Determination of toxicity

The determination of toxicity is understood to mean the determination of the toxicity or harmfulness - the toxicity - of a substance . The determination of toxicity is based on methods that have been recognized and recommended by the responsible international bodies (in particular the OECD , see OECD guidelines for the testing of chemicals ). In most cases, toxicity determinations are carried out using animal experiments . In general, toxicity determinations must be carried out according to the rules of Good Laboratory Practice .

Toxicological Endpoints

Toxicity studies include the chemical concentrations or amounts of the substances tested, information on observed effects and duration of exposure and usually relate to a specific toxicological endpoint.

Some examples of such endpoints are:

EC ₅₀ - Effective Concentration 50%: dose that triggers a defined effect other than death in 50% of a test population.
LC ₅₀ - Median Lethal Concentration: Lethal concentration in water, soil or air at which 50% of the test organisms die within a certain observation period.
LD ₅₀ - Median Lethal Dose: Lethal dose at which 50% of all test animals given a certain amount of poison die.
TD ₅₀ - Median Toxic Dose: Toxic dose at which 50% of the test organisms show signs of toxicity within a certain observation period.
LOAEL - Lowest Observed Adverse Effect Level: Lowest dose of an administered chemical substance for which damage was still observed in animal experiments .
LOEL - Lowest Observed Effect Level: Lowest dose of an administered chemical substance for which effects werestillobservedin animal experiments.
NOAEL - No Observed Adverse Effect Level: Highest dose of a substance that does not leave any recognizable or measurable damage , even with continuous intake.
NOEL - No Observed Effect Level: Highest dose of a substance that does not leave any recognizable or measurable effects , even with continuous exposure.

The difference between effect and damage is important for drugs , for example ; a drug should show a pharmacological effect at a dose at which no toxic damage is to be feared.

acute toxicity

Acute toxicity comprises the harmful effects that occur within a specified time (usually 14 days) after administration of a single dose of a substance .

The acute toxic effects as well as the organ or system toxicity of a substance can be assessed using a series of toxicity tests, which allow initial conclusions to be drawn about the toxicity after a single dose.

Depending on the toxicity of the substance, a limit test or a full test can be considered.

Acute oral toxicity: Fixed dose method

Fixed doses of 5, 50, 300 and 2,000 mg / kg are administered to groups of test animals of one sex in a multi-step process in accordance with OECD Guideline 420. The starting dose is chosen on the basis of a preliminary study that is likely to cause certain signs of toxicity without causing severe toxic effects or mortality . Additional groups of experimental animals may be given higher or lower fixed doses depending on whether signs of toxicity or mortality are evident. This procedure is continued until the dose is determined which is obviously toxic or which has caused the death of at most one test animal, or until no effects can be recognized at the highest dose or until the death of test animals occurs at the lowest dose.

Acute Oral Toxicity: Acute Toxic Class Method

The experimental principle of the Acute Toxic Class according to OECD Guideline 423 consists in gaining sufficient information about the acute toxicity of the test substance in a multi-step process using a minimum number of animals per individual step to enable its classification. The substance is administered orally to a group of test animals at one of the specified doses. The substance is tested in a multi-step process, with three animals of the same sex (usually females) being used for each step. The occurrence or non-occurrence of test substance-related deaths in the animals treated in one step is determined by the next step, i. H.:

whether no further tests are required,
whether three other animals should be treated with the same dose,
whether the next step should be carried out on three other animals with the next higher or next lower dose.

The method enables a test substance to be classified within a series of toxicity classes that are defined by fixed LD ₅₀ limit values.

Acute toxicity (inhalation)

According to OECD Guideline 403, several groups of test animals are exposed to the test substance in graduated concentrations for a certain period of time, one concentration per group. The observed effects and deaths are then recorded. Animals that die during the experiment and those that survived at the end of the experiment are dissected.

Acute toxicity (dermal)

According to OECD guideline 402, the test substance is applied to the skin in graduated doses of several groups of test animals, one dose per group. The symptoms of poisoning and deaths observed are then recorded. Animals that die during the experiment and those that survived at the end of the experiment are dissected.

