Document MJ0En5bpZZwZMdMpdk3JarYex

AR226-3109 FILE No. 715 02/12 '99 09:19 ID:DUPONT JACKSON LAB RflllO 609 540 4463 CONFIDENTIAL FEB -1999 y PAGE : :^ ^iiNOTE- L -esis^y4?BeBrxo. be-tbe `final draft1- It h aitetttau d iteA 'b y Hie. Huptingddn Ltfe Sciebcei Qis3l^!Assitraii;c&; D..e..p..a.r. t:m..e..n..t..:. . . ,- ' - r . .. . ->::'!: j;The SponsorIis; requested:to. review.this=dacuraentand:.<oinniiw icate^igi_cniMBnK.:M ;rto -S h ^ -D g ^ o c s:soon . :AS po3sible..:iiWhen:thetae comments fcaveibeen received,:th e.FINAlLKEM fiX containing pjx4?JTc?*9r *Od UA iiStatemBntswiltbeiESBued.- ....... " . ~ ~1\. . " I PIEASEiNOITEi: . " ' ' ....................., , IIS compliance with GLP any changes to the final report after the date o f issue will,fee in the form o f a separate :amendmec.t;t&fhe report-, ''' " i Please coininumcale:any icomment:to':;... : , , ::Lew fe-X.- MOUu F ax: + 4 4 (0)1480892039: E +nanrM cR aeL U K O is.H untingttoa;cm n: :: ;. / . . ::Pate: 7;TanuarV:il999- -______ ACUTE ORAL TOXICITY TO THE RAT (FIXED DOSE METHOD) Sponsor DuPont Speciality Chemicals Jackson Laboratory Chambers Works Deepwater NJ 08023 USA Research Laboratory Huntingdon Life Sciences Ltd P.O. Box 2 Huntingdon Cambridgeshire PE 18 6ES Page 1 o f 17 Report issued Comparty Sanitized. Does not contain TSCA CBF FILE No. 715 02/12 '99 09:20 ID:DUPONT JACKSON LAB RtlllO 609 540 4463 PAGE 3 % DPT 429/984523/AC CONTENTS Page COMPLIANCE WITH GOOD LABORATORY PRACTICE STANDARDS.................... 3 QUALITY ASSURANCE STATEMENT.............................................................................. 4 RESPONSIBLE PERSONNEL............................................................................................... S U M M A R Y .............................................................................................................................. INTRODUCTION.................................... .............................. ;............................................... 3 6 7 TEST SUBSTANCE................................................................................................................ EXPERIMENTAL PROCEDURE.......................................................................................... 8 9 RESULTS........................................................................................ ;........................................ 12 C O N C L U S IO N ........................................... -............................................................................. Ij TABLES 1. Mortality d a ta ............................................................................. 2. Signs of reaction to treatment - preliminary investigations....... 3. Individual bodyweights & changes - preliminary investigations 4. Signs of reaction to treatment - main study............................... 5. Individual and group mean bodyweights.................................... 6. Individual bodyweight changes.................................................. 14 15 15 16 17 17 :2: Company Sanif:-sd. Doss not contain TSCA CBS FILE No. 715 02/12 '99 09=20 ID=DUP0NT JACKSON LAB RN110 609 540 4463 PAGE 4 DPT 429/984523/AC COMPLIANCE WITH GOOD LABORATORY PRACTICE STANDARDS ZThe study described this tepori wat, conducted in compUanco t h < U r i PraCt" and rii, the exception ofdun ned belo I conside, dtc data generated to be vabd. The UK Good Laboratory Practice Regulations 1997 ( S i OECD Principles of Good Laboratory Practice (as revise || 1 654). C/CHEM(98)17. EC Council Directive 87/18/EEC of 18 D e c e m ^ P ^ 0 Ify I k ' Chemical analysis o f formulated test articljy||| HnnsasHB ot was not underraken for thisujttii& o o/29). ogeneity and concentration Lewis A. McRae, M .LSc.1f|.C.'