Aromatic hydrocarbons, C6-10, C8-rich

Immunotoxicity

Administrative data

Endpoint:

immunotoxicity: short-term inhalation

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study, GLP status not known, published in peer reviewed literature, fully adequate for assessment

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

publication

Title:

Oral gavage subchronic neurotoxicity and inhalation subchronic immunotoxicity studies of ethylbenzene in the rat.

Author:

Li, AA, Maurissen, JPJ, Barnett, JF, Foss, J, Freshwater, L, Garman, RH, Peachee, VL, Hong, SJ, Stump, DG and Bus, JS.

Year:

2010

Bibliographic source:

NeuroToxicology, 31, 247-258.

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.7800

Principles of method if other than guideline:

T-cell dependent antibody responses (assessed using a splenic antibody-forming cell assay) were determined in female rats exposed to ethylbenzene by inhalation for 28-days. The assay measures the activity of IgM antibody-forming cells against sheep red blood cells.

GLP compliance:

not specified

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

Constituent 2

Reference substance name:

101-41-4

IUPAC Name:

101-41-4

Details on test material:

Ethylbenzene, 99.96% pure by GC-FID, was supplied by The Dow Chemical Company, Midland, MI via Ashland Chemical, Morrisville, PA.

Test animals

Species:

rat

Strain:

other: Crl:CD(SD)

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Inc., Raleigh, NC USA
- Age at study initiation: approx. 45 d

ENVIRONMENTAL CONDITIONS
- Animals maintained in AAALAC International accredited facilities in accordance with US-NRC (1996) guidelines.

Administration / exposure

Route of administration:

inhalation: vapour

Vehicle:

air

Details on exposure:

EXPOSURE CONDITIONS
Animals housed individually in stainless steel-glass inhalation chambers (volume = 2 m3) and exposed (whole body) 6 hr/d for 28 consecutive days.

SELECTION OF DOSE LEVELS:
The highest dose level (500 ppm, equivalent to 2.175 mg/l) was selected based on earlier reproduction and developmental neurotoxicity studies. In addition, EPA OPPTS 870.7800 guideline recommends that the high dose should not exceed 2 mg/l.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Achieved exposure concentration confirmed using GC-FID (no further details).

Duration of treatment / exposure:

28-d

Frequency of treatment:

daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 25, 100 and 500 ppm
Basis:
nominal conc.
nominal in air

No. of animals per sex per dose:

10 (females only)

Control animals:

yes, concurrent vehicle

Details on study design:

IMMUNISATION WITH SHEEP RED BLOOD CELLS:
Animals were immunized using sheep red blood cells (sRBC; 2 x 10^8 cells in 0.5 ml Earle's Balanced Salt Solution; EBSS) administered by daily injection into a lateral tail vein on the 4 days prior to sacrifice.

Examinations

Observations and clinical examinations performed and frequency:

CAGE SIDE OBSERVATIONS:
Animals were observed at least twice daily before and after exposure.

BODY WEIGHT and FOOD CONSUMPTION:
Data collected twice weekly for the duration of the study.

Sacrifice and pathology:

NECROPSY PROCEDURES:
Animals were euthanized by carbon dioxide inhalation.
Blood was collected from the vena cava for haematological evaluation.
Organ weight data were collected for: brain, kidney, liver, lung, spleen, thymus.
The spleens were removed, placed in EBSS with 15mM HEPES supplemented with gentamicin, and single cell suspensions prepared.

Cell viabilities:

PREPARATION OF SPLEEN CELL SUSPENSIONS:
Splenocyte viability was determined using propidium iodide staining and flow cytometry (no further details)

Specific cell-mediated immunity:

SPLENIC ANTIBODY-FORMING CELL (AFC) ASSAY
The AFC assay was performed using a modification of the Jerne plaque assay (Jerne, NK et al. (1963) In Amos et al. (eds) "Cell Bound Antibodies", Wistar Institute Press, Philadelphia, PA) (no further details).
The AFC data were expressed as specific activity (IgM AFC/10^6 spleen cells) and total spleen activity (IgM AFC/spleen).

Positive control:

POSITIVE CONTROL SUBSTANCE:
- cyclophosphamide (50 mg/kg) given by daily i.v. injection on the 4 d immediately prior to sacrifice.