Acute toxicity: Skin irritation / corrosion

According to OECD Guideline 404, the test substance is applied to the skin of a test animal in a single dose; untreated skin areas serve as controls. The degree of irritation / corrosion is determined and assessed at predetermined intervals and then described in order to be able to make a comprehensive assessment of the effect. The observation period should be sufficient to fully capture the reversibility or irreversibility of the effects.

Acute toxicity: Eye irritation / corrosion

According to OECD guideline 405 (see also Draize test ), the test substance is applied in a single dose to one eye of each test animal; the untreated eye serves as a control. The degree of irritation / corrosion is determined by assessing damage to the conjunctiva , cornea and iris using a point scale at predetermined time intervals . In addition, other eye reactions and systemic damage are also described in order to fully assess the effects. The observation period should be sufficient to fully capture the reversibility or irreversibility of the effects.

Repeated dose toxicity / subacute, subchronic and chronic toxicity

Toxicity repeated dose / subchronic toxicity comprises the adverse effects as a result of repeated experimental animals daily administration or exposure to a chemical substance occur. The duration of treatment extends over a short time in relation to the species-specific lifetime.

The repeated dose toxicity test evaluates the toxic effects of repeated exposure . The necessity of careful clinical observation of the animals must be emphasized in order to obtain as much data as possible. These tests should help identify the target organs of the toxic effects and the toxic and non-toxic doses . Further in-depth studies of these aspects may be required in the long-term studies.

Sub-Chronic Oral Toxicity Study: 90-day repeated oral toxicity study in rodents

When assessing and evaluating the toxic characteristics of a chemical substance, the determination of the subchronic toxicity for repeated oral administration of active substances can be carried out after initial toxicity data based on tests for acute toxicity or 28-day tests according to OECD Guideline 407 for repeated toxicity Administration were achieved. The 90-day study according to OECD Guideline 408 (rodents) or 409 (non-rodents) provides information on possible health damage that can result from repeated exposure over a longer period, including development from weaning to adulthood. The study also provides information on the most important toxic effects, shows the target organs and possible bioaccumulation and can contribute to the derivation of a NOAEL that can be used to select the doses for studies of chronic toxicity and to establish safety criteria for human exposure.

The method also focuses on neurological endpoints and provides evidence of immunological and reproductive effects. Furthermore, the need for careful clinical observation of the animals is emphasized in order to obtain as much information as possible. The aim of this study was to identify chemical substances with potential neurotoxic and immunological effects as well as effects on the reproductive organs, which may require further investigations.

The test substance is administered daily over a period of 90 days in graduated doses to several groups of test animals, one dose level per group. During the period of administration, the animals are carefully observed for signs of toxicity. Animals that died or were killed during the test are dissected. At the end of the test, all animals still alive are killed and also dissected.

Mutagenicity (genotoxicity)

Mutagenicity or genotoxicity refers to the induction of permanent heritable changes in the amount or structure of the genetic material of cells or organisms. These changes, so-called mutations , can affect a single gene or gene segments, a gene block, or entire chromosomes . The effects on whole chromosomes can be structural and / or numerical in nature .

Reverse mutation test using bacteria

In the reverse mutation test on bacteria, also known as the Ames test , according to OECD guideline 471, strains of Salmonella typhimurium and Escherichia coli , which require a specific amino acid , are used to detect point mutations that involve the substitution, addition or deletion of one or more DNA base pairs . The reverse mutation test carried out using bacteria is based on the detection of mutations, which reverse mutations in the respective strains and restore the functional capacity of the bacteria to synthesize an essential amino acid. The revertant bacteria can be recognized by their ability to grow without the amino acid required by the parent strain.

Point mutations are the cause of numerous human genetic diseases . There is some evidence that point mutations in oncogenes and tumor suppressor genes of somatic cells are involved in the development of cancer in humans and laboratory animals. The reverse mutation test on bacteria is not very time-consuming, inexpensive and relatively easy to carry out.

Many test strains have several characteristics that make them more sensitive to mutation detection, including reactive DNA sequences at the reversion sites, increased cell permeability to large molecules, and elimination of DNA repair systems or an increase in defective DNA repair processes. The specificity of the test strains can provide valuable information about the types of mutations caused by genotoxic agents. A very large body of results is available for reverse mutation testing using bacteria for a wide variety of structures, and well-proven methods have been developed to test chemicals with different physico-chemical properties, including volatile compounds.

