Toxicology Letters, 62 (1992) 25-3 1 0 1992 Elsevier Science Publishers B.V. All rights reserved 0378-4274/92/$ 5.00
25
TOXLET 02751
Inhalation toxicity of chromium from Whetlerite dust in rats
Roger Hilaski”, Sidney Katzb and Harry Salema “U.S. Army Chemical Research, Development and Engineering Center, Toxicology Division, SMCCR-RST-I,
Aberdeen Proving Ground, MD (USA) and bDepartment of Chemistry, Rutgers
University, Camden, NJ (USA)
(Received 11 February 1992) (Accepted 3 April 1992) Key words: Chromium; Copper; Whetlerite dust; OECD Limit Test; Inhalation; Rat
SUMMARY The acute inhalation toxicity and metabolic fate of chromium and copper from Whetlerite dust in rats were investigated. Groups of male and female, Sprague-Dawley rats were exposed to Whetlerite dust and base carbon dust as outlined in the OECD Limit Test guidelines. At 14,28 and 180 days post-exposure, rats were evaluated for gross pathological changes and tissues were collected for chromium and copper determination. Four deaths occurred during or post-exposure, but did not appear to be compound related. No gross pathological changes were observed at necropsy in either group. Organ chromium concentrations were below detection limits of 0.5 fig Cr/g dry tissue in both exposure groups. According to OECD guidelines, neither Whetlerite dust nor base carbon dust demonstrated an acute inhalation toxicity.
INTRODUCTION
Whetlerite (ASC carbon) is a granular, activated carbon impregnated with compounds of copper, silver and chromium to enhance its ability to absorb and destroy toxic gases such as hydrogen cyanide (AC) and cyanogen chloride (CK). Typical impregnations are: 6.&8.0% copper; O.lLO.2% silver; 2.53.5% chromium. When used in respiratory protective devices, small amounts of chromium-containing dust are sometimes released from the impregnated carbon. The possible exposure to those using protective masks equipped with canisters containing Whetlerite has been estimated [l]. Information on the bioavailability and systemic distribution of
Correspondence to: R. Hilaski, U.S. Army Chemical Research, Development and Engineering Center, Toxicology Division, SMCCR-RST-I, Aberdeen Proving Ground, MD 21010-5423, USA.
26
chromium and copper has been obtained from rats that received Whetlerite dust intratracheally [2]. No information, however, is available on the inhalation toxicity of chromium from this source. Chromium is both an essential micronutrient and a chemical carcinogen. Its role as a nutrient or carcinogen is determined by the chemical form and dosage. Oxidation state and solubility are particularly important factors. These also influence the bioavailability and systemic distribution of chromium. The chemical speciation of the chromium in the AX carbon has been determined [I]. The biochemical importance of chromium in glucose metabolism was reported more than a quarter century ago [3], and the glucose tolerance factor (GTF) was subsequently identified as a complex of trivalent chromium [4]. Rhinehart and Gad [5] stated, “. . . there is no evidence to implicate Cr (III) as a carcinogenic risk from any route of exposure, . . ,“. The carcinogenicity of hexavalent chromium compounds, on the other hand, is well documented [&lo]. The American Conference of Governmental Industrial Hygienists (ACGIH) Threshold Limit Values [ 1l] reflect the different allowable exposure levels and carcinogenic potential of the various oxidation states of chromium: 0.5 mg/m3 for chromium metal; 0.5 mgim’ for divalent chromium compounds; 0.5 mg/m3 for trivalent chromium compounds; 0.05 mg/m3 for hexavalent chromium compounds. The National Institute for Occupational Safety and Health (NIOSH) recommendations [I21 for airborne chromium concentration are specifically directed to the carcinogenicity of hexavalent compounds. According to NIOSH criteria documents 1131, noncar~inogeni~ chromium (VI) is the chromium in the mono- and dichromates of hydrogen, lithium, sodium, potassium, rubidium, cesium, and ammonium and in chromium trioxide (CrO,). The chromium in all other hexavalent chromium compounds is identified as carcinogenic chromium (VI). NIOSH identified the water soluble hexavalent chromium compounds