• The study was performed according to the OECD Guideline 439. (europa.eu)
  • The potential of the test material to induce eye irritation was investigated in a human cornea model in an in-vitro study, performed according to the OECD Guideline 492. (europa.eu)
  • The key skin irritation/corrosion study with dodecamethylcyclohexasiloxane (D6) (NOTOX, 1999c), conducted according to OECD Test Guideline 404 and in compliance with GLP, concluded that D6 was not irritating to skin. (europa.eu)
  • Modified EpiDerm SIT is now implemented into the draft EU method B46 and draft OECD guideline as stand alone assay for prediction of skin irritating and non-skin irritating test substances. (mattek.com)
  • A substance can be considered as causing serious eye damage (Category 1) based on positive results in the BCOP test (OECD 437). (europa.eu)
  • The test item (20 % (w/v) in physiologic saline) was tested in the BCOP test according to OECD TG 437. (europa.eu)
  • An example of an in-vitro method that has full potential in replacing an original eye irritancy test known as the Draize eye test is called the Bovine Corneal Opacity Test (BCOP). (edubirdie.com)
  • The BCOP (Bovine Corneal Opacity and Permeability) assay is an in vitro eye irritation test method developed by Gautheron et al. (iivs.org)
  • The absorption, distribution and excretion of test substance was determined by performing in vivo study in rats. (europa.eu)
  • In April 2007, ECVAM endorsed 2 alternative test methods (EPISKIN and EpiDerm Skin Irritation Tests (SIT)) as replacements of the in vivo rabbit skin irritation test. (mattek.com)
  • The Short Time Exposure (STE) assay, developed by Kao Corporation (Japan), is an in vitro assay used to assess acute eye irritation potential as an alternative to the traditional in vivo Draize test. (iivs.org)
  • An acute in vivo skin irritation / corrosion test according to OECD 404 (Hansen, 2013) was performed with vanadium dioxide, and results indicate that it is not irritant to skin. (europa.eu)
  • Animal testing , also known as animal experimentation , animal research , and in vivo testing , is the use of non-human animals in experiments (although some research about animals involves only natural behaviors or pure observation, such as a mouse running a maze or field studies of chimp troops ). (wmflabs.org)
  • The terms animal testing, animal experimentation , animal research, in vivo testing , and vivisection have similar denotations but different connotations . (wmflabs.org)
  • [ 1 ] It includes pure research (such as genetics , developmental biology , and behavioral studies ) as well as applied research (such as biomedical research , xenotransplantation , drug testing, and toxicology tests , including cosmetics testing ). (wmflabs.org)
  • Ultrastructurally, the EpiDerm Skin Model closely parallels human skin, thus providing a useful in vitro means to assess dermal irritancy and toxicology. (cedii.net)
  • Chemicals already shown to have adverse effects in vitro are not currently used in a Draize test, thereby reducing the number and severity of tests that are carried out. (wikipedia.org)
  • Animal testing in the cosmetic industry consists of chemicals intentionally being seeped in numerous places of an animal's body to monitor any damage to the body that may occur (Doyle). (edubirdie.com)
  • For this reason, the skin is often exposed to potentially hazardous agents, including chemicals, which may contribute to the onset of a spectrum of adverse health effects ranging from localized damage (e.g., irritant contact dermatitis and corrosion) to induction of immune-mediated responses (e.g., allergic contact dermatitis and pulmonary responses), or systemic toxicity (e.g., neurotoxicity and hepatoxicity). (cdc.gov)
  • Understanding the hazards related to skin contact with chemicals is a critical component of modern occupational safety and health programs. (cdc.gov)
  • This document provides the scientific rationale and framework for the assignment of multiple hazard-specific skin notations (SK) that clearly distinguish between the systemic effects, direct (localized) effects, and immune-mediated responses caused by skin contact with chemicals. (cdc.gov)
  • In meeting this objective, this Skin Notation Profile intends to inform the audience--mostly occupational health practitioners, researchers, policy- and decision- makers, employers, and workers in potentially hazardous workplaces--so that improved risk- management practices may be developed to better protect workers from the risks of skin contact with the chemicals of interest. (cdc.gov)
  • b) the temperature during the exposure was increased to 37°C. With these modifications, when testing chemicals from the pre-validation and validation studies, a significant increase in sensitivity (84%) was obtained, while maintaining an acceptable specificity of the method. (mattek.com)
