Mutagenicity testing methods

L. Jacobs and R. DeMars, Chemical mutagenesis with diploid human fibroblasts, in Handbook of Mutagenicity Test Methods (B. Kilbey et ai, eds.), pp. 193-220, Elsevier, Amsterdam (1978). 

Nishigaki, K. Nikami, M. Gautum, S. G. Kamiseki, M. Mutagenicity test method using mammalian cells. Jpn. Kokai Tokkyo Koho JP 2012139169, 2012. 

Ames B.N., McCann J. Yamasaki E. (1975) Methods for detecting carcinogens and mutagens with the Salmonella/mammalian microsome mutagenicity test. MutatRes, 31, 347-364. 

Maron DM, BN Ames (1983) Revised methods for the Salmonella mutagenicity test. Mutat Res 113 173-215. 

Table 4. Plant species positive to the Feulgen method, mutagenicity coumpounds used and mutagenic tests applied ...

Some modifications to current in vivo testing methods both can and should be adopted. A current example of this would be in medical devices where a substantial portion of the requirements under the governing regulatory (ISO 10993) can be met with in vitro alternatives (cytogenicity, muscle cell implantation, the limulus test for pyrogens, and in vitro mutagenicity assays). 

Ames, B. N., McCann, J. and Yamasaki, E., Methods for Detecting Carcinogens and Mutagens with the Salmonella/Mammalian Microsome Mutagenicity Test , Mutat. Res. (1975) 31, 347. 

Certain substances may need special consideration, such as highly electrophilic substances, which give positive results in vitro, particularly in the absence of metabolic activation. Although these substances may react with proteins and water in vivo and thus be rendered inactive toward many tissues, they may be able to express their mutagenic potential at the first site of contact with the body. Consequently, the use of test methods that can be applied to the respiratory tract, upper gastrointestinal tract, and skin may be appropriate. 

The in vitro and in vivo test methods available to study combined actions and toxicological and biochemical interactions of chemicals in mixtures are essentially the same as those used for the study of single chemicals in order to examine their potential general toxicity and special effects such as mutagenicity, carcinogenicity, and reproductive toxicity. 

Araki A, Noguchi T, Kato F, et al Improved method for mutagenicity testing of gaseous compounds by using a gas sampling bag. Mutat Res 307(l) 335-44, 1994... 

The panel meeting participants unanimously agreed that the air particulates protocol is a proven method with an adequate data base to demonstrate applicability to the preparation of air particulates for mutagenicity testing. However, the participants agreed that several areas require method validation. 

Salmonella Mutagenicity Test (Ames Test). The methods of bacterial culture, the verification of genetic markers, and the plate incorporation assay were essentially the same as described previously (14, 15). Petri dishes (90 mm) containing about 20 mL of 1.2 Noble agar in minimal Vogel Bonner Medium E supplied with excess biotine and... 

Figure 1. Effect of resin type on the mutagenic activity of drinking water concentrates in the Ames test. The sampling, 7000-fold concentration with either XAD-2 or XAD-4/8, DMSO elution (20 mL, neutral fraction), and subsequent mutagenicity testing were as described in Materials and Methods. Similar concentrates of The Hague tap water were used as controls. Each point represents the average of four plates, and 0.50 mL of concentrate corresponds to 3.5 L of water per plate.

Figure 4. Molecular weight determination of a drinking water concentrate with Sephadex LH20. Sampling, 10 -fold concentration of drinking water before and after chlorination (L5 mg/L of CI2, Meuse River source) on XAD-4/8, elution with DMSO (neutral fraction), and subsequent gel filtration were as described in Materials and Methods. After measuring the absorbance at 263 mm, the fractions were pooled as indicated. After dilution in water, the fractions were reconcentrated on XAD-4/8, eluted with DMSO, and assayed in the Salmonella mutagenicity test (strain TA98 S9).

Figure 6. Fractionation of a mutagenic drinking water concentrate with Sephadex LH20. On a Sephadex LH20 column, 1.6 mL of a drinking water concentrate (IX 106-fold concentrated, neutral fraction) was separated by using stepwise isopropyl alcohol (ISOP) and dioxane/water (D/W) elution as described in Material and Methods. Fractions were pooled as indicated. After reconcentration the fractions were assayed for mutagenic activity in the Salmonella mutagenicity test and CHO cells (Table I).

Figure 8. Mutagenic activity of a fractionated LH20 drinking water concentrate (neutral fraction) as detected with nitroreductase-deficient bacterial strains. LH20 fractionated drinking water concentrates were tested in the Salmonella mutagenicity test as described in Materials and Methods. Each point represents the average value of three plates.

Figure 10. Influence of a chlorine treatment on the mutagenic activity detectable with TA98NR and TA100NR strains. Sampling, 7000-fold concentration of water samples before and after a chlorine treatment (1.5 mg/L of Ch) on XAD-4/8, elution with DMSO (neutral fraction)> and subsequent testing of the DMSO concentrate in the Salmonella mutagenicity test were as described in Materials and Methods. Each value represents the average of three plates, and 0.2 mL of concentrate corresponds to 1.4 L of...

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