as noncarcinogenic chromium (VI), and the water insoluble hexavalent chromium compounds were defined as carcinogenic chromium (VI). This has been demonstrated with implants of various hexavalent chromium compounds in the bronchi of rats [ 141. Whetlerite has been shown to contain both trivalent and hexavalent chromium 1151, and the latter was present in both soluble and insoluble forms [ 161.To fully assess the hazard potential of chromium in Whetlerite, info~ation on its acute inhalation toxicity is needed. This inhalation exposure was conducted in accordance with the Organization for Economic Cooperation and Development (OECD) Limit Test [I 71 which specifies an exposure concentration of 5 mg per liter with a total exposure duration of 4 h. This differs from the EPA Limit Test in which the duration of exposure is 4 h after equilibration of chamber concentration [ 181. MATERIALS
AND METHODS
Whetlerite (ASC Carbon, Calgon lot No. 1462) and the base carbon (CWS Carbon, Calgon lot No. 6266) were used in this study. These materials were reduced to dusts
27
in a micronizing mill (Markson Scientific). The total copper, total chromium, total hexavalent chromium, and soluble hexavalent chromium of the Whetlerite and of the base carbon were determined by methods described earlier [19]. The base carbon (CWS) was found to be free of copper and chromium and was used as the control material. The composition of the Whetlerite (ASC) was: 7.20 & 0.70% total copper: 3.86 ? 0.50% total chromium: 0.89 + 0.74% insoluble chromium (III) 1.64 + 0.99% insoluble chromium (VI) 1.33 ? 0.82% soluble chromium (VI) Thirty-six female and 36 male Sprague-Dawley rats from the Charles River Laboratories, Inc. were used. These animals were sexually mature, and had attained average body weights of 300 and 430 g, respectively, by the time of exposure. The rats were housed in individual wire cages. Access to food and water was ad libitum. The housing environment was 70 & 5°F and 50 ? 20% relative humidity. The dark/ light cycle was 12 h. The research protocol was approved by the CRDEC Laboratory Animal Use Review Committee and the study conducted in compliance with the Guide for the Care and Use of Laboratory Animals [20], and consistent with Good Laboratory Practices. Animal weights were taken weekly before and biweekly after exposure until the end of the post-exposure (PE) periods. Daily observations were made for clinical signs and behavior. Post-exposure observations included checking for toxic signs. Two groups of six female and six male rats were exposed to the CWS and three groups (six female, six male) were exposed to the ASC dust. The CWS and the ASC groups were exposed to the same target concentration. Another group of control animals (three female, three male) were exposed to clean air only in order to evaluate possible stresses associated with the nose-only restrainers. The rats were exposed in a 250-liter nose-only chamber. The animals were restrained in body tubes with their noses protruding. The body tubes were secured to the 250-l chamber allowing only rodent noses to be exposed in the chamber. An ACCU-RATE feeder metered the test materials into a Metronics blower which dispersed the dusts into a mixing bowl on top of the exposure chamber. The exposure chamber, feeder, and blower were enclosed in a 20 m3 containment chamber. The feed air to the containment chamber was conditioned to 75 f. 5°F and 50 ? 10% relative humidity and air flow through the exposure chamber was maintained at 1790 l/min. Air from the exposure chamber was passed through a particulate filter before discharging to the atmosphere. Chamber concentrations were measured gravimetrically by collecting samples on glass fiber filter pads. Samples were collected 5 min after the exposure started and then every 30 min throughout the 4-h exposure period. Particle size distributions were measured with a Sierra Model 2210 lo-stage cascade impactor (Anderson Instruments, Atlanta, GA). Particle size measurements were made at 120 min into the exposure period.
28
300
0
50
-
MAL-ASC
-
MAL-CWS
-
FEM-ASC
-
FEM-CWS
100
150
200
CAYS
Fig. 1. Mean body weight after exposure to Whetlerite and base carbon dust.
