Document xOVKyydD856pE10q8jgzj9B1

CONFIDENTIAL AR226-3132 ZpOISL I DPT 443/984760 TOXICITY STUDY BY ORAL ADM INISTRATIO N TO CD RATS FOR 4 W EEKS Sponsor DuPont Specialty Chemicals. Jackson Laboratory, Chambers Works, Deepwater, . NJ 08023, U.S.A. Page 1 o f 293 Research Laboratory Huntingdon Life Sciences Ltd., P.O. Box 2, Huntingdon, Cambridgeshire, PE18 6ES, 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 S U M M A R Y ............................................................................................................................... 7 INTRODUCTION................................................................................................................... 10 RELEVANT STUDY DATES................................................................................................. 11 TEST SUBSTANCE....................................................................................................................... 12 EXPERIMENTAL PROCEDURE................................................................................................ 13 RESULTS......................................................................................................................................... 24 DISCUSSION AND CONCLUSION........................................................................................... 30 REFERENCES................................................................................................................................ 31 FIGURES - group data 1. Bodyweight........................................................................................................................... 32 :2: Company Sanitized. Does not contain TSCA CBI m 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. APPENDICES - individual data 1. Bodyweight.......................................................................... 2. Haematology...................................................................... 3. Biochemistry....................................................................... 4. Organ weights..................................................................... 5. Clinical and pathological findings for individual animals BEHAVIOURAL SCREENING. FORMULATION CHEMISTRY REPORT.. PROTOCOL AND AMENDMENTS. %ISEVEN DAY ORAL TOXICITY STUDY IN THE RAT (DPT 442/99230:51- DPT 443/984760 Page 34 35 36 37 39 41 43 45 52 54 60 64 66 119 182 199 236 :3 : Company Sanitized. Does not contain TSCA CBI 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 Good Laboratory Practice (as revised in 1997), ENV/MC/CHEM(98) 17. 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.I.Biol., Study Director, Huntingdon Life Sciences Ltd. 3 \. Date :4 : e 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 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 Report 13 August 1999 14 December 1998 14 December 1998 14 December 1998 14 December 1998 14 December 1998 15 January 1999 15 January 1999 15 January 1999 15 January 1999 13 August 1999 Protocol: An audit o f 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 o f 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 o f study were carried out. These were promptly reported to appropriate Company Management. R eport 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. :5: Date Company Sanitized. Does not contain TSCA CBI CONTRIBUTORY SCIENTISTS STUDY MANAGEMENT Helen A. Palmer, B.Sc. (Hons.), 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 o f Pharmacy and Formulation Chemistry. BEHAVIOURAL SCREENING Elizabeth W. Hughes, B.A., M.Sc., Behavioural Scientist, Pharmacology and Short Term Studies Division. DPT 443/984760 Company Sanitized. Does not contain TSCA CBI SUMMARY DPT 443/984760 This study was performed to assess the systemic toxicity oi followed was outlined in: the ra t The method OECD Guideline for Testing of'Chemicals Guideline No.407, `Repeated Dose 28-day Oral Toxicity Study in Rodents', Adopted 27 July 1995. Iw ^ administered by oral gavage, once daily, to three groups o f 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 o f 1.5, 5.0 or 15 mg/ml and administered at a dosage volume o f 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 o f 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 o f the study. All animals were killed at the end o f the 4 week treatment period (Day 29) and examined macroscopically. A range o f organs was weighed and tissues preserved for subsequent light microscopy examinations. The following comments in relation to the principal tradings are made in summary: M ortality There were no unscheduled deaths on the study. Clinical signs No clinical signs considered to be of toxicological 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 o f controls. Behavioural screening No change attributable to treatment wil ras noted for any behavioural parameter. :7 : Company Sanitized. Does not contain TSCA CBI DPT 443/984760 Haematology . Male and female rats dosed with 150 mg/kg/day had toxicologically 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 toxicologically insignificant. Biochemistry Male and female rats dosed with 150 mg/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 o f 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 o f 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 o f 1 male, pallor of the liver, a reduction in adipose tissue and a reduction in the size o f the ovaries and uterus in 1 female. :8 : Company Sanitized. Does not contain TSCA CBI DPT 443/984760 Microscopie pathology Evidence o f nephrotoxicity was detected at 150 mg/kg/day and to a lesser extent at 50 mg/kg/day, and was characterised by interstitial inflammation in th 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 ^ t h e rat when administered orally for 28 days. According to the EEC Council ' Directive 92/69/EEC Annex VI, Part 11(D) as described in Commission Directive 93/21/EEC, labelling with the R48 risk phrase is appropriate. H e n c e f if l^ l^ H I ^ R S labelled R48/22: Harmful if swallowed. :9 : Company Sanitized. Does not contain TSCA CBI INTRODUCTION DPT 443/984760 The study was designed to assess the systemic toxicity of surfactant in the polymerisation o f fluoromonomers, 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 o f rat used was chosen on account o f the availability o f background data. . The rats were dosed orally as the test substance may be ingested accidentally. The dosage levels were selected on the basis o f 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 o f 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 Week 1 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: DuPont Specialty Chemicals 23 June 1998 Batch number Expiry date: Date of manufacture: 2 years from manufacture 8 March 1998 Purity: Appearance: Storage conditions: Room temperature A 1 g sample o f the test substance was retained in the Huntingdon Life Sciences Archive. All dosages reported herein are expressed in terms o f 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 Crl :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 further. No macroscopic abnormalities were noted in any o f 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 o f 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 o f the study. Permanent weekly recordings o f 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 o f 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 o f 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 the start o f 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 Fun 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 7 mm 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 o f treatment. On the day of commencement o f 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 1 2 3 4 Health check Animal numbers MF 1-5 6-10 11 - 1 5 16-20 41 - 4 4 , 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 The test substance, was administered as a solution in distilled water. A correction factor of x4 was applied to correct for the fact that the test material as supplied was a 25% aqueous slurry. A series o f solutions were prepared by serial dilution of die test substance. The test material as supplied was warmed 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 o f 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: Yellow 3: Blue 4: Pink Control () 15 50 150 # Expressed as solids, not as material supplied 0 1.5 5 15 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 o f the formulations was satisfactory under the conditions of the study. Samples o f 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, 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 o f 9 days, treatment continued until completion of 4 weeks. : 15 : Company Sanitized. Does not contain TSCA CBI DPT 443/984760 OBSERVATIONS AND MEASUREMENTS Dated and signed records o f all activities relating to the day by day running and maintenance o f 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 o f the study. Dated and signed records o f 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 earned out during the working period o f that day. B odyw eight The weight o f each rat was recorded one week prior to the commencement o f treatment, on the day of commencement o f 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 o f 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* 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 o f death an animal does not eat : 16 : Company Sanitized. Does not contain TSCA CBI 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 Food 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. W ater consumption Daily monitoring by visual appraisal was maintained throughout the dosing period. NEUROBEHAVTOURAL SCREENING Functional observational battery The functional observational battery and motor activity was performed at approximately the same time of day, before initiation o f 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 functional observational battery is fully detailed in the BEBLAVIOURAL 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 o f activity are recorded: the time spent in no movement, locomotor and non-locomotor activity. The number of occurrences (events) o f each category is also recorded. Normally in reporting this data only the locomotor activity is presented. For testing, designated animals were placed singly into observation cages. Once all animals had been placed into the cage, the test session was started. The test s ession 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 isoflurawne/nitrous oxide anaesthesia, from the orbital sinus o f all rats. The blood samples collected were divided into tubes as follows: 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 Units The following estimations were performed using a Bayer-Technicon HIE haematology analyser: Packed cell volume (PCV) % Haemoglobin concentration (Hb) g/dl Erythrocyte count (RBC) x 10I2/1 Mean cell haemoglobin concentration (MCHC) g/dl Mean cell volume (MCV) A Mean cell haemoglobin (MCH) pg Total leucocyte count (WBC total) x 109/I Differential leucocyte count Neutrophils Lymphocytes Eosinophils Basophils Monocytes Large unstained cells (N) (L) (E) (B) (M) (LUC) ) ) ) ) ) ) x l0 9/l : 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 (Choi) - (Enzymatic assay) : 19 : DPT 443/984760 Units x 109/I s s 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 aminotransferase (GPT) also known as `glutamic-pyruvic transaminase' Reaction temperature 37C Aspartate aminotransferase (GOT) also known as `glutamic-oxaloacetic transaminase' Reaction temperature 37C Gamma-glutamyl transpeptidase (yGT) 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 o f 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 CBI 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 o f 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 o f any macroscopically abnormal tissues were routinely preserved, along with samples o f 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 ZymbaPs 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 o f 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 ;.c 4 - 5 micrometres were stained with haematoxylin and eosin. : 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 o f 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 earned 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 o f 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). 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 o f variance was carried out. If significant heterogeneity o f variance was present, and could not be removed by a transformation, an analysis o f ranks was used, KruskalWallis (1952/3). Analyses o f variance was followed by Student's t 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 non parametric 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 o f the study at Huntingdon Life Sciences, together with a copy o f 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 o f 5 years from the date o f 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 RESULTS DPT 443/984760 FORMULATION CHEMISTRY (page 179) Prior to treatment, tfa^s^rilit^dm um ambient storage for 2 days and refrigerated storage for 15 days was confirmed aqueous formulations, at nominal concentrations of 5 and 400 mg/ml. The mean concentrations test formulations analysed during the study were within 7% o f nominal concentrations confirming the accuracy o f 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 mg/kg/day occasionally. As this finding was transient in nature, it is considered likely that the salivation is a reaction to poor palatability o f the test substance rather than any toxicological response. No significance is attached to this finding. BODYW EIGHT (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, the 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 o f the controls. FOOD CONSUM PTION (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 o f 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 O F 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 tq HAEMATOLOGY (Table 4, Appendix 2) In male and female rats dosed with 150 m g /k g /d a y v ^ ^ |m H H h a e m a to lo g ic changes indicated microcytic anaemia and inflammation. Male and female rats dosed with 150 mg/kg/day had toxicologically 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 toxicologically 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 o f blood loss in urine. Renal insufficiency may have caused decreased production of erythropoietin, decreasing the rate o f 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 toxicologically significant because the degree of change was small or the change was in a direction not considered toxicologically 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 o f 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 hyperbilirubinaemia. 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 o f 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 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 o f 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 i 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 o f sufficient degree to cause hyperbilirubinaemia, ALP inhibition by fluoride may have masked an increase. These changes were minimal but considered toxicologically significant. Statistically significant changes in other clinical chemistry parameters were not considered toxicologically significant because the degree of change was small, the change was in a direction not considered toxicologically 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 o f 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. U reters and urinary bladder: Unilateral distension o f 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 o f both was observed in 1/5 female rats receiving 150 mg/kg/day compared with 0/5 female control rats. The incidence and distribution o f all the other findings were considered to fall within the expected background range of macroscopic changes. : 27 : Company Sanitized. Does not contain TSCA CBI 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 mg/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 Minimal Slight Moderate Inflammatory casts in tubules and collecting ducts Cortical tubular dilatation Total Minimal Slight Moderate Cortical tubular necrosis Basophilia and hyperplasia in tubules and collecting ducts Total Minimal Slight Moderate Marked Medullary tubular dilatation Total Minimal Moderate Marked Papillary tubular dilatation Total Minimal Slight Moderate Papillary necrosis Collecting duct hyperplasia Urothelial hyperplasia Number ofkidneys examined 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 5 Male 15 50 150 0 0 0 5** 0 00 10 00 3 0 00 10 0 0 5** 0 0 2 5** 0 02 00 0 0 5** 0 00 0 0 00 3 0 0 4* 5** 0 0 4* 0 0 00 0 0 0 0 4* 0 00 10 0 1 5** 0 0100 0 0 4* 0 00 10 0 2 5** 0 02 0 0 0 0 4* 0 00 10 00 00 00 2 0 0 0 5** 0 55 55 Female 15 50 150 00 3 002 00 1 00 0 0 0 4* 0 4* 5** 03 0 0 1 4* 00 1 00 0 0 5** 5** 03 0 02 0 0 0 5** 00 0 0 1 5** 0 10 0 0 5** 00 0 0 1 5** 011 0 0 4* 00 0 00 3 03 1 0 1 5** 55 5 ** <0.01 *p<0.05 with Fisher's Exact Test These changes were considered associated with the increased organ weights, macroscopic findings o f pale/in-egular cortical scarring ere; and the increased number o f circulating neutrophils. : 28 : Company Sanitized. Does not contain TSCA CBI DPT 443/984760 Liver - Hepatocyte hypertrophy and fine vacuolation were detected at 150 mg/kg/day and correlated with the macroscopic pale appearance and the increased bodyweight-adjusted liver weight. Dosage level (mg/kg/day) Hepatocyte hypertrophy Total Minimal Slight Hepatocyte fine vacuolation Total Minimal Slight Number of livers examined 0 0 0 0 0 0 0 5 Male 15 50 150 0 0 0 5** 0 0020 0 0 30 0 0 5** 0 00 30 0020 5 5 55 Female 15 50 00 00 00 00 00 00 55 150 5** 4* 1 5** 4* 1 5 ** ><0.01 *><0.05 with Fisher's Exact Test Bone m arrow (Fem ur/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 o f possible perturbation o f 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 o f nephrotoxicity 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 s u b s t a n c e ,a ^ m P ^ P j w a s administered by oral gavage once daily to groups o f five male and five female rats for twenty eight consecutive days at dosage levels o f 15, 50 or 150 mg/kg/day (expressed as solids, not as material supplied). A further group o f 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 o f no biological significance. Other findings attributable to treatment were only noted at 150"or 50 mg/kg/day. PPw ai^oncIuded "that 15 mg/kg/day represent the no observed adverse effect level (NOAEL) f o r f l m j ^ j j j ^ P p l i the rat when administered orally for 28 days. ^ Nephrotoxicity lesions noted at microscopic examination reflect an increased kidney weight at necropsy, and were often graded as o f 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 o f 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 Council Directive 92/69/EEC Annex VI, Part 11(D), as described imCommission Directive 93/21/EEC, labelling with the R48 risk phrase is appropriate. Hence s labelled: R48/22: Harmful if swallowed. k : 30 : Company Sanitized. Does not contain TSCA CBI REFERENCES DPT 443/984760 BARTLETT, M.S. (1937) Properties of sufficiency and statistical te st Proc. Roy. Soc., A 160,268. FISHER, R.A. (1950) in: (Eds). Statistical Methods fo r 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 o f 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. Clin. Path., 36,212. QUICK, A.J. (1942) in: (Eds). The Haemorrhagic Diseases and the Physiology o fHaemostasis, 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, D A . (1971) A test for differences between treatment means when several dose levels are compared with a zero dose control. Biometrics, 27, 103 - 117. WILLIAMS, D A . (1972) The comparison of several dose levels with a zero dose control. Biometrics, 28, 519 -531. : 31 : Company Sanitized. Does not contain TSCA C8I Bodyweight (g) FIGURE 1 Bodyweight - group mean values - males DPT 443/984760 Company Sanitized. Does not contain TSCA CBI Week Bodyweight (g) FIGURE 1 Bodyweight - group mean values - females DPT 443/984760 Company Sanitized. Does not contain TSCA CBI Week TABLEI Bodyweights - group mean values (g) DPT 443/984760 Week -i 0 1 2 3 4 1M 2M Control 15 91 143 199 255 308 339 92 145 198 254 298 325 Group and dosage (mg/kg/day) 3M 4M IF 2F 3F 50 150 Control 15 50 90 141 193 233 278 300 93 151 176 207 217 226 92 135 169 192 215 229 91 136 169 193 212 225 90 132 160 183 197 210 4F 150 93 131 143 156 164 170 1 O Gain (g/rat) sd % of control 195 15.1 - 180 24.4 92 * 158 21.0 81 * 75 28.3 38 sd Standard deviation * /><0.05, **p<0.01 94 11.3 - 90 8.2 96 * 77 7.9 82 + lip 39 13.5 41 : 34 : Company Sanitized. Does not contain TSCA CBI TABLE 2 Food consumption - group mean vaines (g/rat/week) DPT 443/984760 Week 1M 2M Control 15 Group and dosage (mg/kg/day) 3M 4M IF 2F 3F 50 150 C o n t r o l 15 50 -1 145 154 148 158 136 137 119 1 181 190 172 134 146 145 140 2 198 206 176 147 139 138 134 3 210 203 199 136 148 146 139 4 182 173 163 116 132 126 121 Total 1 - 4 % of control 771 -- 772 710 533 100 92 69 565 No statistical analysis performed (only one cage/sex/group) 555 534 98 95 4F 150 125 105 103 108 94 410 73 35 : Company Sanitized. Does not contain TSCA CBI TABLE 3 Food conversion ratios - group mean vaines DPT 443/984760 Heek 1M 2M Control 15 Group and dosage (mg/kg/day) 3M 4M IF 2F 3F 50 150 Control 15 50 4F 150 1 3.3 3.6 3.4 5.2 4.3 4.3 5.1 8.5 2 3.5 3.7 4.3 4.8 6.0 5.8 5.7 7.9 3 4.0 4.6 4.4 12.6 6.5 7.5 10.3 13.2 4 6.0 6.3 7.6 13.6 9.4 9.7 9.5 17.3 1-4 4.0 4.3 4.5 7.1 6.0 6.2 6.9 10.5 Food conversion ratio - food consumption/bodyweight gain : 36 : Company Sanitized. Does not contain TSCA CBI TABLE 4 Haematology - group mean values DPT 443/984760 Day 29 Group/ dosage mg/kg/day PCV Hb R B C M C H C M C V MCH- Pit % g/dl 101 2 /1 g/dl fl pg 109 /1 PT APTT ss 1M Control 44.3 15.2 7.86 34.2 56.4 19.3 1067 13.0 18.4 2M 15 43.9 15.0 7.72 34.3 56.9 19.5 1034 13.5 18.5 3M 50 4M 150 42.6 14.6 . 