Document DMQYX3mpgpdXKG193gBnVxakn
MIN 312/014272
PERFLUOROOCTANESULFONYL FLUORIDE (POSF; T-7661.1) PRELIMINARY TOXICITY STUDY BY INHALATION ADMINISTRATION TO CD RATS FOR 1 WEEK
Sponsor 3M Center, 3M Corporate Toxicology, Building 220-2E-02, St Paul, MN 55133-3220, USA.
Research Laboratory Huntingdon Life Sciences Ltd., Woolley Road, Alconbury, Huntingdon, Cambridgeshire, PE28 4HS, ENGLAND. Report issued: 7 October 2005
Page 1 of 340
MIN 312/014272 CONTENTS
Page CONTENTS....................................................................................................................................... 2 COMPLIANCE WITH GOOD LABORATORY PRACTICE STANDARDS................................. 4 QUALITY ASSURANCE STATEMENT......................................................................................... 5 CONTRIBUTING SCIENTISTS....................................................................................................... 6 SUMMARY ....................................................................................................................................... 7 INTRODUCTION.............................................................................................................................. 8 RELEVANT STUDY DATES........................................................................................................... 9 TEST SUBSTANCES...................................................................................................................... 10 EXPERIMENTAL PROCEDURE................................................................................................... 11 RESULTS......................................................................................................................................... 17 DISCUSSION .................................................................................................................................. 20 CONCLUSION ................................................................................................................................ 20 REFERENCES................................................................................................................................. 21
FIGURES 1. Bodyweight - group mean values (g)...........................................................................................22
TABLES 1. Bodyweight - group mean values (g)...........................................................................................23 2. Food consumption - group mean values (g/rat) ...........................................................................24 3. Water consumption - group mean values (g/rat) ........................................................................25 4. Organ weights - group mean values (g) ......................................................................................26 5. Macroscopic pathology - incidence summary .............................................................................30 6. Microscopic pathology - incidence summary..............................................................................31
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MIN 312/014272 Page
APPENDICES 1. Daily dose observations - individual findings .............................................................................32 2. Bodyweights - individual values (g) ...........................................................................................34 3. Absolute organ weights - individual values (g) ...........................................................................35 4. Individual pathological findings...................................................................................................37 5. pH values for terminal urine - individual values .........................................................................58 6. PCNA staining in the urinary bladder - individual and mean values..........................................59 ADMINISTRATION OF POSF BY INHALATION TO RATS .........................................................60 PROTOCOL AND PROTOCOL AMENDMENTS .............................................................................87 ANALYTICAL PHASE REPORT ......................................................................................................121 SCANNING ELECTRON MICROSCOPE EXAMINATION AND X-RAY MICROANALYSIS................................................................................................................287 HUNTINGDON RESEARCH CENTRE GLP COMPLIANCE STATEMENTS ............................338
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sion COMPLIANCE WITH GOOD LABORATORY PRACTICE STANDARDS
TPhciseyandesdcrsibenddncsonpsrthevdasocfoendrurcetdn0cvomepllice wih the living Gd Laboratory Say ne S04 5 The UK Good Laboratory Practice regulations 1999 (Statutory Instrument No 3106) as amended
`OECD PrinciplesofGood Laboratory Practice (as revised in 1997), ENV/MC/CHEM (98)17.
EC Commission Directive, 1999/11/EC of 8 March 1999 (Official Journal No L 77/8), as amended by EC Commission Directive 2004/I0/EC of 11 February 2004 (Official Journal No. L 50/44).
UThenseiperinsciopfeAonfGeorodiLdipbaroonPhreievtessosfsgsopvebyrhenemlietnrtsures adte Anxpi diteforthestbtnwsotslid, Theserivssame eslor
the duration ofthe study.
No lim ofcompli made wi rea 1te sain cls mirage camiuion
and X-ray microanalysis.
Sty Dre, TerencJe.Kenny, B.Sc. (Hons),
Huntingdon Life Sciences Ltd.
FOde200
Date
QUALITY ASSURANCESTATEMENT MIN 312014272
"The followinghave been inspected oraudited in relation tothisstudy:
Study Phases Inspected Date ofInspection
DateofReporting.
Protocol Audit
21 August 2001
21 August 2001
ExSptuodsyuPrreeparation Sampling CTleisnticiatlemsicgonnstrol disposition PSoEstMMXo-rtReAmY - EM
1133 SSeepptteemmbbeerr22000011 13 September2001 1133 SSeepptteemmbbeerr22000011 2187 SSeepptteemmbbeerr22000011
1144 SSeepptteemmbbeerr 22000011 14 September 2001 1144 SSeepptteemmbbeerr22000011 2188 SSeepptteemmbbeerr22000011
Report Audit
102-817SDeeptceemmbbeerr20200501 2197DSeecpetmembbeerr22000015
Protocol: An audit of the protocol for his study was conducted and reported to the Study Director and Company Management as indicated above.
`Study based inspections: Inspections and auditsofphasesofthis study wereconducted and reported the Study DirectorandCompany Managementas indicated above.
Process based inspections: At or about the time this study was in progress inspections and audits of optrhoemrptrlouytrineepoarnteddtreopaeptpirtiovperipartoeceCdourmepsaenmypMlaonyaegdeomnentth.is typeofstudy were carried out. These were
Report Audit: This report has been audited by the Quality Assurance Department. This audit was `conductedand reportedtothe Study Directorand Company Managementa indicatedabove.
The methods, procedures and observations were found to be accurately described and the reported resultos reflect the raw data.
AmnoanliyttoircianlgpahnadseasudoifttahcicsosrtduidnygctoonEdxuycgteend'bsyoEwxnygpreonceRdeusreeasr.chDewtearielssaurbejepcrteesdenttoedQuianlitthye AAnsasluyrtainccael
CONTRIBUTING SCIENTISTS
STUDY MANAGEMENT Terence J. Kenny, B.Sc. (Hons.), Study Director. Rhiannon Davies, B.Sc. (Hons.), Senior Study Supervisor.
TOXICOLOGY Derek W. Coombs, B.Sc., MSc., Senior Toxicologist.
AEROSOL TECHNOLOGY AND ANALYSIS Ian S. Gilkison, M.A., Ph.D., Section Head, Aerosol Technology and Analysis.
PATHOLOGY Samuel McCormick, M.V.B., M.R.C.V.S., Ph.D., F.R.C.Path., Director of Pathology. BIOANALYSIS Richard A. Grazzini, Exygen Research, 3058 Research Drive, State College, PA 16800, USA.
SEM MICROSCOPY AND X-RAY ANALYSIS Roy Moate, Plymouth Electron Microscope Unit, University of Plymouth, Drake Circus, Plymouth, PL4 8AA, ENGLAND.
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MIN 312/014272
SUMMARY
MIN 312/014272
One group of rats (each of 5 males and 5 females) of the Crl:CDBR strain were exposed to POSF, 6 hours a day for 5 consecutive days using a snout-only exposure system. A second group, acting as control, was exposed to air only. The study mean analysed concentration was 316.95 ppm. The following comments are made in summary: Mortality and clinical signs There were no unscheduled deaths or any treatment related signs seen during the course of the study. Bodyweight, food and water consumption Following 5 days of treatment with POSF there was reduced bodyweight gain, food consumption and water consumption in treated males. Organ weights Bodyweight adjusted liver weights were higher in treated animals, with statistical significance (p 0.05) being attained in females. Absolute lungs and bronchi weights were higher in treated animals with statistical significance being achieved in males and in bodyweight adjusted female lung and bronchi weights. Macroscopic and microscopic pathology There were no treatment-related macroscopic or microscopic findings. Conclusion A no-effect level was not established for this study.
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INTRODUCTION
MIN 312/014272
The purpose of this study performed at Huntingdon Life Sciences Limited, Huntingdon, England was the assessment of systemic toxic potential in a 1-week inhalation study in rats, by snout-only administration of the test substance POSF, for 6 hours a day, for 5 consecutive days. The test substance was administered by inhalation, a possible route of accidental exposure in man. The rat was the species of choice due to requirement for a rodent species by regulatory agencies and the strain was selected on account of the availability of comprehensive background data, relating to clinical and pathological parameters, at our laboratories.
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RELEVANT STUDY DATES
Approved by: Study Director: HRC Management: Study Sponsor:
17 August 2001 17 August 2001 20 August 2001
Animals arrived at HRC: Exposures commenced: Serum/urine sampling: Terminal kill: Experimental completion date:
5 September 2001 13 September 2001 18 September 2001 18 September 2001 28 September 2002
MIN 312/014272
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TEST SUBSTANCES
MIN 312/014272
Sponsor's identification:
Perfluorooctanesulfonyl fluoride
Other names:
POSF, T-7661.1, FX-8B
Storage conditions:
In a refrigerator (ca 4C)
Date received:
14 June 2001
Supplier:
Sponsor
Batch number:
040227
Expiry date:
Assumed to be stable for the duration of the study
Purity:
>99.5%
A small sample (1 ml) was sealed in a suitable contained and stored in Archives at an appropriate temperature.
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EXPERIMENTAL PROCEDURE
MIN 312/014272
ANIMALS Twenty rats (10 male and 10 female) aged approximately 6 weeks, of the Crl:CDBR, a caesarean derived strain of Sprague-Dawley origin, were obtained from Charles River (UK) Limited, Manston Road, Margate, Kent, on 5 September 2001.
The latest Health Screen Report published by the animal supplier was provided to Huntingdon Life Sciences (HLS). In addition, the additional consignments of animals included a health screen relating to the current status of the breeding colony. These documents were sent to HLS Veterinary Services immediately upon receipt for review and subsequent archiving.
Random assignment to experimental groups took place on arrival. The animals were then uniquely identified by numbers tattooed on the tail.
The identification of individual rats in the 2 groups together with the target exposure level were as follows:
Group 1 (Air control) 2 (POSF)
Target exposure level
(ppm) -
300
Animal numbers
Male
Female
1-5
11-15
6-10
16-20
ACCOMMODATION The rats were housed 5 of the same sex to a cage in suspended stainless steel cages fitted with mesh front, back and floor with stainless steel sheet sides. Plastic trays lined with absorbent paper were placed below each cage to collect animal excreta and the paper was changed daily. Each cage had a coloured label identifying the group and the numbers of the animals contained within it. The rats were kept in a single room and, additionally, after the start of the exposure period, each group was positioned on an individual cage battery. Exposure took place in the same room. The temperature and relative humidity of the holding room were recorded using a Kent Clearspan recorder. The study holding room temperature and relative humidity were set to be maintained within limits of 21 2C and 55 15% respectively. Recorded ranges were 19.0 to 21.0C and 40 to 72% humidity. Minor deviations from these ranges were of relatively short duration and considered not to have affected the scientific integrity of the study. Lighting was controlled to give 12 hours light (0600 - 1800 hours) and 12 hours dark per 24 hours.
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MIN 312/014272 DIET While in their cages, all rats had access to a weighed quantity of standard quality-controlled laboratory rat food (SDS Rat and Mouse No. 1 SQC modified maintenance diet, Special Diets Services, Witham, Essex). There was no information available to indicate that any non-nutrient substance likely to influence the effect of the test compound could reasonably be expected to be present in the diet. The analytical data have been lodged in Huntingdon Life Sciences Archives. Tap water was available from moulded polypropylene water bottles at all times while the rats were in the cages. The water bottles were rinsed and refilled daily and thoroughly cleaned at intervals during the study. There was no information available to indicate that any substance likely to influence the effect of the test system could reasonably be expected to be present in the drinking water. Results of the routine physical and chemical analyses of water at source (sampling point, Grafham Final Water) as conducted by the supplier, Anglian Water Services Ltd, have been made available to Huntingdon Life Sciences. Anglian Water takes its guidelines on water quality from the EEC directive relating to water for human consumption, viz. Council Directive 80/778/EEC. The analytical data have been lodged in Huntingdon Life Sciences Archives.
ADMINISTRATION The test substance was administered for 6 hours a day, for 5 consecutive days. The test material was delivered to an all glass vapouriser and generated as a droplet atmosphere, into a stream of air for administration to the rats by inhalation from snout only exposure chambers. The test substance was metered to the vaporiser from an infusion pump. The vapour/air mixture passed directly into the exposure chamber. The target chamber concentration was achieved by using different liquid feed rates controlled by the infusion pump and using syringes of an appropriate value. The target concentrations for treated rats was 300 ppm. Control rats received air only. The rats were exposed to the control/test atmosphere using ADG snout-only exposure chambers (ADG Developments Ltd, Hitchin, Hertfordshire, England) of a modular construction in aluminium alloy comprising a base unit, 3 sections each having 20 exposure ports, and a top section incorporating a central inlet with a tangential air inlet. All animals (including reserves) were subjected to restraint procedures and exposed to air only (`Sham dosing'), for 5 consecutive days, in order to accustom animals to the restraining procedure prior to study initiation. The rats were restrained once per day, increasing the `Sham dosing' period progressively (0.5, 1, 2, 4 and 6 hours for Days -5 to -1 respectively). Details of administration and analysis of the test atmospheres together with the results obtained are presented in ADMINISTRATION OF POSF BY INHALATION TO RATS appended to this report.
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MIN 312/014272 CLINICAL INVESTIGATIONS Dated and signed records of all activities relating to the day to day running and maintenance of the study, as well as to the group observations and examinations outlined in this procedure were recorded in the Study Daybook. Individual dated and signed records noting nature and severity, date and time of onset and duration and progress of observed clinical signs were maintained for each animal. Mortality Throughout the study, all cages were checked in the morning and again at the end of the normal working day for dead or moribund animals. Clinical signs Dated and signed records of appearance, change and disappearance of clinical signs were maintained. Individual animal records were maintained on the basis of:
- any observation, considered to be of possible importance, made at any time during the study; - any observation, considered to be of possible importance, made during transfer to
restraining tubes (prior to exposure), during exposure (although severely restricted due to tube restraint), on return to holding cages (after exposure) and as late a possible in the working day. During the acclimatisation period, observations of the animals and their cages were recorded at least once a day.
