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Altogen Labs provides acute oral toxicity testing per OECD Test Guideline No. 425, utilizing the Up-and-Down Procedure (UDP). Our test procedure provides a statistically robust method for estimating the acute oral toxicity of chemical substances while minimizing the number of test animals required. In addition to an estimation of LD50 and confidence intervals, the UDP is particularly advantageous for evaluating substances that produce observable toxicity, allowing for a flexible dose selection process that maximizes efficiency in data collection. The methodology is designed to provide accurate data for hazard classification under the Globally Harmonized System (GHS) and to support regulatory compliance in toxicity risk assessment.
Before initiating any study at Altogen Labs, we evaluate all available information on the test substance, including its physical chemical properties, toxicological data from structurally related substances, results of other in vitro or in vivo toxicology tests, and the anticipated use of the substance. This initial assessment is essential for determining the relevance of the test for human health and environment protection, as well as, most appropriate starting dose to select the best approach for toxicity testing. If no preliminary information is available to estimate the LD50 or dose-response curve, our computer simulations indicate that starting around 175 mg/kg with half-log units (equivalent to a dose progression factor of 3.2) between doses provides the best results. However, for highly toxic substances, the starting dose is adjusted accordingly. The half-log spacing improves the efficiency of animal use and enhances the accuracy of the LD50 prediction. Since the method is biased toward the starting dose, Altogen Labs ensures that initial dosing is always conducted below the estimated LD50 to prevent unnecessary suffering. For substances with high variability, where lethal dose estimates may exhibit significant statistical error, our testing protocol includes a stopping rule that is based on statistical properties of the estimate rather than a fixed number of test observations.
Our UDP testing approach is most applicable to substances that cause mortality within one or two days, and it is not suitable for compounds that may lead to delayed death (five days or longer). We utilize computer-assisted calculations to determine dosing sequences and final LD50 estimates on an animal-by-animal basis. Substances expected to cause severe pain or distress due to corrosive or highly irritating properties are not administered. If an animal becomes moribund or exhibits clear signs of severe distress, it is humanely euthanized, and its data is included in the test results just as it would be for animals that die during testing. Altogen Labs also utilizes a limit test to identify chemicals with low toxicity potential efficiently.
Principles of the Limit Test and Main Test
At Altogen Labs, we employ two primary approaches for acute oral toxicity assessment: the limit test and the main test. The limit test is conducted when preliminary data suggest that the substance is of low toxicity. A single animal is initially dosed at 2000 mg/kg, and if it survives, up to four additional animals are tested sequentially. If three or more animals survive, the LD50 is concluded to be above 2000 mg/kg, and further testing is unnecessary. If three or more animals die, the main test must be conducted to refine the LD50 estimate. In situations requiring a limit test at 5000 mg/kg due to regulatory requirements, Altogen Labs implements additional safeguards to ensure animal welfare. The likelihood of accurately classifying a compound diminishes as the actual LD50 approaches the limit dose.
The main test employs a sequential dosing strategy in which animals are dosed one at a time at 48-hour intervals, with each subsequent dose determined based on the survival of the previous animal. The first animal receives a dose slightly below the estimated LD50. If the animal survives, the dose is increased by a factor of 3.2 the original dose. If the animal dies, the next dose is reduced accordingly by a similar progression. The test is terminated when specific stopping criteria are met, including three consecutive survivals at the highest tested dose, five reversals in response patterns within six consecutive animals, or 4 animals have been tested after the initial reversal, and the specified likelihood ratios surpassed the critical value. Once testing concludes, the LD50 is calculated using the maximum likelihood estimation method, ensuring statistical robustness. Where possible, Altogen Labs derives confidence intervals using profile-likelihood methods to quantify uncertainty in the LD50 estimate.
Description of Method
Animal Model Selection and Housing Conditions
The preferred test species for acute oral toxicity studies at Altogen Labs is the rat, although other rodent species may be used when necessary. Female rats are typically selected due to their slightly greater sensitivity to toxicants compared to males. However, if existing toxicokinetic data indicate that males exhibit a higher susceptibility, then males may be used with appropriate justification. All animals must be healthy young adults, aged between 8 and 12 weeks, with body weights within ±20% of the mean initial weight of the study cohort.
