Document jg080XZyV8ReYzrKz5MDX37OZ

OCT 5 1 1H/3 MANUFACTURING CHEMISTS ASSOCIATION 1825 CONNECTICUT AVENUE, N.W. WASHINGTON, D. C. 20009 (202) 483-6126 October 30, 1973 To: TECHNICAL TASK GROUP ON VINYL CHLORIDE RESEARCH Subject: First Quarterly Progress Report from Industrial BIO-TEST Laboratories, Inc. Gentlemen: Distributed herewith are copies of the subject report. This describes the physical features of the exposure equipment and monitoring procedures as they related to the present exposure program. Sincerely, KDJ:mb Attachment cc : Dr. D. P. Duffield Mr. D. M. Elliott Dr. Tiziano Garlanda Secretary Technical Task Group on Vinyl Chloride Research AS I 000016644 TOXICOLOGY ENVIRONMENTAL SCIENCES CHEMISTRY PLANT SCIENCES MEDICAL SCIENCES BIO-TEST DECATUR RESEARCH 1800 EAST PERSHING ROAD DECATU R, I LLI NOIS 62526 October 13, 1973 Sac. AREA CODE 217 TELEPHONE S77-925A Dr. Kenneth D. Johnson Manufacturing Chemists Association 1825 Connecticut Avenue N.W. Washington, D. C. 20009 Dear Dr. Johnson: Enclosed is a report on equipment and methods being used for the chronic inhalation toxicity study with vinyl chloride. I apologize for the delay in getting this to you. If you have any questions about the report, please call either Dr. John Goode or myself. Sincerely KS/dm enc. /' Ken Schadeberg, B.S. Senior Group Deader Inhalation and Pharmacology Siiduii'Ual 6 I O - T E 5 T Jtabosxd&U&L, 9*tc. REPORT TO MANUFACTURING CHEMISTS ASSOCIATION ON METHODS AND EQUIPMENT FOR CHRONIC INHALATION TOXICITY STUDY WITH VINYL CHLORIDE IBT NO. 663-03222 I. Introduction At this date we are into the fifth week of inhalation exposures of rats, mice and hamsters to Vinyl Chloride. Herewith are reported information on experimental animals, equipment, and techniques with additional detail on the problems which have been encountered in conducting the project. ASI 00016646 SiuLtit\ial BIO-TSST J.aA&iatoAis6, 9kc. 2 II. Experimental Animals and Caging All test animals were supplied by Charles River Breeding Laboratories and were outbred in origin. Charles River does not keep records to deter mine exactly how many parents were involved in producing animals; however, the number of parents involved in producing the test animals can be approximated from historical average litter sizes. The Charles River laboratory has found that the average litter sizes of their rats, mice and hamsters are 10, 11, and 9.5 respectively. Below is a table presenting birth data ranges for each species use for the study: Species Birth Date Ranges Rats 7-18-73 to 7-22-73 Mice 7-4-73 to 7-10-73 Hamsters 8-1-73 to 8-8-73 The rats used for the study came from two separate lots. One lot of 800 animals is being used for the exposures without food or bedding in the chamber, while a second lot of 100 is being used for the exposure with food and litter in the chamber. All animals have undergone an ear punch and toe removal procedure so that each animal can be individually identified. Thus, in the eve.it that an animal does escape, it can be returned to the proper cage. This system also allows identification for individual body weight determination. AS I 000016647 B I O - T S T 2al&iat<yU&i, 9m. 3 All animals are housed in groups of three to five in stainless steel cages. All cages meet the latest space recommendations for laboratory animals put out by the United States Department of Health, Education and Welfare. The rat, mouse, and hamster cages are eight, five, and six inches high respectively. All cages aford at least 43, 36, and 36 square inches of floor space for rats, mice, and hamsters respectively. The animals are on an automatic watering system made by Unifab Corp. in Kalamazoo, Michigan. All animals receive Purina Rat Chow ad libitum, with the exception of the groups which have the food removed during the inhalation exposure period. ASI 000016648 UrtJMSbiial B I O - T E S T J.aLc'iai&Ue, 9itc. 4 III. Experimental Equipment A. Chambers Four chambers are being used for the major study, i, e. where animals receive exposures without food or bedding in the chamber. Each chamber is all stainless steel and glass in construction and has a volume of 9,200 liters. A diagram revealing basic design of the chambers is shown in