* Global Toxicology, Dow AgroSciences, LLC, 9330 Zionsville Road, Indianapolis, Indiana 46268;
Department of Developmental Toxicology, Eli Lilly & Company, 2001 W. Main Street, Greenfield, Indiana 46140; and
Toxicology and Environmental Research and Consulting, The Dow Chemical Company, Midland, Michigan 48674
Received June 26, 2001; accepted January 2, 2002
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ABSTRACT |
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Key Words: spinosad; reproduction; rats; safety assessment; natural insecticide; fermentation product.
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INTRODUCTION |
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The acute toxicity of spinosad is low, with oral LD50s in rats and mice, and the dermal LD50 in rabbits ranging from 3783 mg/kg to greater than 5000 mg/kg (Thompson et al., 2000). Subchronic toxicity studies were conducted in the Fischer 344 rat at dietary concentrations ranging from 0.003 to 0.4% (dosages of approximately 2.4300 mg/kg/day). Dosage-related increases in liver, kidneys, spleen, and thyroid weights, and microscopic changes in the adrenals, liver, lymphoid tissue, reproductive tissues (oviduct, uterus, vagina, epididymides, and prostate), kidneys, thyroid, stomach, lung, and skeletal muscle were observed in rats given
0.05% (approximately 35 mg/kg/day). Morbidity and mortality occurred at 0.4% (Yano et al., 2002
). Histologic changes consisted of vacuolation, degeneration/regeneration, and/or inflammation and intracellular accumulation of lamellar bodies consistent with effects seen from exposure to cationic amphiphilic drugs. A subchronic NOEL of 0.012% was established. Exposure of rats to spinosad concentrations of 0.005 to 0.05% (approximately 2.463 mg/kg/day) for up to 24 months produced similar results, with no indication of a carcinogenic response in either males or females (Yano et al., 2002
). Developmental toxicity studies with spinosad in rats at dosages of 10200 mg/kg/day, and rabbits at 2.550 mg/kg/day reported no evidence of developmental effects even at dosages that produced maternal toxicity (Breslin et al., 2000
). Evaluation of spinosad in a battery of acute, subchronic, and chronic neurotoxicity studies, as well as extensive histopathologic evaluation from standard studies, produced no evidence of a neurotoxic effect.
The purpose of the current study was to evaluate the effects of continuous dietary exposure to spinosad on the reproductive performance of rodents over 2 generations. The current study was part of a program conducted according to regulatory guidelines established to support pesticide registrations globally. This study was conducted under GLP procedures according to test guidelines established by the U.S. EPA (U.S. EPA, 1984), the European Economic Community (EEC, 1988
), Organisation for Economic Co-operation and Development (OECD, 1983
), and Japan (MAFF, 1985
).
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MATERIALS AND METHODS |
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Test material, diet preparation and analyses.
Spinosad is composed of numerous spinosyns (Fig. 1), of which two are the active factors (spinosyn A and spinosyn D). Lot no. AGR 293707, with a purity of approximately 88.0% (76.1% spinosyn A and 11.9% spinosyn D) was obtained from Dow AgroSciences, LLC for use in this study. The identity of spinosad was confirmed using a combination of elemental analysis, high performance liquid chromatography, infrared and nuclear magnetic resonance, mass spectrometry, and ultraviolet spectroscopic techniques.
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Spinosad was stable in basal rodent feed for at least 32 days, and homogeneity of mixing procedure was determined prior to the start of the study. Analyses of the diets conducted 35 times per generation indicated dietary concentrations within 98109% of targeted levels.
Study design.
Groups of male and female rats were maintained continuously on diets containing spinosad targeted to provide dosage levels of 0, 3, 10, or 100 mg/kg/day over 2 generations. P1 adults (30/sex/dose level) were maintained on diets for approximately 10 weeks prior to breeding to produce the F1a litters. Following weaning of the F1a litters, 30 males and 30 females from each treatment group were selected as the P2 generation and maintained on the appropriate diet for 12 weeks from weaning of the last litter. Following 12 weeks of treatment, the P2 adults were bred to produce the F2 litters. Approximately 1 week following the weaning of the last F1a litter, the P1 adults were again mated to produce the F1b litters. Body weights and feed consumption were recorded for all P1 and P2 animals weekly during the prebreeding periods. Body weights for males were recorded weekly throughout the study. Each rat was examined daily for clinical signs of toxicity and changes in behavior or demeanor. In addition, a daily check for morbidity and mortality, and the availability of feed and water was conducted. All animals found dead or moribund were submitted for a gross pathologic examination as soon as possible. Pups found dead during lactation were examined grossly to the extent possible.
