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Abstract for IMM98008, IMM98006, and IMM98007

Assessment of Contact Hypersensitivity to Dinitrochlorobenzene, Potassium Dichromate, and Methyl Salicylate in BALB/c Female Mice

Substances:

  • Dinitrochlorobenzene (CASRN 97-00-7)
  • Potassium Dichromate (CASRN 7778-50-9)
  • Methyl Salicylate (CASRN 119-36-8)

Report Date: December 1997

Abstract

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 The findings have not been peer reviewed and were not evaluated in accordance with the levels of evidence criteria established by NTP in March 2009.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

Introduction

The objective of this study was to determine the sensitizing potential of Dinitrochlorobenzene, Potassium Dichromate and Methyl Salicylate when applied dermally to female BALB/C mice and to compare results obtained in this laboratory with those obtained in the international Local Lymph Node Assay (LLNA) validation studies.

Design

Dinitrochlorobenzene, potassium dichromate and methyl salicylate were obtained from Sigma Chemical Company (St. Louis, MO.). Dilutions of methyl salicylate were made in acetone/olive oil (4:1 potassium dichromate in dimethyl sulfoxide (DMSO) and dinitrochlorobenzene in acetone/olive oil (4:1 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 the test articles. The chemical concentrations and experimental groups used in the assays are shown in the table below.

For irritancy testing, three mice per group using both ears (N=6) were used to test the designated concentrations of test article in vehicle, vehicle alone and untreated controls. Animals were dosed on 4 consecutive days and checked daily for signs of morbidity. The lowest concentration of test article demonstrating significant irritation when compared to vehicle controls, as determined by the percent ear swelling at 24 hrs post last treatment, was considered the MIC. The highest concentration not to demonstrate a significant irritation over the vehicle controls was considered the MNC. Challenge concentrations for the MEST were chosen based on the MIC. For the MEST pre-, 24 and 48 hr post treatment ear measurements were taken and used to calculate the percent ear swelling. Measurements from animals dosed with varying concentrations of test article were compared to the test article background control for significance. In the LLNA, following 3 consecutive days of dosing, animals were rested for 1 day and then injected with 3H-thymidine. Draining lymph nodes were dissected out 5 hours later and radioassayed. Chemical exposed groups were compared to vehicle controls for statistical significance.

Results

TNo test article related mortality was seen in animals dosed with DNC, PDC or MSC. There were no statistically significant changes in body weight of test groups of animals as compared to vehicle groups during the course of these studies The Minimal Irritating Concentrations of DNC and PDC were determined to be 0.5%. MSC was found to be non-irritating at the highest dose tested. DNC and PDC were identified as potential c0ontact sensitizers by both the MEST and the LLNA. MSC did not test positive in the MEST and only tested positive at the highest concentration tested, 20%, in the LLNA. Results from the MEST and LLNA were similar for the strong sensitizer, DNC. In the case of the weaker sensitizers PDC and MSC, the LLNA was a more sensitive indicator. In comparing results from our laboratory with those from labs involved in the Local Lymph Node international validation studies, our results were consistent with the other labs performing statistical analysis. For DNC,.05% was the lowest concentration showing statistical significance here at MCV, at Proctor & Gamble and IITRI. DuPont detected statistical significance at a concentration of .1%. For PDC, .05% was the lowest concentration detected as significant at MCV. DuPont and IITRI detected significance with PDC at a concentration of 0.10%, and Proctor & Gamble at 0.25%. MSC showed statistical significance at the highest concentration tested, 20%, in the labs at MCV, IITRI and Proctor & Gamble. This compound tested negative in all other labs.

Conclusions

TThe Minimal Irritating Concentrations of both DNC and PDC were determined to be 0.5%. MSC was found to be non-irritating at the highest dose tested. DNC and PDC were identified as potential contact sensitizers by both the MEST and the LLNA. MSC did not test positive in the MEST and only tested positive at the highest concentration tested, 20%, in the LLNA. Results from the MEST and LLNA were similar for the strong sensitizer, DNC. In the case of the weaker sensitizers PDC and MSC, the LLNA was a more sensitive indicator. In comparing results from our laboratory with those from labs involved in the Local Lymph Node international validation studies, our results were consistent with the other labs performing statistical analysis. For DNC, .05% was the lowest concentration showing statistical significance here at MCV, at Proctor & Gamble and IITRI. DuPont detected statistical significance at a concentration of .1%. For PDC, .05% was the lowest concentration detected as significant at MCV. DuPont and IITRI detected significance with PDC at a concentration of .10%, and Proctor & Gamble at 0 .25%. MSC showed statistical significance at the highest concentration tested, 20%, in the labs at Medical College or Virginia, IITRI and Proctor & Gamble. This compound tested negative in all other labs.

Studies

Test Article Dose
Group
Vehicle Test Article Concentration
      Irritancy LLNA MEST
Sensitization
MEST
Challenge
Dinitrochlorobenzene VH acetone:olive oil 0% 0% 0% 0%
Dinitrochlorobenzene BC acetone:olive oil NA NA 0%; 0.5%
Dinitrochlorobenzene D1 acetone:olive oil 0.01% 0.01% 0.01% 0.5%
Dinitrochlorobenzene D2 acetone:olive oil 0.025% 0.025% 0.025% 0.5%
Dinitrochlorobenzene D3 acetone:olive oil 0.05% 0.05% 0.05% 0.5%
Dinitrochlorobenzene D4 acetone:olive oil 0.1% 0.1% 0.1% 0.5%
Dinitrochlorobenzene D5 acetone:olive oil 0.25% 0.25% 0.25% 0.5%
Dinitrochlorobenzene D6 acetone:olive oil 0.5% 0.5% 0.5% 0.5%
 
Potassium Dichromate VH DMSO 0% 0% 0% 0%
Potassium Dichromate BC DMSO NA NA 0% 0.5%
Potassium Dichromate D1 DMSO 0.025% 0.025% 0.025% 0.5%
Potassium Dichromate D2 DMSO 0.05% 0.05% 0.05% 0.5%
Potassium Dichromate D3 DMSO 0.10% 0.10% 0.10% 0.5%
Potassium Dichromate D4 DMSO 0.25% 0.25% 0.25% 0.5%
Potassium Dichromate D5 DMSO 0.50% 0.50% 0.50% 0.5%
 
Methyl Salicylate VH acetone:olive oil 0% 0% 0% 0%
Methyl Salicylate BC acetone:olive oil NA NA 0% 20.0%
Methyl Salicylate D1 acetone:olive oil 1.00% 1.0% 1.0% 20.0%
Methyl Salicylate D2 acetone:olive oil 2.5% 2.5% 2.5% 20.0%
Methyl Salicylate D3 acetone:olive oil 5.0% 5.0% 5.0% 20.0%
Methyl Salicylate D4 acetone:olive oil 10.0% 10.0% 10.0% 20.0%
Methyl Salicylate D5 acetone:olive oil 20.0% 20.0% 20.0% 20.0%
Abbreviations: VH=Vehicle, BC=Background control; D=Dose