急性吸入毒性试验

经合组织TG 403、经合组织TG 433和经合组织TG 436

急性吸入毒性:呼吸危害的科学性和规范性评估

吸入是化学接触的重要途径,因为气体,蒸汽,气溶胶,尘埃和雾能与呼吸道直接相互作用. 根据试验品的物理化学性质,沉积规律,溶解性,活性与清除等不同,吸入接触可能造成局部呼吸道效应,系统毒性,或两者兼而有之. 因此,当吸入是一种相关的接触途径时,即使用急性吸入毒性研究,而现有的信息、替代方法、重述或接受的豁免无法充分满足适用的监管数据要求。

选择适当的急性吸入试验方法应具有科学上的合理性,并应反映管制目标、可获得的毒性信息、预期的人类接触、试验物品的物理形式、产生适当大气的技术可行性以及更换、减少和完善的原则(3R)。 目前的经合组织TG 403特别强调,在进行传统的TG 403研究之前,应考虑现有资料和更精细的急性吸入方法。 如果分类和标签是主要监管目标,经合组织TG 436一般由TG 403推荐,但需符合接收当局的需要。

急性吸入毒性测试的科学依据

急性吸入毒性测试评估了短期吸入后产生的不利影响。 相关终点取决于选定的经合组织试验准则。 精确的LC50或其他定量浓度反应估计主要与经合组织TG 403有关。 经合组织TG 433取而代之的是使用明显的毒性作为关键决定点,而经合组织TG 436则采用分步骤的急性毒性类程序,在这种程序中,死亡率或死亡决定了预定浓度水平之间的进展。

被吸入的物质不一定直接而迅速地从活体进入系统循环。 呼吸道沉降和吸收取决于空气动力粒子大小、蒸汽压力、水和脂溶性、活性、区域沉降、黏膜清除和高活性清除等因素。 因此,应把急性吸入研究解释为局部呼吸道毒性和系统毒性,而不是纯粹的系统毒性试验。

经合组织急性吸入试验准则:正确的监管区分

经合组织 TG 403 - 急性吸入毒性

经合组织TG 403包含两种不同的研究设计:传统LC50协议和浓度×时间(C×t)协议. 传统议定书一般使用固定的接触期,通常为4小时,在主要研究中至少使用3个浓度级. 一项主要研究通常是每浓度有5名男性和5名女性,或者在知道这种易感性时有5名较易发生性行为的动物。 该设计旨在产生强大的定量集中-反应关系,并可能提供LC50和其他有置信限度的致死性估计.

C×t协议也是经合组织TG 403的一部分. 它暴露出动物的多浓度和多接触期,通常使用只靠鼻子的系统. 如果存在管理或科学上的需求,对不同接触期的毒性进行定性,例如在应急反应或土地使用规划中,可以选择这种办法。 这不是经合组织TG 436程序。

经合组织 TG 433 - 急性吸入毒性:固定浓度程序

经合组织TG 433是固定浓度程序。 其核心改进是使用明显的毒性,而不是死亡或死亡作为预定终点,同时避免预期会导致致死或明显疼痛和痛苦的浓度。 该程序是分步骤的,使用预定义的与气体、蒸汽或气溶胶相适应的固定浓度。 一项目击研究可用来确定最敏感的性行为和适当的起始浓度。 主要研究通常使用被调查的每个浓度级的五只一性动物,此外还有目击研究中使用的动物.

建议进行4小时的接触,但可能需要其他时间,以满足具体的管理要求。 该方法主要是为了支持急性毒性危害评估和分类,而不是得出LC50的精确分数估计.

经合组织 TG 436 – 急性吸入毒性:急性毒性分类方法

经合组织TG 436是急性毒性类别方法。 这是一个分步骤的分类程序,每个步骤使用三种性别的动物,以鼠为首选物种. 动物接触预定义浓度达4小时,从与全球统一制度急性吸入毒性类别相对应的固定水平中选择。 与试验品有关的死亡或死亡的发生与否决定了下一步。

因此,TG 436不是一个浓度x时间方法,没有使用明显的毒性作为其主要决定终点。 其主要目的是使用比经典定量LC50设计更少的动物进行危险分类.

