The organic compound 4-methylcyclohexanemethanol (MCHM) is sold as a mixture and is used to reduce impurities in mined coal. On January 9, 2014, an estimated 10,000 gallons of a mixture containing 75% MCHM leaked into the Elk River upstream of the intake for West Virginia American Water Company’s Elk River plant. Upon review of the available toxicity literature for MCHM, the Centers for Disease Control and Prevention and the Agency for Toxic Substances and Disease Registry set a drinking water advisory level of 1 ppm for MCHM and nominated MCHM and other chemicals present in the Elk River spill to the National Toxicology Program (NTP) for toxicity evaluation. Because of the potential for exposure of pregnant women to MCHM and the absence of adequate developmental toxicity data, NTP conducted studies to characterize the toxicity of MCHM in an accepted regulatory in vivo rat model system that assesses the potential harm to the developing conceptus and pregnant rat. Time-mated pregnant Sprague Dawley (Hsd:Sprague Dawley SD) rats received MCHM (99.8% pure) in corn oil via gavage from implantation on gestation day (GD) 6 to GD 20, the day before expected parturition. The potential for MCHM to induce overt maternal and fetal toxicity was examined in a dose range-finding study followed by a prenatal developmental toxicity study. The guideline prenatal developmental toxicity studies discussed in this report provide important animal data that can be used to address the adequacy of the 1 ppm advisory level in protecting sensitive human populations.
Dose range-finding prenatal developmental toxicity study
Time-mated female rats (n = 10/dose level) were administered 0, 150, 300, 600, or 900 mg MCHM/kg body weight per day (mg/kg/day) in corn oil by gavage (2 mL/kg) from GD 6 to GD 20. Control females (0 mg/kg) received corn oil vehicle.
All dams in the 900 mg/kg group were euthanized on GD 7 or 8 due to clinical observations indicating overt toxicity (ataxia, cold to touch, clear ocular discharge, excessive salivation, lethargy/hypoactivity, and/or piloerection); three dams from the 600 mg/kg group displayed similar clinical observations and were removed from study. Body weight gain from GD 6 to 21 in the 600 mg/kg group was 44% lower than that of the vehicle control and was associated with a 13% reduction in feed consumption during the same interval. No signs of maternal toxicity were observed in the 150 or 300 mg/kg dose groups.
Dams administered 600 mg/kg displayed higher postimplantation loss (53%) and lower gravid uterine weight. MCHM exposure did not affect the number of live fetuses per litter or fetal sex ratio; however, fetal weights were 12% and 39% lower in the 300 and 600 mg/kg exposure groups, respectively. No external malformations or variations were attributed to MCHM exposure.
Prenatal developmental toxicity study
Due to the maternal toxicity observed at 600 and 900 mg/kg in the dose range-finding study, time-mated female rats (n = 25/dose level) were administered 0, 50, 100, 200, or 400 mg/kg/day in corn oil (2 mL/kg) by gavage from GD 6 to GD 20. Vehicle control animals (0 mg/kg) received corn oil vehicle.
No clinical observations of toxicity were observed in dams in any dose group. Dams administered 400 mg/kg had significantly lower (11%) mean body weight gains compared to vehicle control dams. Dams administered MCHM had slightly higher feed consumption. Alterations in dam clinical chemistry included reductions in total protein and globulin concentrations that occurred in a dose-related manner in dams administered ≥100 mg/kg.
Dams administered 400 mg/kg exhibited lower gravid uterine weight. No exposure-related effects were found on the number of live fetuses per litter or fetal sex ratio. Fetal body weight was lower (15%) in the 400 mg/kg group. Visceral and skeletal examination identified several anomalies that were attributed to MCHM exposure. Misshapen adrenal glands (malformation) and discolored adrenal glands and kidneys (variations) were observed in fetuses in the 400 mg/kg group. Malformations and variations of the ribs, sternebrae, and vertebrae were also present in the same exposure group. Findings of misaligned costal cartilage (variation); seventh, right costal cartilage not fused to the sternum (malformation); and an increase in short, cervical supernumerary ribs (SNRs) and full, thoracolumbar SNRs (malformations) were significantly higher in the 400 mg/kg group. Together, the total incidence of all malformations of the ribs, sternebrae, SNRs, and vertebrae were present in 1.0%, 1.1%, 2.2%, 2.8%, and 15.8% of fetuses from the 0, 50, 100, 200, and 400 mg/kg groups; these findings were present in 13%, 14%, 14%, 26%, and 57% of litters, respectively.
The maternal no-observed-effect level (NOEL) was 50 mg/kg based on MCHM-related changes in clinical chemistry at doses ≥100 mg/kg, reduced maternal body weight gain at 400 mg/kg, and overt toxicity observed at doses ≥600 mg/kg in the dose range-finding study. The minimal MCHM-related changes in maternal clinical chemistry would not be expected to affect fetal development. MCHM-related effects (lower fetal weight and specific and total axial skeletal malformations) were observed in fetuses exposed to 400 mg/kg, indicating a fetal NOEL of 200 mg/kg. These findings suggest a significant margin of exposure (>1,000-fold) exists between both the maternal and fetal NOELs in the rat and the estimated exposure of 0.04 mg/kg/day in pregnant women at the 1 ppm MCHM advisory level.
Conclusions
Under the conditions of the prenatal study, there was clear evidence of developmental toxicity of MCHM in Sprague Dawley (Hsd:Sprague Dawley SD) rats based on reduced fetal weight, adrenal malformations, and increased malformations of the axial skeleton (short cervical SNR, full thoracolumbar SNR, and costal cartilage not fused to the sternum). These findings occurred in fetuses of dams administered 400 mg/kg and in the absence of overt maternal toxicity.
National Toxicology Program (NTP). 2020. NTP developmental and reproductive toxicity technical report on the prenatal development studies of 4-methylcyclohexanemethanol (CASRN 34885-03-5) in Sprague Dawley (Hsd:Sprague Dawley SD) rats (gavage studies). Research Triangle Park, NC: National Toxicology Program. DART Report 02. https://doi.org/10.22427/NTP-DART-02