Diethylbenzene

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This is a GLP guideline study.

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 Type:

study report

Title:

Unnamed

Year:

1993

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: EPA Guidelines

Principles of method if other than guideline:

Method: T26-16:

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Diethylbenzene

EC Number:

246-874-9

EC Name:

Diethylbenzene

Cas Number:

25340-17-4

Molecular formula:

C10H14

IUPAC Name:

diethylbenzene

Details on test material:

IUCLID4 Test substance: as prescribed by 1.1 - 1.4

MCS 2313 DEB, mixed isomers
Supplier: Monsanto Chemical Group
Reference No.: lot number 3629297, EHL substance identification code: T910095
Appearance: Clear, white liquid

Purity:Not stated

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Species: Alblno rat
Strain: Spragus-Dawley (CD)
Source: Charles River Breedlng Laboratory, Portage MI
Date of Arrival at Environmental Health Laboratory: October 22, 1991
Acclimation Perlod: 19 days
Number Used In Study: 80 (40 males, 40 females). Any unhealthy animals were excluded from assignment to the study.
Test Group Size: 10/sex
Method of Assignment: Computer randomizatlon by weight
Method of Identification: lndividual eartag, bar-coded cage card
Age at Study Start: Approximately seven weeks
Weight at Study Start: Males: Approximately 283 g
Famales: Approximately 192 g
Type of Housing: Individual stainless steel cages wIth wlre mesh bottoms suspended over paper beddlng
Water Availability; ad libitum (St. Louis publIc water supply) except durlng exposure, when water was withheld
Food Availability: ad libitum (Purine Mllls RODENT CHOW No. 5002) except during exposure, when food was withheld
Note: Animal housing and husbandry were in accordance with the provisions of 'Guide to the Care and Use of Laboratory Animals: USPHS-NIH
Publication No. 86-23.

Administration / exposure

Route of administration:

inhalation

Type of inhalation exposure:

whole body

Vehicle:

other: test material (as received) in air

Remarks on MMAD:

MMAD / GSD: No data

Details on inhalation exposure:

Four groups of 10 male and 10 female Sprague-Dawley rats  were exposed for 6 hours per day, five days per week to MCS 2313 at mean  analytical concentrations of 0, 190, 610, and 1400 mg/cubic meter in air,  respectively, for 62 to 64 exposures over a three month period. The  target concentrations were 0 (Control, Group N), 200 (Low, Group 1), 600  (Mid, Group 2) and 1200 (High, Group 3) mg MCS 2313/cubic meter of air.  

Exposure Chamber: Four 10 m3 inhalation chambers Test Atmosphere Generation System: Low exposure level test atmospheres  were generated using a Laskin-style nebulizer/spraybar positioned in the  supply air chamber inlet. Test material was delivered using a syringe  pump. The mid and high exposure level test atmospheres were generated  using a spray nozzle directed down from the top of the chamber. Test  material was delivered for the mid and high level chambers with a  valveless pump. The concentration of test material in the chambers was  controlled by regulating the flow rate of the material from the pumps.

Monitoring of Environmental Measures: Chamber airflow, temperature and %  relative humidity were monitored continuously and recorded approximately  every 30 minutes.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Procedures for Monitoring Amount of Test Material in Chamber
Atmospheric Analytical Sampling: 4 samples (per exposure) at  approximately equal intervals.
Sampling Method: Test atmosphere was drawn at a known rate through  acetonitrile contained in one 25-ml glass impinger
Volume of Test Atmosphere Sampled: 5 to 15 liters
Sampling Location: In the animal breathing area from a port halfway down  on the chamber door
Analytical Method: High Pressure Liquid Chromatography 
Nominal Concentration: Calculated once per exposure by determining the  total amount of test material delivered to the chamber and dividing this  amount by the total air volume passing through the chamber during the  exposure. 
Uniformity of Distribution in Chamber: Performed once pretest and monthly  during the study for all levels

Duration of treatment / exposure:

62 to 64 exposures over a three month period

Frequency of treatment:

six hr/day, five days per week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

200 mg/m³ air (nominal)

Doses / concentrations 2

Dose / conc.:

600 mg/m³ air (nominal)

Doses / concentrations 3

Dose / conc.:

1 200 mg/m³ air (nominal)

Doses / concentrations 4

Dose / conc.:

190 mg/m³ air (analytical)

Doses / concentrations 5

Dose / conc.:

610 mg/m³ air (analytical)

Doses / concentrations 6

Dose / conc.:

1 400 mg/m³ air (analytical)

No. of animals per sex per dose:

10/sex/dose

Control animals:

yes, concurrent no treatment

Details on study design:

Four groups of 10 male and 10 female Sprague-Dawley rats  were exposed for 6 hours per day, five days per week to MCS 2313 at mean  analytical concentrations of 0, 190, 610, and 1400 mg/cubic meter in air,  respectively, for 62 to 64 exposures over a three month period. The  target concentrations were 0 (Control, Group N), 200 (Low, Group 1), 600  (Mid, Group 2) and 1200 (High, Group 3) mg MCS 2313/cubic meter of air.  Each exposure level was sampled four times daily and the control chamber  was sampled weekly for DEB concentration. Ophthalmic examinations were  performed pretest and at the end of the study. Each animal was checked  twice daily for mortality and following each exposure for gross signs of  toxicity. During exposure, visible animals were observed for toxicity.  Body weights and detailed clinical examinations were performed weekly.  Clinicopathologic examinations, gross necropsy, organ weight  determinations and tissue from all animals in the control and highest  exposure groups were examined microscopically.

Positive control:

None

Examinations

Observations and examinations performed and frequency:

Ophthalmic examinations were  performed pretest and at the end of the study. Each animal was checked  twice daily for mortality and following each exposure for gross signs of  toxicity. During exposure, visible animals were observed for toxicity.  Body weights and detailed clinical examinations were performed weekly.  


Animals Examined: All at pretest; control and high exposure level groups  just prior to terminal sacrifice

Mydriatic: 1% atropine Method of Examination: Indirect ophthalmoscopy

Sacrifice and pathology:

Clinicopathologic examinations [hematology (white blood cell count, red blood cell count, hemoglobln. hematocrit, mean corpuscular volume, mean corpuscular hemaglobin, mean corpuscular hemagtobin concentration, platelet count, leukocyte differential and reticulocyte count) and clinical chemistry (Alkallna phosphatase, creatlne phosphoklnase, glutamlc pyruvic transamlnase, glutamic oxaloacetic transamlnase, gamma glutamyl transpeptidase, direct bilirubln, total bilirubin, globulin, cholesterol, glucose, blood urea nitrogen, creatinine, total protein, albumln, calcium, sodium, chloride,
potassium and phosphorus],  gross necropsy and organ weight determinations[adrenals (combined), brain, heart, kidneys (combined).
liver, spleen and testes (combined)] . Tissues [aorta, adrenals, bone and bone marrow (sternum), braln, cecum, colon, duodenum, esophagus, eyes, exorbital lachrymal glands, heart, ileum, jejunum, kldneys, lesions (with possible histopathological correlates) and masses, Iiver, lung (wlth
mainstem bronchi), lymph nodes (mesenteric, submandibular), muscle (quadrlceps femoris),ovaries,nasal turbinates, pancreas,
pituitary, prostate, sciatic nerve,seminal vesicles, skln (with mammary tissue), spinal cord(cervical, thorax, lumbar), spleen,
stomach, submaxillary salivary gland,testes wlth epididymides, thymus, thyroid/parathyroid, trachea, uterus (corpus and cervix) and
urinary bladder] from all animals in the control and highest  exposure groups and lungs, liver, kidneys and gross lesions with possible
histopathological correlations from the low and mid levals were examined microscopically.

Clinical Pathology Frequency: At termination
Number of Animals: Ten/sex/level
Blood Collection Method: Posterior vena cava of CO2 -anesthetized, fasted  rats
Hematology Determinations: White blood cell count, red blood cell count,  hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular  hemoglobin, mean corpuscular hemoglobin concentration and platelet count:  Whole blood was treated with anticoagulant (EDTA pretreated commercial  tubes) and processed on a COULTER S Plus II blood cell counter using the  manufacturer's methods. Leukocyte differential: Thin blood smears on labeled glass slides were  prepared, stained with Wright's stain, and examined microscopically. Reticulocyte count: A portion of the EDTA-treated sample was mixed with a  vital stain (ethylene blue), and a slide was prepared and examined  microscopically.

