Document Gknv1wodD9Yjbx8r9LoJvbLN
ROB WEST ASSOC IATES, INC,
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P.O.BOX 2001 . STAMF0R0, CONNECTICUT 06906 203-322-4466
CONFIDENTIAL
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Review and Audit of IBT No. 663-03222, i.nroiui. Vapor Inhalation Toxicity Study with Vinyl Chloride (Ethylene Derived) in Albino Rats, Albino Mice and Golden Hamsters for the Manufacturing Chemists Association
The review and audit of this study was conducted at IBT's offices in Northbrook, IL, April 14 to April 18, 1980. The review and audit on this study concentrated on the con duct and monitoring of the exposures. However, attention was also devoted to the source of the animals and body weight data as they affected fate and mortality. In addi tion, the review and audit included inspection of hand written observation books reflecting exposure concentrations, as well as gas chromatographic charts where they were avail able, hand written and computer body weight printouts, hand written mortality logs, hand written observations and cor respondence . CORRESPONDENCE, PROTOCOL AND TIMETABLE
The timetable indicates the exposures were to be ini tiated on 9/10/73 for the T-I, T-II and T-III groups and on 10/1/73 for the T-XV groups. The termination of the exposures for the first three groups was to be at 9/8/74 and for the T-IV group at 9/27/74. The animals were then to be held for a year prior to terminal sacrifice. A letter dated June 15,
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1973 from Dr. Torkelson of Dow reflects a site visit was made by the MCA Task Force, visiting the Decatur research labs of IBT where, the study was conducted. Questions were raised about the adequacy of equipment, the exposures main tained, the exposure concentrations, the question of contam ination and record keeping. A major concern was raised about the adequacy of the analyses and record keeping.- Hand written notes alongside the margin of this letter indicate that certain corrective actions were taken at IBT. I was unable to tell whether there were any other documented site visits. In addition, a progress report issued October 13, 1973 after about a month of exposure, reflects adjustments . that were made to compensate for the issues that were raised during the MCA site visit. ANIMALS, HOUSING AND HUSBANDRY
The animals for this study were supplied by the Charles River Breeding Laboratories. Unfortunately, we are not able to identify specific animal receipts with this project. However, we were able to identify from September invoices receipts of hamsters as replacements without charge for ani mals that were dead on arrival in the August shipment. We were able to identify an additional receipt of hamsters in early November, 1973, two months after the study started, which indicated 130 males and 130 females were received at that time.
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Inspection of the body weights early in the study in dicates despite the report not reflecting the mortalities that there was a rather high mortality in the hamsters early in the study. At four and five months there were extensive replacements of the hamster groups.
The animals were ear punched and the toes removed so the animals could be individually identified. This was to allow for identification for individual body weight determin ation and identification of when the animals died, because the animals were group housed in groups of 3-5 in stainless steel cages. Inspection of some of the hand written notes shows the layout of the rats in the rack and indicates that . extra animals were to be spaced evenly throughout the rack. In addition, another hand written sheet shows the arrangement of the gang cages in the chambers. There were IS gang cages, 8 rats each, to equal 144 rats. However, the protocol only calls for 100 rats at each level. This procedure allows for ample opportunities for replacement and substitution.
Despite the fact that the animals were identified by ear punching and toe removal, most of the time the cages were not weighed individually but rather by the cage, e.g., 3-5 animals. Numerous hand written notes showing the arrangement of some of the animals in the racks reflect the animals excaped or were cannibalized, along with mortality .dates.for the remain ing individual animals.
