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P16: Potent carcinogenicity of madder-color-related alizarin and rubiadin in a rat medium-term multi-organ bioassay
ARTICLE IN PRESS Abstracts / Experimental and Toxicologic Pathology 61 (2009) 387–413
(4) intermediate type of astrocytoma and malignant reticulosis: 2 cases (SD: 1 case, F344: 1 case); neoplastic cells are infiltrated more diffusely in meninges and perivascular space with obvious parenchymal infiltration. (5) malignant reticulosis: 2 cases (both in F344); neoplastic cells are infiltrated diffusely in meninges and perivascular space without obvious parenchymal infiltration. The neoplastic cells from all categories showed various immunoreactivities for vimentin, RM-4 (antirat macrophages and interdigitating cells) and ED-1. However, no distinct difference among these categories was observed. The positive cells for RM-4 mainly corresponded to those of ED-1. The reactivity for vimentin was more intense than that of RM-4 and ED-1. A few neoplastic cells from the common and glioblastoma types demonstrated positive reaction for nestin. No reactivity for GFAP or S-100 was observed in any case of any category. Perivascular or intercellular reticulin fibers which were stained with Watanabe’s silver impregnation method were observed in some or many cases in each category except for the early stage of astrocytoma. The results of the malignant reticulosis generally coincided with those of astrocytomas. In conclusion, no difference in the cell origin between astrocytoma and malignant reticulosis could be found. doi: 10.1016/j.etp.2009.02.100
P15 Use of rasH2 transgenic mice for carcinogenesis testing of medical implants Xavier Palazzi, Sylvie Kergozien-Framery BIOMATECH-NAMSA, 115 rue Pasteur, ZI De L’Islon, 38670 CHASSE SUR RHONE – France Several transgenic mice models are accepted by regulatory agencies to determine the carcinogenic potential and predict the human response to exposure of chemicals, as an alternative to the conventional 2-year rodent bioassay. Regarding medical device evaluation, ISO 10993-3 lists several situations that require carcinogenicity testing: absorbable materials, materials that obtained positive results in genetic toxicity on mammalian cells or materials with a permanent or cumulative contact of 30 days or longer in the body, except when significant and adequate human-use history is available. The rasH2 transgenic mouse model has been proposed to evaluate the carcinogenic potential of medical devices, but very few data are currently available regarding study
409
design-namely appropriate positive and negative controls to be used-, as well as pathology historical data. From in-house pre-existing experience and active collaboration with our rasH2 mouse breeder, BIOMATECH-NAMSA recently conducted a 26 week carcinogenicity study following subcutaneous implantation in the transgenic rasH2 mouse model. This poster depicts the study design and the main results obtained in the positive and negative control groups. The survival rate statistical (Kaplan-Meier) analysis showed that the survival rate was significantly affected by the occurrence of tumors in the positive control group when compared to the negative control group, in both genders. Incidence tables were obtained and discussed for hyperplastic and neoplastic findings in control groups. The positive control group revealed an increase in the incidence of neoplastic lesions. Thymic malignant lymphomas and squamous cell papillomas were reported to occur at a higher incidence in rasH2 mice exposed to a known chemical carcinogen, for terminally sacrificed animals as well as for unscheduled and terminally sacrificed animals considered together. Background and age-related lesions were few. Taken together, these data confirmed the reliability and usefulness of the rasH2 transgenic model in the assessment of carcinogenic properties of medical devices. A major beneficial property of this animal model consisted in the ability to demonstrate chemical carcinogenesis response without the solid-state tumorigenesis response seen in traditional 2-year rodent bioassays. doi: 10.1016/j.etp.2009.02.101
P16 Potent carcinogenicity of madder-color-related alizarin and rubiadin in a rat medium-term multi-organ bioassay Kaoru Inouea, Midori Yoshidaa, Miwa Takahashia, Hitoshi Fujimotoa, Makoto Shibutania,b, Masao Hirosea,c, Akiyoshi Nishikawaa a Div. Pathol., Natl. Inst. Health Sci., Tokyo 158-8501, Japan b Lab. Vet. Pathol., Tokyo University Agri. Tech., Tokyo 185-8509, Japan c Food Safety Commission, Tokyo 100-8989, Japan Madder color (MC), a food coloring extracted from roots of Rubia tinctorum L. and used in Japan, proved to have carcinogenic potential on the rat kidney and liver. In addition, madder root is reported to induce DNA adducts in the kidney, liver and large intestine (Westendorf et al., 1998). MC is composed of some
