Nonclinical Toxicology Studies with Zidovudine: Reproductive Toxicity Studies in Rats and Rabbits

Zidovudine (ZDV) was evaluated for adverse effects on reproductionand fetal development in animal test species. Standard preclinicaltests for reproduction and fertility, developmental toxicity,and postnatal toxicity were conducted in CD (Sprague-Dawley)rats and a developmental toxicity study was conducted in NewZealand white rabbits. In an additional study, reproductiveoutcome was characterized in female rats given ZDV before, during,or after mating and drug levels in the plasma and milk of lactatingrats were determined. Finally, drug exposure data includingobserved peak plasma concentrations (C_max) and area under theconcentration-time curve (AUC) were evaluated for pregnant ratsand rabbits. In a reproduction/fertility study in CD rats, toxicityto the early rat embryo, manifested as an increase in earlyresorptions and a decrease in litter size, was noted followingdosage of the parental animals with 75 or 225 mg ZDV/kg bid.A dose of 25 mg/kg bid was a no-effect level in rats. At thetime of mating, male rats had been dosed for 85 days, and femaleshad been dosed for 26 days. To further evaluate the effectsof ZDV on reproduction, dosing of male rats was continued to149 days when they were mated a second time to virgin, untreatedfemales. All reproductive parameters were normal in the untreatedfemales from this second mating, indicating that the embryotoxiceffect of the drug was not likely mediated by a genotoxic orother effect in the male. A separate study in female CD ratsgiven 225 mg/kg bid for various periods pre- or postconceptionsuggests that the toxic effect of ZDV is primarily to the earlyrodent embryo. Early embryo death did not occur in rats or rabbitsin standard developmental (teratology) studies; however, pregnantNew Zealand white rabbits given 250 mg/kg bid during gestationDays 6–18 showed reduced weight gain, anemia, and an increasein late fetal deaths. No other evidence of developmental toxicitywas noted in either species, and ZDV was not teratogenic inrats or rabbits given up to 250 mg/kg bid during the periodof major organogenesis. At this dose, C_max, values in rats andrabbits were approximately 234 and 150 times higher, respectively,than the mean steady-state serum concentration in adults followingchronic oral administration of 250 mg every 4 hr. In both thereproduction/fertility study and a periand postnatal study inrats, liveborn offspring showed no adverse effects on survival,growth, or developmental measurements.     

Nonclinical Toxicology Studies with Zidovudine: Genetic Toxicity Tests and Carcinogenicity Bioassays in Mice and Rats

Zidovudine (ZDV), an antiviral drug active in the treatmentof acquired immunodeficiency syndrome (recommended human dose,100 mg every 4 hr while awake), was evaluated for mutagenicand carcinogenic potential in a battery of short-term in vitroand in vivo assays and in lifetime studies in mice and rats.In L5178Y mouse lymphoma cells (tk^+/– locus), a weak positiveresult was obtained only at the highest concentrations tested(4000 to 5000 µg/ml) in the absence of metabolic activation.In the presence of metabolic activation, the drug was weaklymutagenic at concentrations of 1000 µg/ml and higher.Following 24 hr treatment in the absence of metabolic activation,ZDV was moderately mutagenic at concentrations up to 600 µg/ml;dose-related structural chromosomal alterations were seen atconcentrations of 3 µg/ml and higher in cultured humanlymphocytes. Such effects were not noted at the two lowest concentrationstested, 0.3 and 1 µg/ml, and BALB/c-3T3 cells were transformedat concentrations of 0.5 µg/ml and higher. No effectswere seen in the Ames Salmonella plate incorporation and preincubationmodification assays (possibly due to bacteriocidal activityof ZDV at low concentrations) at concentrations ranging from0.01 to 10 µg/plate or in a single-dose intravenous bonemarrow cytogenetic assay in CD rats. In multidose micronucleusstudies, increases in micronucleated erythrocytes were seenin mice at doses of 100 to 1000 mg/kg/day. Similar results wereseen in rats and mice after 4 or 7 days of dosing at 500 mg/kg/day.In carcinogenicity bioassays, adjusted doses of 20, 30, or 40mg/kg/day and 80, 220, and 300 mg/kg/day were given to CD-1mice and CD rats, respectively, for up to 22 months in miceand 24 months in rats. ZDV caused a macrocytic, normochromicanemia in both species. No evidence of carcinogenicity was seenin male mice or rats. In female mice, five malignant and twobenign vaginal epithelial neoplasms occurred in animals given40 mg/kg/day. A single benign vaginal epithelial tumor was seenin a mouse given 30 mg/kg/day. In rats, two malignant vaginalepithelial neoplasms were seen in animals given 300 mg/kg/day.In a 7-day study in mice, ZDV was shown to be devoid of estrogenicactivity. In an oral pharmacokinetics study, the AUC was 17and 144 µg/ml hr in female mice and rats given 40 or 300mg/kg of ZDV, respectively. In contrast, the average steady-stateconcentration in humans at the recommended daily dose is 0.62µg/ml. Twenty-four hour urine concentrations were 1245and 4417 µg/ml in female mice and rats given 40 or 300mg/kg of ZDV, respectively. These values were approximately26-and 136-fold higher than the human urine concentration atthe recommended daily dose. In a one- to three-day study withintravenously administered sodium fluoroscein in rats and mice,retrograde flow of urine into the vagina was demonstrated. Ina subsequent lifetime carcinogenicity bioassay in mice in whichZDV was given intravaginally at concentrations of 5 or 20 mgZDV/ml in saline, 13 vaginal squamous cell carcinomas were seenat the highest concentration tested. It was concluded that thevaginal tumors seen in the oral carcinogenicity studies werethe result of chronic local exposure of the vaginal epitheliumto high urine concentrations of ZDV.     

