D.2.1. Bacterial Mutagenicity Test Protocol

Testing procedures were modified from those originally reported by Zeiger et al.¹³⁸ Coded samples of the same chemical lots used in the 3-month studies of ionic liquids (ILs; 1-ethyl-3-methylimidazolium chloride [Emim-Cl], 1-butyl-3-methylimidazolium chloride [Bmim-Cl], 1-butyl-1-methylpyrrolidinium chloride [Bmpy-Cl], or n-butylpyridinium chloride [NBuPy-Cl]) were incubated with the Salmonella typhimurium (TA98, TA100) or Escherichia coli WP2 uvrA (pKM101) tester strains, either in buffer or S9 mix (metabolic activation enzymes and cofactors from Aroclor 125-induced male Sprague Dawley rat liver [Moltox, Boone, NC]) for 20 minutes at 37°C. Top agar supplemented with L-histidine (or tryptophan for the E. coli strain) and d-biotin was added, and the contents of the tubes were mixed and poured onto the surfaces of minimal glucose agar plates. Histidine- or tryptophan-independent mutant colonies arising on these plates were counted after incubation for 2 days at 37°C.

Each trial consisted of triplicate plates of concurrent positive and negative controls and five doses of each IL. The highest concentration tested was 10,000 μg/plate for each IL. All trials were repeated.

In this assay, a positive response is defined as a reproducible, dose-related increase in histidine-independent (revertant) colonies in any one strain/activation combination. An equivocal response is defined as an increase in revertants that is not dose-related, is not reproducible, or is not of sufficient magnitude to support a determination of mutagenicity. A negative response is obtained when no increase in revertant colonies is observed after chemical treatment. No minimum percentage or fold increase is required for a chemical to be judged positive or weakly positive, although positive calls are typically reserved for increases in mutant colonies that are at least twofold over background.

D.2.2. Results

The four ILs were tested for induction of gene mutations in the bacterial mutation (Ames) assay. All four ILs—Emim-Cl, Bmim-Cl, Bmpy-Cl, and NBuPy-Cl—were negative for induction of gene mutations in three strains of bacteria (S. typhimurium strains TA98 and TA100, and E. coli strain WP2 uvrA [pKM101]), tested with and without induced rat liver S9 mix at doses up to 10,000 μg/plate (Table D-1, Table D-2, Table D-3, Table D-4).

D.3.1. Peripheral Blood Micronucleus Test Protocol

Upon termination of the 3-month toxicity studies of each of the four ILs (Emim-Cl, Bmim-Cl, Bmpy-Cl, and NBuPy-Cl), blood samples (approximately 200 μL) were collected from male and female rats and mice, placed in ethylenediaminetetraacetic acid (EDTA)-coated tubes, and shipped overnight to the testing laboratory. Upon arrival, blood samples were fixed in ultracold methanol using a MicroFlowPLUS Kit (Litron Laboratories, Rochester, NY) according to the manufacturer’s instructions. Fixed samples were stored in a −80°C freezer until analysis. Thawed blood samples were analyzed for frequency of micronucleated immature erythrocytes (i.e., reticulocytes or polychromatic erythrocytes [PCEs]) and mature erythrocytes (i.e., normochromatic erythrocytes [NCEs]) using a flow cytometer¹³⁹; both the mature and immature erythrocyte populations can be analyzed separately by employing special cell surface markers to differentiate the two cell types. Because the very young reticulocyte subpopulation (CD71+ cells) can be targeted using this technique, rat blood samples can be analyzed for damage that occurred in the bone marrow within the past 24–48 hours, before the rat spleen appreciably alters the percentage of PCEs in circulation.¹⁴⁰ In mice, both the mature and immature erythrocyte populations can be evaluated for micronucleus frequency because the mouse spleen does not sequester and eliminate damaged erythrocytes. Damaged erythrocytes achieve steady state in the peripheral blood of mice after 4 weeks of continuous exposure. Approximately 20,000 PCEs and 1 × 10⁶ NCEs were analyzed per animal for the frequency of micronucleated cells, and the percentage of immature erythrocytes (% PCE) was calculated as a measure of bone marrow toxicity resulting from chemical exposure.

Prior experience with the large number of cells scored using flow cytometric scoring techniques¹⁴¹ suggests it is reasonable to assume that the proportion of micronucleated reticulocytes is approximately normally distributed. The statistical tests selected for trend and for pairwise comparisons with the control group depend on whether the variances among the groups are equal. The Levene test at α = 0.05 is used to test for equal variances. In the case of equal variances, linear regression is used to test for a linear trend with exposure concentration and the Williams test is used to test for pairwise differences between each exposed group and the control group. In the case of unequal variances, the Jonckheere test is used to test for linear trend and the Dunn test is used for pairwise comparisons of each exposed group with the control group. To correct for multiple pairwise comparisons, the p value for each comparison with the control group is multiplied by the number of comparisons made. In the event that this product is >1.00, it is replaced with 1.00. Trend tests and pairwise comparisons with the controls are considered statistically significant at p ≤ 0.025.

In the micronucleus test, it is preferable to base a positive result on the presence of both a positive trend as well as at least one significantly elevated exposed group compared to the corresponding control group. In addition, historical control data are used to evaluate the biological significance of any observed response. Both statistical significance and biological significance are considered when arriving at a call. The presence of either a positive trend or a single significant exposed group generally results in an equivocal call. The absence of both a trend and any significant differences between exposed groups and the control group results in a negative call. Ultimately, the scientific staff determines the final call after considering the results of statistical analyses, reproducibility of any effects observed (in acute studies), and the magnitudes of those effects.

D.3.2. Results

The four ILs were tested for induction of structural or numerical chromosomal damage in the rodent peripheral blood erythrocyte micronucleus assay following 3 months of exposure via dosed water.

Emim-Cl did not increase the frequency of micronucleated erythrocytes in male or female rats or in male mice exposed via dosed water for 3 months (Table D-5, Table D-9). In female mice, the micronucleus assay response was judged to be negative, although a small, significant increase was observed in micronucleated NCEs; this response was downgraded because it was not considered of sufficient magnitude to be biologically relevant and was well within the laboratory historical control range for female B6C3F1 mice.

Bmim-Cl did not increase the frequency of micronucleated erythrocytes in male or female rats or mice exposed via dosed water for 3 months (Table D-6, Table D-10).

Bmpy-Cl was positive in the peripheral blood micronucleus test in male rats following 3 months of exposure via dosed water, although the observed increases in micronucleated PCEs were within the laboratory historical control 95% confidence interval. Bmpy-Cl did not induce an increase in micronucleated erythrocytes in female rats or in male or female mice (Table D-7, Table D-11).

A clearly significant increase in micronucleated NCEs was observed in male rats exposed to NBuPy-Cl for 3 months via dosed water, whereas no increase was seen in micronucleated PCEs in these same animals (Table D-8). The response was judged to be equivocal because (a) this response pattern is in direct contrast to what would be expected in rats for a truly positive compound, (b) the increases observed were within the laboratory 95% confidence interval for male rats, and (c) there is no biological explanation for the observation (such as splenic malfunction). NBuPy-Cl was negative in female rats and male and female mice in the micronucleus assay (Table D-8, Table D-12).

Occasional small increases in % PCE were observed in some of the animal groups, but none was associated with an effect on micronucleus frequencies and all were within the laboratory historical control 95% confidence interval. They do not suggest an effect on erythropoiesis.