3H - 0hrs - Administration of 20m Ci ³HTdR.

C - ³HTdR incorporation into pooled LNC determined.

The method is based upon the assay developed some years ago at the Central Toxicology Laboratory, Zeneca (see References, section 2.15).

Mice, housed in groups of four, are treated by topical application of the test substance to the dorsum of each ear one time per day over three consecutive days to induce sensitization. Control mice are treated with the vehicle alone. Five days after the first topical application, the mice are injected with radiolabelled thymidine (³H-methyl thymidine). Approximately five hours after injection, the mice are sacrificed and the draining auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node cells (LNC) are prepared from pooled lymph nodes which are subsequently washed and incubated with trichloroacetic acid (TCA) overnight. The proliferative capacity of pooled LNC is then determined by the incorporation of ³H-methyl thymidine measured on a b -scintillation counter. Each stage of the method is described below:

2.3 Animals

CBA/Ca strain mice, purchased from Harlan UK Ltd or Charles River UK Ltd, are housed in groups of four in cages lined with `Lignocel' animal bedding RS Grade 3/4. Diets consists of SDS PCD 3/8" SQC pellets and water ad libitum. The mice are acclimated for at least six days before initiation of a study. At the start of a study, 4 young female adults (approximately 8-12 weeks) per test group are housed according to treatment.

All clinical signs, especially at the treatment sites (ie. skin reactions), should be recorded for the animals during a study. Details concerning the care and maintenance of mice can be found in the testing facility’s SOPs. Cage and bottle washing procedures can be found in the testing facility’s SOPs.

More information concerning animal maintenance (including diet batch numbers) are detailed on the 'Animal Log' sheet archived separately from the Study Report.

2.4 Test substance

Handling and characterisation of test substances must comply with the principles established in the testing facility’s GLP policy documents and SOPs. Subsample archiving is conducted by the sample processing unit of the testing facility.

The amount of sample used is recorded on the Sample Accountability form (APPENDIX 1). Details of the subsample (including date received, appearance and the subsample identification) are filled in when the sample is received from the sample processing unit. The amount of sample actually weighed out and the weight of sample + container before and after removing a sample is recorded. Significant deviations of amount of sample used from the difference in weight of sample container before and after must be noted and commented upon on the back of the form. If a sample is sent for analysis, this should also be recorded on the form. At the end of the test, record the amount of material returned to the sample processing unit and the date returned. The Sample Accountability form must be signed by the test operator and archived with the final Study Report.

2.5 Solvent vehicle selection and preparation

When preparing solutions, a suitable solvent vehicle is selected from the following list or according to instructions from the Study Director:

4:1 v/v Acetone/Olive oil (AOO)

Acetone

Methyl ethyl ketone (MEK)

4:1 v/v Methyl ethyl ketone/paraffin oil (MEKPO)

Dimethyl sulfoxide (DMSO)

N,N-Dimethylformamide (DMF)

Propylene glycol (PG)

Physiological saline (0.9%)

50% v/v acetone saline

The vehicles AOO, MEKPO and acetone saline are prepared as follows:

AOO - add 160ml of acetone to 40ml of olive oil.

MEKPO - add 160ml of MEK to 40ml of paraffin oil.

Acetone saline - add 100ml of acetone to 100ml of physiological saline (0.9%).

All vehicles are labelled with "Name" of contents, date of preparation, expiry date/condition, storage/handling and the name/initials of the operator who prepared it.

Where possible the following vehicles should be used (in order of preference): A00 > DMF > MEK > PG > DMSO.

2.6 Test solution preparation

Safety glasses and gloves must be worn during solution preparation and all procedures must be carried out in a fume cupboard where the test substance and/or vehicle is known to present an inhalation hazard.

The test substance is normally assayed at three to five consecutive concentrations from within the following range:

100%, 50%, 25%, 10%, 5%, 2.5%, 1.0%, 0.5%, 0.25%, 0.1%,

using a suitable vehicle. Test concentrations are primarily based upon previous experience in guinea pig tests, structure analysis and solubility factors. In the event of no such support data optimal test concentrations will be prepared based upon the solubility of the test substance in the vehicle.

Solids and liquids are weighed and solutions prepared on a weight upto (-->) a volume basis (this must be specified in the record of solution preparation as w/v). 0.2ml graduated stoppered 10ml measuring cylinders, stoppered 5ml/10ml volumetric flasks and disposable 1.0ml syringes are used in the preparation of solutions. Such measuring cylinders/volumetric flasks are deemed sufficiently accurate for solution preparation . 1.0ml syringes are also sufficiently accurate for solution preparation. Details of solution preparation are recorded in the data sheets for the particular study and archived with the Study Report (APPENDIX 1).

Substances of low solubility can be mixed using a mechanical agitator or using a magnetic stirrer. Heat above 38^oC is not used unless the substance is known to be heat stable.

