Document QJ0pE28njGeRnwR6jrZ6Ev55E

REPORT Study Title ASSESSMENT OF ACUTE ORAL TOXICITY WITH T-7868 IN THE RAT (ACUTE TOXIC CLASS METHOD) Author Drs. M.S. Teunissen Study completion date 20 April 2004 Test Facility NOTOX B.V. Hambakenwetering 7 5231 DD `s-Hertogenbosch The Netherlands Laboratory Project Identification NOTOX Project 389598 NOTOX Substance 132003/A - Page 1 of 15 - v5 T-7868 NOTOX Project 389598 1. CONTENTS 1. CONTENTS........................................ 2, 2. STATEMENT OF GLP COMPLIANCE.................................................................................... 3 3. QUALITY ASSURANCE STATEMENT...................................................................................4 4. SUMMARY................ Th............................................................................................................5 5. INTRODUCTION......................................................................................................................6 5.1. Preface..............................................................................................................................6 5.2. Aims of the study.............................................................................................................. 6 5.3. Guidelines.........................................................................................................................7 5.4. Storage and retention of records and materials...............................................................7 6. MATERIALS AND METHODS................................................................................................. 8 6.1. Test Substance.................................................................................................................8 6.1.1. Test Substance...................................................................................................... 8 6.1.2. Test substance preparation.................................................................. 8 6.2. Test System......................................................................................................................8 6.3. Animal husbandry............................................................................................................ 9 6.4. Study design......................................................................................................................9 6.5. Treatment..........................................................................................................................9 6.6. Observations...................................................................................................................10 6.7. Interpretation.......................................................................... 10 6.8. List of protocol deviations............................................................................................... 10 7. RESULTS......................................................... .................................................................... 11 7.1. Mortality (Table 1 ) ...........................................................................................................11 7.2. Clinical Signs (Table 2 ) ................... ............................................................................. 11 7.3. Body Weight (Table 3 ) .................................................................................................. 11 7.4. Macroscopic Findings (Table 4 )....................................................................................11 8. CONCLUSION........................................................................................................................11 Tables TABLE 1 : MORTALITY DATA..................................................................................................... 12 TABLE 2 : CLINICAL SIGNS........................................................................................................ 