NADA 106-964

A. Toxicity Studies

The toxicity studies listed below are in addition to studies contained in the FOI Summaries for NADA 106-964 and 126-050.

1. A One-Year Chronic Toxicity Study of Apramycin Administered Orally to Beagle Dogs

a. Report Number: D-3088

b. Study Completion: February 27, 1979

c. Investigator:

L.C. Howard
Toxicology Division, Lilly Research Laboratories
Division of Eli Lilly & Company
Greenfield, Indiana 46140

d. Substance and Dosage Form: Apramycin base was administered daily by the oral route.

e. Species and Strain of Animal: Beagle dogs

f. Number of Animals per Group: Animals were allocated 4/sex/dose.

g. Levels and Duration of Dosing: Beagle dogs, between 86 and 142 days of age (males) and 86 to 115 days of age (females) at initiation of the study, were given doses of 0, 25, 50, or 100 mg apramycin activity per kilogram of body weight per day for one year.

h. Route of Administration: Apramycin was given orally on a daily basis.

i. Parameters: Study parameters included general physical condition and behavior, ophthalmoscopic examination, body weight, auditory responsiveness, hematology, bone marrow evaluation, clinical chemistry, urinalysis, absolute and relative organ weight, and gross and microscopic examination of tissues.

j. Toxicities Observed: None

k. No-Observed-Effect Level (NOEL): The NOEL for this study was 100 mg/kg/day.

l. Conclusions: Statistically significant changes in hematology parameters were not considered related to apramycin administration because they were either isolated, not dose related, or were not observed at subsequent sampling times. Terminal myeloid:erythroid ratios were not significantly different from those of control dogs.

Because of a lack of histopathological changes in selected organs, statistically significant changes in mean absolute and relative (to body weight) organ weights were not considered to be of toxicological importance. Additionally, when these tissues were compared relative to brain weights, significant differences were not observed. Statistical analysis of the parameters studied revealed no significant pathological effects of oral apramycin administration to beagle dogs at doses of 25, 50, or 100 mg/kg/day for one year.

2. A Multi-Generation Reproduction Study with Apramycin in the Fischer 344 Rat

a. Report Numbers: R-887, R-728, R-1318

b. Study Completion: R-887, October 18, 1978; R-728, January 5, 1979;
R-1318, June 11, 1979

c. Investigator:

L.C. Howard
Toxicology Division, Lilly Research Laboratories
Division of Eli Lilly and Company
Greenfield, Indiana 46140

d. Substance and Dosage Form: Apramycin was administered continuously in feed.

e. Species and Strain of Animal: F_1b offspring of Fischer 344 rats from the Multi-generation Reproduction Study

f. Number of Animals per Group: Rats were allocated 25 rats/sex/dose for each parent generation.

g. Levels and Duration of Dosing: Fischer rats were provided 0, 0.25, 0.5, or 1.0 percent apramycin in feed to determine the effect of apramycin on growth and reproduction through three generations. Based on feed intake and body weights, these levels translate to 0, 194, 388, and 785 mg/kg/day, respectively.

h. Route of Administration: Apramycin was provided in feed.

i. Parameters: Study parameters included diet assays, parental mortality, physical signs of toxicity, body weight, and food consumption.

Reproduction Data and Progeny Observations: Fertility, liveborn litter size, gestation length and survival, progeny weight and sex. Progeny observations, gross internal examinations (all generations), and microscopic examinations (final generation).

Reproduction Data and Fetal Observations, Teratology Segment: Fertility, live litter size, corpora lutea, implantation and resorption number, gestation length and survival, and fetal weight and sex. External, visceral and skeletal examinations of the fetuses were performed.

j. Toxicities Observed: There were no treatment effects noted for any study parameters for the duration of the studies.

k. No-Observed-Effect Level (NOEL): The NOEL for this study was 1.0% of the diet, or 785 mg/kg/day.

l. Conclusions: Apramycin was well-tolerated with no significant observations for parental mortality, body weights, physical signs of toxicity, or treatment-related conditions. The reproductive capacity of the animals, including fertility, litter size, gestation length and survival, progeny survival and weight, and sex distribution, was unaffected. Apramycin at doses up to 785 mg/kg daily was not teratogenic and produced no adverse reproductive effects.

