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Assessment of oral acute toxicity “Green Health herbal mixture” in Wistar albino rats
S344
Abstracts / Toxicology Letters 238S (2015) S56–S383
talities and significant reduction in body weight were observed hence dose was decreased to 150 mg/kg from PND 25. Weakness and lethargy were observed in 150/200 mg/kg QNL group. Body weight, body weight gain and feed consumption of the 100 and 150 mg/kg QNL groups were statistically significant decreased compared to the vehicle control group. Body weight, body weight gain and feed consumption of the positive control group were comparable with the vehicle control group. Out of 15 rats/group – 13 (G1), 5 (G2), 9 (G3) and 4 (G4) rats had complete vaginal opening. Terminal body weight of the 150 mg/kg QNL groups was statistically significant decreased as compared to the vehicle control group. Absolute liver weight of CNB and QNL treated groups were statistically significant increased compared to the vehicle control group. Absolute organ weights (pituitary, adrenals, thyroid and parathyroid) of QNL treated groups were comparable to the vehicle control group. Increase in urea and BUN was observed in CNB treated group as compared to vehicle control group. Thyroid hormone levels (T4 and TSH) of QNL treated groups were comparable. Based on the result of study, QNL had not altered pubertal development and thyroid function in peripubertal rats. http://dx.doi.org/10.1016/j.toxlet.2015.08.981
P16-012 Endocrine disruptor effects of quinoline (CAS: 91-22-5) in the intact juveniles/peripubertal male rats D. Ujawane ∗ , M. Poshiya, K. Patel, J. Mistry, H. Rana, M. Patel Jai Research Foundation, Toxicology, Valvada, India The potential effects of the quinoline on pubertal development and thyroid function were quantified in the peripubertal male Wistar rats. A total of 75 rats were divided in to 5 groups comprised of 15 rats. Vehicle control (corn oil – 0 mg/kg), positive control – vinclozolin (VCZ – 30 and 100 mg/kg) and two groups of quinoline (QNL – 100 and 150/200 mg/kg) were treated from postnatal day (PND) 23–53 at the dose volume of 2.5 mL/kg. Blood was collected for clinical pathology and thyroid hormone analysis. Organs as defined in study guidelines were excised, weighed and processed for microscopic examination. All animals were sacrificed approximately 2 h following the last dose on PND 53. No treatment related mortality was observed in VCZ and 100 mg/kg QNL treated groups. Two mortalities and significant reduction in body weight was observed at 200 mg/kg of QNL treated group hence dose was decreased to 150 mg/kg from PND 25. Weakness and lethargy were observed in 150 mg/kg QNL group. Body weight, body weight gain and feed consumption of the 100 and 150 mg/kg QNL groups were statistically significant decreased compared to the vehicle control group. Increase in urea and BUN was observed in all test dose levels of VCZ and QNL as compared to vehicle control group. Terminal body weight of the QNL (150 mg/kg) group was statistically significant decreased as compared to the vehicle control group. Decrease in seminal vesicle coagulating gland with and without fluid, ventral prostate, LABC, epididymes was observed in VCZ at 100 mg/kg and QNL at 150 mg/kg as compared to vehicle control group. Increase in testes (left and right) weight was observed in VCZ at 100 mg/kg. Increase in liver weight was observed in QNL treated groups as compared to vehicle control group. Thyroid hormone (T4 and TSH) and testosterone analysis were not affected by treatment. Results of this study revealed that QNL showed no evidence of endocrine disruptor activity. http://dx.doi.org/10.1016/j.toxlet.2015.08.982
P16-013 Cerebrospinal fluid (CSF) sampling and intrathecal/subarachnoidal test article administration in juvenile cynomolgus monkeys J. Luft ∗ , F. Runge, A. von Keutz Covance, Münster, Germany Introduction: Conventional drugs and biologics delivered by the intrathecal route avoid the blood brain barrier in juvenile macaques. Method: Single bolus or repeated intrathecal doses have been administered successfully for up to 53 weeks in juvenile cynomolgus monkeys (ca 11 months old, BW 1–2 kg). The animals are anaesthetized during all sampling and dosing procedures. Sampling and dose administration via an intrathecal spinal needle is accomplished by lumbar puncture at level L3–L4 or L4–L5 