gms | German Medical Science

GMS Current Topics in Otorhinolaryngology - Head and Neck Surgery

Deutsche Gesellschaft für Hals-Nasen-Ohren-Heilkunde, Kopf- und Hals-Chirurgie e.V. (DGHNOKHC)

ISSN 1865-1011

Toxicological Assessment of Noxious Inhalants

Review Article

  • corresponding author N. H. Kleinsasser - Klinik und Poliklinik für Hals-Nasen-Ohrenkranke der Universität Regensburg
  • A. W. Sassen - Klinik und Poliklinik für Hals-Nasen-Ohrenkranke der Universität Regensburg
  • B. W. Wallner - GenPharmTox BioTech AG, Martinsried
  • R. Staudenmaier - Klinik und Poliklinik für Hals-Nasen-Ohrenkranke der Universität Regensburg
  • U. A. Harréus - Klinik und Poliklinik für Hals-Nasen-Ohrenkranke der Ludwig-Maximilians-Universität München
  • E. Richter - Walther Straub-Institut für Pharmakologie und Toxikologie der Ludwig-Maximilians-Universität München

GMS Current Topics in Otorhinolaryngology - Head and Neck Surgery 2004;3:Doc03

Die elektronische Version dieses Artikels ist vollständig und ist verfügbar unter:

Veröffentlicht: 28. Dezember 2004

© 2004 Kleinsasser et al.
Dieser Artikel ist ein Open Access-Artikel und steht unter den Creative Commons Lizenzbedingungen ( Er darf vervielfältigt, verbreitet und öffentlich zugänglich gemacht werden, vorausgesetzt dass Autor und Quelle genannt werden.


In the past centuries mankind has been exposed to various forms of air pollution not only at his occupational but also in his social environment. He mainly gets exposed with these pollutants through the respiratory organs and partially absorbs them into the body. Many of these airborne substances can be harmful for humans and some of them may account for tumorigenic effects.

The following essay describes the main features of toxicological assessment of inhalative environmental and workplace xenobiotics. The essay also explains relevant characteristics and limit values of noxious compounds and gases and depicts modern testing methods. To this end, emphasis is given on methods characterizing the different stages of tumorigenic processes. Various test systems have been developed which can be used in vivo, ex vivo or in vitro. They are to a great part based on the evidence of changes in DNA or particular genes of cells. Among others they have highlighted the impact of interindividual variability on enzymatic activation of xenobiotics and on susceptibility of the host to tumor diseases.

Unfortunately, for many inhalative environmental noxious agents no sufficient risk profiles have been developed. The completion of these profiles should be the goal of toxicological assessment in order to allow reasonable socioeconomic or individual-based risk reduction.

Keywords: xenobiotics, toxicity, genotoxicity, risk analysis, testing methods, limit values, work-related cancer, environmental medicine

1. Introduction

Aerum corumpere non licet

It is prohibited to pollute the air

(After Justinian, Century A.D., narrated in Corpus juris civilis)

Over the past two centuries industrialization has brought numerous additional harmful substances into the atmosphere of a person's life. Today people are particularly exposed to airborne particles and gases, which until now have played no or just a minor role. The beginning of this era has been dominated by mining dusts, gases and aerosols mainly from smelting. Later on, various manufacturing processes releasing considerably harmful substances into the atmosphere have come along. In the same time frame, the social attitude towards stimulants such as alcohol and tobacco has changed dramatically. The upper respiratory airways are particularly exposed to these harmful substances. They are either damaged directly or serve as a port of entry and/or barrier. In the middle of the 19th century Max von Pettenkofer and his students already reported on the gas exchange between the environment and the human organism [1]. The correlation between exposure to volatile occupational and environmental noxious inhalants and human diseases soon became apparent through epidemiological observations as well as through control of exposed laborers. Already in 1903 more than 50 cases of bladder cancer have been reported in workers of the BASF company exposed to aromatic amines during manufacture of aniline. About that time company medical officers in large scale-industries have been established, in order to prevent adverse health effects on laborers in the working process. Another cornerstone has been the awareness of the importance of latency of possibly harmful and tumorigenic effects of occupational - and environmental pollutants. This can be placed in the mid 20th century [1]. The consideration of latency made it possible to show correlations between occupational and social exposure to harmful substances and diseases, to take into account other concomitant circumstances such as diet and lifestyle (e.g. [2], [3], [4], [5]), and to depict diseases as consequences of these factors (e.g. [6], [7], [8], [9]). The identification of specific effects of harmful substances is of particular importance, e.g. in order to recognize correlations between certain tumor entities and specific exposures to harmful substances [10], [11], [12], [13]. The discovery of the mechanisms of damage by toxic substances, including supporting factors such as special pH conditions in certain mucous membrane compartments, is in progress. In recent years, prevention of these diseases by describing possible risk factors of the compounds themselves and by the higher individual susceptibility to disease development has gained increasing importance [14], [15], [16], [17], [18].

1.1. Toxicity

"Toxic" or harmful inhalative substances cause various different effects. Local changes in the skin and mucous membranes can occur which are caused by direct exposure or sensitization. Changes in the genes of somatic cells of the epithelium may lead to mutations which eventually develop to local tumors. These are to be discerned from systemic effects in distant parts of the body part which require uptake of the harmful substances. On the one side one can think of germline mutations leading to impairment or disease in following generations. On the other side, somatic mutations may give rise to cancer, e.g. in the urinary bladder after exposure to aryl amines. The individual enzyme configuration plays an important role in development of this cancer [19], [20], [21], [22], [23], [24], [25], [26], [27].

1.1.1. Skin and Mucous Membrane Irritation and Sensitization

Toxic skin diseases and irritations of mucous membranes require different forms of inflammatory reactions. An acute, non allergic irritation of the epithelium can occur. Also cumulative effects or deeper irritations of the epithelium with necrosis and scar formation may be seen. Sensitizations can occur which consequently lead to contact urticaria or to an irritative contact urticaria or mucositis. While the symptoms of an allergic genesis range from slight skin pruritus to generalized urticaria with rhinitis, asthma and anaphylactic shock, the irritative inflammation reactions are characterized by direct tissue damage. No specific allergic reactions can be detected in this connection. Inflammations by toxic compounds can be considered special forms if they do not require sensitizations but still cause changes of the epithelium without a visible superficial irritation. Lichenification could be as possible consequence. At this point attention should be focused also on corrosions, caused by substances and liquids with extremely low or high pH values. In regard to acute changes in the epithelium, where the caustic substances are still on the surface, water application may result in the release of large quantities of energy thereby leading to further damage [28].

