Approval Date: January 21, 1988
I. GENERAL INFORMATION:
American Cyanamid Co.
P.O. Box 400
Princeton, N.J. 08540
II. INDICATIONS FOR USE
TRAMISOL levamisole Pour-On is a broad spectrum anthelmintic which is effective against the following nematode infections in cattle when used in accordance with label directions:
Stomach worms: (Haemonchus, Trichostrongylus, Ostertagia)
Intestinal worms: (Trichostrongylus, Cooperia, Nematodirus, Bunostomum, Oesophagostum, Chabertia)
III. DOSAGE FORM, ROUTE OF ADMINISTRATION AND RECOMMENDED DOSAGE
TRAMISOL Pour-On is a liquid formulation for topical application to the skin on the back of cattle. The recommended dose is 2.5 mL per 110 lbs (50 kg) of body weight, representing a dosage rate of 10 mg levamisole per kg of body weight.
A. Dose Titration Data
Three dose-range studies (one pivotal and two corroborative) were conducted to establish the optimum effective dose of levamisole pour-on as an anthelmintic in cattle. The optimum effective dose of the pour-on formulation was concluded to be a single topical application of 10 mg levamisole per kg of body weight.
1. Pivotal Dose Titration Study - United States
Forty naturally parasitized yearling cattle were used in a dose titration study of levamisole pour-on conducted by Pitman-Moore, Inc. under the supervision of J. Guerrero, D.V.M., PhD. The animals were randomly divided into groups of ten cattle each and given a single treatment of levamisole pour-on at doses of either 0 (untreated control), 7.5, 10.0 or 12.5 mg levamisole/kg body weight applied to the unprepared skin of the dorsal mid-line. All cattle were necropsied seven days after treatment for a determination of controlled anthelmintic efficacy. The results are given below.
(Eds. note: The following table consists of 8 columns.)
|Dose mg/kg||No. Treated Cattle||Percent Removal - Abomasum - Hp||Percent Removal - Abomasum - Oo||Percent Removal - Abomasum - Ta||Percent Removal - Intestine - Co||Percent Removal - Intestine - Nh||Percent Removal - Intestine - Or|
Haemonchus placei (Hp), Ostertagia ostertagi (Oo), Trichostrongylus axei (Ta), Cooperia oncophora (Co), Nematodirus helvetianus (Nh), Oesophagostomum radiatum (Or)
2. Corroborative Dose Titration Trial - Brazil A dose-range evaluation of levamisole 20% pour on in naturally infected cattle was conducted by Alvimar Jose da Costa, D.V.M. at the Veterinary School of Jaboticabal, Sao Paulo, Brazil. Twenty-four crossbred calves were divided into four groups with five calves per group and treated with levamisole pour-on applied topically to the back of the animals at doses of 0 (untreated control), 5.0, 7.5, 10 or 15 mg levamisole/kg body weight. The animals were sacrificed seven days after treatment for a determination of anthelmintic efficacy using controlled techniques. No adverse reaction was reported. The results are given below:
(Eds. note: The following table consists of 9 columns.)
|Dose mg/kg||No. Treated Cattle||Percent Removal - Abomasum - Hc||Percent Removal - Abomasum - Hs||Percent Removal - Abomasum - Ta||Percent Removal - Intestine - C. spp.||Percent Removal - Intestine - Bp||Percent Removal - Intestine - Or||Percent Removal - Lungs - Dv|
Haemonchus contortus (Hc), Haemonchus similis (Hs), Trichostrongylus axei (Ta), Cooperiaspecies (C. spp.), Bunostomum phlebotomum (Bp), Oesophagostomum radiatum (Or), Dictyocaulus viviparus (Dv)
3. Corroborative Titration and Comparative Trial - Scotland An experiment was conducted by J. Bogan and J. Armour, University of Glasgow, Scotland, in which the efficacy of levamisole pour-on at three dose rates against artificially infected Ostertagia ostertagiin calves was compared with that of levamisole hydrochloride (NILVERM®, ICI, Ltd.) injected subcutaneously. Twenty calves were randomly divided into five groups of four calves each on the basis of body weight and each group was infected orally with 70,000 O. ostertagi third stage larvae (L3). Twenty one days after infection the animals were treated as described below and slaughtered seven days later. Post mortem worm counts and fecal samples taken before treatment and at slaughter were examined for the presence of O. ostergati. The results are given below:
(Eds. note: The following table consists of 6 columns.)
|No. of Calves||Average Weight (kg)||Treatment||Dose mg/kg||O. ostertagi - Avg. No. at necropsy||O. ostertagi - % Reduction|
|4||95||LEV HCl Inj.||7.5||825||95.07|
B. Dose Confirmation Studies Five controlled trials (three pivotal and two corroborative) were conducted to confirm the effective dose of levamisole pour-on at 10 mg/kg as a topical anthelmintic for cattle.
An individual summary of each controlled trial is presented below.
1. Pivotal Dose Confirmation Study
A comparative study was conducted under the supervision of J. Guerrero, D.V.M., Ph.D., Pitman-Moore, Inc., Washington Crossing, New Jersey. A total of 42 naturally parasitized cattle approximately six months of age or less and weighing 142 to 381 lbs (64.5 to 173.2 kg) were randomly allocated to three groups with 14 cattle per group. Group I served as non-treated controls. Group II received levamisole pour-on at a dose of 10 mg levamisole/kg of body weight applied directly to one spot on the normal skin of the dorsal mid-line of the lumbar region of the animal's back, utilizing a hypodermic syringe without a needle. Group III was given a single injection of an approved 13.65% levamisole phosphate injectable solution at a dose of 6 mg levamisole/kg body weight administered subcutaneously in the mid neck region. All animals were necropsied seven to eight days after treatment. The results are given below:
(Eds. note: The following table consists of 4 columns.)
|Parasite (adult)||Mean No. Parasites Post mortem (control)||% Controlled Efficacy - lev Pour-On 10 mg/kg||% Controlled Efficacy - lev Injection 6 mg/kg|
Since the anthelmintic efficacy was comparable with each dosage form of levamisole, it was concluded that the levamisole pour-on is equally as effective as the injectable form. No signs of local irritation or systemic side effects were observed in any of the treated cattle.
