Two-generation reproductive toxicity study of aluminium sulfate in rats

In a two-generation reproductive toxicity study, male and female rats were given aluminium sulfate (AS) in drinking water at 0, 120, 600 or 3000 ppm. AS reduced water consumption in all treatment groups, and body weight was transiently decreased in the 3000 ppm group. In the F1 and F2 pups, preweaning body weight gain was inhibited at 3000 ppm, and the liver and spleen weight was decreased at weaning. At this dose, vaginal opening was slightly delayed. There were no compound-related changes in other reproductive/developmental parameters, including developmental neurobehavioral endpoints. The data indicated that the NOAEL of AS in this two-generation study is 600 ppm for parental systemic toxicity and reproductive/developmental toxicity. The total ingested dose of aluminium from drinking water and food (standard rat diet, containing 25-29 ppm of aluminium) combined for this 600 ppm group was calculated to be 8.06 mg Al/kg bw/day.     

INHALATION DEVELOPMENTAL TOXICITY AND REPRODUCTION STUDIES WITH CYCLOHEXANE

The reproductive and developmental toxicity of cyclohexane was assessed in a two-generation reproduction study with Crl:CD® BR rats and in developmental toxicity studies with Crl:CD®BR rats and Hra:(NZW)SPF rabbits. The animals were exposed whole-body to atmospheric concentrations of 0, 500, 2000, or 7000 ppm cyclohexane. In the two-generation reproduction study, parental effects included statistically significantly lower mean body weight, overall mean body weight gain, and overall mean food efficiency for P₁ and F₁ females of the 7000 ppm level and statistically significantly lower mean body weight for F¹ males of that level. Adult rats exposed to 2000 ppm cyclohexane and above exhibited a transient diminished or absent response to a sound stimulus while in the chambers during exposure. Mean pup weight was statistically significantly lower than control from lactation day 7 throughout the remainder of the 25-day lactation period for both F₁ and F₂ 7000 ppm litters. Changes observed at 500 ppm were either considered not to be compound related or not adverse. Therefore, the systemic-toxicity no-observed-effect level (NOEL) was 500 ppm and the reproductive NOEL was 2000 ppm. The reproductive NOEL was based solely on the decreased pup weights in both the F₁ and F₂ generations observed at 7000 ppm. In the developmental toxicity studies, only the rats showed evidence of maternal toxicity. For rats in the 7000 ppm group, statistically significant reductions were observed in overall maternal body weight gain and overall maternal food consumption for the treatment period. Rats exposed to 2000 ppm cyclohexane and above again exhibited a transient diminished or absent response to a sound stimulus while in the chambers during exposure. Therefore, for rats, the maternal no-observed-effect level (NOEL) was 500 ppm. In the rabbit developmental toxicity study, no compound-related maternal effects were observed at concentration levels of 7000 ppm and below. Therefore, the maternal NOEL for rabbits was 7000 ppm. No compound-related evidence of developmental toxicity was observed at any test concentration in either species. Therefore, the developmental NOEL for both species was 7000 ppm, the highest concentration tested.     

Inhalation developmental toxicity and reproduction studies with cyclohexane

The reproductive and developmental toxicity of cyclohexane was assessed in a two-generation reproduction study with Crl:CD BR rats and in developmental toxicity studies with Crl:CD BR rats and Hra:(NZW)SPF rabbits. The animals were exposed whole-body to atmospheric concentrations of 0, 500, 2000, or 7000 ppm cyclohexane. In the two-generation reproduction study, parental effects included statistically significantly lower mean body weight, overall mean body weight gain, and overall mean food efficiency for P1 and F1 females of the 7000 ppm level and statistically significantly lower mean body weight for F1 males of that level. Adult rats exposed to 2000 ppm cyclohexane and above exhibited a transient diminished or absent response to a sound stimulus while in the chambers during exposure. Mean pup weight was statistically significantly lower than control from lactation day 7 throughout the remainder of the 25-day lactation period for both F1 and F2 7000 ppm litters. Changes observed at 500 ppm were either considered not to be compound related or not adverse. Therefore, the systemic-toxicity no-observed-effect level (NOEL) was 500 ppm and the reproductive NOEL was 2000 ppm. The reproductive NOEL was based solely on the decreased pup weights in both the F1 and F2 generations observed at 7000 ppm. In the developmental toxicity studies, only the rats showed evidence of maternal toxicity. For rats in the 7000 ppm group, statistically significant reductions were observed in overall maternal body weight gain and overall maternal food consumption for the treatment period. Rats exposed to 2000 ppm cyclohexane and above again exhibited a transient diminished or absent response to a sound stimulus while in the chambers during exposure. Therefore, for rats, the maternal no-observed-effect level (NOEL) was 500 ppm. In the rabbit developmental toxicity study, no compound-related maternal effects were observed at concentration levels of 7000 ppm and below. Therefore, the maternal NOEL for rabbits was 7000 ppm. No compound-related evidence of developmental toxicity was observed at any test concentration in either species. Therefore, the developmental NOEL for both species was 7000 ppm, the highest concentration tested.     