ARTICLE IN PRESS 410
Abstracts / Experimental and Toxicologic Pathology 61 (2009) 387–413
anthraquinones including lucidin-3-O-primeveroside (LuP) and alizarin (Alz). To clarify which component is responsible for carcinogenicity, a rat medium-term multi-organ carcinogenesis bioassay was performed, mainly focusing on the kidney, liver and large intestine. Male 6-week-old F344 rats receiving 5 different carcinogens were fed a diet containing either 0.008% or 0.04% of Alz or rubiadin (Rub), a metabolite of LuP, for 23 weeks. In the kidney, 0.04% Rub increased the incidences of atypical tubules and hyperplasias significantly (po0.01 and po0.05, respectively) in the renal outer medulla, a carcinogenic target site of MC, and also induced renal cell adenomas and carcinomas (15% and 10% of animals involved, respectively). The incidences of atypical hyperplasias and renal cell tumors were also increased with 0.04% Alz compared to control, despite being lower than those with Rub. In the liver, the numbers of GST-P-positive foci and incidence of hepatocellular adenomas were also increased with Rub with or without significance (po0.01), but not with Alz. In the large intestine, both incidence and multiplicity of dysplasias were significantly increased only with 0.04% Rub. These results indicate the kidney as a carcinogenic target of both Rub and Alz, the carcinogenic potential of the latter being weaker. Rub may also target liver and large intestine, suggesting that Rub plays a major role in MC-induced carcinogenicity. doi: 10.1016/j.etp.2009.02.102
Miscellaneous P17 Lethal rhinitis/sinusitis in rodents by aspiration of formulation in gavage studies: Importance of evaluation of the nose S. De Jonghe, L. Lammens, A. Raoof, K. Steemans, F. Broeckaert, J. Verbeeck, F. Van Goethem, G. Hanton J&J Pharmaceutical Research and Development, Turnhoutseweg 30, 2340 Beerse, Belgium For repeated dose oral gavage studies in rodents, OECD guidelines do not include the nasal turbinates in the list of recommended tissues for histopathologic examination. During the past few years, unexpected high mortality was noted with two different compounds in rodents for which the cause of death was mainly based on histological evaluation of the nose. For compound A (a solution in PEG 400), unexpected high mortality was encountered early in the carcinogenicity studies. The dose-related increase in mortality
correlated with an increase in inflammatory and necrotic or ulcerative changes within the respiratory tract of the preterminal deaths, often involving the nose and sinuses. For several animals rhinitis/sinusitis (mainly involving the lower half of the nose and most prominently affecting the respiratory epithelium) was the most important finding. The respiratory tract findings were consistent with irritation. The irritant nature of the formulation was confirmed in a BCOP-test. It was concluded that a part of the irritant formulation entered into the nose/airways via aspiration during gavagedosing. This was considered to be related to the high viscosity of the vehicle (PEG400). The problem was further prevented by adapting the gavage procedure and by reducing the volume and/or concentration of the formulation. Compound B (also a solution in PEG 400) resulted in high mortality at the high dose in the 3 m mouse study, from the 1st week onwards. At necropsy, the preterminally dead/sacrificed animals showed gastrointestinal dilatation with test formulation being present in the stomach. Acute, necrotizing rhinitis/sinusitis was the cause of death for the majority of these mice. Only a minority of these animals showed lesions in other parts of the respiratory tract. The test article and PEG 400 (to a lesser extent) were demonstrated to delay gastric emptying in a separate pharmacology study. This effect, together with the high viscosity of the vehicle and the irritant nature of the compound formulation resulted in entry of the formulation in the respiratory tract (probably due to regurgitation/aspiration) with subsequent inflammatory/necrotizing changes. For the majority of mice, the cause of death would not have been evident without evaluation of the nose. In conclusion, evaluation of the nose can be valuable to determine the cause of death in rodents. doi: 10.1016/j.etp.2009.02.103
P18 Relationship between osteoid formations and iron deposition induced by chronic cadmium exposure in ovariectomized rats Yumi Wakoa, Hideaki Hiratsukaa, Yoshimasa Kurataa, Minoru Tsuchitania, Takashi Umemurab a Mitsubishi Chemical Safety Institute Ltd., Ibaraki, Japan b Hokkaido University Graduate School Of Veterinary Medicine, Hokkaido, Japan Itai-Itai disease (IID) like osteomalacia is induced by long-term administration of cadmium (Cd) in rat; however the pathological mechanism is still unclear. Hiratsuka et al. reported that iron deposition was