Breeding procedure.
Each breeding consisted of 3 consecutive 7-day cohabitation periods, with 1 male and 1 female from the respective treatment group cohoused. Females that failed to mate during the first 7-day period were placed with an alternate male from the same group for each successive breeding period, as necessary. During each breeding period, daily vaginal lavage samples were evaluated for the presence of sperm. The day of observing the presence of a sperm-positive sample or an in situ copulatory plug (considered evidence of mating) was identified as gestation day 0 (GD 0). Body weights for mated females were recorded on GD 0, 7, 14, and 21. For the P2 matings, cohabitation of siblings was avoided. Following completion of the mating periods, any female that apparently failed to mate was placed in a nesting cage as a precautionary measure in the event that breeding had gone undetected. Following the mating periods, the males were maintained on the appropriate diet until necropsied.
Litter data.
All litters were examined as soon as possible after delivery. Litter size and the number of live and dead pups were determined on lactation days (LD) 0 (date of birth), 1, 4, 7, 14, and 21, and maternal body weights and the sex and weight of each pup were measured on LD 1, 4, 7, 14, and 21. Any visible physical abnormalities or demeanor changes were noted during lactation.
To reduce the variability in the growth of pups, litters with a total number of pups exceeding 8 were culled to 4 males and 4 females, where possible, on LD 4. Culled pups were selected using a computer generated randomization procedure, and preferential culling of runts was avoided. All culled pups were examined grossly and euthanized with Beuthanasia-D (Schering Corporation, Kenilworth, NJ). Weaning of each litter was conducted on LD 21. Any weanling not selected for the succeeding generation or for necropsy evaluation was given a thorough external examination prior to euthanasia by CO2 inhalation.
Pathology
Adults.
A complete necropsy was conducted on all P1 and P2 adults by a veterinary pathologist. Scheduled necropsies were performed after the last litter of the respective generation was weaned. Adults were fasted overnight, weighed, anesthetized with methoxyflurane, and euthanized. The eyes were examined in situ by gently pressing a moistened glass slide against the cornea and observing the eye under fluorescent light. Liver, kidney, heart, spleen, and thyroid weights were recorded, and organ to body weight ratios calculated. A complete set of tissues from all animals was preserved in neutral, phosphate-buffered 10% formalin, except for testes and epididymides, which were preserved in Bouin's fixative. Histologic examination of a complete set of tissues from 5 rats/sex from P1 control and high-dosage group animals, and liver, kidneys, heart, spleen and reproductive and endocrine organs on all remaining P1 and P2 control and high-dosage animals was performed. Examination of tissues from the low- and middle-dosage groups was limited to liver, kidneys, lungs, mesenteric lymph node, stomach, urinary bladder, reproductive, and endocrine organs. Serum samples were collected at necropsy from 10 P2 adults/sex/dosage group and analyzed by an enzyme-linked immunoassay using a Monarch® 2000 system (Instrumentation Laboratory, Inc., Lexington, MA) for thyroid hormone (T4) levels.
Weanlings.
At weaning, 10 pups/sex/dosage group were randomly selected from the F1a, F1b, and F2 litters for a complete necropsy examination. Gross pathologic examination and the tissues saved on weanlings were the same as described for adults except that tissues were not microscopically examined.
Statistics.