using a 25G, 25 or 50 mm pediatric Pencan Paed® pencil-point needle. After CSF withdrawal, 0.75–1.5 ml of test article is administered by slow manual infusion over one minute. For more frequent, repeated doses, a port catheter system (MID-LOVOL ports with a 3FR intrathecal PU-catheter) is aseptically implanted in an anesthetized animal 3–4 weeks prior study start. An 18G Touhy spinal needle is inserted between L2 and L3. The catheter is inserted through the needle into the subarachnoid space and advanced cranially ca 6 cm. The port is inserted subcutaneously, caudal to the shoulder blade. At the lumbar catheter entry position a small loop is made and the catheter is fixed with tissue glue. Patency is tested with a Huber-, Gripper- or SFN-needle using ca. 200 l sterile 0.9% NaCl solution or artificial CSF. Analgesia and prophylactic antibiotics are administered for 5 days post-surgery. Prior to dosing, the needle insertion site is clipped and disinfected. Fluid from the dead space is slowly withdrawn with a needle attached to a 1 ml syringe, followed by infusion of 0.6 ml test article plus 0.7 ml PBS to flush using a calibrated syringe pump over 11 min. Conclusion: The spinal needle works well for weekly CSF sampling and dose administration. For more frequent test article administration, the port catheter system is used. Necropsy confirmed catheter tip placement at T11/12 with no evidence of pronounced tissue reactions. Changes at the catheter tip included slight to minimal fibrosis, adhesion to the overlying dura, and slight compression of the spinal cord. This is consistent with the tissue reaction reported in other species following placement of intrathecal catheters (Butt, 2011). Sampling CSF via the port-catheter-system is not feasible, because the lumen of the subarachnoidal space collapses, blocking the catheter tip, due to suction. http://dx.doi.org/10.1016/j.toxlet.2015.08.1104 P17 – Safety Assessment of Mixtures P17-001 Assessment of oral acute toxicity “Green Health herbal mixture” in Wistar albino rats O. Adumanya Imo State Polytechnic, Umuagwo, Science Lab. Tech., Owerri, Nigeria “Green Health herbal mixture” is a pharmacological product of Green Healthcare Limited, Aboh, Ohi, Imo State, Nigeria, approved by National Agency for Food and Drug Administration and Control (NAFDAC) with anti-microbial, anti-bacterial and anti-fungal properties. However, its oral lethal dose is yet to be reported. Thus, this
Abstracts / Toxicology Letters 238S (2015) S56–S383
work was aimed at assessing its oral acute toxicity in wistar albino rats. The results of phase one and phase two of the study showed no mortality in any of the groups of rats in 24 h, 72 h and up to two weeks after oral administration of 5000 mg per kg body weight (b.w) of the herbal mixture, but the histology (photomicrograph) of the liver sections revealed multiple dose-dependent necrosis, histological lesions, and abnormal sinusoids. Therefore, oral intake of the herbal mixture at dose less than or equal to 5000 mg/kg b.w. is safe, but may not be advisable, having noted its deleterious effects on the hepatocytes via the photomicrograph (H & E 400). Topical use is therefore recommended. http://dx.doi.org/10.1016/j.toxlet.2015.08.984
P17-002 Cytotoxicity of biofuels produced by esterification of waste materials: Vegetable oils or animal fats on A431 skin cells J. Skowron´ ∗ , K. Miranowicz-Dzierzawska, L. Zapor Central Institute for Labour Protection – NRI, Chemical, Aerosol and Biological Hazard, Warsaw, Poland The use of second-generation biofuels is an environmentfriendly operation. It will reduce greenhouse gas emissions, particularly carbon dioxide from combustion engines by 80–85% compared with conventional fuels. The cytotoxicity of biofuels produced by esterification of frying vegetable oils and waste animals fats on A431 skin cells was evaluated through the MTT reduction method. Both biofuels were produced by methanol transesterification. Identification of the composition of biofuels was carried out using gas chromatography techniques conjugated with mass spectrometric detection. The concentration of methyl ester fatty acid in frying vegetable oils was about 30% lower than in biofuel from waste animals fats. The soluble fraction of tested biofuels was prepared by mixing them with culture medium and agitating on an orbital shaker for 18 h at room temperature. Immediately