Integration of mechanistic and pharmacokinetic information to derive oral reference dose and margin-of-exposure values for hexavalent chromium

Abstract

The current US Environmental Protection Agency (EPA) reference dose (RfD) for oral exposure to chromium, 0.003 mg kg^-1 day^-1 , is based on a no-observable-adverse-effect-level from a 1958 bioassay of rats exposed to ≤25 ppm hexavalent chromium [Cr(VI)] in drinking water. EPA characterizes the confidence in this RfD as "low." A more recent cancer bioassay indicates that Cr(VI) in drinking water is carcinogenic to mice at ≥30 ppm. To assess whether the existing RfD is health protective, neoplastic and non-neoplastic lesions from the 2 year cancer bioassay were modeled in a three-step process. First, a rodent physiological-based pharmacokinetic (PBPK) model was used to estimate internal dose metrics relevant to each lesion. Second, benchmark dose modeling was conducted on each lesion using the internal dose metrics. Third, a human PBPK model was used to estimate the daily mg kg^-1 dose that would produce the same internal dose metric in both normal and susceptible humans. Mechanistic research into the mode of action for Cr(VI)-induced intestinal tumors in mice supports a threshold mechanism involving intestinal wounding and chronic regenerative hyperplasia. As such, an RfD was developed using incidence data for the precursor lesion diffuse epithelial hyperplasia. This RfD was compared to RfDs for other non-cancer endpoints; all RfD values ranged 0.003-0.02 mg kg^-1 day^-1 . The lowest of these values is identical to EPA's existing RfD value. Although the RfD value remains 0.003 mg kg^-1 day^-1 , the confidence is greatly improved due to the use of a 2-year bioassay, mechanistic data, PBPK models and benchmark dose modeling.

Keywords: benchmark dose (BMD) modeling; hexavalent chromium Cr(VI); margin of exposure (MOE); mode of action; reference dose (RfD); risk assessment.

Figure 1

Depiction of flux values estimated with the physiological‐based pharmacokinetic model. (1) pyloric flux (mg Cr(VI) kg⁻¹ total SI day⁻¹) used for interspecies extrapolation. (2–4) SI segment flux (mg Cr(VI) kg⁻¹ SI segment day⁻¹) used for dose–response modeling of SI effects. (5) portal flux (mg Cr(V) kg⁻¹ bodyweight day⁻¹) used for interspecies extrapolation; calculated as the mass of Cr(VI) transferred from all three SI sections, per kg bodyweight. SI, small intestine [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 2

Dose–response modeling of oral tumor incidence in F344 rats. Tumor incidence in males and females combined (P = 0.78). (inset) tumor incidence in females only (P = 0.75). Both curves relflect predictions of the multistage model. Data adapted from NTP (2008). BMD, benchmark dose; BMDL, benchmark dose (with corresponding 95% lower confidence limit) [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 3

Dose–response modeling of intestinal tumor incidence in B6C3F1 mice. Combined incidence of adenomas and carcinomas in male and female mice in the duodenum, jejunum and ileum (multistage model; P = 0.2644). BMDL₁₀ values are shown in Table 3. Data adapted from NTP (2008). BMD, benchmark dose; BMDL, benchmark dose (with corresponding 95% lower confidence limit); SI, small intestine [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 4

Dose–response modeling of the diffuse epithelial hyperplasia incidence in B6C3F1 mice. (A) Combined diffuse epithelial hyperplasia incidence in the duodenum and ileum of male and female mice (Hill; P = 0.0679). (B) Diffuse epithelial hyperplasia after dropping the three highest dose groups in the response plateau (Hill and log‐logistic; P = 0.0986). (C) Diffuse epithelial hyperplasia after omitting single dose at 7.6 SI flux units (Hill; P = 0.1992). All three models result in BMD₁₀ and BMDL₁₀ values of 2.1 and 1.7 SI flux units (mg Cr(VI) l⁻¹ SI) (Table 3). Incidence data are adapted from NTP (2008). BMD, benchmark dose; BMDL, benchmark dose (with corresponding 95% lower confidence limit); SI, small intestine [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 5

Dose and temporal concordance of intestinal diffuse epithelial hyperplasia, adenomas and carcinomas in mice. (A) Dose concordance (based on Cr(VI) SI flux): As evidenced by multistage models from the US EPA's benchmark dose software v.2.6 for three endpoints using data from male and female mice. Incidence data are adapted from NTP (2008). Tumor responses are based on all three intestinal sections from male and female mice. DEH responses are based on duodenum and ileum data from male and female mice. These models are not used for quantitative dose–response analysis, but rather serve to compare visually the responses progressing from non‐neoplastic hyperplasia to adenomas and carcinomas. (B) Temporal concordance (7 days of exposure): As evidenced by crypt hyperplasia in female mice in the absence of neoplastic lesions. Bars represent incidence in hematoxylin and eosin‐stained sections (Thompson, Proctor, et al., 2011). Note: The short bars indicate empirical observations with 0% incidence. Blue line represents mean ± SD for counted cells in a second study (Thompson, Wolf, et al., 2015). (C) Temporal concordance (90 days of exposure): As evidenced by crypt hyperplasia in female mice in the absence of neoplastic lesions. Bars represent incidence in hematoxylin and eosin‐stained sections (Thompson, Proctor, et al., 2011). Note: The short bars indicate empirical observations with 0% incidence. Blue line represents mean ± SD for counted cells (O'Brien et al., 2013). DEH, diffuse epithelial hyperplasia; SI, small intestine

Figure 6

Dose–response modeling of the systemic effects. (A) BMD plot of incidence of chronic liver inflammation in female rats (P = 0.64). (B) BMD plot of incidence of histiocytic cellular infiltration into the liver of female mice (P = 0.25). (C) BMD plot of cytoplasmic alteration of the acinus pancreas in female mice (P = 0.13). BMDL₁₀ values are shown in Table 4. Incidence data are adapted from NTP (2008). BMD, benchmark dose; BMDL, benchmark dose (with corresponding 95% lower confidence limit) [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 7

Basis for EF_HK. Bimodal distribution for baseline gastric pH in humans. Data adapted from Ayazi et al. (2009)

Figure 8

Candidate RfD array. Open and closed circles represent female and male mice, respectively. Half‐filled circles represent male and female data modeled together. Open triangles represent female rat data. Red and black symbols are based on benchmark response values of 5% and 10% extra risk, respectively. Cyto. Alt., cytoplasmic alteration; D, duodenum; DEH, diffuse epithelial hyperplasia; HCI, histiocytic cellular infiltration; Inflamm., inflammation; RfD, reference dose; SI, small intestine