- Email: [email protected]
Ranking of epoxy resin compounds based on their sensitising potency
Abstracts / Toxicology Letters 221S (2013) S59–S256
alternative data, followed, if necessary, by in vivo testing. The latter does not need to be conducted if there is information indicating that the substance should be classified for sensitisation or corrosivity, if the substance is a strong acid or base or if the substance is flammable in air at room temperature. In terms of in vivo testing, the Murine Local Lymph Node assay (LLNA) is considered to be the first choice. This presentation will describe approaches as currently applied by REACH consortia to the skin sensitization assessment of chemicals. It will discuss the ‘tool box’ of non-animal and animal methodologies available for chemical registrations under REACH and look at its practical application based on case studies. Focus will be on illustrating how non-animal approaches (e.g., QSAR; readacross; grouping/category assessments; WoE analyses) are used to avoid unnecessary animal testing but examples will also be provided where animal testing is still unavoidable. http://dx.doi.org/10.1016/j.toxlet.2013.05.494
P18-19 Non-experimental alternative methods for the hazard evaluation of cosmetics and chemicals Carole Charmeau ∗ , Nathalie Ledirac, Laëtitia Fiévez-Fournier, Muriel Danten, Aurélie Sevestre CEHTRA, France Since 11th March 2013, the use of animal testing on vertebrates for the evaluation of cosmetic ingredients has been prohibited in Europe. Only a few alternative methods, which fully exclude the use of living animals, have been validated by the ECVAM and could be used in regulatory submissions, such as cosmetic or REACH regulations. However, these validated methods do not provide all the requested data needed for regulatory hazard assessments. In this context, non-experimental alternative methods, which take advantage of existing data, without the use of additional animals, are promising. Quantitative Structure Activity Relationships (QSAR) methods use large databases of known substances to predict the toxicity of untested chemicals. They can be used as key studies or to support other scientific approaches like read-across, which consist of using the properties of substances with similar biological activities or modes of action to extrapolate to other substances. These QSAR methods are complex and rely on databases which must be evaluated in terms of chemical structure, relevance and study quality, and require specific expertise to provide robust predictions. However, animal testing is still required to evaluate the effect of substances on complex systems (especially when no experimental data is available on similar substances), or on specific substances such as nanomaterials. Despite these limitations, the development of in silico models, together with the progress of in vitro toxicology, will be a key element to conduct hazard evaluation within the framework of the regulations. http://dx.doi.org/10.1016/j.toxlet.2013.05.495
P18-20 Overview of the existing regulations and testing programs for endocrine active chemicals Simon Warren 1,∗ , Elaine Freeman 1 , Julian Reddy 2 1 Center for Chemical Regulations and Food Safety, Exponent Inc., Washington, DC, USA, 2 Center for Chemical Regulations and Food Safety, Exponent Inc., Derby, UK
S209
We present a global perspective of the regulation of endocrineactive chemicals, giving an overview of the existing global regulations and testing requirements from several authorities. The World Health Organization defines an endocrine disruptor as “an exogenous substance or mixture that alters function(s) of the endocrine system and consequently causes adverse health effects in an intact organism, its progeny, or (sub)-populations” and this is widely accepted. The majority of national regulatory authorities have policy statements which state that endocrine disruption is considered during the risk assessment process (including but not limited to US, Canada, Hong Kong, and Australia). The European Union has stringent legislation covering the regulatory acceptability of endocrine disrupting chemicals, but has not yet agreed the precise circumstances in which the legislation will apply. Japan has issued a list of potential endocrine disruptors; however legislation is not available to determine the testing paradigm necessary to test for endocrine disruptors. The United States (US) Endocrine Disruptor Screening Program (EDSP) is currently the only program requiring specific protocols for potential endocrine testing; other jurisdictions accept OECD guideline rodent studies as appropriate screening procedures. The US has stated that the results of the TIER I EDSP testing are strictly for screening purposes and will not be utilized for risk management. However, the TIER I EDSP test results will be available to other regulatory authorities so may potentially trigger stringent regulation of potential endocrine active compounds. The implications of EDSP testing in regard to global chemical registrations are discussed. http://dx.doi.org/10.1016/j.toxlet.2013.05.496
P18-21 Ranking of epoxy resin compounds based on their sensitising potency Ulrike Schuhmacher-Wolz 1,∗ , Karin Heine 1 , Johannes Geier 2 , Fritz Kalberlah 1 1
FoBiG, Freiburg, Germany, 2 IVDK, Göttingen, Germany
Occupational contact allergy to compounds in epoxy resin systems is frequently observed. A priority approach to reduce the risk of contact allergies would be the substitution of strong skin sensitising compounds by substances with a lower sensitising potency, if technically feasible. Thus, a research project aimed at developing a ranking method regarding the sensitising potency of epoxy resin components. The project was funded by the German Social Accident Insurance. A semi-quantitative assessment of 51 sensitising compounds commonly used in epoxy resin systems was performed using a weight-of-evidence approach. A minimal data set required to classify the sensitisation potency was defined based on the current mechanistic understanding of contact allergy. There were three categories: “very high sensitising potency” (SHS), “high sensitising potency” (HS) and “low or moderate sensitising potency” (GMS). The project focussed on data from in vitro experiments and in silico calculations, due to the lack of sufficient in vivo data in a lot of cases (e.g., LLNA studies or human experience/testing). Up to now, approximately 50% of the substances could be assigned to a specific sensitising potency category, whereas for the remaining substances only default assumptions could be made based on insufficient data (leading to default “HS” classification). In order to facilitate substance selection in epoxy resin product formulations based on toxicological aspects, the results will be publicly accessible on an Internet platform.
