Lipid-soluble green tea extract: Genotoxicity and subchronic toxicity studies

To assess the potential safety of lipid soluble green tea extract, also referred to as lipid soluble tea polyphenols (LSTP), a series of genotoxicity tests were conducted, including an Ames, in vivo mouse micronucleus, and in vivo mouse sperm abnormality test. The toxicity of LSTP was evaluated in 90- and 30-day feeding studies. LSTP did not show mutagenic activity in the Ames test and no genotoxic potential in the in vivo assays at doses up to 10 g/kg body weight (bw). In the 90-day feeding study, LSTP was given in the diet at levels providing 0, 0.125, 0.25, or 0.50 g/kg bw/day. No significant effects were noted on body weight, food consumption, hematology, clinical chemistry, organ weights, and histopathological examination. The no-observed-adverse-effect level (NOAEL) was therefore considered to be 0.50 g/kg bw/day, the highest dose tested. Likewise, dosing of SD rats by gavage for 30 days also showed no adverse effects of growth, hematology, clinical chemistry, organ weights, or histopathology at doses of 0.58, 1.17, and 2.33 g/kg bw/day. The NOAEL in the 30-day study was considered to be the highest dose tested. These data provide evidence to support the safe use of LSTP in food.     

Safety evaluation of AMP deaminase from Aspergillus oryzae

Adenosine-5′-monophosphate (AMP) deaminase is an enzyme used to increase concentrations of 5’-inosine monophosphate in certain foods and beverages for flavoring purposes. One commercial source of this enzyme is Aspergillus oryzae, a filamentous fungus with a history of safe use in Asia as a fermentation organism used in the production of miso sauce and sake liquors. Noting the use of the enzyme in food intended for human consumption and potential presence at trace levels in finished goods, a series of safety studies including an in vitro Ames test and chromosome aberration assay with Chinese hamster lung fibroblasts were conducted along with a 90-day oral toxicity study in rats. AMP deaminase showed no evidence of genotoxicity in the in vitro tests. Following gavage administration of Sprague–Dawley rats at dosages of 19.8, 198.4, or 1984 mg total organic solids (TOS)/kg body weight (bw)/day for 90 days, no adverse effects on body weight gain, food consumption, hematology, clinical chemistry, urinalysis, ophthalmological and histopathological examinations were observed. The no-observed-adverse-effect level was considered to be 1984 mg TOS/kg bw/day, the highest dose tested. Results of the genotoxicity studies and subchronic rat study support the safe use of AMP deaminase produced from A. oryzae in food production.     

A subchronic toxicity study of Radix Dipsaci water extract by oral administration in F344 rats

Radix Dipsaci, the dried root of Dipsacus asperoides C.Y. Cheng & T.M.Ai, has therapeutic effects on various disorders, and in particular, bone and joint disease. Despite such ethnomedicinal benefits, there is very little information regarding its in vivo toxicity or adverse effects. This study was conducted to evaluate the potential toxicity of the Radix Dipsaci water Extract (RD-wE) by using F344 rats. The RD-wE was administered orally to rats at doses of 0, 125, 250, 500, 1000, and 2000 mg/kg body weight (bw)/day for 13 weeks. During the treatment period there were no mortalities attributed to RD-wE. Moreover, no toxic effects were observed with regard to body weight, clinical pathology (hematology, clinical biochemistry, and urinalysis), and anatomic pathology (gross findings, organ weight, and microscopic examination). The changes related to the treatment were excessive salivation at the mouth and soft feces, observed in male and female rats at 1000 or 2000 mg/kg bw/day, but these were not accompanied by any microscopic correlate or other pathophysiological changes. Based on these results, the oral no-observed-adverse-effect level of the RD-wE was considered to be 2000 mg/kg bw/day in both genders, although the target organs were not determined under the current experimental conditions.     

