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An efficient direct exposure method for studying the effects of native atmospheres
Abstracts / Toxicology Letters 189S (2009) S57–S273
Small heterocycles, especially epoxides have a wide industrial application, and it is known that some of them are able to react with nucleophilic groups in proteins and DNA. So far the toxicity of these heterocycles towards ciliates is difficult to predict, and only few experimental data are available. For the systematic scrutiny of their reactive toxicity we used the growth impairment assay with the ciliates Tetrahymena pyriformis at different exposure times. By means of testing also substances exerting baseline toxicity, we calibrate the corresponding exposure-time-dependent baseline levels that in turn are used as reference to quantify excess toxicity. The discussion includes a comparison with known structural alerts for identifying excess toxicity potentials. Acknowledgements: Financial support from NOMIRACLE (EU contract no. 003956) and OSIRIS (EU contract no. 037017) is gratefully acknowledged. doi:10.1016/j.toxlet.2009.06.309
M20 Application of developmental neurotoxicology: In vivo and postmortem practical approaches Germano Oberto 1,∗ , R.A. Manno 1 , E. Vezzali 1 , L. Andreini 1 , R. Cicalese 1 , G. Krinke 2
S63
with the medium. Systems which rely on the solution of the gas in the overlaying medium do not resemble the exposure conditions in vivo, and may not be suitable to study, for example, the effects of poorly soluble gases. The most promising approach is based on a biphasic cell culture technique, where cells are grown on microporous membranes at an air–liquid interface. In this way, the cells can be nutrified from the basal side of the membrane whereas the apical part with the cultivated cells is in direct contact with the test atmosphere, meaning gaseous and/or particulate compounds. This type of exposure requires (1) a special exposure device and conditions, (2) close contact between the cultivated cells and the inhalable substances without interfering with the medium—conditions which are represented by the CULTEX® technology. Specially designed exposure modules allow the treatment of the cultivated cell monolayer as well as tissue constructs at the air–liquid interface under defined conditions. This type of treatment results in dose-dependent reactions with regard to cyto- and genotoxicity or cell activation associated, e.g. with the release of bioactive mediators. Thus, in vitro studies using such a strategy in combination with relevant and efficient exposure devices may open up new ways to test native complex gases and aerosols not only under laboratory conditions, but also in real indoor and outdoor situations. doi:10.1016/j.toxlet.2009.06.311
Research Toxicology Centre S.p.A., Pomezia, Italy, 2 Independent Consultant, Frenkendorf, Switzerland
1
M22 Developmental Neurotoxicity Testing (DNT) are required by the EPA and OECD guidelines as an essential tool to detect developmental neurotoxicants and prevent neural risk with particular attention to effects on learning and memory in paediatric population. However, these guidelines only provide flexible recommendations for the conduction of such studies. This presentation describes RTC’s procedures in conducting developmental neuropathology studies, discussing the examination time point, approach for tissue handling, fixation, collection, processing, embedding and staining and, finally, the qualitative and computer-assisted quantitative methods of evaluation adopted. In summary, this is an overview on RTC’s in vivo and post-mortem approaches in this field, according to the guideline requirements, based on a broad literature review and as a result of the experience acquired during different experimental developmental neurotoxicity studies. Keywords: Developmental neuropathology; DNT studies; Guidelines; Practical approaches doi:10.1016/j.toxlet.2009.06.310
M21 An efficient direct exposure method for studying the effects of native atmospheres Michaela Aufderheide CULTEX Laboratories GmbH, Hannover, Germany In vitro studies in the field of inhalation toxicology suffer a number of problems due to the difficulties in exposing cells of the respiratory tract directly to inhalable substances, i.e. in a way comparable to the in vivo situation. The exposure of cells to test atmospheres requires the precise control of the pollutant levels, as well as the close contact of cells and gas without interfering
