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https://ntp.niehs.nih.gov/go/dart02abs

Abstract for DART-02

Prenatal Developmental Toxicity Studies of 4-Methylcyclohexanemethanol in Sprague Dawley (Hsd:Sprague Dawley SD) Rats (Gavage Studies)

CASRN: 34885-03-5
Chemical Formula: C8H16O
Molecular Weight: 128.21
Synonyms/Common Names: Cyclohexanemethanol, 4-methyl-; MCHM
Peer Review Date: July 31, 2019

DRAFT REPORT PDF

Abstract

NOTE: The following abstract is from the draft NTP report for external peer review. The final report will be available in PDF format.

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 parts per million (ppm) for MCHM, and nominated MCHM and other chemicals present in the Elk River spill to the National Toxicology Program (NTP) for toxicity evaluation. Due to the potential for exposure of pregnant women to MCHM and the absence of adequate developmental toxicity data, the NTP conducted studies to characterize the toxicity of MCHM in a regulatory accepted in vivo rat model system that assesses the potential harm to the developing conceptus and pregnant rat. Time-mated pregnant Harlan Sprague Dawley rats (Hsd:Sprague Dawley SD) 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.

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 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 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 post-implantation 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. There were no external malformations or variations 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 MCHM/kg body weight per 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 MCHM 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. There were no exposure-related effects 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 (SNR) and full, thoracolumbar SNR (malformations) were significantly higher in the 400 mg/kg group. Together, the total incidence of all malformations of the ribs, sternebrae, SNR, and vertebrae were present in 1.0, 1.1, 2.2, 2.8 and 15.7% 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 impact 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 (>10³ 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 this prenatal study, there was clear evidence of developmental toxicity of MCHM in Hsd:Sprague Dawley 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.

Studies

Summary of Exposure-Related Findings in Rats in the Prenatal Developmental Toxicity Gavage Study of 4-Methylcyclohexanemethanol