12 TABLE 3 : BODY WEIGHTS (GRAM)..........................................................................................14 TABLE 4 : MACROSCOPIC FINDINGS........................................................................................15 - Page 2 - T-7868 NOTOX Project 389598 2. STATEMENT OF GLP COMPLIANCE NOTOX B.V., 's-Hertogenbosch, The Netherlands The study described in this report has been correctly reported and was conducted in compliance with: The Organization for Economic Cooperation and Development (OECD) Good Laboratory Practice Guidelines (1997). Which essentially conform to: The United States Food and Drug Administration Good Laboratory Practice Regulations. The United States Environmental Protection Agency Good Laboratory Practice Regulations. NOTOX B.V. Drs. M.S. Teunissen Study Director W.J.A.M. Frieling DVM Director of Toxicology Date: .X.i - Page 3 - T-7868 NOTOX Project 389598 3. QUALITY ASSURANCE STATEMENT NOTOX B.V., `s-Hertogenbosch, The Netherlands This report was inspected by the NOTOX Quality Assurance Unit to confirm that the methods and results accurately and completely reflect the raw data. The dates of Quality Assurance inspections are given below. During the on-site process inspections procedures applicable to this type of study were inspected Type of inspections Phase / Section Protocol (Study) On-site (Process) Report (Study) SPF Unit Pathology Start Inspection date(s) 27 AUG 03 07 JUL 03 11 AUG 03 01 DEC 03 End Inspection date(s) 27 AUG 03 11 JUL 03 25 AUG 03 01 DEC 03 Reporting date 27 AUG 03 15 JUL 03 25 AUG 03 01 DEC 03 Head of Quality Assurance C.J.Mitchell B.Sc. 'y"'> - Date: . . Q ^ . . . < ? . h ^ ?.*?.. 4-sokt X - Page 4 - <S T-7868 NOTOX Project 389598 4. SUMMARY Assessment of acute oral toxicity with T-7&68 in the rat (Acute Toxic Class Method). The study was carried out based on the guidelines described in: "Acute Toxicity-Oral, Acute Toxic Class Method", OECD No.423 (1996); "Acute Oral Toxicity"; EC Commission Directive 96/54/EC, Part B.1 tris (1996); Environmental Protection Agency (EPA): Health Effects Test Guidelines OPPTS 870.1100 (1996), "Acute Oral Toxicity - Acute Toxic Class Method" and JMAFF Japanese test guidelines (2000). Initially, T-7868 was administered by oral gavage to three female Wistar rats at 2000 mg/kg body weight. In a stepwise procedure additional groups of animals were dosed at 200 (males and females) mg/kg body weight. All animals were subjected to daily observations and weekly determination of body weight. Macroscopic examination was performed on the day of death or after terminal sacrifice (day 15). Two females dosed at 2000 mg/kg were found dead on days 1 or 2. No further mortality occurred. At 2000 mg/kg the females showed lethargy, ventro-lateral recumbency, hunched posture, uncoordinated movements, flat gait, shallow respiration, piloerection and salivation between days 1 and 4. At 200 mg/kg the females showed lethargy, hunched posture, uncoordinated movements, piloerection and/or ptosis on days 1 and 2. Two males showed hunched posture immediately after treatment only. The body weight gain shown by the remaining animals over the study period was considered to be normal. Macroscopic post mortem examination of one animal that was found dead revealed a stomach distended with gas and dark red discolouration of the mucosa. Macroscopic post mortem examination of the remaining animals did not reveal any test substance related abnormalities. The oral LD50value of T-7868 in Wistar rats was established to be within the range of 200-2000 mg/kg body weight. Based on these results and according to the EC criteria for classification and labelling requirements for dangerous substances and preparations (Council Directive 67/548/EEC), T-7868 should be labelled as: harmful if swallowed (R22). - According to the OECD 423 test guideline, the LD50 cut-off values were considered to be within 300-2000 mg/kg body weight. - According to these LD50 cut off values and based on the OECD Harmonized Integrated Hazard Classification System for Human Health and Environmental Effects of Chemical Substances (OECD, 1998), T-7868 should be classified in Class 4 for acute toxicity by the oral route. - Page 5 - T-7868 5. INTRODUCTION 5.1. Preface Sponsor Study Monitor Test Facility NOTOX Project 389598 3M Center Building 234-1B-10 ST. PAUL, MINNESOTA 55144 U.S.A. Dr. P.H. Lieder 3M Corporate Toxicology 3M Center, Building 220-2E-02 P.O. Box 33220 ST. PAUL, MINNESOTA 55133-3220 U.S.A. NOTOX B.V. Hambakenwetering 7 5231 DD 's-Hertogenbosch The Netherlands Study Director Study Plan Drs. M.S. Teunissen Start : 10 September 2003 Completion : 30 September 2003 5.2. Aims of the study The objective of this study was to assess the toxicity of the test substance when administered in a single dose to rats of both sexes at one or more defined dosages. Furthermore, the results of the study allowed the test substance to be ranked according to most classification systems, currently in use. This study should provide a rational basis for risk assessment in man. The oral route was selected as it is a possible