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Endpoint:
specific investigations: other studies
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1996
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: This study is classified as reliable with restrictions because there was no GLP statement provided, and limited data on methods were reported, but the study seemed to be well-conducted.
Qualifier:
no guideline followed
Principles of method if other than guideline:
This study examined the cardiotoxic effects of n-hexane. Groups of 15 rats were administered 0.1 ml of test substance in olive oil (1:1 v/v) subcutaneously for 30 days. After the exposure, the animals were sacrificed. The hearts of one group were used to determined the effect of exposure of test substance on the ventricular fibrillation threshold (VFT). The other group was used to determine effect of exposure on magnesium and potassium levels. Another group of rats was given the same exposure, but with supplemental magnesium or potassium in the diet as well. The hearts of these animals were also examined using light and electron microscopy.
GLP compliance:
no
Type of method:
in vivo
Endpoint addressed:
not applicable
Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: 6 months
- Weight at study initiation: 250-300 g
- Housing: standard housing conditions
- Diet (e.g. ad libitum): standardized diet
- Water (e.g. ad libitum): ad libitum
Route of administration:
subcutaneous
Vehicle:
olive oil
Details on exposure:
Test animals were injected with 0.1 ml of n-hexane in olive oil (1:1 v/v) daily for 30 days.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
30 days
Frequency of treatment:
daily
Post exposure period:
none
Remarks:
Doses / Concentrations:
0.1 ml
Basis:
nominal conc.
No. of animals per sex per dose:
15 animals per group
Control animals:
yes, concurrent vehicle
Examinations:
Animals were anesthetized and perfused after the end of exposure. Animals were sacrificed in pairs and the hearts examined. The heart of one animal was immediately placed in cold Krebs-Henseleit solution. This heart was then placed on a Langendorff aparatus. After a 15 min rest period, the heart rate was measured using a universal digital rate counter. Outflow was collected in a graduated cylinder to record coronary output. Ventricular fibrillation threshold (VFT) was measured using an ECG with platinum electrodes placed 1 cm apart. One electrode was attached to the metal aortic perfusion cannula. The other was inserted into the pericardium of the left ventricle. 200 msec pulse trains with 10 msec delays and a 2 msec width of 60 Hz and increasing intensity, was applied during the vulnerable period of the cardiac cycle until fibrillation occurred. VFT was defined as repetitive, irregular ventricular rhythm, with a changing pattern that persisted for more than six cycles.

Heart tissue from the second animal was used to determined magnesium and potassium levels. The heart tissue was dissolved in nitric and perchloric acids. Magnesium was analyzed using atomic adsorption spectrophotometry. Potassium was determined using a flame photometer. Serum levels were determined by Natal Regional Laboratories.

Another group of rats were exposed similary, with rats being given magnesium chloride (15 mmol/day) or potassium chloride (12 mmol/day) in the diet in addition to the exposure to n-hexane. At the end of the 30-day exposure period, the rats were sacrificed, and the heart tissue examined with light microscopy and electron microscopy.
Details on results:
The VFT and potassium and magnesium levels were significantly reduced in n-hexane treated rats. The reduction in VFT was also seen in animals given supplemental magnesium and potassium in the diet. No difference in serum levels of magnesium and potassium was seen in exposure groups vs. control groups. Also, no difference was seen in histopathology of heart tissue examined with a light microscope. The electron microscope study found mitochondria elongated across four sarcomeres in n-hexane exposure groups. Swollen mitochondria were also observed, some necrotic. Four mitochondria were enclosed in double membranes and a few inter calated disks appeared abnormal. Myofibers showed Z-band dissolution and myofibrillar disarray. Intermyofibrillar edema was seen. Also fewer glycogen granules were seen.

Control

n-hexane

Control + Mg + K

Hexane + Mg + K

VFT (mA) - study 1

9.3 ± 1.2

6.1 ± 2.7

-

-

VFT (mA) - study 2

9.75 ± 2.23

5.81 ± 1.85

11.2 ± 3.1

7.82 ± 0.81

Magnesium  (micromol/l) - study 1

9.27 ± 0.62

8.13 ± 0.35

-

-

Magnesium  (micromol/l) - study 2

8.93 ± 0.84

7.58 ± 0.72

9.45 ± 1.89

9.13 ± 0.45

Potassium (micromol/l) - study 1

83.51 ±  4.40

76.13 ± 3.14

-

-

Potassium (micromol/l) - study 2

85.18 ± 3.15

75.83 ± 4.28

86.15 ± 3.21

85.78 ± 3.81
Conclusions:
30 days subcutaneous exposure to the test substance at 0.1 ml/day lowered the ventricular fibrillation threshold even with supplemental magnesium and potassium.
Executive summary:

This study examined the cardiotoxic effects of n-hexane. Groups of 15 rats were administered 0.1 ml of test substance in olive oil (1:1 v/v) subcutaneously for 30 days. After the exposure, the animals were sacrificed. The hearts of one group were used to determined the effect of exposure of test substance on the ventricular fibrillation threshold (VFT). The other group was used to determine effect of exposure on magnesium and potassium levels. Another group of rats was given the same exposure, but with supplemental magnesium or potassium in the diet as well. The hearts of these animals were also examined using light and electron microscopy. There was a significent reduction in VFT and myocardial magnesium and potassium levels in all exposure groups. Histopathological changes were seen in heart tissue of exposure groups when examined with an electron microscope. Exposure to 0.1 ml of n-hexane for 30 days subcutaneously resulted in decreased VFT even when supplemental magnesium and potassium was given in the diet.

Endpoint:
specific investigations: other studies
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1992
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: This study is classified as reliable with restrictions because there was no GLP statement provided, and limited data on methods were reported, but the study seemed to be well-conducted.
Qualifier:
no guideline followed
GLP compliance:
no
Type of method:
in vivo
Endpoint addressed:
not applicable
Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: University of Natal
- Weight at study initiation: 200-250 g
Route of administration:
subcutaneous
Vehicle:
olive oil
Details on exposure:
Test animals were injected with 0.1 ml of n-hexane in olive oil (1:1 v/v, total volume of 0.2 ml) daily for 90 days.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
90 days
Frequency of treatment:
daily
Post exposure period:
none
Remarks:
Doses / Concentrations:
0.1 ml
Basis:
nominal conc.
No. of animals per sex per dose:
20
Control animals:
yes, concurrent vehicle
Examinations:
Animals were anesthetized and perfused after the end of exposure. Animals were sacrificed in pairs and the hearts examined. The heart of one animal was immediately placed in cold Krebs-Henseleit solution. This heart was then placed on a Langendorff aparatus. After a 15 min rest period, the heart rate was measured using a universal digital rate counter. Outflow was collected in a graduated cylinder to record coronary output. Ventricular fibrillation threshold (VFT) was measured using an ECG with platinum electrodes placed 1 cm apart. One electrode was attached to the metal aortic perfusion cannula. The other was inserted into the pericardium of the left ventricle. 200 msec pulse trains with 10 msec delays and a 2 msec width of 60 Hz and increasing intensity, was applied at a 30 s delay interval during the vulnerable period of the cardiac cycle until fibrillation occurred. VFT was defined as repetitive, irregular ventricular rhythm, with a changing pattern that persisted for more than six cycles.

Heart tissue from the second animal was used to determined magnesium, potassium, and zinc levels. The heart tissue was dissolved in nitric and perchloric acids. Magnesium and zinc were analyzed using atomic adsorption spectrophotometry. Potassium was determined using a flame photometer.

Histopathology was also done on excised hearts. Body weights were also measured.
Details on results:
No clinical signs were seen in exposure groups, additionally, no significant histopathological effects were noted. Body weight gains were similar in exposure and control groups. There were no significant change in coronary flow or heart rate, however, the VFT was significantly reduced in exposure groups. Also, levels of magnesium, potassium, and zinc were reduced in exposure groups.

Control

Hexane

VFT (mA)

9.48 (2.98)

4.27 (1.87)

Magnesium (microg/g)

208 (18)

164 (28)

Potassium (microg/g)

2968 (218)

2586 (162)

Zinc (microg/g)

33.8 (6.8)

19.6 (4.0)
Conclusions:
90 days subcutaneous exposure to 0.1 ml of hexane resulted in lowered ventricullar fibrillation threshold and lowered levels of magnesium, potassium, and zinc in rats.
Executive summary:

This study examined the cardiotoxic effects of 90 days exposure to hexane on rats. Groups of 20 rats were administered 0.1 ml of test substance subcutaneously for 90 days. After the exposure period, the hearts of one-half of the exposure animals were examined for coronary flow, heart rate, and ventricular fibrillation threshold (VFT). The hearts of the other exposure animals were examined for magnesium, potassium, and zinc levels. The VFT and magnesium, potassium, and zinc levels were significantly reduced in exposure groups as compared to controls.

