Immunotoxicity of N-butylbenzenesulfonamide: impacts on immune function in adult mice and developmentally exposed rats

Abstract

N-butylbenzenesulfonamide (NBBS) is a high-production volume plasticizer that is an emerging contaminant of concern for environmental and human health. To understand the risks and health effects of exposure to NBBS, studies were conducted in adult-exposed mice and developmentally exposed rats to evaluate the potential for NBBS to modulate the immune system. Beginning between 8 and 9 weeks of age, dosed feed containing NBBS at concentrations of 0, 313, 625, 1250, 2500, and 5000 ppm was continuously provided to B6C3F1/N female mice for 28 days. Dosed feed was also continuously provided to time-mated Harlan Sprague Dawley (Sprague Dawley SD) rats at concentrations of 0-, 250-, 500-, and 1000-ppm NBBS from gestation day 6 to postnatal day 28 and in F1 rats until 11–14 weeks of age. Functional assessments of innate, humoral, and cell-mediated immunity were conducted in adult female mice and F1 rats following exposure to NBBS. In female mice, NBBS treatment suppressed the antibody-forming cell (AFC) response to SRBC with small increases in T-cell responses and natural killer (NK)-cell activity. In developmentally exposed rats, NBBS treatment-related immune effects were sex dependent. A positive trend in NK-cell activity occurred in male F1 rats while a negative trend occurred in female F1 rats. The AFC response to SRBC was decreased in female F1 rats but not in male F1 rats. These data provide evidence that oral exposure to NBBS has the potential to produce immunomodulatory effects on both innate and adaptive immune responses, and these effects appear to have some dependence on species, sex, and period of exposure (developmental vs adult).

N-butylbenzenesulfonamide (NBBS) belongs to the sulfonamide class of plasticizers and is recommended for use in polyamide (e.g., Nylon 6, 11, and 12), polycarbonate, copolyester, melamine, urea, and phenolic polymers (Wypych, 2017). It can be difficult to identify the formulated plastic products which contain specific plasticizers due to the lack of a publicly accessible registry and restrictions on the use of commercial data (Groh et al., 2019). The Handbook of Plasticizers lists the main applications for sulfonamide-containing plastics as medical devices, films, fishing lines, and automotive parts (Wypych, 2017). However, NBBS has been detected as a migrant from both cooking utensils (>10 μg/l) (Skjevrak et al., 2005) and baby bottles (38–108 μg/kg) (Simoneau et al., 2012). The production volume of NBBS in the United States from 2012 to 2020 was reported to be 1–<10 million lbs (ChemView UE, 2023). NBBS has been detected in many environmental water samples in the US and Europe (Di Carro et al., 2018; Dsikowitzky et al., 2004a,b; Grigoriadou et al., 2008; Huppert et al., 1998; Oros et al., 2003; Pedersen et al., 2005; Plumlee et al., 2012), typically in the ng/l to μg/l range. Evidence suggests that NBBS is persistent and mobile in groundwater (Blum et al., 2018) and contamination has been detected in drinking water sources (Soliman et al., 2007) making oral exposure a likely route in humans.

Although NBBS has yet to be included in widespread biomonitoring efforts, such as the National Health and Nutrition Examination Survey in the United States (Groh et al., 2019), it has been detected in nontargeted exposure assessments. For example, NBBS was recently detected in house dust (Castro et al., 2019), which has implications for exposure in young children via hand-to-mouth behavior (Hammel et al., 2019). It was also detected in human feces in a survey of volatile organic compounds (Garner et al., 2007). Furthermore, NBBS was identified as one of 212 chemicals with potential emerging risk in the food chain by the European Food Safety Authority (Oltmanns et al., 2019).

Despite the potential for human exposure, either occupationally or through contact with contaminated water or food, there are limited NBBS toxicity data for hazard characterization. The first reports of NBBS toxicity resulted from the accidental leaching of the chemical into saline from a plastic holding container. Researchers observed progressive spastic myelopathy in a control group of New Zealand white rabbits provided with intracisternal saline and identified NBBS as the culprit (Strong et al., 1990). Following that unexpected discovery, several publications described NBBS as a neurotoxicant (Duffield et al., 1994; Kumar et al., 2007; Nerurkar et al., 1993). However, neurotoxicity was not observed in Sprague Dawley rats dosed up to 300 mg/kg bodyweight via oral gavage for 27 days (Rider et al., 2012). In a short-term perinatal toxicity study in Harlan Sprague Dawley (Hsd: Sprague Dawley SD) rats, pregnant dams and their offspring were exposed to NBBS via dosed feed at concentrations of 625–10 000 ppm from gestation day (GD) 6 through postnatal day (PND) 28 (Rider et al., 2020). Overt toxicity was observed in dams in the 10 000-ppm NBBS group. While NBBS did not impact pregnancy or littering at concentrations up to 5000 ppm, pup survival was decreased in the 5000-ppm group and pup weights displayed a dose-responsive decrease with significant decreases observed as early as PND 1 and progressing through PND 25 in both male and female pups exposed to 5000-ppm NBBS (Rider et al., 2020).

In a 28-day study evaluating the immunotoxicity of NBBS in mice following dermal exposure to 100% NBBS, significant increases in liver and kidney weights were reported, although no effects on antibody responses, immune cell population numbers, or evidence of allergic hypersensitivity were observed (Marrocco et al., 2015). To fully assess immunotoxicity following exposure to NBBS through feed (313–5000 ppm), we conducted studies in adult B6C3F1/N mice using a comprehensive immune testing panel (Luster et al., 1988) designed to determine effects on innate, humoral, and cell-mediated immune cells and functions. Immunotoxicity studies conducted in adults are not always predictive of the impacts on the developing immune system. The maturing immune system represents a vulnerable target for toxicants as it progresses through a series of novel developmental milestones that are critical for later-life host defense against a wide array of diseases. As such, developmental immunotoxicity studies were also conducted to address susceptibility to NBBS employing the same comprehensive immune testing panel using a life course exposure design. Sprague Dawley rats were developmentally exposed to NBBS through feed (250–1000 ppm) during gestation, lactation, and through adulthood of the F1 offspring, at which time immune assessments were performed. These studies were part of a comprehensive toxicology assessment for NBBS conducted by the National Toxicology Program (National Toxicology Program, 2020).

Materials and methods

 

 

These studies were conducted in compliance with the U.S. Food and Drug Administration Good Laboratory Practices for Nonclinical Laboratory Studies (Title 21 of the Code of Federal Regulations, Part 58).

Test chemical and dose formulation

NBBS and vehicle chemistry activities were conducted at Battelle Columbus (Columbus, OH). NBBS was obtained from Ivy Fine Chemicals (CAS RN 3622-84-2, Lot IF10505; Cherry Hill, NJ). The identity of NBBS was confirmed by ¹H and ¹³C NMR, Fourier infrared spectroscopy, and gas chromatography (GC)-mass spectrometry and the purity (>99.9%) was determined by high-performance liquid chromatography with ultraviolet detection (HPLC-UV) and using GC with flame ionization detection.

Dosed feed was prepared in NTP-2000 and NIH-07 Open Formula (certified, irradiated) meal rodent chow (Zeigler Brothers Inc., Gardners, PA). Test article was mixed with the feed using a twin-shell V blender for approximately 15 min. Feed formulations were analyzed using a validated HPLC-UV method (r > 0.99; relative standard deviation [RSD], ±10%; relative error [RE], ±10%) prior to exposing animals. The dose formulations were stored under ambient conditions and were used within 42 days of preparation. Prior to study initiation, the stability of NBBS in feed formulations stored at ambient temperature up to 42 days was confirmed (RE ≤±10% of day 0 value). Animal room samples were collected from formulations following administration and were analyzed for concentration of NBBS.