Acute and Subacute Toxicity Studies of Erodium guttatum Extracts by Oral Administration in Rodents

The present study aimed to evaluate the acute and subacute toxicity profiles of Erodium guttatum extracts in mice using the methods described in the guidelines of the OECD. In the acute toxicity study, the LD₅₀ value was greater than 2000 mg/kg. The subacute toxicity study of E. guttatum extracts showed no significant changes in body or organ weights. The administration of E. guttatum extracts to mice at a dose of 200 mg/kg led to an increase in white blood cells, platelets and hemoglobin. Moreover, the aqueous extract of E. guttatum only decreased liver aspartate aminotransferase (ASAT) levels at a dose of 200 mg/kg, and creatinine and urea levels did not show any significant alterations compared to the control group. Our results showed that the extracts of E. guttatum caused a slight increase in alanine aminotransferase (ALAT) and triglycerides. The histological study showed that mice treated with E. guttatum extracts experienced some histopathological changes in the liver, particularly with the methanolic extract, and slight changes in the kidneys and pancreas. Regarding the renal profile, no toxicity was observed. These results provide basic information on the toxicological profile of E. guttatum used in traditional medicine.     

Preclinical Toxicology Studies with Nizatidine, A New H2-Receptor Antagonist: Acute, Subchronic, and Chronic Toxicity Evaluations

Nizatidine (NIZ), a new antiulcer drug, was evaluated for toxicityin acute, subchronic, and chronic tests. Acute toxicity studieswere conducted in rats, mice, dogs, and monkeys. Median lethaldoses (MLD) in rodents were greater than 1600,230, and 1000mg/kg by oral (po), iv, and sc administration, respectively.No deaths occurred in dogs given single doses of 800 mg/kg (po),75 mg/kg (iv), or 225 mg/kg (im) or in monkeys given 1200 mg/kg(po) or 200 mg/kg (iv). Rats survived up to 1.0% dietary NIZ(daily intake ranging from 24 to 800 mg/kg/day) for 1 year.Slight decreases in body weight gain and increases in liverand kidney weights occurred. Slight decreases in erythrocyticparameters at 3 months were not present at 6 or 12 months. Micesurvived up to 1.5% dietary NIZ for 3 months and effects werelimited to slight decreases in body weight gain and increasesin relative liver weight Dogs survived oral doses up to 800mg/kg/day for 3 months but had numerous clinical signs of toxicityand body weight loss. All dogs given oral NIZ doses up to 400mg/kg/day survived except for one high-dose dog that was killedin a moribund condition following convulsions in the 41st weekof treatment Effects in dogs included miosis, body weight loss,increased thrombocyte counts, and decreased hepatic micro-somalenzyme activity and P450 content. The increase in thrombocytecounts was unaccompanied by changes in thrombocyte functionand did not reoccur in a subsequent study. A decrease in plasmatestosterone in two of three surviving male dogs given 400 mg/kg/dayfor 1 year was unaccompanied by effects on the size or morphologyof testes or prostate. Peak plasma levels of NIZ in all speciestested were in excess of human plasma levels after therapeuticdoses. In conclusion, there was no evidence of significant toxicityin organs or tissues including those sites (gastric mucosa,male sex organs, and liver) that have been affected by someagents of this therapeutic Class.     