7.57 * 36.1 12.5 ** 6.66 34.3 56.3 19.3 k 34.7 54.2 18.8 1094 * 1277 13.1 19.5 (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 3F 50 4F 150 41.6 14.4 k 39.7 14.0 + * ** 34.9 12.4 7.54 34.7 55.2 19.2 1069 * 7.25 35.4 54.9 19.4 1175 * * * 6.75 35.5 51.8 18.4 1304 14.1 16.6 14.0 15.5 (13.3)(14.3) * /7<0.05, **p<0.01 () 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 mg/kg/day WBC Total 109 /1 N L E B M LUC 109 /1 1 0 9 /1 1 0 9 /1 1 0 9 /1 1 0 9 /1 1 0 9 /1 1M Control 13.07 1.66 10.69 0.11 0.04 0.32 0.25 2M 15 12.69 1.90 10.15 0.11 0.05 0.29 0.20 3M 50 4M 150 11.16 * 19.74 1.62 8.99 ** 6.55 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 **<0.05, ** p<0.01 0.87 7.97 1.32 5.15 1.41 *+ 3.79 7.17 8.62 0.09 0.03 0.07 0.02 0.11 ' 0.03 * 0.14 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/ dosage mg/kg/day 1M Control 2M 15 3M 50 4M 150 Glu cose mg/dl Protein g/dl A/G Total Alb Glob Orea Nitr mg/dl Creat inine mg/dl AP mO/ ml 88 6.3 3.3 3.0 1.08 11 ** 79 6.3 3.3 3.1 1.07 15 + 84 6.4 3.3 3.0 1.11 19 ** 89 6.4 3.3 3.1 1.06 64 0.5 491 0.5 521 0.6 ** 1.2 553 573 GPT mU/ ml 52 56 52 58 GOT mU/ ml 95 89 92 91 IF Control 107 6.2 3.4 2.9 1.17 18 2F 15 3F 50 4F 150 + 93 6.4 3.5 2.9 1.18 17 + ** 90 6.4 3.4 2.9 1.17 25 * * 98 6.3 3.3 3.0 1.07 90 */t<0.05, ** p<0.01, Williams' test +p<0.05, Student's t test 0.6 377 47 81 0.6 331 42 86 0.6 ** 1.5 298 43 449 47 95 91 : 39 : Company Sanitized. Does not contain TSCA CBI TABLES (Biochemistry - continued) DPT 443/984760 Day 29 1 Group/ dosage mg/kg/day -- yGT Bili- Na mU/ rubin mEq/ ml mg/dl 1 K Ca P Cl Choi Tri- mEq/ mEq/ mEq/ mEq/ giyc 1 1 1 1 mg/dl mg/dl 1M Control <1 0.1 143 3.6 5.3 4.8 99 74 49 2M 15 <1 0.1 143 3.5 5.3 4 .8 99 77 55 3M 50 4M 150 <1 0.1 142 3.4 5.3 4.8 100 77 59 it + ** ** <1 0.4 142 4.0 5.8 6.2 99 71 34 IF Control <1 0.1 142 2F 15 <1 0.1 143 3F 50 4F 150 <1 0.1 143 + ** <2 0.2 138 *p<0.05, ** p<0.01, Williams' test ++p<0.01, Student's /test , 3.6 5.2 3.8 102 3.4 5.2 4.0 101 3.5 5.2 3.9 102 * * ** 4.5 5.8 ** 6.5 ** 97 65 67 ++ 104 79 33 23 27 30 : 40 : Company Sanitized. Does not contain TSCA CBI TABLE Organ weights - group mean values DPT 443/984760 Terminal kill Group/ dosage mg/kg/day Body Brain Thymus Heart Liver Spleen Kidneys Adrenals Testes Epididy wt mides gg gg gg g mg gg 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 * 4M 150 206 1.77 0.290 0.87 11.0 0.47 4.93 40.9 2.90 0.589 Adjusted msans 1M 2M 3M 4M - 1.86 1.89 1.89 1.84 0.435 1.10 0.440 1.01 0.454 1.05 0.476 1.11 11.5 0.50 11.6 0.55 12.9 0.52 15.3 0.68 - 47.2 46.0 45.1 58.5 2.96 3.07 3.10 3.14 0.653 0.688 0.686 0.660 *p<0.05, **p<0.01 : 41 : Company Sanitized. Does not contain TSCA CBI TABLE (Organ weights - continued) DPT 443/984760 Terminal kill Group/ dosage mg/kg/day Body Brain Thymus Heart Liver Spleen Kidneys Adrenals wt gg g9 gg g mg Unadjusted means IF Control 217 1.80 0.501 0.90 8.8 0.45 1.85 64.2 2F 15 214 1.86 0.488 0.89 9.7 0.50 1.92 69.2 3F 50 196 1.81 0.442 0.81 9.0 0.45 2.05 57.2 4F ** ** ** 150' 157 1.78 0.233 0.69 8.7 0.37 4.05 40.3 Adjusted means IF - 1.68 0.428 - iH CO -" 2F - 1.76 0.427 - 9.1 - - - 3F - 1.81 0.441 - 9.0 - - - * 4F - 2.00 0.368 - 10.0 " # *p<0.05, **p<0.01 Company Sanitized. Does not contain TSCA CBI TABLE 7 Macroscopic pathology incidence summary DPT 443/984760 Company Sanitized. Does not contain TSCA CBI Remot 1 reason: Terminal Group 1 Group 2 Group 3 Group 4 Group 1 Group 2 Group 3 Group 4 Skin Alopecia Animals on study 5 5 5 5 5 5 5 5 Animals completed 5 5 55 5 5 5 5 0000 0 0 0 1 Tail Tip missing w Incisors Lower pale 0001000 0 00 10 0 0 0 0 Thymus Small 00000 0 0 1 Lungs Congested 0 0 o. 1 1 0 0 3 Adipose Tissue Minimal 00050 00 5 Liver Median cleft, pale subcapsular area 2 3 0 0 0 0 0 2 Pale 0003 0 0 0 4 Lobular markings accentuated 0001000 0 Stomach Antrum Mucosa White nodule 00000 10 0 Kidneys Increased pelvic dilatation 01000 00 0 TABLE 7 (Macroscopic pathology incidence summary - continued) DPT 443/984760 Company Sanitized. Does not contain TSCA CBI Removal reason: Terminal Animals on study Animals completed Kidneys Pale Irregular cortical Enlarged Swollen Pale cortical foci Misshapen scarring Ureters Distended Contained blood stained urine Urinary Bladder Contained blood stained urine Ovaries Small Uterus Fluid distension Thin Group 1 Group 2 5 5' 5 5 (Continued) 00 00 00 00 00 00 Group 3 5 5 0 0 0 0 0 0 Group 4 5 5 5 2 5 5 3 1 Group 1 5 5 0 0 0 0 0 0 Group Group 2 --, 3 55 55 00 00 00 00 00 00 Group 4 5 5 5 5 5 5 2 0 00010 0 0 0 00010 0 0 0 0001000 0 00000 0 0 1 00003 13 0 0000 00 0 1 # DPT 443/984760 Company Sanitized. Does not contain TSCA CBI TABLE 8 Microscopic pathology incidence summary Removal reason: Terminal Animals on study Animals completed Trachea Examined No abnormalities detected Lungs(including Bronchi) Examined No abnormalities detected Pneumonitis (Total) Minimal Vascu l a r congestion (Total) Minimal Heart Examined No abnormalities detected Thymus Examined No abnormalities detected Involution/atrophy (Total) Minimal Lymph Nodes - Mandibular Examined No abnormalities detected Lymph Nodes - Mesenteric Examined No abnormalities detected Spleen Examined Missing No abnormalities detected Liver Examined Group 1 Group 2 Group 3 Group 4 Group 1 Group 2 Group 3 Group 4 5555 55 5 5 55555 5 5 5 5005 5 0 0 5 5005 5 0 0 5 50055 0 0 5 4004 40 0 2 10 00 0 0 0 0 10 00 0 0 0 0 0001 10 0 3 00 01 1 0 0 3 50055 0 0 5 5005 50 0 5 50055 0 0 5 50055 0 0 4 00 00 0 00 1 0000 0 0 0 1 50055 0 0 5 50055 0 0 5 50 05 5 0 0 5 5005 50 0 5 50055 0 0 4 00 000 0 0 1 50055 0 0 4 55555 5 5 5 TABLE 8 (Microscopic pathology incidence summary - continued) DPT 443/984760 Company Sanitized. Does not contain TSCA CBI Group Group Group Group Group Group Group Group Removal reason: Terminal 1234 1 2 3 4 ----- M a l e s ------- ---- F e m a l e s ------ Animals on study 5 5 5 5 5 5 5 5 Animals completed 5 5 5 5 5 5 5 5 Livor No abnormalities detected (Continued) 44S0 4 5 5 0 Parenchymal inflammatory cell foci (Total) 01000 0 0 0 Minimal 01000 0 0 0 Hepatocyte vacuolation - median cleft 1 0 0 0 0 0 0 0 Hepatocyte hypertrophy - generalised (Total) Minimal n. Os Slight Hepatocyte fine vacuolation - generalised(Total) Minimal Slight Haemorrhage (Total) Minimal 00050 0 0 5 0002 0 0 0 4 00030 0 0 1 0 0 0 5 0 0 0 5 00030 0 0 4 00020 0 0 1 0000 10 0 0 000010 0 0 Kidneys Examined No abnormalities detected 55555 55 5 22 103 2 0 0 Interstitial inflammation in cortex,medulla and papilla(Total) Minimal Slight Moderate 00050 0 0 3 00010 0 0 2 00030 00 1 00010 0 0 0 Basophilia and hyperplasia of tubules and collecting ducts(Total) 00450 0 5 5 Minimal 004000 3 0 Slight 0000002 0 Moderate 00040 0 0 5 Marked 00010 0 0 0 Cortical tubular dila tation (Total) 00250 0 4 5 Minimal 002000 3 0 Slight 000500 1 4 Moderate 000000 0 1 TABLE 8 (Microscopic pathology incidence summary - continued) DPT 443/984760 Company Sanitized. Does not contain TSCA CBI Removal reason: Terminal Group 1 Group 2 Group 3 Group 4 Group 1 Group 2 Group 3 Group 4 Animals on study 5 5 5 5 5 5 5 5 Animals completed 5 5 5 5 5 5 5 5 Kidneys (Continued) Cortical fibrosis and tubular collapse with basophilia(Total) Minimal 1100 00 0 0 1100 0 0 0 0 P a p i llary necrosis (Total) Minimal Slight Pelvic d i l a t a t i o n (Total) Slight Urothelial hyperplasia (Total) 0000 0 0 0 3 00000 0 0 2 00000 0 0 1 0100 0 0 0 0 0 1 0 0 0 0 0 0 00 05 0 0 1 5 Slight 000300 1 1 Moderate 00010 0 0 4 Marked 00010 0 0 0 M e d u l l a r y tubular dilata t i o n (Total) 0 0 1 5 0 0 1 5 Minimal 00 100 0 1 0 Moderate 00040 0 0 5 Marked 00010 0 0 0 Papillary tubular dilatation (Total) 0 0 2 50 0 1 5 Minimal 00200 0 1 1 Slight 00040 0 0 4 Moderate 000 10 0 0 0 Cortical tubular necrosis (Total) 000 30 0 0 0 Minimal 00010 0 0 0 Slight 000200 0 0 Cortico-medullary mineralisation (Tot!'1 00002 2 5 0 Min. mal 00000 2 1 0 Sligi.'- 00002 0 1 0 Moderate 00000 03 0 Inflammatory casts in tubules and collecting ducts 0005000 4 0P a p i l l a r y c o l l e c t i n g d u c t h y p e r p l a s i a 0 0 0 2 03 1 TABLE 8 (Microscopic pathology incidence summary - continued) DPT 443/984760 Company Sanitized. Does not contain TSCA CBI Removal reason: Terminal Animals on study Animals completed Kidneys Cortical basoph i l i c tubules (Total) Minimal Interstitial inflammation in papilla (Total) Minimal Urinary Bladder Examined No abnormalities detected Ureters Examined Luminal dilatation Slight (Total) Uterus Examined No abnormalities detected Luminal dilata t i o n (Total) Moderate Marked Myometrial/endometrial atrophy (Total) Minimal Cervix Examined No abnormalities detected Epithelial mucification Vagina Examined No abnormalities detected Ovaries Examined Group 1 Group 2 Group 3 Group 4. Group 1 Group 2 Group 3 Group 4 5555 5 5 5 5 55 55 5 5 5 5 (Continued) 320012 0 0 320012 0 0 00000 0 3 0 00000 0 3 0 5005500 5 50055 00 5 00010 0 0 0 00010 0 0 0 0001000 0 0000513 5 0000200 3 0000 3 1 3 0 00002 12 0 0000 1 0 1 0 00000 00 2 00000 0 0 2 000050 0 5 00005 0 0 3 00000 0 0 2 00005 0 0 5 0000500 5 00005 0 0 5 TABLE 8 (Microscopic pathology incidence summary - continued) DPT 443/984760 Company Sanitized. Does not contain TSCA CBI Removal reason: Terminal Animals on study Animals completed Ovaries No abnormalities detected Sparse/few corpora lutea Prostate Examined No abnormalities detected Seminal Vesicles Examined No abnormalities detected Epididymides Examined No abnormalities detected Testes Examined No abnormalities detected Thyroids Examined No abnormalities detected Parathyroids Examined No abnormalities detected Adrenals Examined No abnormalities detected Cortii-. 1 v a c u o l a t i o n (Total) Minimal Stomach Examined No abnormalities detected Group 1 Group 2 Group 3 Group 4 Group 1 Group 2 Group 3. Group 4 55555 5 5 5 55555 55 5 (Continued) 0000500 3 00000 0 0 2 50050 0 0 0 50050 0 0 0 50050 0 0 0 50050 0 0 0 50050 0 0 0 50050 00 0 50050 0 0 0 500 50 0 0 0 500 55 0 0 5 500 55 0 0 5 50 055 0 0 5 50 055 0 0 5 5005500 5 50045 0 0 5 00010 0 0 0 00010 0 0 0 50055 00 5 500 55 0 0 5 W DPT 443/984760 Company Sanitized. Does not contain TSCA CBI TABLE 8 (Microscopic pathology incidence summary - continued) Removal reason: Terminal Animals on study Animals completed Duodenum Examined No abnormalities detected Jejunum Examined . No abnormalities detected Ileum(including Payer's Patch) Examined No abnormalities detected Caecum Examined No abnormalities detected Colon Examined No abnormalities detected Rectum Examined No abnormalities detected Spinal Cord Examined No abnormalities detected Va c u o l a t i o n (Total) Minimal Sciatic Nerve Examined No abnormalities detected Group 1 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 0 0 5 5 Group Group 23 Males 55 55 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 Group 4 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 4 1 1 5 5 Group 1 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 0 0 5 4 Group 2 5 5 0 0 o 0 o 0 o 0 o 0 0 0 0 0 0 0 0 0 Group 3 5 5 0 0 o 0 o 0 o 0 o 0 o 0 0 o 0 0 0 0 Group 4 5 5 5 5 5 5 5 5 5 5 5 5 5 5 0 0 5 5 TABLE 8 (Microscopic pathology incidence summary - continued) DPT 443/984760 Company Sanitized. Does not contain TSCA CBI Removal reason: Terminal J3roup 1 Group 2 Group 3 Group 4 Group 1 Group 2 Group 3 Group 4 Animals on study 5 5 5 5 5 5 5 5 Animals completed 5 5 5 5 5 5 5 5 Sciatic Nerve Degenerate fibres (Total) Minimal (Continued) 000010 0 0 000010 0 0 Brain Examined No abnormalities detected 50 05 5 0 0 5 50 05 5 0 0 5 Femur/joint Examined No abnormalities detected Osteoarthrosis (Total) Minimal Marrow - prominent adipocytes 55555 5 5 5 55524 5 5 1 00010 0 0 0 00010 0 0 0 000310 0 4 APPENDIX 1 Bodyweights - individual values (g) G roup 1M: C o n tro l Cage Animal number number 11 2 3 4 5 Week -1 0 1 2 3 4 95 152 212 266 314 336 94 147 208 270 328 363 86 133 185 239 291 323 92 143 195 256 315 350 89 142 195 247 295 322 Group 2M: 1 5 m g/kg/day Cage Animal number number 26 7 8 9 10 Week -1 0 1 2 3 4 89 142 198 253 297 321 94 148 198 258 300 329 89 145 202 270 329 366 99 151 203 252 290 315 88 138 188 236 273 296 DPT 443/984760 Company Sanitized. Does not contain TSCA CBI Group 3M: 50 mg/kg/day Cage Animal number number 3 11 12 13 14 15 Week -1 0 1 2 3 4 88 138 188 228 270 293 90 140 190 230 275 288 93 149 210 261 308 335 88 145 200 243 293 317 92 135 177 204 245 2 67 Group 4M: 150 m g/kg/day Cage Animal number number 4 16 17 18 19 20 Week -1 0 1 2 3 4 97 149 161 183 174 178 95 147 173 203 2 19 222 90 148 17 6 200 222 235 95 162 204 245 253 267 88 148 167 202 218 227 G roup IF: C o n tro l Cage Animal number number 5 21 22 .23 24 25 APPENDIX 1 (Bodyweights - continued) Group 2F: 15 m g/kg/day Week -1 0 1 2 3 4 88 129 163 175 204 223 90 138 175 202 225 232 92 137 174 196 212 231 94 138 175 202 231 248 95 135 158 187 205 213 Cage Animal number number 6 26 27 28 29 30 Week -1 0 1 2 3 4 93 140 174 204 220 237 84 128 160 180 207 224 90 137 174 201 2 19 228 91 135 161 182 2 00 211 95 138 179 198 217 228 DPT 443/984760 Company Sanitized. Does not contain TSCA CBI Group 3F: 50 mg/kg/day Cage Animal number number 7 1031 32 33 34 35 Week -1 0 1 2 3 4 91 130 157 174 181 198 90 126 155 181 197 215 86 123 150 172 184 200 95 142 173 199 211 217 90 140 163 190 211 219 Group 4F: 150 m g/kg/day Cage Animal number number 8 36 37 38 39 40 Week -1 0 1 2 3 4 94 135 146 166 179 181 96 138 148 154 162 164 96 138 145 155 159 162 87 122 142 157 163 177 92 121 136 150 160 1 6 6 APPENDIX 2 Haematology - individual values DPT 443/984760 Day 29 (8 January 1999) Group/ Animai PCV Hb RBC MCHC MCV MCH Pit PT APTT dosage mg/kg/day no. % g /dl l O ' V l g / d l fl pg 10 9 /1 S s 1M Control 1 42.7 14.9 7.90 34.8 54.0 18.8 1000 13.0 18.4 2 44.3 15.1 7.74 34.2 57.2 19.5 970 13.2 15.9 3 46.1 15.6 7.92 33.9 58.2 19.7 1164 12.8 20.7 4 44.5 15.2 7.87 34.1 56.5 19.3 1051 12.9 18.4 5 44.1 15.1 7.89 34.2 56.0 19.1 1150 ctd 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 il 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. 7 6 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 150 16 33.8 12.0 6.54 35.5 51.7 18.4 1434 13.3 12.4 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 2 (Haematology - continued) DPT 443/984760 Day 29 (8 January 1999) Group/ dosage mg/kg/day Animal no. WBC Total 109 /1 N L E B M LOC 10 9 /1 109 /1 1 0 9 /1 10 9 /1 1 0 9 /1 1 0 9 /1 1M Control 1 13.26 1.05 11.22 0.10 0.05 2 14.45 1.95 11.87 0.12 0.03 3 13.89 1.45 11.75 0.07 0.05 4 12.66 1.61 10.29 0.18 0.05 5 11.07 2.22 8.31 0.08 0.02 0.46 0.24 0.27 0.35 0.28 0.38 0.22 0.30 0.19 0.16 Mean sd 13.07 1.66 10.69 0.11 0.04 0.32 0.25 1.302 0.452 1.468 0.044 0.014 0.088 0.OB9 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 il 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 150 16 30.42 9.93 18.64 0.27 0.17 0.83 0.59 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/ Animal PCV Hb RBC MCHC MCV MCH Pit PT APTT dosage mg/kg/day no. % g/dl 1 0 1 2 /1 g/dl fl p g 109 /1 S S IF Control 21 41.8 14.5 7.24 34.7 57.7 20.0 1123 11.8 13.9 22 42.7 15.1 7.92 35.4 54.0 19.1 1097 14.4 20.7 23 42.3 14.9 7.98 35.3 53.0 18.7 1088 14.4 16.4 24 44.6 15.5 7.71 34.7 57.9 20. 1 1054 13.8 12.4 25 43.1 15.1 8.19 34.9 52.7 18.4 1158 ctd 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 5 3.9 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 150 36 37 38 39 40 Mean sd 39.2 33.6 32.4 36.2 33.3 13.9 12.2 11.5 12.7 11.7 7.43 6.73 6.25 7.08 6.24 35.4 36.3 35.4 35.0 35.2 52.8 49.9 51.7 51.2 53.3 18.7 18.1 18.3 17.9 18.8 1130 1226 1768 1182 1215 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 ctd ctd ctd ctd 14.3 ctd ctd ctd ctd 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 mg/kg/day Animal no. Anis 1M Control i 2 3 4 5 2M 6 15 7 8 9 10 3M 11 50 12 13 14 15 4M 150 16 17 18 19 20 ctd Clotted sample - - _ - ~ - - _ - - - ctd - Micro Macro Var Hypo Hyper - - - - - - _ - - ctd + - - - - " - * - - - ctd -- - - - " - - - - - - - ctd -+ + --+ "+ -+ + + -+ + - -' ctd + + + + ctd +_-" - " ++ + + ++ ++ LS Atyp Blast - " " - - - ctd - ~ " " " " " ctd " - - -- -- ctd - " -- : 57 : Company Sanitized. Does not contain TSCA CBI APPENDIX 2 (Haematology - continued) DPT 443/984760 Week 5 (8 January 1999) Group/ dosage mg/kg/day Animal no. Anis IF Control 21 22 23 24 25 _ - - - 2F 26 . _ 15 27 - 28 - 29 - 30 - 3F 1031 _ 50 32 - 33 34 35 - 4F 150 36 _ 37 38 39 40 - Micro Macro Var Hypo Hyper __ -- '-- - __ --- __ --_- " -_ + --" - __ - -+ - - ++ -- + + _+ -- + -- + - - ++ - - ++ _ ++ - - ++ - - ++ - - ++ " - ++ _ _ ++ - - ++ + - ++ - - ++ - ++ LS _ " - - _ - Atyp _ - - _ * - - - - Blast _ - - - ~ - " ~ - - ~ - Company Sanitized. Does not contain TSCA CBI APPENDIX 2 (Haematology - continued) DPT 443/984760 Day 29 (8 January 1999) Group/ dosage mg/kg/day Animal no. WBC Total 109 /1 IF Control 21 5.82 22 14.71 23 9.37 24 9.26 25 7.23 N L E B M LOC i o V i 1 0 9 /1 1 0 9 /1 109 /1 1 0 9 /1 1 0 9 /1 0.69 1.07 1.26 0.64 0.71 4.88 12.95 7.66 8.16 6.18 0.05 0.13 0.12 0.06 0.09 0.01 0.06 0.03 0.04 0.01 0.13 0.27 0.16 .0.21 0.15 0.06 0.23 0.14 0.16 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 8.37 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 4F 150 Mean sd 36 37 38 39 40 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 0.02 9 12.58 11.09 21.24 11.27 8.85 3.14 3.09 6.85 3.98 1.88 8.81 7.52 13.43 6.73 6.59 0.12 0.12 0.14 0.19 0.13 0.04 0.03 0.07 0.02 0.02 0.30 0.22 0.45 0.21 0.14 0.16 0.10 0.30 0.15 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 (8 January 1999) ' Group/ Animal dosage no. mg/kg/day 1M Control 1 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 150 . 16 17 18 19 20 Mean sd Glu- Protein g/dl A/G cose mg/dl Total Alb Glob Urea Crt- AP GPT GOT Nitr inine m/ m/ m/ mg/dl mg/dl ml ml ml 90 6.2 3.2 3.0 1.07 13- 0.5 519 47 103 79 6.4 3.2 3.2 1.00 9 0.5 557 51 82 83 6.3 3.4 2.9 1.17 11 0.5 Sil 48 94 100 6.3 3.3 3.0 1.10 10 0.5 421 54 97 86 6.2 3.2 3.0 1.07 il 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 0.5 491 52 95 0.00 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 90 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 l.ii 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 6.4 3.3 3.1 1.06 64 14.0 0.24 0.04 0.22 0.077 13.3 1.2 573 58 91 0.31 70.0 6.7 8.0 sd Standard deviation 60 : Company Sanitized. Does not contain TSCA CBI APPENDIX 3 (Biochemistry - continued) DPT 443/984760 Week 5 (8 January 1999) Group/ Animal dosage no. mg/kg/day 1M Control 1 2 3 4 5 Mean sd 2M 6 15 7 8 9 10 Mean sd 3M 11 50 12 13 14 15 Mean sd m 16 150 17 18 19 20 Mean sd sd Standard deviation yGT Bili- Na K Ca P Cl Choi Tri- mO/ rubin mq/ mEq/ mEq/ mEq/ mEq/ glyc ml mg/dl 1 1 1 1 1 mg/dl mg/dl <1 0.1 143 3.7 5.2 5.1 99 65 41 <i 0.1 143 3.7 5.3 4.6 99 68 42 <i 0.1 144 3.5 5.3 5.0 100 89 47 <i 0.1 143 3.7 5.4 4.7 100 75 54 i 0.1 143 3.3 5.1 4.7 97 73 63 <i 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 0.1 142 3.5 5.3 4.6 98 64 60 <1 0.1 142 3.4 5.5 4.5 99 68 54 <1 0.1 145 3.6 5.4 4.9 101 81 69 <1 0.1 144 3.7 5.2 4.8 100 79 35 <i 0.1 142 3.5 5.3 5.3 99 93 56 <1 0.1 143 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 <1 0.1 141 3.4 5.2 5.1 99 72 1 0.1 143 3.3 5.3 4.7 100 58 <1 0.1 142 3.4 5.3 4.9 99 72 <1 0.1 143 3.8 5.3 4.8 102 62 78 43 65 55 52 <1 0.1 142 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 <1 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 <1 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 <i 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 (8 January 1999) Group/ Animal dosage no. mg/kg/day 1M Control 21 22 23 24 25 Mean sd 2M 26 15 27 28 29 30 Mean sd 3M 1031 50 32 33 34 35 sd 4M 36 150 37 38 39 40 Mean sd GlU- Protein g/dl A/G cose mg/dl Total Alb Glob Urea Crt- AP GPT GOT Nitr inine mO/ mU/ mU/ mg/dl mg/dl ml ml ml 103 6.3 3.4 2.9 1.17 18 101 6.0 3.3 2.7 1.22 21 91 6.1 3.2 2.9 1.10 16 118 .6.5 3.5 3.0 1.17 14 121 6.2 3.4 2.8 1.21 19 0.6 368 40 0.6 389 35 0.6 331 34 0.5 357 43 0.6 441 85 85 75 84 83 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 82 6.2 3.3 2.9 1.14 15 103 6.5 3.5 3.0 1.17 15 85 6.5 3.4 3.1 1.10 18 91 6.5 3.6 2.9 1.24 16 0.6 247 39 0.5 399 43 0.6 422 51 0.6 357 39 0.6 228 36 75 90 91 94 81 93 9.6 6.4 3.5 2.9 1.18 17 0.14 0.11 0.11 0.064 2.2 0.6 331 42 0.04 88.4 5.8 86 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 24 102 5.8 3.3 2.5 1.32 34 0.6 303 51 0.7 245 37 85 84 90 7.9 6.4 3.4 2.9 1.17 25 0.32 0.08 0.27 0.101 5.5 0.6 298 43 95 0.04 74.1 6.4 11-8 109 6.5 3.4 3.1 1.10 64 .i 359 49 99 6.5 3.4 3.1 1.10 103 1.9 464 54 91 96 95 96 91 6.3 3.2 3.1 1.03 98 6.0 3.1 2.9 1.07 105 6.2 3.2 3.0 1.07 81 1.8 534 38 113 1.6 576 47 76 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 sd Standard deviation 62 : Company Sanitized. Does not contain TSCA CBI APPENDIX 3 (Biochemistry - continued) DPT 443/984760 Day 29 (8January 1999) Group/ Animal dosage no. mg/kg/day IF Control 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 Bili- Na K Ca P Cl Choi Tri- mD/ m b i n m Eq/ mEq/ mEq/ mEq/ mEq/ giyc ml mg/dl 1 1 1 1 1 mg/dl mg/dl <i 0.1 142 3.6 5.1 3.4 103 77 26 <i 0.1 141 3.8 5.3 4.0 101 69 54 <1 0.1 143 3.4 5.1 4.0 101 59 25 <i 0.2 144 3.6 5.4 3.6 103 62 38 <i 0.2 142 3.6 5.2 3.9 102 56 20 <1 0.1 142 3.6 5.2 3.8 102 65 33 0.05 1.1 0.14 0.13 0.27 1.0 8.4 13.7 1 0.1 144 3.5 5.2 4.4 102 58 19 <i 0.1 141 3.6 5.2 4 .2 . 