Acute systemic toxicity testing is the estimation of the human hazard potential of a substance by determining its systemic toxicity in a test system (currently animals) following an acute exposure. Its assessment has traditionally been based on the median lethal dose (LD50) value – an estimate of the dose of a test substance that kills 50% of the test animals. For a substance to have systemic toxic effects it must be absorbed by the body and distributed by the circulation to sites in the body where it exerts toxic effects. The liver may transform a circulating drug or chemical to another form (biotransformation), and this new metabolite may be the one causing the observed toxicity.
Acute systemic toxicity is assessed following oral, dermal, and/or inhalation exposure(s) – depending upon the anticipated routes of human exposure to the substance. The Globally Harmonized System (GHS), which is scheduled for implementation in 2008, defines acute toxicity as “those adverse effects occurring following oral or dermal administration of a single dose of a substance, or multiple doses given within 24 hours, or an inhalation exposure of 4 hours” (UNECE, 2004, p. 109).
The traditional LD50 test provides an estimate of the 50% lethal dose of a test substance administered via the oral, dermal, or inhalation route. The median lethal dose or LD50 is the endpoint for oral or dermal dosing, and the lethal concentration or LC50 the endpoint for dosing via inhalation (Gribaldo, et al., 2005). The preferred species for oral and inhalation testing is the rat, and for dermal testing, the rat or rabbit (UNECE, 2004). Oral administration is the most common form of acute systemic toxicity testing.
Spielmann and colleagues at the Centre for Documentation and Evaluation of Alternative Methods to Animal Experiments (ZEBET) developed the Up-and-Down Procedure (UDP) to reduce the number of animals used in acute toxicity testing (Spielmann, et al., 1999). ZEBET scientists also refined and used a cytotoxicity database to predict the starting dose for the UDP. The Registry of Cytotoxicity (RC), developed by Halle, contains data on the in vitro cytotoxicity endpoint IC50 and the in vivo LD50 values for 347 chemicals. The IC50/LD50 regression model based on the RC data was used to predict the in vivo LD50 from the IC50 cytotoxicity results. The predicted LD50 value was then used as the starting dose for the UDP. The researchers proposed that this tiered approach be used to reduce animal use for acute systemic toxicity testing. The Registry of Cytotoxicity, originally published in German, was recently republished in English (Halle, 2003).
The Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) later validated the UDP as a reduction alternative for acute oral toxicity testing, and it was accepted by US regulatory agencies in 2004. In 2007, ICCVAM endorsed two in vitro basal cytotoxicity assays, the Neutral Red Uptake (NRU) test with rodent cells (3T3 NRU assay) and the NRU test with normal human keratinocyte (NHK) cells (NHK NRU assay), as “reduction alternatives to estimate the starting dose in the UDP and Fixed Dose Procedure (FDP) for assessing acute oral toxicity.” The European Centre for the Validation of Alternative Methods (ECVAM) partnered with ICCVAM in the international cytotoxicity assay validation study.
Five Organisation for Economic Cooperation and Development (OECD) Test Guidelines (TGs 402, 403, 420, 423, and 425) describe acute systemic testing. The OECD TG 401 for Acute Oral Toxicity was deleted in December 2002. Three reduction alternatives to the oral LD50 test have been developed and validated: the Fixed Dose Procedure (OECD TG 420), the Acute Toxic Class method (OECD TG 423), and the Up-and-Down Procedure (OECD TG 425). Each of these methods uses preset doses, with the starting dose based on a small range-finding study, cytotoxicity screens, or preexisting data (Whitehead & Stallard, 2004). Following dosing (typically by gavage), the animals are monitored for overt toxicological signs until death (Acute Toxic Class or Up and Down Method) or “evident toxicity” (Fixed Dose Procedure).
Because the Fixed Dose Procedure (OECD 420) does not rely on death as an endpoint, it is a refinement as well as a reduction alternative to the LD50 test (OECD TG 401). In recent years, using death as an endpoint has been discouraged in all testing contexts, so the use of the reduction alternatives is now mandatory for acute systemic toxicity testing (Schlede, et al., 2005). Schlede, et al. (2005) reported that about 50% of all oral toxicity testing in the EU is conducted using the oral Acute Toxic Class test.