We house animals under standardized conditions to maintain consistency across studies. The environmental parameters include a temperature range of 22°C ± 3°C, relative humidity maintained at 50–60%, and a 12-hour light/dark cycle. Conventional rodent laboratory diets and an unlimited supply of drinking water are provided to ensure adequate nutrition and hydration.
Dose Preparation
At Altogen Labs, we prepare dosing formulations immediately prior to administration unless stability data confirms the acceptability of prolonged storage. Whenever possible, we prioritize the use of aqueous solutions, suspensions, or emulsions as vehicles for administration. If aqueous formulations are unsuitable, we consider oil-based preparations, such as corn oil solutions. We administer the test substance in a constant volume across all dose levels by adjusting the concentration of the preparation. In rodents, the maximum dose volume does not exceed 1 mL per 100 g of body weight, except for aqueous solutions, which may be administered at up to 2 mL per 100 g.
Procedure
Dose Administration
Altogen Labs administers the test substance via oral gavage using a stomach tube or suitable intubation cannula to ensure accurate delivery. Prior to dosing, we ensure that animals undergo a fasting period to standardize gastrointestinal absorption, food but not water withheld overnight for rats and food but not water withheld for three to four hours for mice. Following fasting administration, animals are weighed, test substance administered, and food remains restricted for an additional three to four hours in rats or one to two hours in mice. If the test substance must be administered in fractions due to solubility limitations, the total dose is completed within a 24-hour period.
Limit Test
Altogen Labs utilizes the limit test in situations where prior knowledge suggests that the test substance is unlikely to be highly nontoxic. We refer to toxicity information for the test material, derived from data on similar tested compounds, mixtures, or products, considering the identity and concentration of components known to be toxicologically significant. In cases where limited or no toxicity information is available, or if the test material is expected to be toxic, we conduct the main test.
At Altogen Labs, the procedure begins by dosing a single animal at 2000 mg/kg. If the animal survives, an additional four animals are dosed sequentially. If three or more of these animals survive, the test is concluded with an LD50 greater than 2000 mg/kg. If three or more animals die, the main test must be conducted to determine the LD50 value. If an animal unexpectedly dies later in the study while other animals are still alive, dosing is halted, and all remaining are observed to determine if additional deaths occur during a similar observation period. We consider late deaths in the same way as other fatalities.
In rare cases where the limit test must be conducted at 5000 mg/kg due to specific regulatory requirements, we take additional precautions. The procedure begins with the administration of a single dose of 5000 mg/kg to one animal. If the animal survives, two additional animals are sequentially dosed at the same level. If both of these animals survive, the LD50 is considered to be greater than 5000 mg/kg, and we conclude testing after the mandatory 14-day observation period. However, if one or both of these additional animals die, two more animals are sequentially tested at the same dose level. If at any point three or more animals die, the test is terminated, and Altogen Labs conducts the main test to determine the LD50. If an animal unexpectedly dies later in the study while others survive, dosing is stopped, and all remaining animals are observed to determine if additional deaths occur during a similar observation period. Late deaths are treated the same as other deaths. Altogen Labs discourages the limit test at 5000 mg/kg is due to ethical considerations and is only be performed if there is a compelling scientific justification that the results will have a direct impact on human or environmental health protection.
Main Test
At Altogen Labs, the main test follows an adaptive, sequential dosing procedure at 48-hour intervals designed to refine the LD50 estimate while minimizing animal use. The first animal is dosed at an estimated level below the anticipated LD50. If the animal survives, the next dose is increased by a progression factor of 3.2. If the animal dies, the dose is reduced accordingly. This stepwise progression allows for efficient data collection while limiting unnecessary exposure to excessive doses.