BODYWEIGHT The weight of each rat was recorded a week prior to the start of exposures. During the treatment period, bodyweight was recorded on the day that treatment commenced, daily thereafter, and also prior to necropsy.
FOOD CONSUMPTION The quantity of food consumed by each cage of rats was recorded on a daily basis. Food intake per rat (g/rat/day) was calculated using the total amount of food given to and left by each cage in each group and the number of rats surviving in each cage.
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MIN 312/014272 WATER CONSUMPTION The quantity of water consumed by each cage of rats was recorded on a daily basis, commencing 1 week before the start of exposures. Water intake per rat (g/rat/day) was calculated using the total amount of water given to and left by each cage in each group and the number of rats surviving in each cage.
TERMINAL STUDIES Pre-terminal urine sampling Individual urine samples were collected from animals within 2 hours following lights on in the animal holding room where possible. For animals failing to produce a specimen during this interval, urine was collected at necropsy. The urine samples were immediately assayed for pH using a microelectrode, and prepared SEM stubs despatched to Plymouth University for calculi and crystal analysis by SEM X-ray element identification. Test substance/metabolite analyses Samples of blood (for serum) were obtained at necropsy by cardiac/aorta puncture while the rats were held under terminal sodium pentobarbitone anaesthesia. The blood samples (up to 4 ml) were collected, run into tubes, allowed to clot at room temperature and the serum separated and frozen prior to despatch to the Sponsor for subsequent analysis at Exygen Research. Necropsy All animals were killed on Day 6 of the study, following 5 days of exposure. Animals were killed by an intraperitoneal injection of sodium pentobarbitone followed by exsanguination from the brachial arteries. All study rats were subjected to a macroscopic post mortem examination. The following procedures applied:
All superficial tissues were examined visually and by palpation and the cranial roof removed to allow observation of the brain, pituitary gland and cranial nerves. After ventral mid-line incisions and skin reflection, all subcutaneous tissues were examined. The condition of the thoracic viscera was noted, with due attention to the thymus, lymph nodes and heart. The abdominal viscera were examined before and after removal; the urinary bladder was examined externally and by palpation.
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MIN 312/014272
The gastro-intestinal tract was examined as a whole and the stomach, caecum and portions of duodenum, jejunum, ileum, colon and oesophagus were incised and examined. The lungs were removed and all pleural surfaces examined. The liver was sectioned at intervals of a few millimetres; the kidneys were incised and examined. Any abnormalities in the appearance and size of the gonads, adrenals, uterus, intra-abdominal lymph nodes and accessory reproductive organs were recorded.
Sections of liver samples were immediately frozen by immersion in liquid nitrogen and stored at -70C prior to dispatch to the Sponsor for subsequent analysis at Exygen Research.
The following organs from all surviving animals were dissected free of fat and weighed. For bilateral organs, left and right organs were weighed together.
adrenals
liver
heart
kidneys
lungs
Preservation of tissues
During post mortem examination, samples or the whole of the tissues listed below, from all study animals, were preserved in 10% neutral buffered formalin; except the eyes which were preserved in Davidson's fixative and testes/epididymides were fixed in Bouin's solution and then transferred to 70% alcohol. The lungs were infused with fixative prior to immersion. The nasal cavity was flushed with fixative prior to immersion.
abnormalities adrenals aorta (thoracic) brain caecum colon duodenum epididymides eyes femur (longitudinal
section through joint) harderian glands head heart ileum jejunum
kidneys (with pelvic region)*
lachrymal glands larynx (2 levels) liver lungs (section from all
lobes including bronchi) lymph nodes: mandibular mesenteric tracheobronchial mammary area (caudal) nasal turbinates oesophagus optic nerves ovaries
pancreas pituitary prostate rectum salivary glands
(submandibular/ sublingual) sciatic nerves seminal vesicles skeletal muscle (thigh) skin spinal cord (transverse and longitudinal sections at the cervical, lumbar and thoracic levels) spleen
sternum stomach testes thymus thyroid with
parathyroids tongue trachea (2 levels) urinary bladder* uterus with cervix vagina
* To be examined microscopically
Histopathology examination
The tissues in the list above were examined using a light microscope as annotated. Tissues were embedded in paraffin wax and sections approximately 4 - 5 micrometres were cut, processed and stained with haematoxylin and eosin.
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MIN 312/014272 PCNA Staining for cell proliferation Sections of urinary bladder were taken from all animals for PCNA immunostaining in order to assess the degree of cell proliferation present. A total of 3,000 cells were counted from 3 separate sections (1,000 cells/section) in order to determine the cell proliferation index. Positive PCNA staining cells were counted, and examined by the pathologist to assess the sections and count S-phase positive cells, if practicable. In addition, sections of the duodenum from each animal were taken and stained to act as positive controls for the immunostaining methodology.
STATISTICAL ANALYSIS For categorical data, the proportion of animals were analysed using Fisher's exact test for each treated group versus the control. For continuous data, Bartlett's test was first applied to test the homogeneity of variance between the groups. Using tests dependent on the outcome of the Bartlett's test, treated groups were then compared with the control group, incorporating adjustment for multiple comparisons where necessary.
ARCHIVING All specimens, raw data and study-related documents generated during the course of the study at Huntingdon Life Sciences, together with a copy of the final report were lodged in Huntingdon Life Sciences, Archives. Such specimens and records will be retained for a minimum period of five years, from the date of issue of the final report. At the end of the five-year retention period the Sponsor will be contacted and advice sought on the future requirements. Under no circumstances will any item be discarded without the Sponsor's knowledge.
DEVIATION FROM PROTOCOL Due to some slight tissue damage it was not possible to count 1000 cells/section on a number of urinary bladder sections stained for PCNA analysis. The number of sections affected was low and there was no effect on the outcome of the study.
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RESULTS
MIN 312/014272
CHAMBER ATMOSPHERE CONDITIONS
Chamber analysed concentration of POSF
The data are presented in ADMINISTRATION OF POSF TO RATS BY INHALATION appended to this report.
The mean chamber concentrations (ppm) are summarized below:
Exposure No. Concentration
1 2 3 4a 5 Mean sd cv
ppm 316.04 313.69 323.33 314.74 316.95 4.361
1.4
Sd Standard deviation
CV Coefficient of variation (%)
a
No values recorded due to hardware
failure,
however
nominal
concentrations were similar for all
days therefore the actual exposure
levels were deemed to be on target
Study mean analysed concentration was 316.95 ppm which was in good agreement with the target concentration of 300.
CLINICAL OBSERVATIONS Mortality There were no unscheduled deaths. Clinical signs The data are presented as follows:
Appendix 1 - individual findings There were no treatment-related signs. Signs observed post dose included red staining around eyes, brown staining on head and body and wet fur. These signs were seen in a proportion of Test and Control animals during the shamming and treatment periods and were associated with the method of restraint used.
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MIN 312/014272
Bodyweight The data are presented as follows:
Figure 1 - group mean values (g) Table 1 - group mean values (g) Appendix 2 - individual values (g) Over the five days of treatment, treated rats gained less weight than controls. On Days 2 and 3 of exposure the male rats lost weight. Food consumption The data are presented as follows: Table 2 - group mean values (g/rat) Over the five days of treatment, a small reduction in food consumption was seen in treated males, the greatest effect being on Days 2 and 3 of exposure. Water consumption The data are presented as follows: Table 3 - group mean values (g/rat) On Days 2 and 3 of exposure water consumption in treated males was reduced. At other times consumption was comparable with that of control rats.
TERMINAL INVESTIGATIONS Organ weights The data are presented as follows:
Table 4 - group mean values Appendix 3 - individual values Bodyweight adjusted liver weights were higher in treated animals, with statistical significance (p 0.05) being attained in females. Absolute lungs and bronchi weights were higher in treated animals with statistical significance being achieved in males and in bodyweight adjusted female lung and bronchi weights.
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MIN 312/014272 Macroscopic pathology The data are presented as follows:
Table 5 - incidence summary Appendix 4 - individual pathological findings The macroscopic examination performed at termination revealed no changes attributable to treatment with POSF. The incidence and distribution of all the findings were considered to fall within the background range of macroscopic changes. Microscopic pathology The data are presented as follows: Table 6 - incidence summary Appendix 4 - individual pathological findings No treatment related microscopic findings were reported. All microscopic findings were considered to be incidental and of no toxicological importance. Urinary pH The data are presented as follows: Appendix 5 - individual values Differences between the groups were minimal and considered to be of no toxicological significance. Cell proliferation The data are presented as follows: Appendix 6 - individual and mean values. The mean cell proliferation index (% positive cells) value for treated males was greater than the control value, this was principally due to differences at levels 1 and 2 only. However, intragroup differences were large such that a proportion of the individual values for the treated group lay within the range of values seen for the control group. Differences between control and treated males at level 3 and at all levels in females were minimal. It is considered that the differences seen were not of toxicological significance. SEM and X-ray examination of urine and bladder tissue The results of examination together with SEM photomicrographs are presented in the Scanning Electron Microscope Examination and X-ray Microanalysis report appended to this main report. Test substance and metabolite analysis The methods and results of analyses are presented in the ANALYTICAL PHASE REPORT appended to this main report.
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DISCUSSION
MIN 312/014272
Administration of a POSF to rats at a dosages of 300 ppm a day over 5 consecutive days produced no mortalities and transient clinical effects which were considered to be treatment-related. There was a reduction in bodyweight gain, food and water consumption amongst treated males principally on Days 2 and 3 of exposure. There were no macroscopic pathology changes. Lung weights were higher in treated animals as were bodyweight adjusted liver weights. The toxicological importance of the organ weight changes is unclear given the absence of macroscopic and microscopic findings. There were no differences in urinary pH or cell proliferation index considered to be of toxicological importance.
CONCLUSION
A no-effect level was not established for this study.
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REFERENCES
MIN 312/014272
BARTLETT, M.S. (1937), Properties of sufficiency and statistical test, Proc. Roy. Soc A 160: 268 -282. FISHER, R.A., Statistical Methods for Research Workers, Para. 21.02, Oliver and Boyd, Edinburgh.