101 62 25 <1 0.1 144 3.2 5.2 3.4 103 93 32 <1 0.1 142 3.5 5.3 4.3 100 63 17 <1 0.1 142 3.4 5.2 3.7 101 60 24 <1 0.1 143 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 <1 0.1 144 3.8 5.2 4.0 103 110 <i 0.1 144 3.7 5.2 4.3 102 96 <1 0.1 143 3.2 5.2 3.7 101 79 1 0.2 143 3.7 5.2 4.5 103 105 30 22 27 26 29 <i 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 <i 0.2 138 4.5 5.6 5.5 99 42 <1 0.3 134 4.7 5.9 7.2 92 87 2 0.3 138 4.8 5:9 7.1 96 86 2 0.2 137 4.4 5.7 6.4 96 103 <1 0.1 142 3.9 5.8 6.1 102 76 28 32 27 35 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/ Arti n a i dosage no. m g /kg/day 1M Control 1 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 ' 150 16 17 18 19 20 Mean sd Body wt 9 B rain Thymus H e a rt 9 99 L iv e r Spleen Kidneys A drenals T e stes Epididymides 9 9 9 mg 9 9 309 1.87 325 1.94 295 1.91 319 1.92 298 1.86 0.569 0.587 0.495 0.639 0.362 1.17 1.32 1.10 1.25 1.26 13.6 14.8 12.5 14.7 12.5 0.58 0.77 0.54 0.63 0.54 2.82 2.85 2.S9 2.50 2.09 72.9 58.5 51.0 52.5 45.7 2.95 3.11 3.08 2.99 3.26 0.759 0.670 0.676 0.644 0.696 309 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 295 1.87 309 2.00 333 1.94 298 1.94 275 1.87 0.476 0.605 0.638 0.421 0.437 1.13 1.11 1.27 1.02 1.01 12.1 12.3 15.2 12.7 14.5 0.67 0.61 0.74 0.65 0.51 2.75 2.59 3.06 2.45 2.37 51.6 47.6 67.9 56.7 41.8 2.93 3.12 3.30 3.37 3.10 0.760 0.652 0.753 0.731 0.686 302 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 278 1.91 268 1.96 308 1.90 299 1.96 248 1 .76 0.335 0.405 0.573 0.594 0.442 1.07 1.09 1.13 1.11 0.93 11.9 11.8 16.1 14.4 12.0 0.48 0.49 0.64 0.64 0.45 2.23 2.44 2.64 2.76 2.38 45.1 49.1 44.9 55.4 3B.3 3.18 3.07 3.38 3.19 2.80 0.670 0.703 0.732 0.757 0.597 280 1.90 0.4 7 0 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 162 1.79 203 1.71 217 1.70 246 1.80 202 1.74 0.241 0.309 0.315 0.341 0.244 0.70 0.84 0.93 0.97 0.91 8.1 9.8 12.3 14.2 10.8 0.48 0.35 0.45 0.67 0.41 3.64 3.74 4.65 7.49 5.13 29.1 39.8 48.7 48.6 38.5 2.74 2.90 3.00 2.05 3.02 0.566 0.599 0.594 0.637 0.549 206 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/ Animal dosage no. mg/kg/day IF Control 21 22 23 24 25 Mean sd 2F 26 15 2*7 28 29 30 Mean sd 3F 50 * 1031 32 33 34 35 Mean sd 4F 36 150 37 38 39 40 Mean sd Body Vft 9 208 224 217 237 200 B r a in Thymus H e a rt 9 1.73 1.80 1.87 1.89 1.72 99 0.504 0.564 0.475 0.522 0.438 0.90 0.85 1.03 0.91 0.83 Liver Spleen Kidneys A drenals 99 8.6 0.30 8.4 0.50 8.8 0.56 9.6 0.46 8.8 0.37 9 1.96 1.69 2.00 1.92 1.70 mg 63.3 57.8 74.5 60.1 65.5 217 1 .8 0 0.5 0 1 0 .9 0 14.3 0.075 0.0476 0.077 8.8 0.45 1.85 0.46 0.081 0.148 64.2 6.45 223 2.01 210 1.89 211 1.82 211 1.68 215 1.92 0.543 0.548 0.413 0.352 0.583 0.86 0.94 0.88 0.85 0.94 10.2 9.1 10.8 9.1 9.6 0.40 0.52 0.53 0.49 0.55 2.02 1.91 1.69 1.92 2.08 61.9 62.8 80.1 73.1 68.2 214 1 .8 6 0 .4 8 8 0.B9 5.3 0.122 0.0997 0.044 9.7 0.50 1.92 0.72 0.059 0.149 69.2 7.57 188 1 .6 9 201 1.93 181 1 .7 6 207 1.88 204 1.81 0.470 0.440 0.366 0.509 0.425 0.73 0.61 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 60.4 196 1.81 0.442 0.8 1 11.3 0.096 0.0532 0.078 9.0 0.45 2.05 1.05 0.010 0.199 57.2 8.38 166 1.74 151 1 .8 6 151 1.6 4 163 1 .86 153 1 .80 0.275 0.153 0.223 0.284 0.232 0.67 0.75 0.59 0.72 0.74 8.7 0.40 8.8 0.39 8.6 0.37 9.0 0.36 8.4 0,32 3.72 2.83 3.88 5.47 4.36 44.0 48.4 32.3 31.2 45.5 157 1.78 0.233 0 .6 9 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, the results o f 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 Peyefs Patch); Caecum; Colon; Rectum; Spinal Cord; Sciatic Nerve; Brain; Femur/joint Pathologist D.JJLewis : 67 : Company Sanitized. Does not contain TSCA CBI APPENDIX 5 (Pathology - continued) DPT 443/984760 Compound: Dosage Level: Rat No/Sex: Control 2M (Terminal) CLINICAL FINDINGS No signs o f 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: DJ.Lewis : 68 : Company Sanitized. Does not contain TSCA CBI % APPENDIX 5 (Pathology - continued) DPT 443/984760 Compound: Dosage Level: Rat No/Sex: Control 3M (Terminal) CLINICAL FINDINGS No signs o f ill health or behavioural change were noted. MACROSCOPIC FINDINGS No abnormalities detected M ICROSCOPIC FINDINGS The following observations were noted: Lungs(including 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 Peyefs 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 - continaed) DPT 443/984760 Compound: Dosage Level: Rat No/Sex: Control 4M (Terminal) CLINICAL FINDINGS No signs o f ill health or behavioural change were noted. MACROSCOPIC FINDINGS Liver Median cleft, pale subcapsular area: 1mm All the other organs and tissues appeared normal. MICROSCOPIC FINDINGS The following tissues were considered normal: Trachea; Lungs(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 PeyePs Patch); Caecum; Colon; Rectum; Spinal Cord; Sciatic Nerve; Brain; Femur/joint Pathologist: DJ.Lewis I : 70 : Company Sanitized. Does not contain TSCA CBI APPENDIX 5 (Pathology - contained) DPT 443/984760 Compound: Dosage Level: Rat No/Sex: Control 5M (Terminal) CLINICAL FINDINGS No signs o f ill health or behavioural change were noted. MACROSCOPIC FINDINGS Liver Median cleft, pale subcapsular area: 2mm 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: (M inim al) 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; Parathyroid^; Adrenals; Stomach; Duodenum; Jejunum; Ileum(including Peyefs 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 Compound: Dosage Level: Rat No/Sex: 15 mg/kg/day 6M (Terminal) . CLINICAL FINDINGS No signs o f 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 fibrosis and tubular collapse with basophilia: (Minimal) The following tissues were considered normal: L iv er: (W.N.L.); Femur/joint Pathologist: D.J.Lewis : 72 : Company Sanitized. Does not contain TSCA CBI APPENDIX 5 (Pathology - continued) DPT 443/984760 Compound: Dosage Level: Rat No/Sex: 15 mg/kg/day 7M (Terminal) CLINICAL FINDINGS No signs o f 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: L iver: (W.N.L.); Femur/joint Pathologist: D.J.Lewis : 73 : Company Sanitized. Does not contain TSCA CBI % APPENDIX 5 (Pathology - continued) DPT 443/984760 Compound: Dosage Level: Rat No/Sex: 15 mg/kg/day 8M (Terminal) CLINICAL FINDINGS No signs o f 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: L iver: (W.N.L.); Femur/joint Pathologist DJ-Lewis : 74 : Company Sanitized. Does not contain TSCA CBI APPENDIX 5 (Pathology - continued) DPT 443/984760 Compound: Dosage Level: Rat No/Sex: 15 mg/kg/day 9M (Terminal) CLINICAL FINDINGS No signs o f ill health, behavioural change or reaction to treatment were noted. MACROSCOPIC FINDINGS No abnormalities detected M ICROSCOPIC 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 Compound: Dosage Level: RatNo/Sex: 15 mg/kg/day 10M (Terminal) CLINICAL FINDINGS No signs o f 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 Compound: Dosage Level: RatNo/Sex: SO mg/kg/day 11M (Terminal) . CLINICAL FINDINGS No signs o f ill health, behavioural change or reaction to treatment were noted. MACROSCOPIC FINDINGS No abnormalities detected MICROSCOPIC FINDINGS Tne following observations were noted: Kidneys Basophilia and hyperplasia of tubules and collecting ducts: (Minimal, Focal) Medullary tubular dilatation: (Minimal) Papillary tubular 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) Compound: Dosage Level: Rat No/Sex: 50 mg/kg/day 12M (Terminal) 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: DJ.Lewis DPT 443/984760 : 78 : Company Sanitized. Does not contain TSCA CBI % APPENDIX 5 (Pathology - continued) DPT 443/984760 Compound: Dosage Level: RatNo/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: Liven Kidneys; Femur/joint Pathologist D JLew is : 79 : Company Sanitized. Does not contain TSCA CBI APPENDIX 5 (Pathology - continued) DPT 443/984760 Compound: Dosage Level: Rat No/Sex: 50 mg/kg/day 14M (Terminal) CLINICAL FINDINGS No signs o f ill health, behavioural change or reaction to treatment were noted. ' MACROSCOPIC FINDINGS Incisors Lower pale All die other organs and tissues appeared normal. MICROSCOPIC FINDINGS The following observations were noted: Kidneys Basophilia and hyperplasia o f 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 Compound: Dosage Level: Rat No/Sex: 50 mg/kg/day 15M (Terminal) CLINICAL FINDINGS No signs o f ill health, behavioural change or reaction to treatment were noted. M ACROSCOPIC FINDINGS No abnormalities detected M ICROSCOPIC FINDINGS The following observations were noted: Kidneys Basophilia and hyperplasia o f 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 Compound: Dosage Level: Rat No/Sex: 150 mg/kg/day 16M (Terminal) 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 die other organs and tissues appeared normal. M ICROSCOPIC 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; IIeum(including PeyePs 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 Compound: Dosage Level Rat No/Sex: 150 mg/kg/day 17M (Terminal) CLINICAL FINDINGS No signs o f 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) Hepaiocyte 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 F em ur/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 Peyefs 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) DPT 443/984760 Compound: Dosage Level: Rat No/Sex: 150 mg/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) : 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 o f 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 Luminal 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 Peyer'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) Compound: Dosage Level: RaNo/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 lg 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 Company Sanitized. Does not contain TSCA CBI APPENDIX 5 (Pathology - continued) DPT 443/984760 Rat No/Sex: 19M - continued M ICROSCOPIC FINDINGS - continued Kidneys Interstitial inflammation in cortex,medulla and papilla: (Slight) Basophilia and hyperplasia o f 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 Peyefs Patch); Caecum; Colon; Rectum; Spinal Cord; Sciatic Nerve; Brain; Femur/joint Pathologist: D.J.Lewis : 89 : Company Sanitized. Does not contain TSCA CBI m APPENDIX 5 (Pathology - continued) DPT 443/984760 Compound: Dosage Level: Rat No/Sex: 150 mg/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: (Minimal) Enlarged: 5.125g Swollen Pale cortical foci: (A few) 1mm 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) : 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) F em ur/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; 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 Compound: Dosage Level: RatNo/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(including Bronchi); Heart; Thymus; Lymph Nodes - Mandibular; Lymph Nodes Mesenteric; Spleen; Liver; 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.J.Lewis : 92 : Company Sanitized. Does not contain TSCA CBI APPENDIX 5 (Pathology - continued) DPT 443/984760 Compound: Dosage Level: Rat No/Sex: Control 22F (Terminal) CLINICAL FINDINGS No signs o f ill health or behavioural change were noted. MACROSCOPIC FINDINGS No abnormalities detected MICROSCOPIC FINDINGS The following observations were noted: Kidneys Cortico-medullary mineralisation: (Slight) Femnr/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 Company Sanitized. Does not contain TSCA CBI APPENDIX 5 (Pathology - continued) DPT 443/984760 Compound: 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. M ICROSCOPIC FINDINGS The following observations were noted: Lungs(including Bronchi) Vascular congestion: (Minimal) Uterus Luminal dilatation: (Moderate) The following tissues were considered normal: Trachea; Heart; Thymus; Lymph Nodes - Mandibular; T.ymph Node? . Mesenteric; Spleen; Liver; 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 : 94 : Company Sanitized. Does not contain TSCA CBI APPENDIX 5 (Pathology - continued) DPT 443/984760 Compound: 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: (M inimal, Focus) Uterus Luminal dilatation: (Marked) Sciatic Nerve Degenerate fibres: (Minimal) The following tissues were considered normal: Trachea; Lungs(inciuding Bronchi); Heart; Thymus; Lymph Nodes - Mandibular; Lymph Nodes Mesenteric; Spleen; Kidneys; Urinary Bladder, Cervix; Vagina; Ovaries; Thyroids; Parathyroids; Adrenals; Stomach; Duodenum; Jejunum; Ileum(including Peyeds 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 Compound: Dosage Level: Rat No/Sex: Control 25F (Terminal) CLINICAL FINDINGS No signs o f 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; ileum(including Peyefs 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 Compound: Dosage Level: R at N o/Sex: 15mg/kg/day 26F (Terminal) CLINICAL FINDINGS No signs o f 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: Uterus Luminal dilatation: (Moderate) The following tissues were considered nonnal: Liver, Kidneys; Femur/joint Pathologist DJ.Lewis : 97 : Company Sanitized. Does not contain TSCA CBI APPENDIX 5 (Pathology - continued) DPT 443/984760 Compound: Dosage Level: Rat No/Sex: 15 mg/kg/day 27F (Terminal) CLINICAL FINDINGS No signs o f 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/5oint Pathologist D.J.Lewis : 98 : Company Sanitized. Does not contain TSCA CBI APPENDIX 5 (Pathology - continued) DPT 443/984760 Compound: Dosage Level: Rat No/Sex: 15mg/kg/day 28F (Terminal) CLINICAL FINDINGS No signs o f ill health, behavioural change or reaction to treatment were noted. MACROSCOPIC FINDINGS No abnormalities detected MICROSCOPIC FINDINGS The following observations were noted: Kidneys Cortico-medullary mineralisation: (Minimal) Cortical basophilic tubules: (Minimal) . The following tissues were considered normal: Liver; Femur/joint Pathologist DJ.Lewis : 99 : Company Sanitized. Does not contain TSCA CBI APPENDIX 5 (Pathology - continued) DPT 443/984760 Compound: Dosage Level: Rat No/Sex: 15 mg/kg/day 29F (Terminal) CLINICAL FINDINGS No signs o f 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 Cortico-medullary mineralisation: (Minimal) The following tissues were considered normal: Liver; Femur/joint Pathologist: DJLLewis 100 : Company Sanitized. Does not contain TSCA CBI APPENDIX 5 (Pathology - continued) DPT 443/984760 Compound: Dosage Level: Rat No/Sex: 15 mg/kg/day 3OF (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 D.J.Lewis : 101 : Company Sanitized. Does not contain TSCA CBI APPENDIX 5 (Pathology - continued) DPT 443/984760 Compound: Dosage Level: Rat No/Sex: 50 mg/kg/day 103 IF (Terminal) CLINICAL FINDINGS No signs o f 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 o f tubules and collecting ducts: (Minimal) Cortical tubular dilatation: (Minimal) Cortico-medullary mineralisation: (Moderate) Uterus Luminal dilatation: (Moderate) The following tissues were considered normal: Liver; Femur/joint Pathologist D.JXewis 102 : Company Sanitized. Does not contain TSCA CBI APPENDIX 5 (Pathology - continued) DPT 443/984760 Compound: Dosage Level: Rat No/Sex: 50 mg/kg/day 32F (Terminal) CLINICAL FINDINGS No signs o f 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 o f tubules and collecting ducts: (Slight) Cortical tubular dilatation: (Minimal) Urothelial hyperplasia: (Slight) Medullary tubular dilatation: (Minimal) Cortico-medullary mineralisation: (Moderate) Papillary collecting duct hyperplasia Interstitial inflammation in papilla: (Minimal) Uterus Luminal dilatation: (Marked) The following tissues were considered normal: Liven Femur/joint Pathologist: D.J.Lewis 103 : Company Sanitized. Does not contain TSCA CBI APPENDIX 5 (Pathology - continued) DPT 443/984760 Compound: 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 o f 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: DJ.Lew is : 104 : Company Sanitized. Does not contain TSCA CBI APPENDIX S (Pathology - continued) DPT 443/984760 Compound: Dosage Level: Rat No/Sex: 50 mg/kg/day 34F (Terminal) CLINICAL FINDINGS No signs o f 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 o f tubules and collecting ducts: (Minimal) Cortical tubular dilatation: (Minimal) Papillary tubular dilatation: (Minimal) Cortico-medullary mineralisation: (Minimal) Interstitial inflammation in papilla: (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 Compound: Dosage Level: Rat No/Sex: 50 mg/kg/day 35F (Terminal) CLINICAL FINDINGS No signs o f 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) Compound: Dosage Level: R at N o/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 normal. 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 inflam m ation in cortex,medulla and papilla: (Minimal) Basophilia and hyperplasia o f 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 Peyefs Patch); Caecum; Colon; Rectum; Spinal Cord; Sciatic Nerve; Brain; Femur/joint Pathologist: DJ.Lewis 108 : Company Sanitized. Does not contain TSCA CBI # APPENDIX 5 (Pathology - continued) DPT 443/984760 Compound: Dosage Level: RatNo/Sex: 150 mg/kg/day 37F (Terminal) CLINICAL FINDINGS No signs o f 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 - generalised; (Minimal) Hepatocyte fine vacuolation - generalised: (Minimal) : 109 : Company Sanitized. Does not contain TSCA CBI APPENDIX 5 (Pathology - continued) DPT 443/984760 Rat No/Sex: 37F - continued MICROSCOPIC FINDINGS - continued Kidneys Basophilia and hyperplasia o f tubules and collecting ducts: (Moderate) Cortical tubular dilatation: (Slight) Urothelial hyperplasia: (Moderate) Medullary tubular dilatation: (Moderate) Papillary tubular dilatation: (Slight) F em ur/joint Marrow - prominent adipocytes The following tissues were considered normal: Trachea; Lungs(including 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: D.J.Lewis 110 : Company Sanitized. Does not contain TSCA CBI APPENDIX 5 (Pathology - continued) DPT 443/984760 Compound: 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 o f hair loss was noted. MACROSCOPIC FINDINGS Skin Alopecia Left cervical region: (Minimal) Right scapular region: (Minimal) Lungs(inclnding 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) RatNo/Sex: 38F - continued MICROSCOPIC FINDINGS The following observations were noted: Lungs(including 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 Fem ur/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: 38F - 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 PeyePs 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 Compound: Dosage Level: Rat No/Sex: 150 mg/kg/day 39F (Terminal) CLINICAL FINDINGS No signs o f ill health, behavioural change or reaction to treatment were noted. MACROSCOPIC FINDINGS Lungs(including Bronchi) Congested: (Patchy) 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 the 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) L iver 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) Papillaiy 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 O varies Sparse/few corpora lutea , F em ur/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; IIeum(including Peyeris 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 DJ.Lewis DPT 443/984760 116 : Company Sanitized. Does not contain TSCA CBI APPENDIX 5 (Pathology - continued) DPT 443/984760 Compound: Dosage Level: Rat NO/Sex: 150 mg/kg/day 40F (Terminal) CLINICAL FINDINGS No signs o f 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: 4359g Swollen All the other organs and tissues appeared normal. M ICROSCOPIC FINDINGS The following observations were noted: Lnngs(including Bronchi) Vascular congestion: (Minimal) Liver Hepatocyte hypertrophy - generalised: (Minimal) Hepatocyte fine vacuolatioh - generalised: (Minimal) i 117 Company Sanitized. Does not contain TSCA CBI APPENDIX 5 (Pathology - continued) DPT 443/984760 Rat No/Sex: 40F - continued MICROSCOPIC FINDINGS - continued Kidneys Basophilia and hyperplasia of tubules and collecting ducts: (Moderate) Cortical tubular dilatation: (Moderate) Papillary necrosis: (M inimal, Unilateral) Urothelial hyperplasia: (Moderate) Medullary tubular dilatation: (Moderate) Papillary tubular dilatation: (Slight) Inflammatory casts in tubules and collecting ducts 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 : A nthor E. W. Hughes Company Sanitized. Does not contain TSCA CBI DPT 443/984760 CONTENTS Page EXPERIMENTAL PROCEDURE............................................................................................ 121 RESULTS................................................................................................................................... 124 TABLES - group data 1. Summary of functional observational battery................................................................. 2. Activity counts................................................................................................................. 3. Rearing counts.................................................................................................................. 4. Grip strength forelimb...................................................................................................... 5. Grip strength hindlimb..................................................................................................... 6. Landing footsplay............................................................................................................ 7. Rectal temperature..................................... -.................................................................... 8. Coulboum locomotor activity.......................................................................................... 125 130 131 132 133 134 133 136 APPENDICES - individual data 1. Functional observational battery --pre-dose, Week 1 ,2 ,3 ,4 .......................................... 2. Coulboum locomotor activity - pre-dose, Week 4 ......................................................... 137 180 : 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 tbe 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 bill 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 o f 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 DPT 443/984760 Manipulations*: 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 o f any possible effects o f 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 o f activity are recorded: the time spent in no movement, locomotor and non-locomotor activity. The number of occurrences (events) of each categoty 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 datr 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 0 DPT 443/984760 The reporting o f 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 o f occurrence for each group. Although during recording, some responses were classified in terms o f the degree or type o f response (ie startle: no reaction, an ear twitch, a flinch, etc.), for the purposes o f 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 o f 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 die 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. G rip strength (Tables 4,5; Appendix 1) During Week 4, there were no statistically significant differences in fbrelimb or hindlimb grip strength. Landing footsplay (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 w i t i ^ P H H H s f o r 28 that were considered indicative o f neurotoxicity. was not assoc'ated with ^ behavioural changes : 124 : Company Sanitized. Does not contain TSCA CBI DPT 443/984760 TABLE 1 Summary o f functional observational battery Pre-dose G roup No. o f anim als O B S E R V A T IO N S : REMOVAL FROM CAGE rem oving, easy handling, easy salivation vocalising IN THE ARENA tremors groom ing arousal, alert defecation urine G A IT walking on toes unable to assess M A N IPU L A T IO N S touch, a reaction startle (present) righting, immediately tail pinch, a reaction pupil reflex M ales 12 3 555 4 5 555 5 555 4 100 0 000 0 000 000 555 000 1 10 0 0 5 0 1 12 1 2 10 0 0 555 5 555 5 555 5 555 5 555 5 N um bers reflect the num ber o f animals showing the response F em ales 12 34 5555 5354 3355 10 00 00 11 0 00 0 0000 5555 0000 000 1 132 2 0 10 0 5 555 5555 5555 5555 5555 125 : Company Sanitized. Does not contain TSCA CBI DPT 443/984760 TABLE 1 (Summary o f functional observational battery - continued) W eekl M ales G roup No. o f animals 12 3 4 5555 O B SE R V A T IO N S : REMOVAL FROM CAGE removing, easy 5555 handling, easy 54 55 salivation 0000 vocalising ' 0 0 0 0 IN THE ARENA tremors grooming arousal, alert defecation urine 0000 00 10 4 5 54 0 10 1 0 1 10 G A IT w alking on toes hunched posture 33 32 2000 unable to assess 000 1 N um bers reflect the num ber o f anim als show ing the response F em ales 12 34 5 5 55 54 54 5455 12 2 0 122 1 0 000 0 000 5 54 5 0000 0000 5 553 1 13 1 0001 : 126 : Company Sanitized. Does not contain TSCA CBI DPT 443/984760 TABLEI (Summary o f functional observational battery - continued) Week 2 G roup No. o f anim als O B S E R V A T IO N S : REMOVAL FROM CAGE rem oving, easy handling, easy salivation vocalising IN THE ARENA trem ors groom ing arousal, alert defecation urine G A IT walking on toes hunched posture unable to assess M ales 12 3 555 4 5 5555 5555 5555 0000 000 0 00 11 44 5 5 0000 00 10 4354 000 1 1 10 0 N um bers reflect the num ber o f anim als showing the response Females 1234 5 55 5 5 555 4 34 5 5 555 0 10 1 0 000 0 000 5455 0 000 0 000 5 555 0 02 1 0 000 Company Sanitized. Does not contain TSCA CBI DPT 443/984760 TABLEI (Summary o f functional observational battery --continued) Week 3 Group . No. o f anim als O B S E R V A T IO N S : REMOVAL FROM CAGE removing, easy handling, easy salivation vocalising IN THE ARENA trem ors groom ing arousal, alert defecation urine GAIT w alking on toes hunched M ales 12 3 555 4 5 555 5 555 5 12 10 0000 0000 0000 555 5 0000 1 10 0 3 14 3 0 00 0 N um bers reflect the num ber o f anim als showing the response Fem ales 12 34 5555 5545 5 4 54 12 00 0 1 10 0000 0000 5 5 55 0 000 0 0 10 5 555 1 100 : 128 : Company Sanitized. Does not contain TSCA CBI DPT 443/984760 TABLE 1 (Summary o f functional observational battery - continued) W eek 4 G roup N o. Of animals O B SE R V A T IO N S : REMOVAL FROM CAGE rem oving, easy handling, easy salivation vocalising IN THE A RENA tremors groom ing arousal, alert defecation urine G A IT w alking on toes hunched M ales 12 3 4 5555 5555 5534 2 110 110 0 0000 0000 5 55 5 0000 1 110 2 0. 