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Toxicological information

Specific investigations: other studies

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Administrative data

specific investigations: other studies
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: This study was conducted according to GLP and sufficient information is available for the interpretation of results.
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study

Data source

Referenceopen allclose all

Reference Type:
study report
Reference Type:
Evaluation of potential modes of actions of inhaled ethylbenzene in rats and mice
Stott, W. T.; Johnson, K.A.; Bahnemann, R.; Day, S.J.; McGuirk, R.L.
Bibliographic source:
Toxicol. Sci. 71:53-66

Materials and methods

Test guideline
no guideline available
Principles of method if other than guideline:
The study was undertaken to examine several parameters potentially involved in the mode of tumorigenesis of ethylbenzene in rodents
GLP compliance:
Type of method:
in vivo
Endpoint addressed:
repeated dose toxicity: inhalation

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Details on test material:
- Name of test material (as cited in study report): Ethylbenzene
- Analytical purity: 100%
- Lot/batch No.: AT-4OH-091498

Test animals

other: Rat and mice
other: Fischer 344 rats and B6C3F1 mice
Details on test animals or test system and environmental conditions:
- Source: Charles River
- Age at study initiation: approximately 7.5 weeks
- Fasting period before study: none
- Housing: individual
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least one week prior to start of the study

- Temperature (°C): 22 ± 3°C
- Humidity (%): 40-70%
- Air changes (per hr): 12-15 times/hour
- Photoperiod (hrs dark / hrs light): 12-hour light/dark photocycle

Administration / exposure

Route of administration:
inhalation: vapour
unchanged (no vehicle)
Details on exposure:
- Exposure apparatus: 14.5 cubic meter inhalation chambers (2.4 meter high by 2.4 meter wide by 2.4 meter deep cube with pyramidal top) under dynamic airflow conditions.
- Method of holding animals in test chamber: individual
- Source and rate of air: ambient
- Method of conditioning air: not specified in the report
- Temperature, humidity, pressure in air chamber: The temperature and relative humidity in each chamber was controlled by a system designed to maintain temperature at approximately 22 ± 3°C and relative humidity at approximately 40-70%.
- Air flow rate: 2900 liters/minute
- Air change rate: 12 air changes/hour
- Treatment of exhaust air: not specified in the report

- Brief description of analytical method used: Test atmosphere concentrations, sampled from the chamber reference point of the three test chambers, were determined at least six times per exposure period by the analytical system coupled to a Camile Data Acquisition and Control System. A Hewlett Packard 5890 gas chromatograph with a flame ionization detector and integrator was calibrated with standards of EB in Tedlar sampling bags (SKC, Eighty Four, PA) to cover the range of exposure concentrations that were tested. The GC conditions were as follows: injector temperature, 190°C; oven temperature, 75°C; detector temperature, 250°C; airflow, 300 ml/min, hydrogen, 30 ml/min; helium, ~15 ml/min; split vent, 150 ml/min; column head pressure, 40 kPa and septum purge, 1 ml/min. A Hewlett Packard 50% phenylmethyl silicone column measuring 10 meters x 0.53 mm with a 2.65 micron film thickness was used. An HP3396 integrator (Hewlett Packard Company, Wilmington, DE) was used for integration of the chromatograms.
- Samples taken from breathing zone: yes

