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Comparative Inhalation Toxicity of Ethyltoluene Isomers in Rats and Mice

Georgia K. Roberts, Cynthia J. Willson, Dorian S. Olivera, David E. Malarkey, Daniel L. Morgan.
Inhal Toxicol (2017) DOI: https://doi.org/10.1080/08958378.2018.1430190 PMID: 29374990


Publication


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 hours/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 select tissues revealed 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.

Figures


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 hours 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 hours 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.

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 3-10; 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 2 groups, p-value ≤ 0.05: ‘a’ for control vs. high concentration, ‘b’ for control vs. low concentration, ‘c’ for low vs. high concentration. The grey 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.

Microscopic lesions in the respiratory epithelium (RE) of the nose from male SD rats or female B6C3F1/N mice exposed via nose-only inhalation to 2-ethyltoluene.
(A) Normal respiratory epithelium in the nose of a control mouse from the 2-ethyltoluene study.
(B) The respiratory epithelium 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 respiratory epithelium in the nose of a female mouse exposed to 1000 ppm 2-ethyltoluene. 40x, H&E

Figure 4. Microscopic lesions in the olfactory epithelium (OE) of the nose.

Microscopic lesions in the olfactory epithelium (OE) of the nose from male SD rats or female B6C3F1/N mice exposed via nose-only inhalation to 2-ethyltoluene (0, 1000, or 2000 ppm).
(A) Normal olfactory epithelium in the nose in a control mouse from the 2-ethyltoluene study.
(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 the nose of a mouse exposed to 2000 ppm 2-ethyltoluene for 2 days. 40x, H&E

Figure 5. Microscopic lesions in the bronchiolar epithelium of the lung.

Microscopic lesions in the bronchiolar epithelium of the lung from female B6C3F1/N mice exposed via nose-only inhalation to 2- or 3-ethyltoluene (0, 1000, or 2000 ppm). (A) Normal bronchiolar epithelium in the lung in a control mouse from the 3-ethyltoluene study. (B) The bronchiolar epithelium is thickened and crowded with increased stratification and mitotic figures (hyperplasia) in the lung of a mouse exposed to 1000 ppm 3-ethyltoluene by inhalation 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 the lung of a mouse exposed to 2000 ppm 2-ethyltoluene by inhalation for 2 days. 40x, H&E

Figure 6. Microscopic lesions in the liver.

Microscopic lesions in the liver from male SD rats or female B6C3F1/N mice exposed via nose-only inhalation to 2-ethyltoluene (0, 1000, or 2000 ppm). (A) Normal liver in a control rat at 14 days. 20x. (B) Centrilobular hepatocellular hypertrophy in the liver of a mouse exposed to 2000 ppm 2-ethyltoluene by inhalation for 2 days. 20x. 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 by inhalation for 14 days. 40x. H&E

