Document JdvDDR8Y4Gy4q3XqzZ4bvdYe

CONFIDENTIAL AR226-3132 -J^/SJ 3^ DPT 443/984760 TOXICTTY STUDY BY ORAL ADMINISTRATION TO CD RATS FOR 4 WEEKS Sponsor DuPont Specialty Chemicals; Jackson Laboratory, Chambers Works, Deepwater, NJ 08023, U.S.A. Page 1 of 293 Research Laboratory Huntingdon Life Sciences Ltd., P.O. Box 2, Huntingdon, Cambridgeshire, PE186ES, ENGLAND. Report issued 1 September 1999 Company Sanitized. Does not contain TSCA CBI DPT 443/984760 CONTENTS Page COMPLIANCE WITH GOOD LABORATORY PRACTICE STANDARDS.................... 4 QUALITY ASSURANCE STATEMENT............................................................................ 5 CONTRIBUTORY SCIENTISTS......................................................................................... 6 7 SUMMARY.......................................................................................................................... 10 INTRODUCTION................................................................................................................. RELEVANT STUDY DATES.............................................................................................. 11 TEST SUBSTANCE............................................................................................................. 12 EXPERIMENTAL PROCEDURE........................................................................................ 13 RESULTS.............................................................................................................................. 24 DISCUSSION AND CONCLUSION................................................................................... 30 31 REFERENCES...................................................................................................................... FIGURES - group data 1. Bodyweight................................................................................................................. 32 2 : Company Sanitized. Does not contain TSCA CBI DPT 443/984760 TABLES - group data 1. Bodyweight................................................................................................................. 2. Food consumption........... :.........................................................--......--.--.....--.......... 3. ' Food conversion ratios..................................................................--..--...................... 4. Haematology............................................................................................................... 5. Biochemistry............................................................................................................... 6. Organ weights............................................................................................................. 7. Macroscopic pathology incidence summary............................................................... 8. Microscopic pathology incidence summary............................................................... Page 34 35 36 37 39 41 43 45 APPENDICES - individual data 1. Bodyweight................................................................................................................. 52 2. Haematology............................................................................................................... 54 3. Biochemistry............................................................................................................... 60 4. Organ weights............................................................................................................. 64 5. Clinical and pathological findings for individual animals.......................................... 66 BEHAVIOURAL SCREENING........................................................................................... 119 FORMULATION CHEMISTRY REPORT.......................................................................... 182 PROTOCOL AND AMENDMENTS................................................................................... 199 |:ISEVENDAY ORAL TOXICITY STUDY IN THE RAT _(_DPT 442/992305)........................................................................................-..-.-................ 236 Company Sanitized. Does not contain TSCA CBI ^^ ^wr DPT 443/984760 COMPLIANCE WITH GOOD LABORATORY PRACTICE STANDARDS The study described in this report was conducted in compliance with the following Good Laboratory Practice standards and I consider the data generated to be valid. The United Kingdom Good Laboratory Practice Regulations 1997, Statutory Instrument No.654. EC Council Directive 87/18/EEC of 18 December 1986 (Official Journal No. L 15/29). OECD Principles of ENV/MC/CHEM(98) 17. Good Laboratory Practice (as revised in 1997), No claim for GLP compliance is made for the other study report included within the same cover: DPT 442/992305. Helen A. Palmer, B.Sc. (Hons.), M.Sc., C.BioL, M.LBiol, Study Director, Huntingdon Life Sciences Ltd. ?.L..^.?A^J=;...l.cl<?l.c1.. Date \^J 9 4 : Company Sanitized. Does not contain TSCA CBI QUALITY ASSURANCE STATEMENT DPT 443/984760 The following have been inspected or audited in relation to this study Study Phases Inspected Protocol Date of Inspection 14 December 1998 Date of Reporting 14 December 1998 Study Based Inspections Study Preparation Dose Administration Clinical Signs Formulation Records Data Review 11 December 1998 11 December 1998 11 December 1998 11 December 1998 11 December 1998 14 December 1998 14 December 1998 14 December 1998 14 December 1998 14 December 1998 Functional Observation Battery Blood Sampling Post Mortems Data Review 8-12 January 1999 8-12 January 1999 8-12 January 1999 8-12 January 1999 15 January 1999 15 January 1999 15 January 1999 15 January 1999 Report 13 August 1999 13 August 1999 Protocol: An audit of the protocol for this study was conducted and reported to the Study Director and Company Management as indicated above. Study based inspections: Inspections and audits of phases of this study were conducted and reported to the Study Director and Company Management as indicated above. Process based inspections: At or about the time this study was in progress inspections and audits of other routine and repetitive procedures employed on this type of study were carried out. These were promptly reported to appropriate Company Management. Report Audit: This report, excluding the seven day oral toxicity study DPT 442/992305 report also within this cover, has been audited by the Quality Assurance Department. This audit was conducted and reported to the Study Director and Company Management as indicated above. The methods, procedures and observations were found to be accurately described and the reported results to reflect the raw data. Kevin P. de-Salis, B.A. (Hons.), C.BioL, M.I.BioL, Dip.R.Q.A., Group Leader, Department of Quality Assurance, Huntingdon Life Sciences Ltd. Company Sanitized. Does not contain TSCA CBI CONTRIBUTORY SCIENTISTS STUDY MANAGEMENT Helen A. Palmer, B.Sc. (Rons.), M.Sc., C.Biol., M.I.Biol., Study Director, Toxicological Sciences. William N. Hooks, A.I.B.M.S., B.Sc. (Hons.), Monitoring Toxicologist, Toxicological Sciences. PATHOLOGY Chirukandath Gopinath, B.V.Sc., M.V.Sc., Ph.D., F.R.C.Path.. Director of Pathology. FORMULATION ANALYSIS I. Suzanne Dawe, M.Sc., C.Chem., M.R.S.C., Scientific Manager, Formulation Chemistry, Department of Pharmacy and Formulation Chemistry. BEHAVIOURAL SCREENING Elizabeth W. Hughes, B.A., M.Sc., Behavioural Scientist, Pharmacology and Short Term Studies Division. DPT 443/984760 6 : Company Sanitized. Does not contain TSCA CBI SUMMARY DPT 443/984760 Ovi^^f^^Vf0 This study was performed to assess the systemic toxicity followed was outlined in: L the rat. The method OECD Guideline for Testing of" Chemicals Guideline No.407, 'Repeated Dose 28-day Oral Toxicity Study in Rodents', Adopted 27 July 1995. f^^Hmjwas administered by oral gavage, once daily, to three groups of 5 male and 5 female rats for 28 consecutive days, at dosage levels of 15, 50 or 150 mg/kg/day. The test substance was prepared as a solution in distilled water at concentrations of 1.5, 5.0 or 15 mg/ml and administered at a dosage volume of 10 ml/kg/day. All dosages were corrected for purity as the material was supplied as a 25% slurry in water. Dosages are reported herein in terms of solids. Control animals received the vehicle alone at the same dose volume. During the study, clinical signs, bodyweight and food consumption data were recorded. Neurobehavioural screening was performed at intervals during the study and blood samples were taken from all animals on Day 29 of the study. All animals were killed at the end of the 4 week treatment period (Day 29) and examined macroscopically. A range of organs was weighed and tissues preserved for subsequent light microscopy examinations. The following comments in relation to the principal findings are made in summary: Mortality There were no unscheduled deaths on the study. Clinical signs No clinical signs considered to be of lexicological significance were noted. Bodyweight, food consumption and food conversion ratios Markedly lower bodyweight gain, food consumption and inferior food conversion ratios were seen for males and females receiving 150 mg/kg/day. The bodyweight gain of males and females receiving 50 mg/kg/day was also lower than that of controls. Behavioural screening ^ No change attributable to treatment witlaH^JBJBsvas noted for any behavioural parameter. Company Sanitized. Does not contain TSCA CBI DPT 443/984760 Haematology Male and female rats dosed with 150 nig/kg/day had lexicologically significant anaemia, as indicated by decreased mean red cell mass parameters (RBC, PCV and Hb) and decreased mean MCV. Male and female rats in this group also had leucocytosis, evidenced by increased total mean leucocytes. These changes are consistent with inflammation, and with the renal changes observed microscopically. Statistically significant changes in other haematologic parameters were considered to be lexicologically insignificant. Biochemistry Male and female rats dosed with 150 nig/kg/day had evidence of decreased renal function. Increased urea nitrogen, creatinine and inorganic phosphorus indicated decreased glomerular filtration rate. Male and female rats dosed with 50 mg/kg/day also had increased urea nitrogen; this change was of sufficient magnitude to be considered toxicologically significant. Increased urea nitrogen for males which had received 15 mg/kg/day was considered to be of no biological significance. Mild changes in potassium, calcium, sodium and chloride also occurred in male and female rats in the high dose group. These effects' are consistent with altered kidney function and are considered to be a result of kidney toxicity. Hyperbilirubinemia was also noted in rats dosed with 150 mg/kg/day. These effects are considered secondary to and consistent with liver alterations noted during microscopic examination. Organ weights A higher absolute kidney weight was apparent for males and females receiving 150 mg/kg/day, in comparison with the controls. In males receiving 150 mg/kg/day, a lower absolute liver but a higher bodyweight-adjusted liver weight was seen. A higher bodyweight-adjusted liver weight was apparent for females receiving 150 mg/kg/day, compared to the controls. For females receiving 150 mg/kg/day, a lower absolute heart, spleen and adrenal weight were noted, in comparison with the controls. Macroscopic pathology The macroscopic examination performed at termination revealed the following changes in animals receiving 150 mg/kg/day: enlargement, pallor and swollen appearance of the kidneys and pale cortical foci and irregular cortical scarring of the kidneys, unilateral distension of the ureter and blood stained urine in the ureter and urinary bladder of 1 male, pallor of the liver, a reduction in adipose tissue and a reduction in the size of the ovaries and uterus in 1 female. Company Sanitized. Does not contain TSCA CBI DPT 443/984760 Microscopic pathology Evidence of nephrotoxicity was detected at 150 mg/kg/day and to a lesser extent at 50 nig/kg/day, and was characterised by interstitial inflammation in the cortex, medulla and papilla, inflammatory casts in tubules and collecting ducts, cortical tubular necrosis, basophilia and hyperplasia in tubules and collecting ducts, tubular dilatation, papillary necrosis, collecting duct and urothelial hyperplasia. No effect was seen at 15 mg/kg/day.. In addition, hepatocyte hypertrophy and fine vacuolation and an increased incidence of prominent adipocytes in the bone marrow were also detected at 150 mg/kg/day. Conclusion It was concluded that 15 mg/kg/day represents the no observable adverse effect lev.el (NOAEL) for ll^BIIBBR11 me rat wnen administered orally for 28 days. According to the EEC Council Directive 92/69/EEC Annex VI, Part n(D) as described in Comnnssion Directive 93/2 I/EEC, labelling with the R48 risk phrase is appropriate. HencqU^|^U|s labelled R48/22: Harmful if swallowed. Company Sanitized. Does not contain TSCA CBI INTRODUCTION DPT 443/984760 of^ffff^ffa. The study was designed to assess the systemic toxicity surfactant in the polymerisation offluoromonomers, to the rat when repeatedly administered orally for a period of 28 consecutive days. The procedure used is described in this report and complied with that described in the Organisation for Economic Co-operation and Development, Testing of Chemicals Guideline No. 407, 'Repeated Dose 28-day Oral Toxicity Study in Rodents', Adopted 27 July 1995. The albino rat was chosen as the test species as it has been shown to be a suitable model for this type of study and is the species recommended in the test guidelines. The strain of rat used was chosen on account of the availability of background data. The rats were dosed orally as the test substance may be ingested accidentally. The dosage levels were selected on the basis of a one week preliminary oral toxicity study performed at this laboratory, where dosage levels of 500 and 750 mg/kg/day caused all animals to be prematurely sacrificed or found dead. 250 mg/kg/day caused decreased bodyweight gain and food consumption, increased kidney and liver weights, decreased spleen weight and kidney enlargement/pallor. This dosage was considered too high for this subsequent 4 week study, so a high dosage level of 150 mg/kg/day was chosen in reference to the key dosage relative to OECD labelling requirements. (Huntingdon Life Sciences report number DPT 442/992305, which is appended to this report). To compensate for the purity of the test material as supplied a correction factor was applied. All dosage levels in this report are stated as solids not as material supplied as the test compound was supplied as a 25% slurry in water. 10 : Company Sanitized. Does not contain TSCA CBI RELEVANT STUDY DATES Protocol approved by: Study Director Management Study Sponsor Animals arrival at Huntingdon: Commencement of treatment: Haematology and biochemistry: Day 29 Functional Observational Battery: Pre-dose Weekl Week 2 Week 3 Week 4 Terminal kill (after completion of 4 weeks of treatment) 11 November 1998 12 November 1998 1 December 1998 2 December 1998 11 December 1998 8 January 1999 5-6 December 1998 17 December 1998 23 December 1998 28 December 1998 5 - 6 January 1999 8 January 1999 DPT 443/984760 11 : Company Sanitized. Does not contain TSCA CBI TEST SUBSTANCE DPT 443/984760 Identity: Intended use: Received from: Date received at Huntingdon: 0 c' DuPont Specialty Chemicals 23 June 1998 Batch number Expiry date: Date of manufacture: Purity: 2 years from manufacture 8 March 1998 Appearance: Storage conditions: Room temperature A 1 g sample of the test substance was retained in the Huntingdon Life Sciences Archive. All dosages reported herein are expressed in terms of solids, rather than test substance as supplied. 12 : Company Sanitized. Does not contain TSCA CBI EXPERIMENTAL PROCEDURE DPT 443/984760 ANIMAL MANAGEMENT A total of 29 male and 29 female Cri :CD(SD)BR rats approximately 28 days old and within a weight range of 9 g for males and 14 g for females, was received from Charles River (UK) Ltd, Margate, Kent, England. For those animals selected for the study, their estimated age at the start of treatment was 5 weeks and their bodyweights were in the range 133 g to 162 g for males and 121 g to 142 g for females. On arrival 5 males and 4 females selected at random were used for health check purposes. These animals were killed within 24 hours after arrival at Huntingdon and subjected to routine macroscopic examination. Lungs, liver, kidneys, spleen and heart were preserved in fixative, but not processed farther. No macroscopic abnormalities were noted in any of the health check animals. The remaining rats were placed at random in suspended cages with wire mesh floors with stainless steel wire tops, according to sex, so that each cage contained 5 rats of the same sex. Each cage measured 36.5 cm wide, 55 cm deep and 25 cm high. Animal room temperature and relative humidity controls were set at 21 2C and 55 10% respectively; the actual ranges recorded were 22 to 25C and 25 to 50% respectively. The transient deviations from the set limits were considered not to have affected the integrity of the study. Permanent weekly recordings of these parameters were made by a Foster Clearspan M206 recorder and these are archived with all other raw data for this study. Artificial lighting was controlled to give 12 hours continuous light and 12 hours continuous dark per 24 hours. All rats had free access to tap water and pelleted SDS Rat and Mouse No. 1 maintenance diet, except as noted under LABORATORY INVESTIGATIONS. There was no information available to the Study Director to indicate that any non-nutrient substance likely to influence the effect of the test substance was present in the diet, or the drinking water, both of which were routinely subjected to regular chemical analyses, results of which are lodged in Huntingdon Life Sciences Archives. A period of acclimatisation of 9 days was allowed between allocation of animals to groups and the commencement of treatment. During this period a review of animal health was undertaken by a veterinary officer. The spare animals were retained during this acclimatisation period to replace any rat showing signs of ill health. Prior to me start of treatment, animal number 31 (female) was noted to be emaciated and to have piloerection, hunched posture and yellow staining in the urogenital region. The animal was considered to be unsuitable for treatment and was therefore, sent for post mortem examination which revealed the findings described overleaf: 13 : Company Sanitized. Does not contain TSCA CBI DPT 443/984760 Tissue Observation Fur Thoracic cavity: Incisors: Liver: Lumbar lymph nodes: Adipose tissue: Ileum: Kidney: Spleen: Stained - genital region, minimal, brown Moist - genital region Contained clear serous fluid Pale-lower A few pale capsular areas, punctate Adhesions to diaphragm Left - enlarged Mesentery - multiple, pale, punctate nodules (following course of blood vessels) Mesovarian, omental and mesometrium - multiple, pale nodules, up to 7mm Pale nodule, serosal aspect (2 mm) Peyers Patches prominent A pale area (5 mm) Capsule roughened The liver, kidneys, spleen, heart, lungs and macroscopic abnormalities were processed for histopathological examination. Examination of these tissues indicated that the original lesion was a bacterial endocarditis and the widespread lesions were due to suppurative emboli coming from the original lesion. This was considered not to be infectious to the other animals. This animal was replaced with a spare animal, which was given the unique identification number 1031. The remaining spare animals were discarded from the study on the first day of treatment. On the day of commencement of treatment, the group mean bodyweights were reviewed to ensure that variations in bodyweight did not exceed 20% of the mean for each sex. Throughout the study the animals were housed in the Department of Rodent Toxicology, Barriered Rodent Building No. 1, Room 18. ANIMAL IDENTIFICATION Group Health check Animal numbers M F 1-5 6-10 11-15 16-20 41-44,49 21-25 26-30 1031,32-35 36-40 45-48 The rats were housed 5 to a cage. Each cage was identified by a coloured label according to group and each label was uniquely numbered with cage and study number. The cage number was tattoo .d on the leg of each rat in the cage and within each cage, identification was by earmark. The cages were distributed on the battery so that possible environmental influences arising from their spatial distribution were equilibrated, as far as possible. 14 Company Sanitized. Does not contain TSCA CBI DPT 443/984760 PREPARATION OF FORMULATIONS l|^^BHi The test substance, was administered as a solution in distilled water. A correction factor ofx4 was appliedto correct for the fact that the test material as supplied was a 25% aqueous slurry. A series of solutions were prepared by serial dilution of me test substance. The test material as supplied was wanned to 35-40C in a water bath to ensure a complete solution as the diluent was added. The solutions were dispersed by gentle stirring, care was taken not to shake the formulations. The concentrations were chosen to give a constant dosage volume of 10 ml/kg bodyweight Control animals received the vehicle alone at the same dosage volume. The formulations were prepared weekly and then divided into 7 equal daily aliquots which were stored at +4C until the day of use. The formulations were allowed to equilibrate to room temperature prior to use and were checked to ensure that they were clear solutions. Group/colour code 1: White 2: YeUow 3: Blue 4: Pink Control (0) 15 50 150 Concentration^ ^^^m^^^ 0 1.5 5 15 # Expressed as solids, not as material supplied FORMULATION SAMPLING AND ANALYSIS Prior to the commencement of the study the proposed formulation procedure was checked by chemical analysis to confirm that the method was acceptable and that the stability of the formulations was satisfactory under the conditions of the study. Samples of formulations prepared for Week 1 were also analysed to check the accuracy of preparation. Chemical analysis was carried out by Huntingdon Life Sciences Department of Analytical Chemistry and the results are presented in this report. ADMINISTRATION OF FORMULATIONS The test substance, SS^SS^S was administered as a solution. Control animals received the vehicle alone. The animals were dosed at approximately the same time each day, where possible, using a suitably graduated syringe and a rubber catheter (Ch 10) inserted via the mouth into the stomach. The dosage volume administered to each animal was calculated according to the most recent recorded bodyweight and was adjusted to the nearest 0.1 ml. A constant dosage volume of 10 ml/kg was used. Treatment in this manner continued once a day, seven days a week, for a total period of 4 weeks. DURATION OF TREATMENT Following a total acclimatisation period of 9 days, treatment continued until completion of 4 weeks. Company Sanitized. Does not contain TSCA CBI DPT 443/984760 OBSERVATIONS AND MEASUREMENTS Dated and signed records of all activities relating to the day by day running and maintenance of the study within the animal unit as well as to the group observations and examinations outlined in this procedure were recorded in the Study Daybook. In addition, observations relating to individual animals made throughout the study were recorded. The following observations were made during the course of the study: Clinical signs and mortality Individual animals were observed at least once daily for any signs of behavioural changes, reaction to treatment or ill health. A detailed examination including palpation for the presence of masses was performed weekly. In addition, detailed observations were made in association with dosing daily for Week 1 and twice weekly for Weeks 2-4 of the study. Dated and signed records of appearance, change and disappearance of clinical signs were maintained on clinical history sheets for individual animals. Further checks were made early in each working day and again in the afternoon to look for dead or moribund animals. This allowed post mortem examination to be carried out during the working period of that day. Bodyweight The weight of each rat was recorded one week prior to the commencement of treatment, on the day of commencement of treatment and once a week thereafter (last scheduled bodyweight recorded on Day 28). Food consumption The quantity of food consumed by each cage of rats was recorded on a weekly basis. Food intake per rat (g/rat/week) was calculated using the total amount of food given to and left by the cage in each group and the number of rats surviving in each cage. The following formula was used: Weekly food consumption Total food given - Total food left _ (g/rat/week) ~" Number of animal days*x The results using this formula (presented in Table 2) were subject to rounding to the nearest whole number. * The term 'animal day' counts one animal day for each animal alive for a whole day. Thus, for example, in a 5-animal cage with total survival there are 35 (5 animals x 7 days) animal days of consumption. It is assumed that on the day of death an animal does not eat 16 Company Sanitized. Does not contain TSCA CBI  6 DPT 443/984760 Efficiency of food utilisation Food conversion ratios were calculated, where possible, over the period Weeks 1 to 4, from the bodyweight and food consumption data as weight of food consumed per unit gain in bodyweight The following formula was used: Food consumed F_ ood, . conversion . ratio = Bodyweight gain The 'food consumed' was calculated as indicated in the Food consumption section. The 'bodyweight gain' was calculated from the gain of each animal and uses the mean gain in the formula. Water consumption Daily monitoring by visual appraisal was maintained throughout the dosing period. NEUROBEHAVIOURAL SCREENING Functional observational battery The functional observational battery and motor activity was performed at approximately the same time of day, before initiation of treatment and during Week 4 of treatment. Not all rats were tested in one day, but time of testing was balanced across the groups. Observations made during the treatment period were made prior to dosing. In addition, observations in Week 4 were performed prior to any laboratory investigations. In addition, a shortened battery was performed during Weeks 1, 2 and 3. The fimctional observational battery is fully detailed in the BEHAVIOURAL SCREENING report. Motor activity Motor activity was monitored using a Coulboum Infra-Red Activity Monitoring System (system supplied by Coulboum Instruments, Leigh Valley, PA, USA). This system uses an infra-red detector to monitor activity. The following categories of activity are recorded: the time spent in no movement, locomotor and non-locomotor activity. The number of occurrences (events) of each category is also recorded. Normally in reporting this data only the locomotor activity is presented. For testing, designated animals were placed singly into observ-ition cages. Once all animals had been placed into the cage, the test session was started. The test s -ssion for each animal was 1 hour. Data was collected every 2 minutes and stored on a floppy disk. 17 Company Sanitized. Does not contain TSCA CBI DPT 443/984760 LABORATORY INVESTIGATIONS On Day 29 samples of blood were withdrawn, under isoflurane/nitrous oxide anaesthesia, from the orbital sinus of all rats. The blood samples collected were divided into tubes as follows: EDTA anticoagulant..................................... for haematological investigations Citrate anticoagulant..................................... for coagulation tests Heparin anticoagulant................................... for biochemical tests Food was removed overnight from animals to be sampled for laboratory investigations. The estimations performed on blood samples are listed overleaf, together with an abbreviated title (for use in appendices and tables). Haematology The following estimations were performed using a Bayer-Technicon HIE haematology analyser: Units Packed cell volume (PCV) Haemoglobin concentration (Hb) Erythrocyte count (RBC) Mean cell haemoglobin concentration (MCHC) Mean cell volume (MCV) Mean cell haemoglobin (MCH) Total leucocyte count (WBC total) % g/dl x 10'2/! g/dl fl pg x 109/! Differential leucocyte count Neutrophils (N) ) Lymphocytes (L) ) Eosinophils (E) ) Basophils (B) ) Monocytes (M) ) Large unstained cells (LUC) ) xl09/! 18 : Company Sanitized. Does not contain TSCA CBI Cell morphology: the most common morphological changes (anisocytosis, micro/macrocytosis, variation in colour, hypo/hyperchromasia, left shift, atypical/blast cells) were recorded as follows: = + = ++ = +++ = no abnormalities detected slight moderate marked In the case of atypical/blast cells, or other abnormalities, confirmation or a written description from a blood film was made. Platelet count (Pit) The following were performed using the appropriate methodology as described below: Prothrombin Time (PT) - Quick, A.J. (1942) Activated Partial Thromboplastin Time (APTT) Proctor, R.R. and Rapaport, S.I. (1961) Biochemistry The following parameters were analysed with a Hitachi 917 Clinical Chemistry Analyser: Total protein (Protein Total) Albumin (Alb) Globulin by subtraction (Glob) Albumin/Globulin ratio (A/G) Urea Nitrogen (Urea Nitr) Creatinine Sodium (Na) Potassium (K) Calcium (Ca) Inorganic Phosphorous (P) Chloride (Cl) Total Cholesterol (Chol) - (Enzymatic assay) : 19 : DPT 443/984760 Units xl09/! g/dl g/dl g/dl g/dl mg/dl mg/dl mEq/1 mEq/1 mEq/1 mEq/1 mEq/1 mg/dl Company Sanitized. Does not contain TSCA CBI Alkaline phosphatase (AP) Reaction temperature 37C Total bilirubin (Bili-rubin) Glucose (Hexokinase mediated assay) Alanine ammotransferase (GPT) also known as 'glutamic-pynivic transaminase' Reaction temperature 37C Aspartate aminotransferase (GOT) also known as 'glutamic-oxaloacetic transaminase' Reaction temperature 37C Gamma-glutamyi transpeptidase (jGT) Reaction temperature 37C DPT 443/984760 Units mU/ml mg/dl mg/dl mU/ml mU/ml mU/ml TERMINAL STUDIES Necropsy On completion of 4 weeks of treatment, all animals were killed (Day 29). All animals were killed by carbon dioxide asphyxiation and subjected to the following detailed necropsy procedure: 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 incision 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. The gastrointestinal tract was examined as a whole and the stomach and caecum were incised and examined. The lungs were removed and all pleural surfaces examined under suitable illumination. 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. 20 : Company Sanitized. Does not contain TSCA CBt DPT 443/984760 The following organs from all animals killed at the scheduled sacrifice were dissected free of fat and weighed: adrenals brain epididymides heart kidneys liver spleen testes thymus Preservation of tissues Samples of all the tissues listed below from all animals were preserved in buffered 10% formalin (except eyes, which were preserved in Davidson's fixative and testes and epididymides, which were preserved initially in Bouin's fluid). In addition, samples of any macroscopically abnormal tissues were routinely preserved, along with samples of adjacent tissue where appropriate. adrenals alimentary tract (oesophagus*, stomach duodenum, jejunum, ileum, caecum, colon, rectum) brain epididymides femur (with joint) head* (to preserve nasal cavity, paranasal sinuses, oral cavity, nasopharynx, middle ear, teeth and Zymbal's gland) heart kidneys liver lung (including bronchi) lymph nodes (mandibular and mesenteric) ovaries pancreas* prostate sciatic nerves seminal vesicles spinal cord spleen sternum* testes thymus thyroids (with parathyroids) trachea urinary bladder uterus (with cervix) vagina * Preserved only Histopathological examination Histopathological examination was performed on: The above specified list of tissues, including all macroscopically abnormal tissues from all animals in Groups 1 and 4. Liver, kidney, bone marrow and macroscopically abnormal tissues from all animals in Groups 2 and 3. The required tissues were embedded in paraffin wax and sections cut -.1 4 stained with haematoxylin and eosin. 