The various concentrations of EB vapor were generated using a glass J-tube method. Liquid EB was metered into a glass J-tube assembly through which a preheated stream of compressed air (approximately 150 L/min) was passed to vaporize the test material. The compressed air was heated to the minimum extent necessary to facilitate complete vaporization of the test material. The compressed air and EB vapors were diluted and mixed with supply air to achieve a total flow rate of 2900 L/min and the desired concentration of EB vapors.
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
The overall time-weighted average (TWA) analytical concentrations of EB vapors in the exposure chambers were within a few percent of targeted concentrations; 0.0, 75.2 r0.3 ppm (range, 73.4-76.3) and 738.6 r56.0 ppm (range, 361.4-804.0) for targeted concentrations of 0, 75 and 750 ppm, respectively.
Duration of treatment / exposure:
6 hours/day for 5 consecutive days
Frequency of treatment:
6 hours/day for 5 consecutive days
Post exposure period:
not applicable
Doses / concentrationsopen allclose all
Doses / Concentrations:
0 ppm (nominal)
nominal conc.
Doses / Concentrations:
75 ppm (nominal)
other: time-weighted average 75.2 ± 0.3 ppm
Doses / Concentrations:
750 ppm (nominal)
other: time-weighted average 738.6 ± 56.0 ppm
No. of animals per sex per dose:
12 rats and 36 mice/sex/dose group
Control animals:
yes, sham-exposed
Details on study design:
Group A: Cytology
Osmotic mini-pumps containing 5-bromo-2-deoxyuridine (BrdU) were implanted subcutaneously into 6 animals/species/sex/dose group for nuclear labeling of cells undergoing DNA synthesis. Pumps were implanted on Test Day -1 of the study and these groups of animals (rats were fasted overnight) were sacrificed on Test Day 6 following 5 consecutive daily exposures to EB vapor. Animals were anesthetized with methoxyflurane, sacrificed by decapitation, exsanguinated and the abdominal and thoracic cavities of rats and mice were exposed and major organ systems examined. The liver, kidneys and lungs of all animals were excised, weighed and relative organ weight to body weight ratios were subsequently calculated. A central portion of the right kidney, the left lobe of the lungs, and left lobe of the liver of the first three animals/dose group (rats and mice) were sectioned and preserved in paraformaldehyde/glutaraldehyde fixative for possible EM analyses. The remaining tissues were then preserved in 10% phosphate buffered formalin. Selected tissues were subsequently processed by appropriate methods and cell proliferation and apoptosis rates were quantitated in specific areas of the rat kidney (left) and mouse liver and lung (see below). An adjacent section of these mounted tissues was also processed for histopathologic evaluation.
Group B: Enzymology
Groups of 6 rats and 30 mice/sex/dose group were utilized. On Test Day 6, all animals were anesthetized with methoxyflurane and blood samples were obtained via orbital sinus puncture from all rats and from a subset of 10 mice/sex/dose group. The trachea of all animals were exposed and clamped. Animals were then decapitated, briefly exsanguinated, and the abdominal and thoracic cavities of rats and mice were exposed and major organ systems examined. The liver, kidneys and lungs of all rats were excised and snap frozen in liquid nitrogen and stored at -80qC. The liver and kidneys of 6 mice/sex/dose group and the lungs of all surviving mice were excised, snap frozen in liquid nitrogen, and stored at -80qC. The lungs of all mice were combined into groups of 4-5/sex/dose group. Frozen tissues were subsequently processed by appropriate methods to obtain microsomal fractions from each animal or, in the case of mouse lungs, 6 pooled samples of 4-5 lungs each. The death of several mice during the exposure period resulted in a smaller lung pool size for some dose groups. The relative activities of several MFOs (CYP1A, CYP2B1/2, CYP2E1) and activity of the Phase II enzyme, glucuronosyl transferase (UDPGT) were measured. For economy, enzymes from animals of all exposure groups were evaluated. UDPGT activity in female mouse lungs was measured in a limited number of animals within each exposure group (3-4/group) due to limited amounts of this tissue available. Serum was harvested from collected blood and clinical chemistry evaluations were conducted for all rats and a subset of 10 mice/sex/dose group. Parameters indicative of tissue injury and renal function were evaluated. For economy, clinical chemistry parameters from animals of all exposure groups were evaluated.


