A method for predicting the toxicity of a test substance in living tissues, a method for identifying a toxin, products or kits used for testing toxicity, for identifying toxic chemicals, or for identifying dermal corrosives.

Description

METHOD FOR PREDICTING THE TOXICITY OF A TEST SUBSTANCE In living tissues, a method for identifying a toxin, products or kits used to test toxicity, for To identify toxic chemicals, or to identify DERMAL CORROSIVES CROSS-REFERENCE TO RELATED REQUESTS

[001] This application claims the benefit of Provisional Application No. U.S.63 / 448,157, filed February 24, 2023, the contents of which are incorporated herein by reference. BACKGROUND

[002] Chemical toxicity testing often involves live animals. However, live animals have limitations regarding the costs of studies, acquisition and duration of studies, subjective assessment, variability in animal responses, and concerns about ethics, animal suffering, and new laws prohibiting animal testing for many applications.

[003] For example, many different animal species have been used to determine toxicity to different target organs. Eye toxicity studies have been performed on rabbits (Draize et al., 1944), skin toxicity studies have also been performed on rabbits (Draize et al., 1944), skin sensitization studies have been performed on guinea pigs (Robinson et al., 1990), pulmonary inhalation toxicity studies have been performed on rats, guinea pigs, dogs, monkeys, and hamsters (Phalen, 1976; Robinson et al., 1990), acute toxicity studies have been performed on rats (Bartsch et al., 1976), studies of Petition 870260062914, dated 06 / 26 / 2026, page 6 / 157 2 / 147 Hepatotoxicity studies were performed in different species of rats (Kikkawa et al., 2006), renal toxicity studies were performed in rabbits and rats (Mengs and Stotzem, 1993; Pettersson et al., 2002), cardiotoxicity studies were performed in rabbits, dogs and monkeys (Lamberti et al., 2014) and neurotoxicity studies were performed in rats (Costa, 1998).

[004] Modern toxicity classification systems are based on reference animal toxicity data to develop toxicity categories. Modern classification schemes include the European Union (EU), the Globally Harmonised System of Classification and Labelling of Chemicals (GHS), and the Environmental Protection Agency (EPA) systems.

[005] GHS system for eye: With regard to ocular toxicity, the GHS categories include NC (not classified as an irritant), Category 2A (reversal in 7 days), Category 2B (reversal in 14 days) and Category 1 (no reversal in 21 days) (EC, 2008b; UN, 2021).

[006] GHS Skin System: Regarding skin toxicity, the GHS categories include Category 3 (mild irritation; mean score of > 1.5 and < 2.3 for erythema / eschar or edema in at least 2 of 3 animals at 24, 48, and 72 hours), Category 2 (irritation; mean score of > 2.3 and < 4.0 for erythema / eschar or edema in at least 2 of 3 animals tested at 24, 48, and 72 hours or inflammation that persists until the end of 14 days), and Category 1 (corrosive; necrosis in at least one animal tested after exposure for < 4 hours) (UN, 2021). Category 1 can be divided into three subcategories: Category 1A (corrosive responses in fur). Petition 870260062914, dated 06 / 26 / 2026, page 7 / 157 3 / 147 minus one animal during the exposure period of < 3 min), Category 1B (corrosive responses in at least one animal during an exposure period of < 1 hour), Category 1C (corrosive responses in at least one animal during an exposure period of ^ 4 hours) (UN, 2021). For skin sensitization, the GHS categories include Category 1, which is divided into Category 1A (substances that have a high frequency of occurrence in humans or animals) or Category 1B (substances that have a low to moderate frequency of occurrence in humans or animals) (UN, 2021).

[007] GHS System for Lungs: For acute toxicity (inhalation), the GHS categories include Category 1 (gases [ppmV]: estimated acute toxicity [ATE] < 100; vapors [mg / l]: ATE < 50; dusts and mists (mg / l): ATE < 0.05), Category 2 (gases [ppmV]: 100 < ATE < 500; vapors [mg / l]: 0.5 < ATE < 2.0; dusts and mists (mg / l): 0.05 < ATE < 0.5), Category 3 (gases [ppmV]: 500 < ATE < 2,500; vapors [mg / l]: 2.0 < ATE < 10.0; dusts and mists (mg / l): 0.5 < ATE < 0.1), Category 4 (gases [ppmV]: 2,500 < ATE < 20,000; Vapors [mg / l]: 10 < ATE < 20.0; dusts and mists (mg / l): 1.0 < ATE < 5.0), and Category 5 (gases [ppmV]: ATE > 20,000; Vapors [mg / l]: ATE > 20.0; dusts and mists (mg / l): ATE > 5.0) (UN, 2021). For respiratory sensitizers, the GHS classes include Category 1, which is divided into Category 1A (substances that occur frequently in humans or animals) or Category 1B (substances that occur frequently in humans or animals at low to moderate frequency). (UN, 2021).

[008] GHS system for acute toxicity: For acute toxicity (oral; mg / kg body weight), the categories Petition 870260062914, dated 06 / 26 / 2026, page 8 / 157 4 / 147 of GHS include Category 1 (ATE < 5), Category 2 (5 < ATE < 50), Category 3 (50 < ATE < 300), Category 4 (300 < ATE < 2,000) and Category 5 (2,000 < ATE < 5,000) (UN, 2021). For acute toxicity (dermal; mg / kg body weight), the GHS categories include Category 1 (ATE < 50), Category 2 (50 < ATE < 200), Category 3 (200 < ATE < 1,000), Category 4 (1,000 < ATE < 2,000) and Category 5 (2,000 < ATE < 5,000) (UN, 2021). 2021).

[009] The toxicity classification is used to meet U.S. Food and Drug Administration and international safety labeling requirements and plays an important role in commercial product liability and consumer product satisfaction. Guidance documents produced by the Organization for Trade and The Organization for Economic Development (OECD) is available to coordinate international trade. The OECD describes standard toxicity tests, which are required for the documentation of the safety data sheet that accompanies chemicals and hazardous products. OECD-accepted toxicity tests are separated by target tissue and test type: in vivo (live animal), ex vivo (animal tissue, e.g., eyes, skin, or lungs from the meat industry), in vitro (cell culture, including monolayer and three-dimensional cultures of primary or immortalized cells), and in chemico (cell-free, test matrix is ​​purified and semi-purified chemicals and macromolecules).

[0010] In vivo testing methods include the Draize eye and skin irritation test (Draize et al., 1944; OECD, 2015a, 2021a), the 4-h Human Patch Test (HPT) (York et al., 1996), and the Local Lymph Node Assay (LLNA) (Gerberick et al., 1996). Petition 870260062914, dated 06 / 26 / 2026, page 9 / 157 5 / 147 al., 2007; OECD, 2010), Guinea Pig Maximization Test (GPMT) and Buehler Test (Phalen, 1976; Robinson et al., 1990; OECD, 2022a)

[0011] Ex vivo testing methods include bovine corneal opacity and permeability (BCOP) (OECD, 2020a), isolated chicken eye (ICE) (OECD, 2018), chicken egg-chorioallantoic membrane (HET-CAM) test (ICCVAM, 2010), ex vivo human skin (Eberlin et al., 2021), porcine corneal ocular reversibility (Piehl et al., 2011), and ex vivo ocular irritation test (Spoler et al., 2015).

[0012] In vitro testing methods include Transcutaneous Electrical Resistance (TER) Test (OECD, 2015b), Human Skin Model Test (OECD, 2004), Neutral Red Capture 3T3 (NRU) (OECD, 2019a), Reconstructed Human Epidermis (RhE; EpiSkin™, EpiDerm™ Skin Irritation Test [SIT], SkinEthic™ RHE, LabCyte EPI-MODEL24 SIT, epiCS®, Skin+®, KeraSkin™ SIT) (OECD, 2021b), Leakage of Fluorescein (FL) (OECD, 2017), Short-Term Exposure (STE) (OECD, 2020b), Reconstructed Human Corneal-Like Epithelium (RhCE); EpiOcular™ Eye Irritation Test [EIT], SkinEthic™ Human Corneal Epithelium [HCE] EIT, LabCyte CORNEA-MODEL24EIT, MCTT HCE™ EIT] (OECD, 2019b), ARE-Nrf2 Luciferase Assay (OECD, 2022c) and Human Cell Line Activation Assay (h-CLAT) (OECD, 2022d)

[0013] In-chemical testing methods include Direct Peptide Reactivity Assay (DPRA) (OECD, 2022b), Eye Irritation® (OECD, 2019c), OptiSafe™ (Choksi et al., 2020; Lebrun et al., 2021a, 2021b, 2022, 2023a, 2023b) and Corrositex® (OECD, 2015c; Ulmer and Wang, 2017). IN VIVO TOXICITY TESTS Petition 870260062914, dated 06 / 26 / 2026, page 10 / 157 6 / 147

[0014] The Draize in vivo eye test is used to predict the potential for ocular irritation and corrosion by exposing the eyes of live New Zealand white rabbits to a test substance (Draize et al., 1944; OECD 2021a). The test substance is applied to the conjunctival sac of one eye, while the other serves as an untreated control. Assessments of the rabbit's conjunctiva, cornea, and iris are then performed 1, 24, 48, and 72 hours after exposure, and sometimes at 7 and 21 days if necessary (OECD, 2021a).

[0015] The Draize in vivo skin test is used to predict skin irritation and corrosion potential by exposing a test substance to the skin of live New Zealand white rabbits (Draize et al., 1944; OECD, 2015). The test substance is applied to the shaved skin of the rabbits (usually 3-6) and covered with a gauze adhesive for 4 hours, then assessed at 60 minutes, 24, 48, and 72 hours after adhesive removal to check the potential of a chemical to damage the skin, measuring the clinical rating of erythema and eschar formation and edema formation based on a severity scale (OECD, 2015). Dermal irritant chemicals cause reversible redness and swelling after application of a test substance for up to 4 hours (OECD, 2015). Dermal corrosive chemicals result in necrosis through the epidermis to the dermis in at least one animal after exposure for up to 4 hours.

[0016] The Human Patch Test (4-h HPT) is a clinical test in humans. To test for skin irritation, the test substance is applied via an adhesive patch to the upper arm of the volunteer. The potential for irritation is Petition 870260062914, dated 06 / 26 / 2026, page 11 / 157 7 / 147 evaluated and a positive test is defined as a localized erythema reaction, scoring according to the convention 24, 48 and 72 hours after patch removal; + (weak: erythema, possibly papules), ++ (strong: vesicles, infiltration) or ++ (extreme: bullous) excluding ?+ (doubtful: only weak erythema) (York et al., 1996). For ethical reasons, the 4-hour HPT test is not used to identify dermal corrosives.

[0017] The Local Lymph Node Assay (LLNA) evaluates the skin sensitization potential of a test substance as an alternative to guinea pig assays (Guinea Pig Maximization Test or Buehler Test). The test substance is applied to the animal's ears (back) for 3 consecutive days and monitored daily for any response, then the animals are rested for 2 days and thymidine is injected into the tail and they are returned to their cases to rest for 5 hours before euthanasia. The lymph nodes are excised for processing and the results are calculated by measuring the total disintegrations per minute for each lymph node (Gerberick et al., 2007; OECD, 2010). IN TOXICITY TESTS IN CHEMICAL (CELL-FREE)

[0018] Current chemical tests include the Direct Peptide Reactivity Assay (DPRA) for the identification of skin sensitizers (an allergic response after skin contact with the tested chemical) and the Macromolecular Eye Irritation Test (which includes the OptiSafe™ Eye Irritation Test and another eye test, Ocular Irritation®) (OECD, 2019c; Choksi et al., 2020) and the Corrositex® test for skin corrosives. None of these use enzymes or measure enzymatic activity (OECD, 2015c, 2019c; Choksi et al., 2020). Petition 870260062914, dated 06 / 26 / 2026, page 12 / 157 8 / 147

[0019] The Direct Peptide Reactivity Assay (DPRA) in chemical testing models the molecular initiation event of skin sensitization by measuring the binding of chemicals to synthetic model peptides containing lysine or cysteine. When these peptides are bound by the chemical under evaluation, the peptides have different HPLC elution profiles. The remaining concentration of unmodified cysteine ​​or lysine (not bound to the chemical under evaluation) after a 24-hour incubation is used to predict sensitization (Roberts, 2022; OECD, 2022c).This test predicts the ability to trigger an allergic response, presumably because the modified amino acids are no longer recognized and the immune system becomes stimulated and responds to modified proteins, but since some of these proteins are part of the organism, the immune system begins to attack its own tissues, resulting in an allergic response after exposure to the chemical (Roberts, 2022; OECD, 2022c). The concentration of unmodified peptides is measured by high-performance liquid chromatography (HPLC) with elution at 220 nm; no enzymatic activity is measured or involved in the test. The depletion values ​​of cysteine ​​and lysine peptides are then used in a predictive model that predicts whether the chemical is a sensitizer or non-sensitizer (Roberts, 2022; OECD, 2022c). No enzymes are evaluated or used for the DPRA.

[0020] Macromolecular eye irritation testing methods are in-chemical methods that use a suite of multiplexed biochemical tests to assess eye irritation potential. The multiplex design allows for the identification of chemicals within 24 hours. To conduct the Petition 870260062914, dated 06 / 26 / 2026, page 13 / 157 In the 9 / 147 assay, the test chemical is applied to macromolecules and the effects are quantified using a spectrophotometer. Optical density (OD) values ​​are used to provide estimates of the chemical's potential to cause eye injury (Choksi et al., 2020; Lebrun et al., 2021a, 2021b, 2022, 2023a, 2023b). No enzymes are evaluated during macromolecular tests.

[0021] The Corrositex® test measures the time it takes for a test chemical to pass through a synthetic biobarrier (OECD, 2015c). This test system consists of two components: a synthetic macromolecular biobarrier and a chemical detection system (CDS). The time to move through the biobarrier is determined by setting a timer and waiting until the pH indicator on the other side of where the chemical was applied changes color. The corrosiveness potential of a test chemical is determined by its ability to destroy the biobarrier, which can be observed through a color change as the pH changes to less than 4.5 or above 8.5. Three factors are taken into account: acid or base strength, diffusion rate, and biobarrier destruction rate (Ulmer and Wang, 2017). No enzymes are used or evaluated for the Corrositex® test. ENZYME TESTS AND LIVE CELL ACTIVITY

[0022] The MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) can be used to measure the metabolic activity of living cells to determine if a cell is alive. If there are sufficient reduced cofactors related to energy metabolism (NADH, NADPH), the MTT is reduced, resulting in a positive MTT reaction (forms a purple precipitate inside the cells). Petition 870260062914, dated 06 / 26 / 2026, page 14 / 157 A value of 10 / 147 indicates that the cell is viable (it is actively metabolizing and producing reduced cofactors). The higher the MTT signal, the more viable (alive) the cell culture is. Therefore, MTT is a positive correlate with cell viability and inversely related to toxicity. MTT is a positively charged monotetrazolium salt that, when reduced, forms a blue-violet molecule called formazan, which can be quantified using a spectrophotometer at an optical density wavelength of 570 nm (Ghasemi et al., 2021). Although cellular enzymes are involved, the critical variable is whether the entire cell is alive and actively metabolizing and producing stored energy as reduced cofactors (NADH, NADPH).

[0023] The Neutral Red assay can be used to measure cell viability and toxicity by quantifying the ability of a viable cell to accumulate neutral red with a spectrophotometer at an optical density wavelength of 540 nm (Repetto et al., 2008). The accumulation of neutral red depends on the active pumping (requiring cellular energy stored in the form of ATP) of neutral red into the cell. This test determines whether the cell is viable. As long as the enzyme is included, the test depends on the viable cell being able to accumulate neutral red within its membranes by active pumping.

[0024] The resazurin assay can be used to monitor viable cells by reducing resazurin to resorufin, which turns fluorescent pink and can be quantified using a spectrophotometer at an optical density wavelength of 590 nm (Riss et al., 2013). Like the MTT assay, this test determines the reducing power of the cell and, Petition 870260062914, dated 06 / 26 / 2026, page 15 / 157 11 / 147 therefore, is related to energy metabolism and occurs only in viable (living) cells.

[0025] The Feasibility Marker trial Protease activity can be used to measure viable cells by adding a substrate that can selectively detect protease activity in viable cells, called glycylphenylalanineaminofluorocoumarin (GF-AFC). This substrate penetrates living cells and is converted by cytoplasmic aminopeptidase to form a fluorescent aminofluorocoumarin that can be quantified using a spectrophotometer at an optical density wavelength of 505 nm (Riss et al., 2013). This test only works with living cells.

[0026] Dermal corrosion is defined as direct chemical reactivity in living skin that results in its disintegration and necrosis through the epidermis and into the dermis. Dermal corrosion likely results from a wide range of chemical mechanisms; however, the details of how chemicals disintegrate the skin, resulting in corrosion, are not fully defined. Symptoms of dermal corrosion are clinically referred to as chemical burns and include vesication (blisters), desiccation (fluid loss), necrotic tissue, scarring, ulcers, bleeding, bloody crusts, discoloration, and alopecia (OECD, 2015a; Koh et al., 2017). On the other hand, skin irritants are substances that cause temporary changes in the skin with symptoms that include itching, burning sensation, and erythema (OECD 2015; Kose et al., 2018).

[0027] Chemicals that are corrosive to the skin are classified in Category 1 (corrosive; necrosis in Petition 870260062914, dated 06 / 26 / 2026, page 16 / 157 12 / 147 at least one animal tested after exposure for < 4 hours) (UN, 2021), but contain three subcategories: Category 1A (corrosive responses in at least one animal during an exposure period of ^3 min), Category 1B (corrosive responses in at least one animal during an exposure period of ^1 hour), Category 1C (corrosive responses in at least one animal during an exposure period of ^4 hours) (UN, 2021).

[0028] Exposure to dermal corrosives occurs during the production, transportation, use, and disposal of chemicals and products. Materials are manufactured and transported from around the world to different destinations, and the transportation of hazardous goods increases the likelihood of an accident that can cause skin injuries due to improper chemical release. During 1999 to 2008, 57,975 chemical release incidents were reported to the Hazardous Substances Emergency Events Surveillance (HSEES) system operated by the Agency for Toxic Substances and Disease Registry of the Centers for Disease Control (CDC), and during that time period, 13,196 people reported injuries related to chemical releases (Orr et al., 2015). The U.S. Bureau of Labor Statistics reports that chemical burns occurred in 1 out of every 10 cases.000 full-time workers and were responsible for 0.6% of all occupational injuries (Koh et al., 2017). In a 2015 study on the severity and prevalence of chemical burns, it was found that despite representing only 3% of admissions to a particular burn center, chemical burns were responsible for up to 30% of burn-related deaths. Petition 870260062914, dated 06 / 26 / 2026, page 17 / 157 13 / 147 (Robinson and Chhabra, 2015).

[0029] Testing products and chemicals to determine dermal corrosion potential reduces human suffering and saves lives because testing allows for correct safety labeling so people can take appropriate precautions to avoid injury. Current testing methods for identifying dermal corrosives are all laboratory tests that take days - weeks (are these all laboratory tests, are there other tests, any quick field tests for dermal corrosion?).

[0030] With additional tests, corrosiveness can be divided into three subcategories: 1A (responses after up to 3 minutes of exposure and up to 1 hour of observation), 1B (responses for exposures between 3 minutes and 1 hour and observations up to 14 days), and 1C (responses that occur after exposures between 1 hour and 4 hours and observations up to 14 days) (UN, 2021).

[0031] According to the Code of Regulations Under Federal Classification Rules (CFR), corrosive substances are classified as Class 8, which is defined as a corrosive material that causes total destruction of human skin at the point of contact and are assigned as I, II, or III. Packing Group I is designated to test substances that cause irreversible damage to intact skin tissue from 3 minutes of exposure or less (49 CFR Part 173 Subpart D, 2023). Packing Group II is designated to test substances that cause irreversible damage to intact skin tissue after 3 minutes of exposure but not more than 60 minutes (49 CFR Part 173 Subpart D, 2023). Packing Group III is designated to test substances that cause irreversible damage to tissue Petition 870260062914, dated 06 / 26 / 2026, p. 18 / 157 14 / 147 intact skin after 60 minutes of exposure, but not more than 4 hours (49 CFR 173 Subpart D). Packing Groups I, II and III are equivalent to GHS Categories 1A, 1B and 1C, respectively (Alépée et al., 2014).

[0032] Corrosive substances are labeled as Category 1. This category contains three optional subcategories that correspond to UN Packing Groups I, II and III for the transport of goods. The subcategories are implemented in the EU. They differ in relation to the exposure times required to cause skin corrosion in rabbits and are referred to as 1A (strong corrosive), 1B (moderate corrosive) and 1C (mild corrosive) (UN, 2021).

[0033] Currently, there are a limited number of non-animal tests for dermal corrosion that have been recognized by the Organisation for Economic Co-operation and Development (OECD) for which test guidelines have been established. Each of these tests has been validated by comparing the in vitro corrosivity prediction with the results of Draize data for the same chemicals. These tests include the in vitro membrane barrier (OECD 435), transcutaneous electrical resistance (TER; OECD 430) and reconstructed human epidermis (RhE; OECD 431, 439).

[0034] The transcutaneous electrical resistance (TER) assay measures the skin corrosion potential of a study chemical by assessing the transcutaneous electrical resistance of rat skin and its ability to produce a loss of normal stratum corneum and barrier function (OECD, 2015b). The study chemical (150 μΣ) is applied to three rat skin discs for a maximum period of 24 hours at 20–23 °C and then removed with tap water. Petition 870260062914, dated 06 / 26 / 2026, page 19 / 157 15 / 147 and the results are quantified using a low-voltage, alternating-current Wheatstone bridge (OECD, 2015b). Additionally, if the resistance is less than 5 kΩ, the dye sulforhodamine B is added to the skin to determine if the stratum corneum is disrupted by measuring penetration (OECD, 2015b). A validation study (with modifications to reduce false positives) demonstrated broad applicability to a variety of chemicals and could distinguish between non-corrosive and corrosive substances. Predictability statistics include 94% sensitivity (51 / 54), 71% specificity (48 / 68), 81% accuracy (99 / 122), and balanced accuracy of 82.5% (OECD, 2015b). Like the in vitro membrane barrier test (OECD, 2015c), ordering rats, preparing skin discs, and performing the test has a response time of days to weeks. This is not a quick field test, it is not a chemical test, and no enzymes are involved in this test.

[0035] The reconstructed human epidermis (RhE) test method models the epidermis of human skin. Keratinocytes on a solid support are induced to differentiate at the air / liquid interface using a variety of differentiation factors and methods. The 5 commercially available skin corrosion RhE test methods are EpiSkin™, EpiDerm™ Skin Corrosion Test, SkinEthic™ RHE, epiCS®, and LabCyte EPI-MODEL24 SCT. Accuracy is 78.8% for EpiSkin™, 74.2% for EpiDerm™ SCT, 70.0% for SkinEthic™ RHE, 69.8% for epiCS®, and 76.4% for LabCyte EPI-MODEL24 SCT (OECD, 2019). For all RHE tests, tissues are exposed for 3 minutes and 1 hour, with an additional exposure time of 4 hours in the EpiSkin model. The MTT viability assay is used for Petition 870260062914, dated 06 / 26 / 2026, page 20 / 157 16 / 147 quantify cell viability and the results are compared to a set of standard chemicals to determine corrosion potential. However, this is only a model of the epidermis which may explain the low accuracy (see above). FC To order the tissues and perform the test (or send the material to be tested to a laboratory), there is a turnaround time of days to weeks. This is not a rapid field test and uses the viability of live cells as a measure of toxicity; it is not an in chemico test.

[0036] Current in chemico tests and toxicity prediction strategies do not measure acellular or dead cell enzyme activity to predict toxicity. SUMMARY

[0037] The current revelation provides methods and kits and compositions that overcome the limitations of the state of the art.

[0038] Current cell-based tests use enzymes produced in real time by living cells, and if this activity is reduced, it indicates that the cell is less viable. In other words, these tests measure the transition from alive to dead. Cell- and tissue-based assays do not start with dead cells or purified or semi-purified enzymes.

[0039] One of the needs for animal-free safety testing stemmed from bans or pending bans on the use of animals for the safety of cosmetics and other products. The EU banned animal testing of finished cosmetic products in 2004, ingredients tested on animals 4 years later, and the transport and sale of cosmetics containing ingredients tested on animals in 2013, committing to Petition 870260062914, dated 06 / 26 / 2026, page 21 / 157 17 / 147 to pressure other parts of the world to accept alternatives (Kanter, 2017). As of 2014, there have been severe bans or limitations in Norway, Israel, India, and Brazil (Senate Joint Resolution 22, 2014), and in 2017, the list of countries grew to 37, according to the Humane Society of the USA (Human Society, 2017).

[0040] The United States has been slow to ban animal testing or mandate the use of animal-free alternatives in the product testing industry; however, recent legislation will prohibit animals for a wide range of testing applications that traditionally use live animals. The Humane Cosmetics Act, bill HR2790, was introduced on June 6, 2017, and would prohibit animal testing of cosmetics within 1 year and the sale or transportation of cosmetics tested on animals within 3 years of enactment, which is now supported by over 200 cosmetic companies and stakeholders (HR4148, 2014). Additionally, the Frank R. Lautenberg Chemical Safety Act for the 21st Century Act - S. 697, which revises the Substances Control Act... Toxic Substances Act of 1976 (TSCA) – passed on June 22, 2016. The TSCA now requires the EPA to evaluate new and existing chemicals to determine whether regulatory control of a given chemical is warranted and whether it presents an unreasonable risk of harm to health or the environment, so as to reduce the risks to a reasonable level. The law also requires the EPA to reduce and replace, to the extent possible... the use of vertebrate animals in chemical testing to provide information of equivalent or superior scientific quality and relevance to the assessment of the health or environmental risks of substances. Petition 870260062914, dated 06 / 26 / 2026, p. 22 / 157 18 / 147 or chemical mixtures... and develop a strategic plan within 2 years of enactment or by June 2018 (S.697, 2016). Section 4 of the new law includes specific guidance on the use of non-animal testing where available for initial screening and layered testing of chemical substances and mixtures (S.697, 2016). Therefore, an accurate and internationally accepted non-animal test for eye irritation is required.

[0041] In light of these issues, growing interest has focused on the development of animal-free testing methods and strategies to replace live animal testing. To this end, the Interagency Coordinating Committee for the Validation of Alternative Methods (ICCVAM) and the European Centre for the Validation of Alternative Methods (ECVAM) conducted retrospective assessments of available data on animal-free testing methods. Based on these retrospective assessments, the predictive performance of all individual testing methods was not considered sufficient for any test, or group of tests, to fully replace live animal testing (ICCVAM, 2009). ICCVAM and ECVAM, however, accepted Acute Toxicity, BCOP, Cytosensor Microphysiometer (CM), FL, HET-CAM, ICE, Isolated Rabbit Eye (IRE), EpiSkin™, EpiDerm™, EpiOcular™, Transcutaneous Electrical Resistance (TER) SkinEthic™ HCE and Corrositex®.

[0042] No single animal-free test, or combination of animal-free tests, can currently detect all toxicity levels classified by the GHS (Wilson et al., 2015). There have been further advances with layered testing that suggest combining different validated testing methods. Petition 870260062914, dated 06 / 26 / 2026, p. 23 / 157 19 / 147 to accurately classify test substances and replace bottom-up or top-down testing strategies (Scott et al., 2010; Valadares et al., 2021), such as STE and BCOP (Hayashi et al., 2012; Alépée et al., 2019a), RhCE and BCOP methods (Alépée et al., 2019b), or a combination of the three (Hayashi et al., 2012b).

[0043] In general, there are a limited number of types of tests that do not require the use of animals. These assays include assays based on animal or microbial cell culture (in vitro), assays based on excised animal tissue (ex vivo), assays based on eggs (organotypic), and non-enzymatic assays (the current in chemico assays). The in vitro, ex vivo, and organotypic test arrays are black box systems. Most of the time, the molecular events that result in the measured response are not known, and the relationship to the different responses observed in vivo is unclear; and based on correlation. Furthermore, the lack of understanding of the underlying reasons why some substances are much more harmful than others has hampered the development of animal-free tests for eye safety testing.Those familiar with the state of the art strongly support the use of differentiated living tissues because these in vitro testing systems resemble the tissues being evaluated in vivo. In general, the most significant developments in toxicity testing over the past 20 years have focused on in vitro approaches based on living cells. However, in vitro cell-based approaches are still largely correlative because clinical observations for reference in vivo data are not available and, instead, another source is used. Petition 870260062914, dated 06 / 26 / 2026, page 24 / 157 20 / 147 measurement is made (e.g., cell viability), cells cultured or differentiated in the laboratory may have very different expression profiles and viability, because cells and tissues cultured in the laboratory are maintained under high, artificial growth stimulation by a variety of hormones and cofactors of ill-defined functionality and, most importantly, in vitro cell and tissue tests are not shelf-stable and are generally slow and expensive. However, as mentioned above, those familiar with the state of the art have focused almost exclusively on ex vivo, in vitro live cell and tissue culture approaches, and those familiar with the technique would likely argue against the use of purified or semi-purified enzymes to test general toxicity parameters (versus specific pharmaceutical receptors or regulatory binding studies), as disclosed in this patent document.Therefore, the use of nonspecific reduction of enzymatic activity as a basis for toxicity tests was not found in the literature and represents an unexpected finding that may enable low-cost, stable, and rapid field testing for toxicity testing purposes.

[0044] Enzymes are used to assess health status. Common enzymes that are used to correlate positively with toxicity include alkaline phosphatase, lactate dehydrogenase, alanine aminotransferase, aspartate aminotransferase, beta-glucuronidase, proteases, and antiproteases (Asmis et al., 2008). Important to this invention, it should be noted that abnormal levels of these enzymes are used to determine toxicity based on enzyme production by living cells, tissues, or organs, and this is typically a Petition 870260062914, dated 06 / 26 / 2026, page 25 / 157 21 / 147 positive correlation between more enzyme produced and more toxicity; enzymatic activity depends on living cells; these are not in-chemical tests of free or dead cells (Ambali et al., 2007). Phosphatases are a class of enzymes that catalyze the removal of phosphate groups. They exist in the blood, mostly in tissues, including the liver, heart, brain, intestines, skin, and eyes, etc. In humans, there are several isoforms (tissue-nonspecific, intestinal, placental, and germ cell) that play a role in human metabolic processes, and above-normal levels are used in the diagnosis of diseases and disorders such as bone disease, diabetes, acute kidney injury, inflammatory bowel disease (IBD), necrotizing enterocolitis, sepsis, and metabolic syndrome (Peters et al., 2014; Fawley and Gourlay, 2016; Bover et al., 2018; Danikowski and Cheng, 2019).

[0045] Elevated phosphatase levels in bronchoalveolar lavage have been used to indicate pulmonary toxicity. In one study, this activity is attributed to increased Type II cellular secretions in response to toxins (Henderson, 2005). In several other examples, higher-than-normal blood phosphatase levels are used to determine if a drug or chemical has a toxic effect on the body (Amato et al., 2009; Kartheek and David, 2018). High levels of alkaline phosphatase (ALP) may be a sign of a liver problem or a bone disorder (MedlinePlus, 2022).

[0046] A cell-free, in chemico method is provided in this document for predicting the toxicity in living tissue of a test substance.

[0047] A method for predicting the toxicity of a test substance in living tissues, wherein the method Petition 870260062914, dated 06 / 26 / 2026, page 26 / 157 22 / 147 includes:

[0048] apply the test substance to a predefined enzyme or enzyme mixture to effect a reaction in chemico, wherein the test substance does not bind preferentially or specifically to an active or regulatory site of the predefined enzyme or enzyme mixture;

[0049] measure any reduction in the enzymatic activity of the predefined enzyme or enzyme mixture on a predefined substrate; and,

[0050] compare the measured reduction in enzyme activity with a control activity value or a previously established activity value, and

[0051] predict the extent or classification of the toxicity of the test substance in living tissues based on the measured comparative reduction in enzymatic activity.