^pi|i''M .I.Biol., Study Director, Jl Huntingdon Life Sciences Ltav Date :3: Company Sanitized. Dogs not contain TSCA D31 FILE No. 715 02/12 '99 09=21 ID=DUP0NT JACKSON LAB RH110 609 540 4463 PAGE 5 QUALITY ASSURANCE STATEMENT DPT 429/984523/AC The following have been inspected or audited in relation to this study Study Phases Inspected Generic Standard Protocol Review Process Based Inspections Husbandry Housing/Environment ^gutssnsstb* Weighing of animals Treatment P r o c e ^ ^ ^ / Clinical Signs \ Post Mortem Records Audit Training Records Report Date of Inspectioi 21 November l? nber 1998 uwJsber 1998 n _ Jrtmber 1998 ^y 14 September 1998 4 January 1999 ;____ ber 1998 ^ jeptember 1998 7 September 1998 17 September 1998 17 September 1998 17 September 1998 17 September 1998 17 September 1998 6 January 1999 Protocol: An audit of the standard protocol generated for this type of study design was conducted and reported to Company Management as indicated above. and repetitive procedures employ ed on this type o f study were earned out. These were conducteo reported to appropriate Company Management as indicated above Report Audit: This report has been audited by the Quality Assurance D e p *** 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. Margaret Blows, Quality Assurance Group Leader, Department of Quality Assurance, Huntingdon Life Sciences Ltd. Date 4; Comoeny Sanitized- Does no. contain T3CA CBf FILE No. 715 02/12 '99 09=21 ID DUPONT JACKSON LAB RM110 609 540 4463 PAGE 6 RESPONSIBLE PERSONNEL DPT 429/9&4523/AC Lewis A. McRae. M.I.Sc.T., C.Biol., M.I.Biol., Study Director, Department of Acute Toxicology. :5 : Company Sanlt'-nd. 0<-`- .-5 contain ".nmnaRV Sanitized. Doss not contain TSCA CBi FILE No. 715 02/12 '99 09=21 ID=DUPQNT JACKSON LAB RN110 609 540 4463 PAGE 7 SUMMARY DPT 429/984523/AC . . This studv was performed to assess the acute oral toxicity was based on that described in. thp rat The method followed EEC Methods for foe defornnnatioo o f toxicity. Annex to Difootive ^ E C (M Beia Jo u r , No. L383A, 29.12.92). Part B, Method B. I bis. Acute toxicity (oral) - fixed dose method. OECD Guideline for Testing o f Chemicals No.420 `Acute Oml Toxicity - Fixed Dose Method' Adopted 17 July 1992 equated to 500 mg/kg bodyweight (all d o s a g e | J | f ^ ^ _ J B | ^ doSC level selection was after adjustment for the 75% water content ^ !TM y supported by preliminary study findings and iniSmphance widrAe test gu.dehnes. There wereno deaths in foe main phase of foe study tn which a group of fon **--- " -- " I t a dose level of 500 mg/kg bodyweight. foceived a dosage of "clinical * . o f reacuon ro -- ^ salivation and ungroomed : , ^ on (al, rats) ^ S posture'(males only) had resolved / S iS w"fo piloerendon (a,, , U only evident Eon, Day 4, resolving in all instances hy Day 8. All rats were considered to bave achieved satisfactory bodyweight gains dunng foe study. No macroscopic abnonnalilies were obseived in animals lolled at study leoninanou on Day 15. The discriminating (non-lethal) oral dose to rats of( body-weight. bi i 6* sfoe Directive 93/21/EEC. vas indicated to be 500 mg/kg f ________ fill not ission ;6 ; Company Sanitize-- Does ne ordain TSCACBI FILE No. 715 02/12 '99 09:22 ID:DUP0NT JACKSON LAB RflllO 609 540 4463 PAGE 8 INTRODUCTION DPT 429/984523/AC The studv was designed to assess (he toxicity following a single oral dose to the rat. The rats were dosed by oral gavage as the test substance may be ingested accidentally. The study was conducted in