  • The Eye Irritation Test (EIT) is an OECD-approved in vitro non-animal test method for identifying chemicals and mixtures that may be irritating to the corneal epithelium. (iivs.org)
  • The test method utilizes an in vitro reconstructed human corneal epithelium (RhCE) model (EpiOcular™, MatTek Corp. or HCE, SkinEthic™), in an acute exposure assay to support international regulatory labeling requirements, according to the United Nations Globally Harmonized System of Classification and Labelling of Chemicals (UN GHS). (iivs.org)
  • Assessment of the human epidermis model SkinEthic RHE for in vitro skin corrosion testing of chemicals according to new OECD TG 431. (episkin.com)
  • Understanding and evaluating the irritation potential of chemicals or formulations is an important consideration to avoid exposing human volunteers to potentially irritating materials during clinical testing. (loft-stair.com)
  • 2018). CON4EI: Epi眼 Eye Irritation Test (Epi眼 EIT) for hazard identification and labeling of eye irritating chemicals . (loft-stair.com)
  • Lebrun Labs LLC has been developing and improving the in vitro depth of injury (IVD) ocular irritation test method to serve as an accurate, reliable way to test consumer products and chemicals for their irritancy potential. (lebrunlabs.com)
  • The calculated IVIS (in vitro irritancy score) was 84.29 which is within the range for classification (according to the classification criteria of OECD 437) of the substance as causing serious eye damage. (europa.eu)
  • Based on these data, in comparison to the vehicle control, the In Vitro Irritancy Score (IVIS) value was calculated to be 5.1 which is below the IVIS cut-off threshold of 55 and thus identifying the test item as not inducing serious eye damage. (europa.eu)
  • Effective in-vitro studies include the Bovine Corneal Opacity test and the reconstructed human epidermis (RhE). (edubirdie.com)
  • 1992), which uses living bovine corneal tissue, obtained as a by-product from abattoirs, to evaluate the potential ocular irritancy of a test article. (iivs.org)
  • The test method evaluates the cytotoxicity induced by a series of test chemical dilutions in a monolayer of rabbit corneal fibroblasts (Statens Seruminstitut Rabbit Cornea - SIRC) after a single five-minute exposure. (iivs.org)
  • The Ocular Irritection Assay is an in chemico eye irritation assay that detects, ranks, and predicts the corneal irritation potential of a test material. (iivs.org)
  • However, no current validated non-animal ocular irritation test methods or prediction tools are able to predict corneal recovery in a manner consistent with the animal tests historically employed. (iivs.org)
  • We can also provide phototoxicity and photosensitisation screening to test for irritancy and/or allergy potential following exposures to UV light. (intertek.com)
  • The Draize test is an acute toxicity test devised in 1944 by Food and Drug Administration (FDA) toxicologists John H. Draize and Jacob M. Spines. (wikipedia.org)
  • Because of its controversial nature, the use of the Draize test in the U.S. and Europe has declined in recent years and is sometimes modified so that anaesthetics are administered and lower doses of the test substances used. (wikipedia.org)
  • The following year Draize joined the FDA, and was soon promoted to head of the Dermal and Ocular Toxicity Branch where he was charged with developing methods for testing the side effects of cosmetic products. (wikipedia.org)
  • This work culminated in a report by Draize, his laboratory assistant, Geoffrey Woodard, and division chief, Herbert Calvery, describing how to assess acute, intermediate, and chronic exposure to cosmetics by applying compounds to the skin, penis, and eyes of rabbits. (wikipedia.org)
  • In 1971, before the implementation in 1981 of the modern Draize protocol, toxicologists Carrol Weil and Robert Scala of Carnegie Mellon University distributed three test substances for comparative analysis to 24 different university and state laboratories. (wikipedia.org)
  • A 2004 study by the U.S. Scientific Advisory Committee on Alternative Toxicological Methods analyzed the modern Draize skin test. (wikipedia.org)
  • In the Draize test for skin irritancy, the test substances are applied to skin that is shaved and abraded (several layers of skin are removed with sticky tape), then covered with plastic sheeting. (wikipedia.org)
  • According to the British Research Defence Society, the Draize eye test is now a "very mild test", in which small amounts of substances are used and are washed out of the eye at the first sign of irritation. (wikipedia.org)
  • The Draize eye cosmetic animal testing method continues to raise moral concerns to people around the world due to the past studies that involved harm to rabbits, which were the most frequently used animal in an eye irritancy test (Luco et al 451). (edubirdie.com)
