Phenolic benzotriazoles: a class comparison of toxicokinetics of ultraviolet-light absorbers in male rats

Abstract

Phenolic benzotriazoles are ultraviolet-light absorbers used in a variety of industrial and consumer applications. We investigated the toxicokinetic behavior of 9 compounds, covering unsubstituted, monosubstituted, disubstituted, and trisubstituted compounds, following a single gavage (30 and 300 mg/kg) and intravenous (IV) (2.25 mg/kg) administration.

Following IV administration, no distinct pattern in plasma elimination was observed for the compounds with half-lives ranging from 15.4–84.8 h. Systemic exposure parameters, maximum concentration (C_max) and area under the concentration time curve (AUC), generally increased with the degree of substitution.

Following gavage administration, C_max and AUC of unsubstituted compound were lower compared to the substituted compounds. C_max and AUC increased ≤7-fold with a 10-fold increase in the dose except for the AUC of the unsubstituted compound where the increase was 30-fold. Plasma elimination half-lives for the class ranged from 1.57 to 192 h with the exception of 30 mg/kg drometrizole.

Oral bioavailability was low with ~ 6% estimated for unsubstituted compound and 12.8–23% for others at 30 mg/kg dose. Bioavailability was lower following administration of the higher dose.

Taken collectively, these data point to low oral absorption of phenolic benzotriazoles. The absorption decreased with increasing dose. Substituted compounds may be less metabolized compared to the unsubstituted.

Introduction

Phenolic benzotriazoles are a class of ultraviolet (UV)-light absorbers and are used within products to increase stability of them to light. The main use of these compounds is as industrial additives for polymers and light-stabilized coatings (NTP, 2011; Cantwell et al., 2015). Other applications include use in sunscreens, cosmetics, products, and fragrances. The annual production volumes of the compounds in the class vary and generally range from < 500,000 to 1,000,000 pounds according to U.S. EPA’s Chemical Data Reporting (https://chemview.epa.gov/chemview). The compounds in this class are environmentally persistent and have been detected in a variety of samples including indoor dust, raw sewage and sewage treatment plant effluents, landfills, and marine sediments (NTP, 2011; Cantwell et al., 2015). For example, in the United States, drometrizole levels in wastewater from chemical plants were 0.5–7 ppm and in sediments were 2–670 ppm (Reddy et al., 2000). In addition, due to the high lipophilicity (log K_ow 4–9) of the compounds in this class, there is potential for bioaccumulation in organisms (NTP, 2011; Brandt et al., 2016). Studies have reported detection of these compounds in wildlife and seafood (Nakata et al., 2009b; Nakata et al., 2009a; Kim et al., 2011; Brandt et al., 2016). Hence, there is potential for human exposure to these compounds via oral, dermal or inhalation routes in occupational settings or from consumer products and consumption of contaminated marine organisms.

Despite the potential widespread human exposure, comprehensive toxicity data for phenolic benzotriazole class are limited. The oral and dermal LD₅₀ values, respectively, were ≥1000 mg/kg and >2000 mg/kg in rodents depending on the compound (Thomas et al., 1995; NTP, 2011). Short-term and subchronic oral exposure studies of phenolic benzotriazoles have consistently shown liver as a target (Thomas et al., 1995; Hirata-Koizumi et al., 2007; Ema et al., 2008; Hirata-Koizumi et al., 2008a; Hirata-Koizumi et al., 2009). Chronic exposure to drometrizole via feed (mouse, 5–500 ppm; rat 100–3000 ppm) and 2-(2H-benzotriazol-2-yl)-4-tert-butylphenol (ditBu-BZT) following gavage administration (rat 0.1–12.5 mg/kg) reported liver effects (CIR, 2008; Hirata-Koizumi et al., 2008b; NTP, 2011). At ≤500 ppm drometrizole exposure in mice for 24 months, treatment related benign and malignant liver tumors were observed (CIR, 2008; NTP, 2011). Following exposure of rats to ditBu-BZT for 13 and 52 weeks, enlarged livers accompanied by histopathological changes were noted. Increased relative organ weights (e.g., brain and testes) and hematological effects were also observed after ditBu-BZT treatment at the highest dose in rats (Hirata-Koizumi et al., 2008b).

Due to the limited data available for the class to evaluate the potential adverse effects on human health, the National Toxicology Program (NTP) has initiated toxicity studies in rats following gavage administration (30–1000 mg/kg) (Testing Status). Nine compounds were selected for testing based on exposure, potential for accumulation, production volume, structural differences, and/or potential toxicity. The compounds selected represent 1 unsubstituted, 3 monosubstituted, 3 disubstituted including an ester, and 2 trisubstituted phenolic benzotriazoles. Compound names and abbreviations used are shown in Figure 1.

Figure 1.

Structures of phenolic benzotriazoles

Toxicokinetic (TK) data are important to interpret toxicity data. However, there is a paucity of TK data for this class. The only report we identified in the literature investigated the TK of 2 esters, 3-(2H-benzotriazol-2-yl)-5-(1,1-dimethylethyl)-4- hydroxybenzenepropanoic acid, methyl ester (tBuPrMeEst-BZT) and 3-(2H-benzotriazol-2-yl)-5-(1,1-dimethylethyl)-4-hydroxybenzenepropanoic acid, 1,6- hexanediyl ester (tBuPrHexEst-BZT) in male rats (n=2) following a single 10 mg/kg dose of [¹⁴C]-labelled compound (Thomas et al., 1995). The maximum blood concentration (C_max) for tBuPrMeEst-BZT (<2 μg/g) was achieved between 1–2 h. The apparent half-life was less than 12 h. Although the reported C_max of tBuPrHexEst-BZT was lower (<0.13 μg/g) than tBuPrMeEst-BZT, the half-life (~12 h) was similar. Hydrolysis to the corresponding acid played a major role in the metabolism of these esters and was the main compound in blood with the parent constituting only a minor fraction. tBuPrMeEst-BZT was metabolized to the acid by rat serum, liver homogenate, and small intestine homogenate with metabolism in the small intestine reported to be less efficient than in the liver (Thomas et al., 1995).

