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Acute Toxicity: other routes

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Endpoint:
acute toxicity: other routes
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study well documented, meets generally accepted scientific principles, acceptable for assessment
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
publication
Title:
The biochemical and pathological changes produced by the intratracheal instillation of certain components of zinc-hexachloroethane smoke
Author:
Richards RJ, Atkins J, Marrs TC, Brown RFR & Masek L
Year:
1989
Bibliographic source:
Toxicol 54: 79-88

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
Biochemical and pathological study in lungs-
The potential of the test material to induce respiratory adverse effects/damage to the lungs in Porton Wistar rats was investigated using intra-tracheal instillation of the test material as a single dose. Oedema resulting from the treatment was assessed by increases in lung wet weight together with elevation in the levels of a purified alveolar surface protein fraction derived from the perfused lungs. The formation of a protein rich oedematous fluid is a common early response to toxic substances.
The toxicity of the test material can be correlated with pulmonary oedema inducing capacity, since protein rich oedematous fluid is a common early response to toxic substances.
Assessment of oedematous reaction is carried out by determination of increased in lung wet weight together with elevated levels of a purified alveolar surface protein fraction derived from lungs which had been totally perfused of blood.
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Zinc chloride

Test animals

Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals and environmental conditions:
- Weight at study initiation: 150 -200 g
- Diet (e.g. ad libitum): Commercial diet
- Water (e.g. ad libitum): Tap water

Administration / exposure

Route of administration:
other: intratracheal
Vehicle:
water
Doses:
1. Investigation of rat lung response at different dose levels: 0.25, 0.5, 1.0, 2.0, 4.0 and 5.0 mg/kg bw
2. Investigation dose levels which produce early alveolar oedema: 0.25, 0.5, 1.0 or 1.5 mg/kg bw
3. Investigation of the effects of the perfusion procedure: 1.5 mg/kg bw
4. Investigation of the time course of oedema and any signs of fibrogenesis: 2.5 mg/kg bw
5. Histopatholgical examination of lungs: 2.5 mg/kg bw

No. of animals per sex per dose:
1. Investigation of lung response at low dose: 4
2. Investigation of rat lung response at different dose levels: Not reported
3. Investigation of dose level which produces early alveolar oedema: 6
4. Investigation of the effects of the perfusion procedure: 10
5. Investigation of the time course of oedema and any signs of fibrogenesis: 6
6. Histopatholgical examination of lungs: 10
Control animals:
yes
Details on study design:
- Duration of observation period following administration:
1. To investigate lung response at low dose: 2 and 4 wk
2. To investigate rat lung response at different dose levels: 12 h and 7 d
3. To investigate dose level which produces early alveolar oedema: 20 h
4. To investigate the effects of the perfusion procedure: 20 h
5. To investigate the time course of oedema and any signs of fibrogenesis: 6, 18, 28, 48, 72 h, 7 d and 14 d
6. Histopatholgical examination of lungs: 6, 28, 72 h, 7 d and 14 d

- Necropsy of survivors performed: yes

EXAMINATION
- Examination performed for different groups:
1. To investigate lung response at low dose: Lung wet weight and alveolar surface protein levels
2. To investigate rat lung response at different dose levels: Histopathological examination
3. To investigate dose level which produces early alveolar oedema: Concentration of protein in the lavage fluid
4. To investigate the effects of the perfusion procedure: Lung wet weight and alveolar surface protein levels
5. To investigate the time course of oedema and any signs of fibrogenesis: Lung wet weight, alveolar surface protein levels after all time intervals (6, 18, 28, 48, 72 h, 7 dand 14 d) and lung dried weight and hydroxyproline in the total lavaged lung after 7 and 14 d
6. Histopatholgical examination of lungs
- Other examinations: Biochemical analysis using standard techniques
Statistics:
Statistical analysis of the data was carried out using an unpaired Student's t-test and differences between mean values were considered significant when P < 0.05.

Results and discussion

Effect levelsopen allclose all
Sex:
male
Dose descriptor:
other: Elevated alveolar surface protein and lung wet weight
Effect level:
0.3 mg/kg bw
Remarks on result:
other: exposure duration: 14d
Sex:
male
Dose descriptor:
other: intra-alveolar oedema with charecteristic eosinophilic fluid accumulation and fibrin clots
Effect level:
> 0.5 mg/kg bw
Remarks on result:
other: exposure duration: 12h
Sex:
male
Dose descriptor:
other: interstitial fibrosis
Effect level:
2.5 mg/kg bw
Remarks on result:
other: exposure duration: 14d
Sex:
male
Dose descriptor:
other: Fibrogenic response
Effect level:
2.5 mg/kg bw
Remarks on result:
other: exposure duration: 7d
Mortality:
not reported
Clinical signs:
not observed
Body weight:
not observed
Gross pathology:
not observed
Other findings:
- Other observations:
1. Results for investigation of lung response at low dose after 2 or 4 wks: See Table 1
2. Results for investigation of rat lung response at different dose levels:
- Histologic (intra-alveolar oedema) evaluation after 12 h of instillation: Very slight, moderate and severe distribution of intra-alveolar oedema was observed at 1, 4 and 5 mg/kg bw respectively. Interstitial oedema was observed.
3. Results for investigation of dose level which produces early alveolar oedema and alveolar surface protein, after 20 h of instillation:
- Lung wet weight: Slight increase was observed, except for 0.25 mg/kg bw (not significant)
- Alveolar suface protein: Dose dependent increase, ranging from 5-50 mg, was observed
- Others: Upto 1.5 mg/kg bw, no visible signs of lung haemorrhage and red blood cells were observed in the lavage
4. Results for investigation of effects of perfusion procedure after 20 h: No significant changes in lung weight or in the alveolar surface protein were observed for perfused and non-perfused rat lungs
5. Results for investigation of time course of oedema and any signs of fibrogenesis: See Table 2
6. Results of histopatholgical examination of lungs at 6, 28, 72 h, 7 d and 14 d post-instillation: Interstitial fibrosis was observed in all the lungs after 14 d post-instillation