  • 的 Epi眼 Ultra-mildness Test is an 在体外 risk assessment assay routinely utilized for mild materials for which the Draize test is insensitive. (loft-stair.com)
  • Based on the ET-50, the test article is categorized into one of 4 classifications ranging from non-irritating to severe/extreme which correspond to groupings of Rabbit Draize Eye Scores (MMAS). (loft-stair.com)
  • The objective of this study was to assess the relative level of primary dermal skin irritation of a test material on rabbits under semioccluded conditions. (europa.eu)
  • Our dermal safety studies, from our clinical study centre in Paris, France, are focused on supporting your product development through evidence-based dermatological efficacy testing and claims substantiation studies. (intertek.com)
  • Based on the tissue viability obtained, the substance is not considered to be a skin irritant. (europa.eu)
  • Under the conditions of the test system, the test substance is considered as eye irritant or inducing serious eye damage in the EpiOcular TM Eye Irritation Test. (europa.eu)
  • In order to distinguish between eye irritant and induing serious eye damage, the substance was further evaluated in-vitro according to the OECD 437. (europa.eu)
  • Based on the tissue viability obtained during the OECD 492 study, the substance can be considered as an eye irritant or inducing eye damage. (europa.eu)
  • The test material was also considered to be irritant according to EU labelling regulations Commission Directive 93/21/EEC. (europa.eu)
  • As neither a corrosive effect nor a severe irritant effect was observed after a four-hour exposure, the test was completed using two additional animals, each with one patch only, for an exposure period of 4 hours. (europa.eu)
  • The data are then quantified and related to a prediction model that results in an ocular irritancy classification, including the GHS 2A/2B reversible irritant classification. (lebrunlabs.com)
  • In 2008 ESAC concluded that the performance of this assay met the criteria outlined to be considered to have sufficient accuracy and reliability for prediction of R38 skin irritating and non-skin irritating test substances (5). (mattek.com)
  • The CAMVA is an in vitro assay that measures the presence and degree of vascular effect to the chorioallantoic membrane (CAM) of 10-day fertilized eggs following exposure to test article. (iivs.org)
  • 的 在体外 Epi眼 MTT ET-50 assay is used as a screen to assign expected 在活的有机体内 irritancy responses based on the time to toxicity (ET-50) results obtained with Epi眼. (loft-stair.com)
  • Chemical reactivity and cytotoxicity testing were performed using the commercially available EpiOcularTM assay. (mattek.com)
  • The in vitro- EpiOral (buccal) and EpiGingival (gingival) MTT ET-50 assay is used as a screen to assign expected irritancy responses compared to benchmark controls based on the time to toxicity (ET-50) results obtained with EpiOral and EpiGingival. (gcorponline.net)
  • An in vitro study for assessing ocular irritation properties of the solid test item was performed using the reconstructed human cornea-like epithelium (RhCE) cell model EpiOcularTM. (europa.eu)
  • The solid test item was applied topically to the RhCE tissue surface in duplicate for 6 hours, followed by an 18 hour post-treatment incubation period. (europa.eu)
  • These "ready-to-use" tissues are cultured on specially prepared cell culture inserts using serum free medium, and attain levels of differentiation on the cutting edge of in vitro skin technology. (cedii.net)
  • The study was performed to assess the irritancy potential of the test material to the eye of the New Zealand White rabbit. (europa.eu)
  • It is a test method developed to assess the reproductive toxicity of chemical substances and is the preferred test method to address the standard information requirement defined in Column 1 of Point 8.7.3 of Annexes IX and X to the REACH Regulation instead of the Two-Generation Reproductive Toxicity study. (europa.eu)
  • When cosmetic products are tested, there are a lot of factors that need to be deliberated, yet in-silico and in-vitro methods are intricately developed to assess all of those necessary aspects of a cosmetic product more efficiently than the trusted animal testing methods. (edubirdie.com)
  • Morphologically, the tissue models closely parallel native buccal human tissues, thus providing a useful in vitro means to assess irritancy, disease and other basic oral biology phenomena. (swedishwebagency.com)
  • The distribution of the 14C-labelled test substance was studied using collected samples of blood, organs and tissues by radioassay. (europa.eu)
  • Minimal to mild findings indicative of irritancy in the nasal tissues were noted in the repeated dose 90-day inhalation toxicity study with dodecamethylcyclohexasiloxane (D6) (Dow Corning Corporation, 2013). (europa.eu)
