## 4.3.9. Human toxicity testing - I. General aspects

Authors: Theo Vermeire, Marja Pronk

Reviewers: Frank van Belleghem, Timo Hamers

Learning objectives:

You should be able to:

- describe the aim and scope of human toxicity testing and which organizations are important in the development of tests
- mention alternative, non-animal testing methods
- describe the key elements of human toxicity testing
- mention the available test guidelines

Keywords: toxicity, toxicity testing, test guidelines, alternative testing, testing elements

Introduction

Toxicity is the capacity of a chemical to cause injury to a living organism. Small doses of a chemical can in theory be tolerated due to the presence of systems for physiological homeostasis (i.e., the ability to maintain physiological stability) or compensation (i.e., physiological adaptation). Above a given chemical-specific threshold, however, the ability of organisms to compensate for toxic stress becomes saturated, leading to loss of homeostasis and adverse effects, which may be reversible or irreversible, and ultimately fatal.

Toxicity testing serves two main aims, i.e. to identify the potential adverse effects of a chemical on humans (i.e., hazard identification), and to establish the relationship between the dose or concentration and the incidence and severity of an effect. The data from toxicity testing thus needs to be suitable for classification and labelling and should allow toxicologists to determine safe levels of human exposure (section 6.3.3), to predict and evaluate the risks of these chemicals to humans and to prioritize chemicals for further risk assessment (section 6.1) and risk management (section 6.6).

Toxicologists gather toxicity data from the scientific literature and selected databases or produce these data in experimental settings, mostly involving experimental animals, but more and more also alternative test systems with cells/cell lines, tissues or organs (see section 4.3.9.II). Toxicity data are also obtained from real-life exposures of humans in epidemiological research (section 4.3.10.I) or in experiments with human volunteers under strict ethical rules. This chapter will focus on experimental animal testing.

The scope of toxicity testing depends on the anticipated use, with route, duration and frequency of administration as representative as possible for human exposure to the chemical during normal use. The oral, dermal or inhalation routes are the routes of preference, and the time scale can vary from single exposures up to repeated or continuous exposure over parts or the whole of the lifetime of the experimental organism. In toxicity testing, specific toxicity endpoints such as irritation, sensitization, carcinogenicity , mutagenicity, reproductive toxicity, immunotoxicity and neurotoxicity need to be addressed (see respective subchapters in section 4.2, and section 4.3.9.III). These toxicity endpoints can be investigated at different time scales, ranging from acute exposure (e.g., single dose oral testing) up to chronic exposure (e.g., lifelong testing for carcinogenicity) (see also under ‘test duration’ below).

Other useful tests are tests designed to investigate the mechanisms of action at the tissue, cellular, subcellular and receptor levels (section 4.2), and toxicokinetic studies, investigating the uptake, distribution, metabolism and excretion of the chemical. Such data helps in the design of the testing strategy (which tests, which route of exposure, the order of the tests, the dose levels) and the interpretation of the results.

International cooperation and harmonization

The regulation of chemicals is more and more an international affair, not in the least to facilitate trade, transport and use of chemicals at a global scale. This requires strong international cooperation and harmonization. For instance, guidelines for protocol testing and assessment of chemicals have been developed by the World Health Organization (WHO) and the Organisation for Economic Co-operation and Development (OECD). These WHO and OECD guidelines often are the basis for regulatory requirements at regional (e.g., EU) and national scales (e.g., USA, Japan).

Of prime Importance for harmonization is the OECD Mutual Acceptance of Data (MAD) system. This system is built on two instruments for ensuring harmonized data generation and data quality: the OECD Guidelines for the Testing of Chemicals and the OECD Principles of Good Laboratory Practice (GLP). Under MAD, laboratory test results related to the safety of chemicals that are generated in an OECD member country in accordance with these instruments are to be accepted in all OECD member countries and a range of other countries adhering to MAD.

The OECD test guidelines are accepted internationally as standard methods for safety testing by industries, academia, governments and independent laboratories. They cover tests for physical-chemical properties, effects on biotic systems (ecotoxicity), environmental fate (degradation and accumulation) and health effects (toxicity). These guidelines are regularly updated, and new test guidelines are developed and added, based on specific regulatory needs. This happens in cooperation with experts from regulatory agencies, academia, industry, environmental and animal welfare organizations.

The OECD GLP principles provide quality assurance concepts concerning the organization of test laboratories and the conditions under which laboratory studies are planned, performed, monitored, and reported.

