New (But Old) Advice on Dose Level Selection for DART Studies Under REACH

How do these specific updates impact safety assessment for the chemical industry?

This week, the European Chemicals Agency (ECHA) released a new advice document on dose-level selection for conducting reproductive toxicity studies (OECD TGs 414, 421/422, and 443) under REACH. This document, first released in January 2022 and updated in June 2026, is available on the ECHA website. This updated advice is intended to help registrants comply with the revised legal text in EU Commission Regulation 2021/979 (17 June 2021) which states the following:

“Where a test method offers flexibility in the study design, for example, in relation to the choice of dose levels, the chosen study design shall ensure that the data generated are adequate for hazard identification and risk assessment. To this end, testing shall be performed at appropriately high dose levels. If dose (concentration) selection is limited by the physicochemical properties or biological effects of the test substance, justification shall be provided.”

So, what does this mean? In order to be compliant, the selected dose levels should ensure conclusive data generation for classification and labelling (CLP) and risk assessment i.e., the highest dose level should be sufficiently high to be able to conclude on hazardous properties for the tested parameters, and the lowest dose level should show no toxicity to allow selection of a no-observed-adverse-effect level (NOAEL). 

While at first glance, these recommendations appear reasonable, a deeper dive reveals that the goal here is only to produce conclusive evidence for the purposes of classification and labeling, i.e., the tested doses should be sufficiently high to conclude that a substance “does” or “does not” have reproductive or developmentally toxic properties, warranting a classification as Repr. 1B (either F (Fertility) or D (Development) or both). In the case of substances that fail to provide unequivocal evidence of systemic or reproductive toxicity, the limit dose concept shall be applied, i.e., a high dose of 1000 mg/kg/day will be selected. This approach should be applied even in instances where the substance causes intrauterine or postnatal mortality, i.e., dose levels cannot be lowered even if the number of offspring is reduced, which is counterintuitive to the goal of multigeneration reproductive toxicity studies such as the Extended One-Generation Reproductive Toxicity Study (EOGRTS, OECD TG 443). Not surprisingly, this advice has resulted in considerable debate within the Development and Reproductive Toxicology (DART) scientific community (Lewis et al., 2024; Sewell et al., 2022; Moxon et al., 2023, Beekhuijzen et al 2024).

Despite overwhelming disagreement within the scientific community, this updated document, released four years after the original, provides little new information and shows no obvious change in the agency’s recommended approach to dose selection for DART studies. Key highlights are included below:

•    Testing should still be performed at appropriately high doses to elicit effects on sexual function and fertility, without severe suffering or death, or 1000 mg/kg/day should be selected as the high dose.

•    Experimental data are needed for dose level selection i.e. dose range-finding (DRF) studies are a necessary prerequisite. Dose levels should not be lowered to ensure sufficient numbers of pups for the second filial generation.

•    Dose level spacing mentioned in previously published Test Guidelines (e.g., 2- to 4-fold) may not always be appropriate for setting the highest dose level below a dose level causing severe suffering or mortality. In such cases, additional dose groups or additional DRF studies should be considered to identify a dose that induces toxic effects without severe suffering.

•    Toxicokinetic (TK) data should be considered for dose selection; however, dose setting based on “TK considerations only” is not allowed under REACH (dose-level selection should be based on toxicity to ensure that the data generated are adequate for hazard identification).

•    Because pregnant animals may show differences in toxicity, TK and sensitivity, compared to non-pregnant animals, dose selection based on data from non-pregnant animals alone is not recommended; DRF studies in pregnant animals should be conducted where relevant.

•    Where different sensitivities are noted between male and female animals, different dose levels should be selected for males vs females i.e., the less sensitive sex should be tested at higher doses. During the mating period, both sexes may receive the test substance at doses selected to match the more sensitive sex, to avoid severe suffering and death in these animals.

•    All DRF (and preliminary tolerability) studies should be reported in separate endpoint study records of the IUCLID dossier. The endpoint records for the main studies (i.e., OECD TGs 414 and 443) must state the scientific rationale for dose selection based on the results of the DRF studies. Results of the DRF studies should be detailed in order to be useful to interpret dose-response relationships.

•    Dose level selection for rabbit OECD 414 studies should be based on existing data in the same species, i.e., based on studies performed in rabbits. If no prior information is available in this species, preliminary tolerability/palatability studies should be conducted in non-pregnant rabbits and a DRF study in pregnant rabbits to understand the dose response relationship and select dose levels for the main studies.

•    Because of significant inter-species differences between rat and rabbit, information gathered for the rat cannot be used to inform on dose-level selection for the rabbit and vice versa.

•    If available, information from existing studies including DRF studies show clear evidence of an adverse effect on reproduction (sexual function and fertility) or development, which meet CLP criteria, then the substances should be self-classified.

•    Dose-level selection for EOGRTS (OECD TG 443) studies should be based on actual evidence, not theoretical considerations. e.g., it is not acceptable to assume that a longer exposure duration might result in severe suffering or death and to use this as justification to lower the highest dose.

•    The EOGRTS (OECD TG 443) is intended to ensure assessment of the effects on sexual function and fertility – hence, dose levels should not be reduced to get enough offspring for the assessment of developmental toxicity. Even if the number of offspring would be reduced due to effects on sexual function and fertility, any offspring available at that dose level should be investigated for adverse effects on development (as well as those at lower dose levels), i.e., recommended endpoints should be evaluated despite a statistically insufficient number of pups in the affected groups. 

The European Union has a long-standing policy of replacing, reducing, and refining animal testing (3Rs). In June 2026, the European Commission also adopted the Roadmap towards phasing out animal testing for chemical safety assessments to facilitate the transition to alternative testing methods. Recommendations included in this week’s advice on dose level selection do take animal welfare into consideration (when discussing systemic toxicity and severe suffering).

However, the emphasis on experimental data from pregnant animals of the same species appears to contradict efforts to reduce animal testing and to incorporate alternative methods and approaches, such as species-to-species extrapolation, read-across, and PBPK modeling. Undoubtedly, the discourse regarding dose selection for DART studies will continue to evolve over the coming months/years. The Charles River DART team has conducted an extensive review of the new advice, the specific test guidelines, and their associated regulatory impact. 

For any questions, including specific questions relating to these recent changes, call or email us at [email protected].

Prägati Coder, PhD, DABT, ATS is Global Lead of Developmental and Reproductive Toxicology (DART, C&C) at Charles River, and Senior Director, DART, of Juvenile and Neurotoxicology.