Session E4: Acute Systemic Toxicity Testing: Validation and Implementation of Alternative Methods

Chairs: Philip Botham (UK) and Kathy Stitzel (USA)

E4: Acute Systemic Toxicity Testing: Overview of Recent Key Activities
Philip Botham. Syngenta Central Toxicology Laboratory, Alderley Park, Macclesfield, SK10 4TJ, UK. phil.botham@syngenta.com.

Use of the test that aimed to identify the single lethal dose of a substance that kills half the animals in a test group (the LD50 test) should finally be discontinued by the end of 2002, after many years of controversy and debate. In its stead are three recently developed alternative animal tests that significantly improve animal welfare: the Fixed Dose Procedure, the Acute Toxic Class Method, and the Up and Down Procedure. These tests have already undergone revision, both to improve their scientific performance and, importantly, to increase their regulatory acceptance. They can now be used within a strategy of acute toxicity testing for all types of test substances and for all regulatory and in-house purposes. To aid the implementation of the use of these revised alternative methods, a training workshop was held in February, 2002, under the auspices of ICCVAM/NICEATM, ILSI, and the US-EPA, which was attended by representatives of both the toxicology testing and regulatory communities. The next activity in this area will be the conduct of a validation study on two in vitro basal cytotoxicity tests (the BALB/c 3T3 and the Normal Human Keratinocyte Neutral Red Uptake Assays). Jointly managed by NICEATM and ECVAM, the primary goal of this study is to evaluate the usefulness and effectiveness of these assays for reducing and refining animal use in the three new in vivo acute oral toxicity tests by enabling the prediction of optimal starting doses. The total replacement of animal tests for assessing acute systemic toxicity will, however, require a considerable amount of further test development, followed by validation, which is likely to take at least another ten years.

E4: Evaluation of Cytotoxicity Models for Predicting Acute Oral Toxicity Test Starting Doses
Rodger D. Curren, Greg Mun, Robin Ruppalt, and John Harbell. Institute for In Vitro Sciences, Inc., Gaithersburg, MD, USA. rcurren@iivs.org.

In vitro cytotoxicity methods have long been proposed as predictors of acute lethality in vivo. Recently Halle has compiled a Registry of Cytotoxicity (RC), which compares the LD₅₀ (in mmol/kg; rat or mouse) of 347 chemicals with an average in vitro IC₅₀ (in mmol/l; various cell types). Analysis of these data shows that there exists a reasonably good correlation between lethality in vivo and cytotoxicity in vitro; 73% of the data points lie within a +/- log 5 interval of the regression line. Therefore, Spielmann et al. have proposed that, at a minimum, the results of in vitro basal cytotoxicity tests can be used to estimate the starting dose for in vivo LD₅₀ tests. It has also been suggested that new cytotoxicity methods be first qualified for use in this scheme by testing them with RC chemicals having a range of toxicities and which fit closely to the RC regression line. To qualify, the new results should have a similar slope and lie within +/- log 5 of the RC regression line. We investigated whether two commonly used cytotoxicity tests (neutral red uptake in BALB/c 3T3 [3T3] and normal human keratinocytes [NHK]) would qualify under these conditions. Eleven chemicals from the RC whose in vivo LD₅₀'s ranged from ~0.1 mmol/kg to ~100 mmol/kg were chosen for evaluation. All chemicals were tested under code using a well-defined standard protocol. Cytotoxicity in the treated cells relative to that of control cells was determined after 24 hour-72 hour (3T3) or 48-72 hr (NHK) exposure. The toxicity of most chemicals increased with increased exposure time. When the resulting data were overlaid on the original RC graph, all new data points lay within a +/- log 5 interval around the RC regression line. It appeared that a 48-hour exposure was the optimum time for both cell systems. We conclude that the proposed qualification criteria are reasonable and that both of these candidate tests have met those criteria.

E4: Is it Really Possible to Bear Test Guideline 401 to Burial?
Herman Koëter. OECD Environment, Health and Safety Division, Paris, France. herman.koeter@oecd.org.