Chemistry activities for the positive control, cyclophosphamide (CPS), were conducted at RTI International (Durham, NC). CPS was obtained from Sigma Aldrich (CAS RN 6055-19-2, Lot MKBS0021V; St. Louis, MO). The identity of CPS was confirmed by infrared and nuclear magnetic resonance spectra (proton, ¹³C and ³¹P) and the purity (>99%) was determined by high-performance liquid chromatography with evaporative light scattering detection (HPLC/ELSD). Stability of the CPS formulation was determined to be 52 days when stored at 2–8°C. Formulations of CPS were analyzed for concentration prior to administration to the study animals and following treatment (animal room samples) using HPLC/ELSD.

Analyses for NBBS confirmed the absence of the compound in the vehicle feed formulation. All NBBS and CPS dose formulations analyzed prior to administration to the animals were within 10% of the target concentration and homogeneous (RSD ≤±5). The postadministration concentrations for the animal room samples from all preparations of NBBS and CPS dose formulations were within 10% of the target concentration except the 313 and 625 ppm (12.5% and 11.9% below target concentration, respectively) formulations from a single preparation (out of 8 preparations). These slight deviations in a single batch did not warrant dose adjustment calculations and the nominal target concentrations are referenced for the study.

Experimental animals and study design

The studies were conducted at Burleson Research Technologies which is an AAALAC accredited research facility with the Office of Laboratory Animal Welfare Assurance. Animal procedures were approved by the Burleson Research Technologies Institutional Animal Care and Use Committee prior to conducting these studies.

Female adult B6C3F1/N mice were obtained from Taconic Biosciences, Inc (Germantown, NY). There is evidence that female rodents have higher sensitivity and greater dynamic range than male rodents, especially mice, for many of the innate and adaptive immune responses that are measured as part of immunotoxicity safety assessment (Klein and Flanagan, 2016). Testing only adult female mice reduced animal use while evaluating the effects of NBBS in the more sensitive sex. Following randomization, mice were housed in groups of 4 mice per cage. Beginning between 8 and 9 weeks of age, dosed feed containing NBBS at concentrations of 0, 313, 625, 1250, 2500, and 5000 ppm were continuously provided ad libitum for 28 days. A previous 14-day mouse toxicity study demonstrated that exposure of adult mice to concentrations up to 5000-ppm NBBS showed no evidence of overt toxicity (unpublished data). Vehicle controls received NTP-2000 meal diet from the same lots used for preparation of the dose formulations. An immunomodulatory positive control group of adult female mice was included which received 50 mg/kg (5 mg/ml formulation) CPS via intraperitoneal (IP) injection. Doses were selected to maximally challenge the immune system in the absence of overt toxicity, which could cause immune effects secondary to stress. Time-mated Sprague Dawley (Hsd: Sprague Dawley SD) rats were obtained from Envigo (Indianapolis, IN). NBBS-dosed NIH-07 feed (diet developed specifically for pregnant and nursing rodents) was provided to F0 dams ad libitum containing 0-, 250-, 500-, or 1000-ppm NBBS starting on GD 6. The high dose of 1000-ppm NBBS was selected based on a previous modified 1 generation study, in which decreased bodyweights, possibly indicating overt toxicity, were observed in rats treated developmentally with 2000-ppm NBBS (unpublished data). Litters were maintained with corresponding dams and culled to 4 females and 4 males per litter on PND 4. Feeding was continued until weaning of the F1 generation on PND 28, following which the F0 rats were euthanized. F1 rats were assigned to cohorts (Table 1) at weaning on study day (SD) 0 and switched to dose formulations prepared using the NTP-2000 base diet and remained on the same dose levels until euthanized at approximately 11–14 weeks of age. Vehicle controls received NIH-07 (F0 dams and litters) or NTP-2000 (F1 animals) meal diet from the same lots used for preparation of the respective dose formulations. An immunomodulatory positive control group was also included with rats receiving 15 mg/kg (5 mg/ml formulation) of CPS via IP injection for 4–14 days (administered 4 days before terminal sacrifice or daily starting on the day of immunization/infection; see Table 1) prior to scheduled euthanasia. Five (5) cohorts of female mice and F1 rats (female and male) were used in this comprehensive assessment of immune cells and functions (Table 1). Utilization of the adult female mice provided a comparator group for the purpose of discerning effects on the developing immune system from those that also occur following exposure to NBBS in adults.

Table 1.

Immunization/infection and endpoints examined for each adult mouse or F1 rat cohort

Cohort (N)a	Study Endpoints	Immunization/Infection
1 Mouse (8) F1 male rats (11–12) F1 female rats (12)	Food consumption Clinical observations Body and organ weights Hematology (CBC with differential) Immunopathology	N/A
2 Mouse (8) F1 male rats (12) F1 female rats (11–12)	Food consumption Clinical observations Body and organ weights Antibody response to SRBC	SRBC immunization: 7.5 × 107/mouse, SD 24 (4 days prior to terminal euthanasia) 1 × 108/F1 rat, 11–14 weeks of age (4 days prior to terminal euthanasia)
3 Mouse (8) F1 male rats (12) F1 female rats (12)	Food consumption Clinical observations Body and organ weights Antibody response to KLH—in life, 5 days following immunization (IgM) and terminal, 14 days following immunization (IgG)	KLH immunization: 300 µg/mouse, SD 14 (14 days prior to terminal euthanasia) 300 µg/F1 rat, 11–14 weeks of age (14 days prior to terminal euthanasia)
4 Mouse (8) F1 male rats (11–12) F1 female rats (12)	Food consumption Clinical observations Body and organ weights T-cell proliferation NK cell activity Immunophenotyping of the spleen	N/A
5 Mouse (7–8) F1 male rats (11–12) F1 female rats (12)	Food consumption Clinical observations Body and organ weights CTL response to influenza infection	Influenza infection: ∼4 × 104 PFU/mouse, SD 20 (8 days prior to terminal euthanasia) ∼2 × 105 PFU/F1 rat, 11–14 weeks of age (8 days prior to terminal euthanasia)

Abbreviations: N/A, not applicable; CBC, complete blood count; SRBC, sheep red blood cells; KLH, keyhole limpet hemocyanin; Ig, immunoglobulin; NK, natural killer; CTL, cytotoxic T-lymphocyte.

 ^a Intended group size was 8 mice and 12 rats for NBBS treatment groups and 8 mice and rats for CPS treatment groups. If the n is smaller, it was due to an unscheduled death. Refer to CEBS Summary Tables I01 and I02 for removal reasons for each cohort.

General toxicology

Cageside morbidity/mortality checks were performed at least twice daily. Detailed clinical observations were performed weekly for the duration of the studies. Bodyweights were recorded before NBBS administration and then at least weekly thereafter until scheduled study termination. Weekly bodyweights were collected on the same day of the week within each cohort. Feed consumption was determined weekly in mice and rats with the period corresponding to the collection of bodyweights. At necropsy, the liver, spleen, lungs, thymus, kidneys, and adrenal glands were weighed and then fixed in 10% neutral-buffered formalin (NBF). Additional organs preserved in 10% NBF included bone marrow (femur), gastrointestinal tract with Peyer’s patches (rats only), and mesenteric and popliteal lymph nodes (LN). Tissues were sectioned at 4–6 µm and stained with hematoxylin and eosin (H&E) for histopathological evaluations in the rat study. The lymphoid organs were evaluated using enhanced histopathology guidelines (Elmore, 2012); nonlymphoid organs were evaluated by traditional histopathology. All evaluations were conducted in accordance with the NTP Immunotoxicity Study Pathology Specifications (National Toxicology Program, 2016).