1.1.2. Genotoxicity, Cytotoxicity, Reproductive Toxicity and Chemical Carcinogenesis

"The acclimatization of mankind to his environment over the years of evolution has lead to xenobiotic-induced ... mutations with almost solely negative consequences" [29]. Modulating effects by industrial and environmental pollutants on DNA and genes of the cells can be defined as genotoxic. The term genotoxicity therefore describes various impacts on the genetic material. Aberrations of the chromatid type can be differentiated which include single and double strand breaks, gaps and base exchanges within the DNA. Aberrations of the chromosome type lead to, e.g., ring and acentric fragmentation [30]. Mutations can also be distinguished whether they affect the germline or somatic cells. The former cause hereditary diseases or fertility limitations and therefore possess reproductive toxic effects. Somatic cells may be affected either at the site of entry into the body or in the entire organism and thereby possibly suffering tremendously by this reallocation of harmful substances into certain compartments. This difference is of utmost relevance as a multiplicity of harmful substances undergo metabolic changes not only in the cells of the port of entry but also in the entire organism. So-called metabolic activation can even occur before the compounds reach their actual target cells. In this context interactions enzymes involved in phase I and phase II metabolism play a superior role. Depending on the interplay of these enzymes, initially nontoxic substances get activated and become toxic substances, whereas other noxious substances may be partly deactivated and therefore become harmless. Therefore, the availability and physiological function of both phase I and phase II metabolism is essential for the choice and assessment of testing systems for mutation research. Another important aspect is the existence of enzyme polymorphisms for phase I enzymes such as the cytochrome P 450 system and the alcohol and aldehyde dehydrogenases as well as phase II enzymes, e.g. glutathione-S-transferases, sulfotransferases, UDP glucuronosyl transferases and N-acetyltransferases [19], [23], [25], [31], [32], [33]. A lesion in DNA integrity can either be repaired, cause a change in the inherited information or leads to cell death. If the damaged genetic material gets only partly or not at all repaired, mutagenic changes will persist and result in subsequent transformation [34].

Further stages in tumor development, promotion and progression following tumor initiation, are also influenced by exogenous and endogenous factors such as the presence of specific repair enzymes [35]. Tumor promotors accelerate cancer development but the previous tumor initiation is obligatory. Although strong carcinogens can drive the organism through all tumor stages, promotors are mostly different noxious substances acting synergistically with the initiators. They are also organ specific but the effects are dose-dependent and reversible [36]. Some promoters can support tumor development in certain organs but may be inhibitor of carcinogenesis in others. Progression is defined as a change in the growth habit leading to autonomy and to a tendency to metastasize. In some cases genotoxic carcinogens do not only lead to initiation but can act as progressors.

Besides genetic causes of a tumor initiation, epigenetic initiation is possible. Here the phenotype of a differentiated somatic cell will be passed onto the daughter cell. This also accounts for the transfer of regulatory proteins, DNA methylation patterns and development of cell to cell interactions, which possibly result in initiation [36]. Other possible constituents of chemical carcinogenesis are co-, syn- and anticarcinogenesis. In cocarcinogenesis the sensitivity to an initiating noxious substances will be aggravated through another one given immediately prior or along with the carcinogen. Anticarcinogenesis takes place if the opposite is true, that is a lowering of sensitivity towards the carcinogens. Two compounds act syncarcinogenic when the second noxious substance has a carcinogenic effect in itself, e.g., tobacco-specific nitrosamines and ethanol [37].

1.1.3. Organ Toxicity

Besides the mucosa of the upper aero digestive tract, also the lungs should be mentioned belonging to the first organs in contact with and portal of entry for inhalative noxious substances. After uptake into the body, higher concentrations and extended residence times can be reached by reallocation into specific body compartments. Here, local factors come into play, e.g. a specific pH-milieu which can promote genotoxic effects. The consequences are specific tumors in typical locations, defined by the carcinogens [38]. Organ specific tumors may also develop independently from the route of administration. The development of lung tumors after exposure to the tobacco-specific nitrosamine 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a good example in this regard. Within the lung specific target cells could be identified, which contribute to the activation of NNK due to their specific enzyme configuration [39], [40], [41]. The goal of tests for chemoprevention of specific tumors is the suppression of this activation [42], [43], [44], [45], [46], [47].

1.2. Toxicokinetics

Toxicokinetics describes "what the body does to the toxic substances" [48]. It may be specifically defined as the quantitative change of substance concentrations in different body compartments. This requires knowledge about absorption, distribution, metabolism, storage and excretion of the compounds. Metabolism, storage and excretion can be combined to the term of elimination.

1.3. Toxicodynamics

The effects of toxic substances on the organism can be defined as toxicodynamics. The mechanisms of action and the potency of single substances are particularly of interest. Effects can be characterised as either acute or chronic, local or systemic, reversible or irreversible and primary or secondary. The illustration of effects using dose-response curves is an important application in toxicodynamics. These curves allow a quantitative registration of either effects at target site within the organism or effects on populations. In the half logarithmic diagram they show an S-shaped course. In the lower concentration range only a slightly increasing effect can be seen. In the middle, almost linear increasing area, a strong increase of the effect after dose cumulation becomes apparent. At higher concentrations a flattening of the curve can be seen as a result of the saturation of the effect of the toxic compound.

1.4. Epidemiology and International Studies

The correlation between the risks of harmful effects or carcinogenesis and the exposure to air pollution, e.g., to tobacco smoking, but also to different particles and volatile substances has been reported in multiple epidemiological studies. The European Community Respiratory Health Survey (ECRHS) and the Swiss Study on Air pollution and Lung Diseases in Adults (SAPALDIA) are examples of international studies. In that way it could be demonstrated that the risk of developing asthma and chronic bronchitis is increased in a passive smoking environment [49]. An international study of asthma and allergies in childhood (ISAAC) has the goal of symptom exploration and description of asthma, allergic rhino conjunctivitis and atopic eczema [50]. The study has been initiated to improve epidemiological research in these diseases by establishing standard methods, strengthening international co-operation and by augmenting data- exchange. Amongst other countries, research facilities of Germany, Ireland, the US, New Zealand, Oman and China participate in this study. The study is divided into 3 phases. In the first phase data on frequency and severity of asthma and allergy related diseases are gathered. On the basis of these data, correlations regarding causes of diseases are illustrated in the second phase. Additionally lung, blood and skin tests are conducted. The third phase contains a repetition of the first phase after 5 years. German studies evaluated so far [51], [52], show a strong correlation between exposure to exhaust emissions and increase of asthma, rhinitis and atopic eczema. A comparison of children living in Greifswald and Muenster showed regional differences in the distribution of symptoms which could be related to the use of different heating devices [53]. Overall the ISAAC study shows multiple indications of possible determinants of asthma and allergy triggering in children.

In the first half of the 90s of the last century, 140.000 participants of 22 countries have been interviewed and analyzed in the course of the European Community Respiratory Health Survey (ECHRS I). The reason for this explorative study was the search for risk factors explaining the observed increase of asthmatic diseases in many different countries which could not be traced back to genetic sources. From 1999 to 2001 the participants of the study have been contacted a second time (ECRHS II) to determine their symptomatic status and exposure to different factors such as tobacco smoke, animal hair, atmospheric load at work and air pollution. To some extent, dust has been analyzed for house dust mites and blood samples have been taken. The not yet finished analyses show a significant relationship between passive smoking and nocturnal or activity related dyspnea as well as bronchial reagibility. The impact of occupational exposures in causing asthma is strongly emphasized [54]. The ECRHS- Committee discovered large geographic differences regarding the incidence of asthma, atopy and bronchial hyperreagibility with a high distribution in the English-speaking area and a small incidence in the Eastern European and Mediterranean area [54].