S210
Abstracts / Toxicology Letters 221S (2013) S59–S256
The ranking procedure for sensitising potency is outlined and the overall results of the research project are summarised. http://dx.doi.org/10.1016/j.toxlet.2013.05.497
P18-22 Relationship between mortality and dosage scheme as key issue in the design of regulatory toxicological studies of different types of human insulin analogs Joanna Kupny 1,∗ , Inga Mrzyk 1 , Robert Sornat 1 , Agnieszka Drzewiecka 1 , Aneta Kropidlo 1 , Katarzyna Gruszka 1 , Aleksandra Szewczyk 1 , Monika Pawlowska 2 , Monika Bogiel 2 , Piotr Borowicz 2 1 2
Institute of Industrial Organic Chemistry Branch, Pszczyna, Poland, Institute of Biotechnology and Antibiotics, Warsaw, Polen
In toxicological studies of new human insulin analogs performed on healthy, non-diabetic rats mortality is crucial issue to be considered that can allow to maintain a sufficient number of animals and make it possible to obtain a complete view of potentially harmful effects. Toxicological studies of long-acting analogs developed at the Institute of Biotechnology and Antibiotics were made on the basis of OECD and EMA guidelines within GLP. Acute toxicity studies were performed for 6 compounds with an altered primary structure. One analog was selected for 28-day, 3-month and 6-month repeated dose toxicity study. The studies were conducted on Wistar or Sprague-Dawley rats. Different levels of dosage ranging from 125 IU/kg b.w. to 2500 IU/kg b.w. were used in the acute toxicity studies. In the 28day study the doses of 4.9, 22.2 and 100 IU/kg b.w. given twice daily were used. In the 3-month and 6-month study the doses of 5, 10 and 20 IU/kg b.w. given once daily were used. The LD50 values in the acute toxicity studies amounted to over 1000 IU/kg b.w. No mortality was stated in the 28-day and 3-month study. The 6-month study is under way but some mortality was stated with the 4th month of exposure. A full outcome will be presented after the study termination. The results indicate a direct relationship between the animal mortality and the duration of repeated administration. The level of dosage and the increased frequency of repeated dosing seem not to have such a clear influence. http://dx.doi.org/10.1016/j.toxlet.2013.05.498
P18-23 Reproductive toxicity of azole fungicides from a regulatory perspective K. Myöhänen 1,∗ , F. Broeckaert 1 , D. Court Marques 2 , S. Lapenna 1 , A. Maggiore 1 , L. Spjuth 1 , H. Huuskonen 1 ECHA, Annankatu 18, FI-00121 Helsinki, Finland, 2 EFSA, Via Carlo Magno 1A, 43126 Parma, Italy
1
Azole fungicides are a large group of active substances, having a common mechanism in inhibiting the activity of CYP51 in fungi. These compounds may be subject to non-approval under the new Plant Protection Products and Biocidal Products Regulations, if the active substance is classified and labelled according to CLP Regulation (EC) No 1272/2008, e.g. in Reproductive toxicity Category 1A or 1B. The ECHA Risk Assessment Committee (RAC) is responsi-
ble for adopting opinions on classification proposals under the CLP Regulation. We have reviewed three adopted RAC opinions for azoles; epoxiconazole (Repr. 1B; based on cleft palates and postimplantation loss), penconazole (Repr. 2; based on hydrocephalus and microphthalmia) and fuberidazole (no classification; incidental microphthalmia), with an aim to identifying key scientific and regulatory factors that contribute to different classifications for reproductive toxicity. Proposed mechanisms behind the developmental toxicity, including inhibition of aromatase and CYP26 in the retinoic acid signalling pathway, are evaluated in the context of the classification. The key outcome of this review is that the magnitude, incidence, severity and type of developmental effects together with potential maternal toxicity directly affect classification, and may lead to a different classification although the mechanisms may seem similar. The legal basis for the classification for reproductive toxicity is the appropriate CLP criteria and an assessment of the total weight of evidence, which may include information on modes of action. Many other azoles, such as triflumizole, fluquinconazole, imazalil, etridiazole and tebuconazole will be processed by RAC in the near future. http://dx.doi.org/10.1016/j.toxlet.2013.05.499
