Subchronic (13-week) oral toxicity study, preceded by an in utero exposure phase, with arachidonate-enriched triglyceride oil (SUNTGA40S) in rats.

Polyunsaturated fatty acids (PUFAs), such as arachidonic acid (ARA) and docosahexaenoic acid (DHA) are natural constituents found in human milk, fish oil or egg yolk. Until recently, infant formulas, though providing the essential fatty acid precursors for these PUFAs, did not contain preformed ARA or DHA. In this study the safety of SUNTGA40S as source of ARA, not only for use in infant formulas but also for nutritional products or food supplements, was evaluated in a subchronic study in Wistar rats, preceded by a 4-week pretreatment period of parental (F(0)) rats and exposure of the F(0) dams throughout mating, gestation and lactation. SUNTGA40S was administered at dietary levels of 0.5%, 1.5% and 5% (wt/wt) adjusted with corn oil to 5.76% added fat. An additional group received 3.65% (wt/wt) SUNTGA40S in conjunction with 2.11% (wt/wt) high DHA Tuna oil, providing an ARA:DHA ratio of 2.7:1. High-fat and low-fat controls received basal diet with or without 5.76% corn-oil supplement. The content, stability and homogeneous distribution of the test substances in the diet were confirmed under study conditions. The administration of SUNTGA40S, with or without DHA oil, did not affect health, growth, fertility or reproductive performance of the parental rats, nor pup characteristics (condition, weight gain, viability, number per litter or sex ratio). In the subchronic study with the offspring (F(1)) rats, no significant differences were found in condition, neurobehavioural observations, ophthalmoscopy, growth, urinalysis or macroscopic and microscopic findings between the test groups and the low-fat or the high-fat controls. In males of the 5% SUNTGA40S and the SUNTGA40S/DHA group, red blood cell counts, haemoglobin concentration and packed cell volume were lower and reticulocytes were slightly higher than in the high-fat and low-fat control groups. Cholesterol, triglycerides and phospholipids in plasma were lower than in the high-fat controls in both sexes in the 5% SUNTGA40S and the SUNTGA40S/DHA group and (for triglycerides only) in the 1.5% SUNTGA group. Due to the administration of extra dietary fat, food intake and prothrombin time (males only) were lower and alkaline phosphatase activity was higher in all the high-fat groups, including the corn-oil controls, as compared to the low-fat controls. The weight of the spleen was higher in males of the 5% SUNTGA40S and the SUNTGA40S/DHA group compared to both the low-fat and the high-fat controls. The effects noted in this study at high dose levels of SUNTGA40S are consistent with previously reported physiological responses to dietary intake of high PUFA containing oils. The present results provide evidence that SUNTGA40S is a safe source of arachidonic acid. Except during lactation when the intake in dams doubled, 5% Suntga40S in the diet was equivalent to an overall intake of approximately 3g/kg body weight/day in F(0) and F(1) animals.     

Evaluation of single-cell sources of docosahexaenoic acid and arachidonic acid: 3-month rat oral safety study with an in utero phase.

Owing to the presence of the polyunsaturated fatty acids (PUFA) docosahexaenoic acid (DHA) and arachidonic acid (ARA) in human milk and their important biological function, several authorities recommend that they be added to infant formulas. This study assessed the safety of an algal oil rich in DHA and a fungal oil rich in ARA, blended to provide a DHA to ARA ratio similar to human milk. The oil blend was incorporated into diets and fed to rats such that they received 3, 11 and 22 times the anticipated infant exposure to DHA and ARA. Low-fat and high-fat control groups received canola oil. Rats received experimental diets over a premating interval and throughout mating, gestation and lactation. Pups born during this period (F1) consumed treatment diets from weaning for 3 months. Physical observations, ophthalmoscopic examinations, body weight, food intake, clinical chemistry, neurobehavioural evaluations and postmortem histopathology of selected tissues were performed. No statistically significant, dose-dependent adverse effects were seen in reproductive performance or fertility, nor in the neonates from birth to weaning. Mid- and high-dose treated F1 animals exhibited increased white cell count, neutrophil count and blood urea nitrogen; increased liver and spleen weights (absolute and relative to body weight) also were observed. There were no corresponding microscopic findings. The clinical pathology and organ weight differences at these treatment levels represent physiological or metabolic responses to the test substance rather than adverse responses. These single-cell oils produced no adverse effects in rats when administered in utero and for 90 days at dietary levels resulting in exposures up to 22 or 66 times higher than those expected in infant formulas when extrapolated on the basis of diet composition (g/100 Cal) or intake (g/kg body weight), respectively.     

