Spinosad Insecticide: Subchronic and Chronic Toxicity and Lack of Carcinogenicity in CD-1 Mice

K. E. Stebbins*,1, D. M. Bond*, M. N. Novilla{dagger} and M. J. Reasor{ddagger}

* Toxicology and Environmental Research and Consulting, The Dow Chemical Company, 1803 Building, Midland, Michigan 48674; {dagger} Lilly Research Laboratories, Eli Lilly and Company, Greenfield, Indiana 46268; and {ddagger} Department of Pharmacology and Toxicology, West Virginia University, Morgantown, West Virginia, 26506

Received August 17, 2001; accepted November 6, 2001


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The potential toxicologic and oncogenic effects of spinosad, a natural fermentation product with insecticidal properties, were investigated. The 13-week toxicity study consisted of groups of 10 CD-1 mice/sex provided diets containing 0, 0.005, 0.015, 0.045, or 0.12% spinosad (Study 1). The 0.12% group was terminated on Test Day 44 due to mortality and overt clinical signs of toxicity. An 18-month chronic oncogenicity study consisted of groups of 50 CD-1 mice/sex provided diets containing 0, 0.0025, 0.008, or 0.036% spinosad (Study 2). Two interim groups of 10 mice/sex/group were terminated after 3 and 12 months. Females given 0.036% were terminated on Day 455 due to markedly lower body weights and feed consumption, as well as excessive mortality. Because of the early termination of the female high-dose group, additional groups of 10 male and female mice (12-month interim necrospy) and 50 male and female mice (18-month necropsy) were provided diets containing 0, 0.0008, or 0.024% spinosad (Study 3) to fully assess potential chronic toxicity and oncogenicity. Standard toxicologic parameters were evaluated consistent with existing regulatory guidelines. The primary effect in the 13-week and 18-month studies was intracellular vacuolation of histiocytic and epithelial cells in numerous tissues and organs at doses of >= 0.015%. The histological vacuolation corresponded to ultrastructural lysosomal lamellar inclusion bodies. This alteration was consistent with phospholipidosis, a condition that results from accumulation of polar lipids in lysosomes. Lesions with no apparent direct relation to vacuolation were hyperplasia of the glandular mucosa of the stomach, skeletal muscle myopathy, bone marrow necrosis, and anemia with associated splenic hematopoiesis. The incidence of tumors in mice given spinosad was not increased relative to controls at any dose level. The no observed effect level for the 13-week study was 0.005% (6 mg/kg/day) spinosad, and for the chronic toxicity/oncogenicity study was 0.008% (11 mg/kg/day) spinosad for male and female CD-1 mice.

Key Words: spinosad; subchronic toxicity; carcinogenicity; lysosomes; phospholipidosis; cationic amphiphilic compounds; mice.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Spinosad is a marcolide insect control agent derived from a naturally occurring actinomycete bacterium, Saccharopolyspora spinosa (Mertz and Yao, 1990Go). Fermentation of this bacterium produces numerous metabolites (spinosyns), some of which have insecticidal activity. Spinosad provides highly efficacious control of many caterpillars of lepidopteran insects on numerous crops. Favorable properties of this insecticide include a novel mode of action (Salgado, 1998Go; Salgado et al., 1998Go), low toxicity to beneficial insects (DeAmicis et al., 1997Go; Kirst et al., 1992Go), and rapid degradation in the environment. Spinosad also has low acute mammalian toxicity (oral LD50 3738 mg/kg for male rats and > 5000 mg/kg for female rats and male and female mice, rabbit dermal LD50 > 5000 mg/kg, rat inhalation LC50 > 5.18 mg/l air). In addition, spinosad does not cause significant dermal or ocular irritation in rabbits and is not a skin sensitizer in guinea pigs.

This article describes the results from a 13-week dietary study (Study 1), and 2 18-month dietary oncogenicity studies (Studies 2 and 3) in CD-1 mice that provided data defining the toxicity and carcinogenicity potential of spinosad, consistent with the regulatory guidelines (EEC, 1988Go; EPA, 1982Go; OECD, 1981Go; JMAFF, 1985Go).


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Study design.
The 13-week dietary toxicity study (Study 1) consisted of groups of 10 mice/sex/dose that were given feed formulated to provide 0, 0.005, 0.015, 0.045, or 0.12% spinosad at Eli Lilly and Company (Greenfield, IN). These dose levels corresponded to 0, 6.0, 17.9, 57.2, or 109.7 mg/kg/day for males and 0, 8.1, 23.1, 71.5, or 141.9 mg/kg/day for females, respectively, based on actual body weight data, and an estimated normal feed consumption value of 4.2 g/day for male and female mice in the 0, 0.005, 0.015, and 0.045% dose groups. An estimated feed consumption value of 50% normal was used for calculating test material intake for the high-dose animals (0.12% spinosad), which were terminated on Day 44 in a moribund condition as a result of their cachectic state and decreased body weights.