Preclinical Toxicology Studies with Acyclovir: Carcinogenicity Bioassays and Chronic Toxicity Tests

Preclinical Toxicology Studies with Acyclovir: CarcinogenicityBioassays and Chronic Toxicity Tests. Tucker, W.E., Jr., Krasny,H.C., de Miranda, P., Goldenthal, E.I., Elion, G.B., Hajian,G. and Szczech, G.M. (1983). Fundam. Appl. Toxicol. 3:579–586.Acyclovir (ACV), a nucleoside analog that is a new herpes-specificantiviral drug, was given by gavage at 50, 150 and 450 mg/kg/dayto Sprague Dawley rats and Swiss mice for most of their lifetimeto assess chronic toxicity and carcinogenicity. Treatment withACV did not shorten the lifespan of either rats or mice. Infact, female mice given 150 and 450 mg/kg/day had significantlylonger mean durations of survival than control female mice whenanalyzed by the life table technique. There were no signs oftoxicosis produced by chronic exposure to ACV in either therats or mice, and there was no drug-related increase in neoplasmsin either species. Four groups of Beagle dogs were initiallygiven daily oral doses of 15, 45 or 150 mg/kg ACV in a 1 yearchronic toxicity study. Dogs treated at 150 mg/ kg/day vomited,had diarrhea, consumed less feed and lost weight within 2 weeks.Dogs treated at 45 mg/kg/day also had minimal signs of gastrointestinaltoxicosis. These dose levels were then decreased to 60 and 30mg/kg/day for the rest of the one year test period. With theexception of occasional and inconsistent emesis and diarrhea,the 60 mg/kg/day dose level was well tolerated. Some mid andhigh dose dogs had sore paws due to erosion of footpads andcracking, splitting and loosening of the nails first becomingevident during the 13th week of the study. Several of thesedogs subsequently lost the keratin from some claws. There wasnail regeneration and healing of footpads as the study progressed,with all claws appearing essentially normal at the end of 1year. Nails and footpads of dogs given 15 mg/kg/day were normalthroughout the study.     

Chronic Toxicity Studies with Thiram in Wistar Rats and Beagle Dogs

Chronic Toxicity Studies with Thiram in Wistar Rats and BeagleDogs. Maita, K., Tsuda, S., and Shirasu, Y. (1991). Fundam.Appl. Toxicol. 16, 667–686. Groups of 64 male and 64 femaleWistar rats were given thiram at constant dietary doses of 0,3, 30, and 300 ppm (0, 0.1, 1.2, and 11.6 mg/kg/day for malesand 0, 0.1, 1.4, and 13.8 mg/kg/day for females) for 104 weeks.Eight males and eight females in each group were killed afterWeeks 13, 26, and 52. For the dog study, four male and fourfemale beagle dogs were allotted to each group and treated withthe compound at 0, 0.4, 4, and 40 mg/kg/day for 104 weeks. Thedogs in the 40 mg/kg/day group had severe toxic signs, includingnausea or vomiting, salivation, and occasional clonic convulsion,and all were subjected to unscheduled necropsy before Day 203of treatment. The dogs also had ophthalmological changes suchas fundal hemorrhage, miosis, and desquamation of the retinawhich were consistent with the retinal lesions shown by histopathology.The rats of the high-dose group had retarded growth with a slightlydecreased food intake. Anemia was evident in high-dose femalerats and in middle- and high-dose dogs. Liver failure in maleand female dogs and kidney damage in female dogs were detectedin middle- and high-dose groups by blood biochemistry and/orhistopathology. Regressive changes of the sciatic nerve accompaniedby atrophy of the calf muscle were seen in female rats of thehigh-dose group but not in male rats. In high-dose rats, progressionof myocardial lesions of the heart and chronic nephrosis ofthe kidney were depressed in males and females, respectively.Female rats of the middle- and high-dose groups had decreasedoccurrences of mammary fibroadenoma and decreased developmentof skin masses.     

Worms as a Substitute for Rodents in Toxicology: Acute Toxicity of Three Nickel Compounds

In a continuing investigation of the use of the common earthworm (Lumbricus terrestris) as test subjects for metal toxicity, three nickel salts were evaluated. Saline solutions of either the chloride, the sulfate, or the acetate were injected into the coelom of the worm. Care was taken not to enter the gastrointestinal tract. The acute toxicities were determined by graphical methods using probit paper. The most consistent results were found when lethality was measured at 48 h rather than 24 h. The 48-h LD₅₀ values for the different salts of nickel are as follows: chloride, 52 mg/kg; sulfate, 54 mg/kg; and acetate, 69 mg/kg. The ratios of toxicity of nickel acetate to the chloride and sulfate salts in the worms are similar to those ratios noted in mice. The European Economic Commission (EEC) now suggests using earthworms for toxicity testing for legislation purposes.     

Oral Toxicity of Carbon Tetrachioride: Acute, Subacute, and Subchronic Studies in Rats

