90-day Toxicity Study Research Articles

Scientific Opinion on the Safety of smoke flavour Primary Product Zesti Smoke Code 10 ‐ 2011 Update

The EFSA CEF Panel updated the safety assessment of the smoke flavouring Primary Product Zesti Smoke Code 10 based on evaluation of a new 90-day dietary rat toxicity study. In 2009, the Panel concluded that the margins of safety (range: 5–32) for this Primary Product were insufficient and that the proposed uses and use levels were of safety concern. In this new application, the petitioner proposed to reduce upper use levels and to avoid use in composite foods. The chemical data provided demonstrate that the material tested in the new 90-day study is representative of the Primary Product evaluated by EFSA in 2009. From the new 90-day study the Panel identified a NOAEL for Zesti Smoke of 664 mg/kg bw/day, based on the reduced weight gain and white blood cell changes in female rats elicited by a high dose level. The Panel considers that this NOAEL supersedes that of 134 mg/kg bw/day identified in an earlier 90-day study, since the new study's experimental design permits a more accurate identification of the NOAEL. When use of Zesti Smoke based on upper use levels is considered in all food sources (18 categories), estimated dietary intake ranges from 6.7 to 8.4 mg/kg bw/day, depending on the exposure assessment model used. When use (upper use levels) is assumed in traditionally smoked food only, exposure is estimated to be 5.0-7.7 mg/kg bw/day. The margins of safety for this Primary Product, i.e. ratio between the NOAEL from the new 90-day study and the intake estimates, are either 79–99 (all food sources) or 86–133 (traditionally smoked sources only). The Panel considers that these margins of safety are insufficient, since they are based on a 90-day toxicity study, and data are lacking on reproductive, developmental and long-term toxicity. In line with its conclusions of 2009, the Panel concludes that the Primary Product Zesti Smoke Code 10 at the proposed uses and use levels is of safety concern.

Scientific opinion on the safety of smoke flavour Primary Product Fumokomp ‐ 2011 Update

The EFSA CEF Panel updated the safety assessment of the smoke flavouring Primary Product Fumokomp based on evaluation of a new 90-day dietary toxicity study. In 2009, EFSA concluded that the toxicological data then available did not enable the safety of Fumokomp to be established. The new chemical data provided, though limited, indicate that the composition of test material Fumokomp no. 6482 is comparable to that of the Primary Product and of the material previously evaluated by EFSA. From the new 90-day toxicity study in rats the Panel identified a NOAEL for Fumokomp of 400 mg/kg bw/day, since the changes in kidney and liver weight, urine volume and specific gravity in males receiving either 100 or 400 mg/kg bw/day, together with some changes in females receiving 400 mg/kg bw/day, and coupled with minor haematological and clinical chemistry changes were considered to be adaptive in nature and non-adverse. Based on the 2009 estimates and depending on the exposure assessment model used, dietary exposure (in mg/kg bw/day) is 0.13-0.20 (upper use levels) or 0.08-0.13 (normal use levels) from use of Fumokomp in all food sources (18 categories), and 0.05-0.09 (upper use levels) or 0.03-0.06 (normal use levels) from its use in traditionally smoked foods only. If Fumokomp is present in all food sources, the margins of safety as compared to the NOAEL of 400 mg/kg bw/day amount to 2,000–3,077 or 3,077- 5,000 for the intake estimates based on the upper or normal use levels, respectively. When assuming the use of Fumokomp in traditionally smoked products only, the margins of safety amount to 4,400–8,000 or 6,666–13,333 for the intake estimates based on the upper or normal use levels, respectively. Given the margin of safety of at least 2,000 and the fact that this margin of safety is based on a conservative exposure estimate and a NOAEL that is the highest dose level tested, the Panel concludes that the use of Primary Product Fumokomp at the proposed uses and use levels is not of safety concern.