2 3 10 10 M ANIPULATIONS approach, a reaction touch, a reaction startle (present) righting, im m ediately tail pinch, a reaction pupil reflex 5555 5254 555 5 5555 5555 5555 N um bers reflect the num ber o f animals show ing the response F em ales 12 3 4 5555 4 54 5 5455 2 110 10 0 1 0000 0000 554 5 0000 0 0 10 5555 100 1 5555 5544 5555 5555 5555 5555 : 129 : Company Sanitized. Does not contain TSCA CBI TABLE 2 Activity counts - group mean values Pre-dose G roup /d o sag e (m g/kg/day) 1 -0 2-1 5 3-5 0 4-150 M ean activity counts M ales F em ales 8 13 9 11 12 11 89 W eek 1 G roup/dosage (m g/kg/day) 1 -0 2-1 5 3 -5 0 4-150 M ean activity counts M ales F em ales 7 12 9 10 9 14 99 W eek 2 G ro u p /d o sag e (m g/kg/day) 1 -0 2 -1 5 3 -5 0 4-1 5 0 M ean activity counts M ales Females 9 14 10 18 14 18 11 14 W eek 3 G ro u p /d o sag e (m g/kg/day) 1 -0 2-1 5 3 -5 0 4-150 M ean activity counts M ales F em ales 14 15 14 17 14 19 13 17 W eek 4 G roup/dosage (m g/kg/day) 1 -0 2-1 5 3 -5 0 4 -1 5 0 M ean activity counts M ales F em ales 11 15 10 16 12 16 7 14 N o statistical significancep > 0.05 DPT 443/984760 : 130 : Company Sanitized. Does not contain TSCA CBI TABLE 3 Rearing counts - group mean values Pre-dose G roup/dosage (mg/kg/day) 1 -0 2 - 15 3 - 50 4 - 150 M ean rearing counts M ales F em ales 26 46 66 35 W eek 1 G roup/dosage (m g/kg/day) 1 -0 2 - 15 3 - 50 4 - 150 M ean rearing counts M ales F em ales 82 74 94 64 Week 2 G ro u p /d o sag e (m g/kg/day) 1 -0 2 - 15 3 - 50 4 - 150 M ean rearing counts M ales F em ales 37 58 69 56 W eek 3 G roup/dosage (m g/kg/day) 1 -0 2 -1 5 3 -5 0 4-150 M ean rearing counts M ales F em ales 49 68 78 58 W eek 4 G roup/dosage (m g/kg/day) 1 -0 2 -1 5 3 -5 0 4-150 M ean rearing counts M ales F em ales 68 69 78 37 N o statistical significancep > 0.05 DPT 443/984760 : 131 : Company Sanitized. Does not contain TSCA CBI TABLE 4 Forelimb grip strength - group mean values Pre-dose G ro u p /d o sag e (m g/kg/day) 1 -0 2-1 5 3 -5 0 4 -1 5 0 M ean forelim b grip strength (kg) M ales F em ales 0.25 0.24 0.24 0.23 0.27 0.22 0.26 0.29 Week 4 G roup/dosage (m g/kg/day) 1 -0 2 -1 5 3 -50 4 - 150 M ean forelim b grip strength (kg) M ales F em ales 0.85 0.83 0.72 0.71 0.83 0.70 0.65 0.69 No statistical significance p > 0 .0 5 DPT 443/984760 : 132 : Company Sanitized. Does not contain TSCA CBI DPT 443/984760 m TABLE 5 Hindlimb grip strength - group mean values Pre-dose G roup/dosage (m g/kg/day) 1 -0 2 -1 5 3 -5 0 4 - 150 M ean hindlimb grip strength (kg) M ales Fem ales 0.25 0.26 0.26 0.27 0.27 0.27 0.31 0.34 Week 4 G roup/dosage M ean hindlimb grip strength (kg) (m g/kg/day) M ales Fem ales 1 - 0 ' 0.90 0.88 2 -1 5 0.95 0.88 3 -5 0 0.89 0.84 4 - 150 0.77 0.75 N o statstica! significance p > 0 .0 5 : 133 : Company Sanitized. Does not contain TSCA CBI TABLE Landing footsplay - group mean values Pre-dose G roup/dosage (m g/kg/day) 1 -0 2 -1 5 3 -5 0 4 -150 M ean splay values (cm) M ales F em ales 7.6 6.5 7.6 5.4 7.2 6.8 7.8 7.7 Week 4 G roup/dosage (m g/kg/day) 1 -0 2 -1 5 3 -5 0 4 -150 M ean splay values (cm) M ales Fem ales 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 Company Sanitized. Does not contain TSCA CBI TABLE 7 Rectal temperature - group m ean values Pre-dose G roup/dosage (m g/kg/day) 1 -0 2 -1 5 3 -5 0 4 - 150 M ean tem perature (C) M ales 37.8 37.7 37.5 37.7 F em ales 38.3 38.0 38.0 38.2 W eek 4 G ro u p /d o sag e (m g/kg/day) 1 -0 2 -1 5 3 -5 0 4 - 150 M ean tem perature (C) M ales F em ales 38.6 38.7 38.2 38.4 38.8 38.7 37.9 . 38.4 N o statistical significance p > 0 .0 5 DPT 443/984760 : 135 : Company Sanitized. Does not contain TSCA CBI TABLE 8 Locomotor activity - group mean values Pre-dose G roup/dosage (m g/kg/day) 1 -0 2 -1 5 3 -5 0 4-150 M ean large movements (in secs) during 1 hour observation period M ales F em ales 545 274 489 342 369 498 423 465 W eek 4 G roup/dosage (m g/kg/day) 1 -0 2 -1 5 3 -5 0 4-150 M ean large movements (in secs) during 1 hour observation period M ales F em ales 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 the 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 O 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/tums away Walks past probe Other reaction - see additional comments ; Touch 1 2 3 4 5 6 O No reaction Turns Walks away Freezes Turns to opposite side Walks backwards Other reaction - see additional comments Startle .1 2 3 4 5 0 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, piloerection 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 reflex observed both eyes L/R reflex observed in left/right eye only N 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 1 2 3 4 5 OBSERVATIONS IN THE HAND Removing 2 Handling Salivation 2 N degree Vocalising N degree IN THE ARENA Activity count Arousal Rearing count 12 4 5 Bolus count Urine present 0 S Gait MANIPULATIONS Approach Touch Startle 3 5 3 Righting reflex 1 Tail pinch 3 turns vocalises Y degree 1 Pupil reflex B Temperature(C) 37.7 B o d y w e i g h t (g) 1 0 0 GRIP STRENGTH (kg)# Forelimb 0.23 Hindlimb F O O T S P L A Y (cm) # 0.21 6.5 2 2 N N 7 4 0 0 S 6 3 3 1 3 Y 2 B 38.1 100 0.28 0.29 7.1 2 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 22 22 NN NN 12 3 44 32 00 NN 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 L o w e r t e e t h pale' In the arena: limited walking loss right flank Company Sanitized. Does not contain TSCA CBI DPT 443/984760 APPENDIX 1 (Functional observational battery - continued) Pre-dose ' Group 2 male, 15 m g / k g / d a y Animal 6 78 9 10 OBSERVATIONS IN THE HAND Removing 2 Handling 2 Salivation N degree Vocalising N degree IN THE ARENA Activity count 10 Arousal 4 Rearing count 4 Bolus count 0 Urine present N Gait MANIPULATIONS Approach 3 Touch 3 Startle 3 Righting reflex 1 Tail pinch 3 turns vocalises Y degree 2 Pupil reflex B Temperature(C) 37.8 B o d y w e i g h t (g) 94 GRIP STRENGTH (kg)# Forelimb 0.25 Hindlimb 0.26 F O O T S P L A Y (cm) # 7.2 2 2 Y 1 N 11 4 9 0 N T1 3 2 3 1 3 Y 2 B 37.9 98 0.18 0.18 6.7 2 2 N N 5 4 0 0 N 3 3 3 1 3 Y 2 B 37.7 94 0.31 0.31 8.9 2 2 N N 9 4 3 0 N T1 3 3 3 1 3 Y 2 B 37.5 104 0.21 0.33 9.0 2 2 N N 8 4 3 0 N 3 3 3 1 3 Y 2 B 37.6 92 0.27 0.24 6.2 # Values represent the mean of two trials Additional comments Animal number 6 7 8 10- Slight brown nasal staining, lower teeth pale Lower teeth pale Lower teeth pale In the arena: stretching occasionally Lower teeth pale . 141 Company Sanitized. Does not contain TSCA CBI DPT 443/984760 APPENDIX 1 (Functional observational battery - continued) Pre-dose Group 3 male, 50 m g / k g / d a y A n i m a l 1 1 1 2 13 14 15 OBSERVATIONS IN THE HAND Removing 2 Handling 2 Salivation N degree Vocalising N degree IN THE ARENA Activity count 12 Arousal 4 Rearing count 5 Bolus count 0 Urine present M Gait MANIPULATIONS Approach 3 Touch 3 Startle 3 Righting reflex 1 Tail pinch 3 turns vocalises Y degree 2 Pupil reflex B Temperature(C) 37.5 B o d y w e i g h t {g) 93 GRIP STRENGTH (kg)# Forelimb 0.29 Hindlimb 0.35 F O O T S P L A Y (cm) # 8.3 2 2 N N 10 4 8 0 N 3 3 2 1 6 Y 2 B 37.5 95 0.24 0.25 4.5 2 2 N N 12 4 5 0 N 3 5 4 1 3 Y 2 B 37.4 98 0.23 0.26 6.6 2 2 N N 14 4 6 0 N T1 3 3 3 1 3 Y 2B 38.1 90 0.32 0.23 9.8 2 2 N N 10 4 4 0 N 5 3 3 1 3 Y 2 B 37.1 95 0.25 0.27 7.1 # Values represent the mean of two trials Additional comments 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 Company Sanitized. Does not contain TSCA CBI DPT 443/984760 APPENDIX 1 (Functional observational battery --continued) Pre-dose Group 4 male, 150 mg/kg/day A n i m a l 16 17 18 19 2 0 OBSERVATIONS IN THE HAND Removing 2 Handling 2 Salivation N degree Vocalising - N degree IN THE ARENA Activity count 6 Arousal 4 Rearing count 1 Bolus count 0 Urine present N Gait MANIPULATIONS Approach 3 Touch 3 Startle 2 Righting reflex 1 Tail pinch 6 turns vocalises Y degree 3 Pupil reflex B Temperature(C) 37.6 B o d y w e i g h t (g) 103 GRIP STRENGTH (kg)# Forelimb 0.30 Hindiimb 0.41 F O O T S P L A Y (cm) # 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 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 # Values represent the mean of two trials Additional comments Animal number 16 18 19 20 Lower teeth pale Slight hairloss rump Lower teeth pale Lower teethpale, patchy tail missing hair loss rump, tip . of 143 : Company Sanitized. Does not contain TSCA CBI DPT 443/984760 APPENDIX 1 (Functional observational battery --continued) Pre-dose Group 1 female, Control A n i m a l 2 1 2 2 23 24 25 OBSERVATIONS IN THE HAND Removing 2 Handling 3 Salivation N degree Vocalising N degree IN THE ARENA Activity count 21 Arousal 4 Rearing count 5 Bolus count 0 Urine present N Gait T1 MANIPULATIONS Approach 3 Touch 3 Startle 3 Righting reflex 1 Tail pinch 3 turns vocalises Y degree 2 Pupil reflex B Temperature(C) 38.5 B o d y w e i g h t (g) 97 GRIP S T RENGTH (kg)# Fore limb 0.32 Hindlimb 0.31 F O O T S P L A Y (cm) # 9.8 2 3 Y 1 N 16 4 4 0 N 3 0 3 1 3 Y 1 B 39.1 100 0.31 0.28 6.2 22 22 NN NN 9 10 44 67 00 NN 36 33 32 11 36 YY 22 BB 38.4 37.7 1 0 2 103 0.21 0.15 4.8 0.20 0.31 6.6 2 2 N N 11 4 9 0 M 3 3 3 1 3 Y 2 B 37.6 102 0.20 0.28 5.3 # Values represent the mean of two trials Additional comments 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 tdeth pale : 144 : Company Sanitized. Does not contain TSCA CBI DPT 443/984760 APPENDIX 1 (Functional observational battery --continued) Pre-dose Gro u p 2 female, 15 m g / k g / d a y A n i m a l 26 27 28 29 30 OBSERVATIONS IN THE HAND Removing 3 Handling 2 Salivation' N degree Vocalising N degree IN THE ARENA Activity count 14 Arousal 4 Rearing count 4 Bolus count 0 Urine present N Gait T2 MANIPULATIONS Approach 3 Touch 2 Startle 3 Righting reflex 1 Tail pinch 6 turns vocalises Y degree 2 Pupil reflex B T e m p e r a t u r e (C) 3 8 . 2 B o d y w e i g h t (g) 104 GRIP STRENGTH (kg)# Forelimb 0.25 Hindiimb 0.29 F O O T S P L A Y (cm) # 6.1 2 2 N N 4 4 4 0 N U 5 3 3 1 3 Y 2 B 38.3 93 0.14 0.29 6.0 3 3 N N 9 4 5 0 N T1 3 3 3 1 2 2 Y 2 B 38.0 101 0.27 0.30 5.0 22 32 NN NN 20 4 9 0 S. T1 9 4 8 0 N 33 33 22 11 33 Y 1 B 37.2 98 Y 1 B 38.1 104 0.28 0.26 5.1 0.23 0.21 4.8 # Values represent the mean of two trials Additional comments 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 t h e A r e n a : s i t t i n g alo".g e d g e of arena, w a l k i n g along edge of arena During grip strength: head shake 145 : Company Sanitized. Does not contain TSCA CBI DPT 443/984760 APPENDIX 1 (Functional observational battery --continued) Pre-dose Group 3 female, 50 mg/kg/day A n i m a l 31 32 33 34 35 OBSERVATIONS IN THE HAND Removing 2 Handling 2 Salivation N degree Vocalising N degree IN THE ARENA Activity count 10 Arousal 4 Rearing count 8 Bolus count 0 Urine present N Gait MANIPULATIONS Approach 3 Touch 0 Startle 2 Righting reflex 1 Tail pinch 3 turns vocalises Y degree 2 Pupil reflex B Temperature(C) 38.2 B o d y w e i g h t (g) 1 0 2 GRIP STR E N G T H (kg)# Forelimb 0.26 Hindlirab 0.32 F O O T S P L A Y (cm) # 7.3 2 2 N N 11 4 4 0 N T1 3 3 2 1 3 Y 2 B 38.3 98 0.22 0.27 6.1 2 2 N N 14 4 6 0 N 3 3 3 1 2 1 Y 2 B 37.8 94 0.18 0.22 5.3 2 2 N Y 2 12 4 7 0 N T1 3 3 3 1 3 Y 2 B 37.5 107 0.22 0.22 6.0 2 2 N N 8 4 3 0 N 3 2 3 1 3 1 Y 2 B 38.1 101 0.21 0.30 9.3 # Values represent the mean of two trials Additional comments . Animal number 31 32 33 3435 Slight hair loss neck and rump, lower teeth pale Touch response: rears Lower teeth pale Slight brown nasal staining. lower teeth pale Slight brown nasal staining, lower teeth pale Lower 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 mg/kg/day Animal 36 37 38 39 40 OBSERVATIONS IN THE HAND Removing Handling Salivation degree Vocalising degree IN THE ARENA Activity count 8 Arousal Rearing count Bolus count 4 6 0 Urine present Gait MANIPULATIONS Approach Touch Startle Righting reflex Tail pinch turns vocalises degree Pupil reflex B Temperature(C) 37.9 B o d y w e i g h t (g) 102 GR I P STRENGTH (kg)# Forelimb Hindlimb F O O T S P L A Y (cm) # 0.25 0.27 7.8 2 2 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 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 12 4 6 0 N 5 2 3 1 6 Y 2 B 38.1 96 0.27 0.32 7.4 # Values represent the mean of two trials Additional comments Animal number 36 Lower teeth pale 38 Touch response: rears 39 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 G r oup 1 male, Cont Animal 1 2 3 4 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 N N N 12 4 4 0 N T1HU1 2 2 N N N 4 5 0 0 N 22 22 NN NN NN 84 44 20 00 NN T1 T1HU1 ZO U ^^JZ Z Z M K> Additional comments _ Animal number 1 Right lower tooth pale 2 Lower teeth pale 148 : Company Sanitized. Does not contain TSCA CBI DPT 443/984760 APPENDIX 1 (Functional observational battery --continued) Week 1 Group 2 male, 15 m g / k g / d a y Animal 6 7 8 9 10 OBSERVATIONS IN THE HAND Removing Handling Salivation 2 2 22 2 2 2 22 3 N N NN N degree Vocalising NN NNN degree IN THE ARENA Grooming NNNN N Activity count 8 8 96 12 Arousal 4 4 44 4 . Rearing count 3 3 2 6 8 Bolus count 2 0 00 0 Urine present N N N S N Gait T1 T1 T1 Additional comments Animal - number 6 7 9 io 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 G r o u p 3 m a l e 1/ 50 m g / k g / d a y A n i m a l 1 1 1 2 13 14 15 OBSERVATIONS IN THE HAND Removing Handling Salivation 22222 222 2 2 N NNN N degree Vocalising NNNNN degree IN THE ARENA Grooming NNYNN Activity count 9 7 4 13 1 0 Arousal 4 44 44 Rearing count 5 4 1 6 3 Bolus count .0 0 0 0 0 Urine present N N S N N Gait T1 T1 T1 Additional comments Animal number 11 Slight h a i r loss head, lower te e t h 12 Mod e r a t e ha i r loss head, neck 14' Slight brown nasal staining, lower 15 Lower t e e t h pale pale teeth pale : 150 : Company Sanitized. Does not contain TSCA CBI DPT 443/984760 APPENDIX 1 (Functional observational battery - continued) : 1 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 Grooming Activity count Arousal Rearing count Bolus count Urine present Gait 2 2 N N N 1 3 2 2 N U 2 222 2 222 N NNN N NNN N NNN 6 1 0 1 2 14 4 4 4 4 3871 0 000 N NNN T2 T1 Additional comments Animal number 16 17 19 20 In the arena: limited walking Slight brown nasal staining Slight hair loss head, lower teethpale Slight brown nasal staining, slight hair tip of 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 i Group 1 female. Control . Ani m a l 2 1 2 2 23 24 25 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 N N N 10 4 5 0 N T1 222 222 Y N .N 1 NN N NN 11 12 44 95 00 NN T1 T1HU1 N 11 4 9 0 N T1 2 2 N Y 1 N 14 4 11 0 N T1 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 i . Group 4 female, 150 mg/kg/day A n i m a l 36 37 38 39 40 OBSERVATIONS IN THE HAND Removing Handling Salivation 2 2 2 32 22 222 N N NNN degree Vocalising degree NN NYN 2 IN THE ARENA Grooming N N N N N Activity count 8 3 15 8 13 Arousal 44 444 Rearing count 4 1 97 10 Bolus count 00 000 Urine present N N N N N Gait U T1 T1 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) NM 2 Week 2 Group 1 male. Control Animal 1 2 3 4 OBSERVATIONS IN THE HAND Removing Handling Vocalising degree IN THE ARENA Grooming Activity count Arousal Rearing count Bolus count Urine present Gait 2 2 N N 11 4 5 0 N T1 22 22 NN NN 14 6 44 52 00 NN T1 T1 2 2 N N 3 3 2 0 N U Additional comments Animal number 1 Right lower tooth pale 4 In the arena: limited walking 5 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 m g / k g / d a y A n i m a l 26 27 28 29 30 OBSERVATIONS IN THE HAND Removing Handling Salivation degree Vocalising 22 32 2 22 32 2 NN YN Y 11 NY YN N degree 11 IN THE ARENA Grooming NN NNN Activity count Arousal Rearing count 7 4 4 5 4 4 1 0 17 1 2 44 4 6 11 9 Bolus count 00 00 0 Orine present N N N N N 'Gait T1 T1 Tl Tl T1HU1 Additional comments Animal number 26 27 29 30 Slight brown nasal staining Slight brown nasal staining In the arena: stretching occasionally Slight brown nasal staining, lower teeth Slight 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 Gro u p 3 female, 50 mg/kg/day A n i m a l 3 1 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 2 222 2 222 Y NNN 1 N YYN 12 N NNN 1 3 14 16 13 4 4 .5 4 11 7 10 7 0 000 N NNN T1 T2HU+ T1HU1 T2HU1 2 2 Y 1 N N 15 4 9 0 N T1 * Degree not recorded in error Additional comments Animal number 32 33 34 35 Lower teeth pale Lower teeth pale Slight brown nasal staining Lower teeth pale ' : 154 : Company Sanitized. Does not contain TSCA CBI DPT 443/984760 a ppen d ix 1 (Functional observational battery - continued) Wee]c i Group 4 female, 150 mg/kg/day A n i m a l 36 37 38 39 40 OBSERVATIONS IN THE HAND Removing Handling Salivation _ 2 2 2 32 2 2 22 2 N N NNN degree Vocalising degree N N N 1N 2 IN THE ARENA Grooming N NNNN A c t i v i t y count 8 3 15 8 13 Arousal 4 4 444 Rearing count 4 1 97 10 Bolus count 0 0000 Urine present N N N N N Gait U T1 T1 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) zmm ZOW^HZ Meek 2 Group 1 male, Control Animal 1 2 3 4 OBSERVATIONS IN THE HAND . Removing Handling Vocalising 2 22 2 22 2 2 NNN N degree IN THE ARENA Grooming NNN A c t i v i t y c o u n t 1 1 14 6 Arousal 444 Rearing count 5 5 2 N 3 3 2 Bolus count 0 00 0 Urine present N N N N Gait T1 T1 T1 U Additional comments Animal number 1 Right lower too^h pale 4 In t h e a r e n a : ^.^jtiited w a l k i n g 5 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 78 9 10 OBSERVATIONS IN THE HAND Removing 2 222 Handling Vocalising 2 222 NNNN degree IN THE ARENA Grooming N NNN Activity count 6 15 1 0 2 Arousal 4 443 Rearing count 4 10 3 0 Bolus count 0 0 00 Urine present N N N N Gait T1 T1 U 2 2 N N 15 4 8 0 N T1 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 222 22 Handling Vocalising 222 22 NNN NN degree THE ARENA Grooming NNN NY A c t i v i t y c o u n t 19 14 15 1 1 13 Arousal 444 44 Rearing count 8 6 8 55 Bolus count 000 00 Urine present S NN NN Gait T2 T1 T1 T1 T2 Additional comments Animal number 11 13 14 15 Lower teeth pale Moderate brown nasal staining Slight brown nasalstaining, lower Lower teeth pale teeth pale : 158 : Company Sanitized. Does not contain TSCA CBI DPT 443/984760 APPENDIX I (Functional observational battery --continued) Week 2 Group 4 male, 150 mg/kg/day A n i m a l 16 17 18 19 2 0 OBSERVATIONS IN THE HAND Removing 2222 2 Handling 22 22 2 Vocalising NN NN N degree IN THE ARE N A Grooming Activity count N 5 NNN Y 1 1 1 2 1 1 14 Arousal <4 4 4 4 4 Rearing count 2 4 8 2 8 Bolus count 0000 0 Urine present N N N N N Gait T1 T1 T2 T2HU1 Additional comments Animal number 19 20 Slight brown nasal staining Moderate brown nasal staining, lower teeth pale . tip of tail missing, 159 : Company Sanitized. Does not contain TSCA CBI DPT 443/984760 APPENDIX 1 (Functional observational battery - continued) Week 2 . Group 1 female, Control A n i m a l 2 1 2 2 23 24 25 OBSERVATIONS IN THE HAND Removing Handling Vocalising 2 222 2 3222 2 N NN N N degree IN THE ARENA Grooming N NNNN Activity count 14 16 12 11 15 Arousal 4 444 4 Rearing count 7 78 6 9 Bolus count 0 00 0 0 Urine present N NN N N Gait T1 T1 T1 T1 T1 Additional comments Animal number 21 22 24 Lower teeth Lower teeth Lower teeth pale pale pale : 160 : Company Sanitized. Does not contain TSCA CBI DPT 443/984760 APPENDIX 1 (Functional observational battery --continued) Week 2 Group 2 female, 15 mg/k g / d a y A n i m a l 26 27 28 29 30 OBSERVATIONS IN THE HAND Removing Handling Vocalising 2 2 222 2 3 2 32 YN NNN degree 2 IN THE ARENA Grooming N N NNN Activity count 20 15 19 18 16 Arousal 44 544 Rearing count 7 7 8 9 8 Bolus count 00 000 Urine present N N N N N Gait T2 T1 T2 T1 T1 Additional comments Animal number 27 28 29 In the arena: walking along edge with Slight matted fur lower jaw In the arena: walking along edge Lower teeth pale forepaws 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 A n i m a l 31 32 33 34 35 OBSERVATIONS IN THE HAND Removing Handling Vocalising degree IN THE A R E N A Grooming Activity count Arousal Rearing count Bolus count Urine present Gait 22 22 NN NN 18 17 44 88 00 NN T2 T2HU1 22 23 NN NN 2 1 19 44 1 0 13 00 NN T1 T1H01 2 2 N N 16 4 6 0 N 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 b a tte n ' --continued) Week 2 Group 4 female, 150 mg/kg/day A n i m a l 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 2 2 N N 14 4 6 0 N T1 22 22 NN NN 1 2 18 44 58 00 NN T1 T1HU1 2 2 Y 2 N 9 4 4 0 N T1 2 2 N N 16 4 8 0 N T1 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 Group 1 male, Control Animal 1 2 3 4 5 OBSERVATIONS IN THE HAND Removing 2 22 2 Handling Salivation 2 22 2 NYNN degree Vocalising 1 NNNN degree IN THE ARENA Activity count 16 20 11 8 Arousal 4 44 4 Rearing count 6 8 2 3 Bolus count 0 00 Urine present S N N Gait T1 T1 0 N 2 2 N N 15 4 3 0 N T2 : 164 : Company Sanitized. Does not contain TSCA CBI DPT 443/984760 APPENDIX 1 (Functional observational battery --continued) Week 3 Group 2 male, 15 m g / k g / d a y Animal 6 7 8 9 z zww OBSERVATIONS IN THE HAND Removing 22 22 Handling 22 22 Salivation NYNY degree Vocalising 11 NN NN degree IN THE ARENA Activity count 13 16 14 13 Arousal 4444 Rearing count 4 6 3 8 Bolus count 0000 Urine present S N N N Gait T1 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 m g / k g / d a y A n i m a l 1 1 1 2 13 14 15 OBSERVATIONS IN THE HAND Removing Handling Salivation 2 222 2 2222 2 NN YN N degree Vocalising 1 NNNNN degree IN THE ARENA Activity count 14 13 13 15 14 Arousal 44444 Rearing count 7 6 8 8 6 Bolus count 0 0000 Urine present N N N N N Gait T2 T1 T2 T2 Additional comments Animal number 14 Slight b r o w n na s a l staining, low e r tee t h p a l e 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 A n i m a l 16 17 18 19 2 0 OBSERVATIONS IN THE HAND Removing Handling " Salivation 22 22 2 22 2 22 NNN NN degree Vocalising NNN N N degree IN THE ARENA Activity count 8 14 17 1 1 13 Arousal 44 4 4 4 Rearing count 2 5 6 7 5 Bolus count 00 0 00 Urine present N N N N N Gait T1 T1 T2 Additional comments Animal number 20 ,. Slight brown nasal staining, tip small area matted fur urogenital teeth pale of tali region, missing, lower 167 : Company Sanitized. Does not contain TSCA CBI APPENDIX 1 DPT 443/984760 (Functional observational battery - continued) Week 3 Group 1 female, Control , Animal 2 1 2 2 23 24 25 OBSERVATIONS IN THE HAND Removing 22 22 2 Handling 22 22 2 Salivation NN YNN degree 1 Vocalising N N NNN degree IN THE ARENA Activity count 17 15 2 2 9 12 Arousal 44 44 4 Rearing count 7 14 1 1 6 7 Bolus count 0000 0 Urine present N N N N N Gait T1 T1HU1 T2 T1 T1 Additional comments Animal number 21 In the arena: walking along edge of arena 23 Slight brown nasal staining 24 Lower teeth pale Company Sanitized. Does not contain TSCA CBI DPT 443/984760 (Functional observational battery --continued) Week 3 Group 2 female. 