Subchronic and developmental toxicity studies in rats with Ac-Di-Sol croscarmellose sodium

Studies were conducted to evaluate the subchronic and developmental toxicity of Ac-Di-Sol (croscarmellose sodium). In the subchronic study, groups of Sprague-Dawley rats (20/sex/group) received 0 (control), 10000, or 50000 ppm Ac-Di-Sol in the diet for 90 consecutive days (equivalent to 757 and 893 mg/kg/day for males and females fed 10000 ppm, respectively, and to 3922 and 4721 mg/kg/day for males and females fed 50000 ppm, respectively). No mortality, clinical signs of toxicity, or adverse toxicological effects on hematology or serum chemistry parameters, feed consumption, or ophthalmologic examinations were noted in any treatment group. Body weight gain was depressed in high-dose males during the final 3 weeks. The only treatment-related histological lesion noted was moderate renal mineralization at the corticomedullary junction in one high-dose female. This lesion was not considered a specific effect of Ac-Di-Sol, but rather a secondary effect resulting from a potential increase in urinary pH and renal excretion of sodium due to the high intake of sodium associated with Ac-Di-Sol. In the developmental toxicity study, groups of pregnant Sprague-Dawley rats (25/group) received 0 (control), 10000, or 50000 ppm Ac-Di-Sol in the diet on gestational days 6 to 15. No evidence of maternal, fetal, or embryo toxicity was noted. The no-observed-adverse-effect level (NOAEL) for Ac-Di-Sol in both studies exceeds 50000 ppm in the diet, which represents doses of 3922 and 4712 mg/kg/day, for males and females, respectively. The results of these studies demonstrate the low subchronic oral toxicity and developmental toxicity of Ac-Di-Sol, and support the safe use of Ac-Di-Sol in oral applications such as pharmaceuticals, dietary supplements, and sweetener tablets.     

Preclinical safety evaluation in rats using a highly purified ethyl ester of algal-docosahexaenoic acid

Preclinical studies have shown that docosahexaenoic acid (DHA) derived from microalgae (DHASCO®) is neither mutagenic nor toxic in acute, subchronic or developmental tests. DHASCO®, triglyceride oil from the fermentation of Crypthecodinium cohnii, contains 40–50% (400–500 mg/g) of DHA by weight. Martek Biosciences Corporation has developed a concentrated ethyl ester of DHA (900 mg/g) from DHASCO® (MATK-90). A 90-day subchronic safety study with a one-month recovery period using Sprague–Dawley rats included clinical observations, ophthalmic examination, hematology, clinical chemistry, toxicokinetic evaluation, and pathological assessments. Effects of MATK-90 were compared with those produced from DHASCO® and control (corn oil). Doses of MATK-90 (1.3, 2.5 and 5.0 g/kg/day) and DHASCO® (5.0 g/kg/day = 2 g of DHA) were administered once-daily by oral gavage at a volume of 10 mL/kg. The corn oil was also administered by oral gavage (10 mL/kg/day). There were no treatment-related adverse effects in any of the parameters measured at doses of ?2.5 g/kg/day of MATK-90 or at 5.0 g/kg/day of DHASCO®. In the mesenteric lymph node, marked macrophage infiltration was observed in 3 of 19 animals in the 5.0 g/kg/day MATK-90 treatment group and was considered to be adverse. The no-observable-adverse-effect level (NOAEL) for MATK-90 under the conditions of this study was 2.5 g/kg/day.     

One-generation reproductive toxicity study of DHA-rich oil in rats

Polyunsaturated fatty acids, including docosahexaenoic acid (DHA), are natural constituents of the human diet. DHA-algal oil is produced through the use of the marine protist, Ulkenia sp. The reproductive toxicity of DHA-algal oil was assessed in a one-generation study. Rats were provided diets containing DHA-algal oil at concentrations of 1.5, 3.0, or 7.5%, and the control group received a diet containing 7.5% corn oil. Males and females were treated for 10 weeks prior to mating and during mating. Females continued to receive test diets during gestation and lactation. In parental animals, clinical observations, mortality, fertility, and reproductive performance were unaffected by treatment. Differences in food consumption, body weight, and liver weight in the treated groups were not considered to be due to an adverse effect of DHA-algal oil. Spleen weight increases in treated animals were associated with extramedullary hematopoiesis. Yellow discoloration of abdominal adipose tissue was observed in rats from the high-dose group, and histological examination revealed steatitis in all treated parental groups. Exposure to DHA-algal oil did not influence the physical development of F(1) animals. These results demonstrate that DHA-algal oil at dietary concentrations of up to 7.5% in rats does not affect reproductive capacity or pup development.     