2. Pivotal Dose Confirmation Study - Cold Weather
A controlled efficacy study of levamisole pour-on, applied during cold weather, was conducted by J. Guerrero, D.V.M., Ph.D., at Pitman-Moore, Inc., Washington Crossing, New Jersey. A total of 20 naturally parasitized beef type cattle of various breeds and breed mixtures, one year old or less, and weighing 198 to 470.8 lbs (90 to 214 kg) were used. Ten calves received a single topical application of levamisole pour-on at a dosage of 10 mg levamisole/kg of body weight and ten calves served as non-treated controls. Levamisole pour-on was applied directly on the unprepared skin on the back (lumbar region) using a hypodermic syringe without a needle when the ambient temperature was approximately 24.8°F to 44.6°F (-4°C to +7°C). Necropsies were performed seven to nine days after treatment and included equal numbers of treated and control animals. The results are shown in the summary table located at the end of this section. It was concluded in this study that cold weather did not adversely effect the anthelmintic efficacy of levamisole pour-on. No side effects or skin irritation were observed at any time after treatment.
3. Dose Confirmation Study - Pilot Trial
In a pilot trial, the controlled anthelmintic efficacy of levamisole pour-on in cattle was determined by J. Guerrero, D.V.M., Ph.D., Pitman-Moore, Inc., Washington Crossing, New Jersey. Six yearling cattle naturally parasitized and weighing 227 to 312 lbs (103.2 to 141.8 kg) were used. Three cattle were treated with levamisole pour-on at a dose of 10 mg levamisole per kg of body weight directly of the skin of the back utilizing a hypodermic syringe without a needle. Three cattle were similarly given a placebo treatment of the vehicle only (controls) at the recommended rate, 5 mL/100 kg of body weight. Necropsies were performed seven or eight days after treatment. The results are given in the summary table located at the end of this section. No local or systemic side effects were observed.
4. Corroborative Study - Belgium
A controlled efficacy trial of levamisole pour-on on calves artificially infected with Dictyocaulus viviparouswas conducted under the supervision of D. Thienpont, D.V.M. at Janssen Pharmaceutica, Beerse, Belgium. Twenty eight days post infection the calves were treated with levamisole pour-on applied mid-back at a dose of 10 mg levamisole/kg body weight (three calves) and a group of five calves served as untreated controls. The animals were necropsied 14 days after treatment for a determination of anthelmintic efficacy. The results are included in the summary table located at the end of this section.
5. Corroborative Study - South Africa
A controlled trial was conducted under the supervision of Dr. B. Van Der Westhuizen, Janssen Pharmaceutica, South Africa. Eighteen young six to ten week old Friesian type calves were artificially infected with Haemonchus placei (L3) and Ostertagi ostertagi, Oesophagostomum radiatum (L4) and Cooperiaspp. Twenty eight days after the initial infection, levamisole pour-on at 10 mg levamisole/kg body weight was applied topically to the backs of eleven calves while seven calves served as untreated controls. Twenty one days post-treatment the animals were sacrificed and the efficacy against each parasite was determined. The percent of parasite load before and after treatment with levamisole pour-on at 10 mg/kg was H. placei67.8%, O. ostertagi 76.3%, Oesophagostomum radiatum 88.9% and Cooperia spp. 96.8%.
A summary table of all dose confirmation studies is presented below.
(Eds. note: The following table consists of 11 columns.)
Summary of Dose Confirmation Trials with Levamisole Pour-On in Cattle Treated at 10 mg levamisole/kg Body Weight
|Study||No. Control Cattle||No. Treated Cattle||Percent Removal - Abomasum - Hp||Percent Removal - Abomasum - Oo||Percent Removal - Abomasum - Ta||Percent Removal - Intestine - C. spp.||Percent Removal - Intestine - Bp||Percent Removal - Intestine - N. spp.||Percent Removal - Intestine - Or||Percent Removal - Lungs - Dv|
|1. Pivotal U.S.||14||14||100||73.8||97.8||99.9||100||96.3||100||-|
|2. Pivotal U.S.||10||10||100||90.1||82.9||99.9||100||100||99.4||-|
|3. Pilot U.S.||3||3||100||99.5||100||100||100||-||99.6||-|
|4. Corroborative Belgium||5||3||-||-||-||-||-||-||-||96.3|
|5. Corroborative S. Africa||7||11||67.8||76.3||-||96.8||-||-||88.9||-|
Haemonchus placei(Hp), Ostertagia ostertagi(Oo), Trichostrongylus axei(Ta), Cooperiaspp. (C. spp.), Bunostomum phlebotomum(Bp), Nematodirusspp. (N. spp.), Oesophagostomum radiatum(Or), Dictyocaulus viviparus(Dv)
C. Clinical Trials 1. Pivotal U.S. Clinical Trial
Nine veterinarians in different geographic locations of the United States participated in clinical field investigations of the use of levamisole pour-on in beef cattle. The trial, conducted under the general supervision of Daniel T. Ruth, V.M.D., Pitman-Moore, Inc. consisted of the following investigators: J. Brown, D.V.M., Univ. of Illinois, Urbana, Illinois; J. Clark, D.V.M., Brawley, California; H. Lloyd, D.V.M., Arcadia, Florida; L. Moxey, D.V.M., Sheridan, Wyoming; R. Pedersen, D.V.M., Beatrice, Nebraska; D. Ruth, V.M.D., Washington Crossing, New Jersey; W. Vollmar, D.V.M., Montgomery, Minnesota; D. Waters, D.V.M., McMinnville, Oregon; S. White, D.V.M., Arkansas City, Kansas.
Healthy beef cattle requiring routine anthelmintic treatment were selected for this study. The cattle, representing a variety of breeds ranged in age from one month to 13 years and in weight from 110 to 2,200 lbs (50 to 1,000 kg). Levamisole pour-on was applied topically to the backs of 1,084 cattle at the rate of 5 mL/100 kg (10 mg levamisole per kg body weight) while 218 cattle served as placebo controls and received the vehicle only, topically, at an equivalent volume. Treatments were administered by the investigators and cattle owners under a wide variety of environmental conditions. Ambient temperatures at the time of treatment ranged from 0 °F (Minnesota, February 1984) to 110 °F (California, August 1983). A total of 410 cattle (331 drug treated and 79 placebo control) naturally infected with parasites were selected to evaluate anthelmintic activity by fecal egg per gram counts (EPG) determined before treatment and seven to 16 days after treatment.
