Timothy E. Lawlor

Comparison of SYBR® Green I nucleic acid gel stain mutagenicity and ethidium bromide mutagenicity in the Salmonella/mammalian microsome reverse mutation assay (Ames test)

SYBR Green I nucleic acid gel stain is an unsymmetrical cyanine dye developed for sensitive detection of nucleic acids in electrophoretic gels. Its mechanism of nucleic acid binding is not known, whereas the most commonly used nucleic acid gel stain, ethidium bromide, is a well-characterized intercalator. We compared the mutagenicity of SYBR Green I stain with that of ethidium bromide in Salmonella/mammalian microsome reverse mutation assays (Ames tests). As expected [J. McCann, E. Choi, E. Yamasaki, B.N. Ames, Proc. Natl. Acad. Sci. USA, 72 (1975) 5135-5139], ethidium bromide showed high revertant frequencies in several frameshift indicator strains (averaging 68-fold higher than vehicle controls in TA98, 80-fold higher in TA1538, 15-fold higher in TA1537, and 4.4-fold higher in TA97a), only in the presence of rat liver extracts (S9). Small increases in revertant frequencies were observed for ethidium bromide in the base-substitution indicator strain TA102 both in the presence and absence of S9 (averaging 2.0- and 1.8-fold higher than vehicle controls, respectively) and in base-substitution indicator strain TA100 in the presence of S9 (averaging 1.6-fold higher than vehicle controls). A small mutagenic effect was detected for SYBR Green I stain in frameshift indicator strain TA98 (averaging 2. 2-fold higher than vehicle controls) only in the absence of S9 and in base-substitution indicator strain TA102, both in the presence and absence of S9 (averaging 2.2- and 2.7-fold higher than vehicle controls, respectively). Thus, SYBR Green I stain is a weak mutagen and appears to be much less mutagenic than ethidium bromide. These results suggest that SYBR Green I stain may not intercalate, and if it does, that its presence does not give rise to point mutations at a high frequency.

Ames assays and unscheduled DNA synthesis assays on 2,4-dichlorophenoxyacetic acid and its derivatives

2,4-dichlorophenoxyacetic acid and several of its derivatives (collectively known as 2,4-D) are herbicides used to control a wide variety of broadleaf and woody plants. The genetic toxicity in vitro of 2,4-D and seven of its salts and esters were examined by employing gene mutation in bacteria (Ames test) and induction of DNA damage and repair in rat hepatocytes. In addition, an in vivo unscheduled DNA synthesis (UDS) assay was performed on 2,4-D. There were no indications of genotoxic potential for 2,4-D acid, or any of its derivatives, in these assays. These results are consistent with the reported lack of carcinogenic potential for 2,4-D in both mice and rats.

Integration of Pig‐a, micronucleus, chromosome aberration, and comet assay endpoints in a 28‐day rodent toxicity study with 4‐nitroquinoline‐1‐oxide

As part of the Stage III Pig‐a multilaboratory validation trial, we examined the induction of CD59‐negative reticulocytes and total red blood cells (RETCD59− and RBCCD59−, respectively) in male Sprague Dawley® rats treated with 4‐nitroquinoline‐1‐oxide (4NQO), for 28 consecutive days by oral gavage, at doses of 1.25, 2.50, 3.75, 5.00, and 7.50 mg kg−1 day−1 (the high dose group was sacrificed on Day 15 due to excessive morbidity/mortality). Animals also were evaluated for: micronucleated reticulocytes (mnRET) by flow cytometry; DNA damage in peripheral blood, liver, and stomach using the Comet assay; and chromosome aberrations (CAb) in peripheral blood lymphocytes (PBL). All endpoints were analyzed at two or more timepoints where possible. Mortality, body and organ weights, food consumption, and clinical pathology also were evaluated, and demonstrated that the maximum tolerated dose was achieved at 5.00 mg kg−1 day−1. The largest increases observed for the genetic toxicology endpoints (fold‐increase compared to control, where significant; all at 5.00 mg kg−1 day−1 on Day 29) were: RETCD59− (21X), RBCCD59− (9.0X), and mnRET (2.0X). In contrast, no significant increases were observed for the CAb or Comet response, in any tissue analyzed, at any timepoint. Because 4NQO is a well known mutagen, clastogen, and carcinogen, the lack of response for these latter endpoints was unexpected. These results emphasize the extreme care that must betaken in dose and endpoint selection when incorporating genotoxicity endpoints into routine toxicity studies as has been recommended or is under consideration by various regulatory and industrial bodies. Environ. Mol. Mutagen., 2011.

Absence of mutagenic effects of sodium dichloroacetate.

Sodium dichloroacetate (DCA) is a drug with potential for treating patients with stroke and head injury. Conflicting evidence has been published on the mutagenic potential of DCA. A series of genetic tests for mutagenicity and clastogenicity was carried out on pharmaceutical grade DCA. Four types of mutagenicity test were included, with and without metabolic activation where appropriate. These studies included: (i) Salmonella and Escherichia coli mutation (Ames) test, (ii) thymidine kinase locus forward mutation in L5178Y mouse lymphoma cells, (iii) tests for chromosomal aberrations in Chinese hamster ovary cells, and (iv) and in vivo rat bone marrow erythroid micronucleus test. In each study, there was no evidence of mutagenic activity attributable to DCA. It is possible that the present test material, of pharmaceutical grade, has fewer impurities than materials studied in previous reports. These data extend, and in some cases contradict, previous published reports on DCA.

