|Home | About | Journals | Submit | Contact Us | Français|
Acute systemic toxicity studies are carried out in many sectors in which synthetic chemicals are manufactured or used and are among the most criticized of all toxicology tests on both scientific and ethical grounds. A review of the drivers for acute toxicity testing within the pharmaceutical industry led to a paradigm shift whereby in vivo acute toxicity data are no longer routinely required in advance of human clinical trials. Based on this experience, the following review was undertaken to identify (1) regulatory and scientific drivers for acute toxicity testing in other industrial sectors, (2) activities aimed at replacing, reducing, or refining the use of animals, and (3) recommendations for future work in this area.
This review has been carried out under the auspices of the European Partnership for Alternative Approaches to Animal (EPAA) Testing, an unprecedented collaboration between the European Commission (EC), European industry trade associations, and companies from seven industrial sectors. The partners are committed to pooling knowledge and resources to accelerate the development, validation, and acceptance of alternative approaches to further the reduction, refinement, and replacement (3Rs) of animal use in regulatory testing.
The term “acute toxicity” is used to describe the adverse effects of a substance that may result from a single exposure or multiple exposures within a 24-h period. Acute effects may be local (e.g., skin or eye irritation) and/or systemic in nature. This review focuses on the latter, with emphasis on regulatory required high-dose studies carried out via oral, dermal, and inhalation routes of exposure for the purpose of identifying or estimating doses that cause lethality. Other types of acute studies such as nonlethal single-dose studies (e.g., for derivation of an acute reference dose), acute ecotoxicological studies in fish and avian species, testing for marine biotoxins, and safety/potency testing of vaccines are not explored in this paper.
Acute systemic toxicity studies are rooted in the post-World War I era concept of the “LD50,” which was defined by Trevan (1927) as the single dose of a substance that can be expected to cause death in 50% of the animals in an experimental group. Initially developed to provide a relative index of toxicity for plant and biological extracts, LD50-type studies achieved general acceptance as a basis of comparing and classifying the toxicities of chemicals (FDA, 1988) and have become a routine testing requirement in a number of regulatory sectors (Botham, 2004). According to EC (2007) animal use statistics, acute toxicity studies remain the most prevalent class of toxicological test in use today.
Acute lethality studies have been among the most heavily criticized of all regulatory toxicity tests, both on scientific and on ethical grounds (Ekwall et al., 1998; Langley, 2005; Lorke, 1983; Zbinden and Flury-Roversi, 1981). In response to criticisms, there has been a gradual evolution in study designs for acute systemic toxicity consistent with the 3Rs principle (Russell and Burch, 1959), coupled with increasingly sophisticated efforts to move away from animal testing altogether (Table 1). Notably, reduction and in part refinement methods have been introduced as Organization for Economic Cooperation and Development (OECD) Test Guidelines for oral and inhalation routes, although no such approach for dermal exposure is currently available. And despite efforts over many years, acute toxicity testing remains a core regulatory requirement in many sectors.
In 2003, a working group comprised 18 international pharmaceutical companies and contract testing laboratories, together with the U.K. National Centre for the Replacement, Refinement, and Reduction of Animals in Research (NC3Rs), was established to evaluate the utility of acute systemic toxicity studies in the development of new medicines. The expert group determined that “the information obtained from acute toxicity studies is of little or no value in the pharmaceutical development process,” a conclusion subsequently considered and endorsed by pharmaceutical regulators and scientists via the International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) process (ICH, 2009; Robinson et al., 2008). In light of these findings, and in view of the requirement of acute toxicity testing across multiple industry sectors, the EPAA established a task force to examine scientific and regulatory drivers for such testing and to promote the use of 3Rs approaches that the task force considers are currently available. This publication is one of several products of that effort.
A questionnaire designed to gather information regarding current practices in the conduct of acute toxicity studies and companies’ experiences in this area was sent to all EPAA members except those in the pharmaceutical sector, which has already participated in such an exercise (Robinson et al., 2008). The EPAA survey questions covered the scientific and regulatory objectives of the studies, routes of administration, preferred test guideline, parameters examined, dose limit, and regulatory experience (a link to the EPAA questionnaire is included under “Supplementary data”). Seventeen companies responded, and the number of companies responding per sector is outlined as follows: agrochemicals (four companies), animal health (two companies), consumer products/cosmetics (nine companies), industrial chemicals (four companies), and together with two contract research organizations that conduct studies to support the various sectors. The total number of responding companies appears greater than 17 because some companies represent more than one sector. The aggregated responses are not detailed in this publication because the number of companies responding within each sector was relatively small. The limited nature of the survey means that generalized qualitative responses rather than quantitative data are used to support the points made in relevant sections of this publication. Reference to the earlier pharmaceutical company survey is also made when relevant.