At intervals of 14, 28 and 180 days post-exposure, eight rats (four female, four male) from the CWS exposed group and 12 rats (six female, six male) from the ASC exposed group were euthanized and necropsied. Kidneys, livers and lungs were collected for the determination of their copper and chromium contents [21]. These measurements were made by atomic absorption spectroscopy after acid digestion of the organ [22]. Since Salem and Katz [2] examined chromium distribution at 4 h to 48 h post-lung instillation, it was decided to investigate 14 to 180 days post-exposure. RESULTS
The gravimetric measurements showed that the average exposure concentration was 5 mg/l for all exposures. For the Whetlerite exposures, the mass median aerodynamic diameter was found to be 3 ,um, with a geometric standard deviation of 3.5. A mass medium aerodynamic diameter of 4 pm with a geometric standard deviation of 3.5 was measured for both exposures to the CWS. The rats did not exhibit any adverse effects from the nose-only restraints. Both control and exposed rats displayed similar behavior upon removal from the holders. Generally, body weights were lower at 24 h PE but these losses were regained during the next 48 to 72 h. Figure 1 shows that a normal weight gain was observed for test and control groups throughout the post-exposure period. Two males of the 36 rats exposed to the Whetlerite dust died. The first of these died at the end of the 4-h exposure period, while the second rat died 2 days after the exposure. The deaths did not appear to be compound related, as no gross pathologi-
29
cal changes were observed during necropsy. The first fatality may have been a resolved pneumonia, and the second may have resulted from a self-inflicted injury during exposure. Animals exposed to Whetlerite dust showed no gross pathological changes when necropsied at 14,28 and 180 days PE. Two males of the 24 rats exposed to the base carbon dust died during exposure. As with the ASC exposed rats, post mortem examinations showed no gross pathological changes and did not appear compound related. Within 4 h after exposure, all animals that survived the exposure to the base carbon dust appeared normal with respect to behavior and clinical signs. No gross pathological changes were observed in the animals necropsied at 14,28 and 180 days PE. Organ chromium concentrations were below the instrument detection limits of approx. 0.5 ,ug Cr/g dry tissue for both groups. The organ copper concentrations are summarized in Table I. No significant difference between the experimental and control groups was observed. DISCUSSION
Acute inhalation toxicity is often the initial step for the assessment of health hazards associated with aerosols. Information on toxic characteristics is summarized as the total adverse effect caused by a substance following a single, uninterrupted exposure by inhalation for a short period of time. The OECD Limit Test exposure is 4 h, TABLE I COPPER CONCENTRATIONS IN RAT ORGANS AFTER INHALATION AND BASE CARBON DUSTS (g Cu/g DRY TISSUE) Tissue
Whetlerite
OF WHETLERITE
Base carbon
females
males
females
males
Kidney 14 days PE 28 days PE 180 days PE
24.3 f 13.3 39.4 Xk17.9 51.3 k 13.9
20.4 f 5.6 23.6 f 9.4 29.2 * 6.6
23.9 f 4.5 30.5 + 13.1 43.7 f 6.6
27.5 f 13.3 26.3 k 9.2 24.5 2 5.0
Liver 14 days PE 28 days PE 180 days PE
9.9 + 3.5 9.3 f 3.0 12.4 f 2.8
7.8 f 1.6 8.6 f 2.1 9.4 t 3.1
9.3 f 0.8 12.3 + 1.3 8.7 + 3.0
8.9 f 4.2 9.4 ?r 2.6 10.2 f 1.0
Lung 14 days PE 28 days PE 180 days PE
9.3 f 2.3 16.5 !z 4.1 19.6 f 1.3
8.6 + 1.7 7.7 f 1.5 6.1 f 1.2
8.7 k 2.9 4.6+ 1.2 6.6 + 2.1
6.2 + 2.5 5.9 + 0.4 5.9 f 2.4
PE = post-exposure.