Two-generation reproductive toxicity study of the rubber accelerator N,N-dicyclohexyl-2-benzothiazolesulfenamide in rats

Male and female Crl:CD(SD) rats were fed a diet containing rubber accelerator N,N-dicyclohexyl-2-benzothiazolesulfenamide (DCBS) at 0, 80, 600 or 4500ppm throughout the study beginning at the onset of a 10-week pre-mating period and continuing through the mating, gestation, and lactation periods for two generations. At 4500ppm, decreases in the body weight, body weight gain, and food consumption were found in F0 males and females. No changes in the estrous cyclicity, copulation index, fertility index, gestation index, delivery index, number of implantations, precoital interval, or gestation length were observed in any generation at any dose of DCBS. Delayed preputial separation at 4500ppm as well as delayed vaginal opening and higher body weight at the age of vaginal opening at 600 and 4500ppm were found in the F1 generation. A transient change in performance in a water-filled multiple T-maze was found at 600 and 4500ppm in F1 females. There were no compound-related changes in number of pups delivered, sex ratio of pups, viability of pups, anogenital distance, surface righting reflex, negative geotaxis reflex, mid-air righting reflex, pinna unfolding, incisor eruption, or eye opening in the F1 and F2 generations. The body weight of F1 and F2 male and female pups was lowered at 4500ppm. Reduced uterine weight of the weanlings was noted in the F1 generation at 4500ppm and in the F2 generation at 600 and 4500ppm. The data indicate that the NOAEL of DCBS for two-generation reproductive toxicity is 80ppm (5.2mg/kgbw per day) in rats.     

Tetrahydrofuran: two-generation reproduction toxicity in Wistar rats by continuous administration in the drinking water.

In a two-generation reproduction toxicity study, 25 male and 25 female Wistar rats per dose group and generation were exposed continuously to tetrahydrofuran in the drinking water for at least 70 days prior to and during mating, gestation, parturition and lactation to weaning, at concentrations of 0, 1000, 3000 or 9000 ppm (approximately 100, 300 and 700 mg/kg/day in males and females premating, 100, 300 and 800 mg/kg/day in females during gestation, and 200, 500 and 1300 mg/kg/day in females during lactation) through two successive generations. In both generations and sexes, water consumption was dose-relatedly reduced at all doses; food consumption and body weight were reduced at 9000 ppm. Necropsy kidney weights were increased in 9000 ppm F0 males. Pup body weight gain during lactation was reduced in both generations (F1 and F2 pups) and eye opening delayed in the first generation (F1 pups) at 9000 ppm; there were no treatment-related malformations. The NOAEL of tetrahydrofuran in drinking water is 9000 ppm for parental fertility and reproductive performance, and 3000 ppm for systemic parental and developmental toxicity.     

Rat two-generation reproductive toxicity study of bisphenol A.

This study was conducted to determine the low-dose effects of bisphenol A (BPA) in a rat two-generation reproduction study. Groups of 25 male and 25 female Crj: CD (SD) IGS rats were given BPA at 0.2, 2, 20, or 200 microg/kg/day by gastric intubation throughout the study beginning at the onset of a 10- and 2-week premating period, in F0 males and females, respectively, and continuing through the mating, gestation, and lactation periods, for two generations. There were adult (F0, F1, F2) and postnatal day (PND) 22 (F1, F2) necropsies: the oldest F2 males and females being killed at postnatal weeks 7 and 14, respectively. No compound-related clinical signs or effects on body weight or food consumption were observed in any generation. There were no compound-related changes in surface righting reflex, negative geotaxis reflex, mid-air righting reflex, pinna detachment, incisor eruption, eye opening, testes descent, preputial separation, or vaginal opening in F1 and F2 generations, or behavior in the open field or water filled multiple T-maze in the F1 generation. No test compound-related changes in estrous cyclicity, copulation index, fertility index, number of implantations, gestation length, litter size, pup weight, pup sex ratio, pup viability, or other functional reproductive measures were noted in any generation. A few significant changes in the anogenital distance (AGD) per cube root of body weight ratio were found at 0.2 and 20 microg/kg in F1 males, at 2, 20, and 200 microg/kg in F1 females, and at 20 and 200 microg/kg in F2 females. However, the changes in the AGD were consistently small (within 5% of control values), and no continuous changes in the AGD or AGD/cube root of body weight ratio were detected. There were no compound-related changes in epididymal sperm counts or motility in F0 and F1 males. No compound-related necropsy findings or effects on organ weight including the reproductive organs were found in any generation. Histopathologic examinations revealed no evidence of compound-related changes in any organs including the reproductive organs of both sexes. The data indicate that oral doses of BPA of between 0.2 and 200 microg/kg over 2 generations did not cause significant compound-related changes in reproductive or developmental parameters in rats.     

