Repeated Dose Toxicity

    1. ECVAM validated test methods

    2. Test methods under validation by ECVAM

    3. Development and optimisation of alternative methods

    4. Workshop Reports and Relevant Publications



Repeated dose toxicity comprises the adverse general toxicological effects occurring as a result of repeated daily dosing with, or exposure, to a substance for a specified period up to the expected lifespan of the test species. The studies yield information on general characteristics of the toxicity, the target organs of toxicity, the dose–response (curve) for each toxicity endpoint, responses to toxic metabolites formed in the organism, delayed responses, cumulative effects, the margin between toxic/non-toxic dose, information on reversibility/irreversibility of the effect, and NOAEL (No Observed Adverse Effect Level), NOEL (No Observed Effect Level) for toxicity. 

The repeated dose study is an integral part of the data package produced to perform quantitative risk assessment (QRA) of industrial chemicals, cosmetic ingredients, biocides, pesticides, pharmaceuticals. The point of departure most commonly used for systemic toxicity safety assessment is the NOAEL which is used in the calculation of the MoS (Margin of Safety) or MoE (Margin of Exposure).

The table below shows the in vivo repeated dose toxicity tests available.

Test method


(EC) No 440/2008


Test Method

OECD Test Guideline


in vitro/in vivo

28-day Oral Toxicity Study in Rodents


TG 407

Repeated dose

in vivo

90-Day Oral Toxicity Study in Rodents


TG 408

Repeated dose

in vivo

90-Day Oral Toxicity Study in Non-Rodents


TG 409

Repeated dose

in vivo

Dermal Toxicity: 21/28-day Study (rat, rabbit or guinea pig)


TG 410

Repeated dose

in vivo

Dermal Toxicity: 90-day Study (rat, rabbit or guinea pig)


TG 411

Repeated dose

in vivo

Inhalation Toxicity: 28-Day Study in Rodents


TG 412

Repeated dose

in vivo

Inhalation Toxicity: 90-day Study in Rodents


TG 413

Repeated dose

in vivo

Chronic Toxicity Studies in Rodents


TG 452

Repeated dose

in vivo


Alternative test methods and approaches


1. ECVAM Validated test methods

There are not test methods validated.


2. Test methods under validation by ECVAM

Currently there are not test methods under validation.


3. Development/optimisation/improvement of alternative methods

3.1. Involvement in competitive activities

3.1.1 EU FP6 Predictomics project: Short-term models assays for long-term toxicity (EU contract no 504761)

The aim of this European project was to develop a novel strategy for predicting chronic toxicity in the two most important target organs affected by drugs and xenobiotics, the liver and the kidney. Advanced culture technology, genomic, proteomic and cytomic analysis were combined to detect the early events of cellular injury. The data generated were examined in view of the known pathophysiological changes linked to the chronic disease.

ECVAM was part of the executive board and had an advisory role to ensure that methods were sufficiently well developed so that they could be considered for entering, eventually, future validation process.


3.1.2 EU FP6 Marie Curie Actions Research Training Network PULMO-NET: Pathogenesis of pulmonary disease (EU contract no MRTN-CT-2004-512229)

Pulmonet was created with the aim to improve methodological approaches to study lung function (such as fluid transport, secretion of surfactant, mucous or inflammatory mediators, and barrier/ transport functions) and dysfunction on a cellular and molecular level, gaining a better understanding of normal lung function, toxicity of environmental compounds, cell and tissue damage and generation of pulmonary disease. The network consisted of experts from diverse fields, such as on lung proteins required for host defense and reduction of surface tension (surfactant proteins), lung cell isolation and culture, endotoxins and cell signalling, cytokines, inflammation and immune response, and use of cell lines (such as Calu-3) for barrier function and toxicity tests.

EURL ECVAM contributed to the network by setting up appropriate in vitro model(s) to study chemically induced damage to pulmonary barrier after exposure to substances of various kinds (see below, Forti et al., 2010; Forti et al., 2011).


3.1.3 EU FP7 Predict-IV project: Profiling the toxicity of new drugs: a non animal-based approach integrating toxico-dynamics and biokinetics (EU Contract no. 202222)

The ultimate goal of Predict-IV is to deliver integrated test system/test strategy with specific biomarkers to predict repeated dose toxicity based on in vitro data before entering in vivo testing. JRC-IHCP is a partner in this EU project and leads activities on in vitro neurotoxicity testing, and contributes to OMICs profiling methods and on monitoring European efforts towards other non animal-based integrative approaches (See: Predict-IV website).


3.1.4 EU FP7 SEURAT-1: Towards the replacement of in vivo repeated dose systemic toxicity testing

This Research Initiative is a first step to address the long term strategic target of "Safety Evaluation Ultimately Replacing Animal Testing (SEURAT)". It is composed of six complementary research projects (SCR&Tox, HeMiBio DETECTIVE, COSMOS, NOTOX, ToxBank) and a coordination and support action (COACH).

The European Commission Joint Research Centre, Institute for Health and Consumer Protection (JRC-IHCP) is a partner in SCR&Tox, DETECTIVE, COSMOS, and COACH.

For more information, see SEURAT-1 website.

4. Workshop Reports and Relevant Publications

available here