In vitro test for chromosomal aberrations in mammalian cells

The in vitro test for chromosome aberrations according to OECD guideline 473 is used to detect agents that cause structural chromosome aberrations in mammalian cell cultures. A distinction must be made between structural chromosome type and chromatid type aberrations. In the majority of chemical mutagens , the aberrations are of the chromatid type, but chromosome type aberrations also occur. An increase in polyploidy may be an indication that a chemical is causing numerical aberrations . However, this method is not intended for measuring numerical aberrations and is therefore not routinely used for it.

Chromosome mutations and similar processes are the cause of numerous human genetic diseases. There is some evidence that chromosome mutations and similar processes that trigger changes in oncogenes and tumor suppressor genes in somatic cells are involved in the development of cancer in humans and laboratory animals.

Cultures from established cell lines and cell strains or primary cell cultures can be used in the in vitro chromosome aberration test . The cells used are selected from the point of view of their ability to grow in culture, their karyotype stability, the number of chromosomes, the number of chromosomes and the spontaneous frequency of chromosome aberrations. Tests carried out in vitro usually require the addition of an exogenous foreign substance metabolism system such as the S9-mix . With this system, however, the in vivo conditions in mammals cannot be fully understood. It is essential to avoid conditions where positive results occur which do not reflect intrinsic mutagenicity and may from changes in pH or osmolality or high levels of cytotoxicity originate.

This test is used to detect possible mutagens and carcinogens in mammalian cells. Many chemical compounds that test positive have carcinogenic effects in mammals, but there is no absolute correlation between test and carcinogenicity. The correlation depends on the chemical class, and there is increasing evidence that certain carcinogens are undetectable by this test, as their effects appear to be due to mechanisms other than direct DNA damage.

The treatment of the cell cultures with the test substance takes place with and without the addition of a foreign substance metabolism system. After a predetermined period of time, the cell cultures are treated with a spindle poison (e.g. colcemid or colchicine ), harvested and stained, after which the metaphase cells are microscopically examined for chromosomal aberrations.

In vitro test for gene mutations in mammalian cells

The in vitro test for gene mutations according to OECD guideline 476 is used to detect agents that can produce gene mutations in mammalian cell cultures. Cell lines frequently used in this test are mouse lymphoma cells and CHO cells as well as human lymphoblastoid cells. Usually, mutations in the genes of thymidine kinase (TK), hypoxanthine-guanine phosphoribosyltransferase (HPRT) or xanthine-guanine phosphoribosyltransferase (XPRT) are examined. The TK, HPRT and XPRT tests detect a different spectrum of genetic events. Cells that lack thymidine kinase due to a mutation are resistant to the cytotoxic effect of the pyrimidine analog trifluorothymidine (TFT). Mutant cells survive when treated by TFT, while unmutated cells are killed. Similarly, cells with mutations in HPRT or XPRT are resistant to the purine analogues 6-thioguanine (TG) or 8-azaguanine (AG), while non-mutated cells are killed. The most common and fastest test combination examines mutations in the TK gene in mouse lymphoma cells ( ML / TK test ).

The treatment of the cell cultures with the test substance takes place with and without the addition of a foreign substance metabolism system. The viability of the cells is measured. The culture conditions are chosen so that normally almost all cells die after treatment with the cell toxins TFT, TG or AG. After prolonged culture, surviving cells form visible colonies that can be counted; the mutagenicity of the test substance can be derived from the number of colonies. The size of the colonies is often used for evaluation.

In this test, too, conditions should be avoided in which false positive results occur which do not reflect the intrinsic mutagenicity of the test substance and which may result from changes in the pH value or osmolality or from severe cytotoxicity.

In vivo test for chromosomal aberrations in mammalian bone marrow cells

The in vivo test for chromosome aberrations in mammalian cells according to OECD guideline 475 is used to detect structural chromosomal aberrations that are triggered by the test substance in the bone marrow of mammals, usually rodents. A distinction must be made between structural chromosome and chromatid type aberrations. An increase in polyploidy may be an indication that a chemical is causing numerical aberrations. In the majority of chemical mutagens, the aberrations are of the chromatid type, but chromosome type aberrations also occur. Chromosome mutations and similar processes are the cause of numerous human genetic diseases. There is some evidence that chromosome mutations and similar processes that trigger changes in oncogenes and tumor suppressor genes are involved in the development of cancer in humans and in experimental systems.

