National Toxicology Program

The Immunotoxicity of Meta-nitrotoluene (CAS No. 99-08-1)in Female B6C3F₁ Mice

NTP Report Number IMM95002

Summary

The mono-nitrotoluenes are synthesized by the nitration of toluene. Meta-nitrotoluene (m-nitrotoluene) is a clear, yellow liquid at ambient temperature. The mono-nitrotoluenes are slightly soluble in aqueous solution but are readily soluble in organic solvents. The nitrotoluenes have been detected in air, water, and soil.^{1, 2} Meta- and para-nitrotoluene (p-nitrotoluene) have been identified by the National Toxicology Program (NTP) for detail study. M-nitrotoluene was the second mono-nitrotoluene to be investigated for its potential adverse actions on the immune system.

The objective of the studies reported here was to determine the potential immunotoxicity of m-nitrotoluene in mice exposed by the oral route. The studies were divided into three areas: the more standard toxicology studies provided the base against which to compare the immunology studies; the immunology studies provided data on specific cellular targets of the xenobiotic; and the host resistance studies were used for a global assessment of the ability of the exposed animal to resist pathogenic challenge.

Executive Summary Table 1 (ES-1) shows a summary of the standard toxicology studies. Mice exposed to m-nitrotoluene by gastric gavage gained body weight over the 2 week experimental period to a slightly greater extent than the control groups. Of the selected organs weighed, the liver and kidney of mice exposed to m-nitrotoluene were increased in weight while the thymus weight was decreased. The liver of mice exposed to m-nitrotoluene, but not ortho-nitrotoluene (o-nitrotoluene), showed slight to moderate swelling of the hepatocytes adjacent to the central veins. The hepatocyte swelling appeared to be reversible and there was no evidence of necrosis. The selected hematological and serum chemistries were unaffected by m-nitrotoluene exposure. Bone marrow cellularity and the number of CFU/M and CFU/GM were unaffected by m-nitrotoluene exposure.

Executive Summary Table 2 (ES-2) summarizes the immunology studies of mice exposed to m-nitrotoluene. M-nitrotoluene suppressed the IgM response to sRBC and the DHR response to KLH. There was a slight (13%) decrease in the percentage of T lymphocytes in the spleen. The response to the T cell mitogens was suppressed by as much as 39.2%. FC mediated adherence and phagocytosis of chicken erythrocytes were increased dose-dependently in mice exposed to m-nitrotoluene. All of the other immune parameters were unaffected by exposure to m-nitrotoluene. These parameters included natural killer cell activity, response to mitogens and allogeneic cells, and serum complement and interferon levels.

Executive Summary Table 3 (ES-3) summarizes the host resistance studies in mice exposed to m-nitrotoluene. Exposure of mice to m-nitrotoluene decreased resistance to Listeria monocytogenes. The decreased resistance to Listeria monocytogenes may be related to the decrease in the DHR to KLH. Resistance to Streptococcus pneumoniae and Plasmodium yoelii were unaffected. Resistance to the two tumor models the PYB6 tumor and the B16F10 melanoma was increased.

The three phases of these studies indicate that the liver and, to an equal or greater extent, the immune system are a target for toxicity of m-nitrotoluene. The decreased host resistance to Listeria monocytogenes can be attributed to the decrease in T lymphocytes and, as seen in the number and activity as expressed, to the decreased delayed hypersensitivity response to Keyhole limpet hemocyanin. The decrease in IgM response did not correlate with a host resistance model, most likely because no effect was seen on the IgG response. The increased resistance seen in the tumor models may be related to the increased macrophage activity; i.e., increase ectoenzyme activity and increased FC mediated adherence of chicken erythrocytes.

With the completion of the studies on m-nitrotoluene, three mono-nitrotoluenes were investigated. O-nitrotoluene was used as a comparative control with the dose-response studies on m-nitrotoluene, and p-nitrotoluene was evaluated previously. Meta- and para-nitrotoluene produced greater actions on the immune system than ortho-nitrotoluene. Humoral and cell mediated immunity were affected. Figure ES-1 below shows the structure activity of the three isomers for one humoral immune response to the T-dependent antigen sheep erythrocytes. The delayed hypersensitivity response to Keyhole limpet hemocyanin was suppressed with m- and p-nitrotoluene. All three isomers produced a state of decreased resistance to Listeria monocytogenes.Because these actions were at doses which produced no overt toxicity and only minor actions on the liver, the immune system for the mono-nitrotoluenes must be considered an important target which is at least as equivalent as the liver and the central nervous system.

The studies were conducted at the Medical College of Virginia Immunotoxicology Program under NTP Contract No. ES 55094. The in-life phase of the studies was conducted between 7 January 1987 and 3 July 1987. The animals were housed in the animal facility of the Strauss building.

 

Parameter	Result	Maximum Effect	Dose	Comment
Periodic Weights	Increase in weight gain	26% (weight gain)	600 mg/kg
Gross Pathology	None
Histopathology	Moderate to mild swelling of hepatocytes		400 mg/kg
Organ Weights
Liver	Increased	27%	600 mg/kg
Spleen	No Effect
Lungs	No Effect
Thymus	Decreased	18%	600 mg/kg
Kidney	Increased	11%	600 mg/kg
Brain	No Effect
Hematology
RBCs	No effect
Retic's	Increased	49%	600 mg/kg
Leukocytes	No Effect
Leukocyte Diff
Lymphocytes	No Effect
Neutrophils	No Effect
Serum Chemistries
SGPT	No Effect
Albumin	No Effect
BUN	No Effect
Glucose	No Effect
Total Protein	No Effect
Bone Marrow
Cellurlarity	No effect
DNA Synthesis	No Effect
Stem Cells
CFU-GM	No Effect
CFU-M	No Effect

Parameter	Results	Maximum Effect	Dose Dependent
T Cells	No Effect
B Cells	Decreased	8.3%
IgM AFC to SRBC	Decreased	30%	Yes
IgG AFC to SRBC	No Effect
DHR to KLH	Decreased	43%	Yes
Mitogens
Con A	No Effect
PHA	Decreased	39.2%	Yes
LPS	No Effect
Medium	No Effect
MLR	No Effect
NK Cell Activity	No Effect
Serum Complement
CH50	No Effect
C3	Increased	7%	Yes
Macrophage (RES)	Spleen phagocytosis decreased dose dependently (did not reach significance; all other parameters were unaffected)
Macrophage (Peritoneal)
Cell #	No Effect
Differential
Lymphocytes	No Effect
Poly's	No Effect
Monocytes	No Effect
Phagocytosis Covaspheres	No Effect
Phagocytosis cRBC	No Effect		Dose dependent increase
Adherence of cRBC	Increased	92%	Yes
Ectoenzymes	DNA content of macrophages decreased altering specific activities ectoenzymes and b Glucuronidase
5'ND	No effect based on total activity
LAP	No effect based on total activity
PDE	No effect based on total activity
b Glucuronidase	No effect based on total activity

 

 

¹Pellizzari, E.D. (1978): Quantitation of Chlorinated Hydrocarbons in Previously Collected Air Samples. EPA 450/3-78-112.
²Howard, P.H., Santodonato, J., Saxena, J., Malling, J., and Greninger, D. (1976): Investigation of Selected Potential Environmental Contaminants: Nitroaromatics. U.S. EPA, RTP, NC. p.1407 EPA 560/2-76-010.

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Report Date: October 1994

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