The objectives of the SAPALDIA study (Swiss Study on Air Pollution and Lung Diseases in Adults) initiated in 1991 are the determination of the importance of most common symptoms of respiration dysfunction and to find out the best combinations of symptoms for diagnosing asthma. An extremely high specificity for the most significant symptoms such as dyspnea, chronic phlegm or cough became apparent. The results of the study suggest that certain combinations of symptoms allow a reliable diagnosis of asthma; their investigation is therefore of clinical importance [55]. Furthermore, in contrast to the results of various other study centers a clear correlation could be established between the mean lung function of participants and air pollution. Admittedly, this correlation varies due to different composition of air pollutants, e.g., regional NO2 content [56].

In a Meta analysis of more than 7600 affected patients, the relationship between lung cancer and tobacco consumption was investigated [57]. The correlation between exposure to wood dust and the increased incidence of sinusoidal adenocarcinomas has been summarized by the International Agency for Research on Cancer (IARC) [58]. This relationship was confirmed by multiple studies regarding wood dust but also in employees of the leather industry [59]. The increased exposure to diesel exhaust could be correlated to an increased risk of lung cancer [60], [61] and prostate cancer [62]. Other authors questioning these evidences [63], where not able to exclude partial interests of certain groups in the first place. Epidemiological studies have also demonstrated an increased incidence of multiple myelomas [64] and testicular cancer [65] in workers exposed to phthalates during polyvinyl chloride manufacture.

2. Test Methods for Toxicological Assessment of Noxious Inhalants

In principle, qualitative, semi-quantitative and quantitative testing can be differentiated. These can be conducted in vitro using isolated organ systems, single cells or functional elements, such as enzymes and mitochondria. Alternatively, tests are performed in vivo in the intact, living organism. Regarding a possible tumor development, attention should especially be turned to genes of somatic cells. Another criterion for the choice of test methods is the time period necessary for the experiments and the way of exposure to be analyzed. According to the choice of test methods acute toxic and chronic toxic effects on the targets can be differentiated. Additional parameters to be considered are cumulation and reversibility. Methodical overlapping between in vivo and in vitro settings may also occur. To evaluate several noxious substances, not only single tests but multiple complementary tests are used, also known as test batteries. The following is a description of basic assessments of a possible toxicological risk.

2.1. Dose Dependency of a Defined Toxicological Effect

A toxicity assay of a substance should give information about different criteria: at first it is essential to recognize possible signs of intoxication and to observe their time-dependent development in terms of documentation of first appearance, potentially delayed course, decay and reversibility of symptoms. Furthermore, dose-effect relationships and possible sex and organ specific differences should be depicted [66]. For effect description of a substance the following are considered standards:

2.1.1. No Observed Effect Level (NOEL or NEL)

Highest dose or maximal exposure-concentration without detectable signs of effect.

2.1.2. No Observed Adverse Effect Level (NOAEL)

Highest dose or maximal exposure-concentration without detectable harmful effects.

2.1.3. Mean Lethal Concentration in Room Air (LC 50 [mg/m 3 ])

In inhalation experiments on acute toxicity, the LC50 displays the concentration of a harmful substance in an experimental atmosphere, which causes the death of half of experimental animals. Therefore, the LC50 value is often used as a comparative measure of effect of toxic inhalants.

2.1.4. Mean Lethal Dose (LD 50 [mg/kg body weight])

To determine the LD50, defined amounts of harmful substances are administered to laboratory animals by either gavage or injection. The values for LD50 and LC50 represent statistically calculated single doses or concentrations leading to the death of half of the laboratory animals in a defined time frame of an acute toxicity test. This often used parameter is of limited significance as it does not give information about the steepness of the dose-dependent increase of toxicity.

2.1.5. Mean Effective Dose (ED 50 [mg/kg body weight])

Tests of acute effects with non-letal toxic end points can be described by ED50 values analogical to the LD50 values. This parameter is defined as the dose, which causes a defined non-lethal effect seen in half of the laboratory animals.

2.1.6. Mean Toxic Dose (TD 50 [mg/kg body weight/d])

This value also corresponds to non-lethal toxic effects, e.g., a chronic daily dose of a genotoxic compound leading to tumors in half of the animals.

2.2. In vivo Methods

The examinations are usually conducted in intact organisms, animals or humans. From these, ex vivo examinations have to be distinguished which are intermediate between in vivo and in vitro methods. Ex vivo methods take samples from treated or untreated animals or animals are sacrificed after exposure in order to examine complete organs or cell systems. Examples for these methods will be presented in chapter on in vitro methods.

Furthermore, a multiplicity of in vivo test methods conducted on laboratory animals is available including experiments on acute, subacute, subchronic and chronic toxicity, on reproductive toxicity and carcinogenicity. The time-dependent toxicity of substances is usually tested in laboratory animals. They are exposed to various concentrations/doses over certain time periods. Regarding inhalative toxicity, standardized chamber systems are implemented which are charged daily with a preferably constant respiratory air concentration of the test compounds over uniform time periods, e.g., 6 hours/day. The chamber volume should at least be 20 times larger than the laboratory animal. In these experiments possible damage to the fur, skin, eyes and mucous membranes, changes in respiratory, circulatory and motor function, body weight and behavior are recorded. Laboratory parameters include blood count, coagulation, serum enzymes and urinary parameters. In long-term experiments usually four groups are formed which are exposed to different concentrations. The highest concentration will be chosen so that clear toxic effects but a low lethality is expected. For adequate control, monitoring should be extended to defined time periods after completion of exposure and may include the administration of additional compounds or treatments. After termination of the experiments, the animals are dissected and organs are processed for histopathology.

To answer the question of reliability of the extrapolation of these animal experiments to the human situation, very often the mode of gassing is being discussed.

Roughly two forms can be differentiated: at first whole-body exposure by gassing the chamber in which the animal are kept or secondly nose-only exposure. By whole-body exposure the animal could take the compound not only by additional skin absorption but also orally by licking the contaminated fur. This is clearly different from the human situation as mankind covers and washes himself [67], [68] and should be prevented by nose-only exposure. On the other hand restricting the animal movement by exposing them in small tubes is more stressful and depending on the toxicants investigated may outweigh the advantage of this type of exposure.

2.2.1. Acute Toxicity (4 Hour Test)

To test acute toxic effects of noxious inhalants, single exposures over a defined period of time, usually 4 hours are chosen. During this time, the animals can be left without food supply, but the control of atmospheric oxygen content, air humidity and temperature goes without saying.