Inhalation two-generation reproductive toxicity study of methyl isobutyl ketone in rats

To evaluate whether methyl isobutyl ketone (MIBK) affects reproductive performance, a two-generation reproduction study was conducted. MIBK was administered to 30 Sprague-Dawley rats/sex/group via whole-body inhalation at concentrations of 0, 500, 1000, or 2000 ppm, 6 h daily, for 70 days prior to mating. F(0) and F(1) females were exposed from mating through gestation day 20 and from postnatal day 5; F(2) litters were maintained through postnatal day 21. No treatment-related mortality of adult animals occurred. There was a dose-related increase in adult animals with no or a decreased response to a sound stimulus at 1000 and 2000 ppm; however, no adverse clinical signs occurred 1 h after exposure, suggesting this was a transient sedative effect. Clinical signs of central nervous system (CNS) depression in the pups were observed and one F(1) pup died after initial exposure to 2000 ppm on postnatal day 22; subsequently exposure was delayed until postnatal day 28. Decreased body weight gain and slight decreased food consumption were observed during the first 2 weeks of exposure in both generations at 2000 ppm. There were no adverse effects on male and female reproductive function or landmarks of sexual maturation. Increased F(0) and F(1) liver weights with associated centrilobular hypertrophy occurred in rats at 2000 ppm, indicative of an adaptive response. Increased male kidney weights at all exposure concentrations, associated with hyaline droplets, were indicative of male rat-specific nephropathy. Other than acute sedative effects, the no-observed-adverse-effect level (NOAEL) for parental systemic effects (excluding male rat kidney) was 1000 ppm, based on transient decreased body weight and food consumption; for reproductive effects, 2000 ppm, the highest concentration tested; and for neonatal toxicity, 1000 ppm (based on acute CNS depressive effects).     

Two-generation reproduction study and immunotoxicity screen in rats dosed with phenol via the drinking water

This study evaluated the potential reproductive toxicity of phenol in a rat two-generation reproduction study, which included additional study endpoints, such as sperm count and motility, developmental landmarks, histological evaluation of suspect target organs (liver, kidneys, spleen, and thymus), weanling reproductive organ weights, and an immunotoxicity screening plaque assay. Phenol was administered to 30 Sprague-Dawley rats/sex/group in the drinking water at concentrations of 0, 200, 1000, or 5000 ppm. Parental (P1) animals were treated for 10 weeks prior to mating, during mating, gestation, lactation, and until sacrifice. The F1 generation (P1 offspring) was treated using a similar regimen, while the F2 generation was not treated. After mating, 10 P1 males/group were evaluated using standard clinical pathology parameters and an immunotoxicity screening plaque assay. Significant reductions in water and food consumption were observed in the 5000-ppm group in both generations; corollary reductions in body weight/body weight gain were also observed. Mating performance and fertility in both generations were similar to controls, and no adverse effects on vaginal cytology or male reproductive function were observed. Vaginal opening and preputial separation were delayed in the 5000-ppm group, and were considered to be secondary to the reduction in F1 body weight. Litter survival of both generations was reduced in the 5000-ppm group. Absolute uterus and prostate weights were decreased in the F1 generation at all dose levels; however, no underlying pathology was observed and there was no functional deficit in reproductive performance. Therefore, these findings were not considered to be adverse. No evidence of immunotoxicity was noted in the 5000-ppm group. The effects noted at the high concentration were presumed to be associated with flavor aversion to phenol in the drinking water. Based on a comprehensive examination of all parameters, the no-observable-adverse-effect level (NOAEL) for reproductive toxicity of phenol administered in drinking water to rats is 1000 ppm. The corresponding daily intake of phenol for an adult rat at the NOAEL of 1000 ppm is equivalent to about 70 mg/kg/day for males and 93 mg/kg/day for females.     

