The OECD 101 test, or “UV-VIS Absorption Spectra”, is a standardized method for measuring the absorption of light by a substance in the ultraviolet and visible ranges. This test provides essential data on the wavelengths at which a substance is likely to react to light, particularly through photodegradation. Integrated into the OECD guidelines , it is one of the internationally recognized regulatory tools for assessing the photochemical behavior of chemicals. Used in REACH registrations and cosmetic or pharmaceutical safety dossiers, it is an important prerequisite for other phototoxicity tests such as the OECD 432 test .
Table of Contents
OECD 101 test definition: UV-VIS absorption spectra
Main objective: to predict the photodegradation of substances
The OECD 101 test aims to identify the wavelengths at which a chemical substance absorbs light in the UV-VIS range, i.e., from 200 to 800 nanometers. This information is crucial for assessing a molecule's photostability photochemical degradation when exposed to natural or artificial light. For example, a substance that absorbs strongly at 290 nm (the UVB range) is considered photosensitive, which may require additional precautions during its storage, transport, or use.
UV-VIS spectroscopy thus makes it possible to characterize how a molecule interacts with photons and to determine whether it is at risk of bond breaking, oxidation, or other transformations related to light energy. This has a direct impact on product safety , whether the products are intended for human, environmental, or industrial use.
Scope of the OECD 101 test
The OECD 101 test is applicable to a wide range of pure or formulated chemicals. It is commonly used in:
- REACH registrations ( European regulation no. 1907/2006), in particular for substances produced or imported in quantities exceeding 1 tonne per year;
- safety assessments of active ingredients in medicines, cosmetics, plant protection products or biocides;
- of the physico-chemical characterization of ingredients in industrial products;
- of environmental risk assessment , in connection with the stability of compounds in air, water or soil.
The test is often required in IUCLID dossiers , used for regulatory submissions in Europe, and can be carried out under GLP (Good Laboratory Practice) to ensure compliance with the quality requirements of the authorities.
Scientific and analytical interest
Beyond its regulatory importance, the OECD 101 test is a valuable source of scientific information. UV-VIS absorption is directly linked to the electronic structures of molecules. Electrons located in π or non-bonding (n) orbitals can be excited to antibonding (π*) orbitals by light. These electronic transitions are characteristic of certain chemical functional groups :
- Conjugated double bonds (as in aromatic compounds) often absorb in the UV or visible range;
- Carbonyl, nitro, azo, amine or halogenated groups also exhibit distinctive spectra.
By analyzing the absorption spectrum, one can deduce certain structural properties , identify chromophore groups , and even assess the purity or stability of a sample. In the context of comparative tests (before/after irradiation, pH variation, etc.), this method also allows for monitoring photochemical transformations.
Finally, the OECD 101 test is a fundamental tool for selecting additional tests : when significant absorption is detected in the 290–700 nm range, it is often necessary to carry out additional tests, such as OECD 432 (3T3 NRU in vitro phototoxicity), to assess the potential biological effects of this absorption.
Standardized experimental conditions of the OECD 101 test
The OECD 101 test is based on a rigorously standardized method to ensure the reproducibility and comparability of results from one laboratory to another. The experimental conditions to be met include:
- Measurement temperature : 25 °C ± 1 °C.
- Equipment type : a double-beam UV-VIS spectrophotometer , allowing real-time comparison of absorption between the control cell (blank) and the cell containing the substance being tested.
- White : solution containing the solvent used, without the substance of interest (eliminates interferences due to the solvent).
- Concentration of the test solution : adjusted to obtain at least a maximum absorbance between 0.5 and 1.5 units , which ensures a reliable reading without saturation of the detector.
In most cases, several solvents are used to evaluate the behavior of the substance in different physicochemical environments :
- Acidic medium (pH < 2): often 0.1 N HCl.
- Basic medium (pH ≥ 10): often 0.1 N NaOH.
- Neutral medium (pH ≈ 7).
- Organic solvent : usually methanol , recommended by ICH S10 guidelines for pharmaceutical substances.
These variations allow the identification of potential ionized or tautomeric forms of the molecule, which may exhibit different absorption spectra. The test also aims to determine the molar extinction coefficient (ε) , a fundamental quantitative parameter for characterizing the absorption strength of a substance at a given wavelength.
Complementarity with other OECD tests
The OECD 101 test is part of a comprehensive risk assessment strategy . When a substance shows significant absorption in the UV-VIS range (particularly between 290 and 700 nm), this may trigger the need for further studies.