For the OECD TG 402, Acute Dermal Toxicity, a test substance is applied to no less than 10% of the area of the skin of rats, rabbits, or guinea pigs, followed by 14 days of observation. Death of the animals is used to determine an LD50 value. Also, gross pathological changes are used to estimate the relative toxicity of a substance. The test may be useful in indicating dermal absorption and is used to determine dosing for other studies such as subchronic dermal testing. Stallard, et al. (2004) described a reduction/refinement alternative method and its validation, the dermal fixed dose procedure (dermal FDP), which is based on using only moderately toxic doses and a nonlethal endpoint. The OECD has a proposed Draft Guideline 434 for the Fixed Dose Procedure, which is pending acceptance.
Acute inhalation toxicity is assessed according to OECD TG 403. Rats are typically exposed by inhalation of the test substance for no less than four hours and are monitored for 14 days to determine the LC50. The OECD Draft Guideline 433, Fixed Concentration Procedure (FCP) is being prepared as a reduction/refinement to TG 403. Stallard, et al. (2003) described the FCP and its validation using statistical simulation.
The regulatory testing requirements for acute systemic toxicity testing in the European Union, by the US Environmental Protection Agency (EPA), and in the GHS are described in a recent ECVAM workshop report (Gennari, et al., 2004).
In vitro methods and (quantitative) structure-activity relationship ((Q)SAR) models for the prediction of acute systemic toxicity have been reviewed in several recent workshops and articles (ICCVAM, 2001; ECVAM, 2002; Gribaldo, et al., 2005).
A non-animal replacement method for acute systemic toxicity will have to provide information on many complex biological processes, including toxicokinetics and organ toxicity. A test battery or batteries and decision-making schemes will be required to model these pathophysiological processes.
In vitro basal cytotoxicity will be an important component of any test battery used to predict acute systemic toxicity. Cytotoxicity assays have been developed and evaluated against in vivo data for this purpose for some time. An ECVAM report reviewed the many assessments that have attempted to correlate basal cytotoxicity endpoints using various cells lines and assay endpoints with acute systemic toxicity (ECVAM, 2002). Some of the in vitro methods were reported to correlate well enough with the in vivo data “to predict LD50 values with a reasonable degree of precision” (ECVAM, 2002).
The Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) program in the 1990s demonstrated a good correlation between cytotoxicity data and human lethal blood concentrations. A list of publications resulting from the MEIC program and the MEIC database can be accessed on the MEIC website. The Evaluation-guided Development of new In vitro Test batteries (EDIT) program, which followed MEIC, focused on developing and evaluating in vitro test batteries for both acute and chronic systemic toxicity testing (Clemedson, et al., 2002). For example, the predictive capability of the MEIC results was improved by the addition of assessments for protein binding and the partition of chemicals (Gülden, et al., 2001; Seibert, et al., 2002; Clemedson, et al., 2003). A prediction model has been developed that can estimate the equivalent concentrations of a chemical in serum from the in vitro effective concentrations (Gülden, et al., 2006).
ECVAM proposed a tiered testing strategy for acute systemic toxicity (Siebert, et al., 1996; ECVAM, 2002). In this testing strategy, a substance would be sequentially evaluated by (Q)SAR and in vitro assays [(Q)SAR → cytotoxicity testing → computational model for metabolism → biotransformation assays → cell-specific toxicity tests], and when classified as “very toxic” at any step, the testing would end with the classification of the substance. A limited in vivo acute toxicity test would be performed only if all of the prior assessments indicated the substance to be “not very toxic.”
The proposed ECVAM tiered strategy approach has been superseded by the integrated testing approach being developed in the ACuteTox program, which is attempting to develop a total in vitro/in silico replacement strategy for the prediction of human acute systemic toxicity (Clemedson, et al., 2007). The data and results of the MEIC and EDIT programs are the foundation for the ACuteTox program. “The project aims to improve the already established correlation (over 70%) between in vitro basal cytotoxicity and rodent LD50 values, as well as human lethal blood concentrations [MEIC program], to a level sufficient enough to ensure a valid prediction of human acute toxicity. This will be achieved by integrating cytotoxicity data with additional information on biokinetics, metabolism and target organ toxicity.” Current results of the research program are available on the ACuteTox website.
