This phase requires three pairs of injections administered intradermally, with the test and control injection of each pair on opposite sides intrascapularly. Each injection should contain 0.1 mL, with injection pairs 1 and 2 administered nearer to the head, and injection pair 3 administered slightly farther towards the tail. The pairs are nominally within an area of 8 cm2. The pairs of injections consist of the following:

Seven days (±1 day) after completion of the Intradermal Injection Induction Phase, administer the test sample by topical application to the intrascapular region of each animal. For both test and control animals, if the Test Article Preparation does not cause skin irritation, apply 10% sodium lauryl sulfate in petrolatum approximately 24 hours before the start of the Topical Application Induction Phase to induce a local irritation.

Test animals should have 2- × 4-cm pieces of filter paper or absorbent gauze fully loaded with the Test Article Preparation (prepared within 24 hours of use) using the concentration selected in Determination of Test Article Concentration applied to each injection site. The filter paper or absorbent gauze should be secured to the animals using occlusive dressings. Control animals receive the same treatment, except that the appropriate solvent/vehicle is used instead of the test article.

Remove the dressings and patches approximately 48 hours after application.

This phase should occur 14 ± 1 days after the Topical Application Induction Phase. Hair should be removed from the test application sites. Filter paper patches or chambers are soaked with a freshly prepared Test Article Preparation in the concentration specified in Determination of Test Article Concentration. This is done for all test and control animals. The patches or chambers are secured with an occlusive dressing and removed after 24 ± 2 hours.

Apply 0.4 mL of the Test Article Preparation in an appropriate solvent/vehicle at the dose identified in Determination of Test Article Concentration. Use patches similar to those used in Determination of Test Article Concentration. The patches should be applied to one flank (hair clipped off) and held in place occlusively for 6 hours. The animals may need to be restrained to ensure occlusion. Patches and any visible residues should be removed after 6 hours. Control animals also receive patches, but these contain only the appropriate solvent/vehicle. This process should be repeated three times a week for both test and control animals on the same site for three consecutive weeks (weekly intervals are used in the modified Buehler Test).

At 22 ± 2 hours after removal of the patches, the application sites should have the animal's fur removed via clipping or depilation. After approximately 2 more hours, grade the sites (Tables 4, 5 or 6 may be employed). All signs of reactivity should be recorded, with particular attention paid to signs of erythema and edema. Repeat the grading once again after 24 to 48 hours more have elapsed. The response of the test group versus the control group can be compared statistically. (The Mann-Whitney U test can be used for the comparison.)

The results should be submitted for a statistical analysis (e.g., chi-square contingency table) to determine if the differences in scores between treated and control animals are significant.

See Interpretation in the Magnusson & Kligman Guinea Pig Maximization Test (GPMT).

See Rechallenge in the Magnusson & Kligman Guinea Pig Maximization Test (GPMT).

This test requires that the test article be in the form of a solution that may be directly applied to the animal's skin. Therefore, extracts of the material would need to be made. See Biological Reactivity Tests, In Vivo 88 for information on the preparation procedure.

One flank of each of 20 guinea pigs is shaved, then 0.05 mL of a 0.1% solution of test article is injected into the anterior flank. The next day, and then every other day thereafter up to day 20, 0.1 mL of the test article is injected into a new site on the same flank.

This phase begins 2 weeks after the final injection of the Induction Phase. The untreated flank is shaved, then 0.05 mL of test article is injected into each of the 20 guinea pigs. Twenty previously untreated animals serve as the controls, and receive injections of the test article as well.

The test sites of all control and test animals are evaluated for erythema at 24 and 48 hours after the challenge injections. The degree of reaction in test animals is compared to the reaction in control animals. A larger and/or more intense response by the test animals versus the control animals is indicative of sensitization.

This test requires that the test article be in the form of a solution that may be directly applied to the animal's skin. Therefore, extracts of the material need to be made. See Biological Reactivity Tests, In Vivo 88 for information on the preparation procedure.

A series of concentrations of test article is applied to 2 cm2 areas of skin on the anterior flank of 6 to 8 guinea pigs (0.025 mL per application). The test sites should be examined for erythema 24 hours after test article administration. The highest concentration that does not cause irritation (maximum nonirritant concentration) and the lowest concentration causing erythema in approximately 25% of the animals (minimum irritant concentration) are determined.

The test article (or control vehicle) is applied to 8 cm2 areas of the flank skin of 6 to 8 guinea pigs daily for 3 weeks, or five times a week for 4 weeks. The amount per application is 0.01 mL. A set of increasing concentrations is again employed, ranging from the minimum irritant concentration using a stepwise progression. The test article should be applied to the same sites each time, unless irritation develops, in which case a new site on the same flank should be used. Control animals receive the same series of treatments using the vehicle instead of the test article.

Each animal is challenged on the untreated flank 24 to 72 hours after the last Induction Phase treatment using 0.025 mL applied to 2 cm2 areas. A set of increasing concentrations is used, from minimum irritant concentration to the maximum nonirritant concentration, and five lower concentrations are also used.

The test sites are evaluated at 24, 48, and 72 hours post-treatment. The maximum concentration that does not cause irritation in the control group is determined. Animals from the test groups that develop inflammatory responses at concentrations lower than the maximum nonirritating concentration in the controls should be considered to be sensitized.

Because this test uses intradermal injections, extracts of the test material need to be made in order to use this procedure. See Biological Reactivity Tests, In Vivo 88 for information on the extraction procedure.

The minimum irritating and the maximum nonirritating concentrations are determined in the same manner as for Preliminary Testing in the Open Epicutaneous Test.

The test area consists of six 2 cm2 areas across the shoulders of the guinea pigs. Two groups of 10 to 20 guinea pigs each should be used. The test group animals are injected intradermally with 0.1 mL of a 5% solution of the test article extract in FCA/water. Control animals receive injections with FCA/water without the test article. These injections are repeated every 4 days until a total of three injections have been given.

This phase should begin 2 weeks after the last injection of the Induction Phase. Topical applications of 0.025 mL of test article at the minimum irritating and the maximum nonirritating concentrations, plus two lower concentrations, are administered to 2 cm2 areas of the shaved flank. The test sites should remain uncovered.

The test sites are examined for the presence of erythema 24, 48 and 72 hours after the topical applications. The minimum nonirritating concentration in the control animals should be determined. Those test animals that display erythema at concentrations lower than the minimum nonirritating concentration in the control animals should be considered to be sensitized.

As with other test procedures that incorporate intradermal injections, the test article needs to be in a form suitable for injection. See Biological Reactivity Tests, In Vivo 88 for information on the extraction procedure.

Twenty test and 20 control guinea pigs are used. A total of 10 intradermal injections should be given to each animal. Test animals receive 0.1 mL of a mixture of 0.1% test article and 0.9% saline (50:50) on day 1, with one injection into a shaved flank, and another into a portion of shaved dorsal skin. Two and 4 days later, one intradermal injection of the test article in saline is given to eight new dorsal sites. Every other day during weeks 2 and 3, the test article is injected intradermally into 10 sites over the shoulders in a 50:50 mixture of saline and FCA. The same sequence of injections is given to the 20 control animals, except that no test article is included with the saline or saline/FCA injections.

Thirty-five days after the first injection, the animals are challenged topically with 0.1 mL of the 0.1% solution of test article in saline (for test animals). The control animals receive saline injections only. At 45 days after the first injection, a second topical challenge is given. A nonirritating concentration of test article (0.05 mL) is applied topically to a 1 cm2 area of untreated skin. This site should then be covered with a 2 cm2 piece of filter paper, after which an occlusive dressing should be applied. The patch should be removed after 24 hours.

Twenty-four hours after each injection during week 1, the thickness of a fold of skin over the injection sites for each animal should be measured using a caliper (mm), and the two largest cross-diameters of each erythematous reaction should be recorded (mm). The reaction volumes are calculated by multiplying the fold thickness by the products of the two cross-diameters (expressed as µL). The mean reaction (+1 SD) volume during week 1 should be calculated for each animal.