Two-generation reproductive toxicity study of the flame retardant hexabromocyclododecane in rats

Male and female rats were fed a diet containing flame retardant hexabromocyclododecane (HBCD) at 0, 150, 1500 or 15,000 ppm 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. The mean daily intakes of HBCD during the whole period of administration were 10.2, 101 and 1008 mg/kg bw in F0 males, 14.0, 141 and 1363 mg/kg bw in F0 females, 11.4, 115 and 1142 mg/kg bw in F1 males, and 14.3, 138 and 1363 mg/kg bw in F1 females for 150, 1500 and 15,000 ppm, respectively. The incidence of rats with decreased thyroid follicles size was increased in F0 and F1 males and females at 1500 ppm and higher. Serum TSH levels were increased in F0 and F1 females at 1500 ppm and higher, and serum T4 levels were decreased in F0 males and females at 15,000 ppm. The number of the primordial follicles in the ovary of F1 females was reduced at 1500 ppm and higher. There were increases in the absolute and relative weights of the liver in male adults and male and female weanlings at 1500 ppm and higher, and in female adults at 15,000 ppm, and of the thyroid in male and female adults at 15,000 ppm. Decreased body weight and body weight gain associated with reduced food consumption were found in F1 males and females at 15,000 ppm. Decreases were found in the viability index of F2 pups and the body weight of male F1 and F2 pups and female F2 pups at 15,000 ppm. In F2 pups, there were low incidences of the completion of eye opening in males at 15,000 ppm and in females at 1500 ppm and higher, and of completed mid-air righting in females at 15,000 ppm. The data indicate that the NOAEL of HBCD in this study was 150 ppm (10.2mg/kg bw/day). The estimated human intake of HBCD is well below the NOAEL in the present study.     

One-generation reproductive toxicity study of DHA-rich oil in rats

Polyunsaturated fatty acids, including docosahexaenoic acid (DHA), are natural constituents of the human diet. DHA-algal oil is produced through the use of the marine protist, Ulkenia sp. The reproductive toxicity of DHA-algal oil was assessed in a one-generation study. Rats were provided diets containing DHA-algal oil at concentrations of 1.5, 3.0, or 7.5%, and the control group received a diet containing 7.5% corn oil. Males and females were treated for 10 weeks prior to mating and during mating. Females continued to receive test diets during gestation and lactation. In parental animals, clinical observations, mortality, fertility, and reproductive performance were unaffected by treatment. Differences in food consumption, body weight, and liver weight in the treated groups were not considered to be due to an adverse effect of DHA-algal oil. Spleen weight increases in treated animals were associated with extramedullary hematopoiesis. Yellow discoloration of abdominal adipose tissue was observed in rats from the high-dose group, and histological examination revealed steatitis in all treated parental groups. Exposure to DHA-algal oil did not influence the physical development of F₁ animals. These results demonstrate that DHA-algal oil at dietary concentrations of up to 7.5% in rats does not affect reproductive capacity or pup development.     

Toxicity evaluation of petroleum blending streams: reproductive and developmental effects of light catalytic cracked naphtha distillate in rats