(4) intermediate type of astrocytoma and malignant reticulosis: 2 cases (SD: 1 case, F344: 1 case); neoplastic cells are infiltrated more diffusely in meninges and perivascular space with obvious parenchymal infiltration. (5) malignant reticulosis: 2 cases (both in F344); neoplastic cells are infiltrated diffusely in meninges and perivascular space without obvious parenchymal infiltration. The neoplastic cells from all categories showed various immunoreactivities for vimentin, RM-4 (antirat macrophages and interdigitating cells) and ED-1. However, no distinct difference among these categories was observed. The positive cells for RM-4 mainly corresponded to those of ED-1. The reactivity for vimentin was more intense than that of RM-4 and ED-1. A few neoplastic cells from the common and glioblastoma types demonstrated positive reaction for nestin. No reactivity for GFAP or S-100 was observed in any case of any category. Perivascular or intercellular reticulin fibers which were stained with Watanabe’s silver impregnation method were observed in some or many cases in each category except for the early stage of astrocytoma. The results of the malignant reticulosis generally coincided with those of astrocytomas. In conclusion, no difference in the cell origin between astrocytoma and malignant reticulosis could be found. doi: 10.1016/j.etp.2009.02.100
P15 Use of rasH2 transgenic mice for carcinogenesis testing of medical implants Xavier Palazzi, Sylvie Kergozien-Framery BIOMATECH-NAMSA, 115 rue Pasteur, ZI De L’Islon, 38670 CHASSE SUR RHONE – France Several transgenic mice models are accepted by regulatory agencies to determine the carcinogenic potential and predict the human response to exposure of chemicals, as an alternative to the conventional 2-year rodent bioassay. Regarding medical device evaluation, ISO 10993-3 lists several situations that require carcinogenicity testing: absorbable materials, materials that obtained positive results in genetic toxicity on mammalian cells or materials with a permanent or cumulative contact of 30 days or longer in the body, except when significant and adequate human-use history is available. The rasH2 transgenic mouse model has been proposed to evaluate the carcinogenic potential of medical devices, but very few data are currently available regarding study
409
design-namely appropriate positive and negative controls to be used-, as well as pathology historical data. From in-house pre-existing experience and active collaboration with our rasH2 mouse breeder, BIOMATECH-NAMSA recently conducted a 26 week carcinogenicity study following subcutaneous implantation in the transgenic rasH2 mouse model. This poster depicts the study design and the main results obtained in the positive and negative control groups. The survival rate statistical (Kaplan-Meier) analysis showed that the survival rate was significantly affected by the occurrence of tumors in the positive control group when compared to the negative control group, in both genders. Incidence tables were obtained and discussed for hyperplastic and neoplastic findings in control groups. The positive control group revealed an increase in the incidence of neoplastic lesions. Thymic malignant lymphomas and squamous cell papillomas were reported to occur at a higher incidence in rasH2 mice exposed to a known chemical carcinogen, for terminally sacrificed animals as well as for unscheduled and terminally sacrificed animals considered together. Background and age-related lesions were few. Taken together, these data confirmed the reliability and usefulness of the rasH2 transgenic model in the assessment of carcinogenic properties of medical devices. A major beneficial property of this animal model consisted in the ability to demonstrate chemical carcinogenesis response without the solid-state tumorigenesis response seen in traditional 2-year rodent bioassays. doi: 10.1016/j.etp.2009.02.101
P16 Potent carcinogenicity of madder-color-related alizarin and rubiadin in a rat medium-term multi-organ bioassay Kaoru Inouea, Midori Yoshidaa, Miwa Takahashia, Hitoshi Fujimotoa, Makoto Shibutania,b, Masao Hirosea,c, Akiyoshi Nishikawaa a Div. Pathol., Natl. Inst. Health Sci., Tokyo 158-8501, Japan b Lab. Vet. Pathol., Tokyo University Agri. Tech., Tokyo 185-8509, Japan c Food Safety Commission, Tokyo 100-8989, Japan Madder color (MC), a food coloring extracted from roots of Rubia tinctorum L. and used in Japan, proved to have carcinogenic potential on the rat kidney and liver. In addition, madder root is reported to induce DNA adducts in the kidney, liver and large intestine (Westendorf et al., 1998). MC is composed of some
ARTICLE IN PRESS 410