compliance with: EEC Methods for the determination of toxicity. Annex to Directive 92/69/EEC (Official Journal No. L383A, 29.12.92). Part B. Method B. 1 bis. Acute toxicity (oral) - fixed dose method. OECD Guideline for Testing o f Chemicals No.420 `Acute Oral Toxicity - Fixed Dose MethodAdopted 17 July 1992. The rat was chosen as it has been shown to be a suitable model for this type of study and is the animal recommended in the test guidelines. The dose levels used in this study were chosen to help define the toxicity of the test substance and to be in compliance with the test guidelines, f i e protocolivas approved by Huntingdon Life Sciences Management on 7 July 1998. by the Sponsor on 17 July 1998 and by the Study Director on 27 August 1998. The experimental phase of the study was conducted between the 2 September and 13 November 1998. :7: not contain TSOA CBl FILE No. 715 02/12 '99 09:22 ID:DUPQNT JACKSON LAB RflllO 609 540 4463 PAGE 9 TEST SUBSTANCE DPT 429/984523/AC Identity: Chemical name: Intended use: Appearance: Storage conditions: Lot number: Expiry: Purity: Sample received: Pale yellow slurry Room temperature Two vears from date of receipt 25% acid/salts. 75% Water 23 June 1998 :8: Company Sanitized. Docs not contain TSCA CBF FILE No. 715 02/12 '99 09:22 ID:DUP0NT JACKSON LAB RflllO 609 540 4463 PAGE 10 EXPERIMENTAL PROCEDURE DPT 429/984523/AC ANIMAL MANAGEMENT Animals chosen for this study were selected from a stock supply o f healthy male and female CD rats of Sprague - Dawley origin (Hsd:Sprague-Dawley(CD)) obtained from Harlan UK Ltd, Bicester, Oxon, England. j , ' \ Animals in the main studv were in the weight range of 78 to 97 g and approximately fiveJo seven weeks o f age prior to dosing (Day I). All the iats in the main study were acclimatised to the experimental environment for a period of five days prior to the start of the study. The rats were allocated without conscious bias to cages within fee treatment groups. They were housed in groups o f up to five rats of the same sex in metal cages (RS Biotech Sub-Dividable Rodent Cages polished stainless steel (20cm high x 39cm wide x 39cm long)). The cages were fitted with gnd floors to ensure rapid removal of waste material to undertravs. The cages were suspended in mobile stainless steel racks in Room 6 o f Building R14. A standard laboratory rodent diet (Special Diet Services RM1(E) SQC expanded pellet) and drinking water were provided' ad libitum. Access to food only was prevented overnight prior to and tor approximately 4 hours after dosing. The batch of diet used for fee study was analysed for certain nutrients, possible contaminants and micro-organisms. Results of routine physical and chemical examination of drinking water, as conducted by the supplier, are made available to Huntingdon Lire Sciences Ltd. as quarterly summaries. " Thermostatic controls were set to maintain a temperature of 22 3C. Relaiive humidity was not folly controlled but was anticipated to be in fee range 30 - 70%. Temperature and humidity were recorded continuously using a seven day recorder. Actual measurement of these parameters revealed feat animal room temperature was in the range 21 to 24C and relative humidity was in fee range 08 Permanent daily recordings of these parameters were made and these are archived with other Departmental raw data. Lighting was controlled by means of a time switch to provide 12 hours o artificial light (0700 - 1900 hours) in each 24-hour period. . Each animal was identified by cage number and ear punching. Each cage was identified by a coloured label displaying the dose level, study schedule