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Altogen Labs adheres to with the OECD Guideline for Testing of Chemicals No. 404, our protocols ensure compliance with integrated testing strategies that emphasize the use of existing data and in vitro methodologies to minimize unnecessary in vivo testing. Our testing framework incorporates the latest recommendations on sequential application of test substances and weight-of-evidence approaches, ensuring both scientific rigor and ethical responsibility in dermal irritation and corrosion assessments.
Initial Considerations
Before conducting in vivo testing, all relevant data pertaining to the potential dermal irritation and corrosivity of a test chemical are evaluated using a structured weight-of-evidence approach. This ensures that unnecessary animal testing is avoided whenever possible. Altogen Labs ensures the assessment process involves a thorough review of human data, in vivo studies, in vitro models, physicochemical properties, and computational predictive methods. The integration of these data sources allows for a comprehensive hazard assessment while prioritizing the reduction of live animal testing. If the available data remain inconclusive, further testing is conducted, beginning with validated in vitro models, followed by in vivo testing as a last resort. The implementation of this strategy minimizes the need for animal-based studies while ensuring that regulatory safety requirements are met.
Principle of the In Vivo Test
When in vivo testing is deemed necessary, the test substance is applied in a single dose to a designated area of an experimental rabbit’s skin. The untreated skin of the same rabbit serves as a control to allow for direct comparison. The test aims to determine the degree of irritation or corrosion by evaluating observable effects at specified time intervals following exposure. The study duration is designed to assess whether any observed effects are reversible or irreversible. If the rabbit exhibits persistent signs of severe distress or pain, it is humanely euthanized, and the test chemical is classified accordingly. These humane endpoints align with international standards for ethical animal research and ensure that undue suffering is prevented.
Preparations for the In Vivo Test
Prior to testing, careful selection of the animal model and preparation of the test site are performed to ensure reliable results. The albino rabbit is the preferred species for dermal irritation studies due to its well-characterized skin response and historical use in regulatory toxicology. Only healthy, young adult rabbits with intact skin is used for testing, and a scientific rationale is provided if an alternative species is selected. Approximately 24 hours before testing, the fur is removed from the dorsal area of the trunk using a precise clipping technique to avoid skin abrasion. Special consideration is given to seasonal variations in fur density, as areas with unusually dense hair growth may interfere with test substance application.
Environmental conditions are carefully controlled to maintain test validity. Rabbits are housed individually in temperature-controlled rooms set at 20°C with an allowable fluctuation of ±3°C. The relative humidity is maintained at a minimum of 30 percent and does not exceed 70 percent, with an optimal range between 50 and 60 percent. A strict light cycle of 12 hours light and 12 hours dark is maintained, and rabbits have unrestricted access to a conventional laboratory diet and drinking water.
Test Procedure
Application of Test Chemical
The test substance is applied to a six-square-centimeter area of shaved skin and covered with a semi-occlusive gauze patch secured with non-irritating tape. Depending on the physical characteristics of the test substance, different application methods are used. Liquid chemicals are typically applied undiluted, while solid and paste formulations are moistened with the minimal amount of water or another suitable vehicle to ensure uniform contact with the skin. If direct application is not feasible, the substance is first placed onto the gauze patch before being affixed to the test site. The patch remains in place for four hours, after which residual material is gently removed using water or an appropriate solvent.
Initial Test
At Altogen Labs when a chemical has been classified as corrosive, an irritant, or non-classified based on a comprehensive weight-of-evidence analysis or prior in vitro studies, additional in vivo testing is typically unnecessary. However, in cases where further data is deemed essential, an in vivo assessment is conducted using a stepwise approach with a single test subject.
Initially, up to three test patches are applied sequentially. The first patch is removed after three minutes; if no significant dermal reaction is observed, a second patch is applied to a different site and removed after one hour. If observations indicate that an extended exposure can be ethically permitted, a third patch is applied and removed after four hours, followed by response evaluation. If a corrosive effect is detected at any stage, the test is immediately terminated. In the absence of corrosion after the final patch removal, the test subject is monitored for an additional 14 days unless corrosive effects manifest earlier. For test substances anticipated to be non-corrosive but potentially irritating, a single patch is applied for a four-hour exposure to assess dermal response.
Confirmatory Test and Observation Period
If no corrosive effects are observed in the initial in vivo dermal irritation test, the response is confirmed using up to two additional animals, each exposed to a single patch for four hours. If an irritant effect is detected in the initial test, confirmation may proceed sequentially or simultaneously with two additional test subjects. In cases where no initial test is conducted, two or three animals may undergo a single four-hour exposure. If two animals exhibit the same response, further testing is unnecessary; otherwise, a third animal is assessed. Equivocal results may require additional testing.
To fully evaluate the reversibility of observed effects, the observation period extends up to 14 days post-exposure. However, the study is terminated immediately if an animal exhibits ongoing severe pain or distress. If full reversibility occurs before the 14-day mark, the experiment is concluded accordingly.
Irritation and Corrosion Assessment
Following exposure, erythema and edema formation are evaluated at 60 minutes and then at 24, 48, and 72 hours. If necessary, observations continue for up to 14 days to assess reversibility. The severity of erythema is graded from very slight redness to severe eschar formation, while edema formation is assessed based on the extent of swelling and its spread beyond the application site. In cases where the reaction is unclear, histopathological analysis is performed to differentiate between irritation and corrosion.
The classification of the test substance is based on the observed effects. If the skin exhibits irreversible damage, such as necrosis, ulceration, or significant scarring, the chemical is classified as corrosive. If the observed reactions are transient and resolve within 14 days, the test substance is classified as an irritant.
Sequential Testing and Confirmatory Assessments
If the initial in vivo test using a single animal does not indicate corrosive effects, the irritation potential is further assessed in up to two additional animals. Testing is performed sequentially or simultaneously based on the initial response. If an irritant reaction is observed, confirmatory testing involves exposing two additional animals under the same conditions. If both animals exhibit the same response, further testing is unnecessary. If results are inconsistent, an additional animal is included in the study to clarify the response. When uncertainty remains, additional animals is required to establish a definitive classification.
Data Reporting and Evaluation
A complete and detailed report is generated for each study conducted at Altogen Labs. Study results are summarized in tabular format, providing a comprehensive evaluation of all relevant parameters. The report includes a detailed justification for conducting in vivo testing, outlining the weight-of-evidence assessment of pre-existing data, in vitro findings, and regulatory considerations. A full characterization of the test chemical is provided, including chemical identity, structural composition, purity, solubility, and batch information. The report also details all experimental conditions, including test site preparation, exposure method, and patch application techniques.
All observed effects are systematically documented, including individual irritation and corrosion scores, lesion descriptions, and any systemic toxicity indicators. The classification of the test chemical is determined based on the severity and reversibility of the observed dermal effects. If responses such as alopecia, hyperkeratosis, hyperplasia, or persistent scaling are observed at the end of the 14-day period, the test substance is classified as an irritant. The irritancy threshold is established using OECD grading criteria for erythema and edema.
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