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In vitro method for medical risk assessment of laser fumes
In vitro method for medical assessment of laser fumes W. MALKUSCH,
B. REHN,
risk
J. BRUCH
Laser processing of different materials may produce toxic fumes. In preventive occupational medicine it is necessary to evaluate valid hygienic standards for work places. The basis for such hygienic standards is the classification of laser fumes by their fibrogenic, emphysematous, immunological or other harmful potencies in biological assay systems. This paper is part of a European project on laser safety. Our part in this project is the development of a method for the investigation of lung responses using in vitrocell assays. The appropriate laser fume samples will be supplied by other groups in this European project. In contrast to the cell assays usually used in risk assessment, our method is based on isolated target cells in the lung, such as alveolar macrophages. The test criteria are mediator release, surfactant reactions, release of reactive oxygen species and cell proliferation. As demonstrated in the lung response to other dusts (minerals, fibres etc) these parameters are medically relevant factors in the pathogenic alveolar dust response. The paper gives basic information about the method using lung cell assays and the results of known substances, in comparison with a dust generated by laser processing. KEYWORDS:
laser fumes, medical risk assessment, cell assays, dust, lungs
Introduction The lung as the respiratory organ is always affected by foreign bodies entering with the air stream. Large particles are usually filtered in the nose or cleared from the trachea or main bronchi by coughing up. The lung structure is divided into the conducting airways (bronchi) and the respiratory unit (alveoli). Finer particles and aerosols reaching the alveoli first get into contact with ‘eating cells’ (macrophages) on the alveolar surface. The interaction between substances and alveolar cells may cause different diseases (Table 1). Here, ‘dust’ means solid particles of a mineral or organic origin spread in the air, and is distinguishable from vapours, fumes and smoke, which are collectively known as aerosols. These substances are being investigated by other groups in the project. Dust is produced in mechanical treatments like drilling, milling, sawing, polishing, blasting, squeezing and grinding. Fumes mainly consist of metal oxides produced during the heating of metals above boiling point. The size distribution of the condensed particles ranges from 0.1 to 1.0 urn diameter, but often they The authors are at the Institute of Hygiene and Occupational Medicine (IHA), University Hospitals Essen, Hufelandstrasse D-45122 Essen. Germany. Received 14 June 1993. Revised November 1993
0030-3992/95/$10.00 Optics & Laser Technology Vol 27 No 1 1995
55 17
aggregate-these are the particles we find in laser cutting or welding after recondensation. The size distribution of potentially dangerous particles ranges from those just penetrating the upper airways (> 10 urn aerodynamic diameter) down to gas molecules. We distinguish between inorganic mineral dusts not damaging cells (inert dusts), and dusts that are damaging cells (toxic dusts). Inert dusts (for example corundum) are incorporated into small digestive vacuoles in macrophages (phagocytosis) and remain inside the cells. Dusts incorporated in intact macrophages are possibily eliminated (the clearance function of the lung). Toxic dusts (e.g. quartz) are also incorporated into macrophages by phagocytosis. These dusts destroy the surrounding membranes of the vacuoles. Thus, dissolving (lytic) enzymes are released into the cell fluid (cytoplasm), destroying the cells. Dust stimulates macrophages to release mediators into the alveoli: that is, proteins (i.e. tumour necrose factor), activated oxygen species (i.e. H,O,) and lipids (i.e. prostaglandine). The released substances have a retroactive effect with the macrophages. In addition, they interact with other alveolar cell types. The tumour necrose factor operates by stimulating, and prostaglandine by inhibiting. Excessive production causes cell damage.
@ 1995 Elsevier Science. All rights
reserved
39
Medical
risk assessment
Table
1. Different diseases alveolar macrophages
with Type
of laser fumes:
Lung
of dust
inert
foreign
Mineral
1
W. Malkusch
et al.
