Peer Reviewers

• William J. Brock, Ph.D., DABT, Fellow ATS
  Owner
  Brock Scientific Consulting, LLC
  Montgomery Village, Maryland, USA
• Joshua Kellogg, Ph.D.
  Postdoctoral Research Fellow
  University of North Carolina at Greensboro
  Greensboro, North Carolina, USA
• Kristini K. Miles, Ph.D., DABT
  President
  Venture Chemical Consulting, LLC
  Atlanta, Georgia, USA

Crumb Rubber Handling and Characterization

OEHHA provided the crumb rubber used in these studies. The material, manufactured by either an ambient or a cryogenic manufacturing process, is fresh crumb rubber. Three lots were received in multiple 1-L glass jars. First, the material from one facility and one type of processing was combined to produce three lots, each approximately 5 kg. Subsequently, all material was combined into one lot (Lot No. CRM06092016). Physical and chemical characterization of this lot is described elsewhere.¹⁰

Previous studies were performed to determine the feasibility of exposing animals to crumb rubber by various routes of exposure. The material was size fractionated (mesh sizes 400, 80, 40, and 14) to optimize exposure via various routes.¹⁰ Following completion of the feasibility studies, the three larger particle size fractions (mesh sizes 80, 40, and 14) were combined, resulting in two fraction lots [Lot No. CRM12052016 (combined mesh; >170 µm) and Lot No. CRM09132016 (400 mesh; 37–170 µm)]; both lots were stored refrigerated. Chemical profiling of the two lots was conducted in comparison to the bulk material.¹⁰

For the current study, particle size was evaluated on both lots (Table 1). Size evaluation of Lot No. CRM12052016 (combined mesh) was determined by optical microscopy using an Olympus (Tokyo, Japan) SZK12 stereomicroscope. Particle size evaluation of Lot No. CRM09132016 (400 mesh) was determined by scanning electron microscopy (SEM) using a JEOL (Peabody, MA) JSM-7600F. These results are consistent with the estimated particle size ranges following sieving. Representative digital images of each lot are shown in Figure 1 and Figure 2.

Table 1

Crumb Rubber Particle Size Summary^a.

Figure 1

Image of Crumb Rubber Particles: Lot CRM09132016, 400 Mesh

Figure 2

Image of Crumb Rubber Particles: Lot CRM 12052016, Combined

Chemical analysis (metals, volatiles) of the combined and 400 mesh fractions was conducted and compared to the bulk material, and is summarized in Research Report 11 on the Chemical and Physical Characterization¹⁰ of the materials used in the NTP studies.

Formulations

Formulations of crumb rubber for the oral gavage study (Lot No. CRM09132016, 400 mesh) were prepared in corn oil at concentrations of 0 (corn oil only) and 125 mg/mL. Corn oil was purchased from Spectrum Chemical Manufacturing Corporation (New Brunswick, NJ; Lot No. 2FA0334). Corn oil was analyzed for peroxide content and levels were below 1.5 meq/kg. Gavage formulations were prepared weekly, stored under refrigeration (2 to 8°C), and used within 10 days after formulation. Prior to dosing, gavage formulations were allowed to come to ambient temperature and mixed for 15 minutes using a stir bar and stir plate; formulations were constantly stirred during the time of dose administration.

Formulations of crumb rubber for the dosed feed and mixed bedding studies (Lot No. CRM12052016, combined material) were prepared on a per-cage basis. For the dosed feed study, formulations were prepared at concentrations of 0 (feed only) or 50,000 ppm in irradiated NTP-2000 meal feed (Zeigler, Gardners, PA). Formulations for the mixed bedding study were prepared at concentrations of 0:100 or 50:50 (wt:wt) crumb rubber/bedding in irradiated hardwood bedding (Sani-Chips, P.J. Murphy Forest Products Corporation, Montville, NJ). Formulations for dosed feed and mixed bedding were prepared fresh on Days 0, 4, 7, and 11 by manually mixing until visually homogenous and then immediately presented to the animals.

