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AR226-3139 CONFIDENTIAL SPONSOR Elf Atochem S.A. Cours Michelet La Dfense 10 92091 Paris-la-Dfense CEDEX France IFM recherche SNC AUCPTM. Dt i SSO 000f TEST SUBSTANCE STUDY TITLE ACUTE ORAL TOXICITY IN RATS STUDY DIRECTOR Xavier Manciaux STUDY COMPLETION DATE 13 December 1999 PERFORMING LABORATORY CIT Centre International de Toxicologie BP 563 - 27005 Evreux - France !Snot contain TSCA CB LABORATORY STUDY NUMBER Company Sanitized. Do 18745 TAR CENTRE INTERNATIONAL DE TOXICOLOGIE 5. P. 563 27005 Evreux Cedex France TAI . O" ->Q V X l / U l U U J H l/ X I/ T J 3 CONTENTS STATEMENT OF THE STUDY DIRECTOR 4 OTHER SCIENTISTS INVOLVED IN THIS STUDY 4 STATEMENT OF QUALITY ASSURANCE UNIT 5 SUMMARY 6 RESUME 7 1. INTRODUCTION 8 2. MATERIALS AND METHODS 2.1 TEST SUBSTANCE 2.1.1 Identification 2.1.2 Formulation procedure 2.2 TEST SYSTEM 2.2.1 Animals 2.2.2 Environmental conditions 2.2.3 Food and water 2.3 TREATMENT 2.3.1 Fasting of the animals 2.3.2 Administration of the test substance 2.3.3 Chronology of the study 2.4 CLINICAL EXAMINATIONS 2.4.1 Clinical signs and mortality 2.4.2 Body weight 2.5 NECROPSY 2.6 DATA EVALUATION ' 2.7 PROTOCOL ADHERENCE 2.8 ARCHIVING . 8 8 8 8 9 9 9 9 10 10 10 10 10 10 10 11 11 ' 11 11 3. RESULTS 12 3.1 CLINICAL EXAMINATIONS 3.1.1 Clinical signs and mortality (table 1) 3.1.2 Body weight (figures 1 and 2, tables 2 and 3) 3.2 PATHOLOGY (table 4) 12 12 12 12 4. CONCLUSION 12 Company Sanitized. Does not contain TSCA CB Figure 1: Body weight o f treated rats Figure 2: Body weight of CIT historical control rats T ablet: Individual clinical signs and mortality Table 2: Individual and mean body weight and weekly body weight change (g) Table 3: Mean body weight and weekly body weight changeof CIT historical control rats Table 4: Individual macroscopic examinations at necropsy 13 14 IS 16 17 18 APPENDICES 1. Test article description and analytical certificate 2. Diet formula 19 20 23 and 24 Company Sanitized. Does not contain TSCA CBI V ^ A 1/ O I U U J i i y , i O / t J 1 STATEMENT OF THE STUDY DIRECTOR The study was performed in compliance with the principles of Good Laboratory Practice as described in: . OECD Principles on Good Laboratory Practice (as revised in 1997), ENV/MC/CHEM (98) 17. . Dcret N 90-206 du 7 mars 1990 concernant les Bonnes Pratiques de Laboratoire (Journal Officiel du 9 mars 1990), Ministre de l'Industrie et de l'Amnagement du Territoire. . Council Directive 87/18/EEC of 18 December 1986 on the harmonization of laws, regulations or administrative provisions relating to the application of the Principles of Good Laboratory Practice and the verification of their applications for tests on chemical substances (OJ No. L 15 of 17.1.87). I declare that this report constitutes a true and faithful record of the procedures undertaken and the results obtained during the performance of the study. This study was performed at CIT, Centre International de Toxicologie, BP 563, 27005 Evreux, France. I) / .Toxicology X. Manciaux Study Director Doctor of Pharmacy Date: 13 December 1999 OTHER SCIENTISTS INVOLVED IN THIS STUDY For Pharmacy: P.O. Guillaumat Doctor of Pharmacy For Toxicology: C. Pelcot Study Supervisor Company Sanitized. Doe:s not contain TSCACBi STATEMENT OF QUALITY ASSURANCE UNIT Type of inspections Protocol Report Inspections 4 June 1999 10 November 1999 Dates Reported to Study Director (*) 4 June 1999 9 December 1999 Reported to Management (*) 4 June 1999 10 December 1999 In addition to the above-mentioned inspections, at about the same time as the study described in the present report, "process-based" and routine facility