Ethylbenzene

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: NTP study (comparable to Guideline study) performed under GLP conditions. Study well documented and meets generally accepted scientific principles. No circumstances occurred that would have affected the quality or integrity of the data.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

1999

Reference 2

Reference Type:

publication

Title:

Significance of the renal effects of ethyl benzene in rodents for assessing human carcinogenic risk

Author:

Hard, G.C

Year:

2002

Bibliographic source:

Toxicol. Sci. 69:30-41

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Ethylbenzene

EC Number:

202-849-4

EC Name:

Ethylbenzene

Cas Number:

100-41-4

Molecular formula:

C8H10

IUPAC Name:

ethylbenzene

Details on test material:

as prescribed by 1.1 - 1.4

Test animals

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Species/strain: Rat F344/N
- Number of animals: 50M+50F/dose level
- Source: Simonsen Laboratories, Inc., Gilroy, CA
- Age at study initiation: 6 weeks at start of study
- Weight at study initiation: M 121-124 g; F 100-104 g (mean of average weights reported for week 1)
- Housing: individually housed in stainless steel cages
- Diet: NIH-07 open formula pelleted diet (Zeigler Brothers Inc., Gardners, PA), ad libitum except during exposure
- Water: untreated course-filtered City of Chicago water, ad libitum except during exposure
ENVIRONMENTAL CONDITIONS
- Temperature: controlled to 21-28 ºC
- Humidity: 37-76% relative humidity
- Photoperiod: 12 hour light/dark cycle
- Air changes/hour: 15

Administration / exposure

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

other: Filtered air

Remarks on MMAD:

MMAD / GSD: None

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE/CHAMBER DESCRIPTION
- Chambers: Stainless steel chambers
- Exposure apparatus: Flash evaporator units
- Chamber concentrations: Actual mean exposure concentrations achieved in the chambers throughout the study were 74.8, 250 and 749 ppm ethylbenzene.

TEST SUBSTANCE
Ethylbenzene was used from two lots: A060989 and A051890; A060989 had an overall purity > 99% and contained 62 +/- 3.1 ppm cumene; lot A051890 also had an overall purity of > 99%. Concentration of peroxide ranged from 1.12 to 10.7 ppm

TEST ATMOSPHERE
- Concentration: monitored by an automatic sampling system coupled to a gas chromatograph equipped with a flame ionization detector. Each study chamber atmosphere was analyzed hourly during the 6 hour exposure.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

No data

Duration of treatment / exposure:

104 week

Frequency of treatment:

6 hours/day 5 days/week

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
75 ppm (325 mg/m3)
Basis:
nominal conc.

Doses / concentrations 2

Remarks:

Doses / Concentrations:
250 ppm (1084 mg/m3)
Basis:
nominal conc.

Doses / concentrations 3

Remarks:

Doses / Concentrations:
750 ppm (3251 mg/m3)
Basis:
nominal conc.

No. of animals per sex per dose:

50/sex/dose

Control animals:

yes

Details on study design:

- Rationale for animal assignment: Random
- Post-exposure recovery period in satellite groups: None

Positive control:

Not applicable

Examinations

Observations and examinations performed and frequency:

CLINICAL OBSERVATIONS AND FREQUENCY
- Body weight: weekly for 13 weeks and monthly from week 16 to the end of the study.
- Clinical signs: twice daily
- Mortality: twice daily

Sacrifice and pathology:

ORGANS EXAMINED AT NECROPSY (MACROSCOPIC AND MICROSCOPIC):
All organs and tissues were examined for grossly visible lesions and all major tissues were fixed and preserved in 10% neutral buffered formalin, and processed for microscopic examination. Adrenals, aorta, bone (including marrow), brain, bronchial lymph nodes, caecum, colon, duodenum, epididymis/seminal vesicle/prostate/testes or ovaries/uterus, oesophagus, gross lesions and tissue masses with regional lymph nodes, heart, ileum, jejunum, kidneys, larynx, liver, lungs, mammary gland, mandibular and mesenteric lymph nodes, mediastinal lymph nodes, nose, pancreas, parathyroids, pituitary, rectum, salivary glands, spinal cord and sciatic nerve (if neurologic symptoms) spleen, stomach (glandular and forestomach), thigh muscle, thymus, thyroid, trachea and urinary bladder