caused by the (after Parkes’)
interaction
soot iron (siderosis) tin (stannosis)
nodular
beryllium disease quartz (silicosis) mixed dust fibrosis
diffuse interstitial
asbestos (asbestosis) hard metal disease
granulomas
collagenous fibrosis
bird fanciers lung farmers lung (pneumonitis) bird fanciers lung farmers lung (alveolitis)
no fibrosis interstitial pneumonia Organic collagenous
fibrosis
The final aim of the investigation is the risk assessment of emissions produced during the laser processing of materials. The experiments will concentrate on particle emissions. Tn all probability, their effects cannot be derived from other industrial areas simply by analogous conclusions. As a result of the experiments, an estimation of the health effects of the dusts on the lung should be possible, with regard to the type and severity. Materials produced during laser processing have very complex natures. Some papers have already described volatile chemicals’ (and their effects’) produced during laser processing of composite materials. However, there are only very few investigations of lung diseases in connection with dust from epoxy resins. In particular. no data are available to date concerning dusts generated by laser processing. In an earlier case study, six labourers developed symptoms of an upper respiratory illness after being involved in the destructive removal of epoxy resin concrete3. In a study of inhalation exposures of taxidermists, of dusts and vapours from using epoxy or other resin systems, the conclusion was no likely overdose4. Finally, some painters have complained of chest pains when working in areas with respirable dusts, even after applying epoxy resin surfaces5.
Material
and
methods
The laser processing dusts to be investigated were obtained from the co-operation with other groups in the European project. To establish the method, dust from a fibre-reinforced epoxy resin with 40% aramid fibres, cut with a CO, laser, was used. The dust was sampled with a ten-step cascade impactor (Hanke LPI 25-4/0.015-0.015 pm through 16 pm). Additionally, dusts with known effects, like quartz dust, corundum dust and a dust from a conventionally cut fibre-reinforced epoxy resin, were investigated to prove the efficiency of the method.
substances
Examples
reaction
(no fibrosis)
body
of different
The target cells for the cell assay were collected from the washed lung fluid of guinea pigs by bronchoalveolar lavage (BAL). The cells were extracted with a sequence of centrifugation and resuspending. The count rate of 300000 cells per well was determined with a Coulter Counter (Coulter Electronics) and filled into a microplate. After two hours growing, the plates were washed and dusts and control substances were added. After 10 hours of incubation the plates were washed again. The test of the dust potential to damage cells and a simultaneous check on the influence of the pre-treatment of the dusts were performed in two cell experiments.
(4 Measurement
of the H,O,-release as a measure of cell damage. After the addition of cell mix, the H,O,-release was measured with a fluorescence reader (Fluoroscan 2, Lab Systems). of cell viability. (b) Determination After staining the cells with fluorescence diacetate (FDA, Sigma), they appeared a fluorescent green. The fluorescence was measured with the Fluoroscan 2. The particles of the laser-treated epoxy resin samples were found to be extremely aggregated. This aggregation happened during the collection on the impactor foils, for, in the air, the particles are not aggregated. For the toxicological test of the dust, the particles therefore had to be deagglomerated first. A suspension in physiological saline followed by ultrasonic treatment (Vibra-Cell, Sonics & Materials) did not separate the agglomerates. The diameters of the particle agglomerates were measured under a light microscope (Orthoplan, Leitz) in transmission illumination, with an image analysing instrument (Quantimet 970, Cambridge Instruments) and were distributed by size. Optics
40
& Laser Technology Vol 27 No 1 1995
Medical To separate the agglomerates, a lung surfactant from the BAL-fluid, was used. The surfactant is a surface active agent in the lung, covering the dust when it enters the alveoli. For the experiments, Fetal calf serum (FCS, ICN Flow) was used to deaggregate the agglomerates. FCS has a similar deagglomerating effect, but it is easier to obtain than surfactant. To test the influence of FCS on the dust effects, different concentrations (O%, 0.05%, 0.1% and 0.2% FCS) were added to the cells in combination with a dust of known toxic effect.