30
and its exposure concentration is 5 mgil. Again, the EPA Limit Test differs only by beginning the 4-h exposure after equilibration of the chamber concentration. The failure to detect chromium in the livers and kidneys of the rats exposed to Whetlerite dust is consistent with the observations of Kalman and his research group [23, 241 who demonstrated poor absorption of lead chromate in particulates inhaled from paint aerosols. A copper dose of approx. 15 mgikg can be expected from a Whetlerite dose of 140 mg/kg. This copper dose appears to be in excess of the lethal dose by injection of soluble copper compounds to several laboratory animals: i.e., 12.7 mg/kg for the mouse, 0.5 1 mglkg for the guinea pig, and 1.I5 mgikg for the rabbit [25]. These levels are considerably higher than the copper concentrations determined in this study. Keller and Kaminski ]26], however, did observe signi~~ant increases in liver copper concentrations for weeks after the implantation of dental amalgams into the peritoneal cavities of rats. The deaths in the control (CWS) group (2124) and the experimental (ASC) group (2136) did not appear to be compound related and should not be a factor in the evaluation of the toxicity of these materials. The two carbon dusts did not produce any observable adverse changes in body weight, behavior or gross pathology. According to the OECD Limit Test, neither material demonstrated an acute inhalation toxicity. ACKNOWLEDGEMENTS
The technical expertise of Charles Crouse in designing and operating the exposure chamber is gratefully acknowledged as are Jeffrey Bergmann’s measurements of the particle size distributions. Special thanks go to John Callahan for his patient instruction in observing toxic signs, and to Dr. Stanley Liebenberg for supervising the necropsies.
REFERENCES 1 Katz, S.A. (1985) The chemical speciation and bioavailability of chromium from ACS carbon. U.S. Army Material Command Report, Contract No. DAAG29-81-D-0100, September. 2 Salem, H. and Katz. S.A. (1989) Speciation, bioavailab~lity and systemic distribution of chromium from Whetlerite dust. Sci. Total Environ. 86, 59-64. 3 Schwartz, K. and Mertz, W. (1959) Chromium (III) and glucose tolerance factor. Arch. Biochem. Biophys. 85, 292. 4 Toepfer, E.W. (1974) Separation from yeast of chromium containing material possessing Glucose Tolerance Factor (GTF). Fed. Proc. 33, 659. 5 Rinehart, W.E. and Gad, S.C. (1986) Current concepts in occupational health: metals-chromium. Am. Indust. Hyg. Assoc. J. 47,696. 6 Schechtman, L.M. et al. (1986) Analysis of the interlaboratory and intralaboratory reproducibility of the enhancement of simian adenovirus SA 7 transformation of Syrian hamster embryo cells by model carcinogenic and noncarcinogenic compounds. Environ. Mutagens 8,495.
31 7 Persson, D., Osterberg, R. and Bjursell, G. (1986) Mechanism of chromium carcinogenesis. Acta Pharmacol. Toxicol. 59, 260. 8 Adachi, S., Yoshimura, M., Katayama, M. and Takemoto, K. (1986) Effects of chromium compounds on the respiratory system. Sangyo Igtaku 28,283. 9 Korallus, U. (1986) Chromium compounds: occupational health, toxicology, and biological monitoring. Toxicol. Environ. Chem. 12, 47. 10 Levy, L.S. and Venitt, S. (1986) Carcinogenicity and mutagenicity of chromium compounds: the association between bronchial metaplasia and neoplasia. Carcinogenesis 7, 83 1. 11 Guide to Occupational Exposure Values (1989) American Conference of Governmental Industrial Hygienists, Inc. 12 United States Department of Health (1975) Education and Welfare, Occupational Exposure to Chromium (VI), HEW Publication NIOSH 79-129, US GPO, Washington DC. 13 National Institute for Occupational Safety and Health (1975) Criteria for a Recommendation Standard: Occupational Exposure to Chromium (VI), United States Department of Health, Education, and Welfare, Washington DC. 14 Levy, L.S., Martin, P.A. and Bidstrup, P.L. (1986) Investigation of potential carcinogenicity of a range of chromium containing materials on a rat lung. Br. J. Indust. Med. 43,243. 