Subchronic toxicity 90 days – OECD 408

trial 408 is the reference study for characterizing the toxicity of a substance administered orally daily for 90 days in rodents. This duration represents approximately one-tenth of the animal's lifespan: it allows time for accumulation effects, metabolic adaptations, and slowly evolving lesions to manifest.

This is the test that regulatory agencies expect when it comes to establishing a NOAEL in a quantitative risk assessment. Its current version, revised in 2018 and then reissued in 2025, has incorporated a series of measures sensitive to endocrine function, with particular attention paid to thyroid function.

Objective of the analysis

The 90-day subchronic study pursues an objective that shorter trials cannot achieve: to produce a threshold value robust enough to serve as a starting point for calculating occupational exposure limit values ​​and safety margins.

• Determine the NOAEL and LOAEL with statistical power adapted to small amplitude effects.
• Precisely identify the target organs and the nature of the induced lesions.
• To characterize the progression of effects over time, and not their mere presence.
• Detect endocrine , hematological, neurological and ophthalmological signals
• Document reversibility via a satellite group maintained without treatment after exposure.

OECD Methodology 408

Doubling the sample size compared to a 28-day study is not a mere administrative formality. It is what gives the trial the statistical power necessary to distinguish a real effect from normal biological variation — precisely the kind of discrete effect that escapes detection in a short study.

Parameters evaluated

Clinical follow-up — daily monitoring of mortality and morbidity, weekly clinical examination, weighing and monitoring of food and fluid intake throughout the thirteen weeks.

Functional observations — reactivity to stimuli and motor activity, allowing the detection of neurological damage before it becomes histologically visible.

Ophthalmological examination — performed before the start of the trial and then at its completion.

Biological analyses — complete hematology and clinical biochemistry at the end of exposure.

Endocrine parameters —the 2018 revision introduced measures related to the endocrine system, with thyroid function being the primary focus. These data constitute an early warning signal integrated into a general toxicity study; they do not replace tests specifically designed for endocrine disruption, but can trigger their implementation.

Necropsy, organ weighing and histopathology — complete macroscopic examination, then detailed histopathological reading on the control and high dose groups, extended to other groups when lesions are found.

Interpretation and regulatory consequences

The NOAEL determined by test 408 directly feeds into two separate processes: the calculation of reference values ​​for risk assessment, and the classification of the substance under the CLP Regulation.

The CLP guideline values ​​for the STOT-RE hazard statement (specific target organ toxicity, repeated exposure) are precisely established on the basis of a 90-day rat study:

Unlike a 28-day study, whose results must be extrapolated by multiplying the thresholds by three, the 408 trial applies directly to these values. This is an underestimated advantage: it eliminates a step of interpretation, and therefore a source of dispute by the evaluator.

When is OECD 408 required?

Under REACH, the 90-day subchronic study becomes a standard reporting requirement when the threshold of 100 tonnes per year per registrant. Below this threshold, between 10 and 100 tonnes, the 28-day study applies.

The test is also expected in the approval dossiers for biocidal and phytopharmaceutical active substances, in the evaluations of food and animal feed additives, and in the biological evaluation of certain medical devices.

Is it necessary to carry out an OECD 407 beforehand?

It depends on what you already know about the substance. If no repeated-dose data exist, a preliminary study— such as a 28-day OECD 407 or dose-finding study—will ensure the correct choice of exposure levels for the main study. A high, poorly calibrated dose that causes excessive mortality or, conversely, no effect, renders the 90-day trial uninterpretable.

If your tonnage puts you in the top 100 tonnes per year from the outset, however, combining the two studies is more expensive than directly ordering the 408 trial with a preliminary phase of short dose research.

Sectors and applications concerned

• Chemical industry — REACH registration beyond 100 tonnes per year.
• Biocides and plant protection products — active substance approval dossiers.
• Nutraceuticals and food supplements — safety assessment of new ingredients and Novel Food dossiers.
• Animal feed and health — additives, premixes and raw materials.
• Medical devices — biological evaluation under prolonged exposure.

The cosmetics sector is an exception: as animal testing is prohibited for the purposes of regulation 1223/2009, this test cannot be carried out for the purpose of assessing cosmetic safety in the European Union.

Why use YesWeLab?

A 90-day study involves several hundred animals, six to twelve months of planning, and a significant budget. It cannot be repeated. The quality of the laboratory and the relevance of the study design are the only two factors that determine whether the investment will result in a viable application.

We connect you with partner laboratories selected for their mastery of OECD guidelines. Our partners operate according to Good Laboratory Practices, a requirement for the study to be accepted in a regulatory dossier.

Our added value lies upstream of the trial: arbitrating between 28 and 90 days, choosing the exposure route based on the actual exposure scenario, developing the dose plan, and anticipating the parameters to be integrated from the design stage rather than after the fact. Our platform then centralizes your requests, quotes, and reports, with a single point of contact from the initial exchange to the delivery of the final report.

Frequently Asked Questions

What timeframe should be expected for an OECD 408 study?

The exposure phase lasts thirteen weeks. This is followed by analytical characterization of the substance, a possible dose-finding study, histopathology, and report writing. An overall timeframe of six to twelve months is realistic, depending on the complexity of the substance and the laboratory's availability.

Can a 28-day study replace OECD 408?

No, not when 90 days is required by regulations. The small sample size and short observation window of trial 407 do not allow for the detection of slowly evolving effects, and a negative result over 28 days does not constitute proof of safety under prolonged exposure.

What happens if an effect is observed at all doses?

The study then does not allow for the establishment of a NOAEL, only a LOAEL. This situation generally reflects a dose plan that is too high and frequently leads to having to repeat all or part of the study — hence the importance of a preliminary calibration phase.

Does test 408 cover inhalation exposure?

No. It is specific to the oral route. For primarily respiratory exposure — powders, aerosols, volatile substances — the applicable guideline is OECD 413 over 90 days, or OECD 412 over 28 days.

Are thyroid parameters mandatory?

The 2018 revision incorporated them into the guideline. They are expected in a study conforming to the current version, and their absence may prompt a request for additional information from the evaluator.

See also

• OECD 408: The detailed, step-by-step process of the study
• 28-day subacute toxicity study – OECD 407
• Chronic toxicity study – OECD 452
• Overview of subacute and subchronic toxicity trials
• Our complete catalogue of analyses