Rodents are routinely used in this test. The target tissue is the bone marrow, as it is a vascular tissue with a population of rapidly proliferating cells that can be easily isolated and processed. Other experimental animals and target tissues are not the subject of this method.

This chromosome aberration test is of particular relevance for the assessment of the mutagenic properties, as it enables factors of in vivo metabolism, pharmacokinetics and DNA repair processes to be taken into account , even if these differ in the individual species and tissues. An in vivo test is also useful for further investigation of a mutagenic effect found in in vitro tests.

If there is evidence that the test substance or a reactive metabolite is not reaching the target tissue, this test is not appropriate.

The animals receive the test substance by means of a suitable application form and are sacrificed at a suitable point in time after the treatment. Before being killed, the animals are treated with a spindle poison (e.g. colchicine or colcemid). Chromosomes are then prepared from the bone marrow cells and stained, and the metaphase cells are examined for chromosomal aberrations.

In vivo mammalian red cell micronucleus test

The in vivo micronucleus test in mammals according to OECD guideline 474 is used to detect damage caused by the test substance in the chromosomes or in the mitotic apparatus of erythroblasts by analyzing the erythrocytes from the bone marrow and / or the peripheral blood of animals in the Usually rodents.

The purpose of the micronucleus test is to detect substances that cause cytogenetic damage that lead to the formation of micronuclei with remaining chromosome fragments or entire chromosomes.

When a bone marrow erythroblast develops into a polychromatic red blood cell, the main nucleus is expelled. However, any micronucleus that may have formed can remain in the otherwise enucleated cytoplasm. The visualization of the micronuclei is made easier in these cells because they do not have a main nucleus. An increase in the incidence of polychromatic micronucleated erythrocytes in treated animals suggests that chromosome damage has been caused. Rodent bone marrow is routinely used in this test because polychromatic erythrocytes are produced in this tissue. For the determination of non-mature (polychromatic) micronucleated erythrocytes in the peripheral blood, however, any species can be used for which the spleen has been proven to be unable to break down micronucleated erythrocytes or if there is sufficient sensitivity for the detection of agents that cause structural or numerical chromosome aberrations . Micronuclei can be differentiated using a number of criteria. This includes the determination of the presence or absence of a kinetochore or centromere DNA in the micronuclei. The main endpoint is the frequency of non-mature (polychromatic) micronucleated red blood cells. If the animals are treated continuously over a period of 4 weeks or longer, the proportion of mature (normochromatic) erythrocytes containing micronuclei in a certain number of mature erythrocytes in the peripheral blood can also be considered as the endpoint of the test.

The in vivo micronucleus test in mammals is of particular importance for the assessment of the mutagenic risk, as it enables factors relating to in vivo metabolism, pharmacokinetics and DNA repair processes to be taken into account, even if these are specific to the individual species or tissue types and genetic endpoints are different. An in vivo test is also useful for further investigations of the mutagenic effects determined by means of the in vitro system. If there is evidence that the test substance or a reactive metabolite is not reaching the target tissue, this test is not suitable.

The test substance is administered to the animals via a suitable application form . If bone marrow is used, they are killed at an appropriate time after treatment. The bone marrow is removed, dissected and stained. If peripheral blood is used, it is drawn at appropriate times after treatment and smears are prepared and stained. In experiments with peripheral blood, as little time as possible should elapse between the last exposure and cell collection. The preparations are examined for the presence of micronuclei.

Reproductive toxicity, carcinogenicity and developmental toxicity

Reproductive toxicity

The two-generation study to test reproduction over two generations according to OECD Guideline 416 is designed to provide general information about the effects of a test substance on the integrity and performance of the male and female reproductive systems; These include the function of the gonads , the oestrus cycle, mating behavior, conception, pregnancy , the birth process, suckling and weaning as well as the growth and development of the offspring. The study can also provide information on the effects of the test substance on neonatal morbidity and mortality, as well as preliminary data on pre- and postnatal developmental toxicity, and serve as a guide for follow-up studies. In addition to examining the growth and development of the F1 generation, this test method is also intended to assess the integrity and performance of the male and female reproductive systems as well as the growth and development of the F2 generation. Additional study segments may be included in this protocol for further information on developmental toxicity or functional deficits, using the method for the prenatal developmental toxicity test and / or the developmental neurotoxicity study as appropriate; however, these endpoints could also be examined in separate studies with the aid of suitable test methods.