5 micrometres were 21 : Company Sanitized. Does not contain TSCA CBI DPT 443/984760 The macroscopic and microscopic findings are presented in the Appendix by an automated data collation system. Particular care is taken during tissue removal and processing to ensure recovery and sectioning of all protocol-scheduled tissues. Understandably, omissions or irregularities can occasionally occur, in rodents the most vulnerable tissues in this regard being parathyroid, thymus, male mammary gland and autolysed portions of the gastrointestinal tract. For each animal, any tissue so affected is listed as not seen. The abbreviation 'WNL' indicates that a macroscopically abnormal tissue was within normal limits upon histopathological examination. STATISTICAL ANALYSIS All statistical analyses were carried out separately for males and females. For all parameters, the analyses were carried out using the individual animal as the basic experimental unit. Bodyweight data were analysed using weight gains. The following sequence of statistical tests was used for bodyweight, clinical pathology and organ weight data: If the data consisted predominantly of one particular value (relative frequency of the mode exceeded 75%), the proportion of animals with values different from the mode was analysed, Fisher (1950) and Mantel (1963). Otherwise: A test was applied to test for heterogeneity of variance between treatments, Bartlett (1937). n Where significant (at the 1% level) heterogeneity was found, a logarithmic transformation was tried to see if a more stable variance structure could be obtained. If no significant heterogeneity was detected (or if a satisfactory transformation was found), a one-way analysis of variance was carried out. If significant heterogeneity of variance was present, and could not be removed by a transformation, an analysis of ranks was used, Kruskal- Wallis (1952/3). Analyses of variance was followed by Student's (test and Williams test (Williams 1971/2) for a dose-related response, although only the one thought most appropriate for the response pattern observed was reported. The Kruskal-Wallis analyses were followed by the nonparametric equivalents of these tests (Shirley, 1977). For organ weight data, analysis of variance was performed using terminal bodyweight as covariate when the within group relationship between organ weight and bodyweight was significant at the 10% level. Summary statistics (eg means and standard deviations) presented in the report were calculated from computer-stored individual raw data. The summary statistics and the individual data were stored in the computer to a certain number of decimal places, different for each parameter. For presentation purposes, however, they are usually rounded to fewer places. It will therefore, not in general be possible to reproduce the presented means and standard deviations exactly using the presented individual data. 22 Company Sanitized. Does not contain TSCA CBI DPT 443/984760 LOCATION OF STUDY RECORDS 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 have been lodged in the Huntingdon Life Sciences Ltd Archives, England. Such specimens and records will be retained for a minimum period of 5 years from the date of issue of the final report. At the end of the 5 year retention period the Client will be contacted and advice sought on the future requirements. Under no circumstances will any item be discarded without the Client's knowledge. PROCEDURES The procedures used during the study were those documented in the relevant Huntingdon Life Sciences Procedures Manuals. DEVIATIONS FROM PROTOCOL There were no deviations from the protocol or subsequent amendments that were considered to have affected the integrity of the study. However, the following deviation occurred: On arrival, 5 male animals were selected at random and used for health check purposes rather than the protocol specified 4 animals. 23 Company Sanitized. Does not contain TSCA CBI 9 RESULTS DPT 443/984760 FORMULATION CHEMISTRY (page 179) Prior to treatment, the stability during ambient storage for 2 days and refrigerated storage for 15 days fwnfl^fUfwa was confirmed 400 mg/ml. ' ^ aqueous formulations, at nominal concentrations of 5 and ' The mean concentrations Ei^lU^BUB111 test formulations analysed during the study were within 7% of nominal concentrations confirming the accuracy of formulations. MORTALITY (Appendix 5) There were no unscheduled deaths during the study. CLINICAL SIGNS (Appendix 5) Salivation, immediately after dosing, was noted for males and females receiving 150 ing/kg/day occasionally. As this finding was transient in nature, it is considered likely that the salivation is a reaction to poor palatability of the test substance rather than any lexicological response. No significance is attached to this finding. BODYWEIGHT (Figure 1, Table 1, Appendix 1) Markedly lower group mean bodyweight gains were noted over the treatment period for males and females receiving 150 mg/kg/day, compared with controls with statistical significance attained. A statistically significant lower bodyweight gain was also noted for males and females receiving 50 mg/kg/day. At 150 mg/kg/day, the decrease in group mean bodyweight gain of approximately 60% for both sexes indicated that this dosage was approaching the MTD for rats over 4 weeks. As gains were recorded in Week 4, tfae MTD was considered not to have been exceeded, particularly as no severe clinical signs were noted. The group mean bodyweight gain for males and females receiving 15 mg/kg/day was considered to be comparable with that of the controls. FOOD CONSUMPTION (Table 2) The cumulative food intake over the 4 week treatment period of the males and females receiving 150 mg/kg/day was lower than that of the controls. The food intake of the males and females receiving 15 or 50 mg/kg/day was considered to be comparable to that of controls. : 24 : Company Sanitized. Does not contain TSCA CBI DPT 443/984760 EFFICIENCY OF FOOD UTILISATION (Table 3) Overall efficiencies of food utilisation over the 4 weeks of treatment were inferior for males and females receiving 150 mg/kg/day, in comparison with controls, reflecting the markedly lower bodyweight gain noted for these groups. NEUROBEHAVIOURAL SCREENING (page 116) There were no indications of adverse effects on parameters attributable 1 BAEMATOLOGY (Table 4, Appendix 2) In male and female rats dosed with 150 mg/kg/da: microcytic anaemia and inflammation. iaematologic changes indicated Male and female rats dosed with 150 mg/kg/day had lexicologically significant microcytic anaemia, indicated by decreased mean red cell mass parameters (Hb, PCV and RBC) and decreased mean MCV. Statistically significant decreased mean red cell mass parameters also occurred in female rats dosed with 50 mg/kg/day; these changes were minor and were not lexicologically significant. Generally, anaemia occurs when haemoglobin synthesis is depressed below the level needed to maintain red cell mass. Decreased MCH was also observed (statistically significant in females dosed with 150 mg/kg/day); this calculated parameter generally decreases concurrently with MCV when decreased red cell mass is present. Renal damage may have exacerbated the anaemia seen in this study by three mechanisms. Renal damage may have resulted in small amounts of blood loss in urine. Renal insufficiency may have caused decreased production of erythropoietin, decreasing the rate of red cell production. Inflammation probably also contributed to the decreased red cell mass by mechanisms included in the term "anaemia of chronic disease". Inflammation was evident in the leukogram (leukocytosis with neutrophilia) and histologically (interstitial nephritis). Male and female rats dosed with 150 mg/kg/day had increased total mean leukocytes (statistically significant only in male rats). The leukocytosis was due primarily to statistically significant increased neutrophils. These leukocyte changes are consistent with inflammation, and are consistent with renal changes observed microscopically. Statistically significant changes in other haematologic parameters were not considered lexicologically significant because the degree of change was small or the change was in a direction not considered lexicologically significant. These included: Increased Pit in males (150 mg/kg/day) Increased Eosinophils in females (150 mg/kg/day) The NOEL for haematology was 50 mg/kg/day based on the presence of anaemia at 150 mg/kg/day in both male and female rats. 25 Company Sanitized. Does not contain TSCA CBI DPT 443/984760 BIOCHEMISTRY (Table 5, Appendix 3) In male and female rats dosed with 150 or 50 mg/kg/day, clinical chemistry parameters indicated decreased renal function and hyperbilimbinaemia. Male and female rats dosed with 150 mg/kg/day had evidence of decreased renal function. Increased urea nitrogen, creatinine, and inorganic phosphorus are indicated of decreased glomerular filtration rate. Male and female rats dosed with 50 mg/kg/day also had increased urea nitrogen; this change was of sufficient magnitude to be considered lexicologically significant. Increased urea nitrogen for males which had received 15 mg/kg/day achieved statistical significance but was considered to be of no biological significance. Decreased glomerular filtration can be caused by renal damage or can be secondary to extrarenal changes such as hypovolaemia or dehydration. Renal damage is most likely responsible for the decreased glomerular filtration rate in this study, based on the presence of microscopic lesions in the kidneys, and the lack of supporting evidence of dehydration. There were mild changes in potassium, calcium, sodium and chloride that occurred in male and female rats in the high dose group (Text Table). Although the changes were minor and probably no adverse, they are suggestive of alterations in acid/base and electrolyte homeostasis by the kidneys, and are consistent with other changes noted in this study. Sex Sodium Potassium Calcium Chloride Male No change T T No change Female 4' T t 4- Hyperbilirubinaemia was noted in male and female rats dosed with 150 mg/kg/day. Rats in this group also had hepatocellular vacuolation and hypertrophy (see anatomic pathology report). Hyperbilirubinaemia was most likely caused by decreased biliary excretion secondary to the hepatocyte changes. Although ALP generally increases with cholestasis of sufficient degree to cause hyperbilirubinaemia, ALP inhibition by fluoride may have masked an increase. These changes were minimal but considered lexicologically significant. Statistically significant changes in other clinical chemistry parameters were not considered lexicologically significant because the degree of change was small, the change was in a direction not considered lexicologically significant, or the change was not dose-related. These included: Decreased glucose in females (15 and 50 mg/kg/day) Increased cholesterol in females (50 mg/kg/day) Decreased A/G ratio in females (150 mg/kg/day) The NOEL for chemistry was 15 mg/kg/day for both male and female rats based on the elevation in urea nitrogen in rats dosed with 50 mg/kg/day. 26 Company Sanitized. Does not contain TSCA CBI DPT 443/984760 ORGAN WEIGHTS (Table 6, Appendix 4) A higher and statistically significant group mean absolute kidney weight was apparent for males and females receiving 150 mg/kg/day, in comparison with the controls. In males receiving 150 mg/kg/day, a lower absolute group mean liver but a higher bodyweightadjusted group mean liver weight was seen. A higher group mean bodyweight-adjusted liver weight was apparent for females receiving 150 mg/kg/day, compared to the controls. For females receiving 150 mg/kg/day, a lower absolute heart, spleen and adrenal weight were noted, in comparison with the controls. In the absence of corroborative pathological findings, these variations in organ weight are of uncertain toxicological importance. The higher bodyweightadjusted brain weight seen for females receiving 150 mg/kg/day is unlikely to be related to treatment, since this organ weight is not affected by bodyweight changes. MACROSCOPIC PATHOLOGY (Table 7, Appendix 5) The macroscopic examination performed at termination revealed the following changes: Kidneys: Enlargement, pallor and swollen appearance was noted in 5/5 males and 5/5 female rats receiving 150 mg/kg/day compared with 0/5 male and 0/5 female control rats. Pale cortical foci were observed in 3/5 male and 2/5 female rats receiving 150 mg/kg/day compared with 0/5 male and 0/5 female control rats. Irregular cortical scarring was observed in 2/5 male and 5/5 female rats receiving 150 mg/kg/day compared with 0/5 male and 0/5 female control rats. Ureters and urinary bladder: Unilateral distension of the ureter and blood stained urine in the ureter and urinary bladder were seen in 1/5 male rats receiving 150 mg/kg/day compared with 0/5 male control rats. Liver: Pallor was observed in 3/5 male and 4/5 female rats receiving 150 mg/kg/day compared with 0/5 male and 0/5 female control rats. Adipose tissue: A reduction in adipose tissue was noted in 5/5 male and 5/5 female rats receiving 150 mg/kg/day compared with 0/5 male and 0/5 female control rats. Ovaries and uterus: A reduction in size of both was observed in 1/5 female rats receiving 150 mg/kg/day compared with 0/5 female control rats. The incidence and distribution of all the other findings were considered to fall within the expected background range of macroscopic changes. 27 : Company Sanitized. Does not contain TSCA CBI 9 DPT 443/984760 MICROSCOPIC PATHOLOGY (Table 8, Appendix 5) Treatment-related changes Kidneys - Evidence of nephrotoxicity was detected, at 150 mg/kg, day and to a lesser extent at 50 ing/kg/day, and was characterised by interstitial inflammation in the cortex, medulla and papilla, inflammatory casts in tubules and collecting ducts, cortical tubular necrosis, basophilia and hyperplasia in tubules and collecting ducts, tubular dilatation, papillary necrosis, collecting duct and urothelial hyperplasia. No effect was seen at 15 mg/kg/day. Dosage level (mg/kg/day) Interstitial inflammation in cortex, medulla and papilla Total 0 Minimal 0 Slight 0 Moderate 0 Inflammatory casts in tubules and collecting ducts Cortical tubular dilatation Total 0 Minimal 0 Slight 0 Moderate 0 Cortical tubular necrosis 0 Basophilia and hyperplasia in tubules and collecting ducts Total 0 Minimal 0 Slight 0 Moderate 0 Marked 0 Medullary tubular dilatation Total 0 Minimal 0 Moderate 0 Marked 0 Papillary tubular dilatation Total 0 Minimal 0 Slight 0 Moderate 0 Papillary necrosis 0 Collecting duct hyperplasia 0 Urothelial hyperplasia 0 Number of kidneys examined 5 Male 15 50 150 0 0 5** 0 0 0 1 0 0 0 3 0 0 0 1 0 5** 0 2 5** 0 0 2 0 0 0 0 5** 0 0 0 0 0 0 0 3 0 0 4* 5** 0 0 4* 0 0 0 0 0 0 0 0 4* 0 0 0 1 0 0 1 5** 0 0 1 0 0 0 0 4* 0 0 0 1 0 0 2 5** 0 0 2 0 0 0 0 4* 0 0 0 1 0 0 0 0 0 0 0 2 0 0 0 5** 0 5 5 5 5 Female 15 50 150 0 0 3 0 0 2 0 0 1 0 0 0 4* 0 4* 5** 0 3 0 0 1 4* 0 0 1 0 0 0 0 5** 5** 0 3 0 0 2 0 0 0 5** 0 0 0 0 1 5** 0 1 0 0 0 5** 0 0 0 0 1 5-* 0 1 1 0 0 4* 0 0 0 0 0 3 0 3 1 0 1 5** 5 5 5 ** p<0.01 * p<0.05 with Fisher's Exact Test These changes were considered associated with the increased organ weights, macroscopic findings of pale/irregular cortical scarring etc; and the increased number of circulating neutrophils. Company Sanitized. Does not contain TSCA CBI DPT 443/984760 Liver - Hepatocyte hypertrophy and fine vacuolation were detected at 150 nig/kg/day and con-elated with the macroscopic pale appearance and the increased bodyweight-adjusted liver weight. Male Female Dosage level (mg/kg/day) 0 15 50 150 0 15 50 150 Hepatocyte hypertrophy Total 0 Minimal 0 0 5** 0 0 05*4** Slight Hepatocyte fine vacuolation 0 0 0 2 0 0 0 Total 0 0 0 0 3 0 0 0 1 Minimal 0 0 5** 0 0 05*4** Slight Number of livers examined 00500500532500500500515 ** /><0.01 * /?<0.05 with Fisher's Exact Test Bone marrow (Femur/joint) - An increased incidence of prominent adipocytes was detected and 150 mg/kg/day and may have been related to the decreased erythrocyte parameters recorded. Other findings Ovaries/Uterus -- There was some evidence of possible perturbation of the oestrus cycle with sparse/few corpora lutea and myometrial/endometrial atrophy recorded for some animals. These changes are likely to have been related to the decreased food consumption, suppressed bodyweight gain and reduced adipose tissue noted macroscopically. Incidental findings All other changes described in the individual animal reports were considered spontaneous in origin and therefore of no toxicological importance. Conclusion Evidence ofnephrotoxicity was detected at 150 mg/kg/day and to a lesser extent at 50 mg/kg/day. In addition, hepatocyte hypertrophy and fine vacuolation and an increased incidence of prominent adipocytes in the bone marrow were also detected at 150 mg/kg/day. 29 : Company Sanitized. Does not contain TSCA CBI DISCUSSION AND CONCLUSION DPT 443/984760 The test substance,!----miyjwas administered by oral gavage once daily to groups of five male and five female rats for twenty eight consecutive days at dosage levels of 15, 50 or 150 mg/kg/day (expressed as solids, not as material supplied). A further group of control rats was administered the vehicle, distilled water, alone. Adverse changes in bodyweight gain, food consumption, haematology, biochemistry, organ weights and pathology were noted for animals receiving 150 mg/kg/day, and to a lesser extent for animals receiving 50 mg/kg/day. No behavioural changes attributable to treatment were noted. At 15 mg/kg/day, male blood urea nitrogen values were higher than control, consistent with a dosage related response although at a level considered to be of no biological significance. Other findings attributable to treatment were only noted at Y50~6f 50' ing/kg/day. Q, was joncluaed'tnat 15 fot'lHiUBf'F mg/kg/day represent the no observed adverse effect level (NOAEL) when administered orally for 28 days. l- me rat ~ Nephrotoxicity lesions noted at microscopic examination reflect an increased kidney weight at necropsy, and were often graded as of moderate severity. It is considered that these lesions were likely to be related to alterations in biochemical parameters (increased blood urea nitrogen, creatinine, bilirubin, potassium, calcium and phosphorus, and lower sodium and chloride concentrations). In addition, it is possible to speculate that the degree of renal damage may have resulted in impaired haemopoietin production, which would account for the general decrease in erythrocyte parameters and increased prominent adipocytes in the bone marrow at 150 mg/kg/day. The findings at 150 mg/kg/day were considered to be adverse in nature. According to the EEC f m j | ^ | s Council Directive 92/69/EEC Annex VI, Part n(D), as described in Commission Directive 93/2 I/EEC, labelling with the R48 risk phrase is appropriate. Hence labelled: R48/22: Harmful if swallowed. 30 Company Sanitized. Does not contain TSCA CBI REFERENCES DPT 443/984760 BARTLETT, M.S. (1937) Properties of sufficiency and statistical test Proa. Roy. Soc., A 160,268. FISHER, R.A. (1950) in: (Eds). Statistical Methods/or Research Workers. Oliver and Boyd, Edinburgh. KRUSKAL, W. H. and WALLIS, W.A. (1952) Use of ranks in one-criterion variance analysis. J. Amer. Statist. Ass., 47,583 - 621. KRUSKAL, W. H. and WALLIS, W.A. (1953) Errata. J. Amer. Statist. Ass., 48,907 - 912. MANTEL, N. (1963) Chi-square tests with one degree of freedom : Extensions of the Mantel Haenszel procedure. J. Amer. Stat. Ass., 58,690. PROCTOR, R.R. and RAPAPORT, S.I. (1961) The partial thromboplastin time with kaolin. Am. J. din. Path., 36,212. QUICK, A.J. (1942) in: (Eds). The Haemorrhagic Diseases and the Physiology ofHaemostasis. p.24. Lea & Fibiger, Philadelphia. SHIRLEY, E. (1977) A non-parametric equivalent of Williams' test for contrasting increasing dose levels of a treatment. Biometrics, 33,386 - 389. WILLIAMS, DA. (1971) A test for differences between treatment means when several dose levels are compared with a zero dose control. Biometrics, 27,103 - 117. WILLIAMS, DA. (1972) The comparison of several dose levels with a zero dose control. Biometrics, 28, 519 - 531. 31 : Company Sanitized. Does not contain TSCA CBI DPT 443/984760 s ^ e as o> 9 A g 5 I 611 o 0 n 32 Company Sanitized. Does not contain TSCA CBI DPT 443/984760 a ff a P E I U a 5 BO an "3 a o PQ 33 Company Sanitized. Does not contain TSCA CBI TABLE 1 Bodyweighte - group mean values (g) DPT 443/984760 Week -1 0 1 2 3 4 IM 2M Control 15 91 92 143 145 199 198 255 254 308 298 339 325 Group and dosage (mg/kg/day) 3M 4M IF 2F 3F 50 150 Control 15 50 90 93 141 151 193 176 233 207 278 217 300 226 92 91 90 135 136 132 169 169 160 192 193 183 215 212 197 229 225 210 4F 150 93 131 143 156 164 170 Gain (g/rat) 0-4 195 sd 15.1 % of control sd Standard deviation *pSQ.05,**p<0.0\ 180 24.4 92 Jr 158 21.0 81 ** 75 28.3 38 * ** 94 90 77 39 11.3 8.2 7.9 13.5 96 82 41 34 : Company Sanitized. Does not contain TSCA CBI TABLE 2 Food consumption - group mean values (g/rat/week) DPT 443/984760 Week Group and dosage (nig/kg/day) 1M 2M 3M 4M IF 2F 3F 4F Control 15 50 150 Control 15 50 150 -1 145 154 148 158 136 137 119 125 1 181 190 172 134 146 145 140 105 2 198 206 176 147 139 138 134 103 3 210 203 199 136 148 146 139 108 4 182 173 163 116 132 126 121 94 1-4 Total 771 % of control 772 710 533 100 92 69 565 No statistical analysis performed (only one cage/sex/group) 555 534 410 98 95 73 35 : Company Sanitized. Does not contain TSCA C81 TABLES Food conversion ratios - group mean values DPT 443/984760 Week 1M 2M Control 15 Group and dosage (mg/kg/day) 3M 4M IF 2F 3F 50 150 Control 15 50 1 3.3 3.6 3.4 5.2 4.3 4.3 5.1 2 3.5 3.7 4.3 4.8 6.0 5.8 5.7 3 4.0 4.6 4.4 12.6 6.5 7.5 10.3 4 6.0 6.3 7.6 13.6 9.4 9.7 9.5 1-4 4.0 4.3 4.5 7.1 6.0 6.2 6.9 Food conversion ratio = food consumption/bodyweight gain 4F 150 8.5 7.9 13.2 17.3 10.5 36 : Company Sanitized. Does not contain TSCA CBI TABLE 4 Haematology - group mean values DPT 443/984760 Day 29 Group/ dosage rag/kg/day 1M Control 2M 15 3M 50 PCV Hb % g/dl 44.3 15.2 43.9 15.0 42.6 14.6 RBC MCHC MCV MCH 1012/! g/dl fl pg 7.86 34.2 56.4 19.3 7.72 34.3 56.9 19.5 . 7.57 34.3 56.3 19.3 Pit 109/I 1067 1034 1094 PT s 13.0 13.5 13.1 APTT s 18.4 18.5 19.5 4M ** ** ** * * 150 36.1 12.5 6.66 34.7 54.2 18.8 1277 (13.5)(13.9) IF Control 42.9 15.0 7.81 35.0 55.1 19.3 1104 13.6 15.9 2F 15 41.6 14.4 7.54 34.7 55.2 19.2 1069 14.1 16.6 3F * * * 50 39.7 14.0 7.25 35.4 54.9 19.4 1175 14.0 15.5 4F ** ** ** * * 150 34.9 12.4 6.75 35.5 51.8 18.4 1304 (13.3)(14.3) *p<Q.05,**p<0.0\ () Figures in parentheses - mean of only 2 values for Group 4 M and result of only one analysis for Group 4F 37 : Company Sanitized. Does not contain TSCA CBI TABLE 4 (Haematology - continued) DPT 443/984760 Day 29 Group/ dosage ing/kg/day WBC Total 109/! 1M Control 2M 15 3M 50 4M 150 13.07 12.69 11.16 * 19.74 M 109/! L 109/! E 109/! B 109/! M 109/! LUC 109/! 1.66 10.69 0.11 0.04 0.32 0.25 1.90 10.15 0.11 0.05 0.29 0.20 1.62 * * 6.55 8.99 12.16 0.08 0.15 0.04 0.08 0.27 0.47 0.17 0.33 IF Control 9.28 2F 15 6.83 3F 50 9.06 4F 150 13.01 *p0.05,**p0.0l 0.87 1.32 1.41 ** 3.79 7.97 5.15 7.17 8.62 0.09 0.07 0.11 * 0.14 0.03 0.02 0.03 0.04 0.18 0.18 0.22 0.26 0.14 0.08 0.13 0.16 38 : Company Sanitized. Does not contain TSCA CBI TABLES Biochemistry - group mean values DPT 443/984760 Day 29 Group/ Glu- Protein g/dl A/G Urea Creat- AP GPT GOT dosage mg/kg/day cose mg/dl _T_o_t_al__A_lb__G_lo_b Nitr inine m0/ mU/ mU/ mg/dl mg/dl ml ml ml 1M Control 88 6.3 3.3 3.0 1.08 11 0.5 491 52 95 2M 15 79 6.3 3.3 3.1 1.07 15 0.5 521 56 89 3M 50 84 6.4 3.3 3.0 1.11 19 0.6 553 52 92 4M 150 89 6.4 3.3 3.1 1.06 64 1.2 573 58 91 IF Control 107 6.2 3.4 2.9 1.17 18 2F + 15 93 6.4 3.5 2.9 1.18 17 3F + 50 90 6.4 3.4 2.9 1.17 25 4F * JT-* 150 98 6.3 3.3 3.0 1.07 90 *p<0.05, **p<0.01, Williams' test +pS0.05, Student's (test 0.6 377 47 81 0.6 331 42 86 0.6 298 43 95 1.5 449 47 91 39 : Company Sanitized. Does not contain TSCA CBI TABLES (Biochemistry - continued) DPT 443/984760 Day 29 Group/ dosage mg/kg/day 1M Control 2M 15 3M 50 4M 150 yGT Bili- Na mU/ rubin mEq/ nO mg/dl 1 <1 0.1 143 K Ca P Cl 1 1 1 1 mEq/ mEq/ BEq/ mEq/ 3.6 5.3 4.8 99 Chol mg/dl 74 Tri- glyc mg/dl 49 <1 O.I 143 3.5 5.3 4.8 99 77 55 <1 O.I 142 3.4 5.3 4.8 100 77 59 * * ** *-* <! 0.4 142 4.0 5.8 6.2 99 71 34 IF Control <1 O.I 142 3.6 5.2 3.8 102 65 33 2F 15 <! 0.1 143 3.4 5.2 4.0 101 67 23 3F 50 <! 0.1 143 3.5 5.2 3.9 102 104 27 4F * ir* * it ** ** ** 150 <2 0.2 138 4.5 5.8 6.5 97 79 30 *p.0.05, **pSO.Ol, Williams' test ++p<0.01. Student's? test , 40 : Company Sanitized. Does not contain TSCA CBI TABLE 6 Organ weights - group mean values DPT 443/984760 Terminal kill Group/ dosage ing/kg/day Body wt g Brain Thymus Heart 9 g g Liver Spleen Kidneys Adrenals Testes Epididy- nu.des g g g mo g g Unadjusted means 1M Control 309 1 .90 0.530 1.22 13.6 0.61 2.57 56.1 3.08 0.689 2M 15 302 1.92 0.515 1.11 13.3 0.63 2.64 53.1 3.16 0.716 3M 50 280 1 .90 0.470 1.07 13.2 0.54 2.49 46.6 3.12 0.692 m ** 150 206 1 .77 0.290 0.87 11.0 0.47 4.93 40.9 2.90 0.589 Adjusted means 1M 1.86 0.435 1.10 11.5 0.50 - 47.2 2.96 0.653 2M 1.89 0.440 1.01 11.6 0.55 - 46.0 3.07 0.688 3M - 1 .89 0.454 1.05 12.9 0.52 * 4M - 1 .84 0.476 1.11 15.3 0.68 45.1 58.5 3.10 3.14 0.686 0.660 p<0.05,"p<0.01 41 : Company Sanitized. Does not contain TSCA CBI TABLE 6 .(Organ weights - continued) DPT 443/984760 Terminal kill Group/ dosage ing/kg/day Body wt g Brain Thymus Heart g g a Liver Spleen Kidneys Adrenals g g g mg Unadjusted means IF Control 217 2F 15 214 3F 50 196 4F 150 157 Adjusted means 1.80 1 .86 1 .81 1.78 0.501 0.90 0.488 0.89 0.442 0.233 0.81 ** 0.65 8.8 0.45 1.85 9.7 0.50 1.92 9.0 0.45 * 8.7 0.37 2.05 ** 4.05 64.2 69.2 57.2 ** 40.3 IF - 1 .68 0.428 8.1 - - 2F - 1 .76 0.427 9.1 - - 3F 1.81 0.441 - 9.0 - - - 4F - 2 .00 0.368 10.0 *pS0.05,**p<Q.Ol 42 : Company Sanitized. Does not contain TSCA CBI TABLE 7 Macroscopic pathology incidence summary Group Group Group Group Group Group G Remo\. 1 reason: Terminal 1 2 3 4 1 2 3 Animals on study Animals completed ------------ Males ------------ ----------- Females Skin 5555 555 Alopecia 0 0 0 0 Tail Tip missing 000 ^ w Incisors Lower pale 0 0 1 0 0001 000 Thyrous Small 0 0 0 Lungs Congested 0 0 0. 1 0 0 0 0 0 0 0 Adipose Tissue Minimal 1 0 0 Liver 0 0 0 5 0 0 0 Median cleft, pale subcapsular area Pale Lobular markings accentuated 20 30 00 03 0 Stomach Antrum Mucosa 1 0 0 0 White nodule Kidneys 000100 00 001000 Increased pelvic dilatation TABLE 7 (Macroscopic pathology incidence summary - continued) Group Group Group Group Group Group Gr Removal reason: Terminal 1 2 3 4 1 2 3 Animals on study Animals completed ------------ Males -------------- 5 5 5 - 5 Kidneys 5555 555 Pale t Irregular cortical scarring Enlarged Swollen 0000 00 52 0 0 0 Pale cortical foci Misshapen (Continued) 5 0 0 0 Ureters 00031 000 Distended Contained blood stained urine Urinary Bladder 0 0 0 1 0 0 0 Contained blood stained urine Ovaries Small 0001 000 Uterus Fluid distension 00000000 030010030 Thin TABLE 8 Microscopic pathology incidence summary Group Group Group Group Group Group Gr Removal reason: Terminal 1 2 3 4 1 2 3 Animals on study Animals completed ----------- Females Trachea 5 5 5 5 Examined 5 5 5 No abnormalities detected Lungs(including Bronchi) 5 0 0 5 Examined 5 0 0 No abnormalities detected Pneumonitis (Total) Minimal 540100004150 5040 0 Vascular congestion (Total) u^ i Minimal 0 0 Heart Examined 1 0 0 No abnormalities detected 1 0 0 0 1 0 Thymus 5005 500 Examined No abnormalities detected Involution/atrophy (Total) Minimal 5 0 0 5 5 Lymph Nodes - Mandibular 0000 000 Examined 5 00 0 No abnormalities detected Lymph Nodes - Mesenteric 5 0 0 5 Examined 5 0 0 No abnormalities detected Spleen 5 0 0 5 500 Examined Missing N6 abnormalities detected 50500055 5000 Liver 5555 550505 Examined TABLE 8 (Microscopic pathology incidence summary - continued) Group Group Group Group Group Group G Removal reason; Terminal 1 2 3 4 Animals on study 1 2 3 Animals completed -------------- Males -------------- ---------- Females Liver (Continued) 5 5 5 5 5 5 5 No abnormalities detected j> Parenchyroal inflammatory cell foci (Total) Minimal 40 41 50 00 4 5 5 Hepatocyte vacuolation - median cleft Hepatocyte hypertrophy - generalised 0 0 0 (Total) Minimal t^. 1000 000 o\ Slight Hepatocyte fine vacuolation - 000-000 00 52 0 generalised(Total) 3 0 0 0 Minimal Slight Haemorrhage (Total) Minimal 0 5 000000320 0000 Kidneys 1 0 0 Examined No abnormalities detected 5 5 cInotrteerxs,mtietidaulllainfalnadmmpaatpioilnla(iTnotal) 2210 53525 Minimal 0 Slight Moderate 00 00 00 1351 0 0 0 Basophilia and hyperplasia of tubules and collecting ducts(Total) 0 0 0 Minimal Slight Moderate 0000450 005 Marked 3 Cortical tubular dilatation (Total) 00000241 0002 Minimal Slight 0005050 00004130 Moderate 5 5 TABLE 8 (Microscopic pathology incidence summary - continued) Group Group Group Group Group Group G Removal reason: Terminal 1 2 3 4 1 2 3 Animals on study Animals completed ----------- Males ------------ ----------- Females Kidneys (Continued) 5 5 5 5 5 5 5 Cortical fibrosis and tubular collapse with basophilia(Total) Minimal Papillary necrosis (Total) Minimal 1100 000 Slight 00010010000 000 ^ Pelvic dilatation (Total) Slight 0 0 Urothellal hyperplasia (Total) 0 0 0 Slight Moderate 000035 000 Marked Medullary tubular dilatation (Total) 0001 001 Minimal 0015 0 00 Moderate Marked 0 0 0 40 0 0 1 Papillary tubular dilatation (Total) Minimal 00024105 000 Slight Moderate 0 1 Cortical tubular necrosis (Total) Minimal 0000113 00000 Slight Cortico-medullary mineralisation (Toti.'' 2 0 0 0 Min. -nal Sligi.- Moderate 00 00 00 00 20 2 5 Inflammatory casts in tubules and 20 0 1 collecting ducts 0 0 0 5 0 0 033 Papillary collecting duct hyperplasia 0 0 0 2 TABLE 8 (Microscopic pathology incidence summary - continued) Group Group Group Group Group Group G Removal reason: Terminal 1 2 3 4 . Animals on study 1 2 3 Animals completed ------------ Males ------------ ----------- Females Kidneys 5 5 5 5 5 5 5 Cortical basophilic tubules (Total) Minimal I(nTtoetarls) titial inflammation in papilla Minimal (Continued) 3 2 0 0 1 2 0 Urinary Bladder 0 0 0 0 Examined 0 0 3 No abnormalities detected ^ 00 Ureters 5005 500 Examined Luminal dilatation (Total) Slight 00000011 000 Uterus Examined 0 0 0 No abnormalities detected 0 0 Luminal dilatation (Total) Moderate 00000000000000 5223103 Marked 0 Myometrial/endometrial atrophy 1 10 23 (Total) Minimal 1 Cervix 0000 000 Examined No abnormalities detected Epithelial mucification 0000 500 Vagina Examined 0 0 0 0 0 0 0 No abnormalities detected Ovaries 00000000 550000 Examined TABLE 8 (Microscopic pathology incidence summary - continued) Group Group Group Group Group Group Removal reason; Terminal 1 2 3 4 Animals on study Animals completed Ovaries 5 5 5 5 5 5 5 No abnormalities detected Sparse/few corpora lutea (Continued) Prostate 0 0 0 0 Examined 50 0 0 No abnormalities detected Seminal Vesicles Examined 5005 000 ^ No abnormalities detected Epididyraides 5005 0 0 0 Examined No abnormalities detected Tastes 5005 000 Examined No abnormalities detected Thyroids 5 0 0 5 Examined 0 0 0 No abnormalities detected Parathyroids 5005 500 Examined No abnormalities detected Adrenals 5005 500 Examined No abnormalities detected Cortii-. T. vacuolation (Total) Minimal 50045 500 Stomach 0 0 0 1 0 Examined 5 0 0 5 5 00 00 No abnormalities detected 1 2 ------------ Females 0 0 TABLE 8 (Microscopic pathology incidence summary - continued) Group Group Group Group Group Group . G Removal reason; Terminal 1 2 3 4 1 2 3 Animals on study Animals completed ------------ Males ------------ ----------- Females Duodenum Examined 5 5 5 5 5 5 5 Mo abnormalities detected Jejunum 5 0 0 5 5 0 0 Examined. No abnormalities detected g Ilaum(including Payer's Patch) 5005 500 Examined No abnormalities detected Caacum 5 0 0 5 5 Examined 0 0 No abnormalities detected Colon 5 0 0 5 500 Examined No abnormalities detected 5 0 Rectum 5 0 0 5 5 0 0 Examined No abnormalities detected Spinal Cord 5005 5 0 0 Examined No abnormalities detected Vacuolation (Total) Minimal 050000415 5000 Sciatic Nerve Examined 5 0 0 5 54 00 00 No abnormalities detected TABLE 8 (Microscopic pathology incidence summary - continued) Removal reason: Terminal Animals on study Animals completed Sciatic Nerve Degenerate fibres (Total) Minimal Brain Examined No abnormalities detected Femur/joint Examined No abnormalities detected Osteoarthrosis (Total) Minimal Marrow - prominent adipocytes Group Group Group Group ( 15 5Continue 25d) 5 355 455 055000000 055 5005005002513 Group Group G 152535 150000 5401500500 -a &S a T3 t o g ^SP "s o B i-i a\ w m ID ^* CM CM y? r-i en co m n n CM on r^ o w m en o CM cn r~~ CM fn en CM CM M 00 0 CM l> A CM in in r" in m CS] CM CM CM CM <D S co co CM n co r-i 0\ 0) 0 0 CD .-d r^ CM CM r-1 CM 00 m t-1 CO 0 q* ^' ^p LO fO T-l T-l f-1 1-1 (-1 *-( Cn ^' (T> CTl CO t oo cn co CT) co S' p it 52 '-1 Ll o a ^ E c : < c L| 0 d) Oi .Q re E 0 ; e ^ r- co or o r-t CM ^D n m o CM ^r n ^o CM in CM r^l n n ro n rt ^p co r-i in in m r-f CM en T-I ch n m CM (M u? o en \o r~- A! CM ^D r^ ("') in ^' 0) CM CM CM CM CM 01 B CM co in if) m r-f i-^ o 03 en en CM CM r-< r-l i-l CM f1- f0 fQ CM 0 tH v C^l w ^' *-) i-l *-( <-( ^ .-1 in ^r \D (M cr^ I CT^ CT^ CO ^ 00 s "Q.2 s >- 0 0 U i-l H in a) 6 - ^ E c : < c u <B (B o'.c 10 E U 3 C *-< CM fi <r in r-i 52 DPT 443/984760 co <M m "P~ r- ^* c\j n us CM r-f< CM CM CM CM ^" cr CM n co m r"- -< CM in 1-1 -1 CM CM CM CM n n o m CM A; CM CD 0 0 -W 0 < .-1 CM CM CM CM (1) s f-l n k0 ^r t^- *-t ^D r- r- o ^o -1 r-l r-l CM f-l en r* co CM co 0 .g* ^ ^t \o ^f -t t-i -i -<-< t-1 r^- 10 0 in co 1 0^ ^ 0\ 0^ GO ^s^ a3 . c -- 0 0 o2 -< n a) <u E J: ^ E c ; tt C n w <u oM 'aE U ; C ID r- co cn o T-< -( .-I -1 CM T r- m co in r" T OTt CO P") <--t ^D CM CM n n ' CM r- o in co n m m r- o on T CM CM r0 CM CM ^' 00 0 t-1 r^> ^3- ^c CM CM n ^o o IS CM (M CM CM CM <0 s r- oo o o o 0) 1-1 CO r-i 0 r-- .-< .-1 CM CM r-< CD o en in in 0 m ^* 'a' ^r n *-) i-i r-i *- <-i -( co o m oo CM 1 00 0^ 0"t 00 O't s^, & 00 E 0^ 1-1 t-1 10 0) -Si c ; ft C m <a <u BiX 10 E: U 3 C 1-1 CM m 'y in m Company Sanitized. Does not contain TSCA CBI DPT 443/984760 -q" CD f-1 CD ' t-n CM CM r-t CM CM CM CM CM CM o r^ en o r^ f0 CM 0 r-4 0 .-* CM CM CM CM CM a' 0 i-t CM CD A! <M o co o co 01 (1) CM .-1 CM r-1 r-t 0) s <3' o ^p -< cn *-t [-. \D r^ ^0 |^. r-t ^-( r-t r-i r-1 r- o co i^ CD 0 ^r CM m ro n <-t r-f *-t -< r-1 *-1 m o* o -( 10 ' ff\ CD 0\ 0^ 0^ 1-1 >--< ia aD e A -< E -I SE 0.5) B fe '5b 1--1 ai a 1 u Cf-C 3 n3 E u = I,5 0 ^ c X! I (I & I CL. < T a o CO n \D t-- CD Cn 0 CM CM CM CM n ^D cn CM v-t co en CM n en ^r t-i CM CM CM CM CM <3" in CM r-1 in 0 CM <-4 m 0 CM CM CM CM CM t0 CM ^0 CM r- ^< CM r^ o cn o CD <u <-t CM <-1 CM r-i 0) s m in ^' in co r-4 u? r- r- r- in a\ co t^- oo in 0 CM ro m rn m -( CO 0 CM <T 10 CO 01 d 01 01 gI -2 0 (-) 11 r-1 ^ 10 d] < C ^ <0 HI Cn.I 'O E u : C r-< CM n o* in CM CM CM (M CM m 53 -1 O' C*J P- W ^3* CO \D \D l^ ^0 *-1 i--l f-l i-l r-( w CM <r* M o en r^- u> in \D w r-f r-l t-f *-1 r-< w <3' in r** o ^ 0) ^D io in io io <u r-l .-1 T-f r-l .-1 <u s ^D CD in CM ^0 i-i ^r ^r ^> ^p r^) i-l *-1 T-f i-l r-t m CD oo CM t-i 0 ro m n CM CM r-l -) T-t r-l r-l 0\ T-l <3* y> 'xi r- CM ' 0^ (T\ 01 CD f.c.^^,1) ^fl) 0 0 0^ -1 ^-1 Hi < ) -^ i C ; rf C 1--1 (V tD 3!0'X C y? r- CD fT\ o ro cn co n T 00 r- CD in o en 'q' 0% 1-1 0 1-1 T-t r-1 CM CM CM CM i-i r~- v r-f r-1 m CD en co r^ T-I r-1 r-1 .H CM CM T T-I CM 01 0 ^ CM r^- co r'- 01 en B(U r-4 r-l i-| i-l r-1 s r- in o o ro i-l m in in r~ ^o t-t v-l -( r-t -< o vo n CM o ft 0 CM CM ^* ^* r-t r-l *-< r-1 -( t-f 1-1 o us in o ' 01 01 CD 01 01 I- u.' ^b o.'