In Chemico Test for Toxicity — U.S. Patent US12559784B2
A cell-free, enzyme-activity-based platform that measures biochemical impact across nine human tissue compartments simultaneously. No animals. No cell culture. GHS-compliant results in 6 hours.
What the Patent Covers
The invention describes a cell-free, reagent-based assay in which a test material is exposed to a defined enzyme or enzyme mixture. The degree to which the test material reduces enzymatic activity is used to predict the extent and classification of toxicity to living tissue. Because the assay uses no animal tissues, cells, or cell cultures, it is classified as an in chemico method — the most shelf-stable and field-deployable category of nonanimal test under OECD guidelines.
Shelf-stable reagent kit format suitable for field use and high-throughput laboratory screening, applicable to acids, alkalis, oxidizers, and surfactants.
Applying a test substance to a predefined enzyme or enzyme mixture to effect an in chemico reaction
Measuring any reduction in enzymatic activity on a predefined substrate
Comparing the measured reduction to a control or previously established activity value
Predicting the extent or classification of living tissue toxicity based on the measured reduction
Nine Human Tissue Compartments
The enzyme-activity reduction platform is not limited to skin. The WIPO abstract and patent specification disclose applicability to the full spectrum of human tissue toxicity assessment.
Skin (Dermal)
The most developed application, commercialized as DermaSafe SCT (In Chemico Skin Corrosion Test). Resolves GHS Category 1 into subcategories 1A, 1B, and 1C — a key differentiator over RhE models which typically resolve only Category 1 as a whole. Correlates quantitatively with depth of injury for DOT packing group assignment.
Ocular (Eye)
The enzyme-activity reduction principle extends directly to ocular toxicity prediction. The eye contains lysozyme, lactate dehydrogenase, and corneal esterases that can be modeled in an in chemico format. Provides a pathway to a fully cell-free ocular irritation test complementing the OECD TG 496-listed OptiSafe platform.
Pulmonary (Lung)
The lung presents surfactant-associated proteins, proteases, and oxidoreductases that can be modeled using the enzyme-activity reduction platform. Applications include assessment of aerosols, vapors, and respirable particulates for occupational and consumer safety.
Hepatic (Liver)
The liver is the primary site of xenobiotic metabolism. Liver-specific enzymes including ALT, AST, and CYP450 isoforms are well-characterized biomarkers of hepatotoxicity. The platform can be configured with hepatic enzyme panels to provide a rapid, cell-free screen for hepatotoxic potential before IND-enabling studies.
Neurological
The nervous system relies on tightly regulated enzymatic processes including acetylcholinesterase (AChE) — the target of organophosphate and carbamate pesticides. AChE inhibition is a validated endpoint for predicting cholinergic toxicity. The A/B ratio (AChE vs BChE inhibition) distinguishes true cholinesterase inhibitors from non-selective inhibitors.
Developmental / Reproductive
Key enzymatic processes involved in retinoic acid signaling, folate metabolism, and steroidogenesis can be disrupted by toxic chemicals. CYP19A1 (aromatase) inhibition is a primary endpoint for endocrine disruptor identification under EPA EDSP screening.
Acute Systemic
The platform can be configured as a broad-spectrum acute toxicity screen using panels of enzymes representative of multiple organ systems, providing a rapid tier-1 assessment before more targeted tissue-specific testing.
Renal (Kidney)
The kidney is a major target of chemical toxicity due to its role in concentrating and excreting xenobiotics. Renal tubular enzymes including NAG, alkaline phosphatase, and GGT are sensitive biomarkers of nephrotoxicity. The platform can predict renal tubular injury from industrial chemicals, heavy metals, and pharmaceutical compounds.
Cardiac
Cardiotoxicity is a leading cause of drug withdrawal from the market. Cardiac-specific enzymes including CK-MB and LDH-1 isoforms can be incorporated into the platform to provide a rapid, cell-free screen for chemicals with cardiotoxic potential.
High-Throughput Workflow: Chemical to Report in 6 Hours
Using standard 96-well or 384-well microplate formats, automated liquid handling, and a high-throughput spectrophotometric plate reader, a single operator can test up to 30 different chemicals simultaneously across the full human tissue panel.
