Syrian hamster embryo (SHE) cell transformation assay with conditioned media (without X-ray irradiated feeder layer) using 2,4-diaminotoluene, 2,6-diaminotoluene and chloral hydrate.

The Syrian hamster embryo (SHE) cell transformation assay has traditionally been conducted with a feeder layer of X-ray irradiated cells to provide growth support to the target cells seeded in low numbers. The feeder layer of cells consists of X-ray irradiated cells which are still viable but unable to replicate. We have tried seeding the target cells in conditioned media prepared from the stock culture flasks in lieu of plating them on a feeder layer. Three SHE cell isolates were tested to investigate the feasibility of this approach. With freshly prepared conditioned medium (LeBoeuf's Dulbecco's Modified Eagle's Medium with 2 mM L-glutamine and 20% fetal bovine serum), used within 2 weeks of preparation, there was essentially no difference in the number of target cell colonies in the conditioned medium and in the plates with the X-ray irradiated feeder cell layer. The plating efficiencies of the vehicle controls were within the historical range for the standard SHE cell transformation assay. In each experiment, the positive control benzo(a)pyrene [B(a)P] elicited a significant increase in morphological transformation frequency (MTF), with or without feeder cells. Three compounds, 2,4-diaminotoluene (2,4-DAT), 2,6-diaminotoluene (2,6-DAT), and chloral hydrate were tested in the SHE cell transformation assay without an X-ray irradiated feeder layer and using a 7-day exposure regimen. The results were comparable to those reported in the published literature using the standard methodology with feeder cells, with 2,4-DAT and chloral hydrate eliciting a significant increase in MTF, and 2,6-DAT not eliciting any increase in MTF. The results of this study demonstrate the feasibility of conducting the SHE cell transformation assay without the use of an X-ray irradiated feeder layer, thereby simplifying the test procedure and facilitating the scoring of morphologically transformed colonies.

Evaluation of the butter flavoring chemical diacetyl and a fluorochemical paper additive for mutagenicity and toxicity using the mammalian cell gene mutation assay in L5178Y mouse lymphoma cells.

Diacetyl (2,3-butanedione) is a yellowish liquid that is usually mixed with other ingredients to produce butter flavor or other flavors in a variety of food products. Inhalation of butter flavoring vapors was first associated with clinical bronchiolitis obliterans among workers in microwave popcorn production. Recent findings have shown irreversible obstructive lung disease among workers not only in the microwave popcorn industry, but also in flavoring manufacture, and in chemical synthesis of diacetyl, a predominant chemical for butter flavoring. It has been reported that perfluorochemicals utilized in food packaging are migrating into foods and may be sources of oral exposure. Relatively small quantities of perfluorochemicals are used in the manufacturing of paper or paperboard that is in direct contact with food to repel oil or grease and water. Because of recent concerns about perfluorochemicals such as those found on microwave popcorn bags (e.g. Lodyne P208E) and diacetyl in foods, we evaluated both compounds for mutagenicity using the mammalian cell gene mutation assay in L5178Y mouse lymphoma cells. Lodyne P208E was less toxic than diacetyl and did not induce a mutagenic response. Diacetyl induced a highly mutagenic response in the L5178Y mouse lymphoma mutation assay in the presence of human liver S9 for activation. The increase in the frequency of small colonies in the assay with diacetyl indicates that diacetyl causes damage to multiple loci on chromosome 11 in addition to functional loss of the thymidine kinase locus.

Evaluation of commercial kava extracts and kavalactone standards for mutagenicity and toxicity using the mammalian cell gene mutation assay in L5178Y mouse lymphoma cells.

Kava (Piper methysticum) is a member of the pepper family and has been cultivated by South Pacific islanders for centuries and used as a social and ceremonial drink. Traditionally, kava extracts are prepared by grinding or chewing the rhizome and mixing with water and coconut milk. The active constituents of kava are a group of approximately 18 compounds collectively referred to as kavalactones or kava pyrones. Kawain, dihydrokawain, methysticin, dihydromethysticin, yangonin, and desmethoxyyangonin are the six major kavalactones. Kava beverages and other preparations are known to be anxiolytic and are used for anxiety disorders. Dietary supplements containing the root of the kava shrub have been implicated in several cases of liver toxicity in humans, including several who required liver transplants after using kava supplements. In order to study the toxicity and mutagenicity, two commercial samples of kava, Kaviar and KavaPure, and the six pure kavalactones including both D-kawain and DL-kawain, were evaluated in L5178Y mouse lymphoma cells. Neither the kava samples nor the kavalactones induced a mutagenic response in the L5178Y mouse lymphoma mutation assay with the addition of human liver S9 activation.