A distillate of light catalytic cracked naphtha (CAS number 64741-55-5, LCCN-D), administered by inhalation, was tested for reproductive and developmental toxicity in Sprague-Dawley rats, following a modified OECD Guideline 421, Reproductive/Developmental Toxicity Screening Protocol. LCCN-D was administered as a vapor, 6 h/d, 7 d/wk at target concentrations of 0, 750, 2500 or 7500 ppm to female rats for approximately 7 wk from 2 wk prior to mating, during mating through gestational d 19, and to males beginning 2 wk prior to mating for 8 consecutive weeks. Dams and litters were sacrificed on postnatal d 4, and males were sacrificed within the following week. Parental systemic effects observed at the 7500 ppm exposure level were increased kidney weights and relative liver weights in males and increased spleen weights in high-dose females. Livers and spleens from rats in the high-dose group were normal in appearance at necropsy. IncreaSed kidney weights in high-dose males were indicative of male-rat-specific light hydrocarbon nephropathy. No test-related microscopic changes were observed in the reproductive organs or nasal turbinate tissues of either sex. Reproductive performance was unaffected by treatment with LCCN-D. Fertility index was > or =90% in all dose groups. There were no exposure-related differences in implantation sites and live pups per litter, and no gross abnormalities were observed. Pups born from treated dams showed comparable body weights and weight gains to controls. The viability index on postpartum d 4 was > or =97%; the high-dose group had more male than female pups at birth and at d 4 postpartum. Under the conditions of this study, the no-observable-adverse-effect level (NOAEL) for exposure to light catalytic cracked naphtha distillate for parental toxicity was 2500 ppm and the NOAEL for reproductive performance and developmental toxicity was 7500 ppm.     

Genotoxicity and subchronic toxicity studies of DHA-rich oil in rats

Polyunsaturated fatty acids, including docosahexaenoic acid (DHA), are natural constituents of the human diet. DHA-algal oil is produced through the use of the non-toxigenic and non-pathogenic marine protist, Ulkenia sp. The safety of DHA-algal oil was assessed in a subchronic toxicity study and in genotoxicity studies. In a 90-day study, rats were orally administered water or DHA-algal oil at concentrations of 0, 500, 1000, and 2000 mg/kg in combination with 2000, 1500, 1000 or 0 mg/kg DHA-containing fish oil, respectively. Additional animals were administered water, 2000 mg/kg DHA-algal oil, or 2000 mg/kg fish oil for 90 days, followed by a 4-week recovery phase. No treatment-related effects were observed in clinical observations, food and water consumption, mortality, gross pathology, and histopathology. Increased body weights and liver weights in oil-treated groups were attributed to the large lipid load and were not regarded as toxicologically significant. Furthermore, no treatment-related differences in the measured parameters between the DHA-algal oil and fish oil groups were detected. In genotoxicity experiments, DHA-algal oil exerted no mutagenic activity in various bacterial strains, nor did it induce chromosomal aberrations in Chinese hamster fibroblast cells. These results support the safety of DHA-algal oil as a dietary source of DHA.     

TOXICITY EVALUATION OF PETROLEUM BLENDING STREAMS: REPRODUCTIVE AND DEVELOPMENTAL EFFECTS OF LIGHT CATALYTIC CRACKED NAPHTHA DISTILLATE IN RATS

A distillate of light catalytic cracked naphtha (CAS number 64741-55-5, LCCN-D), administered by inhalation, was tested for reproductive and developmental toxicity in Sprague-Dawley rats, following a modified OECD Guideline 421, Reproductive/ Developmental Toxicity Screening Protocol. LCCN-D was administered as a vapor, 6 h/ d, 7 d/wk at target concentrations of 0, 750, 2500 or 7500 ppm to female rats for approximately 7 wk from 2 wk prior to mating, during mating through gestational d 19, and to males beginning 2 wk prior to mating for 8 consecutive weeks. Dams and litters were sacrificed on postnatal d 4, and males were sacrificed within the following week. Parental systemic effects observed at the 7500 ppm exposure level were increased kidney weights and relative liver weights in males and increased spleen weights in highdose females. Livers and spleens from rats in the high-dose group were normal in appearance at necropsy. Increased kidney weights in high-dose males were indicative of male-rat-specific light hydrocarbon nephropathy. No test-related m icroscopic changes were observed in the reproductive organs or nasal turbinate tissues of either sex. Reproductive performance was unaffected by treatment with LCCN-D. Fertility index was 90% in all dose groups. There were no exposure-related differences in implantation sites and live pups per litter, and no gross abnormalities were observed. Pups born from treated dams showed comparable body weights and weight gains to controls. The viability index on postpartum d 4 was 97% ; the high-dose group had more male than female pups at birth and at d 4 postpartum. Under the conditions of this study, the no-observable-adverse-effect level (NOAEL) for exposure to light catalytic cracked naphtha distillate for parental toxicity was 2500 ppm and the NOAEL for reproductive performance and developmental toxicity was 7500 ppm.     