  • In-vitro testing methods use 'whole cells, part of cells or reconstructed tissues' (Luco et al 452) to perform the required tests that accurately assert the safety of a cosmetic product. (edubirdie.com)
  • The test item was incubated on the cornea for 10 minutes at 32 ± 1 °C. After removal of the test item and 2 hours post-incubation, opacity and permeability values were measured. (europa.eu)
  • Types of injury caused by exposure to the test article are quantitatively measured by changes in opacity and permeability to fluorescein. (iivs.org)
  • The test item, as well as controls, were tested for their ability to impair cell viability after an exposure period of 15 minutes followed by a 42 ± 1 hour recovery period. (europa.eu)
  • The final mean percent tissue viability recorded for the test item is 105 % (rounded). (europa.eu)
  • A study was performed for the assessment of the skin irritancy of the test item with reconstructed human epidermis (RhE). (europa.eu)
  • Evaluation of the sensitization phase as an endpoint stimulate keratinocytes present in the epidermis of the skin results in a reduced time for animals to be on study and to release inflammatory mediators such as interleukins 1, 6 eliminates the discomfort associated with the development of and 18, granulocyte-macrophage colony-stimulating factor, inflammation in the elicitation phase of the response. (cdc.gov)
  • Reconstructed epidermis versus human and animal skin in skin absorption studies. (episkin.com)
  • According to the Guidance on the application of CLP criteria (v.5.0), the OECD 439 can reliably distinguish non-classified from classified substances. (europa.eu)
  • No symptoms of systemic toxicity were observed in the animals during the test period and no mortality occurred. (europa.eu)
  • The Extended One-Generation Reproductive Toxicity Study (EOGRTS) (OECD TG443, EU B.56) in its basic test design is a standard information requirement at REACH Annex IX (where concerns regarding reproductive toxicity are evident) and X levels. (europa.eu)
  • The requirement to conduct complex mammalian toxicity tests at Annex IX which potentially involves the use of hundreds of vertebrates (normally rats) needs to be carefully considered to ensure that it satisfies Article 25(1) of the REACH Regulation that any testing on vertebrate animals is undertaken only as a last resort. (europa.eu)
  • Non-animal testing alternatives that are considered to be in-vitro and in-silico are advanced in identifying the toxicity level of a substance, which is why the dependability of alternative methods has the potential to increase because toxicity is significant in assuring the successfulness of a cosmetic product. (edubirdie.com)
  • The test subject is commonly an albino rabbit, though other species are used too, including dogs. (wikipedia.org)
  • The test material produced irreversible effects and was considered to be corrosive to the rabbit eye. (europa.eu)
  • Test protocols such as the OECD test guidelines and standard methods like ET50 for skin irritancy are great for assessing products for regulatory submission. (x-cellr8.com)
  • Our dermatology experts work to Product Test Guidelines for the Assessment of Human Skin Compatibility, the European Cosmetic, Toiletry and Perfumery Association (COLIPA) guidelines including the Guidelines for the Evaluation of the Efficacy of Cosmetic Products, Guidelines on the Management of Undesirable Event Reports and the Guidelines for the Safety Assessment of a Cosmetic Product. (intertek.com)
  • The test item was applied to a three-dimensional human cornea tissue model in duplicate for an exposure time of 30 minutes. (europa.eu)
  • 50 μl of the liquid test item was applied to each tissue. (europa.eu)
  • To address this limitation, we have developed prediction models that can be applied to an in vitro food source tissue test used measure depth of injury. (lebrunlabs.com)
  • Both of the most trustworthy alternatives that are in-silico and in-vitro methods contain techniques that generate the needed results for the assessment of a particular cosmetic product, which means that cosmetic animal testing will sooner than later no longer be in effect. (edubirdie.com)
  • If this hypothesis is correct, a major limitation of alternative tests is the lack of the assessment of stromal damage needed to detect reversible irritants. (lebrunlabs.com)
  • While the EPISKIN SIT was recognized as a stand alone method, the EpiDerm SIT was endorsed for use in a tiered testing strategy, where irritating results are accepted and non-irritating results may require further testing by another method (e.g. (mattek.com)
  • NUMBER OF TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION: the optical density of the isopropanol-extracts of 3 insert was determined by duplicate per insert = 6 OD values. (europa.eu)
