This Test Guideline (TG) describes the IL-2 Luc Assay test method to evaluate the potential immunotoxic effects of chemicals on T lymphoblastic cell line. This cell line allows quantitative measurement of luciferase gene induction by detecting luminescence from well-established light producing luciferase substrates as indicators of the activity of IL-2, IFN-γ and GAPDH in cells following exposure to immunotoxic chemicals. The method is intended to be used as a part of a battery to determine immunotoxic potential of chemicals.

This Test Guideline is designed to provide an evaluation of reproductive and developmental effects that may occur as a result of pre- and postnatal chemical exposure as well as an evaluation of systemic toxicity in pregnant and lactating females and young and adult offspring. In the assay, sexually-mature males and females rodents (parental (P) generation) are exposed to graduated doses of the test substance starting 2 weeks before mating and continuously through mating, gestation and weaning of their pups (F1 generation). At weaning, pups are selected and assigned to cohorts of animals for reproductive/developmental toxicity testing (cohort 1), developmental neurotoxicity testing (cohort 2) and developmental immunotoxicity testing (cohort 3). The F1 offspring receive further treatment with the test substance from weaning to adulthood. Clinical observations and pathology examinations are performed on all animals for signs of toxicity, with special emphasis on the integrity and performance of the male and female reproductive systems and the health, growth, development and function of the offspring. Part of cohort 1 (cohort 1B) may be extended to include an F2 generation; in this case, procedures for F1 animals will be similar to those for the P animals.

This Test Guideline is designed to provide an evaluation of reproductive and developmental effects that may occur as a result of pre- and postnatal chemical exposure as well as an evaluation of systemic toxicity in pregnant and lactating females and young and adult offspring. In the assay, sexually-mature males and females rodents (parental (P) generation) are exposed to graduated doses of the test substance starting 2 weeks before mating and continuously through mating, gestation and weaning of their pups (F1 generation). At weaning, pups are selected and assigned to cohorts of animals for reproductive/developmental toxicity testing (cohort 1), developmental neurotoxicity testing (cohort 2) and developmental immunotoxicity testing (cohort 3). The F1 offspring receive further treatment with the test substance from weaning to adulthood. Clinical observations and pathology examinations are performed on all animals for signs of toxicity, with special emphasis on the integrity and performance of the male and female reproductive systems and the health, growth, development and function of the offspring. Part of cohort 1 (cohort 1B) may be extended to include an F2 generation; in this case, procedures for F1 animals will be similar to those for the P animals.

The present Key Event based Test Guideline (TG) addresses the human health hazard endpoint skin sensitisation, following exposure to a test chemical. Skin sensitisation refers to an allergic response following skin contact with the tested chemical, as defined by the United Nations Globally Harmonized System of Classification and Labelling of Chemicals (UN GHS). This TG is proposed to address the activation of dendritic cells, which is one Key Event on the Adverse Outcome Pathway (AOP) for Skin Sensitisation. It provides four in vitro test methods addressing the same Key Event on the AOP: (i) the human cell Line Activation Test or h-CLAT method, (ii) the U937 Cell Line Activation Test or U-SENS, (iii) the Interleukin-8 Reporter Gene Assay or IL-8 Luc assay and (iv) the Genomic Allergen Rapid Detection for assessment of skin sensitisers (GARD™skin). All of them are used for supporting the discrimination between skin sensitisers and non-sensitisers in accordance with the UN GHS. The test methods described in this TG either quantify the change in the expression of cell surface marker(s) CD54 and CD86, the cytokine IL-8, or a series of genes (genomic biomarker signature) that are associated with the process of activation of monocytes and DC following exposure to sensitisers.

The present Key Event based Test Guideline addresses the human health hazard endpoint skin sensitisation, following exposure to a test chemical. Skin sensitisation refers to an allergic response following skin contact with a tested chemical, as defined by the United Nations Globally Harmonized System of Classification and Labelling of Chemicals (UN GHS). This Test Guideline is proposed to address a Key Event leading to skin sensitisation, namely keratinocyte activation. This Key Event on the Adverse Outcome Pathway (AOP) leading to skin sensitisation takes place in the keratinocytes and includes inflammatory responses as well as gene expression associated with specific cell signalling pathways such as the antioxidant/electrophile response element (ARE)-dependent pathways. This Test Guideline provides three in vitro test methods addressing the same Key Event on the AOP for skin sensitisation: (i) the ARE-Nrf2 luciferase KeratinoSens™ test method, (ii) the ARE-Nrf2 luciferase LuSens test method and (iii) the Epidermal Sensitisation Assay – EpiSensA. The KeratinoSens and the Lusens are in vitro ARE-Nrf2 luciferase-based test methods and the EpiSensA is based on gene expression quantification using Reverse Transcription- quantitative PCR in reconstructed human epidermis models. The proposed test methods are used for supporting the discrimination between skin sensitisers and non-sensitisers in accordance with the UN GHS. Performance standards have been developed to enable the validation of similar test methods.

