Intralaboratory and interlaboratory evaluation of the EpiDerm™ 3D human reconstructed skin micronucleus (RSMN) assay

Hu, Ting; Kaluzhny, Yulia; Mun, Greg C.; Barnett, Brenda; Karetsky, Viktor; Wilt, Nathan; Klausner, Mitchell; Curren, Rodger; Aardema, Marilyn J.

doi:10.1016/j.mrgentox.2008.12.003

Cited by 56 publications

(35 citation statements)

References 4 publications

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“…These data confirm the positive findings with MMS and 4NQO in skin observed by others in the micronucleus assay (27,39,40). As previously reported for ex vivo human skin tissues by Reus et al (36), MMS was also considered a suitable positive control for the comet assay in EpiDerm™.…”

Section: Resultssupporting

confidence: 92%

Comet assay in reconstructed 3D human epidermal skin models--investigation of intra- and inter-laboratory reproducibility with coded chemicals

Reus¹,

Reisinger

Downs

et al. 2013

Mutagenesis

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Reconstructed 3D human epidermal skin models are being used increasingly for safety testing of chemicals. Based on EpiDerm™ tissues, an assay was developed in which the tissues were topically exposed to test chemicals for 3h followed by cell isolation and assessment of DNA damage using the comet assay. Inter-laboratory reproducibility of the 3D skin comet assay was initially demonstrated using two model genotoxic carcinogens, methyl methane sulfonate (MMS) and 4-nitroquinoline-n-oxide, and the results showed good concordance among three different laboratories and with in vivo data. In Phase 2 of the project, intra- and inter-laboratory reproducibility was investigated with five coded compounds with different genotoxicity liability tested at three different laboratories. For the genotoxic carcinogens MMS and N-ethyl-N-nitrosourea, all laboratories reported a dose-related and statistically significant increase (P < 0.05) in DNA damage in every experiment. For the genotoxic carcinogen, 2,4-diaminotoluene, the overall result from all laboratories showed a smaller, but significant genotoxic response (P < 0.05). For cyclohexanone (CHN) (non-genotoxic in vitro and in vivo, and non-carcinogenic), an increase compared to the solvent control acetone was observed only in one laboratory. However, the response was not dose related and CHN was judged negative overall, as was p-nitrophenol (p-NP) (genotoxic in vitro but not in vivo and non-carcinogenic), which was the only compound showing clear cytotoxic effects. For p-NP, significant DNA damage generally occurred only at doses that were substantially cytotoxic (>30% cell loss), and the overall response was comparable in all laboratories despite some differences in doses tested. The results of the collaborative study for the coded compounds were generally reproducible among the laboratories involved and intra-laboratory reproducibility was also good. These data indicate that the comet assay in EpiDerm™ skin models is a promising model for the safety assessment of compounds with a dermal route of exposure.

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Section: Resultssupporting

confidence: 92%

Comet assay in reconstructed 3D human epidermal skin models--investigation of intra- and inter-laboratory reproducibility with coded chemicals

Reus¹,

Reisinger

Downs

et al. 2013

Mutagenesis

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“…It is important to note however that there remains a void in understanding how truly alike the currently available in vitro systems are to the in vivo scenario. Whilst some of the literature eluded to in the present manuscript (52, 69) suggests that currently used in vitro systems exhibit similarities to in vivo , it is abundantly clear within the field that more efforts must be given towards actually comparing and confirming the proximity of such models to in vivo . Whilst this would begin to achieve the visions of the 3Rs in nano(geno)toxicology research (30), emphasis must also be towards enabling in vitro systems to mimic the human body and not just a replacement for animal experimentation.…”

Section: Discussionmentioning

confidence: 86%

“…Despite this, there has been an extensive international effort to adapt the CBMN assay for the use with the EpiDerm™ model, now known as the 3D reconstructed skin micronucleus assay (RSMN). The RSMN assay has been undergoing pre-validation for the last 8 years with a standardised protocol in place and very good reproducibility (69–71). Efforts are now being placed on increasing throughput of the RSMN assay by the use of automated techniques such as the Metasystems Slide Scanning Platform (Metafer) (72).…”

Section: D Reconstructed Tissue Modelsmentioning

confidence: 99%

Critical review of the current and future challenges associated with advancedin vitrosystems towards the study of nanoparticle (secondary) genotoxicity