15 Hammarstrom, J.L. and Sacco, A. (1986) Investigation of hydrogen reactivity and its use as a surface probe on high surface area copper-chromium-silver impregnated charcoal. J. Catalysis 100, 293. 16 Salem, H. and Katz, S.A. (1986) Chemical speciation of chromium from ASC carbon, Presented at the Eastern Analytical Symposium, New York, October. 17 OECD Guidelines for Testing Chemicals (1981) Acute Inhalation Toxicity - Limit Test ~ 403, Organization for Economic Cooperation and Development, May. 18 Toxic Substances Control Act Test Guidelines (1985) Code of Federal Regulations, 40, Parts 796 to 798, Section 798.1150. 19 Katz, S.A. and Salem, H. (1986) Comparison of spectrophotometric methods for the determination of hexavalent chromium in Whetlerite. Spectroscopy 12, 35. 20 Guide for the Care and Use of Laboratory Animals (1985) U.S. Department of Health and Human Services, National Institute of Health, NIH Publication 85-23. 21 Masironi, R. and Parr, R.E. (1977) Collection of post mortem samples for trace metal analysis, Presented at the International Symposium on Biological Specimen Collection, Luxembourg, April. 22 Katz, S.A. and Jenniss, S.W. (1983) Regulatory Compliance Monitoring by Atomic Absorption Spectroscopy, Verlag Chemie International, Deerfield Beach, FL. 23 Kalman, D., Schumacher, R., Covert, D. and Eaton, D. (1984) Biological availability of lead in a paint aerosol: 1. physical and chemical characterization of a lead paint aerosol. Toxicol. Lett. 22, 301. 24 Eaton, D., Kalman, D., Garvey, D., Morgan, M. and Omenn, G. (1984) Biological availability of lead in a paint aerosol: 2. absorption, distribution and excretion of intratracheally instilled lead paint particles in the rat. Toxicol. Lett. 22, 307. 25 Venugopal, B. and Luckey, T.P. (1975) Toxicology of nonradioactive heavy metals and their salts. In: T.P. Luckey, B. Venugopal and D. Hutcheson (Eds.), Heavy Metal Toxicology, Safety and Hormology, Academic Press, New York, p, 15. 26 Keller, J.C. and Kaminski, E.J. (1984) Toxic effects of Cu implants on liver. Fund. Appl. Toxicol. 4, 778.
25
TOXLET 02751
Inhalation toxicity of chromium from Whetlerite dust in rats
Roger Hilaski”, Sidney Katzb and Harry Salema “U.S. Army Chemical Research, Development and Engineering Center, Toxicology Division, SMCCR-RST-I,
Aberdeen Proving Ground, MD (USA) and bDepartment of Chemistry, Rutgers
University, Camden, NJ (USA)
(Received 11 February 1992) (Accepted 3 April 1992) Key words: Chromium; Copper; Whetlerite dust; OECD Limit Test; Inhalation; Rat
SUMMARY The acute inhalation toxicity and metabolic fate of chromium and copper from Whetlerite dust in rats were investigated. Groups of male and female, Sprague-Dawley rats were exposed to Whetlerite dust and base carbon dust as outlined in the OECD Limit Test guidelines. At 14,28 and 180 days post-exposure, rats were evaluated for gross pathological changes and tissues were collected for chromium and copper determination. Four deaths occurred during or post-exposure, but did not appear to be compound related. No gross pathological changes were observed at necropsy in either group. Organ chromium concentrations were below detection limits of 0.5 fig Cr/g dry tissue in both exposure groups. According to OECD guidelines, neither Whetlerite dust nor base carbon dust demonstrated an acute inhalation toxicity.
INTRODUCTION
Whetlerite (ASC carbon) is a granular, activated carbon impregnated with compounds of copper, silver and chromium to enhance its ability to absorb and destroy toxic gases such as hydrogen cyanide (AC) and cyanogen chloride (CK). Typical impregnations are: 6.&8.0% copper; O.lLO.2% silver; 2.53.5% chromium. When used in respiratory protective devices, small amounts of chromium-containing dust are sometimes released from the impregnated carbon. The possible exposure to those using protective masks equipped with canisters containing Whetlerite has been estimated [l]. Information on the bioavailability and systemic distribution of
Correspondence to: R. Hilaski, U.S. Army Chemical Research, Development and Engineering Center, Toxicology Division, SMCCR-RST-I, Aberdeen Proving Ground, MD 21010-5423, USA.
