Principle
The NRU-Test can be used to test for cytotoxic effects of chemical substances and environmental samples on cell membranes. It is based on a dyeing reaction which allows identification of dead and live cells. Usually the test is performed with the adherent permanent cell lines HEP-G2 (human hepatocyte cells) and V79 (lung epithelium of the Chinese hamster). In addition, several other cell lines like human keratinocytes or fish cells can also be used.

Abstract
In the microwell plate version, a monolayer cell culture, which has been grown over a period of 24 hours, is exposed over a defined period of time (usually between 2 and 24 hours) to different concentrations of the test substance. By adding a rat liver homogenate (so-called S9-mix) the metabolic activation of toxic substances in the organism can be simulated. At the end of the exposure period, the cells are washed and the neutral red dye is added. It passes the cell membrane and binds to intracellular phosphate and carboxyl groups. The accumulation of the dye takes place mainly in the lysosome. Dead cells or such with membrane damage cannot accumulate the dye, so that during the following washing and fixation steps the dye is not retained intracellularly. Wells with dead or damaged cells or with a reduced number of cells due to growth inhibitive effects are less coloured than those with vital cells. The evaluation of the test is done by resolving the intracellular bound dye and measuring the intensity photometrically in comparison to an untreated control sample. The toxicity is expressed as percent inhibition of neutral red dye retention in the sample. It is a measure for cell damage. EC50 can be calculated from the dose-response curve.

Principle
The "Mouse Lymphoma Assay“ enables the testing for genetic mutations caused by chemical substances and assesses the two endpoints gene mutation and chromosome damage.

Abstract
The permanent Mouse Lymphoma cell line L5178Y TK+/- 3.7.2C is usually applied.

Its thymidine-kinase locus (tk1) is heterocygotically expressed on chromosome 11. The inactivation of the tk+ allele is inducing a trifluorothymidine (TFT) resistance. In this way, the tk-/- mutants can be selected in a background of tk+/- non-mutated cells. Mutated cell strains show a bimodal size distribution with strains that grow at the normal growth rate of tk+/- cells, and strains that grow considerably more slowly. It could be shown that the small colonies are frequently associated with aberrations of chromosome 11, while mutants of the bigger colonies show no cytogenetic changes, but are due to genetic mutations.

In our laboratory the „Mouse Lymphoma Assay“ is applied as a micro-well method. According to standard guidelines, at least 8 significant concentrations or 4 concentrations each as duplicate, a double negative control line as well as a positive control, each with and without metabolic activation (S9) are tested. Negative or non-consistent results are independently repeated. Incubation time with regard to the test substance varies usually between 3 and 4 hours. The incubation time for the testing of pharmaceuticals has to be extended to 24 hours by an additional test without metabolic activation (according to ICH S2B). By this, an improved detection of nucleoside and base analogues as well as of aneuploidy is obtained.

Criteria
A positiv effect is an increase of mutants of at least 100 per 10⁶ cells compared to the rate of the control. Additional a significance test with ANOVA has to be performed.

Test substance properties
The Mouse Lymphoma Assay is applicable for solid, soluble substances as well as liquid substances and water samples.

Principle
Genotoxicity test with micronuclei induction with V79 (lung tissue of Chinese hamsters)

Abstract
The micronucleus test allows detecting chromosome damage caused by clastogenic (chromosome breaking) and aneugenic (disrupting the spindle apparatus) substances. During cell division chromosome fragments are no longer integrated into the nuclei of the daughter cells. They remain in the cytoplasm, generating micronuclei, which can be detected by light-optical microscopy. Incidence of increased micronuclei frequency means irreparable damage of the DNA and thus manifested genetic damage which suggests a risk for succeeding cell generations.

Cells are sowed in well defined density and are allowed to adhere over a period of 6 hours to the surface of a microscope slide. The test item is applied in different dilutions and incubated for 4 hours (with S9-mix) or 24 hours (without S9-mix) together with the cells. The addition of the so-called S9-mix, a rat liver fraction, enables the simulation of metabolic activation of pro-mutagenic substances, as may happen in the mammalian liver. Giemsa staining of the cells is performed after washing and fixation. Using light-optical microscopy at least 1,000 cells per test concentration and controls are analysed for the occurrence of micronuclei.

Criteria
A genotoxic effect of the sample can be assumed if a significant increase in micronuclei frequency is detected after treatment of the cell culture with the test item.

Test substance properties
The Micronucleus Test is applicable for solid, soluble substances as well as liquids and water samples.

Principle
Genotoxicity test with eukaryote cells on the base of DNA damage

Abstract
The comet assay (single-cell gel electrophoresis) is a simple method for measuring DNA strand breaks in eukaryotic cells. The test design can be performed with almost every kind of cell typ and is independent from the cell cycle. The DNA damage can be proven in proliferating as well as in non-proliferating cells. Cells embedded in agarose on a microscope slide are lysed with detergent and high salt to form nucleoids containing supercoiled loops of DNA linked to the nuclear matrix. Electrophoresis at high pH results in structures resembling comets, observed by fluorescence microscopy; the intensity of the comet tail relative to the head reflects the number of DNA breaks. The likely basis for this is that loops containing a break lose their supercoiling and become free to extend toward the anode. This is followed by visual analysis with staining of DNA and calculating fluorescence to determine the extent of DNA damage. This can be performed by manual scoring or automatically by imaging software.

Criteria
A positive effect shows a significant increase of DNA fragmentation. The statistical verification of the significance can be performed with statistic tests as Kruskall-Wallis or H-Test.

Test substance properties
The Comet Assay is applicable for solid, soluble substances as well as for liquids and water samples.