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Comparison of acetaminophen toxicity in rat and mouse hepatocytes in vitro
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Abstracts / Toxicology Letters 229S (2014) S40–S252
measured. In sepsis-induced rats, MDA levels were increased and SOD and GSH-Px activities, GSH and TNF-␣ levels were decreased. FA treatment decreased the MDA levels and increased the levels of GSH and TNF-␣ and SOD and activities of GSH-Px in the sepsisinduced rats. According to our results, FA has ameliorative effects against oxidative stress caused by sepsis.
P-4.126 Comparative effect of different chelators and vitamin C on liver and kidney zinc levels following lead intoxication Oluwatosin Dosumu 1,∗ , Beno Onunkwor 1 , Oladipo Ademuyiwa 1 , Elizabeth Balogun 2 , Toyin Arowolo 1,2 , Regina Ugbaja 1
http://dx.doi.org/10.1016/j.toxlet.2014.06.812 University of Agriculture, Abeokuta, Nigeria, 2 University of Ilorin, kwara, Nigeria
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P-4.125 An animal model to explore efavirenz toxicokinetics and its relation to neurological phenotype Nádia M. Grilo 1,∗ , Vânia L. Batalha 2 , Luisa V. Lopes 2 , M. Matilde Marques 3 , Emília C. Monteiro 1 , Alexandra M.M. Antunes 3 , Sofia A. Pereira 1 1 Centro de Estudos de Doenc¸as Crónicas (CEDOC), Faculdade de Ciências Médicas, Universidade NOVA, Lisbon, Portugal, 2 Instituto de Medicina Molecular (IMM), Faculdade de Medicina, Universidade de Lisboa, Lisbon, Portugal, 3 Centro de Química Estrutural, Instituto Superior Técnico (CQE-IST), Universidade de Lisboa, Lisbon, Portugal
Efavirenz (EFV) is a drug of choice for patients infected with the immunodeficiency virus (HIV). Notably, 40–70% of the patients suffer from neurological side effects as attention deficit and memory impairment. In vitro studies have suggested the role of the major EFV metabolite, 8-hydroxy-EFV, in the mechanism underlying its neurotoxicity (NT). To obtain further insights into the molecular mechanism of EFV-induced NT, we explored if Wistar rats exposed to EFV could have a toxicokinetics profile and neurological phenotype similar to those observed in man. Male Wistar rats (6 weeks old, 230 ± 21 g, n = 8) were treated orally with EFV (9 mg/kg, once daily for 36 days), and compared with a control group (n = 8). Their behaviour was evaluated through memory performance and stressrelated activity, with consolidated behavioural tests, Morris Water Maze (MWM) and Elevated Plus Maze (EPM). On day 36, rats were sacrificed and EFV and its phase I metabolites, 7-hydroxy-EFV, 8hydroxy-EFV and 8,14-dihydroxy-EFV, were measured in plasma by HPLC. EFV exposed-rats displayed a slower learning curve during the acquisition of the MWM (p = 0.004) and lower anxiety levels in the EPM (p = 0.003). HPLC analyses allowed the detection of 8hydroxy-EFV (4.02 ± 0.51 g/L/g bw) in all samples. The other two EFV metabolites were not detected (<50 g/L). These results attest that EFV-treated Wistar rats can mimic both the neurological phenotype and toxicokinetics profile observed in man. Therefore, it is a good model to identify key players in its NT mechanism, with the ultimate goal for translation into personalized toxicological assessment. Funding: FCT(EXPL/DTP-FTO/0204/2012;PEst-OE/QUI/UI0100/ 2013;SFRH/BD/86791/2012). http://dx.doi.org/10.1016/j.toxlet.2014.06.813
Lead toxicity resulting from different exposure has been treated with chelating agents and/or antioxidants. This study compared the efficacy of conventional chelators (Succimer, d-Penicillamine and CaNa2 EDTA) with vitamin C in relation to zinc depletion following lead intoxication. Eighty four rats divided into 2 groups were used; control (n = 18), administered normal saline and lead group (n = 66), administered 150 mg/kg body weight for 12 weeks. The control and a part of lead group (n = 18) was sub-divided into 3 groups: 12 weeks (A), 12 weeks 5 days (B) and 12 weeks 10 days (C).The remaining lead group were divided into 8 groups (n = 6) and were treated with Succimer and d-Penicillamine (Oral, 30 mg/kg body weight); vitamin C (oral, 150 mg/kg body weight) and CaNa2 EDTA (iv, 30 mg/kg body weight) in 2 divided doses of 5 days each. Twenty-four hours after treatment, liver and kidney of animals were removed. Metal (Pb and