Other examinations:

None

Statistics:

Statistical analyses for possible dose-related effects on survival used Cox's method for testing two groups for equality and Tarone's life table test to identify dose-related trends. The incidences of neoplasms and nonneoplastic lesions were calculated as were the survival-adjusted neoplasm rate for each group and each site-specific neoplasm. The majority of the neoplasms in these studies were considered incidental to the cause of death or not rapidly lethal. Thus the primary statistical method used was logistic regression analysis, which assumed that the diagnosed neoplasm were discovered as the result of death from an unrelated cause and thus did not affect the risk of death. Neoplastic prevalence was modeled as a logistic function of chemical exposure and time. The neoplasm incidences of exposed and control groups were compared on the basis of the likelihood score test for the regression coefficient of dose. In addition to logistic regression other methods employed were the life table test appropriate for rapidly lethal neoplasms, and the Fisher exact test and the Cochran-Armitage trend test, procedures based on the overall proportion of neoplasm-bearing animals. Tests of significance included pair wise comparisons of each exposed group with controls and a test for an overall dose related trend. For the analysis of nonneoplastic lesions, the primary statistical analysis used was a logistic regression analysis in which nonneoplastic lesion prevalence was modeled as a logistic function of chemical exposure and time. Average severity values were analyzed for significance with the Mann-Whitney test.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

effects observed, treatment-related

Details on results:

MORTALITY AND TIME TO DEATH: At 750 ppm, survival in males was significantly reduced (animals surviving to study termination 2/50 for 750 ppm (p<0.001), 250 ppm 13/50; 75 ppm 14/50; 15/50 in controls). In females survival was increased (not significant). The survival of male rats followed a negative trend, decreasing with increasing dose. Mean survival for male rats was 651 days in controls and 639, 651 and 604 days for 75, 250 and 750 ppm groups respectively. In females mean survival was 661, 690, 696 and 706 days for controls, 75, 250 and 750 ppm groups respectively.
BODY WEIGHT GAIN: Mean body weights of 250 ppm and 750 ppm males were generally lower than those of the chamber controls (up to 5 and 15%, respectively) from week 20. Mean body weights of all exposed groups of females were generally lower (5-6%) than those of the chamber controls during the second year of the study.
CLINICAL FINDINGS: None attributable to ethylbenzene exposure.

HISTOPATHOLOGY: Test article-related organ pathology was present in the kidney and testes of ethylbenzene exposed rats.

Kidney
The incidence of renal tubule hyperplasia in 750 ppm males was significantly greater than that in the chamber control group (12/50 vs. 2/50 in controls; p=0.01). The findings from an extended evaluation (step section) of the kidney showed a significant increase in the incidence of renal tubule hyperplasia in 750 ppm males and females (17/50 vs. 10/50 in controls for males, p=0.05, and 8/49 vs. 1/50 in controls for females, p=0.05). The severity of nephropathy was significantly increased relative to the chamber controls in 750 ppm male (3.5 vs. 2.3 in controls; p=0.01) and all exposed female rats (2.3, 1.7, 1.6 and 1.3 for 750, 250, 75 ppm and control groups; p=0.05 or p=0.01). The enhanced nephropathy was more severe in males than in females. The associated increase in tumour incidence is described in Chapter 5.7 carcinogenicity.

Testis
In the testis, the incidence of interstitial cell and bilateral testicular adenoma was increased in top dose males (see Chapter 5.7 carcinogenicity for details). However the incidence of interstitial cell hyperplasia was significantly decreased at this concentration level (8/50 vs. 14/50 in controls; p=0.05).

OTHER ORGANS:
Lungs: 750 ppm males exhibited increased lesion incidences of oedema (1/50, 0/50, 0/50, 6/50 for chamber control, 75, 250, and 750 ppm), congestion (1/50, 2/50, 0/50, 6/50 for chamber control, 75, 250, and 750 ppm), and hemorrhage (0/50, 2/50, 1/50, 8/50 for chamber control, 75, 250, and 750 ppm) in the lungs.