number, animal mark and fee initials o f the Study Director and Home Office licensee. TEST SUBSTANCE PREPARATION In fee main studyl-- a B ^ ^ ^ ------------------------------- * Toconcentration of 20% v/v in distilled water and administered at a dose volume or vas formulated at a ml/kg bodyweight in order to achieve the desired dosage. The test substance was prepared on fee day o f dosing. The absorption and characterisation of the homogeneity, stability in vehicle and purity of the test substance was not undertaken in this study and remains the responsibility o f fee Sponsor. :9 : Company Sanitized. oes not contain TSC OBI FILE Wo. 715 02/12 '99 09=23 ID:DUP0NT JACKSON LAB RT1110 609 540 4463 PAGE 11 DPT 429/984523/AC TREATMENT PROCEDURE Preliminary investigations In the absence o f precise toxicological information a preliminary study was conducted to help define the toxic pnrpnriai of ihe test substance and aid in selection/validation of the dosage selected for use in the main study. Initially, one female rat was treated with the test substance as s u n ^ ^ ^ a ^ ^ ^ m c /k g , but as a result of a marked toxic response thought to be associated with the pH interests of animal welfare, a further female was treated at 125 mg/kg with the test substance formulated in distilled water and for further clarification of.oral toxicity a female rat was dosed at 500 mg/kg bodyweight and finally one male and one female (per dosage) treated at 1000 and 2000 mg/kg. Main study A group of ten rats (five males and five females) received a single oral gavage dose of the test substance at a dose level o f 500 mg/kg bodyweight. This dose level was chosen in compliance with the test guidelines. No control animals were included in this study. ADMINISTRATION OF THE TEST SUBSTANCE The appropriate dose volume of the test substance was administered to each rat by oral gavage using a syringe and cannula of the appropriate gauge. The day of dosing was designated Day 1. OBSERVATIONS Mortality Cages of rats were checked at least twice daily for mortalities. Clinical signs Animals were observed soon after dosing and at frequent intervals for the remainder o f Day l. On subsequent davs, animals were observed once in the morning and again at the end of the experimental dav (with the exception o f the last day of observation - morning only). The nature and severity of the clinical signs and time were recorded at each observation. Animals in the preliminary arid main study were observed for up to 7 or 14 days respectively after dosing. Bodyweight The bodyweight o f each rat in the preliminary study was recorded on Days t (prior to dosing) and 8 (or at death) and in the main study on Days 1 (prior to dosing), 2, 3, 4, 8 and 15. : 10 : Company Sanitized. D ;t contain TSC C3I FILE No. 715 02/12 '99 09=23 ID:DUP0NT JACKSON LAB RT1110 609 540 4463 PAGE 12 DPT 429/984S23/AC TERM INAL STUDIES Termination The animals (that survived treatment) in the preliminary study were killed on Day 8 (study termination) and all animals in the main study were killed on Day 15 by carbon dioxide asphyxiation. . Macroscopic pathology t ? ' "'* All animals were subjected to a macroscopic examination which consisted of opening the cranial, thoracic and abdominal cavities. The macroscopic appearance of all examined organs was recorded. ARCHIVES All raw data and study related documents generated during the course of the study at Huntingdon, together with a copy of the final report will be lodged in the Huntingdon Life Sciences Archives, Huntingdon. Such records will be retained for a minimum period of five years from the dale of issue