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      • modules/BinViews/Models/Library.php:65
      • modules/BinViews/Models/Library.php:64
      • view::binviews::layouts.manuscript:54
    • OracleBuilder.php#159CEBSBIN18.86msselect * from "BIN_REVIEW" where "BIN_REVIEW"."FILE_ID" = '1de240ff5df84b08015e81351dd60300' and "BIN_REVIEW"."FILE_ID" is not null
      Bindings
      • 0: 1de240ff5df84b08015e81351dd60300
      Hints
      • Use SELECT * only if you need all columns from table
      Backtrace
      • vendor/yajra/laravel-oci8/src/Oci8/Query/OracleBuilder.php:159
      • modules/BinViews/Models/File.php:63
      • modules/BinViews/Models/Library.php:65
      • modules/BinViews/Models/Library.php:64
      • view::binviews::layouts.manuscript:54
    • OracleBuilder.php#159CEBSBIN3.87msselect * from "BIN_REVIEW" where "BIN_REVIEW"."FILE_ID" = '1de240ff5df84b08015e81351e040301' and "BIN_REVIEW"."FILE_ID" is not null
      Bindings
      • 0: 1de240ff5df84b08015e81351e040301
      Hints
      • Use SELECT * only if you need all columns from table
      Backtrace
      • vendor/yajra/laravel-oci8/src/Oci8/Query/OracleBuilder.php:159
      • modules/BinViews/Models/File.php:63
      • modules/BinViews/Models/Library.php:65
      • modules/BinViews/Models/Library.php:64
      • view::binviews::layouts.manuscript:54
    • OracleBuilder.php#159CEBSBIN6.64msselect * from "BIN_REVIEW" where "BIN_REVIEW"."FILE_ID" = '1de240ff5df84b08015e81351e240302' and "BIN_REVIEW"."FILE_ID" is not null
      Bindings
      • 0: 1de240ff5df84b08015e81351e240302
      Hints
      • Use SELECT * only if you need all columns from table
      Backtrace
      • vendor/yajra/laravel-oci8/src/Oci8/Query/OracleBuilder.php:159
      • modules/BinViews/Models/File.php:63
      • modules/BinViews/Models/Library.php:65
      • modules/BinViews/Models/Library.php:64
      • view::binviews::layouts.manuscript:54
    • OracleBuilder.php#159CEBSBIN3.59msselect * from "BIN_REVIEW" where "BIN_REVIEW"."FILE_ID" = '1de240ff5df84b08015e81351e430303' and "BIN_REVIEW"."FILE_ID" is not null
      Bindings
      • 0: 1de240ff5df84b08015e81351e430303
      Hints
      • Use SELECT * only if you need all columns from table
      Backtrace
      • vendor/yajra/laravel-oci8/src/Oci8/Query/OracleBuilder.php:159
      • modules/BinViews/Models/File.php:63
      • modules/BinViews/Models/Library.php:65
      • modules/BinViews/Models/Library.php:64
      • view::binviews::layouts.manuscript:54
    • OracleBuilder.php#159CEBSBIN3.46msselect * from "BIN_REVIEW" where "BIN_REVIEW"."FILE_ID" = '1de240ff5df84b08015e81351e620304' and "BIN_REVIEW"."FILE_ID" is not null
      Bindings
      • 0: 1de240ff5df84b08015e81351e620304
      Hints
      • Use SELECT * only if you need all columns from table
      Backtrace
      • vendor/yajra/laravel-oci8/src/Oci8/Query/OracleBuilder.php:159
      • modules/BinViews/Models/File.php:63
      • modules/BinViews/Models/Library.php:65
      • modules/BinViews/Models/Library.php:64
      • view::binviews::layouts.manuscript:54
    • OracleBuilder.php#159CEBSBIN3.39msselect * from "BIN_REVIEW" where "BIN_REVIEW"."FILE_ID" = '1de240ff5df84b08015e81351e910305' and "BIN_REVIEW"."FILE_ID" is not null
      Bindings
      • 0: 1de240ff5df84b08015e81351e910305
      Hints
      • Use SELECT * only if you need all columns from table
      Backtrace
      • vendor/yajra/laravel-oci8/src/Oci8/Query/OracleBuilder.php:159
      • modules/BinViews/Models/File.php:63
      • modules/BinViews/Models/Library.php:65
      • modules/BinViews/Models/Library.php:64
