Preweaning behaviors,developmental landmarks,and acrylamide and glycidamide levels after pre- and postnatal acrylamide treatment in rats

At high levels of exposure, acrylamide monomer (AA) is a known neurotoxicant (LoPachin, 2004 [23]). The effects of lower levels of exposure, such as those experienced via a typical human diet, have not been widely investigated. Data at these levels are particularly relevant given the widespread human exposure through carbohydrate-containing foods cooked at high temperatures. Additionally, daily AA intake is estimated to be higher for infants and children. Earlier, we described behavioral alterations in preweaning rats resulting from developmental AA treatment (0.5–10.0 mg/kg/day) (Garey et al., 2005 [14]). In the present study, the effects of lower doses were measured as well as serum AA and glycidimide (GA) levels in dams, fetuses, and young pups. Pregnant Fischer 344 dams (n = 48–58/treatment group) were gavaged with 0.0, 0.1, 0.3, 1.0, or 5.0 mg AA/kg/day beginning on gestational day 6 and ending on the day of parturition. Beginning on postnatal day 1 (PND 1) and continuing through PND 21, all pups/litter were gavaged with the same dose as their dam. There were no AA treatment effects on offspring fur development, pinnae detachment, or eye opening. Offspring body weight was somewhat decreased by 5.0 mg/kg/day, particularly in males. However, righting reflex (PNDs 4–7), slant board (i.e., negative geotaxis) (PNDs 8–10), forelimb hang (PNDs 12–16), and rotarod behavior (PNDs 21–22) were not significantly altered by AA treatment. Male and female offspring of the 5.0 mg/kg/day group were 30–49% less active in the open field at PNDs 19–20 (p < 0.05). Serum AA levels of GD20 dams and their fetuses were comparable, indicating the ability of AA to cross the placental barrier. AA levels of pups were not affected by age (PND 1 and 22) or sex. In all rats, serum AA and GA levels exhibited a dose–response relationship. These data extend those of our previous study (Garey et al., 2005 [14]) and demonstrate that overt preweaning neurobehavioral effects are apparent in rats exposed to acrylamide pre- and postnatally, but only at the highest doses tested.     

Chronic oral treatment with isotretinoin alters measures of activity but not anxiety in male and female rats

Use of the anti-acne drug, Accutane® (ACC) (isotretinoin, 13-cis-retinoic acid), has been associated with neuropsychiatric events ranging from depression in animal models to depression and suicide ideation in humans. Our studies, however, have consistently indicated few effects on measures of depression in male and female rats. Still, the comorbidity of depression and anxiety suggests that anxiety assessments in ACC-treated rats could be informative. Such assessments must be balanced with measures of activity since drug-induced activity alterations may impact the expression of anxiety-like behaviors. Here, Sprague–Dawley rats (n = 15/sex/dose) were gavaged daily with 0 (soy oil), 7.5, or 30 mg/kg/day ACC beginning on postnatal day (PND) 59. Blood ACC levels similar to humans taking recommended ACC doses are produced by 7.5 mg/kg/day. Short-term activity was assessed in open fields prior to ACC treatment (PND 51) and again at PNDs 129 and 164 and in a complex environment at PNDs 66 and PND 184. Long-term residential activity was measured in running wheels (PNDs 85–92) and figure 8 mazes (PNDs 99–106). Anxiety-like behavior was assessed via elevated plus maze (EPM) activity on PND 98 and in a black/white apparatus on PND 125. The typical sex differences in most behaviors were exhibited (i.e., increased EPM open arm entries and overall activity in most measures in females); however, there were no significant effects of ACC treatment on open field activity, complex environment activity, residential running wheel activity, or EPM activity. Residential figure 8 maze activity indicated that male and female rats treated with 30 mg/kg/day were less active on all nights (p < 0.05) and females treated with 7.5 or 30 mg/kg/day were less active than same-sex controls on most days (p < 0.05). Similarly, rats of both sexes treated with 30 mg/kg/day were significantly less active in the black/white apparatus (p < 0.05), entering the darkened area less frequently (p < 0.05), although duration in the darkened area did not differ. These data indicate that at blood levels typically achieved by humans (i.e., the 7.5 mg/kg group), there are no significant anxiogenic effects associated with ACC treatment. At higher ACC levels, there are mild effects on activity but these appear to be apparatus- and/or age-specific.     

