All chemicals, triclosan (CAS# 3380-34-5), α-hexylcinnamaldehyde (HCA) [CAS# 101-86-0] and cyclophosphamide [CP; CAS# 50-18-0] were purchased from Aldrich Chemical Company, Inc. (Milwaukee, WI).

Female BALB/c and B₆C₃F₁ mice were used in these studies. BALB/c mice have a T-helper (T_H)-2 bias and are commonly used to evaluate potential IgE-mediated sensitization and were, therefore, used in the hypersensitivity studies (Woolhiser et al., 2000). B₆C₃F₁ mice are the strain of choice for immunotoxicity studies and were used to evaluate the IgM response to sheep red blood cells (SRBC) (Luster et al., 1992).

All mice were purchased from Taconic (Germantown, NY) at 6–8-weeks-of-age. On arrival, the animals were allowed to acclimate for a minimum of 5 days. Each shipment of animals was randomly assigned to treatment group, weighed and individually identified via tail tattoo or marking using a permanent marker. A preliminary analysis of variance on body weights was performed to ensure an homogeneous distribution of animals across treatment groups. The mice were housed at a maximum of five mice/cage in ventilated plastic shoebox cages with hardwood chip bedding. NIH-31 modified 6% irradiated rodent diet (Harlan Teklad, Indianapolis, IN) and tap water was provided from water bottles, ad libitum. Temperature in the facility was maintained at 68–72°F and relative humidity at 36–57%; a light/dark cycle was maintained at 12-h intervals. All animal experiments were performed in an Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) accredited National Institute for Occupational Safety and Health (NIOSH) animal facility in accordance with an animal protocol approved by the Institutional Animal Care and Use Committee (IACUC).

The concentrations of triclosan used in these studies were based on findings from previous studies (Anderson et al., 2013b). For the hypersensitivity study, BALB/c mice (five mice/group) were topically treated with acetone vehicle, increasing concentrations of triclosan or positive control [30% HCA (v/v; sensitization positive control)] on the dorsal surface of each ear (25 µl/ear) once a day for 3 consecutive days. For the immune phenotyping and hematology studies, B₆C₃F₁ mice (n = 5) were topically exposed to acetone or increasing concentrations of triclosan on the dorsal surface of each ear (25 µl/ear) once a day for 28 consecutive days. For analysis of the IgM response to SRBC, B₆C₃F₁ mice (n = 6) were topically exposed to acetone or increasing concentrations of triclosan on the dorsal surface of each ear (25 µl/ear) once a day for 28 consecutive days. Cyclophosphamide (20 mg/kg in isotonic sterile saline) was included as positive control for analysis of the IgM response to SRBC and was injected intraperitoneally for 4 consecutive days prior to sacrifice.

To determine the sensitization potential of triclosan, a local lymph node assay (LLNA) was conducted. Triclosan dosing concentrations (0.75–3.0%) and vehicle (acetone) were selected based on findings from previous studies (Anderson et al., 2013b). The murine LLNA was performed according to the methods previously described by Anderson et al. (2013a).

Animals were euthanized by CO₂ inhalation 24 h after the final exposure, weighed and examined for gross pathology. Blood was collected in EDTA-coated Vacutainer tubes following transection of the abdominal aorta and hematological analysis was conducted (see below). The liver, spleen, kidneys and thymus were removed, cleaned of connective tissue and weighed. Left and right auricular draining lymph nodes (DLN; drain site of chemical application) and spleen cell suspensions were prepared by mechanical disruption of tissues between frosted microscope slides in phosphate-buffered saline (PBS, pH 7.4) and counted on a Cellometer (Nexcelom, Lawrence, MA). Cells were then aliquoted (1–2 × 10⁶) into a 96-well U-bottom plate and washed in staining buffer (PBS + 1% bovine serum albumin+0.1% sodium azide). Cells were re-suspended in staining buffer containing anti-mouse CD16/32 antibody (clone 2.4G2) for blocking of F_c receptors (BD Biosciences, San Jose, CA). Cells were next re-suspended 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, San Diego, CA). Cells were then washed in staining buffer and fixed in Cytofix buffer (BD Biosciences). Within 24 h, cells were re-suspended 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., Ashland, OR). A minimum of 50 000 events was captured for each sample. Leukocytes were first identified by their expression of CD45 and the subsets 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⁺).

Select hematological parameters were evaluated using a Hemavet 950 automatic hematology analyzer (Drew Scientific, Waterbury, CT). Endpoints analyzed included peripheral erythrocyte and leukocyte counts, leukocyte differentials (lymphocytes, neutrophils, monocytes, basophils, and eosinophils), platelet counts, hematocrit, hemoglobin levels, mean corpuscular hemoglobin (MCH) and hemoglobin concentration (MCHC), mean platelet volume (MCV) and platelet distribution width (PDW).

The primary IgM response to SRBC was enumerated using a modified hemolytic plaque assay. Four days prior to euthanasia (i.e. Day 29), the mice were immunized with 7.5 × 10⁷ SRBC (in 200 µl volume) by intravenous injection. All SRBC for these studies were drawn from a single donor animal (Lampire Laboratories, Pipersville, PA). On the day of sacrifice, mice were euthanized by CO₂ inhalation, body and organ weights were recorded and spleens were collected in 3ml Hank’s balanced salt solution (HBSS). Blood was also retrieved in serum collection tubes following transection of the abdominal aorta and stored at −20 °C for subsequent analysis of serum anti-SRBC IgM levels (see below). Single cell suspensions of the spleens from individual animals were prepared in HBSS by disrupting the spleen between the frosted ends of microscope slides. To identify the total number of spleen cells, 20 µl of cells were added to 10 ml Isoton II diluent (1:500; Beckman Coulter, Indianapolis, IN) and two drops of Zap-o-globin II Lytic Reagent (Beckman Coulter) were added to lyse red blood cells. Cells were then counted using a Coulter Counter.