The present studies were undertaken to generate TK data in male rats for compounds being tested by the NTP following gavage administration, which together provide an assessment of various substituted phenolic benzotriazoles. Doses of 30 and 300 mg/kg were selected representing a low and mid dose used in these toxicology studies (Testing Status). Male rats were used in the toxicity studies based on the indications in the literature that they were more sensitive compared to females (Hirata-Koizumi et al., 2009). Studies following a single intravenous (IV) administration of 2.25 mg/kg were also conducted to aid in the interpretation of oral data and to estimate oral bioavailability of compounds.

Materials and methods

Chemicals and reagents.

Phenolic benzotriazoles were procured from multiple sources as given in Table 1. Chemical identity was confirmed using nuclear magnetic resonance spectroscopy and mass spectrometry. The purity of the lots was determined using high performance liquid chromatography (HPLC) with UV detection at 332 nm and corresponding data are shown in Table 1. Determined purity values were ≥98 % and agreed with the values given in the manufacturer’s Certificate of Analysis. 3-(2H-benzotriazol-2-yl)-5(1,1-dimethylethyl)-4-hydrobenzenepropanoic acid (tBuPrA-BZT, Lot No. GC-08152011) was procured from Adesis (New Castle, DE). Sprague Dawley rat plasma (blood collected using potassium ethylenediaminetetracetic acid (K₃EDTA) as an anticoagulant) to be used as a matrix to prepare calibration standards and quality control (QC) samples was obtained from BioIVT (Westbury, NY). Phenomenex Strata impact 96-well protein precipitation plate (Torrance, CA). All other reagents were obtained from commercial sources.

Table 1.

Chemical information of phenolic benzotriazoles.

Chemical Name	Abbreviation	CASRN	Supplier	Lot Number	CoA Purity (%)	Determined purity (%)
2-(2H-Benzotriazol-2-yl)phenol	P-BZT	10096–91-0	Richman Chemical, Lower Gwynedd, PA	373PAL021	> 99	≥99.5
2-(2H-Benzotriazol-2-yl)-4-methylphenol	Drometrizole	2440–22-4	Gojira Fine Chemicals, LLC, Bedford Heights, OH	120935	99.2	≥99.9
2-(2H-Benzotriazol-2-yl)-4-tert-butylphenol	tBu-BZT	3147–76-0	TCI America, Tokyo, Japan	IDRWA	99.6	≥99.9
2-(2H-Benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol	Octrizole	3147–75-9	Gojira Fine Chemicals, LLC, Bedford Heights, OH	120932	99.0	≥98.4
2-(2H-Benzotriazol-2-yl)-4,6-bis(1,1-dimethylpropyl)phenol	ditPe-BZT	26973–555-1	Gojira Fine Chemicals, LLC, Bedford Heights, OH	120933	99.2	≥99.8
2-(2H-Benzotriazol-2-yl)-4,6-bis(1-methyl-1-phenylethyl)phenol	diMeEtPh-BZT	70321–86-7	Gojira Fine Chemicals, LLC, Bedford Heights, OH	120934	99.2	≥99.6
3-(2H-Benzotriazol-2-yl)-5-(1,1-dimethylethyl)-4-hydroxybenzenepropanoic acid, octyl ester	tBuPrOcEst-BZT	84268–23-5	Richman Chemical, Lower Gwynedd, PA	354PAL67	98.4	≥96.6
2-(5-chloro-2H-1,2,3-benzotriazol-2-yl)-6-(1,1-dimethylethyl)-4-methylphenol	Bumetrizole	3896–11-5	Gojira Fine Chemicals, LLC, Bedford Heights, OH	120936	99.1	≥99.5
2-(5-Chloro-2H-benzotriazol-2-yl)-4,6-bis(1,1-dimethylethyl)phenol	ditBuCl-BZT	3864–99-1	Gojira Fine Chemicals, LLC, Bedford Heights, OH	120937	99.2	≥99.5

Animals and animal maintenance.

Studies were conducted at Battelle (West Jefferson, OH) and were approved by the Institutional Animal Care and Use Committee. Animals were housed in facilities that are fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International. Animal procedures were in accordance with the “Guide for the Care and Use of Laboratory Animals” (Council, 2011). Animal studies were conducted in compliance with the Food and Drug Administration Good Laboratory Practice Regulation.

Male Hsd:Sprague Dawley® SD® rats (9 weeks old at receipt) were obtained from Envigo (Indianapolis, IN). All animals were surgically implanted with a catheter placed in the carotid artery for blood collection prior to receipt. Rats for IV administration were also surgically implanted with a catheter placed in the jugular vein for dose administration. Animals were quarantined for at least for 2 d before being used in a study and were randomized into dosing groups using Provantis (Version 8.6.1.2, Instem, Stone, UK). Rats were individually housed in solid bottom polycarbonate cages suspended on stainless steel racks with Sani-Chips® hard wood bedding (P.J. Murphy Forest Products Corp., Montville, NJ) and had ad libitum access to certified, irradiated NTP 2000 feed (Ziegler Bros, Inc., Gardners, PA) and city (West Jefferson, OH) tap water. Water was analyzed annually per Battelle’s standard operating protocol. No known contaminants that would interfere with the study were found in feed or water. During the quarantine and study periods, room temperature was maintained between 69 to 75 °F and relative humidity was maintained within 35 to 65%. A 12-h light/dark cycle was maintained during the quarantine and study period. Animals body weights were 267–310 g at the time of study initiation.

Study design and dose administration.

Study design is given in Supplemental Table1. Gavage dose formulations of compounds at concentrations of 6 and 60 mg/mL were prepared in 0.5% methyl cellulose. IV dose formulations at 1.5 mg/mL were prepared in water:Cremophor:ethanol (6:3:1). To determine formulation concentration, aliquots were diluted and analyzed using a validated HPLC method with UV detection at 332 nm (linear range, 0.008 to 0.24 mg/mL; r ≥0.99; RSD ≤5%; RE ≤±10%). All formulations were within 10% of target concentration. Prior to study initiation, stability (determined as % of day 0; ≤ 10%) in gavage and IV formulations was confirmed for up to 42 d at refrigerated conditions.

