Threshold of Toxicological Concern: Extending the chemical space by inclusion of a highly curated dataset for organosilicon compounds.

The Threshold of Toxicological Concern (TTC) for non-genotoxic substances, a risk assessment tool to establish safe exposure levels for chemicals with insufficient toxicological data, is based on the 5th percentile of cumulated distributions of Point of Departures in a high amount of repeat-dose, developmental and reproductive toxicity studies, grouped by Cramer Classes. The lack of organosilicon compounds in this dataset has resulted in regulatory concerns over the applicability of the TTC concept for this chemistry. We collected publicly available, scientifically robust oral repeat-dose and DART studies for 71 organosilicon substances for inclusion in the existing TTC dataset, using criteria for evaluation of studies and derivation of points of departure analogous to the Munro and COSMOS TTC publications. The resulting 5th percentile of this dataset was 13-fold higher than the 5th percentile for Cramer Class III compounds reported by Munro (which is the default for silicon-containing substances). Both the existing TTC for Cramer Class III compounds from Munro (1.5 µg/kg bw/day) and the COSMOS TTC (2.3 µg/kg bw/day), recommended by the SCCS for cosmetics-related substances, provide a conservative and sufficiently protective approach for this class of chemistry.

Bolstering the existing database supporting the non-cancer Threshold of Toxicological Concern values with toxicity data on fragrance-related materials.

The use of threshold of toxicological concern (TTC) supports the safety assessment of exposure to low levels of chemicals when toxicity data are limited. The Research Institute for Fragrance Materials (RIFM) delivers safety assessments for fragrance materials that result in safe products for consumer use. A major goal for the RIFM safety assessment program is to invest in alternative methods to animal testing for use in assessment of fragrance materials. This includes use of TTC, which provides a pragmatic approach for safety evaluation of fragrance materials in the absence of chemical-specific toxicity data and reduces the need to generate new animal data. To bolster the TTC approach for support of fragrance materials and specifically to strengthen the Cramer class II threshold, the RIFM database was reviewed with a goal of identifying fragrance materials with data that can be added to the existing TTC databases. The RIFM database identified a total of 476 chemicals that were added to the existing TTC databases. The chemicals were then individually assigned a Cramer class and 238, 76 and 162 chemicals in Cramer class I, II and III respectively were identified. The RIFM-TTC dataset was then combined with the COSMOS-Federated TTC dataset for a total of 421, 111 and 795 chemicals in Cramer class I, II and III respectively. The combined dataset further expands the chemical space thereby providing more robust 5th percentile thresholds. Moreover, the combined dataset bolsters the threshold for Cramer class II to include a total of 111 chemicals which is an improvement over the original (Munro) TTC dataset which only included 28 chemicals in Cramer Class II and the COSMOS Federated dataset which had 40 chemicals. This allows for a more reliable and robust 5th percentile NOAEL value for Cramer class II chemicals of 1.27 mg/kg bw/day. The 5th percentile NOAELs for Cramer class I, II and III from the combined dataset are 4.91, 1.27 and 0.29 mg/kg bw/day, which supports the threshold values derived from the original Munro dataset. This work confirms the adequacy of the existing TTC values and provides further support for the use of TTC as a tool to conduct safety assessments for fragrance materials. It further opens the future possibility of updating the existing values with more robust TTC values for fragrance and cosmetic materials.

Structure activity relationship (SAR) toxicological assessments: The role of expert judgment.