夽 The COLIPA strategy for animal-free genotoxicity testing Walter Diembeck 1,∗ , Paul Carmichael 2 , Marilyn Aardema 3 , Rolf Fautz 4 , James Harvey 5 , Nicky Hewitt 1 , Alain Latil 6 , Monique Marrec-Fairley 7 , Stefan Pfuhler 8 , Gladys Quedraogo 9 , Kerstin Reisinger 10 1
Beiersdorf AG, Hamburg, Germany, 2 Unilever, Sharnbrook, United Kingdom, 3 The Procter & Gamble Co., Cincinnati, OH, United States, 4 KPSS-Kao Professional Salon Services, Darmstadt, Germany, 5 GSK, Ware, United Kingdom, 6 Pierre Fabre, Toulouse, France, 7 COLIPA, Brussels, Belgium, 8 Procter & Gamble-Cosmital SA, Marly, Switzerland, 9 L’Oréal Life Sciences Research, Aulnay sous Bois, France, 10 Henkel AG & Co. KGaA, Duesseldorf, Germany
From the 11th March this year, the first of the two deadlines to the 7th Amendment to the Cosmetics Directive came into effect. This implemented a ban on genotoxicity testing of chemical ingredients in cosmetics using animals, and has perhaps had less focus from an animal alternatives standpoint because sensitive in vitro genetic toxicology assays already exist. However, it has become clear that the regulatory-required battery of these in vitro genotoxicity tests has a low specificity (i.e. a high percentage of irrelevant positive results for non-carcinogens). Where, in the past, this would have created the need for unnecessary in vivo follow up testing, the cosmetics industry is now left with the erroneous rejection of valuable new ingredients. To address this problem, the EU Cosmetics Association (COLIPA) Genotoxicity Task Force has been funding, directing and conducting a major program to develop approaches for genotoxicity testing of cosmetic ingredients. The program consists of three main projects: (i) a three-year project performed at Covance Laboratories (UK) to optimize current mammalian cell assays in order to improve specificity, (ii) a two and a half year project led by member companies of the cosmetics industry which aims to establish and validate new methods for genotoxicity testing using reconstructed human 3D
Small heterocycles, especially epoxides have a wide industrial application, and it is known that some of them are able to react with nucleophilic groups in proteins and DNA. So far the toxicity of these heterocycles towards ciliates is difficult to predict, and only few experimental data are available. For the systematic scrutiny of their reactive toxicity we used the growth impairment assay with the ciliates Tetrahymena pyriformis at different exposure times. By means of testing also substances exerting baseline toxicity, we calibrate the corresponding exposure-time-dependent baseline levels that in turn are used as reference to quantify excess toxicity. The discussion includes a comparison with known structural alerts for identifying excess toxicity potentials. Acknowledgements: Financial support from NOMIRACLE (EU contract no. 003956) and OSIRIS (EU contract no. 037017) is gratefully acknowledged. doi:10.1016/j.toxlet.2009.06.309
M20 Application of developmental neurotoxicology: In vivo and postmortem practical approaches Germano Oberto 1,∗ , R.A. Manno 1 , E. Vezzali 1 , L. Andreini 1 , R. Cicalese 1 , G. Krinke 2
S63
with the medium. Systems which rely on the solution of the gas in the overlaying medium do not resemble the exposure conditions in vivo, and may not be suitable to study, for example, the effects of poorly soluble gases. The most promising approach is based on a biphasic cell culture technique, where cells are grown on microporous membranes at an air–liquid interface. In this way, the cells can be nutrified from the basal side of the membrane whereas the apical part with the cultivated cells is in direct contact with the test atmosphere, meaning gaseous and/or particulate compounds. This type of exposure requires (1) a special exposure device and conditions, (2) close contact between the cultivated cells and the inhalable substances without interfering with the medium—conditions which are represented by the CULTEX® technology. Specially designed exposure modules allow the treatment of the cultivated cell monolayer as well as tissue constructs at the air–liquid interface under defined conditions. This type of treatment results in dose-dependent reactions with regard to cyto- and genotoxicity or cell activation associated, e.g. with the release of bioactive mediators. Thus, in vitro studies using such a strategy in combination with relevant and efficient exposure devices may open up new ways to test native complex gases and aerosols not only under laboratory conditions, but also in real indoor and outdoor situations. doi:10.1016/j.toxlet.2009.06.311