20 kGy 감마선으로 조사된 겨우살이 냉수 추출물의 90일 반복투여 독성평가

Traditionally, mistletoes have been used as immunostimulant for the management of certain diseases such as cancer with high profile immune depleting potentials. In order to examine the safety of the 20 kGy irradiated cold water extract powder of mistletoes, we performed a 90-day repeated-dose toxicity study with ICR mice. The mice were treated with daily doses of the 20 kGy irradiated cold water extract powder of mistletoes by gavage at 0, 20, 100, and 500 mg/kg/day for 90 consecutive days. We recorded clinical signs of toxicity, body weight, organ weights, histological changes in target organs, hematology, and clinical blood chemistry analysis data for all mice. There were no significant changes in body and organ weights during the experimental period. The hematological analysis and clinical blood chemistry data revealed no toxic effects from the 20 kGy irradiated cold water extract powder of mistletoes. Pathologically, neither gross abnormalities nor histopathological changes were observed between the control and treated mice of both sexes. Collectively, these data suggest that the 20 kGy irradiated cold water extract powder of mistletoes have a high margin of safety.

A Molecular and Phenotypic Integrative Approach to Identify a No-Effect Dose Level for Antiandrogen-Induced Testicular Toxicity

The safety assessment of chemicals for humans relies on identifying no-observed adverse effect levels (NOAELs) in animal toxicity studies using standard methods. With the advent of high information content technologies, especially microarrays, it is pertinent to determine the impact of molecular data on the NOAELs. Consequently, we conducted an integrative study to identify a no-transcriptomic effect dose using microarray analyses coupled with quantitative reverse transcriptase PCR (RT-qPCR) and determined how this correlated with the NOAEL. We assessed the testicular effects of the antiandrogen, flutamide (FM), in a rat 28-day toxicity study using doses of 0.2-30 mg/kg/day. Plasma testosterone levels and testicular histopathology indicated a NOAEL of 1 mg/kg/day. A no-effect dose of 0.2 mg/kg/day was established based on molecular data relevant to the phenotypic changes. We observed differential gene expression starting from 1 mg/kg/day and a deregulation of more than 1500 genes at 30 mg/kg/day. Dose-related changes were identified for the major pathways (e.g., fatty acid metabolism) associated with the testicular lesion (Leydig cell hyperplasia) that were confirmed by RT-qPCR. These data, along with protein accumulation profiles and FM metabolite concentrations in testis, supported the no-effect dose of 0.2 mg/kg/day. Furthermore, the microarray data indicated a dose-dependent change in the fatty acid catabolism pathway, a biological process described for the first time to be affected by FM in testicular tissue. In conclusion, the present data indicate the existence of a transcriptomic threshold, which must be exceeded to progress from a normal state to an adaptative state and subsequently to adverse toxicity.

Hepatic effects of a highly purified 2,2′,3,4,4′,5,5′-heptachlorbiphenyl (PCB 180) in male and female rats

PCB 180 (2,2′,3,4,4′,5,5′-heptachlorobiphenyl) is a persistent and accumulating polychlorinated biphenyl abundantly present in food and the environment. In this study, we used highly purified PCB 180 (dioxinlike impurities: 2.7 ng TEQ WHO/g PCB 180) in a 28-day toxicity study in young adult Sprague-Dawley rats. Male and female rats were given total doses of 3, 10, 30, 100, 300, 1000 or 1700 mg/kg b.w. PCB 180 by gavage. Increased liver weights were observed at ≥300 mg/kg b.w. in males and females. No increases in serum ALT or ALP activities were found. A significant increase in liver pentoxyresorufin O-dealkylase (PROD) activity was found in males at ≥10 mg/kg b.w. and in females at ≥30 mg/kg b.w. In both genders, a significant induction of hepatic 7-ethoxyresorufin O-deethylase (EROD) activity was also observed in males at ≥10 mg/kg b.w. and in females at ≥300 mg/kg b.w. Western blotting showed that mainly cytochromes P450 (CYPs) 2B1/2 and 3A1 were induced while slight effects were seen on CYP1A1, CYP1A2 and CYP1B1. However, no induction of CYP1A1, 1A2 and 1B1 was found on the mRNA level, except for a slight effect in females at 1000 mg/kg b.w. Furthermore, hepatic UDP-glucuronosyltransferases (UGTs) 1A1 and 1A6 were markedly induced in males and slightly induced in females. The hepatic concentrations of apolar retinoids were decreased in males at ≥30 mg/kg b.w. and in females at ≥300 mg/kg b.w. Taken together our findings show that pure PCB 180 leads to hepatic changes in a dose range which did not cause CYP1A1 induction but causes centrilobular liver hypertrophy, affects drug-metabolizing enzymes involved in the metabolism of exogenous and endogenous substrates and leads to changes in liver retinoid levels. A benchmark dose (BMD) approach is presented in order to model lowest effective dose levels for these effects. Comparison of PCB 180 liver level related to BMDL 5 for hepatic hypertrophy in rats with human data on ‘total′ hepatic PCB levels in individuals without history of specific exposure suggests a relatively small margin of tissue burden in the range of 37-fold. Our results show that the highly pure non dioxin-like PCB 180 exerted strong effects different to dioxin-like compounds and that the low TEQ contamination allowed a characterization of the PCB as non-dioxinlike.