Figure I. A fifth chamber made of all clear Plexiglas is being used for the exposure where food, water, and bedding are present in the inhala tion chamber at all times. This chamber measures 76 x 36 x 82 inches high, and has a capacity of 3, 743 liters. B. Air Supply System All chambers receive air from a common purification system connected to the chambers by 4 x 6 inch galvanized steel duct work. The supply air is obtained from a stack which rises 12 feet above the roof of the building housing the inhalation chambers. This source of air should be free of any contaminants which may be expelled by exhaust blowers located at the roof level. All supply air is filtered by a dust, carbon, and the absolute Cambridge filter system. The air is also humidified, dehumidified, heated, and cooled to obtain air which is 72F (2F) and 50% relative humidity (10%). AS! 000016649 jr*C iu'Ucl BIO 5 3 Jta^AaiG'V&, 9^. Figure 1 ASI 000016650 Sitduit/uaL S J O 7 C 5 T Jlzbyiai&u&i., !)s-u-,, '6 The chamber air is pulled through the purification system with a variable speed blower (max. 3,200 cfm). The air is blown through the duct work feeding the inhalation chambers, thus, the ducts are under positive pressure to prevent contamination of chamber supply air by room air. It should be noted that a small squirrel cage blower (100 cfm) was installed in the supply duct which feeds air to the inhalation chamber housing rats with food, water, and bedding in the chamber at all times. This small blower was necessary due to the limited static pressure available at the location of this chamber. A pressure test of the duct work near this blower (using a 0-0. 1" H2 magnehelic) indicated that the duct was still under positive pressure, and therefore not drawing contaminated room air into the supply duct. C. Chamber Exhaust System The exhaust system on all MCA chambers is common to one fan which is separate from other inhalation chambers in the department. The fan is located at roof level at opposite ends of the inhalation building from the supply source for chamber air. The fan has a maximum capacity of 1, 800 cfm. All inhalation chambers are operated under a negative pressure of 0. 01 inches of water. This prevents cross contamination of inhalation chambers. The four large chambers are each supplied with enough air to permit ten complete air changes per hour, i. e. 1, 533 L/mln. The smaller chamber ASl 000 `SeSl Sf.dadi'ual BIO- I E S T JiaMosiatosUeA., 9ac. 7 which houses only 100 rats with food, etc. in the chamber, also has ten air changes per hour, which is accomplished by a flow of 624 L/min. D. Vinyl Chloride Metering System The Vinyl Chloride supply tanks (one full tank always stored in reserve) are kept in an explosion room equipped with a blow-out wall. The test material is piped to the test chambers through thin wall stainless steel tubing. A Matheson regulator (Model No. 130290) is attached to the Vinyl Chloride tank itself to maintain constant operating pressure of 20 psi. An adaptor connects the regulator to a valve connected to the pipe work which directs the test vapor through a wall into the inhalation chamber room. Before the pipe divides to the four test chambers, the vapor must also pass through a second safety valve positioned in the chamber room itself. The thin-wall tubing delivering Vinyl Chloride to the chambers is connected to a micro metering valve and then a glass rotometer (using steel or sapphire beads) for introducing a constant, controlled, and easily moni tored supply of vapor to each test chamber* Vinyl Chloride which leaves the rotameter is delivered to each chamber air supply hose by a short length of stainless steel tubing. Vinyl Chloride then mixes with the chamber supply air in the chamber supply air delivery line. The resulting vapor-air mixture then enters the top center of the inhalation chamber, circulates through the chamber, and is exhausted at the bottom. Ail pipe, valves, connectors, elbows, etc. are all stainless steel in construction. ASI 000016652 .iaj, BIO-TEST JlaluviaicyU&i, 9kc. E. Flow Calibration of Air and Vinyl Chloride Chamber supply air is measured by use of limiting flow orifices and pressure gauges. Regular calibration checks are made on both the pressure gauges and rate of flow into the chamber. Air flow rate into the chambers is measured with both a Matheson Mass Flow Meter (Model AHL.