inspections of critical procedures relevant to this study type were also made by the Quality Assurance Unit. The findings of these inspections were reported to the Study Director and to CIT Management. The inspections were performed in compliance with CIT Quality Assurance Unit procedures and the Good Laboratory Practice. The reported methods and procedures were found to describe those used and the results to constitute an accurate and complete reflection of the study raw data. Doctor of Biochemistry ' Head of Quality Assurance Unit and Scientific Archives (*) The dates indicated correspond to the dates of signature of audit reports by Study Director and Management. ri0lM ,,lalnTSC/vCBl 4 . Does ppm panySan^' SUMMARY At the re a u e s ^ fE lfA to c h e n ^ A ^ a ris ^ a -D e fc n s e , France, the acute oral toxicity of the test s u b s t a n c e j H ^ ^ ^ H j ^ ^ P i m m ^ U K v ' a s evaluated in rats according to OECD (No. 401,2 4 3 ^ ^ b m a ^ ^ 7 ^ n ^ ^ 9 2 / 6 9 / E E C r ,B .l) 31st July 1992) guidelines. The study was conducted in compliance with the principles of Good Laboratory Practice Regulations. M ethods The test substance was administered by oral route (gavage) to one group of ten fasted Spfague-Dwl rats (five males and five females). The test substance was administered undiluted at the dose o f 2000mg/kg, taking into consideration that its specific gravity was 1.05 g/ml. Clinical signs, mortality and body weight gain were checked for a period of up to 14 days following the single administration of the test substance. All animals were subjected to necropsy. Results No deaths occurred at 2000 mg/kg. The general behaviour and body weight gain of the animals were not affected by treatment with the test substance. No apparent abnormalities were observed at necropsy in all animals. Conclusion Under our experimental conditions, the oral LDo of the test substance! equal to or higher than 2000 mg/kg in rats. No signs o f toxicity were observed at this dose? . According to the classification criteria laid down in Commission Directive 93/21/EEC (27th April 1993) adapting to technical progress for the eighteenth time Council Directive 67/548/EEC, the test substance does not present a significant acute toxic risk if swallowed. Company noi contain TSCACBl RESUME Paris-la-Dfense, France, la toxicit aigu du produit Prs administration unique par voie orale chez le Rat a t value conformment aux lignes directrices de l'OCDE (n 401, 24 fvrier 1987) et de la CEE (92/69/EEC, B. 1,31 juillet 1992). L'tude a t ralise conformment aux rgles de Bonnes Pratiques de Laboratoire. M thode Le produit a t administr par^voie orale (gavage) un groupe de 10 rats Sprague-Dawley (5 mles et 5 femelles) mis la dite hydrique. L'administration a t effectue avec le produit non dilu la dose de 2000 mg/kg, en tenant compte de sa densit (d - 1,05 g/ml). ' Les signes cliniques, la mortalit et l'volution pondrale des animaux ont t suivis pendant une priode de 14 jours aprs l'administration unique du produit. Un examen anatomopathologique a t effectu sur tous les animaux. Rsultats La mortalit est nulle la dose de 2000 mg/kg. Aucun signe clinique n'est observ pendant l'tude. L'volution pondrale des animaux n'est pas influence par le traitement. L'autopsie des animaux ne met en vidence aucune anomalie apparente. Conclusion _ D a n ^ jio ^ ^ o n d itio n s exprimentales, la DL 0 orale du produit i fcst suprieure ou gale 2000 mg/kg chez le Rat. Aucun signe de to x ic it^ e s t "observ cette dose. Selon les critres de classification dcrits dans la Directive 93/21/CEE (27 avril 1993) portant dix-huitime adaptation au progrs technique de la