OECD TG 405: Scientific and Ethical Approach to the Assessment of Acute Eye Irritation and Corrosion
The eye is one of the body’s most sensitive and vulnerable organs when exposed to chemicals. Ocular tissues such as the cornea, iris, and conjunctiva can suffer irreversible damage upon chemical contact, leading to serious consequences that may even result in permanent vision loss. Therefore, determining the acute irritant and corrosive effects that chemicals, pesticides, cleaning products, and industrial formulations can have on the eye is an indispensable requirement for both user safety and GHS (Globally Harmonized System) classification. Our laboratory conducts acute eye irritation and corrosion tests in accordance with the OECD TG 405 guideline, using the most up-to-date scientific approaches and in full compliance with the 3R principles.
OECD TG 405: Test Principle and Scope
OECD TG 405 is an internationally recognized standard in vivo test protocol for evaluating the potential for acute irritation and corrosion that chemical substances and mixtures may cause to eye tissues. The basic principle of the test involves applying a specific amount of the test substance (typically 0.1 mL of liquid or 100 mg of solid) to the conjunctival sac of the test eye in a single application and grading the ocular reactions at specific time points (1, 24, 48, and 72 hours). The evaluation is based on four main parameters: corneal opacity, iris damage, conjunctival redness (erythema), and conjunctival edema (chemosis). Each parameter is scored on a scale from 0 (absent) to 4 (severe), and the time-dependent course of these scores is critical in determining whether the substance’s effects on the eye are reversible.
The most important feature of OECD TG 405 is the stepwise testing strategy that is required prior to initiating the test. This strategy was developed to prevent unnecessary use of animals and to protect animal welfare to the highest degree. The stepwise approach follows this sequence:
- Evaluation of available data: Human and animal data, structure-activity relationship (QSAR) analyses, physicochemical properties (particularly pH).
- pH screening: Substances with a pH ≤2 or ≥11.5 are directly classified as “Eye Corrosive” (Category 1) due to their strong acid/base characteristics, and in vivo testing is not performed.
- Validated in vitro / ex vivo tests: OECD TG 437 (BCOP – Bovine Corneal Opacity and Permeability Test), TG 438 (Isolated Chicken Eye Test), TG 491 (Short-Term Exposure In Vitro Test), or TG 492 (Reconstituted Human Cornea-Like Epithelial Test) are used to screen for the potential for corrosion and serious damage in advance.
- In vivo test: However, when the above steps are insufficient to definitively classify a substance’s potential for eye irritation, the in vivo OECD TG 405 test is performed as a last resort.
Evaluation Parameters and Scoring System
The table below details the four main ocular parameters evaluated under OECD TG 405, the scoring criteria, and the clinical significance of each score:
| Parameter | Score | Definition | Clinical Equivalent |
|---|---|---|---|
| Corneal Opacity | 0 | No opacity | Healthy, transparent cornea |
| 1 | Scattered or diffuse opacity; iris details are clearly visible | Mild cloudiness; vision is minimally affected | |
| 2 | Easily detectable semi-transparent area; iris details are slightly blurred | Moderate corneal damage | |
| 3 | Opaque areas; iris details are not visible; the pupil border is barely discernible | Severe corneal damage; recovery is unlikely | |
| 4 | Completely opaque cornea; neither the iris nor the pupil is visible | Corneal erosion, high likelihood of permanent damage | |
| Iris Damage | 0 | Normal | Healthy iris, normal pupillary reflex |
| 1 | Marked deep hyperemia, congestion, swelling, slowed pupillary reflex | Mild-to-moderate iritis, usually reversible | |
| 2 | Absence of light reflex, hemorrhage, marked structural damage | Severe iritis, risk of permanent damage to iris tissue | |
| Conjunctival Redness (Erythema) | 0 | Normal | Healthy, pale pink conjunctiva |
| 1 | Mild congestion, marked hyperemia in some vessels | Mild conjunctivitis | |
| 2 | Widespread dark red color; individual blood vessels are difficult to distinguish | Moderate conjunctival inflammation | |
| 3 | Pronounced, widespread, and intense dark red color | Severe conjunctivitis, marked inflammatory response | |
| Conjunctival Edema (Chemosis) | 0 | No edema | Normal conjunctiva |
| 1 | Mild swelling slightly above normal | Mild chemosis | |
| 2 | Prominent swelling accompanied by partial eversion of the eyelids | Moderate edema, mild impairment of eyelid function | |
| 3 | Severe swelling causing the eyelids to be half-closed | Severe chemosis, may cause the eyelids to close | |
| 4 | Extreme swelling causing the eyelids to close completely | Very severe edema; the eye is completely closed; requires emergency intervention |
GHS Classification and Interpretation
OECD TG 405 results are classified into three main categories according to the Globally Harmonized System (GHS) criteria. The table below summarizes these classification criteria and their regulatory implications:
| GHS Category | Classification | Criteria | Labeling | Precautionary Statements |
|---|---|---|---|---|
| Category 1 | Serious Eye Damage / Eye Corrosive | Irreversible ocular tissue damage or permanent impairment of the cornea, iris, or conjunctiva in at least one test subject within 21 days | Pictogram: Corrosive (GHS05), Signal Word: Danger, H318 | P280 (protective goggles/face shield), P305+P351+P338, P310 |
