Document NpB0M5qyZKjz837MJxDkrQyg

AR226-3109 FILE No. 715 02/12 '99 09:19 ID:DUPOMT JACKSON LflB RUllO 609 540 4463 ltd FEBfl-1999 CONFIDENTIAL ^Jn<<fc,hi ^0^. ^Th^$TeI>&rt:is:c!OB(aife'^l^:iytl^ft..S fciiaeifiticwafUiijt'jlK t is|HB rtpg.ttQlC.I^! Science^Qua.IiiyAsBurance. DBpiirtincflt.." . ^'^^^ p;:"':''" ':"";"::T.:;: :j::;:'s^""::^::'i;:jii::|"';;! Ip:|iTi:';'::' f :' :'"" i'TtJE SpnrianT::ig:n>qii^gM>:tr. rpvj^^ ^^^tt:^f^^'rp^t^BilB;:$'l^^mivT'i^e;flgy::C(^Im^gltg:tO::^fa^ ;:a.:pobl\^i"WliBB:^ieia6coB^nc^havcbeen"rBci^ive4,:^;FINA'R^^ !:StiafanMtE?flt:b<Bi;iaro(id.; " ":;;" ' --"-------- --;,.-,.,.....,,-.,-.-,,,,",--...,. ;;raE^ae|MCHFij^' : ^iBleofflpliBaceTwBai.^iSlLPianiy-cbantgbe'tsh;,f&a^:r^p<ntlafie^:ffif!!;a'afe":oflasue.viIl& ^amp.nf<ment:t:&,t)ite;reptt'., " " '"" "-- -;' .-"."'-...,".., :=^..:::-,.:-".:.:;:::;":---. -............ Please eoa^umcate!aDy:coininencs^tQ::i. LeYW:^;:W(;,jf|alx^r,- +44:{0)il4M;8S>2W: E^aa^MdiaA iPatei-yjanumyij^ ACUTE ORAL TOXICITY TO THE RAT (FIXED DOSE METHOD) Sponsor DuPont Specialily Chemicals Jackson Laboratory Chambers Works Dsepwater NJ 08023 USA Page 1 of 17 Research Laboratory Huntingdon Life Sciences Ltd P.O. Box 2 Huntingdon Cambridgeshire PE186ES ENGLAND Report issued Company Sanitized. Does r=ot contain TSCA CBi FILE No. 715 02/-12 '99 09:20 ID:DUPONT JflCKSDN LAB RflllO 609 540 4463 PAGE 3 "DPT 429/984523/AC CONTENTS COMPLIANCE WITH GOOD LABORATORY PRACTICE STANDARDS................... Page 3 QUALITY ASSURANCE 4 STATEMENT........................................................................ RESPONSIBLE PERSONNEL. 5 .......,,,,,,.,,,,.,,,,..,,.,....,,.,.,,,,,,,,,,.,,..,,,,,,.,,...,,.................... 6 SUMMARY..................................................................................................................... 7 INTRODUCTION.......................................................................................................... ) TEST 8 SUBSTANCE........................................................................................................ EXPERIMENTAL 9 PROCEDURE..................,....,...,.,.,.,,,,,,..,,......,....,.,..,.,...................... 12 RESULTS...,.,,,,,....,.,,,.......,.,..,.,,,,,...,..,.,,...,,.....,.........................,..........,...,..,,...,........... 13 CONCLUSION................................................................................................................ TABLES 1. Mortality data 14 2. Signs of r e a c t i ... on . . t .. o ... tr ....... eatm ..... ent . - . . p.. r..e. l.i.m...i.n..a..r.y.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 investigations............................................. 3. Individual bodyweights &. changes - preliminary investigations...................................... 15 4. Signs of reaction to treatment - main 16 Individual and group mean study.................................................................... 17 5. bodyweights........................................................................ 6. Individual bodyweight changes 17 ................................................................................... 