Oral Toxicity of Carbon Tetrachloide: Acute, Subacute, and SubchronicStudies in Rats. BRUCKNER, J. V., MACKENZIE, W. F., MURALIDHARA,S., LUTHRA, R., KYLE, G. M., AND ACOSTA, D. (1986). Fundam.Appl. Toxicol. 6, 16–34. This investigation was conductedto characterize the acute, subacute, and subchronic toxic potencyof ingested carbon tetrachloride (CCl₄) In the first acute andsubacute toxicity study, male Sprague-Dawley rats of 300–350g were gavaged with 0, 20, 40, or 80 mg CCl₄/kg once daily for5 consecutive days, rested for 2 days, and dosed once dailyfor 4 additional days. Rats of 200–250 g were gavagedwith 0, 20, 80, or 160 mg CCl₄/kg according to the same dosageregimen in the second acute and subacute study. In the firstand second studies one group of rats at each dosage level wassacrificed for clinical chemistry and histopathological evaluationat 24 hr, 4 days, and 11 days after initiation of dosing. Single20- and 40-mg/kg doses had no apparent toxic effect at 24 hr,although 80 mg/kg caused mild hepatic centrilobular vacuolizationand significant increases in some serum enzyme levels. In general,there was progressively severe hepatic injury at each dosagelevel over the 11-day period. CCl₄ was more hepatotoxic to the200–250-g rats than to the 300–350-g rats. In thesubchronic study, rats initially 200–250 g were gavaged5 times weekly for 12 weeks with 0, 1, 10, or 33 mg CCl4/kgBody weight and clinical chemistry indices were monitored duringthe 12 weeks of dosing and 2 weeks after cessation of dosing,A dose of 1 mg/kg had no apparent adverse effect; 10 mg/kg producedslight, but statistically significant increases in sorbitoldehydrogenase activity and mild hepatic centrilobular vacuolization;33 mg/kg caused marked hepatotoxicity. Serum enzyme levels remainedelevated during the 12-week dosing period, but returned towardnormal within 13 days of cessation of CCl₄ exposure. Microscopicexamination of livers of the 33-mg/kg rats revealed cirrhosis,characterized by bile duct proliferation, fibrosis, lobulardistortion, parenchymal regeneration, hyperplastic nodules,and single-cell necrosis. The fibrosis was not reversed withinthe 13-day recovery period.     

Chronic Dietary Toxicity/Oncogenicity Studies on 2,4-Dichlorophenoxybutyric Acid in Rodents

2,4-Dichlorophenoxybutyric acid (2,4-DB) is principally usedin the United States on peanuts, soybeans, and alfalfa. In Europe,it is used on cereals, undersown cereals, lucerne (alfalfa),clover, and clover mixtures. Doses in the 2-year chronic/oncogenicityrat study were 0, 60, 600, and 1800 ppm. No evidence of an oncogenicpotential for 2,4-DB was evident and the study clearly establisheda NOEL of 2.48 mg/kg/day (60 ppm, males) and 3.23 mg/kg/day(60 ppm, females), as well as an MTD of 78.0 (1800 ppm, males)and 110.6 mg/kg/day (1800 ppm, females), for chronic effectsof 2,4-DB in the rat Doses in the 18-month mouse oncogenicitystudy were 0, 25, 250, and 750 ppm. No oncogenic effect wasnoted in the study. In summary, the findings of these studiesindicate low chronic toxicity of 2,4-DB and the lack of oncogenicresponse to 2,4-DB following chronic dietary exposure of 2,4-DBin the rat and mouse.     

Chronic Dietary Toxicity/Oncogenicity Studies on 2,4-Dichlorophenoxyacetic Acid in Rodents

Forms of 2,4-dichlorophenoxyacetic acid (collectively knownas 2,4-D) are herbicides used to control a wide variety of broadleafand woody plants. Doses in the 2-year chronic/oncogenicity ratstudy were 0, 5, 75, and 150 mg/kg/day. The chronic toxicityparalleled subchronic findings, and a NOEL of 5 mg/kg/day wasestablished. A slight increase in astrocytomas observed (inmales only) at 45 mg/kg/day in a previously conducted chronicrat study was not confirmed in the present study at the highdose of 150 mg/kg/day. Doses in the 2-year mouse oncogenicitystudies were 0, 5, 150, and 300 mg/kg/day for females and 0,5, 62.5, and 125 mg/kg/day for males. No oncogenic effect wasnoted in the study. In summary, the findings of these studiesindicate low chronic toxicity of 2,4-D and the lack of oncogenicresponse to 2,4-D following chronic dietary exposure of 2,4-Din the rat and mouse.     

Preclinical Studies on Human Insulin. I. Acute and Subacute Toxicity Studies

Abstract: Human insulin (prepared from porcine insulin) and porcine insulin were tested for acute toxicity by subcutaneous administration to unfasted mice and rats and fasted mice. The animals were observed for signs of reaction and post mortem examination was performed. LD50 values were calculated when possible. The LD50 values in the unfasted animals were several times higher than in the fasted mice, but similar for the two insulin preparations. The deaths and the signs observed were most probably caused by hypoglycaemia. No dose-related macroscopic organ changes were found. In a 28 day toxicity study rats were given human or porcine insulin subcutaneously at dosages of 2, 20 or 200 U/kg/day. The criteria examined included mortality, body-weight change, food consumption and utilization, haematology, blood chemistry, urinalysis, opthalmoscopy, organ weights, gross- and histopathology. A few deaths occurred because of hypoglycaemia. In surviving rats from dosage groups 20 and 200 U/kg/day of either insulin preparation higher plasma glucose levels than in the control were observed 24 hours after dosing. Higher food intake and body-weight gains, lower plasma protein concentrations and higher urinary volumes with associated low specific gravity were observed in animals given either human or porcine insulin at 200 U/kg/day. No other adverse effects were registered, and no overt difference was found between the effect of human and porcine insulin.     

