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1. OECD Guidelines for Testing of Chemicals are reviewed periodically in light of scientific progress and animal welfare considerations. Guideline 406 (1992) describes methods for assessing skin sensitization potential of chemicals in animals (1). While this Guideline mentions certain alternative screening tests, it relies on guinea pigs tests, notably the Guinea Pig Maximization Test and the Buehler Assay, for the hazard identification of skin sensitizers and non-sensitizers.

2. The details that follow in this Guideline describe the Local Lymph Node Assay (LLNA), an alternative procedure using the mouse (2-4). The LLNA provides advantages with regard to animal welfare (both reduction and refinement) and scientific aspects (specifically, the objective and quantitative nature of the endpoint measured). This method was mentioned in Guideline 406 (1) as a screening test, but has now undergone sufficient validation that it should be considered as a stand-alone method. The details of this validation and a review of the associated work have been published (5-8). In addition, it should be noted that the mild/moderate sensitizers recommended as suitable positive control substances for guinea pig test methods are also appropriate for use with the LLNA (6, 8, 9, 10).

3. Prior to modification of this protocol, changes should be adequately validated and determined to be acceptable (11).

GENERAL PRINCIPLE OF DETECTION OF SKIN SENSITIZATION USING THE LOCAL LYMPH NODE ASSAY

4. The basic principle underlying the LLNA is that sensitizers induce proliferation of lymphocytes in the lymph node draining the site of chemical application. Generally, under appropriate test conditions, this proliferation is proportional to the dose applied, and provides a means of obtaining an objective, quantitative measurement of sensitization. The test measures cellular proliferation as a function of in vivo radioisotope incorporation into the DNA of dividing lymphocytes. The LLNA assesses this proliferation in the draining lymph nodes proximal to the application site (see Appendix 1). This effect occurs as a dose-response in which the proliferation in test groups is compared to that in concurrent vehicle-treated controls. A positive control is added to each assay to provide an indication of appropriate

5. Young adult female mice (nulliparous and non-pregnant) of the CBA/Ca or CBA/J strain should be used at age 8-12 weeks. All animals should be age-matched (preferably within a one-week time frame). Females are used because the existing database is predominantly based on this gender. Other strains and males should not be used until it is sufficiently demonstrated that significant strain- and/or gender-specific differences in the LLNA response do not exist.

Preparation of animals

6. The temperature of the experimental animal room should be 21^oC (± 3^oC) and the relative humidity 30-70%. When artificial lighting is used, the light cycle should be 12 hours light:12 hours dark. For feeding, standard laboratory mouse diets should be used with an unlimited supply of drinking water. The mice should be acclimatised for at least 5 days prior to the start of the test. Animals may be housed individually, or caged in small groups of the same sex. Healthy animals are randomly assigned to the control and treatment groups. The animals are uniquely identified prior to being placed on study. Although a variety of techniques exist to uniquely mark mice, any method that involves identification via ear marking (e.g., ear tags) should not be used.

10. Positive controls are used to ensure the appropriate performance of the assay. The positive control should produce a positive LLNA response at an exposure level expected to give an increase in the stimulation index (SI) >3 over the negative control group. The positive control dose should be chosen such that the induction is clear but not excessive. Preferred positive control substances are hexyl cinnamic aldehyde (HCA) and mercaptobenzothiazole. There may be circumstances where, given adequate justification, other positive control substances may be used.

13. Results for each treatment group are expressed as the mean SI. The SI is the ratio of the mean dpm/mouse within each test substance treatment group and the positive control treated group against the mean dpm/mouse for the solvent/vehicle treated control group. However, the investigator should be alert to possible "outlier" responses for individual animals within a group that may necessitate the use of an alternative measure of response (e.g., median rather than mean) or elimination of the outlier. Each SI should include an appropriate measure of variability that takes into account the inter-animal variability in both the dosed and control groups (8).

• identification data and CAS no., if known;

• physical nature and purity;

• physiochemical properties relevant to the conduct of the study;

• stability of the test substance, if known; and

• lot number of the test substance.

Solvent/vehicle:

• use of the regulatory relevant vehicle;

• justification for choice of solvent/vehicle; and

• solubility and stability of the test substance in the solvent/vehicle.