      • view::binviews::layouts.manuscript:54
    • OracleBuilder.php#159CEBSBIN3.42msselect * from "BIN_REVIEW" where "BIN_REVIEW"."FILE_ID" = '1de240ff5df84b08015e813520a40306' and "BIN_REVIEW"."FILE_ID" is not null
      Bindings
      • 0: 1de240ff5df84b08015e813520a40306
      Hints
      • Use SELECT * only if you need all columns from table
      Backtrace
      • vendor/yajra/laravel-oci8/src/Oci8/Query/OracleBuilder.php:159
      • modules/BinViews/Models/File.php:63
      • modules/BinViews/Models/Library.php:65
      • modules/BinViews/Models/Library.php:64
      • view::binviews::layouts.manuscript:54
    • OracleBuilder.php#159CEBSBIN2.59msselect "BIN_FILE".*, "BIN_LIBRARY_FILES"."LIBRARY_ID" as "LARAVEL_THROUGH_KEY" from "BIN_FILE" inner join "BIN_LIBRARY_FILES" on "BIN_LIBRARY_FILES"."FILE_ID" = "BIN_FILE"."FILE_ID" where "BIN_LIBRARY_FILES"."LIBRARY_ID" = 6778 and "IS_DELETED" != 'T'
      Bindings
      • 0: 6778
      • 1: T
      Hints
      • The != operator is not standard. Use the <> operator to test for inequality instead.
      Backtrace
      • vendor/yajra/laravel-oci8/src/Oci8/Query/OracleBuilder.php:159
      • view::binviews::layouts.manuscript:52
      • vendor/laravel/framework/src/Illuminate/Filesystem/Filesystem.php:124
      • vendor/laravel/framework/src/Illuminate/View/Engines/PhpEngine.php:58
      • vendor/laravel/framework/src/Illuminate/View/Engines/CompilerEngine.php:75
    • OracleBuilder.php#159CEBSBIN4.6msselect * from "BIN_REVIEW" where "BIN_REVIEW"."FILE_ID" = '1de240ff5df84b08015e7b895a9502c9' and "BIN_REVIEW"."FILE_ID" is not null
      Bindings
      • 0: 1de240ff5df84b08015e7b895a9502c9
      Hints
      • Use SELECT * only if you need all columns from table
      Backtrace
      • vendor/yajra/laravel-oci8/src/Oci8/Query/OracleBuilder.php:159
      • modules/BinViews/Models/File.php:63
      • modules/BinViews/Models/Library.php:65
      • modules/BinViews/Models/Library.php:64
      • view::binviews::layouts.manuscript:54
    • OracleBuilder.php#159CEBSBIN2.29msselect "BIN_FILE".*, "BIN_LIBRARY_FILES"."LIBRARY_ID" as "LARAVEL_THROUGH_KEY" from "BIN_FILE" inner join "BIN_LIBRARY_FILES" on "BIN_LIBRARY_FILES"."FILE_ID" = "BIN_FILE"."FILE_ID" where "BIN_LIBRARY_FILES"."LIBRARY_ID" = 6779 and "IS_DELETED" != 'T'
      Bindings
      • 0: 6779
      • 1: T
      Hints
      • The != operator is not standard. Use the <> operator to test for inequality instead.
      Backtrace
      • vendor/yajra/laravel-oci8/src/Oci8/Query/OracleBuilder.php:159
      • view::binviews::layouts.manuscript:52
      • vendor/laravel/framework/src/Illuminate/Filesystem/Filesystem.php:124
      • vendor/laravel/framework/src/Illuminate/View/Engines/PhpEngine.php:58
      • vendor/laravel/framework/src/Illuminate/View/Engines/CompilerEngine.php:75
    • OracleBuilder.php#159CEBSBIN4.5msselect * from "BIN_REVIEW" where "BIN_REVIEW"."FILE_ID" = '1de240ff5df84b08015e7b895ab402ca' and "BIN_REVIEW"."FILE_ID" is not null
      Bindings
      • 0: 1de240ff5df84b08015e7b895ab402ca
      Hints
      • Use SELECT * only if you need all columns from table
      Backtrace
      • vendor/yajra/laravel-oci8/src/Oci8/Query/OracleBuilder.php:159
      • modules/BinViews/Models/File.php:63
      • modules/BinViews/Models/Library.php:65
      • modules/BinViews/Models/Library.php:64
      • view::binviews::layouts.manuscript:54
    • OracleBuilder.php#159CEBSBIN3.32msselect "BIN_FILE".*, "BIN_LIBRARY_FILES"."LIBRARY_ID" as "LARAVEL_THROUGH_KEY" from "BIN_FILE" inner join "BIN_LIBRARY_FILES" on "BIN_LIBRARY_FILES"."FILE_ID" = "BIN_FILE"."FILE_ID" where "BIN_LIBRARY_FILES"."LIBRARY_ID" = 6780 and "IS_DELETED" != 'T'