Zn) levels, were determined spectrophotometrically. A 4-fold increase in hepatic lead of the 12 weeks lead- treated group (5.02 ± 1.07 g/g) over that of control (1.52 ± 0.49 g/g) was observed. The kidney however showed 8, 19 and 17-fold increase after A, B and C treatments respectively. The hepatic and renal Zn of CaNa2 EDTA-treated animals were significantly decreased by 56% and 62% respectively in B and 68% and 46% in C. While Succimer and d-Penicillamine caused a significant increase in renal zinc level, only Succimer significantly improved the hepatic Zn. Vitamin C treatment however, significantly increased Zn level in both tissues. http://dx.doi.org/10.1016/j.toxlet.2014.06.814 P-4.127 Comparison of acetaminophen toxicity in rat and mouse hepatocytes in vitro Otto Kucera ∗ , Tomas Rousar, Vojtech Mezera, Zuzana Cervinkova Charles University in Prague, Faculty of Medicine in Hradec Kralove, Hradec Kralove, Czech Republic Introduction: Acetaminophen (APAP) hepatotoxicity is often studied in primary cultures of hepatocytes of different species (mouse, rat, hamster, human), but there are only few works comparing interspecies susceptibility of hepatocytes to APAP. The aim of our work was to compare hepatotoxicity of APAP on rat and mouse hepatocytes in primary cultures. Materials and methods: Hepatocytes from male Wistar rats and ICR male mice were isolated by two-step collagenase perfusion. Isolated cells were cultured in William’s E medium on collagen-coated well plates (5% CO2 , 37 ◦ C). After cell attachment, hepatocytes were exposed to APAP (0.25–5 mmol/l) for 24 h. Results: In mouse hepatocytes, 0.75 mM APAP induced a decrease in viability of hepatocytes (WST-1) and an increase in caspase-3 activity in cell lysate. Damage to the cell membrane (LDH leakage), steep decrease in mitochondrial membrane potential (MMP) and increased ROS production (DCFDA) were firstly shown at APAP concentration of 1.25 mmol/l at which the highest activity of caspase-3 was observed. In rat hepatocytes, decreased
Abstracts / Toxicology Letters 229S (2014) S40–S252
viability, increased ROS production and caspase-3 activity were found at 2.5 mM concentration of APAP. An abrupt decrease in MMP and increased LDH leakage were induced by 3.75 mM APAP. Conclusion: APAP displayed dose-dependent toxicity in hepatocytes of both species. Mouse hepatocytes in primary culture exerted approximately 3–4× higher susceptibility to the hepatotoxicity of APAP when compared to rat hepatocytes. Cellular injury parameters exerted very similar course in hepatocytes of both species. Acknowledgements: The study was supported by grants PRVOUK P37/02 and NT/14320-3/2013. http://dx.doi.org/10.1016/j.toxlet.2014.06.815 P-4.128 Localisation of novel miRNA biomarkers of doxorubicin-induced cardiotoxicity in rats Claire Barnes 1 , Janet Kelsall 1 , Susan Smith 1 , Laura Cove-Smith 2 , Howard Mellor 3 , Adam Hargreaves 4 , Sally Price 1,∗ AstraZeneca, Macclesfield, UK, 2 CRUK, University of Manchester, Manchester, UK, 3 Vertex Pharmaceuticals, Oxford, UK, 4 PathCelerate, Macclesfield, UK 1
Cardiotoxicity is the largest cause of attrition in both preclinical and clinical drug development. Discovery of novel biomarkers and understanding the pathological processes in which they are involved could lead to earlier detection of cardiotoxicity and reduce drug attrition. The aim of the current study was to investigate the potential of miRNAs as biomarkers of cardiotoxicity in a rat model of progressive doxorubicin-induced cardiomyopathy. miRNA profiling was carried out on hearts from rats treated with doxorubicin (1.25 mg/kg) weekly for 8 weeks followed by 4 weeks recovery. miRNAs that were up or down regulated with doxorubicin treatment were subsequently investigated by in situ hybridization on hearts from rats treated for either 1, 2, 4, 6 or 8 weeks (±4 weeks recovery). miRNA profiling showed that upregulated miRNAs included miR21, miR221, miR222, miR34A, miR199a-5p and miR551b. Those downregulated included miR30c, miR126a-5p and miR208a. Using in situ hybridization we confirmed that miR21 was absent from control animals but detectable in cardiomyocytes following 6 weeks doxorubicin treatment, with expression increasing with duration of treatment. This study confirmed some of the changes identified by miRNA