3. A Chronic Toxicity-Oncogenicity Study in Fischer 344 Rats Maintained for Two Years on Diets Containing Apramycin

a. Report Numbers: R478, R488

b. Study Completion: May 30, 1980

c. Investigator:

L.C. Howard
Toxicology Division, Lilly Research Laboratories
Division of Eli Lilly & Company
Greenfield, Indiana 46140

d. Substance and Dosage Form: Apramycin was provided in feed.

e. Species and Strain of Animal: Fischer 344 rats

f. Number of Animals per Group: 50 rats/sex/dose were used.

g. Levels and Duration of Dosing: Diets were fed for two years and contained 0, 0.25, 0.5, 1.0, and 5.0 percent apramycin, which provided time weighted averages of 0, 124, 245, 488, and 2,772 mg apramycin activity/kg for males and 0, 154, 301, 610, and 3,451 mg apramycin activity/kg for females.

h. Route of Administration: Orally, via feed.

i. Parameters: Study parameters included diet assays, survival, physical signs of toxicity, auditory response, body weight, food consumption, efficiency of food utilization, hematology, clinical chemistry, organ weights, and pathology.

j. Toxicities Observed: Body weights were significantly decreased for rats in both sexes in the 5.0-percent-apramycin-treated group. The body weight effect was not associated with a detrimental effect on survival, nor was there evidence of chronic toxicity. Decreased organ weights for the 5.0-percent-apramycin-treated group were attributed to the significant reduction in growth. No other parameters were adversely affected by apramycin administration.

k. No-Observed-Effect Level (NOEL): Based on the significant body weight reduction at 5.0 percent apramycin, the NOEL 1.0 percent apramycin (approximately 500 mg/kg body weight when averaged across both sexes).

l. Conclusions: Because the rats in this study were progeny from rats utilized in the reproduction/teratology studies (R-887, R-728, R-1318), it is concluded that the exposure of rats to 1.0 percent apramycin during in utero development and up to 5.0 percent apramycin throughout lifetime produced significant weight gain impairment, but neither chronic toxicity nor carcinogenicity.

4. A Chronic Toxicity-Carcinogenicity Study in B6C3F1 Mice Given Apramycin in the Diet for Two Years

a. Report Numbers: M00679, M00779, M00780, M00880

b. Study Completion: M00679, July 17, 1981; M00779, July 24, 1981 M00780, March 10, 1982; M00880, March 12, 1982

c. Investigator:

G.D. Williams
Toxicology Division, Lilly Research Laboratories
Division of Eli Lilly & Company
Greenfield, Indiana 46140

d. Substance and Dosage Form: Apramycin was administered in feed.

e. Species and Strain of Animal: B6C3F1 mice

f. Number of Animals per Group: 60 mice/sex/dose.

g. Levels and Duration of Dosing: Mice were fed 0, 0.15, 0.5, 1.5, or 4.5 percent apramycin, which provided 0, 189, 623, 1,928, or 7,183 mg/kg for males and 0, 213, 668, 2,043, or 7,570 mg/kg for females, for two years.

h. Route of Administration: Orally, via feed.

i. Parameters: Study parameters included diet assays, survival, physical signs of toxicity, body weight, hematology, clinical chemistry, and organ weights.

j. Toxicities Observed: Apramycin resulted in a decrease in both mean body weight and cumulative body weight gain at termination for the female mice fed 0.5, 1.5, and 4.5 percent apramycin, and for the male mice fed 4.5 percent apramycin. Mild increases in erythrocyte count, hemoglobin concentration, and packed cell volume were limited to mice fed diets containing 4.5 percent apramycin.

Serum glucose concentrations were moderately decreased and urea nitrogen concentrations mildly increased in mice of both sexes fed diets containing 4.5 percent apramycin. Serum alkaline phosphatase was mildly elevated in female mice fed 0.5, 1.5, and 4.5 percent apramycin.

Organ weights were consistent with the expected changes associated with a compound-related decrease in mean body weight. A punctate cytoplasmic basophilia involving renal tubular epithelium of mice fed 0.5 to 4.5 percent apramycin, but not 0.15 percent apramycin, occurred after two years on treatment. The incidence and grade of this finding were dose related and the lesion was more pronounced in females than males. The lesion was considered minimal and of no clinical significance.

There was a marked reduction in the incidence of lymphosarcomas occurring in females fed diets containing 4.5 percent apramycin possibly due to their slower growth rate. No treatment related increase in neoplasia was observed.

k. No-Observed-Effect Level (NOEL): The NOEL for this study was 0.15 percent of the diet (approximately 200 mg/kg).

l. Conclusions: Apramycin fed to mice for two years at 0.15 percent of the diet produced no adverse effects. Levels of 0.5 to 4.5 percent produced lower weights and slight kidney histopathology.

B. Safe Concentration of total residues

1. No-Observed-Effect Level (NOEL):

The Safe Concentration of total residue was determined from the lowest NOEL in the most sensitive species from the various toxicology studies conducted. A summary of the studies which can be used in determination of the Acceptable Daily Intake (ADI) follows in Table 6.1.

Table 6.1. Summary of ADI toxicology studies for apramycin

Study Type Species Doses mg/kg/day NOEL mg/kg/day

One Year Oral Dog 0, 25, 50, 100 100

Chronic Toxicity & Oncogenicity Rat 0, 135, 275, 500, 3000* 500

Oncogenicity Mouse 0, 200, 640, 2000, 7350* 200

Three-generation Reproduction Rat 0, 194, 388, 785 785

*Doses approximate based on apramycin concentration in feed, feed intake data and averaged across sexes.