Acute and 90-day subchronic toxicity studies of Silk peptide E5K6, in Sprague-Dawley rats

Acute and 90-day subchronic oral toxicity studies of Silk peptide E5K6 were performed in Sprague-Dawley rats. In the acute toxicity study, Silk peptide E5K6 was administered orally to male and female rats at a single dose of 2000 and 5000 mg/kg. Mortality, clinical signs and body weight changes were monitored for 14 days. There were no treatment-related changes in these parameters. Therefore, the Approximate Lethal Dose (ALD) of Silk peptide E5K6 in male and female rats is higher than 5000 mg/kg. In the subchronic toxicity study, Silk peptide E5K6 was administered orally to male and female rats for 90 days at a single dose of 500, 1000, and 2000 mg/kg. There were no toxicologically significant changes in clinical signs, body weight, food and water consumptions, ophthalmoscopic examination, urinalysis, hematological and serum biochemical examinations, necropsy findings, organ weights and histopathological examination of all of the animals treated with Silk peptide E5K6. These results suggest that the oral No Observed Adverse-Effect Level (NOAEL) of Silk peptide E5K6 is greater than 2000 mg/kg/day in both sexes and the target organs were not established.     

A two-generation reproductive toxicity study of decamethylcyclopentasiloxane (D5) in rats exposed by whole-body vapor inhalation

This two-generation reproduction study assessed the reproductive hazard potential of decamethylcyclopentasiloxane (D(5)). Sprague-Dawley rats (30/sex/group) were exposed by whole-body vapor inhalation to a target concentration of 30, 70, or 160 ppm D(5) or filtered air for 6h/day. Exposures for the F(0) and F(1) generations started at least 70 days prior to mating and lasted through weaning of the respective pups on postnatal day (PND) 21. Female exposures were interrupted from gestation day (GD) 21 through PND 4 to allow for parturition and to permit continuous maternal care for the early neonates. F(2) pups were not directly exposed to D(5). There were no exposure-related mortalities, clinical signs of toxicity, or effects on body weight or food consumption. There were no treatment-related gross findings or organ weight effects at the F(0) and F(1) necropsies. Other than minimal alveolar histiocytosis in all exposed groups, there were no noteworthy microscopic findings. Reproductive parameters (number of days between pairing and mating, mating and fertility indices, gestation length, and parturition), spermatogenic parameters and ovarian primordial follicle counts and numbers of corpora lutea in the F(0) and F(1) parental animals were not significantly changed between treated and control groups. Mean live litter sizes, number of pups born, sex ratios, pup body weights, postnatal pup survival and general physical condition of offspring in each generation were not affected. The slight, but statistically significant, increase in the mean F(1) male pup AGD in the 160 ppm group was not considered to be related to treatment. Vaginal patency and balanopreputial separation were unchanged compared to controls. Thus, the No-Observed-Adverse-Effect-Level (NOAEL) for parental and reproductive toxicity was determined to be 160 ppm D(5).     

Chronic Toxicity and Oncogenicity Study with Vinyl Acetate in the Rat: In Utero Exposure in Drinking Water

The purpose of this study was to evaluate vinyl acetate forpotential chronic toxicity and oncogenicity when given to ratsin drinking water from the time of gestation. Target concentrationswere 0, 200, 1000, and 5000 ppm (v/v). Drinking water solutionswere prepared daily and analyzed at approximately 4-week intervals.F₀ rats were given solutions of vinyl acetate for 10 weeks andthen mated. Offspring (F₁ rats) were culled to equal group sizesof 60 main study rats and 30 rats for satellite groups. F₁ ratswere treated for up to 104 weeks with interim kills of satellitegroups at 52 and 78 weeks. Body weights and clinical signs oftoxicity were monitored in F₀ and F₁ rats. Food and water consumptionwere measured in F₁ rats. At Weeks 52 and 78 of the test, clinicalpathology and urine analysis examinations were conducted on10 rats per group from satellite animals. A complete gross andhistopathological examination of F₁ rats was conducted at theinterim kills and on main study rats at Week 104. Average vinylacetate consumption over the course of the study in male ratsof the 200, 1000, and 5000 ppm groups was 10, 47, and 202 mg/kg/day,respectively. Female rats consumed an average of 16, 76, and302 mg/kg/day, respectively. Compound-related effects observedduring the study included a concentration-related decrease inwater consumption among rats of the 1000 and 5000 ppm groupsand a decrease in food consumption among rats of the 5000 ppmgroups. Concurrent body weight decrement was observed only inthe 5000 ppm groups. There were no compound-related effectson clinical chemical, hematological, or urinalysis parameters.The pathological evaluations revealed no compound-related effectson organ weight, nonneoplastic lesions, or neoplastic lesions.The no-observable-effect level was 200 ppm while the no-observable-adverse-effectlevel was 1000 ppm. Under the conditions of this study, vinylacetate showed no evidence of systemic target organ toxicityand was not oncogenic when administered to rats in the drinkingwater.     