profiling, including an upregulation of miR21, and localized this change to cardiomyocytes, particularly in the atria. This would support studies linking increased miR21 expression to increased fibrosis in the heart since the changes preceded the onset of fibrosis (8 weeks). The study also suggests that miRNAs may represent a novel class of biomarkers for cardiotoxicity; further investigation may identify certain miRNAs as biomarkers of specific pathological processes. http://dx.doi.org/10.1016/j.toxlet.2014.06.816 P-4.129 Effect of chlorpromazine on rat placenta development Satoshi Furukawa 1,∗ , Seigo Hayashi 1 , Masayoshi Abe 1 , Souichiro Hagio 1 , Kota Irie 1 , Yusuke Kuroda 1 , Izumim Ogawa 1 , Akihiko Sugiyama 2 Nissan Chemical Insustris, LTD., Shiraoka, Saitama, Japan, 2 Tottori University, Tottori, Japan
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We examined the sequential histopathological changes in the placentas from rats exposed to chlorpromazine. Chlorpromazine was intraperitoneally administered on GD 14 at 50 and 100 mg/kg and the placentas were sampled on GDs 14.5, 15, 17 and 21. The incidence of dams with complete fetal resorption was increased from GD 17 up to 20% at 50 mg/kg and 44.4% at 100 mg/kg. The embryo/fetal weights reduced on GDs 15 and 17 at 50 mg/kg and during GDs 15–21 at 100 mg/kg. The placental weights reduced on GD 17 at 50 mg/kg and during GDs 14.5–21 at 100 mg/kg. Histopathologically, in the labyrinth zone, apoptotic cells were scattered in the trophoblastic septa without inhibition of cell proliferation on GDs 14.5 and 15 at 50 and 100 mg/kg in a dosedependent manner. A decrease in trophoblasts led to labyrinth zone hypoplasia. In the basal zone, apoptotic cells were scattered on GDs 14.5 and 15 at 100 mg/kg, and most of them appeared to be glycogen cells. A decrease in glycogen cells induced the delayed development of glycogen cell islands and the subsequent remaining glycogen cell islands, and led to the cystic degeneration of glycogen cells. In addition, failure of development of the glycogen cell islands led to the impaired interstitial invasion of the glycogen cells, and then metrial gland hypoplasia occurred. http://dx.doi.org/10.1016/j.toxlet.2014.06.817 P-4.130 Target organ profiles in toxicity studies supporting human dosing: An assessment of recovery and chronic dosing Steve Horner ∗ , David Lees, Sally Robinson, Richard Callander, Ruth Roberts AstraZeneca, Macclesfield, UK We have previously reported the profile of target organs in rodent and non-rodent toxicity studies conducted prior to first time in man (FTiM) for 77 AstraZeneca candidate drugs (CDs) across a range of therapy areas. Here, we present a further analysis of which target organs (defined as organs showing histopathological changes) observed in these studies subsequently recovered after a dose free period, and whether that profile differed for CDs that progressed into man and by therapy area. We also report which additional target organs were observed in subsequent chronic (≥3 month) studies conducted to support later stage clinical trials. The analysis showed that >86% of findings in studies supporting FTiM either fully or partially resolved at the end of the recovery period, with profiles of recovery that were similar whether the CD progressed into man or not and across different therapy areas. Compared to observations in FTiM studies, chronic studies identified toxicities in an additional 39% of target organs. Overall these data demonstrate that chronic studies in both rodents and nonrodents provide valuable information for the risk assessment for longer term dosing in humans. In addition, the high levels of recovery demonstrated in this analysis suggest that inclusion of recovery assessments on FTiM studies should be on a case-by-case basis driven by a positive indication of need. http://dx.doi.org/10.1016/j.toxlet.2014.06.818
Abstracts / Toxicology Letters 229S (2014) S40–S252
measured. In sepsis-induced rats, MDA levels were increased and SOD and GSH-Px activities, GSH and TNF-␣ levels were decreased. FA treatment decreased the MDA levels and increased the levels of GSH and TNF-␣ and SOD and activities of GSH-Px in the sepsisinduced rats. According to our results, FA has ameliorative effects against oxidative stress caused by sepsis.