Results Upon testing the cell assay model with an epoxy resin dust from laser cut material, the first problem was the high tendency of these particles to aggregate and form agglomerates. To test the possible effects of the particles on alveolar macrophages it was therefore necessary to suspend them in a nutrient fluid in order to bring all the particles equally into contact with the cells. An analysis of the size distribution of the suspended particles, with the aid of quantitative image analysis, showed that even an ultrasonic treatment of the dust suspended in physiological saline did not result in a separation of the agglomerates (see Fig. 1, distribution maximum at 50 urn: NaCl). Using a surfactant material received from the washed lung fluid by bronchoalveolar lavage (BAL) a good separation of agglomerates was produced after suspending and ultrasonic treatment (see Fig. 1, distribution maximum at 5 urn: BAL). As surfactant material in sufficient quantities can only be received from a large number of freshly killed animals we looked for an alternative material for the agglomerate separation of the epoxy resin dust, which can be obtained easier. Finally, we found Fetal calf serum (FCS) to be the appropriate material as, regarding the particle separation. it produced results as good. Next, a check was necessary of whether the addition of FCS would have any influence on the cell damaging potency of a prospective material, either in an
risk assessment of laser fumes: W. Malkusch
inhibiting or in a stimulatory manner and at what concentration the addition of FCS already shows an agglomerate resolving effect and, simultaneously, is therefore still without an influence on the toxic dust effects. The dust of a conventionally cut fibre-reinforced epoxy resin (CFE) was suspended in different concentrations of FCS (O%, 0.05%, O.l%, 0.2%) and then added to macrophage cultures. From earlier experiments this dust was known to have a toxic effect. The concentration used was 120 ug per million cells. The dust without the addition of FCS (Fig. 2, 0% FCS) caused a significant reduction of HzO,-release as a measure of the cell damage. The addition of 0.05% or 0.1% FCS caused an insignificant increase in the damaging effect. However, the highest tested concentration of 0.2% FCS again showed a reduced H,O,-release (Fig. 2). Apart from a small reduction in the 0.1% FCS group there was no measurable influence on the cell viability (Fig. 3). In both tests, the control is a mean value of cell assays added only with those FCS concentrations which contain no dust. Based on these results a concentration of 0.1% FCS was chosen for the fine suspension of the aramid fibre-enforced epoxy resin dust from laser cutting (LFE). The ultrasonic treatment resulted in a good particle separation. To have a direct comparison, other different dusts were tested in the experiments, with the exception of the control (FCS without dust). Other materials, additionally tested, were quartz (as a known toxic material), corundum (as an inert dust) and CFE. All dusts were examined in two concentrations: 60 ug and 120 ug per million cells. The their (Fig. large inert
substances showed remarkable differences in effect on the cells with regard to the H,Q,-release 4). Quartz resulted in a concentration-dependent reduction of the H,O,-release. Corundum, as an dust, caused no change, even in different
300 -
13
0 1
10
diameter
100
(pm)
1000
Fig. 1 Size distrrbutton (diameter) of dust from laser-processed aramid fibre remforced epoxy resin (40% fibre content). Area measurement (Y-axis) with quantitative image analysis scanned under transmissron light macroscopic (magnification approximately 400 x ). 0 partrcles suspended in physiological salrne (NaCI) and treated with ultrasound; 0 particles suspended in BAL-flurd and treated with ultrasound
Optics & Laser Technology Vol 27 No 1 1995
et al.
LL
Ii
LL
g
8
P
LL
In
5 d
6
Fig. 2 H,O,-release (Y-axis) of alveolar macrophages after addition of 120 pg per million cells dust of conventionally cut fibre- enforced epoxy resin suspended in different concentrations of FCS. Control: mean value of all FE-concentrations without dust; FCS: different FCS-concentrations with 120 ltg dust per million cells
41
Medical
risk assessment
of laser fumes:
W. Malkusch
Frg. 3 FDA-determrnatron of alveolar macrophages cell vrability (Y- axrs) after addition of 120 pg per million cells dust of conventionally cut frbre-enforced epoxy resin suspended rn different concentrations of FCS. Control, mean value of all FCS-concentratrons without dust; FCS different FCS-concentratrons wrth 120 frg dust per million cells
et al.
Fig. 5 FDA-determination of alveolar macrophages’ cell viability (Y-axis) after addition of different dust samples (60 respectively 120 frg dust per million cells in 0.1% FCS). Control: 0.1% FCS wrthout dust; epoxy: dust of conventionally cut fibre-enforced epoxy resin; laser: dust of laser cut aramid frbre-enforced epoxy resin
enveloped with the surface active agent (surfactant) covering the surface of the alveoli. This agent reduces the surface tension and thus prevents agglomeration. During the collection on filters the dust particles come into contact with each other and form agglomerates (Fig. I). These agglomerates can be separated using either lung surfactant or, alternatively, FCS, which is easier to obtain. There are FCS-concentrations that have no reducing influence on the damaging dust effects (approximately up to 0.1%).
