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      The BlueScreen HC assay to predict the genotoxic potential of fragrance materials

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          Abstract

          BlueScreen HC is a mammalian cell-based assay for measuring the genotoxicity and cytotoxicity of chemical compounds and mixtures. The BlueScreen HC assay has been utilized at the Research Institute for Fragrance Materials in a safety assessment program as a screening tool to prioritize fragrance materials for higher-tier testing, as supporting evidence when using a read-across approach, and as evidence to adjust the threshold of toxicological concern. Predictive values for the BlueScreen HC assay were evaluated based on the ability of the assay to predict the outcome of in vitro and in vivo mutagenicity and chromosomal damage genotoxicity assays. A set of 371 fragrance materials was assessed in the BlueScreen HC assay along with existing or newly generated in vitro and in vivo genotoxicity data. Based on a weight-of-evidence approach, the majority of materials in the data set were deemed negative and concluded not to have the potential to be genotoxic, while only a small proportion of materials were determined to show genotoxic effects in these assays. Analysis of the data set showed a combination of high positive agreement but low negative agreement between BlueScreen HC results, in vitro regulatory genotoxicity assays, and higher-tier test results. The BlueScreen HC assay did not generate any false negatives, thereby providing robustness when utilizing it as a high-throughput screening tool to evaluate the large inventory of fragrance materials. From the perspective of protecting public health, it is desirable to have no or minimal false negatives, as a false-negative result may incorrectly indicate the lack of a genotoxicity hazard. However, the assay did have a high percentage of false-positive results, resulting in poor positive predictivity of the in vitro genotoxicity test battery outcome. Overall, the assay generated 100% negative predictivity and 3.9% positive predictivity. In addition to the data set of 371 fragrance materials, 30 natural complex substances were evaluated for BlueScreen HC, Ames, and in vitro micronucleus assay, and a good correlation in all three assays was observed. Overall, while a positive result may have to be further investigated, these findings suggest that the BlueScreen HC assay can be a valuable screening tool to detect the genotoxic potential of fragrance materials and mixtures.

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          Most cited references67

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          Recommendations for conducting the in vivo alkaline Comet assay. 4th International Comet Assay Workshop.

          The in vivo alkaline single cell gel electrophoresis assay, hereafter the Comet assay, can be used to investigate the genotoxicity of industrial chemicals, biocides, agrochemicals and pharmaceuticals. The major advantages of this assay include the relative ease of application to any tissue of interest, the detection of multiple classes of DNA damage and the generation of data at the level of the single cell. These features give the Comet assay potential advantages over other in vivo test methods, which are limited largely to proliferating cells and/or a single tissue. The Comet assay has demonstrated its reliability in many testing circumstances and is, in general, considered to be acceptable for regulatory purposes. However, despite the considerable data published on the in vivo Comet assay and the general agreement within the international scientific community over many protocol-related issues, it was felt that a document giving detailed practical guidance on the protocol required for regulatory acceptance of the assay was required. In a recent meeting held in conjunction with the 4th International Comet Assay Workshop (Ulm, Germany, 22-25 July 2001) an expert panel reviewed existing data and recent developments of the Comet assay with a view to developing such a document. This paper is intended to act as an update to the more general guidelines which were published as a result of the International Workshop on Genotoxicity Test Procedures. The recommendations are also seen as a major step towards gaining more formal regulatory acceptance of the Comet assay.
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            Structure-based thresholds of toxicological concern (TTC): guidance for application to substances present at low levels in the diet.