Descriptive statistics (means and SDs) were reported for feed consumption. Body weights and weight gains, and serum T4 levels were first evaluated by Bartlett's test for equality of variances (Winer, 1971). Based on the outcome of Bartlett's test, a parametric (Steel and Torrie, 1960
) or nonparametric (Hollander and Wolfe, 1973
) ANOVA was performed. If the ANOVA was significant, a Dunnett's test (Winer, 1971
) or the Wilcoxon Rank-Sum test (Hollander and Wolfe, 1973
) with Bonferroni's correction (Miller, 1966
) was performed. Gestation length, average time to mating, and litter size were analyzed using a nonparametric ANOVA, followed by the Wilcoxon Rank-Sum test with Bonferroni's correction if the ANOVA was significant. Fertility indices were analyzed by the Fisher exact probability test (Siegel, 1956
) with Bonferroni's correction. Neonatal sex ratios were analyzed using a binomial distribution test (Steel and Torrie, 1960
). Survival indices among neonates were analyzed by the Wilcoxon test as modified by Haseman and Hoel (1974) using the litter as the experimental unit. Statistical outliers were identified using a sequential method (Grubbs, 1969
), but, except for feed consumption were not excluded unless justified by sound scientific reasons unrelated to treatment. The critical level of significance for all analyses was set a priori at
= 0.05.
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RESULTS |
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The only observation at necropsy of the P1 and P2 adults associated with treatment was an increase in the incidence of pale foci in the lungs of P1 males and females given 100 mg/kg/day (data not shown). This lesion was not observed grossly in the P2 adults; however, histopathologic changes in the lungs noted in both P1 and P2 adults (discussed below) were consistent with the interpretation of this as a treatment-related effect. Organ weights for the P1 and P2 adults are presented in Tables 1 and 2, respectively. In general, statistically significant, treatment-related increases in absolute and relative liver, kidney, heart, spleen, and thyroid weights were observed in both P1 and P2 males and females given 100 mg/kg/day. The only exceptions were absolute kidney weights in P1 males, and absolute liver weights in P1 females, which failed to reach statistical significance. No treatment-related effects on organ weights were noted at 3 or 10 mg/kg/day in either males or females in either generation. Statistical increases in absolute liver and heart weights at 10 mg/kg/day in P2 females were not accompanied by significant changes in relative weights or any histopathologic correlate, and were similar to P1 control values. Thus, these differences were considered spurious.
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The most common treatment-related histologic change was the presence of histiocytosis, or an accumulation of macrophages/reticuloendothelial cells in the lungs, spleen, and mesenteric lymph nodes. In the lymph nodes, and to a lesser extent in the lungs, this was a reflection of a shift from a very slight degree observed in some or all 0, 3, and 10 mg/kg/day dosage animals to a slight to moderate degree in 100 mg/kg/day dosage males and females of both generations. In the spleen, while the degree was considered very slight to slight in the high dosage animals, histiocytosis was not seen in controls. Also associated with the histiocytosis in the lungs of 100 mg/kg/day rats was an increase in the incidence and degree of inflammatory reactions in the interalveolar septae. A slight increase in the number of females of both generations with dilitation of the glandular crypts of the stomach mucosa of the pyloric region at 100 mg/kg/day was also considered treatment related. The only other histologic change of note was focal areas of very slight to moderate chronic, active inflammation of the prostate in more than 1/3 of the 100 mg/kg/day males in both generations. However, inflammation is recognized as the most frequently occurring lesion in the rat prostate, with a spontaneous incidence of 1923% reported in Sprague-Dawley controls (Bosland, 1992). There were no treatment-related histologic changes in any other reproductive organ in either males or females at this dosage. There were no histologic changes in any organ or tissue in either males or females given 3 or 10 mg spinosad/kg/day that were considered treatment-related.
Reproductive Performance
Reproductive indices for the 3 matings (F1a, F1b, and F2) across the 2 generations are summarized in Table 5. There were no treatment-related effects on male or female mating indices. Fertility indices in both males and females in treated groups were generally lower than the concurrent control values, but there was no evidence of a dose response, and none were statistically significantly different than the control values. In addition, these values were comparable to laboratory historical control fertility indices in Sprague-Dawley rats, which ranged from 63100%. These differences were thus not considered treatment related. There were no effects on gestation length or time to mating in either generation.