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FIGURE 1 Bodyweight - group mean values (g)
Bodyweight (g)
MIN 312/014272 : 22 :
300
M ales 280
260
240
220
200
180 F em ales
160
140
120
100
-7
-6
-5
-4
-3
-2
-1
0
1
2
3
4
5
D ay
G roup 1 : C ontrol G roup 2 : 300 ppm
TABLE 1
MIN 312/014272 : 23 :
Bodyweight - group mean values (g)
Print No: 0001
GROUP COMPOUND
:
1
2
:
CONTROL
POSF
Printed: 21-SEP-01
DOSAGE (PPM)
:
0
300
Xybion protocol number: MIN 312
------------------------------------------------------------------------------------------------------------------------------------
SEX: ---------MALE--------- --------FEMALE--------
GROUP:
1
2
1
2
DAY
------------------------------------------------------------------------------------------------------------------------------------
-7
202
198
148
149
-6
214
211
153
156
-5
223
218
159
159
-4
229
224
162
164
-3
238
232
166
168
-2
244
236
167
170
-1
253
243
173
174
0
254
245
173
175
1
257
249
175
176
2
264
245
175
177
3
266
241
178
177
4
273
250
180
181
5
276
254
180
180
-----------------------------------------------------------------------------------------------------------------------------------++
Gain Day 0-5
22
9
7
5
% of Control
-
41
-
71
------------------------------------------------------------------------------------------------------------------------------------
Level of significance - all comparisons made with Vehicle Control:
Student's `t' test: ++ p 0.01
TABLE 2
MIN 312/014272 : 24 :
Food consumption - group mean values (g/rat)
Print No: 0002
GROUP COMPOUND DOSAGE (PPM)
:
1
2
:
CONTROL
POSF
:
0
300
Printed: 21-SEP-01
Xybion protocol number: MIN 312 ------------------------------------------------------------------------------------------------------------------------------------
SEX: ---------MALE--------- --------FEMALE--------
GROUP:
1
2
1
2
DAY
------------------------------------------------------------------------------------------------------------------------------------
-7
29
27
19
21
-6
29
28
20
21
-5
30
28
19
23
-4
30
28
19
21
-3
28
28
18
23
-2
36
26
19
25
-1
19
25
16
13
1
26
24
18
18
2
27
17
17
17
3
27
15
19
18
4
28
22
18
19
5
29
25
18
19
------------------------------------------------------------------------------------------------------------------------------------
Cumulative
Days 1-5
137
103
90
91
% of Control
-
75
-
101
TABLE 3 Water consumption - group mean values (g/rat/)
MIN 312/014272
Day
-7 -6 -5 -4 -3 -2 -1 1 2 3 4 5 Cumulative 1 to 5 % of Control
1M (Control)
30 31 32 33 34 32 33 31 32 31 35 32 161 -
Group/dosage
2M
1F
(POSF) (Control)
30
21
30
21
32
23
30
25
31
22
30
21
29
22
32
21
21
20
19
21
32
24
32
23
135
110
84
-
2F (POSF)
22 20 23 24 24 21 23 23 21 21 27 23 116 106
: 25 :
TABLE 4
Organ weights - group mean values (g)
Print No: 0004
GROUP COMPOUND DOSAGE (PPM)
:
1
2
:
CONTROL
POSF
:
0
300
Printed: 21-SEP-01
Xybion protocol number: MIN 312 ------------------------------------------------------------------------------------------------------------------------------------
MIN 312/014272 : 26 :
SEX: GROUP:
ABSOLUTE VALUES -------MALE---------1--- ---2---
BODYWEIGHT ADJUSTED VALUES MALE
---1--- ---2---
NUMBER:
5
5
5
5
------------------------------------------------------------------------------------------------------------------------------------
TERMINAL BODY WEIGHT (g)
N
:
5
MEAN : 274.3
5 252.3
sd
: 14.4
12.7
------------------------------------------------------------------------------------------------------------------------------------
ADRENALS
N
:
MEAN :
sd
:
5 0.050 0.005
5 0.054 0.007
-----------------------------------------------------------------------------------------------------------------------------------HEART
N
:
5
5
MEAN :
1.173
1.062
sd
:
0.103
0.186
------------------------------------------------------------------------------------------------------------------------------------
KIDNEYS
N
:
5
5
MEAN :
2.04
1.94
sd
:
0.14
0.08
------------------------------------------------------------------------------------------------------------------------------------
LIVER
LIVER
N
:
MEAN :
5 12.91
5 12.65
N
:
5
MEAN : 12.29
5 13.27
sd
:
1.00
0.82
------------------------------------------------------------------------------------------------------------------------------------
No differences of statistical significance
TABLE 4
(Organ weights - continued)
Print No: 0004
GROUP COMPOUND
:
1
2
:
CONTROL
POSF
Printed: 21-SEP-01
DOSAGE (PPM)
:
0
300
Xybion protocol number: MIN 312
------------------------------------------------------------------------------------------------------------------------------------
MIN 312/014272 : 27 :
SEX:
ABSOLUTE VALUES -------MALE-------
GROUP: ---1--- ---2---
NUMBER:
5
5
------------------------------------------------------------------------------------------------------------------------------------
LUNGS & BRONCHI
+++
N
:
5
5
MEAN :
1.112
1.828
sd
:
0.128
0.204
------------------------------------------------------------------------------------------------------------------------------------
Level of significance - all comparisons made with Vehicle Control:
Student's `t' test:
+++ p 0.001
TABLE 4
(Organ weights - continued)
Print No: 0005
GROUP COMPOUND DOSAGE (PPM)
:
1
2
:
CONTROL
POSF
:
0
300
Printed: 21-SEP-01 Xybion protocol number: MIN 312
------------------------------------------------------------------------------------------------------------------------------------
MIN 312/014272 : 28 :
SEX: GROUP:
ABSOLUTE VALUES ------FEMALE--------1--- ---2---
BODYWEIGHTS ADJUSTED VALUES FEMALE
---1--- ---2---
NUMBER:
5
5
5
5
------------------------------------------------------------------------------------------------------------------------------------
TERMINAL BODY WEIGHT (g)
N
:
5
MEAN : 179.5
sd
:
8.7
5 180.4
12.5
-----------------------------------------------------------------------------------------------------------------------------------ADRENALS
N
:
5
5
MEAN :
0.054
0.056
sd
:
0.004
0.006
------------------------------------------------------------------------------------------------------------------------------------
HEART
HEART
N
:
5
5
N
: 5
5
MEAN :
0.814
0.893
MEAN : 0.816
0.891
sd
:
0.114
0.091
------------------------------------------------------------------------------------------------------------------------------------
KIDNEYS
KIDNEYS
N
:
MEAN :
5 1.36
5 1.44
N
: 5
MEAN : 1.37
5 1.44
sd
:
0.11
0.14
------------------------------------------------------------------------------------------------------------------------------------
LIVER
LIVER
+
N
:
MEAN :
sd
:
5 7.97 0.77
5 9.16 1.31
N
: 5
MEAN : 8.00
5 9.12
-----------------------------------------------------------------------------------------------------------------------------------Level of significance - all comparisons made with Vehicle Control:
Student's `t' test:
+ p 0.05
TABLE 4
(Organ weights - continued)
Print No: 0005
GROUP COMPOUND
:
1
2
:
CONTROL
POSF
Printed: 21-SEP-01
DOSAGE (PPM)
:
0
300
Xybion protocol number: MIN 312
------------------------------------------------------------------------------------------------------------------------------------
MIN 312/014272 : 29 :
SEX:
ABSOLUTE VALUES ------FEMALE------
BODYWEIGHTS ADJUSTED VALUES FEMALE
GROUP: ---1--- ---2---
---1--- ---2---
NUMBER:
5
5
5
5
------------------------------------------------------------------------------------------------------------------------------------
LUNGS & BRONCHI
N
:
5
5
LUNGS & BRONCHI
+++
N
: 5
5
MEAN :
0.891
1.478
MEAN : 0.895
1.474
sd
:
0.070
0.159
------------------------------------------------------------------------------------------------------------------------------------
Level of significance - all comparisons made with Vehicle Control:
Student's `t' test:
+++ p 0.001
TABLE 5
MIN 312/014272 : 30 :
Macroscopic pathology - incidence summary
Print No: 0006
GROUP
:
1
2
Printed: 21-SEP-01
COMPOUND
:
CONTROL
POSF
DOSAGE (PPM)
:
0
300
Xybion protocol number: MIN 312
-------------------------------------------------------------------------------------------------------------------------------------- N U M B E R - O F - A N I M A L S - A F F E C T E D ---
SEX: --MALE-- -FEMALEGROUP: -1- -2- -1- -2-
ORGAN AND KEYWORD(S) OR PHRASE
NUMBER: 5 5 5 5
------------------------------------------------------------------------ -=- -=- -=- -=** TOP OF LIST ** KIDNEYS ............................................... NUMBER EXAMINED: 5 5 5 5
PELVIC DILATION
0 0 1 0
LN MANDIBULAR ......................................... NUMBER EXAMINED: 5 5 5 5
CONGESTED
1 0 0 0
ENLARGED
0 1 0 0
LN MESENTERIC ......................................... NUMBER EXAMINED: 5 5 5 5
ENLARGED
0 0 0 1
SKIN .................................................. NUMBER EXAMINED: 5 5 5 5
SCAB(S)
0 0 3 0
STOMACH ............................................... NUMBER EXAMINED: 5 5 5 5
ANTRUM WHITE NODULE(S)
2 1 1 0
TEETH ................................................. NUMBER EXAMINED: 5 5 5 5
INCISOR(S) PALE
0 0 0 2
UTERUS ................................................ NUMBER EXAMINED: 0 0 5 5
FLUID DISTENTION
0 0 1 1
** END OF LIST **
TABLE 6
MIN 312/014272 : 31 :
Microscopic pathology - incidence summary
Print No: 0011
GROUP COMPOUND DOSAGE (PPM)
:
1
2
:
CONTROL
POSF
:
0
300
Printed: 02-NOV-01
Xybion protocol number: MIN 312 ------------------------------------------------------------------------------------------------------------------------------------
--- N U M B E R - O F - A N I M A L S - A F F E C T E D ---
SEX: --MALE-- -FEMALEGROUP: -1- -2- -1- -2-
ORGAN AND FINDING DESCRIPTION
NUMBER: 5 5 5 5
------------------------------------------------------------------------ -=- -=- -=- -=-
** TOP OF LIST **
KIDNEYS ............................................... NUMBER EXAMINED: 5 5 5 5
--CORTICAL TUBULAR BASOPHILIA
3 3 3 3
--DILATED TUBULE WITH EPITHELIAL HYPERPLASIA
0 1 0 1
--CORTICAL TUBULES WITH HYALINE DROPLETS --CORTICAL INFLAMMATORY CELL INFILTRATE --CORTICAL MINERALISATION --HYPERPLASIA, PELVIC EPITHELIUM
2 2 0 0 1 2 0 1 0 1 0 0 0 0 1 0
--PELVIC DILATATION
0 0 1 0
URINARY BLADDER ....................................... NUMBER EXAMINED: 5 5 5 5
--REFLUXED SEMINAL COLLOID PLUG ** END OF LIST **
1 3 0 0
APPENDIX 1 Daily dose observations - individual findings
MIN 312/014272
Group
1M (Control)
Sign No. Wet fur
Red staining around eyes
Brown staining head
2M (POSF)
Brown staining muzzle Wet fur
Red staining around eyes Brown staining head
Brown staining muzzle
Only animals showing signs are presented
sign present
Animal
Day
No. -3 -2 -1 1 2 3 4 5
1
2
3
4
5
1
2
3
4
5
1
2
3
5
5
6 7 8 9 10
7
9
6
7
8
6
7
: 32 :
APPENDIX 1 (Daily dose observations - continued)
MIN 312/014272
Group 1F
(Control)
Sign No. Wet fur
Red staining around eyes
Brown staining head
2F (POSF)
Brown staining muzzle Wet fur
Brown staining head
Animal
Day
No. -3 -2 -1 1 2 3 4 5
11
12
13
14
15
11
12
13
14
15
11
12
13
14
15
11
16
17
18
19
20
17
19
20
Brown staining muzzle
17
18
20
Only animals showing signs are presented
sign present
: 33 :
APPENDIX 2
MIN 312/014272 : 34 :
Bodyweights - individual values (g)
Print No: 0007
GROUP
:
1
2
Printed: 08-OCT-01
COMPOUND
:
CONTROL
POSF
DOSAGE (PPM)
:
0
300
Xybion protocol number: MIN 312
------------------------------------------------------------------------------------------------------------------------------------
GROUP
DAY
DAY
DAY
DAY
DAY
DAY
DAY
DAY
DAY
DAY
DAY
DAY
DAY
ANIMAL -7
-6
-5
-4
-3
-2
-1
0
1
2
3
4
5
------------------------------------------------------------------------------------------------------------------------------------
1M
1
210
228
237
245
251
254
264
263
265
270
275
281
282
2
194
208
215
215
222
226
233
233
235
241
246
252
254
3
195
206
217
223
233
237
247
248
251
262
263
273
276
4
204
209
220
227
240
248
258
261
265
270
271
279
284
5
206
221
228
236
245
256
261
264
269
276
275
282
283
2M
6
200
212
215
222
233
235
243
240
246
233
221
232
240
7
190
203
213
213
217
220
228
228
236
241
233
241
244
8
207
222
232
240
248
255
260
264
263
262
259
268
269
9
196
208
210
219
228
231
238
243
246
241
240
249
250
10
197
210
220
227
236
240
246
252
252
250
252
259
265
1F 11
152
152
155
159
163
161
169
167
169
166
170
172
171
12
152
159
164
168
172
175
182
182
184
188
191
192
190
13
143
151
156
158
160
164
169
169
170
172
172
177
178
14
140
149
160
162
165
165
170
172
173
170
174
174
175
15
154
156
160
162
170
170
173
176
177
181
181
184
185
2F 16
151
158
163
169
175
177
179
182
179
185
185
183
188
17
153
159
165
168
171
174
180
183
181
178
186
190
189
18
153
162
163
170
174
177
181
185
186
184
184
190
188
19
147
153
153
159
161
164
166
166
167
170
170
173
172
20
142
148
153
154
159
160
165
160
165
167
163
168
165
APPENDIX 3
MIN 312/014272 : 35 :
Absolute organ weights - individual values (g)
Print No: 0010
GROUP
:
1
2
Printed: 08-OCT-01
COMPOUND DOSAGE (PPM)
:
CONTROL
POSF
:
0
300
Xybion protocol number: MIN 312
------------------------------------------------------------------------------------------------------------------------------------
TERMINAL
GROUP ANIMAL BODY WT (g) ADRENALS
HEART
KIDNEYS
LIVER LUNGS & BR
------------------------------------------------------------------------------------------------------------------------------------
1M
1
2
3
281.1 249.8 273.6
0.052 0.045 0.047
1.250 1.012 1.206
2.23 1.94 2.02
13.56 11.29 12.78
1.161 0.908 1.161
4
285.5
0.057
1.263
2.12
13.87
1.246
5
281.5
0.051
1.133
1.88
13.06
1.082
------------------------------------------------------------------------------------------------------------------------------------
2M
6
239.0
0.051
0.973
1.90
11.66
1.600
7
244.1
0.048
1.392
1.90
11.96
2.095
8
268.7
0.058
1.019
2.05
12.81
1.900
9
247.2
0.064
0.963
1.85
13.26
1.901
10
262.7
0.049
0.963
1.99
13.56
1.645
------------------------------------------------------------------------------------------------------------------------------------
APPENDIX 3
MIN 312/014272 : 36 :
(Absolute organ weights - continued)
Print No: 0010
GROUP COMPOUND DOSAGE (PPM)
:
1
2
:
CONTROL
POSF
:
0
300
Printed: 08-OCT-01
Xybion protocol number: MIN 312 ------------------------------------------------------------------------------------------------------------------------------------
TERMINAL
GROUP ANIMAL BODY WT (g) ADRENALS
HEART
KIDNEYS
LIVER LUNGS & BR
------------------------------------------------------------------------------------------------------------------------------------
1F
11
12
169.5 189.3
0.050 0.059
0.798 1.008
1.33 1.52
7.28 8.96
0.816 1.006
13
176.7
0.052
0.785
1.23
7.10
0.891
14
174.3
0.055
0.769
1.44
8.34
0.869
15
187.9
0.053
0.709
1.31
8.15
0.871
------------------------------------------------------------------------------------------------------------------------------------
2F
16
188.1
0.058
0.994
1.57
9.72
1.455
17
190.5
0.063
0.980
1.55
10.10
1.584
18
189.0
0.050
0.866
1.48
10.45
1.690
19
171.6
0.051
0.845
1.38
7.96
1.343
20
162.8
0.061
0.782
1.24
7.55
1.318
------------------------------------------------------------------------------------------------------------------------------------
APPENDIX 4 Individual pathological findings
MIN 312/014272
The initial examination was undertaken by the study pathologist, the results of which were then subjected to a routine peer review by a second pathologist. The diagnoses reported here represent the consensus opinions of both pathologists.
