Local Lymph Node Assay

Table of Contents

  1. Test Submission
    1. Cover Letter
    2. Submission
  2. Appendices
    1. Local Lymph Node Assay Bibliography
    2. List of Chemicals
      1. Chemicals Tested in Local Lymph Node Assay
      2. Discordant Results Between Local Lymph Node Assay and Guinea Pig or Human Test Methods
      3. Disintegrations Per Minute Data and Stimulation Indices for Discordant Results
    3. Key Local Lymph Node Assay Papers
    4. Sample Local Lymph Node Assay Protocol
    5. ICCVAM Local Lymph Node Assay Test Submission Guidelines

APPENDIX D

Sample Local Lymph Node Assay Protocol Test Sheets

THE LOCAL LYMPH NODE ASSAY - STUDY NUMBER

  Sample Accountability
Test substance :
Sub-sample ref. no. :
Appearance of sub-sample :
Active ingredient level :

Data sample received:

Procedure Wt. Sample + Container Amount Used Operator Date
Before After
Initial Weight
To Archive
To Analytical
Solvent Determination
Topical Application
Returned to Sample Processing

Comments:

THE LOCAL LYMPH NODE ASSAY - STUDY NUMBER

Test substance :
Sub-sample ref. no. :
Active ingredient level :
Storage :
Handling :

1. Description of test solutions and preparations
Solvent vehicle:

Test Conc. (%) Preparation Description Operator Date
 
 
 
 
 

2. Method of test solution preparation

Test Conc. (%) Method of Preparation Storage Conditions Other Comments
 
 
 
 
 

 

THE LOCAL LYMPH NODE ASSAY - STUDY NUMBER

Reagent Preparation

  1. Phosphate Buffered slaine (PBS) - 1 sachet of PBS powder ----> 1000 ml distilled water. Stored at + 4°C. Prepared.
  2. Trichloroacetic acid (TCA) - 7.5g TCA ----> 150 ml tap water. Stored at + 4°C. Prepared.
  3. 3H-methyl thymidine (3HTdR), specific activity 2.0 Ci/mMol (Concentration 1.0 mCi/ml). Stored at + 4°C. 'Radiochemical Batch Analysis' sheets received with each batch of 3HTdR are recorded separately from this study. 80 µCi/ml activity 3HTdR was prepared as follows:
3HTdR Code No. In-Use Activity Preparation Operator Date
  80 µCi/ml* ml of 1mCi/ml 3HTdR + ml of sterile PBS.

*Dilution activity of 3HTdR confirmed by removing a 80 µl aliquot, diluting to 200 ml with tap water and removing two 1 ml aliquots (0.032 µCi) and counting these on the β-scintillation counter:

β-Counter printout inserted here

Mean Count : DPM

Since 1.0 µCi = 2220000 DPM (37000 Bq)
then 0.032 µCi - 71040 DPM

Therefore, DPM = DPM X 80 µCi/ml / 71040 DPM = µCi/ml

More information concerning 3HTdR preparation, use, disposal and monitoring during this study are detailed on the 'Radioactive Log' and 'Radioactive Monitoring Swabs' sheets recorded separately from this study.

THE LOCAL LYMPH NODE ASSAY - STUDY NUMBER
GROUP-POOLED LYMPH NODES

Background and control raw data retrieved from the β-scintillation counter

Results:

  1. Background and Control Results
Background 3HTdR in two 1 ml TCA samples was determined and 3HTdR incorporation into control LNC determined days after the first vehicle topical application.

β-Counter printout inserted here

Rack/Sample Position Sample Description No. Lymph Nodes Sample DPM
 
 
 
 

Mean background count : DPM

THE LOCAL LYMPH NODE ASSAY - STUDY NUMBER
GROUP-POOLED LYMPH NODES

Test raw data retrieved from β-scintillation counter

Test substance :
Sample ref. no. :

3HTdR incorporation into test LNC determined days after the first test substance topical applications.