Table 2 compares current and proposed protocols for acute toxicity studies and identifies study designs used to determine LD50 point estimates versus range estimates, as well as how many animals are typically used under each protocol. A full statistical breakdown of animal use across industry sectors and in various classes of acute toxicity studies is available elsewhere (EC, 2007).
Contemporary test guidelines offer greater flexibility for generating data fit for purpose, potentially using fewer animals than the older guidelines, such as the now-deleted OECD Test Guideline 401 (OECD, 2009c). It is also possible to use clinical signs such as “evident toxicity” rather than death as an end point for classification, e.g., in the U.K.-pioneered Fixed Dose Procedure (OECD 420).
Choice of test guideline is driven in large part by national and sector-specific regulatory requirements but can also be influenced by what the LC50 or LD50 might reasonably be expected to be. For example, if there is reason to expect that the acute toxicity will be greater than the limit dose for classification, OECD 420 would be a suitable choice in using the fewest animals to achieve this end. If this is not absolutely certain, the German-developed Acute Toxic Class Method (OECD 423) may ultimately use the fewest animals. If on the other hand a point estimate of the oral LD50 is required, the U.S.-developed Up-And-Down Procedure (OECD 425) would be required. According to EPAA’s survey of members, many European companies and contract research organizations default to OECD 423 unless a specific regulatory authority requires a more humane method or a point estimate of the LD50.
For acute dermal toxicity, the only guideline currently available is the classic dermal LD50 study (OECD 402). An OECD dermal fixed dose guideline was proposed in 2004 but has since been withdrawn. Acute dermal studies are normally performed after oral or inhalation testing, and as discussed later in this publication, dermal toxicity is rarely greater than what is observed in oral or inhalation studies. Thus, a limit test is normally sufficient.
For acute inhalation toxicity, a revised version of the classic mammalian LC50 study (OECD 403) has recently been adopted, together with a new Acute Toxic Class guideline (OECD 436) as an animal reduction measure. An inhalation Fixed Concentration Procedure has also been proposed, and work to develop the scientific evidence needed to support the adoption of this method is currently ongoing (see Table 1).
Most countries examined have enacted legislation and regulations governing the testing and marketing of agricultural and industrial chemicals, biocides, cosmetics, food additives, medicinal products, and other substances for the protection of human health and the environment. A multisector and multiregional overview of regulatory data requirements for acute systemic toxicity is presented in Table 3. This illustrates the complexity of the regulatory arena across sectors and countries and the challenges this creates for those seeking to reduce the numbers of animals used in acute toxicity studies while generating globally acceptable registration data packages.
For “agrochemicals and biocides,” acute data for three routes of administration (oral, dermal, and inhalation) are generally required for all active substances and in many cases for formulated products and certain other chemical ingredients as well (EPA, 2007b, 2008a; FAMIC, undated; GC, 2006; MOA, 2001; OJ, 1992, 1998). Requirements for “industrial chemicals” are generally less rigid, with most countries examined requiring testing by a single route or possibly two routes for higher tonnage substances (GC, 2005; MEP, 2004a; OJ, 2007). Some countries currently impose no specific data requirement for acute toxicity testing of industrial chemicals (EPA, 2007a; METI, 2005) or no testing below a specified production volume, e.g., one metric ton in the European Union (EU) (OJ, 2007). Within the EU, the only officially recognized methods for the determination of acute oral toxicity of industrial chemicals are OECD TG 420 and OECD 423 (OJ, 2004, 2008), which is a consideration when determining a test to be used across geographical regions and regulatory frameworks. The EPAA survey confirmed implementation of these regulatory requirements in practice.
