Forward and reverse dosimetry for aniline and 2,6-dimethylaniline in humans extrapolated from humanized-liver mouse data using simplified physiologically based pharmacokinetic models

Abstract

Although human urinary aniline and 2,6-dimethylaniline were unexpectedly detected in biomonitoring data, little is known about the daily intake doses of aniline and 2,6-dimethylaniline in the living environment or their relation to tolerable daily intake (TDI) values in humans. In the current study, to evaluate the daily oral intake of aniline and 2,6-dimethylaniline in humans, forward and reverse dosimetry was carried out using simplified in silico physiologically based pharmacokinetic (PBPK) modeling established using in vivo experimental pharmacokinetic data. These data were from humanized-liver mice after single oral doses of 100 mg/kg aniline (previously determined) and 116 mg/kg 2,6-dimethylanine (currently investigated). The in vivo elimination rates of 2,6-dimethylaniline from plasma in humanized-liver mice were generally slow compared with those of aniline. Faster in vitro metabolic elimination rates of aniline mediated by liver 9000 × g supernatant fractions from rats than those from humans may suggest the existence of higher first-pass effects in rats than in humanized-liver mice. In silico aniline and 2,6-dimethylaniline concentration curves in human urine after virtual oral administrations were estimated by human PBPK models created with data from humanized-liver mice. Reverse dosimetry analysis using human PBPK models estimated the daily intake of aniline, based on reported human urinary concentrations in biomonitoring data, to be roughly similar to the aniline TDI level. These results suggest that forward and reverse dosimetry using simplified human PBPK models founded on data from humanized-liver mice can be used to evaluate possible higher than expected exposures of aniline and 2,6-dimethylaniline in humans.

INTRODUCTION

Aromantic amines, such as aniline and 2,6-dimethylaniline, are often used as industrial intermediates and in the production of textile dyes, cosmetics, and several pharmaceutical drugs (Pauluhn, 2004; Bugosen et al., 2020; Stabbert et al., 2003; Chinthakindi and Kannan, 2021, 2022b, 2022a). Aniline itself is produced globally on a large scale (e.g., 8.4 million tons in 2020) (Chinthakindi and Kannan, 2022a; Bugosen et al., 2020), although it reportedly has carcinogenic potential in humans. Nonetheless, the tolerable daily intake (TDI) value of aniline has been set by the United States Environmental Protection Agency at 7.0 µg/kg/day (Chinthakindi and Kannan, 2022b). Aniline and 2,6-dimethylaniline are both metabolized to hematotoxic N-hydroxylated metabolites and excretable glucuronide conjugates of their amino groups (Miura et al., 2021a; Lewalter and Korallus, 1985; Gonçalves et al., 2001). Aromatic amines, including aniline and 2,6-dimethylaniline, have been detected in indoor air, cigarette smoke, and indoor dust collected in several countries (Chinthakindi and Kannan, 2021; Stabbert et al., 2003; Zhang et al., 2017; Palmiotto et al., 2001; Chinthakindi and Kannan, 2022b, 2022a). These aromatic amines can migrate from food contact materials such as containers, packaging, cutlery, and kitchen equipment (Chinthakindi and Kannan, 2022a; Perez et al., 2019). Among several aromatic amines, aniline and 2,6-dimethylaniline have been commonly detected in urine samples from humans (Chinthakindi and Kannan, 2022b), dogs, and cats (Chinthakindi and Kannan, 2022a) and in feces from dogs and cats (Chinthakindi and Kannan, 2022a).

Physiologically based pharmacokinetic (PBPK) modeling is a generally recognized method for estimating toxicokinetics (Wambaugh et al., 2015, 2018; Sayre et al., 2020). Simplified PBPK models constructed using chemical receptor, metabolizing, excreting, and central compartments have been developed for a range of substances and species (Miura et al., 2021a, 2020b, 2021b). The elimination rate of 2,6-dimethylaniline from rat plasma was slower than that of aniline, and we previously demonstrated that differences in the pharmacokinetics of aniline and its dimethyl derivatives in rats estimated by PBPK modeling were related to the existence of a methyl group at the C₂-position (Miura et al., 2021a). However, it is not known whether the slow elimination of dimethyl derivatives compared with that of aniline itself would also be seen in humans. Humanized-liver mice generally have an advantage in investigating liver metabolism and urinary/bile excretion as model animals for humans (Miura et al., 2019a, 2021b).

