* Toxicology and Environmental Research and Consulting, The Dow Chemical Company, 1803 Building, Midland, Michigan, 48674;
Lilly Research Laboratories, Division of Eli Lilly and Company, Greenfield, Indiana 46268; and
Department of Pharmacology and Toxicology, West Virginia University, Morgantown, West Virginia, 26506
Received August 17, 2001; accepted November 6, 2001
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ABSTRACT |
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Key Words: spinosad; rat; toxicity; carcinogenicity.
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INTRODUCTION |
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This article highlights the results from 2 subchronic dietary studies, and a dietary chronic toxicity and oncogenicity study in rats that provided data defining the toxicity and carcinogenic potential of spinosad, consistent with regulatory guidelines (EEC, 1988; EPA, 1982
; OECD, 1981
; JMAFF, 1985
).
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MATERIALS AND METHODS |
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Groups of 60 rats/sex/dose were also given feed providing 0, 0.005, 0.02, 0.05, or 0.1% spinosad for up to 2 years (Study 3, The Dow Chemical Company, Midland, MI). These dose levels corresponded to 0, 2.4, 9.5, 24.1, or 49.4 mg/kg/day for males and 0, 3.0, 12.0, 30.3, or 62.8 mg/kg/day for females. Ten randomly selected rats/sex/dose were necropsied at 12 months, and the remaining 50 rats/sex/dose were given spinosad for an additional year, or until they died, were euthanized in a moribund condition, or termination of a dose level (0.1%).
Test material.
Technical grade spinosad (CAS# 131929-60-7), an off-white powder, was supplied by Dow AgroSciences (Lot # ACD13453 for Study 1 and Lot # ACD13651 for Studies 2 and 3), Indianapolis, IN. Fermentation of the bacterium produces numerous metabolites (spinosyns), some of which have insecticidal activity. Spinosad was primarily 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. 1).
The identity of spinosad was confirmed by elemental analysis, high performance liquid chromatography, infrared, nuclear magnetic resonance, mass spectroscopy, 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). The remaining components in spinosad consisted 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.
Test species and animal husbandry.
Male and female CDF Fischer 344 rats, approximately 4 to 5 weeks of age, were obtained from Taconic Laboratory Animals and Services (Germantown, NY; Study 1) and Charles River Laboratories, Inc. (Kingston, NY; Studies 2 and 3). Animals were individually housed in suspended, stainless steel cages with wire floors in animal rooms that were maintained in environmental conditions acceptable for rats (fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International [AAALAC]). Rats were fed Purina Certified Rodent Chow #5002 (Richmond, IN, mill of Ralston Purina Co., St. Louis, MO) ad libitum. Municipal water was provided ad libitum via a pressure-activated automatic watering system.
Test diets.
Test diets were prepared weekly. Concentrations of spinosad in rodent chow were analyzed using high performance liquid chromatography with ultraviolet detection and external standards. Dietary concentrations were within 78 and 3% of the targeted concentrations for the subchronic and 2-year studies, respectively. Spinosad was stable in rodent chow for at least 40 days, and homogeneously distributed within the diets.
Clinical observations, feed consumption, and body weight.
All rats 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. Feed consumption and body weights were measured weekly for the 13-week studies. These parameters were also measured weekly for the first 13 weeks, and for approximately a 1-week period each month thereafter for the 2-year study.
Clinical pathology.
Blood was obtained from the orbital sinus of fasted (Study 1) or nonfasted (Studies 2 and 3) rats that were lightly anesthetized with ether (Study 1) or methoxyflurane (Studies 2 and 3) at the end of the 13 weeks, and at 6, 12, and 24 months. Standard hematologic parameters were evaluated consistent with toxicology regulatory guidelines (EEC, 1988; EPA, 1982
; OECD, 1981
; JMAFF, 1985
).
