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Altogen Labs conducts acute inhalation toxicity studies in alignment with OECD Test Guideline No. 403, ensuring compliance with international regulatory frameworks and scientific best practices. Altogen Labs test procedures evaluate the potential health risks associated with airborne chemical exposures. These studies provide critical data for regulatory compliance, industrial safety assessments, and emergency response planning. The laboratory employs advanced inhalation exposure systems, precise analytical techniques, and a comprehensive approach to data collection and interpretation to ensure accurate and reproducible results. Two primary study methodologies are available: the Traditional LC50 protocol, which determines the median lethal concentration (LC50) following a fixed-duration exposure, and the Concentration x Time (C x t) protocol, which assesses toxicity across multiple exposure durations to establish concentration-time relationships during studies with animal testing over multiple time durations.
Initial Considerations
Before conducting inhalation toxicity testing at Altogen Labs, all available information on the test substance is evaluated to determine whether additional in vivo testing is necessary. Existing data from structurally related compounds, in vitro studies, and computational models are reviewed to minimize unnecessary animal testing. Consideration is given to the identity, chemical structure, and physicochemical properties of the test substance, as well as anticipated human exposure scenarios.
Principle of the Test
Acute inhalation toxicity testing at Altogen Labs is designed to establish dose-response relationships for airborne substances by exposing test animals to controlled concentrations and assessing adverse health effects. The study determines key toxicological parameters such as LC50, LC01, and mortality slope, while also identifying potential sex-based differences in sensitivity. The methodology is designed to balance regulatory requirements with ethical considerations, ensuring that testing is performed only when alternative data sources, including in vitro models, computational predictions, and structurally related compounds, are insufficient. Studies are conducted under strict quality control measures to ensure reliability, and all test conditions, exposure concentrations, and environmental factors are carefully monitored to maintain consistency. Animals that are moribund, in evident pain, or experiencing severe and persistent distress are humanely euthanized. These animals are treated the same as those that die during testing when interpreting results.
Description of the Method
Selection of Animal Species and Preparation of Animals
At Altogen Labs, rats are the preferred test species due to their well-characterized respiratory physiology, historical toxicological data, and widespread regulatory acceptance. When an alternative species is required, a scientific justification must be provided. Selected animals must be healthy young adults, aged between 8 to 12 weeks, with body weights within ±20% of the sex-specific mean. They are housed in controlled environments and are observed daily for any pre-existing health conditions. Randomization is performed to ensure unbiased group assignments.
Animal Husbandry
Animals are maintained in an environment with a temperature at 22±3°C, relative humidity between 30 and 70 percent, and a 12-hour light/dark cycle. They receive a standardized laboratory diet and unrestricted access to drinking water, except during the exposure period. If nose-only exposure is used, animals may be acclimated to restraining tubes before testing to minimize stress and ensure procedural consistency.
Inhalation Chambers
Altogen Labs utilizes advanced inhalation exposure systems selected based on the physicochemical properties of the test substance and the specific study objectives. Nose-only exposure is preferred for liquid or solid aerosols and vapors that may condense to form aerosols, as it limits dermal and oral exposure and ensures targeted delivery to the respiratory system. Whole-body exposure may be used when systemic effects are relevant or when required for specific regulatory applications. Altogen Labs whole-body exposure chambers are designed to maintain a stable test atmosphere, with the total volume of animals limited to five percent of the chamber volume to ensure uniform exposure conditions. Airflow dynamics are optimized to prevent rebreathing of exhaled air, and careful control measures are implemented to maintain consistent test article distribution throughout the exposure period.
Exposure Conditions
Administration of Concentrations
Test substances are administered in the form of gases, vapors, aerosols, or mixtures, depending on their physical properties and anticipated human exposure scenarios. At Altogen Labs, nose-only exposure in rats can last up to 6 hours, while in mice, it is typically limited to 4 hours unless a longer duration is scientifically justified. When animals are exposed to aerosols in whole-body chambers, they are housed individually to prevent ingestion of the test substance through grooming of cage mates. Food is withheld during exposure, but water is provided throughout whole-body exposure.
Animals are exposed to the test substance in the form of gas, vapor, aerosol, or a combination of these. Altogen Labs’ tested physical state is determined by the test substance’s physicochemical properties, chosen concentration, and the form most likely encountered during handling and use. Hygroscopic and chemically reactive substances are tested under dry air conditions, with precautions taken to prevent the formation of explosive concentrations.
