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https://ntp.niehs.nih.gov/go/imm001abs

Abstract for IMM-01

Immunotoxicity Technical Report on the Dermal Hypersensitivity and Irritancy Studies of 4-Methylcyclohexanemethanol and Crude
4-Methylcyclohexanemethanol Administered Topically to Female BALB/c Mice

Substances:

  • 4-Methylcyclohexanemethanol (CASRN 34885-03-5)
  • Crude 4-Methylcyclohexanemethanol (CASRN CRUDEMCHM)

Report Date: July 2020

FULL REPORT PDF

Abstract

4-Methylcyclohexanemethanol (MCHM) and crude MCHM were nominated to the National Toxicology Program (NTP) for toxicity evaluation by the Centers for Disease Control and Prevention and the Agency for Toxic Substances and Disease Registry following the Elk River, West Virginia, chemical spill. The 10,000-gallon spill was a mixture of chemicals that contained approximately 75% MCHM. Due to reports of reddened skin and erythema following dermal exposure to contaminated water from the spill, NTP conducted studies in female BALB/c mice to assess the potential dermal hypersensitivity and irritancy of MCHM and crude MCHM.

MCHM is a clear, colorless oil with low estimated water solubility and a reported alcohol or licorice-like odor. MCHM is commonly sold as a crude mixture, which can contain 68% to 89% MCHM with additional components. It is used as a flotation reagent in fine coal beneficiation. Human exposure can occur during handling or use of the chemical.

In the initial study evaluating whether MCHM and crude MCHM have similar immunotoxicologic profiles, groups of five mice were administered a vehicle control (acetone/olive oil, 4:1 v/v [AOO]); one of two positive controls (1 fluoro-2,4-dinitrobenzene [0.15% in AOO] or isoeugenol [5% in AOO]); 2%, 20%, or 100/50% formulations of MCHM in AOO; or 1%, 2%, 5%, 20%, 40%, or 100/80% formulations of crude MCHM in AOO. The 100/50% group was administered 100% on day 1 and 50% on days 2 and 3; the 100/80% group was administered 100% on day 1 and 80% on days 2 and 3. The 100% dose was lowered to 50% (MCHM) and 80% (crude MCHM) on days 2 and 3 due to signs of morbidity. A repeat study of crude MCHM was undertaken to more clearly establish levels at which the test article induced sensitization in the absence of clinical toxicity. In the repeat study of crude MCHM, groups of female mice were administered the AOO vehicle control; a positive control (1 fluoro-2,4-dinitrobenzene [0.15% in AOO]); or crude MCHM at concentrations of 1%, 5%, 25%, 50%, or 75% in AOO. The mice were treated for three consecutive days by direct epicutaneous application of 25 µL of the test or vehicle control article to the dorsum of each ear.

In the MCHM study, all mice administered 2% or 20% survived to study termination with no clinical observations of toxicity. Mice in the 100/50% group demonstrated signs of morbidity on exposure day 1, resulting in two mice being euthanized moribund. Clinical observations noted in surviving 100/50% mice included eye squinting and hypoactivity. Concentration-dependent increases in mean ear thickness were observed in the 20% and 100/50% groups on day 3 and day 6, relative to the vehicle control group. Treatment with MCHM did not significantly modulate mean group disintegrations per minute (DPM), compared to that of the vehicle control group or the stimulation index (SI) (ratio of treated group mean DPM/vehicle control group mean DPM) values in the local lymph node assay.

In the initial study of crude MCHM, all mice survived to study termination. Clinical signs of toxicity were only observed in the 100/80% group and included eye squinting, hypoactivity, and isolation from cage mates. Mean ear thickness was significantly increased on day 6 in the 20% group relative to the vehicle control group. It was also increased in the 100/80% group on day 3, though not significantly, and there was a significant concentration-related trend on day 3. In the local lymph node assay, mice treated with 5% crude MCHM or greater had significantly increased DPM values relative to vehicle controls, and this proliferative response followed a significant concentration-related trend. Groups of mice treated with 5% crude MCHM or greater had significantly increased mean SI values, with SI values of more than 3.0 occurring at 40% and 100/80%. The concentration that would result in a SI of 3.0 (EC3) was calculated to be 34.6%.

In the repeat study of crude MCHM, all mice survived to study termination. Clinical signs of toxicity were limited to the 50% and 75% groups and included eye squinting and isolation from cage mates. Ear thickness in the 75% group was significantly increased relative to that of the vehicle control group on days 3 and 6. No changes in ear thickness were observed in the remaining dosed groups. Significant increases in lymph node cell proliferation as measured by DPM and SI values were observed at 50% and 75% crude MCHM. The concentration that would result in a SI of 3.0 (EC3) was calculated to be 60%.

Under the conditions of these dermal hypersensitivity and irritancy studies, MCHM induced dermal irritation in female mice, as indicated by increases in ear swelling at concentrations of 20% and 100/50%. Crude MCHM was identified as a skin sensitizer, with an EC3 value in the concentration range of 35% to 60%. Dermal exposure to crude MCHM produced minimal irritant effects, indicated by increases in ear swelling at concentrations of 20% and 75%, which were inconsistent across repeat studies.

National Toxicology Program (NTP). 2020. NTP immunotoxicity technical report on the dermal hypersensitivity and irritancy studies of 4-methylcyclohexanemethanol (CASRN 34885-03-5) and crude 4-methylcyclohexanemethanol administered topically to female BALB/c mice. Research Triangle Park, NC: National Toxicology Program. Immunotoxicity Report 01.

Studies

Summary of Findings Considered Immunotoxicologically Relevant in Female BALB/c Mice Topically Administered 4-Methylcyclohexanemethanol and Crude 4-Methylcyclohexanemethanol
  Pure
4‑Methylcyclohexanemethanol
Crude
4‑Methylcyclohexanemethanol
Crude
4‑Methylcyclohexanemethanol
(Repeat Study)[a][b]
Concentrations by topical application 0%, 2%, 20%, or 100/50% 0%, 1%, 2%, 5%, 20%, 40%, or 100/80% 0%, 1%, 5%, 25%, 50%, or 75%
Survival rates 5/5, 5/5, 5/5, 3/5 5/5, 5/5, 5/5, 5/5, 5/5, 5/5, 5/5 13/13, 13/13, 13/13, 13/13, 13/13, 13/13
Body weights Treated groups similar to the vehicle control group Treated groups similar to the vehicle control group Treated groups similar to the vehicle control group
Clinical observations Isolation; eye shut, bilateral; hunched posture; and hypoactivity in 100/50% group Isolation; eye shut, bilateral; and hypoactivity in 100/80% group Isolation in 50% and 75% groups and eye shut, bilateral in 75% group
Irritancy ↑ Mean ear thickness in the 20% and 100/50% groups ↑ Mean ear thickness in the 20% group ↑ Mean ear thickness in the 75% group
Hypersensitivity[*] None ↑ DPM values in the 5%, 20%, 40%, and 100/80% groups
↑ SI values in the 5%, 20%, 40%, and 100/80% groups
↑ DPM values in the 50% and 75% groups
↑ SI values in the 75% group
Calculated EC3 NA 34.6% 60.0%
SI>3 No Yes (40% and 100/80% groups) Yes (75% group)

*DPM = disintegrations per minute (as measured by 125I-UdR incorporation); SI = stimulation index; N/A = not applicable because SI did not reach 3.
a Irritancy studies were conducted using groups 1 through 7 (as described in the Materials and Methods section).
b Hypersensitivity studies were conducted using groups 8 through 14 (as described in the Materials and Methods section).