For hazard classifications of chemicals, continuous data from animal-or nonanimal testing methods... more For hazard classifications of chemicals, continuous data from animal-or nonanimal testing methods are often dichotomized into binary positive/negative outcomes by defining classification thresholds (CT). Experimental data are, however, subject to biological and technical variability. Each test method's precision is limited resulting in uncertainty of the positive/negative outcome if the experimental result is close to the CT. Borderline ranges (BR) around the CT were suggested, which represent ranges in which the study result is ambiguous, that is, positive or negative results are equally likely. The BR reflects a method's precision uncertainty. This article explores and compares different approaches to quantify the BR. Besides using the pooled standard deviation, we determine the BR by means of the median absolute deviation (MAD), with a sequential combination of both methods, and by using nonparametric bootstrapping. Furthermore, we quantify the BR for different hazardous effects, including nonanimal tests for skin corrosion, eye irritation, skin irritation, and skin sensitization as well as for an animal test on skin sensitization (the local lymph node assay, LLNA). Additionally, for one method (direct peptide reactivity assay) the BR was determined experimentally and compared to calculated BRs. Our results demonstrate that (i) the precision of the methods is determining the size of their BRs, (ii) there is no "perfect" method to derive a BR, alas, (iii) a consensus on BR is needed to account for the limited precision of testing methods.
The availability of reference data is a key requirement for the development of new approach metho... more The availability of reference data is a key requirement for the development of new approach methods (NAM), i.e., in vitro, in chemico and in silico methods and integrated approaches, like defined approaches (DA), which combine these data sources. Reference data are of even greater importance for regulatory acceptance. In contrast to most other adverse effects, human skin sensitization data on many chemicals are available, next to data from animal studies, such as the local lymph node assay (LLNA). Skin sensitization NAM data can therefore be compared to different reference datasets. Recent publications and validation at the OECD focused on human and LLNA reference data. The "2 out of 3" DA (2o3 DA) is the first DA for skin sensitization solely based on experimental data from validated tests and was recently adopted as an OECD test guideline. Here we review the predictivity of the 2o3 DA on multiple human and LLNA reference datasets. Concomitantly, we compare the predictivity of the LLNA for human data within the same datasets. Comparing predictivity of methods not only bilaterally (NAM or DA vs. animal method) but including human data in a triangle "NAM data-animal data-human data" offers a comprehensive assessment of the NAM's and DA's predictivity. In all these assessments, the 2o3 DA was superior to the LLNA in predicting human skin sensitization hazard. This highlights the importance of a holistic view of reference data instead of limiting validation of NAMs and DAs to data from a single animal test only.
The molecular initiating event in the adverse outcome pathway for skin sensitization is the coval... more The molecular initiating event in the adverse outcome pathway for skin sensitization is the covalent binding of the sensitizer to skin proteins, and a first method to address this key event was adopted as OECD TG 442C in 2015. This method, the direct peptide reactivity assay (DPRA), uses two synthetic peptides (one containing a cysteine, one containing a lysine residue) that are incubated with a single concentration of the test substance. After 24 hours incubation, the concentrations of remaining, non-depleted peptide are determined using HPLC. All currently adopted non-animal OECD TGs to assess skin sensitization, including the DPRA, provide information on the
ated in the peer-review of the validation studies, nor has a formalized way to use this quantitat... more ated in the peer-review of the validation studies, nor has a formalized way to use this quantitative information reached test guideline status. Nevertheless, multiple studies have investigated application of quantitative in vitro data from the validated assays for potency prediction (
In regulatory toxicology, in vivo studies are still prevailing, and human-derived in vitro models... more In regulatory toxicology, in vivo studies are still prevailing, and human-derived in vitro models are mostly used in testing for local toxicity to the skin and the eyes. A single in vitro model may be limited to address one or few molecular or cellular events leading to adverse outcomes. Hence, in many instances their regulatory use involves the combination of several in vitro models to assess the hazard potential of test substance. A so-called defined approach combines different testing methods and a 'data interpretation procedure' to obtain a comprehensive overall assessment which is used for the regulatory hazard classification of the test substance.Validation is a prerequisite of regulatory acceptance of new testing methods: This chapter provides an overview of the method development from an experimental method to a test guideline via application of GIVIMP (good in vitro method practice), standardization, validation to the regulatory adoption as an OECD test guidelines. Quandaries associated with the validation towards reference data from in vivo animal studies with limited accuracy and limited human relevance are discussed, as well as uncertainty and limitations arising from restricted applicability and technical and biological variance of the in vitro methods.This chapter provides an overview of human-derived in vitro models currently adopted as OECD test guidelines: From the first skin corrosion tests utilizing reconstructed human epidermis models (RhE), to models to test for skin irritation, phototoxicity, eye irritation, and skin sensitization. The latter is using a battery of different methods and defined approaches which are still under discussion for their regulatory adoption. They will be a vanguard of future applications of human-derived models in regulatory toxicology. RhEs for testing of genotoxicity and of dermal penetration and absorption, have been developed, underwent validation studies and may soon be adopted for regulatory use; these are included in this chapter.