Study Design

Several aspects of the study design, including sex/species and exposure concentrations, were selected based on logistical considerations relating to test article availability. Mice, rather than rats, were selected for the gavage studies to conserve test material. For the dosed feed and mixed bedding studies, female mice (can be group housed) were selected, instead of male mice (require individual housing) or rats (group housed except with larger cage area and feed requirements). For consistency across routes of exposure, female mice were selected for all studies. The dose for the gavage studies was selected based on availability of 400-mesh crumb rubber (Lot No. CRM09132016). For dosed feed, 50,000 ppm was selected as the exposure concentration as a limit dose; 50,000 ppm or 5% of the diet is considered the highest concentration, or limit dose, that should be used to retain adequate nutritional value of the feed. For the bedding studies, a 50:50 (wt:wt) bedding mixture was selected to provide a high environmental exposure, while still including absorbent bedding; this mixture extended the length of time between cage changes, from daily to twice weekly, conserving crumb rubber material.

The studies presented here were conducted at Battelle Memorial Institute (West Jefferson, OH). Animal care and use was in accordance with the U.S. Public Health Service policy on humane care and use of laboratory animals and the Guide for the Care and Use of Laboratory Animals.¹⁴ All animal studies were conducted in an animal facility accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International. Studies were approved by the Battelle Animal Care and Use Committee and conducted in accordance with all relevant National Institutes of Health (NIH) and NTP animal care and use policies, the Specifications for the Conduct of Studies to Evaluate the Toxic and Carcinogenic Potential of Chemical, Biological and Physical Agents in Laboratory Animals for the National Toxicology Program¹⁵, and applicable federal, state, and local regulations and guidelines.

Female B6C3F1/N mice were supplied from the NTP colony at Taconic Biosciences (Germantown, NY). Upon arrival, animals were quarantined for 8 days and provided ad libitum access to irradiated NTP-2000 wafer feed (Zeigler Brothers, Inc., Gardeners, PA) and tap water. Following quarantine, 8- to 9-week-old animals were randomized into one of six groups by body weight. Animals were assigned to the control or exposed group for each route of exposure: oral gavage, dosed feed, or mixed bedding. Animals were uniquely identified by tail tattoo upon randomization and housed five per cage. Housing conditions were maintained at temperatures of 72° ± 3° F and relative humidity of 50% ± 15%. Fluorescent lighting was controlled on an automatic light cycle of 12 hours on/off per day.

Groups of 15 female B6C3F1/N mice were exposed to crumb rubber by oral gavage (0 or 1250 mg/kg), dosed feed (0 or 50,000 ppm), or mixed bedding (0:100 or 50:50 wt:wt crumb rubber/bedding) for 14 consecutive days. Ten animals in each group were assigned to the core study and five were assigned to a biological sampling cohort; these group sizes were selected based on historical experience in detecting effect and interpreting data for the respective endpoints. Beginning on Day 0, animals in the oral gavage study were dosed with corn oil (vehicle, 0 mg/kg) or crumb rubber (1,250 mg/kg/day in corn oil); doses were calculated based on the animal’s most recent weight. Animals in the dosed feed study were given ad libitum access to NTP-2000 meal feed, containing 0 ppm crumb rubber, or 50,000 ppm crumb rubber; animals in the oral gavage and mixed bedding studies remained on NTP-2000 wafer feed. Animals in the mixed bedding study were placed on 0:100 or 50:50 wt:wt. crumb rubber/bedding. Animals were observed for moribundity/mortality and clinical observations twice daily. Body weights and food consumption were recorded at study start (Day 0), twice weekly, and at study termination.

On Day 6, animals in the biological sampling cohort were transferred to metabolism cages and individually housed for overnight urine collection. Feces were not saved or analyzed due to the likely presence of undigested crumb rubber particles. While in metabolism cages, animals in the dosed feed study (50,000-ppm group) were provided control meal feed to prevent contamination of collected samples. Following overnight urine collection, animals were returned to their home cages and continued exposure. On Day 13, immediately following lights on for bedding and dosed feed groups and 2 hours post-gavage for gavage groups, animals in the biological sampling cohort were anesthetized with 70%/30% CO₂/O₂, and blood was collected via cardiac puncture into tubes containing K₃EDTA. Blood was processed to collect plasma. Plasma and urine were analyzed as described below to assess internal exposure