Sample Intake
Up to 30 test materials (industrial chemicals, drug candidates, consumer product formulations, or environmental samples) are received and logged. Each material is prepared at a defined concentration range in aqueous buffer.
Automated Plate Setup
An automated liquid handling robot aspirates and dispenses test materials, enzyme reagents, and substrate solutions into the microplate wells. Each well receives a precisely metered volume (10–50 µL per well in 384-well format), enabling all 30 test chemicals to be run in triplicate across the full tissue enzyme panel on a single plate.
Incubation: Enzyme-Chemical Reaction
Plates are incubated at 37°C for a defined period (typically 1–4 hours depending on the tissue enzyme panel). The test chemical reacts with the enzyme reagent. Chemicals toxic to a given tissue type reduce the activity of the corresponding enzyme — the greater the toxicity, the greater the reduction.
Spectrophotometric Plate Reader
After incubation, the substrate solution is added and the plate is read by a high-throughput spectrophotometric plate reader (e.g., BioTek Synergy Neo2, Molecular Devices SpectraMax). Reduced absorbance relative to the enzyme-only control indicates enzyme inhibition and predicted tissue toxicity. A full 384-well plate is read in under 2 minutes.
384-Well Microplate Format
The 384-well format enables all 30 test chemicals to be run in triplicate across 9 tissue enzyme panels on a single plate, with sufficient wells remaining for positive and negative controls. Low reaction volumes (10–50 µL per well) minimize reagent consumption and allow testing of precious or limited-quantity samples.
Report Generation
Raw absorbance data are processed to calculate percent enzyme activity remaining for each test chemical × tissue panel combination. Results are expressed as a quantitative toxicity score and a GHS/regulatory classification. A complete multi-tissue toxicity report for all 30 chemicals is generated within 6 hours of sample receipt.
Full Human Tissue Panel Report
Example output for a strong oxidizing agent (10% NaOCl, CASRN 7681-52-9) tested across all nine tissue enzyme panels. Up to 30 test chemicals in a batch receive an equivalent multi-tissue profile within the same 6-hour run.
| Tissue Panel | Enzyme Marker | Wells | % Act. Remaining | Classification |
|---|---|---|---|---|
| Skin (Dermal) | Biomimetic skin enzyme substrate | A1–A3 | 4.2% | GHS Cat. 1A: Corrosive (DOT PG I) |
| Ocular (Eye) | Corneal esterase / lysozyme | B1–B3 | 7.1% | GHS Cat. 1: Serious Eye Damage |
| Pulmonary (Lung) | Surfactant protein / lung esterase | C1–C3 | 11.3% | Acute Inhalation Hazard: Cat. 1 |
| Hepatic (Liver) | ALT / CYP3A4 activity | D1–D3 | 38.4% | Moderate hepatotoxic potential |
| Neurological | Acetylcholinesterase (AChE) | E1–E3 | 82.1% | Not classified, low neurotoxic potential |
| Developmental / Repro. | RALDH / aromatase (CYP19A1) | F1–F3 | 44.0% | Potential endocrine disruptor, flag |
| Acute Systemic | Multi-organ enzyme panel | G1–G3 | 9.0% | GHS Acute Tox. Cat. 1: Oral/Dermal |
| Renal (Kidney) | NAG / GGT activity | H1–H3 | 51.2% | Moderate nephrotoxic potential |
| Cardiac | CK-MB / LDH-1 isoforms | I1–I3 | 76.3% | Not classified, low cardiotoxic potential |
Applications of the Full Human Tissue Panel
Drug Development
Screen 30 structural analogs simultaneously to identify the candidate with the most favorable multi-tissue safety profile. Hepatic enzyme panel results (ALT, CYP3A4) flag DILI potential before IND-enabling studies. CK-MB and LDH-1 isoform data provide early cardiotoxicity signals complementing hERG channel assays.
Regulatory Toxicology
Multi-tissue results directly support GHS hazard classification across multiple endpoints from a single test run. Supports REACH registration, OECD test guideline submissions, DOT packing group assignment, and FDA cosmetic/OTC drug safety substantiation under MoCRA.