Regulatory Toxicology and Pharmacology, Apr 1, 2013
Skin corrosion or irritation refers to the production of irreversible or reversible damage to the... more Skin corrosion or irritation refers to the production of irreversible or reversible damage to the skin following the application of a test substance, respectively. Traditionally, hazard assessments are conducted using the in vivo Draize skin test, but recently in vitro tests using reconstructed human epidermis (RhE) models have gained regulatory acceptance. In this study, skin corrosion (SCT) and irritation tests (SIT) using a RhE model were implemented to reduce the number of in vivo tests required by regulatory bodies. One hundred and thirty-four materials were tested from a wide range of substance classes included 46 agrochemical formulations. Results were assessed according to UN GHS, EU-CLP, ANVISA and US EPA classification schemes. There was high correlation between the two in vitro tests. Assessment of the SCT sensitivity was not possible due to the limited number of corrosives in the data set; SCT specificity and accuracy were 89% for all classification systems. Accuracy (63-76%) and sensitivity (53-67%) were low in the SIT. Specificity and concordance for agrochemical formulations alone in both the SCT and SIT were comparable to the values for the complete data set (SCT: 91% vs. 89% specificity, 91% vs. 89% accuracy and SIT: 64-88% vs. 70-85% specificity, 56-75% vs. 63-76% accuracy).
Skin sensitization is a key endpoint in both hazard and risk assessments. The pathways involved h... more Skin sensitization is a key endpoint in both hazard and risk assessments. The pathways involved have recently been formally described in the OECD adverse outcome pathway (AOP). Currently, no single non-animal test method is sufficient to fully address this AOP and therefore the use of a battery of tests is necessary to cover this endpoint. Test methods addressing AOP key events include the direct peptide reactivity assay (DPRA) (molecular initiating event: 'peptide reactivity'; OECD TG 442C), the LuSens and KeratinoSens TM (cellular response: 'keratinocyte activation'; OECD TG 442D), and the human cell-line activation test (h-CLAT; draft OECD TG) as well as the (modified) myeloid U937 skin sensitization test ((m)MUSST; 2nd cellular response: 'dendritic cell activation'). To assess the overall skin sensitizing potentials of the substances, a simple '2 out of 3' integrated testing strategy (ITS) was applied (Urbisch et al., 2015, PMID: 25541156). In order to facilitate regulatory acceptance of these methods, test results of 213 substances were compared to both local lymph node assay (LLNA) and human data, where available. The individual test methods provide predictivities between 73 and 76% or 78 and 84%, when compared to LLNA or human data, respectively. The '2 out of 3' ITS correctly predicts 79% of the human and 90% of the human data. These results demonstrate that the non-animal test methods correlate better with human data than with LLNA data. In addition, different mechanistic domains were identified by probable protein-binding mechanisms of the 213 substances. This approach shows that all assays predict Michael acceptors with the highest accuracies (≥80%). In the domain of acylating agents, the keratinocyte based assays show lower predictivities of ≤58%. In summary, taking mechanistic domains into account offers a more accurate estimation of the predictive performance of the individual non-animal test methods as well as the overall ITS prediction.
P-09-03 / Toxicology Letters 280S (2017) S280-S290 are available which limits the practical aspec... more P-09-03 / Toxicology Letters 280S (2017) S280-S290 are available which limits the practical aspect of testing a compound. The poster will also describe data and knowledge sharing schemes that have been developed to enhance the expert alerts and provide more detailed structure activity relationship (SAR) information. This methodology utilizes proprietary information without revealing any confidential data and incorporates SAR information from 35,000 compounds with mutagenicity data. The results have been used to improve the overall performance of the Leadscope expert alerts system as well as continued improvements to specific classes including aromatic amines, aromatic amides, aryl boronic acids, and alkyl halides.
