Sensitivities Testing In Guinea Pigs Alternatives For Medical Devices & Implants
What is sensitization testing?
Skin sensitization tests on animals evaluate the ability of leachables and unknown antigens to cause hypersensitivity. These biocompatibility sensitization tests and dermal sensitization 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. The most common test is a Magnusson & Kligman sensitivities tests in guinea pigs.
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 can 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 (e.g., 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?
A summary of the top nine sensitization testing methods is detailed in Table 1. Most of these tests utilize guinea pigs for sensitivity evaluations. However, the gold standard for sensitization testing is the Magnusson & Kligman Guinea Pig Maximization Test (GPMT). All other tests are alternatives to the GPMT gold standard. Although, standard Beuhler testing is a close second to GPMT. Some sensitivities testing in guinea pigs 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 guinea pigs alternatives are used instead of Magnusson & Kligman Maximization?
Standard Buehler Test
Preliminary Testing (Test Article Concentration Determination)
See our article on the Magnusson & Kligman test for details on determining the nontoxic dose of the test extract. While alternatives to Magnusson & Kligman test are give below, Magnusson & Kligman tests are still considered a gold standard. Two or three animals are used to determine the nontoxic dosing amount. The concentration that causes only mild to moderate irritation should be used for the induction phase. The highest test extract concentration that does not cause redness is used for the challenge phase.
Induction Phase
Apply patches containing the test extract or diluent/vehicle to one flank of each animal. Control animals receive patches with the diluent/vehicle, and test animals receive patches with the test extract. Patches for dermal sensitization testing are held in place for 6-hours before removal. Patch application is repeated three times a week for both test and control animals on the same site for three consecutive weeks.
Challenge Phase
The challenge phase for biocompatibility sensitization testing occurs 14-days after the last application of the induction phase. For the challenge, apply patches containing the test extract or diluent/vehicle to the untested areas of the test and control animals, respectively. Keep the patches in place for 6-hours before removal.
Observations & Results Interpretation
At 24- and 48-hours after patch removal, grade the application sites (see article on Magnusson & Kligman for grading options). All signs of reactivity, especially swelling and redness, are recorded. The response of the test group versus the control group can be compared statistically (e.g., Mann-Whitney U test can be used for the comparison).
Draize Test
The Draize test was the first sensitization test accepted by regulatory agencies. The Draize test uses intradermal injections.
Induction Phase
The Draize test uses test extracts for solid materials. First, a side of twenty guinea pigs is shaved and given an intradermal injection for dermal sensitization testing with the test extract. The animals receive a single injection of the test article once a day for twenty days.
Challenge Phase
The challenge phase starts 2-weeks after the final induction-phase injection. The untreated side of all twenty guinea pigs is then injected with the test article for biocompatibility sensitization testing. Twenty untreated guinea pigs (controls) also receive a single injection of the test article.
Observations & Results Interpretation
The test sites of control and test animals are assessed for redness 24- and 48-hours after the challenge injection. The reaction in the test animals is compared to the reaction in control animals statistically. A greater response in the test animals indicates sensitization.
Open Epicutaneous Test
The goal is to determine the dose required to induce sensitization by simulating human usage via topical application.
Preliminary Testing
For epicutaneous testing, the test article must be in extract form. A series of test extract concentrations are applied to areas of skin on the anterior side flank of six or eight guinea pigs. The test sites are assessed for redness 24-hours after extract application. The highest extract concentration that does not irritate is the maximum nonirritant concentration. The test site with the lowest concentration causing redness in twenty-five percent of the animals is the minimum irritant concentration.
Induction Phase
The test article (or control vehicle) is applied to the skin of six to eight guinea pigs daily for 3-weeks. Increasing concentrations up to the minimum irritant concentration for dermal sensitization testing are used for daily applications. Unless irritation develops, the test article is applied to the same sites for each application. Control animals receive the same series of treatments using the vehicle instead of the test article.
Challenge Phase
For the challenge, the untreated side of each animal is exposed to increasing concentrations of test extract 24- to 72-hours after the last induction phase treatment for biocompatibility sensitization testing. The increasing concentrations are a set of seven and move from minimum irritant concentration to the maximum nonirritant concentration.
Observations & Results Interpretation
Animal test sites are evaluated at 24-, 48-, and 72-hours post-treatment. The test group animals that show inflammatory responses at concentrations lower than the maximum nonirritating concentration in the controls are considered sensitized.
Freund’s Complete Adjuvant Test
This test uses intradermal injections with Freund’s complete adjuvant (FCA).