15 mg/kg/day A n i m a l 26 . 27 28 29 30 OBSERVATIONS IN THE HAND Removing Handling Salivation degree Vocalising degree 22 2 22 2 2 2 32 NN Y YN 1 1 NN N YN 1 IN THE ARENA Activ i t y count 16 20 14 19 18 Arousal 444 44 Rearing count 9 7 10 8 7 Bolus count 00 0 0 0 Urine present N N N N N Gait T2 T1HU1 T1 T2 T1 # 0 Company Sanitized. Does not contain TSCA CBI DPT 443/984760 APPENDIX 1 (Functional observational battery --continued) Group 3 female, 50 mg/kg/day A n i m a l 31 32 33 34 35 OBSERVATIONS IN THE HAND Removing Handling Salivation ,, 22 1 22 222 N N NN N degree Vocalising N , N Y N IN degree ' 2 IN THE ARENA A c t i v i t y c o u n t 19 24 18 1 2 2 0 Arousal 44 44 4 Rearing count 10 9 8 4 8 Bolus count 00 00 0 Urine present N S N N N Gait T2 T1 T2 T1 T2 Additional comments Animal number 33 Low e r 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 2 2 2 22 2 2 2 32 NN NNN degree Vocalising N N NNN degree IN THE ARENA A c t i v i t y count 19 13 16 16 2 0 Arousal 4 4 444 Rearing count 10 6 8 10 7 Bolus count 0 0 000 Urine present N N N N N Gait T1 T1 T1 T1 T2 Additional comments Animal number 38 M o derate hair loss ne c k 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 Animal 1 2 3 4 5 OBSERVATIONS IN THE HAND Removing Handling 22 22 Salivation degree Y Y- 11 Vocalising NN degree IN THE ARENA Grooming NN Activity count 15 14 Arousal 44 Rearing count 9 7 Bolus count 00 Urine present S N Gait 'r 2 HU1 MANIPULATIONS Approach 33 Touch Startle Righting reflex Tail pinch turns 3 3 1 2 2 3 2 1 3 vocalises degree Y 2 Y 2 Pupil reflex B B Temperature(C) 38.9 38.8 B o d y w e i g h t (g) 3 2 6 353 GRIP STRENGTH (kg)# Forelimb 0.86 0.83 Hindlimb F O O T S P L A Y (cm) # 0.94 0.69 12.9 12.2 2 2 N Y 1 N 7 4 4 0 N 3 3 3 1 3 Y 2 B 38.4 314 0.85 0.97 15.2 2 2 N N N 9 4 4 0 N T1 2 4 3 1 6 Y 3 B 38.5 337 0.91 0.92 11.6 2 2 N N N 12 4 4 0 N 3 3 3 1 3 Y 1 B 38.2 315 0.81 0.99 15.0 # Values represent the mean of two trials Additional comments Animal number 1 Slight brown nasal staining In the arena: walking along edge forepaws 3 Slight brown nasal staining 4 Slight lack of grooming rump and back 5 In the arena: head shake Company Sanitized. Does not contain TSCA CBI DPT 443/984760 APPENDIX 1 (Functional observational battery - continued) Week 4 Group 2 male, 15 mg/kg/day Animal 6 7 8 9 10 OBSERVATIONS IN THE HAND Removing 2 Handling 2 Salivation N degree Vocalising N degree IN THE ARENA Grooming N Activity count 10 Arousal 4 Rearing count 6 Bolus count 0 Urine present M Gait MANIPULATIONS Approach 3 Touch 1 Startle 3 Righting reflex 1 Tail pinch 6 turns vocalises Y degree 2 Pupil reflex B Temperature(C) 38.9 B o d y w e i g h t (g) 309 GRIP STRENGTH (kg)# Forelimb 0.89 Hindlimb 1.08 F O O T S P L A Y (cm) # 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 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 # Values represent the mean of two trials Additional comments Animal number 6 Slight brown nasal staining, lower teeth pale 10 L o w e r t e e t h pale. : 173 : Company Sanitized. Does not contain TSCA CBI DPT 443/984760 . APPENDIX 1 (Functional observational battery --continued) Week 4 Group 3 male, 50 mg/kg/day A n i m a l 1 1 1 2 13 14 15 OBSERVATIONS IN THE HAND Removing 2 Handling 3 Salivation N degree Vocalising N degree IN THE ARENA Grooming N Activity count 12 Arousal 4 Rearing count 12 Bolus count Urine present Gait 0 N 'n h u i MANIPULATIONS Approach 3 Touch Startle 3 4 Righting reflex 1 Tail pinch 3 turns vocalises Y degree 3 Pupil reflex B T e m p e r a t u r e (C) 3 8 . 6 B o d y w e i g h t (g) 288 GRIP STRENGTH (kg)# Forelimb 1.03 Hindiimb F O O T S P L A Y (cm) # 0.98 13.7 2 2 N N N 14 4 8 0 N T1 3 2 3 1 3 Y 2 B 38.2 277 0.83 1.06 7.6 2 2 Y 1 N N 15 4 8 0 N 3 2 2 1 3 Y 2 B 38.4 323 0.77 0.95 12.6 2 3 N N N 12 4 5 0 N 3. 3 2 1 5 Y 2 B 38.8 309 0.89 0.80 13.7 2 2 N N N 8 4 4 0 S 3 3 2 1 3 Y 1 B 38.2 258 0.64 0.68 11.0 # Values represent the mean of two trials Additional comments Animal number 11 Slight l a c k of g r ooming rump 13 Slight b r o w n n a s a l staining 14 S l ight l a c k of g r o o m i n g rump, t i p 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 --continued) Week 4 Group 4 male, 150 mg/kg/day A n i m a l 16 17 18 . 19 2 0 OBSERVATIONS IN THE HAND Removing 2 Handling 3 Salivation N degree Vocalising N degree IN THE ARENA Grooming N Activity count 7 Arousal 4 Rearing count 3 Bolus count 0 Urine present N Gait T2 MANIPULATIONS Approach 3 Touch 1 Startle 3 Righting reflex 1 Tail pinch 5 turns vocalises Y degree 3 Pupil reflex B T e m p e r a t u r e (C) 3 7 . 7 B o d y w e i g h t (g) 174 GRIP STRENGTH (kg)# Forelimb 0.76 Hindiimb 0.77 F O O T S P L A Y (cm) # 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 2 X8 0.62 0.71 12.2 # Values represent the mean of two trials Additional comments Animal number 16 Slight l a c k of gro o m i n g back 18 Patchy h a i r loss r u m p ... 20 Slight b r own staining ears, tip of tail missing 175 : Company Sanitized. Does not contain TSCA CBI DPT 443/984760 APPENDIX 1 (F u n c tio n a l o b se rv a tio n a l b a tte r y --co n tin u ed ) Week 4 Group 1 female, Control A n i m a l 2 1 2 2 23 24 25 OBSERVATIONS IN THE HAND Removing 3 Handling 2 Salivation N degree Vocalising N degree IN THE ARENA Grooming N A c t i v i t y c o u n t 14 Arousal 4 Rearing count 5 Bolus count 0 Urine present N Gait T1 MANIPULATIONS Approach 5 Touch 2 Startle 2 Righting reflex 1 Tail pinch 3 turns vocalises Y degree 2 Pupil reflex B Temperature(C) 38.3 B o d y w e i g h t (g) 215 GRIP STRENGTH (kg)# Forelimb 0.64 Hindlimb 0.75 F O O T S P L A Y (cm) # 14.1 2 2 Y 1 Y 1 N 16 4 10 0 N T1 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 22 22 NN NN NN 1 1 13 44 99 00 NN T1 T2 HU1 53 35 33 11 53 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 A p p r o .ch r e s p o n s e : b i t p r o b e : 176 : Company Sanitized. Does not contain TSCA CBI DPT 443/984760 APPENDIX 1 (Functional observational battery --continued) Week 4 Gro u p 2 female, 15 m g / k g / d a y A n i m a l 26 27 28 29 30 OBSERVATIONS i n 'T H E H A N D Removing 2 Handling 2 Salivation N degree Vocalising N degree IN THE ARENA Grooming N Activity count 2 0 Arousal 4 Rearing count 8 Bolus count 0 Urine present N Gait T2 MANIPULATIONS Approach 3 Touch 0 Startle 3 Righting reflex 1 Tail pinch 3 turns vocalises Y degree 2 Pupil reflex B Temperature(C) 39.2 B o d y w e i g h t (g) 224 GR I P S T RENGTH (kg)# Forelimb 0.66 Hindiimb 1.03 F O O T S P L A Y (cm) # 10.3 2 22 2 2. 3 N NN N NN N NN 15 ' 15 17 4 4 -4 7 9 13 0 00 N NN T2 T2 T2 3 33 3 33 3 32 1 11 6 30 Y 2 B 38.9 217 Y 2 B 38.8 221 Y 1 B 38.8 215 0.58 0.98 12.8 0.97 1.05 11.5 0.67 0.77 13.4 2 2 Y 1 N N 14 4 9 0 N T1 3 3 3 1 3 Y 1 B 38.4 225 0.66 0.60 8.6 # Values represent the mean of two trials Additional comments Animal number 26 Touch response: rears During grip strength: moderate vocalisation 28 29 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 S l i g h t l a c k of g r o o m i n g rump, l o w e r left too^ii pale Touch response: delayed walk away During footsplay: slight red discharge from urethra noted 30 Slight brown nasal staining : 177 Company Sanitized. Does not contain TSCA CBI DPT 443/984760 APPENDIX 1 (Functional observational battery --continued) Week 4 Group 3 female, 50 mg/kg/day A n i m a l 31 32 33 34 35 OBSERVATIONS IN THE HAND Removing 2 Handling 2 Salivation Y degree 1 Vocalising N degree IN THE ARENA Grooming N Activity count 16 Arousal 4 Rearing count 10 Bolus count 0 Urine present N Gait T1 MANIPULATIONS Approach 3 Touch 3 Startle 2 Righting reflex 1 Tail pinch 3 turns 1 vocalises degree Pupil reflex B Temperature(C) 38.5 B o d y w e i g h t (g) 193 GRIP STRENGTH (kg)# Forelimb 0.56 Hindiimb 0.69 F O O T S P L A Y (cm) # 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 T1 3 2 2 1 6 Y 3 B 38.8 194 0.80 0.90 10.3 2 .2 N N N 14 4 8 0 N T1 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 T1 3 3 2 1 5 1 Y 2 B 38.7 217 0.59 0.93 13.0 # Values represent the mean of two trials Additional comments Animal number 32 Touch r e s p o n s e : bit p r o b e 33 Lower teeth pale Approach response: bit probe 35 Slight lack of g r o o m i n g rump 178 : Company Sanitized. Does not contain TSCA CBI D PT 443/984760 APPENDIX 1 (Functional observational battery --continued) Week 4 Group 4 female, 150 mg/kg/day A n i m a l 36 37 38 39 40 OBSERVATIONS IN THE HAND Removing 2 Handling 2 Salivation N degree Vocalising N degree IN THE ARENA . Grooming N Activity count 14 Arousal 4 Rearing count 6 Bolus count 0 Urine present N Gait T1 MANIPULATIONS Approach 3 Touch 2 Startle 2 Righting reflex 1 ' Tail pinch 3 turns vocalises Y degree 2 Pupil reflex B Temperature(C) 38.3 B o d y w e i g h t (g) 1 7 6 G R I P STRENGTH (kg)# Forelimb 0.65 Hindlimb 0.81 F O O T S P L A Y (cm) # 11.8 2 22 222 NNN NNY 1 NNN 1 1 15 13 4 44 5 96 000 NNN T1 T2 HU1 T1 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 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 # Values represent the mean of two trials Additional comments Animal number 36 Approach response: bit probe 38 Moderate hai r los s n e c k 39 During grip strength: moderate vocalisation 179 : Company Sanitized. Does not contain TSCA CBI APPENDIX 2 Locomotor activity Animal no. Total time spent in locomotor activity (secs) Pre-dose Week 4 Group 1 Sex male. Control 1 874 2 484 3 498 4 571 5 299 708 384 362 766 455 Group 2 Sex male, 15 mg/kg/day 6 624 7 347 8 581 9 281 10 610 703 498 872 658 843 Group 3 Sex male, 50 mg/kg/day 11 443 12 403 13 231 14 625 15 142 777 713 632 695 468 Group 4 Sex male, 150 mg/kg/day 16 522 17 524 18 332 19 322 20 414 351 531 569 389 197 DPT 443/984760 : 180 : Company Sanitized. Does not contain TSCA CBI APPENDIX 2 (Locomotor activity - continued) Animal no. Total time spent in locom o t o r a c t i v i t y (secs) Pre-dose Week 4 Group 1 Sex female. Control 21 202 22 120 23 377 24 475 25 197 711 479 617 633 374 G r o u p 2 S e x f e m a l e , 15 m g / k g / d a y 26 207 27 334 28 225 29 539 30 406 923 741 848 780 642 G r o u p 3 S e x female, 50 m g / k g / d a y 31 776 32 160 33 533 34 800 35 2 2 2 1131 871 899 1317 447 Group 4 Sex female, 150 mg/kg/day 36 406 37 172 38 520 39 598 40 628 310 292 452 865 899 DPT 443/984760 : 181 : Company Sanitized. Does not contain TSCA CBI D PT 443/984760 FORMULATION CHEMISTRY 182 : Authors: I. Suzanne Dawe, Paul Mann, Emma Buckland. Company Sanitized. Does not contain TSCA CBI CONTENTS INTRODUCTION........................................... DPT 443/984760 Page 184 EXPERIMENTAL PROCEDURE........................................................................................... 185 RESULTS.................................................................................................................................... 189 DISCUSSION............................................................................................................................ 189 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 : Formulation 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 o f concentrations o study. in test formulations analysed during the The determination o f the stability o: in aqueous formulations. The validation of the analytical procedure for the determination ofj formulations. aqueous The formulations for this study were prepared as solutions o ^ ^ l H H ^ ^ i n 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 ' ^ g M ^ H l u p p l i e d in a letter dated 23rd j u|y 1998) adapted fo r u s ^ M Iu n tin g d o n Life Sciences (HRC/FA/M80/98 issue 01/171198). The method of analysis aqueous solutions involved dilution, initially using water and subsequently solvent A', and injection of the diluted test samples onto j high performance liquid chromatograph (HPLC) with conductivity detection. The amount die test samples was quantified by external calibration with reference to five linearly related staneferds of known concentrations. 1Solvent A Aceronitrile / aqueous 0.01M sodium hydroxide (25/75 v/v). 184 Formulation Chemistry Company Sanitized. Does not contain TSCA CBI EXPERIMENTAL PROCEDURE D PT 443/984760 ANALYTICAL PROCEDURE Apparatus and instrumentation High performance liquid chromatograph (HPLC): As detailed under chromatographic conditions. Balances: 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: [M l m 5Dupont Specialty Chemicals. Control: Distilled water. Acetonitrile: Sigma-Aldrich, Riedel de Han, Far UV for HPLC. Ammonia solution (SG 0.88): Fisher Scientific UK Ltd, Analytical Reagent Sodium hydroxide: Fisher Scientific UK Ltd, Analytical Reagent. Sodium carbonate: Fisher Scientific UK Ltd, Analytical Reagent. Sulphuric acid: Fisher Scientific UK Ltd, Analytical Reagent Buffer solution: Sodium carbonate (2.12 g) was dissolved in water (120 ml), ammonia solution (2.5 ml) was added and the solution was diluted to volume (200 ml) using water. The resulting solution was diluted (10 : 1000 ml) using water. Solvent A: Acetonitrile / aqueous 0.01M sodium hydroxide (25 / 75 v/v). 185 Formulation Chemistry Company Sanitized. Does not contain TSCA CBI D PT 443/984760 Regenerant (0.025M sulphuric acid): 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. Mobile phase: Acetonitrile / buffer solution (25 / 75 v/v). W ater Elgastat UHP-4, deionised reverse osmosis. Sample process An exact volume (1 ml) of test formulation was appropriatel^iluted, initially using water and finally using solvent A, to provide a solution c o n t a i n i n g a n expected concentration in the range 100 pg/ml - 200 (ig/ml. The concentration o f p H H H V j v a s 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: Flow rate: Detection: Chemical Suppression: Suppresser type: Regenerant: Flow rate: Injection volume: Integrator attenuati- -n: Retention volume: , Ambient, nominally 21C. Acetonitrile / buffer solution (25 / 75 v/v). 1.0 ml/minute. Conductivity. Anionic. 0.025M Sulphuric acid. 2.5 ml/minute 100 pi. 64. Approximately 6.5 ml. 186 ; Formulation Chemistry Company Sanitized. Does not contain TSCA CBI D PT 443/984760 Calibration A primary standard solution was prepared fgr each analytical occasion by dissolving an accurately weighed nnantifv /SO mo~l r>f ^ j j p W p n water (50 ml). Solutions for instrument calibration, c o n t a i n i n g U ^ B i f l l f l i t concentrations o f 50 pg/ml, 100 pg/ml, 150 pg/ml, 200 pg/ml and 250 pg/m vw ere prepared oy 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 each calibration chromatogram was measured and calibration curves were constructed by linear regression o f standard response versus standard .concentration. The height response o f the peak observed at the characteristic n ntion volume of m m m m sample chromatograms was measured and the concentration o: as ' determined using the following equation: Concentration, mg / ml = Y - I x V x 10" Where Y I S V Peak height response for Zonyl FS-62 in test chromatogram Intercept derived from linear regression o f calibration data Slope derived from linear regression o f calibration data Dilution factor of sample VALIDATION OF THE ANALYTICAL PROCEDURE The analytical procedure was validated by determining the specificity o f the chromatographic analysis, linearity o f detector response, precision of injection, limit of detection, method accuracy and precision. Specificity Chromatogramstibtained for control extracts were examined for peaks which might interfere with the quantitation of Linearity The linearity o f the standard curve was examined by preparing a series of standard solutions (50 pg/ml, 100 pg/ml, 150 pg/ml, 200 pg/ml and 250 pg/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 pg/ml) and the highest standard solution (250 jig/ml). Limit of detection The limit o f detection, defined as the concentration ofl control matrix producing a signal to noise ratio o f at least 3, was determined by fortifying an ex ict of control vehicle (distilled water) w i t t ^ f l H H ^ I B a provide a peak of suitable size. Method accuracy and precision Prior to the start of treatment, specimen formulations c o n t a i n i n g f i ^ H H I H i n 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 o f analytical results generated for the analysis of six replicate samples from the prepared formulations. STABILITY IN AQUEOUS FORMULATIONS Prior to treatment, specimen aqueous formulations (400 ml) c o n ta in in o J ^ m ^ B ^ ^ ^ ^ t nominal concentrations o f 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 o f 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. D ie 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 o f 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 D P T 443/984760 The mean concentrations o ^ f l j j j H H R ' n test formulations analysed during the study and the deviation of mean results froJtfnominal values are detailed in Table 1. The results obtained for the stability o ^ f l B m H ^ F b r m u l a t e d distilled water formulations are presented in Table 2. The results for the validation o f 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 ___________ Jin test formulations analysed during the study were within -7% o f nominal concenl ations, confirming the accuracy of formulation. Prior to treatment, the stability for j J H j j j U H l t 11 aqueous formulation at nominal concentrations o f 5 mg/ml and 400 m g^^w a^confbm eu with respect to the level o f 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 o f detector response was confirmed for 50 pg/ml - 250 pg/ml. over the concentration range 'injection was confirmed for six replicate injections of standard solutions containing -,a nominal concentration o f 50 jig/ml and 250 pg/ml, for which the coefficients of variation were less than 1.5%. The accuracy and precision of the analytical procedure was confirmed: a mean procedural recovery value o f 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 o f detection was determined as 0.375 mg/ml using the operating parameters defined in this procedure. The specificity o f th^H P L ^^gsa^w as demonstrated by the absence of a peak at the characteristic retention volume f o n H H ^ ^ H v i the control sample chromatogram. 189 Formulation Chemistry Company Sanitized. Does not contain TSCA CBI D PT 443/984760 % CONCLUSION The analytical procedure was validated f o r ^ P ^ ^ H ^ ^ H Q 11 distilled water with respect to the specificity o f the chromatographic analysis, linearity o f detector response, precision o f injection, limit o f detection, method accuracy and precision. Prior to treatment, the stability during ambienMemDeratm-e storage for 2 days and refrigerated storage for 15 days was confirmed f o r a9ueous form ulations,. at nominal concentrations o f 5 mg/ral and 400 mg/ml.*The storage period represented the maximum time from preparation to completion of use. The mean concentrations o f test formulations analysed during the study were within -7% of nominal concentrations confirming the accuracy o f formulation. : 190 Formulation Chemistry Company Sanitized. Does not contain TSCA CBI TABLE 1 Concentrations o) [n test formulations DPT 443/984760 W eek ' ` of dosing 1 G roup C o n tro l 2 3 4 N o m in al inclusion (m g/m l) 0 6 20 60 A nalysed concentration (m g/m l) Analysis 1 ND 5.84 19.9 58.3 Analysis 2 ND 5.88 20.0 53.3 M ean ND 5.86 19.9 55.8 N D N one detected (<0.375 mg/ml) RME Relative m ean 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 in aqueous formulations DPT 443/984760 N o m in al inclusion (m g/m l) B o ttle no. 51 Storage conditions Day H our Temp,C 00 -4 2- +21 +21 +21 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 22 38 4 15 - - +4 +4 +4 4.76 4.90 4.73 4.77 4.89 . 4.69 4.76 4.90 4.71 400 10 0 +21 370 - 4 +21 371 2 - +21 394 378 382 396 22 38 .4 15 - - +4 397 +4 385 +4 384 399 389 385 RM E R elative mean error, representing the deviation from nom inal Mean analysed concentrations were calculated using unrounded figures 374 377 395 398 387 385 00 r? RME (% ) -1 .4 -3 .0 -4 .8 -2 .0 -5 .8 -6 .5 -5 .8 -1 .3 -0 .5 --3.3 -3 .8 : 192 Formulation Chemistry Company Sanitized. Does not contain TSCA CBI m TABLE 3 Validation o f the analytical procedure fori DPT 443/984760 aqueous formulations 3.1 L inearity determ ination ol Concentration (pg/m l) Peak height 49.740 99.480 149-22 198.96 248.70 7647 15383 22150 29006 35472 CD G radient - Intercept Q CD Coefficient o f determination n N umber o f determinations 0.9990 13927 1149.7 5 standard solutions 3.2 System precision o f C o n cen tra tio n 50 pg/m l Peak height 7395 7608 7587 7432 7428 7414 M ean CV (%) n 7477 126 6 CV Coefficient o f variation n N umber o f determinations indard solutions 250 pg/ml 40253 40319 39824 40472 39693 39181 39957 1.21 6 193 Formulation Chemistry Company Sanitized. Does not contain TSCA CBI TABLE 3 (continued) DPT 443/984760 3-3 A ccuracy and precision d ata fo: Analytical phase N om inal fortification (mg/m l) 5 400 V alid atio n 98.2 98.0 96.8 96.1 97.4 98.7 96.6 97.3 95.7 92.7 92.1 92.7 Mean 97.5 94.5 CV (%) 0.99 2.41 Range 96.1 - 9 8 .7 92.1 - 9 7 .3 n 6 16 CV Coefficient o f variation, n Number o f determinations. in aqueous form ulations Results are expressed as percent recovery and calculated using the following equation: Analysed concentration (m g/m l) %Recoveiy = ------------------- -----:-- ------:-- -- x 100 Nominal concentration (mg / ml) : 194 Formulation Chemistry Company Sanitized. Does not contain TSCA CBI FIG U R E I Typical calibration standard graph (W eek 1) 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 1 50000 . . t ;a~'' 'v 45000 . ;; ' ':: -- " ' Ht:... - ' .1.\ '.:''' t'.-.-i. .'H ; '1, - -.7;4 I -r, r. kj;- -a.l la il / r r / . - /`_- L / y r< 30000 11 :3 -M | I 5 S-fi-r-sa ilia w m y : f S T' 1" ; -(p y =;'- .Li-rill" : S I I B - u s f M t p " ' :l 'iv.