Acute Oral Toxicity Testing: Understanding OECD TG 420, 423 and 425
Choosing the Appropriate OECD Approach for Acute Oral Toxicity Assessment
Acute oral toxicity testing is an important component of the non-clinical safety assessment of chemicals and other regulated test items. It provides information on adverse effects that may occur following a single oral exposure, or multiple exposures occurring within a short period, and can contribute to hazard identification, classification and regulatory risk assessment.
Historically, acute oral toxicity assessment was strongly associated with determination of a numerical median lethal dose (LD₅₀). Modern OECD approaches have moved away from routine use of large numbers of animals solely to establish a precise LD₅₀. Instead, current OECD methods are designed to obtain the information required for hazard assessment while incorporating the principles of Reduction and Refinement of animal use.
Three important OECD Test Guidelines used for in vivo acute oral toxicity assessment are:
- OECD TG 420 — Acute Oral Toxicity: Fixed Dose Procedure
- OECD TG 423 — Acute Oral Toxicity: Acute Toxic Class Method
- OECD TG 425 — Acute Oral Toxicity: Up-and-Down Procedure
Although all three methods evaluate acute toxicity following oral administration, they are not interchangeable. Their experimental designs, decision criteria and expected outputs differ, and selection of the appropriate method should be based on the regulatory purpose of the study and the available information on the test item.
What Is Acute Oral Toxicity?
Acute oral toxicity refers to adverse effects occurring following administration of a single oral dose of a substance or following multiple oral doses given within a short period.
Acute oral toxicity information can support:
- identification of acute toxic hazards;
- classification and labelling;
- estimation of an acute toxic range;
- selection of appropriate precautions for handling;
- broader chemical safety and risk assessment;
- regulatory submissions where acute oral toxicity information is required.
The information generated may support classification under the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) or jurisdiction-specific systems implementing GHS principles.
The applicable regulatory requirement must nevertheless be evaluated for the specific substance, product category and jurisdiction.
Why Are There Three Different OECD Acute Oral Toxicity Methods?
OECD TG 420, TG 423 and TG 425 were developed to generate scientifically useful acute toxicity information while reducing reliance on the historical conventional LD₅₀ approach.
The appropriate method depends largely on the type of information required.
OECD Test Guideline | Method | Fundamental Approach | Principal Output |
|---|---|---|---|
TG 420 | Fixed Dose Procedure | Fixed doses selected with emphasis on identifying evident toxicity rather than mortality | Hazard classification based on toxic signs and mortality |
TG 423 | Acute Toxic Class Method | Groups of three animals tested sequentially at predefined dose levels | Acute toxicity class / LD₅₀ cut-off range for classification |
TG 425 | Up-and-Down Procedure | Individual animals dosed sequentially; dose increases or decreases according to the preceding animal’s response | LD₅₀ estimate with associated confidence interval |
The OECD Guidance Document on Acute Oral Toxicity Testing should be considered when determining which method is most appropriate for a particular regulatory objective.
OECD TG 420: Fixed Dose Procedure
Principle
OECD TG 420 is the Fixed Dose Procedure (FDP).
Its distinguishing feature is that the method places particular emphasis on identifying evident signs of toxicity rather than using mortality as the primary endpoint.
The fixed dose levels normally used are:
- 5 mg/kg body weight
- 50 mg/kg body weight
- 300 mg/kg body weight
- 2000 mg/kg body weight
An exceptional dose of 5000 mg/kg may be considered only under the circumstances described in the guideline.
The method is intended primarily for use in rats, and animals of a single sex, normally females, are generally used.
Sighting Study
Before the main study, a sighting study is conducted to assist in selecting an appropriate starting dose.
The objective is to identify a dose expected to produce evidence of toxicity without unnecessarily causing severe toxicity or death.
Selection of the starting dose should therefore incorporate all available information concerning the test item, including where available:
- physicochemical properties;
- structural relationships;
- existing in vitro or in vivo information;
- information on related substances;
- anticipated human exposure;
- existing toxicological information.
Main Study
Animals are subsequently treated at an appropriate fixed dose according to the outcome of the sighting study.
Further testing at a higher or lower fixed dose may be required depending on the observed toxicity or mortality.
A total of five animals of one sex will normally be used for each dose level investigated, according to the OECD guideline.
When Is TG 420 Particularly Useful?
