National Toxicology Program

National Toxicology Program
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Abstract for IMM95002 - Meta-nitrotoluene (CASRN 99-08-1)

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.


Figure ES-1
Structure Activity of Mono-nitrotoluenes

Structure activity chart

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.

 

Table ES-1

SUMMARY TABLE FOR TOXICOLOGY STUDIES

m-Nitrotoluene
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

 

 

 

  Table ES-2

SUMMARY TABLE FOR IMMUNOLOGY STUDIES

2,4-Diaminotoulene

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

 

 

 

Table ES-3

SUMMARY TABLE FOR HOST RESISTANCE STUDIES

m-Nitrotoluene
Model Result Maximum Effect Dose Comment

Listeria monocytogenes

Decreased resistance

70% increase in mortality

600 mg/kg

 

Streptococcus pneumoniae

No effect

 

 

 

PYB6

Increased resistence

Decreased tumor incidence

 

 

B16F10

Decreased resistance

39.3% decrease in pulmonary burden

600 mg/kg

 

Plasmodium yoelii

No effect

 

 

 

 

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.

Report Date: October 1994

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