Statistics:

Body weight change, food consumption, haematology data and organ weights were analysed using one-way ANOVA with Dunnett's test. The AFC assay data were tested for homogeneity of variances using Bartlett’s Chi Square test, with homogenous data evaluated using ANOVA followed by Dunnett's test. Non-homogenous data were analysed a non-parametric ANOVA followed by Gehan-Wilcoxon test. Dose-related trends were identified using Jonckheere's test. The positive control group was compared to the vehicle control group using Student's T-test.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Haematological findings:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

liver and spleen weights increased

Details on results:

GENERAL OBSERVATIONS AND CLINICAL SIGNS:
All animals survived to scheduled necropsy, with no adverse test substance-related clinical signs noted.

BODY WEIGHTS:
Body weight unaffected by treatment; body weight decreased by 16% in positive control group.

FOOD CONSUMPTION:
Food consumption unaffected by treatment (data not reported).

HAEMATOLOGY
Haematology parameters unaffected by treatment (data not reported); white cell and platelet counts, mean corpuscular concentration, absolute and percent lymphocyte and neutraphil counts all decreased in positive control group (data not reported).

ORGAN WEIGHTS:
Liver and kidney weights increased in the 500 ppm treatment group, brain lung, spleen and thymus weights unremarkable.

Selected results by treatment level for female rats exposed to 0, 25, 100 and 500 ppm ethylbenzene or 50 mg/kg bw/d cyclophosphamide:
Absolute spleen weight (mg): 610, 548, 539, 562, 271 **
Relative spleen weight (mg/100 g bw): 0.25, 0.24, 0.23, 0.25, 0.13 **
Absolute thymus weight (mg): 486, 424, 455, 432, 107 **
Relative thymus weight (mg/100 g bw): 0.20, 0.19, 0.20, 0.19, 0.05 **
*** P<0.01 (Dunnet's test; effects present only in positive control group)

SPLENIC ANTIBODY-FORMING CELL ASSAY:
No treatment-related effects on spleen cell number or IgM AFC response to T-cell dependent antigen sRBC, whereas specific and total IgM AFC clearly and significantly reduced after cyclophosphamide treatment.

Results by treatment level for female rats exposed to 0, 25, 100 and 500 ppm ethylbenzene or 50 mg/kg bw/d cyclophosphamide:
Spleen cells (x10^7 per spleen): 78.7, 66.0, 63.8, 67.9, 9.7**
Specific activity - IgM AFC/10^6 spleen cells: 1473, 1546, 1718, 2145, 40 **
Total activity - IgM AFC/10^3 per spleen: 1134, 1027, 1168, 1462, 4 **
***P<0.01 (Dunnet's test; effects present only in positive control group)

Specific immunotoxic examinations

Cell viabilities:

no effects observed

Specific cell-mediated immunity:

no effects observed

Effect levels

Dose descriptor:

NOAEC

Effect level:

500 ppm (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no effect on the activity of IgM antibody-forming cells present in spleen at the highest dose level tested.

Applicant's summary and conclusion

Conclusions:

Exposure to up to 500 ppm ethylbenzene vapour for 28-d did not affect the humoral component of the rat immune system.

Executive summary:

Female SD rats, age approx. 7 weeks, were exposed (whole body) to ethylbenzene vapour (0, 25, 125 and 500 ppm) for 28-d and humoral immune function determined using splenic antibody-forming cell (AFC) plaque-forming assay following sensitisation of the animals to sheep red blood cells (sRBC). A positive control group received cyclophosphamide.The assay assesses the interaction of complement and antibodies (produced in response to immunization) that is directed against sRBC. The study design and conduct was OPPTS 870.7800 compliant, with the results providing a measure of the effect of ethylbenzene on humoral immune responses. The results showed that spleen and thymus weights from ethylbenzene-exposed animals were unremarkable, with no treatment-related effects on spleen cell number or IgM AFC responses in sRBC-sensitised animals. In contrast spleen and thymus weights, together with specific and total IgM AFC, were clearly and significantly reduced after cyclophosphamide treatment. The results demonstrate that sub-acute exposure to up to 500 ppm ethylbenzene vapour for 28 -d was without effect on the humoral component of the rat immune system.