[0052] A method in chemico is disclosed for predicting the toxicity of a test substance to a tissue or cell, wherein the method comprises: applying a test substance that does not specifically bind to a predefined enzyme and / or that does not specifically bind to an active site of a predefined enzyme to the enzyme in a reaction system, measuring any reduction in enzymatic activity on a predefined substrate; and, comparing the measured reduction in enzymatic activity with a control value or previously established activity value, and predicting the toxicity of the test substance based on the compared measured reduction in enzymatic activity after treatment with more toxic test substances.

[0053] In these modalities, the enzyme is purified or semi-purified from non-viable cells or non-viable tissue, Petition 870260062914, dated 06 / 26 / 2026, page 27 / 157 23 / 147 and remnants of cells or tissues remain in the reaction system.

[0054] In these modalities, the reaction system does not comprise living cell(s).

[0055] In certain embodiments, the test substance irreversibly reduces enzymatic activity and / or reduces enzymatic activity in a non-competitive manner.

[0056] In some embodiments, the test substance reduces enzymatic activity by chemically destroying or denaturing the enzyme, and the chemistry or binding is not specific to the enzyme's active or regulatory sites.

[0057] In some embodiments, the test substance is applied in concentrations that are toxic and not therapeutic.

[0058] In these modalities, the substance to be tested is applied in concentrations of mg / ml and not in ug / ml femtograms / ml.

[0059] In these modalities, the test substances are a diverse group of toxins with no, or unknown to have, specific pharmaceutical activity.

[0060] In modalities, the tissue or cell comprises dermis.

[0061] In the various forms, the enzyme is either an alkaline phosphatase or an acid phosphatase.

[0062] In certain modalities, the toxicity is corrosive and the method predicts whether or not the test substance is corrosive.

[0063] In certain embodiments, corrosiveness is indicated if enzymatic activity is reduced to 40% or less of the control by said test substance.

[0064] In the modalities, non-corrosiveness is Petition 870260062914, dated 06 / 26 / 2026, page 28 / 157 24 / 147 indicated if the enzymatic activity is not reduced by at least 40% of the control value by said test substance.

[0065] In the modalities, the tissue or cell comprises an eye tissue or cell.

[0066] In modalities, the enzyme is an esterase.

[0067] In certain embodiments, toxicity is irritation, and the method specifies whether the test substance is irritating or non-irritating.

[0068] In the embodiments, the test substance is indicated as an irritant if the enzymatic activity is reduced by > 60%.

[0069] In the embodiments, the test substance is indicated as non-irritating if the enzymatic activity is reduced by < 60%.

[0070] In modalities, the tissue or cell comprises a tissue of the nervous system.

[0071] In embodiments, the enzyme is one of the following: 12-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylic acid synthase, 3-mercaptopyruvate sulfurtransferase, 4-hydroxytetrahydrodipicolinate reductase, 4-hydroxyphenylpyruvate dioxygenase, 6-oxide hydrolase, acetaldehyde dehydrogenase, acetolactate synthase, acetylcholinesterase, acid hydrolase, adenosine deaminase, adenosylmethionine hydrolase, adenylate cyclase, alanine aminopeptidase, alanine transaminase, alcohol dehydrogenase, alkaline phosphatase, acid phosphatase, nonspecific phosphatase, alkenylglycerophosphocholine hydrolase, alkenylglycerophosphoethanolamine hydrolase, aminolevulinic acid synthase, aminopropanol oxidoreductase, amylase, arginase, aromatase, arsenate reductase, aspartate Petition 870260062914, dated 06 / 26 / 2026, page 29 / 157 25 / 147 transaminase, aspartate transcarbamoylase, ATPase, ATP synthase, beta-carotene isomerase, beta-galactosidase, beta-lactamase, biliverdin reductase, biotin synthase, butyrate kinase, caffeine dehydrogenase, carbon disulfide hydrolase, carbonic anhydrase, carbonyl sulfide hydrolase, catalase, catechol-O-methyltransferase, catechol oxidase, chitinase, cholesterol-5, choline acetyltransferase, cholinesterase, coenzyme Q - cytochrome c reductase, cyclamate sulfohydrolase, cypridin-luciferin 2-monooxygenase, cystathionine beta-lyase, cystathionine gamma-lyase, cysteine ​​desulfurase, cytochrome c oxidase, cytochrome c peroxidase, cytochrome P450 oxidase, D-xylulose reductase, deiodinase, deoxyribonuclease, diacetyl dehydrogenase, dichloromethane dehalogenase, dihydrobenzophenanthridine oxidase, dihydrofolate reductase, DNS methyltransferase, elastase, endonuclease, exonuclease, farnesol 2-isomerase, firefly luciferase, flavin prenyltransferase, fructose-bisphosphate aldolase,Fructose bisphosphatase, furfoylmuramine isomerase, gamma-glutamyl transpeptidase, glucose oxidase, glutathione peroxidase, glutathione reductase, glutathione S-transferase, glyceraldehyde 3-phosphate dehydrogenase, glycerol-3-phosphate dehydrogenase, glycerol dehydrogenase, guanylate cyclase, haloacetate dehalogenase, halohydrin dehalogenase, helicase, hepoxylin epoxide hydrolase, histone methyltransferase, HMG-CoA reductase, homoserine dehydrogenase, hyaluronidase, hypoxanthine-guanine phosphoribosyltransferase, iodothyronine deiodinase, iodotyrosine deiodinase, isochorismatase, isocitrate dehydrogenase, isopenicillin N synthase, L-gluconolactone oxidase, L-xylulose reductase, laccase, lactase, lactate dehydrogenase, leucoanthocyanidin reductase, leukotriene-A4, Petition 870260062914, dated 06 / 26 / 2026, page 30 / 157 26 / 147 hydrolase, leukotriene C4 synthase, linolease isomerase, lipoprotein lipase, lipoyl synthase, lysozyme, malate dehydrogenase, maleate isomerase, maleiacetoacetate isomerase, maltase, mandelate racemase, metalloendopeptidase, methylenetetrahydrofolate reductase, microsomal epoxide hydrolase, molybdenum cofactor sulfurtransferase, molybdopterin synthase, molybdopterin synthase sulfurtransferase, monoamine oxidase, myeloperoxidase, N-sulfoglucosamine sulfohydrolase, NADH dehydrogenase, nicotinate hydrogenase, nitrate reductase, nitric oxide dioxygenase, nitric oxide synthase, nitrilase, nitrite reductase, nitrogenase, nuclease, oplophoro-luciferin 2-monooxygenase, ornithine decarboxylase, ornithine transcarbamoylase, oxoglutarate dehydrogenase, phenylalanine hydroxylase, phosphatase, phosphoamidase, phospholipase A, phospholipase C, phospholipase D, phosphonoacetaldehyde hydrolase, phosphonoacetate hydrolase, phosphonopyruvate hydrolase, photoisomerase, photoporphyrinogen oxidase, prolyl isomerase,propanediol phosphate dehydrogenase, peroxidase, arginine phosphatase, proteinase K, pyruvate dehydrogenase, renilla luciferin 2-monooxygenase, retinal isomerase, retinol isomerase, ribonuclease, ribonucleotide triphosphate reductase, ribonucleotide reductase, RNase, S-adenosyl-L-homocysteine ​​hydrolase, sarcosine oxidase, separase, serine protease, serine racemase, sucrase, sulfite oxidase, superoxide dismutase, tetrahydrocannabinolic acid synthase, thiaminase, thiamine oxidase, thiazole synthase, thioredoxin reductase, thiosulfate-dithiol sulfurtransferase, thiosulfate-thiol sulfurtransferase, thiosulfate sulfurtransferase, thyroid peroxidase, thyroxine 5-deiodinase, transpetition 870260062914, dated 06 / 26 / 2026, page 31 / 157, 27 / 147 epoxysuccinate hydrolase, transaldolase, transglutaminase, transketolase, trithionate hydrolase, tRNA-5-methyluridine(54) 2-sulfurtransferase, tRNA-5-taurinomethyluridine 2-sulfurtransferase, tRNA-uridine-3-sulfurtransferase, tRNA uracil 4-sulfurtransferase, tryptophan synthase, tyrosinase, ubiquitin carboxy-terminal hydrolase, UDP-sulfoquinovose synthase, urate oxidase, urease, uridine monophosphate synthetase, vitamin K epoxide reductase, watasenia-luciferin 2-monooxygenase, xanthine dehydrogenase, xanthine oxidase.

[0072] In some embodiments, the substrate used to determine the level of enzymatic activity is one of the following: 1-(3-bromophenyl)ethanol, 1-(3-chlorophenyl)ethanol, 1-(4'-chlorophenyl)ethanol, 1-(4'-fluorophenyl)ethanol, 1-(4-bromophenyl)ethanol, 1-(4-chlorophenyl)ethanol, 1-(4-fluorophenyl)ethanol, 1-(4-methylphenyl)ethanol, 1-(p-tolyl)ethanol, 1-butanal, 1-butanol, 1-chloro-5-acetylfuro[2], 1-decalone, 1-decanol, 1-dichloroacetone, 1-dodecanol, 1-heptanol, 1-hexanal, 1-hexanol, 1-hydroxy-2-butanone, 1hydroxymethyl-6-methylpyrene, 1-hydroxymethyl-8-methylpyrene, 1hydroxymethylpyrene, 1-indanol, 1-indanone, 1-nonanol, 1octanol, 1-pentanol, 1-phenyl-1-butanol, 1-phenyl-1-propanol, 1phenyl-2-butanol, 1-phenyl-2-propanol, 1-phenylethanol, 2, 2butanediol, 2-butanol, 2-heptanol,2-hexanediol, 2-hexanol, 2methylbutan-1-ol, 2-methylpent-2-enal, 2-octanol, 2pentanediol, 2-pentanol, 2-phenylpropanol, 2-propanediol, 3,3',3'-diaminobenzidine (DAB), 3-butanediol, 3-c]pyridine, 3-methylcyclohexanol, 3-methylcyclohexanone, 3b]thiopyran-4-one, 7,4-butanediol, 4-hydroxynon-2-enal, 4-methylumbelliferyl acetate, potassium salt of 4-methylumbelliferyl sulfate, 4-nitrophenyl acetate, Petition 870260062914, dated 06 / 26 / 2026, page 32 / 157 28 / 147 Nitrophenyl beta-D-maltoside, 4-nitrophenyl decanoate, 4-nitrophenyl palmitate, 4-nitrophenyl stearate, 4-phenyl-2-butanol, 5,5,5'-tetramethylbenzidine (TMB), 5-bromo-4-chloro-3'-indolphosphate, 5-pentanediol, 6-dichloro-4-nitrophenyl phosphate, 6-dienal, 6-dihydro-6-methyl-4H-thieno[2,7-dimethylocta-2,7-dioxide], 11-cis-retinal, 11-cis-retinol, 12-hydroxydodecanoate, 12-hydroxylaric acid methyl ester, 12-oxolauric acid methyl ester, 13-cis-retinal, 13-cisretinol, 16-hydroxyhexadecanoate, alpha, alpha-linolenic acid, alpha-Naphthol, alpha-tetralol, amylose, arachidonic acid, bicarbonate, but-2-en-1-ol, but-2-enal, carbohydrates, carvone, cellulose, blue cellulose, blue chitin, bovine collagen-fluorescein, colloidal chitin, cyclitol phosphate, dec-2-enal, methyl green deoxyribonucleic acid, dihomo-gamma-linolenic acid, disodium salt, docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), fat, gelatin, glutamine, hex-2-en-1-ol, hex-2-enal, indanol, lactose, laminarin,methylene blue, nicotinamide adenine dinucleotide (NADH), tetrazolium nitroblue, oct-2-enal, p-nitrophenyl phosphate, peptide bond, perillyl alcohol, phospholipids, pyrophosphate, tributyrin, tyrosine, urea, p-nitroanilide acetate salt Z-Gly-Pro-Arg, disodium salt of 4-nitrophenyl phosphate hexahydrate, 3,3'-diaminobenzidine tetrachloride, o-phenylenediamine dihydrochloride, o-phenylenediamine, o-dianisidine, 3,3',5,5'-tetramethylbenzidine dihydrochloride, 4-chloro-1-naphthol, 3-amino-9-ethylcarbazole, disodium salt of 4-nitrophenyl phosphate, 10-acetyl-3,7-dihydroxyphenoxazine acetate, casein, alpha-naphthyl acetate.,

[0073] In some modalities, before application Petition 870260062914, dated 06 / 26 / 2026, page 33 / 157 29 / 147 of the test substance, the enzyme is diluted in one or more of the following solvents: butanol, acetonitrile, acetone, diethylene glycol, diethyl ether, 1,2-dimethoxyethane, dimethylformamide, dimethyl sulfoxide, ethanol, ethyl acetate, ethylene glycol, glycerin, heptane, hexane, methanol, pentane, propanol, tetrahydrofuran, toluene, water, xylene or similar.

[0074] In some embodiments, the enzyme comprises alkaline phosphatase, acid phosphatase, nonspecific phosphatase, lactate dehydrogenase, peroxidase, esterase, alanine aminotransferase and aspartate aminotransferase, beta-glucuronidase, proteases or an antiprotease.

[0075] In the modalities, the enzyme is a phosphatase with units ranging from (500 to 25,000).

[0076] In some embodiments, the activity is measured by the disodium salt of p-nitrophenyl phosphate.

[0077] In some modalities, a reduction in enzymatic activity is used to predict dermal, ocular, pulmonary, nervous system, liver, kidney, intestinal or acute developmental toxicity.

[0078] In modalities, the method is used to predict skin corrosion.

[0079] A method for identifying a toxin, wherein the method comprises:

[0080] add the material to be tested to:

[0081] a. a solution of water (W) and salt with the enzyme (WE), and

[0082] b. an enzyme-free saline solution (WB) to be used as a raw block (B), and

[0083] c. a solution of alcohol or organic solvent (S) with enzyme (SE), and Petition 870260062914, dated 06 / 26 / 2026, page 34 / 157 30 / 147

[0084] d. an alcohol or organic solvent solution without enzyme (SB) to be used as a raw block.

[0085] incubation for a defined period of time;

[0086] to measure enzymatic activity and blank values, for example, to measure or observe changes in color or turbidity with the naked eye or with a spectrophotometer;

[0087] subtract the measured value of the saline solution from the measured value of the saline enzyme (WE - WB), and

[0088] subtract the measured value of the blank organic solvent solution from the measured value of the organic solvent enzyme (SE - SB), and;

[0089] identify the lowest value, (WE - WB) or (SE - SB), and compare it with a defined cutoff value or results from known standards used to define a cutoff value;

[0090] predict that the tested chemical is toxic if the lower value is less than a cutoff value or the value measured for a control chemical with known toxicity and / or;

[0091] relate the measured enzymatic activity to a toxicity index, which can be categorized according to established toxicity classes, in order to identify the tested material as a toxin or as a non-toxin.

[0092] A method in which the enzyme comprises one or more compounds selected from phosphatase, peroxidase, lactate dehydrogenase, alanine aminotransferase and aspartate aminotransferase, beta-glucuronidase or protease.

[0093] A method for assessing toxicity, in which the enzyme is a phosphatase. Petition 870260062914, dated 06 / 26 / 2026, page 35 / 157 31 / 147

[0094] A method for evaluating toxicity, in which activity is measured by the disodium salt of p-nitrophenyl phosphate.

[0095] A method in which the method is used to test and predict irritation or corrosion of the dermis, eyes, lungs, nervous system, liver, kidneys, or intestines.

[0096] A method in which the method is used for testing is used to predict dermal corrosion.

[0097] A method in which established toxicity classes are selected from non-irritant, irritant, and corrosive.

[0098] A method in which a water-based saline solution and an alcohol or organic solvent are combined.

[0099] A product or kit used for toxicity testing, which includes;

[00100] a. tubes with an enzyme diluted in a solvent (test and control tubes)

[00101] b. tubes with the solvent alone (empty tube)

[00102] c. a substrate for the solvent

[00103] d. instructions for

[00104] a. adding the material to be tested to a tube with enzyme and solvent (test tube) and to a second tube Use only the solvent (empty tube) and keep a 3rd tube with enzyme and solvent, but do not add the material to be tested (control tube).

[00105] b. Incubate the 3 tubes for a defined time.

[00106] c. transfer a portion of each tube to an enzymatic substrate Petition 870260062914, dated 06 / 26 / 2026, page 36 / 157 32 / 147

[00107] d. to visually or quantitatively measure the amount of enzymatic substrate converted into product during a defined period of time.

[00108] e. Subtract the value of the empty tube (solvent only) from the value of the test tube (solvent plus enzyme).

[00109] f. classify the tested material as toxic if the amount of substrate converted into product (test tube) minus the solvent alone (empty tube) is less than a defined value or less than a defined value or a percentage of the control tube (enzymatic solvent without the material to be tested).

[00110] A product or kit used to identify toxic chemicals that includes;

[00111] a. a cell-free enzymatic solution diluted in one or more suitable solvents, such as water, ethanol or an organic solvent.

[00112] b. a non-toxic chemical to serve as a negative control.

[00113] c. A substrate for the enzyme, which, when the enzyme solution is added, allows the evaluation of the enzyme's activity.

[00114] A product or kit used for high-throughput testing to identify dermal corrosives, which includes;

[00115] a. 4 types of *filled tubes with A1 (buffered saline solution with alkaline phosphatase), B1 (buffered saline solution blank), A2 (ethanol with alkaline phosphatase), B2 (empty ethanol). *Can also be supplied in bulk bottles and the user can divide into portions in tubes.

[00116] b. Negative control (water)

[00117] c. Positive control (acetic acid) Petition 870260062914, dated 06 / 26 / 2026, page 37 / 157 33 / 147

[00118] d. Detection Reagent (DR) solution supplied (p-nitrophenyl phosphate disodium salt).

[00119] In embodiments, the method includes applying the study substance to a cell-free or dead assay system under conditions in which the assay substance may interact with the enzyme, including when the enzyme is diluted in: (1) a saline solution, (2) an organic solvent, (3) or both (1) and (2); adding substrate to the enzyme and then; measuring the enzyme activity product as the substrate is acted upon by the enzyme; and predicting the toxicity of the study substance based on the response of the assay system.

[00120] In some embodiments, the enzyme is in an aqueous solution. In other embodiments, the enzyme is in a solvent, such as alcohol or an organic solvent. In other embodiments, both the enzyme in a water-based solution and the enzyme in a solvent such as alcohol are tested, and a combination or the result with the strongest response is used to predict toxicity.

[00121] In some embodiments, the water-based solution and another solvent solution are tested without the enzyme, and this value is used to subtract any background introduced by the chemical being tested.

[00122] In one embodiment, the predicted toxicity is for skin, eye, lung, heart, brain, kidney, intestine, or other target tissue found in the human body.

[00123] Method for determining the toxicity of a test chemical using a stable enzyme and its substrate, which can be stored and used in the laboratory or in the field. Petition 870260062914, dated 06 / 26 / 2026, page 38 / 157 34 / 147

[00124] In some embodiments of the method, the components are assembled as a kit, which may include, for example, 4 small tubes each with; 1) enzyme in aqueous solution, 2) aqueous solution alone, 3) enzyme in alcohol or organic solvent, alcohol or organic solvent alone and 4 large tubes containing the enzymatic substrate.To use the kit, the user adds a drop of the material to be tested to the small tube, incubates for a defined period of time, and transfers a drop of each incubated material to each large tube, incubates for a defined period of time, and observes a color change or measures the optical density using a laboratory spectrophotometer or plate reader; or measures the optical density using a field pipette spectrophotometer, subtracts the background by subtracting the OD for the enzyme-free condition from the corresponding condition with enzyme, identifies the lowest reading, compares that reading to a defined value, and if the reading is below the defined value, predicts that the tested material is toxic.

[00125] In some embodiments, toxins lacking binding activity to a specific enzymatic active site or binding activity to a regulatory site may include one or more of the following: dodecanammonium, N-(2-hydroxy-3-sulfopropyl)-N,N-dimethyl-,1-naphthaleneacetic acid, 1-octanol, 1,2,4-triazole, sodium salt, 1,3-diisopropylbenzene, 1,3-diiminobenz(f)-isoindoline, 1,5-hexadiene, 2-benzyl-4-chlorophenol, 2-benzyloxyethanol, 2-ethoxyethyl acetate (cellosolve acetate), 2-ethyl-1-hexanol, ethyl ester of 2-hydroxyisobutyric acid, 2-hydroxyisobutyric acid, 2-methyl-1-pentanol, 2-methylbutyric acid, 2-naphthalenesulfonic acid, formaldehyde, monosodium salt of Petition 870260062914, dated 06 / 26 / 2026, page 39 / 157 35 / 147 hydroxymethylbenzene sulfonic acid, 2-nitro-4-thiocyananiline, 2,2-dimethyl-3-pentanol, 2,2-dimethylbutanoic acid, 2,5-dimethyl-2,5-hexanediol, 2,6-dichlorobenzoyl chloride, pyridine 2,6-dichloro-5-fluoro-beta-oxo-3-propanoate, 3-chloropropionitrile, 3,3-dithiodipropionic acid, 3,4-dichlorophenyl isocyanate, 4-(1,1,3,3-tetramethylbutyl)phenol, 4-tert-butylcatechol, 4-carboxybenzaldehyde, 4-chloromethane, 6-methylpurine, p-tert-butylphenol, acetic acid, acetone, acid blue 40, acid red 92, acid alpha-ketoglutaric acid, ammonia, aluminum chloride, gamma-aminopropyltriethoxysilane, ammonium nitrate, antimony oxide, benzalkonium chloride, benzalkonium chloride (10%), benzenesulfonyl chloride, benzethonium chloride (10%), benzene, 1,1'-oxybis-, tetrapropylene derivatives, sulfonates, sodium salts, benzotrichloride, benzyl alcohol, beta-resorcylic acid, bis-(3-aminopropyl)tetramethyldisiloxane, butanol, butyl acetate, butyl cellosolve,butyldipropasol solvent, sodium salt of butylnaphthalenesulfonic acid, butyrolactone, calcium thioglycolate, captan 90-concentrate (solid), camphene, cetylpyridinium bromide (10%), cetylpyridinium chloride (10%), cetyltrimethylammonium bromide (10%), chlorhexidine, chloroform, cyclohexanol, cyclohexanone, cyclohexyl isocyanate, cyclopentanol, sodium salt of deoxycholic acid (10%), sodium di(2-ethylhexyl) sulfosuccinate (10%), di(propylene glycol) propyl ether, dibenzoyl-L-tartaric acid, dibenzyl phosphate, diethylaminopropionitrile, domifo bromide (10%), ethanol, 2-methyl ethyl acetoacetate, trimethyl ethyl acetate, glycidyl methacrylate, Granular, hydroxyethyl acrylate, imidazole, isobutanal, isobutyl alcohol, isopropyl alcohol, lactic acid, acid, Petition 870260062914, dated 06 / 26 / 2026, page 40 / 157 36 / 147 lauric, lauryldimethylamine oxide, lime, m-phenylenediamine, magnesium hydroxide, maneb, methoxyethyl acrylate, methyl acetate, methyl cyanoacetate, methyl cyclopentane, methyl ethyl ketone (2-butanone), methyl isobutyl ketone, methylpentinol, methyl thioglycolate, myristyl alcohol, n-acetylmethionine, n-butanol, n-hexanol, n-lauryl sarcosine sodium salt (10%), n-octylamine, N,N,N',N'-tetramethylhexanediamine, naphthalenesulfonic acid, 2-naphthalenesulfonic acid, sodium salt, nitric acid, organofunctional silane 45-49, phosphorodyloric acid, hydrogenated tallow amine, polyoxyethylene lauryl ether (23), potassium laurate (10%), potassium oleate, promethazine hydrochloride, potassium hydroxide, protectol PP, pyridine, benzyl-C12-16 alkyldimethyl, silver nitrate, sodium 2-naphthalenesulfonate, sodium hydrogen difluoride, sodium hydrogen sulfate, sodium hydroxide (10%), sodium lauryl sulfate, sodium lauryl sulfate (15%), sodium monochloroacetate,sodium oxalate, sodium perborate tetrahydrate, sodium polyoxyethylene(3) lauryl ether sulfate, sodium salicylate, stearyltrimethylammonium chloride, sulfuric acid, tetra-N-octylammonium bromide, tetraethylene glycol diacrylate, tetrahydrofuran, trichloroacetic acid (30%), trichloroacetyl chloride, triethanolamine, triethanolamine polyoxyethylene(3.0) lauryl ether sulfate, Triton X-100, Triton X-100 (5%), Triton X-100 (10%).

[00126] In some embodiments, known non-irritating and non-toxic, some with toxin-binding activity at the active site or regulatory site may include one or more of the following: 1-bromo-4-chlorobutane, styrene, 1,9-decadiene, 2-ethylhexyl p-dimethylaminobenzoate, 2Petition 870260062914, dated 06 / 26 / 2026, page 41 / 157 37 / 147 methylpentane, 2-(n-dodecylthio)-ethanol, 2,2-dimethyl-3-pentanol, 2,4-difluoronitrobenzene, 2,4-pentanediol, 3-methoxy-1,2-propanediol, 3-methylhexane, 3,3-dimethylpentane, acrylic acid homopolymer sodium salt, di-n-propyl disulfide, diisobutyl ketone, ethylhexyl salicylate, glycerol, isooctyl acrylate, isopropyl bromide, isopropyl myristate, iso-octylthioglycolate, methyl trimethyl acetate, n-hexyl bromide, n-octyl bromide, n,n-dimethylguanidine sulfate, polyethylene glycol 400, polyethylene glycol monolaurate (10 EO), polyoxyethylene hydrogenated castor oil (60E.O.), polyoxyethylene tribenzyl phenyl ether(14), polyoxyethylene sorbitan triisostearate(160), polyoxyethylene hydrogenated castor oil(40), potassium tetrafluoroborate, propylene glycol, sodium lauryl sulfate (3%), sorbitan monolaurate, tetraaminopyrimidine sulfate, toluene, triton X-100 (1%) and tween 80.

[00127] In some embodiments, enzymes destroyed or inactivated by toxin chemicals that are not specific to active or regulatory sites may include one or more of the following: 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylic acid synthase, 3-mercaptopyruvate sulfurtransferase, 4-hydroxytetrahydrodipicolinate reductase, 4-hydroxyphenylpyruvate dioxygenase, 6-oxide hydrolase, acetaldehyde dehydrogenase, acetolactate synthase, acetylcholinesterase, acid hydrolase, adenosine deaminase, adenosylmethionine hydrolase, adenylate cyclase, alanine aminopeptidase, alanine transaminase, alcohol dehydrogenase, alkaline phosphatase, alkenylglycerophosphocholine hydrolase, alkenylglycerophosphoethanolamine hydrolase, Petition 870260062914, dated 06 / 26 / 2026, page 42 / 157 38 / 147 aminolevulinic acid synthase, aminopropanol oxidoreductase, amylase, arginase, aromatase, arsenate reductase, aspartate transaminase, aspartate transcarbamoylase, ATPase, ATP synthase, beta-carotene isomerase, beta-galactosidase, beta-lactamase, biliverdin reductase, biotin synthase, butyrate kinase, caffeine dehydrogenase, carbon disulfide hydrolase, carbonic anhydrase, carbonyl sulfide hydrolase, catalase, catechol-O-methyltransferase, catechol oxidase, chitinase, cholesterol-5, choline acetyltransferase, cholinesterase, coenzyme Q - cytochrome c reductase, cyclamate sulfohydrolase, cypridin-luciferin 2-monooxygenase, cystathionine beta-lyase, cystathionine gamma-lyase, cysteine desulfurase, cytochrome c oxidase, cytochrome c peroxidase, cytochrome P450 oxidase, D-xylulose reductase, deiodinase, deoxyribonuclease, diacetyl dehydrogenase, dichloromethane dehalogenase, dihydrobenzophenanthridine oxidase, dihydrofolate reductase, esterase, DNS methyltransferase, elastase, endonuclease,exonuclease, farnesol 2-isomerase, firefly luciferase, flavin prenyltransferase, fructose bisphosphate aldolase, fructose bisphosphatase, furfoylmuramine isomerase, gamma-glutamyl transpeptidase, glucose oxidase, glutathione peroxidase, glutathione reductase, glutathione S-transferase, glyceraldehyde 3-phosphate dehydrogenase, glycerol 3-phosphate dehydrogenase, glycerol dehydrogenase, guanylate cyclase, haloacetate dehalogenase, halohydrin dehalogenase, helicase, hepoxylin-epoxide hydrolase, histone methyltransferase, HMGCoA reductase, homoserine dehydrogenase, hyaluronidase, hypoxanthine-guanine phosphoribosyltransferase, iodothyronine deiodinase, iodotyrosine deiodinase, isochorismatase, isocitrate dehydrogenase, isopenicillin N synthase, L, Petition 870260062914, dated 06 / 26 / 2026, page 43 / 157 39 / 147 gluconolactone oxidase, L-xylulose reductase, laccase, lactase, lactate dehydrogenase, leucoanthocyanidin reductase, leukotriene-A4 hydrolase, leukotriene C4 synthase, linolease isomerase, lipoprotein lipase, lipoyl synthase, lysozyme, malate dehydrogenase, maleate isomerase, maleiacetoacetate isomerase, maltase, mandelate racemase, metalloendopeptidase, methylenetetrahydrofolate reductase, microsomal epoxide hydrolase, molybdenum cofactor sulfurtransferase, molybdopterin synthase, molybdopterin synthase sulfurtransferase, monoamine oxidase, myeloperoxidase, N-sulfoglucosamine sulfohydrolase, NADH dehydrogenase, nicotinate hydrogenase, nitrate reductase, oxide nitric dioxygenase, nitric oxide synthase, nitrilase, nitrite reductase, nitrogenase, nuclease, oplophoro-luciferin 2-monooxygenase, ornithine decarboxylase, ornithine transcarbamoylase, oxoglutarate dehydrogenase, peroxidase, phenylalanine hydroxylase, phosphatase, phosphoamidase, phospholipase A, phospholipase C, phospholipase D,phosphonoacetaldehyde hydrolase, phosphonoacetate hydrolase, phosphonopyruvate hydrolase, photoisomerase, photoporphyrinogen oxidase, prolyl isomerase, propanediol phosphate dehydrogenase, arginine protein phosphatase, proteinase, pyruvate dehydrogenase, renillaluciferin 2-monooxygenase, retinal isomerase, retinol isomerase, ribonuclease, ribonucleotide triphosphate reductase, ribonucleotide reductase, RNase, S-adenosyl-L-homocysteine ​​hydrolase, sarcosine oxidase, separase, serine protease, serine racemase, sucrase, sulfite oxidase, superoxide dismutase, tetrahydrocannabinolic acid synthase, thiaminase, thiamine oxidase, thiazole synthase, thioredoxin reductase, thiosulfate-dithiol sulfurtransferase, thiosulfate-thiol, Petition 870260062914, dated 06 / 26 / 2026, page 44 / 157 40 / 147 sulfurtransferase, thiosulfate sulfurtransferase, thyroid peroxidase, thyroxine 5-deiodinase, trans-epoxysuccinate hydrolase, transaldolase, transglutaminase, transketolase, trithionate hydrolase, tRNA-5-methyluridine(54) 2-sulfurtransferase, tRNA-5-taurinomethyluridine 2-sulfurtransferase, tRNA-uridine-3-sulfurtransferase, tRNA uracil 4-sulfurtransferase, tryptophan synthase, tyrosinase, ubiquitin carboxy-terminal hydrolase, UDP-sulfoquinovose synthase, urate oxidase, urease, uridine monophosphate synthetase, vitamin K epoxide reductase, watasenia-luciferin 2-monooxygenase, xanthine dehydrogenase, xanthine oxidase.