2.2.2. Subacute Toxicity (28 Days Test)

Testing for subacute toxicity particularly targets delayed effects, reversibility, adaptation and effects on specific organs. In this context, determination of the toxicokinetics of the substance including metabolism and enzyme induction/inhibition is of importance.

2.2.3. Subchronic Toxicity (90 Days Test)

In testing subchronic toxicity, special attention is given to possible target organs including the eyes, cumulative or adaptive effects, and towards the maximal tolerated dose (MTD) as well as the maximal dose tolerated without any visible effects (NOEL). The prolonged duration of the test requires larger test groups and longer post treatment observation periods to demonstrate possible reversibility or delayed appearance and persistence of effects.

2.2.4. Chronic Toxicity (Long-Term Test)

In such test arrangements animals are exposed in appropriate chambers for most of their lives but at least for 12 months. These experiments are performed in order to describe effects of substances or mixtures of substance, to which humans are exposed over extended time periods of years up to life time. This may apply to xenobiotics released from industrial processes, air polluting substances in exhaust emissions and voluntary as well as involuntary tobacco smoke exposure. Needless to say, target organs of toxic effects, maximal tolerated doses (MTD) and doses without noticeable effects (NOEL) are of particular interest in these long term tests. Tests for carcinogenicity of noxious substances are usually designed as long term experiments.

2.2.5. Test for Carcinogenicity

In vivo tests for possible carcinogenicity are of particular interest, if in vitro tests make such a harmful effect probable. A methodical challenge is the well known difference between the high tissue concentrations often used in in vitro tests which cannot be achieved in vivo. For example, the high toxicity of nicotine and carbon monoxide in tobacco smoke prevents the use of high concentrations in vivo making it difficult to produce lung tumors in animal experiments [44]. Nevertheless, a harmful effect in the human organism may be supposed, if target cells or tissues are reached by distribution of the noxious substances. Most studies of carcinogenicity are applied to long term test of toxicity.

2.2.6. Reproductive Toxicity

Tests for possible damages in the germline can either be controlled in the first or in the second following generation. For this, paternal and maternal toxicological effects are examined in comprehensive test arrangements. To obtain an exact breakdown after the damage, more differentiated tests are usually necessary which often do not allow more than the statement that progeny damage is possible. Further targets of investigation are the fertility of the parental generation, the course of pregnancy and lactation period and the progression of the succeeding generation. Time of birth and weight related to the time of birth are documented here as well as stillborn and killed offspring. If the observation is expanded to a second generation, the offspring of the first generation may also be exposed to contaminants and its following generation will be investigated in the same way.

2.2.7. Skin and Mucous Membrane Irritation Test

Examinations for irritation of the skin and mucous membranes in connection with possible noxious inhalants search for redness, ulceration and edema development in terms of acute effects. A classic experiment is the Draize test performed in the eye of a rabbit. Due to the possibly painful procedure for the animals it has become a controversial test and today it has been partially replaced by in vitro test methods. However, tests which only determine the cytotoxic capacity in cell cultures, are of little relevance for detecting irritating effects. A better consistency can be achieved using a second method which is conducted on the chorion allantois membrane of fertilized and incubated hen eggs (HET-CAM): after lifting off the egg shell, the chorion-allantois membrane is exposed to the test compounds. Evidence of possible toxicity such as membrane instability, protein and vascular changes are documented and listed on a chart allowing semi-quantitative evaluation of the stimulating potency of the test compound. Standardized reference compounds are included in this method [69], [70].

2.3. In vitro Methods

Nowadays, more and more methods requiring laboratory animals have been replaced. Test methods for description of toxicological risks in vitro are constantly improved and new methods are developed. Among these, are the Ames tests, other bacterial and cellular test systems, including mini-organ cultures and perfusion models of organs or organ slices.

2.4. Testing Methods for Evidence of Genotoxicity

In this section a variety of test methods for the description of cell damage and transformation, gene damage and mutation and also of chromosomal changes of bacteria, mammal cells and organ cultures will be described.

These test systems are applied not only in in vitro but also in vivo and ex vivo examinations.

2.4.1. DNA Adducts

Primary lesions of DNA the can be displayed by using radioactive labeled isotopes of the toxic compounds. Here, the degree of covalent bonds, a stable bond developed under separation of water, between a single nucleotide and a noxious substance is described. The degree is quantified by the Covalent Binding Index (CBI) relating the amount of non-extractable DNA adducts to the unmodified DNA and to the dose of the test compound.

A more universal method for testing DNA binding of unlabeled compounds or mixture of compounds is the introduction of radioactivity into the isolated monophosphate nucleotides bearing those adducts by phosphorylation with γ-32P-ATP. These radioactively labeled adducts are subsequently separated and radiographically quantified. This method is commonly referred to as 32P-postlabeling [71].

Adducts can not only be identified in single-cells in vitro. Another possibility consists also of ex vivo methods, in order to be able to determine DNA adducts for example as a cause of tobacco consumption in a human lymphocyte or mucosa cell of the upper aerodigestive tract of hemoglobin adducts as surrogate markers of exposure to possibly DNA damaging agents [72], [73], [74], [75]. The determination of adducts after in vivo exposure gives information not only about the correlation of concentrations of contaminants in the occupational and social environment but also about the individual susceptibility to either activate or inactivate genotoxic compounds.

2.4.2. DNA-Elution Test

Alkaline elution techniques allow the demonstration of DNA strand disruptions induced by test compounds. After dissolving the double-stranded DNA into single-strands the DNA is filtered through an appropriate fine-pored filter. Fragments of single-strand DNA can pass the membrane allowing subsequent quantification by UV detection. These techniques are conducted partially ex vivo on non-proliferating cells of most animal and human organs or organ systems.

2.4.3. Single Cell Microgelelectrophoresis Assay

The alkaline version of the single cell-microgelelectrophoresis is based on a similar principle as the alkaline elution techniques. In recent years this version has gained increased significance for risk assessment in ecogenotoxicology [76], [77], [78], [79], [80] and for determination of the effectiveness of chemotherapy [81]. This assay is also known as Comet assay, as the electrophoretically unsealed DNA looks like comets. As a short-time test method, it allows the illustration of DNA damages, which have been induced by biologically, physically and chemically toxic substances/environments on human cells. This test system was established to evaluate possible genotoxic effects of environmental and occupational pollutants as well as radiation-induced DNA damages. Furthermore it defines groups of individuals at increased risk for such damages [17]. All types of tissues which may be separatet into single cells can be used. Among human cells, lymphocytes of peripheral blood are most often applied because they are available by simple vein puncture and can easily be isolated using density gradient centrifugation.

Human mucosa of the upper aerodigestive tract, which is exposed to contaminants of respiratory air and food [82], to mastication and sucking activity [15], [79] and to oral hygiene and dental care devices, respectively, are also extremely useful for this type of analysis in terms of ex vivo testing. Investigating these possible target cells is of particular interest because mutagen-sensitivity tests with lymphocytes have proven to be unsatisfactory predictors of activities at the point of origin of malignant mucous membrane tumors [16].