Study pathologist: Peer review:
Takahito Kambara, B.V.Sc., M.V.Sc., Ph.D., Pathologist Department of Pathology Samuel G. McCormick, M.V.B., M.R.C.V.S., Ph.D., F.R.C.Path., Director of Pathology Department of Pathology
: 37 :
APPENDIX 4
MIN 312/014272 : 38 :
(Individual pathological findings - continued)
Print No: 0012
GROUP COMPOUND DOSAGE (PPM)
:
1
2
:
CONTROL
POSF
:
0
300
Printed: 02-NOV-01
Xybion protocol number: MIN 312
------------------------------------------------------------------------------------------------------------------------------------
ANIMAL NUMBER: 0001
SEX: MALE
DOSE GROUP: 1
SACRIFICE STATUS: SCHEDULED, TERMINAL SACRIFICE
DATE OF DEATH: 18-SEP-01
STUDY DAY OF DEATH: 6
STUDY WEEK OF DEATH: 1
TERMINAL BODY WEIGHT: 281.1 GRAMS
------------------------------------------------------------------------------------------------------------------------------------
PATHOLOGY OBSERVATIONS
NECROPSY
HISTOPATHOLOGY
----------------------------------------------------------------
---------------------------------------------------------------KIDNEYS :
-CORTICAL TUBULAR BASOPHILIA,-MINIMAL, FOCAL
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - ------------------------------------------------------------------------------------------------------------------------------------
*** ANIMAL HAS NO GROSS OBSERVATIONS RECORDED ***
------------------------------------------------------------------------------------------------------------------------------------
APPENDIX 4
MIN 312/014272 : 39 :
(Individual pathological findings - continued)
Print No: 0012
GROUP COMPOUND DOSAGE (PPM)
:
1
2
:
CONTROL
POSF
:
0
300
Printed: 02-NOV-01
Xybion protocol number: MIN 312
------------------------------------------------------------------------------------------------------------------------------------
ANIMAL NUMBER: 0002
SEX: MALE
DOSE GROUP: 1
SACRIFICE STATUS: SCHEDULED, TERMINAL SACRIFICE
DATE OF DEATH: 18-SEP-01
STUDY DAY OF DEATH: 6
STUDY WEEK OF DEATH: 1
TERMINAL BODY WEIGHT: 249.8 GRAMS
------------------------------------------------------------------------------------------------------------------------------------
NECROPSY
P A T H O L O G Y O B S E R V A T I O N S HISTOPATHOLOGY
----------------------------------------------------------------
---------------------------------------------------------------KIDNEYS :
-CORTICAL TUBULAR BASOPHILIA,-MINIMAL, FOCAL
URINARY BLADDER : -REFLUXED SEMINAL COLLOID PLUG,-PRESENT
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - ------------------------------------------------------------------------------------------------------------------------------------
*** ANIMAL HAS NO GROSS OBSERVATIONS RECORDED ***
------------------------------------------------------------------------------------------------------------------------------------
APPENDIX 4
MIN 312/014272 : 40 :
(Individual pathological findings - continued)
Print No: 0012
GROUP COMPOUND
:
1
2
:
CONTROL
POSF
Printed: 02-NOV-01
DOSAGE (PPM)
:
0
300
Xybion protocol number: MIN 312
------------------------------------------------------------------------------------------------------------------------------------
ANIMAL NUMBER: 0003 DATE OF DEATH: 18-SEP-01
SEX: MALE
DOSE GROUP: 1
SACRIFICE STATUS: SCHEDULED, TERMINAL SACRIFICE
STUDY DAY OF DEATH: 6
STUDY WEEK OF DEATH: 1
TERMINAL BODY WEIGHT: 273.6 GRAMS
-----------------------------------------------------------------------------------------------------------------------------------PATHOLOGY OBSERVATIONS
NECROPSY ----------------------------------------------------------------
HISTOPATHOLOGY ---------------------------------------------------------------KIDNEYS :
-CORTICAL TUBULAR BASOPHILIA,-MINIMAL, FOCAL -CORTICAL INFLAMMATORY CELL INFILTRATE,-MINIMAL, FOCAL
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - ------------------------------------------------------------------------------------------------------------------------------------
*** ANIMAL HAS NO GROSS OBSERVATIONS RECORDED ***
------------------------------------------------------------------------------------------------------------------------------------
APPENDIX 4
MIN 312/014272 : 41 :
(Individual pathological findings - continued)
Print No: 0012
GROUP
:
1
2
Printed: 02-NOV-01
COMPOUND DOSAGE (PPM)
:
CONTROL
POSF
:
0
300
Xybion protocol number: MIN 312
------------------------------------------------------------------------------------------------------------------------------------
ANIMAL NUMBER: 0004
SEX: MALE
DOSE GROUP: 1
SACRIFICE STATUS: SCHEDULED, TERMINAL SACRIFICE
DATE OF DEATH: 18-SEP-01
STUDY DAY OF DEATH: 6
STUDY WEEK OF DEATH: 1
TERMINAL BODY WEIGHT: 285.5 GRAMS
------------------------------------------------------------------------------------------------------------------------------------
PATHOLOGY OBSERVATIONS
NECROPSY
HISTOPATHOLOGY
---------------------------------------------------------------- ----------------------------------------------------------------
KIDNEYS : -CORTICAL TUBULES WITH HYALINE DROPLETS,-MINIMAL
LN MANDIBULAR : -CONGESTED
STOMACH : -ANTRUM WHITE NODULE(S);
RIDGE, 1MM.
MUCOSA, ONE, NEAR TO LIMITING
------------------------------------------------------------------------------------------------------------------------------------
APPENDIX 4
MIN 312/014272 : 42 :
(Individual pathological findings - continued)
Print No: 0012
GROUP
:
1
2
Printed: 02-NOV-01
COMPOUND DOSAGE (PPM)
:
CONTROL
POSF
:
0
300
Xybion protocol number: MIN 312
------------------------------------------------------------------------------------------------------------------------------------
ANIMAL NUMBER: 0005
SEX: MALE
DOSE GROUP: 1
SACRIFICE STATUS: SCHEDULED, TERMINAL SACRIFICE
DATE OF DEATH: 18-SEP-01
STUDY DAY OF DEATH: 6
STUDY WEEK OF DEATH: 1
TERMINAL BODY WEIGHT: 281.5 GRAMS
------------------------------------------------------------------------------------------------------------------------------------
PATHOLOGY OBSERVATIONS
NECROPSY
HISTOPATHOLOGY
---------------------------------------------------------------- ----------------------------------------------------------------
KIDNEYS : -CORTICAL TUBULES WITH HYALINE DROPLETS,-MINIMAL
STOMACH : -ANTRUM WHITE NODULE(S); RIDGE, 1MM.
MUCOSA, ONE, NEAR TO LIMITING
------------------------------------------------------------------------------------------------------------------------------------
APPENDIX 4
MIN 312/014272 : 43 :
(Individual pathological findings - continued)
Print No: 0012
GROUP COMPOUND
:
1
2
:
CONTROL
POSF
Printed: 02-NOV-01
DOSAGE (PPM)
:
0
300
Xybion protocol number: MIN 312
------------------------------------------------------------------------------------------------------------------------------------
ANIMAL NUMBER: 0006
SEX: MALE
DOSE GROUP: 2
SACRIFICE STATUS: SCHEDULED, TERMINAL SACRIFICE
DATE OF DEATH: 18-SEP-01
STUDY DAY OF DEATH: 6
STUDY WEEK OF DEATH: 1
TERMINAL BODY WEIGHT: 239.0 GRAMS
------------------------------------------------------------------------------------------------------------------------------------
------------------------------------------------------------------------------------------------------------------------------------
*** ANIMAL HAS NO GROSS OBSERVATIONS RECORDED ***
*** ANIMAL HAS NO MICROSCOPIC FINDINGS RECORDED ***
------------------------------------------------------------------------------------------------------------------------------------
APPENDIX 4
MIN 312/014272 : 44 :
(Individual pathological findings - continued)
Print No: 0012
GROUP COMPOUND DOSAGE (PPM)
:
1
2
:
CONTROL
POSF
:
0
300
Printed: 02-NOV-01
Xybion protocol number: MIN 312
------------------------------------------------------------------------------------------------------------------------------------
ANIMAL NUMBER: 0007
SEX: MALE
DOSE GROUP: 2
SACRIFICE STATUS: SCHEDULED, TERMINAL SACRIFICE
DATE OF DEATH: 18-SEP-01
STUDY DAY OF DEATH: 6
STUDY WEEK OF DEATH: 1
TERMINAL BODY WEIGHT: 244.1 GRAMS
------------------------------------------------------------------------------------------------------------------------------------
NECROPSY
P A T H O L O G Y O B S E R V A T I O N S HISTOPATHOLOGY
----------------------------------------------------------------
---------------------------------------------------------------KIDNEYS :
-CORTICAL TUBULAR BASOPHILIA,-MINIMAL, FOCAL
URINARY BLADDER : -REFLUXED SEMINAL COLLOID PLUG,-PRESENT
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - ------------------------------------------------------------------------------------------------------------------------------------
*** ANIMAL HAS NO GROSS OBSERVATIONS RECORDED ***
------------------------------------------------------------------------------------------------------------------------------------
APPENDIX 4
MIN 312/014272 : 45 :
(Individual pathological findings - continued)
Print No: 0012
GROUP
:
1
2
Printed: 02-NOV-01
COMPOUND DOSAGE (PPM)
:
CONTROL
POSF
:
0
300
Xybion protocol number: MIN 312
------------------------------------------------------------------------------------------------------------------------------------
ANIMAL NUMBER: 0008
SEX: MALE
DOSE GROUP: 2
SACRIFICE STATUS: SCHEDULED, TERMINAL SACRIFICE
DATE OF DEATH: 18-SEP-01
STUDY DAY OF DEATH: 6
STUDY WEEK OF DEATH: 1
TERMINAL BODY WEIGHT: 268.7 GRAMS
-----------------------------------------------------------------------------------------------------------------------------------PATHOLOGY OBSERVATIONS
NECROPSY ----------------------------------------------------------------
HISTOPATHOLOGY ----------------------------------------------------------------
KIDNEYS : -CORTICAL TUBULES WITH HYALINE DROPLETS,-MINIMAL
LN MANDIBULAR : -ENLARGED; RIGHT, ONE.
STOMACH : -ANTRUM WHITE NODULE(S); RIDGE, 1MM.
MUCOSA, ONE, NEAR TO LIMITING
URINARY BLADDER : -REFLUXED SEMINAL COLLOID PLUG,-PRESENT
------------------------------------------------------------------------------------------------------------------------------------
APPENDIX 4
MIN 312/014272 : 46 :
(Individual pathological findings - continued)
Print No: 0012
GROUP
:
1
2
Printed: 02-NOV-01
COMPOUND DOSAGE (PPM)
:
CONTROL
POSF
:
0
300
Xybion protocol number: MIN 312
------------------------------------------------------------------------------------------------------------------------------------
ANIMAL NUMBER: 0009
SEX: MALE
DOSE GROUP: 2
SACRIFICE STATUS: SCHEDULED, TERMINAL SACRIFICE
DATE OF DEATH: 18-SEP-01
STUDY DAY OF DEATH: 6
STUDY WEEK OF DEATH: 1
TERMINAL BODY WEIGHT: 247.2 GRAMS
------------------------------------------------------------------------------------------------------------------------------------
PATHOLOGY OBSERVATIONS
NECROPSY
HISTOPATHOLOGY
---------------------------------------------------------------- ----------------------------------------------------------------
KIDNEYS : -CORTICAL TUBULAR BASOPHILIA,-MINIMAL, FOCAL -CORTICAL INFLAMMATORY CELL INFILTRATE,-MINIMAL, FOCAL
-CORTICAL MINERALISATION,-MINIMAL, FOCAL
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - ------------------------------------------------------------------------------------------------------------------------------------
*** ANIMAL HAS NO GROSS OBSERVATIONS RECORDED ***
------------------------------------------------------------------------------------------------------------------------------------
APPENDIX 4
MIN 312/014272 : 47 :
(Individual pathological findings - continued)
Print No: 0012
GROUP COMPOUND DOSAGE (PPM)
:
1
2
:
CONTROL
POSF
:
0
300
Printed: 02-NOV-01 Xybion protocol number: MIN 312
------------------------------------------------------------------------------------------------------------------------------------
ANIMAL NUMBER: 0010
SEX: MALE
DOSE GROUP: 2
SACRIFICE STATUS: SCHEDULED, TERMINAL SACRIFICE
DATE OF DEATH: 18-SEP-01
STUDY DAY OF DEATH: 6
STUDY WEEK OF DEATH: 1
TERMINAL BODY WEIGHT: 262.7 GRAMS
------------------------------------------------------------------------------------------------------------------------------------
PATHOLOGY OBSERVATIONS
NECROPSY
HISTOPATHOLOGY
---------------------------------------------------------------- ----------------------------------------------------------------
KIDNEYS : -CORTICAL TUBULAR BASOPHILIA,-MINIMAL, FOCAL -DILATED TUBULE WITH EPITHELIAL HYPERPLASIA,-MINIMAL, FOCAL
-CORTICAL TUBULES WITH HYALINE DROPLETS,-MINIMAL -CORTICAL INFLAMMATORY CELL INFILTRATE,-MINIMAL, FOCAL >NOTE:>FIBROSIS ALSO SEEN IN THE LESION OF TUBULAR
HYPERPLASIA
URINARY BLADDER : -REFLUXED SEMINAL COLLOID PLUG,-PRESENT
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - ------------------------------------------------------------------------------------------------------------------------------------
*** ANIMAL HAS NO GROSS OBSERVATIONS RECORDED ***
------------------------------------------------------------------------------------------------------------------------------------
APPENDIX 4
MIN 312/014272 : 48 :
(Individual pathological findings - continued)
Print No: 0012
GROUP COMPOUND DOSAGE (PPM)
:
1
2
:
CONTROL
POSF
:
0
300
Printed: 02-NOV-01
Xybion protocol number: MIN 312
------------------------------------------------------------------------------------------------------------------------------------
ANIMAL NUMBER: 0011
SEX: FEMALE
DOSE GROUP: 1
SACRIFICE STATUS: SCHEDULED, TERMINAL SACRIFICE
DATE OF DEATH: 18-SEP-01
STUDY DAY OF DEATH: 6
STUDY WEEK OF DEATH: 1
TERMINAL BODY WEIGHT: 169.5 GRAMS
------------------------------------------------------------------------------------------------------------------------------------
PATHOLOGY OBSERVATIONS
NECROPSY
HISTOPATHOLOGY
---------------------------------------------------------------UTERUS :
-FLUID DISTENTION, MODERATE
----------------------------------------------------------------
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - ------------------------------------------------------------------------------------------------------------------------------------
*** ANIMAL HAS NO MICROSCOPIC FINDINGS RECORDED ***
------------------------------------------------------------------------------------------------------------------------------------
APPENDIX 4
MIN 312/014272 : 49 :
(Individual pathological findings - continued)
Print No: 0012
GROUP COMPOUND
:
1
2
:
CONTROL
POSF
Printed: 02-NOV-01
DOSAGE (PPM)
:
0
300
Xybion protocol number: MIN 312
------------------------------------------------------------------------------------------------------------------------------------
ANIMAL NUMBER: 0012
SEX: FEMALE
DOSE GROUP: 1
SACRIFICE STATUS: SCHEDULED, TERMINAL SACRIFICE
DATE OF DEATH: 18-SEP-01
STUDY DAY OF DEATH: 6
STUDY WEEK OF DEATH: 1
TERMINAL BODY WEIGHT: 189.3 GRAMS
------------------------------------------------------------------------------------------------------------------------------------
PATHOLOGY OBSERVATIONS
NECROPSY ---------------------------------------------------------------SKIN :
HISTOPATHOLOGY ----------------------------------------------------------------
-SCAB(S); TAIL, A FEW, 1MM.
