Local Tolerance
Assessing Local Tolerance Without Testing in Animals
Assessing local tolerance is important for any new drugs, whether for the site of administration (human toxicology), or other sites where unintended or accidental contact may happen. The assessment of the site of administration should be done before any clinical trials begin, but changes to the formulation or new route of exposure could trigger another assessment, especially if a novel excipient is introduced. Sites for possible accidental exposure should be assessed before larger clinical studies are started.
As stated in ICH M3(R2), in vivo stand-alone tests for local tolerance should be avoided. Testing can however be done in in vivo pivotal studies. More recent guidelines, e.g. EMA/CHMP/SWP/2145/2000 Rev. 1, Corr. 1, state that in vivo tests shouldn’t be performed, before alternative sources of data, e.g. from literature, in silico evaluations or in vitro/ex vivo tests, have been taken into consideration. This is in line with the 3Rs Principle (Refine, Reduce, Replace) for the use of animal model alternatives. In vitro and ex vivo tests are also becoming more accepted for screening active substances, impurities and formulations already before nonclinical testing.
In vitro test methods should be internationally validated and preferably adopted by the OECD. They could also be part of defined testing approaches, to allow for a more comprehensive assessment of a drug’s potential for causing irritation.
There are today a wide range of in vitro tests and for different sites of administration or contact. We are offering many of these tests and regularly introducing new test systems to allow for more flexible testing strategies and to cope with varying physicochemical properties of active substances, impurities and excipients.
If you need support to find the most appropriate testing strategy for your active substance, impurities or formulation, then.
Skin Sensitization – Complex Process That Can Be Assessed In Vitro
Skin sensitization (delayed-type reaction) is a complex process in several steps as described in an Adverse Outcome Pathway (AOP). After the penetration of an allergenic substance into the skin, the AOP is divided into several so-called Key Events:
- Reaction of a substance with components in the skin (proteins) under the formation of complexes
- Uptake of the complexes by Langerhans cells, a sub-type of dendritic cells (DCs), maturation of DCs and systemic migration of DCs
- Activation and proliferation of T cells through interaction with mature DCs
- Release of proinflammatory cytokines upon re-exposure to the allergen, triggering localized tissue inflammation and the clinical manifestation of allergic contact dermatitis
- Inflammatory cascade
Over the last few years, new approach methodologies (NAMs) for the assessment of potential for sensitization by chemicals were developed and published in the OECD test guidelines 442C/D/E as well as OECD 497. We offer you testing packages that cover all the key events:
- The Direct Peptide Reactivity Assay (DPRA) and the Amino Acid Derivative Reactivity Assay (ADRA) address the covalent binding of a chemical to proteins
- The ARE-Nrf2-Luciferase test method (LuSens) measures the activation of keratinocytes
- The human Cell Line Activation Test (h-CLAT) and the U-SENS™ Assay examine the activation of DCs
The potential for sensitization can be determined from the results of these tests, but sometimes the applicability domains of the tests may limit testing of substances with certain physicochemical properties.
If the in chemico or in vitro methods cannot be applied, the Local Lymph Node Assay (LLNA) can be used as an alternative. This in vivo test assesses the proliferation of T cells in lymph nodes in mice following dermal application. The LLNA provides robust quantitative data suitable for a dose-response analysis. Also, a threshold value can be determined for supporting risk assessment.
Phototoxicology
Phototoxicology assessment of a new active substance, excipient or formulation is important when a drug may distribute to tissues exposed to light, like skin or eyes. Such an assessment should be performed before clinical trials to prevent unnecessary protective precautions during the trials and to limit the risk to have a phototoxic drug in the market.
However, for a substance to show any phototoxicity, it must absorb sunlight and generate reactive (cytotoxic) species. These prerequisites are detailed in the ICH S10 guideline. Typically, nonclinical assessment starts through measuring the Molar Extinction Coefficient (MEC) for the substance. If within the UV spectrum 290–700 nm, one of the strategies consists of assessing it by a cytotoxicity test in Balb/c 3T3 cells under irradiation. The MEC measurement and the cytotoxicity test are normally combined in one study, but an insufficient MEC would abort that study prematurely.
The cytotoxicity test under irradiation should follow the OECD test guideline 432. It’s recommended for soluble active substances and excipients, while topically-applied formulations could instead be tested in a skin model (following e.g. the OECD test guideline 498). This could also be the case for substances absorbing UVB light or for poorly-soluble substances. Important is that any positive result from the cytotoxicity test wouldn’t always be regarded as clinically relevant. Also, due to lower sensitivity for tests in skin models, higher concentrations or longer exposure times than in the clinical trial should be considered.
We’re happy to support you with in vitro phototoxicity tests in our GLP-certified laboratories.
Substances in the Pharmaceutical Industry May Have To Be Assessed for Chemical Safety
Within the European Union, drugs (e.g. active substances, excipients, impurities and formulations) are exempted from registration as chemicals. Further, drugs, as well as substances in research and development and non-isolated intermediates, don’t need to be classified (in line with CLP, EC 1272/2008) for any chemical hazards.
Still chemicals used as raw materials and imported into Europe, or isolated intermediates (if the volume is above 1 metric ton per year), may require both classification and registration. Further, pharmaceutical producers may voluntarily assess and classify their intermediates and drugs , e.g. to ensure occupational health standards or to comply with national legislation.
To support chemical risk assessments, we offer a broad range of toxicological tests and regulatory advisory support. For more information on testing contact us: info@iccr-rossdorf.de
Tissue - Skin
- Skin Irritation: Reconstructed Human Epidermis Test method
- OECD 439 / ISO 10993-23
- In-Vitro Skin Corrosion (Reconstructed Human Epidermis Test method)
- OECD 431
- Corrositex (In-Vitro Membrane Barrier Test method for Skin Corrosion)
- OECD 435
Tissue - Eye
- Eye Irritation: Reconstructed human Cornea-like Epithelium Test method
- OECD 492 / OECD 492B
- Bovine Cornea Opacity and Permeability Test (BCOP)
- OECD 437
- Short Time Exposure In-Vitro Test Method for Identifying Eye Damage (STE)
- OECD 491
- Hen’s Egg Test – Chorioallantoic Membrane (HET-CAM)
- INVITTOX Protocol No. 47
Tissue - Mucous Membrane
- EpiOral / EpiGingival Test
- EpiVaginal Test
Phototoxicity
In-Vitro 3T3 NRU Phototoxicity Test
- OECD 432 / ICH S10
In vitro Phototoxicity Test with Reconstructed Human Epidermis
- OECD 498 / ICH S10
In chemico / In Vitro Skin Sensitization
Direct Peptide Reactivity Assay (DPRA)
- OECD 442C (Appendix I)
Amino Acid Derivative Reactivity Assay (ADRA)
- OECD 442C (Appendix II)
The ARE-Nrf2 luciferase LuSens test method (LuSens)
- OECD 442D (Appendix IB)
Human Cell Line Activation test (h-CLAT)
- OECD 442E (Annex I)
U-SENS™ Assay
- OECD 442E (Annex II)
In vivo Skin Sensitization
- Local Lymph Node Assay, individual approach
- OECD 429
