Publications by authors named "Susanne Kolle"

44 Publications

A triangular approach for the validation of new approach methods for skin sensitization.

ALTEX 2021 Jul 8. Epub 2021 Jul 8.

BASF SE Experimental Toxicology and Ecology, Ludwigshafen, Germany.

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), combining these data sources. Reference data are of even higher 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 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 on reference data instead of limiting validation of NAM and DA to data from a single animal test only.
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http://dx.doi.org/10.14573/altex.2105111DOI Listing
July 2021

Effectiveness of an electronic hand hygiene monitoring system to increase compliance and reduce healthcare-associated infections.

J Hosp Infect 2021 May 28. Epub 2021 May 28.

Department of Clinical Microbiology, Vejle Hospital, University Hospital of Southern Denmark, Vejle, Denmark.

During an interventional study in a nephrology department, we investigated the effect of an electronic hand hygiene monitoring system on the hand hygiene compliance of healthcare workers (n=99) and hospital-acquired bloodstream infections. The doctors and nurses improved their hand hygiene compliance significantly during the intervention phase when they received group and individual feedback based on actionable insights from the electronic hand hygiene monitoring system. The improvements in hand hygiene compliance were associated with a significant reduction in the number of hospital-acquired bloodstream infections.
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http://dx.doi.org/10.1016/j.jhin.2021.05.011DOI Listing
May 2021

How a GIVIMP certification program can increase confidence in in vitro methods.

ALTEX 2021 ;38(2):316-318

Institute for In Vitro Sciences, Inc., Gaithersburg, MD, USA.

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http://dx.doi.org/10.14573/altex.2102261DOI Listing
January 2021

Accounting for Precision Uncertainty of Toxicity Testing: Methods to Define Borderline Ranges and Implications for Hazard Assessment of Chemicals.

Risk Anal 2020 Dec 9. Epub 2020 Dec 9.

BASF SE, Ludwigshafen am Rhein, Germany.

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.
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http://dx.doi.org/10.1111/risa.13648DOI Listing
December 2020

Human-Derived In Vitro Models Used for Skin Toxicity Testing Under REACh.

Handb Exp Pharmacol 2021 ;265:3-27

BASF SE, Experimental Toxicology and Ecology, Ludwigshafen am Rhein, Germany.

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.
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http://dx.doi.org/10.1007/164_2020_368DOI Listing
March 2021

Predictivity of the kinetic direct peptide reactivity assay (kDPRA) for sensitizer potency assessment and GHS subclassification

ALTEX 2020 19;37(4):652-664. Epub 2020 Aug 19.

BASF SE Experimental Toxicology and Ecology, Ludwigshafen, Germany.

Several in vitro OECD test guidelines address key events 1-3 of the adverse outcome pathway for skin sensitization, but none are validated for sensitizer potency assessment. The reaction of sensitizing molecules with skin proteins is the molecular initiating event and appears to be rate-limiting, as chemical reactivity strongly correlates with sensitizer potency. The kinetic direct peptide reactivity assay (kDPRA), a modification of the DPRA (OECD TG 442C), allows derivation of rate constants of the depletion of the cysteine-containing model peptide upon reaction with the test item. Its reproducibility was demonstrated in an inter-laboratory study. Here, we present a database of rate constants, expressed as log kmax, for 180 chemicals to define the prediction threshold to identify strong sensitizers (classified as GHS 1A). A threshold of log kmax -2 offers a balanced accuracy of 85% for predicting GHS 1A sensitizers according to the local lymph node assay. The kDPRA is proposed as a stand-alone assay for identification of GHS 1A sensitizers among chemicals identified as sensitizers by other tests or defined approaches. It may also be used for the prediction of sensitizer potency on a continuous scale, ideally in combination with continuous parameters from other in vitro assays. We show how the rate constant could be combined with read-outs of other in vitro assays in a defined approach. A decision model based on log kmax alone has, however, a high predictivity and can be used as stand-alone model for identification of GHS 1A sensitizers among chemicals predicted as sensitizers.
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http://dx.doi.org/10.14573/altex.2004292DOI Listing
August 2020

Replacing the refinement for skin sensitization testing: Considerations to the implementation of adverse outcome pathway (AOP)-based defined approaches (DA) in OECD guidelines.

Regul Toxicol Pharmacol 2020 Aug 17;115:104713. Epub 2020 Jun 17.

Givaudan Schweiz AG, Kemptpark 50, 8310, Kemptthal, Switzerland.

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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http://dx.doi.org/10.1016/j.yrtph.2020.104713DOI Listing
August 2020

The kinetic direct peptide reactivity assay (kDPRA): Intra- and inter-laboratory reproducibility in a seven-laboratory ring trial

ALTEX 2020 10;37(4):639-651. Epub 2020 Jun 10.

Givaudan Schweiz AG, Kemptthal, Switzerland.