Directive 67/548/CEE, le produit ne prsente pas de risque toxique aigu significatif en cas d'ingestion. Dos note' Cll/Study INO. 18/4D 1 5 1. INTRODUCTION The objective of this study was to evaluate the toxicity of the test substance following a single oral administration in rats. In the assessment of the toxic characteristics of a test substance, determination of acute oral toxicity is an initial step. It provides information on health hazards likely to arise following a short-term exposure by the oral route in humans. The study was conducted in compliance with: . OECD guideline No. 401, 24th February 1987, . EC Directive No. 92/69/EEC, B. 1, 31st July 1992. 2. MATERIALS AND METHODS 2.1 TEST SUBSTANCE 2.1.1 Identification The test substance ised in the study was supplied by Elf Atochem S.A. The tesf'substance was identified as follows: . name: - protocol and labelling:] . batch number: - protocol and labelling]] . Elf Atochem filing numb . description] . container: on plastic tlasK . date o f receipt: 5 May 1999 . storage conditions: at room temperature and protected from light . expiry date: May 2000. Data relating to the characterization of the test substance are documented in a test article description and in an analytical certificate (presented in appendix 1) provided by the Sponsor. 2.1.2 Formulation procedure The test substance was used undiluted. Company Sanitized. Does not contain TSCA CBJ v~xi/tjiuujr mu. i ]y 2.2 TEST SYSTEM 2.2.1 Animals Species, strain: rat, Sprague-Dawley ICO: OFA-SD (IOPS Caw). Reason for this choice: rodent species generally accepted by regulatory authorities for this type o f study. Breeden Iffa Credo, 69210 L'Arbresle, France. Number and sex: one group of ten animals (five males and five females). Age/weight: on the day of treatment, the animals were approximately 6 weeks old, and had a mean body weight standard deviation of 177 4 g for the males and 145 7 g for the females. Acclimatization: at least 5 days before the beginning of the study. Identification of the animals: the animals were identified individually by earmarks or eamotches. 2.2.2 Environmental conditions During the acclimatization period and throughout the study, the conditions in the animal room were set as follows: . temperature: 21 2C . relative humidity: 30 to 70% . light/dark cycle: 12 h/12 h . ventilation: approximately 12 cycles/hour of filtered, non-recycled air. The temperature and relative humidity were under continuous control and recording. The records were checked daily and filed. In addition to these daily checks, the housing conditions and corresponding instrumentation and equipment are verified and calibrated at regular intervals. The animals were housed in polycarbonate cages (48 cm x 27 cm x 20 cm). Each cage contained one to seven animals of the same sex during the acclimatization period and five rats of the same sex during the treatment period. Each cage contained dust-free sawdust (SICSA, 94142 Alfortville, France). Bacteriological and chemical analyses of the sawdust, including the detection of possible contaminants (pesticides, heavy metals), are performed regularly by external laboratories. The results of these analyses are archived at CIT. 2.2.3 Food and water All the animals had free access to A 04C pelleted diet (UAR, 91360 Villemoisson-sur-Orge, France), except as noted in "2.3.1 Fasting of the animals". Each batch of food was analysed by the supplier for composition and contaminant levels. The diet formula is presented in appendix 2. Drinking water filtered by a FG Millipore membrane (0.22 micron) was provided ad libitum. Bacteriological and chemical analyses of the water and