The present Key Event based Test Guideline addresses the human health hazard endpoint skin sensitisation, following exposure to a test chemical. Skin sensitisation refers to an allergic response following skin contact with the tested chemical, as defined by the United Nations Globally Harmonized System of Classification and Labelling of Chemicals (UN GHS). This Test Guideline is proposed to address the Molecular Initiating Event leading to skin sensitisation, namely protein reactivity, by quantifying the reactivity of test chemicals towards model synthetic peptides or amino acid derivatives containing either lysine or cysteine. This Test Guideline provides three in chemico test methods addressing the same Key Event on the Adverse Outcome Pathway for Skin Sensitisation: (i) the Direct Peptide Reactivity Assay – DPRA, (ii) the Amino Acid Derivative Reactivity Assay – ADRA and (iii) the kinetic Direct Peptide Reactivity Assay – kDPRA. The DPRA and ADRA are used for supporting the discrimination between skin sensitisers and non-sensitisers in accordance with the UN GHS. In contrast, the kDPRA allows discrimination of UN GHS subcategory 1A skin sensitisers from those not categorised as subcategory 1A, i.e. subcategory 1B or no category but does not allow to distinguish sensitisers from non-sensitisers.

The Local Lymph Node Assay: DA (LLNA: DA) is a non-radioactive modification to the LLNA method for identifying potential skin sensitizing test substances and measuring the proliferation of lymphocytes they induce in the auricular lymph nodes. The method, described in mouse (CBA/J strain), is based on measurement of the adenosine triphosphate (ATP) content by bioluminescence as an indicator of this proliferation. A minimum of four animals is used per dose group, with a minimum of three concentrations of the test substance, plus a concurrent negative control group and, if appropriate, a positive control group. The experimental schedule is during 8 days. The time from animal sacrifice to measurement of ATP should not exceed 30 min. The procedure from excision of lymph nodes to ATP measurement should be kept uniform for each animal and completed within 20 minutes. The luciferin/luciferase method is applied to measure the bioluminescence in Relative Luminescence Units (RLU). This study includes: measurements (weighing, RLU), and clinical daily observations. The results are expressed as the Stimulation Index (SI) obtained by calculation. The SI should be ¡Ý1.8 before further evaluation of the test material as a potential skin sensitizer is warranted.

The Hershberger Bioassay is an in vivo short–term screening test. It evaluates the ability of a chemical to elicit biological activities consistent with androgen agonists, antagonists or 5 á-reductase inhibitors. The current bioassay is based on the changes in weight of five androgen-dependent tissues in the castrate-peripubertal male rat: the ventral prostate, seminal vesicle (plus fluids and coagulating glands), levator ani-bulbocavernosus muscle, paired Cowper’s glands and the glans penis. In order to establish whether a test substance can have androgenic or antiandrogenic action, two – respectively three - dose groups of the test substance, plus positive and vehicle (negative) controls are normally sufficient. The test substance is administered by gavage or subcutaneous injection daily for 10 consecutive days. To test for antiandrogens, the test substance is administered together with a reference androgen agonist. Each treated and control group should include a minimum of 6 animals.  The animals are necropsied approximately 24 hours after the last administration of the test substance. The tissues are excised and their fresh weights determined.  A statistically significant increase (androgenic) or decrease (antiandrogenic) in the weights of two of the five tissues indicates a positive response in this assay.

This Test Guideline describes an in vitro procedure that may be used for the hazard identification of irritant chemicals (substances and mixtures) in accordance with the UN Globally Harmonized System of Classification and Labelling (GHS) Category 2.  It is based on reconstructed human epidermis (RhE), which in its overall design closely mimics the biochemical and physiological properties of the upper parts of the human skin. Cell viability is measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that is quantitatively measured after extraction from tissues. Irritant test substances are identified by their ability to decrease cell viability below defined threshold levels (below or equal to 50% for UN GHS Category 2). Coloured chemicals can also be tested by used of an HPLC procedure. There are three validated test methods that adhere to this Test Guideline. Depending on the regulatory framework and the classification system in use, this procedure may be used to determine the skin irritancy of test substances as a stand-alone replacement test for in vivo skin irritation testing, or as a partial replacement test, within a tiered testing strategy.