[00128] In some embodiments, the enzymatic solvents for diluting the enzyme and performing the test may include one or more of the following: 1-(3-bromophenyl)ethanol, 1-(3-chlorophenyl)ethanol, 1-(4'-chlorophenyl)ethanol, 1-(4'-fluorophenyl)ethanol, 1-(4-bromophenyl)ethanol, 1-(4-chlorophenyl)ethanol, 1-(4-fluorophenyl)ethanol, 1-(4-methylphenyl)ethanol, 1-(p-tolyl)-ethanol, 1-butanal, 1-butanol, 1-chloro-5-acetylfuro[2], 1-decalone, 1-decanol, 1-dichloroacetone, 1-dodecanol, 1-heptanol, 1-hexanal, 1-hexanol, 1-hydroxy-2-butanone, 1-hydroxymethyl-6-methylpyrene, 1hydroxymethyl-8-methylpyrene, 1-hydroxymethylpyrene, 1-indanol, 1-indanona, 1-nonanol, 1-octanol, 1-pentanol, 1-fenil-1butanol, 1-fenil-1-propanol, 1-fenil-2-butanol, 1-fenil-2propanol, 1-feniletanol, 2, 2-butanodiol, 2-butanol, 2heptanol,2-hexanodiol, 2-hexanol, 2-metilbutan-1-ol, 2metilpent-2-enal, 2-octanol, 2-pentanodiol, 2-pentanol, 2fenilpropanol,2-propanodiol, 3,3',3'-diaminobenzidina (DAB),3-butanodiol,3-c]piridina, 3-metilciclohexanol, 3metilciclohexanona,3b]tiopiran-4-ona-7,4-butanodiol, 4Petição 870260062914, de 26 / 06 / 2026, pág. 45 / 157 41 / 147 hydroxynon-2-enal, 4-methylumbelliferyl acetate, potassium salt of 4-methylumbelliferyl sulfate, 4-nitrophenyl acetate, 4-Nitrophenyl beta-D-maltoside, 4-nitrophenyl decanoate, 4-nitrophenyl palmitate, 4-nitrophenyl stearate, 4-phenyl-2-butanol, 5,5,5'-tetramethylbenzidine (TMB), 5-bromo-4-chloro-3'-indolphosphate, 5-pentanediol, 6-dichloro-4-nitrophenyl phosphate, 6-dienal, 6-dihydro-6-methyl-4H-thieno[2,7-dimethylocta-2,7-dioxide, 11-cis-retinal, 11-cisretinol, 12-hydroxydodecanoate, methyl ester of 12-hydroxylaric acid, ester methyl 12-oxolauric acid, 13-cisretinal, 13-cis-retinol, 16-hydroxyhexadecanoate, alpha, alpha-linolenic acid, alpha-naphthol, alpha-tetralol, amylose, arachidonic acid, bicarbonate, but-2-en-1-ol, but-2-enal, carbohydrates, carvone, cellulose, blue cellulose, blue chitin, bovine collagen-fluorescein, colloidal chitin, corn starch, cyclitol phosphate, dec-2-enal, methyl green deoxyribonucleic acid, dihomo-gamma-linolenic acid, disodium salt,docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), fat, gelatin, glutamine, hex-2-en-1-ol, hex-2-enal, hydrogen peroxide, indanol, lactose, laminarin, linoleic acid, lipids, methylene blue, nicotinamide adenine dinucleotide (NADH), nitro-tetrazolium blue, oct-2-enal, p-nitrophenyl phosphate, peptide bond, perillyl alcohol, phospholipids, potato starch, protein, pyrophosphate, starch, sucrose, tributyrin, tyrosine, urea, p-nitroanilide Z-Gly-Pro-Arg acetate salt, disodium salt of 4-nitrophenyl phosphate hexahydrate, 3,3'-Diaminobenzidine tetrachloride, o-Phenylenediamine dihydrochloride, oPhenylenediamine, o-Dianisidine, 3,3',5,5'Tetramethylbenzidine dihydrochloride, 4-Chloro-1-naphthol, 3-Amino-9, Petition 870260062914, dated 06 / 26 / 2026, page 46 / 157 42 / 147 ethylcarbazole, disodium salt of 4-nitrophenyl phosphate, 10-acetyl-3,7-dihydroxyphenoxazine acetate, casein or alpha-naphthyl acetate.

[00129] In some embodiments, the solvents used to dilute the enzyme and conduct the reaction to identify potential toxins may include one or more of the following: acetic acid, acetone, acetonitrile, anisole, benzene, 1-butanol, 2-butanol, 2-butanone, tert-butyl alcohol, carbon tetrachloride, chlorobenzene, chloroform, cumene, cyclohexane, 1,2-dichloroethane, 1,1-dichloroethene, 1,2-dichloroethane, dichloromethane, 1,2-dimethoxyethane, diethylene glycol, diethyl ether, diglyme, 1,2-dimethoxyethane, dimethylformamide, dimethyl sulfoxide, 1,4-dioxane, ethanol, 2-ethoxyethanol, ethyl acetate, ethylene glycol, ethyl ether, ethyl formate, formamide, formic acid, glycerin, heptane, hexamethylphosphoramide, hexamethylphosphorus triamide, hexane, isobutyl acetate, isopropyl acetate, methanol, 2-methoxyethanol, methyl acetate, 3-methyl-1-butanol, methylbutyl ketone, methyl tert-butyl ether, methylene chloride, N-methyl-2-pyrrolidinone, methyl ethyl ketone, methyl isobutyl ketone, 2-methyl-1-propanol, nitromethane, pentane, 1-pentanol, petroleum ether, 1-propanol, 2-propanol, propyl acetate, pyridine, sulfolane, tetrachloroethylene, tetrahydrofuran, tetralin, toluene,1,1,1-trichloroethane, 1,1,2-trichloroethene, triethylamine, water, o-xylene, m-xylene and p-xylene.

[00130] As used in this document, unless otherwise indicated by the context, toxicity is used to refer to the ability of a substance to Petition 870260062914, dated 06 / 26 / 2026, page 47 / 157 43 / 147 damage, disintegrate, irritate, or negatively affect body tissue. Toxicity may be evidenced by pain, irritation, swelling, cloudiness, redness, and discharge. Such effects may be temporary or permanent. Consequently, the word toxicity is broadly defined to include any discomfort or adverse experience associated with the presence of a substance in contact with an eye. As used in this document, toxicity or toxin is used to cover the spectrum from irritant to highly corrosive. BRIEF DESCRIPTION OF THE DRAWINGS

[00131] Figure 1 depicts the test results for 3 skin corrosives (clear vials on the right, GHS Cat 1) compared with 6 non-corrosives (GHS NC and Cat 2) and a negative control (water, on the far left). The enzyme was diluted and the chemicals were aliquoted into glass tubes at a 1:10 ratio (100 mg / ml). The chemical / enzyme solutions were incubated for 4 hours at room temperature. After incubation, 2 ml of an enzymatic substrate that changes from clear to yellow was added to each tube, and then the tubes were incubated at room temperature for 1 hour. The results for corrosives show almost complete enzymatic inhibition, as indicated by the clear tube with no observable yellow reaction product versus the other tubes with enzyme incubated with non-corrosives which have an obviously high concentration of yellow reaction product.Using this method, a test material can be tested for toxicity in a field environment without equipment.

[00132] Figure 2 depicts how to conduct and Petition 870260062914, dated 06 / 26 / 2026, page 48 / 157 44 / 147 Quantify a modality of an in chemico test in the laboratory. The material to be tested, plus the controls, are added directly to the enzymatic solution. After incubation, aliquots are added to the detection reagent (enzymatic substrate), which absorbs strongly when active. This reaction is quantified using a plate reader, the backgrounds are subtracted, the resulting net DO values ​​are normalized by dividing by the net DOs of the corresponding negative controls with background subtracted, and the result with the lowest percentage is the In Chemico (IC) value, which is used to determine if the tested material is toxic.

[00133] Figures 3A-3B depict the raw optical density readings from the initial alkaline phosphatase studies. Figure 3A depicts the results of the procedure performed with discs. Figure 3B depicts the results of the procedure performed without discs. The top three lines in both figures show the readings of p-nitrophenyl phosphate (PNPP), and the bottom three lines show the buffered saline blank. The buffered saline blank was conducted to account for any chemical interference. The test materials included C1 = (2-bromoethyl)benzene, C2 = triethylene glycol, C3 = 2-methyl-4-phenyl-2-butanol, C4 = cyclamen aldehyde, C5 = heptanal, C6 = di-n-propyl disulfide, C7 = ethanolamine, C8 = dimethyldipropylenetriamine, and C9 = acetic acid.

[00134] Figure 4 depicts the raw optical density readings from a repeat of the procedure (Figure 3B). The top three lines in both figures show the p-nitrophenyl phosphate (PNPP) readings, and the bottom three lines show the buffered saline blank. A Petition 870260062914, dated 06 / 26 / 2026, page 49 / 157 A 45 / 147 buffered saline blank was conducted to account for any chemical interference. Test materials included C1 = 1-Bromo-4-chlorobutane, C2 = 1,5-hexadiene, C3 = isopropanol, C4 = isopropyl myristate, C5 = di-n-propyl disulfide, C6 = cyclamen aldehyde, C7 = dimethyldipropylnetramine, C8 = potassium hydroxide (10%), and C9 = 2-methylbutyric acid.

[00135] Figures 5A-5D depict the raw optical density readings from the study that evaluated a shorter exposure time and different incubation times of p-nitrophenyl phosphate disodium salt (PNPP). Figure 5A depicts the results of a 10-minute p-nitrophenyl phosphate disodium salt (PNPP) reading. Figure 5B depicts the results of a 1-hour p-nitrophenyl phosphate disodium salt (PNPP) reading. Figure 5C depicts the results of a 4-hour p-nitrophenyl phosphate disodium salt (PNPP) reading. Figure 5D depicts the results of an 18-hour p-nitrophenyl phosphate disodium salt (PNPP) reading. The top three lines in both figures show the p-nitrophenyl phosphate (PNPP) readings, and the bottom three lines show the blank buffered saline solution. The buffered saline blank was prepared to account for any chemical interference.The test materials include C1 = 1-Bromo-4-chlorobutane, C2 = 1,5-hexadiene, C3 = isopropanol, C4 = isopropyl myristate, C5 = di-n-propyl disulfide, C6 = cyclamen aldehyde, C7 = dimethyldipropylnetramine, C8 = potassium hydroxide (10%), and C9 = 2-methylbutyric acid.

[00136] Figures 6A-6F depict the raw optical density readings from the study that evaluated different sources. Petition 870260062914, dated 06 / 26 / 2026, page 50 / 157 46 / 147 and alkaline phosphatase concentrations in the reaction reagent using 30 chemicals. Figure 6A depicts the results of the controls (C1 = Water, C2 = Water / 1% Ink, C3 = Acetic Acid, C4 = Acetic Acid / 1% Ink) for each condition (Original Alkaline Phosphatase [1:1], Original Alkaline Phosphatase [1:10], Original Alkaline Phosphatase [1:100], New Alkaline Phosphatase, Blank [buffered saline solution]). Three readings were conducted for each chemical. Figure 6B depicts the results for the original alkaline phosphatase concentration (1:1). Figure 6C depicts the results for the original alkaline phosphatase concentration (1:10). Figure 6D depicts the results for the original alkaline phosphatase concentration (1:100). Figure 6E depicts the results for the new alkaline phosphatase concentration (1:1).Figure 6F depicts the results for the blank condition (buffered saline solution), which was conducted to account for any chemical interference. The chemicals under study were: C5 = 1-Bromo-4-chlorobutane, C6 = 1,5-hexadiene, C7 = isopropanol, C8 = 4-(methylthio)benzaldehyde, C9 = isopropyl myristate, C10 = benzyl salicylate, C11 = phenylethyl alcohol, C12 = hydroxycitronellal, C13 = benzyl acetate, C14 = dipropylene glycol, C15 = di-n-propyl disulfide, C16 = 1-bromopentane, C17 = cyclamen aldehyde, C18 = cis-3-methyl-2-(2-pentenyl)-2-cyclopenten-1-one, C19 = pmeta-1,8-dien-7-ol, C20 = heptanal, C21 = trans-3,7-Dimethyl-2,6-octadien-1-ol, C22 = alpha-terpineol, C23 = 1-decanol, C24 = nonyl aldehyde, C25 = dimethyldipropylenetramine, C26 = potassium hydroxide (10%), C27 = 2-methylbutyric acid, C28 = octanoic acid, C29 = dimethylisopropylamine, C30 = 2-tert-butylphenol, C31 = 5-isopropyl-2-methylphenol, C32 = Petition 870260062914, dated 06 / 26 / 2026, page 51 / 157. 47 / 147 heptylamine, C33 = methoxy-3-propylamine and C34 = N,Ndimethylbenzylamine.

[00137] Figures 7A-7B depict the raw optical density readings from the study that evaluated different dosage concentrations and solvents. Figure 7A depicts the results of a 3-hour exposure to the test chemical with a 30-minute incubation of p-nitrophenyl phosphate (PNPP) disodium salt. Figure 7B depicts the results of a 3-hour test chemical exposure with a 1-hour incubation of p-nitrophenyl phosphate (PNPP) disodium salt. The top three lines are results from the 10 μL dosage concentration (in 90 pL) and the bottom three lines are from the 50 pL dosage concentration (in 50 pL). Condition A is the buffered saline reaction reagent, condition B is the ethanol reaction reagent, and condition C is the acetone reaction reagent. The chemicals tested were water, methanol, mineral oil, 1-bromo-4-chlorobutane, 1,5-hexadiene, di-n-propyl disulfide, nonyl aldehyde, octanoic acid, dimethylisopropylamine, 5-isopropyl-2-methylphenol, n-heptylamine, and N,N-dimethylbenzylamine.

[00138] Figures 8A-8C depict the raw optical density readings from the study that evaluated different dosage concentrations and solvents. Figure 8A depicts the results of a 22-hour exposure to the test chemical with a 10-minute incubation of pnitrophenyl phosphate (PNPP) disodium salt. Figure 8B depicts the results of a 22-hour exposure to the test chemical with a 30-minute incubation of pnitrophenyl phosphate (PNPP) disodium salt. Figure 8C depicts the results of a 22-hour exposure to the test chemical. Petition 870260062914, dated 06 / 26 / 2026, page 52 / 157 48 / 147 hours with a 1-hour incubation of disodium nitrophenyl phosphate (PNPP) salt. The top three lines are results from a dosage concentration of 10 μL (in 90 pL) and the bottom three lines are from a dosage concentration of 50 pL (in 50 pL). Condition A is the buffered saline reaction reagent, condition B is the ethanol reaction reagent, and condition C is the acetone reaction reagent. The chemicals tested were water, methanol, mineral oil, 1-bromo-4-chlorobutane, 1,5-hexadiene, di-n-propyl disulfide, nonyl aldehyde, octanoic acid, dimethylisopropylamine, 5-isopropyl-2-methylphenol, n-heptylamine, and N,N-dimethylbenzylamine.

[00139] Figures 9A-9B depict the raw optical density readings from the study that evaluated different dilutions of alkaline phosphatase reaction reagents (1:100 and 1:1000) at room temperature and 37°C. Figure 9A depicts the 4-hour exposure to the test chemical with a 30-minute incubation with p-nitrophenyl phosphate disodium salt (PNPP). Figure 9B depicts the 4-hour exposure to the test chemical with a 1-hour incubation of p-nitrophenyl phosphate disodium salt (PNPP). The chemicals studied were water, 4-(methylthio)benzaldehyde, 1-bromo-4-chlorobutane, 1,5-hexadiene, alpha-terpineol, dimethyl disulfide, nonyl aldehyde, octanoic acid, dimethylisopropylamine, 5-isopropyl-2-methylphenol, heptylamine, and N,N-dimethylbenzylamine.

[00140] Figures 10A-10B depict the raw optical density readings from the study that evaluated different dilutions of alkaline phosphatase reaction reagents (1:100 and 1:1000) at room temperature and 37°C. Figure 10A depicts Petition 870260062914, dated 06 / 26 / 2026, page 53 / 157 Figure 10A depicts a 22-hour exposure to the chemical under study with a 30-minute incubation with disodium salt of p-nitrophenyl phosphate (PNPP). Figure 10B depicts a 22-hour exposure to the chemical under test with a 1-hour incubation of disodium salt of p-nitrophenyl phosphate (PNPP). The chemicals under study were water, 4-(methylthio)benzaldehyde, 1-bromo-4-chlorobutane, 1,5-hexadiene, alpha-terpineol, di-n-propyl disulfide, nonyl aldehyde, octanoic acid, dimethylisopropylamine, 5-isopropyl-2-methylphenol, n-heptylamine, and N,N-dimethylbenzylamine.

[00141] Figures 11A-11E depict the raw optical density readings from the study that evaluated the performance of 4 ml glass vials with screw caps using thirty chemicals. Figure 11A depicts the results of the controls for each condition. Figure 11B depicts the results of reaction reagent R1+ (active buffered saline solution). Figure 11C depicts the results of reaction reagent R1- (blank buffered saline solution; no addition of alkaline phosphatase). Figure 11C depicts the results of reaction reagent R2+ (active ethanol). Figure 11D depicts the results of reaction reagent R2 (blank ethanol; no addition of alkaline phosphatase).The chemicals under study were: C5 = 1-Bromo-4-chlorobutane, C6 = 1,5-hexadiene, C7 = isopropanol, C8 = 4-(methylthio)benzaldehyde, C9 = isopropyl myristate, C10 = benzyl salicylate, C11 = phenylethyl alcohol, C12 = hydroxycitronellal, C13 = benzyl acetate, C14 = dipropylene glycol, C15 = di-n-propyl disulfide, C16 = 1-bromopentane, C17 = cyclamen aldehyde, C18 = cis-3-methyl-2-(2-pentenyl)-2-cyclopenten-1-one, C19 = pmeta-1,8-dien-7-ol, C20 = heptanal, C21 = trans-3,7-DimethylPetido 870260062914, dated 06 / 26 / 2026, page 54 / 157. 50 / 147 2,6-octadien-1-ol, C22 = alpha-terpineol, C23 = 1-decanol, C24 = nonyl aldehyde, C25 = dimethyldipropylenetramine, C26 = potassium hydroxide (10%), C27 = 2-methylbutyric acid, C28 = octanonic acid, C29 = dimethylisopropylamine, C30 = 2-tert-butylphenol, C31 = 5-isopropyl-2-methylphenol, C32 = heptylamine, C33 = methoxy-3-propylamine and C34 = N,N-dimethylbenzylamine.

[00142] Figures 12A-12D depict the raw optical density readings from the study that evaluated the performance between 0.5 ml microcentrifuge tubes and 4 ml glass vials with screw caps. Figure 12A shows the results from the 0.5 ml microcentrifuge tubes (upper half) and the results from the 20 μL aliquot from the 4 ml glass vial in 200 μL of p-nitrophenyl phosphate (PNPP) disodium salt substrate solution with a 30-minute p-nitrophenyl phosphate (PNPP) disodium salt incubation. Figure 12B depicts the results from the 0.5 ml microcentrifuge tubes (upper half) and the results from the 20 μL aliquot from the 4 ml glass vial in 200 μL of p-nitrophenyl phosphate (PNPP) disodium salt substrate solution with a 1-hour p-nitrophenyl phosphate (PNPP) disodium salt incubation. Figure 12C depicts the results of adding 2.6 ml of p-nitrophenyl phosphate (PNPP) disodium salt substrate solution directly to 4 ml glass vials followed by a 30-minute p-nitrophenyl phosphate (PNPP) disodium salt incubation. Figure 12D depicts the results of adding 2.6 ml of p-nitrophenyl phosphate (PNPP) disodium salt substrate solution directly to 4 ml glass vials followed by a 1-hour p-nitrophenyl phosphate (PNPP) disodium salt incubation.The chemicals tested. Petition 870260062914, dated 06 / 26 / 2026, page 55 / 157 51 / 147 were C1 = Water, C3 = Acetic acid, C20 = Heptanal, C29 = Dimethylisopropylamine, C32 = N-Heptylamine and C34 = N,NDimethylbenzylamine.

[00143] Figures 13A-13E depict the raw optical readings from the study that combined all the finalized parameters from previous studies. Figure 13A depicts the results of the controls for each condition. Figure 13B depicts the results of the active buffered saline reaction reagent. Figure 13C depicts the results of the blank buffered saline reaction reagent. Figure 13D depicts the results of the active ethanol reaction reagent. Figure 13E depicts the results of the blank ethanol reaction reagent. The chemicals tested were C5 = 1-Bromo-4-chlorobutane, C6 = 1,5-Hexadiene, C7 = Isopropanol, C8 = 4-(Methylthio)benzaldehyde, C9 = Isopropyl myristate, C10 = Benzyl salicylate, C11 = Phenylethyl alcohol, C12= Hydroxycitronellal, C13 = Benzyl acetate, C14= Dipropylene glycol, C15 = Di-n-propyl disulfide, C16 = 1Bromopentane, C17 = Cyclamen aldehyde, C18 = cis-3-Methyl-2(2-pentenyl)-2-cyclopenten-1-one, C19 = p-Meta-1,8-dien-7-ol, C20 = Heptanal, C21 = trans-3,7-Diemethyl-2,6-octadien-1-ol, C22 = Alpha-terpineol, C23 = 1-Decanol, C24 = Nonyl aldehyde, C25 = Dimethyldipropylenetriamine, C26 = Potassium hydroxide (10%), C27 = 2-Methylbutyric acid, C28 = Octanonic acid, C29 = Dimethylisopropylamine, C30 = 2-tert-Butylphenol, C31 = 5Isopropyl-2-methylphenol, C32 = n-Heptylamine, C33 = Methoxy-3propylamine, and C34 = N,N-Dimethylbenzylamine.

[00144] Figure 14 depicts the results of the study that combined all the finalized parameters. Blanks are subtracted from the raw readings and analyzed with a model of Petition 870260062914, dated 06 / 26 / 2026, page 56 / 157 52 / 147 prediction for estimating dermal corrosives versus non-corrosives.

[00145] Figure 15 shows the test kit. The test kit includes the active test matrices (A1 and A2) and the blank test matrices (B1 and B2), the negative control (NC), and the detection reagent. The test kit has a shelf life of 1 year. 6 kits were used to test 60 chemicals in triplicate.

[00146] Figure 16 depicts a list of 60 chemicals tested in triplicate using the test kit. The different classifications include not classified as irritant or corrosive (NC), not classified as corrosive (Category 2), classified as corrosive (Category 1BC), and classified as extremely corrosive (Category 1A).

[00147] Figure 17 depicts a guide sheet for the kit. Steps for using the test kit. For each material to be tested:

[00148] 1. Label four 0.5 ml reaction tubes uniquely to distinguish the four reagents of the test matrix (i.e., A1, B1, A2, B2). Add 180 μL of the appropriate test matrix reagent to each of the correspondingly labeled reaction tubes.

[00149] 2. Add 20 ml of test substance to each of the 4 reaction tubes (A1, B1, A2, B2). Mix the contents of each reaction tube using a pipette, aspirating and dispensing 5 times. Close the caps.

[00150] 3. Incubate the capped tubes at room temperature for 4 hours ± 10 min.

[00151] 4. While the reaction tubes are in Petition 870260062914, dated 06 / 26 / 2026, page 57 / 157 53 / 147 incubation, prepare a 96-well ELISA plate by aliquoting 200 μL of detection reagent solution into each well.

[00152] 5. After incubation for 4 hours, mix the contents of each reaction tube again using a pipette, aspirating and dispensing 5 times. Add 20 μL aliquots of each reaction (in triplicate wells) to the ELISA plate.

[00153] 6. Incubate the ELISA plate at room temperature for 1 hour ± 10 min.

[00154] 7. Measure the optical density (OD) at 405 nm using a plate reader.

[00155] 8. Calculate the percentage of remaining activity for the chemical being tested. If the remaining activity is less than 40%, classify the material as a dermal corrosive.

[00156] Figure 18 depicts the results of 60 chemicals tested in triplicate using the test kit. Average percentage of Negative Control for three independent replicates. Error bars represent standard error. The chemical numbers are the same as listed in Figure 16, which includes the names with the CAS RN in parentheses.

[00157] Figure 19 describes the predictability data for 60 chemicals tested in triplicate using the test kit. DETAILED DESCRIPTION

[00158] As revealed in this document, the inventors found that a cell-free chemical enzyme solution can be used to predict the toxicity of a test material.

[00159] A method for predicting the toxicity of a Petition 870260062914, dated 06 / 26 / 2026, page 58 / 157 54 / 147 test substance in living tissues, wherein the method comprises:

[00160] apply the test substance to a predefined enzyme or enzyme mixture to effect an in chemico reaction, wherein the test substance does not bind preferentially or specifically to an active or regulatory site of the predefined enzyme or enzyme mixture;

[00161] measure any reduction in the enzymatic activity of the predefined enzyme or enzyme mixture on a predefined substrate; and,

[00162] compare the measured reduction in enzyme activity with a control activity value or a previously established activity value, and

[00163] predict the extent or classification of the toxicity of the test substance in living tissues based on the measured comparative reduction in enzymatic activity.

[00164] In some embodiments, the enzyme is purified or semi-purified or is in a mixture composed of non-viable cells and / or non-viable tissue.

[00165] In some embodiments, the test substance irreversibly reduces enzymatic activity and / or reduces enzymatic activity in a non-competitive manner.

[00166] In some embodiments, the test substance reduces enzymatic activity by chemically destroying or denaturing the enzyme and / or the chemistry or binding is not specific to the enzyme's active or regulatory sites.

[00167] In some forms, the test substance is applied in toxic and non-therapeutic concentrations.

[00168] In some embodiments, the mass of the test substance is applied to the enzyme in a 1:1 ratio. Petition 870260062914, dated 06 / 26 / 2026, p. 59 / 157 55 / 147 1:100 parts of test substance to the volume of enzyme solution.

[00169] In some embodiments, the mass of the test substance is applied to the enzyme in a ratio of 1:101:1,000 parts of test substance to the volume of the enzyme solution.

[00170] In some embodiments, the test substance is applied in concentrations of mg / ml and not in pg / ml or lfg / ml.

[00171] In some embodiments, the test substances are a diverse group of toxins without having specific binding affinity or binding to the regulatory or active sites of the enzyme.

[00172] In some modalities, the test predicts toxicity to living skin, and the accuracy of the prediction is 85% or more.

[00173] In modalities, the enzyme is a phosphatase.

[00174] In embodiments, the toxicity is corrosive. In some embodiments, the method predicts whether or not the test substance is corrosive with an accuracy of at least 85%.

[00175] In some embodiments, corrosiveness is predicted if enzymatic activity is reduced to 40% or less of the control or value previously established by said test substance.

[00176] In some embodiments, non-corrosiveness is predicted if the remaining enzymatic activity is greater than 40% of the control value or previously established by the said test substance.

[00177] In some embodiments, the accuracy in predicting whether the test substance is a dermal corrosive is equal Petition 870260062914, dated 06 / 26 / 2026, p. 60 / 157 56 / 147 or higher than 90%.

[00178] In some embodiments, a predefined enzyme or enzyme mixture is sequentially exposed to each of a large number of known toxins (at least 10 toxins) and sequentially exposed to each of a large number of known non-toxins (at least 10 non-toxins), and the results of enzyme activity after exposure to the toxin are calculated to form an activity class, and the results of enzyme activity after exposure to the non-toxin are calculated to form an activity class, and the measurement of the activity class for the toxin class is significantly reduced relative to the enzyme activity for the non-toxic activity class, and there is a statistically significant separation of the activity values ​​of the toxin class relative to the activity values ​​of the non-toxic class (P-value < 0.01),These results are then used to build a predictive model that identifies toxins based on a specific reduction in enzymatic activity, with the intended use of a predictive model for the identification and classification of the toxicity of unknown materials, and when used to test unknowns for toxicity, with a predictive accuracy of at least 85%.

[00179] In embodiments where a mixture of enzymes is used, the activity is determined as an average activity. For example, there may be several enzymes in a cell extract that act on the same substrate - and these enzymes may not be defined, but the average activity can be measured.

[00180] In some modalities, the lack of specificity of connection to the active location is verified with the Petition 870260062914, dated 06 / 26 / 2026, page 61 / 157 57 / 147 use of a chemical analytical technique. In embodiments, the technique includes mass spectrometry, HPLC or gas chromatography.

[00181] In the modalities, the tissue to be evaluated comprises ocular tissue. In the modalities, the prediction accuracy is at least 85%.

[00182] In modalities, the enzyme is an esterase.

[00183] In embodiments, toxicity is irritation. In some embodiments, the method predicts the test substance as irritant or non-irritant with an accuracy of at least 85%.

[00184] In some embodiments, the toxicity is corrosion or irritation, and the method specifies the test substance as corrosive, non-corrosive, irritant, or non-irritant.

[00185] In embodiments, the enzyme is one of the following: 12-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylic acid synthase, 3-mercaptopyruvate sulfurtransferase, 4-hydroxytetrahydrodipicolinate reductase, 4-hydroxyphenylpyruvate dioxygenase, 6-oxide hydrolase, acetaldehyde dehydrogenase, acetolactate synthase, acetylcholinesterase, acid hydrolase, adenosine deaminase, adenosylmethionine hydrolase, adenylate cyclase, alanine aminopeptidase, alanine transaminase, alcohol dehydrogenase, alkaline phosphatase, acid phosphatase, nonspecific phosphatase, alkenylglycerophosphocholine hydrolase, alkenylglycerophosphoethanolamine hydrolase, aminolevulinic acid synthase, aminopropanol oxidoreductase, amylase, arginase, aromatase, arsenate reductase, aspartate transaminase, aspartate transcarbamoylase, ATPase, ATP synthase, beta-carotene isomerase, beta-galactosidase, beta-lactamase, biliverdin reductase, biotin synthase, butyrate Petition 870260062914, dated 06 / 26 / 2026, page 62 / 157 58 / 147 kinase, caffeine dehydrogenase, carbon disulfide hydrolase, carbonic anhydrase, carbonyl sulfide hydrolase, catalase, catechol-O-methyltransferase, catechol oxidase, chitinase, cholesterol-5, choline acetyltransferase, cholinesterase, coenzyme Q-cytochrome c reductase, cyclamate sulfohydrolase, cypridin-luciferin 2-monooxygenase, cystathionine beta-lyase, cystathionine gamma-lyase, cysteine ​​desulfurase, cytochrome c oxidase, cytochrome c peroxidase, cytochrome P450 oxidase, D-xylulose reductase, deiodinase, deoxyribonuclease, diacetyl dehydrogenase, dichloromethane dehalogenase, dihydrobenzophenanthridine oxidase, dihydrofolate reductase, DNS methyltransferase, elastase, endonuclease, exonuclease, farnesol 2-isomerase, firefly luciferase, flavin prenyltransferase, fructose bisphosphate aldolase, fructose bisphosphatase, furfoylmuramine isomerase, gamma-glutamyl transpeptidase, glucose oxidase, glutathione peroxidase, glutathione reductase, glutathione S-transferase,glyceraldehyde 3-phosphate dehydrogenase, glycerol-3-phosphate dehydrogenase, glycerol dehydrogenase, guanylate cyclase, haloacetate dehalogenase, halohydrin dehalogenase, helicase, hepoxylin epoxide hydrolase, histone methyltransferase, HMG-CoA reductase, homoserine dehydrogenase, hyaluronidase, hypoxanthine-guanine phosphoribosyltransferase, iodothyronine deiodinase, iodotyrosine deiodinase, isochorismatase, isocitrate dehydrogenase, isopenicillin N synthase, L-gluconolactone oxidase, L-xylulose reductase, laccase, lactase, lactate dehydrogenase, leucoanthocyanidin reductase, leukotriene-A4 hydrolase, leukotriene C4 synthase, linolease isomerase, lipoprotein lipase, lipoyl synthase, lysozyme, malate dehydrogenase, maleate isomerase, maleiacetoacetate isomerase, Petition 870260062914, dated 06 / 26 / 2026, page 63 / 157 59 / 147 maltase, mandelate racemase, metalloendopeptidase, methylenetetrahydrofolate reductase, microsomal epoxide hydrolase, molybdenum cofactor sulfurtransferase, molybdopterin synthase, molybdopterin synthase sulfurtransferase, monoamine oxidase, myeloperoxidase, N-sulfoglucosamine sulfohydrolase, NADH dehydrogenase, nicotinate hydrogenase, nitrate reductase, nitric oxide dioxygenase, nitric oxide synthase, nitrilase, nitrite reductase, nitrogenase, nuclease, oplophoro-luciferin 2-monooxygenase, ornithine decarboxylase, ornithine transcarbamoylase, oxoglutarate dehydrogenase, phenylalanine hydroxylase, phosphatase, phosphoamidase, phospholipase A, phospholipase C, phospholipase D, phosphonoacetaldehyde hydrolase, phosphonoacetate hydrolase, phosphonopyruvate hydrolase, photoisomerase, photoporphyrinogen oxidase, prolyl isomerase, propanediol-phosphate dehydrogenase, peroxidase, protein arginine phosphatase, proteinase K, pyruvate dehydrogenase, Renilla-luciferin 2-monooxygenase, retinal isomerase,retinol isomerase, ribonuclease, ribonucleotide triphosphate reductase, ribonucleotide reductase, RNase, S-adenosyl-L-homocysteine ​​hydrolase, sarcosine oxidase, separase, serine protease, serine racemase, sucrase, sulfite oxidase, superoxide dismutase, tetrahydrocannabinolic acid synthase, thiaminase, thiamine oxidase, thiazole synthase, thioredoxin reductase, thiosulfate-dithiol sulfurtransferase, thiosulfatethiol sulfurtransferase, thiosulfate sulfurtransferase, thyroid peroxidase, thyroxine 5-deiodinase, transepoxysuccinate hydrolase, transaldolase, transglutaminase, transketolase, trithionate hydrolase, tRNA-5-methyluridine(54) 2-sulfurtransferase, tRNA-5-taurinomethyluridine 2, Petition 870260062914, dated 06 / 26 / 2026, page 64 / 157 60 / 147 sulfurtransferase, tRNA-uridine-3-sulfurtransferase, tRNA uracil 4-sulfurtransferase, tryptophan synthase, tyrosinase, ubiquitin carboxy-terminal hydrolase, UDP-sulfoquinovose synthase, urate oxidase, urease, uridine monophosphate synthetase, vitamin K epoxide reductase, Watasenia-luciferin 2-monooxygenase, xanthine dehydrogenase or xanthine oxidase.