While the skin sensitization hazard of substances can be identified using non-animal methods, the classification of potency into UN GHS sub-categories 1A and 1B remains challenging. The kinetic direct peptide reactivity assay (kDPRA) is a modification of the DPRA wherein the reaction kinetics of a test substance towards a synthetic cysteine-containing peptide are evaluated. For this purpose, several concentrations of the test substance are incubated with the synthetic peptide for several incubation times. The reaction is stopped by addition of monobromobimane, which forms a fluorescent complex with the free cysteine of the model peptide. The relative remaining non-depleted amount of peptide is determined. Kinetic rate constants are derived from the depletion vs concentration and time matrix and used to distinguish between UN GHS sub-category 1A sensitizers and test substances in sub-category 1B/not classified test substances. In this study, we present a ring trial of the kDPRA with 24 blind-coded test substances in seven laboratories. The intra- and inter-laboratory reproducibility were 96% and 88%, respectively (both for differentiating GHS Cat 1A sensitizers from GHS Cat 1B/not classified). Following an independent peer review, the kDPRA was considered to be acceptable for the identification of GHS Cat 1A skin sensitizers. Besides GHS Cat 1A identification, the kDPRA can be used as part of a defined approach(es) with a quantitative data integration procedure for skin sensitization potency assessment. For this aim, next to reproducibility of classification, the quantitative reproducibility and variability of the rate constants were quantified in this study.
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http://dx.doi.org/10.14573/altex.2004291DOI Listing
June 2020

A review of substances found positive in 1 of 3 in vitro tests for skin sensitization.

Regul Toxicol Pharmacol 2019 Aug 18;106:352-368. Epub 2019 May 18.

BASF SE, Experimental Toxicology and Ecology, Ludwigshafen, Germany.

There has been significant progress in recent years in the development and application of alternative methods for assessing the skin sensitization potential of chemicals. The pathways involved in skin sensitization have been described in an OECD adverse outcome pathway (AOP). To date, a single non-animal test method is not sufficient to address this AOP so numerous approaches involving the use of 2 or more assays are being evaluated for their performance. The 2 out of 3 approach is a simple approach that has demonstrated very good sensitivity, specificity and overall accuracy numbers for predicting the skin sensitization potential of chemicals. Chemicals with at least two positive results in tests addressing Key events 1-3 are predicted sensitizers, while chemicals with none or only one positive outcome are predicted non-sensitizers. In this report we have thoroughly reviewed the discordant results of 29 chemicals with 1 out of 3 positive results to understand better what led to the results observed and how this information might impact our hazard assessments of these chemicals. We initially categorized each chemical using a weight of evidence approach as positive, negative or indeterminate based on review of available human and animal data as well as what skin sensitization alerts were triggered using two versions of OECD Toolbox and DEREK Nexus. We determined that 4 of the 29 chemicals should be classified as indeterminate and not included in analysis of method performance based on insufficient, borderline and/or conflicting data to confidently categorized the chemicals as allergens or non-allergens. Of the 29 chemicals included in this analysis, 17 were classified as negative and would be correctly identified using a 2 out of 3 approach while 8 chemicals were classified as positive in vivo and would be false-negative with this approach. For some of these chemicals, the outcomes observed can be explained by in vitro borderline results (13 chemicals) or in some instances there is mechanistic understanding of why a chemical is positive or negative in a particular assay (9 chemicals). Thus, when comparing the performance of different defined approaches, one should attempt to only include chemicals which demonstrate clear evidence to be categorize as allergens or non-allergens. Finally, when interpreting the results obtained for an individual unknown chemical it is critical that the in vitro skin sensitization data is reviewed critically and there is a good understanding of the variance and applicability domain limitations for each assay being used.
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http://dx.doi.org/10.1016/j.yrtph.2019.05.016DOI Listing
August 2019

Corrigendum to "LuSens: a keratinocyte based ARE reporter gene assay for use in integrated testing strategies for skin sensitization hazard identification." by Ramirez et al. [Toxicol In Vitro. 2014 Dec; 28(8):1482-97. doi: 10.1016/j.tiv.2014.08.002].

Authors:
Susanne N Kolle

Toxicol In Vitro 2019 08 11;58:264. Epub 2019 Feb 11.

Experimental Toxicology and Ecology, BASF SE, Carl-Bosch-Str. 38, 67056 Ludwigshafen, Germany. Electronic address:

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http://dx.doi.org/10.1016/j.tiv.2019.02.008DOI Listing
August 2019

GHS additivity formula: can it predict the acute systemic toxicity of agrochemical formulations that contain acutely toxic ingredients?

Regul Toxicol Pharmacol 2018 Feb 4;92:407-419. Epub 2018 Jan 4.

BASF SE Experimental Toxicology and Ecology, Ludwigshafen, Germany.

In vivo acute systemic testing is a regulatory requirement for agrochemical formulations. GHS specifies an alternative computational approach (GHS additivity formula) for calculating the acute toxicity of mixtures. We collected acute systemic toxicity data from formulations that contained one of several acutely-toxic active ingredients. The resulting acute data set includes 210 formulations tested for oral toxicity, 128 formulations tested for inhalation toxicity and 31 formulations tested for dermal toxicity. The GHS additivity formula was applied to each of these formulations and compared with the experimental in vivo result. In the acute oral assay, the GHS additivity formula misclassified 110 formulations using the GHS classification criteria (48% accuracy) and 119 formulations using the USEPA classification criteria (43% accuracy). With acute inhalation, the GHS additivity formula misclassified 50 formulations using the GHS classification criteria (61% accuracy) and 34 formulations using the USEPA classification criteria (73% accuracy). For acute dermal toxicity, the GHS additivity formula misclassified 16 formulations using the GHS classification criteria (48% accuracy) and 20 formulations using the USEPA classification criteria (36% accuracy). This data indicates the acute systemic toxicity of many formulations is not the sum of the ingredients' toxicity (additivity); but rather, ingredients in a formulation can interact to result in lower or higher toxicity than predicted by the GHS additivity formula.
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http://dx.doi.org/10.1016/j.yrtph.2017.12.024DOI Listing
February 2018

Prediction of skin sensitization potency sub-categories using peptide reactivity data.

Toxicol In Vitro 2017 Dec 4;45(Pt 1):134-145. Epub 2017 Sep 4.

BASF SE, Experimental Toxicology and Ecology, Ludwigshafen, Germany. Electronic address:

While the skin sensitization hazard of substances can already be identified using non-animal methods, the classification of potency sub-categories GHS-1A and 1B is still challenging. Potency can be measured by the dose at which an effect is observed; since the protein-adduct formation is determining the dose of the allergen in the skin, peptide reactivity was used to assess the potency. The Direct Peptide Reactivity Assay (DPRA; one concentration and reaction-time) did not sufficiently discriminate between sub-categories 1A and 1B (56% accuracy compared to LLNA data, n=124). An extended protocol termed 'quantitative DPRA' (three concentrations and one reaction-time), discriminated sub-categories GHS 1A and 1B with an accuracy of 81% or 57% compared to LLNA (n=36) or human (n=14) data, respectively. The analysis of the Cys-adducts was already sufficient; additional analysis of Lys-adducts did not improve the predictivity. An additional modification, the 'kinetic DPRA' (several concentrations and reaction-times) was used to approximate the rate constant of Cys-peptide-adduct formation. 35 of 38 substances were correctly assigned to the potency sub-categories (LLNA data), and the predictivity for 14 human data was equally high. These results warrant the kinetic DPRA for further validation in order to fully replace in vivo testing for assessing skin sensitization including potency sub-classification.
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http://dx.doi.org/10.1016/j.tiv.2017.08.015DOI Listing
December 2017

Regulatory accepted but out of domain: In vitro skin irritation tests for agrochemical formulations.

Regul Toxicol Pharmacol 2017 Oct 19;89:125-130. Epub 2017 Jul 19.

BASF SE Experimental Toxicology and Ecology, Ludwigshafen, Germany.

Several in vitro methods have gained regulatory acceptance for the prediction of skin irritation and corrosion. However, the test guidelines for the majority of in vitro methods do not state whether they are applicable to agrochemical formulations. Hence, we would like to share the results from our routine skin corrosion and irritation testing of agrochemical formulations in which both in vitro (according to OECD TG 431 and OECD TG 439) and in vivo (according to OECD TG 404) tests were conducted as regulatory requirements. The in vitro skin irritation test did not correlate well with the CLP classification by in vivo results (44% sensitivity, 60% specificity, and 54% accuracy, based on 65 data pairs). This indicates a lack of applicability of the current protocol of the in vitro skin irritation test for agrochemical formulations. The data set did not contain formulations which were skin corrosive in vivo and hence its applicability could not be assessed. The correlation of in vitro skin corrosion testing to formulations which were not corrosive in vivo was, however, high (95% specificity based on 81 data pairs).
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http://dx.doi.org/10.1016/j.yrtph.2017.07.016DOI Listing
October 2017

The borderline range of toxicological methods: Quantification and implications for evaluating precision.

ALTEX 2017 23;34(4):525-538. Epub 2017 Feb 23.

BASF SE, Experimental Toxicology and Ecology, Ludwigshafen, Germany.

Test methods to assess the skin sensitization potential of a substance usually use threshold criteria to dichotomize continuous experimental read-outs into yes/no conclusions. The threshold criteria are prescribed in the respective OECD test guidelines and the conclusion is used for regulatory hazard assessment, i.e., classification and labelling of the substance. We can identify a borderline range (BR) around the classification threshold within which test results are inconclusive due to a test method's biological and technical variability. We quantified BRs in the prediction models of the non-animal test methods DPRA, LuSens and h-CLAT, and of the animal test LLNA, respectively. Depending on the size of the BR, we found that between 6% and 28% of the substances in the sets tested with these methods were considered borderline. When the results of individual non-animal test methods were combined into integrated testing strategies (ITS), borderline test results of individual tests also affected the overall assessment of the skin sensitization potential of the testing strategy. This was analyzed for the 2-out-of-3 ITS: Four out of 40 substances (10%) were considered borderline. Based on our findings we propose expanding the standard binary classification of substances into "positive"/"negative" or "hazardous"/"non-hazardous" by adding a "borderline" or "inconclusive" alert for cases where test results fall within the borderline range.
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http://dx.doi.org/10.14573/altex.1606271DOI Listing
August 2018

Lacking applicability of in vitro eye irritation methods to identify seriously eye irritating agrochemical formulations: Results of bovine cornea opacity and permeability assay, isolated chicken eye test and the EpiOcular™ ET-50 method to classify according to UN GHS.

Regul Toxicol Pharmacol 2017 Apr 2;85:33-47. Epub 2017 Feb 2.

BASF SE Experimental Toxicology and Ecology, Ludwigshafen, Germany.

In vitro methods have gained regulatory acceptance for the prediction of serious eye damage (UN GHS Cat 1). However, the majority of in vitro methods do not state whether they are applicable to agrochemical formulations. This manuscript presents a study of up to 27 agrochemical formulations tested in three in vitro assays (three versions of the bovine corneal opacity and permeability test (BCOP, OECD TG 437) assay, the isolated chicken eye test (ICE, OECD TG 438) and the EpiOcular™ ET-50 assay). The results were compared with already-available in vivo data. In the BCOP only one of the four, one of five in the ICE and six of eleven tested formulations in the EpiOcular™ ET-50 Neat Protocol resulted in the correct UN GHS Cat 1 prediction. Overpredictions occurred in all assays. These data indicate a lack of applicability of the three in vitro methods to reliably predict UN GHS Cat 1 of agrochemical formulations. In order to ensure animal-free identification of seriously eye damaging agrochemical formulations testing protocols and/or prediction models need to be modified or classification rules should be tailored to in vitro testing rather than using in vivo Draize data as a standard.
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http://dx.doi.org/10.1016/j.yrtph.2017.01.013DOI Listing
April 2017

Assessment of skin sensitization under REACH: A case report on vehicle choice in the LLNA and its crucial role preventing false positive results.

Regul Toxicol Pharmacol 2017 Apr 31;85:25-32. Epub 2017 Jan 31.

Experimental Toxicology and Ecology, BASF SE, 67056 Ludwigshafen am Rhein, Germany.

In the EU, chemicals with a production or import volume in quantities of one metric ton per year or more have to be tested for skin sensitizing properties under the REACH regulation. The murine Local Lymph Node Assay (LLNA) and its modifications are widely used to fulfil the data requirement, as it is currently considered the first-choice method for in vivo testing to cover this endpoint. This manuscript describes a case study highlighting the importance of understanding the chemistry of the test material during testing for 'skin sensitization' of MCDA (mixture of 2,4- and 2,6-diamino-methylcyclohexane) with particular focus on the vehicle used. While the BrdU-ELISA modification of the LLNA using acetone/olive oil (AOO) as vehicle revealed expectable positive results. However, the concentration control analysis unexpectedly revealed an instability of MCDA in the vehicle AOO. Further studies on the reactivity showed MCDA to rapidly react with AOO under formation of various imine structures, which might have caused the positive LLNA result. The repetition of the LLNA using propylene glycol (PG) as vehicle did not confirm the positive results of the LLNA using AOO. Finally, a classification of MCDA as skin sensitizer according to the Globally Harmonized System (GHS) was not justified.
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http://dx.doi.org/10.1016/j.yrtph.2017.01.010DOI Listing
April 2017

Local tolerance testing under REACH: Accepted non-animal methods are not on equal footing with animal tests.

Altern Lab Anim 2016 Jul;44(3):281-99

BASF SE, Experimental Toxicology and Ecology, Ludwigshafen, Germany.

In general, no single non-animal method can cover the complexity of any given animal test. Therefore, fixed sets of in vitro (and in chemico) methods have been combined into testing strategies for skin and eye irritation and skin sensitisation testing, with pre-defined prediction models for substance classification. Many of these methods have been adopted as OECD test guidelines. Various testing strategies have been successfully validated in extensive in-house and inter-laboratory studies, but they have not yet received formal acceptance for substance classification. Therefore, under the European REACH Regulation, data from testing strategies can, in general, only be used in so-called weight-of-evidence approaches. While animal testing data generated under the specific REACH information requirements are per se sufficient, the sufficiency of weight-of-evidence approaches can be questioned under the REACH system, and further animal testing can be required. This constitutes an imbalance between the regulatory acceptance of data from approved non-animal methods and animal tests that is not justified on scientific grounds. To ensure that testing strategies for local tolerance testing truly serve to replace animal testing for the REACH registration 2018 deadline (when the majority of existing chemicals have to be registered), clarity on their regulatory acceptance as complete replacements is urgently required.
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http://dx.doi.org/10.1177/026119291604400311DOI Listing
July 2016

Eye irritation testing of nanomaterials using the EpiOcular™ eye irritation test and the bovine corneal opacity and permeability assay.

