LLNA BrdU-ELISA Test Method: ICCVAM Recommended Protocol

ICCVAM-Recommended Test Method Protocol:

The Murine Local Lymph Node Assay:

2-Bromodeoxyuridine-ELISA Test Method (LLNA: BrdU-ELISA), a

Nonradioactive Alternative Test Method for Assessing the Allergic Contact

Dermatitis Potential of Chemicals and Products

Originally published as Appendix B of “ICCVAM Test Method Evaluation Report on the Murine

Local Lymph Node Assay: BrdU-ELISA”

A Nonradioactive Alternative Test Method to Assess the Allergic Contact Dermatitis Potential of

Chemicals and Products

NIH Publication No. 10-7552 – Published 2010

Available at: http://iccvam.niehs.nih.gov/methods/immunotox/llna-ELISA/TMER.htm

ICCVAM LLNA: BrdU-ELISA Evaluation Report

B-2

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Appendix B – ICCVAM-Recommended Protocol

B-3

1.0 General Principle of Detection of Skin Sensitization Using the

Nonradiolabelled Murine Local Lymph Node Assay: 2-

Bromodeoxyuridine-ELISA Test Method (LLNA: BrdU-ELISA)

The basic principle underlying the murine local lymph node assay (LLNA) is that sensitizers induce

proliferation of lymphocytes in the lymph nodes draining the site of substance application. 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 cell

proliferation as a function of in vivo radioisotope (

H-methyl thymidine or

125

I-iododeoxyuridine)

incorporation into the DNA of dividing lymphocytes, and assesses this proliferation in the draining

lymph nodes proximal to the application site (see Annex I). Due to the use of radioactivity, the

LLNA has limited use in regions where the acquisition, use, or disposal of radioactivity is

problematic. The LLNA: BrdU-ELISA was therefore developed as a nonradioactive modification to

the LLNA (Takeyoshi 2001), which uses nonradiolabelled 5-bromo-2-deoxyuridine (BrdU)

(Chemical Abstracts Service Registry Number [CASRN] 59-14-3) with detection by an enzyme-

linked immunosorbent assay (ELISA) to assess lymphocyte proliferation. The ability to detect skin

sensitizers without the necessity of using a radioactive label for DNA eliminates the potential for

occupational exposure to radioactivity and waste disposal issues. Similar to the LLNA, the

LLNA: BrdU-ELISA studies the induction phase of skin sensitization and provides quantitative data

suitable for dose-response assessment. Lymphocyte proliferation in test groups is compared to that in

the concurrent vehicle-treated control group. The proliferation is proportional to the dose and to the

potency of the applied allergen and provides a simple means of obtaining a quantitative measurement

of sensitization. The LLNA: BrdU-ELISA assesses this proliferation as the proliferation in test groups

compared to that in vehicle treated controls. The ratio of the proliferation in treated groups to that in

concurrent vehicle treated controls, termed the stimulation index (SI), is determined, and should be

≥1.6 before a test substance can be considered as a skin sensitizer, with specific limitations for

borderline positive results (i.e., SI between 1.6 and 1.9) as described in Section 3 of this Test Method

Evaluation Report.

The methods, described here are based on the use of measuring BrdU content to indicate an increased

number of proliferating cells in the draining auricular lymph nodes. BrdU is an analog of thymidine

and is similarly incorporated into the DNA of proliferating cells. The incorporation of BrdU is

measured by ELISA, which utilizes an antibody specific for BrdU that is also labeled with

peroxidase. When the substrate is added, the peroxidase reacts with the substrate to produce a colored

product that is quantified at a specific absorbance using a microtiter plate reader. A concurrent

positive control is added to each assay to provide an indication of appropriate assay performance.

2.0 Description of the LLNA: BrdU-ELISA

2.1 Sex and strain of animals

The mouse is the species of choice for the LLNA: BrdU-ELISA assay. Validation studies were

conducted exclusively with the CBA/JN strain, but other CBA substrains can be used. Young adult

female mice (nulliparous and non-pregnant) are used because most data in the existing database were

generated using mice of this gender.

Male mice may be used if it is sufficiently demonstrated that these animals perform as well as female CBA

mice in the LLNA: BrdU-ELISA.