STOMACH : -ANTRUM WHITE NODULE(S); RIDGE, 1MM.
MUCOSA, ONE, NEAR TO LIMITING
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - ------------------------------------------------------------------------------------------------------------------------------------
*** ANIMAL HAS NO MICROSCOPIC FINDINGS RECORDED ***
------------------------------------------------------------------------------------------------------------------------------------
APPENDIX 4
MIN 312/014272 : 50 :
(Individual pathological findings - continued)
Print No: 0012
GROUP COMPOUND
:
1
2
:
CONTROL
POSF
Printed: 02-NOV-01
DOSAGE (PPM)
:
0
300
Xybion protocol number: MIN 312
------------------------------------------------------------------------------------------------------------------------------------
ANIMAL NUMBER: 0013
SEX: FEMALE
DOSE GROUP: 1
SACRIFICE STATUS: SCHEDULED, TERMINAL SACRIFICE
DATE OF DEATH: 18-SEP-01
STUDY DAY OF DEATH: 6
STUDY WEEK OF DEATH: 1
TERMINAL BODY WEIGHT: 176.7 GRAMS
------------------------------------------------------------------------------------------------------------------------------------
PATHOLOGY OBSERVATIONS
NECROPSY ----------------------------------------------------------------
HISTOPATHOLOGY ---------------------------------------------------------------KIDNEYS :
-CORTICAL TUBULAR BASOPHILIA,-MINIMAL, FOCAL
SKIN : -SCAB(S); TAIL, MULTIPLE, 1MM.
------------------------------------------------------------------------------------------------------------------------------------
APPENDIX 4
MIN 312/014272 : 51 :
(Individual pathological findings - continued)
Print No: 0012
GROUP COMPOUND DOSAGE (PPM)
:
1
2
:
CONTROL
POSF
:
0
300
Printed: 02-NOV-01
Xybion protocol number: MIN 312 ------------------------------------------------------------------------------------------------------------------------------------
ANIMAL NUMBER: 0014 DATE OF DEATH: 18-SEP-01
SEX: FEMALE
DOSE GROUP: 1
SACRIFICE STATUS: SCHEDULED, TERMINAL SACRIFICE
STUDY DAY OF DEATH: 6
STUDY WEEK OF DEATH: 1
TERMINAL BODY WEIGHT: 174.3 GRAMS
------------------------------------------------------------------------------------------------------------------------------------
PATHOLOGY OBSERVATIONS
NECROPSY
HISTOPATHOLOGY
---------------------------------------------------------------KIDNEYS :
-PELVIC DILATION, MINIMAL; RIGHT.
---------------------------------------------------------------KIDNEYS :
-CORTICAL TUBULAR BASOPHILIA,-MINIMAL, FOCAL -HYPERPLASIA, PELVIC EPITHELIUM,-MINIMAL, FOCAL
-PELVIC DILATATION,-SLIGHT
------------------------------------------------------------------------------------------------------------------------------------
APPENDIX 4
MIN 312/014272 : 52 :
(Individual pathological findings - continued)
Print No: 0012
GROUP COMPOUND
:
1
2
:
CONTROL
POSF
Printed: 02-NOV-01
DOSAGE (PPM)
:
0
300
Xybion protocol number: MIN 312
------------------------------------------------------------------------------------------------------------------------------------
ANIMAL NUMBER: 0015
SEX: FEMALE
DOSE GROUP: 1
SACRIFICE STATUS: SCHEDULED, TERMINAL SACRIFICE
DATE OF DEATH: 18-SEP-01
STUDY DAY OF DEATH: 6
STUDY WEEK OF DEATH: 1
TERMINAL BODY WEIGHT: 187.9 GRAMS
------------------------------------------------------------------------------------------------------------------------------------
PATHOLOGY OBSERVATIONS
NECROPSY ----------------------------------------------------------------
HISTOPATHOLOGY ---------------------------------------------------------------KIDNEYS :
-CORTICAL TUBULAR BASOPHILIA,-MINIMAL, FOCAL
SKIN : -SCAB(S); TAIL, A FEW, 1MM.
------------------------------------------------------------------------------------------------------------------------------------
APPENDIX 4
MIN 312/014272 : 53 :
(Individual pathological findings - continued)
Print No: 0012
GROUP COMPOUND
:
1
2
:
CONTROL
POSF
Printed: 02-NOV-01
DOSAGE (PPM)
:
0
300
Xybion protocol number: MIN 312
------------------------------------------------------------------------------------------------------------------------------------
ANIMAL NUMBER: 0016
SEX: FEMALE
DOSE GROUP: 2
SACRIFICE STATUS: SCHEDULED, TERMINAL SACRIFICE
DATE OF DEATH: 18-SEP-01
STUDY DAY OF DEATH: 6
STUDY WEEK OF DEATH: 1
TERMINAL BODY WEIGHT: 188.1 GRAMS
------------------------------------------------------------------------------------------------------------------------------------
------------------------------------------------------------------------------------------------------------------------------------
*** ANIMAL HAS NO GROSS OBSERVATIONS RECORDED ***
*** ANIMAL HAS NO MICROSCOPIC FINDINGS RECORDED ***
------------------------------------------------------------------------------------------------------------------------------------
APPENDIX 4
MIN 312/014272 : 54 :
(Individual pathological findings - continued)
Print No: 0012
GROUP COMPOUND DOSAGE (PPM)
:
1
2
:
CONTROL
POSF
:
0
300
Printed: 02-NOV-01
Xybion protocol number: MIN 312
------------------------------------------------------------------------------------------------------------------------------------
ANIMAL NUMBER: 0017
SEX: FEMALE
DOSE GROUP: 2
SACRIFICE STATUS: SCHEDULED, TERMINAL SACRIFICE
DATE OF DEATH: 18-SEP-01
STUDY DAY OF DEATH: 6
STUDY WEEK OF DEATH: 1
TERMINAL BODY WEIGHT: 190.5 GRAMS
------------------------------------------------------------------------------------------------------------------------------------
PATHOLOGY OBSERVATIONS
NECROPSY
HISTOPATHOLOGY
----------------------------------------------------------------
---------------------------------------------------------------KIDNEYS :
-CORTICAL TUBULAR BASOPHILIA,-MINIMAL, FOCAL
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - ------------------------------------------------------------------------------------------------------------------------------------
*** ANIMAL HAS NO GROSS OBSERVATIONS RECORDED ***
------------------------------------------------------------------------------------------------------------------------------------
APPENDIX 4
MIN 312/014272 : 55 :
(Individual pathological findings - continued)
Print No: 0012
GROUP COMPOUND DOSAGE (PPM)
:
1
2
:
CONTROL
POSF
:
0
300
Printed: 02-NOV-01
Xybion protocol number: MIN 312
------------------------------------------------------------------------------------------------------------------------------------
ANIMAL NUMBER: 0018
SEX: FEMALE
DOSE GROUP: 2
SACRIFICE STATUS: SCHEDULED, TERMINAL SACRIFICE
DATE OF DEATH: 18-SEP-01
STUDY DAY OF DEATH: 6
STUDY WEEK OF DEATH: 1
TERMINAL BODY WEIGHT: 189.0 GRAMS
------------------------------------------------------------------------------------------------------------------------------------
PATHOLOGY OBSERVATIONS
NECROPSY
HISTOPATHOLOGY
---------------------------------------------------------------TEETH :
-INCISOR(S) PALE; LOWER, RIGHT.
----------------------------------------------------------------
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - ------------------------------------------------------------------------------------------------------------------------------------
*** ANIMAL HAS NO MICROSCOPIC FINDINGS RECORDED ***
------------------------------------------------------------------------------------------------------------------------------------
APPENDIX 4
MIN 312/014272 : 56 :
(Individual pathological findings - continued)
Print No: 0012
GROUP
:
1
2
Printed: 02-NOV-01
COMPOUND DOSAGE (PPM)
:
CONTROL
POSF
:
0
300
Xybion protocol number: MIN 312
------------------------------------------------------------------------------------------------------------------------------------
ANIMAL NUMBER: 0019
SEX: FEMALE
DOSE GROUP: 2
SACRIFICE STATUS: SCHEDULED, TERMINAL SACRIFICE
DATE OF DEATH: 18-SEP-01
STUDY DAY OF DEATH: 6
STUDY WEEK OF DEATH: 1
TERMINAL BODY WEIGHT: 171.6 GRAMS
------------------------------------------------------------------------------------------------------------------------------------
PATHOLOGY OBSERVATIONS
NECROPSY
HISTOPATHOLOGY
---------------------------------------------------------------- ----------------------------------------------------------------
KIDNEYS : -CORTICAL TUBULAR BASOPHILIA,-MINIMAL, FOCAL -DILATED TUBULE WITH EPITHELIAL HYPERPLASIA,-MINIMAL, FOCAL
-CORTICAL INFLAMMATORY CELL INFILTRATE,-MINIMAL, FOCAL >NOTE:>FIBROSIS ALSO SEEN IN THE LESION OF TUBULAR
HYPERPLASIA
TEETH : -INCISOR(S) PALE; LOWER.
------------------------------------------------------------------------------------------------------------------------------------
APPENDIX 4
MIN 312/014272 : 57 :
(Individual pathological findings - continued)
Print No: 0012
GROUP
:
1
2
Printed: 02-NOV-01
COMPOUND DOSAGE (PPM)
:
CONTROL
POSF
:
0
300
Xybion protocol number: MIN 312
------------------------------------------------------------------------------------------------------------------------------------
ANIMAL NUMBER: 0020
SEX: FEMALE
DOSE GROUP: 2
SACRIFICE STATUS: SCHEDULED, TERMINAL SACRIFICE
DATE OF DEATH: 18-SEP-01
STUDY DAY OF DEATH: 6
STUDY WEEK OF DEATH: 1
TERMINAL BODY WEIGHT: 162.8 GRAMS
------------------------------------------------------------------------------------------------------------------------------------
PATHOLOGY OBSERVATIONS
NECROPSY
HISTOPATHOLOGY
---------------------------------------------------------------- ----------------------------------------------------------------
KIDNEYS : -CORTICAL TUBULAR BASOPHILIA,-MINIMAL, FOCAL
LN MESENTERIC : -ENLARGED, MINIMAL; ONE.