Diuron exposure induces systemic and organ-specific toxicity following acute and sub-chronic exposure in male Wistar rats

Diuron [3-(3,4-dichlorophenyl)-1,1-dimethylurea] is a substitute urea herbicide widely used on agricultural crops with potential mutagenic, teratogenic, reproductive and carcinogenic effects. Nonetheless, its toxic potential on the immune system needs a detailed assessment. Thus, in order to evaluate the adverse effect of this herbicide on lymphohematopoietic organs and macrophage activity, male Wistar rats were orally treated with Diuron at 125, 1250 and 2500 ppm for 14, 28 or 90 days. General signs of toxicity were observed in Diuron-treated groups (1250 and 2500 ppm), including reduced food intake and body weight gain, as well as higher relative weights for spleen, kidneys and liver (28 and 90-day toxicity studies) and elevated serum levels of ALT, albumin, total protein, creatinine and urea (28-day toxicity study). Diuron exposure caused a severe depletion of splenic white pulp compartments and cellularity, followed by a decreased number of CD4 + T lymphocytes, increased extramedullary hematopoiesis and deposition of hemosiderin in red pulp. Despite alteration in macrophage spreading, the macrophagic activity was not significantly affected by the herbicide. Under these experimental conditions, the results suggest that Diuron exerts systemic and target-organ toxicity, mainly at higher concentration.

Chronic Progressive Nephropathy in Male F344 Rats in 90-Day Toxicity Studies

The occurrence and severity of spontaneous chronic progressive nephropathy (CPN) in control male F344 rats as well as the frequency of treatment-related CPN exacerbation were histopathologically reevaluated. A series of 43 National Toxicology Program (NTP) 90-day toxicity studies comparing the influence of NIH-07 or NTP-2000 diets was examined. Relationships between the histopathologic findings at 90 days and renal tubule proliferative lesions recorded in subsequent 2-year bioassays for 24 chemicals were statistically analyzed. CPN lesions were observed in 100% of the control male rats regardless of diet, but CPN was more severe in control rats fed NIH-07. Approximately one-third of the 90-day studies demonstrated a treatment-related exacerbation of CPN severity, which was independent of diet. For chemicals that proceeded to 2-year bioassays, all studies with a statistically significant increase in renal tubule tumors (RTT) at 2 years had treatment-related exacerbation of CPN in the 90-day and 2-year studies. These findings indicate that CPN occurs ubiquitously in young male F344 rats and that treatment-related exacerbation of CPN in 90-day studies is a relatively common occurrence, having the potential to be predictive of an increased incidence of RTT in subsequent 2-year bioassays.

Influence of coefficient of variation in determining significant difference of quantitative values obtained from 28-day repeated-dose toxicity studies in rats

In order to understand the influence of coefficient of variation (CV) in determining significant difference of quantitative values of 28-day repeated-dose toxicity studies, we examined 59 parameters of 153 studies conducted in accordance with Chemical Substance Control Law in 12 test facilities. Sex difference was observed in 12 parameters and 10 parameters showed large CV in females. The minimum CV was 0.74% for sodium. CV of electrolytes was comparatively small, whereas enzymes had large CV. Large differences in CV were observed for major parameters among 7-8 test facilities. The changes in CV were grossly classified into 11. Our study revealed that a statistical significant difference is usually detected if there is a difference of 7% in mean values between the groups and the groups have a CV of about 7%. A parameter with a CV as high as 30% may be significantly different, if the difference of the mean between the groups is 30%. It would be ideal to use median value to assess the treatment-related effect, rather than mean, when the CV is very high. We recommend using CV of the body weight as a standard to judge the adverse effect level.