Tolerability and toxicological profile of pixantrone (Pixuvri®) in juvenile mice. Comparative study with doxorubicin

The tolerability of pixantrone dimaleate (Pixuvri^®), an aza-anthracenedione for non-Hodgkin lymphoma, was assessed in juvenile mice after intraperitoneal injection.Twenty animals/sex/dose received pixantrone 15 or 27 mg/kg/day on Post-Natal-Days (PND) 10, 13, 17, 20, 35, 39 and 42 in comparison with doxorubicin, 3 mg/kg/day. Animals were sacrificed on PND 42, 73 and 96.All pixantrone animals survived, while doxorubicin induced 52.5% mortality and the surviving animals were sacrificed early due to severe toxicity. Recoverable bone marrow toxicity (pixantrone), and toxicity to thymus and reproductive organs (pixantrone, doxorubicin) were observed without nephro- or hepatotoxicity. Pixantrone was measurable in plasma up to 2 h (occasionally 6 h) post-dose. At PND 42, mean C_max and AUC values increased proportionally with dose, without gender difference or accumulation.Pixantrone showed minimal cardiotoxicity in males and negligible in females at PND 96. Doxorubicin induced significant heart weight reduction at PND 42, however early sacrifice impeded further cardiac assessments.     

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).     

Effects of maternal exposure to di-n-butyl phthalate during pregnancy and breastfeeding on ovarian development and function of F1 female rats

This study aimed to investigate the effects of maternal exposure to di-n-butyl phthalate (DBP) during pregnancy and breastfeeding on F1 ovarian development and function. A rat model of maternal exposure to DBP during pregnancy and breastfeeding was established by gavage feeding female Sprague Dawley rats with 0, 10, 100, or 600 mg/kg/day DBP from gestational day (GD) 12 to postnatal day (PND) 21. F1 offspring were weaned on PND21 and were not exposed to DBP afterward. The age of vaginal opening and estrus onset, estrous cyclicity, c-Kit-ligand expression on ovarian granulosa cells, and the weight of ovaries and uterus of F1 female offspring were not affected, whereas serum levels of estradiol and progesterone were increased significantly by maternal exposure to 10 mg/kg/day DBP from GD12 to PND21. Although F1 ovarian function may not be adversely affected by maternal exposure to DBP, the increased reproductive hormone levels may interfere in F1 rat fertility.     

Agmatine reduces ultrasonic vocalization deficits in female rat pups exposed neonatally to ethanol

Rat pups, in isolation, produce ultrasonic vocalizations (USVs). These USVs have been used as a diagnostic tool for developmental toxicity. We have shown that neonatal ethanol (ETOH) exposure produces deficits in this behavior. The current study was designed to examine whether agmatine (AG), which binds to imidazoline receptors and modulates n-methyl-d-aspartate receptors (NMDAR), could reduce these deficits. In addition, this study examined critical periods for ETOH's effects on USVs by administering ETOH during either the 1st or 2nd postnatal week. Neonatal rats received intragastric intubations of either ETOH (6 g/kg/day), ETOH and AG (6 g/kg/day and 20 mg/kg/day), AG (20 mg/kg/day), or maltose on postnatal days (PND) 1–7 or 8–14. A non-intubated control was also included. Subjects were tested on PND 15. Neonatal ETOH exposure significantly increased the latency to vocalize for females and reduced the rate of USVs in both males and females exposed to ETOH on PND 1–7. Agmatine reduced these deficits, in female but not male pups. Subjects exposed to ETOH on PND 8–14 showed no evidence of abnormal USVs. These findings suggest that there may be gender differences in response to AG following neonatal ETOH exposure and also provide further support that the first neonatal week is a particularly sensitive time for the developmentally toxic effects of ETOH in rodents.     