Based on results of the 13-week study, an 18-month oncogenicity study (Study 2) was conducted at The Dow Chemical Company (Midland, MI). Groups of 70 mice/sex/dose were given feed formulated to provide 0, 0.0025, 0.008, or 0.036% spinosad for up to 18 months. These dose levels corresponded to 0, 3.4, 11.4, or 50.9 mg/kg/day for males, and 0, 4.2, 13.8, or 67.0 mg/kg/day for females, respectively, based on body weight and feed consumption data. Groups of 10 randomly selected mice/sex/dose were terminated at 3 or 12 months, and the remaining 50 mice/sex/dose were on test for 18 months, with the exception of the high-dose females that were terminated on Day 455 due to markedly lower body weights and feed consumption, as well as excessive mortality. Because of the early termination of the female high-dose group, another 18-month chronic toxicity/oncogenicity study (Study 3) was conducted with groups of 10 male and female mice (12-month interim group) and 50 male and female mice (18-month termination) provided diets containing 0, 0.0008, or 0.024% spinosad to assess oncogenic potential. These dose levels corresponded to 0, 1.1, or 32.7 mg/kg/day for males, and 0, 1.3, or 41.5 mg/kg/day for females, respectively, based on body weight and feed consumption data.

Test material.
Technical grade spinosad (CAS# 131929-60-7), an off-white powder, was supplied by Dow AgroSciences (Lot # ACD 13453, Study 1; Lot # ACD 13651, Studies 2 and 3; Indianapolis, IN). Spinosad was primarily (77.6%, Study 1; 88%, Studies 2 and 3) composed of spinosyns A and D that only differ very slightly in chemical structure from each other and represent approximately 100% of the insecticidal activity of spinosad (Fig. 1Go).



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FIG. 1. The structure of spinosad.

 
The remaining components in spinosad consist of trace quantities of a number of structurally related spinosyns that have minor substitutions at various locations in the molecule, as well as amino acids and salts.

The identity of spinosad was confirmed by elemental analysis, high performance liquid chromatography, nuclear magnetic resonance, mass spectroscopy, infrared, and ultra violet spectroscopic techniques. In addition, spinosad was characterized by thermal gravimetric analysis, differential thermal analysis, and x-ray powder diffraction. Test material analyses indicated a purity of 77.6% (65.7% spinosyn A and 11.9% spinosyn D, Study 1) and 88.0% (76.1% spinosyn A and 11.9% spinosyn D, Studies 2 and 3).

Test species and animal husbandry.
Male and female CD-1 mice, approximately 5–6 weeks of age, were obtained from Charles River Laboratories Inc. (Portage, MI) for all studies. Upon arrival at the laboratories, which were fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC), the mice were examined by a veterinarian and were acclimated to the laboratory environment for at least 7 days prior to the initiation of dosing. A weight randomization computer program designed to ensure homogeneity of body weights was used to select and assign the animals to the experimental groups. The animals in the 13-week study were housed in groups of 3 or 4 per cage, and animals in the 18-month studies were housed individually. All studies used elevated stainless steel, wire mesh cages. Animals were maintained in environmental conditions acceptable for mice. A basal diet of Purina Certified Rodent Chow #5002 (Richmond, IN, mill of Ralston Purina Co., St. Louis, MO), the vehicle for the test material, and tap water (municipal water supply) were available ad libitum during the acclimation and dosing periods.

Test diets.
Test diets were prepared weekly. Spinosad was stable in rodent chow for at least 40 days, and homogeneously distributed within the diets. Concentrations of spinosad in rodent chow were within 10% of the targeted concentrations for the 13-week study, and within 5% of the targeted concentrations for the 18-month studies, indicating that the mice received the targeted spinosad concentrations.

Clinical observations, feed consumption, and body weight.
All mice were observed at least once daily for general appearance, behavior, signs of toxicity, moribundity, mortality, and feed wastage. Clinical examinations were performed weekly and evaluated the skin, fur, mucous membranes, respiration, and nervous system functions. Body weights (but not feed consumption) were measured weekly during the 13-week study. In the 18-month studies, body weight data were collected and feed consumption calculated once during the pre-study period, weekly for the first 13 weeks of the study, and for approximately a 1-week period each month thereafter.

Clinical pathology.
Blood for hematology and clinical chemistry determinations was obtained from the orbital sinus, or by cardiac puncture, of fasted or nonfasted (0.12% group) mice following light anesthesia with ether (Study 1), or from the orbital sinus of nonfasted mice following light anesthesia with methoxyflurane at 3, 12, and 18 months (Study 2) or 12 and 18 months (Study 3). Standard hematologic and clinical chemistry parameters consistent with general toxicology guidelines (EEC, 1988Go; EPA, 1982Go; JMAFF, 1985Go; OECD, 1981Go) were evaluated.