  • It provides several benefits over other tests for sensitization because it provides a quantitative endpoint, dose-responsive data, and allows for prediction of potency. (cdc.gov)
  • All four methods of the OECD 439 are valid for the classification of substances for skin irritation according to CLP criteria. (europa.eu)
  • WHO is not responsible, and does not accept any liability, for the testing of pesticides for compliance with the specifications, nor for any methods recommended and/or used for testing compliance. (who.int)
  • Understanding that there are technically sound and effective non-animal testing methods present today initiates a struggle in grasping the concept that cosmetic companies continue to utilize animal testing in assuring that their products are safe for human use. (edubirdie.com)
  • Specific alternatives like in-silico and in-vitro methods are considered to be the most widely validated and precise strategies when testing a cosmetic product's efficiency. (edubirdie.com)
  • Thus, animal testing within the cosmetic industry in the United States should be diminished because there are effective alternative testing methods. (edubirdie.com)
  • The determination of a cosmetic product being toxic is significant when attempting to figure out the 'application methods (ingestion, inhalation, or topical application to the skin or mucous membrane), amount and frequency of use of these cosmetics, and the amount of a particular ingredient used in the manufacture of different products' (Luco et al 452). (edubirdie.com)
  • On the other hand, in silico testing methods have the same goal of developing strategies that generate accurate results, but in-silico methods refer to models that are constructed to precisely analyze a chemical's functions and potential severity the substance may cause (Gellatly and Sewell). (edubirdie.com)
  • Computational techniques, including predictive algorithms and mathematical models that describe a selected process (e.g., skin permeation) by means of analytical or numerical methods. (cdc.gov)
  • IIVS possesses extensive experience with numerous test methods and testing strategies for commercial, industrial and regulatory applications. (iivs.org)
  • The good news is that the use of this animal testing method has been largely replaced with validated alternative methods. (vipermax.ca)
  • Recently the LLNA has been designated as the initial re- the importance of developing rapid and sensitive methods quirement for sensitization testing with the new registration, for hazard identification of chemical sensitizers. (cdc.gov)
  • The key step within assignment of the hazard-specific SK is the determination of the hazard potential of the substance, or its potential for causing adverse health effects as a result of skin exposure. (cdc.gov)
  • Whether evaluating ultra-mild cosmetics and personal care products, or rank ordering the irritation potential of candidate formulations and ingredients, we can provide custom Ocular Screening protocols to best meet your testing goals. (iivs.org)
  • Scientists with CPSC , FDA , and NIEHS and collaborators compiled such a data set for human skin sensitization potential by collecting data from the scientific literature for human predictive patch tests that used the human maximization or human repeated insult patch test protocols. (nih.gov)
  • According to the Guidance on the application of CLP criteria (v.5.0), the OECD 492 can be used for identifying substances not causing serious eye damage/eye irritation. (europa.eu)
  • The data collection identified 2,255 tests that were deemed to be sufficiently reliable for the analysis, including reports for 232 substances with at least two test results. (nih.gov)
  • The study was conducted in accordance with OECD TG 439 and EU Test Method B.46. (europa.eu)
  • This poses a significant public health burden with com- by the Organization for Economic Co-operation and De- bined annual costs of up to $1 billion for medical costs, work- velopment (OECD) as a standalone method (OECD 429). (cdc.gov)
  • [ 22 ] Avenzoar , an Arabic physician in 12th-century Moorish Spain who also practiced dissection, introduced animal testing as an experimental method of testing surgical procedures before applying them to human patients. (wmflabs.org)
  • Although this test method is still in development, we welcome collaborations with interested parties. (lebrunlabs.com)
  • The purpose of this test method is to detect the full range of ocular irritants, including those that are reversible, which will enhance our ability to advise and protect consumers from ocular injury without using animals. (lebrunlabs.com)
  • In this blog, we'll be looking at the importance of testing oral care products to make sure they are sufficiently mild for the intended use, and how other claims regarding their benefits can be supported with in vitro tests. (x-cellr8.com)