&i S 0^ .11 .-1 14- ia a1> ; C S d; c t.-1 ai aD o10 '.iE ' 0 : C i-l m (M m o n n 1-1 ^" in n ro r- Company Sanitized. Does not contain TSCA CBI APPENDIX 2 Haematology - individual values DPT 443/984760 Day 29 (8 January 1999) Group/ dosage ing/kg/day 1M Control Animal no. 1 2 3 4 5 PCV Hb % g/dl 42.7 44.3 46.1 44.5 44.1 14.9 15.1 15.6 15.2 15.1 RBC MCHC MCV MCH 1012/! g/dl fl pg 7.90 7.74 7.92 7.87 7.89 34.8 34.2 33.9 34.1 34.2 54.0 57.2 58.2 56.5 56.0 18.8 19.5 19.7 19.3 19.1 Pit 109/! 1000 970 1164 1051 1150 PT s 13.0 13.2 12.8 12.9 ctd APTT s 18.4 15.9 20.7 18.4 ctd Mean sd 44.3 15.2 7.86 34.2 56.4 19.3 1067 1.21 0.26 0.072 0.34 1.57 0.35 87.3 13.0 18.4 0.17 1.96 2M 6 44.9 15.4 8.07 34.4 55.7 19.1 1096 13.0 18.2 15 7 43.1 14.8 7.74 34.4 55.7 19.1 1199 13.5 20.7 8 44.9 15.0 7.84 33.4 57.3 19.1 1014 14.3 18.2 9 42.2 14.7 7.26 34.8 58.2 20.2 923 ctd ctd 10 44.2 15.2 7.68 34.4 57.5 19.8 940 13.3 16.9 Mean sd 43.9 15.0 7.72 34.3 56.9 19.5 1034 1.18 0.29 0.296 0.52 1.13 0.51 114.7 13.5 18.5 0.56 1.59 3M 11 43.5 14.9 7.80 34.3 55.8 19.1 1062 12.0 16.2 50 12 41.0 14.2 7.23 34.5 56.7 19.6 1104 13.6 23.2 13 42.4 14.4 7.47 34.1 56.7 19.3 1224 13.8 19.2 14 43.4 14.8 7.76 34.2 55.9 19.1 985 ctd ctd 15 ctd ctd ctd ctd ctd ctd ctd ctd ctd Mean sd 42.6 14.6 7.57 34.3 56.3 19.3 1094 1.16 0.33 0.267 0.17 0.49 0.24 99.8 13.1 19.5 0.99 3.51 4M 16 33.8 12.0 6.54 35.5 51.7 18.4 1434 13.3 12.4 150 17 38.4 13.4 6.95 34.8 55.3 19.2 1075 ctd ctd 18 38.0 13.2 6.85 34.8 55.5 19.3 1220 13.6 15.4 19 36.5 12.6 6.68 34.5 54.6 18.8 1133 ctd ctd 20 34.0 11.5 6.30 33.8 53.9 18.2 1524 ctd ctd Mean sd 36.1 12.5 6.66 34.7 54.2 18.8 1277 2.17 0.80 0.257 0.61 1.53 0.48 193.9 13.5 13.9 0.21 2.12 sd Standard deviation ctd Clotted sample 54 : Company Sanitized. Does not contain TSCA CBI APPENDIX! (Haematology - continued) DPT 443/984760 Day 29 (8 Januay 1999) Group/ dosage mg/kg/day Animal no. WBC Total 109/! N 109/! L 109/! E 109/! B 109/! M 109/! LUC 109/I 1M Control 1 2 3 4 5 Mean sd 13.26 14.45 13.89 12.66 11.07 13.07 1.302 1.05 1.95 1.45 1.61 2.22 1.66 0.452 11.22 11.8-7 11.75 10.29 8.31 10.69 1.468 0.10 0.12 0.07 0.18 0.08 0.11 0.044 0.05 0.03 0.05 0.05 0.02 0.04 0.014 0.46 0.24 0.27 0.35 0.28 0.32 0.088 0.38 0.22 0.30 0.19 0.16 0.25 0.089 2M 6 9.57 1.26 7.91 0.05 0.02 0.21 0.13 15 7 13.28 2.40 10.17 0.10 0.06 0.31 0.23 8 18.33 2.38 14.92 0.19 0.09 0.40 0.36 9 8.68 1.30 6.98 0.05 0.02 0.24 0.09 10 13.59 2.14 10.77 0.17 0.05 0.29 0.19 Mean sd 12.69 1.90 10.15 0.11 0.05 0.29 0.20 3.833 0.572 3.090 0.066 0.029 0.073 0.104 3M 11 9.50 1.72 7.00 0.08 0.03 0.41 0.26 50 12 9.43 1.49 7.61 0.03 0.03 0.18 0.10 13 14.13 1.49 12.02 0.11 0.06 0.27 0.18 14 11.58 1.77 9.32 0.09 0.03 0.21 0.14 15 ctd ctd ctd ctd ctd ctd ctd Mean sd 11.16 1.62 8.99 0.08 0.04 0.27 0.17 2.217 0.149 2.248 0.034 0.015 0.102 0.068 4M 16 30.42 9.93 18.64 0.27 0.17 0.83 0.59 150 17 13.69 3.01 10.26 0.03 0.04 0.17 0.17 18 15.18 4.21 10.05 0.07 0.06 0.51 0.29 19 18.51 6.23 11.35 0.25 0.07 0.34 0.27 20 20.88 9.39 10.49 0.11 0.06 0.49 0.34 Mean sd 19.74 6.55 12.16 0.15 0.08 0.47 0.33 6.601 3.066 3.657 0.108 0.051 0.244 0.157 sd Standard deviation ctd Clotted sample 55 : Company Sanitized. Does not contain TSCA CBI APPENDIX 2 (Haematology - continued) DPT 443/984760 Day 29 (8 January 1999) Group/ dosage mg/kg/day IF Control Ani-mal no. PCV Hb % g/dl 21 41.8 14.5 22 42.7 15.1 23 42.3 14.9 24 44.6 15.5 25 43.1 15.1 RBC MCHC MCV MCH 1012/! g/dl fi pg 7.24 7.92 7.98 7.71 8.19 34.7 35.4 35.3 34.7 34.9 57.7 54.0 53.0 57.9 52.7 20.0 19.1 18.7 20.1 18.4 Pit 109/I 1123 1097 1088 1054 1158 PT s 11.8 14.4 14.4 13.8 ctd APTT s 13.9 20.7 16.4 12.4 ctd Mean sd 42.9 15.0 7.81 35.0 55.1 19.3 1104 1.07 0.36 0.361 0.33 2.55 0.76 39.0 13.6 15.9 1.23 3.63 2F 26 42.4 14.7 7.64 34.7 55.5 19.2 1122 14.7 16.7 15 27 42.2 14.5 7.48 34.5 56.4 19.4 1109 14.5 16.0 28 41.5 14.3 7.34 34.4 56.6 19.5 908 ctd ctd 29 39.8 13.9 7.60 34.9 52.4 18.3 1100 13.0 17.1 30 42.1 14.8 7.64 35.2 55.1 19.4 1108 ctd ctd Mean sd 41.6 14.4 7.54 34.7 55.2 19.2 1069 1.06 0.36 0.130 0.32 1.68 0.49 90.6 14.1 16.6 0.93 0.56 3F 1031 39.6 14.0 7.31 35.5 54.1 19.2 1208 ctd ctd 50 32 40.4 14.4 7.44 35.7 54.3 19.4 1442 13.5 14.1 33 42.4 14.8 7.72 35.0 54.9 19.2 1102 14.1 18.4 34 38.9 13.8 7.23 35.5 53.9 19.1 1036 ctd ctd 35 37.3 13.2 6.53 35.4 57.1 20.2 1088 14.3 14.1 Mean sd 39.7 14.0 7.25 35.4 54.9 19.4 1175 1.88 0.61 0.441 0.26 1.31 0.45 161.7 14.0 15.5 0.42 2.48 4F 36 39.2 13.9 7.43 35.4 52.8 18.7 1130 13.3 14.3 150 37 33.6 12.2 6.73 36.3 49.9 18.1 1226 ctd ctd 38 32.4 11.5 6.25 35.4 51.7 18.3 1768 ctd ctd 39 36.2 12.7 7.08 35.0 51.2 17.9 1182 ctd ctd 40 33.3 11.7 6.24 35.2 53.3 18.8 1215 ctd ctd Mean sd 34.9 12.4 6.75 35.5 51.8 18.4 1304 2.77 0.96 0.520 0.50 1.34 0.38 261.9 13.3 14.3 sd Standard deviation ctd Clotted sample 56 : Company Sanitized. Does not contain TSCA CBI APPENDIX 2 (Haematology - continued) DPT 443/984760 Week 5 (8 January 1999) Group/ dosage nig/kg/day 1M Control Animal no. 1 2 3 4 5 2M 6 15 7 8 9 10 3M 11 50 12 13 14 15 4M 16 150 17 18 19 20 ctd Clotted sample Anis Micro Macro Var Hypo Hyper LS Atyp Blast + - - - -----+--- --+ -.- + - + - - - - - - - - - ---+-- --.++ - - - --+--- + - - - ctd ctd ctd ctd ctd ctd ctd ctd ctd -----+--- --+ - -------++----- -+-+---++++ -- ; - - ++ 57 : Company Sanitized. Does not contain TSCA CBI APPENDIX 2 (Haematology - continued) DPT 443/984760 Week 5 (8 January 1999) Group/ Animal Anis Micro Macro Var Hypo Hyper LS dosage no. ing/kg/day Atyp Blast --------++------ IF 21 -- Control 22 23 24 25 - - + ----+--- 2F 26. 15 27 --+--- 28 29 --------+++------ 30 + - - - ++ - - - 3F 1031 ++ - - - ------------++++++-------- 50 4F 150 32 33 34 35 ++ - - 36 ++ - - - 37 - + - - - ++ - . 38 39 40 ---+- ++-- -+++ +- - - -- 58 : Company Sanitized. Does not contain TSCA CBI APPENDIX! (Haematology - continued) DPT 443/984760 Day 29 (8 January 1999) Group/ dosage mg/kg/day Animal no. WBC Total 109/! N lO9/! L 109/! E 109/! B 109/! M 109/! LOC 109/! IF Control 21 5.82 0.69 4.88 0.05 0.01 0.13 0.06 22 14.71 1.07 12.95 0.13 0.06 0.27 0.23 23 9.37 1.26 7.66 0.12 0.03 0.16 0.14 24 9.26 0.64 8.16 0.06 0.04 .0.21 0.16 25 7.23 0.71 6.18 0.09 0.01 0.15 0.09 Mean sd 9.28 0.87 7.97 0.09 0.03 0.18 0.14 3.379 0.275 3.069 0.035 0.021 0.056 0.066 2F 26 5.31 0.73 4.24 0.05 0.01 0.22 0.06 15 27 6.73 1.11 5.10 0.08 0.02 0.30 0.12 28 7.00 1.61 5.06 0.09 0.01 0.14 0.08 29 7.65 1.09 6.27 0.07 0.02 0.13 0.07 30 7.44 2.04 5.09 0.08 0.02 0.13 0.08 Mean sd 6.83 1.32 5.15 0.07 0.02 0.18 0.08 0.921 0.512 0.724 0.015 0.005 0.075 0.023 3F 1031 10.30 1.03 8.81 0.09 0.04 0.21 0.13 50 32 6.84 1.27 5.20 0.14 0.01 0.13 0.09 33 10.23 1.78 7.75 0.15 0.04 0.35 0.17 34 8.87 2.51 5.92 0.07 0.02 0.22 0.13 35 9.06 0.46 8.17 0.08 0.02 0.20 0.14 Mean 5d 9.06 1.41 7.17 0.11 0.03 0.22 0.13 1.403 '0.777 1.539 0.036 0.013 0.080 0.029 4F 36 12.58 3.14 8.81 0.12 0.04 0.30 0.16 150 37 11.09 3.09 7.52 0.12 0.03 0.22 0.10 38 21.24 6.85 13.43 0.14 0.07 0.45 0.30 39 11.27 3.98 6.73 0.19 0.02 0.21 0.15 40 8.85 1.88 6.59 0.13 0.02 0.14 0.09 Mean sd 13.01 3.79 8.62 0.14 0.04 0.26 0.16 4.794 1.868 2.832 0.029 0.021 0.118 0.084 sd Standard deviation 59 : Company Sanitized. Does not contain TSCA CBI APPENDIX 3 Biochemistry - individual values DPT 443/984760 Day 29 (S January 1999) Group/ dosage ing/kg/day IM Control Animal no. 1 2 3 4 5 Mean sd 2M 6 15 7 8 9 10 Mean sd 3M 11 SO 12 13 14 15 Mean sd 4M 16 150 17 18 19 20 Mean sd sd Standard deviation Glu- Protein g/dl A/G cose mg/dl Total Alb Glob Urea Great- AP GET GOT Mitr inine m0/ m0/ mU/ mg/dl mg/dl ml ml ml 90 6.2 3.2 3.0 1.07 13- 79 6.4 3.2 3.2 1.00 9 S3 6.3 3.4 2.9 1.17 11 100 6.3 3.3 3.0 1.10 10 86 6.2 3.2 3.0 1.07 11 0.5 519 47 103 0.5 557 51 82 0.5 511 48 94 0.5 421 54 97 0.5 446 59 98 88 6.3 3.3 3.0 1.08 11 8.0 0.08 0.09 0.11 0.061 1.5 0.5 0.00 491 52 95 55.8 4.9 7.9 79 6.4 3.3 3.1 1.06 13 89 6.5 3.3 3.2 1.03 17 74 6.4 3.3 3.1 1.06 12 68 6.1 3.2 2.9 1.10 17 87 6.3 3.3 3.0 1.10 18 0.5 508 48 83 0.6 573 57 85 0.5 504. 59 85 0.6 515 52 90 0.5 507 65 104 79 6.3 3.3 3.1 1.07 15 8.8 0.15 0.04 0.11 0.030 2.7 0.5 521 56 89 0.05 29.1 6.5 8.6 83 6.3 3.3 3.0 1.10 20 93 6.5 3.4 3.1 1.10 17 94 6.2 3.3 2.9 1.14 20 76 6.5 3.2 3.3 0.97 23 76 6.3 3.5 2.8 1.25 16 0.5 437 49 ?0 0.6 546 51 97 0.6 466 56 79 0.6 689 53 97 0.6 629 50 97 84 6.4 3.3 3.0 1.11 19 8.8 0.13 0.11 0.19 0.100 2.8 0.6 553 52 92 0.04 1 06.5 2.8 7.9 90 6.1 3.3 2.8 1.18 67 91 6.2 3.2 3.0 1.07 45 72 6.5 3.3 3.2 1-03 58 82 6.7 3.3 3.4 0.97 79 110 ^6.4 3.3 3.1 1.06 73 1.1 569 59 93 0.8 648 62 104 1.1 616 59 91 1.6 463 46 85 1.4 567 62 84 89 14.0 6.4 3.3 3.1 1.06 64 0.24 0.04 0.22 0.077 13.3 1.2 573 58 91 0.31 70.0 6.7 8.0 60 : Company Sanitized. Does not contain TSCA CBI APPENDIX 3 (Biochemistry - continued) DPT 443/984760 Week 5 (S January 1999) Group/ dosage eg/kg/day ftnimal no. 1M 1 Control 2 3 4 5 Mean sd 2M 6 15 7 8 9 10 Mean sd 3M 11 50 12 13 14 15 Mean sd 4M 16 150 17 18 19 20 Mean sd sd Standard deviation 1 1 1 1 1 yGT Bili- Ha m0/ rubin mEq/ ml mg/dl K Ca P Cl Cbol mEq/ mEq/ mEq/ mEq/ Tri- glyc mg/dl mg/dl <1 0.1 143 3.7 5.2 5.1 99 65 41 <1 O.I 143 3.7 5.3 4.6 99 68 42 <1 O.I 144 3.5 5.3 5.0 100 89 47 <1 O.I 143 3.7 5.4 4.7 100 75 54 1 0.1 143 3.3 5.1 4.7 97 73 63 <! 0.1 143 3.6 5.3 4.8 99 74 49 0.00 0.4 0.18 0.11 0.22 1.2 9.3 9.2 <1 O.I 142 3.5 5.3 4.6 98 64 60 <1 O.I 142 3.4 5.5 4.5 99 68 54 <! 0.1 145 3.6 5.4 4.9 101 81 69 <1 O.I 144 3.7 5.2 4.8 100 79 35 <! 0.1 142 3.5 5.3 5.3 99 93 56 <1 O.I 143 3.5 5.3 4.8 99 77 55 0.00 1.4 0.11 0.11 0.31 1.1 11.5 12.5 1 0.1 143 3.3 5.5 4.6 98 122 78 <1 O.I 141 3.4 5.2 5.1 99 72 43 1 0.1 143 3.3 5.3 4.7 100 58 65 <1 O.I 142 3.4 5.3 4.9 99 72 55 <1 O.I 143 3.8 5.3 4.8 102 62 52 <1 O.I 142 3.4 5.3 4.8 100 77 59 0.00 0.9 0.21 0.11 0.19 1.5 25.8 13.4 <! 0.3 143 3.7 5.7 6.9 100 68 49 1 0.2 143 3.6 5.4 5.2 101 64 37 <! 0.3 141 4.4 5.9 6.2 97 40 9 1 0.4 141 4.0 6.0 6.1 96 96 27 1 0.6 143 4.1 5.8 6.4 99 85 46 <! 0.4 142 4.0 5.8 6.2 99 71 34 0.15 1.1 0.32 0.23 0.62 2.1 21.4 16.2 61 : Company Sanitized. Does not contain TSCA CBI APPENDIX 3 (Biochemistry - continued) DPT 443/984760 Day 29 (S January 1999) Group/ dosage mg/kg/day Animal no. 1M 21 Control 22 23 24 25 Mean sd 2M 26 15 27 28 29 30 Mean sd 3M 1031 50 32 33 34 35 Mean sd M 36 150 37 38 39 40 Mean sd sd Standard deviation Glu- Protein g/dl A/G cose mg/dl Total Alb Glob Urea Great- AP GPT GOT Nitr inine m0/ m0/ mil/ mg/dl mg/dl ml ml ml 103 6.3 3.4 2.9 1.17 18 0.6 368 40 85 101 6.0 3.3 2.7 1.22 21 0.6 389 35 75 91 6.1 3.2 2.9 1.10 16 0.6 331 34 84 118 .6.5 3.5 3.0 1.17 14 0.5 357 43 83 121 6.2 3.4 2.8 1.21 19 0.6 441 85 79 107 6.2 3.4 2.8 1.17 18 0.6 377 47 81 12.5 0.19 0.11 0.11 0.047 2.7 0.04 41.3 21.3 4.1 102 6.3 3.5 2.8 1.25 20 0.6 247 39 75 82 6.2 3.3 2.9 1.14 15 0.5 399 43 90 103 6.5 3.5 3.0 1.17 15 0.6 422 51 91 85 6.5 3.4 3.1 1.10 18 0.6 357 39 94 91 6.5 3.6 2.9 1.24 16 0.6 228 36 81 93 6.4 3.5 2.9 1.18 17 0.6 331 42 86 9.6 0.14 0.11 0.11 0.064 2.2 0.04 88.4 5.8 7.9 90 6.6 3.4 3.2 1.06 19 0.6 424 48 108 81 6.5 3.4 3.1 1.10 24 0.6 269 37 90 85 6.4 3.5 2.9 1.21 24 0.6 249 41 107 91 6.5 3.5 3.0 1.17 24 0.6 303 51 85 102 5.8 3.3 2.5 1.32 34 0.7 245 37 84 90 6.4 3.4 2.9 1.17 25 0.6 298 43 95 7.9 0.32 0.08 0.27 0.101 5.5 0.04 74.1 6.4 11.8 109 6.5 3.4 3.1 1.10 64 1.1 359 49 91 99 6.5 3.4 3.1 1.10 103 1.9 464 54 96 95 6.3 3.2 3.1 1.03 98 1.8 534 38 113 96 6.0 3.1 2.9 1.07 105 1.6 576 47 76 91 6.2 3.2 3.0 1.07 81 1.3 313 45 78 98 6.3 3.3 3.0 1.07 90 1.5 449 47 91 6.8 0.21 0.13 0.09 0.029 17.4 0.34 112.0 5.9 15.0 62 : Company Sanitized. Does not contain TSCA CBI APPENDIX 3 (Biochemistry - continued) DPT 443/984760 Day 29 (8 January 1999) Group/ dosage mg/kg/day IF Control animal no. 21 22 23 24 25 Mean sd 2F 26 15 27 28 29 30 Mean sd 3F 1031 50 32 33 34 35 Mean sd 4F 36 150 37 38 39 40 Mean sd sd Standard deviation yGT Bill- Na m0/ rubin mEq/ ml mg/dl 1 <1 0.1 142 <! 0-1 141 <1 O.I 143 <! 0.2 144 <! 0.2 142 K Ca P Cl 1 1 1 1 mEq/ mEq/ niEq/ raEq/ 3.6 5.1 3.4 103 3.8 5.3 4.0 101 3.4 5.1 4.0 101 3.6 5.4 3.6 103 3.6 5.2 3.9 102 Chol mg/dl 77 69 59 62 56 Tri- glyc mg/dl 26 54 25 38 20 <1 O.I 142 3.6 5.2 3.8 102 0.05 1.1 0.14 0.13 0.27 1.0 65 33 8.4 13.7 1 0.1 144 3.5 5.2 4.4 102 58 19 <1 O.I 141 3.6 5.2 4.2. 101 62 25 <1 O.I 144 3.2 5.2 3.4 103 93 32 <1 O.I 142 3.5 5.3 4.3 100 63 17 <1 O.I 142 3.4 5.2 3.7 101 60 24 <1 O.I 143 3.4 5.2 4.0 101 67 23 0.00 1.3 0.15 0.04 0.43 1.1 14.5 5.9 1 0.1 141 3.1 5.4 3.1 100 131 30 <1 O.I 144 3.8 5.2 4.0 103 110 22 <1 O.I 144 3.7 5.2 4.3 102 96 27 <1 O.I 143 3.2 5.2 3.7 101 79 26 1 0.2 143 3.7 5.2 4.5 103 105 29 <! 0.1 143 3.5 5.2 3.9 102 104 27 0.04 1.2 0.32 0.09 0.55 1.3 19.1 3.1 <! 0.2 138 4.5 5.6 5.5 99 42 28 <! 0.3 134 4.7 5.9 7.2 92 87 32 2 0.3 138 4.8 5.9 7.1 96 86 27 2 0.2 137 4.4 5.7 6.4 96 103 35 <1 O.I 142 3.9 5.8 6.1 102 76 29 <2 0.2 138 4.5 5.8 6.5 97 79 30 0.08 2.9 0.35 0.13 0.71 3.7 22.7 3.3 63 : Company Sanitized. Does not contain TSCA CBI APPENDIX 4 Organ weights - individual values DPT 443/984760 Terminal kill Group/ dosage mg/kg/day Animal no. 1H 1 Control 2 3 4 5 Body Brain Thymus Heart Liver Spleen wt gggggg 309 1.87 325 1.94 0.569 l.n 0.587 1.32 13.6 0.58 14.8 0.77 295 1.91 0.495 1.10 12.5 0.54 319 1.92 0.639 1.25 14.7 0.63 298 1.86 0.362 1.26 12.5 0.54 Kidneys g 2.82 2.85 2.59 2.50 2.09 Adrenals ng 72.9 58.5 51.0 52.5 45.7 Testes Epididymides g g 2.95 0.759 3.11 0.670 3.0a 0.676 2.99 0.644 3.26 0.696 Mean sd 309 1.90 0.530 1.22 13.1 0.035 0.1074 0.084 13.6 0.61 2.57 1.12 0.095 0.306 56.1 10.43 3.08 0.689 0.121 0.0433 2M 6 295 1.87 0.476 1.13 12.1 0.67 2.75 51.6 2.93 0.760 15 7 309 2.00 0.605 1.11 12.3 0.61 2.59 47.6 3.12 0.652 8 333 1.94 0.638 1.27 15.2 0.74 3.06 67.9 3.30 0.753 9 298 1.94 0.421 1.02 12.7 0.65 2.45 56.7 3.37 0.731 10 275 1.87 0.437 1.01 14.5 0.51 2.37 41.8 3.10 0.686 Mean sd 302 1.92 0.515 1.11 21.3 0.054 0.0996 0.105 13.3 0.63 2.64 1.37 0.083 0.273 53.1 9.90 3.16 0.716 0.176 0.0462 3M 11 278 1.91 0.335 1.07 11.9 0.48 2.23 45.1 3.18 0.670 50 12 268 1.96 0.405 1.09 11.8 0.49 2.44 49.1 3.07 0.703 13 308 1.90 0.573 1.13 16.1 0.64 2.64 ,, 44.9 3.38 0.732 14 299 1.96 0.594 1.11 14.4 0.64 2.76 55.4 3.19 0.757 15 248 1.76 0.442 0.93 12.0 0.45 2.38 38.3 2.80 0.597 Mean sd 280 1.90 0.470 1.07 24.0 0.084 0.1109 0.080 13.2 0.54 2.49 1.94 0.091 0.212 46.6 6.28 3.12 0.692 0.214 0.0622 4M 16 162 1.79 0.241 0.70 8.1 0.46 3.64 29.1 2.74 0.566 150 17 203 1.71 0.309 0.84 9.8 0.35 3.74 39.8 2.90 0.599 18 217 1.78 0.315 0.93 12.3 0.45 4.65 48.7 3.00 0.594 19 246 1.80 0.341 0.97 14.2 0.67 7.49 48.6 2.85 0.637 20 202 1.74 0.244 0.91 10.8 0.41 5.13 38.5 3.02 0.549 Mean sd 206 1.77 0.290 0.87 30.6 0.036 0.0450 0.107 11.0 0.47 4.93 2.35 0.123 1.561 40.9 8.16 2.90 0.589 0.115 0.0338 sd Standard deviation 64 : Company Sanitized. Does not contain TSCA CBI APPENDIX 4 (Organ weights continued) DPT 443/984760 Terminal kill Group/ dosage ng/kg/day IF Control Aninial no- 21 22 23 24 25 Body Brain Tnyrous Heart Liver Spleen wt 999999 208 1.73 224 1.80 217 1.87 237 1.89 200 1.72 0.504 0.564 0.475 0.522 0.438 0.90 0.85 1.03 0.91 0.83 8.6 0.38 8.4 0.50 8.8 0.56 9.6 0.46 8.8 0.37 Kidneys 9 1.96 1.69 2.00 1.92 1.70 Adrenals "9 63.3 57.8 74.5 60.1 65.5 Mean sd 217 1.80 0.501 0.90 14.3 0.075 0.0476 0.077 8.8 0.45 1.85 0.46 0.081 0.148 64.2 6.45 2F 26 223 2.01 0.543 0.86 10.2 0.40 2.02 61.9 15 27 210 1.89 0.548 0.94 9.1 0.52 1.91 62.8 28 211 1.82 0.413 0.88 10.8 0.53 1.69 80.1 29 211 1.68 0.352 0.85 9.1 0.49 1.92 73.1 30 215 1.92 0.5B3 0.94 9.6 0.55 2.08 68.2 Mean sd 214 1.86 0.488 0.89 5.3 0.122 0.0997 0.044 9.7 0.50 1.92 0.72 0.059 0.149 69.2 7.57 BESO C 1031 32 33 34 35 188 1.69 201 1.93 161 1.76 207 1.88 204 1.81 0.470 0.440 0.366 0.509 0.425 0.73 0.81 0.74 0.90 0.87 7.9 8.6 8.6 10.7 9.1 0.44 0.44 0.46 0.45 0.44 1.79 2.15 2.10 2.30 1.92 43.5 64.5 55.3 62.3 60.4 Mean sd 196 1.81 0.442 0.81 11.3 0.096 0.0532 0.078 9.0 0.45 2.05 1.05 0.010 0.199 57.2 8.38 IF 36 166 1.74 0.275 0.67 8.7 0.40 3.72 44.0 150 37 151 1.86 0.153 0.75 8.8 0.39 2.83 48.4 38 151 1.64 0.223 '0.59 8.6 0.37 3.88 32.3 39 163 1.86 0.284 0.72 9.0 0.36 5.47 31.2 40 153 1.80 0.232 0.74 8.4 0.32 4.36 45.5 Mean sd 157 1.78 0.233 0.69 7.1 0.094 0.0521 0.068 8.7 0.37 4.05 0.23 0.031 0.968 40.3 7.96 sd Standard deviation 65 : Company Sanitized. Does not contain TSCA CBI APPENDIX 5 Individual clinical and pathological findings DPT 443/984760 In this appendix the clinical, macroscopic and microscopic findings relating to each animal are listed. The initial examination was undertaken by the study pathologist, die 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: David J Lewis, Ph.D., F.R.C.Path., Consultant Pathologist Department of Pathology Peer Review: John M Offer, Ph.D., C.BioL, M.I.BioL, Consultant Pathologist Department of Pathology 66 : Company Sanitized. Does not contain TSCA CBI APPENDIX 5 (Pathology - continued) DPT 443/984760 Compound: Dosage Level: Rat No/Sex: Control 1M (Terminal) CLINICAL FINDINGS No signs of ill health or behavioural change were noted. MACROSCOPIC FINDINGS No abnormalities detected MICROSCOPIC FINDINGS The following observations were noted: Kidneys Cortical basophilic tubules: (Minimal) The following tissues were considered normal: Trachea; Lungs(including Bronchi); Heart; Thymus; Lymph Nodes - Mandibular; Lymph Nodes Mesenteric; Spleen; Liver, Urinary Bladder, Prostate; Seminal Vesicles; Epididymides; Testes; Thyroids; Parathyroids; Adrenals; Stomach; Duodenum; Jejunum; Ileum(including Peyer's Patch); Caecum; Colon; Rectum; Spinal Cord; Sciatic Nerve; Brain; Femur/joint Pathologist D.J.Lewis 67 : Company Sanitized. Does not contain TSCA CBI APPENDIX 5 (Pathology - continued) DPT 443/984760 Compound: Dosage Level: Rat No/Sex: Control 2M (Tenninal) CLINICAL FINDINGS No signs of ill health or behavioural change were noted. MACROSCOPIC FINDINGS No abnormalities detected MICROSCOPIC FINDINGS The following tissues were considered normal: , Trachea; Lungs(including Bronchi); Heart; Thymus; Lymph Nodes - Mandibular, Lymph Nodes Mesenteric; Spleen; Liver, Kidneys; Urinary Bladder; Prostate; Seminal Vesicles; Epididymides; Testes; Thyroids; Parathyroids; Adrenals; Stomach; Duodenum; Jejunum; Ileum(including Peyer"s Patch); Caecum; Colon; Rectum; Spinal Cord; Sciatic Nerve; Brain; Femur/joint Pathologist: D-LLewis 68 Company Sanitized. Does not contain TSCA CBI APPENDIX 5 (Pathology - continued) DPT 443/984760 Dosage Level: Rat No/Sex: Control 3M (Terminal) CLINICAL FINDINGS No signs of ill health or behavioural change were noted. MACROSCOPIC FINDINGS No abnormalities detected MICROSCOPIC FINDINGS The following observations were noted: Lungs(inctuding Bronchi) Pneumonitis: (Minimal) Kidneys Cortical basophilic tubules: (Minimal) The following tissues were considered normal: Trachea; Heart; Thymus; Lymph Nodes - Mandibular, Lymph Nodes - Mesenteric; Spleen; Liver, Urinary Bladder; Prostate; Seminal Vesicles; Epididymides; Testes; Thyroids; Parathyroids; Adrenals; Stomach; Duodenum; Jejunum; Ileum(including Peyer's Patch); Caecum; Colon; Rectum; Spinal Cord; Sciatic Nerve; Brain; Femur/joint Pathologist: DJ-Lewis 69 : Company Sanitized. Does not contain TSCA CBI APPENDIX 5 (Pathology - continued) DPT 443/984760 Dosage Level: Rat No/Sex: Control 4M (Terminal) CLINICAL FINDINGS No signs of ill health or behavioural change were noted. MACROSCOPIC FINDINGS Liver Median cleft, pale subcapsular area: I mm All the other organs and tissues appeared normal. MICROSCOPIC FINDINGS The following tissues were considered normal: Trachea; Limgs(including Bronchi); Heart; Thymus; Lymph Nodes - Mandibular, Lymph Nodes Mesenteric; Spleen; Liver : (W.N.L.); Kidneys; Urinary Bladder; Prostate; Seminal Vesicles; Epididymides; Testes; Thyroids; Parathyroids; Adrenals; Stomach; Duodenum; Jejunum; Ileum(including Peyer's Patch); Caecum; Colon; Rectum; Spinal Cord; Sciatic Nerve; Brain; Femur/joint Pathologist: D.J.Lewis 70 : Company Sanitized. Does not contain TSCA C81 APPENDIX 5 (Pathology - continued) DPT 443/984760 Dosage Level: Hat No/Sex: Control 5M (Terminal) CLINICAL FINDINGS No signs of ill health or behavioural change were noted. MACROSCOPIC FINDINGS Liver Median cleft, pale subcapsular area: 2mni All the other organs and tissues appeared normal. MICROSCOPIC FINDINGS The following observations were noted: Liver Hepatocyte vacuolation - median cleft Kidneys Cortical fibrosis and tubular collapse with basophilia: (Minimal) Cortical basophilic tubules: (Minimal) The following tissues were considered normal: Trachea; Lungs(including Bronchi); Heart; Thymus; Lymph Nodes - Mandibular, Lymph Nodes Mesenteric; Spleen; Urinary Bladder, Prostate; Seminal Vesicles; Epididymides; Testes; Thyroids; Parathyroids; Adrenals; Stomach; Duodenum; Jejunum; Ileum(including Foyer's Patch); Caecum; Colon; Rectum; Spinal Cord; Sciatic Nerve; Brain; Femur/joint Pathologist D.J.Lewis 71 : Company Sanitized. Does not contain TSCA CBI APPENDIX 5 (Pathology - continued) DPT 443/984760 Dosage Level: Rat No/Sex: 15 ing/kg/day 6M (Terminal) CLINICAL FINDINGS No signs of ill health, behavioural change or reaction to treatment were noted. MACROSCOPIC FINDINGS Liver Median cleft, pale subcapsulararea: 1mm All the other organs and tissues appeared normal. MICROSCOPIC FINDINGS The following observations were noted: Kidneys Cortical fibrosis and tubular collapse with basophilia: (Minimal) The following tissues were considered normal: Liver: (W.N.L.); Femur/joint Pathologist D.J.Lewis 72 : Company Sanitized. Does not contain TSCA CBI APPENDIX 5 (Pathology - continued) DPT 443/984760 Dosage Level: Rat No/Sex; 15 mg/kg/dxy 7M (Terminal) CLINICAL FINDINGS No signs of ill health, behavioural change or reaction to treatment were noted. MACROSCOPIC FINDINGS Liver Median cleft, pale subcapsular area: 1mm All the other organs and tissues appeared normal. MICROSCOPIC FINDINGS The following observations were noted: Kidneys Cortical basophilic tubules: (Minimal) The following tissues were considered normal: Liver: (W.N.L.); Femur/joint Pathologist: DJ.Lewis 73 : Company Sanitized. Does not contain TSCA CBI APPENDIX 5 (Pathology - continued) DPT 443/984760 Dosage Level: Rat No/Sex: 15mg/kg/day 8M (Terminal) CLINICAL FINDINGS No signs of ill health, behavioural change or reaction to treatment were noted. MACROSCOPIC FINDINGS Liver Median cleft, pale subcapsular area: 1mm Kidneys Increased pelvic dilatation: (Right, Minimal) All the other organs and tissues appeared normal. MICROSCOPIC FINDINGS The following observations were noted: Kidneys Pelvic dilatation: (Slight, Unilateral) Cortical basophilic tubules: (Minimal) The following tissues were considered normal: Liver: (W.N.L.); Femur/joint Pathologist DJ-Lewis 74 : Company Sanitized. Does not contain TSCA CBI APPENDIX 5 (Pathology - continued) DPT 443/984760 Dosage Level: Rat No/Sex: 15 mg/kg/day 9M (Terminal) CLINICAL FINDINGS No signs of ill health, behavioural change or reaction to treatment were noted. MACROSCOPIC FINDINGS No abnormalities detected MICROSCOPIC FINDINGS The following tissues were considered normal: Liver; Kidneys; Femur/joint Pathologist DJ-Lewis 75 : Company Sanitized. Does not contain TSCA CBI APPENDIX 5 (Pathology - continued) DPT 443/984760 Dosage Level: Rat No/Sex: 15 nig/kg/day 10M (Terminal) CLINICAL FINDINGS No signs of ill health, behavioural change or reaction to treatment were noted. MACROSCOPIC FINDINGS No abnormalities detected MICROSCOPIC FINDINGS The following observations were noted: Liver Parenchymal inflammatory cell foci: (Minimal) The following tissues were considered normal: Kidneys; Femur/joint Pathologist: DJ-Lewis 76 : Company Sanitized. Does not contain TSCA CBI APPENDIX 5 (Pathology - continued) DPT 443/984760 Dosage Level: Rat No/Sex: 50 ing/kg/day 11M (Terminal) CLINICAL FINDINGS No signs of ill health, behavioural change or reaction to treatment were noted. MACROSCOPIC FINDINGS No abnormalities detected MICROSCOPIC FINDINGS The following observations were noted: Kidneys Basophilia and hyperplasia of tubules and collecting ducts: (Minimal, Focal) Medullary tabular dilatation: (Minimal) Papillary tabular dilatation: (Minimal) The following tissues were considered normal: Liver; Femur/joint Pathologist: D.J.Lewis 77 : Company Sanitized. Does not contain TSCA CBI APPENDIX 5 (Pathology - continued) DPT 443/984760 Dosage Level: Rat No/Sex: 50 mg/kg/day 12M (Tenninal) CLINICAL FINDINGS Incidental finding of hair loss was noted. MACROSCOPIC FINDINGS No abnormalities detected MICROSCOPIC FINDINGS The following observations were noted: Kidneys Basophilia and hyperplasia of tubules and collecting ducts: (Minimal) Cortical tubular dilatation: (Minimal) Papillary tubular dilatation: (Minimal) The following tissues were considered normal: Liver; Femur/joint Pathologist: D-J.Lewis 78 : Company Sanitized. Does not contain TSCA CBI APPENDIX 5 (Pathology - continued) DPT 443/984760 Dosage Level: Rat No/Sex: 50 mg/kg/day 13M (Terminal) CLINICAL FINDINGS No signs of ill health, behavioural change or reaction to treatment were noted. MACROSCOPIC FINDINGS No abnormalities detected MICROSCOPIC FINDINGS The following tissues were considered normal: Liver; Kidneys; Femur/joint Pathologist DJ.Lewis 79 : Company Sanitized. Does not contain TSCA CBI APPENDIX 5 (Pathology - continued) DPT 443/984760 Dosage Level: Rat No/Sex: 50 mg/kg/day 14M (Terminal) CLINICAL FINDINGS No signs of ill health, behavioural change or reaction to treatment were noted.' MACROSCOPIC FINDINGS Incisors Lower pale All the other organs and tissues appeared normal. MICROSCOPIC FINDINGS The following observations were noted: Kidneys Basophilia and hyperplasia of tubules and collecting ducts: (Minimal) Cortical tubular dilatation: (Minimal) The following tissues were considered normal: Liver, Femur/joint Pathologist: D.J.Lewis 80 : Company Sanitized. Does not contain TSCA CBI APPENDIX 5 (Pathology - continued) DPT 443/984760 Dosage Level: Rat No/Sex: 50 nig/kg/day 15M (Terminal) CLINICAL FINDINGS No signs of ill health, behavioural change or reaction to treatment were noted. MACROSCOPIC FINDINGS No abnormalities detected MICROSCOPIC FINDINGS The following observations were noted: Kidneys Basophilia and hyperplasia of tubules and collecting ducts: (Minimal) The following tissues were considered normal: Liver; Femur/joint Pathologist D.J.Lewis 81 : Company Sanitized. Does not contain TSCA CBI APPENDIX 5 (Pathology - continued) DPT 443/984760 Dosage Level: Rat No/Sex: 150 mg/kg/day 16M (Tenninal) CLINICAL FINDINGS Salivation immediately after dosing was noted on one occasion. Hunched posture was noted in Week 3. MACROSCOPIC FINDINGS Adipose Tissue Minimal Kidneys Pale Enlarged: 3.643g Swollen Pale cortical foci: (Multiple) 1mm All me other organs and tissues appeared normal. MICROSCOPIC FINDINGS The following observations were noted: Liver Hepatocyte hypertrophy - generalised: (Slight) Hepatocyte fine vacuolation - generalised: (Slight) Kidneys Interstitial inflammation in cortex^nedulla and papilla: (Moderate) Basophilia and hyperplasia of tubules and collecting ducts: (Moderate) Cortical tubular dilatation: (Slight) Urothelial hyperplasia: (Moderate) Medullary tubular dilatation: (Moderate) Papillary tubular dilatation: (Slight) Cortical tubular necrosis: (Minimal) Inflammatory casts in tubules and collecting ducts Papillary collecting duct hyperplasia 82 : Company Sanitized. Does not contain TSCA CBI APPENDIX 5 (Pathology - continued) DPT 443/984760 Rat No/Sex: 16M - continued MICROSCOPIC FINDINGS - continued Adrenals Cortical vacuolation: (Minimal) Femur/joint Marrow - prominent adipocytes The following tissues were considered normal: Trachea; Lungs(including Bronchi); Heart; Thymus; Lymph Nodes - Mandibular; Lymph Nodes Mesenteric; Spleen; Urinary Bladder, Prostate; Seminal Vesicles; Epididymides; Testes; Thyroids; Parathyroids; Stomach; Duodenum; Jejunum; Ileum(including Peyer's Patch); Caecum; Colon; Rectum; Spinal Cord; Sciatic Nerve; Brain Pathologist: DJ-Lewis 83 : Company Sanitized. Does not contain TSCA CBI APPENDIX 5 (Pathology - continued) DPT 443/984760 Dosage Level: Rat No/Sex: 150 ing/kg/day 17M (Tenninal) CLINICAL FINDINGS No signs of ill health, behavioural change or reaction to treatment were noted. MACROSCOPIC FINDINGS Adipose Tissue Minimal Kidneys Pale: (Minimal) Irregular cortical scarring: (Minimal) Enlarged: 3.742g Swollen All the other organs and tissues appeared normal. . MICROSCOPIC FINDINGS The following observations were noted: Liver Hepatocyte hypertrophy - generalised: (Slight) Hepatocyte fine vacuolation - generalised: (Slight) Kidneys Interstitial inflammation in cortex,medulla and papilla: (Slight) Basophilia and hyperplasia of tubules and collecting ducts: (Moderate) Cortical tubular dilatation: (Slight) Urothelial hyperplasia; (Marked) Medullary tubular dilatation: (Moderate) Papillary tubular dilatation: (Slight) Inflammatory casts in tubules and collecting ducts Papillary collecting duct hyperplasia 84 : Company Sanitized. Does not contain TSCA CBI APPENDIX 5 (Pathology - continued) DPT 443/984760 Rat No/Sex: 17M - continued MICROSCOPIC FINDINGS - continued Femur/joint Osteoarthrosis: (Minimal) Marrow - prominent adipocytes The following tissues were considered normal: Trachea; Lungs(including Bronchi); Heart; Thymus; Lymph Nodes - Mandibular, Lymph Nodes Mesenteric; Spleen; Urinary Bladder, Prostate; Seminal Vesicles; Epididymides; Testes; Thyroids; Parathyroids; Adrenals; Stomach; Duodenum; Jejunum; Ileum(including Peyer's Patch); Caecum; Colon; Rectum; Spinal Cord; Sciatic Nerve; Brain Pathologist: D.J.Lewis 85 : Company Sanitized. Does not contain TSCA CBI APPENDIX 5 (Pathology - continued) Dosage Level: Rat No/Sex: 150mg/kg/day 18M (Terminal) CLINICAL FINDINGS Walking on toes immediately after dosing was noted on one occasion. MACROSCOPIC FINDINGS Adipose Tissue Minimal liver Pale Kidneys Pale: (Minimal) Enlarged: 4.652g Swollen Ureters Distended: (Left) 2mm Contained blood stained urine: (Left) Urinary Bladder Contained blood stained urine All the other organs and tissues appeared normal. MICROSCOPIC FINDINGS The following observations were noted: Liver Hepatocyte hypertrophy - generalised: (Minimal) Hepatocyte fine vacuolation - generalised: (Minimal) DPT 443/984760 86 : Company Sanitized. Does not contain TSCA CBI APPENDIX 5 (Pathology - continued) DPT 443/984760 Rat No/Sex: 18M - continued MICROSCOPIC FINDINGS - continued Kidneys Interstitial inflammation in cortex,medulla and papilla: (Minimal) Basophilia and hyperplasia of tubules and collecting ducts: (Moderate) Cortical tubular dilatation: (Slight) Urothelial hyperplasia: (Slight) Medullary tubular dilatation: (Moderate) Papillary tubular dilatation: (Slight) Inflammatory casts in tubules and collecting ducts Ureters Lmninal dilatation: (Slight) The following tissues were considered normal: Trachea; Lungs(including Bronchi); Heart; Thymus; Lymph Nodes - Mandibular, Lymph Nodes Mesenteric; Spleen; Urinary Bladder : (W.N.L-); Prostate; Seminal Vesicles; Epididymides; Testes; Thyroids; Parathyroids; Adrenals; Stomach; Duodenum; Jejunum; Ileum(including Foyer's Patch); Caecum; Colon; Rectum; Spinal Cord; Sciatic Nerve; Brain; Femur/joint Pathologist: D.J.Lewis 87 : Company Sanitized. Does not contain TSCA CBI APPENDIX 5 (Pathology - continued) Dosage Level: Rat No/Sex: 150 mg/kg/day 19M (Terminal) CLINICAL FINDINGS Salivation immediately after dosing was noted on one occasion. MACROSCOPIC FINDINGS Lungs(inclnding Bronchi) Congested: (Patchy) Adipose Tissue Minimal Liver Pale Lobular markings accentuated Kidneys Pale Enlarged: 7.49 Ig Swollen Pale cortical foci: (A few) up to 3mm Misshapen: (Right) All the other organs and tissues appeared normal. MICROSCOPIC FINDINGS The following observations were noted: Lungs(including Bronchi) Vascular congestion: (Minimal) Liver Hepatocyte hypertrophy - generalised: (Slight) Hepatocyte fine vacuolation - generalised: (Minimal) DPT 443/984760 88 Company Sanitized. Does not contain TSCA CBI APPENDIX 5 (Pathology - continued) DPT 443/984760 Rat No/Sex: 19M - continued MICROSCOPIC FINDINGS - continued Kidneys Interstitial inflammation in cortex,medulla and papilla: (Slight) Basophilia and hyperplasia of tubules and collecting ducts: (Marked) Cortical tubular dilatation: (Slight) Urothelial hyperplasia: (Slight) Medullary tubular dilatation: (Marked) Papillary tubular dilatation: (Moderate) Cortical tubular necrosis: (Slight) Inflammatory casts in tubules and collecting ducts The following tissues were considered normal: Trachea; Heart; Thymus; Lymph Nodes - Mandibular; Lymph Nodes - Mesenteric; Spleen; Urinary Bladder; Prostate; Seminal Vesicles; Epididymides; Testes; Thyroids; Parathyroids; Adrenals; Stomach; Duodenum; Jejunum; Ileum(including Foyer's Patch); Caecum; Colon; Rectum; Spinal Cord; Sciatic Nerve; Brain; Femur/joint Pathologist: D.J.Lewis 89 : Company Sanitized. Does not contain TSCA CBI APPENDIX 5 (Pathology - continued) DPT 443/984760 Dosage Level: Rat No/Sex: 150mg/kg/day 20M (Terminal) CLINICAL FINDINGS Salivation immediately after dosing was noted on occasions. Hunched posture was noted from Week 3. MACROSCOPIC FINDINGS Tail Tip missing Adipose Tissue Minimal Liver Pale Kidneys Pale: (Minimal) Irregular cortical scarring: Enlarged: 5.125g Swollen Pale cortical foci: (A few) (Minimal) 1mm All the other organs and tissues appeared normal. MICROSCOPIC FINDINGS The following observations were noted: Liver Hepatocyte hypertrophy - generalised: (Minimal) Hepatocyte fine vacuolarion - generalised: (Minimal) 90 : Company Sanitized. Does not contain TSCA CBI APPENDIX 5 (Pathology - continued) DPT 443/984760 Rat No/Sex: 20M - continued MICROSCOPIC FINDINGS - continued Kidneys Interstitial inflammation in cortex^nedulla and papilla: (Slight) Basophilia and hyperplasia of tubules and collecting ducts: (Moderate) Cortical tubular dilatation: (Slight) Urothelial hyperplasia: (Slight) Medullary tubular dilatation: (Moderate) Papillary tubular dilatation: (Slight) Cortical tubular necrosis: (Slight) Inflammatory casts in tubules and collecting ducts Spinal Cord Vacuolation: (Minimal) Femur/joint Marrow - prominent adipocytes The following tissues were considered normal: Trachea; Lungs(mcluding Bronchi); Heart; Thymus; Lymph Nodes - Mandibular, Lymph Nodes Mesenteric; Spleen; Urinary Bladder, Prostate; Seminal Vesicles; Epididymides; Testes; Thyroids; Parathyroids; Adrenals; Stomach; Duodenum; Jejunum; Ileum(including Peyer's Patch); Caecum; Colon; Rectum; Sciatic Nerve; Brain Pathologist: DJ-Lewis 91 : Company Sanitized. Does not contain TSCA CBI APPENDIX 5 (Pathology - continued) DPT 443/984760 Dosage Level: Rat No/Sex: Control 2 IF (Terminal) CLINICAL FINDINGS No signs of ill health or behavioural change were noted. MACROSCOPIC FINDINGS Uterus Fluid distension All the other organs and tissues appeared normal. MICROSCOPIC FINDINGS The following observations were noted: Uterus Luminal dilatation: (Moderate) The following tissues were considered normal: Trachea; Lungs(includmg Bronchi); Heart; Thymus; Lymph Nodes - Mandibular; Lymph Nodes Mesenteric; Spleen; Liver; Kidneys; Urinary Bladder, Cervix; Vagina; Ovaries; Thyroids; Parathyroids; Adrenals; Stomach; Duodenum; Jejunum; Ileum(includuig Foyer's Patch); Caecum; Colon; Rectum; Spinal Cord; Sciatic Nerve; Brain; Femur/joint Pathologist: D.J.Lewis 92 : Company Sanitized. Does not contain TSCA CBI APPENDIX 5 (Pathology - continued) DPT 443/984760 Dosage Level: Rat No/Sex: Control 22F (Terminal) CLINICAL FINDINGS No signs of ill health or behavioural change were noted. MACROSCOPIC FINDINGS No abnormalities detected MICROSCOPIC FINDINGS The following observations were noted: Kidneys Cortico-medullary mineralisation: (Slight) Femur/joint Marrow - prominent adipocytes The following tissues were considered normal: Trachea; Lungs(including Bronchi); Heart; Thymus; Lymph Nodes - Mandibular; Lymph Nodes Mesenteric; Spleen; Liver, Urinary Bladder; Uterus; Cervix; Vagina; Ovaries; Thyroids; Parathyroids; Adrenals; Stomach; Duodenum; Jejunum; Ileum(including Peyer's Patch); Caecum; Colon; Rectum; Spinal Cord; Sciatic Nerve; Brain Pathologist DJ-Lewis 93 : Company Sanitized. Does not contain TSCA CBI APPENDIX 5 (Pathology - continued) DPT 443/984760 Dosage Level: Rat No/Sex: Control 23F (Terminal) CLINICAL FINDINGS No signs of ill health or behavioural change were noted. MACROSCOPIC FINDINGS Lnngs(including Bronchi) Congested: (Minimal, Patchy) Uterus Fluid distension All the other organs and tissues appeared normal. MICROSCOPIC FINDINGS The following observations were noted: Lungs(inclnding Bronchi) Vascular congestion: (Minimal) Uterus Luminal dilatation: (Moderate) The following tissues were considered normal: Trachea; Heart; Thymus; Lymph Nodes - Manriihnlar; I .ymph Node? - Mesenteric; Spleen; Livar; Kidneys; Urinary Bladder; Cervix; Vagina; Ovaries; Thyroids; Parathyroids; Adrenals; Stomach; Duodenum; Jejunum; Ileum(including Peyer's Patch); Caecum; Colon; Rectum; Spinal Cord; Sciatic Nerve; Brain; Femur/joint Pathologist D.JJLewis 6 94 : Company Sanitized. Does not contain TSCA CBI APPENDIX 5 (Pathology - continued) DPT 443/984760 Dosage Level: Rat No/Sex: Control 24F (Terminal) CLINICAL FINDINGS No signs of ill health or behavioural change were noted. MACROSCOPIC FINDINGS Uterus Fluid distension: (Minimal) All the other organs and tissues appeared normal. MICROSCOPIC FINDINGS The following observations were noted: Liver Haemorrhage: (Minimal, Focus) Uterus Luminal dilatation: (Marked) Sciatic Nerve Degenerate fibres: (Minimal) The following tissues were considered normal: Trachea; Lungs(including Bronchi); Heart; Thymus; Lymph Nodes - Mandibular; Lymph Nodes Mesenteric; Spleen; Kidneys; Urinary Bladder, Cervix; Vagina; Ovaries; Thyroids; Parathyroids; Adrenals; Stomach; Duodenum; Jejunum; Ileum(including Fever's Patch); Caecum; Colon; Rectum; Spinal Cord; Brain; Femur/joint Pathologist: DJ.Lewis 95 : Company Sanitized. Does not contain TSCA CBI APPENDIX 5 (Pathology - continued) DPT 443/984760 Dosage Level: Rat No/Sex: Control 25F (Terminal) CLINICAL FINDINGS No signs of ill health or behavioural change were noted. MACROSCOPIC FINDINGS No abnormalities detected MICROSCOPIC FINDINGS The following observations were noted: Kidneys Cortico-medullary mineralisation: (Slight) Cortical basophilic tubules: (Minimal) The following tissues were considered normal: Trachea; Lungs(including Bronchi); Heart; Thymus; Lymph Nodes - Mandibular; Lymph Nodes Mesenteric; Spleen; Liver, Urinary Bladder, Uterus; Cervix; Vagina; Ovaries; Thyroids; Parathyroids; Adrenals; Stomach; Duodenum; Jejunum; Deum(including Foyer's Patch); Caecum; Colon; Rectum; Spinal Cord; Sciatic Nerve; Brain; Femur/joint Pathologist DJ.Lewis 96 : Company Sanitized. Does not contain TSCA CBI APPENDIX 5 (Pathology - continued) DPT 443/984760 Dosage Level: Rat No/Sex: ISmg/kg/day 26F (Terminal) CLINICAL FINDINGS No signs of ill health, behavioural change or reaction to treatment were noted. MACROSCOPIC FINDINGS Uterns Fluid distension All the other organs and tissues appeared normal. MICROSCOPIC FINDINGS The following observations were noted: Uterus Luminal dilatation: (Moderate) The following tissues were considered normal: Liver, Kidneys; Femur/joint Pathologist: DJ.Lewis 97 : Company Sanitized. Does not contain TSCA CBI APPENDIX 5 (Pathology - continued) DPT 443/984760 Dosage Level: Rat No/Sex: 15 mg/kg/day 27F (Terminal) CLINICAL FINDINGS No signs of ill health, behavioural change or reaction to treatment were noted. MACROSCOPIC FINDINGS No abnormalities detected MICROSCOPIC FINDINGS The following observations were noted: Kidneys Cortical basophilic tubules: (Minimal) The following tissues were considered normal: Liver, Femur/joint Pathologist DJ.Lewis 98 : Company Sanitized. Does not contain TSCA CBI APPENDIX 5 (Pathology - continued) DPT 443/984760 Dosage Level: Rat No/Sex: 15 mg/kg/day 28F (Terminal) CLINICAL FINDINGS No signs of ill health, behavioural change or reaction to treatment were noted- MACROSCOPIC FINDINGS No abnormalities detected MICROSCOPIC FINDINGS The follov/ing observations were noted: Kidneys Cortico-medullary mineralisation: (Minimal) Cortical basophilic tubules: (Minimal) The following tissues were considered normal: Liver; Femur/joint Pathologist D.J.Lewis 99 : Company Sanitized. Does not contain TSCA CBI APPENDIX 5 (Pathology - continued) DPT 443/984760 Dosage Level: Rat No/Sex: 15 mg/kg/day 29F (Terminal) CLINICAL FINDINGS No signs of ill health, behavioural change or reaction to treatment were noted. MACROSCOPIC FINDINGS Stomach Antrum Mucosa White nodule, near to limiting ridge: (Punctate) All the other organs and tissues appeared normal. MICROSCOPIC FINDINGS The following observations were noted: Kidneys Corrico-medullary mineralisation: (Minimal) The following tissues were considered normal: Liver; Femur/joint Pathologist: DJ-Lewis 100 : Company Sanitized. Does not contain TSCA CBI APPENDIX 5 (Pathology - continued) DPT 443/984760 Dosage Level: Rat No/Sex: 15 ing/kg/day 30F (Terminal) CLINICAL FINDINGS No signs of ill health, behavioural change or reaction to treatment were noted. MACROSCOPIC FINDINGS No abnormalities detected MICROSCOPIC FINDINGS The following tissues were considered normal: Liver, Kidneys; Femur/joint Pathologist DJ-Lewis 101 : Company Sanitized. Does not contain TSCA CBI APPENDIX 5 (Pathology - continued) DPT 443/984760 Dosage Level: Rat No/Sex: 50 mg/kg/day 1031 F (Terminal) CLINICAL FINDINGS No signs of ill health, behavioural change or reaction to treatment were noted. MACROSCOPIC FINDINGS Uterus Fluid distension All the other organs and tissues appeared normal. MICROSCOPIC FINDINGS The following observations were noted: Kidneys Basophilia and hyperplasia of tubules and collecting ducts: (Minimal) Cortical tabular dilatation: (Minimal) Cortico-medullary mineralisation: (Moderate) Uterus Luminal dilatation: (Moderate) The following tissues were considered normal: Liver, Femur/joint Pathologist D.J.Lewis 102 : Company Sanitized. Does not contain TSCA CBI APPENDIX 5 (Pathology - continued) DPT 443/984760 Dosage Level: Rat No/Sex: 50 mg/kg/day 32F (Terminal) CLINICAL FINDINGS No signs of ill health, behavioural change or reaction to treatment were noted. MACROSCOPIC FINDINGS Uterus Fluid distension All the other organs and tissues appeared normal. MICROSCOPIC FINDINGS The following observations were noted: Kidneys Basophilia and hyperplasia of tubules and collecting Cortical tubular dilatation: (Minimal) Urothelial hyperplasia: (Slight) Medullary tubular dilatation: (Minimal) Cortico-medullary mineralisation; (Moderate) Papillary collecting duct hyperplasia Interstitial inflammation in papilla: (Minimal) ducts: (Slight) Uterus Luminal dilatation: (Marked) The following tissues were considered normal: Liver; Femur/joint Pathologist: D.J.Lewis 103 : Company Sanitized. Does not contain TSCA CBI APPENDIX 5 (Pathology - continued) DPT 443/984760 Dosage Level: Rat No/Sex: 50 mg/kg/day 33F (Terminal) CLINICAL FINDINGS No signs of ill health, behavioural change or reaction to treatment were noted. MACROSCOPIC FINDINGS Uterus Fluid distension All the other organs and tissues appeared normal. MICROSCOPIC FINDINGS The following observations were noted: Kidneys Basophilia and hyperplasia of tubules and collecting ducts: (Slight) Cortical tubular dilatation: (Slight) Cortico-medullary mineralisation: (Slight) Papillary collecting duct hyperplasia Interstitial inflammation in papilla: (Minimal) Uterus Luminal dilatation: (Moderate) The following tissues were considered normal: Liver, Femur/joint Pathologist: D-LLewis 104 : Company Sanitized. Does not contain TSCA CBI APPENDIX 5 (Pathology - continued) DPT 443/984760 Dosage Level: Rat No/Sex: 50 mg/kg/day 34F (Terminal) CLINICAL FINDINGS No signs of ill health, behavioural change or reaction to treatment were noted. MACROSCOPIC FINDINGS No abnormalities detected MICROSCOPIC FINDINGS The following observations were noted: Kidneys Basophilia and hyperplasia of tubules and collecting Cortical tubular dilatation: (Minimal) Papillary tubular dilatation: (Minimal) Cortico-medullary mineralisation: (Minimal) Interstitial inflammation in papilla: (Minimal) ducts: (Minimal) The following tissues were considered normal: Liver, Femur/joint Pathologist: DJ-Lewis 105 : Company Sanitized. Does not contain TSCA CBI APPENDIX 5 (Pathology - continued) DPT 443/984760 Dosage Level: Rat No/Sex: 50 mg/kg/day 35F (Terminal) CLINICAL FINDINGS No signs of ill health, behavioural change or reaction to treatment were noted. MACROSCOPIC FINDINGS No abnormalities detected MICROSCOPIC FINDINGS The following observations were noted: Kidneys Basophilia and hyperplasia of tubules and collecting ducts: (Minimal) Cortico-medullary mineralisation: (Moderate) Papillary collecting duct hyperplasia The following tissues were considered normal: Liver, Femur/joint Pathologist DJ-Lewis 106 : Company Sanitized. Does not contain TSCA CBI APPENDIX 5 (Pathology - continued) Dosage Level: Rat No/Sex: 150 mg/kg/day 36F (Terminal) CLINICAL FINDINGS Salivation immediately after dosing was noted on one occasion. MACROSCOPIC FINDINGS Adipose Tissue Minimal Liver Pale Kidneys Pale Irregular cortical scarring: (Moderate) Enlarged: 3.723g Swollen Pale cortical foci: (A few) 1mm All the other organs and tissues appeared nonnal. MICROSCOPIC FINDINGS The following observations were noted: Liver Hepatocyte hypertrophy - generalised: (Minimal) Hepatocyte fine vacuolation - generalised: (Minimal) DPT 443/984760 107 : Company Sanitized. Does not contain TSCA CBI APPENDIX 5 (Pathology - continued) DPT 443/984760 Rat No/Sex: 36F - continued MICROSCOPIC FINDINGS - continued Kidneys Interstitial inflammation in cortex,medulla and papilla: (Minimal) Basophilia and hyperplasia of tubules and collecting ducts: (Moderate) Cortical tubular dilatation: (Slight) Urothelial hyperplasia: (Moderate) Medullary tubular dilatation: (Moderate) Papillary tubular dilatation: (Minimal) Inflammatory casts in tubules and collecting ducts The following tissues were considered normal: Trachea; Lungs(including Bronchi); Heart; Thymus; Lymph Nodes - Mandibular, Lymph Nodes Mesenteric; Spleen; Urinary Bladder, Uterus; Cervix; Vagina; Ovaries; Thyroids; Parathyroids; Adrenals; Stomach; Duodenum; Jejunum; Ileum(including Peyer's Patch); Caecum; Colon; Rectum; Spinal Cord; Sciatic Nerve; Brain; Femur/joint Pathologist: DJLLewis 108 : Company Sanitized. Does not contain TSCA CBI APPENDIX 5 (Pathology - continued) DPT 443/984760 Dosage Level: Rat No/Sex: 150mg/kg/day 37F (Terminal) CLINICAL FINDINGS No signs of ill health, behavioural change or reaction to treatment were noted. MACROSCOPIC FINDINGS Thymus Small Adipose Tissue Minimal Liver Median cleft, pale subcapsular area: 1mm Pale Kidneys Pale Irregular cortical scarring: (Moderate) Enlarged: 2.825g Swollen All the other organs and tissues appeared normal. MICROSCOPIC FINDINGS The following observations were noted: Thymus Involution/atrophy: (Minimal) Liver Hepatocyte hypertrophy - generalise (Minimal) Hepatocyte fine vacuolation - generalised: (Minimal) 109 : Company Sanitized. Does not contain TSCA CBI APPENDIX 5 (Pathology - continued) DPT 443/984760 Rat No/Sex: 3 7F - continued MICROSCOPIC FINDINGS - continued Kidneys Basophilia and hyperplasia of tubules and Cortical tubular dilatation: (Slight) Urothelial hyperplasia: (Moderate) Medullary tubular dilatation: (Moderate) Papillary tubular dilatation: (Slight) collecting ducts: (Moderate) Femur/joint Marrow - prominent adipocytes The following tissues were considered normal: Trachea; Lungs(includmg Bronchi); Heart; Lymph Nodes - Mandibular, Lymph Nodes Mesenteric; Spleen; Urinary Bladder, Uterus; Cervix; Vagina; Ovaries; Thyroids; Parathyroids; Adrenals; Stomach; Duodenum; Jejunum; Ileum(including Peyer's Patch); Caecum; Colon; Rectum; Spinal Cord; Sciatic Nerve; Brain Pathologist: DJ-Lewis 110 : Company Sanitized. Does not contain TSCA CBI APPENDIX 5 (Pathology - continued) DPT 443/984760 Dosage Level: Rat No/Sex: 150 mg/kg/day 38F (Terminal) CLINICAL FINDINGS Salivation immediately after dosing was noted on occasions. Hunched posture was noted from Week 4. Incidental finding of hair loss was noted. MACROSCOPIC FINDINGS SIdn Alopecia Left cervical region: (Minimal) Right scapular region: (Minimal) Limgs(including Bronchi) Congested Adipose Tissue Minimal Liver Pale Kidneys * Pale Irregular cortical scarring: (Moderate) Enlarged: 3.879g Swollen All the other organs and tissues appeared normal. Ill : Company Sanitized. Does not contain TSCA CBI APPENDIX 5 (Pathology - continued) Rat No/Sex: 3 8F - continued MICROSCOPIC FINDINGS The following observations were noted: Lungs(inclnding Bronchi) Vascular congestion: (Minimal) Liver Hepatocyte hypertrophy - generalised: (Slight) Hepatocyte fine vacuolation - generalised: (Slight) Kidneys Interstitial inflammation in cortex,medulla and papilla: (Slight) Basophilia and hyperplasia of tubules and collecting ducts: (Moderate) Cortical tubular dilatation: (Slight) Papillary necrosis: (Slight, Unilateral) Urothelial hyperplasia: (Moderate) Medullary tubular dilatation: (Moderate) Papillary tubular dilatation: (Slight) Inflammatory casts in tubules and collecting ducts Papillary collecting duct hyperplasia Uterus Myometrial/endometrial atrophy: (Minimal) Cervix Epithelial mucification Ovaries Sparse/few corpora lutea Femur/joint Marrow - prominent adipocytes DPT 443/984760 112 : Company Sanitized. Does not contain TSCA CBI APPENDIX 5 (Pathology - continued) DPT 443/984760 Rat No/Sex: 3 8F - continued MICROSCOPIC FINDINGS - continued The following tissues were considered normal: Trachea; Heart; Thymus; Lymph Nodes - Mandibular; Lymph Nodes - Mesenteric; Spleen; Urinary Bladder, Vagina; Thyroids; Parathyroids; Adrenals; Stomach; Duodenum; Jejunum; Ileum(including Foyer's Patch); Caecum; Colon; Rectum; Spinal Cord; Sciatic Nerve; Brain Pathologist: DJ.Lewis 113 : Company Sanitized. Does not contain TSCA CBI APPENDIX 5 (Pathology - continued) DPT 443/984760 Dosage Level: Rat No/Sex: 150 mg/kg/day 39F (Terminal) CLINICAL FINDINGS No signs of ill health, behavioural change or reaction to treatment were noted. MACROSCOPIC FINDINGS Langs(including Bronchi) Congested: (Pafchy) Adipose Tissue Minimal Liver Median cleft, pale subcapsular area: 1mm Kidneys Pale Irregular cortical scarring: (Moderate) Enlarged: 5.469g Swollen Pale cortical foci: (A few, Punctate) Ovaries Small Uterus Thin All me other organs and tissues appeared normal. 114 : Company Sanitized. Does not contain TSCA CBI APPENDIX 5 (Pathology - continued) DPT 443/984760 Rat No/Sex: 39F - continued MICROSCOPIC FINDINGS The following observations were noted: Lungs(including Bronchi) Vascular congestion: (Minimal) Liver Hepatocyte hypertrophy - generalised: (Minimal) Hepatocyte fine vacuolation - generalised: (Minimal) Kidneys Interstitial inflammation in cortex,medulla and papilla: (Minimal) Basophilia and hyperplasia of tubules and collecting ducts: (Moderate) Cortical tubular dilatation: (Slight) Papillary necrosis: (Minimal, Unilateral) Urothelial hyperplasia: (Slight) Medullary tubular dilatation: (Moderate) Papillary tubular dilatation: (Slight) Inflammatory casts in tubules and collecting ducts Uterus Myometrial/endometrial atrophy: (Minimal) Cervix Epithelial mucification Ovaries Sparse/few corpora lutea Femur/joint Marrow - prominent adipocytes The following tissues were considered normal: Trachea; Heart; Thymus; Lymph Nodes - Mandibular; Lymph Nodes - Mesenteric; Urinary Bladder; Vagina; Thyroids; Parathyroids; Adrenals; Stomach; Duodenum; Jejunum; Ileum(including Peyer's Patch); Caecum; Colon; Rectum; Spinal Cord; Sciatic Nerve; Brain 115 : Company Sanitized. Does not contain TSCA CBI APPENDIX 5 (Pathology - continued) Rat No/Sex: 39F - continued MICROSCOPIC FINDINGS - continued Tissues not available for examination were: Spleen: (Not seen) Pathologist D.J.Lewis DPT 443/984760 116 : Company Sanitized. Does not contain TSCA CBI APPENDIX 5 (Pathology - continued) DPT 443/984760 Dosage Level: Rat No/Sex: 150 mg/kg/day 40F (Terminal) CLINICAL FINDINGS No signs of ill health, behavioural change or reaction to treatment were noted. MACROSCOPIC FINDINGS Lungs(including Bronchi) Congested Adipose Tissue Minimal liver Pale Kidneys Pale Irregular cortical scarring: (Moderate) Enlarged: 4J59g Swollen All the other organs and tissues appeared normal. MICROSCOPIC FINDINGS The following observations were noted: Lungs(including Bronchi) Vascular congestion: (Minimal) Liver Hepatocyte hypertrophy - generalised: (Minimal) Hepatocyte fine vacuolariori - generalised: (Minimal) 117 : Company Sanitized. Does not contain TSCA CB1 APPENDIX 5 (Pathology - continued) DPT 443/984760 Rat No/Sex: 40F - continued MICROSCOPIC FINDINGS - continued Kidneys Basophilia and hyperplasia of tubules and collecting Cortical tubular dilatation: (Moderate) Papillary necrosis: (Minimal, Unilateral) Urothelial hyperplasia: (Moderate) Medullary tubular dilatation: (Moderate) Papillary tubular dilatation: (Slight) Inflammatory casts in tubules and collecting ducts ducts: (Moderate) Femur/joint Marrow - prominent adipocytes The following tissues were considered normal: Trachea; Heart; Thymus; Lymph Nodes - Mandibular, Lymph Nodes - Mesenteric; Spleen; Urinary Bladder; Uterus; Cervix; Vagina; Ovaries; Thyroids; Parathyroids; Adrenals; Stomach; Duodenum; Jejunum; Ileum(including Peyer's Patch); Caecum; Colon; Rectum; Spinal Cord; Sciatic Nerve; Brain Pathologist: D.J.Lewis 118 : Company Sanitized. Does not contain TSCA CBI DPT 443/984760 BEHAVIOURAL SCREENING 119 : Author E. W. Hughes Company Sanitized. Does not contain TSCA CBI DPT 443/984760 CONTENTS Page EXPERIMENTAL PROCEDURE............................................................................................ 121 124 RESULTS................................................................................................................................... TABLES - group data 1. Summary of functional observational battery................................................................. 125 2. Activity counts................................................................................................................. 130 3. Rearing counts.................................................................................................................. 131 4. Grip strength foreumb....................................................................................... 132 5. Grip strength hindlimb..................................................................................................... 133 6. Landing footsplay............................................................................................................ 134 7. Rectal temperature........................................................................................................... 135 8. Coulboum locomotor activity.......................................................................................... 136 APPENDICES - individual data 1. Functional observational battery-pre-dose. Week 1,2,3,4......................................... 137 2. Coulboum locomotor activity - pre-dose. Week 4......................................................... 180 120 : Company Sanitized. Does not contain TSCA CBI EXPERIMENTAL PROCEDURE DPT 443/984760 NEUROBEHAVIOURAL SCREENING During the study, a functional observational battery and motor activity were performed at approximately the same time of day. Not all rats were tested in one day, but time of testing was balanced across the groups. Observations made during the treatment period were made prior to dosing. In addition observations in Week 4 were performed prior to any laboratory investigations. A full functional observational battery was performed during the pre-dose period, and during Week 4. A shortened battery was performed during Weeks 1, 2 and 3. The functional observational battery is detailed below: The battery comprised 3 sets of observations. The first set was performed when initially handling the animal. The second set of observations was performed in the test arena and the third set comprised handling/specific testing of the animal. All these observations were made with the observer blind to the treatment condition of the animal. Observations in the hand: Ease of removing the animal from the cage Reactivity to handling (ease of handling) Salivation/lacrimation Exophthalmus Piloerection Fur appearance Vocalisation on handling Observation in the arena: Occurrence of convulsions, tremors, twitches Activity counts Level of arousal Rearing count Grooming Assessment of gait/posture Palpebral closure Record presence of faecal boluses, urine 121 : Company Sanitized. Does not contain TSCA CBI Manipulations*: DPT 443/984760 Approach response Touch response Auditory startle response Righting reflex Tail pinch response Pupil reflex Grip strength (fore and hindlimb) Landing footsplay Body temperature (C) Bodyweight (g) At any point during the observations, additional comments were made as free text where considered appropriate. * The manipulations were only made during the pre-dose period and Week 4. Although bodyweight was recorded, no discussion of any possible effects of treatment on bodyweight is presented in this report as this has been covered in the main report For the observations, if all animals in all groups failed to show a given sign such as lacrimation this sign has been omitted from presentation in the report although it has been recorded on the raw data sheet Motor activity was performed before initiation of treatment and during the 4th week of treatment and was monitored using a Coulboum Infra-Red Activity Monitoring System. (System supplied by Coulboum Instruments, Lehigh Valley, PA, U.S.A.). This system uses an infra-red detector to monitor activity. The following categories of activity are recorded: the time spent in no movement, locomotor and non-locomotor activity. The number of occurrences (events) of each category is also recorded. For reporting this data, only the time spent in locomotor activity is presented. For testing, designated animals were placed singly into observation cages. Once all animals had been placed into the cages, the test session programme was started. The test session for each animal was 1 hour. Data was collected every 2 minutes and written onto a floppy disk. The functional observational battery was performed in Room 27 and the motor activity monitoring was performed in Room 26. ANALYSIS AND PRESENTATION OF THE BEHAVIOURAL SCREENING DATA The following datf were routinely subjected to statistical analysis: rearing and activity counts, grip strength, hind limb splay, bodyweight and temperature. These data were analysed using a one-way analysis of variance followed by Williams' test (Williams 1971/2) for a dose-related response. Pre-dose data was analysed by analysis of variance followed by Student's 'f test 122 Company Sanitized. Does not contain TSCA CBI DPT 443/984760 The reporting of the categorical data for the observational battery has been handled in the following manner. The observational endpoints such as ease of handling, arousal, etc., have been tabulated for frequency of occurrence for each group. Although during recording, some responses were classified in terms of the degree or type of response (ie startle: no reaction, an ear twitch, a flinch, etc.), for the purposes of reporting, as there were no remarkable differences between the groups, for a given endpoint the response has been reported as being either present or absent As there were no remarkable differences in the incidence of observations, no statistical analyses were performed on the categorical data. The Coulboum activity data were analysed using a one-way analysis of variance followed by Williams' test (Williams 1971/2) for a dose-related response. Pre-dose data were analysed by analysis of variance followed by Students 'f test REFERENCES HOLLANDER M, WOLFE DA (1973) Nonparametric Statistical Methods. John Wiley & Sons, New York. WILLIAMS, D.A, (1971/2), Biometrics, 27:103 - 117 and 28: 519-531. 123 : Company Sanitized. Does not contain TSCA CBI RESULTS DPT 443/984760 FUNCTIONAL OBSERVATIONAL BATTERY DATA Observational endpoints (Table 1, Appendix 1) There were no remarkable differences in the incidence of various observations between treated and controls groups during the course of the study. Activity counts (Table 2, Appendix 1) There were no statistically significant differences between treated and control groups on any occasion of testing. Rearing counts (Table 3, Appendix 1) There were no statistically significant differences between treated and control groups on any occasion of testing. Grip strength (Tables 4,5; Appendix 1) During Week 4, there were no statistically significant differences in forelimb or hindlimb grip strength. Landing footeplay (Table 6, Appendix 1) During Week 4, there were no statistically significant differences in landing footsplay. Temperature (Table 7, Appendix 1) During Week 4, there were no statistically significant differences in mean rectal temperature. Coulbourn activity monitoring (Table 8, Appendix 2) During Week 4, there were no statistically significant differences in the locomotor activity between treated and control groups. DISCUSSION/CONCLUSION To conclude: treatment wit^n^--HI^^B^B}for 28 days was not associated with any behavioural changes that were considered indicative ofneurotoxicity. 124 : Company Sanitized. Does not contain TSCA CBI DPT 443/984760 TABLE 1 Summary of functional observational battery Pre-dose Group No. of animals OBSERVATIONS: REMOVAL FROM CAGE removing, easy handling, easy salivation vocalising IN THE ARENA iremors grooming arousal, alert defecation GAIT urine walking on toes unable to assess MANIPULATIONS touch, a reaction startle (present) righting, immediately tail pinch, a reaction pupil reflex Milies 1 2 3 4 5 5 5 5 5 5 5 5 5 5 5 4 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 5 5 5 5 0 0 0 0 1 1 0 1 1 2 1 2 1 0 0 0 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 Numbers reflect the number of animals showing the response Ferniales 1 2 3 4 5 5 5 5 5 3 5 4 3 3 5 5 1 0 0 0 0 0 1 1 0 0 0 0 0 0 0 0 5 5 5 5 0 0 0 0 0 0 0 1 1 3 2 2 0 1 0 0 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 125 : Company Sanitized. Does not contain TSCA CBI DPT 443/984760 TABLE 1 (Summary of functional observational battery - continued) Weekl Group No. of animals OBSERVATIONS: REMOVAL FROM CAGE removing, easy handling, easy salivation vocalising IN THE ARENA tremors grooming arousal, alert defecation GAIT urine walking on toes hunched posture unable to assess Milies 1 2 3 4 5 5 5 5 5 5 5 5 5 4 5 5 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 4 5 5 4 0 1 0 1 0 1 1 0 3 3 3 2 2 0 0 0 0 0 0 1 Numbers reflect the number of animals showing the response Feniales 1 2 3 4 5 5 5 5 5 4 5 4 5 4 5 5 1 2 2 0 1 2 2 1 0 0 0 0 0 0 0 0 5 5 4 5 0 0 0 0 0 0 0 0 5 5 5 3 1 1 3 1 0 0 0 1 126 : Company Sanitized. Does not contain TSCA CBI DPT 443/984760 TABLE 1 (Summary of functional observational battery - continued) Week 2 Group No. of animals OBSERVATIONS: REMOVAL FROM CAGE removing, easy handling, easy salivation vocalising IN THE ARENA tremors grooming arousal, alert defecation urine GAIT walking on toes hunched posture unable to assess Mslies 1 2 3 4 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 0 0 0 0 0 0 0 0 0 0 1 1 4 4 5 5 0 0 0 0 0 0 1 0 4 3 5 4 0 0 0 1 1 1 0 0 Numbers reflect the number of animals showing the response Fentales 1 2 3 4 5 5 5 5 5 5 5 5 4 3 4 5 5 5 5 5 0 1 0 1 0 0 0 0 0 0 0 0 5 4 5 5 0 0 0 0 0 0 0 0 5 5 5 5 0 0 2 1 0 0 0 0 127 : Company Sanitized. Does not contain TSCA CBI DPT 443/984760 TABLE 1 (Summary of functional observational battery - continued) Week 3 Group No. of animals OBSERVATIONS: REMOVAL FROM CAGE removing, easy handling, easy salivation vocalising IN THE ARENA tremors grooming . arousal, alert defecation urine GAIT walking on toes hunched Milies 1 2 3 4 5 5 5 5 5 5 5 5 5 5 5 5 1 2 1 0 0 0 0 0 0 0 0 0 0 0 0 0 5 5 5 5 0 0 0 0 1 1 0 0 3 1 4 3 0 0 0 0 Numbers reflect the number of animals showing the response Fentales 1 2 3 4 5 5 5 5 5 5 4 5 5 4 5 4 1 2 0 0 0 1 1 0 0 0 0 0 0 0 0 0 5 5 5 5 0 0 0 0 0 0 1 0 5 5 5 5 1 1 0 0 128 : Company Sanitized. Does not contain TSCA CBI DPT 443/984760 TABLE 1 (Summary of functional observational battery - continued) Week 4 Group NO. of animals OBSERVATIONS: REMOVAL FROM CAGE removing, easy handling, easy salivation vocalising IN THE ARENA tremors grooming arousal, alert defecation GAIT urine walking on toes hunched &/lales 1 2 3 4 5 5 5 5 5 5 5 5 5 5 3 4 2 1 1 0 1 1 0 0 0 0 0 0 0 0 0 0 5 5 5 5 0 0 0 0 1 1 1 0 2 0 2 3 . 