Developmental and reproductive toxicity evaluation of toluene vapor in the rat. I. Reproductive toxicity

The reproductive toxicity of toluene was evaluated in a 2-generation test in which male and female Sprague–Dawley rats, parental (F0) and first generation (F1), were exposed to toluene via whole body inhalation, 6 h/day, 7 days/week for 80 days premating and 15 days of mating at concentrations of 0, 100, 500 and 2000 ppm (0, 375, 1875 and 7500 mg/m³). Toluene was administered at 2000 ppm to both sexes, or to females or males only to be mated with untreated partners. Pregnant females at all dose levels were exposed from gestation day (GD) 1–20 and lactation day (LD) 5–21. At LD5, females were removed from their litters for daily exposure and returned when 6 h of exposure was completed. F1 pups selected to produce the F2 generation were treated for 80 days beginning immediately after weaning (LD21) and initially mated at a minimum of 100 days of age. F2 pups were not exposed to toluene by inhalation.

Toluene exposure did not induce adverse effects on fertility, reproductive performance, or maternal/pup behaviors during the lactation period in males and females of the parental or first generation, but did inhibit growth in F1 and F2 offspring in the 2000 ppm (both sexes treated) and 2000 ppm (females only treated) groups. Caesarean section of selected 2000 ppm (both sexes treated) dams at GD20 showed reduced fetal body weight and skeletal variations. Exposure to toluene caused decreased pup weights throughout lactation in F1 and F2 2000 ppm (both sexes treated), and 2000 ppm (females only treated) groups. Exposure at 2000 ppm to male parents only did not induce similar weight inhibition in offspring. The toluene offspring NOAEL is 500 ppm in groups in which maternal animals were exposed, and 2000 ppm for male only treated groups.     

Three-generation reproductive toxicity study of dietary bisphenol A in CD Sprague-Dawley rats.

Bisphenol A (BPA) was evaluated at concentrations of 0, 0.015, 0.3, 4.5, 75, 750, and 7500 ppm ( approximately 0.001, 0.02, 0.3, 5, 50, and 500 mg/kg/day of BPA) administered in the diet ad libitum to 30 CD((R)) Sprague-Dawley rats/sex/dose for 3 offspring generations, 1 litter/generation, through F3 adults. Adult systemic toxicity at 750 and 7500 ppm in all generations included: reduced body weights and body weight gains, reduced absolute and increased relative weanling and adult organ weights (liver, kidneys, adrenals, spleen, pituitary, and brain), and female slight/mild renal and hepatic pathology at 7500 ppm. Reproductive organ histopathology and function were unaffected. Ovarian weights as well as total pups and live pups/litter on postnatal day (PND) 0 were decreased at 7500 ppm, which exceeded the adult maximum tolerated dose (MTD). Mating, fertility, gestational indices; ovarian primordial follicle counts; estrous cyclicity; precoital interval; gestational length; offspring sex ratios; postnatal survival; nipple/areolae retention in preweanling males; epididymal sperm number, motility, morphology; daily sperm production (DSP), and efficiency of DSP were all unaffected. At 7500 ppm, vaginal patency (VP) and preputial separation (PPS) were delayed in F1, F2, and F3 offspring, associated with reduced body weights. Anogenital distance (AGD) on PND 0 was unaffected for F2 and F3 males and F3 females (F2 female AGD was increased at some doses, not at 7500 ppm, and was considered not biologically or toxicologically relevant). Adult systemic no observed adverse effect level (NOAEL) = 75 ppm (5 mg/kg/day); reproductive and postnatal NOAELs = 750 ppm (50 mg/kg/day). There were no treatment-related effects in the low-dose region (0.001-5 mg/kg/day) on any parameters and no evidence of nonmonotonic dose-response curves across generations for either sex. BPA should not be considered a selective reproductive toxicant, based on the results of this study.     

Developmental toxicity and genotoxicity studies of 1,1,1,3,3,3-hexachloropropane (HCC-230fa) in rats.