For “cosmetics,” acute toxicity testing of both finished products and raw ingredients is now prohibited in the EU (OJ, 2003) and not specifically required in the United States or Canada, although information on systemic effects may be obtained using other methods to ensure the legally required safety of the product. In Japan, for cosmetics consisting of ingredients already on an approved list, there is no requirement for additional testing. In contrast, China and certain South American countries require premarket registration of cosmetic finished products, which may entail some level of acute toxicity testing above and beyond the safety assessment of raw ingredients (RPA, 2004). Additionally, some of these countries do not consistently accept foreign data, which may result in cosmetic products produced by foreign companies being subject to duplicate testing.
For “food additives, flavorings, and food-contact materials,” a specific requirement to generate acute systemic toxicity data could not be found in applicable legislation, regulations, or guidance in any of the countries surveyed (EC, 2001a, 2001b; FDA, 2002, 2006; MHLW, 2009).
For the development of new “human medicines,” the requirement for acute toxicity tests is now largely historic because the revised text of ICH Test Guideline “M3 R2” was adopted last year (ICH, 2009). All that remains is for the regional guidelines in Europe, the United States, and Japan to be updated to reflect the text of the revised ICH M3. Many pharmaceutical companies have not conducted acute toxicity studies for new medicines for some time because data generated from other more refined study types (e.g., in vivo genetic toxicology studies, safety pharmacology studies, and dose-range finding studies), which are already conducted as part of the development of new medicines, are considered to provide a better assessment of potential human safety risks in advance of clinical trials. The same is true regarding the protection of workers in manufacturing and production plants, such that most companies are now using data from other studies to inform Material Safety Data Sheets and other worker protection measures.
With respect to “veterinary medical products,” acute toxicity studies are not specifically required for the demonstration of safety either to target animals or to human consumers (EMEA, 2009; OJ, 1990; VICH, 2008). However, acute studies may be carried out on a voluntary basis to obtain information on other aspects of safety for veterinary medical product (e.g., worker protection), though as above, other available data could be used for these purposes.
Across all sectors and countries examined, it is generally accepted that acute toxicity studies may be waived if a substance is known to be corrosive or if there is a low risk of human exposure (ECHA, 2008b). Route-specific waivers may be granted on the basis of physicochemical properties, such as volatility, particle size, molecular weight and volume, and log Kow (ECHA, 2008c). A notable exception is for agrochemical and biocide active substances in the EU, where acute toxicity studies must usually be carried out for hazard classification of the active substances regardless of the expected exposure. For formulations, waivers may be granted in cases where a scientifically sound case can be made, e.g., when the outcome of the study is highly predictable based on the properties and concentration of individual ingredients (EPA, 2001; OJ, 1999). Weight-of-evidence and read-across approaches might also be used to estimate acute toxicity (discussed further in the “Alternative Approaches” section below).
Classification and labeling of substances and products is relevant to various sectors. Regulatory authorities across the globe have also developed frameworks for the classification and labeling of chemical hazards for the protection of workers, consumers, and the environment. In many cases, the regulatory requirement for acute toxicity data is for classification and labeling purposes only, a fact confirmed by the EPAA survey, with the majority of companies identifying classification and labeling as a primary reason for conducting acute toxicity testing.
When testing is conducted solely to meet classification and labeling requirements, precise LD50/LC50 values are not necessary because testing to the upper boundary of a hazard category (i.e., limit dose) is sufficient to establish a regulatory classification. Therefore, there is no scientific necessity to establish a dose-response curve for mortality.
The Globally Harmonized System of Classification and Labeling (GHS) was developed under the auspices of the United Nations (UN, 2007) to promote increased consistency among diverse national and sectoral frameworks. To date, the GHS has been or is being implemented in the EU, New Zealand, Korea, China, India, Japan, and the United States (OECD, 2007a), although in certain cases, the flexibility provided by the GHS modular design has led to continued differences in implementation. For example, European authorities and the U.S. Occupational Safety and Health Administration accept a limit dose of 2000 mg/kg (i.e., GHS category 4), beyond which a substance or product is not required to bear an acute hazard label (OJ, 2008; OSHA, 2009), whereas other authorities require testing to a limit dose of 5000 mg/kg (i.e., GHS category 5) to support a no-label designation. Figures 1 and and22 illustrate the different hazard class cutoffs between the GHS, EU, and U.S. pesticide (EPA, 2004) classification schemes.