Animal observations - twice a day (cageside observations)
Body weights - on the day prior to exposure and on the day of sacrifice
Cell Proliferation - Levels of DNA replication were determined in test animals using standard 5-bromo-2’-deoxyuridine (BrdU) immunohistochemical labeling methodology. Briefly, BrdU labeled nuclei were quantitated utilizing immunohistochemical methods. A labeling index (proportion of nuclei that have undergone DNA replication in the presence of BrdU) based upon a total count of nuclei examined and counted was calculated in the following tissues - kidney (rat), lungs (mouse) and liver (mouse). A small section of duodenum was also collected from all rats and mice and processed to serve as a control for confirming systemic availability of the implanted BrdU.
Mitotic figures (Group A) - Mitotic figures within the three regions of the liver lobule, portal, midzonal and central, were then counted and normalized(number of mitotic figures/1000 nuclei) based upon a total count of nuclei examined.
Apoptosis evaluation (Group A) - Organs from high dose and control animals were processed and immunohistochemically stained for identification of apoptotic cells. Labeled and unlabelled cortical and medullary (inner and outer stripe) kidney cells, centrilobular and periportal liver parenchyma, and lung epithelium of the lower airways and alveoli were counted microscopically. An apoptosis index (proportion of apoptotic cells) based upon a minimum total count of cells similar to those used for BrdU labeling was calculated for each animal.
Hepatic Phase I and II Enzyme activities (Group B) - Post microsome isolation and estimation of total microsomal proteins, the activities of selected Phase I and II enzymes were measured in vitro. CYP1A1/2 and CYP2B1/2 activities were measured as ethoxyresorufin (EROD) and pentoxyresorufin (PROD) O-dealkylation, respectively, using the standard fluorometric methods. CYP2E1 activity was measured as U-nitrophenol hydroxylase (U-NPH) activity using the spectrophotometric method. In addition, ethoxyfluorocoumarin-O-dealkylase (EFCOD) activity was measured using the fluorometric method (which provides a measure of the combined net activities of several MFOs, including CYP2E1, CYP1A and CYP2B. Microsomal UDPGT activity was measured after a modification of the fluorometric method.
Necropsy, Tissue collection and organ weights - liver, kidneys and lungs of all animals weighed and processed for EM examination
Clinical Pathology - Parameters assayed were: Alanine Transaminase (ALT), Aspartate Transaminase (AST), Alkaline Phosphatase (AP), Creatinine (CREAT), Blood Urea (UN) and g-Glutamyl Transpeptidase (GGT)
Histopathology and EM
Positive control:
not applicable

Results and discussion

Details on results:
Exposure of Fisher 344 rats and B6C3F1 mice to ethylbenzene vapors for 5 days resulted in a number of apparent treatment-related changes in the parameters evaluated - A); selected organ weights, histopathology, cell proliferation or the in vitro activities of specific Phase I enzymes, and possibly apoptosis.

In the kidneys of rats, focal increased hyaline droplet deposition and degenerative changes in the proximal tubular epithelium of males was consistent with an alpha-2uglobulin based pathogenesis. Elevations in cell proliferation in these foci reflected corresponding tissue degeneration and regeneration. Changes in cell proliferation rates and, possibly, apoptosis in renal medullary cells of treated male rats may or may not have been related to the development of hyaline droplet nephropathy. Likewise the significance of decreased cell proliferation in the cortex of females was not clear; however, minimal increases in renal pNPH primarily in high-exposure males, PROD in high-exposure group females and UDPGT activity in both sexes of high-exposure rats suggested at least a minimal potential to induce CYP2E1, CYP2B and Phase II enzymes. These data suggest a mechanism of tumorigenic action of ethylbenzene in the kidneys of male rats chronically exposed to this compound. An D-2u-globulin based nephropathy is generally believed to be a male rat specific effect of chemical exposure which may lead to chronic tissue damage and tumors. A clear mechanism of action in female rats was not suggested by the data, but may be related to chronic CYP2B induction upon prolonged exposure to ethylbenzene.

Increases in the liver weights of male and female mice inhaling 750 ppm ethylbenzene vapor resulted from increases in cell proliferation, especially in centrilobular and midzonal hepatocytes, as supported by increases in both BrdU uptake, averaged over the entire exposure period, and the incidence of mitoses at sacrifice (test day 6). The latter finding in particular suggests a possible longer term increase in cell proliferation in liver. Changes in liver weights were also accompanied by a minimal induction of CYP1A and, in males, CYP2B activities consistent with the adaptation of liver tissues to the increased metabolic load placed upon it by absorbed ethylbenzene. In both enzymological and cytological parameters, males appeared to be more affected than females despite the larger organ weight changes in females. Enhanced cell proliferation, either on an acute or chronic basis, in this tissue, along with other factors such as genetic predisposition, represents a plausible mechanism of tumor formation.