Structure activity relationships (SAR) and read-across are widely used animal alternative approaches for filling toxicological data gaps. A framework describing the use of expert judgment in evaluating analogs for SAR has been published and widely cited, however, reliance on expert judgment can introduce inconsistent results across experts and hinder transparency. Here we explore the use of a quantitative similarity score between an analog and a Structure of Interest (SOI) to see if these scores correlate with the expert judgement-based suitability rankings. We find these global similarity scores representing a "whole-molecule" view of similarity to be insensitive to differences in local structure which may be important for toxicity, and, therefore, cannot be substituted for expert judgement-based similarity rankings. In this paper, we suggest that the next step in the progression of SAR approaches retains the insights from expert judgment, but facilitates consistency and transparency through the development of rating "rules". This report outlines and defines analog rating rules for several compound categories. While not comprehensive, the exercises demonstrate the development of rules for categories with a large spread in molecular weight and alkyl chain length and explains the advantages that we see in this approach compared to relying solely on a computational approach or an unstructured expert judgement approach. These rules may be incorporated into analog searching work flows to define boundaries for analogs "suitable" for read-across.

Framework for identifying chemicals with structural features associated with the potential to act as developmental or reproductive toxicants.

Developmental and reproductive toxicity (DART) end points are important hazard end points that need to be addressed in the risk assessment of chemicals to determine whether or not they are the critical effects in the overall risk assessment. These hazard end points are difficult to predict using current in silico tools because of the diversity of mechanisms of action that elicit DART effects and the potential for narrow windows of vulnerability. DART end points have been projected to consume the majority of animals used for compliance with REACH; thus, additional nonanimal predictive tools are urgently needed. This article presents an empirically based decision tree for determining whether or not a chemical has receptor-binding properties and structural features that are consistent with chemical structures known to have toxicity for DART end points. The decision tree is based on a detailed review of 716 chemicals (664 positive, 16 negative, and 36 with insufficient data) that have DART end-point data and are grouped into defined receptor binding and chemical domains. When tested against a group of chemicals not included in the training set, the decision tree is shown to identify a high percentage of chemicals with known DART effects. It is proposed that this decision tree could be used both as a component of a screening system to identify chemicals of potential concern and as a component of weight-of-evidence decisions based on structure-activity relationships (SAR) to fill data gaps without generating additional test data. In addition, the chemical groupings generated could be used as a starting point for the development of hypotheses for in vitro testing to elucidate mode of action and ultimately in the development of refined SAR principles for DART that incorporate mode of action (adverse outcome pathways).

Correlation of chemical structure with reproductive and developmental toxicity as it relates to the use of the threshold of toxicological concern.

In the absence of toxicological data on a chemical, the threshold of toxicological concern (TTC) approach provides a system to estimate a conservative exposure below which there is a low probability of risk for adverse health effects. The original toxicology dataset underlying the TTC was based on NOELs from repeat dose studies. Subsequently there have been several efforts to assess whether or not these limits are also protective for reproductive/developmental effects. This work expands the database of chemicals with reproductive and developmental data, presents these data in a comprehensive and transparent format and groups the chemicals according to the TTC "Cramer Class" rules. Distributions of NOAELs from each of these classes were used to assess whether the previously proposed TTC values based on repeat dose data are protective for reproductive/developmental toxicity endpoints as well. The present analysis indicates that, for each Cramer Class, the reproductive and developmental endpoints would be protected at the corresponding general TTC tiers derived by Munro et al. (1996).

Synthesis and evaluation of unsaturated caprolactams as interleukin-1beta converting enzyme (ICE) inhibitors.

Peptidomimetic compounds possessing a caprolactam ring constraint were prepared and evaluated as interleukin-1beta converting enzyme (ICE) inhibitors. The caprolactam ring was used to constrain the P3 region of our inhibitors. This strategy proved to be effective for the synthesis of ICE inhibitors, maintaining key hydrogen bond interactions with the enzyme and invoking a preferred conformation for binding. Several compounds exhibited IC(50) values less than 10nM in a caspase-1 enzyme assay and less than 100nM in a THP-1 whole cell assay measuring IL-1beta production. Two compounds, 13c and 13j, were found to have good oral bioavailability (>50%) in rats when administered as prodrugs.

Synthesis and evaluation of novel 8,5-fused bicyclic peptidomimetic compounds as interleukin-1beta converting enzyme (ICE) inhibitors.