The potential developmental toxicity and the in vitro and in vivo genotoxicity of HCC-230fa were assessed. In the developmental toxicity study, groups of 25 mated Crl:CD(R)(SD)BR rats were exposed (whole body) by inhalation to HCC-230fa over days 7-21 of gestation; the day of confirmed mating was designated as gestation day 1 (GD1). Exposures were 6 h per day at concentrations of 0, 0.5, 2.5, or 25 ppm. Body weight, food consumption, and clinical observation data were collected during the study. On day 22 of gestation, the dams were euthanized and examined grossly. The fetuses were removed and subsequently weighed, sexed, and examined for external, visceral, head, and skeletal alterations. Evidence of maternal and developmental toxicity was observed at 25 ppm and was noted as significant, compound-related reductions in mean maternal body weight, weight change, and food consumption. Significant fetal effects also were observed at 25 ppm as compound-related reductions in mean fetal weight and increased fetal malformations (filamentous tail, situs inversus, absent vertebrae) and variations (rudimentary cervical ribs, delayed sternebral ossification). There was no evidence of either maternal or developmental toxicity at 0.5 or 2.5 ppm. The genotoxicity of HCC-230fa was examined in a bacterial reversion assay and in erythrocyte micronucleus studies in two species by different routes of administration. No increases in the number of revertants were observed in the bacterial reversion assay. In one micronucleus study, HCC-230fa was administered by inhalation to rats as part of a 90-day study at doses indicated above. For the second study, ICR mice were given a single ip dose at 0, 166, 330, or 660 mg/kg. In both micronucleus studies, a significant increase in micronucleated erythrocytes was observed. The results of these studies suggest that HCC-230fa affects rapidly dividing cells and may have long-term consequences for occupational exposures.     

Toxicologic evaluations of DHA-rich algal oil in rats: Developmental toxicity study and 3-month dietary toxicity study with an in utero exposure phase

DHA-rich algal oil ONC-T18, tested for subchronic, reproductive, and developmental toxicity in the rat, did not produce any significant toxicologic manifestations. Based on the absence of maternal or developmental toxicity at any dosage level, a dosage level of 2000 mg/kg/day was considered to be the no observed adverse-effect level (NOAEL) for maternal toxicity and embryo/fetal development when DHA-rich algal oil was administered orally by gavage to pregnant Crl:CD(SD) rats during gestation days 6–19. In a dietary combined one-generation/90-day reproductive toxicity study in rats, the NOAEL for F0 male and female and F1 male systemic toxicity was considered to be 50,000 ppm (highest concentration administered) and 25,000 ppm for F1 female systemic toxicity (higher mean body weight, body weight gain, and food consumption). F0 reproductive performance values, estrous cycle length, gestation length, or the process of parturition, and the numbers of former implantation sites and unaccounted-for sites were unaffected by algal oil exposure. Postnatal survival and developmental parameters in the F1 generation were unaffected by algal oil exposure at all dietary concentrations. There were no neurotoxic effects noted at any algal oil exposure level. The results support the safety of DHA-rich algal oil for its proposed use in food.     

Multigeneration study of FD & C Blue No. 2 in rats

In a three-generation reproduction study, groups of ten male and 20 female Charles River CD rats were fed FD & C Blue No. 2 at dietary levels providing intakes of 0.0, 2.5, 25, 75 and 250 mg/kg body weight/day. Slightly bluish-coloured fur was noted in rats at the 250-mg/kg/day dose level and bluish-green-coloured faeces were produced by rats in the 75- and 250-mg/kg/day groups. The gestation, viability and lactation indices of all litters were comparable for the control and treated groups. The fertility indices for female rats in the 2.5- and 25-mg/kg/day groups were significantly lower than those for control females in the case of the F2 litters. However, there was no reduction in the female fertility indices for the F2 litters at the two higher dosage levels, nor for the F1 and F3 litters at any dosage level. Although fertility indices were reduced for some groups of male rats in the F2b and F2c litters, these changes were not considered to be compound-related. Examination of the ovaries and uteri of all dams killed on day 19 of gestation of the F2c and F3c litters revealed no gross anatomical abnormalities. No unusual changes were observed in the stillborn pups or in pups dying during the study. There were no compound-related gross or microscopic pathological lesions in any of the F1 or F3a rats that were killed and necropsied, and no compound-related organ-weight variations were recorded in the F1 parental rats.     

Reproductive and developmental toxicity screening test of tetrahydrofurfuryl alcohol in rats

Twelve male and female rats per group were given tetrahydrofurfuryl alcohol (THFA) by gavage at 0, 15, 50, 150 or 500 mg/kg/day. Males were dosed for 47 days, beginning 14 days before mating, and females were dosed for 42–52 days beginning 14 days before mating to day 4 of lactation throughout the mating and gestation period. Changes in locomotor activity, inhibition of body weight gain, and/or histopathological changes in the thymus, spleen, testes and/or epididymides were observed in males and females at 150 mg/kg and above. No effects of THFA were found on the copulation index, fertility index, or the number of corpora lutea and implantations in pregnant females. At 500 mg/kg, no pregnant females delivered any pups. At 150 mg/kg, gestation length was prolonged, and the total number of pups born and the number of live pups on postnatal days 0 and 4 was markedly decreased. No effects of THFA were found on the sex ratio and body weight of live pups, or the incidence of pups with malformations or variations. Based on these findings, the NOAELs for parental and reproductive/developmental toxicity of THFA were concluded to be 50 mg/kg/day in rats.     