Blood for serum chemistry determinations was obtained from the abdominal aorta (Study 1) following anesthesia of fasted rats or from the orbital sinus (Studies 2 and 3) following anesthesia and at 6, 12, 18, and 24 months from fasted rats. Standard parameters were evaluated (EEC, 1988; EPA, 1982
; OECD, 1981
; JMAFF, 1985
).
A urinalysis was conducted on 5 (Study 1) or 10 (Study 2) rats/sex/dose at the end of the 13-week studies; and from 10 rats/sex/dose at 6, 12, and 18 months and from 20 rats/sex/dose at 24 months. Standard parameters were evaluated (EEC, 1988; EPA, 1982
; OECD, 1981
; JMAFF, 1985
).
Gross pathology and organ weights.
A complete gross examination was performed on all animals. The adrenals, brain, heart, kidneys, liver, ovaries, testes, spleen, thyroids with parathyroids, prostate (Study 1), uterus (Study 1), and thymus (Studies 2 and 3) were weighed from all surviving animals. Standard tissues were collected and preserved in neutral, phosphate-buffered 10% formalin (EEC, 1988; EPA, 1982
; JMAFF, 1985
; OECD, 1981
).
Histopathology.
A histopathologic examination was performed on all tissues collected from all rats (Study 1), or from the control and high-dose rats (Study 2) from the 13-week studies. The lungs, liver, kidneys, and target organs were examined from low- and intermediate-dose groups (Study 2). All tissues collected at 12 months from the control and 0.1% rats, and from the control and 0.05% rats at 24 months were examined histologically. Tissues evaluated from the low- and intermediate-dose group rats at 12 and 24 months (Study 3) consisted of target organs and gross lesions. In addition, lungs from selected rats given 0.1% spinosad for up to 21 months that had gross pathologic observations suggestive of a mass were also examined. Tissues were prepared by conventional techniques and were examined by a veterinary pathologist.
Electron microscopy.
Tissue samples of the thyroid, spleen, liver, kidney, and lungs from 3 rats/sex from the control and 0.2% groups, and the liver and kidneys from the 0.4% group (Study 1) were fixed in a modified Karnovsky's fixative. Specimens were postfixed in 2% osmium tetroxide, embedded in epon plastic, and processed for transmission electron microscopic evaluation by conventional procedures.
Statistical Analyses
Study 1.
Dunnett's test was used in the analyses of body weights/gains, feed consumption, feed efficiency, hematology, clinical chemistry, urine volume, and organ weight data. The homogeneity of variances was tested by the method of Bartlett (Steel and Torrie, 1980).
Studies 2 and 3.
Body weights, clinical chemistry, hematologic, absolute and relative organ weights, and urine specific gravity parameters were evaluated by Bartlett's test for equality of variances ( = 0.01; Winer, 1971
). Based upon the outcome of Bartlett's test, exploratory data analysis was performed by a parametric (
= 0.10; Steel and Torrie, 1960
) or nonparametric analysis of variance (
= 0.10; Hollander and Wolfe, 1973
), followed respectively by Dunnett's test (
= 0.05, 2-sided; Winer, 1971
) or the Wilcoxon Rank-Sum test (
= 0.05, 2-sided; Hollander and Wolfe, 1973
) with Bonferroni's correction for multiple comparisons (Miller, 1966
).
Differences in mortality patterns were tested by the Gehan-Wilcoxon procedure ( = 0.05; Breslow, 1970
) for all animals scheduled for the 2-year necropsy. The incidences of specific histopathologic observations were first tested for linearity using ordinal spacing of the doses (
= 0.01, 2-sided; Armitage, 1971
) 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 (
= 0.02, 2-sided; Armitage, 1971
). 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 (
= 0.05, 2-sided; Fleiss, 1981
).
Statistical analysis was limited to the pairwise comparison of control and high dose for tissues that were evaluated only from control and high-dose rats. 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.
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RESULTS |
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Body weight and feed consumption.
