The OECD 408 test is a reference method for assessing the effects of prolonged oral exposure to a chemical substance. Internationally recognized, this subchronic toxicity study is an essential step in assessing hazard and risk to human health.
Developed by the Organisation for Economic Co-operation and Development (OECD), Guideline 408 is integrated into the regulatory protocols required for the registration or evaluation of chemical, pharmaceutical, food, cosmetic, and material substances. By simulating repeated oral exposure over 90 days in rodents, it allows for the characterization of a compound's potentially harmful medium-term effects and the definition of critical thresholds such as the No Observed Adverse Effect Level (NOAEL). Recognized globally, this harmonized method is part of a graded approach to regulatory toxicology and informs REACH dossiers, notifications to health agencies, and product safety assessments.
Table of Contents
Objectives and general principle of the OECD 408 test
Identify the systemic effects of repeated exposure
The main objective of the OECD 408 test is to determine the adverse effects that could occur following daily oral exposure to a chemical substance over a 90-day period. This duration corresponds to subchronic, or medium-term, exposure, allowing the identification of effects that would not be observable during short-term exposure (such as in 14- or 28-day studies).
Daily repetition of exposure allows for the detection of any accumulation of the substance or its metabolites in the body. It also helps to identify progressive or delayed effects, which only a prolonged study can reveal. The test allows observation of the substance's impact on metabolism, internal organs, biochemical parameters, and the animals' overall health.
Determine a NOAEL and a dose-response relationship
One of the major expected results of this study is the determination of a NOAEL (No Observed Adverse Effect Level), that is, the maximum dose at which no significant toxic effect is observed in the tested animals. This parameter is essential in assessing risks to human health, as it allows for the calculation of safety margins and the establishment of tolerable exposure limits.
The protocol is designed to establish a dose-response relationship. Three groups receive increasing doses of the compound under investigation, in addition to a control group that receives no treatment or only the vehicle (neutral carrier). Comparative analysis between these groups allows for the detection of dose-related effects and the assessment of their intensity and frequency. This dose-response relationship is a fundamental criterion for confirming the toxicity of a substance.
Target the affected organs and physiological systems
The OECD 408 test is not limited to a general observation of health status. It also aims to identify the target organs of toxicity. Through a combination of clinical, biological, histopathological, and behavioral examinations, researchers can detect the specific effects of the substance on precise physiological systems: liver, kidneys, nervous system, digestive system, endocrine glands, reproductive system, etc.
Experimental implementation of the test
Animal species used and breeding conditions
The OECD 408 test is primarily performed on rodents, with a preference for rats, whose physiological responses are well-documented and widely used as a predictive model for humans. Mice may be used in certain cases, but only if scientifically justified.
Each treatment group must include at least 10 males and 10 females to allow for the analysis of sex-related differences. The animals must belong to standardized strains, originating from healthy colonies without pre-existing disease, and must be of similar age and weight.
The rearing conditions must be strictly controlled throughout the study. The temperature is generally maintained between 22°C ± 3°C, with relative humidity between 30% and 70%, and a 12-hour day/12-hour night light cycle. The animals are housed in suitable cages with absorbent bedding and access to a standardized phytoestrogen-free diet, as well as ad libitum drinking water. These conditions ensure physiological stability and minimize experimental bias.
Methods and procedures of administration
The substance to be tested is administered orally on a daily basis for the entire duration of the study, i.e., 90 consecutive days. Three routes of administration are permitted: gavage (direct introduction into the stomach using a tube), incorporation into food, or dilution in drinking water.
The choice of administration method depends on the compound's physicochemical properties, but also on the realistic exposure scenario a human being might face. Forced feeding is often preferred to ensure precise dosing, particularly with liquid or unstable formulations. Food or drinking water are more suitable for simulating chronic exposure to low doses.
The administered volume must not exceed the animal's physiological capacity, and solutions must be prepared according to rigorous procedures, with homogeneity and stability verified at each stage. Particular attention is paid to the traceability of the substance: batch number, storage conditions, and concentrations prepared and consumed are systematically recorded.