Treatment-related effects on the offspring were restricted to the 100 mg/kg/day dosage level. A consistent pattern of smaller litter size was observed on LD 0, 1, and 4 prior to culling in the F1a, F1b, and F2 litters from dams given 100 mg/kg/day. Statistically lower gestation survival indices (percentage of pups born live) noted in the F1a litters at 3 and 10 mg/kg/day, but not at 100 mg/kg/day, or in the F1b litters at any dosage, were considered unrelated to treatment. Gestation survival in the F1b litters at 100 mg/kg/day was substantially lower than the controls, but this decrease reflected the death of 16 pups from a single litter (of a total of 19 dead pups at this dosage) and thus did not reach statistical significance. Pup survival was significantly decreased in the F2 litters on LD 0, 1, and 4 at 100 mg/kg/day. These differences were reflective of effects across a number of litters, and thus were considered treatment related. Clinical observations of the litters during lactation also revealed increases in the incidence of pups with stomachs that were void of milk, were cold to the touch, and appeared thin and lethargic at 100 mg/kg/day in all 3 sets of litters across the 2 generations. These clinical signs suggested that the early neonatal effects were secondary to the effects on maternal animals around the time of parturition noted above. In many cases, the debilitated condition of the dams following dystocia led to poor maternal care and was reflected in the decreased early postnatal survival and pup growth.
Pup body weights and body weight gains in both males and females in most instances were lower than in controls throughout lactation in the 100 mg/kg/day litters, though they reached statistical significance only on LD 14 and LD 21 in the F1a and F1b litters, and LD 0 and 21 in F2 males. Gross pathologic examination of 10 pups/sex/dosage level from the F1a, F1b, and F2 litters revealed no treatment-related changes. Throughout the course of the 2 generations, there was no evidence of any treatment-related effect on pups at 3 or 10 mg/kg/day.
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DISCUSSION |
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Fertility indices in both males and females in treated groups were generally lower than the concurrent control values; but there was no evidence of a dose response, none was statistically significantly different from the control values, and most were within the historical control range. Thus, these differences were not considered treatment related. Treatment-related signs of dystocia and perineal soiling and/or vaginal bleeding were noted during parturition and early lactation in both P1 and P2 females at 100 mg/kg/day. In many cases, the debilitated condition of the dams following dystocia led to poor maternal care and was reflected in early postnatal effects. Gestation survival was reduced in the F2 litters, and smaller litter sizes were observed in the PND 14 interval in all generations at 100 mg/kg/day. Decreased early postnatal survival at 100 mg/kg/day was accompanied by clinical observations of thin pups with stomachs void of milk, cold to the touch, with lethargic movements. In contrast, developmental toxicity studies with rats and rabbits failed to produce any effects on embryonal or fetal development, even at levels causing maternal effects (Breslin et al., 2000). This suggests that the effects on early postnatal survival may have been maternally mediated or an effect of late in utero exposure, or a combination of these factors.
Pup body weight and weight gains were reduced throughout lactation, with statistically identified lower weights on PND 21 in all generations at 100 mg/kg/day. The effects noted on pup body weights during the last half of lactation were not surprising in light of what is known about feed consumption during lactation. Cripps and Willams (1975) measured feed consumption during lactation in Sprague-Dawley rats and found an approximately 3- to 5-fold increase in daily feed consumption between PND 1 and PND 21. Similar results were reported by Shirley (1984) and Arnold et al. (2000). Hanley and Watanabe (1985), reporting similar results in Fischer 344 rats, were able to quantitate the onset and extent of solid feed consumption in pups and demonstrated that pups rapidly begin solid feed consumption prior to weaning. The timing of the maximum effect on pup body weights and weight gains in this study correlates with the time at which pups are known to consume feed directly, and is consistent with the noted decreases in body weights in the P1 and P2 males at 100 mg/kg/day.
The organ weight and histologic changes noted in adults in the present study were consistent with effects reported in rats following subchronic or chronic exposure to spinosad. Yano et al. (2002) reported similar organ weight and histologic changes in Fischer 344 rats exposed to spinosad via the diet at dosages 34 mg/kg/day for 13 weeks. In the present study, other than the prostate, no treatment-related histologic changes were noted in reproductive organs of either males or females.