P-4.126 Comparative effect of different chelators and vitamin C on liver and kidney zinc levels following lead intoxication Oluwatosin Dosumu 1,∗ , Beno Onunkwor 1 , Oladipo Ademuyiwa 1 , Elizabeth Balogun 2 , Toyin Arowolo 1,2 , Regina Ugbaja 1
http://dx.doi.org/10.1016/j.toxlet.2014.06.812 University of Agriculture, Abeokuta, Nigeria, 2 University of Ilorin, kwara, Nigeria
1
P-4.125 An animal model to explore efavirenz toxicokinetics and its relation to neurological phenotype Nádia M. Grilo 1,∗ , Vânia L. Batalha 2 , Luisa V. Lopes 2 , M. Matilde Marques 3 , Emília C. Monteiro 1 , Alexandra M.M. Antunes 3 , Sofia A. Pereira 1 1 Centro de Estudos de Doenc¸as Crónicas (CEDOC), Faculdade de Ciências Médicas, Universidade NOVA, Lisbon, Portugal, 2 Instituto de Medicina Molecular (IMM), Faculdade de Medicina, Universidade de Lisboa, Lisbon, Portugal, 3 Centro de Química Estrutural, Instituto Superior Técnico (CQE-IST), Universidade de Lisboa, Lisbon, Portugal
Efavirenz (EFV) is a drug of choice for patients infected with the immunodeficiency virus (HIV). Notably, 40–70% of the patients suffer from neurological side effects as attention deficit and memory impairment. In vitro studies have suggested the role of the major EFV metabolite, 8-hydroxy-EFV, in the mechanism underlying its neurotoxicity (NT). To obtain further insights into the molecular mechanism of EFV-induced NT, we explored if Wistar rats exposed to EFV could have a toxicokinetics profile and neurological phenotype similar to those observed in man. Male Wistar rats (6 weeks old, 230 ± 21 g, n = 8) were treated orally with EFV (9 mg/kg, once daily for 36 days), and compared with a control group (n = 8). Their behaviour was evaluated through memory performance and stressrelated activity, with consolidated behavioural tests, Morris Water Maze (MWM) and Elevated Plus Maze (EPM). On day 36, rats were sacrificed and EFV and its phase I metabolites, 7-hydroxy-EFV, 8hydroxy-EFV and 8,14-dihydroxy-EFV, were measured in plasma by HPLC. EFV exposed-rats displayed a slower learning curve during the acquisition of the MWM (p = 0.004) and lower anxiety levels in the EPM (p = 0.003). HPLC analyses allowed the detection of 8hydroxy-EFV (4.02 ± 0.51 g/L/g bw) in all samples. The other two EFV metabolites were not detected (<50 g/L). These results attest that EFV-treated Wistar rats can mimic both the neurological phenotype and toxicokinetics profile observed in man. Therefore, it is a good model to identify key players in its NT mechanism, with the ultimate goal for translation into personalized toxicological assessment. Funding: FCT(EXPL/DTP-FTO/0204/2012;PEst-OE/QUI/UI0100/ 2013;SFRH/BD/86791/2012). http://dx.doi.org/10.1016/j.toxlet.2014.06.813
Lead toxicity resulting from different exposure has been treated with chelating agents and/or antioxidants. This study compared the efficacy of conventional chelators (Succimer, d-Penicillamine and CaNa2 EDTA) with vitamin C in relation to zinc depletion following lead intoxication. Eighty four rats divided into 2 groups were used; control (n = 18), administered normal saline and lead group (n = 66), administered 150 mg/kg body weight for 12 weeks. The control and a part of lead group (n = 18) was sub-divided into 3 groups: 12 weeks (A), 12 weeks 5 days (B) and 12 weeks 10 days (C).The remaining lead group were divided into 8 groups (n = 6) and were treated with Succimer and d-Penicillamine (Oral, 30 mg/kg body weight); vitamin C (oral, 150 mg/kg body weight) and CaNa2 EDTA (iv, 30 mg/kg body weight) in 2 divided doses of 5 days each. Twenty-four hours after treatment, liver and kidney of animals were removed. Metal (Pb and Zn) levels, were determined spectrophotometrically. A 4-fold increase in hepatic lead of the 12 weeks lead- treated group (5.02 ± 1.07 g/g) over that of control (1.52 ± 0.49 g/g) was observed. The kidney however showed 8, 19 and 17-fold increase after A, B and C treatments respectively. The hepatic