diet, including the detection of possible contaminants (pesticides, heavy metals and nitrosamines), are performed regularly by external laboratories. The results of these analyses are archived at CIT. No contaminants were known to have been present in the diet, drinking water or bedding material at levels which may be expected to have interfered with or prejudiced the outcome of the study. Company Sanitized. Does not contain TSC CBf: 2 3 TREATMENT 23.1 Fasting of the animals The animals were fasted for an overnight period of approximately 18 hours before dosing, but had free access to water. Food was given back approximately 4 hours after administration of the test substance. 2.3.2 Administration of the test substance As the test substance was anticipated to be non-toxic at 2000 mg/kg, a limit test was performed by administering 2000 mg/kg of the test substance to one group of ten animals (five males and five females). The test substance was administered undiluted, taking into consideration its specific gravity (1.05 g/ml). The administration was performed in a single dose by oral route using a metal gavage tube fitted to a 1 ml plastic syringe (0.01 ml graduations). The volume administered to each animal was adjusted according to body weight determined on the day of treatment. 2.3.3 Chronology of the study The single administration was performed on 13 July 1999 in the morning (day 1) and was followed by a 14-day observation period until 27 July 1999 (day 15). 2.4 CLINICAL EXAMINATIONS 2.4.1 Clinical signs and mortality The animals were observed frequently during the hours following administration of the test substance, for detection of possible treatment-related clinical signs. Thereafter, observation of the animals was made at least once a day until day 15. Type, time of onset and duration of clinical signs were recorded for each animal individually. Time of death was recorded individually, in terms of the number of hours or days after dosing. 2.4.2 Body weight The animals were weighed individually just before administration of the test substance on day 1 and then on days 8 and 15. The body weight gain of the treated animals was compared to that o f CIT control animals with the same initial body weight. , nnTSCACBl v ix /iJiu u jr iiu . io /* r j ir u v 11 % 2.5 NECROPSY On day 15, all animals were killed by carbon dioxide asphyxiation and a macroscopic examination was performed. After opening the thoracic and abdominal cavities, a macroscopic examination of the main organs (digestive tract, heart, kidneys, liver, lungs, pancreas, spleen and any other organs with obvious abnormalities) was performed. In case of macroscopic lesions, organ samples were taken and preserved in 10% buffered formalin. No microscopic examination was performed. 2.6 DATA EVALUATION Evaluation of the toxicity of the test substance following a single oral administration in rats should include the relationship, if any, between the animals'exposure to the test substance and the incidence and severity of all abnormalities including behavioural and clinical abnormalities, macroscopic lesions, body weight changes, mortality and any other toxic effects. 2.7 PROTOCOL ADHERENCE The study was performed in accordance with Study Protocol No. 18745 TAR and subsequent amendments, with the following deviations from the agreed Study Protocol: . the temperature and relative humidity recorded in the animal room were sometimes outside of the target ranges specified in the protocol. These minor deviations were not considered to compromise the validity or integrity of the study. 