Registration Dossier

Administrative data

Endpoint:
repeated dose toxicity: other route
Remarks:
other: no regulatory test guideline followed
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study is a mechanistic investigation for inhibition of trypsin and elastase enzymes due to Chloramine T treatment in order to develop a model for emphysema in genetic diseases.

Data source

Referenceopen allclose all

Reference Type:
review article or handbook
Title:
Pulmonary effects of intravenously injected chloramine T in the dog
Author:
King, R.R., R.F. Henderson, R.K. Wolff, and J.L. Mauderly
Year:
1983
Bibliographic source:
Toxicol 3, 109
Reference Type:
publication
Title:
A model of decreased functional 1-proteinase inhibitor. Pulmonary pathology of dogs exposed to chloramine
Author:
Abrams W.R., A.B. Cohen, V.V. Damiano, A. Eliraz, P. Kimbel, D.R. Meranze, and G. Weinbaum
Year:
1981
Bibliographic source:
T. J Clin Invest 68:1132-1139

Materials and methods

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
Gelatin capsules, containing 300 mg Chloramine T (CT)

Test animals

Species:
dog
Strain:
Beagle
Sex:
male

Administration / exposure

Route of administration:
infusion
Vehicle:
other: deionised water
Details on exposure:
IV infusion, oral and combinations. See further details on the study design.
Details on study design:
IV treatment with Chloramine T (CT) was given in 4 dogs, routinely anesthetized with sodium pentobarbital and infusion of the sequential administration of methylene blue (10% wt/vol), 0.9% sodium chloride (saline), 300 mg of CT in 200 mL saline delivered slowly over 1 h, 5 mL saline, 1 mL of 10 mg/mL methylene blue intravenously, and, finally, 1 mL i.m. of 200 mg/mL DL-alpha tocopherol. Samples of serum were taken for analysis before each CT exposure. Treatment was continued at a rate of 3 infusions/wk for 5 wks for two dogs (A and B). Two animals were observed for 6 wk additional, and were anesthetized only for blood sampling. These animals were not intubated during the IV procedure.

Gelatin capsules, containing 300 mg Chloramine T, were given to two dogs (C and D) 10 min before morning and evening feedings.
During the initial exposure period (1-2 wk), the dogs regurgitated their food at several feedings, but there were no further side effects from the exposure. In one animal the treatment continued for 4 months (dog C). In dog D, after 4 months, the dose was increased from 600 mg/d to 1200 mg/d for 2 months more. Samples of serum were obtained periodically throughout the treatment.

A third regimen was tried with 200 mg CT in 200 mL saline was given IV to dogs E and F three times per week. Dogs E and F were also treated orally with 400 mg CT on the other 4 d of the week. After 14 d the IV frequency was reduced to twice weekly with an increase in concentration of CT to 400 mg/200 mL. The IV dose was subsequently increased to 600 and 800 mg/200 mL on days 51 and 94, respectively. The oral dose of 400 mg CT was increased to 600 mg/d.

Finally, two dogs, G and H, were treated only intravenously in a similar fashion to dogs A and B but for only 3 wks. Morphometric analysis was performed on lungs from these dogs.

Examinations

Observations and examinations performed and frequency:
Bronchial lavage:
Dogs A through F were anesthetized and bronchial lavage performed at least twice during each experiment. The lavages consisted of six instillations of 20 mL sterile, pyrogen free, 0.9% NaCl through an Olympus fiberoptic bronchoscope wedged in a single lobe (Olympus Corp. of America, New Hyde Park, N. Y.). Total recovery averaged - 100 mL. The lavage was centrifuged at 200 g for 15 min to remove cells. The supernate was filtered through multiple layers of cheesecloth to remove mucus, lyophilized, resuspended in 5-10 mL saline, dialyzed overnight against 1 liter of saline at 4°C, relyophilized, and suspended with H2O to a 2-mL final volume. The cellular pellet from the lavage was examined for the distribution of cell types and the concentrated supernate was assayed for both total protein and a-l-PI. EIC was determined in the lavages from an untreated dog and from dog F.

