1,4-bis(p-tolylamino)anthraquinone

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

repeated dose toxicity: inhalation, other

Type of information:

experimental study

Adequacy of study:

disregarded due to major methodological deficiencies

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

unsuitable test system

Remarks:

The study is performed with a mixture of Solvent Yellow and Solvent Green

Data source

Reference

Reference Type:

review article or handbook

Title:

Unnamed

Year:

1987

Materials and methods

Principles of method if other than guideline:

Review: Subacute and subchronic dermal toxicity studies.

GLP compliance:

no

Test material

Test material information

Constituent 1

Reference substance name:

Reference substance 001

Cas Number:

8003-22-3

Constituent 2

Reference substance name:

1,4-bis(p-tolylamino)anthraquinone

EC Number:

204-909-5

EC Name:

1,4-bis(p-tolylamino)anthraquinone

Cas Number:

128-80-3

Molecular formula:

C28H22N2O2

IUPAC Name:

1,4-bis[(4-methylphenyl)amino]-9,10-anthraquinone

Test animals

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Administration / exposure

Route of administration:

inhalation: aerosol

Results and discussion

Any other information on results incl. tables

In an inhalation toxicity study, Henderson et al. (1984b) exposed male and female Fischer 344 rats to aerosols of Solvent Yellow 33/Solvent Green 3 mixture (30:70) for 4 weeks. The mean measured aerosol concentrations were 11 ± 5 (low dose), 49 ± 11 (medium dose), and 210 ± 50 mg/m³ (high dost), with particle sizes of 3,2 ± 0.4, 3.7 ± 0.5, and 4.9 4± 0.6 µm, respectively. The animals were observed for clinical signs of toxicity before initiation of exposure, 2 weeks after, and after termination of exposure. Body weights and measurements of respiratory function were taken before and after termination of exposure; lung biochemistry, hematology tests, serum chemistry tests, and gross and histopathological evaluations were performed after termination of exposure (Henderson et al. 1984b).

No adverse gross clinical effects were observed. Male and female animals exposed to the high dose gained significantly less weight than controls. Male and female rats exposed to the medium and low doses, however, gained slightly more weight than controls.

Respiratory function tests were performed on 16 control and 16 high-dose animals. Absolute expiratory rates were significantly decreased, but the expiratory rates normalized against the forced vital capacity were not significantly altered. Other parameters significantly altered by exposure to the dye mixture were vital capacity normalized against total lung capacity (increased); residual volume, both absolute and normalized against total lung capacity (decreased); and diffusing capacity normalized against body weight or against alveolar volume (decreased). Henderson et al. (1984b) concluded that the dye mixture caused a decrease in lung volume, a reduction in gas exchange efficiency, and a slight airflow obstruction, but only in those animals exposed to the highest dose.

Evaluation of lung biochemistry by analysis of bronchoalveolar lavage (BAL) fluid showed that the following parameters were significantly elevated in high-dose rats: lactate dehydrogenase, ß-glucuronidase, alkaline phosphatase, glutathione reductase, glutathione peroxidase, acid proteinase, protein content, macrophages, and neutrophils.  Most of the acid proteinase activity was resistant to inhibition by leupeptin, indicating that the activity was cathepsin D. Protein content and neutrophils were elevated in medium-dose rats; macrophages and neutrophils were also elevated in low-dose rats.

Henderson et al.,(1984b) suggested that the elevation in protein content and enzymes and the increase in macrophages and ncutrophils in BAL fluid were indicative of an inflammatory response in high-dose animals. A mild inflammatory response in medium-dose animals was indicated by the increase in neutrophils. Henderson et al. (1984b) further suggested that the high level of cathepsin D, along with the more modest increase in cathepsin B, indicated that cleanup of lung particles and cellular debris was more important than turnover of pulmonary architecture.

Acid proteinase activity in lung tissue was elevated in animals exposed to the high dose of Solvent Yellow 33/Solvent Green 3 mixture. This activity was also peristant to leupeptin, indicating that it was cathepsin B; cathepsin B was not elevated in lung tissue. The neutralproteinases (plasminogen and cathepsin 6-polymonrphonuclear  leucocyte elastase were moderately increased. According to Henderson et al. (1984b) these results were also indicative of an inflammatory response.