Research Toxicology
Rapidly map the toxicological SAR of a chemical series across nine tissue types. Test complex mixtures for multi-tissue hazard in a single run. Use tissue-specific enzyme inhibition patterns to generate mechanistic hypotheses. Compatible with HTS workflows for large compound library screening.
Industrial Chemical Safety
Provides a cost-effective alternative to in vivo studies for REACH Annex VII–X endpoints. Dermal and systemic toxicity data support DOT packing group assignment and proper shipping name determination for hazardous materials transport.
AI-Driven Analysis of Multi-Tissue OD Data
The nine-tissue OD matrix is a high-dimensional dataset ideally suited for machine learning. AI analysis produces predictions of human toxicity that are more accurate, more mechanistically informative, and more directly applicable to human risk assessment than any existing approach.
Supervised GHS Classification
Gradient-boosted tree models (XGBoost) or random forests trained on reference datasets predict GHS hazard categories directly from the OD matrix. Published benchmarks for single-tissue in chemico assays achieve 85–92% concordance with in vivo GHS classifications; the nine-tissue panel substantially exceeds this by providing cross-tissue context.
Deep Learning for Mechanism-of-Action
A CNN or transformer architecture trained on the full OD matrix, treating the 9 × N concentration-response matrix as an image, learns to classify compounds by mechanism of action without explicit feature engineering — covering membrane disruption, reactive metabolite formation, cholinesterase inhibition, endocrine disruption, and mitochondrial toxicity.
QSAR Integration
The OD matrix combined with molecular descriptors (RDKit, Mordred) trains multi-output QSAR models that predict tissue-specific toxicity from chemical structure alone. A multi-output neural network with nine output nodes enables virtual screening of large compound libraries for multi-tissue hazard before any physical synthesis.
PBPK Model Integration
Tissue-specific EC₅₀ values from the OD matrix are directly usable as input parameters for physiologically based pharmacokinetic (PBPK) models. Coupling in chemico data with PBPK enables prediction of target tissue concentration, margin of exposure, route-of-exposure sensitivity, and sensitive subpopulation risk.
Bayesian Uncertainty Quantification
Bayesian network models propagate uncertainty from raw OD measurements through dose-response fitting into the final GHS classification, producing a probability distribution over hazard categories. A submission reporting '95% probability of GHS Category 1 skin corrosion' is more informative and defensible than a point prediction.
Transfer Learning & Cross-Species Extrapolation
Pre-trained toxicology models (EPA ToxCast: 9,000+ chemicals, 700+ assays; NIH Tox21: 12,000+ chemicals, 72 assays) can be fine-tuned on the nine-tissue OD matrix using transfer learning. Fine-tuning on 200–500 compounds produces highly accurate predictions and enables cross-species extrapolation for regulatory submissions.
Development Outlook
Five-Year Outlook (2026–2031)
Benchtop unit integrating robotic liquid handling, nine-tissue reagent dispensing, microplate reader, and AI analysis software. Validated against the reference dataset from NIH/NIEHS R44 ES036065. Target: 50 compounds/week, full GHS report in 6 hours.
Submission of a prevalidation dossier to the OECD Test Guidelines Programme, building on OECD TG 431 and TG 496 precedents. Parallel submission to EPA under the Alternative Test Method (ATM) program. Estimated timeline to OECD acceptance: 3–5 years from submission.
Release of the first public nine-tissue QSAR model, trained on 500+ compounds. Contribution of the reference dataset to EPA DSSTox and NIH Tox21 databases. First peer-reviewed publications on AI-driven multi-tissue prediction accuracy versus in vivo outcomes.
Integration into pharmaceutical lead optimization workflows at 10+ major pharma companies. Adoption by chemical manufacturers for REACH registration support. First regulatory submissions (EPA, ECHA) citing nine-tissue in chemico data as primary evidence for multi-endpoint GHS classification.
Twenty-Year Vision (2026–2046)
OECD acceptance of the nine-tissue panel as a validated test method. EPA, ECHA, and FDA issue guidance recognizing multi-tissue in chemico data packages as sufficient for multi-endpoint GHS classification. Estimated impact: elimination of 500,000+ animal tests per year globally.
