selected on the basis of the safe concentrations for residues in cattle (and swine) identified in the paper (i.e., 50 ppb and 75 ppb, respectively, for cattle and swine liver at that time of the publication), rather than the codified tolerances, even though the tolerances for the marker residue in the target tissues were stated in the publication (i.e., 15 ppb and 20 ppb, respectively, for cattle and swine liver at that time of the publication) and are available for all edible tissues under 21 CFR 556.344. We note that standard method validation would include a demonstration that the method performs satisfactorily at ½ the tolerance for the marker residue in the target tissue (i.e., for bison liver, half the tolerance of 15 ppb or 8 ppb). There are several samples, including all of the muscle samples, where the concentrations reported are less than the 25 ng/mL standard on the standard curve. The analytical report also lacks LOD/LOQ information and supporting data. You should identify and provide data to support the selected values for LOD and LOQ for this study.

Although your laboratory SOPs indicate that samples will be rerun if the %RSD is greater than 10%, we find several samples where these SOPs do not appear to have been followed (i.e., ABFATSUM #15, 26; LIVER # 19, 5, 6, 8, 11, 22; RENAL FAT # 9, 10, 6, 26, 15, 22, 11; THORASIC FAT # 9, 5, 19, 6, 2, 10, 26, 15, 11; STERNAL FAT # 9, 5, 10, 26, 16, 15,11, 22; TREATMENT SITE FAT # 11; MUSCLE # 5, 6, 8, 11, 22). You should provide an explanation for this apparent deviation from the laboratory SOP.

Conversely, there are a number of report sheets where the analyst indicates that “peak heights were greater than the highest standard” and samples were “diluted and re-injected”, although from the submitted materials we see no indication that a dilution has occurred or that it was needed. Additionally, we note that the values for the reinjected samples are nearly identical to those of the original samples. Until the chromatographic raw data are submitted for our review, we are precluded from accepting this conclusion and we are unable to determine which of the residue values should be included in our withdrawal analysis.

Reported recoveries from spiked tissues are 10-20% less in bison than in the reported values for cattle. You have not provided parallel recovery data in cattle tissues. Therefore, it is not possible to determine whether the lower recovery is related to a difference between cattle and bison tissues or represents a lack of “readiness to perform” on the part of your laboratory. We again remind you of the need to demonstrate a “readiness to perform” before undertaking the analysis of incurred study samples.

We will be able to continue our review of your residue depletion study in bison following submission of this additional information:

1. An explanation for the presence of treatment site residue values from animals identified in the in-life laboratory notebook as not having adipose tissue at the treatment site.
2. Confirmation that the weights of animals recorded on the examination sheets are in “pounds” and that the body temperatures are in “°F”. You should also note the absence of this information as a protocol deviation.
3. Corrected report identifying the location of the NRSP-7 sponsor and data storage as Michigan State University.
4. An explanation for the lack of entry in the in-life laboratory notebook if the QA unit inspected on February 20, 1996.
5. The QA Statement form does not indicate who conducted the specific inspection phases. If the inspections were conducted by QA personnel other than Ms. Ogletree, the inspection report should be annotated with the individual inspectors’ names. Finally, Ms. Ogletree’s affiliation is not indicated on the QA statement. To ensure an independent QA audit, the affiliation of the Quality Assurance Coordinator should be identified.
6. An explanation for the lack of QA inspection of the analytical phase of the study.
7. The submission of an independent QA audit of the analytical phase of the study will be necessary before any data from the analytical laboratory can be accepted. As noted above, the QA inspector/coordinator should be identified and their affiliation noted. Since the QA audit will occur after the fact, the entire analytical phase should be audited retrospectively and all deviations from GLP noted.
8.  Storage stability data for all samples held for more than 1 week between the time of collection and the time of analysis.
9. A minimum of 10% of the chromatograms/uV response printouts for individual animal samples. This should be a random sampling of all animals, time points and tissues. You should annotate the submitted chromatograms/uV response printouts to allow ready identification and cross reference to the individual animal data. For GLP purposes, these annotations should be initialed and dated.
10. Identification of the LOD and LOQ for the analysis used in this study and the data to support the selected values. Please note that none of the residue values less than the LOQ should be used in the calculation of any mean residue values.
11. An explanation for the apparent deviation from your laboratory SOP regarding the rerunning of samples with %RSD greater than 10%.
12. An explanation for the annotations regarding samples with “peak heights were greater than the highest standard” and “diluted and re-injected”. Also, you should identify which of the values should be used in our subsequent withdrawal calculations and an explanation for their selection.
13. You should confirm the dose of ivermectin Pour-On administered to treated animals. The study report or the FOI Summary should be revised accordingly.