Overall efficacy (calculated by averaging the percent efficacy of all cases) was 92% for levamisole pour-on versus 12% for the formulation vehicle (placebo control). There was no significant difference in efficacy between levamisole pour-on administered under cold or warm weather conditions. Each investigator, excluding Dr. Ruth due to vested interest or bias, rated the levamisole pour-on treatment as good to excellent with regard to clinical acceptability. Transient, self-limiting reactions were observed in both the drug treated and placebo treated groups; however, none of these effects was considered by the investigators to be clinically significant. These side effects were predominantly flaking skin, 8% and salivation, 4%.
2. Pivotal Clinical Study - Simulated Heavy Rain
A clinical trial was conducted by L.A. Hanrahan, D.V.M., Auburn University, Auburn, Alabama, to examine the effects of simulated rain representing a worst case severe rain condition on levamisole pour-on treatment. One hundred and forty-two cattle were randomly divided into three groups and were treated as follows: one group of 46 cattle received a single application of levamisole pour-on on the dry skin of the back at a dose of 10 mg levamisole/kg body weight; a second group of 49 cattle were likewise treated with levamisole pour-on on the dry skin of the back and immediately exposed to six hours of non-stop simulated rain; a third group of 47 cattle were wet down using a water hose prior to treatment with levamisole pour-on at 10 mg levamisole/kg body weight applied to the wet skin of the back and immediately exposed to six hours of simulated continuous rain. Fecal material was sampled twice before treatment and seven and 14 days after treatment for a determination of efficacy by the fecal egg per gram count method.
The results of this study indicated that the average efficacy of levamisole pour-on, 95%, is reduced when dry animals are treated and immediately exposed to heavy rain of six hours duration (average efficacy 65%) and further reduced (to 38%) if the animals are wet when treated and immediately exposed to heavy rain for six hours.
3. Pivotal Clinical Study - Water Immersion
A clinical trial was conducted by L.A. Hanrahan, D.V.M., Auburn University, Auburn, Alabama, in which the effects of levamisole pour-on in cattle dipped in water was determined. One group of 48 cattle was treated with levamisole pour-on topically along the dorsal mid line of the lumbar region at the recommended dose of 10 mg levamisole/kg body weight and was immediately required to swim a distance of 25 feed in water six feet deep. This treatment simulated the dilution and agitation that could occur in a dip vat during routine feed yard in-processing. Another group of 46 cattle were similarly treated with levamisole pour-on at the recommended dose of (10 mg/kg) but were not dipped and allowed to remain dry. Efficacy was determined in these 92 cattle by comparing fecal nematode egg per gram counts sampled twice before treatment and seven to 14 days after treatment.
The results of the study indicted that the efficacy of levamisole pour-on was reduced (72% efficacy) in those animals immediately dipped in water following treatment as compared to those animals that were not dipped (95% efficacy).
4. Corroborative Clinical Studies
Studies conducted in Mexico, Belgium, and Australia corroborate the high degree of efficacy obtained in the pivotal U.S. clinical trials. No side effects were reported in the Belgian or Australian studies while in trials conducted in Mexico, the only side effect reported was dry hair at the treatment site in 37 of 355 treated cattle (10.4%). The animals observed two months after treatment showed no visible lesions or signs of treatment. There was no evidence of systemic toxicity, skin irritation or discomfort in any of the treated animals.
A summary of the clinical field trial results is shown below.
C. Clinical Trials
(Eds. note: The following table consists of 6 columns.)
Summary of Clinical Field Trials with Levamisole pour-on in Cattle Treated at 10 mg levamisole/kg Body Weight
|Trial Location||No. Control Cattle||No. Treated Cattle||Average EPG Before Treatment||Average EPG After Treatment||Percent Efficacy|
|United States - Alabama||0||45||319||8||95|
|United States - California||12||49||614||2||100|
|United States - Florida||8||40||259||101||60|
|United States - Illinois||24||86||316||8||97|
|United States - Kansas||6||33||161||2||100|
|United States - Minnesota||6||33||43||0||100|
|United States - Montana||5||16||44||0||100|
|United States - Nebraska||8||40||178||31||82|
|United States - New Jersey||4||15||215||13||92|
|United States - Oregon||6||19||43||1||95|
|Mexico - Tapachula||0||44||435||0||100|
|Mexico - Guadalajara||0||10||555||0||100|
|Mexico - Ramos Arispe||0||20||256||0||100|
|Mexico - Monterrey||0||18||104||0||100|
|Mexico - Poza Rica||15||15||300||2||100|
|Belgium - Beerse||0||20||405||95||77|
|Australia - Albury||11||11||49||0||100|
|Australia - Emu Plains||11||11||101||0||100|
|Australia - Cumberlans||6||10||440||40||91|
V. ANIMAL SAFETY
A series of studies were conducted in the United States and abroad to identify the toxicologic profile of levamisole pour-on in cattle.
A. Pivotal Toxic Syndrome Cattle
A safety study of levamisole pour-on was conducted in cattle at Pitman-Moore, Inc., Washington Crossing, New Jersey, under the supervision of B.P. Seibert, V.M.D. Twenty Hereford or Hereford/Angus cattle (10 heifers and 10 steers) approximately one year old and weighing 393 to 611 lbs (178.7 to 277.6 kg) were used. The cattle were allocated into five groups, two heifers and two steers per group and given a single topical application of levamisole pour-on to the mid-backline at doses of 0 (non-treated control), 10, 30, 50 or 100 mg/kg corresponding to 1x, 3x, 5x, or 10x the recommended rate of 10 mg of levamisole per kg of body weight. The cattle were observed on a blind basis for any clinical signs of side effects continuously for eight hours after treatment and twice the following day. The cattle were also observed on days three, seven, ten and 14 after treatment for any signs of local reactions at the treatment site (dorsal mid-line).