The majority of European companies surveyed reported using 2000 mg/kg as the default limit dose, and the GHS itself expressly discourages testing beyond 2000 mg/kg for animal welfare reasons (UN, 2007). Regulatory guidance is also available to support extrapolation of data gained with a limit dose of 2000 to GHS category 5 without retesting (OJ, 2004). However, ongoing geopolitical differences continue to inspire duplicative animal testing (e.g., to retain a no-label designation in a country or sector where GHS category 5 is considered mandatory).
There have been several scientific reasons proposed for conducting acute toxicity studies. Potential drivers have been gathered from the 2003 pharmaceutical industry initiative, as well as the more recent EPAA survey of member companies in other sectors, and appear to be common across industrial sectors. These are listed below, together with a discussion of their merit.
Each of the above statements has some merit when acute lethality studies are conducted for regulatory purposes anyway (e.g., to support hazard classification and labeling). However, it is not necessary to conduct an acute toxicity study to address these scientific objectives per se. In fact, in terms of dose setting for a repeated dose study, the use of lethality as a specific end point is counterintuitive. Other study types with more refined end points (e.g., a dose escalation study to identify maximum tolerated dose) can equally address these objectives. This also holds true in cases where repeated dose toxicity data are available.
This statement assumes that the data obtained from acute toxicity studies provide information on the likely effects of acute overdose or accidental exposure in humans. However, the EPAA and pharmaceutical industry surveys demonstrated that these studies do not normally include clinical pathology, microscopic pathology, or toxicokinetic evaluation, which would provide useful information to aid in risk assessment. In addition, clinical observations seen in rodents at doses above 1000 mg/kg are often nonspecific and do not add information that would support measures to be taken in overdose or accidental exposure situations in humans.
A pilot survey of European and U.S. poison centers conducted by the NC3Rs and AstraZeneca indicated that 6 of 10 do not use the acute toxicity data in animals to manage cases of overdose in humans (Robinson and Chapman, 2009). Four centers stated that they do use animal acute toxicity data. However, the data that these poison centers thought were useful, such as target organ or mode of toxicity, are not normally provided by conventional acute toxicity studies. To explore this issue further, the NC3Rs held a workshop in January 2010, bringing together representatives from international poison centers, the pharmaceutical and chemical industries, and regulatory bodies to discuss whether and how acute toxicity data are used to assess and treat cases of pharmaceutical overdose and chemical poisoning. The discussions from this workshop are currently being written up for publication elsewhere.
The EPAA and pharmaceutical industry surveys have shown that microscopic pathology is not routinely performed during acute toxicity studies, which essentially negates their value in identifying target organs or mechanisms of toxic action.
In conclusion, it is evident that the scientific drivers listed above may have some merit when acute toxicity tests are conducted for regulatory purposes, such as classification and labeling. However, in the absence of a specific regulatory requirement, the scientific objectives can equally be met by other study designs that do not include lethality as the end point and that include parameters that could assist risk assessment (e.g., histopathology, clinical pathology, and measures of systemic exposure).
This section outlines accepted and emerging strategies with the potential to affect an immediate and substantial reduction in the number of animals used in regulatory acute toxicity testing (Table 3). A more extensive listing of ongoing and historic activities aimed at refinement, reduction, and replacement of animal use in acute toxicity studies is provided in Table 1.
Retrospective data analyses have been undertaken by Creton et al. (2010) and Seidle, Prieto, and Bulgheroni (submitted for publication elsewhere) to ascertain the value of regulatory requirements prescribing multiroute testing for acute systemic toxicity. These analyses have examined the concordance among regulatory classifications for acute oral, dermal, and/or inhalation toxicity for ~500 agrochemical and biocidal active substances and nearly 2000 industrial chemicals. The findings from these two independent reviews have revealed that acute dermal studies of pure substances do not add value above and beyond oral data for hazard classification of pesticides, biocides, or chemicals. Follow-up work is currently under way by Seidle to ascertain whether this conclusion holds true for multicomponent formulations. Concordance between oral and inhalation data sets was also reasonably high for certain substance classes, suggesting that it may be possible to develop waiver criteria for inhalation testing, subject to further review and analysis including consideration of factors, such as physicochemical properties, bioavailability, etc. An international workshop to discuss the findings on redundancy of the dermal route with industry and regulators is planned for September 2010.
A range of nontesting approaches, including chemical grouping and read across, weight of evidence, exposure-based waiving, and various calculation methods, could be put to immediate use to satisfy regulatory requirements for acute toxicity data without new testing. These approaches are commonly accepted under most regulatory frameworks including EU and U.S. pesticide and chemical regulations and international regulations implementing the GHS.