The selection of reference and proficiency chemicals is an important component of method validati... more The selection of reference and proficiency chemicals is an important component of method validation and proficiency evaluations. Reference chemicals are a set of test substances used by a method developer to evaluate the reliability and relevance of a new method, in comparison to reference data (usually to a validated reference method). Proficiency chemicals, as defined in OECD Guidance Document on Good In Vitro Method Practices, are defined post validation as a subset of the reference chemicals, or other chemicals with sufficient supporting data, that are used by naïve laboratories to demonstrate technical competence with a validated test method. Proficiency chemicals should cover different physical states, several chemical classes within the applicability domain of the method and yield the full range of responses (in the validated reference method and in vivo). They shall be commercially available (at non-prohibitive costs) and have high quality reference data. If reference and subsequent proficiency chemicals are chosen without sufficient evidence for their inclusion, both test method evaluation and demonstration of technical proficiency can be hampered. In this report we present cases in which the selection of reference chemicals led to problems in the reproduction of the reference results and demonstration of technical proficiency.
Letter to the editor: "Evaulation of radioisotopic and non-radioisotopic versions of local lymph ... more Letter to the editor: "Evaulation of radioisotopic and non-radioisotopic versions of local lymph node assays for subcategorization of skin sensitiers compliant to UN GHS rev 4" by Ha et al., 2017
Toxicology in vitro : an international journal published in association with BIBRA, 2016
The molecular initiating event (MIE) of skin sensitization is the binding of a hapten to dermal p... more The molecular initiating event (MIE) of skin sensitization is the binding of a hapten to dermal proteins. This can be assessed using the in chemico direct peptide reactivity assay (DPRA) or in silico tools such as the QSAR Toolbox and TIMES SS. In this study, the suitability of these methods was analyzed by comparing their results to in vivo sensitization data of LLNA and human studies. Compared to human data, 84% of non-sensitizers and sensitizers yielded consistent results in the DPRA. In silico tools resulted in 'no alert' for 83%-100% of the non-sensitizers, but alerted only 55%-61% of the sensitizers. The inclusion of biotic and abiotic transformation simulations yielded more alerts for sensitizers, but simultaneously dropped the number of non-alerted non-sensitizers. In contrast to the DPRA, in silico tools were more consistent with results of the LLNA than human data. Interestingly, the new "DPRA profilers" (QSAR Toolbox) provided unsatisfactory results. Add...
Toxicology in vitro : an international journal published in association with BIBRA, 2016
In acute inhalation toxicity studies, animals inhale substances at given concentrations. Without ... more In acute inhalation toxicity studies, animals inhale substances at given concentrations. Without additional information, however, appropriate starting concentrations for in-vivo inhalation studies are difficult to estimate. The goal of this project was the prevalidation of precision-cut lung slices (PCLS) as an ex-vivo alternative to reduce the number of animals used in inhalation toxicity studies. According to internationally agreed principles for Prevalidation Studies, the project was conducted in three independent laboratories. The German BfR provided consultancy in validation principles and independent support with biostatistics. In all laboratories, rat PCLS were prepared and exposed to 5 concentrations of 20 industrial chemicals under submerged culture conditions for 1h. After 23h post-incubation, toxicity was assessed by measurement of released lactate dehydrogenase and mitochondrial activity. In addition, protein content and pro-inflammatory cytokine IL-1α were measured. For...
While single non-animal methods have been adopted in OECD test guidelines, combinations of method... more While single non-animal methods have been adopted in OECD test guidelines, combinations of methods (so called defined approaches, DA) are not. Hardly any animal study can be replaced by a single non-animal method, rather DA are needed. The OECD published the Adverse Outcome Pathway (AOP) on skin sensitization in 2012 and is currently discussing the implementation of DA into a guideline. Obviously, it takes thorough considerations and evaluations to validate such DA. Currently we see four preconditions for a proper and expedient implementation of DA in a guideline: (i) The reference data should be selected to allow meaningful evaluations and must not replicate the limitations of the murine local lymph node assay (LLNA) (ii) Methods and prediction models should be validated before they are used in an OECD-adopted DA, (iii) An OECD-adopted DA should follow the respective AOP and (iv) acknowledge regulatory needs and successful toxicological practice. These points still need to be considered in the current discussion at the OECD. A guideline for skin sensitization DA is setting the scene for regulatory acceptance of all new approaches (for any toxicological endpoint) in the future. In this commentary, we are expounding these preconditions to allow a scientifically valid and sustainable application of modern (no-animal) approaches in regulatory toxicology.
