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Assessment of pre- and pro-haptens using non-animal test methods for skin sensitization
S306
Abstracts / Toxicology Letters 258S (2016) S62–S324
ies, nasal tissue is the first site of contact. Due to the size of the nasal tissue, collection and preparation of single cell population requires skilled technicians and validated technique, and hence Comet assay in nasal tissues is less routinely performed. To satisfy the validation requirements as per the OECD Comet assay guideline 489 and in order to build historical database, male Sprague Dawley rats were dosed with vehicle (saline) and the positive control ethyl methanesulfonate (EMS) at 200 mg/kg using oral gavage. Five independent experiments with two groups of five animals in each were conducted. Nasal tissues were collected and processed to prepare single cell suspensions as per the Test Guidelines. The comet assay results from these five experiments demonstrated that oral dosing of EMS resulted in significant DNA damage in the nasal tissue (% tail DNA range from 9.63 to 20.55) and the response is significantly higher than the vehicle control (% tail DNA range from 0.18 to 0.39). The work presented in this poster is a proof of concept experiment to validate nasal tissue extraction, cell suspension preparation in detail and to demonstrate that Comet assay can detect the DNA strand breaks in nasal tissues. In summary, this technique can be used to prepare single cell population to perform Comet assay on nasal tissues for inhalation studies. http://dx.doi.org/10.1016/j.toxlet.2016.06.2045 P22-002 Assessment of pre- and pro-haptens using non-animal test methods for skin sensitization D. Urbisch 1 , N. Honarvar 1,∗ , S.N. Kolle 1 , B. Wareing 1 , M. Becker 1 , A. Mehling 2 , W. Teubner 3 , R. Landsiedel 1 1
Experimental Toxicology and Ecology, BASF, SE, Germany BASF Personal Care and Nutrition GmbH, Düsseldorf, Germany 3 BASF Schweiz AG, Basel, Switzerland 2
Due to ethical and regulatory reasons, several non-animal test to assess skin sensitization potential of chemicals have been developed and validated. In contrast to animal tests, they lack or provide limited metabolic capacity. For this reason, identification of pro-haptens but also pre-haptens, which require molecular transformations to gain peptide reactivity, is a challenge for these methods. In this study, 27 pre- and pro-haptens were tested using nonanimal tests. Of these, 18 provided true positive results in the DPRA, although lacking structural alerts for direct peptide reactivity. The reaction mechanisms leading to peptide depletions were therefore elucidated using mass spectrometry. Hapten-peptide adducts were identified for 13 of the 18 chemicals indicating that these prehaptens were activated and that peptide binding occurred. Positive results for five of the 18 chemicals can be explained by dipeptide formations or the oxidation of the sulfhydryl group of the peptide. Nine of the 27 chemicals were tested negative in the DPRA. Of these, four yielded true positive results in cell-based assays. Likewise, 16 of the 18 chemicals tested positive in the DPRA were also positive in either one or both of the cell-based assays. A combination of DPRA, KeratinoSensTM and h-CLAT used in a ‘2 out of 3’ WoE approach identified 22 of the 27 pre- and pro-haptens correctly (sensitivity of 81%), exhibiting a similar sensitivity as for directly acting haptens. This analysis shows that the combination of in chemico and in vitro test methods is suitable to identify pre-haptens and the majority of pro-haptens. http://dx.doi.org/10.1016/j.toxlet.2016.06.2046
P22-003 Determination of thyroxine (T4) in mouse plasma by microsampling P. Singh ∗ , J. Perron, R. Michaud, M. Fonsi, Y. Lambert, R. Forster CiToxLAB France, Evreux, France The evaluation of thyroxine (T4) levels is now routinely incorporated into regulatory toxicology studies, due to concerns over potential impacts of drug or chemical exposures on thyroid hormone homeostasis. Blood volume and sampling frequency are often limiting factors for the evaluation of thyroid hormone levels in toxicology studies. The objective of our study was to improve blood sampling and bioanalytical techniques in order to allow multiple sampling of individual mice over the full duration of the study. The experiment included 20 untreated CD1 mice sampled for T4 levels (6 timepoints over 24 h) on 2 occasions separated by 6 weeks and at one additional