National Toxicology Program

National Toxicology Program

IMM89029: NTP Assessment of Contact Hypersensitivity of 4-Chloro-o-phenylenediamine (CASRN 95-83-0) in Female B6C3F1 Mice


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 The findings have not been peer reviewed and were not evaluated in accordance with the levels of evidence criteria established by the NTP on March 2009 (see The findings and conclusions for this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


4-chloro-o-phenylenediamine (CPD) was selected for evaluation as a sensitizing agent for contact hypersensitivity in mice. 4-chloro-o-phenylenediamine is used in dyes and is, therefore, a dyestuff intermediate. It is also used in the manufacture of 5-chlorobenzotriazole.

The objective of this study was to determine the sensitizing potential of 4-chloro-o-phenylenediamine when applied dermally to female B6C3F1 mice.

4-chloro-o-phenylenediamine was tested on female B6C3F1 mice. The doses of 4-chloro-o-phenylenediamine ranged from 1% to 10% in a solution of 4 parts acetone to one part olive oil (4:1) for sensitization and 10% for challenge. Mice received 20 ┬Ál by direct dermal application for 5 consecutive days to a prepared site. DNFB (1-fluoro-2,4-dinitrobenzene) (99.6%, Sigma Chemical Co.) was used as a positive control at a concentration of 0.5%. Measurement of allergic contact hypersensitivity was accomplished by a radioisotopic assay and the mouse ear swelling test.

A dose-dependent contact hypersensitivity response to 4-chloro-o-phenylenediamine was demonstrated in mice by the radioisotopic assay whether or not the site of sensitization was prepared using Freund's complete adjuvant. However, a statistically significant response was observed only in animals prepared using adjuvant and sensitized with 10% 4-chloro-o-phenylenediamine. The mouse ear swelling test detected a dose-dependent contact hypersensitivity response without adjuvant pretreatement at one and two days after challenge. A statistically significant response was detected only on the first day following challenge in mice sensitized and challenged with 10% 4-chloro-o-phenylenediamine.

Summary of Irritancy and Contact Hypersensitivity Results
Type of Response Day of
Method Used Adjuvant Dose Response
(P < 0.05)*
Positive Response
(P < 0.05)*
No No No
Day +1 MEST No Yes 10%

Day +2 MEST No Yes No

Yes Yes 10%
Abbreviations: MEST - Mouse Ear Swelling Test
*When a positive response is detected the minimum sensitizing concentration is listed

The studies were conducted at the Medical College of Virginia Immunotoxicology Laboratory under NTP Contract No. ES 55094. The in-life phase of the studies was conducted between 1 October 1987 and 16 November 1987. The animals were housed in the animal facility of the Strauss Building. To the best of our knowledge, no significant deviations from Good Laboratory Practices occurred that affected the quality of the data and the ability to interpret the data with respect to the sensitizing potential of 4-chloro-o-phenylenediamine.

NTP is located at the National Institute of Environmental Health Sciences, part of the National Institutes of Health.