      Bindings
      • 0: 6780
      • 1: T
      Hints
      • The != operator is not standard. Use the <> operator to test for inequality instead.
      Backtrace
      • vendor/yajra/laravel-oci8/src/Oci8/Query/OracleBuilder.php:159
      • view::binviews::layouts.manuscript:52
      • vendor/laravel/framework/src/Illuminate/Filesystem/Filesystem.php:124
      • vendor/laravel/framework/src/Illuminate/View/Engines/PhpEngine.php:58
      • vendor/laravel/framework/src/Illuminate/View/Engines/CompilerEngine.php:75
    • OracleBuilder.php#159CEBSBIN4.26msselect * from "BIN_REVIEW" where "BIN_REVIEW"."FILE_ID" = '1de240ff5df84b08015e7b895ad402cb' and "BIN_REVIEW"."FILE_ID" is not null
      Bindings
      • 0: 1de240ff5df84b08015e7b895ad402cb
      Hints
      • Use SELECT * only if you need all columns from table
      Backtrace
      • vendor/yajra/laravel-oci8/src/Oci8/Query/OracleBuilder.php:159
      • modules/BinViews/Models/File.php:63
      • modules/BinViews/Models/Library.php:65
      • modules/BinViews/Models/Library.php:64
      • view::binviews::layouts.manuscript:54
    • OracleBuilder.php#159CEBSBIN2.7msselect "BIN_FILE".*, "BIN_LIBRARY_FILES"."LIBRARY_ID" as "LARAVEL_THROUGH_KEY" from "BIN_FILE" inner join "BIN_LIBRARY_FILES" on "BIN_LIBRARY_FILES"."FILE_ID" = "BIN_FILE"."FILE_ID" where "BIN_LIBRARY_FILES"."LIBRARY_ID" = 6781 and "IS_DELETED" != 'T'
      Bindings
      • 0: 6781
      • 1: T
      Hints
      • The != operator is not standard. Use the <> operator to test for inequality instead.
      Backtrace
      • vendor/yajra/laravel-oci8/src/Oci8/Query/OracleBuilder.php:159
      • view::binviews::layouts.manuscript:52
      • vendor/laravel/framework/src/Illuminate/Filesystem/Filesystem.php:124
      • vendor/laravel/framework/src/Illuminate/View/Engines/PhpEngine.php:58
      • vendor/laravel/framework/src/Illuminate/View/Engines/CompilerEngine.php:75
    • OracleBuilder.php#159CEBSBIN3.9msselect * from "BIN_REVIEW" where "BIN_REVIEW"."FILE_ID" = '1de240ff5df84b08015e7b895b4102cc' and "BIN_REVIEW"."FILE_ID" is not null
      Bindings
      • 0: 1de240ff5df84b08015e7b895b4102cc
      Hints
      • Use SELECT * only if you need all columns from table
      Backtrace
      • vendor/yajra/laravel-oci8/src/Oci8/Query/OracleBuilder.php:159
      • modules/BinViews/Models/File.php:63
      • modules/BinViews/Models/Library.php:65
      • modules/BinViews/Models/Library.php:64
      • view::binviews::layouts.manuscript:54
    • OracleBuilder.php#159CEBSBIN2.63msselect * from "BIN" where "BIN_ID" = 3169
      Bindings
      • 0: 3169
      Hints
      • Use SELECT * only if you need all columns from table
      Backtrace
      • vendor/yajra/laravel-oci8/src/Oci8/Query/OracleBuilder.php:159
      • modules/BinViews/Services/BreadcrumbService.php:32
      • modules/BinViews/Routes/breadcrumbs.php:55
      • vendor/diglactic/laravel-breadcrumbs/src/Generator.php:64
      • vendor/diglactic/laravel-breadcrumbs/src/Generator.php:42
    Modules\BinViews\Models\File
    14File.php#?
    Modules\BinViews\Models\Review
    14Review.php#?
    Modules\BinViews\Models\Library
    7Library.php#?
    Modules\BinViews\Models\LibraryGroup
    7LibraryGroup.php#?
    Modules\BinViews\Models\Characteristic
    5Characteristic.php#?
    Modules\BinViews\Models\PublicationManuscript
    3PublicationManuscript.php#?
      web
      null
        _token
        uCx9mrC2thI98UkJrGW87HceC184PnLoXFM3ETQL
        ClearShow all
        Date ↕MethodURLData
        #12025-04-05 19:43:00GET/cebs/paper/31691803350