  • The EpiOcularTM Eye Irritation Test (EIT) was conducted in accordance with OECD 492. (europa.eu)
  • ALTBIB is the Bibliography on Alternatives to the Use of Live Vertebrates in Biomedical Research and Testing. (nih.gov)
  • To distinguish between these two classifications, the substance was further tested in the OECD 437. (europa.eu)
  • The results were reported as radioactivity (% of administered dose of test substance). (europa.eu)
  • The results of the positive (20 % imidazole solution) and vehicle (physiologic saline solution) controls confirmed the validity of the test. (europa.eu)
  • The animals are euthanized after testing if the test renders irreversible damage to the eye or skin. (wikipedia.org)
  • No skin irritation was observed by 4 hours exposure to the test substance. (europa.eu)
  • Where testing is proposed it is also important to ensure that in the studies conducted the fewest number of animals possible are used to satisfy the objective pursued as required by Directive 2010/63/EU of the European Parliament and of the Council on the protection of animals used for scientific purposes (OJ L 276, 20.10.2010, p. 33). (europa.eu)
  • Because humans are the primary subject of interest for regulatory safety testing, it is advantageous to have human reference data available for evaluation of NAMs for assessing chemical safety. (nih.gov)
  • An initial thought of animal testing in the cosmetic industry is that the companies are required to utilize animals as subjects in testing their products, but the US Food and Drug Administration (FDA) does not demand cosmetic companies to have their products be tested on animals first (Seeker 01:40-01:46). (edubirdie.com)
  • The controversy of cosmetic animal testing continues to stand because sacrificing animal lives remains to be seen around the world even when other strategies are accessible. (edubirdie.com)
  • To see a decrease in testing cosmetic products on animals, consumers need to become more consciously aware of what they buy. (edubirdie.com)
  • Many personal care and cosmetic products accidentally contact the eye, which is very sensitive to numerous compounds that would otherwise be benign when applied to the skin. (iivs.org)
  • Initially used for testing cosmetics, the procedure involves applying 0.5 mL or 0.5 g of a test substance to the eye or skin of a restrained, conscious animal, and then leaving it for set amount of time before rinsing it out and recording its effects. (wikipedia.org)
  • other: Adjacent areas of skin of each animal served as controls. (europa.eu)
  • The test material produced only a very slight erythema reaction in one animal. (europa.eu)
  • You asked us some questions about animal-free testing and we've answered… Can you claim a product is 'Cruelty-Free' even if animal-derived components have been used in the testing process? (x-cellr8.com)
  • Here is what she had to say following her time in the animal-free testing world… What made you want to do work experience at XCellR8? (x-cellr8.com)
  • As it was expected that the test item would not produce any severe irritancy or corrosion, the test was started using at first only one animal, receiving a single patch for an exposure period of 4 hours. (europa.eu)
  • None of the three rabbits exposed for 4 hours to 500 mg vanadium dioxide/animal showed any skin reactions 60 minutes, 24, 48 and 72 hours after the application. (europa.eu)
  • Animal Test Alternatives. (cdc.gov)
  • The research is conducted inside universities, medical schools, pharmaceutical companies, farms, defense establishments, and commercial facilities that provide animal-testing services to industry. (wmflabs.org)
  • The earliest references to animal testing are found in the writings of the Greeks in the 2nd and 4th centuries BCE. (wmflabs.org)
  • NLM developed ALTBIB to provide access to PubMed citations for users seeking information on alternatives to animal testing. (nih.gov)
  • Furthermore, the extent to which the test information will improve the quantification and management of risks to human health and the environment based on the Risk Characterisation Exercise needs to be demonstrated. (europa.eu)
  • The animals are observed for up to 14 days for signs of erythema and edema in the skin test, and redness, swelling, discharge, ulceration, hemorrhaging, cloudiness, or blindness in the tested eye. (wikipedia.org)
  • The submission substance is considered not classified with regards to skin irritation/corrosion. (europa.eu)
  • The test item was thus shown to be not irritating to reconstructed human skin in vitro. (europa.eu)
  • The test item was brought onto the cornea of a bovine eye which had been incubated with cMEM without phenol red at 32 ± 1 °C for 1 hour and whose opacity had been measured. (europa.eu)