Male rats were approximately 10 weeks old at the time of dosing. Each animal was weighed prior to dosing and its weight was recorded to determine the dosing volume. Single gavage doses were administered at 30 and 300 mg/kg for 3 animals per dose group. Dose formulations were administered in a volume of 5 mL/kg for rat via intragastric gavage. A single IV dose of 2.25 mg/kg was administered via catheters placed in jugular vein in a volume of 1.5 mL/kg. Prior to dose administration, individual animals were placed in Culex® Automated In Vivo Sampling System (BASI, West Lafayette, IN) system for blood collection. Approximately 0.25 mL blood was collected via catheters into tubes containing K₃EDTA from each animal for all 16 time points (0.0333 to 72 h post administration) as given in Supplemental Table 1. The actual times for blood collection were recorded. Blood was maintained at 5°C for approximately an hour until plasma was collected. All plasma samples were stored at −70 °C until analysis. Immediately following final blood collection each animal was humanely terminated.

Analyte quantitation.

A method using protein precipitation followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis was developed and qualified to quantitate the parent in male rat plasma, except for tBuPrOcEst-BZT. For tBuPrOcEst-BZT, the concentration of the parent was low and hence the total concentration of the corresponding acid, tBuPrA-BZT, was quantified.

Calibration standards and quality control (QC) samples were prepared in rat plasma to demonstrate linearity, sensitivity, precision, and accuracy. Accuracy was evaluated as percent relative error (%RE) and precision as percent relative standard deviation (%RSD). Precision and accuracy were evaluated at two concentration levels using three independent QCs prepared on the same day. The experimental limit of quantitation (LOQ) was assessed by preparing six replicates at the lowest concentration of the calibration curve. The limit of detection (LOD) was defined as three times the standard deviation of the experimental LOQ response expressed as concentration. Absence of carryover was confirmed by analyzing three blank rat plasma samples immediately following a high matrix standard.

Calibration concentration ranges used are given in Table 2. Internal standards (ditBuCl-BZT for quantitation of all analytes except DitBuCl-BZT where bumetrizole was used for quantitation) were prepared at 40 ng/mL in 5% formic acid in acetonitrile. Three stock solutions of individual analytes were prepared at 500 μg/mL in acetonitrile except octrizole and tBuPrA-BZT which were prepared in methanol. Spiking standards were prepared using two alternate stock solutions by diluting in the same solvent. Plasma calibration standards and QC samples were made from the spiking standards at desired concentrations. Plasma blanks with and without internal standard were also prepared.

Table 2.

Summary of analytical method used for the quantitation of phenolic benzotriazoles in rat plasma.

Analyte abbreviationa	Calibration concentration range (ng/mL)	Plasma QC concentrations (ng/mL)	Multiple reaction monitoring transition used for quantitation (m/z)	Retention time (min)
P-BZT	5.25–50	8, 40	212 → 65	5.0
Drometrizole	2–100	4.5, 75	226 → 120	5.7
tBu-BZT	1–100	2, 50	268 → 212	7.4
Octrizole	1–100	2, 50	324 → 92	8.9
ditPe-BZT	10.5–100	16, 80	352 → 212	10.4
diMeEtPh-BZT	5–100	10, 80	448 → 370	10.1
tBuPrA-BZTb	1–100	1.5, 80	340 → 224	6.8
Bumetrizole	5–100	10, 80	316 → 260	9.4
ditBuCl-BZT	5–100	10, 80	358 → 302	10.2

^aP-BTZ, 2-(2H-Benzotriazol-2-yl)phenol; tBu-BZT, 2-(2H-Benzotriazol-2-yl)-4-tertbutylphenol; ditPe-BZT, 2-(2H-Benzotriazol-2-yl)-4,6- bis(1,1-dimethylpropyl)phenol; diMeEtPh-BZT, 2-(2H-Benzotriazol-2-yl)-4,6-bis(1-methyl-1-phenylethyl)phenol; tBuPrOcEst-BZT, 3-(2H-Benzotriazol-2-yl)-5-(1,1-dimethylethyl)-4-hydroxybenzenepropanoic acid, octyl ester; ditBu-ClBZT, 2-(5-Chloro-2H-benzotriazol-2-yl)-4,6-bis(1,1dimethylethyl)phenol. tBuPrA-BZT, 3-(2H-benzotriazol-2-yl)-5(1,1-dimethylethyl)-4-hydrobenzenepropanoic acid.

^bFor tBuPrOcEst-BZT, tBuPrA-BZT was used as the analyte for quantitation.

Samples were prepared for analysis as follows. Fifty microliter plasma aliquots were transferred to individual wells in 96-well protein precipitation plates. For analysis of tBuPrA-BZT, 50 μL of plasma was first hydrolyzed with 100 μL of 6N HCl for 2 h at 100 °C and neutralized with 100 μL of 6N NaOH prior to transferring to wells. To each well, 200 μL of respective internal standard solution was added. For blank plasma samples without internal standard, 200 μL of 5% formic acid in acetonitrile was added. The plate was covered, vortexed for 2 min, and allowed to stand for 10 min following which pressure (8 psi) was applied for 2 min using a pressure manifold. The eluate was collected for analysis.

All samples were analyzed using a Shimadzu Prominence liquid chromatograph (Kyoto, Japan) coupled to a Sciex Triple Quad 5500 or API 5000 mass spectrometer (Framingham, MA). Phenomenex Kinetex phenyl hexyl column (2.6 μM, 50 mm x 2.1 mm) (Torrance, CA) was used for analyte separation, with mobile phases A (0.1% formic acid in water) and B (0.1% formic acid in acetonitrile) run at a gradient of 30% B to 95% B in 10 min using a flow rate of 0. 3 mL/min. The electrospray ion source was operated in negative mode with a source temperature of 500˚C and an ion spray voltage of −5500 V. Transitions monitored for quantitation and retention times of analytes are shown in Table 2. Samples with responses above the calibration range were diluted with blank plasma to bring the concentrations within the calibrated range. The peak area response ratio of analyte to internal standard was plotted against analyte concentration. A linear regression with 1/X weighting was used to relate response ratio of analyte to internal standard to analyte concentration. The concentration of analyte in each sample was calculated using response ratio, regression equation, and any applicable dilution factors.