- OECD 432 3T3 NRU in vitro phototoxicity test , evaluating photo-induced cytotoxicity on mouse fibroblast cells.
- OECD 495 : ROS assay , testing the production of reactive oxygen under the effect of light (photoactivity).
- OECD 498 : Phototoxicity test on reconstituted human epidermis .
This step-by-step approach minimizes animal use by starting with in vitro and in chemico methods.
Produced in an accredited laboratory
For the results to be admissible in a regulatory framework (REACH dossier, biocide registration, application for marketing of cosmetic or pharmaceutical products), the OECD 101 test must be carried out in a laboratory accredited according to the ISO 17025 standard , preferably under the Good Laboratory Practices (GLP) regime .
This guarantees, in particular:
- Complete traceability of testing steps;
- Quality assurance of measurements;
- Regulatory archiving of raw data and reports;
- Compliance with export formats (including IUCLID ).
YesWeLab facilitates access to this type of analysis by leveraging a network of over 200 accredited partner laboratories in France and Europe. Through its platform, manufacturers can order an OECD 101 test and track each step of the process, from sample logistics to the retrieval of validated results.
Scientific and industrial applications of the OECD 101 test
The OECD 101 test is more than just a spectrophotometric analysis; it is a key step in evaluating the photoreactive profile of a compound. By identifying wavelengths of significant absorption, manufacturers can anticipate the risks of degradation, phototoxicity, or transformation of the molecule into potentially harmful byproducts. This second part of the article examines the main areas of application of the OECD 101 test, as well as its regulatory and technical uses in the laboratory.
Safety of chemical substances with respect to light
The primary function of the OECD 101 test is to assess a chemical's susceptibility to photodegradation . A molecule absorbing in the UV (100–400 nm) or visible (400–700 nm) spectrum is potentially unstable when exposed to light. This can lead to:
- The loss of product effectiveness (in the case of a cosmetic or pharmaceutical active ingredient sensitive to light);
- The formation of degradation by-products , sometimes more toxic than the initial molecule;
- Instability in commercial formulations (discoloration, precipitation, phase separation…).
The OECD 101 test makes it possible to identify these risks from the R&D and formulation phase, upstream of marketing.
REACH dossier and hazard assessment
Under the European REACH (EC No. 1907/2006), manufacturers and importers of chemical substances must provide data on the physicochemical profile of their products. The UV-VIS spectrum is among the data required, even for small quantities (≥ 1 tonne/year).
The OECD 101 test is therefore required in the applications:
- To assess the stability of a substance in the face of natural or artificial light;
- To determine the relevance of additional tests such as phototransformation in water (OECD 316) or phototoxicity (OECD 432);
- To justify the absence of photoactivity , if no absorption is detected above 290 nm.
The data obtained is integrated into the IUCLID format , the standard for submitting European regulatory dossiers.
Photostability of pharmaceutical substances (ICH S10)
In the pharmaceutical field, the photostability of active ingredients is a major regulatory requirement. The ICH S10 (International Council for Harmonisation) guidelines recommend UV-VIS testing as a first step to assess an active ingredient's sensitivity to light.
Laboratories then use the OECD 101 test to:
- Check that the active ingredient does not absorb in the risk zone of 290 to 700 nm ;
- Define the conditions for forced photostability to be put in place (exposure to artificial light);
- Validate opaque packaging or storage conditions (UV protection).
A significant absorbance result usually triggers a series of additional tests (degradation profile, identification of products formed, etc.).
Phototoxicity of cosmetic products
In cosmetics, phototoxicity is a key issue whenever an active molecule is likely to absorb light . UV-VIS absorption can cause adverse effects on the skin such as redness, irritation, or photosensitivity reactions.
The OECD 101 test is used here as an early screening , particularly for:
- Sun filters (e.g. oxybenzone, avobenzone);
- Essential oils or plant extracts;
- Aromatic compounds or colorings.
An absorption spectrum in the critical zone (320–400 nm) guides the choice towards phototoxicity tests such as the OECD 432 test , or towards a reformulation.
Packaging material compatibility
Some plastic materials or packaging films contain chemical additives, colorants, or stabilizers that can interact with UV light. In the food and pharmaceutical industries, it is essential to ensure that these substances do not undergo photo-induced degradation , which could alter the quality of the contents.
The OECD 101 test is used for:
- Evaluate the photostability of additives in contact with light;
- Detect photo-induced migration towards food or medicine;
- Justify the use of protective or UV-absorbing films in sensitive packaging.
In these sectors, the test is sometimes combined with migration tests according to EC Regulation No. 1935/2004 for materials in contact with foodstuffs.