-ar W 01 ill; pi-ii is 0- a; f `% : vjfc'Vaiti: -i-y- ii: 1 r : 0.000 50.000 100.000 150.000 200.000 ' Concentration (ug/ml) 'I i;l 250.000 300.000 i i 195 Formulation Chemistry Company Sanitized. Does not contain TSCA CBI FIGURE 2 Typical analytical chromatograms (W eek 1) DPT 443/984760 Calibration standard, 250 pg/ml CHANNEL A INJECT 0 9 - 1 2 - 9 8 1 7 : 5 1 : 2 7 STORED TO BIN # 1 1 6 Group 1, Control (Dilution factor 1:50) CHANNEL A 196 Formulation Chemistry Company Sanitized. Does not contain TSCA CBI FIGURE 2 (continued) G roup 2 ,6 mg/ml (Dilution factor 1:50) CHANNEL A INJECT 0 9 -1 2 - 9 8 1 9 :4 7 :0 8 STORED TO BIN 8 126 DPT 443/984760 G roup 3 ,2 0 mg/ml (Dilution factor 1:100) CHANNEL A INJECT 0 9 - 1 2 - 9 8 1 8 :4 9 :1 1 STORED TO BIN 8 121 197 Formulation Chemistry Company Sanitized. Does not contain TSCA CBI FIGURE 2 (continued) DPT 443/984760 Group 4 ,6 0 mg/ml (Dilution factor 1:500) CHANNEL A INJECT 0 9 -1 2 - 9 8 1 8 :1 4 :3 2 STORED TO BIN 8 118 198 Formulation Chemistry Company Sanitized. Does not contain TSCA CBI Study Num ber DPT 443/984760 PROTOCOL AND AMENDMENTS : DPT/443 Huntingdon Life Sciences CONFIDENTIAL PROTOCOL TOXICITY STUDY BY ORAL ADMINISTRATION TO CD RATS FO R 4 WEEKS Sponsor DuPont Specialty Chemicals Jackson Laboratory Chambers Works Deepwater NJ 08023 USA Research Laboratory Huntingdon Life Sciences Ltd PO Box 2 Cambridgeshire PE18 6ES ENGLAND Total number o f pages: 27 Final Protocol H m abtgim U JcSaentxiU d. n g iiu n d in England: I8IS730 : 199 : Company Sanitized. Does not contain TSCA CBI Study Number : DPT/443 CONTACT DETAILS DPT 443/984760 Huntingdon Life Sciences Sponsor's Monitoring Scientist : Dr K. Dastur. Final Protocol Page ii : 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 Bttmley, B.Sc. (Hobs), M.Sc., C 3 io U M J-Biol. Study Director, Huntingdon L ife Sciences Ltd. Date The signature o f the Study Director confirms this protocol as th e w orking document fo r the study. A ny changes m ade subsequent to the date o f th e Study D irector's signature will be docum ented in formal amendment; 1 M v `tv R J. Sortwell M anagement, Huntingdon Life Sciences Ltd. D ate D rK . Dastur. Sponsor, DuPont Specialty Chemicals ^ '/ > D ate P lea se sig n both co p ies o f th is p age, retain onef o r y o u r reco rd s a n d retu rn one to the Stu dy D irecto r a t H untingdon Life Sciences. Final Protocol Page iii : 201 : Company Sanitized. Does not contain TSCA CBI % Study Natnber : DPT/443 DPT 443/984760 Huntingdon Life Sciences TOXICITY STUDY BY ORAL ADMINISTRATION TO CD RATS FOR 4 WEEKS Enquiry Number 175560 Number of pages for internal distribution: 24 Primarylocation of study Huntingdon Research C entre Huntingdon Cambridgeshire Building N u m b er 1BRB All procedures to be perform ed at the above site. D ate Final Protocol Page 1 : 202 : Company Sanitized. Does not contain TSCA CBI Study Number :DPT/443 ' CONTENTS 1. INTRODUCTION 2. STUDY SCHEDULE AND STRUCTURE 2.1. D uration o f treatment 2 3 . Scheduled tim e plan 2 2 . Identity o f treatment groups 3. TEST SUBSTANCE AND FORMULATION 3.1. Test substance 32. Formulation 3 3 . Q uality control o f dosage form 4. ANIMAL MANAGEMENT 4.1. Animals - supply, acclimatisation and allocation 4 2 . Animals -housing, diet and water supply 4 3 . Animals - procedures 4.4. Animals - termination 5. FUNCTIONAL OBSERVATIONAL BATTERY 5.1. In the hand and standard arena observations 5 3 . M anipulations 5.3. M otor activity 6. CLINICAL PATHOLOGY 6.1. Haematology, peripheral blood 6 3 . Blood Chemistry 7. NECROPSY AND HISTOLOGY 7.1. M ethod o f kill 7 3 . M acroscopic Pathology 7 3 . Organ weights 7.4. Fixation 7.5. Histology . 8. PATHOLOGY 8.1. Light microscopy 8 3 . Extension o f initial examination 9. DATA TREATMENT 9.1. Food conversion efficiency 9.2. Statistical analysis 10. REPORTING 11. QUALITY ASSURANCE AND ARCHIVING PROCEDURES 11.1. Q uality Assurance 113. Archives DPT 443/984760 H lin tin g d O n Life Sciences Page 3 4 4 4 4 5 6 6 7 8 8 9 11. 14 14 14 15 13 16 16 16 17 17 17 17 17 18 21 21 21 22 22 22 23 23 23 24 Final Protocol Page 2 Company Sanitized. Does not contain TSCA CBI Study Number : DPT/443 DPT 443/984760 Huntingdon Life Sciences 1. INTRODUCTION M anagem eat o f etudy Study Director M onitoring Toxicologist In the temporary absence of the Study D irector, die scientific responsibilities will be taken over by die Monitoring Toxicologist; other H unt o f routine study m anagement should be referred to the follow ing person in the first instance. V : S_M. Bottomlcy. : W N. Hooks. : MJL Barker. O bjective Assessm ent o f systemic toxic potential in a 4 week oral gavage study in C D rats. R egulatory com pliance The study will be performed in accordance w ith the following regulations o r guidelines: Organisation for Economic Co-operation and Development, Testing o f Chemicals Guideline N o. 407 (revised 1995). Good L aboratory Practice The study will be conducted in compliance with principles o f Good Laboratory Practice Standards as set fordi in: The UK Good Laboratory Practice Regulations 1997 (Statutory Instrum ent N o 654). OECD Principles o f Good Laboratory Practice (as revised in 1997), ENV/M C/CHEM (98)17. EC Council D irective 87/18/EEC o f 18 December 1986 (Official Journal N o L 15/29). A nim al m odel C D rat, accepted by regulatory agencies, background data available. Route Oral gavage, to simulate the conditions o f potential human exposure. Treatm ent groups and dosages G roup Compound Dosage (mg/kg/day)t : : : Control 0 2 Low 3 In term ed iate 4 H igh t Expressed in terms o f solids. T h e test substance as supplied i Final Protocol Page 3 : 204 : Company Sanitized. Does not contain TSCA CBI DPT 443/984760 Study Number : DPT/443 H untingdon Life Sciences 2. STUDY SCHEDULE AND STRUCTURE 2.1. D uration o f treatm ent M inim um period : 28 days. All surviving anim als will b e killed on D ay 29. 2 3 . Scheduled tim e pian Sample o f ^ ^ ^ ^ ^ ^ ^ ^ h r r iv e d A nim als toarrive Treatment to commence Term inal sacrifice to commence Histopathology to be completed D raft report to be issued 23 June 1998 4 December 1998 11 D ecem ber 1998 8 January 1999 5 March 1999* March 1999 (estim ated) (estim ated) 2 3 . Identity o f treatm ent groups (to be selected from 58 animals ordered) G roup Treatm ent 1 Control K2 3 Un 4 D osage (m g/kg/day) t 0 Low Interm ediate H igh Dosage (m g/k g /d ay )* # 0 Low Interm ed iate H ig h N um ber o f anim ats M ain study M ale Fem ale 55 55 55 55 t Expressed in to m s o f solids. The test substance as supplied i # E xpressed in term s o f the test substance as supplied. * Dosages (mg/kg/day) to be selected on the basis o f a prelim inary study undertaken at H untingdon L ife Sciences (DPT/442). Final Protocol Page 4 : 205 : Company Sanitized. Does not contain TSCA CBI Study Number : DPT/443 G roup 1 2 3 4 Cage num bers M ale F em ale 1S 26 37 48 Health screen DPT 443/984760 Huntingdon Life Sciences Animal aum bers M ale Fem ale 1*5 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 W ork etc. A ct 1974, and the Control o f Substances H azardous to Health Regulations 1994, it is a condition o f 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 o f any substance supplied by the Sponsor to Huntingdon Life Sciences. The Sponsor shall also comply w ith all current legislation and regulations concerning shipment o f substances by road, rail, sea or air. Such inform ation in the form o f a completed Huntingdon Life Sciences test substance data sheet m ust be received by Safety M anagement Services at Huntingdon Life Sciences before the test substance can be handled in the laboratory. A t the discretion o f Safety M anagement Services at H untingdon Life Sciences, other documentation containing the equivalent information may be acceptable. Information received will be used to set the Huntingdon Life Sciences Hazard Class, w hich determines safety precautions taken in die workplace. Huntingdon Life Sciences H azard Class: 2 Final Protocol Page 5 206 Company Sanitized. Does not contain TSCA CBI % stud?Nber DPT/3 DPT 443/984760 Huntingdon Life Sciences 3.1. Test c a ta ta Sponsor's identification Storage conditions Sponsor's responsibilities At room temperature. Documentation o f methods o f synthesis, fabrication o r derivation. . Stability data. Certificate o f analysis. Certificate o f analysis details : Test substance identity. Batch n u m b er (L o t# 3). Purity. C om position. O ther appropriate characteristics. Current expiry date: 2 years from manufacture. 3.2. Formulation T reatm ent G roup 1, Control Group 2 Group 3 Group 4 Conversion factor V ehicle M ethod o f preparation Frequency o f preparation V ehicle. low mg/ml. ediatem g/m L high mg/ml. The test s u te ta n c ^ iM e u s e ^ s s u i substance been correcte^foM h^rate^m rtem T led. The test losagcs have Distilled water. Will be documented. Will depend upon die availability o f supporting stability data. W here sufficient stability data is available, batches will cover one week o f dosing and m ay be prepared up to three days in advance o f the first day o f dosing. Where stability data does not support this length o f use period, a more frequent mixing regime will be initiated. Final Protocol Page 6 : 207 : Company Sanitized. Does not contain TSCA CBI DPT 443/984760 Stndy Nimber : DPT/443 Huntingdon Life Sciences 3 4 Quality control o f dosage form Liquid formulation : Before commencement o f treatment, the suitability o f the proposed mixing procedures will be determined and specimen formulations w ill be analysed to assess the stability o f the test substance in the liquid matrix. A t specified intervals during treatment, the test formulations will be analysed for achieved concentration o f th e test substance. A nalysis : The formulated samples will be analysed using a method validated with respect to the determination o f the specificity o f analysis, limits o f quantitation and/or detection, linearity o f detector response, reproducibility, m ethod accuracy and precision. S ta b ility Preparation : Specim en formulations (nom inally 400 m l) w ill be prepared at the anticipated highest and lowest concentrations and equally distributed between four screw-capped amber bottles. Sampling and determination : B efore sampling, each formulation will be m ixed by inversion. At each time-point duplicate samples will be taken for assay from the m iddle o f the formulation. Storage conditions : A m bient temperature (nom inally 21C) for 0 ,4 and 4 8 h o u rs (B ottle 1). Refrigeration (nom inally 4C ) for 2, 8 and 15 days (Bottles 2 ,3 and 4). A chieved concentration Sam pling and determination : D ay 1. O ther sampling regimens m ay be specified by the Sponsor. One sample (nominally 20 m l) from all groups; 2 assays from each sample. Final Protocol Page 7 : 208 : Company Sanitized. Does not contain TSCA CBI % Study Number : DPT/443 DPT 443/984760 Huntingdon Life Sciences 4. ANIMAL MANAGEMENT 4.1. Ani-- U - apply, 4.1.1. Animals aad Hocatmn Species Strain A ge ordered W eight range ordered Supplier R at Crl.CD BR_ 28 2 days. 11 g/sex (m inim um 75 g). Charles R iver (U K) Limited. 4.1.2. Health screen An additional four males (animal numbers 41-44) and four fem ales (animal numbers 45-48) will be ordered from the supplier. These will be killed, bled and subjected to macroscopic examination immediately upon receipt Serum sam ples w ill be retained frozen pending possible future serology investigations (samples discarded after tw o months). Lungs, liver, kidneys, spleen and heart w ill be preserved in fixative, but not processed further unless macroscopically abnormal. M acroscopic abnormalities will be immediately processed and examined microscopically. Results o f die health screen w ill be reviewed before comm encement o f treatm ent 4.1.3. A cclim atisation D uration : 1 to 2 weeks (animals will be approximately 5 weeks o f age a t commencement). Husbandry conditions : R eferto Section 4 2 . 4.1.4. A llocation to treatm ent groups A llo catio n M ethod : Approximately 1 w eek before commencement o f treatm ent : Random allocation to cages to equalise variation in bodyw eight 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 TMn!s from the sam e group w ill be arranged in blocks within cage-racks. The position o f the racks within the room w ill be exchanged a t intervals during the study. 4.1.5. Identification N um bering Method Unique for each animal w ithin study. Cage number by foot tattoo. Animal number by earmark. Cage labels Uniquely identifying the occupants. 4.1.6. Precomm encement anim al replacem ent 10 spare anim als w ill be ordered to replace any individuals rejected during the acclim atisation period. Replacement before treatment : Ill-health. A b norm alities. Bodyweight range extremes. . On Day 1 (before dosing) variations in bodyweight o f animals should not exceed 20% o f the m ean for each sex. Replacement during treatment : N one scheduled. 42. Animals - housing, diet and water supply 4 2 .1 . Environm ental control Rodent facility Limited access - to minim ise entry o f external biological and chemical agents. Air supply : Filtered, not recirculated. T em p eratu re : T arget range 19-23C. Relative humidity : Target range 40-70%. M onitored continuously. Excursions outside these ranges docum ented in the study data. Lighting 12 hours light : 12 hours dark. Final Protocol Page 9 : 210 : Company Sanitized. Does not contain TSCA CBI % a.,TM* :dpt/443 DPT 443/984760 Huntingdon Life Sciences A larm systems Electricity supply Activated on ventilation failure and when ternperature/hnmidity lim its exceeded. Public supply with autom atic stand-by generators. 4.2.2. Animal accommodation Anim als per cage Cage material Cage flooring - : Five o f same sex, unless reduced by mortality or isolation. : Stainless steel. : Stainless steel grid. The cages w ill be suspended above absorbent paper. The latter w ill be changed at appropriate intervals each week; cages, cage-trays, food hoppers and w ater bottles w ill be changed at appropriate intervals. Precise details o f caging will be included in the final report 4 2 3 . D iet and water supply C opies o f all certificates o f analysis are stored in the archives. D iet supply Diet name D iet type A vailability C ertificatio n : R at and Mouse No. 1 Maintenance D iet : Pelleted d ie t : Non-restricted. : B efore delivery each batch o f diet is analysed b y the supplier for various nutritional components and chemical and microbiological contaminants. Supplier's analytical certificates are scrutinised and approved before any batch o f diet is released for use. T his diet contains no added antibiotic o r other chemotherapeutic o r prophylactic ag e n t W ater supply Supply Regulatory agency A vailability Public drinking water. U JC Department o f the Environm ent Non-restricted via polyethylene or polycarbonate bottles w ith sipper tubes. C e rtific a tio n : Certificates o f analysis are routinely received from th ' supplier. Final Protocol Page 10 Company Sanitized. Does not contain TSCA CBI % Study Number : DPT/443 DPT 443/984760 Huntingdon Life Sciences 4 4 4 . Contaminants a n y It is the Sponsor's responsibility to advise Huntingdon Life Sciences o f any specific contam inants likely to prejudice die outcome o f die study. Analyses for such contaminants may be performed if requested by the Sponsor. 4-3. Animals - procedure* Investigations noted as occurring on specified Day numbers (where quoted) will not be varied. 43.1. A dministration Route Treated at Volume dosage Individual dose volume Controls (Group 1) Frequency Sequence Formulation Oral gavage. Constant dosages in mg/kg/day. 10 ml/kg/day. Calculated from the most recently recorded scheduled bod y w eig b t Vehicle at die same volume dosage as treated groups. Once daily at approximately the same time each day. By group. A daily record o f the usage o f 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 stirrer before and throughout the dosing procedure. 4-3-2. C linical o bservations Animals and their cages Inspected at least twice daily for evidence o f reaction to treatment or ill-health. Deviations from normal recorded at th e tim e in respect of Nature and severity. Date and time of onset Duration and progress o f the observed condition. Physical examination : 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 w ill be made in association w ith dosing according to the follow ing schedule and frequency: M inim um schedule : 1. W eek 1 - daily. 2. W eeks 2 to 4 - tw ice weekly (middle and end o f week). Frequency : 1. Pre-dose observation. 2. A s is returned to its hom e cage. 3. A t the end o f dosing each group. 4. Between 1 and 2 bouts after completion o f dosing all groups. 5. As late as possible in the working day. T he above schedule w ill be amended, a s necessary, in the light o f signs observed. D uring the aw lim atication period, observations o f the anim als and their cages w ill be recorded at least once per day. 4 3 3 . M ortality TVhilitatwl animals : Observed carefully, m ay be isolated to prevent can n ib alism . Premature sacrifice : Animals may be killed on humane grounds or if considered in extrem is. Animals found dead, killed m extrem is o r on hum ane grounds 4 3 .4 . Bodyweight A necropsy is performed as soon as possible. Animals found outside the normal w orkday will be preserved in a refrigerator (approxim ately 4C ) provided for tins purpose. Bodyweight recording : D ay that treatment commences. W eekly (last scheduled bodyweight recorded on Day 28) Before necropsy. M ore frequent weighings may be performed to aid the m onitoring o f the condition o f animals displaying ill-health. These data will be retained in the archives. 4.3.5. Food consum ption Food consumption recording Food supplied : : W eekly. At intervals each week. Final Protocol Page 12 Company Sanitized. Does not contain TSCA CBI DPT 443/984760 Study Number : DPT/443 Huntingdon Life Sciences Food spilled Food remaining 4 3 .6 . W a te r consum ption : Estimated at cage cleaning. : Recorded at end o f study week. ' Fluid intake will be assessed by daily visual observation. Precise measurem ents m ay be undertaken if treatm ent-related changes are suspected. . 4 3 .7 . Functional observational b attery A nim als will be subject to procedures as specified in Section 5. Investigations w ill be perform ed in the follow ing w eeks: Exam ination In the hand Standard arena M anipulations W eek Pretreatment, 1 ,2 ,3 ,4 Pretreatment, 4 Animals All animals All animals Automated M otor Activity 4.3.8. Biosampling Investigations will be performed as follows: B lood sam p les- Haem atology/B lood C h e m istry Day A nim als 29 All animals. Conditions Sample site A n ticoagulant/ Sample volume Analysis Following overnight deprivation o f 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 (B lood chem istry). Sections 6.1 and 6 2 . Final Protocol Page 13 : 214 : Company Sanitized. Does not contain TSCA CBI DPT 443/984760 SntdyN onber :W I43 H u n t in g d o n Life Sciences 4.4. Animals - termination All anim als will be subject to term inal investigations (Section 7). The sequence in which the anim als are killed after completion o f treatment period w ill allow satisfactory inter-group com p ariso n . 5. FUNCTIONAL OBSERVATIONAL BATTERY Each anim al will be subject to the procedures detailed below on the specified occasions. The functional observational battery will be performed at the sam e time o f day on each occasion and the observer w ill be unaw are o f the experimental group to which the animal belongs. Animals will not necessarily all be tested on the sam e day but the number o f animals will be balanced across the groups on each day o f testing. A ny deviations from normal will be recorded with respect to nature and, w here appropriate, degree o f severity. Further details on test procedures and definitions will b e documented in the report. 5.1. In the hand and standard arena observations O bservations will be perform ed in th e hand and then during a 1 minute recording period in a standard arena as follows: W eek Pretreatment, 1 ,2 ,3 ,4 Animals A ll animals. After removal from the home cage die following param eters w ill be assessed: In the hand E xophthalm os Fur appearance L acrim ation Piloerection Reactivity to handling Ease o f removal from cage Salivation ' Vocalisation on handling Standard arena Activity counts Arousal Convulsion Defecation count G ait Grooming Palpebral closure 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 1 2 . Manipulation! W eek Pretreatment, 4 ' A nimals All animals. ' The following measurem ents, reflexes and responses will be recorded: Approach response Auditory startle reflex Body temperature Bodyweight Grip strength - forelimbs and hindlimbs Landing footsplay Tail pinch response Pupil reflex Righting reflex Touch response 5.3. M otor activity W eek Pretreatment, 4 A nim als All animals M otor activity w ill be measured by automated infra-red sensor equipm ent, recording individual animal activity over a one hour period. Final Protocol Page 15 : 216 : Company Sanitized. Does not contain TSCA CBI  Stady Number DPT/443 DPT 443/984760 H U n tin g d O fl Life Scien ces 6. CLINICAL PATHOLOGY 6.1. Haematology, peripheral blood Blood sample analysis w ill be performed on the following occasions): D ay 29 A nim als All animals. A ll sam ples will be examined for the following characteristics: 1) U sing ED TA a s anticoagulant - Packed cell volume Haemoglobin concentration Erythrocyte count Total leucocyte count Differential leucocyte count Abnorm alities o f the blood film Platelet count M ean cell haemoglobin M ean cell volum e M ean cell haemoglobin concentration 2) Using citrate as anticoagulant - Prothrombin time Activated partial thromboplastin tim e 6.2. B lood C hem istry Blood sam ple analysis will be performed on sam ples obtained from the same animals and at the sam e tim e as for haematology. All. sam ples will be examined for the following characteristics: U sing lithium heparin as anticoagulant Alkaline phosphatase Alanine amino-transferase (G PT)' Aspartate amino-transferase (GOT) G amma glutamyl transpeptidase G lu co se Bilirubin - total Cholesterol - total Final Protocol Page 16 : 217 : Company Sanitized. Does not contain TSCA CBI Study Number : DPT/443 Triglycerides Creatinine Urea nitrogen Total protein Albumin by chemical assay Albumin/globnlin ratio Sodium Potassium Chloride C alcium Phosphorus DPT 443/984760 Huntingdon Life Sciences 7. NECROPSY AND HISTOLOG Y 7.1. M ethod o f kill M ethod : Carbon dioxide. M acroscopic Pathology (T able 1) C om plete Checks 7 3 . O rg an weights (T able 1) D ata collection D ata presentation 7.4. F ixation (T able 1) Standard O thers A ll animals. Retained tissues. For bilateral organs, left and right organs w ill be weighed together unless otherwise specified on the Pathology Procedures Table. Organ weights are not routinely recorded for anim als killed or dying prematurely. Absolute. Adjusted for terminal bodyw eight 10% Neutral Buffered Formalin. Testes pnd epididymides: Initially in B ouin's fluid. Final Protocol Page 17 : 218 : ' Company Sanitized. Does not contain TSCA CBI DPT 443/984760 Stndy N um ber : DPT/443 Huntingdon Life Scien ces 7.5. Histology (T able 1 and Section 8.1) Processing-Full List Processing - Abnormalities only Routine staining Special staining : All animals killed or dying prematurely. A ll terminal animals o f Groups 1 and 4. : A ll terminal animals o f Groups 2 and 3. : 4-5 pm 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 Study Nunber : DPT/443 Huntingdon Life Sciences TABLE 1-Pathology procedures T im e Abnormalities Adrenals Brain Caecum Colon Duodenum Epididymides Femur withjoint Head Heart Ileum (including Pcyer's patch) Jejunum Kidneys Liver Lungs (including bronchi) Lymph nodes - mandibular -mesenteric Oesophagus Ovaries Pancreas Prostate Rectum Sciatic nerves Seminal vesicles Spinal cord Spleen Sternum Stomach Testes Thymus Thyroid with parathyroids Trachea Urinary bladder Uterus with cervix Vagina Weigh * * Fra Light microscopy 0 b) a) * 0 0 * 0 0 0 0 0 0 # 0 0 0 0 0 0 0 # # 0 0 t # 0 a) Including nasal cavity, paranasal sinusesand nasopharynx. b) Both hindlimbs retained, one sectioned whereappropriate. * Organs weighed, samples fixed or sectionsexamined microscopically. # Examined if effects suspected duringthe study, t Only one examined. ' Final Protocol Page 19 : 220 : Company Sanitized. Does not contain TSCA CBI m Study Number : DPT/443 DPT 443/984760 Huntingdon Life Sciences The tissues subjected to histolgica] processing w ill include the following regions: T issu e Regions to be exam ined A d ren als Brain Femur with joint Heart Ileum K idneys Liver Lungs Spinal cord cortex and medulla. '. cerebellum, cerebrum and midbrain. longitudinal section including th e bone marrow, including auricular and ventricular regions, including Peyer's patch w here possible, including cortex, m edulla and papilla regions, section from all m ain lobes, section from two m ajor lobes, to include bronchi. transverse and longitudinal sections at the cervical level. S tom ach Thyroid U terus : keratinised, glandular and antrum. : includes parathyroid in section, w here possible. : uterus section separate from cervix section. For bilateral organs sections o f both f te left and right organs will be exam ined, unless otherwise specified on the Pathology Procedures Table. A single section will be prepared from each o f the remaining tissues required for microscopic pathology. Final Protocol Page 20 : 221 : Company Sanitized. Does not contain TSCA CBI Stndy Number : DPT/443 DPT 443/984760 Huntingdon Life Sciences 8. PATHOLOGY 8.1. lig h t microscopy C ategory Prem ature deaths Terminal sacrifice Term inal sacrifice Animals All from all groups. All animals o f Groups 1 and 4. All anim als o f G roups 2 and 3. Tissues A ll specified in T able 1. A ll specified in T able 1. Abnormalities only. Peer Review : Carried out by a reviewing pathologist to Internationally accepted standards. 