On Day 14, blood was collected via the retro-orbital sinus from core study animals anesthetized with 70/30% CO₂/O₂ into tubes containing K₃EDTA. The following analyses were performed using an Advia 120 hematology analyzer (Siemens Medical; City, State): erythrocyte, reticulocyte, and platelet counts; total white blood cell count and differential; hematocrit; hemoglobin concentration; mean cell volume; and mean cell hemoglobin concentration. A manual hematocrit was performed with the use of a microcentrifuge and capillary reader. Blood smears were prepared and stained with a Wright-Giemsa stain and the morphology of erythrocytes, white blood cells and platelets evaluated; nRBCs were enumerated when present. Following blood collection, a gross necropsy then was performed on all core animals. Organ weights were recorded for liver, thymus, kidney, ovary, heart, and lungs. Tissues were collected for histopathology (adrenal glands, brain, clitoral glands, esophagus, eyes, femur-left, gallbladder, Harderian glands, heart and aorta, large and small intestine, kidneys, liver, lungs, lymph nodes, mammary gland, muscle, nerve, nose, oral cavity, pancreas, parathyroid glands, pituitary glands, salivary glands, spinal cord, spleen, stomach, thymus, thyroid gland, tongue, trachea, urinary bladder, uterus/cervix/vagina/ovaries, and Zymbal’s gland). All collected tissues were preserved in 10% neutral-buffered formalin, trimmed and processed, embedded in paraffin, sectioned at 5 µm and stained with hematoxylin and eosin. Bone marrow was flushed from the right femur using Hank’s Balanced Salt Solution that was supplemented with 0.15% K₃EDTA and 5% bovine serum albumin. A total nucleated cell count of the bone marrow single cell suspension was determined using an Advia 120 hematology analyzer. From the suspension, cytospin slide preparations were made for the cytological evaluation of the bone marrow. Included in this evaluation was the morphologic assessment of all the bone marrow cell lines, a differential cell count of 500 cells, and calculation of a myeloid to erythroid (M:E) ratio. Bone marrow cytology is not a standard endpoint evaluated in NTP studies, but was included here due to relevance to endpoints of concern in humans.

In-life data (body weights, food consumption, clinical observations), histopathology, hematology, and bone marrow cytology data were analyzed by first using the Levene test for homogeneity of variances and the Shapiro-Wilk test for normality of distributions. Assuming normal distribution, Dunnett’s pairwise test was used to determine a statistical difference between groups. If the Levene test or Shapiro-Wilk test failed, a Wilcoxon/Bonferroni-Holm test was run.

Assessment of Internal Exposure

Plasma samples (n = 4–5) and urine samples (n = 3–5) were each classified into six groups according to the exposure route and treatment group: oral gavage (0 or 1,250 mg/kg), dosed feed (0 or 50,000 ppm) or mixed bedding (bedding only or 50:50 wt:wt). All samples were vortexed 2 minutes and centrifuged for 2 minutes. Pooled samples were made for each of the six groups for plasma and urine by mixing equal volumes of individual animal samples from a given group and processed as described above. A total pool representing all groups, for each plasma and urine sample, was made by mixing volumes of each of the six respective pools. The total pool served as a measure of overall variability. Buffer was added to precipitate protein, and supernatant was dried down and reconstituted in 95:5% volume water:methanol.

Samples were vortexed and supernatant was analyzed using Waters Acquity Ultra Performance Liquid Chromatography (UPLC) and a Synapt G2-Si ESI-Q-TOF mass spectrometer. Chromatographic separation was accomplished on an Acquity HSST3 C18 column (2.1 × 100 mm, 1.8 µm) at 50°C using a gradient elution involving mobile phase A: 0.1% formic acid-water (v/v), and mobile phase B: 0.1% formic acid-methanol (v/v), using a flow rate of 400 µL/min. The injection volume was 5 µL. The gradient consisted of 99% A and 1% B for 1 minute, a linear gradient from 1% B to 99% at 16 minutes, hold at 99% B until 20 minutes, with a linear gradient to return to 99% A at 20.5 minutes.

Spectra of samples were imported into Progenesis QI software (Nonlinear Dynamics, UK) according to sample matrix, exposure route, and mode (positive or negative). Progenesis QI (Nonlinear Dynamics, UK) aligned samples to an alignment reference, and picked unique peaks observed for the appropriate project.

Differentiating compounds (p < 0.1, fold change >2) were determined from a data reduction method listed below, and the compounds flagged within Progenesis QI (Nonlinear Dynamics, UK). Identification of compounds was attempted using two approaches, untargeted and suspect screening. For the untargeted approach, compounds were searched against In-house Retention Time library, In-house Exogenous database, as well as the HMDB, T3DB, EPA ToxCast, and EPA DSSTox databases. Identifications were accepted for compounds based on their exact mass, isotope ratio, fragmentation, and retention time (if available). For the suspect screening approach, a special database was created to encompass compounds anticipated to be found in crumb rubber, based on analysis of the bulk material⁵ and other published information. Compounds were then search against this compiled database and identifications were accepted using the same criteria listed above.

