Incorporating new approach methodologies in toxicity testing and exposure assessment for tiered risk assessment using the RISK21 approach: Case studies on food contact chemicals

Abstract

Programs including the ToxCast project have generated large amounts of in vitro high‒throughput screening (HTS) data, and best approaches for the interpretation and use of HTS data, including for chemical safety assessment, remain to be evaluated. To fill this gap, we conducted case studies of two indirect food additive chemicals where ToxCast data were compared with in vivo toxicity data using the RISK21 approach. Two food contact substances, sodium (2-pyridylthio)-N-oxide and dibutyltin dichloride, were selected, and available exposure data, toxicity data, and model predictions were compiled and assessed. Oral equivalent doses for the ToxCast bioactivity data were determined by in-vitro in-vivo extrapolation (IVIVE). For sodium (2-pyridylthio)-N-oxide, bioactive concentrations in ToxCast assays corresponded to low- and no-observed adverse effect levels in animal studies. For dibutyltin dichloride, the ToxCast bioactive concentrations were below the dose range that demonstrated toxicity in animals; however, this was confounded by the lack of toxicokinetic data, necessitating the use of conservative toxicokinetic parameter estimates for IVIVE calculations. This study highlights the potential utility of the RISK21 approach for interpretation of the ToxCast HTS data, as well as the challenges involved in integrating in vitro HTS data into safety assessments.

Keywords: Dibutyltin dichloride; Food additive; High-throughput screening; In-vitro in-vivo extrapolation; RISK21; Sodium (2-pyridylthio)-N-Oxide (pyrithione sodium); ToxCast.

Fig. 1.

DBTC Estimate of Exposure. Estimates of exposure for DBTC were generated by modeling the migration of dibutyltin from plastic food packing into foods based on physicochemical parameters and initial amounts of the chemical in the packaging. Human exposure to foods containing dibutyltin was estimated using food diaries from NHANES. The 1st, 25th, 50th, 75th, and 99th percentiles of exposure were calculated for the total population and for various subgroups. Estimates were computed and plotted separately when considering different uses for DBTC as A) a catalyst or B) a heat stabilizer. C) Exposure values for the total population were plotted for use of DBTC as a catalyst, heat stabilizer, or combined use.

Fig. 2.

RISK21. Estimates of exposure from Table 1, and the most sensitive NOEL/NOAEL value from Table 2 were used to develop a RISK21 plot using the RISK21 webtool. The ExpoCast estimates of exposure are used as the lower estimate of exposure for both DBTC and SPO, and the upper estimates of exposure are the CEDI value for SPO and the SHEDS-HT value for DBTC. Estimates of toxicity (a NOEL of 0.5 mg/kgbw/d for SPO and a NOAEL of 0.025mg/kgbw/d for DBTC) were plotted as point estimates with an uncertainty factor of 100 to define the reference dose or tolerable daily intake established limit for each compound. The top of the colored boxes per chemical denote the reference dose or the tolerable daily intake, and the bottom of the box is the dose given to the animal used to determine that limit.

Fig. 3.

Summary of the ToxCast data for DBTC and SPO. Assay-specific data for both chemicals were pulled from the ToxCast Dashboard. A) ToxCast hit calls were used to define negative and active assays. Cytotoxicity filtering was done by applying the cytotoxicity limit as a cutoff value wherein assays having an AC₅₀ below the cytotoxicity limit remained active after filtering. B, C) All assays determined to be active for either compound were plotted as AC₅₀ versus the modlTp (maximum efficacy of modeled concentration-response curve) parameter from the ToxCast data, for each assay, with the cytotoxicity limit plotted as a straight line.

Fig. 4.

Comparison of ToxCast and Traditional Toxicology Data. ToxCast assay AC₅₀ values were converted to oral equivalent doses (OED) and plotted as a distribution across the 5th, 25th, 50th, 75th, and 95th percentiles for all active assays for SPO and DBTC. The LOAEL/NOAEL values from the in vivo animal data, the cytotoxicity center, and cytotoxicity limit OEDs are plotted as point estimates. A) OEDs for SPO were determined using available toxicokinetic data. B) OEDs for DBTC were determined using the worst-case toxicokinetic parameter estimates. C) For DBTC, a range of toxicokinetic parameter estimates were used to determine OEDs from the AC₅₀ values for the 5th, 25th, 50th, 75th, and 95th percentiles of all active assays, the cytotoxicity limit, and the cytotoxicity center. The in vivo NOAEL and LOAEL data are plotted as point estimates shown as green and purple lines respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)