The Comet assay in its alkaline version mainly verifies single-strand breaks (SSB), compared to double-strand disruptions (DSB). SSB are more often induced by genotoxic compounds. In addition, a small part of the measured DNA fragments can derive from an excision repair of the DNA [83]. Primarily this technique has been developed to evaluate the migration activity of single cells after radiation and under electrophoresis conditions [84]. Here, the DNA shows a migration towards the direction of the anode which is proportional to the degree of the induced damage.

Migration is made possible through damage of nuclear contents and the unwinding of the super coiling of DNA. DNA fragments possess an electrical polarization and migrate towards the anode. The higher the fragmentation, the larger is the DNA fraction arranged at the end of electrophoresis around the comet tail in relation to unoffended DNA located in the crown [Fig. 1]. The amount of DNA in the tail is multiplied by the mean stretch of way going into the Olive Tail Moment, which therefore demonstrates a quantitative degree of DNA strand disruptions. The Comet assay is a well established test method, which is available in multiple versions [85], [86].

The application of this test method for human epithelia of nose and oropharynx has been established in the German Cancer Research Center in Heidelberg [87], [88]. Without exposure to additional noxious substances in the acute test, this assay can also be used as a test method in terms of biomonitoring for the assessment of individual chronic exposure in vivo [89]. Furthermore, the assay provides information about specific sensitivities to contaminants, also known as mutagen sensitivity [82], [90], [91]. For this reason this test is applicable also ex vivo.

Besides the analysis of substance-induced strand disruptions, the ability of the cells for repair of such damage can be analyzed in a quantitative and chronological way with the Comet assay. In this context, intervals of different length should be maintained between incubation with the contaminant and lysis of the cell membranes. Depending on the in vitro capacity of the cell to repair, an adynamic comet tail will develop. These test methods have been presented for the target organ to come first in contact with most xenobiotics in humans, the mucous membranes of the upper aerodigestive tract [77]. In the presence of malign tumors, a decreased individual ability to repair DNA became apparent [92]. Possible intraindividual differences of DNA repair ability in mucosa cells and lymphocytes have been demonstrated as well as group-specific differences between patients with a squamous cell carcinoma of the oropharynx and tumor-free donors [72]. In addition, patients with a history of nasopharyngeal carcinoma have been compared to a control group without malignancy in regard to DNA repair ability after exposure to nitrosamine. No significant differences could be described [93].

A further improvement of the method represents its adaptation for use in organ cultures of human mucosa cells of the aerodigestive tract. This new model allows not only the prolonged and repeated incubation of mucosal cells within cell formations with genotoxic compounds but also to follow-up phases of repair. It therefore is able to simulate occupational exposure. This model approaches the in vivo situation of mucosa of the upper aerodigestive tract [Fig. 2] [94], [95]. First tests in our study group on metabolic competence of such mini-organ cultures verified the activity of cytochrome P450 enzymes over a extended period of time of 11 days (unpublished data).

2.4.4. Fluorescence in situ Hybridization (FISH)

By using the fluorescence in situ hybridization staining of specific gene loci with testing probes it is made possible allowing either complete chromosomes or single regions to be stained. Preliminary results showed the possibility of combining this method with methods detecting DNA strand disruptions. Particularly, a combination of FISH and Comet assay has been established in human mucosa cells [96]. It seems that this refined method allows a more accurate determination of the localization of strand disruptions and also allows the detection of particularly sensitive gene loci. Therefore, it is a promising extension of existing test batteries for the evaluation of the genotoxic potential of pollutants in human target cells such as mucosa cells and lymphocytes [78].

2.4.5. Unscheduled DNA Synthesis (UDS)

The test for unscheduled DNA synthesis takes place during a DNA repair. It allows the differentiation from planned DNA duplication within the normal cell cycle. The test method measures the increased incorporation of radiolabeled thymidine into the DNA which is due to repair damaged parts by excision and de novo synthesis by specific enzymes. The natural DNA replication is suppressed enzymatically to prevent false positive results by excessive incorporation of thymidine. This test is usually conducted on human lymphocytes, cell cultures or on hepatocytes from rodents. A detailed description of this method is available from the European Community guidelines [97].

2.4.6. Micronucleus Test

Micronuclei are defined as chromatin-containing particles outside the cell nucleus. If complete chromosomes or fragments are not incorporated during mitosis into the new cell nuclei, but appear in the following interphase as chromatin-particles in the cytoplasm [Fig. 3], micronuclei will develop. They are the result of genomic instability and contaminant-induced damage [98]. The micro nucleus test is used in cytogenetic studies, to determine chromosomal changes such as acentric chromosome or chromatid fragments or the lack of chromosomes in the anaphase. Lymphocytes of single individuals respond very differently to stimulation by phytohemagglutinine (PHA) which results in differing amounts of cell divisions in culture. Therefore, micronuclei are often counted only in cells appearing large and binuclear because of blocked cell division which is achieved by the addition of cytochalasin B. This compound represses cell division but does not produce micronuclei nor does it cause chromosomal damage. With this modification the micronucleus assay can be used for biomonitoring exposure to genotoxins in vivo [99], [100] but also for a determination of the mutagen-induced damage of the chromosomes in vitro [101]. Details of a standardized protocol is given in a European Community guideline [102], [103].

2.4.7. Ames Test (Salmonella Mutagenicity Test)

The detection of genotoxic effects of a compound in this test is based on backward mutations. The test can similarly be performed with different bacterial strains. Initially, the strains of bacteria used were not able to synthesize the amino acid histidine (histidine-deficient-mutants, his-). Through point mutations induced by genotoxic compounds these bacteria regain the ability so produce histidine and are thus independent of histidine supply in the nutrient medium. Because of a compromised DNA repair they indeed become more sensitive to further mutations and also form more unstable cell membranes. The quantitative evaluation of this test is based on the mount of colonies developing in the absence of added histidine. Only the backward mutated bacteria are able to produce colonies by multiplication. If the rate of backward mutation is higher compared to the spontaneous rate of mutation in controls, the test compound is classified as a mutagen and therefore regarded to be a potential carcinogen. The drawback of the basic methods was the inability of bacteria to metabolically activate most of the test substances. Therefore, bacteria were mixed with liver homogenates which take over this function. Lately, bacteria strains equipped with a different array of enzyme have become available making them more sensitive to certain contaminants. Also human enzymes such as those of the cytochrome P 450 system have been inserted into bacteria. This test is established as a quick and cheap assay for examining a large array of different chemicals from industry and environment for their genotoxic potential. However, false positive and false negative results make the test not sufficiently reliable. The test is also not feasible with human cells. A detailed description is given in a European Community guideline [102], [103], [104], [105].