Data were exported from Progenesis QI (Nonlinear Dynamics, UK) and imported into SIMCA 14.1 (Umetrics, Umeå, Sweden) to observe and identify any differentiating metabolites between treated and control groups. Unsupervised principal component analysis (PCA) was conducted on the datasets.

Data were exported from Progenesis QI (Nonlinear Dynamics, UK) for SAS analysis, and p-values calculated using an Exact Wilcoxon Rank Sum Test. Fold changes were calculated for each compound by comparing the median values of the treated and untreated group. In addition, where fold changes were not available for a compound (due to the presence of a near-zero median abundance in either the treated or untreated group), the median values of the non-zero group served as a “pseudo” fold change to capture changes.

Survival, Body Weights, and Clinical Observations

Crumb rubber exposure had no effect on survival, body weight, or clinical observations following oral gavage, dosed feed, or mixed bedding exposure. Body weights were within 2% of the respective control group throughout the study for each route of exposure (Figure 3, Figure 4, and Figure 5). Transient observations of soiled or ruffled coat were noted in some gavage animals (treated and control), and was likely related to the corn oil vehicle used in those animals, and was not considered to be related to crumb rubber exposure.

Figure 3

Body Weight; Oral Gavage Study

Figure 4

Body Weight; Dosed Feed Study

Figure 5

Body Weight; Mixed Bedding Study

Qualitatively, the feces from animals exposed to crumb rubber in feed and by oral gavage appeared darker in color (dark brown to black) compared to feces from control animals. In oral gavage animals, a few occurrences of dark feces were observed at several time points throughout the study. Feces from animals in the 50,000-ppm group appeared black on study Day 4. On subsequent observations, feces from these animals remained darker than those for bedding or gavage animals, but much lighter than Day 4 feces, suggesting an active avoidance of the crumb rubber within feed compared to early in the study. Feces color from animals in the mixed bedding study was unchanged.

Food and Material Consumption

Some statistically significant differences in food consumption were observed for each route of exposure, but none of these changes was considered biologically relevant. The differences observed were within an expected range of variability for this endpoint.

Quantifying the consumption of crumb rubber was attempted in the 50,000-ppm feed group. Traditionally in feed studies, the chemical of interest is adsorbed to feed particles and homogeneously distributed, such that chemical consumption can be calculated by changes in feed weight, before and after administration of dosed feed. Due to the larger size of crumb rubber particles compared to milled feed, animals could have actively avoided consuming the crumb rubber particles. Further, the crumb rubber did not adsorb to the feed, thereby making traditional methods to measure compound consumption impractical. To address the consumption issue, an attempt was made to separate the crumb rubber from feed post-administration and weigh the fractions separately. Prior to preparing feed formulations, aliquots of feed and crumb rubber were weighed separately and then when feeders were changed (Days 4, 7, 11), the contents were sieved to separate the feed and crumb rubber and re-weighed. Table 2 shows the weigh-in and weigh-back weights of feed and crumb rubber for the three cages in the 50,000-ppm group for each of the 3-day intervals that correspond with feeder changes. Because of fecal and bedding contamination of feeders, there were consistently inaccurate weigh-back weights for crumb rubber due to the size similarity of the feces, bedding, and crumb rubber. Based on this complication, quantitation of crumb rubber consumption was deemed infeasible. Qualitatively, based on fecal observations described above, animals in the 50,000-ppm feed group appeared to consume some crumb rubber; however, the change in fecal color from Days 0–4 and later in the study suggests an active avoidance of the crumb rubber particles.

Table 2

Consumption of Crumb Rubber in 50,000 ppm Dosed Feed Group.

Assessment of Internal Exposure

Based on an unsupervised PCA, of the plasma and urine data for each study matrix tested, separation between control and treated groups was not apparent for any route of exposure or matrix (Figure 6, Figure 7, and Figure 8).