Frg 4 H,O*-release (Y-axis) of alveolar macrophages after addition of different dust samples (60, respectively 120 frg per mrllron cells rn 0.1% FCS). Control: 0.1% FCS wrthout dust; epoxy: dust of conventronally cut frbre-enforced epoxy resrn; laser: dust of laser cut aramid fibre-enforced epoxy resin
concentrations. The CFE-dust had, corresponding to the pre-experiments, a significant reducing effect on the H,O,-release, while the laser dust (LFE) astonishingly produced no remarkable change in H,O,-release compared with the control. In the cell viability test only the addition of quart7 dust resulted in a measurable difference in comparison with the control. All other substances did not show a real influence on cell viability (Fig. 5). Conclusions
and
future
Dust from laser-processed to aggregate a great deal. recondensation in the air, separated, and it is in this enter the respiratory tract.
experiments
epoxy resin material tends However, after the particles arc still condition that they will Inside the lung they will be
Compared with the control (which is a mean value from all the FCS concentrations used added to the cells without dust), the dust alone resulted in a reduction of H,Oz-release from 259 to 166 nmol. This corresponds to a cell damage of 36%. After suspension of the dust in 0.05% FCS or 0.1% FCS the small increase in cell damage is possibily caused by the deagglomeration effect of the FCS. Higher FCS concentrations (above 0.1 “/(I) already form a protective protein envelope around the dust particles that inhibits the damaging effect of the dust (Fig. 2). As the cell assay indicates the damaging potency of a dust, a FCS concentration of 0. I % was chosen for the experiments. A similar result was also found in the cell viability test, where only the dust treated with 0.1 ‘%IFCS showed a measurable reduction (Fig. 3). In the final irr tjitro cell assay, the control result (only 0.1 “/r, FCS added to the cells. no dust) was used as the 0”/1, damage value (Fig. 4). Quartz dust, a known toxic agent, caused the expected high dose-dependent damage, corresponding to a 70% (respectively 87%) reduction in H,Oz-release. Corundum dust as a known material with no damaging effect caused no change in H,Oz-release. Both dust types were suspended in 0.1% FCS, which proved the cell model worked properly. Dust from conventionally resulted in approximately
cut epoxy resin (CFE) 45% cell damage in the in Optrcs
42
& Laser Technology Vol 27 No 1 1995
Medical
ritro cell assay. Dust from laser processed epoxy resin (LFE), on the other hand, had no cell damaging effect. Next, more samples, representatives of other processes and other materials have to be tested, to find the reason for this result. Further dusts have to be investigated to see if these different behaviours of dusts, from conventionally and laser-treated materials, can be corroborated. In other investigations it was found that freshly ground silica dusts were more cytotoxic than comparably sized aged silicah. A comparison of quartz and crocidolite asbestos effects on rat peritoneal macrophages resulted in differences caused by catalytic properties of the dust surfaces’. The toxicity of a dust is dependent on its potential to induce an enhanced generation of free radicals. For toxic dusts. such as silica, amosite, chrysotile and crocidolite, the potential for oxygen radical generation is enhanced by their surface properties, physical dimensions, and the surface-based radical generating redox site?. The decrease of toxicity could be explained by a different surface structure of the particles. One dust type was mechanically produced, the other by evaporation and recondensation. This can be tested with scanning electron microscopy. As mentioned by Kwan’, there is a possibility that volatile substances with damaging potency may be absorbed on particle surfaces. This can be tested in surpernatant experiments. In these experiments the absorbed substances have to be dissolved from the particles and measured in similar cell assays for their damaging potency.
Optics & Laser Technology Vol 27 No 1 1995
risk assessmenr
of laser fumes:
W. Malkusch
et al.
Acknowledgements This study was supported by grants from the EEC and the German BMFT as part of the Eureka project EU643.