Preclinical Toxicology Studies with Acyclovir: Genetic Toxicity Tests

Preclinical Toxicology Studies with Acyclovir: Genetic ToxicityTests. Clive, D., Turner, N.T., Hozier, J., Batson, A.G. andTucker, W.E., Jr. (1983). Fundam. Appl. Toxicol 3: 587–602.Acyclovir (ACV), an antiviral drug active in the treatment oforal and genital Herpes infections, has been evaluated for mutagenicand carcinogenic potential in a battery of in vitro and in vivoshort-termassays. Negative results were obtained in the following in vitrotests: Ames Salmonella, plate incorporation and preincubationmodification assays; E. coli polA⁺/polA^– DNA repair; yeast(S. cerevisiae D4) gene conversion; Chinese hamster ovary cells(HGPRT, APRT loci and ouabain-resistance marker); L5178 Y mouselymphoma cells (HGPRT locus and ouabain-resistance marker);and C3H/10Tmouse fibroblast neo-plastic transformation assay.All except the last assay were performed in the presence andabsence of an exogenous metabolic activation system. ACV waspositive at high concentrations x exposure times in the absenceof exogenous metabolic activation in the following in vitrosystems and at the indicated concentrations: BALB/c-3T3 neoplastictransformation (50 /µg/mL, 72 h exposure); human lymphocytecytogenetics (250–500 µg/mL, 48 h exposure); andL5178Y mouse lymphoma cells (TK locus, 400–2400 µg/mL,4 h exposure; predominantly small colony mutants of chromosomalorigin produced). No effects were seen in vivo (mouse dominantlethal assay; rat and Chinese hamster bone marrow cytogenetics)at up to maximum tolerated doses (MTD). An unusual clastogeniceffect was seen in Chinese hamsters at 5 times the MTD. Overall,positive effects were seen only at either high concentrations(250 µg/mL in vitro or plasma levels) or prolonged exposure(72 hr in the BALB/ c-3T3 neoplastic transformation assay).These studies support the view that ACV is a chromosomal mutagen,i.e., one which causes multi-locus damage but not single geneeffects. The significance of these results for the genetic riskof ACV to man is discussed.     

Isolation and Use of Primary Adrenocortical Cells from Guinea Pigs,Dogs and Monkeys forIn Vitro Toxicity Studies

A method was developed to obtain enriched populations of zona fasci-culata cells from the adrenal glands of guinea pigs, dogs, and monkeys. Adrenocortical cells (ADC) in primary culture were shown to maintain viability and cellular morphology, with an estimated ≥80% of cultures being zona fasciculata cells. Three known adrenal toxicants, PD 132301–2, 1-(o-chlorophenyl)-1-(p-chloro-phenyl)-2, 2-dichloroethane (o, p′-DDD), and aminoglutethimide (AG) were tested in this in vitro system. Neutral red (NR) uptake was used as a marker of cell viability and cortisol production was measured to assess ADC function. NR uptake following 24 h of treatment with PD 132301–2 (10 μM) was 32, 31, and 53% of control in guinea pig, dog, and monkey cultures, respectively. Similarly, o, p'-DDD (100 μM) decreased NR uptake to 32, 40, and 69% of control. AG (300 μM) decreased NR uptake by 50% only in dog ADC. Cortisol production was evident in cells from all three species with rates being highest in monkey, followed by the dog and guinea pig. Cortisol production was decreased following treatment with all three toxicants. Decreases paralleled loss of viability in PD 132301–2-treated cultures from all three species and in o, p'-DDD-treated cultures from guinea pig and dog. In contrast, decreased cortisol production preceded any change in viability in o, p'-DDD-treated cultures from monkey, and in AG-treated cultures from all species. Cytotoxic responses to adrenal toxicants of varied structure and mechanisms of action suggests that the ADC cultures from these three species may be useful in toxicologic screening or for investigating mechanisms of adrenocortical toxicity. Key Words: Adrenocortical cells—-Cytotoxicity—Cortisol—o, p'DDD—Aminoglutethimide—PD 132301–2.     