At the start of the study, mice should be 8-12 weeks of age. All

ICCVAM LLNA: BrdU-ELISA Evaluation Report

B-4

mice should be age matched (preferably within a one-week time frame). Weight variations between

the mice should not exceed 20% of the mean weight.

2.2 Preparation of animals

The temperature of the experimental animal room should be 22°C (±3°C) and the relative humidity

30%-70% (although the aim is for 50%-60%). Lighting should be artificial, the sequence being 12

hours light, 12 hours dark. For feeding, an unlimited supply of standard laboratory mouse diets and

drinking water should be used. The mice should be quarantined/acclimatized for at least five days

prior to the start of the test (ILAR 1996). Mice should be allocated to small groups by a stratified

randomization or other appropriate methods before the start of the study unless adequate scientific

rationale for housing mice individually is provided (ILAR 1996). Four animals per cage is the

recommended housing arrangement. The mice are uniquely identified prior to being placed in the

study. The method used to mark the mice should not involve identification via the ear (e.g., marking,

clipping, or punching of the ear). Colored marks on the tail or other appropriate methods should be

used. All mice should be examined (e.g., clinical signs, body weights, observation of excrement) prior

to the initiation of the test to ensure good health and the absence of skin lesions.

2.3 Preparation of doses

Solid test substances should be dissolved or suspended in appropriate solvents/vehicles and diluted, if

appropriate, prior to dosing of the mice. Liquid test substances may be dosed directly (i.e., applied

neat) or diluted prior to dosing. Insoluble materials, such as those generally seen in medical devices,

should be subjected to an exaggerated extraction in an appropriate solvent to extract all extractable

constituents for testing prior to dosing. Fresh preparations of the test substance should be prepared

daily unless stability data demonstrate the acceptability of storage.

2.4 Test conditions

2.4.1 Solvent/vehicle

The solvent/vehicle should not interfere with or bias the test result and should be selected on the basis

of maximizing the solubility in order to obtain the highest concentration achievable while producing a

solution/suspension suitable for application of the test substance. Recommended vehicles are acetone:

olive oil (4:1 v/v) (AOO), N,N-dimethylformamide (DMF), methyl ethyl ketone (MEK), propylene

glycol, and dimethyl sulfoxide (DMSO) (Van Och et al. 2000; Kimber et al. 1994), but others may be

used if sufficient scientific rationale is provided (Kimber and Basketter 1992). Particular care should

be taken to ensure that hydrophilic materials are incorporated into a vehicle system that incorporates

appropriate solubilizers (e.g., 1% Pluronic L92) that wet the skin and does not immediately run off.

Thus, wholly aqueous vehicles may need to be avoided. In certain situations, it may be necessary for

regulatory purposes to test the substance in the clinically relevant solvent or product formulation.

2.4.2 Controls

Concurrent negative (solvent/vehicle) and positive controls should be included in each test to ensure

that the test system is functioning properly and that the specific test is valid. In some circumstances

(e.g., when using a solvent/vehicle not recommended in Section 2.4.1), it may be useful to include a

naïve control. Except for treatment with the test substance, the mice in the negative control groups

should be handled in an identical manner to the mice of the treatment groups.

Positive controls are used to demonstrate appropriate performance of the assay by responding with

adequate and reproducible sensitivity to a sensitizing substance for which the magnitude of the

response is well characterized. Inclusion of a concurrent positive control is recommended because it

Appendix B – ICCVAM-Recommended Protocol

B-5

demonstrates competency of the laboratory to successfully conduct each assay and allows for an

assessment of intra- and interlaboratory reproducibility and comparability. The positive control

should produce a positive LLNA: BrdU-ELISA response at an exposure level expected to give an

increase in the SI ≥ 1.6 over the negative control group. The positive control dose should be chosen

such that the induction is reproducible but it does not cause excessive skin irritation or systemic

toxicity. Preferred positive control substances are 50% hexyl cinnamic aldehyde (HCA; CASRN 101-

86-0) and 50% eugenol (CASRN 97-53-0) in AOO. There may be circumstances in which, given

adequate justification, other positive control substances meeting the above criteria may be used.

Although the positive control substance should be tested in the vehicle that is known to elicit a

consistent response (e.g., AOO), there may be certain regulatory situations in which testing in a non-

standard vehicle (clinically/chemically relevant formulation) will also be necessary. In such

situations, the possible interaction of a positive control with this unconventional vehicle should be

tested. If the concurrent positive control substance is tested in a different vehicle than the test

substance, then a separate vehicle control for the concurrent positive control should be included.