            The threshold of toxicological concern (TTC) is a pragmatic risk assessment tool that is based on the principle of establishing a human exposure threshold value for all chemicals, below which there is a very low probability of an appreciable risk to human health. The concept that there are levels of exposure that do not cause adverse effects is inherent in setting acceptable daily intakes (ADIs) for chemicals with known toxicological profiles. The TTC principle extends this concept by proposing that a de minimis value can be identified for many chemicals, in the absence of a full toxicity database, based on their chemical structures and the known toxicity of chemicals which share similar structural characteristics. The establishment and application of widely accepted TTC values would benefit consumers, industry and regulators. By avoiding unnecessary toxicity testing and safety evaluations when human intakes are below such a threshold, application of the TTC approach would focus limited resources of time, cost, animal use and expertise on the testing and evaluation of substances with the greatest potential to pose risks to human health and thereby contribute to a reduction in the use of animals. An Expert Group of the European branch of the International Life Sciences Institute-ILSI Europe-has examined the TTC principle for its wider applicability in food safety evaluation. The Expert Group examined metabolism and accumulation, structural alerts, endocrine disrupting chemicals and specific endpoints, such as neurotoxicity, teratogenicity, developmental toxicity, allergenicity and immunotoxicity, and determined whether such properties or endpoints had to be taken into consideration specifically in a step-wise approach. The Expert Group concluded that the TTC principle can be applied for low concentrations in food of chemicals that lack toxicity data, provided that there is a sound intake estimate. The use of a decision tree to apply the TTC principle is proposed, and this paper describes the step-wise process in detail. Proteins, heavy metals and polyhalogenated-dibenzodioxins and related compounds were excluded from this approach. When assessing a chemical, a review of prior knowledge and context of use should always precede the use of the TTC decision tree. The initial step is the identification and evaluation of possible genotoxic and/or high potency carcinogens. Following this step, non-genotoxic substances are evaluated in a sequence of steps related to the concerns that would be associated with increasing intakes. For organophosphates a TTC of 18microg per person per day (0.3 microg/kg bw/day) is proposed, and when the compound is not an OP, the TTC values for the Cramer structural classes III, II and I, with their respective TTC levels (e.g. 1800, 540 and 90 microg per person per day; or 30, 9 and 1.5 microg/kg bw /day), would be applied sequentially. All other endpoints or properties were shown to have a distribution of no observed effect levels (NOELs) similar to the distribution of NOELs for general toxicity endpoints in Cramer classes I, II and III. The document was discussed with a wider audience during a workshop held in March 2003 (see list of workshop participants).
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              Evaluation of the ability of a battery of three in vitro genotoxicity tests to discriminate rodent carcinogens and non-carcinogens I. Sensitivity, specificity and relative predictivity.

              The performance of a battery of three of the most commonly used in vitro genotoxicity tests--Ames+mouse lymphoma assay (MLA)+in vitro micronucleus (MN) or chromosomal aberrations (CA) test--has been evaluated for its ability to discriminate rodent carcinogens and non-carcinogens, from a large database of over 700 chemicals compiled from the CPDB ("Gold"), NTP, IARC and other publications. We re-evaluated many (113 MLA and 30 CA) previously published genotoxicity results in order to categorise the performance of these assays using the response categories we established. The sensitivity of the three-test battery was high. Of the 553 carcinogens for which there were valid genotoxicity data, 93% of the rodent carcinogens evaluated in at least one assay gave positive results in at least one of the three tests. Combinations of two and three test systems had greater sensitivity than individual tests resulting in sensitivities of around 90% or more, depending on test combination. Only 19 carcinogens (out of 206 tested in all three tests, considering CA and MN as alternatives) gave consistently negative results in a full three-test battery. Most were either carcinogenic via a non-genotoxic mechanism (liver enzyme inducers, peroxisome proliferators, hormonal carcinogens) considered not necessarily relevant for humans, or were extremely weak (presumed) genotoxic carcinogens (e.g. N-nitrosodiphenylamine). Two carcinogens (5-chloro-o-toluidine, 1,1,2,2-tetrachloroethane) may have a genotoxic element to their carcinogenicity and may have been expected to produce positive results somewhere in the battery. We identified 183 chemicals that were non-carcinogenic after testing in both male and female rats and mice. There were genotoxicity data on 177 of these. The specificity of the Ames test was reasonable (73.9%), but all mammalian cell tests had very low specificity (i.e. below 45%), and this declined to extremely low levels in combinations of two and three test systems. When all three tests were performed, 75-95% of non-carcinogens gave positive (i.e. false positive) results in at least one test in the battery. The extremely low specificity highlights the importance of understanding the mechanism by which genotoxicity may be induced (whether it is relevant for the whole animal or human) and using weight of evidence approaches to assess the carcinogenic risk from a positive genotoxicity signal. It also highlights deficiencies in the current prediction from and understanding of such in vitro results for the in vivo situation. It may even signal the need for either a reassessment of the conditions and criteria for positive results (cytotoxicity, solubility, etc.) or the development and use of a completely new set of in vitro tests (e.g. mutation in transgenic cell lines, systems with inherent metabolic activity avoiding the use of S9, measurement of genetic changes in more cancer-relevant genes or hotspots of genes, etc.). It was very difficult to assess the performance of the in vitro MN test, particularly in combination with other assays, because the published database for this assay is relatively small at this time. The specificity values for the in vitro MN assay may improve if data from a larger proportion of the known non-carcinogens becomes available, and a larger published database of results with the MN assay is urgently needed if this test is to be appreciated for regulatory use. However, specificity levels of <50% will still be unacceptable. Despite these issues, by adopting a relative predictivity (RP) measure (ratio of real:false results), it was possible to establish that positive results in all three tests indicate the chemical is greater than three times more likely to be a rodent carcinogen than a non-carcinogen. Likewise, negative results in all three tests indicate the chemical is greater than two times more likely to be a rodent non-carcinogen than a carcinogen. This RP measure is considered a useful tool for industry to assess the likelihood of a chemical possessing carcinogenic potential from batteries of positive or negative results.
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                Author and article information