Evans

Clift

Singh

et al. 2016

MUTAGE

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With the need to understand the potential biological impact of the plethora of nanoparticles (NPs) being manufactured for a wide range of potential human applications, due to their inevitable human exposure, research activities in the field of NP toxicology has grown exponentially over the last decade. Whilst such increased research efforts have elucidated an increasingly significant knowledge base pertaining to the potential human health hazard posed by NPs, understanding regarding the possibility for NPs to elicit genotoxicity is limited. In vivo models are unable to adequately discriminate between the specific modes of action associated with the onset of genotoxicity. Additionally, in line with the recent European directives, there is an inherent need to move away from invasive animal testing strategies. Thus, in vitro systems are an important tool for expanding our mechanistic insight into NP genotoxicity. Yet uncertainty remains concerning their validity and specificity for this purpose due to the unique challenges presented when correlating NP behaviour in vitro and in vivo. This review therefore highlights the current state of the art in advanced in vitro systems and their specific advantages and disadvantages from a NP genotoxicity testing perspective. Key indicators will be given related to how these systems might be used or improved to enhance understanding of NP genotoxicity.

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“…Further, following the 7th Amendment to the Cosmetics Directive in 2009, use of laboratory animals in cosmetics testing has been banned in the European Union (1). Due to the fact that in vivo testing has previously been heavily relied upon to confirm positive results of in vitro genotoxicity assays, it is now vital that in vitro tests alone can accurately identify human carcinogens (2). However, in vitro tests often exaggerate toxic effects when compared with in vivo results, possibly due to lack of normal human tissue structure or dosing strategies being unrealistic.…”

Section: Introductionmentioning

confidence: 99%

Automation and validation of micronucleus detection in the 3D EpiDerm™ human reconstructed skin assay and correlation with 2D dose responses

et al. 2014

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Recent restrictions on the testing of cosmetic ingredients in animals have resulted in the need to test the genotoxic potential of chemicals exclusively in vitro prior to licensing. However, as current in vitro tests produce some misleading positive results, sole reliance on such tests could prevent some chemicals with safe or beneficial exposure levels from being marketed. The 3D human reconstructed skin micronucleus (RSMN) assay is a promising new in vitro approach designed to assess genotoxicity of dermally applied compounds. The assay utilises a highly differentiated in vitro model of the human epidermis. For the first time, we have applied automated micronucleus detection to this assay using MetaSystems Metafer Slide Scanning Platform (Metafer), demonstrating concordance with manual scoring. The RSMN assay’s fixation protocol was found to be compatible with the Metafer, providing a considerably shorter alternative to the recommended Metafer protocol. Lowest observed genotoxic effect levels (LOGELs) were observed for mitomycin-C at 4.8 µg/ml and methyl methanesulfonate (MMS) at 1750 µg/ml when applied topically to the skin surface. In-medium dosing with MMS produced a LOGEL of 20 µg/ml, which was very similar to the topical LOGEL when considering the total mass of MMS added. Comparisons between 3D medium and 2D LOGELs resulted in a 7-fold difference in total mass of MMS applied to each system, suggesting a protective function of the 3D microarchitecture. Interestingly, hydrogen peroxide (H2O2), a positive clastogen in 2D systems, tested negative in this assay. A non-genotoxic carcinogen, methyl carbamate, produced negative results, as expected. We also demonstrated expression of the DNA repair protein N-methylpurine-DNA glycosylase in EpiDerm™. Our preliminary validation here demonstrates that the RSMN assay may be a valuable follow-up to the current in vitro test battery, and together with its automation, could contribute to minimising unnecessary in vivo tests by reducing in vitro misleading positives.

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Intralaboratory and interlaboratory evaluation of the EpiDerm™ 3D human reconstructed skin micronucleus (RSMN) assay

Cited by 56 publications

References 4 publications

Comet assay in reconstructed 3D human epidermal skin models--investigation of intra- and inter-laboratory reproducibility with coded chemicals

Comet assay in reconstructed 3D human epidermal skin models--investigation of intra- and inter-laboratory reproducibility with coded chemicals

Critical review of the current and future challenges associated with advancedin vitrosystems towards the study of nanoparticle (secondary) genotoxicity

Automation and validation of micronucleus detection in the 3D EpiDerm™ human reconstructed skin assay and correlation with 2D dose responses

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