[00186] In some embodiments, the substrate used to determine the level of enzymatic activity is one of the following: 1-(3-bromophenyl)ethanol, 1-(3-chlorophenyl)ethanol, 1-(4'-chlorophenyl)ethanol, 1-(4'-fluorophenyl)ethanol, 1-(4-bromophenyl)ethanol, 1-(4-chlorophenyl)ethanol, 1-(4-fluorophenyl)ethanol, 1-(4-methylphenyl)ethanol, 1-(p-tolyl)ethanol, 1-butanal, 1-butanol, 1-chloro-5-acetylfuro[2], 1-decalone, 1-decanol, 1-dichloroacetone, 1-dodecanol, 1-heptanol, 1-hexanal, 1-hexanol, 1-hydroxy-2-butanone, 1hydroxymethyl-6-methylpyrene, 1-hydroxymethyl-8-methylpyrene, 1hydroxymethylpyrene, 1-indanol, 1-indanone, 1-nonanol, 1octanol, 1-pentanol, 1-phenyl-1-butanol, 1-phenyl-1-propanol, 1phenyl-2-butanol, 1-phenyl-2-propanol, 1-phenylethanol, 2, 2butanediol, 2-butanol, 2-heptanol,2-hexanediol, 2-hexanol, 2methylbutan-1-ol, 2-methylpent-2-enal, 2-octanol, 2pentanediol, 2-pentanol, 2-phenylpropanol, 2-propanediol, 3,3',3'-diaminobenzidine (DAB), 3-butanediol, 3-c]pyridine, 3-methylcyclohexanol, 3-methylcyclohexanone, 3b]thiopyran-4-one, 7,4-butanediol, 4-hydroxynon-2-enal, 4-methylumbelliferyl acetate, potassium salt of 4-methylumbelliferyl sulfate, 4-nitrophenyl acetate, 4-nitrophenyl beta-D-maltoside, 4-nitrophenyl decanoate, 4-nitrophenyl palmitate, 4-nitrophenyl stearate, 4-phenyl-2-butanol, 5,5,5'-tetramethylbenzidine (TMB), 5-bromo-4-Petição 870260062914, dated 06 / 26 / 2026, page 65 / 157 61 / 147 chloro-3'-indolephosphate, 5-pentanediol, 6-dichloro-4-nitrophenyl phosphate, 6-dienal, 6-dihydro-6-methyl-4H-thieno[2,7-dimethylocta-2,7-dioxide, 11-cis-retinal, 11-cis-retinol, 12-hydroxydodecanoate, 12-hydroxylaric acid methyl ester, 12-oxolauric acid methyl ester, 13-cis-retinal, 13-cisretinol, 16-hydroxyhexadecanoate, alpha, alpha-linolenic acid, alpha-naphthol, alpha-tetralol, amylose, arachidonic acid, bicarbonate, but-2-en-1-ol, but-2-enal, carbohydrates, carvone, cellulose, blue cellulose, blue chitin, bovine fluorescent collagen, colloidal chitin, cyclitol phosphate, dec-2-enal, deoxyribonucleic acid, methyl green, dihomo-gamma-inolenic acid, disodium salt, docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), fat, gelatin, glutamine, hex-2-en-1-ol, hex-2-enal, indanol, lactose, laminarin, methylene blue, nicotinamide adenine dinucleotide (NADH), nitro blue tetrazolium, oct-2-enal, p-nitrophenyl phosphate, peptide bond, perillyl alcohol, phospholipids,pyrophosphate, tributyrin, tyrosine, urea, p-nitroanilide acetate salt Z-Gly-Pro-Arg, 4nitrophenyl phosphate disodium salt hexahydrate, 3,3'diaminobenzidine tetrahydrochloride, o-phenylenediamine dihydrochloride, ophenylenediamine, o-dianisidine, o-diaminobenzidine dihydrochloride 3,3',5,5'tetramethylbenzidine, 4-chloro-1-naphthol, 3-amino-9ethylcarbazole, 4-nitrophenyl phosphate disodium salt, 10-acetyl-3,7-dihydroxyphenoxazine acetate, casein or alpha-naphthyl acetate.,

[00187] In some embodiments, prior to application of the test substance, the enzyme is diluted in one or more of the following solvents: butanol, acetonitrile, acetone, diethylene glycol, diethyl ether, 1,2-dimethoxyethane, Petition 870260062914, dated 06 / 26 / 2026, page 66 / 157 62 / 147 dimethylformamide, dimethyl sulfoxide, ethanol, ethyl acetate, ethylene glycol, glycerin, heptane, hexane, methanol, pentane, propanol, tetrahydrofuran, toluene, water, xylene or similar.

[00188] In some embodiments, before applying the test substance, the enzyme is diluted in 1) ethanol or similar and 2) water or similar, and the test substance is applied to either 1 or 2, and the greater reduction in enzymatic activity in 1 or 2 is used to predict the toxicity of the test substance or material.

[00189] In some embodiments, the enzyme comprises alkaline phosphatase, acid phosphatase, nonspecific phosphatase, lactate dehydrogenase, peroxidase, esterase, alanine aminotransferase and aspartate aminotransferase, beta-glucuronidase, protease or an antiprotease.

[00190] In some embodiments, the enzyme is a phosphatase and 500 to 25,000 units are applied to each 1) salt water and 2) ethanol.

[00191] In some embodiments, the activity is measured by the disodium salt of p-nitrophenyl phosphate.

[00192] In some modalities, a reduction in enzymatic activity is used to predict dermal, ocular, pulmonary, nervous system, liver, kidney, intestinal or acute developmental toxicity.

[00193] In some forms, the method is used for testing to be used to predict skin corrosion.

[00194] The test developed in this disclosed document has predictive value. In the embodiments, the control values ​​are either derived from a validation dataset or from a set of validation data. The validation data may be derived, for example, from testing performed using known standard toxicants. Petition 870260062914, dated 06 / 26 / 2026, page 67 / 157 63 / 147 (e.g., irritants, corrosives) and / or confirming substances identified as toxic to living tissue (e.g., irritants, corrosives) by other known secondary tests that conform to the same identity.

[00195] A method for identifying a toxin, wherein the method comprises:

[00196] add the material to be tested to:

[00197] a. a solution of water (W) and salt with the enzyme (WE), and

[00198] b. an enzyme-free saline solution (WB) to be used as a raw block (B), and

[00199] c. a solution of alcohol or organic solvent (S) with enzyme (SE), and

[00200] d. an alcohol or enzyme-free organic solvent solution (SB) to be used as a raw block;

[00201] incubation for a defined period of time;

[00202] measurement of enzymatic activity and blank values, for example, by measuring or observing changes in color or turbidity with the naked eye or with a spectrophotometer;

[00203] subtract the measured value of the water-bottle saline solution from the measured value of the water-bottle saline enzyme (WE-WB);

[00204] subtract the measured value of the blank organic solvent solution from the measured value of the organic solvent enzyme (SE-SB);

[00205] identify the lowest value, (WE-WB) or (SE-SB), and compare it with a defined cutoff value or results from known standards used to define a value Petition 870260062914, dated 06 / 26 / 2026, page 68 / 157 64 / 147 cut;

[00206] predict that the tested chemical is toxic if the lower value is less than a cutoff value or the value measured for a control chemical with known toxicity, and / or;

[00207] relate the measured enzymatic activity to a toxicity index, which can be categorized according to established toxicity classes, in order to identify the tested material as a toxin or non-toxin.

[00208] In some embodiments, the enzyme comprises one or more compounds selected from phosphatase, peroxidase, lactate dehydrogenase, alanine aminotransferase and aspartate aminotransferase, beta-glucuronidase or protease.

[00209] In modalities, the enzyme is a phosphatase.

[00210] In some embodiments, the activity is measured by the disodium salt of p-nitrophenyl phosphate.

[00211] In some applications, the method is used to test for and predict irritation or corrosion of the skin, eyes, lungs, nervous system, liver, kidneys, or intestines.

[00212] In some embodiments, the method is used for testing and is used to predict dermal corrosion.

[00213] In some embodiments, the established toxicity classes are selected from non-irritant, irritant and corrosive.

[00214] In some embodiments, the water-based saline solution and alcohol or organic solvent are combined.

[00215] A product or kit used for toxicity testing, which includes;

[00216] a. tubes with an enzyme diluted in a Petition 870260062914, dated 06 / 26 / 2026, p. 69 / 157 65 / 147 solvent (test and control tubes) (tube

[00217] b. tubes with solvent alone empty)

[00218] c. a substrate for the solvent

[00219] d. instructions for:

[00220] a. adding the material to be tested to a tube with enzyme and solvent (test tube) and to a second tube Keep only the solvent (empty tube) and preserve a third tube with enzyme and solvent, but do not add the material to be tested (control tube);

[00221] b. incubate the three tubes for a defined time;

[00222] c. transfer a rise from each tube to an enzymatic substrate;

[00223] d. measure visually or quantitatively the amount of enzymatic substrate converted into product during a defined period of time;

[00224] e. subtract the value of the empty tube (solvent only) from the value of the test tube (solvent plus enzyme); and

[00225] f. classify the tested material as toxic if the amount of substrate converted into a product (test tube) minus the solvent alone (empty tube) is less than a defined value or less than a defined value or a percentage of the control tube (enzymatic solvent without the material to be tested).

[00226] A product or kit used to identify toxic chemicals, which includes:

[00227] a. a cell-free enzymatic solution diluted in one or more suitable solvents, such as water, ethanol or an organic solvent. Petition 870260062914, dated 06 / 26 / 2026, page 70 / 157 66 / 147

[00228] b. a non-toxic chemical to serve as a negative control.

[00229] c. a substrate for the enzyme, which, when the enzyme solution is added, allows the enzyme activity to be evaluated.

[00230] A product or kit used for high-throughput testing to identify dermal corrosives, which includes:

[00231] a. four types of *filled tubes with A1 (alkaline phosphatase-buffered saline solution), B1 (blank buffered saline solution), A2 (ethanol with alkaline phosphatase), B2 (empty ethanol). *Also available in bulk bottles, which the user can divide into portions in tubes.

[00232] b. negative control (water)

[00233] c. positive control (acetic acid)

[00234] d. Detection reagent (DR) solution (p-nitrophenyl phosphate disodium salt).

[00235] Previously, an in chemico test for eye irritants and corrosives (called OptiSafe Eye Irritation Test™) was developed (Choksi et al., 2020; Lebrun, 2021a, 2021b; Lebrun et al., 2021a, 2021b, 2022, 2023a, 2023b). The OptiSafe macromolecular test measures turbidity resulting from protein denaturation by chemical irritants. No enzymatic activity is involved. Initially, the OptiSafe test was evaluated for its ability to identify other types of toxins, including dermal corrosives, but little or no correlation was found with OptiSafe results for dermal corrosives; clearly, the skin and the eye have very different histological, protective, and macromolecular compositions. The eye has a thin layer (5-7 cells) of epithelium covering a transparent cornea, Petition 870260062914, dated 06 / 26 / 2026, page 71 / 157 67 / 147 composed primarily of collagen. The skin has a comparatively thick epidermis, with epithelial cells in various stages of terminal differentiation with the apical layer fully keratinized, resulting in a water-resistant protective barrier, and below the epidermis, the dermal layer with connective tissue. Initially, it was assumed that the significant structural differences between the eye and the skin likely explained why the OptiSafe test did not accurately predict skin toxicity. Therefore, to try to identify additional molecular targets or markers that could be used to develop other shelf-stable toxicity tests, over the past four years, a series of studies have been conducted in which tissues from rabbits or pigs were dosed and, after incubation, a wide range of immunohistochemistry experiments were performed on post-exposure cryosections.Conjugated antibodies or other probes were used for a large number of inflammatory markers, cytokines, and markers for connective tissue breakdown (collagen, actin, etc.). The main reason why work was carried out for so many years on ex vivo and 3D tissue models was that the inventors were convinced that an accurate toxicity test required, and especially a skin toxicity test requires, full-thickness skin. Histopathological studies using ex vivo buttons (5 mm biopsy), punctures followed by sectioning and antibody probing have found that full-thickness buttons of ex vivo tissue have considerable heterogeneity (random occurrence of vessels, glands, hair follicles, etc.) that stain differentially and interfere with quantification and reproducibility. Furthermore, both... Petition 870260062914, dated 06 / 26 / 2026, page 72 / 157 68 / 147 ex vivo tissue models, as well as those from differentiated cultures (produced in-house and purchased from external suppliers, e.g., Mattek, USA), used in the studies, had low accuracy, sensitivity, and specificity for detecting dermal irritants or corrosives. Furthermore, it was not conceivable how living tissues could be developed into an easy-to-use and shelf-stable test kit. Although there have been years of effort without developing an accurate corrosion test for ex vivo or 3D tissue construction, these experiments were not a total loss because, for some of these studies, attempts were made to identify potential corrosion markers using enzyme-conjugated antibodies. Typically, tissues fixed with paraformaldehyde (which cross-links proteins and inactivates most enzymes) are used for this type of histology. However, since the aim was to identify native protein markers, unfixed rabbit and pig skin was used.Significant background endogenous phosphatase activity was observed for skin treated with non-corrosive dermis, but this activity was not observed for skin treated with corrosives; the skin has significant endogenous phosphatase activity, however, the skin treated with dermal corrosives did not continue to exhibit endogenous phosphatase activity. EXPERIMENTAL DETAILS AND EXAMPLES

[00236] Initial studies involved the use of fresh and frozen pigskin buttons. These buttons were punched into pigskin (Spear Products, Coopersburg, PA) using a 0.6 mm biopsy punch tool. Three different types of skin buttons were prepared from fresh pigskin, frozen pigskin, and pigskin. Petition 870260062914, dated 06 / 26 / 2026, page 73 / 157 69 / 147 frozen with the fatty layer removed. Nine chemicals ((2-Bromoethyl)benzene, triethylene glycol, 2-methyl-4-phenyl-2-butanol, heptanal, di-n-propyl disulfide, cyclamen aldehyde, ethanolamine, dimethyldipropylenetriamine, and acetic acid) and two controls (water and naive) were tested. Each skin button was placed in a 96-well plate and dosed with 140 μL of the test substance for 30 minutes, then transferred to tubes containing 2 mL of phosphate-buffered saline (PBS) with added sodium azide and incubated for 18 hours at 37°C. The following day, the tubes were removed and subjected to vortices, and 500 μL of the solution were divided into aliquots in cuvettes for reading at three different optical density wavelengths (OD190, OD290, OD300) to evaluate the best response.

[00237] Protocol: SKIN BUTTON EXPERIMENT FIRST WEEK LIST OF CHEMICAL PRODUCTS TABLE 1 Condition Chemical Name CASRN GHS in vivo Virgin Water C1 (2-bromoethyl)benzene 103-63-9 NC C2 Triethylene glycol 112-27-6 NC C3 2-Methyl-4-phenyl-2-butanol 103-05-9 NC C4 Heptanal 111-71-7 Cat. 2 C5 Di-n-propyl disulfide 629-19-6 Cat. 2 C6 Cyclamen aldehyde 103-95-7 Cat. 2 C7 Ethanolamine 141-43-5 Cat. 1 Petition 870260062914, dated 06 / 26 / 2026, page 74 / 157 70 / 147 C8 Dimethyldipropyletriamine 10563-29- 8 Cat. 1 C9 Acetic acid 64-19-7 Cat. 1 SKIN PREPARATION

[00238] 1. Thaw 2 pieces of frozen pork skin

[00239] 2. Perforated buttons: 24 buttons in each tube filled with 40 ml of PBS-Azide

[00240] Preparation of the 96-well plate

[00241] 1. Dosing plate model:

[00242] TABLE 2 Virgin Water in C1 C2 C3 C4 C5 C6 C7 C8 C9 Original Tissue Frozen Tissue Frozen Fat Removed PROCEDURE

[00243] 1. Dose each tissue with 140 μL of the test chemical.

[00244] 2. Place in the incubator for 3 hours, then transfer to 7 ml tubes filled with 2 ml of PBSAzide, and then place back in the incubator for 18 hours. READING RESULTS

[00245] 1. Remove from incubator and vortex pulse Petition 870260062914, dated 06 / 26 / 2026, page 75 / 157 71 / 147 3x

[00246] 2. Aliquot 500 μL into cuvette and read results at OD190, OD290 and OD300

[00247] The results are shown in Tables 3 - 5 below. TABLE 3. RESULTS OF INITIAL STUDIES USING SKIN FRESH PORK Chemical Name CASRN GHS in vivo R OD190 OD290 OD300 Water n / dn / d 1 0.124 0.147 0.108 2 0.170 0.129 0.097 Virgin n / dn / d 1 0.141 0.154 0.119 2 0.313 0.250 0.213 (2- 103- NC 1 0.128 0.125 0.080 bromoethyl)benzene 63-9 2 0.304 0.133 0.094 Triethylene glycol 112- NC 1 0.221 0.153 0.118 27-6 2 0.372 0.105 0.068 2-Methyl-4-phenyl- 103- NC 1 0.210 0.339 0.272 2-butanol 05-9 2 0.559 0.426 0.371 Heptanal 111- Cat. 1 0.486 0.170 0.123 71-7 2 2 0.365 0.165 0.123 di-629- Cat. 1 disulfide 0.419 0.139 0.105 n-propyl 19-6 2 2 0.400 0.137 0.096 103- Cat. 2 0.716 0.477 0.447 Ethanolamine 141- Cat. 1 >3A 0.848 0.711 43-5 1BC 2 >3A 1.169 1.044 Dimethyldipropilet 10563- Cat. 1 >3A 0.922 0.819 ryamine 29-8 1A 2 >3A 1.016 0.887 Acetic acid 64-19- Cat. 1 >3A 1.122 1.065 Petition 870260062914, dated 06 / 26 / 2026, p. 76 / 157 72 / 147 7 1A 2 >3A 0.931 0.857 TABLE 4. RESULTS OF INITIAL STUDIES OF THE BUTTON CUTANEOUS USING FROZEN PORK SKIN Chemical Name CASRN GHS in vivo R OD190 OD290 OD300 Water n / dn / d 1 0.204 0.186 0.098 2 0.184 0.177 0.080 Virgin n / dn / d 1 0.299 0.19 0.092 2 0.185 0.397 0.274 (2-bromoethyl)benzene 103-63-9 NC 1 0.129 0.279 0.137 2 0.272 0.254 0.128 Triethylene glycol 112-27-6 NC 1 0.231 0.156 0.072 2 0.474 0.257 0.149 2-Methyl-4-phenyl- 2-butanol 103-05-9 NC 1 0.338 0.496 0.342 2 0.362 0.637 0.497 Heptanal 111-71-7 Cat. 2 1 0.362 0.245 0.154 2 0.371 0.241 0.137 Dimpropyl disulfide 629-19-6 Cat. 2 1 0.314 0.294 0.161 2 0.396 0.240 0.119 Cyclamen aldehyde 103-95-7 Cat. 2 1 >3A 1.023 0.901 2 0.675 0.955 0.852 Ethanolamine 141- Cat. 43-5 Cat. 1A 1 >3A 1.002 0.935 2 >3A 0.873 0.859 TABLE 5. RESULTS OF INITIAL BUTTON STUDIES Petition 870260062914, dated 06 / 26 / 2026, page 77 / 157 73 / 147 CUTANEOUS USING FROZEN PORK SKIN WITH A LAYER OF FAT REMOVED Chemical Name CASRN GHS in vivo R OD190 OD290 OD300 Water n / dn / d 1 0.094 0.042 0.017 2 0.106 0.065 0.027 Virgin n / dn / d 1 0.105 0.069 0.020 2 0.201 0.046 0.016 (2-bromoethyl)benzene 103-NC 1 0.131 0.071 0.030 63-9 2 0.144 0.080 0.034 Triethylene glycol 112-NC 1 0.101 0.054 0.016 27-6 2 0.201 0.050 0.016 2-Methyl-4-phenyl- 103- NC 1 0.215 0.079 0.026 2-butanol 05-9 2 0.322 0.094 0.052 Heptanal 111- Cat. 1 0.144 0.111 0.040 71-7 2 2 0.149 0.140 0.050 di-629- Cat. 2 0.214 0.153 0.119 Ethanolamine 141- Cat. 1 0.761 0.168 0.099 43-5 1BC 2 0.800 0.093 0.048 Dimethyldipropylet 10563- Cat. 1 0.610 0.238 0.162 Riamine 29-8 1A 2 0.594 0.170 0.118 Acetic acid 64-19- Cat. 1 0.591 0.054 0.040 7 1A 2 0.599 0.064 0.047 CASRN = Abstract Service Registration Number Chemicals; GHS = Globally Harmonized System of Classification and Labelling of Chemicals; n / a = Not applicable; NC = Not classified; >3A = Reading outside the range Petition 870260062914, dated 06 / 26 / 2026, page 78 / 157 74 / 147 readable from the spectrophotometer. [0024 8] Interpretation of Tables 3–5: Nine chemicals ((2-Bromoethyl)benzene, triethylene glycol, 2-methyl-4-phenyl-2-butanol, heptanal, di-n-propyl disulfide, cyclamen aldehyde, ethanolamine, dimethyldipropylenetriamine, and acetic acid) and two controls (water and virgin) were tested on three different pig hides (fresh, frozen, and frozen with the fat layer removed). Three readings were taken with optical densities of 190, 290, and 300 nm to evaluate the best response. At an optical density of 190 nm, the spectrophotometer readings were unstable and fluctuating, while at 290 nm and 300 nm, the readings were stable without fluctuations; therefore, the optical density wavelength for the next study was chosen to be 300 nm. These results represent the presence of proteins, with higher readings indicating a greater protein response and lower readings indicating a smaller protein response.As shown in Tables 3 and 4, the response to dermal corrosive chemicals was greater than to dermal irritant and non-irritant chemicals. However, as shown in Table 5, the response to dermal corrosive, dermal irritant, and non-irritant chemicals was variable and therefore inconclusive. The responses from this study led us to think critically about the surface area of ​​skin buttons and how the test chemical (some viscous, others non-viscous) sits on the epidermis during the exposure period, such as whether it remains strictly on the epidermis or runs off to the sides of the skin button and potentially changes the way the chemical is inducing toxicity to the button. Petition 870260062914, dated 06 / 26 / 2026, page 79 / 157 75 / 147 skin from the sides. To address varying exposure conditions, subsequent studies involved placing a larger section of pig skin on a support that exposes only the epidermal side without potential exposure to the test chemical from the back or sides.

[00249] Subsequent studies involved applying the test substance (500 pL) directly to the epidermal side of previously frozen pig skin (Spear Products, Coopersburg, PA) placed on a support exposing only the epidermal side for 30 minutes, adding phosphate-buffered saline (PBS) with added sodium azide (a metabolic inhibitor ensuring no viable tissue), and then aliquoting into cuvettes for reading at an optical density of 300 nm. The results were inconclusive, as the solution divided into aliquots in the cuvettes was opaque and gave the same reading of >3A (outside the readable range of the spectrophotometer). Next, the Lowry method was attempted for a different method of protein determination, but the results were also inconclusive because there was no clear difference in color change for the test substances and controls.After several studies attempting to identify skin damage, changes in viability, released factors, and damage, and finding nothing significant, as an afterthought following a failed experiment, the previous experience (described above) was recalled, involving histology of cross-sections of skin treated with dermal corrosives, where it was noted that perhaps phosphatase activity was reduced after exposure to skin corrosives. Thus, at the end of the initial studies described above, almost out of frustration, an attempt was made to probe the activity of phosphatase released using salt. Petition 870260062914, dated 06 / 26 / 2026, page 80 / 157 76 / 147 disodium p-nitrophenyl phosphate (PNPP; Sigma Aldrich, St. Louis, MO) was used to detect any presence of phosphatase. After removing the chemical, 1 ml of PNPP was added and incubated for 48 hours. Then, the incubated disodium p-nitrophenyl phosphate (PNPP) salt was recovered from each of the different skin sections and the optical density was read at 405 nm, and the results are shown in Table 7 below.

[00250] Protocol: SKIN EXPERIMENT SECOND WEEK LIST OF CHEMICAL PRODUCTS TABLE 6 Condition Chemical Name CASRN GHS in vivo Water 01 (2-bromoethyl)benzene 103-63-9 NC 02 Triethylene glycol 112-27-6 NC 03 2-Methyl-4-phenyl-1,2-butanol 103-05-9 NC 04 Heptanal 111-71-7 Cat. 2 05 Dipropyl disulfide 629-19-6 Cat. 2 06 Cyclamen aldehyde 103-95-7 Cat. 2 07 Ethanolamine 141-43-5 Cat. 1 08 Dimethyldipropyltriamine 10563-298 Cat. 1 09 Acetic acid 64-19-7 Cat. 1 Virgin Note: If there are sufficient tissue discs PREPARATION

[00251] 1. Thaw 2 pieces of frozen pork skin Petition 870260062914, dated 06 / 26 / 2026, page 81 / 157 77 / 147

[00252] a. Punch out as many skin discs as possible and mount them on stands

[00253] 2. Label the cuvettes (3 for each chemical) PROCEDURE

[00254] 1. Add 500 μL of the chemical under study to the support; incubate at room temperature for 30 minutes.

[00255] 2. After 30 minutes, add 2 ml of PBSazide to the support containing the chemical under study; pipette forcefully and pipette again 10 times.

[00256] 3. From the support, aliquot 400 µL into cuvettes; pipette forcefully and repipette 3 times before dispensing into cuvettes. READING RESULTS

[00257] 1. Measure at OD300 for all cuvettes TABLE 7. RESULTS OF INITIAL SKIN SUPPORT STUDIES WITH FROZEN SKIN SECTIONS Chemical Name CASRN GHS in vivo Corrosive Reading 1 Reading 2 Water n / dn / d No >3A >3A Virgin n / dn / d No >3A >3A (2-bromoethyl)benzene 103- 63-9 NC No >3A >3A Triethylene glycol 112- 27-6 NC No 0.886 0.845 2-Methyl-4-phenyl-2-butanol 103- 05-9 NC No 2.957 >3A Heptanal 111- 71-7 Cat. 2 No >3A 2.831 di-n- 629- disulfide Cat. No 2.044 2.148 Petition 870260062914, dated 06 / 26 / 2026, page 82 / 157 78 / 147 Propyl 19-6 2 Cyclamen aldehyde 103-95-7 Cat. 2 No >3A >3A Ethanolamine 141-43-5 Cat. 1BC Yes 0.603 0.587 Dimethyldipropyltriamine 1056329-8 Cat. 1A Yes 0.706 0.531 Acetic acid 64-19-7 Cat. 1A Yes 0.574 0.559 CASRN = Chemical Abstracts Service Registration Number; GHS = Globally Harmonized System of Classification and Labelling of Chemicals; n / a = Not applicable; NC = Not classified; >3A = Reading outside the readable range of the spectrophotometer.

[00258] Interpretation of Table 7: Nine chemicals ((2-Bromoethyl)benzene, triethylene glycol, 2-methyl-4-phenyl-2-butanol, heptanal, di-n-propyl disulfide, cyclamen aldehyde, ethanolamine, dimethyldipropylenetriamine, and acetic acid) and two controls (water and virgin) were added to the epidermis of previously frozen pig skin sections to evaluate the skin corrosion potential after a 30-minute exposure. Using the measured optical density values ​​compared to the naive condition to obtain the measured phosphatase percentage, the three GHS category 1 chemicals (ethanolamine: 20.1%, 19.6%, mean 19.9%; dimethyldipropylenetramine: 23.5%, 17.7%, mean 20.6%; acetic acid: 19.1%, 18.6%, mean 18.9%) showed a significantly lower response than the dermal irritants (Heptanal: 100.0%, 94.4%, mean 97.2%; di-n-propyl disulfide: 68.1%, 71.6%, mean 69.9%; aldehyde of Petition 870260062914, dated 06 / 26 / 2026, page 83 / 157 79 / 147 cyclamen: 100.0%, 100.0%, average 100.0%) or non-irritating ((2-bromoethyl)benzene: 100.0%, 100.0%, average 100.0%; 2-Methyl-4-phenyl-2-butanol: 98.6%, 100.0%, average 99.3%), with the exception of triethylene glycol (29.5%, 28.2%, average 28.9%) which had a low response. As shown in Table 7, surprisingly, there was significantly less phosphatase activity for the dermal corrosives (ethanolamine: 0.603, 0.587; Dimethyldipropylenetramine: 0.706, 0.531; Acetic acid: 0.574, 0.559) compared to dermal irritants (Heptanal: >3A, 2.831; di-n-propyl disulfide: 2.044, 2.148; Cyclamen aldehyde: >3A, >3A) or non-irritating ((2-Bromoethyl)benzene: >3A, >3A; Triethylene glycol: 0.886, 0.845; 2-Methyl-4-phenyl-2-butanol: 2,957, >3A). Skin corrosion by GHS Category 1 dermal corrosive chemicals results in the disintegration of the epidermis into the dermis. This was the first experiment in which it was found that dermal corrosives inhibited enzymatic activity. Based on the preliminary findings shown in Table 7, it was proposed to further explore and optimize the study with a greater focus on the phosphatase substrate system and disodium salt of p-nitrophenyl phosphate (PNPP).