Abstracts / Experimental and Toxicologic Pathology 61 (2009) 387–413
anthraquinones including lucidin-3-O-primeveroside (LuP) and alizarin (Alz). To clarify which component is responsible for carcinogenicity, a rat medium-term multi-organ carcinogenesis bioassay was performed, mainly focusing on the kidney, liver and large intestine. Male 6-week-old F344 rats receiving 5 different carcinogens were fed a diet containing either 0.008% or 0.04% of Alz or rubiadin (Rub), a metabolite of LuP, for 23 weeks. In the kidney, 0.04% Rub increased the incidences of atypical tubules and hyperplasias significantly (po0.01 and po0.05, respectively) in the renal outer medulla, a carcinogenic target site of MC, and also induced renal cell adenomas and carcinomas (15% and 10% of animals involved, respectively). The incidences of atypical hyperplasias and renal cell tumors were also increased with 0.04% Alz compared to control, despite being lower than those with Rub. In the liver, the numbers of GST-P-positive foci and incidence of hepatocellular adenomas were also increased with Rub with or without significance (po0.01), but not with Alz. In the large intestine, both incidence and multiplicity of dysplasias were significantly increased only with 0.04% Rub. These results indicate the kidney as a carcinogenic target of both Rub and Alz, the carcinogenic potential of the latter being weaker. Rub may also target liver and large intestine, suggesting that Rub plays a major role in MC-induced carcinogenicity. doi: 10.1016/j.etp.2009.02.102
Miscellaneous P17 Lethal rhinitis/sinusitis in rodents by aspiration of formulation in gavage studies: Importance of evaluation of the nose S. De Jonghe, L. Lammens, A. Raoof, K. Steemans, F. Broeckaert, J. Verbeeck, F. Van Goethem, G. Hanton J&J Pharmaceutical Research and Development, Turnhoutseweg 30, 2340 Beerse, Belgium For repeated dose oral gavage studies in rodents, OECD guidelines do not include the nasal turbinates in the list of recommended tissues for histopathologic examination. During the past few years, unexpected high mortality was noted with two different compounds in rodents for which the cause of death was mainly based on histological evaluation of the nose. For compound A (a solution in PEG 400), unexpected high mortality was encountered early in the carcinogenicity studies. The dose-related increase in mortality
correlated with an increase in inflammatory and necrotic or ulcerative changes within the respiratory tract of the preterminal deaths, often involving the nose and sinuses. For several animals rhinitis/sinusitis (mainly involving the lower half of the nose and most prominently affecting the respiratory epithelium) was the most important finding. The respiratory tract findings were consistent with irritation. The irritant nature of the formulation was confirmed in a BCOP-test. It was concluded that a part of the irritant formulation entered into the nose/airways via aspiration during gavagedosing. This was considered to be related to the high viscosity of the vehicle (PEG400). The problem was further prevented by adapting the gavage procedure and by reducing the volume and/or concentration of the formulation. Compound B (also a solution in PEG 400) resulted in high mortality at the high dose in the 3 m mouse study, from the 1st week onwards. At necropsy, the preterminally dead/sacrificed animals showed gastrointestinal dilatation with test formulation being present in the stomach. Acute, necrotizing rhinitis/sinusitis was the cause of death for the majority of these mice. Only a minority of these animals showed lesions in other parts of the respiratory tract. The test article and PEG 400 (to a lesser extent) were demonstrated to delay gastric emptying in a separate pharmacology study. This effect, together with the high viscosity of the vehicle and the irritant nature of the compound formulation resulted in entry of the formulation in the respiratory tract (probably due to regurgitation/aspiration) with subsequent inflammatory/necrotizing changes. For the majority of mice, the cause of death would not have been evident without evaluation of the nose. In conclusion, evaluation of the nose can be valuable to determine the cause of death in rodents. doi: 10.1016/j.etp.2009.02.103
P18 Relationship between osteoid formations and iron deposition induced by chronic cadmium exposure in ovariectomized rats Yumi Wakoa, Hideaki Hiratsukaa, Yoshimasa Kurataa, Minoru Tsuchitania, Takashi Umemurab a Mitsubishi Chemical Safety Institute Ltd., Ibaraki, Japan b Hokkaido University Graduate School Of Veterinary Medicine, Hokkaido, Japan Itai-Itai disease (IID) like osteomalacia is induced by long-term administration of cadmium (Cd) in rat; however the pathological mechanism is still unclear. Hiratsuka et al. reported that iron deposition was