Lymph nodes: 750 ppm males also exhibited hemorrhage in mesenteric (3/49, 5/50, 4/50, 8/50 for chamber control, 75, 250, and 750 ppm) and renal (0/9, 0/8, 1/9, 8/14 for chamber control, 75, 250, and 750 ppm) lymph nodes were slightly increased. These circulatory lesions were considered to be agonal changes in moribund animals and not directly related to chemical toxicity.

Liver: The incidences of cystic degeneration of the liver was also increased in 750 ppm males (15/50, 12/50, 19/50, 30/49 for chamber control, 75, 250, and 750 ppm); the biologic significance of this increase in the absence of other hepatotoxic changes was deemed unclear.

Prostate: Compared to the chamber control group, the incidences of prostate gland inflammation in all exposed groups of males were significantly increased (11/50, 29/50, 22/50, 25/50 for chamber control, 75, 250, and 750 ppm). This inflammatory change consisted of infiltration by predominately mononuclear inflammatory cells with glandular acini and interstitium, increased interstitial fibrosis, and loss of secretory material in affected areas.

Bone marrow: Relative to chamber controls, males exposed to 75 or 750 ppm exhibited increased incidences of hyperplasia of the bone marrow characterized by hypercellularity due to the increased numbers of erythroid and myeloid precursor cells (7/49, 16/49, 9/50, 19/50 for chamber control, 75, 250, and 750 ppm). The relationship of these changes to ethylbenzene exposure is uncertain due to the lack of clear concentration-dependent responses.

Effect levels

1

Dose descriptor:

NOAEC

Effect level:

250 ppm

Sex:

male

Basis for effect level:

other: decreased survival, reduced body weight and significantly increased incidence of nephropathy at 750 ppm

2

Dose descriptor:

LOAEC

Effect level:

75 ppm

Sex:

female

Basis for effect level:

other: decreased survival, reduced body weight, and significantly increased incidence of nephropathy in all groups

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:

According to NTP there was clear evidence of carcinogenicity in male rats due to increased incidences of kidney (renal tubule neoplasms) and testes tumors (testicular adenoma) and some evidence of carcinogenicity in female rats that also showed kidney tumors (renal tubule adenomas), but in a lower incidence and only detected after extended evaluation by step sections.
Chronic Toxicity - A decrease in survival and body weight and an increase in kidney pathology (renal tubule hyperplasia and nephropathy) were observed in rats that inhaled >= 75 ppm ethylbenzene for 2 years. NOAEL 250 ppm (males), <75 ppm females.
Cancer - At 750 ppm ethylbenzene, male rats exhibited increased incidences of kidney (renal tubule neoplasms) and testes tumors (testicular adenoma) and female rats also showed kidney tumors (renal tubule adenomas), but in a lower incidence and only detected after extended evaluation. NOAEL 250 ppm.

Executive summary:

Fischer-344 rats (50/sex/exposure) were exposed to 0.0, 75, 250, 750 ppm (0, 325, 1084, 3251 mg/m3) of ethylbenzene for 6 hr/day, 5 days /week for 104 weeks.

Rats were received from Simonsen Laboratories, Inc. At study initiation rats were of approximately 6 weeks old. Rats were housed 1/cage in stainless were housed in stainless steel cages with wire bottoms. A standard laboratory dietNIH-07 open formula pelleted diet (Zeigler Brothers Inc.), ad libitum except during exposure supplied to rats. Rats were supplied untreated course-filtered City of water, ad libitum except during exposure.

Monitored for effects included clinical observations, body weights, gross pathology and histopathology.

At 750 ppm, survival in males was significantly reduced (animals surviving to study termination 2/50 for 750 ppm (p<0.001), 250 ppm 13/50; 75 ppm 14/50; 15/50 in controls). In females survival was increased (not significant). The survival of male rats followed a negative trend, decreasing with increasing dose. Mean survival for male rats was 651 days in controls and 639, 651 and 604 days for 75, 250 and 750 ppm groups respectively. In females mean survival was 661, 690, 696 and 706 days for controls, 75, 250 and 750 ppm groups respectively.

Mean body weights of 250 ppm and 750 ppm males were generally lower than those of the chamber controls (up to 5 and 15%, respectively) from week 20. Mean body weights of all exposed groups of females were generally lower (5-6%) than those of the chamber controls during the second year of the study.