2 : Company SaniS^sri. Doss not contain TSCA CB? FILE No. 715 02/12 '99 09:20 ID:DUPDNT JACKSON LflB RTlllO 609 540 4463 PAGE 4 DPT 429/984523/AC COMPLIANCE WITH GOOD LABORATORY PRACTICE STANDARDS The study described in this report was conducted in compliance with the following Good Laboratory. Practice standards and with the exception of that noted below I consider the data generated to be valid. The UK Good Laboratory Practice Regulations 1997 (^^ly^^ent No 654). OECD Principles of Good Laboratory Practice (as revise^,l^^^\WlC/CHEM(98)I7. EC Council Directive 87/18/EEC of 18 Decemha^:^>^ (0^^tlIounia^o^S5/29). Chemical analysis of formulated tea article ^^tie%nB^|^Sadf sta^iy^^tnogeneity and concentration \) Company Sanitized. Doss not contain TSCA C.W FILE No. 715 02/12 '99 09:21 ID:DUPONT JACKSON LAB RT1110 609 540 4463 QUALITY ASSURANCE STATEMENT OPT 429/984523/AC The following have been inspectedor audited in relation to this study Study Phases Inspected Generic Standard Protocol Review Process Based Inspections Husbandry Housing/Environment Weifibinfi of animals Treatment Procfcd Clinical Signs Post Mortem Records Audit Trainins Records Report if Reporting 1997 1998 ^September 1998 17 September 1998 17 September 1998 17 September 1998 17 September 1998 17 September 1998 17 September 1998 Protocol: An audit of the standard protocol generated for this type of study design was conducted and reported to Company Management as indicated above. Process based inspections: At or about the time this study was in progress inspectionsand audits of routine and repetitive procedures employed on this type of study were carried out. These were conducted and reported to appropriate Company Management as indicated above Report Audit: This report 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. Margaret Blows, Quality Assurance Group Leader. Department of Quality Assurance, Huntingdon Life Sciences Ltd, Date Company Sanit^s-S- Doss r'f'i confam T3CA CBf FILE No. 715 02/12 '99 09:21 ID:DUPONT JACKSON LAB RDllO 609 540 4463 PAGE 6 DPT429/9&4523/AC RESPONSIBLE PERSONNEL Lewis A. McRae, M.I.Sc.T., C.BioL, M.I.Biol., Study Director, Department of Acute Toxicology. 5 : Company San^od. D'-^-! -'^ c-ip^a'n TSr-fi ~";? c-nmnanv Samfced. Doss not cor.tain 7SCA CBi FILE No. 715 02/12 '99 09:21 ID:DUPONT JACKSON LAB RMllO 609 540 4463 PAGE 7 SUMMARY DPT 429/984523/AC This study was performed to assess the acute oral toidcity (^y"UHIl^fJ0 was based on that described in: f^^^^^^J ^e Tat- The method followed EEC Methods for the determination oftoxicity, 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 of Chemicals No.420 'Acute Oral Toxicity Fixed Dose Method' Adopted 17 July 1992 A group of ten fasted rats (five males and five females) received a single oral gavage dose of the test substance, formulated in distilled water. The administered dose after adjustment for water content (75%) equated to 500 nig/kg body-weight (all dosageyBferencedhetmfler are expressed as dose concentrations cgUU^B after adjustment for the 75% water content The rnam study dose level selection was supported by preliminary study findings and in compliance witn"Ae test guidelines. re were jo deaths in the main phase of the study in which a group of ten rats received a dosage of it a dose level of 500 mg/kg bodyweight. Clinical signs of reaction to treatment were characterised by piloerection, hunched posture, increased ! salivation and imgroomed appearance notable in all rats, with waddling/unsteady gait and thin appearance less commonly observed. All but piloerection (all rats) and hunched posture (males only) had resolved ; within 24 hours of dosing v-ilh piloerection (all rats) only evident from Day 4, resolving in all instances by ' Day 8. All rats were considered to have achieved satisfactory bodyweight gains during the study. No macroscopic abnormalities v."ere observed in animals killed at study termination on Day 15. The discrirmnanng (non-lethal) oral dose to rats ofJUBIBHil^215 indicated to be 500 