While inclusion of a concurrent positive control group is recommended, there may be situations in

which periodic testing (i.e., at intervals ≤6 months) of the positive control substance may be adequate

for laboratories that conduct the LLNA: BrdU-ELISA regularly (i.e., conduct the LLNA: BrdU-

ELISA at a frequency of no less than once per month) and have an established historical positive

control database that demonstrates the laboratory’s ability to obtain reproducible and accurate results

with positive controls. Adequate proficiency with the LLNA: BrdU-ELISA can be successfully

demonstrated by generating consistent results with the positive control in at least 10 independent tests

conducted within a reasonable period of time (i.e., less than one year).

A concurrent positive control group should always be included when there is a procedural change to

the LLNA: BrdU-ELISA (i.e., change in trained personnel, change in test method materials and/or

reagents, change in test method equipment, change in source of test animals), and such changes

should be documented in laboratory reports. Consideration should be given to the impact of these

changes on the adequacy of the previously established historical database in determining the necessity

for establishing a new historical database to document consistency in the positive control results.

Investigators should be aware that the decision to conduct a positive control on a periodic basis

instead of concurrently has ramifications on the adequacy and acceptability of negative study results

generated without a concurrent positive control during the interval between each periodic positive

control study. For example, if a false negative result is obtained in the periodic positive control study,

all negative test substance results obtained in the interval between the last acceptable periodic positive

control study and the unacceptable periodic positive control study may be questioned. Implications of

these outcomes should be carefully considered when determining whether to include concurrent

positive controls or to only conduct periodic positive controls. Consideration should also be given to

using fewer animals in the concurrent positive control group when this is scientifically justified and if

the laboratory demonstrates, based on laboratory-specific historical data, that fewer mice can be used

without substantially increasing the failure rate of the positive control (i.e., the rate at which SI < 1.6

and the frequency with which studies will need to be repeated due to positive control failure

[Appendix A of ICCVAM 2009a]).

In instances where substances of a specific chemical class or range of responses are being evaluated,

benchmark substances may be useful to demonstrate that the test method is functioning properly for

detecting the skin sensitization potential of a test substance. Appropriate benchmark substances

should have the following properties:

• Structural and functional similarity to the class of the substance being tested

• Known physical/chemical characteristics

• Supporting data from the LLNA: BrdU-ELISA

ICCVAM LLNA: BrdU-ELISA Evaluation Report

B-6

• Supporting data on known effects in animal models and/or from humans

2.5 Methodology

A minimum of four animals is used per dose group, with a minimum of three concentrations of the

test substance, plus a concurrent negative control group treated only with the vehicle for the test

substance, and a concurrent positive control. The processing of lymph nodes from individual mice

allows for the assessment of interanimal variability and a statistical comparison of the difference

between test substance and vehicle control group measurements. In addition, evaluating the

possibility of reducing the number of mice in the positive control group is only feasible when

individual animal data are collected.

Test substance treatment dose levels should be based on the recommendations given in Kimber and

Basketter (1992) and in the ICCVAM Panel Report (ICCVAM 1999). Consecutive doses are

normally selected from an appropriate concentration series such as 100%, 50%, 25%, 10%, 5%, 2.5%,

1%, 0.5%, etc. Adequate scientific rationale should accompany the selection of the concentration

series used. All existing toxicological information (e.g., acute toxicity and dermal irritation) and

structural and physicochemical information on the test material of interest (and/or structurally related

test materials) should be considered, where available, in selecting the three consecutive

concentrations so that the highest concentration maximizes exposure while avoiding systemic toxicity

and/or excessive local skin irritation (Kimber et al. 1994; OECD 2002). In the absence of such

information, an initial prescreen test may be necessary (Annex II).

The LLNA: BrdU-ELISA experimental procedure is performed as follows:

Day 1. Individually identify and record the weight of each animal and any clinical

observations. Apply 25 µL of the appropriate dilution of the test substance, the vehicle alone,

or the concurrent positive control to the dorsum of each ear.

Days 2 and 3. Repeat the application procedure as carried out on Day 1.

Day 4. No treatment.