of the final report. At the end of the five year retention period the Sponsor will be contacted and advice sought on the future requirements. Under no circumstances will any item be discarded without the Sponsors knowledge. ' DEVIATIONS FROM PROTOCOL There were no deviations from the protocol that were considered to have affected either the validity or the integrity o f this study. However, the following deviation did occur; to assist in further defining the toxicity o f the test substance one male and one female (per dosage) were treated at 1000 and 2000 mg/kg each dosage. These animals were also slightly above the upper weight limit specified in che study protocol. : 11 : Company Santfesd. Doss not contain TSCA Cbi FILE No. 715 02/12 '99 09:23 IDDUPONT JACKSON LAB Rill10 609 540 4463 PAGE 13 RESULTS DPT 429/984523/AC PRELIMINARY STUDY (Tables 1. 2 and 3) Two females r e c e i v e d f ^ ^ ^ B ^ t a dosage of 125 mg/kg (administered either as supplied or as an aqueous f o r m u la tilS ^ U u r tK e m a le was treated at 500 mg/kg and finally one male and one female (per dosage) was treated at 1000 and 2000 mg/kg bodyweight. Results were as follows. Deaths were recorded between Day 3 and Day 4 for one female dosed at 1000 mg/kg and one male and one female treated at 2000 mg/kg. All other animals were killed as scheduled at study termination (Day 8). Clinical signs comprised piloerection, hunched posture, waddling/unsteady gait and dark colouring to eyes observed in rats at all dosages. In addition, ungroomed/thm appearance lethargy, abnormal faeces, protruding abnormal respiration, pallid extremities, walking on toes and blue/cold extremities eyes increased salivation, increased lacrimation and body tremors were observed m rats at one or dosages. Bodyweight gains were considered to be within acceptable limits for a study of this nature and duration. Macroscopic examination of animals treated at 125 and 500 mg/kg killed at ^ termination revealed no abnormalities. Examination of the one rat that survived treatment at 1000 mg/kg revealed pallor of the brain and kidneys, slightly enlarged liver, kidneys and darkened spleen. Findings in decedents were primarily congestive in nature (characterised by darkened tissues, prominent blood vessels and enlarged tissues/organs) in subcutaneous tissue, brain heart, lungs, liver, spleen and along the alimentary tract. Also observed in the latter was fluid/food retention. Findings from this phase of the study demonstrated that administration o f of 1000 and 2000 mg/kg resulted in >50% mortality. dosages MAIN STUDY There were no administration o B B of tn rats (five males and five females) following a single oral a dose level of 500 mg/kg bodyweight. CLINICAL SIGNS (Table 4) Piloerection increased salivation and ungroomed appearance were observed in all rats within seven minutes of dosing and accompanied later on Day 1 or thereafter by hunched posture notable in all rats with waddling/unsteady gait and thin appearance less commonly observed. All but ,pdof f f nJ " ! rats) and hunched posture (males only) had resolved within 24 hours of dosing with hunched posture resolving in all instances bv Dav and piloerection resolving in all instances by Day 8. 