2.7 Topical application

Gloves must be worn during this operation.

Each group of mice are treated by topical application to the dorsal surface of each ear with a different selected concentration of the test substance. A further group of mice is treated with the vehicle alone. The application volume, 25m l, is administered using a 0-50m l positive displacement pipette and is spread over the entire dorsal surface of the ear. For treatment, one mouse is removed from the home cage, treated and placed in an empty cage. When all mice from that group have been treated they are returned to the home cage. Topical application is performed once daily over three consecutive days. After the final topical application each group of mice are transferred into plastic disposable cages.

After treatments excess sample or the empty container is returned to the sample processing unit. Excess solutions, in small quantities, can normally be emptied down the drains using plenty of cold water. Hazardous solutions, however, must be returned to the sample processing unit for correct disposal.

2.8 Working with radiation

All work with radionuclides is conducted in a room which is a designated area approved by the test facility’s Radiation Safety Office. The workstation has a 'Designated Workstation Log' in which details of the work undertaken and monitoring data is recorded.

Only suitably trained and approved staff will be allowed to work with unsealed radioactive sources.

Bench surfaces where radionuclides are handled are lined with absorbent plastic-lined paper, such as 'Benchkote' and plastic 'lipped' trays are used to confine contamination in the event of spills. Personal protection must be used when handling radionuclides, these include a labcoat, plastic gloves and safety glasses.

2.9 Preparation of ³H-methyl thymidine

The radionuclide ³H-methyl thymidine (³HTdR) is used in the LLNA. ³HTdR is purchased from Amersham International, catalogue Code No. TRA.310 (specific activity, 2.0Ci/mMol; concentration 1.0mCi/ml). 'Radiochemical Batch Analysis' sheets received with each batch of 3HTdR are recorded separately from the Study Report.

The ³HTdR is diluted to a working concentration of 80m Ci/ml on a volume to volume basis using sterile phosphate buffered saline (PBS). ³HTdR is prepared in sterile 30ml disposable `Universal' containers and is prepared fresh prior to the study. A disposable B-D plastipak 1ml syringe + 26G ³/8" hypodermic needle and disposable B-D plastipak 1ml/10ml/30ml syringes + 0.2mm micropore filter are used for the measurement of volumes of ³HTdR and PBS respectively.

The concentration of 80mCi/ml of ³HTdR is confirmed by removing a 80m l aliquot, diluting to 200ml with tap water and 'counting' two 1ml aliquots of this dilution in a b -Scintillation Counter after adding 10mls of 'Optiphase-mp' scintillant.

Details of ³HTdR preparation and confirmation of the concentration are recorded in the data sheets for the particular Study and archived with the Study Report (APPENDIX 1, Section 3). Further details concerning ³HTdR preparation and use are also detailed on `Radioactive Log' sheets archived separately from the Study Report.

2.10 Incorporation of ³H-methyl thymidine in vivo

Five days after the first topical application treatment, all mice are administered ³H-methyl thymidine (³HTdR). Several minutes prior to ³HTdR administration mouse tail veins are visualised by placing the mice in a warm air environment. This is achieved using a 'Thermacage' (Beta medical and Scientific; Datesand Ltd) which consists of four separate compartments each fitted with a lid, catch and vent control enabling temperature adjustment of each chamber. 20m Ci ³HTdR is administered per mouse by injecting intravenously via tail vein with 250m l of 80m Ci/ml ³HTdR using B-D Plastipak 1.0ml disposable syringes + 26G ³/₈" hypodermic needles. 1.0ml disposable syringes are deemed sufficiently accurate for the measurement of volumes in the range 0.2-1.0ml.

2.11 Preparation of single cell suspensions

Approximately five hours after ³HTdR injection all mice are sacrificed by carbon dioxide asphyxiation, the draining auricular lymph nodes rapidly excised and pooled for each experimental group (8 nodes per group). Pooled lymph nodes are collected into 7ml disposable bijou bottles containing 1.0ml of phosphate buffered saline (PBS). A single cell suspension (SCS) of pooled lymph node cells (LNC) is prepared and collected into the base of a 90mm plastic Petri dish by gentle mechanical disaggregation of pooled lymph nodes through stainless steel gauze (200 mesh size) using the plunger of a B-D `Discardit' 5.0ml disposable syringe (catalogue code no. 309050). The gauze is washed with 4-5mls of PBS into the base of the Petri dish, and the

SCS transferred into a 10ml graduated plastic round-bottomed Sarstedt centrifuge tube. The SCS is finally made up to 10 mls with 4-5mls of PBS used to rinse the Petri dish. This procedure is repeated for each group of pooled lymph nodes.