2.4.8. Forward Mutation Tests

Mutations can cause an increased or diminished conversion of substances or a changed resistance potential of control cell subsequent to modification by substance-related toxicity. Some of the many tests are the guanine analog resistance test, the thymidine kinase test, the ouabain resistance test and the hypoxanthine phosphoribosyltransferase test (HPRT). For these tests also human cell lines such as fibroblasts can be used but mostly specificly designed cell lines are used which show adequate enzyme and protein patterns [102]. For the HPRT test V79 cells derived from lung tissue of a Chinese hamster are increasingly applied. HPRTransferase is an enzyme which converts free purine bases into nucleoside monophosphates and therefore makes them available for nucleic acid synthesis. Incubation with a modified nucleoside, 6-thioguanine, results in high toxicity of the cells. Mutant cells which after incubation with a genotoxin have lost HPRTransferase activity are being selected in this assay as they are resistant to 6-thioguanine toxicity [106].

2.4.9. Chromosome Aberration Test

Chromosome aberrations are defined as structural or numeric changes in chromosomes, which are visible by microscopic examination. Substances causing this effect are so-called clastogens. If these modified cells replicate, mutations develop. Possible reasons for chromosome aberrations are DNA double-strand disruptions, which are either generated directly, independent of replication, or are generated by dimer formation which depends on replication. Chromatid-type aberrations affecting only one sister-chromatid can be differentiated from the chromosome-type damage. Tests are performed in various proliferating cell lines including those of humans. Details are specified in a European Community guideline [102], [103].

2.4.10. Sister Chromatid Exchange Test (SCE)

During a sister chromatid exchange, a reciprocal DNA exchange between two sister-chromatids of a replicated in a double form existing chromosome occurs. The frequency of spontaneous SCE can be increased by the impact of genotoxic compounds. This test verifies DNA damages such as base alkylations and crosslinks but it does not show strand disruptions. In contrast to the Comet assay, this test depends on cell proliferation. Human lymphocytes and standardized cell lines of the Chinese hamster (V79 or CHO cells) are commonly used. The basic principle of this method is an alteration in the staining with bromdesoxyuridine which has inserted into only one DNA strand during replication but appears in the opposite strand together with a loss of staining in the parent strand after SCE [102]. This test is often used as a screening test and is standardized in a European Community guideline [97].

2.4.11. Cell Transformation Test

Compared to the so far described test systems dealing with changes in cells for example in the DNA section, this test analyzes malignant transformations. These are determined by means of morphological changes and by an increased loss of growth control in the confluence of colonies. Additional parameters are the development of surface proteins, changes in enzyme patterns and signal transduction chains. By showing even further endpoints compared to the previously described test methods, these transformation tests are important elements of many test batteries. Standardized fibroblast cell lines are mostly used for this test.

2.5. Dose Dependency of Various Toxicological Manifestations of a Substance

Toxic substances usually posses a dose-dependent spectrum of effects. Most single effects reach a maximum when the dose/concentration of the noxious substance is increased. Beyond this point no further increase in effect is possible. This can be explained for example with a receptor saturation. Subsequently other effects may become apparent [107]. Considering this, it becomes clear that there are no fatal substances per se, but almost for all substances there is a fatal dose. This supports Paracelsus (1493-1541) in his concept that "dosis sola facit venenum" ("the dose alone makes a thing poisonous") [1].

2.6. Exposure to Multiple Substances

The risk evaluation for exposure to environmental contaminants has proven difficult as the individual or the population usually is exposed to a multitude of chemicals. This problem is aggravated by possible antagonistic and non-specific dose ranges with potentially different effects. Usually only single effects are studied.

3. Risk Asessment of Various Toxicological Manifestations of a Substance

The assessment of a possible toxicological risk of a noxious inhalant has two important aspects. On the one hand, the substance-induced effects on the individual should be considered. On the other hand, a limitation of risk for the society as a whole should be achieved. For the latter a threshold of exposure must be defined which largely excludes health risk. Unfortunately, in most cases there are not enough data on humans available for a scientifically based risk assessment of airborne pollutants. Therefore, experimental data have to be used and extrapolated to the human situation [9].

Because political arguments are often involved in the social facets, meaningful scientific evaluations are oftentimes of no great concern. Newer temporary fashions are generally short-lived in our today's media and are seldom helpful. This is why at this point some fundamental considerations for a toxicological risk assessment of noxious inhalants are described.

3.1. Definition of a Toxicological Risk

According to the WHO, a risk can be defined as the expected frequency of undesirable effects which are initiated by an exposure to contaminants. A health hazard is considered as the probability that a chemical substance causes undesired effects on the health under actual conditions of production or application [107], [108].

3.2. Basic Principles of Risk Assessment

Chemical substances which proved to be carcinogenic in in vitro, ex vivo and in vivo in an animal experiment raise the question on how far the human can be exposed. Because there is no threshold value for genotoxic substances but only an extension of latency, probabilities are estimated when undesired effects of a substance appear. An increase of the spontaneous rate of a certain cancer type can be exemplified. In this connection, the undesired effects and the dimension of the exposed population should be known to be able to estimate the number of individuals who probably are affected by a certain type of cancer. In this calculation highly sensitive individuals in the population are not considered. Indications about individual susceptibilities are found in various sources and research establishments. Possible approaches could be specific mutagen sensitivities and DNA repair capacities [77], [93], and also varying enzyme equipments and activities, which are responsible for toxication/detoxication.

These causes make it clear that a risk assessment of air pollutants can not be a single-stage procedure but includes many individual steps and a multiplicity of information. That involves data on the chemistry of the substance, findings from animal experiments in vivo and in vitro, data on pharmacodynamics and pharmacokinetics, and as far as possible experiments on biological and toxicological effects in humans including mock experiments. At this point, the Integrated Risk Information System (IRIS) of carcinogen and not-carcinogen effects of chemicals and environmental contaminants should be mentioned. This system has been made available by the American National Library (NLM) and by the US Environmental Protection Agency (EPA) [109], [110].

3.2.1. Risk Assessment after Observation in the Human

As far as available, observations or studies on the human possess the highest significance. Especially double-blind, placebo controlled or prospective, controlled cohort studies are highlighted working with defined exposures and endpoints. It is obvious that such data are not available for most environmental pollutants.

Data coming from epidemiological investigations are far more comprehensive. However, cause effect relationship can seldom be proved and mostly only associations can be described. It is legitimate to create hypotheses, which deserve a revision in independent test methods. It is often not correct to calculate a risk in terms of a relative risk from epidemiological observations because the associations accounted for may be modulated by further possibly independent parameters [107]. Such parameters are called confounders. Thus it concerns factors, which also have influence on the test compound and its presumed effect. These factors should always be considered. At this point, the risk of hepatic carcinoma (effect) after exposure to aflatoxin (test compound) can serve as an example. The development of liver cancer not only depends on aflatoxin exposure but is also strongly dependent on chronic liver disease (confounder). The possibility of useless, but statistically presentable pseudo-correlations should also be considered in the context of testing procedures.