Gross pathology and organ weights.
A veterinary pathologist conducted a complete gross examination on all animals. The following tissues were weighed from all animals that survived to the scheduled termination of each study: brain, heart, kidneys, liver, ovaries (Study 1), testes, and spleen. Organ weight data were not collected for male and female animals in the 0.12% group necropsied on Day 44 (Study 1), or from female animals in the 0.036% group necropsied on Day 455 (Study 2). In addition, animals that died or were euthanized in a moribund condition did not have organ weight data collected. A complete set of tissues, consistent with general toxicology guidelines (EEC, 1988Go; EPA, 1982Go; JMAFF, 1985Go; OECD, 1981Go), were collected and preserved in neutral, phosphate-buffered 10% formalin for all animals.

Histopathology.
A histopathologic examination was performed on all tissues collected from all mice (Study 1), from all controls and high-dose mice (Study 2: 3- and 12-month necropsies); from all controls, intermediate-dose females, and high-dose males (Study 2: 18-month necropsy), or from all control females and high-dose females (Study 3). Tissues evaluated from the low- and/or intermediate-dose group mice (Study 2) consisted of cervix, epididymides, kidneys, liver, lungs, mesenteric and mediastinal lymph nodes, ovaries, oviducts, pancreas, parathyroids, skeletal muscle, spleen, stomach, thymus, tongue, uterus, vagina, and gross lesions. Tissues were prepared by conventional techniques, sectioned approximately 6 microns thick, stained with hematoxylin and eosin, and were examined by a veterinary pathologist using a light microscope.

Electron microscopy.
Electron microscopic evaluations were conducted on the liver, kidney, and lungs from 3 mice/sex from the 0 and 0.45% groups (Study 1). Tissues were placed in a modified Karnovsky's fixative, postfixed in 2% osmium tetroxide, embedded in epon plastic, and processed for transmission electron microscopic examination by conventional procedures.

Statistical analysis, Study 1.
Dunnett's test (Dunnett, 1964Go) was used in the analysis of body weights, body weight gains, hematology, clinical chemistry, and organ weight data. The homogeneity of variances was tested by the method of Bartlett (Steel and Torrie, 1960Go). All references to statistical significance corresponded to p <= 0.05.

Statistical analysis, Studies 2 and 3.
Body weights, clinical chemistry parameters, hematologic parameters, absolute and relative organ weights were evaluated by Bartlett's test for equality of variances (Winer, 1971Go). Based upon the outcome of Bartlett's test, exploratory data analysis was performed by a parametric (Steel and Torrie, 1960Go) or nonparametric ANOVA (Hollander and Wolfe, 1973Go), followed respectively by Dunnett's test or the Wilcoxon Rank-Sum test with Bonferroni's correction for multiple comparisons (Miller, 1966Go).

Differences in mortality patterns were tested by the Gehan-Wilcoxon procedure for all animals scheduled for the 18-month necropsy. The incidences of specific histopathologic observations were first tested for linearity using ordinal spacing of the doses for tissues that were examined from all animals and doses. If linearity was not rejected, the data were then tested for a dose-response relationship using the Cochran Armitage trend test. If the trend was statistically significant, or if significant deviation from linearity was found, incidences for each dose were compared to that of the control using a pairwise chi-square test with Yates correction. Statistical analysis was limited to the pairwise comparison of control and high dose for tissues that were evaluated only from control and high-dose mice. Observations from tissues or organs from the low or mid doses that were examined only because of a grossly observed lesion, or because of mortality/morbidity, were not analyzed statistically.

The nominal alpha levels used were as follows: Bartlett's test = 0.01 (Winer, 1971Go), parametric ANOVA = 0.10 (Steel and Torrie, 1960Go), nonparametric ANOVA = 0.10 (Hollander and Wolfe, 1973Go), Dunnett's test = 0.05, 2-sided (Winer, 1971Go), Wilcoxon rank-sum test = 0.05, 2-sided (Hollander and Wolfe, 1973Go), Bonferroni correction (Miller, 1966Go), outlier test = 0.02, 2-sided (Grubbs, 1969Go), Gehan-Wilcoxon = 0.05 (Breslow, 1970Go), chi-square test for linearity = 0.01, trend test = 0.02, 2-sided (Armitage, 1971Go), and the pairwise comparison, Yates chi-square test = 0.05, 2-sided (Fleiss, 1981Go).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Male and female mice in the 13-week and 18-month studies exhibited significant toxicity at the highest dose levels. A large number of dependent variables were examined, and the salient findings are summarized in Tables 1 and 2GoGo.


