Assessment of mutagenicity and environmental safety: SYBR® Safe DNA gel stain
Assay for morphological transformation in primary cultures
An independent laboratory investigated the ability of SYBR® Safe DNA gel stain for inducing an increase in morphological transformation of Syrian hamster embryo (SHE) cells, relative to vehicle control cultures, following a 7-day exposure period. None of the three treatment groups (0.0500, 0.150, and 0.300 µg/mL) induced a significant increase in morphological transformation compared to the concurrent vehicle control. In addition, a significant increase of the morphological transformation frequency was also obtained from the positive control treatment with benzo pyrene at 5.0 mg/mL. The test article (SYBR® Safe DNA gel stain) was therefore evaluated as negative in the screening SHE cell transformation assay under 7-day exposure conditions of this study.
Conclusion—SYBR® Safe DNA gel stain does not induce transformations in primary cultures of Syrian hamster embryo (SHE) cells when compared with solvent alone, strongly indicating that the SYBR® Safe DNA gel stain is noncarcinogenic. In contrast, ethidium bromide tests positive in the SHE assay, consistent with its known activity as a strong mutagen.
Assays for chromosomal aberations and forward mutations
An independent laboratory investigated the ability of SYBR® Safe DNA gel stain to induce chromosomal aberrations in cultured peripheral blood lymphocytes with and without exogenous metabolic activation. No significant increases in the number of cells with structural aberrations, polyploidy, or endoreduplication were observed in test either with or without S9 activation. Similarly, no increases in the mutant frequency were observed that exceeded the minimum criteria in L5178Y TK+/- mouse lymphoma cells.
Conclusion—SYBR® Safe DNA gel stain does not cause mutations in mouse lymphoma cells at the TK locus, nor does it induce chromosomal aberrations in cultured human peripheral blood lymphocytes, with or without S9 metabolic activation, using standardized tests against appropriate controls.
Summarized data: mammalian genotoxicity analysis of SYBR® Safe DNA gel stain
|In vitro test*
||Result with S9 activation†
||Result without S9 activation†
||Syrian hamster embryo (SHE) cells
||Cultured human peripheral blood lymphocytes
||L5178YTK+/- mouse lymphoma cells
* In vitro tests were performed by Covance Laboratories Inc., Vienna, VA. † Mammalian S9 fraction obtained from Aroclor 1254 induced rat liver. NA = Not applicable. 1Fundamental and Molecular Mechanisms of Mutagenesis 356, 1 (1996); 2Evans, H.J., in Chemical Mutagens, Principles and Methods for Their Detection, A. Hollaender, Ed., Vol. 4, (1976) pp. 129; 3Mutation Res 72, 447 (1980); 4Mutation Res 59, 61 (1979).
Assay for reverse mutations (Ames test)
Samples were pretreated with a mammalian S9 fraction and then tested. With S. typhimurium strains TA97a, TA98, TA100 and TA102, an increase in revertants of more than twofold over background indicates a positive result for mutagenicity in this test. With strains TA1535, TA1537 and TA1538, an increase in revertants of more than threefold over background indicates a positive result.
Conclusion—Compared to ethidium bromide, SYBR® Safe DNA gel stain causes fewer mutations in the standard Ames test, as measured in several different strains of Salmonella typhimurium. Weakly positive results for SYBR® Safe DNA gel stain in this test occurred in four out of seven strains and only with activation by a mammalian S9 fraction (see figure).
Assay for oral toxicity
A single oral administration of SYBR® Safe DNA gel stain in 0.5X TBE at a limit dose of 5,000 mg/kg to three female rats produced no mortalities or toxic signs. The procedure is designed to determine that acute oral toxicity of the material under test. A Limit Screen test was performed using three female Sprague Dawley rats, which received an oral limit dose of 5,000 mg/kg of SYBR® Safe DNA gel stain. The animals were observed for mortality, weight change, and toxic signs for a two-week period. Since all three rats survived for two weeks after the dose administration, the LD50 for the test article was considered to be greater than the Limit Dose and no additional testing was required.
Conclusion—A single oral administration of SYBR® Safe DNA gel stain produces no signs of mortality or toxicity at a limit dose of 5,000 mg/kg.
Summarized data: oral toxicity
||Fathead minnow CA Title 22 acute screening
||Not hazardous or toxic to aquatic life
||EPA Acute Oral Toxicity Test OPPTS 870.1100
||LD50 > 5000 mg/kg
* Performed by AMEC Earth and Environmental San Diego Bioassay Laboratory, San Diego, CA.
** Performed by Northview Pacific Laboratories, Inc., Hercules, CA.