Research Toxicology Centre S.p.A., Pomezia, Italy, 2 Independent Consultant, Frenkendorf, Switzerland
1
M22 Developmental Neurotoxicity Testing (DNT) are required by the EPA and OECD guidelines as an essential tool to detect developmental neurotoxicants and prevent neural risk with particular attention to effects on learning and memory in paediatric population. However, these guidelines only provide flexible recommendations for the conduction of such studies. This presentation describes RTC’s procedures in conducting developmental neuropathology studies, discussing the examination time point, approach for tissue handling, fixation, collection, processing, embedding and staining and, finally, the qualitative and computer-assisted quantitative methods of evaluation adopted. In summary, this is an overview on RTC’s in vivo and post-mortem approaches in this field, according to the guideline requirements, based on a broad literature review and as a result of the experience acquired during different experimental developmental neurotoxicity studies. Keywords: Developmental neuropathology; DNT studies; Guidelines; Practical approaches doi:10.1016/j.toxlet.2009.06.310
M21 An efficient direct exposure method for studying the effects of native atmospheres Michaela Aufderheide CULTEX Laboratories GmbH, Hannover, Germany In vitro studies in the field of inhalation toxicology suffer a number of problems due to the difficulties in exposing cells of the respiratory tract directly to inhalable substances, i.e. in a way comparable to the in vivo situation. The exposure of cells to test atmospheres requires the precise control of the pollutant levels, as well as the close contact of cells and gas without interfering
夽 The COLIPA strategy for animal-free genotoxicity testing Walter Diembeck 1,∗ , Paul Carmichael 2 , Marilyn Aardema 3 , Rolf Fautz 4 , James Harvey 5 , Nicky Hewitt 1 , Alain Latil 6 , Monique Marrec-Fairley 7 , Stefan Pfuhler 8 , Gladys Quedraogo 9 , Kerstin Reisinger 10 1
Beiersdorf AG, Hamburg, Germany, 2 Unilever, Sharnbrook, United Kingdom, 3 The Procter & Gamble Co., Cincinnati, OH, United States, 4 KPSS-Kao Professional Salon Services, Darmstadt, Germany, 5 GSK, Ware, United Kingdom, 6 Pierre Fabre, Toulouse, France, 7 COLIPA, Brussels, Belgium, 8 Procter & Gamble-Cosmital SA, Marly, Switzerland, 9 L’Oréal Life Sciences Research, Aulnay sous Bois, France, 10 Henkel AG & Co. KGaA, Duesseldorf, Germany
From the 11th March this year, the first of the two deadlines to the 7th Amendment to the Cosmetics Directive came into effect. This implemented a ban on genotoxicity testing of chemical ingredients in cosmetics using animals, and has perhaps had less focus from an animal alternatives standpoint because sensitive in vitro genetic toxicology assays already exist. However, it has become clear that the regulatory-required battery of these in vitro genotoxicity tests has a low specificity (i.e. a high percentage of irrelevant positive results for non-carcinogens). Where, in the past, this would have created the need for unnecessary in vivo follow up testing, the cosmetics industry is now left with the erroneous rejection of valuable new ingredients. To address this problem, the EU Cosmetics Association (COLIPA) Genotoxicity Task Force has been funding, directing and conducting a major program to develop approaches for genotoxicity testing of cosmetic ingredients. The program consists of three main projects: (i) a three-year project performed at Covance Laboratories (UK) to optimize current mammalian cell assays in order to improve specificity, (ii) a two and a half year project led by member companies of the cosmetics industry which aims to establish and validate new methods for genotoxicity testing using reconstructed human 3D