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TABLE 1 Summary of Treatment-Related Findings (13 Weeks)
 

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TABLE 2 Summary of Treatment-Related Findings (Chronic Toxicity and Oncogenicity Studies)
 
Thirteen-Week Study
Clinical observations.
Signs of toxicity in mice given 0.12% spinosad were nonspecific, and included rough hair coat, decreased activity, thinness, and rapid respiration. Three males and 2 females given 0.12% spinosad died during the first 6 weeks of treatment, and all surviving mice in this dose level were euthanized (Day 44) because the maximum tolerated dose (MTD) was exceeded. Males given 0.015 or 0.045% spinosad had ventral or perineal soiling.

Body weights.
Male and female mice given 0.12% spinosad had progressive weight loss, and body weights of males given 0.045% spinosad were significantly lower than controls. After 6 weeks of treatment, the mean body weight gain of males and females from the 0.12% dose group was decreased 230 and 165%, respectively, relative to controls. By study termination, the mean body weight gain of males given 0.045% spinosad was 33% lower than controls.

Hematology.
Mice given 0.12% spinosad had decreases in mean hematocrit (males 32%, females 22% decrease), hemoglobin concentration (males 33%, females 29% decrease), and RBC count (males 19%, females 7% decrease), relative to controls, with corresponding decreases in RBC indices for both sexes. Mice given 0.045% spinosad had less pronounced decreases in mean hematocrit (males 11%, females 3% decrease), hemoglobin concentration (males 12%, females 4% decrease), and RBC indices relative to controls. Male and female mice given 0.12% spinosad had markedly higher mean total leukocyte counts (12–16 times higher than controls), with elevations in mean neutrophil, lymphocyte, and monocyte counts. Neutrophils in mice given 0.12% spinosad had cytoplasmic basophilia and nuclear hypersegmentation. Males given 0.045% spinosad had a significant decrease in mean lymphocyte count (43% decrease) and females given 0.045% spinosad had a significant increase in mean neutrophil count (73% increase), relative to controls. The alterations in leukocyte counts were attributed to hepatic inflammation with necrosis, and inflammation of the glandular mucosa of the stomach.

Clinical chemistry.
Male and female mice given 0.12% spinosad had increases in mean alkaline phosphatase (AP) (2–3 times higher than controls), alanine aminotransferase (ALT) (11–15 times higher than controls), and aspartate aminotransferase (AST) (5–8 times higher than controls) enzyme activities; minor increases in mean globulin levels; and minor decreases in mean albumin levels. Males also had minor decreases in mean glucose, blood urea nitrogen, bilirubin, cholesterol and triglyceride levels, and females had minor decreases in mean glucose and bilirubin levels. Males given 0.045% spinosad had significant increases in mean AP (37% higher than controls), ALT and AST (2 times higher than controls) enzyme activities, and a significant decrease in mean albumin (10% lower than controls). Females given 0.045% spinosad had significant increases in mean ALT (2.5 times higher than controls) and AST (2 times higher than controls) enzyme activities. The alterations in AP, ALT, and AST were attributed to hepatic necrosis and inflammation. In addition, degeneration of skeletal muscle may have contributed to the elevated AST.

Gross pathology.
Mice given 0.12% spinosad were thin and cachectic. Other treatment-related gross observations consisted of pale or necrotic liver, pale kidneys, and enlargement of the spleen and mesenteric lymph nodes.

Organ weights.
Mice given 0.045% spinosad had treatment-related increases in absolute and relative weights of the kidneys, liver, and spleen. The mean relative kidney weights of males and females were increased 16 and 13%, respectively, relative to controls. The mean relative liver weights were increased 25% for both sexes, and the mean relative spleen weights of males and females were increased 26 and 59%, respectively, relative to controls. Females given 0.015% spinosad had a treatment-related increase in mean relative spleen weight (17%).

Histopathology.
Treatment-related effects occurred in multiple tissues and were characterized by the following morphologic findings: (1) cytoplasmic vacuolation of cells, (2) aggregates of histiocytes/macrophages within an organ, (3) degeneration, regeneration, or necrosis, and (4) combinations of these lesions (Table 3Go). Additional treatment-related effects consisted of hyperplasia of the glandular mucosa of the stomach, and extramedullary hematopoiesis of the spleen.


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TABLE 3 Histopathology: Effects and Target Organs at 13 Weeks
 
The majority of mice given 0.045 or 0.12% spinosad had cytoplasmic vacuolation of various combinations of the following tissues: liver (hepatocytes and Kupffer cells), lymphoid tissue (lymph nodes, spleen, and thymus), renal tubular epithelial cells, myocardium, skeletal muscle, pancreatic acinar cells, ovary (follicles, corpora lutea, and interstitial cells), and epithelial cells of the oviduct, uterus, cervix, vagina, and epididymides. Other organs with less frequent occurrence of cytoplasmic vacuolation were the adrenal glands (males only from the 0.45 and 0.12% groups), and the pituitary gland (0.12% group only). In addition, a few mice given 0.015% spinosad had vacuolation of lymphoid tissue, liver, and/or kidneys.

