Sensitivities Testing In Mice Alternatives For Medical Devices & Implants
What is sensitization testing?
Sensitization tests are in vivo tests that evaluate the ability of leachables and unknown antigens to cause hypersensitivity. The tests are designed to determine if a patient will develop a reaction with repeated exposure to a medical device. A few important factors to consider when selecting a sensitivity test include the type and extent of contact with the body, the chemical composition of the product or materials, the product’s manufacturing process, and the product’s sterilization process.
What is needed for sensitization testing?
Sensitization testing will require a certified animal care facility, trained technicians, animals, and an injectable formulation of the cosmetic, drug, or device extract undergoing testing. Most companies that provide in-vivo testing services will be able to cover all facility, animal, staffing, and documentation needs for sensitivities testing. However, obtaining and filling an injectable formulation for your cosmetic, drug, or medical device extracts will require additional outsourcing. Companies, like MycoScience, can support cost-effective syringe filling and vial filling for sensitization testing and other regulatory tests in animals.
What tests are used for sensitization testing?
Table 1 summarizes the top nine methods used for regulatory sensitization testing. Most of these tests utilize guinea pigs for sensitivity evaluations. The Magnusson & Kligman Guinea Pig Maximization Test (GPMT) is the gold standard for sensitization testing. All other tests are alternatives to the GPMT gold standard. Although, Standard Beuhler testing is a close second to GPMT. Some sensitivities testing in mice alternatives evaluate implanted solid articles, while others only evaluate extracts of solid articles. In some medical device and implant applications, toxicology requirements may be satisfied with data available from previously marketed products.
What sensitivities testing in mice alternatives are used instead of Magnusson & Kligman Maximization?
Local Lymph Node Test
Mice exposed to sensitizers have an overgrowth of T cells within their lymph nodes. Local lymph node tests do not have a challenge phase and require the use of radioisotopes.
Animals
Four groups of four mice (at least) are used. These mice can be all male or all female CBA/ca mice between the ages of 8- to 12-weeks.
Preliminary Testing
Solid test articles (products) could be used, but extracts of solid materials work best for local lymph node testing. USP 88, which covers in-vivo cytotoxicity testing, provides a process for preparing test extracts. A nontoxic concentration of the test extract should be used. A preliminary test for toxicity may be needed to establish a nontoxic dose.
Induction Phase
Twenty-five microliters of the nontoxic concentration, or diluent (negative control), should be applied to the outside of each ear (pinna) for three consecutive days. Five days after the first treatment, the animals are given a tail vein injection with phosphate-buffered saline containing 3H-methyl thymidine. Five hours after the injection, the animals are euthanized and the auricular lymph nodes removed from each test and control animal. A single-cell suspension is prepared from the nodes of all animals within a given group (test or control). Each control and test cell suspension are placed in scintillation vials with ten milliliters of scintillation fluid. Then the disintegrations per minute (dpm) are counted with a β-counter.
Observations & Results Interpretation
The ratio of dpm for each test group is compared to the dpm for the control group. If the dmp ratio equals or is greater than three for any test group, the test article (product) is considered sensitizing at that concentration.
Mouse Ear Swelling Test
Advantages to using mice versus guinea pigs for sensitization methods include less time, lower cost, and both topical exposures and injections can be used.
Animals
Female, 6- to 8-week-old CF-1, Balb/c, or Swiss mice can be used. The ear thickness of each animal is measured and recorded before sensitization testing.
Preliminary Testing
The test article needs to be suitable for injection (e.g., extract form). The minimally irritating and maximally nonirritating concentrations of test extracts should be determined. Dosing concentrations can be achieved by using four groups of two mice and examining the effects of at least four concentrations of the test article.
Induction Phase
The mouse abdomens are shaved and tape-stripped with surgical tape for injection preparation. An intradermal injection of 0.05 milliliters of Freund’s Complete Adjuvant (FCA) is subdivided into two injection sites within the abdomen of each mouse. Then test animals are given injections of test extract (using the minimally irritating concentration), and controls are given vehicle (diluent) to the center of the shaved area. Tape stripping and application of test extract (or vehicle) is repeated each day for three consecutive days.