Particle-Size Distribution
Particle size distribution is analyzed at least twice during each exposure session using cascade impactors or aerodynamic particle sizers. If vapors are suspected to condense into aerosols, additional assessments are performed to determine the mixed-phase distribution and ensure consistency in inhalation exposure. Altogen Labs’ aerosolized test articles meet strict particle size criteria to ensure proper deposition within the respiratory tract. The mass median aerodynamic diameter (MMAD) is maintained between 1 and 4 micrometers, with a geometric standard deviation between 1.5 and 3.0.
Test Article Preparation in a Vehicle
When a vehicle is needed to generate an appropriate test atmosphere, water is preferred whenever possible. Altogen Labs ensures that any mechanical processing used to achieve the required particle size must not alter the test substance’s chemical composition. In cases where granular materials are formulated to be non-inhalable, attrition testing may be required to determine whether respirable particles are generated before proceeding with inhalation toxicity testing.
Control Animals
At Altogen Labs, concurrent negative control group exposed only to air is generally unnecessary unless specifically required for a study. When a vehicle is used, a vehicle control group is only included if historical data on the vehicle’s inhalation toxicity are unavailable.
Monitoring of Exposure Conditions
Chamber Airflow
Chamber airflow is continuously monitored to ensure stable exposure conditions. Special precautions are taken in nose-only exposures to prevent rebreathing of exhaled air, and oxygen concentrations are maintained at a minimum of 19 percent, while carbon dioxide concentrations are kept below one percent. Altogen Labs ensures the oxygen concentrations are measured if there is reason to believe that these standards cannot be met.
Chamber Temperature and Relative Humidity
Chamber temperature is maintained at 22±3°C, with humidity monitored at least three times per four-hour exposure session. Adjustments are made to account for test substances that interfere with humidity measurements.
Test Article: Nominal and Actual Concentrations
The nominal concentration of the test article, calculated as the total mass of the substance divided by the chamber airflow, provides an estimate of generation efficiency but is not used for exposure characterization. Actual concentrations are determined using validated analytical methods, including spectrometry, chromatography, and gravimetric techniques, ensuring precise quantification of exposure levels. To ensure the reliability of the test system, Altogen Labs calibrates all analytical instruments regularly and follows stringent quality control procedures. Deviations from expected concentrations are documented and assessed to determine potential impacts on study outcomes.
Test Article: Particle Size Distribution
Aerosols and condensable vapors are analyzed to ensure that respirable fractions meet defined particle size criteria. Any deviations from target values must be justified based on expert evaluation of the test article properties and exposure conditions. A vehicle may be used to achieve the desired concentration and particle size of the test substance in the atmosphere, with water being the preferred choice whenever possible. Particulate materials can undergo mechanical processing to attain the required particle size distribution, but care must be taken to prevent decomposition or alteration of the test substance. If mechanical processing is suspected to have changed the composition, such as through excessive heat from milling, analytical verification is conducted. At Altogen Labs, precautions are also taken to avoid contamination of the test material. Non-friable granular materials specifically designed to be non-inhalable are not tested as they do not require testing. However, an attrition test is conducted to confirm that respirable particles are not generated during handling. If respirable particles are detected, an inhalation toxicity study is performed.
Procedure
Traditional Protocol
General Considerations
At Altogen Labs, the Traditional LC50 protocol evaluates the acute toxicity of a test substance by exposing animals to a fixed concentration for a predetermined duration. This method provides robust data for regulatory classification and risk assessment. The study may include a sighting study, a limit test, and a main study, depending on the available data and study objectives. The Traditional protocol is ideal when precise LC50 values are needed for classification under regulatory guidelines.
Sighting Study
The sighting study is a preliminary phase used to estimate test substance potency and assess potential sex-based differences in susceptibility. In this phase, a small number of animals, typically one to three per sex, are exposed to a single concentration for four hours. If no mortality occurs, a higher concentration may be tested. If severe toxicity is observed, lower concentrations are selected for subsequent testing. This phase helps refine dose selection for the main study and reduces unnecessary animal use.
Limit Test
The limit test is conducted when a test substance is expected to have low acute toxicity. For this study at Altogen Labs, a single group of three males and three females is exposed to a predefined regulatory limit concentration, such as 5 mg/L for aerosols under the GHS Classification System. If no mortality occurs, no further testing is required, and the substance is classified as having low acute inhalation toxicity. If mortality is observed, a full main study is conducted to determine precise dose-response relationships.
Main Study
The main study is conducted when the sighting study or limit test indicates significant toxicity. This phase involves exposing multiple groups of animals, typically five per sex, to at least three different concentrations in a stepwise approach. The objective is to generate LC50 and LC01 values, establish the dose-response curve, and characterize the toxicological profile of the test substance.