TG 420 may be appropriate when the regulatory objective is primarily acute toxicity classification and when identifying a dose producing evident toxicity provides sufficient information.
Unlike methods focused principally on mortality, the Fixed Dose Procedure incorporates non-lethal signs of toxicity directly into its decision process.
OECD TG 423: Acute Toxic Class Method
Principle
OECD TG 423 is the Acute Toxic Class Method.
It is a stepwise procedure in which three animals of a single sex are used at each step.
The predefined dose levels are generally:
- 5 mg/kg body weight
- 50 mg/kg body weight
- 300 mg/kg body weight
- 2000 mg/kg body weight
Progression to a higher or lower dose depends on the mortality and/or moribund status observed in the animals treated at the preceding step.
The OECD guideline indicates that, depending on the outcome, an average of approximately two to four steps may be necessary.
Importantly, TG 423 is therefore a group-sequential method. It should not be confused with TG 425, in which individual animals are dosed sequentially.
How Does the Method Work?
Three animals are administered the selected starting dose.
The subsequent decision may involve:
- testing an additional group at the same dose;
- testing at a higher dose;
- testing at a lower dose; or
- stopping the study and assigning the appropriate classification.
The decision is determined according to the decision rules specified in the OECD guideline.
What Information Does TG 423 Provide?
TG 423 is primarily a classification-oriented method.
It permits a substance to be assigned to an acute toxicity category and allows determination of an LD₅₀ cut-off value or range, rather than attempting to determine an exact numerical LD₅₀.
When May TG 423 Be Appropriate?
TG 423 may be particularly useful where the principal regulatory objective is:
- hazard classification;
- assignment of an acute toxicity category; or
- identification of an LD₅₀ cut-off range.
OECD TG 425: Up-and-Down Procedure
Principle
OECD TG 425 uses a fundamentally different experimental design.
The Up-and-Down Procedure (UDP) administers the test item to one animal at a time.
The response of the preceding animal determines the dose given to the next animal.
In simplified terms:
- if an animal survives, the next animal generally receives a higher dose;
- if an animal dies, the next animal generally receives a lower dose.
The main test continues according to predefined sequential rules until one of the statistical stopping criteria specified in the guideline has been satisfied.
Animals are normally dosed at intervals allowing the outcome of the previous animal to be established before the next animal is treated. OECD describes an interval of approximately 48 hours as usual, although the appropriate interval depends on the onset, duration and severity of toxic signs.
What Makes TG 425 Different?
Unlike TG 420 and TG 423, TG 425 is specifically capable of generating:
- an estimated LD₅₀, and
- an associated confidence interval.
The LD₅₀ is calculated using the statistical methodology specified for the Up-and-Down Procedure.
OECD also provides statistical software associated with TG 425; laboratories should use the applicable methodology and understand the assumptions and limitations of the statistical procedure.
Limit Test
TG 425 also contains a limit-test approach that may be appropriate when existing information suggests that the test item is likely to have relatively low acute toxicity.
The decision to use a limit test should be scientifically justified based on available information.
When May TG 425 Be Appropriate?
TG 425 may be particularly useful when a regulatory programme requires a numerical estimate of the LD₅₀ rather than only assignment to a toxicity class.
TG 420 vs TG 423 vs TG 425: Key Differences
Parameter | OECD TG 420 | OECD TG 423 | OECD TG 425 |
|---|---|---|---|
Official name | Fixed Dose Procedure | Acute Toxic Class Method | Up-and-Down Procedure |
Study design | Stepwise fixed-dose | Group-sequential | Individual sequential |
Animals per dosing step | Fixed-dose groups; normally five animals per dose level investigated | 3 animals per step | 1 animal sequentially |
Main decision basis | Evident toxicity and mortality | Mortality/moribund status | Survival/mortality sequence |
Typical fixed doses | 5, 50, 300, 2000 mg/kg | 5, 50, 300, 2000 mg/kg | Sequential dose progression |
Primary purpose | Classification based strongly on evident toxicity | Toxicity-class assignment | Numerical LD₅₀ estimation |
Numerical LD₅₀ | Not the principal objective | Not the principal objective | Yes |
Confidence interval for LD₅₀ | No | No | Yes |
Sequential individual dosing | No | No | Yes |
No single method should automatically be considered superior to the others. The appropriate method depends on the regulatory question being addressed.
Animal Selection
The OECD acute oral toxicity guidelines generally identify the rat as the preferred species, with females normally used.