[00259] The next study focused on an in vitro method for detecting the potential of dermal corrosives to inhibit nonspecific phosphatases that may be released from the skin. It was decided to test pure phosphate instead of relying on endogenous skin phosphatase. Alkaline phosphatase (Sigma Aldrich, St. Louis, MO) was purchased and suspended to make the reaction reagent (10,000 units). An initial pilot study was conducted by suspending the alkaline phosphatase in saline solution and then adding the chemicals to be tested in a ratio of 1 part chemical to 9 parts phosphate. Petition 870260062914, dated 06 / 26 / 2026, page 84 / 157 80 / 147 parts of enzyme suspension. It was observed that the ratio used was 0.1 g / ml or 10% of chemicals – which is far superior to any pharmaceutical application. It was further observed that pharmaceutical enzyme inhibition tests, studies and use involve highly specific active site binding and these assays are conducted in microgram-femtogram per ml ratios. These studies are conducted in these ranges because these are realistic concentrations for drug and therapeutic applications. For example, the desired working dose – a single tablet to be consumed by a human being – may contain 5 mg of drug. Thus, when a 50 kg human consumes the tablets, the tissues will be exposed to 5 mg / 50 kg = 0.1 mg / kg = 0.0000001 g / ml.Therefore, although both the method described here and pharmaceutical uses can inhibit enzymatic activity, the concentration used for the method described here is approximately 6 orders of magnitude higher than that used for specific drug effects at the active site or regulatory site of the drug. On the other hand, the method disclosed here is nonspecific, and the active site, regulatory sites, and the entire enzyme are destroyed or inactivated as a result of a very high concentration of the chemical being tested and the resulting nonspecific widespread molecular damage caused by the toxin being tested.Figure 1 shows the pilot results where 3 dermal corrosives and 6 non-dermal corrosives were added to an enzymatic solution at a ratio of 0.1 g of chemical enzymatic solution / ml, incubated for 4 hours at room temperature, and then a transparent enzymatic substrate that turns yellow when the enzyme is active was added; the enzyme remains. Petition 870260062914, dated 06 / 26 / 2026, page 85 / 157 81 / 147 is active against non-corrosive substances (yellow tubes) and inactive against dermal corrosive substances (transparent tubes).

[00260] Next, two quantitative methods were evaluated: 1) using disc pieces from our OptiSafe™ test method and 2) directly in the 24-well plate. For the first exposure method, 100 pL of the study chemical were added to the center of the eye disc, placed in a 24-well plate containing 1.25 ml of alkaline phosphatase reaction reagent (10,000 units), and incubated at 37 °C for 18 hours. For the second exposure method, 100 pL of the study chemical were added to the corresponding well of a 24-well plate containing 1.25 ml of alkaline phosphatase reaction reagent, mixed with the same pipette tip, wrapped, and incubated at 37 °C for 18 hours.The following day, 20 pL of alkaline phosphatase reaction reagent in the wells was added to a 96-well plate containing 200 pL of p-nitrophenyl phosphate disodium salt (PNPP) substrate solution, incubated for 1 hour, and the results were read at 405 nm using a spectrophotometer.

[00261] Protocol: Experiment with Alkaline Phosphatase - Third Week - List of Chemical Products - Table 8 Condition Chemical Name CASRN GHS in vivo PBS Cl (2-bromoethyl)benzene 103-63-9 NC C2 Triethylene glycol 112-27-6 NC Petition 870260062914, dated 06 / 26 / 2026, page 86 / 157 82 / 147 C3 2-Methyl-4-phenyl-2-butanol 103-05-9 NC C4 Cyclamen aldehyde 103-95-7 Cat. 2 C5 Heptanal 111-71-7 Cat. 2 C6 di-n-propyl disulfide 629-19-6 Cat. 2 C7 Ethanolamine 141-43-5 Cat. 1 C8 Dimethyldipropyletriamine 10563-298 Cat. 1 C9 Acetic acid 64-19-7 Cat. 1 Virgin PREPARATION

[00262] 1. Alkaline phosphatase reaction reagent

[00263] a. Take 1 vial of AP, add 1 ml of PBS, mix with a pipette / repipette 10 times.

[00264] b. In a 50 ml conical tube, add 1 ml of the AP solution prepared above and 40 ml of PBS.

[00265] 2. Prepare two 24-well plates (1 with discs, 1 without discs)

[00266] 3. Prepare two 96-well plates for PNPP reading (3 wells for each chemical under study)

[00267] a. Rate 200 pL PNPP and PBS PROCEDURE

[00268] 1. Plate 1 (without discs)

[00269] a. Add 1.25 ml of the prepared PBS-AP conical tubing to each well (11 wells)

[00270] b. Add 100 pL of the test material to the well, mix with the same pipette tip, discard, and repeat for the remaining test materials.

[00271] c. Wrap the dish in plastic wrap and place it in the container. Petition 870260062914, dated 06 / 26 / 2026, page 87 / 157 83 / 147

[00272] 2. Plate 2 (Discs)

[00273] a. Configure the 11 disks on the board so that the board cover is stable.

[00274] b. Add 1.25 ml of the prepared PBS-AP conical tubing to each well (11 wells)

[00275] c. Add 100 μL of the test material to the center of the disc and repeat for the remaining test materials.

[00276] d. Place the plate cover and put it in the container.

[00277] 3. Incubate for 18 hours at 37° C

[00278] 4. To the 96-well plates prepared with 200 μL PNPP and PBS, add 20 μL of the 24-well plate.

[00279] 5. Incubate for 1 hour READING RESULTS

[00280] 1. Measure in OD405

[00281] The results are shown in Figures 3A and 3B.

[00282] Interpretation of Figures 3A and 3B: The same nine chemicals (C1: (2-bromoethyl)benzene, C2: Triethylene glycol, C3: 2-Methyl-4-phenyl-2-butanol, C4: Cyclamen aldehyde, C5: Heptanal, C6: Di-n-propyl disulfide, C7: Ethanolamine, C8: Dimethyldipropyltriamine, and C9: Acetic acid) were tested in a new study to find the best exposure method, comparing the results with and without eye discs. As shown in Figures 3A and 3B, there was no significant difference in readings between the two methods, and therefore the method without discs was chosen to keep the test method less complicated. In Figure 3A, the results for the three dermal corrosives (C7 [ethanolamine]: 0.170, 0.166, Petition 870260062914, dated 06 / 26 / 2026, page 88 / 157 84 / 147 0.172; C8 [Dimethyldipropylenetramine]: 0.152, 0.159, 0.140; C9 [Acetic acid]: 0.106, 0.102, 0.103) were much lower than the dermal irritants and non-irritants (C1 [(2-bromoethyl)benzene], C2 [triethylene glycol], C3 [2-methyl-4-phenyl-2-butanol], C4 [cyclamen aldehyde], C5 [heptanal], C6 [di-n-propyl disulfide]) which were all above 3.000, suggesting that there was no inhibition of alkaline phosphatase. In Figure 3B, the results for the three dermal corrosives (C7 [ethanolamine]: 0.174, 0.185, 0.175; C8 [Dimethyldipropylenetramine]: 0.138, 0.176, 0.133; C9 [Acetic acid]: 0.104, 0.129, 0.101) were lower than the dermal irritants and non-irritants (C1 [(2-bromoethyl)benzene], C2 [triethylene glycol], C3 [2-methyl-4-phenyl-2-butanol], C4 [cyclamen aldehyde], C5 [heptanal], C6 [di-n-propyl disulfide]) which were all above 3.000, suggesting that there was no inhibition of alkaline phosphatase.These results expanded upon the inventors' findings, as dermal corrosives had the potential to inhibit purified alkaline phosphatase outside the tissue; therefore, the nitrophenyl phosphate disodium salt (PNPP) substrate system failed to detect the presence of alkaline phosphatase. For the blank reaction reagents, no alkaline phosphatase was added to the reagent to account for any chemical interference in the OD value. Two conditions (with and without discs) were tested to assess the need for a controlled input of the chemical under study into the reaction reagent; however, based on the results of this experiment, the results show that the discs were not necessary. For analytical purposes, if the corrosive cutoff is set to less than approximately 0.500, this... Petition 870260062914, dated 06 / 26 / 2026, page 89 / 157 Study 85 / 147 shows that the test system correctly identified dermal corrosives. With these results, it was decided to repeat this study and proceed to the next study to optimize the exposure period and incubation period of p-nitrophenyl phosphate disodium salt (PNPP).

[00283] The next study included repeating the previous study (method without eye discs) and introducing a new study with an exposure period to the test chemical of 10 minutes and incubation periods of p-nitrofentill phosphate disodium salt (PNPP) of 10 minutes, 1 hour, 4 hours, and 18 hours. For the new study, alkaline phosphatase was suspended in phosphate-buffered saline (PBS) to make the reaction reagent. Then, 100 μL of the study chemical was added directly to the corresponding wells in a 24-well plate, with 1.25 mL of already aliquoted alkaline phosphatase reaction reagent. After 10 minutes, the wells are mixed with the tip of the pipette and 20 μL of the reaction reagent are divided into aliquots in a 96-well plate containing 200 μL of p-nitrophenyl phosphate disodium salt (PNPP) and phosphate-buffered saline (PBS; blank).The results for the disodium salt of p-nitrophenyl phosphate (PNPP) are read after 10 minutes, 1 hour, 4 hours, and 18 hours of incubation at 37 °C.

[00284] Protocol: Experiment with Alkaline Phosphatase - Fourth Week LIST OF CHEMICAL PRODUCTS TABLE 9 Condition Chemical Name CASRN GHS in vivo Petition 870260062914, dated 06 / 26 / 2026, page 90 / 157 86 / 147 PBS C1 1-Bromo-4-chlorobutane 6940-78-9 NC C2 1,5-hexadiene 592-42-7 NC C3 Isopropanol 67-63-0 NC C4 Isopropyl myristate 110-27-0 NC C5 Di-n-propyl disulfide 629-19-6 Cat. 2 C6 Cyclamen aldehyde 103-95-7 Cat. 2 C7 Dimethyldipropyltriamine 10563-29-8 Cat. 1 C8 Potassium hydroxide (10%) 1310-58-3 Cat. 1 C9 2-Methylbutyric acid 116-53-0 Cat. 1 Virgin PREPARATION

[00285] 1. Alkaline phosphatase reaction reagent

[00286] a. Take 1 bottle of AP, add 1 ml of PBS, mix with a pipette / repipette 10 times.

[00287] b. In a 50 ml conical tube, add 1 ml of the AP solution prepared above and 40 ml of PBS.

[00288] 2. Prepare two 24-well plates

[00289] 3. Prepare two 96-well plates for PNPP reading (3 wells for each chemical under study)

[00290] a. Aliquot with 200 μL PNPP and PBS

[00291] 4. Prepare glass jars

[00292] Repeat procedure 1-3-23

[00293] 1. 24-well plate

[00294] a. Add 1.25 ml of the prepared PBS-AP conical tube to each well (11 wells)

[00295] b. Add 100 μL of the test material to the well, mix with the same pipette tip, discard and Petition 870260062914, dated 06 / 26 / 2026, page 91 / 157 87 / 147 Repeat for the remaining test materials.

[00296] c. Wrap the dish in plastic film and place it in the container.

[00297] 2. Incubate for 18 hours at 37°C

[00298] 3. To the prepared 96-well plate containing 200 μL of PNPP or PBS, add 20 μL of the 24-well plate.

[00299] 4. Incubate for 1 hour

[00300] 5. Measure at 405 nm PROCEDURE

[00301] 1. 24-well plate

[00302] a. Add 1.25 ml of the prepared PBS-AP conical tube to each well (11 wells)

[00303] b. Add 100 μL of the test material Add the solution to the well, mix with the same pipette tip, discard, and repeat for the remaining test materials.

[00304] c. After 10 minutes, mix each well

[00305] 2. To the prepared 96-well plate containing 200 μL of PNPP or PBS, add 20 μL of the 24-well plate.

[00306] 3. Measure in 10 minutes, 1 hour, 4 hours, hours TEST TUBE PROCEDURE

[00307] 1. Aliquot 200 μL from the 24-well plate into glass vials filled with 2 ml of PNPP.

[00308] The results of the repeated study are shown in Figure 4, the results for the different incubation periods of the disodium salt of p-nitrophenyl phosphate (PNPP) are shown below in Figures 5A - 5D, and the results of the visual study of the test tube are shown in Figure 1.

[00309] Interpretation of Figure 1, Figure 4 and Petition 870260062914, dated 06 / 26 / 2026, page 92 / 157 88 / 147 Figures 5A - 5D: In repeating the previous study, it was concluded that the results were reproducible and the best exposure method was to add directly to the well containing the alkaline phosphatase reaction reagent. For example, as shown in Figure 4, the results for the dermal corrosives (C7 [Dimethyldipropylenetramine]: 0.132, 0.128, 0.127); C8 [Potassium hydroxide (10%)]: 0.123, 0.124, 0.124; C9 [2-methylbutyric acid]: 0.138, 0.138, 0.137) were still significantly lower than the dermal irritants or non-irritants (C1 [1-Bromo-4-chlorobutane], C2 [1,5hexadiene], C3 [isopropanol], C4 [isopropyl myristate], C5 [di-n-propyl disulfide], C6 [cyclamen aldehyde]) which had readings above 3.000. For the 10-minute chemical exposure test study, it was concluded that this exposure period was too short and was not long enough for the dermal corrosives to interact with the alkaline phosphatase reaction reagent.This study concluded that the ideal exposure period was 18 hours with a 1-hour incubation period of p-nitrophenyl phosphate disodium salt (PNPP). For example, for dimethyldipropylenethramine, the 10-minute PNPP incubation readings were 3.449, 3.502, and 3.500 (Figure 5A), the 1-hour PNPP incubation readings were 0.352, 0.283, and 0.333 (Figure 5B), the 4-hour PNPP incubation readings were 0.122, 0.116, and 0.114 (Figure 5C), and the 18-hour PNPP incubation readings were 0.112, 0.105, and 0.110 (Figure 5D). Therefore, based on these results, the most ideal incubation period for PNPP was 1 hour. As shown in Figure 1, a visual study was performed by adding 200 μL to 2 ml of p-nitrophenyl phosphate disodium salt (PNPP) substrate solution to see the... Petition 870260062914, dated 06 / 26 / 2026, page 93 / 157 89 / 147 color reaction and no color was observed for the dermal corrosive chemicals, while the dermal irritants and non-irritants turned yellow (Figure 1). Therefore, for the next study, another source of alkaline phosphatase was obtained (Sigma Aldrich, St. Louis, MO; Catalog No. P7640) to try to optimize the concentration of alkaline phosphatase present in the reaction reagent to mediate the color intensity of the reaction of the disodium salt of p-nitrophenyl phosphate (PNPP).

[00310] The next study compared different dilutions of the original alkaline phosphatase source (1:10 [Activity: 1,000 units] and 1:100 [100 units]) and two alkaline phosphatase sources, both purchased from Sigma Aldrich (St. Louis, MO). The first alkaline phosphatase (Catalog No. P0114) is an aqueous glycerol-buffered solution with 10,000 units, a specific activity of >5,700 DEA units / mg of protein, and a concentration of >10 mg / ml; this was the original alkaline phosphatase used for the previous studies. The second alkaline phosphatase (Catalog No. P7640) is a lyophilized powder with a specific activity of >10 DEA units / mg solid; this was prepared by adding 400 mg to 40 ml of phosphate-buffered saline (PBS). The five conditions for this study included original alkaline phosphatase (1:1), original alkaline phosphatase (1:10), original alkaline phosphatase (1:100), new alkaline phosphatase (1:1), and the blanks (phosphate-buffered saline (PBS)).Four controls (Water, Water / 1% Ink, Acetic Acid, Acetic Acid / 1% Ink) and thirty chemicals (1,5-Hexadiene, Isopropanol, 4(Methylthio)benzaldehyde, Isopropyl myristate, Benzyl salicylate, Phenylethyl alcohol, Hydroxycitronellal, Benzyl acetate, Dipropylene glycol, Di-n-propyl disulfide, 1Petition 870260062914, dated 06 / 26 / 2026, page 94 / 157. 90 / 147 Bromopentane, cyclamen aldehyde, cis-3-methyl-2-(2-pentenyl)2-cyclopenten-1-one, p-metha-1,8-dien-7-ol, heptanal, trans-3,7-dimethyl-2,6-octadien-1-ol, alpha-terpineol, 1-decanol, nonyl aldehyde, dimethyldipropylenetramine, potassium hydroxide (10%), 2-methylbutyric acid, octanoic acid, dimethylisopropylamine, 2-tert-butylphenol, 5-isopropyl-2-methylphenol, n-heptylamine, methoxy-3-propylamine, N,N-dimethylbenzylamine) were tested. The two controls contained ink to determine if there would be any background effect. In a 24-well plate, 1 ml of the reaction reagent for each condition was added to the corresponding well, followed by 100 μL of the chemical under study, mixed with the tip of a pipette, and incubated for 18 hours at room temperature.The following day, 96-well plates were prepared with 200 μL of p-nitrophenyl phosphate disodium salt and phosphate-buffered saline solution (blanks), and then 20 μL from the 24-well plates were added and incubated for 1 hour with results read at 405 nm.

[00311] Protocol: Experiment with Alkaline Phosphatase - Fifth Week - List of Chemical Products - Table 10 Chemical No. Chemical Name CASRN GHS in vivo 1 NC1: Water 2 NC2: Water / 1% ink 3 PC1: Acetic acid 4 PC2: Acetic acid / 1% ink 5 1-Bromo-4-chlorobutane 6940-78-9 NC Petition 870260062914, dated 06 / 26 / 2026, page 95 / 157 91 / 147 6 1,5-hexadiene 592-42-7 NC 7 Isopropanol 67-63-0 NC 8 4-(Methylthio)benzaldehyde 3446-89-7 NC 9 Isopropyl myristate 110-27-0 NC 10 Benzyl salicylate 118-58-1 NC 11 Phenylethyl alcohol 60-12-8 NC 12 Hydroxycitronellal 107-75-5 NC 13 Benzyl acetate 140-11-4 NC 14 Dipropylene glycol 25265-71-8 NC 15 Di-n-propyl disulfide 629-19-6 Cat. 2 16 1-Bromopentane 110-53-2 Cat. 2 17 Cyclamen aldehyde 103-95-7 Cat. 2 18 cis-3-methyl-2-(2-pentenyl) 2-cyclopentene-1-one 488-10-8 Cat. 2 19 p-Metha-1,8-dien-7-ol 18457-55-1 Cat. 2 20 Heptanal 111-71-7 Cat. trans-3,7-dimethyl-2,6octadiene-1-ol 106-24-1 Cat. 2 22 alpha-Terpineol 98-55-5 Cat. 2 23 1-Decanol 112-30-1 Cat. 2 24 Nonyl aldehyde 124-19-6 Cat. 2 25 Dimethyldipropyletriamine 10563-29-8 Cat. 1A 26 Potassium hydroxide (10%) 1310-58-3 Cat. 1B 27 2-Methylbutyric acid 116-53-0 Cat. 1B / 1C 28 Octanoic acid 124-07-02 Cat. 1B / 1C 29 Dimethylisopropylamine 996-35-0 Cat. 1B / 1C 30 2-tert-Butylphenol 88-18-6 Cat.1B / 1C 31 5-Isopropyl-2-methylphenol 499-75-2 Cat. 1B / 1C. Petition 870260062914, dated 06 / 26 / 2026, page 96 / 157 92 / 147 32 n-heptylamine 111-68-2 Cat. 1B / 1C 33 Methoxy-3-propylamine 5332-73-0 Cat. 1B / 1C 34 N,N-Dimethylbenzylamine 103-83-3 Cat. 1C PREPARATION

[00312] 1. Alkaline phosphatase reaction reagent

[00313] a. Original AP

[00314] i. Add 1 ml of PBS to the AP bottle, pipette again 10 times and invert with the cap on to mix well. Take this 1 ml and add it to a 50 ml conical tube filled with 40 ml of PBS. Invert to mix 10 times, vortex mixing, let stand for 2 minutes, invert to mix again 10 times.

[00315] ii. Prepare 2 more 50 ml conical tubes

[00316] 1. 1:10

[00317] a. Take 4 ml of the solution made above, add it to 36 ml of PBS. Vortex the mixture and then invert to mix 10 times.

[00318] 2. 1:100

[00319] a. Take 4 ml of the solution prepared above, add it to 36 ml of PBS. Mix using a vortex mixer and then invert to mix 10 times.

[00320] b. New AP

[00321] i. Add 400 mg to a 50 ml conical tube filled with 40 ml of PBS. Invert to mix 10 times, vortex, let stand for 2 minutes, invert to mix 10 more times. Check that all are suspended. If not, mix and vortex until Petition 870260062914, dated 06 / 26 / 2026, page 97 / 157 93 / 147 that is totally suspended.

[00322] 2. Prepare 24-well plates

[00323] 3. Prepare six 96-well plates for PNPP reading (3 wells for each chemical under study)

[00324] CONDITIONS a. Aliquot । with 200 μL of PNPP

[00325] 1. Old AP

[00326] 2. Old AP (1:10)

[00327] 3. Old AP (1:100)

[00328] 4. New AP

[00329] 5. Blank (PBS only) PROCEDURE

[00330] 1. 24-well plates

[00331] a. Add 1 ml (using P1000, blue tips) of the prepared PBS-AP to the plate (for each condition)

[00332] b. Add 100 μL of the test material Add the solution to the well, mix with the same pipette tip, discard, and repeat for the remaining test materials.

[00333] c. Wrap the dish in plastic wrap and place it in the container.

[00334] d. Incubate for 18 hours at room temperature

[00335] 2. To the prepared 96-well plate containing 200 μL of PNPP or PBS, add 20 μL of the 24-well plate.

[00336] 3. Incubate for 1 hour

[00337] 4. Reading at 405 nm

[00338] The results of this study are shown in Figures 6A - 6F. From this study, it was concluded that a good source of alkaline phosphatase was the original source (Sigma Petition 870260062914, dated 06 / 26 / 2026, page 98 / 157 94 / 147 Aldrich, St. Louis, MO; Catalog No. P0114), but both will work and the best reaction reagent concentration was 1:100 (100 units).

[00339] Interpretation of Figures 6A — 6F: As shown in Figure 6B, the 1:1 alkaline phosphatase concentration appears to be saturated and a very high concentration because it resulted in maximum or near-maximum OD405 readings for all chemicals except for dimethyldipropylenetriamine No. 25 and potassium hydroxide No. 26 (10%). The other dermal corrosives (No. 27 2-methylbutyric acid, No. 28 octanoic acid, No. 29 dimethylisopropylamine, No. 30 2-tert-butylphenol, No. 31 5-isopropyl-2-methylphenol, No. 32 n-heptylamine, No. 33 methoxy-3-propylamine and No. 34 N,N-dimethylbenzylamine) were incorrectly predicted based on the high OD405 values. As shown in Figure 6C, for the 1:10 dilution, there was still a high OD405 reading for most dermal corrosives; however, the results for dimethyldipropylenetriamine No. 25 were now poorly predicted. This dilution was corrected for 2-methylbutyric acid No. 27 and methoxy-3-propylamine No. 33.As shown in Figure 6D, the 1:100 dilution correctly predicted No. 25 Dimethyldipropylenetriamine, No. 26 Potassium hydroxide (10%), No. 27 2-Methylbutyric acid, No. 30 2-tert-Butylphenol, and No. 33 Methoxy-3-propylamine. As shown in Figure 6D, for the chemicals that did not cause a significant reduction in enzymatic activity (No. 28 Octanoic acid [3.462, 3.491, 3.333]; No. 29. Dimethylisopropylamine [3.466, 3.502, 3.502]; No. 31 5Isopropyl-2-methylphenol [0.991, 0.974, 1.167]; No. 32 nHeptylamine [3.244, 3.057, 3.152]; No. 34 N,NExclusion 870260062914, dated 06 / 26 / 2026, page 99 / 157 95 / 147 Dimethylbenzylamine [3.431, 3.502, 3.61]), the chemical product was studied and the solubility of these chemicals in phosphate-buffered saline (PBS) was questioned, and further research into their properties revealed that they were mostly insoluble in water, and therefore, in subsequent experiments, some different solvents were tried.

[00340] In our next study, different solvents (dimethyl sulfoxide [DMSO], methanol, ethanol, acetone, vegetable oil, and mineral oil) were selected by adding 200 μL of solvent to 2 mL of p-nitrophenyl phosphate disodium salt (PNPP) in a 24-well plate and allowing the plate to stand for 1 hour. The results were not quantified with the spectrophotometer, but the color change was observed visually. The solvents with the brightest yellow intensity, similar to water, were ethanol, acetone, and vegetable oil, while the lowest yellow intensity was methanol and mineral oil, and no (clear) response was observed with dimethyl sulfoxide (DMSO). With these results, the main experiment was carried out with two chosen solvents, ethanol and acetone. Three alkaline phosphatase reaction reagents (1:10 concentration) were tested: phosphate-buffered saline (PBS), ethanol, and acetone.Twelve chemicals were selected (water, methanol, mineral oil, 1-Bromo-4-chlorobutane, 1,5-hexadiene, di-n-propyl disulfide, nonyl aldehyde, octanoic acid, dimethylispropylamine, 5-isopropyl-2-methylphenol, heptylamine, N,N-dimethylbenzylamine). Two different dosage concentrations were evaluated: 10 μL of the chemical under study added to 90 μL of alkaline phosphatase reaction reagent and 50 μL of the chemical under study. Petition 870260062914, dated 06 / 26 / 2026, pp. 100 / 157 96 / 147 were added to 50 μL of alkaline phosphatase reaction reagent; all were divided into aliquots in 96-well plates. After dosing, the 96-well plates were incubated for 3 hours and 22 hours at room temperature, and 20 μL were added to a different 96-well plate containing 200 μL of p-nitrophenyl phosphate disodium salt (PNPP) and incubated for 30 minutes and 1 hour at room temperature for the 3-hour exposure, and then 10 minutes, 30 minutes, and 1 hour for the 22-hour exposure. It was observed that some wells evaporated or melted and could not be recovered for quantification using the spectrophotometer at an optical density of 405 nm, as shown in Figures 7A-7B and Figures 8A-8C.

[00341] Protocol: Experiment with Alkaline Phosphatase, Sixth Week, List of Chemical Products, Table 11 Chemical No. Chemical Name CASRN GHS in vivo 1 Water n / dn / d 2 Methanol 67-56-1 n / d 3 Mineral Oil 8042-47-5 n / d 4 1-Bromo-4-chlorobutane 6940-78-9 NC 5 1,5-hexadiene 592-42-7 NC 6 Di-n-propyl disulfide 629-19-6 Cat. 2 7 Nonyl aldehyde 124-19-6 Cat. 2 8 Octanoic acid 124-07-02 Cat. 1B / 1C 9 Dimethylisopropylamine 996-35-0 Cat. 1B / 1C Petition 870260062914, dated 06 / 26 / 2026, pp. 101 / 157 97 / 147 10 5-Isopropyl-2-methylphenol 499-75-2 Cat. 1B / 1C 11 n-heptylamine 111-68-2 Cat. 1B / 1C 12 N,N-Dimethylbenzylamine 103-83-3 Cat. 1C PREPARATION

[00342] 1. Alkaline phosphatase reaction reagent (fresh, 1:100)

[00343] a. Take 4 ml of AP stock solution (1:10) and add to 36 ml of PBS. Mix in a vortex and then invert to mix 10 times.

[00344] 2. Prepare the alkaline phosphatase reaction reagent for each condition.

[00345] a. 200 μL AP (1:10) in 1.8 ml of PBS

[00346] b. 200 μL AP (1:10) in 1.8 ml of ethanol

[00347] c. 200 μL AP (1:10) in 1.8 ml of acetone

[00348] 3. Prepare 96-well plates for dosing

[00349] 4. Prepare 96-well plates for PNPP reading (3 wells for each chemical under study)

[00350] a. Aliquot with 200 μL of PNPP CONDITIONS

[00351] 1. 1:10 in PBS (A)

[00352] 2. 1:10 in ethanol (B)

[00353] 3. 1:10 in acetone (C) PROCEDURE

[00354] 1. Set 1

[00355] a. 10 μL of chemical product added to μL of reaction reagent AP

[00356] 2. Set 2

[00357] a. 50 μL of chemical product added to Petition 870260062914, dated 06 / 26 / 2026, pp. 102 / 157 98 / 147 μL of AP reaction reagent

[00358] 3. Incubate for 3 hours

[00359] a. To the 96-well plate prepared with 200 μL of PNPP divided into aliquots, add 20 μL

[00360] b. Read the results at 405 nm at 30 minutes and 1 hour.

[00361] 4. Keep the remainder for 18 hours

[00362] a. To the 96-well plate prepared with 200 μL of PNPP divided into aliquots, add 20 μL

[00363] b. Read the results at 405 nm at 10 minutes, 30 minutes, and 1 hour.

[00364] Interpretation of Figures 7A - 7B and Figures 8A - 8C: Twelve chemicals were selected to screen three solvents (buffered saline, ethanol, and acetone) to try to solve problems with dermal corrosive chemicals in the previous study that did not have the expected low results. The twelve chemicals include water, methanol, mineral oil, 1-Bromo-4-chlorobutane, 1,5-hexadiene, di-n-propyl disulfide, nonyl aldehyde, octanoic acid, dimethylisopropylamine, 5-isopropyl-2-methylphenol, n-heptylamine, and N,N-dimethylbenzylamine. Two different dosage concentrations were evaluated (10 μL in 90 μL of reaction reagent and 50 μL in 50 μL of reaction reagent) and it was found that the results for the 10 μL dose concentration were better because the results for the 50 μL dose concentration were low for many dermal irritants and non-irritants.For example, as shown in Figure 7A, in the solvent ethanol, the concentration of 4 1-Bromo-4-chlorobutane was 3.349 (10 pL) compared to 0.700 (50 pL). Furthermore, it was concluded that leaving the reaction of the disodium salt of... Petition 870260062914, dated 06 / 26 / 2026, pp. 103 / 157 99 / 147 p-nitrophenyl phosphate (PNPP) incubation for only 30 minutes was too short, and the readings for dermal irritants and non-irritants were lower than if the reaction were incubated for a full hour. For example, as shown in Figure 7A, for a dose of 10 μL in ethanol solvent, at 30 minutes, on° 5 1,5-hexadiene was 1.702 and on° 7 nonyl aldehyde was 1.589. However, after a full 1-hour incubation, as shown in Figure 7B, on° 5 1,5-hexadiene was... The concentration of nonylaldehyde in 2,947 eon° 7 was 3,078. As shown in Figures 8A-8B, after 18 hours of incubation, acetone was not the ideal solvent because most of the wells evaporated or melted. However, ethanol appeared to be a good solvent because it resolved the problems with previous dermal corrosives that did not provide low readings. The dermal corrosive chemicals from the previous experiment now had a significant reduction in enzymatic activity, as shown in Figures 8A-8B. Based on this finding, there was a focus on refining the alkaline phosphatase reaction reagent to be made from buffered saline and ethanol and confirming whether a 1:100 dilution of alkaline phosphatase in ethanol works.

[00365] In our next study, ethanol and buffered saline solutions (potassium chloride, monobasic potassium phosphate, sodium chloride, dibasic sodium phosphate) were used as solvents. First, two different concentrations (1:100 and 1:1000) of the alkaline phosphatase reaction reagent were compared to optimize the color intensity response of the alkaline phosphatase substrate disodium salt of p-nitrophenyl phosphate (PNPP), and incubation was performed at room temperature or 37°C. Twelve products Petition 870260062914, dated 06 / 26 / 2026, pp. 104 / 157 100 / 147 chemicals (4-(Methylthio)benzaldehyde, 1-Bromo-4-chlorobutane, 1,5-Hexadiene, alpha-Terpineol, di-n-propyl disulfide, nonyl aldehyde, octanoic acid, dimethylisopropylamine, 5-isopropyl-2-methylphenol, n-heptylamine, and N,N-dimethylbenzylamine) were tested. The chemicals under study (20 μΣ) were added to two sets of 0.5 ml microcentrifuge tubes containing 180 μΣ of alkaline phosphatase reaction reagent and then incubated at room temperature or 37 °C for 4 hours and 18 hours. After the exposure period, 20 μΣ of the reaction reagent were added to 200 μΣ of p-nitrophenyl phosphate disodium salt (PNPP) substrate solution in a 96-well plate, for readings at 30 minutes and 1 hour at 405 nm.