For hazard classifications of chemicals, continuous data from animal-or nonanimal testing methods... more For hazard classifications of chemicals, continuous data from animal-or nonanimal testing methods are often dichotomized into binary positive/negative outcomes by defining classification thresholds (CT). Experimental data are, however, subject to biological and technical variability. Each test method's precision is limited resulting in uncertainty of the positive/negative outcome if the experimental result is close to the CT. Borderline ranges (BR) around the CT were suggested, which represent ranges in which the study result is ambiguous, that is, positive or negative results are equally likely. The BR reflects a method's precision uncertainty. This article explores and compares different approaches to quantify the BR. Besides using the pooled standard deviation, we determine the BR by means of the median absolute deviation (MAD), with a sequential combination of both methods, and by using nonparametric bootstrapping. Furthermore, we quantify the BR for different hazardous effects, including nonanimal tests for skin corrosion, eye irritation, skin irritation, and skin sensitization as well as for an animal test on skin sensitization (the local lymph node assay, LLNA). Additionally, for one method (direct peptide reactivity assay) the BR was determined experimentally and compared to calculated BRs. Our results demonstrate that (i) the precision of the methods is determining the size of their BRs, (ii) there is no "perfect" method to derive a BR, alas, (iii) a consensus on BR is needed to account for the limited precision of testing methods.
The availability of reference data is a key requirement for the development of new approach metho... more The availability of reference data is a key requirement for the development of new approach methods (NAM), i.e., in vitro, in chemico and in silico methods and integrated approaches, like defined approaches (DA), which combine these data sources. Reference data are of even greater importance for regulatory acceptance. In contrast to most other adverse effects, human skin sensitization data on many chemicals are available, next to data from animal studies, such as the local lymph node assay (LLNA). Skin sensitization NAM data can therefore be compared to different reference datasets. Recent publications and validation at the OECD focused on human and LLNA reference data. The "2 out of 3" DA (2o3 DA) is the first DA for skin sensitization solely based on experimental data from validated tests and was recently adopted as an OECD test guideline. Here we review the predictivity of the 2o3 DA on multiple human and LLNA reference datasets. Concomitantly, we compare the predictivity of the LLNA for human data within the same datasets. Comparing predictivity of methods not only bilaterally (NAM or DA vs. animal method) but including human data in a triangle "NAM data-animal data-human data" offers a comprehensive assessment of the NAM's and DA's predictivity. In all these assessments, the 2o3 DA was superior to the LLNA in predicting human skin sensitization hazard. This highlights the importance of a holistic view of reference data instead of limiting validation of NAMs and DAs to data from a single animal test only.
The molecular initiating event in the adverse outcome pathway for skin sensitization is the coval... more The molecular initiating event in the adverse outcome pathway for skin sensitization is the covalent binding of the sensitizer to skin proteins, and a first method to address this key event was adopted as OECD TG 442C in 2015. This method, the direct peptide reactivity assay (DPRA), uses two synthetic peptides (one containing a cysteine, one containing a lysine residue) that are incubated with a single concentration of the test substance. After 24 hours incubation, the concentrations of remaining, non-depleted peptide are determined using HPLC. All currently adopted non-animal OECD TGs to assess skin sensitization, including the DPRA, provide information on the
ated in the peer-review of the validation studies, nor has a formalized way to use this quantitat... more ated in the peer-review of the validation studies, nor has a formalized way to use this quantitative information reached test guideline status. Nevertheless, multiple studies have investigated application of quantitative in vitro data from the validated assays for potency prediction (
In regulatory toxicology, in vivo studies are still prevailing, and human-derived in vitro models... more In regulatory toxicology, in vivo studies are still prevailing, and human-derived in vitro models are mostly used in testing for local toxicity to the skin and the eyes. A single in vitro model may be limited to address one or few molecular or cellular events leading to adverse outcomes. Hence, in many instances their regulatory use involves the combination of several in vitro models to assess the hazard potential of test substance. A so-called defined approach combines different testing methods and a 'data interpretation procedure' to obtain a comprehensive overall assessment which is used for the regulatory hazard classification of the test substance.Validation is a prerequisite of regulatory acceptance of new testing methods: This chapter provides an overview of the method development from an experimental method to a test guideline via application of GIVIMP (good in vitro method practice), standardization, validation to the regulatory adoption as an OECD test guidelines. Quandaries associated with the validation towards reference data from in vivo animal studies with limited accuracy and limited human relevance are discussed, as well as uncertainty and limitations arising from restricted applicability and technical and biological variance of the in vitro methods.This chapter provides an overview of human-derived in vitro models currently adopted as OECD test guidelines: From the first skin corrosion tests utilizing reconstructed human epidermis models (RhE), to models to test for skin irritation, phototoxicity, eye irritation, and skin sensitization. The latter is using a battery of different methods and defined approaches which are still under discussion for their regulatory adoption. They will be a vanguard of future applications of human-derived models in regulatory toxicology. RhEs for testing of genotoxicity and of dermal penetration and absorption, have been developed, underwent validation studies and may soon be adopted for regulatory use; these are included in this chapter.