ENVIRONMENTAL - Complete

This action qualifies for a categorical exclusion from the requirement of preparing an environmental assessment under 21 CFR 25.33 (d)(4). Therefore, neither an environmental assessment nor an environmental impact statement is required.

FREEDOM OF INFORMATION SUMMARY - Incomplete

The Effectiveness section of the FOI was reviewed and accepted in the C-0001 submission. The following changes were made to the Target Animal Safety section of the FOI summary. In section C(1) the parenthetical dosage was changed from "500 mcg ivermectin/kg body weight" to "2500mcg…". In the "Agency Conclusions" section, "hypodermosis" was replaced with "ostertagiasis" and "Hypoderma bovis" was replaced with "Ostertagia ostertagi".

With regards to the Human Food Safety section of the FOI, the text indicates that control animals received a dose of 1 mL vehicle/10 kg while treated animals received 5 mL IvomecÒ/10 kg (2500 mg/kg body weight). We note that the text of the study report and the treatment records indicate that both treated and control animals received 1 mL/10 kg body weight (500 mg/kg body weight). We believe the language in the FOI Summary has been copied in error from the Target Animal Safety section in which a 5X-safety study was described. You should confirm the dose of ivermectin administered to treated animals and revise either the study report or the FOI Summary (text and residue table), as appropriate.

You also should redraft the section describing the collection of tissues. As currently written, this section indicates that all animals were sacrificed via captive bolt and exsanguination whereas the in-life report indicates that one of the animals in the 1 day withdrawal group was sacrifice via pistol shot

Your tissue residue table should be condensed to show only Mean±SD for each of the tissues, by withdrawal period. Only residue values above the LOQ should be used in these calculations.

Finally, the last paragraph referring to the possible withdrawal assignment should be deleted in its entirety. A decision regarding the withdrawal period will be made at the time the NADA is submitted for this supplemental species claim.

You should submit a revised FOI Summary that:

1. Correctly identifies the sacrifice of animals used in this study.
2. Contains a revised mean residue table. The revised table should contain only means ± SD and these should be calculated using only residue values above the LOQ.
3. Deletes all reference to the calculated withdrawal period.

We will defer final acceptance of this FOI summary until all issues (Human Food Safety) have been addressed

Future correspondence regarding this letter should reference the date of this correspondence, your file number, PMF 005484 C 0002, and be addressed to the Document Control Unit, HFV-199. Please include only one request per submission, clearly stating the request in the first paragraph of the submission.

If you have any questions regarding this correspondence, please telephone Dr. Janis R. Messenheimer, Team Leader, Antiparasitic and Physiologic Drugs Team at
(301) 827-7578.

Sincerely yours,

/s/
Steven D. Vaughn, D.V.M.
Director, Division of
Therapeutic Drugs for Food Animals
Office of New Animal Drug Evaluation
Center for Veterinary Medicine
 

¹ Markus, John, and Joseph Sherma (1992) Method I. Liquid Chromatography/Fluorescence Determination of Ivermectin in Animal Tissue and Plasma. Journal AOAC International 75(4):757-67.