National Toxicology Program

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Abstract from Report I88486 on 3,3',4,4'-Tetrachloroazobenzene

Range-Finding Report on the Immunotoxicity of 3,3',4,4'-Tetrachloroazobenzene in Female Sprague Dawley Rats (CAS No. 14047-09-7)

Report Date: November 2012

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 The findings have not been peer reviewed and were not evaluated in accordance with the levels of evidence criteria established by NTP in March 2009.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.


3,3',4,4'-Tetrachloroazobenzene is not commercially manufactured but is present as a contaminant of 3,4-dichloroaniline and its herbicidal derivatives Propanil, Linuron, and Diuron (Sundstrom et al., 1978; Bunce et al., 1979; Carey et al., 1980; Hill et al., 1981). In addition, environmental contamination occurs when TCAB is formed by the photolysis and biolysis of 3,4-dichloroaniline. TCAB was nominated by the United States Environmental Protection Agency for toxicity testing, based on concerns over the potential for human exposure, the structural resemblance to the potent immunotoxic and carcinogenic agent 2,3,7,8-tetrachlorodibenzo-p-dioxin, and reports that TCAB demonstrated dioxin-like effects (van Birgelen et al., 1999). The toxicity of TCAB was evaluated in 13-week gavage studies in male and female F344/N rats and B6C3F1 mice (van Birgelen et al., 1999; NTP, 1998). Results of these studies demonstrated decreased thymus weights, thymic atrophy, and macrocytic anemia in male and female rats following exposure to TCAB at doses ≥ 10 mg/kg. Thymic atrophy has also been reported 10 days following a single intraperitoneal administration of TCAB in Sprague Dawley rats (Hsia et al., 1982). These effects suggest that TCAB may be immunotoxic. A comparison of various dioxin-like effects with those reported in the literature indicate that TCAB is two to six orders of magnitude less potent than TCDD (van Birgelen et al., 1999; NTP, 1998).

The National Toxicology Program requested that a dose range-finding study be performed to establish the potential effects of TCAB on the immune system. These studies were conducted in female Sprague Dawley rats. Five dose levels of TCAB (0.1, 0.5, 1, 5, and 10 mg/kg) were administered by oral gavage for 28 days, using acetone/corn oil (1/99 v/v) as the vehicle.

Female Sprague Dawley rats exposed to TCAB by oral gavage demonstrated no overall effects on terminal body weights, however, body weight gain was decreased (11-15%) at the 10 mg/kg dose level throughout the 28-day exposure period. No effects were observed on the weights of major organs of the immune system, i.e., the thymus and the spleen. Absolute and relative liver weights were significantly increased in a dose-related manner following TCAB exposure. Effects on several hematological parameters suggesting that TCAB exposure resulted in anemia were also observed, primarily at the 10 mg/kg dose. Specifically, hemoglobin concentration, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration were significantly decreased, while reticulocyte percentages were significantly increased, each in a dose-dependent manner.

Total splenocyte number and absolute splenic phenotypes were unaffected by TCAB exposure, with the exception of significant decreases in natural killer cell numbers at several dose levels. No effects were observed in either the antibody-forming cell assay or the keyhole limpet hemocyanin enzyme-linked immunosorbent assay, suggesting that TCAB exposure does not adversely affect humoral immunity. Effects on cell-mediated immunity were varied. No overall effects were observed on the anti-CD3-mediated proliferative response, while decreases in the delayed-type hypersensitivity response to Candida albicans were observed. Minimal effects on innate immunity were also observed. The activity of the mononuclear phagocytic system was decreased in the liver and increased in both the spleen and the lung. NK cell activity was not affected by TCAB exposure.

In conclusion, based on the weight of evidence, these results suggest that TCAB has minimal immunosuppressive activity when administered for 28 days by oral gavage to female Sprague Dawley at doses up to 10 mg/kg.


Sundstrom G., Jansson B., and Renberg L. (1978). Determination of the toxic impurities 3,3',4,4'-tetrachloroazobenzene and 3,3',4,4'-tetrachloroazoxybenzene in commercial diuron, linuron, and 3,4-dichloroaniline samples. Chemosphere 12:973-9.

Bunce N.J., Corke C.T., Merrick R.L., and Bright J.H. (1979). 3,3'4,4'-Tetrachloroazobenzene as a contaminant in commercial propanil. Chemosphere 8:283-4.

Carey A.E., yang H.S., Wiersma G.B., Tai H., Maxey R.A., Dupuy A.E. Jr. (1980). Residual concentrations of propanil, TCAB, and other pesticides in rice-growing of soils in the United States, 1972. Pestic Monit J, 14(1):23-5.

Hill R.H. Jr., Rollen Z.J., Kimbrough R.D., Groce D.F., Needham L.L. (1981). Tetrachloroazobenzene in 3,4-dichloroaniline and its herbicidal derivatives: propanil, diuron, linuron, and neburon. Arch Environ Health, 36(1):11-4.

van Birgelen A.P.J.M., Hebert C.D., Wenk M.L., Grimes L.K., Chapin R.E., Mahler J., Travlos G.S., and Bucher J.R. (1999). Toxicity of 3,3',4,4'-tetrachloroazobenzene in rats and mice. Toxicol Appl Pharmacol 156:147-59.

NTP (1998). NTP Technical Report on the Toxicity Studies of 3,3',4,4'-Tetrachloroazobenzene (CAS No. 14047-09-7) Administered by Gavage to F344/N Rats and B6C3F1 Mice. Toxic Rep Ser. 65:1-F6.

Hsia M.T., Burant C.F., Kreamer B., Schrankel K.R. (1982). Thymic atrophy induced by acute exposure of 3,3',4,4'-tetrachloroazobenzene and 3,3',4,4'-tetrachloro-azoxybenzene in rats. Toxicology, 24(3-4):231-44.

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