Metabonomics study of transgenic Bacillus thuringiensis rice (T2A-1) meal in a 90-day dietary toxicity study in rats

Rice is one of the most important staple foods in the world. The Cry2A gene was inserted into the rice genome to help the plant combat insects. As the unintended effects of the genetically modified (GM) organisms are the most important barriers to the promotion of GM organisms, we have carried out a useful exploration to establish a new in vivo evaluation model for genetically modified foods by metabonomics methods. In this study, the rats were fed for 90 days with the GM and NON-GM rice diets. The changes in metabolites of the urine were detected using (1)H-NMR. The metabonomics were analyzed to see whether the GM rice can induce the metabolite changes in the rats' urine when compared with the NON-GM rice group. The multivariate analysis and ANOVA were used to determine the differences and the significance of differences respectively, and eventually we concluded that these differences did not have a biological significance. The conclusion of the metabonomics was comparable with that from the traditional method. As a non-invasive and dynamic monitoring method, metabonomics will be a new way of assessing the food safety of GM foods.

A toxicological investigation of a celery seed extract having anti-inflammatory activity

An extract of the seed from celery (Apium graviolens) (CSE), and fractions thereof, have been found to possess anti-inflammatory activity, gastro-protective activity, and anti-Helicobacter pylori activity. In view of the potential for employing these extracts for therapeutic use, toxicological investigations were undertaken with an alcoholic extract (A-CSE) which has previously been shown to have the above pharmacological activities. A 28-day toxicity study was performed in rats according to Good Laboratory Practice (GLP) conditions. Eighteen adult male and 18 adult female rats were randomly assigned to 3 treatment groups of 6 rats/sex/group and were dosed orally with A-CSE of 0, 150 or 5,000mg/kg per day. Daily observations of vital signs and body weights were recorded and ophthalmological investigations were performed. At autopsy, the principal organs were weighed and sections collected for histological analysis. Serum and urine samples were collected at termination for routine clinical chemistry. Under non-GLP conditions alpha-2-μ-globulin immunohistochemistry was performed on kidney tissues and hepatic cytochrome P450 protein was determined, as well as, the enzymatic activities of the principal isoforms. All animals survived treatments with no visible or behavioral signs of toxicity being observed during the study. There were no statistically significant differences in body weight gains, body weight gains per day or cumulative absolute body weight gains, for either sex, in any treatment groups when compared with controls. Slightly increased liver weight and liver to body and brain weight ratios were observed in female rats and in liver to body weight ratios in male rats given high dose A-CSE which was a test article effect, but the absence of any microscopic correlates for the liver weight increases suggests that these were not toxicologically significant. Treatment related macroscopic changes were not observed at necropsy and microscopic findings were limited to minimal increases in gastric eosinophils in several male and female rats in the 5,000mg/kg per day treatment groups. Minimal focal degeneration of renal tubules was observed sporadically in both sexes assigned to all treatment groups including control and was consistent with early spontaneous nephropathy of laboratory rats and thus was not considered to represent a pathologic change associated with the test article. Increased serum globulin and phosphorus levels were observed in male rats given 5,000mg/kg per day A-CSE and decreased serum triglycerides levels in female animals given 150 or 5,000mg/kg per day A-CSE. The increase in serum globulin and phosphorus in male animals was small in magnitude and not considered toxicologically significant. The mechanism for the decrease in serum triglycerides in female rats was not apparent. Changes in urinalysis parameters were limited to small decreases in urine pH in female animals in the 150 and 5,000mg/kg per day groups and were not deemed toxicologically significant. Alpha-2-μ-globulin immunohistochemistry was performed on kidney tissues from all animals and found to be within normal physiologic limits. Minor corneal mineralization occurred in some animals from all treatment groups. Cataracts were observed in one in the control and one in an animal that had 5,000mg/kg per day but since the cataracts occurred in the metabolically inactive region of the lens, these were not considered indicative of test article related lesions. There were no changes in total hepatic microsomal protein or in total cytochrome P450 protein. Although male rats appeared to have to higher levels of total microsomal protein than female rats, there appeared to be no treatment effect in either male or female animals. As regards the activity of the various isoforms tested (CYP2B1/2, CYP1A1/2, CYP3A1/2), with the large range of activities detected for each P450 isoform, no clear change in activity or protein were observed, however, these data were not statistically analyzed. These results suggest that there are no toxicologically significant sub-chronic effects of oral A-CSE in rats. The no adverse effect level for systemic toxicity would appear to be 5,000mg/kg per day.