Chromium picolinate does not produce chromosome damage in CHO cells.

Chromium picolinate (CrPic, Chromax) is a dietary supplement that has been commercially available for the past two decades. CrPic has potential benefits for reducing insulin dependence in diabetics by increasing sensitivity of insulin receptors and in stimulating insulin binding. In this study, CrPic was tested for its ability to produce chromosomal aberrations in vitro using Chinese hamster ovary K1 (CHO) cells. CHO cells were exposed to a range of cytotoxic to non-cytotoxic concentrations of CrPic for 4 or 20h in the absence of metabolic (S9) activation or for 4h in the presence of S9 activation. CrPic was solubilized with dimethyl sulfoxide (DMSO) to attain the highest possible solubility for maximizing the test doses. Cells were treated with 96.25, 192.5, 385 or 770 microg/mL of CrPic for 4 h in the presence of S9 activation, and for 4 or 20 h in the absence of S9 activation. A distinct precipitate of CrPic was evident in the cell culture medium at 770 microg/mL, which was the highest dose tested. Results showed no statistically significant increases in structural or numerical chromosome aberrations were produced at any test dose level with CrPic in 4-h treatments up to a precipitating dose of 770 microg/mL in either the presence or absence of S9 activation. Additionally no aberrations were observed up to 385 microg/mL (the maximum analyzable dose) following treatment for 20 h in the absence of S9 activation. The percentage of cells with structural or numerical aberrations in CrPic treated cultures was not statistically different (p>0.05) from that quantified in controls at any dose level. The absence of significant differences from control levels demonstrates that CrPic did not induce structural or numerical chromosome aberrations up to doses that were insoluble in the culture medium.

Mutagenicity of chromium picolinate and its components in Salmonella typhimurium and L5178Y mouse lymphoma cells.

Chromium picolinate is one of the most commonly used chromium dietary supplements available in the United States, and it has been marketed to consumers for use in weight loss, increasing muscle mass, and lowering serum cholesterol. Chromium picolinate is a synthetic compound that provides a bioavailable form of Cr(III) that is absorbed better than dietary chromium. However, there are several reports that it can have adverse effects. In order to study the mechanism of observed cellular toxicity and mutagenicity, chromium picolinate and its component compounds, chromium (III) chloride and picolinic acid, were evaluated in Salmonella typhimurium and L5178Y mouse lymphoma cells. Neither chromium picolinate nor chromium chloride induced a mutagenic response in S. typhimurium. However, in the L5178Y mouse lymphoma mutation assay, chromium picolinate induced mutagenic responses without and with the addition of S9.

Lack of mutagenicity of chromium picolinate in the hypoxanthine phosphoribosyltransferase gene mutation assay in Chinese hamster ovary cells.

Chromium picolinate (CrPic, Chromax) is a dietary supplement that is stable and more bioavailable than other commercially available forms of chromium. Chromium supplementation is known to enhance the action of insulin, particularly in insulin resistance and type 2 diabetes mellitus. A previous study reported that CrPic produced increases in mutations of the hypoxanthine phosphoribosyltransferase (Hprt) gene in Chinese hamster ovary (CHO) cell mutation tests. This study, however, evaluated CrPic produced by the testing laboratory and used an atypical 48 h exposure period for this test system. The current study evaluated the mutagenic potential of the most widely utilized commercial form of CrPic in CHO/Hprt mutation tests following International Conference on Harmonisation (ICH) Guidelines (+/-S9 metabolic activation with a 5h exposure) in addition to repeating the test with a 48 h exposure period -S9 activation. CrPic was suspended in dimethyl sulfoxide (DMSO) up to a concentration of 50 mg/mL; exposures were conducted under conditions in which precipitate was not evident and under conditions in which some precipitate of CrPic was visually evident in the cell culture medium at the highest concentrations (500 microg/mL). The concentrations evaluated for mutagenicity ranged from 15.6 to 500 microg/mL (+S9 and -S9) for the 5 h exposure and 31.3-500 microg/mL for the 48 h exposure (-S9). Only a slight degree of cytotoxicity was seen in the standard tests up to the limit of solubility in the medium. Toxicity, i.e., cloning efficiency < or =50% of the solvent control, but no mutagenic increases were observed at 500 microg/mL following a 48 h exposure period. The results of these studies showed that CrPic was non-mutagenic in two independent CHO/Hprt assays and in an assay using a 48 h exposure period.