Preliminary Testing
For complete adjuvant testing, the test article must be in solution-form (extract form). The test’s minimum irritating and maximum nonirritating concentrations can be determined the same way as concentrations for epicutaneous testing.
Induction Phase
Two groups of ten to twenty guinea pigs are used. The test group animals are injected intradermally across the shoulders with the test extract in FCA and water for dermal sensitization testing. Control animals receive injections with FCA and water alone. These injections are repeated every 4 days until three injections (total) have been administered to each animal.
Challenge Phase
The challenge phase begins 2-weeks after the last induction-phase injection. For the challenge, the test extract is applied topically in four concentrations to the shaved side of each guinea pig for biocompatibility sensitization testing. The four concentrations are a minimum irritating dose, a maximum nonirritating dose, and two concentrations lower than the minimum irritating dose.
Observations & Results Interpretation
The test sites are examined for redness at 24-, 48- and 72-hours after topical challenge. The test group animals that display redness at concentrations lower than the minimum nonirritating concentration in the control animals are considered sensitized.
Optimization Test
Optimization tests have similarities to the Draize test. However, the optimization test uses adjuvant for some injections and both intradermal and topical treatments.
Induction Phase
The test article needs to be in extract form for optimization testing. This study uses the test and control group guinea pigs (twenty each). Ten total intradermal injections are administered to each animal. Test animals receive a saline and test extract injection on day 1 into a shaved flank and shaved dorsal skin for dermal sensitization testing. The 2nd and 4th days after the initial injection, one intradermal injection of the test extract in saline is administered and spread amongst eight new dorsal sites. Every other day (beginning at 2-weeks), the test extract (with saline and FCA) is injected intradermally into ten shoulder sites. Injections every other day occur until the remaining seven intradermal injections are complete. The control animals receive the same injection sequence of injections without test extract (i.e., saline or saline/FCA injections).
Challenge Phase
On the 35th day after the first injection, the test group animals are challenged topically with the test extract for biocompatibility sensitization testing. The control group animals received saline injections instead on the same day. On the 45th day after the first injection, a second topical challenge is applied to the test group animals at a nonirritating concentration to untreated skin. The second topical challenge is removed after 24-hours.
Observations & Results Interpretation
Twenty-four hours after each injection during the first seven days, the thickness of the skin over the injection sites for each animal should be measured using a caliper. Additionally, the sizes of the two largest cross-diameters of each skin rash are recorded. The reaction volumes are calculated by multiplying the skin thickness by the products of the two cross-diameters. The mean reaction volume for week 1 is calculated for each animal. Next, challenge reaction volumes are calculated for each animal following the injections at day 35. An animal with a challenge reaction volume greater than its mean reaction volume is considered sensitized. After the patch testing challenge, the test sites are graded based on erythema and edema formation. The number of sensitized animals is compared statistically with the control animals. This comparison is completed for the injection and patch test results using a Fisher exact test. The comparison results can classify a test article as a strong sensitizer, moderate sensitizer, weak sensitizer, or non-sensitizer.
Split Adjuvant Test
This test uses both FCA and skin damage through topical application of the test article.
Preliminary Testing
The test article can be either in solid or liquid form for split adjuvant testing.
Induction Phase
Ten to twenty guinea pigs are used for both test and control groups. An area of skin behind the scapula is shaved to glistening and treated with dry ice for ten seconds. Next, the test article (liquids or solid) is placed within an opening in an applied dressing on top of the iced skin for dermal sensitization testing. The test article is secured with paper and adhesive tape. Two days later, the test article will be removed and reapplied to the same site. An additional two days later, the old test article is removed, two injections of FCA are administered into the edges of the test site, and the test article is reapplied once more. On the 7th day, the test article is reapplied once more and removed two days later (on day 9).
Challenge Phase
On the 22nd day following induction treatment, half a milliliter of test material (or the solid article) is applied to a shaved midback area of each control and test animal for biocompatibility sensitization testing. Items can be held in place with an elastic bandage and adhesive tape. The challenge materials are removed after 24-hours of exposure.
Observations & Results Interpretation
Test sites are assessed for redness and swelling 24-, 48-, and 72-hours after challenge treatment removal. A grading system based on erythema and edema formation is used for test site assessment.
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. This article details sensitivities testing in guinea pigs alternatives to the Magnusson & Kligman sensitization studies. The available alternative guinea pig studies for sensitization testing are the standard Buehler test, the open epicutaneous test, the Draize test, Freund’s complete adjuvant test, the optimization test, and the split adjuvant 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>).