Day 5. Inject 0.5 mL (5 mg/mouse) of 10 mg/mL BrdU in physiological saline

intraperitoneally.

Day 6. Record the weight of each animal and any clinical observations. Approximately 24

hours (24 h) after BrdU injection, humanely kill the animals. To further monitor the local skin

response in the experimental study, additional parameters such as scoring of ear erythema or

ear thickness measurements (obtained either by using a thickness gauge, or ear punch weight

determinations at necropsy) may be included in the study protocol.

Excise both bilateral draining auricular lymph nodes from each mouse ear (see diagram and

description of dissection in Annex I) and store in a 1.5 mL centrifuge tube at -20

C until

BrdU is measured by ELISA.

For BrdU measurement, a single-cell suspension of lymph node cells (LNC) from each

mouse is prepared by adding a small volume of physiological saline (approximately 0.3 mL)

to the excised lymph nodes, crushing the lymph nodes with a disposable plastic pestle, and

passing through a #70 nylon mesh or another acceptable technique for mechanical

disaggregation (e.g., passing through 200 micron-mesh stainless steel gauze) to generate a

single-cell suspension. The procedure for preparing the LNC suspension is a critical step of

this assay; it is most important to crush the lymph node and suspend the LNC completely.

Every technician should establish the skill in advance. The lymph nodes in negative control

animals are small, so careful operation is required to avoid an artificial effect on SI values.

Appendix B – ICCVAM-Recommended Protocol

B-7

In each case, the target volume of the LNC suspension should be adjusted to a pre-determined

optimized volume (approximately 15 mL) based on achieving a mean absorbance of the

negative control group within 0.1-0.2. Because this absorbance depends on the assay

apparatus and the target volume of cell suspension, every laboratory should decide their own

optimal volume of LNC suspension in advance.

The incorporation of BrdU into lymph node cells should be determined using a commercial

cell proliferation assay kit (Roche Diagnostics GmbH, Roche Applied Science, 68298

Mannheim, Germany; Cat. No. 11 647 229 001) after they are crushed and suspended in

physiological saline. The absorbance is defined as the BrdU labeling index. Follow the

instructions in the assay kit. Briefly, 100 µL of the LNC suspension is added to the wells of a

flat-bottom microplate in triplicate. After fixation and denaturation of the LNC, anti-BrdU

antibody is added to each well and allowed to react. Subsequently the anti-BrdU antibody is

removed by washing and the substrate solution is then added and allowed to produce

chromogen. Absorbance at 370 nm with a reference wavelength of 492 nm is then measured.

2.6 Reduced LLNA

Using this test method protocol, there is also the opportunity to perform a reduced

LLNA: BrdU-ELISA (rLLNA: BrdU-ELISA). Use of the rLLNA: BrdU-ELISA has the potential to

reduce the number of animals by omitting the middle and low dose groups from the

LLNA: BrdU-ELISA (Kimber et al. 2006; ESAC 2007; ICCVAM 2009b). This is the only difference

between the LLNA: BrdU-ELISA and the rLLNA: BrdU-ELISA. Thus, the test substance

concentration evaluated in the rLLNA: BrdU-ELISA should be the maximum concentration that does

not induce overt systemic toxicity and/or excessive local skin irritation in the mouse (Annex II). The

rLLNA: BrdU-ELISA should be used for the hazard classification of skin sensitizing substances if

dose-response information is not needed, provided there is adherence to all other

LLNA: BrdU-ELISA protocol specifications.

2.7 Observations

Mice should be carefully observed at least once daily for any clinical signs, either of local irritation at

the application site or of systemic toxicity (Annex II). Weighing mice prior to treatment and at the

time of necropsy will aid in assessing systemic toxicity. All observations are systematically recorded

with records maintained for each individual mouse. Animal monitoring plans should include criteria

to promptly identify those mice exhibiting systemic toxicity or excessive irritation, or corrosion of

skin for euthanasia (OECD 2000).

3.0 Calculation of Results

Results for each treatment group are expressed as the mean SI. The SI is derived by dividing the mean

BrdU labeling index/mouse within each test substance group and the concurrent positive control

group by the mean BrdU labeling index for the solvent/vehicle control group. The average SI value

for vehicle treated controls is then equal to one.