2.8 ARCHIVING The study documentation and specimens generated during the course of the study are archived at CIT, 27005 Evreux, France, for 10 years after the end of the in vivo phase of the study. The archived study materials include: . protocol and possible amendments, . raw data, . correspondence, . final report and possible amendments. On completion of this period, the archived study materials will be returned to the Sponsor, or may be archived at CIT for a further period. m TscA Cta e r 0 tc o o lal . ooos Com? my S a n A ^ ' -' VL 3. RESULTS 3.1 CLINICAL EXAMINATIONS 3.1.1 Clinical signs and mortality (table 1) No clinical signs and no deaths were observed during the study. 3.1.2 Body weight (figures 1 and 2, tables 2 and 3) The body weight gain of the treated animals was similar to that of CIT historical control animals. 3.2 PATHOLOGY (table 4) Macroscopic examination of the main organs of the animals revealed no apparent abnormalities. 4. CONCLUSION Under our experimental conditions, the oral LDo of the test substancel is equal to or higher than 2000 mg/kg in rats. No sighs o f toxicity were observed at this dose According to the classification criteria laid down in Commission Directive 93/21/EEC (27th April 1993) adapting to technical progress for the eighteenth time Council Directive 67/548/EEC, the test substance does not present a significant acute toxic risk if swallowed. ____ _ ishl ,nQicoirvia-jnn lS:C A cB* .,,,, A .O aS % Figure 1: Body weight of treated rats j n c s,,s-(AConiain SanB&ed* Figure 2: Body weight of CIT historical control rats e.g. CIT Historical data of animals dosed by the oral route: results of control animals / ^ S C A C B l October 1995 to December 1997. . 0^ c n 3 W A A / W /klAw* V i I V i 1 U / T ^ A 15 Table 1: Individual clinical signs and mortality Dose (mg/kg) Time Animals Males Females Mortality Clinical signs 2000 lh - 2h - 4h } 01-02-03-04-05 06-07-08-09-10 D 2 t o D 15 J min : minutes h : hour D : day No None not u c k C 8 SaWSB*-0 0 * G prnP ^- / W 'tU U J 1U Table 2: Individual and mean body weight and weekly body weight change (g) Dose mg/kg Volume ml/kg Sex Animals 1 Days (1) 8 (1) 2000 1.91 Male 01 174 64 238 62 02 173 80 253 69 03 177 73 250 56 04 180 74 254 58 05 182 72 254 48 M 177 73 250 59 SD 4 6 7 8 2000 1.91 Female 06 144 39 183 8 07 136 51 187 14 08 151 43 194 37 09 142 32 174 46 10 154 44 198 33 M 145 42 187 28 SD 7 7 9 16 ( I) - Body weight gain M -Mean SD - Standard Deviation 15 300 322 306 312 302 308 9 191 201 231 220 231 215 18 C p T flP ^^ .1 OOGS ^ % Table 3: Mean body weight and weekly body weight change of CIT historical control rats BODY WEIGHT OF CONTROL RATS (g) Dose mg/kg Volume ml/kg Sex Days 18 0 10 Male M 182 SD 10 n 45 261 12 45 0 10 Female M 147 SD 10 n 45 190 15 45 M : mean SD : standard deviation n : number of animals 15 315 21 45 215 17 45 Dose mg/kg BODY WEIGHT CHANGE OF CONTROL RATS (g) Volume ml/kg Sex Days 1 to 8 8 to 15 0 10 Male M SD 79 54 7 13 0 10 Female M SD 43 25 87 M : mean SD : standard deviation e.g.: CIT Historical data of animals dosed by the oral route: results of control animals from October 1995 to December 1997. Sanitized. Doss not contain TSCACBl Company Sanmz-a- v ~ i i / o u u i y iNU. i o / h j i / \ j v m Table 4: Individual macroscopic examinations at necropsy 18 Dose Time mg/kg Animals Males Females Macroscopic abnormalities 2000 D: day D 15 01-02-03-04-05 06-07-08-09-10 No apparent abnormalities ontPanXSS aanto- - . ^ l M c W a T S O i C ^; iy APPENDICES San' Co>nPan^ tscacb^ n0t contain 1. Test article description and analytical certificate TSCft C3'L oS no* C O O ^ TOXICOLOGY DEPARTMENT CONFIDENTIAL April 99 elf atochem s.a. La dfense 10, cours Michelet 92091 