Dose selection and limit testing
The determination of dose levels is based on the results of previous tests, such as the acute toxicity test or the OECD 407 28-day test. The aim is to cover a range of responses, including a high dose causing toxic effects, an intermediate dose, and a low dose that ideally induces no measurable effect.
Three test groups are formed, in addition to a control group receiving either no treatment or only the vehicle (e.g., water, oil, neutral solution). The protocol also provides for the possibility of performing a limit test at 1000 mg/kg/day in cases where no toxic effects are expected due to the known properties of the substance or structural analogs.
The limit test is useful for simplifying the study when human exposure is low or controlled, but it does not replace a full protocol if the results indicate potential effects or if environmental exposure is significant. In all cases, dose selection must avoid unnecessary suffering and adhere to the ethical principles of reduction, refinement, and replacement (3Rs) in animal experimentation.
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Parameters evaluated throughout the study
Daily and weekly clinical monitoring
The animals are observed at least once a day, at a fixed time, to detect any signs of suffering, deterioration of general condition, or changes in behavior. Clinical signs looked for include abnormal posture, locomotor disorders, respiratory difficulties, loss of reflexes, seizures, or prostration.
Body weight is measured once a week, as are food and water intake. This data allows us to monitor growth and metabolism, and to detect any reductions in appetite or dehydration, which can be early indicators of toxicity.
Weekly functional assessments may include tests of reactivity, motor skills, and balance. They complement general observations by providing information about the central and peripheral nervous systems.
Functional and biological analyses
Several types of analyses are carried out during the study, particularly in the final phase. They aim to document the effects of the compound on blood parameters, liver, kidney, electrolyte and metabolic functions.
Hematological analyses include the measurement of hematocrit, hemoglobin, red and white blood cell count, leukocyte differential, as well as platelet count and the study of coagulation parameters.
Biochemical analyses are performed on serum or plasma, with a full panel including: glucose, urea, creatinine, liver transaminases (ALT, AST), alkaline phosphatase (ALP), total cholesterol, HDL and LDL fractions, triglycerides, total protein, albumin, sodium, potassium, calcium and phosphorus.
A urine analysis is often added to investigate kidney function. It may include measurements of pH, specific gravity, the presence of glucose, protein, ketones, blood or leukocytes, as well as a microscopic examination of the sediment.
Endocrine and reproductive parameters
Since 2018, OECD guideline 408 has included endocrine parameters, particularly those related to thyroid function. The hormones measured are thyroxine (T4), triiodothyronine (T3), and thyroid-stimulating hormone (TSH). These measurements allow for the detection of early hormonal disturbances, which could reflect the endocrine activity of the tested substance.
In some cases, particularly depending on the toxicological profile of the substance, additional hormone tests are performed to assess reproductive function. These may include tests for testosterone, estradiol, FSH (follicle-stimulating hormone), and LH (luteinizing hormone). These tests are carried out at specific times to minimize variations due to the circadian rhythm, stress, or the estrous cycle in females.
Necropsy, histopathology and target organ analysis
End-of-study necropsy procedures
All animals, including those in the control group, are euthanized ethically according to current protocols, minimizing stress and suffering. Autopsies are performed within hours of euthanasia to preserve tissue integrity.
The procedure involves opening the thoracic and abdominal cavities, as well as macroscopic examination of the major organs. The organs are visually inspected for obvious abnormalities: changes in size, color, or consistency, the presence of lesions, tumors, hemorrhages, or necrosis.
A precise record is made for each organ, accompanied by photographs if necessary. The observations are then correlated with the biological, behavioral, and clinical results obtained during the study.
Weighing and macroscopic examination of the organs
Certain organs are routinely removed and weighed to assess changes in mass that may indicate hypertrophy, atrophy, or inflammation. This weighing is performed using precision scales after excision, and the organs are handled carefully to avoid any loss of tissue.
The organs weighed include the liver, kidneys, testicles, epididymides, ovaries, spleen, heart, thymus, adrenal glands, and thyroid. Both absolute and relative weight (compared to body weight) are calculated for each individual.