3.2.2. Risk Assessment Using Experimental Data

An absolute requirement for risk assessment using experimental data is that they are available to the public and nor buried within the borders of research facilities of the chemical industry. Their quality is another concern. A common problem is that high doses are used concomitantly with low case numbers. The necessity of higher case numbers in animal experiments is in direct opposite to the claim of animal protectionists. Despite this fact, studies which create conclusions about effects of environmental contaminants are legitimate even when it is not clear from the data, if these effects would also appear in the human after appropriate exposure. One example is the inability to prove in animal experiments the epidemiologically clear correlation between lung cancer and tobacco smoke [111]. At present, it is also impossible and may be even more difficult to prove tumors in the oral cavity caused by the use of alcohol and tobacco.

If the observed differences between exposed and non-exposed experimental groups are definite, they easily can be illustrated. A clear differentiation is more challenging if there are small doses of the test compound and little differences between the observed effects. Similar consequences as small differences in effects have large variations and high background values e.g. by spontaneous changes in the DNA.

Dose effect relationships are the best predictors for substance-induced effects and a threshold where no effect is expected. After completing pilot experiments, usually a range of at least three doses is tested. One of the doses should be without effect, one with a clear noticeable effect and another one with a strong toxicological effect. In experiments where at the high dose laboratory animals experience a strong constraint in their general condition with a remarkable decrease of body weight but survival in most animals a so-called Maximal Tolerated Dose (MTD) can be defined [107]. For a possible risk description, dose effect relationships of acute toxicity are differences documented. These should not be restricted on LD50/LC50 values because they represent only a single value and do not describe the steepness of increase of the effect, NOEL and serious toxicological but not deadly dose ranges. Therefore, the interpretation of the entire curve is considerably more interesting. A disadvantage of most acute toxicity test methods is their limitation on simple conclusions about the toxic effect, very often the death of the animals. More differentiated conclusions are usually attained from long-term studies. Special attention should be paid to dose effect relationships in the context of demonstration of reproductive toxic effects [107], as here a multiplicity of examination endpoints could be affected. Here, primary effects to the mother animal should also be considered which can be harmful to the progeny before or even without the toxic substance reaches the progeny.

3.2.3. Statistical Implications

Results from animal experiments usually suggest a non-linear relationship between the dose and effect over the whole range of doses. For the most part, a linear relation exists only in the median concentration range but not in a high or low range. However, exposure results from epidemiological tests almost always suggest a linear relation between the examined effects and the dose. That is why linear extrapolations are oftentimes conducted from experimental data of high and low doses.

3.3. Substance Classification Depending on Toxic Potential

There are different classifications regarding toxic effects arising from various considerations. The protection of the individual and its possible increased susceptibility is an important guiding principle. In this connection the classification of harmful substances according to the guidelines of the European Community should be mentioned [Tab. 1].

4. Threshold Values for Airborne Environmental Pollutants

The determination of threshold values of exposure to air pollutants is scientifically justified when available data make it possible. This accounts, e.g., for workplace and consumer protection. Threshold values which pursue an ecological intention are usually based on social considerations and constraints. This is important to differentiate because exceeding a scientifically justified threshold value can cause unfavorable consequences for the individual, whereas exceeding of an ecological, socio-politically raised threshold value is usually not harmful for the individual.

4.1. Scientific Justification of Threshold Values

The objective here is to obtain a scientifically justified risk limitation [107]. Exposure, concentrations or doses should be defined, where no health risk is expected. Such threshold values oftentimes integrate safety factors accommodating for the uncertainty of scientifically justified values. Most prominent examples for scientifically justified threshold values are the Maximal Workplace Concentrations (MAK values), listed by the Deutsche Forschungsgemeinschaft [112]. The MAK-values have their counterpart in the Permissible Exposure Limits (PEL) of the U.S. Occupational Safety and Health Administration (OSHA) and will be replaced in the future by Indicative Limit Values of the European Union [113]. These threshold limits serve as a protection of health regarding the employment, are scientifically justified for each single value [114] and are updated every year. They are based mainly on inhalation studies. Nonetheless, they do not allow definite conclusions about overall risk reduction for the population as a whole. That is because they only consider the average occupational exposure time and disregard the possible increased susceptibility of certain population groups such as elderly and children.

4.2. Social Implications of Threshold Values and Precautionary Hazard Limitation

If the data from scientific examinations defining the potential risk of an environmental contaminant are insufficient, social, political and scientific considerations could still make it essential to establish threshold values. Similar consideration have lead to the definition of various classifications of toxicity which have already been reviewed.

Another important aspect which should be considered in defining threshold values are possible beneficial effects of a substance. Here, a benefit-risk assessment becomes necessary. For airborne pollutants in most cases this assessment cannot be justified scientifically but is directed by social and economic interests. The latter are not necessarily conform; very often they are contrary to each other. The most extreme threshold value, which is usually imposed because of political but not scientific reasons, is the absolute ban of a substance. Such a ban can mostly be seen as a precautious reduction of a any potential risk. Another social aspect regarding the toxicological evaluation of environmental inhalants is animal protection, which has largely different legislative restriction in different countries and even different states of Germany. They determine where and to what extent animal experiments can be reduced. At the same time it became apparent that future examinations for chronic toxicity and carcinogenicity will be essential. Therefore, it would be wrong to enact a general ban for animal tests in a country. On the other hand discussion of the necessity to introduce every year a myriad newly developed substances of purely economical value should not be neglected.

4.3. Established Threshold Values and their Applications

This chapter describes relevant established threshold values which additionally can be used as references for general risk limitation. They have been developed using divergent requirements by different boards. For carcinogenic substances no ineffective concentration can be specified; this is why no threshold values are used but values are recommended which contribute to risk minimization.

4.3.1. Maximal Workplace Concentration (MAK)

The MAK is defined as the maximum concentration of a substance in form of gases, steams or atmospheric particles which does not endanger or annoy the health of workers after chronic exposure for 8 hours per day and 40 hours per week. The MAK values are published and updated annually by a committee of the Deutsche Forschungsgemeinschaft (DFG) on the basis of substance-specific scientifically acquired data [112]. The publication of these values also serves to provide information about the present state research and information of the committee. These values cannot be taken for legal decision on an individual case of a suspected work-related cancerous disease. This requires a drafting of an expert's report. The MAK values proved the basis for occupational air monitoring and risk limitation for the workers. As far as possible, undulating exposure concentrations will be converted into an 8 hour day average. Limitations in exposure spikes (category I to IV) are determined for certain substances e.g. irritating gases. Most of the MAK values are explicitly established for to exposure to pure substances and not to mixtures of substances. In fact, they consider some worker-specific factors such as age and physique but not sex-dependant different sensitivities [112].

For determination of a possible progeny damage of a pregnant woman this list is arranged into groups A to D. Group A substances are definitely harmful to the progeny. In Group B substances can be found where such an effect is only assumed. Group C contains substances which by maintaining the MAK- and BAT values do not cause damage to the progeny. Group D encompass the many compounds for which up to now no such classification is possible.

Furthermore, the list gives information about possible effects on the cells of the germline. Here, substances for which a proof of increased mutation rates exists in the offspring of humans or experimental animals are differentiated from substances known or suspected to be mutagenic in for germline cells in human and/or animals and from those which are to weak to be an additional mutagenic risk over the background in humans [112].