Endpoint:
specific investigations: other studies
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1983
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: This study is classified as reliable with restrictions because there was no GLP statement provided, and limited data on methods were reported, but the study seemed to be well-conducted.
Qualifier:
no guideline followed
Principles of method if other than guideline:
This study examined the cardiotoxic effects of hexane exposure to rats. 10 rats were exposed to 0.1 ml of test substance subcutaneously for 30 days. At the end of the exposure period, the animals were sacrificed, and their heart tissue examined via light microscopy and TEM. The morphometry of the cardiac tissue was also examined.
GLP compliance:
no
Type of method:
in vivo
Endpoint addressed:
not applicable
Species:
rat
Strain:
Wistar
Sex:
male
Route of administration:
subcutaneous
Vehicle:
olive oil
Details on exposure:
Test animals were injected with 0.1 ml of n-hexane in olive oil (1:1 v/v) daily for 30 days.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
30 days
Frequency of treatment:
daily
Post exposure period:
none
Remarks:
Doses / Concentrations:
0.1 ml
Basis:
nominal conc.
No. of animals per sex per dose:
10 males
Control animals:
yes, concurrent vehicle
Examinations:
At the end of the exposure period, animals were immediately sacrificed. Hearts were removed while still beating, and immediately fixed. Tissue was examined using light microscopy and TEM. Morphometry was analyzed by having each myofiber represented by a cross-nuclear fiber diameter (FD) and nuclear diameter (ND). The FD and ND were determined using a VIDS image analyzer and the least diameter method. A regression is then done to determine the normal FD/ND means with an ellipse that indicates the P < 0.05 distribution. Myocardial pathology is detected by FD/ND results outside these normal limits.
Details on results:
Light microscopy showed little difference between control and exposure groups. The FD/ND analysis showed a trend in a reduction in mean FD and ND in exposure groups, in addition there were some myofiber abnormalities. The TEM analysis showed some elongated mitochondria with densely packed cristae in the exposure group. Swollen mitochondria were frequent in the exposure group as well. Double membranes were seen as well, and myofilaments were disrupted in the area of the Z-band. Z-band streaming and dissolution was seen in the myofibers as well. Intermyofibrillar edema, intercalated discs, and intermyofibrillar glycogen granules were also seen.

F/D

Control: 15.4

Experimental: 14.0

N/D

Control: 5.25

Experimental: 5.05

Conclusions:
Exposure of 0.1 ml of hexane subcutaneously for 30 days caused ultrastructural changes in the mitochondria and myofibrils that mimic myocardial ischaemia.
Executive summary:

This study examined the cardiotoxic effects of hexane exposure to rats. 10 rats were exposed to 0.1 ml of test substance subcutaneously for 30 days. At the end of the exposure period, the animals were sacrificed, and their heart tissue examined via light microscopy and TEM. The morphometry of the cardiac tissue was also examined. The exposure did not cause chronic myofiber pathology. However, ultrastructural changes were noted in the mitochondria and myofibrils that mimic myocardial ischaemia. These changes could lead to myocardial failure.

Description of key information

Cardiotoxicity (30-day subcutaneous; rat) – decreased ventricular fibrillation threshold (VFT)

 

Cardiotoxicity (90-day subcutaneous; rat) – decreased VFT, magnesium, potassium and zinc

 

Cardiotoxicity (30-day subcutaneous; rat) – mitochondria and myofibril ultrastructural changes

Additional information

Cardiotoxicity

n-hexane

In a key 30 day cardiotoxic effects of n-hexane was determined (Khedun, 1996; Klimisch score =2). Groups of 15 rats were administered 0.1 ml of test substance in olive oil (1:1 v/v) subcutaneously for 30 days. After the exposure, the animals were sacrificed. The hearts of one group were used to determine the effect of exposure of test substance on the ventricular fibrillation threshold (VFT). The other group was used to determine effect of exposure on magnesium and potassium levels. Another group of rats was given the same exposure, but with supplemental magnesium or potassium in the diet as well. The hearts of these animals were also examined using light and electron microscopy. There was a significant reduction in VFT and myocardial magnesium and potassium levels in all exposure groups. Histopathological changes were seen in heart tissue of exposure groups when examined with an electron microscope. Exposure to 0.1 ml of n-hexane for 30 days subcutaneously resulted in decreased VFT even when supplemental magnesium and potassium was given in the diet.

In a key subcutaneous exposure study the cardiotoxic effects of 90 days exposure to hexane on rats was examined (Khedun, 1992; Klimisch score =2). Groups of 20 rats were administered 0.1 ml of test substance subcutaneously for 90 days. After the exposure period, the hearts of one-half of the exposure animals were examined for coronary flow, heart rate, and ventricular fibrillation threshold (VFT). The hearts of the other exposure animals were examined for magnesium, potassium, and zinc levels. The VFT and magnesium, potassium, and zinc levels were significantly reduced in exposure groups as compared to controls.

In a key subcutaneous 30 day study the cardiotoxic effects of hexane exposure to rats were examined (Maharaj, 1982; Klimisch score =2). 10 rats were exposed to 0.1 ml of test substance subcutaneously for 30 days. At the end of the exposure period, the animals were sacrificed, and their heart tissue examined via light microscopy and TEM. The morphometry of the cardiac tissue was also examined. The exposure did not cause chronic myofiber pathology. However, ultrastructural changes were noted in the mitochondria and myofibrils that mimic myocardial ischaemia. These changes could lead to myocardial failure.