Hematology tests in 12 control rats and 12 rats exposed at each of the three dose levels revealed no changes. Serum chemistry tests showed that serum alkaline phosphatase activity, total bilirubin, and creatinine were significantly elevated in all exposure groups, whereas inorganic phosphorus was elevated in animals exposed to the high dose. Cholesterol and glucose were elevated, but not significantly. The absence of histopathological changes in the liver, however, indicated that these changes in serum chemistry were not physiological significant.

Histopathological evaluation of animals exposed to the highest dose showed a mild reaction around the terminal airways of the lungs that consisted of minimal to slight proliferation of foamy alveolar macrophages and minimal to slight hyperplasia of Type II pulmonary epithelial cells.

This reaction was observed more often in males than in females, and was even observed in some medium-dose animals. Also in high-dose animals, clusters of reticuloendothelial cells with lymphoid hyperplasia were observed in the tracheobronchial lymph nodes, suggesting that, even in the absence of phagocytized particles, the dye had moved into the lymph nodes.

A yellowish-brown pigment was found below the respiratory epithelium of the nasal septum and turbinates, but not in the larynx, trachea, or bronchi. No exposure-related lesions were observed in other organs (Henderson et al. 1984b). No exposure-related microscopic lesions were observed in rats exposed to Solvent Yellow 33 alone; therefore, the lesions observed in animals exposed to Solvent Yellow 33/Solvent Green 3 mixture may be caused by Solvent Green 3.

From these studies, Henderson et al. (1984b) concluded that the lowest observed- effect level (LOEL) for aerosols of Solvent Yellow 33/Solvent Green 3 mixture was => 50 mg/m³ ; the no-observed-effect level (NOEL) was 11 mg/m³.

Male and female Fisher 344 rats were also exposed to aerosols of Solvent Yellow 33/Solvent Green 3 mixture for 13 weeks (90 days) (Henderson et al. 1985b). Concentrations were 0 (control), 1.1 ± 0.5 (low-dose), 10.2 ± 3.1 (medium-dose), and 101 ± 23 mg/m³ (high-dose), and particle sizes, expressed as MMAD, were 2.8 ± 0.4, 3.0 ± 0.2, and 4.2 ± 0.4 pm, respectively. Measurements and observations were made during exposure, immediately after exposure, and 30 days after termination of exposure.

Clinical observations 6 weeks after initiation of exposure, at termination of exposure, and after a 30-day recovery period showed no gross clinical effects or mortality. Body weights measured immediately after termination of exposure showed that high-dose males weighed 8.0 percent less than control males, and high-dose females weighed 9.2 percent less than control females. At the end of the 30-day recovery period, the body weight of high-dose male rats remained significantly lower than control males, whereas the body weight of high-dose female rats was similar to controls.

The parameters of respiratory function were the same as those measured in animals exposed for 4 weeks. There were no significant differences between values of absolute functions in control and exposed animals.

Because body weights in high-dose animals were lower than control, there was a trend for variables normalized against body weight to be higher than those in control animals. Nevertheless, carbon monoxide diffusing capacity normalized against body weight was the only variable significantly altered (increased). At the end of the 30-day recovery period, the only variable significantly affected by exposure

was a lower carbon monoxide diffusing capacity normalized against alveolar volume (0.016 ml/min/mm Hg/ml in high dose-animals, 0.020 in controls).

These results demonstrated that exposure to Aerosols of Solvent Yellow 33/Solvent Green 3 mixture had very little effect on respiratory function in rats.

Lung biochemistry was evaluated by analysis of BAL fluid 6 weeks after initiation of exposure, immediately after termination of exposure, and after the 30-day recovery period. Lactate dehydrogenase, L-glucuronidase, protein content, and the number of macrophages and neutrophils were significantly affected by exposure to Solvent Yellow 33/Solvent Green 3 mixture. In high-dose animals killed 6 weeks after initiation of exposure, these effects did not become more severe but became less severe with continued treatment and recovery. Except for the number of macrophages, the values for the parameters were lower immediately after termination of exposure than at 6 weeks after initiation of exposure.

Acid proteinase activity in BAL fluid was not elevated. Acid proteinase activity in lung tissue, however, was significantly elevated in rats exposed to the high dose and killed immediately after termination of exposure. Although the level of activity decreased during recovery, it remained significantly higher than that in control animals. The changes observed in BAL fluid and lung tissues were indicative of an inflammatory reaction that did not clear up by the end of the recovery period. Henderson et al. (1985b) attributed the inflammation to Solvent Green 3 in the mixture and not to Solvent Yellow 33.