Three-generation reproduction study with caprolactam in rats

In a three-generation reproduction study, rats were given caprolactam in the diet of 0, 1000, 5000 and 10,000 ppm. No treatment-related effects were observed in the parental animals with respect to mortality, clinical signs, reproductive performance or gross pathology findings. Consistently lower body weights were noted in the P2 and P3 mid- and high-dose males and females. Consistently lower mean food consumption values were noted in the P2 and P3 mid- and high-dose males and the high-dose females. These differences were generally significant (P less than or equal to 0.05) in the high-dose group of both sexes. Compound-related histopathologic findings noted in the high-dose P1 males consisted of a slight increase in the severity of spontaneous nephropathies, occasionally accompanied by granular casts. The offspring data revealed no treatment-related effect with respect to gross appearance, gross pathology, survival, number of pups, percentage of male pups or kidney weight. Analysis of the offspring body weights on Days 1, 7 and 21 of lactation revealed consistently and generally significant lower mean values in the high-dose male and female animals of all filial generations. The mean body weights of both sexes in the mid-dose group were generally lower than those of the controls. The effects on mean body weight, mean food consumption and the group increases in the severity of nephropathy, accompanied by the presence of granular casts in some animals, are considered to be related to the administration of caprolactam.     

Safety evaluation of Algal Oil from Schizochytrium sp.

The safety of Algal Oil from Schizochytrium sp. was evaluated by testing for gene mutations, clastogenicity and aneugenicity, and in a subchronic 90-day Sprague-Dawley rat dietary study. The results of all genotoxicity tests were negative. The 90-day study involved dietary exposure to 0.5, 1.5, and 5 wt.% of Algal Oil and two control diets: a standard low-fat basal diet and a basal diet supplemented with 5 wt.% menhaden oil (the fish oil control). There were no treatment-related effects of Algal Oil on clinical observations, body weight, food consumption, behavior, hematology, clinical chemistry, coagulation, or urinalysis parameters. Increased mean liver weights and alveolar histiocytosis were observed in both the fish oil control and the high-dose Algal Oil-treated animals and were not considered to be adverse. Algal Oil was bioavailable as demonstrated by the dose-related increase of DHA and EPA levels in tissues and plasma. The no observable adverse effect level (NOAEL) for Algal Oil under the conditions of this study was 5 wt.% in the diet, equivalent to an overall average Algal Oil intake of 3250 mg/kg bw/day for male and female rats. Based on the body surface area, the human equivalent dose is about 30 g Algal Oil/day for a 60 kg adult.     

Influence of corn oil and diet on reproduction and the kidney in female Sprague-Dawley rats.

The purpose of this study was to investigate the influence of corn oil administration on gestation, parturition, and lactation in rats, in conjunction with diets differing in composition of nutrients. Rats were divided into two groups, each fed different commercial pellets for rodents, CA-1 or CE-2, different from each other mainly in the source of protein. Female Sprague-Dawley rats in both diet groups were administered 0 (untreated control), 2, or 10 ml corn oil/kg body weight by gavage during the premating period (2 weeks), the mating period, the gestation period, and the lactation period (until day 3 of lactation). Food consumption of both the 10 ml/kg corn oil groups was significantly reduced throughout the study. Body weight gain in the 10 ml/kg corn oil group fed the CA-1 diet was significantly reduced on days 0 through 4 of lactation. Neither mating nor fertility indices were affected, and no clinical signs were observed during the gestation period in any groups. Several dams in the 10 ml/kg corn oil group fed the CA-1 diet, however, showed abnormal conditions after parturition, and three dams became moribund. Pup viability was also reduced in this group. Histopathologic examination of the kidneys of dams in the 10 ml/kg corn oil group fed the CA-1 diet revealed severe lesions in the proximal tubular epithelium, i.e., necrosis and fatty degeneration. Females in any group fed the CE-2 diet showed neither abnormal condition after parturition nor any severe lesions in the kidney. These data show that the combination of corn oil and diet with a particular constitution may cause adverse effects on the renal tubules in pregnant and/or lactating rats, suggesting that corn oil gavage as a vehicle can be a confounding factor in the reproductive toxicity studies, depending on the diet.     