The purpose of the current study was to evaluate the daily oral intake of aniline and 2,6-dimethylaniline in humans by reverse dosimetry based on reported human urinary concentrations from biomonitoring data. Using our simplified PBPK models, the daily oral doses estimated on the basis of reported human urinary concentrations were comparable to the TDI level for aniline.

MATERIALS AND METHODS

Chemicals, materials, and animals

Aniline (CAS no. 62-53-3) and 2,6-dimethylaniline (CAS no. 87-62-7) were purchased from Tokyo Chemical Industry (Tokyo, Japan). Pooled liver microsomes from male Sprague−Dawley rats (8-week-old) were produced as described previously (Kamiya et al., 2022b; Miura et al., 2020a). Pooled mouse and pooled human liver microsomes were obtained from Corning Life Sciences (Woburn, MA, USA). Pooled rat and pooled human liver 9000 × g supernatant fractions (S9) and intestinal microsomes from male CD-1 mice (11-week-old), male Sprague−Dawley rats (8-week-old), and humans were obtained from Xenotech (Kansas City, KS, USA). An enhanced TK-NOG mouse (NOG-TKm30, Central Institute for Experimental Animals, Kawasaki, Japan) (Uehara et al., 2021) was utilized in the current study under the approval of the Animal Ethics Committee of the Central Institute for Experimental Animals (Permit Number: 20060A). Humanized-liver NOG-TKm30 mice (females weighing ~20–30 g) underwent single oral administrations of 2,6-dimethylaniline (116 mg/kg) based on the reported lowest observed effect levels of dimethylaniline derivatives (Kamiya et al., 2022a). Pharmacokinetic data for aniline (100 mg/kg) were taken from our previous study using male humanized-liver TK-NOG mice (Kamiya et al., 2022a).

In vivo and in vitro metabolic studies of aniline and 2,6-dimethylaniline

Blood samples (~30 µL) were taken at intervals in the period 0.5–24 hr following single oral doses of 2,6-dimethylaniline (116 mg/kg) given by gavage to five humanized-liver mice. Plasma samples (~20 µL) were deproteinized by adding 40 µL of methanol and then centrifuged at 20,000 × g for 15 min at 4°C.

The in vitro metabolic elimination rates of aniline and 2,6-dimethylaniline mediated by liver or intestinal microsomes or S9 fractions from mice, rats, and humans were determined for 30 min at 37°C. Typical incubation mixtures contained liver or intestinal microsomes or S9 fractions (0.50 mg protein/mL), aniline or 2,6-dimethylaniline (10 µM), an NADPH-generating system and/or 1.0 mM acetyl Co-A in a final volume of 0.10 mL of potassium phosphate buffer (100 mM, pH 7.4).

To measure the concentrations of aniline and 2,6-dimethylaniline, supernatant samples (10–20 µL) were analyzed using a liquid chromatography system with an analytical C₁₈ reversed-phase column (5 µm, 4.6 × 150 mm, Mightysil RP-18 GP 2, Kanto Chemicals, Tokyo, Japan) consisting of a multiwavelength UV detector and pump (Shimazu, Kyoto, Japan). 2,6-Dimethylaniline was eluted with 30% CH₃CN at 35°C at a flow rate of 1.0 mL/min and monitored at a wavelength of 254 nm with retention times of 11.1 min. For aniline determination, 20% CH₃CN was used as the eluent with a retention time of 6.5 min.

Statistical analyses of in vitro aniline and 2,6-dimethylaniline elimination rates by liver S9 fractions from rats and humans were carried out using GraphPad Prism (GraphPad Prism Software, La Jolla, CA, USA).

PBPK modeling of aniline and 2,6-dimethylaniline

Simplified PBPK models comprising gut, liver, kidney, and central compartments were established for aniline and 2,6-dimethylaniline (Miura et al., 2021b, 2020b, 2021c, Kamiya et al., 2022a, 2021). The values used in this study for the hepatic/renal volumes and the hepatic/renal blood flow rates (Q_h/Q_r), respectively, were 0.85 mL, 0.34 mL, and 0.160 L/hr in mice and 8.5 mL, 3.7 mL, and 0.853 L/hr in rats (Miura et al., 2021b, 2021a). Urine volumes of mice and humans were 1.0 mL/day and 1.5 L/day, respectively. Initial values for the fraction absorbed × intestinal availability (F_a·F_g), the hepatic clearance (CL_h), and the renal clearance (CL_r) for PBPK modeling were obtained from the elimination constants derived using one-compartment models (Miura et al., 2020b, 2019a).

The values of the plasma unbound fraction (f_u,p), octanol–water partition coefficient (logP), blood-to-plasma concentration ratio (R_b), and the liver-to-plasma and kidney-to-plasma concentration ratios (K_p,h and K_p,r) for aniline and 2,6-dimethylaniline were taken from our previous study (Miura et al., 2021a). The PBPK model input parameters for aniline and 2,6-dimethylaniline shown in Table 1, i.e., the absorption rate constants (k_a), the volumes of the systemic circulation (V₁), and the hepatic intrinsic clearances (CL_h,int), were generated utilizing a nonlinear least squares algorithm (simplex and modified Marquardt methods) to make the model results consistent with the experimental plasma substrate concentrations measured in this and our previous study (Miura et al., 2021b). The general ratios of CL_h to CL_r were set at 9:1 (Kamiya et al., 2020, 2019). F_g values were estimated from the gut extraction ratios as one-tenth of the hepatic extraction ratios in the hepatic well-stirred model. The resulting differential equations were solved to carry out the modeling of substrate concentrations:

where X_g, V_h, V_r, C_h, C_r, C_u, and C_b are the amount of compound in the gut; the liver and kidney volumes; and the hepatic, renal, urinary, and blood substrate concentrations, respectively.

To establish simplified human PBPK models founded on humanized-mouse or rat models, the k_a, V₁, and CL_h,int values for humans, as shown in Table 1, were estimated using scale-up strategies from humanized-liver mice or rats to humans. A multiplicative factor of 0.744 was applied to the rodent k_a value to generate the human k_a value. V_1,human was obtained using a scale-up strategy based on body weights using known values of the human liver volume (V_h,human, 1.50 L) and blood volume (V_b,human, 4.90 L):

where V_ss is the steady-state volume of distribution. The expression corresponds to the ratio of f_u,p to the tissue fraction unbound. The in vivo hepatic intrinsic clearance (CL_h,int) in humans was estimated based on that of humanized liver-mice, with no application of interspecies factors in vitro, as previously described (Miura et al., 2021b, 2019a, 2019b). CL_r was generated using rodent body weights of 0.025 kg (mouse) and 0.25 kg (rat) and a human body weight of 70 kg as follows (Kamiya et al., 2020):

In vitro hepatic intrinsic clearance (CL_{h,int,in vitro}) values were calculated from aniline and 2,6-dimethylaniline elimination rates, and the unbound fractions for aniline and 2,6-dimethylaniline in liver or intestinal microsomes (0.95 and 0.86, respectively) were determined using a commercial software (Simcyp, Certara, Sheffield, UK; Jamei et al., 2009), by extrapolation, using the following values: 40 mg microsomal protein per 1 g organ and 10 g liver weight per 0.25 kg of rat bodyweight or 1.5 kg liver weight per 70 kg of human bodyweight (Takano et al., 2010).

Table 1. Experimental parameters for PBPK models of aniline and 2,6-dimethylaniline.