Regulatory Toxicology and Pharmacology, Apr 1, 2013
Skin corrosion or irritation refers to the production of irreversible or reversible damage to the... more Skin corrosion or irritation refers to the production of irreversible or reversible damage to the skin following the application of a test substance, respectively. Traditionally, hazard assessments are conducted using the in vivo Draize skin test, but recently in vitro tests using reconstructed human epidermis (RhE) models have gained regulatory acceptance. In this study, skin corrosion (SCT) and irritation tests (SIT) using a RhE model were implemented to reduce the number of in vivo tests required by regulatory bodies. One hundred and thirty-four materials were tested from a wide range of substance classes included 46 agrochemical formulations. Results were assessed according to UN GHS, EU-CLP, ANVISA and US EPA classification schemes. There was high correlation between the two in vitro tests. Assessment of the SCT sensitivity was not possible due to the limited number of corrosives in the data set; SCT specificity and accuracy were 89% for all classification systems. Accuracy (63-76%) and sensitivity (53-67%) were low in the SIT. Specificity and concordance for agrochemical formulations alone in both the SCT and SIT were comparable to the values for the complete data set (SCT: 91% vs. 89% specificity, 91% vs. 89% accuracy and SIT: 64-88% vs. 70-85% specificity, 56-75% vs. 63-76% accuracy).
Skin sensitization is a key endpoint in both hazard and risk assessments. The pathways involved h... more Skin sensitization is a key endpoint in both hazard and risk assessments. The pathways involved have recently been formally described in the OECD adverse outcome pathway (AOP). Currently, no single non-animal test method is sufficient to fully address this AOP and therefore the use of a battery of tests is necessary to cover this endpoint. Test methods addressing AOP key events include the direct peptide reactivity assay (DPRA) (molecular initiating event: 'peptide reactivity'; OECD TG 442C), the LuSens and KeratinoSens TM (cellular response: 'keratinocyte activation'; OECD TG 442D), and the human cell-line activation test (h-CLAT; draft OECD TG) as well as the (modified) myeloid U937 skin sensitization test ((m)MUSST; 2nd cellular response: 'dendritic cell activation'). To assess the overall skin sensitizing potentials of the substances, a simple '2 out of 3' integrated testing strategy (ITS) was applied (Urbisch et al., 2015, PMID: 25541156). In order to facilitate regulatory acceptance of these methods, test results of 213 substances were compared to both local lymph node assay (LLNA) and human data, where available. The individual test methods provide predictivities between 73 and 76% or 78 and 84%, when compared to LLNA or human data, respectively. The '2 out of 3' ITS correctly predicts 79% of the human and 90% of the human data. These results demonstrate that the non-animal test methods correlate better with human data than with LLNA data. In addition, different mechanistic domains were identified by probable protein-binding mechanisms of the 213 substances. This approach shows that all assays predict Michael acceptors with the highest accuracies (≥80%). In the domain of acylating agents, the keratinocyte based assays show lower predictivities of ≤58%. In summary, taking mechanistic domains into account offers a more accurate estimation of the predictive performance of the individual non-animal test methods as well as the overall ITS prediction.
P-09-03 / Toxicology Letters 280S (2017) S280-S290 are available which limits the practical aspec... more P-09-03 / Toxicology Letters 280S (2017) S280-S290 are available which limits the practical aspect of testing a compound. The poster will also describe data and knowledge sharing schemes that have been developed to enhance the expert alerts and provide more detailed structure activity relationship (SAR) information. This methodology utilizes proprietary information without revealing any confidential data and incorporates SAR information from 35,000 compounds with mutagenicity data. The results have been used to improve the overall performance of the Leadscope expert alerts system as well as continued improvements to specific classes including aromatic amines, aromatic amides, aryl boronic acids, and alkyl halides.