Safety studies on products from whole coffee fruit

The fruit of the coffee plant, Coffea arabica, has high phenolic antioxidant and phytonutrient content and could be a beneficial food ingredient. However, the fruit has historically been discarded for the favored harvesting of the coffee bean alone. CoffeeBerry® products are derived from the whole fruit and include a ground whole powder, a water extract, and a more recently developed water–ethanol extract. The safety of CoffeeBerry® products was evaluated in three genotoxicity studies, three short-term oral toxicity studies, and a 90-day dietary toxicity study. Bacterial mutagenicity studies and a micronucleus test using murine peripheral cells demonstrated that none of the three products showed mutagenic or genotoxic potential. In the short-term studies, despite palatability issues, female rats showed a tolerance for whole powder and ethanol extract at doses up to 8800 mg/kg bw/day. Male rats also exhibited palatability issues and tolerated lower doses of approximately 4000 mg/kg bw/day ethanol extract via gavage and approximately 2100 mg/kg bw/day whole powder or water extract in the diet. When fed in the diet to Sprague–Dawley rats for 90 days, ethanol extract showed no adverse effects at dietary concentrations of up to 5% (approximately 3446 and 4087 mg/kg bw/day for male and female rats, respectively).

Scientific Opinion on the use of calcium lignosulphonate (40–65) as a carrier for vitamins and carotenoids

The Panel on Food Additives and Nutrient Sources added to Food provides a scientific opinion on the safety of calcium lignosulphonate (40–65) when used as a carrier for vitamins and carotenoids intended to be added to foods for colouring and nutrient purposes. Calcium lignosulphonate (40–65) has been evaluated by JECFA and an ADI of 20 mg/kg bw/day was established. Calcium lignosulphonate (40–65) is poorly absorbed following oral administration. From the results obtained in vitro from one bacterial reverse mutation assay and one mammalian chromosomal aberration assay it can be concluded that there is no indication for a genotoxic potential of calcium lignosulphonate (40–65). In a short-term 28-day toxicity study, a NOAEL of 1500 mg/kg bw/day was identified for calcium lignosulphonate (40–65) based on minimal focal/multifocal chronic inflammation in the rectum of male rats. In a 90-day subchronic toxicity study the petitioner identified a NOAEL of 2000 mg/kg bw/day for calcium lignosulphonate (40–65), the highest dose tested. The Panel, however, considered this study inadequate for evaluating the safety of calcium lignosulphonate (40–65) due to the high incidence of lymphoid hyperplasia and lymphoid infiltration in the mandibular and mesenteric lymph nodes, in the Peyer's patches and in the liver in all animals, including controls. Therefore, the Panel considers that available data on calcium lignosulphonate (40–65) are insufficient to establish an ADI. Furthermore, the Panel considers that long-term toxicity studies are needed to elucidate whether the histiocytosis in the mesenteric lymph nodes of the rats observed in the inadequate 90-day toxicity study may progress into a more adverse state with time. Overall, based on the available information, the Panel concludes that the safety of use of calcium lignosulphonate (40–65), as a carrier for vitamins and carotenoids intended to be added to foods for colouring and nutrient purposes, cannot be assessed.