Parameter	Abbreviation (unit)	Chemical	Humanized-liver mouse			Human (parameters from humanized-liver mouse) by scale-up	Human (parameters from ratb) by scale-up
Fraction absorbed × intestinal availability	Fa·Fg	Aniline	1			1	0.824
		2,6-Dimethylaniline	0.442			0.442	0.292
Absorption rate constant	ka (1/hr)	Aniline	3.62	±	2.62a	2.69	14.0
		2,6-Dimethylaniline	4.09	±	1.47a	3.04	5.22
Volume of systemic circulation	V1_substrate (L)	Aniline	0.0407	±	0.0199a	116	113
		2,6-Dimethylaniline	0.469	±	0.061a	1316	105
	V1_substrate (L/kg)	Aniline	1.63			1.66	1.61
		2,6-Dimethylaniline	18.8			18.8	1.50
Hepatic intrinsic clearance	CLh,int_substrate (L/hr)	Aniline	0.0922	±	0.0221a	92.2	515
		2,6-Dimethylaniline	0.0509	±	0.0222a	50.9	68.1
Hepatic clearance	CLh,_substrate (L/hr)	Aniline	0.0379			32.8	71.7
		2,6-Dimethylaniline	0.0147			13.8	17.6
Renal clearance	CLr,_substrate (L/hr)	Aniline	0.0040			0.811	2.69
		2,6-Dimethylaniline	0.0015			0.303	0.542

^aData are means ± standard deviations by fitting to measured concentrations. ^bPhysiological parameters of aniline and 2,6-dimethylaniline were taken from our previous study (Kamiya et al., 2022a; Miura et al., 2021a). Parameters such as K_p,h, R_b, and f_u,p were assumed to be the same for humans and for rodents. The bioavailabilities of aniline and 2,6-dimethylaniline were 0.8 and 0.4.

RESULTS

In vivo and in vitro metabolic studies of aniline and 2,6-dimethylaniline

In this study, humanized-liver mice were treated with single oral administrations of 2,6-dimethylaniline (116 mg/kg). Pharmacokinetic data for aniline in humanized-liver mice were taken from our previous study (Kamiya et al., 2022a). The elimination of 2,6-dimethylaniline from plasma was generally slow in humanized-liver mice compared with that of aniline (Fig. 1). The maximum concentration (C_max) values in plasma for aniline and 2,6-dimethylaniline in humanized-liver mice were 26 and 2.4 µg/mL, respectively, and the areas under the curve (AUC)_{0–4 hr} and AUC_{0–7 hr} were 45 and 12 µg·hr/mL, respectively. The plasma C_max of aniline in the humanized-liver mouse (Kamiya et al., 2022a) was one order of magnitude greater than that measured in the rat in our previous study (2.6 µg/mL) (Miura et al., 2021a). To investigate these differences, the elimination rates of aniline and 2,6-dimethylaniline mediated in vitro by pooled liver or intestinal microsomes or S9 fractions from mice, rats, and humans were investigated (Table 2). When microsome fractions from intestines and livers were used as enzyme sources for mice, rats, and humans, there were no apparent differences in elimination rates. In contrast, the elimination rate of aniline mediated by rat liver S9 fractions was significantly higher than that by S9 fractions from humans (p < 0.05), suggesting possible extensive conjugating potential of rat livers to aniline.

Fig. 1

Plasma concentrations of aniline (triangles) and 2,6-dimethylaniline (circles) in humanized-liver mice after single oral doses. Mean observed plasma concentrations of aniline and 2,6-dimethylaniline are shown with standard deviations (bars) for four or five mice. The pharmacokinetic data for aniline were taken from our previous study (Kamiya et al., 2022a). Dashed and solid lines show the PBPK model results for aniline and 2,6-dimethylaniline, respectively, after single virtual oral doses of 100 and 116 mg/kg.

Table 2. In vitro aniline and 2,6-dimethylaniline elimination rates by liver/intestinal microsomes and S9 fractions from mice, rats, and humans.

Species	Chemical	Metabolic
		Intestine				Liver
		Microsomal fractions				Microsomal fractions			S9 fractions
Mouse	Aniline	0.082	±	0.039		0.36	±	0.01	Not determined
	2,6-Dimethylaniline	0.059	±	0.010		0.20	±	0.01	Not determined
Rat	Aniline	0.15	±	0.12		0.29	±	0.03	0.45	±	0.02*
	2,6-Dimethylaniline	0.069	±	0.038		0.26	±	0.02	0.066	±	0.012
Human	Aniline	0.17	±	0.04		0.33	±	0.01	0.097	±	0.028
	2,6-Dimethylaniline	0.076	±	0.015		0.14	±	0.01	0.072	±	0.008

Aniline and 2,6-dimethylaniline (10 µM) were incubated with liver/intestine microsomes or S9 fractions (0.50 mg/mL) at 37°C for 30 min in triplicate determinations. The NADPH generating system was added to both liver/intestine microsomes and S9 fractions, but acetyl-CoA was fortified to the liver S9 fractions. Data are means and standard deviations.