A 28-day oral toxicity study of echimidine and lasiocarpine in Wistar rats

Pyrrolizidine alkaloids (PAs) are a class of naturally-occurring plant toxins. Echimidine is one of the predominant PAs found in honeys produced in Australia and New Zealand. There is a lack of information on the oral toxicity of echimidine on which to base regulatory decisions concerning the risk to humans of these honeys. This GLP study was conducted to assess the subchronic dietary toxicity of echimidine to rats compared to that of lasiocarpine as a positive control. Wistar rats, 10/sex, were fed diets containing 0, 0.6, 1.2 or 2.5 mg/kg bw echimidine. Positive control groups, 10/sex, were fed diets containing 0.6, 1.2 or 2.5 mg/kg bw lasiocarpine.Neither PA had any effect on survival, food consumption, clinical signs, gross lesions, or histopathology. Consumption of lasiocarpine, but not echimidine, decreased bodyweight gain in males at ≥ 1.2 mg/kg bw, and in females at 2.5 mg/kg bw. Slight alterations in white cell counts and serum ALT concentrations at 2.5 mg/kg bw of both PAs were not clinically significant, had no histological correlates, and were considered to be of equivocal relevance.In conclusion, the subchronic No Observed Adverse Effect Level (NOAEL) for echimidine is 2.5 mg/kg bw/day, whereas, on the basis of a treatment-related decrease in bodyweight gain in males at 1.2 mg/kg bodyweight, the NOAEL for lasiocarpine is 0.6 mg/kg bw/day.     

Chemical-specific adjustment factors (inter-species toxicokinetics) to establish the ADI for steviol glycosides

The acceptable daily intake (ADI) of commercially available steviol glycosides is currently 0–4 mg/kg body weight (bw)/day, based on application of a 100-fold uncertainty factor to a no-observed-adverse-effect-level value from a chronic rat study. Within the 100-fold uncertainty factor is a 10-fold uncertainty factor to account for inter-species differences in toxicokinetics (4-fold) and toxicodynamics (2.5-fold). Single dose pharmacokinetics of stevioside were studied in rats (40 and 1000 mg/kg bw) and in male human subjects (40 mg/kg bw) to generate a chemical-specific, inter-species toxicokinetic adjustment factor. T_max values for steviol were at ∼8 and ∼20 h after administration in rats and humans, respectively. Peak concentrations of steviol were similar in rats and humans, while steviol glucuronide concentrations were significantly higher in humans. Glucuronidation in rats was not saturated over the dose range 40–1000 mg/kg bw. The AUC_0-last for steviol was approximately 2.8-fold greater in humans compared to rats. Chemical-specific adjustment factors for extrapolating toxicokinetics from rat to human of 1 and 2.8 were established based on C_max and AUC_0-last data respectively. Because these factors are lower than the default value of 4.0, a higher ADI for steviol glycosides of between 6 and 16 mg/kg bw/d is justified.     

Oral subchronic toxicity evaluation of a novel antitumor agent 25-methoxydammarane-3, 12, 20-triol from Panax notoginseng in Sprague–Dawley rats

Panax notoginseng and its main active ingredients ginsenosides have long been used as medicines and food additives in China. Comparing with the extensive uses and active researches of P. notoginseng and its products, the side effect and probable toxicity were rare. 25-Methoxydammarane-3,12,20-triol (25-OCH₃-PPD), a novel dammarane-type triterpene sapogenin that was first isolated from the extract of P. notoginseng, was proven to have strong antitumor activities against prostate cancer, breast cancer and lung cancer. The aim of the present study was to investigate the potential subchronic toxicity of 25-OCH₃-PPD after it was repeatedly orally administered to Sprague–Dawley rats (5/sex/group/each time-point) at dose levels of 0, 150, 300 or 600 mg/kg/day for 13 weeks and 4-week recovery. No mortality and treatment-related toxicity effects were observed as a result of the administration of 25-OCH₃-PPD at any dose level (150, 300 and 600 mg/kg) for 92 consecutive days. Although there were some statistical changes, such as increased weights in female rats and decreased organ weights and coefficients of the liver, spleen, kidney, and adrenal gland compared with the control group at the corresponding time, the autopsy and histopathological examination of the target organs did not show any abnormal responses. As a result, 25-OCH₃-PPD was well tolerated by SD rat at doses of up to 600 mg/kg and that it is a potential candidate for therapeutic use.     

Safety evaluation of oleic-rich triglyceride oil produced by a heterotrophic microalgal fermentation process

Numbers of macro- and microalgae have been used as food sources in various cultures for centuries. Several microalgae are currently being developed as modern food ingredients. The dietary safety of oleic-rich microalgal oil produced using a heterotrophic fermentation process was assessed in a 13-week feeding trial in rats with genotoxic potential evaluated using in vitro and in vivo assays. In the genotoxicity assays, the test oil was not mutagenic in Salmonella typhimurium or Escherichia coli tester strains (⩽5000 μg/plate) with or without metabolic activation. Further, no clastogenic response occurred in chromosome aberration assays in the bone marrow of mice administered a single intraperitoneal dose (2000 mg/kg). In the subchronic study, rats consumed feed containing 0, 25,000, 50,000 or 100,000 ppm oleic-rich oil for 90 days. No treatment-related mortalities or adverse effects occurred in general condition, body weight, food consumption, ophthalmology, urinalysis, hematology, clinical chemistry, gross pathology, organ weights or histopathology. Although several endpoints exhibited statistically significant effects, none were dose-related or considered adverse. Taking all studies into consideration, the NOAEL for the oleic-rich oil was 100,000 ppm, the highest concentration tested and equivalent to dietary NOAELs of 5200 mg/kg bw/day and 6419 mg/kg bw/day in male and female rats, respectively.     