Aggregates of histiocytes/macrophages were present in the lymph nodes, lungs, stomach, uterus, and cervix of numerous mice given 0.045% or 0.12% spinosad. In addition, 1 male given 0.015% spinosad had histiocytosis of lymph nodes. The histiocytes/macrophages in all affected tissues had foamy-appearing cytoplasm with faintly visible cytoplasmic vacuoles.

Degenerative changes occurred in the kidneys and skeletal muscles of mice from the 0.045 and 0.12% groups. The degeneration of the kidneys was characterized by clusters of cortical tubules undergoing vacuolar degeneration with accompanying regenerative changes. Skeletal muscle myopathy was characterized by individual degenerative or regenerative muscle fibrils of the tongue and quadriceps.

Necrosis of the liver was noted in most of the mice given 0.12% spinosad and was considered to be the proximate cause of death in 5 animals. The liver necrosis was characterized by foci of coagulation necrosis and fibrosis surrounded by thick bands of necrotic inflammatory cells. The majority of mice given 0.12% spinosad had extensive necrosis in the mesenteric lymph nodes. Necrosis of lymphocytes was noted in the lymph nodes, spleen, and thymus of mice given 0.015, 0.045, or 0.12% spinosad. In addition, 1 male given 0.12% spinosad had lymphocytic necrosis of Peyer's patches of the ileum. Mild necrosis occurred in the bone marrow of 1 of 20 and 11 of 19 mice given 0.015% or 0.045% spinosad, respectively. One female from the 0.12% group had an area of necrosis involving the entire right half of the lung, with secondary adhesive pleuritis.

All mice given 0.45 or 0.12%, and 7 of 20 mice given 0.15% spinosad had hyperplasia of the glandular mucosa of the stomach. The hyperplasia was characterized by a multifocal distribution of marked glandular dilation at various depths of the mucosa. In addition, there were combinations of the following changes: inflammation in the basilar portion of the mucosa; hyaline droplets, mineralized debris, and sloughed cells within the dilated glands; and necrosis of superficial epithelium usually in areas of hyaline droplet formation. Histiocytic cells with foamy cytoplasm accompanied the gastric lesions in some mice given 0.045 or 0.12% spinosad.

Splenic involvement, other than the changes in lymphoid cells already discussed, consisted of extramedullary hematopoiesis. This occurred in 2 of 19 and 15 of 20 mice given 0.045 or 0.12% spinosad, respectively.

Electron microscopy.
Vacuolation identified by light microscopy was ultrastructurally characterized primarily by the presence of numerous lysosomes that contained membranous whorls, and irregular electron dense aggregates in mice given 0.045% spinosad. In the liver, cytoplasmic lamellar inclusion bodies occupied up to one-quarter of the cytoplasmic volume of hepatocytes (Fig. 2Go). In the kidney, cytoplasmic lamellar inclusion bodies occupied up to one-half of the cytoplasmic volume of some proximal and distal convoluted tubular epithelial cells. Some lamellar material also was present in the lumen of renal tubules. Changes in the lung consisted of lamellar inclusions in the cytoplasm of pneumocytes and alveolar macrophages. Vascular endothelial cells and fibrocytes in the interstitium of each tissue also had cytoplasmic lamellar inclusion bodies, although the extent of the change was substantially less than the changes in epithelial cells. Occasional cytoplasmic lamellar inclusion bodies occurred in tissues of the control mice but these inclusion bodies were small and infrequent.



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FIG. 2. Electron micrograph of a hepatocyte from a male mouse given 0.045% spinosad for 13 weeks demonstrates numerous lysosomal inclusion bodies that contain a whorled pattern of lipid membranes.

 
Chronic Toxicity and Oncogenicity Studies
Clinical observations.
In Study 2, mortality for females given 0.036% spinosad increased relative to controls around Day 280 and was 60% by Day 448. All surviving females given 0.036% spinosad were euthanized on Day 455 because of excessive mortality. There were no statistically identified differences in overall mortality for the remaining groups of mice in Studies 2 and 3. Mortality rates at the end of Study 2 (18 months) for the remaining dose groups were 24, 35, 29, and 44% for males given 0, 0.0025, 0.008, or 0.036% spinosad, respectively; and 18, 26, and 13% for females given 0, 0.0025, or 0.008% spinosad, respectively. Mortality rates at the end of Study 3 (18 months) were 20, 14, and 20% for males, and 22, 16, and 22% for females given 0, 0.0008, or 0.024% spinosad, respectively.

Treatment-related clinical observations in mice given 0.036% spinosad consisted of an increased incidence of thin appearance, roughened haircoat, dermatitis of the ears, lacrimation, and perineal soiling. These findings were consistent with the markedly lower body weights, excessive mortality, and general debilitated condition of these mice.