Pooled LNC are pelleted with a relative centrifugal force (RCF) of 190 x g (RCF calculated to bottom of centrifuge tube) for 10 minutes in a centrifuge set at 4^oC. After centrifugation each supernatant is removed by aspiration using disposable plastic pipettes leaving 1-2mls of supernatant above each pellet. Each pellet is gently agitated before making up to 10mls with PBS and resuspending the LNC. This washing procedure is repeated twice.

2.12 Determination of incorporated ³H-methyl thymidine

Safety glasses and gloves must be worn when handling TCA and 'Optiphase mp' scintillation fluid.

After the final wash each supernatant is removed leaving just a small volume (<0.5ml) of supernatant above each pellet. Each pellet is gently agitated before resuspending the LNC in 3mls of 5% TCA for precipitation of macromolecules. After incubation with 5% TCA at +4^oC overnight, each precipitate is recovered by centrifugation at 190 x g for 10 minutes, removing each supernatant and resuspending in 1ml of 5% TCA. Each precipitate is transferred to a 25ml glass scintillation vial with 10mls of 'Optiphase mp' scintillation liquid and thoroughly mixed. The vials are loaded into a b -scintillation counter, and after approximately 30 minutes ³HTdR incorporation is measured. The b -counter expresses ³HTdR incorporation as the number of radioactive disintegrations per minute (DPM), the results of which are produced on a printout. Similarly, background ³HTdR levels are also measured in two 1ml aliquots of 5% TCA.

2.13 Radioactive contamination monitoring

After completing an otherwise uneventful work routine the workplace must be thoroughly monitored. Such monitoring must be carried out regardless of the level of activity at which the work is done. Monitoring data is recorded in the 'Designated Workstation Log' and on 'Radioactive Monitoring Swabs' sheets which are archived separately from the Study Report. If necessary these will be made available to the Radiation Safety Officer. If contamination has been detected then the area contaminated must be decontaminated immediately using a suitable detergent such as 'Decon 90'.

In addition personal exposure to ³HTdR is monitored by monthly urine analysis.

Prompt whole body examination will be compulsory for staff who have been exposed to radionuclides as a result of accidents and major spillages.

Accidental contamination of personnel and equipment must be immediately reported to the local Radiation Safety Officer and medical department. Decontamination measures must be undertaken without delay. Contaminated protective clothing may be laundered in a 'Hot Lab' and personal contamination must be reduced by washing and scrubbing. Success of decontamination measures must be assessed by monitoring.

2.14 Disposal of radioactive waste

All contaminated solid waste from each experiment including animal carcasses is placed in biohazard plastic bags lined with plastic bin liners, sealed, labeled `Radioactive material' and sent for incineration. If radioactive carcasses cannot be incinerated immediately then they must be placed in double plastic bags and frozen until it is convenient to do so.

Contaminated liquid waste is temporarily stored in a 2.5 litre impact resistant bottle and the contents sent for incineration when full.

Contaminated waste should not be allowed to accumulate and should be sent for incineration as soon as practically possible.

The quantity of radioactivity present within the waste is recorded on the 'Radioactive Log' sheet and archived separately from the Study Report. The quantity of radioactivity incinerated each week is submitted to the Radiation Safety Officer.

2.15 References

Kimber, I. and Weisenberger, C. 1989. A modified murine local lymph node assay for the identification of contact allergens. In "Current Topics in Contact Dermatitis". pp 592-595. Eds. Frosch, P.J. et al., Springer-Verlag Berlin Heidelberg.

Kimber, I. et al. 1989. The murine local lymph node assay for the identification of contact allergens: a preliminary evaluation of in situ measurement of lymphocyte proliferation. Contact Dermatitis, 21, 215-220.

Kimber, I. et al. 1991. The murine local lymph node assay: results of an inter-laboratory trial. Toxicology Letters, 55, 203-213.

Kimber, I. and Basketter, D.A. 1992. The murine local lymph node assay. A commentary on collaborative studies and new directions. Fd. Chem. Toxic., 30, 165-169.

3.1 Interpretation/treatment of results

The proliferative response of lymph node cells (LNC) is expressed as the number of radioactive disintegrations per minute per lymph node (DPM/NODE) and as the ratio of ³HTdR incorporation into LNC of test lymph nodes relative to that recorded for control lymph nodes (TEST/CONTROL RATIO). Before DPM/NODE values are determined, background ³HTdR is subtracted from test and control raw DPM data.

A substance is regarded as a sensitizer in the LLNA if at least one concentration of the test substance results in a 3-fold or greater increase in ³HTdR incorporation into LNC of test lymph nodes relative to that recorded for control lymph nodes, as indicated by the TEST/CONTROL RATIO. The data should also not be incompatible with a biological dose response, although allowance must be made, especially at high topical application concentrations, for either local toxicity or immunological suppression.

3.2 Example

Raw data: Background ³HTdR in two 1ml TCA samples - 90 DPM

100 DPM