Mode of action based risk assessment of the botanical food-borne alkenylbenzene apiol from parsley using physiologically based kinetic (PBK) modelling and read-across from safrole

The present study developed physiologically-based kinetic (PBK) models for the alkenylbenzene apiol in order to facilitate risk assessment based on read-across from the related alkenylbenzene safrole. Model predictions indicate that in rat liver the formation of the 1′-sulfoxy metabolite is about 3 times lower for apiol than for safrole. These data support that the lower confidence limit of the benchmark dose resulting in a 10% extra cancer incidence (BMDL₁₀) that would be obtained in a rodent carcinogenicity study with apiol may be 3-fold higher for apiol than for safrole. These results enable a preliminary risk assessment for apiol, for which tumor data are not available, using a BMDL₁₀ value of 3 times the BMDL₁₀ for safrole. Based on an estimated BMDL₁₀ for apiol of 5.7–15.3 mg/kg body wt per day and an estimated daily intake of 4 × 10⁻⁵ mg/kg body wt per day, the margin of exposure (MOE) would amount to 140,000–385,000. This indicates a low priority for risk management. The present study shows how PBK modelling can contribute to the development of alternatives for animal testing, facilitating read-across from compounds for which in vivo toxicity studies on tumor formation are available to compounds for which these data are unavailable.     

Genotoxicity,acute and subchronic toxicity evaluation of savory food ingredients

The potential toxicity of two savory food ingredients produced by fermentation of enzymatically hydrolyzed corn starch (Savory Base 100 and Savory Base 200) was evaluated individually in a bacterial reverse mutation assay, an in vitro mammalian cell gene mutation assay, an acute oral study and as a mixture in a 90-day dietary study. In the bacterial reverse mutation and in vitro mammalian cell gene mutation assays, neither ingredient was mutagenic at concentrations up to 5000 μg/plate and 5000 μg/mL, respectively in the presence and absence of metabolic activation. In the acute study, the no-observed-adverse-effect level (NOAEL) for each Savory Base 100 and Savory Base 200 in male and female rats was 2000 mg/kg body weight. In the 90-day study, the hematology and clinical chemistry findings and histopathological changes noted in the liver, heart and kidneys were deemed to be of no toxicological significance, as the mean values were within the historical control range, were not dose-dependent, occurred at a similar frequency in control groups, or only occurred in the control group. Considering these findings, the NOAEL for Savory Base 100 and Savory Base 200 was 2333 and 1167 mg/kg body weight, respectively, the highest dose tested in each case.     

A comprehensive study on in vitro and in vivo toxicological evaluation of Artemisia capillaris

Artemisia capillaris (AC) has been used as an alternative therapy in obesity, atopic dermatitis, and liver diseases through several biological activity including anti-steatotic, antioxidant, and anti-inflammatory activities. Despite its ethnomedicinal benefits, no sufficient background information is available about the long-term safety and genotoxicity of the AC extract. Therefore, the present study was carried out to investigate the 13-week subchronic toxicity and genotoxicity of the AC extract according to the test guidelines published by the Organization for Economic Cooperation and Development. In the 13-week toxicity study using doses of 25, 74, 222, 667, and 2000 mg/kg body weight, oral administration of the AC extract in male and female rats did not result in any significant adverse effects in food/water consumption, body weight, mortality, hematology, serum biochemistry, organ weight and histopathology. Accordingly, the no-observed-adverse-effect level in rats of both genders was established for the AC extract at 2000 mg/kg/day, the highest dose level tested. In addition, the AC extract was not genotoxic in a battery of tests including Ames test, in vitro chromosome aberration assay and in vivo micronucleus assay. In conclusion, we demonstrated that the AC extract is considered as a safe traditional medicine for human consumption.     

Toxicity and oxidative stress induced by T-2 toxin and HT-2 toxin in broilers and broiler hepatocytes

T-2 and HT-2 toxins belong to mycotoxins which are found in human foods and animal chow. We investigated the toxicity and oxidative stress induced by T-2/HT-2 in broilers and chicken hepatocytes. Maize cultures of Fusarium poae was fed to broilers for 42 d, and the physiological index, biochemical index and expression of mRNAs related to oxidative stress were analyzed. Chicken hepatocytes were treated with different levels of T-2/HT-2, and the following parameters were detected: cell viability, GSH and MDA concentration, LDH leakage, activities of ALT/AST, ROS, GSH-PX, SOD and CAT, and expression of mRNA related to oxidative stress. In vivo, high levels of mycotoxins (4 mg/kg T-2 and 0.667 mg/kg HT-2) in feed caused significant reductions in body weight, weight gain, and serum total protein, and significant increases in feed conversion ratio, ALP, ALT/AST activities, and expression of mRNA related to oxidative stress. In vitro, cells treated with T-2/HT-2 showed reductions of GSH concentration and significant increases in LDH leakage, ALT/AST ROS, GSH-PX, SOD and CAT activities, MDA concentration, and expression of mRNA related to oxidative stress. Consequently, F. poae culture material and T-2/HT-2 induced toxicity and oxidative stress in vivo and in vitro, respectively.     

Cyto-genotoxicity and oxidative stress induced by zinc oxide nanoparticle in human lymphocyte cells in vitro and Swiss albino male mice in vivo

ZnO-np has immense potential and application in cosmetic and health care sectors. Hence it was imperative to assess the toxicity/safety of these nanoparticles. In this study, we have evaluated the effects of ZnO-np in human peripheral blood mononuclear cells (PBMCs) in vitro and in Swiss albino male mice in vivo for cyto-genotoxicity and oxidative damage. In vitro results showed that ZnO-nps were weakly genotoxic, induced significant decrease in mitochondrial membrane potential and was capable of ROS generation, leading to apoptosis. In bone marrow cells in vivo, reduction of mitochondrial membrane potential (MMP), increased oxidative stress and G0/G1 cell cycle arrest was observed along with chromosome aberrations and micronuclei formation. In liver cells DNA damage and induction of oxidative stress with concurrent decrease in inhibition of antioxidant enzymes were noted. These in vitro and in vivo results demonstrated that ZnO-np induced genotoxic response and ROS production leading to apoptotic cell death and established a good co-relation between the two biological systems. More importantly, the results stress on the need of multiple endpoint assay-approaches, with an in vitro-in vivo study design to assess nanoparticle toxicology.     

In vivo and in vitro evaluation for nutraceutical purposes of capsaicin, capsanthin,lutein and four pepper varieties

The purpose of this study is to determine the nutraceutic potential of different Capsicum sp, capsaicin, capsanthin and lutein and provide data in order to clarify the conflicting results obtained for capsaicin by different authors. To achieve these objectives, in vivo (geno/antigenotoxicity and lifespan assays in the animal model Drosophila) and in vitro (cytotoxicity and DNA-fragmentation assays in HL60 promyelocytic cell line) assays were carried out. Results showed that i) none of the tested substances were genotoxic except green hot pepper and capsaicin at the highest tested concentration (5 mg/mL and 11.5 μM respectively), ii) all tested substances except green hot pepper are antimutagenic against H₂O₂-induced damage, iii) only red sweet pepper significantly extend the lifespan and healthspan of D. melanogaster at 1.25 and 2.5 mg/mL, iv) all pepper varieties induce dose-depended cytotoxic effect in HL60 cells with different IC₅₀, and v) all pepper varieties and capsaicin exerted proapoptotic effect on HL60 cells. In conclusion: (i) sweet peppers could be suggested as nutraceutical food, (ii) hot peppers should be moderately consumed, and (iii) supplementary studies are necessary to clarify the synergic effect of the carotenoids and capsaicinoids in the hot pepper food matrix.     

Estimation of in vivo and in vitro exposure to bisphenol A as food contaminant

The goal of this cross-sectional study was to examine the occurrence of bisphenol A (BPA) in the morning spot urine taken from 145 female volunteers of various ages. Total urine BPA concentration was detected in 38.6% samples in the 0.92–70.96 μg/g Cr range. The majority of BPA + women belonged to the 25 + body mass index (BMI) group (54.5% were overweight and 43.4% were obese women). Occurrence of BPA in the urine samples was higher at 40 + ages. The maximum BPA concentration of 70.96 μg/g Cr was detected in the urine sample of an obese woman. It is known that BPA is highly toxic in vitro. In this study BPA impaired significantly the growth of all investigated cell lines, i.e. the EC₅₀ values were reached at very low concentrations, in the range from 3.24 to 34.85 μg/mL. The obtained in vivo results suggest that a higher exposure to BPA could contribute to weight problems in women and the absence of the BPA in vitro selective toxicity studies indicates to its general toxic mode of action and raises awareness of the health risks associated with its ubiquitous presence in the environment.     

