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Toxicological information

Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Non GLP

Data source

Reference
Reference Type:
publication
Title:
Chronic inhalation of carbon monoxide: Effects on the respiratory and cardiovascular system at doses corresponding to tobacco smoke
Author:
Søhaug, S., Steinshamn, S., Nilsen, O.G., Waldum, H.L
Year:
2006
Bibliographic source:
Toxicology Vol. 228., pp 280-290

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
Female rats exposed to carbon monoxide for 20hrs/day, 5 days/wk for 72 weeks. This study replaces the requirement for a 90-day study as no suitable sub chronic studies with CO are available.
GLP compliance:
no
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Carbon monoxide (AGA, Oslo, Norway)
- Physical state: gas
- Analytical purity: Not stated, however mixed with hospital medical quality air
- Impurities (identity and concentrations): not stated
- Lot/batch No.: Not stated
- Expiration date of the lot/batch: Not stated
- Stability under test conditions: Not stated
- Storage condition of test material: Not stated

Test animals

Species:
rat
Strain:
Wistar
Sex:
female

Administration / exposure

Route of administration:
inhalation: gas
Type of inhalation exposure:
whole body
Vehicle:
air
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
72 weeks
Frequency of treatment:
20hr/day, 5 days/week
Doses / concentrations
Dose / conc.:
200 ppm (nominal)
Remarks:
Doses / Concentrations:
200 ppm
Basis:
nominal conc.
No. of animals per sex per dose:
CO treated = 51 animals
Control (air) = 26 animals
Control animals:
yes

Results and discussion

Effect levels

Dose descriptor:
LOAEC
Effect level:
200 ppm
Based on:
other:
Sex:
female
Basis for effect level:
other: Based on cardiac hypertrophy (increases in right ventricle heart weights)

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Results: In total 3 animals exposed to CO (at weeks 20, 60 and 68 weeks) and 1 control animal (68 weeks) were withdrawn from the study due to illness.

There were no observed differences in weight gain between CO exposed and control animals, with group mean weights of 275±4g and 270±6g (p=0.544) obtained respectively. The only difference in organ weights was seen in cardiac weights (see below)  

Effects on the cardiovascular system

Organ

Control (n=23)

CO Exposed (n=43)

Right ventricle (mg)

112 ± 5.2

134 ± 3.2*

Left ventricle (mg)

561 ± 14.7

642 ± 12.4*

Right ventricle/bodyweight (mg/g)

0.42 ± 0.02

0.48 ± 0.01*

Left ventricle/bodyweight (mg/g)

2.09 ± 0.04

2.33 ± 0.03*

± SEM *p≤0.001 vs. Control group  

Chronic CO exposure for 72 weeks induced a 20% (p=0.001) increase in right ventricular weight and a 14% increase in left ventricular + interventricular septum (p=0.001) increase in right ventricular weight and a 14% increase in left ventricular + interventricular septum (p<0.001). This same trend was observed after only 6 months of exposure, where analysis of some CO exposed animals showed an increase in left ventricular + interventricular septum compared to the controls (556 ± 33mg (n=4) vs. 510±14mg (n=2) respectively.

No macroscopic changes in the heart were observed in either of the two groups. Furthermore, no histopathological changes like oedema, inflammation or signs of scarring were observed in the right or left ventricular wall.

No structural signs of hypertension were observed in pulmonary arteries. In addition, no differences in the proportion of small muscularised arteries to non-muscularised arteries were observed in CO exposed and control animals (47.9±2.3% vs. 47.6±1.9% [p=0.936] respectively).

Signs of atherosclerotic lesions in the femoral and three sections of the distal part of the thoracic aorta were absent in the CO exposed group. Of note of the 15 control animals examined showed plaque-like lesions in the femoral artery with thickening of the lamina interna. No abnormality was observed in the thoracic aorta in the control group.

Effects on the respiratory system

No morphological signs of smoking associated pathology (i.e. emphysema, inflammation, bronchial/peribronical thickening, fibrosis, pulmonary hypertension) were observed in CO exposed animals. No distinct morphological differences between CO exposed and control animal were observed. No ultrasound differences in alveolar epithelial cells or alveolar septas between the two groups were observed with the use of electron microscopy.

The thickness of the fused basal laminas of the alveolar epithelial ad endothelial cells of the blood-air barrier did not differ significantly between CO exposed and control animals (89.21±2.6nm vs. 85.22±2.32nm [p=0.252] respectively). Chronic CO exposure was not associated with any significant morphological changes in the pulmonary neuroendocrine system.