Finally, exposure to ethylbenzene vapors resulted in a dose-related decrease in CYP1A and CYP2B activities in the lungs of male and female mice. The absence of a concomitant loss of pulmonary CYP2E1 activities in these animals suggests a selectively toxic mechanism, possibly via metabolic inactivation of the enzyme. Accompanying increases in cell proliferation and possible elevations in apoptosis in one or both types of epithelia examined in the absence of histopathological changes suggested possible alterations in cell populations early in the exposure period.

Any other information on results incl. tables


Applicant's summary and conclusion

Exposure of Fisher 344 rats and B6C3F1 mice to ethylbenzene vapors for 5 days resulted in a number of apparent treatment-related changes in the parameters evaluated (selected organ weights, histopathology, cell proliferation or in vitro activities of specific Phase 1 enzymes and possibly apoptosis.
Executive summary:

Groups of male and female rats and mice (12 rats and 36 mice/sex/dose group) were exposed to 0 (control), 75 or 750 ppm ethylbenzene vapors 6 hours/day for 5 consecutive days. Parameters evaluated in all or selected subgroups of animals included: body weights, organ weights (liver, kidneys, lungs), gross pathology, and selected clinical chemistry endpoints. Parameters evaluated in rat kidneys and mouse liver and lungs included: apoptosis, cell proliferation, mitotic figure incidence (liver only), histopathology (including electron microscopy), activities of the Phase I enzymes ethoxy- and pentoxy- resorufin O-dealkylases (EROD, PROD), U-nitrophenol hydroxylase (UNPH) and ethoxyfluorocoumarin O-dealkylase (EFCOD), and activity of the Phase II enzyme glucuronosyl transferase (UDPGT). A number of treatment-related effects were observed.

Rat Kidney Effects: Rat kidney weights were increased in high-exposure group males and females. In high-exposure males, increases in hyaline droplet deposition and degeneration of cortical tubules were observed; this correlated with a focal increase (1.4-fold) in cortical cell proliferation. A 3-fold increase in apoptosis of medullary cells was also noted, but due to variability in these measurements, was of unknown significance. No histopathologic changes occurred in high exposure female rats and cortical cell proliferation was decreased in nearly 50% in these animals. Activities of renal PROD in females and U-NPH in males were minimally increased (1.7-1.9 fold), indicating a minimal induction of CYP2B and CYP2E1, respectively. The activity of UDPGT was also minimally increased (1.3-fold) in both sexes of rats. These results are consistent with alpha-2u-globulin nephropathy in the male rat kidney which is generally regarded as a nongenotoxic and male-specific mode of tumorigenesis. Under the conditions of the study, a mode of action in females was not readily apparent, but may be associated with the chronic iduction of CYP2B.

Mouse Liver Effects: Mouse liver weights were elevated in both males and females. Increases in the proliferation of centilobular and midzonal hepatocytes, as evidenced by increases in BrdU uptake (2-12 fold) and increased incidence of mitotic figures, occurred in both sexes of high-exposure mice. Males were more affected than females. Lesser effects were observed in mice inhaling 75 ppm ethylbenzene. Increased liver weights also correlated with minimal increases (1.4-1.6 fold) in EROD activity in both sexes and PROD and EFCOD activities in males indicative of a minimal induction of CYP1A and/or CYP2B. Histopathological changes consisting of increased mitotic figures in centrilobular and midzonal areas accompanied these changes. Enhanced cell proliferation in the livers of high-exposure mice suggests a generally accepted nongenotoxic mode of tumorigenic action.

Mouse Lung Effects: In mouse lungs, EROD, PROD and EFCOD activities were decreased as much as 45%, in a dose related

manner in males and females inhaling ethylbenzene suggesting a loss of CYP1A and CYP2B. The latter was not accompanied by histopathologic changes in pulmonary tissue. However, changes in cell population dynamics were suggested by observed increases (3-fold) in cell proliferation in the bronchiolar epithelium and, possibly, in apoptosis of bronchiolar and alveolar epithelia of high-exposure males and/or females. Elevated cell proliferation, as modified by other factors such as genetic predisposition, suggests a possible nongenotoxic mode of tumorigenic activity of ethylbenzene in the mouse lung.