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Evaluation of 4-methylcyclohexanemethanol (MCHM) in a Combined Irritancy and Local Lymph Node Assay (LLNA) in Mice

Johnson VJ, Auerbach SS, Luster MI, Waidyanatha S, Masten SA, Wolfe MS, Burleson FG, Burleson GR, Germolec DR.
Food and Chemical Toxicology (2017) DOI: https://doi.org/10.1016/j.fct.2017.03.034 PMID: 28343035

Publication

Abstract

4-Methylcyclohexanemethanol (MCHM) is a flotation reagent used in fine coal beneficiation. On January 9, 2014, crude MCHM, a mixture containing predominantly MCHM, was inadvertently released into the Elk River, a municipal water source that serves about 300,000 people in the Charleston, WV area, resulting in temporary contamination of 15 percent of the state's tap water and causing significant dermal exposure. The current studies were undertaken to determine whether crude MCHM or MCHM has the potential to produce dermal irritancy and/or sensitization. BALB/c female mice were treated daily for 3 consecutive days by direct epicutaneous application of 25 μL of various concentrations of crude MCHM or MCHM to the dorsum of each ear. A mouse ear-swelling test was used to determine irritancy potential and was undertaken in combination with the standardized Local Lymph Node Assay (LLNA) to determine skin sensitizing potential. MCHM was found to produce skin irritation at concentrations above 20% and did not produce sensitization. Crude MCHM also produced irritation, although weaker, and in addition was found to be a weak to moderate skin sensitizer. The results are discussed in terms of potential human health hazard.

Figures

Figure 1. Impact of dermal treatment with MCHM on ear swelling in BALB/c mice.

(Study 1). Treatment induced changes in ear thickness were measured using a calibrated micrometer approximately 2 h following the final application of MCHM on Day 3 (A) and again prior to euthanasia on Day 6 (B). Data are expressed as relative change from the vehicle control group mean ear thickness. DNFB - 1-Fluoro-2,4-dinitrobenzene; MCHM - 4-methylcyclohexanemethanol; AOO – acetone:olive oil (4:1 v/v) Each bar represents the mean ± SE (N = 5 except 100/50% MCHM where N = 3). *Significantly different from AOO control group (P < 0.05, Dunnett's T-Test). 1Significant dose-response trend for crude MCHM and MCHM (P < 0.05, Jonckheere's Trend Test). 2Significant dose-response trend for MCHM (P < 0.05, Jonckheere's Trend Test).

Figure 2. Impact of dermal treatment with MCHM on lymphocyte proliferation.

Impact of dermal treatment with MCHM on lymphocyte proliferation in the draining lymph node (Study 1). Lymphocyte proliferation in the draining lymph node was determined by quantifying the incorporation of 125IUdR into the DNA of the proliferating cells and data are presented at DPM values. The Stimulation Index (SI) was determined for each animal relative to the mean lymphocyte proliferation in the vehicle control group and was used to determine the concentration of MCHM that increased proliferation 3-fold (EC3). SI values for the AOO vehicle control group were 1.00 ± 0.22. SI values for crude MCHM treatments were 1.83 ± 0.64, 1.73 ± 0.15, 2.29 ± 0.27, 2.95 ± 0.33, 3.09 ± 0.58, and 4.83 ± 0.81 for groups treated with 1%, 2%, 5%, 20%, 40%, and 100/80%, respectively. SI values for MCHM treatments were 0.11 ± 0.05, 0.28 ± 0.06, and 0.88 ± 0.29, respectively for groups treated with 2%, 20%, and 100/50%, respectively. SI values for the DNFB positive control group were 10.02 ± 1.19. DNFB - 1-Fluoro-2,4-dinitrobenze; MCHM - 4-methylcyclohexanemethanol; AOO - acetone:olive oil (4:1 v/v). Each bar represents the mean +/− SEM (N = 5 except 100/50% MCHM where N = 3). *Significantly different from AOO control group (P < 0.05, Dunnett's T-Test). 1Significant dose-response trend for crude MCHM (P < 0.05, Jonckheere's Trend Test).

Figure 3. Impact of dermal treatment with crude MCHM on ear swelling in BALB/c mice.

(Study 2). Treatment induced changes in ear thickness were measured using a calibrated micrometer approximately 2 h following the final application of MCHM on Day 3 (A) and again prior to euthanasia on Day 6 (B). Data are expressed as relative change from the vehicle control group mean ear thickness. DNFB - 1-Fluoro-2,4-dinitrobenzene; MCHM - 4-methylcyclohexanemethanol; AOO - acetone:olive oil (4:1 v/v). Each bar represents the mean ± SE (N = 8). *Significantly different from AOO control group (P < 0.05, Dunnett's T-Test). 1Significant dose-response trend for crude MCHM (P < 0.05, Jonckheere's Trend Test).

Figure 4. Impact of dermal treatment with crude MCHM on lymphocyte proliferation.

Impact of dermal treatment with crude MCHM on lymphocyte proliferation in the draining lymph node (Study 2). Lymphocyte proliferation in the draining lymph node was determined by quantifying the incorporation of 125IUdR into the DNA of the proliferating cells and data are presented at DPM values. The Stimulation Index (SI) was determined for each animal relative to the mean lymphocyte proliferation in the vehicle control group and was used to determine the concentration of MCHM that increased proliferation 3-fold (EC3). SI values for the AOO vehicle control group were 1.00 ± 0.24. SI values for crude MCHM treatments were 1.10 ± 0.28, 0.73 ± 0.10, 1.45 ± 0.27, 2.24 ± 0.46, and 4.22 ± 0.54 for groups treated with 1%, 5%, 25%, 50%, and 75%, respectively. SI values for the DNFB positive control group were 76.02 ± 12.50. DNFB - 1-Fluoro-2,4-dinitrobenzene at 0.15%; MCHM - 4-methylcyclohexanemethanol; AOO - acetone:olive oil (4:1 v/v). Each bar represents the mean+/− SEM (N = 8). *Significantly different from AOO control group (P < 0.05, Dunnett's T-Test). 1Significant dose-response trend (P < 0.05, Jonckheere's Trend Test).