[00366] Protocol Experiment with Alkaline Phosphatase, Seventh Week, List of Chemical Products, Table 12 Chemical No. Chemical Name CASRN GHS in vivo 1 Water n / dn / d 2 4-(Methylthio)benzaldehyde 3446-89-7 NC 3 1-Bromo-4-chlorobutane 6940-78-9 NC 4 1,5-hexadiene 592-42-7 NC 5 alpha-Terpineol 98-55-5 Cat. 2 6 Di-n-propyl disulfide 629-19-6 Cat. 2 7 Nonyl aldehyde 124-19-6 Cat. 2 8 Octanoic acid 124-07-02 Cat. 1B / 1C 9 Dimethylisopropylamine 996-35-0 Cat. 1B / 1C Petition 870260062914, dated 06 / 26 / 2026, pp. 105 / 157 101 / 147 10 5-Isopropyl-2-methylphenol 499-75-2 Cat. 1B / 1C 11 n-heptylamine 111-68-2 Cat. 1B / 1C 12 N,N-Dimethylbenzylamine 103-83-3 Cat. 1C PREPARATION

[00367] 1. Alkaline phosphatase reaction reagent

[00368] a. 1:100

[00369] i. PBS: Take 400 μL of AP stock solution (1:10) and add to 3.6 ml of PBS. Mix in a vortex and then invert to mix 10 times.

[00370] ii. Ethanol: Take 400 μL of AP stock solution (1:10), add 3.6 ml of ethanol. Mix in a vortex and then invert to mix 10 times.

[00371] b. 1:1000

[00372] i. PBS: Take 400 μL of AP stock solution (1:100) and add to 3.6 ml of PBS. Mix in a vortex and then invert to mix 10 times.

[00373] ii. Ethanol: Take 400 μL of AP (1:10)0 stock solution, add 3.6 ml of ethanol. Mix in a vortex and then invert to mix 10 times.

[00374] 2. Prepare microcentrifuge tubes of 0.5 ml for dosage

[00375] a. One set for room temperature, one set for 37 °C

[00376] 3. Prepare 96-well plates for PNPP reading (3 wells for each chemical under study)

[00377] a. Aliquot with 200 μL of PNPP CONDITIONS Petition 870260062914, dated 06 / 26 / 2026, pp. 106 / 157 102 / 147

[00378] 1. 1:100 in PBS at RT

[00379] 2. 1:100 in ethanol at RT

[00380] 3. 1:100 in PBS at 37°C

[00381] 4. 1:100 in ethanol at 37°C

[00382] 5. 1:1000 in PBS at RT

[00383] 6. 1:1000 in ethanol at RT

[00384] 7. 1:1000 in PBS at 37°C

[00385] 8. PROCEDURE 1:1000 in ethanol at 37°C

[00386] 1. In each microcentrifuge tube for each condition, add 180 μL of the corresponding AP reaction reagent.

[00387] 2. Add 20 μL of the chemical under study to each microcentrifuge tube.

[00388] 3. Incubate for 4 hours (one hour at room temperature; one hour at 37 °C)

[00389] 4. For 96-well plates prepared with 200 μL of PNPP aliquots, add 20 μL from each microcentrifuge tube to the corresponding well.

[00390] 5. Read at 30 minutes and 1 hour at 405 nm.

[00391] The results are shown in Figures 9A - 9B and in Figures 10A - 10B.

[00392] Interpretation of Figures 9A - 9A and 10A - 10B: Twelve chemicals were tested to evaluate the different alkaline phosphatase reaction reagents diluted to 1:100 and 1:1000, the results using ethanol as solvent, and whether the incubation temperature affects the results. Based on the results shown in Figures 9A-9B and 10A-10B, the 1:1000 dilution of the alkaline phosphatase reaction reagent was too dilute and the results were inconclusive. Petition 870260062914, dated 06 / 26 / 2026, pp. 107 / 157 103 / 147 because the readings were variable and did not indicate the normal trend observed in previous studies for dermal corrosives, irritants, and non-irritants. For example, in Figure 9B for the 1:1000 ethanol solvent condition, No. 3 1-Bromo4-chlorobutane, a non-irritant dermally, was 0.799, while No. 12 N,N-dimethylbenzylamine, a dermal corrosive, was 0.587. Furthermore, the results for incubation at 37°C were not as good as the results for incubation at room temperature. For example, in Figure 9D for the 1:100 ethanol solvent condition, No. 2 4-(methylthio)benzaldehyde was 2.243 at room temperature and 0.857 at 37°C. Therefore, for our next study, the most ideal conditions of this study were chosen, which was to use a 1:100 dilution of the alkaline phosphatase reaction reagent with buffered saline solution and ethanol, followed by a 4-hour incubation at room temperature.

[00393] February 1, 2023: In our next study, we focused on a 1:100 concentration of the alkaline phosphatase reaction reagent and explored the option of using a 4 ml glass vial with a screw cap instead of the 0.5 ml plastic microcentrifuge tubes. Thirty chemicals were tested (1,5-hexadiene, isopropanol, 4-(methylthio)benzaldehyde, isopropyl myristate, benzyl salicylate, phenylethyl alcohol, hydroxycitronellal, benzyl acetate, dipropylene glycol, di-n-propyl disulfide, 1-bromopentane, cyclamen aldehyde, cis-3-methyl-2-(2-pentenyl)2-cyclopenten-1-one, p-meta-1,8-dien-7-ol, heptanal, trans-3,7-dimethyl-2,6-octadiene-1-ol, alpha-terpineol, 1-decanol, nonyl aldehyde, dimethyldipropylentriamine, potassium hydroxide (10%), 2-methylbutyric acid, octanoic acid, Petition 870260062914, dated 06 / 26 / 2026, pp. 108 / 157 104 / 147 dimethylisopropylamine, 2-tert-butylphenol, 5-isopropyl-2-methylphenol, n-heptylamine, methoxy-3-propylamine, N,N-dimethylbenzylamine). The dosage of the chemical under study was changed to 30 μL and the alkaline phosphatase reaction reagent to 300 μL, but the incubation was maintained at 4 hours at room temperature. The four reaction reagents are R1+ (alkaline phosphatase in phosphate-buffered saline solution), R1- (phosphate-buffered saline solution only), R2+ (alkaline phosphatase in ethanol), and R2- (ethanol only). Another change was to add 3 ml of the p-nitrophenyl phosphate (PNPP) disodium salt substrate solution directly to the glass vial and let it stand for 1 hour, then add 200 pL to a 96-well plate for quantification at 405 nm using a spectrophotometer.

[00394] Protocol Experiment with Alkaline Phosphatase, Eighth Week, List of Chemical Products, Table 13 Chemical No. Chemical Name CASRN GHS in vivo 1 NC1: Water 2 NC2: Water / 1% ink 3 PC1: Acetic acid 4 PC2: Acetic acid / 1% ink 5 1-Bromo-4-chlorobutane 6940-78-9 NC 6 1,5-hexadiene 592-42-7 NC 7 Isopropanol 67-63-0 NC 8 4-(Methylthio)benzaldehyde 3446-89-7 NC 9 Isopropyl myristate 110-27-0 NC Petition 870260062914, dated 06 / 26 / 2026, pp. 109 / 157 105 / 147 10 Benzyl salicylate 118-58-1 NC 11 Phenylethyl alcohol 60-12-8 NC 12 Hydroxycitronellal 107-75-5 NC 13 Benzyl acetate 140-11-4 NC 14 Dipropylene glycol 25265-71-8 NC 15 Di-n-propyl disulfide 629-19-6 Cat. 2 16 1-Bromopentane 110-53-2 Cat. 2 17 Cyclamen aldehyde 103-95-7 Cat. 2 18 cis-3-methyl-2-(2-pentenyl)2-cyclopenten-1-one 488-10-8 Cat. 2 19 p-Metha-1,8-dien-7-ol 18457-55-1 Cat. 2 20 Heptanal 111-71-7 Cat. 2 21 trans-3,7-dimethyl-2,6-octadien-1-ol 106-24-1 Cat. 2 22 alpha-Terpineol 98-55-5 Cat. 2 23 1-Decanol 112-30-1 Cat. 2 24 Nonyl aldehyde 124-19-6 Cat. 2 25 Dimethyldipropyltriamine 10563-29-8 Cat. 1A 26 Potassium hydroxide (10%) 1310-58-3 Cat. 1B 27 2-Methylbutyric acid 116-53-0 Cat. 1B / 1C 28 Octanoic acid 124-07-02 Cat. 1B / 1C 29 Dimethylisopropylamine Cat. 5332-73-0 Cat. 1B / 1C Petition 870260062914, dated 06 / 26 / 2026, pp. 110 / 157 106 / 147 34 N,N-Dimethylbenzylamine 103-83-3 Cat. 1C PREPARATION

[00395] 1. Reaction reagents

[00396] a. AP reaction reagent 1:100

[00397] i. PBS: Take 4 ml of stock solution AP (1:10) and add to 36 ml of PBS. Mix in a vortex and then invert to mix 10 times.

[00398] ii. Ethanol: Take 4 ml of AP stock solution (1:10), add to 36 ml of ethanol. Mix in a vortex and then invert to mix 10 times.

[00399] b. PBS (1x)

[00400] c. Ethanol

[00401] 2. Prepare 4 ml glass vials for dosing.

[00402] a. Four sets

[00403] i. R1+ (PBS-AP)

[00404] ii. R1- (PBS)

[00405] iii. R2+ (Ethanol-AP)

[00406] iv. R2- (Ethanol)

[00407] 3. Prepare 96-well plates for PNPP reading (3 wells for each chemical under study) PROCEDURE

[00408] 1. Aliquot 300 μL of each reaction reagent into 4 ml glass vials.

[00409] 2. Add 30 μL of the test sample to each glass bottle contains 4 ml.

[00410] 3. Incubate at room temperature for 4 hours.

[00411] 4. Aliquot of 3 ml of PNPP for each Petition 870260062914, dated 06 / 26 / 2026, page 111 / 157 107 / 147 4 ml glass vial, let stand for 1 hour.

[00412] a. Divide into 750 μL aliquots in the 4 ml glass vial to stop the reaction.

[00413] 5. Incubate for 1 hour and then read the results at 405 nm.

[00414] The results are shown in Figures 11A - 11E.

[00415] Interpretation of Figures 11A - 11E: Thirty chemicals were tested to evaluate the use of 4 ml glass vials with screw caps instead of 0.5 ml microcentrifuge tubes. Based on the results, as shown in Figures 11B and 11D, the dissolved dermal corrosives now showed high readings. For example, in Figure 11B, for the buffered saline alkaline phosphatase reaction reagent, No. 28 (octanoic acid; 1.427, 1.415, 1.416), No. 29 (dimethylisopropylamine; 3.354, 3.466, 3.502), No. 31 (5-isopropyl-2-methylphenol; 1.990, 1.990, 1.926) and No. 34 (N,N-dimethylbenzylamine; 3.360, 3.238, 3.273) were high readings. For example, in Figure 11D, for the ethanol alkaline phosphatase reaction reagent, on° 28 (octanoic acid; 3.283, 3.408, 3.220), on° 29 (dimethylisopropylamine; 2.926, 2.922, 2,852), no. 31 (5-isopropyl-2-methylphenol; 3,444, 3,465, 3.398) eon° 34 (N,N-dimethylbenzylamine; 3.429, 3.249, 3.164) were high readings. In previous studies, these dermal corrosives were corrected by the ethanol solvent condition. The experiment and which variables could have caused these inconsistent results were discussed, and it was concluded that the glass vial was a new variable. How the reagent inside the vial was mixed was also discussed. It is believed that by tilting the vial to mix by pipette and pipetting again, Petition 870260062914, dated 06 / 26 / 2026, page 112 / 157 108 / 147 the alkaline phosphatase enzyme may have become trapped on the side of the vial and therefore, upon adding 3 ml of p-nitrophenyl phosphate (PNPP) solution, the untouched alkaline phosphatase was interacting with the substrate solution. Therefore, for our next study, we compared the results between the 0.5 ml microcentrifuge tubes used previously with the 4 ml glass vials with screw caps.

[00416] February 2, 2023: In our next study, we wanted to compare the difference between 0.5 ml microcentrifuge tubes and 4 ml glass vials, since the results from the glass vials did not correspond to what was observed in previous studies. The assay chemicals included water, acetic acid, heptanal, dimethylisopropylamine, n-heptylamine, and N,N-dimethylbenzylamine. It is believed that when tilting the glass vial to mix by pipette / repipette, alkaline phosphatase is sticking to the sides and therefore, when the disodium salt of p-nitrophenyl phosphate (PNPP) is added, it immediately detects the presence of alkaline phosphatase and turns intensely yellow.Furthermore, by adhering to the side of the vial for injection, since the volume of the chemical under study added is low, the probability of the chemical under study interacting with alkaline phosphatase may be lower, thus resulting in an intensely yellow enzymatic reaction product. This study focused on two stocks (one from a previous experiment and one newly made) of ethanol alkaline phosphatase reaction reagent at a concentration of 1:100. For 0.5 ml microcentrifuge tubes, 20 μL of the chemical under study were added to 180 μL of the alkaline phosphatase reaction reagent and incubated for 4 hours. Petition 870260062914, dated 06 / 26 / 2026, pp. 113 / 157 109 / 147 room temperature. After incubation for 4 hours, remove 20 μL from the 0.5 ml microcentrifuge tube and add to 200 μL of p-nitrophenyl phosphate disodium salt (PNPP) in a 96-well plate, let stand for 1 hour and then read the results at 405 nm at 30 minutes and 1 hour. For 4 ml vials, add 30 μL of the test chemical to 300 μL of the alkaline phosphatase reaction reagent and incubate for 4 hours at room temperature. After a 4-hour incubation, add 3 ml of p-nitrophenyl phosphate (PNPP) disodium salt substrate solution and let stand for 30 minutes, then divide into aliquots onto a 96-well plate to read the results at 405 nm, and then repeat this step again with a 1-hour incubation.

[00417] Protocol Experiment with Alkaline Phosphatase, Ninth Week, List of Chemical Products, Table 14 Chemical No. Chemical Name CASRN GHS in vivo 1 NC1: Water 3 PC1: Acetic acid 20 Heptanal 111-71-7 Cat. 2 29 Dimethylisopropylamine 996-35-0 Cat. 1B / 1C 32 n-Heptylamine 111-68-2 Cat. 1B / 1C 34 N,N-Dimethylbenzylamine 103-83-3 Cat. 1C CONDITIONS

[00418] 1. Ethanol-AP (1:100) [used during the Petition 870260062914, dated 06 / 26 / 2026, pp. 114 / 157 110 / 147 experiment 2.1.23]

[00419] 2. AP-Ethanol (1:00) - Freshly manufactured before this experiment PROCEDURE (0.5 ml MICROCENTRIFUGE TUBE)

[00420] 1. Divide each reaction reagent into 180 μL aliquots in the corresponding tube.

[00421] 2. Add 20 μL of each sample of assay, mix with pipette / repipette.

[00422] 3. Incubate at RT for 4 hours.

[00423] 4. Prepare the 96-well plate by dividing 200 μL of PNPP into aliquots and then adding 20 μL of 0.5 ml microcentrifuge tube.

[00424] 5. Read OD405 in 30 min and 60 min. PROCEDURE (4 ml BOTTLES)

[00425] 1. Divide 300 μL of each reaction reagent

[00426] into aliquots in the corresponding vial. 2. Add 30 μL of each sample to test, mix with pipette / repipette.

[00427] 3. Incubate at room temperature for 4 hours.

[00428] 4. Add 3 ml of PNPP to each vial.

[00429] 5. Within 30 minutes, divide into 200 μL aliquots in a 96-well plate for reading on OD405

[00430] 6. At 60 minutes, read the sign again. PNPP.

[00431] The results of this study are shown in Figures 12A - 12D.

[00432] Interpretation of Figures 12A - 12D: Four chemicals and two controls were tested to evaluate the differences between using 0.5 ml microcentrifuge tubes and 4 ml glass vials with screw caps and whether Petition 870260062914, dated 06 / 26 / 2026, pp. 115 / 157 111 / 147 There was some problem with the stock of ethanol alkaline phosphatase reaction reagents. The three dermal corrosives that gave inconsistent results were tested (dimethylisopropylamine, n-heptylamine and n-dimethylbenzylamine). As shown in Figure 12B, for n-heptanal the results were different between microcentrifuge tubes (2.360, 2.539, 2.690) and glass vials (0.804, 0.852, 0.901), indicating that this dermal irritant was inhibiting alkaline phosphatase when previous studies had not shown this. Therefore, it was concluded that the glass vials would not be used and the 0.5 ml microcentrifuge tubes were used again for the next study.

[00433] For our next study, we proceeded with testing 30 chemicals using a 0.5 ml microcentrifuge tube, since the previous experiment determined that glass can confound the results. New stocks were made from 1:100 alkaline phosphatase reaction reagents. For exposure to the chemical under study, 20 μL were added to the 0.5 ml microcentrifuge tube divided into aliquots with 180 μL of the corresponding reaction reagent, incubated for 4 hours, and then 20 μL from the 0.5 ml microcentrifuge tube to a 96-well plate with 200 μL of p-nitrophenyl phosphate disodium salt (PNPP) substrate solution and incubated for 1 hour with the results read at 405 nm.

[00434] Protocol EXPERIMENT WITH ALKALINE PHOSPHATASE TENTH WEEK LIST OF CHEMICAL PRODUCTS TABLE 15 Petition 870260062914, dated 06 / 26 / 2026, pp. 116 / 157 112 / 147 Chemical No. Chemical Name CASRN GHS in vivo 1 NC1: Water 2 NC2: Water / 1% ink 3 PC1: Acetic acid 4 PC2: Acetic acid / 1% ink 5 1-Bromo-4-chlorobutane 6940-78-9 NC 6 1,5-hexadiene 592-42-7 NC 7 Isopropanol 67-63-0 NC 8 4-(Methylthio)benzaldehyde 3446-89-7 NC 9 Isopropyl myristate 110-27-0 NC 10 Benzyl salicylate 118-58-1 NC 11 Phenylethyl alcohol 60-12-8 NC 12 Hydroxycitronellal 107-75-5 NC 13 Benzyl acetate 140-11-4 NC 14 Dipropylene glycol 25265-71-8 NC 15 di-n-propyl disulfide 629-19-6 Cat. 2 16 1-Bromopentane 110-53-2 Cat. 2 17 Cyclamen aldehyde 103-95-7 Cat. 2 18 cis-3-methyl-2-(2-pentenyl)- 2-cyclopentene-1-one 488-10-8 Cat. 2 19 p-Metha-1,8-dien-7-ol 18457-55-1 Cat. 2 20 Heptanal 111-71-7 Cat. 2 21 trans-3,7-dimethyl-2,6octadiene-1-ol 106-24-1 Cat. 2 22 alpha-Terpineol 98-55-5 Cat. 2 23 1-Decanol 112-30-1 Cat. 2 24 Nonyl aldehyde 124-19-6 Cat. 2 25 Dimethyldipropyletriamine 10563-29-8 Cat. 1A 26 Potassium hydroxide (10%) 1310-58-3 Cat.1B 27 2-Methylbutyric acid 116-53-0 Cat. 1B / 1C. Petition 870260062914, dated 06 / 26 / 2026, pp. 117 / 157 113 / 147 28 Octanoic acid 124-07-02 Cat. 1B / 1C 29 Dimethylisopropylamine 996-35-0 Cat. 1B / 1C 30 2-tert-butylphenol 88-18-6 Cat. 1B / 1C 31 5-Isopropyl-2-methylphenol 499-75-2 Cat. 1B / 1C 32 n-heptylamine 111-68-2 Cat. 1B / 1C 33 Methoxy-3-propylamine 5332-73-0 Cat. 1B / 1C 34 N,N-Dimethylbenzylamine 103-83-3 Cat. 1C PREPARATION

[00435] 1. Reaction reagents

[00436] a. AP reaction reagent 1:100

[00437] i. PBS: Take 4 ml of stock solution AP (1:10) and add to 36 ml of PBS. Mix in a vortex and then invert to mix 10 times.

[00438] ii. Ethanol: Take 4 ml of AP stock solution (1:10), add to 36 ml of ethanol. Mix in a vortex and then invert to mix 10 times.

[00439] b. Blank PBS

[00440] c. Blank ethanol

[00441] 2. Prepare microcentrifuge tubes of 0.5 ml for dosage

[00442] a. Check if they can be securely closed.

[00443] b. Label according to each condition and chemical product

[00444] 3. Prepare 96-well plates for reading Petition 870260062914, dated 06 / 26 / 2026, pp. 118 / 157 114 / 147 of PNPP (3 wells for each chemical under study)

[00445] a. Divide 200 pL of PNPP into well aliquots PROCEDURE

[00446] 1. Divide each reaction reagent

[00447] into 180 pL aliquots in the corresponding tube. 2. Add 20 pL of each sample of test, mix with pipette / repipette.

[00448]

[00449] 3. 4. Incubate at RT for 4 hours. Prepare the 96-well plate by dividing into 200 pL aliquots of PNPP before the end of the 4-hour incubation.

[00450] microcentrifuge

[00451]

[00452]

[00453] 5. 0.5 ml. 6. 7. Add 20 pL of PNPP. Incubate in PNPP for 60 min. Read the results at 405 nm. tube Two protocols were developed: NO SKIN CORROSION TEST IN CHEMICAL HIGH-YIELD PROCEDURE

[00454] This protocol is for testing 6 materials plus controls per 96-well plate.

[00455] 1. Add 20 pl (or 20 mg if solid) of the material to be tested to each of the 4 types of 1 / 2 ml pre-filled tubes labeled A1, B1, A2, B2. Mix with a pipette / repipette 5 times. NOTE: Use a new pipette tip for each aliquot in A1, B1, A2, B2. Close the caps. Repeat for each additional test material. * It can also be supplied in bulk in 20 ml vials, and the user can divide it into 200 µl aliquots for 1 / 2 ml tubes. Petition 870260062914, dated 06 / 26 / 2026, pp. 119 / 157 115 / 147

[00456] 2. Add 20 μL of the negative control to the pre-filled 0.5 ml vials labeled NC-A1, NC-B1 and NC-A2, NC-A2. Mix with a pipette / repipette 5 times. Close the caps.

[00457] 3. Add 20 μL of the positive control to the pre-filled 0.5 ml vials labeled PC-A1, PC-B1, PC-B1, PC-B2. Mix with a pipette / new pipette 5 times. Close the caps.

[00458] 4. Incubate the capped tubes at room temperature for 4 hours + / - 10 min.

[00459] 5. Pre-fill a 96-well ELISA plate with 200 μL per well with the supplied Detection Reagent (DR) solution. Note: Triplicate detection reactions will be performed per 0.5 ml tube.

[00460] 6. After incubation for 4 hours, add μl of triplicate aliquots from each 1 / 2 ml reaction tube to the ELISA plate. Note: Mix with a pipette and re-pipette 5 times before the first aliquot.

[00461] 7. Incubate at room temperature for an hour + / - 10 min.

[00462] 8. Measure the OD at 405nm using a plate reader.

[00463] 9. Calculate the corrosion score IC:

[00464] a. Subtract the average Gross Block (of the tripled rates) (B) from the average of the active OD results (A) to obtain the NET OD value. The minimum value is 0.001.

[00465] (The license plate reader only reads thousandths places; additional fractions are insignificant) NET1 = A1 - B1 ; NET2 = A2 - B2

[00466] b. Take the result of NET and divide by Petition 870260062914, dated 06 / 26 / 2026, pp. 120 / 157 116 / 147 negative control OD value NET to obtain the NET ratio. Round the net ratio to the nearest thousandth, the minimum value is 0.001. Ratio NET1 = NET1 / NC1 ; Ratio NET2 = NET2 / NC2

[00467] c. Take the inverse of the NET Ratio (Ratio 1 / NET).

[00468] Ratio 1 / NET1; Ratio 1 / NET2. (Round to the nearest whole number)

[00469] d. The IC corrosion score is the larger value for the inverse of NET1 or the inverse of NET2.

[00470] 10. Apply the IC Corrosion score to the prediction model.

[00471] 11. Predict whether the material is corrosive by applying the IC corrosion score to the prediction model.

[00472] TABLE 16 Corrosion Score Prediction IC Non-corrosive < 3 Corrosive > 3

[00473] 12. Confirm that the A1 and A2 positive control scores are greater than 3; otherwise, the test has failed quality assurance. FIELD TEST PROCEDURE

[00474] This protocol is for testing 1 material plus controls in a non-laboratory environment.

[00475] 1. Using the calibrated spoons provided, apply 1 level spoonful (~25 mg) of the material to be tested to each of the small pre-filled tubes labeled A1, B1, A2, B2. Use a separate spoon for Petition 870260062914, dated 06 / 26 / 2026, pp. 121 / 157 117 / 147 per tube. Cap and mix by inverting.

[00476] 2. Add a level scoop of negative control to the small pre-filled tube labeled NC-A1 and NC-A2. Cap and mix by inversion.

[00477] 3. Add a level scoop of the positive control to the small pre-filled tube labeled NC-A1 and NC-A2. Cap and mix by inversion.

[00478] 4. Incubate the capped tubes at room temperature for 4 hours + / - 10 min.

[00479] 5. Retrieve the larger filled glass reaction flasks labeled A1, B1, A2, B2.

[00480] 6. Remove the cap from each small tube and place it upside down over the opening of the large tube, so that all the contents move from the small tube to the large tube.

[00481] 7. Cap the small tube and discard it safely. Replace the cap on the large bottle and mix by inverting it 3 times.

[00482] 8. Incubate at room temperature for 1 hour + / - 10 min.

[00483] 9. Using a spectrophotometric pipette, measure the OD at 405nm.

[00484] a. Open the pipette software application and tap Auto Zero to blind the pipette using the supplied obturation solution.

[00485] b. Tap on Measurement to read the material.

[00486] c. The measurements will be saved on the Datasets screen.

[00487] 10. Calculate the corrosion score of IC.

[00488] a. Subtract the Gross Block (B) from Petition 870260062914, dated 06 / 26 / 2026, pp. 122 / 157 118 / 147 results from Active OD (A) to obtain the NET OD value. The minimum value is 0.001.

[00489] (The spectrophotometer only reads thousandths of a millimeter; additional fractions are insignificant) NET1 = A1 - B1 ; NET2 = A2 - B2

[00490] b. Take the NET result and divide it by the negative control OD value NET to obtain the NET ratio. Round the net ratio to the nearest thousandth place, the minimum value is 0.001. Ratio NET1 = NET1 / NC1 ; Ratio NET2 = NET2 / NC2

[00491] c. Take the inverse of the NET Ratio (Ratio 1 / NET).

[00492] Ratio 1 / NET1; Ratio 1 / NET2. (Round to the nearest whole number)

[00493] d. The IC corrosion score is the larger value for the inverse of NET1 or NET2.

[00494] 11. Predict whether the material is corrosive by applying the IC corrosion score to the prediction model.

[00495] TABLE 17 Corrosion Score Prediction IC Non-corrosive < 3 Corrosive > 3

[00496] 12. Confirm that the A1 and A2 positive control scores are greater than 3; otherwise, the test has failed quality assurance.