Part Fibre Toxicol 2016 Apr 15;13:18. Epub 2016 Apr 15.

BASF SE, Experimental Toxicology and Ecology, GB/TB - Z470, 67056, Ludwigshafen, Germany.

Background: Assessment of eye irritation hazard has long been a core requirement in any chemical legislation. Nevertheless, publications focussing on the eye damaging potential of nanomaterials are scarce. Traditionally, eye irritation testing was performed using rabbits. The OECD Test Guideline 437 Bovine Corneal Opacity and Permeability (BCOP) test method allows determining severely irritating substances without animals, and the recently adopted OECD Test Guideline 492 Reconstructed human cornea-like epithelium test method allows identifying chemicals that neither induce eye irritation nor serious eye damage. For substances applicable to these tests, huge progress has been made in replacing animal testing.

Methods: The in vitro eye irritation potential of 20 nanosized and 3 non-nanosized materials was investigated in a 2-tier EpiOcular™ Eye Irritation Test (EpiOcular™-EIT) and BCOP testing strategy including histopathology of the bovine corneas. Furthermore, applicability of the testing strategy for nanomaterials was assessed. Test materials encompassed OECD representative nanomaterials (metals (Ag), metal oxides (ZnO, TiO2, CeO2), amorphous SiO2 and MWCNTs), three organic pigments, quartz, and talc.

Results: None of the dry-powder nanomaterials elicited eye irritation in either the EpiOcular™-EIT or the BCOP assay. Likewise, an amorphous SiO2 nanomaterial that was supplied as suspension was tested negative in both assays. By contrast, in the EpiOcular™-EIT, the silver nanomaterial that was supplied as dispersion was tested positive, whereas its surfactant-containing dispersant was borderline to negative. In the BCOP assay, the silver nanomaterial elicited highly variable results and dark-brown patches remained on the corneal surface, whereas the results for its dispersant alone were borderline to positive, which was assessed as inconclusive due to high inter-assay variability.

Conclusion: The present study points to the low eye irritation potential of a spectrum of nanomaterials, which is consistent with available in vivo data for the same test materials or for nanosized or bulk materials of the same composition.
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http://dx.doi.org/10.1186/s12989-016-0128-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4833924PMC
April 2016

Assessment of Pre- and Pro-haptens Using Nonanimal Test Methods for Skin Sensitization.

Chem Res Toxicol 2016 05 29;29(5):901-13. Epub 2016 Apr 29.

Experimental Toxicology and Ecology, BASF SE , Ludwigshafen, Germany.

Because of ethical and regulatory reasons, several nonanimal test methods to assess the skin sensitization potential of chemicals have been developed and validated. In contrast to in vivo methods, 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 test methods. Of these, 18 provided true positive results in the direct peptide reactivity assay (DPRA; sensitivity of 67%), although lacking structural alerts for direct peptide reactivity. The reaction mechanisms leading to peptide depletion in the DPRA were therefore elucidated using mass spectrometry. Hapten-peptide adducts were identified for 13 of the 18 chemicals indicating that these pre-haptens 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 the keratinocyte and dendritic 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, KeratinoSens, and h-CLAT used in a 2 out of 3 weight of evidence (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.
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http://dx.doi.org/10.1021/acs.chemrestox.6b00055DOI Listing
May 2016

The effect of communication and implicit associations on consuming insects: An experiment in Denmark and Italy.

Appetite 2016 11 6;106:30-6. Epub 2016 Feb 6.

Department of Management, Aarhus University, Bartholins Allé 10, building 1328, DK-8000, Aarhus C, Denmark. Electronic address:

It has been widely noted that the introduction of insects in Westerns' diet might be a promising path towards a more sustainable food consumption. However, Westerns' are almost disgusted and sceptical about the eating of insects. In the current paper we report the results of an experiment conducted in two European countries-Denmark and Italy-different for food culture and familiarity with the topic of eating insects. We investigated the possibility to foster people's willingness to eat insect-based food through communication, also comparing messages based on individual vs. societal benefits of the eating of insects. Communication proved to be effective on intention and behaviour, and the societal message appeared to be more robust over time. The communication effect is significant across nation, gender, and previous knowledge about the topic. In addition, we investigated the impact of non-conscious negative associations with insects on the choice to eat vs. not eat insect-based food. Implicit attitudes proved to be a powerful factor in relation to behaviour, yet they did not impede the effectiveness of communication.
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http://dx.doi.org/10.1016/j.appet.2016.02.006DOI Listing
November 2016

The EpiOcular™ Eye Irritation Test is the Method of Choice for the In Vitro Eye Irritation Testing of Agrochemical Formulations: Correlation Analysis of EpiOcular Eye Irritation Test and BCOP Test Data According to the UN GHS, US EPA and Brazil ANVISA Classification Schemes.