Paris-la-Dfense, France TEST ARTICLE DESCRIPTION I PHYSICAL AND CHEMICAL PROPERTIES Appearance Melting point Flash point Solubility TOXICOLOGICAL INFORMATIONS AND USE SAFETY See safety data sheet___________________________________ STORAGE AND DISPOSAL Storage Expiry date Disposal : in dark and at room temperature : may 2000 . : incineration not contain T S C & C B I Compaq Sanfflzed. Does ^ n /o tu ay Ino. i s /^ d i elf atochem Z) ) Elf Atochem S.A. Usina de VBarj-SiitM-PuJ ZA Villen Sein! Paul Rleui B.P. 20 60870 a e u x (Fiance) T4I : 44.74.44.7i - Fa* : Jl.74.12.07 - T4te. : 140 429 ATO VSP 1 VilJcr S In| Paul, Ic 9 N ovem bre 1999 iv: Lot N* Extrait Scc Point clair pH Taille des Particules Test d'application Cuir olophobie et hydrophobie Unit Rsultat Spcification Mthode de d'analyse fabrication % IAT LCU 622 m mC LCU057 m (* )________ -VLCU 642 nm LCU 672 m m LCU 674 Le Cbcf de Service du Laboratoire SIGNATURE: NOM : Roher! Gmppo NuvcjoC'ioifjJ 1"ur. ii inotconlalnTSCACI C o m p a n y . S a n if a s d . Do- LTI'/Study No. 18745 1, % ) 23 2. Diet formula Sanv ttcontam w * w i w 24 Ref: A04 COMPLETE DIET RAT AND MOUSE MAINTENANCE DIET Appearance: 15 mm diameter pellets or powder Conditioning: 25 kg double paper bag with aluminium on the outside Daily portion: Rat 18-25 g, Mouse 5-10 g, water ad libitum. FORMULA % Cereals and cereal byproducts..... Vegetable protein (soya bean meal, yeast)................................... Animal protein (fish)......................... Vitamin and mineral mixture....... 88 7 2 3 AVERAGE ANALYSIS % Calorific value (KCal/kg)............. 2900 Moisture............................................ 12 Proteins............................................. 17 Lipids................................................. 3 Carbohydrates (N .F.E .)............... 58.7 Fibre................................................... 4 Minerals (ash).................................... 5 MINERALS (calculated in mg/kg) Nat CMV vai. vai. Total p ........ .... 5900 C a ..... .... 3300 K ........ .... 6700 N a..... .... 300 M g.... .... 1900 M n.... .... 50 Fe....... .... 90 C u..... .... 15 Z n ...... .... 40 C o...... ..:. T I .............. 0.3 ' 0 5000 0 1600 100 40 150 15 45 1.5 0 5900 8300 6700 1900 2000 90 240 30 85 1.5 0.3 AMINO ACID VALUES (calculated in mg/kg) VITAMINS (calculated per kg) Nat CMV vai. vai. Total Arginine........................................ 9800 Cystine.......................................... 2300 Lysine............................................. 8500 Methionine.................................... 3200 Tryptophan.................................... 1900 Glycine.......................................... 8100 FATTY ACID VALUES (calculated in mg/kg) Palmitic acid.................................. 2600 Palmitoleic acid.............................. Traces Stearic acid.................................... 500 Oleic acid...................................... 8000 Linoleicacid.................................. 14500 Linolenic acid................................. Traces Vitamin A Traces Vitamin D3 Traces Vitamin B 1 6 mg Vitamin B2 2 mg Vitamin B3 10 mg Vitamin B6 1.3 mg Vitamin B 12 0.01 mg Vitamin E 15 mg Vitamin K3 0.25 mg Vitamin PP 60 mg Folic acid 0.5 mg Biotin 0.04 mg Choline 1200 mg 7500 IU 1500 r u 1 mg 4.5 mg 6.5 mg 1.3 mg 0.01 mg 15 mg 2.25 mg 15 mg 0 mg 0 mg 400 mg 7500 IU 1500IU 7 mg 6.5 mg 16.5 mg 2.6 mg 0.02 mg 30 mg 2.5 mg 75 mg 0.5 mg 0.04 mg 1600 mg Available under quality "Control Ref.: A04 C " UAR, 7 rue Gallini, 91360 Villemoisson - Tel: 01.69.04.03.57 - Fax : 01.69.04.81.97 (Ref. Doc. UAR: 1992) tsc a c s* r.at c o n ta i e o m p s i .y Sanili: ;ed.C O C S