In vitro genotoxicity testing of ARASCO® and DHASCO® oils

ARASCO and DHASCO oils are microbially-derived triglycerides rich in arachidonic (20:4n-6) and docosahexaenoic (22:6n-3) acids, respectively. Both oils were tested for mutagenic activity in three different in vitro mutagenesis assays. All assays were conducted with and without metabolic activation. Neither ARASCO nor DHASCO oil was mutagenic in the Ames reverse mutation assay using five different Salmonella histidine auxotroph tester strains, nor were the oils mutagenic in the mouse lymphoma TK(+/-) forward mutation assay. The oils showed no clastogenic activity in chromosomal aberration assays performed with Chinese hamster ovary cells. Based on these assays, neither ARASCO nor DHASCO oils appear to have any genotoxic potential.

Assessment of the Potential Genotoxicity of Perfluorodecanoic Acid and Chlorotrifluoroethylene Trimer and Tetramer Acids

Perfluoro-n-decanoic acid (PFDA) is a perfluorinated fatty acid that produces hepatomegaly and increased peroxisomal beta-oxidation when administered to rodents. Chlorotrifluoroethylene (CTFE) trimer acid and CTFE tetramer acid are metabolites of the six- and eight-carbon oligomers of CTFE, respectively. They are structurally related to PFDA, and CTFE tetramer acid has caused toxic effects in rodents that are similar to those observed following PFDA administration. Because of the correlation between peroxisome proliferation and hepatocarcinogenesis, CTFE trimer acid, CTFE tetramer acid, and PFDA were evaluated in in vitro and in vivo/in vitro bioassays to assess their potential genotoxic activity. The assays conducted were the Ames Salmonella/microsomal mutagenicity assay, the hypoxanthineguanine phosphoribosyltransferase (HGPRT) locus Chinese hamster ovary gene mutation assay, the sister chromatid exchange (SCE) assay, chromosomal aberration assay, and an in vivo/in vitro unscheduled DNA synthesis (UDS) and S-phase DNA synthesis assay. All test articles were negative in the Ames assay, the HGPRT assay, and the SCE assay. In the chromosomal aberration assay CTFE trimer acid and CTFE tetramer acid were negative in cultures with and without S9 metabolic activation. PFDA was also negative in the absence of metabolic activation, but chromosomal aberrations were observed when PFDA was incubated in the presence of S9 fraction. All test articles were negative for inducing UDS but all induced S-phase replicative DNA synthesis 16 hr after administration of the test article to the test animals; only CTFE tetramer acid and PFDA induced S-phase synthesis 48 hr after dosing: the usual timepoint examined for this response.

Evaluation of the in vitro genetic toxicity of 4-(2,4-dichlorophenoxy)butyric acid

The herbicide 4-(2,4-dichlorophenoxy)butyric acid (2,4-DB) is principally used in the USA on peanuts, soybeans and alfalfa. In Europe, it is used on undersown spring barley and grassland (with clover). The genetic toxicity in vitro of the dimethylamine salt of 2,4-DB was examined by employing a range of end points including gene mutation in bacteria (Ames test) and mammalian cell cultures (CHO/HGPRT assay), cytogenetic abnormalities in mammalian cells (CHO/chromosomal aberration assay), and induction of DNA damage and repair in rat hepatocytes. There were no indications of genotoxic potential for 2,4-DB in the first three of these assays. One of the two criteria for a positive response in the UDS assay was exceeded but the increases did not exceed the second criteria for a positive response. The test material was therefore evaluated as weakly active in this assay. The weight of the evidence clearly indicates that 2, 4-DB is not genotoxic to mammals and are consistent with the reported lack of carcinogenic potential for 2,4-DB in both mice and rats.

Lack of genotoxic effects of sucrose acetate isobutyrate (SAIB)

Sucrose acetate isobutyrate (SAIB) was tested for potential genotoxic activity in four different in vitro assay systems. Two independent trials of a Salmonella reverse mutation assay (using strains TA98, TA100, TA1535, TA1537 and TA1538) showed no increases in revertant frequencies at doses up to 10,000 microg/plate which was non-toxic but exceeded the solubility limit. Similarly, no mutagenic response was observed at doses up to 1000 microg/ml at the HGPRT locus in cultured CHO cells; SAIB was toxic and its solubility limit was exceeded at 50 microg/ml. No clastogenic activity was detected in cultured CHO cells at concentrations up to 2000 microg/ml. All three preceding in vitro tests were conducted both in the presence and absence of Aroclor 1254-induced rat liver S-9 metabolic activation systems. An unscheduled DNA synthesis assay also was performed using rat primary hepatocyte cultures with doses up to 1000 microg/ml, and no DNA repair was detectable. Thus, SAIB was stringently tested at doses exceeding the solubility limit in culture medium and causing toxicity to CHO cells without obtaining any evidence for genotoxic activity as a mutagen, clastogen, or DNA-damaging agent.