The original OECD Test Guideline 401 was adopted in May 1981, as one of the first set of 48 Test Guidelines. At the time, coordinated national expert input and commenting rounds were not considered. Nonetheless, the Guideline was extensively used as one of the first tests to be conducted for the hazard identification of chemicals. Already, after five years, because of animal welfare concerns, the Guideline was substantially modified, and the revised version of this method was adopted in February 1987. Unfortunately, the updated Guideline was slightly more complicated and, consequently, a substantial number of regulatory submissions of acute toxicity data were based on the old method. Last year, in December, the OECD Council agreed to the deletion of Guideline 401 and allowed exactly one year for data submitters to introduce the available alternative methods (Guidelines 420, 423, or 425) in their testing facilities. These alternative methods, earlier adopted in 1992, 1996, and 1998, respectively, had been extensively discussed and revised in order to meet all current regulatory requirements and were adopted by the Council at the same date Guideline 401 was made obsolete. Details will be provided of the process of updating the alternative methods and of the follow-up actions to ensure that Guideline 401 will no longer be used. The pros and cons of refusing data by regulatory authorities obtained by conducting Guideline 401 will be discussed.

E4: The Use of In Vitro Data to Estimate Starting Doses for Acute Oral In Vivo Studies
M. Liebsch, E. Genschow, W. Halle, H. Spielmann, R. Curren and W. Stokes. Center for Documentation and Evaluation of Alternative Methods to Animal Experiments (ZEBET), 12277 Berlin, Germany. liebsch.zebet@bgvv.de.

The Register of Cytotoxicity (RC) currently contains in vitro cytotoxicity data (IC₅₀) and in vivo oral toxicity data (LD₅₀) for 500 chemicals. A linear regression of the RC data was used to develop a prediction model (PM) that we propose for prediction of starting doses for in vivo studies in order to further reduce animal use. The total number of animals used in the 3 oral toxicity testing methods, Fixed Dose Procedure (FDP), Acute Toxicity Class Method (ATC), and Up and Down Procedure (UDP), is dependent on the distance between the starting dose and true LD50. For the UDP, for example, if no information is available, a starting dose of 175 mg/kg is recommended. Applying the PM will, in many instances, estimate a starting dose that is closer to the actual LD50 than the 175 mg/kg figure. This typically will result in fewer animals that need to be used before achieving the first reversal in the UDP test. The greatest reduction in animal use occurs for those chemicals that have an LD50 above the limit dose, especially 5 g/kg. Thus, if the in vitro test predicts an LD50 above 5 g/kg, 3 animals would be used, rather than 6 animals, if dosing were started at 175 mg/kg. In this example, a 50% reduction in animal use would be achieved, as well as a time-savings of 6 days. The procedure to check suitability of a cytotoxicity test protocol and the potential reduction in animal use for the FDP, ATC, and UDP will be presented.

E4: Comparison and Validation of Novel Pyrogen Tests Based on the Human Fever Reaction
Thomas Hartung and Human(e) Pyrogen Test Study Group (No. QLRT-1999-00811). Steinbeis-Technology-Transfer-Center for In Vitro Pharmacology and Toxicology (STZ InPuT) at the University of Konstanz, D-78464 Konstanz, Germany. Thomas.hartung@uni-konstanz.de.

The absence of pyrogens in injectable drugs is a crucial safety control, because pyrogens pose a life-threatening risk to patients. The field of applications for pyrogen testing is becoming more diverse, due to innovative high-tech products, such as medical devices (implants, medical plastic materials, dialysis machines), cellular therapies, and species-specific agents (e.g., recombinant proteins). The overall aim is to develop and validate a method based on the human fever reaction to replace the rabbit pyrogen test and to complement the Limulus assay. The network brings together the six most prominent test systems developed within the last years for trans-national comparison and subsequent validation of the most promising models. The consortium has evaluated the six different tests, defined Standard Operation Procedures (SOP's) and checked test performance and intra-laboratory variances. Each test was established in 2 to 3 partner laboratories, and currently inter-laboratory variation of individual tests is being determined. This comparison will help identify the most promising models for subsequent validation, which will be completed by the end of the year. A method for introduction into Pharmacopoeia will be deduced as a replacement for the rabbit pyrogen test for end-product control.