before the experiment, the tubes were vortexed vigorously and the emulsion layers were carefully removed by aspiration, leaving the clear media. On the basis of the MTT test, biofuel concentration decreasing cell viability by 50% compared to the control (IC50 value) was calculated after 24 h cells exposure to soluble solutions of tested biofuels in the medium. Based on IC50 values, a more powerful cytotoxic activity on A431 cells was shown by a biofuel which is the product of esterification of vegetable fat frying, than biofuel from waste animals fats. The higher cytotoxicity of this biofuel may be due to the contamination present in the raw material i.e. vegetable frying oil. This paper has been based on the results of a research task carried out within the scope of the third stage of the National Programme “Improvement of safety and working conditions” partly supported in 2014–2016—within the scope of research and development—by the Ministry of Science and Higher Education/National Centre for Research and Development. The Central Institute for Labour Protection–National Research Institute is the Programme’s main co-ordinator. http://dx.doi.org/10.1016/j.toxlet.2015.08.985
S345
P17-003 Chemical mixtures: Application of a tiered approach M. Krishan 1,∗ , G. Rice 2 1
ILSI North America, Washington, DC, United States U.S. Environmental Protection Agency, National Center for Environmental Assessment, Cincinnati, OH, United States 2
Evaluating potential health risks posed by exposures to chemical mixtures is challenging for toxicology research and risk assessment. Considering the current challenges in food mixtures, a hypothetical case study was conducted using a tiered screening approach that utilized simple conservative screening assumptions in lower tiers to focus resources on assessing chemicals of greater concern in higher tiers. Noncancer hazards associated with chronic oral exposures were evaluated using the hazard index (HI) method and the target organ toxicity dose approach (TTD). A hypothetical new bean product is being considered to replace pinto beans in a food program. Comparing old and new beans (NB) there is a 10% decrease in the following: cadmium (Cd), deltamethrin (De) and cyfluthrin. The What We Eat in America Food Commodity Intake Database (WWEIAFCID) was used to estimate pinto bean consumption rates (PBCR) in the general U.S. population in Tier 1. This tier provided a crude filter using recent, conservative health reference values for the mixture components. As the HI exceeded 1, in Tier 2, we refined PBCR distributions across different age groups and assessed exposures among Mexican Americans and children <12 years of age, groups exhibiting the highest PBCRs. HI estimates and the Risk 21 model were used to prioritize refinements in Tier 3; Cd and De were the largest contributors to HI in Tier 2. In Tier 3, De and Cd exposure estimates were refined using biomonitoring data and recent Cd intake estimates from Total Diet Study (2014), respectively. Between Tiers 2 and 3 the HI estimate decreased from 35 to 0.08 in the 95th percentile youth NB consumers. In Tier 4, constituents were grouped based on specific health effects, resulting in a HI estimate of 0.01 for NB among 95th percentile youth consumers using the TTD approach. Tiered approaches are useful and resource conserving as they can be implemented to effectively screen the effects of chemical mixtures in a health protective manner. This abstract does not necessarily reflect U.S. EPA policy. http://dx.doi.org/10.1016/j.toxlet.2015.08.986
P17-004 SH-SY5Y human cell line as a sensitive in vitro model to assessment oxidative-stress induced by a mixture of pyretroids A. Anadón ∗ , M. Martínez, I. Ares, E. Ramos, V. Castellano, M. ˜ Martínez, M. Martínez-Larranaga, A. Romero Universidad Complutense de Madrid, Department of Toxicology and Pharmacology, Faculty of Veterinary Medicine, Madrid, Spain Research has indicated that humans can be exposed to insecticides pyrethroids or mixtures of pyrethroids in several different types of environmental media (i.e., dust and floor wipes) at home and in consumed foods and beverages. The objective of this work was to evaluate the hypothesis of dose-additive effects of the mixture of 6 Type II pyrethroids (␣-cypermethrin, cyfluthrin, cyhalothrin, deltamethrin, cyphenothrin and esfenvalerate) on levels of nitric oxide (NO) and lipid peroxides measured as malondialdehyde (MDA) on the basis that induction of oxidative