“Chemical grouping and read across” is based on the recognition that substances with similar molecular structures often share similar toxicological profiles, and where end point data are available for one member of a chemical family, these data may be used to bridge a gap for another member of the same chemical family. This approach requires expert judgment, which may be augmented by “in silico” tools, such as the OECD (quantitative) structure-activity-relationship ((Q)SAR) toolbox (OECD, 2009b) or the Ambit 2.0 database (“http://ambit.sourceforge.net”).
“Weight of evidence” recognizes that data exist which on their own would not be sufficiently robust or reliable for regulatory purposes but that when relevant data from different sources (e.g., animal studies that were not performed to current standards, in vitro data, (Q)SARs predictions, and threshold considerations) are combined using expert judgment, sound regulatory conclusions can be drawn. Further information on how read-across and weight-of-evidence approaches may be implemented can be found in ECHA (2008a) guidance documents and elsewhere (OECD, 2007b; Worth et al., 2007).
For formulated products containing mixtures of chemicals, a number of organizations, and regulations, including the GHS, provide guidance on the use of calculation methods to determine the toxicity and appropriate classification, thus avoiding the need for acute toxicity testing (UN, 2007; WHO, 2005). Classification can be determined on the basis on the toxicological properties of the individual ingredients and their relative proportions within the mixture or formulation.
Where exposure can be demonstrated to be negligible, or the risk of exposure is low, it could be argued that hazard characterization, i.e., an acute toxicity study, is unnecessary.
In 2008, European Centre for the Validation of Alternative Methods performed an investigation to explore whether it is possible to identify nontoxic compounds (LD50 > 2000 mg/kg) using information from 28-day repeated dose toxicity studies. Taking into account the high prevalence of nontoxic substances (87% of 4219) in the EU’s New Chemicals Database (Bulgheroni et al., 2009), a No Observed Adverse Effect Level threshold was set that allowed the correct identification of 63% of nontoxic compounds, while less than 1% of harmful compounds were misclassified as nontoxic. The proposed approach could permit the waiving of acute oral testing of more than 50% of chemical substances. Although the research focused on using the proposed approach for cosmetic ingredients, it could potentially also be applied for chemicals in other sectors where 28-day studies are performed.
Based on an analysis showing strong concordance between in vitro cytotoxicity data and human lethal blood concentrations, i.e., R2 = 0.77–0.83 (Ekwall et al., 1998), it was recommended in 2000 that basal cytotoxicity tests be put to immediate use in establishing starting doses for acute oral toxicity studies in animals as a means of reducing animal use, e.g., by up to 40% in relation to OECD 425 (ICCVAM/NICEATM, 2001b). The following year, U.S. validation authorities published a guidance document on the use of in vitro data to estimate oral starting doses (ICCVAM/NICEATM, 2001a), although to date, this approach does not appear to have been widely taken up in practice. More recently, the OECD (2009a) has undertaken to update this guidance for an international audience to promote wider awareness and use of this animal reduction strategy.
In the following, conclusions and recommendations are listed in hierarchical order according to the authors’ perspective:
As this paper highlights, the regulatory landscape across industry sectors and geographical regions is complex, and multiple efforts are ongoing to promote the 3Rs in acute toxicity testing across sectors and parts of the globe. However, there remains a need for greater cross-sector and international cooperation to ensure that developments that can reduce, refine, and ultimately replace the use of animals in acute toxicity testing, while assuring safety, are fully implemented.
This work was supported by the authors’ affiliated institutions, together with a grant for the lead author from the Doerenkamp-Zbinden Foundation, Switzerland.
The authors are members of the EPAA Acute Toxicity Task Force and would like to thank the other members for their input and advice: David Dreher (Covance), Nigel Moore (Dow), Sally Old (Sanofi Aventis), Andrea Paetz (Bayer), Andreas Schnurstein (Evonik), Thomas Skripsky (Novartis Animal Health), and Susanne Thun-Battersby (Solvay). The authors would also like to acknowledge support from members of EPAA working group 4 (dealing with implementation of 3Rs in legislation), as well as additional support from representatives of industry sectors and the EPAA steering committee. Further information about the EPAA and its current initiatives can be found at “http://www.epaa.eu.com.”