Example of laboratory application: spectral analysis of a plant extract
A laboratory wishing to test a chamomile extract used in a cosmetic lotion can use OECD 101 to verify the absence of phototoxic risk. By analyzing the absorption spectrum between 200 and 800 nm, chemists detect several peaks above 300 nm.
These results point towards a further phototoxic evaluation (OECD 432), or even towards a reformulation of the lotion or an adjustment of the conditions of use (e.g., do not expose the skin to the sun after application).
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Experimental implementation of the OECD 101 test in the laboratory
The OECD 101 test follows a rigorous analytical protocol designed to ensure reliable, reproducible, and usable results within a regulatory framework. This third part details the key steps of the protocol, the instrumental requirements, and the experimental conditions to be met. It also highlights the criteria for interpreting the results and their integration into a regulatory dossier.
Preparation of test solutions
Before starting the spectral measurement, it is essential to prepare test solutions at appropriate concentrations . The goal is to obtain a spectrum with at least one absorbance peak between 0.5 and 1.5 units .
- Preliminary calibration : a concentration determination phase is often necessary to adjust the dilution. This helps prevent detector saturation or areas of unmeasurable absorbance.
- Solvents used : Methanol is generally recommended, in accordance with ICH S10 guidelines. It is compatible with the majority of organic substances and does not interfere with the measured wavelengths.
- Analysis media : To detect sensitivity to the environment, the analysis is carried out in three distinct media:
- Acid (pH < 2, e.g. 0.1 N HCl),
- Neutral (distilled water or pH 7 buffer),
- Basic (pH ≥ 10, e.g., 0.1 N NaOH).
This multifactorial approach makes it possible to evaluate the ionic form of the compound under different conditions, which is essential to anticipate its environmental or physiological behavior.
UV-VIS spectrophotometer configuration
The test requires a double-beam spectrophotometer , capable of comparing the absorption of the tested solution to that of a blank (solvent alone). This type of instrument is standard in physicochemical analysis laboratories.
- Spectral range analyzed : from 200 to 800 nm, covering the far UV, near UV and visible ranges.
- Spectral resolution : it must be sufficient to detect fine peaks, generally on the order of 1 nm.
- Tank length : between 0.1 and 10 cm depending on the expected absorbance, to adjust the sensitivity of the measurement.
- Temperature conditions : the test is carried out at 25°C to avoid any variation due to evaporation or thermal denaturation.
The device must be calibrated before each series of tests , and be subject to regular metrological checks within the framework of a quality system (ISO 17025).
Spectrum recording and processing
Once the experimental parameters are defined, the UV-VIS spectrum is recorded for each solution:
- Raw spectrum : the curve represents absorbance as a function of wavelength, highlighting the characteristic maxima (λmax) of the compound.
- Determination of the molar extinction coefficient (MEC) : this quantitative data indicates the absorption intensity of one mole of a substance. It is expressed in L·mol⁻¹·cm⁻¹ and calculated according to Beer-Lambert's law.
A=ε⋅c⋅lA = \varepsilon \cdot c \cdot lA=ε⋅c⋅l
Or :
- AAA is the measured absorbance.
- ε\varepsilonε is the molar extinction coefficient (MEC),
- ccc is the concentration (mol/L),
- lll is the optical path length (cm).
- Comparative analysis : the spectra obtained in the three media allow us to detect possible variations in absorption as a function of pH, which indicates a photochemical instability or a change in ionic form .
Interpretation criteria and triggering of additional tests
The results of the OECD 101 test are interpreted according to the position and intensity of the absorbance peaks:
- No absorption > 290 nm : the substance is not likely to induce phototoxicity or photochemical degradation under normal light exposure conditions. No further testing is required.
- Significant absorption between 290 and 700 nm : the substance may be photoactive. This triggers:
- An in vitro phototoxicity test (OECD 432) or
- A phototransformation test (OECD 316, if the substance is discharged into water).
These interpretations are incorporated into regulatory reports such as IUCLID , SDS (safety data sheet) or REACH Annexes .
Quality requirements and regulatory compliance
The OECD 101 test can be performed under good laboratory practices (GLP) , particularly for substances intended for REACH registration or regulatory submission:
- ISO 17025 accreditation : a guarantee of the laboratory's technical competence.
- Traceability : all steps must be documented (weighing sheet, analysis conditions, calibration).
- Archiving : source documents and samples must be kept for 5 to 10 years according to the regulatory framework.