No mortality occurred during the study. Side effects were not observed throughout the evaluation period in any of the cattle in the 0, 1x (except for one isolated occurrence of salivation) or the 3x groups. In the 5x and 10x groups, salivation, restlessness and muscle fasciculations were the most prevalent clinical signs occurring at three hours or more after treatment and lasting for five or more hours. Diarrhea, tail raising, incoordination and excitability occurred less frequently in these groups. All of the clinical side effects subsided within 24 hours post treatment. Superficial epidermal flakes along the dorsal midline were observed in 11 of 16 levamisole pour-on treated cattle and also in one untreated control animal beginning seven to 14 days after treatment and lasting seven days or less.
There were no signs of erythema, edema, changes in hair coat quality or discomfort associated with this flaking, and the incidence and severity did not appear to be dose related. It was concluded that the overdosage of levamisole pour-on in cattle are likely to cause mild to severe signs of toxicity, similar to those seen with other dosage forms of levamisole. These reactions are generally dosage related, occur three hours or more after treatment, may persist for five hours or longer, and will completely subside within 24 hours.
B. Pivotal Organophosphate Compatibility
The effect of levamisole pour-on and a concomitantly applied organophosphate insecticide on erythrocyte cholinesterase activity was determined in a study conducted under the supervision of B.P. Seibert, V.M.D., Pitman-Moore, Inc. While levamisole does not inhibit cholinesterase activity, the purpose of this experiment was to determine whether the pour-on formulation would enhance the absorption of an organophosphate resulting in a greater depression of cholinesterase activity. Eight beef cattle weighing 370 to 552 lbs (168 to 251 kg) were divided into two groups of four cattle each. One group was treated with WARBEX® (13.2% famphur, American Cyanamid), a commonly used cattle grubicide, applied topically on the mid-back at the recommended rate (1 oz/200 lb). The second group was similarly treated with WARBEX plus levamisole pour on at the rate of 10 mg levamisole/kg body weight, applied at the same site. All cattle were observed at varying intervals for up to three weeks after treatment. Whole blood samples were collected approximately one week before treatment and 1, 2, 4, 8, 24, 48 hours and seven days after treatment for determination of erythrocyte cholinesterase activity using the colorimetric method of Ellman, et al, (Biochem Pharmacol7:88-95, 1961).
All cattle remained in good general health throughout the experiment. There were no signs of organophosphate toxicosis or local side effects in any of the treated cattle. No significant (P less than .05) erythrocyte cholinesterase inhibition occurred after treatment in either group and there were no significant (P less than 0.05) differences in cholinesterase activity between the two treatment groups at any sampling time. It was concluded from this study that the application of levamisole pour-on does not increase the absorption of a topically applied organophosphate insecticide in cattle as measured by erythrocyte cholinesterase activity.
C. Acute Toxicity Trials - Scotland
Four acute toxicity trials were conducted by Robert Young & Co. Ltd., Glasgow, Scotland, using a total of 34 cattle of different weights and breeds (Blue-Grey, Fresian, Simmental cross, Angus, Hereford, Luing and Shorthorn). The animals were treated topically on the back with the levamisole pour-on formulation at doses of 60, 80, 100, 150, and 170 mg/kg body weight and were observed for 24 hours following treatment for physical behavior and side effects. It was concluded from these trials that levamisole pour-on has an adequate safety margin and that the symptoms of toxicity were typical of those associated with overdosing with levamisole.
D. Acute Toxicity Trials - South Africa
An acute toxicity trial was conducted under the supervision of Dr. B. Van Der Westhuizen, Janssen Pharmaceutica, South Africa, to determine the minimum lethal dose and the maximum tolerated dose of levamisole pour-on in calves of both sexes. Two calves each (one male, one female) were given levamisole pour on at doses of 60, 70, 100, 120, 140 and 160 mg levamisole/kg body weight and examined clinically every hour for the first 24 hours after treatment and then three times per day for the duration of the trial. It was concluded that levamisole pour-on has a wide margin of safety between the therapeutic dose (10 mg/kg) and the dose at which symptoms of toxicity were first observed (70 mg/kg) and a lethal dose (160 mg/kg). All symptoms of toxicity e.g., tailraising, salivation, diarrhea, muscle fasciculations and staggering were typical of those associated with overdosing of other formulations of levamisole.
E. Double Dose Field Trial - South Africa
A safety trial was conducted under the general supervision of Dr. B. Van Der Westhuizen, Janssen Pharmaceutica, South Africa, in which a total of 459 cattle were treated with levamisole pour-on at double the recommended dose, 20 mg levamisole/kg body weight, applied topically on the back. All animals were observed for at least six hours post treatment for any signs of toxicity. Thereafter, the owners were requested to observe the animals daily and record any side effects or mortalities for at least 30 days posttreatment. The treated animals included 158 cows, 147 calves, 6 bulls and 148 castrated males at six different trial locations representing a variety of management practices. No adverse reactions were noted during the 30 observation period in any of the treated animals. Thus, it is concluded that levamisole pour-on at double the dose was safe for all classes of cattle irrespective of age, sex or management condition.
F. Skin Irritation - South Africa
A skin irritation trial of levamisole pour-on after dermal application in cattle was conducted under the supervision of Dr. B. Van Der Westhuizen, Janssen Pharmaceutica, South Africa. Levamisole pour-on was administered topically via an automatic syringe at dose rates of 10 mg levamisole/kg body weight (169 cattle) and 20 mg/kg (264 cattle). All animals were observed at bi-weekly intervals for up to eight weeks (56 days) after treatment. Although five of 169 (2.9%) of the animals treated showed signs of skin irritation, the reactions were mild and of a passing nature.
G. Skin Irritation - Scotland
A study was conducted by Robert Young & Co., Ltd., Glasgow, Scotland, to determine the effects of high doses of levamisole on the skin of cattle. A total of 22 cattle were used and given levamisole pour-on administered topically to the back at doses of 60, 80, 100, 150 or 170 mg levamisole/kg body weight. It was concluded that at six, eight or ten times the recommended therapeutic dose rate, only slight superficial skin reactions (dandruff) were observed. At 15 or 17 times the recommended rate, the reaction was more pronounced, but had nearly disappeared two weeks after treatment. No hair loss was observed.