8.2. Extension of initial examination A t the discretion o f the pathologist, further processing and staining techniques m ay be used to evaluate individual lesions. Details o f these techniques will be documented and retained in die archives. Light m icroscopy may be extended, following consultation with the Sponsor, as follows: from all animals o f Groups 2 and 3 killed at terminal sacrifice for tissues considered to exhibit a reaction to treatm ent in G roup 4. Any such requirement will be documented in an amendment to the protocol. Tissues displaying treatment-related change may be further examined using additional processing o r staining techniques. Final Protocol Page 21 : 222 : Company Sanitized. Does not contain TSCA CBI DPT 443/984760 Study Ntunber : DPT/443 H u n ting d on Life Sciences 9. DATA TREATMENT 9.1. Food conversion efficiency The group mean food conversion efficiency will be calculated, where appropriate, from food conversion ratios using weight o f food consumed per unit gain in bodyweigbt 94. Statistical analysis Datatypes T he follow ing data types will be analysed at each tim epoint separately:- bodyweight, using gains over appropriate study periods. food consum ption, over appropriate study periods, using cage totals. functional observational battery. blood chemistry, haematology and urinalysis. organ w eights, both absolute and adjusted feu terminal bodyweight, where appropriate, pathological findings, for the number o f animals w ith and without each finding. M ethods F o r categorical data, th e proportion o f anim als w ill be analysed using Fisher's E xact test fo r each treated group versus the control. F o r continuous data, Bartlett's test will first be applied to test the homogeneity o f variance betw een die groups. Using tests dependent on the outcom e o f Bartlett's test, treated groups will then be com pared w ith the control group, incorporating adjustment for multiple com parisons where necessary. Final Protocol Page 22 : 223 : Company Sanitized. Does not contain TSCA CBI % DPT 443/984760 Study Number : DPT/443 H u n tin g d o n Life Sciences 10. REPORTING Study progress D raft final report Authorised final report Periodic verbal and written updates on study progress w ill be provided by th e Study Director. A synopsis report will be sent a t term ination o f the ttHlife phase. For review by th e Sponsor. After approval from the Sponsor. Routinely reports are supplied on A4 paper. The following numbers o f reports are supplied. Type o f report D raft report A uthorised final P rin tin g D ouble-sided D ouble-sided Single-sided N um ber o f copies Bound Unbound 02 10 01 A ny 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. Q uality Assurance Protocol check Procedure inspections Study audit Report review (Final report) R eport o f QA findings Authorised protocol and any amendments. Critical phases o f this study (study based) and routine procedures on representative studies (process based). T he GLP aspects o f the management and conduct o f this study. Following issue o f the draff report to the Sponsor. To Study Director and management promptly on completion o f each QA action. Final Protocol Page 23 : 224 : Company Sanitized. Does not contain TSCA CBI % Stndy N um ber :D P T /443 DPT 443/984760 H u n tin g d o n Life Sciences 11.2. A rchives AH experim ental data arising from th e study (including docum entary raw d ata, specim ens, records, other materials; collectively defined as the "materials") w ill rem ain the property o f the Sponsor. Huntingdon Life Sciences shall retain the materials in its archive for a period o f 5 years from the date o f issue o f the final report A lter such tim e, the Sponsor w ill be contacted and their advice sought on the return, disposal or further retention o f the materials. I f requested, Huntingdon Life Sciences w ill 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 N um ber : DPT/443 Protocol A m endm ent N u m b er : 1 (one) DPT 443/984760 Huntingdon Life Scien ces TOXICITY STUDY BY ORAL ADMINISTRATION TO CD RATS FOR 4 WEEKS Total number of pages: 4 Number of pages for internal distribution: 4 Study D irector : S M Bottomley, B.Sc. (Hons), M .Sc,, C-Biol., MJLBiol. # The signature o f the Study D irector authorises the implementation o f this amendm ent to protocol. In this amendment, deleted statements are struck through and new statem ents are underlined. Any changes to the study design after the date o f this authorising signature will be documented in a further formal am endm ent AMENDMENT APPROVAL F o r Huntingdoi Authorised by; (Study Director)' F o r die Sponsor A pproved by: ___ 1Date: Date: & U . 7f I ? ? ? Page 1 : 226 : Company Sanitized. Does not contain TSCA CBI Study Number : DPT/443 Protocol A n o d M it Number : 1 (ose) DPT 443/984760 H untingdon Life Sciences TOXICITY STUDY BY ORAL ADMINISTRATION TO CD RATS FOR 4 WEEKS Reasons for am endm ents : 1. C ollection o f th e anim ai arrival date. 2. A ddition o f dosage levels. 3. Correction to tuning o f allocation o f animals to treatment groups. 4 . A ddition o f W eek -1 bodyw eight Amendments Treatment groups and dosages Group :1 2 3 4 Compound Dosage (m g/kg/day)t Control 0 t Expressed in term s o f solids. T he test substance as supplied is 25% solids in 75% water. 2 2 Scheduled tim e plan Sample o f Zonyl FS-62 arrived Animals to arrive Treatment to commence Terminal sacrifice to commence H istopathology to be completed Draft report *j be issued 23 June 1998 2 -4 December 1998 11 D ecem ber 1998 8 January 1999 5 March 1999 March 1999 (estim ated) (estim ated) Page 2 : 227 : Company Sanitized. Does not contain TSCA CBI D PT 443/984760 Study Number : DPT/443 Protocol Amendmeat Number : 1 (one) H u n ting d on Life Sciences 2-3 Identity o f treatm ent group (to be selected from 58 n"nlg ordered) Group Treatment 1 Control Dosage (mg/kg/day) t 0 2 I J5 Lew 3 4 150 High JDosage (mg/kg/day)* # 0 60 Lew *)A rt A V v tlH W IlW O lW w 600 High Number o f animals Main study M ale Fem ale 55 55 55 55 t Expressed in term s o f solids. The te s t substance as supplied i^ # Expressed in term s o f the test substance as supplied. * D osages (m g/kg/day) to be selected on th e basis o f a prelim inary study undertaken at H untingdon L ife Sciences (DPT/442). U Formillation T reatm ent G roup 1, Control Group 2 Group 3 Group 4 Conversion factor V ehicle M ethod o f preparation Frequency o f preparation Vehicle. 64ew m g/m l. A interniodiate mg/ml. 60 high mg/ml. T h e te s t snubl stanc^w ilU ^ise^^uD D l[ilieedd. T he te st substance is above concentraittiionlts fm e/m ll dooaasea haavvee--fe n corre c te d for the water content D istilled water. : W ill be documented. : W ill depend upon the availability o f supporting stability data. Where sufficient stability data is available, batches will cover one week o f dosing and m ay be prepared up to three days in advance o f the first day o f dosing. Where stability data does not support this length o f use period, a more frequent mixing regim e w ill be initiated. Page 3 : 228 : Company Sanitized. Does not contain TSCA CBI DPT 443/984760 Study Number :DPT/443 Protocol Ameadmeat Number : 1 (one) H untingdon Life Sciences 4.1 A Allocation to treatment groups A llocation : On arrival. Appronimata ly 1woali befo-- i M ethod Cage distribution Random allocation to cages to equalise variation in bodyw eight C ages housing " "! from th e sam e group w ill be arranged in Mocks within cage-racks. The position o f the racks within the rotan w ill b e exchanged at intervals during the study. 4.3.4 B odyw eight Bodyweight recording : One week prior to commencem ent Day that treatm ent commences. W eekly Oast scheduled bodyweight recorded on Day 28) Before necropsy. More frequent weighings may be performed to aid the m onitoring o f the condition o f animals displaying ill-health. These data will be retained in the archives. Page 4 : 229 : Company Sanitized. Does not contain TSCA CBI Stody Number : DPT/443 Protocol Amendment Number : 2 (two) DPT 443/984760 H untingdon Life Sciences TO X ICITY STUDY BY O RA L A DM INISTRATION T O CD R A TS F O R 4 W EEK S Total num ber of pages: 4 N um ber of pages for internal distribution: 4 Study Director : S M Botlomley, B.Sc. (Hons), M.Sc., C.Biol., Mi-Biol. The signature of the Study Director authorises the implementation o f this amendment to protocol. In this amendment, deleted statements are struck through and new statements are underlined. Any rhangpc to the study design after the date o f this authorising signature will be documented in a further formal amendment. AMENDMENT APPROVAL F o r Huntingdoi Authorised (Study Director) F or the Sponsor Approved by: Date: . Date: /4 9 f *4 f? ? 7 Pa ,e I : 230 : Company Sanitized. Does not contain TSCA CBI Study N um ber : DFT/443 Protocol A m e n d m e n t N u m b er : 2 (two) DPT 443/984760 H u n tin g d o n Life Sciences TOX ICITY STUDY BY ORAL A DM INISTRATIO N T O CD RATS F O R 4 W EEKS Reasons fo r amendm ents : 1. Change o f Study Director due to maternity leave. 2. Addition o f the examination o f the kidneys, liver and bone marrow from all animals from the low and intermediate dosage group in order to further evaluate the effects seen in the high dose level animals. Addition o f information to the scheduled time plan because o f the additional work. A m endm ents 1 INTRODUCTION M anagement o f study Study Directoiforiginal): 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 ofthis amendment to protocol. Any changes to the study design after the date o f this approval signature will be documented in a further formal amendment. Page 2 : 231 : Company Sanitized. Does not contain TSCA CBI DPT 443/984760 Stndy Num ber : DPT/443 Protocol A m endm ent N um ber : 2 (two) H untingdon Life Sciences Reason for amendment: Declaration Orianal Study Director Replacement Study Director The original Study Director (S M Bottomley) is taking maternity leave 1am satisfied with the conduct o f the study to date. js........... Date: I am satisfied with the conduct o f the study to date. From the date of my signature on this amendment, I assume responsibilities o f the Stndy Director. S ig n a tu re :...... ................................................................. Date: For Huntingdon Life Sciences Approved by:_ (Replacement Study Director) ________________ Date: 7 / Y W f k F ft0 * . Released by:_ Date: 2 - U t j c L . K f f 2. STUDY SCHEDULE AND STRUCTURE Z 2 Scheduled time plan Sample o; ived . Animals to arrive Treatment to commence Terminal sacrifice to commence Histopathology to be completed Additional histonathology to be completed Draft report to be issued 23 June 1998 2 December 1998 11 December 1998 8 January 1999 5 March 1999 16 April 1999 Morph 30 April 1999 (estimated) (estim ated ) Page 3 : 232 : Company Sanitized. Does not contain TSCA CBI Study Number : DPT/443 Protocol Amendment Number : 2 (two) DPT 443/984760 H untingdon Life Sciences 8. PATHOLOGY 8.1 L ight m icroscopy . Category Premature deaths Terminal sacrifice Terminal sacrifice Terminal sacrifice A nim als All from all groups. All animals o f Groups 1 and 4. All animals o f Groups 2 and 3. All animals o f Grouns 2 and 3. Tissues All specified in Table 1. All specified in Table 1. Abnormalities only Kidnevs. liver and bone marrow Peer review : Carried out by a reviewing pathologist to internationally accepted standards. Pag' 4 233 Company Sanitized. Does not contain TSCA CBI DPT 443/984760 % Study Num ber : DPT/443 Protocol Amendment Num ber : 3 Huntingdon Life Sciences TOXICITY STUDY BY ORAL ADMINISTRATION T O CD RATS FO R 4 WEEKS Total number o f pages: 2 Num ber of pages for internal distribution: 2 Study Director : Helen A Palmer The signature o f the Study Director authorises the implementation o f 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 F or Huntingdon Life Sciences Ltd Authorised by: TAmp-7 ____________ Date: ZT2. T o J 'J - lt~fCH<~l (Study Director) For the Sponsor Approved b y :__ Date: 3 o i* m Page 1 234 : Company Sanitized. Does not contain TSCA CBI DPT 443/984760 % Study Number : DPT/443 Protocol Amendment Number : 3 H u n tin g d o n Life Sciences TOXICITY STUDY BY ORAL ADMINISTRATION TO CD RATS FO R 4 W EEKS frrgm -m W nt : Addition o f 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 haematology. All samples will be examined for the following characteristics: Using lithium heparin as anticoagulant - Alkaline phosphatase Alanine amino-transferase (GPT) Aspartate amino-transferase (GOT) Gamma glutamyl transpeptidase Glucose Bilirubin - total Cholesterol-total Triglycerides Creatinine Urea nitrogen Total protein Albumin by chemical assay Globulin - bv subtraction Albumin/globuiin ratio 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 R EPO RT NUM BER : DPT 442/992305 : 236 : Company Sanitized. Does not contain TSCA CBI D PT 442/992305 SEVEN DAY ORAL TOXICITY STUDY IN THE RAT EXPERIMENTAL PROCEDURE Groups o f six rat (three males and three females) were treated w i t h ^ m ^ ^ H ^ p t dosages o f 250, 500 or 750 m g/kg/day (Groups 2 to 4 respectively) formulated as 25, 50 and 75 mg/ml solutions in distilled water at a dosage volume o f 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) o f three males and three females received the vehicle alone at the same dose volume. Group Cage label/ colour code Treatment Dosages mg/kg/day No. of rats MF Rat numbers MF 1 White 2 Yellow 3 Blue 4 Red 3 3 1-3 13-15 250 3 3 4 -6 16-18 500 3 3 7 - 9 19-21 750 3 3 10-12 2 2 -2 4 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 o f commencement of treatment (Day 1), on Day 4 and 8. Food consumption was recorded on a weekly basis during the treatment period. A t p o st m ortem organ weights and the macroscopic appearance o f 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 Novem ber 1998 following the completion o f seven days of treatment. : 237 : Company Sanitized. Does not contain TSCA CBI DPT 442/992305 SEVEN DAY ORAL TOXICITY STUDY IN THE RAT RESULTS M O RTA LITY (Appendix 3) ' All o f the animals receiving 500 or 750 mg/kg/day were sacrificed or found dead in the period Days 3 - 6 o f the study. There were no unscheduled deaths amongst the animals receiving 250 mg/kg/day or the controls. CLIN ICA L SIGNS (Appendix 3) Salivation, immediately after dosing was noted in all treated animals. B O D Y W E IG H T (Table 1, Appendix 1) A group mean bodyweight loss over the first 3 days o f treatment was seen for all treated groups of anim al^ 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. FO O D CONSUM PTION (Table 2) Lower food consumption values were noted for all treated groups o f animals, when compared with the controls. O R G A N W EIG H TS (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 bodyweightaajusted 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 TH E RAT M A C R O SC O PIC PA TH O LO G Y (Table 4, Appendix 3) The macroscopic examinations performed revealed the following changes: K idneys - 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 m g/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 m g/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 m g/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 o f 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 mg/kg/day (a labelling point under the OECD guidelines). The next dosage level in relation to the labelling criteria is 15 mg/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 DPT 442/992305 SEVEN DAY ORAL TOXICITY STUDY IN THE RAT TABLE 1 Bodyweights - g roup m ean values (g) ' Day Group and dosage (mg/kg/day) 1M 2M Control 500 3M 750 4M 1000 IF 2F Control 500 3F 750 4F 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 1 2 2 123 105 8 190 142 167 129 Gain (g/rat) 1-4 1-8 24 - 8 -27 -38 59 1 0 " " 18 - 1 -2 -19 37 6 -- V : 241 : Company Sanitized. Does not contain TSCA CBI DPT 442/992305 SEVEN DAY ORAL TOXICITY STUDY IN THE RAT TABLE 2 Food consumption - group mean values (g/rat/day) Day 1 Group and dosage (mg/kg/day) 1M 2M Control 500 3M 750 4M 1000 IF Control 2F 500 3F 4F 750 1000 25 15 6 6 22 13 11 8 : 242 : Company Sanitized. Does not contain TSCA CBI DPT 442/992305 SEVEN DAY ORAL TOXICITY STUDY IN THE RAT TABLE 3 O rgan weights - group m ean values Terminal kill Group/ dosage mg/kg/day Body wt g Liver Spleen Kidneys gg g 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 Liver Spleen Kidneys gg g Unadjusted means IF Control 162 10.7 0.49 1.83 2F 500 128 8.5 0.38 2.94 Adjusted means IF - 8.8 - - 2F - 10.3 - - : 243 : Company Sanitized. Does not contain TSCA CBI D PT 442/992305 SEVEN DAY ORAL TOXICITY STUDY IN THE RAT TABLE 4 M acroscopic pathology incidence sum m ary - Term inal Removal reason: Texminal Incisors Lower pale Animals on study Animals completed Kidneys Pale Pale subcapsular area/s Enlarged Group Group 12 --- M a l e s -- ^ 3 '3 33 Group Group 12 -- Females -- 33 33 0 0 11 0 1 02 0 1 oo 0 303 : 244 : Company Sanitized. Does not contain TSCA CBI DPT 442/992305 SEVEN DAY ORAL TOXICITY STUDY IN THE RAT TABLE 4 M acroscopic pathology incidence sum m ary - Decedents Removal reason: Intercurrent Animals on study Animals completed Pur Stained - perinasal region Stained - periorbital region/s Stained - perioral region Moist - genital region Skin Alopecia Thymus Congested Adipose Tissue Minimal Spleen Small Stomach Contents dark Contents watery Forestomach Thickened Roughened Stomach Corpus Mucosa Haemorrhagic depression/s Group Group 34 --- M a l e s --- 33 3 ,3 Group Group 34 -- Females -- 33 33 00 01 02 00 2 .0 00 0 0 0 0 1 1 1 0 1 2 2 0 1 02 0 2 20 2 0 10 o 0 20 0 0 o. 0 n 0 0 1 1 1 20 0 : 245 : Company Sanitized. Does not contain TSCA CBI D P T 442/992305 SEVEN DAY ORAL TOXICITY STUDY IN THE RAT TABLE 4 (M acroscopic pathology incidence sum m ary - Decedents - continued) : 246 : Company Sanitized. Does not contain TSCA CBI DPT 442/992305 SEVEN DAY ORAL TOXICITY STUDY IN THE RAT APPENDIX 1 Bodyweights - individual values (g) Group 1M: Control Cage Animal number number i1 2 3 Day -7 1 4 g 73 128 155 194 77 138 161 196 79 128 148 178 Group 3M: 750 mg/kg/day Cage Animal number number 37 8 9 Day -7148 77 136 113 80 147 125 74 133 98 Group IF: Control Cage Animal number number 5 13 14 15 Day -7 1 48 77 128 142 160 71 134 157 180 78 126 144 162 Group 2M: 500 mg/kg/day Cage Animal number number 24 5 |6 Day "7 1 4 8 80 129 131 151 76 138 127 146 74 130 115 129 Group: 4M .1000 mg/kg/day Cage Animal number number 4 10 11 | 12 Day -71 80 139 75 132 76 143 48 100 Group: 2F 500 mg/kg/day Cage Animal number number 6 16 17 18 Day -7 1 48 72 118 119 131 80 129 131 139 75 121 115 117 Group 3F: 750 mg/kg/day Cage Animal number number 7 19 20 21 Day -7 1 4 73 126 127 73 119 113 78 131 128 8 Group 4F: 1000 mg/kg/day Cage Animal number number 8 22 23 24 ________ 1 Day -7 1 4 82 132 106 73 127 108 72 112 103 8 : 247 : Company Sanitized. Does not contain TSCA CBI D PT 442/992305 SEVEN DAY ORAL TOXICITY STUDY IN THE RAT APPENDIX 2 O rgan weights - individual values Terminal kill Group/ dosage mg/kg/day Animal no. Body wt g Liver Spleen Kidneys gg g 1M Control 1 191 11.0 0.51 2.03 2 196 10.6 0.50 1.81 3 174 9.2 0.59 1.66 Mean sd 187 11.4 10.2 0.54 1.83 0.98 0.049 0.185 2M 500 4 148 10.6 0.38 2.50 5 144 11.3 0.42 2.49 6 127 9.0 0.33 3.03 Mean sd 140 10.9 10.3 0.38 2.67 1.15 0.047 0.306 Group/ dosage mg/kg/day Animal no. IF Control 13 14 15 Body wt g Liver Spleen Kidneys gg g 156 172 157 9.3 12.7 10.1 0.49 0.53 0.44 1.80 2.06 1.63 Mean sd 162 9.0 10.7 0.49 1.83 1.76 0.043 0.217 2F 500 16 128 8.9 0.40 2.74 17 139 8.7 0.39 2.49 18 118 7.8 0.34 3.60 Mean sd sd Standard deviation 128' 10.2 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 m DPT 442/992305 SEVEN DAY ORAL TOXICITY STUDY IN THE RAT APPENDIX 3 (Pathology - continued) Compound: Dosage Level: RatN/Sex: Control M (Terniinal) CLINICAL FINDINGS No signs of ill health or behavioural change were noted. M ACROSCOPIC FINDINGS No abnormalities detected 250 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 2M (Terminal) CLINICAL FINDINGS No signs o f ill health or behavioural change were noted. M ACROSCOPIC FINDINGS No abnormalities detected : 251 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 3M (Terminal) CLINICAL FINDINGS No signs o f ill health or behavioural change were noted. M ACROSCOPIC FINDINGS No abnormalities detected 252 Company Sanitized. Does not contain TSCA CBI D P T 442/992305 SEVEN