                Journal
                Mutagenesis
                Mutagenesis
                mutage
                Mutagenesis
                Oxford University Press (UK )
                0267-8357
                1464-3804
                January 2022
                18 March 2022
                18 March 2022
                : 37
                : 1
                : 13-23
                Affiliations
                [1 ] Research Institute for Fragrance Materials, Inc. , 50 Tice Blvd, Woodcliff Lake, NJ 07677, United States
                [2 ] Firmenich, Inc. , 250 Plainsboro Rd, Plainsboro Township, NJ 08536, United States
                [3 ] Global Product Safety, Colgate-Palmolive Company , 909 River Rd, Piscataway, NJ 08854, United States
                [4 ] Firmenich, Inc. , Rue de la Bergère 7, 1242 Satigny, Switzerland
                [5 ] Innovations in Food & Chemical Safety Programme, Agency for Science, Technology and Research (A*STAR) , 1, #20-10 Fusionopolis Way, Connexis, North Tower, 138632, Singapore
                [6 ] Singapore Institute of Food & Biotechnology Innovation, A*STAR , 1, #20-10 Fusionopolis Way, Connexis, North Tower, 138632, Singapore
                [7 ] Symrise AG, Mühlenfeldstr 1 , 37603, Holzminden, Niedersachsen, Germany
                [8 ] Gentronix, Alderley Edge , Macclesfield SK10 4TG, United Kingdom
                [9 ] SC Johnson , 1525 Howe St, Racine, WI 53403, United States
                [10 ] L'Oreal Life Sciences Research , 1, Av Eugene Schueller 93600 Aulnay sous Bois, France
                [11 ] The Procter & Gamble Company, Mason Business Centre , Mason, OH, United States
                [12 ] Takasago , 4 Volvo Dr, Rockleigh, NJ 07647, United States
                [13 ] New York Medical College , 40 Sunshine Cottage Rd, Valhalla, NY 10595, United States
                [14 ] Technical University of Munich , Arcisstraße 21, 80333 München, Germany
                [15 ] University of Kaiserslautern, Erwin-Schrödinger-Straße 52 , 67663 Kaiserslautern, Germany (Retired)
                [16 ] Department of Pharmacology and Toxicology of the University of Würzburg , Sanderring 2, 97070 Würzburg, Germany
                Author notes
                Corresponding author. Research Institute for Fragrance Materials, Inc., 50 Tice Boulevard, Woodcliff Lake, NJ 07677-7654, United States. E-mail: ythakkar@ 123456rifm.org
                Present address: 15211 North Kierland Blvd Scottsdale, AZ 85254, United States
                Article
                geac004
                10.1093/mutage/geac004
                8976226
                35302169
                dc074d98-7348-469e-9087-70032b77c01a
                © The Author(s) 2022. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License ( https://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

                History
                : 08 June 2021
                : 04 February 2022
                Page count
                Pages: 11
                Funding
                Funded by: National Research Foundation Singapore, DOI 10.13039/501100001381;
                Award ID: W20W3D0002
                Categories
                Original Manuscripts
                AcademicSubjects/SCI01140
                AcademicSubjects/MED00305

                Molecular biology
                bluescreen,genotoxicity,fragrance materials
                Molecular biology
                bluescreen, genotoxicity, fragrance materials

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