P18-24 Role of post-translational modifications in a function of pregnane X receptor A. Vavrova ∗ , R. Vrzal, Z. Dvorak Department of Cell Biology and Genetic, Faculty of Science, Palacky University Olomouc, Slechtitelu 11, 783 71 Olomouc, Czech Republic The pregnane X receptor (PXR) is a key xenobiotic receptor that regulates the expression of numerous drug-metabolizing enzymes. Ligand binding is the primary mode of PXR activation, but several signaling pathways that are activated by various developmental or physiological signals also interface with PXR and affect its overall responsiveness to environmental stimuli, likely by altering the posttranslational modification status of PXR and subsequent interaction with its associated protein partners. Acetylation has already been established as important in the regulation of NR biology. While it is clear that PXR is basally acetylated and that it deacetylates upon activation, it remains unclear as to which lysine residues are important for acetylation. Based on the prediction algorithm and the preliminary mass spectrometry results performed by our colleagues, we designed single mutants having acetylation-site mutations. For each residue, we have generated acetylation-deficient mutation (K62R, K210R, K210Q and K109R) and an acetylation-mimicking mutation (K62Q, K210Q, K234Q and K109Q), in order to begin studying the functional consequences of acetylation on PXR activity. Moreover, there are examples of crosstalk between phosphorylation and acetylation, and other modifications for NRs. These findings led us to found specific sites, which can be modified by phosphorylation or acetylation and affect one another or work in combination and thus forming dynamic regulatory programs. We used these mutants to see how they may affect PXR’s various functions as transactivation of target genes, ability to bind its DNA response elements, ability to recruit cofactors and others. http://dx.doi.org/10.1016/j.toxlet.2013.05.500
alternative data, followed, if necessary, by in vivo testing. The latter does not need to be conducted if there is information indicating that the substance should be classified for sensitisation or corrosivity, if the substance is a strong acid or base or if the substance is flammable in air at room temperature. In terms of in vivo testing, the Murine Local Lymph Node assay (LLNA) is considered to be the first choice. This presentation will describe approaches as currently applied by REACH consortia to the skin sensitization assessment of chemicals. It will discuss the ‘tool box’ of non-animal and animal methodologies available for chemical registrations under REACH and look at its practical application based on case studies. Focus will be on illustrating how non-animal approaches (e.g., QSAR; readacross; grouping/category assessments; WoE analyses) are used to avoid unnecessary animal testing but examples will also be provided where animal testing is still unavoidable. http://dx.doi.org/10.1016/j.toxlet.2013.05.494
P18-19 Non-experimental alternative methods for the hazard evaluation of cosmetics and chemicals Carole Charmeau ∗ , Nathalie Ledirac, Laëtitia Fiévez-Fournier, Muriel Danten, Aurélie Sevestre CEHTRA, France Since 11th March 2013, the use of animal testing on vertebrates for the evaluation of cosmetic ingredients has been prohibited in Europe. Only a few alternative methods, which fully exclude the use of living animals, have been validated by the ECVAM and could be used in regulatory submissions, such as cosmetic or REACH regulations. However, these validated methods do not provide all the requested data needed for regulatory hazard assessments. In this context, non-experimental alternative methods, which take advantage of existing data, without the use of additional animals, are promising. Quantitative Structure Activity Relationships (QSAR) methods use large databases of known substances to predict the toxicity of untested chemicals. They can be used as key studies or to support other scientific approaches like read-across, which consist of using the properties of substances with similar biological activities or modes of action to extrapolate to other substances. These QSAR methods are complex and rely on databases which must be evaluated in terms of chemical structure, relevance and study quality, and require specific expertise to provide robust predictions. However, animal testing is still required to evaluate the effect of substances on complex systems (especially when no experimental data is available on similar substances), or on specific substances such as nanomaterials. Despite these limitations, the development of in silico models, together with the progress of in vitro toxicology, will be a key element to conduct hazard evaluation within the framework of the regulations. http://dx.doi.org/10.1016/j.toxlet.2013.05.495