DAY ORAL TOXICITY STUDY IN THE RAT APPENDIX 3 (Pathology - continued) Compound: Dosage Level: Rat-No/Sex: 500 mg/kg/day 4M (Terminal) CLINICAL FINDINGS Salivation immediately after dosing on Days 2 - 7 and walking on toes 1 hour after dosing on Day 5 only were noted. M ACROSCOPIC 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 CBI DPT 442/992305 SEVEN DAY ORAL TOXICITY STUDY IN THE RAT APPENDIX 3 (Pathology - continued) Compound: Dosage Level: RatNo/Sex: 500 mg/kg/day 5M (Trminal) CLINICAL FINDINGS Salivation immediately after dosing on Days 1,2, 6 and 7 was noted. M ACROSCOPIC 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: RatNo/Sex: 500 mg/kg/day 6M (Terminal) 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: RatNo/Sex: 1 750 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 TOXICITY STUDY IN THE RAT APPENDIX 3 (Pathology - continued) Compound: Dosage Level: Rat No/Sex: 750 mg/kg/day 8M (Intercurrent) 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) Compound: 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 Stom ach Corpus M ucosa Haemorrhagic depression/s: (A few) 1mm Caecum Dark: (Minimal) K idneys 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) Compound: Dosage Level: Rat No/Sex: 1000 mg/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 (Intercurrent) 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 aroundjaw 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 UP 1 442/992305 3 SEVEN DAY ORAL TOXICITY STUDY IN THE RAT APPENDIX 3 (Pathology - continued) Compound: Dosage Level: Rat No/Sex: 1000 mg/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: RatNo/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 u r i 44z/yy^ius SEVEN DAY ORAL TOXICITY STUDY IN THE RAT APPENDIX 3 (Pathology - continued) Compound: Dosage Level: Rat No/Sex: Control 14F (Terminal) 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: RatNo/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 mg/kg/day 16F (Terminal) C U B IC A L FIN D IN G S Salivation immediately after dosing on Days 2, 3 and 5 - 7 , unsteady gait approximately 1 hour after dosing on Day 5 only and paddling of forelimbs 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) clM cal find ing s Salivation immediately after dosing on Day 6 only was noted. Incidental finding o f hair loss was noted. m acroscopic finding s 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: 500 mg/kg/day 18F (Terminal) CLINICAL FINDINGS Salivation immediately after dosing on Days 2,3 and 6 was noted. Piloerection was noted from Day 6. Incidental finding o f 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) Compound: Dosage Level: Rat No/Sex: 750 mg/kg/day 19F (Intercurrent) CLINICAL FINDINGS Day of death 6 Salivation immediately after dosing on Days 2 ,3 and 5 and walking on toes and paddling of forelimbs 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 IN THE RAT APPENDIX 3 (Pathology - continued) Compound: Dosage Level: Rat No/Sex: 750 mg/kg/day 20F (Intercurrent) 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, puoerection 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 TOXICITY STUDY IN THE RAT APPENDIX 3 (Pathology - continued) Compound: Dosage Level: Rat No/Sex: 750 mg/kg/day 2 IF (Intercurrent) 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 after 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: 3.335g All the other organs and tissues appeared normal. : 270 : Company Sanitized. Does not contain TSCA CBI DPT 442/992305 a SEVEN DAY ORAL TOXICITY STUDY IN THE RAT APPENDIX 3 (Pathology - continued) Compound: Dosage Level: Rat No/Sex: 1000 mg/kg/day 22F (Intercurrent) CLINICAL FINDINGS Day o f death 5 Salivation immediately after dosing on Days 2 - 4 was noted. Partiallyxlosed 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) Compound: Dosage Level: Rat No/Sex: 1000 mg/kg/day 23F (Intercurrent) 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 Forestomach Roughened Adrenals Congested ' : 272 : Company Sanitized. Does not contain TSCA CBI f1 SEVEN DAY ORAL TOXICITY STUDY IN THE RAT DPT 442/992305 APPENDIX 3 (Pathology - continued) Rat No/Sex: 23F - continued MACROSCOPIC FINDINGS - continued Kidneys Pale: (Minimal) Enlarged: (Minimal) 1.981g 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: 1000 mg/kg/day 24F (Intercurrent) 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 oi 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 tady N um ber : DPT/442 C O N F ID E N T IA L PROTOCOL Huntingdon Life Sciences PROTOCOL PRELIM IN A R Y T O X IC IT Y STUDY BY O RA L A D M IN ISTR A TIO N T O CD RATS FO R 7 DAYS Sponsor DuPont Specialty Chemicals Jackson Laboratory Chambers Works Deepwater NJ 08023 USA 1> .al number of pages: 16 Final Protocol Research Laboratory Huntingdon Life Sciences Ltd POBox2 Huntingdon Cambridgeshire PE18 6ES ENGLAND Paget Huntingdon L ift Sciences Ltd. registered In E n g la a t 18/3730 : 275 : Company Sanitized. Does not contain TSCA CBI . DPT 442/992305 SEVEN DAY ORAL TOXICITY STUDY IN THE RAT Study Number :DPT/442 CONTACT DETAILS H U n tn g d O V 1 Life Sciences Sponsor's Monitoring Scientist DrK.Dastur. F inti Protocol Page it : 276 : 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 PROTOCOL APPROVAL PRELIMINARY TOXICITY STUDY BY ORAL ADMINISTRATION TO CD RATS FOR 7 DAYS Study Director, Huntingdon Life Sciences Ltd. *7 4/ O /Z jJtJiM v ( ct ctiT Date The signature o f the Study Director confirms this protocol as the working document for die study. Any changes made subsequent to the date o f the Study Director's signature will be documented in formal amendments. R J. Sortwell M anagem ent, Huntingdon Life Sciences Ltd. Date __________ Dr K. Dasmr. Sponsor, DuPont Specialty Chemicals ?uw3o, ftir D" 6 Please sign both copies o fthispage, retain onefo r your records and return one to the Study Director at Huntingdon Life Sciences. Final Protocol Page iii 277 : Company Sanitized. Does not contain TSCA CBI D PT 442/992305 SEVEN DAY ORAL TOXICITY STUDY IN THE RAT Study Number : DPT/442 PRELIMINARY TOXICITY STU D Y BY ORAL ADMINISTRATION TO CD RATS FOR7 DAYS Enquiry Number 17556N Number o f pages for internal distribution: 13 This working document is approved for circulation and use: P rim ary location o f study Huntingdon Research Centre Huntingdon Cambridgeshire Building Number. 1BRB All procedures to be performed at the above site. Date Final Protocol Page I : 278 : Company Sanitized. Does not contain TSCA CBI D PT 442/992305 SEVEN DAY ORAL TOXICITY STUDY IN THE RAT Stady N um ber : DPT/442 CONTENTS 1. INTRODUCTION 2. STUDY SCHEDULE AND STRUCTURE 2.1. Duration o ftreatment 2 2 . Scheduled tim e pirn 2 J . Identity o ftreatment groups 3. TEST SUBSTANCE AND FORMULATION 3.1. Test substance 3 2 . Formulation 2 3 . Quality control ofdosage form 4. ANIMAL MANAGEMENT 4.1. Animals - supply, scclimatisatioo end allocation 4 2 . Animals - bousing, diet and water supply 4.3. Animals-procedures 4.4. Animals - termination 5. NECROPSY AND HISTOLOGY 5.1. Method o f kill 5 2 . Macroscopic Pathology 53 . Organ weights 5.4. Fixation 6. REPORTING 7. QUALITY ASSURANCE AND ARCHIVING PROCEDURES 7.1. Quality Assurance 12. Archives Huntingdon Life Sciences Page 3 4 4 4 4 5 6 6 6 7 7 8 9 11 12 12 12 12 12 13 13 13 13 F inal Protocol Page 2 : 279 : Company Sanitized. Does not contain TSCA CBI DPT 442/992305 SEVEN DAY ORAL TOXICITY STUDY IN THE RAT S tody N um ber : DPT/442 Huntingdon Life Sciences 1. INTRODUCTION M anagem ent o f study Study D irector Monitoring Toxicologist In the temporary absence o f the Study Director, the scientific responsibilities will be taken over by the Monitoring Toxicologist; other hems of routine study management should be referred to the following person in the first instance. : S.M. Bottomley. : W N. Hooks. : M il Barker. O bjective Assessment of systemic toxic potential in a 7 day oral gavage study in CD rats, to select a suitable high dosage for a subsequent 4 week study. Good L aboratory P ractice 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 (OfficiaUoumal 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. j t o nte : Oral gavage, to simulate the conditions o f potential human exposure. Final Protocol Page 3 : 280 : Company Sanitized. Does not contain TSCA CBI DPT 442/992305 SEVEN DAY ORAL TOXICITY STUDY IN THE RAT Study Num ber : DPT/442 Huntingdon Life Sciences Treatm ent gronpa and dosage* Group Compound " Dosage (mg/kg/day)t : : 1 Control 0 250 500 750 t Expressed in tenns o f solids. The test substance as supplied is 25% solids in 75% water. 2. STUDY SCHEDULE AND STRUCTURE 2.1. D uration of treatm ent Minimum period: 7 days oftreatment. All surviving animals will be killed on Day 8. 2 2 . Scheduled time plan Sample o f Zooyl FS-62 anived Animais to arrive Tieatment to commence Terminal sacrifice to commence 23 June 1998 4 November 1998 11 November 1998 17November 1998 7 a Identity o f treatm ent groups (to be selected from 38 animals ordered) G roup Treatm ent 1 Vehicle control Dosage (m g/ltg/day)t 0 2 250 3 M M iiP 500 4 750 Dosage* (mg/kg/day)# 0 1000 2000 3000 N um ber o f animals M ale F em ale 33 33 33 33 Expressed in terms of the test substance'as supplied. _ | terms o f solids. Die test substance as supplied ii^ Dosages (mg^g/day) selected with reference to existing toxicological data. Final Protocol Page 4 : 281 : Company Sanitized. Does not contain TSCA CBI DPT 442/992305 SEVEN DAY ORAL TOXICITY STUDY IN THE RAT Study N um ber : DPT/442 Huntingdon Life Sciences G roup -1 Cage anmbers M ale Female 15 2 26 3 37 4 48 Health screen Animal numbers Male Female 1-3 13-15 4-6 16-18 7-9 19-21 10-12 22-24 25-28 29-32 3. TEST SUBSTANCE AND FORMULATION In order for Huntingdon Life Sciences to comply wife fee Health and Safety at Work etc. Act 1974, nHfee Control o f Substances Hazardous to Health Regulations 1994, it is a condition o f undertaking the study that the Sponsor shall provide Huntingdon Life Sciences wife all information available to it regarding known or potential hazards associated wife fee handling and use o f any substance supplied by fee Sponsor to Huntingdon Life Sciences. The Sponsor shall also comply with all curent legislation and regulations concerning shipment of substances by road, rail, sea or air. Such information in the form o f a completed Huntingdon Life Sciences test substance data sheet must be received by Safety Management Sendees at Huntingdon Life Sciences before fee test substance can be handled in fee laboratory. At fee discretion of Safety Management Services at Huntingdon Life Sciences, other documentation containing fee equivalent information may be acceptable. Information received will be used to set fee Huntingdon Life Sciences Hazard Class, which determines safety precautions taken in fee workplace. Huntingdon Life Sciences Hazard Class: 2 Final Protocol Page 5 : 282 : Company Sanitized. Does not contain TSCA CBI DPT 442/992305 SEVEN DAY ORAL TOXICITY STUDY IN THE RAT S tudy N m nber : DPT/442 Huntingdon Life Sciences 3 J . T est substance Sponsor's identification Storage conditions Sponsor's responsibilities C ertificate o f analysis deoils At room tem perature. Documentation o f methods o f synthesis, fabrication o r derivation. _ . Stability data. . C ertificate o f analysis. Test substance identity. Batch num ber (Lot S 3). Purity. C om position. O ther appropriate characteristics. Current expiry date: (2 Years from manufacture). 3 3 . Form ulation Treatm ent Croup 1, Vehicle control Group 2 Group 3 Group 4 Conversion factor V ehicle M ethod o f preparation Frequency o f preparation V ehicle. The test sub substance i: been 00 mg/ml. mg/ml. 100 mg/ml. icc w ill be used as a led. The test es have for me w ater content D istilled water. W ill be documented. D aily. 3 3 . Q uality control ofdosage form Liquid formulation Before commencement o f treatm ent, the suitability o f the proposed mixing procedures will be determined by visual assessm ent. Final Protocol Page 6 : 283 : Company Sanitized. Does not contain TSCA CBI DPT 442/992305 SEVEN DAY ORAL TOXICITY STUDY IN THE RAT Huntingdon Life Sciences 4. ANIMAL MANAGEMENT 4.1. Animals nppiyi acclimatisation and allocation 4X 1. Animals Species Strain A ge ordered W eight range ordered Supplier nat CrltCD BR. 28 + 2 days. To be w ithin 11 g range for each sex (minimum 75 g). Charles River (UK) Limited. 4.1X H ealthscrcea An pAAMn n .l four m ales (animal numbers 25-28) and four fem ales (anim al num bers 29-32) w ill be ordered from the supplier. These w ill be killed, bled and subjected to macroscopic . exam ination immediately upon receipt Serum sam ples w ill be remined frozen pending possible future serology investigations (sam ples discarded after tw o months). Lungs, liver, kidneys, spleen and heart w ill be preserved in fixative, but not processed further unless m aeroscopieally abnorm al. M acroscopic abnorm alities will be immediately processed and exam ined microscopically. R esults o f the health screen w ill be reviewed before commencement o f treatm ent 4.13. A cclim atisation D uration : A t least 5 days (animals w ill be approxim ately 5 weeks o f age at commencement). Husbandry conditions : R efer to Section 4 2 . 4X 4. A llocation to treatm ent groups A llocation M ethod Approxim ately 1 week before commencement o f treatm ent Random allocation to cages to equalise variation in bodyw eight Final Protocol Page 7 284 : Company Sanitized. Does not contain TSCA CBI DPT 442/992305 SEVEN DAY ORAL TOXICITY STUDY IN THE RAT Study N u nber : DPT/442 Huntingdon Life Sciences 4.15. Identification Numbering Method Cage labels Unique for each animal within study. Cage number by foot tattoo, animal number by earmark. Uniquely identifying the occupants. 4.15. Precommencement animal replacement 6 spare animals will be ordered to replace any individuals rejected during the acclimatisation period. Replacement before treatment : Ill-health. Abnormalities. Bodyweight range extremes. On Day 1 (before dosing) variations in bodyweight of animals should not exceed 20% ofthe mean for each ~ Replacement during treatment : None scheduled. 4.2. Animals housing, diet and water sapply 4.2.1. Environm ental 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 lig h t: 12 hours dark. Activated on ventilation failure and when temperature/bumidity 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 TOXICITY STUDY IN THE RAT S tudy N um ber : DPT/442 Huntingdon Life Sciences 4 3 3 . Anim al accommodation Animals per cage Three ofsame sex, unless reduced by mortality or isolation. Cage materia] Cage flooring : Stainless steel. : Stainless steel grid. 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 dunged at appropriate intervals. Precise details o f caging will be indnded in the final report. 4.23. D iet and w ater (apply Copies o f all certificates of analysis ate stored in the archives. D iet (apply D iet name D iet type Availability Certification Rat and Moose No. 1 Maintenance D iet Pelleted diet N on-icstricted. 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 o f diet is released for use. This diet contains no added antibiotie or other chemotherapeutic or prophylactic agent W ater supply Supply Regulatory agency A vailability Public drinking water. UJC. Departmento f rite Environment Non-restricted via polyethylene or polycarbonate bottles with sipper tubes. Certification Certificates o f analysis are routinely received from the supplier. 4 3 .4 . Contam inants assay It is the Sponsor's responsibility to advise Huntingdon Life Sciences o f any specific contaminants likely to prejudice the outcome of the study. Analyses for such contaminants may be performed if requested by the Sponsor. 4 3 . Anim als - procedures Investigations noted as occurring on specified Day numbers (w jere quoted) will not be varied. Final Protocol Page 9 286 : Company Sanitized. Does not contain TSCA CBI DPT 442/992305 SEVEN DAY ORAL TOXICITY STUDY IN THE RAT Study Num ber : DPT/442 Huntingdon Life Sciences O l Administration Route Treated at Volume dosage Individual dose volume Controls (Group 1) Frequency Sequence Formulation -_ . 4 JJL Clinical obaervatioas Oral gavage. Constantdosages m mg/kg/day. lOml/kg. rlni1fwt fmm tlitm M t mcmtly recorded scheduled bodyweighL Vehicle at the same volume-dosage as treated groups. Once daily at approximately die same time each day. By group. A daily record ofthe usage o f formulation w ill be maintained based on weights. This balance is compared with the expected usage as a check o f correct administration. Sn^pgrcif)ftg are stirred using t magnetic stirrer before and throughout the dosing procedure. Animals and their cages : Inspected at least twice daily for evidence o f reaction to treatment or ill-health. Deviations from normal : recorded at the tim e in respect of . Nature and severity. Date and time o fonset Duration and progress o f the observed condition. Physical examination : Once during treatment 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-dose observation. 2. As each animal is returned to its home cage. 3. At the end o f dosing each group. 4. Between 1and 2 hours after completion o f 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. Final Protocol Page 10 : 287 : Company Sanitized. Does not contain TSCA CBI DPT 442/992305 SEVEN DAY ORAL TOXICITY STUDY IN THE RAT Stndy N um ber : DPT/442 Huntingdon Life Sciences 4 3 3 . M ortality Debilitated animals : Observed carefully, may be isolated to prevent cannibalism. Premature sacrifice : Animals may be killed on humane grounds or if consideredm extremis. Animals found dead, killed in : extremis or on humane grounds 4 3A . Bodyweigkt A necropsy is performed as soon as possible. Animals found outside the normal workday will be preserved in a refrigerator (approximately 4C) provided for this purpose. Bodyweight recording : Once immediately before treatment, once during the treatment week and on Day 8. More frequent weighing may be performed to aid the monitoring o f the condition o f animals - displaying iU-beahh. These data will be retained in the archives. 4 3 3 . Food consumption Food consumption recording : Food supplied : Food spOled : Food remaining : Week 1. At intervals. Estimated at cage paper dunging. Recorded at end o f study week. 4.3.6. W ater consumption Fluid intake will be assessed by daily visual observation. 4.4. A nim als - term ination All animals will be subject to terminal investigations (Section S). Final Protocol Page 11 : 288 : Company Sanitized. Does not contain TSCA CBI DPT 442/992305 SEVEN DAY ORAL TOXICITY STUDY IN THE RAT Study Number :DPT/442 , H lffltin g d O n Life Sciences 5. NECROPSY ANDHISTOLOGY 5.1. Medrad of lull Method S J. Macroscopic Pathology Carbon dioxide. Necropsy Checks Special requirements S 3 . O rgan weights All animals, thoracic t abdominal cavities opened, crania] cavity opened only if observations indicate possible neurotoxic action. Retained tissues. Macroscopic abnormalities retained and fixed. Organs to be weighed Data presentation 5.4. fixation Liver, kidneys and spleen. Organ weights are not routinely recorded for animals killed or dying prematurely. Absolute. Adjusted for terminal bodyweight . Standard 10% Neutral Buffered Formalin. Final Protocol Page 12 : 289 : Company Sanitized. Does not contain TSCA CBI DPT 442/992305 SEVEN DAY ORAL TOXICITY STUDY IN THE RAT Stndy Number : DPT/442 Huntingdon Life Sciences 6. REPORTING Study progress Reporting A summary of findings will be sent to the Study Monitor by fix on completion o f the in-life phase o f this study. The results will be reported in an appendix to tbe report fbr the subsequent 28-day study. 7. QUALITY ASSURANCE AND ARCHIVING PROCEDURES 7.1. Q uality A ssurance No formal study-based Quality Assurance procedures will be performed on this study. These may be included if requested by the Sponsor. 72. Archives A ll experimental data arising from the study (including documentary raw data, specimens, records, other materials; collectively defined as the "materials") will remain the property o f the Sponsor. ' Huntingdon Life Sciences shall retain the materials in its archive for a period o f 5 years after completion o f the study. After such time, the Sponsor will be contacted and their advice sought on the return, disposal or further retention of die 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 13 : 290 : Company Sanitized. Does not contain TSCA GBI DPT 442/992305 SEVEN DAY ORAL TOXICITY STUDY IN THE RAT Study Namber : DPT/442 Protocol Amendment Number : 1 (One) Huntingdon Life Sciences PRELIM IN A RY T O X IC IT Y STUDY BY O RA L A D M IN ISTRA TIO N T O C D RATS FO R 7 DAYS Total number of pages: 3 Number ofpages for internal distribution: 3 Study Director : S M Bottomley, B-Sc. (Hons), M.SC-, C B iol, M-I-BioL #. The signanue o f the Study Director authorises the implementation ofthis amendment to protocol. In this amendment, deleted statements are struck through and new statements are underlined. Any rhangee to the study design after the date of this authorising signature will be documented in a farther formal amendment AMENDMENT APPROVAL For Huntingdon Authorised b (Study Director) F or the Sponsor Approved by:__ Date: - IA.CU.CM ( Ct `j Ct Date: Iw u t/L . t&; / ? T? Page 1 : 291 : Company Sanitized. Does not contain TSCA CBI DPT 442/992305 SEVEN DAY ORAL TOXICITY STUDY IN THE RAT Study Number : DPT/442 Protocol Amendment Number : 1 (one) Huntingdon Life Sciences P R E U M E 'IS K Y TO X IC ITY 'ST D Y BY O RA L A D M IN ISTRA TIO N TO CD RATS FO R 7 DAYS Reasons for amendments : Change of Study Director due to maternity leave. Amendments i INTRODUCTION Management ofstudy Study Directoiforiginal): S M Bottomley Study Director(replacement): H A Palmer This amendment formally registers the assignment ofa replacement Study Director. The signature ofthe replacement Study Director approves the implementation ofthis amendmentto protocol. Any changes to the study design alter the date ofthis approval signature will be documented in a further formal amendment. Reason for amendment: Declaration The original Study Director (S M Bottomley) is talcing maternity leave Original Study Director I am satisfied with the conductofthe study to date. Sii Replacement Study Director 'ricif.......... Date:!. f I am satisfied with the conduct of the study to date. From the Hat*ofmy signature on this amendment, 1 assume responsibilities ofthe Study Director. Signature: ............................... Date: .. ..f l t a c k .. k t ? i . Page 2 : 292 : Company Sanitized. Does not contain TSCA CBI DPT 442/992305 SEVEN DAY ORAL TOXICITY STUDY IN THE RAT Stndy N um ber : DPT/442 Protocol Am endm ent Number : 1 (one) Huntingdon Life Sciences For Huntingdon Life Sciences Approved by:_ J A Q 'VaM L / (ReplacementStudy Director) Released by:_ Date: ~1 VYi&yrL l0 ! * ^ Dae: 3. Page 3 : 293 : Company Sanitized. Does not contain TSCA CBI