Multigenerational reproductive study of genistein (Cas No. 446-72-0) in Sprague-Dawley rats (feed study)

Genistein is a naturally occurring isoflavone that interacts with estrogen receptors and multiple other molecular targets. Human exposure to genistein is predominantly through consumption of soy products, including soy-based infant formula and dietary supplements. Consumption of soy and genistein has been associated with a variety of beneficial effects in animals and humans, but concerns have also been raised concerning potential adverse effects of genistein, particularly with regard to reproductive toxicity and the induction or potentiation of carcinogenesis, due primarily to its weak estrogenic activity. Because of these concerns, genistein was selected as one of the compounds to be examined in a protocol utilizing Sprague-Dawley rats to evaluate the effects of multigenerational and long-term exposures to doses of estrogenic agents that produce subtle reproductive tract lesions in developmentally exposed Sprague-Dawley rat pups. Results from the multigenerational reproductive toxicology feed study are reported in this report, and results of the 2-year feed study are reported separately (NTP, 2008a). Data from a preliminary reproductive dose range-finding feed study (NTP, 2007) that utilized exposure concentrations of up to 1,250 ppm genistein were used to select dietary exposure concentrations of 0, 5, 100, and 500 ppm for the current study. These dietary doses resulted in ingested genistein doses of approximately 0, 0.3, 7, or 35 mg genistein/kg body weight per day for males and 0, 0.5, 10, or 51 mg/kg per day for females during the time that the rats were directly consuming dosed feed. The current study was a multigenerational study (F(0) through F(4), with F(5) litters terminated at weaning) focused on reproductive endpoints. Animals were continuously exposed to genistein from the time that the F(0) generation was 6 weeks old through weaning of the F(3) generation, and animals of the F(0) through F(4) generations were sacrificed and necropsied on postnatal day 140 (PND 140). Dosed feed was removed from the F(3) pups at the time of weaning, and this generation and subsequent generations were maintained on control feed for the remainder of the study. For this study, 140 animals of each sex were obtained from the NCTR CD (Sprague-Dawley) rat colony at weaning and placed on a soy- and alfalfa-free diet that was used throughout the study in an attempt to maintain consistently low background exposure to phytoestrogens. Thirty-five animals per sex were assigned to exposure groups by a weight-ranked randomization procedure prior to the start of dietary exposure of the parental (F(0)) generation at 6 weeks of age. At the time of mating, males were paired with females from the same exposure group, and they were housed together until evidence of successful mating was detected or for a maximum of 14 days. Litters were randomly standardized to four males and four females on PND 2, and 25 litters per exposure group and their associated sires and dams were randomly selected to continue on study to produce the next generation and then necropsied at termination at 20 weeks of age (PND 140). Similar procedures were used to produce each generation. Results of the current study are summarized below. In the postweaning period, exposure to 500 ppm genistein reduced body weights predominantly in females of generations in which rats were ingesting the compound throughout adulthood (F(0) through F(2)). In the unexposed F(4) generation, female body weight was also depressed, although to a lesser extent than in the earlier generations. In the F(1) generation, postweaning body weights were reduced in all 100 and 500 ppm groups, with a more pronounced effect in the females. While pup birth weights were not significantly affected by genistein in the F(1) through F(4) generations (with the exception of 100 ppm males in the F(1) generation), both sexes showed depressed body weight gains during the preweaning period in the 500 ppm groups in all of these generations. Male pup preweaning body weight gains were also depressed in the 5 and 100 ppm groups in the F(1) generation. In the unexposed F(5) generation, pup birth weights in all exposed groups of both sexes were significantly lower than those in the controls, although it seems likely that this is a chance observation rather than a carryover effect from exposures in earlier generations. Measures of fertility were not adversely affected by genistein except for litter size. Litter size of the 500 ppm group in the F(2) generation was significantly smaller than that in the corresponding control group. The litter sizes in the F(1), F(2), and F(3) generations showed negative exposure concentration trends. Male and female 500 ppm pups in the F(1) generation had slightly reduced anogenital distances (AGDs) relative to controls when covaried by body weight. Female pups also had reduced AGDs in the F(2) (500 ppm) and F(3) (100 ppm) generations, although the statistical significance was dependent on the analysis method applied. Females exposed to 500 ppm showed an accelerated time of vaginal opening (approximately 3 days) in the F(1) and F(2) generations, while the 5 ppm group showed an earlier time of vaginal opening (1.3 days) in the F(3) generation. Body weight at vaginal opening was lower in 500 ppm females of the F(1) through F(3) generations and in the 5 ppm females of the F(1) generation. When examined shortly after vaginal opening, estrous cycles of 500 ppm females in the F(1) and F(2) generations