Arch Toxicol (1992) 66:750-753
Archives of
Toxicology 9 Springer-Verlag 1992
Short communication
Acute and subacute inhalation toxicity of sUane 1000 ppm in mice Kazuyuki Omae 1, Tohru Sakai 1, Haruhiko Sakurai 1, Kazuto Yamazaki 2, Toshikatsu Shibata3, Koji Mori 4, Mitsuhiro Kudo 5, Hirokazu KanohS, and Masatomo Tati5 1Department of Preventive Medicine and Public Health, School of Medicine, Keio University, Tokyo, Japan 2 Department of Pathology, School of Medicine, Keio University 3 Second Department of Pathology, Saitama Medical School, Saitama, Japan 4 University of Occupational and Environmental Health, Kita-Kyushu,Japan 5 Occupational Health Service Center, Japan Industrial Safety and Health Association, Tokyo, Japan Received 26 March 1992/Accepted 10 August 1992
Abstract. Male ICR mice were exposed to silane 1000 ppm, a concentration 200 or 2000 times higher than the recommended occupational exposure limits by many countries and academic associations, for 1, 2, 4, and 8 h (phase I study) and for 6 h/day, 5 days/week, over 2 and 4 weeks (phase II study). Hematological and biochemical studies were performed, and the animals were examined for histopathological lesions of the cornea, nasal cavity, respiratory tract, lung, liver, kidney, spleen, pancreas, thymus, thyroid, bone marrow, salivary glands, esophagus, and testis. All mice in both studies survived until they were sacrificed. In the phase I study, no exposure-related changes were found as a result of the hematological, biochemical, or histopathological examinations. In the phase II study, hematological and biochemical examinations failed to reveal any exposure-related changes, but mild irritation, manifested in the form of a small amount of exudate (eight out of ten animals), and inflammatory cells and/or necrotic cells on the nasal mucosa (six out of ten animals) was observed in the mice exposed to silane for 4 weeks. These findings suggest that silane toxicity and irritation are not severe.
Key words: Silane - S e m i c o n d u c t o r - Inhalation - Toxicity - Mouse
Introduction Silane (Sill4, silicon tetrahydride) is the major specialty gases used to produce ingots of crystalline silicon, silicon semiconductors, amorphous silicon membranes for photoelectric cells, and silicon fine particles for certain fine
Correspondence to: K. Omae, Department of Preventive Medicine and Public Health, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160, Japan
ceramics. Silane consumption in Japan was approximately 70 tons in 1990. Silane is spontaneously combustible in room air at room temperature and is explosive when it steadily accumulates to levels of 1.37% v/v or above, having a minimal oxygen requirement for autoignition of 0.2%. Standard or recommended m a x i m u m occupational exposure levels of silane are 5 p p m (TWA) in Australia, Belgium, France, and by the American Conference of Governmental and Industrial Hygienists (ACGIH; United States), and the National Institute of Occupational Safety and Health/Occupational Safety and Health Agency (United States), and 0.5 p p m (TWA) in Denmark, Finland, Switzerland, and the United Kingdom (ILO 1991). Scientific evidence in support of these values, however, is very poor. A paper by MacEwen and Vernot (1972) is the only published report on silane toxicity. Although they exposed rats and mice to silane and observed the death of six out of ten male C F I mice exposed to 9600 p p m silane for 4 h, no gross pathological, histopathological, biochemical, or hematological studies were carried out. Information on silane toxicity from the Union Carbide Corporation and Matheson Gas Products are quoted in the A C G I H documentation (ACG1H 1985), but this information is in the form of a communication to a committee and unpublished reports. The present study was designed to determine the acute and subacute inhalation toxicity of silane in mice.
Materials and methods Animals
Four-week-old, male, SPF-grade ICR mice were purchased from Charles River, Japan9 The mice were"acclimated to laboratory conditions for 1 week prior to the exposure experiment. Five mice each were housed in transparent plastic cages with a stainless steel wire-line ceiling in filtered air ventilated, stainless steel housing for small animal breeding during periods of nonexposure (lsorack, Shinano Seisakusho). The animal room was maintained at a temperature of about 22~C and a relative humidityof 60%. The light/dark cycle consisted of 12 h each. Mice were fed pelleted rodent food (CRF-1, Oriental Kobo) and distilled water ad libitum.
751
Table 1. Summary of silane concentrations (ppm) in the phase I and II studies Phase I
II
Exposure length
n
Mean
SD
Range
1 hour 2 hours 4 hours 8 hours
5 11 22 43
1085 1094 957 977
34 64 51 50
1038-1124 973- 1211 876- 1067 876- 1086
2 weeks 4 weeks
294 590
973 966
61 62
855- 1045" 881 - 1061 *
9 5th percentile - 95th percentile
Table 2. ADWG in the phase II study: Monday morning-Friday morning (mean _ S D )
Exposed Nonexposed
1st week (n = 20)
2nd week (n = 20)
3rd week (n = 10)
4th week (n = 10)
0.24+0.22 0.33___0.23
0.15-1-0.22 0.21 -+0.14
---0.06--+0.35 0.16--+0.25
0.01 -+0.13 0.10-+0.19
Reagent Silane 10000 ppm, diluted with pure nitrogen and compressed in a gas cylinder, was supplied by Nippon Sanso (Oyama, Tochigi Prefecture). This concentration was considered the maximum that could be safely handled in our laboratory.
glands, esophagus, and testis were examined histopathologically. The organs were placed in 10% buffered neutral formalin solution except for one testis. The skull was decalcificated in 10% formic acid solution for histopathological examination of the nasal cavity. All tissues were embedded in paraffin, sectioned, and stained with hematoxylin and eosin using standard histological procedures. One testis from each pair was fixed in Bouin's solution and was stained with PAS-hematoxylin.
Experimental design The exposure level was set at 1000 ppm for the following reasons: (a) because this concentration is 200 or 2000 times higher than the recommended TWA exposure limits by many countries and academic associations (ILO 1991), the authors believed that 1000 ppm was sufficiently high to adversely affect the health of mice; and (b) the effect of oxygen deficiency on mice did not need to be taken into consideration, because dynamic flow of air containing 1000 ppm silane was created by ten fold dilution of 10000 ppm silane with filtered room air using a HEPA filter, and contained approximately 19% oxygen. Ten mice in each experiment were exposed to silane 1000 ppm for 1, 2, 4, and 8 h (phase I study), and 6 h/day, 5 days/week, for 2 and 4 weeks (phase II study) in a tightly sealed 550-1 stainless steel inhalation chamber with glass windows. Nonexposed mice as a control group were exposed to filtered room air in the same way as the mice exposed to silane. Silane concentrations were monitored at 10-rain intervals using a gas chromatograph (GCC-001, Gastec).
Animal observations The behavior and external appearance of the mice were checked daily for evidence of any exposure-related effects. In the phase Ii study, body weight was measured on Monday, Wednesday, and Friday morning prior to exposure to silane.
Biochemical and hematological measurements Biochemical and hematologic items assessed were alkaline phosphatase (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), cholinesterase (ChE), blood urea nitrogen (BUN), sodium (Na), potassium (K), red blood cell count (RBC), and total and differential white blood cell counts (WBC).
Results
Exposure levels T h e silane c o n c e n t r a t i o n s in the e x p o s u r e c h a m b e r are s u m m a r i z e d in T a b l e 1. In t h e p h a s e I s t u d y t h e m e a n s i l a n e e x p o s u r e c o n c e n t r a t i o n s w e r e 1085 p p m in the 1-h e x p o s u r e g r o u p , 1094 p p m in the 2 - h e x p o s u r e g r o u p , 957 p p m in the 4 - h e x p o s u r e g r o u p , a n d 977 p p m in the 8-h e x p o s u r e g r o u p . In the p h a s e II s t u d y the m e a n c o n c e n t r a tions w e r e 977 p p m in the 2 - w e e k e x p o s u r e g r o u p a n d 9 6 6 p p m in the 4 - w e e k e x p o s u r e group.
Pathological examination
Animal observations
Mice in the phase I study were sacrificed 3 days after the conclusion of silane exposure, and in the phase II study on the day after the final exposure. The mice were anesthetized with sodium pentobarbiturate and sacrificed by exsanguination from the axillary artery. Their organs were examined grossly, and the cornea, nasal cavity, respiratory tract, lung, liver, kidney, spleen, pancreas, thymus, thyroid, bone marrow, salivary
I m m e d i a t e l y after b e i n g e x p o s e d to s i l a n e o n e a c h d a y o f e x p o s u r e , m o s t m i c e b e g a n to p e r f o r m f a c e w a s h i n g m o v e m e n t s and l i c k the l o w e r a b d o m e n f o r short p e r i o d s m o r e f r e q u e n t l y t h a n n o n e x p o s e d m i c e . N o m i c e d i e d in e i t h e r p h a s e o f the study.
752 Table 3. ADWG in the phase II study: Friday morning-Monday morning (mean ___SD)
Exposed Nonexposed
1st- 2nd week (n = 20)
2ud- 3rd week (n = 10)
3rd - 4th week (n = 10)
0.35 -4-0.26 0.46_+0.14
0.44 +_0.25 0.47_+0.14
0.59 +_0.17* 0.27-+0.17
9 P
Archives of
Toxicology 9 Springer-Verlag 1992
Short communication
Acute and subacute inhalation toxicity of sUane 1000 ppm in mice Kazuyuki Omae 1, Tohru Sakai 1, Haruhiko Sakurai 1, Kazuto Yamazaki 2, Toshikatsu Shibata3, Koji Mori 4, Mitsuhiro Kudo 5, Hirokazu KanohS, and Masatomo Tati5 1Department of Preventive Medicine and Public Health, School of Medicine, Keio University, Tokyo, Japan 2 Department of Pathology, School of Medicine, Keio University 3 Second Department of Pathology, Saitama Medical School, Saitama, Japan 4 University of Occupational and Environmental Health, Kita-Kyushu,Japan 5 Occupational Health Service Center, Japan Industrial Safety and Health Association, Tokyo, Japan Received 26 March 1992/Accepted 10 August 1992
Abstract. Male ICR mice were exposed to silane 1000 ppm, a concentration 200 or 2000 times higher than the recommended occupational exposure limits by many countries and academic associations, for 1, 2, 4, and 8 h (phase I study) and for 6 h/day, 5 days/week, over 2 and 4 weeks (phase II study). Hematological and biochemical studies were performed, and the animals were examined for histopathological lesions of the cornea, nasal cavity, respiratory tract, lung, liver, kidney, spleen, pancreas, thymus, thyroid, bone marrow, salivary glands, esophagus, and testis. All mice in both studies survived until they were sacrificed. In the phase I study, no exposure-related changes were found as a result of the hematological, biochemical, or histopathological examinations. In the phase II study, hematological and biochemical examinations failed to reveal any exposure-related changes, but mild irritation, manifested in the form of a small amount of exudate (eight out of ten animals), and inflammatory cells and/or necrotic cells on the nasal mucosa (six out of ten animals) was observed in the mice exposed to silane for 4 weeks. These findings suggest that silane toxicity and irritation are not severe.
Key words: Silane - S e m i c o n d u c t o r - Inhalation - Toxicity - Mouse
Introduction Silane (Sill4, silicon tetrahydride) is the major specialty gases used to produce ingots of crystalline silicon, silicon semiconductors, amorphous silicon membranes for photoelectric cells, and silicon fine particles for certain fine
Correspondence to: K. Omae, Department of Preventive Medicine and Public Health, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160, Japan
ceramics. Silane consumption in Japan was approximately 70 tons in 1990. Silane is spontaneously combustible in room air at room temperature and is explosive when it steadily accumulates to levels of 1.37% v/v or above, having a minimal oxygen requirement for autoignition of 0.2%. Standard or recommended m a x i m u m occupational exposure levels of silane are 5 p p m (TWA) in Australia, Belgium, France, and by the American Conference of Governmental and Industrial Hygienists (ACGIH; United States), and the National Institute of Occupational Safety and Health/Occupational Safety and Health Agency (United States), and 0.5 p p m (TWA) in Denmark, Finland, Switzerland, and the United Kingdom (ILO 1991). Scientific evidence in support of these values, however, is very poor. A paper by MacEwen and Vernot (1972) is the only published report on silane toxicity. Although they exposed rats and mice to silane and observed the death of six out of ten male C F I mice exposed to 9600 p p m silane for 4 h, no gross pathological, histopathological, biochemical, or hematological studies were carried out. Information on silane toxicity from the Union Carbide Corporation and Matheson Gas Products are quoted in the A C G I H documentation (ACG1H 1985), but this information is in the form of a communication to a committee and unpublished reports. The present study was designed to determine the acute and subacute inhalation toxicity of silane in mice.
Materials and methods Animals
Four-week-old, male, SPF-grade ICR mice were purchased from Charles River, Japan9 The mice were"acclimated to laboratory conditions for 1 week prior to the exposure experiment. Five mice each were housed in transparent plastic cages with a stainless steel wire-line ceiling in filtered air ventilated, stainless steel housing for small animal breeding during periods of nonexposure (lsorack, Shinano Seisakusho). The animal room was maintained at a temperature of about 22~C and a relative humidityof 60%. The light/dark cycle consisted of 12 h each. Mice were fed pelleted rodent food (CRF-1, Oriental Kobo) and distilled water ad libitum.