Chronic Toxicity, Oncogenic Potential, and Reproductive Toxicity of p-Nitroaniline in Rats

Chronic Toxicity, Oncogenic Potential, and Reproductive Toxicityof p-Nitroaniline in Rats. NAIR, R. S., AULETTA, C. S., SCHROEDER,R. E., AND JOHANNSEN, F. R. (1990). Fundam. Appl. Toxicol 15,607–621. Dose levels for these studies were selected mainlyon the basis of subchronic studies, although consideration wasalso given to workplace exposure levels and proposed mechanismof tumor formation with structurally similar compounds. Forthe chronic study, groups of 60 male and 60 female Sprague—DawleyCD (Registered Trademark of Charles River Breeding Laboratories,Portage, Ml) rats were given 0, 0.25, 1.5, or 9.0 mg/kg/daypara-nitroaniline (PNA) by gavage in corn oil for a period of2 years. Parameters monitored included clinical observations,ophthalmoscopic exams, body weights, food consumption, hematology,clinical chemistry, and urinalysis at regular intervals throughoutthe study. All gross lesions and over 40 tissues were examinedhistologically for all control and high-dosage-level animals.Gross lesions, spleens, and livers of low- and mid-dosage groupswere also examined histologically. For the reproduction study,groups of 15 male and 30 female rats, designated as F₀ generation,were given PNA at the same levels as the chronic study for 14weeks prior to mating and during mating, gestation, and lactation.Selected groups of 15 male and 30 female rats of the F₁ generationreceived the same dose of PNA for 18 weeks prior to mating andduring mating, gestation, and lactation. F₂ pups were observedthrough weaning at which time they were euthanized. Observationsmade during the study included body weights, food consumption,mating and fertility indices, pup and litter survival indices,and histopathology of selected tissues. In the chronic study,except for a slight decrease in survival of high-dose male ratslate in the study, survival in all treated groups was comparableto controls. Blood methemoglobin levels were elevated in themid- and high-dosage groups, while slight anemia was observedin the high-dosage group also. Spleen weights were significantlyincreased in the high-dosage groups. An accumulation of brownpigment was observed in the cytoplasm of the sinusoidal macrophagesor littoral cells of the liver and in the reticuloendothelialcells of the spleen. No treatment-related increase in tumorincidence was observed. In the reproduction study, no consistentpattern of effect from treatment between the F₀ and F₁ generationwas seen in mating, pregnancy, or fertility indices. Thus, administrationof PNA at levels which produced significant methemoglobin-emiaand low-level anemia in the rat and histological changes inthe spleen produced no tumors or reproducible effects on reproductiveperformance.     

Safety evaluation of sources of docosahexaenoic acid and arachidonic acid for use in infant formulas in newborn piglets.

Human milk provides small quantities of preformed docosahexaenoic acid (DHA) and arachidonic acid (ARA), usually less than 1% of total fatty acids. Vegetable oil blends commonly used in infant formulas have, until recently, provided the essential fatty acid precursors for these long-chain polyunsaturated fatty acids (LCPUFA), but no preformed DHA and ARA. This study evaluated the safety of ingredient sources of DHA and ARA for use in infant formulas in a neonatal piglet model. Newborn piglets were allowed to suckle for 3 days and then divided into 4 feeding groups of 6 males and 6 females. Piglets were bottle-fed at frequent feeding intervals until 19 days of age. The composition of the piglet formulas was modeled after standard milk-based formulas for human infants while meeting nutritional requirements for piglets. Formulas were a control formula (no added DHA or ARA), a DHA formula providing 55 mg DHA/100 Cal, an ARA formula providing 96 mg/100 Cal ARA, and a DHA+ARA formula providing 34 mg DHA and 62 mg ARA/100 Cal. All formulas were equal in fat content and provided approximately 1000 Cal/l. The ARA-rich oil was from a fermentation product of Mortierella alpina (40 wt.% fatty acids as ARA) and DHA was from high DHA tuna oil (25 wt.% fatty acids as DHA). There were no test article related effects of DHA and/or ARA indicative of an adverse health consequence to the animals seen in the clinical signs, body weights, food consumption, clinical chemistry, hematology, organ weights or gross or histopathology. The findings in this neonatal animal study support the safety of these ingredient oil sources of DHA and ARA for use in infant formulas.     

A combined subchronic (90-day) toxicity and neurotoxicity study of a single-cell source of docosahexaenoic acid triglyceride (DHASCO oil).

