Document pm29ZwMREokkXrDzqx79mzmrX

SPONSOR Elf Atochem S.A. Cours Michelet La Dfense 10 92091 Paris-la-Dfense CEDEX ' France AR226-3098 STUDY TITLE ACUTE DERMAL IRRITATION IN RABBITS TEST SUBSTANCE STUDY DIRECTOR Xavier Manciaux STUDY COMPLETION DATE 20 May 1998 PERFORMING LABORATORY Centre International de Toxicologie (C.I.T.) Miserey - 27005 Evreux - France LABORATORY STUDY NUMBER 16046 TAL Company San?t'**d. Do no o"*-" Company Sanitized. Doss not contain TSCA CBI J 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 8 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 Preparation and selection of the animals 2.3.2 Study design 2.3.3 Application of the test substance 2.3.4 Date of treatment 2.4 CUTANEOUS EXAMINATIONS 2.5 DESCRIPTION AND EVALUATION OF CUTANEOUS REACTIONS 2.6 INTERPRETATION OF RESULTS AND CLASSIFICATION OF SUBSTANCES 2.6.1 Interpretation of the results 2.6.1.1 Criteria for irritation 2.6.1.2 Criteria for corrosion 2.6.2 Classification o f the test substances 2.6.2.1 Irritant substances 2.6.2.2 Corrosive substances ' * . 2.7 PROTOCOL ADHERENCE 8 8 8 9 9 9 9 10 10 10 10 10 10 11 11 11 11 11 11 11 12 12 2.8 ARCHIVING 12 3. RESULTS (table 1) 4. CONCLUSION fjO.I 13 s S i i S ' 5 ^ ` " " 'a," 'rscA fieiBpsfty 13 Table 1: Individual cutaneous examinations and mean values of the scores recorded at each reading (24, 48 and 72 hours) for each animal 14 APPENDICES 1. Test article description 2. Diet formula 16 17 19 and 20 J STATEMENT O F THE STUDY DIRECTOR The study was performed in compliance with the principles of Good Laboratory Practice as described in: . O.E.C.D. principles o f Good Laboratory Practice, Decision Concerning Mutual Acceptance of Data in the Assessment of Chemicals, C(81)30(final) Annex 2. May 12,1981. . Dcret N 90-206 du 7 mars 1990 concernant les Bonnes Pratiques de Laboratoire (Journal Offcil du 9 mars 1990), Ministre de l'Industrie et de l'Amnagement du Territoire. . Council Directive 87/18/E.E.C. 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 (O.J. 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 the Centre International de Toxicologie (C.I.T.), Miserey, 27005 Evreux, France. ' Toxicology X. Manciaux Date: 20 May 1998 Study Director Doctor of Pharmacy OTHER SCIENTISTS INVOLVED IN THIS STUDY For Pharmacy: P. O. Guillaumat Doctor of Pharmacy For Toxicology: C. Pelcot Study Supervisor not containTSCACBI STATEMENT OF QUALITY ASSURANCE UNIT Type of inspections ' Protocol Report Inspections 28 August 1997 11 May 1998 Dates Reported to Study Director (*) 2 September 1997 12 May 1998 Reported to Management (*) 2 September 1997 12 May 1998 At about the same time as the study described in this report, "process-based" and routine facility inspections of critical procedures relevant to this study type were made by the Quality Assurance Unit. The findings o f these inspections were reported to the Study Director and to C.I.T. Management. The inspections were performed in compliance with C.I.T. 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. L. Valette-Talbi Date: 20 May 1998 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. fee'd.tfoestio* SUMMARY At the request of E lf Atochem S.A., Paris-ja-Dfense, France, the potential of the test substance ____________________ [to induce skin irritation was evaluated in rabbits ! according to O.E.C.D. (No. 404, 17th July 1992) and E.C. (92/69/E.E.C., B4, 31st July 1992) guidelines. The study was conducted in compliance with the Principles of Good Laboratory Practice Regulations. Methods The study design was established according to available information on the test substance and the above guidelines. The test substance was applied for 4 hours to three male New Zealand White rabbits. A single dose of 0.5 ml of the undiluted test substance was applied to the closely-clipped skin of one flank. , The test substance was held in contact with the skin by means of a semi-occlusive dressing. Cutaneous reactions were observed approximately 1 hour, 24, 48 and 72 hours after removal of the dressing and then daily until the end of the observation period. The mean values o f the scores for erythema and oedema were calculated for each animal. Results Well-defined to severe cutaneous reactions were observed in all animals from day 1. These reactions persisted up to day 11 at the latest. Dryness of the skin was noted in all animals from day 5 or 6 up to day 13 or 15. Mean scores