The Draize Rabbit Eye Test (DRET) is the regulatory ‘Gold Standard’ for assessing and categorizing ocular irritation/corrosivity. The DRET focuses attention on damage to a number of different ocular structures, which are scored and weighted based on toxicological importance. The structures are as follows: Cornea (CO) – 80 points, Conjunctiva (Conj) – 20 points, and Iris (IR) – 10 points.
The heaviest weighting is on corneal damage, which is 80 points out of the total 110-point scale devised by Draize. Corneal irritation scoring is based on assessment of opacities on the cornea, Conj irritation is assessed by increased vascularization, and iridial damage is assessed by function of the iris (ability to constrict or dilate pupil)and deepening of the rugae.
PorCORA – Corneal Irritation Focus
Since the CO scores have the heaviest weight, and most often are the drivers of eye irritation, we developed the Porcine Cornea Opacity Reversibility Assay (PorCORA), an ex vivo corneal model. The PorCORA can distinguish between a material’s potential to cause severe (reversible) versus corrosive (irreversible) damage. Excised cultured porcine corneas are topically dosed with a test material and kept in culture for 21 Days. Corneal damage is visually assessed and scored. If CO reverse and are not visible by Day 21, the test material is categorized as a non-corrosive material and deemed to not cause permanent ocular damage.
In internal validation studies, we tested 56 chemicals and dilutions of chemicals ranging from corrosive (GHS category 1) to non-irritating (GHS not categorized). Using Cooper Statistics, we arrived at an accuracy of 89% with a positive and negative predictivity of 85% (cat. 1) and 93% (not cat.1), respectively.
To determine if these Cooper Statistics could be improved, we used the drivers of classification concept based on Barroso et al., 2016*. Upon re-examination of our data based on this published database and methodology, we found that four chemicals (1,2,4-Triazole, N-Butanol, 2,5-Dimethyl-2,5-Hexanediol, and Potassium Cyanate) had invalid tests (animals euthanized prior to day 21) or produced irritation not driven by corneal opacities. These chemicals were removed from our dataset. Without these four chemicals the accuracy improved to 92%. Moreover, the major change was in our positive predictivity, which increased to 91%. The negative predictivity for this subset of chemicals remained the same.
Lastly, PorCORA’s predictivity was assessed based on CO persistence; i.e., materials that produced low CO scores (from Draize Rabbit Eye Tests). Of the 56 chemicals tested, 40 had low severity (i.e., CO mean scores < 3). The accuracy for persistence of low CO scores was 88% with positive and negative predictivity of 79%, and 92%, respectively.
* Barroso, J., Pfannenbecker, U., Adriaens, E., Alépée, N., Cluzel, M., De Smedt, A., Hibatallah, J., Klaric, M., Mewes, K., Millet, M., Templier, M., McNamee, P. 2016. Cosmetics Europe compilation of historical serious eye damage/irritation in vivo data analysed by drivers of classification to support selection of chemicals for development and evaluation of alternative methods/strategies: the Draize eye test Reference Database (DRD). Arch. Toxicol. 91:521-547.
Based on these data, the PorCORA was proven to be a valid test to assess a material’s potential to distinguish reversible versus irreversible eye damage.
Appropriate choices of validation chemicals, based on the drivers of classification, have proven valuable and should be applied to replacement ocular assays that previously failed to meet acceptance criteria.
PorCORA testing results are encouraging and future studies are planned that will pursue mixtures and chemical substances of unknown or variable composition, complex reaction products and biological materials. PorCORA
If you would like to find out more about PorCORA and other in vitro/alternative toxicology testing methods, please click here.
Corrositex® is an in vitro method used to determine the dermal corrosive potential of chemicals and chemical mixtures.
Corrositex® has been designed as a replacement for the dermal corrosivity rabbit test based upon proprietary biomembrane and chemical detection technology. Corrositex® is also OECD approved – OECD Test Guideline 435: Non-Animal Dermal Corrosivity Test for Packing Group Classification.