Cocaine responsiveness or anhedonia in rats treated with methylphenidate during adolescence

Methylphenidate (MPH) treatment in boys diagnosed with ADHD is reported to decrease the risk of drug abuse in adulthood. Similarly, MPH treatment appears to decrease the cocaine preference of male rats during conditioned place preference (CPP) tests. However, the effects of MPH treatment on later drug use of girls/women or CPP in female rodents have not been fully examined, nor have a clinically-relevant MPH dose and/or administration route been thoroughly studied. Here, Sprague–Dawley rats (n = 34/sex/treatment) were treated orally 3×/day on postnatal days (PNDs) 29–50 with water or 3 mg MPH/kg, a dose producing serum levels within the human clinical range. CPP assessments to cocaine (10 mg/kg, ip) (PNDs 62–71) indicated MPH-treated rats were less active during pre- and postconditioning sessions (p < .04), but there were no significant MPH-related differences in conditioning strength. Baseline open field activity at PND 84 indicated that MPH-treated females were more active than same-sex controls (p < .05). A cocaine challenge (10 mg/kg, ip) elevated activity similarly in MPH-treated and controls of both sexes. As an anhedonia measure, saccharin solution intake on PNDs 87–90 indicated no significant MPH effects. Estrous cycle phase did not appear to affect cocaine response during CPP or open field assessments. Hormonal levels at PND 90 indicated 63% higher corticosterone levels in MPH-treated females relative to same-sex controls (p < .05), a finding that deserves further investigation. These results address some of the major issues surrounding animal models of MPH treatment and provide additional support for a lack of severe long-term behavioral effects of adolescent MPH.     

Maternal in utero exposure to the endocrine disruptor di-(2-ethylhexyl) phthalate affects the blood pressure of adult male offspring

Di-(2-ethylhexyl) phthalate (DEHP) is used industrially to add flexibility to polyvinyl chloride (PVC) polymers and is ubiquitously found in the environment, with evidence of prenatal, perinatal and early infant exposure in humans. In utero exposure to DEHP decreases circulating testosterone levels in the adult rat. In addition, DEHP reduces the expression of the angiotensin II receptors in the adrenal gland, resulting in decreased circulating aldosterone levels. The latter may have important effects on water and electrolyte balance as well as systemic arterial blood pressure. Therefore, we determined the effects of in utero exposure to DEHP on systemic arterial blood pressure in the young (2 month-old) and older (6.5 month-old) adult rats. Sprague-Dawley pregnant dams were exposed from gestational day 14 until birth to 300 mg DEHP/kg/day. Blood pressure, heart rate, and activity data were collected using an intra-aortal transmitter in the male offspring at postnatal day (PND) 60 and PND200. A low (0.01%) and high-salt (8%) diet was used to challenge the animals at PND200. In utero exposure to DEHP resulted in reduced activity at PND60. At PND200, systolic and diastolic systemic arterial pressures as well as activity were reduced in response to DEHP exposure. This is the first evidence showing that in utero exposure to DEHP has cardiovascular and behavioral effects in the adult male offspring.     

Safety profile of Hoodia gordonii extract: Mouse prenatal developmental toxicity study

Hoodia gordonii extract (0, 5, 15 or 50 mg/kg body weight/day, n = 24 mice/group) was orally administered by gavage to female CD-1 mice from gestation days 5–17. On gestation day 18 the females were euthanized and examined. Treatment at 50 mg/kg/day caused a marked reduction in feed intake and body weight gain. Feed consumption was sporadically reduced at 15 mg/kg/day. At 50 or 15 mg/kg/day fetal weights, ossification of some bones and full and empty uterus weights were reduced. There were no clear maternal or fetal effects at 5 mg/kg/day. Reproductive indices were unaffected at all doses and there were no treatment-related malformations, anomalies or variations. The overall study no-observed-adverse-effect level was set at 5 mg/kg/day.In summary, at doses that reduced maternal feed consumption, H. gordonii extract delayed fetal development. The fetal effects seen could be consequent to reduced maternal feed consumption, the desired biological activity of the test item.     

Thyroid hormone disruption and cognitive impairment in rats exposed to PBDE during postnatal development

Polybrominated diphenyl ether flame-retardants (PBDEs) are thyroid-disrupting environmental chemicals. We investigated the effects of postnatal exposure to DE-71 (a mixture of tetra- and penta-brominated congeners), n-propylthiouracil (PTU) and thyroxine (T4) replacement on open-field (OF) and radial maze (RAM) tests. Wistar rats (5 males/5 females per litter, 32 litters) were treated orally (PND 5–22) with PTU (4 mg/kg bw/d), DE-71 (30 mg/kg bw/d), with and without co-administration of T4 (15 μg/kg bw/d, sc). PTU depressed T4 serum levels and body weight gain and enlarged thyroid gland. Although decreasing T4 levels, DE-71 did not change thyroid and body weights. PTU-treated rats showed hyperactivity (PND 42 and 70), and working and reference memory learning deficits (RAM, PND 100). Although not altering motor activity and working memory, DE-71 caused a reference memory deficit (females only). T4 co-administration averted hypothyroxinemia and long-term cognitive deficits caused by PTU and DE-71.     