-50) and with a hot-wire anemometer made by Alnor. Both devices have given repeatable data; however, it is felt that the hot-wire anemometer is more reliable in this high flow system because of the sizable amounts of back pressure created by the mass flow meter. In addition, calculations are made regularly to compute air flow in the chamber from vinyl chloride flow and analytical concentration data. The flow of Vinyl Chloride through the rotameters is checked by use of a bubble meter (graduated cylinder and soap bubble). The continued flow of the test material through the valves and rotameters have caused no observable change in calibrated readings. 0000tfi Stubidtiial B I O - T E S T lalomfouei-. 9*c. 9 IV. Analytical Techniques A. Manual Sampling Procedure 1. Equipment A Hewlett-Packard 7614A*gas chromatograph is being used for the vinyl chloride analyses during the manual sampling procedure. The gas chromatograph (GC) is equipped with a dual flame ionization detector. It utilizes 6 foot, 3. 5 mm I. D. , glass, u-tube columns packed with 10% (w/w) Carbowax 20 M on 60/80 mesh Chromosorb W^. The Chromosorb is acid washed and dimethyl-dichlorosilane treated. The temperature conditions of the GC are as follows; injection post 150C, oven 1Q0C, and detector 300C. The flow rates of the carrier gas (helium), hydrogen, and air are 40, 40, 240 ml/min respectively. 1 Hewlett-Packard, Skokie, Illinois. 2 Applied Science Laboratories, Inc., State College, Pa. AS I 000016G54 BIO - 1 :ST JlaL&iaic-i.ie.i, Sac. 10 2. Calibration Calibration standards are made using 500 ml, Pyrex, gas 3 collecting flasks . The flasks are flushed with filtered air for approxi mately ten minutes and then evacuated using a vacuum pump. Two vinyl chloride samples are withdrawn from the stock vinyl chloride tanks^ under positive pressure. The vinyl chloride samples are allowed to equilibrate to room temperature fob ten minutes before venting them to the air. A rubber serum cap3 is placed over one arm of the flask. A 5 ml sample is withdrawn through the serum cap with a gas-tight syringe^* and injected through the serum cap of an evacuated flask. The stopcock of the evacuated flask is opened to the air for 1-2 seconds. This diluted sample is allowed to equilibrate for approximately ten minutes. Then duplicate injections (0. 5 ml) are made into the GC. This dilution pro cedure is performed for both pure vinyl chloride samples. If the two diluted samples do not agree when injected into the GC, new pure vinyl chloride samples are taken. Five different dilutions of the pure vinyl chloride are made for each test level using the above procedure. Duplicate samples are injected into the GC and the results plotted on graph paper. All samples are coriected to 25C and 1 atmosphere of pressure. 3 Curtis-Matheson, Maryland Heights, Mo. 4 Pittsburgh Plate Glass Co. , Pittsburgh, Pa. 5 Fisher Scientific, Chicago, 111. 6 Precision Sampling, Baton Rouge, La. AsI 00001 6655 Sndui&Ual B I O - T S T Jtalwuzbvuei, Sac. 11 3. Sampling the Chambers Samples are withdrawn from the test chambers by inserting a glass tube into the test chamber. The tube is flushed with a vacuum pump. One end of a segment of rubber tubing is placed on the glass tube, and the other end is connected to the evacuated gas collecting flask. The stopcock is slowly opened to the test air. The samples are then taken to the laboratory and allowed to equilibrate before 0. 5 ml is in jected into the GC. Six ports are sampled from each chamber at various depths as shown by stars in Figure II. ASI 000016656 vB30-THS7 jfpt v:. Figure II 12' ASI 000016657 n .* FIGURE 1 Automatic Sequential Sampler 13 AS I 000016G58 St'Mtai'Md B I O - T E 5 T Jlalmai&ii&i,, 9kc. 14 The valve system consists of a model 4300 valve oven, models 2025 and 2026 valves, model 4200 valve actuator and a model 4150 valve minder. Figure 1 illustrates the various connections from the valve system to the GC, vacuum source, carrier gas, and test chambers. The lines to the test chamber are made of Type 304, Q 1/8 inch O D. commercial 8269 stainless steel tubing0. All connec tions are Swagelok ^ stainless steel fittings. 8 Tube Sales Warehouse, Berkeley, 111. 9 St. Louis Valve Si Fitting Co. , Ferguson, Mo. AS I 00001GG59 J%idutiUzl B I O - 7 3 7 9m. 15 2. Calibration Calibration standards are made up as previously described. The stainless steel line which was attached to the control chamber is fitted with a three-way valve. A needle is attached to the valve and inserted through the rubber cap of the gas collecting flask. The valve minder is manually actuated. All standards are monitored by this procedure and the results are plotted on graph paper. The column which is not attached to the valve system is used to check various locations in the test chambers. AS I 000J6660 9riAaitiial B I O - T E 5 T Jiakvuitaru&i, !)nc. 16 3. Sampling the Chambers When the valve minder is placed in the automatic mode, the lines to the test chambers are sequentially monitored from #1 to #11 at five minute intervals. Each line is flushed out at a rate of 25 ml/minutes for five minutes before the sample is drawn into the valve system. Three sampling lines are connected to each of the three large inhalation chambers. The three points which these lines sample are shown by circled stars in Figure II. The control chamber and the chamber housing 100 rats with food in cages at all times, each have only one sampling port. AS I 000016661 4 jr:-:Ucii'ual B 3 O - 7 E 5 7 Jlak&ud&u&L, Hxc. 17 V. Problems Encountered. A. Animal Feeding System One of the requirements of MCA's protocol was that food should be removed from all animals in specified groups (Control, TE-1, TE-II, and TE-III as in revised August 31, 1973 protocol). A feeder, designed to attach and detach to the outside of the animal cages, was constructed for this purpose. A problem was encountered with the initial design of the feeders in that although food was accessible to the animals, the food (approximately 1/2 x 1/2 x 1" pellets) was separated from the wire mesh of the animal cages by thin metal slits. The slits were to serve to con tain the food when attached it to the animal cage, yet still allow animals to feed through them. After one to two weeks of feeding by this system, body weight effects and mortality were noted in all groups of animals. Therefore, a decision was made to terminate the exposures, redesign the feeders, and restart exposures with a new set of experimental animals. The new feeder design is one which presents food pellets to the animals immediately against the mesh of the cage. This method has been employed for approximately six weeks and no adverse effects on body weight gains have been observed. B, Air Supply and Exhaust System AS I 000016662 During a \'isit of the steering committee of MCA for this in halation study, it was pointed out that the air supply source for the inhalation chamber was room air. Jt would therefore be possible 9*'jdu.iiiizl 310-TEST Jialwjzfenisi, 9*ic. 1 18 '' #- for impurities from other inhalation studies (carcinogens, etc. ) to enter * _ the air supply system and contaminate the chambers being used for MCA's project. To alleviate this problem, a stack reaching 12 feet above the roof of the inhalation building was installed and connected to the air supply inlet of the air purification and conditioning system for the inhalation chambers. It was also pointed out that since the exhaust system was common among all inhalation chambers in the department, it would be possible for contaminants from other chambers to back up through the ducts and possibly into the chambers being used for the MCA project. This prob lem was taken care of by installing a separate exhaust blower and separ ate duct work for the five chambers being used for the study. C. Analytical Method Another request of the MCA committee was that the analytical data obtained during the study would be more desirable if a gas chroma tograph connected to a sequential sampling device (for sampling a few points from each inhalation chamber) would be employed to turn out daily chart records on the chamber concentration. This request was fulfilled, and details of the methods and equipment for this procedure were expressed in previous pages (section IV, B). AS I 000016