and renal Zn of CaNa2 EDTA-treated animals were significantly decreased by 56% and 62% respectively in B and 68% and 46% in C. While Succimer and d-Penicillamine caused a significant increase in renal zinc level, only Succimer significantly improved the hepatic Zn. Vitamin C treatment however, significantly increased Zn level in both tissues. http://dx.doi.org/10.1016/j.toxlet.2014.06.814 P-4.127 Comparison of acetaminophen toxicity in rat and mouse hepatocytes in vitro Otto Kucera ∗ , Tomas Rousar, Vojtech Mezera, Zuzana Cervinkova Charles University in Prague, Faculty of Medicine in Hradec Kralove, Hradec Kralove, Czech Republic Introduction: Acetaminophen (APAP) hepatotoxicity is often studied in primary cultures of hepatocytes of different species (mouse, rat, hamster, human), but there are only few works comparing interspecies susceptibility of hepatocytes to APAP. The aim of our work was to compare hepatotoxicity of APAP on rat and mouse hepatocytes in primary cultures. Materials and methods: Hepatocytes from male Wistar rats and ICR male mice were isolated by two-step collagenase perfusion. Isolated cells were cultured in William’s E medium on collagen-coated well plates (5% CO2 , 37 ◦ C). After cell attachment, hepatocytes were exposed to APAP (0.25–5 mmol/l) for 24 h. Results: In mouse hepatocytes, 0.75 mM APAP induced a decrease in viability of hepatocytes (WST-1) and an increase in caspase-3 activity in cell lysate. Damage to the cell membrane (LDH leakage), steep decrease in mitochondrial membrane potential (MMP) and increased ROS production (DCFDA) were firstly shown at APAP concentration of 1.25 mmol/l at which the highest activity of caspase-3 was observed. In rat hepatocytes, decreased
Abstracts / Toxicology Letters 229S (2014) S40–S252
viability, increased ROS production and caspase-3 activity were found at 2.5 mM concentration of APAP. An abrupt decrease in MMP and increased LDH leakage were induced by 3.75 mM APAP. Conclusion: APAP displayed dose-dependent toxicity in hepatocytes of both species. Mouse hepatocytes in primary culture exerted approximately 3–4× higher susceptibility to the hepatotoxicity of APAP when compared to rat hepatocytes. Cellular injury parameters exerted very similar course in hepatocytes of both species. Acknowledgements: The study was supported by grants PRVOUK P37/02 and NT/14320-3/2013. http://dx.doi.org/10.1016/j.toxlet.2014.06.815 P-4.128 Localisation of novel miRNA biomarkers of doxorubicin-induced cardiotoxicity in rats Claire Barnes 1 , Janet Kelsall 1 , Susan Smith 1 , Laura Cove-Smith 2 , Howard Mellor 3 , Adam Hargreaves 4 , Sally Price 1,∗ AstraZeneca, Macclesfield, UK, 2 CRUK, University of Manchester, Manchester, UK, 3 Vertex Pharmaceuticals, Oxford, UK, 4 PathCelerate, Macclesfield, UK 1
Cardiotoxicity is the largest cause of attrition in both preclinical and clinical drug development. Discovery of novel biomarkers and understanding the pathological processes in which they are involved could lead to earlier detection of cardiotoxicity and reduce drug attrition. The aim of the current study was to investigate the potential of miRNAs as biomarkers of cardiotoxicity in a rat model of progressive doxorubicin-induced cardiomyopathy. miRNA profiling was carried out on hearts from rats treated with doxorubicin (1.25 mg/kg) weekly for 8 weeks followed by 4 weeks recovery. miRNAs that were up or down regulated with doxorubicin treatment were subsequently investigated by in situ hybridization on hearts from rats treated for either 1, 2, 4, 6 or 8 weeks (±4 weeks recovery). miRNA profiling showed that upregulated miRNAs included miR21, miR221, miR222, miR34A, miR199a-5p and miR551b. Those downregulated included miR30c, miR126a-5p and miR208a. Using in situ hybridization we confirmed that miR21 was absent from control animals but detectable in cardiomyocytes following 6 weeks doxorubicin treatment, with expression increasing with duration of treatment. This