timepoint every 2 weeks in between. Fifty uL of blood/timepoint was collected using a heparinised capillary. The capillary was placed in a conical tube for centrifugation, and spun to separate the plasma from the blood. T4 plasma levels ranged from 8 to 45 ng/ml. The results indicate that a slight decrease in T4 concentrations appears at the beginning of the nocturnal cycle. Levels of T4 showed both individual variation throughout the day and inter-individual variations during the study. The results are indicative of circadian variation in T4 levels in mice between age 6 and 13 weeks. In addition, this approach is in line with 3R principles and could reduce the number of animals utilized for bioanalysis in toxicology studies without limiting the statistical interpretation of the data. http://dx.doi.org/10.1016/j.toxlet.2016.06.2047 P22-004 A novel methodology to test dry dislodgeable foliar residue of agrochemical spray with in OECD 428 test guideline M. Aggarwal 1,∗ , W. Maas 2 , P. Fisher 3 , N. Morgan 4 , R. Parr Dobrzanski 4 , M. Soufi 5 , C. Strupp 6 , C. Wiemann 7 1
Dow AgroSciences Ltd., UK TNO Triskelion, Netherlands 3 Bayer CropScience, France 4 Syngenta Ltd., UK 5 DuPont de Nemours GmbH, Germany 6 ADAMA MAH BV Amsterdam NL Schaffhausen Branch, Switzerland 7 BASF Oesterreich GmbH, Austria 2
During agrochemical use, dermal exposure may occur to the undiluted product and/or the diluted spray. Once the spray has dried, re-entry workers can enter the field and may be exposed to dried dislodgeable foliage residues (DFR) present on contact surfaces. To determine systemic exposure following dermal exposure, dermal absorption (DA) studies are typically performed using the product and representative in-use spray(s). However, a methodology to test DFR for DA is currently lacking. In this study, a novel methodology was developed to create 14Clabeled DFR and its transfer on to the human skin in vitro to perform a standard OECD 428 study. A pre-determined volume of spray containing 14C-labeled substance was transferred onto a PTFE-coated septum for air drying. The septum was placed onto the wet skin surface mounted in a flow-through Franz diffusion chamber without applying manual pressure (avoiding skin damage) and was rotated three-times to transfer the dose. A pre-test was also performed to
Abstracts / Toxicology Letters 258S (2016) S62–S324
ies, nasal tissue is the first site of contact. Due to the size of the nasal tissue, collection and preparation of single cell population requires skilled technicians and validated technique, and hence Comet assay in nasal tissues is less routinely performed. To satisfy the validation requirements as per the OECD Comet assay guideline 489 and in order to build historical database, male Sprague Dawley rats were dosed with vehicle (saline) and the positive control ethyl methanesulfonate (EMS) at 200 mg/kg using oral gavage. Five independent experiments with two groups of five animals in each were conducted. Nasal tissues were collected and processed to prepare single cell suspensions as per the Test Guidelines. The comet assay results from these five experiments demonstrated that oral dosing of EMS resulted in significant DNA damage in the nasal tissue (% tail DNA range from 9.63 to 20.55) and the response is significantly higher than the vehicle control (% tail DNA range from 0.18 to 0.39). The work presented in this poster is a proof of concept experiment to validate nasal tissue extraction, cell suspension preparation in detail and to demonstrate that Comet assay can detect the DNA strand breaks in nasal tissues. In summary, this technique can be used to prepare single cell population to perform Comet assay on nasal tissues for inhalation studies. http://dx.doi.org/10.1016/j.toxlet.2016.06.2045 P22-002 Assessment of pre- and pro-haptens using non-animal test methods for skin sensitization D. Urbisch 1 , N. Honarvar 1,∗ , S.N. Kolle 1 , B. Wareing 1 , M. Becker 1 , A. Mehling 2 , W. Teubner 3 , R. Landsiedel 1 1
Experimental Toxicology and Ecology, BASF, SE, Germany BASF Personal Care and Nutrition GmbH, Düsseldorf, Germany 3 BASF Schweiz AG, Basel, Switzerland 2