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VINYL CHLORIDE, SOURCE, IDENTITY Vinyl, chloride (ethylene based) appears to have been
supplied as, e.g., containers in excess of 500 pounds, by PPG Industries, Industrial Chemical Division, Lake Charles, LA. A letter from W. B. Braybill of PPG to Dr. John Goode of Bio-Test in Decatur indicates that one cylinder would be required per month. Heel sample analyses from the BiojrTest residues were also performed by PPG at the Lake Charles, LA operation and those that were available appeared to be in good agreement with the new sample analyses supplied to BioTest. Unfortunately, there was no inventory log at IBT for vinyl chloride. PPG personnel should be asked for account-, ability of the vinyl chloride sent to IBT. In addition, they would also have records of the returns for heel analyses. GENERATION OF VINYL CHLORIDE VAPORS
The vinyl chloride supply tanks were kept in an explos ion room. The material was piped to the test chambers through thin-walled stainless steel tubing. Before the thin-walled tubing delivered the vinyl chloride to the chambers, it was connected to a micrometering valve and a glass rotometer to control and monitor the supply of vapor to each chamber. Vapor-air mixture entered the top of the inhalation chamber, circulated through the chamber and was exhausted at the bot tom. Unfortunately, we were unable to find a daily log for
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the weighing or measuring of the vinyl chloride that was dispensed .during each daily run. CHAMBER MONITORING
There are records of standardizations being conducted in September of 1973, covering the range from 20 to 100 ppm standardization and from 50 to 400 ppm for the 200 ppm standardization and from 400-4000 ppm for the 2500 ppnw level. Standardizations were performed at approximately weekly in tervals prior to that point where the automatic sampling system was hooked up to the on-line gas chromatograph record er. The initial samples from the chambers for the first part of the study (six weeks) were obtained by use of grab samples which were then analyzed by the gas chromatograph. We were unable to find any record of daily sample analyses being taken in the research books for the month of October, 1973, the second month of the study, other than ranges and mean concentrations. There also was no evidence of chamber temp erature or humidity being recorded. General Observations
The following observations were obtained by inspection of research log books which present means and ranges of exposure concentrations, time interval sampling for the gas chromatographic concentrations and charts, as well as the hand written observations in the research log books.
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1. Most of the time the chambers were not on for six hours. 2. Most of the time the chambers were not even monitored
for five hours, in some instances they were only moni tored for one to two hours. 3. In some instances there was selected data for the daily exposure concentration, e.g., values on the low side may have been eliminated or values on the high side may have been eliminated. 4. Periodically, there were mechanical problems associated with the rotometer, e.g., broken or clogged or unclean lines. 5. Some days there was no exposure because of equipment failure or TV filming or only a one-hour exposure was conducted. 6. There was evidence that the chambers did not have an adequate clearance time, since measurements made 45 minutes after termination of the exposure still showed rather high levels of vinyl chloride present. 7. There was evidence of contamination in the solvents room and the pilot plant which were in close proximity to the exposure room. 8. Some days there was no analytical data recorded for the exposure concentrations. 9. In some cases, the research log book showed means and ranges for exposure concentrations, with no raw supportive
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data shown for periodic sampling with the gas chromato graph. _ 10. In some instances, the 50 ppm nominal concentration may have reached a concentration as high as 100 ppm and, in one instance, as high as 400 ppm which is not reflected in the means or ranges presented in the interim report. 11. In some instances, the control chamber indicated Levels of vinyl chloride in a range of 5-11.5 ppm. 12. In September of 1974, near the end of the year of expos ure, there was some research book data reflecting exposure concentrations but no supportive gas chromato graphic data. 13. In some cases there is no data available for one or two levels out of the four exposure chambers being run. 14. There were periodic problems with the gas chromatograph itself, either with the Ignitor, Detector or the Recorder. 15. In some instances there were means and ranges presented in the research book but no interval data were available. 16. In some instances, the chamber may have been on for four or five hours yet gas chromatographic readings were only taken for two hours. 17. There were a number of instances where the data presented in the research book do not agree with the data shown on the gas chromatographic chart.
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Specific Exposure Observations
The following notations were associated with the spe
cific exposures and may also be used together with the inhalation chamber concentrations presented in Table I. The exposure concentrations presented in Table I are those presented by IBT and one can see with the use of these spe cific notations that the values presented in Table I are not
t an accurate reflection, in many instances, of the exposure concentrations or are based on minimal supportive analytical
data.