The number of single PNEC in the airway was slightly higher in CO exposed animals compared to control animals, but this did not achieve statistical significance (1.9±0.2 cells/cm2vs. 1.7±0.3 cells/cm2[p=0.579] respectively). Similarly, no statistically significant difference was found between the CO exposed group and control group regarding the size of NEBs located in the alveolobronchiolar (1.7±0.3 cells/cm2vs. 1.8±0.6 cells/cm2[p=0.837], respectively) and bronchial (1.9±0.1 cells/cm2vs. 2.1±0.3 cells/cm2[p=0.530), respectively). No difference between CO exposed and control animals was seen regarding the size of aNEBs (5.6±0.6 cells vs. 5.2±0.7 cells [p=0.696], respectively) or bNEBs (7.0±0.5 cells vs. 7.1±0.2 cells [p=0.902], respectively).

Tumourgenesis

Control (n=25)

CO Exposed (n=49)

Pituitary gland

- adenoma

 

4

 

6

Mammary gland

- fibroadenoma

 

0

 

3

Ovary

- Adenocarcinoma

- Haemangioma

 

0

1

 

1

0

Uterus

- leiomyoma

 

0

 

1

Salivary gland

- Adenocarcinoma

 

1

 

0

Haematopoietic system

- leukaemia

 

0

 

1

Liver

- Metastasis from adenocarcinoma of the uterus

 

0

 

1

Lung

- Adenocarcinoma

- Tumourlets/NE hyperplasia

 

0

1

 

1

1

Total number of animals with tumours

7 (28%)

14 (29%)

 

Tumours of the anterior pituitary gland were the most frequent neoplasia, observed in 12 and 16% of CO exposed and control animals respectively. These were all classified as benign adenomas, with a neuroendocrine morphology without atypia and with a low rate of mitosis.

Mammary gland tumours were observed only in rats exposed to CO. However the level of animals exhibiting mammary tumours (6%) is consistent with published data which report that not only do nulliparous rats have a higher incidence of mammary tumours (Nagaokaet al) but rats (in particular females) frequently develop various tumours. In the case of females, the majority of these are in the mammary glands (Gross and Dreyfuss).

The proportion of total number of animals with tumours did not differ significantly between groups (28% vs. 29%,p=0.959). This data is consistent with published data where tumour incidences in Sprague Dawley and Long Evans female rats have been reported as 22% in both cases (Gross and Dreyfuss).

Only one of the CO exposed animals died spontaneously during the exposure period, suffering from leukaemia. In addition, two CO exposed rats were sacrificed after 15 and 17 months, respectively, and one control rat after 17 months due to signs of illness. Examination of the animals revealed a large mammary tumour, an adenocarcinoma of the ovary and a pituitary adenoma, respectively.

COHb levels During the study, levels of COHb and Hb were measured. In CO exposed animals COHb levels ranged from 11 to 14.7%. COHB levels of control animals were increased in the CO exposed animals compared to control animals from week 12 until the end of the study.

 

Time
(weeks)

COHb (%)

Hb (g/dL)

Control

CO Exposed

Control

CO Exposed

n

 

n

 

n

 

n

 

0

-

-

-

 -

8

13.2±0.2

8

13.7±0.2

2

41

0.1

2

11.0±1.0

-

-

-

-

12

-

-

-

-

8

13.4±0.3

16

14.5±0.6*

24

2

0.1±0.1

4

12.6±0.7

2

11.8±0.2

4

15.0±0.9

72

22

0.3±0.1

43

14.7±0.3*

22

13.1±0.2

43

14.7±0.1*

Data presented as means± SEM *p<0.02 vs. Control Group

References: Gross, L. Yolande, Y. (1979). Spontaneous tumors in Sprague-Dawley and Long-Evans rats in F1 hybrids: Carcinogenic effect of total-body x-irradiation. Proc. Natl. Acad. Sci., vol. 76 (11), pp 5910 -5913

Nagaoka, T., Takegawa, T., Takeuchi, M., Maekawa, A. (2000). Effects of reproduction on spontaneous development of endometrial adenocarcinomas and mammary tumors in Donryu rats. Jpn. J. Cancer Res., vol. 91. pp 375 -382.

Applicant's summary and conclusion

Conclusions:
No definitive NOAEC could be identified. Therefore, the LOAEC is 200 ppm (based on evidence of cardiac hypertrophy).
Executive summary:

Except for cardiac hypertrophy, no structural changes in the respiratory and cardiovascular systems or tumourgenic effects in animals exposed to CO were observed.

Chronic exposure to CO did not induce morphological changes in the lungs. Whilst changes in the pulmonary neuroendocrine system with hyperplasia of the PNEC/NEBs have been described in susceptible smokers and diseases associated with tobacco smoke, no such effect was observed in this study.