UTERUS : -FLUID DISTENTION, MODERATE
------------------------------------------------------------------------------------------------------------------------------------
APPENDIX 5 pH values for terminal urine - individual values
MIN 312/014272
Group
Males
Females
Animal
pH
Animal
pH
number
number
1
1
NS
6
INS
(Control)
2
8.7
7 a
8.6
3
8.6
8 a
8.8
4
9.1
9
8.9
5
9.3
10
INS
2
11a
9.0
16 a
8.7
(POSF)
12 a
8.9
17
7.4
13
8.3
18 a
9.1
14 a
8.1
19
6.9
15
8.4
20 a
8.8
a
Samples obtained in animal room following procedure
provided by Sponsor, remaining samples obtained immediately
prior to or during post mortem examination
INS Sample volume < 8 l required to obtain accurate pH reading
NS No sample obtained
: 58 :
APPENDIX 6
MIN 312/014272 : 59 :
PCNA staining in the urinary bladder - individual and mean values
Level 1
Animal Positive Negative Total C.P.I.*
No
1
14
644 658 2.1
2
63
937 1000 6.3
3
18
982 1000 1.8
4
5
995 1000 0.5
5
6
994 1000 0.6
mean
2.3
6
4
996 1000 0.4
7
65
935 1000 6.5
8
47
953 1000 4.7
9
17
983 1000 1.7
10
98
902 1000 9.8
mean
4.6
11
3
997 1000 0.3
12
9
991 1000 0.9
13
7
904 911 0.8
14
6
994 1000 0.6
15
2
998 1000 0.2
mean
0.6
16
1
999 1000 0.1
17
1
999 1000 0.1
18
6
994 1000 0.6
19
11
475 486 2.3
20
6
863 869 0.7
mean
0.8
*: cell proliferation index (%positive cells)
Positive 15 53 20 5 3 11 77 49 15 83 4 16 6 5 7 1 0 2 4 2
Level 2 Negative Total
837 852 947 1000 980 1000 995 1000 997 1000 989 1000 923 1000 951 1000 985 1000 917 1000 996 1000 984 1000 994 1000 995 1000 993 1000 999 1000 926 926 998 1000 382 386 696 698
C.P.I.* 1.8 5.3 2.0 0.5 0.3 2.0 1.1 7.7 4.9 1.5 8.3 4.7 0.4 1.6 0.6 0.5 0.7 0.8 0.1 0.0 0.2 1.0 0.3 0.3
Positive 6 37 16 4 5 6 31 27 7 41 2 9 1 1 1 3 1 4 4 4
Level 3 Negative Total
248 254 870 907 984 1000 996 1000 995 1000 994 1000 969 1000 973 1000 993 1000 959 1000 998 1000 991 1000 864 865 999 1000 999 1000 997 1000 999 1000 763 767 416 420 557 561
C.P.I.* 2.4 4.1 1.6 0.4 0.5 1.8 0.6 3.1 2.7 0.7 4.1 2.2 0.2 0.9 0.1 0.1 0.1 0.3 0.3 0.1 0.5 1.0 0.7 0.5
Combined Levels Positive Negative Total
11.7 576.3 588.0 51.0 918.0 969.0 18.0 982.0 1000.0 4.7 995.3 1000.0 4.7 995.3 1000.0 7.0 993.0 1000.0 57.7 942.3 1000.0 41.0 959.0 1000.0 13.0 987.0 1000.0 74.0 926.0 1000.0 3.0 997.0 1000.0 11.3 988.7 1000.0 4.7 920.7 925.3 4.0 996.0 1000.0 3.3 996.7 1000.0 1.7 998.3 1000.0 0.7 974.7 975.3 4.0 918.3 922.3 6.3 424.3 430.7 4.0 705.3 709.3
C.P.I.* 2.1 5.2 1.8 0.5 0.5 2.0 0.7 5.8 4.1 1.3 7.4 3.9 0.3 1.1 0.5 0.4 0.3 0.5 0.2 0.1 0.4 1.4 0.6 0.5
Mean cell proliferation index (%positive cells)
Group Sex Mean SD CV(%)
1
Male
2.0
1.95
97.1
2
3.9
2.86
74.2
CV (%) Coefficient of variation (sd 100 / mean)
Group 1 2
Sex Female
Mean 0.5 0.5
SD CV(%)
0.34
63.9
0.53
99.8
MIN 312/014272
ADMINISTRATION OF POSF BY INHALATION TO RATS
Author Simon Moore
: 60 :
Administration of POSF by inhalation to rats
CONTENTS
MIN 312/014272
Page
TEST SUBSTANCE AND ADMINISTRATION
Test substance .................................................................................................................................. 62 Administration.................................................................................................................................. 62 Test atmosphere generation.............................................................................................................. 62 Exposure chambers .......................................................................................................................... 63 Procedure.......................................................................................................................................... 64 Aerosol analysis ............................................................................................................................... 65 Chamber monitoring system ............................................................................................................ 65 Target concentrations ....................................................................................................................... 66 Exposure chamber conditions .......................................................................................................... 66
RESULTS Vapour concentration ....................................................................................................................... 68 Discussion ........................................................................................................................................ 69 Calculations...................................................................................................................................... 70
FIGURES A. Schematic of a rodent inhalation dosing system................................................................... 71 B. Schematic of a Fourier Transform Infrared Spectrophotometer.......................................... 72
TABLES A. Operating conditions of the inhalation exposure system..................................................... 73 B. Chamber concentrations of POSF (ppm) - daily mean values ............................................ 74 C. Nominal concentrations of POSF(ppm) - individual exposure values ................................ 75 D. Chamber temperature - exposure mean values.................................................................... 76
APPENDICES A. Methods of sample collection and analysis for POSF......................................................... 77 B. Individual POSF concentration measurements.................................................................... 85
: 61 :
Administration of POSF by inhalation to rats
TEST SUBSTANCE AND ADMINISTRATION
MIN 312/014272
TEST SUBSTANCE The test substance, POSF, is a liquid with boiling point of 154C. A consignment of the Test Article (4 x 20 kg, Lot number 040227), was received from the Sponsor on 14 June 2001. The test substance was stored securely in the original containers at room temperature until it was transferred to the atmosphere generation system. The stated purity was >95.5%. Information regarding the purity and stability of the test substance is the responsibility of the Sponsor.
ADMINISTRATION The test material was administered to the rats by snout-only exposure chambers described below: The chamber atmosphere was produced by metering the liquid test substance into a glass vapour generator through which dried air was passed at a flow rate of 29 l/minute for administration to the rats, by inhalation from snout only exposure chambers. The target chamber concentration was achieved by metering the test substance from polypropylene syringes mounted on a syringe driver. This atmosphere gave the final chamber concentration of POSF. The setting of the test substance metering system required to obtain the target chamber concentration was determined during preliminary generation trials without animals present and based on the Fourier Transform Infrared (FT-IR) analysis of chamber atmosphere samples. Minor adjustments were made to the test material delivery rates in order to maintain the chamber concentration close to target. Animals assigned to Group 1 (Air control) received an exposure to air only, from the same compressed air source as used for the generation of the test atmospheres. The duration of administration was a single 6-hour exposure, daily, for 5 days. The usage of POSF was determined, for each day of treatment, for the lone test group.
TEST ATMOSPHERE GENERATION The vapour delivery system for the dose group comprised a polypropylene syringe located on a syringe driver (Precidor, Model 5003), which delivered the liquid test material to a glass frit contained in a glass vessel via PolyTetraFluoroEthylene (PTFE) tubing. The syringe size and syringe driver settings required to achieve the target concentrations were established during the preliminary phase of the study. Air was passed through the vapouriser at a rate of 29 l/minute. The vapour/air mixture passed out of the vapouriser into the chamber inlet ducting through a 22 mm diameter flexible pipe. All equipment was housed in an extracted cabinet. The air control group received clean air only at a rate of 29 l/minute.
: 62 :
Administration of POSF by inhalation to rats
EXPOSURE CHAMBERS (Figure A)
MIN 312/014272
The inhalation exposure system comprised a snout-only inhalation exposure chamber and rats restraining tubes. Accessories included air supply and extract lines, which attached to the top and bottom of the chamber respectively. A filtration system was incorporated into the extract line.
A schematic diagram of an exposure system is shown in Figure A. The component parts of the system are described in further detail below:
Inhalation chamber
ADG snout-only inhalation chamber (ADG Developments Ltd, Hitchin, Hertfordshire, England). This is a modular apparatus of aluminium alloy construction comprising of a base unit, a variable number of animal exposure sections, each having 20 exposure ports, and a top section incorporating a central aerosol inlet surrounded by a tangential air inlet.
The chambers used on this study were assembled using 3 rodent exposure sections, identified as levels 1 (top) to 3 (bottom). All exposure sections had 20 ports and formed a 28 cm diameter cylinder with a volume of approximately 47 litres. During dosing each chamber was housed in an enclosed ventilated cabinet.
Rats restraining tubes
Moulded polycarbonate tubes tapered at one end to allow the snout only to project from the tapered end. The other end is normally closed by insertion of an expanded plastic bung. A push rod passes through the centre of the bung and is adjustable to maintain the position of the rats during restraint. Tubes are attached to a chamber by means of push-fit "O" ring seals located in the exposure ports of the animal exposure sections. The restraining tubes were attached to chamber level two.
Chamber level two was used to expose the animals. All exposure ports not in use were sealed with an expanded plastic bung.
Air supply and extract
Air supplies were provided by a compressor. The air was filtered to remove any residual particulate and was dried (dew point ~2C). A 1 l/minute differential was maintained between the inlet and outlet airflows to provide a small negative pressure within the exposure system. The in-line flowmeters were calibrated daily against high quality tapered tube rotameters measuring the free flow of air at points of attachment of the supply and extract lines to each chamber. The airflows used for each group are detailed in Table A.
The calibrated exhaust airflow of each exposure system was passed through a trapping system comprising a Fluosorber carbon vapour filter and a silica gel column before it was passed to atmosphere. The exhaust flows used for each group are detailed in Table A.
: 63 :
Administration of POSF by inhalation to rats
PROCEDURE
MIN 312/014272
Two exposure systems were used, one for each group. The procedure followed for each group was similar except that, for the control group, the rats in the control group received air only. Therefore in the following description the comments relating to the sampling and the Fourier Transform Infrared spectrophotometer apply to the test group only.
The FT-IR data capture programme was initialised and the path difference was inspected to ensure the value was zero, subsequently, a background scan was recorded and the cell path length was calibrated.
A syringe was filled with the test substance, the weight of liquid was recorded and the syringe was mounted on the syringe driver. The syringe was connected to the vapouriser by PTFE tubing. The pump was turned on briefly in order to engage the gearing and to move the liquid along the PTFE tubing almost to the surface of the glass frit. The injection rate to be used was then set.
The rats were removed from their cages and placed into restraining tubes, which were then attached to their assigned level of the chamber. Unused exposure ports were sealed with blanking plugs. The rats were removed from their cages and placed into restraining tubes, which were colour coded for the treatment group and numbered for each animal. The tubes were then attached to level 2 of the chamber utilising 5 ports on either side of the 20 port section. Unused exposure ports were sealed with blanking plugs.
The inlet and outlet airflows of the exposure system were calibrated using precision made tapered glass tube flowmeters.
The connections of the generation and extract systems to the chamber were checked to ensure they were correct and working.
The barometric pressure was recorded manually.
Generation commenced as the syringe driver was turned on and 15 minutes into the exposure, the chamber sampling system was activated (see below). At intervals of 30 minutes, any reactions by the rats to exposure, together with checks of generation and chamber operational parameters including temperature and inlet and outlet air flowrate were manually recorded. The airflows were measured throughout the exposures using in-line flowmeters.
A sample pump continually delivered the chamber atmosphere from the test chamber to the FT-IR system at a flow rate of 1 l/minute. Analysis for the determination of POSF concentration occurred at exactly 30 mimute intervals throughout each exposure. The chamber atmosphere was returned to the chamber downstream of the sampling point.
At the end of six hours generation, the syringe driver was turned off and the weight of the remaining syringe contents was recorded. Generation airflow was turned off and the chamber extract allowed to clear the vapour for 5 - 10 minutes (equilibration time t99 is 7 minutes). The data capture program halted automatically after collecting a specific number of samples.
At the end of this time, the rats were unloaded from the chambers and returned to their respective holding cages.
The chambers were washed with hot water.
The IR cell path length was retested to ensure that the initial value prior to the exposure was consistent throughout the exposure.
: 64 :
Administration of POSF by inhalation to rats
ANALYSIS OF THE TEST ATMOSPHERE (FIGURE B)
MIN 312/014272
The concentration of POSF in air within the test inhalation chamber was measured using a Fourier Transform Infrared (FT-IR) Spectrophotometer. Definitive details of the FT-IR, its standardisation and validation are given in Appendix A.
The Spectrophotometer was located adjacent to the exposure chambers.
CHAMBER MONITORING SYSTEM A PC running the AutoQuant 3.01 software was used to monitor and record the system performance during each exposure. The data collection sequence and display were controlled by a personal computer (PC) and all information collected was displayed on a monitor. Simultaneously, the data was stored electronically. This program was composed of three basic stages of operation: an initial setting up (pre-exposure) phase, an exposure monitoring phase and the post exposure data collation and presentation phase. The program and FTIR hardware were loaned by the Sponsor for the duration of the study. Raw data printed as a hard copy. Setting-up phase In the initial phase, prompted by the program screen display, the instrument response was checked, followed by a background scan. An ultra pure nitrogen cylinder was used for this purpose. The pathlength was then calibrated using a certified cylinder of ethylene. A regulated flowrate of 2 l/minute was used for the nitrogen and ethylene cylinders, the analyser drew 1 l/minute of the gas stream and the remainder was vented to waste. The flow meters used in conjunction with the cylinders were monitored using a calibrated in-line tapered tube gas flowmeter. The study details including the daily pathlength, number of scans and samples and frequency of samples are entered and stored into the data capture program prior to the exposure. Exposure monitoring phase This phase was started 15 minutes later than the start of atmosphere generation. The test chamber's environment was monitored during a 30-minute cycle when the analysed concentration was recorded. The data were displayed on screen, printed and stored on the local hard drive of the computer and an external zip disc. A total of twelve sample points were recorded, each of which represented 32 coadded scans.
: 65 :
Administration of POSF by inhalation to rats
Post exposure phase
MIN 312/014272
At the end of the exposure, the data collected during exposure was collated and printed. The mean values, together with standard deviation were calculated for each parameter recorded. The first set of chamber concentration data was included in the calculation of the mean and standard deviation because chamber conditions stabilised within the 15 minutes from the start of exposure (equilibration time, t99 was 7 minutes) before the analysis was started.
Midac Grams 32 version 4.11 software was solely used to generate a hard copy of the infrared data.
The IR cell path length calibration of the FT-IR was reassessed to ensure that the initial value prior to the exposure was consistent throughout the exposure.
Details of the analytical methodologies used are given in Appendix A.
TARGET CONCENTRATIONS
The target concentrations of POSF was:
Group 2
Designation Test Group
Concentration (ppm) 300
The target concentrations were selected in consultation with the Sponsor, following the review of available data.
EXPOSURE CHAMBER CONDITIONS Chamber analysed concentration of POSF The test atmosphere was sampled from one point within the test atmosphere chamber. This was continually drawn through a transfer line, which was therefore in equilibrium with the mean concentration from the test chamber. The chamber atmosphere was returned to the exposure chamber downstream of the sampling point. Every 30 minutes, the software for automated analysis and data logging activated the FT-IR. The methodology is presented in Appendix A. Chamber spatial distribution All the animals and the analytical sampling point were situated on level two of the three level exposure chamber.
: 66 :
Administration of POSF by inhalation to rats
Nominal concentration of chamber atmospheres
MIN 312/014272
The chamber nominal concentrations were calculated from the amount of liquid used over the six-hour exposure period, the mass of the liquid and the exposure mean airflow. The formulae used were as follows:
Concentration = V 1,000,000 ppm
(1)
Va + V
V = W R T 760 mm Hg
(2)
M
Atm
where
V
=
W =
M =
R
=
T
=
Atm =
Va
=
gaseous volume of POSF (L) mass of POSF (g) molecular weight of POSF (502.14 g/mole) Gas constant (0.08205 L atm mmol-1 K-1) temperature (K) atmospheric pressure (mmHg) volume of air (L)
Airflow and temperature These parameters were recorded manually, as described above under the Test Atmosphere generation and Procedure sections.