Serum chemistry and hematology tests revealed that alkaline phosphatase activity was significantly decreased, and cholesterol, glucose, inorganic phosphorus, total protein, and albumin were significantly increased in rats exposed to the high dose. Glucose, inorganic phosphorus, total protein, and albumin were elevated in medium-dose animals, and glucose, total protein, and albumin were elevated in low-dose animals.

Because blood urea nitrogen (BUN), serum glutamic pyruvic transaminase (SGPT), and creatinine levels were normal, indicating no damage to the kidneys and liver. Henderson et al. (1985b) concluded that these changes were not clinically significant. All serum chemistry parameters returned to normal by the end of recovery, indicating that the changes were also reversible.

Histopathological evaluation of animals exposed to Solvent Yellow 33/Solvent Green 3 mixture showed that in almost all high-dose animals, pigment was deposited in the submucosa of the nasal epithelium, the cortical tubules in the kidneys, and the bile duct epithelium or in hepatocytes adjacent to the bile duct. Lung lesions consisted of slight to moderate accumulation of foamy alveolar macrophages (containing pigment), accompanied by slight to moderate hyperplasia of Type II cells. Reticuloendothelial and lymphoid cells in the tracheobronchial lymph nodes were moderately hyperplastic.

In medium-dose animals, pigment was observed in the submucosal nasal epithelium. Minimal lesions in the lungs (alveolar macrophages and Type II cell hyperplasia) were observed. Reticuloendothelial and lymphoid cell hyperplasia with pigment deposition were also observed. No exposure-related lesions was observed in low-dose animals.

After the 30-day recovery period, lesions in the lungs in high-dose animals were slightly less severe than those observed immediately after exposure. In the nasal cavity and cortical tubules in the kidney pigment

deposition was less severe, but in the liver it was unchanged. Reticuloendothelial cell hyperplasia was more severe and lymphoid hyperplasia was unchanged.

In medium-dose animals, minimal lesions in the lungs were observed. Pigment deposition was observed in the nasal cavity, it was comparable to that of the controls in the kidney, but it was absent in the liver.

Lymphoid hyperplasia was observed in one animal, but reticutoendothelial cell hyperplasia was absent in all animals. No exposure-related lesions were observed in the low-dose animals (Henderson et al. 1985b).

In this study, the findings related to pigment deposition were similar to those observed after exposure to Solvent Yellow 33 alone; therefore, these effects could not be attributed to Solvent Green 3 in the mixture.

Because aerosols of Solvent Yellow 33/Solvent Green 3 mixture caused microscopic lesions in rats at the medium concentration (10 mg/m³ ) and no exposure related lesions at the low concentration, Henderson et al. (1985b) concluded that the NOAEL was 1 mg/m³.

Applicant's summary and conclusion

Executive summary:

In an inhalation toxicity study, Henderson et al. (1984b) exposed male and female Fischer 344 rats to aerosols of Solvent Yellow 33/Solvent Green 3 mixture (30:70) for 4 weeks. The mean measured aerosol concentrations were 11 ± 5 (low dose), 49 ± 11 (medium dose), and 210 ± 50 mg/m³ (high dost), with particle sizes of 3,2 ± 0.4, 3.7 ± 0.5, and 4.9 4± 0.6 µm, respectively. The animals were observed for clinical signs of toxicity before initiation of exposure, 2 weeks after, and after termination of exposure. Body weights and measurements of respiratory function were taken before and after termination of exposure; lung biochemistry, hematology tests, serum chemistry tests, and gross and histopathological evaluations were performed after termination of exposure (Henderson et al. 1984b).

No adverse gross clinical effects were observed. Male and female animals exposed to the high dose gained significantly less weight than controls. Male and female rats exposed to the medium and low doses, however, gained slightly more weight than controls.

Respiratory function tests were performed on 16 control and 16 high-dose animals. Absolute expiratory rates were significantly decreased, but the expiratory rates normalized against the forced vital capacity were not significantly altered. Other parameters significantly altered by exposure to the dye mixture were vital capacity normalized against total lung capacity (increased); residual volume, both absolute and normalized against total lung capacity (decreased); and diffusing capacity normalized against body weight or against alveolar volume (decreased). Henderson et al. (1984b) concluded that the dye mixture caused a decrease in lung volume, a reduction in gas exchange efficiency, and a slight airflow obstruction, but only in those animals exposed to the highest dose.