[00497] The results of this study are shown in Figures 13A - 13E. TABLE 18. RESULTS FOR SALINE REACTION REAGENT Petition 870260062914, dated 06 / 26 / 2026, pp. 123 / 157 119 / 147 ACTIVE BUFFERED Chemical No. Chemical Name CASRN A1 - Results R1 R2 R3 AVG SE 1 NC1: Water 3.50 2 3.50 2 3.50 2 3.50 2 0.00 0 2 NC2: 1% water / ink 3.50 2 3.50 2 3.42 5 3.47 6 0.02 6 3 PC1: Acetic acid 0.09 6 0.09 6 0.09 6 0.09 6 0.00 0 4 PC2: 1% acetic acid / ink 0.13 0 0.12 5 0.12 5 0.12 7 0.00 2 5 1-Bromo-4-chlorobutane 694078-9 3.38 7 3.50 2 3.50 2 3.46 4 0.03 8 6 1.5-hexadiene 592-427 3.50 2 3.48 9 3.49 8 3.49 6 0.00 4 7 Isopropanol 67-63-0 3.48 4 3.48 3.30 5 3.42 3 0.05 9 8 4(Methylthio)benzal dehyde 344689-7 3.35 4 3.44 1 3.23 8 3.34 4 0.05 9 9 Isopropyl myristate 110-270 3.37 3 3.49 6 3.48 4 3.45 1 0.03 9 10 Benzyl salicylate 118-581 3.50 2 3.44 4 3.44 5 3.46 4 0.01 9 11 Phenylethyl alcohol 60-12-8 3.50 2 3.50 2 3.40 3 3.46 9 0.03 3 12 Hydroxycitronellal l 107-755 3.41 2 3.50 2 3.15 4 3.35 6 0.10 4 13 Benzyl acetate 140-114 3.40 1 3.48 5 3.50 2 3.46 3 0.03 1 14 Dipropylene glycol l 2526571-8 3.50 2 3.50 2 3.49 9 3.50 1 0.00 1 15 Di-n-propyl disulfide 629-196 4.46 7 3.47 4 3.39 5 3.44 5 0.02 5 16 1-Bromopentane 110-532 3.42 8 3.29 8 3.05 6 3.26 1 0.10 9, Petition 870260062914, dated 06 / 26 / 2026, pp. 124 / 157 120 / 147 17 Cyclamen aldehyde 103-95-7 3.40 5 3.50 2 3.50 2 3.47 0 0.03 2 18 cis-3-methyl-2(2-pentenyl)-2-cyclopenten-1-one 488-10-8 3.50 2 3.50 2 3.50 2 3.50 2 0.00 0 19 p-Metha-1,8-dien-7-ol 1845755-1 3.50 2 3.47 6 3.45 3 3.47 7 0.01 4 20 Heptanal 111-717 3.40 9 3.13 1 3.04 9 3.19 6 0.10 9 21 trans-3,7dimethyl-2,6octadiene-1-ol 106-24- 1 3.40 6 3.50 2 3.50 2 3.47 0 0.03 2 22 alpha-Terpineol 98-55-5 3.50 2 3.50 2 3.50 2 3.50 2 0.00 0 23 1-Decanol 112-30- 1 3.50 2 3.46 6 3.38 0 3.44 9 0.03 6 24 Nonyl aldehyde 124-196 3.39 3 3.44 5 3.05 0 3.29 6 0.12 4 25 Dimethyldipropyl triamine 1056329-8 0.11 8 0.12 1 0.12 4 0.12 1 0.00 2 26 Potassium hydroxide (10%) 131058-3 0.11 8 0.11 1 0.11 2 0.11 4 0.00 2 27 2-Methylbutyric acid 116-530 0.10 1 0.11 7 0.13 5 0.11 8 0.01 0 28 Octanoic acid 124-0702 0.74 1 0.79 1 0.86 0 0.79 7 0.03 4 29 Dimethylisopropylamine 996-350 3.42 3 3.46 5 3.48 5 3.45 8 0.01 8 30 2-tertiarybutylphenol 88-18-6 0.43 7 0.52 8 0.60 7 0.52 4 0.04 9 31 5-Isopropyl-2methylphenol 499-752 0.21 1 0.24 2 0.27 2 0.24 2 0.01 8 32 n-heptylamine 111-68- 2 2.14 1 2.66 8 2.86 2 2.55 7 0.21 5 33 Methoxy-3propylamine 533273-0 0.13 0 0.13 2 0.13 3 0.13 2 0.00 1, Petition 870260062914, dated 06 / 26 / 2026, pp. 125 / 157 121 / 147 34 N,N- Dimethylbenzylamine 103-83- 3 3.26 5 3.22 5 3.23 0 3.24 0 0.01 3 TABLE 19. RESULTS FOR SALINE REACTION REAGENT WHITE TAMPONADED Chemical No. Chemical Name CASRN B1 - Results R1 R2 R3 AVG SE 1 NC1: Water 0.13 0 0.12 9 0.13 1 0.13 0 0.00 1 2 NC2: 1% water / ink 0.14 4 0.14 4 0.14 8 0.14 5 0.00 1 3 PC1: Acetic acid 0.09 6 0.09 4 0.09 5 0.09 5 0.00 1 4 PC2: 1% acetic acid / ink 0.12 6 0.12 7 0.12 5 0.12 6 0.00 1 5 1-Bromo-4-chlorobutane 694078-9 0.16 6 0.18 4 0.19 1 0.18 0 0.00 7 6 1.5-hexadiene 592- 42-7 0.16 1 0.15 2 0.15 0 0.15 4 0.00 3 7 Isopropanol 67-630 0.12 3 0.12 2 0.12 0 0.12 2 0.00 1 8 4(Methylthio)benzaldehyde 344689-7 0.17 5 0.17 0 0.16 9 0.17 1 0.00 2 9 Isopropyl myristate 110- 27-0 0.13 1 0.13 4 0.12 7 0.13 1 0.00 2 10 Salicylate Benzyl 118-58-1 0.15 1 0.14 4 0.14 9 0.14 8 0.00 2 11 Phenylethyl alcohol 60-128 0.13 0 0.12 2 0.14 0 0.13 1 0.00 5 12 Hydroxycitronellal 107-75-5 0.12 3 0.12 1 0.12 6 0.12 3 0.00 1 13 Benzyl acetate 140-11-4 0.13 3 0.12 1 0.13 8 0.13 1 0.00 5 Petition 870260062914, dated 06 / 26 / 2026, pp. 126 / 157 122 / 147 14 Dipropylene glycol 2526571-8 0.13 0 0.13 3 0.13 3 0.13 2 0.00 1 15 Di-n-propyl disulfide 629-19-6 0.13 9 0.13 7 0.14 8 0.14 1 0.00 3 16 1-Bromopentane 110-53-2 0.12 3 0.12 4 0.12 5 0.12 4 0.00 1 17 Cyclamen aldehyde 103-95-7 0.18 0 0.12 2 0.12 1 0.14 1 0.02 0 18 cis-3-methyl-2- (2-pentenyl)-2cyclopentene-1-one 488- 10-8 0.13 1 0.13 1 0.14 0 0.13 4 0.00 3 19 p-Metha-1,8dien-7-ol 1845755-1 0.12 7 0.12 2 0.23 3 0.16 1 0.03 6 20 Heptanal 111- 71-7 0.15 3 0.16 1 0.13 6 0.15 0 0.00 7 21 trans-3,7dimethyl-2,6octadiene-1-ol 106- 24-1 0.13 2 0.11 9 0.12 0 0.12 4 0.00 4 22 alpha-Terpineol 98-55- 5 0.12 1 0.11 9 0.11 9 0.12 0 0.00 1 23 1-Decanol 112-30-1 0.13 1 0.12 6 0.11 8 0.12 5 0.00 4 24 Nonyl aldehyde 124-19-6 0.14 6 0.13 1 0.12 4 0.13 4 0.00 6 25 Dimethyldipropyl etriamine 1056329-8 0.12 4 0.11 6 0.12 0 0.12 0 0.00 2 26 Potassium hydroxide (10%) 131058-3 0.11 0 0.11 1 0.11 2 0.11 4 0.00 2 27 2Methylbutyric Acid 116- 53-0 0.10 3 0.11 7 0.13 5 0.11 8 0,01 0 28 Octanoic acid 124- 07-02 0.12 9 0.79 1 0.86 0 0.79 7 0.03 4 29 Dimethylisopropylamine 996- 35-0 0.11 5 3.46 5 3.48 5 3.45 8 0.01 8 30 2-tertiarybutylphenol 88-186 0.12 7 0.52 8 0.60 7 0.52 4 0.04 9, Petition 870260062914, dated 06 / 26 / 2026, pp. 127 / 157 123 / 147 31 5-Isopropyl-2methylphenol 499- 75-2 0.12 2 0.24 2 0.27 2 0.24 2 0.01 8 32 n-heptylamine 111- 68-2 0.12 4 2.66 8 2.86 2 2.55 7 0.21 5 33 Methoxy-3propylamine 533273-0 0.11 5 0.13 2 0.13 3 0.13 2 0.00 1 34 N,N- Dimethylbenzylamine 103- 83-3 0.12 3 3.22 5 3.23 0 3.24 0 0.01 3 TABLE 20. RESULTS FOR THE REACTION REAGENT OF ACTIVE ETHANOL Chemical No. Chemical Name CASRN A2 - Results R1 R2 R3 AVG SE 1 NC1: Water 3.45 1 3.50 2 2.95 4 3.30 2 0.17 5 2 NC2: 1% water / ink 3.50 2 3.44 4 3.32 6 3.42 4 0.05 2 3 PC1: Acetic acid 0.99 6 0.72 4 0.82 9 0.85 0 0.07 9 4 PC2: 1% acetic acid / ink 0.46 6 0.58 1 0.47 0 0.50 6 0.03 8 5 1-Bromo-4-chlorobutane 694078-9 2.94 8 2.73 1 2.86 9 2.84 9 0.06 3 6 1.5-hexadiene 592- 42-7 2.69 2 2.76 7 2.69 3 2.71 7 0.02 5 7 Isopropanol 67-630 2.29 4 2.90 2 2.38 9 2.52 8 0.18 9 8 4(Methylthio)benzaldehyde 344689-7 3.37 9 3.35 5 3.26 6 3.33 3 0.03 4 9 Isopropyl myristate 110- 27-0 3.41 3 3.24 4 3.36 0 3.33 9 0.05 0 10 Salicylate benzyl 118- 58-1 3.34 5 3.27 2 3.33 3 3.31 7 0.02 3 Petition 870260062914, dated 06 / 26 / 2026, pp. 128 / 157 124 / 147 11 Phenylethyl alcohol 60-12-8 3.34 2 3.28 0 3.28 7 3.30 3 0.02 0 12 Hydroxycitronellal 107-75-5 2.51 3 2.15 4 3.08 9 2.58 5 0.27 2 13 Benzyl acetate 140-11-4 3.42 3.25 2 3.48 4 3.38 5 0.06 9 14 Dipropylene glycol 2526571-8 2.34 3 2.46 7 2.68 5 2.49 8 0.10 0 15 Di-n-propyl disulfide 629-19-6 2.64 9 2.39 8 2.36 8 2.47 2 0.08 9 16 1-Bromopentane 110-53-2 2.59 0 2.87 0 2.59 8 2.68 6 0.09 2 17 Cyclamen aldehyde 103-95-7 2.73 6 2.75 2.84 5 2.77 7 0.03 4 18 cis-3-methyl-2(2-pentenyl)-2-cyclopenten-1-one 488-10-8 2.40 5 2.47 1 2.70 4 2.52 7 0.09 1 19 p-Metha-1,8-dien-7-ol 1845755-1 2.75 7 2.75 4 2.86 8 2.79 3 0.03 8 20 Heptanal 111- 71-7 2.73 0 2.39 5 2.09 8 2.40 8 0.18 3 21 trans-3,7dimethyl-2,6octadiene-1-ol 106- 24-1 1.87 6 1.85 1 1.95 6 1.89 4 0.03 2 22 alpha-Terpineol 98-55- 5 2.22 3 2.75 1 2.79 3 2.58 9 0.18 3 23 1-Decanol 112- 30-1 2.20 5 2.44 1 2.47 2 2.37 3 0.08 4 24 Nonyl aldehyde 124-19-6 2.49 6 2.06 7 2.11 7 2.22 7 0.13 5 25 Dimethyldipropyl etriamine 1056329-8 0.11 9 0.11 8 0.11 4 0.11 7 0.00 2 26 Potassium hydroxide (10%) 131058-3 0.10 7 0.10 9 0.10 8 0.10 8 0.00 1 27 2-Methylbutyric acid 116- 53-0 2.30 5 2.03 5 3.33 9 2.56 0 0.39 7, Petition 870260062914, dated 06 / 26 / 2026, pp. 129 / 157 125 / 147 28 Octanoic acid 124-07-02 2.12 2.77 6 2.12 9 2.34 2 0.21 7 29 Dimethylisopropylamine 996-35-0 0.22 6 0.21 2 0.29 5 0.24 4 0.02 6 30 2-tertiarybutylphenol 88-18-6 2.82 9 2.56 7 2.44 8 2.61 5 0.11 3 31 5-Isopropyl-2-methylphenol 499-75-2 2.46 2 2.55 8 2.77 2 2.59 7 0.09 2 32 n-heptylamine 111-68-2 0.17 0 0.16 1 0.19 3 0.17 5 0.01 0 33 Methoxy-3propylamine 533273-0 0.12 1 0.11 8 0.12 7 0.12 2 0.00 3 34 N,N- Dimethylbenzylamine 103- 83-3 0.58 8 0.58 3 0.53 9 0.57 0 0.01 6 TABLE 21. RESULTS FOR REACTION REAGENT OF BLANK ETHANOL Chemical No. Chemical Name CASRN B2 - Results R1 R2 R3 AVG SE 1 NC1: Water 0.10 3 0.10 0 0.09 8 0.10 0 0.00 1 2 NC2: 1% water / ink 0.11 9 0.11 9 0.12 0 0.11 9 0.00 0 3 PC1: Acetic acid 0.09 7 0.09 7 0.10 0 0.09 8 0.00 1 4 PC2: 1% acetic acid / ink 0.11 2 0.12 0 0.11 9 0.11 7 0.00 3 5 1-Bromo-4-chlorobutane 694078-9 0.35 0 0.31 0 0.32 5 0.32 8 0.01 2 6 1.5-hexadiene 592- 42-7 0.10 8 0.11 0 0.10 8 0.10 9 0.00 1 7 Isopropanol 67-630 0.10 2 0.10 5 0.10 7 0.10 5 0.00 1 8 4- 3446- 0.20 0.29 0.24 0.24 0.02 Petition 870260062914, dated 06 / 26 / 2026, pp. 130 / 157 126 / 147 (Methylthio)benzaldehyde 89-7 6 1 0 6 5 9 Isopropyl myristate 110-27-0 0.13 1 0.12 6 0.11 1 0.12 3 0.00 6 10 Benzyl salicylate 118-58-1 0.39 7 0.48 8 0.50 9 0.46 5 0.03 4 11 Phenylethyl alcohol 60-12-8 0.11 6 0.12 0 0.12 0 0.11 9 0.00 1 12 Hydroxycitronellal 107-75-5 0.12 4 0.12 3 0.11 9 0.12 2 0.00 2 13 Benzyl acetate 140-11-4 0.19 3 0.15 3 0.13 7 0.16 1 0.01 7 14 Dipropylene glycol 2526571-8 0.11 4 0.11 1 0.11 0 0.11 2 0.00 1 15 Di-n-propyl disulfide 629-19-6 0.19 4 0.29 6 0.17 4 0.22 1 0.03 8 16 1-Bromopentane 110-53-2 0.23 2 0.23 9 0.25 4 0.24 2 0.00 6 17 Cyclamen aldehyde 103-95-7 0.13 8 0.15 3 0.14 4 0.14 5 0.00 4 18 cis-3-methyl-2-(2-pentenyl)-2cyclopentene-1-one 488- 10-8 0.14 3 0.14 4 0.14 3 0.14 3 0.00 0 19 p-Metha-1,8dien-7-ol 1845755-1 0.20 4 0.19 8 0.11 7 0.17 3 0.02 8 20 Heptanal 111- 71-7 0.14 6 0.14 9 0.15 4 0.15 0 0.00 2 21 trans-3,7dimethyl-2,6octadiene-1-ol 106- 24-1 0.10 4 0.10 5 0.10 6 0.10 5 0.00 1 22 alpha-Terpineol 98-55- 5 0.10 6 0,10 6 0.10 8 0.10 7 0.00 1 23 1-Decanol 112-30-1 0.10 7 0.10 6 0.10 9 0.10 7 0.00 1 24 Nonyl aldehyde 124-19-6 0.15 3 0.17 6 0.16 0 0.16 3 0.00 7, Petition 870260062914, dated 06 / 26 / 2026, pp. 131 / 157 127 / 147 25 Dimethyldipropyl etriamine 1056329-8 0.10 9 0.10 8 0.10 9 0.10 9 0.00 0 26 Potassium hydroxide (10%) 131058-3 0.10 8 0.10 7 0.10 8 0.10 8 0.00 0 27 2-Methylbutyric acid 116-53-0 0.11 1 0.11 1 0.11 1 0.11 1 0.00 0 28 Octanoic acid 124-07-02 0.15 4 0.13 5 0.13 7 0.14 2 0.00 6 29 Dimethylisopropylamine 996-35-0 0.10 8 0.10 7 0.10 5 0.10 7 0.00 1 30 2-tertiarybutylphenol 88-18- 6 0.11 1 0.10 9 0.11 5 0.11 2 0.00 2 31 5-Isopropyl-2methylphenol 499- 75-2 0.11 3 0.10 8 0.12 1 0.11 4 0.00 4 32 n-heptylamine 111- 68-2 0.13 1 0.12 4 0.16 0 0.13 8 0.01 1 33 Methoxy-3propylamine 533273-0 0.10 8 0.10 7 0.10 6 0.10 7 0.00 1 34 N,N-Dimethylbenzylamine 103-83-3 0.11 5 0.11 0 0.11 3 0.11 3 0.00 1 CASRN = Abstract Service Registration Number Chemicals; R = Reading; AVG = Average; EP = Standard Error; NC = Negative Control; PC = Positive Control; A1 = Active Buffered Saline Reaction Reagent; B1 = Blank Buffered Saline Reaction Reagent; A2 = Active Ethanol Reaction Reagent; B2 = Blank Ethanol Reaction Reagent. TABLE 22. PREDICTABILITY STATISTICS Accuracy 100.0% (30 / 30) Balance Accuracy 100.0% Responsiveness 100.0% (10 / 10) Specificity 100.0% (20 / 20) False Rate 0.0% (0 / 20) Petition 870260062914, dated 06 / 26 / 2026, pp. 132 / 157 128 / 147 Positives False negative rate 0.0% (0 / 10) Bal Acc = Balanced accuracy Corrosion Score Prediction IC Non-corrosive < 3 Corrosive > 3 TABLE 23. IC = In-chemico PREDICTION MODEL

[00498] Interpretation of Figures 13A -13E, Tables 18-21, Figure 14, Table 22, and Table 23: After reviewing all previous studies, thirty chemicals were tested. The conditions included: 1:100 dilution of the alkaline phosphatase reaction reagent with two solvents (buffered saline and ethanol), dosing in 0.5 ml microcentrifuge tubes with 20 μL of chemical added to 180 μL of reaction reagent, and incubation with p-nitrophenyl phosphate (PNPP) disodium salt substrate for 1 hour. Figures 13A-13E show the raw data from the study, while Tables 18-21 calculate the mean and standard error. Figure 14 shows the blank-corrected data and calculates the readings as a score that can be interpreted based on the prediction model (Table 23). Table 22 shows the predictability statistics. This study was highly successful with 100% sensitivity, 100% specificity, and 100% accuracy.Compared to other available non-animal tests, this is a performance level. Petition 870260062914, dated 06 / 26 / 2026, pp. 133 / 157 129 / 147 very high (unprecedented level of accuracy, other tests have accuracies around 80%); and this test is much faster and easier to perform than other animal-free tests. It was concluded that a novel testing method has been found that is a quick and simple way to test the potential toxicity of chemicals. Despite extensive database searches, including patent databases, peer-reviewed publications, and the internet, no examples of other individuals or groups attempting this approach could be found. It is believed that the inventors are the first to discover this approach to toxicity testing, which is unexpected, and given its ease of use, shelf stability, and applications in on-site field testing, this is a novel and potentially useful invention.Once again, it is emphasized that no other test adopts the approach of using lower activity of purified or semi-purified enzymes as a method to identify toxins, and no other animal-free test has such high predictability.

[00499] As shown in Figure 15, test kits were created and the test kits were used to study in order to better evaluate the invention and confirm the accuracy and repeatability of the test method. The ECETOC skin toxin database (ECETOC, 1995) lists Draize data from rabbits in vivo, including clinical observations after exposure to a large number of readily available chemicals. In addition, there are OECD and ICCVAM publications that provide guidance on assay method validation and include requirements on chemical selection (ICCVAM & NICEATM, 2003; OECD GD 34, 2005, ICCVAM, 1997, ICCVAM, 2023). For preliminary studies, 60 chemicals from the database were used. Petition 870260062914, dated 06 / 26 / 2026, pp. 134 / 157 Data from ECETOC (ECETOC, 1995), published ICCVAM & NICEATM reference databases (ICCVAM & NICEATM, 2004), OECD Testing Guidelines (OECD TG 431, 2019; OECD TG 439, 2021), and published validation studies (Alépée et al., 2019; Han et al., 2021) were selected. Chemicals were selected based on the diversity of chemical groups and physicochemical properties that fit the OECD chemical selection guidelines (OECD GD 34, 2005). Selection of chemicals with confirmatory in vivo secondary GHS classifications accepted by the OECD was ensured because they were used for OECD validation studies for OECD-accepted skin corrosion tests (OECD TG 431, 2021). (2019; OECD TG 439, 2021, Alépée et al., 2019; Han et al., 2021). The classifications are listed in Figure 16 (colored bars). Each chemical name is followed by its CAS RN (in parentheses) and has a number that is used to identify the chemical and classification in Figure 16. The instruction sheet for the test kit is shown in Figure 17. In summary, the test kit procedure involves adding 20 μL of the test substance to 180 μL of each test matrix reagent with the kit (Figure 16, A1, A2, B1, B2) and incubating for 4 h ± 10 min. After a 4 h incubation, 20 μL of the reaction tube are added to a pre-prepared 96-well plate containing 200 μL of the detection reagent (PNPP) and incubated for 1 h ± 10 min, then read at 405 nm using a microplate reader.

[00500] The 60 chemicals tested with the kit included different levels of in vivo skin damage, including NC, Category 2, Category 1BC, and Category 1A. The results are shown in Figure 18. Average results and error Petition 870260062914, dated 06 / 26 / 2026, pp. 135 / 157 131 / 147 Standard results from three independent runs on different days are shown as the average percentage with the background subtracted and normalized to the control. Figure 18 has a cutoff line showing where, if the normalized result (negative control percentage) is less than 40%, the chemical is predicted to be corrosive. For the chemicals tested that are: Non-Irritant or Corrosive (NC) (blue), the normalized results (negative control percentage) ranged from 79.6% to 99.0% with a standard error range of 0.1 to 6.9; For Irritant (Non-Corrosive / Category 2) (green), the normalized results (negative control percentage) ranged from 69.9% to 95.2% (except for 18.0% for Chemical No. 30, which is a false positive). For Corrosive (Category 1BC; shown in yellow), the normalized result ranged from 0.1% to 30.7% with a standard error range of 0.0 to 6.6. For Extreme Corrosive (Category 1A; shown in red), the normalized results ranged from 0.1% to 28.1% with a standard error range of 0.0 to 1.8. As shown in Figure 19, the consensus of independent triplicate results (each repetition performed on a different day, with different reagents) indicates that the method has excellent predictability. It is highly sensitive (100%; all corrosives were identified without NFs), specific (96.7%; (only one false positive); and accurate (98.3%) with high repeatability within the laboratory (100% repeatability) based on corrosive versus non-corrosive predictions compared to in vivo. The procedure and these results were presented in a webinar to a large audience of skin corrosion specialists, and they agreed on the shelf stability, ease of use, and accuracy. Petition 870260062914, dated 06 / 26 / 2026, pp. 136 / 157 The 132 / 147 accuracy of 98% and the 100% repeatability of the test make it something that has been seen as a highly innovative finding and can be used to test products and chemicals for labeling purposes. In fact, commercial opportunities for the invention have already emerged.

[00501] Novelty and Intellectual Property: To date, results suggest that chemical toxicity can be modeled using this simple and stable laboratory test. This hypothesis is based on the consideration that the epidermis is a complex protective matrix composed primarily of a highly cross-linked protein (keratin), but also includes other proteins and protective factors, one of which is PE. Therefore, PE possesses structural and distributional properties in the skin that make it representative (a biomarker) of skin vulnerability to dermal corrosives. Specifically, strong corrosives degrade the epidermis into the dermis, and since the PE enzyme is located in this tissue, it is also degraded by dermal corrosives. Therefore, PE activity decreases as a function of exposure to dermal corrosives.As shown in Figure 18, a reduction in EP activity identified all tested dermal corrosives with 100% sensitivity, indicating that the corrosion mechanisms of these chemicals are accounted for. Since chemicals were selected based on guidelines to be as diverse as possible, the test will be widely applicable to the identification of dermal corrosives and may be generalizable to all corrosive chemicals. The very high accuracy of the preliminary studies means that even if further studies identify some erroneous predictions, the current accuracy will remain. Petition 870260062914, dated 06 / 26 / 2026, pp. 137 / 157 133 / 147 will likely still be higher than that of the alternatives. Furthermore, our test has an additional competitive advantage because it is very easy to use and can remain on the shelf (possibly for years) and be ready to identify toxic materials quickly and accurately. Based on the ease of use and predictability demonstrated so far, this test has the potential to introduce a new and unexpected paradigm in toxicity testing that could expand test development.

[00502] A database search was conducted using Google Scholar, PubMed, USPTO Patent Search Database, Google Patents, and NIH Report. Despite extensive research, no cell-free test using enzymatic activity to predict dermal corrosion could be identified. To our knowledge, the data presented in this proposal are novel, and the inventors are the first to discover and apply this approach to identify dermal corrosives.

[00503] Hypothesis Although the most notable proteins of the epidermis are keratins (intermediate filaments that protect epithelial cells from stressors that can cause rupture and cell death) and of the dermis, collagen (a protein that strengthens the skin), there is a wide range of other macromolecules, such as elastin and amino acids (Gu and Coulombe, 2007; Brown and Krishnamurthy, 2022), enzymes, etc. The hypothesis that explains our discovery is that the ability of a chemical to destroy molecules and disintegrate skin or other tissues is not very specific to the target when the chemical is at high concentrations compared to the enzyme to be inactivated during the toxin identification test procedure. Based on preliminary findings, it is proposed that the Petition 870260062914, dated 06 / 26 / 2026, pp. 138 / 157 134 / 147 Enzymatic destruction by a chemical can be used to model (or as a marker) the potential of a toxin to destroy molecules and thus disintegrate or rupture tissues. As noted above, it was assumed that an accurate test of toxicity, irritation, or corrosion would require evaluation of a full-thickness tissue model, and it has been found that those skilled in the art assume that full-thickness tissues or cells are necessary to determine toxicity, and the use of a single enzyme to model toxicity for a larger, complex tissue or organ may be counterintuitive to some or even seemingly lacking in rigor to others.However, it was also noted that a true invention is, by definition, counter-intuitive and not expected by those skilled in the art; and that the aim of experimentation is to demonstrate results with a level of rigor that, if consistent with the hypothesis being tested, can overcome intuition and thus advance the field of study; once it has been demonstrated that an enzyme can be used to accurately model the toxicity of tissues and organs, it is relatively simple to apply the same discovery to different enzymes and target systems, this has been accomplished in the following prophetic examples:. PROPHETIC EXAMPLE: LUNG

[00504] In a prophetic example, an in chemico test for pulmonary toxins was developed by diluting 25 units of horseradish peroxidase enzyme in a reaction solution 1 (10 ml of 100mM potassium chloride solution) and also a reaction solution 2 (50 units of horhardal peroxidase enzyme diluted in a second 10 ml vial of dimethyl sulfoxide). After a mixing step, 1 / 2 ml of Petition 870260062914, dated 06 / 26 / 2026, pp. 139 / 157 135 / 147 each (reaction solution 1 and reaction solution 2) were divided into aliquots in separate tubes and 10 µl of negative control (water) or pulmonary toxins (lewisite, nitrogen mustard, phosgene oxime, sulfur mustard, ammonia, bromine, chlorine, hydrogen chloride, methyl bromide, methyl isocyanate, osmium tetroxide, phosgene, phosphine, phosphorus (elemental, white, yellow), sulfur fluoride, hydrogen sulfide, cyanide, carbon monoxide, nitrogen, sulfur oxide) and non-toxic chemicals to the lungs were added to form complete reaction 1 and complete reaction 2. In another proactive step, unknown chemicals were tested. After mixing, the tubes were incubated for 1 hour at 37°C. 200 µl of 3,3',5,5'-tetramethylbenzidine (TMB) substrate were added to each well of a 96-well plate, and 10 µl aliquots of complete reaction 1 were added to 3 wells, and 10 µl aliquots of complete reaction 2 were added to another 3 wells.The reaction was incubated for 20 minutes, and then 50 µl of 0.16 M sulfuric acid was added to each well to stop the reaction, and the optical density (OD 450) was measured. The measured optical density of the pulmonary irritants and the non-irritant was compared to the control (water) by dividing the measured value by the control value. Materials, including known and unknown ones, were identified as pulmonary irritants if the resulting value was less than 0.6 (60% of control or less). Prophetic example: Nervous system

[00505] In a prophetic example, an in chemico test for neurotoxins was developed by diluting semi-purified nonspecific peroxidase in a solution of Petition 870260062914, dated 06 / 26 / 2026, pp. 140 / 157 136 / 147 diethylene glycol.

[00506] After a mixing step, 100 µl were aliquoted into 1 / 2 ml tubes and 10 µl of negative control (water) or neurotoxins (sarin, soman, tabun, methylphosphonothioic acid, cyclosarin, S-(diethylamino)ethyl O-ethyl ethylphosphonothioate, O,O-diethyl-S-[2-(diethylamino)ethyl]phosphorothioate, phosphonothioic acid, methyl-, S-(2(diethylamino)ethyl)O-ethyl ester, O-ethyl-S[2(diisopropylamino)ethyl]methylphosphonothioate, lead, ethanol, glutamate, nitric oxide, botulinum toxin, tetanus toxin, gamma-butyrolactone, 2-chloropropanoic acid, amphetamine, nicotine, 1,2,3-trimethylbenzene, 1,2,4-trimethylbenzene, 1,2,5,6,9,10-hexabromocyclodedecane, 1,3-dinitrobenzene, 1-bromopropane, l-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, 2,5-hexanedione, 3,4-dihydroxymandelaldehyde, 3,4-methylenedioxymethamphetamine, 3-nitropropanoic acid, 4-hydroxybutyric acid, N-butylbenzenesulfonamide, N-hydroxy-PhIP, N-methyl-4-phenylpyridinium, N-methylnorsalsolinol, racgabaculin, acromelic acid A, acrylamide, aetoktonotoxin,allyl cyanide, bicuculline, butane-1,4-diol, carbon monoxide, ammonia, decabromodiphenyl ether, descarbamoyl saxitoxin, dexormaplatin, diethyl phthalate, domoic acid, edifenfos, endrin, hexane, hydrogen peroxide, ibotenic acid, ketamine, lead chromate, leptofos, mancozeb, maneb, mercury, methamphetamine, neosatitoxin, oltipraz, ormaplatin, oxidopamine, paralytic shellfish poisoning, phencyclidine, potassium cyanide, resiniferatoxin, salsolinol, saxitoxin, tetrodotoxin, thallium(I) acetate, tinyatoxin, toluene, trimethyltin) and chemicals that are not toxic to the nervous system for, Petition 870260062914, dated 06 / 26 / 2026, pp. 141 / 157 137 / 147 to form a complete reaction. In further steps, materials with no known neurotoxicity were tested for neurotoxicity by adding them to additional complete reactions. After mixing, the tubes were incubated at 30°C for 3 hours. Then, 100 µl of 2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt were added to each well of a 96-well plate, and 100 µl aliquots of complete reaction 1 were added to 3 wells. The reaction was incubated for 60 minutes, and then the optical density at 405 nm was measured. The measured optical density of the neurotoxins and non-neurotoxins and unknowns was compared to the control (water) by dividing the measured value by the control value. The material was identified as a toxin if the resulting value was less than 0.6 (60% of control or less). PROPHETIC EXAMPLE: HEART

[00507] In a prophetic example, a preparation of dried bovine hearts, supplied as a light, fine powder, was used as a source of enzymes. The dried powder was added at 1% w / v to normal saline solution and also to an organic solvent (e.g., DMSO or ethanol). After the solutions were mixed, in one condition an unknown material or in another condition a known cardiotoxin, for example, anthracyclines (daunorubicin, doxorubicin, epirubicin, idarubicin), organophosphates, carbamates, pyrethroids, organochlorines, phosphides, triazoles, triazines, dipyridyls, platinum, cobalt, mercury, nanoparticles (titanium, zinc, silver, carbon, silica, iron oxide), ethanol, or in a third condition a non-cardiotoxic chemical was added. Furthermore, each material to be tested was added to the saline or organic solvent that did not contain the Petition 870260062914, dated 06 / 26 / 2026, pp. 142 / 157 138 / 147 bovine heart powder. After incubation at a defined temperature for a defined period of time, aliquots of each were added to substrates for phosphatase (disodium salt of 4-nitrophenyl phosphate [PNPP]), peroxidase (10-Acetyl-3,7-dihydroxyphenoxazine [ADHP]), protease (casein), or esterase (alpha-naphthyl acetate). The amount of each product for phosphatase, peroxidase, protease, and esterase was quantified at (405 nm, 590 nm, 660 nm, 390-440 nm, respectively), and the toxicity of the test material was identified based on the reduction of one or more of the enzymatic product levels compared to a standard non-cardiotoxic chemical, such as water. PROPHETIC EXAMPLE: KIDNEY

[00508] In a prophetic example, the human kidney cell line HEK 293 was cultured to 200 mg or more of cells. Cells were collected by scraping plates with a rubber policeman and transferred to a conical tube in phosphate-buffered saline solution. Centrifuged, resuspended in saline and centrifuged again to wash. The cells were then suspended in 10 ml of high-salt buffered solution (for 100 ml of deionized water and 0.14 g of Na2HPO4, 0.24 g of KH2PO4, 8 g of sodium chloride to the solution, 1 g of potassium chloride, 0.14 g of calcium chloride, pH to 7.4 + / - 0.005). After incubation for 30 minutes on ice (to allow the cells to absorb salt), the cells are centrifuged and resuspended in 10 L of low-salt solution to lyse the cells (for 100 mL of deionized water and 0.14 g of Na2HPO4, 0.24 g of KH2PO4, no sodium chloride in the solution, 0.1 g of potassium chloride, no calcium chloride, pH to 7.4 + / - 0.005).After 10 minutes,. Petition 870260062914, dated 06 / 26 / 2026, pp. 143 / 157 139 / 147 Collect the proteins by adding 1 gram of ammonium sulfate and spinning the capped tube for 30 minutes at 4-8°C. Collect pellets by centrifugation. Resuspend the pellet in 2 ml of phosphate-buffered saline (PBS) and transfer to a dialysis membrane with a pore size of 10 kD. Dialyze with refrigeration 3 times against 1 liter of PBS. The resulting solution is a crude cell-free enzyme preparation.