Acute, Subchronic, and Chronic Toxicity Studies with Felbamate, 2-Phenyl-1,3-propanediol Dicarbamate

Felbamate, 2-phenyl-1,3-propanediol dicarbamate, is a novelanticonvulsant that is effective against both chemically andelectrically induced seizures in laboratory animals. Acute,subchronic, and chronic studies were conducted in mice, rats,and dogs to establish a preclinical safety profile for thisdrug. Clinical signs following single intraperitoneal dosesincluded hypoactivity, tremors, decreased muscle tone, ataxia,prostration, and labored breathing. Death was observed afterintraperitoneal but not oral administration. A consistent drug-relatedeffect noted in al multiple-dose studies with this compoundwas decreased body weight and food consumption. The only otherconsistent change noted in multiple-dose studies with felbamatewas an increase in liver weight (relative and absolute) in therat and dog which was accompanied in some cases by increasesin serum enzyme levels. No histopathological changes were observedin the liver that could explain these elevated serum enzymelevels. Based on the results of these studies it was concludedthat long-term administration of felbamate in himan clinicaltrials was warranted.     

Preclinical Toxicology Studies with Acyclovir: Acute and Subchronic Tests

Preclinical Toxicology Studies with Acyclovir: Acute and SubchronicTests. Tucker, W.E., Jr., Macklin, A.W., Szot, R.J., Johnston,R.E., Elion, G.B., de Miranda, P. and Szczech, G.M. (1983).Fundam. Appl. Toxicol. 3:573–578. Acyclovir (ACV), a newantiherpes drug, was evaluated for toxicity in a series of acuteand subchronic toxicity tests. Oral LD₅₀ values were greaterthan 10 000 mg/kg in male ICR mice and greater than 20 000 mg/kgin male Long Evans rats. When ACV was given iv, the LD₅₀ was405 mg/kg for male mice and greater than 600 mg/kg for malerats. Additionally, LD₅₀ values for male rats treated sc were1070, 790, 678, and 650 mg/kg in rats that were respectively,3, 10, 28 and 71 days old indicating that very young rats werenot more sensitive to acute toxic effects of ACV. There wereno signs of toxicosis in CD-1 mice given ACV by gavage at doselevels of 50, 150 and 450 mg/kg/day for 1 month. Obstructivenephropathy occurred in rats given 20, 40 and 80 mg/kg/day onceeach day by rapid iv injection for 3 weeks. Both 5 and 10 mg/kg/daywere no effect dose levels. Renal damage caused by precipitationof drug crystals in renal tubules and collecting ducts in ratsgiven ACV by rapid iv injection was readily reversible within2 weeks. Beagle dogs were given doses of 10, 20, 25, 50 and100 mg/kg b.i.d. by rapid iv injection for 1 month. All 8 dogsgiven 100 mg/kg b.i.d. died by the 8th day of treatment; 5 of8 dogs given 50 mg/kg b.i.d. died after 21 to 31 days of treatment.At 50 and 100 mg/kg b.i.d. the clinical signs of toxicosis werenumerous and mainly resulted from the underlying morphologicaland functional changes associated with hypoplasia of the esophagealand gastrointestinal mucosa, lymphoid tissue, and bone marrow.At the 20 and 25 mg/kg b.i.d. dose levels the kidney was thetarget organ; the principal indications of altered renal functionwere increased water intake and hyposthenuria. The dose levelof 10 mg/kg b.i.d. was a no effect level for Beagle dogs treatediv. Thus, in subchronic experiments, the rapid iv injectionof acyclovir caused precipitation of crystals in the renal tubules,resulting in obstructive nephropathy in rats and dogs. Primarytoxicity occurred only in the dog where high doses of acyclovircaused hypoplasia of certain tissues with rapid cell turnover.     

Issues in the Design and Interpretation of Chronic Toxicity and Carcinogenicity Studies in Rodents: Approaches to Dose Selection

For more than three decades chronic studies in rodents have been the benchmark for assessing the potential long-term toxicity, and particularly the carcinogenicity, of chemicals. With doses typically administered for about 2 years (18 months to lifetime), the rodent bioassay has been an integral component of testing protocols for food additives, pesticides, pharmaceuticals, industrial chemicals, and all manner of byproducts and environmental contaminants. Over time, the data from these studies have been used to address an increasing diversity of questions related to the assessment of human health risks, adding complexity to study design and interpretation. An earlier ILSI RSI working group developed a set of principles for the selection of doses for chronic rodent studies (). The present report builds on that work, examining some of the issues that arise and offering new perspectives and approaches for putting the principles into practice. Dose selection is considered both from the prospective viewpoint of the choosing of dose levels for a study and from the retrospective interpretation of study results in light of the doses used. A main theme of this report is that the purposes and objectives of chronic rodent studies vary and should be clearly defined in advance. Dose placement, then, should be optimized to achieve study objectives. For practical reasons, most chronic studies today must be designed to address multiple objectives, often requiring trade-offs and innovative approaches in study design. A systematic approach to dose selection should begin with recognition that the design of chronic studies occurs in the context of a careful assessment of the accumulated scientific information on the test substance, the relevant risk management questions, priorities and mandates, and the practical limitations and constraints on available resources. A stepwise process is described. The aim is to increase insofar as possible the utility of an expensive and time-consuming experiment. The kinds of data that are most commonly needed for dose selection and for understanding the dose-related results of chronic rodent studies, particularly carcinogenicity studies, are discussed as “design/interpretation factors.” They comprise both the inherent characteristics of the test substance and indicators of biological damage, perturbation or stress among the experimental animals. They may be primary toxicity endpoints, predictors or indicators of appropriate dose selection, or indicators of conditions to be avoided in dose selection. The application and interpretation of design/interpretation factors is conditioned by the study objectives–what is considered desirable will depend on the strategy for choice of doses that is being followed. The challenge is to select doses that accommodate all of the issues raised by the relevant design/interpretation factors. Three case studies are presented here that illustrate the interplay between study objectives and the design and selection of doses for chronic rodent studies. These examples also highlight issues associated with multiple plausible modes of action, multiple pathways for biotransformation of the chemical, extraneous high-dose effects, the use of modeling in dose selection, and the implications of human exposure levels. Finally, looking to the future, the report explores seven potential paradigm shifts for risk assessment that will significantly impact the design and interpretation of toxicity and carcinogenicity studies.     

