National Toxicology Program

National Toxicology Program

IMM87099: Immunotoxicity 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 The findings have not been peer reviewed and were not evaluated in accordance with the levels of evidence criteria established by the NTP on March 2009 (see The findings and conclusions for this study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), one of the substituted benzenediamines of the oxidative bases, is used in dyes and is, therefore, a dyestuff intermediate. It has received attention as a potential carcinogen because of the high incidence of bladder cancer reported among dye manufacturing industry workers1, 2. There are no studies conducted on the potential immunotoxicity of CPD. As a result of the usage of the benzenediamines in the work place and the fact that the class of chemicals is implicated as a carcinogen, it was selected by the National Toxicological Proogram (NTP) for immunotoxicological assessment.

The doses selected for evaluation of the immune system as a target for toxicity were 50, 200 and 400 mg/kg. From probe studies, higher doses of 400 mg/kg/day for 14 days had lethal actions in the B6C3F1 mouse; thus, the high dose used in the immunotoxicity evaluation is considered to be a maximum tolerated dose for 14 days of oral exposure. However, this maximum tolerated dose produced no major toxicities. Thus, the dose response curve for lethality from 14 days exposure is between 400 and 600 mg/kg.

CPD at doses between 50 and 400 mg/kg per day for 14 days by gavage lack immunotoxicity as well as toxicity to other organ systems. The toxicology studies are summarized in Table ES-1. Mice exposed to CPD in doses up to 400 mg/kg for 14 days by gastric gavage showed no change in body weight or body weight gain. There was a slight increase in liver and kidney weight in mice exposed to CPD. No gross pathology or histopathology was observed in the liver, spleen, lungs, thymus, kidney or mesenteric lymph nodes. There were no biologically meaningful changes in selected hematological or serum chemistry parameters. The immunology studies are summarized in Table ES-2. CPD, administered by gastric gavage in doses between 50 and 400 mg/kg, did not suppress any of the immunological parameters measured and the immune system is not considered a target for toxicity. Serum complement levels were elevated dose-dependently. There was no effect on the IgM response to sRBC and the DHR response to KLH. FC mediated macrophage adherence and phagocytosis of chicken erythrocytes were unaffected. The percentage of T & B lymphocytes was not altered. Responses to mitogens and allogeneic cells were unaffected as was macrophage status and natural killer cell activity. The host resistance studies are summarized in Table ES-3. One bacterial and one tumor host resistance model were employed in these studies. CPD exposure for 14 days did not alter host resistance to the bacterial challenge, Streptococcus pneumoniae, the pulmonary tumor model, B16F10, or the PYB6 Fibrosarcoma.

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 10 September 1987 and 17 November 1987. The animals were housed in the animal facility of the Strauss Building.

Table ES-1
Parameter Results Maximum Effect Dose Comments
Body Weight No effect

Gross Pathology None

Histopathology None

Organ Weights
Liver Increased 16.2% 400mg/kg Dose Dependent
Spleen No effect

Lungs No effect

Thymus No effect

Kidney Increased 12% 400mg/kg Dose Dependent
Brain No effect

RBC's No effect

Rede's No effect

Leukocytes No effect

Leukocyte Diff

Lymphocytes No biologically meaningful change
NeutrophUs No biologically meaningful change
Serum Chemistries
SGPT No effect

Albumin No effect

BUN No effect

Glucose No biologically meaningful change
Protein No effect

Table ES-2
Parameter Results Maximum Effect Dose
T Cells No effect

B Cells No effect

IgM AFC to sRBC No effect

IgG AFC to sRBC No effect

DHR to KLH No effect

Mitogens- Con A No effect

-PHA No effect

-LPS No effect

-Medium No effect

MLR No effect

NK Cell Activity No effect

Serum Complement
CH50 Increased 33% Yes
C3 Increased 12% Yes
Macrophage (Peritoneal)
Phagocytosis cRBC No biologically meaningful effect
Adherence of cRBC No biologically meaningful effect

Table ES-3
MODEL Results Maximum Effect Dose Comments
Streptococcus pneumoniae No effect

PYB6 No effect

B16F10 No effect

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