H. Semen Quality - Belgium
The effect of levamisole pour-on on semen quality was evaluated in a study conducted by R. Debruyne, D.V.M. and L. Desplenter, D.V.M., Janssen Pharmaceutica, Belgium. Five bulls in good clinical health, with a proven history of normal fertility and semen quality, were given levamisole pour-on topically at the therapeutic dose, 10 mg levamisole/kg of body weight. Semen quality evaluated twice before treatment and four to five times after treatment for about two months showed no adverse effect on any of the following parameters measures: sperm, mass movement of sperm cells, percent of dead sperm cells, morphology and after deep freezing, percent motility at thawing. No local side effects or skin irritation were observed following treatment.
I. Safety to Pregnant Cattle - Belgium
Data regarding the safety of levamisole pour-on in pregnant cattle were compiled by L. Desplenter, D.V.M., Janssen Pharmaceutica, Belgium, from the results of one pilot trial and three previously conducted experiments investigating the immunomodulating activity of levamisole and its beneficial effects on the immunomodulating activity of levamisole and its beneficial effects on the survival and morbidity of calves during the neo and postnatal period. In a pilot trial, ten cows were treated once per week with levamisole pour-on at 10 mg levamisole/kg body weight topically during the last four weeks of pregnancy. No local or general side effects were observed and delivery and health status of the calves at birth were without complications.
In three other larger experiments, levamisole in the pour-on formulation was administered topically at 5 mg/kg, an immunomodulating dose level, to a total of 496 cattle and compared to 556 placebo treated controls. Treatments were given once weekly during the last four weeks of pregnancy. There was no harmful effect as a result of levamisole pour-on treatment on pregnancy, delivery, or the health status of the calves at birth. Further, mortality and morbidity of the newborn calves were significantly reduced in the drug treated as compared to the placebo treated group.
VI. HUMAN SAFETY
A. Data to Support Human Safety
The underlying human safety data supporting the use of levamisole in cattle are described in the Freedom of Information Summary under NADA 102-437, for TRAMISOL levamisole phosphate Injectable Solution, 13.65% (21 CFR 522.1244). Results of a comparative study indicated that plasma levels of levamisole in the pour-on formulation were bioequivalent to the injectable formulation at the recommended therapeutic dose levels in cattle. Thus, it was concluded that the use of levamisole pour-on will not significantly increase human exposure to residues of the drug. Additional toxicity studies of levamisole pour-on and the vehicle, diethylene glycol monbutyl ether (DGME), are contained in the subject New Animal Drug Application and are summarized below.
The blood bioequivalency of two formulations of levamisole in cattle was determined in a cross-over trial conducted by Janssen Pharmaceutica, Beerse, Belgium. Also measured in the same study was the plasma depletion of DGME.
Ten cattle, divided into two groups of five, each group consisting of two heifers, two dairy cows and one bull, were treated with either levamisole pour-on, applied by pouring on the mid-backline of the animal at a rate of 10 mg levamisole/kg, or the approved 13.65% levamisole phosphate injectable solution administered subcutaneously at the recommended dose of 6 mg levamisole phosphate/kg (equivalent to 5.1 mg levamisole/kg). Plasma was sampled before and at 0.5, 1, 2, 3, 4, 6, 8, 24, 32 and 48 hours after treatment. After a ten day interval, treatments were crossed over and the study repeated. Plasma samples were analyzed for levamisole using a gas chromatographic method. The validated sensitivity of the method was 0.01 ppm.
The area under the plasma concentration-time curve (AUC 0-48h) values of 6.29 ± 1.74 µg·hour/mL for pour-on and 6.91 ± 1.20 µg·hour/mL for subcutaneous administration were not significantly different (p is greater than .05). Thus, the systemic availability was similar for either treatment, demonstrating that the two dosage forms of levamisole were bioequivalent at the given therapeutic dose levels.
DGME was analyzed using a specific gas chromatographic procedure which was shown to have a validated sensitivity of 0.1 ppm. Maximum plasma levels, amounting to an average of 1.44 ± 0.90 ppm were attained 1 to 4 hours (2.7 ± 1.2 hours) after application. Subsequent elimination from plasma proceeded with an apparent half life of 1.3 to 3.9 hours (2.05 ± 0.83 hours). In most animals, plasma DGME levels were lower than 0.1 ppm within 24 hours after application. No sex nor age related influences on the systemic absorption of DGME was apparent. It was concluded in this evaluation that the systemic availability of DGME was very limited and that its elimination from plasma was rapid.
1. Acute Oral Toxicity - Mice
The acute oral toxicity of 10% levamisole pour-on and its vehicle DGME were tested in male and female mice in a study conducted by Robert Young & Co., Ltd., Glasgow, Scotland. In male mice, the maximum tolerated dose of levamisole was 40 mg/kg. At higher dose levels mortality was observed. In female mice, the maximum tolerated oral dose level was 80 mg/kg. At higher dose levels, there appeared to be an individual difference in susceptibility to the formulation.
The maximum tolerated dose level of DGME corresponded with 1.89 mL/kg (1.8 g/kg). At 3.41 mL/kg (3.24 g/kg) mortality was observed. The toxic symptoms observed before death included gasping, twitching, incoordination and tremors.
2. Acute Oral Toxicity - Rats
The acute oral toxicity of 10% levamisole pour-on and DGME was tested in female rats by Robert Young & Co., Ltd., Glasgow, Scotland. Four female rats per treatment group (average weight of 105 g) were anesthetized with ether and treated by oral gavage with 10% levamisole pour-on at the following doses: 100, 150, 200, 250 and 300 mg/kg. Four rats per treatment group were administered DGME orally by gavage at doses of 1.5, 2.0, 2.5, 3.0 and 3.8 mL/kg corresponding to 1.43, 1.9, 2.38, 2.85 and 3.61 g/kg. The rats were observed for mortality up to four days after treatment.
3. Dermal Toxicity - Rats
The acute dermal toxicity of 20% levamisole pour-on and DGME were determined by Robert Young & Co., Ltd., Glasgow, Scotland. SD-01a female rats weighing approximately 150g, were used in these tests. The maximum tolerated dose level of levamisole in the pour-on formulation after dermal application was 267 mg/kg. At higher dose levels, marked but transient skin lesions were observed. With DGME, no local or general side effects were observed even at the highest dose administration 5 mL/kg (4.75 g/kg).