route of human exposure during manufacture, handling or use of the test substance. - Page 6 - 8 T-7868 NOTOX Project 389598 5.3. Guidelines As required by the Dutch Act on Animal Experimentation, the study protocol was reviewed and agreed by the Article 14-functionary and the Ethical Committee of NOTOX (DEC NOTOX 97-0314) as required by the Dutcfi'Act on Animal Experimentation (February 1997). The study procedures described in this report were based on the following guidelines: Organisation for Economic Co-operation and Development (OECD), OECD Guidelines for Testing of Chemicals, Section 4, Health Effects. No.423, "Acute Oral Toxicity - Acute Toxic Class Method", Paris Cedex, 1996. European Community (EC), Council Directive 67/548/EEC, Annex V, Part B, Methods for the Determination of Toxicity, as last amended by Commission Directive 96/54/EC, Annex IV B, B.1 tris: "Acute Toxicity (Oral) - Acute Toxic Class Method". Official Journal of the European Communities No. L 248, 1996. United States Environmental Protection Agency (EPA). Health Effects Test Guidelines, OPPTS 870.1100, Acute Oral Toxicity. Office of Prevention, Pesticides and Toxic Substances (7101), EPA 712-096-190, June 1996. Japanese Ministry of Agriculture, Forestry and Fisheries (JMAFF), 12 Nousan, Notification No 8147, November 2000, including the most recent partial revisions. 5.4. Storage and retention of records and materials Records and materials pertaining to the study including protocol, raw data, specimens (except specimens requiring refrigeration or freezing) and the final report are retained in the NOTOX archives for a period of at least 10 years after finalization of the report. After this period, the sponsor will be contacted to determine whether raw data and specimens should be returned to them, retained or destroyed on their behalf. Those specimens requiring refrigeration or freezing will be retained by NOTOX for as long as the quality of the specimens permits evaluation but no longer than three months after finalization of the report. NOTOX will retain a test substance sample until the expiry date, but no longer than 10 years after finalization of the report. After this period the sample will be destroyed. - Page 7 ? T-7868 NOTOX Project 389598 6. MATERIALS AND METHODS 6.1. Test Substance 6.1.1. Test Substance The sponsor is responsible for all test substance data unless determined by NOTOX. Identification Molecular formula Molecular weight Description Batch Composition Test substance storage Stability under storage conditions Expiry date Specific Gravity Stability in vehicle Water 1% Aq. Carboxymethyl cellulose Corn oil Propylene glycol Polyethylene glycol Methyl ethyl ketone Dimethyl sulphoxide Ethanol Acetone Olive oil Dimethyl formamide T-7868 346 Light yellow hazy liquid Lot 1 78 % (22 % water) At room temperature in the dark Stable 07 November 2005 1.4 At least 96 h Not indicated Not indicated Not indicated Not indicated Not Indicated Not indicated Not indicated Not indicated Not indicated Not indicated 6.1.2. Test substance preparation Preparation The test substance was dosed undiluted as delivered by the sponsor. 6.2. Test System Species Number of animals Age and body weight Identification Rat, Wistar strain Crl:(WI) BR (outbred, SPF-Quality). Recognised by international guidelines as the recommended test system (e.g. OECD, EC). Source: Charles River Deutschland, Sulzfeld, Germany. 9 Animals. Each dose group consisted of 3 animals of one sex (females were nulliparous and non-pregnant). Young adult animals (approx. 8 weeks old) were selected. Body weight variation did not exceed +/- 20% of the sex mean. Earmark. - Page 8 - T-7868 NOTOX Project 389598 6.3. Animal husbandry Conditions Animals were housed in a controlled environment, in which optimal conditions were considered to be approximately 15 air Changes per hour, a temperature of 21.0 3.0C (actual range: 18.5 - 22.8C), a relative humidity of 30-70% (actual range: 34 - 73%) and 12 hours artificial fluorescent light and 12 hours darkness per day. Cleaning procedures in the room might have caused the temporary fluctuations above the optimal maximum level of 70% for relative humidity. Based on laboratory historical data, these fluctuations were considered not to have affected the study integrity. Accommodation Group housing of 3 animals per sex per cage in labelled Macrolon cages (type IV; height 18 cm.) containing purified sawdust as bedding material (SAWI, Jelu Werk, Rosenberg, Germany). Certificates of analysis were examined and then retained in the NOTOX archives. Acclimatisation period was at least 5 days before start of treatment under laboratory conditions. Diet Free access to standard pelleted laboratory animal diet (from Altromin (code VRF 1), Lage, Germany). Certificates of analysis were examined and then retained in the NOTOX archives. Water Free access to tap-water. Certificates of quarterly analysis were examined and then retained in the NOTOX archives. 