P18-20 Overview of the existing regulations and testing programs for endocrine active chemicals Simon Warren 1,∗ , Elaine Freeman 1 , Julian Reddy 2 1 Center for Chemical Regulations and Food Safety, Exponent Inc., Washington, DC, USA, 2 Center for Chemical Regulations and Food Safety, Exponent Inc., Derby, UK
S209
We present a global perspective of the regulation of endocrineactive chemicals, giving an overview of the existing global regulations and testing requirements from several authorities. The World Health Organization defines an endocrine disruptor as “an exogenous substance or mixture that alters function(s) of the endocrine system and consequently causes adverse health effects in an intact organism, its progeny, or (sub)-populations” and this is widely accepted. The majority of national regulatory authorities have policy statements which state that endocrine disruption is considered during the risk assessment process (including but not limited to US, Canada, Hong Kong, and Australia). The European Union has stringent legislation covering the regulatory acceptability of endocrine disrupting chemicals, but has not yet agreed the precise circumstances in which the legislation will apply. Japan has issued a list of potential endocrine disruptors; however legislation is not available to determine the testing paradigm necessary to test for endocrine disruptors. The United States (US) Endocrine Disruptor Screening Program (EDSP) is currently the only program requiring specific protocols for potential endocrine testing; other jurisdictions accept OECD guideline rodent studies as appropriate screening procedures. The US has stated that the results of the TIER I EDSP testing are strictly for screening purposes and will not be utilized for risk management. However, the TIER I EDSP test results will be available to other regulatory authorities so may potentially trigger stringent regulation of potential endocrine active compounds. The implications of EDSP testing in regard to global chemical registrations are discussed. http://dx.doi.org/10.1016/j.toxlet.2013.05.496
P18-21 Ranking of epoxy resin compounds based on their sensitising potency Ulrike Schuhmacher-Wolz 1,∗ , Karin Heine 1 , Johannes Geier 2 , Fritz Kalberlah 1 1
FoBiG, Freiburg, Germany, 2 IVDK, Göttingen, Germany
Occupational contact allergy to compounds in epoxy resin systems is frequently observed. A priority approach to reduce the risk of contact allergies would be the substitution of strong skin sensitising compounds by substances with a lower sensitising potency, if technically feasible. Thus, a research project aimed at developing a ranking method regarding the sensitising potency of epoxy resin components. The project was funded by the German Social Accident Insurance. A semi-quantitative assessment of 51 sensitising compounds commonly used in epoxy resin systems was performed using a weight-of-evidence approach. A minimal data set required to classify the sensitisation potency was defined based on the current mechanistic understanding of contact allergy. There were three categories: “very high sensitising potency” (SHS), “high sensitising potency” (HS) and “low or moderate sensitising potency” (GMS). The project focussed on data from in vitro experiments and in silico calculations, due to the lack of sufficient in vivo data in a lot of cases (e.g., LLNA studies or human experience/testing). Up to now, approximately 50% of the substances could be assigned to a specific sensitising potency category, whereas for the remaining substances only default assumptions could be made based on insufficient data (leading to default “HS” classification). In order to facilitate substance selection in epoxy resin product formulations based on toxicological aspects, the results will be publicly accessible on an Internet platform.