The in vitro methods that have been or are being developed and/or validated for consideration as part of an integrated testing scheme for the prediction of acute systemic toxicity fall into three broad categories: cytotoxicity assays, organ-specific toxicity assays, and biokinetics/metabolism methods. A few examples of relevant cytotoxicity assays are listed in Table 1; organ-specific and biokinetic/metabolism models are discussed in other sections of AltTox.org. Resources that describe additional assays include: ICCVAM, 2001; ECVAM, 2002; Gennari, et al., 2004; Gribaldo, et al., 2005; ACuteTox website.
Table 1. Examples of cytotoxicity assays for acute systemic toxicity (Gribaldo, et al., 2005).
|BALB/c 3T3 – mouse fibroblast cell line||Neutral red uptake||Cell viability/cytotoxicity|
|Normal human keratinocytes||Neutral red uptake||Cell viability/cytotoxicity|
|LLC-PK1 kidney proximal tubule cell line||Transepithelial resistance (TER) and paracellular permeability||Barrier integrity/cell damage|
|MDCK dog kidney epithelial cell line||Transepithelial resistance (TER) and paracellular permeability||Barrier integrity/cell damage|
|HepG2 liver cell line (hepatoma)||Protein content||Cell growth|
|HL-60 human acute promyelocytic leukemia cell line||Adenosine triphosphate (ATP) content||Energy production and metabolism|
|Change liver cell line||Morphology change followed by pH change||Cell growth/cytotoxicity|
An International Workshop on In Vitro Methods for Assessing Acute Systemic Toxicity, convened by ICCVAM in October 2000, reviewed the results of prior research and programs that assessed in vitro cytotoxicity assays for the prediction of acute systemic toxicity. None of the in vitro assays had yet been validated. A validation study was proposed for evaluation of two basal cytotoxicity assays for determining starting doses for the in vivo oral toxicity test. Although the validation was not for the purpose of replacement, the cytotoxicity assays could further reduce animal use.
Following a peer review process, ICCVAM endorsed two in vitro basal cytotoxicity assays for use in acute systemic toxicity testing: the NRU test with mouse 3T3 fibroblasts (3T3 NRU assay) and the NRU test with normal human epidermal keratinocytes (NHK) (NHK NRU assay) (Table 2). The ICCVAM endorsement stated: “In 2007, both in vitro test methods recommended as reduction alternatives to estimate the starting dose in the UDP and Fixed Dose Procedure (FDP) for assessing acute oral systemic toxicity.” The NRU test methods have not been validated for determining the hazard classification of chemicals. Documents on the ICCVAM website describe the limitations of the uses of these in vitro methods.
Table 2. Non-animal test methods for acute systemic toxicity testing.
|In vitro basal cytotoxicity assay: Neutral red uptake (NRU) test with rodent cells (mouse 3T3 fibroblasts) (3T3 NRU assay)||Adjunct to in vivo acute oral toxicity tests for determining starting doses|
|In vitro basal cytotoxicity assay: Neutral red uptake (NRU) test with human cells (normal human epidermal keratinocytes (NHK)) (NHK NRU assay)||Adjunct to in vivo acute oral toxicity tests for determining starting doses|
|Colony Forming Unit-Granulocyte/Macrophage (CFU-GM) Assay||Haematotoxicity test for acute neutropenia|
The 3T3 NRU and the NHK NRU assays are cytotoxicity assays in which the dye neutral red is taken up by living cells. Toxic chemicals can decrease the amount of absorbed neutral red by altering cell membranes via cytotoxicity and/or inhibition of cell growth. The protocols and basis for the selection of the assays and cell types are explained in the ICCVAM Background Review Document.
The ECVAM Scientific Advisory Committee (ESAC) has reviewed and endorsed a cell-based assay, the Colony Forming Unit-Granulocyte/Macrophage (CFU-GM) assay, for the prediction of human acute neutropenia (one possible manifestation of systemic toxicity). It can be used as a substitute for regulatory testing in a second species, which is commonly a dog test.
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