study confirmed some of the changes identified by miRNA profiling, including an upregulation of miR21, and localized this change to cardiomyocytes, particularly in the atria. This would support studies linking increased miR21 expression to increased fibrosis in the heart since the changes preceded the onset of fibrosis (8 weeks). The study also suggests that miRNAs may represent a novel class of biomarkers for cardiotoxicity; further investigation may identify certain miRNAs as biomarkers of specific pathological processes. http://dx.doi.org/10.1016/j.toxlet.2014.06.816 P-4.129 Effect of chlorpromazine on rat placenta development Satoshi Furukawa 1,∗ , Seigo Hayashi 1 , Masayoshi Abe 1 , Souichiro Hagio 1 , Kota Irie 1 , Yusuke Kuroda 1 , Izumim Ogawa 1 , Akihiko Sugiyama 2 Nissan Chemical Insustris, LTD., Shiraoka, Saitama, Japan, 2 Tottori University, Tottori, Japan
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We examined the sequential histopathological changes in the placentas from rats exposed to chlorpromazine. Chlorpromazine was intraperitoneally administered on GD 14 at 50 and 100 mg/kg and the placentas were sampled on GDs 14.5, 15, 17 and 21. The incidence of dams with complete fetal resorption was increased from GD 17 up to 20% at 50 mg/kg and 44.4% at 100 mg/kg. The embryo/fetal weights reduced on GDs 15 and 17 at 50 mg/kg and during GDs 15–21 at 100 mg/kg. The placental weights reduced on GD 17 at 50 mg/kg and during GDs 14.5–21 at 100 mg/kg. Histopathologically, in the labyrinth zone, apoptotic cells were scattered in the trophoblastic septa without inhibition of cell proliferation on GDs 14.5 and 15 at 50 and 100 mg/kg in a dosedependent manner. A decrease in trophoblasts led to labyrinth zone hypoplasia. In the basal zone, apoptotic cells were scattered on GDs 14.5 and 15 at 100 mg/kg, and most of them appeared to be glycogen cells. A decrease in glycogen cells induced the delayed development of glycogen cell islands and the subsequent remaining glycogen cell islands, and led to the cystic degeneration of glycogen cells. In addition, failure of development of the glycogen cell islands led to the impaired interstitial invasion of the glycogen cells, and then metrial gland hypoplasia occurred. http://dx.doi.org/10.1016/j.toxlet.2014.06.817 P-4.130 Target organ profiles in toxicity studies supporting human dosing: An assessment of recovery and chronic dosing Steve Horner ∗ , David Lees, Sally Robinson, Richard Callander, Ruth Roberts AstraZeneca, Macclesfield, UK We have previously reported the profile of target organs in rodent and non-rodent toxicity studies conducted prior to first time in man (FTiM) for 77 AstraZeneca candidate drugs (CDs) across a range of therapy areas. Here, we present a further analysis of which target organs (defined as organs showing histopathological changes) observed in these studies subsequently recovered after a dose free period, and whether that profile differed for CDs that progressed into man and by therapy area. We also report which additional target organs were observed in subsequent chronic (≥3 month) studies conducted to support later stage clinical trials. The analysis showed that >86% of findings in studies supporting FTiM either fully or partially resolved at the end of the recovery period, with profiles of recovery that were similar whether the CD progressed into man or not and across different therapy areas. Compared to observations in FTiM studies, chronic studies identified toxicities in an additional 39% of target organs. Overall these data demonstrate that chronic studies in both rodents and nonrodents provide valuable information for the risk assessment for longer term dosing in humans. In addition, the high levels of recovery demonstrated in this analysis suggest that inclusion of recovery assessments on FTiM studies should be on a case-by-case basis driven by a positive indication of need. http://dx.doi.org/10.1016/j.toxlet.2014.06.818