Alternative testing

The use of animal testing for risk assessment has been a matter of debate for a long time, first of all for ethical reasons, but also because of the costs of animal testing and the difficulties in translating the results of animal tests to the human situation. Therefore, there is political and societal pressure to develop and implement alternative methods to replace, reduce and refine animal testing. In some legal frameworks such as the EU cosmetics regulation, the use of experimental animals is already banned. Under the EU chemicals legislation REACH, experimental animal testing is a last resort option. In 2017, the number of animals used for the first time for research and testing in the EU was just below 10 million. Twenty-three percent of these animals were for all regulatory uses, of which approximately one-third was for toxicity, pharmacology and other safety testing (850,000 animals) for industrial chemicals, food and feed chemicals, plant protection products, biocides, medicinal products and medical devices (European Commission, 2020).

Alternative methods include the use of (quantitative) structure-activity relationships ((Q)SARs; i.e., theoretical models to predict the physicochemical and biological (e.g. toxicological) properties of molecules from the knowledge of chemical structure), in vitro tests (section 4.3.9.II; preferably with cells/cell lines, organs or tissues of human origin) and read-across methods (using toxicity data on structurally related chemicals to predict the toxicity of the chemical under investigation). In Europe, the European Union Reference Laboratory for alternatives to animal testing (EURL_ECVAM) has an important role in the development, validation and uptake of alternative methods. It is an important contributor to the OECD Test Guideline Programme; a number of OECD test guidelines are now based on non-animal tests.

Since alternative methods do not always fit easily in current regulatory risk assessment and standard setting approaches, there is also a huge effort to develop testing strategies in which the results of alternative tests are combined with toxicokinetic information and information on the mechanism of action, adverse outcome pathways (AOPs), genetic information (OMICS), read-across and in vitro in vivo extrapolation (IVIVE). Such methods are also called: Integrated Approaches to Testing and Assessment (IATA) or intelligent testing strategies (ITS). These will help in making alternative methods more acceptable for regulatory purposes.

Core elements of toxicity testing

Currently, there are around 80 OECD Test guidelines for human health effects, including both in vivo and in vitro tests. The in vivo tests relate to acute (single exposures) and repeated dose toxicity (28 days, 90 days, lifetime) for all routes of exposure (oral, dermal, inhalation), reproductive toxicity (two generations, (extended) one generation, developmental (neuro)toxicity), genotoxicity, skin and eye irritation, skin sensitization, carcinogenicity, neurotoxicity, endocrine disruption, skin absorption and toxicokinetics. The in vitro tests concern skin absorption, skin and eye irritation and corrosion, phototoxicity, skin sensitization, genotoxicity and endocrine disruption.

Important elements of these test guidelines include the identity, purity and chemical properties of the test substance, route of administration, dose selection, selection and care of animals, test duration, environmental variables such as caging, diet, temperature and humidity, parameters studied, presentation and interpretation of results. Other important issues are: good laboratory practice (GLP), personnel requirements and animal welfare.

Test substance

The test substance should be accurately characterized. Important elements here are: chemical structure(s), composition, purity, nature and quantity of impurities, stability, and physicochemical properties such as lipophilicity, density, vapor pressure.

Route of administration

The three main routes of administration used in experimental animal testing are oral, dermal and inhalation. The choice of the route of administration depends on the physical and chemical characteristics of the test substance and the predominant route of exposure of humans.

Dose and dose selection

The selection of the dose level depends on the type of study. In general, studies require careful selection and spacing of the dose levels in order to obtain the maximum amount of information possible. The dose selection should also consider and ensure that the data generated is adequate to fulfill the regulatory requirements across OECD countries as appropriate (e.g., hazard and risk assessment, classification and labelling, endocrine disruption assessment, etc.).

To allow for the determination of a dose-response relationship, the number of dose levels is usually at least three (low, mid, high) in addition to concurrent control group(s). Increments between doses generally vary between factors of 2 and 10. The high dose level should produce sufficient evidence of toxicity, however without severe suffering of the animals and without excess mortality (above 10%) or morbidity. The mid dose should produce slight toxicity and the low dose no toxicity. Toxicokinetic data and tests already performed, such as range-finding studies and other toxicity studies, can help in dose selection. Measurement of dose levels and concentrations in media (air, drinking water, feed) is often recommended, in order to know the exact exposure and to detect mistakes in the dosing.

Animal species

Interspecies and intraspecies variation is a fact of life even when exposure route and pattern are the same. Knowledge of and experience with the laboratory animal to be used is of prime importance. It provides the investigator with the inherent strengths and weaknesses of the animal model, for instance, how much the model resembles humans. Although the guiding principle in the choice of species is that it should resemble humans as closely as possible in terms of absorption, distribution, metabolic pattern, excretion and effect(s) at the site, small laboratory rodents (mostly rats) of both sexes are usually used for economic and logistic reasons. They additionally provide the possibility of obtaining data on a sufficient number of animals for valid statistical analysis. For specialized toxicity testing guinea pigs, rabbits, dogs and non-human primates may be used as well. Most test guidelines specify the minimum number of animals to be tested.