*Significantly different from those in human liver S9 fractions (p < 0.05).

The set of equations that constitute the PBPK models were solved to generated virtual plasma concentration curves of aniline and 2,6-dimethylaniline for humanized-liver mice. The resulting in silico concentration curves are shown in Fig. 1. These PBPK-generated curves were consistent with the measured in vivo plasma data plots. Because the pharmacokinetic input parameters for PBPK models for aniline and 2,6-dimethylaniline in humanized-liver mice were calculated to achieve the best fit to experimentally established blood substrate concentrations (Table 1), the calculated C_max and AUC_0-4 or AUC_0-7 values of aniline (30 µg/mL and 46 µg·hr/mL) and 2,6-dimethylaniline (2.6 µg/mL and 16 µg·hr/mL) in plasma extrapolated using the PBPK models were consistent (within roughly twofold) with the observed mean values (26 µg/mL and 45 µg·hr/mL; and 2.4 µg/mL and 12 µg·hr/mL) in humanized-liver mice (Fig. 1).

Using simplified PBPK models to estimate the pharmacokinetics of repeated doses of aniline and 2,6-dimethylaniline in humans

The first human PBPK models for aniline and 2,6-dimethylaniline were established on the basis of the humanized-liver mouse PBPK models by utilizing allometric scaling methods without the application of interspecies factors between in vitro liver clearances. The second human PBPK models for aniline and 2,6-dimethylaniline were set up based on the rat PBPK models also by using allometric scaling methods without interspecies factors between in vitro liver clearances in rats and humans (Table 2). Pharmacokinetic data and pharmacokinetic input parameters for aniline and 2,6-dimethylaniline in rats were taken from our previous study (Miura et al., 2021a). The urinary concentrations of aniline and 2,6-dimethylaniline over 28 days (Fig. 2) were computed using human PBPK models based on the humanized-liver mouse PBPK models for virtual oral administrations of aniline and 2,6-dimethylaniline. Unlike the case of 2,6-dimethylaniline (Fig. 2C and 2D), no accumulation of urinary excretions of aniline was seen (Fig. 2A and 2B), because of the rapid clearance of aniline from the human blood. The virtual oral doses used in this 28-day modeling were generated by reverse dosimetry based on the 50^th and 95^th percentile urinary levels from biomonitoring data.

Fig. 2

Human urinary concentrations (solid lines) of aniline (A and B) and 2,6-dimethylaniline (C and D) after repeated virtual oral doses for 28 days. The doses of aniline and 2,6-dimethylaniline were calculated by reverse dosimetry to obtain the daily intake (A, 11; B, 26; C, 8.3; and D, 29 µg/kg/day, respectively) that corresponded to the reported 50th (A and C) and 95th (B and D) percentiles of human urinary biomonitoring data (dashed lines) (A, 6.0; B, 13.7; C, 3.8; D, 13.3 ng/mL, respectively) (Chinthakindi and Kannan, 2022b). The human PBPK model input parameters were obtained by scale-up from humanized-liver mice data, as shown in Table 1.

Reverse dosimetry analyses using the two human PBPK models to estimate the daily intake of aniline and 2,6-dimethylaniline based on reported human urinary concentrations of aniline and 2,6-dimethylaniline from biomonitoring data (Chinthakindi and Kannan, 2022b) are shown in Table 3. The repeated dose levels of aniline (11 and 26 µg/kg/day) and 2,6-dimethylaniline (8.3 and 29 µg/kg/day) were reversely calculated using the 50th and 95th percentiles of human urinary concentration levels of aniline (6.0 and 13.7 ng/mL) and 2,6-dimethylaniline (3.8 and 13.3 ng/mL) published in a biomonitoring report (Chinthakindi and Kannan, 2022b) (Table 3). Modeling repeated daily administrations of aniline and 2,6-dimethylaniline indicated that exposures of aniline and 2,6-dimethylaniline should be apparent in humans (Fig. 2).