Safety and toxicological evaluation of a novel anti-obesity formulation LI85008F in animals

LI85008F is a novel synergistic composition of Moringa oleifera, Murraya koenigi, and Curcuma longa. These herbs are well recognized and widely used in ayurvedic system of medicine for treating a variety of diseases and are also have been used for culinary purposes for thousands of years. LI85008F inhibits preadipocyte differentiation and potentiates lipid breakdown in mature adipocytes. In diet-induced obese rats, LI85008F significantly reduced weight gain and improved serum adiponectin levels. These findings motivated the authors to determine the broad-spectrum safety of LI85008F. Acute oral toxicity, acute dermal toxicity, primary skin irritation, primary eye irritation, and dose-dependent 28-day sub-acute toxicity studies were conducted. The acute oral LD50 of LI85008F was greater than 5000 mg/kg in female SD rats and no changes in body weight or adverse effects were observed following necropsy. Acute dermal LD50 of LI85008F was greater than 2000 mg/kg. LI85008F was classified as non-irritating to skin in a primary dermal irritation study conducted using New Zealand Albino rabbits. LI85008F caused minimal irritation to eyes in a primary eye irritation test conducted on New Zealand Albino rabbits. A dose-dependent 28-day sub-acute toxicity study demonstrated no significant changes in selected organ weights. Evaluations on hematology, clinical chemistry, and histopathology did not show any significant adverse changes. The NOAEL of LI85008F was found to be greater than 2500 mg/kg body weight. These results demonstrate the broad spectrum safety of LI85008F in animal models.

Scientific Opinion on the safety of smoke flavour Primary Product Scansmoke R909

This opinion concerns the safety of the smoke flavouring Primary Product Scansmoke R909. The Panel considered the technical and analytical data provided acceptable to characterise the Primary Product and to demonstrate its batch-to-batch variability and stability. Two in vivo genotoxicity tests were negative and sufficient to eliminate the concerns over the in vitro genotoxicity. The Panel derived a NOAEL of 1250 mg/kg bw/day from a 90-day study in rats. Based on this NOAEL and on the intake data calculated with the use levels of the Primary Product Scansmoke R909 provided by the applicant for the 18 food categories, the margins of safety would amount to 100 and 160 for the intake estimates based on the upper use levels, and to 350 and 420 when normal use levels are considered. When assuming the use in traditionally smoked products only, the margins of safety would amount to 210 and 300 for the intake estimates based on the upper use levels, and to 520 and 735 when normal use levels are considered. The fact that these margins of safety based on a 90-day toxicity study are inadequate, and in addition, data on reproduction and developmental toxicity and long term studies are absent, it is concluded that the uses and use levels for the Primary Product in a wide range of product categories would require a larger margin of safety. The Panel concludes that the margins of safety are insufficient and that the use of Primary Product Scansmoke R909 at the proposed uses and use levels is of safety concern.

Scientific Opinion on safety of smoke flavour Primary Product – TRADISMOKE™ A MAX

This opinion concerns the safety of the smoke flavouring Primary Product TRADISMOKETM A MAX. The Panel considered the technical and analytical data provided acceptable to characterise the Primary Product and to demonstrate its batch-to-batch variability and stability. Two in vivo genotoxicity tests were negative and sufficient to eliminate the concerns over the in vitro genotoxicity. The Panel derived a NOAEL of 1000 mg/kg bw/day from a 90-day study in rats. Based on this NOAEL and on the intake data calculated with the use levels of the Primary Product TRADISMOKETM A MAX provided by the applicant for the 18 food categories, the margins of safety would amount to 30 and 61 for the intake estimates based on the upper use levels, and to 139 and 294 when normal use levels are considered. When assuming the use in traditionally smoked products only, the margins of safety would amount to 83 and 120 for the intake estimates based on the upper use levels, and to 417 and 588 when normal use levels are considered. The fact that these margins of safety based on a 90-day toxicity study are inadequate, and in addition, that data on reproduction and developmental toxicity and long term studies are absent, it is concluded that the uses and use levels for the Primary Product in a wide range of product categories would require a larger margin of safety. The Panel concludes that the margins of safety are insufficient and that the use of Primary Product TRADISMOKETM A at the proposed uses and use levels is of safety concern.