These measurements are sensitive indicators of physiological imbalance. For example, a significant increase in liver weight may indicate enzyme induction or toxic overload, while a reduction in testicular weight may reflect impaired spermatogenesis.
Histopathological examination: lesions, accumulation, dose-response effects
The harvested organs are fixed in a buffered formalin solution, embedded in paraffin, sectioned into thin slices, and stained using standard techniques (usually hematoxylin and eosin). They are then examined under a light microscope by an experienced pathologist.
The aim of histopathological examination is to identify characteristic microscopic alterations: inflammation, cell necrosis, vacuolization, degeneration, fibrosis, hyperplasia, or cell infiltration. These observations allow us to confirm the nature of the observed effects, to specify their location, and to assess their severity.
When effects are detected, their frequency and intensity are compared between the different treatment groups. This allows us to verify whether there is a dose-response relationship, and whether the effects are reversible, persistent, or progressive.
Histopathology thus constitutes a valuable source of data for identifying the target organs of toxicity and guiding future investigations. It also contributes to the interpretation of the mechanisms of action of the tested substance, particularly when a specific impairment is observed reproducibly in several studies.
Data processing and statistical analysis
Statistical tools used
The data collected during the study are subjected to a series of statistical analyses to detect any significant differences between the groups exposed to the tested substance and the control group. Depending on the nature of the data (parametric or non-parametric), different tests may be applied.
Analysis of variance (ANOVA) is commonly used to compare means between several groups when the data are normally distributed. In the case of significant differences, post-hoc tests (such as Dunnett's or Tukey's test) can identify the groups involved. For non-parametric data, the Kruskal-Wallis or Mann-Whitney tests are preferred.
Regression analyses can also be used to assess the linearity of the dose-effect relationship, while the chi-square test is suitable for comparing frequencies of discrete events (such as the occurrence of lesions).
The choice of methods depends on the type of variable being measured (quantitative, qualitative), the data distribution, the group size, and whether or not multiple comparisons need to be corrected. All analyses must be performed using validated software (e.g., SAS, R, SPSS), according to pre-established protocols.
Interpretation of the data in relation to the control groups
The results of the exposed groups are systematically compared to those of the control group to determine whether the observed differences are related to the tested substance or to normal biological variability. This comparison is essential to definitively attribute an effect to the exposure.
Particular attention is paid to statistically significant differences, but also to their biological relevance. An effect may be statistically significant but remain within the limits of historical values observed for the species and strain used. Conversely, a moderate but constant variation may indicate a toxicological trend that should be monitored.
To strengthen the robustness of the results, the data obtained can be compared to internal or external reference databases, which compile the results of previous trials conducted under the same conditions. These databases allow for the assessment of the consistency of the effects and the detection of isolated anomalies.
Determination of the NOAEL and the LOAEL
One of the main objectives of the OECD 408 test is to identify a NOAEL (No Observed Adverse Effect Level), that is, the highest dose that does not cause any observable toxic effects. This threshold is fundamental for risk assessments, as it serves as the starting point for calculating the acceptable daily intake (ADI) or the occupational exposure limit (OEL).
When toxic effects are observed, the lowest dose at which these effects appear is designated as the LOAEL (Lowest Observed Adverse Effect Level). These two values define the toxicological safety level of the substance.
The determination of the NOAEL relies on an integrated synthesis of all data: clinical signs, body weight, biochemical parameters, and histopathological lesions. It must take into account both the statistical results, their biological significance, and their reproducibility.
In cases of uncertainty or lack of effect, a provisional value may be proposed, provided that the absence of harmful effects under the test conditions is clearly justified. This conservative approach is often accepted within a regulatory framework, particularly in the case of high-dose limit tests with no observed toxicity.
The relevance of the OECD 408 test within the regulatory framework
Registration of chemical substances
The OECD 408 test is required under numerous regulatory frameworks, particularly the REACH (Registration, Evaluation, Authorisation and Restriction of Chemicals) regulation applicable in the European Union. It is required for substances produced or imported in tonnages exceeding 100 mg/kg/year, in order to document their medium-term toxicity.