Exposure Number
5 20 & 35
53 56
80 83
Comment The values for the T-I mean and the range for the raw data in the report do not agree. In the daily intervals, there were problems with sampling and the curve does not appear to be of value. 200 ppra equaled 400 ppm. The gas chromatographic charts are no good and the research book concentration is not shown or recorded. The gas chromatographic chart agrees with the data in the research book. There are no gas chromatographic charts but there is data in the research book.
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Exposure Number
Comment
35 ` There are no T-I or T-II data on the chart or in
the book. T-III data is present in the research
book but not on the gas chromatographic chart.
86 The gas chromatographic chart and the data in the
research book agree but the exposure concentra
tions are on the low side.
108-110
The gas chromatographic charts do not match the
data in the log book. Observations were noted
that some of the data was off the GC chart and
there were some operator errors and missing data.
134&135
There were no GC charts to go with the data pre
sented in the research books. No charts available
for the exposures 110-136.
141 A line was drawn through the first hour values and
apparently discarded.
144 There are gas chromatographic charts and research
book data available but another notebook says that
exposures were not run. In addition, the control
line shows contamination.
145 A notation was made that some of the readings were
off the chart -- wide excursions on both the low
and high sides. In addition, the controls were
contaminated.
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Exposure Number
Comments_________________
146 *Some of the data from the charts has been dis
carded.
150 The T-II value at the first sampling interval does
not agree with the data presented in the research
book. The negative control line also shows con
tamination. The T-III value is on the high side
and reaches 3420.
152 A broken rotometer on line T-I. The values for the
T-III line indicate they are not working and they
have been deleted. The control background is high.
Selected data has been used for the tabulated data
presented.
158 Analytical samples are present for four hours. There
are missing data when one compares the GC charts
versus that present in the research book.
159 The chambers were only on for two hours. There
are no exposure concentrations for T-II, T-III and
T-IV at the first sampling. There are incomplete
values for the second sample at two hours.
165 The chamber was operated for 4 1/2 hours. Zero
time was taken ten minutes after the start of the
exposure, at which time the gas chromatographic
readings were taken. Environmental samples on the
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Exposure Number
Comments
165 '`roof appeared high. Hood efficiency, where the (cont'd)
syringes were cleaned was poor. Vinyl chloride
was still detectable in the T-IV chamber 45 min
utes after the exposure was terminated. The
solvent room and the pilot plant both showed
vinyl chloride contamination.
166 The first reading on the gas chromatographic
chart is 3:55 pm. The readings continue until
4:40 pm at sharply lower concentration. The
chamber was turned on at 10 o'clock in the morn
ing.
169 Sampling was only conducted for 4 hours. The
T-III value was 3710 ppm. For the T-I and r-II
lines, there was no sample taken for the second
sampling interval or the fourth interval.
180 Basically no analytical data on exposure concen
tration.
196 Exposures were sampled for five hours. Selected
data in the daily range and no noon samples. The
control line was contaminated.
197 The chambers ran for five hours and GC data are
available for three.
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Exposure Number
Comment
201 * A seven-hour exposure is indicated as having been
run. The sampling for GC analysis was run for
somewhat less than five hours. Selected data has
been taken at the initial reading and then spor
adic data is missing throughout the run.
206 The chambers were not monitored and no data are
available.
210 No sampling. Exposures were only on for one hour
with a notation of TV filming.
218 There was one reading for each line for the first
two hours, obtained manually. Half of the samp-'
lings were done on automatic, but two, rather than
three, samples were obtained.
224 Notation is made of CBS filming tomorrow; expos
ures were run for 4 1/2 hours. The line control
had a reading of 6. The GC chart shows no agree
ment with the research book, which is sparse.
However, it does show T-III range from 3-320 ppm.
The T-I 50 ppm range equals 445 ppm.
225 No gas chromatographic charts to support the mini
mal data in the research book. The chambers were
turned on at 9:00 am and were off at 11:40 am.
GC readings were taken only at 10:00 and 11:00 am.