The selection of reference and proficiency chemicals is an important component of method validati... more The selection of reference and proficiency chemicals is an important component of method validation and proficiency evaluations. Reference chemicals are a set of test substances used by a method developer to evaluate the reliability and relevance of a new method, in comparison to reference data (usually to a validated reference method). Proficiency chemicals, as defined in OECD Guidance Document on Good In Vitro Method Practices, are defined post validation as a subset of the reference chemicals, or other chemicals with sufficient supporting data, that are used by naïve laboratories to demonstrate technical competence with a validated test method. Proficiency chemicals should cover different physical states, several chemical classes within the applicability domain of the method and yield the full range of responses (in the validated reference method and in vivo). They shall be commercially available (at non-prohibitive costs) and have high quality reference data. If reference and subsequent proficiency chemicals are chosen without sufficient evidence for their inclusion, both test method evaluation and demonstration of technical proficiency can be hampered. In this report we present cases in which the selection of reference chemicals led to problems in the reproduction of the reference results and demonstration of technical proficiency.
Letter to the editor: "Evaulation of radioisotopic and non-radioisotopic versions of local lymph ... more Letter to the editor: "Evaulation of radioisotopic and non-radioisotopic versions of local lymph node assays for subcategorization of skin sensitiers compliant to UN GHS rev 4" by Ha et al., 2017
Toxicology in vitro : an international journal published in association with BIBRA, 2016
The molecular initiating event (MIE) of skin sensitization is the binding of a hapten to dermal p... more The molecular initiating event (MIE) of skin sensitization is the binding of a hapten to dermal proteins. This can be assessed using the in chemico direct peptide reactivity assay (DPRA) or in silico tools such as the QSAR Toolbox and TIMES SS. In this study, the suitability of these methods was analyzed by comparing their results to in vivo sensitization data of LLNA and human studies. Compared to human data, 84% of non-sensitizers and sensitizers yielded consistent results in the DPRA. In silico tools resulted in 'no alert' for 83%-100% of the non-sensitizers, but alerted only 55%-61% of the sensitizers. The inclusion of biotic and abiotic transformation simulations yielded more alerts for sensitizers, but simultaneously dropped the number of non-alerted non-sensitizers. In contrast to the DPRA, in silico tools were more consistent with results of the LLNA than human data. Interestingly, the new "DPRA profilers" (QSAR Toolbox) provided unsatisfactory results. Add...
Toxicology in vitro : an international journal published in association with BIBRA, 2016
In acute inhalation toxicity studies, animals inhale substances at given concentrations. Without ... more In acute inhalation toxicity studies, animals inhale substances at given concentrations. Without additional information, however, appropriate starting concentrations for in-vivo inhalation studies are difficult to estimate. The goal of this project was the prevalidation of precision-cut lung slices (PCLS) as an ex-vivo alternative to reduce the number of animals used in inhalation toxicity studies. According to internationally agreed principles for Prevalidation Studies, the project was conducted in three independent laboratories. The German BfR provided consultancy in validation principles and independent support with biostatistics. In all laboratories, rat PCLS were prepared and exposed to 5 concentrations of 20 industrial chemicals under submerged culture conditions for 1h. After 23h post-incubation, toxicity was assessed by measurement of released lactate dehydrogenase and mitochondrial activity. In addition, protein content and pro-inflammatory cytokine IL-1α were measured. For...
While single non-animal methods have been adopted in OECD test guidelines, combinations of method... more While single non-animal methods have been adopted in OECD test guidelines, combinations of methods (so called defined approaches, DA) are not. Hardly any animal study can be replaced by a single non-animal method, rather DA are needed. The OECD published the Adverse Outcome Pathway (AOP) on skin sensitization in 2012 and is currently discussing the implementation of DA into a guideline. Obviously, it takes thorough considerations and evaluations to validate such DA. Currently we see four preconditions for a proper and expedient implementation of DA in a guideline: (i) The reference data should be selected to allow meaningful evaluations and must not replicate the limitations of the murine local lymph node assay (LLNA) (ii) Methods and prediction models should be validated before they are used in an OECD-adopted DA, (iii) An OECD-adopted DA should follow the respective AOP and (iv) acknowledge regulatory needs and successful toxicological practice. These points still need to be considered in the current discussion at the OECD. A guideline for skin sensitization DA is setting the scene for regulatory acceptance of all new approaches (for any toxicological endpoint) in the future. In this commentary, we are expounding these preconditions to allow a scientifically valid and sustainable application of modern (no-animal) approaches in regulatory toxicology.
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Papers by Susanne Kolle