National Toxicology Program

National Toxicology Program

Abstract for IMM94008 - Methadone (CASRN 1095-90-5)

The following abstract presents results of a study conducted by a contract laboratory for the National Toxicology Program (NTP). The findings have not been peer reviewed and were not evaluated in accordance with the levels of evidence criteria established by the NTP on March 2009 (see The findings and conclusions for this study should not be construed to represent the views of the NTP or the US Government.

The Immunotoxicity of Methadone (CAS No.1095-90-5) in Female B6C3F1 Mice

NTP Report Number IMM94008


Methadone was nominated to the NTP for toxicological evaluationand was selected for immunotoxicity studies by the chemical manager.Thus, the purpose of these studies was to determine the effectof methadone on the immune system and host resistance to selectedmicrobial models and to compare the effects to those of previouslypublished work on morphine sulfate.

The studies were conducted in female B6C3F1mice. The animals were administered methadone on day 1 at dosesof 10, 20 and 30 mg/kg. Methadone was administered by subcutaneousadministration as a solution in sterile distilled water. In range-findingstudies, serum methadone and corticosterone levels peaked onehour following a single subcutaneous injection of 20 mg/kg methadoneHCl (Fig 3). After a single injection with 20 mg/kg methadone,the pharmacokinetics revealed a serum half-life of nearly equal to 2 hours (Fig2). Following five injections over a 24 hour period (every 6 hours),methadone levels were elevated as would be expected; however,corticosterone levels were not increased (Fig 4). This suggestedthat the ability of methadone to elevate corticosterone becameuncoupled following repeated dosing, indicative of a tolerancemechanism. Moreover, dosing every 6 hours for five days inducedan increase in the catabolism of methadone itself (Fig 5). Therefore,all assays were begun one hour after subcutaneous administrationof methadone HCl, a time at which both methadone and corticosteroneserum levels were elevated.

Executive Summary Table 1 (ES-1) shows a summaryof the standard toxicology studies. Body weight, body weight change,organ weights and hematological parameters were measured. Therewas a 48% decrease in body weight change five days after the highdose of 30 mg/kg. None of the organ weights were significantlyaltered with the exception of the lungs which increased in weightby 15%. There was a dose-dependent increase in red blood cellnumber of 9% at a dose level of 30 mg/kg. Additionally, MCV andMCH were decreased at the 30 mg/kg dose level, suggesting a decreasein red blood cell size while the hematocrit remained stable. Therewas no change in the leukocyte differential; however, there wasa 33% increase in leukocyte number at the low dose of 10 mg/kg.This increase in leukocyte number was not apparent at the mediumand high dose exposure to methadone.

Executive Summary Table 2 (ES-2) summarizesthe immunologic studies of mice administered methadone HCl. Immunologicassays were performed one hour after exposure to methadone duringthe peak in both serum methadone and corticosterone. Therefore,the changes in immune function may, in part, be associated withelevated corticosterone levels. Based on absolute values, therewas a decrease in both CD4+CD8+ and CD4-CD8+ T lymphocytes followingexposure to 30 mg/kg methadone. The proliferative response toallogeneic cells was decreased, while CTL activity was increasedat the lower effector-to-target ratios of 1.5:1 and 0.75:1. Naturalkiller cell activity was increased following administration of30 mg/kg methadone at effector-to-target ratios of 1:100 and 1:25.The most pronounced of the immunologic effects of methadone wasa dose-dependent 59% decrease in Kupffer cell phagocytic activity.

In the two studies conducted, there was no change in host resistanceto either Listeria monocytogenes or Streptococcus pneumoniaefollowing exposure to methadone HCl [Executive Summary Table 3 (ES-3)].

In conclusion, methadone appears to have several effects on theimmune system of female B6C3F1 mice. While there was a decreasedability of splenocytes to proliferate in response to the alloantigen,DBA/2 cells, there was a significant increase in the activityof cytotoxic T lymphocytes at the lower effector:target ratiosone hour after exposure to methadone. The most pronounced effectof methadone was a dose-dependent decrease in the phagocytic activityof Kupffer cells of the liver. There was also a significant decreasein the ability of resident macrophages of the spleen to phagocytizesRBC. Although the animals had a compromised ability to phagocytize,host resistance to two bacterial models was unaltered. This suggeststhat the decrease in phagocytic capacity, induced by methadoneexposure, was not sufficient to adversely effect the immunocompetenceof the system.


Table ES-1
Parameter Result Maximum Effect Dose Comment
Body Weight        
Day 1 No Effect      
Day 5 No Effect      
Weight Changes        
Day 5-1 Decrease 48%  30 mg/kg   
Gross Pathology No Effect      
Histopathology Not Done      
Organ Weights        
Brain Decreased 6%  30 mg/kg   
Liver No Effect      
Spleen No Effect      
Lungs Increased 15%  20 mg/kg   
Thymus No Effect      
Kidney No Effect      
RBCs Increased 9%  30 mg/kg 
Dose Dependent
Hemoglobin Increased 4%  10 mg/kg   
Hematocrit No Effect      
MCV Decreased 6%  30 mg/kg   
MCH Decreased 5%  30 mg/kg   
MCHC No Effect      
Reticulocytes No Effect      
Leukocytes Increase 33%  10 mg/kg   
Leukocyte Diff No Effect      
Lymphocytes No Effect      
Neutrophils No Effect      
Eosinophils No Effect      



Table ES-2


Surface Markers
Parameter Result Maximum Effect Dose Comment
Ig+ Decreased 17%  10 mg/kg   
CD3+ No Effect      
CD4+CD8- No Effect     Based on Absolute Values
CD4-CD8+ Decrease 17%  30 mg/kg   
CD4+CD8+ Decrease 31%  30 mg/kg   
Spleen IgM Antibody-Forming Cell Response to Sheep Erythrocytes
Parameter Result Maximum Effect Dose Comment
IgM AFC to sRBC No Effect      
Proliferation Assay, Mixed Leukocyte Response
Parameter Result Maximum Effect Dose Comment
MLR Decrease     Dose Dependent
Stimulation Index Decrease 50% 30 mg/kg   
Cytotoxic T Lymphocyte Activity
Parameter Result Maximum Effect Dose Comment
CTL Increased 90%
20 mg/kg  1.5:1


Reticuloendothelial System
Parameter Result Maximum Effect Dose Comment
RES Decreased 59% 30 mg/kg  Dose Dependent
Liver Phagocytosis
NK Cell Activity
Parameter Result Maximum Effect Dose Comment
1:100 Increased 22% 30 mg/kg   
1:50 No Effect      
1:25 Increased 17% 30 mg/kg   



Table ES-3


Parameter Results Maximum Effect Dose Comment
Listeria monocytogenes No Effect       
Streptococcus pneumoniae No Effect       

Report Date: March 1994

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