The use of females is based on evidence considered during guideline development indicating that, where sex differences occur, females are frequently slightly more sensitive.
Where males are used instead, the scientific rationale should be documented.
Animals should be healthy, appropriately acclimatized and within the age and body-weight specifications described in the applicable guideline.
Administration of the Test Item
The test item is normally administered orally as a single dose by gavage, using a stomach tube or suitable intubation cannula.
Animals are generally fasted before dosing in accordance with the applicable guideline.
Where a vehicle is required, its selection should consider:
- solubility or dispersibility of the test item;
- stability;
- homogeneity;
- compatibility with the test item;
- potential biological effects of the vehicle;
- achievable dosing concentration and volume.
The formulation strategy should be scientifically justified and documented.
Clinical Observations
Careful clinical observation is essential in acute toxicity studies.
Animals should be observed individually after administration, including:
- at least once during the first 30 minutes;
- periodically during the first 24 hours;
- with particular attention during the first four hours; and
- daily thereafter throughout the observation period.
The observation period is generally 14 days, although its duration may be adapted where scientifically justified based on the onset and reversibility of toxic effects.
Clinical observations should systematically document relevant signs involving, where applicable:
- skin and fur;
- eyes and mucous membranes;
- respiratory system;
- circulatory system;
- autonomic nervous system;
- central nervous system;
- somatomotor activity;
- behavioural pattern.
Attention should also be given to tremors, convulsions, salivation, diarrhoea, lethargy, sleep, coma and other relevant findings.
Animals showing severe and enduring pain or distress, or meeting established humane-endpoint criteria, should be humanely euthanized in accordance with applicable animal-welfare procedures.
Body Weight
Individual animal body weights should be recorded according to the applicable OECD guideline.
Body-weight changes can provide useful supportive evidence of systemic toxicity and should be considered together with:
- clinical observations;
- survival;
- time of onset of toxicity;
- recovery or persistence of findings;
- gross pathological observations.
Necropsy and Gross Pathology
All animals used in the study should be subjected to gross necropsy, including:
- animals surviving to scheduled termination;
- animals found dead; and
- animals euthanized for humane reasons.
Gross pathological abnormalities should be documented.
Where indicated by the applicable guideline and the nature of the findings, microscopic examination of tissues showing gross abnormalities may also be considered.
Acute Oral Toxicity and GHS Classification
Acute oral toxicity information may contribute to classification according to the UN Globally Harmonized System of Classification and Labelling of Chemicals.
The current UN GHS oral acute toxicity categories are based on acute toxicity estimates expressed in mg/kg body weight.
GHS Acute Oral Toxicity Category | Acute Toxicity Estimate |
|---|---|
Category 1 | ≤5 mg/kg |
Category 2 | >5–50 mg/kg |
Category 3 | >50–300 mg/kg |
Category 4 | >300–2000 mg/kg |
Category 5 | >2000–5000 mg/kg |
Category 5 is an additional category intended for substances of comparatively low acute toxicity where specific criteria for inclusion are met.
The regulatory implementation of GHS differs among jurisdictions. Therefore, classification should always be confirmed against the legally applicable national or regional framework rather than assuming that the UN GHS text itself constitutes the applicable legislation.
The 3Rs in Acute Oral Toxicity Testing
The development of TG 420, TG 423 and TG 425 represents an important evolution from traditional acute lethality testing.
The 3Rs are:
Replacement
Use non-animal approaches instead of animals wherever scientifically valid and regulatorily acceptable alternatives can provide the required information.
Reduction
Use the minimum number of animals necessary to obtain scientifically and regulatorily meaningful information.
The sequential and classification-oriented designs used in modern acute oral toxicity methods are important examples of the Reduction principle.
Refinement
Design and conduct studies to minimize avoidable pain, suffering and distress.
Examples include:
- appropriate starting-dose selection;
- avoidance of doses expected to cause unnecessary severe toxicity where possible;
- frequent clinical observation;
- predefined humane endpoints;
- timely veterinary intervention and humane euthanasia where necessary.
It is important not to describe reduced animal numbers as “Replacement.” Reduction and Replacement are distinct concepts within the 3Rs framework.
Importance of Existing Information Before Starting an Animal Study
An acute oral toxicity study should not begin simply because a guideline exists.
Before initiating an in vivo study, all reasonably available information should be reviewed.