were significantly longer (approximately 3 days and 1 day, respectively) than those of their respective control groups. Other estrous cycle disturbances (with the exception of decreased time in diestrus for 100 ppm females in the F(4) generation) were confined to the 500 ppm group of the F(1) generation and included reduced time in proestrus and an increase in the number and percentage of aberrant cycles. When the estrous cycles of older animals were examined prior to termination, the sole significant effects were a decreased time in estrus and increased time in diestrus in 5 ppm females of the F(2) generation and an increased number of abnormal cycles in 500 ppm females of the F(3) generation. No effects of genistein on male sexual development were noted with the exception of an increased time to testicular descent in 500 ppm males of the F(3) generation. Significant organ weight effects in both sexes were largely confined to single exposed groups in single generations; no clear patterns indicating toxicity to reproductive or nonreproductive organs were observed. Exposure-related microscopic lesions were confined to males, with the mammary gland and kidney affected. Incidences of mammary gland alveolar/ductal hyperplasia were significantly increased in 500 ppm males in the F(0) through F(2) generations and in 100 ppm males in the F(1) and F(2) generations. In the F(3) generation, a significant positive linear exposure concentration trend in the incidences of mammary gland hyperplasia occurred, but no exposed group differed significantly from the controls in pairwise comparisons. The more pronounced effect of genistein on the incidences of male mammary gland hyperplasia in the continuously exposed F(1) and F(2) generations as compared to the late adolescent and adult exposures of the F(0) generation and the preweaning-only exposure of the F(3) generation indicates that both developmental and adult exposures contribute to the maintenance of this effect into adulthood. Statistically significant effects of genistein on the incidences of generally minimal to mild kidney lesions in males were confined to the continuously exposed F(1) and F(2) generations. Incidences of renal tubule mineralization were significantly increased in 100 and 500 ppm males in the F(1) and F(2) generations, and incidences of inflammation and renal tubule regeneration were significantly increased in 500 ppm males in the F(1) generation. In addition to the results reported above for animals from the main study, ancillary studies were conducted with pups derived from the current study or from animals treated under similar conditions. These results have been reported elsewhere (Appendix P) and are not presented in detail in this report. Of particular importance are the data on blood and tissue genistein concentrations obtained from adult animals in the F(1) generation (Chang et al., 2000), from dams and fetuses (Doerge et al., 2001), and from dams and nursing pups (Doerge et al., 2006). These data provide measures of the internal dose resulting from the dietary exposure concentrations used in the current study and indicate that while fetal and adult exposures to genistein were at concentrations relevant to the full range of human exposures, only very low exposures were achieved during the early neonatal period when the pups were receiving exposures exclusively from the milk. The minimal exposure to genistein during this critical developmental period must be considered in the interpretation of the data derived from the current study. In summary, although genistein did show adverse effects with dietary exposures of 100 or 500 ppm, there were no clear adverse effects on the reproductive or developmental parameters measured at genistein concentrations ranging from less than 1 ppm (control diet) to 100 ppm, a range of doses producing serum concentrations achievable from the phytoestrogen content of human diets. There were few clear, overtly toxic effects that carried over across directly exposed generations or appeared to be imprinted to carry over into unexposed descendents under the conditions of exposure in this study. (ABSTRACT TRUNCATED).     

Safety assessment of methanol extract of red dragon fruit (Hylocereus polyrhizus): Acute and subchronic toxicity studies

Recently, the fruits of Hylocereus polyrhizus, known as red dragon fruit, have received much attention from growers worldwide. However, there is little toxicological information regarding the safety of repeated exposure to these fruits. The present study evaluated the potential toxicity of a methanol extract of H. polyrhizus fruit after acute and subchronic administration in rats. In the acute toxicity study, single doses of fruit extract (1250, 2500 and 5000 mg/kg) were administered to rats by oral gavage, and the rats were then monitored for 14 days. In the subchronic toxicity study, the fruit extract was administered orally to rats at doses of 1250, 2500 and 5000 mg/kg/day for 28 days. There was no mortality or signs of acute or subchronic toxicity. There was no significant difference in body weight, relative organ weight or hematological parameters in the subchronic toxicity study. Biochemical analysis showed some significant changes, including creatinine, globulin, total protein and urea levels. No abnormality of internal organs was observed between treatment and control groups. The lethal oral dose of the fruit extract is more than 5000 mg/kg and the no-observed-adverse-effect level (NOAEL) of the extract for both male and female rats is considered to be 5000 mg/kg per day for 28 days.