Test concentrations are selected to cover a range from non-lethal to lethal doses, ensuring an accurate statistical modeling of toxicity thresholds. The exposure is performed under controlled environmental conditions, with continuous monitoring of temperature, humidity, chamber airflow, and test article concentration. The study design allows for the identification of concentration-dependent trends, including delayed onset of toxicity, time-to-death relationships, and potential sex-specific differences in susceptibility.
Post-exposure, animals are observed for 14 days to document delayed toxicity effects. Clinical signs such as labored breathing, ataxia, tremors, or convulsions are recorded. Moribund animals or those in severe distress are euthanized, and necropsy is performed to assess gross pathological changes, particularly in the respiratory tract. Histopathological evaluation may be conducted to further investigate tissue damage and identify potential systemic toxicity. Data from this phase are used to determine the LC50, slope of the dose-response curve, and potential target organ effects.
Concentration x Time (C x t) Protocol
General Considerations
The Concentration x Time (C x t) protocol is designed to assess the time-dependent toxicity of inhaled substances. This method is particularly useful for deriving acute exposure thresholds, such as those needed for emergency response planning and land-use assessments. Unlike the Traditional LC50 protocol, which focuses on a single fixed-duration exposure, the C x t protocol examines how toxicity varies across multiple exposure durations.
Sighting Study
The sighting study in the C x t protocol is used to estimate test substance potency and guide selection of exposure concentrations and durations for the main study. Typically, one to three animals per sex are exposed to a single concentration for 240 minutes. If mortality occurs, lower concentrations or shorter durations are selected for subsequent testing. If no toxicity is observed, a higher concentration may be tested. This phase helps refine dose selection and minimizes unnecessary animal use.
Initial Concentration Testing
The first exposure session, tests a single concentration over multiple durations. Groups of one animal per sex are exposed for multiple durations at the initial concentration level, for a total of 10 animals. This approach provides preliminary concentration-response data and helps determine the exposure conditions for subsequent testing. The primary goal of this phase is to establish the time-dependent toxicity profile of the test substance.
Main Study
The main study expands upon the data generated in Exposure Session I by testing additional concentrations across multiple durations. Typically, three to five different concentrations are tested using a geometric spacing approach, where each concentration is tested at multiple time points. The study aims to derive LC50 values for different time durations and to model the concentration-time-mortality relationship.
Each test group consists of animals exposed to a fixed concentration for varying durations, allowing researchers to compare the effects of short-term high-concentration exposures versus longer-term low-concentration exposures. This method enables the development of predictive models for acute toxicity under different exposure scenarios, which are crucial for establishing safety limits in occupational and environmental health settings.
Animals are monitored throughout exposure and for 14 days post-exposure. Clinical signs are carefully recorded, with emphasis on respiratory distress, central nervous system impairment, and behavioral changes. The time-to-death data are analyzed in relation to exposure concentration and duration, providing a detailed understanding of the kinetics of toxicity onset.
Post-mortem examinations include gross necropsy and histopathological evaluation of respiratory tissues. The lungs are examined for signs of edema, hemorrhage, inflammation, or structural damage. If necessary, other organs such as the liver, kidneys, and brain may also be assessed to determine potential systemic effects. The collected data are statistically analyzed to derive the concentration-time-response relationship, which is then used to establish safe exposure limits and hazard classifications.
Observations
Body weights are recorded before exposure, on post-exposure days 1, 3, and 7, and weekly thereafter. A sustained weight loss of more than 20 percent triggers additional monitoring. All Altogen Labs test animals undergo gross necropsy, with a focus on respiratory tract examination. Histopathological evaluation may be conducted in cases of suspected irritation or systemic toxicity. Lung weights may be measured as an additional parameter to assess pulmonary effects.
Data and Reporting
Data Collection and Analysis
Altogen Labs study data, including animal observations, mortality rates, body weights, and necropsy findings, are systematically recorded. Clinical signs are monitored throughout the exposure period and post-exposure observation phase. Airflow rates, temperature, humidity, and test atmosphere concentration data are continuously recorded to ensure stable and reproducible exposure conditions. The collected data are analyzed using validated statistical methods to determine LC50 and LC01 values. When applicable, confidence intervals and dose-response slopes are calculated to provide a comprehensive assessment of toxicity.
Test Report and Study Documentation
The final study report at Altogen Labs includes detailed information on all aspects of the study, ensuring full transparency and reproducibility. It includes a description of test article characterization, exposure concentrations, environmental conditions, and test methodology. Toxicological findings, including mortality rates, clinical observations, and pathological findings, are presented in a structured format. Regulatory interpretation is provided based on the observed toxicity profile, allowing for proper classification of the test substance. The report also includes a discussion and interpretation of results as well as records of any deviations from standard protocols, along with a justification for such modifications.
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