The LLNA-BrdU ELISA is conducted at MB Research Labs and is based upon the OECD 442B Testing Guideline. The LLNA is an alternative toxicology assay for dermal sensitization using CBA/J mice. The LLNA is an alternative to guinea pig studies that require more animals and long test durations.
To determine the sensitizing potential of topically applied test material utilizing the LLNA, and measurement of lymphocyte proliferation by BrdU incorporation detected by ELISA.
Irritation Screen Test:
An initial Irritation Screen is performed using 3-5 highest achievable concentrations. The screen consists of 2 animals per concentration that will be dosed in an identical manner to that of the main test. Ear measurements will be taken prior to dosing on Study Days 1 and 3, and prior to euthanasia on Study Day 6. Measurements taken on Study Days 3 and 6 will be compared to pre- dosing measurements. If there is no effect, or an increase of <25% in ear thickness, the same concentrations will be assayed in definitive test.
However, if significant ear swelling (ES) is present (>25% over control), then an extended screen (Quantitative Irritation Test; QIT) is recommended. The QIT is similar to the Irritation Screen except 4-6 concentrations will be assayed to determine the highest non-irritating concentration.
Definitive Sensitization Test:
The main test will be conducted by topically applying test material to the dorsum of the ears at the 3-5 highest achievable concentrations (100 [liquid only], 50, 25, 10, 5, 2.5, 1, 0.5, etc.), which do not cause severe irritation or overt systemic effects. Ear measurements will be taken on Study Days 1 (prior to first dosing), 3 (approximately 48 hours after initial dosing) and 6 (prior to euthanasia). Animals will be injected with BrdU on Study Day 5, approximately 24 hours prior to euthanasia. Auricular lymph nodes will be collected on Study Day 6, a single-cell suspension will be created and lymph node cells (LNC) will be assayed for BrdU incorporation via ELISA.
A Positive Response is when a Stimulation Index (SI) is greater than or equal to 1.6 (relative to that of control lymph nodes).
A “Borderline Positive Response” is defined as an SI from 1.6 to 1.9; additional information and testing may be needed.
Over 45 Years of Toxicology Testing – Chemicals, Pharmaceuticals, Cosmetics, Household Products, Personal Care Products
MB Research Laboratories, a GLP contract research Toxicology laboratory, has been conducting acute, subchronic / chronic toxicological and pharmacological studies since 1972. MB Research provides industry-leading general toxicology services for the pharmaceutical, biotechnology, medical device and agrochemical industries and government agencies. Routine and non-traditional toxicology studies are performed, custom testing services to meet specific testing needs is available.
Study Directors and technicians strive to consistently deliver exemplary results to support both Good Laboratory Practices(GLP) and non-GLP Toxicology studies. With a wide range of in vivo and in vitro test protocols available, MB Research Labs offers comprehensive Toxicology Testing capabilities to address your unique needs and goals. With expertise in many areas of in vivo and in vitro testing, research, and safety evaluations, MB Research serves the testing needs of both government and industry.
Complete support services including consultation, protocol development, quality assurance, analytical chemistry and archive facilities are integral components of studies performed at MB. Our technical and support procedures are in full compliance with OECD, FDA, and EPA.
MB Research Labs is a leader in the US for development and validation of in vitro alternative test methods that reduce/replace the need for using live animals in product safety testing of consumer products, chemicals, pharmaceuticals and cosmetics.
MB Research Labs is a leading in vitro toxicology laboratory that offers toxicological testing services to the cosmetic, chemical and pharmaceutical industries.
One of the more important assays offered in the 3T3 Neutral Red Uptake Phototoxicity (3T3 NRU PT) Assay. The 3T3NRUPT is used to screen test materials for the potential of causing photoirritation after exposure to the material and sunlight.
The 3T3 NRU PT was developed and validated in a joint EU/COLIPA project from 1992-1997. In 1996, the 3T3 NRU PT was recommended by OECD as an in vitro approach for the assessment of chemicals phototoxicity potential. In April 2004, the 3T3 NRU PT protocol was finalized and adopted as the OECD 432 protocol, In Vitro 3T3 NRU phototoxicity test.
The 3T3 NRU PT can be utilized to identify the phototoxic effect of a test substance induced by the combination of test substance and light and is based on the comparison of the cytotoxic effect of a test substance when tested after the exposure and in the absence of exposure to a non-cytotoxic dose of UVA/vis light. Cytotoxicity is expressed as a concentration-dependent reduction of the uptake of the vital dye – Neutral Red.
Substances that are phototoxic in vivo after systemic application and distribution to the skin, as well as compounds that could act as phototoxicants after topical application to the skin can be identified by the test. The reliability and relevance of the 3T3 NRU PT have been evaluated and has been shown to be predictive when compared with acute phototoxicity effects in vivo in animals and humans.
Cosmetics are required to be safe when consumers use them according to directions in the labeling, or in the customary or expected way.
To ensure the safety of a cosmetic product, manufacturers may conduct cosmetic safety testing.
Cosmetic products and ingredients (except for color additives) do not require FDA approval before being released on the market.
The FDA does not have a list of mandatory tests required for any particular cosmetic product or ingredient. Action can be taken against the manufacturer of a cosmetic on the market if we have reliable
information to show that a cosmetic does not meet the legal requirement for safety.
Color additives are treated differently. Unlike other cosmetic ingredients, color additives (other than coal-tar hair dyes and related materials)
must be approved by the FDA for the specific intended use before they are permitted in cosmetic products.
Your Cosmetic Safety Testing Partner
MB Research has been conducting product safety assessments for the cosmetics, personal care, chemical and pharmaceutical industries for over 40 years and is often sought out by Cosmetics Industry Leaders to ensure the safety their ingredients and aid in the rapid development of products.
We are a leader in the use and development of In Vitro and Alternative Toxicology Tests and continue to introduce new testing methods regularly.
Cosmetic, Beauty and Healthcare product industries are subject to global regulatory requirements such as the EU Cosmetics Directive (76/768/EEC) in response to growing concern over the safety of cosmetic products and their ingredients.
As Regulators begin to initiate laws and regulations that require the reduction of using live animals for safety studies, more and more companies are looking for valid substitutes that are economical and thorough.
Since the late 1980s, MB Research has been a pioneer in the used and development of in vitro & alternative test methods, reducing the use of live animals in toxicological research.
What are the 3 Rs?
The principle of the 3Rs was first mentioned by William Russell and Rex Burch in 1959. In their book “The Principles of Humane Experimental Technique”, they classified humane techniques under the headings Refinement, Reduction, and Replacement, commonly known now as the 3Rs.
MB Research Laboratories prides itself on pursuing in vitro and alternative toxicological methods that Reduce, Refine and Replace the use of animals in laboratory research and has worked with many leaders in this field to advance the development, validation and regulatory acceptance of in vitro and alternative tests with a goal on replacing laboratory animals in toxicological research.
Hazardous shipping labels can be complex. There are kits that can be purchased that can satisfy regulatory requirements, but getting a kit alone does not provide they well-being you get when the test is performed by an experienced laboratory with a excellent quality assurance follow up and an acutal report.
Weigh of Evidence (WoE) testing with in vitro test methods is now being accepted by European chemical agencies to satisfy REACH requirements. This is especially helpful when specific regulatory guidelines require complex safety testing but no validated in vitro test or alternative assay exists.
WoE must be accompanied with substantial background data to aid in acceptance of our provided safety data. GLPs are a must.
MB Research Labs offers many in vitro test methods that are directly in-line with some regulatory testing requirements as well as novel in vitro test that have not been accepted yet but can still provide a strong case for safety assessments.