Hematology

At study termination, the animals in cohort 1 were anesthetized with CO₂ and a microhematocrit tube used for retro-orbital blood collection followed immediately by euthanasia with CO₂ inhalation overdose. The first drop or 2 of blood was discarded prior to collection into a K₂EDTA collection tube, and the blood was analyzed on the day of collection using an Advia 120 hematology analyzer running Advia 120 v6.3.2-MS software (Siemens Medical Solutions USA, Inc.). The following parameters were assessed: erythrocyte count, hematocrit, hemoglobin concentration, mean cell volume, mean cell hemoglobin, mean cell hemoglobin concentration, platelet count, reticulocyte count, and leukocyte count and differential.

Assessment of humoral immunity to T-dependent antigens

The T-dependent antibody response (TDAR) was stimulated by immunization with 2 antigens, sheep red blood cells (SRBC) and Keyhole Limpet Hemocyanin (KLH), and assessed using cellular functional assays as well as serum levels of antigen-specific antibodies. This approach provided a comprehensive assessment of the humoral immune response and provided overlap between rodent immunotoxicity studies that typically use SRBC and nonhuman primate immunotoxicity studies that typically use KLH as the antigen.

SRBC T-dependent antigen (cohort 2)

SRBC in Alsever’s Solution (Colorado Serum Company, Denver, CO) were washed 3 times in phosphate-buffered saline (PBS). Mice were intravenously immunized via the tail vein with 0.2 ml of a suspension containing 3.75 × 10⁸ SRBC/mL (7.5 × 10⁷ SRBC/mouse) on SD 24. F1 rats were intravenously immunized via the tail vein with 0.5 ml of an SRBC suspension containing 2 × 10⁸ SRBC/mL (1 × 10⁸ SRBC/rat). Four days after immunization, animals were euthanized with CO₂ and weighed. A maximum amount of blood was collected by cardiac puncture, serum isolated, and stored at ≤−70°C until evaluated for anti-SRBC IgM antibodies.

For the antibody-forming cell (AFC) response to SRBC, spleens were processed to single-cell suspensions in HBSS + HEPES and cell concentration and viability were determined. Spleen cells (1:30 and 1:120 dilution for mouse or 1:50 and 1:150 dilution for rat, in 100 µl volume) and SRBCs (25 µl of ∼50% suspension in HBSS) were added to 500 µl of molten agar media at 44 ± 1°C and mixed with Guinea pig complement (1/3 dilution of stock in HBSS with 0.1 ml of 50% SRBC suspension; Cedarlane Laboratories, Burlington, NC) in duplicate tubes. The resulting suspensions were poured onto the center of a petri dish in duplicate and covered with a cover glass. Once the agar solidified, the petri dish was placed in an incubator set to maintain 37°C for at least 3 h followed by visual enumeration of the AFC plaques. The number of AFC plaques was expressed per million spleen cells and per spleen.

The same spleen single-cell suspensions that were used for the AFC assay were used to determine the impact of NBBS exposure on B-cell production of IgM antibodies specific for SRBC using an ELISpot technique. Briefly, spleen cells were added to an ELISpot plate that was coated overnight with SRBC membranes (Life Diagnostics, St. Petersburg, FL). Spleen cells were incubated for approximately 3 h, following which, the cells were decanted, membranes washed and mouse or rat-specific HRP-labeled anti-IgM detection antibody (Jackson ImmunoResearch, West Grove, PA) added to the plate. The plate was incubated with detection antibody for approximately 72 (mouse) or 45 (rat) h followed by washing and addition of TMB (3,3′, 5,5″-tetramethylbenzidine) substrate for approximately 5 min. The plate was washed with deionized water to stop the color development followed by drying at room temperature in the dark. The spots/well were quantified using the CTL Immunospot S6 Macro Analyzer (using software CTL Switchboard v. 2.5.4.0, CTL ImmunoCapture v. 6.4.77.0 Series 5, CTL ImmunoSpot DC v. 5.1.33). Each spot was interpreted as corresponding to a single anti-SRBC IgM-producing cell. Both anti-SRBC IgM-producing cells/10⁶ spleen cells and per spleen were calculated.

Serum samples were stored at ≤−70°C until evaluated for anti-SRBC IgM antibody titers using a commercial ELISA technique (Life Diagnostics) as validated according to the manufacturer’s instructions. Following the addition of the stop solution, the optical density was determined spectrophotometrically at 450 nm using a Spectramax i3 spectrophotometer and Softmax Pro GxP v6 software (Molecular Devices, Sunnyvale, CA).

KLH T-dependent antigen (cohort 3)

KLH (Stellar Biotechnologies, Inc.) whole protein was used as a second T-dependent antigen. Mice or F1 rats from cohort 3 were immunized 14 days prior to the scheduled euthanasia with KLH via IP injection of 300 µg protein/animal. Serum from an in-life blood collection (venipuncture of the saphenous vein for mice and tail vein for rats) performed on day 5 following immunization was used to determine the primary anti-KLH IgM antibody response. Serum collected from the terminal blood collection, 14 days following immunization with KLH, was used to determine the isotype switching to anti-KLH IgG antibody production. Serum was stored at ≤−70°C until analyzed for anti-KLH IgM and IgG antibody titers using an ELISA procedure (Life Diagnostics) as validated and per the manufacturer’s instructions. Following the addition of the stop solution, the optical density was determined spectrophotometrically at 450 nm using a Spectramax i3 spectrophotometer and Softmax Pro GxP v6 software (Molecular Devices, Sunnyvale, CA).

Lung cytotoxic T-lymphocyte activity (cohort 5)

To stimulate an in vivo cell-mediated immune response, mice and F1 rats in cohort 5 were infected 8 days prior to scheduled termination (SD 20 for mice and PND 83–87 for rats) with influenza virus (∼4 × 10⁴ plaque-forming units [pfu]/mouse or ∼2 × 10⁵ pfu/rat) via intranasal instillation. Single-cell suspensions were isolated from the bronchoalveolar lavage fluid (BALF) of mice or from collagenase digested lung tissue (following Ficoll-Paque Plus isolation, GE Healthcare, Chicago, IL) of F1 rats. Adherent cells were removed by plating on plastic, and effector cells were evaluated for the impact of NBBS treatment on cytotoxic T-lymphocyte (CTL) killing activity toward target cells as described previously (Burleson et al., 2018). Briefly, effector cells from the BALF or lung of influenza-infected animals were combined with influenza-infected autologous target cells (EL-4 cells for mouse and UMR 106 cells for rat; ATCC, Manassas, VA) labeled with Chromium-51 (⁵¹Cr) at various effector cell:target cell (E:T) ratios in round-bottomed microtiter plates and incubated for 4 h. Release of ⁵¹Cr was measured using a gamma counter. Percent specific ⁵¹Cr release (CTL lysis) was calculated using the formula ([E − S]/[T − S]) × 100; where E is the ⁵¹Cr release from target cells in the presence of effector cells, S is the spontaneous release of ⁵¹Cr from target cells alone, and T is the maximum release of ⁵¹Cr from target cells in the presence of Triton X-100.