Docosahexaenoic acid (DHA), a 22-carbon long-chain polyunsaturated fatty acid of the omega-3 family, is a major structural component of neural membranes and is a particularly important nutrient during infant development. New safe and well-defined sources of DHA are required for infant formula fortification and dietary supplementation. DHASCO oil is an algal-derived triglyceride containing 40-50% DHA. Previous studies have shown that DHASCO oil is neither mutagenic nor toxic in acute or 28-day subchronic tests. To further establish the safety of this oil, a 90-day subchronic toxicity study in rats which included haematology, clinical chemistry, pathology and ophthalmologic, neurobehavioural and neuropathological assessments, using doses of 0.5 and 1.25g/kg body weight/day was performed. There were no treatment-related adverse effects in any of the parameters measured at either dose. Based on these results, the no-adverse-effect level (NOAEL) for DHASCO oil under the conditions of this study corresponds to the highest dose level. The DHA in the DHASCO oil was bioavailable, resulting in significant elevations in the levels of this fatty acid in liver, heart and brain after 90 days of administration. In conclusion, this 90-day subchronic toxicity study provides additional evidence that DHASCO oil is a safe and bioavailable source of dietary DHA.     

Potentiation of the teratogenic effects and altered disposition of diphenylhydantoin in mice fed a purified diet

The effect of a standardized purified diet (AIN-76) on the teratogenic response to diphenylhydantoin (DPH) was studied in mice. Mice were fed either the purified diet or Purina Rodent Laboratory Chow for 2-4 weeks prior to mating, and were treated with either saline or 50 mg/kg of DPH on Days 12, 13, and 14 of gestation (copulatory plug = Day 0). The teratogenic response to DPH was found to be markedly potentiated in mice fed the purified diet (75% cleft palate) as compared to mice fed rodent chow (21% cleft palate). The potentiated teratogenic response to DPH correlated with markedly higher plasma DPH levels in pregnant mice fed the purified diet, indicating that the disposition of DPH was impaired. These effects were attributed to a decreased basal level of drug-metabolizing enzymes in mice fed the purified diet, as indicated by markedly prolonged hexobarbital sleeping times. Modifications of the purified diet, which included the replacement of soluble carbohydrate (50% sucrose) in the purified diet with either cornstarch or casein, did not alter the high incidence of cleft palate. A reduction in the incidence of cleft palate was observed, however, when corn oil in the purified diet was replaced with linseed oil. The replacement of corn oil with linseed oil in the purified diet also restored the hexobarbital sleeping times to those observed in mice fed rodent chow. It is concluded that mice fed purified diets have decreased basal levels of drug-metabolizing activity that alter the disposition of DPH and, as a consequence, potentiate its teratogenic effects.     

Safety evaluation of DHA-rich Algal Oil from Schizochytrium sp.

The safety of DHA-rich Algal Oil from Schizochytrium sp. containing 40–45 wt% DHA and up to 10 wt% EPA was evaluated by testing for gene mutations, clastogenicity and aneugenicity, and in a subchronic 90-day Sprague–Dawley rat dietary study with in utero exposure, followed by a 4-week recovery phase. The results of all genotoxicity tests were negative. In the 90-day study, DHA-rich Algal Oil was administered at dietary levels of 0.5, 1.5, and 5 wt% along with two control diets: a standard low-fat basal diet and a basal diet supplemented with 5 wt% of concentrated Fish Oil. There were no treatment-related effects of DHA-rich Algal Oil on clinical observations, body weight, food consumption, behavior, hematology, clinical chemistry, coagulation, or urinalysis. Increases in absolute and relative weights of the liver, kidney, spleen and adrenals (adrenals and spleen with histological correlates) were observed in both the Fish Oil- and the high-dose of DHA-rich Algal Oil-treated females and were not considered to be adverse. The no observed adverse effect level (NOAEL) for DHA-rich Algal Oil under the conditions of this study was 5 wt% in the diet, equivalent to an overall average DHA-rich Algal Oil intake of 4260 mg/kg bw/day for male and female rats.     