References Levensen, K., Emmrich, M., Kock. H., Priess, B., Sollinger, S., Trasser, F.J. Organic emissions during laser cutting of fibre-reinforced plastics, S/~&J Reinhcrltumy tlw Luff, 51 ( 1991) 365 -372 Kwan, J.K. Toxicological characterization of chemical produced from laser irradiation of graphite-composite materials. International Laser Safety Conference, Cincinnati, 3.69-3.96 (1990) Joyner, R.E., Pegues, W.L. A health hazard associated with epoxy resin-concrete dust, J O~~c~u/ionrrl Mcdic~rrc,. 3 (1961 ) 21I-214 Boiano, J.M. Potential health hazards in taxidermy shops, Anr Tu.YidWnzi.\r, 19 (1983) 3438 Salibury, S., Egilman, D. Health hazard evaluation Report No. HETA-83-132-1508, Grand Gulf Nuclear Power Plat. Port Gibson. Mississippi. Hazard Evaluations and Technical Assistance Branch, NIOSH, US Department of Health and Human Services, Cincinnati, Ohio (1984). Vallyathan, V., Kang, J.H., Van Dyke, K., Dalal, N.S., Castranova, V. Response of alveolar macrophages to in vitro exposure to freshly fractured versus aged silica dust: the ability of prosil 28. an organosilane material, to coat silica and reduce its biological reactivity, J Tosicolo~y~ crnd tiu~irorrmmtal Health. 33 (1991) 3033315 Soodaeva, SK., Korkina, L.C., Velichovski, B.T., Klegeris, A.M. Formation of active forms of oxygen by rat peritoneal macrophages under effect of cytotoxic dust, Biulk~trn Ek‘k.vperirnrmtrrlnoi Bioloyii i Meditsin~~, I I2 ( 1991) 32 254 Vallyathan, V., Mega, J.F., Shi, X., Dalal, Y.S. Enhanced generation of free radicals from phagocytes induced by mtneral dusts, Am J Rrspirczforr Cell Molecular Biolqqv. 6 ( 1992) 404413 Parkes, W.R. Owprt~onul Lung llisord~w. Chapter I, Butterworths. London (1982)
43
B. REHN,
risk
J. BRUCH
Laser processing of different materials may produce toxic fumes. In preventive occupational medicine it is necessary to evaluate valid hygienic standards for work places. The basis for such hygienic standards is the classification of laser fumes by their fibrogenic, emphysematous, immunological or other harmful potencies in biological assay systems. This paper is part of a European project on laser safety. Our part in this project is the development of a method for the investigation of lung responses using in vitrocell assays. The appropriate laser fume samples will be supplied by other groups in this European project. In contrast to the cell assays usually used in risk assessment, our method is based on isolated target cells in the lung, such as alveolar macrophages. The test criteria are mediator release, surfactant reactions, release of reactive oxygen species and cell proliferation. As demonstrated in the lung response to other dusts (minerals, fibres etc) these parameters are medically relevant factors in the pathogenic alveolar dust response. The paper gives basic information about the method using lung cell assays and the results of known substances, in comparison with a dust generated by laser processing. KEYWORDS:
laser fumes, medical risk assessment, cell assays, dust, lungs
Introduction The lung as the respiratory organ is always affected by foreign bodies entering with the air stream. Large particles are usually filtered in the nose or cleared from the trachea or main bronchi by coughing up. The lung structure is divided into the conducting airways (bronchi) and the respiratory unit (alveoli). Finer particles and aerosols reaching the alveoli first get into contact with ‘eating cells’ (macrophages) on the alveolar surface. The interaction between substances and alveolar cells may cause different diseases (Table 1). Here, ‘dust’ means solid particles of a mineral or organic origin spread in the air, and is distinguishable from vapours, fumes and smoke, which are collectively known as aerosols. These substances are being investigated by other groups in the project. Dust is produced in mechanical treatments like drilling, milling, sawing, polishing, blasting, squeezing and grinding. Fumes mainly consist of metal oxides produced during the heating of metals above boiling point. The size distribution of the condensed particles ranges from 0.1 to 1.0 urn diameter, but often they The authors are at the Institute of Hygiene and Occupational Medicine (IHA), University Hospitals Essen, Hufelandstrasse D-45122 Essen. Germany. Received 14 June 1993. Revised November 1993
0030-3992/95/$10.00 Optics & Laser Technology Vol 27 No 1 1995
55 17
aggregate-these are the particles we find in laser cutting or welding after recondensation. The size distribution of potentially dangerous particles ranges from those just penetrating the upper airways (> 10 urn aerodynamic diameter) down to gas molecules. We distinguish between inorganic mineral dusts not damaging cells (inert dusts), and dusts that are damaging cells (toxic dusts). Inert dusts (for example corundum) are incorporated into small digestive vacuoles in macrophages (phagocytosis) and remain inside the cells. Dusts incorporated in intact macrophages are possibily eliminated (the clearance function of the lung). Toxic dusts (e.g. quartz) are also incorporated into macrophages by phagocytosis. These dusts destroy the surrounding membranes of the vacuoles. Thus, dissolving (lytic) enzymes are released into the cell fluid (cytoplasm), destroying the cells. Dust stimulates macrophages to release mediators into the alveoli: that is, proteins (i.e. tumour necrose factor), activated oxygen species (i.e. H,O,) and lipids (i.e. prostaglandine). The released substances have a retroactive effect with the macrophages. In addition, they interact with other alveolar cell types. The tumour necrose factor operates by stimulating, and prostaglandine by inhibiting. Excessive production causes cell damage.