Any other information on results incl. tables

Table 1:Results forinvestigation of lung response at low dose (0.3 mg/kg bw) after 2 or 4 wks:

Time after

Instillation (weeks)

Treatment

 

n

 

Lung wet wt (g) as mean± SD 

Alveolar surface protein (mg/rat) as mean± SD

2

Distilled water

4

1.27 ± 0.08

3.5 ± 1.4

Zinc chloride

4

1.27 ± 0.04

4.6 ± 3.9

4

Distilled water

4

1.47 ± 0.15

4.3 ± 1.4

Zinc chloride

4

1.72 ± 0.32

34.4 ± 50.5

n = No. of animals

* = Significantly different from controls by Student's t-test for unpaired samples

Table 2: Results for effect of2.5 mg/kg bw zinc chloride on lungoedema and alveolar surface protein at different time intervals and lavaged lung dry weights, and hydroxyproline levels (indicator of fibrogenesis) after 7 and 14 d post-exposure

Treatment

Time after instillation

n

Lung wet weight (g) asmean± SD

Alveolar surface protein (mg) as 

mean± SD

Dry lung weight (g) as 

mean± SD 

Hydroxyproline (mg/lung) asmean± SD

Control

6 h

6

1.29 ± 0.38

5.41 ± 2.43

-

-

Zinc chloride

6 h

6

1.97 ± 0.74

51.17 ± 40.09*

-

-

Zinc chloride

18 h

6

1.95 ± 0.34

30.09 ± 13.28

-

-

Control

28 h

4

1.10 ± 0.17

5.59 ± 2.46

-

-

Zinc chloride

28 h

6

2.80 ± 0.71*

71.97 ± 14.88*

-

-

Zinc chloride

2 d

5

2.21± 0.66

37.76 ± 25.85

-

-

Control

3 d

4

1.09 ± 0.04

3.25 ± 0.28

-

-

Zinc chloride

3 d

6

2.43 ± 0.64*

36.91± 1.76*

-

-

Control

7 d

4

1.13 ± 0.08

5.18 ± 2.08

0.188 ± 0.010

2.12 ± 0.26

Zinc chloride

7 d

6

2.05 ± 0.47*

9.99 ± 4.02

0.222 ± 0.023*

3.59 ± 0.88*

Control

14 d

5

1.17 ± 0.12

4.16 ± 0.69

0.166 ± 0.069

1.80 ± 0.25

Zinc chloride

14 d

6

1.57 ± 0.42

13.77 ± 7.83*

0.202 ± 0.069

3.46 ± 1.56

n = No. of animals

* = Significantly different from controls by Student's t-test for unpaired samples

Applicant's summary and conclusion

Conclusions:
Significant lung damage, in terms of oedematous reaction, was induced in rats on single intra-tracheal instillation of zinc chloride
Executive summary:

A number of single intra-tracheal instillation studies were performed in Wistar rats to investigate the delayed respiratory adverse-effects, and the potential of zinc chloride to damage the lungs.

 

In the study for investigation of pulmonary effects at low dose (0.3 mg/kg bw), a delayed elevated alveolar surface protein, with accompanying increased lung wet weight were observed after 4 wks. These effects correlated histologically with the quantitative biochemical assay for alveolar surface protein in a lavage fluid fraction.

Histopathological studies confirmed the induction intra-alveolar oedema at 12 h post-instillation of different dose levels (0.25-5 mg/kg bw) and interstitial fibrosis in the lungs at 2.5 mg/kg bw, after 14 d. The oedematous reaction in individual animals, after 20 h at 0.25-1.5 mg/kg bw, were variable, but the overall effect was dose dependent.  

 

The onset of oedema, was rapid with maximal effects being noted within 3 days when high doses (2.5 mg/kg bw) of test material were instilled. However, in majority of the animals the severe oedematous reaction regressed between 3 and 7 d. In some animals, adjudged by histochemistry and elevated lung hydroxyproline, evidence of a fibrogenic response was observed 7 d post-exposure.

 

Hence, significant lung damage, in terms of oedematous reaction was induced in rats, on single intra-tracheal instillation of zinc chloride.