* For two (2) or more samples sent together; Single (1) sample price: $995
The FD&C Act prohibits the marketing of adulterated or misbranded cosmetics in interstate commerce and EU Regulation 1223/2009 aims to protect human health, reduce and regulate animal testing as well as make information more available to consumers, leading to the introduction of the product ingredient file concept.
In 2013, the European Union (EU) enacted the Directive 76/768/EC, the primary law that outlines cosmetics safety requirements. This Directive includes the 7th amendment which mandates prohibition of animal testing for cosmetic products and ingredients.
In accordance with European regulations, a Product Information File (PIF) must be authored for each cosmetic product and submitted to the competent authorities on demand.
A key part of the PIF is a safety assessment or Cosmetic Product Safety Report, which is a safety assessment that provides:
Toxicological profile of each substance in the finished product (including Hazard Identification)
Chemical and physical specifications of the substances
Exposure level for each substance
A risk characterization for each substance
Expert Cosmetic Safety Testing Partner
MB Research Labs has been the premier choice in conducting product safety assessments for the cosmetics, personal care, chemical and pharmaceutical industries for over 45 years. Our expertise is sought out to ensure the safety our partners’ cosmetic ingredients. MB Research is a leader in the use and development of In Vitro and Alternative Toxicology Tests and continue to introduce new testing methods regularly.
Evaluate the mutagenic potential of a test article based on the reversion of selective growth mutations
The purpose of this study is to evaluate the mutagenic potential of a test article based on the reversion of selective growth mutations in several strains of Salmonella typhimurium bacteria and in Escherichia coli WP2 uvrA bacteria, in the presence and absence of S9 activation. This protocol is based on OECD Guideline for Testing of Chemicals: No. 471 – Bacterial Reverse Mutation Test and U.S. EPA Health Effects Test Guidelines OSCPP/OPPTS 870.5100 – Bacterial Reverse mutation Test. MB Research Labs utilizes the test guideline recommended bacterial strains (E. coli WP2 uvrA , S. typhimurium TA97a, S. typhimurium TA98, S. typhimurium TA100, and S. typhimurium TA1535). More information available here.
Basis of the Method:
The test system will be exposed to the test article via the plate incorporation method, which has been shown to effectively detect a wide variety of mutagenic compounds. This assay is based on the methodology originally described by Ames, et al. (1975) and updated by Maron and Ames (1983) and complies with the Guidelines.
In general, a 2-fold increase with or without metabolic activation will be considered a positive response. Dose-related increases approaching a 2-fold increase will be deemed equivocal and a repeat test will be recommended.
Negative Results will be determined by the absence of a dose-related increase in all five tester strains, again taking into account toxicity of the test article as well as the quality checks of the assay.
Positive Results from the bacterial reverse mutation test indicate that the substance induces point mutations by base substitutions or frame shifts in the genome of either Salmonella typhimurium and/or Escherichia coli. Negative results indicate that under the test conditions, the test substance is not mutagenic in the tested species.
This study is conducted in accordance with the Good Laboratory Practices of the EPA, 40 CFR 160 and 792, FDA 21 CFR Part 58, and as specified in the OECD, Principles on Good Laboratory Practice, revised 1997.
1. B. N. Ames, J. McCann, and E. Yamasaki. Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity test. Mutat Res 31 (6):347-364, 1975.
2. D. M. Maron and B. N. Ames. Revised methods for the Salmonella mutagenicity test. Mutat Res 113 (3-4):173-215, 1983.
3 L. D. Claxton, J. Allen, A. E. Auletta, K. Mortelmans, E. Nestmann, and E. Zeiger. Guide for the Salmonella typhimurium/mammalian microsome tests for bacterial mutagenicity. Mutat Res 189 (2 (Oct)):83-91, 1987.