Anti-CD3-induced T-cell proliferation (cohort 4)

T-cell proliferation in response to ex vivo treatment with monoclonal anti-CD3 antibodies was determined in adult female mice and F1 rats following treatment with NBBS. Briefly, microtiter plates were coated overnight with anti-CD3 (100 µl/well of 1 µg/ml solution of Clone G4.18; BD Biosciences) and washed. Spleen cells in RPMI complete medium (containing 10% Fetal Bovine Serum and 1% penicillin/streptomycin; Gibco ThermoFisher Scientific, Grand Island, NY) were added to the appropriate wells and incubated at 37°C and 5% CO₂ for up to 96 h. Cell proliferation was determined using the Click-iT EdU Proliferation Assay (ThermoFisher Scientific, Waltham, MA) according to the manufacturer’s instructions. EdU incorporation into DNA during proliferation results in a fluorescence signal that is directly proportional to single-cell DNA content. Data were collected using an Accuri C6 flow cytometer and CFlow Plus software v1.0.264.21 (BD Biosciences).

Natural killer cell activity (cohort 4)

Spleen effector cells were separated from red blood cells and adherent cells and the single-cell suspensions were adjusted to achieve the desired effector-to-target ratios. Effector cells (100 µl) were added to wells of round-bottom microtiter plates containing 100 µl of YAC-1 target cells (1 × 10⁵ cells/ml; ATCC) labeled with ⁵¹Cr at 100 µCi per 1 × 10⁶ target cells for 90 min. Spontaneous-release (S) and total ⁵¹Cr release (T) controls were prepared separately by adding 100 µl of YAC-1 target cells to appropriate wells containing 100 µl of RPMI 1640 or Triton X-100 for spontaneous and total release, respectively. The plates were centrifuged at 250 × g for 5 min and then incubated at 37°C/5%CO₂ for 4 h. Culture supernatants were harvested and release of ⁵¹Cr was determined using a Cobra II Auto-Gamma counter (Packard). Percent specific ⁵¹Cr release (natural killer [NK] lysis) was calculated using the formula ([E − S]/[T − S]) × 100; where E is the ⁵¹Cr release from target cells in the presence of effector cells, S is the spontaneous release of ⁵¹Cr from target cells alone, and T is the maximum release of ⁵¹Cr from target cells in the presence of Triton X-100.

Immunophenotyping (cohort 4)

Spleen cells obtained from cohort 4 animals were subjected to ammonium chloride red blood cell lysis. The mononuclear cells (2.5 × 10⁵ cells) were suspended in 50 µl of stain buffer (1× PBS/2%Bovine Serum Albumin/0.1%NaN3) and were incubated for 5–30 min on ice in the presence of Fc Block (BioLegend, San Diego, CA). Following the blocking step, antibody (all BioLegend, San Diego, CA) cocktails containing antibodies to: (1) CD3, CD161a, CD45, and CD45RA; (2) CD8a, CD3, CD45, and CD4; or (3) for mouse CD11b/CD11c, Ly6G, and NKp46, or rat CD11b/c, CD103, RP-1, and CD161a were added to the appropriate tubes. Antibody staining titers were previously optimized for the experimental conditions and equipment used. Control tubes contained cells only, cells with a single antibody from the list above, or cells with a single isotype control antibody. The tubes were incubated on ice, protected from light, for 20–50 min. Following incubation, the samples were fixed using 2% paraformaldehyde for at least 30 min followed by centrifugation and resuspension in stain buffer. Samples were stored at 2–8°C, protected from light until analyzed. Sample acquisition was performed on an Accuri C6 flow cytometer using CFlow Plus v 1.0.264.15 (BD Biosciences, San Jose, CA).

Lymphocyte gating was performed on the CD45⁺ population. The following lymphocyte subsets were identified: T cells (CD3⁺CD45RA⁻), B cells (CD3⁻CD45RA⁺), NK cells (CD3⁻CD161a⁺), T-helper cells (CD3⁺CD4⁺), and cytotoxic T lymphocytes (CD3⁺CD8⁺).

Myeloid cells were gated based on being positive for CD11b with low to mid-intensity staining for CD11c (mouse) or CD103 (rats). Myeloid populations were differentiated from NK cells based on lack of NKp46 expression (mouse), or lack of CD161a (rats). Further differentiation was based on the expression of Ly6G (mouse) or RP-1 (rat) with positive cells being neutrophils and negative cells differentiated using side scatter into monocytes/macrophages with low granularity and eosinophils with high granularity.

Data collection and statistical analysis

Data were collected into Provantis v9.3.2.1 (Instem, Philadelphia, PA) and calculation of endpoints was performed within this validated electronic data collection and management system. Results are presented as mean ± SEM. Jonckheere’s test was used to test for dose-related trends (Jonckheere, 1954). Bodyweight and organ weight data, which typically exhibit a normal distribution, were analyzed using a parametric multiple comparison procedure. If a significant trend was detected at p ≤ .01, Williams’ test was used (Williams, 1986); if the trend was not significant Dunnett’s test was used (Dunnett, 1955). Data for other endpoints were analyzed using a nonparametric multiple comparison procedure. If a significant trend was observed, Shirley’s test was used (Shirley, 1977); if the trend was not significant, Dunn’s test was used (Dunn, 1964). Positive control group data were compared to the vehicle control group using the Kruskal-Wallis test. Data that were different from control at p ≤ .05 were considered statistically significant. Extreme values were identified by the outlier test of Dixon and Massey (1957). All flagged outliers were examined, and implausible values were eliminated from the final analyses.

Results

 

Summary findings pertinent for evaluating immunotoxicity are presented below. All study findings including results that are discussed in this section but not presented in the manuscript and individual animal data can be found in the NTP Chemical Effects in Biological Systems (CEBS) database (https://doi.org/10.22427/NTP-DATA-500-005-003-000-3).

Feed consumption

Feed consumption was collected to determine the impact of NBBS on palatability as well as to calculate NBBS intake. Food consumption in adult mice showed small increases in the higher-dosed treatment groups in some cohorts while others showed small decreases following NBBS exposure (CEBS Summary Table I06). There were no effects of NBBS treatment on feed consumption in F0 rats during gestation. Minor fluctuations in feed consumption occurred in F0 rats during late lactation between lactation days (LD) 14–28. Minor changes in feed consumption were observed in F1 offspring; however, it was variable between cohorts (CEBS Summary Table I06). Overall, these effects were minor and suggest that NBBS did not significantly affect feed palatability at the dose levels tested.

Chemical intake was calculated using feed consumption data and animal bodyweight. Based on these calculations, the average delivered doses over the study across all cohorts (SD 0–28) were 0, 84.7–86.7, 147.4–199.4, 290.9–385.9, 562.7–714.1, and 1108.9–1515.7 mg/kg NBBS, for mice treated with 0-, 313-, 625-, 1250-, 2500-, and 5000-ppm NBBS in the feed, respectively. The mean NBBS intake for dams during GD6-21 was 0, 17.0, 32.6, and 65.1 mg/kg for the vehicle, 250-, 500-, and 1000-ppm NBBS treatment groups, respectively. The mean NBBS intake for dams during LD 1–28 was higher than during gestation being 0, 56.0, 108.1, and 222.0 mg/kg in the vehicle, 250-, 500-, and 1000-ppm NBBS treatment groups, respectively. F1 offspring were weaned on PND 28 (SD 0) and provided the same dose levels of NBBS formulated in NTP-2000 meal feed. Generally, there was a small, but statistically significant, dose-dependent decrease in feed consumption in male and female F1 rats. The mean weekly ranges for NBBS intake from feed in male F1 rats were 0, 15.2–33.0, 30.2–69.1, and 62.0–137.0 mg/kg in the 0-, 250-, 500-, and 1000-ppm treatment groups, respectively. The mean ranges for NBBS intake from feed in female F1 rats were 0, 16.2–31.4, 33.6–65.8, and 72.6–133.3 mg/kg for the 0-, 250-, 500-, and 1000-ppm NBBS treatment groups, respectively. The ranges of chemical intake for mice and rats are based on the individual cohorts (cohort-specific chemical intake is provided in CEBS Summary Table I08).