Altern Lab Anim 2015 Jul;43(3):181-98

BASF SE Experimental Toxicology and Ecology, Ludwigshafen, Germany.

The Bovine Corneal Opacity and Permeability (BCOP) test is commonly used for the identification of severe ocular irritants (GHS Category 1), but it is not recommended for the identification of ocular irritants (GHS Category 2). The incorporation of human reconstructed tissue model-based tests into a tiered test strategy to identify ocular non-irritants and replace the Draize rabbit eye irritation test has been suggested (OECD TG 405). The value of the EpiOcular™ Eye Irritation Test (EIT) for the prediction of ocular non-irritants (GHS No Category) has been demonstrated, and an OECD Test Guideline (TG) was drafted in 2014. The purpose of this study was to evaluate whether the BCOP test, in conjunction with corneal histopathology (as suggested for the evaluation of the depth of the injury( and/or the EpiOcular-EIT, could be used to predict the eye irritation potential of agrochemical formulations according to the UN GHS, US EPA and Brazil ANVISA classification schemes. We have assessed opacity, permeability and histopathology in the BCOP assay, and relative tissue viability in the EpiOcular-EIT, for 97 agrochemical formulations with available in vivo eye irritation data. By using the OECD TG 437 protocol for liquids, the BCOP test did not result in sufficient correct predictions of severe ocular irritants for any of the three classification schemes. The lack of sensitivity could be improved somewhat by the inclusion of corneal histopathology, but the relative viability in the EpiOcular-EIT clearly outperformed the BCOP test for all three classification schemes. The predictive capacity of the EpiOcular-EIT for ocular non-irritants (UN GHS No Category) for the 97 agrochemical formulations tested (91% sensitivity, 72% specificity and 82% accuracy for UN GHS classification) was comparable to that obtained in the formal validation exercise underlying the OECD draft TG. We therefore conclude that the EpiOcular-EIT is currently the best in vitro method for the prediction of the eye irritation potential of liquid agrochemical formulations.
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http://dx.doi.org/10.1177/026119291504300307DOI Listing
July 2015

Assessing skin sensitization hazard in mice and men using non-animal test methods.

Regul Toxicol Pharmacol 2015 Mar 23;71(2):337-51. Epub 2014 Dec 23.

Safety Science Research Laboratories, Kao Corporation, 2606 Akabane, Ichikai, Haga, Tochigi 321-349, Japan.

Sensitization, the prerequisite event in the development of allergic contact dermatitis, is a key parameter in both hazard and risk assessments. The pathways involved have recently been formally described in the OECD adverse outcome pathway (AOP) for skin sensitization. One single non-animal test method will not be sufficient to fully address this AOP and in many cases the use of a battery of tests will be necessary. A number of methods are now fully developed and validated. In order to facilitate acceptance of these methods by both the regulatory and scientific communities, results of the single test methods (DPRA, KeratinoSens, LuSens, h-CLAT, (m)MUSST) as well for a the simple '2 out of 3' ITS for 213 substances have been compiled and qualitatively compared to both animal and human data. The dataset was also used to define different mechanistic domains by probable protein-binding mechanisms. In general, the non-animal test methods exhibited good predictivities when compared to local lymph node assay (LLNA) data and even better predictivities when compared to human data. The '2 out of 3' prediction model achieved accuracies of 90% or 79% when compared to human or LLNA data, respectively and thereby even slightly exceeded that of the LLNA.
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http://dx.doi.org/10.1016/j.yrtph.2014.12.008DOI Listing
March 2015

Systematic evaluation of non-animal test methods for skin sensitisation safety assessment.

Toxicol In Vitro 2015 Feb;29(1):259-70

Henkel AG & CO KGaA, Henkelstr. 67, 40191 Düsseldorf, Germany.

The need for non-animal data to assess skin sensitisation properties of substances, especially cosmetics ingredients, has spawned the development of many in vitro methods. As it is widely believed that no single method can provide a solution, the Cosmetics Europe Skin Tolerance Task Force has defined a three-phase framework for the development of a non-animal testing strategy for skin sensitization potency prediction. The results of the first phase – systematic evaluation of 16 test methods – are presented here. This evaluation involved generation of data on a common set of ten substances in all methods and systematic collation of information including the level of standardisation, existing test data,potential for throughput, transferability and accessibility in cooperation with the test method developers.A workshop was held with the test method developers to review the outcome of this evaluation and to discuss the results. The evaluation informed the prioritisation of test methods for the next phase of the non-animal testing strategy development framework. Ultimately, the testing strategy – combined with bioavailability and skin metabolism data and exposure consideration – is envisaged to allow establishment of a data integration approach for skin sensitisation safety assessment of cosmetic ingredients.
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http://dx.doi.org/10.1016/j.tiv.2014.10.018DOI Listing
February 2015

Immunophenotyping does not improve predictivity of the local lymph node assay in mice.