4-Week repeated dose oral toxicity study of N-ethyl-2-pyrrolidone in Sprague Dawley rats

The solvent N-ethyl-2-pyrrolidone (NEP) was evaluated in a 4-week repeated dose study in rats. NEP diluted in distilled water was orally administered by gavage to male and female Sprague-Dawley rats at doses of 0 (vehicle control), 5, 50, and 250 mg/kg/day for 28 consecutive days. Transient decreases in the body weight and in the body weight gain of the males was observed during the first days of treatment at the 50 and 250 mg/kg/day doses. There was a marked increase in urine volume at the beginning of treatment in males and female rats at doses of 50 and 250 mg/kg/day. No biologically significant differences were observed in hematological and clinical chemistry values in males and females at necropsy. Histological examination revealed an increase in hyaline droplets in the renal tubules of the kidneys and hepatocellular centrilobular hypertrophy in the liver of males at 250 mg/kg/day. Cytochrome P450 concentration in liver microsomes was slightly increased at 250 mg/kg/day in males. The results of this study demonstrate that NEP has mild to no effects at doses up to 250 mg/kg/day when administered orally to rats for 28 days with males being more susceptible than females.     

In vitro and in vivo safety evaluation of Nephure™

Nephure™ is a proprietary oxalate decarboxylase (OxDC) enzyme being developed as a food ingredient. In this study, the safety of Nephure™ was evaluated in a bacterial mutagenicity assay and in a sub-chronic (13-week) oral toxicity study in rats. Nephure™ did not show any mutagenic properties in the mutagenicity assay. In the 13-week sub-chronic oral toxicity study in which 10 Sprague Dawley rats per sex were administered 0, 118, 235 and 475 mg/kg bw/day (8260, 16450 and 33,250 Units/kg bw/day, respectively) of Nephure™ by gavage, male and female rats did not show any test article-related clinical observations or effects on body weight, body weight gain, food consumption, food efficiency, ophthalmology, functional observational battery parameters or motor activity. Furthermore, there were no changes in coagulation, clinical chemistry, urinalysis or hematology parameters, macroscopic/microscopic findings or organ weights that could be attributed to the test article. Based on these results, Nephure™ was not mutagenic and the no-adverse-effect level (NOAEL) in the 13-week study was determined to be 475 mg/kg bw/day (33,250 Units/kg bw/day). Evaluation of the estimated consumption of Nephure™, generation of the metabolite formate, and the current safety studies resulted in a conclusion of a tolerable upper limit of 3450 Units of OxDC activity/day (57.5 Units activity/kg bw/day), when Nephure™ is added to food to decrease dietary oxalate.     

A two-year dietary carcinogenicity study of cyadox in Sprague-Dawley rats

To investigate the potential carcinogenicity of cyadox, an antimicrobial agent, four groups of Sprague-Dawley rats (50 rats/sex/group) were fed diets containing cyadox (0, 200, 600 or 2000 mg/kg) for up to two years. There were significant decreases in body weight, feed intake and feed efficiency in both genders during most of the period in the 2000 mg/kg group. Significant decreases in serum ALT were observed in the 2000 mg/kg group at weeks 52, 78 and 104. For the control, 200, 600, and 2000 mg/kg groups, the tumor incidence in females was 33.3%, 37.2%, 40.0% and 19.0%, while it in males it was 18.9%, 2.6%, 17.1% and 13.6%, respectively. At histopathology, no increases in tumor incidence were attributed to treatment with cyadox. The mild swelling and fatty degeneration in hepatocytes, and mild swelling and tubular necrosis in the kidney were observed in 2000 mg/kg group. The no-observed-effect-level (NOEL) for carcinogenicity of cyadox fed to rats was 2000 mg/kg diet (132.18–156.28 mg/kg b.w./day). In conclusion, cyadox was not carcinogenic to rats with the liver and kidney as the target organs, and the side chain may be involved in toxicity and carcinogenicity mediated by QdNOs.     