mg/kg bodyweight. On the basis of findings in this study and in accord mm EU hazard clas3scifaictiaQtiiorfi^l|lBlUBJHUIBp*rn^ not require labelling with the risk phrase R22 "Harmful if swallowed", in accordance with CBommission Directive 93/21/EEC. 6 ; COW S^t^. Doe. r.c- contain TSCA CB1 FILE No. 715 02/12 '99 09:22 ID:DUPONT JACKSON LAB RH110 609 540 4463 PAGE 8 INTRODUCTION DPT429/984523/AC The study was designed to assess (he toxicity cMI^U^^jfollowing a single oral dose to the rat. The rars were dosed by oral gavage as the test substance may be Rl|estedaccidentally. 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). Pan B. Method B. I bis. Acute toxicity (oral) fixed dose method, OECD Guideline for Testing of Chemicals No.420 'Acute Oral Toxicily - Fixed Dose Method' Adopted 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. ' y,' The dose levels used in this studv were chosen to help define the toxicity of the test substance and to be in compliance with the iesi guidelines, TRe protocol"was 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. ? ,-S Company Sanded. Does not contain TSCA CB1 FILE No. 715 02/12 '99 09:22 ID:DUPONT JACKSONLAB RtlllO 609 540 4463 PAGE 9 TEST SUBSTANCE DPT 429/984523/AC Identity: Chemical name: Intended use: Appearance: Storage conditions: Loc number: Expiry; PuritySample received: Pale yellow slurry Room temperature W Two years from date of receipt 25% acid/salts, 75% Water 23 June 1998 8 : Company Sanitized. Coas nst contain 7SCA CS! FILE Mo. 715 02^12 '99 09:22 ID:DUPDNT JACKSON LAB R11110 609 540 4463 PAGE 10 EXPERIMENTAL PROCEDURE DPT429/984523/AC ANIMAL MANAGEMENT ? , . Animals chosen for this study were selected from a stock supply of healthy male and female CD rats of Sprague - Dawley origin (Hsd:Sprague-Dawley(CD)) obtained from Harlan UK Ltd, Bicester, Oxon, End and. .' > p. . ^ Animals in the main study were in the weight range of 78 to 97 g and approximately five to seven weeks of age prior to dosing (Day 1). All the rats 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 the treatment groups. They were housed in groups of 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 grid floors to ensure rapid removal of waste material to undertrays. The cages were suspended in mobile stainless steel racks in Room 6 of Building R14, A standard laboratory rodent diet (Special Diet Services RMl(E) SQC expanded pellet) and drinking water were provided ad libitum, Access to food only was prevented overnight prior to and for approximately 4 hours after dosing. The batch of diet used for the 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 Life Sciences Ltd. as quarterly summaries. Thermostatic controls were set to maintain a temperature of223C. Relative humidity was not fully controlled but was anticipated to be in the range 30 - 70%, Temperature and humidity were recorded continuously using a seven day recorder, Actual measurement of these parameters revealed that animal room temperature was in the range 21 to 24C and relative humidity was in the range 38 - 61%. 