Evaluation of lung biochemistry by analysis of bronchoalveolar lavage (BAL) fluid showed that the following parameters were significantly elevated in high-dose rats: lactate dehydrogenase, ß-glucuronidase, alkaline phosphatase, glutathione reductase, glutathione peroxidase, acid proteinase, protein content, macrophages, and neutrophils.  Most of the acid proteinase activity was resistant to inhibition by leupeptin, indicating that the activity was cathepsin D. Protein content and neutrophils were elevated in medium-dose rats; macrophages and neutrophils were also elevated in low-dose rats.

Henderson et al.,(1984b) suggested that the elevation in protein content and enzymes and the increase in macrophages and ncutrophils in BAL fluid were indicative of an inflammatory response in high-dose animals. A mild inflammatory response in medium-dose animals was indicated by the increase in neutrophils. Henderson et al. (1984b) further suggested that the high level of cathepsin D, along with the more modest increase in cathepsin B, indicated that cleanup of lung particles and cellular debris was more important than turnover of pulmonary architecture.

Acid proteinase activity in lung tissue was elevated in animals exposed to the high dose of Solvent Yellow 33/Solvent Green 3 mixture. This activity was also peristant to leupeptin, indicating that it was cathepsin B; cathepsin B was not elevated in lung tissue. The neutralproteinases (plasminogen and cathepsin 6-polymonrphonuclear  leucocyte elastase were moderately increased. According to Henderson et al. (1984b) these results were also indicative of an inflammatory response.

Hematology tests in 12 control rats and 12 rats exposed at each of the three dose levels revealed no changes. Serum chemistry tests showed that serum alkaline phosphatase activity, total bilirubin, and creatinine were significantly elevated in all exposure groups, whereas inorganic phosphorus was elevated in animals exposed to the high dose. Cholesterol and glucose were elevated, but not significantly. The absence of histopathological changes in the liver, however, indicated that these changes in serum chemistry were not physiological significant.

Histopathological evaluation of animals exposed to the highest dose showed a mild reaction around the terminal airways of the lungs that consisted of minimal to slight proliferation of foamy alveolar macrophages and minimal to slight hyperplasia of Type II pulmonary epithelial cells.

This reaction was observed more often in males than in females, and was even observed in some medium-dose animals. Also in high-dose animals, clusters of reticuloendothelial cells with lymphoid hyperplasia were observed in the tracheobronchial lymph nodes, suggesting that, even in the absence of phagocytized particles, the dye had moved into the lymph nodes.

A yellowish-brown pigment was found below the respiratory epithelium of the nasal septum and turbinates, but not in the larynx, trachea, or bronchi. No exposure-related lesions were observed in other organs (Henderson et al. 1984b). No exposure-related microscopic lesions were observed in rats exposed to Solvent Yellow 33 alone; therefore, the lesions observed in animals exposed to Solvent Yellow 33/Solvent Green 3 mixture may be caused by Solvent Green 3.

From these studies, Henderson et al. (1984b) concluded that the lowest observed- effect level (LOEL) for aerosols of Solvent Yellow 33/Solvent Green 3 mixture was => 50 mg/m³ ; the no-observed-effect level (NOEL) was 11 mg/m³.

Male and female Fisher 344 rats were also exposed to aerosols of Solvent Yellow 33/Solvent Green 3 mixture for 13 weeks (90 days) (Henderson et al. 1985b). Concentrations were 0 (control), 1.1 ± 0.5 (low-dose), 10.2 ± 3.1 (medium-dose), and 101 ± 23 mg/m³ (high-dose), and particle sizes, expressed as MMAD, were 2.8 ± 0.4, 3.0 ± 0.2, and 4.2 ± 0.4 pm, respectively. Measurements and observations were made during exposure, immediately after exposure, and 30 days after termination of exposure.

Clinical observations 6 weeks after initiation of exposure, at termination of exposure, and after a 30-day recovery period showed no gross clinical effects or mortality. Body weights measured immediately after termination of exposure showed that high-dose males weighed 8.0 percent less than control males, and high-dose females weighed 9.2 percent less than control females. At the end of the 30-day recovery period, the body weight of high-dose male rats remained significantly lower than control males, whereas the body weight of high-dose female rats was similar to controls.

The parameters of respiratory function were the same as those measured in animals exposed for 4 weeks. There were no significant differences between values of absolute functions in control and exposed animals.

