Poster Presentation

SOT 64th Annual Meeting and ToxExpo

Orlando - March 16-20 2025

Abstract:

The introduction of ECHA’s dose level setting advice for repeated dose and reproductive toxicity testing in 2022 led to a significant change in expectations and a number of associated challenges for dose-level setting, with implications beyond Europe. Via two case studies, we demonstrate how ECHA’s advice can lead to different outcomes, depending on interpretation of the advice and application of individual laboratory welfare limits, even when the labs are regulated by the same national legislation.

Both case studies concern the dose range finding phase of the OECD 414 prenatal developmental toxicity study, undertaken to support substance registration under EU REACH. Both laboratories were based in the same nation and were therefore regulated by the same national legislation. For each, we present a summary of testing information on key dose levels from the dose range finding investigations, and discuss the subsequent rationale used to select a high dose for the definitive OECD 414. 

Case Study A:

An OECD 414 study was commissioned following issue of a Testing Proposal Decision by ECHA (October 2022). Dose setting investigations included tolerability and dose range finding studies. In the tolerability study, 3 non-pregnant females were dosed with test material via oral gavage for up to 10 days at doses of 300, 375, 450 and 600 mg/kg bw/day based on dose level information from repeated dose studies. 300 mg/kg bw/day was well tolerated, with no test material associated clinical signs, body weights and food consumption within the expected range, and all animals microscopically normal. Dose levels of ≥ 375 mg/kg bw/day were not tolerated. In the 375 mg/kg bw/day group, 2/3 animals had consecutively low food consumption from day 1–8, corresponding with a body weight loss of 9-11%. This resulted in early termination of the group based on the laboratory’s welfare limits.

In the dose range finding study, groups of 6 pregnant females were dosed from Gestation Day (GD) 6-28. A high dose of 335 mg/kg bw/day was selected based on the results of the tolerability study, as a mid-point between doses with no toxicity vs. severe toxicological effects (300 mg/kg bw/day and 375 mg/kg bw/day, respectively). At 335 mg/kg bw/day there was higher initial mean body weight loss from GD 6-12 vs. control (-3% treated vs -2% control). After this initial body weight loss, a similar body weight gain of 9% treated vs 11% control was recorded from GD 12-29. Corresponding lower food consumption was noted in earlier stages of gestation (-34% GD 6-14 and -21% GD 14-23), returning to levels comparable with control from GD 23-29. There were no macroscopic effects on the F0 animals or foetuses, and no effects on corpora lutea, pre- and post-implantation loss, resorptions or number of foetuses/live foetuses. 

Case Study B:

An OECD 414 study was commissioned following issue of a Testing Proposal Decision by ECHA (August 2022). Based on available information, a dose range finding study was commenced whereby groups of 6 pregnant females were dosed via oral gavage 0, 400, 650 and 850 mg/kg bw/day. As this did not reveal toxicity considered sufficient to meet ECHA’s dose setting requirements, a further dose level of 1000 mg/kg bw/day was also examined. Below we detail results of two key dose levels.

1000 mg/kg bw/day was not tolerated, with 2 animals undergoing unscheduled euthanisa due to severe clinical signs (irregular respiration, erect fur, cold to touch, low carriage, decreased activity and subdued behaviour).  At 850 mg/kg bw/day, clinical signs (salivation, ploughing and fur loss) were observed alongside a slight increase in body weight gain and food consumption vs. control. There were no macroscopic effects on the F0 animals or foetuses, and no effects on corpora lutea, pre- and post-implantation loss, resorptions or number of foetuses/live foetuses. 

Discussion:

In both case studies, the situation is similar: there is a dose level that is clearly not tolerated and another which has little to no toxicological effect on the maternal animals or developing foetuses (375 mg/kg bw/day not tolerated vs. 300 mg/kg bw/day tolerated in Case Study A, and 1000 mg/kg bw/day not tolerated vs. 850 mg/kg bw/day tolerated in Case Study B). The difference between the tolerated and non-tolerated dose level is also small at 10% (35 mg/kg bw) and 15% (150 mg mg/kg bw) for Case Studies A and B, respectively. 

ECHA’s dose setting advice states that for the OECD 414 the highest dose level should be selected with the aim to induce “some developmental and/or material toxicity (clinical signs of a decrease in body weight) but not severe suffering or death”. For the high dose level, it is further stipulated that this should be the “highest possible dose…not causing severe suffering or death”, and that the dose range selected is sufficiently high to “conclude on a lack of clear evidence for reproductive toxic properties warranting a classification as Repr. 1B for the tested parameters”. The advice also refers to additional guidance on severe suffering, including OECD guidance document 19 and national legislation on animal experiments. 

Notable in ECHA’s advice is a lack of guidance on how close to a non-tolerable dose level studies must go to demonstrate achievement of the aforementioned criteria. For these case studies, although the situations were similar, this lack of guidance led to the two Study Directors (SD) having contrasting approaches when ultimately electing the high dose for the OECD 414. In Case Study A, the SD concluded that despite only a 10% difference between tolerated and non-tolerated dose levels, the mild toxicity observed did not met ECHA’s dose level criteria to demonstrate ‘sufficient’ maternal or developmental toxicity. Consequently, they considered there was insufficient information to conclude the highest possible dose level had been reached, adding a further dose level at 355 mg/kg bw/day to meet ECHA’s criteria. Contrastingly, the SD for Case Study B concluded that although at 850 mg/kg bw/day there was little to no evidence of maternal or developmental toxicity; due to the small difference between the tolerated and non-tolerated dose levels (15%) and the mortalities observed at 1000 mg/kg bw/day, 850 mg/kg bw/day was the highest achievable dose.

These case studies demonstrate how the dose setting advice can be interpretated differently by laboratories even when subjected to same national legislation. Primarily, this is due to lack of i) clarity on what is considered as proof that the highest possible dose has been achieved, and ii) criteria to define a ‘sufficient difference’ between tolerated and non-tolerated dose levels without causing unnecessary pain and suffering to animals on study. This increasingly results in a move away from traditional 2-4-fold dose spacings, which when combined with the need to ‘prove’ the highest possible dose level has been achieved often leads to increased animal numbers and severe impacts on welfare. This moves such studies firmly away from the principles of the 3Rs in practice. This is particularly evidenced in cases where there is a steep dose response and therefore very small difference between a well-tolerated and toxicologically adverse dose level, as we have demonstrated.

We consider it as imperative that more in-depth guidance is provided on how to prove the highest dose , and when a dose level is considered as sufficiently close to known severe toxicity to allow for main study dose levels to be established without negatively impacting animal welfare. Such guidance would also allow a standardised approach to be established for selection of high dose levels between laboratories, both within and out with the same nation, as guidance provided by ECHA is used to produce local & national legislation from which individual welfare limits are established by labs. This additional guidance will also allow balance to be established between maintaining animal welfare and achieving the highest dose possible, to ensure robust, reliable data is generated for hazard identification and classification.

Presenter Bio: Dr. Susan Watters

Susan is a senior consultant toxicologist within the Blue Frog Scientific human safety team, specialising in reproductive and developmental toxicology and the assessment of potential human endocrine disruptors. Susan also provides expert regulatory toxicology consulting services including data gap analysis, integrated testing strategies, study monitoring, interpretation of datasets, and technical dossier compilation.