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 of 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 the initials of the Study Director and Home Office licensee. TEST SUBSTANCE PREPARATION concentration of 20% v/v in distilled waierand administered at a dose volume oflO ml/kg bodyweight in order to achieve the desired dosage. The test substance was prepared on the day of 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 of the Sponsor. ^ Company Sar^zec. Goes n3t eantain TSCA C31 FILE No. 715 02/12 '99 09:23 ID:DUPOMT JACKSON LAB Rill 10 609 540 4463 PAGE 11 DPT 429/984523/AC TREATMENT PROCEDURE Preliminary investigations In the absence of precise toxicological information a preliminary study was conducted to help define the toxic potential of the test substance and aid in selection/validation of the dosageselected for use in the ofHHi^Hgid main study. Initially, one female rat was treated with the test substance as sumliodj^23^^kg, but as a result of a marked toxic response thought to be associated with the pH in the interests of animal welfare, a further female was treated at 125 ing/kg wmmciesffsubstance 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 of 500 mg/kg bodyweight. This dose level was chosen in compliance with the lest guidelines. No control animals were included in this study. ADMINISTRATION OF THE TEST SUBSTANCE The appropriate dose volume of me 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 of Day I. On subsequent days, animals were observed once in the morning and again at the end of the experimental day (with the exception of 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 of each rat in the preliminary study was recorded on Days 1 (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. DOES r.st canta-nTSCACSl FILE No. 715 02/12 '99 09:23 ID:DUPONT JflCKSON LAB RIlllO 609 540 4463 PflGE 12 DPT 429/984523/AC TERMINAL 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 asphyxiarion. i ? ^ Macroscopic pathology 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 Sponsor's knowledge. DEVIATIONS FROM PROTOCOL There were no deviations from the protocol that were considered to have affected either the validity or the integrity of this study. However, the following deviation did occur; to assist m further defining the toxicity of the test substance one male and one female (per dosage) were treated at 1000 and 2000 me/kg each dosage. These animals were also slightly above the upper weight limit specified in the study protocol. 11 Company Sanffissd. Doss not contain TSCA Cb; FILE No. 715 02^12 '99 09:23 ID:DUPONT JACKSON LAB RDllO 609 540 4463 PAGE 13 RESULTS DPT 429/984523/AC PRELIMINARY STUDY (Tables 1. 2 and 3) ^ ^ ^ _ 1 Two females receivedf^^^BKt a dosage of 125 mg/kg (administered either as supplied or as an aqueous formularioi^^^irtnSwemale was treated at 500 ing/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 ar 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 racs at all dosages. In addition, ungroomed/thin 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 in rats atone 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 study termination revealed no abnormalities. Examination of the one rat thai 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 tatter was fluid/food retention. o f J U H d | p Findings from this phase of the study demonstrated that administration of 1000 and 2000 mg/kg resulted in ^50% mortality. dosages MAIN STUDY OHUHH^f There were no derthsu_^mup of ten rats (five males and five females) following a single oral administration ^^BH^^HH^H^^^^^BH a dose level of 500 mg/kg bodyweight. CLINICAL SIGNS (Table 4) Piloerection increased salivation and ungroomcd 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 piloereclion (all rats) and hunched posture (males only) had resolved within 24 hours of dosing with hunched posture resolving in all instances by Day and piloerection resolving in all instances by Day 8. 12 Company Sanitszsd. 