The test results complete the toxicological dossier of a substance by providing precise data on systemic effects, target organs, dose-response relationships, and NOAEL and LOAEL thresholds. This information is essential for determining hazard classifications (specific target organ toxicity, repeated-dose toxicity) and for assessing risks to human health based on exposure scenarios.
Beyond REACH, the OECD 408 test is also used in the context of the CLP (Classification, Labelling and Packaging) regulation, the biocides regulation (BPR), plant protection product evaluation procedures or applications for authorization of new substances at the international level.
Acceptance by regulatory agencies
The OECD 408 methodology is widely recognized internationally. It is accepted by regulatory agencies in all OECD member countries, as well as by other jurisdictions through mutual data recognition agreements.
In the United States, it corresponds to the OCSPP 870.3100 study defined by the Environmental Protection Agency (EPA) as part of chemical safety assessments under the TSCA law. This equivalence allows manufacturers to generate a single study valid in multiple regions of the world, reducing costs and avoiding unnecessary duplication of animal testing.
The international acceptability of this method rests on its standardization, its statistical robustness, and its integration into harmonized risk assessment frameworks. It ensures consistent interpretation of results by the competent authorities, facilitating the processing of regulatory dossiers.
Complementarity with new approaches (NAM)
Despite its importance, the OECD 408 test is increasingly associated with alternative methods grouped under the term NAM (New Approach Methodologies). These approaches include in vitro assays, in silico models (QSAR), toxicokinetic simulations (PBPK), and extrapolation strategies such as read-across.
The use of these tools allows for better targeting of in vivo tests and a reduction in the number of animals used, in accordance with the 3Rs principle (Replace, Reduce, Refine). Following this logic, the OECD 408 test is often integrated into IATA (Integrated Approaches to Testing and Assessment) type approaches, combining several data sources to assess a hazard or risk.
Thus, a PBPK model can be used to simulate the distribution of the substance in the body, guide dose selection, and interpret observed effects. Similarly, an alert from a QSAR model regarding a given chemical structure can justify targeted testing using OECD 408 to validate or refute a toxicological hypothesis.
OECD 408 test performed with YesWeLab
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YesWeLab relies on a network of over 200 partner laboratories, located throughout France and Europe, all selected for their high level of technical expertise and compliance with international standards. The majority of these laboratories are accredited according to ISO 17025 and recognized by COFRAC or their European equivalents.
For the OECD 408 test, YesWeLab collaborates exclusively with laboratories specializing in regulatory toxicology, equipped with facilities adapted for animal experimentation and compliant with the requirements of Directive 2010/63/EU on animal welfare. These partners are proficient in OECD guidelines and Good Laboratory Practices (GLP), ensuring data production that meets the requirements of regulatory authorities.
YesWeLab offers comprehensive scientific support, from defining the protocol to interpreting the results, integrating the specific constraints of each sector: chemistry, cosmetics, biocide, animal health, etc.
Support for the chemical, cosmetics and animal health industries
Conducting an OECD 408 test is often integrated into a comprehensive toxicological assessment strategy. YesWeLab is involved at every stage of this strategy, guiding manufacturers in selecting the necessary studies and prioritizing tests based on risk profiles, regulatory requirements, and existing data.
For chemical manufacturers, the OECD 408 test ensures compliance with REACH regulations. YesWeLab guarantees the proper integration of results into the registration dossier, with complete documentation compatible with regulatory platforms such as IUCLID and REACH-IT.
In the cosmetics sector, YesWeLab collaborates with safety managers and toxicologists to document the potential effects of ingredients used for extended periods. OECD study 408 can thus contribute to the cosmetic product safety report, particularly when dealing with new active ingredients or controversial substances.
In animal health, the OECD 408 test is integrated into the authorization processes for additives for animal feed or veterinary substances, with particular attention paid to the selection of parameters relevant to the target species.
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