Each animal is observed individually for at least 48 hours before dosing the next subject. The time between doses is determined by the observed onset, severity, and duration of toxicity. If no signs of toxicity are observed within the 48-hour period, we proceed with dosing according to the predetermined progression. However, if an inconclusive response occurs, the dosing interval may be adjusted accordingly.
If no lethality estimate for the substance is available, we begin dosing at 175 mg/kg. This dose is typically sublethal and helps minimize pain and suffering. If animal tolerance to the chemical is expected to vary significantly, Altogen Labs dose progression factor is adjusted before starting the test. Likewise, for substances known to have very steep dose-response slopes, smaller dose progression factors than the default are considered.
The test is terminated once one of the following conditions is met: at least three consecutive animals survive at the highest tested dose, five reversals occur in any six consecutive animals, or 4 animals must have been tested after the initial reversal, and the specified likelihood ratios must surpass the critical value. The likelihood-ratio method evaluates the relative support for the estimated LD50 against alternative values, ensuring that the test concludes once a sufficiently precise estimate has been obtained.
Animals that exhibit moribund conditions or severe distress are euthanized humanely and considered equivalent to test subjects that succumb to toxicity. If an animal unexpectedly dies late in the study and other survivors are present at the same or higher dose, we halt further dosing to observe whether additional mortality occurs. If subsequent animals at similar or higher doses survive, we maintain the original dose progression. However, if a pattern of delayed mortality is observed, the study is restarted at a lower initial dose to ensure accurate estimation of the LD50.
Following study completion, Altogen Labs calculates the LD50 using the maximum likelihood method. Confidence intervals are generated where feasible, with narrower intervals indicating higher precision in the estimate.
Observations
Following administration of the test substance, we observe each animal individually at least once during the first 30 minutes post-dosing, periodically throughout the next 24 hours, and daily for up to 14 days unless humane euthanasia is necessary due to excessive distress. Our observations include changes in skin, fur, eyes, mucous membranes, respiration, circulation, autonomic and central nervous system function, and behavior patterns. Symptoms such as tremors, convulsions, lethargy, diarrhea, and coma are also carefully recorded.
Altogen Labs records body weights prior to dosing, weekly thereafter, and at study termination. All animals, including those that die or are euthanized due to distress, undergo a necropsy to determine gross pathological changes at Altogen Labs. The presence of hemorrhages, ulcers, and damage to major organs is also documented. Histopathological evaluations may be conducted where necessary to provide further insight into the mechanisms of toxicity.
Data and Reporting
Data
Altogen Labs records all individual animal data, presenting it in a tabular format that includes the number of animals used at each dose level, observed toxic effects, survival rates, time of death, and necropsy findings. We also provide justification for the selected starting dose, dose progression, and any supporting data in our study documentation.
Computation of Confidence Interval
Altogen Labs computes confidence intervals for the LD50 to assess the reliability of the estimate. If three or more dose levels have been tested and the middle dose results in both survival and mortality, we determine the confidence interval using a profile-likelihood method, ensuring a 95% probability that the true LD50 falls within this range. If all animals survive at or below a given dose and all animals die at the next higher dose, the LD50 is estimated between those two values, and the interval is classified as approximate. However, because this response typically occurs only with a steep dose-response curve, we expect the true LD50 to fall within or very close to this interval.
Test Report
Altogen Labs compiles all study data into a comprehensive report detailing the experimental conditions, observations, and analytical findings. Our report includes detailed information on the test substance, such as its purity, physical properties, and vehicle used, along with justification for dose selection and progression. We analyze individual and summary data on survival rates, toxic effects, body weight changes, necropsy findings, and histopathological results in relation to dose levels.
We calculate LD50 values using the maximum likelihood estimation method, providing confidence intervals where applicable. All statistical analyses, including computational routines used, are documented in detail. Our observations report summarizes clinical signs, time-course trends, and reversibility of toxic effects. Our interpretation of results is aligned with regulatory classification criteria and relevant toxicological findings. Altogen Labs ensures that all study records are maintained according to regulatory guidelines to ensure data integrity, traceability, and reproducibility.
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