经合组织急性吸入方法比较

试验大气的生成和特性

吸入大气的产生和特性是研究有效性的核心。 所测试的物理形态应反映试验物品的物理化学特性,并酌情反映在处理或使用过程中可能遇到的形态。 气体和蒸汽可能由受控制的稀释或蒸发所产生,而粉尘和液体气溶胶则需要能够产生稳定和可呼吸大气的适当生成系统。

应在动物呼吸区使用经适当验证或科学合理的分析方法确定试验品的实际浓度。 就经合组织TG 403而言,接触大气应尽可能保持恒定。 如果在4小时的研究中采用断断续续的取样方法,一般预计至少有两个试验室样品,如果观察到有很大的可变性,则会有更多的取样方法。 气体和蒸汽的单个浓度样本一般不应偏离平均室内浓度的±10%,液体或固体气溶胶的±20%。

For OECD TG 433, actual concentration is likewise measured in the breathing zone. When intermittent sampling is used, the guideline specifies at least five samples at approximately hourly intervals where feasible. The same ±10% criterion for gases/vapours and ±20% criterion for liquid/solid aerosols applies to individual chamber concentration samples relative to the mean.

Aerosol Particle-Size Characterization

Particle-size characterization should be expressed using aerodynamic parameters, not simply as the percentage of particles in a nominal size band. Under OECD TG 403, aerosols with a mass median aerodynamic diameter (MMAD) of 1-4 µm and a geometric standard deviation (GSD) of 1.5-3.0 are recommended to permit exposure of relevant regions of the respiratory tract. Particle-size distribution should be determined at least twice during each 4-hour exposure.

Under OECD TG 433, an aerosol MMAD of ≤4 µm with a GSD of 1.0-3.0 is recommended, and particle-size distribution should also be determined at least twice during each 4-hour exposure. Any scientifically justified deviation from the recommended particle-size range should be explained in the study documentation.

Exposure Systems: Nose/Head-Only and Whole-Body

Both nose/head-only and whole-body exposure systems may be appropriate depending on the test article, study design and regulatory objective. Nose/head-only systems can reduce non-inhalation exposure and generally provide tighter control over the inhaled atmosphere, but restraint can introduce stress and requires careful acclimatization and monitoring. Whole-body systems avoid restraint but may increase test-article consumption and can permit additional dermal exposure or oral exposure through grooming after deposition on the fur.

Accordingly, nose-only exposure should not be described as completely preventing dermal or ocular exposure. A more accurate statement is that it minimizes exposure by non-inhalation routes.

GHS and EU CLP Classification of Acute Inhalation Toxicity

Acute inhalation toxicity classification depends on the physical form of the test material and the 4-hour acute toxicity estimate (ATE) or equivalent LC50 information. For EU CLP, Categories 1-4 are used. The UN GHS also includes an optional Category 5, but Category 5 is not an acute toxicity category under EU CLP.

Note: The numerical criteria shown are the EU CLP acute inhalation toxicity categories for 4-hour exposure. For other jurisdictions, the applicable adopted GHS edition and national/regional implementation should be checked.

Conduct of a Regulatory Acute Inhalation Study

A scientifically defensible acute inhalation program normally begins with review of all available information, including test-item identity and composition, purity, physicochemical properties, volatility, particle-size characteristics where relevant, flammability/explosibility, existing toxicology data, anticipated use and exposure, and the analytical method for atmosphere measurement.

Technical feasibility should be demonstrated before animal exposure. This includes generation of the intended atmosphere, confirmation of stability and homogeneity, appropriate sampling at the animals’ breathing zone, and verification that environmental conditions such as airflow, temperature, relative humidity, oxygen and carbon dioxide remain within acceptable ranges.