Body weights and feed consumption.
Body weights of males given 0.024% or 0.036% spinosad were statistically lower than controls (3 to 8% and 3.0 to 11%, respectively) for the majority of the 18-month dosing period. Females given 0.036% spinosad had lower body weights (5 to 11%) that were statistically identified from Day 182 until their termination on Day 455. Body weights of males given <= 0.008% spinosad and females given <= 0.024% spinosad were comparable to the controls. Mean feed consumption of males given 0.036% spinosad was 2 to 11% lower than controls from Days 21 to 312. The mean feed consumption of this group was comparable to controls from Day 340 through the remainder of the study. Mean feed consumption of females given 0.036% spinosad was 5 to 17% lower than controls from Day 147 through the remainder of the dosing period (Day 455).

Hematology.
Erythrocytic parameters (RBC count, hematocrit, and hemoglobin concentration) were decreased approximately 10–20% in males given 0.036% spinosad at 3 and 12 months, and in females given 0.036% spinosad at 3 months. The WBC counts of males and females given 0.036% spinosad, and females given 0.024% spinosad, were 2–2.5 times higher than the controls at 12 months. The higher WBC counts were likely related to inflammation of the stomach observed in these mice. At 18 months, there were no significant hematologic alterations in mice from any dose level.

Clinical chemistry.
AST activity was 50% higher than controls in males given 0.036% spinosad for 3 months. This difference was consistent with microscopic degenerative lesions seen in skeletal muscle. Total protein and albumin concentrations were 6–13% lower than controls in mice given 0.036% spinosad for 12 and 18 (males only) months. Calcium levels were 5% lower than controls in males given 0.036% spinosad for 12 and 18 months, and phosphorus levels were 27% higher than controls in females given 0.036% spinosad for 12 months.

Gross pathology.
The glandular mucosa of the stomach was thickened in the majority of mice given 0.024 or 0.036% spinosad for >= 12 months. Decreased body fat was a nonspecific sign of toxicity in mice given 0.036% spinosad for >= 12 months that reflected general stress and malnutrition.

Organ weights.
Increased spleen weights (absolute and relative) were identified in males and females given 0.036% spinosad for 3 months and were attributed to extramedullary hematopoiesis of the spleen. Absolute and relative liver weights were increased by 38 and 26%, respectively, in females given 0.036% spinosad for 3 months. Relative liver weights were 5–21% higher in the following groups: males given 0.036% spinosad for 3, 12, and 18 months; females given 0.036% spinosad for 12 months; and males and females given 0.024% spinosad for 18 months. These weight differences were not associated with any treatment-related microscopic effects. Relative brain weights were 11–21% higher in males given 0.036% spinosad for 18 months, females given 0.036% spinosad for 12 months, and males given 0.024% spinosad for 12 months. Absolute heart weights were 10% lower in males given 0.036% spinosad for 18 months, and 15% lower in males given 0.024% spinosad for 12-months. The differences in brain and heart weights were attributed to lower body weights of animals given 0.024% or 0.036% spinosad, and were not associated with any treatment-related microscopic alterations.

Histopathology.
Treatment-related microscopic findings are tabulated in Tables 3 and 4GoGo. The majority of mice given 0.036% spinosad for >= 3 months had treatment-related cytoplasmic vacuolation of combinations of the following tissues: pancreas (acinar cells), parathyroid gland (parenchymal cells), lymph nodes (macrophages), ovary (follicles, corpora lutea, and interstitial cells), and epithelial cells of the uterus (Fig. 3Go), cervix, vagina, and epididymides. Approximately 50% of the females given 0.024% spinosad for >= 12 months had vacuolation of parenchymal cells of the parathyroid gland. There was no significant progression in the severity or incidence of vacuolation in any tissue from 3 or 12 months until study termination at 18 months.


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TABLE 4 Histopathology: Effects and Target Organs at 12 and 18 Months
 


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FIG. 3. Photomicrographs of H & E stained uterus from mice given 0 (A) or 0.036% (B) spinosad for 18 months demonstrate significant intracytoplasmic vacuolation of endometrial gland epithelial cells of the spinosad treated mouse.

 
Aggregates of histiocytes/macrophages were present in the lungs and lymph nodes of numerous mice given 0.024 or 0.036% spinosad. The histiocytes/macrophages in the lungs and lymph nodes had foamy cytoplasm. Some mice given 0.036% spinosad for 3 or 12 months had vacuolation of histiocytes in mesenteric lymph nodes.