0 mg/kg 50 mg/kg 100 mg/kg 200 mg/kg 400 mg/kg
Maternal Parameters
Animals on study 25 25 25 25 25
Number pregnant 23 21 22 19 21
Number died or euthanized moribund 0 0 0 0 0
Clinical Observations None None None None None
Body Weight and Feed Consumption[a]
Necropsy body weight 370.9 ± 5.7** 381.5 ± 4.3 370.1 ± 5.8 368.8 ± 5.6 356.9 ± 4 .9
Body weight change GD 6 to 21 131.4 ± 3.9** 141.5 ± 4.3 130.1 ± 4.8 129.6 ± 4.9 116.5 ± 4.1*
Feed consumption GD 6 to 21 20.7 ± 0.26** 21.2 ± 0.35 21.2 ± 0.26 21.9 ± 0.23** 22.1 ± 0.29**
Necropsy Observations None None None None None
Clinical Pathology
Hematology None None None None None
Clinical chemistry None None ↓ Total protein (6%) ↓ Total protein (5%) ↓ Total protein (8%)
↓ Globulin (10%) ↓ Globulin (9%) ↓ Globulin (10%)
Developmental/Fetal Parameters
Number of litters examined 23 21 22 19 21
Number of live fetuses evaluated 296 283 279 247 254
Number of live fetuses per litter[b] 12.87 ± 0.64 13.48 ± 0.58 12.68 ± 0.71 13.00 ± 0.81 12.10 ± 0.79
Number of early resorptions 21 16 12 14 20
Number of late resorptions 1 2 0 0 0
Number of dead fetuses 0 0 0 0 0
Number of whole litter resorptions 0 0 1 0 0
Percent post-implantation loss 8.02 ± 2.47 6.54 ± 3.13 8.09 ± 4.50 5.18 ± 1.96 7.76 ± 2.51
Fetal body weight per litter[a] 5.14 ± 0.07** 5.16 ± 0.08 5.14 ± 0.07 4.98 ± 0.09 4.39 ± 0.09**
Male fetal body weight per litter 5.28 ± 0.06** 5.30 ± 0.08 5.28 ± 0.07 5.12 ± 0.09 4.46 ± 0.09**
Female fetal body weight per litter 4.99 ± 0.07** 5.00 ± 0.08 4.98 ± 0.07 4.82 ± 0.09 4.34 ± 0.12**
Gravid uterine weight[a] 91.76 ± 4.05** 96.88 ± 3.61 90.41 ± 4.83 88.57 ± 4.88 75.58 ± 4.20**
External Findings None None None None None
Visceral Findings[c]
Abdominal Viscera
Adrenal, total, discolored — [V]
Fetuses 0   (0.0)* 1   (0.4) 0   (0.0) 0   (0.0) 3   (1.2)
Litters 0   (0.00)* 1   (4.76) 0   (0.00) 0   (0.00) 3   (14.29)
Adrenal, total, misshapen — [M]
Fetuses 0   (0.0)* 1   (0.4) 0   (0.0) 0   (0.0) 3   (1.2)
Litters 0   (0.00)* 1   (4.76) 0   (0.00) 0   (0.00) 3   (14.29)
Urinary Tract
Kidney, total, discolored — [V]
Fetuses 0   (0.0) 1   (0.4) 0   (0.0) 0   (0.0) 3   (1.2)
Litters 0   (0.00)* 1   (4.76) 0   (0.00) 0   (0.00) 3   (14.29)
Skeletal Findings
Ribs
Costal cartilage, total, misaligned — [V]
Fetuses 0   (0.0)** 0   (0.0) 0   (0.0) 0   (0.0) 3   (1.2)
Litters 0   (0.00)* 0   (0.00) 0   (0.00) 0   (0.00) 3   (9.52)
Costal cartilage, 7th right, not fused to sternum — [M]
Fetuses 0   (0.0)** 0   (0.0) 0   (0.0) 2   (0.8) 4   (1.6)*
Litters 0   (0.00)** 0   (0.00) 0   (0.00) 1   (5.26) 4   (19.05)*
Sternebrae
Sternebra, total, unossified or misaligned — [V]
Fetuses 0   (0.0)** 1   (0.4) 0   (0.0) 0   (0.0) 7   (2.8)**
Litters 0   (0.00)** 1   (4.76) 0   (0.00) 0   (0.00) 6   (28.57)**
Vertebrae
Thoracic centrum, total, incomplete ossification or unossified — [V]
Fetuses 0   (0.0)** 1   (0.4) 0   (0.0) 0   (0.0) 4   (1.6)*
Litters 0   (0.00)** 1   (4.76) 0   (0.00) 0   (0.00) 3   (14.29)
Supernumerary rib
Cervical, total, short — [M]
Fetuses 0   (0.0)** 0   (0.0) 0   (0.0) 0   (0.0) 6   (2.4)**
Litters 0   (0.00)** 0   (0.00) 0   (0.00) 0   (0.00) 3   (14.29)
Thoracolumbar, total, full — [M]
Fetuses 2   (0.7)**## 1   (0.4) 6   (2.2) 5   (2.0) 26   (10.2)**##
Litters 2   (8.70)** 1   (4.76) 3   (14.29) 4   (21.05) 7   (33.33)

Level of evidence of developmental toxicity: Clear evidence


*    Statistically significant (P≤0.05) trend (denoted in vehicle control column) or pairwise comparison (denoted in dosed group column);
**   P≤0.01.
##  Statistically significant (P≤0.01) trend (denoted in vehicle control column) or pairwise comparison (denoted in dosed group column) in litter-based analysis of fetuses
[a]   
Results given in grams. Data are displayed as mean ± standard error.
[b]   
Data are displayed as mean ± standard error.<p>
[c]   
Upper row denotes the number of affected fetuses and (%) and lower row the number of affected litters and (%)
GD = Gestation Day
[M] = Malformation
[V] = Variation