[00509] Add the unknown material to be tested, known renal toxins (benzene, organic solvents, degreasing agents, glyphosate, paraquat, 1,2-Dibromo-3-chloropropane, cadmium, uranium, lead, mercury, ethylene glycol, methanol, isopropyl alcohol, cannabinoids, amphetamines, organophosphates), materials known to be non-toxic to the kidneys to separate the tubes from the cell-free enzymatic material. Include a water control. Incubate for 18 hours at 37°C. Measure esterase activity, transfer an aliquot of each to a 96-well plate containing 200 µl of a naphthol chloroacetate AS-D solution. Allow the mixture to incubate for 2 hours. Measure the optical density (OD). Compare the resulting OD of the toxins and unknown material with the OD of the negative control and non-toxins. Identify materials as toxic if the dissolved oxygen (DO) value is significantly lower than the negative control. Prophetic example: Liver

[00510] Obtain a section of bovine liver. Cut into small pieces, add to a small volume of PBS and homogenize using a Dounce homogenizer. Transfer to the centrifuge tube and conduct a low-speed centrifuge (200 rpm). Remove the top half, leaving any solid material in the bottom half. Separate into different Petition 870260062914, dated 06 / 26 / 2026, pp. 144 / 157 140 / 147 aliquots, add control, unknowns to be tested and known hepatotoxins (amoxicillin-clavulanate, flucloxacillin, erythromycin, diclofenac, sulfamethoxazole / trimethoprim, isoniazid, disulfram, ibuprofen, flutamide, vinyl chloride, carbon tetrachloride, paraquat, polychlorinated biphenyls, 2-nitropropane, 4,4'-methylenedianiline, 2,4,6-trinitrotoluene, ethylene dichloride, propylene dichloride, carbon tetrabromide, acetylene tetrabromide, ethylene dibromide, hexachloronaphthalene, octachloronaphthalene, trichloronaphthalene, Ntitrosodimethylamine, methylformamide, tetrahydrofuran, dimethylacetamide, diphenyl) and unknown hepatotoxins. Incubate for 4 hours.Transfer an aliquot to an alkaline phosphatase substrate (e.g., disodium salt of 4-nitrophenyl phosphate hexahydrate), incubate for 1 hour, measure the OD (405 nm), predict that it is a liver toxin if unknown or known liver toxins reduce the OD by 50% compared to the negative control. PROPHETIC EXAMPLE: EYE

[00511] In a prophetic example, corneas from food-source cattle are collected; the cells are killed by freezing and thawing 3 times. 5 mm corneal buttons are collected using a biopsy puncture. The corneal buttons are placed in a 96-well plate, and 150 µl of a negative control, known ocular irritants and known non-ocular irritants are added to different wells with buttons. After a 10-minute incubation, the buttons are removed, washed 3 times in PBS, and transferred to the AS-D naphthol chloroacetate esterase substrate solution. After 1 hour of incubation, the buttons are removed and the oD is Petition 870260062914, dated 06 / 26 / 2026, pp. 145 / 157 141 / 147 measurement. Materials are identified as eye irritants or corrosives based on the fact that the OD is significantly lower than the value for the control. Prophetic example: acute toxicity

[00512] In a prophetic example, the bacterium E. coli was used as a peroxidase source. An E. coli (Escherichia coli) culture was grown in Luria-Bertani (LB) broth to the late logarithmic phase in a 2-liter flask. Cells were collected by centrifugation, washed with PBS, and killed and lysed by grinding in a Dounce homogenizer and 3 rounds of freezing at -80 °C followed by thawing at 37 °C. The resulting lysate was used as is to identify acute toxins. 100 µl of lysate were divided into aliquots in each well of a 96-well plate. The test materials were diluted as follows: A 10-fold serial dilution (from 1:10 to 1:10 million) of a negative control (water) and a positive control (5% sodium dodecyl sulfate, CASRN). 151-21-3), 1 very toxic chemical, 1 moderately toxic chemical, and 1 low toxic chemical, and 1 essentially non-toxic material and 1 unknown. 10 µl of each concentration of each test material were added to a well of the 96-well plate containing the lysate and mixed by pipette repipette. The 96-well plate was covered and incubated at 37 °C for 1 hour. After incubation, 200 µl of a solution with the peroxidase substrate 2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) was added to 396-well plates. Then, triplicate aliquots of 20 µl of the lysate with serial dilutions of test materials were added to each well and mixed by pipette repipette. The plate was transferred to 37 °C from an incubation of Petition 870260062914, dated 06 / 26 / 2026, pp. 146 / 157 142 / 147 hour. After incubation, the OD at 405 nm was measured using a plate reader or spectrophotometry. The unknown materials were judged as having a toxicity similar to the best OD match for the controls: best OD fit within the range of very toxic, moderately toxic, slightly toxic, essentially non-toxic based on which An unknown OD was set for each of them. In a further step, the mg / kg of each of them needed to result in an effective dose (ED) of 20, 50 and 80 or a lethal dose (LD) of 20, 50 and 80 were calculated by comparing an extrapolated curve using Graphpad's Prism software. Prophetic example: Skin irritation

[00513] 5-Bromo-4-chloro-3-indolyl β-D-galactopyranoside was diluted in salt water and 200 µl added to each well of a 96-well plate. Serial dilutions of known dermal irritants, known non-irritants, and unknown water and chemical controls were performed. A 20 µl aliquot of galactosidase substrate was added to each well, and unknowns were considered irritants if the OD was less than the known irritant positive controls. PROPHETIC EXAMPLE: SKIN CORROSION

[00514] Prepare reaction reagents (enzyme in buffered saline solution and ethanol) using the chosen enzymatic substrate system, such as disodium salt of 4-nitrophenyl phosphate hexahydrate (phosphatase), 3,3'-diaminobenzidine tetrachloride hydrochloride (peroxidase), phenylenediamine dihydrochloride (peroxidase), o-phenylenediamine (peroxidase), o-dianisidine (peroxidase), 3,3',5,5'-tetramethylbenzidine dihydrochloride (peroxidase), 4-chloro-1-naphthol Petition 870260062914, dated 06 / 26 / 2026, pages 147 / 157 143 / 147 (peroxidase), 3-amino-9-ethylcarbazole; (peroxidase). For 0.5 ml microcentrifuge tubes, add 180 μL of each corresponding reaction reagent. Then, add 20 μL of the test material and mix with a pipette and pipette again. Incubate at room temperature for 1 minute, 1 hour, and 4 hours. After each time point, add 20 μL to 200 μL of the chosen substrate solution and read the results at the corresponding wavelength. The results from the different time points will be indicative of the different subcategories of Category 1 (Category 1A, Category 1B, and Category 1C).

[00515] The results of enzyme activity after exposure to the toxin will be calculated to form a toxic activity class, and the results of enzyme activity after exposure to the non-toxic exposure will be calculated to form a non-toxic activity class. The results are then used to define an activity cutoff value that separates the toxic activity class from the non-toxic activity class, and this value is used to define a predictive model. Substances with reference or reference value data are then tested and the results are applied to the predictive model. This data is analyzed to determine the predictability of the test, including accuracy. It is generally accepted that tests with an accuracy of 80% or more are useful.

[00516] Abstract It has been found that tissue toxicity for a wide range of diverse and widespread toxic chemicals can be identified by measuring the inactivation of a representative enzyme(s). Although the proposed widespread toxins are highly reactive, this reactivity is not specific to a single molecule or enzyme or active site or enzyme regulator, and is not specific to Petition 870260062914, dated 06 / 26 / 2026, pp. 148 / 157 144 / 147 the enzyme used to assess generalized loss of function and subsequent toxicity.

[00517] The invention readily identifies widespread loss-of-function toxins by measuring a decrease in enzymatic activity resulting from exposure to the test material or substance, and the change in enzymatic activity serves only as a functional marker or representative of the widespread toxicity of the test material or substance.

[00518] Purified or semi-purified enzymes can be stored as lyophilized powders or stabilized (e.g., frozen). As such, a product based on this technology will have a shelf life of months to years, which is a clear advantage compared to many toxicological tests that require cells or living organisms.

[00519] Identifying relevant generalized chemical toxic reactivity would be complex without an enzymatic marker to assess functional status. Simply measuring generalized denaturation without a defined enzymatic marker or measuring toxin binding to specific protein sequences requires complex assay design and analytical methods and is not as simple, fast, and useful as measuring a reduction in enzymatic activity. Furthermore, while measuring toxin binding to specific peptides may indicate specific endpoints, such as allergic response or sensitization potential, this will not assess a generalized loss of functionality and resulting toxicity for a wide range of toxic chemicals, as described for this invention. Similarly, measuring turbidity related to protein denaturation may be relevant to a specific endpoint in ocular toxicity: Petition 870260062914, dated 06 / 26 / 2026, pp. 149 / 157 145 / 147 Corneal opacity. However, corneal opacity can be transient or permanent and is distinct from other ocular toxicity outcomes, including redness, swelling, pain, discharge, and lesions. Corneal opacity is a narrow, tissue-specific toxic endpoint that does not reflect the overall state of ocular tissue activity. Therefore, it is taught not to measure the binding of toxins to peptides, small molecules, and non-enzymatic macromolecules, as this would be analytically complex and not amenable to the simple, easy-to-use type of test described here. In addition to being complex, measuring protein denaturation or specific binding reactions does not indicate overall functional status. The use of an enzymatic marker provides a direct and easy-to-perform assessment of overall functional status.

[00520] For this invention, many different enzymes can be used to assess the functional state which, when activity decreases to predefined levels, indicates generalized toxicity. Since the effects are not specific to a single enzyme, the choice of enzyme(s) to use can be based on enzyme availability, analytical methods and required characteristics, including methods of activity quantification and robustness of quantification methods, range and clarity of measurable activities, potential for cutoff values ​​resulting in high precision and repeatability, reliability, cost, ease of use and practicality of the assay system used to measure changes in representative enzymatic activity.

[00521] It is proposed that widespread toxins reduce tissue function, and nonspecific enzyme inactivation is one of the many outcomes of toxin exposure. Petition 870260062914, dated 06 / 26 / 2026, pp. 150 / 157 146 / 147 chemical. As revealed in the examples, the concentration or ratio of test material to the amount of enzyme used as a toxicity marker exceeds by orders of magnitude the ratio used for drug screening or therapeutic use; even if these drug screening tests incidentally use dead, inactivated, or purified cell-free systems for high-throughput screening, screening assays to identify drugs or small molecules that specifically alter enzymatic activity are conducted at concentrations orders of magnitude lower than what is required for the generalized toxicity test of this invention. For example, the in chemico test is performed at concentrations of mg / ml (toxin / enzyme solution) versus drug screening or therapeutic use, where the drug-to-body ratio will be in the range of pg / g or fg / g or even more diluted than fg / g.

[00522] In certain embodiments, the invention will use cells or tissues, but these cells or tissues may not be viable because the cells can produce the enzyme, thus confounding the interpretation of reduced enzymatic activity as a measure of toxicity. It is recognized that this is unexpected due to the fact that existing enzymatic measures of toxicity use regulation by the cell as a measure of toxicity, and the invention uses the opposite of the standard: dead or inactivated cells or a cell-free system.

[00523] Although standardized diagnostic tests largely measure enzyme levels in biological fluids, these tests measure dynamic changes in enzyme levels produced by cells, tissues, or the body; these tests compare enzyme levels to reference levels in biological fluids to determine whether a level is higher or lower than a given level. Petition 870260062914, dated 06 / 26 / 2026, pp. 151 / 157 147 / 147 a certain enzyme is produced. Such diagnostic techniques are inconsistent with the method proposed here because, for the disclosed invention, it is essential that the enzyme mass or activity be standardized and that there be no active increase or decrease related to living cells in the mass or activity of the enzymes. The invention requires a procedure for testing acellular or non-viable cells to allow the measurement of activity changes as a result of the direct interaction of the toxin with a defined enzyme mass or activity. For these reasons, in vivo and living cells are excluded from teachings.

[00524] Although several aspects and modalities have been revealed in this document, other aspects and modalities will be apparent to those skilled in the art.

[00525] The various aspects and modalities disclosed in this document are for illustrative purposes only and are not intended to be limiting, the true scope and spirit being indicated by the following claims.

[00526] A person skilled in the art will find that this and other processes and methods can be implemented in a different order. Furthermore, the steps and operations described are given only as examples, and some of the steps and operations may be optional, combined into fewer steps and operations, or expanded into additional steps and operations without affecting the essence of the embodiments disclosed.

Claims

1. METHOD FOR PREDICTING THE TOXICITY OF A TEST SUBSTANCE IN LIVING TISSUES, the method being characterized by comprising: applying the test substance to a predefined enzyme or enzyme mixture to effect an in chemico reaction, wherein the test substance does not preferentially or specifically bind to an active or regulatory site of the predefined enzyme or enzyme mixture; measuring any reduction in the enzymatic activity of the predefined enzyme or enzyme mixture on a predefined substrate; and comparing the measured reduction in enzymatic activity with a control activity value or a previously established activity value, and predicting the extent or classification of the toxicity of the test substance in living tissues based on the compared measured reduction in enzymatic activity.

2. METHOD, according to claim 1, characterized in that the enzyme is purified or semi-purified or is in a mixture composed of non-viable cells and / or non-viable tissue.

3. METHOD, according to claim 1 or 2, characterized by the test substance irreversibly reducing enzymatic activity and / or reducing enzymatic activity in a non-competitive manner.

4. METHOD, according to any one of claims 1 to 3, characterized by the test substance reducing enzymatic activity by chemically destroying or denaturing the enzyme and / or the chemical or non-specific bonding of the enzyme's active or regulatory sites.

5. METHOD, according to any one of claims 1 to 4, characterized in that the test substance is applied in toxic and non-therapeutic concentrations.

6. METHOD, according to any one of claims 1 to 4, characterized in that the mass of the test substance is applied to the enzyme in a ratio of 1:1-1:100 parts of test substance to the volume of the enzyme solution.

7. METHOD, according to any one of claims 1 to 4, characterized in that the mass of the test substance is applied to the enzyme in a ratio of 1:10-1:1,000 parts of test substance to the volume of the enzymatic solution.

8. METHOD, according to any one of claims 1 to 7, characterized in that the test substance is applied in concentrations of mg / ml and not in pg / ml-fg / ml.

9. METHOD, according to any one of claims 1 to 8, characterized in that the test substances are a diverse group of toxins without having specific binding affinity or binding to the regulatory or active sites of the enzyme.

10. METHOD, according to any one of claims 1 to 9, characterized by the test predicting toxicity to living skin and the accuracy of the prediction being 85% or more.

11. METHOD, according to any one of claims 1 to 10, characterized in that the enzyme is a phosphatase.

12. METHOD, according to claim 11, characterized by the toxicity being corrosive and the method provides Petition 870250074402, dated 08 / 22 / 2025, p. 9 / 27 3 / 13 the corrosiveness or non-corrosiveness of the test substance within at least 85%.

13. METHOD, according to claim 12, characterized in that corrosiveness is predicted if the enzymatic activity is reduced to 40% or less of the control or value previously established by said test substance.

14. METHOD, according to claim 11 or 12, characterized in that non-corrosiveness is expected if the remaining enzymatic activity is greater than 40% of the control value or previously established by said test substance.

15. METHOD, according to claim 13 or 14, characterized by the accuracy in predicting whether the test substance is a dermal corrosive being equal to or greater than 90%.

16. METHOD, according to any one of claims 1 to 15, characterized by a predefined enzyme or enzyme mixture being sequentially exposed to each of a large number of known toxins (at least 10 toxins) and sequentially exposed to each of a large number of known non-toxins (at least 10 non-toxins), and the results of the enzyme activity after exposure to the toxin are calculated to form an activity class, and the results of the enzyme activity after exposure to the non-toxin are calculated to form an activity class, and the measurement of the activity class for the toxin class is significantly reduced in relation to the enzyme activity for the non-toxic activity class, and there is a statistically significant separation of the activity values ​​of the toxin class in relation to the activity values ​​of the non-toxic class (P-value < 0.01), and these results are then Petition 870250074402, dated 22 / 08 / 2025, p.10 / 27 4 / 13 used to build a predictive model that identifies toxins based on a specific reduction in enzymatic activity with the intended use of a predictive model for the identification and classification of the toxicity of unknown materials and when used to test unknowns for toxicity, and a predictive accuracy of at least 85%.

17. METHOD, according to claim 1, characterized by the lack of specificity of binding to the active site being verified using a chemical analytical technique.

18. METHOD, according to any one of claims 1 to 9, characterized in that the tissue to be evaluated comprises an ocular tissue and the accuracy of the prediction is at least 85%.

19. METHOD, according to any one of claims 1 to 9 or 18, characterized in that the enzyme is an esterase.

20. METHOD, according to any one of claims 18-19, characterized by the toxicity being irritation and the method predicting the test substance as irritant or non-irritant within at least 85%.

21. METHOD, according to any one of claims 1 to 9, characterized by the toxicity being corrosion or irritation, and the method providing the test substance as corrosive, non-corrosive, irritant or non-irritant.

22. METHOD, according to any one of claims 1 to 21, characterized in that the enzyme is one of the following: 12-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylic acid synthase, 3-mercaptopyruvate sulfurtransferase, 4-hydroxy-tetrahydrodipicolinate reductase, Petition 870250074402, dated 08 / 22 / 2025, p. 11 / 27 5 / 13 4-hydroxyphenylpyruvate dioxygenase, 6-oxide hydrolase, acetaldehyde dehydrogenase, acetolactate synthase, acetylcholinesterase, acid hydrolase, adenosine deaminase, adenosylmethionine hydrolase, adenylate cyclase, alanine aminopeptidase, alanine transaminase, alcohol dehydrogenase, alkaline phosphatase, acid phosphatase, nonspecific phosphatase, alkenylglycerophosphocholine hydrolase, alkenylglycerophosphoethanolamine hydrolase, aminolevulinic acid synthase, aminopropanol oxidoreductase, amylase, arginase, aromatase, arsenate reductase, aspartate transaminase, aspartate transcarbamoylase, ATPase, ATP synthase, beta-carotene isomerase, beta-galactosidase, beta-lactamase,biliverdin reductase, biotin synthase, butyrate kinase, caffeine dehydrogenase, carbon disulfide hydrolase, carbonic anhydrase, carbonyl sulfide hydrolase, catalase, catechol-O-methyltransferase, catechol oxidase, chitinase, cholesterol-5, choline acetyltransferase, cholinesterase, coenzyme Q-cytochrome c reductase, cyclamate sulfohydrolase, cypridin-luciferin 2-monooxygenase, cystathionine beta-lyase, cystathionine gamma-lyase, cysteine ​​desulfurase, cytochrome c oxidase, cytochrome c peroxidase, cytochrome P450 oxidase, D-xylulose reductase, deiodinase, deoxyribonuclease, diacetyl dehydrogenase, dichloromethane dehalogenase, dihydrobenzophenanthridine oxidase, dihydrofolate reductase, DNS methyltransferase, elastase, endonuclease, exonuclease, farnesol 2-isomerase, firefly luciferase, flavin prenyltransferase, fructose bisphosphate aldolase, fructose bisphosphatase, furfoylmuramine isomerase, gamma-glutamyl transpeptidase, glucose oxidase, glutathione peroxidase, glutathione reductase,glutathione S-transferase, glyceraldehyde Petition 870250074402, dated 08 / 22 / 2025, page. 12 / 27 6 / 13 3-phosphate dehydrogenase, glycerol-3-phosphate dehydrogenase, glycerol dehydrogenase, guanylate cyclase, haloacetate dehalogenase, halohydrin dehalogenase, helicase, hepoxylin epoxide hydrolase, histone methyltransferase, HMG-CoA reductase, homoserine dehydrogenase, hyaluronidase, hypoxanthine-guanine phosphoribosyltransferase, iodothyronine deiodinase, iodotyrosine deiodinase, isochorismatase, isocitrate dehydrogenase, isopenicillin N synthase, L-gluconolactone oxidase, L-xylulose reductase, laccase, lactase, lactate dehydrogenase, leucoanthocyanidin reductase, leukotriene-A4 hydrolase, leukotriene C4 synthase, linolease isomerase, lipoprotein lipase, lipoyl synthase, lysozyme, malate dehydrogenase, maleate isomerase, maleyacetoacetate isomerase, maltase, mandelate racemase, metalloendopeptidase, methylenetetrahydrofolate reductase, microsomal epoxide hydrolase, molybdenum cofactor sulfurtransferase,molybdopterin synthase, molybdopterin synthase sulfurtransferase, monoamine oxidase, myeloperoxidase, N-sulfoglucosamine sulfohydrolase, NADH dehydrogenase, nicotinate hydrogenase, nitrate reductase, nitric oxide dioxygenase, nitric oxide synthase, nitrilase, nitrite reductase, nitrogenase, nuclease, oplophoro-luciferin 2-monooxygenase, ornithine decarboxylase, ornithine transcarbamoylase, oxoglutarate dehydrogenase, phenylalanine hydroxylase, phosphatase, phosphoamidase, phospholipase A, phospholipase C, phospholipase D, phosphonoacetaldehyde hydrolase, phosphonoacetate hydrolase, phosphonopyruvate hydrolase, photoisomerase, photoporphyrinogen oxidase, prolyl isomerase, propanediol phosphate dehydrogenase, peroxidase, arginine phosphatase, proteinase K, pyruvate dehydrogenase, Petition 870250074402, dated 08 / 22 / 2025, page 13 / 27 7 / 13 Renilla-luciferin 2-monooxygenase, retinal isomerase, retinol isomerase, ribonuclease, ribonucleotide triphosphate reductase, ribonucleotide reductase, RNase,S-adenosyl-L-homocysteine ​​hydrolase, sarcosine oxidase, separase, serine protease, serine racemase, sucrase, sulfite oxidase, superoxide dismutase, tetrahydrocannabinolic acid synthase, thiaminase, thiamine oxidase, thiazole synthase, thioredoxin reductase, thiosulfate-dithiol sulfurtransferase, thiosulfatethiol sulfurtransferase, thiosulfate sulfurtransferase, thyroid peroxidase, thyroxine 5-deiodinase, transepoxysuccinate hydrolase, transaldolase, transglutaminase, transketolase, trithionate hydrolase, tRNA-5-methyluridine(54) 2-sulfurtransferase, tRNA-5-taurinomethyluridine 2-sulfurtransferase, tRNA-uridine-3-sulfurtransferase, tRNA uracil 4-sulfurtransferase, Tryptophan synthase, tyrosinase, ubiquitin carboxy-terminal hydrolase, UDP-sulfoquinovose synthase, urate oxidase, urease, uridine monophosphate synthetase, vitamin K epoxide reductase, Watasenia-luciferin 2-monooxygenase, xanthine dehydrogenase or xanthine oxidase.

23. METHOD, according to any one of claims 1 to 22, characterized in that the substrate used to determine the level of enzymatic activity is one of the following: 1-(3-bromophenyl)ethanol, 1-(3-chlorophenyl)ethanol, 1-(4'-chlorophenyl)ethanol, 1-(4'-fluorophenyl)ethanol, 1-(4-bromophenyl)ethanol, 1-(4-chlorophenyl)ethanol, 1-(4-fluorophenyl)ethanol, 1-(4-methylphenyl)ethanol, 1-(p-tolyl)ethanol, 1-butanal, 1-butanol, 1-chloro-5-acetylfuro[2], 1-decalone, 1-decanol, 1-dichloroacetone, 1-dodecanol, 1-heptanol, 1-hexanal, 1-hexanol, 1-hydroxy-2-butanone, 1-hydroxymethyl-6-methylpyrene, 1-hydroxymethyl-8-methylpyrene, 1Petition 870250074402, dated 08 / 22 / 2025, page. 14 / 27 8 / 13 hydroxymethylpyrene, 1-indanol, 1-indanone, 1-nonanol, 1octanol, 1-pentanol, 1-phenyl-1-butanol, 1-phenyl-1-propanol, 1phenyl-2-butanol, 1-phenyl-2-propanol, 1-phenylethanol, 2,2butanediol, 2-butanol, 2-heptanol,2-hexanediol, 2-hexanol, 2methylbutan-1-ol, 2-methylpent-2-enal, 2-octanol,2pentanediol, 2-pentanol, 2-phenylpropanol,2-propanediol, 3,3',3'-diaminobenzidine (DAB), 3-butanediol, 3-c]pyridine, 3-methylcyclohexanol, 3-methylcyclohexanone, 3b]thiopyran-4-one, 7,4-butanediol, 4-hydroxynon-2-enal, 4-methylumbelliferyl acetate, potassium salt of 4-methylumbelliferyl sulfate, 4-nitrophenyl acetate, 4-nitrophenyl beta-D-maltoside, 4-nitrophenyl decanoate, 4-nitrophenyl palmitate, 4-nitrophenyl stearate, 4-phenyl-2-butanol, 5,5,5'-tetramethylbenzidine (TMB), 5-bromo-4-chloro-3'-indolephosphate, 5-pentanediol, 6-dichloro-4-nitrophenyl phosphate, 6-dienal, 6-dihydro-6-methyl-4H-thieno[2,7-dimethylocta-2,7-dioxide, 11-cis-retinal, 11-cis-retinol, 12-hydroxydodecanoate, 12-hydroxylaric acid methyl ester, 12-oxolauric acid methyl ester, 13-cis-retinal, 13-cisretinol, 16-hydroxyhexadecanoate, alpha, alpha-linolenic acid, alpha-naphthol, alpha-tetralol, amylose, arachidonic acid, bicarbonate, but-2-en-1-ol, but-2-enal, carbohydrates, carvone, cellulose, blue cellulose, blue chitin, bovine fluorescent collagen, colloidal chitin, cyclitol phosphate,dec-2-enal, deoxyribonucleic acid, methyl green, dihomo-gamma-inolenic acid, disodium salt, docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), fat, gelatin, glutamine, hex-2-en-1-ol, hex-2-enal, indanol, lactose, laminarin, methylene blue, nicotinamide adenine dinucleotide (NADH), tetrazolium nitroblue, oct-2-enal, p-nitrophenyl phosphate, Petition 870250074402, dated 08 / 22 / 2025, page. 15 / 27 9 / 13 peptide bond, perillyl alcohol, phospholipids, pyrophosphate, tributyrin, tyrosine, urea, p-nitroanilide acetate salt Z-Gly-Pro-Arg, disodium salt of 4-nitrophenyl phosphate hexahydrate, 3,3'-diaminobenzidine tetrahydrochloride, o-phenylenediamine dihydrochloride, o-phenylenediamine, o-dianisidine, 3,3',5,5'-tetramethylbenzidine dihydrochloride, 4-chloro-1-naphthol, 3-amino-9-ethylcarbazole, disodium salt of 4-nitrophenyl phosphate, 10-acetyl-3,7-dihydroxyphenoxazine acetate, casein or alpha-naphthyl acetate.

24. METHOD, according to any one of claims 1 to 9, characterized, before the application of the test substance, by the enzyme being diluted in one or more of the following solvents: butanol, acetonitrile, acetone, diethylene glycol, diethyl ether, 1,2-dimethoxyethane, dimethylformamide, dimethyl sulfoxide, ethanol, ethyl acetate, ethylene glycol, glycerin, heptane, hexane, methanol, pentane, propanol, tetrahydrofuran, toluene, water, xylene or similar.

25. METHOD, according to claim 24, characterized, before applying the test substance, by diluting the enzyme in 1) ethanol or similar and 2) water or similar, and applying the test substance to either 1 or 2, and using the greater reduction in enzymatic activity in 1 or 2 to predict the toxicity of the test substance or material.

26. METHOD, according to any one of claims 1 to 25, characterized by the enzyme comprising alkaline phosphatase, acid phosphatase, nonspecific phosphatase, lactate dehydrogenase, peroxidase, esterase, alanine aminotransferase and aspartate aminotransferase, beta-glucuronidase, protease or an antiprotease. Petition 870250074402, dated 22 / 08 / 2025, pp. 16 / 27 10 / 13 27. METHOD, according to claim 26, characterized in that the enzyme is a phosphatase and 500 to 25,000 units are applied to each 1) salt water and 2) ethanol.

28. METHOD, according to claim 27, characterized in that the activity is measured by the disodium salt of p-nitrophenyl phosphate.

29. METHOD, according to any one of claims 1 to 9, characterized by a reduction in enzymatic activity, to be used to predict dermal, ocular, pulmonary, nervous system, liver, kidney, intestinal or acute developmental toxicity.

30. METHOD, according to claim 29, characterized by the test to be used to predict skin corrosion.

31. METHOD FOR IDENTIFYING A TOXIN, the method being characterized by comprising: adding the material to be tested to: a. a solution of water (W) and salt with the enzyme (WE), and b. a saline solution without enzyme (WB) to be used as a bulk block (B), and c. a solution of alcohol or organic solvent (S) with enzyme (SE), and d. a solution of alcohol or organic solvent without enzyme (SB) to be used as a bulk block; incubation for a defined period of time; measurement of enzymatic activity and bulk block values, for example, by measuring or observing changes in color or turbidity with the naked eye or with a spectrophotometer; subtracting the measured value of the saline solution from the water of the Petition 870250074402, dated 22 / 08 / 2025, page.17 / 27 11 / 13 measured value of the water-saline enzyme (WE-WB); subtract the measured value of the organic solvent blank solution from the measured value of the organic solvent enzyme (SE-SB); identify the lower value, (WE-WB) or (SE-SB), and compare it with a defined limit value or results from known standards used to define a limit value; predict that the tested chemical is toxic if the lower value is lower than a limit value or the measured value for a control chemical with known toxicity, and / or; relate the measured enzymatic activity to a toxicity index, which can be categorized according to established toxicity classes, in order to identify the tested material as a toxin or non-toxin.

32. METHOD, according to claim 31, characterized by the enzyme comprising one or more compounds selected from phosphatase, peroxidase, lactate dehydrogenase, alanine aminotransferase and aspartate aminotransferase, beta-glucuronidase or protease.

33. METHOD, according to claim 31, characterized in that the enzyme is a phosphatase.

34. METHOD, according to claim 31, characterized in that the activity is measured by the disodium salt of p-nitrophenyl phosphate.

35. METHOD, according to claim 31, wherein the method is characterized by being used for testing to predict irritation or corrosion of the dermis, eyes, lungs, nervous system, liver, kidneys, and intestines.

36. METHOD, according to claim 31, in Petition 870250074402, dated 08 / 22 / 2025, pp. 18 / 27 12 / 13, which is characterized by being used to test and predict dermal corrosion.

37. METHOD, according to claim 31, characterized in that the established toxicity classes are selected from non-irritating, irritating and corrosive.

38. METHOD, according to claim 31, characterized by combining a water-based saline solution and an alcohol or organic solvent.

39. PRODUCT OR KIT USED FOR TOXICITY TESTING characterized by including: a. tubes with an enzyme diluted in a solvent (test and control tubes); b. tubes with the solvent alone (empty tube); c. a substrate for the solvent; d. instructions for: a. adding the material to be tested to a tube with enzyme and solvent (test tube) and to a second tube with only the solvent (empty tube) and keeping a third tube with enzyme and solvent, but without adding the material to be tested (control tube); b. incubating the three tubes for a defined time; c. transferring a portion from each tube to an enzymatic substrate; d. visually or quantitatively measuring the amount of enzymatic substrate converted into product during a defined period of time; e. subtracting the value from the empty tube (solvent only) from the value from the test tube (solvent plus enzyme); and f. classify the tested material as toxic Petition 870250074402, dated 08 / 22 / 2025, page.19 / 27 13 / 13 if the amount of substrate converted into a product (test tube) minus the solvent alone (empty tube) is less than a defined value or less than a defined value or a percentage of the control tube (enzymatic solvent without the material to be tested).

40. PRODUCT OR KIT USED TO IDENTIFY TOXIC CHEMICALS characterized by including: a. a cell-free enzyme solution, diluted in one or more suitable solvents, such as water, ethanol or an organic solvent; b. a non-toxic chemical to serve as a negative control; c. a substrate for the enzyme, which, when the enzyme solution is added, allows the evaluation of the enzyme activity.

41. PRODUCT OR KIT USED FOR HIGH-YIELD TESTING TO IDENTIFY DERMAL CORROSIVES characterized by including: a. four types of pre-filled tubes with A1 (buffered saline solution with alkaline phosphatase), B1 (buffered saline solution blank), A2 (ethanol with alkaline phosphatase), B2 (empty ethanol); It can also be supplied in bulk bottles and the user can divide it into portions in tubes; b. negative control (water); c. positive control (acetic acid); d. Detection reagent (DR) solution (disodium salt of p-nitrophenyl phosphate).

Images