F0 survival and reproductive outcomes

There were no NBBS treatment-related effects on survival in F0 rats as all treated dams survived to scheduled euthanasia following weaning (CEBS Summary Tables I01 and I02). There were no NBBS treatment-related effects on reproductive performance other than a small increase in gestational length in all NBBS treatment groups (CEBS Summary Table R02). Littering parameters, including pup number and survival, showed no remarkable treatment effects (CEBS Summary Table R03).

General toxicology findings

There were no consistent significant effects on bodyweights from NBBS treatment in mice, although minor changes occurred in bodyweight gains including a negative dose-response trend. There was evidence of temporal negative dose-response trends in bodyweights for several periods in F0 rats during gestation and lactation although none of the individual treatment groups were statistically significant. Bodyweight gains showed similar decreasing trends with increasing dose of NBBS during gestation that were statistically significant in F0 rats treated with ≥500-ppm NBBS for the overall treatment period. In contrast, bodyweight gain was unaffected during lactation in F0 rats (CEBS Summary Tables I04 and I04G).

Pup bodyweights were largely unaffected until PND 28 where a significant decrease was noted in both male (3.3%) and female (3.4%) rats from the 1000-ppm treatment group (CEBS Summary Table R19). Pup bodyweight gains were also decreased at PND 21–28 and 4–28 in the 1000-ppm treatment group (CEBS Summary Table R19G). F1 offspring were weaned and assigned to cohorts (Table 1) on PND 28 and provided the same dose levels of NBBS formulated in NTP-2000 meal feed. Bodyweights, particularly in male F1 rats, showed a negative dose-response trend with statistically significant decreases occurring in both the 500- and 1000-ppm treatment groups that generally remained within 10% of vehicle control levels (CEBS Summary Table I04; note that SD in CEBS Summary Tables I04 and I04G refer to days postweaning). Bodyweight gains also tended to be lower in F1 rats with the most pronounced effects occurring in males (CEBS Summary Table I04G).

Clinical observations were sporadic and did not appear to be related to NBBS treatment in mice, F0 rats during gestation or lactation, or F1 rats prior to or postweaning. Most of the clinical observations were head and facial alopecia in mice which is a common occurrence in this strain and age (CEBS Summary Tables I05 and I05P).

There were no gross or microscopic changes identified in lymphoid tissues (spleen, thymus, LN, bronchus-associated lymphoid tissue, bone marrow) or in nonlymphoid tissues examined (liver, lung, right kidney, right adrenal gland) in female mice. Examination of the popliteal LN in control and NBBS-treated F1 rats revealed the accumulation of pigmented material most likely originating from the tattoo application of the permanent ID on the tail. The prevalence and degree of pigment accumulation in the medullary cords and sinuses in the popliteal LN of male and female F1 rats decreased with increasing the dose of NBBS (Table 2 and Figs. 1A and B). There was a decreasing trend in pigment accumulation and an increased incidence of neutrophils in the same region of the popliteal LN (Figure 1C). The pigment was dark brown to black and granular and was mostly observed within the cytoplasm of cells in the LN (Figure 1B). Mice treated with NBBS did not show this effect as they were identified using ear tags rather than tail tattoos. There were several other gross and histopathological findings identified but these were considered sporadic, or background findings based on their low incidence, minimal severity, and/or similar incidence between control and treated groups (CEBS Summary Tables PA05, PA18, and PA46).

Table 2.

Pigment accumulation and neutrophil influx into the popliteal lymph nodes of F1 rats treated with N-butylbenzenesulfonamide (NBBS)

	NBBS (ppm in Feed)
	0 ppm	250 ppm	500 ppm	1000 ppm
No. rats	12	12	12	12
Female F1 rats
Pigment in popliteal LN
Minimal	2	3	5	2
Mild	7	6	3	1
Moderate	1	0	0	0
Marked	0	0	0	0
Total	10	9	8	3
Popliteal LN medulla, increased neutrophilsa
Minimal	0	0	0	5
Mild	0	0	0	3
Moderate	0	0	0	0
Total	0	0	0	8
Male F1 rats
Pigment in popliteal LN
Minimal	9	5	4	5
Mild	2	4	2	0
Moderate	0	0	0	0
Total	11	9	6	5

Abbreviation: LN, lymph node.

 ^a Only females showed increased neutrophils in the medullas of the popliteal lymph nodes.

Figure 1.

Popliteal lymph node histology in female F1 rats following developmental exposure to NBBS. A, H&E section of popliteal lymph node from a vehicle control female rat (227) showing scattered black pigment (arrows) and mast cells (arrowheads) within the medullary sinuses and cords. B, H&E section of popliteal lymph node from a female rat (333) treated with 1000-ppm NBBS. There is a lack of black pigment within the medullary sinuses and cords. C, H&E section of popliteal lymph node from a female rat (334) treated with 1000-ppm NBBS. The medullary cords and sinuses are filled with pale foamy macrophages and numerous neutrophils (arrows). Abbreviations: NBBS, N-butylbenzenesulfonamide; H&E, Hematoxylin and Eosin.

In unimmunized/uninfected mice, NBBS exposure resulted in a small decrease in spleen weights (5000-ppm treatment group) and increases in absolute (≥2500-ppm NBBS) and relative (≥625-ppm NBBS) liver weights and relative kidney weights (5000 ppm) compared to the vehicle control group. There was a dose-related increasing trend in absolute and relative kidney weights. There were no treatment-related effects on thymus or spleen weights in unimmunized/uninfected mice (cohorts 1 and 4); however, spleen weights from mice immunized with SRBC (cohort 2) and from mice infected with influenza (cohort 5) were significantly decreased in the 5000-ppm NBBS group relative to the vehicle control group. In unimmunized/uninfected rats, relative, but not absolute, liver and kidney weights were increased in both males (≥500 ppm) and females (1000 ppm). Absolute thymus weights were decreased in male F1 rats (≥500 ppm) immunized with SRBC (cohort 2) and relative thymus weights were increased in male F1 rats (≥500 ppm) infected with influenza (cohort 5), when compared to the respective control groups. Relative lung weights were increased in male F1 rats at all dose levels. The absolute lung weights were also increased in these animals, but the changes were not statistically significant. Therefore, decreased body weight in male F1 rats contributed to the increased relative lung weights. Lung weights were not significantly affected in female F1 rats. Overall, the organ weight changes in mice and rats were more evident in animals that received an immunization or infection suggesting the possibility that challenge of the immune system may influence the effects of NBBS. There were no histological changes that correlated to these differences in organ weight. Any other effects on organ weights were minor and not consistent between cohorts and sexes (CEBS Summary Table PA06).

Hematology

Compared to vehicle controls, treatment of mice with NBBS resulted in a mild increase in mean corpuscular volume (MCV) following treatment with ≥625-ppm NBBS and a decrease in mean corpuscular hemoglobin concentration (MCHC) in ≥2500-ppm NBBS treatment groups. In addition, there was an increasing trend observed in the hematocrit percent and a decreasing trend in MCHC. Although there were no remarkable changes in white blood cell differentials, there was a negative dose-response trend in large unstained cells that was significant in the 5000-ppm NBBS group. In male F1 rats, there were significant decreasing trends in hemoglobin, MCHC, and total leukocyte counts. In these rats, MCHC and total leukocyte counts were significantly decreased in the ≥500-ppm NBBS and ≥250-ppm NBBS treatment groups, respectively, relative to the vehicle-treated rats. The decrease in total leukocyte counts was attributable to decreases in lymphocyte and monocyte counts. In female F1 rats, neutrophil counts were increased in all NBBS treatment groups and showed a dose-response relationship. All other observed hematological and white blood cell changes were minimal or inconsistent and considered to be due to biological variability (CEBS Summary Tables M03 and M04).