Subchronic and developmental toxicity studies of n-butyl propionate vapor in rats

Two inhalation studies were conducted to evaluate the possible subchronic and developmental toxic effects of n-butyl propionate. In the subchronic study, Sprague-Dawley rats (15/sex/group) were exposed to 0, 250, 750, or 1500 ppm vapor for 6 h/d, 5 d/wk for 13 wk. Five of the rats per sex per group were held after the final exposure for an 8-wk recovery period. Standard parameters of subchronic toxicity were measured throughout the study, and at the end of exposure and recovery periods, necropsies were performed, organs weighed, and tissues processed for microscopic examination. Exposure did not produce marked treatment-related deaths or adversely affect clinical signs, hematology, clinical chemistries, organ weights, or the histology of major visceral organs. The only systemic toxic effects were significant decreases in body weight, body weight gain, and feed consumption that occurred in 1500 ppm group rats. Morphologic changes were limited to the nasal cavity as evidenced by a concentration-related increased incidence and severity of olfactory epithelium degeneration in rats of the 750 and 1500 ppm groups. These degenerative microscopic alterations were primarily confined to the olfactory epithelium within the dorsal portion of the medial meatus, with lesser involvement of the olfactory mucosae overlying the tips of some of the adjacent ethmoturbinates. Both the systemic and nasal cavity effects appeared reversible after exposure ceased. In the developmental toxicity study, pregnant Sprague-Dawley rats (24/group) were exposed to 0, 500, 1000, or 2000 ppm vapor for 6 h/d on gestation d 6-15 and sacrificed on gestation d 20. All treatment-group dams exhibited significant reductions in body weight, body weight gain, and feed consumption. Gestational parameters were equivalent across all groups and there were no treatment-related developmental or teratogenic effects. The no-observed-adverse effects levels (NOAELs) determined for nbutyl propionate were 250 ppm for subchronic toxicity (based on the olfactory epithelium degeneration) and 22000 ppm for developmental toxicity (no developmental effects at top dose tested). Under the conditions of this study, a NOAEL was not determined for maternal toxicity.     

Reproductive Toxicity Evaluation of Acetaminophen in Swiss CD-1 Mice Using a Continuous Breeding Protocol

Acetaminophen (APAP) was evaluated for reproductive toxicityin Swiss CD-1 mice using a continuous breeding protocol. APAPwas administered in the diet at 0, 0.25, 0.5, and 1.0% (w/w),which represented average daily intakes of 0, 357, 715, and1430 mg APAP/kg/day, respectively. Exposure of parental (P)breeding pairs to 1% APAP in the diet for 14 weeks during cohabitationsignificantly decreased the number of litters per pair, andreduced, although not significantly, the number of live pupsper litter. Importantly, 6 of 19 high-dose P pairs failed toproduce a fifth litter, and this fully accounted for the diminishednumber of litters in this group. In addition, the fifth litterthat was produced by the 13 high-dose P pairs averaged onlyabout 9 live pups per litter, which correspondingly reducedthe overall group average for this parameter. In comparison,the control and two lower-dose P pairs produced 11 or 12 livepups per litter on average. Although the birth weights for F₁pups in the final litter were unaffected by prenatal APAP exposure,postnatal growth was adversely affected as evidenced by retardedweight gain as measured at 28 and 74 ± 10 days of agefor all three dietary levels. At 1% APAP this weight gain effectwas more pronounced at Day 28 than at Day 74 ± 10, suggestingthat nursing pups may have been exposed to higher concentrationsor may be more sensitive to APAP and/or an active metabolitethan were the young adults. A mating trial of F₁ pairs at 74± 10 days of age indicated that mating, fertility, andreproductive performance were normal, except that the birthweight of F₂ pups was significantly depressed at 1% APAP. Thislatter effect may have been attributable to maternal toxicityin that body weight and relative pituitary weight were significantlydecreased, and relative brain and liver weight significantlyincreased, in high-dose F₁ females. In addition, body weightwas significantly reduced in the high-dose F₁ males at necropsy.No treatmentrelated effects on reproductive organ weights andno gross or histological changes in the reproductive tissuesof the high-dose F₁ male and female mice were observed. Continuousexposure of F₁ males at 1% APAP, however, significantly increasedthe percentage of abnormal epididymal sperm, while sperm densityand percentage of motile sperm were unaffected. Collectively,these data indicated that continuous exposure to 1% APAP viathe diet (1.43 g/kg/day) led to cumulative effects on reproductionin the P pairs, to retarded growth and abnormal sperm in theF₁ mice, and to reduced birth weight of F₂ pups.