Blood Chemistry Determinations: Alkaline phosphatase, creatine  phosphokinase (CPK), glutamic pyruvic transaminase, glutamic oxaloacetic  transaminase, gamma glutamyl transpeptidase, direct bilirubin, total  bilirubin, globulin, cholesterol, glucose, blood urea nitrogen,  creatinine, total protein, albumin, calcium, sodium, chloride, potassium  and phosphorus: Serum harvested by centrifugation of samples submitted in  commercial clot tubes was assayed using the manufacturers' methods.  Globulin was determined by subtraction of the albumin value from the  total protein value. These determinations were performed on a Hitachi 717  clinical analyzer. 

Gross Pathology: Animals Examined: All
Scheduled Sacrifice: Day following final test exposure
Extent of Examination: External and internal. Internal cavities were  opened, and organs were examined in situ and then removed. Hollow organs  were opened and examined.
Organs Weighed: Adrenals (combined), brain, heart, kidneys (combined),  liver, spleen and testes (combined)
Tissues Retained: Aorta, adrenals, bone and bone marrow (sternum), brain,  caecum, colon, duodenum, esophagus, eyes, exorbital lachrymal glands,  heart, lieum, jejunum, kidneys, lesions (with possible histopathological  correlates) and masses, liver, lung (with mainstem bronchi), lymph nodes  (mesenteric, submandibular), muscle (quadriceps femoris), ovaries, nasal  turbinates, pancreas, pituitary, prostate, sciatic nerve, seminal  vesicles, skin (with mammary tissue), spinal cord (cervical, thorax,  lumbar), spleen, stomach, sub maxillary salivary gland, testes with  epididymides, thymus, thyroid/parathyroid, trachea, uterus (corpus and  cervix), and urinary bladder
Fixatives: Eyes- 5% buffered neutral formalin/0.5% glutaraldehyde
Remaining Tissues: 10% buffered neutral formalin
Histopathology Tissue Examined: All retained tissues from the control and high level  animals and lungs, liver, kidneys and gross lesions with possible  histopathological correlations from the low and mid levels  Tissue Preparation: Fixed tissues were washed, dehydrated, embedded in  paraffin, sectioned at approximately 5 microns, and stained with  hematoxylin and eosin. 
Examination: Light microscopy

Other examinations:

None

Statistics:

Statistics The following statistical procedures were used to detect statistically  significant differences between treated animals and their respective  controls: Dunnett's Multiple Comparison Test (two-tailed): In-life body weights. Decision-Tree Analysis: Hematology data, clinical chemistry data,  terminal body weights, absolute organ weights and organ/body weight  ratios were evaluated by decision-tree statistical analyses which,  depending on the results of tests for normality and homogeneity of  variances [Bartlett's Test], utilized either parametric [Dunnett's Test  and Linear Regression] or nonparametric [Kruskal-Wallis, Jonokheere's  and/or Mann-Whitney Tests] routines to detect differences and analyze for  trend. Fisher's Exact Test (one-tailed): Incidence of microscopic lesions Grubb's test was used to detect outliers in the organ weight data.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

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:

no effects observed

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

Body Weights
Group mean body welghts of both sexes at the high exposure level were lower, compared to thelr controls, throughout most of the study. However,
group mean body weights of males at the high exposure level were statistically significantly lower only in four of the first seven weeks (Weeks 1, 3, 4 and 7), Except for the tenth week, group mean body weights of females at the high level were statistically significantly lower from the eighth through the fourteenth weeks of the study. At the mid exposure level, group mean body weights of female animals were lower than the controls throughout the study and the male animals' group mean body weights were reduced for the first nine weeks of the study. The only statistically significant change at this exposure level occured in females at week four.

Animal Observations
Loss of balance and head tilt was observed durlng exposure In one hlgh exposure level male anlmal. Both observatlona were transient, lasting one to three days before disappearlng. Tnls animal also had a weight decrease compared to other anlmals in its group during this period. However, one week later, the anlmal's weight returned to wlthin 10% of the group mean body weight. It is felt that these observations were not caused by exposure to the test material. Abnormal observations following exposures were limited to a few animals and did not appear to be compound-related. Abnormal observations, when the animals were weighed, weekly, prior to exposure, also did not appear compound-related. These observations were limited in the number of animals affected and the number of occurrences. Observations which occurred in multiple anlmals included perlnasal encrustatlon (four animals, all in one week) and periorbital encrustation (two animals).