An 8,5-fused bicyclic peptidomimetic ring system generated by a stereoselective ring metathesis reaction was elaborated into potent inhibitors of interleukin-1beta converting enzyme (ICE, caspase-1). Multiple compounds were found that exhibited ICE IC50 values < 10 nM and were selective over caspase-3 and caspase-8. These active analogs generally possessed good activity (IC50 values < 100 nM) in a whole cell assay measuring IL-1beta production. Pharmacokinetic analysis of the ethyl acetal prodrug form of a selected active lead revealed a compound with a reasonable plasma half-life (1.1 h) and good oral bioavailability (30%).

Design and synthesis of novel N-sulfonyl-2-indole carboxamides as potent PPAR-gamma binding agents with potential application to the treatment of osteoporosis.

The synthesis and structure-activity relationships of a novel series of N-sulfonyl-2-indole carboxamides that bind to peroxisome proliferator-activated receptor gamma (PPAR-gamma) are reported. Chemical optimization of the series led to the identification of 4q (IC(50)=50 nM) as a potent binding agent of PPAR-gamma. Also reported is preliminary cell based data suggesting the use of these compounds in the treatment of osteoporosis.

Synthesis and evaluation of thiazepines as interleukin-1beta converting enzyme (ICE) inhibitors.

A series of monocyclic thiazepine inhibitors of interleukin-1beta converting enzyme (ICE) were synthesized in eight steps from commercially available intermediates. In vitro biological evaluation showed the thiazepines to be moderately potent ICE inhibitors, with the most active compound exhibiting an IC50 value of 30 nM in an enzyme inhibition assay. Compounds of this class possessed good selectivity against the related enzymes caspase-3 and caspase-8.

Synthesis and evaluation of novel 1-(2-acylhydrazinocarbonyl)-cycloalkyl carboxamides as interleukin-1beta converting enzyme (ICE) inhibitors.

Novel 1-(2-acylhydrazinocarbonyl)cycloalkyl carboxamides were designed as peptidomimetic inhibitors of interleukin-1beta converting enzyme (ICE). A short synthesis was developed and moderately potent ICE inhibitors were identified (IC(50) values <100 nM). Most of the synthesized examples were selective for ICE versus the related cysteine proteases caspase-3 and caspase-8, although several dual-acting inhibitors of ICE and caspase-8 were identified. Several of the more potent ICE inhibitors were also shown to inhibit IL-1beta production in a whole cell assay (IC(50) < 500 nM).

Synthesis and evaluation of novel 8,6-fused bicyclic peptidomimetic compounds as interleukin-1beta converting enzyme inhibitors.

Two novel 8,6-fused bicyclic peptidomimetic ring systems were synthesized utilizing olefin metathesis as the key reaction for the formation of the eight-membered ring. Both peptidomimetic scaffolds were further elaborated into potent ICE inhibitors, with numerous compounds exhibiting caspase-1 IC(50)s less than 10nM.

Discovery of novel conformationally restricted diazocan peptidomimetics as inhibitors of interleukin-1beta synthesis.

A novel diazocan containing dipeptide mimetic was synthesized via reductive N-N bond cleavage of a pyrazolidino-pyrazolidine using Raney-Ni and evaluated as an ICE inhibitor. This versatile 8-membered ring containing scaffold possesses an N-5 ring nitrogen that was used to explore structure-activity relationships in a cell-based assay measuring inhibition of interleukin-1beta.

Synthesis and evaluation of tricyclic pyrrolopyrimidinones as dipeptide mimetics: inhibition of interleukin-1beta-converting enzyme.

The application of a tricyclic pyrrolopyrimidinone scaffold for the synthesis of peptidomimetic inhibitors of interleukin-1beta-converting enzyme (ICE) is reported. The synthesis of the tricyclic scaffold and conversion of it to a variety of target ICE inhibitors were accomplished in 4-5 steps. In vitro biological evaluation of the tricyclic pyrrolopyrimidinones revealed fair to good ICE inhibitors, with the most active compound exhibiting an IC50 of 14 nM in a caspase-1 enzyme binding assay.