Challenge reaction volumes are calculated for each animal following the injections at day 35. If an animal develops a challenge reaction volume greater than its mean reaction volume + 1 SD, it should be considered sensitized.

Following the patch testing challenge, the test sites are evaluated for erythema and edema. Evaluations should be made using Table 5.

The number of positive animals should be compared statistically with the pseudopositive control animals. This should be done for both intradermal injection results and patch testing results. The Fisher exact test may be used.

The results from the intradermal injections and the patch testing, following separate statistical analysis, may be combined and evaluated using Table 7 in order to classify a test article as a strong, moderate, or weak sensitizer; or not a sensitizer.

Table 7. Classification Scheme for Test Articles Based on the Optimization Test

Because this test employs topical test article applications, the article can be either in solid or liquid form. If extracts are to be made, see chapter Biological Reactivity Tests, In Vivo 88 for extraction procedures.

Ten to 20 guinea pigs are used for both test and control groups. An area of back skin immediately behind the scapulas should be shaved to the extent that the skin becomes glistening. The shaved areas should then be treated with dry ice for 5 to 10 seconds. A dressing made of loose mesh gauze with stretch adhesive and a 2- × 2-cm opening should be placed over the treated area, then secured with adhesive tape. The test article (0.2 mL of viscous materials, 0.1 mL of liquids, or solid material) is placed within the opening in the dressing on top of the treated skin. Two layers of #2 filter paper should be placed over the test article, then backed by occlusive tape. Then the filter paper/occlusive backed material should be secured to the surrounding dressing with adhesive tape. After 2 days have passed, the filter paper should be lifted from the test sites, and the test article reapplied on the same site. The filter paper and backing should be secured once again. After 2 more days, the filter paper should be lifted and two injections of 0.075 mL of FCA should be administered into the edges of the test site. Then the test material is once again applied, and the filter paper/backing resecured. The test article should be reapplied once more on day 7 and the filter paper/backing resealed. On day 9, the filter paper and all associated dressing material should be removed.

On day 22 following the induction treatment, 0.5 mL of test material (or the solid article) should be applied to a 2-× 2-cm area of shaved midback. The test sites should be covered by filter paper and backed by adhesive tape. This should be held in place with an elastic bandage secured with adhesive tape. Control animals receive the same challenge phase treatment. The preparation should be removed after 24 hours.

Twenty-four, 48, and 72 hours after the removal of the challenge phase preparation, the test sites should be evaluated for erythema and edema. The grading scheme of Table 5 could be employed.

Female, 6- to 8-week old CF-1, Balb/c, or Swiss mice should be used. They may be group housed in direct bedding cages. Acclimatization should be for at least 5 to 7 days. Food (appropriate mouse feed) and water should be available ad libitum. No animals with damaged pinnae should be used in the study. The thickness of both ears of each animal should be measured and recorded at this time.

As with other test procedures that incorporate intradermal injections, the test article needs to be in a form suitable for injection. See Biological Reactivity Tests, In Vivo 88 for information on the extraction procedure.

The minimally irritating and maximally nonirritating concentrations of test article for this procedure should be determined. This is done by using four groups of two mice and examining the effects of at least four concentrations of test article.

The abdomens of the animals should be shaved, then tape-stripped using a surgical adhesive tape until the test area is glistening. A single injection of 0.05 mL of FCA is subdivided into two injection sites administered intradermally within the shaved/stripped area, but along the borders. After the adjuvant injections, 100 µL of test article (using the minimally irritating concentration) or vehicle (controls) is applied to the center of the shaved test areas. After the test areas dry, the mice should be returned to their cages. The tape stripping and application of test article (but not FCA) is repeated each day for the next 3 days.

This phase should occur 7 days after the final topical induction application. The test article (highest nonirritating concentration) should be applied topically (20 µL) to one ear, while the opposite ear receives 10 µL of vehicle alone. This should be done for both test and control animals.