Effects of in utero exposure to di-n-hexyl phthalate on the reproductive development of the male rat

Recently, the plasticizer di-n-hexyl phthalate (DnHP) has been demonstrated to be teratogenic and adversely affect the reproductive tract in male rat fetuses. This study was undertaken to determine the long-term effects of an in utero exposure to DnHP on the reproductive development of the male offspring. Di-2-ethylhexyl phthalate (DEHP), another phthalate ester known to disrupt the androgen-dependent sexual differentiation in the male rat, was used as a positive control. Pregnant Sprague-Dawley rats were administered DnHP or DEHP, by gavage on gestation Days 12–21, at doses of 0, 50, 125, 250, or 500 mg DnHP/kg-d and 500 mg DEHP/kg-d. DnHP had no significant effect on maternal body weight gain and pup weights during lactation. The proportion of live pups on postnatal day 1 was slightly, but not significantly, lower than control at 250 and 500 mg DnHP/kg-d. Male offspring displayed reduced anogenital distance on postnatal day 1 (PND) at 125 mg DnHP/kg-d and above, and areola/nipple retention before weaning and at adulthood at 250 and 500 mg DnHP/kg-d. At necropsy on PND 70–78 or PND 111–120, severe malformations of the reproductive tract were observed in young adult males at 125 mg DnHP/kg-d and higher doses. They mainly consisted of hypospadias, underdeveloped testis, and undescended testis. Additionally, histopathological examination revealed seminiferous tubule degeneration at the two high doses. Our results showed that prenatal exposure to DnHP caused permanent and dose-related alterations of the male rat reproductive development, with a similar profile as DEHP.     

In vivo preliminary investigations of the effects of the benzimidazole anthelmintic drug flubendazole on rat embryos and fetuses

Flubendazole, in a new formulation with high systemic bioavailability, has been proposed as a macrofilaricide against filarial diseases.To investigate embryotoxic activity, the new flubendazole formulation was administered orally to Sprague Dawley rats at 2, 3.46, 6.32 mg/kg/day on gestation day (GD) 9.5 and 10.5. Embryos/fetuses were evaluated on GD 11.5, 12.5 or 20.At 6.32 mg/kg/day (C_max = 0.801 μg/mL after single administration), flubendazole initially induced an arrest of embryonic development followed by a generalized cell death that led to 100% embryolethality by GD 12.5.At 3.46 mg/kg/day (C_max = 0.539 μg/mL after single administration), flubendazole markedly reduced embryonic development by GD 12.5 without causing cell death. On GD 20, 80% of fetuses showed malformations.At 2 mg/kg/day (C_max = 0.389 μg/mL after single administration), it did not interfere with rat embryofetal development.     

Developmental treatment with bisphenol A causes few alterations on measures of postweaning activity and learning

Widespread bisphenol A (BPA) exposure necessitates increased knowledge of its potential effects for better risk assessment and regulatory guidance. Here, female Sprague–Dawley rats, reared in low exogenous estrogen environments and bred at adulthood, were gavaged on gestational days 6–21 with vehicle (VEH), 2.5 or 25.0 μg/kg/day BPA, or 5.0 or 10.0 μg/kg/day ethinyl estradiol (EE₂). Offspring were orally treated on postnatal days (PNDs) 1–21 with the same dose their dam received. A naïve control group (NC) was not gavaged. Post-weaning, one offspring/sex/litter (n = 11–12/sex/group) was assessed for the typical behaviors measured in developmental neurotoxicology studies. At PND 29, novelty preference was unaffected by treatment; however, relative to the VEH group, males and females of both EE₂ groups were more active. VEH males appeared somewhat hypoactive in open field assessments at PNDs 40–42 and, as a result, males of the BPA and EE₂ groups were significantly more active. Latency to locate the Barnes maze escape box at PNDs 47–50 was increased in males and females of the 5.0 μg/kg/day EE₂ group. Relative to other male groups, VEH males exhibited an increased startle response on the first trial block at PND 54 and thus, males of both BPA groups and the 10.0 μg/kg/day EE₂ group exhibited a significantly decreased startle response. PNDs 43–44 motor coordination and PNDs 75–79 water maze performance were unaffected by treatment. These results indicate few consistent or dose-related effects resulting from developmental treatment with BPA at these doses. Few of these behaviors, however, were sexually dimorphic which may prove more sensitive.     