A 90-day Feeding Toxicity Study of L-Serine in Male and Female Fischer 344 Rats

A subchronic feeding study of l-serine (l-Ser) was conducted with groups of 10 male and 10 female Fischer 344 rats fed a powder diet containing 0, 0.06, 0.5, 1.5 or 5.0% concentrations of l-Ser for 90 days. There were no toxicologically significant, treatment-related changes with regards to body weight, food intake, water intake or urinalysis data. In several of the hematology, serum biochemistry and organ weight parameters, significant changes were observed between some of the treated groups and the controls. All these changes, however, were subtle and lacked any corresponding pathological findings. In addition, the increased or decreased values remained within the range of the historical control values. In fact, histopathological assessment revealed only sporadic and/or spontaneous lesions. In conclusion, the no-observed-adverse-effect-level (NOAEL) for l-Ser was, therefore, determined to be at least a dietary dose of 5.0% (2765.0 mg/kg body weight/day for males and 2905.1 mg/kg body weight/day for females) under the present experimental conditions.

Inhalation toxicity study of disk-shaped potassium octatitanate particles (Terracess TF) in rats following 90 days of aerosol exposure

Since fibrous particles such as asbestos and some man-made fibers (MMF) have been known to produce carcinogenic or fibrogenic effects, disk-shaped potassium octatitanate (POT) particles (trade name: Terracess TF) were manufactured as nonfibrous particles. A 90-day inhalation toxicity study of Terracess TF was performed to evaluate comparative inhalation toxicity of the disk shape with a fibrous shape that was previously evaluated. Four groups of 20 male and 15 female rats each were exposed to Terracess TF aerosols at concentrations of 0, 2, 10, or 50 mg/m3 for 90 days. Ten male and 10 female rats per group were sacrificed at 90 days of exposure. After 90 days of exposure, 5 male rats per group were sacrificed at 3 wk of recovery period and 4–5 male rats per group or 5 female rats per group were sacrificed at 15 wk of recovery for lung clearance and histopathology. The mass median aerodynamic equivalent diameter (MMAED) of the aerosols of test materials ranged from 2.5 to 2.9 μm. There were no test-substance-related adverse effects on clinical observations. At the end of the 90-day exposure, a slight increase in lung-to-body weight ratios was observed at 50 mg/m3 in male but not in female rats. However, lung weights were within normal limits after 3- or 15-wk recovery periods. Microscopically, inhaled Terracess TF particles were mostly phagocytized by free alveolar macrophages (AMs) in the alveolar airspaces and alveolar walls maintained normal structure at 2 and 10 mg/m3. At 50 mg/m3, some alveoli were distended and filled with aggregates of particle-laden AMs. The alveolar walls showed slight type II pneumocyte hyperplasia, but neither proliferative inflammation nor alveolar fibrosis was present at 50 mg/m3. The clearance half-times for Terracess TF were estimated to be in the order of 6 to 9 mo for the 50-mg/m3 group and 2 to 3 mo for the 10- and 2-mg/m3 groups. The lung responses and lung clearance rate were comparable to those of “nuisance” type dusts at these concentrations. Based on interpretation that aggregated particle-laden AMs in alveoli was considered to be an early histopathological sign of lung overloading, an effect level was considered to be 50 mg/m3 and no-observed-adverse-effect level (NOAEL) was 10 mg/m3. This experiment clearly demonstrated that particle morphology was considered to be an important factor to determine inhaled particle toxicity.

A 28-Days Sub-Acute Toxicity Study in Swiss Albino Mice to Evaluate Toxicity Profile of Neurotol Plus (Mannitol and Glycerol Combination)

Osmotic agents are still the most common treatment options available for controlling intracranial pressure (ICP). Combining Mannitol and Glycerol provides a better alternative and is currently available as best therapy used for increased ICP. The aim of this work was to study the effects of repeated dosing (28 days) of Mannitol 20% and Glycerol 10% combined formulation, Neurotol plus on safety profile. A twenty eight days sub-acute toxicity study was conducted at three different dose levels of 5ml/kg, 10 ml/kg and 20 ml/kg. Mice were randomly divided into four groups of six animals each. Physical parameters, biochemical parameters related to liver toxicity & nephrotoxicity and hematological parameters were studied as end point of evaluation. We also carried out histopathlogical studies to assess any organ specific toxicity. The present study demonstrated that there were either no or very minimal changes (at high dose) were observed on various physical, biochemical and hematological parameters between Neurotol plus and control group. In conclusion the data of present study suggest that the combination of Mannitol and Glycerol is not associated with any serious adverse effects and is safe therapeutic choice for ICP reduction.

