Jul. 29, 2019
Alternative methods implementation is a worldwide emerging trend, as an effort to reduce animal suffering and the generation of redundant data. Since 2014, the Brazilian council of animal experimentation (Concea) has recognized the alternative methods validated by OECD. In 2015, the regulatory agency ANVISA (Agência Nacional de Vigilância Sanitária) approved these methods and determined that Brazilian laboratories have 2019 September as deadline to replace current in vivo assays. In this context, and committed to the principle of 3Rs (refinement, reduction and replacement), NSF International Brazil are working to put into practice approaches that can combine existing data review, in silico, in chemico and in vitro assays for agrochemicals and pharmaceuticals formulations toxicological evaluation.
Integrated Approaches to Testing and Assessment - IATA
OECD has recommended that, before starting new toxicological tests, especially in vivo tests, all available data must be considered, to avoid redundant results and unnecessary animal distress. In this way, an Integrated Approach to Testing and Assessment (IATA) can be conducted(1). An IATA starts with the search for existing data (Part 1), followed by a weight of evidence (WoE) analysis (Part 2). For this WoE analysis, all available information must be collected and evaluated from relevant databases. Quantitative structure-activity relationship (QSAR) predictions is an effective resource for this evaluation. An adequate WoE analysis can be used for making a regulatory conclusion. If WoE analysis are not enough for classification of the test substance, new assays must be performed (Part 3), as described in Figure 1.
Figure 1. Parts of an integrated approach to testing and assessment
NSF International Brazil have a perspective to offer this approach, starting with Parts 1 and 2, in the attempt to make a regulatory conclusion without new tests. In this phase, a QSAR analysis will be performed by NSF International to support the approach. If no conclusions can be made, a new phase of the study with new tests is proposed. It is important to mention that, in the Part 3 of the IATA, new tests should start with alternative methods and the use of animals must be considered only as a last resource. In the case of Brazilian resolution, the alternative methods must replace the current in vivo tests for eye irritation/corrosion, skin irritation/corrosion, skin sensitization and micronucleus assays.
Alternative methods in the context of an IATA
When new tests are required for the test substance safety assessment, the following guidelines may be conducted:
OECD TG 491
This assay uses a monolayer of rabbit cornea cells (SIRC) on a 96-well microplate. The cells are treated with two concentrations of the chemical test (5% and 0.05%). After a short term of exposure (5 minutes) with the chemical test and solvent control, an MTT assay is performed to measure the cell viability. In the case of cell viability ≤ 70% with the two concentrations, the test substance can be classified as Category 1 (potential to be corrosive). The substance is predicted as No Category (no need for classification) when the two concentrations result in > 70% of cell viability(2).
OECD TG 492
The assay makes use of a reconstructed human cornea-like epithelium. After the exposure of the epithelium with the test substance and solvent controls, the viability of the tissue can be evaluated with an MTT assay. If the viability is > 60% the test chemical is predicted as No Category (no need for classification)(3).
OECD TG 431
It uses a three-dimensional human skin model to which the test substance is applied. After treatment for a short period with the chemical test and controls, the MTT assay is performed, allowing the identification of chemicals with corrosive potential(4).
OECD TG 439
It uses the same three-dimensional human skin model described in OECD 431, but in a different protocol. The major difference between the two test guidelines is the time of exposure with the test substance(5).
Allergic dermatitis is the clinical manifestation of an adaptive immune system response after the exposure to a sensitizing substance. Chemical and biological key events in the skin sensitization are well stablished and known as the Adverse Outcome Pathway (AOP). The initial molecular event is the covalent binding of the sensitizing substance with skin proteins (haptenation) which can be detected by the in chemico assay Direct Peptide Reactivity Assay, described in OECD 442C (DPRA). The second event is the activation of epidermis keratinocytes and can be detected by KeratinoSens method (ARE-Nrf2 Luciferase) described in OECD 442D. The third key event is the activation (maturation) and mobilization of Langerhans and dendritic cells which can be assessed by the methods described in OECD 442E (h-CLAT, U-SENS and IL-8 LUC). Events 2 and 3 occur at cellular level. The fourth event occurs at the organ level and is evaluated by the T-cell antigen presence mediated by dendritic cells, which can be evaluated by LLNA (Local Lymph Node Assay, OECD 429). The activation of these events leads to inflammation, which is the response at the organism level (Figure 2).
Figure 2. The adverse outcome pathway and validated assays to assess each key event.
Defined approaches in the context of an IATA for skin sensitization
The OECD document 256(6) includes some examples of define approaches to be used in the context of an IATA for skin sensitization. A defined approach consists in a fixed procedure of data interpretation applied to results generated from available information sources, are based on rules, and can be used alone or with other sources of information. These approaches were developed by corporations and can identify the test substance risk potential. One example is a sequence strategy developed by Kao Corporation (Figure3), that evaluates key events 1 and 3 described previously. The approach starts with key event 3. In the case of a positive result, it can be concluded that the test substance is a sensitizer. If the result is negative, the key event 1 should be assessed. A positive result in key event 1 is enough to classify the substance as a sensitizer. Two negative results indicate a non-sensitizer.
Figure 3. Sequential test approach for sensitizing potential prediction
About NSF International Brazil
Located in Brazil, NSF Brazil Labs is the global GLP Testing Facility of NSF International. With extensive experience in GLP data generation, NSF provides clients with expertise in physicochemical, residue, ecotoxicological, genotoxicity, toxicological (in vivo and in vitro), pathogenicity, 5-Batch Analysis and in silico studies. By following Brazilian and International guidelines such as NBR, OECD and EPA, sponsors enjoy the benefit of applying NSF high quality reports for global registration, including USA, Europe, Asian and LATAM countries. Accreditations include: GLP by Brazilian INMETRO, ISO 17025, REBLAS, MAPA and ANVISA Brazilian agencies, among others.
About NSF International
With world headquarters in Michigan, USA, NSF International is a leading global organization committed to protecting and improving human health worldwide. Founded in 1944, it operates in 150 countries and counts with over 2.500 employees, including microbiologists, toxicologists, chemists, engineers, and environmental and public health professionals to carry out its mission.
1. OECD Test No. 263 - Guidance Document on an Integrated Approach on Testing and Assessment (IATA) for Serious Eye Damage and Eye Irritation. 2017.
2. OECD Test No. 491 - Short Time Exposure In Vitro Test Method for Identifying Chemicals Inducing Serious Eye Damage and Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage. 2018.
3. OECD Test No. 492 - Reconstructed human Cornea-like Epithelium (RhCE) test method for identifying chemicals not requiring classification and labelling for eye irritation or serious eye damage. 2018.
4. OECD Test No. 431 - In Vitro Skin Corrosion: Reconstructed Human Epidermis (Rhe) Test Method. 2014.
5. OECD Test No. 439 - In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method. 2015.
6. OECD Test No. 256 - on the Reporting of Defined Approaches and Individual Information Sources to be Used within Integrated Approaches to Testing and Assessment (IATA) for Skin Sensitization. 2016.
This story was initially published in AgroPages '2019 Latin America Focus' magazine. Download the PDF version of the magazine to read more stories.