Ames Test

Overview

Ames Test is a standard short-term bacterial reverse mutation test widely used as an initial screen of the mutagenicity potential of new chemical entities. Although in vitro Ames assay is not a replacement of in vivo DNA damage/carcinogenicity studies, any compound that exhibits positive Ames test results is very likely to be carcinogenic in vivo.

Ames Test module provides fast and accurate predictions of mutagenic potential of candidate compounds expressed as probabilities of exhibiting positive Ames Test results. The predictive algorithm enables the researcher to quickly identify and eliminate potentially hazardous substances and thus, may be highly useful as an aid for compound selection and prioritization of genotoxicity testing in risk assessment. It may also serve as partial replacement of in vitro bacterial mutagenicity assays in the early stages of development.

The predictive model of Ames genotoxicity is Trainable, meaning that its Applicability Domain may be expanded by addition of ‘in-house’ experimental Ames test data. Training of the model with new data allows obtaining reliable predictions for the compounds synthetized in your company.

Features

• Calculates probability of positive Ames Test results for the compound of interest supplemented by the Reliability Index (RI) of prediction.
• RI values represent a quantitative evaluation of prediction confidence. High RI shows that the calculated value is likely to be accurate, while low RI indicates that no similar compounds with consistent data are present in the training set.
• Visualizes the genotoxic potential of different parts of the molecule by color/mapping the contributions of different atoms (or fragments) onto the structure (red – associated with genotoxicity, green – not involved in genotoxic effect).
• Overall experimental Ames test results with references for up to 5 similar structures from the training set are displayed along with each prediction. Additionally, access to a fully browsable Ames Test DB is available, providing information about individual studies conducted with each compound corresponding to various bacterial strains tested, presence or absence of metabolic activation, as well as other experimental conditions.
• User-defined data can be added to the Self-training Library of the model for an instant improvement of accuracy and reliability of calculations for similar compounds.

Interface

File:genotoxicity ames test.png

1. Indicated mutagenicity potential.
2. Calculated probability of compound beeing mutagenic.
3. Indication of the prediction reliability along with the Reliability Index value. Reliability index (RI):
   RI < 0.3 – Not Reliable,
   RI in range 0.3-0.5 – Borderline Reliability,
   RI in range 0.5-0.75 – Moderate Reliability,
   RI >= 0.75 – High Reliability
4. Up to 5 similar structures in the training set with names, CAS numbers and results (positive, negative, weakly positive, inconclusive)
   

Technical information

Experimental data

Modeling was per formed using a standardized Ames genotoxicity data set containing more than 8500 compounds that was compiled from well known public databases. The main data sources were:

• Chemical Carcinogenesis Research Information (CCRIS)
• Genetic Toxicology Data Bank (GENE-TOX)

The results of Ames genotoxicity assays were collected for several strains of S. typhimurium that are most frequently used for testing (TA97, TA98, TA100, TA102, TA104, TA1535, TA1537, TA1538 and also E. coli strain WP2 uvrA), with or without metabolic activation.

Data interpretation and assignment of qualitative categories

In the Ames Genotoxicity database, compounds were classified as Ames positive if it demonstrated clear positive results in at least one tested strain with or without metabolic activation. Compounds that did not increase the frequency of revertants in all tested strains were considered safe (Ames negative). Some chemicals that consistently exhibited weak mutagenic activity were marked weakly positive while in those cases when the results of different studies were discrepant the corresponding compounds were labeled inconclusive.

Ames genotoxicity hazards

Ames Test Hazards (Genotoxicity\Hazards module in Tox Boxes) is a knowledge-based expert system that identifies and highlights structural elements that may be responsible for the mutagenic activity of the analyzed molecules. Ames Hazards system searches through a list of 27 predefined 'genotoxicophores' collected from toxicological literature. Most 'genotoxicophores' comprising this list are not defined by a single hazardous fragment, but represent groups of similar substructures sharing common properties in terms of causing DNA damage.

Each found hazard is provided with textual comments regarding its mode of action and bar charts showing the distribution of compounds with a particular hazardous fragment in various bacterial strains. The bar charts may also give valuable insight on the possible mechanism of action and need for metabolic activation for genotoxic effect to become evident. Different types of mutations are detected by different strains (e.g. TA98 – frameshift mutations, TA100 – base pair substitutions). If a substructure becomes hazardous after biotransformation, the distribution pattern in the corresponding chart is usually highly shifted towards Ames positive compounds while no such difference is evident for direct acting mutagens. For an example please refer to the Section: Demonstration scenario.

Model features & prediction accuracy

The model was developed with Algorithm Builder using a novel methodology consisting of two parts:

• Global baseline statistical model employing binomial PLS with multiple bootstrapping using a predefined set of fragmental descriptors.
• Local correction to baseline prediction based on analysis of experimental data for similar compounds.

The underlying methodology enables obtaining an intrinsic evaluation of prediction confidence by the means of Reliability Index (RI) values calculated for each prediction. RI ranging from 0 to 1 serves as an indication whether a submitted compound falls within the Model Applicability Domain. Two criteria influence the calculation od Reliability Index of a prediction:

• Similarity of the analyzed molecule to compounds in the Self-training Library (prediction is unreliable if no similar compounds have been found in the Library).
• Consistency of experimental data for similar compounds (discrepant data for similar molecules, i.e. alternating Ames positive and Ames negative compounds lead to lower RI values).

Classification of compounds as genotoxic or non-genotoxic performed by Tox Boxes is highly accurate: model validation results demonstrate that less than 5% of test set compounds (~20% of the overall data set) are mispredicted if inconclusive (0.2 < p < 0.8) and unreliable (RI < 0.3) predictions are not considered. If probability threshold value of 0.5 is used the number of mispredicts still only slightly exceeds 10% of the test set. The percentages of misclassified compounds and inconclusive predictions (calculated probabilities falling in the range 0.2-0.8) also decrease significantly if only predictions of moderate and high reliability (RI > 0.5) are considered (see Table below).

Futhermore, more than 90% of genotoxic compounds from the entire data set are covered by the list of hazardous substructures used in Genotoxicity Hazards module.

Accuracy testing		Calculated probability (P)
		<0.5	>0.5	<0.2	0.2-0.8	>0.8
Test set (RI > 0.3) N = 1,483	Safe	392 (26.4%)	96 (6.5%)	310 (20.9%)	130 (8.8%)	48 (3.2%)
	Genotoxic	67 (4.5%)	928 (62.6%)	22 (1.5%)	125 (8.4%)	848 (57.2%)
Test set (RI > 0.5) N = 1,117	Safe	257 (23.0%)	51 (4.6%)	225 (20.1%)	51 (4.6%)	32 (2.9%)
	Genotoxic	23 (2.1%)	786 (70.4%)	11 (0.9%)	38 (3.4%)	760 (68.0%)