Ophthalmic Examinations
There were no ocular abnormalities which were attributed to admlntstration of the test material.

Toxic response/effects:  Decreased mean body weights in the high-dose  group animals throughout the study were indicative of toxicity.  There  were no abnormal clinical observations considered to be  treatment-related.  There were no ocular abnormalities attributed to  administration of the test material. 

Treatment-related changes in hematologic parameters included moderate  decreases in total white cell and lymphocyte counts in the mid- and  high-exposure level males.  Abnormal sera color (blue or blue-gray) was  observed in high-exposure level animals of both sexes.  Treatment-related  changes in serum chemistry parameters included decreases in ALT, AST, and  CPK in high-exposure level females and increases in potassium in  high-level males and phosphorus in males from the high-exposure group and  females from the mid- and high-exposure groups.  The reason(s) for the changes in hematologic and clinical chemistry  parameters was not determined. The decreased CPK, ALT, and AST levels  observed in high exposure level females were unusual since changes in  these enzymes usually occur as increases. 

An abnormal blue-gray color was observed in most tissues from all but one  high-exposure animal.  At the mid-exposure level, the same color was  observed in brains of eight males and all females and in the urinary  bladders of five females and one male.  This abnormal color probably  resulted from the presence of the parent chemical or a metabolite in  these tissues.  However, there were no other gross or microscopic changes  attributed to the test material.

Liver weights relative to terminal body weights and brain weights were  slightly increased in male animals from all three treated groups. The  degree of change was similar among all groups and there were no  treatment-related changes observed microscopically. Therefore, the organ  weight changes were apparently the result of slightly lower hepatic  weights in control rats.

There were no treatmant-related microscopic lesions.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

190 mg/m³ air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

haematology

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:

NOAEL: 190 mg/m3 Repeated exposures to Mixed Diethylbenzenes did not result in any target organ toxicity.

Executive summary:

Four groups of 10 male and 10 female Sprague-Dawley rats were exposed for 6 hours per day, five days per week to MCS 2313 at mean analytcal concentrations of 0, 190, 610, and 1400 mg MCS 2313/cubic meter in air, respectively, for 62 to 64 exposures over a three month period, The target concentrations were 0 (Control, Group N), 200 (LOW, Group 1), 600 (Mid, Group 2) and 1200 (High, Group 3) mg MCS 2313/cubic meter of air. Each exposure level was sampled four times daily and the control chamber was sampled weekly for MCS 2313 concentration. No test material was detected in the control chamber. The chamber distribution of test

material for each of the three target levels was checked monthly. Ophthalmic examinations were perfoemed pretest and at the end of the study. Each animal was checked twice daily for mortality and following each exposure for gross signs of toxicity. During exposure, visible animals were observed for toxicity. Body weights and detailed clinical examinations were performed weekly. Clinicopathologlc examinations, gross nscropsy, organ weight determinations and tissue sampling were conducted at study termination. All retained tissues from all animals in the control and highest exposurs groups were examined microscopically.

Decreased mean body weights in high level animals throughout the study were indicative of toxicity. There were no abnormal clinical observations whlch were considered to be treatment-related. There were no ocular abnormalities attributed to administration of the test material.

An abnormal blue-grey color was observed in most tissues from all but one high exposure level animal. At the mid exposure level, the same color was observed in brains of eight males and all females and in the urinary bladders of five females and one male. This abnormal color probably resulted from the presence of the parent chemical or a metabolite in these tissues. However, there were no other gross or microscopic changes attributed to the test material.

Treatment-related changes in hematologlc parameters included moderate decreases in total white cell and lymphocyte counts in mid and high exposure level male animals. Abnormal sera color (blue or blue-gray) was observed in high exposure level animals of both sexes. Treatment-related changes in serum chemistry parameters included decreases in ALT, AST, and CPK in high exposure level female animals and increases in potassium in high level males and phosphorus in males from the high exposure level and females from the two highest exposure levels. The reason(s) for these changes was not determined. Also, the effect of the abnormal serum

coloration on the validity of the analytical results was not detemined.

The no-observed-effect- level (NOEL) for MCS 2313, based on the results of this study, is considered to be 190 mg/m3.