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Exposure Number 228
231
263S237
233S239 243
244 251
255
Comment For T-I and T-II, there are missing data. For T-III, there are no values for the first and second samplings. One-third of the values which were on the low side were deleted from the calculation of the averages, one of which was off the graph. Se lected data is shown. The control shows a peak height of 7.5, indicating some contamination. The gas chromatograph was not working; no data but means have been reported for one value at one interval. The research book shows a mean and ranges but no supportive data were available. The line control shows a peak height of 8. Onethird of the data from the GC charts were not used in the calculations or averages in the research book. Samples are only shown for three hours. The GC chart data is sparse. The line control is shown as 6.5. The 50 ppm equaled 400 ppm and a notation of major problems. Air recirculated was causing high concentrations in the control chamber. The chambers were turned
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Exposure Number
Comment
255 (cont'd)
`off. The control chamber had a level equal to 3.4 ppm of vinyl chloride. A notation of definite
leakage into the room was recorded. Selected data
only for two hours of the exposure was used.
257 The T-IV chambers were turned on two hours after
the rest of the chambers. The line control shows
vinyl chloride contamination. One of the 50 ppm
values is equal to at least 100 ppm. In addition,
one-third of the values were unavailable and se
lected data were used.
258 The first exposure concentration readings were
taken at 1:15 pm and also at 2:15, despite the
fact the chambers were turned on at 11:30 in the
morning. Most of the data were incomplete.
This appeared to be the last exposure where gas chromatographic chart data were available. The research book has data for exposures 259, 260, 261, 262, but there are no sup portive gas chromatographic data.
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body weights
Body weights were available, either as hand written calculations or as computer printouts. Unfortunately, many of the computer printout body weight tabulations reflected the animals being weighed on a gang-cage basis. Therefore, it becomes more difficult to determine substitutions. A tabulation beginning with month 5 and proceeding through month 13 is shown below for the body weight data. This com pares the data presented in the interim IBT reports versus that obtained when one inspects the computer printout itself. These data have only been examined for the T-III group.
MEM BODY WEIGHTS FROM PRINTOUTS VINYL CHLORIDE RATS, T-III - 2,000 PPM
Month
Male Female
Month
Male Female
5 Report
549 321
Printout
550.5 312
9 Report Printout
681 359 627 359
6 Report
567 329
Printout
567.3 339
10 Report Printout
663 356 629 356
7 Report
565
Printout* 714
691
702
606
346 436
11 Report Printout
12 Report Printout
696 374 680 374
736 378 718 378
8 Report
585
Printout** 581
359 359
13 Report Printout
740 420 729 . 420
*A11 values shown on printout
**Subtracted 329 grams from printout mean
Inspection of these data indicates that during the seventh month there
is a major difference between the body weights in the printouts and those
presented in the final report, suggesting replacements.
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MORTALITY AND FATE Hand written sheets reflecting tabulations of mortalix4
ties are available. These sheets bear various dates at periodic intervals. Additional tabulation reflects animals that are missing or cannibalized, based upon the gang hous ing. However, there are numerous errors present and inconsis tencies in these tabulations. A chart of missing rats, pre pared near the end of the third month of the study, shows numerous animals not accounted for in Dr. Shah's tabulation. These differences are as follows:
Untreated control male rats - 2 animals missing; none shown on Dr. Shah's tabulation.
Untreated control females - 3 missing; none shown on Dr. Shah's tabulation.
T-I male rats - 7 missing; 1 shown on Dr. Shah's tabulation. T-II male rats - 6 missing; none shown on Dr. Shah's tab ulation. T-III male rats - 4 missing; none shown on Dr. Shah's tab ulation. T--1 female rats - 6 missing; none shown on Dr. Shah's tab ulation. T-II female rats - 2 missing; none shown on Dr. Shah's tab ulation . T-III female rats - 4-5 missing; none shown on Dr. Shah's tabulation.
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There are numerous evidences of errors in the animal numbers orx 4 the animals being missexed, e.g., animal 751 female should be 638 male in the T-III group; animal 787 female should be male 691 in the T-III group; animal 788 female should be 692 male in the T-III group; animal 689, T-III male rat should be 686; 605, T-III female rat, is a male* Autopsy sheets that reflect extra animals were avail able .