Male and female rats given 0.2% spinosad had body weights that were significantly lower than the controls, dose related, and statistically identified (Study 1; Fig. 2
). Feed consumption of males and females from the 0.2 and 0.4% groups was decreased approximately 10 and 7%, and 44 and 51% respectively, compared to the controls throughout the study.
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Clinical chemistry.
Male and/or female rats given 0.1 or 0.2% spinosad had minor increases in aspartate aminotransferase, alanine aminotransferase, gamma glutamyl transferase, and alkaline phosphatase activities, urea nitrogen, inorganic phosphorus, cholesterol, and triglyceride levels. These alterations were attributed to decreased body weights and feed consumption, as well as treatment-related degenerative effects in the liver, cardiac and skeletal muscles. Similar alterations in these parameters were also noted in rats given 0.4% spinosad.
Urinalysis.
Male and female rats given 0.2% spinosad had urine pH values that were 1.8 and 2.8 units lower than the controls, respectively. Females given 0.1% spinosad also had a lower urine pH (1.6 units). These differences in pH were possibly related to alterations in acid-base balance, or the excretion of acid metabolites.
Gross pathology.
Rats from the 0.4% group that died or were euthanized were thin, and had enlarged/fluid-filled ceca, and mottled, firm-brown lungs. Enlarged spleens and mesenteric lymph nodes, pale kidneys, and a proliferation of the gastric ridge were noted in rats given 0.2% spinosad. Rats given 0.1% spinosad only had enlarged lymph nodes.
Organ weights.
Rats from the 0.2% group had increased absolute and relative kidney, liver, heart, spleen, and thyroid gland weights. The greatest alteration in organ weight was an increase in the relative thyroid gland of 144 and 113% in males and females, respectively. Rats given 0.1% spinosad had increased kidney, spleen, heart, and thyroid gland weights of lesser magnitude.
Histopathology.
Treatment-related microscopic effects occurred in multiple tissues and were characterized by (1) cytoplasmic vacuolation of cells, (2) accumulation of aggregates of macrophages within the parenchyma, (3) degenerative or regenerative changes, and (4) combinations of these lesions. Cytoplasmic vacuolation was the primary effect of spinosad and occurred in rats given 0.05% spinosad (Table 3
). Vacuolation was dose related in severity and the number of tissues affected. Parenchymal cell vacuolation occurred in the adrenal gland (females only), myocardium, pancreas, and hepatocytes. Vacuolation also occurred in macrophages in the liver, lung (Fig. 3
), spleen, thymus, lymph nodes, and lymphoid tissue of the ileum; and epithelial cells of the epididymides, prostate, oviduct, uterus, cervix, vagina, thyroid, and glandular stomach. Vacuoles within thyroid follicular epithelial cells consisted of a mixture of large and fine vacuoles within the cytoplasm (Fig. 4
), and was representative of the appearance of vacuolation in other tissues. Vacuolation of thyroid epithelial cells and macrophages within lymphoid tissue were the most sensitive treatment-related effect in the subchronic studies, and defined the lowest observed effect level (LOEL) of 0.05% (33.9 mg/kg/day) spinosad.
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Electron microscopy.
Cytoplasmic vacuolation in numerous tissues was ultrastructurally characterized by the presence of numerous lysosomes that contained membranous whorls and irregular electron dense aggregates in rats given 0.2% spinosad. These lysosomes were prominent in the follicular epithelial cells of the thyroid (Fig. 5
) and the renal epithelial cells of the proximal and distal convoluted tubules, compared to the infrequent and small cytoplasmic lamellar inclusion bodies seen in tissues of control animals. Affected lysosomes were also occasionally seen in hepatocytes, pneumocytes, macrophages in the lung and lymphoid tissue, and splenic endothelial cells.
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Hematology.
The white blood cell count of females from the 0.1% group was 39% higher than the controls at 18 months. This difference was likely related to inflammation of the lung and thyroid gland in these rats.