26
chromium and copper has been obtained from rats that received Whetlerite dust intratracheally [2]. No information, however, is available on the inhalation toxicity of chromium from this source. Chromium is both an essential micronutrient and a chemical carcinogen. Its role as a nutrient or carcinogen is determined by the chemical form and dosage. Oxidation state and solubility are particularly important factors. These also influence the bioavailability and systemic distribution of chromium. The chemical speciation of the chromium in the AX carbon has been determined [I]. The biochemical importance of chromium in glucose metabolism was reported more than a quarter century ago [3], and the glucose tolerance factor (GTF) was subsequently identified as a complex of trivalent chromium [4]. Rhinehart and Gad [5] stated, “. . . there is no evidence to implicate Cr (III) as a carcinogenic risk from any route of exposure, . . ,“. The carcinogenicity of hexavalent chromium compounds, on the other hand, is well documented [&lo]. The American Conference of Governmental Industrial Hygienists (ACGIH) Threshold Limit Values [ 1l] reflect the different allowable exposure levels and carcinogenic potential of the various oxidation states of chromium: 0.5 mg/m3 for chromium metal; 0.5 mgim’ for divalent chromium compounds; 0.5 mg/m3 for trivalent chromium compounds; 0.05 mg/m3 for hexavalent chromium compounds. The National Institute for Occupational Safety and Health (NIOSH) recommendations [I21 for airborne chromium concentration are specifically directed to the carcinogenicity of hexavalent compounds. According to NIOSH criteria documents 1131, noncar~inogeni~ chromium (VI) is the chromium in the mono- and dichromates of hydrogen, lithium, sodium, potassium, rubidium, cesium, and ammonium and in chromium trioxide (CrO,). The chromium in all other hexavalent chromium compounds is identified as carcinogenic chromium (VI). NIOSH identified the water soluble hexavalent chromium compounds as noncarcinogenic chromium (VI), and the water insoluble hexavalent chromium compounds were defined as carcinogenic chromium (VI). This has been demonstrated with implants of various hexavalent chromium compounds in the bronchi of rats [ 141. Whetlerite has been shown to contain both trivalent and hexavalent chromium 1151, and the latter was present in both soluble and insoluble forms [ 161.To fully assess the hazard potential of chromium in Whetlerite, info~ation on its acute inhalation toxicity is needed. This inhalation exposure was conducted in accordance with the Organization for Economic Cooperation and Development (OECD) Limit Test [I 71 which specifies an exposure concentration of 5 mg per liter with a total exposure duration of 4 h. This differs from the EPA Limit Test in which the duration of exposure is 4 h after equilibration of chamber concentration [ 181. MATERIALS
AND METHODS
Whetlerite (ASC Carbon, Calgon lot No. 1462) and the base carbon (CWS Carbon, Calgon lot No. 6266) were used in this study. These materials were reduced to dusts
27
in a micronizing mill (Markson Scientific). The total copper, total chromium, total hexavalent chromium, and soluble hexavalent chromium of the Whetlerite and of the base carbon were determined by methods described earlier [19]. The base carbon (CWS) was found to be free of copper and chromium and was used as the control material. The composition of the Whetlerite (ASC) was: 7.20 & 0.70% total copper: 3.86 ? 0.50% total chromium: 0.89 + 0.74% insoluble chromium (III) 1.64 + 0.99% insoluble chromium (VI) 1.33 ? 0.82% soluble chromium (VI) Thirty-six female and 36 male Sprague-Dawley rats from the Charles River Laboratories, Inc. were used. These animals were sexually mature, and had attained average body weights of 300 and 430 g, respectively, by the time of exposure. The rats were housed in individual wire cages. Access to food and water was ad libitum. The housing environment was 70 & 5°F and 50 ? 