A mortality tabulation prepared from the animals avail able in each group, as determined by the body weight print outs, beginning with month five and continuing through month 13 is shown in the accompanying Table. You will note a sub stantial difference in the numbers available as tabulated between the printout and that presented in the report for the male T-III group, month 7, 12 and 13. Similarly substantial differences in the females in months 12 and 13, as well, also suggests substitution of the animals was occurring.
Close attention was paid to determining the specific fate of the five animals examined in detail by Dr. Bussey in the T-III male group, as reported to have brain tumors. Inspection of the hand written post mortem tabulations by IBT staff, as opposed to the data presented on the autopsy sheets and the mortality data presented in the final report indicates substantial discrepancies.
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Animal #641, T-III male, was found dead at 369 days, according to the report. Hand written P.M. tabulation sheet shows the animal to be dead by 8/6/74, which is about 330 days into the study. The necropsy sheet shows the animal's date of death as 9/3/74 (359 days). These differences have not been explained.
In the case of animal #649, the necropsy sheet and the report shows that the animal died at 402 days. The sheet presenting post mortem tabulations, dated 9/11/74, however, shows the animal as being dead. This date would be approxi mately one year into the study.
Animal #664 was reported as having died at 373 days. This animal is shown as being dead 7/17/74 on the necropsy sheet and as being dead on a hand written tabulation on 8/9/74. Therefore, the 373 day mortality presented in the report is incorrect and was probably 311 days.
Animal #672, shown in the report as having died at 310 days, is also shown on a post mortem mortality tabulation dated 7/26/74 as being dead by that time. A necropsy date of 7/16/74 for this animal is noted. Therefore, the fate of this animal appears to be correct.
In the case of animal #667, the report indicates days on test as 383 but the necropsy sheet is dated 6/27/74, re flecting less than a year on test. A post mortem log dated 6/26/74 also shows the animal as being dead. Therefore, the date presented as days on test in the report is in error.
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CONCLUSIONS An intensive audit of certain aspects of IBT No. 633-03222,
chronic vapor inhalation toxicity study with vinyl chloride, devoted to inspection of exposure concentrations and pertin ent body weight and mortality logs indicates the following: (a) There are numerous evidences of replacement of animals
in this study. There appears to be an exceptionally high replacement of the hamsters. Also, because of the dif ferences in body weights and mortalities noted in the T-III rats, it is possible that there was major substi tution of the animals between the sixth and seventh months of the study and perhaps at 12 and 13 months. There are evidences of extra animals being available for the various species in the study. The accountability for the animals and the mortalities show substantial discrepancies. (b) The monitoring of the exposure concentrations and the conduct of the exposures shows poor technique and selec tion of data in many instances but does seem to indicate that a three-level study was conducted, even though the swings in exposure concentrations periodically may have been t 50-100%. The instances of actual overlap between exposure concentrations at the T-I level and the T-II level were relatively infrequent and there do not appear
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to be any real instances of overlap between the T-II and T,, -* III levels. (c) The animals reported to have brain tumors indicate discrepancies as to their actual dates of death versus those reported in interim reports and in some cases on the necropsy sheets.
In summary, the siudy was conducted in an extremely sloppy fashion and one would have to look at any of the re sults more from a qualitative standpoint than a quantitative standpoint.