Immune function

A comprehensive immune testing battery was employed with the goal of determining the impact of NBBS ingestion on humoral, cellular, and innate immunity. NBBS treatment caused a dose-related decrease in spleen weight and cellularity in immunized mice (Figure 2A). In addition, there was a significant decrease in the number of AFCs/10⁶ spleen cells (2500- and 5000-ppm NBBS treatment groups) and AFCs/total spleen cells (1250, 2500, 5000 ppm) relative to the vehicle control group (Figs. 2B and C). Like the effect of NBBS in mice, oral exposure in male F1 rats resulted in a decreasing trend in splenic cellularity, significant in the 500- and 1000-ppm NBBS treatment groups (Figure 2D). However, the AFC response to SRBC was unaffected by NBBS treatment in F1 male rats (Figs. 2E and F). Consistent with the effects in female mice, spleen cell numbers (Figure 2G) and the number of AFCs (Figs. 2H and I) were reduced in female F1 rats but only in the 1000-ppm NBBS treatment group (Figs. 2G–I). There were no NBBS treatment-related effects on serum antibody titers to SRBC, SRBC antibody-producing B cells measured by ELISpot or serum antibody titers to KLH in either mice or F1 rats. See CEBS Summary Tables (M07, M08, M09, and M19) for additional data on the effects of NBBS on humoral immunity.

Figure 2.

NBBS treatment suppresses the AFC response to SRBC. Data are expressed as (A) spleen cellularity in mice, (B) AFC per 10⁶ splenocytes in mice, (C) AFC per spleen in mice, (D) spleen cellularity in male F1 rats, (E) AFC per 10⁶ splenocytes in male F1 rats, (F) AFC per spleen in male F1 rats, (G) spleen cellularity in female F1 rats, (H) AFC per 10⁶ splenocytes in female F1 rats, and (I) AFC per spleen in female F1 rats. ^↓Indicates a significant negative trend with increasing NBBS dose (p < .05). Significantly different from the vehicle control group at *p < .05 or **p < .01. Abbreviations: AFC, antibody-forming cell; CPS, cyclophosphamide (50 mg/kg in mice or 15 mg/kg in F1 rats); NBBS, N-butylbenzenesulfonamide.

Cell-mediated immune function was assessed by determining the ability of CTL isolated from the lungs of influenza-infected mice and F1 rats to destroy influenza-infected autologous target cells (CEBS Summary Table M12). CTL activity in mice showed a positive dose-related trend that was significant at the 5:1 and 20:1 E:T ratios and was significantly different from the vehicle control in mice treated with ≥1250-ppm NBBS at the 5:1 E:T cell ratio (Table 3). Consistent with the increased CTL response, treatment with NBBS resulted in a significant increasing trend in T-cell proliferation index following ex vivo stimulation with anti-CD3 antibodies, although only the 625-ppm NBBS group was significantly increased relative to the vehicle control group (CEBS Summary Table M11). Excluding a small, but significant, increase in male F1 rats in the 250-ppm NBBS group, there were no treatment-related effects on CTL activity in either male or female F1 rats following developmental exposure to NBBS, although there was a positive dose-response trend in female rats (Table 3). NBBS treatment did not impact T-cell proliferation in F1 rats following stimulation with anti-CD3 antibodies (CEBS Summary Table M11).

Table 3.

Cytotoxic T-lymphocyte (CTL) activity in adult female mice and F1 rats exposed to N-butylbenzenesulfonamide (NBBS)

	NBBS (ppm in Feed)
	0	313		625	1250	2500	5000	50 mg/kg CPS
CTL activity in female mice
CTL activity (5:1)a	18.81 ± 3.41 (7)**	27.79 ± 3.36 (8)		24.54 ± 4.82 (7)	38.12 ± 2.36 (8)**	35.91 ± 7.51 (7)*	37.65 ± 4.37 (7)**	1.19 ± 0.98 (2)*
CTL activity (10:1)	38.12 ± 6.44 (7)	37.93 ± 3.20 (8)		41.37 ± 6.71 (7)	45.77 ± 2.28 (8)	46.05 ± 6.76 (7)	50.31 ± 3.91 (7)	NR
CTL activity (20:1)	51.50 ± 9.53 (6)*	52.54 ± 3.52 (8)		56.02 ± 12.48 (4)	60.97 ± 2.56 (7)	64.67 ± 6.17 (6)	66.33 ± 5.15 (7)	NR
	NBBS (ppm in Feed)
	0		250		500	1000	15 mg/kg CPS
CTL activity in F1 male rats
CTL activity (12.5:1)a	3.38 ± 0.33 (12)		5.15 ± 0.92 (11)		3.16 ± 0.50 (12)	3.69 ± 0.46 (12)	0.57 ± 0.15 (8)**
CTL activity (25:1)	7.77 ± 0.96 (12)		12.93 ± 1.74 (11)*		9.91 ± 1.44 (12)	8.89 ± 0.83 (12)	0.91 ± 0.17 (8)**
CTL activity (50:1)	26.33 ± 2.47 (12)		30.55 ± 2.64 (11)		30.67 ± 2.06 (12)	28.38 ± 2.38 (12)	1.15 ± 0.29 (8)**
CTL activity in F1 female rats
CTL activity (12.5:1)a	7.26 ± 0.53 (12)*		5.27 ± 0.43 (12)		7.81 ± 0.60 (12)	8.71 ± 0.72 (12)	2.63 ± 0.38 (7)**
CTL activity (25:1)	15.08 ± 1.21 (12)*		11.01 ± 0.92 (12)		16.73 ± 1.32 (12)	18.51 ± 1.76 (12)	3.23 ± 0.53 (7)**
CTL activity (50:1)	46.58 ± 3.24 (12)		40.17 ± 2.34 (12)		45.16 ± 2.97 (12)	49.99 ± 3.07 (12)	3.72 ± 0.68 (7)**

Data are displayed as mean ± SEM (N) unless otherwise noted. Data displayed as a mean of (effector:target cell ratio). Statistical analysis performed by Jonckheere (trend) and Shirley or Dunn (pairwise) tests. Statistical analysis for the positive control group compared to the vehicle control group was performed using the Kruskal-Wallis test. Statistical significance for the control group indicates a significant trend test. Statistical significance for a treatment group indicates a significant pairwise test compared to the vehicle control group. NR not recorded due to insufficient number of effector cells.

Abbreviation: CPS, cyclophosphamide.

^aCTL activity is expressed as % target cell killing calculated as (sample Cr⁵¹ release − spontaneous Cr⁵¹ release/total Cr⁵¹ release − spontaneous Cr⁵¹ release).

^*Statistically significant at p ≤ .05.

^**Statistically significant at p ≤ .01.

The impact of oral exposure to NBBS on innate immunity was investigated using an NK cell assay (CEBS Summary Table M15). In adult female mice, there was a significant positive trend in NK cell activity following NBBS exposure with a significant increase in almost all treatment groups when compared to vehicle controls (Figs. 3A–C). There was also a positive dose-response trend at all E:T cell ratios in male F1 rats but a statistically significant increase in NK activity only occurred in the 1000-ppm NBBS treatment group, relative to the vehicle control group (Figs. 3D–F). In contrast to mice and male F1 rats, NK-cell activity trended lower in female F1 rats following developmental exposure to NBBS, with a statistically significant decrease relative to the vehicle control group occurring in the 500-ppm treatment group at the 25:1 E:T cell ratio and in all NBBS-treated groups tested at a 12.5:1 E:T cell ratio (Figs. 3G–I).