: 67 :
Administration of POSF by inhalation to rats
RESULTS
MIN 312/014272
VAPOUR CONCENTRATION
Analysed concentration of POSF
The data are presented as follows:
Daily mean values Individual values
Table B Appendix B
The study mean concentration (the mean of daily mean values) for each group exposed to POSF are presented below:
Group 2 (Test Group)
Chamber concentration (ppm)
Target
Analysed
300
317
Analysed concentration was in good agreement with target concentration. The coefficient of variation of the daily mean was 1.4 % for Group 2.
The uncertainties relating to the analysed and room air concentrations were estimated by the data capture program based on the least squares statistics.
Nominal concentration of POSF
The data are presented in Table C and is summarised below:
Group 2 (Test Group)
Nominal concentration (ppm)
283
A/N ratio (%) 111.5
A/N
=
Analysed
concentration
100
Nominal concentration
For Group 2, the nominal concentration for each exposure was calculated from the following parameters:
The mass of POSF delivered into each vapour generator; The mean chamber temperature; The barometric (atmospheric) pressure; The molecular weight of POSF; The gas constant; The chamber airflow; The exposure duration. The equations used for the calculation of the nominal concentration are detailed in Table C.
: 68 :
Administration of POSF by inhalation to rats
MIN 312/014272 The nominal concentration for each exposure period was calculated, for the test group from the mass of POSF delivered into the generator. The daily ratios of analysed to nominal concentration (A/N), expressed as a percentage, were between 109 and 115%, with a coefficient of variation of only 2.1%. Possible reasons for this unexpectedly high A/N ratio are discussed below. Chamber Temperature The daily mean chamber temperatures are presented in Table D. The chamber temperatures were similar for both groups on each day of the study.
DISCUSSION Control of the POSF vapour delivery to the exposure chambers was excellent, with a coefficient of variation of less than 1.5%, and a study mean concentration, which was within 6% of the target value for the test group. As no analytical data was available for exposure 4 due to a computer problem, it seems reasonable (using the consistent A/N ratio) that the animals were dosed with a concentration around ca. 313 ppm. The ratio between the average analysed and the nominal chamber concentrations (A/N ratio) showed a definite discrepancy between these two values, with a mean of 111.5%. The coefficients of variation for the analysed and nominal values are 1.4% and 1.5% respectively, which shows that the discrepancy is consistent throughout the five day long exposure. The estimate of the nominal chamber concentration was lower than the analysed value and this is attributed to a combination of uncertainties as follows:
- POSF reference spectra and subsequent AutoQuant method calculations. These have errors of 5% associated with them (responsibility of the Sponsor);
- The cylinder of ethylene standard has certified errors of 2%; - Uncertainties in airflow measurements. A 1 l/minute difference in the airflow would
produce a 3% change in the nominal concentration; - The test material has a stated purity of >95.5% POSF. The other components of the test
material were various perfluoroalkyl sulfonyl fluorides (<4.5%) and their absorbance may interfere with that of POSF; - The effect of using ultra pure nitrogen for the background scans (rather than the control chamber atmosphere) is unknown.
: 69 :
Administration of POSF by inhalation to rats
CALCULATIONS
MIN 312/014272
In order to minimise the cumulative errors, which result from repeated rounding of numbers, much of the data in this report has been calculated continuously using unrounded numbers and only rounded for printing. Consequently, these rounded numbers may include rounding errors in the last significant figure, possibly leading to small apparent discrepancies with other data in the report.
: 70 :
Administration of POSF by inhalation to rats
FIGURE A Schematic of a rodent inhalation dosing system
MIN 312/014272
Key a Polypropylene syringe b Syringe driver c Test compound feed line d Glass Vapouriser e Air supply (29 l/minute) f Sinter diameter (114 mm) g Exposure chamber
h Observation port i Blanking plugs j Rodent restraining tubes (standard sections
with 20 exposure ports on levels 1 to 3 k Exhaust plenum l Filtration m Air extract (30 l/minute)
: 71 :
Administration of POSF by inhalation to rats
FIGURE B
MIN 312/014272
Schematic of a Fourier Transform Infrared spectrophotometer
Key a Nitrogen cylinder b Ethylene cylinder c Flowmeter regulated to 1 l/minute d Test compound sample line from
chamber
e 10 cm IR cell f FT-IR spectrophotometer g Flowmeter regulated to 1 l/minute h Diaphragm pump i Sample line return to chamber
: 72 :
Administration of POSF by inhalation to rats
TABLE A Operating conditions for the inhalation exposure system
MIN 312/014272
Parameter
Target concentration of POSF (ppm)
Chamber airflows (l/minute) Elutriator output (chamber inlet) Chamber extract
Vapour generator settings Test material feed Syringe size (ml)
Syringe pump setting (Speed, mm/min):
Exposure 1 Exposures 2-5 N/A Not applicable
1 (Air control)
0
29 30
N/A N/A
Group
2 (Test Group)
300
29 30
Precidor 5003 syringe pump 50
N/A
0.145
N/A
0.140
: 73 :
Administration of POSF by inhalation to rats
TABLE B
MIN 312/014272
Chamber concentration of POSF (ppm) - daily mean values
Exposure No.
1 2 3 4 b 5 Mean sd CV (%)
a
b
sd CV
Analysed Concentration
ppm
Error a
316.04
0.85
313.69
0.83
323.33
0.82
Room Air
ppm
Error
0.398 0.373 0.289
0.004 0.004 0.003
Concentration minus Room Air
(ppm) 315.65
313.32
323.04
314.74
0.83
0.428
0.004
314.31
316.95
0.83
0.372
0.004
316.58
4.361
0.009
0.0598 0.0007
4.414
1.4
1.1
16.1
19.9
1.4
Error value reported by Autoquant software.
No analytical data collected for exposure 4 due to computer problem with data capture program
Standard deviation
Coefficient of variation (sd 100/mean)
: 74 :
Administration of POSF by inhalation to rats
TABLE C
MIN 312/014272
Nominal concentrations of POSF (ppm) - individual exposure values
Group 2 (Test Group) - Target concentration 300 ppm
Exposure Barometric POSF usage Chamber
No.
Duration pressure (min) (mmHg)
(g) airflow (l/min)
Chamber concentration
Nominal c
Analysed
(ppm)
(ppm)
1
360
753
64.6
30.0
289
316
2
360
763
63.0
30.0
278
313
3
360
757
4
360
762
63.5
30.0
63.3
30.0
283
323
280
a
5
360
766
64.8
30.0
285
314
Mean of
Means sd
360 0.0
760 5.2
63.8
30.0
0.81
0.0
283 4.4
317 4.4
CV (%)
a c
0.0
0.7
1.3
0.0
1.5
1.4
No analytical data collected due to computer problem with data capture program Nominal concentrations were calculated from the following equations:
Concentration (ppm) = V 106 Va + V
A/N ratio (%)
109.1 112.6 114.2
a
110.3
111.5
2.32 2.1
V = W R T 760 mm Hg
M
Atm
where
V
=
W
=
M
=
R
=
T
=
Atm =
Va
=
gaseous volume of POSF mass of POSF molecular weight POSF (502.14 g/mole) gas constant (0.08205 l atm mol-1 K-1) temperature (K), = temperature (C, see Table D) + 273 atmospheric pressure (mmHg) volume of air (litres) passing through the chamber during the exposure
A/N
Analysed/nominal concentration ratio expressed as a percentage
sd
Standard deviation
CV
Coefficient of variation (sd 100/mean)
: 75 :
Administration of POSF by inhalation to rats
TABLE D Chamber temperature - exposure mean values
Temperature (oC)
Exposure
Group 1
Group 2
(Control) (Test Group)
1
20.0
20.2
2
20.0
20.0
3
20.0
20.0
4
19.9
20.0
5
19.9
19.9
Mean
20.0
20.0
sd
0.06
0.11
CV (%)
0.3
0.5
sd standard deviation
CV Coefficient of variation (sd 100/mean)
MIN 312/014272
: 76 :
Administration of POSF by inhalation to rats
APPENDIX A Methods of sample collection and analysis for POSF
MIN 312/014272
SAMPLE COLLECTION Chamber concentration The atmosphere sample was continually drawn from the test chamber to the FT-IR system at a regulated flow rate of 1 l/minute using a laboratory pump positioned downstream of the IR cell. The concentration of POSF was determined at exactly 30 minute intervals throughout each exposure. The sampled atmosphere was returned to the exposure chamber downstream of the sampling point. The airflow to the spectrophotometer was monitored throughout each of the exposures using a calibrated in-line tapered tube gas flowmeter. Gas sampling lines were PTFE tubing (0.6 cm diameter).
METHOD OF ANALYSIS Chamber atmosphere samples were analysed by extractive FT-IR. The method of sample analysis is detailed, together with a summary of the method validation, in the Inhalation Analytical Procedure at the end of this Appendix.
: 77 :
Administration of POSF by inhalation to rats
APPENDIX A
MIN 312/014272
(Methods of sample collection and analysis for POSF - continued)
CALCULATIONS FT-IR analysis The samples of chamber atmosphere were passed through the IR cell, which was calibrated using nominal vapour standards. The method for calculating the concentration of POSF from the mass used to prepare each vapour standard using the nominal feed rate is given below in equations 1 and 2.
Concentration = V 1,000,000 ppm
(1)
Va + V
V = W R T 760 mm Hg
(2)
M
Atm
where
V
=
W =
M =
R
=
T
=
Atm =
Va
=
gaseous volume of POSF (ml) mass of POSF (g) molecular weight of POSF (502.14 g/mole) Gas constant (0.08205 ml atm mmol-1 K-1) temperature (K) atmospheric pressure (mmHg) volume of air (ml)
: 78 :
Administration of POSF by inhalation to rats
APPENDIX A
MIN 312/014272
(Methods of sample collection and analysis for POSF - continued)
COMPOUND SPECIFIC INHALATION ANALYTICAL PROCEDURE FOR POSF
The analysis of POSF (Perfluorooctane sulfonyl fluoride) in air sample substrate
The method outlined in this document has been validated and is considered fit for the purpose of monitoring conditions in an Inhalation Toxicology study.
This document details the basic procedures for the analysis of POSF sampled by extractive FT-IR from test atmospheres. The resulting samples, of approximate concentration 240 to 360 ppm, are analysed using the MIDAC I-Series spectrophotometer and AutoQuant software. Study specific amendments and additions will be detailed within a supplementary document.
NOTE Throughout this document, the symbol indicates that the relevant information is not available at present, but will be included in a Study specific supplement.
EFFECTIVE FROM: 29 August 2001
Test substance
POSF, perfluorooctane sulfonyl fluoride has the following formula: C8F17SO2F.
Appearance
Clear liquid
Storage
Ambient temperature
Reagents
Air
In House
Compressed
Ethylene
Sponsor
Calibration gas (certified 2%)
Nitrogen
Sponsor
Calibration gas (certified >99.9995%)
Liquid nitrogen
BOC
Cooling Medium
: 79 :
Administration of POSF by inhalation to rats
APPENDIX A
MIN 312/014272
(Methods of sample collection and analysis for POSF - continued)
Equipment
Balance and Data printer
Sartorius
R160P with YDP-01
Flow meter
Gilmont Instruments
0-4 Lpm
FT-IR
Midac Corporation
I2001 series (serial no. 151)
Computer
Compaq
LTE5400
Sotware
Midac Corporation
AutoQuant Version 3.01 Midac Grams 32 Version 4.11
Chamber
ADG
3 level snout only
Syringe Driver
Precidor
Type 5003
UV Quartz Cells
Midac Corporation
ZnSe Gas Cell
General laboratory glassware
Accessories also employed are: a vacuum pump; cylinder regulators, power connectors; regulated exhaust pumps.
Consumables
Syringes
Aldrich
50 ml polypropylene
Method of sample extraction
A volume of POSF is dispensed from the test drum into a 50ml syringe. The syringe driver is set to give a concentration of 300ppm in an airstream of 30 l/minute airflow, mixed thoroughly in the vapouriser and fed directly into the chamber. The test atmosphere is drawn along the sample line at a flow rate of 1 l/minute directly through the IR cell.
Production of a Background Scan
A flowrate of 1l/minute ultra-pure nitrogen (cylinder No. C0C7700219 (nitrogen >99.9995 2%) is drawn though the IR cell. After a period of 3-5 minutes, an FT-IR single beam spectrum is generated approximately every 2 seconds and continuously collected for 32 scans to produce a more definitive spectrum.
: 80 :
Administration of POSF by inhalation to rats
APPENDIX A
MIN 312/014272
(Methods of sample collection and analysis for POSF - continued)
Storage of standards and samples Stability experiments were not required for on-line analysis.
Calibration and Quantification For each sample measurement, the absorbance determines the amount present in the sample using the equation below (Beer's Law):
Where
Amount( ppm) = a A b
A = absorbance at a given frequency of POSF in the sample spectrum a = absorption coefficient (absorptivity) of POSF in the sample spectrum, which is
derived from the reference calibration transfer standard spectrum b = path length of the cell derived from calibration data
Calibration of Standards Calibration is performed daily before each exposure (see "MIN/312 Operating Procedure"). A certified concentration of ethylene (cylinder No. C0C9700874 (ethylene = 2020ppm 2%) is passed through the sampling system prior to sample collection to check instrument response and determine the calibrated cell path-length. Cell path-length is a critical input to the analytical method used to perform each spectral analysis. The ethylene value is read from the test certificate on the calibration cylinder. A test atmosphere of ethylene at a flowrate of 1 l/minute is drawn through the IR cell. After a period of 3-5 minutes, a FT-IR single beam spectrum is generated approximately every 2 seconds and continuously collected for 32 scans to produce a less noisy spectrum. This produces a calibrated path length. This value is then entered into the POSF method file and manually recorded. QC Path length Limits: -The calibration will be repeated by the user if no peak is present in the
1400-650 cm-1 region or if the path length value is outside the range 0.100 to 0.115 m. If the calibration still does not conform, all of the calibration set up parameters will be checked. QA Instrument Calibration Check: The calibration is reverified and subsequently recorded at the end of the exposure. Instrument calibration checks are within 5%. Repetition of two four hour periods showed that the final calibration gave the path length values differed by less than 2% from the initial calibration value of the ethylene cylinder (cylinder No. C0C9700874 (ethylene = 2020ppm 2%). The 2% value is inside the 5% tolerance limits allowed as quality check standards. The temperature of the test gas stream and the barometric pressure will be monitored manually.