Integration with human genomic databases enables prediction of individual-level chemical sensitivity based on genetic variants in metabolic enzymes (CYP2D6, CYP2C19, NAT2). The nine-tissue PBPK model, parameterized with individual genomic data, predicts personalized occupational exposure limits.
Miniaturized, field-deployable versions integrated into smartphones, wearable sensors, and environmental monitoring networks provide real-time chemical hazard assessment for drinking water, air quality, food safety, and consumer product safety.
Accumulated nine-tissue OD profiles for hundreds of thousands of compounds, combined with molecular generative AI, enables design of new chemicals optimized simultaneously for desired function and minimal multi-tissue toxicity — making 'safe by construction' the default paradigm for new chemical development.
Test Kits Based on This Technology
The patented enzyme-activity reduction platform is commercially available as validated test kits through InChemico — a wholly owned subsidiary of Lebrun Labs.
OptiSafe EIT™ Main Assay
- 100% sensitivity — zero false negatives for nonirritants
- GHS NC or refers for further testing
- No aseptic technique required
- Shelf-stable vegan matrix
- Results in under 24 hours
- Kit tests 3 samples
IVD EIT™ Main Kit
- Discriminates all 4 GHS eye categories (NC, 2B, 2A, 1)
- >90% accuracy per GHS classification
- Highest mechanistic relevance for ocular drug studies
- Uses food-source animal eyes — qualifies as nonanimal
- Only method to correctly discriminate GHS 2B vs. 2A
- Requires IVD Viability or TUNEL kit (sold separately)
DermaSafe SCT™ High-Throughput
- GHS 1A / 1B+1C / Not Corrosive classification
- DOT packing group assignment (I, II+III, IV)
- Colorimetric plate reader, high-throughput format
- Results in under 6 hours at room temperature
- Shelf-stable, no animals, no cell culture
- Lab and field-deployable formats available
Submit samples directly to the Lebrun Labs affiliate lab for GHS classification reports and regulatory data packages. OptiSafe EIT™, IVD EIT™, and DermaSafe SCT™ testing available.
Eye Irritation & Skin Corrosion Testing for Cosmetic Manufacturers
The patented in chemico platform underpins OptiSafe (eye irritation test, OECD TG 496) and DermaSafe SCT (skin corrosion test) — validated nonanimal methods used by cosmetic manufacturers, ingredient suppliers, and CROs for GHS safety classification and regulatory labeling compliance.
Cosmetic Eye Irritation Testing (OECD TG 496)
OptiSafe is the only fully cell-free, in chemico eye irritation test listed in OECD Test Guideline 496. Cosmetic manufacturers use it to classify finished formulations, rinse-off products, eye-area products, and raw ingredients for GHS eye irritation hazard — without animal testing. Results in under 24 hours.
Cosmetic Skin Corrosion Testing
DermaSafe SCT classifies cosmetic ingredients and formulations for GHS skin corrosion hazard (1A, 1B+1C, Not Corrosive) and DOT packing groups. 98% global accuracy — the highest of any validated method. Shelf-stable, high-throughput, no cell culture, no animals.
Cosmetic Ingredient Safety Screening
Screen new cosmetic ingredients — surfactants, preservatives, actives, fragrances — for ocular and dermal safety before formulation. The high-throughput platform tests up to 30 chemicals per run in under 6 hours across 9 human tissue types simultaneously.
GHS Regulatory Compliance & Labeling
Both OptiSafe and DermaSafe SCT generate GHS classification data accepted for regulatory submissions under REACH, GHS, and DOT. OptiSafe is listed in OECD TG 496 — the international standard for nonanimal eye irritation testing. Full regulatory data packages available from LebrunLabs.com.
Need eye irritation or skin corrosion testing for your cosmetic formulation?
OptiSafe (OECD TG 496) and DermaSafe SCT are available as test kits at InChemico.com or as contract testing services at LebrunLabs.com.
Patent & IP Details
Contract Research & Licensing
Lebrun Labs offers contract research services and licensing opportunities built on the patented enzyme-activity reduction platform. Whether you need GHS-compliant toxicity testing for a single chemical or are interested in licensing the technology for your own testing program, our team can help.
Research supported by NIH/NIEHS under Award Number R44 ES036065. Content does not represent official NIH views.