Toxicokinetic modeling.

All samples above LODs were used in the TK analysis. All data sets were evaluated for aberrant concentration and time point values, as well as for any evidence of mis-dosing. The actual blood collection times were within 5% of nominal values for time points ≤4 h and within 15 min for time points >4 h and hence, the actual collection time was used for plasma TK analysis. Q-tests were performed on potential outliers for evaluation of plasma concentration agreement for a given time point. One animal at 72 h following administration of 300 mg/kg ditBu-ClBZT was considered an outlier and was not used in analysis.

WinNonlin software (Version 8.0, Certara L.P., Princeton, NJ) was used for TK analysis. Mean plasma concentration versus time data sets following IV and gavage administration were fitted using one-, two-, or three-compartment models. To account for the variability of the individual animal data and allow for the best estimation of TK parameters, concentrations were averaged at each time point prior to be used in a model. For each model, each data set was analyzed without and with weighting factors (1/Y, 1/Y², and/or 1/Y^∧2). The model and the weighting factor that resulted in the best goodness of fit (evaluated using the Akaike Information Criterion and Schwarz Bayesian Criterion) was selected as the final model. Based on this, IV data sets were modeled using a three-compartmental model with bolus Input, first order output, and 1/Y^∧2 weighting (model 18, equation 1) and gavage data sets were modeled using a two-compartmental model with first order Input, first order output, and 1/Y^∧2 weighting (model 13, equation 2). Glossary terms used for parameters calculated using compartmental models are given in Supplemental Table 2.

$${\text{C}}_{(\text{t})}=\text{A}{\text{e}}^{-\text{αt}}+\text{B}{\text{e}}^{-\text{βt}}+\text{C}{\text{e}}^{-\text{γt}}$$ (1)

$${\text{C}}_{(\text{t})}=\text{A}{\text{e}}^{-\text{αt}}+\text{B}{\text{e}}^{-\text{βt}}+\text{C}{\text{e}}^{-\text{k}}{{}_{01}}^{\text{t}}$$

Where C_(t) is the concentration at time t; A, B, and C are the intercepts of the distribution and elimination phases; α, β, and γ are the first order hybrid rate constants for distribution and elimination phases; k₀₁ is the absorption rate constant.

Results

All individual animal and full set of TK parameters are available in NTP Chemical Effects of Biological Systems (CEBS) data base at

https://doi.org/10.22427/NTP-DATA-002-03291-0028-0000-7

Due to the large number of data available for the class, only the selected parameters are summarized here.

Analytical method qualification.

We modified a method, previously developed using solid phase extraction or liquid-liquid extraction, to quantitate phenolic benzotriazoles. The use of protein precipitation for sample cleanup and incorporation of 96-well format increased the efficiency of the method and sample throughput. For all compounds, except the ester, the parent concentrations were quantified. For the ester, the corresponding acid, tBuPrA-BZT, was used. Analytical method qualification data for the quantitation of analytes are summarized in Table 3. Standard curves were linear over the concentration ranges evaluated with coefficients of determination >0.98 for all analytes. Estimated LOD values ranged from 0.166 to 1.16 ng/mL depending on the analyte (Table 3). The %RE values estimated based on calibration standards were ≤±18.9 and QC samples were ≤±22.7. The %RSD values were ≤13.7 for all except for octrizole which was ≤24.5 Taken collectively, the data show that the method is suitable for the quantitation of analytes in plasma from the investigation on TK behavior of the class.

Table 3.

Summary of analytical method qualification data for the quantitation of phenolic benzotriazoles in rat plasma.

Analyte abbreviationa	Experimental LOQc (ng/mL)	LODc (ng/mL)	Accuracy (%RE)d,f	Accuracy (% RE)e,f	Precision (%RSD)e
P-BZT	5.25	1.02	≤±13.7	≤±15.7	≤8.3
Drometrizole	2.00	0.552	≤±18.9	≤±12.0	≤6.9
tBu-BZT	1.00	0.166	≤±15.0	≤±19.0	≤7.8
Octrizole	1.00	0.315	≤±16.0	≤±18.3	≤24.5
ditPe-BZT	10.5	0.906	≤±10.4	≤±20.5	≤10.0
diMeEtPh-BZT	5.00	1.04	≤±13.9	≤±15.7	≤6.2
tBuPrA-BZTb	1.00	0.345	≤±18.6	≤±22.7	≤10.9
Bumetrizole	5.00	1.16	≤±12.0	≤±17.8	≤8.6
ditBuCl-BZT	5.00	1.06	≤±14.9	≤±20.3	≤13.7

^aP-BZT, 2-(2H-Benzotriazol-2-yl)phenol; tBu-BZT, 2-(2H-Benzotriazol-2-yl)-4-tertbutylphenol; ditPe-BZT, 2-(2H-Benzotriazol-2-yl)-4,6- bis(1,1-dimethylpropyl)phenol; diMeEtPh-BZT, 2-(2H-Benzotriazol-2-yl)-4,6-bis(1-methyl-1-phenylethyl)phenol; tBuPrOcEst-BZT, 3-(2H-Benzotriazol-2-yl)-5-(1,1-dimethylethyl)-4-hydroxybenzenepropanoic acid, octyl ester; ditBu-ClBZT, 2-(5-Chloro-2H-benzotriazol-2-yl)-4,6-bis(1,1dimethylethyl)phenol. tBuPrA-BZT, 3-(2H-benzotriazol-2-yl)-5(1,1-dimethylethyl)-4-hydrobenzenepropanoic acid.

^bFor tBuPrOcEst-BZT, tBuPrA-BZT was used as the analyte for quantitation.

^cLOQ, limit of quantitation; LOD, limit of detection.

^dValues given are for calibration standards.

^eValues given are for 2 concentrations of QC samples (refer to Table 2).

^fUp to 5 samples out of a total of 30 standard concentrations or total of 60 QC samples run with each analyte were considered potential outliers and hence were not used.

Toxicokinetic analysis following a single IV administration.