The BrdU labeling index is defined as:

BrdU labeling index = (ABS

– ABS blank

) – (ABS

ref

– ABS blank

ref

)

where ABS = absorbance, em = emission wavelength and ref = reference wavelength.

The decision process regards a result as positive when SI ≥ 1.6 (see Section 3 of this Test Method

Evaluation Report). However, the strength of the dose response, chemical toxicity, solubility, and,

where appropriate, statistical significance should be considered together with SI values to arrive at a

final decision (Basketter et al. 1996; ICCVAM 1999; EPA 1998; Kimber et al. 1998).

ICCVAM LLNA: BrdU-ELISA Evaluation Report

B-8

Collecting data at the level of the individual mouse will enable a statistical analysis for presence and

degree of dose response in the data. Any statistical assessment could include an evaluation of the

dose-response relationship as well as suitably adjusted comparisons of test groups (e.g., pair-wise

dosed group versus concurrent solvent/vehicle control comparisons). Statistical analyses may include,

for instance, linear regression or Williams’s test to assess dose-response trends, and Dunnett’s test for

pairwise comparisons. In choosing an appropriate method of statistical analysis, the investigator

should maintain an awareness of possible inequalities of variances and other related problems that

may necessitate a data transformation or a nonparametric statistical analysis. In any case, the

investigator may need to carry out SI calculations and statistical analyses with and without certain

data points (sometimes called “outliers”).

4.0 Evaluation and Interpretation of Results

Consideration should be given to the possibility of borderline positive results when SI values between

1.6 and 1.9 are obtained. This is based on the validation database of 43 substances using an SI ≥ 1.6

for which the LLNA: BrdU-ELISA correctly identified all 32 LLNA sensitizers, but incorrectly

identified two of 11 LLNA nonsensitizers with SI values between 1.6 and 1.9 (i.e. borderline positive)

(see Section 3.0 of this Test Method Evaluation Report). If an SI value between 1.6 and 1.9 is

obtained, other available information such as the nature of the dose-response, evidence of systemic

toxicity or excessive local skin irritation, and, where appropriate, statistical significance together with

SI values should be considered to confirm that such borderline positive results are potential skin

sensitizers (see Section 3.0 of this Test Method Evaluation Report). Consideration should also be

given to various properties of the test substance, including whether it has a structural relationship to

known skin sensitizers. These and other considerations are discussed in detail elsewhere (Basketter et

al. 1998).

Employing the optimized assay condition described previously, the mean SI value for the positive

control group (50% HCA) should be equal to or greater than 1.6. If not, data derived from the

experiment should not be used for evaluation.

5.0 Data and Reporting

5.1 Data

Data should be summarized in tabular form showing the individual animal BrdU labeling index

values, the group mean BrdU labeling index/animal, its associated error term (e.g., standard deviation

[SD], standard error of the mean [SEM]), and the mean SI value for each dose group compared

against the concurrent solvent/vehicle control group.

5.2 Test report

The test report should contain the following information:

Test Substances and Control Substances

• Identification data (e.g., CASRN, if available; source; purity; known impurities; lot

number)

• Physical nature and physicochemical properties (e.g. volatility, stability, solubility,

physicochemical properties relevant to the conduct of the study)

• Composition and relative percentages of components, if formulation

Solvent/Vehicle

• Identification data (CASRN; purity; concentration, where appropriate; volume used)

Appendix B – ICCVAM-Recommended Protocol

B-9

• Justification for choice of vehicle

Test Animals

• Source of CBA mice, housing conditions, diet, etc.

• Microbiological status of the animals, when known

• Number and age of animals

Test Conditions

• Details of test substance preparation and application

• Justification for dose selection (including results from prescreen test, if conducted)

• Vehicle and test substance concentrations used, and total amount of substance

applied

• Details of food and water quality (including diet type/source, water source)

• Details of treatment and sampling schedules

• Methods for measurement of toxicity

• Criteria for considering studies as positive or negative

• Details of any protocol deviations and an explanation on how the deviation affects

the study design and results

Reliability Check

• Summary of results of latest reliability check, including information on substance,

concentration and vehicle used

• Concurrent and/or historical positive and negative (solvent/vehicle) control data for

testing laboratory

• Date and laboratory report for the most recent periodic positive control and a report

detailing the historical positive control data for the laboratory justifying the basis for

not conducting a concurrent positive control, if a concurrent positive control was not

included

Results

• Individual weights of mice at start of dosing and at scheduled kill; as well as mean

and associated error term (e.g., SD, SEM) for each treatment group

• Time course of onset and signs of toxicity, including dermal irritation at site of

administration, if any, for each animal

• Table of individual mouse BrdU labeling indices and SI values for each treatment

group

• Mean and associated error term (e.g., SD, SEM) for BrdU labeling index/mouse for

each treatment group and the results of outlier analysis for each treatment group

