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Use of three-dimensional (3D) tissue equivalents in toxicology has been increasing over the last decade as novel preclinical test systems and as alternatives to animal testing. In the area of genetic toxicology, progress has been made with establishing robust protocols for skin, airway (lung) and liver tissue equivalents. In light of these advancements, a "Use of 3D Tissues in Genotoxicity Testing" working group (WG) met at the 7th IWGT meeting in Tokyo in November 2017 to discuss progress with these models and how they may fit into a genotoxicity testing strategy. The workshop demonstrated that skin models have reached an advanced state of validation following over 10 years of development, while liver and airway model-based genotoxicity assays show promise but are at an early stage of development. Further effort in liver and airway model-based assays is needed to address the lack of coverage of the three main endpoints of genotoxicity (mutagenicity, clastogenicity and aneugenicity), and information on metabolic competence. The IWGT WG believes that the 3D skin comet and micronucleus assays are now sufficiently validated to undergo an independent peer review of the validation study, followed by development of individual OECD Test Guidelines.

Previous work with a diverse set of reference chemicals suggests that an in vitro multiplexed flow cytometry-based assay (MultiFlow™ DNA Damage Kit-p53, γH2AX, Phospho-Histone H3) can distinguish direct-acting clastogens and aneugens from nongenotoxicants (Bryce SM et al. []: Environ Mol Mutagen 57:171-189). This work extends this line of investigation to include compounds that require metabolic activation to form reactive electrophiles. For these experiments, TK6 cells were exposed to 11 promutagens and 37 presumed nongenotoxicants in 96 well plates. Unless precipitation or foreknowledge about cytotoxicity suggested otherwise, the highest concentration was 1 mM. Exposure occurred for 4 hr after which time cells were washed to remove S9 and test article. Immediately following the wash and again at 24 hr, cell aliquots were added to wells of a microtiter plate containing the working detergent/stain/antibody cocktail. After a brief incubation, robotic sampling was employed for walk-away flow cytometric data acquisition. Univariate logistic regression analyses indicated that γH2AX induction and p53 activation provide the greatest degree of discrimination between clastogens and nongenotoxicants. Multivariate prediction algorithms that incorporated both of these endpoints, in each combination of time points, were evaluated. The best performing models correctly predicted 9 clastogens out of 11 and 36 nongenotoxicants out of 37. These results are encouraging as they suggest that an efficient and highly scalable multiplexed assay can effectively identify clastogenic chemicals that require bioactivation. More work is planned with a broader range of chemicals, additional cell lines, and other laboratories to further evaluate the merits and limitations of this approach. Environ. Mol. Mutagen. 57:546-558, 2016. © 2016 Wiley Periodicals, Inc.

ESAC, the EURL ECVAM Scientific Advisory Committee, advises EURL ECVAM on scientific issues. Its main role is to conduct independent peer review of validation studies of alternative test methods and to assess their scientific validity for a given purpose. The committee reviews the appropriateness of study design and management, the quality of results obtained and the plausibility of the conclusions drawn. ESAC peer reviews are formally initiated with a EURL ECVAM Request for ESAC Advice, which provides the necessary background for the peer-review and establishes its objectives, timelines and the questions to be addressed. The peer review is normally prepared by specialised ESAC Working Groups. These are typically composed of ESAC members and other external experts relevant to the test method under review. These experts may be nominated by ESAC, EURL ECVAM and partner organisations within the International Cooperation on Alternative Test Methods (ICATM). ESAC ultimately decides on the composition of these Working Groups. ESAC's advice to EURL ECVAM is formally provided as 'ESAC Opinions' and 'Working Group Reports' at the end of the peer review. ESAC may also issue Opinions on other scientific issues of relevance to the work and mission of EURL ECVAM but not directly related to a specific alternative test method. The ESAC Opinion expressed in this report relates to the peer-review of the BASF-coordinated Performance Standards-based validation of the LuSens test method for skin sensitisation testing., JRC.F.3-Chemicals Safety and Alternative Methods

ESAC, the EURL ECVAM Scientific Advisory Committee, advises EURL ECVAM on scientific issues. Its main role is to conduct independent peer review of validation studies of alternative test methods and to assess their scientific validity for a given purpose. The committee reviews the appropriateness of study design and management, the quality of results obtained and the plausibility of the conclusions drawn. ESAC peer reviews are formally initiated with a EURL ECVAM Request for ESAC Advice, which provides the necessary background for the peer-review and establishes its objectives, timelines and the questions to be addressed. The peer review is normally prepared by specialised ESAC Working Groups. These are typically composed of ESAC members and other external experts relevant to the test method under review. These experts may be nominated by ESAC, EURL ECVAM and partner organisations within the International Cooperation on Alternative Test Methods (ICATM). ESAC ultimately decides on the composition of these Working Groups. ESAC's advice to EURL ECVAM is formally provided as 'ESAC Opinions' and 'Working Group Reports' at the end of the peer review. ESAC may also issue Opinions on other scientific issues of relevance to the work and mission of EURL ECVAM but not directly related to a specific alternative test method. The ESAC Opinion expressed in this report relates to the peer-review of the Ocular Irritection® test method for prediction of serious eye damage/eye irritation potential of chemicals., JRC.F.3-Chemicals Safety and Alternative Methods