Abstracts / Toxicology Letters 238S (2015) S56–S383
talities and significant reduction in body weight were observed hence dose was decreased to 150 mg/kg from PND 25. Weakness and lethargy were observed in 150/200 mg/kg QNL group. Body weight, body weight gain and feed consumption of the 100 and 150 mg/kg QNL groups were statistically significant decreased compared to the vehicle control group. Body weight, body weight gain and feed consumption of the positive control group were comparable with the vehicle control group. Out of 15 rats/group – 13 (G1), 5 (G2), 9 (G3) and 4 (G4) rats had complete vaginal opening. Terminal body weight of the 150 mg/kg QNL groups was statistically significant decreased as compared to the vehicle control group. Absolute liver weight of CNB and QNL treated groups were statistically significant increased compared to the vehicle control group. Absolute organ weights (pituitary, adrenals, thyroid and parathyroid) of QNL treated groups were comparable to the vehicle control group. Increase in urea and BUN was observed in CNB treated group as compared to vehicle control group. Thyroid hormone levels (T4 and TSH) of QNL treated groups were comparable. Based on the result of study, QNL had not altered pubertal development and thyroid function in peripubertal rats. http://dx.doi.org/10.1016/j.toxlet.2015.08.981
P16-012 Endocrine disruptor effects of quinoline (CAS: 91-22-5) in the intact juveniles/peripubertal male rats D. Ujawane ∗ , M. Poshiya, K. Patel, J. Mistry, H. Rana, M. Patel Jai Research Foundation, Toxicology, Valvada, India The potential effects of the quinoline on pubertal development and thyroid function were quantified in the peripubertal male Wistar rats. A total of 75 rats were divided in to 5 groups comprised of 15 rats. Vehicle control (corn oil – 0 mg/kg), positive control – vinclozolin (VCZ – 30 and 100 mg/kg) and two groups of quinoline (QNL – 100 and 150/200 mg/kg) were treated from postnatal day (PND) 23–53 at the dose volume of 2.5 mL/kg. Blood was collected for clinical pathology and thyroid hormone analysis. Organs as defined in study guidelines were excised, weighed and processed for microscopic examination. All animals were sacrificed approximately 2 h following the last dose on PND 53. No treatment related mortality was observed in VCZ and 100 mg/kg QNL treated groups. Two mortalities and significant reduction in body weight was observed at 200 mg/kg of QNL treated group hence dose was decreased to 150 mg/kg from PND 25. Weakness and lethargy were observed in 150 mg/kg QNL group. Body weight, body weight gain and feed consumption of the 100 and 150 mg/kg QNL groups were statistically significant decreased compared to the vehicle control group. Increase in urea and BUN was observed in all test dose levels of VCZ and QNL as compared to vehicle control group. Terminal body weight of the QNL (150 mg/kg) group was statistically significant decreased as compared to the vehicle control group. Decrease in seminal vesicle coagulating gland with and without fluid, ventral prostate, LABC, epididymes was observed in VCZ at 100 mg/kg and QNL at 150 mg/kg as compared to vehicle control group. Increase in testes (left and right) weight was observed in VCZ at 100 mg/kg. Increase in liver weight was observed in QNL treated groups as compared to vehicle control group. Thyroid hormone (T4 and TSH) and testosterone analysis were not affected by treatment. Results of this study revealed that QNL showed no evidence of endocrine disruptor activity. http://dx.doi.org/10.1016/j.toxlet.2015.08.982
P16-013 Cerebrospinal fluid (CSF) sampling and intrathecal/subarachnoidal test article administration in juvenile cynomolgus monkeys J. Luft ∗ , F. Runge, A. von Keutz Covance, Münster, Germany Introduction: Conventional drugs and biologics delivered by the intrathecal route avoid the blood brain barrier in juvenile macaques. Method: Single bolus or repeated intrathecal doses have been administered successfully for up to 53 weeks in juvenile cynomolgus monkeys (ca 11 months old, BW 1–2 kg). The animals are anaesthetized during all sampling and dosing procedures. Sampling and dose administration via an intrathecal spinal needle is accomplished by lumbar puncture at level L3–L4 or L4–L5 using a 25G, 25 or 50 mm pediatric Pencan Paed® pencil-point needle. After