Challenge Phase
The Challenge phase occurs 7-days after the final topical application. Twenty microliters of the highest nonirritating test extract concentration are applied topically to one ear. The opposite ear receives ten microliters of diluent (vehicle control). Both test and control animals receive the twenty and ten microliter doses.
Observations & Results Interpretation
The ear thickness of both ears of each animal is measured with calipers and recorded 24- and 48-hours after the challenge. A sensitized animal will have a 20% increase in ear thickness for the test extract-treated ear compared to its opposite control ear. The test article-treated ears of control animals should not be more than 10% thicker than the opposite ears for a valid test. If the control animal ears do not meet the requirements, the mouse ear swelling test should be repeated using lower concentrations of test extract.
Vitamin A Enhancement Test
This test is similar to the Mouse Ear Swelling Test in that test extracts are applied topically to the abdomen and challenges applied to the ears. The principal difference for vitamin A enhancement is that the mice receive mouse feed supplemented with vitamin A acetate. Vitamin A acetate increases the immune system’s reactivity and increases the potential for sensitization.
Animals
Male, 3- to 4-week old Balb/c mice are given a diet supplemented with vitamin A acetate for at least 3-weeks. Thus, the mice at the time of the sensitization study should be 7- to 10- weeks old. The ear thickness of all animals is measured and recorded before sensitization testing.
Preliminary Testing
An extract of solid test articles should be used if liquid or topical formulations are unavailable. The maximally nonirritating dose and minimally irritating concentration of the extract or product formulation can be determined as described in the mouse ear swelling test.
Induction Phase
Ten mouse abdomens per group are shaved. Then one hundred microliters of test extract (at the minimally irritating concentration) are applied to the prepared abdominal areas on days 0, 2, 4, 7, and 11. Control animals are given one hundred microliters of diluent (vehicle control) on the same schedule.
Challenge Phase
The Challenge phase occurs 4-days after the final application of the induction phase. For the challenge, twenty-five microliters of the test article are applied to both ears of the test and control animals. The maximally nonirritating concentration of test extract is used for ear application.
Observations & Results Interpretation
The ear thickness of both ears of each animal is measured with calipers and recorded 24- and 48-hours after the challenge. The percent increase in ear thickness is calculated for each ear by subtracting the pretreatment measurement from the post-treatment measurement, dividing the result by the pretreatment measurement, and multiplying by a hundred. Test and control groups are statistically (e.g., the Mann-Whitney U test at p < 0.01). There is sensitization if an increase in ear thickness for an animal from the test group is at least 50% greater than the largest increase of a control animal.
Summary
Overall, sensitization tests evaluate the ability of leachables and unknown antigens to cause hypersensitivity. Sensitization tests are designed to determine if a patient will develop a reaction with repeated exposure to a medical device or implant. All sensitization tests are in-vivo animal studies. Most sensitization studies (including the Magnusson & Kligman gold standard) utilize guinea pigs. However, this article details the sensitivities testing in mice alternatives. Studies in mice offer time and cost benefits compared to guinea pig studies. The available mouse studies for sensitization testing are the local lymph node assay, the mouse ear swelling test, and the vitamin A enhancement test. All in all, ensure you choose a contract testing organization that can support you with appropriate toxicity testing for your unique medical device or product needs.
MycoScience is a contract manufacturing organization specializing in sterile syringe and vial filling for animal studies & cosmetics. In addition, MycoScience offers testing services, including Preservative Efficacy Testing, Cytotoxicity Testing, Bioburden Testing, Cleaning Validations, Microbial Aerosol Challenge Testing, Accelerated Aging, Microbiology Testing, EO Residual Testing, Bacterial Endotoxin Testing, Package Integrity Testing, Sterilization Validations & Environmental Monitoring services medical devices and allied industries. MycoScience is an ISO 13485 certified facility.
References
United States Pharmacopeial Convention. <1184> Sensitization Testing. Rockville, MD, USA. 2021. (USPC <1184>).