Ultrastructural examination of thyroid tissue from studies by Yano et al. (2002) identified vacuolation associated with lysosomal accumulation of concentric membranous whorls. These changes are consistent with lesions induced by cationic amphiphilic drugs (CADs), which belong to diverse pharmacological classes (Schneider, 1992). These CADs share a common structural feature within the group, as well as with spinosad, in that they contain a hydrophobic domain in close proximity to a hydrophilic domain. Irrespective of the mechanisms of intracellular phospholipid accumulation induced by CADs (as reviewed by Halliwell, 1997
), induction of phospholipidosis is consistent with a threshold phenomenon, which has been shown to be reversible following withdrawal of the CADs (Reasor and Castranova, 1981
; Reasor and Walker, 1982
; Reasor et al., 1988
).
No effects on adults, or their offspring, were noted with spinosad at dosage levels of 3 or 10 mg/kg/day.
In summary, dietary administration of spinosad to rats at a dosage of 100 mg/kg/day over 2 generations produced parental toxicity and associated effects on the offspring. Among adults, body weights and weight gains were decreased, absolute and relative organ weights were increased, and histologic changes were noted in numerous tissues of P1 and P2 males and females. In females, increased incidences of dystocia, and vaginal bleeding and mortality during parturition and lactation also occurred at 100 mg/kg/day. Adverse effects on the offspring (decreased litter size and survival) were limited to the high-dosage group. Clinical signs indicative of poor maternal care of the pups (stomachs void of milk, cold, thin, etc.) were also observed at 100 mg/kg/day. Early postnatal effects on the offspring were considered secondary to the effects noted above on maternal animals around the time of parturition. Decreased pup body weights and weight gains were noted at 100 mg/kg/day during lactation. There were no treatment-related effects on adults or their offspring at 3 or 10 mg/kg/day in either generation. Based on these results, spinosad is not considered a selective reproductive toxicant, and the NOEL for both parental and reproductive/perinatal toxicity was 10 mg/kg/day.
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NOTES |
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REFERENCES |
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Bosland, M. C. (1992). Lesions in the male accessory sex glands and penis. In Pathology of the Aging Rat (U. Mohr, D. L. Dungworth, and C. C. Capen, Eds.), pp. 443467. ILSI Press, Washington, DC.
Breslin, W. J., Marty, M. S., Vedula, U. V., Liberacki, A. B., and Yano, B. L. (2000). Developmental toxicity of spinosad administered by gavage to CD rats and New Zealand white rabbits. Food Chem. Toxicol. 38, 11031112.[ISI][Medline]
Cripps, A. W., and Williams, V. J. (1975). The effect of pregnancy and lactation on food intake, gastrointestinal anatomy and the absorptive capacity of the small intestine in the albino rat. Br. J. Nutr. 33, 1732.[ISI][Medline]
DeAmicis, C. V., Dripps, J. E., Hatton, C. J., and Karr, L. L. (1997). Physical and biological properties of the spinosyns: Novel macrolide pest-control agents from fermentation. In Phytochemicals for Pest Control, Symposium Series 658 (P. A. Hedin, R. M. Hollingworth, E. P. Masler, J. Miyamoto, and D. G. Thompson, Eds.), pp. 144154. American Chemical Society, Washington, DC.
EEC (1988). Methods for the determination of toxicity. European Economic Community. Off. J. Eur. Communities 31(L133), 4750.
Grubbs, F. E. (1969). Procedures for detecting outlying observations in samples. Technometrics 11, 121.[ISI]
Halliwell, W. H. (1997). Cationic amphiphilic drug-induced phospholipidosis. Toxicol. Pathol. 25, 5360.[ISI][Medline]
Hanley, T. R., Jr., and Watanabe, P. G. (1985). Measurement of solid feed consumption patterns in neonatal rats by 141Ce-radiolabeled microspheres. Toxicol. Appl. Pharmacol. 77, 496500.[ISI][Medline]
Haseman, J. K., and Hoel. D. G. (1974). Tables of Gehan's generalized Wilcoxon test with fixed point censoring. J. Stat. Comput. Simul. 3, 117135.
Hollander, M., and Wolfe, D. A. (1973). Nonparametric Statistical Methods. John Wiley and Sons, New York.