ESAC, the EURL ECVAM Scientific Advisory Committee, advises EURL ECVAM on scientific issues. Its main role is to conduct independent peer review of validation studies of alternative test methods and to assess their scientific validity for a given purpose. The committee reviews the appropriateness of study design and management, the quality of results obtained and the plausibility of the conclusions drawn. ESAC peer reviews are formally initiated with a EURL ECVAM Request for ESAC Advice, which provides the necessary background for the peer-review and establishes its objectives, timelines and the questions to be addressed. The peer review is normally prepared by specialised ESAC Working Groups. These are typically composed of ESAC members and other external experts relevant to the test method under review. These experts may be nominated by ESAC, EURL ECVAM and partner organisations within the International Cooperation on Alternative Test Methods (ICATM). ESAC ultimately decides on the composition of these Working Groups. ESAC's advice to EURL ECVAM is formally provided as 'ESAC Opinions' and 'Working Group Reports' at the end of the peer review. ESAC may also issue Opinions on other scientific issues of relevance to the work and mission of EURL ECVAM but not directly related to a specific alternative test method. The ESAC Opinion expressed in this report relates to the peer-review of the L'Oréal-coordinated study on the transferability and reliability of the U-SENS™ test method for skin sensitisation testing., JRC.F.3-Chemicals Safety and Alternative Methods

ESAC, the EURL ECVAM Scientific Advisory Committee, advises EURL ECVAM on scientific issues. Its main role is to conduct independent peer review of validation studies of alternative test methods and to assess their scientific validity for a given purpose. The committee reviews the appropriateness of study design and management, the quality of results obtained and the plausibility of the conclusions drawn. ESAC peer reviews are formally initiated with a EURL ECVAM Request for ESAC Advice, which provides the necessary background for the peer-review and establishes its objectives, timelines and the questions to be addressed. The peer review is normally prepared by specialised ESAC Working Groups. These are typically composed of ESAC members and other external experts relevant to the test method under review. These experts may be nominated by ESAC, EURL ECVAM and partner organisations within the International Cooperation on Alternative Test Methods (ICATM). ESAC ultimately decides on the composition of these Working Groups. ESAC's advice to EURL ECVAM is formally provided as 'ESAC Opinions' and 'Working Group Reports' at the end of the peer review. ESAC may also issue Opinions on other scientific issues of relevance to the work and mission of EURL ECVAM but not directly related to a specific alternative test method. The ESAC Opinion expressed in this report relates to the peer-review of the validation study of the epiCS® Skin Irritation Test (SIT) based on the EURL ECVAM/OECD Performance Standards for in vitro skin irritation testing using Reconstructed human Epidermis (RhE)., JRC.F.3-Chemicals Safety and Alternative Methods

ESAC, the EURL ECVAM Scientific Advisory Committee, advises EURL ECVAM on scientific issues. Its main role is to conduct independent peer review of validation studies of alternative test methods and to assess their scientific validity for a given purpose. The committee reviews the appropriateness of study design and management, the quality of results obtained and the plausibility of the conclusions drawn. ESAC peer reviews are formally initiated with a EURL ECVAM Request for ESAC Advice, which provides the necessary background for the peer-review and establishes its objectives, timelines and the questions to be addressed. The peer review is normally prepared by specialised ESAC Working Groups. These are typically composed of ESAC members and other external experts relevant to the test method under review. These experts may be nominated by ESAC, EURL ECVAM and partner organisations within the International Cooperation on Alternative Test Methods (ICATM). ESAC ultimately decides on the composition of these Working Groups. ESAC's advice to EURL ECVAM is formally provided as 'ESAC Opinions' and 'Working Group Reports' at the end of the peer review. ESAC may also issue Opinions on other scientific issues of relevance to the work and mission of EURL ECVAM but not directly related to a specific alternative test method. The ESAC Opinion expressed in this report relates to the peer-review of the SkinEthic™ Human Corneal Epithelium (HCE) Eye Irritation Test (EIT)., JRC.F.3-Chemicals Safety and Alternative Methods