This may include:
- physicochemical characteristics;
- existing toxicity data;
- structure-activity information;
- data from related substances;
- available in vitro information;
- previous animal data;
- human exposure information, where relevant;
- regulatory classification already available;
- information relevant to corrosivity or severe irritation.
This review serves two important purposes:
- determining whether additional animal testing is scientifically and regulatorily necessary; and
- selecting the most appropriate OECD method and starting dose where testing is required.
A substance known to produce severe pain or distress because of corrosive or strongly irritating properties should not be administered merely to generate additional acute oral toxicity data where such testing is scientifically unnecessary.
Conducting Acute Oral Toxicity Studies Under GLP
When an acute oral toxicity study is intended for regulatory submission and GLP is applicable, the study should be conducted according to the OECD Principles of Good Laboratory Practice and the relevant OECD Test Guideline.
GLP provides a quality system addressing how non-clinical safety studies are:
- planned;
- performed;
- monitored;
- recorded;
- reported; and
- archived.
Important elements include:
- an approved study plan;
- clearly defined Study Director responsibility;
- qualified and trained personnel;
- characterized test and reference items;
- controlled equipment and computerized systems;
- contemporaneous raw-data recording;
- independent Quality Assurance activities;
- documented deviations;
- controlled final reporting;
- retention and archiving of records and specimens.
Where studies are generated within the OECD Mutual Acceptance of Data (MAD) framework, applicability also depends on the GLP compliance-monitoring status and scope of the test facility and participating country.
Selecting the Appropriate OECD Guideline
The choice between TG 420, TG 423 and TG 425 should be made before study initiation based on the regulatory objective and available scientific information.
A practical conceptual approach is:
Consider TG 420 when:
the regulatory objective can be satisfied through a fixed-dose procedure emphasizing evident toxicity and hazard classification without requiring a precise numerical LD₅₀.
Consider TG 423 when:
classification into an acute toxicity class or establishment of an LD₅₀ cut-off range is the principal requirement.
Consider TG 425 when:
a numerical LD₅₀ estimate and associated confidence interval are specifically required.
These are general considerations rather than universal selection rules.
The final method should be selected after considering the applicable regulatory data requirement, OECD Guidance Document No. 24, existing information on the test item and animal-welfare considerations.
Conclusion
OECD TG 420, TG 423 and TG 425 provide different scientifically validated strategies for evaluating acute oral toxicity.
The major distinction is not simply the number of animals used:
- TG 420 is a Fixed Dose Procedure emphasizing evident toxicity.
- TG 423 is an Acute Toxic Class Method using three animals per sequential step to support classification.
- TG 425 is an Up-and-Down Procedure using individual sequential dosing to estimate an LD₅₀ and its confidence interval.
Correct selection and execution of the method require consideration of the regulatory purpose, existing toxicological information, animal welfare, dose-selection strategy and applicable GLP requirements.
Modern acute toxicity assessment should therefore be viewed not as the routine determination of a lethal dose, but as a regulatory decision-making process designed to generate the necessary hazard information using a scientifically justified and ethically responsible study design.
References
- OECD (2002). Test No. 420: Acute Oral Toxicity – Fixed Dose Procedure. OECD Guidelines for the Testing of Chemicals, Section 4. OECD Publishing, Paris. DOI: 10.1787/9789264070943-en.
- OECD (2002). Test No. 423: Acute Oral Toxicity – Acute Toxic Class Method. OECD Guidelines for the Testing of Chemicals, Section 4. OECD Publishing, Paris. DOI: 10.1787/9789264071001-en.
- OECD (2022). Test No. 425: Acute Oral Toxicity: Up-and-Down Procedure. OECD Guidelines for the Testing of Chemicals, Section 4. OECD Publishing, Paris. DOI: 10.1787/9789264071049-en.
- OECD (2002). Guidance Document on Acute Oral Toxicity Testing. OECD Series on Testing and Assessment No. 24. OECD Publishing, Paris. DOI: 10.1787/9789264078413-en.
- United Nations (2025). Globally Harmonized System of Classification and Labelling of Chemicals (GHS), Eleventh Revised Edition. ST/SG/AC.10/30/Rev.11.
- OECD (1998). OECD Principles on Good Laboratory Practice. OECD Series on Principles of Good Laboratory Practice and Compliance Monitoring No. 1. DOI: 10.1787/9789264078536-en.