12 Company Sanitized. Does not contain TSCA CB1 FILE No. 715 02/12 '99 09=24 ID=DUPONT JACKSON LAB RM110 609 540 4463 PAGE 14 DPT 429/984523/AC BODYW EIGHT (Tables 5 and 6) All rats were considered to have achieved satisfactory bodyweight gains during the study. MACROSCOPIC EXAMINATION No macroscopic abnormalities were observed for animals killed at study termination on Day 15. CONCLUSION The acute lethal oral dose to rats oj bodyweight. -as demonstrated to be greater than 500 mg/kg fev : 13 : Company Sanii' :d. Doss not contain TSC Crs; FIUE No. 715 02/12 '99 09:24 II>:DUP0NT JACKSGN LAB ROllO 609 540 4463 PAGE 15 TABLE 1 Mortality data DPT 429/984523/AC 1 "TM" Phase of Dose No. of study (rag/kg) deaths 125 F+ 0/1 125 F 0/1 Preliminarv 500 F 0/1 1000 M 0/1 1000 F 1/1 2000 M 1/1 2F 1/1 Main 500 M 500 F 0/5 0/5 1 &2 0 0 0 0 0 0 0 0 0 Dav of studv 3 4 3 to 8 00 0 00 0 00 0 00 0 0l ' 01 ' 1- - 00 0 00 0 9 to 15 * 0 0 administered as supplied Not applicab! : 14 : Company Sanitised. Doss not contain TSCA CB1 FILE No. 715 02/12 '99 09:24 ID:DUPONT JACKSON LAB RTIllO 609 540 4463 PAGE 16 DPT 429/984523/AC TABLE 2 Signs of reaction to treatment - preliminary investigations Signs of reaction Piloerection Hunched posture Waddiing/unsteady gait <* Increased salivation Ungroomed appearance Thin appearance Lethargy / Abnonnai respiration9 Pallid extremities Walking on toes Abnormal faeces'1 Protruding eyes Blue/cold extremities Dark colouring to eyes Increased tacrimation Body tremors Prostration - No. of rats in eroup of 1` or showing signs Dose (mg/kg) 125'+ 125' 500' 10001 20001 F F F MF M F 1# 1 1 1 1 1 l 1# 1 I l 1 1 1 1 1 0 11 l 1 0 0 0 01 1 0 1 0 0 11 1 1 l 0 11 1 1 0 0 00 1 1 1# 1 0 0 0 1 1 1 0 0 00 1 1 1 0 0 00 1 1 1 0 0 11 1 1 1 0 0 11 1 1 1 0 0 00 1 1 l 0 0 11 1 1 0 0 0 00 1 1 0 0 00 1 0 0 0 0 00 1 0 1. Preliminaiy study 2 i study +; _______ las administered as supplied #: Still evident aretudy termination a: Characterised by increased, decreased or gasping/noisy breathing . . . , b: Characterised by soiled/stained fur around the face/muzzle or ano/gemtal region or wet rur c: Characterised bv soft to liquid faeces/few TABLE 3 Individual bodyweights and gains (g) - preliminary investigations Dose (mg/kg) 125+ 125 500 1000 2000 Animal No. & Sex 1F 2F 3F 23 M 24 F 25 M 26 F Bodvweight (g) at Day 1* 8 Death 116 124 f 8 1 120 165 (45) - 122 161 197 206 [39]____ (9] - 185 - 1-1 173 235 _ [-1 186 199 - _ U ____ 191 *: r P rio n ^ o sin t. +: administered as supplied | ): Weight gain shown in parenthesis : 15 : Company Sanitized. lo ss not lontsin TSCA CBl FILE No. 715 02/12 '99 09:24 ID:DUP0NT JACKSON LAB Rill10 609 540 4463 PAGE 17 ' DPT 429/984523/AC TABLE 4 Signs of reaction to treatment - main study Signs of reaction Piloerection * Hunched posture Waddling/unsteady gait / increased salivation Ungroomed appearance* Thin appearance No. of rats in group of S per sex showing signs Dose ( 500 mg/kg) M 55 55 5 O'55 55 11 rJ y77 3: Characterised by soiled/stained fur around the faee/muzzie or ano/genital region or wet fur M; Male rats F: Female rats ; 16 : Sanitized. Doss not contain TSCA CBI FILE Wo. 715 02/12 '99 09:25 ID:DUPGNT JACKSON LAB Rill10 609 540 4463 PAGE 18 DPT 429/984523/AC TABLES Individual and group mean bodyweights (g) - main study Dose (ms/ks) 500 Animal No. &. Sex Il M 12 M 13 M 14 M 15 M Mean 16 F 17F 18 F 19F 20 F Mean *: Prior to dosing 1* 86 89 78 86 85 85 95 88 84 97 94 92 Bodvweieht (fi) at Day 23 4 8 86 88 98 127 94 102 116 142 87 94 99 122 95 103 113 146 91 101 112 144 31 98 108 136 109 118 117 139 99 108 93 102 108 120 117 109 123 136 124 142 100 112 118 136 102 112 117 135 15 187 193 174 210 204 194 154 156 145 172 160 157 TABLE 6 Individual and group mean bodyweight changes (g) - main study Dose fmg/kg) 500 Animal No. &. Sex 11 M 12 M 13 M 14 M 15 M Mean 16F 17 F 18 F 19 F 20 F Mean Bodvweiehi sains (b) Day 1-8 Day 8-15 41 60 55 51 44 52 60 64 59 60 51 57 44 15 48 20 40 21 45 30 42 24 44 22 17 : Doss not contain T S C A CBi Company Sanitizes.