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BOB WEST ASSOC 1 ATKS. INC.
MflY 2 0 mo
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AiOFORC, CONNECTICUT 06S05 203 322-46
Review and Audit of IBT No. 663-03222, Chronic Vapor Inhalation Toxicity Study with Vinyl Chloride (Ethylene Derived) in Albino Rats, Albino Mice and Golden Hamsters for the Manufacturing Chemis~t~s~~Association
The review and audit of this study was conducted at IBT's offices in Northbrook, IL, April 14 to April 13, 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 13 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 animals 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, 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. 3. 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
I ;I
*
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
5 20 & 35
53 56
30 33
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 ppm 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
85 86 108-110
134&135
141 144
145 1r
Comment
There are no T-X 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.
The gas chromatographic chart and the data in the
research book agree but the exposure concentra
tions are on the low side.
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.
There were no GC charts to go with the data pre
sented in the research books. No charts available
for the exposures 110-136.
A line was drawn through the first hour values and
apparently discarded.
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.
A notation was made that some of the readings were
off the chart -- wide excursions on both the low
and high sides. contaminated.
In addition, the controls were ^^
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Exposure Number i
Comments , |
j,
146
Some of the data from the charts has been dis
carded.
150
The T-IX 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.
15 8
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 165 (cont'd)
166
169
180 196 197 r
Comments
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.
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.
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.
Basically no analytical data on exposure concen
tration .
Exposures were sampled for five hours. Selected
data in the daily range and no noon samples. The
control line was contaminated.
The chambers ran for five hours and GC data are
available for three.
!<
I
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Exposure Number l
201
206 210
218
224
225
Commentj . (
j,
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.
The chambers were not monitored and no data are
available.
No sampling. Exposures were only on for one hour
with a notation of TV filming.
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.
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.
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
26 3&237
238S239 243
244 251
255
1t
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. One-
third 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
i "
1
in the control chamber. The chambers were turned
' r-
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Exposure Number 255
(cont'd)
257
25 8
Comment. , j ____________________
i,
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 chroma
tographic 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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30DY 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.
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
U 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 |duting the seventh montl^ 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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MQRTALITY AND FATE Haifd written sheets feillecting tabulations of mortali- 1
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-X 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-XII 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 missexed, e.g., animal 751
female should be633 male in the T-III group; animal 737
female should bemale 691 in the T-III group; animal 733
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
1 Vindicates substantial discrepancies.
f
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Animal #641, T-III male, was found dead at 369 days, according to the report. ,;Ha|nd written P.M. tabulation sheieti 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 50-100%. The instances of actual overlap between
'f
exposure concentrations at the T-I level and the T-II level were relatively inirequent and there dj not appear
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to be any real instances of overlap between the- T-XI
ani, T-IXX 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.
TAB LI.'
Date: May 10, 1980
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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 Humber Humber
TE-I Ranae
Mean
Chamber Concentration
TE-I I
Ranae
Mean
(ppm)
TE-III
Range
Mean
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
,
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
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 43
54 51
56 52
63 60
66 58
55 43
114
84
58 49
60 54
31 - 30
46 45
63 53
58 49
62 44
110
58.9
55 48
53 49.6
55.5 1 ' 50.7
55 45.1
56 49
55.5
48
54.5
51.4
64.5
48.1
52.5
45
55 49.2
46 49.4
71 52.3
51 47.6
130 24
182 54
192 188 172 178 220 258 168 180
90 150 170 186 184 180 178 178 174 170 154 138 130 190 184 196
180 162 237 266 248 216 4 52 211 364 310 232 228 288 234 232 210 224 262 216 310 212 242 282 250 138 216 228 215
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 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
CO CO O
cc
<
co
U
Day Exposure Number Number.
TE-I Range
Mean
Chamber Concentration (ppm)
TE- II
TE-III
Range
Mean
Range
Mean
T-IV***
Range
Mean
248 274 275 281 304 312 315 316 318 323 332 333 339 345 351 359 360
178 196 197 201 218 224 225 226 228 231 238 239 243 247 251 257 248
47.5 44.5 38 40 45.5 39 37 46 46.5 49.4 . 56 47.5 40 49.5 35 46 44.5
52.5 51 56 56 50.5 46 52.5 55 52.0 62 56 55 60 51 45.5 71 59.5
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
186 188 174 168 202 148 146 182 170 176 206 185 184 190 180 202 170
222 216 242 232 226 172 196 220 280 250 208 210 234 210 246 208 216
203 202 198 204 218 160 174 208 192 208 208 202 203 203 228 206 202
2420 2180 2160 2360 2140 1800 1800 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
2573 2377 2386 2515 2288 1920 2073 2473 2022 2475 2300 2556 2607
2525 2783 2487 2602
~
*Previously reported by IBT as 1970-2770, with a mean of 2474
**0nlv gross inspections were made of the TE--IV concentrations but the same problems existed with this concentration as with the others.
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*
8 Report Printout
9 Report Printout
10 Report Printout
11. Report Printout
12 Report Printout
13 Report Printout**
Male
97 96
9 5. 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
I1
I
** All over the lot, 5 body weights shown
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