• Calculated SI and an appropriate measure of variability that takes into account the

interanimal variability in both the test substance and control groups

• Dose response relationship

• Statistical analysis, where appropriate

Discussion of the Results

• Brief commentary on the results, the dose-response analysis, and statistical analyses,

where appropriate, with a conclusion as to whether the test substance should be

considered a skin sensitizer

ICCVAM LLNA: BrdU-ELISA Evaluation Report

B-10

Conclusion

A Quality Assurance Statement for GLP-compliant Studies

• Indicate all inspections made during the study and the dates any results were reported

to the Study Director; confirm that the final report reflects the raw data

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Appendix B – ICCVAM-Recommended Protocol

B-11

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728.

Woolhiser MR, Hayes BB, Meade BJ. 1998. A combined murine local lymph node and irritancy

assay to predict sensitization and irritancy potential of chemicals. Toxicol Meth 8:245-256.

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ICCVAM LLNA: BrdU-ELISA Evaluation Report

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Annex I:

An Approach to Dissection and Identification of the Draining

(“Auricular”) Lymph Nodes

1.0 Background

Although minimal technical training of the LLNA: BrdU-ELISA is required, extreme care must be

taken to ensure appropriate and consistent dissection of the lymph nodes. It is recommended that

technical proficiency in the dissection and identification of the lymph nodes draining the ear be

achieved by practice on mice that have been (a) injected with a colored agent (dye) and/or (b)

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: BrdU-ELISA 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: BrdU-ELISA.

2.0 Training and Preparation for Node Identification

2.1 Identification of the Draining Node – Dye Treatment

Several methods 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 might be used effectively.

Evan’s Blue Dye treatment:

Inject approximately 0.1 mL of 2% Evan’s Blue Dye (prepared in sterile saline) intradermally

into the pinna of an ear. Euthanize the mouse after several minutes and continue with the

dissection as noted below.

Colloidal carbon and other dye treatments:

Colloidal carbon and India ink are examples of other dye treatments that may be used (Tilney

1971).

2.2 Identification of the Draining Node – Application of Strong Sensitizers

For the purpose of node identification and training, a strong sensitizer is recommended. This agent

should be applied in the standard AOO vehicle. Suggested sensitizers 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 identification and location of the node.

Using a procedure similar to that described in the test method protocol, apply the agent to the dorsum

of both ears (25 µL/ear) for three consecutive days. On the fourth day, euthanize the mouse.

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 assay performance. They should only be used for training and node identification

purposes.

Appendix B – ICCVAM-Recommended Protocol

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3.0 Dissection Approach

3.1 Lateral Dissection (Figure B-I-1)

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.

Perform this approach bilaterally (on both sides of the mouse). After euthanizing the mouse, place it

in a lateral position. Wet the face and neck with 70% ethanol. Use scissors and forceps to make an

initial cut from the neck area slightly below the ear. Carefully extend the incision toward the mouth

and nose. Angle the tip of the scissors slightly upward during this procedure to prevent the damage of

deeper tissue. Gently retract the glandular tissue in the area using the forceps. Using the masseter

muscle, facial nerves, blood vessels, and the bifurcation of the jugular vein as landmarks, isolate and

remove the draining node (Figure B-I-1). The draining node (“auricular”) will be positioned adjacent

to the masseter muscle and proximal to and slightly above the jugular bifurcation.

3.2 Ventral Dissection (Figure B-I-2)

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, wet the neck and abdomen with 70% ethanol. Use scissors and forceps to

carefully make the first incision across the chest and between the arms. Make a second incision up the

midline 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. Take care to avoid salivary tissue at the midline and nodes

associated with this tissue. The nodes draining the ear (“auricular”) are located distal to the masseter

muscle, away from the midline, and near the bifurcation of the jugular veins.