Figure 3.

NBBS treatment modulated NK cell activity in mice and rats. NK cells isolated from the spleen were mixed at E:T ratios with YAC-1 target cells of (A) 6.25:1, (B) 12.5:1, and (C) 25:1 in female mice, (D) 12.5:1, (E) 25:1, and (F) 50:1 in male F1 rats, and (G) 12.5:1, (H) 25:1, and (I) 50:1 in female F1 rats. NK killing activity was expressed as % cytotoxicity of the total target cells. Indicates a significant ^↑positive or ^↓negative trend with increasing NBBS dose (p < .05). Significantly different from the vehicle control group at *p < .05 or **p < .01. Abbreviations: E:T, effector cell:target cell ratio; NBBS, N-butylbenzenesulfonamide; NK, natural killer.

Treatment-related changes in spleen cell immunophenotypes following NBBS exposure were unremarkable (CEBS Summary Table M06). No treatment-related effects in absolute spleen cell numbers of any cell phenotype occurred in mice, while male F1 rats revealed a small decrease in neutrophils counts in the 500- and 1000-ppm NBBS treatment group compared to vehicle controls. Treatment-related changes in absolute cell numbers in female F1 rats included a significant increase in eosinophils numbers and a positive dose-response trend in CD4⁺:CD8⁺ T cell ratios. When relative percentages were analyzed, some minor shifts in cell phenotypes occurred following NBBS exposure that were considered inconsequential. In mice, there was a significant decrease in the relative percentages of lymphocytes in the 2500- and 5000-ppm treatment groups relative to the vehicle control group. In addition, there was a decrease in the percentage of CD4⁺ T cells and monocytes/macrophages in the 5000-ppm NBBS group, and negative dose-response trends in the relative percentages of lymphocytes, CD4⁺ T cells, and monocytes/macrophages. In male F1 rats, there was a small decrease in the relative percentage of lymphocytes and neutrophils and an increase in eosinophils compared to the vehicle control group. Consistent with the increased CD4⁺:CD8⁺ T-cell ratios, the relative percentage of CD4⁺ T cells was increased while the percentage of CD8⁺ T cells was decreased in female F1 rats treated with 1000-ppm NBBS relative to the vehicle control group. Additionally, a negative dose-related trend in neutrophils and a positive dose-response trend in monocytes/macrophages and eosinophils were noted in female F1 rats. With the exception of the decreasing trend in NK cells, most changes were small and the biological relevance is not known.

Cyclophosphamide-positive control groups

The effects of the positive control, CPS, were consistent with previous studies from this laboratory with significant decreases observed in humoral and cell-mediated immune function tests that involve cell proliferation. The effect of CPS on NK cell activity was inconsistent as these cells do not proliferate and any observed change was usually associated with a change in the relative percentage of total spleen cells as observed previously (Watson et al., 2021).

Discussion

The prevalence of plastic-associated chemicals combined with their potential to elicit toxicity represents a significant public health concern (Groh, 2019). While much of the attention has focused on their endocrine-disrupting potential (Flaws et al., 2020), various plastic-associated chemicals have also been implicated in immune-related diseases (Nowak et al., 2019). The current studies were undertaken to characterize immunotoxicity associated with exposure to NBBS in adult female mice and developmentally exposed male and female rats. NBBS intake increased during the lactation period in rats which was likely a result of the increased consumption per cage once the F1 pups started consuming food around PND 12. The ingested dose levels were below the lowest observable effect level for dams from a previous short-term perinatal toxicity study (Rider et al., 2020). In F1 rats, bodyweight decreases in the 10% range occurred in the higher NBBS treatment groups (up to 1000-ppm NBBS). A decrease in pup bodyweights was also observed in a previous study, in which male and female pup bodyweights were up to 25% and 52% lower than the control group in the 2500- and 5000-ppm groups, respectively, through PND 25 (Rider et al., 2020).

Similar to observations in other toxicity studies of NBBS, albeit to a lesser degree, kidney, and liver weights were significantly elevated in female mice and male and female rats treated with NBBS, indicating that they could be potential target organs for NBBS toxicity at higher exposure concentrations (Marrocco et al., 2015; unpublished data). NBBS is excreted mainly in urine (Waidyanatha et al., 2020a), which could increase the likelihood of toxicity to the kidneys. However, significant increases were mainly confined to relative kidney weights, which unlike absolute kidney weight increases, are not correlated to kidney injury (Craig et al., 2015). NBBS is extensively metabolized in the liver by phase I and II enzymes to hydroxylated and glucuronidated metabolites (Waidyanatha et al., 2020a). Amacher et al. (2006) found that increases in liver weight below 20% can be associated with the initiation of metabolic processes without accompanying histopathological changes. Therefore, it is possible that metabolism in the liver accounts for increases in liver weight following NBBS exposure rather than cell injury and inflammation. This is consistent with the lack of histopathological changes in the liver in the current study. It was interesting that alterations in immune organ weights, namely spleen and thymus, were mainly impacted in animals that received either an immunization with antigen or an infection with virus. Immunization with SRBC resulted in an increase in spleen weight in vehicle-treated animals and the decrease in spleen weight observed in female mice and female F1 rats is consistent with the suppression of the anti-SRBC AFC response following NBBS exposure. NBBS exposure also decreased spleen weights in female mice infected with influenza which may reflect increased migration of CTL from the spleen to the lung and is consistent with the increased CTL activity in the lung of influenza-infected mice treated with NBBS.

Developmental exposure of female F1 rats impacted neutrophils in several compartments. There was an increased infiltration of neutrophils into the popliteal LN of female F1 rats from the 1000-ppm NBBS group, which correlated with lower accumulation of tattoo pigment. Previous investigations in mice demonstrated that tattoo ink migrated to skin-draining LN and was associated with infiltration of polymorphonuclear cells (Gopee et al., 2005), consistent with the results in the F1 rats. Female F1 rats also showed a dose-related increase in neutrophils in the blood and a decrease in the relative number of neutrophils in the spleen. These results suggest that NBBS may have an effect on mobilization and migration of neutrophils to sites of inflammation, in this case, the tattoo pigment-ladened popliteal LN.

Several mild hematological changes were observed in the mice and rats. In the female mice, the MCV was increased and paired with a decrease in MCHC. These changes indicate there was an increase in the average volume of the erythrocytes (increased MCV), which lead to an overall decrease of hemoglobin within the cells (decreased MCHC). The increase in the volume of the erythrocytes caused the observed increasing trend in the hematocrit relative to controls because larger cells occupy more space; that is, hematocrit is the percentage of blood volume filled by erythrocytes. A minimal decrease in MCHC was also observed in the male F1 rats, though MCV was unchanged. The changes in MCV may be a direct effect of NBBS on the erythrocytes or may indicate an effect on erythropoiesis. While the MCV and MCHC changes are biologically interesting, the erythrocyte count and hemoglobin concentration were unchanged and mild increases in MCV alone are not considered to be adverse. In male F1 rats, the lymphocyte and monocyte counts were decreased in several dose groups. As indicated previously, these decreases are consistent with mild toxicity and may even be mediated by increased secretion of endogenous corticosterone (Everds et al., 2013).