Toxicity and Carcinogenicity Studies of Nalidixic Acid in Rodents

ABSTRACT

Toxicity and carcinogenicity studies of nalidixic acid, an antimicrobial agent used to treat bacterial infections of the urinary tract, were conducted in F344/N rats and B6C3F mice of each sex for 13 weeks or 2 years. In the 13–week studies, nalidixlc acid was administered at dietary concentrations ranging from 1,000 to 16,000 pp. Body weights of both rats and mice were reduced in the groups receiving diet containing 8,000 and 16,000 ppm, and feed consumption of rats in the highest treatment groups was approximately tw-thirds that of controls. Degeneration of the germinal epithelium in the seminiferous tubules of the testis was observed in male rats that received 16,000 ppm; no other compound-related histopathologic effects were observed in either species. TWo-year studies were conducted by feeding diets containing 0, 2,000, or 4,000 ppm nalidixic acid to groups of 50 rats and mice/sex/group. The average daily feed consumption was slightly reduced compared to control groups and resulted in approximate daily doses of 82 or 175 mg nalidixic acidfig for low dose and high dose rats, and 220 or 475 mg/kg for low dose and high dose mice. Mean body weights of dosed rats and mice were lower than those of controls, except for groups of low dose female rats and male mice. The incidences of preputial gland neoplasms in dosed male rats and of clitoral gland neoplasms in dosed female rats were significantly increased compared to those in controls; responses in low dose groups were similar to those in high dose groups. There were decreased incidences of leukemia and mammary gland neoplasms in dosed female rats and of pituitary gland neoplasms in dosed male rats. Subcutaneous tissue fibrosarcomas were marginally increased in dosed male mice. There were no increased incidences of neoplasms in dosed female mice. under the conditions of these studies, the dietary administration of nalidixic acid was carcinogenic for rats, causing preputial gland or clitoral gland neoplasms in males and females, respectively. The association of subcutaneous neoplasms with administration of nalidixic acid to male mice was equivocal.     

Chronic Toxicity, Reproductive, and Teratogenic Studies with Oxamyl

Chronic Toxicity, Reproductive, and Teratogenic Studies withOxamyl. KENNEDY, G. L., JR., (1986). Fundam. Appl Toxicol. 7,106-118. Oxamyl (methy1N',N'-dimethy1-N'-[(methylcarba-moyl)oxy]-l-thiooxamimidate;CAS 23135-22-0) was tested for oral toxicity in the rat anddog (90-day and 2-year feeding studies) and in the mouse (2-yearfeeding study). Teratogenic potential was evaluated in the ratand rabbit and functional reproductive capacity was studiedin the rat in a one- and a three-generation reproduction study.Rats fed a diet containing oxamyl at 500 ppm showed clinicalsigns of cholinesterase inhibition and body weight loss within2 days. Feeding of either 100 or 150 ppm oxamyl for 90 daysproduced a reduced rate of weight gain without other signs ofresponse, and no effects were detected at 50 ppm. An oxamylfeeding period of 2 years also showed depressed body weightgains in rats fed either 100 or 150 ppm. Cholinesterase activitywas depressed only during the first week of feeding and onlyin the 150-ppm group. All other indices of response, includingthe type and distribution of tumors, were similar in the testand control rats and it was concluded that the no-observed-effectlevel was 50 ppm (equivalent to approximately 5 mg/kg). Micefed oxamyl at 100 ppm for 6 weeks showed signs of cholinesteraseinhibition and some mortalities, so the dietary concentrationwas reduced to 75 ppm in the 2-year study. Body weights of micefed oxamyl at 50 or 75 ppm were lower than controls during thefirst 6 months of the study. No other signs of a toxic responseto oxamyl were seen in mice and a no-observed-effect level of25 ppm (approximately 2.5 mg/kg) was assigned to this compound.No evidence of a tumorigenic response was obtained. Dogs fedoxamyl at 150 ppm for 2 years showed marginal increases in serumalkaline phosphatase activity and cholesterol concentrationbut no tissue pathology was seen. No evidence of cholinesteraseinhibition was seen. It was concluded that the no-observed-effectlevel for oxamyl in the dog was 100 ppm (approximately 2.5 mg/kg).In the one- and three-generation reproduction studies, littersizes were somewhat lower in rats fed oxamyl at 100 or 150 ppmoxamyl with normal values seen at 50 ppm. Weanling body weightswere normal in rats in the 50-ppm group for three generationsbut were reduced in the one-generation study. Pup body weightswere lower in rats in both the 100- or 150-ppm groups. Transferof F3b pups, derived from dams in the 150-ppm group, to controldiets following delivery improved the weight gains. No evidenceof a teratogenic response was seen in the rat although a reducedrate of weight gain was seen in pregnant rats fed oxamyl at100 ppm or greater. In the rabbit, no evidence of a teratogenicresponse was found even at dose levels in which maternal toxicitywas encountered.     