Bob West, Ph.D. BW/am
Date: May 10, 1980
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081 0896
TABLE I
TEST MATERIAL: Vinyl Chloride (Ethylene Derived)
Chronic Vapor Inhalation Toxicity Study - Albino Rats, Albino Mice, and Golden Hamsters
Chamber Concentration Data
Day Exposure Number Number
1 4 5 16 29 39 56 73 80 99
120 130 150 151 152 171 186 187 197 200 201 208 212 220 221 229 232 235
1 4 5 12 21 29 48 53 58 71 86 94 108 109 110 123 134 135 141 144 145 150 152 158 159 165 166 169
TE-I ___ Range
Mean
39 45 50 42 48 42 48 42 45 29 45 42 35 32
41.5 35 47 46.5 40 38.5 37.5 48 36 36.5 44.5 46.5 47 44
45 54 56 63 66 55 114 58 60 31 46
63 58 62 110 55 53 55.5 55 56 55.5 54.5 64.5 52.5 55 46 71 51
43 51 52 60 58 43 84 49 54 -- 30 45
53 49 44 58.9 48 49.6 50.7 45.1 49 48 51.4 48.1 45 49.2 49.4 52.3 47.6
Chamber Concentration (ppm)
TE-I I
TE-III
Range
Mean
Range
Mean
130 24
182 54
192 188 172 178 220 258 168
180 90
150 170 186 184 180 178 178 17 4 170 154 138 130 190 184 196
180 162 237 266 248
216 452 211 364 310 232 228 288 234 252 210 224 262 216 310 212 242 282 2 50 138 216 228 215
173 1820 2610
87 1190 3600
218 1160 3100
156 2400 3100
219 1090 2490
209 1900 3010*
298 2240 4000
202 2380 2700
243 2020 2760
277 2740 3940
199
1960 2960
201 2680 3100
200
1780 2920
193 1280 2800
198 2300 2800
197 2320 3020
209 2040 2680
194 2180 3400
196 2400 2700
223 2340 3120
197 2340 2740
200 2260 3420
194 2160 3200
192 1440 2600 '
134 2460 2620
200 2420 2800
201 * 2100 2820
207 2440 3710
2510 2540 2202 2653 1561 2455 2910 2565 2404 3185 2565 2883 2556 2349 2497 2708 2419 2550 2535 2561 2500 2627 2583 2240 2540 2583 2520 2932
' TE-IV**
Range
Mean
TABLE I continued
TEST MATERIAL: Vinyl Chloride (Ethylene Derived)
Chronic Vapor Inhalation Toxicity Study - Albino Rats, Albino Mice and Golden Hamsters
Chamber Concentration Data
Day Number
Chamber Concentration (ppm)
Exposure
TE-I
TE-II
TE-III
T-IV**
NumberRangeMeanRange
MeanRangeMeanRange
Mean
248 178 47.5 52.5 51.2 186 222 203 2420 2700 2573
274
196 44.5 51
47.9 188
216
202
2180 2580
2377
275
197 38 56
49.5 174 242 198
2160 2640
2386
281
201 40 56
47.5 168 232 204
2360 2740
2515
304 218 45.5 50.5 48.2 202 226 218 2140 2460 2288
312
224 39 46
42.8 148 172 160
1800 2040
1920
315 225 37 52.5 44.2 146 196 174 1800 2440 2073
316 226 46 55 54 182 220 208 2080 2500 2473
318 228 46.5 52.0 49.2 170 280 192 1960 2380 2022
323
231 49.4 62
54.9 176 2 50 208
2160 2840
2475
332 238 56 56 56 206 208 208 2260 2380 2300
333
239 47.5 55
51.3 185 210 202
2160 2600
2556
339
243 40 60
48.9 184 234 203
2380 2960
2607
345 247 49.5 51 50 190 210 203 2140 2620 2525
351 251 35 45.5 39.5 180 246 228 2640 2900 2783
359 257 46 71 56 202 208 206 2420 2560 2487
360 248 44.5 59.5 50.7 170 216 202 2360 2840 2602
Previously reported by IBT as 1970-2770, with a mean of 2474
f **Only gross inspections were made of the TE-IV concentrations but the same problems existed
with this concentration as with the others.
CMA 000898
TABLE II IBT No. 663-03222 VINYL CHLORIDE RATS - SURVIVAL
T-III - 2,000 ppm
Month
5 Report Printout
6 Report Printout
7 Report Printout*
3 Report Printout
9 Report Printout
10 Report Printout
11 Report Printout
12 Report Printout
13 Report Printout**
Male
97 96
95 96
94 89 or 92
90 89
84 82
76 74
66 62
42 37
27 23
Female
96 95
96 94
95 92
92 90
85 81
72 68
48 46
34 15 (?)
17 5 (?)
* None of #'s or weights agree with report * All over the lot, 5 body weights shown
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