In rats following a single IV administration of P-BZT, concentrations were measurable at the earliest post-dose plasma collection time point of 0.0333 through 24 h. All samples at 48 and 72 h timepoints were below LOD of the method. For all other compounds, concentrations were measurable at the earliest post-dose sample collection time through 72 h. Plasma concentration versus time profiles for all phenolic benzotriazoles following a single 2.25 mg/kg dose showed an apparent triphasic decline. Data were fitted using a three-compartment model with bolus input, first order output, and 1/Y^∧2 weighting. Model fits are shown in Figure 2 and summary of selected key TK parameters estimated are given in Table 4.

Figure 2.

Plasma concentration versus time profiles following a single intravenous administration of 2.25 mg/kg phenolic benzotriazoles in male rats. A) unsubstituted B) monosubstituted C) disubstitued D) trisubstituted. Lines shown are for model fits based on a three-compartmental model with bolus Input and first order output and 1/Y^² weighting (model 18). P-BZT, 2-(2H-Benzotriazol-2-yl)phenol; tBu-BZT, 2-(2H-Benzotriazol-2-yl)-4-tertbutylphenol; ditPe-BZT, 2-(2H-Benzotriazol-2-yl)-4,6- bis(1,1-dimethylpropyl)phenol; diMeEtPh-BZT, 2-(2H-Benzotriazol-2-yl)-4,6-bis(1-methyl-1-phenylethyl)phenol; tBuPrOcEst-BZT, 3-(2H-Benzotriazol-2-yl)-5-(1,1-dimethylethyl)-4-hydroxybenzenepropanoic acid, octyl ester; ditBuCl-BZT, 2-(5-Chloro-2H-benzotriazol-2-yl)-4,6-bis(1,1dimethylethyl)phenol. For tBuPrOcEst-BZT, data for the corresponding acid, 3-(2H-benzotriazol-2-yl)-5(1,1-dimethylethyl)-4-hydrobenzenepropanoic acid (tBuPrA-BZT) is shown.

Table 4.

Systemic exposure parameters and plasma elimination half-lives following a single intravenous administration of 2.25 mg/kg phenolic benzotriazoles in male rats^a.

	Parameterb
Compound/abbreviationc	Cmax (ng/mL)	Half-life (h)d	AUC (h*ng/mL)
Unsubstituted
P-BZT	886 ± 46	22.4 ± 4.4	284 ± 10
Monosubstituted
Drometrizole	4510 ± 116000	84.8 ± 20.5	10500 ± 2200
tBu-BZT	3050 ± 450	30.5 ± 5.9	1650 ± 100
Octrizole	20600 ± 7800	15.4 ± 2.4	3510 ± 440
Disubstituted
ditPe-BZT	37400 ± 2700	22.4 ± 1.8	36500 ± 1100
diMeEtPh-BZT	45900 ± 1700	25.1 ± 2.0	71100 ± 1700
tBuPrOcEst-BZTe	22400 ± 900	19.2 ± 1.1	13800 ± 300
Trisubstituted
Bumetrizole	22400 ± 4700	31.7 ± 7.3	10200 ± 600
DitBuCl-BZT	36400 ± 1600	17.3 ± 0.6	35000 ± 600

^aBased on a three-compartmental model with bolus Input and first order output and 1/Y^² weighting (model 18). Values given are mean ± standard error for 3 animals.

^bValues given are mean ± standard error for 3 animals. For IV studies C_max = C_0.

^cP-BZT, 2-(2H-Benzotriazol-2-yl)phenol; tBu-BZT, 2-(2H-Benzotriazol-2-yl)-4-tertbutylphenol; ditPe-BZT, 2-(2H-Benzotriazol-2-yl)-4,6- bis(1,1-dimethylpropyl)phenol; diMeEtPh-BZT, 2-(2H-Benzotriazol-2-yl)-4,6-bis(1-methyl-1-phenylethyl)phenol; tBuPrOcEst-BZT, 3-(2H-Benzotriazol-2-yl)-5-(1,1-dimethylethyl)-4-hydroxybenzenepropanoic acid, octyl ester; ditBuCl-BZT, 2-(5-Chloro-2H-benzotriazol-2-yl)-4,6-bis(1,1dimethylethyl)phenol.

^dGamma half-life is given.

^eData for the corresponding acid, 3-(2H-benzotriazol-2-yl)-5(1,1-dimethylethyl)-4-hydrobenzenepropanoic acid (tBuPrA-BZT) is shown.

Unsubstituted phenolic benzotriazole, P-BZT, was eliminated from plasma with a terminal elimination half-life (gamma half-life) of 22.4 h. In comparison, the half-lives for monosubstituted compounds ranged from 15.4 to 84.8 h, disubstituted compounds ranged from 19.2 to 25.1 h and for trisubstituted compounds ranged from 17.3 to 31.7 h demonstrating that there is no clear pattern in plasma elimination half-life with the increasing degree of substitution following IV administration of the class compounds.

C_max following administration of 2.25 mg/kg of P-BZT was 886 ng/mL. The value increased for the substituted compounds with 3050–20600 ng/mL for mono-, 22400–45900 ng/mL for di- and trisubstituted compounds. A similar pattern was observed for area under the concentration versus time curve (AUC) with lowest values observed for P-BZT (284 ng/mL*h ). The value increased with the degree of substitution with 1650–10500 ng/mL*h for monosubstituted and 10200–71100 ng/mL*h for di- and trisubstituted compounds.

Toxicokinetic analysis following a single gavage administration.

In rats following a single gavage administration, analyte concentrations were measurable for both 30 and 300 mg/kg groups except the following where concentrations were below LOD of analytical methods; P-BZT ≥12 h for 30 mg/kg group; octrizole at 0.0333 h for 300 mg/kg group; diMeEtPh-BZT 0.0333–0.0833 h for 30 and 300 mg/kg groups; bumetrizole 0.0333 h for 30 mg/kg; ditBu-ClBZT 0.0333–0.167 h for 30 and 300 mg/kg groups.

Plasma concentration versus time profiles for all compounds, in general, showed a biphasic decline. Some of the deviations are as follows. In the P-BZT 30 mg/kg group, the terminal elimination phase was not well-defined due to undetectable levels in samples ≥12 h (Supplemental Figure 2A) and timepoints ≥12 h for 300 mg/kg showed high variability with a slow terminal phase. Drometrizole 30 mg/kg showed a slow terminal phase.