1 0 1 0 MANIPULATIONS approach, a reaction tonch, a reaction startle (present) righting, immediately tail pinch, a reaction pupil reflex 5 5 5 5 5 2 5 4 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 Numbers reflect the number of animals showing the response Fenlales 1 2 3 4 5 5 5 5 4 5 4 5 5 4 5 5 2 1 1 0 1 0 0 1 0 0 0 0 0 0 0 0 5 5 4 5 0 0 0 0 0 0 1 0 5 5 5 5 1 0 0 1 5 5 5 5 5 5 4 4 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 129 : Company Sanitized. Does not contain TSCA CBI TABLE 2 Activity counts - group mean values Pre-dose Group/dosage (mg/kg/day) 1-0 2-15 3-50 4-150 Mean activity counts Males Females 8 13 9 11 12 11 8 9 Weekl Group/dosage (mg/kg/day) 1-0 2-15 3-50 4-150 Mean activity counts Males Females 7 12 9 10 9 14 9 9 Week 2 Group/dosage (mg/kg/day) 1-0 2-15 3-50 4-150 Mean activity counts Males Females 9 14 10 18 14 18 11 14 Week 3 Group/dosage (mg/kg/day) 1-0 2-15 3-50 4-150 Mean activity counts Males Females 14 15 14 17 14 19 13 17 Week 4 Group/dosage (mg/kg/day) 1-0 2-15 3-50 4-150 Mean activity counts Males Females 11 15 10 16 12 16 7 14 No statistical significance p> 0.05 DPT 443/984760 130 : Company Sanitized. Does not contain TSCA CBI TABLES Rearing counts - group mean values Pre-dose Group/dosage (mg/kg/day) 1-0 2-15 3-50 4-150 Mean rearing counts Males 2 Females 6 4 6 6 6 3 5 Weekl Group/dosage (mg/kg/day) 1-0 2-15 3-50 4-150 Mean rearing counts Males Females 8 2 7 4 9 4 6 4 Week 2 Group/dosage (mg/kg/day) 1-0 2-15 3-50 4-150 Mean rearing counts Males 3 Females 7 5 8 6 9 5 6 Week 3 Group/dosage (mg/kg/day) 1-0 2-15 3-50 4-150 Mean rearing counts Males Females 4 9 6 8 7 8 5 8 Week 4 Group/dosage (mg/kg/day) 1-0 2-15 3-50 4-150 Mean rearing counts Males 6 Females 8 6 9 7 8 3 7 No statistical significance p > 0.05 DPT 443/984760 131 Company Sanitized. Does not contain TSCA CBI TABLE 4 Forelimb grip strength - group mean values Pre-dose Group/dosage (mg/kg/day) 1-0 2-15 3-50 4-150 Mean forelhnb grip strength (kg) Males Females 0.25 0.24 0.24 0.27 0.26 0.23 0.22 0.29 Week 4 Group/dosage (mg/kg/day) 1-0 2-15 3-50 4- 150 Mean forelimb grip strength (kg) Males Females 0.85 0.72 0.83 0.71 0.83 0.70 0.65 0.69 No statistical significance p > 0.05 DPT 443/984760 Company Sanitized. Does not contain TSCA CBI TABLES Hindlimb grip strength - group mean values Pre-dose Group/dosage (mg/kg/day) 1-0 2-15 3-50 4-150 Mean hindlimb grip strength (kg) Males Females 0.25 0.26 0.26 0.27 0.27 0.27 0.31 0.34 Week 4 Group/dosage (mg/kg/day) 1-0 2-15 3-50 4- 150 Mean hindlimb grip strength (kg) Males Females 0.90 0.88 0.95 0.88 0.89 0.84 0.77 0.75 No statistical significance p> 0.05 DPT 443/984760 133 : Company Sanitized. Does not contain TSCA CBI TABLE 6 Landing fbotsplay - group mean values Pre-dose Group/dosage (mg/kg/day) 1-0 2-15 3-50 4-150 . Mean splay values (cm) Males Females 7.6 6.5 7.6 5.4 7.2 6.8 7.8 7.7 Week 4 Group/dosage (mg/kg/day) 1-0 2-15 3-50 4-150 Mean splay values (cm) Males Females 13.4 10.8 13.0 11.3 11.7 9.7 11.3 9.6 No statistical significance p> 0.05 DPT 443/984760 134 : Company Sanitized. Does not contain TSCA CBI TABLE 7 Rectal temperature - group mean values Pre-dose Group/dosage (mg/kg/day) 1-0 2-15 3-50 4- 150 Mean temperature (C) Males Females 37.8 37.7 37.5 37.7 38.3 38.0 38.0 38.2 Week 4 Group/dosage (mg/kg/day) 1-0 2-15 3-50 4- 150 Mean temperature (C) Males Females 38.6 38.7 38.2 38.8 38.4 38.7 37.9 38.4 No statistical significance p> 0.05 DPT 443/984760 135 : Company Sanitized. Does not contain TSCA CBI TABLES Locomotor activity - group mean values Pre-dose Group/dosage (mg/kg/day) 1-0 2-15 3-50 4- 150 Mean large movements (in sees) during 1 hour observation period Males Females 545 274 489 342 369 498 423 465 Week 4 Group/dosage (mg/kg/day) 1-0 2-15 3-50 4-150 Mean large movements (in sees) during 1 hour observation period Males Females 535 563 715 787 657 933 407 564 No statistical significance p > 0.05 DPT 443/984760 136 : Company Sanitized. Does not contain TSCA CBI APPENDIX 1 Functional observational battery DPT 443/984760 KEY Tremors blank no tremor observed B Body the numbers associated with tremors indicate me degree of effect 1,2,3 increasing degree of effect Ease of removal from cage 2 easy (little resistance) 3 slightly awkward Ease of handling 2 easy (little resistance) 3 slightly awkward Salivation (only scored if present) Y sign observed with 1 being slight N sign not observed Arousal 2,3,4,5 increasing levels of arousal with 4 being alert Gait T Walking on toes Hu Hunched A Swaying/lurching gait H Hindlimbs splayed F Front limbs dragging, unable to support weight 0 Unusual gait - see additional comments U Unable to assess - see additional comments blank normal gait the numbers associated with gait indicate the degree of effect 1,2,3 increasing degree of effect Mobility impaired - is the mobility of the rat impaired due to gait abnormalities 137 : Company Sanitized. Does not contain TSCA CBI APPENDIX 1 (Functional observational battery - continued) DPT 443/984760 Approach 1 2 3 4 5 6 0 No reaction Sniffs only Approaches and sniffs Freezes Back/turns away Walks past probe Other reaction - see additional comments Touch No reaction Turns Walks away Freezes Turns to opposite side Walks backwards Other reaction - see additional comments Startle No reaction Ear twitch only Normal flinch Noticeable response Exaggerated response Other reaction - see additional comments Tail pinch 1 2 3 4 5 6 0 no reaction turns 2 turns immediately 3 violent turn walks away freezes jumps forward runs away Other reaction - see additional comments A 1 with a response that is not a turn indicates that the response included a turn such as walks away with a turn Righting reflex 1 immediate reaction 2 reaction slow 138 Company Sanitized. Does not contain TSCA CBI APPENDIX 1 (Functional observational battery - continued) DPT 443/984760 Vocalising, grooming, piloerecdon Y sign observed N sign not observed the numbers associated with vocalising indicate the degree of effect 1,2,3 increasing loudness of vocalising Pupil reflex B L/R N reflex observed both eyes reflex observed in left/right eye only no reflex both eyes Urine N S M L none observed small amount observed moderate amount observed large amount observed Rearing and activity counts Counts were made of rearing and activity when the animals were in the arena. A count for rearing was counted every time the animal lifted both fore feet clear of a supporting surface. The floor of the arena was marked off into 6 equal areas ("squares"), A count for activity was made whenever the animal moved all four feet into one of these squares. 139 : Company Sanitized. Does not contain TSCA CBI DPT 443/984760 APPENDIX 1 (Functional observational battery - continued) Pre-dose Group 1 male. Control Animal OBSERVATIONS IN THE HAND Removing Handling Salivation degree Vocalising degree IN THE ARENA Activity count Arousal Rearing count Bolus count Urine present Gait MANIPULATIONS Approach Touch Startle Righting reflex Tail pinch turns vocalises degree Pupil reflex Temperature(C) Bodyweight (g) GRIP STRENGTH (kg)# Forelimb Hindlimb FOOTS PLAY (cm) # 1 2 2 2 2 2 N N N N 12 7 4 4 5 0 0 0 S S 3 6 5 3 3 3 1 1 3 3 .Y 1 B 37.7 100 Y 2 B 38.1 100 0.23 0.21 6.5 0.28 0.29 7.1 32 2 N N 4 4 2 0 N 3 3 3 1 3 Y 2 B 37.3 90 0.21 0.22 8.2 4 52 2 2 2 N N N N 12 3 4 4 3 2 0 0 N N T1 U 3 3 3 1 3 1 Y 2 B 38.2 95 3 3 3 1 3 Y 2 B 37.6 96 0.28 0.30 7.1 0.25 0.27 9.2 # Values represent the mean of two trials Additional comments Animal number 1 2 3 4 5 Lower teeth pale Lower teeth pale, slight hair Patchy hair loss rump Lower teeth pale' In the arena: limited walking loss right flank 140 : Company Sanitized. Does not contain TSCA CBI DPT 443/984760 APPENDIX 1 (Functional observational battery - continued) Pre-dose Group 2 male, 15 mg/kg/day Animal 6 7 8 9 10 OBSERVATIONS IN THE HAND Removing Handling Salivation degree Vocalising degree IN THE ARENA Activity count Arousal Rearing count Bolus count Urine present Gait MANIPULATIONS Approach Touch Startle Righting reflex Tail pinch turns vocalises degree Pupil reflex Temperature(C) Bodyweight (g) GRIP STRENGTH (kg)# Forelimb Hindlimb FOOTSPLAY (cm) # 10 11 5 9 N044049N 400N 403N Tl Tl Y 2 B 37.8 94 Y 2 B 37.9 98 Y 2 B 37.7 94 Y 2 B 37.5 104 0.25 0.26 7.2 0.18 0.18 6.7 0.31 0.31 8.9 0.21 0.33 9.0 <t Values represent the mean of two trials Additional comments Y 2 B 37.6 92 0.27 0.24 6.2 Animal number 6 7 8 10- Slight brown nasal staining, lower teeth Lower teeth pale Lower teeth pale In the arena: stretching occasionally Lower teeth pale pale 141 : Company Sanitized. Does not contain TSCA CBI DPT 443/984760 APPENDIX 1 (Functional observational battery - continued) Pre-dose Animal Group 3 male, 50 mg/kg/day 11 12 13 14 15 OBSERVATIONS IN THE HAND Removing Handling Salivation degree Vocalising degree IN THE ARENA Activity count Arousal Rearing count Bolus count Urine present Gait MANIPULATIONS Approach Touch Startle Righting reflex Tail pinch turns vocalises degree Pupil reflex Temperature(C) Bodyweight (g) GRIP STRENGTH (kg)# Fore limb Hindlimb FOOTSPLAY (cm) ft 12 4 5 0 M 3 3 3 1 3 Y 2 B 37.5 93 0.29 0.35 8.3 10 4 8 0 N 3 3 2 1 6 Y 2 B 37.5 95 0.24 0.25 4.5 12 4 5 0 N 3 5 4 1 3 Y 2 B 37.4 98 0.23 0.26 6.6 # Values represent the mean of two trials Additional comments 14 4 6 0 N T1 3 3 3 1 3 Y 2 B 38.1 90 0.32 0.23 9.8 10 4 4 0 N 5 3 3 1 3 Y 2 B 37.1 95 0.25 0.27 7.1 Animal number 11 12 13 14 15 Slight brown nasal staining, Patchy hair loss back Slight brown nasal staining, Lower teeth pale Slight hair loss neck, lower lower lower teeth teeth teeth pale pale pale 142 : Company Sanitized. Does not contain TSCA CBI DPT 443/984760 APPENDIX 1 (Functional observational battery - continued) Pre-dose Group 4 male, 150 ing/kg/day Animal 16 17 18 19 20 OBSERVATIONS IN THE HAND Removing Handling Salivation degree Vocalising degree IN THE ARENA Activity count Arousal Rearing count Bolus count Urine present Gait MANIPULATIONS Approach Touch Startle Righting reflex Tail pinch turns vocalises degree Pupil reflex Temperature(C) Bodyweight (g) GRIP STRENGTH (kg)# Forelimb Hindiimb FOOTSPLAY (cm) # 2 2 N N 6 4 1 0 N 3 3 2 1 6 Y 3 B 37.6 103 0.30 0.41 9.3 2 2 N N. 8 4 3 0 N 3 3 2 1 6 1 Y 1 B 38.4 100 0.25 0.32 6.1 2 3 N N 5 4 3 0 N 3 3 2 1 3 Y 2 B 37.8 95 0.36 0.32 8.0 # Values represent the mean of two trials Additional comments 2 2 N N 9 4 5 0 N T1 3 3 3 1 6 Y 2 B 37.2 103 0.23 0.29 6.6 2 2 N N 13' 4 4 0 N T1 3 3 3 1 6 Y 2 B 37.6 94 0.16 0.24 9.0 Animal number 16 Lower teeth pale 18 Slight hair loss rump 19 Lower teeth pale 20 Lower teeth pale, patchy hair loss runp, tip of tail missing 143 : Company Sanitized. Does not contain TSCA CBI DPT 443/984760 APPENDIX 1 (Functional observational battery - continued) Pre-dose Group 1 female. Control Animal 21 22 23 24 25 OBSERVATIONS IN THE HAND Removing Handling Salivation degree Vocalising degree IN THE ARENA Activity count Arousal Rearing count Bolus count Urine present Gait MANIPULATIONS Approach Touch Startle Righting reflex Tail pinch turns vocalises degree Pupil reflex Temperature(C) Bodyweight (g) GRIP STRENGTH (kg)# Forelimb Hindiimb FOOTSPLAY (cm) ft 21 4 5 0 N Tl Y 2 B 38.5 97 0.32 0.31 9.8 16 4 4 0 N Y 1 B 39.1 100 0.31 0.28 6.2 Y 2 B 38.4 102 0.21 0.15 4.8 # Values represent the mean of two trials Additional comments 10 4 7 0 N Y 2 B 37.7 103 0.20 0.31 6.6 11 4 9 0 M Y 2 B 37.6 102 0.20 0.28 5.3 Animal number 21 22 23 24 25 Patchy hair loss back, lower teeth pale During grip strength and temperature: moderately vocalising Lower teeth pale Touch response: rears Lower teeth pale, slight hair loss right hindlimb Lower teeth pale During manipulations: soft faeces Lower teeth pale 144 Company Sanitized. Does not contain TSCA CBI DPT 443/984760 APPENDIX 1 (Functional observational battery - continued) Pre-dose Group 2 female, 15 nig/kg/day Animal 26 27 28 29 30 OBSERVATIONS IN THE HAND Removing Handling Salivation degree Vocalising degree IN THE ARENA Activity count Arousal Rearing count Bolus count Urine present Gait MANIPULATIONS Approach Touch Startle Righting reflex Tail pinch turns vocalises degree Pupil reflex Temperature (C) Bodyweight (g) GRIP STRENGTH (kg)# Forelimb Hindiimb FOOTSPLAY (cm) # 14 4 4 0 N T2 3 2 3 1 6 Y 2 B 38.2 104 0.25 0.29 6.1 4 4 4 0 N U 5 3 3 1 3 Y 2 B 38.3 93 0.14 0.29 6.0 9 4 5 0 N T1 3 3 3 1 2 2 Y 2 B 38.0 101 0.27 0.30 5.0 # Values represent the mean of two trials Additional comments 20 4 9 0 S . T1 3 3 2 1 3 Y 1 B 37.2 98 0.28 0.26 5.1 9 4 8 0 N 3 3 2 1 3 Y 1 B 38.1 104 0.23 0.21 4.8 Animal number 27 28 29 30 Lower teeth pale In the arena: stretching occasionally, sitting on edge of arena, walking along edge of arena, limited walking Lower teeth pale In the arena: sitting along edge of arena Lower teeth pale Lower teeth pale In the arena: sitting alo-.g edge of arena, walking along edge of arena During grip strength: head shake 145 Company Sanitized. Does not contain TSCA CBI APPENDIX 1 DPT 443/984760 (Functional observational battery - continued) Pre-dose Group 3 female, 50 mg/kg/day Animal 31 32 33 34 35 OBSERVATIONS IN THE HAND Removing Handling Salivation degree Vocalising degree IN THE ARENA Activity count Arousal Rearing count Bolus count Urine present Gait MANIPULATIONS Approach Touch Startle Righting reflex Tail pinch turns vocalises degree Pupil reflex Temperature(C) Bodyweight (g) GRIP STRENGTH (kg)# Forelimb Hindlimb FOOTSPLAY (cm) ft 10 4 8 0 N 3 0 2 1 3 y 2 B 38.2 102 0.26 0.32 7.3 11 4 4 0 N T1 3 3 2 1 3 Y 2 B 38.3 98 0.22 0.27 6.1 14 4 6 0 N 3 3 3 1 2 1 Y 2 B 37.8 94 0.18 0.22 5.3 # Values represent the mean of two trials Additional comments 12 4 7 0 N T1 3 3 3 1 3 Y 2 B 37.5 107 0.22 0.22 6.0 8 4 3 0 N 3 2 3 1 3 1 Y 2 B 38.1 101 0.21 0.30 9.3 Animal number 31 32 33 34. 35 Slight hair loss neck and rump, lower teeth pale Touch response: rears Lower teeth pale Slight brown nasal staining, Slight brown nasal staining, Lower teeth pale lower lower teeth pale teeth pale 146 : Company Sanitized. Does not contain TSCA CBI DPT 443/984760 APPENDIX 1 (Functional observational battery - continued) Pre-dose Group 4 female, 150 ing/kg/day Animal 36 37 38 39 40 OBSERVATIONS IN THE HAND Removing Handling Salivation degree Vocalising degree IN THE ARENA Activity count Arousal Rearing count Bolus count Orine present Gait MANIPULATIONS Approach Touch Startle Righting reflex Tail pinch turns vocalises degree Pupil reflex Temperature(C) Bodyweight (g) GRIP STRENGTH (kg)# Forelimb Hindlimb FOOTSPLAY (cm) # 2 2 N N 8 4 6 0 N 3 2 3 1 3 B 37.9 102 0.25 0.27 7.8 2 2 N N 7 4 1 0 N 3 3 3 1 6 Y 1 B 38.3 104 0.37 0.41 7.9 2 2 N N 11 4 10 0 N T1 5 0 2 1 6 Y 2 B 38.5 105 0.29 0.35 6.5 3 2 N Y 2 9 4 4 0 N T1 3 3 3 1 2 2 Y 2 . B 38.2 94 0.29 0.36 9.0 2 2 N N 12 4 6 0 N 5 2 3 1 6 Y 2 B 38.1 96 0.27 0.32 7.4 ft Values represent the mean of two trials Additional comments Animal number 36 38 39 Lower teeth pale Touch response: rears During grip strength and vocalising footsplay: moderate 147 : Company Sanitized. Does not contain TSCA CBI DPT 443/984760 APPENDIX 1 (Functional observational battery - continued) Week 1 Group 1 male, Control OBSERVATIONS IN THE HAND Animal 1 Removing 2 Handling 2 Salivation N degree Vocalising N degree IN THE ARENA Grooming N Activity count 12 Arousal 4 Rearing count 4 Bolus count 0 Urine present N Gait T1HU1 2 3 N 8 4 2 0 N Tl 4 5 N 7 4 3 0 N T1HU1 Additional comments -. Animal number Right lower tooth pale Lower teeth pale 148 : Company Sanitized. Does not contain TSCA CBI DPT 443/984760 APPENDIX 1 (Functional observational battery - continued) Week 1 Animal Group 2 male, 15 ing/kg/day 6 7 8 9 10 OBSERVATIONS IM THE HAND Removing Handling Salivation 2 2 2 2 N N degree Vocalising N N degree IN THE ARENA Grooming N N N Activity count 8 8 12 Arousal 4 4 4 Rearing count 3 3 8 Bolus count 2 0 0 Urine present N N N Gait Tl Tl Tl Additional comments Animal number 6 7 9 10 Slight hair loss head, neck, lower teeth pale Lower teeth pale In the arena: sitting on edge of arena, walking along edge Slight hair loss head, slight brown nasal staining Slight brown nasal staining,' lower teeth pale 149 : Company Sanitized. Does not contain TSCA CBI DPT 443/984760 APPENDIX 1 (Functional observational battery - continued) Week 1 Group 3 male, 50 ing/kg/day Animal 11 12 13 14 15 OBSERVATIONS IN THE HAND Removing 2 Handling 2 Salivation M degree Vocalising N degree IN THE ARENA Grooming N Activity count 9 Arousal 4 Rearing count 5 Bolus count 0 . Urine present N Gait Tl N N 13 10 4 4 6 3 0 0 N N Tl Tl Additional comments Animal number 11 12 14' 15 Slight hair loss head, lower teeth Moderate hair loss head, neck Slight brown nasal staining, lower Lower teeth pale pale teeth pale 150 : Company Sanitized. Does not contain TSCA CBI DPT 443/984760 APPENDIX 1 (Functional observational battery - continued) Week 1 Group 4 male, 150 ing/kg/day Animal 16 17 18 19 20 OBSERVATIONS IN THE HAND Removing Handling Salivation degree Vocalising degree IN THE ARENA Grooming Activity count Arousal Rearing count Bolus count Urine present Gait N N N 10 12 14 4 4 8 1 0 0 N N T2 T1 Additional comments Animal number 16 17 19 20 In the Slight Slight Slight tip of arena: limited walking brown nasal staining hair loss head, lower teeth pale brown nasal staining, slight hair tail missing, lower teeth pale loss head, 151 : Company Sanitized. Does not contain TSCA CBI DPT 443/984760 APPENDIX 1 (Functional observational battery -- continued) Week 1 Group 1 female. Control Animal 21 22 23 24 25 OBSERVATIONS IN THE HAND Removing 2 2 2 2 2 Handling 2 2 2 2 2 Salivation degree N Y N N N 1 Vocalising degree N N N N Y 1 IN THE ARENA Grooming N N N N N Activity count 10 11 12 11 14 Arousal 4 4 4 4 4 Rearing count 5 9 5 9 11 Bolus count 0 0 0 0 0 Urine present N N N N N Gait Tl Tl T1HU1 Tl Tl Additional comments Animal number . 21 22 23 24 Lower teeth pale In the arena: climbing edge of arena Lower teeth pale Lower teeth pale Slight brown nasal staining, lower teeth pale In the arena: sitting along edge of arena 152 : Company Sanitized. Does not contain TSCA CBI DPT 443/984760 APPENDIX 1 (Functional observational battery -- continued) Week 1 Group 4 female, 150 mg/kg/day Animal 36 37 38 39 40 OBSERVATIONS IN THE HAND Removing Handling Salivation degree Vocalising degree IN THE ARENA Grooming Activity count Arousal Rearing count Bolus count Urine present Gait N N N 15 13 4 4 9 10 0 0 N N Tl Tl T1HU1 Additional comments Animal number 36 37 38 39 Lower teeth pale In the arena: head shake, limited Slight brown nasal staining Slight hair loss neck walking 155 : Company Sanitized. Does not contain TSCA CBI DPT 443/984760 APPENDIX 1 (Functional observational battery - continued) Week 2 Group 1 male. Control Animal OBSERVATIONS IM THE HAND Removing Handling Vocalising degree IM THE ARENA Grooming Activity count Arousal Rearing count Bolus count Urine present Gait 12345 N2222N 22N N N N N 11 14 6 10 4 3 444 0 552 N N N N 0 0 0 Tl Tl Tl Tl Additional comments Animal number Right lower tooth pale In the arena: limited walking Slight brown nasal staining 156 : Company Sanitized. Does not contain TSCA CBI DPT 443/984760 APPENDIX 1 (Functional observational battery - continued) Week 1 Group 2 female, 15 ing/kg/day Animal 26 27 28 29 30 OBSERVATIONS IN THE HAND Removing Handling Salivation degree Vocalising degree IN THE ARENA Grooming Activity count Arousal Rearing count Bolus count ' Urine present Gait 2 2 2 2 N N N Y 1 N N N N N 7 5 10 17 12 O4O4O44O4O 4 4 6 11 9 N N N N N Tl Tl Tl Tl T1HU1 Additional comments Animal number 26 27 29 30 Slight Slight In the Slight Slight brown nasal staining brown nasal staining arena: stretching occasionally brown nasal staining, lower teeth brown nasal staining, lower teeth pale pale 153 : Company Sanitized. Does not contain TSCA CBI DPT 443/984760 APPENDIX 1 (Functional observational battery -- continued) Week 1 Group 3 female, 50 mg/kg/day Animal 31 32 33 34 35 OBSERVATIONS IN THE HAND Removing Handling Salivation degree Vocalising degree IN THE ARENA Grooming N N N N N Activity count 13 14 16 13 15 Arousal 4 4 5 4 4 Rearing count 11 7 10 7 9 Bolus count 0 0 0 0 0 Urine present Gait N N N N N Tl T2HU* T1HU1 T2HU1 Tl * Degree not recorded in error Additional comments Animal number 32 33 34 35 Lower teeth pale Lower teeth pale Slight brown nasal Lower teeth pale staining 154 : Company Sanitized. Does not contain TSCA CBI DPT 443/984760 APPENDIX 1 (Functional observational battery - continued) Week 1 Group 4 female, 150 nig/kg/day Animal 36 37 38 39 40 OBSERVATIONS IN THE HAND Removing Handling Salivation degree Vocalising degree IN THE ARENA Grooming Activity count Arousal Rearing count Bolus count Urine present Gait N N 15 13 4 4 9 10 0 0 N N Tl Tl T1HU1 Additional comments Animal number 36 37 38 39 Lower teeth pale In the arena: head shake, limited Slight brown nasal staining Slight hair loss neck walking : 155 : Company Sanitized. Does not contain TSCA CBI DPT 443/984760 APPENDIX 1 (Functional observational battery - continued) Week 2 Group 1 male. Control Animal OBSERVATIONS IN THE HAND Removing Handling Vocalising degree IN THE ARENA Grooming Activity count Arousal Rearing count Bolus count Urine present Gait 12345 N2222N 22N 22N 22N N N N N N 11 14 6 3 10 5040540423024 N N N N N 03 Tl Tl Tl U Tl Additional comments Animal number 1 4 5 Right lower too*-^ pale In the arena: ^-^nited walking Slight brown nasal staining : 156 : Company Sanitized. Does not contain TSCA CBI DPT 443/984760 APPENDIX 1 (Functional observational battery - continued) Week 2 Group 2 male, 15 mg/kg/day Animal 6 7 8 9 10 OBSERVATIONS IN THE HAND Removing Handling Vocalising degree IN THE ARENA N2222N 22M Grooming N N N N Activity count 6 15 10 15 Arousal 4 Rearing count 4 10 3 8 4 4 4 Bolus count 0 Urine present N N N N 0 0 0 Gait Tl Tl Tl Additional comments Animal number 6 7 9 10 Slight brown nasal staining Lower teeth pale, slight brown nasal staining In the arena: sitting in corner Slight brown nasal staining, lower teeth pale 157 : Company Sanitized. Does not contain TSCA CBI DPT 443/984760 APPENDIX 1 (Functional observational battery - continued) Week 2 Group 3 male, 50 mg/kg/day Animal 11 12 13 14 15 OBSERVATIONS IN THE HAND Removing Handling Vocalising degree IN THE ARENA Grooming N N N N Y Activity count 19 14 15 11 13 Arousal 4 4 4 4 4 Rearing count 8 6 8 5 5 Bolus count 0 0 0 0 0 Urine present S N N N N Gait T2 T1 T1 T1 T2 Additional comments Animal number 11 13 14 15 Lower teeth pale Moderate brown nasal staining Slight brown nasal staining, lower Lower teeth pale teeth pale 158 : Company Sanitized. Does not contain TSCA CBI DPT 443/984760 APPENDIX 1 (Functional observational battery-continued) Week 2 Animal Group 4 male, 150 mg/kg/day 16 17 18 19 20 OBSERVATIONS IN THE HAND Removing Handling Vocalising degree IN THE ARENA Grooming N Activity count 5 Arousal 4 Rearing count 2 Bolus count 0 Urine present N Gait Tl N N 11 12 4 4 4 8 0 0 N 'N Tl N Y 11 14 4 4 2 8 0 0 N N T2 T2HU1 Additional comments Animal number 19 Slight brown nasal staining 20 Moderate brown nasal staining, tip of tail missing, lower teeth pale 159 : Company Sanitized. Does not contain TSCA CBI DPT 443/984760 APPENDIX 1 (Functional observational battery - continued) Week 2 Group 1 female. Control Animal 21 22 23 24 25 OBSERVATIONS IN THE HAND Removing Handling Vocalising degree IN THE ARENA Grooming Activity count Arousal Rearing count Bolus count Urine present Gait 2322222222 N N N N N N N N N N 14 16 12 11 15 4 07N Tl 4 07N Tl 080644 N N Tl Tl 4 09N T l Additional comments Animal number 21 22 2 4 Lower Lower Lower teeth pale teeth-pale teeth pale 160 : Company Sanitized. Does not contain TSCA CBI DPT 443/984760 APPENDIX 1 (Functional observational battery - continued) Week 2 Group 2 female, 15 ing/kg/day Animal 26 27 28 29 30 OBSERVATIONS IN THE HAND Removing Handling Vocalising degree IN THE ARENA Grooming Activity count Arousal Rearing count Bolus count Urine present Gait 22Y 2 32N 22N 32N 22N N N N N N O4O4O5O4O4 20 15 19 18 16 7 7 8 9 8 N N N N N T2 Tl T2 Tl Tl Additional comments Animal number 27 28 29 In the arena: walking along Slight matted fur lower jaw In the arena: walking along Lower teeth pale edge edge with forepaws 161 : Company Sanitized. Does not contain TSCA CBI DPT 443/984760 APPENDIX 1 (Functional observational battery -- continued) Week 2 Group 3 female, 50 mg/kg/day Animal 31 32 33 34 35 OBSERVATIONS IN THE HAND Removing Handling Vocalising degree IN THE ARENA Grooming N N N N N Activity count 18 17 21 19 16 Arousal 4 4 4 4 4 Rearing count 8 8 10 13 6 Bolus count 0 0 0 0 0 Orine present Gait N N N N N T2 T2H01 T1 T1HU1 T1 Additional comments Animal number 31 32 33 34 35 Slight brown nasal on ears Lower teeth pale Lower teeth pale Slight brown nasal Slight brown nasal staining, staining staining slight brown staining 162 : Company Sanitized. Does not contain TSCA CBI DPT 443/984760 APPENDIX 1 (Functional observational battery -- continued) Week 2 Group 4 female, 150 mg/kg/day Animal 36 37 38 39 40 OBSERVATIONS IN THE HAND Removing Handling Vocalising degree IN THE ARENA Grooming Activity count Arousal Rearing count Bolus count Urine present Gait 2222222222 N N N Y N 2 N N N N N 14 12 18 9 16 460N Tl 450N Tl 480N T1HU1 440N Tl 480NT l Additional comments Animal number 37 Slight brown nasal staining 38 Slight brown nasal staining 163 : Company Sanitized. Does not contain TSCA CBI DPT 443/984760 APPENDIX 1 (Functional observational battery - continued) Week 3 Animal OBSERVATIONS IN THE HAND Removing Handling Salivation degree Vocalising degree IN THE ARENA Activity count Arousal Rearing count Bolus count Urine present Gait Group 1 male. Control 12345 N Y N N N 2222222222 1 N N N N N 16 20 11 8 15 0460480420434 S N N N N 03 Tl Tl T2 164 : Company Sanitized. Does not contain TSCA CBI DPT 443/984760 APPENDIX 1 (Functional observational battery - continued) Week 3 Group 2 male, 15 mg/kg/day Animal 6 7 8 9 10 OBSERVATIONS IN THE HAND Removing Handling Salivation degree Vocalising degree IN THE ARENA Activity count Arousal Rearing count Bolus count Urine present Gait 2222222222 N Y N Y N 1 1 M N N N N 4446434847 13 16 14 13 13 0 0 0 0 0 S N N N N Tl Additional comments Animal number 6 Lower teeth pale 10 Lower teeth pale 165 : Company Sanitized. Does not contain TSCA CBI DPT 443/984760 APPENDIX 1 (Functional observational battery - continued) Week 3 Group 3 male, 50 mg/kg/day Animal 11 12 13 14 15 OBSERVATIONS IN THE HAND Removing Handling Salivation degree Vocalising degree IN THE ARENA Activity count Arousal Rearing count Bolus count Urine present Gait 14 13 13 15 14 7040640484084 N N N 06 T2 Tl N N T2 T2 Additional comments Animal number 14 Slight brown nasal staining, lower teeth pale 15 Lower teeth pale 166 : Company Sanitized. Does not contain TSCA CBI DPT 443/984760 APPENDIX 1 (Functional observational battery - continued) Week 3 Group 4 male, 150 mg/kg/day Animal 16 17 18 19 20 OBSERVATIONS IN THE HAND Removing Handling " Salivation degree Vocalising degree IN THE ARENA Activity count Arousal Rearing count Bolus count Urine present Gait 22 N 22 N 222N 2N 2 2 N N N N N N 8 14 17 11 13 4204054064074 N N N N N 05 Tl Tl T2 Additional comments Animal number 20 Slight brown nasal staining, tip of tail missing, small area matted fur urogenital region, lower teeth pale 167 : Company Sanitized. Does not contain TSCA CBI DPT 443/984760 APPENDIX 1 (Functional observational battery - continued) Week 3 Group 1 female. Control Animal 21 22 23 24 25 OBSERVATIONS IN THE HAND Removing Handling Salivation degree Vocalising degree IN THE ARENA Activity count Arousal Rearing count Bolus count Urine present Gait 2222222222 N N Y N N 1 N N N N N 17 15 22 9 12 4O4O4O4O4O 7 14 11 6 7 N N N N N Tl T1HU1 T2 Tl Tl Additional comments Animal number 21 In the arena: walking along edge of arena 23 Slight brown nasal staining 24 Lower teeth pale 168 : Company Sanitized. Does not contain TSCA CBI DPT 443/984760 APPENDIX 1 (Functional observational battery - continued) Week 3 Group 2 female, 15 mg/kg/day Animal 26 . 27 28 29 30 OBSERVATIONS IN THE HAND Removing Handling Salivation degree Vocalising degree IN THE ARENA Activity count Arousal Rearing count Bolus count Urine present Gait 2222223222 N N Y Y N 1 1 N N N Y N 1 16 20 14 19 18 O4O4O4O44O 9 7 10 8 7 N N N N N T2 T1HU1 Tl T2 Tl : 169 : Company Sanitized. Does not contain TSCA CBI DPT 443/984760 APPENDIX 1 (Functional observational battery - continued) Week 3 Group 3 female, 50 ing/kg/day Animal 31 32 33 34 35 OBSERVATIONS IN THE HAND Removing Handling Salivation degree Vocalising degree ' IN THE ARENA Activity count Arousal Rearing count Bolus count Urine present Gait 2222322222 N N M N N N N Y N N . 2 19 24 18 12 20 O4O4O44O4O 10 9 8 4 8 N S N N N T2 Tl T2 Tl T2 Additional comments Animal number 33 Lower teeth pale 35 In the arena: walking along edge of arena 170 : Company Sanitized. Does not contain TSCA CBI DPT 443/984760 APPENDIX 1 (Functional observational battery - continued) Week 3 Group 4 female, 150 mg/kg/day Animal 36 37 38 39 40 OBSERVATIONS IN THE HAND Removing Handling Salivation degree Vocalising degree IN THE ARENA Activity count 19 13 16 16 20 Arousal 4 4 4 4 4 Rearing count 10 6 8 10 7 Bolus count 0 0 0 0 0 Urine present N N N N N Gait Tl Tl Tl Tl T2 Additional comments Animal number 38 Moderate hair loss neck 39 Slight brown nasal staining 171 : Company Sanitized. Does not contain TSCA CBI DPT 443/984760 APPENDIX 1 (Functional observational battery - continued) Week 4 Group 1 male. Control 1 2 3 4 5 OBSERVATIONS IN THE HAND Animal Removing Handling Salivation degree Vocalising degree IN THE ARENA Grooming N N N Activity count 15 14 7 Arousal 4 4 4 Rearing count 9 7 4 Bolus count 0 0 0 Urine present S N N Gait T2 HU1 N N 9 12 4 4 4 4 0 0 N N T1 MANIPULATIONS Approach Touch Startle Righting reflex; Tail pinch turns vocalises degree Pupil reflex Temperature(C) Bodyweight (g) 3 3 3 1 2 2 Y 2 B 38.9 326 3 3 2 1 3 Y 2 B 38.8 353 3 3 3 1 3 Y 2 B 38.4 314 2 4 3 1 6 Y 3 B 38.5 337 3 3 3 1 3 Y 1 B 38.2 315 GRIP STRENGTH (kg)# Forelimb Hindlimb FOOTSPLAY (cm) # 0.86 0.94 12.9 0.83 0.69 12.2 0.85 0.97 15.2 0.91 0.92 11.6 0.81 0.99 15.0 # Values represent the mean of two trials Additional comments Animal number 1 3 4 . 5 Slight In the Slight Slight In the brown nasal staining arena: walking along edge brown nasal staining lack of grooming rump and arena: head shake forepaws back 172 : Company Sanitized. Does not contain TSCA CBI DPT 443/984760 APPENDIX 1 (Functional observational battery - continued) Week 4 Group 2 male, 15 nig/kg/day Animal 6 7 8 9 10 OBSERVATIONS IN THE HAND Removing Handling Salivation degree Vocalising degree IN THE ARENA Grooming Activity count Arousal Rearing count Bolus count Urine present Gait MANIPULATIONS Approach Touch Startle Righting reflex Tail pinch turns vocalises degree Pupil reflex Temperature(C) Bodyweight (g) GRIP STRENGTH (kg)ft Forelimb Hindlimb FOOTSPLAY (cm) tt 2 2 N N N 10 4 6 0 M 3 1 3 X 6 Y 2 B 38.9 309 0.89 1.08 13.9 2 2 Y 1 Y 2 N 14 4 8 0 N 3 1 2 1 3 Y 1 B 38.3 314 0.73 0.75 14.1 2 2 N N N 10 .4 4 0 N 3 1 3 1 3 B 38.3 352 0.82 0.90 11.1 # Values represent the mean of two trials Additional comments 2 2 N N N 5 4 3 0 N 3 3 2 1 3 Y 2 B 37.6 305 0.95 1.03 14.3 2 2 N N N 9 4 7 0 N 3 3 3 1 3 Y 2 B 38.1 288 0.75 1.00 11.5 Animal number 6 Slight brown nasal staining, lower teeth pale 10 Lower teeth pale. 173 : Company Sanitized. Does not contain TSCA CBI DPT 443/984760 APPENDIX 1 (Functional observational battery -- continued) Week 4 Group 3 male, 50 ing/kg/day Animal 11 12 13 14 15 OBSERVATIONS IN THE HAND Removing Handling Salivation degree Vocalising degree IN THE ARENA Grooming Activity count Arousal Rearing count Bolus count Urine present Gait MANIPULATIONS N 12 4 12 0 N Tl HU1 Approach 3 Touch 3 Startle Righting reflex Tail pinch turns vocalises degree Pupil reflex Temperature(C) Bodyweight (g) GRIP STRENGTH (kg)# 4 1 3 Y 3 B 38.6 288 Forelimb Hindiimb FOOTSPLAY (cm) # 1.03 0.98 13.7 N 14 4 8 0 N Tl 3 2 3 1 3 Y 2 B 38.2 277 0.83 1.06 7.6 N 15 4 8 0 N 3 2 2 1 3 Y 2 B 38.4 323 0.77 0.95 12.6 # Values represent the mean of two trials Additional comments N 12 4 5 0 N 3 3 2 1 5 Y 2 B 38.8 309 0.89 0.80 13.7 N 8 4 4 0 S 3 3 2 1 3 Y 1 B 38.2 258 0.64 0.68 11.0 Animal number 11 Slight lack of grooming rump 13 Slight brown nasal staining 14 Slight lack of grooming rump, tip of tail missing, lower teeth pale 15 Slight lack of grooming on rump 174 : Company Sanitized. Does not contain TSCA CBI DPT 443/984760 APPENDIX 1 (Functional observational battery - contmned) Week 4 Group 4 male, 150 mg/kg/day Animal 16 11 18 19 20 OBSERVATIONS IN THE HAND Removing Handling Salivation degree Vocalising degree IN THE ARENA Grooming Activity count Arousal Rearing count Bolus count Urine present Gait MANIPULATIONS Approach Touch Startle Righting reflex Tail pinch turns vocalises degree Pupil reflex Temperature(C) Bodyweight (g) GRIP STRENGTH (kg)ft Forelimb Hindiimb FOOTS PLAY (cm) # 2 3 N N N 7 4 3 0 N F2 3 1 3 1 5 Y 3 B 37.7 174 0.76 0.77 12.9 2 2 N N N 9 4 3 0 N 3 3 3 1 2 3 Y 2 B 38.8 214 0.51 0.80 8.8 2 2 N N N 8 4 3 0 N T1 3 5 3 1 6 1 Y 2 B 38.1 232 0.58 0.74 9.4 2 2 N N N 8 4 3 0 N T2 3 3 3 1 3 Y 2 B 37.7 265 0.77 0.87 13.2 2 2 N N N 5 4 4 0 N 3 3 3 1 3 1 Y 2 B 37.4 218 0.62 0.71 12.2 # Values represent the mean of two trials Additional comments Animal number 16 Slight lack of grooming back 18 Patchy hair loss rump 20 Slight brown staining ears, tip of tail missing 175 : Company Sanitized. Does not contain TSCA CBI DPT 443/984760 APPENDIX 1 (Functional observational battery - continued) Week 4 Animal Group 1 female. Control 21 22 23 24 25 OBSERVATIONS IM THE HAND Removing Handling Salivation degree Vocalising degree IN THE ARENA Grooming Activity count Arousal Rearing count Bolus count Urine present Gait MANIPULATIONS Approach Touch Startle Righting reflex Tail pinch turns vocalises degree Pupil reflex Temperature(C) Bodyweight (g) GRIP STRENGTH (kg)# Forelimb Hindlimb FOOTS PLAY (cm) ft 3 2 N N N 14 4 5 0 N Tl 5 2 2 1 3 Y 2 B 38.3 215 0.64 0.75 14.1 2 2 Y 1 Y 1 N 16 4 10 0 N Tl 3 0 3 1 3 1 Y 2 B 39.4 233 0.88 1.01 9.4 2 2 Y 1 N N 19 4 9 0 N T2 3 0 3 1 3 Y 2 B 38.8 221 0.61 0.80 8.4 2 2 2 2 N N N N N N 11 13 4 4 9 9 0 0 N N Tl T2 HU1 5 3 3 5 3 3 1 1 5 3 Y 1 B 38.7 244 Y 2 B 38.4 212 0.72 0.96 11.1 0.76 0.89 11.1 # Values represent the mean of two trials Additional comments Animal number 21 22 23 24 25 During grip strength: moderate vocalisation During footsplay: moderate vocalisation and slightly awkward to handle During touch response: rears During pupil response, grip strength and footsplay: moderate vocalisation Touch response: rears During grip strength: moderate vocalisation Appro .ch response: bit probe 176 Company Sanitized. Does not contain TSCA CBI DPT 443/984760 APPENDIX 1 (Functional observational battery - continued) Week 4 Group 2 female, 15 mg/kg/day Animal 26 27 28 29 30 OBSERVATIONS IN THE HAND Removing Handling Salivation degree Vocalising degree IN THE ARENA Grooming Activity count Arousal Rearing count Bolus count Urine present Gait MANIPULATIONS Approach Touch Startle Righting reflex Tail pinch turns vocalises degree Pupil reflex Temperature(C) Bodyweight (g) GRIP STRENGTH (kg)# Forelimb Hindiimb E-OOTSPLAY (cm) # N 20 4 8 0 N T2 3 0 3 1 3 Y 2 B 39.2 224 0.66 1.03 10.3 N 15 4 7 0 N T2 3 3 3 1 6 Y 2 B 38.9 217 0.58 0.98 12.8 N N 15 17 4. 4 9 13 0 0 N N T2 T2 3 3 3 3 3 2 1 1 3 0 Y 2 B 38.8 221 Y 1 B 38.8 215 0.97 1.05 11.5 0.67 0.77 13.4 # Values represent the mean of two trials Additional comments N 14 4 9 0 N T1 3 3 3 1 3 Y 1 B 38.4 225 0.66 0.60 8.6 Animal number 26 28 29 30 Touch response: rears During grip strength: moderate vocalisation In the arena: sitting on edge of arena Approach response: bit probe During grip strength: moderate vocalisation During footsplay and bodyweight: slightly awkward to handle, moderate vocalisation Slight lack of grooming rump, lower left too4-!! pale Touch response: delayed walk away During footsplay: slight red discharge from urethra noted Slight brown nasal staining 177 Company Sanitized. Does not contain TSCA CB1 DPT 443/984760 APPENDIX 1 (Functional observational battery -- continued) Week 4 Group 3 female, 50 mg/kg/day Animal 31 32 33 34 35 OBSERVATIONS IN THE HAND Removing Handling Salivation degree Vocalising degree IN THE ARENA Grooming Activity count Arousal Rearing count Bolus count Urine present Gait MANIPULATIONS Approach Touch Startle Righting reflex Tail pinch turns vocalises degree Pupil reflex Temperature(C) Bodyweight (g) GRIP STRENGTH (kg)# Forelimb Hindi inib FOOTS PLAY (cm) ft 2 2 Y 1 N N 16 4 10 0 N Tl 3 3 2 1 3 1 B 38.5 193 0.56 0.69 7.2 3 2 N N N 22 5 11 0 S T2 3 1 3 1 6 1 Y 1. B 39.. 