- Auditability : the tests must be able to be verified by competent authorities (ANSES, ECHA, etc.).
YesWeLab works exclusively with accredited partner laboratories to guarantee the reliability of results, in strict compliance with GLP, ISO 17025 and REACH standards.
Relationship between OECD Test 101 and other regulatory guidelines
OECD 101 testing is only a first step in understanding the photochemical behavior of a substance. If significant absorption is observed in the UV or visible range, further testing should be considered to complete the hazard assessment, particularly regarding phototoxicity, environmental transformation, and end-user safety. This section explores the links between OECD 101 and other relevant guidelines.
OECD 432: In vitro phototoxicity by the 3T3 NRU method
OECD 432: In vitro phototoxicity by the 3T3 NRU method
When the UV-VIS spectrum of a substance reveals absorption above 290 nm, the OECD recommends conducting a phototoxicity test to determine whether exposure to sunlight can amplify its toxicity.
The OECD 432 test, entitled “3T3 NRU Phototoxicity Test” , is based on the exposure of mouse fibroblast cells (Balb/c 3T3 cell line) to the test substance, in the presence or absence of UVA light (320–400 nm). Cell viability is then measured by the absorption of the neutral red dye.
- Phototoxicity identified : if the substance is significantly more toxic under light than in darkness, it is considered phototoxic.
- Phototoxicity excluded : if the dose-response curve shows no difference between the two conditions, no risk is identified.
This test is particularly relevant for substances used in cosmetics (creams, lotions, UV filters) or in cutaneous pharmacology.
OECD 316: Phototransformation in aquatic environments
In the case where a UV-VIS absorbing substance is intended to be released into the aquatic environment , OECD 316 proposes a protocol to simulate photochemical degradation in natural water .
The test assesses:
- The rate of degradation under irradiation (natural or simulated),
- Identification of transformation products (metabolites),
- Calculating half-life as a function of light intensity.
ecotoxicological assessments , particularly for biocidal active substances, plant protection products, or drug residues in water.
Integration into regulatory dossiers (REACH, BPR, CLP)
The OECD 101 test, combined with complementary tests such as OECD 432 or 316, provides a solid basis for the risk assessments required by European regulations :
- REACH (EC No. 1907/2006) : OECD 101 is included in section 7.1.1 of Annex VII, mandatory for substances produced in quantities exceeding 1 tonne/year. The result guides phototoxicity or photodegradation tests.
- BPR (Biocides Regulation EC No. 528/2012) : for biocidal active substances, the assessment of photostability is essential to predict persistence in the environment.
- CLP (Regulation EC No 1272/2008) : Although the classification of substances is not directly based on OECD 101, the results may support a request for classification or the justification of absence of hazard.
The data from the OECD 101 test must be included in the IUCLID dossier , along with all traceability elements, the complete study report and, if applicable, a GLP certification.
Summary of interactions and decision tree
The diagram below summarizes the logic of interpretation based on OECD essay 101:
- No absorption > 290 nm
→ No further phototoxicity testing required
→ No photolytic transformation expected
→ End of photochemical evaluation - Absorption > 290 nm
→ OECD 432 test to assess phototoxicity
→ OECD 316 test if potential emission into the environment
→ Integration into regulatory annexes (IUCLID, BPR, etc.)
This interconnection between the guidelines ensures a coherent and graduated approach to the safety of chemical substances, respecting the 3Rs principle (reduction, refinement, replacement of animal testing) and international regulations.
Performing the OECD 101 test in the laboratory: techniques, requirements and best practices
The OECD 101 test relies on a rigorous methodology that requires specific analytical equipment, precise technical skills, and compliance with internationally recognized quality standards. This section describes in detail how specialized laboratories perform this test, from sample preparation to the issuance of the final report.
Equipment used: the double-beam UV-VIS spectrophotometer
The central tool of the OECD 101 test is the dual-beam UV-VIS spectrophotometer , capable of measuring the absorption of light by a solution in wavelength ranges from 200 to 800 nm .
- Double beam : one passes through the cuvette containing the test solution, the other passes through a cuvette containing the blank (solvent alone).
- Sensitive detectors : generally photodiodes or photomultipliers allow a very precise measurement of absorbance.
- Spectral resolution : it is often set at 1 to 2 nm , which allows for precise detection of absorption peaks.
The system must be calibrated regularly with certified standards (e.g., potassium nitrate or potassium permanganate solutions) to ensure the accuracy of measurements.