This Test Guideline describes an in vitro procedure that may be used for the hazard identification of irritant chemicals (substances and mixtures) in accordance with the UN Globally Harmonized System of Classification and Labelling (GHS) Category 2. It is based on reconstructed human epidermis (RhE), which in its overall design closely mimics the biochemical and physiological properties of the upper parts of the human skin. Cell viability is measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that is quantitatively measured after extraction from tissues. Irritant test chemicals are identified by their ability to decrease cell viability below defined threshold levels (below or equal to 50% for UN GHS Category 2). This Test Guideline also includes a set of Performance Standards for the assessment of similar and modified RhE-based test methods. There are four validated test methods that adhere to this Test Guideline. Depending on the regulatory framework and the classification system in use, this procedure may be used to determine the skin irritancy of test substances as a stand-alone replacement test for in vivo skin irritation testing, or as a partial replacement test, within a tiered testing strategy.

This Test Guideline describes an in vitro procedure that may be used for the hazard identification of irritant chemicals (substances and mixtures) in accordance with the UN Globally Harmonized System of Classification and Labelling (GHS) Category 2.  It is based on reconstructed human epidermis (RhE), which in its overall design closely mimics the biochemical and physiological properties of the upper parts of the human skin. Cell viability is measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that is quantitatively measured after extraction from tissues. Irritant test substances are identified by their ability to decrease cell viability below defined threshold levels (below or equal to 50% for UN GHS Category 2). This Test Guideline also includes a set of Performance Standards for the assessment of similar and modified RhE-based test methods. There are three validated test methods that adhere to this Test Guideline. Depending on the regulatory framework and the classification system in use, this procedure may be used to determine the skin irritancy of test substances as a stand-alone replacement test for in vivo skin irritation testing, or as a partial replacement test, within a tiered testing strategy.

The Isolated Chicken Eye  (ICE) test method is an in vitro test method that can be used to classify substances as “ocular corrosives and severe irritants”. The ICE method uses eyes collected from chickens obtained from slaughterhouses where they are killed for human consumption, thus eliminating the need for laboratory animals. The eye is enucleated and mounted in an eye holder with the cornea positioned horizontally. The test substance and negative/positive controls are applied to the cornea. Toxic effects to the cornea are measured by a qualitative assessment of opacity, a qualitative assessment of damage to epithelium based on fluorescein retention, a quantitative measurement of increased thickness (swelling), and a qualitative evaluation of macroscopic morphological damage to the surface. The endpoints are evaluated separately to generate an ICE class for each endpoint, which are then combined to generate an Irritancy Classification for each test substance.

The Bovine Corneal Opacity and Permeability test method (BCOP) is an in vitro test method that can be used to classify substances as 'ocular corrosives and severe irritants'. The BCOP uses isolated corneas from the eyes of cattle slaughtered for commercial purposes, thus avoiding the use of laboratory animals. Each treatment group (test substance, negative/positive controls) consists of a minimum of three eyes where the cornea has been excised and mounted to a holder. Depending on the physical nature and chemical characteristics of the test substance, different methods can be used for its application since the critical factor is ensuring that the test substance adequately covers the epithelial surface. Toxic effects to the cornea are measured as opacity and permeability, which when combined gives an In Vitro Irritancy Score (IVIS) for each treatment group. A substance that induces an IVIS superior or equal to 55.1 is defined as a corrosive or severe irritant.

The method described by this Test Guideline provides information that allows hazard assessment for short-term exposure to a test article by inhalation, and allows the substance to be classified according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS). The test method is based on a stepwise procedure, each step using 3 animals of each sex (the preferred species is rat). Animals are exposed in  inhalation chambers to a pre-defined concentration for 4 hours. Absence or presence of compound-related mortality of the animals at one step will determine the next step. Animals in severe pain or distress should be humanely killed. The starting concentration is selected from one of four fixed levels corresponding to GHS categories 1-4 for gases, vapours or aerosols. Animals are observed daily for clinical signs of toxicity for a total of at least 14 days. Animals' body weights should be determined at least weekly. All the animals should be subjected to gross necropsy.

This updated Test Guideline 435 provides an in vitro membrane barrier test method that can be used to identify corrosive chemicals. The test method utilizes an artificial membrane designed to respond to corrosive chemicals in a manner similar to animal skin in situ.