CSF withdrawal, 0.75–1.5 ml of test article is administered by slow manual infusion over one minute. For more frequent, repeated doses, a port catheter system (MID-LOVOL ports with a 3FR intrathecal PU-catheter) is aseptically implanted in an anesthetized animal 3–4 weeks prior study start. An 18G Touhy spinal needle is inserted between L2 and L3. The catheter is inserted through the needle into the subarachnoid space and advanced cranially ca 6 cm. The port is inserted subcutaneously, caudal to the shoulder blade. At the lumbar catheter entry position a small loop is made and the catheter is fixed with tissue glue. Patency is tested with a Huber-, Gripper- or SFN-needle using ca. 200 l sterile 0.9% NaCl solution or artificial CSF. Analgesia and prophylactic antibiotics are administered for 5 days post-surgery. Prior to dosing, the needle insertion site is clipped and disinfected. Fluid from the dead space is slowly withdrawn with a needle attached to a 1 ml syringe, followed by infusion of 0.6 ml test article plus 0.7 ml PBS to flush using a calibrated syringe pump over 11 min. Conclusion: The spinal needle works well for weekly CSF sampling and dose administration. For more frequent test article administration, the port catheter system is used. Necropsy confirmed catheter tip placement at T11/12 with no evidence of pronounced tissue reactions. Changes at the catheter tip included slight to minimal fibrosis, adhesion to the overlying dura, and slight compression of the spinal cord. This is consistent with the tissue reaction reported in other species following placement of intrathecal catheters (Butt, 2011). Sampling CSF via the port-catheter-system is not feasible, because the lumen of the subarachnoidal space collapses, blocking the catheter tip, due to suction. http://dx.doi.org/10.1016/j.toxlet.2015.08.1104 P17 – Safety Assessment of Mixtures P17-001 Assessment of oral acute toxicity “Green Health herbal mixture” in Wistar albino rats O. Adumanya Imo State Polytechnic, Umuagwo, Science Lab. Tech., Owerri, Nigeria “Green Health herbal mixture” is a pharmacological product of Green Healthcare Limited, Aboh, Ohi, Imo State, Nigeria, approved by National Agency for Food and Drug Administration and Control (NAFDAC) with anti-microbial, anti-bacterial and anti-fungal properties. However, its oral lethal dose is yet to be reported. Thus, this
Abstracts / Toxicology Letters 238S (2015) S56–S383
work was aimed at assessing its oral acute toxicity in wistar albino rats. The results of phase one and phase two of the study showed no mortality in any of the groups of rats in 24 h, 72 h and up to two weeks after oral administration of 5000 mg per kg body weight (b.w) of the herbal mixture, but the histology (photomicrograph) of the liver sections revealed multiple dose-dependent necrosis, histological lesions, and abnormal sinusoids. Therefore, oral intake of the herbal mixture at dose less than or equal to 5000 mg/kg b.w. is safe, but may not be advisable, having noted its deleterious effects on the hepatocytes via the photomicrograph (H & E 400). Topical use is therefore recommended. http://dx.doi.org/10.1016/j.toxlet.2015.08.984
P17-002 Cytotoxicity of biofuels produced by esterification of waste materials: Vegetable oils or animal fats on A431 skin cells J. Skowron´ ∗ , K. Miranowicz-Dzierzawska, L. Zapor Central Institute for Labour Protection – NRI, Chemical, Aerosol and Biological Hazard, Warsaw, Poland The use of second-generation biofuels is an environmentfriendly operation. It will reduce greenhouse gas emissions, particularly carbon dioxide from combustion engines by 80–85% compared with conventional fuels. The cytotoxicity of biofuels produced by esterification of frying vegetable oils and waste animals fats on A431 skin cells was evaluated through the MTT reduction method. Both biofuels were produced by methanol transesterification. Identification of the composition of biofuels was carried out using gas chromatography techniques conjugated with mass spectrometric detection. The concentration of methyl ester fatty acid in frying vegetable oils was about 30% lower than in biofuel from waste animals fats. The soluble fraction of tested biofuels was prepared by mixing them with culture medium and agitating on an orbital shaker for 18 h at room temperature. Immediately before the experiment, the tubes were vortexed vigorously and the emulsion