Kirst, H. A., Michel, K. H., Mynderse, J. S., Chio, E. H., Yao, R. C., Nakatsukasa, W. M., Boeck, L. D., Occlowitz, J. L., Paschal, J. W., Deeter, J. B., and Thompson, G. D. (1992). Discovery, isolation, and structure elucidation of a family of structurally unique, fermentation-derived tetracyclic macrolides. In Synthesis and Chemistry of Agrochemicals III (D. R. Baker, J. G. Fenyes, and J. J. Steffans, Eds.), pp. 214225. American Chemical Society, Washington, DC.
MAFF (1985). Requirements for safety evaluation of agricultural chemicals, Japan Ministry of Agriculture, Forestry and Fisheries, 59 NohSan, Notification No. 4200, Agricultural Production Bureau.
Mertz, F. P., and Yao, R. C. (1990). Saccharopolyspora sinosa sp. Nov. isolated from soil collected in a sugar mill rum still. Int. J. Syst. Bacteriol. 40, 3439.
Miller, R. G., Jr. (1966). Simultaneous Statistical Inference. McGraw-Hill, New York.
OECD (1983). Guidelines for testing of chemicals, Section 4Health Effects, Guideline No. 416. Organization for Economic Co-Operation and Development, May 26, 1983.
Reasor, M. J., and Castranova, V. (1981). Recovery from chlorphentermine-induced phospholipidosis in rat alveolar macrophages: Biochemical and cellular features. Exp. Mol. Pathol. 35, 359369.[ISI][Medline]
Reasor, M. J., Ogle, C. L., Walker, E. R., and Kacew, S. (1988). Amiodarone-induced phospholipidosis in rat alveolar macrophages. Am. Rev. Respir. Dis. 137, 510518.[ISI][Medline]
Reasor, M. J., and Walker, E. R. (1982). Recovery from chlorphentermine-induced phospholipidosis in rat alveolar macrophages: Morphological features. Exp. Mol. Pathol. 35, 370379.[ISI]
Salgado, V. L. (1998). Studies on the mode of action of spinosad: Insect symptoms and physiological correlates. Pestic. Biochem. Physiol. 60, 91102.[ISI]
Salgado, V. L., Sheets, J. J., Watson, G. B., and Schmidt, A. L. (1998). Studies on the mode of action of spinosad: The internal effective concentration and the concentration dependence of neural excitation. Pestic. Biochem. Physiol. 60, 103110.[ISI]
Schneider, P. (1992). Drug-induced lysosomal disorders in laboratory animals: New substances acting on lysosomes. Arch. Toxicol. 66, 2333.[ISI][Medline]
Shirley, B. (1984). The food intake of rats during pregnancy and lactation. Lab. Anim. Sci. 34, 169172.[Medline]
Siegel, S. (1956). Non-Parametric Statistics for the Behavioral Sciences, pp. 96104. McGraw-Hill, New York.
Steel, R. G. D., and Torrie, J. H. (1960). Principles and Procedures of Statistics. McGraw-Hill, New York.
Thompson, G. D., Dutton, R., and Sparks, T. C. (2000). Spinosada case study: An example from a natural products discovery programme. Pest Manage. Sci. 56, 696702.[ISI]
U. S. EPA (1984). Environmental Protection AgencyPesticide Assessment Guidelines, Subdivision F, Hazard Evaluation: Human and Domestic Animals, Series 834. U.S. Department of Commerce, National Technical Information Service PB86108958, Revised Edition, November 1984.
U.S. EPA (1984). Pesticide assessment guidelines, subdivision F, hazard evaluation: Human and domestic animals. U.S. Environmental Protection Agency, NTIS report PB86108958, pp. 126130.
Winer, B. J. (1971). Statistical Principles in Experimental Design, 2nd ed., McGraw-Hill, New York.
Yano, B. L., Bond, D. M., Novilla, M. N., McFadden, L. G., and Reasor, M. J. (2002). Spinosad insecticide: Subchronic and chronic toxicity and lack of carcinogenicity in Fischer 344 rats.Toxicol. Sci. 65, 288298.