Document LKJ1JORDn4gwkvkN6ExR347jQ
BOB WEST ASSOCIATES, INC. '
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P.O.BOX 2001 STAMFORD, CONNECTICUT 06906 203-322-4466
CONFIDENTIAL
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Review and Audit of IBT No. 663-03222, Chronic Vapor Inhalation Toxicity Study with Vinyl Chloride (Ethylene Derived) in Albino1 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-IV 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 18 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 Bio-Test 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 ppm 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, ether 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. 6j There was evidence that the chambers did not have an
\_y 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.
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.
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 .
Aw
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 an accurate reflection, in many instances, of the exposure concentrations or are based on minimal supportive analytical data.
Exposure Number
Comment
5 The values for the T-I mean and the range for the
raw data in the report do not agree.
20 & 35
In the daily intervals, there were problems with
sampling and the curve does not appear to be of
value.
200 ppm equaled 400 ppm. 56 The gas chromatographic charts are no good and
the research book concentration is not shown or
recorded.
80 The gas chromatographic chart agrees with the data
in the research book.
33 There are no gas chromatographic charts but there
is data in the research book.
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Exposure Number
Comment
85 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
146
Comments 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 (cont'd)
roof appeared high. Hood efficiency, where the 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 T-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 C3S 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
Comment
228 For T-I end T-II, there are missing data. For
T-III, there are no values for the first and
second samplings.
231 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.
263&237
The gas chromatograph was not working; no data
but means have been reported for one value at one
interval.
23S&239
The research book shows a mean and ranges but no
supportive data were available.
243 The line control shows a peak height of 8. One-
third of the data from the GC charts were not used
in the calculations or averages in the research
book.
244 Samples are only shown for three hours.
251 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.
255 Air recirculated was causing high concentrations
in the control chamber. The chambers were turned
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Exposure Number 255 (cont'd)
257
258
Comment 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. 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. 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 chromatograohic 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 itsel: These data have only been examined for the T- III group
MEAN 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** 531
359 359
13 Report Printout
740 420 729 420
*All 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 mortali
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-l 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 or the animals being missexea,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 prir.zout 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. 3ussey 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 study 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.
3ob West, ?h.D. BW/am
Date: Mav 10, 1980 CTL031313
BOH WEST ASSOCIATES. INC.
TABLE I
TEST MATERIAL: Vinyl Chloride (Ethylene Derived) Chronic Vapor Inhalation Toxicity Study - Albino Rats, Albino Mice, and Golden Hamsters
Chamber Concentration Data
Day umber
1 4 5 16 29 39 66 73 80 99 120 130 150 151 152 171 186 187 197 200 201 208 212 220 221 229 232 235
Exposure Number
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 Ranee
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 50 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
Mean
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
Ranee
Mean
Range
Mean
130 24
182 54
192 188 172 178 220 250 168
180 90
150 170 186 184
180 178 178 174 170 154 138 130 190 184 196
180 162 237 266 248 216 452 211 364 310 232 228 288 234 252 210 224 262
216 310 212 242 282 250 138 216 228 215
173 87
218 156 219 209 298 202 243 277 199
201 200 193 , 198 197
209 194
196 223 197 200 194 192 134 200 201 207
1820 1190 1160 2400 1090 1900 2240 2380 2020 2740 1960 2680 1780 1280 2300 2320 2040 2180 2400 2340 2340 2260 2160 1440 2460 2420 2100 2440
2610 3600 3100 3100 2490 3010* 4000 2700 2760 3940 2960 3100 2920 2800 2800 3020 2680 3400 2700 3120 2740 3420 3200 2600 2620 2800 2820 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 2503 2520 2932
TE--IV**
Range
Mean
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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 Exposure dumber Number
TE-I Range
240 170 47.5 52.5 274 196 44.5 51
275 197 38 56
281 201 40 56
304 210 45.5 50.5
312 224 39 46
315 22 5 37 52.5
316 226 46 55
310 228 46.5 52.0
323
231
49.4 62
332 238 56 56
333 239 47.5 55
339 243 40 60
345 247 49.5 51
351 251 35 45.5
359 257 46 71
360 248 44.5 59.5
Mean
51.2 47.9 49.5 47.5 48.2 42.8 44.2 54 49.2 54.9 56 51.3 48.9 50 39.5 56 50.7
Chamber Concentration (ppm)
TE- II
TE-III
Range
Mean
Range
Mean
186 222 203 188 216 202 174 242 198 168 232 204 202 226 218 140 172 160 146 196 174 182 220 208 170 280 192 176 250 208 206 208 208 185 210 202 184 234 203 190 210 203 180 246 ' 228 202 208 206 170 216 202
2420 2180 2160 2360 2140 1800 1000 2080 1960 2160 2260 2160 2380 2140 2640 2420 2360
2700 2580 2640 2740 2460 2040 2440 2500 2380 2840 2380 2600 2960 2620 2900 2560 2840
2 57 3 2377 2386 2515 2288 1920 2073 2473 2022 2475 2300 2556 2607 2525 2783 2487 2602
T-IV* *
Range
Mean
*Previously reported by IBT as 1970-2770, with a mean of 2474
**Only gross inspections were made of the TE-IV concentrations but the same problems existed with this concentration as with the others.
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TABLE II IBT No. 663-03222 VINYL CHLORIDE RATS - SURVIVAL
T-III - 2,000 ppm
f
Month
5 Report Printout
6 Report Printout
7 Report Printout*
8 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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