4.0 Accuracy in Identification

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. Application of sensitizing agents (especially the

strong sensitizers used in training) will cause enlargement of the node size. If a dye is injected for

training purposes, the node will take on the tint of the dye.

ICCVAM LLNA: BrdU-ELISA Evaluation Report

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Figure B-I-1 Lateral Dissection

Credit: Dee Sailstad, U.S. EPA

Figure B-I-2 Ventral Dissection

Credit: Dee Sailstad, U.S. EPA

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ICCVAM LLNA: BrdU-ELISA Evaluation Report

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Annex II:

Evaluating Local Irritation and Systemic Toxicity in the LLNA: BrdU-

ELISA

As noted in the ICCVAM LLNA: BrdU-ELISA test method protocol, the maximum dose tested

should be the maximum possible concentration that does not produce systemic toxicity or excessive

local irritation after topical application in the mouse. In the absence of information to determine this

concentration (e.g., acute toxicity and dermal irritation data, and/or structural and physicochemical

information on the test material and/or structurally related test materials), a prescreen test should be

performed using three dose levels of the test substance, in order to define the appropriate dose to test

in the LLNA: BrdU-ELISA.

The prescreen test is conducted under identical conditions as the main LLNA: BrdU-ELISA study,

except there is no assessment of lymph node cell proliferation. The maximum dose tested should be

100% of the test material for liquids or the maximum possible concentration for solids or suspensions.

One or two animals per dose group are suggested. All mice will be observed daily for any clinical

signs of systemic toxicity or local irritation at the application site. Body weights are recorded pre-test

and prior to termination (Day 6). Both ears of each mouse are observed for erythema and scored using

Table B-II-1. Ear thickness measurements are taken using a thickness gauge (e.g., digital micrometer

or Peacock Dial thickness gauge) on Day 1 (predose), Day 3 (approximately 48 hours after the first

dose), and Day 6 (termination). Additionally on Day 6, ear thickness could be determined by ear

punch weight determinations, which must be performed after the animals are humanely killed.

Excessive local irritation is indicated by an erythema score ≥3 and/or an increase in ear thickness of

≥25% on any day of measurement (Reeder et al. 2007; ICCVAM 2009c). The highest dose selected

for the main LLNA: BrdU-ELISA study will be the next lower dose in the prescreen concentration

series that does not induce systemic toxicity and/or excessive local skin irritation.

Table B-II-1 Erythema Scores

Observation

Value

No erythema

Very slight erythema (barely perceptible)

Well-defined erythema

Moderate to severe erythema (beet redness)

Severe erythema (beet redness) to eschar

formation preventing grading of erythema

In addition to a 25% increase in ear thickness (Reeder et al. 2007; ICCVAM 2009c), a statistically

significant increase in ear thickness in the treated mice compared to control mice has also been used

to identify irritants in the traditional LLNA (Hayes et al. 1998; Homey et al. 1998; Woolhiser et al.

1998; Hayes and Meade 1999; Ehling et al. 2005; Vohr and Jürgen 2005). While statistically

significant increases can occur when ear thickness is less than 25%, they have not been associated

specifically with excessive irritation (Woolhiser et al. 1998; Hayes and Meade 1999; Ehling et al.

2005; Vohr and Jürgen 2005; Patterson et al. 2007).

Test guidelines for assessing acute dermal toxicity recommend a number of clinical observations for

assessing systemic toxicity (OECD 1987; EPA 1998). The following clinical observations, which are

Appendix B – ICCVAM-Recommended Protocol

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based on test guidelines and current practices (ICCVAM 2009d), may indicate systemic toxicity when

used as part of an integrated assessment and therefore may indicate the maximum dose level to use in

the main LLNA: BrdU-ELISA:

• Changes in nervous system function (e.g., piloerection, ataxia, tremors, and convulsions)

• Changes in behavior (e.g., aggressiveness, change in grooming activity, marked change

in activity level)

• Changes in respiratory patterns (i.e., changes in frequency and intensity of breathing such

as dyspnea, gasping, and rales)

• Changes in food and water consumption

• Lethargy and/or unresponsiveness

• Any clinical signs of more than slight or momentary pain and distress

• Reduction in body weight >5% from Day 1 to Day 6

• Mortality

Moribund animals or animals showing signs of severe pain and distress should be humanely killed

(OECD 2000).