4. Ninety Day Topical Levamisole Pour-On - Dogs
A three month subacute toxicity study of 20% levamisole pour-on applied topically to the backs of dogs was conducted by Janssen Pharmaceutica, Beerse, Belgium. Twenty-four young and healthy purebred Beagle dogs weighing between 4.2 and 14.6 kg were used. The animals were divided into four groups with three males and three females per group. Levamisole pour-on was applied topically on the back of the dogs daily for 90 consecutive days at doses of 2.5, 10 and 40 mg/kg. One group of three males and three females served as untreated controls. The parameters measured were mortality, behavior and appearance, body weight, heart rate, electrocardiogram, blood pressure, hematology, serum analysis, urinalysis, gross pathology, organ weights and histopathology.
All 24 dogs survived the three month study. Behavior and appearance were normal in all groups. Body weight was comparable between the control dogs and those of the 2.5 and 10 mg/kg dosage groups. At 40 mg/kg, a weight loss (p less than .05) was noted during the last three to four weeks of dosing. No adverse effect was seen for heart rate, electrocardiogram, blood pressure, hematology and urinalysis. All serum chemical values were within acceptable limits except for a slight terminal increase of alkaline phosphatase and serum glutamic pyruvic transaminase (SGPT) in the 40 mg/kg dosage group. No adverse effect was noted in gross pathology, organ weights or histopathology. The no observable effect level in this study was concluded to be 10 mg/kg.
Diethylene glycol monobutyl ether (DGME), Vehicle
1. Mutagenicity Assays
The mutagenicity potential of DGME was evaluated in three short-term genotoxicity assays performed for Pitman-Moore, Inc. The results of this Ames Salmonella/Microsome, L5178Y Mouse Lymphoma and Unscheduled DNA Repair testing and a corroborative study from the published literature conducted independently by E.D. Thompson, et al. are summarized below:
Ames Salmonella/Microsome Test
The mutagenic potential of DGME was examined in the Ames reverse mutation test by NOTOX Pathobiology Research, The Netherlands. DGME, at concentrations up to 24 µL per plate, did not reveal an increase in the number of revertant colonies with the strains TA1537, TA 1538, TA98 and TA100 either in the presence or in the absence of a metabolic activation system (S9-mix). However, in the same study, a slight increase in the number of revertant colonies was found with a strain TA1535 only in the absence of rat liver S9-mix, and only in two out of four tests. As the increase in revertant colonies found with TA 1535 could not be reproduced in all the tests, and as the more sensitive strain TA 100 did not reveal an increase in the number of revertant colonies, the authors concluded that DGME is not mutagenic towards Salmonella typhimurium .
As the increase in the number of revertant colonies found with TA 1535 in some tests was a point of concern for the Food and Drug Administration, a supplementary Ames reverse mutation test was conducted by Janssen Pharmaceutica, Belgium, for definite determination of mutagenic potential of DGME towards the strain TA 1535. The supplemental Ames test was conducted with DGME in triplicate using the standard strain TA 1535 in the presence and in the absence of a metabolic activation system (S9-mix). Induction of rat liver enzymes was obtained by intraperitoneal injection of 500 mg/kg Aroclor to five male rats. Top agar, 0.1 mL of bacteria, S9-mix (20 and 50 µL S9/plate) if needed and 2, 4, 6, 8, 10, 12, 14, 18, 20, 22, 24, 26, 28 and 30 µL DGME/plate were sequentially added, shaken and poured onto agar plates. After incubation in the dark at 37 degrees C during 48 hours, the number of revertant colonies were counted. DGME did not induce a significant increase in the number of revertant colonies beyond the solvent control incidence with or without a metabolic activation system.
L5178Y Mouse Lymphoma Test
DGME was screened for mutation at the thymidine kinase (TK) locus and at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus on L5178Y TK +/- mouse lymphoma cells in tests conducted by Microbiological Associates, Inc., Bethesda, Maryland. DGME produced a positive response at the TK locus both in the presence and absence of Aroclor induced rat liver S-9. The increases in mutant frequency were approximately three-fold in the absence of activation, but only at concentrations of the test article greater than 3,000 µg/mL. In the presence of activation, increases in mutant frequency were approximately 2.7 fold and occurred only at concentrations greater than 5,000 µg/mL. No significant induction of mutation occurred at the HGPRT locus when the cultures were treated with the test article either in the presence or absence of S-9 activation.
Since the response was seen only at very high concentrations, viz 3,000 µg/L and above, it was postulated that the mutagenic activity might be due to a contaminant in the DGME, possibly an oxidation product. Therefore, the mouse lymphoma assay at the TK locus was repeated employing DGME containing 90 ppm BHT (butylated hydroxytoluene) as antioxidant. No increase in mutant frequency was noted with DGME containing BHT at the same doses which produced a positive response of DGME alone.
Unscheduled DNA Synthesis Assay
The potential of DGME to induce unscheduled DNA synthesis (UDS) in primary cultures of rat hepatocytes was determined by Microbiological Associates, Inc., Bethesda, Maryland. Based on the results of a preliminary cytotoxicity test, DGME was tested at eight dose levels ranging from 0.1 to 333 µg/mL. The top five doses were fully evaluated for unscheduled DNA synthesis. The results of the UDS assay demonstrated that DGME did not evoke a significant increase in the mean number of net nuclear grain counts (i.e., an increase of at least five counts over the control) at any dose level. Therefore, DGME was considered negative in this assay.
Mutagenicity Testing of DGME - Independent Study
The nonmutagenic potential of DGME was confirmed in an independent study reported in the scientific literature (Thompson, et al, Environmental Health Perspective57:105-112, 1984). DGME was examined with a Tier I battery of in vitroassays followed by a Tier II in vivoDrosophila sex-linked recessive lethal assay. The in vitrobattery consisted of: Salmonellamutagenicity test, the L5178Y mouse lymphoma test, a cytogenetics assay using Chinese hamster ovary cells and the unscheduled DNA synthesis (UDS) assay in rat hepatocytes. Results of the Salmonellamutagenicity test, the cytogenetics test and the rat hepatocyte assay were negative. A very weak positive response in the mouse lymphoma test was found not to have mutagenic significance because of a clear negative finding obtained in the in vivoDrosophila sex-linked recessive lethal assay. It was concluded in this battery of tests that DGME does not present any genetic risk to humans.
