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Description of key information

The most sensitive endpoint available today to assess chemical induced alteration to the immune system is used to evaluate BBSA for immunosuppression in the murine B6C3F1 model. Immunosuppression of BBSA was evaluated by examining the antibody plaque-forming cell (PFC) response to sheep red blood cells (SRBC). The antibody plaque-forming cell assay quantifies production of specific antibody through enumeration of antibody-producing cells following immunization with sheep red blood cells. This is based upon the antibody response to SRBC, a T-dependent antigen that requires functional integration of several cell populations including macrophages, T-helper cells, and B cells. Alterations in function or numbers of any of these populations may result in a suppressed immune response to pathogens. No suppression of the immune response was noticed after a 28 day dermal exposure with BBSA to the mice. In addition results showed in the LD group an increase in the IgM response in the spleen. However the biological significance of this finding is questionable due to the lack of change in spleen cellularity and absence of dose response. Furthermore immunophenotyping of the lymph node resulted in no marked changes in immune cell markers in the lymph node. Due to a technical issue, evaluation of the splenic immune markers could not be conducted. These results demonstrate that dermal exposure to BBSA, did not induce immunotoxicity in a murine model.

Key value for chemical safety assessment

Effect on immunotoxicity: via oral route

Link to relevant study records
Reference
Endpoint:
immunotoxicity: short-term dermal
Remarks:
28 day
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Principles of method if other than guideline:
In this study the potential for hypersensitivity and immune suppression was evaluated following dermal exposure to NBBS using a murine model.
GLP compliance:
not specified
Limit test:
no
Species:
mouse
Strain:
B6C3F1
Sex:
female
Details on test animals or test system and environmental conditions:
B6C3F1 mice are commonly uszed to evaluate potential immunotoxicioty and were used to evaluate the IgM response to sheep red blood cells (SRBC).
Route of administration:
dermal
Vehicle:
acetone
Details on exposure:
25 µl per dorsal surface of each ear.
Groups of mice (5/group) were exposed on one of the three concentrations of test agents or acetone vehicle over a 28- d period for each mouse.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
Groups of mice (5/group) were exposed on one of the three concentrations of test agents or acetone vehicle over a 28- d period for each mouse.
Frequency of treatment:
B6C3F1 mice were exposed over a 28-d period dermally (25µl per dorsal surface of each ear). This exposure schedule is based on immune-toxicity guidelines set forth by the NTP.
Dose / conc.:
25 other: %
Dose / conc.:
50 other: %
Dose / conc.:
100 other: %
No. of animals per sex per dose:
Five female animals per dose group
Control animals:
yes, concurrent vehicle
Details on study design:
THe primary IgM response to sheep red blood cells (SRBC) was enumerated using a modified hemolytic plaque-forming cell (PFC) assay of Jerne and Nordin (1963). B6C3F1 mice were exposed over a 28-d period dermally( 25µl per dorsal surface of each ear). This exposure schedule is based on immune-toxicity guidelines set forth by the NTP. Groups of mice(5/group) were exposed to one of three concentrations of test agents or acetone vehicle over a 28-d period for each mouse.Results were expressed as specific activity (IgM PFC per 10E6 spleen cells) and total activity (IgM PFC per spleen).
Observations and clinical examinations performed and frequency:
Systemic toxicity was evaluated weekly by clinical observation and change in body weight. The plaque-forming cell assay was conducted. Results were expressed as specific activity (IgM per 10E6 spleen cells) and total activity (IgM PFC spleen).
Sacrifice and pathology:
Animals were euthanized by CO2 inhalation 24 h after the final exposure, weighed, and examined for gross pathology. Blood was collected in ethylenediamine tetraacetic acid (EDTA)-coated vacutainer tubes following transection of the abdominal aorta, and hematological analysis was conducted . The liver, spleen, kidney,and thymus were removed, cleaned of connective tissue,and weighed. Spleen and DLN cell suspensions were prepared by mechanical disruption of tissues between frosted microscope slides in phosphate-buffered saline (PBS) and counted on a cellometer( Nexcelom).
Humoral immunity examinations:
Immunoglobulin IgM response was evaluated to shep red blood cells.
Other functional activity assays:
Immune cell phenotyping