20% relative humidity. The dark/ light cycle was 12 h. The research protocol was approved by the CRDEC Laboratory Animal Use Review Committee and the study conducted in compliance with the Guide for the Care and Use of Laboratory Animals [20], and consistent with Good Laboratory Practices. Animal weights were taken weekly before and biweekly after exposure until the end of the post-exposure (PE) periods. Daily observations were made for clinical signs and behavior. Post-exposure observations included checking for toxic signs. Two groups of six female and six male rats were exposed to the CWS and three groups (six female, six male) were exposed to the ASC dust. The CWS and the ASC groups were exposed to the same target concentration. Another group of control animals (three female, three male) were exposed to clean air only in order to evaluate possible stresses associated with the nose-only restrainers. The rats were exposed in a 250-liter nose-only chamber. The animals were restrained in body tubes with their noses protruding. The body tubes were secured to the 250-l chamber allowing only rodent noses to be exposed in the chamber. An ACCU-RATE feeder metered the test materials into a Metronics blower which dispersed the dusts into a mixing bowl on top of the exposure chamber. The exposure chamber, feeder, and blower were enclosed in a 20 m3 containment chamber. The feed air to the containment chamber was conditioned to 75 f. 5°F and 50 ? 10% relative humidity and air flow through the exposure chamber was maintained at 1790 l/min. Air from the exposure chamber was passed through a particulate filter before discharging to the atmosphere. Chamber concentrations were measured gravimetrically by collecting samples on glass fiber filter pads. Samples were collected 5 min after the exposure started and then every 30 min throughout the 4-h exposure period. Particle size distributions were measured with a Sierra Model 2210 lo-stage cascade impactor (Anderson Instruments, Atlanta, GA). Particle size measurements were made at 120 min into the exposure period.
28
300
0
50
-
MAL-ASC
-
MAL-CWS
-
FEM-ASC
-
FEM-CWS
100
150
200
CAYS
Fig. 1. Mean body weight after exposure to Whetlerite and base carbon dust.
At intervals of 14, 28 and 180 days post-exposure, eight rats (four female, four male) from the CWS exposed group and 12 rats (six female, six male) from the ASC exposed group were euthanized and necropsied. Kidneys, livers and lungs were collected for the determination of their copper and chromium contents [21]. These measurements were made by atomic absorption spectroscopy after acid digestion of the organ [22]. Since Salem and Katz [2] examined chromium distribution at 4 h to 48 h post-lung instillation, it was decided to investigate 14 to 180 days post-exposure. RESULTS
The gravimetric measurements showed that the average exposure concentration was 5 mg/l for all exposures. For the Whetlerite exposures, the mass median aerodynamic diameter was found to be 3 ,um, with a geometric standard deviation of 3.5. A mass medium aerodynamic diameter of 4 pm with a geometric standard deviation of 3.5 was measured for both exposures to the CWS. The rats did not exhibit any adverse effects from the nose-only restraints. Both control and exposed rats displayed similar behavior upon removal from the holders. Generally, body weights were lower at 24 h PE but these losses were regained during the next 48 to 72 h. Figure 1 shows that a normal weight gain was observed for test and control groups throughout the post-exposure period. Two males of the 36 rats exposed to the Whetlerite dust died. The first of these died at the end of the 4-h exposure period, while the second rat died 2 days after the exposure. The deaths did not appear to be compound related, as no gross pathologi-
29
cal changes were observed during necropsy. The first fatality may have been a resolved pneumonia, and the second may have resulted from a self-inflicted injury during exposure. Animals exposed to Whetlerite dust showed no gross pathological changes when necropsied at 14,28 and 180 days PE. Two males of the 24 rats exposed to the base carbon dust died during exposure. As with the ASC exposed rats, post mortem examinations showed no gross pathological changes and did not appear compound related. Within 4 h after exposure, all animals that survived the exposure to the base carbon dust appeared normal with respect to behavior and clinical signs. No gross pathological changes were observed in the animals necropsied at 14,28 and 180 days PE. Organ chromium concentrations were below the instrument detection limits of approx. 0.5 ,ug Cr/g dry tissue for both groups. The organ copper concentrations are summarized in Table I. No significant difference between the experimental and control groups was observed. DISCUSSION