2. Two Generation Reproductive Study - Rats
An oral two generation reproductive study of DGME in Wistar rats was conducted by Janssen Pharmaceutica, Beerse, Belgium. DGME was administered daily through the drinking water to male and female Wistar rats during 60 days prior to mating and throughout mating, pregnancy and lactation for two successive generations, each producing one litter. The dilutions of drinking water used were 0.01 , 0.04 and 0.16 mL DGME (stock solution) per 50 mL providing approximately doses of 0.04, 0.16 and 0.63 mL DGME per kg of body weight, respectively. Control rats were given drinking water without DGME. Under the conditions of this study, the reproductive performance in Wistar rats was not affected at the doses used. In the parents, body weight gain, food consumption, mortality, mating, pregnancy rate and duration of gestation were not affected. Litter size, percentage of live fetuses, weight at birth and during a three week lactation period and survival rate were comparable between groups or considered to be within normal limits. No teratogenicity was seen.
3. Primary Eye Irritation - Rabbits
The eye irritancy potential of DGME was investigated by B. Ballantyne, M.D., D.Sc., Ph.D., J. Toxicol. - Cut. and Ocular Toxicol. 3(1):7-15 (1984). A summary of these studies are as follows.
Rabbit eye irritation studies, using instillation volumes of 0.1 mL, showed that undiluted DGME causes moderately severe conjunctivitis, with mild blepharitis and slightly detectable to mild diffuse keratitis. These effects were most marked during the first 24 hours postinstillation, and thereafter, spontaneously subsided with no residual tissue injury by 14 days. The lowest concentration producing a detectable keratitis was DGME (25% v/v), and that causing minor transient conjunctivitis was 10%. DGME (5%) did not produce local inflammation. Instilling 0.1 mL of solution resulted in measured peak increases in intraocular tension at ten minutes ranging from 3% (DGME, 1%) to 63% (DGME, 20%). Varying the volume of DGME (10%) caused measured peak increases of intraocular pressure at 10 minutes ranging from 1% (0.01 mL) to 33% (.10 mL). Intraocular pressure returned to control values by one hour postinstillation.
4. Ninety-Day Oral DGME - Rats
A 90 day subchronic oral toxicity study of DGME in Wistar rats was conducted by Janssen Pharmaceutica, Beerse, Belgium. One hundred and sixty Wistar rats, less than six weeks of age at the start of the study, were used. The rats were divided into four groups with 20 males and 20 females per group. DGME was administered orally by gavage at doses of 0.04, 0.16, and 0.63 mL/kg of body weight corresponding to 38, 152 and 598.5 mg/kg. One group of control rats (20 males and 20 females) received only water orally by gavage at the rate of 1 mL per 100 g body weight. All treatments were administered daily for 90 consecutive days. The parameters measured were mortality, clinical observations, food consumption, body weight, hematology, serum analysis, urinalysis, gross pathology, organ weights and histopathology. From this study it was concluded that repeated dosing of DGME in rats at 0.04 and 0.16 mL/kg is without toxic effects. Dosing with 0.63 mL/kg resulted in slight toxic effects especially in females consisting of marginal hematologic changes (decreased HCT, Hgb and RBC), slight urinary effects (increased incidence of calcium oxalate crystals) and slight liver changes (more hydropic aspect in perilobular area). Therefore, the highest no-observable effects level (NOEL) in rats determined in this study was 0.16 mL/kg/day.
5. Ninety-Day Oral DGME - Dogs
A 90 day subchronic oral toxicity study of DGME in dogs was conducted by Janssen Pharmaceutica, Beerse, Belgium. Thirty two purebred Beagle dogs approximately five months of age were used. The dogs were divided into four groups with four males and four females per group. DGME was administered to three groups of dogs orally by gavage once daily for 90 consecutive days at doses of 0.04, 0.16 or 0.63 mL/kg of body weight. These dose levels were equivalent to 38, 152, and 598.5 mg/kg. One group of four males and four females served as a control and received water only (1 mL/kg) at the same frequency. The parameters measured were mortality, behavior and appearance, food consumption, body weight, hematology, serum analysis, urinalysis, gross pathology, organ weights and histopathology. From this study it was concluded that repeated dosing of dogs with DGME at 0.04 mL/kg did not result in toxic effects. Dosing at 0.16 mL/kg was a marginally toxic dose, whereas 0.63 mL/kg was clearly toxic and fatal in half of the animals. The target organs for toxicity were the kidneys, the liver and the gastrointestinal tract. Therefore, 0.04 mL/kg was determined as the no-effect level from this study.
B. Safe Concentrations of Residues
The safe concentration of total levamisole residues in edible tissues of cattle are described in the Freedom of Information Summary and were previously established in the currently approved NADAs for levamisole bolus (NADAs 91-826 and 39-356; 21 CFR 520.1242b), drench (NADAs 112-051 and 39-357; 21 CFR 520.1242a), injectable (NADAs 126-742 and 102-437; 21 CFR 522.1244) and gel (NADA 126-237; 21 CFR 520.1242f) formulations. The tolerance for negligible levamisole residues in edible tissues of cattle has been established at 0.1 ppm (21 CFR Section 556.350).
C. Metabolism Studies
Metabolism studies which were used to establish human safety of levamisole are described in the Freedom of Information Summary under NADAs 126-742 and 102-437 (21 CFR 522.1244). Additional metabolism studies of the vehicle, DGME in rats and cattle were conducted by Janssen Pharmaceutica, Beerse, Belgium, and are summarized below.