: 81 :
Administration of POSF by inhalation to rats
APPENDIX A
MIN 312/014272
(Methods of sample collection and analysis for POSF - continued)
FT-IR analyser specification The Midac I2000 series FT-IR spectrophotometer has a wavelength accuracy of 0.5cm-1. A MCT detector monitors the absorbance of various chemical bonds. AutoQuant version 3.01 (MIDAC, Irvine, CA) software is used for all infrared data acquisition and quantification.
The length of the IR cell or path length (approximately 10 cm) dictates the concentration range that will give a linear relationship between absorbance and concentration.
The optimum flow rate for the IR cell is 1 l/minute, which is set by a flow meter and all the analysis is conducted at that flow rate to avoid differences of cell pressure. Further details can be obtained from the operating procedure stored proximal to the FT-IR system. The analyser draws 1 l/minute from the gas stream and the remainder is returned to the exposure system exhaust. All exhaust lines are monitored by precision stainless steel and glass in-line rotameters.
Raw Data
Raw data is defined as the first hard copy output obtained immediately following data acquisition by the FT-IR system. This will be signed and dated immediately after printing. An electronic backup copy of this is saved to the local zip drive. If a run time hard copy was not obtained through a printer fault, the electronic copy from the zip drive directory may be used to generate a hard copy, which will be signed and dated. Exposure data is stored in the file format YYMMDD.txt.
Computer hardware:
Computers and VDUs are maintained by the Sponsor.
Printer is maintained by the IT department.
FT-IR System hardware:
The Sponsor has supplied the hardware. Arrangements for servicing and maintenance would be made in consultation with the Sponsor.
Software:
Documentation is the responsibility of the Sponsor.
Safety
The COSHH assessment provides details regarding the toxicity of the test material POSF. It is considered that the standard handling procedures used within the department are suitable to prevent exposure to the test material.
In case of a total power failure, switch off the FT-IR and test compound generation equipment immediately.
: 82 :
Administration of POSF by inhalation to rats
APPENDIX A
MIN 312/014272
(Methods of sample collection and analysis for POSF - continued)
Summary of method validation The raw data for the method validation is located in study MIN/312. Comparison of test blanks and test samples showed that the analyte was well resolved from any potential interfering peak. Repeatability The variability of the POSF level in a dynamic (exposure) system is determined by the airflow and feed rate through the syringe driver being set correctly and checked every 30 minutes (as the flow is pressure dependent). Statistical analysis shows that, with these checks in place, over a four hour exposure in a 3-level ADG snout only exposure system, the Coefficient of variation is typically less than 7.0 at 300 ppm. This figure includes the variability of generation as well as the repeatability of measurement. Specificity This Fourier Transform infrared technique monitors the absorbance of POSF in the region 1400-650 cm-1, specifically between 1400 and 1000 cm-1. Linearity Linear regression analysis of the FT-IR spectrophotometer response to standards was conducted during preliminary trials. This was used to demonstrate that the FT-IR response is linear over the concentration range. Feed rates were selected to achieve a nominal concentration of 240 to 360 ppm (see data file 010912.txt, 010912a.txt and reference sequences 010912 MIN312 and 010912a MIN312) utilising a total extract flow rate of 30 l/minute. Least squares regression analysis with a unweighted linear regression of the predicted concentration from the nominal against FT-IR analysed concentration (240 to 360 ppm) produced a correlation coefficient of 0.999301 and relative errors less than 1.0% in the range 360 to 240 ppm. The Limit of Quantification (LOQ) for POSF will be set by the lowest acceptable nominal feed rate, however, the LOQ and Limit of Detection (LOD) are potentially as low as 12.17 and 4.01 ppm respectively (calculated statistically using the standard deviation obtained for a nominal feed rate to obtain a concentration of 240 ppm). Uncertainty of POSF analysis Uncertainties were said (by the Sponsor) to be less than 5% for the AutoQuant method calculations. The POSF reference spectrum is from the 3M quantitative library and was generated at 3M using EPA guidelines. Other principal uncertainties were: ethylene path length Calibration (2%); interference from alkyl sufonyl fluorides (<4.5% v/v, but interference unknown).
: 83 :
Administration of POSF by inhalation to rats
APPENDIX A
MIN 312/014272
(Methods of sample collection and analysis for POSF - continued)
MIN/312 - STUDY SPECIFIC SUPPLEMENT TO THE INHALATION ANALYTICAL PROCEDURE FOR POSF (PERFLUORO-OCTANE SULFONYL FLUORIDE) This supplement details additions and amendments to the procedure to be used for the FT-IR assay of POSF obtained from air samples collected on the above study. The assay, incorporating the additions and amendments, is suitable for the analysis of POSF, in air, at concentrations within the range of 240 to 360ppm. Details given in this supplement supersede those in the compound specific IAP.
EFFECTIVE DATE : 4 September 2001
Analytical standard
Name Batch number Purity Expiry date Supplier
FX-8B, POSF, T-7661.1, Perfluoro-octane sulfonyl fluoride 040227 >95.5% (Perfluoroalkyl sulfonylfluorides <4.5%) Not Supplied Sponsor
Fourier Transform - Infrared Spectrophotometer
The analysis is performed using a FT-IR analyser supplied by the sponsor.
Preparation of standard solutions
Provide nominal feed rates to the chamber in the range 240 to 360 ppm.
Calibration and Quantification
Calibration of the pathlength by an ethylene standard (cylinder No. C0C9700874 (ethylene = 2020ppm 2%). Quantification using the 3M quantitative library generated at 3M using EPA guidelines for generating reference spectra (v).
: 84 :
Administration of POSF by inhalation to rats
APPENDIX B Individual POSF concentration measurements
MIN 312/014272
Exposure
Date and Time
Concentration
(ppm) Error
1
13/09/01 09:24
272.86 0.71
13/09/01 09:54
345.50 0.88
13/09/01 10:24
323.61 0.85
13/09/01 10:54
315.21 0.83
13/09/01 11:24
316.94 0.83
13/09/01 11:54
310.42 0.84
13/09/01 12:24
312.39 0.84
13/09/01 12:54
316.65 0.86
13/09/01 13:24
318.48 0.87
13/09/01 13:54
316.82 0.87
13/09/01 14:24
322.98 0.88
13/09/01 14:54
320.68 0.88
Mean
316.04 0.85
sd
16.272 0.047
CV (%)
5.1 5.5
2
14/09/01 09:25
276.10 0.72
14/09/01 09:55
320.49 0.81
14/09/01 10:25
321.14 0.82
14/09/01 10:55
314.85 0.81
14/09/01 11:25
310.00 0.81
14/09/01 11:55
312.98 0.83
14/09/01 12:25
317.30 0.85
14/09/01 12:55
315.09 0.85
14/09/01 13:25
321.14 0.87
14/09/01 13:55
315.14 0.86
14/09/01 14:25
319.44 0.87
14/09/01 14:55
320.64 0.87
Mean
313.69 0.83
sd
12.375 0.043
CV (%)
3.9 5.1
sd
standard deviation
CV
Coefficient of variation (sd 100/mean)
Room Air
(ppm) 0.171 0.245 0.309 0.365 0.402 0.413 0.445 0.464 0.475 0.488 0.497 0.5
0.398 0.1066 26.8
0.166 0.233 0.297 0.332 0.357 0.405 0.419 0.439 0.448 0.463 0.457 0.465
0.373 0.0986 26.4
Error 0.003 0.003 0.003 0.003 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.005
0.004 0.0007 17.6
0.003 0.003 0.003 0.003 0.003 0.004 0.004 0.004 0.004 0.004 0.004 0.004
0.004 0.0005 15.0
Concentration Room Air (ppm) 272.69 345.26 323.31 314.85 316.53 310.01 311.95 316.19 318.00 316.33 322.48 320.18
315.65 16.240 5.1
275.93 320.25 320.84 314.52 309.65 312.58 316.88 314.65 320.69 314.68 318.99 320.18
313.320 12.3137 3.9
: 85 :
Administration of POSF by inhalation to rats
APPENDIX B
MIN 312/014272
(Individual POSF concentration measurements - continued)
Exposure a Date and Time
Concentration
Room Air
Concentration Room Air
(ppm) Error (ppm) Error
(ppm)
3
15/09/01 07:55
c
0.18
0.208 0.002
15/09/01 07:59
c
0.18
0.224 0.002
15/09/01 08:02
c
0.19
0.233 0.002
15/09/01 08:06
c
0.2
0.245 0.002
15/09/01 08:10
c
0.03
0.005 0.000
15/09/01 08:13
0.273 c 0.07
0.012 0.000
15/09/01 08:15
c
0.65
0.021 0.000
15/09/01 08:17
318.76 0.75
0.077 0.001
318.69
15/09/01 08:25
350.94 0.82
0.098 0.001
350.84
15/09/01 08:55
319.95 0.78
0.18
0.002
319.77
15/09/01 09:25
318.60 0.79
0.233 0.002
318.36
15/09/01 09:55
323.86 0.82
0.284 0.003
323.58
15/09/01 10:25
327.15 0.84
0.314 0.003
326.84
15/09/01 10:55
318.38 0.84
0.345 0.003
318.04
15/09/01 11:25
318.32 0.83
0.359 0.003
317.96
15/09/01 11:55
321.12 0.85
0.375 0.003
320.75
15/09/01 12:25
321.74 0.85
0.379 0.003
321.36
15/09/01 12:58
322.46 0.86
0.404 0.004
322.05
15/09/01 13:28
318.70 0.86
0.417 0.004
318.29
Mean
323.33 0.82
0.289 0.003
323.04
sd
9.101 0.034 0.117 0.0009
9.154
CV (%)
2.8 4.2
40.5
35.1
2.8
5
17/09/01 09:07
259.59 0.68
0.189 0.003
259.40
17/09/01 09:37
329.96 0.83
0.262 0.003
329.70
17/09/01 10:07
333.40 0.84
0.333 0.004
333.06
17/09/01 10:37
330.72 0.85
0.387 0.004
330.33
17/09/01 11:07
322.77 0.81
0.303 0.003
322.46
17/09/01 11:37
316.42 0.82
0.458 0.004
315.96
17/09/01 12:07
313.36 0.83
0.492 0.004
312.87
17/09/01 12:37
313.05 0.84
0.508 0.005
312.54
17/09/01 13:07
312.63 0.85
0.528 0.005
312.11
17/09/01 13:37
316.11 0.86
0.544 0.005
315.57
17/09/01 14:07
315.02 0.87
0.561 0.005
314.46
17/09/01 14:37
313.82 0.87
0.570 0.005
313.25
Mean
314.74 0.83
0.428 0.004
314.31
sd
18.963 0.051 0.1292 0.0008
18.931
CV (%)
6.0 6.1
30.2
19.5
6.0
sd
standard deviation
CV
Coefficient of variation (sd 100/mean)
a
No data collected for exposure 4 due to computer problem with data capture program
c
Problem with data capture value excluded from mean and standard deviation
: 86 :
Administration of POSF by inhalation to rats
PROTOCOL AND PROTOCOL AMENDMENTS
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MIN 312/014272 : 263 :
MIN 312/014272 : 264 :
MIN 312/014272 : 265 :
MIN 312/014272 : 266 :
MIN 312/014272 : 267 :
MIN 312/014272 : 268 :
MIN 312/014272 : 269 :
MIN 312/014272 : 270 :
MIN 312/014272 : 271 :
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MIN 312/014272 : 273 :
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MIN 312/014272 : 275 :
MIN 312/014272 : 276 :
MIN 312/014272 : 277 :
MIN 312/014272 : 278 :
MIN 312/014272 : 279 :
MIN 312/014272 : 280 :
MIN 312/014272 : 281 :
MIN 312/014272 : 282 :
MIN 312/014272 : 283 :
MIN 312/014272 : 284 :
MIN 312/014272 : 285 :
MIN 312/014272 : 286 :
MIN 312/014272 SCANNING ELECTRON MICROSCOPE EXAMINATION
AND X-RAY MICROANALYSIS
: 287 :
MIN 312/014272 : 288 :
MIN 312/014272 : 289 :
MIN 312/014272 : 290 :
MIN 312/014272 : 291 :
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MIN 312/014272 : 296 :
MIN 312/014272 : 297 :
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MIN 312/014272 : 299 :
MIN 312/014272 : 300 :
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MIN 312/014272 : 305 :
MIN 312/014272 : 306 :
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MIN 312/014272 : 308 :
MIN 312/014272 : 309 :
MIN 312/014272 : 310 :
MIN 312/014272 : 311 :
MIN 312/014272 : 312 :
MIN 312/014272 : 313 :
MIN 312/014272 : 314 :
MIN 312/014272 : 315 :
MIN 312/014272 : 316 :
MIN 312/014272 : 317 :
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MIN 312/014272 : 319 :
MIN 312/014272 : 320 :
MIN 312/014272 : 321 :
MIN 312/014272 : 322 :
MIN 312/014272 : 323 :
MIN 312/014272 : 324 :
MIN 312/014272 : 325 :
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MIN 312/014272 : 335 :
MIN 312/014272 : 336 :
MIN 312/014272 : 337 :
MIN 312/014272 HUNTINGDON RESEARCH CENTRE GLP COMPLIANCE STATEMENT 2001
: 338 :
MIN 312/014272 HUNTINGDON RESEARCH CENTRE GLP COMPLIANCE STATEMENT 2003
: 339 :
MIN 312/014272 HUNTINGDON RESEARCH CENTRE GLP COMPLIANCE STATEMENT 2005
: 340 :