Clinical chemistry.
Aspartate aminotransferase activity was higher in males given 0.1% spinosad for 12 months, and males and females given 0.1% for 18 months. These differences were consistent with microscopic degenerative lesions seen in cardiac and skeletal muscles.
Gross pathology and organ weights.
The lungs of all females and 1 male from the 0.1% group at 12 months had multiple subpleural pale foci. Rats given 0.1% spinosad for > 12 months also had increased incidences of enlarged thyroid glands, pale foci in the heart, left atrial thrombi, pale foci and/or masses in the lung, and hydrothorax. Females given 0.05% spinosad also had pale lung foci. Nonspecific signs of toxicity occurred in rats given 0.1% spinosad for > 12 months and included perineal soiling and decreased body fat. Decreased incidences of mammary gland hyperplasia, pituitary masses, and enlarged spleens were also noted in these rats and were attributed to their decreased body weights and the deteriorating nutritional status.
Increased kidney weights (absolute and relative) were identified in rats given 0.1% spinosad for 12 months and in females given 0.05% spinosad for 24 months. Increased kidney weights were associated with an increase in renal tubular vacuolation in females given 0.1% spinosad for 12 months. Heart weights (absolute and relative) of rats from the 0.1% group were also higher than the controls and may have been related to the microscopic degenerative heart lesions observed at 12 months. Increased spleen weights (absolute and relative) were identified in males and females given 0.1% spinosad and in females given 0.05% spinosad for 12 months. This was associated with a very slight degree of splenic extramedullary hematopoesis in females given 0.1% spinosad.
Histopathology.
Treatment-related microscopic effects at 12 months occurred in the heart, lungs, mesenteric lymph node, liver, kidneys, skeletal muscle (larynx and semimembranosus), spleen, stomach, and thyroid gland of females; and the heart, skeletal muscle (larynx), lungs, mesenteric lymph node, and thyroid gland of males given 0.1% spinosad; the thyroid gland of males and females given 0.05% spinosad and the mesenteric lymph node of females given 0.05% spinosad (Table 4). Microscopic effects in rats from the
0.05% groups consisted of cytoplasmic vacuolation, degeneration/regeneration, and/or inflammation. In general, microscopic effects in tissues were greater in incidence and severity for females than males, at comparable dose levels. Vacuolation of the thyroid gland was similar in appearance to that noted from the rats examined at 3 and 12 months at comparable dose levels. In addition, vacuolation was noted in the renal tubular epithelial cells of females given 0.1% spinosad for 12 months.
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There were no treatment-related increases in the incidence of any neoplastic type from any tissue that was statistically identified and there were no neoplasms that were interpreted to be treatment related.
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DISCUSSION |
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Not all CADs induce phospholipidosis in humans or animals. This is in part related to species and tissue differences in the metabolism among CADs. Only a few of these drugs are known to induce phospholipidosis in humans, including amiodarone, fluoxetine, and perhexiline. This may be because of differences in the metabolism of the drug, the smaller dosages used in humans for the desired therapeutic effect than was used to cause phospholipidosis in animal studies, or that phospholipidosis occurred in humans but was not detected.
The principal light microscopic response to CADs is a cytoplasmic vacuolation of tissues that results in the accumulation of phospholipids, and concentric membranous structures within secondary lysosomes of these cells. The mechanisms of intracellular accumulation of lamellar bodies induced by CADs have been evaluated (Lullmann et al., 1978; Reasor 1989
) and recently reviewed (Halliwell, 1997
). Although the exact mechanism for the accumulation of these phospholipids is unknown, it may be due to the indigestible drug-lipid complex, inhibition of phospholipases, inactivation of lysosomal enzymes, or a combination of these possibilities.