| Category 2A | Eye Irritant | Positive ocular reactions in at least two test subjects, fully reversible within 21 days | Piktogram: Exclamation mark (GHS07), Warning: Caution, H319 | P264, P280, P305+P351+P338, P337+P313 |
| Category 2B | Mild Eye Irritant | Mild and transient effects that do not fully meet Category 2A criteria but resolve completely within 7 days | Pictogram: None, Warning: Caution, H320 (in some regulations) | Depends on regional regulations; some authorities do not use this category |
| Not classified | Not Irritating | No adverse ocular reactions are observed, or the effects are too mild to fall into the categories above | Pictogram: None, Warning: None | GHS label not required |
In our laboratory, we follow the OECD TG 405 Implementation Strategy and the 3R Principles
When planning OECD TG 405 studies in our GLP-certified laboratory, we apply the 3R principles (Replacement, Reduction, Refinement) to the highest degree. Our testing strategy follows the stepwise approach outlined below:
| Stage | Method | Objective | 3R Contribution |
|---|---|---|---|
| 1. Data Screening | Existing human/animal data, QSAR, read-across, in silico prediction | If classification is possible based on existing information, no testing is required | Replacement – complete avoidance of animal testing |
| 2. pH and Physicochemical Assessment | pH measurement, buffering capacity, acid/base reserve | Direct Category 1 classification in cases of extreme pH (≤2 or ≥11.5) | Replacement – no animal testing for corrosive substances |
| 3. In Vitro / Ex Vivo Screening | OECD TG 437 (BCOP), TG 438 (ICE), TG 491 (STE), TG 492 (RhCE) | Determination of corrosion and severe damage potential without the use of animals | Replacement – in vivo testing requirements are significantly reduced |
| 4. In Vivo Validation (Last Resort) | OECD TG 405 – start with a single subject, add additional subjects based on results | Only when definitive classification cannot be achieved with previous steps | Reduction – minimal animal use through a stepwise testing design; Refinement – analgesia and close monitoring |
| 5. Classification and Reporting | Final classification according to GHS criteria, integrated data interpretation | A definitive decision is made by combining data from all stages | The most accurate classification using the Weight of Evidence (WoE) approach |
In our laboratory, in vivo when the OECD TG 405 test is unavoidable, we initiate the test with a single subject and include additional subjects only when necessary, based on the results. This sequential test design ensures scientific accuracy while keeping the number of animals to a minimum. In addition, experienced veterinarians continuously monitor the test throughout its duration; in the event of a severe reaction, the test is immediately terminated, and appropriate analgesic treatment is administered.
Frequently Asked Questions
| Question | Answer |
|---|---|
| For which types of products is the OECD TG 405 test mandatory? | It may be required under GHS/CLP classification and REACH registration for chemicals, pesticides, biocidal products, cleaning agents, industrial chemicals, and all mixtures posing a risk of eye contact. Since animal testing is banned for cosmetics in the EU, in vitro alternatives are mandatory. |
| Is it necessary to conduct TG 405 testing for a substance with an extreme pH value? | No. Substances with a pH ≤2 or ≥11.5 are classified directly as “Serious Eye Damage / Corrosive” (Category 1) without applying OECD TG 405. This approach has been mandated by the OECD to prevent unnecessary use of animals. |
| Can in vitro test results serve as a substitute for OECD TG 405? | Yes, in many cases they can. In particular, validated in vitro tests such as BCOP (TG 437) and RhCE (TG 492) may be sufficient for classifying a substance as “Category 1” (corrosive/serious eye damage) or “non-Category 2” (non-irritating). However, in some cases, an in vivo test may be required to distinguish between Category 2A and 2B distinction, an in vivo test may be required in some cases. |
| How long does the test take? | The in vivo OECD TG 405 test takes approximately 3–4 weeks, including an observation period that can last up to 21 days. In vitro alternative tests, on the other hand, typically yield results within 1–2 weeks. Including reporting and quality assurance review, the total duration is 4–6 weeks. |
| In which regulatory submissions are the test results used? | They are used for chemical safety assessments, Safety Data Sheet (SDS) preparation, and labeling under the EU REACH (Annexes VII/VIII), CLP Regulation (No. 1272/2008), U.S. EPA FIFRA, TSCA, and Turkish KKDIK and SEA regulations. |
| What documents must be provided to the laboratory prior to testing? | The safety data sheet (SDS) for the test substance, its physicochemical properties (pH, solubility), available toxicity data, chemical structure, and purity information, as well as any previously conducted in vitro eye irritation test results, if available, are required. |
The eyes are our most vulnerable organs against chemical hazards, and scientifically demonstrating this sensitivity should be a priority for every responsible manufacturer. Our laboratory determines the ocular safety profile of your products in accordance with the highest scientific standards and in full compliance with the 3R principles, using OECD TG 405 and complementary in vitro alternative tests. For your eye irritation and corrosion assessment needs, contact our GLP-certified laboratory, and let’s plan your customized testing strategy together.