Also the risks of a possible absorption through the skin (H) and of sensitization of airways (Sa) and skin (Sh) are mentioned. Additionally, a possible photosensitization (SP) is listed.

The carcinogenic substances are classified into Groups 1 to 5 [Tab. 2]. No MAK values are defined for all compounds in groups 1 and 2 and most of the compounds in group 3. Here technical guide line values (TRK) are defined (see below). Epidemiological data and information about effect mechanisms indicate that substances of Group 1 tremendously contribute to a cancer risk and even cause cancer in human. Data from chronic animal experiments or from animal experiments and epidemiological investigations exist sufficiently in Group 2 but they are too scarce to be conclusive for humans. In Group 3 substances are listed, where a carcinogenic effect is only assumed but where the data are insufficient for a further classification. The assumption is based upon experimental results; therefore the classification has a tentative character. Group 4 contains such substances where epigenetic mechanisms of cancer development have been described by means of experimental results. For these compounds no noteworthy contribution to human cancer risk is expected when the MAK values are not exceeded. In Group 5 substances are listed which are so weakly carcinogenic or genotoxic that by maintaining the MAK-values no increased cancer risk is to be expected. The example of ethanol which is listed in Group 5, reveals that this classification is only a matter of occupational concentrations in the air but can not be transferred to beverages where already a very low uptake can bee seen very critically concerning a possible carcinogenic effect [37].

4.3.2. Biological Workplace Tolerance Value (BAT)

BAT values are created by the DFG on the basis of scientific data and are published together with the MAK values [112]. They define the maximum concentrations, which are allowed to be proved in a healthy human organism. The requirement of exposure is analog to the MAK values. Herewith, they only can be specified for such substances which are largely taken up into the organisms by respiration or absorption through the skin and mucous membranes of the upper aerodigestive tract and where enough experience/data is available. By definition BAT values are not checked by measuring concentrations in the air of the workplace but by examining the individual worker. Blood or urine for example can serve as test materials. Therefore, these values describe threshold values for parameters of biological monitoring and of internal exposure to toxic inhalants. However, a direct transfer to an exposure of undefined environmental conditions is not possible. The relationship between MAK and BAT values is complex and depend not only on the extent of absorption but also on the individuals ability to metabolize, toxify or detoxify and to eliminate the poison. BAT-values are not specified for possible allergic effects which are apparently concentration-independent. For genotoxic compounds classified in group 1 to 3 of the MAK list no BAT values but biological guide line values (BLW) and exposure equivalents for carcinogenic compounds (EKA) are defined (see below).

4.3.3. Biological Guide Line Value (BLW)

BLW values have been established by the DFG in 2002. They are defined as quantity of a substance or its metabolites, respectively, or as the extent of a change from the norm of a biologic indicator value for carcinogens, suspected carcinogens and substances for which no sufficient data are available to define BAT values. In particular they should be used in the scope of biomonitoring and help taking appropriate precautions. Even by adhering to the BLW value, a health risk cannot be excluded [115].

4.3.4. Technical Guide Line Concentration (TRK)

TRK values for the workplace are established by the DFG for carcinogens and substances where a carcinogenic effect can be assumed but by definition not safe concentrations exists. Therefore, TRK are specified instead of defining MAK values. These help to minimize the risk to a level which is technically achievable [116]. However, an absolute safety, even by maintaining the TRK, cannot be presumed.

4.3.5. Exposure Equivalents for Carcinogenic Substances (EKA)

Similar to the TRK values EKA values are defined for carcinogens instead BAT values reflecting the extent of internal exposure. Regarding the safety, the same restrictions as for TRK values can be applied to EKA values.

4.3.6. Technical Guidance for Clean Air (TA Luft)

For industrial plants underlying legal restriction regarding emission and imission of gases threshold values are established by the German Engineer Alliance (VDI) and described in the TA-Luft. The definition of Maximal Imission Values (MIK values) and reduced MAK values are other fundamental tasks of the same alliance. No risk for the human is assumed from the MIK values. However, they are usually provided with some additional restrictions regarding the length of exposure. Within the TA Luft recommendations for emission limitations are also given [116].

4.3.7. Standard Values of the U.S. Environmental Protection Agency

Besides the already mentioned guidelines, the U.S. Environmental Protection Agency discusses further guidelines: the Lowest Observed Adverse Effect Level (LOAEL) is defined as the smallest concentration, where the first harmful effects can be observed. The Acceptable Daily Intake (ADI) stems from animal experiments defining a NOAEL which is further divided by a security factor most often a factor of hundred. Herewith, a dose, which is defined for human intake (ADI) can be calculated with an experimental determined value (NOAEL) and a random security factor. However, the use of ADI as the tolerable amount of uptake is controversially discussed. Furthermore, a reference doses (RfD) are described, which results from NOAEL and one uncertainty (UF) and one modification factor (MF). Exposures which are under this guide value are possibly harmless for humans [109].

5. Conclusion

Despite their ubiquitous distribution, for many inhalative pollutants there exist no sufficient detailed toxicological risk profiles. Therefore, no scientifically based threshold values can be defined. Wouldn't it be socially desirable to abstain from newly or already established chemicals, as long as the relevant data are insufficient? Would it not be extremely necessary to intensify research on cancer risk factors and their prevention with help of funding by the European Union? The complete support of medical research by the European Union encompassed about 2.4 billions of Euros in the year 2002 [117]. This budget could be increased substantially by canceling the subsidies for cultivation of tobacco in the European Union which accounted for about one billion of Euros in the same year [118].

6. Acknowledgement

The excellence of Dr. Mara Sheehan in translating this manuscript into the English language is highly appreciated.

7. Glossary

ADI Acceptable Daily Intake

BAT Value Biological Workplace Tolerance Value

BL Value Biological Guide Line Value

CBI Covalent Binding Index

DNA Desoxyribonucleic acid

ED 50 Mean Effective Dose

EKA Exposure Equivalents for Carcinogenic Substances

FISH Fluorescence in situ Hybridization

HPRT Hypoxanthine-Phosphoribosyltransferase

LC 50 Mean Lethal Concentration in Room Air

LD 50 Mean Lethal Dose

LOAEL Obeserved Adverse Effect Level (LOAEL Lowest Obeserved Adverse Effect Level (LOAELLowest Observed Adverse Effect Level

MAK Value Maximal Workplace Concentration

MIK Value Maximal Immission-Value

MF Modification Factor

MOC Miniorgan Cultures

MTD Maximal Tolerated Dose

NNK 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone

NOAEL No Observed Adverse Effect Level

NOEL No Observed Effect Level

PHA Phytohemagglutinine

RfD Reference Dose

SCE Sister Chromatid Exchange Test

TA Luft Technical Guidance for Clean Air

TD 50 Mean Toxic Dose

TRK Value Technical Guide Line Concentration

UF Uncertainty Factor

UDS Unscheduled DNA-Synthesis


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