Since phospholipidosis represents a morphological response to CADs and may be associated with tissue injury as seen in these studies, it is appropriate to consider whether phospholipidosis can actually induce tissue injury (i.e., what are the toxicological implications of phospholipidosis). The general opinion in the field is that phospholipidosis is most likely an adaptive response to the inhibition of phospholipid catabolism, and if subsequent toxicity develops in a target tissue, it is unrelated to the phospholipidosis (Reasor, 1989, Reasor and Kacew, 2001
).
The induction of phospholipidosis is consistent with a threshold phenomenon, in that a certain level of the CAD must accumulate in the tissue for lysosomal lamellar body development. Phospholipidosis could theoretically occur in any tissue, and can be species, strain, and age specific. This would indicate that appropriate caution should be used in extrapolating the susceptibility of humans to CADs based on the results from animal toxicology studies. Reversal of phospholipid accumulation in animals has been documented following withdrawal of the CADs (Reasor and Castranova, 1981; Reasor and Walker, 1981
; Reasor et al., 1988
). The reversal of phospholipidosis is dependent upon reducing the diffusion gradient of the CAD, disassociation of the CAD/phospholipid complex, and elimination of the CAD from the cell.
Treatment-related effects in the 13-week studies reported in this article, using spinosad in which the ratio of spinosyn A and D was 5:1, can be compared with the results obtained in another 13-week dietary study in which Fischer 344 rats were fed spinosyns A and D in a ratio of 1:1 (Yano, B., and Liberacki, A., unpublished report for The Dow Chemical Company). The data from these 3 studies indicated that the toxicity of spinosad was not significantly affected by giving spinosad as a 5:1 or 1:1 spinosyn A:D ratio based on identical NOELs, and similarity of target organs and microscopic effects.
There was no evidence of a treatment-related increase in tumors in any tissue, notwithstanding the significant toxicity that was induced at the higher dose levels in the oncogenicity portion of the study. The incidences of a few common tumor types (adrenal pheochromocytoma in females, and Fischer rat leukemia in males and females) were in fact lower in rats given 0.05% spinosad. Lower tumor incidences have been reported for diet restricted control Sprague-Dawley rats (Keenan et al., 1995). Since spinosad-treated rats had lower body weights and feed consumption, dietary restriction was considered to be a likely mechanism for the lower tumor incidence in the rats from this study.
The reference dose (RfD) is an estimated average dose that the general human population could be exposed to for a lifetime without appreciable risk. The RfD for spinosad has been established as 0.027 mg/kg/day by the U.S. EPA (EPA, 2000) and 0.024 mg/kg/day by the European Union (EU), Australia, and Japan (Racke, personal communication).
The RfD for the U.S. EPA was based on a chronic dog study in which a NOAEL of 2.68 mg/kg/day was determined for dogs; whereas the RfD in the EU, Australia, and Japan was based on the chronic rat study with an NOAEL of 2.4 mg/kg/day. The RfD incorporated a 100-fold safety factor to the NOAELs to account for intraspecies and interspecies variations. Human exposure to spinosad could occur by the consumption of food and water that contain low residues of spinosad, and by nondietary routes of exposure. However, significant human exposure to spinosad will not occur due to the low residues of spinosad in treated crops, lack of mobility in soil/water, and low use rates. The existing and proposed uses of spinosad will only use approximately 37% of the RfD for the U.S. population. In addition, there is no information that would indicate that the toxic effects of spinosad would be cumulative with any other pesticide. Thus, there is reasonable certainty that the use of this insect control agent will not pose appreciable risks to human health (EPA, 2000).
In summary, the data from the 13-week and 2-year rat studies indicated that mortality and histopathologic effects consisting of cytoplasmic vacuolation, degeneration, regeneration, and inflammation occurred in rats 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 drug. Similar histopathologic changes were also observed in rats given lower dose levels of spinosad. Lifetime exposure to the highest dose levels of spinosad did not cause a treatment-related increase in the incidence of neoplasms in any tissue.
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NOTES |
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