S210
Abstracts / Toxicology Letters 221S (2013) S59–S256
The ranking procedure for sensitising potency is outlined and the overall results of the research project are summarised. http://dx.doi.org/10.1016/j.toxlet.2013.05.497
P18-22 Relationship between mortality and dosage scheme as key issue in the design of regulatory toxicological studies of different types of human insulin analogs Joanna Kupny 1,∗ , Inga Mrzyk 1 , Robert Sornat 1 , Agnieszka Drzewiecka 1 , Aneta Kropidlo 1 , Katarzyna Gruszka 1 , Aleksandra Szewczyk 1 , Monika Pawlowska 2 , Monika Bogiel 2 , Piotr Borowicz 2 1 2
Institute of Industrial Organic Chemistry Branch, Pszczyna, Poland, Institute of Biotechnology and Antibiotics, Warsaw, Polen
In toxicological studies of new human insulin analogs performed on healthy, non-diabetic rats mortality is crucial issue to be considered that can allow to maintain a sufficient number of animals and make it possible to obtain a complete view of potentially harmful effects. Toxicological studies of long-acting analogs developed at the Institute of Biotechnology and Antibiotics were made on the basis of OECD and EMA guidelines within GLP. Acute toxicity studies were performed for 6 compounds with an altered primary structure. One analog was selected for 28-day, 3-month and 6-month repeated dose toxicity study. The studies were conducted on Wistar or Sprague-Dawley rats. Different levels of dosage ranging from 125 IU/kg b.w. to 2500 IU/kg b.w. were used in the acute toxicity studies. In the 28day study the doses of 4.9, 22.2 and 100 IU/kg b.w. given twice daily were used. In the 3-month and 6-month study the doses of 5, 10 and 20 IU/kg b.w. given once daily were used. The LD50 values in the acute toxicity studies amounted to over 1000 IU/kg b.w. No mortality was stated in the 28-day and 3-month study. The 6-month study is under way but some mortality was stated with the 4th month of exposure. A full outcome will be presented after the study termination. The results indicate a direct relationship between the animal mortality and the duration of repeated administration. The level of dosage and the increased frequency of repeated dosing seem not to have such a clear influence. http://dx.doi.org/10.1016/j.toxlet.2013.05.498
P18-23 Reproductive toxicity of azole fungicides from a regulatory perspective K. Myöhänen 1,∗ , F. Broeckaert 1 , D. Court Marques 2 , S. Lapenna 1 , A. Maggiore 1 , L. Spjuth 1 , H. Huuskonen 1 ECHA, Annankatu 18, FI-00121 Helsinki, Finland, 2 EFSA, Via Carlo Magno 1A, 43126 Parma, Italy
1
Azole fungicides are a large group of active substances, having a common mechanism in inhibiting the activity of CYP51 in fungi. These compounds may be subject to non-approval under the new Plant Protection Products and Biocidal Products Regulations, if the active substance is classified and labelled according to CLP Regulation (EC) No 1272/2008, e.g. in Reproductive toxicity Category 1A or 1B. The ECHA Risk Assessment Committee (RAC) is responsi-
ble for adopting opinions on classification proposals under the CLP Regulation. We have reviewed three adopted RAC opinions for azoles; epoxiconazole (Repr. 1B; based on cleft palates and postimplantation loss), penconazole (Repr. 2; based on hydrocephalus and microphthalmia) and fuberidazole (no classification; incidental microphthalmia), with an aim to identifying key scientific and regulatory factors that contribute to different classifications for reproductive toxicity. Proposed mechanisms behind the developmental toxicity, including inhibition of aromatase and CYP26 in the retinoic acid signalling pathway, are evaluated in the context of the classification. The key outcome of this review is that the magnitude, incidence, severity and type of developmental effects together with potential maternal toxicity directly affect classification, and may lead to a different classification although the mechanisms may seem similar. The legal basis for the classification for reproductive toxicity is the appropriate CLP criteria and an assessment of the total weight of evidence, which may include information on modes of action. Many other azoles, such as triflumizole, fluquinconazole, imazalil, etridiazole and tebuconazole will be processed by RAC in the near future. http://dx.doi.org/10.1016/j.toxlet.2013.05.499
P18-24 Role of post-translational modifications in a function of pregnane X receptor A. Vavrova ∗ , R. Vrzal, Z. Dvorak Department of Cell Biology and Genetic, Faculty of Science, Palacky University Olomouc, Slechtitelu 11, 783 71 Olomouc, Czech Republic The pregnane X receptor (PXR) is a key xenobiotic receptor that regulates the expression of numerous drug-metabolizing enzymes. Ligand binding is the primary mode of PXR activation, but several signaling pathways that are activated by various developmental or physiological signals also interface with PXR and affect its overall responsiveness to environmental stimuli, likely by altering the posttranslational modification status of PXR and subsequent interaction with its associated protein partners. Acetylation has already been established as important in the regulation of NR biology. While it is clear that PXR is basally acetylated and that it deacetylates upon activation, it remains unclear as to which lysine residues are important for acetylation. Based on the prediction algorithm and the preliminary mass spectrometry results performed by our colleagues, we designed single mutants having acetylation-site mutations. For each residue, we have generated acetylation-deficient mutation (K62R, K210R, K210Q and K109R) and an acetylation-mimicking mutation (K62Q, K210Q, K234Q and K109Q), in order to begin studying the functional consequences of acetylation on PXR activity. Moreover, there are examples of crosstalk between phosphorylation and acetylation, and other modifications for NRs. These findings led us to found specific sites, which can be modified by phosphorylation or acetylation and affect one another or work in combination and thus forming dynamic regulatory programs. We used these mutants to see how they may affect PXR’s various functions as transactivation of target genes, ability to bind its DNA response elements, ability to recruit cofactors and others. http://dx.doi.org/10.1016/j.toxlet.2013.05.500