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New Intralaboratory assessment criteria for the UDS-assay in vitro
Bark ofAbsmacrs- EUROlyIX ‘94
IO
New lntraMoratory
Aswssment Criteria for the UDS-Assay in V&o
S.Y. Erendler-Schwaab ‘, H. Mager*. H. Lehn3. 8. Herbold l. l Depeftment of Genotoxicity.InstiMe of fndusttial Bayer AG, Aprather T~icofogJ: Ba@r AG, Apmther Weg, D4ZOg6 ~ppertal, Germany; 2 Depattment of &met& k-t&g*D-42096 wlrppertal. Germany; 3 Department of OuaIity Assurance. Bayer AG, Aprather Weg, 042096 INuppertat. Germany Routinely the UDS-test (Unscheduled DNA Synthesislin vitro with rat hepatocytes is performed using the autoradiographical method. For evaluationthe amount of incorporated 3H-Thymidine (indicating DNA repair) is determined by calculating the nettograin (NNG) value = nuclear grains (NG) - cytoplasmic grains (CG). A conservative estimation of a positive UDS response (fixed limit value = +5 or +3 NNGI was used over a quite long period [?.2]. Since more advanced techniques have been developed the NNG control values decreased. Therefore, newer attempts try to define the limit for a positive result on the basis of intralaboratory considerations 13.41.It was our aim to combine a statistical method adapted to the UDS data with our historical data. This approach is compared to the use of the statistical method based solely on the concurrent vehicle control. An additional step for a sensible evaluation may be not to fully subtract the CG value from the NG value. Alternatively, a correction factor for the CG count rns; be used thus avoiding some overestimation of the CG above the nucleus. This correction factor is based on the intralaboratory historical data. With this approach it seems possible to define a reasonable intralaboratorylimr value for a positive UDS response in vitro. [l ] [2] [3] [4]
Williams G. Cancer Res. 37.1846 ( 2977). Butterworth et al., Mut. Res. 189.113 (1987:. Casciano DA b Gaylor DW, Mut. Res. 122,81 (1983). Brambilla G b Ma&Ii A. Mut. Res. 272,Q il992).
Key words: UDS in vitro; limit value; positive test result; statistical method; historical data
A Serum-Free Neurotoxiclty Screening System Using Embryonic Chick Cells (SCENT) A. Bruinink. J. Miiller. 3. Shah-Derier.C. Sidler, I? Reiser, F.Birchler,T. Rasonyi. G. Zimmerman. tnsf. of Toxicology .E3Yh iiniZti&h. (X-8603 Schwerzenbach Neurotox;dty testing with intact animals have several shortcomings, including slow “throughput*’of compounds in St&c% which do not generate m~hanistic data and high costs. In order to overcome these disa~antages an in vitro modei was developed (SCENT: Serum-free Chicken Embryonic culture system for Neurotoxicity Testing)using combinations of serum-free flat sedimented cultures of embryonic chick neuronal retina, retinal pigment epithelium (RPE), brain and meningeal cells, and reaggregate ceil cultures of the neuronal retina. The validity of the model was tested with eight different compounds of established toxicity for man: bismuth, aluminum, cisplatin, chloroquine. 0RG.2677. l-~~rabinofuranosvl~~os~ne, taurine and ochratoxin A. In order to faithfully reproduce the established in viva toxicity in vitro. the postulated and established mechanisms and sites of action of each compound were considered in the experimental approach. The results obtained were in agreement with in viva data with respect to la) target area and cell type, (b) the toxic concentrations in culture medium compared with concentrations in blood, (cl the area under the m~ium concentration-time curve (AUC) compa~ng critical blood AUK-inducing cisplatin toxicity. (dt mechanisms of action as far as these are known in v&o. Furthermore, additional information concerning to mechanisms of toxicity have been provided by SCENT.The potential application of the present model in predicting human toxicity in a pm-screen procedure is currently being further evaluated.
Work Environment lnftuence on Cytostatlca-induced Genotoxicity in Oncologic Nurses V Brumen. D. Horvat. institute for Medical Research and Occupational Health, Zagreb, Cm&a The aim of the study is to point out the difference in chromosomal damage incidence among nurses handling cytostatics in various workplace conditions. The study comprised two groups of nurses in charge of cytostatics’ preparation and administration and the correspondent control group. The exposed groups, comparable in all the relevant aspects (age. duration of exposure. cytoststics most commonly handled, proportion of subjects with a positive history of some of the inte~ering factors known to affect the ~hromosomal damage inc~ence), except for the extremely different workplace conditions, consisted of 17 oncologic nurses each. The genotoxic effect of such an occupational exposure ‘was established by sister chromatid exchange (SC@ method, using peripheral blood lymphocyte culture as cellular material. In the group of nurses provided with a safe working environment, the SCE-frequencyrate was insignif~antlv increased when compared to the controls, though wide SCE-ranges were obtained. On the contrary, among those provided neither with such an environment nor with the appropriate personal protective equipment, the SCE-incidence was significantly higher not only than in controls (p c 0.001). but also than in the first testgroup (p e 0.001). pointing out the influence of diffeiant workplace conditions on chromosomal damage incidence.