The one-year oral dog study was selected as the most appropriate study with the lowest NOEL for determining the acceptable daily intake (ADI). The NOEL for apramycin in that study was 100 mg/kg/day.

2. Calculation of the Acceptable Daily Intake (ADI) of total residue of apramycin:
                                     Lowest NOEL                                 
 Acceptable Daily Intake (ADI) =    -------------
                                    Safety Factor                               
A Safety Factor of 100 was used because the NOEL was from a chronic study.
 The lowest NOEL is 100 mg/kg, so
                                       100 mg/kg of body weight/day      
                              ADI =    ----------------------------
                                                   100
                                                  
                                  =      1 mg/kg of body weight/day     
However, this NOEL-based ADI exceeds 0.025 mg/kg body weight/day (1.5 mg/day). The Center has concerns about the microbiological safety of apralan at levels above 1.5 mg/person/day. That 1.5-mg/day level should produce no effects on human intestinal microflora. Therefore, in the absence of specific information on the effect of apramycin residues on human gut microflora, the Safe Concentrations were recalculated using the above equations and a weight-independent ADI of 1.5 mg/day and are summarized in Table 6.2.
3. Calculations of Safe Concentrations (SC): The maximum ADI for total microbiologically-active residues of noncarcinogens is limited to 1.5 mg/person/day. The daily consumption values of edible tissues are approximated as 300 g (0.3 kg) for muscle, 100 g (0.1 kg) for liver, 50 g (0.05 kg) for fat/skin, and 50 g (0.05 kg) for kidney.
                               Acceptable Daily Intake (ADI)                    
 Safe Concentration (SC)   =   -----------------------------
                                    Consumption Value   



                  1.5 mg/day                
SC (muscle)   =   ----------   =  5 mg/kg  =  5 ppm                                      
                  0.3 kg/day                                       
                                                                
                  1.5 mg/day              
SC (liver)    =   ----------   = 15 mg/kg  = 15 ppm                                          
                  0.1 kg/day                                       
                                                                
                  1.5 mg/day              
SC (fat/skin) =   ----------   = 30 mg/kg  = 30 ppm                                          
                  0.05 kg/day                                      
                                                                
                  1.5 mg/day              
SC (kidney)   =   ----------   = 30 mg/kg  = 30 pp                                         
                  0.05 kg/day                                      
Table 6.2. Safe Concentrations (SC) for total residues of apramycin in edible tissues using the revised food consumption factors

Edible Tissue Amount Consumed/Day Safe Concentration (SC)

Muscle 300 g 5 ppm

Liver 100 g 15 ppm

Fat/Skin 50 g 30 ppm

Kidney 50 g 30 ppm

C. Total Residue Depletion and Metabolism Studies

Refer to the original FOI Summary for NADA 106-964 for this information.

D. Comparative Metabolism

Refer to the original FOI Summary for NADA 106-964 for this information.

E. Tolerance for Residues

Refer to the original FOI Summary for NADA 106-964 this information. Since this approval does not result in a different tolerance than that currently codified and the sponsor did not petition FDA to change the tolerance for marker residue in swine kidney, that tolerance of 0.1 ppm in swine kidney remains codified.

F. Withdrawal Time

Refer to the original FOI Summary for NADA 106-964. The sponsor is not applying for a change in the withdrawal period at this time. The current withdrawal period for apramycin use in swine is 28 days.

G. Regulatory Method

Refer to the original FOI Summary for NADA 106-964 for this information.

**Table 6.1.** Summary of ADI toxicology studies for apramycin
Study Type	Species	Doses mg/kg/day	NOEL mg/kg/day
One Year Oral	Dog	0, 25, 50, 100	100
Chronic Toxicity & Oncogenicity	Rat	0, 135, 275, 500, 3000*	500
Oncogenicity	Mouse	0, 200, 640, 2000, 7350*	200
Three-generation Reproduction	Rat	0, 194, 388, 785	785
*Doses approximate based on apramycin concentration in feed, feed intake data and averaged across sexes.

**Table 6.2.** Safe Concentrations (SC) for total residues of apramycin in edible tissues using the revised food consumption factors
Edible Tissue	Amount Consumed/Day	Safe Concentration (SC)
Muscle	300 g	5 ppm
Liver	100 g	15 ppm
Fat/Skin	50 g	30 ppm
Kidney	50 g	30 ppm

VII. AGENCY CONCLUSIONS:

NADA	106-964
Sponsor:	Elanco Animal Health A Division of Eli Lilly & Company 2001 West Main Street Greenfield, Indiana 46140
Generic Name:	apramycin sulfate
Trade Name:	APRALAN®
Marketing Status:	Over the Counter (OTC)
Effect of Supplement:	Assigns an increased acceptable daily intake (ADI) and an increased safe concentration for total apramycin residues in edible tissues.