over 24, 48 and 72 hours for each animal were 3.0, 2.0 and 2.0 for erythema and 4.0, 2.0 and 2.7 for oedema. Conclusion Under our experimental conditions, the test substance is irritant when applied topically to rabbits. According to the classification criteria laid down in Commission Directive 93/21/E.E.C. (27th April 1993) adapting to technical progress for the eighteenth time Council Directive 67/548/E.E.C., the test substance was considered irritating to skin. Smilfeed. Does nol contain TSCA cm Company RESUME CA la_deniande_de_Elf Atochem S.A., garis-la-Dfense, France, les proprits irritantes du produit ^ | | [ m | | ^ H H | H H H | m y a p r s application cutane ont t values chez le Lapin selon les lignes directrices de l'O.C.D.E. (No. 404, 17 juillet 1992) et de la C.E.E. (92/69/E.E.C., B4, 31 juillet 1992). L'tude a t ralise conformment aux rgles de Bonnes Pratiques de Laboratoire. M thode L'tude a t ralise selon les informations disponibles sur le produit et les lignes directrices mentionnes ci-dessus. Le produit a t appliqu pendant 4 heures sur 3 lapins mles New Zealand White. Une dose unique de 0,5 ml de produit tel quel a t applique sur une surface de peau tondue d 'un des flancs. Le produit a t maintenu en contact avec la peau au moyen d'un pansement semi-occlusif. Les ractions cutanes ont t observes environ 1 heure, 24, 48 et 72 heures aprs l'enlvement du pansement puis quotidiennement jusqu' la fin de la priode d 'observation. La moyenne des scores a t calcule pour l'rythme et pour l'oedme pour chaque animal. Rsultats Des ractions cutanes bien dfinies svres sont observes chez tous les animaux partir du jour 1. Ces ractions persistent jusqu'au jour 11 au plus. Une scheresse cutane est note chez tous les animaux du jour 5 ou 6 au jour 13 ou 15. Les scores moyens calculs pour chaque animal aprs 24, 48 et 72 heures sont de 3,0 ; 2,0 et 2,0 pour l 'rythme et de 4,0 ; 2,0 et 2,7 pour l 'oedme. Conclusion Dans nos. conditions exprimentales, le produit!! considr irritant par voie cutane chez le Lapin. Selon les critres de classification dcrits dans la Directive 93/21/C.E.E (27 avril 1993) portant dix-huitime adaptation au progrs technique; de la Directive 67/548/C.E.E., le produit est considr irritant pour la peau. V a w m S r flize * 00>>W contain TSM I -- 'J 1. IN TRODUCTION The objective of this study was to evaluate the potential o f the test substanc to induce skin irritation following a single topical application to rabbits. In the assessment of the toxic characteristics of a test substance, determination of the irritant and/or corrosive effects on the skin of mammals is an important initial step. Information derived from this test serves to indicate the possible hazards likely to arise from exposure of the skin to the test substance. This study was conducted in compliance with: . O.E.C.D. guideline No. 4 0 4 ,17th July 1992. . E.C. Directive No. 92/69/E.E.C., B4,31st July 1992. 2. MATERIALS AND METHODS 2.1 TEST SUBSTANCE 2.1.1 Identificatioi The test substan used in the study was supplied by Elf Atochem S.A. The test substance was identified as follows: . name: - protocol and labellingj . batch number: - protocol and labelling . Elf Atochem filing number . description: . at receipt!" . on the test article descriptionj . container: one plastic flask . date of receipt: 23 August 1997 . storage conditions: at room temperature and protected from light . expiry date: August 1998. Data relating to the characterization of the test substance are documented in a test article description (presented in appendix 1) provided by the Sponsor. . At the finalization of the study report, an analytical certificate was not available. Characterization o f the test substance, which appropriately defines the tested batch, is under the responsibility of the Sponsor. The pH of the test substance wa 2.1.2 Formulation procedure The test substance was used undiluted. 'CsmpnfSanitized. Does noi contain TSCA 2.2 TEST SYSTEM 2.2.1 Animals Sex, species, strain: male New Zealand White rabbits. Justification of the test system : species generally accepted by regulatory authorities for this type o f study. Breeder: Elevage Cunicole de Val de Selle, 80160 Prouzel, France. Number of animals and identification: three animals were used, as recommended by the international guidelines and taking into account that a good correlation of results can be obtained with either three or six animals (1). The animals were identified individually with a metal tag in the ear. Weight: on the day of treatment, the animals had a mean body weight of 2.5 0.2 kg. Acclimatization: at least 5 days before the beginning of the study. 2.2.2 Environmental conditions The conditions in the animal room were set as follows: . temperature: 18 3C . 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 retained. In addition to these daily checks, the housing conditions and corresponding instrumentation and equipment were verified and calibrated at regular intervals. The animals were housed individually in polystyrene cages (35 cm x 55 cm x 32 cm or 48.2 cm x 58 cm x 36.5 cm). Each cage was equipped with a food container and a water bottle. 2.2.3 Food and water During the study, the animals had free access to 112 C pelleted diet (U.A.R., 91360 Villemoisson-sur-Orge, France). 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 F.G. Millipore membrane (0.22 micron) was provided ad libitum. Bacteriological and chemical analysis of the water and diet, including the detection of possible contaminants (pesticides, heavy metals and nitrosamines), are performed regularly by external laboratories. The results o f these analyses are archived at C.I.T. _ No contaminants are known to be present in the diet or drinking water at levels which may be expected to interfere with or prejudice the outcome o f the study. t (l) Hatoum, N.S.; Leach, C.L.; Talsma, D.M.; Gibbons, R.D.; Garvin, P.J.: A statistical basis for using fewer Rabbits in dermal irritation testing. Journal o f the American College of Toxicology. 9:49-60 (1990). . ___ jjgttjgy i gaftlifeed 'm m no! contain TSCA CBlf c "J 2.3 TREATM ENT 2.3.1 Preparation and selection of the animals The day before treatment, the flanks of each animal were clipped using electric clippers and the skin of each animal was examined. Only animals with healthy intact skin were used. 2.3.2 Study design The study design was established according to available information on the test substance and the O.E.C.D. (No. 404) and E.C. (92/69/E.E.C., B4) guidelines. The test substance was applied for 4 hours to three animals. 2.3.3 Application of the test substance The test substance was used undiluted. A single dose of 0.5 ml o f the test substance was placed on a 6 cm2 dry gauze pad (Cooprative Pharmaceutique Franaise, 77000 Melun, France), which was then applied to the right flank of the animals for 4 hours. The test substance and the gauze pad were held in contact with the skin by means of an adhesive hypoallergenic aerated semi-occlusive dressing (Laboratoires de Pansements et d'Hygine, 21300 Chenove, France) and a restraining bandage (Coheban), Laboratoires 3M Sant, 92245 Malakoff, France). The untreated skin served as control. No residual test substance was observed on removal o f the dressing. 2.3.4 Date of treatment Animal number 01 02 03 Date of treatment (day 1) 7 October 1997 7 October 1997 -7 October 1997 End of the observation period 20 October 1997 20 October 1997 21 October 1997 . 2.4 CUTANEOUS EXAMINATIONS The skin was examined approximately 1 hour, 24, 48 and 72 hours after removal o f the dressing. Following the O.E.C.D. and'E.C. guidelines: . when there was no evidence of dermal irritation after 72 hours, the study was ended. . when there was persistent cutaneous irritation after 72 hours, the observation period was extended to a maximum .of 14 days (until day 15) in order to determine the progress o f the lesions and their reversibility. . when severe irritant effects were observed, the animals were killed on humane grounds. Any change in the animals' behaviour was noted. Company Sanitized. Does not contain TSCACW 17 J 2.5 DESCRIPTION AND EVALUATION OF CUTANEOUS REACTIONS Dermal irritation was evaluated for each animal according to the following scoring scale: Erythema and eschar formation: . no erythem a.................................................................................................................. . very slight erythema (barely perceptible).................................................................. . well-defined erythema......................... . moderate to severe erythema.................. . severe erythema (beet redness) to slight eschar formation (injuries in depth)...... 0 1 2 3 4 Oedema formation . no oedema..................................................................................................................... . very slight oedema (barely perceptible)..................................................................... . slight oedema (edges of area well-defined by definite raising)..... .................... . moderate oedema (raised approximately 1 millimetre)........................................... . severe oedema (raised more than 1 millimetre and extending beyond area of exposure).................................................................................................................. 0 1 2 3 4 Any other lesions were noted. 2.6 INTERPRETATION OF RESULTS AND CLASSIFICATION OF SUBSTANCES The results obtained were evaluated in conjunction with the nature and the reversibility of the findings observed. 2.6.1 Interpretation of the results 2.6.1.1 Criteria for irritation A substance or a preparation is considered to be irritating to the skin if, when it is applied to healthy intact animal skin for up to 4 hours, significant inflammation is caused and which persists for 24 hours or more after the end of the exposure period. 2.6.1.2 Criteria for corrosion A substance or a preparation is considered to be corrosive if, when it is applied to healthy intact anim al skin, it produces full thickness destruction of skin tissue on at least one animal during the test for skin irritation, or if the result can be predicted, for example: from strongly acid or alkaline reactions. ' , '. 2.6.2 Classification of the test substances 2.6.2.1 Irritant substances - symbol Xi, indication o f danger "irritant", . - phrases indicating the nature of special risks: R 38: "Irritating to skin" Wrtcontain TSC A C flized.Uoes V " C 3 Inflammation is significant if: . the mean value o f the scores is two or more for either erythema and eschar formation or 'i oedema formation. The same will be the case where the test has been completed using three animals if the score for either erythema and eschar formation or oedema formation observed in two or more animals is equivalent to the value of two or more, . it persists in at least two animals at the end of the observation period. Specific effects such as hyperplasia, desquamation, discolouration, fissures, eschar and alopecia should be taken into account. All scores obtained at each reading time (24, 48 and 72 hours) for an effect are used by calculating the respective mean values. 2.6.2.2 Corrosive substances - symbol C, indication o f danger: "corrosive", - phrases indicating the nature of special risks: - R 34: "Causes bums" If, when applied to healthy intact animal skin, full thickness destruction of skin tissue occurs as a result of up to 4 hours exposure, or if this result can be predicted. - R 35: "Causes severe bums" If, when applied to healthy intact animal skin, full thickness destruction of skin tissue occurs as a result of up to 3 minutes exposure, or if this result can be predicted. 2.7 PROTOCOL ADHERENCE The study was performed in accordance with Study Protocol No. 16046 TAL and subsequent amendments, with the following deviation from the agreed Study Protocol: - the relative humidity recorded in the animal room was sometimes outside of the target ranges specified in the protocol. This minor deviation was not considered to compromise the validity or integrity o f the study. 2.8 ARCHIVING * The study documentation and specimens generated during the course o f the study are archived at C.I.T., 27005 Miserey, Evreux, France, for 10 years after the end o f 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 C.I.T. for a further period. CompanySanitize?. Dees netcontain TSna cw 3. RESULTS (table 1) Well-defined to severe cutaneous reactions (well-defined or moderate erythema, grade 2 or 3, and slight or severe oedema, grade 2 or 4) were observed in all animals from day 1. These reactions persisted up to day 11 at the latest. Dryness of the skin was noted in all animals from day 5 or 6 up to day 13 or 15. Mean scores over 24, 48 and 72 hours for each animal were 3.0, 2.0 and 2.0 for erythema and 4.0,2.0 and 2.7 for oedema. 4. CONCLUSION Under our experimental conditions, the test substance is irritant when applied topically to rabbits. According to the classification criteria laid down in Commission Directive 93/2I/E.E.C. (27th April 1993) adapting to technical progress for the eighteenth time Council Directive 67/548/E.E.C., the test substance was considered irritating to skin. pew , TSO&CB, J w' X W T W X i mJ Table 1: Individual cutaneous examinations and mean values of the scores recorded at each reading (24, 48 and 72 hours) for each animal Rabbit number Dermal Irritation lh D1 Scores 24h 48h D2 D3 Mean Interpretation irritation (+) 72h score (1) (-) D4 01 Erythema 2 3 33 3.0 (+) Oedema 4 4 4 4 Other ** ** 4.0 (+) 02 Erythema 2 2 2 2 2.0 (+) Oedema 4 2 2 2 Other ** ** 2.0 (+) 03 j Erythema 2 2 2 2 Oedema 4 4 2 2 Other **** 2.0 2.7 (+) (+) (1) mean o f scores on days 2, 3 and 4 h = hour D = day (+) = irritant according to E.E.C. criteria Sanitized. Does Hotcontain tso a r.m Table 1 (continued) Rabbit number Dermal Scores Irritation ______________________________ D5 D 6 D7 D8 D9 DIO D l l D12 D13 D14 D 15 01 Erythema 32 1 10 00 00 0 - Oedema Other 42000 00 00 0 - *SS S S S S S S * - 02 Erythema l 1110 00 00 0 - Oedema 000 00 00 00 0 - Other SSSSSSs SS *- 03 Erythema 2 2 2 2 2 2 1 0 0 0 0 Oedema 200 0 0 00 00 00 Other *SS S S S S S s s s D = day * - None S = Dryness o f the skin - --Cutaneous examination not performed JZmpafty Sanitizes. Dees fie! eeniafn TSCA 6I APPENDICES C&inpafty Sanitized. Does not contain Tsn* CBI 1. Test article description Company Sanitized Does no! contain TSCA CBf TOXICOLOGY DEPARTMENT CONFIDENTIAL 11August 1997 elf atochem s.a. La dfense 10, Cours Michelet 92091 Paris-la-Dfense cedex, France TEST ARTICLE DESCRIPTION IDENTITY Test article name CAS number EINECS number Purity Origin Batch | Elf Atochem filing number IPHYSICAL AND CHEM ICAL PROPERTIES Appearance Specific gravity Boiling point Vapor pressure Flash point Solubility TOXICOLOGICAL INFORMATIONS AND USE SAFETY see fiche de donnes de scurit STORAGE AND DISPOSAL Storage Expiry date Disposal in dark and at room temperature . August 1998 - incineration fjfflflpgiiy Sanitized. Does not contain TS tw J 2. Diet formula Iwflpiiy 'Sanlttawl. 'does m i contain TSCA CB Ref: 112 COM PLETE DIET RABBIT MAINTENANCE DIET Appearance: 4.5 mm diameter granules Conditioning: bags of 25 kgs Daily portion: in accordance with race and body weight, Rabbits 100-150 g, water ad libitum. FORMULA % MINERALS (calculated in mg/kg) Nat. CMV Cereals........................................ 43.8 val. val. Total Grain biproducts and legum es.. Vegetable protein (soya bean 49 P.............. ......... 3500 Ca .......... .... . 4500 3500 4500 7000 9000 meal, y east)................................ 4.2 K ............ ........ 11600 0 11600 Vitamin and mineral mixture.... 3 N a .......... ........ 400 M g .......... ........ 2100 1600 100 2000 2200 AVERAGE ANALYSIS % Calorific value (Kcal/kg).......... M o istu re...................................... P r o te i n s ....................................... L i p id s .......................................... M n ......... ........ Fe............ ...... 2200 C u .......... ...... 10 Zn .......... ...... 13 C o ........... ...... 2.7 I .............. ....... 40 160 12 30 0.1 O' 40 140 15 45 1.5 0 80 300 27 75 1.6 0 Carbohydrates (N.F.E.) 49.3 C l ............ ...... 500 3000 3500 Fibre............................................ 17 Minerals (ash)............................ 8 VITAMINS (calculated per kg) AMINO ACID VALUES Nat. CMV (calculated in mg/kg) val. val. Total Vitamin A 2850IU 6500IU 9350 IU Arginine................................... .. 6800 Vitamin D3 3 0 IU 1000IU 1030 IU Cystine...................................... .. 2100 Vitamin B1 4.3 mg 0 mg 4.3 mg L ysine....................................... .. 4600 Vitamin B2 3.8 mg Omg 3.8 mg Methionine............................... .. 1600 Vitamin B3 16 mg Omg 16 mg Tryptophan............................... .. 1400 Vitamin B6 1 mg 1 mg 2 mg Glycine...................................... .. 5200 Vitamin B12 0 mg Omg Omg Vitamin E 16 mg 10 mg 26 mg FATTY ACID VALUES (calculated in mg/kg) Vitamin K3 Vitamin PP 6 mg 55 mg 1 mg 5 mg 7 mg 60 mg Folic acid Omg Omg Omg Palmitic acid............................. . 6400 Biotin Omg Omg Omg Palmitoleic acid........................ . 0 Choline 850 mg 200 mg 1050 mg Stearic acid.................................. 600 'Vleso-Inositol Oleic acid................................... . 6400 Omg Omg Omg Linoleic acid.. 12100 Linolenic acid, 2400 Available under quality "Control Ref.: 112 C" U.A.R., 7 rue Gallieni, 91360 Villemoisson - T e l: 01.69.04.03.57 - Fax : 01.69.04.81.97 (Ref. Doc. U A R : 1992) es m i contain TSCA CBl