J Appl Toxicol 2015 Apr 3;35(4):434-45. Epub 2014 Sep 3.

BASF SE, Experimental Toxicology and Ecology, Z470, 67056, Ludwigshafen, Germany.

The local lymph node assay (LLNA) is a regulatory accepted test for the identification of skin sensitizing substances by measuring radioactive thymidine incorporation into the lymph node. However, there is evidence that LLNA is overestimating the sensitization potential of certain substance classes in particular those exerting skin irritation. Some reports describe the additional use of flow cytometry-based immunophenotyping to better discriminate irritants from sensitizing irritants in LLNA. In the present study, the 22 performance standards plus 8 surfactants were assessed using the radioactive LLNA method. In addition, lymph node cells were immunophenotyped to evaluate the specificity of the lymph node response using cell surface markers such as B220 or CD19, CD3, CD4, CD8, I-A(κ) and CD69 with the aim to allow a better discrimination above all between irritants and sensitizers, but also non-irritating sensitizers and non-sensitizers. However, the markers assessed in this study do not sufficiently differentiate between irritants and irritant sensitizers and therefore did not improve the predictive capacity of the LLNA.
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http://dx.doi.org/10.1002/jat.3042DOI Listing
April 2015

LuSens: a keratinocyte based ARE reporter gene assay for use in integrated testing strategies for skin sensitization hazard identification.

Toxicol In Vitro 2014 Dec 27;28(8):1482-97. Epub 2014 Aug 27.

BASF SE, Experimental Toxicology and Ecology, Germany. Electronic address:

Allergic contact dermatitis can develop following repeated exposure to allergenic substances. To date, hazard identification is still based on animal studies as non-animal alternatives have not yet gained global regulatory acceptance. Several non-animal methods addressing key-steps of the adverse outcome pathway (OECD, 2012) will most likely be needed to fully address this effect. Among the initial cellular events is the activation of keratinocytes and currently only one method, the KeratinoSens™, has been formally validated to address this event. In this study, a further method, the LuSens assay, that uses a human keratinocyte cell line harbouring a reporter gene construct composed of the antioxidant response element (ARE) of the rat NADPH:quinone oxidoreductase 1 gene and the luciferase gene. The assay was validated in house using a selection of 74 substances which included the LLNA performance standards. The predictivity of the LuSens assay for skin sensitization hazard identification was comparable to other non-animal methods, in particular to the KeratinoSens™. When used as part of a testing battery based on the OECD adverse outcome pathway for skin sensitization, a combination of the LuSens assay, the DPRA and a dendritic cell line activation test attained predictivities similar to that of the LLNA.
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http://dx.doi.org/10.1016/j.tiv.2014.08.002DOI Listing
December 2014

Applicability of rat precision-cut lung slices in evaluating nanomaterial cytotoxicity, apoptosis, oxidative stress, and inflammation.

Toxicol Appl Pharmacol 2014 Apr 29;276(1):1-20. Epub 2013 Dec 29.

Experimental Toxicology and Ecology, BASF SE, Ludwigshafen, Germany. Electronic address:

The applicability of rat precision-cut lung slices (PCLuS) in detecting nanomaterial (NM) toxicity to the respiratory tract was investigated evaluating sixteen OECD reference NMs (TiO₂, ZnO, CeO₂, SiO₂, Ag, multi-walled carbon nanotubes (MWCNTs)). Upon 24-hour test substance exposure, the PCLuS system was able to detect early events of NM toxicity: total protein, reduction in mitochondrial activity, caspase-3/-7 activation, glutathione depletion/increase, cytokine induction, and histopathological evaluation. Ion shedding NMS (ZnO and Ag) induced severe tissue destruction detected by the loss of total protein. Two anatase TiO₂ NMs, CeO₂ NMs, and two MWCNT caused significant (determined by trend analysis) cytotoxicity in the WST-1 assay. At non-cytotoxic concentrations, different TiO₂ NMs and one MWCNT increased GSH levels, presumably a defense response to reactive oxygen species, and these substances further induced a variety of cytokines. One of the SiO₂ NMs increased caspase-3/-7 activities at non-cytotoxic levels, and one rutile TiO₂ only induced cytokines. Investigating these effects is, however, not sufficient to predict apical effects found in vivo. Reproducibility of test substance measurements was not fully satisfactory, especially in the GSH and cytokine assays. Effects were frequently observed in negative controls pointing to tissue slice vulnerability even though prepared and handled with utmost care. Comparisons of the effects observed in the PCLuS to in vivo effects reveal some concordances for the metal oxide NMs, but less so for the MWCNT. The highest effective dosages, however, exceeded those reported for rat short-term inhalation studies. To become applicable for NM testing, the PCLuS system requires test protocol optimization.
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http://dx.doi.org/10.1016/j.taap.2013.12.017DOI Listing
April 2014

Predictive toxicology of cobalt ferrite nanoparticles: comparative in-vitro study of different cellular models using methods of knowledge discovery from data.