6.4. Study design The toxicity of the test substance was assessed by stepwise treatment of groups of 3 animals. The first group was treated at a dose level of 2000 mg/kg body weight. The absence or presence of mortality of animals dosed at one step determined the next step, based on the test procedure defined in the guidelines. The onset, duration and severity of the signs of toxicity were taken into account for determination of the time interval between the dose groups. 6.5. Treatment A health inspection was performed prior to commencement of treatment, to ensure that the animals were in a good state of health. Method Oral gavage, using a stainless steel stomach tube. Fasting Food was withheld overnight (for a maximum of 20 hours) prior to dosing until 3-4 hours after administration of the test substance. Frequency Single dosage, on day 1. Dose level (volume) 2000 mg/kg (1.43 ml/kg) body weight. 200 mg/kg (0.14 ml/kg) body weight. Dose volume calculated as dose level : specific gravity. - Page 9 - T-7868 NOTOX Project 389598 6.6. Observations MortaiityA/iability Twice daily. The time of death was recorded as precisely as possible. Body weights Days 1 (pre-administration), 8 and 15 and at death (if found dead after day 1). Clinical signs At periodic intervals on the day of dosing (day 1) and once daily thereafter, until day 15. The symptoms were graded according to fixed scales and the time of onset, degree and duration were recorded: Maximum grade 4: grading slight (1) to very severe (4) Maximum grade 3: grading slight (1) to severe (3) Maximum grade 1: presence is scored (1). Necropsy The surviving to the end of the observation period were sacrificed by asphyxiation using a oxygen/carbon dioxide procedure. All animals assigned to the study were subjected to necropsy and descriptions of all internal macroscopic abnormalities recorded. 6.7. interpretation The oral LD50value of the test substance was ranked within the following ranges: 0-25, 25-200 or 200-2000 mg/kg b.w. or as exceeding 2000 mg/kg b.w. No statistical analysis was performed (The method used is not intended to allow the calculation of a precise LD50 value). The results were evaluated according to the OECD Harmonized Integrated Hazard Classification System for Human Health and Environmental Effects of Chemical Substances (OECD, 1998) and the EC criteria for classification and labelling of dangerous substances and preparations (Council Directive 67/548/EEC and all adaptations to technical progress and amendments of this Directive published in the Official Journal of the European Communities). 6.8. List of protocol deviations No protocol deviations were made during this project. - Page 10 - T-7868 NOTOX Project 389598 7. RESULTS 7.1. Mortality (Table 1) The incidence of mortality w&s as follows, presented in chronological order of treatment: Dose level Mortality Sex Date of treatment 2000 mg/kg 2/3 females 10 September 2003 200 mg/kg 0/3 females 12 September 2003 200 mg/kg 0/3 males 16 September 2003 The decedents were found on days 1 or 2. 7.2. Clinical Signs (Table 2) Clinical signs observed during the study period were as follows Dose level Clinical signs 2000 mg/kg (females) Lethargy, ventro-lateral recumbency, hunched posture, uncoordinated movements, flat gait, shallow respiration, piloerection and/or salivation 200 mg/kg (females) Lethargy, hunched posture, uncoordinated movements, piloerection, and/or ptosis. 200 mg/kg (males) Hunched posture. The surviving animals had recovered from the symptoms by between days 1 and 5. 7.3. Body Weight (Table 3) The body weight gain shown by the surviving animals over the study period was considered to be similar to that expected of normal untreated animals of the same age and strain 7.4. Macroscopic Findings (Table 4) At macroscopic post mortem examination a stomach distended with gas and dark red discolouration of the glandular mucosa was found in one animal that died during the study. Macroscopic examination of the other animal that died during the study and of the surviving animals at termination did not reveal any test substance related abnormalities. Severe autolysis was noted in one animal that died during the study. 