Data were fitted using a two-compartment model with first order input, first order output, and 1/Y^² weighting. Model fits are shown in Figure 3 for 300 mg/kg and Supplemental Figure 2 for 30 mg/kg. A summary of TK parameters estimated is given in Table 5.

Figure 3.

Plasma concentration versus time profiles following a single gavage administration of 300 mg/kg phenolic benzotriazoles in male rats. A) unsubstituted B) monosubstituted C) disubstitued D) trisubstituted. Lines shown are for model fits based on a two-compartmental model with first order Input and first order output and 1/Y^² weighting (model 13). P-BZT, 2-(2H-Benzotriazol-2-yl)phenol; tBu-BZT, 2-(2H-Benzotriazol-2-yl)-4-tertbutylphenol; ditPe-BZT, 2-(2H-Benzotriazol-2-yl)-4,6- bis(1,1-dimethylpropyl)phenol; diMeEtPh-BZT, 2-(2H-Benzotriazol-2-yl)-4,6-bis(1-methyl-1-phenylethyl)phenol; tBuPrOcEst-BZT, 3-(2H-Benzotriazol-2-yl)-5-(1,1-dimethylethyl)-4-hydroxybenzenepropanoic acid, octyl ester; ditBuCl-BZT, 2-(5-Chloro-2H-benzotriazol-2-yl)-4,6-bis(1,1dimethylethyl)phenol. For tBuPrOcEst-BZT, data for the corresponding acid is shown.

Table 5.

Systemic exposure parameters and plasma elimination half-lives following a single gavage administration of 30 and 300 mg/kg phenolic benzotriazoles in male rats^a

	Parameterb
Compound/abbreviationc	Tmax (h)		Cmax (ng/mL)		Half-life (h)d		AUC (h*ng/mL)
	30 mg/kg	300 mg/kg	30 mg/kg	300 mg/kg	30 mg/kg	300 mg/kg	30 mg/kg	300 mg/kg
Unsubstituted
P-BZT	0.862 ± 0.300	1.49 ± 0.36	69.7 ± 12.7	237 ± 56	1.57 ± 0.78	192 ± 562	226 ± 34	6720 ± 14900
Monosubstituted
Drometrizole	0.0196 ± 24.6	0.266 ± 0.053	138 ± 1900	358 ± 26	1120 ± 5780	103 ± 72	132000 ± 675000	29800 ± 14700
tBu-BZT	0.863 ± 2.41	2.47 ± 2.13	1070 ± 230	4640 ± 1100	13.6 ± 2.7	16.8 ± 10.6	4140 ± 930	39500 ± 9100
Octrizole	3.71 ± 1.08	2.80 ± 0.64	561 ± 170	3590 ± 850	56.8 ± 233	21.4 ± 18.2	5980 ± 1950	30000 ± 7400
Disubstituted
ditPe-BZT	2.96 ± 1.10	6.29 ± 1.69	7090 ± 3280	11100 ± 4400	13.4 ± 7.2	17.1 ± 53.3	95400 ± 26600	269000 ± 67000
diMeEtPh-BZT	5.18 ± 2.04	7.80 ± 2.34	386 ± 194	697 ± 305	19.8 ± 26.3	42.7 ± 1170	9270 ± 2580	19500 ± 41800
tBuPrOcEst-BZTe	1.07 ± 0.30	1.91 ± 0.56	12800 ± 3100	47800 ± 13500	9.53 ± 1.73	13.6 ± 6.4	42300 ± 11500	294000 ± 85000
Trisubstituted
Bumetrizole	3.08 ± 0.77	5.06 ± 1.24	2020 ± 490	2450 ± 710	48.4 ± 116	37.5 ± 401	21000 ± 5300	41600 ± 11600
DitBuCl-BZT	4.32 ± 4.15	4.53 ± 1.38	3560 ± 7800	6810 ± 2420	14.1 ± 4.3	14.0 ± 13.3	89400 ± 40700	113000 ± 28000

^aBased on a two-compartmental model with first order Input and first order output and 1/Y^² weighting (model 13).

^bValues given are mean ± standard error for 3 animals.

^cP-BZT, 2-(2H-Benzotriazol-2-yl)phenol; tBu-BZT, 2-(2H-Benzotriazol-2-yl)-4-tertbutylphenol; ditPe-BZT, 2-(2H-Benzotriazol-2-yl)-4,6- bis(1,1-dimethylpropyl)phenol; diMeEtPh-BZT, 2-(2H-Benzotriazol-2-yl)-4,6-bis(1-methyl-1-phenylethyl)phenol; tBuPrOcEst-BZT, 3-(2H-Benzotriazol-2-yl)-5-(1,1-dimethylethyl)-4-hydroxybenzenepropanoic acid, octyl ester; ditBu-ClBZT, 2-(5-Chloro-2H-benzotriazol-2-yl)-4,6-bis(1,1dimethylethyl)phenol.

^dBeta half-life is given.

^eData for the corresponding acid, 3-(2H-benzotriazol-2-yl)-5(1,1-dimethylethyl)-4-hydrobenzenepropanoic acid (tBuPrA-BZT) is shown.

P-BZT was absorbed following gavage administration of 30 and 300 mg/kg with T_max values (time to reach C_max) between 0.862 and 1.49 h (Table 5). T_max for drometrizole (0.0196–0.266 h) was shorter than for P-BZT, although T_max for other monosubstituted compounds (t-Bu-BZT and octrizole) were similar to P-BZT and ranged between 0.863–3.71 h. T_max for di- and trisubstituted compounds (2.96–7.80 h), in general were longer than unsubstituted and mono-substituted compounds, except for the ester where total acid was used; T_max of the acid was similar to P-BZT (1.07–1.91 h). In general, there was an apparent increase in T_max following administration of 300 mg/kg (0.266–7.80 h) compared to 30 mg/kg (0.0196–0.862 h) (Table 5).