Test duration

The response of an organism to exposure to a potentially toxic substance will depend on the magnitude and duration of exposure. Acute or single-dose toxicity refers to the adverse effects occurring within a short time (usually within 14 days) after the administration of a single dose (or exposure to a given concentration) of a test substance, or multiple doses given within 24 hours. In contrast, repeated dose toxicity comprises the adverse effects observed following exposure to a substance for a smaller or bigger part of the expected lifespan of the experimental animal. For example, standard tests with rats are the 28-day subacute test, the 90-day semi-chronic (sub-chronic) test and the 2-year lifetime/chronic test.

Diet

Composition of the diet or the nature of a vehicle in which the substance is administered influences physiology and as a consequence, the response to a chemical substance. The test substance may also change the palatability of the diet or drinking water, which may affect the observations, too.

Other environmental variables

Housing conditions, such as caging, grouping and bedding, temperature, humidity, circadian-rhythm, lighting and noise, may all influence animal response to toxic substances. OECD and WHO have made valid suggestions in the relevant guidelines for maintaining good standards of housing and care. The variables referred to should be kept constant and controlled.

Parameters studied

Methods of investigation have changed dramatically in the past few decades. A better understanding of physiology, biochemistry and pathology has led to more and more parameters being studied in order to obtain information about functional and morphological states. In general, more parameters are studied in the more expensive in vivo tests for longer durations such as reproductive toxicity tests, chronic toxicity tests and carcinogenicity tests. Nowadays, important parameters to be assessed in routine toxicity testing are biochemical organ function, physiological measurements, metabolic and haematological information and extensive general and histopathological examination. Some other important parameters that lately gained more interest, such as endocrine parameters or atherogenic indicators, are not or not sufficiently incorporated in routine testing.

Presentation and evaluation of results

Toxicity studies must be reported in great detail in order to comply with GLP regulations and to enable in-depth evaluation by regulating agencies. Electronic data processing systems have become indispensable in toxicity testing and provide the best way of achieving the accuracy required by the internationally accepted GLP regulations. A clear and objective interpretation of the results of toxicity studies is important: this requires a clear definition of the experimental objectives, the design and proper conduct of the study and a careful and detailed presentation of the results. As there are many sources of uncertainty in the toxicity testing of substances, these should also be carefully considered.

Toxicity studies aim to derive insight into adverse effects and possible target organs, to establish dose-response relationships and no observed adverse effect levels (NOAELs) or other intended outcomes such as benchmark doses (BMDs). Statistics are an important tool in this evaluation. However, statistical significance and toxicological/biological significance should always be evaluated separately.

Good laboratory practice

Non-clinical toxicological or safety assessment studies that are to be part of a safety submission for the marketing of regulated products, are required to be carried out according to the principles of GLP, including both quality control (minimizing mistakes or errors and maximizing the accuracy and validity of the collected data) and quality assurance (assuring that procedures and quality control were carried out according to the regulations).

Personnel requirements and animal welfare

GLP regulations require the use of qualified personnel at every level. Teaching on the subject of toxicity has improved tremendously over the last two decades and accreditation procedures have been implemented in many industrialized countries. This is also important because every toxicologist should feel the responsibility to reduce the number of animals used in toxicity testing, to reduce stress, pain and discomfort as much as possible, and to seek for alternatives, and this requires proper qualifications and experience.

Relevant sources and recommendations for further reading:

European Commission (2020). 2019 Report on the statistics on the use of animals for scientific purposes in the Member States of the European Union in 2015-2017, Brussels, Belgium, COM(2020) 16 final

OECD Test guidelines for chemicals. https://www.oecd.org/env/ehs/testing/oecdguidelinesforthetestingofchemicals.htm

OECD Integrated Approaches to Testing and Assessment (IATA). http://www.oecd.org/chemicalsafety/risk-assessment/iata-integrated-approaches-to-testing-and-assessment.htm

Van Leeuwen, C.J., Vermeire, T.G. (eds) (2007). Risk assessment of chemicals: an introduction, Second edition. Springer Dordrecht, The Netherlands. ISBN 978-1-4020-6101-1 (handbook), ISBN 978-1-4020-6102-8 (e-book), Chapters 6 (Toxicity testing for human health risk assessment), 11 (Intelligent Testing Strategies) and 16 (The OECD Chemicals Programme). DOI 10.1007/978-1-4020-6102-8

WHO, International Programme on Chemical Safety (1978). Principles and methods for evaluating the toxicity of chemicals. Part I. World Health Organization, Environmental Health Criteria 6. IPCS, Geneva, Switzerland. https://apps.who.int/EHC_6

World Health Organization & Food and Agriculture Organization of the United Nations (‎2009)‎. Principles and methods for the risk assessment of chemicals in food. World Health Organization, Environmental Health Criteria 240, Chapter 4. IPCS, Geneva, Switzerland. https://apps.who.int/EHC_240_4