Comparative inhalation toxicity of ethyltoluene isomers in rats and mice

Abstract

The C9 alkylbenzenes, composed mostly of ethyltoluenes and trimethylbenzenes, comprise 75-90% of the naphtha fraction of crude oil. Occupational and environmental exposure to C9 alkylbenzenes occur via inhalation. We conducted short-term inhalation studies on the ethyltoluene isomers (2-, 3- or 4-) to select one isomer for more comprehensive studies. Male Hsd:Sprague Dawley rats and female B6C3F1/N mice (n = 10) were exposed by nose-only inhalation to 2-, 3- or 4-ethyltoluene (0, 1000 or 2000 ppm) or cumene (a reference compound: 0, 500 or 1000 ppm) 3 h/day, 5 days/week, for 2 weeks. Clinical observations included abnormal gait and delayed righting reflex. Rats and mice exposed to 2000 ppm 2-ethyltoluene and mice exposed to 2000 ppm 4-ethyltoluene were euthanized early in moribund condition; no exposure-related deaths were observed with 3-ethyltoluene or cumene. Histopathology of selected tissues revealed that the nose and liver (rats and mice) and lung (mice only) to be toxicity targets. In the mouse lung, all compounds except 4-ethyltoluene produced bronchial and bronchiolar hyperplasia. In rats and mice, 2-ethyltoluene was the only compound to produce lesions in the nose and liver: in mice, squamous metaplasia and neutrophilic inflammation of the respiratory epithelium and atrophy and degeneration of the olfactory epithelium were observed in the nose and centrilobular hypertrophy and necrosis were observed in the liver. In rats, 2-ethyltoluene exposure produced atrophy of the olfactory epithelium in the nose and centrilobular necrosis in the liver. Based on mortality, body weight effects and histopathology, the 2-ethyltoluene isomer was the most potent isomer.

Keywords: C9 alkylbenzene; CASRN 25550-14-5; CASRN 611-14-3; CASRN 620-14-4; Ethyltoluene; inhalation toxicity; naphtha; nose-only inhalation.

Figure 1.

Schematic of the exposure regimen. Groups of male SD rats and female B6C3F1/N mice were acclimated to the nose-only exposure conditions for 3 h per day for 3 days prior to exposure. Animals were then exposed to chemical vapor in filtered, conditioned air or filtered, conditioned air alone (controls) for 3 h per day in the morning, Monday through Friday, for up to 12 exposures over 16 days. Day 0 was the first day of exposure. Shaded blocks represent exposure days while white blocks represented acclimation or no-exposure periods during the study.

Figure 2.

Body weight curves for female B6C3F1/N mice and male SD rats exposed to 2-ethyltoluene, 3-ethyltoluene, 4-ethyltoluene or cumene. Study day 0 was the first day of exposure, study day 15 was the final exposure with scheduled necropsy on day 16. Values shown are group averages (±SEM) with group sizes ranging from 3 to 10; lower group sizes are observed for the day(s) following early removal of 2- and 4-ethyltoluene mice. Statistical significance is shown for Tukey’s post hoc test following positive one-way ANOVA, or pair-wise tests for two groups, p value ≤ .05: “a” for control versus high concentration, “b” for control versus low concentration, “c” for low versus high concentration. The gray solid line represents controls, the dashed black line represents the low concentration [1000 ppm for 2-, 3- or 4-ethyltoluene or 500 ppm for cumene] and the solid black line represents the high concentration [2000 ppm for 2-, 3- or 4-ethyltoluene or 1000 ppm for cumene].

Figure 3.

Microscopic lesions in the respiratory epithelium (RE) of the nose from female B6C3F1/N mice exposed to 2-ethyltoluene. (A) Normal RE in the nose of a control mouse. (B) The RE has been replaced by squamous epithelium (squamous metaplasia) in the nose of a mouse exposed to 1000 ppm 2-ethyltoluene. (C) Neutrophilic inflammation in the RE of a mouse exposed to 1000 ppm 2-ethyltoluene. 40×, H&E.

Figure 4.

Microscopic lesions in the olfactory epithelium (OE) of the nose from male SD rats or female B6C3F1/N mice exposed to 2-ethyltoluene. (A) Normal olfactory epithelium in a control mouse. (B) The OE is thinned due to cell loss (atrophy) in the nose of a rat exposed to 1000 ppm 2-ethyltoluene for 14 days. (C) The OE is disrupted and disorganized with gaps and cellular debris (necrosis) in a mouse exposed to 2000 ppm 2-ethyltoluene for 2 days. 40×, H&E.

Figure 5.

Microscopic lesions in the bronchiolar epithelium of the lung from female B6C3F1/N mice exposed to 2- or 3-ethyltoluene. (A) Normal bronchiolar epithelium in a control mouse. (B) The bronchiolar epithelium is thickened and crowded with increased stratification and mitotic figures (hyperplasia) in a mouse exposed to 1000 ppm 3-ethyltoluene for 14 days. (C) The hyperplastic bronchiolar epithelium also contains cytoplasmic clear vacuoles, primarily in the apical cytoplasm, forming blebs that slough into the bronchiolar lumen (vacuolation) in a mouse exposed to 2000 ppm 2-ethyltoluene for 2 days. 40×, H&E.

Figure 6.

Microscopic lesions in the liver from male SD rats or female B6C3F1/N mice exposed to 2-ethyltoluene. (A) Normal liver in a control rat. 20×. (B) Centrilobular hepatocellular hypertrophy in the liver of a mouse exposed to 2000 ppm 2-ethyltoluene for 2 days. 20×. Hepatocytes surrounding central veins (CV) are enlarged, extending into midzonal areas. (C) Pigment (arrowheads) and individual dying hepatocytes (necrosis; arrows) in the liver of a rat exposed to 1000 ppm 2-ethyltoluene for 14 days. 40×. H&E.