1 204 0.74 1.01 8.2 2 2 N N N 18 4 6 0 N Tl 3 2 2 1 6 Y 3 B 38.8 194 0.80 0.90 10.3 # Values represent the mean of two trials Additional comments 2 2 N N N 14 4 8 0 N Tl 3 3 3 1 3 Y 2 B 38.6 218 0.82 0.67 9.9 2 2 N N N 10 4 3 0 N Tl 3 3 2 1 5 1 Y 2 B 38.7 217 0.59 0.93 13.0 Animal number 32 33 35 Touch response: bit probe Lower teeth pale Approach response: bit probe Slight lack of grooming rump 178 : Company Sanitized. Does not contain TSCA CBI DPT 443/984760 APPENDIX 1 (Functional observational battery - continued) Week 4 Group 4 female, 150 mg/kg/day Animal 36 37 38 39 40 OBSERVATIONS IN THE HAND Removing Handling Salivation degree Vocalising degree IN THE ARENA Grooming Activity count Arousal Rearing count Bolus count Urine present Gait MANIPULATIONS Approach Touch Startle Righting reflex Tail pinch turns vocalises degree Pupil reflex Temperature(C) Bodyweight (g) GRIP STRENGTH (kg)ft Forelirob Hindlimb FOOTSPLAY (cm) # 2 2 N N N 14 4 6 0 N Tl 3 2 2 1 3 Y 2 B 38.3 176 0.65 0.81 11.8 2 2 2 2 2 2 N N N N N Y 1 N N N 11 15 13 4 4 4 5 9 6 0 0 0 N N N Tl T2 HU1 Tl 3 3 2 1 3 Y 1 B 38.9 158 3 2 2 1 2 2 Y 1 B 38.6 161 3 1 3 1 3 1 Y 2 B 37.8 157 0.86 0.82 8.9 0.60 0.63 8.5 0.83 0.80 10.8 # Values represent the mean of two trials Additional comments 2 2 N N N 15 4 8 0 N T2 5 3 3 1 6 1 Y 2 B 38.2 163 0.51 0.69 8.1 Animal number 36 38 39 Approach response: bit probe Moderate hair loss neck During grip strength: moderate vocalisation 179 : Company Sanitized. Does not contain TSCA CBI APPENDIX! Locomotor activity Animal no. Total time spent in locomotor activity (sees) Pre-dose Week 4 Group 1 Sex male. Control 874 708 484 384 498 362 571 766 299 455 Group 2 Sex male, 15 nig/kg/day 6 624 703 7 347 498 8 581 872 9 281 658 10 610 843 Group 3 Sex male, 50 ing/kg/day 11 443 777 12 403 713 13 231 632 14 625 695 15 142 468 Group 4 Sex male, 150 ing/kg/day 16 522 351 17 524 531 18 332 569 19 322 389 20 414 197 DPT 443/984760 180 : Company Sanitized. Does not contain TSCA CBI APPENDIX! (Locomotor activity - continued) Animal no. Total time spent in locomotor activity (sees) Pre-dose Week 4 Group 1 Sex female. Control 21 202 711 22 120 479 23 377 617 24 475 633 25 197 374 Group 2 Sex female, 26 207 27 334 28 225 29 539 30 406 15 mg/kg/day 923 741 848 780 642 Group 31 32 33 34 35 Sex female, 50 nig/kg/day 776 1131 160 871 533 899 800 1317 222 447 Group 4 Sex female, 36 406 37 172 38 520 39 598 40 628 150 mg/kg/day 310 292 452 865 899 DPT 443/984760 181 : Company Sanitized. Does not contain TSCA CBI DPT 443/984760 FORMULATION CHEMISTRY 182 : Authors: I. Suzanne Dawe, Paul Mann, Emma Buckland. Company Sanitized. Does not contain TSCA CBI CONTENTS DPT 443/984760 Page INTRODUCTION................................................................................................................. 184 EXPERIMENTAL PROCEDURE........................................................................................ 185 189 RESULTS.............................................................................................................................. 189 DISCUSSION....................................................................................................................... CONCLUSION................................................................................................................ 190 TABLES 1. Concentrations in test formulations............................................................................ 191 2. Stability in aqueous formulations............................................................................... 192 3. Validation of the analytical procedure........................................................................ 193 FIGURES 1. Typical calibration standard graph .............................................................-...--......... 195 2. Typical analytical chromatograms ........................................................... 196 183 : Fonnulaiion Chemistry Company Sanitized. Does not contain TSCA CBI INTRODUCTION DPT 443/984760 This report details the analytical procedure used, the sampling procedure and the results obtained for: The determination of concentrations cylHI^IUfi11 test formulations analysed during the study. ^" The determination of the stability oqUH^^By11 aqueous formulations. ^ ' ^ ^ ^ ^ ^ The validation of the analytical procedure for the determination ^I^I^HHiBf11 aqueous formulations. fc- . The formulations for this study were prepared as solutions oalHUHBRin distilled water by Pharmacy personnel at the Huntingdon Research Centre, Huntingdon Life Sciences Ltd. The samples were analysed using a method supplied by the Sponsor (reference 'Chromatography of _ f|U|||BsuppIied in a letter dated 23^ July 1998) adapted for^use at Huntingdon Life . Sciences (HRC/FA/M80/98 issue 01/171198). The method of analysis fDiBBIBBiD11 ^"so"8 solutions involved dilution, initially using water and subsequently solvent A', and injection of the onllJUHIji diluted test samples onto ^3 high performance liquid chromatograph (HPLC) with conductivity detection. The amount the test samples was quantified by external calibration with reference to five linearly related standards of known concentrations. ' Solvent A Acsronitrile / aqueous 0.01 M sodium hydroxide (25/75 v/v). 184 : Formulation Chemistry Company Sanitized. Does not contain TSCA CBI EXPERIMENTAL PROCEDURE DPT 443/984760 ANALYTICAL PROCEDURE Apparatus and instrumentation High performance liquid chromatograph (HPLC): Balances: As detailed under chroinatographic conditions. Mettler AT261, fitted with a data print LC-P45. Sartorius L2200-P, fitted with a data print YDP01-*D. Autodiluter: Hamilton MicrolabTM 1000. General laboratory glassware and apparatus. These are typical details and equivalent apparatus and instrumentation may have been substituted. Reagents Test substance: Supplier: Batch no: Stated purity: Control: Acetonitrile: Ammonia solution (SG 0.88): Sodium hydroxide: Sodium carbonate: Sulphuric acid: ' Dupont Specialty Chemicals. Distilled water. Sigma-Aldrich, Riedel de Haen, Far UV for HPLC. Fisher Scientific UK Ltd, Analytical Reagent Fisher Scientific UK Ltd, Analytical Reagent. Fisher Scientific UK Ltd, Analytical Reagent. Fisher Scientific UK Ltd, Analytical Reagent. Buffer solution: Solvent A: Sodium carbonate (2.12g) (120 ml), ammonia solution the solution was diluted to water. The resulting (10 : 1000 ml) using water. was dissolved in water (2.5 ml) was added and volume (200 ml) using solution was diluted Acetonitrile / aqueous 0.01M sodium hydroxide (25 / 75 v/v). : 185 Formulation Chemistry Company Sanitized. Does not contain TSCA CBI Regenerant (0.025M sulphuric acid): Mobile phase: Water DPT 443/984760 Sulphuric acid (28 ml) was added to sufficient water (1000 ml) to provide a 0.5M solution, and diluted (50 : 1000 ml) using water to provide the regeneration solution. Acetonitrile / buffer solution (25 / 75 v/v). Elgastat UHP-4, deionised reverse osmosis. Sample process An exact volume (1 ml) of test formulation was appropriately diluted, initially using water and finally using solvent A, to provide a solution containing|HU|BHH^at an expected concentration in the range 100 ug/ml - 200 ug/ml. w ~ The concentration of iHI^B^Hyas quantified by high performance liquid chromatography using conductivity detection as detailed in the following section. Typical chromatographic conditions High performance liquid chromatograph (HPLC): Pump: Dionex GP40. Autosampler: Perkin Elmer ISS200. Detector: Dionex Electrochemical. Data handling: Spectra Physics SP4270. Analytical column: PLRP-S, 250 x 4.6 mm id, Polymer Laboratories. Column temperature: Mobile phase: Ambient, nominally 21C. Acetonitrile / buffer solution (25 / 75 v/v). Flow rate: 1.0 ml/minute. Detection: Conductivity. Chemical Suppression: Suppressor type: Regenerant: Flow rate: Anionic. 0.025M Sulphuric acid. 2.5 ml/minute Injection volume: 100 pi. Integrator attenuati- -n: 64. Retention volume: Approximately 6.5 ml. 186 : Formulation Chemistry Company Sanitized. Does not contain TSCA CBI DPT 443/984760 Calibration A primary standard solution was prepared for each analytical occasion by dissolving an accurately weighed quaiititYf50jng)of|IJfJHu water (50 ml). Solutions for instrument calibration, containinglU^mUit concentrations of 50 ng/ml, 100 ng/ml, 150 Ug/ml, 200 ug/ml and 250 Hg/ml, were prepared By appropriate dilution of the primary standard using solvent A. Calibration solutions were injected onto the HPLC, at the beginning and end of each sample analysis sequence, using the conditions detailed in the previous section. Calculation The peak height response ofjBiiHI^^HR111eac^ calibration chromatogram was measured and calibration curves were constructed by linear regression of standard response versus standard (B^HB^H11 "^^HIB^W^ ^concentration. The height response of the peak observed at the characteristic retention volume of sample chromatograms was measured and the concentration determined using the following equation: ( Concentration, mg / ml = V- T ------ x. 0 V x 10"3 Where Y = Peak height response for Zonyl FS-62 in test chromatogram I = Intercept derived from linear regression of calibration data S = Slope derived from linear regression of calibration data V = Dilution factor of sample VALIDATION OF THE ANALYTICAL PROCEDURE The analytical procedure was validated by determining the specificity of the chromatographic analysis, linearity of detector response, precision of injection, limit of detection, method accuracy and precision. Specificity Chromatograms^btained for control extracts were examined for peaks which might interfere with the quantitation of^----------|f V^^^^^fs Linearity The linearity of the standard curve was examined by preparing a series of standard solutions (50 ug/ml, 100 ng/ml, 150 ug/ml, 200 ug/ml and 250 u.g/ml) to encompass the working range of the assay. The peak height for each standard was plotted against the concentration using least square regression analysis to provide information on the slope, intercept and correlation coefficient. 187 : Formulation Chemistry Company Sanitized. Does not contain TSCA CBI DPT 443/984760 System precision The repeatability of injections was evaluated by determining the precision of six replicate injections of both the lowest standard solution (50 ug/ml) and the highest standard solution (250 (ig/ml). Limit of detection ^^^^J The limit of detection, defined as the concentration of^BI^U^Jun control matrix producing a signal to noise ratio of at least 3, was determined by fortifying an extract of control vehicle (distilled water) ^ilfiUBHBRr P'"0'^'16 a Pea^ of suitable size. Method accuracy and precision Prior to the start of treatment, specimen formulations containing||^BBU9in distilled water at nominal concentrations of 5 mg/ml and 400 mg/ml were prepared by Pharmacy personnel. The analytical procedure was validated at the low and high inclusion levels by determining the accuracy and precision of analytical results generated for the analysis of six replicate samples from the prepared formulations. STABILITY IN AQUEOUS FORMULATIONS ina^B^^Uyt Prior to treatment, specimen aqueous formulations (400 ml) contain nominal concentrations of 5 mg/ml and 400 mg/ml, were prepared and equally subdivided (4 x 100 ml) into four amber screw-top bottles by Pharmacy personnel at the Huntingdon Research Centre and submitted for analysis. On receipt, one bottle of each formulation was retained for immediate analysis (Day 0) and analysis following ambient temperature storage for 4 hours and 48 hours. The remainder were refrigerated (nominally +4C) for 2, 8 and 15 days. At each time point, the appropriate formulations were mixed by inversion and sampled in duplicate from the approximate centre for analysis. The formulations removed from refrigerated storage were allowed to equilibrate to ambient temperature for 1 hour prior to sampling for analysis. The 400 mg/ml formulations were examined for evidence of any precipitate and, if present, the formulation was gently warmed to 35C to achieve solution, cooled to ambient temperature and sampled. At each time-point, the two sub-samples from each formulation were analysed in accordance with the analytical procedure. CONCENTRATION IN TEST FORMULATIONS At specified intervals (V eek 1) during treatment, freshly prepared test formulations were sampled (20 ml) by Pharmacy personnel at the Huntingdon Research Centre and the samples were submitted for analysis. On receipt, each formulation was thoroughly mixed by vigorous shaking and duplicate samples (1 ml) were analysed in accordance with the analytical procedure. 188 : Formulation Chemistry Company Sanitized. Does not contain TSCA CBI RESULTS DPT 443/984760 The mean concentrations Q^HB^^^In'11 test formulations analysed during the study and the deviation of mean results fronTnominal values are detailed in Table 1. C^^^^t The results obtained for the stability ofiUH^Ifbrmulated in distilled water formulations are -- presented in Table 2. c- The results for the validation of the analytical procedure determined with respect to the linearity of detector response, system precision, method accuracy and precision are presented in Table 3. A typical calibration standard graph is presented in Figure 1. Typical analytical chromatograms are presented in Figure 2. DISCUSSION The mean concentrations of fH^SfS^ffm test formulations analysed during the study were within -7% of nominal concentrations, confirming the accuracy of formulation. Prior to treatment, the stability tor j^BIBHIU111 me ^"s0"3 formulation at nominal concentrations of 5 mg/ml and 400 nig/ml was confirmed with respect to the level of concentration. The mean analysed concentration remained close to nominal (within --6.5%) during ambient temperature storage for 2 days and refrigerated storage for up to 15 days. The linearity of detector response was confirmed forl^|^^|^qover the concentration range 50 ug/ml - 250 Hg/ml. . Thejrecision of injection was confirmed for six replicate injections of standard solutions containing Qfmmjrat a nominal concentration of 50 ug/ml and 250 ug/ml, for which the coefficients of variation wereless than 1.5%. The accuracy and precision of the analytical procedure was confirmed: a mean procedural recovery . ' value of 97.5% (CV =0.99%, n = 6) was obtained for 5 mg/ml and 94.5% (CV = 2.41%, n = 6) for 400 mg/ml. The limit of detection was determined as 0.375 mg/ml using the operating parameters defined in this procedure. The specificity of the.HPLC assay s/as demonstrated by the absence of a peak at the characteristic retention volume ^'I^^H^^Hr1 trle control sample chromatogram. : 189 : Formulation Chemistry Company Sanitized. Does not contain TSCA CBI CONCLUSION DPT 443/984760 The analytical procedure was validated forjH^^U^Hnn distilled water with respect to the specificity of the chromatographic analysis,Tinearity of detector response, precision of injection, limit of detection, method accuracy and precision. Prior to treatment, the stability during ambienttemperafaire storage for 2 days and refrigerated storage for 15 days was confirmed forB^|^HK)in aqueous formulations,. at nominal concentrations of 5 mg/ml and 400 mg/mI.^The storage period represented the maximum time from preparation to completion of use. The mean concentrations oflHHHB^11 test formulations analysed during the study were within -7% of nominal concentrations confirming the accuracy of formulation. 190 : Formulation Chemistry Company Sanitized. Does not contain TSCA CBI Concentrations o: TABLE 1 ia test fonnnlations DPT 443/984760 Week of dosing 1 Group Control 2 3 4 Nominal inclusion (mg/ml) 0 6 20 60 Analysed concentration (mg/ml) Analysis 1 ND 5.84 19.9 58.3 Analysis 2 ND 5.88 20.0 53.3 Mean ND 5.86 19.9 55.8 NDNone detected (0.375 mg/ml) RME Relative mean error, representing the deviation from nominal Analysed concentrations were calculated using unrounded figures RME (%) - -2.3 -0.5 -7.0 191 : Formulation Chemistry Company Sanitized. Does not contain TSCA CBI Stability o TABLE 2 n aqueous formulations DPT 443/984760 Nominal inclusion (mg/ml) Bottle no. 5 1 Storage conditions Day 0 2 Hour 0 4 - Temp,C +21 +21 +21 2 2 - +4 3 8 - +4 4 15 - +4 Analysed concentration (mg/ml) Analysis 1 Analysis 2 Mean 4.82 4.93 4.89 4.81 4.92 4.81 4.81 4.93 4.85 4.76 4.90 4.73 4.77 4.89 . 4.69 4.76 4.90 4.71 400 1 0 0 +21 370 4 +21 371 2 - +21 394 378 374 382 377 396 395 2 2 - 3 8 - .4 15 - +4 397 +4 385 +4 384 399 398 389 387 385 385 RME Relative mean error, representing the deviation from nominal Mean analysed concentrations were calculated using unrounded figures RME (%) -3.8 -1.4 -3.0 -4.S -2.0 -5.8 -6.5 -5.8 -1.3 -0.5 s "^ --3.3 -3.8 192 : Formulation Chemistry Company Sanitized. Does not contain TSCA CBI TABLES Validation of the analytical procedure for DPT 443/984760 in aqueous formulations 3.1 Linearity determination o: Concentration (Hg/ml) Peak height 49.740 99.480 149-22 198.96 248.70 7647 15383 22150 29006 35472 CD Gradient Intercept n CD Coefficient of determination n Number of determinations 0.9990 139.27 1149.7 5 ndard solutions 3.2 System precision of| Concentration 50 fig/ml Peak height 7395 7608 7587 7432 7428 7414 Mean CV (%) n 7477 1.26 6 CV Coefficient of variation n Number of determinations .ndard solutions 250 tig/ml 40253 40319 39824 40472 39693 39181 39957 1.21 6 193 : Formulation Chemistry Company Sanitized. Does not contain TSCA CBI TABLES (continued) DPT 443/984760 3-3 Accuracy and precision data for Analytical phase Nominal fortification (mg/ml) 5 400 nn aqueous formulations Validation 98.2 98.0 96.8 96.1 97.4 98.7 Mean CV (%) Range n 97.5 0.99 96.1-98.7 6 CV Coefficient of variation. n Number of determinations. 96.6 97.3 95.7 92.7 92.1 92.7 94.5 2.41 92.1-97.3 6 Results are expressed as percent recovery and calculated using the following equation: %., _ Recovery = Analysed concentration (mg /ml) x 1, 0-- 0 -N--o--mi--na--l c--on--ce--ntr--ati:o--n (,nig/, ml,), 194 : Formulation Chemistry Company Sanitized. Does not contain TSCA CBI FIGURE 1 Typical calibration standard graph (Weekl) Concentration (ug/ml) Peak area Regression analysis 51.580 103.16 154.74 206.32 257.90 8711 17240 25184 33703 40991 Regression Slope Intercept n 0.99967 157.08 858.90 5 DPT 443/984760 DPT/443 Week I 50000 T : . ..; -. ^. ^. :..-. : 45000 ;- ;'' ;:.:::.. :-: ;:-'^ r-- -' -- -: --:!^-. ! 40000 ' . '. ''-.'^ :-u ':' :.-'-';-": ---.-;:-. . m:;\- 35000 ''..'. ' i L ff- "' .'.-' ;~ ! ';:,.MiN ^'.-i '.:':. ;S;SA ''-'.;-:- -'"".'I? ^:'. ^ : '^"'l~'~ "--. -: ' ' / ^'^ r"- ' " '' - /^' -'~- ' ^^m,:- .:1|SiiKII 1 30000 '5. - N^il -'^y ^ ,;"- :; E."- i i|S^': ^wfvr cr'-H'^ ;:,i:"'-. rA-p--";-- 1^4.l;-..- --^:.- -: .,-v . ! '^ S^^ .^.:? ^lij, ' S 25000 pLJIJ-/ ! -t -v'^ :.S. y .y.^ft >:.. :. -i-i.. : ":,: a ^ u ^^^-J^~ ' ' : .; '- - '. : ". ; 20000 SkC^^is . ' r~. ~'^ -- - ^.Ai^ ::,:^iJ -ife.'.. 15000, ZWgKS- wa -; ; : wr. ..,,:'.; i2-'S(' ^'. ^.^i ^w^ te-S^J' : : ": '--'-;.- - ' ": ~!'; 'i '' 10000 . ''^W^&l S-fcj:;;..,, n."s-'/"-y -': ||:^"'. -'. -"-'''."-::..; - 5000 y.. -. ^.y?.'-,7. "w' i-': .<;.''-;'- ! ia'.it;..-^-.;,'', ?N^,'1.'^11 K^';'" 0 " '-.,,''' '--n - .. ' ' - 0.0 00 50.000 100.000 150.000 200.000 250.000 300.000 Concentration (ug/ml) 195 : Formulation Chemistry Company Sanitized. Does not contain TSCA CBI FIGURE 2 Typical analytical chromatograms (Week 1) DPT 443/984760 Calibration standard, 250 fig/ml CHANNEL A INJECT 09-12-98 17:51:27 STORED TO BIN '16 Group 1, Control (Dilution factor 1:50) CHANNEL A INJECT 09-12-98 20:32:53 STORED TO BIN B <-- 196 : Formulation Chemistry Company Sanitized. Does not contain TSCA CBI FIGURE 2 (continued) Group 2,6 ing/ml (Dilution factor 1:50) CHANNEL A INJECT 09-12-98 19:47:08 STORED TO BIN C 126 DPT 443/984760 6.44 Group 3,20 mg/ml (Dilution factor 1:100) CHANNEL A INJECT 09-12-98 18:49:11 STORED TO BIN It 121 197 : Formulation Chemistry Company Sanitized. Does not contain TSCA CBI FIGURE 2 (continued) DPT 443/984760 Group 4,60 mg/ml (Dilution factor 1:500) CHANNEL A INJECT 09-12-98 18:14:32 STORED TO BIN it 118 _ 198 : Formulation Chemistry Company Sanitized. Does not contain TSCA CBI Study Number DPT 443/984760 PROTOCOL AND AMENDMENTS : DPT/443 Huntingdon Life Sciences CONFIDENTIAL PROTOCOL TOXICTTY STUDY BY ORAL ADMINISTRATION TO CD RATS FOR 4 WEEKS Sponsor DuPont Specialty Chemicals Jackson Labor-story Chambers Works Deepwater NJ 08023 USA BaeTcb Laboratory Hinitniprinn Lne Sciences Xjtu POBox2 H^ftttihffiJ^li C-BI n PTiflHes"*rff PEltfflSS ENGLAND Total number ofpages: 27 Final Protocol HiallnpSantijeSaeimLld. rrpsemlm ngtolA 1815730 Page I : 199 : Company Sanitized. Does not contain TSCA CBI Study Number :DPT/443 CONTACT DETAILS DPT 443/984760 Huntingdon Life Sciences Sponsor's Monitoring Scientist : Or K. Dastur. Final Protocol Page U : 200 : Company Sanitized. Does not contain TSCA CBI Study Number : DPT/443 DPT 443/984760 Huntingdon Life Sciences PROTOCOL APPROVAL TOXICITY STUDY BY ADMINISTRATION TO CD RATS FOR 4 WEEKS S.M. BoOomley, B.SC. (Hghs), MA;., C.Biol, MJ-Biol. Study Director, Huntingdon Life Sciences Ltd. 'L^y^^f^..'.^ y Date The signature of the Study Director confirms this protocol as the working document for the study. Any changes made subsequent to the date of the Study Director's signature will be documented in formal amendments. J/L, n- A^cy 're RJ. Sortwell Management, Huntingdon Life Sciences Ltd. Date DrK-Dastur. Sponsor, DuPont Specialty Chemicals J^.ll..^.^ Date Please sign both copies of this page, retain one for your records and return one to the Study Director at Huntingdon Life Sciences. Final Protocol Page ill : 201 : Company Sanitized. Does not contain TSCA CBI Study Nuaber : DPT/443 DPT 443/984760 Huntingdon Life Sciences Toxicrry STUDY BY ORAL ADMINISTRATION TO CD RATS FOR 4 WEEKS Enquiry Number: 175560 Number ofpagea for internal distribution: 24 This working document is approved for circulation and use: Primary location of study Huntingdon Research Centre Huntingdon Cambridgeshire Building Number: 1BRB All procedures to be performed at the above site. /' ftfc^eju^t^ /e} ^ Date Final Protocol Pagel : 202 : Company Sanitized. Does not contain TSCA CBI Study Number :DPT/443 CONTENTS 1. INTRODUCTION i 2. STUDY SCHEDULE AND STRUCTURE 2.1. Duration of treatment 12.. Scheduled time plan 23. Identity of treatment groups 3. TEST SUBSTANCE AND FORMULATION 3.1. Test substance 3.2. Formulation 3-3. Quality control of dosage form 4. ANIMAL MANAGEMENT 4.1. Animals - supply, acclimatisation and allocation 4.2. Animals - housing, diet and water supply 4J. Animals - procedures 4.4. Animals - termination 5. FUNCTIONAL OBSERVATIONAL BATTERY 5.1. In the hand and standard arena observations 5.2.. Manipulations 5.3. Motor activity 6. CLINICAL PATHOLOGY 6.1. Haematology, peripheral blood 6.2. Blood Chemistry 7. NECROPSY AND HISTOLOGY 7.1. Method of kill 7.2. Macroscopic Pathology 73. Organ weights 7.4. Fixation 7.5. Histology 8. PATHOLOGY 8.1. Light microscopy 8.2. Extension of initial examination 9. DATA TREATMENT 9.1. Food conversion efficiency 9.2. Statistical analysis 10. REPORTING 11. QUALITY ASSURANCE AND ARCHIVING PROCEDURES 11.1. Quality Assurance 11.2. Archives DPT 443/984760 Huntingdon Life Sciences 4 4 4 4 5 6 6 7 8 8 9 11 14 14 14 15 15 16 16 16 17 17 17 17 17 18 21 21 21 22 22 22 23 23 23 24 Final Protocol Page 2 203 -: Company Sanitized. Does not contain TSCA CBI Study Nuaber : DPT/443 DPT 443/984760 Huntingdon Life Sciences i. INTRODUCTION Miiafemextofftiidy Study Director Monitoring Toadcotogist In the temporaly absence of the Study Director, the scientific responsibilities will be taken over by tfac Monitoring Toxicologist; other nems of routine study ^^ipgg" i?^ should be referred to the following person in the first instnncfi. Objective v : S-M. Bottomley. : WJ4. Hooks. : M.H- Barker. Assessment of systemic toxic potential in a 4 week oral gavage study in CD rats. Regulatory compliance The study will be performed in accordance with the following regulations or guidelines: Organisation for Economic Co-operation and Development, Testing of Chemicals Guideline No. 407 (revised 1995). Good Laboratory Practice The study will be conducted in compliance with principles of Good Laboratory Practice Standards as set form in: The UK Good Laboratory Practice Regulations 1997 (Statutory Instrument No 654). OECD Principles of Good Laboratory Practice (as revised in 1997), ENV/MC/CHEM(98)17. EC Council Directive 87/18/EEC of 18 December 1986 (Official Journal No L 15/29). Animal modef : CD rat, accepted by regulatory-agencies, background data available. Route : Oral gavage, to simulate the conditions of potential human exposure. Treatment groups and dosages Group : 1 Compound : Control Dosage (mg/kg/day)+ : 0 t Expressed in terms of solids. The test substance as supplied i; Final Protocol Page 3 : 204 : Company Sanitized. Does not contain TSCA CBI DPT 443/984760 Study Number : DPT/443 Huntingdon Life Sciences 2. STUDY SCHEDULE AND STRUCTURE 2.1. DuratioB oftreateieat Mitihnnm period : 28day& All surviving animals will be lulled on Day 29. 12. ScBedwIedthnepiaB |^^"^--T Sample of^^^^^^Hknrived Animals to arrive Treatment to commence snce Histopathology to be compicrted Draft report to be issued 23 June 1998 4 December 1998 11 December 1998 8 January 1999 5 March 1999^ March 1999 (estimated) (estimated) 13. Meatfly oftraitmeat groupM (to be selected from 58 animeils ordered) Group TrcxtnieDt 1 Control ft-- 2 ---- 3 4 Dosage (mg/kg/day) t 0 Low Intermediate High Dosage (nig/kg/day)* # 0 Low Intennediate High Number of animals Main tody Male Female 5 5 5 5 5 5 5 5 '^BB^^^HIUHB \ t Expressed intennsofso: ids. The test substance as supplied # Expresssd m tarns of the test substance as supplied. ^--^-^^^^^^^--^j * Dosagps (mg/kg/day) to be selected on the basis of a preliminary study undertaken at Huntiingdon Life Scieflceis(DPT/442). Final Protocol Page 4 : 205 : Company Sanitized. Does not contain TSCA CBI Study Number :DPT/443 Group 1 2 3 4 Cage numbers Male Female 1 5 2 6 3 7 4 8 Health screen DPT 443/984760 Huntingdon Life Sciences Aoiiaal wben Mak Feflmc 15 21-25 6-10 26-30 11-15 31-35 16-20 36-40 41-44 45-48 3. TEST SUBSTANCE AND FORMULATION In order for Huntingdon Life Sciences to comply with the Health and Safety at Work etc. Act 1974, and the Control of Substances Hazardous to Health Regulations 1994, it is a condition of undertaking the study that the Sponsor shall provide Huntingdon Life Sciences with all information available to it regarding known or potential hazards associated with the handling and use of any substance supplied by the Sponsor to Huntingdon Life Sciences. The Sponsor shall also comply with all current legislation and regulations concerning shipment of substances by road, rail, sea or air. Such information in the form of a completed Huntingdon Life Sciences test substance data sheet must be received by Safety Management Services at Huntingdon Life Sciences before the test substance can be handled in the laboratory. At the discretion of Safety Management Services at Huntingdon Life Sciences, other documentation containing the equivalent information may be acceptable. Information received wilt be used to set the Huntingdon Life Sciences Hazard Class, which determines safety precautions taken in me workplace. Huntingdon Life Sciences Hazard Class: 2 Final Protocol Page 5 ^A)(u\fu\ Company Sanitized. Does not contain TSCA CBI Study Namber : DPT/443 DPT 443/984760 Huntingdon Life Sciences 3.1. Text cnbftuce Sponsor's identification Stoisxe conditions Sponsor's responsibilities Certificate of analysis details 3.2. Formulation Treatment Group 1, Control Group 2 Group 3 Group 4 Conversion factor Vehicle Method of preparation Frequency of preparation At room temperature. Documentation of methods of synthesis, fabrication or derivation. Stability data. Certificate of analysis. Test substance identity. Batch number (Lot#3). Purity. Composition. Other appropriate characteristics. Current expiry date: 2 years from manufacture. Vehicle. HHlowmg/mL ^^^^^^ntiemxeuiate mRrmL ^^^luhizh nignnl. The test substance will be used as sui been (MnecteSforthewatercoSteat. Distilled water. Will be documented. Will depend upon die availability of supporting stability data. Where sufficient stability data is available, batches will cover one week of dosing and may be prepared up to three days in advance of the first day of dosing. Where stability data does not support this length of use period, a more frequent mixing regime will be initiated. Final Protocol Page 6 207 Company Sanitized. Does not contain TSCA CBI StadyNuaber :DPT/443 DPT 443/984760 Huntingdon Life Sciences 3.3. Quality coatrol of donge form Liquid formulation Analysis Stability Preparation Sampling and determination Storage conditions Before commencement of treatment, the suitability of the proposed mixmg procedures will be detenxuoed uxd specimen formulations will be analysed to assess the stability of the test substance in the liquid matrix. At specified intervals during treatment, the test formulations will be analysed for achieved concentration of the test substance. The formulated samples will be analysed using a method validated with respect to the determination of the specificity of analysis, limits ofquamitation and/or detection, linearity of detector response, rcproducibility, method accuracy and precision. Specimen formulations (nominally 400 ml) will be prepared at the anticipated highest and lowest concentrations and equally distributed between four screw-capped amber bottles. Before sampling, each formulation will be mixed by inversion. At each time-point duplicate samples will be taken for assay from the middle of me formulation. Ambient temperature (nominally 21C) for 0,4 and 48 hours (Bottle 1). Refrigeration (nominally 4C) for 2, 8 and 15 days (Bottles 2,3 and 4). Achieved concentration Sampling and determination : Dayl. Other sampling regimens may be specified by the Sponsor. One sample (nominally 20 ml) from all groups; 2 assays from each sample. Final Protocol Page? : 208 : Company Sanitized. Does not contain TSCA CBI Study Number : DPT/443 DPT 443/984760 Huntingdon Life Sciences 4. ANIMAL MANAGEMENT 4.1. AnuMla- MpjJy, fM^Hff..--imf ml allne^w.^ 4.1.1. Aaimab Species Strain Age ordered Weight range ordered Supplier Rat. Cri:CDBR28 2 days. 11 g/sex (minimum 75 g). Charles River (UK) Limited. 4.U. Health screen An additional four males (animal numbers 41-44) and four females (animal numbers 45-48) will be ordered from the supplier. These will be killed, bled and subjected to macroscopic examination immediately upon receipt. Serum samples will be retained frozen pending possible future scrology investigations (samples discarded after two months). Lungs, liver, kidneys, spleen and heart will be preserved in fixative, but not processed further unless macroscopicalty abnormal. Macroscopic abnormalities will be immediately processed and examined microscopically. Results of die health screen will be reviewed before commencement of treatment. 4.U. Acclimatisation Duration : Husbandry conditions : 4.1.4. Allocation to treatment groups 1 to 2 weeks (animals will be approximately 5 weeks of age at commencement). Refer to Section 4.2. Allocation Method : Approximately I week before commencement oftrcatuieuL : Random allocation to cages to equalise variation in bodyweighL Final Protocol Page 8 209 : Company Sanitized. Does not contain TSCA CBI DPT 443/984760 Study Number :DPT/443 Huntingdon Life Sciences Cage distribution Cages bousing minutis from the same group will be arranged in blocks within cage-racks. The position of the racks within the room will be exchanged at intervals during the study. 4.1.5. Identification Numbering Method Cage labels . : Unique for each mimal within study. : Cage number by foot tattoo. Animal number by earmark. : Uniquely identifying die occupants. 4.1.6. PrecoBuneaceuent animal replacement 10 spare animals will be ordered to replace any individuals rejected during the acclimatisation period. Replacement before treatment : Replacement during treatment : Ill-health. Abnormalities. Bodyweight range extremes. On Day 1 (before dosing) variations in bodyweight of animals should not exceed 20% of the mean for each sex. None scheduled. 4.2. Animals - homing, diet and water (apply 4.2.1. Environmental control Rodent facility : Limited access - to minimise entry of external biological and chemical agents. Air supply : Filtered, not recirculated- Temperature ; Target range 19-23-C. Relative humidity : Target range 40-70%. Monitored continuously. Excursions outside these ranges documented in the study data. Lighting : 12 hours light: 12 hours dark. Final Protocol Page 9 210 : Company Sanitized. Does not contain TSCA CBI Study Number :DPT/443 DPT 443/984760 Huntingdon Life Sciences Alarm systems Electricity supply : Activated on ventilation failure and when temperature/humidity limbs exceeded : Public supply with automatic stand-by generators. 4.Z2. Animal accommodation Animals per cage Cage material Cage flooring Five of same sex, unless reduced by mortality or isolation. Stainless steel. Stainless steel grid. 4.23. The cages will be suspended above absorbent paper. The latter will be changed at appropriate intervals each week; cages, cage-trays, food hoppers and water bottles will be changed at appropriate intervals. Precise details of caging will be included in the final report. Diet and water lopply Copies of all certificates of analysis are stored in the archives. Diet supply Diet name Diet type Availability Certification : Rat and Mouse No. 1 Maintenance Diet : Pelleted diet : Non-restricted. : Before delivery each batch of diet is analysed by the supplier for various nutritional components and chemical and microbiological contaminants. Supplier's analytical certificates are scrutinised and approved before any batch of diet is released for use. This diet contains no added antibiotic or other chemotherapeutic or prophylactic agent. Water supply Supply Regulatory agency Availability Public drinking water. UJC. Department of the Environment Non-restricted via polyethylene or polycarbonate bottles with sipper tubes. Certification Certificates of analysis are routinely received from th- supplier. Final Protocol Page 10 211-: Company Sanitized. Does not contain TSCA CBI Study Nunber :DPT/443 DPT 443/984760 Huntingdon Life Sciences 4.1.4. CoBtuninaBti uny It is the Sponsor's responsibility to advise Huntingdon Life Sciences of any specific contaminants likely to prejudice the outcome of die study. Analyses for such contaminants may be performed if requested by the Sponsor. 