Protective effect of artemisinin on chronic alcohol induced-liver damage in mice

The liver disease related to chronic alcohol consumption is one of the leading causes of death for alcoholics. The efficient drug to ameliorate the alcoholic liver injury was needed urgently. The present study was performed to investigate whether artemisinin possessed the protective effect against chronic alcohol consumption. 50 male Kunming mice were divided into 5 groups: control group (C): 10 ml/kg saline + 10 ml/kg saline, alcohol group (A): 10 ml/kg 56%(v/v) alcohol + 10 ml/kg saline, low dose group of artemisinin (L): 10 ml/kg 56%(v/v) alcohol + 30 mg/kg/day artemisinin, medium dose group of artemisinin (M): 10 ml/kg 56%(v/v) alcohol + 60 mg/kg/day artemisinin, high dose group of artemisinin (H): 10 ml/kg 56%(v/v) alcohol + 120 mg/kg/day artemisinin. Drugs were given orally every day. The general state of mice was observed and the levels of serum activities of AST and ALT were detected after treatment with drugs for 30 days. Besides, the liver weight index was calculated and histopathological analysis was performed. We successfully demonstrated that treatment with high dose of artemisinin significantly decreased the elevated levels of AST (p < 0.05) and ALT (p < 0.01) in plasma, as well as the liver weight index (p < 0.01). The loss of body weight, tissue injury, oedema and inflammatory cell infiltration in the hepatocytes were found in the A group. These symptoms were remarkably alleviated in animals treated with artemisinin. Artemisinin can inhibit the activation of NF-кB and the expression of inflammatory cytokines inducible nitric oxide synthase. Besides, it can also enhance the stability of liver cell membrane, and reduce the damage of liver cell membrane and liver cell. Artemisinin showed a protective effect against chronic alcohol poisoning and it has a great potential for the clinical application to treat the liver injury induced by alcohol.     

An antioxidant,N,N′-diphenyl-p-phenylenediamine (DPPD), affects labor and delivery in rats: A 28-day repeated dose test and reproduction/developmental toxicity test

A 28-day repeated dose toxicity test and reproduction/developmental toxicity test for N,N′-diphenyl-p-phenylenediamine (DPPD) were conducted in [Crl:CD(SD)] SPF rats. Male and female rats were dosed with DPPD by gavage for 28 days at 0, 100, 300, or 1000 mg/kg bw/day or for a total of 42–46 days at 0, 8, 50, or 300 mg/kg bw/day. No significant adverse effects were observed in the repeated dose toxicity study up to 1000 mg/kg bw/day in both sexes. In the reproduction/developmental toxicity study, two females showed piloerection, hypothermia, and pale skin; one died and the other showed dystocia on day 23 of pregnancy at 300 mg/kg bw/day. Another female delivered only three live pups at 300 mg/kg bw/day. A significantly prolonged gestation period was observed at 50 and 300 mg/kg bw/day. The NOAELs of repeated dose toxicity and reproduction/developmental toxicity were considered to be 1000 and 8 mg/kg bw/day, respectively.     

Disposition of diiosononyl phthalate and its effects on sexual development of the male fetus following repeated dosing in pregnant rats

Pregnant Sprague-Dawley rats received 50, 250, and 500 mg/kg/day diisononyl phthalate (DiNP) from GD 12 to 19 via corn oil gavage to study the dose response for effects on fetal male rat sexual development as well as metabolite disposition in the dam and fetus. Monoisononyl phthalate (MiNP), mono(carboxy-isooctyl) phthalate (MCiOP), mono(hydroxyl-isononyl) phthalate (MHiNP), mono(oxo-isononyl) phthalate (MOiNP), and monoisononyl phthalate glucuronide (MiNP-G) were found in all measured tissues. MCiOP was the major metabolite, followed in decreasing order by MiNP, MHiNP, MOiNP, and MiNP-G. Percentage of dose absorbed decreased at 750 mg/kg/day. Testosterone concentration in the fetal testes was reduced at 250 and 750 mg/kg/day. Multinucleated germ cells were increased in the testes of rats at 250 and 750 mg/kg/day. The no observed effect level (NOEL) for this study was 50 mg/kg/day based on increased MNGs and reduced testes testosterone concentration in the fetal rat.     