1. Metabolism of 14C DGME in Rats
Male and female rats were dosed orally with 14C labelled DGME at 0.16 mL/kg/day for seven days. The excretion of the radioactivity was extremely rapid, amounting to about 85% of the dose at eight hours after dosing, and was almost complete at 24 hours after dosing. Urinary excretion of the radioactivity accounted for 82 to 89% of the dose, and excretion of 14CO2 in expired air for about 11%. The excretion in the feces was minimal. The main metabolic pathway of DGME in rats was it oxidation to (2-butoxyethoxy)acetic acid, accounting for about 60% of the dose. Other major metabolic pathways were the oxidative O-dealkylation, with further oxidative degradation, finally resulting in 14CO2, and the glucuronidation of DGME. No unchanged DGME could be detected in urine.
After a seven day dosing, the elimination of the radioactivity from the tissues showed an initial very rapid phase and a second slower phase. At 48 hours after the last dose, less than 1% of the total dose could be recovered in the tissues. At that time, the contribution of unchanged DGME and of its major urinary metabolites to the tissue radioactivity was very small, whereas at least 30 to 40% of the tissue radioactivity was incorporated in natural compounds.
2. Metabolism of 14C-DGME in Cattle
14C-labelled DGME was administered subcutaneously to two ruminating cattle (198 - 213 kg) at 4.0 mL/100 kg. They were sacrificed at six and 48 hours after dosing, respectively. Peak plasma levels of unchanged DGME and of its metabolites were reached at about one hour after dosing; then they decreased with half-lives of one to two hours up to to eight hours after dosing, and thereafter, the radioactivity was eliminated with a half-life of 63 hours.
As in orally dosed rats, the radioactivity in cattle was excreted extremely rapidly, mainly with the urine (84% of the dose at six hours after dosing). The metabolite pattern in urine was studied qualitatively by HPLC and mass spectrometry, and quantitatively by radio-HPLC. The major metabolic pathway of DGME in cattle were identical to those in rats: oxidation to (2-butoxyethoxy) acetic acid, the main metabolite which accounted for about 2/3 of the urinary radioactivity in both species, glucuronidation of DGME, and O-dealkylation with further degradation up to CO2. The glycine conjugate of (2-butoxyethoxy) acetic acid accounted for about 5% of the dose in urine of cattle, but could not be detected in that of rats.
Tissue radioactivity levels were already low at six hours after dosing, amounting to 25.5 ppm in kidney, 9.0 ppm in liver, 1.2 ppm in fat and 0.91 ppm in muscle. Just as in rats, the elimination of the radioactivity from bovine tissues was very rapid. At 48 hours after dosing, radioactivity levels were 2.1 ppm in liver, 1.2 ppm in kidney, 0.29 ppm in fat and 0.13 ppm in muscle. DGME and the major urinary metabolites were determined in liver, kidney, and muscle by a radio-HPLC method. Just as in the rats, they accounted for only a small part of the tissue radioactivity, whereas evidence could be presented for the incorporation of a very small part of the administered radioactivity into endogenous constituents.
D. Regulatory Methods
The regulatory analytical method for the determination of residues of levamisole in cattle tissues is described in the Food Additives Manual which is on display in FDA's Freedom of Information Public Room (Room 12A-30, 5600 Fisher's Lane, Rockville, MD 20857).
E. Withdrawal Time
Levamisole tissue residues in cattle following a single topical dose of levamisole pour-on at 10 mg levamisole/kg body weight was determined in a study conducted by Janssen Pharmaceutica, Beerse, Belgium. Twenty two Belgian White-Grey beef breed cattle, 11 steers and 11 heifers, ranging in weight from 843 to 1166 lbs (383 to 530 kg) were used. Four groups of five cattle each were treated topically with the proposed commercial formulation of levamisole pour-on. At 2, 6, 10 and 14 days after treatment, respectively, one group was slaughtered and samples of muscle, liver, kidney and fat were collected from each animal. On the day before treatment, two control animals (one steer and one heifer) had been similarly sacrificed. Levamisole tissue levels were determined using the official regulatory method for the detection of levamisole residues. The detection limit of the gas chromatographic method was 0.01 µg/g (ppm).
The results confirm that liver should be considered as the target tissue for levamisole in cattle. From six days after application, all mean tissue levels were at or below the established tolerance level of 0.1 ppm. Fourteen days after treatment, residual levels of levamisole were undetectable (less than 0.01 ppm) in all tissues except liver in which levels averaged 0.017 ppm. Based on these data, a withdrawal period of nine days is proposed. The results of this withdrawal study are presented below.
(Eds. note: The following table consists of 6 columns.)
Levamisole Tissue Residue Levels (Mean + S.D.) in Cattle After A Single Pour-On Dose of Levamisole at 10 mg/kg
|Levamisole µg/kg (ppm)|
|Necropsy Day||Muscle (Shoulder)||Muscle (Appl. site)||Fat||Kidney||Liver*|
* Mean value of the three liver segments were considered as being representative for the whole organ.
** Not detectable by the GC method (less than 0.010 µg/g or ppm)
F. Safety to Handler
With regard to human safety relative to possession, handling and administration of TRAMISOL levamisole pour-on, the following statement appears on the labeling:
"PRECAUTIONS: Rubber gloves should be used to protect the hands of the operator. Avoid contact with skin, eyes or clothing. Wash thoroughly after use. Wash splashes from exposed skin and eyes immediately."
VII. AGENCY CONCLUSIONS
The data submitted in support of this NADA satisfy the requirements of Section 512 of the Food, Drug and Cosmetic Act and with 21 CFR 514 of the implementing regulations. Those data demonstrate that TRAMISOL levamisole Pour-On Topical Cattle Anthelmintic is safe and effective when used under its labeled conditions of use. Because of the drug's broad spectrum anthelmintic activity, its conditions of use, and its route of administration, the agency has concluded that the TRAMISOL Pour-On labeling contains adequate directions for use by the layperson and, thus, this drug may be marketed over the counter.
So far as Human Food Safety is concerned, we regard this as if it were a Category II supplement (42 FR 64367, December 23, 1977) which did not require the reevaluation of the levamisole data. TRAMISOL Pour-On is an additional dosage form of the drug, and this approval is not the kind that will result in increased human exposure to residues of the drug.
VIII. LABEL (attached):
- TRAMISOL® levamisole Pour-On package label
Copies of these labels may be obtained by writing to the:
Freedom of Information Office
Center for Veterinary Medicine, FDA
7500 Standish Place
Rockville, MD 20855