43. AniBub . procedure* 4.3.1. Investigations noted as occurring on specified Day numbers (where quoted) will not be varied. Administration Route Treated at Volume dosage Individual dose volume Controls (Group 1) Frequency Sequence Formulation Oral gavage. Constant dosages in nig/kg/day. 10 ml/kg/day. Calculated from the most recently recorded scheduled bodyweight. Vehicle at me same volume dosage as treated groups. Once daily at approximately the same time each day. By group. A daily record of the usage of formulation will be maintained based on weights. This balance is compared with the expected usage as a check of correct administration. Suspensions are stirred using a magnetic stincr before and throughout the dosing procedure. 4.3.2. Clinical observations Animals and their cages Deviations from normal recorded at the time in respect of Physical examination Inspected at least twice daily for evidence of reaction to treatment or ill-health. Nature and severity. Date and time of onset Duration and progress of the observed condition. Once each week for all animals. Final Protocol Page 11 212 Company Sanitized. Does not contain TSCA CBI DPT 443/984760 Study Number : DPT/443 Huntingdon Life Sciences In addition detailed observations will be made in association with dosing according to the following schedule and frequency: Minimum schedule : 1. Week I-daily. 2. Weeks2to4 -twicewcckly (middle and end of week). Frequency : 1. Pre-dosc observation. 2. As each animal is returned to its home cage. 3. At the end of dosing each group. 4. Between 1 and 2 hours after completion of dosing all groups. 5. As late as possible in the working day. The above schedule will be amended, as necessary, in the light of signs observed. During the acclimatisation period, observations of the animals and their cages will be recorded at least once per day. 4.3.3. Mortality Debilitated animals : Observed carefully, may be isolated to prevent cannibalism. Premature sacrifice : Animals may be killed on humane grounds or if considered iff extremis. Animals found dead, killed m ; extremis or on humane grounds 43.4. Bodyweight A necropsy is performed as soon as possible. Animals found outside the normal workday will be preserved in a refrigerator (approximately 4C) provided far lias purpose. Bodyweight recording : Day that treatment commences. Weekly (last scheduled bodyweight recorded on Day 28) Before necropsy. More frequent weighings may be performed to aid the monitoring of the condition of animals displaying ill-health. These data will be retained in the archives. 4.3.5. Food consumption Food consumption recording : Food supplied : Weekly. At intervals each week. Final Protocol Page 12 : 213-: uompany Sanitized. Does not contain TSCA CBI DPT 443/984760 Study Number :DPT/443 Huntingdon Life Sciences Food spilled Food remaining 4.3.6. Water comnimptiOD Estimated at cage cleaning. Recorded at end of study week- Fluid intake will be assessed by daily visual observation. undertaken if treatment-reiated changes are suspected. Precise measurements may be 43.7. FunetioBal obiervational battery Animals will be subject to procedures as specified in Section 5. performed in the following weeks: Investigations will be Examination In the hand Standard arena Manipulations Automated Motor Activity Week Pretreatment, 1,2,3,4 Pretreatment, 4 Animals All animals All animals 4.3.8. Bioaampling Investigations will be performed as follows: Blood samples- Haematology/Blood Chemistry Day 29 Anip^ali All animals. Conditions Sample site Anti coagulant/ Sample volume Analysis Following overnight deprivation of food (after the 28th dose). Samples collected under light general anaesthesia. Retro-orbital sinus. EDTA/0.5 ml (Haematology). Citrate/0.5 ml (Coagulation). Lithium heparin/1.0 ml (Blood chemistry). Sections 6.1 and 6.2. Final Protocol Page 13 : 214 : Company Sanitized. Does not contain TSCA C81 DPT 443/984760 Stady Number : DPT/443 Huntingdon Life Sciences 4.4. Axiaia]* - teniUBatioB All animals will be subject to terminal investigations (Section 7). The sequence in which the animals are killed after completion of treatment period will allow satisfactory inter-group comparison. 5. FUNCTIONAL OBSERVATIONAL BATTERY Each animal will be subject to the procedures detailed below on the specified occasions. The functional observational battery will be performed at the same time of day on each occasion and the observer will be unaware of the experimental group to which the animal belongs. Animals will not necessarily all be tested on the same day but the number of animals will be balanced across the groups on each day of testing. Any deviations from normal will be recorded with respect to nature and, where appropriate, degree of severity. Further details on test procedures and definitions will be documented in the report. 5.1. In the h--d and fndud arena observations Observations will be performed in the hand and men during a 1 minute recording period in a standard arena as follows: Week Pretreannent, 1,2,3,4 Ailimak All animals. After removal from the home cage me following parameters will be assessed: In the hand Exophthalmos Fur appearance Lacrimarion Piloerection Reactivity to handling Ease of removal from cage Salivation ' Vocalisation on handling Standard arena Activity counts Arousal Convulsion Defecation count Gait Grooming Palpefaralclosure Posture Rearing count Tremor Twitches Urination Final Protocol Page 14 : 215 : Company Sanitized. Does not contain TSCA CBI DPT 443/984760 Study Number :DPT/443 Huntingdon Life Sciences 5.2. Miuiipalatiou Week Pretreatment, 4 J^iyptpl* All animals ' The following measurements, reflexes and responses will be recorded: Approach response Auditory startle reflex Body temperature Bodywcight Grip stienglli - foielimbs and hindlimbs Landing footsplay Tail pinch response Pupil reflex Righting reflex Touch response 53. Motor activity Week Pretreatment, 4 Animals All aninrals Motor activity will be measured by automated infra-red sensor equipment, recording individual animal activity over a one hour period. final Protocol Page 15 : 216 : Company Sanitized. Does not contain TSCA CBI Stody Number :DPT/443 DPT 443/984760 Huntingdon Life Sciences 6. CLINICAL PATHOLOGY 6.1. Haematology, peripheral blood \ Blood sample analysis will be pcrfonned on the following occasions): Day 29 Aoiimb All animals. All samples will be examined for the following characteristics: 1) Using EDTA as anticoagutant - Packed cell volume Haemoglobin concentration Erythrocytc count Total leucocyte count Differential leucocyte count Abnormalities of the blood film Platelet count Mean cell haemoglobin Mean cell volume Mean cell haemoglobin concentration 2) Using citrate as anticoagulant - Prothrombin time Activated partial thromboplastin time 6.2. Blood Chemistry Blood sample analysis will be performed on samples obtained from the same animals and at the same time as for haematology. All.samples will be examined for the following characteristics: Using lithium heparin as anticoagulant - Alkaline phosphatase Alanine amino-transferase (GFT)' Aspartate amino-transferase (GOT) Gamma glutamyi transpeptidase Glucose Bilirubin - total Cholesterol - total Final Protocol Page 16 : 217 : Company Sanitized. Does not contain TSCA CBI Study Namber : DPT/443 Triglyccrides Crratimne Urea nitrogen Total protein Albumin by chemical assay Albumin/globulin ratio . Sodium pronftf-l.sirsTiiuiimi Chloride Calcium Phosphorus DPT 443/984760 Huntingdon Life Sciences 7. NECROPSY AND HISTOLOGY 7.1. Method of kBI Method Carbon dioxide. 7.2. Macroscopic Pathology (Table 1) Complete Checks 73. Organ weights (Table 1) Data collection Data presentation 7.4. Fixation (Table 1) Standard Others All animal-: Retained tissues. For bilateral organs, left and right organs will be weighed together unless otherwise specified on the Pathology Procedures Table. Organ weights are not routinely recorded for animals killed or dying prematurely. Absolute. Adjusted for terminal bodyweight 10% Neutral Buffered Formalin. Testes fnd epididymides: Initially in Bourn's fluid. Fin^l Protocol Page 17 : 218 : Company Sanitized. Does not contain TSCA CBI Study Number : DPT/443 7.5. Hutolocy (Table 1 and Section 8.1) Processing - Full List Processing-Abnormalities only Routine staining Special staining DPT 443/984760 r Huntingdon Life Sciences All animals killed or dying prematurely. All terminal animals ofGroups 1 and 4. All terminal animals ofGroups 2 and 3. 4-5 fua sections stained with haematoxylin and eosin except testes which are stained using a standard PAS method. None. Final Protocol Page 18 : 219 : Company Sanitized. Does not contain TSCA CBI DPT 443/984760 Stndy Number :DPT/443 Huntingdon Life Sciences TABLE 1-Pathotoiy procedures niuie Abaonnalides Adrenals Brain Cfeeconi Colon Duodenum Feznur with joint Head Heart Ucxnn (including Foyer's patch) Jejunum Kidneys Liver Lungs (including bronchi) ^yxnpb nodes - xnandibular - tMfvntfnc Oesophagus Ovaries Pancreas ^restate IcctuBi Sciatic nerves igmmal vesicles kpuial cord Spleen Sternum Stomach "estcs Thymus Thyroid with parathyroids Trachea Urinary bladder Items with cervix Vagina Weigh * '0 * * b ) a ) 4 4 * * Light wwcopy * * iir 4 A jU * ff Including nasal cavily, paranasal sinuses and nasopharynx. Both hindlimbs retained, one sectioned where appropriate. Organs weighed, samples fixed or sections examined microscopically. Examined if effects suspertrd during the study. Only one examined. Final Protocol Page 19 : 220 : Company Sanitized. Does not contain TSCA CBI DPT 443/984760 StBdy Number :DPT/443 Huntingdon Life Sciences The tissues subjectedto histologica] processing will include the following regions: Tiwne Adrenals Brain Femur with joint Heart Ileum Kidneys Liver Lungs Spinal cord Repou to beexamiacd cortex and medulla. cerebellum, cerebrum and midbrain. longitudinal section including the bone marrow. including auricular and ventricular regions. including Peyer's patch where possible. including cortex, medulla and papilla regions. section from all main lobes. section from two major lobes, to include bronchi. transverse and longitudinal sections at the cervical level. Stomach Thyroid Uterus : keratiniscd, glandular and antrom. : includes parathyroid in section, where possible. : uterus section separate from cervix section. For bilateral organs sections of both Sie left and right organs will be examined, unless otherwise specified on the Pathology Procedures Table. A single section will be prepared from each of the remaining tissues required for microscopic pathology. Final Protocol Page 20 : 221 : Company Sanitized. Does not contain TSCA CBI Stady Nomber :DPT/443 DPT 443/984760 Huntingdon Life Sciences 8. PATHOLOGY 8.1. U(bt microMopy Category Premature deaths Terminal sacrifice Terminal sacrifice Anipinl. All from all groups. All an""^s of Groups 1 and 4. All animals of Groups 2 and 3. Tunes All specified in Table I. All specified in Table 1. Abnonnalitics only. Peer Review : Carried out by a reviewing pathologist to Internationally accepted standards. &2. Extension of initial cumulation At the discretion of the pathologist, further processing and staining techniques may be used to evaluate individual lesions. Details of these techniques will be documented and retained in die archives. Light microscopy may be extended, following consultation with the Sponsor, as follows: from all animals of Groups 2 and 3 killed at terminal sacrifice for tissues considered to exhibit a reaction to treatment in Group 4. Any such requirement will be documented in an amendment to the protocol. Tissues displaying treatment-related change may be farther examined using additional processing or staining techniques. Final Protocol Page 21 : 222 : Company Sanitized. Does not contain TSCA CBI DPT 443/984760 Study Number :DPT/443 Huntingdon Life Sciences 9. DATA TREATMENT 9.1. Food cmnwnion efficiency The group mean food conversion efficiency will be calculated, where appropriate, from food conversion ratios using weight of food consumed per unit gain in bodyweight 93,. Stattetk*! nalyHs Date-types The following data types will be analysed at each timepoint separately:- bodyweight, using gains over appropriate study periods. food consumption, over appropriate study periods, using cage totals. functional observational battery. blood chemistry, haematology and urinalysis. organ weights, both absolute and adjusted for tenninal bodyweight, where appropriate. pathological findings, for the number of animals with and without each finding. Methods For categorical data, the proportion of animals will be analysed using Fisher's Exact test for each treated group versus the control. For continuous data, Bartlett's test will first be applied to test the homogeneity of variance between die groups. Using tests dependent on the outcome of Bartlett's test, treated groups will then be compared with the control group, incorporating adjustment for multiple comparisons where necessary. Final Protocol Page 22 : 223 : Company Sanitized. Does not contain TSCA CBI DPT 443/984760 Study Number :DPT/443 Huntingdon Life Sciences 10. REPORTING Study progress Draft final report Authorised final xepoit Periodic verbal and written updates on study progress will be provided by the Study Director. A synopsis report will be sent at tennination of the in-life phase. For review by the Sponsor. After approval from the Sponsor. Routinely reports are supplied on A4 paper. The following numbers of reports are supplied. Type of report Draft report Authorised final Printine Double-sided Double-sided Single-sided Number of copid Bound UDbonnd 0 2 1 0 0 1 Any additions or corrections to an authorised final report will be documented as a formal addendum/amendment to the final report. 11. QUALITY ASSURANCE AND ARCHIVING PROCEDURES 11.1. Quality Assurance Protocol check Procedure inspections Study audit Report review (Final report) Report ofQA findings Authorised protocol and any amendments. Critical phases of this study (study based) and routine procedures on representative studies (process based). The GLP aspects of the management and conduct of mis study. Following issue of the draft report to the Sponsor. To Study Director and management promptly on completion of each QA action. Final Protocol Page 23 : 224 : Company Sanitized. Does not contain TSCA CBI Study Number :DPT/443 DPT 443/984760 Huntingdon Life Sciences 11.2. Archives All experimental data arising from the study (including documentary raw data, specimens, records, other materials; collectively defined as the "materials") will remain the property of the Sponsor. Huntingdon Life Sciences shall retain the materials in its archive for a period of 5 years from me date of issue of me final report After such time, the Sponsor will be contacted and their advice sought on the return, disposal or further retention of the materials. If requested, Huntingdon Life Sciences will continue to retain the materials subject to a reasonable fee being agreed with the Sponsor. Final Protocol Page 24 : 225 : Company Sanitized. Does not contain TSCA CBI Study Number Protocol Ameodmeat Number :DPT/443 : I (one) DPT 443/984760 Huntingdon Life Sciences TOXIdTY STUDY BY ORAL ADMINISTRATION TO CD RATS FOR 4 WEEKS Total Bomber of page*: 4 Number of page* for biteraaldistribBtioB: 4 Study Director : S M Bottanley, B.Sc. (Hons), M.SC., C.Biol, MJLBiol. The signature of the Study Director authorises die implementation of this amendment to protocol. In this amendment, deleted statements are struck through and new statements are underlined. Any changes to the study design after the date of mis authorising signature will be documented in a further forma] amendment AMENDMENT APPROVAL For Hnntiagdol Authorised by^f y7 JJ//^^L Date: 3. UeCt^^lWf^ (Study Director)^7^7y For die Sponsor Approved by: k^AxJZL Date: f^te. 7 <???' Pagel 226 : Company Sanitized. Does not contain TSCA CBI Study Number Protocol AmeBdBKBt Nmnber : DPT/443 : 1 (ooe) DPT 443/984760 Huntingdon Life Sciences TOXICnY STUDY BY ORAL ADMINISTRATION TO CD RATS FOR 4 WEEKS ReasoBS for ameadaeajs : 1. Correction of the animal arrival date. 2. Addition of dosage levels. 3. Conecdon to timmg of allocation of animals to treatment groups. 4. Addition of Week -1 bodyweight. Amendments Treatment group* and dosages Group : Compound : Dosage (mg/kg/day)t : 1 Control 0 15fcw go Inttnncdinte .ISOffigt* t Expressed in terms of solids. The test substance as supplied is 25% solids in 75% water. 13. Sckedofed time plan Sample of Zonyl FS-62 arrived Animals to arrive Treatment to commence Terminal sacrifice to commence Histopathology to be completed Draft report * > be issued : 23 June 1998 : 2-4-December 1998 ' : 11 December 1998 : 8 January 1999 : 5 March 1999 : March 1999 (estimated) (estimated) Page 2 227 : Company Sanitized. Does not contain TSCA CBI Study Number Protocol Aloendmeat Nunber : DPT/443 : 1 (one) DPT 443/984760 Huntingdon Life Sciences cnmy 01 ucauuem poflpB (to be selected from 58 animals ordered) Group 1 Treatment Control Dosage (mg/kg/day) t 0 JDosage (mgAg/day)* # 0 Number of animals Main tody Male Female 5 5 2 ISfcew 60 few 5 5 3 50 Inttnnodinto ^~^n^nHi--jnR^B^nWi--n--ii--i 5 5 4 600 High 5 5 --------. ------.----------1------------------ t Expressed in terms of solids. The test substance as supplied id # Expressed in terms of the test substance as supplied. * Dosages (nig/kg/day) te-be selected on the basis of a preliminary study undertaken at Huntingdon Life Sciences (DP7/442). 3J Formulation Treatment Group 1, Conirol Group 2 Group 3 Group 4 Conversion factor Vehicle Method of preparation Frequency of preparation Vehicle. U|G tow mg/ml. aintennodmgi/mal. to ^m&'ml. The test suhstence^il^ensedasjumrii substance is|^^^H^^^^^^^^^^B|i concentration faig/ml^eflSshavefe the water content- Distilled water. Will be documented. Will depend upon the availability of supporting stability data. Where sufficient stability data is available, batches will cover one week of dosing and may be prepared up to three days in advance of the first day of dosing. Where stability data does not support this length of use period, a more frequent mixing regime wil! be initiated. Page 3 228 : Company Sanitized. Does not contain TSCA CBI Study Number Protocol AmeadmeBt Namber : DPT/443 : 1 (one) DPT 443/984760 Huntingdon Life Sciences 4.1.4 Allocation to trextmeat gnrnps Allocation : On arrival. >ly 1 w--k brfo Method Cage distribution Random allocation to cages to equalise variation in bodywcight Cages bousing animals from toe same group will be arranged in blocks within cage-racks. The position of the recks within the room will be exchanged at intervals during the study. 4.3.4 Bodyweignt Bodyweight recording : One week prior to .commencement, Day that treatment commences. Weekly Oast scheduled bodyweight recorded on Day 28) Before necropsy. More frequent weighings may be performed to aid the monitoring of the condition of animals displaying ill-health. These data will be retained in the archives. Page 4 229 : Company Sanitized. Does not contain TSCA CBI Study Number Protocol Amendment Number : DPT/443 : 2 (two) DPT 443/984760 Huntingdon Life Sciences TOXIOTY STUDY BY ORAL ADMINISTRATION TO CD RATS FOR 4 WEEKS Total number of pages: 4 Number of pages for internal distribution: 4 Study Director : S M Bottomley, B.Sc. (Hons), M.Sc., C-Biol., MJ.Biol- The signature of the Study Director authorises the implementation of this amendment to protocol- In this amendment, deleted statements are struck through and new statements are underlined. Any changes to the study design after the date of this authorising signature will be documented in a further formal amendment AMENDMENT APPROVAL For Hunt; Director)^J/ I^TZ^L Authorised t^C J^ (Study D^:^^CUC^l W? For the Sponsor Approved by: WiS^^. Date: \\f^i t0, <??7 P?,e ! : 230 : Company Sanitized. Does not contain TSCA CBI Study Number Protocol Amendment Number : DFT7443 : 2 (two) DPT 443/984760 Huntingdon Life Sciences r3 TOXIdTY STUDY BY ORAL ADMINISTRATION TO CD RATS FOR 4 WEEKS Reasons for amendments : 1. Change of Study Director due to maternity leave. 2. Addition of the examination of the kidneys, liver and bone marrow from all animals from the low and intermediate dosage group in order to further evaluate me effects seen in the high dose level animals. Addition of information to the scheduled time plan because of the additional work. Amendments 1 INTRODUCTION Management of study Study Directoi<original): S M Bottomley Study Director (replacement): H A Palmer This amendment formally registers the assignment of a replacement Study Director. The signature of the replacement Study Director approves the implementation of this amendment to protocol. Any changes to the study design after the date of this approval signature will be documented in a further formal amendment. Page 2 231 Company Sanitized. Does not contain TSCA CBI DPT 443/984760 Study Number Protocol Amendment Number : DPT/443 : 2 (two) Huntingdon Life Sciences Reason for amendment: Declaration Original Replacement Study Director Signature: .....UAU^fc^. For Huntingdon Life Sciences 'l^ffmil^ Approved by: ^ (Replacement Study Director) Released by:_ The original Study Director (S M Bottomley) is talcing maternity leave I am satisfied with the conduct of the study to date. ^MC^C^I^ I am satisfied with the conduct of the study to date. From the date of my signature on mis amendment, I assume responsibilities of me Study Director. Date^.^W^...!0^. Date: -? fW^rL ffi^ Dale: 2- Utjet- (W 1. STUDY SCHEDULE AND STRUCTURE 23, Scheduled time plan Sample < . Animals to arrive Treatment to commence Terminal sacrifice to commence Histopathology to be completed Additional histonamologv to be completed Draft report to be issued 23 June 1998 2 December 1998 11 December 1998 8 January 1999 5 March 1999 16ApriH999 Maeb 30 April 1999 (estimated) (estimated) Page 3 232 : Company Sanitized. Does not contain TSCA CBI Study Number Protocol Amendment Number : DPT/443 : 2 (two) DPT 443/984760 Huntingdon Life Sciences 8. PATHOLOGY 8.1 Light microscopy S3. Category Animals Premature deaths All from all groups. Terminal sacrifice Terminal sacrifice All animals of Groups 1 and 4. All animals of Groups 2 and 3. Terminal sacrifice All animals ofGrouos 2 and 3. Tissues All specified in Table 1. All specified in Table 1. Abnormalities only Kidnevs. liver and bone marrow Peer review Canted out by a reviewing pathologist to Internationally accepted standards. Pag' 4 233 Company Sanitized. Does not contain TSCA CBI Study Number Protocol Amendment Number :DPT/443 : 3 DPT 443/984760 Huntingdon Life Sciences Toxrcrry STUDY BY ORAL ADMINISTRATION TO CD RATS FOR 4 WEEKS Total number of pagea: 2 Nmnber of pages for internal distribution: 2 Study Director : Helen A Palmer The signature of the Study Director authorises the implementation of this amendment to protocol. In (his amendment, deleted statements are struck through and new statements are underlined. Any changes to the study design after the date of this authorising signature will be documented in a further formal amendment. AMENDMENT APPROVAL For Huntingdon Life Sciences Ltd Authorised by. 4fM^n^________ (Study Director) Date: :L-2- TuJMi- t^0! For the Sponsor Approved by: \^S^L_ Date: %~- 30, I'??'? Pagcl : 234 : Company Sanitized. Does not contain TSCA CBI Study Number Protocol Amendment Number :DPT/443 :3 DPT 443/984760 IHIuUnI ItUinI iQyd-IVO^InI Life Sciences TOXICTTY STUDY BY ORAL ADMINISTRATION TO CD RATS FOR 4 WEEKS Reasons for amendments : Addition of blood chemistry parameter at the sponsor's request Amendments 6.2 Blood Chemistry Blood sample analysis will be performed on samples obtained from the same animals and at the same time as for hacmatology- All samples will be examined for the following characteristics: Using lithium heparin as anticoagulant - Alkaline phosphatase Alaninc amino-tiansferase (OPT) Aspartate amino-transferase (GOT) Gamma gluamyi transpcptidasc Glucose Bilirubin - total Cholesterol-total Triglycerides Creatininc Urea nitrogen Total protein Albiimin by chemical assay Globulin - bv subtraction Albumin/globulin rntio Sodium Potassium Chloride Calcium Phosphorus Page 2 235 : Company Sanitized. Does not contain TSCA CBI DPT 442/992305 SEVEN DAY ORAL TOXICITY STUDY IN THE RAT REPORT NUMBER : DPT 442/992305 : 236 : Company Sanitized. Does not contain TSCA CBI DPT 442/992305 SEVEN DAY ORAL TOXICTTY STUDY IN THE RAT EXPERIMENTAL PROCEDURE Groups of six rat (three males and three females) were treated with^U^^B^kt dosages of 250, 500 or 750 mg/kg/day (Groups 2 to 4 respectively) formulated as 25, 50 and 75 mg/ml solutions in distilled water at a dosage volume of 10 ml/kg/day. All dosages are reported as solids, not as material supplied. The formulations were prepared on a daily basis and if necessary, the formulations were warmed to 35 - 40C prior to use to ensure that a true solution was obtained. A control group (Group 1) of three males and three females received the vehicle alone at the same dose volume. Group 1 Cage label/ colour code White Treatment Control, distilled water Dosages ing/kg/day - No. of rats M F 3 3 Rat numbers M F 1-313-15 During the study, clinical signs were recorded prior to dosing and at regular intervals following dosing on a daily basis. Bodyweights were recorded prior to dosing, on the day of commencement of treatment (Day 1), on Day 4 and 8. Food consumption was recorded on a weekly basis during the treatment period. At post mortem organ weights and the macroscopic appearance of the tissues were noted. Macroscopic abnormalities were retained for possible future examination. All procedures undertaken were as described in the accompanying report for the 4 week study (DPT 443/984760). The protocol approval page was signed by the Study Director and Huntingdon Management on 9 November 1998 and by the Sponsor on 30 November 1998. Treatment commenced on 11 November 1998 and all surviving animals were sacrificed on 17 November 1998 following the completion of seven days of treatment. 237 Company Sanitized. Does not contain TSCA CBI DPT 442/992305 SEVEN DAY ORAL TOXICITY STUDY IN THE RAT RESULTS MORTALITY (Appendix 3) All of the animals receiving 500 or 750 mg/kg/day were sacrificed or found dead in the period Days 3 - 6 of the study. There were no unscheduled deaths amongst the animals receiving 250 mg/kg/day or the controls. CLINICAL SIGNS (Appendix 3) Salivation, immediately after dosing was noted in all treated animals. BODYWEIGHT (Table 1, Appendix 1) A group mean bodyweight loss over the first 3 days of treatment was seen for all treated groups of animals, with a dosage-relationship apparent. A lower group mean bodyweight gain over the 7-day treatment period, was observed for animals receiving 250 mg/kg/day, when compared with the controls. FOOD CONSUMPTION (Table 2) Lower food consumption values were noted for all treated groups of animals, when compared with the controls. ORGAN WEIGHTS (Table 3, Appendix 2) Higher group mean kidney weights and lower absolute spleen weights were noted for animals receiving 250 mg/kg/day, when compared with the controls. A higher group mean bodyweightadjusted liver weight was apparent for males and females receiving 250 mg/kg/day. 238 : Company Sanitized. Does not contain TSCA CBI DPT 442/992305 SEVEN DAY ORAL TOXICITY STUDY IN TBE RAT MACROSCOPIC PATHOLOGY (Table 4, Appendix 3) The macroscopic examinations performed revealed the following changes: Kidneys - enlargement was observed in 1/3 decedent male and 3/3 decedent female rats treated with 750 mg/kg/day, 2/3 decedent male and 3/3 decedent female rats treated with 500 mg/kg/day and 3/3 terminal male and 3/3 terminal female rats treated with 250 mg/kg/day compared with 0/3 terminal male and 0/3 terminal female control rats. Pallor was noted in 1/3 decedent male and 3/3 decedent female rats treated with 750 mg/kg/day, 3/3 decedent male and 3/3 decedent female rats treated with 500 mg/kg/day and 1/3 terminal male and 2/3 terminal female rats treated with 250 mg/kg/day compared with 0/3 terminal male and 0/3 terminal female control rats. Pale areas were seen in 1/3 terminal male rats treated with 250 mg/kg/day compared with 0/3 terminal male control rats. 239 Company Sanitized. Does not contain TSCA CBI DPT 442/992305 SEVEN DAY ORAL TOXICITY STUDY IN THE RAT CONCLUSION As a no-effect level was not determined in this study and since the purpose of the 4 week study is to determine the labelling criteria for the test substance, the high dosage level for the subsequent 4 week study (Huntingdon Life Sciences schedule number DPT/443) was set at 150 nig/kg/day (a labelling point under the OECD guidelines). The next dosage level in relation to the labelling criteria is 15 nig/kg/day and therefore, this would be suitable for the low dosage level. The intermediate dosage level was set at 50 mg/kg/day. 240 Company Sanitized. Does not contain TSCA CBI [' r3 SEVEN DAY ORAL TOXICITY STUDY IN THE RAT DPT 442/992305 TABLE 1 Bodyweights - group mean values (g) Group and dosage (nig/kg/day) Day 1M 2M 3M 4M IF 2F 3F 4F Control 500 750 1000 Control 500 750 1000 -7 76 77 77 77 75 76 75 76 1 131 132 139 138 130 123 125 124 4 . 155 124 112 100 148 122 123 105 8 190 142 167 129 Gain (g/rat) 1-4 1-8 24 -8 -27 -38 59 10 - 18 -1 -2 -19 37 6 - - ^ 241 Company Sanitized. Does not contain TSCA CBI DPT 442/992305 SEVEN DAY ORAL TOXICTTY STUDY IN THE RAT TABLE 2 Food consumption - group mean values (g/rat/day) Group and dosage (mg/kg/day) Day 1M 2M Control 500 3M 4M 750 1000 IF 2F Control 500 3F 4F 750 1000 1 25 15 6 6 22 13 11 8 242 : Company Sanitized. Does not contain TSCA CBI DPT 442/9923 05 SEVEN DAY ORAL TOXICTTY STUDY IN THE RAT TABLE 3 Organ weights - group mean values Terminal kill Group/ dosage mg/kg/day Body wt <3 Liver Spleen Kidneys 9 <3 <3 Unadjusted means 1M Control 187 10.2 0.54 1.83 2M 500 140 10.3 0.38 2.67 Adjusted means 1M - 8.2 2M - 12.3 Group/ dosage mg/kg/day Body wt g Unadjusted means IF Control 162 2F 500 128 Adjusted means IF 2F Liver Spleen Kidneys g g g 10.7 0.49 1.83 8.5 0.38 2.94 8.8 10.3 Company Sanitized. Does not contain TSCA CBI DPT 442/992305 SEVEN DAY ORAL TOXICITY STUDY IN THE RAT TABLE 4 Macroscopic pathology incidence summary - Terminal Removal reason: Terminal Animals on study Animals completed Incisors Lower pale Kidneys Pale Pale subcapsular Enlarged area/s Group 1 Group 2 ---- Males ---- ' 3 3 3 3 Group 1 Group 2 -- Females -- 3 3 3 3 0 0 1 1 0 1 0 2 0 1 0 0 0 3 0 3 244 : Company Sanitized. Does not contain TSCA CBI ^ SEVEN DAY ORAL TOXICITY STUDY IN THE RAT DPT 442/992305 TABLE 4 Macroscopic pathology incidence summary - Decedents Removal reason: Intercurrent Animals on study Animals completed Fur Stained Stained Stained Moist - - perinasal region - periorbital region/s - perioral region genital region Skin Alopecia Thymus Congested Adipose Tissue Minimal Spleen Small Stomach Contents dark Contents watery Forestomach Thickened Roughened Stomach Corpus Mucosa Haemprrhagic depression/s Group 3 Group 4 ---- Males ---- 3 3 3 3 .. Group 3 Group 4 -- Females -- 3 3 3 3 0 0 0 1 0 1 0 0 0 2 0 1 0 0 0 2 2 0 1 2 . 0 0 1 0 1 0 2 0 2 2 0 2 0 1 0 0 0 2 0 0 0 0 0 1 0 n 0 1 . 