Due to ethical and regulatory reasons, several non-animal test to assess skin sensitization potential of chemicals have been developed and validated. In contrast to animal tests, they lack or provide limited metabolic capacity. For this reason, identification of pro-haptens but also pre-haptens, which require molecular transformations to gain peptide reactivity, is a challenge for these methods. In this study, 27 pre- and pro-haptens were tested using nonanimal tests. Of these, 18 provided true positive results in the DPRA, although lacking structural alerts for direct peptide reactivity. The reaction mechanisms leading to peptide depletions were therefore elucidated using mass spectrometry. Hapten-peptide adducts were identified for 13 of the 18 chemicals indicating that these prehaptens were activated and that peptide binding occurred. Positive results for five of the 18 chemicals can be explained by dipeptide formations or the oxidation of the sulfhydryl group of the peptide. Nine of the 27 chemicals were tested negative in the DPRA. Of these, four yielded true positive results in cell-based assays. Likewise, 16 of the 18 chemicals tested positive in the DPRA were also positive in either one or both of the cell-based assays. A combination of DPRA, KeratinoSensTM and h-CLAT used in a ‘2 out of 3’ WoE approach identified 22 of the 27 pre- and pro-haptens correctly (sensitivity of 81%), exhibiting a similar sensitivity as for directly acting haptens. This analysis shows that the combination of in chemico and in vitro test methods is suitable to identify pre-haptens and the majority of pro-haptens. http://dx.doi.org/10.1016/j.toxlet.2016.06.2046
P22-003 Determination of thyroxine (T4) in mouse plasma by microsampling P. Singh ∗ , J. Perron, R. Michaud, M. Fonsi, Y. Lambert, R. Forster CiToxLAB France, Evreux, France The evaluation of thyroxine (T4) levels is now routinely incorporated into regulatory toxicology studies, due to concerns over potential impacts of drug or chemical exposures on thyroid hormone homeostasis. Blood volume and sampling frequency are often limiting factors for the evaluation of thyroid hormone levels in toxicology studies. The objective of our study was to improve blood sampling and bioanalytical techniques in order to allow multiple sampling of individual mice over the full duration of the study. The experiment included 20 untreated CD1 mice sampled for T4 levels (6 timepoints over 24 h) on 2 occasions separated by 6 weeks and at one additional timepoint every 2 weeks in between. Fifty uL of blood/timepoint was collected using a heparinised capillary. The capillary was placed in a conical tube for centrifugation, and spun to separate the plasma from the blood. T4 plasma levels ranged from 8 to 45 ng/ml. The results indicate that a slight decrease in T4 concentrations appears at the beginning of the nocturnal cycle. Levels of T4 showed both individual variation throughout the day and inter-individual variations during the study. The results are indicative of circadian variation in T4 levels in mice between age 6 and 13 weeks. In addition, this approach is in line with 3R principles and could reduce the number of animals utilized for bioanalysis in toxicology studies without limiting the statistical interpretation of the data. http://dx.doi.org/10.1016/j.toxlet.2016.06.2047 P22-004 A novel methodology to test dry dislodgeable foliar residue of agrochemical spray with in OECD 428 test guideline M. Aggarwal 1,∗ , W. Maas 2 , P. Fisher 3 , N. Morgan 4 , R. Parr Dobrzanski 4 , M. Soufi 5 , C. Strupp 6 , C. Wiemann 7 1
Dow AgroSciences Ltd., UK TNO Triskelion, Netherlands 3 Bayer CropScience, France 4 Syngenta Ltd., UK 5 DuPont de Nemours GmbH, Germany 6 ADAMA MAH BV Amsterdam NL Schaffhausen Branch, Switzerland 7 BASF Oesterreich GmbH, Austria 2
During agrochemical use, dermal exposure may occur to the undiluted product and/or the diluted spray. Once the spray has dried, re-entry workers can enter the field and may be exposed to dried dislodgeable foliage residues (DFR) present on contact surfaces. To determine systemic exposure following dermal exposure, dermal absorption (DA) studies are typically performed using the product and representative in-use spray(s). However, a methodology to test DFR for DA is currently lacking. In this study, a novel methodology was developed to create 14Clabeled DFR and its transfer on to the human skin in vitro to perform a standard OECD 428 study. A pre-determined volume of spray containing 14C-labeled substance was transferred onto a PTFE-coated septum for air drying. The septum was placed onto the wet skin surface mounted in a flow-through Franz diffusion chamber without applying manual pressure (avoiding skin damage) and was rotated three-times to transfer the dose. A pre-test was also performed to