Effects of n-butylparaben on steroidogenesis and spermatogenesis through changed E2 levels in male rat offspring

Parabens are widely used as antibacterial agents, which are concerned recently in the relationship between the use of parabens and reproductive toxicity. So that reassessment of the risk of parabens is needed. In this study, one of parabens, n-butylparaben (n-BP) was orally administered to pregnant Wistar rats (0, 64, 160, 400 and 1000 mg/kg/day) from gestation day (GD) 7 through postnatal day (PND) 21. Reduced anogenital distance (AGD) and delayed preputial separation (PPS) were observed in the male offspring. The weights of the testes were significantly reduced at PND 21–90. The weights of the epididymides were significantly reduced at all monitoring points, except PND 35. Seminal vesicle weights were significantly reduced on PND 21. Serum testosterone (T) was significantly decreased, especially on PND 49. The levels of 17β-estradiol (E₂) showed an increase at each of the tested points except on PND 180. Serum luteinising hormone (LH) and follicle-stimulating hormone (FSH) levels in the n-BP treated groups were lower on PND 21, 35 and 49 but elevated on PND 90 compared to control levels. n-BP reduced epididymal cauda sperm counts and daily sperm production in a dose-dependent manner; this difference was statistically significant at exposure groups of 400 and 1000 mg/kg/day. The present study strongly suggests that exposure to n-BP in utero and during lactation has adverse effects on the reproductive system in male offspring, with a no observed adverse effect level (NOAEL) of 160 mg/kg/day. To our knowledge, this is the first study that reports increased E₂ levels of male rats following n-BP exposure; we suggest that E₂ levels may be considered as biomarkers for some endocrine disrupting chemicals (EDCs).     

Genotoxic and cytotoxic evaluation of Jatropha dioica Sessé ex Cerv. by the micronucleus test in mouse peripheral blood

Jatropha dioica Sessé ex Cerv. is a medicinal plant credited with low cytotoxicity in vitro. Thus, the objective of this work was to evaluate the possible genotoxic and cytotoxic effect in vivo of the J. dioica aqueous extract by means of micronucleus assay in mouse peripheral blood. Four different J. dioica aqueous extract dose-units were evaluated (30, 60, 100, and 300 mg/kg). The extract was administered orally to male Balb-C-strain mice every 24 h during 5 days. Blood samples were taken at 0, 24, 48, 72, 96, and 120 h from the mouse's tail and were performed in duplicate extensions. The number of Polychromatic Erythrocytes (PCE), Polychromatic Micronucleus Erythrocytes (PCEMN), and Micronucleus Erythrocytes (MNE) was determined at the different sampling times in the different study groups. Our results showed that the group that received 60 mg/kg of cyclophosphamide (positive control) presented a significant decrease in the PCE (p = 0.044) proportion and a significant increase in MNE (p = 0.032, p = 0.0001). The groups that received the different J. dioica aqueous extract doses did not present either a PCE decrease or an increase in PCEMN and MNE. J. dioica exerts neither a genotoxic nor a cytotoxic effect on mouse peripheral blood at high doses.     

A dietary two-generation reproductive toxicity study of (2R,4R)-monatin salt in Crl:CD(SD) rats

(2R,4R)-Monatin salt [sodium/potassium 2R,4R-2-amino-4-carboxy-4-hydroxy-5-(3-indolyl) pentanoate] was fed at 5000, 15,000, or 35,000 ppm to Crl:CD(SD) rats over two generations. Reduced body weights were observed at all dose levels. Sustained effect on body weight gain at 35,000 ppm in the F₀ and F₁ parental animals was associated with lower feed efficiency, soft stool, and slightly lower numbers of implantation sites. Lower numbers of pups born and live litter size at 35,000 ppm were considered secondary to slightly lower numbers of former implantation sites in the dams. Spermatogenic endpoints, estrous cyclicity, reproductive performance, mean gestation length, and parturition were unaffected in the F₀ and F₁ generations. There were no effects on F₁ and F₂ generation postnatal survival. Reduced pre-weaning pup body weights at 35,000 ppm resulted in lower F₁ and F₂ body weights at study termination. Slight delays in pubertal landmarks in the F₁ offspring were considered secondary to the reduced pup body weights. The no-observed-adverse-effect level (NOAEL) was 15,000 ppm for systemic, reproductive, and neonatal effects based on test article-related effects on body weight and food efficiency, slight decrease in maternal implantation sites and corresponding reduction in live litter size, and reductions in pre-weaning pup body weights at 35,000 ppm.