In this page, we introduce our core in-vitro core battery tests, Ames, NRU, and MN tests, and also introduce an example of our research
AMES TEST
Ames test can identify chemicals that induce gene mutations (Mutagenicity).
It has been widely used for the purpose of evaluating the mutagenic potential of various chemicals such as food additives and drugs. The test uses several bacterial strains that contain the different mutations in an amino acid biosynthesis gene. Those mutations prevent bacterial growth in the absence of the amino acid in the cell culturegrowth medium. Exposure to mutagenic chemicals may induce a mutation that will restore the wild type DNA sequence and the functional capability of the bacteria to synthesize the essential amino acid, and thus, to grow on the medium without the required amino acid. Bacteria in which this function-restoring mutation has occurred are called “Revertants” and those revertant colonies are counted for the test method.
Vehicle control plate
Mutegenic chemical treated plate
NRU test
The NRU (Neutral Red Uptake) test is a cytotoxicity test that evaluates the life/ death of mammalian cells.
This test takes advantage of the fact that living cells are able to take up surrounding substances i nto the cell, whereas damaged cells are unable to take it up. When cells are given a toxic substance followed by a red dye (NR), cells that are alive will take up the dye into the cell, damaged ones will take up less and those that are dead will not. Therefore, the amount of dye incorporated is evaluated as an indicator of the cytotoxicity.
The plate of NRU test. From left to right, the exposure level increases and the cytotoxicity increases.
MN TEST
The MN (micronucleus) test identifies chemicals that induce chromosomal abnormalities.
It has been widely used for the purpose of evaluating the genotoxic potential of general chemicals such as food additives and drugs. A parent cell divides to produce two identical daughter cells. However, when a chromosome is damaged by genotoxic substance, part of the chromosome is fragmented and appears as a micronucleus (MN) after cell division. The increase of MN frequency is evaluated as an indicator of genotoxicity.
Microscopic image of MN test. Micro nucleus is seen in the lower left cell.
OUR RESEARCH
Recently, we have performed the in-vitro toxicological assessment for IT1 (In-direct heating tobacco system platform 1) – one of our heated tobacco products. As a result, our research has revealed that the vapor of IT1 has much lower mutagenicity and causes significantly less cellular damage in in-vitro assays, compared to conventionally combusted cigarette smoke.
Tobacco vapor generated from IT 1 was exposed to cultured cells and bacteria, and the degree of genetic mutations and cell damage was compared with those of a reference cigarette (3R4F).
Much lower levels of genotoxic, mutagenic and cytotoxic responses were observed in tobacco vapor generated from IT1.
Mutagenicity (Ames assay)
Genotoxicity (MN assay)
Cytotoxicity
This research was conducted in compliance with GLP at an external laboratory.
Regulatory Toxicology and Pharmacology, 2018
Regulatory Toxicology and Pharmacology, 2018