During and after exposure, animals are assessed for mortality/moribundity and clinical signs of toxicity according to the selected guideline. Body weights are recorded at specified intervals. Post-exposure observation is generally continued for approximately 14 days, with the exact duration adjusted when justified by the onset, persistence or recovery of toxic effects. All animals are subjected to gross necropsy as required by the applicable guideline; histopathological examination may be appropriate when indicated by lesions, study objectives or regulatory requirements.

For regulatory submissions, the study should be performed under the applicable Good Laboratory Practice (GLP) framework when required by the receiving authority. The final report should clearly distinguish target, nominal and actual concentrations, provide the atmosphere-generation and analytical methods, document particle-size data for aerosols, report individual-animal observations and body weights, describe necropsy findings, and provide the statistical analysis appropriate to the selected study design.

常见问题

Do all gases, vapours, aerosols, dusts or volatile liquids automatically require an acute inhalation study?

No. The need for testing depends on the applicable regulatory framework, the expected route of human exposure, available acute toxicity information, physicochemical properties, and whether accepted adaptations, read-across, alternative methods or waivers can address the endpoint. Testing should not be presented as universally mandatory for every inhalable material.

Which OECD guideline should be selected when only GHS classification and labelling are required?

The current OECD TG 403 notes that TG 436 is generally recommended when information for classification and labelling is the principal need. TG 433 may also be appropriate where the Fixed Concentration Procedure and its evident-toxicity endpoint meet the regulatory objective. The final choice should be justified against the requirements of the receiving authority.

When is OECD TG 403 appropriate?

TG 403 is appropriate when a quantitative concentration-response relationship, a point estimate such as LC50/LCx, or concentration-time information over multiple exposure durations is scientifically or regulatorily required. TG 403 should not be used automatically when a more refined approach can satisfy the data requirement.

Can a precise LC50 be calculated from TG 433 or TG 436?

These methods are principally classification procedures and are not designed to generate the same precise point estimate of LC50 as a conventional TG 403 quantitative design. They provide information sufficient to assign or bracket acute inhalation hazard categories according to their decision schemes.

How long does an acute inhalation study take?

The animal phase normally includes the exposure followed by an approximately 14-day observation period. Total project duration depends on test-item characterization, analytical method readiness, atmosphere-generation feasibility, study scheduling, pathology requirements, data review, QA review and final reporting. A fixed universal turnaround time should therefore not be stated without study-specific confirmation.

Regulatory Conclusion

Acute inhalation toxicity assessment should be designed around the actual regulatory question rather than a single default method. OECD TG 403 provides quantitative Traditional LC50 and C × t options; OECD TG 433 provides a Fixed Concentration Procedure centred on evident toxicity; and OECD TG 436 provides an Acute Toxic Class procedure based on predefined concentrations and mortality/moribundity. Correct method selection, technically controlled atmosphere generation, guideline-compliant aerosol characterization and accurate interpretation of GHS/CLP criteria are essential for a scientifically valid and regulatorily acceptable study.

Authoritative References

1. OECD (2024). Test No. 403: Acute Inhalation Toxicity. OECD Guidelines for the Testing of Chemicals, Section 4. Corrected 25 June 2024. DOI: 10.1787/9789264070608-en. 链接
2. OECD (2018). Test No. 433: Acute Inhalation Toxicity: Fixed Concentration Procedure. OECD Guidelines for the Testing of Chemicals, Section 4. DOI: 10.1787/9789264284166-en. 链接
3. OECD (2009). Test No. 436: Acute Inhalation Toxicity – Acute Toxic Class Method. OECD Guidelines for the Testing of Chemicals, Section 4. DOI: 10.1787/9789264076037-en. 链接
4. OECD (2018). Guidance Document on Inhalation Toxicity Studies – Second Edition. OECD Series on Testing and Assessment No. 39. DOI: 10.1787/fba59116-en. 链接
5. European Parliament and Council. Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures (CLP), Annex I, Section 3.1 – Acute Toxicity. 链接
6. United Nations Economic Commission for Europe (UNECE). Globally Harmonized System of Classification and Labelling of Chemicals (GHS), 11th revised edition. 链接