Results:

  1. Test Results

β-Counter printout inserted here

Rack/Sample Position Sample Description No. Lymph Nodes Sample DPM
 
 
 
 

THE LOCAL LYMPH NODE ASSAY - STUDY NUMBER
GROUP-POOLED LYMPH NODES

Expression and interpretation of results

Test substance :
Sample ref. no. :
Exposure period (days) :

The proliferative response of lymph node cells (LNC) is expressed as the number of radioactive disintergrations per minute per lymph node (DPM/NODE) and as the ration of 3HTdR incorporation into LNC of test nodes relative to that recorded for control nodes (TEST/CONTROL RATIO). The test substance can be regarded as a 'sensitizer' if at least one test concentration produces a test/control ration equal to or greater than 3.0. The data must also be compatible with a biological dose response, although allowance must be made, especially at high topical application concentrations, for local toxicity and/or immunological suppression. Where the data does not fulfill these criteria, the test substance can be regarded as 'unlikely to be a strong sensitizer'.

Background count : DPM

Sample Description Sample DPM - Background DPM No. Lymph Nodes DPM/NODE TEST/CONTROL RATIO +/-
 
 
 
 

Biological dose response - Yes/No.

Comments:

THE LOCAL LYMPH NODE ASSAY - STUDY NUMBER
INDIVIDUAL ANIMAL LYMPH NODES

Control raw data retrieved from the β-scintillation counter

Results:

  1. Control Results
3HTdR incorporation into control LNC determined days after the first vehicle topical application.

β-Counter printout inserted here

Rack/Sample Position Sample Description No. Lymph Nodes Sample DPM
 
 
 
 

Mean background count : DPM

THE LOCAL LYMPH NODE ASSAY - STUDY NUMBER
INDIVIDUAL ANIMAL LYMPH NODES

Test raw data retrieved from β-scintillation counter

Test substance :
Sample ref. no. :

3HTdR incorporation into test LNC determined days after the first test substance topical applications.

Results:

  1. Test Results

β-Counter printout inserted here

Rack/Sample Position Sample Description No. Lymph Nodes Sample DPM
 
 
 
 

THE LOCAL LYMPH NODE ASSAY - STUDY NUMBER
INDIVIDUAL ANIMAL LYMPH NODES

Expression and interpretation of results

Test substance :
Sample ref. no. :
Exposure period (days) :

The proliferative response of lymph node cells (LNC) is expressed as the number of radioactive disintergrations per minute per lymph node (DPM/NODE) and as the ration of 3HTdR incorporation into LNC of test nodes relative to that recorded for control nodes (TEST/CONTROL RATIO). The test substance can be regarded as a 'sensitizer' if at least one test concentration produces a test/control ration equal to or greater than 3.0. The data must also be compatible with a biological dose response, although allowance must be made, especially at high topical application concentrations, for local toxicity and/or immunological suppression. Where the data does not fulfill these criteria, the test substance can be regarded as 'unlikely to be a strong sensitizer'.

Background count : DPM

Sample Description
Test or Control Group		 Group Mean DPM Group Mean Standard Error TEST/CONTROL RATIO +/-
 
 
 
 

Biological dose response - Yes/No.

Comments:

THE LOCAL LYMPH NODE ASSAY - STUDY NUMBER

Mouse maintenance, treatment record, and task sheet

Strain : CBA/_____
Sex : Female
Age :
Source :
Diet :
Water : Ad libitum
Housing :

Test substance :
Sample ref. no. :

Animal Group Topical Application Admin. of 3HTdR Mice Killed Processing Nodes Samples Counted
Day 0 Day 1 Day 2   No. Mice Injured   No. Nodes Excised
 
 
 
 

More information concerning animal maintenance (including diet batch numbers) are detailed on the 'Animal Log' sheet recorded separately from this study.

Comments: