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SPONSOR Elf Atochem S.A. Cours Michelet La Dfense 10 92091 Paris-la-Dfense CEDEX France TEST SUBSTANCE STUDY TITLE ACUTE ORAL TOXICITY EMRATS STUDYDIRECTOR Xavier Manciaux AR226-3141 CONFIDENTIAL IFMrechercha SN C. AU CAflMl D s H O 00C f SIl a SOOAl ;.MISST l `0 0 i tVRLUX >pj> r,f.n m B z \ t w i n STUDY COMPLETION DATE 13 December 1999 PERFORMING LABORATORY . err Centre'International de Toxicologie BP 563 - 27005 Evreux - France LABORATORY STUDY NUMBER 18748 TAR Sanitized. D oes not contain TSCACffll Company C E N T R E INTERNATIONAL D E T O X IC O LO G IE B. R S63 27005 Evreux Cedex France T!.:+33 2 32 29 26 26 dl/CUUy INO. iO/HO 1 .11 niuiiiciu o.rt. 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 o f the animals 2.3.2 Administration o f the test substance 2.3.3 Chronology o f 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 3. RESULTS 3.1 CLINICAL EXAMINATIONS 3.1.1 Clinical signs and mortality (table i) 3.1.2 Body weight (figures 1 and 2, tables 2 and 3) 3.2 PATHOLOGY (table 4) 4. CONCLUSION n o t contain T S C A j^ i Sanifeed. Doc C om ply L.11/Study NO. 1 8 /4 8 1 ,ll A to cn e m Si.A. 5 Figure 1: Body weight of treated rats Figure 2: Body weight of CIT historical control rats Table 1: 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 change o f CIT historicalcontrol rats Table 4: Individual macroscopic examinations at necropsy 13 14 15 16 17 18 APPENDICES 1. Test article description and analytical certificate 2. Diet formula 19 20 23 and 24 ,s r ,ot c O ^ SC* C" S a n l * 4 - 001 QOfiVPaTV^ CIT/Study No. 18748 T ;if Atochem S.A. 4 STATEMENT OF THE STUDY DIRECTOR The study was performed in compliance with the principles o f Good Laboratory Practice as described in: . OECD Principles on Good Laboratory Practice (as revised in 1997), ENV/M/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 o f 18 December 1986 on the harmonization o f laws, regulations or administrative provisions relating to the application o f the Principles o f Good Laboratory Practice and the verification of their applications for tests on chemical substances (OJ No. L 15 o f 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. Toxicology X. Manciaux Study Director Doctor o f Pharmacy Date: 13 December 1999 OTHER SCIENTISTS INVOLVED IN THIS STUDY For Pharmacy: P.O. Guillaumat Doctor o f Pharmacy For Toxicology: C. Pelcot Study Supervisor rmTSCACBl CIT/Study No. 18748 T [Elf Atochem S.A. 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 8 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 o f critical procedures relevant to this study type were also made by the Quality Assurance Unit. The findings o f 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 o f the study raw data. L. Valette-Talbi Date: 13 December 1999 Doctor of Biochemistry Head of Quality Assurance Unit and Scientific Archives (*) The dates indicated correspond to the dates o f signature o f audit reports by Study Director and Management. contationTSCACBl Got CIT/Study No. 18748 1 ,ir m ocnem o./\. U SUMMARY At the request o f E lf Atochem S^ P a r i s ^ ^ e f e n s e , France, the acute oral toxicity o f the test s u b s t a n c l B i i ^ M l | | H | B H 0 H H R was evaluated in rats according to OECD (No. 401^ 4t h ^ b r u a r ^ 98^andEC ^(92/69/EEc! B .l, 31st July 1992)guidelines. The study was conducted in compliance with the principles of Good Laboratory Practice Regulations. Methods The test substance was administered by oral route (gavage) to one group of ten fasted Sprague-Dawley rats (five males and five females). The test substance was administered undiluted at the dose o f 2000m g/kg, taking into consideration that its specific gravity was 1.1 g/ml. Clinical signs, mortality and body weight gain were checked for a period o f up to 14 days following the single administration o f the test substance. All animals were subjected to necropsy. ' Results No deaths occurred at 2000 mg/kg. The general behaviour and body weight gain o f 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 o f the test s u b s ta n c e ^ P H fe lH M M H H iV ^ ^ `^ R s 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. TSCACBI .niiized.D oss*; \ conta0 Compr'V s u n /s t u a y n o , i /^ks i .h Aiocnem z>./\. / RESUME A l a d e m a n d ^ j ^ l ^ t o h e m S . A ., Paris-la-Dfense, France, la toxicit aigu du produit i 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. Mthode 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 --1,1 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 Dans nos conditions exprimentales, la DL 0 orale du produit|_ est suprieure ou gale 2000 mg/kg chez le Rat. Aucun signe de toxicit n'est 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 aig significatif en cas d'ingestion. ! ordin fSCACBV , so u s no CIT/Study No. 18748 T :if Atochem S.A. 8 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 o f the toxic characteristics o f a test substance, determination of acute oral toxicity is an initial step. It provides information on health hazards likely to anse 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 .l, 31st July 1992. 2. MATERIALS AND METHODS 2.1 TEST SUBSTANCE 2.1.1 Identificatioi The test substanca sed in the study was supplied by Elf Atochem S.A. The test substance was identified as follows: . name: - protocol and labelling:] . batch number: - protocol and labellin g^ . E lf Atochem filing number: CAL 1392/99 . description: amber liquid . container: one smoked glass flask . date of receipt: 5 May 1999 ' . storage conditions: at room temperature and protected from light . expiry date: May 2000. Data relating to the characterization o f the test substance are documented in a test article description and in analytical certificate (presented in appendix 1) provided by the Sponsor. 2.1.2 Formulation procedure The test substance was used undiluted. C o m p aq Saniti28^ - tscacbi contain CIT/Study No. 18748 T, Elf Atochem S.A. 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 Crdo, 69210 L'Arbresle, France. Number and sex: one group o f ten animals (five males and five females). Age/weight: on the day o f treatment, the animals were approximately 6 weeks old, and had a mean body weight standard deviation of 179 8 g for the males and 144 5 g for the females. Acclimatization: at least 5 days before the beginning o f the study. Identification o f the animals: the animals were identified individually by 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 o f 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 o f the same sex during the acclimatization period and five rats of the same sex during the treatment period. Each cage contained dust-free sawdust (SICS, 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 o f 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 o f 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 o f possible contaminants (pesticides, heavy metals and nitrosamines), are performed regularly by external laboratories. The results o f 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. Comp3RV .V V f inot contain TSCA CBl CIT/Study No. 18748T it Atochem .A. iu 23 TREATMENT 2.3.1 Fasting of the animals The animals were fasted for an overnight period o f approximately 18 hours before dosing, but had free access to water. Food was given back approximately 4 hours after administration o f the test substance. 2.3.2 Administration o f 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 o f the test substance to one group o f ten animals (five males and five females). The test substance was administered undiluted, taking into consideration its specific gravity (1.1 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 o f treatment. .. 2.3.3 Chronology o f 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 o f the test substance, for detection o f possible treatment-related clinical signs. Thereafter, observation of the animals was made at least once a day until day 15. .... Type, time o f onset and duration o f clinical signs were recorded for each animal individually. Time o f death was recorded individually, in terms o f the number o f hours or days after dosing. 2.4.2 Body weight . The animals were weighed individually just before administration o f 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. Company Sar.ucci, fm TSCACBI CIT/Study No. 18748 T ilf Atochem S.A. 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 o f 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. N o microscopic examination was performed. 2.6 DATA EVALUATION Evaluation o f the toxicity o f 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 o f 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. 18748 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 o f the study are archived at CIT, 27005 Evreux, France, for 10 years after the end of the in vivo phase o f 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. Company Sanitised. Doss not contain TSCA CBl CnVStudy No. 18 /48 li ,u Aiocnem 8 .a . iz 3. RESULTS 3.1 CLINICAL EXAMINATIONS 3.1.1 Clinical signs and mortality (table 1) N o 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 o f the treated animals was similar to that o f CIT historical control animals. 3.2 PATHOLOGY (table 4) Macroscopic examination o f the main organs o f the animals revealed no apparent abnormalities. 4. CONCLUSION Under our experimental conditions, the oral LD0 o f the test substano__ s 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 ISCA CIT/Study No. 18748 T ilt Atochem S.A. Figure 1: Body weight o f treated rats U 3 not coniata TSCA^Cl ;Q. V" Company S a ^ ` CIT/Study No. 1874S 1 ,ir Atocnem .a . Figure 2: Body weight o f CIT historical control rats 14 e.g.: CIT Historical data of animals dosed by the oral route: results of control animals from October 1995 to December 1997. \ contain TSCACB^ Company CIT/Study No. 18748 1. t i r Atocnem .a . io Table 1: Individual clinical signs and mortality Dose (mg/kg) Time Animals Males Females Mortality Clinical signs 2000 1h 1 2 h -4 h } 01 "02-03-04-05 06-07-08-09-10 D 2 to D 15 J min : minutes h : hour D : day No None ,,01 contain T S C A C ft Company S a n ili 0' D oes CIT/Study No. 18748 TAJ ji Aiocnem o./v 10 t. Table 2: Individual and mean body weight and weekly body weight change (g) Dose Volume Sex Animals mg/kg ml/kg 1 (1) 8 (1) 15 2000 1.82 Male 01 194 84 278 72 350 02 179 74 253 58 311 03 176 76 252 54 306 04 175 73 248 62 310 05 173 68 241 61 302 M 179 75 254 61 316 SD 8 6 14 7 19 2000 1.82 Female 06 142 42 184 31 215 07 146 31 177 34 211 08 137 36 173 . 29 202 09 142 42 184 19 203 10 151 37 188 27 215 M 144 38 181 28 209 SD 5 5 6 6 6 (1) - Body weight gain M - Mean SD - Standard Deviation Com PanV UT/StUdy NO. 18748 li m Atocnem a.A. i/ 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 BODY WEIGHT CHANGE OF CONTROL RATS (g) Dose mg/kg Volume ml/kg Sex 0 # 0 10 10 Male Female M : mean SD : standard deviation M SD M SD Days 1 to 8 79 7 43 8 8 to 15 54 13 25 7 e.g.: CIT Historical data of animals dosed by the oral route: results o f control animals from October 1995 to December 1997. 5 n oK on alCACBl eotnpany Sar ` CIT/Study No. 18748X It Atochem S.A. 18 Table 4: Individual macroscopic examinations at necropsy Dose mg/kg Time Animals Males Females 2000 D: day D 15 01-02-03-04-05 06-07-08-09-10 Macroscopic abnormalities No apparent abnormalities ,n0t contain t s c a c b i Sanitiisd' QOQ` Company u /Mucneiu o./\. APPENDICES 3 ,,01 M m S M I-0 * CoW Pa n ^ CIT/Study No. 18748 T. t i t Atoctiem S.A. V 1. Test article description and analytical certificate not contain TSAB|._ ^ojnpany S anvVe.eS CIT/Study NO. 18748 T, t i t Atochem 8.A. 'Ll TOXICOLOGY DEPARTMENT CONFIDENTIAL April 99 elf atochem s.a. La dfense 10, cours Michelet 92091 Paris-la-Dfense, France TEST ARTICLE DESCRIPTION IDENTITY Test article name Chemical name CAS number EINECS number Purity Origin and batch Batch Elf Atochem filing number t l f Atochem, VSP Ca H 3 9 2 /9 9 PHYSICAL AND CHEMICAL PROPERTIES Appearance pH Density Boiling point Flash point Solubility . amber liquid 5+ 1 1100kg/m3 at 20C 98-100 C no flash point water: insoluble soluble in alcohols, aromatic hydrocarbons TOXICOLOGICAL INFORMATIONS AND USE SAFETY See safety data sheet Company Sanitized. Does not contain t sc a c b i CIT/StudyNo. 18748 T, eiF atochem ilf Atochem S.A. 22 Eli A tochem S.A. Usina da VHervSaint-Paul ZAViDerj Saint Paul ftteux 0.P. 20 60870 Rieux (Flanco) T4J:Xa.74.4a.7A. Fax:44.74.42.07 . Tiax: 140420ATOvsp tf Y illerj Saint Paul, le 9 Novembre 1W Lot N " Extrait See Tertio Butanol Acrylamide Acide acrylique Test d'extinction Unit % Rsultat Spcification de fabrication Mthode d'analyse 30,3 29 31 LCU 622 % 0.95 O 1 LCU 653 % 0,07 O O .l LCU 658 % 0.1 02 LCU 658 / positif p o s it if . SA-1021 Le Chef de S e m a du Laboratoire SIGNATURE : Sanitized.. no^s nn^ot` vcontain TSCA CBi hnui'C.n( apitjl 3ur, 123?n0 Fruorj CIT/Study No. 18748 TAJ % li' Atochem S.A. 23 i . 2. Diet formula S not contain T S C A C ^ Company Sa;ntfized, D2: m / i u a y ino. i /^+ i /\ j 'eu ruu^iicm o.n. 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...... 88 Vegetable protein (soya bean meal, yeast)........................................... 7 Animal protein (fish)........................... 2 Vitamin and mineral mixture....... 3 AVERAGE ANALYSIS % Calorific value (K Cal/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) Arginine........................................... 9800 Cystine............................................. 2300 L y s in e ................................ 8500 Methionine....................................... 3200 Tryptophan....................................... 1900 G lycine............................................. 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 VITAMINS (calculated per kg) Nat CMV vai. vai. Total Vitamin A Traces Vitamin D3 Traces Vitamin B1 6 mg Vitamin B2 2 mg Vitamin B3 10 mg Vitamin B6 1.3 mg Vitamin B12 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 7 5 0 0 IU 1500IU 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 1500 IU 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 Gallieni, 91360 Villemoisson - Tel: 01.69.04.03.57 - Fax : 01.69.04.81.97 (Ref. Doc. UAR: 1992) ^ i<*- r* \* 0 **** C om paq S an---- conlainTSCA CB