layers were carefully removed by aspiration, leaving the clear media. On the basis of the MTT test, biofuel concentration decreasing cell viability by 50% compared to the control (IC50 value) was calculated after 24 h cells exposure to soluble solutions of tested biofuels in the medium. Based on IC50 values, a more powerful cytotoxic activity on A431 cells was shown by a biofuel which is the product of esterification of vegetable fat frying, than biofuel from waste animals fats. The higher cytotoxicity of this biofuel may be due to the contamination present in the raw material i.e. vegetable frying oil. This paper has been based on the results of a research task carried out within the scope of the third stage of the National Programme “Improvement of safety and working conditions” partly supported in 2014–2016—within the scope of research and development—by the Ministry of Science and Higher Education/National Centre for Research and Development. The Central Institute for Labour Protection–National Research Institute is the Programme’s main co-ordinator. http://dx.doi.org/10.1016/j.toxlet.2015.08.985
S345
P17-003 Chemical mixtures: Application of a tiered approach M. Krishan 1,∗ , G. Rice 2 1
ILSI North America, Washington, DC, United States U.S. Environmental Protection Agency, National Center for Environmental Assessment, Cincinnati, OH, United States 2
Evaluating potential health risks posed by exposures to chemical mixtures is challenging for toxicology research and risk assessment. Considering the current challenges in food mixtures, a hypothetical case study was conducted using a tiered screening approach that utilized simple conservative screening assumptions in lower tiers to focus resources on assessing chemicals of greater concern in higher tiers. Noncancer hazards associated with chronic oral exposures were evaluated using the hazard index (HI) method and the target organ toxicity dose approach (TTD). A hypothetical new bean product is being considered to replace pinto beans in a food program. Comparing old and new beans (NB) there is a 10% decrease in the following: cadmium (Cd), deltamethrin (De) and cyfluthrin. The What We Eat in America Food Commodity Intake Database (WWEIAFCID) was used to estimate pinto bean consumption rates (PBCR) in the general U.S. population in Tier 1. This tier provided a crude filter using recent, conservative health reference values for the mixture components. As the HI exceeded 1, in Tier 2, we refined PBCR distributions across different age groups and assessed exposures among Mexican Americans and children <12 years of age, groups exhibiting the highest PBCRs. HI estimates and the Risk 21 model were used to prioritize refinements in Tier 3; Cd and De were the largest contributors to HI in Tier 2. In Tier 3, De and Cd exposure estimates were refined using biomonitoring data and recent Cd intake estimates from Total Diet Study (2014), respectively. Between Tiers 2 and 3 the HI estimate decreased from 35 to 0.08 in the 95th percentile youth NB consumers. In Tier 4, constituents were grouped based on specific health effects, resulting in a HI estimate of 0.01 for NB among 95th percentile youth consumers using the TTD approach. Tiered approaches are useful and resource conserving as they can be implemented to effectively screen the effects of chemical mixtures in a health protective manner. This abstract does not necessarily reflect U.S. EPA policy. http://dx.doi.org/10.1016/j.toxlet.2015.08.986
P17-004 SH-SY5Y human cell line as a sensitive in vitro model to assessment oxidative-stress induced by a mixture of pyretroids A. Anadón ∗ , M. Martínez, I. Ares, E. Ramos, V. Castellano, M. ˜ Martínez, M. Martínez-Larranaga, A. Romero Universidad Complutense de Madrid, Department of Toxicology and Pharmacology, Faculty of Veterinary Medicine, Madrid, Spain Research has indicated that humans can be exposed to insecticides pyrethroids or mixtures of pyrethroids in several different types of environmental media (i.e., dust and floor wipes) at home and in consumed foods and beverages. The objective of this work was to evaluate the hypothesis of dose-additive effects of the mixture of 6 Type II pyrethroids (␣-cypermethrin, cyfluthrin, cyhalothrin, deltamethrin, cyphenothrin and esfenvalerate) on levels of nitric oxide (NO) and lipid peroxides measured as malondialdehyde (MDA) on the basis that induction of oxidative