1 2 0 0 : 245 : Company Sanitized. Does not contain TSCA CBI DPT 442/992305 SEVEN DAY ORAL TOXICITY STUDY IN THE RAT TABLE 4 (Macroscopic pathology incidence summary - Decedents - continued) Removal reason: Intercurrent Animals on study Animals completed Stomach Antjrum Mucosa Haemorrhagic depression/s Small Intestine Contents dark Contents minimal Caecum Dark Adrenals Congested Kidneys Pale Enlarged Group 3 Group . Group 4 3 Group 4 ---- Males ---- 3 3 3 3 -- Females -- 3 3 3 3 0 1 0 0 0 1 0 0 0 1 0 0 1 0 0 1 0 0 0 1 3 1 3 3 2 1 3 3 246 : Company Sanitized. Does not contain TSCA CBI DPT 442/9923 05 SEVEN DAY ORAL TOXICITY STUDY IN THE RAT APPENDIX 1 Bodyweights - individual values (g) Group 1M: Control Cage Animal 1 1 2 3 Day - 7 1 4 8 73 128 155 194 77 138 161 196 79 128 148 178 Group 2M: 500 ing/kg/day Cage Animal 2 4 5 6 Day - 7 1 4 8 80 129 131 151 76 138 127 146 74 130 115 129 Group 3M: 750 mg/kg/day Cage Animal 3 7 S 9 Day -7148 77 136 113 80 147 125 74 133 98 Group: 4M .1000 mg/kg/day Cage Animal 4 10 11 12 Day -7148 80 139 75 132 100 76 143 Group IF: Control Cage Animal 5 13 14 15 Day - 7 1 4 8 77 128 142 160 71 134 157 180 78 126 144 162 Group: 2F 500 mg/kg/day Cage Animal 6 16 17 18 Day - 7 1 4 8 72 118 119 131 80 129 131 139 75 121 115 117 Group 3F: 750 mg/kg/day Cage Animal 7 19 20 21 Day -7148 73 126 127 73 119 113 78 131 128 Group 4F: 1000 mg/kg/day Cage number Animal number 8 22 23 24 Day -71 82 132 73 127 72 112 4 8 106 108 103 : 247 : Company Sanitized. Does not contain TSCA CBI DPT 442/992305 SEVEN DAY ORAL TOXICITY STUDY IN THE RAT APPENDIX 2 Organ weights - individual values Terminal kill Group/ dosage lag/kg/day 1M Control Animal no. 1 2 3 2M 500 Mean sd 4 5 6 Mean sd Body wt g Liver Spleen Kidneys g g g 191 196 174 187 11.4 148 144 127 140 10.9 11.0 10.6 9.2 0.51 0.50 0.59 2.03 1.81 1.66 10.2 0.54 1.83 0.98 0.049 0.185 10.6 11.3 9.0 0.38 0.42 0.33 2.50 2.49 3.03 10.3 0.38 2.67 1.15 0.047 0.306 Group/ dosage mg/kg/day Animal no. IF 13 Control 14 15 Mean sd 2F 16 500 17 18 Mean sd sd Standard deviation Body wt g Liver Spleen Kidneys g g g 156 172 157 162 9.0 128 139 118 128' 10.2 9.3 12.7 10.1 0.49 0.53 0.44 1.80 2.06 1.63 10.7 0.49 1.83 1.76 0.043 0.217 8.9 0.40 8.7 0.39 7.8 0.34 2.74 2.49 3.60 8.5 0.38 2.94 0.60 0.030 0.581 248 Company Sanitized. Does not contain TSCA CBI DPT 442/992305 SEVEN DAY ORAL TOXICITY STUDY IN THE RAT APPENDIX 3 Individual clinical and pathological findings In this appendix the clinical and macroscopic findings relating to each animal are listed. : 249 : Company Sanitized. Does not contain TSCA CBI DPT 442/992305 SEVEN DAY ORAL TOXICITY STUDY IN THE RAT APPENDIX 3 (Pathology - continued) Compound: Dosage Level: Rat No/Sex: CLINICAL FINDINGS No signs of ill health or behavioural change were noted. MACROSCOPIC FINDINGS No abnormalities detected : 250 : Company Sanitized. Does not contain TSCA CBI DPT 442/9923 05 SEVEN DAY ORAL TOXICITY STUDY IN THE RAT APPENDIX 3 (Pathology - continued) Compound: Dosage Level: Rat-No/Sex: CLINICAL FINDINGS No signs of ill health or behavioural change were noted. MACROSCOPIC FINDINGS No abnormalities detected : 251 : Company Sanitized. Does not contain TSCA CBI DPT 442/992305 SEVEN DAY ORAL TOXICTTY STUDY IN THE RAT APPENDIX 3 (Pathology - continued) Compound: Dosage Level: Rat No/Sex: Control 3M (Terminal) CLINICAL FINDINGS No signs of ill health or behavioural change were noted. MACROSCOPIC FINDINGS No abnormalities detected : 252 : Company Sanitized. Does not contain TSCA CBI DPT 442/992305 SEVEN DAY ORALTOXICITY STUDY IN THE RAT APPENDIX 3 (Pathology - continued) Dosage Level: Rat-No/Sex: 500 mg/kg/day 4M (Terminal) CLINICAL FINDINGS 2-7 Salivation immediately after dosing on Days only were noted. and walking on toes 1 hour after dosing on Day 5 MACROSCOPIC FINDINGS Kidneys Pale subcapsular area/s: (A few) up to 2mm Enlarged: 2.500g All the other organs and tissues appeared normal. : 253 : Company Sanitized. Does not contain TSCA C81 DPT 442/992305 SEVEN DAY ORAL TOXICITY STUDY IN THE RAT APPENDIX 3 (Pathology - continued) Dosage Level: Rat-No/Sex: ; 500 mg/kg/day 5M (Terminal) CLINICAL FINDINGS Salivation immediately after dosing on Days 1,2, 6 and 7 was noted. MACROSCOPIC FINDINGS Kidneys Enlarged: 2.493g All the other organs and tissues appeared normal. : 254 : Company Sanitized. Does not contain TSCA CBI DPT 442/992305 SEVEN DAY ORAL TOXICITY STUDY IN THE RAT APPENDIX 3 (Pathology - continued) Compound: Dosage Level: RatNe/Sex: 500 nig/kg/day 6M (Term inal) CLINICAL FINDINGS Salivation immediately after dosing on Days 6 and 7 and paddling of forelimbs immediately after dosing on Day 6 were noted. MACROSCOPIC FINDINGS Kidneys Pale: (Minimal) Enlarged: 3.026g All the other organs and tissues appeared normal. : 255 : Company Sanitized. Does not contain TSCA CBI DPT 442/992305 SEVEN DAY ORAL TOXICITY STUDY IN THE RAT APPENDIX 3 (Pathology - continued) Compound: Dosage Level: RarNo'/Sex: 750 mg/kg/day 7M (Intercurrent) CLINICAL FINDINGS Day of death 5 Salivation immediately after dosing on Days 2-4 was noted. Incidental finding of hair loss was noted. Found dead. MACROSCOPIC FINDINGS Found dead Skin Alopecia Dorsum: (Patchy) Spleen Small: (Minimal) Kidneys Pale: (Minimal) Enlarged: 2.062g All the other organs and tissues appeared normal. : 256 : Company Sanitized. Does not contain TSCA CBI DPT 442/992305 SEVEN DAY ORAL TOXICTTY STUDY IN THE RAT APPENDIX 3 (Pathology - continued) Dosage Level: Rat No/Sex: 750 mg/kg/day 8M (Intercun-ent) CLINICAL FINDINGS Day of death 6 Salivation immediately after dosing on Days 3 and 4 was noted. Piloerection, walking on toes, partially closed eyelids, unsteady gait and hunched posture were noted approximately 1 and 4 hours after dosing on Day 5 only. Poor condition characterised by piloerection from Day 5 and hunched posture from Day 6 was noted. Incidental finding of patchy hair loss was noted. Sacrificed due to poor condition. MACROSCOPIC FINDINGS Adipose Tissue Minimal Kidneys Pale Enlarged: 3.447g All the other organs and tissues appeared normal. : 257 : Company Sanitized. Does not contain TSCA CBI DPT 442/992305 SEVEN DAY ORAL TOXICITY STUDY IN THE RAT APPENDIX 3 (Pathology - continued) Dosage Level: Rat-No/Sex: 750 mg/kg/day 9M-(Intercurrent) CLINICAL FINDINGS Day of death 5 Salivation immediately after dosing on Days 2 and 3 was noted. Incidental finding of hair loss was noted. Found dead. . MACROSCOPIC FINDINGS Found dead Skin Alopecia Left lumbar region Spleen Small Stomach Corpus Mucosa Haemorrhagic depression/s: (A few) 1mm Caecum Dark: (Minimal) Kidneys Pale All the other organs and tissues appeared normal. : 258 : Company Sanitized. Does not contain TSCA CBI DPT 442/992305 SEVEN DAY ORAL TOXICITY STUDY IN THE RAT APPENDIX 3 (Pathology - continued) Dosage Level: Rat No/Sex: 1000 ing/kg/day 10M(Intercurrent) CLINICAL FINDINGS Day of death 3 Salivation immediately after dosing on Day 2 was noted. Poor condition characterised by brown staining around muzzle, irregular breathing, piloerection and state of collapse was noted on Day 3. Sacrificed due to poor condition. MACROSCOPIC FINDINGS Fur Stained - perioral region: (Minimal, Brown) Stomach Contents dark: (Minimal) Stomach Corpus Mucosa Haemorrhagic depression/s: (A few, Punctate) Small Intestine Contents dark Kidneys Pale Enlarged: 2.250g All the other organs and tissues appeared normal. 259 : Company Sanitized. Does not contain TSCA CBI DPT 442/992305 SEVEN DAY ORAL TOXICITY STUDY IN THE RAT APPENDIX 3 (Pathology - continued) Compound: Dosage Level: Rat No/Sex: 1000 mg/kg/day 11M (Intel-current) CLINICAL FINDINGS Day of death 4 Salivation immediately after dosing on Days 2 and 3 was noted. Moribund condition characterised by partially closed eyelids, sunken eyes, red and cold extremities, lethargy, hunched posture, irregular and laboured respiration, piloerection, salivation and brown staining around jaw was noted on Day 4. Sacrificed due to moribund condition. MACROSCOPIC FINDINGS Found dead Fur Stained - periorbital region/s: (Minimal, Red) Spleen Small Stomach Contents watery Stomach Antrum Mucosa Haemorrhagic depression/s: (One) 1mm All the other organs and tissues appeared normal. 260 : Company Sanitized. Does not contain TSCA CBI UK 1 442/992305 SEVEN DAY ORAL TOXICITY STUDY IN THE RAT APPENDIX 3 (Pathology - continued) Dosage Level: Rat No/Sex: 1000 nig/kg/day 12M (Intercurrent) CLINICAL FINDINGS Day of death 4 Salivation immediately after dosing on Days 2 and 3 was noted. Found dead. MACROSCOPIC FINDINGS Found dead Fur Stained - perioral region: (Minimal, Red) Spleen Small Stomach Contents watery Stomach Corpus Mucosa Haemorrhagic depression/s: (A few) 1mm Small Intestine Contents minimal All the other organs and tissues appeared normal. 261 : Company Sanitized. Does not contain TSCA CBI DPT 442/992305 SEVEN DAY ORAL TOXICITY STUDY IN THE RAT APPENDIX 3 (Pathology - continued) Compound: Dosage Level: Rat No/Sex: Control 13F (Terminal) CLINICAL FINDINGS No signs of ill health or behavioural change were noted. MACROSCOPIC FINDINGS Incisors Lower pale All the other organs and tissues appeared normal. 262 : Company Sanitized. Does not contain TSCA CBI ur i 44^/yy2jUS SEVEN DAY ORAL TOXICITY STUDY IN THE RAT APPENDIX 3 (Pathology - continued) Dosage Level: Rat No/Sex: CLINICAL FINDINGS No signs of ill health or behavioural change were noted. MACROSCOPIC FINDINGS No abnormalities detected 263 : Company Sanitized. Does not contain TSCA CBI DPT 442/992305 SEVEN DAY ORAL TOXICITY STUDY IN THE RAT APPENDIX 3 (Pathology - continued) Compound: Dosage Level: Rat No/Sex: Control 15F (Terminal) CLINICAL FINDINGS No signs of ill health or behavioural change were noted. MACROSCOPIC FINDINGS No abnormalities detected 264 : Company Sanitized. Does not contain TSCA CBI DPT 442/992305 SEVEN DAY ORAL TOXICITY STUDY IN THE RAT APPENDIX 3 (Pathology - continued) Compound: Dosage Level: Rat No/Sex: 500 nig/kg/day 16F (Terminal) CLERICALFINDINGS Salivation immediately after dosing on Days 2, 3 and 5-7, unsteady gait approximately 1 hour after dosing on Day 5 only and paddling offorelimbs immediately after dosing on Day 6 only were noted. Incidental finding of patchy hair loss was noted. MACROSCOPIC FINDINGS Kidneys Pale: (Minimal) Enlarged: 2.737g All the other organs and tissues appeared normal. 265 Company Sanitized. Does not contain TSCA CBI DPT 442/992305 SEVEN DAY ORAL TOXICITY STUDY IN THE RAT APPENDIX 3 (Pathology - continued) Compound: Dosage Level: Rat No/Sex: 500 mg/kg/day 17F (Terminal) CLERICAL FINDINGS Salivation immediately after dosing on Day 6 only was noted. Incidental finding of hair loss was noted. MACROSCOPIC FINDINGS Incisors Lower pale Kidneys Enlarged: 2.489g All the other organs and tissues appeared normal. 266 : Company Sanitized. Does not contain TSCA CBI DPT 442/992305 SEVEN DAY ORAL TOXICITY STUDY IN THE RAT APPENDIX 3 (Pathology-continued) Compound: Dosage Level: Rat No/Sex: CLINICAL FINDINGS Salivation immediately after dosing on Days 2,3 and 6 was noted. Piloerection was noted from Day 6. Incidental finding of hair loss was noted. MACROSCOPIC FINDINGS Kidneys Pale: (Minimal) Enlarged: 3.597g All the other organs and tissues appeared normal. 267 : Company Sanitized. Does not contain TSCA CBI DPT 442/992305 SEVEN DAY ORAL TOXICITY STUDY IN THE RAT APPENDIX 3 (Pathology - continued) Dosage Level: Rat No/Sex: 750 mg/kg/day 19F(Intercun-ent) CLINICAL FINDINGS Day of death 6 Salivation immediately after dosing on Days 2,3 and 5 and walking on toes and paddling offorelimbs approximately 1 hour after dosing on Day 5 only were noted. Poor condition characterised by piloerection from Day 5 and brown nasal staining from Day 6 was noted. Incidental finding of patchy hair loss was noted. Sacrificed due to poor condition. MACROSCOPIC FINDINGS Adipose Tissue Minimal Kidneys Pale Enlarged: 3.007g All the other organs and tissues appeared normal. 268 : Company Sanitized. Does not contain TSCA CBI DPT 442/992305 SEVEN DAY ORAL TOXICITY STUDY W THE RAT APPENDIX 3 (Pathology - continued) Dosage Level: Rat No/Sex: 750 ing/kg/day 20F (Intel-current) CLINICAL FINDINGS Day of death 6 Salivation immediately after dosing on Days 1 and 2 was noted. Walking on toes and paddling of forelimbs immediately after dosing and hunched posture, unsteady gait, walking on toes, piloerection and laboured respiration approximately 1 and 4 hours after dosing were noted on Day 5 only. Piloerection was noted from Day 5. Incidental finding of hair loss was noted. Found dead (partially cannibalised). MACROSCOPIC FINDINGS Found dead and partially cannibalised; left side of head Skin Alopecia Dorsum: (Patchy) Thymus Congested Kidneys Pale Enlarged: 2.026g All the other organs and tissues appeared normal. 269 : Company Sanitized. Does not contain TSCA CBI DPT 442/992305 SEVEN DAY ORAL TOXICTTY STUDY IN THE RAT APPENDIX 3 (Pathology - continued) Compound: Dosage Level: Rat No/Sex: 750 mg/kg/day 2 IF (Intel-current) CLINICAL FINDINGS Day of death 6 Salivation immediately after dosing on Days 2 and 3 was noted. Walking on toes immediately after dosing, walking on toes and unsteady gait approximately 1 hour after dosing and walking on toes, unsteady gait and piloerection approximately 4 hours rJfter dosing were noted on Day 5 only. Poor condition characterised by piloerection and hunched posture from Day 5 and walking on toes, eyelids partially closed, slight brown nasal staining and matted fur in the urogenital region from Day 6 was noted. Sacrificed due to poor condition. MACROSCOPIC FINDINGS Adipose Tissue Minimal Kidneys Pale Enlarged: 3335g All the other organs and tissues appeared normal. 270 : Company Sanitized. Does not contain TSCA CBI DPT 442/992305 SEVEN DAY ORAL TOXICITY STUDY IN THE RAT APPENDIX 3 (Pathology - continued) Compound: Dosage Level: Rat No/Sex: 1000 ing/kg/day 22F (Intercurrent) CLINICAL FINDINGS Day of death 5 2-4 Salivation immediately after dosing on Days was noted. Partially closed eyelids, walking on toes and unsteady gait were noted immediately after dosing and approximately 1 and 4 hours after dosing with paddling of forelimbs noted immediately after dosing and piloerection approximately 1 and 4 hours after dosing on Day 4 only. Piloerection, wet urogenital region, cold extremities, hunched posture, lethargy, prominent vertebrae, brown nasal staining and emaciation were noted from Day 4. Found dead. MACROSCOPIC FINDINGS Found dead Fur Stained - perinasal region: (Brown) Moist - genital region Spleen Small: (Minimal) Forestomach Thickened Caecum Dark: (Minimal) Kidneys Pale Enlarged: 1.769g All the other organs and tissues appeared normal. 271 Company Sanitized. Does not contain TSCA CBI DPT 442/992305 SEVEN DAY ORAL TOXICITY STUDY IN THE RAT APPENDIX 3 (Pathology - continued) Dosage Level: Rat No/Sex: 1000 mg/kg/day 23F (Intel-current) CLINICAL FINDINGS Day of death 5 Salivation immediately after dosing on Days 2 - 4 and approximately 1 hour after dosing on Day 4 was noted. Walking on toes and unsteady gait were noted immediately after dosing and approximately 1 and 4 hours after dosing with paddling of forelimbs and partially closed eyelids noted immediately after dosing and piloerection approximately 1 and 4 hours after dosing on Day 4 only. Poor condition characterised by brown staining on cranium, cold extremities, piloerection, hunched posture, emaciation, prominent vertebrae from Day 4 and lethargy and wet urogenital region from Day 5 was noted. Incidental finding of hair loss was noted. Sacrificed due to poor condition. MACROSCOPIC FINDINGS Fur Stained - perioral region: (Brown) Moist - genital region Skin Alopecia Dorsum: (Minimal, Diffuse) Periorbital regions: (Minimal) . Spleen Small Forestomacb Roughened Adrenals Congested 272 Company Sanitized. Does not contain TSCA CBI DPT 442/992305 SEVEN DAY ORAL TOXICITY STUDY IN THE RAT APPENDIX 3 (Pathology - continued) Rat No/Sex: 23F - continued MACROSCOPIC FINDINGS - continued Kidneys Pale: (Minimal) Enlargedf (Minimal) 1.98 Ig All the other organs and tissues appeared normal. 273 : Company Sanitized. Does not contain TSCA CBI DPT 442/992305 SEVEN DAY ORAL TOXICITY STUDY IN THE RAT APPENDIX 3 (Pathology - continued) Compound: Dosage Level: Rat No/Sex: lOOOmg/kg/day 24F (Intel-current) CLINICAL FINDINGS Salivation immediately after dosing on Days 2-4 was noted. Walking on toes and unsteady gait were noted immediately after dosing and approximately 1 and 4 hours after dosing with paddling of forelimbs noted immediately after dosing and piloerection approximately 1 and 4 hours after dosing on Day 4 only. Poor condition characterised by piloerection from Day 4 and emaciation, hunched posture and brown nasal staining from Day 5 was noted. Incidental finding of hair loss was noted. Sacrificed due to poor condition. MACROSCOPIC FINDINGS Skin Alopecia Dorsal cervical region: (Diffuse) Kidneys Pale: (Minimal) Enlarged: 2.444g All the other organs and tissues appeared normal. 274 Company Sanitized. Does not contain TSCA CBI DPT 442/992305 SEVEN DAY ORAL TOXICITY STUDY IN THE RAT s^yN,-^ ^442 CONFIDENTIAL PROTOCOL Huntingdon Life Sciences PROTOCOL PRELIMINARY TOXK1TY STUDY BY ORAL ADMINISTRATION TO CD KATS FOR 7 DAYS Sponsor DuPont Specialty Chemicals Jackson Laboratory Chambers Works Deepwatcr NJ 08023 USA T'-.al number of pages: 16 Research Laboratory Huntingdon Life Sciences Ltd POBox2 HUntIORuOll Cambridgeshire PE186ES ENGLAND Filial Protocol Pagel Huntingdon lift Saasa ltd. reftiKrfrf fti &gtorf 1915730 275 : Company Sanitized. Does not contain TSCA CBI DPT 442/992305 SEVEN DAY ORAL TOXICITY STUDY IN THE RAT Study Number : DPT/442 Huntingdon Life Sciences CONTACT DETAILS Sponsor's Monitoring Scientist : DrK-Dastur. FiesI Protocol Page n : 276 : Company Sanitized. Does not contain TSCA CBI DPT 442/992305 SEVEN DAY ORAL TOXICITY STUDY IN THE RAT stBdyNMber DFTO42 Huntingdon Life Sciences PROTOCOL APPROVAL PRELIMINARY TOXICITY STUDY BY ORAL ADMINISTRATION TO CD RATS FOR 7 DAYS ^M.Sc,CBiol,MJ^ioL Study Director, Huntingdon Life Sciences Ltd. ^Oe^cA^/^.r Date The signature of the Study Director confirms this protocol as the working document for die study. Any changes made subsequent to the date of the Study Director's signature will be documented in formal amendments. RJ. Sortwell Management, Huntingdon Life Sciences Ltd. Date Dr K. Dastur. Sponsor, DuPont Specialty Chemicals ..^..^...Lt17- Date Please sign both copies oflhapage. Wain one for yaw record's anil return one to ihs Study Director at Huntingdon Lift Sciences. Finti Protocol Page I'M 277 : Company Sanitized. Does not contain TSCA CBI DPT 442/992305 SEVEN DAY ORAL TOXICITY STUDY IN THE RAT Study Number :DPT/442 Huntingdon Life Sciences PRELIMINARY TOXICTrY STUDY BY ORAL ADMINISTRATION TO CD RATS FOR 7 DAYS Enquny Number: 17556N Number of pages for intenul distribution: 13 This waking document is approved for circulation and use: Primuy locatioo ofitody Huntingdon Research Cfime Hundngdon Cambridgeshire Building Number. 1BRB All procedures to be performed at the above she. ^^e^&e^^y Date Fhul Protocol Pagcl : 278 : Company Sanitized. Does not contain TSCA CBI DPT 442/992305 SEVEN DAY ORAL TOXICITY STUDY IN THE RAT SttdyNmnber :DPT/442 CONTENTS 1. INTRODUCTION 2. STUDY SCHEDULE AND STRUCTURE 2.I. DurebOD oftreatment 2^. Scheduled time pim Z3* Identity oitxotoent firoup& 3. TEST SUBSTANCE AND FORMULATION 3.1. Test substance 3.2. Fonnulatioo 3.3. Quality control of dosage fonn 4. ANIMAL MANAGEMENT 4.1. Aaimals - supply, gcclimatiattinn end allocation 4.2. Animals - bousing, diet and water supply 4 J. Animals-procedures 4.4. Aninuls tenBination 5. NECROPSY AND HISTOLOGY 5.1. Method of kill 5.2- Macroscopic Pathology 53. Organ weights 5.4. Fixation 6. REPORTING 7. QUALITY ASSURANCE AND ARCHIVING PROCEDURES 7.1. Quality Assurance 7.2. Archives Huntingdon Life Sciences 7 g 9 11 12 12 12 12 12 13 13 13 13 Final Protocol Page 2 : 279 : Company Sanitized. Does not contain TSCA CBI DPT 442/992305 SEVEN DAY ORAL TOXICITY STUDY IN THE RAT Study Nuaber : DPT/442 Huntingdon Life Sciences i. INTRODUCTION Maiu(emeM of (tidy StudyDuector Monitoring Toxicologist In the temporary absence of the Study Director, the scientific responsibilities will be telcen over by the Mooitoriog Toxicologist^ other items of routine study management should be referred to toe following person in the first instance. Objective : SM Bottomley. : WX Hooks. : M.H. Barker. Assessment of systemic toxic potential in a 7 day oral gavage study in CO rats, to select a suitable high dosage for a subsequent 4 week study. Good Laboratory Practice The study will be conducted in compliance with principles of Good Laboratory Practice Standards as set forth in: The UK Good Laboratory Practice Regulations 1997 (Statutory Instrument No 654). OECD Principles of Good Laboratory Practice (as revised in 1997), ENV/MC/CHEM(98)17. EC Council Directive 87/18/EEC of 18 December 1986 (Official Journal No L 15/29). No specific study-related Quality Assurance procedures or analysis of dose form will be performed. Animal model CD rat, accepted by regulatory agencies, background data available. Route Oral gavage, to simulate the conditions of potential human exposure. Final Protocol Page3 : 280 : Company Sanitized. Does not contain TSCA CBI DPT 442/992305 SEVEN DAY ORAL TOXICITY STUDY IN THE RAT Sfdy Number : DPT/442 Huntingdon Life Sciences TreatmeBt group* ud dosage* Group : 1 Compound ' : Control Dosage (mg/kg/day)t : 0 250 500 750 t Expressed in terms of solids. The test substance is supplied is 25% solids in 75% water. 2. STUDY SCHEDULE AND STRUCTURE 2.1. Duration of treatment Minimum period: 7 days of treatment. All surviving animals will be killed on Day 8. 23. Scheduled time plan Sample of ZonylFS-<2 arrived Animals to airive Treatment to commence Terminal sacrifice to commence : 23 June 1998 : 4Novcmberl998 : 11 November 1998 : 17 November 1998 13. Identity of treatment groups (to be selected from 38 animals ordered) Dosage (me/lte/dayjt Dosage* (ag/lcg/day)# Number ofanimab Male Female 250 1000 500 2000 750 3000 Expressed in terms of the test substance'as supplied. S^aj^^a terms of solids. The test substance as supplied i Dosages (mg^cg/day) selected with reference to existing lexicological data. Final Protocol Page4 281 Company Sanitized. Does not contain TSCA CBI DPT 442/9923 05 SEVEN DAY ORAL TOXICITY STUDY IN THE RAT Stedy Number : DPT/442 Huntingdon Life Sciences Group -1 2 Cagenoben Male Female 1 5 2 6 3 3 7 4 4 8 Health screen Male 1-3 4-6 7-9 10-12 25-28 Female 13-15 16-18 19.21 22-24 29-32 3. TEST SUBSTANCE AND FORMULATION In order for Huntingdon Life Sciences to comply with the Health and Safety at Work etc. Act 1974, -and the Control of Substances Hazardous to Health Regulations 1994, it is a condition ofundertalong the stutiy mat the Sponsor shall provide Huntingdon Life Sciences with all information available to it regarding known or potential hazards associated with the handling and use of any substance supplied by the Sponsor to Huntingdon Life Sciences. The Sponsor shall also comply wftfa all current legislation and regulations concenuag shipment of substances by road, nil, sea or air. Such information in the form of a completedHuntingdon Life Sciences test substance data sheet must be received by Safety Management Services at Huntingdon Life Sciences before the test substance can be handled in the laboratory. At the discretion of Safety Management Services at Huntingdon Life Sciences, other documentation containing the equivalent infonnttion may be acceptable. Information received will be used to set the Huntingdon Life Sciences Hazard Class, which determines safety precautions taken in the workplace. Huntingdon Life Sciences Hazard Class: 2 Final Protocol Page5 : 282 : Company Sanitized. Does not contain TSCA CBI DPT 442/992305 SEVEN DAY ORAL TOXICITY STUDY IN THE RAT Study Nmnber : DPT/442 Huntingdon Life Sciences 3J. Test nbtuce Sponsor's identification Storage conditions Sponsor's responsibilities Certificate of analysis details 3.2. FormnlatioB iVCSSSSCOt Group 1, Vehicle control Group 2 Group3 Group4 Conversion factor At room temperature. Documentation of methods of synthesis, fabrication or nfTyygtirtt|, ^ Stability dn*. Certificate of analysis. Test substBxice identity. Batch number: (Lot 93). Purity. Composition. Other appropriate characteristics. Current expiry date: (2 Years from manufacture). Vehicle. lied. The test have Vehicle Method of preparation Frequency of preparation : Distilled water. : Will be documented. : Daily. 33. Quality control of douse form Liouid fonnulation ^ Before commencement of treatment, the suitability of the proposed mixing procedures will be determined by visual assessment. Final Protocol Pagefi : 283 : Company Sanitized. Does not contain TSCA CBI 9 DPT 442/992305 SEVEN DAY ORAL TOXICITY STUDY IN THE RAT Stady Nunber : DPT/442 Huntingdon Life Sciences 4. ANIMAL MANAGEMENT 4.1. Aoinub - npply, edhnliutioa a&d allocation 4JJ. ABiaab Spcdes Strain Age ordered Weight range ordered Supplier : RsL : Cri:CDBR. : 2g2days. : To be within 11 grange for each sex (minimum 75 g). : Charles River (UK) Limited. 4.1.2. BeallbiciceB An additional four mAles (njipyi ouaxbers 25-2X) and four feinales (animal annxbcrs 29-32) will be ordered 6001 the supplier. These will be killed, bled and subjected to macroscopic exmination immediately upoo receipt. Senmi samples will be retained frozen pending possible future serology investigations (samples discarded after two months). Lungs, liver, kidneys, spleen and heart will be preserved in fixative, but not processed further unless inacroscopically abnormal. Macroscopic abnormalities will be immediately processed and exaniiBcdniKroscopicaJly. Results of the health screen will be reviewed before commencement of treatment. 4.13. AcclunataatioB Duration : Husbandry conditions : 4.1.4. Allocation to treatment groaps At least 5 days (animals will be approximately 5 weeks of age at commencement). Refer to Section 4.2. Allocation Method : Approximately I week before commencement oftreatoient. : Random allocation to cages to equalise variation in bodywcight. Final Protocol Page? : 284 : Company Sanitized. Does not contain TSCA CBI DPT 442/992305 SEVEN DAY ORAL TOXICITY STUDY IN THE RAT Stedy Number :DPT/442 Huntingdon Life Sciences 4.LS. IdeatifietioB Numbering Method Cage labels Unique for each animal within study. Cage number by foot tanoo, animal number by eannaAUniqucly identifying the occuprats. 4.1A Pr 6 spare iinim^l; will be ordered to replace any individuals rejected during (he acclimatisation period. Replacement before treatment : Ill-health. Abnormalities. Bodyweight range isj^i cities. On Day 1 (before dosing) variations in bodywcight of inimils should not exceed 20% of thtmetn for each sex. Replacement during treatment : None scheduled. 4.2. Animals-houif, diet aad water rpply 4.2.1. Environmental control Rodent facility ' : Limited access - to minimise entry of external biological and chemical agents. Air supply Filtered, not recirculated. Temperature Target range 19-23C. Relative humidity Target range 40-70%. Monitored continuously. Excursions outside these ranges documented in the study data. Lighting Alarm systems : 12 hours light: 12 hours dark. Activated on ventilation failure and when temperature/humidity limits exceeded. Electricity supply Public supply with automatic stand-by generators. Final Protocol Page 8 : 285 : Company Sanitized. Does not contain TSCA CBI DPT 442/992305 SEVEN DAY ORAL TOXICTTY STUDY IN THE RAT Stady Number : DPT/442 Huntingdon Life Sciences ^ ^ y ABuul ceoflUBodxtioB Animals per cage : Three of same sex, unless reduced by mortality or Cagematerial Gageflooring : Stainless steeL : Stainless steel grid. 4.2J. The cages will be suspended above absorbent paper. The latter will be changed at appropriate intervals eacfa week; cages, cage-trays, food hoppers and water bottles will be changed at appropriate intervals. Precise details of caging will be inclnded in the final report. Diet and water (apply Copies of all certificates of analysis are stored in the archives. Diet (apply Dietname Diet type Availability Certification : Rat and Mouse No. 1 Maintenance Diet- : Pelleted diet. : Non-restricted. : Before delivery each batch of diet is analysed by the supplier ibr-various nutritional components 9W^ chemical and microbiological contaminants. Supplier's analytical certificates are scrutinised and approved before any batch of diet is released for use. This diet contains no added antibiotic or other chemotherapeutic or prophylactic agent. Water supply Supply Regulatory agency Availability : Public drinking water. : UX- Department of die Environment. : Non-reacted via polyethylene or polycarbonate bottles with sipper tubes. Certification : Certificxtec of analysis are routinely received from the supplier. 4.2-4. 43. Contaminants assay It is the Sponsor's responsibility to advise Huntingdon contaminants likely to prejudice die outcome of the study. may be performed if requested by the Sponsor. Life Sciences of any specific Analyses for such contaminants Animals - procedures Investigations noted as occurring on specified Day numbers (ftjere quoted) will not be varied. Final Protocol Page9 286 Company Sanitized. Does not contain TSCA CBI DPT 442/992305 SEVEN DAY ORAL TOXICITY STUDY IN THE RAT Sfdy Number : DPT/442 Huntingdon Life Sciences 4.3.1. AdiBiBictratxoB Route Treated at Volume dosage Individual dose volume Controls (Group 1) Frequency Sequence Formulation Oralgavage. Constant dosagei in mg/kg/day. 10 ml/kg. r.lr.iirt^ frmtl tfc. rr.n<-t r>f^ntly r>fnt>W ^-h>A.lp^ bodyweight. Vehicle at toe same volume-dosage as treated groups. Once daily at approximately the same time each day. By group. A daily record ofthe usage of formulation will be maintained based on weights. This balance is compared with the expectedusage as a check of conect 4.3.2- Clinical obwrvtMHU Suspensionsare stirred using a magnetic stiner before and throughout me dosing procedure. Animals and their cages Inspected at least twice daily for evidence of reaction to treatment or ill-health. Deviations from normal recorded at the time in respect of Physical examination Nature and severity. Date and time of onset. Duration and progress of the observed condition. Once duringtreatment week and on Day 8 prior to despatch to necropsy. In addition detailed observations will be made daily in association with dosing according to the following frequency: Frequency ; 1. Pre-dnse observation. 2. As each animal is returned to its home cage. 3. At the end of dosing each group. 4. Between 1 and 2 hours after completion of dosing all groups. 5. As late as possiblein the working day. The above schedule will be amended, as necessary, in the light of signs observed, During the acclimatisation period, observations of the animals and their cages will be recorded at least once per day. Final Protocol Page 10 287 Company Sanitized. Does not contain TSCA CBI DPT 442/992305 SEVEN DAY ORAL TOXICITY STUDY IN THE RAT Study Nuxber : DPT/442 Huntingdon Life Sciences 4.3.3. Mortality Debilitated animals Prematuie sacrifice : Observed carefully, may be isolated to prevent : Animals mxy be killed on biniMae grounds or if Animals found dead, killed in ; ewwnir or on humane grounds 43.4. Bodywe^kt A necropsy is perfonned as soon as possible. Animals found outside the normal woricday will be preserved in a reftigeraw (approximately 4C) provided for this pufpo&e. Bodyweight recording : Once immediately before treatment, once during the treatment week and on Day 8. More frequent weighing may be performed to aid me monitoring of the condition of animals displaying ill-health. These data will be retained in the archives. 4.3.5. Food eolDmptioo Food consumption recording : Food supplied : Food spilled : Food remaining : 43.6. Water consumption Week 1. At intervals. Estimated at cage paper changing. Recorded at end of study week. Fluid intake will be as'a^sif) by daily visual observation. 4.4* Aninfisis terBOBsstioD All animals will be subject to tennmal investigations (Section S). Final Protocol Page 11 : 288 : Company Sanitized. Does not contain TSCA CBI DPT 442/992305 SEVEN DAY ORAL TOXICTTY STUDY IN THE RAT SfdyNamber :DPT/442 Huntingdon Life Sciences 5. NECROPSY AND HISTOLOGY 5.1. Method of kin Method &2. Macroscopic Patliolocy Cirbon dioxide. Necropsy Checks Special requirements S3. Or|U wrighta Data presentation 5.4. fixation Standard AH animals, tlioncicnd abdominal cavities opened, cmualcjvity opened only if observations indicate possibleoeurotoxxc ftction* Retaised tissues. Macroscopic aboonnalitits retained *nd fined. Liver, kidneys and spleen. Organ weights Be not routinely recorded for animab killed or dying prematnrely. Absolute. Adjusted for tenninal bodywcight 10% Neutral Buffered Formalin. Final Protocol Page 12 : 289 : Company Sanitized. Does not contain TSCA CBI DPT 442/992305 SEVEN DAY ORAL TOXICTTY STUDY IN THE RAT Stedy Nunber : DPT/442 Huntingdon Life Sciences 6. REPORTING Study pi ogress Reporting A summaiy of findings will be sent to the Study Monitor by fax on completion of the in-life phaseof rii?g study- The lesuhs will be reported ia a ppeodix to the report for the subiequcnt 28-day study. 7. QCAUIT ASSURANCE AND ARCHIVING PROCEDURES 7.1. Qoalhy Aunruce No formal study-based Quality Assurance procedures will be performed on this study. These may be included if requested by the Sponsor. 13.. Archives All experimental data arising from the study (including documentaly raw data, specimens, records, other materials; collectively defined as the "materials") will remain the property of the Sponsor. Huntingdon Life Sciences shall retain the materials in its archive for a periodof 5 years after completion of the study. After such time, the Sponsor will be contacted and their advice sought on the return, disposal or further retention of me materials. If requested, Huntingdon Life Sciences will continue to retain the materials subject to a reasonable fee being agreed with the Sponsor. Fmxl Protocol Page 13 : 290 : Company Sanitized. Does not contain TSCA GBI DPT 442/992305 SEVEN DAY ORAL TOXICITY STUDY IN THE RAT Study Number Protocol Amendment Number :DPT/442 :l(0ne) HI IiUjnItMinI laydUOVlnI Life Sciences PRELIMINARY TOXICITY STUDY BY ORAL ADMINISTRATION TO CD RATS FOR 7 DAYS Total number of pages: 3 Number of pages for internal datribntion: 3 Study Director : S M Bottomley, B.SC. (Hons). M.SC, CBiol, M-I-Biot- The signature of the Study Director authorises the implementation of this amendment to protocol. In this amendment, deleted statements are struck through and new statements are underlined. Any changes to the study design after the date of this authorising signature will be documented in a further fbnnaJ amendment. AMENDMENT APPROVAL For HaatiBgdo^sfe-ScuLt^d \^^^/\^ Authorised bv^^y (Study Director) Date: ^ UCUCM C^^ For the Sponsor Approved by: '^^f>-- Date: , ^---------- L*>^ 19, /H? Pagcl 291 : Company Sanitized. Does not contain TSCA CBI 6 DPT 442/992305 SEVEN DAY ORAL TOXICITY STUDY IN THE RAT Study Nnmber Protocol Amendment Number : DPT/442 : I (one) Huntingdon Life Sciences PREUMIMAKYTOXICTn^STUDYBY ORAL ADMINISTRATION TO CD RATS FOR 7 DAYS Reasons for uneBdments : Change of Study Director due to maternity leave. Ameffldmeats 1 INTRODUCTION Management of study Study Dircctoi<original): S M Bottomley Study Director (replacement): H A Palmer This amendment formally registers the assignment of a replacement Study Director. The signature of the replacement Study Director approves the implementation of this amendment to protocol. Any changes to the study design after the date of this approval signature will be documented in a further formal amendment Reason for^nifcudnient: Declaration The original Study Director (S M Boaomky) is talcing maternity teave Original Study Director I am satisfied with the conduct of the study to date. D^^MC^^./^ I am satisfied with the conduct of the study to date. From the date of my signature on mis amendment, 1 assume responsibilities of the Study Director. Signature:. Dale:..2...ro,(W!^...^. Page 2 292 Company Sanitized. Does not contain TSCA CBI DPT 442/992305 SEVEN DAY ORAL TOXICITY STUDY IN THE RAT Study Number Protocol Amendment Nnmber :DPT/442 : 1 (one) For BnntmgdoD Life Sciences Approved by:__JA^A8=Z- (Rcplactment Study Director) Released by:_ Huntingdon Life Sciences D^. -1 ^A^L 10.^ Date: 3. Aft^g^t1?1 Page 3 293 : Company Sanitized. Does not contain TSCA CBI