C_max (69.7–237 ng/mL) for the unsubstituted P-BZT was the lowest for this compound class (Table 5). The values increased as the degree of substitution increased with 138–4640 ng/mL for monosubstituted and 386–11100 ng/mL for di- (with the exception of tBuPrA-BZT) and trisubstituted. C_max for the ester (12800–47800 ng/mL) is vastly higher than the other disubstituted compounds due to use of total acid, tBuPrA-BZT, which is a sum of the total acid and ester concentration in plasma. The pattern for AUC was similar to C_max with lowest value for unsubstituted P-BZT (226–6720 h*ng/mL); AUC increased as the degree of substitution increased with 4140–39500 h*ng/mL for mono-substituted, 9270–294000 h*ng/mL for di- and trisubstituted compounds. An exception to this pattern was monosubstituted drometrizole at 30 mg/kg, for which the estimated AUC value for 30 mg/kg (132000 h*ng/mL) was much higher than other monosubstituted compounds as well as the 300 mg/kg drometrizole (29800 h*ng/mL). This is likely stemming from predicted slow elimination phase leading to an overestimation of AUC (Supplemental Figure 2B).

In general, systemic exposure parameters, C_max and AUC, increased with the dose for the class, although the increase was not dose-proportional (Table 5). The increase in C_max was only 3-fold for P-BZT (69.7–237 ng/mL) and 2- to 6-fold for mono- and disubstituted compounds with a 10-fold increase in the dose. The increase in C_max for trisubstituted compounds was ≤2-fold. The AUC for P-BZT increased 30-fold (226–6720 h*ng/mL) and for monosubstituted compounds (excluding drometrizole) increased 5- to 10-fold with a 10-fold increase in the administered dose. For disubstituted compounds, AUC increased 2- to7-fold with a 10-fold increase in the dose (Table 5). The increase in AUC for trisubstituted compounds was ≤2-fold with a 10-fold increase in the dose.

P-BZT was eliminated from plasma with a half-life (beta half-life) of 1.57 h for 30 mg/kg. However, the estimated half-life following administration of 300 mg/kg was much longer (192 h). In comparison, the half-lives for monosubstituted compounds following administration of 30 and 300 mg/kg ranged from 13.6 to 56.8 h, disubstituted compounds ranged from 13.4 to 42.7 h and trisubstituted compounds ranged from 14.1 to 48.4 h demonstrating that there is no distinct pattern in elimination half-life with the degree of substitution or the dose. However, the monosubstituted phenolic benzotriazole, drometrizole, was distinct in that it had generally longer half-lives (103–1120 h).

Oral bioavailability.

Oral bioavailability (F) was estimated using AUC following oral and IV administration, adjusted for dose administered (%F = AUC/Dose_(oral) ÷ AUC/Dose_(IV) X 100) (Table 6). In general, the oral bioavailability of phenolic benzotriazoles was low. At 30 mg/kg, the estimated bioavailability was ~ 6% for P-BZT; the bioavailability for most substituted phenolic benzotriazoles were higher than P-BZT (with the exception of diMeEtPh-BZT) and ranged from 15 to 23% for all except for drometrizole (~ 94%). The high value estimated for drometrizole was due to slow elimination phase leading to overestimation of AUC. At 300 mg/kg, there was no clear pattern in oral bioavailability with the increasing degree of substitution with values ranging from 2 to 18%. The compound diMeEtPh-BZT behaved differently from other compounds tested with estimated bioavailability values ≤1%.

Table 6.

Estimated oral bioavailability of phenolic benzotriazoles following a single gavage administration of 30 and 300 mg/kg in male rats

Compound/abbreviationa	Oral bioavailability (%)
	30 mg/kg	300 mg/kg
Unsubstituted
P-BZT	5.97	17.7
Monosubstituted
Drometrizole	94.3	2.13
tBu-BZT	18.8	18.0
Octrizole	12.8	6.41
Disubstituted
ditPe-BZT	19.6	5.53
diMeEtPh-BZT	0.978	0.206
tBuPrOcEst-BZTb	23.0	16.0
Trisubstituted
Bumetrizole	15.4	3.06
DitBuCl-BZT	19.2	2.42

^aP-BZT, 2-(2H-Benzotriazol-2-yl)phenol; tBu-BZT, 2-(2H-Benzotriazol-2-yl)-4-tertbutylphenol; ditPe-BZT, 2-(2H-Benzotriazol-2-yl)-4,6- bis(1,1-dimethylpropyl)phenol; diMeEtPh-BZT, 2-(2H-Benzotriazol-2-yl)-4,6-bis(1-methyl-1-phenylethyl)phenol; tBuPrOcEst-BZT, 3-(2H-Benzotriazol-2-yl)-5-(1,1-dimethylethyl)-4-hydroxybenzenepropanoic acid, octyl ester; ditBuCl-BZT, 2-(5-Chloro-2H-benzotriazol-2-yl)-4,6-bis(1,1dimethylethyl)phenol.

^bData for the corresponding acid, 3-(2H-benzotriazol-2-yl)-5(1,1-dimethylethyl)-4-hydrobenzenepropanoic acid (tBuPrA-BZT) is shown.

Discussion

Phenolic benzotriazole are a large class of compounds with some structural diversity and potential human exposures via multiple routes. Here, we investigated the TK behavior of 9 compounds following a single gavage administration. Compounds were selected to cover a variety of structural features including unsubstituted, monosubstituted, disubstituted including an ester and a halogen substitution, and trisubstituted including a halogen substitution. Doses of 30 and 300 mg/kg were selected to overlap with the doses used in rat toxicity studies (Testing Status). Studies were also conducted following a single IV administration of 2.25 mg/kg to generate data to estimate the oral bioavailability of the class. To minimize the animal usage, serial blood samples were collected from 3 animals for a given dose group. Blood sampling was done using Culex® Automated In Vivo Sampling System (BASi, West Lafayette, IN) to minimize animal handling. To the best of our knowledge, this is the first systematic investigation of TK behavior of compounds in this class.