@ 1995 Elsevier Science. All rights
reserved
39
Medical
risk assessment
Table
1. Different diseases alveolar macrophages
with Type
of laser fumes:
Lung
of dust
inert
foreign
Mineral
1
W. Malkusch
et al.
caused by the (after Parkes’)
interaction
soot iron (siderosis) tin (stannosis)
nodular
beryllium disease quartz (silicosis) mixed dust fibrosis
diffuse interstitial
asbestos (asbestosis) hard metal disease
granulomas
collagenous fibrosis
bird fanciers lung farmers lung (pneumonitis) bird fanciers lung farmers lung (alveolitis)
no fibrosis interstitial pneumonia Organic collagenous
fibrosis
The final aim of the investigation is the risk assessment of emissions produced during the laser processing of materials. The experiments will concentrate on particle emissions. Tn all probability, their effects cannot be derived from other industrial areas simply by analogous conclusions. As a result of the experiments, an estimation of the health effects of the dusts on the lung should be possible, with regard to the type and severity. Materials produced during laser processing have very complex natures. Some papers have already described volatile chemicals’ (and their effects’) produced during laser processing of composite materials. However, there are only very few investigations of lung diseases in connection with dust from epoxy resins. In particular. no data are available to date concerning dusts generated by laser processing. In an earlier case study, six labourers developed symptoms of an upper respiratory illness after being involved in the destructive removal of epoxy resin concrete3. In a study of inhalation exposures of taxidermists, of dusts and vapours from using epoxy or other resin systems, the conclusion was no likely overdose4. Finally, some painters have complained of chest pains when working in areas with respirable dusts, even after applying epoxy resin surfaces5.
Material
and
methods
The laser processing dusts to be investigated were obtained from the co-operation with other groups in the European project. To establish the method, dust from a fibre-reinforced epoxy resin with 40% aramid fibres, cut with a CO, laser, was used. The dust was sampled with a ten-step cascade impactor (Hanke LPI 25-4/0.015-0.015 pm through 16 pm). Additionally, dusts with known effects, like quartz dust, corundum dust and a dust from a conventionally cut fibre-reinforced epoxy resin, were investigated to prove the efficiency of the method.
substances
Examples
reaction
(no fibrosis)
body
of different
The target cells for the cell assay were collected from the washed lung fluid of guinea pigs by bronchoalveolar lavage (BAL). The cells were extracted with a sequence of centrifugation and resuspending. The count rate of 300000 cells per well was determined with a Coulter Counter (Coulter Electronics) and filled into a microplate. After two hours growing, the plates were washed and dusts and control substances were added. After 10 hours of incubation the plates were washed again. The test of the dust potential to damage cells and a simultaneous check on the influence of the pre-treatment of the dusts were performed in two cell experiments.
(4 Measurement
of the H,O,-release as a measure of cell damage. After the addition of cell mix, the H,O,-release was measured with a fluorescence reader (Fluoroscan 2, Lab Systems). of cell viability. (b) Determination After staining the cells with fluorescence diacetate (FDA, Sigma), they appeared a fluorescent green. The fluorescence was measured with the Fluoroscan 2. The particles of the laser-treated epoxy resin samples were found to be extremely aggregated. This aggregation happened during the collection on the impactor foils, for, in the air, the particles are not aggregated. For the toxicological test of the dust, the particles therefore had to be deagglomerated first. A suspension in physiological saline followed by ultrasonic treatment (Vibra-Cell, Sonics & Materials) did not separate the agglomerates. The diameters of the particle agglomerates were measured under a light microscope (Orthoplan, Leitz) in transmission illumination, with an image analysing instrument (Quantimet 970, Cambridge Instruments) and were distributed by size. Optics
40
& Laser Technology Vol 27 No 1 1995
Medical To separate the agglomerates, a lung surfactant from the BAL-fluid, was used. The surfactant is a surface active agent in the lung, covering the dust when it enters the alveoli. For the experiments, Fetal calf serum (FCS, ICN Flow) was used to deaggregate the agglomerates. FCS has a similar deagglomerating effect, but it is easier to obtain than surfactant. To test the influence of FCS on the dust effects, different concentrations (O%, 0.05%, 0.1% and 0.2% FCS) were added to the cells in combination with a dust of known toxic effect.