4. K. Mortelmans and E. Zeiger. The Ames Salmonella/microsome mutagenicity assay. Mutat Res 455 (1-2):29-60, 2000.
5. OECD Guideline for Testing of Chemicals: No. 471 – Bacterial Reverse Mutation Test (July 1997)
6. U.S. EPA Health Effects Test Guidelines OPPTS 870.5100 – Bacterial Reverse mutation Test (August 1998).
The California Cruelty-Free Cosmetics Act makes it unlawful for cosmetic manufacturers to sell any cosmetic in California if the final product or any component of the product was tested on animals and goes into effect Jan. 1, 2020.
Products that have undergone testing on animals before that date can still be sold in California. Earlier in the month, the bill passed the state legislature after it was narrowed in scope.
Are You Ready With In Vitro Testing?
MB Research Labs is positioned to help manufacturers with a suite of in vitro and alternative (non-animal) tests for cosmetic safety testing. MB has over 45 years of toxicology testing experience and over 25 years of testing using non-animal test methods.
The Organization for Economic Co-Operation and Development (OECD) has prescribed the use of a weight-of-evidence (WoE) analysis, integrated with a sequential testing strategy, for the classification of acute eye hazards. The October 2017 revision to the Health Effects Test Guideline No. 405 outlines a stepwise strategy that includes Step 6, “Perform validated and accepted in vitro or ex vivo ocular test(s),” specifying only four possible tests: three designed to identify severe eye irritation/ corrosion (GHS Category 1) and one to identify non-irritants (GHS No Category), the top-down and bottom-up approaches, respectively. The GHS Category 2 (eye irritant) classification is impossible by any one bottom-up or top-down test.
The EpiOcular™ Eye Irritation Test; EIT (OECD 492), as a bottom-up test, either determines a material to be No Category, or that it causes eye effects, but cannot discriminate between eye irritation, serious eye damage, and corrosion.
Conversely, the top-down Bovine Corneal Opacity and Permeability; BCOP Test (OECD 437), is used for ruling in or ruling out Category 1 effects (serious eye damage/corrosion). By using a dual-assay/approach system – the combination of the EIT and BCOP test – we have determined, with a high degree of accuracy, GHS Acute Eye Hazard Category 2 chemicals that cause reversible irritation to the eye.
A Double Elimination occurs when a BCOP test rules out GHS Category 1 (severe irritation/corrosion) and the EIT rules out GHS No Category (non-irritant), analysis of the combined results concludes the only other possible designation – Category 2 (irritant). The classification defaults to Category 2A for labeling and handling purposes because at present differentiation between category 2A and 2B cannot be made. Using this approach, we correctly identified 87% of the Category 2A/B chemicals as Category 2 after testing 42 chemicals (nine, 15, and 18 in Category 1, 2A/B, and No Category, respectively).
As a result of this analysis, we are encouraged by the potential of the BCOP-EIT dual-assay system, coupled with WoE evaluation, to correctly assign unknowns into GHS Category 1, Category 2A, and No Category Acute Eye Hazards without the use of less predictive and more expensive in vitro tests. Moreover, in keeping with the OECD’s recommendations, we predict that such a system would greatly reduce reliance on the Draize Rabbit Eye Tests.
MB Research offers Quality In Vitro Testing for Cosmetics.
Visit MB Research @ Booth#1549 for the latest on In Vitro Cosmetic Safety Testing
New York Society of Cosmetic Chemists Supplier’s Day
May 15-16, 2018, Jacob Javits Convention Center, New York, NY
MB Research Labs is a leader in the use and development of in vitro and alternative toxicology test methods. We specialize in protocols for Eye and Skin Irritation, Corrosivity, Skin Sensitization, Genotoxicity, and Phototoxicity.
MB Research Labs is honored to be attending the 10th World Congress on Alternatives and Animal Use in the Life Sciences. We will be presenting several posters focusing on the use and development of in vitro and alternative toxicology methods that are used in our laboratories.
Scientific Posters presented by MB Research Labs on the Use and Development of In Vitro and Alternative Toxicology Test Methods