751
Table 1. Summary of silane concentrations (ppm) in the phase I and II studies Phase I
II
Exposure length
n
Mean
SD
Range
1 hour 2 hours 4 hours 8 hours
5 11 22 43
1085 1094 957 977
34 64 51 50
1038-1124 973- 1211 876- 1067 876- 1086
2 weeks 4 weeks
294 590
973 966
61 62
855- 1045" 881 - 1061 *
9 5th percentile - 95th percentile
Table 2. ADWG in the phase II study: Monday morning-Friday morning (mean _ S D )
Exposed Nonexposed
1st week (n = 20)
2nd week (n = 20)
3rd week (n = 10)
4th week (n = 10)
0.24+0.22 0.33___0.23
0.15-1-0.22 0.21 -+0.14
---0.06--+0.35 0.16--+0.25
0.01 -+0.13 0.10-+0.19
Reagent Silane 10000 ppm, diluted with pure nitrogen and compressed in a gas cylinder, was supplied by Nippon Sanso (Oyama, Tochigi Prefecture). This concentration was considered the maximum that could be safely handled in our laboratory.
glands, esophagus, and testis were examined histopathologically. The organs were placed in 10% buffered neutral formalin solution except for one testis. The skull was decalcificated in 10% formic acid solution for histopathological examination of the nasal cavity. All tissues were embedded in paraffin, sectioned, and stained with hematoxylin and eosin using standard histological procedures. One testis from each pair was fixed in Bouin's solution and was stained with PAS-hematoxylin.
Experimental design The exposure level was set at 1000 ppm for the following reasons: (a) because this concentration is 200 or 2000 times higher than the recommended TWA exposure limits by many countries and academic associations (ILO 1991), the authors believed that 1000 ppm was sufficiently high to adversely affect the health of mice; and (b) the effect of oxygen deficiency on mice did not need to be taken into consideration, because dynamic flow of air containing 1000 ppm silane was created by ten fold dilution of 10000 ppm silane with filtered room air using a HEPA filter, and contained approximately 19% oxygen. Ten mice in each experiment were exposed to silane 1000 ppm for 1, 2, 4, and 8 h (phase I study), and 6 h/day, 5 days/week, for 2 and 4 weeks (phase II study) in a tightly sealed 550-1 stainless steel inhalation chamber with glass windows. Nonexposed mice as a control group were exposed to filtered room air in the same way as the mice exposed to silane. Silane concentrations were monitored at 10-rain intervals using a gas chromatograph (GCC-001, Gastec).
Animal observations The behavior and external appearance of the mice were checked daily for evidence of any exposure-related effects. In the phase Ii study, body weight was measured on Monday, Wednesday, and Friday morning prior to exposure to silane.
Biochemical and hematological measurements Biochemical and hematologic items assessed were alkaline phosphatase (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), cholinesterase (ChE), blood urea nitrogen (BUN), sodium (Na), potassium (K), red blood cell count (RBC), and total and differential white blood cell counts (WBC).
Results
Exposure levels T h e silane c o n c e n t r a t i o n s in the e x p o s u r e c h a m b e r are s u m m a r i z e d in T a b l e 1. In t h e p h a s e I s t u d y t h e m e a n s i l a n e e x p o s u r e c o n c e n t r a t i o n s w e r e 1085 p p m in the 1-h e x p o s u r e g r o u p , 1094 p p m in the 2 - h e x p o s u r e g r o u p , 957 p p m in the 4 - h e x p o s u r e g r o u p , a n d 977 p p m in the 8-h e x p o s u r e g r o u p . In the p h a s e II s t u d y the m e a n c o n c e n t r a tions w e r e 977 p p m in the 2 - w e e k e x p o s u r e g r o u p a n d 9 6 6 p p m in the 4 - w e e k e x p o s u r e group.
Pathological examination
Animal observations
Mice in the phase I study were sacrificed 3 days after the conclusion of silane exposure, and in the phase II study on the day after the final exposure. The mice were anesthetized with sodium pentobarbiturate and sacrificed by exsanguination from the axillary artery. Their organs were examined grossly, and the cornea, nasal cavity, respiratory tract, lung, liver, kidney, spleen, pancreas, thymus, thyroid, bone marrow, salivary
I m m e d i a t e l y after b e i n g e x p o s e d to s i l a n e o n e a c h d a y o f e x p o s u r e , m o s t m i c e b e g a n to p e r f o r m f a c e w a s h i n g m o v e m e n t s and l i c k the l o w e r a b d o m e n f o r short p e r i o d s m o r e f r e q u e n t l y t h a n n o n e x p o s e d m i c e . N o m i c e d i e d in e i t h e r p h a s e o f the study.
752 Table 3. ADWG in the phase II study: Friday morning-Monday morning (mean ___SD)
Exposed Nonexposed
1st- 2nd week (n = 20)
2ud- 3rd week (n = 10)
3rd - 4th week (n = 10)
0.35 -4-0.26 0.46_+0.14
0.44 +_0.25 0.47_+0.14
0.59 +_0.17* 0.27-+0.17
9 P