Results Upon testing the cell assay model with an epoxy resin dust from laser cut material, the first problem was the high tendency of these particles to aggregate and form agglomerates. To test the possible effects of the particles on alveolar macrophages it was therefore necessary to suspend them in a nutrient fluid in order to bring all the particles equally into contact with the cells. An analysis of the size distribution of the suspended particles, with the aid of quantitative image analysis, showed that even an ultrasonic treatment of the dust suspended in physiological saline did not result in a separation of the agglomerates (see Fig. 1, distribution maximum at 50 urn: NaCl). Using a surfactant material received from the washed lung fluid by bronchoalveolar lavage (BAL) a good separation of agglomerates was produced after suspending and ultrasonic treatment (see Fig. 1, distribution maximum at 5 urn: BAL). As surfactant material in sufficient quantities can only be received from a large number of freshly killed animals we looked for an alternative material for the agglomerate separation of the epoxy resin dust, which can be obtained easier. Finally, we found Fetal calf serum (FCS) to be the appropriate material as, regarding the particle separation. it produced results as good. Next, a check was necessary of whether the addition of FCS would have any influence on the cell damaging potency of a prospective material, either in an
risk assessment of laser fumes: W. Malkusch
inhibiting or in a stimulatory manner and at what concentration the addition of FCS already shows an agglomerate resolving effect and, simultaneously, is therefore still without an influence on the toxic dust effects. The dust of a conventionally cut fibre-reinforced epoxy resin (CFE) was suspended in different concentrations of FCS (O%, 0.05%, O.l%, 0.2%) and then added to macrophage cultures. From earlier experiments this dust was known to have a toxic effect. The concentration used was 120 ug per million cells. The dust without the addition of FCS (Fig. 2, 0% FCS) caused a significant reduction of HzO,-release as a measure of the cell damage. The addition of 0.05% or 0.1% FCS caused an insignificant increase in the damaging effect. However, the highest tested concentration of 0.2% FCS again showed a reduced H,O,-release (Fig. 2). Apart from a small reduction in the 0.1% FCS group there was no measurable influence on the cell viability (Fig. 3). In both tests, the control is a mean value of cell assays added only with those FCS concentrations which contain no dust. Based on these results a concentration of 0.1% FCS was chosen for the fine suspension of the aramid fibre-enforced epoxy resin dust from laser cutting (LFE). The ultrasonic treatment resulted in a good particle separation. To have a direct comparison, other different dusts were tested in the experiments, with the exception of the control (FCS without dust). Other materials, additionally tested, were quartz (as a known toxic material), corundum (as an inert dust) and CFE. All dusts were examined in two concentrations: 60 ug and 120 ug per million cells. The their (Fig. large inert
substances showed remarkable differences in effect on the cells with regard to the H,Q,-release 4). Quartz resulted in a concentration-dependent reduction of the H,O,-release. Corundum, as an dust, caused no change, even in different
300 -
13
0 1
10
diameter
100
(pm)
1000
Fig. 1 Size distrrbutton (diameter) of dust from laser-processed aramid fibre remforced epoxy resin (40% fibre content). Area measurement (Y-axis) with quantitative image analysis scanned under transmissron light macroscopic (magnification approximately 400 x ). 0 partrcles suspended in physiological salrne (NaCI) and treated with ultrasound; 0 particles suspended in BAL-flurd and treated with ultrasound
Optics & Laser Technology Vol 27 No 1 1995
et al.
LL
Ii
LL
g
8
P
LL
In
5 d
6
Fig. 2 H,O,-release (Y-axis) of alveolar macrophages after addition of 120 pg per million cells dust of conventionally cut fibre- enforced epoxy resin suspended in different concentrations of FCS. Control: mean value of all FE-concentrations without dust; FCS: different FCS-concentrations with 120 ltg dust per million cells
41
Medical
risk assessment
of laser fumes:
W. Malkusch
Frg. 3 FDA-determrnatron of alveolar macrophages cell vrability (Y- axrs) after addition of 120 pg per million cells dust of conventionally cut frbre-enforced epoxy resin suspended rn different concentrations of FCS. Control, mean value of all FCS-concentratrons without dust; FCS different FCS-concentratrons wrth 120 frg dust per million cells
et al.