Acute inhalation toxicity is often the initial step for the assessment of health hazards associated with aerosols. Information on toxic characteristics is summarized as the total adverse effect caused by a substance following a single, uninterrupted exposure by inhalation for a short period of time. The OECD Limit Test exposure is 4 h, TABLE I COPPER CONCENTRATIONS IN RAT ORGANS AFTER INHALATION AND BASE CARBON DUSTS (g Cu/g DRY TISSUE) Tissue
Whetlerite
OF WHETLERITE
Base carbon
females
males
females
males
Kidney 14 days PE 28 days PE 180 days PE
24.3 f 13.3 39.4 Xk17.9 51.3 k 13.9
20.4 f 5.6 23.6 f 9.4 29.2 * 6.6
23.9 f 4.5 30.5 + 13.1 43.7 f 6.6
27.5 f 13.3 26.3 k 9.2 24.5 2 5.0
Liver 14 days PE 28 days PE 180 days PE
9.9 + 3.5 9.3 f 3.0 12.4 f 2.8
7.8 f 1.6 8.6 f 2.1 9.4 t 3.1
9.3 f 0.8 12.3 + 1.3 8.7 + 3.0
8.9 f 4.2 9.4 ?r 2.6 10.2 f 1.0
Lung 14 days PE 28 days PE 180 days PE
9.3 f 2.3 16.5 !z 4.1 19.6 f 1.3
8.6 + 1.7 7.7 f 1.5 6.1 f 1.2
8.7 k 2.9 4.6+ 1.2 6.6 + 2.1
6.2 + 2.5 5.9 + 0.4 5.9 f 2.4
PE = post-exposure.
30
and its exposure concentration is 5 mgil. Again, the EPA Limit Test differs only by beginning the 4-h exposure after equilibration of the chamber concentration. The failure to detect chromium in the livers and kidneys of the rats exposed to Whetlerite dust is consistent with the observations of Kalman and his research group [23, 241 who demonstrated poor absorption of lead chromate in particulates inhaled from paint aerosols. A copper dose of approx. 15 mgikg can be expected from a Whetlerite dose of 140 mg/kg. This copper dose appears to be in excess of the lethal dose by injection of soluble copper compounds to several laboratory animals: i.e., 12.7 mg/kg for the mouse, 0.5 1 mglkg for the guinea pig, and 1.I5 mgikg for the rabbit [25]. These levels are considerably higher than the copper concentrations determined in this study. Keller and Kaminski ]26], however, did observe signi~~ant increases in liver copper concentrations for weeks after the implantation of dental amalgams into the peritoneal cavities of rats. The deaths in the control (CWS) group (2124) and the experimental (ASC) group (2136) did not appear to be compound related and should not be a factor in the evaluation of the toxicity of these materials. The two carbon dusts did not produce any observable adverse changes in body weight, behavior or gross pathology. According to the OECD Limit Test, neither material demonstrated an acute inhalation toxicity. ACKNOWLEDGEMENTS
The technical expertise of Charles Crouse in designing and operating the exposure chamber is gratefully acknowledged as are Jeffrey Bergmann’s measurements of the particle size distributions. Special thanks go to John Callahan for his patient instruction in observing toxic signs, and to Dr. Stanley Liebenberg for supervising the necropsies.
REFERENCES 1 Katz, S.A. (1985) The chemical speciation and bioavailability of chromium from ACS carbon. U.S. Army Material Command Report, Contract No. DAAG29-81-D-0100, September. 2 Salem, H. and Katz. S.A. (1989) Speciation, bioavailab~lity and systemic distribution of chromium from Whetlerite dust. Sci. Total Environ. 86, 59-64. 3 Schwartz, K. and Mertz, W. (1959) Chromium (III) and glucose tolerance factor. Arch. Biochem. Biophys. 85, 292. 4 Toepfer, E.W. (1974) Separation from yeast of chromium containing material possessing Glucose Tolerance Factor (GTF). Fed. Proc. 33, 659. 5 Rinehart, W.E. and Gad, S.C. (1986) Current concepts in occupational health: metals-chromium. Am. Indust. Hyg. Assoc. J. 47,696. 6 Schechtman, L.M. et al. (1986) Analysis of the interlaboratory and intralaboratory reproducibility of the enhancement of simian adenovirus SA 7 transformation of Syrian hamster embryo cells by model carcinogenic and noncarcinogenic compounds. Environ. Mutagens 8,495.
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