The AFC response to SRBC was inhibited in mice and female F1 rats, but not male F1 rats. Quantification of B cells producing anti-SRBC IgM by ELISpot also showed a decrease in female F1 rats when examined per spleen, although this effect was not statistically significant. It should be noted that there were significant decreasing trends in CD4⁺ T cells and monocyte/macrophage populations in female mice exposed to NBBS which could suggest that decreases in these populations may be responsible in part for the observed decrease in the AFC response to SRBC as these cell types are critical to the T-dependent antibody response. The sex difference observed in rats could be due to the toxicokinetic profile of NBBS, with female rats demonstrating higher oral bioavailability than male rats following exposure to NBBS (Waidyanatha et al., 2020b). In addition, the heightened sensitivity to immunotoxic insults in female rats could contribute to the observed sex differences. The observed decrease in the SRBC antibody response contrasts with a previous study conducted in B6C3F1 mice where no effect on the antibody response was noted (Marrocco et al., 2015). However, that study investigated the effects of dermal exposure to NBBS and therefore, it is not possible to directly compare effects with the present study due to toxicokinetic differences between exposure routes. The authors treated female mice by applying 25 µl of NBBS to the surface of the ear. While the different routes of exposure could result in toxicokinetic differences in exposure to NBBS, it is important to note that liver and kidney weights were significantly increased in that study (up to 28% and 64% increases in absolute kidney and liver weights, respectively) indicating adequate exposure to elicit a biological response. The lack of effects observed in other measures of humoral immunity in the current studies, including serum antibody titers, may be due to their lower sensitivity relative to the SRBC AFC assay (Temple et al., 1993) as well as timing of the assays. The SRBC endpoints were all conducted in the same cohort of animals and the timing was optimized for the AFC assay. The serum IgM is optimal 2 days after the AFC which likely impacted the sensitivity of this endpoint to NBBS.

Examination of cell-mediated immunity showed a general tendency for increased activity resulting from NBBS treatment in female mice and female F1 rats, but not male F1 rats, as evidenced by elevations in the CTL response. T-cell proliferation also showed a dose-related increasing trend in female mice. However, the effects on T cells were modest in nature and not always consistent. As the antibody response was suppressed in female mice and female F1 rats, it is possible that a shift in the Th1 and Th2 profiles was a contributing factor. In general, Th1 cytokines, such as IL-2 and IFN-γ, stimulate cell-mediated immune responses while Th2 cytokines, such as IL-4, IL-5, and IL-10, stimulate humoral immunity. In this respect, various treatments, such as terpenoids (Ku and Lin, 2013) and glucocorticoids (Elenkov, 2004), have been shown to influence the immune system by shifting the normal balance of Th1 and Th2 cytokines. Confirmation of a shift in Th1:Th2 responses, however, would require additional cytokine profiling which was not conducted in these studies.

NK cell activity is an innate immune function that plays critical roles in defense against infectious disease as well as tumor surveillance. Oral exposure to NBBS resulted in elevated NK activity in adult female mice and male F1 rats and a small decrease in NK activity in female F1 rats. As with the TDAR, the impact of NBBS on NK activity appears to be influenced by sex as well as period of exposure (i.e., developmental vs adulthood). Immunophenotyping studies indicated that these changes were not associated with absolute cell numbers and, in fact, the relative percentage of NK cells in mice was lower following NBBS exposure. The clinical consequences of deficient NK cell activity are well established and include fatal viral infections and increased susceptibility to latent viral infections such as cytomegalovirus, influenza virus, hepatitis C virus, and herpes viral infections, to name a few (Jost and Altfeld, 2013; Mace and Orange, 2019). In contrast to decreased NK cell activity, increased NK-cell activity can occur from short-term or even mild stress. NK cells express high levels of β-adrenergic receptors (Landmann, 1992), and their adherence to endothelium and migration into the blood are affected by catecholamine levels (Benschop et al., 1996). However, in the present study, NK activity was increased by NBBS at dose levels well below that which caused minimal evidence of general toxicity. NK cells can also be activated by compounds with stimulatory properties. Apart from interleukins, which belong to the best-characterized group of NK cell-stimulating compounds, vitamins and constituents extracted from plants also display the ability to activate NK cells (Grudzien and Rapak, 2018). Increased activity of NK cells could lead to detrimental cell damage and inflammation and contribute to worse outcomes for certain infections. As an example, NK cells are considered to play more of a pathogenic role than a protective role in chronic hepatitis B infection, likely owing to the preservation of cytolytic properties accompanied by a decay in capacity to produce anti-viral cytokines. At the same time, these NK cells tend to exert negative regulatory pressures on viral T-cell responses that further suppress anti-viral immunity (Fisicaro et al., 2019). Therefore, increased NK-cell activity could result in protective or detrimental effects on the immune response depending on the infectious agent.

The majority of effects from developmental exposure to NBBS in rats showed some level of sex dependency. Suppression of the SRBC TDAR was observed in female F1 rats but not males, measures of cell-mediated immunity tended to increase in female F1 rats but not males, and NK activity was increased in male F1 rats but decreased in females. NBBS has been shown to have antiandrogenic properties and has been suggested as a potential therapy for prostate cancer (Papaioannou et al., 2010). Therefore, it is possible that NBBS exposure may shift the estrogen to androgen balance resulting in higher relative estrogen levels. Previous work demonstrated that pretreatment of mice with estriol resulted in suppression of the delayed-type hypersensitivity response and the primary TDAR to SRBC (Ezaki et al., 1982). Estrogen receptor activation has also been shown to suppress NK killing of tumor targets (Curran et al., 2001). Therefore, antiandrogenic effects of NBBS may, in part, play a role in the sex differences observed for various immune functions, however, measurement of hormone levels would be required to support this.

Calculation of human equivalent doses can aid in the translation of findings from animal studies to human exposure scenarios. Human equivalent doses can be estimated based on allometric scaling by multiplying NBBS intake levels from each study (in this case we used the lowest intake value in the range) by the ratio of the model animal K_m factor (6 for rats, 3 for mice) to the human K_m factor (37) (Reagan-Shaw et al., 2008). Human equivalent doses ranged from 6.9 to 97.5 mg/kg for the adult mouse study, 2.5 to 10.1 mg/kg for the developmentally exposed male rats, and 2.6 to 11.8 mg/kg for the female rats. While we were not able to find human exposure levels in the literature, we anticipate that inclusion of NBBS in future biomonitoring efforts will allow for better understanding of the margin of exposure and aid in risk analysis.

In summary, the findings from the studies reported herein indicate that moderate immunotoxic effects on both innate and adaptive immunity can occur in adult mice and developmentally exposed rats following NBBS treatment. Under the conditions of this study, adult-exposed female mice and developmentally exposed female F1 rats showed compromised humoral immunity relative to male F1 rats, whereas innate immunity was increased in adult-exposed female mice and developmentally exposed male F1 rats but decreased in female F1 rats. To clarify the biological significance of the immune effects and their dependence on sex and timing, additional tests would be required to place the effects into the context of infectious and malignant diseases.

Declaration of conflicting interests

The authors report no real or perceived conflicts of interest. The authors alone are responsible for the content of this article.

Funding

Intramural Research Program of the National Institutes of Health; National Institute of Environmental Health Sciences; Intramural Research projects ES103379-01, ES103316, and ES103319; and National Institute of Environmental Health Sciences, National Institutes of Health, U.S. Department of Health and Human Services, contracts HHSN273201600011C (DHL, Durham, NC), HHSN273201000016C (Battelle Memorial Research Institute, Columbus, OH) and HHSN273201400017C (Burleson Research Technologies, Morrisville, NC).

Data availability

Supplementary data are available at https://doi.org/10.22427/NTP-DATA-500-005-003-000-3.