ESAC, the EURL ECVAM Scientific Advisory Committee, advises EURL ECVAM on scientific issues. Its main role is to conduct independent peer review of validation studies of alternative test methods and to assess their scientific validity for a given purpose. The committee reviews the appropriateness of study design and management, the quality of results obtained and the plausibility of the conclusions drawn. ESAC peer reviews are formally initiated with a EURL ECVAM Request for ESAC Advice, which provides the necessary background for the peer-review and establishes its objectives, timelines and the questions to be addressed. The peer review is normally prepared by specialised ESAC Working Groups. These are typically composed of ESAC members and other external experts relevant to the test method under review. These experts may be nominated by ESAC, EURL ECVAM and partner organisations within the International Cooperation on Alternative Test Methods (ICATM). ESAC ultimately decides on the composition of these Working Groups. ESAC's advice to EURL ECVAM is formally provided as 'ESAC Opinions' and 'Working Group Reports' at the end of the peer review. ESAC may also issue Opinions on other scientific issues of relevance to the work and mission of EURL ECVAM but not directly related to a specific alternative test method. The ESAC Opinion expressed in this report relates to the use of Performance Standards to evaluate test methods similar to a Validated Reference Method., JRC.F.3-Chemicals Safety and Alternative Methods

ESAC, the EURL ECVAM Scientific Advisory Committee, advises EURL ECVAM on scientific issues. Its main role is to conduct independent peer review of validation studies of alternative test methods and to assess their scientific validity for a given purpose. The committee reviews the appropriateness of study design and management, the quality of results obtained and the plausibility of the conclusions drawn. ESAC peer reviews are formally initiated with a EURL ECVAM Request for ESAC Advice, which provides the necessary background for the peer-review and establishes its objectives, timelines and the questions to be addressed. The peer review is normally prepared by specialised ESAC Working Groups. These are typically composed of ESAC members and other external experts relevant to the test method under review. These experts may be nominated by ESAC, EURL ECVAM and partner organisations within the International Cooperation on Alternative Test Methods (ICATM). ESAC ultimately decides on the composition of these Working Groups. ESAC's advice to EURL ECVAM is formally provided as 'ESAC Opinions' and 'Working Group Reports' at the end of the peer review. ESAC may also issue Opinions on other scientific issues of relevance to the work and mission of EURL ECVAM but not directly related to a specific alternative test method. The ESAC Opinion expressed in this report relates to the peer-review of the BASF-coordinated Performance Standards-based validation of the LuSens test method for skin sensitisation testing., JRC.F.3-Chemicals Safety and Alternative Methods

Human testicular germ cell tumors are histologically heterogenous neoplasms with variable malignant potential. Two main types of germ cell tumors occur in men: seminomas and nonseminomas. In the present study a set of genes (protoncogene and tumor suppressor genes) was investigated in testicular cancers. K-ras protooncogene was tested for activating point mutations, and CDH1, APC, p53 and nm23-H1 tumor suppressor genes for loss of heterozygosity (LOH). Three seminomas and eight nonseminomas were analysed by PCR using restriction fragment length polymorphism, dinucleotide or tetranucleotide repeat polymorphism method. The results of our analysis showed that the most frequent changes were LOH of p53. Allelic loss of p53 was detected in three nonseminomas. Allelic losses of APC gene were present in one seminoma and one nonseminoma, whereas another seminoma and one nonseminoma showed LOH of CDH1. Analysis of heterozygosity has shown no consistent structural genetic alterations in tumor tissues. Although limited number of samples have been analysed so far, these findings are in accordance with the results of similar studies performed on testicular cancers.

Leachates from landfill sites are the main origin of water pollution because of continuous exposure and flow of landfills to the water logging system, generates aqueous leachates which contaminate ground and surface water reservoirs. Two samples of landfill leachate from Rovinj landfill site were collected, analyzed and treated with powdered activated carbon in order to decrease concentration of toxic compounds. The optimum purification conditions were as follows: contact time: 20 minutes ; mass of adsorbent 2.5 g per 100 mL of leachate ; pH =8. All parameters in the purified water were significantly lower compared to initial concentrations and maximum allowed values for wastewater suitable for discharge directly into the environment. Nevertheless, due to the complex nature of the leachates and their purified samples, chemical analysis alone is not sufficient for assessing their potential genotoxicity. In this work two test systems were used to evaluate cytotoxicity and genotoxicity of leachates and purified water. In order to determine cytotoxic and mutagenic potential of leachates, two strains of Salmonella typhimurium TA98 and TA100 were used. The genotoxicity of leachates and purified samples was evaluated on human cervical carcinoma cell line, during different incubation period using alkaline comet assay and lipid peroxidation assay. The leachates produced significant dose-dependent and time-dependent increases in DNA damage and lipid peroxidation induction and suggest that the exposure of human populations to leachates may lead to adverse health effects. Purified samples caused insignificant increase in DNA damage and lipid peroxidation only after prolonged exposure of the cells to purified waters, suggesting that purification of landfill leachates with activated carbon is a optimal method for genotoxic compounds removal prior to their release into environment.