Table 3. Reverse dosimetry analyses using human PBPK models for aniline and 2,6-dimethylaniline to determine the daily intake after repeated virtual oral administrations for 28 days based on the reported human urinary 50th and 95th percentile concentrations of aniline and 2,6-dimethylaniline in biomonitoring data.

Analyte in urine	Reported biomonitoringa, 50th and 95th (range) concentrations in urine, ng/mL	Chemical	Estimated daily doses by reverse dosimetry, 50th and 95th percentile doses, µg/kg/day		Tolerable daily intakea, µg/kg/day
			Human model (from humanized-liver mouse)by scale-up	Human model (from rat) by scale-up
Aniline	6.0 and 13.7 (1.2–51)	Aniline	11 and 26	24 and 54	7.0
2,6-Dimethylaniline	3.8 and 13.3 (0.1–77)	2,6-Dimethylaniline	8.3 and 29	8.3 and 29	Not available

^aReported values taken from the literature (Chinthakindi and Kannan, 2022b).

DISCUSSION

In the current study, to evaluate the daily oral intake of aniline and 2,6-dimethylaniline in humans based on urinary biomonitoring data, reverse dosimetry was carried out using simplified in silico PBPK modeling. The PBPK models were based on in vivo and in vitro experimental pharmacokinetic data from humanized-liver mice and rats obtained either previously or in the present study after single oral administrations of 100 and 116 mg/kg of aniline and 2,6-dimethylaniline, respectively (Miura et al., 2021a; Kamiya et al., 2022a). The plasma C_max of aniline (26 µg/mL) in humanized-liver mice(Kamiya et al., 2022a) (Fig. 1) was one order of magnitude higher than that (2.6 µg/mL) in rats observed in our previous study (Miura et al., 2021a). This finding may be partly accounted for by the faster aniline elimination rates by rat liver S9 fractions than those from humans, as one of the possible factors (Table 2).

A previous study by Chinthakindi and the associate (Chinthakindi and Kannan, 2022b) using human body weights suggested that the estimated daily intake of aniline and 2,6-dimethylaniline in humans may be several orders of magnitude below the TDI value (7.0 µg/kg/day). However, the reverse dosimetry approach applied here (based on the reported human urinary concentrations from biomonitoring data) using the current human PBPK models yielded estimated daily oral doses in the range 8.3–54 µg/kg/day, i.e., similar to or greater than the TDI level for aniline, as shown in Table 3. Although there is no TDI value for 2,6-dimethylaniline to the best of our knowledge, the exposure levels for 2,6-dimethylaniline estimated here might be close to tolerable values. The reason for the apparent disagreement between the current estimate and the previous reported low estimate of aniline exposure calculated based on human urine excretion rates per body weight (Chinthakindi and Kannan, 2022b) is not clear at present. However, the current volume of systemic circulation (V₁) values for aniline and 2,6-dimethylaniline were ~1.6 L/kg (Table 1), which is higher than the simple body weights. Despite these discrepancies, the human PBPK models developed using a scale-up strategy based on parameters from both humanized-liver mice and rats gave similar output values in this study (Table 3).

In conclusion, forward and reverse dosimetry using simplified human PBPK models in combination with data from humanized-liver mice has indicated possible higher than expected exposures of aniline and 2,6-dimethylaniline in humans. Human PBPK modeling in combination with data from humanized-liver mice has the potential to predict and evaluate biomonitoring strategies for human exposure of aromatic amines, such as aniline and 2,6-dimethylaniline.

ACKNOWLEDGMENTS

This work was supported in part by the Japan Chemical Industry Association Long-range Research Initiative Program. TM was partly supported by the Japan Society for the Promotion of Science Grants-in-Aid for Young Scientists 202021210. The authors thank Drs. Norie Murayama, Koichiro Adachi, Tasuku Sato, and Masayoshi Utsumi for their assistance and David Smallbones for copyediting a draft of this article.

Conflict of interest

The authors declare that there is no conflict of interest.

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