Preparation of test solutions: pH, solvent, concentration
Preparing the solution being tested is a crucial step in obtaining a usable spectrum:
- Solvent : most often it is methanol , ethanol , or an aqueous buffer , depending on the solubility of the substance.
- Target concentration : adjusted to obtain at least one absorbance peak between 0.5 and 1.5 units . This allows for a linear and reproducible measurement.
- pH conditions :
- Acidic medium (pH < 2, e.g., 0.1 N HCl)
- Basic medium (pH > 10, e.g., 0.1 N NaOH)
- Neutral medium (purified water or phosphate buffer)
These conditions allow us to explore the different ionic forms of a substance and their absorption properties.
Test procedure: acquisition of the UV-VIS spectrum
The absorption spectrum is obtained by scanning the solution between 200 and 800 nm , with typical increments of 1 nm. The following is observed:
- Peak positions (wavelengths λ max)
- Spectrum shape (width, shoulders, plateaus)
- Absorbance values in optical units (OU)
Spectra are usually recorded in three different media (acidic, basic, neutral) in order to detect any transitions specific to an ionic form.
The data are then used to calculate the molar extinction coefficient (ε) , expressed in L·mol⁻¹·cm⁻¹. This value indicates the molecule's ability to absorb light, a key element for predicting photodegradability or phototoxicity.
Good Laboratory Practices and GLP/ISO 17025 Compliance
For the results to be recognized at the regulatory level, the OECD 101 test must be carried out under strict conditions:
- GLP (Good Laboratory Practices) Compliance :
- Complete traceability of handling operations
- Method validation
- Archiving of data and samples
- Supervision by a quality assurance unit
- ISO 17025 Accreditation :
- Demonstration of the laboratory's technical competence
- Regular quality control of equipment
- Participation in interlaboratory trials
- Structured document management system
YesWeLab collaborates only with accredited laboratories that meet these requirements to ensure the reliability and validity of results , particularly in REACH, BPR or biocide registration contexts.
Laboratory analyses by spectrophotometry
The OECD 101 test is a concrete example of spectrophotometry applied to chemical risk assessment . This technique is also used in other analytical fields, such as the quantification of organic compounds in the food, cosmetics, and pharmaceutical sectors.
malic acid analysis :
- UV-VIS spectrophotometry : a simple and quick method to estimate the concentration of acid in juices, jams or creams.
- Acid-base titration : complementary for simple matrices.
- HPLC (liquid chromatography) : more specific, allows the distinction of malic acid isomers.
In all cases, compliance with ISO 17025 or COFRAC standards remains essential to guarantee the regulatory value of the results.
YesWeLab: your partner for OECD testing and regulatory analyses
YesWeLab supports manufacturers in the complete management of their regulatory tests, including OECD tests, through a unique digital platform and a network of more than 200 accredited partner laboratories in France and Europe.
A single platform to centralize your analytical needs
Thanks to its intuitive digital platform, YesWeLab enables quality, R&D, and regulatory professionals to:
- Compare and order OECD tests in just a few clicks
- Track samples and analysis progress in real time
- Download test reports compliant with GLP, REACH or ISO 17025
- Communicate directly with the laboratories to adapt the protocol
This centralized management greatly simplifies analytical processes and reduces regulatory compliance time.
A network of accredited laboratories to meet all your requirements
YesWeLab works exclusively with laboratories certified to ISO 17025, GLP and/or COFRAC, which guarantees:
- The quality and reliability of the results
- Compliance with international regulations (REACH, BPR, CLP, OECD, etc.)
- Trials recognized by health and environmental authorities
Whether it is an OECD 101 test , a subchronic toxicity study (OECD 408), or a phototoxicity test (OECD 432), YesWeLab's partner laboratories have the equipment and expertise to handle the most demanding protocols.
Tailored expert support
Beyond simply connecting you with others, YesWeLab acts as your technical partner , capable of:
- Translate your regulatory requirements into analytical specifications
- Select the right laboratory according to the OECD method, matrix or application sector
- Coordinate testing across multiple sites or analytical disciplines
- Validate the reports and assist you in integrating them into your regulatory files (IUCLID, REACH files, etc.)
This level of support is particularly appreciated by manufacturers, distributors and consultants who manage complex portfolios of substances or formulations.
Multi-sector expertise
YesWeLab operates in many industrial sectors affected by the OECD guidelines:
- Agrifood and animal nutrition
- Cosmetics and medical devices
- Biocidal and phytosanitary products
- Packaging and contact
- Environment , REACH and waste
Whatever your field, YesWeLab helps you find the OECD method best suited to your product, its dosage form and its regulatory obligations.
