National Toxicology Program

National Toxicology Program

NTP Report on the Assessment of Contact Hypersensitivity to Azithromycin in BALB/c Female Mice 
(CASRN: 83905-01-5)

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 The findings have not been peer reviewed and were not evaluated in accordance with the levels of evidence criteria established by the NTP on March 2009 (see The findings and conclusions for this study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.

Assessment of Contact Hypersensitivity to Azithromycin in BALB/c Female Mice
(CASRN: 83905-01-5)

NTP Report Number IMM97010


Introduction: Azithromycin (AZI) has been nominated for testing in the immunotoxicology program in part due to its potential for heightened drug allergy, its increased use in pediatric settings, and its use in the treatment of secondary infections associated with AIDS. AZI is an azilide antibiotic that has improved acid stability and enhanced pharmacokinetic properties. In addition, it induces minimal gastrointestinal irritation and has a wider spectrum of antimicrobial activity than other commonly used macrolide antibiotics such as erythromycin.

Design: Azithromycin (purity approximately 97%) was provided by NIEHS. Dilutions were made in acetone which served as the vehicle. Three assays were used in this assessment, a primary irritancy study to screen for toxicity and determine the minimal irritating concentration (MIC) and the maximal non-irritating concentration (MNC), and two assays, the mouse ear swelling test (MEST) and the local lymph node assay (LLNA), to test the dermal sensitizing potential of AZI.

For irritancy testing four mice per group were used to test six concentrations of test article in vehicle, vehicle alone and untreated controls . The test article did not induce a statistically significant increase in percent ear swelling when compared to the vehicle control when doses as high as 40% AZI were employed. Thus, the irritancy study did not establish an MIC or an MNC. Therefore, 10%, 20%, and 40% AZI (w/v) were the concentrations used for dermal sensitization in the MEST and LLNA.

The design for the MEST is shown in the chart below. Pre-, 24 and 48 hr post-treatment ear measurements were taken and used to calculate the percent ear swelling. Percent ear swelling from animals dosed with varying concentrations of test article were compared to the percent ear swelling for the AZI background control group for significance.



MEST design
Group Exp Group No. of Mice Sensitization Challenge
1 VH 8 Acetone Acetone
2 BC 8 Acetone 40.0% AZI
3 D1 8 10,0% AZI 40.0% AZI
4 D2 8 20.0% AZI 40.0% AZI
5 D3 8 40.0% AZI 40.0% AZI
6 BP 8 Acetone 0.15% DNFB
7 PC 8 0.15% DNFB 0.15% DNFB

Abbreviations: VH=vehicle; BC=Background control; BP=backgound positive;
PC=positive control; AZI=azithromycin, DNFB=2,4-dinitrofluorobenzene

The chemical concentrations and experimental groups used for the LLNA are shown in the chart below. After 3 consecutive days of dosing, animals were rested for I day and then injected with H-thymidine, 5 hours later the draining lymph nodes were dissected out and radioassayed. Chemical exposed groups were compared to the vehicle control group for statistical significance.


LLNA design
Exp Group No. of Mice Induction
VH 6 Vehicle
D1 6 10.0% AZI
D2 6 20.0% AZI
D3 6 40.0% AZI
PC 6 0.15% DNFB

Abbreviations: VH=vehicle; PC=positive control;
AZI=azithromycin, DNFB=2,4-dinitrofluorobenzene

Results: No signs of toxicity were seen in AZI exposed animals. The maximal dose tested, 40.0% was limited by the solubility of the test article. No significant irritation was seen when AZI (at concentrations as high as 40%) was dermally applied to mice. A contact hypersensitivity response was not demonstrated by the MEST or the LLNA in AZI exposed animals. The positive control group in the MEST produced a statistically significant response at a sensitizing concentration of 0.15% and challenge concentration of 0.15% DNFB as compared to the 0.15% DNFB challenge only group. In the LLNA the positive control (0.15% DNFB) produced a significant response as compared to the vehicle treated group.

Conclusions: Azithromycin was not found to produce irritation at concentrations as high as 40%. Testing for sensitizing potential using the mouse ear swelling test and local lymph node assay at a maximum concentration of 40.0% failed to indicate AZI as a contact sensitizer.

Report Date: June 1998

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