Test animals:

• strain of mice used;

• number, age, and sex of mice;

• source, housing conditions, diet, etc.;

• individual weight of the animals at the start and end of the test, including body weight range, mean and associated error term for each group; and

• microbiological status of the mouse

Test conditions:

• positive and negative (vehicle/solvent) control data;

• data from range-finding study, if conducted;

• rationale for dose level selection;

• details of test substance preparation;

• details of the administration of the test substance;

• details of food and water quality;

• detailed description of treatment and sampling schedules;

• methods for measurement of toxicity;

• criteria for considering studies as positive, negative, or equivocal.

Results:

• signs of toxicity;

• dpm/mouse values for each mouse within each treatment group;

• mean and associated error term for dpm/mouse for each treatment group;

• calculated SI and associated error term for each test substance treatment dose group and concurrent positive control group;

• dose-response relationship;

• statistical analyses and method applied;

• concurrent and historical negative control data as established in the testers laboratory;

• concurrent positive control data

1. OECD (Organization for Economic Cooperation and Development). (1992). OECD guideline for testing of chemicals 406: Skin Sensitisation. OECD, Paris.

2. Kimber, I., and Basketter, D.A. (1992). The murine local lymph node assay; collaborative studies and new directions: A commentary. Food and Chemical Toxicology 30, 165-169.

3. Kimber, I., Dearman, R.J., Scholes E.W., and Basketter, D.A. (1994). The local lymph node assay: developments and applications. Toxicology 93, 13-31.

4. Kimber, I., Hilton, J., Dearman, R.J., Gerberick, G.F., Ryan, C.A., Basketter, D.A., Lea, L., House, R.V., Ladies, G.S., Loveless, S.E., and Hastings, K.L. (1998). Assessment of the skin sensitisation potential of topical medicaments using the local lymph node assay: An interlaboratory exercise. Journal of Toxicology and Environmental Health 53, 563-579 (1998).

5. Chamberlain, M., and Basketter, D.A. (1996). The local lymph node assay: status of validation. Food and Chemical Toxicology 34, 999-1002.

6. Basketter, D.A., Gerberick, G.F., Kimber, I., and Loveless, S.E. (1996). The local lymph node assay—A viable alternative to currently accepted skin sensitisation tests. Food and Chemical Toxicology 34, 985-997.

7. Basketter, D.A., Gerberick, G.F., and Kimber, I. (1998). Strategies for identifying false positive responses in predictive sensitisation tests. Food and Chemical Toxicology. (in press) 36, 327-33.

8. ICCVAM (Interagency Coordinating Committee on the Validation of Alternative Methods). (1999). The murine local lymph node assay: A test method for assessing the allergic contact dermatitis potential of chemicals/compounds. NIH Publication No. 99-4494, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina.

9. Basketter, D.A., Selbie, E., Scholes, E.W., Lees, D., Kimber, I., and Botham, P.A. (1993). Results with OECD recommended positive control sensitisers in the maximisation. Buehler and local lymph node assays. Food and Chemical Toxicology 31, 63-67.

10. OECD (Organization for Economic Cooperation and Development). (1996). Report of the OECD workshop on harmonization of validation and acceptance criteria for alternative toxicological test methods. OECD, Paris.

11. Dearman, R.J., Hilton, J., Evans, P., Harvey, P., Basketter, D.A., and Kimber, I. (1998). Temporal stability of local lymph node assay responses to hexyl cinnamic aldehyde. Journal of Applied Toxicology 18, 281-284.

12. Loveless, S. E., Ladics, G. S., Gerberick, G.F., Ryan, C.A., Basketter, D.A., Scholes, E.W., House, R.V., Hilton, J., Dearman, R.J., and Kimber, I.. (1996). Further evaluation of the local lymph node assay in the final phase of an international collaborative trial. Toxicology 108, 141-152.

13. Kimber, I., Hilton, J., Dearman, R.J., Gerberick, G.F., Ryan, C.A., Basketter, D.A., Scholes, E.W., Ladics, G.S., Loveless, S.E., House, R.V., and Guy, A. (1995). An international evaluation of the murine local lymph node assay and comparison of modified procedures. Toxicology 103, 63-73.