8. CONCLUSION The oral LD50value of T-7868 in Wistar rats was established to be within the range of 200-2000 mg/kg body weight. Based on these results and according to the EC criteria for classification and labelling requirements for dangerous substances and preparations (Council Directive 67/548/EEC), T-7868 should be labelled as: harmful if swallowed (R22). - According to the OECD 423 test guideline, the LD50 cut-off values were considered to be within 300-2000 mg/kg body weight. - According to these LD50 cut off values and based on the OECD Harmonized Integrated Hazard Classification System for Human Health and Environmental Effects of Chemical Substances (OECD, 1998), T-7868 should be classified in Class 4 for acute toxicity by the oral route. - Page 11 - /3 T-7868 NOTOX Project 389598 TABLE 1 : MORTALITY DATA TEST DAY HOURS AFTER TREATMENT FEMALES 2000 MG/KG FEMALES 200 MG/KG MALES 200 MG/KG 1 1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 ^ ___________ 0 2 4__________________________________________________ - - 1 1 .................................................................................... ............................................................................................................... ............................................................................................................... TABLE 2 : CLINICAL SIGNS TEST DAY HOURS AFTER TREATMENT 1 1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 MAX 0 2 4 GRADE FEMALES 2000 MG/KG ANIMAL 1 BEHAVIOR LETHARGY POSTURE VENTRO-LATERAL RECUMBENCY HUNCHED POSTURE BREATHING SHALLOW RESPIRATION ANIMAL 2 BEHAVIOR LETHARGY POSTURE VENTRO-LATERAL RECUMBENCY HUNCHED POSTURE BREATHING SHALLOW RESPIRATION SKIN /FUR /PLUMAGE PILOERECTION SECRETION / EXCRETION SALIVATION ANIMAL 3 BEHAVIOR LETHARGY POSTURE HUNCHED POSTURE GAIT / MOTILITY UNCOORDINATED MOVEMENTS FLAT GAIT _ SKIN /FUR /PLUMAGE PILOERECTION (3) * 3 + (1) - 1 + (1) 1 " + (3) 1 + (3) - 3 3 + (1) - 1 1 + (1) 1 - - + (3) - 1 1 + (1) 1 1 1 + (3) 1 + (3) * 1 (1) 1 1 1 1 (3) - 1 (1) - 1 (1) - 1 1 1 1 FEMALES 200 MG/KG ANIMAL 4 BEHAVIOR LETHARGY (3) - 1 ................................... POSTURE HUNCHED POSTURE (1) 1 1 1 1 ......................... SKIN/FUR/PLUMAGE PILOERECTION (1) - 1 1 .............................. - = SIGN NOT OBSERVED / . = OBSERVATION NOT PERFORMED / + = ANIMAL DEAD - Page 12 - T-7868 TABLE 2 : CLINICAL SIGNS NOTOX Project 389598 TESTDAY HOURS AFTER TREATMENT 1 1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 MAX 0 2 4 GRADE FEMALES 200 MG/KG ANIMAL 5 BEHAVIOR LETHARGY POSTURE HUNCHED POSTURE GAIT / MOTILITY UNCOORDINATED MOVEMENTS SKIN / FUR / PLUMAGE PILOERECTION ANIMAL 6 BEHAVIOR LETHARGY POSTURE HUNCHED POSTURE SKIN/FUR/PLUMAGE PILOERECTION VARIOUS PTOSIS (3) - 1 - - - (1) 1 1 1 1 - (3) - 1 - - - ( 1) - 1 1 - - (3) - 1 - - - (1) 1 1 1 1 - (1) - 1 1 - - 1(3) - --- MALES 200 MG/KG ANIMAL 7 POSTURE HUNCHED POSTURE (1) 1 .......................................... ANIMAL 8 NO CLINICAL SIGNS NOTED .............................................. ANIMAL 9 POSTURE HUNCHED POSTURE (1) 1 .......................................... - = SIGN NOT OBSERVED / . = OBSERVATION NOT PERFORMED / + = ANIMAL DEAD - Page 13 - /S T-7868 TABLE 3 : BODY WEIGHTS (GRAM) SEX/DOSE LEVEL FEMALES 2000 MG/KG FEMALES 200 MG/KG MALES 200 MG/KG * animal found dead on day 2. ANIMAL 1 2* 3 MEAN ST.DEV. N 4 5 6 MEAN ST.DEV. N 7 8 9 MEAN ST.DEV. N NOTOX Project 389598 DAY 1 DAY 2 DAY 8 DAY 181 178 161 -- -- 182 219 237 180 219 237 2 ---- 3 11 185 224 234 199 243 255 173 217 235 186 228 241 13 13 12 3 33 303 385 418 282 353 394 292 359 396 292 366 403 11 17 313 3 33 - Page 14 - T-7868 NOTOX Project 389598 TABLE 4 : MACROSCOPIC FINDINGS ANIMAL ORGAN 'FINDING FEMALES 2000 MG/KG 1 Stomach Distended with gas. Glandular mucosa: discolouration, dark red 2 General observations Severe autolysis 3 No findings noted FEMALES 200 MG/KG 4 5 6 No findings noted No findings noted No findings noted MALES 200 MG/KG 7 S 9 No findings noted No findings noted No findings noted DAY OF DEATH Spontaneous death Day 1 after treatment Spontaneous death Day 2 after treatment Scheduled necropsy Day 15 after treatment Scheduled necropsy Day 15 after treatment Scheduled necropsy Day 15 after treatment Scheduled necropsy Day 15 after treatment - Scheduled necropsy Day 15 after treatment Scheduled necropsy Day 15 after treatment Scheduled necropsy Day 15 after treatment - Page 15 - /7