Animals were euthanized by CO2 inhalation 24 h after the final exposure, weighed, and examined for gross pathology. Blood was collected in ethylenediamine tetraacetic acid (EDTA)-coated vacutainer tubes following transection of the abdominal aorta, and hematological analysis was conducted (see later description). The liver, spleen, kidneys, and thymus were removed, cleaned of connective tissue, and weighed. Spleen and DLN cell suspensions were prepared by mechanical disruption of tissues between frosted microscope slides in phosphate-buffered saline (PBS) and counted on a cellometer (Nexcelom). Cells
(1–2 × 106) were aliquoted into a 96-well U-bottom plate and washed in staining buffer (PBS + 1% bovine serum albumin + 0.1% sodium azide). Cells were resuspened in staining buffer containing anti-mouse CD16/32 antibody (clone 2.4G2) for blocking of Fc receptors (BD Biosciences). Cells were next resuspended in staining buffer containing a cocktail of fluorochrome-conjugated antibodies specific for cell surface antigens: CD45-Allophycocyanin (clone 30-F11), CD3e-V500 (500A2), CD4-Allophycocyanin-H7 (GK1.5), CD8a-PE-CF594 (53-6.7), CD45R/B220-Alexa Fluor 700 (RA3-6B2), NK1.1-FITC (PK136) (BD Biosciences), CD11c-eFluor 450 (N418), and CD11b-PerCP-Cyanine5.5 (M1/70) (eBioscience). Cells were washed in staining buffer and fixed in Cytofix buffer (BD Biosciences). Within 24 h, cells were resuspended in staining buffer and analyzed on an LSR II flow cytometer (BD Biosciences). Data analysis was performed with FlowJo 7.6.5 software (TreeStar, Inc.). Leukocytes were first identified by their expression of CD45, and the cells were further identified as follows: CD4 T cells (CD4+ CD3+), CD8 T cells (CD8+ CD3+), B cells (B220+ CD3-), NK cells (NK1.1+, CD3-), and dendritic cells (CD11b+ CD11c+).
Other examinations:
Selected hematological parametrs were evaluated using a Hemavet 950 automatic hematology analyzer (Frew Scientific , Waterbury , CT). Endpoints analyzed included peripheral erythrocyte and leukocyte counts, leukocyte differentials (lyùphocyres, nonocytes, basophols, and eosinophils), platelet counts, hematocrit, hemoglobin levels, mean corpuscular hemoglobin (MCH) and hemoglobin concentration (MCHC), mean corpuscular volume (MCP), mean platelet volume (MCV) and platelet distribution width.
Positive control:
Cyclophosphamide (CP) control for plaque -forming cell assay.
Statistics:
Data were first tested for homogeneity using the Bartlett's chi-squared test.If data were homogeneous, a one way analysis of variance (ANOVA) was conducted. If ANOVA showed significance at p<0.05 or less, Dunnett's multiple range t-test was used to compare treatment groups with the control group. For dose-response studies, linear trend analysis (linear trend test) was performed to detrmine whether BBSA had exposure concentration-related effects for specific endpoints. Differences were considered to be significant if p<0.05 compaire to vehicle controls. Statistical analysis using Graph Pad prism version 5.0( San Diego, Ca).
Clinical signs:
no effects observed
Description (incidence and severity):
Mice exposed to the positive control, cyclophosphamide (CP), showed a significantly reduced specific spleen IgM response and total IgM response compared to vehicle control.
In mice exposed to BBSA no suppression of IgM response to SRBC was observed. Mice exposed to the lowest concentration of BBSA 525 %) demonstrated a significantincrease of PFC/spleen. (87 % compaired with vehicle treated mice), suggesting an elevation in IgM antibody response to SRBC. However the biological significance of this finding is questionable due to the lack of change in spleen cellularity and absence of dose response.
Dermal irritation (if dermal study):
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
A significant elevation in kidney and liver weight as observed. However this was due to the extreme high not relevant exposure dose (25; 50, 100 % of BBSA), in contrast to the body weight which was not changed.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
no effects observed
Description (incidence and severity):
Mice exposed to the positive control, cyclophosphamide (CP), showed a significantly reduced specific spleen IgM response and total IgM response compared to vehicle control.
In mice exposed to BBSA no suppression of IgM response to SRBC was observed. Mice exposed to the lowest concentration of BBSA 525 %) demonstrated a significantincrease of PFC/spleen. (87 % compaired with vehicle treated mice), suggesting an elevation in IgM antibody response to SRBC. However the biological significance of this finding is questionable due to the lack of change in spleen cellularity and absence of dose response.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
not specified
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Humoral immunity examinations:
no effects observed
Description (incidence and severity):
Dermal exposure to BBSA did not suppress the IgM response to SRBC.
Specific cell-mediated immunity:
no effects observed
Non-specific cell-mediated immunity:
no effects observed
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
No suppression of the immune response was noticed after a 28 day dermal exposure with BBSA to the mice
Critical effects observed:
no
Conclusions:
The purpose of this stuidy was to evaluate the potential for immune suppression following dermal exposure to BBSA using a murine model. To estimate the immunosuppressive potential of BBSA , assays that assessed immunotoxicity were performed, including the immunoglobulin (Ig) M response to cell-dependent antigen sheep red blood cells( SRBC),using the plaque-forming cell (PFC) assay and immune cell phenotyping phenotyping. 28-d treatment with BBSA did not induce immunotoxicity in a murine model.
Executive summary:

The most sensitive endpoint available today to assess chemical induced alteration to the immune system is used to evaluate BBSA for immunosuppression in the murine B6C3F1 model. Immunosuppression of BBSA was evaluated by examining the antibody plaque-forming cell (PFC) response to sheep red blood cells (SRBC). The antibody plaque-forming cell assay quantifies production of specific antibody through enumeration of antibody-producing cells following immunization with sheep red blood cells. This is based upon the antibody response to SRBC, a T-dependent antigen that requires functional integration of several cell populations including macrophages, T-helper cells, and B cells. Alterations in function or numbers of any of these populations may result in a suppressed immune response to pathogens. No suppression of the immune response was noticed after a 28 day dermal exposure with BBSA to the mice. In addition results showed in the LD group an increase in the IgM response in the spleen. However the biological significance of this finding is questionable due to the lack of change in spleen cellularity and absence of dose response. Furthermore immunophenotyping of the lymph node resulted in no marked changes in immune cell markers in the lymph node. Due to a technical issue, evaluation of the splenic immune markers could not be conducted. These results demonstrate that dermal exposure to BBSA, did not induce immunotoxicity in a murine model.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

Effect on immunotoxicity: via inhalation route

Endpoint conclusion
Endpoint conclusion:
no study available

Effect on immunotoxicity: via dermal route

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Study duration:
subacute
Species:
mouse

Additional information

Justification for classification or non-classification

Based on the results and according to the EC criteria for classification and labelling requirements for dangerous substances and preparations (Guidelines in Commission Directive 93/21/EEC) and CLP regulation (EC No. 1272/2008 of 16 December 2008), N-butylbenzenesulphonamide is not classified for immunotoxicity.

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