14. Tilney, N.L. (1971). Patterns of lymphatic drainage in the adult laboratory rat. Journal of Anatomy 109, 369-383.

Although minimal technical training of the LLNA is required, extreme care must be taken to obtain appropriate and consistent dissection of the lymph nodes. It is recommended that technical proficiency be achieved by the dissection and identification of the lymph nodes draining the ear by:a) practice dissection on mice that have been injected with a colored agent (dye); and/or b) practice dissection with mice sensitized with a strong positive sensitizer. Brief descriptions of these practice dissections are provided below. Recognizing that nodes from vehicle treated and naïve mice are smaller, laboratories performing the LLNA must also gain proficiency in the dissection of these nodes. It may be helpful for laboratories inexperienced in this procedure to request guidance from laboratories that have successfully performed the LLNA.

There are several methods that can be used to provide color identification of the draining nodes. These techniques may be helpful for initial identification and should be performed to ensure proper isolation of the appropriate node. Examples of such treatments are listed below. It should be noted, that other such protocols may be used effectively.

1. Evan’s Blue Dye treatment:

Inject approximately 0.1 ml of 2% Evan’s Blue Dye (prepared in sterile saline) intradermally into the pinnae of an ear. Euthanize the mouse after several minutes and continue with the dissection as noted below.

2. Colloidal carbon and other dye treatments:

For the purpose of node identification and training, a strong sensitizer is recommended. This agent should be applied in the standard acetone:olive oil vehicle (4:1). Suggested sensitizers used for this training exercise include 0.1% oxazolone, 0.1% (w/v) 2,4-dinitrochlorobenzene, and 0.1% (v/v) dinitrofluorobenzene.After treating the ear with a strong sensitizer, the draining node will dramatically increase in size, thus aiding in the identification and location of the node.

Using a procedure similar to that listed in the protocol, the agent is applied to the dorsum of both ears (25 m L/ear) for three consecutive days. On the fourth day, the mouse is euthanized. Identification and dissection (listed below) of the node should be performed in these animals prior to practice in non-sensitized or vehicle-treated mice, where the node is significantly smaller.

Please note: Due to the exacerbated response, the suggested sensitizers are not recommended as controls for the assay performance. They should only be used for training and node identification purposes.

Although lateral dissection is not the conventional approach used to obtain the nodes draining the ear, it may be helpful as a training procedure when used in combination with the ventral dissection. This approach is performed bilaterally (on both sides of the mouse). After the mouse is euthanized, it is placed in a lateral position. The facial and neck area is wetted with 70% ethanol. Using scissors and forceps, an initial cut is made from the neck area slightly below the ear. This incision is carefully extended toward the mouth and nose. During this procedure, the tip of the scissors should be angled slightly upward to prevent the damage of deeper tissue. The glandular tissue in the area is gently retracted using the forceps. Using the masseter muscle, facial nerves, blood vessels, and the bifurcation of the jugular vein as landmarks, the draining node is isolated and removed (Figure 1). The draining node will be positioned adjacent to the masseter muscle and proximal to and slightly above the jugular bifurcation.

The most commonly used dissection approach is from the ventral surface of the mouse. This approach allows both right and left draining nodes to be obtained without repositioning the mouse. With the mouse ventrally exposed, the neck and abdomen area is wetted with 70% ethanol. Using scissors and forceps, carefully make the first incision across the chest and between the arms. Make a second incision up the mid-line, perpendicular to the initial cut, and then cut up to the chin area. Reflect the skin to expose the external jugular veins in the neck area. Care should be used to avoid salivary tissue at the midline and nodes associated with this tissue. The nodes draining the ear are located distal to the masseter muscle, away from the midline, and near the bifurcation of the jugular veins.

The nodes can be distinguished from glandular and connective tissue in the area by the uniformity of the nodal surface and a shiny translucent appearance. The application of sensitizing agents (especially the strong sensitizers used in training) will cause an enlargement of the node size. If a dye is injected for training purposes, the node will take on the tint of the dye.

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