The test substance is administered to various groups of male and female animals in graduated doses. P-generation males (parent animals) are dosed during growth and for at least one complete cycle of spermatogenesis (approximately 56 days in the mouse and 70 days in the rat) to clarify any adverse effects on spermatogenesis. Effects on the sperm cells are determined using various sperm parameters (for example sperm morphology and motility) and using tissue preparations with a detailed histopathological examination. If data on spermatogenesis are available from a previous study with repeated administration of sufficient duration, for example a 90-day study, males of the P generation need not be included in the assessment. However, it is recommended that samples or digital recordings of P-generation sperm are kept for possible later assessment. P-generation females are dosed during growth and through several complete oestrus cycles to assess any harmful effects of the test substance on the normal oestrus cycle. The test substance is administered to the parent animals (P) during the mating, during the resulting pregnancy and during the weaning of their F1 offspring. After weaning, the compound is administered to the F1 offspring during growth to adulthood, during mating and production of an F2 generation until the F2 generation is weaned.

Clinical observations and pathological examinations for signs of toxicity are made in all animals; A special focus is placed on the effects on the integrity and performance of the male and female reproductive systems as well as on the growth and development of the offspring.

Carcinogenicity

In the long-term carcinogenicity study according to OECD guideline 451, test animals are exposed to the test substance for a large part of their lifetime in order to observe whether the animals develop more tumors as a result of the treatment. Since these studies run over several years and require a large number of animals, such studies must be planned, carried out and statistically evaluated with particular care.

The most common carcinogenicity study is in rats and mice. Mice have to be treated with the test substance daily for one and a half years, rats for two years, and the animals have to undergo regular clinical examinations. At the end of the treatment, all animals are killed and carefully examined pathologically - macroscopically and microscopically. Three dose levels and an untreated control group are prescribed. The highest dose should have a slight toxic effect without reducing the lifespan. At least fifty female and fifty male animals should be treated per dose, so that the minimum number of animals in the rodent carcinogenicity test is 400 animals. The large number of animals is required to obtain the statistical power required to produce a significant result for a relatively rare event such as chemical carcinogenesis.

The problem with the rodent carcinogenicity test is that there are a number of substances that induce cancer only in rodents, but not in other mammals. So not all positive findings are relevant for humans. Conversely, however, all known carcinogens in humans are also carcinogenic in rats, so that if the result is negative in mice and rats, carcinogenicity in humans can be ruled out with certainty.

Developmental toxicity

The term developmental toxicity includes impairments in the development of the child

before and after birth due to exposure of a parent before conception,
due to exposure of the child during pregnancy or
after birth until reaching sexual maturity.

The classification of substances as developmentally toxic serves to warn pregnant women with an appropriate notice.

Essentially, developmental toxicity refers to the adverse effects during pregnancy or as a result of exposure to one of the parents. The corresponding effects can appear at any time in the life of the created organism. Among the most significant manifestations of developmental toxicity are

the death of the developing organism,
Deformities,
Stunted growth as well
Functional disorders.

literature

Klaus Aktories , Ulrich Förstermann, Franz Bernhard Hofmann, Klaus Starke : General and special pharmacology and toxicology . Founded by W. Forth, D. Henschler, W. Rummel, 11th edition, Elsevier, Urban & Fischer, Munich 2013, ISBN 978-3-437-42523-3 .
Hans Marquardt, Siegfried Schäfer, Holger Barth: Toxicology . 3rd edition, Wissenschaftliche Verlagsgesellschaft, Stuttgart 2013, ISBN 978-3-8047-2876-9 .
Catherine A. Harris, Alexander P. Scott, Andrew C. Johnson, Grace H. Panter, Dave Sheahan, Mike Roberts, John P. Sumpter: Principles of Sound Ecotoxicology. In: Environmental Science & Technology . 2014, pp. 3100–3111, doi : 10.1021 / es4047507 ( PDF ).