IO
New lntraMoratory
Aswssment Criteria for the UDS-Assay in V&o
S.Y. Erendler-Schwaab ‘, H. Mager*. H. Lehn3. 8. Herbold l. l Depeftment of Genotoxicity.InstiMe of fndusttial Bayer AG, Aprather T~icofogJ: Ba@r AG, Apmther Weg, D4ZOg6 ~ppertal, Germany; 2 Depattment of &met& k-t&g*D-42096 wlrppertal. Germany; 3 Department of OuaIity Assurance. Bayer AG, Aprather Weg, 042096 INuppertat. Germany Routinely the UDS-test (Unscheduled DNA Synthesislin vitro with rat hepatocytes is performed using the autoradiographical method. For evaluationthe amount of incorporated 3H-Thymidine (indicating DNA repair) is determined by calculating the nettograin (NNG) value = nuclear grains (NG) - cytoplasmic grains (CG). A conservative estimation of a positive UDS response (fixed limit value = +5 or +3 NNGI was used over a quite long period [?.2]. Since more advanced techniques have been developed the NNG control values decreased. Therefore, newer attempts try to define the limit for a positive result on the basis of intralaboratory considerations 13.41.It was our aim to combine a statistical method adapted to the UDS data with our historical data. This approach is compared to the use of the statistical method based solely on the concurrent vehicle control. An additional step for a sensible evaluation may be not to fully subtract the CG value from the NG value. Alternatively, a correction factor for the CG count rns; be used thus avoiding some overestimation of the CG above the nucleus. This correction factor is based on the intralaboratory historical data. With this approach it seems possible to define a reasonable intralaboratorylimr value for a positive UDS response in vitro. [l ] [2] [3] [4]
Williams G. Cancer Res. 37.1846 ( 2977). Butterworth et al., Mut. Res. 189.113 (1987:. Casciano DA b Gaylor DW, Mut. Res. 122,81 (1983). Brambilla G b Ma&Ii A. Mut. Res. 272,Q il992).
Key words: UDS in vitro; limit value; positive test result; statistical method; historical data
A Serum-Free Neurotoxiclty Screening System Using Embryonic Chick Cells (SCENT) A. Bruinink. J. Miiller. 3. Shah-Derier.C. Sidler, I? Reiser, F.Birchler,T. Rasonyi. G. Zimmerman. tnsf. of Toxicology .E3Yh iiniZti&h. (X-8603 Schwerzenbach Neurotox;dty testing with intact animals have several shortcomings, including slow “throughput*’of compounds in St&c% which do not generate m~hanistic data and high costs. In order to overcome these disa~antages an in vitro modei was developed (SCENT: Serum-free Chicken Embryonic culture system for Neurotoxicity Testing)using combinations of serum-free flat sedimented cultures of embryonic chick neuronal retina, retinal pigment epithelium (RPE), brain and meningeal cells, and reaggregate ceil cultures of the neuronal retina. The validity of the model was tested with eight different compounds of established toxicity for man: bismuth, aluminum, cisplatin, chloroquine. 0RG.2677. l-~~rabinofuranosvl~~os~ne, taurine and ochratoxin A. In order to faithfully reproduce the established in viva toxicity in vitro. the postulated and established mechanisms and sites of action of each compound were considered in the experimental approach. The results obtained were in agreement with in viva data with respect to la) target area and cell type, (b) the toxic concentrations in culture medium compared with concentrations in blood, (cl the area under the m~ium concentration-time curve (AUC) compa~ng critical blood AUK-inducing cisplatin toxicity. (dt mechanisms of action as far as these are known in v&o. Furthermore, additional information concerning to mechanisms of toxicity have been provided by SCENT.The potential application of the present model in predicting human toxicity in a pm-screen procedure is currently being further evaluated.
Work Environment lnftuence on Cytostatlca-induced Genotoxicity in Oncologic Nurses V Brumen. D. Horvat. institute for Medical Research and Occupational Health, Zagreb, Cm&a The aim of the study is to point out the difference in chromosomal damage incidence among nurses handling cytostatics in various workplace conditions. The study comprised two groups of nurses in charge of cytostatics’ preparation and administration and the correspondent control group. The exposed groups, comparable in all the relevant aspects (age. duration of exposure. cytoststics most commonly handled, proportion of subjects with a positive history of some of the inte~ering factors known to affect the ~hromosomal damage inc~ence), except for the extremely different workplace conditions, consisted of 17 oncologic nurses each. The genotoxic effect of such an occupational exposure ‘was established by sister chromatid exchange (SC@ method, using peripheral blood lymphocyte culture as cellular material. In the group of nurses provided with a safe working environment, the SCE-frequencyrate was insignif~antlv increased when compared to the controls, though wide SCE-ranges were obtained. On the contrary, among those provided neither with such an environment nor with the appropriate personal protective equipment, the SCE-incidence was significantly higher not only than in controls (p c 0.001). but also than in the first testgroup (p e 0.001). pointing out the influence of diffeiant workplace conditions on chromosomal damage incidence.