Part Fibre Toxicol 2013 Jul 29;10:32. Epub 2013 Jul 29.

Department of Physiology and Pharmacology, Faculty of Medicine and the Marian Gertner Institute for Medical Nanosystems, Tel Aviv University, Tel-Aviv 69978, Israel.

Background: Cobalt-ferrite nanoparticles (Co-Fe NPs) are attractive for nanotechnology-based therapies. Thus, exploring their effect on viability of seven different cell lines representing different organs of the human body is highly important.

Methods: The toxicological effects of Co-Fe NPs were studied by in-vitro exposure of A549 and NCIH441 cell-lines (lung), precision-cut lung slices from rat, HepG2 cell-line (liver), MDCK cell-line (kidney), Caco-2 TC7 cell-line (intestine), TK6 (lymphoblasts) and primary mouse dendritic-cells. Toxicity was examined following exposure to Co-Fe NPs in the concentration range of 0.05 -1.2 mM for 24 and 72 h, using Alamar blue, MTT and neutral red assays. Changes in oxidative stress were determined by a dichlorodihydrofluorescein diacetate based assay. Data analysis and predictive modeling of the obtained data sets were executed by employing methods of Knowledge Discovery from Data with emphasis on a decision tree model (J48).

Results: Different dose-response curves of cell viability were obtained for each of the seven cell lines upon exposure to Co-Fe NPs. Increase of oxidative stress was induced by Co-Fe NPs and found to be dependent on the cell type. A high linear correlation (R2=0.97) was found between the toxicity of Co-Fe NPs and the extent of ROS generation following their exposure to Co-Fe NPs. The algorithm we applied to model the observed toxicity belongs to a type of supervised classifier. The decision tree model yielded the following order with decrease of the ranking parameter: NP concentrations (as the most influencing parameter), cell type (possessing the following hierarchy of cell sensitivity towards viability decrease: TK6 > Lung slices > NCIH441 > Caco-2 = MDCK > A549 > HepG2 = Dendritic) and time of exposure, where the highest-ranking parameter (NP concentration) provides the highest information gain with respect to toxicity. The validity of the chosen decision tree model J48 was established by yielding a higher accuracy than that of the well-known "naive bayes" classifier.

Conclusions: The observed correlation between the oxidative stress, caused by the presence of the Co-Fe NPs, with the hierarchy of sensitivity of the different cell types towards toxicity, suggests that oxidative stress is one possible mechanism for the toxicity of Co-Fe NPs.
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http://dx.doi.org/10.1186/1743-8977-10-32DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3734223PMC
July 2013

Erratum to "Applicability of in vitro tests for skin irritation and corrosion to regulatory classification schemes: substantiating test strategies with data from routine studies" [Regul. Toxicol. Pharmacol. (2012) 402-414].

Regul Toxicol Pharmacol 2013 Apr;65(3):366-78

BASF SE, Experimental Toxicology and Ecology, Carl-Bosch-Strasse 38, 67056 Ludwigshafen, Germany.

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).
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http://dx.doi.org/10.1016/j.yrtph.2013.02.001DOI Listing
April 2013

Vinclozolin: a case study on the identification of endocrine active substances in the past and a future perspective.

Toxicol Lett 2013 Dec 1;223(3):271-9. Epub 2013 Apr 1.

BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany. Electronic address:

In the late 1980s vinclozolin was tested for prenatal developmental toxicity in rats for registration purposes in USA. At 1000mg/kgbw, 95% of all fetuses were female upon visual inspection (ano-genital distance determination). Anti-androgenic effects (AA) were also noted in a subsequent 2-generation study. These findings triggered mechanistic investigations at BASF and at US-EPA. Results published by the latter were the starting point of the endocrine disruption (ED) discussion in the 1990s. AA effects of vinclozolin are mediated by two metabolites, which have an antagonistic effect on the androgen receptor. Currently, determination of ED has become a major end-point in toxicology testing and the US-EPA has set up an elaborated testing paradigm to fulfill this requirement. Future screening for ED can be improved making use of new technologies. ED modes of action can be determined by three alternative (3R) methods. Steroid synthesis in H295R cells (1), androgen-receptor binding in modified yeast (2) and metabolomics (3). Using vinclozolin as a case study, results indicate: (1) an effect on steroid synthesis in vitro, (2) an antagonistic effect on the androgen receptor and (3) that the metabolome profile of vinclozolin is similar to that of other receptor mediated anti-androgens (e.g. flutamide).
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http://dx.doi.org/10.1016/j.toxlet.2013.03.029DOI Listing
December 2013
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