Degenerative changes occurred in the kidneys of mice given 0.036% spinosad at 3 months, and in skeletal muscles of mice given 0.024 or 0.036% spinosad at >= 3 months. The degeneration of the kidneys was characterized by clusters of cortical tubules with basophilic and vacuolated epithelial cell cytoplasm, thickened basement membranes, and occasional proteinaceous casts with accompanying regenerative changes. Mice given spinosad for 12 or 18 months had no treatment-related kidney alterations. Skeletal muscle myopathy was most commonly present in the tongue, and was noted with less frequency in the biceps femoris, and in muscle fibers adjacent to the vertebra, sternum, esophagus, trachea, larynx, and nasal turbinates. The myopathy was characterized by degenerative myofibers that had fragmentation, swelling, or atrophy, and loss of visible striations. Macrophages were at the periphery of some affected myofibers.

The incidence of extramedullary hematopoiesis of the spleen was increased in mice given 0.036% spinosad for 3 months. This effect was associated with decreases in RBC count, hematocrit, and hemoglobin concentration of mice following 3 months of dosing.

Mice given 0.024 or 0.036% spinosad had an increase in the incidence and severity of hyperplasia of the glandular mucosa of the stomach relative to controls. The hyperplasia was first noted at the 3-month evaluation, and progressed in severity with duration of the study. Some mice from the control, low- and intermediate-dose groups had a spontaneous occurrence of very slight to slight, focal or multifocal hyperplasia of the stomach, identified by dilated gastric glands in the mid to lower level of the glandular mucosa. The epithelial cells in these dilated glands were smaller, less differentiated, and increased in number compared to adjacent unaffected glands. The affected mucosal glands were most common in the fundus, particularly in the region close to the forestomach. In mice from the 0.024 and 0.036% groups, there was a spectrum of stomach alterations ranging from hyperplastic foci similar to those observed in control mice, to severe, diffuse hyperplasia involving the entire thickness of the glandular mucosa (Fig. 4Go). As with controls, the hyperplasia was most prominent in the fundic region close to the forestomach and decreased in severity towards the pyloric antrum. Even at 3 months, the lesions in the 0.036% group involved the entire thickness of the mucosa in some mice. The hyperplasia appeared to involve all of the various cell types of the glandular mucosa, with surface mucous cells and neck mucous cells representing the majority of proliferative cell types in most affected mice. In addition, there were increased numbers of relatively undifferentiated cells. These cells did not appear dysplastic or anaplastic, but rather to be immature cells that had not differentiated. In more severely affected mice and primarily later in the study, there was evagination of the proliferative mucosa into the submucosa and, in a few mice, into the subserosa. The incidence of hyperplasia of the glandular mucosa of the stomach was between 90 and 100% in mice given 0.024 or 0.036% spinosad at all evaluation times. Controls generally had less than 50% incidence of gastric hyperplasia at all evaluation times, with significantly less severity than the hyperplasia noted in mice given 0.024 or 0.036% spinosad. Additional treatment-related alterations of the stomach in mice given 0.024 or 0.036% spinosad consisted of increased incidences of chronic inflammation of the glandular mucosa, and hyperplasia with or without hyperkeratosis of the nonglandular mucosa.



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FIG. 4. Photomicrographs of H & E stained stomach from mice given 0 (A) or 0.036% (B) spinosad for 18 months demonstrate diffuse hyperplasia of the glandular mucosa of the spinosad treated mouse.

 
The tumor observations from the oncogenicity portion of Studies 2 and 3 are presented in Tables 5 and 6GoGo. There were no increases in any tumor type from any tissue that were statistically identified, nor were there any tumors that were interpreted to be treatment related.


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TABLE 5 Incidence of Neoplasia in CD-1 Mice Ingesting Spinosad for 18 Months (Study 2)
 

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TABLE 6 Incidence of Neoplasia in Female CD-1 Mice Ingesting Spinosad for 18 Months (Study 3)
 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Decreased body weights and increased mortality occurred in mice given 0.12% spinosad for up to 44 days and in females given 0.036% spinosad for up to 455 days. Necrosis of the liver was considered to be the proximate cause of death in 5 animals given 0.12% spinosad. Marked hyperplasia of the stomach glandular mucosa was interpreted to be a contributory factor for the death or moribund condition of several mice given 0.036% spinosad for up to 18 months. The progressive gastric hyperplasia may have led to decreased feed consumption and eventual debility and death in some animals.