A 28-Day Intrathecal Toxicity Study with Trastuzumab in Nonhuman Primates.

Abstract Introduction: Trastuzumab is indicated for the treatment of patients with breast cancer whose tumors overexpress the HER2 protein. Brain metastases have been observed in breast cancer patients, being the highest in women with HER2-overexpressing tumors. The blood-brain barrier, however, may prevent trastuzumab from reaching appropriate concentrations in the cerebrospinal fluid (CSF). Case studies have been reported in which trastuzumab was intrathecally administered for the treatment of metastatic brain cancer in humans. However, no extensive preclinical safety evaluation has been performed for intrathecal application after repeat-dosing in a trastuzumab cross-reactive species using doses equivalent to or higher than those used in patients. To evaluate the potential of local effects, a 28-day toxicology study with weekly intrathecal administration of trastuzumab was performed in cynomolgus monkeys, a trastuzumab cross-reactive species. Preservative-free trastuzumab was used in this preclinical safety study, as caution has been raised for the intrathecal use of benzyl alcohol-containing solutions. Methods: Five male and five female monkeys were evaluated per dose group, of which two monkeys/sex/group were evaluated after a 4-week recovery period. Animals were assigned to 3 groups which received 4 weekly 30-minute intrathecal infusions at 0, 3, or 15 mg per dose at an infusion rate of 1.5 mL/hour. Endpoints evaluated included clinical signs, body weight, appetite, neurological examinations, clinical pathology, gross pathology, and histopathology of the brain (with inclusion of meninges), spinal cord, and infusion site. In addition, CSF and serum samples were collected for toxicokinetic evaluations. Results and Discussion: No test article-related effects on clinical signs, body weight, appetite, neurological examinations, clinical pathology, gross pathology or histopathology were noted. Of note is the absence of demyelination as was discussed as an uncertainty of intrathecally administered proteins. The doses used of 3 and 15 mg per dose resulted in 0.051 and 0.24 mg/g brain doses, respectively. Doses administered in clinical case reports ranged from 5 to 100 mg per dose. For a 1500 g human brain weight, these intrathecal doses would be equivalent to 0.0033 and 0.067 mg/g brain weight. At the high dose of 15 mg, maximum CSF concentrations (Cmax) were as high as 403 µg/mL. The applied doses and CSF concentrations achieved in the current study greatly exceeded those reported in patients (6.4 µg/mL at an intrathecal dose of 20 mg). Conclusion: The results of this 4-week toxicology study in monkeys with the results of the clinical case studies support future studies to evaluate intrathecal application of trastuzumab in patients with brain metastasis in HER2-positive breast cancer. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5101.

A 90-day intravenous administration toxicity study of CaO-SiO2-P2O5-B2O3 glass-ceramics (BGS-7) in rat

CaO-SiO2-P2O5-B2O3 glass ceramics directly bond to bone and have potential use as a bone substitute material. The present study evaluated the toxicity from subchronic intravenous administration of CaO-SiO2-P2O5-B2O3 glass ceramics to male and female Sprague-Dawley rats. The rats were divided into four groups, each consisting of 10 male and 10 female rats, and administered different amounts of CaO-SiO2-P2O5-B2O3 glass ceramics (aqueous extract 5, 2.5, and 1.25 mL/kg body weight/day and saline) 7 days per week for 90 consecutive days. During the experiment, no deaths were observed in any groups, and there were no remarkable changes in clinical signs, body weight, food and water intake, hematological and serum biochemical parameters, organ weight, and histopathological findings between the control and treated groups. The results show no adverse toxic effects of CaO-SiO2-P2O5-B2O3 glass ceramics (aqueous extract 5 mL/kg body weight/day) to rats of either sex.