Figure 6

Principal Component Analysis of Plasma and Urine Untargeted Data; Oral Gavage Study

Figure 7

Principal Component Analysis of Plasma and Urine Untargeted Data; Dosed Feed Study

Figure 8

Principal Component Analysis of Plasma and Urine Untargeted Data; Mixed Bedding Study

The data for all matrices, exposure routes, and ionization modes (positive ion or negative ion), were subsequently analyzed (post processing in Progenesis) in SAS to calculate fold changes and p-values for well-detected features determined by Progenesis.

To initially filter data, a p-value <0.1 and fold change >2 (or <−2) were used to investigate differences between treated and control groups (https://doi.org/10.22427/NTP-DATA-RR-14). Following an initial screen, the data were filtered further using more strict criteria (p < 0.05, fold change >5) and features with a negative fold change (higher in controls than in treated) were not included. The focus of these studies was to identify potential constituents from the crumb rubber material in plasma and urine, rather than interpreting disruption of endogenous processes. For both the untargeted and suspect screening searches described below, database matching used exact mass and isotope ratio for M = 1, M + 2, etc., fragmentation (which for many databases are predicted fragments), and retention time. For each identified feature, other structures are possible; therefore 100% confidence in identity was not possible as standard reference samples were not used to confirm identification. In the hierarchy of identification for untargeted methods and unknowns, different confidence levels can be assigned depending on the extent of the match.¹⁶

Using an untargeted search against several databases listed above, a summary of the number of features with a p-value <0.05 and a fold change of >5 are shown in Table 3 for all matrices, exposure routes, and analysis modes. The crumb rubber gavage group had the highest number of differentially identified features compared to other routes, and more features were identified in plasma compared to urine for all routes of exposure. One feature was identified in plasma for all three routes of exposure with a p-value <0.05 and a fold change >600; this feature was tentatively identified as 6-acetamido-2-naphthalenesulfonic acid but was not confirmed with an authentic standard. No common features were identified in urine across exposure routes.

Table 3

Untargeted Analysis^a.

For the suspect screening approach, a database was constructed based on predicted mass, isotope ratios and authentic standards for compounds known to be found in crumb rubber. Very few of the features identified had fold changes >5, and only one had p < 0.05. In the gavage group, 1,2-Dihydro-2,2,4-trimethylquinoline (CAS 147-47-4) was detected higher (12.6 fold, p = 0.008) in crumb rubber-exposed animals compared to controls. Several other compounds were detected with p = 0.057 with high fold changes in crumb rubber gavage animals compared to controls, including 2(3H)-benzothiazolethione (CAS 149-30-4; fold change 4,631.2) and neodecanoic acid (CAS 26896-20-8; fold change 3,259.4). These three compounds were all detected in the bulk crumb rubber material.¹⁰ Using the suspect screening approach, there were no compounds identified in the feed or bedding groups that had p < 0.05 and fold change >5.

Organ Weights

Organ weights for animals in the oral gavage study did not significantly change. Animals in the 50,000-ppm dosed feed group had significantly lower mean thymus (−10%) and ovary (−32%) weights compared to controls. Animals in the 50:50 wt:wt mixed bedding group had slightly higher, statistically significant, relative liver weight (6%) compared to controls. These weight changes were not associated with any microscopic finding.

Hematology and Bone Marrow Cytology

Hematology values in the crumb rubber oral gavage study did not change relative to the control values. The neutrophil count was significantly lower in the 50,000-ppm dosed feed group. In the 50:50 wt:wt mixed bedding group, hematocrit was significantly lower, and the white blood cell and lymphocyte count significantly higher. These changes, however, were mild and not considered treatment related.

The M:E ratio of the bone marrow was unchanged in the dosed feed and oral gavage studies. The M:E ratio of the 50:50 wt:wt mixed bedding group was significantly higher but was not considered a treatment-related change, in part because the control group’s M:E ratio was substantially lower than would be normally expected (0.8-2.8 with an average of 1.5).¹⁷

Histopathology

There were no gross observations at necropsy and no histopathologic lesions were observed in animals exposed to crumb rubber via dosed feed (50,000 ppm) or mixed bedding (50:50 wt:wt).

Animals dosed with crumb rubber by oral gavage (1,250 mg/kg/day) for 14 days had occurrences of esophageal inflammation in the control (4/10) and dosed (6/10) groups, however the incidence and average severity (minimal) of this lesion was similar between control and crumb rubber-exposed groups. This lesion was not observed in other groups that were not gavage dosed.