The primary microscopic effect in the 3-month and 18-month studies was intracellular vacuolation of histiocytic and epithelial cells in numerous tissues and organs at doses of >= 0.015%. Also vacuolated, but to a lesser degree, were cardiac and skeletal muscle cells. Ultrastructural characterization of the vacuolation of the liver, kidneys, and lungs from mice given 0.045% spinosad for 13 weeks indicated that the vacuoles consisted of lysosomes that contained concentric membranous whorls (lamellar bodies). The light microscopic and ultrastructural appearances of these cytoplasmic lamellar bodies were consistent with phospholipidosis induced by cationic amphiphilic drugs (CADs) (Reasor, 1989Go; Schneider, 1992Go). These drugs have a common structural feature in that they are cationic and amphiphilic in nature. That is, the drugs contain a hydrophobic domain in close proximity to a hydrophilic domain. The hydrophilic group consists of 1 or more primary or substituted nitrogen groups, of which at least 1 is charged at physiological pH. Spinosad has a structure consisting of a tertiary amine group, 2 sugars (1 on each end of the molecule) and a large hydrophobic multiple ring complex, and can be classified as a cationic amphiphilic compound. CADs belong to a number of pharmacological classes that include anorectic, antiarrhythmic, antidepressant, antihistaminic, antianxiety, antimalarial, cholesterol synthesis inhibitors, antibiotics, and others. Spinosad has no known pharmacological activity in mice (Horii, D., unpublished report). Further discussions on the characteristics and physiologic effects of cationic amphiphilic drugs, along with spinosad reference dose data, are presented in the companion article in this journal (Yano et al., 2002Go).

The gastric mucosal hyperplasia in mice was not accompanied by vacuolar changes and the pathogenesis is unexplained. In untreated control mice, spontaneous hyperplasia of the glandular mucosa of the stomach was present in approximately 50% of males and females at 12 and 18 months. In most affected control mice, the gastric alteration consisted of small hyperplastic foci in the fundic region near the forestomach, interspersed among mostly normal glandular mucosa. The hyperplasia in control mice was consistent with spontaneous age-associated lesions described in mice (Rehm et al., 1987Go; Stewart and Anervont, 1938) and rats (Frantz et al., 1991Go). In mice from the 0.015, 0.045, and 0.12% groups of the 13-week study, and from the 0.024 and 0.036% groups of the 18-month study, there appeared to be a treatment-related exacerbation of the gastric hyperplasia noted in controls. In mice from these higher dose groups there was a spectrum of lesions, ranging from small foci of hyperplasia as noted in control mice, to severe, diffuse hyperplasia involving the entire thickness of the glandular mucosa that worsened with duration of exposure. In more severely affected mice and primarily later in the 18-month study, there was evagination of the proliferative mucosa into the submucosa and, in a few mice, into the subserosa of the stomach. Although florid in nature, these proliferative lesions were hyperplastic. The hyperplastic response at all sacrifice intervals appeared to involve all of the various cell types of the glandular mucosa, with surface mucous cells and neck mucous cells representing the majority of proliferative cell types in most affected mice. Hyperplastic gastric mucosal lesions similar to those seen in this study have been reported in mice under a variety of circumstances including autoimmune factors (Kojima et al., 1980Go; Suzuki et al., 1981Go), nutritional changes (Rehm et al., 1987Go), environmental changes (Greaves and Boiziau, 1984Go), and administration of chemicals (Betton et al., 1988Go; Streett et al., 1988Go).

Other lesions with no apparent direct relation to the vacuolation were splenic hematopoiesis, skeletal muscle myopathy, hepatocellular cytomegaly, bone marrow necrosis, and necrosis in the liver, lymph nodes, and lung. Increased hematopoiesis of the spleen was in response to anemia of high-dose animals following 3 months of dosing. Skeletal muscle myopathy appeared to be distinct from the occasional myocytic vacuolation, and was considered to be a primary lesion induced by treatment. A similar muscle alteration has been reported in rats given chlorphentermine, a compound that interferes with the metabolism of phospholipids (Schmalbruch, 1978Go). When administered to the rat in daily doses for 5 days, chlorphentermine caused skeletal muscle necrosis and the formation of multilayered lipid bodies in secondary lysosomes of nonnecrotic muscle fibers. Hepatocellular cytomegaly, noted only in animals from the 13-week study, was not prominent or frequent, and may have been a precursor to or part of the hepatocellular vacuolation process although enzyme induction is not ruled out. Bone marrow necrosis in animals of the 13-week study was also mild. The bone marrow necrosis did not appear to be a secondary response to inanition or cachexia. Liver, lung, and mesenteric lymph node necrosis was infrequent and affected only the high-dose group of the 13-week study.

In summary, the data from the 13-week and 18-month studies indicated that mortality and histopathologic effects consisting of vacuolation, degeneration/regeneration, and aggregates of histiocytes/macrophages occurred in mice given the highest doses of spinosad. Vacuolation was due to the intralysosomal accumulation of lamellar bodies and is consistent with the effects induced by a cationic amphiphilic compound. The other prominent effect, hyperplasia of the gastric mucosa, was not accompanied by vacuolar changes. Lifetime exposure to the highest dose levels of spinosad did not cause a treatment-related increase in the incidence of neoplasms in any tissue.


    NOTES
 
1 To whom correspondence should be addressed. Fax: (989) 638-9863. E-mail: kestebbins{at}dow.com. Back


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 RESULTS
 DISCUSSION
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