Test article-related organ pathology was present in the kidney and testes of ethylbenzene exposed rats. The incidence of renal tubule hyperplasia in 750 ppm males was significantly greater than that in the chamber control group (12/50 vs. 2/50 in controls; p=0.01). The findings from an extended evaluation (step section) of the kidney showed a significant increase in the incidence of renal tubule hyperplasia in 750 ppm males and females (17/50 vs. 10/50 in controls for males, p=0.05, and 8/49 vs. 1/50 in controls for females, p=0.05). The severity of nephropathy was significantly increased relative to the chamber controls in 750 ppm male (3.5 vs. 2.3 in controls; p=0.01) and all exposed female rats (2.3, 1.7, 1.6 and 1.3 for 750, 250, 75 ppm and control groups; p=0.05 or p=0.01). The enhanced nephropathy was more severe in males than in females. The associated increase in tumor incidence is described in Chapter 5.7 carcinogenicity.
      

In the testis, the incidence of interstitial cell and bilateral testicular adenoma was increased in top dose males (see Chapter 5.7 carcinogenicity for details). However the incidence of interstitial cell hyperplasia was significantly decreased at this concentration level (8/50 vs. 14/50 in controls; p=0.05).
OTHER ORGANS:
Lungs: 750 ppm males exhibited increased lesion incidences of edema (1/50, 0/50, 0/50, 6/50 for chamber control, 75, 250, and 750 ppm), congestion (1/50, 2/50, 0/50, 6/50 for chamber control, 75, 250, and 750 ppm), and hemorrhage (0/50, 2/50, 1/50, 8/50 for chamber control, 75, 250, and 750 ppm) in the lungs.

Lymph nodes: 750 ppm males also exhibited hemorrhage in mesenteric (3/49, 5/50, 4/50, 8/50 for chamber control, 75, 250, and 750 ppm) and renal (0/9, 0/8, 1/9, 8/14 for chamber control, 75, 250, and 750 ppm) lymph nodes were slightly increased. These circulatory lesions were considered to be agonal changes in moribund animals and not directly related to chemical toxicity.
 
Liver: The incidences of cystic degeneration of the liver was also increased in 750 ppm males (15/50, 12/50, 19/50, 30/49 for chamber control, 75, 250, and 750 ppm); the biologic significance of this increase in the absence of other hepatotoxic changes was deemed unclear.
 
Prostate: Compared to the chamber control group, the incidences of prostate gland inflammation in all exposed groups of males were significantly increased (11/50, 29/50, 22/50, 25/50 for chamber control, 75, 250, and 750 ppm). This inflammatory change consisted of infiltration by predominately mononuclear inflammatory cells with glandular acini and interstitium, increased interstitial fibrosis, and loss of secretory material in affected areas.
 
Bone marrow: Relative to chamber controls, males exposed to 75 or 750 ppm exhibited increased incidences of hyperplasia of the bone marrow characterized by hypercellularity due to the increased numbers of erythroid and myeloid precursor cells (7/49, 16/49, 9/50, 19/50 for chamber control, 75, 250, and 750 ppm). The relationship of these changes to ethylbenzene exposure is uncertain due to the lack of clear concentration-dependent responses.

According to NTP there was clear evidence of carcinogenicity in male rats due to increased incidences of kidney (renal tubule neoplasms) and testes tumors (testicular adenoma) and some evidence of carcinogenicity in female rats that also showed kidney tumors (renal tubule adenomas), but in a lower incidence and only detected after extended evaluation by step sections.

Chronic Toxicity - A decrease in survival and body weight and an increase in kidney pathology (renal tubule hyperplasia and nephropathy) were observed in rats that inhaled >= 75 ppm ethylbenzene for 2 years. NOAEL 250 ppm (males), LOAEL 75 ppm (females).

Cancer - At 750 ppm ethylbenzene, male rats exhibited increased incidences of kidney (renal tubule neoplasms) and testes tumors (testicular adenoma) and female rats also showed kidney tumors (renal tubule adenomas), but in a lower incidence and only detected after extended evaluation NOAEL 250 ppm.