5oes r.st coniain TSCA CBi FILE No. 715 02/-12 '99 09:24 ID:DLPDI\IT JACKSON LAB RDllO 609 540 4463 PAGE 14 DPT429/984523/AC BODYWEIGHT (Tables 5 and 6) All rats were considered to have achieved satisfactory bodyweignt 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 C^^^H^HV^ demonstrated to be greater than 500 mg/kg <&? : 13 : Company SaniS'.sseS. Dsss r.st ccntein TSCA C FILE No. 715 02/12 '99 09:24 ID-:DUPONT JACKSON LAB RllllO 609 540 4463 TABLE 1 Mortality data DPT 429/984523/AC Phase of Dose No. of Day of" study study (ing/kg) deaths I&2 3 125 F+ 0/1 125 F 0/1 0 0 0 0 Preliminary 500 F 0/1 0000 1000 M 0/1 0000 1000 F 1/1 0000 2000 M 1/1 001- 2000 F 1/1 001- 500 M 0/5 Main 500 F 0/5 0 00 01000-00- adJTunistcred as supplied a -- | -:'N4oot aappppucliacl ant 4 5 to 8 9 to 15 0 0 14 Comps'aly Sanitised. Doss not contain TSCA CBi FILE No. 715 02/12 '99 09:24 ID:DUPQIMT JACKSON LAB RMllO 609 540 4463 DPT 429/984523/AC TABLE 2 Signs of reaction to treatment - preliminary investigations Signs of reaction Piloerection Hunched posture Waddling/unsteady gait ^ Increased salivation Ungroomed appearance11 Thin appearance Lethargy Y Abnormal respiration'1 Pallid extremities Walking on toes Abnormal faeces'1 Protruding eyes Blue/cold extremities Dark colouring to eyes Increased lamination Body tremors Prostration No. of rats in group of 1" or 2" showing signs Dose(ne/kg) 125'+ 125' 500' 10001 20002 F F F M F M F [# 1 1 1 1 lit 1 1 1 1 1 1 0 1 0 0 0 ( 1 0 1 0 0 1 I 0 0 1 0 1 0 0 0 0 1# 1 0 0 0 1 0 0 0 0 | 0 0 0 0 1 0 0 1 1 1 0 0 1 1 1 0 0 0 0 1 0 0 1 1 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 00 1 0 Prelimmaly study Mainstud_^ +; U^^^^^^as administered as supplied : Sull cvidentaRcudytermination a: Characterised by increased, decreased or gasping/noisy breathing b: Characterised bv soiled/stained far around the faca/muzzle or ano/gcnital region or wet fur c; Characterised by 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 IF 2F 3F 23 M 24 F 25 M 26 F Bodyweight (g) at Day I* 8 Death 116 124 f8t 120 165 [45] 122 161 f39] 197 206 (9] 185 - (-1 173 235 - 199 - [-] 186 [-] 191 ^.fUHH +: administered as supplied Weight gain shown in parenthesis 15 Company Sanitized, ucss no'; contain TSCA CBl FILE No. 715 02/12 "99 09:24 ID:DUPQNT JACKSDN LAB RTIllO 609 540 4463 TABLE 4 Signs of reaction to treatment - main study DPT429/984523/AC Signs of reaction Piloereciion l/ Hunched posture Waddling/unsteady gail ' Increased salivation Ungroomed appearance1 Thin appearance No. of rats in group of 5 per sex showing signs Dose ( 500 mfi/kp) M F 5 5 55 05 < '"" ,r A- 5 5 5 5 1 1 -/'/' .>- y- Characterised by soiled/stained fur around the face/muzzle or ajio/genital region or wot fur M; Male rats F: Female rats : 16 : Company Sanitized. Doss no? contain TSCA CBI FILE No. 715 02/12 '99 09:25 ID:DUPDNT JACKSON LAB RnilO 609 540 4463 DPT 429/984523/AC TABLES Individual and group mean bodyweights (g) - main study Dose Animal No. (ros/ke) &Scx I* 11M 86 12 M 80 Boflvweigbl (g) at Day 2 3 4 8 15 86 8S 98 127 187 94 102 116 142 193 13 M 78 87 94 99 122 174 14 M 86 95 103 113 146 210 15 M 85 91 101 112 144 204 500 Mean 85 31 98 108 136 194 16 F 95 109 118 117 139 154 17F 18 F 88 99 108 117 84 93 102 109 136 156 124 145 19F 20 F 97 108 120 123 94 100 112 118 142 172 136 160 Mean 92 102 112 117 135 157 : Prior to dosing TABLE 6 Individual and group mean bodyweight changes (g) - main study Dose ^mg/ks) 500 Anunal No. A Sex 11M 12 M 13 M 14 M 15 M Mean 16F 17F 18 F 19F 20 F Mean Bodywcighi gains (g) Dav 1-8 41 53 Dav 8-15 60 51 44 52 60 64 59 60 51 57 44 15 48 20 40 21 45 30 42 24 44 22 : 17 : Company S- ar.i.is ze,,c,.:,. r-.^0a- ni^o1t ^contain TSCA C81