A dose response study to assess effects after dietary administration of diisononyl phthalate (DINP) in gestation and lactation on male rat sexual development

Male rat sexual development was evaluated after dietary administration of 0, 760, 3800, 11,400 ppm diisononyl phthalate (DiNP) and 7600 ppm dibutyl phthalate (DBP) from gestation day (GD) 12 to postnatal day (PND) 14. Maternal weight was reduced on GD 20, PND 2 and 14 at 11,400 ppm DiNP. Pup weight was reduced on PND 2 and 14 at 11,400 and 3800 ppm DiNP. DBP induced multinucleated germ cells (MNGs) and Leydig cell aggregates (LCAs) in PND 2 testes. 7600 ppm DBP reduced anogenital distance (AGD) on PND 2 and 14, and increased nipple retention and reproductive tract malformations on PND 49. DiNP induced MNGs (3800 ppm) and LCAs (11,400 ppm) on PND 2, and reduced AGD (11,400 ppm) on PND 14. DiNP did not alter AGD, nipple retention or reproductive tract malformations on PND 49. Global endpoint analysis showed no evidence of a rat “phthalate syndrome” on PND 49 with DiNP administration.     

Safety evaluation of a thermolysin enzyme produced from Geobacillus stearothermophilus

Thermolysin is a zinc metalloprotease that has potential uses in the food industry. The safety of thermolysin has not been demonstrated before, and therefore a series of standard toxicological tests to assess its potential toxicity was undertaken. The thermolysin used in this study was derived from the thermophilic bacterium Geobacillus stearothermophilus, which had undergone chemical mutagenesis to generate strains with increased thermolysin production. Acute toxicity studies in rats and mice showed that thermolysin powder is not acutely toxic with an oral LD₅₀ of more than 18,000 mg/kg (2520 mg/kg thermolysin protein) in rats and more than 24,000 mg/kg (3360 mg/kg protein) in mice. Subchronic feeding studies in rats for 91 days at doses up to 1000 mg/kg (390 mg/kg protein) revealed no significant differences between treated and non-treated groups and a No Observed Effect Level (NOEL) of 1000 mg/kg (390 mg/kg protein) per day was established. Results from genotoxicity tests such as in vitro chromosomal aberration assay and in vivo mouse micronucleus were negative. Allergenicity sequence analysis revealed no evidence suggesting that thermolysin is an allergen. The data presented in this study support the conclusion that thermolysin is safe for use in food production.     

Combination of peramivir and rimantadine demonstrate synergistic antiviral effects in sub-lethal influenza A (H3N2) virus mouse model

Efficacy of combination of the intramuscularly administered neuraminidase (NA) inhibitor, peramivir, and the orally administered M2 ion channel blocker, rimantadine was evaluated in mouse influenza A/Victoria/3/75 (H3N2) model. Mice were challenged with a sub-lethal virus dose (0–40% mortality in placebo group) and changes in body weights were analyzed by three-dimensional effect analysis to assess mode of drug interactions.Compounds were administered in a 5-day treatment course starting 1 h before viral inoculation. The peramivir and rimantadine doses ranged from 0.3–3 mg/kg/d and 5–30 mg/kg/d, respectively. The maximum mean weight loss of 5.19 g was observed in the vehicle-infected group on day 10. In the 1 and 3 mg/kg/d peramivir monotherapy groups, the weight losses were 4.3 and 3.55 g, respectively. In the rimantadine monotherapy group, the weight losses were 3.43, 2.1, and 1.64 g for the 5, 10, and 30 mg/kg/d groups, respectively. Combination of 1 mg/kg/d peramivir with 5 and 10 mg/kg/d rimantadine produced weight losses of 1.69 and 0.69 (p < 0.05 vs. vehicle and individual agent), respectively, whereas the combination of 3.0 mg/kg/d peramivir with 10 and 30 mg/kg/d rimantadine did not show any weight loss (p < 0.05 vs. vehicle and individual agent). The three-dimensional analysis of the weight loss for the majority of the drug combinations of peramivir and rimantadine tested demonstrated synergistic antiviral effects.