The thickness of both ears of each animal should be recorded after 24 and 48 hours postchallenge. The measurements should be made with a caliper (a spring-loaded caliper is preferable). A sensitized animal is one in which the test article-treated ear is at least 20% thicker than its opposite ear. For the test to be valid, the test article-treated ears of control animals should not be more than 10% thicker than the opposite ears. If the control animal ears do not meet the requirements, the test should be repeated using lower concentrations.

Four groups of four mice at least, male or female CBA/ca mice (only one sex in a given test) between the ages of 8 to 12 weeks should be used.

Although in theory one could apply a solid test article to the dorsal surface of the ear of a mouse, in practice an extract of such an article should be used. See Biological Reactivity Tests, In Vivo 88 for information on the extraction procedure.

A nontoxic concentration of test article should be used. If not already established, a preliminary test for overt toxicity may be required to establish a suitable dose.

Twenty-five µL of the appropriate test article concentration, or vehicle (controls), should be applied to the dorsal surface of each pinna for 3 consecutive days. Five days after the first treatment, the animals should be injected, via the tail vein, with 2.5 mL of phosphate buffered saline containing 20 µCi of 3H-methyl thymidine. Five hours after the isotopic injection, the animals should be euthanized. The draining auricular lymph nodes should be removed from each animal of each test and control group. The nodes from all animals within a given group should be combined, such that a single cell suspension can be made from each group of animals. The cell suspension can be made by passing the nodes through a 200-mesh stainless steel gauze using a syringe plunger. The cells should then be centrifuged at 190 × g for 10 minutes, resuspended in 3 mL of 5% trichloroacetic acid (TCA), and held overnight at 4.

The resulting precipitate should be recovered by centrifugation, and the pelleted precipitate should be resuspended in 1 mL of 5% TCA. The suspension should then be placed in scintillation vials with 10 mL of scintillation fluid, and the disintegrations/minute (dpm) counted with a -counter.

The ratio of dpm for each test group should be compared to the dpm for the control group. If the ratio equals or exceeds 3 for any test group, the concentration of test article used with that group may be considered to be sensitizing.

Male, 3- to 4-week old Balb/c mice should be maintained on a diet supplemented with vitamin A acetate. The diet may be prepared by mixing each kg of feed with 0.477 g of gelatinized vitamin A acetate. The feed mixture should be used within 3 weeks of preparation. Mice intended for use in sensitization studies should have been on the supplemented diet for at least 4 weeks. The mice at the time of the sensitization study should therefore be between 7 and 10 weeks old. The thickness of both ears of each animal should be measured and recorded at this time.

Although, in theory, one could apply a solid test article to the dorsal surface of the ear of a mouse, in practice an extract of such an article should be used. See Biological Reactivity Tests, In Vivo 88 for information on the extraction.

The maximally nonirritating dose and minimally irritating concentrations should be determined using separate groups of animals. This could be done as described for Preliminary Testing in the Mouse Ear Swelling Test.

The fur of the abdomen and thorax of 10 mice per group should be shaved. Then 100 µL of test article (at the minimally irritating concentration) should be applied to the test areas on days 0, 2, 4, 7, and 11. Control animals receive 100 µL of vehicle alone on the same schedule.

This phase should occur 4 days after the final application of the Induction Phase. Twenty-five µL of test article (at the maximally nonirritating concentration) should be applied to each ear of each animal in the test and control groups.

Ear thickness for both ears of each animal should be recorded after 24 and 48 hours postchallenge. The measurements should be made with a caliper (a spring-loaded caliper is preferable). The percent increase in ear thickness should be calculated for each ear by subtracting the pretreatment measurement from the post-treatment measurement, dividing the result by the pretreatment measurement, then multiplying by 100. The response of the test group versus the control group should be compared statistically. (The Mann-Whitney U test could be used for the comparison.)

The results of individual animals should also be calculated. 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, that is indicative of sensitization. As an overall evaluation, should the results of the study provide a significant result of the statistical test at p < 0.01 for the control versus test group comparisons, or if at least two test animals have ear thickness increases in excess of 50% of the maximum control thickness changes and the group comparison showed a p < 0.05, sensitization is indicated for the test article.