Because body weights in high-dose animals were lower than control, there was a trend for variables normalized against body weight to be higher than those in control animals. Nevertheless, carbon monoxide diffusing capacity normalized against body weight was the only variable significantly altered (increased). At the end of the 30-day recovery period, the only variable significantly affected by exposure

was a lower carbon monoxide diffusing capacity normalized against alveolar volume (0.016 ml/min/mm Hg/ml in high dose-animals, 0.020 in controls).

These results demonstrated that exposure to Aerosols of Solvent Yellow 33/Solvent Green 3 mixture had very little effect on respiratory function in rats.

Lung biochemistry was evaluated by analysis of BAL fluid 6 weeks after initiation of exposure, immediately after termination of exposure, and after the 30-day recovery period. Lactate dehydrogenase, L-glucuronidase, protein content, and the number of macrophages and neutrophils were significantly affected by exposure to Solvent Yellow 33/Solvent Green 3 mixture. In high-dose animals killed 6 weeks after initiation of exposure, these effects did not become more severe but became less severe with continued treatment and recovery. Except for the number of macrophages, the values for the parameters were lower immediately after termination of exposure than at 6 weeks after initiation of exposure.

Acid proteinase activity in BAL fluid was not elevated. Acid proteinase activity in lung tissue, however, was significantly elevated in rats exposed to the high dose and killed immediately after termination of exposure. Although the level of activity decreased during recovery, it remained significantly higher than that in control animals. The changes observed in BAL fluid and lung tissues were indicative of an inflammatory reaction that did not clear up by the end of the recovery period. Henderson et al. (1985b) attributed the inflammation to Solvent Green 3 in the mixture and not to Solvent Yellow 33.

Serum chemistry and hematology tests revealed that alkaline phosphatase activity was significantly decreased, and cholesterol, glucose, inorganic phosphorus, total protein, and albumin were significantly increased in rats exposed to the high dose. Glucose, inorganic phosphorus, total protein, and albumin were elevated in medium-dose animals, and glucose, total protein, and albumin were elevated in low-dose animals.

Because blood urea nitrogen (BUN), serum glutamic pyruvic transaminase (SGPT), and creatinine levels were normal, indicating no damage to the kidneys and liver. Henderson et al. (1985b) concluded that these changes were not clinically significant. All serum chemistry parameters returned to normal by the end of recovery, indicating that the changes were also reversible.

Histopathological evaluation of animals exposed to Solvent Yellow 33/Solvent Green 3 mixture showed that in almost all high-dose animals, pigment was deposited in the submucosa of the nasal epithelium, the cortical tubules in the kidneys, and the bile duct epithelium or in hepatocytes adjacent to the bile duct. Lung lesions consisted of slight to moderate accumulation of foamy alveolar macrophages (containing pigment), accompanied by slight to moderate hyperplasia of Type II cells. Reticuloendothelial and lymphoid cells in the tracheobronchial lymph nodes were moderately hyperplastic.

In medium-dose animals, pigment was observed in the submucosal nasal epithelium. Minimal lesions in the lungs (alveolar macrophages and Type II cell hyperplasia) were observed. Reticuloendothelial and lymphoid cell hyperplasia with pigment deposition were also observed. No exposure-related lesions was observed in low-dose animals.

After the 30-day recovery period, lesions in the lungs in high-dose animals were slightly less severe than those observed immediately after exposure. In the nasal cavity and cortical tubules in the kidney pigment deposition was less severe, but in the liver it was unchanged. Reticuloendothelial cell hyperplasia was more severe and lymphoid hyperplasia was unchanged.

In medium-dose animals, minimal lesions in the lungs were observed. Pigment deposition was observed in the nasal cavity, it was comparable to that of the controls in the kidney, but it was absent in the liver.

Lymphoid hyperplasia was observed in one animal, but reticutoendothelial cell hyperplasia was absent in all animals. No exposure-related lesions were observed in the low-dose animals (Henderson et al. 1985b).

In this study, the findings related to pigment deposition were similar to those observed after exposure to Solvent Yellow 33 alone; therefore, these effects could not be attributed to Solvent Green 3 in the mixture.

Because aerosols of Solvent Yellow 33/Solvent Green 3 mixture caused microscopic lesions in rats at the medium concentration (10 mg/m³ ) and no exposure related lesions at the low concentration, Henderson et al. (1985b) concluded that the NOAEL was 1 mg/m³.