Fig. 5 FDA-determination of alveolar macrophages’ cell viability (Y-axis) after addition of different dust samples (60 respectively 120 frg dust per million cells in 0.1% FCS). Control: 0.1% FCS wrthout dust; epoxy: dust of conventionally cut fibre-enforced epoxy resin; laser: dust of laser cut aramid frbre-enforced epoxy resin
enveloped with the surface active agent (surfactant) covering the surface of the alveoli. This agent reduces the surface tension and thus prevents agglomeration. During the collection on filters the dust particles come into contact with each other and form agglomerates (Fig. I). These agglomerates can be separated using either lung surfactant or, alternatively, FCS, which is easier to obtain. There are FCS-concentrations that have no reducing influence on the damaging dust effects (approximately up to 0.1%).
Frg 4 H,O*-release (Y-axis) of alveolar macrophages after addition of different dust samples (60, respectively 120 frg per mrllron cells rn 0.1% FCS). Control: 0.1% FCS wrthout dust; epoxy: dust of conventronally cut frbre-enforced epoxy resrn; laser: dust of laser cut aramid fibre-enforced epoxy resin
concentrations. The CFE-dust had, corresponding to the pre-experiments, a significant reducing effect on the H,O,-release, while the laser dust (LFE) astonishingly produced no remarkable change in H,O,-release compared with the control. In the cell viability test only the addition of quart7 dust resulted in a measurable difference in comparison with the control. All other substances did not show a real influence on cell viability (Fig. 5). Conclusions
and
future
Dust from laser-processed to aggregate a great deal. recondensation in the air, separated, and it is in this enter the respiratory tract.
experiments
epoxy resin material tends However, after the particles arc still condition that they will Inside the lung they will be
Compared with the control (which is a mean value from all the FCS concentrations used added to the cells without dust), the dust alone resulted in a reduction of H,Oz-release from 259 to 166 nmol. This corresponds to a cell damage of 36%. After suspension of the dust in 0.05% FCS or 0.1% FCS the small increase in cell damage is possibily caused by the deagglomeration effect of the FCS. Higher FCS concentrations (above 0.1 “/(I) already form a protective protein envelope around the dust particles that inhibits the damaging effect of the dust (Fig. 2). As the cell assay indicates the damaging potency of a dust, a FCS concentration of 0. I % was chosen for the experiments. A similar result was also found in the cell viability test, where only the dust treated with 0.1 ‘%IFCS showed a measurable reduction (Fig. 3). In the final irr tjitro cell assay, the control result (only 0.1 “/r, FCS added to the cells. no dust) was used as the 0”/1, damage value (Fig. 4). Quartz dust, a known toxic agent, caused the expected high dose-dependent damage, corresponding to a 70% (respectively 87%) reduction in H,Oz-release. Corundum dust as a known material with no damaging effect caused no change in H,Oz-release. Both dust types were suspended in 0.1% FCS, which proved the cell model worked properly. Dust from conventionally resulted in approximately
cut epoxy resin (CFE) 45% cell damage in the in Optrcs
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& Laser Technology Vol 27 No 1 1995
Medical
ritro cell assay. Dust from laser processed epoxy resin (LFE), on the other hand, had no cell damaging effect. Next, more samples, representatives of other processes and other materials have to be tested, to find the reason for this result. Further dusts have to be investigated to see if these different behaviours of dusts, from conventionally and laser-treated materials, can be corroborated. In other investigations it was found that freshly ground silica dusts were more cytotoxic than comparably sized aged silicah. A comparison of quartz and crocidolite asbestos effects on rat peritoneal macrophages resulted in differences caused by catalytic properties of the dust surfaces’. The toxicity of a dust is dependent on its potential to induce an enhanced generation of free radicals. For toxic dusts. such as silica, amosite, chrysotile and crocidolite, the potential for oxygen radical generation is enhanced by their surface properties, physical dimensions, and the surface-based radical generating redox site?. The decrease of toxicity could be explained by a different surface structure of the particles. One dust type was mechanically produced, the other by evaporation and recondensation. This can be tested with scanning electron microscopy. As mentioned by Kwan’, there is a possibility that volatile substances with damaging potency may be absorbed on particle surfaces. This can be tested in surpernatant experiments. In these experiments the absorbed substances have to be dissolved from the particles and measured in similar cell assays for their damaging potency.
Optics & Laser Technology Vol 27 No 1 1995
risk assessmenr
of laser fumes:
W. Malkusch
et al.
Acknowledgements This study was supported by grants from the EEC and the German BMFT as part of the Eureka project EU643.
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