Rocket immunoelectrophoresis:
The concentration of alpha-I-PI in serum or concentrated bronchial lavage fluid was measured immunologically by rocket immunoelectrophoresis in 1% agarose containing antibody to dog alpha-I-PI. Rocket heights obtained from six different standard concentrations of alpha-I-PI were fit with the equation for a straight line with a correlation coefficient > 0.98. Experimental values were calculated from the best-fit line.

Chemical assays.
Liver function was monitored before and during CT treatment by analysis for cholesterol, glucose, uric acid, creatine phosphokinase, bilirubin, phosphate, calcium, total protein, albumin, alkaline phosphatase, serum glutamic-aspartic transaminase, and lactate dehydrogenase on freshly prepared dog serum.
The total protein content of bronchial lavages was determined by the method of Lowry et al, with bovine serum albumin as a standard.

Elastase Inhibitory Capacity (EIC) and Trypsin Inhibitory Capacity (TIC):
EIC and TIC were measured with 0.25% hemoglobin, 1% agarose plates. The resulting clear area is proportional to the concentration of active protease. Exogenous protease (porcine pancreatic elastase or bovine trypsin) is incubated in wells in the agarose plates for 20 h at 37°C. Inhibitory capacity is determined from the volume of serum required to completely inhibit a fixed concentration of the control protease. For lavage fluids the inhibitory capacity was the amount of alpha-1-PI, determined immunologically, required for 100% inhibition of SAPNA hydrolysis by a fixed amount (1 ,ug) of elastase. Each inhibitor capacity determination was based on eight individual lavage concentrations. The SAPNA hydrolysis was followed at 410 nm in a Gilford recording spectrophotometer (Gilford Instrument Laboratories, Oberlin, Ohio) maintained at 25°C.

Sacrifice and pathology:
Histological examination:
At the termination of each experiment, the lungs were rapidly excised and fixed in the inflated state in phosphate-buffered 10% formalin under 25cm H20 pressure. Routinely, five tissue cores from each lobe were taken. The cores were dehydrated, embedded, and used for the preparation of 10- and 30-µm-thick sections for histology. The slides were stained by routine hematoxylin and eosin and examined by light microscopy. Randomly selected areas were analyzed for their mean linear intercepts (MLI) by the method of Dunnill.

Scanning electron microscopy (SEM):
200µm-thick paraffin sections were cut adjacent to sections cut for light microscopy. These were transferred to a 1' x 3" microscope slide and allowed to dry. These sections were then deparaffinized by immersion of the slide in xylene for 30-60 min. The section was then postfixed in 1% OsO4 in 0.1 M Sym-collidine buffer for 30 min. They were dehydrated in graded ethanol solutions, and treated with 50% ethanol-50% amyl acetate and then with concentrated amyl acetate. They were then critical-point dried in a Denton DCP-1 critical point drying apparatus (Denton Vacuum Inc., Cherry Hill, N. J.). The sections still attached to the slide were coated with 100-200 A of gold in a Denton Desk-i sputterer. SEM was performed with a JSM-50A SEM (JEOL, Peabody, Mass.) at 25 kV. The samples were scanned at low magnification (x 100).

Results and discussion

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Chronic exposure of dogs to tosylchloramide sodium (Chloramine T) resulted in a persistent mild or moderate anemia and a reduction in EIC in both serum and pulmonary lavage fluids.

Executive summary:

Beagle dogs were dosed orally or intravenously, or through a combination of the two, with doses ranging from approximately 12.8 to 640mg/kg per day for up to six months. In the study, 50% of the animals were sacrificed at 17 weeks. At that time, persistent mild or moderate anemia was observed. A reduction in Elastase Inhibitory Concentration (EIC) was found in the serum from the dosed animals. Furthermore, a reduction in EIC was found in the pulmonary lavage fluid. However the levels of immunologically determined protease inhibitor did not change with treatment, therefore, the enzyme must have been inactivated by chloramine T. The reaction was specific for the protease inhibitor, as the ability to inhibit trypsin was less affected. Emphysema-like alterations in lung morphology were found in both studies. The data suggest that this model parallels the emphysema associated with the genetic alpha-l-proteinase inhibitor deficiency in man.