Dilutions (1:30 and 1:120) of spleen cells were then prepared and 100 µl of each dilution were added to test tubes containing a 0.5 ml warm agar/dextran mixture (0.5% Bacto-Agar, DIFCO; and 0.05% DEAE dextran; Sigma, St. Louis, MO), 25 µl of 1:1 ratio of SRBC suspension and 25 µl of 1:4 dilution (1 ml lyophilized) guinea pig complement (Cedarlane Labs, Burlington, Canada). Each sample was vortexed, poured into a petri dish, cover-slipped and incubated for 3 h at 37 °C. The plaques (representing antibody-forming B-cells or plaque-forming cells [PFC]) were then counted. Results were expressed in terms of both specific activity (IgM PFC per 10⁶ spleen cells) and total activity (IgM PFC per spleen).

Serum samples were analyzed for anti-SRBC IgM using a commercially available ELISA kit (Life Diagnostics, West Chester, PA), according to manufacturer recommendations with modifications. In brief, test serum was diluted (1:200, 1:400, 1:800 and 1:1,600) and incubated in the anti-SRBC coated microtiter wells for 45 min at 25 °C. The wells were subsequently washed, 100 µl horseradish peroxidase-conjugated secondary antibody was added and the plates incubated for an additional 45 min at 25 °C. Thereafter, the wells were washed to remove unbound antibodies and 100 µl tetramethylbenzidine peroxidase (TMB) reagent was added to each well. The plates were incubated for 20 min at room temperature before color development was stopped by addition of 50 µl of kit-provided Stop Solution. Optical density in each well was then measured spectrophotometrically at 450 nm using a Spectra Max M2 plate reader (Molecular Devices, Sunnyvale, CA). The concentration of anti-SRBC IgM in the test samples was determined by comparison to a standard curve generated in parallel using SoftMax Pro software and reported as units of anti-SRBC IgM (U/ml) plotted vs absorbance values at 450 nm.

For analysis of the data generated from the described animal studies, the data were first tested for homogeneity using the Bartlett’s Chi Square test. If homogeneous, a one-way analysis of variance (ANOVA) was conducted. If the ANOVA showed significance at p<0.05 or less, the Dunnett’s Multiple Range t-test was used to compare treatment groups with the control group. Linear trend analysis was performed to determine if triclosan had exposure concentration-related effects for the specified endpoints. Statistical analysis was performed using GraphPad Prism v5.0 (San Diego, CA). Statistical significance was designated at *p ≤ 0.05 and **p ≤ 0.01.

All animals appeared clinically normal throughout the course of the study and there were no changes in body weight. These animals had no visible signs of skin irritation or inflammation during the course of this study following exposure to triclosan (Figure 1). No significant increase in DLN proliferation was identified after treatment with triclosan (Figure 2); however, a slight increase in proliferation (1254±385) was observed following exposure to 3.0% triclosan compared to the vehicle control (607±128), resulting in a stimulation index (SI; foldchange above vehicle control) value of 2.1. HCA (30%) was used as a positive control for these experiments, resulting in an average SI value of 8.9.

There was no loss of body weight or overt signs of toxicity in animals exposed to triclosan for 28 days. However, animals exposed to 3.0% triclosan developed a mild irritation and scabbing/crusting on the ears after ~2 weeks of exposure that resolved by the end of the study. In contrast to body weight data, a statistically significant increase in liver weights was observed following exposure to 1.5% and 3.0% triclosan (11% and 44%, respectively, vs values for vehicle-treated mice) (Table 1). There was also a statistically significant increase in spleen weight following exposure to 3.0% triclosan. While this increase was not significant when expressed as percentage body weight, there was a significant increasing trend for both spleen weights and spleen as a percentage of body weight (Linear Trend Test, p<0.05). No other significant changes in body or organ weight were observed following exposure to any concentration of triclosan (Table 1). There was a dose-dependent increase (Linear Trend Test, p<0.05) in platelets reaching statistical significance at 3.0%, but dermal exposure to triclosan did not alter any other analyzed hematological parameter (Table 2).

Dermal exposure to triclosan resulted in a dose-dependent (Linear Trend Test, p<0.05) increase in DLN cellularity, reaching statistical significance at 1.5% and 3.0% (Figure 3A). Significant increases in the absolute number of B-cells, T-cells (CD4 and CD8), NK cells and dendritic cells were observed in DLN (Figure 4A), reaching statistical significance at 1.5% and 3.0% for all populations. A small but significant increase in the frequency of the dendritic cell population was observed in the DLN following exposure to 3.0% triclosan (Figure 4B). No significant changes were observed in the frequency of the other cell populations in the DLN. No differences in cellularity, absolute number or frequency of cells were identified in the spleen (Figures 3B and 5).

To evaluate if exposure to triclosan was immunosuppressive, the murine IgM response to SRBC was examined following a 28-day exposure to triclosan. No statistically significant reductions in the PFC/spleen and specific (PFC/10⁶ cells) IgM antibody activity against SRBC were observed after exposure to triclosan (Figure 6A and B). However, exposure of mice to 0.75% triclosan resulted in statistically significant increases in PFC/spleen and PFC/10⁶ cells (45 and 54%, respectively, vs values for vehicle-treated mice) (Figure 6). Although not statistically significant, this increase was also observed in the serum anti-SRBC IgM levels (Figure 6C). Mice exposed to positive control cyclophosphamide (CP) had a significantly reduced specific spleen IgM response and total IgM response compared to levels noted in vehicle-treated controls (Figure 6).