For all compounds, except the ester, the parent concentrations in plasma over time were determined. For the ester, the concentration of the parent in plasma was mostly below LOD following oral administration, likely stemming from the first pass intestinal and hepatic metabolism of the ester to the corresponding acid, tBuPrA-BZT. Extensive metabolism of the phenolic benzotriazole esters to corresponding acids has previously been reported for tBuPrMeEst-BZT and tBuPrHexEst-BZT (Thomas et al., 1995). In addition, we found that the parent ester, tBuPrOcEst-BZT, was unstable in plasma during sample preparation. Hence, total acid concentration, after hydrolyzing the plasma with HCl to convert both conjugated acid and parent to free acid, was determined for the ester which should be taken into consideration when the data for the ester is compared to the other compounds investigated in the class. As mentioned above, all study data can be found in the NTP CEBS system (https://doi.org/10.22427/NTP-DATA-002–03291-0028–0000-7). In this report, we focused on key parameters, systemic exposures and plasma elimination half-lives, to compare and contrast of TK behavior of compounds in the class.

Following a single IV administration of 2.25 mg/kg, no distinct pattern based on the type or degree of substitution was observed for elimination kinetics with half-lives ranging from 15.4–31.7 h except for drometrizole where the estimated value was longer (half-life 84.8 h) but was within 3- to 6-fold of the other substituted phenolic benzotriazoles. C_max, for unsubstituted P-BZT was the lowest (886 ng/mL) amongst the 9 compounds investigated. C_max, increased with the degree of substitution with, in general, tri- and disubstituted > monosubstituted >> unsubstituted compounds. A similar pattern was observed for AUC tri- and disubstituted > monosubstituted > unsubstituted. Overall, C_max was 5- to 23-fold and 25- to 52-fold higher and AUC was 6- to 37-fold 36- to 250-fold higher for monosubstituted and di- and trisubstituted compounds, respectively, compared to unsubstituted P-BZT. Taken collectively, the data following IV administration may indicate extensive metabolism of unsubstituted P-BZT relative to substituted compounds and that metabolism decreases as the degree of substitution in phenolic benzotriazoles increases.

Following a single gavage administration, P-BZT was absorbed rapidly with the maximum concentration reached ≤1.49 h. As the degree of substitution and the size of the compound increased, the absorption was slower as evident by the longer T_max (≤7.80 h). As observed following IV administration, the systemic exposure of P-BZT was lowest compared to the substituted compounds following a single gavage administration. C_max, increased with the degree of substitution with, in general, tri- and disubstituted > monosubstituted >> unsubstituted compounds following administration of either 30 or 300 mg/kg dose. A similar trend was observed for AUC except for drometrizole where AUC was highest across the class following administration of 30 mg/kg which is likely an overestimation stemming from the longer elimination phase observed (Supplemental Figure 2). Whether this is a real difference or an anomaly in the study conduct is not clear at the present time. Regardless, the general trend held where C_max was 2- to 20-fold and 3- to 202-fold higher and AUC was 4- to 26-fold and 3- to 422-fold higher (with the exception of 30 mg/kg drometrizole) for monosubstituted and di-/trisubstituted compounds, respectively, compared to unsubstituted P-BZT. It is noteworthy that use of the total acid, tBuPrA-BZT, for the ester overestimates its overall systemic exposure compared to other compounds where only parent was used. These data may indicate extensive intestinal and hepatic first pass metabolism of unsubstituted P-BZT relative to substituted compounds and that metabolism decreases as the degree of substitution increases leading to increased systemic exposure.

Although the systemic exposure increased with the dose following gavage administration, the increase was less than dose proportional for most of the compounds with couple of exceptions. With a 10-fold increase in the dose, C_max increased 2- to 6-fold for mono- and disubstituted compounds whereas for trisubstituted compounds the increase was generally ≤2-fold. The increase in AUC was higher than dose-proportional for P-BZT with a 30-fold increase with a 10-fold increase in the dose suggesting potential saturation of metabolic pathways occurring between 30 and 300 mg/kg for P-BZT. On the other hand, for substituted compounds, similar to C_max, the increase in AUC with the dose was less than dose-proportional with 2- to 7-fold increase for mono- and disubstituted compounds and ≤2-fold for trisubstituted compounds with a 10-fold increase in the dose. The only exception was monosubstituted compound, tBu-BZT, where the increase in AUC was dose-proportional. Overall, the data points to changes in ADME processes for this compound class with the increasing dose, potentially resulting from decreases in absorption for substituted compounds as the dose increases.

Oral bioavailability of phenolic benzotriazoles in general was low. Lowest bioavailability (~6%) was estimated for P-BZT at 30 mg/kg dose supporting that the first pass metabolism lowers the amount of parent entering the systemic circulation. The bioavailability was slightly higher for substituted compounds (12.8–23.0%) despite the potential lower absorption of the substituted compounds compared to lower or unsubstituted compounds, further confirming decreased metabolism with increased substitution. The exception to this was diMeEtPh-BZT where bioavailability was <1%. This could potentially be due to an underestimation stemming from estimated lower and higher AUC, respectively, following gavage and IV administration for this compound. In general, the bioavailability was lower following administration of the higher dose for all except for P-BZT, where the estimated value was ~ 18%. Taken collectively, these data suggest that the oral absorption of phenolic benzotriazoles are low and decreases with the increasing dose. Substituted compounds may be less metabolized compared to unsubstituted P-BZT.

As mentioned previously, the only TK data found in the literature were for the two esters, tBuPrMeEst-BZT and tBuPrHexEst-BZT, following administration of 10 mg/kg [¹⁴C]-labelled compounds. Based on the measurement of total radioactivity in blood, T_max reported was 1–2 h and the half-life was ~12 h (Thomas et al., 1995). These data are consistent with our data for tBuPrOcEst-BZT, following administration of 30 or 300 mg/kg, based on the measurement of the total acid, tBuPrA-BZT (T_max 1.07–1.91 h; plasma half-life 9.53–13.6 h) pointing to the acid as the major metabolite in the systemic circulation following gavage administration of esters of phenolic benzotriazoles.

Conclusion

Our findings showed that the absorption of phenolic benzotriazoles following a single gavage administration in male rats was low and the absorption decreased with the increased substitution and dose. There was no distinct pattern in plasma elimination half-life with the degree of substitution or the dose. The substituted compounds may be less metabolized compared to the unsubstituted compound leading to higher systemic exposure. The estimated oral bioavailability was low with unsubstituted compound having the lowest bioavailability (~6%) compared to substituted compounds (12.8–23.0%).