Acute Toxicity Studies with Oxamyl

Acute Toxicity Studies with Oxamyl. KENNEDY, G. L., JR. (1986).Fundam. Appl. Toxicol 6, 423–429. The acute toxicity ofoxamyl, an insecticide and nematicide, has been evaluated toestablish proper handling guides. The material is highly toxicwhen given as a single oral dose; its LD50 is in fasted rats2.5 to 3.1 mg/kg, 2.3 to 3.3 mg/kg in fasted mice, and 7 mg/kgin guinea pigs. A beagle dog given 30 mg/kg died, while 15 mg/kgwas not lethal. In all species, clinical signs of cholinesteraseinhibition (lacrimation, salivation, tremors) were observed.Cholinesterase activity was depressed in rats treated with asingle oral dose. Atropine, when given immediately after oxamyl,was antidotal. When given by intraperitoneal injection, oxamylwas highly toxic to rats, mice, and guinea pigs. The materialis a mild eye irritant with the reaction limited to the conjunctivaand iris, but systemic absorption via eye contact makes useof protective equipment essential. Oxamyl produces mild skinirritation and the dermal absorption toxicity in rats (LD50is> 1,200 mg/kg) and rabbits (744) mg/kg) is relatively highsuggesting limited absorption. No sensitization was producedwhen tested in guinea pigs. Oxamyl is highly toxic via inhalationwith the 1-hr LC50 value in rats being 0.17 mg/liter (male)and 0.12 mg/liter (female). The corresponding 4-hr value is0.064 mg/liter for male rats which indicates that concentrationX time is a constant through the time periods tested. Repeated-dosestudies, orally in rats and dermally in rabbits, showed oxamylto be noncumulative, with the target system being the nervoussystem mediated through cholinesterase inhibition. No specifictissue or organ pathology was seen in either species tested.     

Subacute Toxicity of a Novel Inhibitor of Acyl-CoA: Cholesterol Acyltransferase in Beagle Dogs

PD 132301–2 is a substituted urea hypolipidemic and antiatheroscleroticagent that is a potent inhibitor of acyl-CoA:cholesterol acyltransferase(ACAT). To determine its subacute toxicity, PD 132301–2was administered orally to beagle dogs at 0, 6, 12, 25, 50,200, 400, or 800 mg/kg/day for 2 weeks. Clinicopathologic evaluationswere completed on all dogs. Liver and adrenal total and esterifiedcholesterol concentrations, adrenocorticotrophic hormone (ACTH)responsiveness, and adrenal ultrastructure were determined at0, 6, 12, and 25 mg/kg. At 12 mg/kg or greater, salivation,epiphora, conjunctivitis, emesis, anorexia or decreased foodconsumption, and soft to mucoid feces and/or diarrhea were noted.Suppression of ACTH response occurred by Day 6 at all doses.Adrenocortical degeneration and/or necrosis in zona fasciculataand reticularis was seen at all doses; adrenal free and esterifiedcholesterol were normal at 6 mg/kg and decreased at 12 and 25mg/kg. Increases in serum alanine aminotransferase (2- to 15-fold),aspartate aminotransferase (2- to 12-fold), and alkaline phosphatase(2- to 7-fold) were noted at 50 mg/kg or greater. Periportalhepatocellular hypertrophy and hypereosinophilia occurred at50 mg/kg or greater; hepatic cholesterol values were not significantlyaffected by treatment. Dose-dependent ultrastructural alterationsin adrenocortical cells included decreased numbers of mitochondriaand smooth endoplasmic reticulum profiles, qualitative and quantitativechanges in lipid globules, and increased numbers of autolysosomes.PD 132301-2 or one of its metabolites has potent adrenocorticolyticproperties and limited hepatotoxic properties by mechanism(s)that are likely independent of systemic ACAT inhibition.